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The present study examined the gonadotropin surge-inducing actions of estradiol (E(2)), both alone and with progesterone (P(4)), in middle-aged, early persistent-estrous (PE) female rats that had become PE within 35 days. In addition, we also assessed the effect of P(4) on the mating-induced gonadotropin surges in these acyclic animals. Early PE rats were ovariectomized and received E(2) implants (Day 0). On Day 4, an s.c. injection of P(4) (0.5 mg/ 100 g body weight) at 1200 h markedly increased plasma P(4) and elicited both LH and FSH surges, whereas vehicle-treated controls displayed no rise in P(4) or gonadotropins. This observation confirms that at middle age, female rats no longer respond to the positive-feedback stimulation of E(2) on gonadotropin surges whenever the estrous cyclicity ceases. As PE continued, such a surge-inducing action of E(2) plus P(4) became diminished after 75 days of PE and disappeared thereafter. When caged with males, vehicle-treated early PE rats display a mating-induced increase in P(4) from the adrenal along with small gonadotropin surges. The amplitude of these mating-induced gonadotropin surges was enhanced by supplementation with exogenous P(4) in early PE rats. Our findings indicate that during the early phase of PE, the surge-inducing action of E(2) and P(4) remains intact but deteriorates as PE continues. Thus, a deficiency in P(4) secretion during aging may contribute to the diminished gonadotropin surge response in the hypothalamic-pituitary axis and the subsequent cessation of estrous cyclicity.

As circulating estrogen levels rise on the afternoon of proestrus, they stimulate the hypothalamo-pituitary axis. This estrogen positive feedback is pivotal to stimulate the luteinizing hormone (LH) surge required for ovulation and luteinization of ovarian follicles. In addition to estrogen, pre-LH surge progesterone is critical for an LH surge as was demonstrated by blocking progesterone synthesis. In ovariectomized (OVX) rats treated with trilostane, a blocker of the enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) that catalyzes the conversion of pregnenolone to progesterone, estrogen did not induce an LH surge. Further, estrogen induced an LH surge in OVX and adrenalectomized (ADX) rats, indicating that the source of progesterone was neither the ovary nor adrenal gland. This estrogen-only LH surge was inhibited by pretreatment with trilostane, indicating that although the adrenal gland and ovary were not necessary for positive feedback, progesterone synthesis was critical for estrogen-induced positive feedback in an OVX/ADX rat. This suggested that the LH surge is dependent on the pre-LH surge synthesis of progesterone. Estrogen-induced progesterone receptors in the hypothalamus are vital for the LH surge, so a potential location for progesterone synthesis is the hypothalamus. OVX/ADX female rats were treated with 17beta-estradiol (50 microg) and progesterone levels were assayed by RIA. Progesterone levels were elevated in hypothalamic tissue following estrogen treatment. No increases in tissue progesterone levels were found in parietal cortex, cerebellum, medulla, pituitary or plasma. Additionally, male rats that do not have an estrogen positive feedback-induced LH surge were examined. Castrated/ADX male rats had no increase in hypothalamic progesterone levels after estrogen treatment. Together, these data strongly suggest that estrogen enhances neuroprogesterone synthesis in the hypothalamus that is involved in the positive feedback regulating the LH surge., (Copyright 2003 S. Karger AG, Basel)

This study examined the effects of aging on LH surge magnitude, ovulation, and ovarian expression of tissue-type plasminogen activator (tPA), a protease implicated in follicular rupture. While mean LH levels and ovulation rates were similar in middle-aged cyclic and young groups, there was a significant correlation between peak LH levels and ovulation rates in individual rats, such that females with lower LH surges ovulated fewer ova. In a separate experiment, proestrous LH levels were characterized in young and middle-aged rats, followed by in situ hybridization analysis of ovarian tPA mRNA. In young proestrous rats, tPA expression was observed in thecal-interstitial cells and oocytes, but not granulosa cells, prior to the LH surge. After the LH surge, there was a marked increase in tPA mRNA levels in granulosa cells of preovulatory, but not smaller follicles, peaking at 0200 hr estrus. By 0500 hr estrus, ovarian tPA expression declined, and ovulation had occurred. In contrast, LH-induced follicular tPA mRNA levels were dramatically lower in middle-aged rats with attenuated LH surges, and persisting preovulatory follicles were common in ovaries of these females on estrus morning. These findings suggest that age-related declines in ovulatory function result in part from altered induction of ovarian tPA expression, likely due to decreased proestrous LH secretion.

