Mammalian SNM1 is required for genome stability.

The protein encoded by SNM1 in Saccharomyces cerevisiae has been shown to act specifically in DNA interstrand crosslinks (ICL) repair. There are five mammalian homologs of SNM1, including Artemis, which is involved in V(D)J recombination. Cells from mice constructed with a disruption in the Snm1 gene are sensitive to the DNA interstrand crosslinker, mitomycin (MMC), as indicated by increased radial formation following exposure. The mice reproduce normally and have normal life spans. However, a partial perinatal lethality, not seen in either homozygous mutant alone, can be noted when the Snm1 disruption is combined with a Fancd2 disruption. To explore the role of hSNM1 and its homologs in ICL repair in human cells, we used siRNA depletion in human fibroblasts, with cell survival and chromosome radials as the end points for sensitivity following treatment with MMC. Depletion of hSNM1 increases sensitivity to ICLs as detected by both end points, while depletion of Artemis does not. Thus hSNM1 is active in maintenance of genome stability following ICL formation. To evaluate the epistatic relationship between hSNM1 and other ICL repair pathways, we depleted hSNM1 in Fanconi anemia (FA) cells, which are inherently sensitive to ICLs. Depletion of hSNM1 in an FA cell line produces additive sensitivity for MMC. Further, mono-ubiquitination of FANCD2, an endpoint of the FA pathway, is not disturbed by depletion of hSNM1 in normal cells. Thus, hSNM1 appears to represent a second pathway for genome stability, distinct from the FA pathway.

Efficacy after sequencing of brain radiotherapy and enhanced antibody targeted chemotherapy delivery in a rodent human lung cancer brain xenograft model.

PURPOSE: The objective of this study was to evaluate the efficacy of sequencing radiation therapy (RT) and antibody targeted chemotherapy (BR96-DOX) in nude rats bearing human lung cancer (B.5 LX-1) intracerebral (i.c.) xenografts. METHODS AND MATERIALS: Our approach was to administer RT using 20 Gy single-fraction cranial irradiation either before, concurrent with, or after BR96-DOX treatment via osmotic blood-brain barrier disruption to enhance immunoconjugate delivery. All rats were inoculated with i.c. B.5 LX-1 tumors and were randomly assigned to treatment groups. RESULTS: BR96-DOX alone on Day 6 or Day 12 significantly increased survival compared to negative control rats receiving no treatment (25.9 +/- 2.1 and 23.3 +/- 2.5 days vs. 14.8 +/- 1.9 days, p < 0.05). Rats that received chemotherapy before radiation (34.0 +/- 2.0 days) lived the longest compared to the other sequences (RT prior, 29.5 +/- 1.9; RT concurrent, 27.1 +/- 2.1). Histopathology of 39 rat brains did not reveal any neuropathology. CONCLUSIONS: Enhanced delivery of immunoconjugates is more effective in combination with RT for the treatment of experimental metastatic brain tumors. Moreover, BR96-DOX administration prior to RT significantly increased survival compared to those receiving RT and chemotherapy concurrently (p < 0.05).

Importance of photoperiodic signal quality to entrainment of the circannual reproductive rhythm of the ewe.

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.

Thyroid hormones act primarily within the brain to promote the seasonal inhibition of luteinizing hormone secretion in the ewe.

In the ewe, thyroid hormones are required for the seasonal suppression of GnRH and LH secretion, thereby maintaining an annual rhythm in reproductive activity. The primary site of action of thyroid hormones is unknown; in particular, there is no evidence to distinguish a central from a peripheral action. In this study, we test the hypothesis that thyroid hormones can act directly within the brain to promote GnRH/LH seasonal inhibition. Ovariectomized estradiol-treated ewes were thyroidectomized late in the breeding season to prevent seasonal LH inhibition. T4 was then infused for 3 months, either peripherally or centrally. Neuroendocrine reproductive state was monitored by assaying the LH concentration in biweekly blood samples. Central infusion of low dose T4, which restored a physiological concentration of the hormone in cerebrospinal fluid of these thyroidectomized ewes, promoted the neuroendocrine changes that lead to anestrus. The serum LH concentration in these animals fell at the same time as the seasonal LH decline in euthyroid controls. Neither this same T4 dose infused peripherally nor vehicle infused centrally was effective; LH remained elevated, signifying blockade of the mechanism for anestrus. Our results provide strong evidence that thyroid hormones can act directly within the brain to promote seasonal inhibition of neuroendocrine reproductive function in the ewe.

