Male Germ Cell Gene Research Articles

Chromatin remodeler CHD4 establishes chromatin states required for ovarian reserve formation, maintenance, and germ cell survival.

The ovarian reserve defines female reproductive lifespan, which in humans spans decades due to the maintenance of meiotic arrest in non-growing oocytes (NGO) residing in primordial follicles. Unknown is how the chromatin state of NGOs is established to enable long-term maintenance of the ovarian reserve. Here, we show that a chromatin remodeler, CHD4, a member of the Nucleosome Remodeling and Deacetylase (NuRD) complex, establishes chromatin states required for formation and maintenance of the ovarian reserve. Conditional loss of CHD4 in perinatal mouse oocytes results in acute death of NGOs and depletion of the ovarian reserve. CHD4 establishes closed chromatin at regulatory elements of pro-apoptotic genes to prevent cell death and at specific genes required for meiotic prophase I to facilitate the transition from meiotic prophase I oocytes to meiotic arrested NGOs. In addition, CHD4 establishes closed chromatin at the regulatory elements of pro-apoptotic genes in male germ cells, allowing male germ cell survival. These results demonstrate a role for CHD4 in defining a chromatin state that ensures germ cell survival, thereby enabling the long-term maintenance of both female and male germ cells.

Expression profile of spermatogenesis associated genes in male germ cells during postnatal development in mice

Spermatogonial stem cells are self-renewal and differentiate into sperm in post-pubertal mammals. There exists a balance between the self-renewal and differentiation in the testes. Spermatogonial stem cells make up only 0.03% of testicular cells in adult mice. These cells maintain sperm production by differentiating after puberty. Therefore, analyzing the expression of genes associated with spermatogenesis is critical for understanding differentiation. The present study aimed to establish the postnatal period of cells in relation to spermatogenesis. To study the expression of differentiated and undifferentiated marker genes in enriched spermatogonial stem cells, in vitro culture was performed and cells from pup (6–8-day-old) and adult (4-months-old) testicular tissues were isolated. As a result, undifferentiated genes, Pax7, Plzf, GFRa1, Etv5 and Bcl6b , were highly increased in cultured spermaotogonial stem cells compared with pup and adult testicular cells. On the other hands, differentiated gene, c-kit was highly increased in adult testicular cells, Also Stra8 gene was highly increased in pup and adult testicular cells. This study provides a better understanding of spermatogenesis-associated gene expression during postnatal periods.

CDK2 regulates the NRF1/Ehmt1 axis during meiotic prophase I

Meiosis generates four genetically distinct haploid gametes over the course of two reductional cell divisions. Meiotic divisions are characterized by the coordinated deposition and removal of various epigenetic marks. Here we propose that nuclear respiratory factor 1 (NRF1) regulates transcription of euchromatic histone methyltransferase 1 (EHMT1) to ensure normal patterns of H3K9 methylation during meiotic prophase I. We demonstrate that cyclin-dependent kinase (CDK2) can bind to the promoters of a number of genes in male germ cells including that of Ehmt1 through interaction with the NRF1 transcription factor. Our data indicate that CDK2-mediated phosphorylation of NRF1 can occur at two distinct serine residues and negatively regulates NRF1 DNA binding activity in vitro. Furthermore, induced deletion of Cdk2 in spermatocytes results in increased expression of many NRF1 target genes including Ehmt1 We hypothesize that the regulation of NRF1 transcriptional activity by CDK2 may allow the modulation of Ehmt1 expression, therefore controlling the dynamic methylation of H3K9 during meiotic prophase.

Epigenetic modulation of Nrf2 and Ogg1 gene expression in testicular germ cells by methyl parathion exposure

