Expression Of Germ Cell-specific Genes Research Articles

Effects of trigonelline, diosgenin, and Cistanche deserticola polysaccharide on the culture of female germline stem cells in vitro

Female germline stem cells (FGSCs) are renewable sources of oocytes that play an indispensable role in re-establishing mammal fertility. Here, we have established FGSCs from neonatal mice, which exhibit characteristics of germline stem cells. We show that compared with monomeric trigonelline and diosgenin, macromolecular compounds Cistanche deserticola polysaccharides (CDPs) in Chinese herbal medicine can enhance the ability of FGSCs to differentiate into oocytes at appropriate concentrations while maintaining self-renewal in vitro. In contrast, trigonelline and diosgenin inhibited the expression of germ cell-specific genes while reducing cell proliferation activity. In summary, CDPs could induce the differentiation and self-renewal of FGSCs in vitro. The comparison of the effects of the active components of different types of Chinese medicine will provide a reference for the development of clinical drugs in the future, and help to elucidate the development process of FGSCs.

Isolation and characterisation of promoters from mouse genome to drive post-meiotic germ cell-specific robust gene expression for functional genomics studies

The generation of spermatozoa from developing germ cells through mitotic and meiotic divisions is a highly regulated and complex process. Any defect in this process, may lead to subfertility/infertility. The role of different transcripts (mRNA/miRNA/lncRNA) in regulation of the pre-meiotic, meiotic, and post-meiotic stages of spermatogenesis are being proposed based on various multiomics based approaches. Such differential gene-expression is regulated by promoter elements that are activated in a stage specific manner. To determine the role of these differentially expressed transcripts in the process of meiosis, a robust post-meiotic germ cell specific promoter is required. In the present study, we have isolated and characterized the expression of the mouse Proacrosin, SP10, and ELP promoters for driving post-meiotic germ cell specific gene-expression. Promoter regions of all these 3 genes were isolated and cloned to generate mammalian expression vector. The transgene expression in post-meiotic germ cells was assessed in mice using the testicular electroporation method in vitro as well as in vivo, using above promoters. It was also validated in goat seminiferous tubules, in vitro. We have also carried out a comparative analysis of the strength of these promoters to confirm their robustness that indicated Proacrosin to be the most robust promoter that can be useful for diving post-meiotic germ cells specific gene-expression. These promoters can be used to alter gene-expression specifically in post-meiotic germ cells for deciphering the role(s) of germ cell genes in spermatogenic progression or for expressing various genome editing tools for engineering the germ cell genome to understand basis of subfertility/infertility.

Cryopreservation of testicular cell in striped catfish (Pangasianodon hypophthalmus) and its effects on apoptosis, germ-cell specific gene expression and germ cell transplantability

Cryopreservation of testicular tissue would provide a useful tool to maintain spermatogonia, a precursor of gametes, for fish regeneration by germ cell transplantation. This study aimed to develop a cryopreservation method for spermatogonia by freezing the testicular tissue of striped catfish (Pangasianodon hypophthalmus) and testing whether cryopreserved spermatogonia exhibit transplantability. Freezing cryomedium containing L-15 medium and 1.3 M DMSO yielded the highest spermatogonia recovery rate after thawing. Supplementation with glucose and non-permeating cryoprotectants was not necessary to improve the recovery rate. We determined that an optimised equilibration time for cryopreserved testes in cryomedium included 60 min of equilibrium on ice before slow freezing (−1 °C/min), and subsequent thawing at 10 °C for 4–8 min. An Annexin V affinity assay and flow cytometry analysis were performed to identify apoptosis in cryopreserved testicular cells. Our results showed that cryoinjury of spermatogonia during cryopreservation could lead to apoptosis. Additionally, to determine the characteristics of cryopreserved spermatogonia at the molecular level, RT-qPCR was performed to quantify the expression of cxcr4b, dazl, dnd1, dnd2, nanos1, nanos2, and vasa. The results showed that compared to fresh testicular cells, frozen cells exhibited lower expression levels of cxcr4b and dazl. However, there were no significant differences in dnd1, dnd2, nanos1, and nanos2 expression levels. Although the expression level of vasa was lower in cryopreserved testes, both fresh and cryopreserved testicular cells showed positive vasa expression using in situ hybridisation. Furthermore, cryopreserved spermatogonia were transplanted into allogeniec recipient larvae and compared with fresh spermatogonia. Although the incorporation rate was lower than that of fresh spermatogonia, cryopreserved spermatogonia could be incorporated into the genital ridge of recipient larvae, demonstrating the transplantability of cryopreserved spermatogonia. Taken together, a method for cryopreservation of testicular tissue of striped catfish was optimised, and the cryopreserved testicular cells retained several spermatogenic stem cell characteristics, as well as transplantability.

