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Primordial germ cells (PGCs) are germline competent cells which form the functional gametes of the animal. The potential usage of avian PGCs in producing genetically modified birds has driven research in the derivation, culturing, and genetic manipulation of PGCs. In chicken blastoderm, approximately 50 PGCs are present which proliferate in both male and female embryos until stage HH34 (day 8) and subsequently follow different differentiation pathways in male and female gonads. I investigated the hypothesis that chicken migratory stage PGCs are not initially determined to an oocyte or spermatogonial fate. To understand the differences in genetic mechanisms between male and female chicken PGCs, I studied the RNA transcriptome of PGCs from chicken. Analysis of RNA-Seq data of chicken PGCs reveals transcriptome divergence between the male and female cells and identified 150 differentially expressed genes (DEGs). The cultured female PGCs showed higher expression of cell adhesion genes like NCAM2 and PCDH9, and SMAD7B than male PGCs and also showed that dosage compensation is not maintained throughout the Z sex chromosome. To identify novel germ cell and stem cell factors in avian PGCs, I compared the transcriptome of chicken PGCs with immortalized chicken cell lines. As a result, a set of genes were identified which are specific to germ cells including DAZL, DDX4, DDX43, PNLDC1, DMRT1, DMRTB1, and FKBP6. This analysis also helped to identify a suite of pluripotency genes expressed in PGCs: NANOG, OCT4, LIN28, SOX3, GNOT1, TGIF2, PRDM14 and many others. Furthermore, a cross-species transcriptome comparison between in vitro cultured chicken and goose PGC transcriptomes revealed that the expression of these sets of germ cell-specific genes and pluripotent genes expression is conserved in PGCs from these two avian species. This study also revealed the contrasting gene regulatory networks involved in the selfrenewal are active in chicken and goose PGCs. Chicken PGCs exhibit expression of both Activin and BMP signalling pathway genes whereas BMP signalling pathway genes are active in goose PGCs. PRDM14 belongs to the family of the transcription factors containing a conserved N-terminal SET regulatory domain. In mouse, Prdm14 gene expression is limited to the pluripotent cells and essential for the development of the germ cell lineage. In chicken, the PRDM14 knockout embryos do not form a primitive streak. I characterized germ cell development in PRDM14 null chicken embryos and found that PRDM14 has a crucial role in the survival and maintenance of germ cells. Extending my transcriptome analysis to wild-type and PRDM14 null embryos identified DEGs and regulatory pathways possibly responsible for the gastrulation phenotype in the null embryos.

DNA is constantly exposed to both exogenous and endogenous sources of DNA damage. Therefore, organisms evolved a plethora of DNA repair pathways to repair these distinct lesions and prevent mutations from arising. Mutations that occur in somatic cells can lead to disease, which may be detrimental to that single individual. However, germ line mutations can be passed on to the next generation. These mutations not only drive genome evolution but can also impair the fitness of the offspring. It has been shown that primordial germ cells (PGCs), the germ cells specified during embryonic development, have the highest mutation frequency compared to germ cells belonging to other stages of gametogenesis. Furthermore, mutations in PGCs are particularly important because mutations in these cells can affect multiple offspring. Evidence shows that during PGC development, which entails extensive genomic and epigenomic transactions, there is the generation of DNA damage. However, the DNA repair pathways used in PGCs and the nature of DNA damage encountered by these cells which can provide mechanistic insight into how mutagenesis is suppressed in the germ line remains largely unexplored. Here we describe the use of an in vitro system that generates PGC-like cells (PGCLCs) from ESCs to discover novel factors required for PGC development. Using this system, we have firstly shown that we can recapitulate the genetic requirement for DNA repair in PGCLCs in vitro. Secondly, we coupled this system with CRISPR/Cas9 screening technology, which permitted us to identify novel factors required during the development of PGCLCs. We identified the translesion synthesis (TLS) factor Rev1 as a key factor involved in PGCLC development. Furthermore, we validated this requirement in vivo and showed that absence of Rev1 causes a numeric reduction in PGCs during the migratory period of PGC development. We also identified additional factors, namely involved in homologous recombination, chromosome cohesion which will be investigated in the future. This screen permitted the identification for the first time of the requirement for TLS in PGCs suggesting that the germ cell lineage may employ this mutagenic pathway to promote genome evolution.

With continuing advances in genome sequencing technology, the chicken genome assembly is now better annotated with improved accuracy to the level of single nucleotide polymorphisms. Additionally, the genomes of other birds such as the duck, turkey and zebra finch have now been sequenced. A great opportunity exists in avian biology to use genome editing technology to introduce small and defined sequence changes to create specific haplotypes in chicken to investigate gene regulatory function, and also perform rapid and seamless transfer of specific alleles between chicken breeds. The methods for performing such precise genome editing are well established for mammalian species but are not readily applicable in birds due to evolutionary differences in reproductive biology. A significant leap forward to address this challenge in avian biology was the development of long-term culture methods for chicken primordial germ cells (PGCs). PGCs present a cell line in which to perform targeted genetic manipulations that will be heritable. Chicken PGCs have been successfully targeted to generate genetically modified chickens. However, genome editing to introduce small and defined sequence changes has not been demonstrated in any avian species. To address this deficit, the application of CRISPR/Cas9 and short oligonucleotide donors in chicken PGCs for performing small and defined sequence changes was investigated in this thesis. Specifically, homology-directed DNA repair (HDR) using oligonucleotide donors along with wild-type CRISPR/Cas9 (SpCas9-WT) or high fidelity CRISPR/Cas9 (SpCas9-HF1) was investigated in cultured chicken PGCs. The results obtained showed that small sequences changes ranging from a single to a few nucleotides could be precisely edited in many loci in chicken PGCs. In comparison to SpCas9-WT, SpCas9-HF1 increased the frequency of biallelic and single allele editing to generate specific homozygous and heterozygous genotypes. This finding demonstrates the utility of high fidelity CRISPR/Cas9 variants for performing sequence editing with high efficiency in PGCs. Since PGCs can be converted into pluripotent stem cells that can potentially differentiate into many cell types from the three germ layers, genome editing of PGCs can, therefore, be used to generate PGC-derived avian cell types with defined genetic alterations to investigate the host-pathogen interactions of infectious avian diseases. To investigate this possibility, the chicken ANP32A gene was investigated as a target for genetic resistance to avian influenza virus in PGC-derived chicken cell lines. Targeted modification of ANP32A was performed to generate clonal lines of genome-edited PGCs. Avian influenza minigenome replication assays were subsequently performed in the ANP32A-mutant PGC-derived cell lines. The results verified that ANP32A function is crucial for the function of both avian virus polymerase and human-adapted virus polymerase in chicken cells. Importantly, an asparagine to isoleucine mutation at position 129 (N129I) in chicken ANP32A failed to support avian influenza polymerase function. This genetic change can be introduced into chickens and validated in virological studies. Importantly, the results of my investigation demonstrate the potential to use genome editing of PGCs as an approach to generate many types of unique cell models for the study of avian biology. Genome editing of PGCs may also be applied to unravel the genes that control the development of the avian germ cell lineage. In the mouse, gene targeting has been extensively applied to generate loss-of-function mouse models to use the reverse genetics approach to identify key genes that regulate the migration of specified PGCs to the genital ridges. Avian PGCs express similar cytokine receptors as their mammalian counterparts. However, the factors guiding the migration of avian PGCs are largely unknown. To address this, CRISPR/Cas9 was used in this thesis to generate clonal lines of chicken PGCs with loss-of-function deletions in the CXCR4 and c-Kit genes which have been implicated in controlling mouse PGC migration. The results showed that CXCR4-deficient PGCs are absent from the gonads whereas c-Kit-deficient PGCs colonise the developing gonads in reduced numbers and are significantly reduced or absent from older stages. This finding shows a conserved role for CXCR4 and c-Kit signalling in chicken PGC development. Importantly, other genes suspected to be involved in controlling the development of avian germ cells can be investigated using this approach to increase our understanding of avian reproductive biology. Finally, the methods developed in this thesis for editing of the chicken genome may be applied in other avian species once culture methods for the PGCs from these species are developed.