Methyl parathion (Me-Pa) is an oxidizing organophosphate (OP) pesticide that generates reactive oxygen species (ROS) through its biotransformation. Some studies have also suggested that OP pesticides have the capacity to alkylate biomolecules, including DNA. In general, DNA methylation in gene promoters represses transcription. NRF2 is a key transcription factor that regulates the expression of antioxidant, metabolic and detoxifying genes through the antioxidant response element (ARE) situated in promoters of regulated genes. Furthermore, DNA repair genes, including 8-oxoguanine DNA glycosidase (OGG1), have been proposed as NRF2 target genes. Me-Pa exposure produces poor semen quality, genetic and oxidative damage in sperm cells, and reduced fertility. However, the Me-Pa effects on the methylation status and the expression of antioxidant (Nrf2) or DNA repair (Ogg1) genes in male germ cells have not been investigated. Therefore, mice were exposed to Me-Pa to evaluate the global (%5-mC) and specific methylation of Nrf2 and Ogg1 genes using pyrosequencing, gene expression, and total protein carbonylation in male germ cells. The results showed that Me-Pa significantly decreased the global DNA methylation pattern and significantly increased the methylation of two CpG sites within Ogg1 promoter and one CpG site within Nrf2 promoter. In addition, Ogg1 or Nrf2 expression did not change after Me-Pa exposure despite the oxidative damage produced. Altogether, our data suggest that Me-Pa toxicity alters Ogg1 and Nrf2 promoter methylation in male germ cells that may be modulating their gene expression.

The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner

BackgroundCTCF is a highly conserved and essential zinc finger protein expressed in virtually all cell types. In conjunction with cohesin, it organizes chromatin into loops, thereby regulating gene expression and epigenetic events. The function of CTCFL or BORIS, the testis-specific paralog of CTCF, is less clear.ResultsUsing immunohistochemistry on testis sections and fluorescence-based microscopy on intact live seminiferous tubules, we show that CTCFL is only transiently present during spermatogenesis, prior to the onset of meiosis, when the protein co-localizes in nuclei with ubiquitously expressed CTCF. CTCFL distribution overlaps completely with that of Stra8, a retinoic acid-inducible protein essential for the propagation of meiosis. We find that absence of CTCFL in mice causes sub-fertility because of a partially penetrant testicular atrophy. CTCFL deficiency affects the expression of a number of testis-specific genes, including Gal3st1 and Prss50. Combined, these data indicate that CTCFL has a unique role in spermatogenesis. Genome-wide RNA expression studies in ES cells expressing a V5- and GFP-tagged form of CTCFL show that genes that are downregulated in CTCFL-deficient testis are upregulated in ES cells. These data indicate that CTCFL is a male germ cell gene regulator. Furthermore, genome-wide DNA-binding analysis shows that CTCFL binds a consensus sequence that is very similar to that of CTCF. However, only ~3,700 out of the ~5,700 CTCFL- and ~31,000 CTCF-binding sites overlap. CTCFL binds promoters with loosely assembled nucleosomes, whereas CTCF favors consensus sites surrounded by phased nucleosomes. Finally, an ES cell-based rescue assay shows that CTCFL is functionally different from CTCF.ConclusionsOur data suggest that nucleosome composition specifies the genome-wide binding of CTCFL and CTCF. We propose that the transient expression of CTCFL in spermatogonia and preleptotene spermatocytes serves to occupy a subset of promoters and maintain the expression of male germ cell genes.

MicroRNA-449 and MicroRNA-34b/c Function Redundantly in Murine Testes by Targeting E2F Transcription Factor-Retinoblastoma Protein (E2F-pRb) Pathway

MicroRNAs (miRNAs) mainly function as post-transcriptional regulators and are involved in a wide range of physiological and pathophysiological processes such as cell proliferation, differentiation, apoptosis, and tumorigenesis. Mouse testes express a large number of miRNAs. However, the physiological roles of these testicular miRNAs remain largely unknown. Using microarray and quantitative real time PCR assays, we identified that miRNAs of the microRNA-449 (miR-449) cluster were preferentially expressed in the mouse testis, and their levels were drastically up-regulated upon meiotic initiation during testicular development and in adult spermatogenesis. The expression pattern of the miR-449 cluster resembled that of microRNA-34b/c (miR-34b/c) during spermatogenesis. Further analyses identified that cAMP-responsive element modulator τ and SOX5, two transcription factors essential for regulating male germ cell gene expression, acted as the upstream transactivators to stimulate the expression of the miR-449 cluster in mouse testes. Despite its abundant expression in testicular germ cells, miR-449-null male mice developed normally and exhibited normal spermatogenesis and fertility. Our data further demonstrated that miR-449 shared a cohort of target genes that belong to the E2F transcription factor-retinoblastoma protein pathway with the miR-34 family, and levels of miR-34b/c were significantly up-regulated in miR-449-null testes. Taken together, our data suggest that the miR-449 cluster and miR-34b/c function redundantly in the regulation of male germ cell development in murine testes.

