Human spermatogonial stem cells patients in two-dimensional(2D)and three-dimensional (3D) culture systems

Azoospermia, which is one of the causes of male infertility, is no sperm count in semen. Spermatogenesis begins in the male with puberty and ocur both mitosis and meiosis stages to result for spermatid formation. During this process, the spermatogonium cells complete their progressive differentiation with the factors controlling the meiosis stage. In azoospermia patient, testicular sperm extraction (TESE) in testes samples retrieval. However, assisted reproductive techniques can not be applied in cases with no sperm in TESE specimens. In the study, it was aimed to differentiate spermatogonial stem cells in different culture media and characterize differentiated cells from TESE specimens in which spermatogonial cells are not observed. It was aimed to investigate the effects of different 3D culture media on this differentiation process by predicting that three-dimensional (3D) culture media could provide a similar microenvironment for in situ organization for the differentiation of spermatogonium cells in terms of their compatibility with in vivo media. The work analyzes the characterization of differentiated cells at both immunohistochemical and molecular levels to determine which cell stage of the spermatogenetic line can be differentiated functionally by comparing the culture systems that provide sperm-like cell differentiation from human spermatogonium cells. The fact that different culture systems are being investigated for their differentiation is also being studied extensively in terms of molecular analyzes, making the project unique from the projects already in the literature. In the project proposal, some of the samples of 8 patients who had non-obstructive azoospermia pre-diagnosis referred to Celal Bayar University, Family Planning Infertility Practice and Research Center and whose sperm were not sperm samples and after TESE were observed, should be routinely light microscopically and DIFF Qick staining and presence of spermatogonium cells. The remaining samples are incubated in a solution of trypsin / EDTA overnight at + 40 °C, then cultured in Ham's F10 on gelatinous culture dishes and cells that do not adhere to the culture chamber collected. Collected cells separated with CD90+ MiniMAcs and they will culture with differentiation culture medium in conventional 2B, Soft Agar (SACS), Collagen foam tissue scaffolds. In RT-PCR gene expression analysis, the complementary DNAs obtained from spermatogonial stem cells were used to express c-kit, Sirtl, Aurora-B, Aurora-C, Jmjdla and Akrozin gene expressions determined. GPR125 CD90, c-kit, α6 integrin, β1 integrin and akrozin distribution analyzed by indirect immunohistochemical method. According to immunohistochemical analysis of forward triggering differentiation in three different culture were evaluated, it was found that collagen foam tissue scaffolds were suitable for the maintenance and proliferation of germ cells, agar was shown to support the differentiation of spermatogenesis from the 15th day. According to the results of differentiation, it is thought to be up to the stage of primary spermatocytes or early secondary spermatocytes. In the agar group, immunoreactivity of GPR125 (days 15 and 22) were silm and Akrozin was strongly positive, suggests that agar, especially effective for triggering the differentiation. Particularly, Acrosine, immunoreactivity in agar and collagen on the 15 th day, while strongly positive positive, On the 22 day, the collagen positivity continued. It suggests that collagen, especially effective for triggering proliferation and differentiation. This positiveness strengthens the hypothesis. Spermatogonium cells obtained from TESE samples cultured with matrigel in the 2nd week of culture decreased c-kit expression, especially, the highest observation of Aurora-C expression was thought to support the differentiation of spermatogonium cells in the culture medium.