Summary. Ovine granulosa cells respond, through transcriptional mechanisms, to preovulatory concentrations of gonadotrophins by secreting an M _r 30 000 polypeptide. To determine the stages of the oestrous cycle at which this polypeptide is secreted, corpora lutea were collected on Days 3, 7, 10, 13 and 16 (Day 0 = oestrus; n = 4 per group), cut into 1-mm slices, and incubated for 6–7 h with [³⁵S]methionine. Radiolabelled polypeptides of intra- and extra-cellular origin were separated by polyacrylamide gel electrophoresis and quantitated by densitometry. The M _r 30 000 polypeptide was secreted at all stages of the luteal phase tested (Days 3–16), and represented approximately 24% of the total labelled polypeptide present in the medium; polypeptides of approximate M _r 14 000, 25 000 and 46 000 accounted for most of the other secreted proteins. Neither pituitary hormones (LH, FSH, prolactin) nor cholera toxin (chosen to activate adenylate cyclase) affected the rate of production of M _r 30 000 polypeptide, indicating that, once secretion has been initiated in the granulosa cells, it is not readily modulated by hormonal intervention after luteinization.

Incubation of luteinized granulosa cells with tunicamycin (inhibits N-linked glycosylation reactions) showed that the secreted polypeptide consists of a heavily glycosylated amino acid backbone of approximately M _r 20 000. Western blot analysis established further that the polypeptide was not an inhibin subunit. However, NH₂-terminal amino acid sequencing of the first 25 amino acids revealed a 68% sequence identity between the secreted polypeptide (M _r 30 000) and a human tissue inhibitor of metalloproteinases.

Keywords: granulosa cell; corpus luteum; polypeptide secretion; metalloproteinase inhibitor; sheep

Summary.

The reasons for low fertility in a flock of inbred sheep were investigated. Of a total of forty-one inbred ewes run with tested rams at normal mating time, sixteen (39%) were unlikely to have carried a lamb to term. Causes included non-ovulation (six ewes), non-cleavage or abnormal cleavage of ova (four ewes) and non-viability of early implanted embryos (six ewes). Out of twenty-six non-inbred half-sibs of similar ages only one potentially infertile ewe was found (non-ovulation).

Summary.

Sexually mature mice were stimulated to superovulate by giving exogenous gonadotrophins at known stages of the oestrous cycle. Untreated animals which ovulated spontaneously served as controls. The number of oocytes ovulated by each female was estimated from counts of the number of CL of pregnancy, and the incidence of embryonic mortality during the pre- and post-implantation stages of pregnancy was assessed from the number of zygotes recovered from the reproductive tract at 2·0 and 4·0 days post coitum and of conceptuses examined at 7·5 and 11·5 days post coitum.

The mean number of oocytes ovulated by treated animals was 39·54, compared with 12·80 in controls: in mice which had superovulated, 44% of the ova were lost before implantation compared with about 10% in the controls. Further losses occurred about the time of implantation and at mid-pregnancy and thus the number of embryos classified as normal rarely exceeded the maximum found in controls. Death at mid-pregnancy seemed to be preceded by developmental retardation.

The possibility that genetic and environmental factors play a role in embryonic loss after superovulation is discussed.