Histone crotonylation specifically marks the haploid male germ cell gene expression program

The haploid male germ cell differentiation program controls essential steps of male gametogenesis and relies partly on a significant number of sex chromosome-linked genes. These genes need to escape chromosome-wide transcriptional repression of sex chromosomes, which occurs during meiosis and is largely maintained in post-meiotic cells. A newly discovered histone lysine modification, crotonylation (Kcr), marks X/Y-linked genes that are active in post-meiotic male germ cells. Histone Kcr, by conferring resistance to transcriptional repressors, could be a dominant element in maintaining these genes active in the globally repressive environment of haploid cell sex chromosomes. Furthermore, the same mark was found associated with post-meiotically activated genes on autosomes. Histone Kcr therefore appears to be an indicator of the male haploid cell gene expression program and a notable element of genome programming in the post-meiotic phases of spermatogenesis.

Expression profile of male germ cell‐associated genes in mouse embryonic stem cell cultures treated with all‐trans retinoic acid and testosterone

Cells that morphologically and functionally resemble male germ cells can be spontaneously derived from ES cells. However, this process is inefficient and unpredictable suggesting that the expression pattern of male germ cell associated genes during spontaneous ES cell differentiation does not mimic the in vivo profiles of the genes. Thus, in the present study, the temporal profile of genes expressed at different stages of male germ cell development was examined in differentiating ES cells. The effect of all-trans retinoic acid (RA) which is a known inducer of primordial germ cell (PGC) proliferation/survival in vitro and testosterone which is required for spermatogenesis in vivo on the expression of these genes was also determined. Each of the 12 genes analyzed exhibited one of four temporal expression patterns in untreated differentiating ES cells: progressively decreased (Dppa3, Sycp3, Msy2), initially low and then increased (Stra8, Sycp1, Dazl, Act, Prm1), initially decreased and then increased (Piwil2, Tex14), or relatively unchanged (Akap3, Odf2). RA-treated cells exhibited increased expression of Stra8, Dazl, Act, and Prm1 and suppressed expression of Dppa3 compared to untreated controls. Furthermore, testosterone increased expression of Stra8 while the combination of RA and testosterone synergistically increased expression of Act. Our findings establish a comprehensive profile of male germ cell gene expression during spontaneous differentiation of murine ES cells and describe the capacity of RA and testosterone to modulate the expression of these genes. Furthermore, these data represent an important first step in designing a plausible directed differentiation protocol for male germ cells.

DNA methylation impairs activation of haploid expressed genes in male germ cells.

DNA methylation impairs activation of haploid expressed genes in male germ cells.

Pre-Messenger RNA Cleavage Factor I (CFIm): Potential Role in Alternative Polyadenylation During Spermatogenesis1

A hallmark of male germ cell gene expression is the generation by alternative polyadenylation of cell-specific mRNAs, many of which utilize noncanonical A(A/U)UAAA-independent polyadenylation signals. Cleavage factor I (CFIm), a component of the pre-mRNA cleavage and polyadenylation protein complex, can direct A(A/U)UAAA-independent polyadenylation site selection of somatic cell mRNAs. Here we report that the CFIm subunits NUDT21/CPSF5 and CPSF6 are highly enriched in mouse male germ cells relative to somatic cells. Both subunits are expressed from spermatogenic cell mRNAs that are shorter than the corresponding somatic transcripts. Complementary DNA sequencing and Northern blotting revealed that the shorter Nudt21 and Cpsf6 mRNAs are generated by alternative polyadenylation in male germ cells using proximal poly(A) signals. Both sets of transcripts contain CFIm binding sites within their 3'-untranslated regions, suggesting autoregulation of CFIm subunit formation in male germ cells. CFIm subunit mRNA and protein levels exhibit distinct developmental variation during spermatogenesis, indicating stage-dependent translational and/or posttranslational regulation. CFIm binding sites were identified near the 3' ends of numerous male germ cell transcripts utilizing A(A/U)UAAA-independent sites. Together these findings suggest that CFIm complexes participate in alternative polyadenylation directed by noncanonical poly(A) signals during spermatogenesis.

