Search

The sterility or inviability of hybrid offspring produced from an interspecific mating result from incompatibilities between parental genotypes that are thought to result from divergence of loci involved in epistatic interactions. However, attributes contributing to the rapid evolution of these regions also complicates their assembly, thus discovery of candidate hybrid sterility loci is difficult and has been restricted to a small number of model systems. Here we reported rapid interspecific divergence at the DXZ4 macrosatellite locus in an interspecific cross between two closely related mammalian species: the domestic cat (Felis silvestris catus) and the Jungle cat (Felis chaus). DXZ4 is an interesting candidate due to its structural complexity, copy number variability, and described role in the critical yet complex biological process of X-chromosome inactivation. However, the full structure of DXZ4 was absent or incomplete in nearly every available mammalian genome assembly given its repetitive complexity. We compared highly continuous genomes for three cat species, each containing a complete DXZ4 locus, and discovered that the felid DXZ4 locus differs substantially from the human ortholog, and that it varies in copy number between cat species. Additionally, we reported expression, methylation, and structural conformation profiles of DXZ4 and the X chromosome during stages of spermatogenesis that have been previously associated with hybrid male sterility. Collectively, these findings suggest a new role for DXZ4 in male meiosis and a mechanism for feline interspecific incompatibility through rapid satellite divergence.

Initiation of spermatogonial differentiation in the mouse testis begins with the response to retinoic acid (RA) characterized by activation of KIT and STRA8 expression. In the adult, spermatogonial differentiation is spatiotemporally coordinated by a pulse of RA every 8.6 days that is localized to stages VII–VIII of the seminiferous epithelial cycle. Dogmatically, progenitor spermatogonia that express retinoic acid receptor gamma (RARG) at these stages will differentiate in response to RA, but this has yet to be tested functionally. Previous single-cell RNA-seq data identified phenotypically and functionally distinct subsets of spermatogonial stem cells (SSCs) and progenitor spermatogonia, where late progenitor spermatogonia were defined by expression of RARG and Dppa3. Here, we found late progenitor spermatogonia (RARGhigh KIT−) were further divisible into two subpopulations based on Dppa3 reporter expression (Dppa3-ECFP or Dppa3-EGFP) and were observed across all stages of the seminiferous epithelial cycle. However, nearly all Dppa3+ spermatogonia were differentiating (KIT+) late in the seminiferous epithelial cycle (stages X–XII), while Dppa3− late progenitors remained abundant, suggesting that Dppa3+ and Dppa3− late progenitors differentially responded to RA. Following acute RA treatment (2–4 h), significantly more Dppa3+ late progenitors induced KIT, including at the midpoint of the cycle (stages VI–IX), than Dppa3− late progenitors. Subsequently, single-cell analyses indicated a subset of Dppa3+ late progenitors expressed higher levels of Rxra, which we confirmed by RXRA whole-mount immunostaining. Together, these results indicate RARG alone is insufficient to initiate a spermatogonial response to RA in the adult mouse testis and suggest differential RXRA expression may discriminate responding cells.

Although reactive oxygen species (ROS) are required for spermatogonial stem cell (SSC) self-renewal, they induce DNA damage and are harmful to SSCs. However, little is known about how SSCs protect their genome during self-renewal. Here, we report that Ogg1 is essential for SSC protection against ROS. While cultured SSCs exhibited homologous recombination-based DNA double-strand break repair at levels comparable with those in pluripotent stem cells, they were significantly more resistant to hydrogen peroxide than pluripotent stem cells or mouse embryonic fibroblasts, suggesting that they exhibit high levels of base excision repair (BER) activity. Consistent with this observation, cultured SSCs showed significantly lower levels of point mutations than somatic cells, and showed strong expression of BER-related genes. Functional screening revealed that Ogg1 depletion significantly impairs survival of cultured SSCs upon hydrogen peroxide exposure. Thus, our results suggest increased expression of BER-related genes, including Ogg1, protects SSCs from ROS-induced damage.Summary sentenceOGG1 suppresses ROS-induced damages in spermatogonial stem cells.

More than a decade ago, the ENCODE and NIH Epigenomics Roadmap consortia organized large multilaboratory efforts to profile the epigenomes of >110 different mammalian somatic cell types. This generated valuable publicly accessible datasets that are being mined to reveal genome-wide patterns of a variety of different epigenetic parameters. This consortia approach facilitated the powerful and comprehensive multiparametric integrative analysis of the epigenomes in each cell type. However, no germ cell types were included among the cell types characterized by either of these consortia. Thus, comprehensive epigenetic profiling data are not generally available for the most evolutionarily important cells, male and female germ cells. We discuss the need for reproductive biologists to generate similar multiparametric epigenomic profiling datasets for both male and female germ cells at different developmental stages and summarize our recent effort to derive such data for mammalian spermatogonial stem cells and progenitor spermatogonia. Summary sentence The ENCODE and NIH Epigenome Roadmap consortia have generated valuable datasets profiling epigenomes of >110 different mammalian somatic cell types, but none of these have included profiles of even a single germ cell type.

A number of environmental factors from nutrition to toxicants have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. This requires alterations in the germline (sperm or egg) epigenome. Previously, the agricultural fungicide vinclozolin was found to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs) termed epimutations that help mediate this epigenetic inheritance. The current study was designed to investigate the developmental origins of the transgenerational DMRs during gametogenesis. Male control and vinclozolin lineage F3 generation rats were used as a source of embryonic day 13 (E13) primordial germ cells, embryonic day 16 (E16) prospermatogonia, postnatal day 10 (P10) spermatogonia, adult pachytene spermatocytes, round spermatids, caput epididymal spermatozoa, and caudal sperm. The DMRs between the control versus vinclozolin lineage samples were determined for each developmental stage. The top 100 statistically significant DMRs for each stage were compared. The developmental origins of the caudal epididymal sperm DMRs were assessed. The chromosomal locations and genomic features of the different stage DMRs were investigated. In addition, the DMR associated genes were identified. Previous studies have demonstrated alterations in the DMRs of primordial germ cells (PGCs). Interestingly, the majority of the DMRs identified in the current study for the caudal sperm originated during the spermatogenic process in the testis. A cascade of epigenetic alterations initiated in the PGCs appears to be required to alter the epigenetic programming during spermatogenesis to modify the sperm epigenome involved in the transgenerational epigenetic inheritance phenomenon.