Comparative effects of retinoic acid, granulosa cells conditioned medium or forskolin in combination with granulosa cell co-culturing on mouse germ cell differentiation.

Devising of an appropriate in vitro culture method for germ cells differentiation in the presence of soluble factors has attracted considerable attention, which results will provide new insight into reproductive biology. In this study, we compared the effects of forskolin, retinoic acid (RA) or granulosa cell-conditioned medium in the presence or absence of granulosa cell co-culturing on germ cell differentiation from embryonic stem cells (ESCs). Embryonic stem cells were differentiated using embryoid bodies (EBs) for 5 days, and then EB-derived cells were co-cultured with or without adult mouse granulosa cells using monolayer protocol and treated with 50 µM forskolin, 1 µM RA and 50% granulosa cell-conditioned medium for 4 days. Granulosa cell-conditioned medium significantly increased the levels of Scp3, Rec8, Mvh and Gdf9 expression in the granulosa cell co-culture method compared to untreated cells. A significant elevation of Stra8, Rec8 and Mvh was observed after treatment with RA in the absence of granulosa cells and there was no significant increase in the levels of expression of germ cell-specific genes after treatment with forskolin compared to control. Furthermore, forskolin and RA significantly increased viability and proliferation of germ-like cells, compared with granulosa cell-conditioned medium. Our study revealed that granulosa cell-conditioned medium and RA effectively can induce germ cell differentiation from ESCs, however combined application of granulosa cell-conditioned medium and co-culturing with granulosa cells had synergic effect on germ cell development in vitro as optimized protocol.

Isolation and in vitro culture of ovarian stem cells in Chinese soft-shell turtle (Pelodiscus sinensis).

Gonadal cell lines provide valuable tools for studying gametogenesis, sex differentiation, and manipulating germ cells in reproductive biology. Female germline stem cells have been characterized and isolated from ovaries of mammalian species, including mice and human, but there has been very few studies on female germline stem cells in reptiles. Here, we described an ovarian stem cell-like line isolated and cultured from the Chinese soft-shell turtle (Pelodiscus sinensis), designated as PSO1. The cells showed high alkaline phosphatase activity with a normal diploid karyotype. As shown by reverse transcription-polymerase chain reaction, the cells were positive for the expression of germ cell-specific genes, vasa and dazl, as well as a stem cell marker, nanog, but negative for the expression of the folliculogenesis-specific gene, figla. Likewise, through fluorescent immunostaining analyses, both the Dazl and Vasa proteins were detected abundantly in the cytoplasm of perinuclear region, whereasNanog and PCNA were dominantly observed in the nuclei in PSO1 cells. Moreover, PSO1 cells transfected with pCS2:h2b-egfp could properly express the fusion protein in the nuclei. Taken together, the findings suggested that the germline stem cells exist in the ovary of juvenile Chinese soft-shell turtle and these cells can be isolated for a long-term in vitro culture under experimental conditions. This study has provided a valuable basis for further investigations on the molecular mechanisms whereby the germline stem cells develop and differentiate into gametes in turtles. Also, it has paved the way for studies on oogenesis in turtles, even in the other reptiles.