In polycystic ovary syndrome (PCOS), increased luteinizing hormone (LH) pulse frequency has been attributed to either the hypothalamic gonadotrophin-releasing hormone (GnRH) pulse generator or ovarian oestrogen feedback. To address this issue, a detailed examination of pulsatile LH secretion was undertaken during recovery from GnRH agonist (GnRHa) suppression. Each of six women with PCOS and six normal ovulatory women received daily GnRHa treatment for 14 weeks. Frequent blood samples were collected and assayed for gonadotrophins, androgens and oestrogens before, during and up to 4 weeks after treatment. Women with PCOS had higher basal LH pulse frequency and amplitude and increased serum concentrations of LH, androstenedione, testosterone and oestrone than controls. After 3 months of GnRHa treatment, all these parameters were suppressed with no differences observed between the two groups. One week after cessation of GnRHa, LH pulse frequency promptly returned to pre-treatment range with no between-group differences noted, whereas LH pulse amplitude, serum gonadotrophins and ovarian steroids remained maximally suppressed and equivalent in the two groups. Subsequent LH pulse frequency remained constant while LH pulse amplitude and circulating concentrations gradually increased in parallel with a return of serum oestrogen to pre-treatment values. Despite comparable resumption of LH secretion in the two groups, corresponding androgen concentrations in women with PCOS were greater than those of normal ovulatory women. Thus, the immediate restoration of LH pulse frequency after discontinuing GnRHa treatment is largely independent of ovarian oestrogen production and reflects primacy of the GnRH pulse generator in determining basal LH pulse frequency. Equivalent LH pulse frequency rates in the two groups during the recovery period do not suggest an intrinsic hypothalamic-pituitary hyperactivity in PCOS.

This study compared the changes in pituitary and serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) at various times following ovariectomy (OVX) between young cyclic and middle-aged persistent-estrous (PE) rats and related these to the relative gene expression of the pituitary gonadotropin subunits. In intact animals, both pituitary and serum levels of LH were similar between these two age groups, while the LH beta mRNA expression was significantly (p < 0.05) greater in young rats. Following OVX in young rats, the serum LH levels markedly increased (p < 0.05) beginning on day 7 and reaching a maximum fourfold increase by day 9. In contrast, the post-OVX increases in serum LH in middle-aged females were significantly delayed. OVX significantly (p < 0.05) increased pituitary LH contents of young rats by day 5, but had no effect on LH contents in middle-aged females until day 30 post-OVX. These changes were associated with increases in LH beta mRNA expression in both young and middle-aged females, but the levels were significantly (p < 0.05) lower in middle-aged females. Both pituitary and serum levels of FSH were significantly (p < 0.05) higher in middle-aged PE than in young rats prior to OVX, while the FSH beta mRNA expression was similar in both age groups. Following OVX in young rats, serum FSH levels rapidly increased (p < 0.05) on day 3 and attained tenfold higher values by day 30.(ABSTRACT TRUNCATED AT 250 WORDS)

The acute gonadotropin and estradiol responses following single subcutaneous injections of 10, 50, 100 and 200 micrograms of [(imBzl)]D-His6,Pro9-NEt]-GnRH were compared to those after 100 micrograms of [D-Trp6, Pro9-NEt]-GnRH. The gonadotropin responses after 50-100 micrograms of the D-His analog of GnRH were equivalent to those following 100 micrograms of the D-Trp analog. The ovarian E2 response, a reflection of the total cumulative secretion of gonadotropins, was similar at 100 micrograms of each analog. The estradiol response paralleled the increasing gonadotropin response accompanying the graded doses of the D-His analog, indicating the lack of a direct inhibitory action of this GnRH agonist on the ovary. Assessment of both gonadotropin and estradiol responses appears to be satisfactory for assessing relative potency among GnRH agonistic analogs.

