Factors Required during Preculture of Rat Oocytes Soon after Sperm Penetration for Promoting Their Further Development in a Chemically Defined Medium

Abstract

Abstract. Blastocyst formation in a chemically defined medium (mR1ECM) of rat oocytes soon after sperm penetration is less frequent than in those undergoing male pronuclear formation. This inhibition is released by preculturing the oocytes for a few hours in modified Krebs-Ringer bicarbonate solution (mKRB). The present study examined the effects of phosphate (Pi), bovine serum albumin (BSA) and osmolarity during preculture of sperm penetrated rat oocytes on their development to blastocysts in mR1ECM in vitro. These are the major factors that differ between mR1ECM and mKRB. When oocytes collected at 0730-0800 h on the day following mating and freed from cumulus cells were precultured for 5 h in mKRB or Pi-free mKRB and then cultured for 127 h in mR1ECM, about 73-74% of oocytes developed to blastocysts. In both media, replacement of BSA with polyvinylalcohol (PVA) or osmolarity of 246 mOsM reduced blastocyst formation compared with media containing BSA or with osmolarity of 304 mOsM; blastocyst formation was greatly inhibited when oocytes were precultured in media with PVA and osmolarity of 246 mOsM. On the other hand, when precultured in mR1ECM or mR1ECM with osmolarity of 304 mOsM or BSA instead of PVA, fewer oocytes developed to blastocysts than those precultured in Pi-free mKRB and mR1ECM with osmolarity of 304 mOsM and BSA. These results indicate that both BSA and osmolarity, but not Pi, are essential factors during preculture of rat oocytes soon after sperm penetration for promoting their further development to blastocysts in a chemically defined medium.

Rats!

“…The Rat Resource and Research Center is now a robust national repository. Methods have been developed that allow the rat embryo to be cultured from one cell into a blastocyst (Iannaccone et al, 2001;Yang et al, 2004;Zhou et al, 2003). A high-quality draft sequence of the rat genome is available.…”

Perspective: One-Cell and Cleavage-Stage Mouse Embryos Thrive in Hyperosmotic Oviductal Fluid Through Expression of a Glycine Neurotransmitter Transporter and a Glycine-Gated Chloride Channel: Clinical and Transgenerational Implications

“…Hence, it could become beneficial to create culture conditions for oocytes and preimplantation embryos in vitro that more nearly reflect conditions in vivo. Such conditions likely differ among species, since one-cell rat embryos develop better in media of 304 than 246 mOsmol/kg (Yang et al, 2004), whereas the reverse is true for the fertilized mouse eggs discussed above. The osmolality of oviductal fluid in the cycling rat was reported to be 287 ± 1 mOsmol/kg (Waring, 1976), but this osmolality has not, to our knowledge, been measured during pregnancy.…”

Advances in transgenic rat production

“…Eggs were washed in M2 and placed in BMOC-3 (Invitrogen, Carlsbad, CA) under embryo tested mineral oil (Sigma) in a 37°C, 5% CO 2 , humidified incubator. For two-phase cell culture, eggs were transferred to mR1ECM prepared as described (Yang et al 2004) after overnight culture in BMOC-3.…”

“…We tested four media used to culture rodent eggs (BMOC-3, M16, KSOM, and mR1ECM) and found that none of them supported development of fertilized rat eggs to the blastocyst stage (data not shown). Yang et al (2004) described a two-phase culture system that supports the development of 74% of fertilized Wistar rat eggs to blastocyst stage. We tested the development of SD and F344 eggs cultured in BMOC-3 overnight and then cultured in mR1ECM for 5 days and found that 85% of tested SD eggs and 52% of tested F344 eggs developed to blastocyst (Table 4).…”