Massive expression of germ cell-specific genes is a hallmark of cancer and a potential target for novel treatment development

Cancer cells have been found to frequently express genes that are normally restricted to the testis, often referred to as cancer/testis (CT) antigens or genes. Because germ cell-specific antigens are not recognized as “self” by the innate immune system, CT-genes have previously been suggested as ideal candidate targets for cancer therapy. The use of CT-genes in cancer therapy has thus far been unsuccessful, most likely because their identification has relied on gene expression in whole testis, including the testicular somatic cells, precluding the detection of true germ cell-specific genes. By comparing the transcriptomes of micro-dissected germ cell subtypes, representing the main developmental stages of human spermatogenesis, with the publicly accessible transcriptomes of 2617 samples from 49 different healthy somatic tissues and 9232 samples from 33 tumor types, we here discover hundreds of true germ cell-specific cancer expressed genes. Strikingly, we found these germ cell cancer genes (GC-genes) to be widely expressed in all analyzed tumors. Many GC-genes appeared to be involved in processes that are likely to actively promote tumor viability, proliferation and metastasis. Targeting these true GC-genes thus has the potential to inhibit tumor growth with infertility being the only possible side effect. Moreover, we identified a subset of GC-genes that are not expressed in spermatogonial stem cells. Targeting of this GC-gene subset is predicted to only lead to temporary infertility, as untargeted spermatogonial stem cells can recover spermatogenesis after treatment. Our GC-gene dataset enables improved understanding of tumor biology and provides multiple novel targets for cancer treatment.

Retinoic acid promotes expression of germline-specific genes in chicken blastoderm cells by stimulating Smad1/5 phosphorylation in a feeder-free culture system

BackgroundProducing transgenic chickens with chicken blastodermal cells (cBCs) is inefficient due to the extremely low germline transmission capacity of cBCs. As chicken primordial germ cells (PGCs) have been reported as an efficient method for producing transgenic chickens, the inefficiency of cBCs could potentially be resolved by inducing them to differentiate into germ cells. However, whether chemical inducers are able to enhance cBCs germline competence in vitro is unknown and the molecular mechanisms of differentiation of chicken pluripotent cells into germ cells are poorly understood.ResultsWe cultured cBCs with a monolayer morphology in E8 medium, a xeno- and feeder-free medium. We showed that retinoic acid (RA) treatment increased expression of germ cell-specific genes in cBCs. Using western blot, we determined that RA stimulated Smad1/5 phosphorylation. Moreover, Smad1/5 activation regulates the expression of germ cell-specific genes, as co-treatment with a Smad1/5 phosphorylation inhibitor or activator alters expression of these genes. We also demonstrate that Smad1/5 is required for RA-induced differentiation by RNA interference knockdown.ConclusionOur results demonstrated that E8 medium is able to maintain cBC growth for weeks and RA treatment induced germ cell differentiation of cBCs through the BMP-Smad1/5 signaling pathway.

Induction of male germ cell-like lineage from chicken fetal bone marrow stem cells with chicken testis extract

Bone marrow-derived stem cells (BMCs) are able to differentiate into multilineage cells such as muscle, bone, cartilage, fat, and nerve cells. In the present study, we investigated the differentiation capability of chicken BMCs into germ cells by using retinoic acid (RA) and chicken testis extract (chTE). The chicken BMCs were isolated from fetal chicken femurs on post-fertilization day 20, cultured in vitro, and treated with RA and chTE, respectively. The cultured chicken BMCs displayed fibroblast-like morphology and were positive for mesenchymal-specific markers such as CD44, CD90, and CD105 at the mRNA level. RT-PCR and immunocytostaining revealed that both RA and chTE treatments induced the expression of early-germ-cell markers such as Stra8, Dazl and DDX4. The increase of germ cell-specific gene expression after chTE treatment indicates that testicular environment-derived proteins may induce in vitro germ-cells. In addition, we performed a microarray analysis to identify differentially expressed genes (DEGs) in RA and chTE, respectively. A total of 1,629 DEGs were obtained and the chTE treatment showed very lower numbers of DEGs than the RA treatment. Collectively, our results indicate that chicken BMCs have the potential to differentiate to male germ cells in vitro with testis derived proteins.

Epigenetic profiles signify cell fate plasticity in unipotent spermatogonial stem and progenitor cells.