Systemic challenge with endotoxin stimulates corticotropin-releasing hormone and arginine vasopressin secretion into hypophyseal portal blood: coincidence with gonadotropin-releasing hormone suppression.

We tested the hypothesis that systemic immune/inflammatory challenge (endotoxin) activates the neuroendocrine stress axis centrally by stimulating the secretion of CRH and arginine vasopressin (AVP) into hypophyseal portal blood. In addition, we examined the temporal association between this stimulation of the stress neuropeptides and the inhibition of pulsatile GnRH and LH secretion. Using alert, normally behaving ewes, hypophyseal portal and peripheral blood were sampled simultaneously at 10-min intervals for 14 h. Temperature was monitored remotely by telemetry at the same interval. Endotoxin (400 ng/kg, i.v. bolus) or saline as a control was injected after a 4-h baseline period. Portal blood was assayed for CRH, AVP, and GnRH, and peripheral blood was assayed for cortisol, progesterone, and LH. In controls, hypophyseal portal CRH and AVP remained just above or at assay sensitivity, and cortisol showed a regular rhythmic pattern unaffected by saline and typical of basal secretion. In contrast, endotoxin potently stimulated CRH and AVP secretion into portal blood, and cortisol and progesterone into peripheral blood. Both CRH and AVP generally rose and fell simultaneously, although the peak of the AVP response was approximately 10-fold greater than that of CRH. The AVP in portal blood was not due to recirculation of hormone secreted into the peripheral circulation by the posterior pituitary gland, because the AVP increase in peripheral blood was negligible relative to the marked increase in portal blood. The stimulation of CRH and AVP coincided with significant suppression of GnRH and LH pulsatile secretion in these same ewes and with the generation of fever. We conclude that endotoxin induces central activation of the neuroendocrine stress axis, stimulating both CRH and AVP release into the hypophyseal portal blood of conscious, normally behaving ewes. This response is temporally coupled to inhibition of pulsatile GnRH and LH release as well as with stimulation of adrenal cortisol and progesterone secretion and generation of fever.

Importance of the gonadotropin-releasing hormone (GnRH) surge for induction of the preovulatory luteinizing hormone surge of the ewe: dose-response relationship and excess of GnRH.

The preovulatory LH surge in the ewe is stimulated by a large sustained surge of GnRH. We have previously demonstrated that the duration of this GnRH signal exceeds that necessary to initiate and sustain the LH surge. The objective of the present study was to determine whether a similar excess exists for amplitude of the GnRH surge. Experiments were performed using an animal model in which GnRH secretion was blocked by progesterone, which in itself does not block the pituitary response to GnRH. To assess the amplitude of the GnRH surge needed to induce the LH surge, we introduced artificial GnRH surges of normal contour and duration but varying amplitudes. Twelve ewes were run through 3 successive artificial follicular phases (total of 36). Six of these artificial follicular phases were positive controls, in which progesterone was removed, the estradiol stimulus was provided, and vehicle was infused. In these control cycles, animals generated endogenous LH surges. In the remaining artificial follicular phases, progesterone was not withdrawn, the estradiol stimulus was provided, and either vehicle (negative control) or GnRH solutions of varying concentrations (experimental) were infused. The circulating GnRH concentrations achieved by infusion were monitored. No LH surges were observed in negative controls, whereas LH surges were induced in experimental cycles provided a sufficient dose of GnRH was infused. A highly significant dose-response relationship was observed between the amplitude of the GnRH surge and both the amplitude of the LH surge and the area under the curve describing the LH response, but no such relationship existed between the amplitude of the GnRH surge and the duration of the LH response. In numerous cases, LH surges similar to those in the positive control animals resulted from infusion of amounts of GnRH estimated to be considerably less than those delivered to the pituitary during the endogenously generated GnRH/LH surge. These findings indicate that, in the ewe, increased GnRH secretion drives the preovulatory LH surge in a dose-dependent fashion, and they provide evidence that the amplitude of the GnRH surge may exceed that needed to generate the LH surge.

