Evidence that desensitization of the negative feedback of oestrogen and priming for the positive feedback of oestrogen depend upon a common mechanism of oestrogen action in rats.

Oestrogen priming of the central nervous system is required for the positive feedback of oestrogen, and the sensitivity of the negative feedback of oestrogen can be reduced by oestrogen itself. Using adult female and male rats we examined the possibility that these effects depend upon a common mechanism of oestrogen action that is mediated by the medial preoptic area (MPOA). Guide cannulae were implanted in the MPOA of 4-day cyclic rats which were ovariectomized during the evening of day 1 of dioestrus. Glass capillary tubes containing different substances were placed in the cannulae between 09.00 and 12.00 h on the presumptive day 2 of dioestrus. The effectiveness of oestrogen priming was evaluated by examining whether an s.c. implant of oestradiol-17 beta (OE2) induced an LH surge, and the inhibitory effect of oestrogen on tonic LH secretion was investigated by injecting the rats with 3 micrograms oestradiol benzoate (OB)/100 g body weight. The priming effect of an s.c. implant of OE2 could be imitated by the bilateral implantation in the MPOA of a mixture of OB and cholesterol at a ratio of 1:360 for 3 h only. Similar medial preoptic oestrogen implantation also significantly reduced the LH-inhibiting effect of OB. In accord with findings obtained in former studies on desensitization of the negative oestrogen feedback, oestrogen priming resulting from the s.c. administration of OE2 could be suppressed by short-term medial preoptic implantation of clomiphene citrate or apomorphine; bilateral electrical stimulation of the medial amygdaloid nucleus induced an increase in the serum concentration of LH in ovariectomized females implanted with OB in the MPOA, but not in castrated males pretreated and implanted with OB.(ABSTRACT TRUNCATED AT 250 WORDS)

[Dopamine antagonists in anovulation?]. / Dopaminolytika bei Anovulation?

Increased hypothalamic dopamine turnover may prevent the ovulatory LH peak by different mechanisms and may in this way result in anovulation. Ten patients with anovulatory menstrual cycles who had been treated with clomiphene citrate without success were given the dopaminolytic drug pimozide (Antalon) at a dose of 5 mg/day orally either from day 7 to day 11 (7 women; group 1) or from day 11 to day 15 of the menstrual cycle (4 women; group 2). One patient from group 2 ovulated during the treatment cycle. A further woman from this group exhibited an LH peak, but did not ovulate, whereas a slow increase of the circulating LH level was recorded in the remaining two patients. Systematic investigations should clarify whether introduction of dopaminolytic therapy in the treatment of hypothalamic anovulation may be justified.

Oestrogen priming for the positive oestrogen feedback: site of action.

The significance of oestrogen priming for efficacy of the positive, ovulation-inducing oestrogen feedback has been known for more than 15 years, but the site and mechanism of oestrogen action in the priming effect have not yet been elucidated. Long-term ovariectomized adult female rats were injected once or twice with 20 micrograms oestradiol benzoate (OB), and the serum LH concentration was estimated. Whereas a single injection of OB induced significant inhibition of LH secretion, high circulating LH levels were recorded in rats injected twice with the hormone at an interval of 48 h. This increase was prevented in ovariectomized females fitted with guide cannulae, if the antioestrogen clomiphene citrate was implanted into the medial preoptic area (MPOA) before the first injection of oestrogen and removed prior to the second. On the other hand, replacement of the first oestrogen administration by the implantation of a very low dose of OB into the MPOA resulted in stimulation of LH secretion. OB implants placed into the hypothalamic ventromedial-arcuate region were ineffective in this regard. Taken together, the findings suggest that the priming effect of oestrogen is mediated in rats, at least in part, by the MPOA.

Partial compensation of sexual receptivity deficits in female rats with bilateral lesions of the hypothalamic ventromedial nucleus by transplants of fetal mediobasal hypothalamic tissue.

