Effect of α-tocopherol in the vitrification medium on the viability, lipid peroxidation, expression of key developmental, apoptotic and stress-related genes in ovine secondary follicles.

The present study aimed to evaluate the effect of α-tocopherol on viability, lipid peroxidation and the expression of apoptosis, stress and development-related genes in the vitrified sheep secondary follicles. Ovarian secondary follicles (200-300 µm) were isolated and distributed separately to the vitrification treatment and supplemented with 5 mM, 10 mM, 20 mM and 30 mM of α-tocopherol (while the control fresh group was without vitrification and supplementation of α-tocopherol). After a week, the follicles were thawed and evaluated for follicular viability by trypan blue dye exclusion method, lipid peroxidation and gene expression studies. The results showed that the vitrification with 10 and 20 mM of α-tocopherol positively affected (p < .05) the viability of vitrified follicles in comparison with vitrified ones without α-tocopherol but the higher concentration of α-tocopherol, i.e., 30 mM negatively affected the viability (p < .05) in comparison with the 10 and 20 mM of α-tocopherol groups. The malondialdehyde (MDA) levels were significantly (p < .05) higher in the vitrified without α-tocopherol group in comparison to the vitrified with 20 mM of α-tocopherol group. The expression of apoptotic-related gene, BCL2L1 was significantly higher in 10 mM α-tocopherol group compared to the control fresh and CASPASE 3, 9 expressions were significantly higher in the vitrified group when compared to the vitrified with 10 mM α-tocopherol group. Expressions of BAX, BAD, BAK, BMP-15 and GDF-9 showed no significant difference among the groups. The mRNA expression of SOD1 was significantly higher in the vitrified without α-tocopherol group when compared to other groups. We conclude that the supplementation of 10 and 20 mM α-tocopherol in vitrification solution was the efficient vitrification procedure for the vitrification of ovine secondary follicles.

Regulatory role of Wnt signal in the oestradiol synthesis of different size categories of ovarian follicles in buffalo (Bubalus bubalis).

The aim of the present study was to understand the role of Wnt signal in ovarian oestradiol synthesis in various size categories of ovarian follicles. A six-day cell culture system was adopted to test the effect of a Wnt inhibitor i.e. Inhibitor of Wnt response (IWR) on the ovarian granulosa cell oestradiol synthesis and associated genes related to oestradiol synthesis and Wnt signalling (CYP19A1, CCND2, WNT2, FZD6, DVL1, APC, AXIN2, CTNNB1) in buffalo. It was conducted with four groups: Group 1: control, Group 2: control + FSH, Group 3: IWR, Group 4: IWR + FSH. No significant effect of IWR was observed on the ovarian granulosa cell proliferation. No significant difference in the oestradiol levels was found in the spent media harvested after six days of in vitro culture among different groups in small and large-sized ovarian follicles. However, the oestradiol level varied significantly (p < .05) among different treatment groups in medium-sized follicles. The oestradiol level was significantly lower (p < .05) in IWR group compared with the control group and was also significantly lower in IWR + FSH group compared with the FSH group. The Wnt inhibitor had significantly (p < .05) reduced the gene expression of CYP19A1 in large ovarian follicles. Varied effects of IWR-1 and FSH on the expression of other genes were observed. The results indicated that there is a positive role of Wnt signal in oestradiol synthesis in buffalo, but the positive role was more discernible in medium- and large-sized follicles.

Effect of different vitrification protocols on post thaw viability and gene expression of ovine preantral follicles.

The aim of the present study was to establish a vitrification protocol for ovine preantral follicles, which can retain viability after thawing and to evaluate the impact of different vitrification treatments on apoptosis and development-related gene expression. Preantral follicles were isolated from cortical slices of ovaries by the mechanical method of isolation. The isolated preantral follicles (200-300 µm) were randomly assigned into four groups. Group1 - Control Fresh preantral follicles (256 follicles); Group 2- Vitrification treatment A (259 follicles) (Vitrification solution 1 (VS1) - Fetal bovine serum (FBS)10%, Ethylene glycol (EG):1.8 M, Dimethyl sulfoxide (DMSO): 1.4 M, Sucrose-0.3 M for 4 min; VS2- FBS10%, EG:4.5 M, DMSO: 3.5 M, Sucrose:0.3 M for 45 s), Group 3 - Vitr. treatment B (235 follicles) (VS1-FBS 20%, EG:1.3 M, DMSO1.05 M for 15 min, VS2- FBS 20%, EG:2.7 M, DMSO:2.1 M for 5 min) and Group 4-Vitrification treatment C (248 follicles) (VS1-Glycerol(Gly):1.2 M for 3 min, VS2- Gly:1.2 M, EG:3.6 M for 3 min, VS3- Gly3M, EG: 4.5 M for 1 min). Preantral follicles were placed in corresponding vitrification treatments and later plunged immediately into liquid nitrogen (-196 °C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue dye exclusion method as well as for gene expression. The results showed that the low concentration of cryoprotectants (vitrification treatment B) negatively affected the viability of preantral follicles in comparison with control follicles. There was no significant difference in the viability rates among the Control (87%), Treatment A (79%) and Treatment C (75%). The percentage of viable preantral follicles (73%) derived from Treatment B was significantly decreased (P<0.05%) in comparison to that of control. The expression of apoptotic gene BAK was higher in the vitrification treatment B group. Expressions of the other apoptosis-related genes i.e. Bcl2L1, BAD, BAX, Caspase 3, and Annexin showed no significant difference among the groups. The expression pattern of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in vitrification treatment A and C, respectively. Expression of NOBOX gene was significantly increased in preantral follicles with Vitrification treatment B compared to the control group. We conclude that both the Vitrification treatment A and Treatment C were the efficient vitrification treatment methods for the vitrification of ovine preantral follicles.

