of the Pituitary Gland in Sheep1

BIOLOGY OF REPRODUCTION 43, 554-558 (99) Evidence for a Direct Negative Effect of Estradiol at the Level of the Pituitary Gland in Sheep T. M. NETF,2 J. A. FLORES, F. CARNEVALI, and J. P. KILE Animal Reproduction and Biotechnology Laboratory, Department of Physiology Colorado State University, Fort Collins, Colorado 8523 ABSTRACT The purpose of this experiment was to determine if pituitary stores of LII could be replenished by administration of when circulating concentrations of both progesterone and estradiol-7 (estradiol) were present at levels observed during late gestation. Ten ovariectomized (OVX) ewes were administered estradiol and progesterone via Silastic implants for 69 days, One group of 5 steroid.treated OVX ewes was given for an additional 42 days (25 ng once every 4 h). Steroid treatment alone reduced (p <.) the amount of UI in the anterior pituitary gland by 77%. Pulsatile administration of to steroidtreated ewes resulted in a further decrease (p <.) in pituitary content of UI. Compared to the OVX ewes, concentrations of mrnas for a- and LH(-subunits were depressed (p <.) in all steroid-treated ewes, whether or not they received GnRl-I. The ability of the dosage of used to induce release of LII was examined by collecting blood samples for analysis of UI at 5 days and 42 days after treatment was initiated. Two of 5 and 3 of 5 steroid-treated ewes that received pulses of responded with increased serum concentrations of LII after administration during the first and second bleedings, respectively. The same dosage of induced release of LH in 5 of 5 ewes treated with during the midluteal phase of the estrous cycle. receptors in the anterior pituitary of the steroid-treated ewes that received pulses of were not downregulated. Therefore, it appears that pulses of are unable to increase the amount of UI in the pituitary gland when serum concentrations of estradiol and progesterone are maintained at levels observed during late gestation. Since the amounts of mrna for a- and LH-subunits were not increased in steroid-treated ewes administered, we speculate that estradiol (and/or progesterone) acts directly at the level of the anterior pituitary gland to inhibit production of mrnas required for synthesis of UI. INTRODUCTION High serum concentrations of progesterone and estradiol-73 (estradiol) have been postulated to be responsible for the reduced pituitary content of LH noted during gestation and the early postpartum period [). There is a general consensus that the negative feedback of progesterone occurs at the level of the hypothalamus to reduce the frequency of pulsatile discharges of [2, 3,4]. However, the effect of progesterone on pituitary content of LH appears to be minimal [5, 6, 7]. At present, it is not clear if estradiol acts directly at the level of the pituitary to decrease content of LH, or indirectly by inhibiting secretion of. Low levels of estradiol administered to ovariectomized ewes during the nonbreeding season inhibit secretion of [3]; however, low levels of estradiol do not decrease amounts of mrna for LHI3-subunit in ovariectomized, hypothalamic-pituitary-disconnected ewes receiving pulses of [8]. Thus, it seemed possible that administration of would increase amounts of mrna for the subunits of LH, and ultimately pituitary content of LH, in animals with high circulating concentrations of estradiol and progesterone. Moreover, the initial phase of postpartum anestrus appears to be due to insufficient se- Accepted May 4, 99. Received March 2, 99. Supported by NIH HD784 and the CSU Experiment Station. JA.F. and J.F.K were supported by NIH Training Grant -)73. Correspondence. cretion of LH [], probably as a result of decreased pituitary content of W during late gestation [9,, ]. Therefore, if pituitary content of LH could be increased during late gestation by treatment of animals with, this might lead to a shorter postpartum anestrous interval. In this study, we used ovariectomized (OVX), steroidtreated ewes to determine if pituitary stores of LH could be replenished by administration of when circulating concentrations of both progesterone and estradiol were experimentally maintained at levels observed during late gestation. Our