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Equine pituitary extract (EPE) has been reported to induce heightened follicular development in mares, but the response is inconsistent and lower than results obtained in ruminants undergoing standard superovulatory protocols. Three separate experiments were conducted to improve the ovarian response to EPE by evaluating: (1) effect of increasing the frequency or dose of EPE treatment; (2) use of a potent gonadotropin-releasing hormone agonist (GnRH-a) prior to EPE stimulation; (3) administration of EPE twice daily in successively decreasing doses. In the first experiment, 50 mares were randomly assigned to one of four treatment groups. Mares received (1) 25 mg EPE once daily; (2) 50 mg EPE once daily; (3) 12.5 mg EPE twice daily; or (4) 25 mg EPE twice daily. All mares began EPE treatment 5 days after detection of ovulation and received a single dose of cloprostenol sodium 7 days postovulation. EPE was discontinued once half of a cohort of follicles reached a diameter of >35 mm and hCG was administered. Mares receiving 50 mg of EPE once daily developed a greater number (P = 0.008) of preovulatory follicles than the remaining groups of EPE-treated mares, and more (P = 0.06) ovulations were detected for mares receiving 25 mg EPE twice daily compared to those receiving either 25 mg EPE once daily and 12.5 mg EPE twice daily. Embryo recovery per mare was greater (P = 0.05) in the mares that received 12.5 mg EPE twice daily than those that received 25 mg EPE once daily. In Experiment 2, 20 randomly selected mares received either 25 mg EPE twice daily beginning 5 days after a spontaneous ovulation, or two doses of a GnRH-a agonist upon detection of a follicle >35 mm and 25 mg EPE twice daily beginning 5 days after ovulation. Twenty-four hours after administration of hCG, oocytes were recovered by transvaginal aspiration from all follicles >35 mm. No differences were observed between groups in the numbers of preovulatory follicles generated (P = 0.54) and oocytes recovered (P = 0.40) per mare. In Experiment 3, 18 mares were randomly assigned to one of two treatment groups. Then, 6-11 days after ovulation, mares were administered a dose of PGF2, and concomitantly began twice-daily treatments with EPE given in successively declining doses, or a dose of PGF2alpha, but no EPE treatment. Mares administered EPE developed a higher (P = 0.0004) number of follicles > or = 35 mm, experienced more (P = 0.02) ovulations, and yielded a greater (P = 0.0006) number of embryos than untreated mares. In summary, doubling the dose of EPE generated a greater ovarian response, while increasing the frequency of treatment, but not necessarily the dose, improved embryo collection. Additionally, pretreatment with a GnRH-a prior to ovarian stimulation did not enhance the response to EPE or oocyte recovery rates.

Steroid hormones have a profound influence on the secretion of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). These effects can occur as a result of steroid hormones modifying the secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, or a direct effect of steroid hormones on gonadotropin secreting cells in the anterior pituitary gland. With respect to the latter, we have shown that estradiol increases pituitary sensitivity to GnRH by stimulating an increase in expression of the gene encoding the GnRH receptor. Since an estrogen response element (ERE) has not been identified in the GnRH receptor gene, this effect appears to be mediated by estradiol stimulating production of a yet to be identified factor that in turn enhances expression of the GnRH receptor gene. However, the importance of estradiol for enhancing pituitary sensitivity to GnRH during the periovulatory period is questioned because an increase in mRNA for the GnRH receptor precedes the pre-ovulatory rise in circulating concentrations of estradiol. In fact, it appears that the enhanced pituitary sensitivity during the periovulatory period may occur as a result of a decrease in concentrations of progesterone rather than due to an increase in concentrations of estradiol. Estradiol also is capable of altering secretion of FSH and LH in the absence of GnRH. In a recent study utilizing cultured pituitary cells from anestrous ewes, we demonstrated that estradiol induced a dose-dependent increase in secretion of LH, but resulted in a dose-dependent decrease in the secretion of FSH. We hypothesized that the discordant effects on secretion of LH and FSH might arise from estradiol altering the production of some of the intrapituitary factors involved in synthesis and secretion of FSH. To examine this hypothesis, we measured amounts of mRNA for activin B (a factor known to stimulate synthesis of FSH) and follistatin (an activin-binding protein). We found no change in the mRNA for follistatin after treatment of pituitary cells with estradiol, but noted a decrease in the amount of mRNA for activin B. Thus, the inhibitory effect of estradiol on secretion of FSH appears to be mediated by its ability to suppress the expression of the gene encoding activin.

Fertility control offers a potential alternative to traditional methods for regulating the growth of overabundant wild ungulate populations. However, current technology is limited due to practical treatment application, undesirable side-effects and economic considerations. A promising non-steroidal, non-immunological approach to contraception involves the use of a potent GnRH agonist. Two experiments were conducted to evaluate the effectiveness of a GnRH agonist (leuprolide) for controlling fertility in captive female wapiti and to assess physiological and behavioural side-effects of the treatment. In Expt 1, the optimum dose of agonist treatment was determined by measuring serum LH response of eight female wapiti to four formulations of leuprolide (0, 45, 90 and 180 mg) administered as a subcutaneous (s.c.) bioimplant. In Expt 2, the effects of leuprolide on wapiti pregnancy rates, duration of suppression of serum LH and progesterone secretion, and short-term behavioural and physiological side-effects were evaluated. All concentrations of leuprolide in Expt 1 were equally effective in reducing serum LH to non-detectable values throughout the 130 day trial. In Expt 2, leuprolide administered before the breeding season was 100% effective at preventing pregnancy in treated females. Serum LH and progesterone were reduced to baseline values by day 92 and remained at this concentration for 195-251 days after treatment, and returned to pretreatment concentrations in the following breeding season. Reproductive behaviour rates were similar for treated and untreated wapiti for all behaviour categories for both the breeding and post-breeding seasons. Haematology and blood chemistry parameters of treated and un-treated females were similar, and seasonal intake and body weight dynamics appeared normal. In conclusion, leuprolide is a safe, effective contraceptive agent and can potentially suppress fertility in female wapiti for one breeding season.

Rates of internalization of the murine GnRH receptor fused via its C-terminus to green fluorescent protein (GnRH-R-GFP) were examined in Chinese hamster ovary cells (CHO cells) and compared to those of native murine GnRH-R in a clonal murine gonadotroph cell line (LbetaT2 cells). The resulting rates of internalization of murine receptors were then compared with those of sheep GnRH-R in ovine gonadotrophs. Cells were incubated with radioiodinated [D-Ala6]GnRH on ice for 4 h to allow binding of the ligand to GnRH-R, then cells were warmed to 37 degrees C to permit internalization. Surface-bound radioligand began to decrease as soon as the cells were warmed and had decreased significantly within 20 min. A steady-state level of surface-bound radioligand was achieved after 60 min in both CHO cells and LbetaT2 cells (38% and 41%, respectively, of initial value; P < 0.05). Internalization of radioligand began immediately after warming the cells to 37 degrees C, and a significant proportion of surface ligand had been internalized by 20 min. A steady-state maximum of internalization was reached after 60 min in both CHO cells and LbetaT2 cells (29% and 28%, respectively, of total cell-associated ligand; P < 0.05). Changes in surface-bound radioligand and internalized radioligand in sheep pituitary cells were similar to those in CHO cells and LbetaT2 cells, but the amount of radioligand internalized after 60 min (40% of total cell-associated ligand) was 1.4 times higher than in CHO cells and LbetaT2 cells (P < 0.05). In a separate experiment, the effect of estradiol on the rate of internalization of GnRH-R in ovine pituitary cells was examined. Although treatment of ovine pituitary cells with estradiol approximately doubled the number of GnRH receptors, it did not alter either the rate or extent of receptor internalization. These results show that rates of internalization of recombinant murine GnRH-R-GFP in CHO cells and native murine and ovine GnRH-R in LbetaT2 cells and in sheep pituitary cells, respectively, are similar, but amounts of ovine GnRH-R internalized are greater than those for murine GnRH-R. Further, the rate of internalization of occupied receptor is similar in gonadotroph and nongonadotroph cells, and the addition of GFP to the C-terminus of the murine GnRH-R does not alter the rate of internalization.

Objective: To evaluate gonadotropin secretion and ovarian function after administration of deslorelin acetate to induce ovulation in mares., Design: Randomized controlled trial., Animals: 16 healthy mares with normal estrous cycles., Procedure: 8 control mares were allowed to ovulate spontaneously, whereas 8 study mares received deslorelin to induce ovulation when an ovarian follicle > 35 mm in diameter was detected. Follicle development and serum concentrations of gonadotropins were monitored daily during 1 estrous cycle. Pituitary responsiveness to administration of gonadotropin-releasing hormone (GnRH) was evaluated 10 days after initial ovulation., Results: Interovulatory intervals of mares treated with deslorelin (mean +/- SD, 25.6 +/- 2.6 days) were longer than those of control mares (22.9 +/- 1.8 days). Diameter of the largest follicle was significantly smaller during 2 days of the diestrous period after ovulation in deslorelin-treated mares than in control mares. Concentrations of follicle-stimulating hormone (FSH) were lower in deslorelin-treated mares on days 5 through 14 than in control mares. Concentrations of luteinizing hormone were not different between groups during most of the cycle. Gonadotropin release in response to administration of GnRH was lower in mares treated with deslorelin acetate than in control mares., Conclusions and Clinical Relevance: Administration of deslorelin was associated with reduction in circulating concentrations of FSH and gonadotropin response to administration of GnRH during the estrous cycle. Low concentration of FSH in treated mares may lead to delayed follicular development and an increased interovulatory interval.

