Successful development in vitro of hamster 8-cell embryos to 'zona-escaped' and attached blastocysts: assessment of quality and trophoblast outgrowth.
The peri-implantation development involves zona escape (hatching) of blastocysts and their attachment and proliferation. These events are difficult to study in vivo, so in this study hamster 8-cell embryos were cultured through the hatched and attached blastocyst stages using different formulations of hamster embryo culture medium (HECM)-2. Supplementation of succinate, amino acids, vitamins (inositol, pantothenate, choline chloride) and bovine serum albumin (BSA) to HECM-2 supported 100% development of 'zona-escaped' blastocysts. In this medium (designated as hatching, i.e. HECM-2h) all blastocysts invariably deflated and escaped from focally lysed zonae, which underwent complete dissolution. In their presence, pre-morula stage embryos also escaped from zonae. Omission of BSA from HECM-2h failed to support zona escape whereas that of vitamins reduced zona escape (34.0% +/- 7.0). Blastocysts with the potential to undergo zona escape in HECM-2h were of high quality as they had a higher mean cell number (MCN) than the MCN of those developing in BSA-free HECM-2h (35.2 +/- 1.6 v. 24.3 +/- 1.1). Cell allocation (i.e. trophectoderm to inner cell mass ratio) in blastocysts remained unaltered in both media (2.6 +/- 0.2 v. 2.7 +/- 0.2). Supplementation of 10% bovine fetal serum (BFS) to HECM-2h was detrimental to the development of blastocysts (22.3% +/- 7.4) and none of them underwent zona escape. Interestingly, BFS was required either as a supplement to the medium or as a coating on dishes for azonal blastocysts to attach (> or = 70%) and exhibit trophoblast (TB) outgrowth (30.3 x 10(3) +/- 2.9 x 10(3) micron 2 at 48 h). These results show that HECM-2h supports maximal development of zona-escaped blastocysts with the potential to attach and exhibit TB outgrowth, and there is a developmental stage-specific requirement for serum during peri-attachment in hamster development.
Mishra
A
A
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India.
Seshagiri
P B
PB
D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000502:physiology; D002448:Cell Adhesion; D002452:Cell Count; D002455:Cell Division; D002478:Cells, Cultured; D006224:Cricetinae; D003470:Culture Media; D046508:Culture Techniques / Q000379:methods; D004622:Embryo, Mammalian / Q000166:cytology; D005314:Embryonic and Fetal Development / Q000502:physiology*; D005260:Female; D008647:Mesocricetus; D013997:Time Factors; D014327:Trophoblasts / Q000166:cytology; D015044:Zona Pellucida / Q000502:physiology
D003470:Culture Media