title: Molecular biological methods for monitoring oocyte maturation and in vitro fertilization in bitches.
abstract: Oocytes were collected from ovaries of bitches, at various stages of the oestrous cycle, after routine sterilization. Cumulus-enclosed oocytes were cultured for 0-72 h in a modified M-199 medium containing 10% oestrous bitch serum and 20 micrograms oestradiol ml-1. For oocytes surrounded by two or more layers of cumulus cells, an increase in the expression of mRNA transcripts for zona pellucida glycoprotein 3 (ZP3) was seen and reached peak levels after 48 h culture in vitro. Histological assessment showed that 39% of these oocytes had extruded their first polar body after 24 h culture in vitro. When these in vitro matured oocytes were transferred to oviduct cell monolayers and inseminated with fresh dog spermatozoa in Medium-199 supplemented with 10% fetal calf serum and 50 mg gentamicin sulfate ml-1, penetration of the zona pellucida started 1 h after insemination for oocytes that had been cultured for 48 and 72 h. At 12 h after insemination both male and female pronuclei were seen in 37.5% and 20% of the oocytes incubated for 48 and 72 h, respectively. No further development was seen.
authors.affiliation: Department of Surgery/Reproduction, University of Glasgow Veterinary School, UK.
authors.forename: D A
authors.initials: DA
authors.lastname: Nickson
authors.forename: J S
authors.initials: JS
authors.lastname: Boyd
authors.forename: P D
authors.initials: PD
authors.lastname: Eckersall
authors.forename: J M
authors.initials: JM
authors.lastname: Ferguson
authors.forename: M J
authors.initials: MJ
authors.lastname: Harvey
authors.forename: J P
authors.initials: JP
authors.lastname: Renton
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004285:Dogs / Q000502:physiology*; D004527:Egg Proteins; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods*; D017403:In Situ Hybridization; D008562:Membrane Glycoproteins / Q000235:genetics*; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D009866:Oogenesis / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis*; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins
chemical_list: D004527:Egg Proteins; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins