Search

Fetal exposure to excess glucocorticoid is associated with low birth weight and increased cardiovascular disease risk in first generation offspring. Such phenotypes can be produced experimentally through the administration of the synthetic glucocorticoid dexamethasone (Dex) to pregnant rats during the last week of gestation. These ‘programmed effects’ can be transmitted to a second generation through both maternal and paternal lines. The overall hypothesis for this thesis was that the transmission of programmed effects through the male line may result from alterations in fetal germ cells, which form sperm in adulthood. Epigenetic reprogramming of germ cells is characterised by the genome-wide erasure and subsequent re-establishment of 5-methylcytosine (5mC), however this process has not previously been described for the rat. Furthermore, the involvement of more recently identified cytosine modifications; 5-hydroxymethylcytosine (5hmC), 5- formylcytosine (5fC) and 5-carboxylcytosine (5caC), has not been characterised during germ cell ontogeny. Using immunofluorescence to study DNA modifications during late gestation I identified that 5hmC, 5fC and 5caC were present between e14.5 and e16.5 but absent thereafter. In contrast, 5mC was absent during this time but remethylation was noted from e19.5 onwards. Prenatal Dex exposure was associated with the presence of significantly more 5mC-positive germ cells at e19.5 relative to controls. This difference did not persist at e20.5 suggesting that Dex exposure promotes premature global remethylation. The mechanisms for this are unclear since there were no differences between groups in the localisation of the DNA methyltransferases DNMT3a and 3b, or in markers of normal testis maturation. To enable the study of gene-specific changes in DNA methylation in the germline a colony of Germ Cell Specific-Enhanced Green Fluorescent Protein (GCS-EGFP) rats was established and characterised. GCS-EGFP rats had a transgenerational decrease in pup weight with Dex exposure, as in Wistar rats. The expression of both established and novel candidate genes was compared between strains. Multiple genes across different pathways had altered expression, with some affected in both Wistar and GCS-EGFP rats, whilst other differences were strain-specific. Enhanced Reduced Representation Bisulfite Sequencing was performed on liver and fetal germ cells from males exposed to Dex in utero to explore effects on DNA methylation. These studies confirm that epigenetic reprogramming occurs in the rat and that this process may be susceptible to modification by prenatal Dex exposure. GCS-EGFP rats also exhibited a Dex programming phenotype, with decreased pup weight and altered liver gene expression. The use of this unique strain of rats will permit dissection of the mechanisms for the transmission of programmed phenotypes across generations.

Oct4, a transcription factor belonging to the fifth class of POU proteins (POUV), plays essential roles in the maintenance of pluripotency, differentiation and the generation of induced pluripotent stem cells (iPSCs). Oct4 regulates two levels of pluripotency, which are distinguished by their gene expression profiles and epigenetic status, namely the naïve and primed state of pluripotency. Embryonic stem cells (ESCs) and embryonic germ cells (EGCs), which are isolated from inner cell mass and primordial germ cells in the embryo, respectively, are in vitro models in which the naïve state is propagated through self-renewal. Epiblast stem cells (EpiSCs) and traditional human ESCs have gene expression profiles that are closest to the post-implantation epiblast, which is closer to embryonic differentiation, and exhibit a primed state of pluripotency. As Oct4 is important for pluripotency in all these cell types, where it regulates different targets, it appears to have two distinct sets of functions, namely germ cell/naïve ESC-like activity and epiblast/primed pluripotency-like activity. Based on protein sequences and syntenic gene analysis, Oct4/POUV homologs of jawed vertebrates can be classified into two subfamilies: POU5F1 and POU5F3, which are thought to originate from a genome duplication event that occurred in a common ancestor. Most extant vertebrates have lost one of these paralogs, while a small fraction, including coelacanths, axolotls, turtles, and marsupials, retains both POUV forms. In my thesis, I investigated the gene duplication event that underlies divergence of POU5F1 and POU5F3 in both expression pattern and specialised function. In particular, I focused on species that have retained both genes and asked whether POUV functional divergence correlates with ancestral origin. To test the function of POU5F1 and POU5F3, I substituted endogenous mouse Oct4/Pou5f1 with different POUV proteins using a cell line in which endogenous Oct4 expression can be silenced with tetracycline (ZHBTc4). Results showed that POU5F1 proteins had a greater capacity to support naïve ESC pluripotency and self-renewal than POU5F3 proteins. Global transcriptome analysis of the POUV-rescued ESC lines revealed that coelacanth POU5F1 protein regulates gene expression in a similar manner to mouse Oct4, in that genes involved in stem cell maintenance, reproduction and development are upregulated in ESCs rescued by POU5F1, but not POU5F3. Coelacanth POU5F3 rescued lines, however, expressed genes involved in various cell differentiation programs, including cell adhesion (e.g. E-cadherin and N-cadherin). This suggests that POU5F3 plays a role in primed pluripotency, while POU5F1 regulates naïve pluripotency. However, there is one POU5F3 factor that rescues ESCs like Oct4, the Xenopus gene Xlpou91 (Pou5f3.1). In Xenopus, a further duplication of POU5F3 gene enabled specialization, and Xlpou91 is expressed specifically in the primordial germ cells. Xlpou25 (Pou5f3.2) exhibits epiblast-specific activities and lacks the capacity to maintain naïve ESC pluripotency, similar to other POU5F3 proteins. This functional distinction between the different Xenopus POUV paralogs enabled us to address how specific Oct4 functions (germ cell-like versus epiblast-like activity) are related to the induction of pluripotency. To address this question, mouse Oct4 was replaced by either Xlpou91 or Xlpou25 in murine cellular reprogramming using a Nanog-GFP reporter line to monitor iPSC generation. Results showed that Xlpou91 and mouse Oct4 were required at similar levels to reprogram somatic cells toward iPSCs and reprogrammed cells emerged with similar kinetics. Conversely, Xlpou25 was required at higher expression levels and the resulting iPSCs appeared at a later timepoint, while the pluripotent population in these cultures appeared to be less stable and more prone to differentiate. I found that this phenotype of enhanced differentiation in Xlpou25 reprogrammed cultures may be a product of a different set of immediate early genes induced at the first stages of differentiation. Global transcriptome analysis of the naïve ESC-like pluripotent subpopulation of these iPSC lines confirmed the capacity of all Xenopus POUVs to drive reprogramming towards the pluripotent state. However, the gene sets induced by both Xlpou91 and mouse Oct4, but not Xlpou25, were somewhat enriched for genes involved in reproduction, emphasizing the segregated role of Xlpou91 as a germ cell specific POUV protein. Lastly, I explored the evolutionary origin of these two POUV paralogs and attempted to identify a POUV-related gene in jawless vertebrate (cyclostomes). Based on in silico analysis of genomic and transcriptome databases, my collaborators and I were able to identify a single POUV gene in the Japanese/arctic lamprey, thus providing the first insight into the origin of gnathosome POUV genes.

With meiosis spanning from embryonic development to the end of reproductive life in females, scientists have faced considerable limitations in studying female meiosis and the effects of toxicants on the developing oocyte. Over the last half century, various culture methods have been developed with the aim of studying the mechanisms of early ovary development, as well as for use in reproductive toxicology. However, very few of the established embryonic ovary culture systems have been used to investigate potential reproductive toxicants on the embryonic ovary, in particular when compared with the vast number of in vitro reproductive toxicity studies on the post-natal ovary. Here, a novel test compound, a topoisomerase II inhibitor: AstraZeneca Test Compound (AZTC), was used to investigate the efficacy and validity of ovarian culture methods when compared with in vivo reprotoxicity studies. AZTC was selected due to preliminary in vivo studies demonstrating its detrimental effects on spermatogenesis in male rats. AZTC targets bacterial type II topoisomerases that might have mammalian homologues involved in meiosis. Topoisomerase-II α was expressed within the female germ cells pre-natally, but became localised to the granulosa and stroma cells post-natally. This occurred both in vivo and in vitro. Ovaries from female rats exposed pre-natally to AZTC in vivo were analysed histologically and a significant increase in the number of primordial follicles was observed within the ovaries, as well as an increase in the number of unhealthy follicles. A novel mouse embryonic ovary culture system was developed by adapting, improving and bridging existing available culture techniques. The culture system supported growth of pre-meiotic mouse germ cells through prophase I of meiosis, the formation of primordial follicles and initiation of follicle growth. Cultured ovaries contained follicles at stages in comparable ratios to those in vivo and appeared morphologically normal and healthy. The culture also supported meiotic progression of oocytes to the pachytene stage, albeit with a slight delay. AZTC was used to validate the novel embryonic ovary culture by comparing the results with those from the in vivo study, where AZTC exposure had also occurred during embryonic development. Similar results were consistently observed between the in vivo and in vitro studies. In vitro effects of AZTC on the post-natal mouse ovary were also investigated, where neonatal mouse ovaries cultured with AZTC had fewer primordial follicles and more unhealthy follicles than did control ovaries. AZTC therefore demonstrated different effects when exposure occurred pre-natally vs. post-natally. The embryonic ovary culture was then used to examine the effects of another topoisomerase II inhibitor, etoposide, on the pre-natal ovary. Etoposide is a chemotherapy agent and has previously been prescribed to pregnant women. A significant reduction in the size of the follicle pool was observed in exposed cultured embryonic ovaries, where primordial and transitional follicles were targeted. Overall, establishment of post-natal culture systems have become a useful addition to in vivo reproductive toxicology studies. The embryonic ovary culture system developed here could become a valuable and powerful tool to screen potential reproductive toxicants, as well as to study the dynamics and regulation of early ovary development.

Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression.

Expression of Cancer Testis (CT) genes is normally restricted to testis and placenta, but these genes are also aberrantly activated in tumours of distinct origins, with PRAME, MAGE, GAGE and ESO/LAGE family members encoding cancer testis antigens (CTAs) that stimulate tumorigenesis and elicit immune response. This makes CT genes/CTAs unique biomarkers and potential anticancer therapeutic targets. In this work, we screened the gene expression profiles of a panel of normal tissues and identified 1345 Testis-biased genes, with the aim of correlating subgroups with tumour expression profiles and identify potential oncogenic functional clusters. We also characterised various functional aspects of a benchmark CT gene, TEX19, namely transcript levels during phases of embryogenesis and spermatogenesis, aiming to establish parallelism with its transcriptional and posttranscriptional regulation in cancer. The findings of this study showed that Testis-biased genes are mostly downregulated in testicular tumours, which comprise seminoma, embryonal carcinoma, yolk sac and teratoma as the major subtypes, while in other solid malignancies, these genes are mostly upregulated when differentially expressed. Since more than 90% of testicular tumours have origin in germ cells, this could suggest impairment of germ-like features either as a cause or consequence of testicular tumours, while the CT gene activation in other solid tumours may not be necessarily linked to an attempt to enter a testis programme. Novel functional aspects involving TEX19 were unravelled, with this protein co-localising with UBR2 in distinct nuclear foci in some cancer cell lines, in addition to the inter-regulation that was previously observed between these two proteins. TEX19 could be involved in DNA damage repair in cancer since the levels of RAD51 and γH2AX were seen to be altered upon TEX19 depletion in cancer cells. In addition, TEX19 was shown to have a reciprocal regulatory relationship with L1 elements. Finally, monoclonal antibodies targeting TEX19 were developed, opening future possibilities of developing assays to detect TEX19 in cancer diagnosis, or to target this protein in anticancer therapy.

Mammalian embryogenesis begins with the fusion of an oocyte and a sperm to create the first cell of the newly developing embryo called the zygote. The mechanisms by which this single cell generates a fully formed organism have long been the subject of study of experimental biologists. Initially, embryonic development is reliant on maternally inherited determinants (e.g., RNA, proteins) generated in the oocyte. Overtime, maternal products are degraded and development is controlled by the zygotic genome. Zygotic genome activation (ZGA) is critical for successful embryogenesis, and yet the precise mechanisms of its regulation remain elusive. The first aim of this thesis was to assess the contribution of inherited cell cycle regulators to the onset of ZGA in the early mouse embryo. Specifically, the role of the maternal cell cycle regulator cyclin B3 was examined through zygote microinjections with mRNA combined with live imaging and pharmacological approaches. The findings presented here show a novel link between cyclin B3 activity and the expression of ZGA-related genes Zscan4, Eif1a and Sp110 in the mouse embryo. Overexpression of a degradation-resistant cyclin B3 resulted in the elongation of the S-phase at the expense of the G2 phase in the 2-cell embryo and a suppression of ZGA-related genes. This effect was partially rescued when embryos were forced through the cell cycle by exposure to a Chk1 inhibitor. Conversely, elongation of the G2 phase through Cdk inhibition resulted in an upregulation of ZGA-related genes, which suggested a close link between the cell cycle progression and ZGA regulation in the early embryo. As embryogenesis proceeds, cells are gradually restricted in potency, and morphogenesis shapes the formation of the tissues and organs in the developing body. Complex signalling interactions between embryonic and extraembryonic tissues are indispensable for successful development. The study of early mammalian post-implantation development is challenging due to a lack of accessibility to the embryo as it grows in the uterus. Recent work has focused on establishing models to study and manipulate developmental stages that are equivalent to post-implantation development in the mouse. Yet, extraembryonic tissues are not appropriately represented in current in vitro models and as such these are incapable of recapitulating the true morphogenetic events and architecture of the embryo developing in vivo. The second aim of this thesis was to extend development of an integrated stem cell model of post-implantation embryogenesis that includes an extraembryonic component. When subjected to culture conditions capable of sustaining natural embryo development beyond gastrulation, iETX synthetic embryos were capable of recapitulating the architecture and morphogenesis of the natural embryo and developing to organogenesis stages in the absence of external signalling cues. Specifically, iETX embryos underwent morphogenesis of headfold structures, a neural tube, somite pairs, specification of primordial germ cells, and generated mesodermal tissues including a beating heart. Furthermore, iETX embryos captured extensive development of the extraembryonic lineages, including the formation of a yolk sac with blood islands, and tissues resembling the chorion and allantois. Finally, iETX embryos generated with Pax6 knockout embryonic stem cells were able to recapitulate known phenotypes of Pax6-/- mouse embryos, such as the expansion of the ventral progenitor domain in the neural tube. Overall, these results highlighted that iETX embryos form a complete in vitro model of the mouse embryo that can be used to study development and disease at stages that were previously inaccessible, particularly to shed light on signalling interactions between the embryo, the yolk sac and the placenta.

Organisms rely on a stable genome in their germ cells to successfully establish cellularly competent offspring. However, the mobilization of many transposons and retroviruses called retrotransposons can interrupt important or essential genes in the host’s genome, rendering the effected germ cells mutagenized. Such attacks are so common that retrotransposon-derived sequences make up nearly 40% of most mammalian genomes. To combat the possibly deleterious effects of these mobile elements, metazoans have evolved a robust genome defense system housed in their germ cells. This defense system is composed of thousands of endogenously encoded small non-coding RNAs called piRNAs. A PIWI Argonaute protein traffics piRNAs to mRNA targets that share sequence complementarity and helps regulate their expression. In the first chapter of this dissertation, I review the current literature on small non-coding RNA pathways in general, and how those pathways function in the C. elegans germline in particular. I then discuss the connection between germline small RNA pathways and phase separated condensates enriched in animal germ cells called germ granules. Finally, I outline what is currently known about how particular mRNAs become targeted and silenced by small RNAs. In the second chapter, I demonstrate that a reporter assay can successfully uncover novel components involved in piRNA mediated gene silencing. I discuss where in the pathway each factor likely functions, and further characterize how each factor likely plays a role in gene silencing. This chapter demonstrates that highly conserved and ancient cellular machinery that is essential for snRNA biogenesis, pre-mRNA splicing, nuclear protein import, and nuclear pore formation has evolved to also contribute to small RNA mediated germline defense. In the third chapter, I discuss the role that the hallmark of animal germ cells, the germ granule, plays in the piRNA pathway. I show that, surprisingly, these highly conserved, phase separated structures are largely dispensable for small RNA-mediated gene silencing. However, without germ granules, competing small RNA pathways lose robustness, allowing a subset of typically silenced genes to become expressed and a subset of typically expressed genes to become silenced. This mis-regulation likely contributes to the deleterious fertility phenotypes associated with germ granule loss described here and in previous work. In the fourth chapter, I characterize signals that determine whether particular germline transcripts will become targeted and silenced by the piRNA pathway. The coding region of germline mRNAs rather than the untranslated regions are uniquely vulnerable to piRNA targeting. This finding suggests that the piRNA pathway has evolved to target regions of mRNAs which are less likely to escape detection due to genetic drift. In the fifth chapter, I present a collaborative effort to make the suite of tools used to perform the complex analyses discussed in this dissertation available to the scientific community. Many analyses involved in parsing next generation sequencing data require advanced domain knowledge to replicate. We have made these tools available in a convenient graphical format for the community to use without specific knowledge of the underlying code required to use them. I hope that the availability of such tools will become a standard in academia to allow for greater transparency and to open barriers closed to many biologists who do not have the bioinformatic expertise necessary to analyze the data already available to the community. In the final chapter, I discuss several important implications of this work for the field of small RNA biology. I address how the findings presented here influence our understanding of piRNA-mediated transcriptome regulation, and I suggest future work that could further clarify our understanding of how self and non-self is determined for germline mRNAs.

The novel reproductive technology, In Vitro Gametogenesis (IVG), includes the process of obtaining mature viable germ cells from pluripotent stem cells. To do so, embryonic stem cells and induced pluripotent stem cells are specified to primordial germ cells and differentiated to form gametes that undergo further maturation, which subsequently may undergo in vitro fertilization to form an embryo. With this capability, IVG holds the power to provide a novel treatment option for human infertility. As of now, research has been conducted on mice successfully, and with limited success in humans. Future research will likely focus on discovering the species-specific differences between mice and humans through studies with nonhuman primates. Research on large mammals such as the rhinoceros will also contribute to animal conservation efforts. While human IVG has not been fully developed yet, several concerns have arisen regarding the ethical and legal implications of this technology. While none of these concerns are tangible due to the current state of research findings and current International Society for Stem Cell Research guidelines, they may inform legislation in the future.

In flowering plants, the male gametophyte produces a pair of functional sperm cells that are transported to the embryo sac for double fertilisation. Asymmetric division of the microspore establishes the germline and division of the germ cell results in two sperm cells, however, the molecular mechanisms governing the germ cell specification and division are yet to be uncovered. DUO1 has been identified as a germline-specific MYB binding transcription factor that coordinates germ cell division with gamete specification. One of the major objectives of this thesis is the characterization of an EMS-induced germ cell division mutant termed as duo pollen 5 (duo5) in Arabidopsis. Mutant duo5 germ cells were shown to elongate and enter mitosis but fail to complete the division cycle. Genetic analysis showed that duo5 is an incompletely penetrant gametophytic mutation that has reduced transmission thorough the male. Map based cloning defined duo5 to a genetic interval of ~250 kb region on the lower arm of chromosome IV. The thesis also explores the expression and regulation of two novel DUO1-activated zinc finger genes, DAZ3 and DAZ3L. The reduction in the activity of both promoters in duo1 germ cells suggested that DUO1 is required for their activation in the male germline. Analysis suggests that DUO1 possibly employ both direct and indirect mechanisms to activate DAZ3 and DAZ3L. Furthermore, analysis of protein fusion constructs demonstrated that DAZ3 and DAZ3L expression is predominantly localized in the sperm cell cytoplasm and this expression pattern persists in the developing pollen tubes. The analysis of DAZ3 expression in germ cell division mutants revealed that late activation of DAZ3 promoter is independent of germ cell division. The work demonstrated, will further add to the knowledge of male gametophyte development and will provide new opportunities to understand molecular and genetic mechanisms involved in the production of two plant sperm cells.

Primordial germ cells (PGCs) are the embryonic precursors of adult gametes. In the mouse, they arise around E7.5 in the allantois, and migrate through the developing hindgut and midline dorsal body wall mesenchyme to colonize the gonad primordia by E11.5. PGC behavior, including proliferation, survival, and motility, is controlled by cellular signaling during migration. Steel factor is known as an essential survival factor for PGCs. It is the protein product of the Steel locus, and the ligand for the receptor tyrosine kinase c-kit, which is expressed by PGCs throughout migration. Steel factor exists in two forms, membrane-bound and the soluble, generated by alternative splicing. This thesis addresses two general questions:1) Is PGC behavior controlled by Steel factor from the beginning of their migration? 2) Do the two different forms of Steel factor control different aspects of PGC behavior?Using the mouse reporter line Stella-GFP, in which PGCs express GFP under the control of the promoter of Stella gene, I demonstrate that PGC number is significantly reduced in Steel-/- embryos at E7.5. Similarly, in the absence of Steel factor, either by null mutation or antibody blockade, PGCs aggregate together and show dramatically decreased motility, but their directionality is maintained. These data indicate an essential role for Steel factor in PGC survival and motility. I then show that Steel factor-expressing cells surround PGCs from the time of their initial specification in the allantois, to the time of their colonization of the gonad primordia, providing a “spatio-temporal niche” that travels with PGCs to regulate their survival, proliferation and motility throughout migration. Further, I show that these functions of Steel factor in PGC behavior are distributed between the membrane-bound and soluble forms by analyzing PGC behavior in Steel-dickie mutant embryos, which make only the soluble form. Soluble Steel factor alone is sufficient for PGC survival at E7.5. However, PGCs in E7.5 Steel-d/d embryos aggregate and migrate at much slower rate, indicating that membrane-bound Steel factor is required for PGC motility. Addition of excess soluble Steel factor to Steel-d/d embryos rescued PGC motility, suggesting that the membrane-bound form provides a higher local concentration of Steel factor that controls the balance between germ cell motility and aggregation. Taken together, this thesis demonstrates that the two forms of Steel factor control different aspects of germ cell behavior, and the membrane-bound Steel factor, by providing higher local ligand concentration, defines a “motility zone” for PGCs. This ensures that PGCs in appropriate locations maintain survival and motility, but ectopic PGCs cease motility and then die by apoptosis.

Genomic imprinting directs the allele‐specific expression of a subset of loci according to their parental origin. Monoallelic expression of these genes is regulated by imprinting control regions (ICRs) and is established in the embryonic germ line through differential DNA methylation. Differentiated cells can be reprogrammed to pluripotency by several strategies including the ectopic expression of specific ‘inducers’ and by transfer of nuclei into enucleated eggs. Cellular fusion of somatic cells with a pluripotent stem cell partner can also lead to dominant pluripotent reprogramming. Although ES cells (derived from the inner cell mass) and embryonic germ cells (EG, derived from primordial germ cells) can both reprogram, EG cells are unique in being able to erase genomic imprints from the somatic partner. In order to characterize the earliest events in successful reprogramming, as well as EG‐specific DNA demethylation, I generated experimental heterokaryons between B‐lymphocytes and mouse stem cell lines. I showed that ES cells that lack Polycomb Repressor Complex 2 (PRC2) failed to reprogram B cells and were unable to induce two early events that characterise successful B cell reprogramming; a global redistribution of HP1α and an increased serine 10 phosphorylation. In the second part of my study I confirmed that EG cells were able to induce DNA demethylation at several ICR in B cells following fusion. I present evidence that this reprogramming of the somatic genome requires Tet1 and Tet2 and is accomplished through a two‐step process involving both DNA synthesis and conversion of 5methylcytosine into 5hydroxymethylcytosine.

The production of viable germ cells during human embryonic development determines adult reproductive success. This is particularly true for females, as development of germ cells (GCs) into primordial follicles before birth is imperative for future fertility. During fetal development GCs migrate to the genital ridge to form the gonad, after which several tightly regulated events, including proliferation, differentiation, and association with somatic cells, must occur to form a functional gonad. In the ovary these processes also include the initiation and subsequent arrest of meiosis. These developmental processes are orchestrated by local autocrine and paracrine factors, many of which remain to be identified in the human. In order to decipher further the pathways by which the gonad and GCs develop, potential regulators including prostaglandin (PG) E2, the interleukin (IL)6-type cytokines, and the prokinetecins (PROKs), were examined in the human fetal ovary and PROKs in the human fetal testis. Patterns of gene expression, protein localisation, function, and interaction of the potential mediators throughout human development (8-20 weeks gestation) were determined. Primary fetal tissue was investigated, in addition to immortalized GCs (T-Cam2 cells) and a murine model of fetal ovarian development. PGE2 interacts with known regulators of GC development in non-reproductive organs. It was postulated PGE2 may regulate GC progression by modulating these factors. Examination of PGE2 receptors and precursor enzymes in the fetal ovary revealed that all were present and some were developmentally regulated, with mRNA expression increasing with gestation. These developmentally regulated components were localised to the GCs. The PGE2 receptors were among those differentially expressed, with one localised solely to mature GCs. Culture of human fetal ovary confirmed that PGE2 regulates known regulators of GC development, increasing expression of survival and anti-apoptotic factors. To test the hypothesis that PGE2 is necessary for female GC development, paracetamol, an inhibitor of PGE2 precursor enzymes, was utilised in a murine model of fetal exposure. Fetal ovaries from this experiment displayed disruption of normal development. The IL6-type cytokines are also postulated to be involved in early gonad development, and are known to regulate proliferation and differentiation of mouse embryonic stem and GCs in vitro. A significant increase in transcript levels of the shared receptor components was determined in second trimester human ovaries, as well as developmental increases of several of the IL6-type ligands. Both common receptor components were located specifically in the GCs identifying them as the target of IL6 action in the human fetal ovary. The PROKs regulate cell migration, proliferation and differentiation, and modulate secretion of PGE2 and expression of some IL6-type cytokines. To-date, PROKs have not been examined in the human fetal gonad. Transcript levels were higher in the fetal testis compared to the ovary, with receptor and ligand components increasing with gestation. Most components also increased with gestation in the ovary. However, location of PROK components was strikingly different between the two tissues, with GCs being the primary target of PROK action in the fetal ovary, and Leydig and interstitial cells being the target in the testis. PROKs interaction with other regulators of gonad development was examined utilising a GC line in the case of the ovary and primary interstitial cell cultures in the case of the testis. These studies have identified new factors involved in human fetal gonad development, and how they interact with known regulatory pathways of development.

Gametogenesis is a highly complex process that requires stringent control of gene expression, in which translational regulation plays an essential role. Deleted in Azoospermia-like (DAZL) belongs to the DAZ family of RNA-binding proteins, which are restricted to germ cells, and regulate mRNA translation. Importantly, loss of function of these proteins results in infertility in both males and females in a wide variety of organisms. A model for the mechanism by which DAZL stimulates translation has been proposed based on work in Xenopus laevis (X. laevis) oocytes. In this model, DAZL functions by recruiting the translation initiation factor poly(A)-binding protein (PABP) to the 3’ untranslated region (UTR) of messenger RNAs. Simultaneous binding of PABP to Dazl and factors at the 5’ end confers a “closed-loop” mRNA conformation, which promotes translation initiation. To examine whether DAZL plays a similar role in mammals, co-expression of Dazl and PABP family members was investigated in fetal and adult mouse gonads. In contrast to X. laevis, mammals encode four cytoplasmic PABPs which share a similar domain organisation: PABP1, tPABP, ePABP and PABP4, of which PABP1 and PABP4 appear to be expressed in a wide range of tissues. Immunohistochemistry revealed that Dazl, Pabp1 and Pabp4 are all expressed in primordial germ cells (PGCs) but these show different expression patterns following germ cell sex differentiation. In adult testes Dazl is expressed in spermatogonia and spermatocytes, coinciding with the peak of Pabp4 expression. In contrast, the peak of Pabp1 expression occurs later than that of Dazl, with these proteins only being co-expressed in late pachytene and secondary spermatocyte phases. In adult ovaries, Pabp1, Pabp4 and Dazl are all expressed in the oocytes of primordial and primary follicles. Since both PABP family members are co-expressed with Dazl, the ability of DAZL to interact with PABP1 and PABP4 was investigated in vitro and in vivo. Surprisingly, these studies showed that DAZL discriminates between different PABP family members, only interacting with PABP1, providing the first report of a PABP-specific protein partner. Several putative DAZL mutations have been identified in patients with impaired fertility. Two of these mutations, I37A and R115G, are located in the RNA recognition motif (RRM), a domain which is found in many RNA-binding proteins and mediates both RNA and protein interactions. Thus, the role of these mutations in the ability of DAZL to stimulate translation was investigated. To this end, a translational target of human DAZL (hDAZL) was sought. The 3’UTR of growth differentiation factor 9 (hGDF9) mRNA was found to confer regulation by hDAZL and thus the ability of mutant DAZLs to stimulate reporter mRNAs containing this 3’UTR was examined. This revealed that both mutations compromised the ability of hDAZL to stimulate hGDF9 translation, suggesting a causative effect. These results were further confirmed in assays in which hDAZL is artificially tethered to mRNAs. The ability of mutant hDAZLs to stimulate translation in this assay was compromised suggesting that loss of function is, at least in part, due to impaired protein-protein interactions rather than altered RNA-binding. This work provides insights into the molecular mechanism by which DAZL stimulates the translation of specific mRNAs during mammalian gametogenesis and provides evidence that this function may play an important physiological role in human reproduction.

