Induction of spermatogenesis under 3-dimentional tissue culture conditions by in vitro transplantation of spermatogonial stem cells isolated from human frozen-thawed testis tissue

Abstract

Sperm production is one of the most complex biological processes in the body. In vitro production of sperm is one of the most important goals of researches in the field of male infertility treatment, which is very important in male cancer patients treated with gonadotoxic methods and drugs. In this study, we examine the progression of spermatogenesis after transplantation of spermatogonial stem cells under conditions of testicular tissue culture. Testicular tissue samples from azoospermic patients were obtained and then these were freeze- thawed. Spermatogonial stem cells were isolated by two enzymatic digestion steps and the identification of these cells was confirmed by detecting the PLZF protein. These cells, after being labeled with DiI, were transplanted in azoospermia adult mice model. The host testes were placed on agarose gel as tissue culture system. After 8 weeks, histomorphometric, immunohistochemical and molecular studies were performed. For each experimental group, 3 to 5 NMRI mouse were used at the age of 4 weeks. These mice are treated with Busulfan 40 mg/kg and after 4 weeks, the Azoospermia model is developed. This study is based on 5 biopsy samples taken from different obstructive azoospermic patients. SSCs were isolated by Mirzapour et al (2012) protocol under two steps of enzymatic digestion. SSCs were transplanted into the host testes below the stereo microscope then they were cut into small pieces and placed under 3-D tissue culture conditions on the agarose support layer. The results of histomorphometric studies showed that the mean number of spermatogonial cells, spermatocytes and spermatids in the experimental group was significantly more than the control group (without transplantation) (P<0.05) and most of the cells responded positively to the detection of DiI. Immunohistochemical studies in host testes fragments in the experimental group express the PLZF, SCP3 and ACRBP proteins in spermatogonial cells, spermatocyte and spermatozoa, respectively, which confirmed the human nature of these cells. Also, in molecular studies of PLZF, Tekt1 and TP1, the results indicated that the genes were positive in the test group, while not in the control group. These results suggest that the slow freezing of SSCs can support the induction of spermatogenesis to produce haploid cells under the 3-Dimentional testicular tissue culture.