title: Development of early bovine embryos to the blastocyst stage in serum-free conditioned medium from bovine granulosa cells.
abstract: Bovine granulosa cell-conditioned medium (BGC-CM) was prepared in a serum-free medium consisting of TCM 199, 5 micrograms/ml insulin, and 0.5 micrograms/ml aprotinin (TCM 199 IAP). Granulosa cells surrounded with embryos were denuded 24 to 30 h after in vitro fertilization. The proportion of denuded granulosa cell-free embryos that developed to the blastocyst stage in BGC-CM (43/219; 20%) as well as in the co-culture system (43/178; 24%) was significantly greater (P less than 0.001) than in fresh TCM 199 IAP medium (FM: 10/191; 5%), whereas the proportion of embryos that developed to the eight-cell stage was similar (P greater than 0.05) in all three culture systems (95/178; 53% in co-culture, 111/219; 51% in BGC-CM, and 86/191; 45% in FM, respectively). Higher rates of hatching and hatched blastocysts 8.5 days after in vitro fertilization were observed in co-culture (13/44; 29.5%) and in conditioned medium (8/39; 20.5%). On the other hand, no hatching or hatched blastocysts were obtained in the fresh medium (0/7; 0%). Cell numbers per blastocyst in BGC-CM (178.3 cells/blastocyst) were approximately two-fold higher than those in FM (97.1 cells/blastocyst). However, higher cell numbers (249.3 cells/blastocyst) were observed in co-culture with BGC than in BGC-CM. The embryotrophic activity in BGC-CM was stable upon freezing and thawing, lyophilization, and heating at 56 degrees C whereas activity was reduced by dilution in fresh medium, dialysis, pronase digestion, and heating at 80 degrees C. These results suggest that BGC cultured in a serum-free medium can synthesize and secrete an embryotrophic factor(s) that supports blastocyst formation in vitro beyond the 8- to 16-cell stage.
authors.affiliation: Research Institute for the Functional Peptides, Yamagata, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kobayashi
authors.forename: Y
authors.initials: Y
authors.lastname: Takagi
authors.forename: T
authors.initials: T
authors.lastname: Satoh
authors.forename: H
authors.initials: H
authors.lastname: Hoshi
authors.forename: T
authors.initials: T
authors.lastname: Oikawa
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D001769:Blood; D002417:Cattle; D002478:Cells, Cultured; D003470:Culture Media; D003956:Dialysis; D005260:Female; D006107:Granulosa Cells / Q000502:physiology*; D006358:Hot Temperature; D011402:Pronase / Q000494:pharmacology
chemical_list: D003470:Culture Media; D011402:Pronase