Androgen receptor (AR) signalling is essential for the development and function of the male reproductive system. Conditional gene ablation using the Cre-loxP system has previously assisted in the elucidation of the role of AR in different cell types. The aim of this study was to examine the effects of the ablation of AR in previously untargeted cell types, with the hypothesis that this will have significant and novel effects on reproductive development and function that have not been previously documented by current models of androgen disruption. In these studies, three Cre recombinase lines were empirically validated for action in the male reproductive system, before being used to ablate AR and the phenotypes of the resulting lines were characterised. Endothelial-specific receptor tyrosine kinase (Tie2)-Cre was shown to target the vascular and endothelial cells of the testis, and used to ablate AR in these cells. The testes of the resulting Tie2-ARKO line were morphologically similar to controls, with normal spermatogenesis and mature spermatozoa present in the cauda epididymis. Aquaporin 2 (Aqp2)-Cre was shown to target the post-meiotic germ cells of the testis, and was used to ablate AR in these cells. The testes of the resulting Aqp2-ARKO line were morphologically similar to controls, with normal spermatogenesis and mature spermatozoa present in the cauda epididymis. It was concluded that the Ar gene was dispensable in the endothelial cells and post-meiotic germ cells of the testis for normal spermatogenesis. Forkhead box protein G1 (FoxG1)-Cre was shown to target the caput epididymal epithelium and pituitary, and used to ablate AR in these cells. d100 FoxG1-ARKO mice had a severe testicular phenotype, with sloughing of the seminiferous epithelium, atrophy of some seminiferous tubules and distension of the rete testis with spermatozoa. Despite the severe testis phenotype, ablation in the testis was incomplete and restricted to a small percentage of Leydig cells, with no ablation in Sertoli cells. Ablation of AR in the embryonic pituitary did not cause adult serum testosterone or LH concentrations to change, nor did it cause changes in other pituitary hormone transcripts. Mosaic ablation of AR in the caput epididymal epithelium was shown to impair epididymal development, with failure of initial segment (segment I) development and a significant decrease in epithelial cell height and lumen diameter in the remaining proximal caput epididymis (segment II). Dysfunction of the caput epididymis resulted in the failure of spermatozoa to transit the efferent ducts into the epididymis correctly: instead they were found to stall in the efferent ducts and produce a block. The testicular phenotype could be explained as the result of fluid backpressure effects resulting from the efferent duct block. Consequently, low concentration of spermatozoa in the cauda epididymis resulted in infertility in the FoxG1-ARKO, which represents a new model of obstructive azoospermia.

Normal germ cell development in the human testis is crucial for subsequent fertility and reproductive health. Disruption of testis development in fetal life can result in deleterious health consequences such as testicular dysgenesis syndrome (TDS), which includes disorders, such as cryptorchidism, hypospadias, infertility and testicular germ cell tumours (TGCT). A rat model of TDS in which rats are exposed to phthalates in utero has been validated, but does result in the development of TGCT. In humans, TGCTs result from transformation of pre-neoplastic carcinoma in-situ (CIS) cells and these CIS cells are believed to arise from human fetal germ cells during their transition from gonocyte to spermatogonia, based on their morphology and protein expression profile. It has been proposed asynchronous differentiation of germ cells in the human fetal testis may predispose fetal germ cells to become CIS cells. Studying the development of these tumours in humans is difficult because of their fetal origins and prolonged duration from initiation of impaired development to invasive disease. For this reason the use of relevant animal models that can mimic normal and abnormal germ cell development may provide new insight into how TGCT develop. The Common Marmoset monkey, a New World primate exhibits many similarities to the human in terms of reproductive biology and could represent such a model. This thesis aimed to further characterise the origins of CIS cells in the human testis by investigating the protein expression profile of CIS cells in patients with TGCT and comparing them to established markers of human fetal germ cell types using immunohistochemistry and immunofluorescence. Quantification of the various subpopulations of CIS and proliferation within these populations was performed. The thesis also investigated the Common Marmoset monkey as a potential model of normal testis and germ cell development by comparing the differentiation and proliferation profile of germ cells with those of the human during fetal and early postnatal life. During the present studies methods were successfully developed that enabled us to use testicular xenografts to recapitulate normal development of immature testes from marmoset and human. This involved grafting pieces of testis tissue subcutaneously under the dorsal skin of immunodeficient mice and retrieving them several weeks later to investigate their development during the grafting period. Xenografts using tissue from fetal, neonatal and juvenile marmosets were performed in addition to testes from first and second trimester human fetuses. Finally the present studies aimed to use the marmoset and the xenografting approach as systems in which to examine the effects of gonadotrophin suppression and phthalate treatment on germ cell differentiation and proliferation, with particular attention to the potential for development of CIS and TGCT. Heterogeneous phenotypes of CIS cells were identified, mostly consistent with those seen in the normal human fetal testis, however some of these CIS cells did not exhibit the same phenotype as germ cells identified in normal fetal testes. In addition it was shown that some of the proteins considered to be ‘classical’ markers of CIS cells, such as the pluripotent transcription factor OCT4, were not expressed in a proportion of the CIS cells. The proliferation index of CIS cells is also significantly higher in those subpopulations with the most ‘undifferentiated’ phenotype (i.e. OCT4+/VASA-). The present studies have generated novel data showing that the marmoset is a good model of fetal and neonatal germ cell development, with similarities to the human in terms of an asynchronous and prolonged period of differentiation and proliferation of germ cells from gonocyte to spermatogonia. This feature is also common to the human, but not a characteristic of the rodent. Fetal, neonatal and pre-pubertal germ cell development can be re-capitulated by xenografting tissue from marmoset and human testes into nude mouse hosts. Human fetal testis grafts produced testosterone and were responsive to hCG stimulation. First trimester human testis xenografts that have not developed fully formed seminiferous cords prior to grafting can complete the process of cord formation whilst grafted in host mice. In addition, germ cells in fetal human and marmoset xenografts can differentiate and proliferate in a similar manner to that seen in the intact non-grafted testis. In the intact neonatal marmoset, suppression of gonadotrophins resulted in a 30% decrease in proliferation, however differentiation of gonocytes is not affected. In-utero treatment of neonatal marmosets with mono-n-butyl phthalate was associated with unusual ‘gonocyte’ clusters, however, di-n-butyl phthalate treatment of mice carrying fetal marmoset xenografts resulted in no visible effects on germ cell differentiation or proliferation and did not result in the development of CIS or TGCT. In conclusion, this thesis has shown that there are many subpopulations of CIS cells of which many have not been previously described. These subpopulations have different characteristics, such as variable proliferation rates and this may indicate the potential for progression or invasiveness. These subpopulations have similar protein expression phenotypes to normal human fetal germ cells although the present studies have identified some CIS cells with phenotypes that are not found in the normal human testis. This thesis has demonstrated that the marmoset is a comparable model to the human in terms of asynchronous fetal germ cell development, which may predispose this species to the development of CIS/TGCT. In addition to the use of intact marmosets, these studies have also demonstrated for the first time that testis xenografting provides a comparable system for testis cord formation, germ cell differentiation and proliferation in fetal/postnatal marmosets and fetal human testis. In addition the marmoset and xenografting models have indicated that phthalates may have minor effects on testis development in the human and marmoset but do not result in CIS or TGCT. These model systems are suitable for further investigation of normal and disrupted testis development.

The germ lineage is unique among cell types in that it must maintain its own cellular identity while simultaneously maintaining totipotency, the ability to generate all other cell types. To achieve this, germ cells package their genome into domains of transcriptionally active euchromatin and transcriptionally inactive heterochromatin to maintain expression of germline-appropriate genes and repression of germline-inappropriate genes. Germ cells must also differentiate between heterochromatin domains that are relatively permanent (constitutive heterochromatin) and heterochromatin domains that are temporary (facultative heterochromatin) and are activated at another time or in a different cell type. Precisely how constitutive and facultative heterochromatin promote germline development and function is not known. In C. elegans, the constitutive heterochromatin mark H3K9me and the facultative heterochromatin mark H3K27me are required for germline development in subsequent generations and have been shown to redundantly repress the single X chromosome in the male germline and promote germline development in offspring. Surprisingly, domains of H3K9me and H3K27me display significant overlap in C. elegans, suggesting that constitutive and facultative heterochromatin may be less distinct in C. elegans than in other organisms. Together, these observations suggest that H3K9me and H3K27me have at least some redundant roles in repressing genes to promote germline development in C. elegans. I determined the relative roles of H3K9me and H3K27me in gene repression in adult C. elegans germlines to promote germline development in offspring. I used RNA-sequencing to identify the gene targets of H3K9me alone, of H3K27me alone, and of the two marks redundantly in adult hermaphrodite and male germlines. I found that oogenesis genes on the X chromosome are a particular focus of repression in the germline and that repression of the X is primarily carried out by H3K27me alone in the hermaphrodite germline and primarily by H3K9me and H3K27me redundantly in the male germline. I utilized these differences in X repression by H3K9me and H3K27me between hermaphrodite and male germlines to identify a set of genes whose upregulation causes sterility in offspring. I found that the activity of the transcription factors LIN-15B, LIN-54, and possibly EFL-1 contribute to sterility in offspring from germlines that lack H3K9me and H3K27me. I synthesize my findings with those in the literature to present a model of X repression through germline development and how the roles of H3K9me and H3K27me in X repression differ between hermaphrodites and males.

The transmission of genetic information across generations depends on the integrity of the germline. Primordial germ cells (PGCs) are the embryonic precursors of mature germ cells, spermatozoa and oocytes, and their proper development is essential for reproductive success. PGCs are initially formed in extraembryonic regions and migrate to the developing gonads. Through a series of mitotic divisions, meiosis, and differentiation, PGCs transform into fully mature gametes that carry the genetic material required for the creation of a new individual upon fertilization. The NANOS gene family has been found to play a critical role in germ cell development across various organisms, including mammals, with specific expression in germ cells. Through murine studies, Nanos3 has been identified as a crucial regulator of germ cell development in both sexes, protecting germ cells from apoptosis during migration and colonization of the gonadal ridge. In contrast, Nanos2 is specifically involved in male germ cell differentiation and maintenance of the spermatogonial stem cell population. These genes exert their control through translational repression of target mRNAs, influencing the expression of key factors involved in meiosis and germ cell differentiation. Recent studies in pigs, sheep, and cattle have shown that NANOS2 knockout (KO) animals replicate the male-specific germline ablation observed in mice, while female germline development remains unaffected. Similarly, NANOS3 KO livestock, including male and female pigs and a female bovine fetus, exhibited a complete loss of germ cells but normal gonadal development. Notably, live NANOS3 KO cattle have not been generated to date, and the specific role of NANOS3 in male cattle has yet to be explored. Collectively, these findings suggest that NANOS3 KO livestock could serve as potential hosts for germline complementation, a technique where donor cells from one genetic background replace the germline of a sterile host with a different genetic background. Germline complementation in livestock offers the potential to generate germ cells from highly genetically valuable donor animals in the gonads of sterile host animals, thereby increasing the availability of gametes from desirable dams and sires (i.e., surrogate sires, or more generally, surrogate hosts). Studies have demonstrated the achievement of germline complementation and the production of live, donor-derived offspring in rodents as well as non-mammalian food species, including chickens and fish. To facilitate the application of germline complementation technology in livestock, it is crucial to develop efficient methods for generating germline-ablated hosts that retain the function and architecture of their somatic cell gonadal support tissues. One promising method involves using genetic tools like gene editing (GnEd) to inactivate essential genes for germline production at the embryo stage. This creates a vacant germline niche within the host, providing an opportunity for donor cells to colonize and develop in the germline in the absence of competition from endogenous germ cells. Among the genes targeted in mammals to achieve germline ablation thus far, the RNA-binding protein gene, NANOS3, stands out as a promising target in cattle for two primary reasons. The disruption of NANOS3, being one of the earliest germline-specific genes expressed, is anticipated to result in the early elimination of PGCs. This would create a favorable environment for exogenous donor cells to migrate to and colonize the gonadal ridge, offering the possibility of germline complementation at an early stage of embryogenesis. Furthermore, if NANOS3 is confirmed to play an essential role in both male and female germ cell development, as hypothesized, the generation of NANOS3 KO cattle could serve as hosts for introducing donor-derived germ cells in both sexes, thus expanding the potential for novel breeding schemes. This study aimed to investigate the consequences of eliminating NANOS3 in bovine germline development and evaluate the potential for NANOS3 KO cattle to serve as hosts for germline complementation. To accomplish this, an approach to achieve a CRISPR/Cas9-mediated KO of NANOS3 via co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes at 6 hours post fertilization in in vitro produced bovine zygotes was successfully optimized. This efficient and repeatable NANOS3 KO method allowed the generation of NANOS3 KO cattle and investigation of the impact of NANOS3 elimination on bovine germline development at different stages of development and at reproductive age. Through embryo transfer, eight pregnancies with NANOS3-presumptively-edited embryos were produced, which were collected at four different timepoints for analysis. These timepoints included during sexual differentiation (41 days of fetal age (d)), after sexual differentiation (90d), at the perinatal stage (283d), and post-puberty (15 months of age (mo)). These samples were comprehensively evaluated using DNA, RNA, protein, and physiological assessments. Long-read sequencing analyses showed the achievement of a remarkable 75% KO rate, with all but one of the edited NANOS3 alleles resulting in a predicted KO (i.e., loss-of-function). Immunofluorescence analyses revealed that male NANOS3 KO bovine gonads exhibited the elimination of PGCs as early as 41d. Further analyses using single-cell RNA sequencing confirmed a complete loss of germ cells in the NANOS3 KO fetal and perinatal testes, while maintaining normal testis cord formation and the presence of somatic support cell populations. Ultimately, three live, healthy NANOS3 edited calves were born without assistance, a heifer calf #854, and two bull calves, #838 and #3964. Heifer #854 and bull #838 were determined to be NANOS3 mosaic KOs. On the other hand, bull #3964 showed editing without complete KO, as he carried an allele with only small, in-frame deletions, that did not result in a complete loss-of-function mutation. The live NANOS3 edited animals exhibited normal growth patterns and were ultimately harvested at 15mo to enable collection of meat samples and their reproductive tracts. The edited bulls both exhibited normal pre-pubertal reproductive hormone profiles. At sexual maturity, KO bull #838 was found to have normal libido and an anatomically normal reproductive tract, although no spermatozoa were present in his ejaculate. Additionally, histological analysis of his testes confirmed the absence of germ cells and the presence of gonadal somatic support cells, indicating successful ablation of the germline while preserving the integrity of the somatic gonad. In contrast, the sexually mature NANOS3 edited bull #3964 exhibited fertility as evidenced by the presence of spermatozoa in his ejaculate and cross-sections of his testes. These findings suggest that bovine NANOS3 is a haplosufficient gene, and that the allele with small, in-frame deletions produced a functional protein. The NANOS3 KO heifer #854 also had a complete loss of germ cells, as confirmed by the absence of oogenesis in her ovarian tissue. However, she also exhibited an anatomically abnormal reproductive tract and irregular gonad development. Additionally, her hormone profiles indicated a lack of reproductive cycling and functional granulosa cells, consistent with the absence of germ cells and follicles in this animal. The relationship between germ cells and supporting somatic cells in the gonad is crucial for the successful development and function of gametes, so any disruption to germ cells can have detrimental effects on the coordinated processes and functions of both germ and somatic cells involved in gametogenesis. In this study, the absence of germ cells in NANOS3 KO cattle had a greater impact on the normalcy of ovarian development as compared to testes development. Finally, the meat composition from these NANOS3 KO cattle at 15mo was within the bounds of the normal variation seen in international meat compositional databases, which is not surprising as NANOS3 is a germline specific gene, and the lack of germ cells would not be expected to have an impact of meat composition. Overall, this study provides insights into the role of NANOS3 in bovine germline development and indicates the potential of NANOS3 KO cattle to serve as hosts for donor-derived exogenous germ cell production in both sexes. This could unlock both an opportunity to reduce the genetic lag between elite seedstock animals and the genetic merit of commercial cattle, and additionally provide an efficient means for the generation and dissemination of genetically improved GnEd donor genetics.