Abnormal follicles can produce oestradiol continuously for up to 20 days and this inhibits the hypothalamic–pituitary axis. The present experimental series was designed to determine the minimum exposure to high or low follicular phase concentrations of oestradiol that were required to exert inhibitory effects on LH surge secretion induced by additional oestradiol administered at the end of continuous exposure to oestradiol. Experiments were also included to establish whether the inhibitory effects of prolonged oestradiol were mediated at the pituitary, and whether the failure of response to the oestradiol challenge could be corrected by exposure to normal luteal phase patterns of progesterone. Treatment of ewes between 2 and 12 days with 1, 2 or 4 oestradiol implants (3 cm) totally blocked the subsequent normal LH surge in response to an oestradiol challenge in 45 of 52 ewes pretreated with oestradiol. In the seven ewes that did have an increase in LH, the response occurred at the expected time but was greatly reduced (14 versus 40 ng ml⁻¹), and occurred only in ewes pretreated with oestradiol implants for 2 or 4 days. We were unable to establish a robust linear time–dose relationship, i.e., when ewes were treated with lower doses of oestradiol (one or two implants) for a reduced time (2,4 or 8 days), there was random distribution of the 7 of 32 animals that had a reduced LH surge after oestradiol challenge (with four implants or 50 μg injection). The present study is the first to show that exposure for only 2–4 days to continuous oestradiol at late follicular phase concentrations can disrupt LH surge release. However, in oestradiol-treated ewes, LH secretion was provoked by high or low doses (0.5 mg or 0.5 μg) of GnRH, although it was reduced by 50%, and a GnRH self-priming effect was still evident. All of these results suggest that inhibitory effects occur at the pituitary and hypothalamus. It remains to be confirmed whether the major effect is at the pituitary by reducing GnRH receptor or LH synthesis, or at the hypothalamus via inhibition of GnRH secretion.

The frequency with which transuterine migration of embryos occurs in sheep has been studied by a number of workers (Casida, Woody & Pope, 1966; Baier & Russe, 1968; Scanlon, 1972). When a single ovum is shed from one or both ovaries, the resultant embryo tends to be located in the associated horn of the uterus. When two or more ova are shed from a single ovary, migration of one embryo to the contralateral horn is the general rule, even when this uterine horn is not associated with a CL. There appears to be a greater loss of embryos in ewes with two CL in one ovary than in ewes with a single CL in each ovary, and although it has been suggested that migration is responsible for higher mortality, Sittmann (1972) considered that the available evidence did not support it. From studies on egg transfer in cattle, Rowson, Lawson &

Summary. The reproductive performance of Coopworth ewes after administration of zearalenone was determined in two trials. In Trial 1, zearalenone was administered to groups of 33 ewes at rates of 0, 1·5, 3·0, 6·0, 12·0 and 24·0 mg/ewe/day for 10 days, starting on Day 7 of the oestrous cycle before mating. There was a linear decline (P < 0·001) in ovulation rate with dose of zearalenone; also cycle length decreased and duration of oestrus increased with increasing dose levels. Reductions in the incidence of ovulation and in fertilization were seen only at doses of 12 and 24 mg. In Trial 2, groups of 50 ewes were given the same range of doses of zearalenone for 10 days, starting 5 days after mating to entire rams. There was no effect of zearalenone treatment after mating on pregnancy rate or embryonic loss. These results indicate that the effects of zearalenone, administered orally, on ewe reproduction, at the dose levels examined, were restricted to ewes exposed before mating. Intakes of zearalenone of 3 mg/ewe/day or more during this period would be reflected as depressed ovulation rates and lower lambing percentages.