Bilateral aspiration as well as bilateral electrolytic lesions of the hypothalamic ventromedial nucleus (VMN) in ovariectomized, adult female rats treated with testosterone or estrogen resulted in a long-lasting decrease of sexual receptivity. Fetal mediobasal hypothalamic (MBH) tissue was grafted successfully to the site of VMN lesions. The following presuppositions for a sufficient rate of long-term survival of the grafts could be defined: (1) careful lesioning and minimalizing of damage to the surrounding tissues, (2) high vitality of the donor fetuses, (3) delay of transplantation after lesioning, and (4) testosterone treatment of the recipient animals at an early stage of graft development. In rats with surviving MBH grafts the receptivity deficits resulting from bilateral aspiration of the VMN were compensated partially.

Sex-specific postnatal development of negative oestrogen feedback in rats.

Experiments were performed in immature and adult male and female rats to study the postnatal development of the negative oestrogen feedback in males and to investigate the mechanisms underlying this development. The following results were obtained: 1. Immature male and neonatally androgenized female rats were less responsive to the LH-inhibiting effect of oestradiol benzoate (OB) than untreated females. In contrast, long-term castrated adult males and females with the androgen-induced persistent oestrus syndrome were distinctively more sensitive than cyclic females to the negative feedback action of oestrogen. 2. The low oestrogen sensitivity in prepubertal males is probably caused by the testicular androgen secretion during the perinatal critical differentiation phase, because after neonatal castration, the LH response to OB injected at 21 days of age did not differ between both sexes. 3. As opposed to the peripubertal desensitization to the negative feedback action of testosterone, the sensitivity to the inhibitory effect of oestrogen on LH secretion did not decrease in males at the time of puberty. 4. Whereas former studies have shown that implantation of OB in the medial preoptic area results in significant reduction of the oestrogen sensitivity in female rats, medial preoptic implants containing oestradiol, testosterone or 5 alpha-dihydrotestosterone did not diminish the LH-suppressing effect of s.c. injected OB in males. The results suggest that 1. a marked sex difference exists in the postnatal development of the negative oestrogen feedback in rats. 2. Higher oestrogen sensitivity in adult male as compared to adult female rats may depend, at least partly, on prepubertal and cyclic desensitization of the negative oestrogen feedback in females and more or less constant sensitivity in males from infancy to adulthood. 3. In males the oestrogen sensitivity seems to be settled for long by the testicular androgen secretion during the perinatal critical differentiation phase. 4. The prepubertal increase of gonadotrophin secretion necessary for the induction of male puberty may mainly be caused by diminution of the gonadotrophin-inhibiting effect of androgens and by the decline of testicular release of inhibin. 5. The oestrogen-induced desensitization of the negative oestrogen feedback mediated by the medial preoptic area is probably a sex-specific mechanism that is only operative in female rats.

Diminished oestrogen sensitivity and increased ovulation rate in adult female rats after prepubertal treatment with oestrogen or pimozide.

Immature female rats were implanted with oestradiol benzoate or cholesterol in the medial preoptic area at different ages, and the inhibition of the ovariectomy-induced increase of LH secretion by s.c. injected oestradiol was investigated. Medial preoptic oestrogen implants reduced the inhibition of LH secretion in 4-week-old rats, but not in younger animals. Elevation of the circulating oestrogen concentration or suppression of the central nervous dopamine activity by daily injections of oestradiol and pimozide, respectively, from Day 26 to the day of vaginal opening, i.e. during the time when the mechanism of the oestrogen-induced desensitization of the negative oestrogen feedback matures, resulted in considerable diminution of the LH-inhibiting effect of oestradiol in ovariectomized adult females. In intact cyclic rats, both prepubertal treatments led to a significant increase of the average number of eggs per ovulation that was mainly caused by reduction of the number of animals with a low ovulation rate.

Effect of medial preoptic oestradiol implants on hypothalamic beta-endorphin concentration.

