title: In vitro development of bovine embryos in conditioned media from bovine granulosa cells and vero cells cultured in exogenous protein- and amino acid-free chemically defined human tubal fluid medium.
abstract: We have investigated the effect of protein- and amino acid-free simple human tubal fluid (HTF) medium conditioned with bovine granulosa cells (BGC) or Vero cells on the development of early bovine embryos to the blastocyst stage. Serum-containing medium (SCM) and serum-free medium (CM) conditioned by BGC (BGC-SCM, BGC-CM) and by Vero cells (VC-SCM, VC-CM) were prepared. Early embryos (1-cell stage and 5- to 8-cell stage) were obtained by in vitro maturation and fertilization of oocytes from slaughtered cows. Embryos were randomly divided into 7 culture groups as follows: culture with BGC-SCM, BGC-CM, VC-SCM, or VC-CM; coculture with BGC or Vero cells; or culture with fresh HTF medium without serum. The proportion of 5- to 8-cell embryos developing to the blastocyst stage in BGC-CM (16%) and VC-CM (12%) was significantly lower (p < 0.05) than in BGC-SCM (41%) and in VC-SCM (29%) and after coculture with BGC (48%) and Vero cells (30%). Similarly, the percentages of 1-cell embryos developing to blastocyst in BGC-CM and VC-CM were significantly lower than in BGC-SCM and VC-SCM and after coculture. Cell numbers per blastocyst developed from 5- to 8-cell embryos in BGC-CM (96.8 cells) and in VC-CM (94.0 cells) were somewhat lower than those in BGC-SCM (128.5 cells) and VC-SCM (117.1 cells) and after coculture with BGC (124.2 cells) and Vero cells (115.3 cells). These results suggest that BGC and Vero cells cultured in a protein- and amino acid-free simple HTF medium synthesize and secrete factor(s) promoting blastocyst formation in vitro. Physiochemical analysis indicated that the embryotrophic substances in BGC-CM were distributed in two molecular size ranges, one between 10 kDa and 30 kDa and another greater than 30 kDa.
authors.affiliation: Department of Obstetrics and Gynecology, Fukui Medical School, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Maeda
authors.forename: F
authors.initials: F
authors.lastname: Kotsuji
authors.forename: A
authors.initials: A
authors.lastname: Negami
authors.forename: N
authors.initials: N
authors.lastname: Kamitani
authors.forename: T
authors.initials: T
authors.lastname: Tominaga
mesh_terms: D000596:Amino Acids / Q000502:physiology; D000818:Animals; D001826:Body Fluids / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002522:Chlorocebus aethiops; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000737:chemistry; D004622:Embryo, Mammalian / Q000502:physiology*; D005187:Fallopian Tubes / Q000502:physiology*; D005260:Female; D006107:Granulosa Cells / Q000502:physiology*; D006801:Humans; D008297:Male; D009865:Oocytes / Q000502:physiology; D011247:Pregnancy; D011489:Protein Denaturation; D014462:Ultrafiltration; D014709:Vero Cells
chemical_list: D000596:Amino Acids; D017077:Culture Media, Conditioned