In several physiological paradigms, secretion of FSH and LH are not coordinately regulated. Because these hormones appear to be produced by a single cell type in the anterior pituitary gland, their discordant regulation must be related to differential intracellular responses to various stimuli. Estradiol-17beta (estradiol) has been shown to influence secretion of both FSH and LH and some of its effects are mediated directly on the gonadotrope. Changes in expression of intrapituitary factors such as activin and follistatin may mediate effects of estradiol and account for discordant patterns of FSH and LH. The aims of this study were 1) to determine if estradiol alters expression of genes encoding activin, follistatin, or both in ovine pituitary cells; and 2) to observe the effects of immunoneutralizing activin B in vitro on gonadotropin secretion. Pituitary cells from five ewes in the anestrous season were cultured for 24 h with estradiol (0.01 or 1.0 nM). Estradiol reduced basal secretion of FSH in a dose-dependent manner (P: < 0.001) and simultaneously increased basal secretion of LH (P: < 0.001). Decreased secretion of FSH in estradiol-treated cultures was accompanied by suppressed levels of FSHbeta subunit mRNA (P: < 0.001). Amounts of mRNA for activin beta(B) were reduced in a dose-dependent manner by estradiol (27% +/- 4.9% at 0.01 nM, P: < 0.02; and 46% +/- 3.9% at 1.0 nM, P: < 0.002). In contrast, mRNA for follistatin was not affected by treatment with estradiol. Treatment of pituitary cells with an antibody to activin B reduced secretion of FSH by 50% (P: < 0.01) without influencing secretion of LH. These data lead us to conclude that discordant secretion of gonadotropins can be induced by estradiol acting directly at the pituitary level. The inhibitory effect of estradiol on FSH secretion may be mediated indirectly through decreased pituitary expression of the activin gene.

Although the ability of estradiol to enhance pituitary sensitivity to GnRH is established, the underlying mechanism(s) remain undefined. Herein, we find that approximately 9,100 bp of 5' flanking region from the ovine GnRH receptor (oGnRHR) gene is devoid of transcriptional activity in gonadotrope-derived cell lines and is not responsive to either estradiol or GnRH. In stark contrast, this same 9,100 bp promoter fragment directed tissue-specific expression of luciferase in multiple lines of transgenic mice. To test for hormonal regulation of the 9,100-bp promoter, ovariectomized transgenic females were treated with a GnRH antiserum alone or in combination with estradiol. Treatment with antiserum alone reduced pituitary expression of luciferase by 80%. Pituitary expression of luciferase in animals receiving both antiserum and estradiol was approximately 50-fold higher than animals receiving antiserum alone. The estradiol response of the -9,100-bp promoter was equally demonstrable in males. In addition, a GnRH analog (D-Ala-6-GnRH) that does not cross-react with the GnRH antiserum restored pituitary expression of luciferase in males passively immunized against GnRH to levels not different from castrate controls. Finally, treatment with both estradiol and D-Ala-6-GnRH increased pituitary expression of luciferase to a level greater than the sum of the individual treatments suggesting synergistic activation of the transgene by these two hormones. Thus, despite the complete absence of transcriptional activity and hormonal responsiveness in vitro, 9,100 bp of proximal promoter from the oGnRHR gene is capable of directing tissue-specific expression and is robustly responsive to both GnRH and estradiol in transgenic mice. To begin to refine the functional boundaries of the critical cis-acting elements, we next constructed transgenic mice harboring a transgene consisting of 2,700 bp of 5' flanking region from the oGnRHR gene fused to luciferase. As with the -9,100 bp promoter, expression of luciferase in the -2,700 lines was primarily confined to the pituitary gland, brain and testes. Furthermore, the passive immunization-hormonal replacement paradigms described above revealed both GnRH and estradiol responsiveness of the -2,700-bp promoter. Thus, 2,700 bp of proximal promoter from the oGnRHR gene is sufficient for tissue-specific expression as well as GnRH and estradiol responsiveness. Given the inability to recapitulate estradiol regulation of GnRHR gene expression in vitro, transgenic mice may represent one of the few viable avenues for ultimately defining the molecular mechanisms underlying estradiol regulation of GnRHR gene expression.

Steroid hormones can act either at the level of the hypothalamus or the pituitary to regulate gonadotropin subunit gene expression. However, their exact site of action remains controversial. Using the bovine gonadotropin alpha-subunit promoter linked to an expression cassette encoding the beta-subunit of LH, we have developed a transgenic mouse model where hypersecretion of LH occurs despite the presence of elevated ovarian steroids. We used this model to determine how hypersecretion of LH could occur when steroid levels are pathological. During transition from the neonatal period to adulthood, the endogenous LHbeta subunit gene becomes completely silent in these mice, whereas the alpha-directed transgene and endogenous alpha-subunit gene remain active. Interestingly, gonadectomy stimulates expression of the endogenous alpha and LHbeta subunit genes as well as the transgene; however, only the endogenous LHbeta gene retains responsiveness to 17beta-estradiol and GnRH. In contrast, LH levels remain responsive to negative regulation by androgen. Thus, alpha-subunit gene expression, as reflected by both the transgene and the endogenous gene, has become independent of GnRH regulation and, as a result, unresponsive to estradiol-negative feedback. This process is accompanied by a decrease in estrogen receptor alpha gene expression as well as an increase in the expression of transcription factors known to regulate the alpha-subunit promoter, such as cJun and P-LIM. These studies provide in vivo evidence that estrogen-negative feedback on alpha and LHbeta subunit gene expression requires GnRH input, reflecting an indirect mechanism of action of the steroid. In contrast, androgen suppresses alpha-subunit expression in both transgenic and nontransgenic mice. This suggests that androgens must regulate alpha-subunit promoter activity independently of GnRH. In addition to allowing the assessment of site of action of sex steroids on alpha-subunit gene expression, these studies also indicate that chronic exposure of the pituitary to LH-dependent ovarian hyperstimulation leads to a heretofore-undescribed pathological condition, whereby normal regulation of alpha, but not LHbeta, subunit gene expression becomes compromised.

The GnRH receptor plays a pivotal role in reproduction. This review summarizes current knowledge of the regulation of GnRH receptor gene expression by endocrine factors in sheep and cattle. Expression of the GnRH receptor gene, measured by steady-state amounts of GnRH receptor messenger RNA (mRNA), is maximal during the preovulatory period. The molecular events leading to maximal GnRH receptor gene expression are probably triggered by decreased circulating concentrations of progesterone at luteolysis. Because GnRH is a positive homologous regulator of its own receptor, increased pulsatile GnRH after removal of negative feedback effects of progesterone stimulates expression of the GnRH receptor gene early in the preovulatory period. Oestradiol is also a positive regulator of GnRH receptor gene expression, and increased serum concentrations of oestradiol from developing follicles probably maintain high abundance of GnRH receptor mRNA later in the preovulatory period. Since increased amount of GnRH receptor mRNA precedes maximal numbers of GnRH receptors before the LH surge, increased expression of the GnRH receptor gene is an important mechanism by which maximal sensitivity of gonadotrophs to GnRH is achieved. Future efforts should be directed towards elucidating the molecular mechanisms underlying transcriptional regulation of the GnRH receptor gene in ruminants by endocrine factors.

GnRH and estradiol are important regulators of GnRH receptors. When delivered to the anterior pituitary gland continuously, GnRH decreases numbers of GnRH receptors on gonadotropes. Treatment with estradiol consistently increases numbers of GnRH receptors. Because estradiol acts via intracellular receptors while GnRH exerts its effects through a membrane receptor, it is likely that these hormones influence GnRH receptor expression via different mechanisms. In this experiment, we tested two hypotheses: 1) continuous infusion of GnRH will decrease expression of the GnRH receptor gene; and 2) estradiol will override the negative effects of continuous infusion of GnRH on GnRH receptor expression. Ovariectomized ewes were administered either GnRH (10 microg/h, n = 10) or saline (n = 10) continuously for 136 h. At 124 h, 5 ewes in each group were administered estradiol (25 microg i.m.) and anterior pituitary glands were collected 12 h later. Treatment with GnRH caused an abrupt increase in circulating concentrations of LH, and the maximal mean concentration was observed 4 h after the start of GnRH infusion. Following this increase, concentrations of LH in GnRH-treated ewes declined and were similar to those in saline-treated ewes from 8 h to 124 h. After injection of estradiol at 124 h, circulating concentrations of LH increased in both GnRH- and saline-treated ewes. However, this response occurred within 6 h in ewes treated with GnRH compared with 9 h in ewes treated with saline (P < 0.05). Compared with saline-treated controls, treatment with GnRH decreased mean steady-state amount of GnRH receptor messenger RNA (mRNA) (P < 0.01) and concentration of GnRH receptors (P < 0.05). Treatment with estradiol caused an increase in concentrations of GnRH receptor mRNA (P < 0.05) and GnRH receptors (P < 0.01). Amounts of GnRH receptor mRNA and numbers of GnRH receptors in ewes treated with both GnRH and estradiol were not different from those in the control group but were higher (P < 0.002) relative to ewes treated with GnRH alone. Treatment with GnRH and estradiol also influenced the expression of genes encoding the LHbeta and FSHbeta subunits. Compared with saline-treated controls, treatment with GnRH reduced steady-state amounts of mRNA encoding LHbeta subunit (P < 0.005) and FSHbeta subunit (P < 0.05). Treatment with estradiol caused a decrease in concentrations of FSHbeta subunit mRNA (P < 0.01) but did not affect amounts of LHbeta subunit mRNA. The combined treatment of GnRH and estradiol reduced concentrations of mRNA encoding LHbeta subunit (P < 0.01) and FSHbeta subunit (P < 0.005). From these data we conclude that 1) reduced numbers of GnRH receptors during continuous infusion of GnRH are mediated in part by decreased expression of the GnRH receptor gene; and 2) estradiol is able to override the negative effect of GnRH by stimulating an increase in GnRH receptor gene expression and GnRH receptor concentrations. Therefore, although the gonadotrope becomes refractory to GnRH during homologous desensitization, this desensitization does not affect the cell's ability to respond to estradiol.