In eumenorrheic women with endometriosis and in oligo-amenorrheic women with polycystic ovarian disease (PCO), chronic administration of a long-acting GnRH agonist (GnRH-a) reduced the circulating concentrations of estrogens and androgens to levels similar to those of castrated women. The concommittant elevation of LH in both groups suggested that the measured immunoreactive LH had reduced bioactivity. In seven women with endometriosis, bioactive LH (BA LH) measured as the in-vitro secretion of testosterone by dispersed Leydig cells, was significantly (p less than 0.001) reduced from 10.8 +/- 1.2 (SEM) to 4.4 +/- 0.2 mIU/ml at the end of 28 days of GnRH-a therapy. In five women with PCO, BA LH decreased from 44.2 +/- 15.5 to 5.7 +/- 0.6 mIU/ml (p = 0.06). These changes of BA LH appeared to be responsible for the suppression of ovarian androgen secretion during GnRH-a treatment and in turn may have contributed to the profound decreases of estrogen production by reducing the amount of precursor androgen available for aromatization. Free alpha subunit levels increased simultaneously with the decrease of BA LH at the end of therapy, suggesting a post-receptor effect of GnRH-a. Beta subunit levels became undetectable. Cross-reaction of alpha subunit in the RIA for LH was sufficient to only partially account for the LH levels measured. On sephadex G-100 chromatography the excess immunoreactive material was detected at and immediately following the alpha subunit tracer. Further studies will be necessary to elucidate the chemical nature of the immunoreactive LH secreted during GnRH-a therapy.

The principal glandular source of increased serum androgens in polycystic ovarian disease (PCO) is controversial), since complete separation of ovarian from adrenal function has not been achieved. The purpose of this study was to determine whether a long-acting GnRH agonist could be used to selectively inhibit ovarian steroid secretion in PCO and ovulatory women. Each of five typical PCO patients and six ovulatory subjects on day 2 of their menstrual cycles received D-Trp6-Pro9-NEt-LHRH (GnRH-a; 100 micrograms) for 28 consecutive days. Their results were compared to basal serum hormone values in eight oophorectomized women. In response to GnRH-a, PCO and normal subjects exhibited sharp and sustained rises of LH and gradual decreases in FSH. These levels were clearly less than basal levels seen in oophorectomized women. Episodic LH release was significantly attenuated in both groups at the end of GnRH-a treatment. After the administration of agonist, serum estradiol (E2), estrone (E1), androstenedione (A), and testosterone (T) were suppressed to castrate levels in both groups. The decrements of E2 and E1 in PCO were gradual and continuous compared to initial dramatic rises, which reached peaks at 14 days, and subsequent abrupt falls in the ovulatory controls. Serum A and T declined steadily in both groups. Basal serum dehydroepiandrosterone and dehydroepiandrosterone sulfate, but not cortisol, levels were elevated in PCO subjects. The 24-h secretion patterns and responses to ACTH of these hormones in PCO and ovulatory subjects were unaltered by GnRH-a administration. These data demonstrate that 1) in PCO subjects, GnRH-a induced complete suppression of ovarian steroid secretion, as circulating levels at the end of treatment were comparable to those seen in our oophorectomy subjects; 2) elevated A and T levels in PCO patients were derived primarily from the ovary; and 3) adrenal steroid secretion was unaltered by GnRH-a in both PCO and normal women.