Summary: Spermatogenesis is a complex and dynamic cellular differentiation process critical to male reproduction and sustained by spermatogonial stem cells (SSCs). Although patterns of gene expression have been described for aggregates of certain spermatogenic cell types, the full continuum of gene expression patterns underlying ongoing spermatogenesis in steady state was previously unclear. Here, we catalog single-cell transcriptomes for >62,000 individual spermatogenic cells from immature (postnatal day 6) and adult male mice and adult men. This allowed us to resolve SSC and progenitor spermatogonia, elucidate the full range of gene expression changes during male meiosis and spermiogenesis, and derive unique gene expression signatures for multiple mouse and human spermatogenic cell types and/or subtypes. These transcriptome datasets provide an information-rich resource for studies of SSCs, male meiosis, testicular cancer, male infertility, or contraceptive development, as well as a gene expression roadmap to be emulated in efforts to achieve spermatogenesis in vitro. : Hermann et al. present single-cell transcriptomes from >62,000 individual spermatogenic cells from immature and adult male mice and adult men. Their analysis facilitates resolution of SSCs and progenitor spermatogonia, elucidates the full range of gene expression changes during male meiosis and spermiogenesis, and derives unique gene expression signatures for eleven mouse and human spermatogenic cell types. Keywords: male germ cells, RNA, heterogeneity, meiosis, spermiogenesis, continuum, human, mouse

Chromatin of male and female gametes undergoes a number of reprogramming events during the transition from germ cell to embryonic developmental programs. Although the rearrangement of DNA methylation patterns occurring in the zygote has been extensively characterized, little is known about the dynamics of DNA modifications during spermatid maturation. Here, we demonstrate that the dynamics of 5-carboxylcytosine (5caC) correlate with active transcription of LINE-1 retroelements during murine spermiogenesis. We show that the open reading frames of active and evolutionary young LINE-1s are 5caC-enriched in round spermatids and 5caC is eliminated from LINE-1s and spermiogenesis-specific genes during spermatid maturation, being simultaneously retained at promoters and introns of developmental genes. Our results reveal an association of 5caC with activity of LINE-1 retrotransposons suggesting a potential direct role for this DNA modification in fine regulation of their transcription., Blythe et al investigate the patterns of oxidized forms of the 5-methylcytosine DNA modification during spermatogenesis. They find that open reading frames of evolutionarily young and transcriptionally active LINE-1 retrotransposons are enriched in 5-carboxylcytosine (5caC) during spermatid maturation, suggesting a link between 5cac and LINE-1 activity.

OBJECTIVE To provide an overview of the history of the North American Testis Workshop (NATW), of its relationship to the American Society of Andrology (ASA), and of the publications that resulted from the first 25 workshops. METHODS The collection of volumes and journal articles that relate to the NATW was searched. DISCUSSION AND CONCLUSION During the first twenty-five meetings of the NATW, a remarkable number of breakthroughs regarding every aspect of the testis were presented. We anticipate that with the acceleration of new genetic, epigenetic, and molecular knowledge of the functions of testicular cells, we will continue to learn about the discovery of new and clinically important aspects of testicular function during the next twenty-five NATWs.

Numerous environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of disease and phenotypic variation. Alterations in the germline epigenome are necessary to transmit transgenerational phenotypes. In previous studies, the pesticide DDT (dichlorodiphenyltrichloroethane) and the agricultural fungicide vinclozolin were shown to promote the transgenerational inheritance of sperm differential DNA methylation regions, non-coding RNAs and histone retention, which are termed epimutations. These epimutations are able to mediate this epigenetic inheritance of disease and phenotypic variation. The current study was designed to investigate the developmental origins of the transgenerational differential histone retention sites (called DHRs) during gametogenesis of the sperm. Vinclozolin and DDT were independently used to promote the epigenetic transgenerational inheritance of these DHRs. Male control lineage, DDT lineage and vinclozolin lineage F3 generation rats were used to isolate round spermatids, caput epididymal spermatozoa, and caudal sperm. The DHRs distinguishing the control versus DDT lineage or vinclozolin lineage samples were determined at these three developmental stages. DHRs and a reproducible core of histone H3 retention sites were observed using an H3 chromatin immunoprecipitation-sequencing (ChIP-Seq) analysis in each of the germ cell populations. The chromosomal locations and genomic features of the DHRs were analyzed. A cascade of epigenetic histone retention site alterations was found to be initiated in the round spermatids and then further modified during epididymal sperm maturation. Observations show that in addition to alterations in sperm DNA methylation and ncRNA expression previously identified, the induction of differential histone retention sites (DHRs) in the later stages of spermatogenesis also occurs. This novel component of epigenetic programming during spermatogenesis can be environmentally altered and transmitted to subsequent generations through epigenetic transgenerational inheritance.

SUMMARY Spermatogonial stem cells (SSCs) sustain spermatogenesis by balancing self-renewal and initiation of differentiation to produce progenitor spermatogonia committed to forming sperm. To define the regulatory logic among SSCs and progenitors, we performed single-cell RNA velocity analyses and validated results in vivo. A predominant quiescent SSC population spawns a small subset of cell-cycle-activated SSCs via mitogen-activated protein kinase (MAPK)/AKT signaling. Activated SSCs form early progenitors and mTORC1 inhibition drives activated SSC accumulation consistent with blockade to progenitor formation. Mechanistically, mTORC1 inhibition suppresses transcription among spermatogonia and specifically alters expression of insulin growth factor (IGF) signaling in early progenitors. Tex14−/− testes lacking intercellular bridges do not accumulate activated SSCs following mTORC1 inhibition, indicating that steady-state mTORC1 signaling drives activated SSCs to produce progenitor clones. These results are consistent with a model of SSC self-renewal dependent on interconversion between activated and quiescent SSCs, and mTORC1-dependent initiation of differentiation from SSCs to progenitor clones., Graphical Abstract, In Brief Suzuki et al. define relationships between subsets of adult mouse SSCs and progenitor spermatogonia using single-cell RNA velocity analyses and in vivo validations. Quiescent SCCs convert to cell-cycle-activated SCCs via MAPK/AKT signaling. Activated SCCs are driven to become early progenitor clones ready to initiate differentiation through mTORC1 signaling.

Epigenetic alterations in the germline can be triggered by a number of different environmental factors from diet to toxicants. These environmentally induced germline changes can promote the epigenetic transgenerational inheritance of disease and phenotypic variation. In previous studies, the pesticide DDT was shown to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs), also called epimutations, which can in part mediate this epigenetic inheritance. In the current study, the developmental origins of the transgenerational DMRs during gametogenesis have been investigated. Male control and DDT lineage F3 generation rats were used to isolate embryonic day 16 (E16) prospermatogonia, postnatal day 10 (P10) spermatogonia, adult pachytene spermatocytes, round spermatids, caput epididymal spermatozoa, and caudal sperm. The DMRs between the control versus DDT lineage samples were determined at each developmental stage. The top 100 statistically significant DMRs at each stage were compared and the developmental origins of the caudal epididymal sperm DMRs were assessed. The chromosomal locations and genomic features of the different stage DMRs were analyzed. Although previous studies have demonstrated alterations in the DMRs of primordial germ cells (PGCs), the majority of the DMRs identified in the caudal sperm originated during the spermatogonia stages in the testis. Interestingly, a cascade of epigenetic alterations initiated in the PGCs is required to alter the epigenetic programming during spermatogenesis to obtain the sperm epigenetics involved in the epigenetic transgenerational inheritance phenomenon.