Prostaglandin F2 alpha (PGF2 alpha) decreases secretion of progesterone from the corpus luteum in domestic ruminants. However, it is less effective during the early part of the oestrous cycle (Louis et al., 1973) and at the time of maternal recognition of pregnancy (Silvia and Niswender, 1984; Lacroix and Kann, 1986). Decreased luteal responsiveness may be due to failure of PGF2 alpha to activate fully its normal second messenger system, protein kinase C (PKC). Alternatively, increased resistance of the corpus luteum to PGF2 alpha might be attributable to greater concentrations of recently identified biological inhibitors of PKC. These possibilities were addressed by measuring steady-state concentrations of mRNA encoding PGF2 alpha receptor and two inhibitors of PKC, protein kinase C inhibitor-1 (PKCI-1) and kinase C inhibitor protein-1 (KCIP-1, brain 14-3-3 protein), in corpora lutea collected from ewes on days 4, 10 and 15 of the oestrous cycle (n = 5 per day) and day 15 of pregnancy (n = 7). There were no differences in mean concentrations of mRNA encoding PGF2 alpha receptor among the groups. However, concentrations of mRNA encoding both inhibitors of PKC were higher (P < 0.01) on day 4 of the oestrous cycle compared with the other groups. Treatment of ewes with a luteolytic dose of PGF2 alpha, which activates PKC, did not change concentrations of mRNA encoding either PKCI-1 or KCIP-I up to 24 h later. Luteal expression of mRNA encoding the PKC inhibitors and PGF2 alpha receptor was also examined in ewes treated with oestradiol in vivo for 16 h in the midluteal phase. High concentrations of oestradiol in serum (20 and 70 pg ml-1) did not influence quantities of any of the mRNAs examined. Therefore, an increase in PKC inhibitors may be involved in resistance of the corpus luteum to PGF2 alpha during the early part of the oestrous cycle but does not appear to mediate the increased resistance of the corpus luteum to PGF2 alpha during maternal recognition of pregnancy. Neither PGF2 alpha nor oestradiol affected steady-state concentrations of mRNAs encoding PKCI-1 or KCIP-I.

The objective of this study was to determine whether progesterone prevents the stimulatory effects of oestradiol on GnRH receptor gene expression. In Expt 1, ewes were treated during the luteal phase (days 10-12 of the oestrous cycle) with either one or five subcutaneous implants containing oestradiol (n = 6 per group). Control ewes received no treatment (n = 6). Anterior pituitary glands were collected 16 h after treatment with oestradiol. Steady-state amounts of GnRH receptor mRNA were similar among all three treatment groups despite increased circulating concentrations of oestradiol in implanted ewes at the time of pituitary collection (4.3 +/- 0.6 and 24.7 +/- 2.6 pg ml-1 in ewes treated with one or five implants, respectively, compared with 0.5 pg ml-1 in controls). Experiment 2 was designed to determine whether progesterone was the ovarian factor preventing the stimulatory effects of oestradiol on expression of the GnRH receptor gene in Expt 1. Twenty-five ewes were ovariectomized on day 6 or day 7 of the oestrous cycle and assigned to one of five treatment groups (n = 5 per group). Control ewes received no further treatment. Endogenous luteal phase concentrations of progesterone were replaced in three groups of ewes at the time of ovariectomy via intravaginal implants. Three days after ovariectomy, one group of progesterone-treated ewes received one oestradiol implant, while another group of progesterone-treated ewes received five oestradiol implants. An additional group was treated with five oestradiol implants only, and anterior pituitary glands were collected from all ewes 16 h later. Compared with untreated ovariectomized ewes, treatment with progesterone alone did not affect amounts of GnRH receptor mRNA. In ewes treated with progesterone and either one or five oestradiol implants, steady-state amounts of GnRH receptor mRNA were increased twofold (P < 0.01). Treatment with oestradiol in the absence of progesterone increased amounts of GnRH receptor mRNA threefold (P < 0.001). These results provide evidence that the stimulatory effects of oestradiol on the expression of the GnRH receptor gene are prevented during the natural luteal phase in ewes. However, progesterone does not appear to act independently to mediate this effect.

We compared pregnancy rates of beef cows subjected to the traditional Syncro-Mate-B protocol or the new Ovsynch protocol and timed insemination. Multiparous Angus cows (n = 436) were stratified by age, postpartum interval, and AI sire and were randomly divided into two treatment groups for synchronization of estrus/ovulation. Approximately half of the cows (n = 216) received the traditional Syncro-Mate-B protocol with 48-h calf removal from the time of implant removal until breeding. The remaining cows (n = 220) received the Ovsynch protocol, which consists of an injection of GnRH (100 microg) on d -10, an injection of PGF2alpha (25 mg) and 48-h calf removal on d -3, another injection of GnRH and calf return on d -1, and timed insemination 24 h later (d 0). Blood samples were collected from all cows before treatment to identify anestrous and cyclic females. Pregnancy rates were higher (P < .025) for Ovsynch-treated cows (54%) than for Syncro-Mate-B-treated cows (42%). Pregnancy rates of cyclic Ovsynch-treated cows (59%) were higher (P < .005) than pregnancy rates of cyclic Syncro-Mate-B-treated cows (38%). Pregnancy rates of anestrous cows also tended to favor synchronization with the Ovsynch protocol. From these data, we conclude that the Ovsynch protocol is capable of inducing a fertile ovulation in cyclic and anestrous beef cows and that pregnancy rates to a timed insemination are higher than those obtained with synchronization of estrus using Syncro-Mate-B.

The objective of this study was to determine if prolonged pulsatile administration of homologous gonadotropin-releasing hormone (GnRH) at therapeutic or 5x therapeutic doses would cause down-regulation of the stallion's hypothalamic-pituitary-testicular axis. Fifteen stallions were randomly assigned to three treatment groups (n=5/group) and received a 0.5 ml subcutaneous dose of saline (group 1), 50 microg GnRH (group 2), or 250 microg GnRH (group 3) every 2 hours for 75 days. Weekly evaluations of follicle stimulating hormone, luteinizing hormone, and testosterone and monthly evaluations of daily sperm output and spermatozoal motility failed to demonstrate any decreased pituitary or gonadal responsiveness within or among treatment groups (P > 0.1) as a result of treatment with GnRH. Results of this study demonstrate that the hypothalamic-pituitary-testicularaxis of the stallion, unlike that of other domestic species, is remarkably refractory to GnRH-induced down-regulation.

The presence of organochlorine pesticides, such as p,p'-DDT[2,2-bis(p-chlorophenyl)-1,1,1-trichloroethanel, and of polychlorinated biphenyls (PCBs) in human serum and adipose tissue has been reported in many studies over the last four decades. Recently, debate has heightened concerning the link of these compounds to breast cancer. To clarify and resolve this issue, accurate analytical residue data must be obtained. Separation of the organochlorine pesticides from the PCBs in breast tissue is critical to obtaining valid residue data. Based on methods refined in the Analytical Laboratory at Colorado State University, accurate residue levels were established for nine individual PCB congeners and eight organochlorine pesticides. The breast adipose tissue method used was a modification of the Mills et al. and de Faubert Maunder et al. methods. The serum method employed was a modification of the Burse et al. method. Both breast adipose tissue and serum from 36 women were analyzed, and correlations of the residues from the two substrates were evaluated. Serum concentrations of p,p'-DDE, the primary metabolite of p,p'-DDT, were correlated (alpha = .05) with the concentrations of p,p'-DDE in human breast adipose tissue (r = .808). Serum concentrations of the PCB congener BZ 153 were also significantly correlated to the human breast adipose tissue concentrations of BZ 153 (r = .377). No significant relationship was found between serum concentrations and tissue residues for 15 of the 17 compounds analyzed. This lack of correlation between breast adipose tissue and serum, as well as an absence of the compound residues in serum, emphasized that adipose tissue should be analyzed in addition to serum to fully understand the relationship of the organochlorine compounds to breast cancer.

In sheep, growth and development of ovarian follicles beyond 2 mm in diameter is acutely dependent on gonadotropin support. As a consequence, following hypophysectomy (HPX) or hypothalamic-pituitary stalk disconnection (HPD), growth of follicles beyond 2 mm is arrested and all follicles > 2 mm undergo atresia. Although administration of exogenous gonadotropins stimulates follicular growth and ovulation in HPD ewes, follicles in HPX ewes remain unresponsive unless growth hormone (GH) is also given. To determine whether the difference in follicular sensitivity to gonadotropins after HPD (gonadotropin sensitive) or HPX (gonadotropin insensitive) is related to the distribution and quantity of binding sites for FSH, LH, and/or insulin-like growth factor I (IGF-I), binding sites for these hormones were localized and quantified using topical autoradiography in healthy follicles from control (pituitary-intact), HPD, and HPX ewes. In addition, in situ hybridization was performed to localize mRNA for GH and FSH receptors. Irrespective of treatment, binding of FSH and mRNA for FSH receptor were greatest (p < 0.05) in the membrana granulosa; LH binding was greatest (p < 0.05) in the theca interna; and IGF-I binding was greatest (p < 0.05) in the theca externa. Although the relative number of binding sites for LH did not differ among treatments, those for FSH and IGF-I were lower (p < 0.05) in HPD and HPX ewes compared to controls. Attempts to quantify binding sites for GH were unsuccessful due to high nonspecific binding. However, mRNA for GH receptor was most abundant (p < 0.05) in the membrana granulosa and oocytes of small antral and preantral follicles. Compared to levels in controls and HPD ewes, the level of GH receptor mRNA was lower (p < 0.05) in follicles obtained from HPX ewes. On the basis of these data, failure of small antral follicles in HPX ewes to respond to exogenous gonadotropins is not due to a reduction in receptors for FSH, LH, or IGF-I. The observed reduction of mRNA for GH receptor in the membrana granulosa of follicles from HPX ewes provides evidence that GH may play an important role in early stages of folliculogenesis and that it is involved in the maintenance of sensitivity to gonadotropins.

