Catecholamine mechanisms in the feedback effects of estradiol benzoate on the release of LH and prolactin

The role of hypothalamic catecholamines and luteinizing hormone releasing hormone (LHRH) in the negative feedback effect of estradiol benzoate (EB) on luteinizing hormone (LH) release was studied in chronic ovariectomized rats. Administration of 10 micrograms EB decreased plasma LH levels and increased LHRH content in the medial basal hypothalamus (MBH) 1 day after injection. Inhibition of dopamine and norepinephrine synthesis with alpha-methyl-p-tyrosine (alpha-MT) reduced the LHRH content in the MBH in both oil- and EB-treated animals and partially reversed the decrease in plasma LH levels. Inhibition of norepinephrine synthesis with fusaric acid decreased LHRH content in both oil- and EB-treated rats but had no effect on plasma LH levels. The results suggest that at least a portion of the inhibitory effect of EB on LH release is due to the stimulation of an inhibitory dopaminergic mechanism which reduces LHRH release from the MBH. This feedback mechanism is apparently not susceptible to dopaminergic receptor blockade since administration of pimozide had no effect on LH levels. The stimulatory feedback effect of EB on prolactin release was studied in the same animals. alpha-MT and EB produced additive effects on plasma prolactin levels whereas fusaric acid blocked the EB-induced increase in plasma prolactin levels. Pimozide appeared to potentiate the effect of EB on prolactin release. The results reconfirm the possible role of noradrenergic neurons in the release of prolactin induced by EB and also suggest that EB stimulates a dopaminergic mechanism which is inhibitory to prolactin release but is normally masked by increased noradrenergic activity.     

Progesterone administration prolongs the inhibitory effects of hyperprolactinemia on luteinizing hormone secretion in acutely ovariectomized rats

Several studies have shown that hyperprolactinemia in rats inhibits the post-gonadectomy rise in plasma luteinizing hormone (LH) for a limited period only. In intact rats the suppression of plasma LH during hyperprolactinemia is more prolonged. In the present study we have examined the possibility that the elevated levels of progesterone brought about by the raised plasma prolactin levels in intact rats are involved in the maintenance of LH inhibition. We have observed the effect of exogenous progesterone administration during the early post-ovariectomy period on plasma LH levels in female rats made hyperprolactinemic by administration of the dopamine antagonist, domperidone. Following ovariectomy of virgin, female rats, plasma LH was determined on each day from Day 3 to Day 10 after ovariectomy. In control rats plasma LH had increased by approximately 5-fold during the period of the experiment. In control rats treated with progesterone the rise in plasma LH was inhibited temporarily but LH had increased to similar levels to the controls by Day 10. In hyperprolactinemic rats LH was suppressed until Day 7, after which significant rises were observed. However, in hyperprolactinemic rats treated with progesterone, LH did not rise in a similar fashion, and remained low throughout the experiment. We conclude that a combination of hyperprolactinemia and raised plasma progesterone concentrations is necessary for the continued inhibition of LH release after ovariectomy.     

Plasma gonadotropins and prolactin in pseudopregnancy in the rat

Radioimmunoassay presented a method of measuring plasma levels of FSH,LH and prolactin in pseudopregnant rats. Plasma prolactin levels doubled 15 minutes following cervical probing (p .01) on the day of estrus. Plasma LH levels were not significantly elevated. Due to the use of ether anesthesia at blood collecting 3 hr before and 15 minutes after stimulation, only 1 of 16 rats developed pseudopregnancy. On Day 4 of pseudopregnancy in rats mated with vasectomized males; plasma LH was lower (p .05) than in normal rats on the first day of diestrus, perhaps due to the suppressive action of ovarian progesterone and some estrogen. FSH was higher than in normal rats (p .05) perhaps due to the lesser sensitivity of FSH to the inhibitory effect of progesterone. Large decidoumata developed by Day 9 in uterine horns traumatized on Day 4 (153 plus or minus 8 mg uterus weight compared to 1510 plus or minus 204 mg). Thus, the corpora remain functional after LH and prolactin are at normal and subnormal levels. On Day 9 plasma prolactin was lower than at Day 1 of diestrus (p .001). Plasma FSH was elevated (p .01). Plasma LH was unchanged. The degree of rise of LH levels 5 days following ovariectomy on Day 4 of psuedopregnancy or on the first day of diestrus was greater in the former group (p .02), perhaps due to rebound of LH from suppression by ovarian steroids. FSH rose equally in both groups. Prolactin remain about the same. Increased prolactin release by the adenohypophysis was briefer than expected.     