Despite the general advice of avoiding medication during pregnancy, the majority of pregnant woman use one or more ‘over the counter’ analgesics. During the last few years there has been growing evidence that analgesic exposure, such as paracetamol, ibuprofen or indomethacin, during pregnancy can have detrimental effects on rodent and human fetal gonads. The majority of previous studies have focused in alterations in testosterone production and male reproductive disorders. However, few studies have analysed the effect of these analgesics on fetal germ cells and possible consequences on fertility. During my thesis, I first focused on the effect of paracetamol and indomethacin exposure during pregnancy on rat fetal gonads. These showed that both paracetamol and indomethacin are able to alter the expression of genes important for fetal gonad and germ cell development. Previous studies on germ cells and analgesics have focused on rat models, but there is a lack of similar studies performed in human models. Therefore, I investigated the consequences of exposure of therapeutically relevant doses of paracetamol and ibuprofen on human gonads, with a special attention to the germ cells. Fetal gonads from the 1st and 2nd trimester were used in two different models: hanging drop cultures for 1st trimester testes and ovaries and a xenograft system for 2nd trimester fetal testes. Fetal gonad culture in the presence of paracetamol or ibuprofen reduced AP2γ+ (gonocyte) GC number in both 1st trimester fetal testes (22-28% reduction) and ovaries (43-49% reduction). 2nd trimester fetal testes were exposed to three different regimes, 1 or 7 days paracetamol and 7 days ibuprofen, which led to reductions of 17% and 30%, respectively in AP2γ+ GC number for paracetamol and a 53% reduction in total germ cell number for ibuprofen.

The prevalence of obesity is increasing each year, likely due to altered diet and a sedentary lifestyle. However, some epidemiological data suggests that parental and grandparental environments may also impact upon weight gain, metabolic health and mortality. This non-genetic inheritance has been reproduced in some experimental animal studies. The mechanisms are, however, unknown. One hypothesis has been that environmental exposures perturb germ cell development. Changes to the germ cell epigenome are an example of one such alteration. In this thesis, epigenetic mechanisms are explored in rodent and primate male germ cells throughout development. The conservation of global methylation patterns, expression of DNA methyltransferases and of key histone modifications is demonstrated in rat, marmoset and human tissue demonstrating mechanisms are in place that might be disrupted by environmental exposures either in utero or during post-natal development. A rat model of parental high fat diet (HFD) exposure is established and phenotype explored in two generations of offspring. Maternal HFD results in weight gain in male and female offspring and paternal HFD in weight gain in just female offspring. Grandpaternal exposure to HFD via the maternal line has the greatest impact on the second generation where, in males, increased weight, adiposity, reduced insulin sensitivity and an increased luteinising hormone to testosterone ratio were found. Given that postnatal exposure in males resulted in adverse metabolic health in grand offspring, the exposed germ cells were interrogated by RNA and smallRNA-sequencing. HFD exposure did not alter the germline transcriptome suggesting an alternative mechanism to be responsible for the intergenerational effects observed. Thus, HFD perturbs metabolic health in two generations of rats in a grand-parent and sex specific manner but does not affect the germ cell transcriptome.

Premature ovarian insufficiency (POI) occurs in 1% of women younger than 40 years of age. The manifestation of this condition varies considerably ranging from ovaries devoid of follicles (afollicular POI) to ovaries containing follicles at various stages of development (follicular POI). No methods exist in mainstream healthcare to restore ovarian function for POI patients and ≥70% of POI cases are idiopathic. The Reaggregated Ovary (RO) method, as established for mice, enables investigation into the developmental potential of oocytes by replacing dysfunctional ovarian cells. This approach could enable us to develop a potential treatment for patients with follicular POI. Investigations carried out in this thesis led to the establishment of key processes required to adapt the RO method for human tissue. A method was developed to dissociate human ovarian tissue remnants (surplus after tissue cryopreservation) and frozen/thawed ovarian cortex obtained from pre-pubertal and post-pubertal patients. Analyses revealed that age affected the efficacy of dissociation of frozen/thawed ovarian cortical tissue. To detect germ cells in a single cell suspension of ovarian cells, a method was developed for flow cytometry using a monoclonal antibody that recognises the (cytoplasmic) N-terminus region of DDX4. To assess the use of an alternative somatic cell source to support oocyte development, human dermal fibroblasts were aggregated with mouse oocytes and follicle-like structures were formed after 7-day in culture. These findings will contribute to developing the use of the RO using human oocytes and a human somatic cell alternative. The RO method once adapted for human tissue could be used to replace the dysfunctional ovarian somatic cells in POI patients and potentially restore follicle development for pre-pubertal and post-pubertal POI patients.

Primordial germ cells (PGCs) are the embryonic precursors of the germ cell lineage. Segregation of the chicken germ line from somatic cells occurs very early in embryonic development. By day two of incubation chicken PGCs can be isolated from the circulating blood. The in vitro culture of chicken PGCs has significant potential as a tool for the investigation of germ cell development and as a cell-based system for the production of genetically modified chickens. The isolation, culture and manipulation of migratory chicken PGCs reported previously have not been independently validated. Initial attempts to isolate and culture chicken PGCs by reproducing a published protocol proved difficult. Key components of the published culture medium are by their nature variable, including the use of BRL-conditioned medium and animal sera. The protocol also stated that addition of SCF to the culture medium is essential but did not identify the source of SCF used. Several components of the culture conditions were tested including sources and batches of bovine and chicken sera and the growth factors FGF2 and SCF. Chicken PGCs from wild type and GFPexpressing chicken embryos were cultured and several cell lines established, proliferating for more than 100 days in culture. After seventy days in culture a single chicken PGC cell line was shown to retain the potential to develop into functional sperm. This was demonstrated by injection of the cultured chicken PGCs into early chick embryos, which were hatched and produced offspring derived from the injected chicken PGCs. To understand and produce a more robust system for the isolation and propagation of chicken PGCs three signalling pathways, AKT, MAPK and JAK/STAT, were investigated. When any of these signalling pathways were blocked, using chemical inhibitors, chicken PGC proliferation in vitro was significantly inhibited, showing the pathways to be essential for chicken PGC proliferation. Chicken PGCs were treated with individual components of the standard culture medium, FGF2, SCF, animal sera, BRL-conditioned medium, LIF and IGF, and the activation status of the key signalling pathways was assessed by western blot. Individual components of the culture medium induced activation of the AKT and MAPK pathways but not the JAK/STAT pathway. These data increase our understanding of PGC biology and are the first steps towards the development of a feeder- and serum-free medium for the growth of chicken PGCs. Published methods for the genetic manipulation of chicken PGCs are inefficient. To improve the efficiency of stable transgene integration, transposable element-derived gene transfer vectors were assessed for their ability to transpose into the genome of chicken PGCs. Comparison of Tol2 and piggyBac transposable elements, carrying reporter transgenes, demonstrated that both can be used to genetically-modify chicken cells. The incidence of stable transposition achieved was higher when using the Tol2 transposable element in comparison to the piggyBac element. The genetically-modified chicken PGCs formed functional gametes, demonstrated by injection of genetically modified chicken PGCs into host embryos which were hatched and produced transgenic offspring expressing the reporter gene construct.

