The observation that circulating thyroxine concentration increases during the breeding season of the ewe, coupled with the finding that thyroid hormones are required for the transition from the breeding season to anestrus in this species, led us to test the hypothesis that the transition to anestrus is driven by a rise in circulating thyroxine. Suffolk ewes were thyroidectomized (THX) late in the anestrous season. Thyroxine was then either not replaced or provided at doses that produced nadir, incremental (simulating the seasonal rise), or mildly hyperthyroid concentrations in serum. Additional ewes remained thyroid-intact. To monitor seasonal changes in reproductive neuroendocrine activity, the ewes were ovariectomized and received implants of constant-release Silastic capsules containing estradiol. Serum concentrations of LH and thyroxine were determined in samples collected twice weekly. In all groups, LH increased in mid-September, signifying that manipulation of thyroid status did not influence onset of the neuroendocrine breeding season. In thyroid-intact controls, LH decreased to low concentrations in mid-January, marking the neuroendocrine transition to anestrus. As expected, LH remained elevated through the end of the study (April) in THX controls not receiving thyroxine, confirming that the neuroendocrine transition to anestrus is dependent on thyroid hormones. The seasonal decrease in LH was seen in all ewes treated with thyroxine. This decrease in LH was neither advanced in mildly hyperthyroid ewes nor delayed in ewes exposed to low serum concentrations of thyroxine. These results lead to the conclusion that the seasonal increase in circulating thyroid hormone in the ewe does not drive the transition from the breeding season to anestrus.(ABSTRACT TRUNCATED AT 250 WORDS)

An endogenous circannual rhythm drives the seasonal reproductive cycle of a broad spectrum of species. This rhythm is synchronized to the seasons (i.e., entrained) by photoperiod, which acts by regulating the circadian pattern of melatonin secretion from the pineal gland. Prior work has revealed that melatonin patterns secreted in spring/summer entrain the circannual rhythm of reproductive neuroendocrine activity in sheep, whereas secretions in winter do not. The goal of this study was to determine if inability of the winter-melatonin pattern to entrain the rhythm is due to the specific melatonin pattern secreted in winter or to the stage of the circannual rhythm at that time of year. Either a summer- or a winter-melatonin pattern was infused for 70 days into pinealectomized ewes, centered around the summer solstice, when an effective stimulus readily entrains the rhythm. The ewes were ovariectomized and treated with constant-release estradiol implants, and circannual cycles of reproductive neuroendocrine activity were monitored by serum LH concentrations. Only the summer-melatonin pattern entrained the circannual reproductive rhythm. The inability of the winter pattern to do so indicates that the mere presence of a circadian melatonin pattern, in itself, is insufficient for entrainment. Rather, the characteristics of the melatonin pattern, in particular a pattern that mimics the photoperiodic signals of summer, determines entrainment of the circannual rhythm of reproductive neuroendocrine activity in the ewe.

Previous studies indicate an elevation of circulating progesterone blocks the positive feedback effect of a rise in circulating estradiol. This explains the absence of gonadotropin surges in the luteal phase of the menstrual or estrous cycle despite occasional rises in circulating estradiol to a concentration sufficient for surge induction. Recent studies demonstrate estradiol initiates the LH surge in sheep by inducing a large surge of GnRH secretion, measurable in the hypophyseal portal vasculature. We tested the hypothesis that progesterone blocks the estradiol-induced surge of LH and FSH in sheep by preventing this GnRH surge. Adult Suffolk ewes were ovariectomized, treated with Silastic implants to produce and maintain midluteal phase concentrations of circulating estradiol and progesterone, and an apparatus was surgically installed for sampling of pituitary portal blood. One week later the ewes were allocated to two groups: a surge-induction group (n = 5) in which the progesterone implants were removed to simulate luteolysis, and a surge-block group (n = 5) subjected to a sham implant removal such that the elevation in progesterone was maintained. Sixteen hours after progesterone-implant removal (or sham removal), all animals were treated with additional estradiol implants to produce a rise in circulating estradiol as seen in the follicular phase of the estrous cycle. Hourly samples of pituitary portal and jugular blood were obtained for 24 h, spanning the time of the expected hormone surges, after which an iv bolus of GnRH was injected to test for pituitary responsiveness to the releasing hormone. All animals in the surge-induction group exhibited vigorous surges of GnRH, LH, and FSH, but failed to show a rise in gonadotropin secretion in response to the GnRH challenge given within hours of termination of the gonadotropin surges. The surges of GnRH, LH, and FSH were blocked in all animals in which elevated levels of progesterone were maintained. These animals in the surge-block group, however, did secrete LH in response to the GnRH challenge. We conclude progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by acting centrally to inhibit the surge of GnRH secreted into the hypophyseal portal vasculature.

