Spermatogenesis is a dynamic developmental process that includes stem cell proliferation and differentiation, meiotic cell divisions and extreme chromatin condensation. Although studied in mice, the molecular control of human spermatogenesis is largely unknown. Here, we developed a protocol that enables next-generation sequencing of RNA obtained from pools of 500 individually laser-capture microdissected cells of specific germ cell subtypes from fixed human testis samples. Transcriptomic analyses of these successive germ cell subtypes reveals dynamic transcription of over 4000 genes during human spermatogenesis. At the same time, many of the genes encoding for well-established meiotic and post-meiotic proteins are already present in the pre-meiotic phase. Furthermore, we found significant cell type-specific expression of post-transcriptional regulators, including expression of 110 RNA-binding proteins and 137 long non-coding RNAs, most of them previously not linked to spermatogenesis. Together, these data suggest that the transcriptome of precursor cells already contains the genes necessary for cellular differentiation and that timely translation controlled by post-transcriptional regulators is crucial for normal development. These established transcriptomes provide a reference catalog for further detailed studies on human spermatogenesis and spermatogenic failure.

Spermatogenesis is a complex developmental process that ultimately generates mature spermatozoa. This process involves a phase of proliferative expansion, meiosis, and cytodifferentiation. Mouse models have been widely used to study spermatogenesis and have revealed many genes and molecular mechanisms that are crucial in this process. Although meiosis is generally considered as the most crucial phase of spermatogenesis, mouse models have shown that pre-meiotic and post-meiotic phases are equally important. Using knowledge generated from mouse models and in vitro studies, the current review provides an overview of the molecular control of rodent spermatogenesis. Finally, we briefly relate this knowledge to fertility problems in humans and discuss implications for future research. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.

Mouse embryonic fibroblasts (MEFs) deficient for pocket proteins (i.e., pRB/p107-, pRB/p130-, or pRB/ p107/p130-deficient MEFs) have lost proper G 1 control and are refractory to Ras V12 -induced senescence. However, pocket protein-deficient MEFs expressing Ras V12 were unable to exhibit anchorage-independent growth or to form tumors in nude mice. We show that depending on the level of pocket proteins, loss of adhesion induces G 1 and G 2 arrest, which could be alleviated by overexpression of the TBX2 oncogene. TBX2-induced transformation occurred only in the absence of pocket proteins and could be attributed to downregulation of the p53/p21 CIP1 pathway. Our results show that a balance between the pocket protein and p53 pathways determines the level of transformation of MEFs by regulating cyclin-dependent kinase activities. Since transformation of human fibroblasts also requires ablation of both pathways, our results imply that the mechanisms underlying transformation of human and mouse cells are not as different as previously claimed.Deregulation of the pRB tumor suppressor pathway is a frequent event in the development of cancer (18, 31). pRB and its close homologs p107 and p130 comprise the family of socalled pocket proteins and are widely known for their role in cell cycle regulation, especially during G 1 phase. In their active, hypophosphorylated form, pocket proteins restrict cell cycle progression by binding to E2F transcription factors. E2F-pocket protein complex formation inhibits the expression of E2F target genes both by blocking E2F's ability to induce transcription and by active repression. As a result, initiation of S phase is inhibited. Upon cell cycle stimulation, cyclin D-CDK4/6 (cyclin-dependent kinase 4 or 6) and cyclin E-CDK2 complexes are activated and hyperphosphorylate the pocket proteins, resulting in liberation and activation of E2F transcription factors and initiation of S phase. Further cell cycle progression requires cyclin A-CDK1/2 activity in S phase and cyclin A-CDK1/2 and cyclin B1-CDK1 activities in G 2 /M phase. The activity of cyclin-CDK complexes can be inhibited by the INK4a (inhibitor of cyclin-dependent kinase 4a) and the CIP/KIP families of CDK inhibitors (reviewed in reference 2).Consistent with a role for pocket proteins in G 1 control, we and others have shown that complete ablation of pocket proteins in mouse embryonic fibroblasts (MEFs) abrogated G 1 arrest in response to growth-inhibitory signals, such as cell-cell contact, growth factor depletion, and DNA damage. Additionally, upon prolonged culturing or expression of constitutively active Ras (Ras V12 ), wild-type MEFs arrested in G 1 and displayed hallmarks of senescence, while MEFs deficient for both pRb and p107 or both pRb and p130 (double-knockout [DKO] MEFs) or all three pocket proteins (triple-knockout [TKO] MEFs) were refractory to replicative and Ras V12 -induced senescence (6,7,32,38). Similar to ablation of pocket proteins, ablation of the tumor suppressor p53 or its upstream regulator p19 ARF also byp...