Summary. The influence of breed and season on the sensitivity of the pituitary gland of sheep to LH-RH was assessed. Ovariectomized ewes of 3 breeds (Finnish Landrace, Scottish Blackface and Tasmanian Merino) with differing normal breeding seasons and with differing ovulation rates were injected (i.v.) with 3 doses of LH-RH (1·56, 6·25 or 25·0 μg) at 3 different times of the year covering the anoestrous and the breeding seasons of intact ewes; 9 ewes of each breed (3 per sub-class) were examined on the first and third occasions, 6 (2 per sub-class) on the second. The response was measured in terms of the concentration of LH in peripheral plasma 20, 40, 60 and 80 min after injection. Time of year, but not the breed of sheep, affected the magnitude of the response; the data indicated that the duration of LH secretion was greater during the breeding season than during anoestrus. It was concluded that changes in the spontaneous activity of the hypothalamus/hypophysis could contribute to seasonal changes in LH secretion independently of the modifying effects of gonadal steroids. Such variation in unmodulated activity apparently does not contribute to the differences in ovulation rate among the 3 breeds.
R. B. Land, W. R. Carr and R. Thompson
R. B. LAND, R. THOMPSON and D. T. BAIRD
The relationship between the duration of induced oestrus and the characteristics of the progesterone-oestrogen treatment used for its induction was investigated and described. The regression of the duration of oestrus on the dose of oestrogen was found to be 0·0032 and 0·0045 days/μg in the Blackface and Finnish Landrace ewes, respectively. The duration of oestrus was also increased by approximately 40% following the division of a particular dose of oestrogen into two units given 24 hr apart.
The mean durations of induced oestrus over all treatments, 1·52 and 1·92 days, respectively, in the Blackface and Finnish Landrace ewes, were significantly different.
It was concluded that the relationship between ewe prolificacy and the duration of natural oestrus arose at least in part from differences in the pattern of oestrogen secretion around the time of ovulation.
R. B. LAND, MARIE-MADELEINE de REVIERS, R. THOMPSON and P. MAULÉON
The ovarian activity of three strains of rat and their crosses was measured in terms of (1) the number of oocytes present at birth, (2) the response to exogenous gonadotrophins as measured by weight and (3) by ovulation rate at 4 weeks of age, and (4) the number of eggs shed at natural oestrus at 12 weeks.
The inheritance of the number of oocytes at birth and the weight of the ovaries did not deviate from an additive genetic model, whereas strain cross interactions contributed to the variation in the two measures of ovulation rate. Induced ovulation was related to ovarian weight, but neither of the two measures of response to exogenous hormones was highly correlated with the natural ovulation rate. Variation in the number of eggs shed at natural oestrus was, however, related to the number of oocytes at birth; strain types with high ovulation rates had low oocyte numbers and vice versa.
These relationships were interpreted as indicating that the ovarian hormonal conditions which lead to the ovulation of a large number of eggs also led to a relatively small population of oocytes in the ovaries at birth.
Marie-Thérèse Hochereau-de Reviers, R. B. Land, Christine Perreau and R. Thompson
Summary. Season but not hemicastration affected the cellular composition of the testis. Despite similar weight, the testicular histology differed markedly with season of birth. The number of Leydig cells and of Sertoli cells was greater in summer- than in winter-born lambs by factors of 2 and 1 ·5 respectively. Similarly the number of spermatogonia and their rate of production increased substantially in summer-born lambs. The rate of spermatid production was affected by both hemicastration and season. Season of birth exerted more modifications to testicular histology than did hemicastration.
