Izadyar F, Zeinstra E, Bevers M M
Department of Herd Health and Reproduction, Veterinary Faculty, Utrecht University, The Netherlands.
Mol Reprod Dev. 1998 Nov;51(3):339-45. doi: 10.1002/(SICI)1098-2795(199811)51:3<339::AID-MRD14>3.0.CO;2-Y.
This study was designed to investigate the effect of follicle-stimulating hormone (FSH) on nuclear maturation, fertilization, and early embryonic development of in-vitro-matured bovine oocytes and to find out whether this effect is exerted through a cyclic adenosine monophosphate (cAMP) signal transduction pathway. In addition the effect of the combination of FSH and growth hormone (GH) on subsequent cleavage and embryo development was studied. Therefore cumulus oocyte complexes were cultured in the presence of FSH (0.05 IU/ml) and the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenyl-indole (DAPI) staining either after 16, 20, or 24 hr of in vitro maturation or 18 hr after the onset of fertilization. To assess the effect of FSH and the combination of FSH and GH added during in vitro maturation on the developmental capacity of the oocytes, cumulus oocyte complexes were incubated in the presence of either FSH (0.05 IU/ml) or FSH (0.05 IU/ml) plus GH (100 ng/ml) for 22 hr, followed by in vitro fertilization and in vitro embryo culture. To investigate whether FSH-induced oocyte maturation is exerted through the cAMP pathway, cumulus oocyte complexes were cultured in M199 supplemented with FSH (0.05 IU/ml) and H-89 (10 microM), a specific inhibitor of cAMP-dependent protein kinase A. After 16 hr of culture, the proportion of oocytes in metaphase II (MII) stage was determined. Cultures with GH and without FSH and H-89 served as controls. The percentage of MII oocytes at 16 hr of incubation was significantly lower (P < 0.001) in the presence of FSH than in the control group, while the number of MII oocytes beyond 20 hr did not differ from the control group. That points to a transient inhibition of nuclear maturation by FSH. Opposite to FSH, addition of GH during in vitro maturation significantly enhanced the number of MII oocytes after 16 hr of culture (P < 0.001), which points to the acceleration of nuclear maturation by GH. Addition of FSH during in vitro maturation significantly enhanced the proportion of normal fertilized oocytes, cleaved embryos and blastocysts (P < 0.001). Similarly, addition of GH during in vitro maturation significantly enhanced the number of cleaved embryos and blastocysts (P < 0.001); however, in vitro maturation in the presence of GH and FSH did not result in an extra enhancement of the embryo development. Both the inhibition of nuclear maturation by FSH and its acceleration by GH was completely abolished by H-89. In conclusion, in vitro maturation of bovine oocytes in the presence of FSH retards nuclear maturation via a cAMP-mediated pathway, while it enhances fertilizability and developmental ability of the oocytes. Supplementation of GH and FSH during in vitro maturation did not result in an extra increase in the number of blastocysts following in vitro fertilization and in vitro embryo culture.
本研究旨在探讨促卵泡激素（FSH）对体外成熟牛卵母细胞核成熟、受精及早期胚胎发育的影响，并确定这种影响是否通过环磷酸腺苷（cAMP）信号转导途径发挥作用。此外，还研究了FSH与生长激素（GH）联合使用对后续卵裂和胚胎发育的影响。因此，将卵丘卵母细胞复合体在FSH（0.05 IU/ml）存在的情况下进行培养，并在体外成熟16、20或24小时后，或受精开始18小时后，使用4,6-二氨基-2-苯基吲哚（DAPI）染色评估卵母细胞的核阶段。为了评估体外成熟过程中添加FSH以及FSH与GH联合使用对卵母细胞发育能力的影响，将卵丘卵母细胞复合体在FSH（0.05 IU/ml）或FSH（0.05 IU/ml）加GH（100 ng/ml）存在的情况下孵育22小时，随后进行体外受精和体外胚胎培养。为了研究FSH诱导的卵母细胞成熟是否通过cAMP途径发挥作用，将卵丘卵母细胞复合体在补充有FSH（0.05 IU/ml）和H-89（10 μM）（一种cAMP依赖性蛋白激酶A的特异性抑制剂）的M199中进行培养。培养16小时后，确定处于中期II（MII）期的卵母细胞比例。以添加GH但不添加FSH和H-89的培养物作为对照。在FSH存在的情况下，孵育16小时时MII期卵母细胞的百分比显著低于对照组（P < 0.001），而20小时后MII期卵母细胞的数量与对照组无差异。这表明FSH对核成熟有短暂抑制作用。与FSH相反，体外成熟过程中添加GH显著增加了培养16小时后MII期卵母细胞的数量（P < 0.001），这表明GH加速了核成熟。体外成熟过程中添加FSH显著提高了正常受精卵母细胞、卵裂胚胎和囊胚的比例（P < 0.001）。同样，体外成熟过程中添加GH显著增加了卵裂胚胎和囊胚的数量（P < 0.001）；然而，在GH和FSH存在的情况下进行体外成熟并没有导致胚胎发育的额外增强。FSH对核成熟的抑制作用及其被GH加速的作用均被H-89完全消除。总之，在FSH存在的情况下，牛卵母细胞的体外成熟通过cAMP介导的途径延迟核成熟，同时增强了卵母细胞的受精能力和发育能力。体外成熟过程中补充GH和FSH并没有导致体外受精和体外胚胎培养后囊胚数量的额外增加。
