The promotory effect of growth hormone on the developmental competence of in vitro matured bovine oocytes is due to improved cytoplasmic maturation.

摘要

To determine the cause of the stimulatory effect of growth hormone (GH) supplementation during in vitro maturation (IVM) on subsequent development of cattle embryos, cumulus-oocyte complexes (COC) were cultured for 22 h in M199 supplemented with 100ng/ml bovine GH (NIH-GH-B18) and fertilized in vitro. COC cultured without GH served as controls. To determine whether GH promotes fertilization by its effect on cumulus expansion or oocyte maturation, cumulus cells were removed from oocytes after IVM and denuded metaphase II (MII) oocytes were selected and fertilized in vitro. IVM and in vitro fertilization were performed at 39deg C in a humidified atmosphere with 5% CO2 in air. 18 h after the onset of fertilization, the nuclear stage of oocytes was assessed by 4,6-diamino-2-phenylindole (DAPI) staining. Oocytes with a metaphase I (MI) or MII nucleus and without a penetrated sperm head were considered unfertilized, oocytes with 2 pronuclei, zygotes and cleaved embryos were considered normally fertilized, and oocytes with >2 pronuclei were considered polyspermic. Cytoplasmic maturation was assessed by the distribution of cortical granules 22 h after the onset of IVM, and sperm aster formation 8 h after the onset of fertilization. To assess sperm-bindingcapacity, COC were fertilized in vitro, and 1 h after the onset of fertilization the number of spermatozoa bound to oocytes was counted. Addition of GH during IVM increased fertilization rate (80.2 vs. 67.6% in controls; P<0.001). Removal of the cumuluscells before fertilization and selection of the MII oocytes did not eliminate the positive effect of GH on fertilization. GH had no effect on the sperm-binding capacity of oocytes. GH supplementation during IVM increased (P<0.001) the migration of cortical granules and sperm aster formation. It is concluded that the stimulatory effect of GH on the developmental competence of the oocyte is due to improved cytoplasmic maturation.