Twenty-four-day-old immature and cyclic female rats in metoestrus were ovariectomized and bilaterally implanted with oestradiol benzoate (OB) and cholesterol at a ratio of 1:360 or with cholesterol alone in the medial preoptic area (MPOA), or, for control, the hypothalamic dorsomedial nucleus. Estimation of beta-endorphin immunoreactivity in the ventromedial-arcuate-median eminence region at 6 and 3 days after implantation, respectively, revealed a significantly higher concentration in rats implanted with OB in the MPOA as compared to those implanted with cholesterol. OB implants placed in the dorsomedial nuclei were ineffective in this regard. The hypothesis is put forward that inhibition of hypothalamic beta-endorphin release as probably resulting from the implantation of OB in the MPOA may be related to desensitization of the negative oestrogen feedback that is induced by similar preoptic oestrogen implantation.

Effects of neonatal intracerebral implantation of sex steroids on sexual behaviour, social play behaviour and gonadotrophin secretion.

Female rats were intracerebrally implanted with testosterone propionate (TP) or 5 alpha-dihydrotestosterone (DHT) on the 3rd day of life. After both treatments, prepuberal social play behaviour was significantly enhanced as compared to control females and did not differ from that recorded in males. In contrast, intracerebral implantation of oestradiol benzoate (OB) at the same day of age had no effect on the frequency with which females engaged in social play. DHT, which is not aromatizable to oestrogen, showed a significant male-type organizational effect also on sexual orientation but not on the organization of gonadotrophin secretion pattern and hence on ovarian weight. On the other hand, OB displayed in a dose-dependent manner a significant male-type organizational effect on gonadotrophin secretion resulting in an anovulatory syndrome with significantly decreased ovarian weights due to failure of corpus luteum formation as well as on male-type sexual orientation. The results suggest that different sex hormones (oestrogens and/or androgens) are responsible for the sex-specific brain differentiation of gonadotrophin secretion, sexual orientation and gender role behaviour.

Differential effects of chronic testosterone treatment on the onset of puberty in male rats.

Immature male rats were daily injected with 5 or 10 micrograms testosterone propionate (TP)/100 g b.w. from day 5 after birth through day 30 or through the day at which spermatozoa appeared in preputial smears (SpA). The age and body weight at preputial separation (PS) and SpA as well as the testes weight on the day of SpA were recorded. Reduced testicular weights as compared to the controls injected with oil were found in all experimental groups. Whereas daily injections of 5 micrograms TP/100 g b.w. did not alter the age and body weight at PS and SpA, administration of the higher TP dose from day 5 through day 30 resulted in highly significant delay of PS and SpA. This effect was almost completely abolished if treatment was continued to the day of SpA. In otherwise untreated males, however, injections of 10 micrograms TP/100 g b.w. from day 30 through the day of SpA did not advance the spontaneous onset of puberty. The findings suggest that slight elevation of the circulating testosterone level during infancy causes impairment of testicular functions resulting in retardation of spermatogenesis and, presumedly, long-lasting reduction of testosterone secretion. The effect can be abolished by substitution of testosterone during the late prepubertal phase, but spontaneous sexual maturation can probably not be accelerated in this way.

Influence of the medial preoptic dopaminergic activity on the efficiency of the negative estrogen feedback in prepubertal and cyclic female rats.