Estradiol requirements for induction and maintenance of the gonadotropin-releasing hormone surge: implications for neuroendocrine processing of the estradiol signal.

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.

Endotoxin inhibits the reproductive neuroendocrine axis while stimulating adrenal steroids: a simultaneous view from hypophyseal portal and peripheral blood.

This study was designed to test the hypothesis that systemic immune challenge with endotoxin inhibits the reproductive axis centrally by suppressing GnRH pulsatile release into hypophyseal portal blood. Using alert, normally behaving, ovariectomized ewes, we sampled hypophyseal portal blood at 10-min intervals beginning 4 h before and continuing 10 h after endotoxin (400 ng/kg, iv bolus, n = 6) or saline (vehicle, iv, n = 6). Simultaneous jugular samples for measurement of LH, cortisol, and progesterone were taken, and core body temperature was monitored by telemetry. Saline had no effect on any of the parameters in control ewes. In contrast, endotoxin dramatically inhibited the reproductive neuroendocrine axis coincident with stimulating the adrenal steroids, cortisol and progesterone, and elevating body temperature. Mean GnRH collection rate and GnRH pulse amplitude were suppressed (pre- vs. 7 h postendotoxin: collection rate 0.93 +/- 0.31 vs. 0.34 +/- 0.13 pg/min; amplitude 4.13 +/- 1.33 vs. 1.30 +/- 0.41 pg/min per pulse; P < 0.05 and P = 0.01). However, endotoxin did not have a significant effect on GnRH pulse frequency. Along with inhibited GnRH secretion, endotoxin significantly suppressed mean LH concentrations (P = 0.001) and LH pulse amplitude (P < 0.05). In addition, endotoxin suppressed LH pulse frequency (P = 0.01). Coincident with reproductive inhibition, endotoxin stimulated cortisol (P < 0.001), progesterone (P < 0.01), and core body temperature (P < 0.001). We conclude that the suppressive effects of endotoxin on the reproductive axis can be mediated centrally through an inhibition of GnRH and thus LH pulsatile secretion. The coincident stimulation of cortisol, progesterone, and temperature raises the possibility that the central inhibition of the reproductive system may be a consequence of any or all of these activated parameters.

A critical period for thyroid hormone action on seasonal changes in reproductive neuroendocrine function in the ewe.

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.

Effect of thyroidectomy on maintenance of seasonal reproductive suppression in the ewe.

Thyroid hormones are essential to end the breeding season in sheep; however, it is not clear whether thyroid hormone action is limited to initiation of seasonal reproductive suppression in the ewe. The purpose of this study was to determine the influence of thyroid hormones on maintenance of anestrus and onset of the subsequent breeding season in the ewe. In both experiments, ewes were thyroidectomized (THX) either soon after they had completed the transition from the breeding season to anestrus or just before the transition into the breeding season (late anestrus). All ewes were ovariectomized and received constant-release silicone elastomer implants of estradiol. Circulating levels of LH were monitored as an index of seasonal changes in reproductive neuroendocrine activity. After thyroidectomy early in anestrus, timing of the subsequent LH rise, indicative of the next neuroendocrine breeding season, was the same among THX and thyroid-intact ewes. As observed previously, LH remained elevated in ewes THX late in anestrus beyond the time associated with development of anestrus. We conclude therefore that thyroid hormones are not needed to maintain suppression of the reproductive neuroendocrine axis once anestrus has been established, nor do they influence the onset of the subsequent breeding season in the ewe. Rather, thyroid hormone action on seasonal alterations of the reproductive neuroendocrine axis in the ewe is restricted to the changes that cause development of anestrus.

