SummaryGlobal transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis.
The exact role of the endocannabinoid system (ECS) during spermatogenesis has not been clarified. We used purified germ cell fractions representative of all phases of spermatogenesis and primary cultures of spermatogonia. This approach allowed the precise quantification of the cannabinoid receptor ligands, anandamide and 2-arachidonoylglycerol, and of the expression at transcriptional and transductional levels of their metabolic enzymes and receptors. Our data indicate that male mouse germ cells possess an active and complete ECS, which is modulated during meiosis, and suggest the presence of an autocrine endocannabinoid signal during spermatogenesis. Mitotic cells possess higher levels of 2-arachidonoylglycerol, which decrease in spermatocytes and spermatids. Accordingly, spermatogonia express higher and lower levels of 2-arachidonoylglycerol biosynthetic and degrading enzymes, respectively, as compared to meiotic and postmeiotic cells. This endocannabinoid likely plays a pivotal role in promoting the meiotic progression of germ cells by activating CB2 receptors. In fact, we found that the selective CB2 receptor agonist, JWH133, induced the Erk 1/2 MAPK phosphorylation cascade in spermatogonia and their progression toward meiosis, because it increased the number of cells positive for SCP3, a marker of meiotic prophase, and the expression of early meiotic prophase genes.cannabinoid receptors ͉ meiosis ͉ TRPV1 S ince its discovery, the endocannabinoid system (ECS) has been shown to be implicated in several fundamental physiological functions as well as in many pathological conditions (1, 2). The ECS is modulated during cell proliferation, differentiation, and apoptosis through alterations of the expression levels of cannabinoid receptors (CNRs) of type 1 (CB 1 ) and 2 (CB 2 ), and of the enzymes involved in the biosynthesis and degradation of the 2 main CNR agonists: anandamide (AEA) and 2-arachidonoylglycerol (2-AG) (1). It has been demonstrated that AEA and synthetic agonists of CNRs exert antitumoral and antimetastatic activities by inhibiting cell proliferation, angiogenesis, and tumor cell migration (2).A central role of CB 1 receptors in the regulation of the pituitarygonad axis has been described by Wenger et al. (3), demonstrating the involvement of CB 1 in testosterone production by Leydig cells. The presence of an active ECS has been described both in testis and isolated spermatozoa of mammals, sea-urchin, and Rana esculenta (4-9). In particular, it has been demonstrated (6, 7) that activation of CB 1 receptors by AEA in both human and boar spermatozoa reduces their motility and the acrosomal reaction (10).Spermatogenesis is a highly coordinated complex process characterized by mitotic (spermatogonia), meiotic (spermatocytes), and differentiative haploid (spermatids) phases. Spermatogenesis is initiated in the basal compartment of the seminiferous epithelium, by spermatogonial stem cells that proliferate and differentiate into type A1 spermatogonia. Type A1 spermatogonia undergo a series of synchronized...
Follicle-stimulating hormone (FSH) and its intracellular mediator, cAMP, increase the mRNA levels for the Steel factor (SLF, the c-kit ligand) in cultured primary mouse Sertoli cells. The inductive effect of cAMP is more evident in cultures from 13-day-old animals than in cultures from 18-day-old animals. Analysis through the polymerase chain reaction (PCR) indicates that (Bu)2cAMP or FSH treatment increases the levels of the mRNAs for both the potentially soluble form and the transmembrane form of SLF in cultured Sertoli cells. The ratio between mRNAs encoding the potentially soluble form and the transmembrane form of SLF increases during postnatal testis development, and it is higher in cultured Sertoli cells with respect to total testis, suggesting that, under the in vitro conditions, SLF could be produced by Sertoli cells mainly as a soluble factor. Soluble recombinant SLF stimulates, in a dose-dependent fashion, thymidine incorporation in cultures of isolated germ cell populations enriched in the mitotic stages (spermatogonia), independently of the presence of serum, whereas cAMP analogs have no effect. Autoradiographic analysis shows that SLF selectively stimulates DNA synthesis in type A spermatogonia.
Sam68 is a KH-type RNA-binding protein involved in several steps of RNA metabolism with potential implications in cell differentiation and cancer. However, its physiological roles are still poorly understood. Herein, we show that Sam68−/− male mice are infertile and display several defects in spermatogenesis, demonstrating an essential role for Sam68 in male fertility. Sam68−/− mice produce few spermatozoa, which display dramatic motility defects and are unable to fertilize eggs. Expression of a subset of messenger mRNAs (mRNAs) is affected in the testis of knockout mice. Interestingly, Sam68 is associated with polyadenylated mRNAs in the cytoplasm during the meiotic divisions and in round spermatids, when it interacts with the translational machinery. We show that Sam68 is required for polysomal recruitment of specific mRNAs and for accumulation of the corresponding proteins in germ cells and in a heterologous system. These observations demonstrate a novel role for Sam68 in mRNA translation and highlight its essential requirement for the development of a functional male gamete.
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