Evaluation of Differentiation of Spermatogonial Stem Cell Derived Non-Obstetric Azoospermic Men in Me-dia with Vitamin C Differentiation Factors In Vitro

Background: Spermatogonial cells (sscs) are responsible for spermatogenesis throughout life in the male.because disrup-tions of spermatogenesis in vitro in order to treat infertility is essential. Thise study was aimed to investigate isolation and enrichment of adult human SSCs, compare the in-vitro effects of collagen and gelatin with growth factors on the proliferation of SSCs and then induction in vitro differentiation of sscs with sertoli cells coculture and hormones in presence and absence of vitamin C.effects of the culture condition on differentiation and expression of the genes involved in spermatogenesis was evalu-ated as well as apoptosis Materials and Methods: TESE samples of nonobstructive azoospermic(NOA) patients, was dissociated with enzymatic digestion to obtain a cell suspention. The CD49f+ cells, as a marker to identify spermatogonial stem cells, were sorted using fluorescence-activated cell sorting (FACS). Isolated testicular cells were cultured IN dmem supplemented with GDNF, EGF and LIF in the presence and absence of collagen and gelatin coated-dish. Colony assay was performed during culture. Pres-ence of spermatogonia in colonies was determined by an immo-nucytochemistry based on OCT4 and α6-integrin expression. On the other hand, sertoli cells were isolated by Lectin DSA ad culture.For in vitro differentiation, the cells that cultured in previous step cultivated on sertol cells in present of hormones with and without vitamin C after 4 weeks.After 2 and 4 weeks the cells ere collected in in different groups and the effects of supplementary media on the differentiation induction and ex-pression of the meiosis and postmeiosis genes including C-KIT, SCP3, TH2b. PRM and acrosin were evaluated by RT-PCR and immunocytochemistry. Also after 2and 4 weeks apoptosis of cells was assessed by annexin V/P1 and flow cytometery Results: SSCs enrichment was up to 75% .the findings indicat-ed that the addition of GDNF, EGF and LIF on collagen-coated dishes significantly increased in vitro spermatogonial cell num-ber and colony formation in comparsion with the other group. The expression of spermatogonial markers include OCT4 and α6-integrin was maintained throughout the proliferation culture period.C-KIT, SCP3,TH2b,TP1,PRM and Acrosin were ex-pressed in both with and without vitamin groups.expression of genes in vitamin group was better than without vitamin groups. Viabillity invitamin group was more than withoutvitamin group (P<0.001) and present study indicate that CD49f is a suitable marker for SSCs isolation.Conclusion: Furthermore concluded that human SSCs obtained From NOA patients had the ability to self-renew in the culture system and collagen coated dish with growth factors can be used for the propagation of a small number of these cells from small biopsies. Coculture of SSCs Obtained from non-obstruc-tive azoospermic (NOA) Patients with sertoli cells in presence of hormones and vitamin C play an important role in meiosis induction and maintain of SSCs. Our systems may provide new window to treatment of maile infertility.

کلیدواژه ها:

Spermatogonia ، Azospermi ، Vitamin C