Characterization of the Genes Essential for Mouse Spermatogenesis
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Spermatogenesis is a complex process that starts with the proliferation of differentiated spermatogonia through mitotic division. Primary spermatocytes produced from differentiated spermatogonia enter the prolonged prophase of meiosis during which DNA is exchanged by homologous recombination. Primary spermatocytes undergo two meiotic divisions to form haploid spermatids. Haploid spermatids differentiate through the elongation phase and eventually form mature spermatozoa through dramatic morphological changes termed spermiogenesis, during which: 1) the Golgi apparatus forms the acrosome, 2) nuclear chromatin undergoes compaction and condensation, 3) sperm tail is formed and 4) the excess cytoplasm of spermatid is eliminated. Given the complexity of spermatogenesis, the normal development of male germ cells is controlled by both protein-coding genes and small non-coding RNAs. Because of the lack of in vitro models, mouse models are currently the most powerful tools used to study these processes.KLHL10 (Kelch-like 10) is a spermatid-specific protein, which belongs to a BTB (Brica-brac, Tramtrack, and Broad-Complex)-Kelch protein superfamily. Haploinsufficiency of <italic>Klhl10</italic> causes male infertility and prevents the genetic transmission of both mutant and wild-type alleles in mice. Therefore, a transgenic rescue strategy was used to overcome the haploinsufficiency of <italic>Klhl10</italic> and knockout (KO) mice were generated for studying the function of <italic>Klhl10</italic> during spermatogenesis. <italic>Klhl10</italic> KO testes exhibited disrupted spermatogenesis, characterized by severe depletion of germ cells, degeneration of spermatids and reduction in the number of late spermatids. In <italic>Klhl10</italic> KO testes, spermatid differentiation was arrested in elongating stage at step 9. Comparison of protein profiles between control and KO testes revealed that many mitochondrial proteins were up-regulated in <italic>Klhl10</italic> KO testes. In addition, COX IV (mitochondrion marker) staining showed enhanced mitochondria signal in KLHL10 depleted germ cells. Our results indicate that KLHL10 might be involved in regulating mitochondrial protein turnover during late spermiogenesis. The BTB domain of KLHL10 was reported to directly interact with CUL3 (an ubiquitin E3 ligase), suggesting KLHL10 is involved in the ubiquitination pathway to regulate protein turnover. Using yeast two-hybrid, we screened an adult mouse testis library to identify testicular proteins that can interact with KLHL10 and spermatogenesis-associated proteins 3 (SPATA3) and 6 (SPATA6) were two proteins identified. <italic>In vitro</italic> co-immunoprecipitation assay revealed that KLHL10 interacts with both SPATA3 and SPATA6 through the Kelch domain, a substrate-recruiting domain in most of the CUL3-BTB/Kelch E3 ligase complexes. Therefore, an <italic>in vivo</italic> ubiquitination assay was performed to examine whether KLHL10 can recruit SPATA3 or SPATA6 for ubiquitination. We found the ubiquitination level of SPATA3, but not SPATA6, was significantly increased upon overexpression of KLHL10, suggesting SPATA3 is a substrate of CUL3-KLHL10 E3 ligase. Our data suggests CUL3-KLHL10 complex is a spermatid-specific ubiquitin E3 ligase that involved in removal of proteins during late spermiogenesis.<italic>Hils1</italic> is another spermatid-specific gene, which encodes a linker histone H1-like protein. The expression of HILS1 overlaps with the expression of transition nuclear proteins (TNP1 and TNP2). While <italic>Hils1<super>-/-</super></italic> males were fertile and <italic>Tnp1<super>-/-</super></italic> males were subfertile, the double KO (<italic>Hils1<super>-/-</super>Tnp1<super>-/-</super></italic>) mice we generated were completely infertile. Electron microscopy revealed severe nuclear condensation defect in both late spermatids and epididymal sperm of <italic>Hils1<super>-/-</super>Tnp1<super>-/-</super></italic> mice. The number of epididymal sperm was highly reduced and most sperm had abnormal morphologies with head-bent-back as predominant defect in <italic>Hils1<super>-/-</super>Tnp1<super>-/-</super></italic> mice. Double KO sperm also had a greater susceptibility of DNA to denaturation and elevated levels of protamine 2 precursors. Injection of mutant cauda epididymal sperm into intact oocyte showed normal fertilization rates, however, most zygotes didn't develop beyond the 2-cell stage. Single cell PCR revealed that the mRNA expression profile was altered in 2PN and 2-cell mutant embryos compared to WT, suggesting the disruption of paternal nuclear condensation can cause abnormal embryo development at pre-implantation stage.In addition to gene functional study using universal KO mouse models, we also used conditional KO mouse models to study the functions of small RNAs during spermatogenesis. microRNAs (miRNAs) are produced from short hairpin structures by the cleavages of DROSHA, a RNase III enzyme, in the nucleus and DICER, another RNase III enzyme, in the cytoplasm. endo-siRNAs are distinguished from miRNAs in that endo-siRNAs are processed from naturally occurring long dsRNAs and the biogenesis of endo-siRNAs is DROSHA-independent but DICER-dependent. To investigate the role of miRNA and/or endo-siRNA in spermatogenesis, we generated <italic>Drosha</italic> or <italic>Dicer</italic> conditional knockout (cKO) mouse lines using Cre-loxP strategy to specifically delete <italic>Drosha</italic> or <italic>Dicer</italic> in spermatogenic cells in postnatal testes. Although both <italic>Drosha</italic> and <italic>Dicer</italic> cKO males are infertile, <italic>Drosha</italic> cKO testes appeared to display more severe spermatogenic disruptions than <italic>Dicer</italic> cKO testes. Microarray analyses revealed transcriptomic differences between <italic>Drosha</italic> and <italic>Dicer</italic>-null pachytene spermatocytes or round spermatids. Although levels of sex-linked mRNAs were mildly elevated, meiotic sex chromosome inactivation appeared to have occurred normally in both <italic>Drosha</italic> and <italic>Dicer</italic> cKO cells. Our data demonstrate that gene regulation mediated by small RNAs is required for the normal development of male germ cells and male fertility.Overall, we demonstrated that both protein-coding genes and small RNAs play roles in regulation of male germ cell development. Investigations using mouse models help us gain deeper understanding of the fundamentals of reproductive biology, which will ultimately benefit the human health.