Post-meiotic Germ Cell Differentiation Research Articles

ADAD2 regulates heterochromatin in meiotic and post-meiotic male germ cells via translation of MDC1.

ABSTRACTMale germ cells establish a unique heterochromatin domain, the XY-body, early in meiosis. How this domain is maintained through the end of meiosis and into post-meiotic germ cell differentiation is poorly understood. ADAD2 is a late meiotic male germ cell-specific RNA-binding protein, loss of which leads to post-meiotic germ cell defects. Analysis of ribosome association in Adad2 mouse mutants revealed defective translation of Mdc1, a key regulator of XY-body formation, late in meiosis. As a result, Adad2 mutants show normal establishment but failed maintenance of the XY-body. Observed XY-body defects are concurrent with abnormal autosomal heterochromatin and ultimately lead to severely perturbed post-meiotic germ cell heterochromatin and cell death. These findings highlight the requirement of ADAD2 for Mdc1 translation, the role of MDC1 in maintaining meiotic male germ cell heterochromatin and the importance of late meiotic heterochromatin for normal post-meiotic germ cell differentiation.

Astragalus membranaceus Ameliorates Reproductive Toxicity Induced by Cyclophosphamide in Male Mice

The root of Astragalus membranaceus B(UNGE) (AM) is a medicinal herb that has been capable of reducing the adverse effects of conventional chemotherapy. To investigate the effects of AM on cyclophosphamide (CP)-induced reproductive toxicity in mouse testes, 5-week-old male imprinting control region mice were divided into five groups; CP was treated on the first day of each week for 5 weeks (100 mg/kg, i.p.), and AM was treated for 5 days a week for 5 weeks. At the end of the treatment period, the testes were taken out, cleared of the adhering tissues, and weighed. Epididymis was taken out and used for sperm analysis. Testis samples were frozen for real-time quantitative PCR and Western blot analysis. AM treatment increased diminished relative testes weight, and sperm count and motility in mice treated with CP. CP treatment has detrimental effects on the expression of cAMP-responsive element modulator (CREM), a transcription factor that is highly expressed in male germ cells and is crucial to post-meiotic germ cell differentiation. AM restored CREM at both the mRNA and protein levels. AM has beneficial influences and appears able to ameliorate relative testes weight, sperm parameters, and CREM expression against CP-induced reproductive toxicity.

Interaction between microRNAs and actin-associated protein Arpc5 regulates translational suppression during male germ cell differentiation

Decoupling of transcription and translation during postmeiotic germ cell differentiation is critical for successful spermatogenesis. Here we establish that the interaction between microRNAs and actin-associated protein Arpc5 sets the stage for an elaborate translational control mechanism by facilitating the sequestration of germ cell mRNAs into translationally inert ribonucleoprotein particles until they are later translated. Our studies reveal that loss of microRNA-dependent regulation of Arpc5, which controls the distribution of germ cell mRNAs between translationally active and inactive pools, results in abnormal round spermatid differentiation and impaired fertility. Interestingly, Arpc5 functions as a broadly acting translational suppressor, as it inhibits translation initiation by blocking 80S formation and facilitates the transport of mRNAs to chromatoid/P bodies. These findings identify a unique role for actin-associated proteins in translational regulation, and suggest that mRNA-specific and general translational control mechanisms work in tandem to regulate critical germ cell differentiation events and diverse somatic cell functions.

Role of β-Catenin in Post-Meiotic Male Germ Cell Differentiation

Though roles of β-catenin signaling during testis development have been well established, relatively little is known about its role in postnatal testicular physiology. Even less is known about its role in post-meiotic germ cell development and differentiation. Here, we report that β-catenin is highly expressed in post-meiotic germ cells and plays an important role during spermiogenesis in mice. Spermatid-specific deletion of β-catenin resulted in significantly reduced sperm count, increased germ cell apoptosis and impaired fertility. In addition, ultrastructural studies show that the loss of β-catenin in post-meiotic germ cells led to acrosomal defects, anomalous release of immature spermatids and disruption of adherens junctions between Sertoli cells and elongating spermatids (apical ectoplasmic specialization; ES). These defects are likely due to altered expression of several genes reportedly involved in Sertoli cell-germ cell adhesion and germ cell differentiation, as revealed by gene expression analysis. Taken together, our results suggest that β-catenin is an important molecular link that integrates Sertoli cell-germ cell adhesion with the signaling events essential for post-meiotic germ cell development and maturation. Since β-catenin is also highly expressed in the Sertoli cells, we propose that binding of germ cell β-catenin complex to β-catenin complex on Sertoli cell at the apical ES surface triggers a signaling cascade that regulates post-meiotic germ cell differentiation.