Spermatogonial stem and progenitor cells (SSCs) generate adult male gametes. During in vitro expansion, these unipotent murine cells spontaneously convert to multipotent adult spermatogonial-derived stem cells (MASCs). Here we investigate this conversion process through integrative transcriptomic and epigenomic analyses. We find in SSCs that promoters essential to maintenance and differentiation of embryonic stem cells (ESCs) are enriched with histone H3-lysine4 and -lysine 27 trimethylations. These bivalent modifications are maintained at most somatic promoters after conversion, bestowing MASCs an ESC-like promoter chromatin. At enhancers, the core pluripotency circuitry is activated partially in SSCs and completely in MASCs, concomitant with loss of germ cell-specific gene expression and initiation of embryonic-like programs. Furthermore, SSCs in vitro maintain the epigenomic characteristics of germ cells in vivo. Our observations suggest that SSCs encode innate plasticity through the epigenome and that both conversion of promoter chromatin states and activation of cell type-specific enhancers are prominent features of reprogramming.

Oocyte Development and Loss

Soon after implantation, cell lineages bifurcate into future somatic and germ cells. Canonical expression of germ cell-specific genes continues during and after their migration to the gonadal ridge, where, after further cell cycles, they enter meiosis, form syncytial clusters, and arrest at diplotene for folliculogenesis. The balance between cell survival and death leaves an ovarian follicular reserve as a legacy.

Large, Male Germ Cell-Specific Hypomethylated DNA Domains With Unique Genomic and Epigenomic Features on the Mouse X Chromosome

To understand the epigenetic regulation required for germ cell-specific gene expression in the mouse, we analysed DNA methylation profiles of developing germ cells using a microarray-based assay adapted for a small number of cells. The analysis revealed differentially methylated sites between cell types tested. Here, we focused on a group of genomic sequences hypomethylated specifically in germline cells as candidate regions involved in the epigenetic regulation of germline gene expression. These hypomethylated sequences tend to be clustered, forming large (10 kb to ∼9 Mb) genomic domains, particularly on the X chromosome of male germ cells. Most of these regions, designated here as large hypomethylated domains (LoDs), correspond to segmentally duplicated regions that contain gene families showing germ cell- or testis-specific expression, including cancer testis antigen genes. We found an inverse correlation between DNA methylation level and expression of genes in these domains. Most LoDs appear to be enriched with H3 lysine 9 dimethylation, usually regarded as a repressive histone modification, although some LoD genes can be expressed in male germ cells. It thus appears that such a unique epigenomic state associated with the LoDs may constitute a basis for the specific expression of genes contained in these genomic domains.

Identification of germ cell-specific genes in mammalian meiotic prophase

BackgroundMammalian germ cells undergo meiosis to produce sperm or eggs, haploid cells that are primed to meet and propagate life. Meiosis is initiated by retinoic acid and meiotic prophase is the first and most complex stage of meiosis when homologous chromosomes pair to exchange genetic information. Errors in meiosis can lead to infertility and birth defects. However, despite the importance of this process, germ cell-specific gene expression patterns during meiosis remain undefined due to difficulty in obtaining pure germ cell samples, especially in females, where prophase occurs in the embryonic ovary. Indeed, mixed signals from both germ cells and somatic cells complicate gonadal transcriptome studies.ResultsWe developed a machine-learning method for identifying germ cell-specific patterns of gene expression in microarray data from mammalian gonads, specifically during meiotic initiation and prophase. At 10% recall, the method detected spermatocyte genes and oocyte genes with 90% and 94% precision, respectively. Our method outperformed gonadal expression levels and gonadal expression correlations in predicting germ cell-specific expression. Top-predicted spermatocyte and oocyte genes were both preferentially localized to the X chromosome and significantly enriched for essential genes. Also identified were transcription factors and microRNAs that might regulate germ cell-specific expression. Finally, we experimentally validated Rps6ka3, a top-predicted X-linked spermatocyte gene. Protein localization studies in the mouse testis revealed germ cell-specific expression of RPS6KA3, mainly detected in the cytoplasm of spermatogonia and prophase spermatocytes.ConclusionsWe have demonstrated that, through the use of machine-learning methods, it is possible to detect germ cell-specific expression from gonadal microarray data. Results from this study improve our understanding of the transition from germ cells to meiocytes in the mammalian gonad. Further, this approach is applicable to other tissues for which isolating cell populations remains difficult.