rationale was that if these steroids act primarily at the hypothalamus to decrease secretion of, then administration of should increase pituitary stores of LH when both steroids are present at high concentrations. Conversely, if the steroids also have a negative effect at the level of the pituitary gland, would not be effective in stimulating synthesis of LH in this model. MATERIALS AND METHODS The experiment was conducted between November and January when intact ewes were exhibiting regular estrous cycles. Twenty mature western range ewes were divided into four groups. Groups, 2, and 3 were ewes that hadbeen OVX for at least 6 days. Group 4 were intact ewes during the midluteal phase of the estrous cycle. Animals in Group received estradiol and progesterone (Sigma Chemical Co., St. Louis, MO) via subcutaneous Silastic implants. Each ewe received six estradiol-containing implants Downloaded from https://academic.oup.com/biolreprod/article-abstract/43/4/554/2762767 by guest on 8 December 28 554

ESTRADIOL INHIBITS LII SYNTHESIS AT THE PITUITARY GLAND 555 [2] designed to maintain serum concentrations of estradiol at approximately 3 pg/ml [3]. Four progesterone-containing implants [4] were inserted subcutaneously in the axillary region of each ewe and provided serum concentrations of about 2 ng/ml [5]. Sixty-nine days after implants were inserted, an interval sufficient to reduce pituitary content of LH by >75% [3], animals were moved indoors, penned indivdually, and administered subcutaneously in pulses of 25 ng once every 4 h. Subcutaneous administration was chosen to prevent the phlebitis associated with prolonged intravenous administration, and because the adsorption of after subcutaneous administration provides a pattern in the circulation approximating that obtained during a 6-mm intravenous infusion [6] (Nett, unpublished observation). Pulses were administered at 4-h intervals since pituitary content of UI in OVX ewes appears to increase more rapidly if the ewes are treated with progesterone [7]. Treatment with progesterone has been shown to decrease frequency of endogenous pulses to one pulse every 4-6 h [3]. Each pulse of lasted for -3 s and was delivered via a programmable autosyringe pump mounted on the back of the sheep on a fiberglass saddle [7]. After 5 days of treatment with GnRT-I, a length of time after parturtion sufficient for pituitary content of LH to return to -5% of normal [], the pumps were turned off for a period of 2 h and the animals were bled once every mm for hr. Pumps were then turned back on to synchronize delivery of a dose of to each of the ewes and they were bled at -mm intervals for an additional h. Administration of was continued for 27 more days to provide a duration of treatment similar to the interval required for complete restoration of pituitary content of LH following parturition [8], and this bleeding regimen was repeated. Serum collected from the two bleedings was assayed for LH. The day after the second bleeding, the animals in Group were euthanized with an overdose of pentobarbital and their pituitary glands were collected and stored at -7#{76}Cuntil analyzed for content of LH, receptors, and mrna for the subunits of LH. Animals in Group 2 received steroid implants as described for Group. After 69 days, these animals were euthanized and their pituitary glands were collected and frozen for analysis at a later time. Ewes in Group 3 received empty implants and were euthanized 69 days later, and their pituitaries were collected and frozen. Ewes in Group 4 (in the midluteal phase of the estrous cycle) were bled at - mm intervals for h. A single subcutaneous injection of (25 ng) was administered and bleeding was continued for an additional hour. One half of each frozen pituitary gland was weighed, minced, and homogenized in Iris buffer ( mm Tris[hydroxymethyl]aminomethane, mm CaCl2,.25 M sucrose,.