Development of Sperm from Spermatogonial Cells of Busulfan-Treated Immature Mice in Three-Dimension Culture System - Israeli Fertility Association Conference-Ayala 2018, May 7-8, Dan Panorama Hotel, Tel-Aviv

Ali AbuMadigem ¹ Ronnie Solomon ² Eitan Lunenfeld ³ Mahmoud Huleihel ⁴

¹The Shraga Segal Dep. of Microbiology, Immunology and Genetics, The Center of Advanced Research and Education in Reproduction (CARER), Faculty of Health Sciences, Ben-Gurion University of the Negev
³Dep. OB/GYN, Soroka Medical Center, The Center of Advanced Research and Education in Reproduction (CARER), Faculty of Health Sciences, Ben-Gurion University of the Negev
⁴The Shraga Segal Dep. of Microbiology, Immunology and Genetics, The Center of Advanced Research and Education in Reproduction (CARER), Faculty of Health Sciences, The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev

Introduction: Aggressive chemotherapy may lead to permanent male infertility. Prepubertal maleas do not generate sperm to be cryopreserved for future fertility preservation. However, their testes contain spermatogonial cells (SPGCs) that could be used for fertility preservation.

Objectives: To examine the capacity of survivor SPGCs from BU-treated immature mice to develop spermatogenesis in vitro.

Materials and Methods: Seven-day-old mice were injected intraperitoneal with BU (45 mg/kg) and sacrificed after 10 days [a time demonstrated by our group to severely affect spermatogenesis compared to control (DMSO)]. Cells were enzymatically isolated from seminiferous tubules (STs) and cultured in methylcellulose as a 3D in vitro culture, in the presence of KSR and growth factors and with/without homogenates (from adult GFP mice, which were frozen/thawed and filtered in 0.22 m filters to exclude the presence of sperm cells).

Results: Isolated tubular cells from STs of BU-treated immature mice showed development of colonies 4 weeks after initiation the in vitro culture in all conditions. We also detected development of meiotic (CFREM-1 and BOULE) and post meiotic (ACROSIN) cells in those cultures. Homogenates from adult GFP mice, which were added to cultures of tubular cells of BU-treated immature mice, were found to induce also the development of sperm-like cells, negative for GFP staining, after 4 weeks of culture.

Conclusions: We demonstrate for the first time, the presence of biologically active SPGCs in testicular tissue of BU-treated immature mice, and their capacity to develop in vitro to different stages of spermatogenesis including the generation of sperm-like cells with the addition of homogenates from adult mice.