Effect of Zinc Ions on Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells to Male Germ Cells and Some Germ Cell-Specific Gene Expression in Rams

This is the first report to describe the effects of zinc (Zn) ions on the expression of germ cell (GC) genes from bone marrow-derived mesenchymal stem cells (BM-MSCs). Zn plays an important role in germinal epithelium maintenance, testosterone secretion, differentiation of GCs, and spermatogenesis. In addition, several studies have suggested that MSCs have the potential for differentiation into numerous cells types, including male GCs. In this study, we have treated passage-3 ram BM-MSCs with 0.14 μg/ml Zn sulfate (ZnSO₄) for a period of 21 days with the intent to determine whether Zn treatment can stimulate MSCs to differentiate into male GCs in vitro. We also sought to determine the type of changes seen in MSCs by Zn treatment. Differentiation into male GCs was evaluated by the assessment of expressions of the following GC-specific markers: VASA, PIWIL2, OCT4, beta1 INTEGRIN (ITG b1), DAZL (by reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR), and PGP 9.5 (by immunocytochemistry). Also studied were morphological characteristics and changes in alkaline phosphatase activity. Interestingly, Zn upregulated the expressions of VASA and ITG b1 but downregulated PIWIL2 and OCT4. DAZL and PGP 9.5 were not expressed in the treatment group. According to our results, Zn ions did not stimulate BM-MSCs to transdifferentiate into male GCs; however, it changed the expression of GC genes in BM-MSCs. It can be concluded that a possible mechanism by which Zn ions can increase male fertility is by regulation of the expression of testis GC-specific genes in the differentiation process and spermatogenesis.

An epigenetic mechanism regulates germ cell-specific expression of the porcine Deleted in Azoospermia-Like ( DAZL) gene

The Deleted in Azoospermia-Like ( DAZL) gene is specifically expressed in fetal and adult gonads. While DAZL is known to play a role during gametogenesis, the mechanisms governing its germ cell-specific expression remain unclear. We identified the 5′ untranslated region (UTR) of the porcine DAZL gene and cloned and characterized 2 kilobase pairs of its TATA-less 5′ flanking region, identifying CpG-rich regions within the proximal promoter. Nine of 18 CpG sites in proximity to one region were largely unmethylated in germ cells but hypermethylated in somatic cells, suggesting that DNA methylation may regulate DAZL promoter activity. Furthermore, DAZL expression was induced in fibroblasts treated with a demethylating agent. Deletion analyses revealed that the minimal 149 base pair promoter region was sufficient to activate transcription. In vitro methylation of a reporter construct corresponding to these 149 base pairs resulted in complete suppression of DAZL promoter activity in primordial germ cells, further supporting a role for methylation in regulating DAZL expression. Interestingly, the differentially methylated region was shown to harbor several putative Sp1-binding sites. Mutation of only the most highly conserved site significantly reduced promoter activity in a reporter assay. Furthermore, gel shift assays revealed that Sp1 was able to specifically bind to this site, and that complex formation was inhibited when CpG dinucleotides within this region were methylated. Chromatin immunoprecipitation (ChIP) assays revealed that in vivo Sp1 binding to the core DAZL promoter region was enriched in germ cells but not in fibroblasts. Our data suggests that DNA methylation may suppress DAZL expression in somatic cells by interfering with Sp1 binding. This study provides insights into the potential mechanisms underlying the regulation of germ cell-specific gene expression.

Germ cell‐specific expression of GFP gene induced by chicken vasa homologue (Cvh) promoter in early chicken embryos