Twenty stallions (3 to 18 yr old) were used in a study between June 1993 and March 1994. The stallions were divided into 5 groups of 4 each, and, within groups, were randomly assigned to 1 of 4 treatments: 1) untreated controls; 2) once-a-day oral altrenogest (0.088 mg/kg BW) treatment for 150 d; 3) daily altrenogest treatment at the same dose for 240 d; and 4) daily oral altrenogest treatment for 240 d plus subcutaneous GnRH (80 microg) every 4 h from Days 151 to 240. Total scrotal width (TSW) was recorded and semen was collected and evaluated for gel free volume, concentration, sperm motility and sperm morphology. Sexual behavior (libido) was measured as times to first erection and ejaculation. Serum LH and testosterone (T) were measured at various periods throughout the study. Altrenogest decreased serum concentrations of LH and T, TSW, daily spermatozoa output (DSO), the percentage of normal spermatozoa and libido. There was a significant decrease in sperm motility in the Alt-240 and Alt-240+GnRH group, but not the ALT-150 group. The suppression appeared to be partially reversible because DSO, TSW and serum concentrations of LH increased after cessation of progestin treatment. Administration of GnRH during altrenogest treatment resulted in increased (P < 0.05) TSW, DSO and serum concentrations of LH but did not alter sperm morphology or behavior. In summary, the suppressive effects of altrenogest were apparently mediated primarily through a negative feedback inhibition of LH secretion.

The regulation of mRNAs encoding insulin-like growth factor I (IGF-I) and the receptor for growth hormone (GH-R) in ovine luteal tissue by luteotrophic and luteolytic hormones was examined. In Expt 1, ewes were hypophysectomized (HPX) on day 5 of the oestrous cycle and administered saline (S), LH, GH, or LH + GH until day 12 of the oestrous cycle (n = 4 ewes per group). Concentrations of luteal mRNA encoding IGF-I in HPX + S ewes and pituitary-intact ewes at day 5 (n = 4) were approximately 60% (P < 0.05) of those in pituitary-intact ewes at day 12 (n = 4). Treatment of HPX ewes with GH or GH + LH, but not LH alone, increased concentrations of mRNA encoding IGF-I to values similar to those in pituitary-intact ewes at day 12. Hypophysectomy also reduced the mean concentration of mRNA encoding GH-R to approximately 60% (P < 0.05) of the values in pituitary-intact ewes (days 5 or 12). Treatment with LH, but not GH, increased (P < 0.05) concentrations of mRNA encoding GH-R to values observed in pituitary-intact ewes. In Expt 2, prostaglandin F2 alpha (PGF2 alpha; 1 mumole) injected into the ovarian artery on day 11 or day 12 of the oestrous cycle had no effect on luteal concentrations of mRNA for either IGF-I or GH-R. In Expt 3, concentrations of mRNA encoding IGF-I increased (P < 0.05) between days 3 and 6 and remained high for the duration (days 9, 12 and 15) of the oestrous cycle while luteal concentrations of mRNA encoding GH-R did not change. In conclusion, responsiveness of the corpus luteum to GH and luteal synthesis of IGF-I are likely regulators of luteal development and function. However, PGF2 alpha-induced luteolysis was not associated with a decrease in concentrations of mRNAs encoding either IGF-I or GH-R.

In cultured ovine pituitary cells, inhibin increases concentrations of mRNA encoding GnRH receptor and numbers of GnRH receptors. The objective of this study was to test the hypothesis that inhibin increases concentrations of ovine GnRH receptor mRNA in vivo. Ovariectomized ewes were used to eliminate effects of endogenous ovarian hormones, and passive immunization against GnRH was employed to avoid possible confounding influences of GnRH on GnRH receptor gene expression. Two groups of ewes (n = 5/group) were treated with 50 ml GnRH antiserum on Days 0 and 3 of the experiment. One group of immunized ewes received 10 ml charcoal-extracted bovine follicular fluid (bFF) as a source of inhibin every 8 h for 48 h on Days 4-6 of the experiment. A third group of ewes was not passively immunized and was treated only with bFF, and control ewes received no treatments. Anterior pituitary glands were collected from all ewes on Day 6. Passive immunization against GnRH, alone or in combination with treatment with bFF, decreased mean concentrations of LH (p < 0.01) and LH pulse amplitude (p < 0.001). In ewes treated only with GnRH antiserum, number of LH pulses was also reduced (p < 0.03). Circulating concentrations of FSH tended to be lower (p = 0.06) in passively immunized ewes compared to controls. Treatment with bFF, alone or in combination with GnRH antiserum, reduced circulating concentrations of FSH (p < 0.02) and amounts of FSHbeta subunit mRNA (p < 0.001) to less than 30% and 10% of control values, respectively. Despite effects of bFF on concentrations of FSHbeta mRNA and secretion of FSH, concentrations of GnRH receptor mRNA were similar among controls, ewes treated with bFF alone, and passively immunized ewes treated with bFF. Passive immunization against GnRH did not affect concentrations of GnRH receptor mRNA but resulted in a reduction (p < 0.05) in amount of LHbeta mRNA. Treatment with bFF did not affect amounts of either alpha subunit or LHbeta subunit mRNA except when combined with treatment with antiserum, when amounts of both alpha and LHbeta subunit mRNA were reduced (p < 0.05). These results do not support the hypothesis that inhibin increases concentrations of GnRH receptor mRNA in the ewe, and they provide evidence that inhibin is not an acute regulator of ovine GnRH receptor gene expression in vivo.

Steroidogenic factor-1 (SF-1) is a transcription factor involved in regulation of steroidogenic enzymes. Recent evidence indicates that SF-1 is also important in the anterior pituitary gland, where it may influence gene expression in gonadotropes. We isolated a cDNA encoding ovine SF-1 and demonstrated that the SF-1 gene is expressed in the anterior pituitary gland of sheep. SF-1 transcripts and luteinizing hormone (LH) were colocalized in gonadotropes by in situ hybridization and immunohistochemistry, respectively. To test the hypothesis that GnRH stimulates pituitary expression of ovine SF-1 mRNA, ewes were ovariectomized to increase endogenous secretion of GnRH. Compared to ovary-intact ewes, ovariectomy resulted in three- and fourfold increases in steady-state amounts of mRNA encoding SF-1 and LH beta subunit, respectively. In ovariectomized ewes in which delivery of GnRH to the anterior pituitary gland was prevented by hypothalamic-pituitary disconnection (HPD), steady-state amounts of mRNA encoding SF-1 and LH beta-subunit were decreased. These results provide evidence that pituitary SF-1 gene expression in sheep is regulated by GnRH. Coordinate regulation of mRNAs encoding SF-1 and LH beta-subunit raises the possibility that SF-1 may be an important transcriptional regulator of LH beta-subunit gene expression in ovine gonadotropes.

Thirty-two nutritionally anestrous cows were used to determine the effect of the frequency of exogenous GnRH pulses on ovarian follicular growth, serum concentrations of LH and FSH, and concentrations of LH, FSH, GnRH receptors (GnRH-R), messenger RNA (mRNA) for GnRH-R, and mRNA for gonadotropin subunits in the pituitary. Cows were randomly assigned to one of four treatments: 2 micrograms GnRH infused (i.v.) continuously during 1 h, 2 micrograms GnRH infused during 5 min once every hour, 2 micrograms GnRH infused during 5 min once every fourth hour, or saline (control) for 13 days. Infusion of GnRH every hour increased LH concentrations in serum (P < 0.05), but FSH concentrations were not affected by GnRH infusion. Luteal activity (LA) was assessed by the presence of corpora lutea and/or serum progesterone greater than 1 ng/ml. Six of eight cows infused with GnRH every hour had LA by day 13, whereas only 25% of cows infused either continuously or with a pulse every fourth hour had LA by day 13. None of the control cows had LA during the experiment (P < 0.01). Concentrations of LH and FSH in the pituitary were significantly reduced when GnRH was infused hourly or continuously. Concentrations of common alpha and FSH beta mRNA were not influenced by treatment. However, continuous infusion of GnRH decreased (P < 0.05) LH beta mRNA subunit. Concentrations of GnRH-R (P < 0.1) and GnRH-R mRNA (P < 0.05) were reduced when GnRH was infused continuously. We concluded that pulsatile secretion of LH is necessary for follicular growth and LA in beef cattle, and GnRH treatment differentially regulates LH and FSH gene transcription and serum concentrations of LH and FSH in cattle.

This study examines the regulation of progesterone receptor (PR) in the inhibition of pituitary luteinizing hormone (LH) secretion. Ovariectomized ewes underwent hypothalamic-pituitary disconnection, were pulsed with gonadotropin-releasing hormone (GnRH) and received 1 of 4 treatments: estradiol alone (E), estradiol priming before progesterone (E+P), E removed and replaced with P (E-P), or no steroids (C). P treatment for 24 h, with E or following E-priming, reduced LH pulse amplitude by 55% (p<0.05). E alone did not affect LH release. E increased pituitary cytosolic P binding capacity fourfold over controls (p<0.01) and P further increased binding to eight times controls (p<0.01). Pituitary PR mRNA increased to 149 and 171% of C in E and E+P groups, respectively (p<0.05), but E removal resulted in PR mRNA levels not different from controls. Pituitary receptors for GnRH were tripled by E alone compared to C (p<0.01), whereas P alone or with E had no effect. These data suggest an E-induced, direct pituitary inhibition of LH secretion by P and that this effect of P is associated with E-enhanced binding of P in the pituitary. Additionally, the direct pituitary effects of P on LH secretion cannot be accounted for by influences on GnRH receptor numbers.