Role of Beta-Catenin in Post-Meiotic Male Germ Cell Differentiation.

Though roles of beta-catenin signaling during testis development have been well established, relatively little is known about its role in postnatal testicular physiology. Even less is known about its role in post-meiotic germ cell development and differentiation. Here, we report that beta-catenin is highly expressed in post-meiotic germ cells and plays an important role during spermiogenesis. Spermatid-specific deletion of beta-catenin resulted in significantly reduced sperm count, increased germ cell apoptosis and impaired fertility. In addition, ultrastructural studies show that the loss of beta-catenin in post-meiotic germ cells led to acrosomal defects, anomalous release of immature spermatids and disruption of adherens junctions between Sertoli cells and elongating spermatids (apical ectoplasmic specialization). Interestingly, These defects are likely due to altered expression of several genes reportedly involved in Sertoli cell-germ cell adhesion and germ cell differentiation, as revealed by gene expression analysis. Taken together, our results suggest that beta-catenin is an important molecular link that integrates Sertoli cell-germ cell adhesion with the signaling events essential for post-meiotic germ cell development and maturation. Since β-catenin is also highly expressed in the Sertoli cells, we propose that binding of germ cell beta-catenin complex to beta-catenin complex on Sertoli cell at the apical ES surface triggers a signaling cascade that regulates post-meiotic germ cell differentiation. (poster)

Regulation of Translational Suppression in Post-Meiotic Germ Cells: Crosstalk Between MicroRNAs and Actin-Associated Proteins.

During male germ cell development, the vast majority of mRNAs is translationally repressed at the post-meiotic stage. While individual components implicated in the transcriptional and posttranscriptional control of specific germ cell gene expression have been identified, the mechanism that coordinates widespread translational suppression during germ cell differentiation remains unclear. In this study, we established that the cross-talk between miRNAs and actin-associated protein sets the tone for the mRNA-specific as well as general uncoupling of transcription and translation during post-meiotic germ cell maturation. Our study reveals that loss of the miRNA-processing enzyme Dicer function in haploid spermatids causes altered translational activation of several post-meiotic germ cell-specific transcripts, resulting in abnormal chromatin compaction and impaired fertility. We identified a specific miRNA-targeted actin-associated protein 2/3 complex (Arpc) subunit that plays a crucial role in this process as it sequesters germ cell-specific transcripts through their 3' UTR into translationally inert RNP complexes, and making them inaccessible to translational machinery. Interestingly, this actin-associated protein mediates translational suppression by interacting with GW182, a component of processing and chromatoid bodies, discrete cytoplasmic foci in the somatic and germ cells, respectively, believed to play an instrumental role in the storage and degradation of translationally suppressed mRNA. Collectively, our findings suggest that miRNAs are important checkpoint regulators that control timely expression of genes critical for post-meiotic germ cell differentiation and add a new dimension to the increasing role of actin and actin-associated proteins in post-transcriptional gene regulation. In light of recent studies showing an association between altered expression of actin-binding proteins and poor semen quality in human males, we propose that miRNA(s) targeting this Arpc subunit may serve as novel biomarkers for defective sperm and impaired male fertility. This research is funded by the NIH (HD053774-03). (platform)