In the present study, we cloned the promoter sequence of the chicken vasa homologue (Cvh) gene, and observed the induction of germ cell-specific gene expression regulated by Cvh promoter in vitro and in vivo. The 2,718-bp Cvh promoter sequence involved putative binding sites of transcription factors such as Oct-1, Nkx2-5, HNF-4 and v-Myb, and a putative CpG island. The reporter vectors, which expressed humanized recombinant green fluorescent protein (hrGFP) under the control of one of three lengths (827, 1,555 and 2,718 bp) of Cvh promoter, were transfected to the dispersed gonadal cells of 7-day incubated embryos in vitro. Every length of promoter-induced germ cell-specific hrGFP expression was verified by immunohistochemistry for germ cell-specific antigens, and the intensity of fluorescence was significantly higher in the transfected gonadal cells using 1,555- and 2,718-bp Cvh promoter compared with 827-bp promoter. These results suggested that the germ cell-specific and enhanced gene expression required at least a 1,555-bp sequence of the 5' flanking region of Cvh gene. In vivo transfection of the reporter vector with 1,555-bp Cvh promoter, which was performed by injecting liposome-DNA complex into the blood vessels of 2-day incubated embryos, also induced germ cell-specific hrGFP expression. The Cvh promoter could provide the basis for a methodology for the visualization, purification and genetical modification of germ cells, and might further contribute to our understanding of the development, proliferation, migration and differentiation of germ cells in the chicken.

CAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis.

Promoters, including neither TATA box nor initiator, have been frequently found in testicular germ cell-specific genes in mice. These investigations imply that unique forms of the polymerase II transcription initiation machinery play a role in selective activation of germ cell-specific gene expression programs during spermatogenesis. However, there is little information about testis-specific core promoters, because useful germ cell culture system is not available. In this study, we characterize the regulatory region of the haploid-specific Oxct2b gene in detail by using in vivo transient transfection assay in combination with a transgenic approach, with electrophoretic mobility shift and chromatin immunoprecipitation assays. Expression studies using mutant constructs demonstrate that a 34 bp region, which extends from −49 to −16, acts as a core promoter in an orientation-dependent manner. This promoter region includes the cAMP-responsive element (CRE)-like sequence TGACGCAG, but contains no other motifs, such as a TATA box or initiator. The CRE-like element is indispensable for the core promoter activity, but not for activator in testicular germ cells, through the binding of a testis-specific CRE modulator transcription factor. These results indicate the presence of alternative transcriptional initiation machinery for cell-type-specific gene expression in testicular germ cells.

Meiotic and epigenetic aberrations inDnmt3L-deficient male germ cells

The DNA methyltransferase-like protein Dnmt3L is necessary for the establishment of genomic imprints in oogenesis and for normal spermatogenesis (Bourc'his et al., 2001; Hata et al., 2002). Also, a paternally imprinted gene, H19, loses DNA methylation in Dnmt3L-/- spermatogonia (Bourc'his and Bestor, 2004; Kaneda et al., 2004). To determine the reason for the impaired spermatogenesis in the Dnmt3L-/- testes, we have carried out a series of histological and molecular studies. We show here that Dnmt3L-/- germ cells were arrested and died around the early meiotic stage. A microarray-based gene expression-profiling analysis revealed that various gonad-specific and/or sex-chromosome-linked genes were downregulated in the Dnmt3L-/- testes. In contrast, expression of retrovirus-like intracisternal A-particle (IAP) sequences was upregulated; consistent with this observation, a specific IAP copy showed complete loss of DNA methylation. These findings indicate that Dnmt3L regulates germ cell-specific gene expression and IAP suppression, which are critical for male germ cell proliferation and meiosis.

Novel role for a sterol response element binding protein in directing spermatogenic cell-specific gene expression.

Sperm are highly specialized cells, and their formation requires the synthesis of a large number of unique mRNAs. However, little is known about the transcriptional mechanisms that direct male germ cell differentiation. Sterol response element binding protein 2gc (SREBP2gc) is a spermatogenic cell-enriched isoform of the ubiquitous transcription factor SREBP2, which in somatic cells is required for homeostatic regulation of cholesterol. SREBP2gc is selectively enriched in spermatocytes and spermatids, and, due to its novel structure, its synthesis is not subject to cholesterol feedback control. This suggested that SREBP2gc has unique cell- and stage-specific functions during spermatogenesis. Here, we demonstrate that this factor activates the promoter for the spermatogenesis-related gene proacrosin in a cell-specific manner. Multiple SREBP2gc response elements were identified within the 5'-flanking and proximal promoter regions of the proacrosin promoter. Mutating these elements greatly diminished in vivo expression of this promoter in spermatogenic cells of transgenic mice. These studies define a totally new function for an SREBP as a transactivator of male germ cell-specific gene expression. We propose that SREBP2gc is part of a cadre of spermatogenic cell-enriched isoforms of ubiquitously expressed transcriptional coregulators that were specifically adapted in concert to direct differentiation of the male germ cell lineage.