Steroidogenic acute regulatory protein (StAR), proposed to be involved in the transport of cholesterol to the inner mitochondrial membrane, has recently been cloned from MA-10 cells. Using reverse transcription-polymerase chain reaction, we generated a complementary DNA encoding 404 base pairs of StAR from ovine luteal tissue to perform studies regarding regulation of the messenger RNA (mRNA) encoding this protein. In Exp 1, ewes were hypophysectomized (HPX) on day 5 of the estrous cycle and administered saline or physiological regimens of LH and/or GH until collection of luteal tissue on day 12 of the estrous cycle (n = 4/group). Luteal concentrations [mean +/- SEM; femtomoles per microgram poly(A)+ RNA] of mRNA encoding StAR were lower (P < 0.05) in the HPX plus saline-treated ewes (26.4 +/- 7.3) than in day 12 pituitary-intact ewes (n = 4; 77.7 +/- 9.3). Replacement of LH (59.1 +/- 13.1), GH (59.1 +/- 12.8), or LH and GH (69.9 +/- 4.5) in HPX ewes increased (P < 0.05) concentrations of mRNA encoding StAR to values not different from those in day 12 controls. In Exp 2, ewes on day 11 or 12 of the estrous cycle were injected with prostaglandin F2 alpha (PGF2 alpha) to induce luteal regression. Corpora lutea were collected 4, 12, or 24 h after injection (n = 4-5/time point) and from untreated control ewes (n = 4) or 24 h after injection of saline (n = 4). Treatment with PGF2 alpha decreased (P < 0.05) concentrations of progesterone in serum 4, 12, and 24 h after injection. Concentrations of StAR mRNA were decreased (P < 0.01) to 47%, 19%, and 8% of control values 4, 12, and 24 h after PGF2 alpha injection, respectively. In Exp 3, ewes received ovarian arterial infusions of saline, PGF2 alpha, or phorbol 12-myristate 13-acetate (PMA), and luteal tissue was collected 0 (no infusion), 4, 12, or 24 h later (n = 3-4/group). Treatment with PGF2 alpha or PMA decreased (P < 0.05) concentrations of progesterone in serum 4, 12, and 24 h postinjection. Steady state concentrations of mRNA encoding StAR (P < 0.05) were 36% and 25% of the control value 12 and 24 h after PGF2 alpha injection. Injection of PMA decreased (P < 0.05) concentrations of StAR mRNA to 75% and 50% of control values at 4 and 12 h, but concentrations of mRNA encoding StAR were not different from control values at 24 h.(ABSTRACT TRUNCATED AT 400 WORDS)

To test the hypothesis that low levels of estradiol are sufficient to increase concentrations of GnRH receptor mRNA in the absence of progesterone, ewes were ovariectomized and immediately treated with estradiol implants for 12 h to achieve circulating concentrations of estradiol typical of the early (n=5) or late (n=4) follicular phase. Five additional ewes underwent lutectomy, and control ewes were untreated. Treatment of ewes with 1/2 or 1 estradiol implant increased concentrations of estradiol in serum to 3.0 ± 0.8 pg/ml or 6.3 ± 0.3 pg/ml, respectively, and concentrations of estradiol in lutectomized ewes (2.4 ± 0.5 pg/ml) were intermediate. Ovariectomy did not alter concentrations of GnRH receptor mRNA or numbers of GnRH receptors. Treatment of ewes with 1 estradiol implant increased concentrations of GnRH receptor mRNA and numbers of GnRH receptors. In ewes treated with 1/2 estradiol implant, concentrations of GnRH receptor mRNA were intermediate between controls and ewes treated with 1 estradiol implant, and numbers of GnRH receptors were greater than controls. Lutectomy increased concentrations of GnRH receptor mRNA but did not affect numbers of GnRH receptors. We conclude that estradiol stimulates expression of the GnRH receptor gene and numbers of GnRH receptors in the absence of progesterone. However, effects of estradiol on expression of the GnRH receptor gene were clearly evident only when concentrations of estradiol were elevated to levels typical of the late follicular phase.

As circulating concentrations of progesterone decrease during the early preovulatory period, concentrations of mRNA encoding ovine GnRH receptor in the anterior pituitary gland increase. The purpose of this study was to determine whether removal of progesterone affects amounts of GnRH receptor mRNA directly or whether withdrawal of progesterone affects GnRH receptor gene expression indirectly by permitting secretion of GnRH to increase. Ovulation was induced in seasonally anestrous ewes, and luteolysis was initiated with prostaglandin F2 alpha (PGF2 alpha) 11 or 12 days later. Anterior pituitary glands were collected 0 h, 4 h, 12 h, or 24 h after treatment with PGF2 alpha, and 24 h after injection of saline (n = 3 or 4 animals/group). Two groups of ewes (n = 3) received infusions of GnRH (250 ng infused over 6 min) hourly for 12 h; luteolysis was induced in one of these groups at the time that treatment with GnRH was initiated, and anterior pituitary glands were collected at the end of the 12-h infusion period. Blood samples were collected at 15-min intervals for 12 h from all ewes treated with GnRH and from animals administered PGF2 alpha and killed 12 h later. No differences in concentrations of GnRH receptor mRNA, numbers of GnRH receptors, or circulating concentrations of progesterone or estradiol were detected between groups of animals at 0 h and 24 h after treatment with saline; therefore, data from these control groups were combined. Concentrations of progesterone in serum decreased in PGF2 alpha-treated ewes and were lower (p < 0.05) than those in controls 24 h after treatment with PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute actions of estradiol and progesterone on synthesis and secretion of gonadotropins without the confounding effects of endogenous steroids and GnRH were examined by using the ovariectomized (OVX), hypothalamic-pituitary-disconnected (HPD) ewe. Thirty-two OVX ewes were subjected to HPD and randomly assigned to eight groups (4 ewes/group). An additional four OVX ewes served as controls. Each of 12 OVX-HPD ewes received 4 s.c. implants of estradiol 24 h after HPD (implantation was designated as Time 0 h). Sixteen OVX-HPD ewes received no treatment and served as contemporary controls. The remaining four OVX-HPD ewes received implants of estradiol for 48 h and were administered 16 mg progesterone i.m. twice daily in the last 24 h of estradiol treatment. Blood samples and pituitary glands were collected from OVX-HPD control and steroid-treated ewes at 0, 12, 24, and 48 h. Amounts of gonadotropin subunit mRNAs and serum concentrations of gonadotropins were decreased at 24 h after HPD and remained unchanged thereafter in OVX-HPD controls. Although treatment with estradiol decreased pituitary content of LH and steady-state levels of mRNA for LH beta-subunit (p < 0.05) compared to contemporary controls, serum concentrations of LH were not affected. Serum concentrations and pituitary content of FSH decreased (p < 0.05) 24 h after initiating treatment with estradiol, but steady-state levels of FSH beta-subunit mRNA were unchanged. There was a transient decrease in pituitary content of FSH at 24 h of estradiol treatment. Steady-state levels of mRNA for alpha-subunit were unaffected by estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)

To study the regulation of ovine GnRH receptors in the absence of GnRH, hypothalamic input was removed by hypothalamic-pituitary disconnection (HPD). Steady-state concentrations of GnRH receptor mRNA and numbers of GnRH receptors were measured after HPD and subsequent treatment with estradiol. Anterior pituitary glands were collected 24 (n = four), 36 (n = two), 48 (n = four), and 72 h (n = four) after HPD. An additional group of ewes received subcutaneous implants of estradiol 24 h after HPD, and pituitary glands were collected 0 (n = four), 12 (n = four), 24 (n = three), and 48 h (n = four) after exposure to estradiol. Pituitary glands were also obtained from four ovariectomized ewes that did not undergo HPD (OVX controls). At 24 h after HPD, mean number of GnRH receptors had decreased (P < .05) by 73%; however, mean concentration of GnRH receptor mRNA was not different from OVX controls. Relative to HPD ewes, treatment with estradiol increased mean concentrations of GnRH receptor mRNA and mean numbers of GnRH receptors (P < .01 and P < .001, respectively). From these data we conclude that 1) acute removal of GnRH decreases the numbers of GnRH receptors but does not affect steady-state concentrations of GnRH receptor mRNA and 2) estradiol increases the numbers of GnRH receptors and steady-state concentrations of GnRH receptor mRNA via direct effects at the level of the pituitary gland.

To test the hypothesis that growth hormone (GH) as well as luteinizing hormone (LH) is required for normal luteal growth and function, 16 western range ewes were hypophysectomized (HPX) on day 5 of the estrous cycle. Ewes were randomly assigned to receive saline (S), LH, GH, or LH + GH (n=4 per group) from the time of HPX until collection of corpora lutea 7 days after HPX (day 12). Corpora lutea were also collected from pituitary-intact ewes on days 5 (day 5 control,n=4) and 12 (day 12 control,n=4) of the estrous cycle. To assess luteal function, concentrations of progesterone in sera, luteal weights and luteal concentrations of mRNA encoding cytochrome P450 side-chain cleavage enzyme (P450(scc)) and 3β-hydroxysteroid dehydrogenase/Δ5,Δ4 isomerase (3β-HSD) were determined. Concentrations of progesterone in sera and luteal weights increased between days 5 and 12 of the estrous cycle in control ewes, but not in HPX + S ewes. In HPX ewes treated with LH, concentrations of progesterone in sera and luteal mRNA for P450(scc) and 3β-HSD increased but luteal weights were unaffected. Treatment with GH increased luteal weight and luteal concentrations of mRNA encoding P450(scc) but did not increase concentrations of mRNA encoding 3β-HSD compared to HPX + S ewes. Concentrations of progesterone in sera of GH-treated, HPX ewes were similar to those of day 12 control ewes but not significantly different from those in HPX + S ewes. Treatment of HPX ewes with LH + GH increased all parameters of luteal function measured to values similar to those in day 12 controls. In conclusion, both GH and LH are necessary for normal luteal development in the ewe.