Infertility affects ~20% of couples in Europe and in 50% of cases the problem lies with the male. The development of assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) and intra-cytoplasmic spermatozoa injection (ICSI) has allowed some couples to overcome male-factor infertility. However concerns remain over the increasing use of ART as elevated levels of DNA damage in sperm from infertile men have been reported and a link between DNA damage in sperm and early embryonic failure has been demonstrated. DNA damage in sperm, caused by oxidative stress may also be passed on from father to child resulting in an increased incidence of childhood cancer. This has led to fears that the use of damaged sperm in ART could contribute to early embryonic failure and/or birth defects. The studies described in this thesis used mouse models to investigate the relationship between DNA integrity in male germ cells and male fertility. This was achieved by studying both the effects of targeted ablation of genes involved in DNA repair and the impact of scrotal heat stress on testicular function and sperm DNA integrity. Three lines of transgenic mice with deletions in genes involved in genomic integrity (Ercc1, Msh2 and p53) were studied. All three genes are expressed in the testis. These studies confirmed and extended studies on Ercc1 knockout (-/-) mice showing reduced germ cell complement, increased apopotosis, an increased percentage of damaged sperm and demonstrated for the first time that depletion of Ercc1 results in an increased incidence of unrepaired double strand DNA breaks (DSB) in pachytene spermatocytes. The persistence of DSBs in spermatocytes and abnormal sperm chromatin structure confirmed that the repair functions of Ercc1 are essential for normal germ cell maturation. In the p53-/- mice these studies showed for the first time that there was an increase in DSBs in spermatocytes and an increase in numbers of sperm with damaged DNA. The level of apoptosis was also increased in the testes suggesting that caspase-3 mediated apoptosis is not entirely p53 dependent as been previously suggested. These studies demonstrated for the first time that targeted ablation of Msh2 compromises germ cell complement and as in the Ercc1-/- this resulted in gaps in the seminiferous epithelium consistent with clonal loss of germ cells. Consistent with a role for MSH2 in mismatch repair no DSBs were detected in spermatocytes from Msh2-/-. Testicular function is temperature dependant and due to their location in the scrotum testes are normally kept between 2ºC and 8ºC below core body temperature. In mice transient scrotal heat stress (30 minutes at 38°C, 40°C and 42°C) disrupted testicular function. Analysis of sperm and testis parameters revealed that stress at 38°C was sufficient to have subtle effects on epididymal function but the higher temperatures had additional consequences for testicular function which resulted in DNA damage in spermatocytes, germ cells loss and increased apoptosis. Further studies into the pathways of apoptosis demonstrated that the mitochondrial/intrinsic pathway plays a role in heat stress response. The fertility of males was altered in those heated to 42°C resulting in reduced pregnancy rate and litter size. Given that the paternal genome is reported to be required for the development of extraembryonic tissues and this will influence growth of the embryo, it was interesting to note an increase in resorption sites in pregnancies using 40°C males. IVF was used to demonstrate that embryos formed using sperm from males stressed at 42°C were compromised between the 4-cell and blastocyst stage suggesting that though sperm with DNA damage are still capable of fertilisation, the paternal DNA was introducing genomic instability to the embryo and having fatal effects on development. These studies have also shown that one possible underlying cause of the disturbance in testicular function is hypoxia, as a marked increase in Hif1 alpha (a marker of hypoxia) mRNA and relocalisation of the protein was observed in the testis. In conclusion, DNA damage in the male germ line caused either by induced stress, or by targeted ablation of DNA repair genes, can disrupt testicular architecture, function and therefore the fertility of mice. These data have demonstrated that deletion of Ercc1, Msh2 and p53 can have differential but overlapping affects on germ cell function and sperm production and that increased scrotal temperature can cause subfertility in male mice. This study has provided further confirmation of possible male-mediated effects on embryo survival and these findings should be taken into consideration when using sperm from infertile men in IVF/ICSI treatments where the normal quality control processes involved in fertilisation are bypassed.

The DAZ (Deleted in Azoospermia) gene is located on the human Y chromosome and is associated with male infertility in humans. An autosomal form of this gene (DAZ-like (Dazl)) is present in all mammals and is expressed in germ cells, the Dazl itself being an RNA binding protein. Transgenic homozygous Dazl knockout (KO) male and female mice are infertile due to loss of almost all germ cells in early neonatal life. However, in heterozygous (het) females there is a significant increase in ovulation rate and litter size in comparison to their homozygous wild type (wt) littermates, in spite of lower levels of plasma Follicle Stimulating Hormone (FSH), suggesting an alteration in follicle senstiviity to FSH. The aims of this study were to assess follicle numbers in ovaries and follicle FSH-sensitivity in het mice compared to wt mice. The present studies suggest that, although Dazl is an oocyte-specific gene, the putative protein(s) that Dazl affects through RNA binding in the oocyte targets granulosa cells. The influence of Dazl on follicle development occurs through the single copy of Dazl enhancing FSH sensitivity, but the exact mechanism of Dazl on the granulosa cells is unknown. The studies presented in this thesis suggest that follicles from the Dazl het mice have accelerated growth because of a reduced threshold for FSH that allows them to remain for a longer duration within the critical FSH threshold for follicle growth. In addition, this study suggests that the percentage of healthy and atretic follicles in the Dazl wt and het mice is similar in untreated and oFF-treated mice, but that after FSH treatment there are more healthy follicles in het mice. It could be concluded that, in the adult after follicle activation and recruitment into the pool of growing follicles, a single copy of Dazl results in maintained follicle growth, because of increased FSH-sensitivity. Thus, more larger follicles achieve dominance and these subsequently ovulate, leading to an increased litter size.

Immunohistochemistry was used on Bouins-fixed marmoset testes from birth to adulthood, including tissue from males treated prepubertally for ten weeks with a GnRH antagonist, to elucidate the role of gonadotrophins. No difference was found between germ cells from control and GNRH antagonist-treated animals in the immunoexpression of MAGE, a germ cell-specific marker, Ki67, a marker of cell proliferation, and Histone H3, a marker of cell mitosis, confirming that spermatogonial proliferation is gonadotrophin-independent. To determine potential regulators, spermatogonial immunoexpression of growth factor receptors was investigated. Receptors for several growth factors were identified in spermatogonia. The expression of receptors for both epidermal Growth Factor and Neurturin was pronounced in a proportion of germ cells in late infancy, thus implicating a role for these factors in spermatogonial development. Prepubertal expression of these receptors was unaffected by prior treatment with a GnRH antagonist, demonstrating their gonadotrophin independence. Studies using confocal microscopy demonstrated that the spermatogonial population expressing these receptors were not actively proliferating, raising the possibility that these factors act to inhibit proliferation. Manipulation of these factors may protect fertility in boys treated for cancer. As survival of stem spermatogonia is essential for future fertility, this study also attempted to identify a marker of stem cells in the primate, in order to investigate their activity prepubertally. GFR-α1, the receptor for Glial Cell Line-Derived Neurotrophic Factor, is expressed by A-spermatogonia in the mouse. In marmoset testis, expression of this receptor was shown in occasional germ cells, in a pattern consistent with stem cells, at all ages studied. Attempts to co-localise GFR-α1 with cell cycle markers suggest these cells are not actively proliferating.

During development, a variety of cell types undertake a guided migration to their target location and, consequently, display motile, proliferative and invasive behaviours. Failure in guided migration can result in severe developmental errors, including the formation of metastatic cancers or, in the case of germ cells, congenital defects. Primordial germ cells (PGCs) are the germ line stem cell progenitors that produce the gametes and pass the genetic material from generation to generation. In Drosophila melanogaster, as in many other organisms, the PGCs are set-aside at the beginning of embryogenesis as a distinct sub-set of cells that are easily detectable by immunochemical means. As such, they provide a good system in which to study the control of cell migration during development. Furthermore, since they are subject to various cell death and cell survival signals during their journey, they provide an opportunity to study the regulation of stem cell survival in vivo. wunen and wunen-2 encode lipid phosphate phosphatases (LPPs) and regulate Drosophila PGC survival and migration. LPP-like activity has been detected in all cell and tissue types so far studied, but remarkably little is known of their biological functions, and their physiological substrates remain elusive. The first aim of this work was to investigate differences in substrate recognition between Drosophila Wunen and the mammalian LPP isoforms, both in vitro, by means of biochemical assays and in vivo, by examining the ability of the mammalian isoforms to recognise the same physiological substrate as Wunen in Drosophila embryos. It also investigates hetero- and homo- oligomerisation of the fly and mammalian LPPs as a possible mechanism of action. In order to fully understand these proteins, it is necessary to identify and characterise their receptors. This work identifies a G protein-coupled receptor involved in Drosophila PGC migration and survival, and investigates its possible relationship to Wunen.

Xenogenesis is a method of reproduction where successive generations differ from each other and no genetic material is transmitted from the parent to the offspring. Xenogenesis can be accomplished by transplanting spermatogonial stem cells (SSCs), oogonial stem cells (OSCs) or primordial germ cells (PGCs) from the desired diploid donor species into a sterilized host. Many beneficial ecological and aquaculture applications are possible from this process. Specifically, the production of embryos from hybridizing channel catfish (Ictalurus punctatus) females with blue catfish (I. furcatus) males could be refined to a simpler and more cost-effective practice of xenogenesis. Xenogenesis could alleviate the reliance on growing blue catfish males to maturity and sacrificing them for gonad extraction and subsequent artificial spawning. Male blue catfish cannot be stripped of milt for artificial fertilization and must be sacrificed for sperm collection. Thus, one objective was to produce xenogenic common carp, Cyprinus carpio, to become biological blue catfish milt factories. Common carp embryos, and larvae were injected with blue catfish oogonial stem cells and spermatagonial stem cells at various points in relation to their development. Fish were injected beginning at 0-degree days, every 23-degree days until 621-degree days. In total, 152 potential common carp xenogens were sampled and 57 of them were confirmed to be hosting blue catfish stem cells through polymerase chain reaction (PCR), giving a total 37.5% success rate of common carp hosting blue catfish cells in the gonads. The highest percentage of positive samples were observed at 0 to 46-degree days and 483-575 degree days, averaging 62.3% and 56.7% success respectively. Rate of xenogenesis was similar for male and female common carp hosts. The mean survival among all the treated groups through the first 34 days was 47%, however, among groups injected during embryonic development, mean survival was 8.6%. During the grow out stage (34-545 dpf), mean survival for all treated groups was 70%. At one and half years of age, xenogenic common carp males produced blue catfish sperm based upon DNA analysis of the expressed sperm. In addition to stronger reproductive control, some targeted genetic alterations were performed to aid in higher growth rates and increase yield within catfish aquaculture. Two induced mutations were investigated, melanocortin-4 receptor (mc4r) and myostatin (mstn) gene in channel catfish. In a earthen pond environment stocked at 18,508 fish per hectare, the mean weight for channel catfish mc4r P1, mc4r F1 X control, control X mc4r F1, and mc4r F1 X mc4r F1 mutants was 361, 266, 521, and 426 grams, respectively, was larger (P>0.05,P>0.05, P

The cell lineage that forms eggs and sperm is referred to as the germ line, and the integrity of this cell lineage is critical for the successful reproduction and the survival of a species. The earliest progenitors of the germ line are known as primordial germ cells (PGCs), and PGCs form during embryonic development. In most animals, germ cells are formed in a location that is distant from the location where gonads (testes or ovaries) will develop, and therefore germ cells must migrate, often long distances, during embryonic development. I investigate the mechanisms for two major processes in early germ line development: specification and migration. To study these processes, I look to insects, the most diverse group of animals, where we can study mechanisms that govern germ line development in a comparative context and generate evolutionary hypotheses about how these mechanisms evolve. While most mechanistic studies of developmental processes have been in holometabolous insects (insects that undergo metamorphosis), hemimetabolous insects have many features that are more representative of an ancestral form of insect development. Here, I study the hemimetabolous insect, the large milkweed bug Oncopeltus fasciatus (Hemiptera), which is in a group sister to the monophyletic Holometabola. It is therefore in an ideal phylogenetic position to make comparisons to mechanisms that have been well-studied in holometabolous insects such as the fruit fly Drosophila melanogaster (Diptera). In Chapter 1, I review evidence in the literature showing the roles of the Bone Morphogenetic Protein (BMP) pathway in reproductive system development and function across animals. I propose that there is an ancient association with BMP signaling in the development of both the germ line and supporting somatic gonad tissue that may have arisen with the segregation of the germ line early in metazoan evolution. In Chapter 2, I investigate whether the BMP pathway has a role in PGC specification in O. fasciatus where the signaling factors specifying PGCs are currently unknown. I provide evidence that O. fasciatus PGCs are competent to receive BMP signal, but future work is needed to assess whether this pathway is required for PGC formation in this insect. In Chapter 3, I characterize the dynamics of PGC migration in O. fasciatus and use a candidate gene approach to find genes involved in regulating PGC migration. I provide evidence that at least one member of the JAK/STAT signaling pathway is involved in PGC migration in O. fasciatus. To my knowledge, this is the first functional genetic study into the mechanism of PGC migration in a hemimetabolous insect.

The work described in this thesis investigated the function of cyclin A1 during murine spermatogenesis, furthering the current understanding of molecular regulation of mammalian meiosis in general. The expression of regulators of the mitotic G2/M transition was characterised during murine spermatogenesis. The expression of cyclin A2 is restricted to the mitotic proliferation phase of germ cells, suggesting the function of cyclin A2 in such mitotic cycles is similar to that in somatic cells. However, subtle differences are observed. In germ cells, cyclin A2 persists until anaphase, whereas in cultured somatic cells, it is being degraded at prometaphase. On the other hand, the expression of cyclin A2 in preleptotene cells implicates that it regulates the pre-meiotic DNA replication. During the meiotic cycle, expression of B-type cyclins and Cdc25C precedes that of cyclin A1. Cyclin B1 and Cdc25C are already present in the nucleus before the onset of cyclin A1 expression. These observations suggest that the temporal and spatial expression of B-type cyclins and Cdc25C is unlikely to be regulated by a cyclin A1-associated function. Cyclin A1, in contrast, only becomes detectable at the end of prophase I indicating that it is dispensable for the meiotic recombination. To define possible function of cyclin A1, the precise point of spermatogenic arrest in Ccna1-null mice was determined. Most spermatocytes arrest at diplotene/diakinesis, while a minority progresses to metaphase/anaphase I with nuclear envelope breakdown, the presence of MPM-2 epitopes, and the phosphorylation of histone H3 on condensed chromosomes. The temporal and spatial expression of B-type cyclins and Cdc25C remains unchanged in the absence of cyclin A1. These findings suggest that cyclin A1 does not regulate the prophase to metaphase transition in meiosis I.

The antero-posterior and dorso-ventral axes of the Drosophila embryo are set up during oogenesis. These axes of polarity are the results of several symmetry-breaking steps that characterise the development of the Drosophila oocyte. During my PhD, I have studied two of these steps. Oocyte determination. During early oogenesis, one cell is selected to become the oocyte within a cyst of 16 germ cells. These 16 germ cells are all siblings and share the same cytoplasm. By using several oocyte-specific markers, I have found that there at least three different pathways to restrict the oocyte fate to one cell. The restriction of cytoplasmic proteins depends on the microtubules and on the activity of the Egl/BicD complex. The restriction of meiosis also depends on the same complex but is independent of the microtubules. Finally, the restriction of the centrosomes is independent of both the microtubules and the Egl/BicD complex. From this analysis, I also concluded that the selection of the oocyte does not depend on the microtubules and that the oocyte is probably selected while the cyst is still dividing, as shown by the asymmetric inheritance of the fusome. By homology with the polarisation of the C. elegans one cell embryo, I have shown that the Drosophila homologues of par-1, par-3 and par-6 are required for the first polarisation of the oocyte. This polarisation is then required to maintain the oocyte fate. Oocyte polarisation. During mid-oogenesis, the A/P and D/V axes of the oocyte are defined by the asymmetric localisation of several mRNAs. I have conducted a germline clone screen to find lethal genes involved in this polarisation. In particular, I report here that one of the major Drosophila hnRNP, hrp48, is required for the localisation of oskar mRNA within the oocyte.