167. MUSASHI FAMILY OF RNA BINDING PROTEINS: CELL CYCLE REGULATORS IN SPERMATOGENESIS

Mammalian meiosis is a tightly regulated process involving specialized cell cycle progression and morphogenetic changes. We have demonstrated that the Musashi family of RNA binding proteins is implicated in the regulation of spermatogonial stem self renewal and germ cell differentiation. Here we describe the novel mechanism by which the Musashi family proteins, Msi1 and Msi2, act to control exit from spermatogonial mitotic amplification and normal entry into meiosis. Gene and protein analysis indicated overlapping Msi1 and Msi2 profiles in enriched populations of isolated germ cells and reciprocal subcellular expression patterns in spermatogonia and pachytene spermatocytes/ round spermatids in testes sections. Recombinant Msi1 protein-RNA pulldown and microarray analysis coupled with in vitro shRNA knockdown studies in spermatogonial culture and subsequent immunoprecipitation and qPCR established that Msi1 targeted Msi2 mRNA for post transcriptional translational repression. Immunoprecipitation of Msi2 target mRNA and subsequent qPCR together with in vitro shRNA knockdown studies inround spermatidculture identified a cell cycle inhibitor protein CDKN1C (p57kip2) as the principal target of Msi2 translational inhibition. Immunolocalisation of CDKN1C protein indicated that expression of this cell cycle regulator coincided with the nuclear import of Msi1 and the appearance of cytoplasmic Msi2 expression in early pachytene spermatocytes. Using a transgenic Msi1 overexpression mouse model in conjunction with quantitative gene and protein expression, we confirmed Msi1 targeting of Msi2 and subsequent Msi2 targeting of CDKN1C for translational repression in vivo. Ectopic overexpression of Msi1 in germ cellsinduces substantial Msi2 downregulation and aberrant CDKN1C expression, resulting in abnormal spermatogenic differentiation, germ cell apoptosis/arrest and sterility. In conclusion, our results indicate a sophisticated molecular switch encompassing cell cycle protein regulation by Musashi family proteins, is required for normal exit from mitotic division, entry into meiosis and post meiotic germ cell differentiation.

Yukmijihwang-tang protects against cyclophosphamide-induced reproductive toxicity

A side effect of cyclophosphamide (CP), an alkylating agent widely used to treat tumors and autoimmune disorders, is the alteration of male reproductive function. Yukmijihwang-tang (YJT) is a multi-herbal medicinal formula that has been used in traditional Asian medicine to treat male reproductive problems. In this study, we investigated the effects of YJT on CP-induced reproductive toxicities in rat testes. Eight-week-old male Wistar rats were divided into three groups: vehicle-treated (control), CP-treated and CP + YJT-treated groups. CP was administered for the first 7 days (20 mg/kg/day, p.o.), and YJT was administered for 56 days consecutively (1.0 g/kg/day, p.o.). The CP-treated group showed significant decreases in the weight of the testes, epididymal sperm count and sperm motility compared to the control group, while the CP + YJT-treated group had significant increases for these variables compared to the CP-treated group. The enhancement of lipid peroxidation by CP in the rat testes was reduced by YJT treatment. CP diminished the expression of cAMP-responsive element modulator (CREM), a transcription factor that is highly expressed in male germ cells and is crucial to post-meiotic germ cell differentiation. YJT restored CREM at both the mRNA and protein levels. These results suggest that YJT has a protective effect against CP-induced reproductive toxicities by inhibiting the increases in lipid peroxidation and enhancing CREM expression.

Crem Activator Isoforms in Normal and Impaired Human Spermatogenesis Analyzed by Real Time RT-PCR

cAMP responsive element modulator (CREM) activator isoforms are involved in mammalian spermatogenesis and spermiogenesis. CREM proteins are highly expressed in postmeiotic germ cells of rodents and primates. Homozygous CREM inactivated mice exhibit round spermatid maturation arrest. The lack of CREM expression at both the mRNA and protein levels is associated with spermatid maturation arrest in infertile patients. Using real-time RT-PCR, we have examined the levels of CREM activator isoform mRNAs: CREMtheta1, CREMtheta2 and CREMt2 + Ex-gamma in gametogenic and interstitial cell fractions from normal human testis, in homogenized tissue samples from spermatogenic arrest and from testicular tumors. We have shown for the first time the presence of CREM activator isoform containing exon gamma (CREMtau2 + Exgamma) in normal human spermatogenesis. Among the three CREM isoforms, CREMtheta1 was expressed in its highest level in the male gonads. In comparison, CREMtheta2 mRNA was significantly less suggesting that the P3 promoter is much more active in human testis than the P4 promoter. Minimal-nill levels of mRNA for either of the CREM activator isoforms were detected in lymphocytes or in gonadal tissues from patients with SCOS (Sertoli Cell Only Syndrome). This data underlines the significance of CREMtheta1 isoform in the regulation of transcription during post-meiotic germ cell differentiation.