Almost 65 years have passed since the discovery of 5 methylcytosine (5mC). DNA methylation has since been shown to be involved in gene expression, genomic imprinting, X chromosome inactivation, the repression of retrotransposons and cell differentiation. In the context of mammalian embryonic development, DNA methylation undergoes genome-wide reprogramming in primordial germ cells (PGCs). Despite a huge research effort, the mechanism underlying DNA demethylation in PGCs is unclear. Limited progress is largely due to technical challenges. Many widely used analytical techniques require large amounts of biological material and are therefore inappropriate for studies using small numbers of cells isolated from developing embryos. Our understanding of the mechanisms behind the developmental reprogramming processes may therefore be greatly enhanced by the use of complementary model systems and alternative technologies. The experiments described herein focus on the development and optimisation of a number of in vitro and in vivo systems to explore the nature of epigenetic DNA modifications, including 5-methylcytosine, and the mechanisms that dictate dynamic changes in the epigenetic landscape. The development and validation of sensitive LC-MS methods facilitates the quantification of DNA modifications, potentially enables the discovery of novel modifications, and by combining LC-MS with isotope tracing, may fundamentally challenge our current understanding of the stable nature of DNA methylation. Ultimately, we envisage that these approaches will form a versatile and informative platform to study genome-wide epigenetic reprogramming processes in an in vivo context. In many respects, loss of 5-methylcytosine in migratory PGCs mirrors the loss of 5-methylcytosine in mESCs upon transition from serum-containing to 2i medium. Accordingly, I used this accessible system to probe the contribution of DNA methyltransferases, putative DNA demethylases, their cofactors and binding partners to the loss of 5-methylcytosine in nascent PGCs. I show that loss of Dnmt3a isoform 2 is necessary for 2i-induced DNA demethylation. Furthermore, this loss is dependent on RNA-induced silencing. More generally, miRNAs appear to be necessary for the epigenetic reprogramming of mESCs to the naive ground state. It is possible that the same miRNAs are involved in DNA demethylation in migratory PGCs. I also show that retinol has an unexpected effect on the rate and extent of DNA demethylation. How this finding translates in the context of primordial germ cell development is unknown. It does, however, highlight both a potential source of variation in the published data and the need to understand the interplay between cell metabolism and epigenetic reprogramming.

Germ cells represent the link between generations. At the time of fertilisation, sperm and egg come together to form a single cell, called the zygote, from which an entire organism is ultimately constructed. In mouse, two unique properties underlie the exceptional potential of gametes: 1) the ability to execute meiosis; and 2) the correct establishment of complementary epigenetic signatures (genomic imprints) in sperm and oocyte that enable post-implantation embryonic development in the next generation. At the molecular level, both the progression through meiosis and the establishment of sex-specific methylation imprints are critically dependent on a wave of global DNA demethylation that is initiated in primordial germ cells (PGCs), the embryonic precursors of the gametes, following their colonisation of the embryonic gonads. The work I present in this thesis focuses on the molecular mechanisms regulating this wave of DNA demethylation. Specifically, I conclusively show that DNA demethylation proceeds independent of the 5-methylcytosine oxygenase Tet1, previously proposed to act as the putative DNA demethylase. Rather, I show that, with respect to DNA demethylation, Tet1 appears to protect the hypo-methylated state in PGCs following the major loss of 5-methylcytosine. Most strikingly, however, with respect to germ cell development, Tet1 appears to play an important role in regulating the timing of the major developmental and transcriptional transitions that occurs in the mouse foetal germ line between embryonic day (E) 10.5 and E14.5, with PGCs derived from Tet1-knockout embryos showing increased stability of the demethylated pre-meiotic germ cell state. Beyond understanding the role of Tet1 in gonadal PGCs, I also present results focusing on the potential relationship between cell signalling, DNA demethylation and licensing for gametogenesis, with preliminary data suggesting a potential role for Akt-mTORC1 signalling in activating the expression of the key meiotic regulator Dazl.

Primordial germ cells (PGC) are the precursors of the gametes. The mechanisms of PGC induction, specification and development are very well characterized in rodents, however recent investigations have demonstrated that the mechanisms of germ cell development differ significantly between mice and humans. Since the knowledge of PGC development in non-rodents is very limited, and early human embryos cannot be accessed it is important to establish a new model for PGC development with relevance to humans. In this thesis, I use pig embryo as a model for investigating PGC development in non-rodent mammals. The expression profile of key transcription factors, epigenetic reprograming and the role of signalling pathways were investigated during specification and development of pig PGCs. The key findings are: A- Specification of porcine PGC occurs after the onset of gastrulation, requiring BMP4 signalling. B- WNT signalling is required for the generation of precursors competent for germline commitment; however it is downregulated after PGCs are specified. WNT downregulation could be modulated by SOX17, the earliest gene expressed in pig PGCs. C- Epigenetic reprogramming of DNA and histone marks starts in pre-migratory porcine PGC. Furthermore, chromatin dynamics in pig gonadal PGCs resemble that of humans but differs to that of mice. D- The expression profile of transcription factors of porcine PGC is similar to that of humans, but different to mouse PGC. In conclusion, this study has highlighted critical differences between mice and humans/pigs during germ cell specification. I provide evidence that the pig embryo is a useful model for the study of human development, and future studies will need to be directed to re-evaluate concepts of cell differentiation and early lineage commitment established in mice that may not apply to humans.

Embryonic stem cells (cESCs) can be isolated from chick embryos, with the ability to contribute to all somatic lineages in chimaeras, but not to the germ line. However, lines of chicken embryonic germ cells (cEGCs), which are able to contribute to the germ line, can be established from chicken primordial germ cells (cPGCs). However very little is known about these cells, or about the changes that accompany the establishment of gonadal cells as self-renewing cell lines. This thesis presents a detailed study of the properties of cPGCs and the parent tissue from which they are derived. Gene expression profiles for 30 genes related to pluripotency and/or differentiation were are compared between gonads at the indifferent stage (stage 26-28HH), in primary gonocytes, established PGCs and cESCs. The results reveal great heterogeneity in the expression of various markers in culture. Several genes associated with pluripotency change dramatically upon culture. The most salient of these changes is that while cSox3 (but not Sox2) is expressed in the gonads, whereas their expression becomes reversed upon culture (becoming more similar to mammalian stem cells). This suggests that these two SoxB1 class genes have swapped functions in chick. In the process of studying the expression pluripotency markers in later (stage HH35) gonads, we made an unexpected discovery: both male and female embryos show left-right asymmetric patterns of expression of some, but not all, of these markers. Expression of pluripotency (cPouV, cNanog, cSox2 and ERNI) in the left gonad is much higher than those in the right gonad of both sexes. The expression of pluripotency markers is irrespective of its colonisation by primordial germ cells, and it appears that this left-right decision is made independently of whether the gonad will regress or be retained. These findings offer a new model system for investigating the roles of pluripotency-related markers during normal development as well as in stem cell lines.

A critical event during embryonic development is the segregation of the germ line from the soma. The germ line founders, or primordial germ cells (PO C), are the precursors of the gametes, which enable the transmission of the genetic material to future generations. In rodents, the PGC precursors are segregated from somatic lineages before the start of gastrulation. In several model organisms segregation of the germ line is accomplished by maternally inherited transcriptional repressors contained in the egg's germ plasm. In mammals, PGC specification is regulated by epigenesis, a mechanism by which pluripotent cells ill the epiblast respond to specification signals according to a temporal and spatial program of differentiation. In the mouse embryo, the transcriptional repressor Blimp] plays a fundamental role in restricting nascent POC to somatic differentiation signals. The aim of this project was to determine whether the mechanism of lineage specification is conserved in humans.