Two experiments were performed to examine the temporal requirements of the estradiol signal for the GnRH and LH surges in the ewe. Hypophyseal portal and jugular blood (to measure GnRH and LH, respectively) were sampled from ewes set up in an artificial follicular phase model. After progesterone withdrawal to simulate luteolysis, circulating estradiol was raised to a preovulatory level by inserting estradiol implants, which then were removed at different times to vary estradiol signal duration. The objective of the first experiment was to assess the effect of withdrawing estradiol at surge onset on development and maintenance of the GnRH/LH surges. Removal of estradiol, before surge onset, neither altered the LH surge in relation to that induced when the estradiol stimulus was maintained nor affected stimulation of a massive and sustained GnRH surge that outlasted the LH surge by many hours. Continued estradiol treatment, however, did prolong the GnRH surge. In the second experiment, the estradiol stimulus was shortened to test the hypothesis that estradiol need not be present for the whole presurge period to induce GnRH/LH surges. Ewes received estradiol either up to the time of surge onset (21 h) or for periods equivalent to the last 14 h, the last 7 h, or the earliest 7 h of the 21-h signal. Shortening the signal to 14 h did not reduce its ability to stimulate a full GnRH surge, but it did reduce the amplitude of the resultant LH surge. Further shortening of the signal to 7 h, however, produced a mixed response. Most animals (8 of 10 combining the two 7-h groups) did not express GnRH surges. In the two ewes that did, GnRH surge amplitude and duration were again within the range observed with the 21-h estradiol signal, but the LH response was greatly reduced. These results indicate that, once the GnRH/LH surges of the ewe have begun, elevated estradiol is not required for surge maintenance. Development of a full GnRH surge requires elevated estradiol for only a portion of the presurge period. More prolonged exposure to estradiol, however, is needed to maximize pituitary responsiveness to GnRH. Since the estradiol signal for the GnRH surge is relatively short (7-14 h) and temporally located well in advance of the surge itself, these results are consistent with the hypothesis that estradiol is required only to activate the steroid-responsive neuronal elements and not for progression of the signal from these elements to the actual surge process of GnRH release.

Thyroid hormones are obligatory for the annually recurring termination of reproductive activity in a spectrum of seasonal breeders, including sheep. Previous studies involving thyroidectomy and T4 replacement have led to the hypothesis that, in the ewe, thyroid hormones are necessary only during a limited interval late in the breeding season for the neuroendocrine processes that cause the transition to anestrus. The present series of experiments tested this hypothesis by assessing the influence of thyroidectomy, with or without T4 replacement for specific durations and at different times of the year, on the transition to anestrus. Seasonal alterations in reproductive neuroendocrine activity were monitored by changes in serum LH concentration in ovariectomized ewes bearing s.c. SILASTIC brand silicon tubing implants containing estradiol. Thyroidectomy in mid-December, just before the putative period of thyroid hormone action, prevented the development of the neuroendocrine anestrous season (fall in LH in this animal model). T4 replacement for 90 days beginning in late December (i.e., during the postulated period of thyroid hormone action) overcame the blockade of anestrus, causing LH to fall in ewes thyroidectomized several months previously. The minimal effective duration of exposure to thyroid hormones required for the transition to anestrus was estimated to be 60-90 days. Further, exposure to T4 for 60-90 days beginning in late December was found to be the only time of the year that thyroid hormones were required to maintain seasonal changes in reproductive neuroendocrine activity. Finally, replacement of T4 for 90 days at a different time of year (beginning in August) failed to provoke development of neuroendocrine anestrus in thyroidectomized ewes. These results support the hypothesis that thyroid hormones are necessary only during a limited interval late in the breeding season to promote seasonal reproductive suppression in the ewe. Further, the reproductive neuroendocrine axis is not equally responsive to thyroid hormone at all times of the year. This suggests there is a critical period of responsiveness during which thyroid hormones must be present for anestrus to develop.