Induced pluripotent stem cells (iPSCs) offer the possibility of cell replacement therapies using patient-matched cells to treat otherwise intractable diseases and debilitations. To successfully realize this potential, several factors must be optimized including i) selection of the appropriate cell type and numbers to transplant, ii) determination of the means of transplantation and the location into which the transplanted cells should be delivered, and iii) demonstration of the safety and efficacy of the cell replacement protocol to mitigate each targeted disease state. A majority of diseases or debilitations likely to be targeted by cell-based therapeutic approaches represent complex conditions or physiologies manifest predominantly in primates including humans. Nonhuman primates afford the most clinically relevant model system for biomedical studies and testing of cell-based therapies. Baboons have 92% genomic similarity with humans overall and especially significant similarities in their immunogenetic system, rendering this species a particularly valuable model for testing procedures involving cell transplants into living individuals. To maximize the utility of the baboon model, standardized protocols must be developed for the derivation of induced pluripotent stem cells from living adults and the long-term maintenance of these cells in culture. Here we tested four commercially available culture systems (ReproFF, mTeSR1, E8 and Pluristem) for competence to maintain baboon iPSCs in a pluripotent state over multiple passages, and to support the derivation of new lines of baboon iPSCs. Of these four media only Pluristem was able to maintain baboon pluripotency as assessed by morphological characteristics, immunocytochemistry and RT-qPCR. Pluristem also facilitated the derivation of new lines of iPSCs from adult baboon somatic cells, which had previously not been accomplished. We derived multiple iPS cell lines from adult baboon peripheral blood mononuclear cells cultured in Pluristem. These were validated by expression of the pluripotency markers OCT4, NANOG, SOX2, SSEA4 and TRA181, as well as the ability to differentiate into tissues from all three germ layers when injected into immunocompromised mice. These findings further advance the utility of the baboon as an ideal preclinical model system for optimizing iPS cell-based, patient-specific replacement therapies in humans.

PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a yet to be identified pathway, which is different from all currently known small RNA biogenetic pathways. In addition, pilRNAs appear to preferentially target the 3'-UTRs of mRNAs in a partially complementary manner. Our data suggest that pilRNAs, as an integral component of the small RNA transcriptome in somatic cell lineages, represent a distinct population of small RNAs that may have functions similar to germ cell piRNAs.

Mammalian reproduction requires that males and females produce functional haploid germ cells through complex cellular differentiation processes known as spermatogenesis and oogenesis, respectively. While numerous studies have functionally characterized protein-coding genes and small noncoding RNAs (microRNAs and piRNAs) that are essential for gametogenesis, the roles of regulatory long noncoding RNAs (lncRNAs) are yet to be fully characterized. Previously, we and others have demonstrated that intergenic regions of the mammalian genome encode thousands of long noncoding RNAs, and many studies have now demonstrated their critical roles in key biological processes. Thus, we postulated that some lncRNAs may also impact mammalian spermatogenesis and fertility. In this study, we identified a dynamic expression pattern of lncRNAs during murine spermatogenesis. Importantly, we identified a subset of lncRNAs and very few mRNAs that appear to escape meiotic sex chromosome inactivation, an epigenetic process that leads to the silencing of the X- and Y-chromosomes at the pachytene stage of meiosis. Further, some of these lncRNAs and mRNAs show a strong testis expression pattern suggesting that they may play key roles in spermatogenesis. Lastly, we generated a mouse knockout of one X-linked lncRNA, Tslrn1 (testis-specific long noncoding RNA 1), and found that males carrying a Tslrn1 deletion displayed normal fertility but a significant reduction in spermatozoa. Our findings demonstrate that dysregulation of specific mammalian lncRNAs is a novel mechanism of low sperm count or infertility, thus potentially providing new biomarkers and therapeutic strategies.

The phosphoglycerate kinase-2 (Pgk2) gene is regulated in a tissue-, cell type-, and developmental stage-specific manner during spermatogenesis and is required for normal sperm motility and fertility in mammals. Activation ofPgk2transcription is regulated by testis-specific demethylation of DNA and binding of testis-specific transcription factors to enhancer and core promoter elements. Here, we show that chromatin remodeling including reconfiguration of nucleosomes and changes in histone modifications is also associated with transcriptional activation of thePgk2gene during spermatogenesis. Developmental studies indicate that the order of events involved in transcriptional activation of thePgk2gene includes demethylation of DNA in T1- and T2-prospermatogonia, binding of a factor to the CAAT box in type A and B spermatogonia, followed by recruitment of chromatin remodeling factors, displacement of a nucleosome from thePgk2promoter region, binding of factors to thePgk2core promoter and enhancer regions, and, finally, initiation of transcription in primary spermatocytes. Transgene studies show thatPgk2core promoter elements are required to direct demethylation of DNA and reconfiguration of nucleosomes, whereas both enhancer and core promoter elements are required to direct changes in histone modifications and initiation of transcription. These results provide novel insight into the developmental order of molecular events required to activate tissue-specific transcription of thePgk2gene, the distinct elements in the 5′-regulatory region of thePgk2gene that regulate each of these events, and the relationship among these events in that each step in this process appears to be a necessary prerequisite for the subsequent step.

In multicellular organisms, germ cells carry the hereditary material from one generation to the next. Developing germ cells are unipotent gamete precursors, and mature gametes are highly differentiated, specialized cells. However, upon gamete union at fertilization, their genomes drive a totipotent program, giving rise to a complete embryo as well as extraembryonic tissues. The biochemical basis for the ability to transition from differentiated cell to totipotent zygote is unknown. Here we report that a set of developmentally critical genes is maintained in an epigenetically poised (bivalent) state from embryonic stages through the end of meiosis. We performed ChIP-seq and RNA-seq analysis on flow-sorted male and female germ cells during embryogenesis at three time points surrounding sexual differentiation and female meiotic initiation, and then extended our analysis to meiotic and postmeiotic male germ cells. We identified a set of genes that is highly enriched for regulators of differentiation and retains a poised state (high H3K4me3, high H3K27me3, and lack of expression) across sexes and across developmental stages, including in haploid postmeiotic cells. The existence of such a state in embryonic stem cells has been well described. We now demonstrate that a subset of genes is maintained in a poised state in the germ line from the initiation of sexual differentiation during fetal development and into postmeiotic stages. We propose that the epigenetically poised condition of these developmental genes is a fundamental property of the mammalian germ-line nucleus, allowing differentiated gametes to unleash a totipotent program following fertilization.

