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B. G. Miller and N. W. Moore

Summary. The secretion and synthesis of protein in vitro by explants of endometrium were examined in entire ewes during the first 10 days of the oestrous cycle and during an equivalent interval in ovariectomized ewes which received injections of oestradiol and progesterone. The schedule of steroid injections given was designed to simulate endogenous ovarian secretion of progesterone during the luteal phase before oestrus, of oestradiol around oestrus and of progesterone during the luteal phase after oestrus.

The rate of protein synthesis and tissue RNA: DNA and protein: DNA ratios in intercaruncular and caruncular endometrium were generally higher in entire than in ovariectomized ewes. In ovariectomized ewes oestradiol increased these activities at 2– 4 days after oestrus, whereas progesterone preceding oestradiol caused increases at oestrus, but not thereafter. In entire ewes and in ovariectomized ewes receiving the full steroid treatment regimen, protein secretion was high at oestrus and declined markedly during the next 4–6 days. In ovariectomized ewes not receiving progesterone before oestradiol, secretion increased between 4 and 6 days after oestrus, or during the equivalent stage of treatment in ewes which did not show oestrus. The omission of this progesterone did not modify secretion by caruncular endometrium. Oestradiol increased protein secretion by both tissues.

The data suggest that progesterone given before oestradiol (or its equivalent in entire ewes) inhibits the secretion, at about 4–7 days after oestrus, of uterine proteins which may impair embryo development in ovariectomized ewes which do not receive this progesterone.

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A. Caraty, D. W. Miller, B. Delaleu and G. B. Martin

Corticotrophin-releasing hormone (CRH) has been proposed as a mediator of the antireproductive effects of stress through an action within the hypothalamus to inhibit GnRH secretion. This hypothesis was tested in sheep by studying the responses to central administration of CRH in both sexes and in both seasons. Sexually mature, Ile-de-France ewes and Romanov rams that had been gonadectomized and implanted with a permanent guide cannula into the third cerebral ventricle were used. Ewes were studied in the presence and absence of exogenous oestradiol plus progesterone, in both the breeding and anoestrous seasons. All rams were treated with testosterone and were studied only during the breeding season. Each observation involved serial samples (every 10 min) of jugular blood for 5 h before (control) and 5 h after an intracerebroventricular (icv) injection of either saline (vehicle) or 5 nmoles CRH in 20 μl vehicle. The saline injections did not affect any of the endocrine variables measured; however, CRH always increased cortisol concentrations in jugular plasma. In the absence of treatment with replacement sex steroids, icv injection of CRH had no effect on pulsatile LH secretion in females either during the breeding season or during anoestrus. However, LH pulse frequency and mean LH concentrations increased significantly on every occasion on which animals were treated with sex steroids. Treatment with CRH also increased LH secretion in the testosterone-treated rams. It is concluded that, contrary to the hypothesized role of CRH as an inhibitor of reproductive activity, this neuropeptide stimulates pulsatile LH (and thus GnRH) secretion, at least in this species. The fact that gonadal steroids seem to be obligatory for the expression of this effect suggests that the protocols used in past studies need to be reassessed.

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U-11555A is oestrogenic in vaginal smear tests in mice with a low slope and a subcutaneous med of not less than 5 mg. Its oestrogenic activity intravaginally was not fully investigated, but is not high. It is weakly anti-oestrogenic in similar tests, with an med against oestradiol of about 200 μg subcutaneously, perhaps a little lower intravaginally. No interactions were seen as they were with U-11100A† (Emmens & Martin, 1965).

U-11555A has an antifertility action in the mouse when injected on Days 1 to 3 or 4 to 6 of pregnancy, with meds of about 100 μg (Days 1 to 3) and 300μg (Days 4 to 6). In rats, U-11555A has a constant subcutaneous or oral med on Days 1 to 3 or 4 to 6 of about 400 μg. In simultaneous tests in rats, U-11100A had meds of about 20 μg (Days 1 to 3) and 60 μg (Days 4 to 6) and was significantly more potent orally than by injection.

From these results, the antifertility action of U-11555A can hardly be attributed to oestrogenic properties, as was that of U-11100A by Emmens & Martin (1965), and its action profile is not very similar to that of the latter. Its antifertility action in mice is in accord with its anti-oestrogenic potency in that species.

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N. W. Moore, B. G. Miller and M. N. Trappl

Summary. Embryos collected from donor ewes 2 or 3 days after oestrus were transferred synchronously to the oviducts or uterine horns of entire or ovariectomized ewes and 2–4 days after transfer the embryos were recovered by flushing the uteri and oviducts. Ovariectomized ewes were treated with all, or part, of a steroid hormone regimen designed to simulate ovarian secretion, in entire ewes of progesterone during the luteal phase before oestrus, of oestradiol around oestrus and of progesterone during the luteal phase after oestrus.

