LH Release In Response Research Articles

Effect of intraventricular infusion of catecholamines on luteinizing hormone release in ovariectomized and ovariectomized, steroid-primed rats.

This study examined alterations in episodic LH release in response to prolonged, slow infusions of dopamine (DA), norepinephrine (NE), or epinephrine (EPIN) into the thire ventricle in adult, ovariectomized (OVX) rats, and the influence of priming with ovarian steroids on the LH response to the catecholamines. Unanesthetized rats with right atrial cannulae were bled continuously at slow rates for 1--1 1/2 h prior to infusion, 1--1 1/2 h during infusion, and up to 1 h afterwards. The amines were protected from oxidation by ascorbic acid, and infused in an artificial cerebrospinal fluid (CSF) vehicle (pH 7.29--7.33) into the third ventricle at a rate of 25--27 microliter/h. Blood samples were analyzed for LH by radioimmunoassay. In unprimed, OVX rats, infusions of artificial CSF, as well as low doses of DA (2--4 micrograms/h) or NE (0.3--0.6 micrograms/h), had no effect on episodic LH release or mean blood LH levels. However, administration of 8.5 and 17 micrograms DA/h, and 5.5 and 11 micrograms NE/h, resulted in a decrease in blood LH levels and, in most animals, prolonged intervals between peak blood LH levels during infusion or inhibitions which began rapidly and lasted for nearly the entire infusion period or longer. In contrast, infusion of 5.7 and 11.5 micrograms EPIN/h had no effect on blood LH levels in uprimed rats. In OVX rats primed 3 days prior to infusion with 50 micrograms estradiol benzoate and 25 mg progesterone (OEP), administration of CSF or the same doses of DA that previously inhibited episodic LH release had no effect on LH secretion. However, these steroids completely reversed the LH response to 11 micrograms NE/h, with increases in LH relase occurring during infusion. EPIN, in doses ineffective in unprimed rats (5.7 and 11.5 micrograms/h), also caused elevations in blood LH levels in EOP rats. Finally, in rats primed with 5 micrograms estradiol benzoate/100 g b.w./day for the 2 days prior to infusion, none of the three neurotransmitters had any effect on LH release.

Role of Ovarian Secretion in the Development of Dysfunction in the Regulation of Luteinizing Hormone Secretion in Female Rats Exposed to Constant Illumination

Female rats in constant illumination (LL) fail to show the facilitation of LH release following steroid administration that is characteristic of animals in normal lighting. To determine whether this effect is mediated through changes in ovarian function, rats were spayed either at the time of placement into different lighting schedules (LL or a 14:10 light-dark (LD) schedule) or 10 weeks later, and their plasma LH responses to steroids were compared after an additional 3-week exposure to the experimental lighting conditions. To test the LH response, estradiol benzoate (EB) was injected at 12.00 h and followed 72 h later by injection of progesterone (P) or a second injection of EB. Neither steroid regime revealed differences in LH release between animals ovariectomized at the time of placement into LL and those spayed 10 weeks later. The duration of castration in animals in LD affected the LH response to a priming dose of EB, but not to a second dose of EB or to P. It is concluded that altered ovarian activity is not the factor which mediates the loss of a facilitatory response of LH release following administration of gonadal steroids to rats under constant illumination.