Differential responsiveness of LH and prolactin to p-tyramine in male and female rats

The effect of p-tyramine, a natural amine which is found in the rat brain in trace amounts, was evaluated for its capacity to influence LH and prolactin secretion in male and female rats under different hormonal conditions. p-Tyramine (40 mg/kg ip) was ineffective in modifying LH levels in either female or male rats which had been gonadectomized for 2 days, but if the animals were injected with 12.5 micrograms of estradiol benzoate (EB) on the day of castration, p-tyramine was able to release LH in female but not in male rats. To evaluate whether early androgenization of brain structures which control LH secretion was involved in the sexual difference observed, p-tyramine was tested in female androgenized rats (200 micrograms of testosterone propionate on the day of birth), and in male rats castrated at birth. The trace amine was ineffective in altering LH levels in both experimental models, even if rats were pretreated with EB as control females. On the other hand, p-tyramine inhibited prolactin secretion in male rats pretreated with EB, and not in similarly treated female rats. The present results suggest that p-tyramine may be involved not only in prolactin regulation as it has been previously shown, but also in LH control, and that the hormonal response to this amine is sexually differentiated in the rat.     

LH and prolactin secretion under constant light in estrogen treated rats (author's transl)

Plasma LH and prolactin levels were studied in ovariectomized adult female rats submitted to light-darkness (L:D) or constant light (L:L) schedules, after the administration of estradiol benzoate (EB), 75 micrograms/day for six consecutive days. Previous to the treatment with EB LH levels were lower and prolactin levels higher in the L:L females. On days 1 and 2 after treatment, L:D females showed circadian variations of LH levels, these being higher at 17 h than at 10 h. This pattern disappeared in the L:D females. Prolactin levels increased similarly in both groups. Nine days after treatment, plasma prolactin levels remained high and the circadian pattern of LH in the L:D group disappeared.     

Chronic estradiol exposure induces oxidative stress in the hypothalamus to decrease hypothalamic dopamine and cause hyperprolactinemia

Estrogens are known to cause hyperprolactinemia, most probably by acting on the tuberoinfundibular dopaminergic (TIDA) system of the hypothalamus. Dopamine (DA) produced by TIDA neurons directly inhibits prolactin secretion and, therefore, to stimulate prolactin secretion, estrogens inhibit TIDA neurons to decrease DA production. However, the mechanism by which estrogen produces this effect is not clear. In the present study, we used a paradigm involving chronic exposure to low levels of estradiol-17β (E(2)) to mimic prolonged exposures to environmental and endogenous estrogens. We hypothesized that chronic exposure to low levels of E(2) induces oxidative stress in the arcuate nucleus (AN) of the hypothalamus that contains TIDA neurons and causes nitration of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA. This results in a significant decrease in DA and consequently, hyperprolactinemia. To investigate this, adult, intact female cycling rats were implanted with slow-release E(2) pellets (20 ng/day) for 30, 60, or 90 days and were compared with old (16-18 mo old) constant estrous (OCE) rats. Chronic E(2) exposure significantly increased the expression of glial fibrillary acidic protein and the concentrations of interleukin-1β (IL-1β) and nitrate in the AN that contains perikarya of TIDA neurons and increased nitration of TH in the median eminence (ME) that contains the terminals. These levels were comparable to those seen in OCE rats. We observed a significant decrease in DA concentrations in the ME and hyperprolactinemia in an exposure-dependent manner similar to that seen in OCE rats. It was concluded that chronic exposure to low levels of E(2) evokes oxidative stress in the AN to inhibit TIDA neuronal function, most probably leading to hyperprolactinemia.     

Gonadotropin secretion during prolonged hyperprolactinemia: basal secretion and the stimulatory feedback effect of estrogen

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.     

Effect of CRF in the median eminence on gonadotropin levels in conscious female rats

To evaluate whether the median eminence (ME) is the site of action of CRF (corticotropin releasing factor) in inhibiting LH levels in female rats, we have injected CRF (1 nmol) directly into the ME and then measured plasma LH and FSH concentrations in conscious ovariectomized (OVX) rats in the absence or presence of a single dose of estradiol benzoate (EB). CRF caused a significant decrease in plasma LH levels in both OVX and OVX + EB rats at 30 min postinjection, in comparison to the values obtained in animals injected with water only. Injection into the ME of water had no effect on plasma LH levels in either OVX or OVX + EB animals. The results suggest that CRF probably inhibits LH secretion, at least in part by a central action on GnRH release in ME.     

