Effect of season on the in-vitro maturation and developmental competence of buffalo oocytes after somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) is a valuable technology tool with various uses in transgenic animals, regenerative medicine, and stem cell research. However, the efficiency of SCNT embryos appears to have poor developmental competency. Environmental issues may adversely affect SCNT embryos in buffalo. Thereafter, the present study aimed to explore the effect of season on the maturation of buffalo oocytes and subsequent developmental capability after parthenogenetic activation and SCNT in buffalo. Buffalo oocytes (n = 6353) were collected from local slaughterhouse at various seasons; spring (March-April), summer (May-August), autumn (September-November), and winter (December-January). A significant increase (p < 0.05) was recorded in the maturation rate (57.07%) at autumn compared with spring, summer, and winter (50.46, 50.93, and 50.66%, respectively). No significant differences were recorded in the fusion and the cleavage rates among all seasons. Blastocyst development rate was higher (p < 0.05) in autumn and winter (16.52 ± 8.45% and 15.98 ± 7.17%, respectively) than in spring and summer (9.47 ± 6.71% and 10.84 ± 6.58%, respectively) seasons. It could be concluded that the season had a significant effect on oocyte development competence which can be used for SCNT in buffalo.

Vitrification alters cell adhesion related genes in pre-implantation buffalo embryos: Protective role of ß-mercaptoethanol.

The objectives of the present study were to investigate the effect of vitrification on the expression of the key genes associated with blastocyst developmental potential (ß-catenin, E-cadherin, Oct-4, Cdx2, Gata3), and whether the presence of ß-mercaptoethanol (ß-ME, 100 µM) in in vitro culture (IVC) media will affect the expression of these genes. Buffalo pre-implantation embryos were divided into three groups: (1) fresh non-vitrified embryos were used as control, (2) vitrified embryos cultured with ß-ME (+), and (3) vitrified embryos cultured without (-) ß-ME. The results showed that all genes were affected by vitrification, however, the presence of ß-ME in IVC media significantly (P < 0.05) modified the expression level of ß-catenin, E-cadherin and Oct-4 in vitrified blastocyst compared to those cultured without ß-ME. Protein expression analysis by immunofluorescence and western blot also revealed that the expression level of ß-catenin and E-cadherin was significantly higher in vitrified embryos cultured with ß-ME than those cultured without ß-ME, which, in turn, was lower than fresh control group. However, there was no significant difference between vitrified groups in the expression level of Cdx2 and Gata3. Furthermore, the reduced rate of apoptosis in embryos cultured with ß-ME confirms its role in protecting vitrified blastocyst against stress. In summary, vitrification alters the expression of the adhesion related genes in vitrified blastocyst, which may explain, at least in part, the reason for the low pregnancy rate following transfer of such embryos into recipient animal, and the supplementation of IVC media with ß-ME significantly improved the quality of vitrified blastocyst evidenced by the modulation of the expression of blastocyst important genes, ß-catenin, E-cadherin and Oct-4, and the ability to protect vitrified blastocyst against apoptosis.

Developmental competence of buffalo (Bubalus bubalis) denuded oocytes cocultured with cumulus cells: Protective role of cumulus cells.

The objectives of the present study were to evaluate the developmental competence of buffalo denuded oocytes (DOs) cocultured with cumulus cells (CCs) during in vitro maturation, and to investigate the mechanisms by which CCs promote oocyte maturation and development. Buffalo oocytes were matured in vitro for 24 h in three groups: (1) intact cumulus-oocyte complexes (COCs) (2) DOs cocultured with CCs (DOsCC), and (3) DOs cultured alone (DOs). Matured oocytes were used to determine the relative mRNA abundance of Gdf-9, Bmp15, Zar1, Caspase-3, Bcl-2, Zp2, Zp3, Cd9 and Pde3a by Rt-qPCR and CASPASE-3 protein expression by immunofluorescence. The intracellular content of cGMP, cAMP and MPF activity and the rate of embryonic development were also assessed. Results of the present study showed that in DOs, the relative mRNA abundance of Gdf-9, Bmp15, and Cd9 significantly (P < 0.05) decreased, whereas Caspase-3 (mRNA and protein levels), Bcl-2, and Pde3a exhibited higher expression than DOsCC and COCs. However, there was no significant difference among the groups in the expression level of Zar-1, Zp2, and Zp3. The intracellular content of cAMP and MPF activity was notably higher (P < 0.05) in DOs compared to COCs and DOsCC. There was no significant difference between COCs and DOsCC in cGMP content, which was significantly lower (P < 0.05) in DOs. Moreover, the cleavage and blastocyst rates were 58.4 ±â€¯1.8%, 43.7 ±â€¯1.1%, 18.4 ±â€¯0.9% and 18.0 ±â€¯1.3%, 11.0 ±â€¯0.9% and 4.5 ±â€¯0.6% in COCs, DOsCC and DOs groups, respectively. In conclusion, the presence of CCs protects buffalo DOs from apoptosis and promotes maturation through regulation of the intracellular content of cAMP and MPF activity and improves the fertilizing capacity of oocytes through modulation of the gamete fusion gene, Cd9.

Buffalo oocyte-secreted factors promote cumulus cells apoptosis and the rate of cGMP production but not steroidogenesis.

The objectives of this study were to investigate the effect of buffalo oocyte-secreted factors (OSFs) on cumulus cells (CCs) functions, apoptosis and cGMP generation, and whether the direct contact between oocyte and CCs is essential for oocyte-mediated regulation of CCs functions. Buffalo CCs were cultured during IVM within three groups: (a) intact cumulus-oocyte complexes (COCs), (b) CCs cocultured with denuded oocytes (DOs) (CCs + DOs) and (c) CCs monolayer cultured alone (CCsM). After 24 hr of IVM, CCs were harvested for evaluation of the relative mRNA abundance of the genes encoding gap junction (GJA1), glycolysis (PFKP and LDHA), apoptosis (CASPASE-3 and BCL-2) and steroidogenesis (ER-ß and PGR) by QRT-PCR, and CASPASE-3 proteins, using western blot. Intracellular cGMP content was also assessed by ELISA. Results showed that the relative abundance of LDHA, PFKP and BCL-2 significantly increased (p < 0.05) in COCs, whereas GJA1 and CASPASE-3 exhibited lower expression (p < 0.05) compared to CCs + DOs and CCsM groups. However, the expression levels of CASPASE-3, both mRNA and protein, were significantly (p < 0.05) downregulated in CCs + DOs compared to CCsM. There was no significant difference in the expression level of PGR and ER-ß between the groups. The intracellular content of cGMP was notably (p < 0.05) higher in COCs compared to CCs + DOs and CCsM groups. In conclusion, this study demonstrated, for the first time, that buffalo OSFs protect CCs against apoptosis and stimulate their cGMP production; however, the regulation of cumulus glycolysis and gap junction is confined to those in close contact with the oocyte. Neither OSFs from COCs nor those from DOs have any effect on CCs steroidogenesis.