The imprinting centre 2 (IC2) on mouse distal chromosome 7 is part of a cluster of genes of which ten are currently known to be monoallelically expressed. All genes in the cluster are paternally silenced, with the exception of the non-coding RNA gene Kcnqlot1, which is maternally silenced. In this study I have been interested to understand how and when allelic expression and allelic histone marks are established in the IC2 cluster, and how they are erased in germ cells. I have used trophoblast stem (TS) and trophoblast giant cells as a model system of placental development to determine lineage specific histone modifications and gene expression. Using this model system, I was able to show that placental allele specific histone modifications are established after the blastocyst stage. Using a mutant for an oocyte-specific form of Dnmt1 (Dnmtlo), I have analysed imprinted expression and levels of transcription of genes in the cluster. This has allowed me to identify a particular time point during development from which expression of Kcnqlot1 can target gene silencing and repressive histone marks in cis to the imprinted genes in the cluster. Finally, I was interested to see if histone marks associated with imprinting are removed during the process of imprint erasure in primordial germ cells (PGCs). To this end I have developed a sensitive chromatin immunoprecipitation (ChIP) technique that can be used in combination with single strand conformational polymorphism (SSCP). This method enabled me to investigate allele specific histone modifications in small numbers of cells (103-104). This has allowed me to show that some allele specific histone modifications remain in primordial germ cells (PGCs) at the IC2 cluster after DNA methylation has been erased at E13.5.

Diseases such as avian influenza (Al) virus can result in the destruction of whole chicken and turkey flocks. Many valuable and rare strains of poultry are maintained on farms worldwide, making them particularly susceptible to outbreaks of such diseases and resulting in a loss of valuable genetic material from the turkey gene pool, with consequences for the poultry Industry. Primordial germ cells (PGCs) are the stem cell precursors to the gametes and they take a complex migratory route throughout the developing avian embryo. starting from the epiblast and moving to the germinal crescent, blood vascular system and eventually settling in their final anatomical location within the embryonic gonads, where they will differentiate into sperm and eggs.

Oligonucleotide arrays identified a global up-regulation of X and Yp linked genes in mouse models with deletions of MSYq. In combination with previous cDNA array data, the oligonucleotide array data identified four genes with little or no previous characterisation. Two of these genes (h2aly and AK006152) were mapped to the short arm of the Y chromosome outside of the Sxrb interval. H2aly is part of a novel histone family, most closely related to the H2A.Bbd family and has multi X linked relatives and an autosomal relative. This protein coding family is expressed specifically in spermatids. No human ortholog was identified. AK006152 is a novel, mouse specific single or low copy Y linked gene. It has a potential open reading frame and is expressed specifically in spermatids. The two multi-copy genes (Orly and CJ175665) were significantly down-regulated in the mouse MSYq deletion models and subsequently mapped to the long arm of the Y chromosome. Orly is a chimeric locus composed of Ssty1, Asty and Sly and generates many variant transcripts that show a spermatid specific expression. No open reading frame was identified in Orly transcripts and anti-sense transcripts may be involved in the regulation of Sl. .CJ175665 is composed of exon 6 of Sly and the surrounding 3’ and 5’ intronic regions and was also expressed in spermatids only. An antibody raised against a peptide designed from the ORF of CJ175665 localised to the nucleus and produced a testis specific band in Western blotting. However, RT-PCR analysis detected other transcripts from the Sly locus containing intronic sequence which could not exclude the possibility of unprocessed transcripts. Anti-sense transcripts also detected from the Sly and CJ175665 loci also indicate a role for self-regulation of transcription.

The aims of this study were to investigate the role of steroid hormones, in particular oestrogens, in murine spermatogenesis. A major focus of these investigations was the role played by ERβ in the modulation of germ cell and somatic cell function. Studies were conducted both using a transformed murine Sertoli cell line (SK11), which has maintained a differentiated Sertoli cell phenotype and spermatogonial stem cells, which were successfully isolated and characterised. ERβ mRNA and protein were shown to be expressed in the SK11 cells both in the undifferentiated and differentiated states. Transient transfections using ERE or ARE-luciferase reporter constructs and stimulation with steroid ligands revealed that the cells contained functional steroid hormone receptors. Knockdown of ERβ mRNA and protein was achieved in the cells after targeted deletion using a short hairpin RNAi containing vector; this blunted the ability of the cells to respond to oestrogen. Isolation of spermatogonial stem cells was carried out using immunomagnetic beads. The stem cell population were shown to express Oct-4 and GFRα-1 mRNAs, both of which are stem cell markers, but not c-kit, which is a marker of differentiated germ cells. Taqman Q-RT-PCR demonstrated that the stem cell population expressed ERβ. Oct-4 mRNA expression was shown to be reduced by RNAi; this induced the cells to undergo differentiation in vitro characterised by increased expression of c-kit. In conclusion, the current studies have extended our understanding of the impact of steroid hormones on testicular function and have revealed for the first time that spermatogonial stem cells are ERβ positive. The SK11 cell line has been found to provide a suitable model system for the study of steroid regulation of Sertoli cell function. In addition, the use of RNAi has provided and exciting new avenue by which to manipulate gene expression levels in testicular germ and somatic cells.

Avian embryos have played an important role in the study of vertebrate development but further understanding of their development may also benefit the commercial poultry industry. Manipulation and use of primordial germ cells (PGCs) as a vehicle for constructed genotypes will be increasingly important for future improvement of commercial poultry including the turkey. However, the manipulation and use of PGCs will depend on a basic understanding of PGC migration. PGCs are known to migrate considerable distances before colonising the gonad. Previous work in the chick and quail have suggested that avian PGCs arise from the ventral surface of the area pellucida before the blastoderm undergoes gastrulation, translocate to the germinal crescent during primitive streak stages and penetrate the extraembryonic blood vessels (stage 10) after they have formed. PGCs are subsequently carried by the blood stream into the embryo proper during stages 12-14 and then leave the blood vessels and migrate to the developing gonads. However, the factors controlling PGC migration are poorly understood. The developmental stages of older turkey embryos are not well documented. Therefore, this study started by classifying them. It was found that turkey embryos at stage 4 and younger were slightly different from chick embryos, but from stages 5 onwards, turkey embryos could be staged using criteria described for the chick (Hamburger and Hamilton, 1951). Avian PGCs can be recognized by their distinctive morphology being large cells with large and eccentrically placed nuclei, and a cytoplasm containing refractile granules. These characteristics were confirmed for turkey PGCs. In order to manipulate PGCs, cells need to be identified using appropriate markers. A panel of markers previously used for chick, quail, mouse, rat and rabbit were tested with turkey PGCs at different stages. It was found that turkey PGCs could be detected by the histochemical stains, periodic acid-Schiff (PAS) and alkaline phosphatase (AP) or using antibodies to stage-specific embryonic antigen-1 (SSEA-1), fucosylated polylactosamine carbohydrate groups (EMA-1) and ovomucin-like protein (OLP). At primitive streak stages, turkey PGCs were located in the area pellucida. During stages 11 - 15, they were found in the blood reaching maximal numbers at stage 14. After stage 15, most PGCs were located outside the circulatory system. From stage 18, PGCs began to settle in gonadal ridges and after stage 28, PGCs were found only in the gonads. Turkey PGCs were isolated from the blood with a micropipette, during visual selection under the microscope, and their Identity confirmed using the above markers. These PGCs could be cultured on a gonadal stromal cell layer or on coverslips coated with rat-tail collagen. Isolated PGCs adhered to coated coverslips were prepared and examined with scanning electron microscope (SEM). Such cells had typical features of PGCs seen in situ and some had the morphology of motile cells. PGC movement in vitro, in response to a variety of potential chemoattractants, was then studied. Conventional chemotaxis assays could not be used to study such small numbers of cells. Therefore, the assay was carried out using a Dunn chemotaxis chamber which allows direct observation of cells and detailed analysis of their movement from a timed series of images. A positive chemotactic response was observed with cells exposed to a gradient of medium conditioned by stromal cells or 10 ng/ml transforming growth factor beta-1 (TGFbeta1). Cells exposed to 100 ng/ml stem cell factor (SCF) or control medium showed no such response. These observations indicate that TGF?l can play a role in directed migration of PGCs in vitro, but further experiments are required to determine whether this factor is similarly involved in vivo.