Immature and adult female rats were bilaterally implanted in the medial preoptic area (MPOA) or hypothalamic ventromedial-arcuate region (VMAR) with the dopamine (DA) antagonist, pimozide, or the DA agonist, apomorphine, and the sensitivity to the LH-inhibiting effect of a subcutaneous injection of estradiol benzoate (EB), the onset of puberty and ovarian cyclicity were investigated. Diminution of the inhibitory effect of EB on LH secretion was recorded in ovariectomized immature and adult females implanted in the MPOA with pimozide. This response was not obtained in rats implanted in the VMAR. In contrast, medial preoptic, but not intrahypothalamic, implantation of agar pellets containing apomorphine resulted in enhanced sensitivity to estrogen both prepubertally and during the ovarian cycle. The sensitizing effect of apomorphine was completely prevented in prepubertal rats by pretreatment with EB for 6 days. Bilateral implantation of pimozide-agar pellets in the MPOA of 28-day-old intact females induced significant advancement of the onset of puberty, whereas sexual maturation was not affected by daily subcutaneous injections of the DA antagonist from day 28 till the day of vaginal opening. In adult 4-day-cyclic rats fitted with guide cannulae, the forthcoming ovulation was delayed for about 7 days as compared to the controls implanted with agar if apomorphine was placed in the MPOA from the morning of metestrus to the morning of diestrus. Similar implants located in the VMAR were ineffective in this regard. The results suggest that: (1) low DA activity in the MPOA reduces and high activity enhances the sensitivity of the negative estrogen feedback in immature and adult female rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Retardation of preovulatory desensitization to negative oestrogen feedback: mechanism of the effect of progesterone in 5-day cyclic rats.

Recent studies have shown that oestrogen can induce desensitization to its own gonadotrophin-inhibiting effect in female rats by an action on the medial preoptic area (MPOA). Probably as a consequence of this action, sensitivity to the negative oestrogen feedback declines markedly between metoestrus and dioestrus of the 4-day ovarian cycle. To study this desensitization process in 5-day cyclic rats, females exhibiting regular 5-day vaginal cyclicity were ovariectomized on consecutive days of the cycle, injected with oestradiol benzoate (OB) or oil on the day of ovariectomy and autopsied 24 h after the injection. Estimation of the serum concentration of LH revealed that desensitization to negative oestrogen feedback occurred only between day 2 of dioestrus and pro-oestrus, i.e. 2 days later than in females with a 4-day cycle. In the latter animals, an injection of progesterone in metoestrus or early dioestrus, which induced lengthening of the ovarian cycle for 1 day, delayed the onset of desensitization to a degree similar to that found in spontaneously 5-day cyclic rats. In acutely ovariectomized females, progesterone implants placed in the MPOA, but not those located in the mediobasal hypothalamus, increased the LH-inhibiting effect of low doses of OB. The results suggest that the prolonged secretion of progesterone recorded in 5-day cyclic rats retards follicle maturation and delays the forthcoming ovulation by acting, at least partly, on the MPOA and antagonizing the desensitizing effect of oestrogen. In this way, inhibition of gonadotrophin secretion by oestrogen is enhanced and the increase in tonic LH secretion necessary for the completion of follicle maturation is retarded.

Sexual differentiation of gonadotrophin secretion, sexual orientation and gender role behavior.

The positive estrogen feedback was found to be a relatively sex-specific reaction of the hypothalamo-hypophyseal system in rats as well as in human beings. It is dependent--most of all--on the estrogen convertible androgen level during sexual brain differentiation, but also on an estrogen priming effect in adulthood. The lower the estrogen convertible androgen or primary estrogen level during brain differentiation, the higher is the evocability of a positive estrogen action on LH secretion in later life. In clinical studies, we were able to induce a positive estrogen feedback on LH secretion in most intact homosexual men in clear-cut contrast to intact hetero- or bisexual men. These findings were strongly confirmed by Gladue and associates. In addition, the evocability of a positive estrogen feedback was also demonstrable in most homosexual male-to-female transsexuals in significant contrast to hetero- or bisexual male-to-female transsexuals. These findings suggest that homosexual males possess, at least in part, a predominantly female-differentiated brain, which may be caused by a low estrogen convertible androgen level during brain organization. Recently, the following relations were found between sex hormone levels during brain differentiation and sex-specific responses in adulthood: (1) estrogens--which are mostly converted, however, from androgens--are responsible for the sex-specific organization of gonadotrophin secretion and hence the evocability of a positive estrogen feedback in later life; (2) estrogens and androgens, occurring during brain differentiation, predetermine synergistically sexual orientation and (3) androgens--without conversion to estrogens--are responsible for the sex-specific organization of gender role behaviour in later life. Furthermore, the organization periods for sex-specific gonadotrophin secretion, sexual orientation and gender role behaviour are not identical but overlapping. Thus, combinations as well as dissociations between deviations of the neuroendocrine organization of sex-specific gonadotrophin secretion, sexual orientation and gender role behaviour are conceivable. Most recently, female-type sexual orientation could be converted to male-type sexual orientation in adult rats by administration of the dopamine agonist and serotonin antagonist lisuride.