In aging, persistently estrous (PE) female rats, there are no estrous cycles or cyclic increases in luteinizing hormone (LH) secretion, but the sexual receptivity to the male is consistently maintained. We recently reported that caging and mating with fertile males elicits an LH surge followed by ovulation in aging PE rats. The present study examined the relationship between the LH surge, the increase in progesterone (P) secretion and ovulation in PE females exposed to males, and assessed whether intromission was essential for the male-induced pre-ovulatory LH surge. PE rats were implanted with intra-atrial cannulae. Six to eight days later, these females were individually caged with a fertile male and repeatedly sampled (once every 30 or 60 min) between 1400 and 1900 h for assays of plasma LH and P. Sexual behavior of the female was recorded and correlated with the changes in plasma LH and P values. Similar experiments were also performed on cannulated PE rats with their vaginal orifice blocked with adhesive tape during the caging and sampling session. In both experiments, over 90% of the PE females displayed a high degree of lordosis response to mounting by the male, and over 60% of those sexually receptive PE females exhibited an LH surge followed by ovulation. The male-induced preovulatory LH surge occurred in PE females without actual intromission. Caging with fertile males also elicited a marked increase in plasma P concentrations in PE rats and in PE females prevented from experiencing intromission.(ABSTRACT TRUNCATED AT 250 WORDS)

In female rats ovariectomy (OVX) on the morning of diestrus day 2 resulted in a prompt (within 8 h) and gradual increase in LH release, whereas a similar operation on estrus did not raise serum LH until 24 h later. In proestrous females, OVX in the morning neither prevented the anticipated LH surge on that evening, nor increased LH release on the next morning. Since circulating concentrations of estradiol (E2) are progressively increased during diestrus day 2 and proestrus, this pattern of E2 secretion may affect the acute increase in LH secretion following OVX. To test such a hypothesis, we examined the effects of large, sustained or large, transient increases in circulating estrogen on the subsequent increase in gonadotropin secretion after OVX. On the morning of diestrus day 1 a subcutaneous injection of 20 micrograms estradiol benzoate (EB) produced prompt and large increases in both serum E2 and estrone (E1) for about 2 days, despite OVX of females at 24 h after the EB injection. An injection of 20 micrograms 17 beta-estradiol (E2) also promptly increased both serum E2 and E1. micrograms E2 injection were twice that observed after 20 micrograms EB, and both E2 and E1 returned to low baseline values within 24 h after the E2 injection. In contrast, administration of 1 microgram EB only produced small and transient rises in serum E2 and E1.(ABSTRACT TRUNCATED AT 250 WORDS)

The progressive cessation of regular ovulatory function in aging female rats is preceded by a significant decrease in the magnitude of the proestrous LH surge during regular estrous cycles. However, our recent study has demonstrated that normal LH secretion and regular estrous cycles can be maintained for an extended period of time in aging females housed with fertile males and allowed to undergo repeated pregnancies. Since progesterone (P) secretion is persistently increased in pregnant rats, the present study examined whether repeated increases in circulating progesterone accounted for these results. Starting at 8 months of age and continuing to 13 months, multiparous rats were grouped and treated as follows: controls: females were housed five per cage; mated: five females were housed with one fertile male and allowed to undergo repeated pregnancies; and P-implanted: females were housed five per cage and implanted sc with Silastic capsules containing P for 3 of every 4 weeks. During the 4.5 months of study, serum concentrations of estradiol (E2) in the P-implanted rats remained between 13 and 27 pg/ml, similar to levels in pregnant females (8-26 pg/ml) of the mated group. These E2 values were less than the preovulatory increase in serum E2 on proestrus (mean +/- SE, 56 +/- 10 pg/ml) in cyclic control females. In contrast, serum P values were persistently elevated in both the pregnant and the P-implanted rats, although the values in the latter (27-55 ng/ml) were about one third to one half of those in the former group (117-125 ng/ ml). All treatments were stopped at 13 months of age, and estrous cycle patterns were determined thereafter. Between 13 and 17 months of age, the percentages of regularly cycling rats were significantly (P less than 0.01) greater in the mated group (50%, 36%, and 15% at 13, 15, and 17 months, respectively) than in the control group (23%, 20%, and 9%, respectively). During this same period, 50% of the females from the P-implanted group continued to display regular cycles. By the age of 11 months, 8 of 21 untreated retired breeder females exhibited attenuated LH surges on proestrus and subsequently ceased to display regular estrous cycles within 2 months, whereas the other 13 rats showed normal LH and FSH surges and continued to maintain regular cycles. In contrast to these, aged female rats from the previously mated (15-month-old) and the P-implanted (19-month-old) groups exhibited normal profiles of proestrous LH and FSH surges during regular estrous cycles.(ABSTRACT TRUNCATED AT 400 WORDS)