How much of the gonadotropin-releasing hormone (GnRH) surge is required for generation of the luteinizing hormone surge in the ewe? Duration of the endogenous GnRH signal.

The preovulatory LH surge in the sheep is accompanied by a massive and sustained surge of GnRH. The objective of this study was to examine the duration of the endogenous GnRH signal required to induce and maintain a LH surge of full amplitude and duration. For this purpose, we assessed the effect of a competitive GnRH receptor antagonist (Nal-Glu), administered at various times relative to the LH surge, on the development and progression of the surge pattern of LH release. All studies were conducted in a physiological model for the follicular phase of the estrous cycle (artificial follicular phase). In this model, as during the natural follicular phase, the onset of the LH surge is coincident with the initiation of a massive and sustained rise in GnRH secretion. The experimental approach was validated in a preliminary study by determination that the GnRH antagonist could block the LH surge without compromising GnRH release, as measured in pituitary portal blood. In the main experiment, 25 ewes were run through five successive artificial follicular phases, during which the antagonist was not given (control) or was administered before the LH surge, during its ascending limb, or during the descending limb. Treatment with antagonist before the expected time of the surge prevented the LH surge. Treatment during the ascending limb of the LH surge interrupted the rise in LH and caused a prompt cessation of the surge. Treatment during the descending limb of the LH surge resulted in a faster decline in circulating LH concentrations than in control cycles and caused premature termination of the LH surge. Our results are consistent with the conclusion that development and progression of the preovulatory LH surge in sheep depend upon GnRH stimulation throughout its entire time course.

Time-course of thyroid hormone involvement in the development of anestrus in the ewe.

It is well established that the thyroid gland is essential for termination of seasonal reproductive activity in a variety of birds and mammals. In the present study, we examined when during the breeding season the thyroid exerts this effect in female sheep. Previous results suggest that the presence of thyroid hormones during the first 4-6 wk (20-25%) of the breeding season is not sufficient for the neuroendocrine changes that lead to anestrus. We therefore hypothesized that thyroid hormone action is exerted at some point during the latter 75-80% of the breeding season. To test this hypothesis, ewes thyroidectomized early in the breeding season received replacement of thyroxine at various times to create gaps during the mid- to late breeding season when thyroid hormones were absent. We then examined the effect, if any, of this absence on development of seasonal neuroendocrine anestrus. Each ewe was ovariectomized and treated with a constant-release Silastic capsule containing estradiol. Serum concentrations of LH were used as an index of seasonal changes in reproductive neuroendocrine activity. We found that when thyroid hormones were removed for a 60-day period in mid- to late breeding season (from mid-Oct. to late Dec., which is approximately 40% of the entire breeding season), anestrus still developed at the normal time. We conclude, therefore, that thyroid hormones need not be present for much of the breeding season (mid-Sept. through late Dec.) for anestrus to develop in the ewe. Rather, we postulate that thyroid hormones need to be present for only a brief period of time near the end of the breeding season for the neuroendocrine changes that lead to anestrus.

Does estradiol induce the preovulatory gonadotropin-releasing hormone (GnRH) surge in the ewe by inducing a progressive change in the mode of operation of the GnRH neurosecretory system.

Estradiol profoundly influences GnRH secretion during the follicular phase of the estrous cycle of the sheep. Estradiol not only regulates the frequency and amplitude of GnRH pulses, but also produces qualitative changes in its pattern of release and induces a sustained GnRH surge during which discrete pulses are not readily evident. In this study, we tested the hypothesis that qualitative changes in GnRH secretion are an integral part of an estradiol-induced change in the mode of operation of the GnRH neurosecretory system that leads to generation of the GnRH surge. This was achieved by the measurement of GnRH in samples of pituitary portal blood collected at 1-min intervals for an 11-h period encompassing the pre- and early surge periods in an artificial follicular phase model. In each of the seven ewes studied, a highly characteristic alteration in the moment to moment pattern of GnRH was observed. This consisted of a progressive change from a strictly episodic pattern of GnRH release to one containing both episodic and nonepisodic components and, after amplification of both components, a period of extremely high values during which individual episodic increases were no longer readily recognizable. Preliminary mathematical modeling of the data suggested that these patterns could be produced by a change in GnRH from a predominantly low to a mixture of low and high amplitude inputs. Similar changes in minute to minute patterns of GnRH secretion were observed during the natural follicular phase. These findings are consistent with the hypothesis that estradiol induces the GnRH surge by altering the mode of neurosecretion, rather than by merely causing quantitative changes in the episodic pattern of release.