To determine the expression pattern of XPAT (Xenopus primordial germ cell associated transcript) protein in Xenopus oocytes, XPAT-GFP fusion proteins were generated. When XPAT was amino-terminally tagged with GFP it became localised to the nuclei of stage VI Xenopus oocytes. However, when carboxy-terminally tagged with GFP, XPAT also translocated to the vegetal pole of stage VI oocytes. XPAT-GFP formed particles (1 to 2.5mm in diameter) which aggregated into large (10 to 50mm) granular structures at the vegetal pole. These particles looked exactly like those seen after in situ hybridisation to germ plasm RNAs. The granules of XPAT-GFP were larger than endogenous germ plasm granules seen in stage VI oocytes; they were more consistent with those observed in 2-cell embryos during germ plasm aggregation. Studies involving the use of the anticytoskeletal drugs colcemid, nocodazole and cytochalasin D and the microtubule stabilising agent taxol indicated that microtubular transport was important in the location of XPAT-GFP. Several attempts were made to raise antibodies to XPAT peptides, but at present the endogenous expression pattern of XPAT protein is unresolved. To investigate possible domain structure of XPAT, one carboxy-terminal and three amino-terminal deletion variants of XPAT-GFP were constructed. An N-terminal deletion protein lacking the first 61 amino acids of XPAT was able to form small particles, but none of the deletion proteins exhibited vegetal localisation or formed large aggregates in Xenopus oocytes. The N-terminal deletion proteins all became predominantly localised to the nucleus; protein motif analysis revealed that XPAT contains a putative bipartite NLS in its carboxy-terminal region. The C-terminal deletion protein, which lacked the putative NLS, was evenly distributed throughout the nucleus and cytoplasm of Xenopus oocytes. XPAT was shown to be able to bind to homopolymeric RNAs in vitro. When Xpat mRNA was depleted from stage VI Xenopus oocytes (by injection of an antisense oligo) levels of DEADSouth and XVLG1 mRNAs decreased substantially.

Spermatogenesis is largely dependent on stage-specific gene expression that takes place within the developing germ cell, and requires the close association between each spermatogenic cell stage and the supporting Sertoli cell. The aim of my thesis was to isolate purified populations of the post-meiotic germ cell (round spermatids) from the rat, and to characterise genes expressed at this cell stage which might encode proteins targeted to the acrosome A novel panning technique was devised which allowed the isolation of 95% pure round spermatids from adult rat testis, exceeding purities obtained by sedimentation through a BSA gradient. Subsequently, two experimental strategies were devised which included the use of antiserum raised against mammalian acrosomal membranes to 1) immunologically isolate specific polysomal mRNA or 2) to screen a human testis cDNA expression library. Although limitations were encountered using the polysome approach, a 2 kb cDNA clone (352) was isolated following library screening, which appeared to derive from a highly conserved, novel gene Low levels of 352 mRNA expression were evident in a range of human tissues, but were clearly up-regulated in the testis Before 15 days of age, 352 mRNA was not apparently expressed in the rat testis. Between 15 and 22 days of age, expression was at a very low level, coinciding with the initial accumulation of pachytene spermatocytes within the seminiferous epithelium. However, in males of 27 days or older, 352 mRNA was significantly elevated. This expression was concomitant with the appearance and subsequent differentiation of stage 1-5 spermatids. Surprisingly, Northern blot analysis indicated that round spermatids were not the cell type responsible for the elevated levels of expression of 352 mRNA in 27 day old rats. It was hypothesized that this up-regulation might be the result of predominant expression in Sertoli cells, such that, the appearance of round spermatids could induce Sertoli cells to synthesize 352 mRNA, and subsequently transfer the protein product to round spermatids where it may be targeted to the acrosome.

A number of methods are currently used for the detection of chemically-induced chromosome damage but all are subject to the criticisms that they are either time-consuming and labour intensive or require a large number of animals and can be insensitive. However, for human risk-assessment, conclusive evidence that compounds possess potential heritable hazards can still only come from mammalian germ cell studies. Thus, there is a need for less demanding germ cell tests that may be used to identify potential germ cell clastogens. One such system involves the use of one-cell mouse embryos. Although not as well validated as the currently available germ cell assays i.e. the dominant lethal test (DLT) and the heritable translocation test (HTT), it has the advantage that both structural and numerical chromosome aberrations can be directly analysed. The aim of this thesis was to i) fully validate the one-cell embryo test system (analysing both male and female germ cell effects) with a number of model clastogens and spindle poisons and ii) investigate the fate/survival of these chemically induced aberrant one-cell embryos through to the F1 generation. Sexually mature male or female mice were administered test compound* as a single ip dose and then mated at different stages of gametogenesis; for males the whole of the spermatogenic cycle was sampled; for females preantral as well as antral/Graafian follicle stages were sampled. The afternoon after mating, one-cell embryos were recovered from mated females, cultured overnight in a mitotic inhibitor and then prepared for chromosomal analysis. In embryos derived from matings involving dosed males, only damage induced in the spermatid and spermatozoa was transmitted to the F1 progeny. There was no significant damage from the spermatocyte or spermatogonial stages. In embryos derived from females dosed with chemical clastogens only damage induced in the preovulatory Graafian follicle stage was transmitted. The survival of these chromosomally aberrant embryos was investigated by analysing 3-day and post-implantation embryos as well as weaned F1 progeny. These studies show that chromosomally aberrant embryos are able to survive up to at least the early post-implantation stages. At implantation and post-implantation stages, when the embryonic genome is required for continued development, those embryos with gross chromosomal aberrations (as induced by MMS, CYC, MMC and VIN) die. However, if the induced damage does not involve deletions and/or duplications of DNA (e.g. balanced reciprocal translocations - as induced by EMS) the embryo inheriting such aberrations will survive to term. One chromosomally aberrant F1 offspring was detected in this system. Thus, the analysis of one-cell embryos is a sensitive and precise method for the detection of mutagenic potential in mammalian germ cells and has the advantage that both structural and numerical chromosome aberrations can be directly analysed. Furthermore, the studies have shown it to be less costly and more sensitive than either the DLT or HTT. However, the technically demanding methods involved in the preparation of one-cell embryos prevents their use for routine screening purposes. *The model compounds selected were methyl methane sulphonate (MMS), ethyl methane sulphonate (EMS), cyclophosphamide (CYC), trenimon (TRN), mitomycin C (MMC), busulphan (BSN), hexamethyl phosphoramide (HMPA), 5-fluorouracil (5FU), colcemid (COL), vinblastine sulphate (VBS) and diethyl stilboestrol (DES)

