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RNA alternative polyadenylation contributes to the complexity of information transfer from genome to phenome, thus amplifying gene function. Here, we report the first X. tropicalis resource with 127,914 alternative polyadenylation (APA) sites derived from embryos and adults. Overall, APA networks play central roles in coordinating the maternal-zygotic transition (MZT) in embryos, sexual dimorphism in adults and longitudinal growth from embryos to adults. APA sites coordinate reprogramming in embryos before the MZT, but developmental events after the MZT due to zygotic genome activation. The APA transcriptomes of young adults are more variable than growing adults and male frog APA transcriptomes are more divergent than females. The APA profiles of young females were similar to embryos before the MZT. Enriched pathways in developing embryos were distinct across the MZT and noticeably segregated from adults. Briefly, our results suggest that the minimal functional units in genomes are alternative transcripts as opposed to genes.

The PIWI-interacting RNA (piRNA) pathway is essential for retrotransposon silencing. In piRNA-deficient mice, L1-overexpressing male germ cells exhibit excessive DNA damage and meiotic defects. It remains unknown whether L1 expression simply highlights piRNA deficiency or actually drives the germ-cell demise. Specifically, the sheer abundance of genomic L1 copies prevents reliable quantification of new insertions. Here, we developed a codon-optimized L1 transgene that is controlled by an endogenous mouse L1 promoter. Importantly, DNA methylation dynamics of a single-copy transgene were indistinguishable from those of endogenous L1s. Analysis of Mov10l1-/- testes established that de novo methylation of the L1 transgene required the intact piRNA pathway. Consistent with loss of DNA methylation and programmed reduction of H3K9me2 at meiotic onset, the transgene showed 1,400-fold increase in RNA expression and consequently 70-fold increase in retrotransposition in postnatal day 14 Mov10l1-/- germ cells compared with the wild-type. Analysis of adult Mov10l1-/- germ-cell fractions indicated a stage-specific increase of retrotransposition in the early meiotic prophase. However, extrapolation of the transgene data to endogenous L1s suggests that it is unlikely insertional mutagenesis alone accounts for the Mov10l1-/- phenotype. Indeed, pharmacological inhibition of reverse transcription did not rescue the meiotic defect. Cumulatively, these results establish the occurrence of productive L1 mobilization in the absence of an intact piRNA pathway but leave open the possibility of processes preceding L1 integration in triggering meiotic checkpoints and germ-cell death. Additionally, our data suggest that many heritable L1 insertions originate from individuals with partially compromised piRNA defense.

Spermatogenesis is a highly organized and regulated process that requires the constant production of millions of gametes over the reproductive lifetime of the mammalian male. This is possible because of an active stem cell pool and an ordered entry into the germ cell developmental sequence. The ordered entry is a result of t he synthesis and action of retinoic acid allowing for the onset of spermatogonial differentiation and an irreversible commitment to spermatogenesis. The periodic appearance and actions of retinoic acid along the seminiferous tubules is a result of the interactions between germ cells and Sertoli cells that result in the generation and maintenance of the cycle of the seminiferous epithelium and is the subject of this review. [ABSTRACT FROM AUTHOR]

Sex in mammals is determined in the fetal gonad by the presence or absence of the Y chromosome gene Sry, which controls whether bipotential precursor cells differentiate into testicular Sertoli cells or ovarian granulosa cells (1). This pivotal decision in a single gonadal cell type ultimately controls sexual differentiation throughout the body. Sex determination can be viewed as a battle for primacy in the fetal gonad between a male regulatory gene network in which Sry activates Soxx9 and a female network involving WNT/β-catenin signalling (2). In females the primary sex-determining decision is not final: loss of the FOXL2 transcription factor in adult granulosa cells can reprogram granulosa cells into Sertoli cells (2). Here we show that sexual fate is also surprisingly labile in the testis: loss of the DMRT1 transcription factor (3) in mouse Sertoli cells, even in adults, activates Foxl2 and reprograms Sertoli cells into granulosa cells. In this environment, theca cells form, oestrogen is produced and germ cells appear feminized. Thus Dmrt1is essential to maintain mammalian testis determination, and competing regulatory networks maintain gonadal sex long after the fetal choice between male and female. Dmrt1 and Foxxl2 are conserved throughout vertebrates (4,5) and Dmrt1-related sexual regulators are conserved throughout metazoans (3). Antagonism between Dmrt1 and Foxxl2 for control of gonadal sex may therefore extend beyond mammals. Reprogramming due to loss of Dmrt1 also may help explain the aetiology of human syndromes linked to DMRT1, including disorders of sexual differentiation (6) and testicular cancer (7)., Human chromosome 9p deletions removing DMRT1 are associated with XY male-to-female sex reversal, and Dmrt1 homologues determine sex in several non-mammalian vertebrates (8-10). In mice, Dmrt1 is expressed and required [...]