Changes in gene regulation frequently underlie changes in morphology during evolution, and differences in chromatin state have been linked with changes in anatomical structure and gene expression across evolutionary time. Here we assess the relationship between evolution of chromatin state in germ cells and evolution of gene regulatory programs governing somatic development. We examined the poised (H3K4me3/H3K27me3 bivalent) epigenetic state in male germ cells from five mammalian and one avian species. We find that core genes poised in germ cells from multiple amniote species are ancient regulators of morphogenesis that sit at the top of transcriptional hierarchies controlling somatic tissue development, whereas genes that gain poising in germ cells from individual species act downstream of core poised genes during development in a species-specific fashion. We propose that critical regulators of animal development gained an epigenetically privileged state in germ cells, manifested in amniotes by H3K4me3/H3K27me3 poising, early in metazoan evolution.

Exposure to environmental factors can induce the epigenetic transgenerational inheritance of disease. Alterations to the epigenome termed “epimutations” include “primary epimutations” which are epigenetic alterations in the absence of genetic change and “secondary epimutations” which form following an initial genetic change. To determine if secondary epimutations contribute to transgenerational transmission of disease following in utero exposure to the endocrine disruptor vinclozolin, we exposed pregnant female rats carrying the lacI mutation-reporter transgene to vinclozolin and assessed the frequency of mutations in kidney tissue and sperm recovered from F1 and F3 generation progeny. Our results confirm that vinclozolin induces primary epimutations rather than secondary epimutations, but also suggest that some primary epimutations can predispose a subsequent accelerated accumulation of genetic mutations in F3 generation descendants that have the potential to contribute to transgenerational phenotypes. We therefore propose the existence of “tertiary epimutations” which are initial primary epimutations that promote genome instability leading to an accelerated accumulation of genetic mutations.

We previously demonstrated that intracytoplasmic sperm injection (ICSI), a type of assisted reproductive technology (ART), can induce epimutations and/or epimutant phenotypes in somatic tissues of adult mice produced by this method. In the present study, we compared the occurrence of epimutations in mice produced by natural conception, ICSI and somatic cell nuclear transfer. Surprisingly, we observed the highest frequency of epimutations in somatic tissues from ICSI-derived mice. We also observed a delay in reprogramming of the maternal allele of the imprinted H19 gene in spermatogonia from juvenile ICSI-derived male mice. These observations led us to hypothesize that the exposure of the maternal gametic genome to exogenous gonadotropins during the endocrine stimulation of folliculogenesis (superovulation) may contribute to the disruption of the normal epigenetic programming of imprinted loci in somatic tissues and/or epigenetic reprogramming in the germ line of ensuing offspring. To test this hypothesis, we uncoupled superovulation from ICSI by subjecting female mice to gonadotropin stimulation and then allowing them to produce offspring by natural mating. We found that mice produced in this way also exhibited epimutations and/or epimutant phenotypes in somatic tissues and delayed epigenetic reprogramming in spermatogenic cells, providing evidence that gonadotropin stimulation contributes to the induction of epimutations during ART procedures. Our results suggest that gonadotropin stimulation protocols used in conjunction with ART procedures should be optimized to minimize the occurrence of epimutations in offspring produced by these methods.

Homeobox genes encode transcription factors that regulate diverse developmental events. The largest known homeobox gene cluster – the X-linked mouse reproductive homeobox (Rhox) cluster – harbors genes whose expression patterns and functions are largely unknown. Here, we report that a member of this cluster,Rhox10, is expressed in male germ cells.Rhox10is highly transcribed in spermatogoniain vivoand is upregulated in response to the differentiation-inducing agent retinoic acidin vitro. Using a specific RHOX10 antiserum that we generated, we found that RHOX10 protein is selectively expressed in fetal gonocytes, germline stem cells, spermatogonia, and early spermatocytes. RHOX10 protein undergoes a dramatic shift in subcellular localization as germ cells progress from mitotically arrested gonocytes to mitotic spermatogonia and from mitotic spermatogonia to early meiotic spermatocytes, consistent with RHOX10 performing different functions in these stages.

Phosphoglycerate kinase 2 (PGK2), an isozyme that catalyzes the first ATP-generating step in the glycolytic pathway, is encoded by an autosomal retrogene that is expressed only during spermatogenesis. It replaces the ubiquitously expressed phosphoglycerate kinase 1 (PGK1) isozyme following repression of Pgk1 transcription by meiotic sex chromosome inactivation during meiotic prophase and by postmeiotic sex chromatin during spermiogenesis. The targeted disruption of Pgk2 by homologous recombination eliminates PGK activity in sperm and severely impairs male fertility, but does not block spermatogenesis. Mating behavior, reproductive organ weights (testis, excurrent ducts, and seminal vesicles), testis histology, sperm counts, and sperm ultrastructure were indistinguishable between Pgk2(-/-) and wild-type mice. However, sperm motility and ATP levels were markedly reduced in males lacking PGK2. These defects in sperm function were slightly less severe than observed in males lacking glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS), the isozyme that catalyzes the step preceding PGK2 in the sperm glycolytic pathway. Unlike Gapdhs(-/-) males, the Pgk2(-/-) males also sired occasional pups. Alternative pathways that bypass the PGK step of glycolysis exist. We determined that one of these bypass enzymes, acylphosphatase, is active in mouse sperm, perhaps contributing to phenotypic differences between mice lacking GAPDHS or PGK2. This study determined that PGK2 is not required for the completion of spermatogenesis, but is essential for sperm motility and male fertility. In addition to confirming the importance of the glycolytic pathway for sperm function, distinctive phenotypic characteristics of Pgk2(-/-) mice may provide further insights into the regulation of sperm metabolism.