J. G. Thompson, R. J. Partridge, F. D. Houghton, C. I. Cox and H. J. Leese
The consumption of oxygen, uptake of pyruvate and glucose and production of lactate were determined for groups of bovine embryos produced in vitro from the one-cell to the blastocyst stage (day 0–6 of culture). Measurements were made in Hepes-buffered synthetic oviduct fluid medium supplemented with 1.0 mmol pyruvate l−1, 10 mmol d,l-lactate l−1 and 1.5 mmol glucose l−1 and also 3 mg BSA ml−1 and, from day 5 of development, 10% (v/v) fetal calf serum. The amount of ATP production was determined from oxygen consumption and the proportion of glucose taken up that could be accounted for by lactate production. The data revealed that oxygen consumption was relatively constant from days 0–4 of culture (0.24–0.27 nl per embryo h−1), but increased with the initiation of compaction (0.39 nl per embryo h−1) and continued to increase with the formation and expansion of the blastocoel (0.9 nl per embryo h−1). Both pyruvate and glucose uptake followed similar patterns. Furthermore, when plotted against oxygen consumption, both pyruvate and glucose uptake increased significantly (P < 0.001) in a linear relationship (R 2 = 0.61 and 0.49, respectively). Lactate production also increased with development and accounted for 40% of glucose uptake at day 0 of culture (putative zygotes), increasing to 70% by day 2 (eight-cell stage) and 100% of glucose uptake from day 4 of culture onwards. ATP production followed a similar pattern to that of oxygen consumption (60–85 pmol per embryo h−1 from day 0 to day 4) increasing with compaction (124 pmol per embryo h−1) and blastulation (221 pmol per embryo h−1). For precompaction stages, 93–96% of ATP production was derived from oxidative phosphorylation, decreasing to 82% with compaction. ATP produced by oxidative phosphorylation could be accounted for by the uptake of pyruvate, suggesting that bovine embryos produced in vitro utilize little endogenous substrates when appropriate exogenous substrates are present in the culture medium. The data revealed that bovine embryos were dependent on oxidative phosphorylation for energy (ATP) production at all stages of pre-elongation development, with perhaps a shift in dependence towards glycolysis in conjunction with compaction. It follows that oxidizable substrates, such as pyruvate and certain amino acids, are preferred in embryo culture medium during development in vitro.
A R Clark, Y M Stokes, M Lane and J G Thompson
Immature oocytes benefit from nutrient modification of the follicular environment by the surrounding cumulus mass. However, the oxygen concentration that the oocyte may be exposed to could be lower than the antral follicular concentration due to the metabolism of surrounding cumulus cells. Using metabolic data previously determined, we have developed a mathematical model of O2 diffusion across the bovine and murine cumulus–oocyte complex. From this we have determined that across a physiological range of external pO2, less than 0.25% and 0.5% O2 is removed by cumulus cells within the bovine and murine cumulus–oocyte complex respectively. Our model differs from others as it: incorporates a term that allows for nonlinear variation of the oxygen consumption rate with oxygen concentration; considers two regions (oocyte and cumulus) sharing a common boundary, both of which consume oxygen at different non linear rates. Cumulus cells therefore remove little O2, thus sparing this essential gas for the oocyte, which is dependent on ATP generation via oxidative phosphorylation.
JUDITH A. MURPHY, J. W. E. WORTHAM, J. MARTAN and MARY R. THOMPSON
Ultrastructural studies of cytoplasmic droplets of the spermatozoa of bulls, rams, rabbits, rats (Bloom & Nicander, 1961) and bats (Fawcett & Ito, 1965) revealed only numerous membranous vesicles and tubules in the matrix of the droplets. These authors agreed that the membranous elements were probably of endoplasmic reticulum and/or Golgi complex origin. The simplicity of the ultrastructure and lack of other organelles and inclusions in the droplet matrix led Fawcett & Ito (1965) to doubt suggestions that cytoplasmic droplets contain endogenous substrates for epididymal sperm survival.
Recent biochemical and cytochemical analyses of cytoplasmic droplets in spermatozoa of different domestic animals (Dott & Dingle, 1968; Harrison & White, 1972; Moniem & Glover, 1972) showed a rather high activity of hydrolytic and glycolytic enzymes. Harrison & White (1972) suggested that at least some
J. G. E. Thompson, A. C. Simpson, P. A. Pugh, P. E. Donnelly and H. R. Tervit
Summary. Two-cell sheep embryos and 2–4-cell and 8-cell cow embryos were cultured for 5 days in stoppered test-tubes in Synthetic Oviduct Fluid supplemented with 32 mg BSA/ml. The medium had been previously equilibrated with one of the following O2 concentrations (sheep: 0, 2, 4, 6, 8, 10, 12, 17, 20%; cow: 0, 4, 8, 12, 17, 20%). At the end of culture embryos were examined for morphology and stained to assess numbers of nuclei. Mean (± s.e.m.) nuclei/embryo was highest at 8% O2 for sheep embryos (23·6 ± 3·1), 4% for 2–4-cell cow embryos (23·2 ± 6·1) and 8% for 8-cell cow embryos 29·6 ± 5·2). The minimum number of nuclei/embryo occurred at 20% O2 in each case (10·3 ± 0·9, 10·3 ± 2·7, 14·5 ± 2·4, respectively) with similar values also recorded at 0% O2 (10·8 ± 1·9, 16·5 ± 6·0, 14·6 ± 2·4, respectively). Analysis of the proportion of embryos reaching at least the morula stage demonstrated a significant quadratic component for the different oxygen concentrations for sheep (P < 0·01) and cow (P < 0·05) embryos. A number of sheep and cow embryos showed abnormalities, suggesting that the culture conditions require further refinement.