% BSA) and centrifuged at 3 X g for 3 mm. receptors were quantified in the membrane fraction by a standard curve technique [9] and supernatants were saved for determining concentrations of LH. Serum and pituitary concentrations of UI were measured by RIA [2]. The intraassay coefficient of variation was.3% and the mnterassay coefficient of variation was 5.8%. Sensitivity of the assay was 2 pg per tube (6 pg/mi). The standard was NIH-LH-S24. Total cellular RNA was isolated from the remainder of each pituitary by centrifugation through a cesium chloride gradient [2]. Concentrations of mrna for a- and LHI3-subunits and growth hormone (GH) were quantified by slotblot analysis. Amounts of mrna for GH were measured to examine the specificity of the steroid feedback. Hybridization intensities of RNA in the samples were compared with those of RNA standards. Standard RNA transcripts of psp64 cdna clones of a- [22] and LHI3-subunit [23] were prepared as described by Melton et al. [24]. Increasing amounts of each RNA transcript (-25 pg) or sample (5 pg total RNA) were heated to 65#{76}C for 2 mm and then applied in triplicate to nylon filters (Gene Screen, New England Nuclear, Boston, MA). RNA on the filters was hybridized with 32Plabeled cdna probes (approximately i7 total cpm for each probe) at 65#{76}C for 48 h. Filters were washed and placed on X-ray film (Kodak XAR-5; Eastman Kodak Co., Rochester, NY) and stored at -7#{76}Cfor 24-48 h. The 32P-cDNA-mRNA heteroduplexes were visualized by autoradiography and the relative density of each band was quantified by use of a GS- 3 scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA). When increasing quantities of total cellular RNA (.5-8 p.g) were subjected to hybridization, the amount of specific mrna detected increased linearly in a manner parallel to the standard curve for each of the subunit mrnas (LHI3-subunit, Fig. ; similar data were obtained for a-subunit but are not shown). Analysis of mrna for GH was performed by using similar procedures except that mrna transcripts for use as standards were unavailable at the time of this study. Therefore, amounts of mrna for GH are reported in arbitrary units. Data were subjected to analysis of variance. When significant treatment effects were observed, differences be- L id2 io 2,3 LH mrna OR TOTAL RNA (ng) FIG.. Intensities of autoradiograms for standard RNA transcript of LH-subunit and for increasing amounts of total cellular ANA from an OVX ewe after hybridization to an LHR-subunit cdna probe. Downloaded from https://academic.oup.com/biolreprod/article-abstract/43/4/554/2762767 by guest on 8 December 28

556 NEU ET AL. tween means were determined by Dunnett s procedure. All statistical procedures were conducted using the SPSS program [25]. RESULTS x > 5 Relative to the OVX ewes, steroid treatment reduced (p <.) pituitary content of UI by 77% (Fig. 2). Further, pituitaries from the steroid-treated OVX ewes administered contained less (p <.) LH than pitumtarmes from OVX ewes treated only with steroid. Steroid treatment also decreased (p <.) the concentrations of mrna for a- and LHI3-subunits in the anterior pituitary gland (Fig. 3). Pulsatile administration of to steroid-treated ewes for 42 days was unable to increase the amount of mrna for either a- or LHI3-subunit. Compared to the OVX control ewes, neither treatment with steroids alone nor steroids plus influenced the amount of mrna for GH in the anterior pituitary (Fig. 4). The number of receptors in OVX ewes treated with estradiol and progesterone and administered pulses 6 C) 4 I...j 2 >< o8 4O. 2 FIG. 2. Concentrations of LH in pituitaries of OVX ewes, OVX ewes bearing implants containing progesterone and estradiol (P + E), and OVX ewes bearing steroid implants and administered pulses of. Values depicted are mean ± SE (n = 5 per group). Means with unlike superscripts differ (p <.). TaT Ia LH b z E C, 5 FIG. 4. Concentrations of mrna for GH in pituitaries of OVX ewes, OVX ewes bearing implants containing progesterone and estradiol (P + E), and OVX ewes bearing steroid implants and administered pulses of. Values depicted are mean ± SE (n = 5 per group). of for 42 days was similar to that in OVX ewes receiving only the steroid treatment (Fig. 5). Thus, pulsatile administration of did not cause a down-regulation of receptors in the steroid-treated ewes. Basal serum concentrations of LH in steroid-treated ewes were 37 ± 7 pg/mi compared to 5 ± 4 pg/ml in ewes during the midluteal phase of the cycle. Two of 5 steroid-treated ewes that received pulses responded to a challenge with