To examine the relationship between the occurrence of menopausal hot flashes and the pulsatile release of LH, we have investigated the serum hormone levels and the occurrence of hot flashes by objective recordings in five women with endometriosis given daily injections of a long-acting GnRH agonist (GnRH-a) for 28 days. Results were compared to the findings made in 25 young women 6-8 weeks after bilateral oophorectomy. Serum levels of estrone and estradiol were similar in the subjects given GnRH-a and the women who underwent a surgical castration. In comparison with values before GnRH-a administration, the mean FSH level was lower whereas the mean LH concentration was significantly higher (P less than 0.01) on the last day of therapy. The coefficients of variations of both gonadotropins measured during 4-h sampling periods at 20-min intervals before and at the end of GnRH-a administration were significantly reduced (P less than 0.01) with therapy. During the total of 20 h of frequent sampling in the 5 subjects, 15 pulses (20% rise from nadir) of LH and 12 pulses of FSH were detected before GnRH-a, whereas only 2 and 8 pulses, respectively, were observed on day 28 of treatment. Hot flashes were observed in both groups of patients. The proportion of women experiencing hot flashes, the rate of occurrence/h and the characteristics of the physiological changes were similar in the 2 groups of women. These data indicate that hot flashes can occur in the absence of prominent LH pulses, suggesting the pulsatile release of this hormone is merely associated with the hot flash rather than being etiological.

The disassociation between serum LH and FSH levels in polycystic ovarian disease (PCO) has been attributed to chronic acyclic estrogen production characterized by a predominance of circulating estrone (E1). This study was designed to determine whether the administration of estrone benzoate (E1B) modulates gonadotropin release in PCO. In five normal women studied during the early follicular phase of a control and subsequent treatment cycle, daily LH and FSH levels were unaltered by E1B administered from days 2 to 6. Gonadotropin responses to LRF given on day 7 were similar during control and treatment cycles. In seven patients with PCO, the mean LH concentration (25.7 +/- 0.7 mIU/ml) and the daily pattern of release were unchanged by E1B administered for 14 days. In contrast, a progressive decline in FSH occurred in each subject. Mean FSH levels decreased significantly from a pretreatment value of 11.3 +/- 0.2 to 9.3 +/- 0.9 mIU/ml by day 2 (P less than 0.05) and 7.2 +/- 1.2 mIU/ml by day 14 (P less than 0.005) of E1B administration. The LH response to LRF in PCO was significantly greater than that observed in the normal subjects, whereas responses before, during, and after E1B administration were similar. The FSH responses to LRF in PCO subjects were comparable to those of the normal subjects. These data indicate that the administration of E1B to PCO subjects reduces FSH levels without altering LH release, thereby enhancing the disparity of gonadotropin secretion encountered in this syndrome. This finding is consistent with the hypothesis that impairment of FSH release by chronic acyclic estrogen production derived from nonglandular aromatization of circulating androgen could in large part be responsible for anovulation in PCO.