Spermatogenesis in adult mammals is highly organized, with the goal being continual sperm production. Vertebrate testes are arranged into recurring cellular associations that vary with time and distance along the tubule. These changes over time and distance are designated the cycle of the seminiferous epithelium and the spermatogenic wave, respectively. In this Review, we briefly outline the roles that follicle-stimulating hormone (FSH) and testosterone play in regulating spermatogenesis and describe our current understanding of how vitamin A regulates germ cell differentiation and how it may lead to the generation of both the cycle of the seminiferous epithelium and the spermatogenic wave., Introduction Normal fertility in males of most heterogametic species (i.e., species in which either males or females have nonidentical sex chromosomes; e.g., in humans where males have an X and [...]

Dmrt1 (doublesex and mab-3 related transcription factor 1) is a conserved transcriptional regulator of male differentiation required for testicular development in vertebrates. Here, we show that in mice of the 129Sv strain, loss of Dmrt1 causes a high incidence of teratomas, whereas these tumors do not form in Dmrt1 mutant C57BL/6J mice. Conditional gene targeting indicates that Dmrt1 is required in fetal germ cells but not in Sertoli cells to prevent teratoma formation. Mutant 129Sv germ cells undergo apparently normal differentiation up to embryonic day 13.5 (E13.5), but some cells fail to arrest mitosis and ectopically express pluripotency markers. Expression analysis and chromatin immunoprecipitation identified DMRT1 target genes, whose missexpression may underlie teratoma formation. DMRT1 indirectly activates the GDNF coreceptor Ret, and it directly represses the pluripotency regulator Sox2. Analysis of human germ cell tumors reveals similar gene expression changes correlated to DMRT1 levels. Dmrt1 behaves genetically as a dose-sensitive tumor suppressor gene in 129Sv mice, and natural variation in Dmrt1 activity can confer teratoma susceptibility. This work reveals a genetic link between testicular dysgenesis, pluripotency regulation, and teratoma susceptibility that is highly sensitive to genetic background and to gene dosage. GDNF | testicular teratoma | germ cell tumor | embryonal carcinoma doi/10.1073/pnas.0905431106

Polyadenylation, the process of eukaryotic mRNA 3' end formation, is essential for gene expression and ceil viability. Polyadenylation of male germ cell mRNAs is unusual, exhibiting increased alternative polyadenylation, decreased AAUAAA polyadenylation signal use, and reduced downstream sequence element dependence. CstF-64, the RNA-binding component of the cleavage stimulation factor (CstF), interacts with pre-mRNAs at sequences downstream of the cleavage site. In mammalian testes, meiotic XY-body formation causes suppression of X-linked CstF-64 expression during pachynema. Consequently, an autosomal paralog, [tau]CstF-64 (gene name Cstf2t), is expressed during meiosis and subsequent haploid differentiation. Here we show that targeted disruption of Cstf2t in mice causes aberrant spermatogenesis, specifically disrupting meiotic and postmeiotic development, resulting in male infertility resembling oligoasthenoteratozoospermia. Furthermore, the Cstf2t mutant phenotype displays variable expressivity such that spermatozoa show a broad range of defects. The overall phenotype is consistent with a requirement for [tau]CstF-64 in spermatogenesis as indicated by the significant changes in expression of thousands of genes in testes of [Cstf2t.sup.-/-] mice as measured by microarray. Our results indicate that, although the infertility in [Cstf2t.sup.-/-] males is due to low sperm count, multiple genes controlling many aspects of germ-cell development depend on [tau]CstF-64 for their normal expression. Finally, these transgenic mice provide a model for the study of polyadenylation in an isolated in vivo system and highlight the role of a growing family of testis-expressed autosomal retroposed variants of X-linked genes. spermatogenesis | oligoasthenoteratozoospemia | meiosis | XY body | meiotic sex chromosome inactivation