The development of in vitro fertilisation (IVF) and intra-cytoplasmic spermatozoa injection (ICSI) techniques has revolutionised treatment for couples with so-called male-factor infertility. However concern over the integrity of the DNA in spermatozoa used for ICSI has been expressed. Poor semen quality and morphologically abnormal spermatozoa are associated with poor embryonic development following IVF. Furthermore, studies have demonstrated that the transmission of defective DNA (e.g. adducts, gene deletions) from the spermatozoa to the developing embryo can occur and that this may lead to developmental failure of the embryo or future health risks for the child. The aims of this project were; a) to develop a Comet assay for the study of DNA integrity of murine spermatozoa, b) to use this assay to assess DNA integrity in spermatozoa from mice with known sub/infertility and to examine the susceptibility of these spermatozoa to heat -induced DNA damage, c) to identify the stages of spermatogenesis in wild type mice which are susceptible to heat-induced DNA damage, and d) to determine whether this damage is present in the mature spermatozoa developed from heat -treated germ cells. The single -cell gel- electrophoresis (Comet) assay was originally developed to study DNA damage in somatic cells and has been modified to study both endogenous and induced DNA damage in human ejaculated spermatozoa. DNA packaging and condensation in mouse spermatozoa is not identical to that in the human. The human spermatozoa Comet assay was therefore not suitable for use on murine spermatozoa. A series of modifications were made to the human spermatozoa Comet assay and a usable assay based on a commercially- available Comet Assay Kit (Trevigen) was developed for the study of murine spermatozoa. The motility, morphology and DNA integrity of motile spermatozoa recovered from the epidiymes of four transgenic lines of mice, all of which suffered from male infertility /subfertility, were examined. Deleted in azoospermia -like autosomal (Dazla)-deficient mice (-/-) are infertile, and males fail to produce any mature spermatozoa; heterozygous (+/-) males are fertile but exhibit reduced numbers of spermatozoa with high incidence of morphological abnormality. Reduced numbers of motile spermatozoa and increased incidence of morphological abnormalities in motile spermatozoa from +l- Dazla mice was observed. Compared to wild type (+/+) mice, the level of DNA damage in the spermatozoa of +/- mice was significantly higher. Levels of DNA damage in both +1+ and +/- were increased following in vitro heat treatment. Similar findings were also observed in mice with excision repair cross - complementation gene 1 (ercc -1) genotypes (+/- and -/-). ERCC1 is involved in the nucleotide excision repair (NER) pathway and mitotic recombination process, and is highly expressed in the testis. Deletion of the Prion protein (PrP) or the PrP- related gene Doppel (PrnD) also resulted in lower numbers of motile spermatozoa with higher levels of DNA damage. However, following in vitro heating, levels of DNA damage in the motile spermatozoa from these mice did not increase. To investigate the effects of heat stress on DNA integrity, wild type mice (Dazia +/+) underwent scrotal heating (42 °C, 30 min) and were then sacrificed at various time points; 1, 2, 4, 6 or 24 hours (h) or 7, 14, 21, 24, 28, and 32 days (d) after heat stress. Testes and epididymides were removed and fixed for histological analysis, and motile epididymal spermatozoa were retrieved. Altered expression of Cirp (a heat -response protein), heat shock protein 105 (HSP105) and Bax (a pro - apoptotic protein), together with increased numbers of TUNEL- positive cells were detected in testes. Expression of Cirp, Bax and the macrophage marker CD68 were also altered in the epididymis. Levels of DNA damage in motile spermatozoa increased significantly within lh of heating, reaching a peak at 4h and then recovering to control levels at 7 and 14d. At 21d after heating, DNA damage increased again reaching a second peak at 28d and failing to recover by 32d. These results indicate that motile spermatozoa in the epididymis are susceptible to heat -induced DNA damage. Furthermore, while DNA integrity of spermatozoa derived from spermatids subjected to heat stress is normal, loss of DNA integrity in pre-meiotic germ cells caused by heat stress is not repaired as these cells mature, resulting in motile spermatozoa with impaired DNA integrity. These findings suggest that it may be necessary for additional criteria to be taken into consideration when selecting spermatozoa for ICSI/IVF treatment.

The aim of this project is to understand how germ cells are specified in the grasshopper Schistocerca gregaria. I made expression constructs of the Schistocerca Vasa protein and the Drosophila Vasa protein, and produced them through bacteria hosts. Affinity purified Schistocerca and Drosophila vasa fusion proteins were sequentially injected into rabbits to induce antibodies cross reacting to conserved vasa-specific motifs. I affinity purified antibodies from two rabbits and named the antibodies Formosa 1 (for most vasa) and Formosa 2. Western blots demonstrate that both of the purified Formosa antibodies recognise protein specifically expressed in the Schistocerca ovary and testis. Both antibodies also recognise germ cells in the pea aphid. Formosa 2 antibody recognises Drosophila pole cells. Furthermore, like the anti-mouse vasa protein antibody, both Formosa antibodies specifically detect a protein in the mouse testis. The size of the protein is very similar to that of the mouse Vasa protein. My immunostaining results show that putative germ cells lie in the outer germ band layer at a dorsal site in embryos from 15% to 30% of development. After 35% of development, they are guided into the bilateral gonadal mesoderm, which migrates dorsally toward the midline of the body. The bilateral gonad primordia coalesce around 70% of development. I conclude that the Schistocerca Vasa protein is a germ-cell marker. Using this marker I have traced the germline lineage through all developmental stages, though the identification of germ cells at early stages remains to be confirmed.

Long interspersed nuclear element 1 (LINE-1) is the only autonomous retrotransposon in humans and represents 17% of the genome. LINE-1 retrotransposition occurs by a "copy and paste" mechanism in which an RNA intermediate is reverse-transcribed and integrates at a new genomic location. LINE-1 elements have shaped the evolution of the human genome but are often deleterious causing insertional mutagenesis, transcriptional dysregulation and genomic instability. De novo LINE-1 insertions are frequently pathogenic causing inherited genetic diseases but also contributing to somatic diseases, such as auto-inflammatory and neurological disorders, and tumorigenesis. Therefore, organisms have evolved multiple mechanisms to restrict LINE-1 element expansion. In somatic cells, LINE-1 elements are usually transcriptionally silenced with both DNA methylation and chromatin modification playing critical roles in restricting LINE-1. Germline-specific restriction factors, such as the piRNA, are critical to restrict LINE-1 retrotransposition preventing the acquisition of novel integrations between generations but also to maintain fertility. However, an emerging body of evidence has implicated DNA repair as a new process contributing to both suppressing and promoting LINE-1 retrotransposition. Roles for factors involved in a wide range of repair processes have been implicated using different cell-based assays, from the repair of bulky adducts (factors involved in nucleotide excision repair) to factors required to maintain DNA replication fork stability (BRCA1 and FANCD2). Despite this, two key questions remain to be elucidated; firstly, how does the DNA repair machinery suppress LINE-1 retrotransposition? And secondly, is DNA repair a physiologically relevant LINE-1 restriction mechanism? Here, using reverse genetics, I show that Fanconi Anaemia DNA inter-strand crosslink (FA ICL) repair factors act in concert to restrict LINE-1 retrotransposition. Moreover, by purifying the recombinant FA ICL repair incision complex (SLX4-XPF-ERCC1), I show that it can cleave intermediates of retrotransposition, aborting new integrations. I show that all stages of FA ICL repair, defective in the human disease FA, are required to prevent retrotransposition in vivo. I find that FA ICL repair-deficient mice accumulate LINE-1 integrations in an array of somatic tissues with male germ cells having very high levels of such events. Finally, I find that early zygotic development is particularly dependent upon FA ICL repair factors to prevent LINE-1 retrotransposition.

Prophase I encapsulates the unique and defining events of meiosis; chromosome pairing, homologous recombination, synapsis, and subsequent segregation, to produce genetically unique haploid germ cells. These essential processes depend on a variety of protein complexes including the meiotic telomere complex (MTC), the synaptonemal complex (SC) and the meiotic recombination machinery. To carry out homology searches chromosomes must be tethered to the nuclear envelope, this is achieved by interplay between the MTC proteins (MAJIN, TERB1, and TERB2) and the shelterin complex protein, TRF1. Once attached, chromosomes undergo rapid prophase movements to find their homologous partner and begin to synapse. This involves the formation of the universally conserved SC structure along the lengths of aligned homologues. The SC provides the essential structural framework for HR and the crossover (CO) pathway. CO formation is dependent on the pro-CO machinery, including the E3 ligase proteins, HEI10 and RNF212. The main focus of this thesis is to use biophysical and structural approaches to deepen our understanding of the roles and mechanisms of the proteins and protein complexes involved in these meiotic processes. Here, we report the structural basis of the mammalian MTC and provide a mechanistic insight into chromosome tethering at the inner nuclear membrane (INM) achieved by the MTC. We show that the MTC recruits telomere-bound TRF1, through the 2:1 TRF1:TERB1 interaction, and undergoes subsequent structural rearrangement to displace TRF1 allowing the MTC to directly bind telomeric DNA and subsequently stabilise telomere-INM connectivity. The core architecture of mammalian SC is provided through the self-assembly of the transverse filament (TF) protein, SYCP1. We provide the first structural analysis of the D. melanogaster SC, specifically the TF protein, C(3)G. Biophysical analysis reveals that the central α-helical domain of C(3)G form dimers in a side-by-side parallel arrangement, but has some propensity to tetramerise, which could serve as building blocks for the recruitment and assembly of the complete SC. We show that HEI10 forms an obligate tetrameric structure and RNF212:RNF212b for a highly stable 2:2 complex and propose a structural model for the human E3 ligase proteins based upon solution scattering studies. Together, these findings provide a solid foundation for elucidating the mechanisms of mammalian meiosis.

The aim of this work was to assess the influence of germ cells on the expression of selected Sertoli cell mRNAs. To this end, adult rats were treated with 650mg/kg methoxyacetic acid (MAA) to induce the specific depletion of >80% of pachytene and later spermatocytes from most tubules, and expression of selected Sertoli cell mRNAs was then assessed at various times after treatment when particular germ cell types were depleted selectively (see Bartlett et al. 1988; Allenby et al., 1991). Studies on the expression of cyclic protein 2(CP-2) mRNA supported the hypothesis that germ cells can influence the cyclic function of Sertoli cells. Expression of CP-2 mRNA was shown by Northern blot analysis to decrease significantly 21 days after MAA treatment. In situ hybridisation showed that CP-2 mRNA expression was decreased or absent from tubules at stages at which CP-2 mRNA is normally expressed (stages IV-VII) when elongate spermatids were depleted selectively from these tubules. This decrease was reflected in loss of CP-2 protein production. These observations lead us to hypothesise that elongate spermatids positively modulate CP-2 expression in the Sertoli cell, with this modulation occurring at the level of transcription. In conclusion, this study demonstrated that germ cells do appear to influence Sertoli cell gene expression. This can occur at the level of transcription as was demonstrated by the effect of elongate spermatids on CP-2 mRNA expression, or may occur post-transcriptionally, as would appear to be the case with ABP.

Early work in this laboratory has shown that after testicular damage is induced in the rat there is an increase in the excretion of creatine in the urine. The aim of this project was to examine the distribution and metabolism of creatine within the rat testis, and to assess urinary creatine excretion as an index of testicular damage. Groups of rats were administered single doses of various cell-specific testicular toxicants: a germ cell toxicant (methoxyacetic acid, MAA), one of two Sertoli cell toxicants (di-n-pentyl phthalate, DPP; 1,3-dinitrobenzene, 1,3-DNB), or a Leydig cell toxicant (ethane-1,2-dimethane sulphonate, EDS). Urinary creatine and creatinine levels were monitored over the following 48 h, after which time the testes were removed, weighed and, after processing, sections were examined by light microscopy. All four treatments resulted in reductions in relative testis weight and produced morphological changes similar to those which have been widely reported in the literature. In addition, MAA, DPP and 1,3-DNB all caused large, transient elevations in urinary creatine excretion and the urinary creatine:creatinine ratio within 24 h. EDS had no such effect. It is inferred from these observations that creatine is associated with the seminiferous epithelium, and may represent a marker for damage to these cells in vivo. The distribution of creatine within the seminiferous epithelium was examined by the use of Sertoli and germ cells isolated from the testes of 4 week old rats. Both cell populations were found to contain creatine and N-phosphorylcreatine (PCr) pools, and to express creatine phosphoryltransferase (CPT) activity. Isolated seminiferous tubules synthesised creatine, and its biosynthetic precursor, guanidinoacetic acid (GAA), from L-arginine as determined by radio-metabolism studies using L-[guanidino-14C]arginine. A crude interstitial cell preparation synthesised neither creatine nor GAA. Cultured Sertoli cells incorporated radioactivity from both L-[guanidino-14C]arginine and [1-14C]glycine into creatine and GAA. Therefore, Sertoli cells have the capacity to carry out both stages of creatine synthesis; transamidination between arginine and glycine, with subsequent methylation of GAA to creatine. Germ cells did not exhibit this activity. Cultured Sertoli cells, maintained in a defined medium, secreted creatine into the overlying incubation medium, in a manner which was linear with time over at least 6 h, but which reached a plateau within 24 h. The secretion of creatine over 24 h was stimulated both by physiological and toxicological modulators of Sertoli cell function. Stimulation of creatine secretion by follicle-stimulating hormone (FSH) and N6,O-2'-dibutyl adenosine 3',5'-cyclic monophosphate (dbcAMP) was enhanced by the inhibition of adenosine 3',5'-cyclic monophosphate phosphodiesterase (cAMP PDE), indicating that creatine secretion is under the influence of intracellular cAMP levels. The secretion of creatine by cultured Sertoli cells, incubated over 4 h in BBSS, was independent of exogenous L-glutamine. However, the dbcAMP-induced stimulation of creatine secretion was dependent upon the presence of L-glutamine in the incubation medium, suggesting that increases in creatine secretion may occur as a consequence of stimulated glutamine oxidation. Isolated germ cells sequestered [1-14C]creatine in a time-dependent manner, which was linear over at least 3 h, by a two component process. One component had a high affinity for creatine (Km = 25 μM), was inhibited both at low temperature and by the absence of sodium ions from the medium, and probably represents an active uptake system. The other component, which had a low affinity for creatine and was independent of temperature and sodium, probably represents passive diffusion. It is concluded that an intercellular pathway for creatine metabolism may exist within the seminiferous epithelium of the rat, by which creatine is synthesised and secreted by the Sertoli cells and is taken up by the germ cells from the interstitial milieu. Creatine is phosphorylated within both the Sertoli and germ cells, but its role is as yet unclear.