Primordial germ cells (PGCs), the precursors of gametes, are specified in early post-implantation human embryos, which are inaccessible for direct investigation. This process can however be modelled using human embryonic stem cells (hESCs) for in vitro derivation of human PGC-like cells (hPGCLCs). hESCs in conventional culture conditions have very low potential for germ cell fate; this increases significantly if the cells are first cultured in PGC competence-promoting media. To address how the PGC-competent state might be gained, I used a new computational tool - Branch-Recombinant Gaussian Processes (B-RGPs) - to identify potentially relevant pathways based on single-cell RNA sequencing of early human embryos, as well as gonadal PGCs and soma. I then tested these predictions experimentally by modulating the candidate signalling pathways in competent hESCs, followed by hPGCLC induction. I found that inhibition of FGF receptor (FGFR) or its downstream effectors (MEK and PI3K) enhanced competency for PGC fate. By contrast, premature MEK/ERK activation in hESCs resulted in aberrant expression of endoderm markers and loss of germ cell potential. Altogether the study showed that FGFR pathway is a negative regulator of cellular competence for human PGCLC fate. PGCLC specification from competent cells occurs in response to BMP signalling and is orchestrated by specific transcription factors (TFs). Emerging evidence points to many differences between TF networks in mouse and human PGCs. For example, SOX17 is essential for human germ cell fate, but is dispensable for mouse PGCs. On the other hand, a key pluripotency factor PRDM14 is crucial for mouse PGC specification, but its role in human PGC development is unclear. I designed genetic approaches to address PRDM14 involvement in human pluripotency versus PGC specification. To achieve inducible loss of function, I deleted endogenous PRDM14 in the background of doxycycline-repressible PRDM14 transgene expression. This approach, however, did not allow fast and homogenous control over protein levels. To circumvent this, I used CRISPR/Cas9 in conjunction with conditional degrons (auxin- and jasmonate-inducible degrons) for rapid and reversible degradation of the endogenous protein. Notably, PRDM14 depletion during differentiation significantly reduced the number of specified hPGCLCs, revealing its role in human PGC fate. Furthermore, RNA sequencing of mutant cells showed aberrant gene expression, with derepression of WNT signalling and somatic genes. Importantly, ectopic PRDM14 expression in mutant cells rescued hPGCLC specification and transcriptional changes, thus proving the specificity of the observed phenotype. Interestingly, loss of PRDM14 in hESCs had a distinct transcriptional effect, indicating a context-dependent role of PRDM14 in PGCs and pluripotent cells. ChIP-sequencing in hESCs and hPGCLCs identified PRDM14 binding to regulatory elements of key pluripotency and germ cell genes and motif analysis suggested its integration in the core TF circuitry in both cell types. Interestingly, the overlap between PRDM14 targets in mouse and human was limited. Altogether, this reveals a key role of PRDM14 in human germ cell fate and exemplifies the importance of complete and fast protein depletion in loss-of-function studies. Overall my research has furthered our understanding of signalling and transcriptional regulators controlling the segregation of human germ cells from soma. It also expanded the toolkit for protein studies in dynamic processes and uncovered hitherto unknown roles of PRDM14 in early human development.

Reproductive containment in farmed fish is highly desired for sustainable aquaculture to prevent genetic introgression with wild conspecifics and enhance productivity by suppressing sexual maturation. A number of strategies have already been implemented or have been tested in commercially important fish (e.g. triploidy, monosexing, hormonal therapies); however, they either do not result in 100% containment, or they cannot be applied to all species. One promising new approach consists in disrupting primordial germ cells (PGCs), at the origin of germline cells, to induce sterility. The work carried out in this doctoral thesis aimed to investigate the genes involved in the survival of germ cells and subsequently conduct a functional analysis of candidate genes using CRISPR/Cas9 gene editing system to ultimately provide the basis for the development of a novel sterilisation technique. Nile tilapia was chosen as the experimental animal as it is a major aquaculture species worldwide and the control of reproduction plays a critical role in the farming productivity in this species. In addition, the species has clear advantages as its whole genome sequence is accessible, the generation time is relatively short and zygotes can be available all year round. Initially, a panel of 11 candidate genes with reported roles in survival of PGCs was investigated during the ontogenic development which led to the selection of piwi-like (piwil) gene as a target for genome editing. Then, high temperature was tested as a means to induce germ cell loss to better understand the mechanism underlying germ cell survival and apoptosis, and this study confirmed the functional importance of piwil genes in relation to germ cell loss and proliferation. In addition, the study suggested potential subfunctionalisation within the Bcl-2 gene family which requires further investigation. The next step aimed to optimise the CRISPR/Cas9 gene editing method by improving the microinjection system and testing different concentrations of sgRNAs. Over 95% of injected embryos showed on-target mutation in piwil2 via zygote injection of CRISPR/Cas9 reagents and complete KO larvae were shown in half of the mutants, producing putative sterile fish. However, there was no clear association between the phenotypes in PGCs and the mutation rate. Further comparative studies of mutant screening methods including T7E1, RGEN, HRMA, fragment analysis and NGS revealed that the genotypes of F0 are highly mosaic, suggesting that deep sequencing is recommended for accurate and high throughput F0 screening and further improvement for predictable genome editing is required for a reliable gene functional analysis in F0. In summary, the current thesis provided new scientific knowledge and supporting evidence for the use of the CRISPR/Cas9 gene editing platform to study gene function associated with sterility, with the ultimate goal to develop an alternative sterilisation method in fish.

More than 1500 proteins have been identified as centrosome or cilia proteins, however it is still unknown how these proteins form networks to control functions such as centrosome orientation, maturation and ciliogenesis. TALPID3 is a centrosomal protein that plays a role in centrosome orientation and migration, ciliogenesis and Hedgehog pathway signal transduction. Although several proteins that interact with TALPID3 have been identified, the TALPID3 protein interacting network has not been established. Loss of TALPID3 leads to a loss of ciliogenesis, as seen in the embryonic lethal talpid3 chicken. Unlike the talpid3 chicken however, human ciliopathy patients with mutations in KIAA0586 (human TALPID3 orthologue) have a range of defects that often do not result in lethality. Human KIAA0586 was modelled in the chicken, in order to understand the functional domains that are affected by mutations that are predicted to be hypomorphic. A construct containing human KIAA0586 was able to rescue Hedgehog-dependent expression patterns, when electroporated into the developing neural tube of talpid3 chicken embryos. An attempt to introduce a fluorescent tag at the endogenous TALPID3 locus to look at specific subcellular localisation, proved unsuccessful; however a super resolution imaging approach confirmed localisation of TALPID3 to the centrosome. Structured Illumination Microscopy showed that TALPID3 localises to the distal end of centrioles with more TALPID3 on one centriole than the other centriole, but no specific regions of localisation elsewhere in the cell. A proteomic approach on isolated centrosomes was undertaken to investigate the TALPID3 protein network. Proteomic studies in the centrosome have previously been carried out in KE37 cells, however in order to establish alternative cell lines, specifically chicken Primordial Germ Cells (PGCs), as models for studying the centrosome and cilia, this study was completed in human Jurkat cells and chicken PGCs. PGCs can be derived from the talpid3 flock, therefore by including chicken PGCs the aim was to construct the centrosomal proteome of talpid3 vs wildtype centrosomes from talpid3 PGCs. Through this study, I have shown that Jurkat cells have a low frequency of ciliogenesis with modifications of the centrioles that correlate with their low ciliogenesis frequency; however PGCS are capable of forming cilia and have modifications of the centrioles that correlate with their ability to form cilia, highlighting PGCs as a suitable cell model for carrying out centrosome and cilia studies. A mass spectrometry screen on centrosomes isolated from Jurkat cells and PGCs failed to identify TALPID3; however, the screen identified CCDC77 and CCDC127 as novel centrosome proteins, which was confirmed by immunofluorescence. CRISPR/Cas9 editing, demonstrated that mutations in CCDC127 resulted in a significant reduction of ciliogenesis and altered centrosome protein localisation patterns in RPE1 cells. Isolation of centrosomes from PGCs did not prove effective for constructing the proteome of talpid3, therefore an alternative approach was taken using protein extracted from whole cell lysate of talpid3 and wildtype PGCs. A quantitative proteomic study was undertaken in talpid3 PGCs with the aim of understanding how protein networks are altered in talpid3. Following a Tandem Mass Tag (TMT) mass spectrometry approach, pathway analysis in Ingenuity Pathway Analysis software (IPA) identified down-regulation of protein pathways linked to regulation of the actin cytoskeleton and showed an overall change to protein pathways associated with cholesterol biosynthesis. Immunofluorescence in talpid3 embryo sections was used to examine changes to proteins involved in actin regulation, including F-actin, Profilin, Cofilin, Twinfilin and RhoC. The main findings of this thesis include evidence that talpid3 embryos can be used to model human ciliopathy mutations, demonstrate that PGCs are a primary cell model that can be used to study the centrosome and identify CCDC127 as a novel centrosome protein that is necessary for ciliogenesis in human RPE1 cells. Additionally, the findings show that protein pathways associated with the regulation of the actin cytoskeleton are downregulated in talpid3. Together the results produced in this thesis provide insight into the centrosome from the perspective of chicken PGCs as well as a better understanding of protein pathways altered in talpid3.