Cloning by somatic cell nuclear transfer (SCNT) circumvents processes that normally function during gametogenesis to prepare the gamete genomes to support development of new progeny following fertilization. One such process is enhanced maintenance of genetic integrity in germ cells, such that germ cells typically carry fewer spontaneously acquired mutations than somatic cells in the same individual. Thus, embryos produced from somatic cells by SCNT could directly inherit more mutations than naturally conceived embryos. Alternatively, they could inherit epigenetic programming that predisposes more rapid accumulation of de novo mutations during development. We used a transgenic mouse system to test these possibilities by producing cloned midgestation mouse fetuses from three different donor somatic cell types carrying significantly different initial frequencies of spontaneous mutations. We found that on an individual locus basis, mutations acquired spontaneously in a population of donor somatic cells are not likely to be propagated to cloned embryos by SCNT. In addition, we found that the rate of accumulation of spontaneous mutations was similar in fetuses produced by either natural conception or cloning, indicating that cloned fetuses do not acquire mutations more rapidly than naturally conceived fetuses. These results represent the first direct demonstration that the process of cloning by SCNT does not lead to an increase in the frequency of point mutations. These results also demonstrate that epigenetic mechanisms normally contribute to the regulation of genetic integrity in a tissue-specific manner, and that these mechanisms are subject to reprogramming during cloning.

Meiotic sex chromosome inactivation (MSCI) during spermatogenesis is characterized by transcriptional silencing of genes on both the X and Y chromosomes in mid to late pachytene spermatocytes1. MSCI is believed to result from meiotic silencing of unpaired DNA because the X and Y chromosomes remain largely unpaired throughout first meiotic prophase2. However, unlike X-chromosome inactivation in female embryonic cells, where 25–30% of X-linked structural genes have been reported to escape inactivation3, previous microarray4- and RT-PCR5-based studies of expression of >364 X-linked mRNA-encoding genes during spermatogenesis have failed to reveal any X-linked gene that escapes the silencing effects of MSCI in primary spermatocytes. Here we show that many X-linked miRNAs are transcribed and processed in pachytene spermatocytes. This unprecedented escape from MSCI by these X-linked miRNAs suggests that they may participate in a critical function at this stage of spermatogenesis, including the possibility that they contribute to the process of MSCI itself, and/or that they may be essential for post-transcriptional regulation of autosomal mRNAs during the late meiotic and early postmeiotic stages of spermatogenesis.

Phosphoglycerate kinase 2 (PGK2) is a germ cell-specific protein whose mRNA is translationally regulated in the mammalian testis. Using RNA affinity chromatography with the 3'-untranslated region (UTR) of Pgk2 mRNA and adult testis extracts, several associated proteins including a novel isoform of the AU-rich element RNA-binding protein and KH-type splicing regulatory protein (KSRP) were identified. KSRP, a protein of approximately 75 kDa, is widely expressed in somatic and germ cells where it is primarily nuclear. In addition to the approximately 75-kDa KSRP, a approximately 52-kD KSRP, t-KSRP, is present in the cytoplasm of a subpopulation of germ cells. t-KSRP binds directly to a 93-nt sequence (designated the F1 region) of the 3'-UTR of the Pgk2 mRNA and destabilizes Pgk2 mRNA constructs in testis extracts and in transfected cells. We conclude that this testicular variant of the multifunctional nucleic acid-binding protein, KSRP, serves as a decay-promoting factor for Pgk2 mRNA in male germ cells.

Comparisons between eutherians and marsupials suggest limited conservation of the molecular mechanisms that control genomic imprinting in mammals. We have studied the evolution of the imprinted IGF2-H19 locus in therians. Although marsupial orthologs of protein-coding exons were easily identified, the use of evolutionarily conserved regions and low-stringency Bl2seq comparisons was required to delineate a candidate H19 noncoding RNA sequence. The therian H19 orthologs show miR-675 and exon structure conservation, suggesting functional selection on both features. Transcription start site sequences and poly(A) signals are also conserved. As in eutherians, marsupial H19 is maternally expressed and paternal methylation upstream of the gene originates in the male germline, encompasses a CTCF insulator, and spreads somatically into the H19 gene. The conservation in all therians of the mechanism controlling imprinting of the IGF2-H19 locus suggests a sequential model of imprinting evolution.

Transcription of the testis-specific Pgk2 gene is selectively activated in primary spermatocytes to provide a source of phosphoglycerate kinase that is critical to normal motility and fertility of mammalian spermatozoa. We examined dynamic changes in protein-DNA interactions at the Pgk2 gene promoter during murine spermatogenesis in vivo by performing genomic footprinting and chromatin immunoprecipitation assays with enriched populations of murine spermatogenic cells at stages prior to, during, and following transcription of this gene. We found that genes encoding the testis-specific homeodomain factor PBX4 and its coactivator, PREP1, are expressed in patterns that mirror expression of the Pgk2 gene and that these factors become bound to the Pgk2 enhancer in cells in which this gene is actively expressed. We therefore suggest that these factors, along with CREM and SP3, direct stage- and cell type-specific transcription of the Pgk2 gene during spermatogenesis. We propose that binding of PBX4, plus its coactivator PREP1, is a rate-limiting step leading to the initiation of tissue-specific transcription of the Pgk2 gene. This study provides insight into the developmentally dynamic establishment of tissue-specific protein-DNA interactions in vivo. It also allows us to speculate about the events that led to tissue-specific regulation of the Pgk2 gene during mammalian evolution.

X-chromosome inactivation (XCI) evolved in mammals to deal with X-chromosome dosage imbalance between the XX female and the XY male. In eutherian mammals, random XCI of the soma requires a master regulatory locus known as the 'X-inactivation center' (XIC/Xic), wherein lies the noncoding XIST/Xist silencer RNA and its regulatory antisense Tsix gene. By contrast, marsupial XCI is imprinted to occur on the paternal X chromosome. To determine whether marsupials and eutherians share the XIC-driven mechanism, we search for the sequence equivalents in the genome of the South American opossum, Monodelphis domestica. Positional cloning and bioinformatic analysis reveal several interesting findings. First, protein-coding genes that flank the eutherian XIC are well-conserved in M. domestica, as well as in chicken, frog, and pufferfish. However, in M. domestica we fail to identify any recognizable XIST or TSIX equivalents. Moreover, cytogenetic mapping shows a surprising break in synteny with eutherian mammals and other vertebrates. Therefore, during the evolution of the marsupial X chromosome, one or more rearrangements broke up an otherwise evolutionarily conserved block of vertebrate genes. The failure to find XIST/TSIX in M. domestica may suggest that the ancestral XIC is too divergent to allow for detection by current methods. Alternatively, the XIC may have arisen relatively late in mammalian evolution, possibly in eutherians with the emergence of random XCI. The latter argues that marsupial XCI does not require XIST and opens the search for alternative mechanisms of dosage compensation.

