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

Abstract

Efforts have been made for the isolation and characterization of human stem spermatogonia (SG) which would be of major interest for fertility preservation in oncologic patients. We evaluated the expression of mammalian SG stem cell markers, KIT, OCT4, integrin alpha 6 (ITGA6), and integrin beta 1 (ITGB1) as possible indicators for the isolation of those cells in humans. Two different types of SG were individually isolated by micromanipulation from testicular biopsies of men with conserved spermatogenesis. Expression of mRNA showed the absence of KIT and ITGB1 markers in SG. By immunocytochemistry (IC), protein expression for KIT and integrins revealed two types of SG populations, negative (type-1) and positive (type-2). By immunohistochemistry (IH), protein expression for KIT and ITGB1 also revealed two kinds of SG populations, negative (SG A-dark) and positive (SG A-pale). Results suggest that in humans it may be possible to obtain pure populations of stem SG by using negative KIT^(-)/ITGB1^(-) sorting.

Keywords:

• Immunocytochemistry
• immunohistochemistry
• RT-PCR, spermatogenesis
• spermatogonia
• stem cell markers

The authors appreciate the contribution of Maria João Pinho, PhD (Department of Genetics, Faculty of Medicine of University of Porto) for CD34+ preparation; Susana Fernandes, PhD (Department of Genetics, Faculty of Medicine of University of Porto) for RT-PCR teaching; Luis Ferraz, MD, Urologist (Director of Department of Urology, Hospital of Vila Nova de Gaia) for treatment testicular biopsies; António Couceiro, MD; Pathologist (Department of Pathology, Hospital of Vila Nova de Gaia) for archive testicular biopsies; Sónia Oliveira, MD, Gynecologist (Department of Gynecology and Obstetrics) for human umbilical cord blood; Joaquina Silva, MD, Senior Clinical Embryologist-ESHRE (CGR A. Barros) for IVF supervision; Paulo Viana, BSc and Mariana Cunha, BSc, Clinical Embryologists-ESHRE (CGR A. Barros) for biopsy preparation, as well as the support provided by a PhD grant to Rosália Sá from the Foundation for Science and Technology, Ministry of Science, Technology and Superior Education, Portugal.

Declaration of interest: PhD grant to RS (SFRH/BD/23616/2005) from the Foundation for Science and Technology, Ministry of Science, Technology and Superior Education, Portugal. The authors report no conflicts of interest. The authors alone account for the content and writing of the manuscript.

Author contributions: Full data acquisition, analysis, interpretation, critical discussion, and manuscript writing: RS; Initial data acquisition, analysis, interpretation, discussion, and text writing: CM; Co-supervision of the PhD of RS and text review: FC; Patient recruitment and ART supervision: AB; Study conception and design, data acquisition, analysis and interpretation, critical discussion, and final text review: MS.