Within the mammalian testis, specialized tight junctions between somatic Sertoli cells create basal and apical polarity within the cells, restrict movement of molecules between cells, and separate the seminiferous epithelium into basal and adluminal compartments. These tight junctions form the basis of the blood--testis barrier, a structure whose function and dynamic regulation is poorly understood. In this study, we used microarray gene expression profiling to identify genes with altered transcript levels in a mouse model for conditional androgen insensitivity. We show that testosterone, acting through its receptor expressed in Sertoli cells, regulates the expression of claudin 3, which encodes a transient component of newly formed tight junctions. Sertoli cell-specific ablation of androgen receptor results in increased permeability of the blood--testis barrier to biotin, suggesting claudin 3 regulates the movement of small molecules across the Sertoli cell tight junctions. These results suggest that androgen action in Sertoli cells regulates germ cell differentiation, in part by controlling the microenvironment of the seminiferous epithelium. Our studies also indicate that hormonal strategies for male contraception may interfere with the blood--testis barrier. androgen receptor | testosterone | tight junctions

Research has been conducted on the clusterin, a heterodimeric glycoprotein found in the body tissues and associated with cellular injury. Results indicate that clusterin binds to different molecules with high affinity and can function as a biological detergent capable of solubilizing bacteriorhodopsin.

Research has been conducted on the cell type-specific microtubules. The identification of the gene that encodes epithelial microtubule-associated protein of 115 kD has been carried out.

The transition of undifferentiated A spermatogonia to differentiated spermatogonia requires the action of retinoic acid (RA). The synthesis of retinoic acid from retinal in the seminiferous epithelium is a result of the action of aldehyde dehydrogenases termed ALDH1A1, ALDH1A2, and ALDH1A3. We used a mouse with a global deletion of the Aldh1a1 gene that is phenotypically normal and the CRE -loxP approach to eliminate Aldh1a2 genes globally and from Sertoli cells and germ cells. The results show that global elimination of Aldh1a1 and Aldh1a2 genes blocks spermatogenesis but does not appear to affect viability. The cell specific elimination of Aldh1a2 gene showed that retinoic acid synthesis by Sertoli cells is required for the initial round of spermatogonial differentiation but that there is no requirement for retinoic acid synthesis by germ cells. In both the global gene deletion and the cell specific gene deletions the maintenance of Aldh1a3 activity could not compensate. [ABSTRACT FROM AUTHOR]

Retinoic acid, an active metabolite of vitamin A, is necessary for many developmental processes in mammals. Much of the field of reproduction has looked toward retinoic acid as a key transcriptional regulator and catalyst of differentiation events. This review focuses on the effects of retinoic acid on male and female gamete formation and regulation. Within spermatogenesis, it has been well established that retinoic acid is necessary for the proper formation of the blood–testis barrier, spermatogonial differentiation, spermiation, and assisting in meiotic completion. While many of the roles of retinoic acid in male spermatogenesis are known, investigations into female oogenesis have provided differing results. [ABSTRACT FROM AUTHOR]

Background: Spermatogenesis in mammals is organized in a manner that maximizes sperm production. The central aspect of this organization is the cycle of the seminiferous epithelium that is characterized by an asynchronous repeating series of germ cell associations. These cell associations are the result of a fixed point of entry into the cycle at regular short time intervals and the longer time required for cells to fully differentiate and exit the cycle. Objective: This review will examine the current information on the action and metabolism of retinoic acid in the testis, the interaction of retinoic acid (RA) with the cycle and the spermatogenic wave, and the mechanisms that can lead to synchronous spermatogenesis. Finally, the unique applications of synchronous spermatogenesis to the study of the cycle and the mass isolation of specific germ cell populations are described. Materials and methods: Retinoic acid metabolism and spermatogonial differentiation have been examined by gene deletions, immunocytochemistry, chemical inhibitors, and mass spectrometry. Results, discussion, and conclusion: Both the Sertoli cells and the germ cells have the capacity to synthesize retinoic acid from retinol and in the mouse the entry into the cycle of the seminiferous epithelium, and the subsequent conversion of undifferentiated spermatogonia into differentiating spermatogonia is governed by a peak of RA synthesis occurring at stages VIII‐IX of the cycle. Normal asynchronous spermatogenesis can be modified by altering RA levels, and as a result the entire testis will consist of a few closely related stages of the cycle. [ABSTRACT FROM AUTHOR]