Neonatal dihydrotestosterone and estrogen stimulation: Effects on sexual behavior of male rats

Male rats castrated on the second day after birth (Day 2) were, for the next 10 days, given daily injections of one of five steroids or steroid combinations: 200 μg of testosterone propionate (TP); 200 μg of dihydrotestosterone propionate (DHTP); 5 μg of estradiol benzoate (EB); 5 μg of estradiol benzoate plus 200 μg of dihydrotestosterone propionate; oil vehicle (VH). Control male rats castrated on Day 90 received a sham castration and oil vehicle in the neonatal period. All animals were given TP in adulthood and tested for male sexual behavior. There was no difference in mounting activity among the subjects. Day 2 DHTP subjects displayed intromissions but were incapable of ejaculating. The more frequent display of intromissions by Day 2 DHTP animals in comparison to Day 2 VH animals could be solely due to their larger and more highly developed penes. On the other hand, the ejaculatory failure of the Day 2 DHTP subjects was attributed to some deficiency in central neural processes controlling ejaculatory mechanisms rather than inadequate penile development. Equivocal results were obtained with the Day 2 EB and Day 2 EB-DHTP animals in that only a few animals in both groups showed an ejaculatory pattern.     

Neuroendocrine control of luteinizing hormone secretion and reproductive function in spontaneously persistent-estrous aging rats

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)     

Effects of third ventricular injection of beta-endorphin on luteinizing hormone surges in female rat: sites and mechanisms of opioid actions in the brain

The effects of third ventricular injection of beta-endorphin (beta-EP) on spontaneous, brain stimulation-induced and estrogen-induced LH surges were studied in the adult female rat. It was found that beta-EP blocked the preovulatory surge of LH release and ovulation, while it did not affect LH release in response to LH-RH injection. The site of the beta-EP blockade of ovulation was proved to be in the brain. Beta-EP completely blocked ovulatory LH release induced by the electrochemical stimulation of the medial amygdaloid nucleus and medial septum-diagonal band of Broca, but failed to block ovulation due to the stimulation of the medial preoptic area (MPO) or median eminence, though serum LH levels after the MPO stimulation were inhibited by beta-EP. In the spayed rats treated with estradiol benzoate (EB) on Day 1 and 4 of experiment, beta-EP given on Day 5 blocked the LH surge that normally occurred on that day and led to a compensatory surge of LH on the following day. Moreover, the LH surge on Day 5 was inhibited by beta-EP given either on Day 1 or Day 4. Present data suggest that beta-EP may act in inhibiting the preovulatory LH surges not only by suppressing the preoptic-tuberal LH-RH activities but also by affecting the initiation and development of stimulatory feedback of estrogen in the central nervous system.     

Chronic hyperprolactinemia depletes hypothalamic dopamine concentrations in male rats

The tuberoinfundibular dopamine (TIDA) system appears to tonically inhibit pituitary prolactin secretion while moderate elevations in serum prolactin levels, in turn, augment the turnover rate of dopamine (DA) without affecting the steady state concentrations of DA in the TIDA neurons (1–5). The present study demonstrates that chronic elevations in serum prolactin, to greater than 2,000 ng/ml, induced by the prolactin secreting MtTW15 tumor, decreased DA concentrations by 47% in the median eminence-arcuate nucleus (ME-ARC) region, by 43% in the medial basal hypothalamus (MBH) and 14% in the preoptic area-anterior hypothalamic region (POA-AH) without influencing the norepinephrine levels in these regions. Thus, chronic stimulation of hypothalamic DA neurons by prolactin may lead to depletion of DA concentrations and this may be an important factor in the reduced DA levels observed in hyperprolactinemia of senescent rats or that produced by chronic estrogen treatment.     