We conducted two studies to determine the pattern of GnRH analog-induced LH secretion in adult female wapiti during the breeding season, anestrus, and pregnancy. In the first study, we measured LH secretion during the breeding season and anestrus in five females challenged with each of five doses of GnRH analog (0.3, 1, 3, 10, and 30 micrograms/50 kg BW). In the second study, LH response was determined in six pregnant females treated with six doses of GnRH analog (0.5, 1, 2, 4, 8, and 16 micrograms/50 kg BW). Animals were fitted nonsurgically with indwelling jugular catheters, and blood samples were collected at 0, 30, 60, 90, 120, 180, 240, 300, 360, 420, and 480 min postinjection. Mean maximum serum concentrations of LH were not different (p = 0.45) between females treated during the breeding season (mean = 29.1 +/- 1.9 [SE] ng/ml) and anestrus (mean = 32.4 +/- 4.3 [SE] ng/ml); however, anestrous females required 3.3 times more GnRH analog (3 vs. 10 micrograms/50 kg BW) to produce the same response. Maximal inducible LH concentrations were lowest (mean = 7.7 +/- 2.5 [SE] ng/ml) for pregnant wapiti, and the magnitude of the response decreased exponentially (r2 = 0.98, p = 0.002, SEb = 0.65) during gestation. Although the magnitude of the response declined over time, maximal release consistently occurred at the same dose (2 micrograms/50 kg BW) (treatment x trial interaction, p = 0.53). We conclude from these studies that the amount of LH released in response to a given dose of GnRH analog depends on reproductive status.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypersecretion of luteinizing hormone (LH) is implicated in infertility and miscarriages in women. A lack of animal models has limited progress in determining the mechanisms of LH toxicity. We have recently generated transgenic mice expressing a chimeric LH beta subunit (LH beta) in gonadotropes. The LH beta chimera contains the C-terminal peptide of the human chorionic gonadotropin beta subunit. Addition of this peptide to bovine LH beta resulted in a hormone with a longer half-life. Furthermore, targeted expression of the LH beta chimera led to elevated LH levels and infertility in female transgenics. These mice ovulated infrequently, maintained a prolonged luteal phase, and developed pathologic ovarian changes such as cyst formation, marked enlargement of ovaries, and granulosa cell tumors. Testosterone and estradiol levels were increased compared to nontransgenic littermates. An unusual extragonadal phenotype was also observed: transgenic females developed hydronephropathy and pyelonephritis. The pathology observed demonstrates a direct association between abnormal secretion of LH and infertility and underscores the utility of the transgenic model for studying how excess LH leads to cyst formation, ovarian tumorigenesis, and infertility.

Four new azasteroid inhibitors of steroid 5 alpha-reductase were compared to the benchmark compound finasteride, each at a dose level of 1 mg/kg/day, as well to placebo and to castration, in seven groups of mature male beagle dogs with enlarged prostates. Prostate volumes were measured repetitively by a volume MRI method over 15 weeks of treatment. The study probed the obverse of the familiar relation between DHT and prostate growth, and provides the first documentation of a tight negative correlation between prostate regression and the prostatic concentration of DHT across a range of treatment regimens (r = -0.982). In this first direct comparison study of structure vs. in vivo activity for several azasteroids in the dog model of BPH, relative efficacy for induction of shrinkage of the dog prostate did not correlate at all with the inhibitor's relative activity against the dog 5 alpha-reductase in vitro. On the basis of the relative IC50 values it would not have been predicted that, at the dose tested, the analogue MK-434 (17 beta-benzoyl-4-aza-5 alpha-androst-1-en-3-one) was distinguished from the other inhibitors with respect to the induction of faster and more complete regression (69%) as well as greater reduction in prostatic DHT (95%), both of which approached the castrated dog levels of 75% prostatic shrinkage and > 98% reduction in DHT. Treatment with any one of the five azasteroids induced two- to five-fold increases in prostatic testosterone. However, total androgen was conserved at the placebo control level. Despite the differences noted, each azasteroid tested induced a highly significant decrease in prostatic volume that correlated tightly with a decreased prostatic DHT level in canine spontaneous BPH.

Transient transfection studies have proven useful in unraveling the molecular mechanisms underlying gonadotrope-specific expression and hormonal regulation of the gene encoding the alpha-subunit of the glycoprotein hormones. In contrast, similar studies performed with the LH beta gene have been less informative. When assayed by transient transfection into alpha T3-1 cells, activity of a 776-basepair bovine LH beta promoter-chloramphenicol acetyltransferase fusion gene (bLH beta CAT) was no greater than that of a promoterless control. To determine whether limited activity in vitro reflected the absence of critical regulatory elements, we examined activity of bovine LH beta fusion genes after stable integration in transgenic mice. In contrast to transient transfection studies, the LH beta promoter targeted high levels of CAT expression specifically to the pituitary. In addition, a bLH beta TK fusion gene was active only in gonadotropes. The bLH beta CAT transgene was also evaluated for responsiveness to gonadal steroids and GnRH. Testosterone and 17 beta-estradiol were capable of suppressing activity 70-80% in castrated males, despite the absence of high affinity binding sites for androgen or estrogen receptors. This suggests that feedback inhibition of LH beta CAT transgene expression by gonadal steroids may occur through an indirect mechanism, possibly at the level of the hypothalamus. To address whether the bLH beta CAT transgene could be regulated by GnRH, we treated ovariectomized females with antide, a GnRH antagonist. Antide suppressed transgene activity by 60%. Thus, the proximal promoter of the bovine LH beta subunit gene directs appropriate patterns of cell-specific expression and retains responsiveness to gonadal steroids and GnRH. In light of the robust activity of the LH beta promoter in transgenic mice, we suggest that this animal model can be exploited further to dissect the complex mechanisms that underlie gonadotrope-specific expression and hormonal regulation of the LH beta gene.

Estradiol increases the number of GnRH receptors in the ewe. Although results from studies conducted in vitro indicate that progesterone may have a negative influence on the number of ovine GnRH receptors, this effect of progesterone has not been documented in vivo. To explore the regulation of GnRH receptors at the level of gene expression, a partial complementary DNA (cDNA) encoding ovine GnRH receptor was isolated using reverse transcription and polymerase chain reaction methodology. This partial cDNA (701 basepairs) was used to isolate a full-length cDNA encoding GnRH receptor from an ovine pituitary cDNA library. Northern blot analysis of RNA from ovine pituitary glands using the partial cDNA as a molecular probe revealed four messenger RNA (mRNA) transcripts at 5.6, 3.8, 2.1, and 1.3 kilobases. In some samples, a fifth transcript at 0.8 kilobases was also evident. GnRH receptor mRNA was not detected in ovine brain, heart, kidney, adrenal, or liver tissues. To examine the regulation of GnRH receptor mRNA and GnRH receptors during the early preovulatory period, relationships among steady state concentrations of GnRH receptor mRNA, numbers of GnRH receptors, and circulating concentrations of progesterone and estradiol during luteolysis were characterized. We hypothesized that during luteolysis, decreased concentrations of progesterone would be associated with increased concentrations of GnRH receptor mRNA and increased numbers of GnRH receptors. On day 11 or 12 of the estrous cycle, luteolysis was induced in 14 ewes by treatment with prostaglandin F2 alpha (PGF2 alpha). Four ewes were treated with saline (saline controls). Anterior pituitary tissue was collected 4 h (n = 4), 12 h (n = 5), and 24 h (n = 5) after treatment with PGF2 alpha or 24 h after treatment with saline and from four untreated ewes on day 11 or 12 of the estrous cycle (untreated controls). Twelve hours after treatment with PGF2 alpha, circulating concentrations of progesterone had decreased (P < 0.05) to 46% of the control values; however, concentrations of estradiol were not different from those in control ewes. Concentrations of GnRH receptor mRNA increased 2-fold during luteolysis and were higher than control values 12 h after PGF2 alpha treatment (P < 0.05). This increase in GnRH receptor mRNA was not accompanied by an increase in the number of GnRH receptors. Twenty-four hours after treatment with PGF2 alpha, concentrations of progesterone in PGF2 alpha-treated ewes had decreased (P < 0.05) to 15% of control values, whereas concentrations of estradiol had increased (P < 0.05) to 321% of control values.(ABSTRACT TRUNCATED AT 400 WORDS)

Although treatment of heifers and ewes with recombinant bovine somatotropin (rbST) does not increase ovulation rate, data for heifers indicate that the number of small antral follicles is approximately doubled. Accordingly, the objectives of this study were to determine whether 1) treatment of ewes with rbST would increase the number of small antral follicles, thereby increasing the number of follicles that could potentially respond to superovulation treatment, and 2) superovulatory responses could be improved in ewes with "synchronized" populations of follicles. Twenty-four ewes were divided into four groups: control, control+rbST, hypothalamic-pituitary stalk disconnected (HPD), and HPD+rbST. Beginning on d 5 of the estrous cycle, ewes were injected once daily for 13 d with either rbST (3 mg) or saline. The superovulatory regimen consisted of a single dose of PMSG followed by twice-daily injections of FSH for four consecutive days. After ovariectomy, ovulation sites and follicles were counted. Twice-daily blood samples were assayed for somatotropin (ST) and IGF-I. The concentrations of ST in rbST-treated ewes were greater (P < .05) than those in controls. Treatment with rbST increased (P < .05) the mean serum concentration of IGF-I in control but not in HPD ewes. There was no increase in ovulation rate or number of small antral follicles in response to rbST. Synchronizing follicle populations also failed to increase ovulation rate or reduce variability of response. We conclude that supplementation with rbST and synchronization of follicles does not increase the superovulatory response in sheep.