Gastrulation occurs between 6.5 and 8.0 days post coitum in the mouse and is associated with a dramatic increase in cell proliferation, being initiated when the embryo consists of some 500-600 cells and resulting in a neural plate stage embryo containing 50,000 cells or more. There is evidence to suggest that some of these areas may be determined with respect to their future development quite early in the process of gastrulation. During this period of development it is clear that the growth rate of the whole embryo, and of its constituent parts, is under intrinsic control and capable of controlled variation aimed presumably at generating an organogenesis stage embryo of a notional target size in which the parts are of appropriate relative size. Investigations in the mouse have shown that growth of the embryo parts and their co-ordination can be profoundly disturbed in early embryonic stages by insult with the cytotoxic agent, Mitomycin C. MMC is an alkylating agent that inhibits DNA synthesis. It causes cell death and reduces pre-organogenesis stage embryos to 10% of normal size as measured by cell number. During the progression of embryogenesis the normal co-ordination of development of different parts of the embryo is abnormal. Considerable and localized growth control is obviously operating in these stages, thus the proto-oncogenes with known growth regulatory functions were looked at as likely candidates controlling the dramatic growth patterns observed. During the gastrulation movements the primordial germ cells are located at the base of the allantois and appear to 'escape' the movements and rearrangements of gastrulation by migrating into the extraembryonic membranes. The primordial germ cells then appear to undergo their own differentiation, proliferate and migrate into the genital ridges. The germ cells multiply as they migrate until some 5000 cells ultimately invade the gonads. The proto-oncogene c-kit expression was analysed, as it has been recently identified with the dominant white spotting (W) locus of the mouse. The W mutations have pleiotropic effects on mouse development, one of which is to cause an impairment of proliferation and/or migration of primordial germ cells. It appears that the c-kit product is necessary for the maintenance of the primordial germ cells during their migratory/mitotically active phase. This finding holds true for overall expression of c-kit in structures of the developing embryo. As the testes develop and become functional, the germ cells undergo further differentiation. There is evidence from Y chromosome deletion mapping that there is a multiple copy gene on the long arm of the mouse Y which is needed for the normal development of the sperm head. A Y-specific genomic DNA sequence (Y353/B) has previously been described which is present in multiple copies on the long arm of the mouse Y and is specifically transcribed in the testis. Using quantitative Northern analysis it was shown that the RNA transcripts are from the multiple copies. In situ hybridization was used to demonstrate that the transcripts are In the germ line and not In the soma, and that the expression is predominantly in round spermatids. This was the first evidence for a Y chromosome gene being transcribed in the testis (and probably anywhere). Y353/B is thus a candidate for being the multiple copy 'sperm morphology gene' on the mouse Y chromosome.

In females, reproduction is a key facet of women’s health, livestock production, and conservation of endangered species. The ovary is the gonad of the female and is a central component of female reproductive physiology as it houses the germ cells (i.e., oocytes) within follicles and produces hormones to coordinate folliculogenesis. The ovary is a target organ for developing novel assisted reproductive technologies (ART) such as safeguarding fertility via cryopreserving ovarian tissue containing follicles and developing strategies to grow early staged preantral follicles in vitro for oocyte production. Moreover, a better understanding of cellular and molecular events constituting early gonad development would be widely valuable to further advance ART that capitalizes on components of the ovary. In this work, we describe three aims using the bovine as a model and collectively expand our current knowledge in 1) ovarian tissue cryopreservation (OTC), 2) in vitro bovine preantral follicle development, and 3) in vitro differentiation of bovine embryonic stem cells (bESCs) into progenitors of bipotential gonad-like cells. In Aim 1, we found that, overall, slow freezing outperformed vitrification when bovine ovarian fragments were thawed and cultured as indicated by higher rates of normal follicle morphology, lower rates of apoptosis in stromal cells, and proper expression of the gap junction protein connexin 37. In addition to the use of OTC and culturing ovarian tissue fragments to rescue folliculogenesis, in Aim 2 we explored the use of bioengineered and proteolytically degradable (poly)ethylene glycol (PEG) hydrogels for in vitro culture of isolated bovine preantral follicles. We also tested co-encapsulation of mesoderm-like cells (MeLCs) from bESCs or native bovine ovarian cells (BOCs) with follicles to determine their propensity to become theca-like cells and improve folliculogenesis in vitro. After customizing a PEG hydrogel to complement bovine preantral follicle extracellular matrix (ECM) enzyme expression, we found that smaller follicles grew better in PEG hydrogels compared to two-dimensional control over ten days. However, MeLCs did not survive PEG hydrogel encapsulation process. Although BOCs could survive in PEG hydrogels throughout the culture period, BOCs did not better support follicle survival and development when co-encapsulated compared to PEG-only control. Moreover, the culture conditions did not maintain expression of selected genes essential for theca cell differentiation. Finally, in Aim 3, we tested the ability of bESCs to be driven towards the intermediate mesoderm (IM), early coelomic epithelium (eCE), and steroidogenic state during in vitro culture using several protocols. Our data indicated that inclusion of the steroidogenic factor-1 (SF1) agonist, RJW100, did not upregulate steroidogenic genes during the differentiation. Similarly, the cytokines basic fibroblast growth factor (bFGF) and bone morphogenic protein-4 (BMP4) were dispensable for inducing IM and eCE gene expression, and WNT activation was a predominant driver of eCE and IM gene expression, high concentrations of BMP4 led to upregulation of lateral plate mesoderm gene expression, and paraxial mesoderm gene expression remained unchanged across all regimens. Collectively, this dissertation work sheds light on several facets of ovarian biology including a better understanding of the impacts of OTC, in vitro preantral folliculogenesis, and use of embryonic stem cells to recapitulate bipotential gonad somatic cell differentiation. Nevertheless, additional research is required to elucidate mechanisms that are pivotal to both early folliculogenesis and early embryonic gonad formation in the bovine model.

Chromosome ends face two problems: the end-protection (end-to-end fusions) and the end-replication (progressive telomere shortening) problems. The protein complex shelterin binds to the telomeric DNA repeats at chromosome ends to protect them from illicit end joining and other unwanted recombination or resection events. The ribonucleoprotein complex telomerase extends chromosome ends in somatic and germline stem cells to overcome the end-replication problem and ensure continued proliferation. Mutations in genes important for telomerase and/or shelterin function often result in diseases termed telomeropathies, the most prominent example of which being dyskeratosis congenita (DC). Severe shortening of telomeres in patients with DC results in depletion of stem cells and bone marrow (BM) failure, the primary cause of death. TPP1 is the only shelterin component known to both protect chromosome ends and recruit telomerase to telomeres. We have previously defined regions of TPP1 that are critical for recruiting telomerase and characterized the consequences of a patient-derived DC mutation in TPP1 (K170Δ) resulting in decreased telomerase activity, impaired recruitment, and short telomeres in cultured human cells. While these studies provide a direct cause-effect relationship, they do not provide insights into stem cell dysfunction in vivo. A DC mutation in TPP1 (K170∆) that specifically compromises telomerase recruitment to telomeres is a valuable tool to evaluate telomerase-dependent telomere length maintenance in mice. In this dissertation, I first present work on how we used CRISPR-Cas9 to generate a mouse knocked in for the equivalent of the TPP1 K170∆ mutation (TPP1 K82∆) and investigated both its hematopoietic (Chapter 2) and germline (Chapter 3) compartments in unprecedented detail. TPP1 K82∆ caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82∆ caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of dysfunctional seminiferous tubules, and a decrease in germ cells. Intriguingly, both TPP1 K82∆ mice and previously characterized telomerase knockout mice show no spontaneous BM failure but rather succumb to infertility at steady-state. Our work suggests a species-specific sensitivity in the germline rather than the soma in mice, which is reversed in human patients with this disease. We speculate that the species-specific differences in the response to severe telomere shortening arises from the distinct proliferation burdens on the mouse and human, soma and germline. Small and short-lived species like mice may boost germline proliferation to increase gamete production and maximize the number of offspring produced in a lifetime, while large and long-lived species like humans prioritize somatic development to survive to the age of reproduction (and nurturing) to produce fewer, but healthy, offspring.

Faithful cell division is crucial for all eukaryotic cells to ensure that daughter cells receive thecorrect number of chromosomes. Errors in chromosome segregation during meiosis in germ cells can result in aneuploid gametes, potentially leading to infertility, genetic disorders like Down Syndrome, and miscarriages. Similarly, chromosome segregation errors during mitosis can produce aneuploid somatic cells, which are often associated with cancer. Although the regulation of these two types of cell division is vital, the mechanisms that ensure their accuracy are still actively being studied. Among the proteins identified as key regulators of both mitotic and meiotic fidelity in various species, including Caenorhabditis elegans (C. elegans), is PCH-2. PCH-2 is a conserved AAA+ ATPase required for timely coordination of the meiotic prophase events pairing, synapsis and recombination, but the mechanism for how it accomplishes this coordination is currently unknown. Additionally, PCH-2 has been shown to promote crossover formation and affect their distribution. Here I use C. elegans as a model system to investigate the conserved meiotic role of PCH-2.In Chapter 1, I begin my investigation of PCH-2’s conserved role by observing it’s localizationto meiotic chromosomes in the mnT12 background, where two chromosomes, X and IV, are fused. In order to fully understand how defects in chromosomal rearrangements can affect PCH-2’s localization, I also studied its localization in heterozygous inversions, mIn1/+ and hIn1/+. I find that PCH-2 localization is extended in the chromosomal fusion background mnT12, but not in mIn1 heterozygotes and hIn1 heterozygotes. I also find that the extension of PCH-2 in mnT12 worms results in the promotion of crossover formation, providing another example of how PCH-2 can affect crossover formation.In Chapter 2, I demonstrate that PCH-2 regulates the number and distribution of crossoversby opposing their formation. This antagonistic action has distinct effects at various stages of meiotic prophase. I show that in the early stages, PCH-2 restricts the conversion of double strand breaks (DSBs) into crossovers, thereby limiting crossover events at the initial sites of DSB formation and homolog interactions. And then later in meiotic prophase, PCH-2 reduces the number of crossover-eligible intermediates, aiding in the reinforcement and designation of these intermediates, and ultimately ensuring crossover assurance. Additionally, I show that PCH-2 exerts this control through the meiotic HORMAD protein, HIM-3. The data I present strongly support a model where PCH-2's conserved function is to remodel meiotic HORMADs throughout meiotic prophase, destabilizing crossover-eligible precursors, coordinating meiotic recombination with synapsis, and contributing to the progressive execution of meiotic recombination to ensure precise crossover regulation.Finally in Chapter 3, I test PCH-2’s role on modulating synapsis. Specifically by observinghow the loading and unloading of a central region component of the synaptonemal complex (SC) is affected in the absence of PCH-2. I show that loss of PCH-2 does not affect how this protein – SYP-3, is loading onto chromosomes during meiotic prophase. Added together, my work establishes a crucial framework for understanding the role of PCH-2 in regulating the number and distribution of crossovers (Chapter 1 and Chapter 2), a function I propose is conserved across species. While specific mechanisms may differ among systems (Chapter 3), I suggest that PCH-2 plays a key role in remodeling meiotic HORMADs throughout meiotic prophase to destabilize crossover-eligible precursors. This process coordinates meiotic recombination with synapsis, contributing to the stepwise progression of recombination and ensuring crossover assurance, interference, and homeostasis.

The investigation into the processes involved in fetal gonadal development has been an area of interest for many years. This thesis investigated the effects of putative diffusible factors, produced by fetal mouse ovaries and testes in culture, on normal gonadal differentiation. It was shown that testis-conditioned media from a specific age in development (13-15dpc) induced a significant reduction in germ cell number within fetal (13dpc) ovaries. Results suggested the involvement of a diffusible factor distinct from Anti-Mullerian Hormone. In addition, a significant reduction in the total surface area covered by testicular cords in section was induced by exposure of 14dpc testes to 15-17dpc ovary-CM for 4 days. Further, adrenal-CM generated from 14-17dpc produced an advance in meiosis with significantly more synaptonemal complexes than in cultured controls. A possible masculinising effect was induced by testis-conditioned media generated from 14dpc onwards, with the formation of areas of tunica albuginea resembling that of a developing testis in some ovaries. It has been suggested that male germ cells located outwith testicular cords and ectopically located within the fetal adrenal gland, enter meiotic prophase at the same time as those within equivalent ovaries (Upadhyay and Zamboni, 1982) . No evidence of precocious entry into meiosis was noted in testes cultured within conditioned media generated from fetal adrenal- or ovary-conditioned media, even when testicular cords were disrupted. In addition, isolated germ cells, cultured upon fibronectin-coated coverslips, were successfully cultured and maintained within gonadal conditioned media for up to 7 days. Electrophoretic analysis revealed proteins of a higher molecular weight in 13-15dpc testis-CM than in eguivalent ovary-CM. Testis-CM generated between 17 and 19dpc, however, contained proteins of a lower molecular weight than in 17-19dpc ovary-CM indicating that distinct differences exist between developing ovaries and testes and gonads of different ages.

The mammalian Y chromosome has a fundamental role in the control of primary sex determination, diverting the undifferentiated bi-potential gonad to form a testis. In addition, the Y chromosome has been implicated in a number of other male-specific functions. This study aims to provide additional details of the function of the Y chromosome in spermatogenesis during development. A quantitative analysis of germ cells in XOSxrb mice compared to their XY±Sxrb sibs, during the first two post-natal weeks, investigated the function of the spermatogenesis gene Spy. The spermatogenesis gene was shown to act on the survival and proliferation of early differentiating A spermatogonia by five days after birth. By a quantitative analysis of germ cells in XOSxra and XYSxra mice - the latter identified by DNA analysis - throughout puberty, it was shown that there was no pre-meiotic cell loss. Cell degeneration was first observed at metaphase I in XOSxra testes, but earlier, at pachytene, in XYSxra mice. XYSxra testes are mosaic for normal and defective germ cell patches. The 'pairing site' hypothesis of Miklos (1974) states that there is a correlation between the number of 'unsaturated' pairing sites and the extent of spermatogenic impairment. It is suggested that the earlier spermatogenic breakdown in XYSxra testes, results from the presence of two unpaired univalents (as opposed to one in XOSxra) with the consequent increased number of 'unsaturated' sites. The XSxra chromosome was provided with a pairing partner - the chromosomal product, derived from XY* mice - which could satisfy at least some of the pairing sites, without adding Y long arm material. The germ cells in XSxraYdel testes overcame the block at Ml seen in XOSxra mice, and proceeded to sperm. All sperm, however, were abnormal, confirming previous findings that a sperm morphology gene (Smy), present on the long arm, is essential for fertility.