[Experimental studies of the significance and mechanism of desensitization to the gonadotropin-inhibiting effect of estrogen. 2. Detection and mechanisms of preovulatory desensitization in the ovarian cycle]. / Experimentelle Untersuchungen zur Bedeutung und zum Mechanismus der Desensibilisierung gegenüber der gonadotropinhemmenden Estrogenwirkung. 2. Mitteilung: Nachweis und Mechanismus einer präovulatorischen Desensibilisierung im Ovarialzyklus.

It has recently been demonstrated in women and several mammalian species that the basal LH secretion increases prior to the ovulation-inducing LH surge in spite of a simultaneous rise of the estrogen level in the blood. The temporary relative inefficiency of estrogen in its negative feedback action may be necessary to make adequate gonadotrophic support of final preovulatory follicle maturation possible. To study the mechanisms underlying this phenomenon, the gonadotrophic response to a single injection of estradiol benzoate (EB) was evaluated in acutely ovariectomized adult rats during the different stages of a 4-day ovarian cycle. The results showed that the sensitivity to the gonadotrophin-inhibiting effect of EB is high during late estrus and early metestrus. Between metestrus and diestrus it suddenly declines, and EB did not inhibit the hypophysial gonadotrophin secretion from diestrus through the morning of the subsequent estrus. The cyclic variation of the sensitivity to the negative estrogen feedback is probably not based upon an endogenous rhythm that is independent of the ovarian hormone secretion, because similar variability of the gonadotrophic response to estrogen was not found in rats that had been castrated three days before the injection of EB. A further experiment demonstrated that bilateral implants of EB placed in the medial preoptic area (MPOA) of ovariectomized rats significantly reduced the gonadotrophin-inhibiting effect of s. c. injected estradiol, whereas similar implants located in the mediobasal hypothalamus were completely ineffective in this regard. Since bilateral lesioning of the MPOA in long-term ovariectomized females also lowered the sensitivity to estrogen, the conclusion may be drawn that preovulatory desensitization to the negative estrogen feedback is probably induced in cyclic female rats by an inhibitory effect on medial preoptic neurones of the increase of circulating estrogen recorded in metestrus. In accordance with this assumption, imitation of the periovulatory diminution of estrogen action on the MPOA of intact rats by the removal of medial preoptic EB implants in castrated females during the afternoon of proestrus, resulted in high sensitivity to estrogen during estrus and metestrus. The possible clinical significance of the hitherto not described preovulatory desensitization is briefly discussed.

Postovulatory sensitization to the negative oestrogen feedback in female rats is probably induced by the preceding decline of the oestrogen concentration in the medial preoptic area.

To examine the question if an endogenous oestrogen-independent rhythm is involved in the cyclic variation of sensitivity to the negative feedback of oestrogen recorded in a former study, adult female rats were ovariectomized on subsequent days of a 4-day ovarian cycle, injected with 3 micrograms oestradiol benzoate (OB)/100 g b.w. or oil three days after castration, and autopsied on the following day. Estimation of the serum LH concentration revealed a similar LH-inhibiting effect of OB in all experimental groups. Female rats were then implanted with OB or cholesterol in the medial preoptic area (MPOA) in metoestrus. In part of the rats, the implants were removed on the presumptive day of pro-oestrus to imitate the periovulatory decline of the circulating oestrogen level acting on the MPOA. Evaluation of the sensitivity to the negative oestrogen feedback during oestrus and metoestrus demonstrated that s.c. injected OB was highly effective in suppressing the LH secretion after removal of the OB implants in pro-oestrus, but not in rats with the implants left in place till autopsy. In a final experiment, the pro-oestrous progesterone surge was inactivated by the injection of specific antibodies. An influence of this treatment on the LH-inhibiting effect of OB examined during oestrus and metoestrus could not be found. Taken together the results suggest that the high sensitivity to the negative oestrogen feedback recorded during the postovulatory period in cyclic female rats is mainly induced by the periovulatory fall of the circulating oestrogen level leading to reduction of the medial preoptic oestrogen concentration.