To determine whether discernible alterations in neuroendocrine and/or ovarian function precede the loss of regular oestrous cycles in ageing female rats, the present study examined the pattern of gonadotrophin secretion near the time of ovulation and the pattern of ovarian steroid secretion in the early morning of pro-oestrus in middle-aged (10-12 months old) females displaying regular oestrous cycles and compared these with young (4 months old) animals. In addition, the subsequent reproductive patterns in these animals were observed and correlations between the changes in hormonal profiles and the decline in regular reproductive cyclicity were established. In middle-aged females which subsequently ceased to display regular oestrous cycles (middle-aged non-regular; M-NR) within 1-2 months, the pro-oestrous surge of LH was significantly reduced in magnitude. There was no difference in the LH surge between young females and middle-aged animals which maintained regular oestrous cycles (middle-aged regular; M-R) for at least 2 months. There also was no difference in the magnitude of the pro-oestrous FSH surge or in the secondary rise in FSH in the early morning of oestrus among young, M-R and M-NR females. In a separate group of middle-aged females which subsequently became M-NR, serum concentrations of both oestradiol and testosterone in the early morning of pro-oestrus were markedly raised over those in the young and M-R groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Middle-aged female rats cease to display estrous cycles and exhibit a state of persistent estrus (PE). Under PE and chronic anovulatory conditions, there is a lack of spontaneous luteinizing hormone (LH) surges, but ovulations often occur after the females are caged with males. This study examined the effects of caging and mating with male rats on LH release in PE females, and assessed their reproductive capacity. Young cyclic rats received intra-atrial cannulae, and subsequently were sampled every 90 min during 1400-2130 h on proestrus for plasma LH measurement. PE females were similarly cannulated and sampled. Two days later, these PE rats received an s.c. injection of estradiol benzoate (EB) and were sampled on the following day. While young females exhibited the proestrous LH surge, PE rats maintained low plasma LH levels persistently and were unable to increase LH release after EB administration. On the other hand, when cannulated PE females were caged with fertile males, 92% displayed lordotic responses, and 75% of those sexually receptive PE females exhibited LH surges followed by ovulation. The initiations of the lordotic response and the LH surge both were more rapid in PE females caged with males beginning at 1500 h than at 1400 h. In contrast, when individual PE rats were placed in clean boxes without males, only one of 13 females showed an increase in LH release followed by ovulation. Separate groups of PE rats were mated with fertile males, and subsequently used for counting the number of blastocysts in the uteri on Day 5 of pregnancy and the number of pups delivered at term.(ABSTRACT TRUNCATED AT 250 WORDS)

Inoculation of cyclic female rats with the prolactin (Prl)/growth hormone-secreting pituitary tumor, MtT.W15, resulted in a cessation of estrous cyclicity within 5--10 days. Associated with this acyclicity was a persistently low serum concentration of estradiol and marked increases in both circulating Prl and progesterone. At Day 26 of acyclicity, basal serum luteinizing hormone (LH) values measured in samples taken every 20 min from 0900--1100 h were significantly reduced when compared to cyclic, nontumor animals on diestrus Day 2. There was no difference in basal follicle-stimulating hormone (FSH) concentrations. In a separate group of acyclic, tumor-bearing females 42--56 days after transplantation, a single s.c. injection of 20 micrograms estradiol benzoate (EB) at 1030 h elicited significant increases in both serum LH and FSH values between 1700 and 1830 h on the next day. The magnitude of the LH surge was reduced and that of FSH was increased in tumor-bearing animals when compared to cyclic, nontumor females given a similar EB injection on diestrus Day 1. These results demonstrate that chronic hyperprolactinemia is associated with inhibition of basal LH secretion and ovarian estrogen production and an increase in circulating progesterone concentrations. Nevertheless, the stimulatory feedback effects of estrogen on LH and FSH release are still present and functioning in acyclic female rats under chronically hyperprolactinemic conditions. These data suggest that the cessation of regular ovulatory cycles associated with hyperprolactinemia may be due to a deficiency of LH and/or estrogen secretion, but not to a lack of central nervous system response to the stimulatory feedback action of estrogen.