Finasteride is a selective inhibitor of the enzyme 5 alpha-reductase which is responsible for the conversion of testosterone (T) to dihydrotestosterone (DHT). Finasteride is indicated for the treatment of benign prostatic hyperplasia in man (approximately 0.1 mg/kg/day). The effect of long-term treatment was studied in mice given high doses (2.5, 25, and 250 mg/kg/day) of finasteride for 83 weeks. In finasteride-treated mice, increased incidences of testicular Leydig cell hyperplasia (52% compared to 24% in control group) at doses equal to or greater than 25 mg/kg/day and Leydig cell adenomas (32% compared to 0.5% in control group) at 250 mg/kg/day were observed. There were no drug-related effects on the seminiferous tubules. Since luteinizing hormone (LH) is a trophic hormone for Leydig cells, short-term studies (5 to 14 weeks) were done to investigate the relationship between Leydig cell hyperplasia and serum LH levels in finasteride-treated mice. In these studies, there was a positive correlation between the drug-related increased incidence of Leydig cell hyperplasia and a statistically significant (p < or = 0.05) increase in serum LH levels in finasteride-treated (250 mg/kg/day) mice. Furthermore, studies in castrated male mice showed that the suppression of serum LH levels by T is reversible by inhibition of conversion of T to DHT with finasteride (250 mg/kg/day), supporting the hypothesis that DHT is involved in the regulation of LH release in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

One hundred anestrous mares (early February) were injected s.c. with implants containing 0, .9, 1.8, 3.6, or 5.4 mg of a GnRH analogue (goserelin acetate) in an attempt to induce ovarian cyclicity. Follicular activity and concentrations of progesterone and LH were determined every 3 d, or daily during estrus. In treated mares that ovulated, the interval to the second ovulation of the season was compared to that for an additional group receiving 16 h/d of light beginning December 16 (positive controls). Of the mares that did not ovulate in 30 d, eight from each dose group were challenged on d 33 or 34 with an i.v. bolus of 950 micrograms of GnRH. Blood collected at -2, -1, and 0 h before GnRH and at 15, 30, 45, 60, and 90 min and 2, 3, and 4 h after injection was assayed for serum LH. More mares (P < .05) ovulated when given 3.6- (n = 7) or 5.4-mg (n = 6) implants than when given .9-mg implants (n = 0) or placebo (n = 0). Mares with initial follicles < or = 15 mm in diameter were less (P < .05) likely to ovulate (10 of 88) than were those with follicles 16 to 20 mm in diameter (5 of 12). Area under the curve (AUC) for LH was greater for mares receiving larger doses of GnRH. The AUC and peak LH were similar between ovulating and nonovulating mares. Luteinizing hormone peaked in all mares on approximately d 12. There was no difference (P > .05) in either peak LH or AUC among treatment groups in response to the GnRH challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

A lack of pituitary LH stores has been implicated as the cause of seasonal anestrus and failure to ovulate during the spring transition period in mares. In this experiment, 40 mares were used to study the effects of GnRH, estrogen, and an estrogen-GnRH combination on increasing releasable pituitary LH. Mares were stratified based on their ability to secrete LH in response to a 950-micrograms challenge of GnRH (n = 10 per group) and then assigned to one of four treatment groups: 1) controls, given no treatment; 2) 1 mg of estradiol-17 beta in oil i.m. daily for 8 d; 3) 200 micrograms of GnRH analogue des-Gly10, [D-ala6]-LHRH ethylamide in saline i.m. twice daily for 8 d; or 4) estradiol for 4 d then estradiol plus GnRH for four subsequent days. Blood was collected on d 1, 3, 5, and 7 of treatment, and serum was assayed for LH. On d 10 after initiation of treatment, mares were again challenged with GnRH (950 micrograms), and blood was collected for 4 h. Concentrations of serum LH did not vary significantly in control, estradiol-treated, or estradiol plus GnRH-treated mares among treatment days. In contrast, administration of GnRH alone increased (P < .05) concentrations of LH on d 5 and 7. Response to GnRH challenge, as measured by area under the LH curve (AUC) and peak LH, was greater (P < .05) for mares administered GnRH (7,307.1, 67.6 ng/mL, respectively) and GnRH plus estradiol (5,691.4, 60.3 ng/mL) than for mares given estradiol alone (1,519.4, 22.1 ng/mL) or no treatment (1,213.8, 19.4 ng/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

A higher dose of GnRH is required to stimulate release of FSH than of LH, both in vivo and in vitro. Therefore, we tested the hypothesis that secretion of FSH may be mediated via a second messenger pathway different from the one that modulates secretion of LH. Pituitary cells from intact ewes were cultured in suspension in DMEM plus 10% wether serum. After 18 h, cell were washed and challenged for 2 h with agents capable of activating protein kinase A (dibutyryl cAMP), protein kinase C (phorbol 12-myristate 13-acetate; PMA), or increasing intracellular calcium (the calcium ionophore A23187). GnRH (0.01-10 nM) and PMA (0.2-20 nM) stimulated dose-dependent increases in secretion of LH. FSH secretion also was stimulated by GnRH and PMA; however, the percentage of total cellular FSH released was lower (p < 0.05) than the percentage of total cellular LH released. Dibutyryl cAMP (10 mM) induced a modest release (p < 0.05) of both LH and FSH. A23187 (1-10 microM) stimulated secretion of LH in a dose-dependent manner but did not influence secretion of FSH; however, GnRH- and PMA-induced secretion of FSH required the presence of intracellular calcium. On the basis of the results of this study, we suggest that secretion of FSH is less than secretion of LH following direct activation of these second messenger systems. Furthermore, we suggest that in contrast to the situation for LH, increased intracellular calcium is not the primary stimulus for inducing secretion of FSH.

The effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C (PKC), and the PKC inhibitor staurosporine on GnRH secretion and mRNA levels were studied in GT1-7 hypothalamic neuronal cells. Dose-response and time-course studies revealed that TPA (10(-8) M) acutely increased GnRH secretion 3-fold at 3-6 h, which then declined to baseline at 24 h, while it progressively decreased GnRH mRNA levels by 50% and 70% at 6 and 24 h, respectively. To ensure that these effects were due to activation and not down-regulation of PKC, cells were treated for 30 min with TPA (10(-8) M). This brief exposure to TPA also resulted in a decrease (60%) in GnRH mRNA levels at 6 h, with a 1.5- to 2-fold increase in GnRH secretion compared to control values, suggesting that activation of PKC decreases the pretranslational expression of GnRH while increasing GnRH secretion. Additional studies measured PKC activity and documented a shift from a cytosolic to a membrane fraction after incubation with TPA, again supporting PKC activation. Exposure of GT1-7 cells to staurosporine (10(-8) M), a PKC inhibitor, resulted in no change in the level of GnRH mRNA or secretion at 6 h. However, incubation with both TPA and staurosporine prevented the decrease in GnRH mRNA levels and partially blocked the increase in GnRH secretion induced by TPA. We conclude that TPA, by activating the PKC pathway, acutely increases GnRH secretion, but dramatically decreases GnRH gene expression. The exact mechanism of these divergent effects on the synthesis and secretion of GnRH remain to be elucidated.

Plasma membrane receptors for prostaglandins (PG) F2 alpha and E2 were quantified in ovine corpora lutea obtained from nonpregnant and pregnant ewes on Days 10, 13, and 15 post-estrus, and from additional ewes on Days 25 and 40 of pregnancy. Regardless of reproductive status or day post-estrus, concentrations of luteal receptors for PGF2 alpha were 7- to 10-fold greater than those for PGE2. In pregnant ewes the concentration of receptors for PGF2 alpha was highest on Day 10 (35.4 +/- 2.8 fmol/mg) and lowest on Day 25 (22.3 +/- 2.5 fmol/mg). A difference in the concentration of luteal receptors for PGF2 alpha between pregnant and nonpregnant ewes was apparent only on Day 15 post-estrus, at which time the concentration of receptors for PGF2 alpha was higher in pregnant ewes than in nonpregnant ewes (27.1 +/- 2.7 vs. 17.7 +/- 2.7 fmol/mg). Concentrations of receptors for PGE2 in pregnant ewes were similar (p > 0.05; 2.8 +/- 0.3 to 3.7 +/- 0.2 fmol/mg) between Days 13 and 40 but were higher (p < 0.05) than in corpora lutea obtained from nonpregnant ewes on Days 10 (5.0 +/- 0.4 vs. 4.1 +/- 0.2 fmol/mg) and 15 (3.7 +/- 0.2 vs. 2.0 +/- 0.4 fmol/mg) post-estrus. Although concentrations of receptors for both PGF2 alpha and PGE2 were lowest in corpora lutea obtained from nonpregnant ewes on Day 15, this was not due to luteal regression since the weights and concentrations of progesterone in corpora lutea on Day 15 were not lower than those for corpora lutea obtained on Days 10 and 13.(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitaries were collected from late follicular phase (n = 5), mid-luteal phase (n = 5), and anestrous ewes (n = 4) to assess changes in intrapituitary LH heterogeneity at selected reproductive states. After homogenization, an aliquot of each pituitary extract was desalted by flow dialysis against water and chromtofocused on a pH 10.5 to 4.0 gradient. Concentrations of LH in pituitary extracts and chromatofocusing fractions were determined by RIA. The LH in pituitary extracts resolved into 13 isoforms during chromatofocusing, which were coded with letters beginning with the most basic isoform. Follicular and mid-luteal phase ewes exhibited similar distributions of intrapituitary LH among its isoforms. Relative to follicular and luteal phase ewes, anestrous ewes had lower percentages of isoforms D and E as well as higher percentages of isoforms G, H, J and K. Isoform F, the predominant molecular form of LH, constituted a similar percentage in all treatment groups (P > .05). Thus, the distribution of intrapituitary LH among its isoforms did not change significantly between the mid-luteal and follicular phases of the estrous cycle, but higher percentages of the weakly basic and acidic forms of LH were present during anestrus. These observations suggest that intrapituitary LH heterogeneity changes minimally throughout the estrous cycle of ewes during the breeding season.

The proximal 5'-flanking region of the alpha-subunit gene from humans and cattle confers pituitary-specific expression to heterologous reporter genes in transgenic mice. To investigate whether these promoter regions also contain the necessary regulatory elements for cell-specific expression and hormonal regulation, we used three independent lines of transgenic mice. Two lines of transgenic mice contained chimeric genes consisting of either 1.6 kilobasepairs (kbp) of human or 3 15 basepairs of bovine alpha-subunit proximal 5'-flanking sequence linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). A third line of transgenic mice contained the proximal 1.6 kbp of 5'-flanking sequence of the human alpha-subunit gene linked to the bacterial lacZ gene encoding beta-galactosidase (beta gal; H alpha beta gal transgenic mice). Hormonal replacement paradigms indicate that both human and bovine alpha CAT transgenes are regulated by GnRH, suggesting that their expression occurs in gonadotropes. Thus, the proximal 5'-flanking regions of both the human and bovine alpha-subunit genes must contain regulatory elements that confer both gonadotrope-specific expression and responsiveness to GnRH. In contrast to the human alpha-subunit promoter, the bovine alpha-subunit promoter lacks a functional cAMP response element, suggesting that transduction of both cell-specific and GnRH transcriptional signals occurs through cAMP response element-independent pathways. Thyrotropes also express the glycoprotein hormone alpha-subunit gene. Yet, hormone replacement paradigms with propylthiouracil and T3 were ineffective in altering CAT activity in the pituitary of human or bovine alpha CAT transgenic mice. Because a thyroid hormone response element has been localized to the proximal 5'-flanking region of the human alpha-subunit gene, these data suggest that the alpha CAT transgenes lack sufficient information to direct expression to thyrotropes. Direct evidence for this possibility was obtained through immunocytochemical studies performed on pituitaries from H alpha beta gal transgenic mice. beta-Galactosidase activity appeared in gonadotropes, but not thyrotropes. We conclude, therefore, that distinct and separable regulatory elements mediate the expression of the alpha-subunit gene in gonadotropes and thyrotropes.

Concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in serum samples obtained from 100 dogs. Groups (n = 25/group) consisted of sexually intact and ovariohysterectomized bitches and sexually intact and castrated male dogs. Mean (+/- SD) concentrations of LH in the serum of sexually intact and ovariohysterectomized bitches were 1.2 (+/- 0.9) and 28.7 (+/- 25.8) ng/ml, respectively. Mean concentrations of FSH in the serum of sexually intact and ovariohysterectomized bitches were 98 (+/- 49) and 1,219 (+/- 763) ng/ml, respectively. Mean concentrations of LH in the serum of sexually intact and castrated male dogs were 6.0 (+/- 5.2) and 17.1 (+/- 9.9) ng/ml, respectively. Mean concentrations of FSH in the serum of sexually intact and castrated male dogs were 89 (+/- 28) and 858 (+/- 674) ng/ml, respectively. In addition to history, physical examination results, and other laboratory values, the measurement of serum gonadotropin concentrations may aid in determining whether dogs have been neutered.

LH, FSH, and TSH are heterodimeric glycoprotein hormones composed of a common alpha-subunit and unique beta-subunits. The alpha-subunit is produced in two distinct specialized cell types of the pituitary gland: gonadotropes, which synthesize LH and FSH, and thyrotropes, which synthesize TSH. We have demonstrated that 313 base pairs of the bovine-alpha subunit promoter direct expression of diphtheria toxin A chain specifically to the gonadotropes in transgenic mice. Animals carrying this transgene generally exhibit reproductive failure and lack of gonadal differentiation, consistent with gonadotrope ablation. Lack of gonadotrope activity was verified by RIA and immunohistochemical staining for LH. The phenotype of these transgenic mice is nearly identical to mice homozygous for the spontaneous mutation, hpg, which is due to a deletion in the gene encoding GnRH. Thyrotrope function was judged normal based on overall growth of the animals, appearance of their thyroids, T4 levels measured by RIA, and immunohistochemical staining for TSH. The ablation of gonadotropes but not thyrotropes suggests that separate cis-acting elements are necessary for expression of the alpha-subunit gene in these two cell types. Pituitary content of ACTH and GH was apparently normal, while PRL synthesis and storage were reduced. Thus, in a pituitary almost completely devoid of gonadotropes, most other pituitary functions were normal. This suggests that most pituitary cells are able to differentiate independently of terminal gonadotrope differentiation and can function in the absence of paracrine signaling provided by gonadotropes.

To test the hypothesis that the synthesis and secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are differentially regulated after depletion by oestradiol, circulating concentrations of oestradiol were maintained at approximately 30 pg/ml for 16 days in each of 35 ovariectomized ewes. Five other ovariectomized ewes that did not receive oestradiol implants served as controls. After treatment with oestradiol, implants were removed and pituitary glands were collected from each of 5 ewes at 0, 2, 4, 8, 12, 16 and 32 days thereafter and amounts of mRNA for gonadotrophin subunits and contents of LH and FSH were quantified. Before collection of pituitary glands, blood samples were collected at 10-min intervals for 6 h. Treatment with oestradiol reduced (P less than 0.05) steady-state concentrations of LH beta- and FSH beta-subunit mRNAs and pituitary and serum concentrations of these hormones. At the end of treatment the amount of mRNA for FSH beta-subunit was reduced by 52% whereas that for LH beta-subunit was reduced by 93%. Steady-state concentrations of mRNA for FSH beta-subunit returned to control values within 2 days of removal of oestradiol, but 8 days were required for concentrations of FSH in the pituitary and serum to return to control values. Steady-state concentrations of mRNA for LH beta-subunit and mean serum concentrations of LH returned to control values by Day 8, but pituitary content of LH may require as long as 32 days to return to control levels. Therefore, replenishment of FSH beta-subunit mRNA preceded increases in pituitary and serum concentrations of FSH.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine the acute and chronic effects of estradiol on synthesis and secretion of LH and FSH, ovariectomized ewes were administered estradiol via silastic capsules for 0 h, 12 h, 1 day, 2 days, 4 days, 8 days, 16 days, or 32 days (n = 5/group). Concentrations of GnRH in the median eminence began to decrease within 12 h and were lower (p less than 0.05) than in control ewes from 1 to 4 days after estradiol administration was begun. Serum concentrations of LH were decreased relative to pretreatment control levels from 1 to 10 h, elevated during a preovulatory-like surge from 11 to 22 h, and then decreased and remained below 1 ng/ml for the duration of the experiment. Serum concentrations of FSH followed a pattern similar to those for LH except that the magnitude of change was smaller. Treatment with estradiol initially (12 h) reduced (p less than 0.05) quantities of mRNA for alpha-, LH beta-, and FSH beta-subunits, after which the quantities of mRNA for the subunits returned to near or above control levels by Day 2. After 8 days of treatment the amounts of mRNAs for gonadotropin subunits were again less (p less than 0.05) than those of controls, and they remained suppressed through Day 32. Pituitary concentrations of LH and FSH decreased (p less than 0.05) during the first day of treatment and remained suppressed for the duration of the experiment. Thus, estradiol had a triphasic effect on secretion of gonadotropins and steady-state levels of mRNA for the gonadotropin subunits, but not on pituitary content of gonadotropins.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic administration of estradiol inhibits transcription of the gene encoding the alpha-subunit of pituitary glycoprotein hormones. Here, we show, using transfection analyses and a filter binding assay, that 1500 basepairs of proximal 5' flanking sequence of the human alpha-subunit gene lack a functional estrogen response element when transfected into heterologous cell lines, and fail to bind estrogen receptor purified from calf uterus. Yet, this same region of the alpha-subunit gene confers estradiol responsiveness (transcriptional suppression) to the bacterial chloramphenicol acetyltransferase gene in transgenic mice. A smaller promoter fragment of the bovine alpha-subunit gene also confers responsiveness to estradiol in transgenic mice, suggesting that the same element may mediate the steroid responsiveness of both promoters. Furthermore, regulation by estradiol of the chimeric human or bovine alpha-chloramphenicol acetyltransferase genes is pituitary specific, underscoring the physiological significance of these studies. Based on these results, we conclude that estradiol regulates expression of the alpha-subunit gene in vivo through a mechanism that does not involve high affinity binding of estrogen receptor to the alpha-subunit gene. Whether this mechanism is manifest at the level of the pituitary or hypothalamus remains to be determined.

Primary cultures of ovine pituitary cells were used to characterize the effects of inhibin and activin on the secretion of gonadotropins and on the regulation of number of GnRH receptors in the presence or absence of estradiol. Number of GnRH receptors was determined by the specific binding of a saturating dose of [125I]des-Gly10-D-Trp6-GnRH-ethylamide (GnRH-A). Recombinant human inhibin-A (rh-inhibin-A) or inhibin in porcine and bovine follicular fluid (pFF and bFF, respectively) decreased secretion of FSH in a dose-dependent manner, with maximum inhibition at an inhibin concentration of approximately 0.1 nM. Neither pFF or bFF affected secretion of LH, although rh-inhibin-A caused a modest decrease (p less than 0.05) in secretion of LH. Treatment of cells with rh-inhibin-A, bFF, or pFF approximately doubled the number of GnRH receptors. Scatchard analysis indicated that increases in GnRH-A binding were due to an increase in receptor number rather than a change in affinity. Additionally, rh-inhibin-A, at a dose that doubled numbers of GnRH receptors, increased GnRH-induced LH release above that caused by GnRH alone, indicating that the increase in receptor number leads to increased responsiveness to GnRH. Recombinant human activin-A (rh-activin-A) increased secretion of FSH but did not affect secretion of LH. Number of GnRH receptors was not affected by lower concentrations of rh-activin-A but was decreased (p less than 0.05) by 3.0 nM activin.(ABSTRACT TRUNCATED AT 250 WORDS)

During prolonged periods of reduced dietary energy, there is a reduction in secretion of LH in females. To test the hypothesis that decreased secretion of LH is due to reduced secretion of GnRH, 18 ovariectomized ewes were fed either a low-energy diet (LOW, 60% of the National Research Council recommendations, n = 12) or a normal diet, (control, n = 6), for 127 days. Each ewe received basal levels (approximately 5 pg/ml) of estradiol via sc Silastic implants. After 127 days, serum concentrations of FSH and LH were reduced (P less than 0.05) by 63% and 77%, respectively in LOW ewes compared to control ewes. Pituitary concentrations of FSH and LH in LOW ewes also were reduced by 56% and 80%, respectively. Compared to levels in control ewes, concentrations of messenger RNAs for alpha-, FSH beta-, and LH beta-subunits were reduced by 75%, 76%, and 91%, respectively. Pulsatile administration of GnRH (250 ng/2 h) for three weeks to LOW ewes restored each of the parameters to levels not different from those in controls. By the end of the study, serum concentrations of GH in all LOW animals had risen dramatically, but not in control ewes. Therefore, it appears that exogenous GnRH is capable of restoring synthesis and secretion of gonadotropins in ewes receiving low-energy diets. These results provide support for the hypothesis that reduced dietary energy results in decreased secretion of GnRH.