245 CONNEXIN 43 mRNA EXPRESSION AT DIFFERENT TIME POINT IN IN VITRO MATURATION OF BUFFALO OOCYTES

Abstract

In advanced technologies of ART, the basic requirement is the production of in vitro-matured oocytes, and embryo production efficiency depends on healthy, matured oocytes. Oocyte growth and development depends on the ability of oocytes and their surrounding cumulus granulosa cells (Eppig et al. 1979 J. Exp. Zool. 208, 111–120). Cumulus cells provide carbohydrate precursors, amino acids, and nucleotides to the oocytes (Brower and Schultz 1982 Dev. Biol. 90, 144–153). Oocytes and cumulus cell gap junctions are required for the coordination of cytoplasmic and nuclear maturation (Carabatsos et al. 2002 Dev. Biol. 226, 167–179). In bovine COC, functional gap junctions are required for the progression of oocyte maturation. Gap junctions allow for metabolic coupling between adjacent granulosa cells. Disruption in the integrity of the gap junction inhibits oocyte maturation (Anderson and Albertini 1976 J. Cell Biol. 71, 680–686). The aim of this study was to analyze the trend of Cx43 mRNA transcript in in vitro-matured oocytes at different times of maturation in the Indian water buffalo to estimate the correlation with expression level. Oocytes collected from slaughterhouse ovaries were matured in TCM-199 medium supplemented with 2.5 mm pyruvate, gentamycin sulfate (10 mg mL^–1), β-estradiol (1000 ng mL^–1), FSH (500 ng mL^–1), LH (500 ng mL^–1), and 10% FBS at 38.5°C in 5% CO₂ in air. Cumulus–oocyte complexes were used after 0, 6, 12, 18, and 24 h of maturation for the cDNA preparation with cells of a cDNA II Kit. Expression of the Cx43 gene was quantified at different time intervals for maturation with real-time PCR. Statistical analysis was performed with one-way ANOVA, followed by Duncan’s multiple pair-wise comparison. Our results showed that Cx43 mRNA abundance was affected by time of maturation. The expression of Cx43 was significantly higher at 6 h than at 18 and 24 h, whereas the 12-h value was intermediate. Our results are in agreement with decreased Cx43 protein contents in the outer cumulus layers of COC at maturation time points (Calder et al. 2003 Reprod. Biol. Endocrinol. 1, 14) and the expression of Cx43 in oocyte development regulation (Granot et al. 2002 Biol. Reprod. 66, 568–573). When Cx43 expression was compared among immature oocytes, denuded oocytes, cumulus cells, and COC at 6 h, there was no significant difference. However, 6-h-matured COC showed significantly higher expression than other groups. Further, our study supported the role of cumulus cells in COC in Cx43-mediated communication (Vozzi et al. 2001 Reproduction 122, 619–628). Differential expression of Cx43 mRNA among varying COC classes indicates that this gene may be a useful marker for oocyte quality to improve in vitro production or somatic cell nuclear transfer rates. Marker genes that predict developmental competence could be used in the optimization of maturation and culture conditions. Understanding the molecular mechanism involved in in vitro oocyte maturation would be an additional advantage in analyzing this complex biological phenomenon to improve embryo production.