Irrespective of treatment regimen in ovariectomized ewes, only ≤ 50% of embryos transferred to the oviducts were recovered and the majority remained in the oviducts. Most of those remaining in the oviducts showed apparently normal development. In ewes on the complete regimen all of 3 embryos recovered from the uterus had developed normally, whereas none of 7 recovered from the uteri of ewes treated with only part of the regimen had developed normally. Most embryos transferred to the oviducts of entire ewes reached the uterus within 3 days of transfer and, irrespective of whether located in the uterus or oviducts, almost all had developed normally. Few 2-day embryos survived transfer to the uterus. When transfer was delayed to Day 3, 70–80% of embryos recovered from entire ewes, and ovariectomized ewes given the full regimen, had developed normally, whereas only 20–30% of embryos recovered from ovariectomized ewes given part of the regimen developed normally.

Delayed transport of embryos from the oviducts to the uteri of ovariectomized ewes seemed to be due to deficiencies of the treatment regimen or residual effects of ovariectomy. However, the steroid hormone requirements for continued development in the oviducts were not critical, whereas requirements both of steroid hormone regimen and of time of transfer were critical for continued development in the uterus.

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S. P. Brinsko, B. A. Ball, P. G. Miller, P. G. A. Thomas and J. E. Ellington

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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A number of 4,4′-dihydroxybibenzyls has been prepared, with resolution of some isomers. Most are pro-oestrogens and all are anti-oestrogens of varying potency. Dextro- and laevo-α,α′-dimethyl-4,4′-dihydroxybibenzyl (d- and l-dma) have the same anti-oestrogenic and antifertility potencies in mice, but threo- and erythro-α-ethyl-α′-methyl-4,4′-dihydroxybibenzyl (threo- and erythro-mea) differ markedly in all properties examined. Erythro-mea, although a fairly potent pro-oestrogen, is highly anti-oestrogenic intravaginally and a potent antifertility agent with an med of less than 1 μg/day. dl-Hexoestrol (dl-dea) is also a potent anti-oestrogen and fairly potent antifertility agent, with an med about 10 μg/day.

This series of compounds contrasts with the stilbene-4,4′-diols in possessing members with exceptional antifertility potency, not readily explained by their oestrogenic activities.

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D. W. Miller, D. Blache, R. Boukhliq, J. D. Curlewis and G. B. Martin

Nutrition influences the reproductive axis via alteration of gonadotrophin secretion. However, a link between nutrition and the secretion of GnRH, which drives the axis, has yet to be established. The aim of the present study was to measure the change in the concentrations of metabolic substances in the cerebrospinal fluid of adult male sheep offered a diet designed to maintain constant gonadotrophin secretion (Group M; n = 6), or a diet known to increase gonadotrophin secretion (Group M + L; n = 6). On days 1, 3 and 10 of the dietary treatments, cerebrospinal fluid and jugular blood were sampled and analysed for metabolic fuels (glucose, amino acids and free fatty acids) and metabolic hormones (insulin, insulin-like growth factor I, GH, prolactin, cortisol and the thyroid hormones). On day 11 of the dietary treatment, LH pulse frequency and mean FSH concentrations in Group M + L had increased relative to Group M and to day 0. Plasma concentrations of prolactin and insulin on days 3 and 10, and glucose and insulin-like growth factor I on day 10, were higher in Group M + L than in Group M, but only cerebrospinal fluid concentrations of insulin, glucose and certain amino acids were affected by the dietary treatments on days 3 and 10. Cerebrospinal fluid, but not plasma, concentrations of aspartate, tyrosine, cystine, phenylalanine and arginine on day 3, and glutamine, γ-aminobutyric acid, threonine, alanine on days 3 and 10, were higher in Group M + L relative to Group M. On day 10, plasma and cerebrospinal fluid concentrations of arginine, phenylalanine, proline, tyrosine, methionine and phosphoserine, but only the plasma concentrations of linoleic acid, aspartate and serine, were higher in Group M + L than in Group M. Concentrations of triiodothyronine, thyroxine, and cortisol in plasma and cerebrospinal fluid were not affected. These results show that the nutritional stimulation of gonadotrophin secretion is accompanied primarily by fluctuations in plasma and cerebrospinal fluid concentrations of insulin and certain amino acids, which suggests that, when nutritional status is improved, insulin, amino acids and possibly glucose interact to modulate GnRH secretion.

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S. M. Skinner, G. J. Killian, L. A. Miller and B. S. Dunbar

The effectiveness of zona pellucida antigens in immunizing white-tailed deer to reduce fertility was evaluated by analysing the constituent deer zona pellucida proteins and their immunogenicity. Does were immunized with porcine zona pellucida antigens. The antibodies were characterized using immunohistochemical and immunoblot analysis, in which zona pellucida proteins were separated by one-dimensional and two-dimensional PAGE. Deer anti-porcine zona pellucida antibodies were found to recognize all the major proteins of the porcine zona pellucida. These antibodies also recognized several proteins of deer zona pellucida, indicating that it is possible to break immune tolerance in the deer using such a protocol. The antibodies were also found to recognize peptides of 55 and 75 kDa that were produced by expressing cDNA clones containing antigens of major glycoproteins of rabbit zona pellucida. Furthermore, antibodies against rabbit zonae pellucidae recognized antigens in zonae of paraffin-embedded deer ovaries. Taken together, these experiments demonstrate the crossreactive nature of a number of zona pellucida epitopes found in deer and in several other species. They also illustrate the immunogenicity possible in such an immunization protocol, and provide valuable probes for the investigation of follicular development in this and other species.