Cyclic adenosine 3',5'-monophosphate-responsive element modulator gene expression in germ cells of normo- and oligoazoospermic men.

In about one third of infertile men the cause of impaired spermatogenesis is not known. Spermatogenesis appears to be mediated at least in part by the pituitary gonadotropins, which activate the cAMP-dependent signaling pathway. The end point of this pathway is the activation of nuclear transcription factors, such as cAMP-responsive element-binding protein and cAMP-responsive element modulator (CREM). These factors, upon binding to gene sequences identified as cAMP response elements, modulate the expression of germ cell-specific genes that, in turn, promote the completion of spermatogenesis. The expressions of the cAMP-responsive element-binding protein and CREM genes create different isoforms, which can be divided into two groups: activators or repressors of gene regulation. Only CREM repressors are expressed in premeiotic germ cells in mice, whereas a switch to the expression of the CREM activator tau is observed from postmeiotic germ cells onward. Completion of germ cell maturation appears to be dependent on this phenomenon. Recently, mice lacking CREM gene expression have been generated. These animals were infertile and presented a developmental arrest of germ cell maturation at the stage of early spermatid. In this report we demonstrate that CREM gene expression also occurs in human germ cells. In particular, we determined by RT-PCR that a switch from the expression of CREM repressors to CREM activators is present in postmeiotic germ cells in normospermic men. Conversely, in oligoazoospermic patients only the expression of CREM repressors was detected. These data were confirmed by in situ hybridization studies in which transcripts for CREM activators were detected in postmeiotic germ cells in testis specimens showing conserved spermatogenesis, but not in specimens showing maturation arrest at the spermatid stage. Thus, our results indicate that the lack of a switch in the expression of CREM gene isoforms may be related to impaired spermatogenesis in humans.

Pre- and Postmeiotic Expression of Male Germ Cell-Specific Genes Throughout 2-Week Cocultures of Rat Germinal and Sertoli Cells1

The present study was aimed at examining, by reverse transcription polymerase chain reaction, the expression of germ cell-specific genes in cocultures of Sertoli cells with either pachytene spermatocytes (PS) or round spermatids (RS). In situ hybridization studies showed that the mRNAs encoding phosphoprotein p19 and the testis-specific histone TH2B were specifically expressed in PS whereas those encoding the transition proteins TP1 and TP2 were specific to RS. This resulted in p19:TP1 and TH2B:TP2 ratios that were much higher in PS fractions than in RS fractions prepared by elutriation. When PS or RS were seeded on Sertoli cell monolayers in bicameral chambers, both the number and the viability of the cells decreased during the coculture. However, both parameters were equal to, or higher than, 60% after 2 wk. In PS-Sertoli cell cocultures, the ratios of p19:TP1 and TH2B:TP2 decreased dramatically during the second week of culture; this was due not only to a decrease in the levels of p19 and TH2B mRNAs but also to an enhancement in the relative amounts of TP1 and TP2 as compared to the amounts present on the first day of the coculture. Conversely, both ratios remained low in RS-Sertoli cell cocultures; this was due to a decrease in the levels of the four mRNAs studied during the coculture period. DNA flow cytometry studies showed the occurrence of a haploid cell population (1C) in PS-Sertoli cell cocultures from Day 2 onward, together with a decrease in the tetraploid cell population (4C). No such changes were observed in Sertoli cell-only cultures. By contrast, the haploid population decreased 3-fold during the first week in RS-Sertoli cell cocultures. Immunocytochemical studies demonstrated further that 5-bromo-2'-deoxyuridine-labeled PS of stages V-VIII were able to differentiate into RS under the present coculture conditions. Hence, although clearly imperfect, the present coculture system should help to clarify the local regulations governing spermatogenesis and should allow easier study of spermatogenic cell gene expression.