Prevention of lesioning-induced precocious puberty in female rats by neonatal implantation of fetal preoptic tissue.

Damage to the medial preoptic area (MPOA) was produced in one-day-old female rats by bilateral electrolytic lesions or by aspiration of preoptic tissue. Both procedures resulted in similar advancement of vaginal opening and the first ovulation. A further group of rats was lesioned or aspirated in the MPOA and immediately bilaterally implanted with medial preoptic tissue collected by the puncture method from 17-19-day-old female rat fetuses. After autopsy, tissue destruction and implants located in the MPOA could be identified in 5 females that showed significantly later onset of puberty than both the lesioned or aspirated and the untreated controls. The findings suggest that the elimination of neurones and not a stimulatory effect of electrolytic lesions on GnRH secretion is responsible for the acceleration of sexual maturation recorded after lesioning of the MPOA. Possible reasons for the failure to identify the grafts in most of the implanted females are discussed.

Short-term implantation of oestrogen into the anterior amygdaloid area of immature rats delays the first pubertal ovulation.

Unilateral implants containing oestradiol benzoate (OB) and cholesterol at the ratio of 1 + 4 were placed for 48 h either in the region of the anterior amygdaloid area (AAA) and nucleus of the lateral olfactory tract (NLOT) or in the anterior or posterior part of the mediocortical amygdala of immature 26-day-old female rats, and the age at vaginal opening and the first ovulation was recorded. Whereas implants located in the mediocortical amygdala did not influence the occurrence of ovulation or advanced it distinctively, implantation of OB into the AAA/NLOT resulted in a highly significant delay of the first pubertal ovulation that was associated with postpubertal persistent vaginal cornification in part of the rats.

Varying sensitivity to the negative oestrogen feedback during the ovarian cycle of female rats: evidence for the involvement of oestrogen and the medial preoptic area.

The gonadotrophic response to a single injection of oestradiol benzoate (OB) was studied in acutely ovariectomized adult rats during the different stages of a 4-day ovarian cycle. The results showed a sudden decline of the sensitivity to the gonadotrophin-inhibiting effect of OB between metoestrus and dioestrus. This desensitization to the negative oestrogen feedback was probably caused by an oestrogen action on the medial preoptic area (MPOA). In rats ovariectomized and implanted with OB in the MPOA in metoestrus, an s.c. injection of OB on the presumptive day of pro-oestrus did not lower the circulating LH and FSH levels, whereas a clear suppression of gonadotrophin secretion was seen in females implanted with cholesterol in the MPOA or implanted with OB in the hypothalamic ventromedial-arcuate region. Similar findings were obtained in rats which had been ovariectomized 3-4 weeks before implantation. A final experiment demonstrated that bilateral lesioning of the MPOA also reduced the sensitivity to the negative feedback action of oestrogen in long-term ovariectomized rats. In all experiments performed, diminution of the oestrogen-induced inhibition of LH secretion was more marked than that of suppression of FSH secretion. It is proposed that desensitization to the negative oestrogen feedback, probably resulting from an inhibitory oestrogen action on medial preoptic neurones, is a prerequisite for adequate gonadotrophic support of preovulatory follicle maturation in the presence of a continuously rising oestrogen concentration in the blood.