Stimulatory effect of growth hormone on in vitro maturation of bovine oocytes is exerted through the cyclic adenosine 3',5'-monophosphate signaling pathway.
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.
Izadyar
F
F
Department of Herd Health and Reproduction, Faculty of Veterinary Medicine, Utrecht University, The Netherlands. fari@bdv.dgk.ruu.nl
Colenbrander
B
B
Bevers
M M
MM
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002934:Cinnamates / Q000494:pharmacology; D003470:Culture Media; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D016048:Dideoxyadenosine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D013006:Growth Hormone / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D015398:Signal Transduction; D013449:Sulfonamides
D000067956:Adenylyl Cyclase Inhibitors; D002934:Cinnamates; D003470:Culture Media; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D013449:Sulfonamides; D016048:Dideoxyadenosine; D004815:Epidermal Growth Factor; C067472:methyl 2,5-dihydroxycinnamate; D005640:Follicle Stimulating Hormone; D013006:Growth Hormone; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide