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This study was carried out to determine the effects of growth factors (epidermal growth factor, transforming growth factors-α and -β1, basic fibroblast growth factor, insulin), gonadotrophins (LH, FSH), and fetal bovine serum added to TCM199 medium on cumulus expansion and fertilization during in vitro maturation, and on subsequent embryonic development of bovine cumulus cell-enclosed oocytes. Epidermal growth factor, transforming growth factor-α, LH and FSH enhanced cumulus expansion and oocyte fertilizability. No significant effect was achieved with transforming growth factor-β1 nor with basic fibroblast growth factor. No additive stimulation on cumulus expansion and oocyte fertilizability was observed when epidermal growth factor was combined with LH or FSH. The addition of either epidermal growth factor or transforming growth factor-α to the maturation medium increased the number of fertilized ova that developed to the blastocyst stage. These results demonstrate the potential use of epidermal growth factor and transforming growth factor-α in obtaining high quality mature bovine oocytes for in vitro fertilization.
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The presence and possible role of protein kinase C in the regulation of fowl sperm functions were investigated. Immunoblot analysis of sperm extract using antibody to protein kinase C revealed a crossreacting protein of approximately 80 kDa. As the concentration of the protein kinase C activators N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9) or 1-oleoyl-2-acetylglycerol (OAG) was increased, the motility of intact spermatozoa at 30°C was reduced. However, this inhibition of motility was reversed by reducing the concentrations of activators. Even in the presence of 1 mmol CaCl2 l−1, the addition of SC-9 and OAG inhibited the motility of intact spermatozoa. In contrast, the motility of demembranated spermatozoa was not inhibited by the addition of SC-9 or OAG at 30°C. However, the addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a protein kinase C inhibitor, did not appreciably affect the motility of either intact or demembranated spermatozoa at 30°C. At 40°C, both intact and demembranated spermatozoa were almost immotile in the presence or absence of the activators or inhibitor. Intracellular free Ca2+ concentrations, measured by means of a fluorescent Ca2+ indicator, fura-2, gradually increased after the addition of SC-9 and OAG, but no changes were observed in H-7-treated spermatozoa. These results suggest that endogenous protein kinase C is present in the cytoplasmic matrix or the membrane, but is not retained in the axoneme, and that the activation of this enzyme may contribute to a decrease in the flagellar movement of fowl spermatozoa.
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Summary. Endometrial glandular cells and mononuclear phagocytic cells dominated the samples of undiluted uterine fluid obtained at different stages of the menstrual cycle whereas granulocytes were present in much lower numbers. Mast cells were occasionally found. The number of each cell type was significantly decreased in the luteal phase as compared to the midcycle and premenstrual phases. Phagocytic activity of the mononuclear cells was also significantly decreased in the luteal phase. Low numbers of inflammatory cells and low phagocytic activity during the luteal phase may be important for survival of the blastocyst in the event of conception.
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