Quantitative Proteomics Reveals the Essential Roles of Stromal Interaction Molecule 1 (STIM1) in the Testicular Cord Formation in Mouse Testis

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, and proteomic approach can provide a systemic view of protein level regulation in a large scale (18). Therefore, this study aimed to obtain a better understanding of male gonadogenesis by establishing a first temporal proteomic profile during the initiation of gonad development in male mice. After confirming the specific time point by immunofluorescence (IF) staining, we performed a comparative proteomic analysis of samples of male mouse gonads obtained at 11.5, 12.5, and 13.5 dpc. Bioinformatics analysis and functional studies demonstrate that reactive oxygen species (ROS) regulation in Sertoli cells may be important for testicular cord formation, and functional characterizing the of stromal interaction molecule 1 (stim1), a Sertoli cell specific protein, supported this hypothesis. Our categorized protein lists can serve as a useful resource for further exploring the molecular mechanisms involved in gonadal development.