Cytological and Expression Studies and Quantitative Analysis of the Temporal and Stage-Specific Effects of Follicle-Stimulating Hormone and Testosterone During Cocultures of the Normal Human Seminiferous Epithelium1

Abstract

In vitro culturing of normal human seminiferous epithelium remains largely unexplored. To study normal human spermatogenesis in vitro, we used a micromethod for the purification and culture of Sertoli cells, spermatogonia A, spermatocytes, and early round spermatids. Cytological quantitative data for Sertoli and premeiotic germ cell cocultures isolated from normal testicular biopsies demonstrated that cells were able to proliferate (4%), complete meiosis (6.7%), and differentiate into late round (54%), elongating (49%), and elongated (17%) spermatids at similar in vivo time delays (up to 16 days) in response to FSH + testosterone stimulation. Cells maintained normal meiotic segregation, chromosome complements, and specific gene expression profiles. Follicle-stimulating hormone + testosterone stimulated spermatogonia proliferation and Sertoli cell survival. Follicle-stimulating hormone and especially FSH + testosterone increased diploid germ cell survival during the first week, whereas only FSH + testosterone was able to inhibit cell death during the second week of culture. Follicle-stimulating hormone and especially FSH + testosterone also stimulated meiosis resumption, although this was restricted to late pachytene and secondary spermatocytes. In contrast, spermiogenesis was only stimulated by FSH + testosterone. Expression studies showed that apoptosis was induced in the nucleus of diploid cells, and in nuclear and cytoplasmic compartments of spermatids, mainly triggered by the Fas pathway. Although junctional complexes between Sertoli and premeiotic germ cells were partially reacquired, the same did not apply to spermatids, suggesting that FSH potentiated by testosterone was unable to render Sertoli cells competent to bind round spermatids.

Expression of stem cell markers: OCT4, KIT, ITGA6, and ITGB1 in the male germinal epithelium

“…However, we have previously demonstrated the specific expression of SC with KITGL and the absence of expression with MAGEA1, MLH1, HSPA2, and SPANXA1 [Sá et al 2008]. Additionally, the purity was confirmed as cells were individually isolated by micromanipulation, and the distinct cell features were evaluated by published morphological criteria [Holstein and Roosen-Runge 1991].…”

“…The suspension was then washed (2x5 min, 500 x g) and finally submitted to enzymatic treatment [Crabbé et al 1998]. The suspension was separated (5 min, 50 x g) into supernatant (haploid germ cells) and pellet (DGC) fractions [Sousa et al 2002b;Sá et al 2008]. The fluid fractions were washed with SPM (2x5 min, 1,000 x g), and the final pellets were resuspended in 100 µl in in-vitro fertilization medium (Universal IVF Medium, Medicult) and incubated at 32°C.…”

“…Individual germ cells were isolated by micromanipulation pursuant to the procedures described previously [Sousa et al 2002a;Sousa et al 2002b;Sá et al 2008]. Cell stages were confirmed by morphological criteria [Holstein and Roosen-Runge 1991;Sousa et al 2002b].…”

“…1). In addition to distinct morphological cell features, the purity of these cell types, which were individually isolated by micromanipulation, were previously characterised by FISH analysis, mRNA expression, and electron microscopy [Sá et al 2008].…”

Immunohystochemical analysis of CFTR in normal and disrupted spermatogenesis

“…We just identified all cell types according to Holstein and Roosen-Runge [1981], Sousa et al [2002], and Sá et al [2008]. Ten sequential sections (3 µm thick) were obtained from each block, and the first was stained with Hematoxylin-Eosin (Merck) to verify the integrity and the amount of the ST. To find the different cell types, all ST (more or less 20) in each section were analyzed.…”