| With the improvement of industrialization, escalation of environmental pollution and increasing pressure from life and work, infertility has increasingly perplexed so many families and became an important problem that all human beings must face to. Morbidity of infertility is raised year by year now, and about 15% couples of childbearing are suffering from infertility, half of whom are males. Meanwhile, more than 60-70% of male infertility remained unexplained, which extremely make us so confused to treat male infertility. So it is so important and essential for us to identify the reason and mechanism of male infertility.Spermatogenesis is a complex progress of germ cell differentiation, including mitosis, meiosis and spermatid deformation, which generates mature sperm. Meiosis I is a very special progress and plays an important role in spermatogenesis, if there is something wrong with this progress, spermatogenesis will be arrested, which will lead to sterility at last. The most important features among this progress are homologous pairing, synapsis and recombination.The mammalian X and Y chromosomes share little homology and are largely unsynapsed during normal male meiosis. This asynapsis triggers inactivation of sex chromosome linked genes, or called meiotic sex chromosome inactivation (MSCI). MSCI plays an essential role in normal male meiosis and is indispensable for male fertility, if MSCI is disrupted, spermatogenesis will be arrested at pachytene stage. As an important regulator in mammalian cell cycle progression, cyclin-dependent kinase 2 (CDK2) also plays an essential role in meiosis, especially in homologous pairing, recombination and sex body formation. While the function of phosphorylation of CDK2 (P-CDK2) in meiosis, especially p-CDK239 (phosphorylation of CDK2 isoform 1 (39 kD) on threonine 160), has not been reported. Here, we show that p-CDK239 can localize to the sites of asynapsis and the sex body, interacting with phosphorylated H2AX (y-H2AX), a very important regulator in MSCI. The interaction between p-CDK2 and y-H2AX is dependent on the phosphorylation site of CDK2 on Thr160, point-mutant version of CDK2 in this site can reduce the interaction. Meanwhile, we find it is only CDK239 can interact with y-H2AX and localizes to sex body. We also find that absence of p-CDK2 level in the sex body leads to failure of meiotic sex chromosome inactivation, and an increase in the expression of the sex-linked genes, which suggests p-CDK2 can participate in MSCI.When the offspring of a cross of different animal species is rare or absent, or presents sterility, it will tend to be of the heterogametic sex, a phenomenon known as Haldane’s rule. Hybrid sterility is a post-zygotic reproductive isolation mechanism that is widespread in animal and plant species. The sterile hybrid mice are often males, and they exhibit spermatogenic disruptions, leading to a decreased number and/or malformation of mature sperm. Although the phenomenon of reproductive barriers by spermatogenic impairment is well known, the underlying genetic mechanism and molecular basis remain elusive. One hypothesis for a force contributing to hybrid male sterility is’meiotic drive’.Here, we studied a cross between a wild-derived inbred stain of PWK/Ph and C57BL/6J in which sterility is asymmetric:F1 males with a PWK mother are sterile, while F1 males with a C57BL/6J mother are fertile. We found that the spermatogenesis of sterile F1 males was arrested at the mid-pachytene stage with extensive apoptosis of germ cells. Meanwhile, other phenotypes, such as asynapsis of autosomes and sex chromosomes and disturbance of the sex body, have also been found in sterile F1 males. Because normal meiosis need a lot of proteins, we isolate spermatocytes from fertile and sterile F1 males to identify the differentially expressed proteins between them to understand molecular mechanisms of hybrid male sterility and how these proteins connect with each other to contribute to hybrid male sterility. Of 4005 proteins we find, 215 were up-regulated and 381 were down-regulated. Bioinformatics analysis of the proteome led to the identification of 43 and 59 proteins known to be essential for male meiosis and spermatogenesis, respectively. Moreover, we find these proteins can potentially interact with and regulate each other. Characterization of the proteome of pachytene spermatocytes of hybrid male sterility provides an inventory of proteins that is useful for understanding meiosis and the mechanisms of hybrid male infertility.Reproduction, fat metabolism and longevity are interconnected with each other, and numerous observations have suggested that reproduction can influences organismal lifespan and aging. In many species, abolished or reduced reproduction system could increase fat storage and lead to weight gain. In C.elegans, signals from reproductive system could regulate longevity and ablation of germ cells can alter fat metabolism and significantly prolong lifespan. Resent researches have provided the most direct molecular evidence in C. elegans for the connection between reproduction, fat metabolism, and lifespan.We found a novel gene, c30f12.4, which could regulate reproduction, fat metabolism and longevity in C.elegans. When knock down of c30f12.4 by RNAi, the brood size was decreased. After knockout this gene by using CRISPR/Cas9, we found that oogenesis of these mutant worms was disrupted and transition zone was gone, which led to reduced offspring. Besides the phenotype in reproduction, we find that the loss of c30f12.4 alter fat metabolism, with decreased fat storage and small lipid droplets. Meanwhile, the c30f12.4 mutant worms display short lifespan. To detect the endogenously produced protein encoded by c30f12.4 and the location of C30F12.4 in worms, we construct a 3×FLAG-GFP:C30F12.4 transgene worm by using Mosl-mediated single-copy insertion (mosSCI) system. We found that the fused GFP::C30F12.4 was expressed very well and could be detected clearly in germ cells and early embryo. In order to identify the mechanism, we found C30F12.4 could interact with many proteins by using IP and MS analysis, which suggested that C30F12.4 might interact with other proteins to couple reproduction, fat metabolism and longevity. |
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