In eutherian mammals, the X and Y chromosomes undergo meiotic sex chromosome inactivation (MSCI) during spermatogenesis in males. However, following fertilization, both the paternally (Xp) and maternally (Xm) inherited X chromosomes are active in the inner cell mass of the female blastocyst, and then random inactivation of one X chromosome occurs in each cell, leading to a mosaic pattern of X-chromosome activity in adult female tissues. In contrast, marsupial females show a nonrandom pattern of X chromosome activity, with repression of the Xp in all somatic tissues. Here, we show that MSCI also occurs during spermatogenesis in marsupials in a manner similar to, but more stable than that in eutherians. These findings support the suggestion that MSCI may have provided the basis for an early dosage compensation mechanism in mammals based solely on gametogenic events, and that random X-chromosome inactivation during embryogenesis may have evolved subsequently in eutherian mammals. genesis 45:696–708, 2007. © 2007 Wiley-Liss, Inc.

Past studies have indicated that transcription of all X-linked genes is repressed by meiotic sex chromosome inactivation (MSCI) during the meiotic phase of spermatogenesis in mammals. However, more recent studies have shown an increase in steady-state levels of certain X-linked miRNAs in pachytene spermatocytes, suggesting that either synthesis of these miRNAs increases or that degradation of these miRNAs decreases dramatically in these cells. To distinguish between these possibilities, we performed RNA-FISH to detect nascent transcripts from multiple miRNA genes in various spermatogenic cell types. Our results show definitively that Type I X-linked miRNA genes are subject to MSCI, as are all or most X-linked mRNA genes, whereas Type II and III X-linked miRNA genes escape MSCI by continuing ongoing, active transcription in primary spermatocytes. We corroborated these results by co-localization of RNA-FISH signals with both a corresponding DNA-FISH signal and an immunofluorescence signal for RNA polymerase II. We also found that X-linked miRNA genes that escape MSCI locate non-randomly to the periphery of the XY body, whereas genes that are subject to MSCI remain located within the XY body in pachytene spermatocytes, suggesting that the mechanism of escape of X-linked miRNA genes from MSCI involves their relocation to a position outside of the repressive chromatin domain associated with the XY body. The fact that Type II and III X-linked miRNA genes escape MSCI suggests an immediacy of function of the encoded miRNAs specifically required during the meiotic stages of spermatogenesis.

Spermatogonial stem cells (SSCs) are a subset of undifferentiated spermatogonia responsible for ongoing spermatogenesis in mammalian testes. Spermatogonial stem cells arise from morphologically homogeneous prospermatogonia, but growing evidence suggests that only a subset of prospermatogonia develops into the foundational SSC pool. This predicts that subtypes of undifferentiated spermatogonia with discrete mRNA and protein signatures should be distinguishable in neonatal testes. We used single-cell quantitative RT-PCR to examine mRNA levels of 172 genes in individual spermatogonia from 6-day postnatal (P6) mouse testes. Cells enriched from P6 testes using the StaPut or THY1(+) magnetic cell sorting methods exhibited considerable heterogeneity in the abundance of specific germ cell and stem cell mRNAs, segregating into one somatic and three distinct spermatogonial clusters. However, P6 Id4-eGFP(+) transgenic spermatogonia, which are known to be enriched for SSCs, were more homogeneous in their mRNA levels, exhibiting uniform levels for the majority of genes examined (122 of 172). Interestingly, these cells displayed nonuniform (50 of 172) expression of a smaller cohort of these genes, suggesting there is substantial heterogeneity even within the Id4-eGFP(+) population. Further, although immunofluorescence staining largely demonstrated conformity between mRNA and protein levels, some proteins were observed in patterns that were disparate from those detected for the corresponding mRNAs in Id4-eGFP(+) spermatogonia (e.g., Kit, Sohlh2, Stra8), suggesting additional heterogeneity is introduced at the posttranscriptional level. Taken together, these data demonstrate the existence of multiple spermatogonial subtypes in P6 mouse testes and raise the intriguing possibility that these subpopulations may correlate with the development of functionally distinct spermatogenic cell types.

Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3'-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3'-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3'-processing characteristics in the testicular samples, compared to control sets of widely used 3'-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3'-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3'-untranslated regions (3'-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3'-UTR truncation and no significant difference in 3'-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3'-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis.

SummaryIn mammals, the X and Y chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during prophase I in the male germline, but their status thereafter is currently unclear. An abundance of X-linked spermatogenesis genes has spawned the view that the X must be active [1–8]. On the other hand, the idea that the imprinted paternal X of the early embryo may be preinactivated by MSCI suggests that silencing may persist longer [9–12]. To clarify this issue, we establish a comprehensive X-expression profile during mouse spermatogenesis. Here, we discover that the X and Y occupy a novel compartment in the postmeiotic spermatid and adopt a non-Rabl configuration. We demonstrate that this postmeiotic sex chromatin (PMSC) persists throughout spermiogenesis into mature sperm and exhibits epigenetic similarity to the XY body. In the spermatid, 87% of X-linked genes remain suppressed postmeiotically, while autosomes are largely active. We conclude that chromosome-wide X silencing continues from meiosis to the end of spermiogenesis, and we discuss implications for proposed mechanisms of imprinted X-inactivation.

Cellular differentiation is mediated by differential gene expression. The cells of the testis are no exception. Indeed, recent studies based on microarray and expressed sequence tag analyses have revealed dynamic changes in gene expression during spermatogenesis. The autosomal phosphoglycerate kinase gene Pgk2 is an example of a gene that is tightly regulated during spermatogenesis, with transcription initiating in primary spermatocytes and ceasing in postmeiotic spermatids. Our studies show that this tissue-specific transcription is regulated at multiple levels, including binding of ubiquitous and testis-specific transcription factors, reconfiguration of nucleosomes, decondensation of chromatin, modifications of specific residues in histones, and demethylation of DNA. Our ability to isolate relatively pure populations of different spermatogenic cell types has allowed us to determine the order of these events that lead up to initiation of transcription of the Pgk2 gene. By examining similar parameters in transgenic mice carrying various portions of Pgk2 regulatory sequence, we have localized specific signals responsible for each of these regulatory events. Together these results demonstrate that activation of testis-specific transcription of the Pgk2 gene involves a multifaceted, ordered cascade of epigenetic events that potentiates this locus in preparation for the initiation of transcription.

Previous reports described the rat synapsin 1 promoter as primarily neuron selective. However, ectopic expression of a transgene under the rat synapsin 1 promoter was also detected in testis from some transgenic mouse lines. Here we investigate which cells within the testis express a transgene consisting of the rat synapsin 1 promoter fused with luciferase. Synapsin 1-luciferase expression vectors were introduced into HeLa cells, into TM3 cells derived from mouse testicular Leydig cells, and into one-cell embryos to make transgenic mice. Indirect immunofluorescence suggests that nontransfected TM3 cells do not express endogenous synapsin 1. TM3 stable transfectants, however, expressed luciferase under the direction of the synapsin 1 promoter, in both promoter orientations. HeLa cells displayed only low levels of activity. Transgenic mice carrying the synapsin 1-luciferase construct displayed high levels of luciferase activity in the brain, spinal cord, and testis. Enriched populations of prepuberal types A and B spermatogonia and adult Leydig cells, pachytene spermatocytes, and round spermatids prepared from transgenic mice all displayed substantial luciferase activity. Thus, the rat synapsin 1 promoter can mediate reporter gene expression in neurons and testicular cell types.