Keywords: zearalenone; ewes; ovulation; oestrus; fertility

The initial aim of the present study was to test whether the stress of transport suppresses LH pulsatile secretion in ewes. In a pilot experiment in the late breeding season, transport resulted in an unexpected response in three out of five transported, ovariectomized ewes pretreated with oestradiol and progesterone. Before transport, seasonal suppression of LH pulses had occurred earlier than anticipated, but LH pulsatility suddenly restarted for the period of transport. This finding was reminiscent of unexplained results obtained in ovariectomized ewes infused centrally with high doses of corticotrophin-releasing hormone after pretreatment with low doses of oestradiol with or without progesterone. Hence, an additional aim of the present study was to examine whether these latter results with corticotrophin-releasing hormone could be reproduced by increasing endogenous corticotrophin-releasing hormone secretion by transport. Subsequent experiments used groups of at least eight ovariectomized ewes at different times of the year with or without prior exposure to steroids to assess whether these unexpected observations were associated with season or the prevailing endocrine milieu. In the mid-breeding season, transport for 4 h in the absence of steroid pretreatment for 8 months reduced LH pulse frequency from 7.5 ± 0.3 to 6.3 ± 0.4 pulses per 4 h (P < 0.05) and LH pulse amplitude from 2.6 ± 0.5 to 1.8 ± 0.3 ng ml⁻¹ (P < 0.05). Similarly, in the mid-breeding season, 34 h after the cessation of pretreatment with oestradiol and progesterone, transport suppressed LH pulse frequency from 6.1 ± 0.4 to 5.5 ± 0.3 pulses per 4 h (P < 0.05) with a tendency of effect on amplitude (6.2 ± 2.7 to 2.61 ± 0.6 ng ml⁻¹; P = 0.07; note the large variance in the pre-transport data). During mid-anoestrus, evidence of a suppressive effect of transport was only observed on LH pulse amplitude (4.7 ± 0.6 versus 3.0 ± 0.5 pulses per 4 h; P < 0.05) in ovariectomized ewes that had not been exposed to ovarian steroids for 4 months. Repetition of the pilot experiment with 12 ewes during the transition into anoestrus resulted in one ewe with LH pulses seasonally suppressed but increased by transport; 11 ewes had a distinct pulsatile LH pattern which was decreased by transport in six ewes. In anoestrus, there was no effect of transport on LH pulse frequency or amplitude in intact ewes, or those ovariectomized 2–3 weeks previously, with or without prior oestradiol and progesterone treatment. However, basal concentrations of cortisol were greater in anoestrus than in the breeding season, and the increment in cortisol during transport was similar in anoestrus and the breeding season but greater during the transition into anoestrus (P < 0.05). Progesterone concentrations increased from 0.31 ± 0.02 ng ml⁻¹ before transport to 0.48 ± 0.05 ng ml⁻¹ during the second hour of transport (P < 0.05). In conclusion, transport reduced LH pulse frequency and amplitude in ovariectomized ewes that had not been exposed to exogenous steroids for at least 4 months. In most animals, the previously observed increase in LH pulsatility induced by exogenous CRH was not reproduced by increasing endogenous CRH secretion by transport. However, in four ewes, transport did increase LH pulsatility, but only during the transition into anoestrus in ewes with seasonally suppressed LH profiles after withdrawal of steroid pretreatment.

Although the decrease of progesterone in serum and in luteal tissue during luteal regression is well characterized, relatively little is known about changes in proteins produced by the corpus luteum during this time. The first objective was to examine changes in patterns of protein secretion that might be associated with functional and structural luteal regression. The second objective was to characterize the expression of two major secretory products of regressing corpora lutea. Thirty normally cyclic heifers were randomly assigned at day 15–16 of the oestrous cycle (oestrus = day 0) to be ovariectomized at 0 h (no PGF_2α; n = 5) or at 4, 8, 12, 24 or 48 h after PGF_2α-induced luteal regression (n = 5 per time point). Total cellular RNA was isolated from tissue frozen at the time of ovariectomy. Thin slices (< 1 mm) of tissue were placed in methionine-deficient minimum essential media with [³⁵S]methionine and placed in a humidified CO₂ incubator at 38°C. Media and tissues were collected 6 h later. Changes in profiles of secreted proteins were analysed by one-dimensional SDS-PAGE. A number of proteins (relative molecular mass ranging from 14 300 to 200 000) were produced by luteal tissue at each time point (0–48 h). The major secretory proteins had relative molecular masses of approximately 21 500, 28 200, 43 700 and 46 000. Secretion of the relative molecular mass 46 000 protein(s) increased (P < 0.05) between 4 and 24 h after PGF_2α injection compared with the 0 h group. Western blot analyses with either tissue inhibitor of metalloproteinase-1 or tissue inhibitor of metalloproteinase-2 antisera detected immunoreactive proteins of relative molecular mass 28 200 and 21 500, respectively. Concentrations of mRNA encoding tissue inhibitor of metalloproteinases-1 increased (P < 0.01) by 8 h after PGF_2α injection, remained stable (P > 0.20) through 24 h and decreased (P < 0.05) by 48 h after PGF_2α. Concentrations of tissue inhibitor of metalloproteinases-2 mRNA were highest (P < 0.05) at 8 h after PGF_2α injection and lowest (P < 0.05) 48 h following induction of luteolysis. In summary, the profile of luteal protein production changed during luteolysis and two secretory products (tissue inhibitor of metalloproteinases-1 and -2) were identified. Metalloproteinase inhibitors may have an important role in tissue remodelling during structural luteolysis.