Effect of retinol as antioxidant on the post-thaw viability and the expression of apoptosis and developmental competence-related genes of vitrified preantral follicles in buffalo (Bubalus bubalis).

The present study evaluated the effect of supplementation of retinol in the vitrification solution on the viability, apoptosis and development-related gene expression in vitrified buffalo preantral follicles. Preantral follicles isolated from cortical slices of ovaries were randomly assigned into three groups: Group1-Control fresh preantral follicles; Group 2-Vitrification treatment (Vitrification solution 1 (VS1) -TCM-199 + 25 mM HEPES + Foetal bovine serum (FBS) 10%, Ethylene glycol (EG): 10%, Dimethyl sulphoxide (DMSO): 10%, Sucrose-0.3 M for 4 min; VS2- TCM-199 + 25 mM HEPES + FBS10%, EG:25%, DMSO: 25%, Sucrose:0.3 M for 45 s); Group3-vitrification treatment +5 µM of Retinol. Preantral follicles were placed in corresponding vitrification medium and plunged into liquid nitrogen (-196°C). After a week, the follicles were thawed and analysed for follicular viability and gene expression. There was no significant difference in the viability rates among the Group 1(Fresh preantral follicles) (91.46 ± 2.39%), Group 2 (89.59 ± 2.46%) and Group 3 (87.19 ± 4.05%). There was a significantly (p < .05) higher mRNA expression of BCL2L1, GDF-9 and BMP-15 in the vitrification + retinol group compared with the control group. There was a significantly (p < .05) higher expression of Caspase-3 and Annexin-5 in the vitrification group and Vitrification + retinol group compared with control group of follicles. It is concluded that the supplementation of 5 µM of Retinol in Vitrification solution was an efficient vitrification procedure for the vitrification of buffalo preantral follicles.

Copper and Selenium stimulates CYP19A1 expression in caprine ovarian granulosa cells: possible involvement of AKT and WNT signalling pathways.

The role of copper and selenium on activation of estradiol synthesis pathways viz. PKA/AKT/WNT is not clearly elucidated. On this background we attempt to elcuiated the role of copper and selenium on mRNA expression of genes associated with estradiol synthesis in caprine ovarian granulose cell models. Ovarian granulosa cells from medium (3-5 mm) sized follicles were aspirated and distributed separately to different groups. Group I: control, Group II: cupric chloride (Cu: 0.5 mM), Group III: sodium selenite (Se: 100 ng/ml), Group IV: Cu + Se. The cells (105/well) were cultured in 96 well plate in the base culture medium of MEMα comprising of nonessential amino acids (1.1 mM), FSH (10 ng/mL), transferrin (5 µg/mL), IGF-I (2 ng/mL), androstenedione (10-6 M), penicillin (100 IU/mL), streptomycin (0.1 mg/mL) and fungizone (0.625 µl/mL) and insulin (1 ng/mL). The cells were incubated in a carbondioxide incubator (38 °C, 5% CO2, 95% RH). The medium was changed on alternate days and cells were harvested on day 6. Day 6 media was used for estimation of estradiol. The RNA isolated form harvested cells was used for qPCR assay. There was no significant (p > 0.05) difference in estradiol concentration between groups. The mRNA expression of AKT1, CYP19A1, WNT2 & 4, FZD6 and APC2 were significantly (p < 0.05) higher in Cu and Cu + Se groups compared to control. Whereas, the mRNA transcript of DVL1 and CSNK1 was significantly (p < 0.05) higher in Cu + Se group compared to control. Incontrast, no significant difference in mRNA expression of PRKAR1A and CTNNB1 was noticed. Our study support a key role of copper and selenium in activation of AKT and WNT signalling pathway that further lead to increase in the mRNA expression of CYP19A1.