Over the past decade many publications attest to the substantial increase in public and scientific concern regarding the potential of industrial and environmental chemicals to interfere with male reproductive function. Spermatogenesis, (the process of sperm formation) depends on a series of complex biological interactions which can be interrupted at various stages by chemical insult. Recently a number of these chemicals have been used experimentally to directly disrupt testicular function to provide an insight into the processes involved in normal spermatogenesis. Based on these investigations the primary objective of the work in this thesis was to study the time course of development of testicular lesions, their stage specificity, and subsequent recovery of normal spermatogenesis using morphological and functional characteristics of function in vivo and in vitro. The chemicals used in these studies included meta dinitrobenzene (mDNB), nitrobenzene (NB), the isomers of mononitrotoluene (mNT) and 2-methoxyacetic acid (MAA). Within 24h of a single oral administration of mDNB or NB to rats there was a progressive decrease in testicular weight and increase in serum follicle stimulating hormone (FSH) levels coincident with widespread germ cell degeneration, as determined by histological examination of the testis. The degree of disruption induced by mDNB and its reversibility were time - but not stage - dependent with a decline in both sperm number and motility in parallel with germ cell degeneration. NB however had a more specific effect causing the initial degeneration of leptotene spermatocytes and subsequently more extensive germ cell loss involving round spermatids at a number of stages. During the recovery of spermatogenesis a considerable change was observed in the frequency of occurrence of spermatogenic stages in comparison to controls. These changes remained significantly different up to at least 70 days post-treatment. The isomers of mNT induced small but statistically significant dose-dependent decreases in testicular weight. Spermatogenesis in these animals remained qualitatively normal, so detailed histological studies were not undertaken. Further investigation into the effects of the testicular toxicants listed above was performed in vitro using Sertoli cell monocultures and Sertoli cell-germ cell co-cultures. mDNB, NB and the isomers of mNT produced dose-dependent increases in germ cell exfoliation (from co-cultures). mDNB and NB also produced histological changes in culture including Sertoli cell vacuolation and phagocytosis of germ cells, and induced dose-dependent increases in the secretion of lactate and pyruvate, two established indices of Sertoli cell function in vitro. Sertoli cells secrete over 100 different proteins, only a handful of which have been characterised and a role in spermatogeneis proposed. One of these proteins is inhibin. The potenial of inhibin secretion as a further index of Sertoli cell function (or dysfunction) in monocultures and co-cultures has been investigated. mDNB, NB, 2-mNT and 3-mNT all induced dose-dependent alterations in the secretion of inhibin in vitro. Historically, FSH has been postulated to control inhibin secretion in a classical negative feedback manner. Recent evidence obtained in vivo and in co-culture suggests that its secretion may be controlled by specific type(s) of germ cells. Seminiferous tubule cultures represent a potentially more useful culture system to investigate this further. They present a unique opportunity to study Sertoli cell-germ cell interactions under more physiological conditions since the integrity of the seminiferous epithelium is maintained in vitro. Previous investigators have demonstrated that MAA specifically destroys pachytene spematocytes in vivoand this specificity of action was utilised to study which germ cel-Sertoli cell interactions control the secretion of inhibin. At selected times after MAA treatment when specific complements of germ cells were absent or grossly depleted, seminiferous tubules were isolated and cultured to measure inhibin secretion under basal or stimulated conditions. The results demonstrate that elongate spematids are the primary germ cell type which exerts major control over inhibin secretion under both basal and FSH-stimulated conditions. In conclusion, these studies demonstrate: Firstly that these compounds can be used as a means to investigate normal spermatogenesis. Secondly, effects of these compounds on spermatogenesis can be demonstrated both in vivo and in vitro. Finally, that the seminiferous tubule culture technique represents a more physiological approach with which to investigate in vivo - in vitro comparative toxicity with respect to chemically induced disruption of spermatogenesis.

In sexually reproducing organisms, germ cell development is vital for the faithful transmission of the genome and epigenome across generations. Recent studies have shown that germ cell development is affected by different environmental toxicants, resulting in a decrease in germ cell health and number. Here, we examine the transgenerational impact and mechanisms of two prevalent toxicants, the plastic manufacturing chemical Bisphenol A and ethanol, in Caenorhabditis elegans. Both have well-described impacts on the developing fetus; however, their effects on developing germ cells and subsequent generations are less explored. We hypothesize that exposure disrupts the epigenetic machinery in germ cells, causing changes in histone modifications, fertility defects, and germline dysfunction in a transgenerational manner. First, we show that BPA exposure causes a transgenerational two-fold increase in germline chromatin desilencing coupled with a reduction and redistribution of histone H3K9me3 and H3K27me3. We show that the alteration of repressive histone levels is required for the observed transgenerational 43% increase in germline apoptosis and 85% increase in embryonic lethality. By performing a genetic rescue using RNAi, we identified the chromatin modifying enzymes H3K9 and H3K27 demethylases (jmjd-2, jmjd-3, and utx-1) to be responsible for the transgenerational effects of BPA, since RNAi alone was able to restore the chromatin into a silenced state and rescue fertility defects. This confirms our idea that epigenetic memory is mediated by histone marks. Next, an increase in apoptosis suggests possible perturbations in the germline checkpoint machinery. To understand which checkpoint BPA perturbs, we used mutants of each checkpoint to rescue the effect. This revealed that BPA perturbs the synapsis checkpoint since a pch-2 mutant decreased BPA induced apoptosis by two-fold whereas mutants of the DNA damage checkpoint, spo-11 and cep-1, did not. Furthermore, visualization of the synaptonemal complex (SC) using a SYP-3::GFP strain revealed perturbations in proper SC assembly and aggregation of SC proteins resulting from ancestral BPA exposure. The formation of these aggregates was further associated with increases in embryonic lethality transgenerationally at the F3. In parallel, we also explored another common toxicant, ethanol, but instead we built the complete transgenerational transcriptional map of ethanol using single cell RNA-seq approaches. Together, my work identified the molecular drivers of environmental transgenerational effects on the germline machinery and reproductive health. We hope to further understand how it induces germline dysfunction, carrying important implications for human reproductive health in the context of environmental exposures.

While physiologic stress has long been known to impair mammalian reproductive capacity through hormonal dysregulation, mounting evidence now suggests that stress experienced prior to or during gestation may also negatively impact the health of future offspring. A growing body of work in recent years has clearly demonstrated that rodent models of physiologic stress can induce a variety of neurologic and behavioral phenotypes that are able to persist for up to three generations, suggesting that stress signals can induce lasting epigenetic changes in the germline. Any perturbations to the proper transmission of genetic information through the germ cells can be detrimental, and thus understanding the mechanism by which stress can lead to epigenetic alterations in the germline remains a crucial unanswered question in the field. Interestingly, treatment with glucocorticoid stress hormones is sufficient to recapitulate the transgenerational phenotypic inheritance seen in physiologic stress models. These hormones are known to bind and activate the glucocorticoid receptor (GR), a ligand-inducible transcription factor, thus implicating GR-mediated signaling as a potential contributor to the transgenerational inheritance of stress-induced phenotypes. It remains unclear, however, whether the heritability of stress-induced phenotypes following glucocorticoid treatment results from direct actions of glucocorticoids on the germ cells of the gonad, or from indirect effects of glucocorticoids elsewhere in the body. Moreover, evidence for the expression of GR in the developing and adult germ cells of both the male and female remains controversial, making it difficult to assess any potential cell-autonomous roles of GR in modulating the epigenome of the germline in response to stress. We therefore set out to definitively characterize the expression of GR in the germline of the developing and adult gonads, and to determine what cell-intrinsic role GR plays in normal germline formation and function. We discovered that GR is expressed in the female germline specifically during fetal development (peaking at approximately E13.5 - E14.5), yet is absent from the adult oocyte. We found that the female germline is, surprisingly, resistant to changes in GR signaling. Both genetic deletion of GR as well as GR agonism with dexamethasone revealed minimal transcriptional changes in the female germ cells, as well as no significant changes in meiotic progression, suggesting the female germline is intrinsically buffered against changes in GR signaling. In contrast we found that GR is expressed in the male germline during the final days of development, with expression maintained in pro-spermatogonia during early postnatal development. This expression becomes restricted to the spermatogonia of the adult, and transcriptomic analysis of germ cells from dexamethasone treated males revealed a potential role of GR in regulating RNA splicing. Together our data confirm the dynamic spatiotemporal expression of GR in both the male and female germ cells of the mouse, and suggest a sexually dimorphic function for this receptor in the mammalian germline.

The gonad is vital for successful sexual reproduction and species’ survival. Gonad development is wellstudied in mammals, but far less is known about its development and subsequent sex determination in teleosts. In mammals, early gonad development begins with the specification of primordial germ cells (PGCs) that migrate to the genital ridge. After the coalescence of PGCs and somatic gonad cell precursors at the genital ridge, the primordial gonad undergoes significant proliferation and eventual sex differentiation. However, in zebrafish, early gonad development and sex determination remain to be understood. There is growing evidence that gap junction proteins have an integral role in supporting early germ cell development in mammals. Gap junctions serve to transfer large molecules between cells for communication as well as small metabolites. In the ovary, there are two known gap junction proteins, Gja1 and Gja4, that connect the oocyte to the somatic granulosa cells in order to facilitate metabolic support. We recently found that the gap junction protein gjc4b is expressed in zebrafish oocytes through single-cell RNAseq. We hypothesize that gjc4b may serve a similar role as Gja4 in connecting the somatic follicle cells to the developing oocytes. During our investigation, I discovered that gjc4b mutants have normal PGC migration and apparent survival but have deformed or absent germ cells past 20 days post fertilization (dpf) that retain a PGC-like state instead of transitioning to a gonocyte. Additionally, there is significant evidence in mammals that Wnt4 is the female sex determination factor. We have previously shown that the zebrafish ortholog, wnt4, is also required for primary female sex determination: mutants develop with a significant male bias and are largely incapable of initiating any oocyte development that would be indicative of the potential for female development. We have recently identified an additional Wnt ligand expressed in the juvenile ovary through single-cell RNAseq analysis called wnt9b. I investigated its role in sex determination and found through wnt9b mutant analysis that wnt9b plays a role in female sex maintenance.

The primary aim of this thesis was to examine the specification of human primordial germ cells (PGCs) and the mechanism regulating the transient gain and loss of competence for germ cell fate. Specification of PGCs in humans occurs during weeks 2 to 3 in gastrulating embryos, which eventually develop into sperm and ova, generate totipotency at fertilisation, and transmit genetic and epigenetic information for development. Since direct studies on early human embryos are not feasible, I used an established human pluripotent stem cell-based in vitro model for early human development. Accordingly, I used human pluripotent stem cells (PSCs) to induce mesendoderm (ME), which at midpoint progresses through a pre-mesendoderm (PreME) state that transiently acquires competence for PGC fate and commits to PGC-like cells (PGCLCs) in response to BMP signalling. To address these questions, I performed highly-resolved transcriptomic profiling and careful epigenomic characterisation of cis-regulatory regions at key steps during the in vitro model. To determine the regulation of the transient gain and loss of competence for germ cell fate, I re-engineered a CRISPR toolkit for activation and inactivation to test candidates for germ cell competence. I found the inhibition of OTX2 increased the efficiency of human PGCLC specification, which is consistent with a transient reduction in OTX2 in the germline competent PreME against rising levels of EOMES. The OTX2 levels continue to increase in ME cells that gain competence for the mesoderm and endoderm fates but are incompetent for germ cell fate. Using the CRISPR toolkit, I further show that SOX17 enhancers are the critical targets of the regulatory circuitry of germline competence. SOX17 and TFAP2C initiate a core germline program, including induction of PRDM1 by SOX17 and maintenance of pluripotency factors, POU5F1 and NANOG. I also explored PGC competence and specification using highly resolved single-cell transcriptomic profiling of our in vitro PGC-like cells (PGCLC). Using an integrative analysis with existing human and primate embryonic datasets demonstrates that our model represents the lineages at the caudal region of the embryo, including mesoderm, endoderm, PGCs and amnion. I found a high degree of fidelity between in vitro PGCLC and bona fide early embryonic PGCs, which map spatially to a transition region in primate embryos in proximity to the amnion. I further identified TFAP2A as a critical early regulator of human PGC fate, with a potential role in suppressing SOX2, which might contribute to initiating the combinatorial role of OCT4-SOX17 for the PGC program. This differs from mouse PGC specification, where no functional relevance for TFAP2A or SOX17 has been described. Finally, to test if SOX17 is a critical regulator of PGC fate in another mammal that develops as a bilaminar disc at the time of PGC specification, I examined early development and PGC specification in bilaminar disc rabbit embryos (in collaboration with Toshihiro Kobayashi). Indeed, SOX17 is a crucial determinant of rabbit PGCs, which are transcriptionally similar to their human PGC counterparts. Accordingly, rabbits are a suitable animal surrogate for investigating early human development and the origin of the germline. This study provides further evidence that development via embryonic disc morphology is correlated with a SOX17-centric PGC network topology, in stark contrast to the PGC network in cup-shaped embryos such as in mice. In this thesis, I revealed several new facets about the origin of the human germline. I demonstrated for the first time that OTX2 has a role in human germline competence, discovered a novel role for TFAP2A during germline formation, and revealed an intricate enhancer-regulated transcription factor (TF) network underpinning human PGC specification and progression.

The core feature of human heritable genome editing ("HGE") is that any modification made on the germ cells (sperms or eggs) or early human embryos is likely to be passed down to subsequent generations. This feature makes the use of this technology highly controversial. Thus, there is a need to adopt a suitable approach to consider future children who are going to live the consequences of this technology. Taking a child-centred perspective, this thesis explores two main concerns: first, whether, and to what extent, the act of HGE is ethically acceptable; and second, how should HGE be regulated if it is deemed ethically acceptable for reproductive use. More specifically, it examines how a child-centred approach should be adopted in answering these questions. This thesis first examines four child-related arguments commonly raised in the existing HGE debate, including the non-identity problem, the open future argument, the purported right to non-modified genomes, and the proposed long-term follow up system for the resulting children. It argues that the existing arguments and proposal have failed to appropriately consider (future) children's interests mainly because there is (1) a lack of solid theoretical framework to justify the recommendation commonly made for a health-related use of HGE, and (2) a lack of detailed analysis on an important but often overlooked concept in the HGE debate, that is the concept of "identity". In order to fill these gaps, this thesis explores the capability approach and argues that Jennifer Ruger's health capability paradigm provides a good philosophical framework in understanding why "health capabilities" can justify and more importantly, also limit the health-related use of the technology. Additionally, this thesis analyses the concepts of "identity" and argues for a multi-faceted understanding of "identity" with narrative identity to be given the greatest weight in the ethico-legal considerations of HGE.

During meiosis in diploid organisms, Mendel's laws suggest that homologous chromosomes should recombine and be randomly distributed to gametes. This is true for meiosis in most organisms, however, in some cases, chromosome transmission is not random, and can be biased depending on a number of factors including the parent of origin of a chromosome or selfish genes located on the chromosome. Why and how non-Mendelian inheritance evolves is not well understood in many cases, but having a good understanding of the evolution of these systems is important for establishing under what scenarios non-Mendelian inheritance might evolve and how meiosis is manipulated by certain chromosomes. This thesis focuses on two insect clades that exhibit non-Mendelian inheritance: scale insects in the family Eriococcidae and fungus gnats in the superfamily Sciaroidea. Both of these families exhibit a non-Mendelian inheritance system known as paternal genome elimination (PGE), where males only transmit chromosomes inherited from their mother to future generations. In Sciaroidea, I focus on one species with an unusual genetic system: Bradysia coprophila, which in addition to PGE has an unusual reproduction system in several other respects. I examine content and evolution of germline restricted chromosomes, which are present in the germ cells in this species but eliminated from somatic cells early in development. I characterise how many genes are on these chromosomes, and explore using genomic methods how these chromosomes evolved. I also conduct a mating preference experiment on B. coprophila to determine whether the chromosome inheritance system in this species affects male mating preferences, as this is one case among animals where we may expect males to evolve mating preferences for certain types of females. In Eriococcidae, how PGE occurs is labile, and transitions between different types of PGE are not well understood. I conduct of survey of species to establish how male meiosis differs in species across this clade, how the type of PGE affects paternal chromosomes in somatic cells, and whether there is any evidence for transitions to new genetic systems (i.e., loss of PGE) in any species within this clade. Studying these two insect systems can provide information about what happens in lineages when non-Mendelian chromosome inheritance evolves. We can determine how this affects a species behaviour, the mechanisms of chromosome inheritance, and genome evolution.