Gonadotropin release as a function of mating and steroid feedback in the female rat

The aim of this investigation was to study possible relationships between mating-induced and steroid-induced luteinizing hormone (LH) release in spayed Long-Evans rats. Large amounts of LH were released approximately 7 hr following progesterone injection in rats primed with estradiol benzoate (EB). The amount of LH release varied widely depending on (1) the interval between the time of the progesterone injection and the EB priming; (2) the progesterone dose; and (3) the time of day when blood samples were collected. These findings provided confirmation of those of Caligaris, Astrada and Taleisnik (1971a). Females, prepared with estrogen-progesterone treatment in a variety of schedules in which the three above-mentioned variables were altered systematically, were allowed to mate with vigorous males. Mating under these various conditions did not significantly increase plasma LH levels even when the females showed high degrees of sexual receptivity. Sodium pentobarbital prevented the afternoon LH rise resulting from progesterone treatment 3 days after EB priming. Pituitary sensitivity to LRF was not enhanced in the afternoon and the mating did not significantly increase plasma LH in these barbiturate-blocked rats. Following administration of 5 large daily doses of EB without progesterone, however, significant increases in LH were produced by mating on the sixth day. Postcopulatory LH release in these circumstances was dependent on a diurnal factor since the effect of mating was greater in the afternoon than in the morning. Thus, although major LH release can be readily induced by mating in estrogen-treated spayed rats, this effect could not be obtained under conditions of progesterone administration to estrogen primed rats.     

Effects of the intraventricular administration of 5,7-dihydroxytryptamine on FSH, LH and prolactin secretion in neonatally estrogenized male rats

The role of the serotoninergic system in the control of LH, FSH and prolactin secretion was analyzed in control and neonatally estrogenized male rats. Animals injected s.c. with 500 micrograms of estradiol benzoate (EB) on day 1 of life, or their corresponding sham-treated controls, were divided on day 75 into the following groups: (1) orchidectomized; (2) injected intraventricularly with 5,7-dihydroxytryptamine (5,7-DHT); (3) orchidectomized and treated with 5,7-DHT, and (4) sham operated. 15 days later, the animals were decapitated and their FHS, LH and prolactin plasma values measured by specific RIA systems. After the treatment with 5,7-DHT, control animals showed a decline in basal prolactin levels but no modification in basal LH and FSH values. After castration, 5,7-DHT-treated animals showed a reduced LH increase and a more marked prolactin decrease. In neonatal estrogen-treated animals, the 5,7-DHT injection did not change FSH, LH or prolactin levels but did partially or completely abolish the post-castration rise in FSH and LH levels, respectively. These data seem to indicate that neonatal estrogenization induced a modification of the serotoninergic role in the control of LH, FSH and prolactin.     

Effects of estradiol on circulating levels of prolactin in female rats bearing ectopic pituitaries

The existence of local mechanisms controlling the prolactin (PRL) release from anterior pituitaries (AP) grafted to an ectopic location has been recently described. To study if these mechanisms are affected by estrogens, pituitary-grafted (GRAFT) and sham-operated (SHAM) rats were injected with a single dose of estradiol benzoate (EB), their plasma PRL levels as well as their hypothalamic and AP contents of norepinephrine (NE) and dopamine (DA) being analyzed. Administration of EB to GRAFT animals produced a small increase in their previously high plasma PRL levels, with both an increased NE and a decreased DA content in the ectopic AP. Since NE enhances the PRL release from ectopic AP and DA partially inhibits this secretion these changes may explain such a small increase in PRL levels. However, an additional increase in the decreased PRL release from the in situ AP of these animals cannot be discarded since EB produced also a decrease of the DA content in this tissue with an unaltered hypothalamic content. Finally, administration of this steroid to SHAM animals produced an important increase in plasma PRL levels. Since this increase was correlative to a decrease in DA and NE hypothalamic contents and unaltered AP contents. EB may be supposed to be able to reduce the DA synthesis in the tuberoinfundibular neurons, while the changes in noradrenergic inputs could be more related to the feedback effects of estrogens on the gonadotrophin release.     

Hypothalamic LHRH and plasma LH levels after electrical stimulation of the deafferented medial basal hypothalamus

The medial basal hypothalamus (MBH) of female rats was surgically isolated on the morning of proestrus and luteinizing hormone (LH) levels in the blood were determined before and after electrochemical stimulation (ECS) of the disconnected arcuate-median eminence (ARC-ME) region either on the same afternoon (Group 1) or on the 5th postoperative day (Group 2). Animals of Groups 3 and 4 were stimulated and sampled for LH on the 5th or 10th postoperative day, respectively, these rats having been primed with 5 micrograms estradiol injected daily throughout the experimental period. ECS resulted in a significant rise of plasma LH level in Group 1 and caused full ovulation as evaluated by the presence of ova found the next morning in the Fallopian tubes. Rats of Groups 2-4 failed to show any changes in plasma LH, and no ovulation was observed following ECS. Immunohistochemical examination of the brains revealed that axons staining for the luteinizing hormone-releasing hormone (LHRH) in the ARC-ME region remained at control levels 3 days after deafferentation (e.g. Group 1), whereas a marked decrease or complete absence of these structures was observed 8 or 13 days following surgical isolation of the MBH (e.g. Groups 2-4). These studies strongly support the view that no LHRH synthesizing perikarya are located within the MBH of the rat.     