The transcription factor CREMτ and cAMP regulate promoter activity of the Na,K‐ATPase α4 isoform

The Na,K-ATPase is an essential enzyme of the plasma membrane that plays a key role in numerous cell processes that depend on the transcellular gradients of Na(+) and K(+). Among the various isoforms of the catalytic subunit of the Na,K-ATPase, alpha4 exhibits the most limited pattern of expression, being restricted to male germ cells. Activity of alpha4 is essential for sperm function, and alpha4 is upregulated during spermatogenesis. The present study addressed the transcriptional control of the human Na,K-ATPase alpha4 gene, ATP1A4. We describe that a 5' untranslated region of the ATP1A4 gene (designated -339/+480 based on the ATP1A4 transcription initiation site) has promoter activity in luciferase reporter assays. Computer analysis of this promoter region revealed consensus sites (CRE) for the cyclic AMP (cAMP) response element modulator (CREM). Accordingly, dibutyryl cAMP (db-cAMP) and ectopic expression of CREMtau, a testis specific splice variant of CREM were able to activate the ATP1A4 promoter driven expression of luciferase in HEK 293 T, JEG-3 and GC-1 cells. Further characterization of the effect of db-cAMP and CREMtau on deleted constructs of the ATP1A4 promoter (-339/+80, and +25/+480), and on the -339/+480 region carrying mutations in the CRE sites showed that db-cAMP and CREMtau effect required the CRE motif located 263 bp upstream the transcription initiation site. EMSA experiments confirmed the CRE sequence as a bonafide CREMtau binding site. These results constitute the first demonstration of the transcriptional control of ATP1A4 gene expression by cAMP and by CREMtau, a transcription factor essential for male germ cell gene expression.

A Developmental Switch in Transcription Factor Isoforms During Spermatogenesis Controlled by Alternative Messenger RNA 3'-End Formation1

Spermatogeniccells elaborate a highly specialized differentiation program that is mediated in part by germ cell-enriched transcription factors. This includes a novel member of the sterol response element-binding factor family, SREBF2_v1/SREBP2gc. Somatic SREBFs are predominantly synthesized as precursor proteins and are critical regulators of cholesterol and fatty acid synthesis. In contrast, SREBF2_v1 bypasses the precursor pathway and has been directly implicated in spermatogenic cell-specific gene expression. During spermatogenesis, SREBF2 precursor transcripts predominate in premeiotic stages, while SREBF2_v1 is highly upregulated specifically in pachytene spermatocytes and round spermatids. In the present study, we demonstrate thatSrebf2_v1mRNAs are present in the testis of several mammalian species, including humans. The basis for the stage-dependent transition in SREBF2 isoforms was also investigated. A 3' rapid amplification of cDNA ends (RACE)-PCR analysis of the rat and human revealed thatSrebf2_v1transcripts are generated by alternative pre-mRNA cleavage/polyadenylation. This involves the use of an intronic, A(A/U)UAAA-independent poly(A) signal within intron 7 of theSrebf2gene. Developmentally regulated competition between germ cell factors that control RNA splicing and pre-mRNA cleavage/polyadenylation may underlie this process. These results define an important role for alternative polyadenylation in male germ cell gene expression and development by controlling a stage-dependent switch in transcription factor structure and function during spermatogenesis. TheSrebf2gene thus provides a useful model to explore the role of alternative polyadenylation in regulating stage-dependent functions of important protein regulators in spermatogenic cells.

Expression of a novel, sterol-insensitive form of sterol regulatory element binding protein 2 (SREBP2) in male germ cells suggests important cell- and stage-specific functions for SREBP targets during spermatogenesis.