Microtubule-independent and Protein Kinase A-mediated Function of Kinesin KIF17b Controls the Intracellular Transport of Activator of CREM in Testis (ACT)

Kinesins are motor proteins that transport their cargos along microtubules in an ATP-dependent manner. The testis-specific kinesin KIF17b was shown to directly regulate cAMP-response element modulator (CREM)-dependent transcription by determining the subcellular localization of the activator of CREM in testis (ACT), the testis-specific coactivator of CREM in postmeiotic male germ cells. CREM is a crucial transcriptional regulator of many important genes required for spermatid maturation, as demonstrated by the complete block of sperm development at the first steps of spermiogenesis in crem-null mice. To better understand the complex regulation of postmeiotic germ cell differentiation, we further characterized the ACT-KIF17b interaction, the function of KIF17b, and the signaling pathways governing its action. In this study, we demonstrated that the abilities of KIF17b to shuttle between the nuclear and the cytoplasmic compartments and to transport ACT are neither dependent on its motor domain nor on microtubules, thus revealing a novel microtubule-independent function for kinesins. We also showed that the cyclic AMP-dependent protein kinase A mediates the phosphorylation of KIF17b, and this modification is important for its subcellular localization. These results indicate that cyclic AMP signaling controls CREM-mediated transcription in male germ cells through modification of KIF17b function.

Germ-line specific variants of components of the mitochondrial outer membrane import machinery in Drosophila

A search of the Drosophila genome for genes encoding components of the mitochondrial translocase of outer membrane (TOM) complex revealed duplication of genes encoding homologues of Tom20 and Tom40. Tom20 and Tom40 were represented by two differentially expressed homologues in the Drosophila genome. While dtom20 and dtom40 appeared to be expressed ubiquitously, the second variants, called tomboy20 and tomboy40, were expressed only in the male germ-line. Transcripts for tomboy20 and tomboy40 were detected in primary spermatocytes as well as post-meiotic stages. Transcription of tomboy20 and tomboy40 in spermatocytes was not dependent on the transcription factor Cannonball, which is responsible for controlling expression of gene products exclusively required for post-meiotic germ cell differentiation. Epitope-tagging and transient expression of dTom20 and Tomboy40 in mammalian cell culture showed proper targeting to mitochondria.

Sensitivity of Sertoli and leydig cells to xenobiotics in in vitro models

Different chemicals are known to cause testicular damage in the human male and experimental animals. However, the ability to assess the potential and mechanism of action leading to chemically-induced damage in men has been hampered by a lack of good predictive models. Although many of these chemicals were found to impair reproductive capacity in various laboratory animals, only some have caused reproductive damage in men. Mammalian spermatogenesis takes place within the avascular seminiferous tubules of the testis. Specialized tight junctions, which form between adjacent Sertoli cells at the time of puberty, divide the tubular space into the basal and adluminal compartments, and create a “blood-testis” barrier that restricts passage of substances and ions from the circulation. Thus, the completion of meiosis and post-meiotic germ cell differentiation, which take place in the adluminal compartment, are isolated from circulating substances unable to cross the blood-testis barrier. It seems feasible, therefore, that damage to the germ cells induced by testicular toxicants may be mediated through other cells in the testis such as the Sertoli, peritubular, or Leydig cells. A recently developed two-compartment system for culture of testicular cells can simulate, to some degree, the normal physiologic conditions. In principle, Sertoli cells isolated from mammalian testes are cultured on a permeable support (that is millipore filter) between two fluid compartments. They form a highly polarized epithelial layer with characteristics tight junctions that restrict the passage of substances between the two compartments, in analogy to the blood-testis barrier. We believe this system provides an excellent in vitro model for determining the ability of chemicals to: a) alter the permeability of the blood-testis barrier, b) impair the secretory function of Sertoli cells, or c) affect their viability, all of which could indirectly affect the germ cells. We have utilized this system for examining the effects of cadmium chloride (CdCl 2) and other toxic substances known to affect the testis. The Leydig cell toxicity was investigated in testicular perfusion system or cultures of isolated Leydig cells.