Serum levels of prolactin, LH and LH-RH after ablation of the medial basal hypothalamus.

4 weeks after surgical ablation (MHA) of the medial basal hypothalamus (MBH) in adult male rats, serum levels of prolactin, LH, and LH-RH were determined by radioimmunoassay. Blood samples were obtained by jugular venipuncture under ether anesthesia (designated 'stress' samples) and 30 min after the ether stress by rapid decapitation (designated 'resting samples). 30 min after ether, serum levels of prolactin, LH, and LH-RH in MHA rats were comparable to those of intact and sham-operated controls. Among intact and sham-operated rats, ether elicited an initial increased in prolactin but not in LH or LH-RH. In the MHA group, prolactin levels were also acutely increased, although the increment was not as great as in control groups. The data indicate that considerable basal prolactin and LH secretion persists after MHA, and that this continued secretion may be regulated by neurohoromones such as LH-RH which arise from areas outside the MBH.     

Depletion of tuberoinfundibular dopamine does not restore gonadotropin secretion in ovariectomized hyperprolactinemic rats

The effect of depleting tuberoinfundibular dopamine (TIDA) on gonadotropin secretion was studied in rats made hyperprolactinemic by implantation of the prolactin and growth hormone secreting Furth MtTW15 tumor. Implants of the pituitary tumor prevented gonadotropin release in response to castration. Daily injection of 100 mg dl-alpha-methyl-para-tyrosine/kg bw which reduced TIDA levels in tumor bearing rats more than 90% did not restore gonadotropin release. It seems likely that the increased activity of the TIDA system does not mediate the suppression of gonadotropins during chronic hyperprolactinemia.     

An increase of pituitary 3', 5' cyclic adenosine monophosphate produced by estradiol benzoate in vitro: possible implication of this increase in the secretion of luteinizing hormone.

In an attempt to study the site and mechanism of action of estrogen in producing positive feedback control, porcine anterior pituitary slices were incubated in vitro in the presence of estradiol benzoate (EB). EB elevated pituitary cyclic AMP concentration within 5 min and augmented pituitary release of luteinizing hormone (LH). The magnitude of increase of cyclic AMP and LH release was related to the doses of EB used. Also, luteinizing hormone releasing hormone (LH-RH) elevated pituitary cyclic AMP concentration and stimulated pituitary release of LH. The magnitude of increase of cyclic AMP and LH release was inversely related to the doses of LH-RH used. EB and LH-RH were additive in increasing cyclic AMP. Progesterone and clomiphene citrate interfered with an increase of pituitary cyclic AMP produced by EB, but did not significantly affect the basal level of pituitary cyclic AMP. Testosterone propionate, human chorionic gonadotropin and hexestrol were without effect on either basal or stimulated level of pituitary cyclic AMP. Since cyclic AMP and dibutyryl cyclic AMP (DBC) stimulated LH release, it is suggested that EB directly stimulates the release of LH by augmenting cyclic AMP synthesis in the anterior pituitary.     

Hormonal induction of incubation behavior in ovariectomized female turkeys (Meleagris gallopavo)

Two experiments were conducted to evaluate the effect of estradiol benzoate (EB), progesterone (P), ovine prolactin (oPrl), or their combinations on temporal patterns of serum luteinizing hormone (LH) and Prl and on nesting behavior in adult ovariectomized female turkeys. Levels of serum LH were initially reduced (p greater than 0.05) by the steroid treatments, while continuation of treatments induced surges of LH to levels comparable to pretreatment levels. Administration of steroid increased (p less than 0.05) levels of serum Prl, which persisted until termination of treatments. Administration of oPrl had no effect on levels of serum LH but blunted the steroid-induced release of Prl. Neither EB, P, nor oPrl treatments alone nor a combination of EB + P elicited nest occupation. Nest occupation was observed after administration of P only in turkeys pretreated with EB. Administration of oPrl maintained and advanced the P-induced nesting to persistent nesting behavior (incubation behavior). Once persistent nesting behavior was established, hormonal treatments were terminated, yet nesting behavior was maintained and serum samples showed increasing levels of Prl and decreasing levels of LH. It is suggested that incubation behavior in the female turkey is facilitated by the combined action of estradiol, P, and Prl.