Circannual alterations in the circadian rhythm of melatonin secretion.

To determine if a circadian rhythm known to be functionally related to the reproductive axis varies on a circannual basis, we monitored the circadian secretion of melatonin at monthly intervals for 2 years in four ovariectomized, estradiol-implanted ewes held in a constant short-day photoperiod. Prior to the study, ewes had been housed in a short-day (8L:16D) photoperiod for 4 years and were exhibiting circannual reproductive rhythms as assessed by serum luteinizing hormone (LH) levels. Three of the four sheep showed unambiguous deviations from the expected nocturnal melatonin secretion at two different times approximately 1 year apart. Nocturnal rises in melatonin, which usually last the duration of the dark phase, were delayed by 3-14 h or were missing. Altogether, five of the seven melatonin alterations observed in these three ewes occurred during the nadir of the circannual LH cycle. In the remaining ewe, we did not observe an altered melatonin secretory pattern during this period, and this ewe also failed to show a high amplitude circannual cycle of LH. The results provide evidence for a circannual change in the circadian rhythm of melatonin secretion. This alteration in melatonin secretion may serve as a "functional" change in daylength, and thereby may influence the expression of the circannual reproductive rhythm of sheep held in a fixed photoperiod for an extended time.

Thyroxine is permissive to seasonal transitions in reproductive neuroendocrine activity in the ewe.

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)

Involvement of thyroid hormones in seasonal reproduction.

This article reviews experiments performed to investigate the importance of thyroid hormones to the expression of the seasonal reproductive cycle of ewes. Thyroidectomy was found to block the transition from the breeding season to anoestrus and to cause ewes to exhibit oestrous cycles all year round. Mechanistically, thyroidectomy produced this effect by preventing the seasonal increase in responsiveness to the negative feedback action of oestradiol on episodic GnRH secretion, thus interfering with a key neuroendocrine process necessary for anoestrus to develop. This response to thyroidectomy was fully prevented by replacement with physiological concentrations of thyroxine. Furthermore, the reproductive response to thyroidectomy was specific to the mechanisms that lead to anoestrus; other aspects of reproductive neuroendocrine function and seasonal and photoperiodic mechanisms were not affected. Evidence is presented to indicate that thyroid hormones have a permissive mode of action on seasonal reproduction, and that their effect is exerted during a restricted 'window' of time during the year. It is concluded that thyroid hormones play a crucial role of physiological significance to generation of the seasonal reproductive cycle of ewes.

A central negative feedback action of thyroid hormones on thyrotropin-releasing hormone secretion.

Two experiments were conducted to test the hypothesis that thyroid hormones exert central negative feedback effects on the secretion of TRH from the hypothalamus in the ewe. In the first experiment, we examined the effects of thyroidectomy on the secretion of TRH and TSH. Thyroidectomy was followed by an unambiguous increase in TRH in pituitary portal plasma and TSH in the peripheral circulation. In the second experiment, we tested the effects of T4 replacement to thyroidectomized ewes. T4 replacement reversed the effects of thyroidectomy on TRH and TSH release. Of interest, TRH secretion in thyroidectomized ewes was continuously elevated during the collection, raising the possibility that TRH is secreted continuously, rather than exclusively in a strictly pulsatile manner indicative of phasic discharges synchronized among TRH neurosecretory elements. Collectively, these results suggest that thyroid hormones can act centrally to inhibit TRH (and thus TSH) release in the ewe, and they support the concept that at least part of the negative feedback action of thyroid hormones is exerted at the hypothalamic level.

Progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by inhibiting the surge of gonadotropin-releasing hormone.

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.