Establishment of a proteomic profile associated with gonocyte and spermatogonial stem cell maturation and differentiation in neonatal mice

Abstract

Initiation of the first wave of spermatogenesis in the neonatal mouse testis is characterized by differentiation of a transient population of germ cells called gonocytes in the center of the seminiferous tubules. After resuming mitotic activity, gonocytes relocate on the basement membrane, giving rise to spermatogonial stem cells (SSCs). These processes begin from birth in mice, and differentiated type A spermatogonia first appear by day 6 postpartum. During these processes, Sertoli cells within the seminiferous tubules and Leydig cells in the interstitial tissue form the stem cell "niche," and influence SSC fate decisions. Thus, we collected whole mouse testis tissues during the first wave of spermatogenesis at specific time points (days 0.5, 1.5, 2.5, 3.5, 4.5, and 5.5 postpartum) and constructed a comparative proteomic profile. We identified 252 differentially expressed proteins classified into three clusters based on expression, and bioinformatics analysis correlated each protein pattern to specific cell processes. Expression patterns of nine selected proteins were verified via Western blot, and cellular localizations of three proteins with little known information in testes were further investigated during spermatogenesis. Taken together, the results provide an important reference profile of a functional proteome during neonatal mouse gonocyte and SSC maturation and differentiation.

Mammalian gonocyte and spermatogonia differentiation: recent advances and remaining challenges

“…The comparison of these data with existing databases led to the identification of several potential candidate proteins possibly involved in gonocyte and SSC maturation. The germ cell expression of some of these proteins was confirmed by immunohistochemical analysis of PND3.5 and adult testes, identifying the presence of NUP153, SUZ12, and scaffold attachment factor B2 (SAFB2) in P3.5 undifferentiated spermatogonia, while whole-testes immunoblot analysis indicated their upregulation between PND3.5-PND5.5, suggesting potential roles in SSC maturation (Zheng et al 2014). NUP153 is a nucleoporin protein that has not been previously shown to be involved in spermatogenesis.…”

Long-term effects of repeated superovulation on ovarian structure and function in rhesus monkeys

“…The aliquots were then combined, and the pooled samples were evaporated in a vacuum. Strong cation-exchange chromatography (SCX) fractionation was performed using an UltiMate 3000 high-pressure liquid chromatography (HPLC) system as previously described elsewhere (15)(16)(17). A total of 21 fractions were collected at 2-minute intervals over the SCX gradient and sequentially detected on an LTQ Orbitrap Velos (Thermo Fisher Scientific) using the data-dependent acquisition mode.…”

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

“…After washing with PBS, secondary antibody was applied for 2 h, and the nucleus was stained with DAPI. For whole mount staining, gonads were fixed with 4% paraformaldehyde for 30 min, blocked with 5% bovine serum albumin for 2 h and incubated with primary antibodies overnight at 4°C, as described previously (16). On the second day, the gonads were washed thrice, and incubated with secondary antibody for 2 h, mounted on slides, and examined.…”

“…The MS3 method was programmed as previously described (16). For each cycle, one full MS scan of mass/charge ratio (m/z) ϭ 350 -1800 was acquired in the Orbitrap at a resolution of 60,000.…”

“…Proteomics technology has been widely used in postnatal testis development and function research in mice (12)(13)(14)(15)(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.…”