In mice, once primordial germ cells (PGCs) are generated, they continue to proliferate and migrate to eventually reach the future gonads. They initiate sexual differentiation after their colonization of the gonads. During this process, retinoic acid (RA) induces meiosis in the female germ cells, which proceeds to the diplotene stage of meiotic prophase I, whereas the male germ cells initiate growth arrest. After birth, meiosis is initiated in mice spermatogonia by their conversion to preleptotene spermatocytes. There are evidences showing the roles of RA in the regulation of spermatogonial differentiation and meiosis initiation. However, it is still not well known on what responds to RA and how RA signaling engages meiosis. Thus, we constructed a proteomic profile of proteins associated with meiosis onset during testis development in mouse and identified 104 differentially expressed proteins (≥ 1.5 folds). Bioinformatic analysis showed proteins functioning in specific cell processes. The expression patterns of five selected proteins were verified via Western blot, of which we found that Tfrc gene was RA responsive, with a RA responsive element, and could be up regulated by RA in spermatogonial stem cell (SSC) line. Taken together, the results provide an important reference profile for further functional study of meiosis initiation. Biological significance Spermatogenesis involves mitosis of spermatogonia, meiosis of spermatocytes and spermiogenesis, in which meiosis is a unique event to germ cells, and not in the somatic cells. Till now, the detailed molecular mechanisms of the transition from mitosis to meiosis are still not elucidated. With high-throughput proteomic technology, it is now possible to systemically identify proteins possibly involved. With TMT-6plex based quantification, we identified 104 proteins differentially between testes without meiosis (day 8.5) and those that were meiosis initiated (day 10.5). And a well-known protein essential for meiosis initiation, stra8, was identified to be differentially expressed in the study. And bioinformatic analysis and functional studies revealed several proteins regulated by retinoic acid, a chemical known to regulate the meiosis initiation. Thus, this quantitative proteomic approach can identify meiosis initiation regulating proteins, and further functional studies of these proteins will help elucidate the mechanisms of meiosis initiation.

Testicular cord formation in male gonadogenesis involves assembly of several cell types, the precise molecular mechanism is still not well known. With the high-throughput quantitative proteomics technology, a comparative proteomic profile of mouse embryonic male gonads were analyzed at three time points (11.5, 12.5, and 13.5 days post coitum), corresponding to critical stages of testicular cord formation in gonadal development. 4070 proteins were identified, and 338 were differentially expressed, of which the Sertoli cell specific genes were significant enrichment, with mainly increased expression across testis cord development. Additionally, we found overrepresentation of proteins related to oxidative stress in these Sertoli cell specific genes. Of these differentially expressed oxidative stress-associated Sertoli cell specific protein, stromal interaction molecule 1, was found to have discrepant mRNA and protein regulations, with increased protein expression but decreased mRNA levels during testis cord development. Knockdown of Stim1 in Sertoli cells caused extensive defects in gonadal development, including testicular cord disruption, loss of interstitium, and failed angiogenesis, together with increased levels of reactive oxygen species. And suppressing the aberrant elevation of reactive oxygen species could partly rescue the defects of testicular cord development. Taken together, our results suggest that reactive oxygen species regulation in Sertoli cells is important for gonadogenesis, and the quantitative proteomic data could be a rich resource to the elucidation of regulation of testicular cord development. Male gonadogenesis is a complex process that requires the formation and assembly of several cell types that come together to form a functional organ. These cell lineages coordinate to maintain testicular cord development but do not differentiate independently (1, 2). Shortly after the activation of Sox9, when the genital ridges are still long and very thin, pre-Sertoli cells start to aggregate around germ cell clusters and form cords; they are then referred to as Sertoli cells. Partitioning of this mass of cells into cord-forming units coincides with endothelial cell invasion and expansion of interstitial space (3, 4). In mice, organization of the testicular cords begins with aggregate of germ cell and Sertoli progenitors in the gonad. Previous studies using confocal analysis and three-dimensional modeling have reported that testicular cord formation involves three basic steps (5, 6): pre-Sertoli cells and germ cells coalesce between 10.5 and 12.5 days post coitum (dpc)1; cords partition at 12.5 dpc with a clear basal lamina surrounding the cords, and all cords are characterized as “external” cords, defined as a single transverse loop located just under the celomic epithelium that surrounds the gonad at this stage; and refinement of cords continues at 13.5 dpc. Although Sertoli cells acting as a organizing center in testicular cord formation has been well known (3) and studies in knockout mouse models have revealed several genes associated with testicular cord formation (7⇓⇓–10), how these cell types assemble into a functional organ remains to be explored (2, 11). Proteomics technology has been widely used in postnatal testis development and function research in mice (12⇓⇓⇓–16). Two proteomics studies have been carried out in the fetal gonads in mice, and identified more than 1000 proteins expressed in gonads (17, 18), however, the temporal proteome changes have not been elucidated during gonadogenesis. Additionally, mRNA abundance may not always predict the quantity of the corresponding functional protein&#

Tandem proteomic strategies based on large‐scale and high‐resolution mass spectrometry have been widely applied in various biomedical studies. However, protein sequence databases and proteomic software are continuously updated. Proteomic studies should not be ended with a stable list of proteins. It is necessary and beneficial to regularly revise the results. Besides, the original proteomic studies usually focused on a limited aspect of protein information and valuable information may remain undiscovered in the raw spectra. Several studies have reported novel findings by reanalyzing previously published raw data. However, there are still no standard guidelines for comprehensive reanalysis. In the present study, we proposed the concept and draft framework for complementary proteomics, which are aimed to revise protein list or mine new discoveries by revisiting published data.

Meiosis is a special type of cellular renovation that involves 2 successive cell divisions and a single round of DNA replication. Two major degradation systems, the autophagy-lysosome and the ubiquitin-proteasome, are involved in meiosis, but their roles have yet to be elucidated. Here we show that autophagy mainly affects the initiation of meiosis but not the nuclear division. Autophagy works not only by serving as a dynamic recycling system but also by eliminating some negative meiotic regulators such as Ego4 (Ynr034w-a). In a quantitative proteomics study, the proteasome was found to be significantly upregulated during meiotic divisions. We found that proteasomal activity is essential to the 2 successive meiotic nuclear divisions but not for the initiation of meiosis. Our study defines the roles of autophagy and the proteasome in meiosis: Autophagy mainly affects the initiation of meiosis, whereas the proteasome mainly affects the 2 successive meiotic divisions.

Although CRISPR/Cas9 has been widely used to generate knockout mice, two major limitations remain: the founders usually carry a mixture of genotypes, and mosaicism harboring multiple genotypes. Therefore, it takes a long time to get homozygous mutants. Recently developed base editing (BE) system, which introduces C-to-T conversion without double strand DNA cleavage, has been used to introduce artificial stop codons (i-STOP) to prematurely terminate translation, providing a cleaner strategy for genome engineering. Using this strategy, we generated CD160 KO and VISTA/CD160 double KO mice by microinjection of a single sgRNA targeting CD160 and a mixture of sgRNAs targeting VISTA and CD160, respectively. The BE system induced STOP efficiently in mouse embryos and consequently in founder mice without detectable off-target. Most interestingly, the majority of the mutants harbor same genetic modifications, indicating we generated isogenic single and multiplex gene mutant mice by BE-induced STOP. We also obtained homozygous mutant mouse in F1 mice, demonstrating the accelerated strategy in generating animal models.