This study was designed to investigate the development of day 2 embryos obtained from young and aged mares, co-cultured with oviductal epithelial cells obtained from mares in each age group in a 2 × 2 crossover design. Young, fertile mares (n = 19; 2–7 years of age) and aged, subfertile, mares (n = 16; 17–24 years of age) were used as embryo and oviductal epithelial cell donors. Embryos (n = 37) were collected from the oviducts 2 days after ovulation and were paired (embryos obtained from young mares with embryos obtained from aged mares) so that eight pairs were co-cultured with young mare oviductal epithelial cells and eight pairs were co-cultured with aged mare oviductal epithelial cells. Five additional embryos obtained from young mares were co-cultured with oviductal epithelial cells from either young mares or aged mares but were not paired. Embryos were co-cultured for 7 days at 38.5°C in 5% CO2 or until morphological degeneration was detected. The proportions of paired embryos that reached the blastocyst stage were similar for embryos obtained from young mares and embryos obtained from aged mares after co-culture with oviductal epithelial cells from young mares (6 of 8 versus 5 of 8) or from aged mares (6 of 8 versus 5 of 8), respectively. Although the overall rate of development of embryos to blastocyst from both young mares and aged mares was similar, blastocysts developing from embryos obtained from aged mares were inferior to blastocysts obtained from young mares in terms of number of cell nuclei, quality score, and diameter at day 7. The results of this experiment indicate that the high rate of early embryonic loss in aged, subfertile mares may be due to inherent developmental defects in their embryos, but does not appear related to the ability of embryos from aged, subfertile mares to reach the blastocyst stage.
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S. P. Brinsko, B. A. Ball, P. G. Miller, P. G. A. Thomas, and J. E. Ellington
R. G. Gosden, J. F. Huntley, A. Douglas, L. Inglis, and H. R. P. Miller
A role for mast cell proteases (RMCP I and II) in the cyclical remodelling of ovarian and uterine tissues of rats was investigated in the oestrous and pregnancy cycles using immunocytochemistry and enzyme-linked immunosorbent assays. The concentrations of RMCP I exceeded that of RMCP II by 100-fold in both tissues, but were always much higher in uteri than ovaries. Most of the protease activity in the uterus was located in the myometrium, whereas it was more focally distributed in the hilus and medulla of the ovary. Protease activity was confined to mast cells identified by metachromatic staining and no single cell contained both proteases. The concentrations of RMCP I and II in the two organs did not fluctuate throughout the 4-day oestrous cycle. Neither were RMCP I concentrations in the uterus altered by administration of diethylstilboestrol to ovariectomized animals, although total amounts per uterus were substantially greater than in controls. Concentrations of RMCP I were substantially reduced in the uterus after day 6 of pregnancy and rose during the puerperium. The reduction was greater in pregnant than in pseudopregnant horns and tended to be lower in the vicinity of conceptuses than between them. The physiological significance of the lower mast cell protease concentrations is unclear, although their absence may contribute to the decreased protein catabolism during pregnancy.
R G Lea, L P Andrade, M T Rae, L T Hannah, C E Kyle, J F Murray, S M Rhind, and D W Miller
This study aimed to determine whether reduced fetal ovary folliculogenesis in ewes undernourished during early/midpregnancy is associated with altered ovarian cell proliferation and/or the expression of apoptosis-regulating genes. Groups of ewes (n = 11–19) were fed either 100% (high; H) or 50% (low; L) of metabolisable energy requirements for live-weight maintenance during selected windows of gestation. All animals were killed at days 50, 65 or 110 of gestation. Between mating and slaughter, control animals were fed the H ration, while animals of other subgroups were fed the L ration from (a) mating to slaughter at 50, 65 or 110 days; (b) 0 to 30 days; (c) 31 to 50 or 65 days; or (d), in the day 110 slaughter group only, from 66 to 110 days. Bouin’s-fixed fetal ovaries were examined for (a) Ki67 immunoexpression (proliferation) and (b) Bax and Mcl-1 (apoptosis-regulating genes) expression by in situ hybridisation (day 110) and immunohistochemistry (days 50, 65 and 110). At day 50, maternal nutrition had no effect on Ki67, predominant in germ cells, or Bax and Mcl-1, predominant in the oocytes. Restricted maternal food intake from 0 to 30 days significantly reduced staining for Ki67 in germ cells at day 65 (P < 0.05) but increased staining in granulosa cells at day 110 (P < 0.05). In animals fed the L ration for 110 days, primordial follicle Bax and Mcl-1 were significantly increased (Bax: P < 0.01; Mcl-1: P < 0.05). Granulosa cell Bax was also increased (P < 0.05). When the L ration was fed from 66 to 110 days, granulosa cell Bax (P < 0.05) and primordial follicle Mcl-1 (P < 0.01) were also significantly increased. In the fetal ovarian vasculature, animals underfed for 0–110 days had significantly elevated perivascular Mcl-1 (P < 0.001) and endothelial Bax expression (P < 0.05). Moreover, at day 110, endothelial Mcl-1 was increased by underfeeding from 0 to 30 days (P < 0.05). These data indicate that maternal undernutrition alters proliferation and the expression of apoptosis-regulating genes in the developing fetal ovary. The precise mechanism depends on the window of maternal food restriction.