Germ cells undergo epigenetic modifications as they develop, which suggests that they may be ideal donors for nuclear transfer (cloning). In this study, nuclei from confirmed embryonic germ cells were used as donors to determine whether they are competent for cloning and at which stage they are most competent. Embryos cloned from migrating 10.5-days-postcoitum (dpc) primordial germ cells (PGCs) showed normal morphological development to midgestation but died shortly thereafter. In contrast, embryos cloned from later-stage germ cells were developmentally delayed at midgestation. Thus, donor germ cell age inversely correlated with the developmental stage attained by cloned embryos. The methylation status of the H19 - and Snrpn -imprinting control regions in germ cell clones paralleled that of the donors, and revealed that demethylation, or erasure of imprints, was already initiated in PGCs at 10.5 dpc and was complete by 13.5 dpc. Similarly, clones derived from male 15.5-dpc germ cells showed increased methylation correlating with the initiation of de novo methylation that resets imprints at this stage, and clones from neonatal germ cells showed nearly complete methylation in the H19 imprinting control region. These results indicate that the epigenetic state of the donor nucleus is retained in cloned embryos, and that germ cells are therefore inadequate nuclear donors for cloning because they are either erasing or resetting epigenetic patterns.

We have cloned and characterized the expression, during spermatogenesis, of three novel zinc finger genes (Zfp94, Zfp95, Zfp96). Analysis of the deduced protein sequences reveals that all three molecules belong to the LeR family (leucine-rich zinc fingers) and that ZFP95 contains a domain homologous to the Krüppel-associated box. All three genes were found expressed at high levels in testis among other tissues, but testis-specific transcripts were observed for Zfp95 and Zfp96. Northern blot analyses of the testis-specific transcripts of Zfp95 and Zfp96 were performed using whole testis RNA as well as RNA isolated from enriched populations of specific spermatogenic cell types. The testis-specific transcript of Zfp95 showed the highest expression in pachytene spermatocytes, while that of Zfp96 was highly expressed in pachytene spermatocytes, in round spermatids and residual bodies. Northern blot analysis of RNA from the testis of mice carrying the atrichosis mutation further validated these expression patterns. In particular, the testis-specific transcripts of Zfp95 and Zfp96 were greatly reduced in heterozygous, and completely absent in homozygous testis RNA from atrichosis mutant mice, further defining the germ cell specificity of these transcripts.

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.

Spermatogonia are the self-renewing, mitotic germ cells of the testis from which sperm arise by means of the differentiation pathway known as spermatogenesis1. By contrast with hematopoietic and other mammalian stem-cell populations, which have been subjects of intense molecular genetic investigation, spermatogonia have remained largely unexplored at the molecular level. Here we describe a systematic search for genes expressed in mouse spermatogonia, but not in somatic tissues. We identified 25 genes (19 of which are novel) that are expressed in only male germ cells. Of the 25 genes, 3 are Y-linked and 10 are X-linked. If these genes had been distributed randomly in the genome, one would have expected zero to two of the genes to be X-linked. Our findings indicate that the X chromosome has a predominant role in pre-meiotic stages of mammalian spermatogenesis. We hypothesize that the X chromosome acquired this prominent role in male germ-cell development as it evolved from an ordinary, unspecialized autosome.

Spermatogenic cells exhibit a lower spontaneous mutation frequency than somatic tissues in a lacI transgene and many base excision repair (BER) genes display the highest observed level of expression in the testis. In this study, uracil-DNA glycosylase-initiated BER activity was measured in nuclear extracts prepared from tissues obtained from each of three mouse strains. Extracts from mixed spermatogenic germ cells displayed the greatest activity followed by liver then brain for all three strains, and the activity for a given tissue was consistent among the three strains. Levels of various BER proteins were examined by western blot analyses and found to be consistent with activity levels. Nuclear extracts prepared from purified Sertoli cells, a somatic component of the seminiferous epithelium, exhibited significantly lower activity than mixed spermatogenic cell-type nuclear extracts, thereby suggesting that the high BER activity observed in mixed germ cell nuclear extracts was not a characteristic of all testicular cell types. Nuclear extracts from thymocytes and small intestines were assayed to assess activity in a mitotically active cell type and tissue. Overall, the order of tissues/cells exhibiting the greatest to lowest activity was mixed germ cells > Sertoli cells > thymocytes > small intestine > liver > brain.

Differences in DNA methylation distinguish the maternal and paternal alleles of many imprinted genes. Allele-specific methylation that is inherited from the gametes and maintained throughout development has been proposed as a candidate imprinting mark. To determine how methylation is established in the germline, we have analyzed the allelic methylation patterns of the maternally expressed, paternally methylated H19 gene during gametogenesis in the mouse embryo. We show here that both parental alleles are devoid of methylation in male and female mid-gestation embryonic germ cells, suggesting that methylation imprints are erased in the germ cells prior to this time. In addition, we demonstrate that the subsequent hypermethylation of the paternal and maternal alleles in the male germline occurs at different times. Although the paternal allele becomes hypermethylated during fetal stages, methylation of the maternal allele begins during perinatal stages and continues postnatally through the onset of meiosis. The differential acquisition of methylation on the parental H19 alleles during gametogenesis implies that the two unmethylated alleles can still be distinguished from each other. Thus, in the absence of DNA methylation, other epigenetic mechanism(s) appear to maintain parental identity at the H19 locus during male germ cell development.

Genomic imprinting is characterized by allele-specific expression of multiple genes within large chromosomal domains1 that undergo DNA replication asynchronously during S phase2,3. Here we show, using both fluorescence in situ hybridization analysis and S-phase fractionation techniques, that differential replication timing is associated with imprinted genes in a variety of cell types, and is already present in the pre-implantation embryo soon after fertilization. This pattern is erased before meiosis in the germ line, and parent-specific replication timing is then reset in late gametogenesis in both the male and female. Thus, asynchronous replication timing is established in the gametes and maintained throughout development, indicating that it may function as a primary epigenetic marker for distinguishing between the parental alleles.

Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase.