DNA methylation entails the addition of a methyl group to the 5-carbon of the cytosine base of the DNA. This modification is important during many biological processes such as imprinting, X-chromosome inactivation, cell differentiation as well as silencing of transposable elements (TEs). DNA methylation is dynamic during early mammalian development, despite being a more static mark in somatic cells. Global hypomethylation is a hallmark of epigenetic reprogramming in mammalian primordial germ cells (PGCs), the early embryo and in naïve embryonic stem cells (ESCs). Genome integrity is crucial during early development, as the germline DNA needs to be protected for future generations. Therefore, epigenetic reprogramming presents a critical phase for TE defence since presumably alternative silencing pathways need to be employed to limit their activity. In this thesis, I investigate the role of small RNAs to control TEs during global waves of DNA demethylation in cellular reprogramming, naïve pluripotency as well as early mammalian development. Following an introduction to the research questions, in chapter 3 I investigate the mechanism of TE regulation in an in vitro model of Dnmt1 deletion in mouse ES cells to recapitulate in vivo epigenetic reprogramming. I find that certain classes of TEs become transcriptionally upregulated and subsequently resilenced by a mechanism independent of DNA methylation. I identify ARGONAUTE 2 (AGO2) bound siRNAs as the prominent mechanism to control certain classes of TEs, while others appear to be regulated by redistribution of repressive histone modifications. In chapter 4, I construct Dicer constitutive and conditional KO ESCs in the background of the Dnmt1f l/f l ESCs using CRISPR-Cas9. I dissect the role of DNA methylation and of DICER dependent small RNAs on transcriptional changes of ESCs. Additionally, I find that DICER dependent small interfering RNAs (siRNAs) re-silence transcriptionally active TE classes. Finally, in chapter 5, I examine the role of small RNAs in TE silencing in different models of global hypomethylation in vivo and in vitro PGCs, during iPSC reprogramming and in a transition from serum to 2i culturing of mouse ESCs.

Primordial germ cells (PGCs) are the precursors of the germ cell lineage that eventually differentiate into mature spermatozoa and oocytes. Although present throughout the animal kingdom, the specification and migration of PGCs differs widely between species. In vertebrates, avians are evolutionary divergent from mammals and therefore allow a comparative system in which to study germ cell development in higher organisms. Unlike mouse, PGCs can be isolated from the chicken embryo, expanded and cultured long term in vitro. Analysis of these cells showed that cultured chicken PGCs maintain the characteristics of their in vivo counterparts, including the expression of key germ cell specific markers and cell surface adhesion proteins, and thus, are an ideal system to study germ cell biology. Further characterisation revealed that an avian homologue of the zinc finger transcription factor PRDM14, essential for the specification of the mammalian germ cell lineage, was expressed in chicken PGCs. cPRDM14 was found to be expressed in PGCs in vitro and in vivo from early developmental stages until expression is lost by embryonic day 10 and subsequently re-expressed in the adult testis. The expression of cPRDM14 suggested that this gene may play a conserved role in the avian germ cell lineage. To investigate the function of cPRDM14, a novel single piggyBac transposon vector containing a reverse tetracycline activator protein and a tetracycline response element-regulated promoter was developed. Testing of the integrated transposon revealed that expression was tightly regulated and it was possible to conditionally express one gene product whilst simultaneously reducing the expression of a second gene, both in vitro and in vivo. This vector system was fully functional in PGCs, and was used to create transgenic founder chickens capable of having gene expression manipulated in germ cells at various developmental stages. Transgenic offspring were produced and the transgene was inducible at early developmental stages in the G1 animals. The un-induced transgene proved to be toxic to early embryos so a transgenic line of birds could not be produced. The inducible transposon was used to knockdown cPRDM14 expression in chicken PGCs. Knockdown of this gene led to reduced PGC numbers and increased cell death, both in vitro and in ovo. Expression of the pluripotency factor cNANOG was also significantly reduced which may explain the increased cell death. The knockdown of cPRDM14 also led to an increased susceptibility of PGCs to spontaneously de-differentiate to pluripotent embryonic germ cells (EGCs). cPRDM14 knockdown PGCs exhibited elevated levels of phosphorylated ERK, a target of the FGF signalling pathway. It was possible to prevent de-differentiation of the knockdown PGCs by removing ectopic FGF from the media. Furthermore, a sustained high level of FGF signalling in the media was sufficient to drive the de-differentiation of control PGCs to EGCs, suggesting that increased FGF signalling was key to the de-differentiation process. Extensive epigenetic remodelling of mouse PGCs occurs during embryonic development and PRDM14 was shown to be involved in this process. Chicken PGCs in vitro, contain several key histone modifications (H3K4me3, H3K9me2 and H3K27me3) and are 5-methyl cytosine (5-mC) positive. Immunohistochemical analysis of these markers in PGCs, at various stages during early embryonic development, suggests that these cells do not undergo the extensive epigenetic remodelling found in their mammalian counterparts. In contrast to the mouse germ cell lineage, knockdown of cPRDM14 in cultured PGCs had no noticeable effect on the epigenetic status of chicken PGCs. Together these results demonstrate that cPRDM14 is essential for the survival and maintenance of germ cell identity in chicken PGCs, but may not be critical for maintaining the epigenetic status of these cells.

