Expression And Function Of Kisspeptin During Bovine Oocyte Maturation In Vitro

Kisspeptin is a polypeptide encoded by Ki SS-1 gene, it expressed in hypothalamus and controlled mammalian reproductive activities has been widely confirmed. Researches show that, Kisspeptin in addition to expressed in hypothalamus, also expressed in the ovary, but there is few research about its function and mechanism. The researches about porcine and rat show that Kisspeptin participate in the oocyte maturation, but its function according to species variation. It is not clear that whether Kisspeptin expressed in bovine oocyte or not and its function and mechanism in vitro maturation yet. This study first utilized immunofluorescence and q RT-PCR to study the location and regulation of expression of Kisspeptin in bovine oocytes in vitro maturation, and then studied the effect and mechanism of Kisspeptin in bovine oocytes in vitro maturation, the results mainly include following 2 parts: 1. Collect the oocytes of different incubation time(0, 6, 12, 18, 24h) and dye by immunofluorescence, then deserve the condition with fluorescence microscope. Results showed that Kp expressed in bovine oocyte at 0, 6, 12, 18, 24 h during in vitro maturation, and expressed at the same location. But the fluorescence intensity of oocytes showed, expression quantity changed continually in vitro maturation. Collect the oocytes of different incubation time(0, 6, 12, 18, 24h) and utilize q RT-PCR to detect the expression quantity of Ki SS-1, GPR54. Results showed that use basal medium to culture oocytes, expression quantity of Ki SS-1 and GPR54 are all the highest in 0h, they changed volatility and similarly during in vitro maturation. 2. According to different concentrations of exogenous Kisspeptin supplementation in medium divided into 5 groups, respectively, TCM-199 as control, 1nmol/L, 10nmol/L, 102nmol/L, 103nmol/L Kisspeptin supplemented as experimental groups, cultured COCs in vitro and statistics maturation rate, according to maturation rate ensure the optimal concentration of Kisspeptin supplementation. The results showed that the maturation rate of oocytes is the highest in 10nmol/L Kisspeptin supplemented, and it differ from others significantly(P <0.01), therefore determined 10nmol/L as the optimal concentration.According to the different treatment, medium could be divided into eight groups, respectively ① TCM-199 ② TCM-199 + Kisspeptin ③ TCM-199 + FSH ④TCM-199 + FSH + Kisspeptin ⑤TCM- 199 + Kisspeptin + FBS ⑥TCM-199 + FBS⑦TCM-199 + Kisspeptin + p234 ⑧TCM-199 + p234 + FSH, cultured COCs in vitro and statistics maturation rate. The results show that Kisspeptin group and Kisspeptin + FSH group have the high oocyte maturation rate, there was no significant difference between them, but were higher than FBS group significantly(P <0.01); the maturation rate of FBS + Kisspeptin group was low, and differed from Kisspeptin group significantly(P <0.01); the maturation rate of Kisspeptin + p234 group was lower than Kisspeptin group significantly(P <0.05); the maturation rate of FSH + p234 had no significant difference with FSH group.Utilize q RT-PCR detect the expression quantity of BMP15 and GDF9, key gene of oocyte maturation, during in vitro maturation(12, 24h). BMP15 and GDF9 have low expression in 12 h and 24 h, but they all increased obviously when supplemented with Kisspeptin in vitro maturation, it indicated that Kisspeptin can promote the expression of maturation relative genes.