title: 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 epithelium.
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.
authors.affiliation: Laboratories of Cell Biology and Cytogenetics, Institute of Biomedical Sciences Abel Salazar (ICBAS), and Department of Genetics, Faculty of Medicine, University of Porto, Porto 4099-003, Portugal.
authors.forename: Rosália
authors.initials: R
authors.lastname: Sá
authors.forename: Rui
authors.initials: R
authors.lastname: Neves
authors.forename: Susana
authors.initials: S
authors.lastname: Fernandes
authors.forename: Cláudia
authors.initials: C
authors.lastname: Alves
authors.forename: Filipa
authors.initials: F
authors.lastname: Carvalho
authors.forename: Joaquina
authors.initials: J
authors.lastname: Silva
authors.forename: Nieves
authors.initials: N
authors.lastname: Cremades
authors.forename: Isabel
authors.initials: I
authors.lastname: Malheiro
authors.forename: Alberto
authors.initials: A
authors.lastname: Barros
authors.forename: Mário
authors.initials: M
authors.lastname: Sousa
mesh_terms: D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D049109:Cell Proliferation; D002470:Cell Survival; D018920:Coculture Techniques; D005640:Follicle Stimulating Hormone / Q000502:physiology*; D015870:Gene Expression; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007365:Intercellular Junctions / Q000648:ultrastructure; D008297:Male; D008540:Meiosis; D012670:Seminiferous Epithelium / Q000166:cytology; D012708:Sertoli Cells / Q000502:physiology; D013091:Spermatogenesis; D013094:Spermatozoa / Q000254:growth & development* / Q000648:ultrastructure; D013739:Testosterone / Q000502:physiology*; D013997:Time Factors
chemical_list: D015415:Biomarkers; D013739:Testosterone; D005640:Follicle Stimulating Hormone