Specification of a germ cell lineage and appropriate maturation are essential for the transfer of genetic information from one generation to the next. Germ cells form from pluripotent precursor cells that migrate into the gonadal ridge and undergo commitment to either the female or male lineage. In the fetal ovary, germ cells enter meiotic prophase I, then arrest at the diplotene stage; in the testis germ cells do not begin meiosis until puberty. Abnormal differentiation of germ cells can result in malignant transformation. Somatic cells play a key role in modulating the developmental fate of the germ cells. Research into germ cell development during fetal life has almost exclusively focused on studies in rodents, but we, and others, have reported several fundamental differences in the expression of germ cell specific markers in the human compared with the mouse. The studies described in this thesis have investigated germ cell-specific gene expression and the possible impact of the somatic cells during development. This was achieved by studying human fetal gonads obtained during the 1st and 2nd trimesters of pregnancy and through the use of both wild-type and mutant mouse ES cell lines. Studies on germ cells in the human fetal testis have extended the findings of others, and confirmed that germ cell populations at different stages of maturation co-exist in the human fetal testis, a situation that is in contrast to that in rodents. For example expression of M2A and AP2γ was restricted to the OCT4-positive gonocyte population, while VASA and NANOS1 were localised exclusively to the to the OCT4-negative prespermatogonia. DAZL was expressed in both populations. Analysis also revealed that both the gonocyte and prespermatogonial populations proliferate throughout the 2nd trimester. Recent studies have implicated retinoic acid (RA) in the control of meiotic entry in germ cells of the fetal mouse ovary. In this study we demonstrated for the first time that two genes implicated in the action of RA in mouse gonad, STRA8 and NANOS2, are also expressed in a similar sexspecific- manner in the human fetal gonads, and that the RA receptors are present in both somatic and germ cells suggesting that RA may regulate germ cell function in the human as well as the mouse. However, whilst the mesonephros appears to be the primary site of RA synthesis in the mouse our initial studies indicate that in the human the gonad itself may be a more likely site of RA biosynthesis. In the fetal mouse testis, RA is degraded by the enzyme Cyp26b1 present in the somatic cells and germ cells do not enter meiosis, our novel findings suggest that CYP26B1 is more abundant in the human fetal ovary than the testis, suggesting that meiotic entry may be controlled by an alternative signalling pathway in the human. One of the methods that can aid our understanding of somatic cell gene expression in the gonad is in vitro culture. To date, there have been no published reports of the successful in vitro culture of somatic cells from the human fetal testis. In the current study, populations of human somatic cells were dissociated and maintained in vitro and characterised. Analysis demonstrated that cells expressing mRNAs characteristic of Sertoli cells, Leydig cells and peritubular myoid (PTM) cells were present initially, but long-term culture resulted in downregulation in expression of mRNAs specific for Sertoli cells and Leydig cells, suggesting that these cells either failed to survive or underwent alterations to their phenotype. In contrast PTM/fibroblast cells proliferated in vitro and initially maintained androgen receptor expression. These cultures therefore hold promise for studies into the signalling or cell-cell interactions in testicular somatic cells especially those relevant to the PTM population. Several studies have claimed differentiation of putative germ cells from ES cells. In the current study, analysis of mouse ES cell lines has expanded on results showing that ES cells and early germ cells express a number of genes in common. Kit signalling was shown to be important for ES cell survival as they differentiate although expression of Kit was heterogeneous. We also demonstrated that ES cells that did not express Kit displayed a decreased expression of the early germ cell genes Blimp1, Fragilis and Stella, implicating Kit signalling in the control of germ cell-associated gene expression in ES cells. This may be important to future studies optimising germ cell derivation from ES cells. In conclusion, this study has demonstrated important differences in protein expression patterns in germ cells of the human fetal testis compared to the mouse, and has raised questions about whether the proposed mechanism controlling meiotic entry of germ cells in the mouse can be applied to the human. The establishment of a system for culturing human fetal gonadal somatic cells may lead to further understanding of gene expression and development in the human fetal testis, and data suggest that the Kit/Kitl signalling system may influence germ cell gene expression in mouse ES cells.

In mammalian development, the process of gametogenesis begins with a group of germline precursors known as primordial germ cells (PGCs), which emerge shortly after embryo implantation. These PGCs undertake a migration through the developing hindgut to ultimately reach and populate the embryonic gonads. In vitro modeling of this complex developmental process offers a non-invasive method to study the molecular mechanisms governing gametogenesis. This is particularly significant for understanding the genetic and epigenetic underpinnings of infertility, which can inform advancements in assisted reproductive technologies (ART), including gene correction techniques and the optimization of in vitro gametogenesis (IVG). My thesis will concentrate on refining this in vitro model for generating mouse PGCs, aiming to make the process more efficient and scalable. An important aspect of my research will involve utilizing CRISPR technology for perturbation screens, designed to identify key epigenetic regulators that play a crucial role in PGC development. By enhancing our understanding of these regulators and the overall process of PGC development, my research could provide valuable insights and tools for improving ART and IVG techniques, ultimately aiding in the treatment of infertility and related reproductive issues. In Chapter 1 of my thesis, I provide a comprehensive overview of the current understanding in several key areas related to mammalian PGC development. This includes an in-depth examination of IVG, the epigenetic mechanisms at play during PGC development, and the various applications of the PGC-like cell (PGCLC) platform. The primary objective of this chapter is to collect and present all relevant information concerning the early stages of embryonic germline development, with a particular focus on the period preceding sex determination. By compiling this information, the chapter aims to offer a clear picture of the current state of research in the field of germline development. This not only enhances our collective understanding of this vital developmental process but also highlights existing gaps and challenges in the current body of knowledge. Identifying these areas is crucial as it sets the direction for future research endeavors, guiding scientists and researchers in their quest to unravel the complexities of germline development and its implications for reproductive health and ART. In Chapter 2 of my thesis, I focus on the development of a transcription factor (TF)-based system for the differentiation of PGCLCs. By inducing the overexpression of four key transcription factors—Prdm1, Prdm14, Tfap2c, and Nanog—I have established an efficient, cost-effective method for the scalable and simple differentiation of PGCLCs. This 4TF-induced system not only generates PGCLCs that show transcriptomic similarities to in vivo PGCs, but also parallels the characteristics of cytokine-induced PGCLCs. Another significant aspect of this work is the demonstration that PGCLC differentiation via this TF-based method can be achieved on a scalable level and sustained over prolonged cultures. Additionally, I have shown the direct differentiation of formative state embryonic stem cells (ESCs) into PGCLCs, further simplifying the PGCLC differentiation process. Overall, the work presented in this chapter establishes a powerful in vitro platform for germline modeling. In Chapter 3 of my thesis, I applied the TF-based system for PGCLC differentiation in combination with CRISPR inhibition (CRISPRi) technology to conduct a comprehensive screen for epigenetic regulators that influence PGC development. Through this approach, I targeted a total of 701 genes to identify those that significantly affect PGCLC formation. From this extensive screen, 53 genes were identified as key influencers in the efficiency of PGCLC formation. Notably, NCOR2, a transcriptional repressor known for its role in recruiting Class I and IIa histone deacetylases (HDACs) to target genes, emerged as an influential factor in inhibiting PGCLC differentiation. This finding aligns with existing evidence that underscores the importance of histone deacetylation in germline differentiation. Further, the study explored the impact of HDAC inhibitors (HDACi) on the differentiation process from ESCs to PGCLCs. Compounds like valproic acid and sodium butyrate were found to suppress this differentiation. Transcriptomic analysis of cultures treated with HDACi during ESC to PGCLC differentiation revealed a suppression of germline-associated pathways and an upregulation of somatic pathways. This work demonstrates the feasibility of conducting large scale screens of genes, chemical agents, and possibly genetic variants that impact germline development and epigenome.

An early event in Drosophila melanogaster gonad formation requires germ cells to move from the developing gut and migrate to the overlying mesoderm. Wunen, a Drosophila lipid phosphate phosphohydrolase (LPP) plays a role in this guidance mechanism by repelling germ cells from the gut. This work identified and cloned the cDNA for Drosophila Tunen a homolog of Wunen. In situ hybridisation analysis showed that Tunen and Wunen share a common expression pattern in developing embryos. Creating a Tunen transcript null tunenΔ4 and Tunen overexpression studies demonstrated that Tunen is also a repulsive guidance factor involved in germ cell migration. Analysis of genomic sequence around the wunen locus revealed that wunen and tunen co-localise and are divergently transcribed. Germ cell migration in the wunen null wunK10201 is much more perturbed than in the Tunen null tunenΔ4. In situ hybridisation analysis of wunK10201 embryos demonstrated that these embryos were also null for Tunen. This finding along with the shared expression pattern, common function and spatial organisation of wunen and tunen suggests these proteins co-operate to regulate germ cell migration.