The germ cell lineages are among the best characterized of all cell lineages in mammals. This characterization includes precise nomenclature that distinguishes among numerous, often subtle, changes in function or morphology as development and differentiation of germ cells proceed to form the gametes. In male rodents, there are at least 41 distinct cell types that occur during progression through the male germ cell lineage that gives rise to spermatozoa. However, there is one period during male germ cell development-that which occurs immediately following the primordial germ cell stage and prior to the spermatogonial stage-for which the system of precise and informative cell type terminology is not adequate. Often, male germ cells during this period are referred to simply as "gonocytes." However, this term is inadequate for multiple reasons, and it is suggested here that nomenclature originally proposed in the 1970s by Hilscher et al., which employs the terms M-, T1-, and T2-prospermatogonia, is preferable. In this Minireview, the history, proper utilization, and advantages of this terminology relative to that of the term gonocytes are described.

Mammalian genomes encode a large number of small noncoding RNAs (sncRNAs) that play regulatory roles during development and adulthood by affecting gene expression. Several sncRNA species, including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), endogenous small interfering RNAs (endo-siRNAs), and small nucleolar RNAs (snoRNAs), are abundantly expressed in the testis and required for normal testicular development and spermatogenesis. To evaluate global changes in sncRNA expression, the next-generation sequencing (NGS)-based sncRNA transcriptomic analysis has become routine, because it allows rapid determination of the small RNA transcriptome of a particular testicular cell type. However, annotation of small RNA NGS reads can be challenging due to the volume of reads obtained, which is usually in the millions. Therefore, we developed a computer-assisted sncRNA annotation protocol that could identify not only known sncRNAs but also previously uncharacterized ones. Using this protocol, we annotated NGS reads of a Sertoli cell sncRNA library, and we report to our knowledge the first comprehensive annotation of the sncRNA transcriptome of immature murine Sertoli cells. Moreover, the computer-assisted sncRNA annotation pipeline that we report is applicable for annotating NGS reads derived from other cell types and/or sequencing platforms.

Meiosis 1 arresting protein (M1ap) is a novel vertebrate gene expressed exclusively in germ cells of the embryonic ovary and the adult testis. In male mice, M1ap expression, which is present from spermatogonia to secondary spermatocytes, is evolutionarily conserved and has a specific spatial and temporal pattern suggestive of a role during germ cell development. To test its function, mice deficient in M1ap were created. Whereas females had histologically normal ovaries, males exhibited reduced testicular size and a myriad of tubular defects, which led to severe oligozoospermia and infertility. Although some germ cells arrested at the zygotene/pachytene stages, most cells advanced to metaphase I before arresting and entering apoptosis. Cells that reached metaphase I were unable to properly align their chromosomes at the metaphase plate due to abnormal chromosome synapses and failure to form crossover foci. Depending on the state of tubular degeneration, all germ cells, with the exemption of spermatogonia, disappeared; with further deterioration, tubules displaying only Sertoli cells reminiscent of Sertoli cell-only syndrome in humans were observed. Our results uncovered an essential role for M1ap as a novel germ cell gene not previously implicated in male germ cell development and suggest that mutations in M1AP could account for some cases of nonobstructive oligozoospermia in men.

Genetically based diseases constitute a major human health burden, and de novo germline mutations represent a source of heritable genetic alterations that can cause such disorders in offspring. The availability of transgenic rodent systems with recoverable, mutation reporter genes has been used to assess the occurrence of spontaneous point mutations in germline cells. Previous studies using the lacI mutation reporter transgenic mouse system showed that the frequency of spontaneous mutations is significantly lower in advanced male germ cells than in somatic cell types from the same individuals. Here we used this same mutation reporter transgene system to show that female germ cells also display a mutation frequency that is lower than that in corresponding somatic cells and similar to that seen in male germ cells, indicating this is a common feature of germ cells in both sexes. In addition, we showed that statistically significant differences in mutation frequencies are evident between germ cells and somatic cells in both sexes as early as mid-fetal stages in the mouse. Finally, a comparison of the mutation frequency in a general population of early type A spermatogonia with that in a population enriched for Thy-1-positive spermatogonia suggests there is heterogeneity among the early spermatogonial population such that a subset of these cells are predestined to form true spermatogonial stem cells. Taken together, these results support the disposable soma theory, which posits that genetic integrity is normally maintained more stringently in the germ line than in the soma and suggests that this is achieved by minimizing the initial occurrence of mutations in early germline cells and their subsequent gametogenic progeny relative to that in somatic cells.

The use of assisted reproductive technologies (ART) has become increasingly common worldwide and is now responsible for 2–3% of children born in developed countries. Multiple reports have suggested that ART-conceived children are more likely to develop rare epigenetic disorders such as Beckwith-Wiedemann Syndrome or Angelman Syndrome, both of which involve dysregulation of imprinted genes. Anecdotal reports suggest that animals produced with ART that manifest apparent epigenetic defects typically do not transmit these epimutations to subsequent generations when allowed to breed naturally, but this hypothesis has not been directly studied. We analyzed allele-specific DNA methylation and expression at three imprinted genes, H19, Snrpn, and Peg3 , in somatic cells from adult mice generated with the use of intracytoplasmic sperm injection (ICSI), a type of ART. Epimutations were detected in most of the ICSI-derived mice, but not in somatic cells of their offspring produced by natural mating. We examined germ cells from the ICSI mice that exhibited epimutations in their somatic cells and confirmed normal epigenetic reprogramming of the three imprinted genes analyzed. Collectively, these results confirm that ART procedures can lead to the formation of primary epimutations, but while such epimutations are likely to be maintained indefinitely in somatic cells of the ART-derived individuals, they are normally corrected in the germ line by epigenetic reprogramming and thus, not propagated to subsequent generations.

Members of the family of nuclear receptors that include peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) are important mediators of selective gene activation during development and cellular differentiation. In this study, developmentally-specific PPAR and RXR patterns of expression that occur in somatic and germ cell populations in the testis were determined using quantative real-time PCR (qRT-PCR) studies on RNAs that were isolated from StaPut purified mouse germ cells and primary rat Sertoli cells. These qRT-PCR studies indicate that transcripts encoding the PPAR-Alpha (α), -Beta (β), and -Gamma (γ) and RXR -Alpha (α), -Beta (β), and -Gamma (γ) are developmentally expressed in both differentiating germ and Sertoli cells. In further experiments aimed at deciphering the physiological role that PPAR-Gamma (γ) plays in Sertoli cells, 15-day primary rat Sertoli cells were infected with recombinant adenoviral vectors containing PPAR-Gamma (γ) cDNA and PPAR-Gamma (γ) RNAi constructs. Affymetrix microarray analysis and qRT-PCR validation studies using total RNA isolated from these transfected cells indicated that PPAR-Gamma regulates the pattern of expression of key lipid metabolic genes in Sertoli cells.