Characterization of the Embryotrophic Activity of Exogenous Protein-Free Oviduct-Conditioned Medium Used in Culture of Cattle Embryos1

Abstract

Bovine zygotes, obtained after in vitro maturation and fertilization of oocytes from slaughtered cow ovaries, were cultured in droplets of nonconditioned or conditioned medium on bovine oviduct cell monolayers. The media tested were Medium 199 alone and Medium 199 supplemented with 10% fetal calf serum (FCS). Oviduct conditioning increased both early cleavage and development to blastocysts. Only the effect on early cleavage was mimicked by FCS. The blastocysts obtained in serum-free conditioned medium (SFCM) appeared morphologically normal and had the same cell number as those produced in conditioned medium containing serum. Their hatching rates did not differ. Transfer of 16 blastocysts developed in SFCM to 16 synchronized recipients resulted in five pregnancies (31%), indicating good embryonal viability. Boiling of SFCM resulted in a total loss of activity, while heating at 56 degrees C for 30 min had no deleterious effect. A 10-kDa ultrafiltration of SFCM removed the blastocyst development-supporting activity from the filtrate but not the early cleavage-favoring activity. This allows us to conclude that at least two different factors are present in SFCM: one of low molecular mass (< approximately 10 kDa), needed to obtain the 5-8 cell stage and mimicked by FCS, and another of higher molecular mass allowing embryos to develop from the 8-cell to blastocyst stage.

Fertilization and early embryology: Differential effect of epithelial cell-conditioned medium fractions on preimplantation mouse embryo development

“…In contrast, the benefits observed using MDBK cells were lost with Vero cells, suggesting either that some cell lines are unable to remove inhibitory factors from a poor medium such as B2 or that inhibiting factors are produced by certain cell lines. In addition, contrary to studies reporting the existence of a low molecular weight molecule which enhanced embryo development (Minami et al, 1992;Mermillod et al, 1993), no beneficial effect was consistently observed in any specific fraction tested in our work. This supports the idea that cells are responsive only to certain media and that feeder cells do not improve embryo development through specific factors secreted into the medium but only act by changing the base medium.…”

“…In the light of this, several studies have argued that a number of different cell lines may produce such a factor(s) Ouhibi et al, 1990;Minami et al, 1992;Bongso et al, 1993;Mermillod et al, 1993). In this study only B2 medium conditioned by MDBK cells improved embryo development, but no blastocyst formation was observed in B2 alone.…”

“…embryo development in coculture and conditioned medium was 22% compared to 3% in control medium. Ellington et al (1990) and Mermillod et al (1993) also examined the use of conditioned medium to improve cattle embryo culture and reported a low molecular weight molecule which enhanced embryo development when medium (Ml99) was conditioned on oviductal cells.…”

Effect of all-trans retinol on in vitro development of preimplantation buffalo embryos

“…Expanded day 8 blastocysts from each treatment groups were fixed and stained as per the method described (Mermillod et al, 1993). These expanded blastocysts were then transferred individually onto glass microscopic slides and after drying at room temperature, fixed with 70% ethanol for 24 h. The fixed blastocysts were stained with 10 mg/ml bisbenzamide (Hoechst 33342) and 2.3% sodium citrate.…”

Effect of vitamin A supplementation at different gaseous environments on in vitro development of pre-implantation sheep embryos to the blastocyst stage

“…Expanded day eight blastocysts from each treatment group were fixed and Vitamin A as antioxidant in ovine in vitro fertilisation stained as per the method described by Mermillod et al (1993). These expanded blastocysts were then individually transferred onto glass microscopic slides and dried at room temperature.…”