after 5 days of administration. The magnitude of the LH response in these 2 ewes was 3 ± 4 pg/mi. After 42 days of treatment with, 3 of 5 ewes responded to the GnRI-I challenge, but the increase in UI in these 3 ewes was only 78 ± 6 pg/ml, When ewes were challenged with a single subcutaneous injection of during the midluteal phase of the estrous cycle, 5 of 5 responded with increased secretion of UI (25 ± 65 pg/mi). Co I- I-. QCI) )2 G) DISCUSSION In the present study, pituitary content of UI in steroidtreated OVX ewes was reduced to values comparable to those Downloaded from https://academic.oup.com/biolreprod/article-abstract/43/4/554/2762767 by guest on 8 December 28 ovx P+E P+E FIG. 3. Concentrations of mrna for u- and LH-subunit in pituitaries of OVX ewes, OVX ewes bearing implants containing progesterone and astradiol (P + E), and OVX ewes bearing steroid implants and administered pulses of. Values depicted are mean ± SE In = 5 per group). Means with unlike superscripts differ (p <.). FIG. 5. Concentrations of receptors in pituitaries of OVX ewes, OVX ewes bearing implants containing progesterone and estradiol (P + E), and OVX ewes bearing steroid implants and administered pulses of. Values depicted are mean ± SE (n = 5 per group).

ESTRADIOL INHIBITS UI SYNTHESIS AT THE PITUITARY GlAND 557 reported for ewes during late gestation and the early postpartum period [, 3]. We had predicted that if the site of action of estradiol to decrease pituitary content of UI was restricted to the hypothalamus and decreased secretion of, then we should see an increase in the pituitary content of UI in steroid-treated OVX ewes administered pulses of for 6 wk, sufficient time for pituitary stores of UI to increase after parturition [, 8]. We did not find this to be the case. On the contrary, the hypophyseal content of UI was lower (p <.) in steroid-treated OVX ewes administered pulses of than in OVX ewes treated only with estradiol and progesterone. Therefore, since administration of at a dosage shown to increase pituitary content of UI [26] and amounts of mrna for subunits of the gonadotropins [27] failed to increase pituitary content of UI in steroid-treated ewes, some of the negative effects of estradiol on content of UI are probably exerted directly at the level of the gonadotroph. A direct feedback effect of estradiol on secretion of UI at the level of the pituitary is in agreement with previous observations [28, 29] reported for ewes and for rhesus monkeys [3]. Likewise, Phillips et al. [3] found that treatment of ovine anterior pituitary cells with estradiol inhibits transcription of the genes encoding a- and FSHI3-subunits. More recently, Shupnik et al. [32] reported the presence of an estrogen response element on the gene encoding the UI3-subunit in rats. Therefore, it seems likely that estradiol may act directly on the UI3-subunit gene in the ovine anterior pituitary to prevent transcription, thereby preventing synthesis of LH. Further support for this hypothesis is provided by the fact that concentrations of mrna for UIt3-subunit were reduced in steroid-treated ewes and that administration of to steroid-treated ewes did not alter steady-state levels of mrna for LHI3-subunit. The negative effect of estradiol on concentrations of mrna for UI3-subunit may be dose-dependent since low levels of estradiol do not inhibit the induced increase in OVX ewes after hypothalamic-pituitary disconnection [8]. Steroid treatment also decreased pituitary concentrations of mrna for a-subunit, which is in agreement with our previous observations [2,33]. Again, administration of, which stimulates an increase in mrna for a-subunit in OVX ewes after hypothalamic-pituitary disconnection, was not able to override the inhibitory effect of steroid treatment on a-subunit mrna. However, to date, the presence of an estrogen response element on the gene for a-subunit has not been reported. Since estradiol does appear to have a negative effect on production of a-subunit at the pituitary (Phillips et al. [29]; this study), it must stimulate the production of another factor that inhibits transcription of the a-subunit gene. The percentage decrease in pituitary concentrations of mrna for a-subunit was similar to that observed for UI3-subunit. This was not surprising since high concentrations of estradiol also decrease mrna for FSHI3- subunit [3,33,34]. Moreover, we have previously postulated that -85% of mrna for a-subunit is associated with UI []. That content of UI in the anterior pituitary of steroidtreated ewes administered pulses of was lower than in steroid-treated ewes further strengthens the interpretation for a direct action of estradiol at the pituitary gland. We take this to mean that the provided in the replacement regimen was able to induce secretion of the small amount of UI stored in gonadotrophs, but replenishment through new synthesis of UI did not occur because of the inhibition of transcription by estradiol. Since the release of UI in response to is an integration of the amounts of UI derived from both stored and newly produced pools [35], the results described herein lead us to speculate that the direct effect of estradiol at the anterior pituitary is related to the newly produced component of the UI response rather than to the stored LH. In agreement with results from previous studies [,2, 27,33], treatment of OVX ewes with steroids or with steroids plus did not influence steady-state levels of mrna for GH. Therefore, it would appear that these treatments do not result in a nonspecific inhibition of hypothalamic and/or pituitary function, but rather specifically influences those cells regulating synthesis and secretion of gonadotropins. The fact that the number of receptors in steroidtreated ewes receiving pulses of was not different from steroid-treated OVX ewes precludes the interpretation that the treatment was down-regulating these receptors, which would cause the anterior pituitary to become refractory to the provided in the treatment. It is also possible that administration of desensitized the gonadotrophs at a post-receptor site [36]. Although this possibility was not examined in the present experiment, we do not believe that the dosage of (25 ng once every 4 h) used resulted in desentization, since the magnitude of UI pulses did not decrease after 4 wk treatment with 5 ng administered hourly to OVX ewes that had been subjected to hypothalamic-pituitary disconnection [37]. Not surprisingly, some of the steroid-treated ewes did not respond to injection of with increased secretion of LH. First, one effect of estradiol is to reduce the amplitude of UI pulses [2, 4]. Second, there is a high correlation between pituitary content of UI and -induced release of UI [3]. Therefore, the provided in the replacement regimen probably induced secretion of UI to the degree that was possible considering the high steroid levels and the low content of LH in these animals. Since transcription of the genes encoding the subunits of LH appeared to be inhibited, secretion of UI induced by the GnRI-I treatment most likely accounts for the lower UI content in the anterior pituitary of the steroid-treated ewes receiving pulses of. In summary, the decreased pituitary content of LH observed at the end of gestation and the reported changes in Downloaded from https://academic.oup.com/biolreprod/article-abstract/43/4/554/2762767 by guest on 8 December 28

558 Nm ET AL morphological characteristics of gonadotrophs [38] represent integrated negative feedback effects of estradiol both at the hypothalamus [3] and at the pituitary level [28]. For this reason, it does not appear that administration of will stimulate replenishment of pituitary LH during late gestation or at other times when circulating concentrations of estradiol are elevated. REFERENCES. Nets TM. Function of the hypothalamic-hyophyseal axis during the post partum period In ewes and cows. J Reprod Fert Suppl 987; 34:2-2)3. 2. Goodman RI, Karsh FJ. Pulsatile secretion of luteinizing hormone: differential suppression by ovarian steroids. Endocrinology 98; 7:286-29. 3. Kai-sch FJ, CumminsJT, Thomas GB, Clarke U. Steroid feedback inhibition of the pulsatile secretion of gonadotropin-releasing hormone in the ewe. Biol Reprod 987; 36:27-28. 4. Tamanini C, Crowder ME, Nett TM. Effect of oestradiol and progesterone on pulsatile secretion of luteinizing hormone in ovariectomized ewes. 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