Cholesterol biosynthesis in somatic cells is controlled at the transcriptional level by a homeostatic feedback pathway involving sterol regulatory element binding proteins (SREBPs). These basic helix-loop-helix (bHLH)-Zip proteins are synthesized as membrane-bound precursors, which are cleaved to form a soluble, transcriptionally active mature SREBP that regulates the promoters for genes involved in lipid synthesis. Homeostasis is conferred by sterol feedback inhibition of this maturation process. Previous work has demonstrated the expression of SREBP target genes in the male germ line, several of which are highly up-regulated during specific developmental stages. However, the role of SREBPs in the control of sterol regulatory element-containing promoters during spermatogenesis has been unclear. In particular, expression of several of these genes in male germ cells appears to be insensitive to sterols, contrary to SREBP-dependent gene regulation in somatic cells. Here, we have characterized a novel isoform of the transcription factor SREBP2, which is highly enriched in rat and mouse spermatogenic cells. This protein, SREBP2gc, is expressed in a stage-dependent fashion as a soluble, constitutively active transcription factor that is not subject to feedback control by sterols. These findings likely explain the apparent sterol-insensitive expression of lipid synthesis genes during spermatogenesis. Expression of a sterol-independent, constitutively active SREBP2gc in the male germ line may have arisen as a means to regulate SREBP target genes in specific developmental stages. This may reflect unique roles for cholesterol synthesis and other functional targets of SREBPs during spermatogenesis.

Where Do Babies Come From?

T his is a question parents inevitably hear from their children. It's hard enough to find an answer that will satisfy a 4-year-old, much less a reproductive biologist. Understanding the basic biology of reproduction, researchers hope, will guide treatments for infertility, pregnancy-related disorders, and diseases of the reproductive tract, as well as aid in the development of contraceptives, hormone replacements, and methods for assisted reproductive technology (ART). This reproductive biology special issue assembles articles from several active areas of research. Brinster (p. [2174][1]) describes male germ-cell transplantation, a method that can be used in the study of stem cell biology and infertility and in transgenesis. Sassone-Corsi (p. [2176][2]) illustrates the special case of male germ-cell gene expression and how this mechanism differs from that seen in all the other cells of the body. Matzuk et al. (p. [2178][3]) shift attention to the female germ cell and describe the communication between the oocyte and surrounding somatic cells. In order to contribute the appropriate chromosomal complement, germ cells undergo meiosis. Hunt and Hassold (p. [2181][4]) explain sexual dimorphism in meiotic disruption and its consequences. ![Figure][5] CREDIT: RUBBERBALL PHOTOGRAPHY Once germ cells are fully differentiated, fertilization can take place. Primakoff and Myles (p. [2183][6]) outline the fertilization process. After egg and sperm fusion, a blastocyst develops; Paria et al. (p. [2185][7]) describe the molecular events of its implantation. When normal fertilization is impeded, couples often turn to ART. Schultz and Williams (p. [2188][8]) outline recent advances but also warn that basic science must be applied to make protocols safer and more effective. In a related Editorial, Braude (p. [2101][9]) cautions that the success of ART should not be measured only in terms of live births; the health of the children must be considered. Reproduction is a perilous process. In the News section, Jon Cohen (p. [2164][10]) examines the most common cause of miscarriage—aneuploidy—and describes some of the latest techniques aimed at addressing the problem. Even successful pregnancies can leave babies at risk of certain diseases; Jennifer Couzin (p. [2167][11]) reports that disruptions in a fetus's environment can reverberate 50 years later in a propensity for heart disease and other adult ailments. Likewise, some of the physical costs of bearing a child aren't apparent for decades. Marcia Barinaga (p. [2169][12]) explores microchimerism: Cells exchanged during pregnancy can live on indefinitely in the mother and child, spurring autoimmune and other diseases. Those who choose not to reproduce are mostly stuck with contraceptive methods that have been around for a generation; Constance Holden (p. [2172][13]) finds that few new strategies are being researched, despite a growing need for appropriate contraceptives, particularly in the developing world. It is unlikely that we will come up with a satisfactory answer to the 4-year-old's question of where babies come from, for any response is sure to be followed by, “But why?” Similarly, as each hypothesis about reproduction is tested, new questions spring to mind. [1]: /lookup/doi/10.1126/science.1071607 [2]: /lookup/doi/10.1126/science.1070963 [3]: /lookup/doi/10.1126/science.1071965 [4]: /lookup/doi/10.1126/science.1071907 [5]: pending:yes [6]: /lookup/doi/10.1126/science.1072029 [7]: /lookup/doi/10.1126/science.1071601 [8]: /lookup/doi/10.1126/science.1071741 [9]: /lookup/doi/10.1126/science.296.5576.2101 [10]: /lookup/doi/10.1126/science.296.5576.2164 [11]: /lookup/doi/10.1126/science.296.5576.2167 [12]: /lookup/doi/10.1126/science.296.5576.2169 [13]: /lookup/doi/10.1126/science.296.5576.2172

Chronic cyclophosphamide treatment alters the expression of stress response genes in rat male germ cells.

