The protein content of rabbit embryos during the first 7 days of development in vivo was determined. The protein content of intact embryos, embryonic cells (intact embryos without mucin coats for developmental stages up to 96 h post-coitum and free of blastocyst coverings for later stages) and blastocyst coverings were determined by the Pierce Micro BCA assay. The mean protein content of intact one-cell or two-cell embryos was 0.16 μg and increased at the four- to six-cell stage with no further increase until the late morula/early blastocyst stages (days 3 to 4). There was a 53-fold increase in protein from the early to late blastocyst stages. The protein content of embryonic cells was stable at a mean value of 0.16 μg until the late morula stage (day 3) and then increased to a mean of 6.85 μg on day 6 and 50.38 μg on day 7. The increase in protein content of intact embryos up to about 72 h appeared to be due solely to an increase in the protein content of the mucin coat. The protein content of the blastocyst coverings increased from a mean of 2 μg on day 5 to a mean of 35 μg on day 7. For blastocyst stages, the total protein content of intact blastocysts and of embryonic cells was correlated with the surface area of the embryos (r 2 = 0.895 and 0.873, respectively) and, thus, an increase in blastocyst size is a true index of blastocyst development.
P. M. Morgan and M. T. Kane
C. W. Gray, P. M. Morgan and M. T. Kane
Summary. A factor of low M r with growth-promoting effects on rabbit embryos was extracted and purified from commercial bovine serum albumin (BSA). This embryotrophic factor was extracted from BSA dissolved in formic acid by membrane filtration (membrane cutoff of M r 10 000) and then freeze-drying of the filtrate. The extract was purified successively by chromatography on G-10 Sephadex, QAE-Sephadex A-25 anion exchange and high-performance liquid chromatography (HPLC) reverse-phase columns. Mass spectrometry of the active reverse-phase material indicated that the major component in this material had an M r of 192. The embryotrophic factor in the low M r extract of BSA was shown to be citrate, because: (i) the mass spectra of the active reverse-phase material and citrate were identical, (ii) the activity was eluted at the identical position to citrate on an analytical HPLC anion-exchange column, (iii) the original BSA sample was shown by enzyme assay to be heavily contaminated by citrate and (iv) citrate stimulated cell proliferation and expansion of blastocysts.
Keywords: citrate; al citrate; albumin; rabbit
G. L. Morgan, R. D. Geisert, J. P. McCann, F. W. Bazer, T. L. Ott, M. A. Mirando and M. Stewart
The progesterone antagonist mifepristone (RU 486) was injected i.m. into ewes during the early luteal phase of the oestrous cycle to test the hypothesis that duration of uterine exposure to progesterone from the corpus luteum initiates luteolysis through the proper timing of endometrial oxytocin receptor expression and pulsatile secretion of PGF2α coincident with release of luteal oxytocin. In Expt 1, duration of cycle, the PGF2α metabolite 15-keto-13,14,-dihydro-PGF2α (PGFM) and oxytocin concentrations were measured in ewes treated on days 5, 6, 7 and 8 of the oestrous cycle with either 2.5 or 5.0 mg RU 486 kg−1 day−1 (n = 4 per group); control ewes (n = 6) were injected i.m. with 80% ethanol (diluent). In Expt 2, the presence of functional uterine oxytocin receptors was determined indirectly on day 12 of the cycle by measuring the plasma PGFM response to oxytocin challenge (20 iu, i.v.) in diluent-treated ewes (n = 3) and in ewes treated with 2.5 mg RU 486 kg−1 day−1 on days 6, 7 and 8 of the oestrous cycle. Duration of the oestrous cycle of control ewes (16 ± 1 days) was extended beyond day 24 (day 0 = oestrus) in 10 of 11 ewes treated with RU 486 as determined by daily exposure of ewes to a ram and by measurement of progesterone concentrations in plasma in the two experiments. Luteolysis (days 14–16) in control ewes was preceded (days 12–15) by pulsatile release of PGFM in plasma and by the presence of functional endometrial oxytocin receptors that responded to oxytocin challenge (day 12) with a significant increase in plasma PGFM concentrations. RU 486 treatment prevented pulsatile PGFM release on days 12–15 and release of PGF2α following oxytocin challenge on day 12 but not on day 20 when oxytocin administration was repeated. The absence of pulsatile PGFM release in ewes treated with RU 486 on days 12, 13, 14 and 15 in Expt 1 was associated with different plasma oxytocin activity in treated and control ewes in that plasma oxytocin increased from days 12 to 14 in mifepristone-treated but not in control ewes. We conclude that adequate progesterone exposure during the early to mid-luteal phase of the oestrous cycle is essential for initiation of ovarian–uterine mechanisms that lead to luteolysis in ewes.
R. D. Geisert, T. C. Fox, G. L. Morgan, M. E. Wells, R. P. Wettemann and M. T. Zavy
Summary. Treatment of recipient cows with 100 mg of progesterone daily from Days 1 to 5 of the oestrous cycle increased plasma progesterone compared with vehicle-treated recipients. Embryo transfer to progesterone-treated recipients which showed oestrus 72 h after the donor cows resulted in pregnancy rates at Day 35 similar to those of synchronous (±12 h) recipients (42 vs. 50%). Only 1 of 22 (4·8%) asynchronous (−72 h) vehicle-treated recipients established pregnancy. Similar treatments of cyclic cows with progesterone shortened (P < 0·01) the interoestrous interval by 3·2 days. When assessed on Day 7 of pregnancy, administration of progesterone to superovulated donor cows on Days 1–4 of pregnancy did not affect early embryo development compared with superovulated cows treated with vehicle alone. Plasma progesterone increased rapidly in superovulated cows compared with cows during the oestrous cycle. The results indicate that administration of progesterone early in the oestrous cycle of the recipient can effectively advance uterine receptivity for the transfer of older asynchronous embryos.
Keywords: bovine; embryo; uterus; progesterone