Izadyar F, Colenbrander B, Bevers M M
Department of Herd Health and Reproduction, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.
Biol Reprod. 1997 Dec;57(6):1484-9. doi: 10.1095/biolreprod57.6.1484.
The aim of this study was to investigate whether the stimulatory effect of growth hormone (GH) on the in vitro maturation and cumulus expansion of bovine oocytes is exerted through the cAMP or the tyrosine kinase pathway. Therefore bovine cumulus-oocyte complexes (COCs) were cultured in Medium 199 without fetal calf serum and gonadotropins, but supplemented with 100 ng/ml bovine GH (bGH; NIH-GH-B18) with or without 10 microM methyl 2,5-dihydroxycinnamate (erbstatin analogue), a specific tyrosine kinase inhibitor; 100 microM 2',3'-dideoxyadenosine (DDA), a specific adenylate cyclase inhibitor; or 10 microM H-89, a specific inhibitor of cAMP-dependent protein kinase A. Epidermal growth factor (EGF; 20 ng/ml) was added as a positive control for tyrosine kinase activation, and FSH (0.05 IU/ml) was added as a positive control for cAMP mediation during in vitro maturation in the absence or presence of the inhibitors. Culture was performed at 39 degrees C in a humidified atmosphere with 5% CO2 in air. To assess the effect on nuclear maturation, the proportion of oocytes in metaphase II stage after 16 h of culture was determined using 4,6-diamino-2-phenylindole staining. To determine the effect on cumulus expansion, the diameter of COCs at the onset and after 24 h of culture was measured. The stimulatory effects of GH on oocyte maturation and cumulus expansion were blocked by DDA and H-89 (p < 0.01). Similarly, FSH-induced cumulus expansion was abolished by DDA and H-89 (p < 0.05), while DDA did not block either EGF-induced oocyte maturation or cumulus expansion. Erbstatin analogue significantly blocked the stimulation of oocyte maturation and cumulus expansion by EGF (p < 0.02) but did not inhibit GH action on the COCs. It is concluded that the stimulatory effect of GH on oocyte maturation and cumulus expansion is mediated by the cAMP signal transduction pathway and not by JAK2 phosphorylation.
本研究的目的是调查生长激素（GH）对牛卵母细胞体外成熟和卵丘扩展的刺激作用是通过环磷酸腺苷（cAMP）途径还是酪氨酸激酶途径发挥的。因此，将牛卵丘-卵母细胞复合体（COCs）培养于不含胎牛血清和促性腺激素的199培养基中，但添加100 ng/ml牛生长激素（bGH；NIH-GH-B18），同时添加或不添加10 μM 2,5-二羟基肉桂酸甲酯（埃布他汀类似物，一种特异性酪氨酸激酶抑制剂）；100 μM 2',3'-二脱氧腺苷（DDA，一种特异性腺苷酸环化酶抑制剂）；或10 μM H-89，一种cAMP依赖性蛋白激酶A的特异性抑制剂。添加表皮生长因子（EGF；20 ng/ml）作为酪氨酸激酶激活的阳性对照，添加促卵泡素（FSH；0.05 IU/ml）作为在有无抑制剂情况下体外成熟过程中cAMP介导的阳性对照。在39℃、含5%二氧化碳的湿润空气中进行培养。为评估对核成熟的影响，使用4,6-二脒基-2-苯基吲哚染色法测定培养16小时后处于中期II期的卵母细胞比例。为确定对卵丘扩展的影响，测量培养开始时及培养24小时后COCs的直径。DDA和H-89阻断了GH对卵母细胞成熟和卵丘扩展的刺激作用（p < 0.01）。同样，DDA和H-89消除了FSH诱导的卵丘扩展（p < 0.05），而DDA既未阻断EGF诱导的卵母细胞成熟也未阻断其卵丘扩展。埃布他汀类似物显著阻断了EGF对卵母细胞成熟和卵丘扩展的刺激作用（p < 0.02），但未抑制GH对COCs的作用。得出的结论是，GH对卵母细胞成熟和卵丘扩展的刺激作用是由cAMP信号转导途径介导的，而非由JAK2磷酸化介导。
