Total protein content and protein synthesis within pre-elongation stage bovine embryos.
Protein content was measured in zona-free bovine oocytes and pre-elongation stage embryos, following in vitro maturation, fertilisation, and then culture in Synthetic Oviduct Fluid medium supplemented with amino acids and 8 mg ml(-1) bovine serum albumin (BSA). Values (ng embryo(-1)) of 122 +/- 7.8, 137 +/- 8.6, 111 +/- 8.8, 115 +/- 10.4, 139 +/- 9.0 and 152 +/- 10.1 were obtained for zona-free mature oocytes, 2-cell (day 2), 8-cell (day 3), compact morula (day 6), blastocyst (day 7), and expanded blastocyst (day 8) stage embryos, respectively. The protein content of day 7 zona-enclosed blastocysts was 337 +/- 58.0 ng embryo(-1). These values suggest that prior to compaction and blastulation, the early cleavage stage bovine embryo has a higher rate of protein degradation than that of synthesis. Net growth is observed only after initiation of compaction. The protein content of day 7 blastocysts was measured in embryos following in vitro production and culture in the same media supplemented with either 0.5% w/v polyvinyl alcohol (PVA), 8 mg ml(-1) BSA, 8 mg ml(-1) BSA and further supplemented with 10% fetal calf serum (FCS) from the beginning of culture (FCS-D1), 8 mg ml(-1) BSA and 10% FCS from the fourth day of culture (day 5 of development) or from in vivo-derived day 7 blastocysts. Protein content was significantly (P < 0.05) lower in PVA-cultured embryos than other treatments. To determine if this difference in PVA-cultured embryos was due to a difference in the rate of protein synthesis, comparisons were made between day 7 embryos derived from BSA-culture and either PVA-culture, FCS-D1 culture or in vivo-derived embryos. Despite differences in diameter, no significant difference was observed in the incorporation of L-[2,3,4,5,6-3H]-phenylalanine into the TCA-precipitable fraction in any of the three comparisons made. However, incubation in the presence of FITC-abelled BSA or beta-casein and examination under either fluorescence or confocal microscopy revealed that protein in the extra-embryonic environment was actively taken up by the trophectoderm of day 7 blastocysts, most likely by endocytosis. These results suggest that exogenous protein is an important nutritive source, probably maintaining intracellular amino acid pools. Results obtained from the production of em bryos in protein-free medium should be viewed with the knowledge that such embryos differ metabolically from those embryos grown in the presence of protein, including in vivo-derived embryos.
Thompson
J G
JG
AgResearch Ruakura Research Centre, Hamilton, New Zealand. thompsonj@agresearch.cri.nz
Sherman
A N
AN
Allen
N W
NW
McGowan
L T
LT
Tervit
H R
HR
D000818:Animals; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D046508:Culture Techniques; D004622:Embryo, Mammalian / Q000378:metabolism*; D005314:Embryonic and Fetal Development; D005260:Female; D008297:Male; D009865:Oocytes / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism
D002364:Caseins; D011506:Proteins; D012710:Serum Albumin, Bovine; D010649:Phenylalanine