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Abstract

Volume 14, Issue 2 (March 2012) 14, 285–293; 10.1038/aja.2011.112

Differentiation of murine male germ cells to spermatozoa in a soft agar culture system

Mahmoud Abu Elhija1, Eitan Lunenfeld2, Stefan Schlatt3 and Mahmoud Huleihel1

1 The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
2 Department of Obstetrics and Gynecology, Soroka University Medical Center, and Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
3 Centre of Reproductive Medicine and Andrology of the University of Münster, Münster 84149, Germany

Correspondence: Professor M Huleihel, (huleihel@bgu.ac.il)

Received 26 January 2011; Revised 3 May 2011; Accepted 26 June 2011; Advance online publication 7 November 2011

Abstract

Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system (SACS), which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum (FCS). The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia (Vasa, Dazl, OCT-4, C-Kit, GFR-α-1, CD9 and α-6-integrin), meiotic cells (LDH, Crem-1 and Boule) and post-meiotic cells (Protamine-1, Acrosin and SP-10). Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.

Keywords: acrosome; agar; in vitro culture; meiosis; spermatogenesis; spermatogonia; spermatozoa; testis

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