Survival and persistence of live matter requires certain flexibility to allow multiple adaptations, yet it is grounded on orderly and stringent regulation of its important functions. An astounding example of natural planning is given by mammalian embryological development where a sophisticated specification program enclosed in a single cell is resolved into a diversity of tissues in a mature organism. Tracing back the developmental timeline is a way to deconvolute advanced biological properties that cells acquire through a series of hierarchical differentiation decisions. One of the primary unspecified states spanning a short period between conception and functional commitment of the cell is known as pluripotency. Pluripotency is the ability of a cell to give rise to the derivatives of the three germ layers and germ cells during differentiation. Naturally, pluripotency is restricted to the early embryo only and represented by two morphologically, physiologically and epigenetically sequential stages - naïve (embryonic stem cells, or ESCs) and primed (post-implantation epiblast stem cells, or EpiSCc). Revolutionary technologies of nuclear reprogramming are now able to artificially revert developmental vector and direct differentiated cells towards naïve pluripotency. Reprogrammed cells not only come as a useful object for fundamental research but also as a flexible biological tool for regenerative therapies. Currently reprogramming is moderately efficient, however reliable and safe medical strategies require decent understanding of the physiological and genetic networks underpinning cell identity change. Importantly, the primed pluripotent cell can be reprogrammed too allowing to focus on the first and often subtle cell identity transitions that cells adopt. In my project, I have performed a forward genome-wide CRISPR-gRNA knockout screen to identify genes working as barriers for reprogramming from EpiSCs to ESCs. Having assembled a comprehensive lentiviral library and established a tractable cell system to target the mouse genome, I screened EpiSCs in the reprogramming setup which allowed selection of effective reprogramming antagonists. Here I discover the JAK/STAT3 pathway regulator Socs3 as a key screen hit and describe this gene as a self-sufficient and powerful roadblock between the pluripotent identities. I show how a spectrum of its attenuated activities displayed in naïve pluripotent and reprogramming cells may be functionally linked to reinforced pluripotency. Exploring the interplay between JAK/STAT3 pathway and other major naïve pluripotency genes across naïve, primed and non-pluripotent cell contexts reveals partially conservative mode of action of SOCS3. I demonstrate how SOCS3 may integrate external signalling with endogenous core pluripotency factors and by thereby feed in the naïve circuity supporting it and instructing its establishment. This work helps understand the mechanisms behind reprogramming and biology of pluripotency which potentially can provide insights into consolidated and robust medical cell technologies.

Genomic imprinting is an epigenetic phenomenon which orchestrates allele specific expression of a subset of genes according to parental origin. Monoallelic expression is directed by imprinting control centres and is established in the germ line through acquisition of differential DNA methylation. Even though they represent a small subset of genes within the mammalian genome, imprinted genes have critical roles in an array of important biological functions including metabolism, growth and behaviour. Even though the evolutionary rationale for the functional haploidy of these gene is uncertain, the consequences of dysregulation of some imprinted genes demonstrate that gene dosage is important. Cdkn1c is a maternally expressed gene which has an important role in foetal development, behaviour and metabolism and has been shown to be sensitive to gene dosage alteration. In this thesis, I first demonstrate that embryonic germ cells, on fusion with somatic mouse B cells can, not only dominantly reprogram them, but can induce imprint erasure at the gametic DMR governing imprinted expression of Cdkn1c. I also describe the development and characterisation of a mouse line in which firefly luciferase was knocked in to endogenous mouse Cdkn1c. I demonstrate that bioluminescence imaging can be used to provide a non-invasive read out of endogenous imprinted gene expression thus providing an opportunity to not only monitor expression longitudinally but also provide an opportunity to study the consequences of imprint perturbation in vivo. Furthermore, the reporter was expressed in a parent-of-origin manner and heterozygotes were found to have wild type levels of transcript and DNA methylation at DMRs. Using this model, transient de-repression of the paternal allele by chromatin modifying drugs could by visualised non-invasively via bioluminescence in vivo in embryos. This work demonstrates that these reporter mouse models can be used to interrogate the effects of in utero environmental stressors on imprinted gene expression.

Alkaptonuria (AKU) is an ultra-rare, autosomal recessive metabolic bone disease characterised by increased urinary and circulating homogentisic acid (HGA) caused by homogentisate 1,2-dioxygenase (HGD) enzyme deficiency. The deposition of a dark, HGA-derived ochronotic pigment in cartilage and other connective tissues causes a severe and early-onset osteoarthropathy, due to ochronotic tissue being stiff and brittle. In addition to joint manifestations in AKU, eye pigmentation, tendon rupture, kidney and prostate stones, and heart valve stenosis are common features. An existing mutagenesis AKU mouse model has previously been used to investigate initial pigmentation of cartilage and to evaluate the effectiveness of the HGA-lowering drug nitisinone. Nitisinone therapy was proven to halt ochronosis in this mouse model and has been the subject of human clinical trials (DevelopAKUre), where it has been shown to reduce HGA and slow disease progression in AKU patients. Nitisinone-induced tyrosinaemia however is an upstream effect of 4-hydroxyphenylpyruvate dioxygenase inhibition that can cause keratopathy. The major aims of this thesis were to phenotype a new Hgd-targeted AKU mouse model, obtained from the KOMP repository, to use the LacZ gene which is expressed as a fusion transcript with the Hgd gene to determine the tissue expression of Hgd via ex vivo LacZ staining, to explore the effect of conditional liver-specific Hgd deletion on the AKU phenotype using double transgenic Hgd tm1d (fl/fl) MxCre mice, and finally to explore the effectiveness of dietary restriction and the potential application of an exogenous tyrosine-degrading enzyme at reducing tyrosine. The level of HGA and the tissue distribution of ochronotic pigmentation were evaluated using the knockout-first Hgd tm1a -/- model. HGA was shown to be increased in the plasma and urine, with the first signs of knee joint pigmentation found at 9 weeks in the knee joint, increasing linearly with age. Pigmentation was only found associated with chondrocytes within the calcified articular cartilage of joints, the calcified endplates of vertebrae, and the calcified knee joint meniscus. Hgd tm1a -/- mice exhibit the early stages of human ochronosis, with pigmentation confined to calcified tissues. Positive LacZ staining was identified in the cytoplasm of hepatocytes and kidney proximal convoluted tubule cells, beginning at E12.5 and E15.5 respectively, in addition to the developing male germ cells within the testis and epididymis, with qPCR showing liver Hgd mRNA expression to be approximately 2000-7,500-fold greater than the Hgd mRNA expression in the testis and epididymis. Germ cell expression was deemed insignificant and unlikely to contribute towards HGA metabolism. Liver-specific Hgd deletion via polyinosinic: polycytidylic acid induction of MxCre in Hgd tm1d (fl/fl) MxCre +/- mice demonstrated that 20% residual liver Hgd mRNA does not reduce circulating HGA leading to joint pigmentation and that kidney Hgd mRNA cannot rescue AKU. It was determined that the minimum level of liver Hgd mRNA required is 26-43% to reduce circulating HGA. The detection of downstream metabolites of intravenously injected 13C6-HGA in Hgd tm1a -/+ plasma revealed that hepatocytes can take up and metabolise HGA. The liver should therefore be the target of future gene therapy in AKU. Nitisinone was shown to reduce HGA and increase tyrosine, phenylalanine, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenyllactic acid in the plasma of AKU mice. Dietary restriction of tyrosine/phenylalanine significantly reduced nitisinone-induced tyrosinaemia, with phenylalanine-only restriction not effective. Evidence of AKU patients reducing tyrosine was observed, although the reductions were not comparable with that of mice. Due to the proof-of-principle provided by the dietary studies, preliminary experiments then demonstrated tyrosine degradation by the bacterial enzyme tyrosine ammonia lyase, which has the potential to be developed into an oral therapeutic for reducing nitisinone-induced tyrosinaemia.

Cancer cells acquire a number of abnormal epigenetic and/or genetic properties. These enable cancer cells to grow, spread and escape the action of therapeutic and immunologic targeting. An important feature of many cancers is the re-activation of a set of genes which are normally expressed during distinct development stages. These genes include the germline genes. Cancer Testis Antigen (CTA) genes are normally expressed in male germ cells in the adult testes and variety of cancer cells. Understanding this group of genes has great significance in cancer biology. This research is aimed at investigation of the functions of two germ specific CTA genes, namely PRDM9 and TEX19, both of which are activated in several kinds of tumours. PRDM9 has histone methyltransferase activity to coordinate meiotic recombination hotspot activation. Activity the murine orthologue of human PRDM9, Meisetz, has been described as a meiotic recombination activator which is also involved in regulation of transcription for other meiosis specific genes. This current research indicates that PRDM9 can affect cancer cell proliferation and may transcriptionally regulate other germline genes in cancer cells. TEX19 functions in the germ line to mediate an array of process. TEX19 has recently been demonstrated to be an oncogenic driver with potential role in self-renewal and proliferation of cancer cells. This current research confirmed that TEX19 depletion affects proliferation of cancer cells. Also, it was found that siRNA depletion of TEX19 caused considerable decrease in histone H3K9Ac, showing a potential contribution of TEX19 to epigenetic regulation of chromatin and / or chromosomal dynamics. Finally, we found that TEX19 might control L1-ORF1p, which is a protein encoded by LINE-1 that is needed for retro-transposition. This feature shows both commonalties and distinctions to the reported activities of murine Tex19.1. This might reflect fundamental species differences, or specific TEX19 functions in cancer cells. We put forward a model in which we propose TEX19 functions in oncogenesis as a nucleic acid mimetic.

The homeodomain transcription factor NANOG promotes self-renewal of embryonic stem cells (ESCs) and specification of primordial germ cells (PGCs). NANOG binds thousands of sites within the ESC genome together with OCT4, SOX2 and RNA polymerase II (RNAPII) regulating transcription of its target genes. Replacing tryptophans within the NANOG tryptophan repeat (WR) region for alanines (W10A) causes NANOG to lose its capacity to homodimerise and to interact with other proteins including RNAPII, SOX2 and RESF1. NANOGW10A mutant is also unable to sustain LIF-independent self-renewal. However, the requirements of the WR to interact with other proteins for NANOG function remains unclear. To characterise the cellular localisation of RESF1, endogenously tagged Resf1 mouse embryonic stem cell lines were produced. RESF1 is a widely expressed nuclear protein which interacts with NANOG in nuclei of ESCs. To quantify the requirements of Resf1 for ESC self-renewal and NANOG function, Resf1-/- ES cell lines were derived. In serum/LIF culture, Resf1 deletion promotes cell differentiation and decreases expression of Pou5f1, Nanog and Esrrb. Episomal expression of NANOG sustains LIF-independent self-renewal independently of Resf1. Resf1-/- ESCs are also capable to differentiate into epiblast stem cells. To investigate the role of Resf1 in PGC specification, Resf1-/- ESCs were differentiated into PGC-like cells (PGCLCs). Deletion of Resf1decreasedPGCLCspecification4-foldandpreventedefficientactivation of Blimp1, Prdm14 and Ap2γ. The effect of NANOG on OCT4, SOX2 and RNAPII chromatin binding was evaluated by inducing NANOG in Nanog-/- mouse ESCs. OCT4, SOX2 and RNAPII occupancy before and shortly after NANOG induction was measuredbyanalysingchromatinimmunoprecipitationfollowedbysequencing data sets. The k-mean clustering of Nanog binding sites revealed seven clusters with different response patterns of OCT4, SOX2 and RNAPII to NANOG. The sites where NANOG increases OCT4, SOX2 and RNAPII chromatin binding are mostly associate with the genes activated by NANOG, whereas sites where RNAPII binding decreases after NANOG induction associate with genes repressed by NANOG. To assess the requirements of the NANOG WR and the NANOG DNA binding for directing OCT4, SOX2 and RNAPII chromatin binding, NANOG variants with debilitating mutations in the WR or the homeodomain were induced in Nanog-/- ESCs and chromatin binding of NANOG, OCT4, SOX2 and RNAPII was assessed. The NANOGW10A has reduced chromatin binding at most of the NANOG binding sites. A reduced DNA binding capacity of recombinant NANOGW10A was also suggested by electrophoretic mobility shift assay. Moreover, NANOGW10A is unable to increase OCT4, SOX2 and RNAPII binding at the sites associated with gene activation. At the sites associated with gene repression, NANOGW10A induction leads to increased OCT4 binding. NANOG variant incapable to bind DNA (NANOGN51A) is unable to direct OCT4 and RNAPII binding. However, SOX2 binding temporarily decreases after NANOGN51A induction. In summary, Resf1 is not essential for ESC self-renewal and NANOG function but it is important for efficient specification of PGCLCs. NANOG directs the core transcription complex regulating self-renewal via the WR. This thesis contributes to understanding of NANOG function in ESC self-renewal and describes the function of previously uncharacterised RESF1 in ESC self-renewal and germ cell specification.

The roles of androgen signalling in the differentiation, maintenance and function of the zebrafish testis are poorly understood. This project aimed to explore this topic by characterising the phenotypes of androgen and cortisol deficient fdx1b and cyp11c1 mutant zebrafish. Homozygous mutant adult zebrafish were found to exhibit female type pigmentation patterns, however these fish could possess either differentiated ovaries or testes. This demonstrates that androgens are dispensable for testis differentiation in zebrafish, as has also been found in androgen receptor mutant zebrafish. Androgen deficient male zebrafish were infertile in standard breeding scenarios, but their sperm could fertilise eggs collected from wild-type female zebrafish in IVF experiments. Various phenotypes in these fish are likely to contribute to their infertility, including perturbed breeding behaviours, disorganised testis organisation, reduced spermatogenesis and impaired sperm release. Targeted gene expression analysis by qPCR revealed downregulation of igf3 and insl3 in the testes of mutant fish, these genes encode important factors for spermatogonial differentiation. Histological examination of the testes and analysis of germ cell marker expression by qPCR revealed an accumulation of early germ cells. Taken together these findings indicate that spermatogonial differentiation is impaired, and that this process is highly dependent on androgens in zebrafish. Further investigations into the molecular mechanisms underlying the assorted phenotypes of these fish were undertaken by characterisation of the impacts of fdx1b mutation on the testis transcriptome. Several miRNAs with roles in repressing spermatogonial differentiation in mammals were upregulated in the testes of Fdx1b deficient zebrafish, indicating a potentially conserved role for these miRNAs in zebrafish and mammalian spermatogenesis. In addition to this, dysregulation of several structural elements of the testes was identified, including the basement membrane and Sertoli cell barrier. This is the first investigation into the mechanisms underlying disorganisation of testicular structure in androgen deficient or resistant zebrafish.