Increases in the survival rate of men treated with chemotherapeutic drugs and their desire to have children precipitate concerns about the effects of these drugs on germ cells. Azoospermia, oligospermia, and infertility are common outcomes resulting from treatment with cyclophosphamide, an alkylating agent. Exposure of male rats to cyclophosphamide results in dose-dependent and time-specific adverse effects on progeny outcome. Elucidation of the effects of chronic low-dose cyclophosphamide treatment on the expression of stress response genes in male germ cells may provide insight into the mechanisms underlying such adverse effects. Male rats were gavaged with saline or cyclophosphamide (6 mg/kg) for 4-5 wk; pachytene spermatocytes, round spermatids, and elongating spermatids were isolated; RNA was extracted and probed on cDNA arrays containing 216 cDNAs. After saline treatment, 125 stress response genes were expressed in pachytene spermatocytes (57% of genes studied), 122 in round spermatids (56%), and 83 in elongating spermatids (38%). Cyclophosphamide treatment reduced the number of genes detected in all germ cell types. The predominant effect of chronic cyclophosphamide exposure was to decrease the expression level of genes in pachytene spermatocytes (34% of genes studied), round spermatids (29%), and elongating spermatids (4%). In elongating spermatids only, drug treatment increased the expression of 8% of the genes studied. The expression profiles of genes involved in DNA repair, posttranslational modification, and antioxidant defense in male germ cells were altered by chronic cyclophosphamide treatment. We hypothesize that the effects of cyclophosphamide exposure on germ cell gene expression during spermatogenesis may have adverse consequences on male fertility and progeny outcome.

Male germ cell gene expression.

Formation of the male gamete occurs in sequential mitotic, meiotic, and postmeiotic phases. Many germ cell-specific transcripts are produced during this process. Their expression is developmentally regulated and stage specific. Some of these transcripts are product of genes that are male germ cell-specific homologs of genes expressed in somatic cells, while some are expressed from unique genes unlike any others in the genome. Others are alternate transcripts derived from the same gene as transcripts in somatic cells but differing from them in size and/or overall sequence. They are generated during gene expression by using promoters and transcription factors that activate transcription at different start sites upstream or downstream of the usual site, by incorporation of alternate exons, by germ cell-specific splicing events, and by using alternate initiation sites for polyadenylation. Male germ cell development consists of an assortment of unique processes, including meiosis, genetic recombination, haploid gene expression, formation of the acrosome and flagellum, and remodeling and condensation of the chromatin. These processes are intricate, highly ordered, and require novel gene products and a precise and well-coordinated program of gene expression to occur. The regulation of gene expression in male germ cells occurs at three levels: intrinsic, interactive, and extrinsic. A highly conserved genetic program "intrinsic" to germ cells determines the sequence of events that underlies germ cell development. This has been underscored by recent studies showing that meiosis involves many genes that have been conserved during evolution from yeast to man. During meiosis and other processes unique to germ cells, the intrinsic program determines which genes are utilized and when they are expressed. In the postmeiotic phase, it coordinates the expression of genes whose products are responsible for constructing the sperm. The process of spermatogenesis occurs in overlapping waves, with cohorts of germ cells developing in synchrony. The intrinsic program operating within a particular germ cell requires information from and provides information to neighboring cells to achieve this coordination. Sertoli cells are crucial for this "interactive" process as well as for providing essential support for germ cell proliferation and progression through the phases of development. The interactive level of regulation is dependent on "extrinsic" influences, primarily testosterone and follicle-stimulating hormone (FSH). Studies during the last 4 years have established that FSH is not essential for germ cell development but instead serves an important supportive role for this process. While testosterone is essential for maintenance of spermatogenesis, it acts on Sertoli cells and peritubular cells and has indirect effects on germ cells. The extrinsic and interactive processes are extremely important for establishing and maintaining an optimum environment within which gametogenesis occurs. Nevertheless, an intrinsic evolutionarily conserved genetic program regulates male germ cell gene expression and development.