Gamal A. Sosa

Evidence of melatonin synthesis in the cumulus oocyte complexes and its role in enhancing oocyte maturation in vitro in cattle

Melatonin is a multifunctional molecule that mediates several circadian and seasonal reproductive processes. The exact role of melatonin in modulating reproduction, however, is not fully understood—especially its effects on the ovarian follicles and oocytes. This study was conducted to investigate the expressions of the ASMT and melatonin‐receptor MTNR1A and MTNR1B genes in bovine oocytes and their cumulus cells, as well as the effects of melatonin on oocyte nuclear and cytoplasmic maturation in vitro. Cumulus‐oocyte complexes (COCs) from abattoir ovaries were cultured in TCM‐199 supplemented with melatonin at concentrations of 0, 10, 50, and 100 ng/ml. The expression of ASMT, MTNR1A, and MTNR1B genes was evaluated by RT‐PCR. Moreover, the effects of melatonin on cumulus cell expansion, nuclear maturation, mitochondrial characteristics and COCs steroidogenesis were investigated. Furthermore, the level of reactive oxygen species (ROS) was evaluated in denuded oocytes. Our study revealed that ASMT and MTNR1A genes were expressed in COCs, while the MTNR1B gene was expressed only in oocytes. Additionally, melatonin supplementation at 10 and 50 ng/ml to in vitro maturation medium significantly enhanced oocyte nuclear maturation, cumulus cell expansion and altered the mitochondrial distribution patterns, but had no effects on oocyte mitochondrial activity and COCs steroidogenesis. Melatonin‐treated oocytes had a significantly lower level of ROS than controls. The presence of melatonin receptors in COCs and its promoting effects on oocyte nuclear and cytoplasmic events, indicate the potentially important roles of this hormone in regulating bovine oocyte maturation. Moreover, the presence of ASMT transcript in COCs suggests the possible involvement of these cells in melatonin biosynthesis. Mol. Reprod. Dev. 78:250–262, 2011.

Effect of using ascorbic acid and cysteamine supplementation on in-vitro development of buffalo embryos

Objective: To improve in vitro embryo production in buffalo by supplementation of L-ascorbic acid during maturation and development (experiment 1) and combination with another antioxidant as cysteamine (experiment 2). Methods: Two experiments were performed, the first one aimed to evaluate the different concentrations (0, 25, 50, 100 μM) of L-ascorbic acid on embryo developmental rate of buffalo oocytes. The L-ascorbic acid was added to the maturation and culture media. In the second experiment, oocytes were cultured in media with two type of antioxidant (ascorbic acid + cysteamine) or ascorbic acid only. Results: There was a significant increase in cleavage rate at 25, 50 μM than 100 μM and control group. But, the blastocyst rate was higher at 50 μM ascorbic acid than other concentrations (0, 25, 100 μM). Supplementation of ascorbic acid and cysteamine to maturation and cultured media improved embryo development than ascorbic acid alone. Conclusions: Using of 50 μM L-ascorbic acid during in vitro maturation and development improve the developmental competence of buffalo oocytes, this effect was increase with the presence of cysteamine.