The results confirm that, under lowered oxygen levels, development of sheep and cattle embryos can occur through the 8- to 16-cell block in a simple defined medium without somatic cell support.
Keywords: sheep; cattle; embryos; oxygen; culture
J. L. Crawford, G. H. Shackell, E. G. Thompson, B. J. McLeod and P. R. Hurst
The common method for synchronizing oestrus in brushtail possums is by removal of their pouch young (RPY). However, there is little information on the ovarian response to this treatment, the timing and incidence of ovulation is poorly defined, and methods of identifying oestrus are unreliable. In this study, the development of preovulatory follicles, ovulation and reproductive tract changes following RPY were monitored by repeated laparoscopic observation. A total of 120 adult female possums underwent laparoscopy at intervals of 1–4 days over the period from 0 to 21 days after RPY. Tissue was collected from a further 30 animals for correlative histology of ovarian structures, and to quantify changes in reproductive tract organs. Only 80 of 120 animals ovulated, and the time of ovulation ranged from 7 to 18 days following RPY. In most animals, enlargement of vaginal cul-de-sac and uterine tissue occurred within 10 days. Correlative histology supported the macroscopic classification of ovarian structures, and healthy and atretic follicles could be identified by laparoscopy. Vaginal smears and plasma progesterone concentrations verified the occurrence of ovulation as observed by laparoscopy. A 'presumptive' preovulatory follicle, first identifiable approximately 5 days before ovulation, was recorded in all animals that ovulated and in none that failed to ovulate. Changes to its surface morphology indicated impending ovulation. This study has enabled the day of ovulation to be identified accurately for the first time in this species. It has also shown that there is wide variation in follicle development, lack of synchrony in the time of ovulation in the brushtail possum, and that some animals fail to ovulate following RPY.
Coleman H Young, Heather M Rothfuss, Philip F Gard, Aaron Muth, Paul R Thompson, Ryan L Ashley and Brian D Cherrington
There are five peptidylarginine deiminase (PAD) isozymes designated as PADs 1, 2, 3, 4 and 6, and many are expressed in female reproductive tissues. These enzymes post-translationally convert positively charged arginine amino acids into neutral citrulline residues. Targets for PAD-catalyzed citrullination include arginine residues on histone tails, which results in chromatin decondensation and changes in gene expression. Some of the first studies examining PADs found that they are localized to rodent uterine epithelial cells. Despite these findings, the function of PAD-catalyzed citrullination in uterine epithelial cells is still unknown. To address this, we first examined PAD expression in uterine cross-sections from pregnant ewes on gestation day 25 (d25). Immunohistochemistry revealed that the levels of PADs 2 and 4 are robust in luminal and glandular epithelia compared with those of PADs 1 and 3. As PADs 2 and 4 have well-characterized roles in histone citrullination, we next hypothesized that PADs citrullinate histones in these uterine cells. Examination of caruncle lysates from pregnant ewes on gestation d25 and an ovine luminal epithelial (OLE) cell line shows that histone H3 arginine residues 2, 8, 17 and 26 are citrullinated, but histone H4 arginine 3 is not. Using a pan-PAD inhibitor, we next attenuated histone citrullination in OLE cells, which resulted in a significant decrease in the expression of insulin-like growth factor-binding protein 1 (IGFBP1) mRNA. As IGFBP1 is important for the migration and attachment of the trophectoderm to uterine endometrium, our results suggest that PAD-catalyzed citrullination may be an important post-translational mechanism for the establishment of pregnancy in ewes.