MORPHOLOGICAL AND MOLECULAR ASPECTS OF EARLY DEVELOPMENT IN THE BOVINE F.L. Barnes, J.J. Parrish, J.L. Susko-Parrish and N.L. First Department of Meat and Animal Science College of Agricultural and Life Sciences University of Wisconsin-Madison Madison, Wisconsin 53706 USA Recent advances in in vitro fertilization, gene injection and nuclear transfer have prompted us to characterize early bovine development in anticipation that a better understanding will facilitate the use of Bovine oocytes were aspirated from L-5 mm follicles new technologies. obtained from abattoir recovered ovaries and matured in vitro for 22 Sperm-egg interaction (penetration) and to 26 h (BOR 34(Supp1.1)286). formation were determined by mounting eggs at 2 h pronuclear (PN) intervals 2 to 12 h postinsemination (hpi), staining with aceto-orecin under phase contrast microscopy examination Sperm and (500x). penetration determined for five bulls occurred 4 to 6 hpi with PN formation occurring 2 to 4 h postpenetration. PN formation was complete 2 h after first observed. An assessment of fertilization was periodically tested at 17-23 hpi to determine the frequency of normal PN present and no polyspermy). fertilized eggs (two In separate embryos were cultured in Hams F-10 with 10% heat-treated experiments, Cultures were terminated at 6 h intervals 14-68 hpi fetal calf serum. 12 h intervals 68-92 hpi. At the time of culture and then at termination the proportion of embryos cleaving was assessed. Embryos and analyzed by 2-dimensional were then incubated with 35S-methionine polyacrylamide gel electrophoresis with subsequent fluorography. To compare in viva versus in vitro development, in vivo cultured embryos were collected from superovulated cows at slaughter from days 1 to 5 of development and prepared for electrophoresis as described above. The frequency of normally fertilized eggs (110/179, 61%) was comparable to the frequency of embryos cleaving (921/1739, 53%). Cleavage to the two-cell stage occurred synchronously between 26 to 32 the highest proportion of hpi with two cells occurring at 32 hpi (49%). Cleavage to the four cell, five to six cell and eight cell stage occurred at 38, 44 and 56 hpi, respectively. If time of ovulation in vivo is equated to the time of insemination in vitro then equivalent to are that observed b these stages viva (J. Anat. 80:194-209). A qualitative change in protein profiles was observed at the eight-cell stage both ti m and in vitro Early eight cell --. embryos (58 hpi) were similar to late four-cell embryos (52 hpi) is This was also observed vitro. in vivo between day 2.5 four-cell embryos and day 3 eight-cell embryos. A qualitative change in protein profiles of eight-cell embryos was observed at 64 hpi in vitro and day 4 in viva. Protein profiles were similar between in vivo and in vitro In conclusion, embryos. in vitro development of in vitro matured, h oocytes appears vitro fertilized to progress at a similar rate as observed in viva. The changes in the pattern of proteins synthesized in the early to mid-eight cell stage, observed here, occur at the same time as increased transcription of rRNA (Gamete Res. 14:65-73), and may suggest the onset of transcription and translation directed by the bovine embryonic genome. Supported by grant 85-CRCR-1-1801 from the U.S. Department of Agriculture.
1987 VOL. 27 NO. 1