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This Article Full Text Full Text (PDF) All Versions of this Article: 79/5/962    most recent biolreprod.107.067546v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sá, R. Articles by Sousa, M. Search for Related Content PubMed PubMed Citation Articles by Sá, R. Articles by Sousa, M. Agricola Articles by Sá, R. Articles by Sousa, M.

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

Laboratories of Cell Biology³ and Cytogenetics,⁴ Institute of Biomedical Sciences Abel Salazar (ICBAS), and Department of Genetics,⁵ Faculty of Medicine, University of Porto, Porto 4099-003, Portugal Centre for Reproductive Genetics Alberto Barros,⁶ Porto 4099-003, Portugal In Vitro Fertilization Unit,⁷ Department of Obstetrics and Gynecology, General University Hospital of Alicante, Alicante 03550, Spain

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..

apoptosis, germ cell differentiation, germ cell proliferation, meiosis, spermatogenesis in vitro

¹Supported in part by Governmental and European Community funds, Foundation for Science and Technology, Ministry of Science, Technology, and Superior Education, for a Ph.D. grant to R.S. (SFRH/BD/23616/2005) and research grants to M.S. (POCI/SAU-MMO/60709/60555/59997/04; UMIB).

Correspondence: ²Mário Sousa, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Largo do Prof Abel Salazar 2, 4099-003 Porto, Portugal. FAX: 351 22 206 22 32; e-mail: msousa{at}icbas.up.pt