THE PROMOTORY EFFECT OF GROWTH-HORMONE ON THE DEVELOPMENTAL COMPETENCE OF IN-VITRO MATURED BOVINE OOCYTES IS DUE TO IMPROVED CYTOPLASMIC MATURATION

In a previous study we have shown that the addition of growth hormone (GH) during in vitro maturation accelerates nuclear maturation, induce s cumulus expansion, and promotes subsequent cleavage and embryonic de velopment. The aim of this study was to investigate whether the promot ory effect of GH on subsequent cleavage and blastocyst formation is du e to an improved fertilization and whether this effect is caused by an improved cytoplasmic maturation of the oocyte. Therefore, bovine cumu lus oocyte complexes (COCs) were cultured for 22 hours in M199 supplem ented with 100 ng/ml bovine GH (NIH-GH-B18). Subsequently the COCs wer e fertilized in vitro. Cultures without GH served as controls. To veri fy whether the promoted fertilization is caused by the effect of GH on cumulus expansion or oocyte maturation, cumulus cells were removed fr om the oocytes after in vitro maturation (IVM) and denuded MII oocytes were selected and fertilized in vitro. Bath IVM and in vitro fertiliz ation (IVF) were performed at 39 degrees C in a humidified atmosphere with 5% CO2 in air. At 18 hours after the onset of fertilization, the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenylin dole (DAPI) staining. Oocytes with either an metaphase I (MI) or MII n uclear stage and without penetrated sperm head were considered unferti lized; oocytes with two pronuclei, zygotes, and cleaved embryos were c onsidered normally fertilized; and oocytes with more than two pronucle i were considered polyspermic. To evaluate cytoplasmic maturation, the distribution of cortical granules 22 hours after the onset of IVM, an d sperm aster formation 8 hours after the onset of fertilization were assessed. In addition, to assess the sperm-binding capacity, COCs were fertilized in vitro, and 1 hour after the onset of fertilization the number of spermatozoa bound to the oocytes was counted. The addition o f GH during IVM significantly (P < 0.001) enhanced the proportion of n ormal fertilized oocytes. Removal of the cumulus cells prior to fertil ization and selection of the MII oocytes did not eliminate the positiv e effect of GH on fertilization. No effect of GH on the sperm-binding capacity of the oocyte was observed. In addition, GH supplementation d uring IVM significantly (P < 0.001) enhanced the migration of cortical granules and sperm aster formation. It can be concluded that the prom otory effect of GH on the developmental competence of the oocyte is du e to a higher fertilization rate as a consequence of an improved cytop lasmic maturation. (C) 1998 Wiley-Liss, Inc.