Propagation of goat putative spermatogonial stem cells under growth factors defined serum-free culture conditions.

In the present study, we used a serum-free culture media to propagate goat putative spermatogonial stem cells (SSCs) and evaluated the effect of crucial growth factors on relative expression of some SSC markers and self-renewal related genes. The enriched SSCs were cultured on a homologous Sertoli cell feeder layer in KO-DMEM supplemented with 10% KOSR. Putative SSC colonies emerged between day 6 and 10 which were then characterized by the expression of numerous spermatogonial and pluripotency related markers. After 15 days of subculture, the relative mRNA expression study revealed that 40 ng/mL concentration of Glial cell line-derived neurotrophic factor (GDNF) upregulated the expression of BCL6B, ID4, PLZF, and UCHL1. Moreover, the supplementation of GDNF + bFGF up-regulated the expression of PLZF and BCL6B. UCHL1 expression was higher after addition of GDNF + LIF while, THY1 overexpressed in response to the addition of GDNF + CSF1. These results demonstrated that the goat SSCs were efficiently propagated using a KOSR based serum-free media and the growth factor supplementation markedly influences their gene expression profile.

Optimization of Serum-Free Culture Conditions for Propagation of Putative Buffalo (Bubalus bubalis) Spermatogonial Stem Cells.

Spermatogonial stem cells (SSCs) self-renew and produce a large number of differentiated germ cells to maintain normal spermatogenesis. However, the growth factors crucial for SSC self-renewal and the mechanism underlying this process remain unclear. In the present study, a serum-free culture media was used to evaluate the effect of several growth factors on the expression of some SSC markers and self-renewal related genes. The putative SSCs were cultured on buffalo Sertoli cell feeder layer in KO-DMEM +10% KOSR. The colony formation was observed between 7 and 10 days. The putative SSC colonies also expressed markers specific for undifferentiated type A spermatogonia and pluripotency markers. After 15 days, relative mRNA expression study revealed that 20 ng/mL concentration of Glial cell line-derived neurotrophic factor (GDNF) upregulated the expression of PLZF, TAF4B, and THY1. Furthermore, supplementation of a combination of 20 ng/mL GDNF, 10 ng/mL basic fibroblast growth factor (bFGF), 1000 IU/mL leukemia inhibitory factor (LIF), and 1 ng/mL colony stimulating factor 1 (CSF1) upregulated the expression of PLZF, TAF4B, BCL6B, and ID4 genes. These results demonstrated that our defined culture media in combination with GDNF, bFGF, LIF, and CSF1 well supported SSC self-renewal.

Supplementation of Glial Cell Line-Derived Neurotrophic Factor, Fibroblast Growth Factor 2, and Epidermal Growth Factor Promotes Self-Renewal of Putative Buffalo (Bubalus bubalis) Spermatogonial Stem Cells by Upregulating the Expression of miR-20b, miR-21, and miR-106a.

In this study, we investigated the effect of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF) 2, and epidermal growth factor (EGF) on the expression of some self-renewal-related microRNAs (miRs) in putative buffalo spermatogonial stem cells (SSCs). The SSCs were cultured on a buffalo Sertoli cell feeder layer, colony formation was observed between 7 and 10 days. The SSC colonies expressed markers specific for undifferentiated type A spermatogonia and pluripotency markers. After 15 days of initial culture, the colonies were subcultured as treatment (supplemented with 20 ng mL-1 GDNF +10 ng mL-1 FGF2 + 10 ng mL-1 EGF) and control groups. The number and area of SSC colonies were significantly (p < 0.05) higher in the treatment group than in the control group. The relative abundance of miR-20b, miR-21, and miR-106a in SSCs supplemented with growth factors was significantly higher (p < 0.001) than that in the control. The results indicate that supplementation of SSC culture medium with growth factors (GDNF, FGF2, and EGF) may promote the expression of miR-20b, miR-21, and miR-106a, which is essential for self-renewal and maintenance of SSCs.

Supplementation of tauroursodeoxycholic acid during IVC did not enhance in vitro development and quality of buffalo IVF embryos but combated endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress, a dysfunction in protein-folding capacity of ER, is involved in many pathologic and physiological responses including embryonic development. This study investigated the effect of supplementation of IVC medium with an ER stress inducer, tunicamycin (TM), and an inhibitor, tauroursodeoxycholic acid (TUDCA), on the developmental competence, apoptosis, and gene expression in buffalo embryos produced by IVF. Treatment of presumed zygotes with TM resulted in a significant (P < 0.01) decrease in the blastocyst rate, whereas TUDCA supplementation did not improve the blastocyst development rate. Further, presence of TUDCA could not ameliorate the adverse effects of TM in terms of the blastocyst rate in combined (TM + TUDCA) treatment. Tunicamycin treatment increased (P < 0.01) the apoptotic index and reduced the total cell number, whereas TUDCA did not affect them significantly. However, TUDCA reduced the extent of TM-mediated apoptosis during combined (TM + TUDCA) treatment. Tunicamycin treatment increased (P < 0.01) and TUDCA treatment decreased (P < 0.01) the expression level of ER chaperones, GRP78 and GRP94. In the combined TM + TUDCA treatment, TUDCA decreased their expression level compared to that in the controls. A similar pattern was observed in the case of proapoptotic gene BAX. We did not find any significant difference in the expression level of BCl-XL, BID, P53, and CASPASE 3 after TM and TUDCA supplementation. In conclusion, our study reported that TM induces ER stress in buffalo embryos produced in vitro resulting in a decrease in the blastocyst rate and an increase in the level of apoptosis and that these actions are mediated by modulating the expression of apoptosis-related genes and ER chaperones. Tauroursodeoxycholic acid did not improve the developmental potential of buffalo embryos; however, it attenuated the TM-induced apoptosis by downregulating BAX and ER chaperones.