Extensive extracellular matrix remodelling occurs within the lifespan of the corpus luteum, particularly during corpus luteum formation and regression. A major mechanism for the regulation of extracellular matrix remodelling is via local production of specific proteinase inhibitors, such as the serine proteinase inhibitor plasminogen activator inhibitor type-1 (PAI-1). The objective of the present study was to characterize the localization, ontogeny and regulation of PAI-1 expression within ovine corpora lutea. Urokinase binding activity was detected within medium conditioned by ovine luteal cells. Production of PAI-1 by ovine luteal cells was confirmed by immunoprecipitating it from labelled proteins in culture medium. mRNA encoding PAI-1 was present within developing (day 3), mature (day 10) and regressing (30 h after prostaglandin F_2α injection on day 10 after the onset of oestrus) corpora lutea as demonstrated by in situ hybridization. The ontogeny of PAI-1 mRNA expression was characterized within corpora lutea collected on days 3, 7, 10, 13 and 16 after the onset of oestrus (n = 4, 4, 4, 3 and 4, respectively). Expression of PAI-1 mRNA did not differ during the luteal phase (P= 0.06), although a trend for an increase in the amount of PAI-1 mRNA was observed on day 16. Expression of PAI-1 mRNA was also examined during luteal regression in corpora lutea collected 0, 6, 12, 24 and 36 h after injection of prostaglandin F_2α on day 10 after the onset of oestrus (n = 4 at each time). Relative PAI-1 mRNA concentrations changed significantly during luteolysis induced by prostaglandin F_2α (P = 0.0002). Administration of prostaglandin F_2α resulted in a transient sevenfold increase in PAI-1 mRNA 6 h after injection (P = 0.0001) but by 12 h the amounts had returned to values similar to those detected on day 10. We conclude that PAI-1 is a major secretory product of ovine luteal cells and that a transient increase in PAI-1 mRNA occurs during luteolysis induced by prostaglandin F_2α. PAI-1 probably plays a key local role in the control of extracellular proteolysis during the luteal phase.

Summary. Mature beef cows were actively immunized pre partum (N = 5) or post partum (N = 10) against a PGF-2α–ovalbumin conjugate or against ovalbumin alone (control; N = 5). All cows in the control group exhibited first oestrous cycles which were of short duration (⩽ 12 days). Mean specific serum binding to [³H]PGF-2α in the control group was consistently < 1%. In the pre-partum PGF-2α-immunized cows, lifespan and progesterone secretion of the first corpus luteum formed post partum was maintained for > 39 days. Specific serum binding to [³H]PGF-2α in pre-partum and post-partum PGF-2α-immunized cows was elevated. Lifespan of the first corpus luteum formed in post-partum PGF-2α-immunized cows was short (<10 days; N = 1), normal (mean = 22 days; N = 4) or maintained (>31 days; N = 5). Luteal lifespan was dependent upon serum PGF-2α antibody titres, with cows exhibiting higher titres frequently having prolonged luteal lifespans after first ovulation. We conclude that active immunization of beef cows against PGF-2α extends the lifespan and progesterone secretion of corpora lutea anticipated to be short-lived. These results support the concept that the shorter lifespan of some corpora lutea in post-partum cows is due to a premature release of PGF-2α from the uterus.

Keywords: active immunization; PGF-2α; corpus luteum; cattle