REFRACTORINESS OF THE PITUITARY GLAND AFTER CONTINUOUS EXPOSURE TO LUTEINIZING HORMONE RELEASING HORMONE

The refractoriness of LH release by pituitary glands from intact female rats was studied during stimulation by luteinizing hormone releasing hormone (LH-RH), monobutyryl cyclic AMP+theophylline or potassium in vitro. Various concentrations of LH-RH (0.1, 0.3 and 10 ng/ml) all caused refractoriness within 24 h. Subsequent exposure to a supramaximally active concentration of LH-RH for 6 h also resulted in a depressed response; the degree of inhibition depended on the concentration of LH-RH to which the glands had been exposed previously. Glands made refractory to LH-RH also showed a depressed response to monobutyryl cyclic AMP+theophylline, although these agents by themselves were unable to induce refractoriness. Incubation in medium containing a high concentration of potassium also resulted in the release of LH, which in all respects was similar to that caused by LH-RH. Glands made refractory to LH-RH showed a decreased response to potassium and, conversely, the release of LH in response to LH-RH was reduced after exposure of glands to potassium. It is concluded that the LH releasing activity of LH-RH, which is mimicked by potassium, deteriorates during continuous exposure to the secretagogue.

Hypothalamic Control of the Testis in the Ram

Adult Soay rams were maintained under an artificial lighting schedule which induced a seasonal cycle in testicular activity every 8 months. At 3 stages of the testis cycle (regressed, developing and active) the episodic nature of LH secretion was assessed from blood samples collected at frequent intervals. At the same time the LH release in response to 3 doses of LH‐RH (50, 250 and 1000 ng) was measured. The endogenous release of LH‐RH was then deduced from the episodic pattern of LH secretion using the dose response to synthetic LH‐RH as a standard. The result showed that in the ram LH‐RH secretion is episodic, and the hypothalamus regulates release by changing both the frequency and magnitude of the episodes of secretion.

Sex difference in LH response to LHRH and DBcAMP and effect of testosterone.

Because luteinizing hormone-releasing hormone (LHRH) stimulates both pituitary cAMP production and LH release, cAMP has been implicated in the action of LHRH on LH release. The effects of LHRH and DBcAMP on LH release were tested in 4-h incubations with pituitary cultures prepared from male or female rats. LH contents in medium and cells were separately determined by radioimmunoassays. LH release in response to 10 nM LHRH was significantly greater in cultures prepared from female rats (female-RPC) than in cultures prepared from male rats (male-RPC), 1,070 and 418% of control, respectively. Addition of DBcAMP (3, 5, or 10 mM) significantly stimulated LH release by female-RPC (212, 206, or 286% of control, respectively) but did not affect LH release in male-RPC. Furthermore, DBcAMP significantly increased the cellular LH content in female- but not in male-RPC. Testosterone pretreatment of female-RPC significantly lowered the LHRH-induced LH release but did not affect the DBcAMP-induced LH release. These data indicate that testosterone may contribute to the sex difference in pituitary LH response to LHRH but not to DBcAMP.

Ovarian function in suckling and non-suckling beef cows post partum.

Two groups, each of 7 crossbred beef cows, which were suckling or not suckling calves, were fed a high quality food ad libitum for 3 months post partum. The non-suckling cows experienced regular ovarian cycles from 10--33 days post partum while the suckling cows did not do so until at least 14 weeks post partum. There was little difference between the groups in growth rate or in plasma glucose concentration. The plasma prolactin concentrations in the non-suckling cows showed a seasonal trend which paralleled ambient temperature and daylight hours; in the suckling cows this trend was less evident. Plasma LH concentrations were lower in suckling cows before Day 30 post partum but were similar thereafter. Most suckling cows also failed to experience oestrus or to exhibit LH release in response to an injection of oestradiol benzoate at about 6 weeks post partum. This failure, together with the earlier lower levels of LH in the suckling cows, is considered to be indicative of malfunction of the hypothalamic mechanism normally responsible for the establishment and maintenance of cyclic ovarian function.

Impaired estrogen-induced luteinizing hormone release in young women with anovulatory dysfunctional uterine bleeding.

Spontaneous ovarian activity, as reflected by the urinary excretion of total estrogen and pregnanediol measured serially (thrice weekly) over a period of 3-4 months, was studied in nine young women (15-27 yr old) with a history of dysfunctional uterine bleeding of at least 2-yr duration. Results were compared to those obtained in sex regularly menstruating women, aged 23-45 yr. All control women had ovulatory cycles, but seven of the nine patients with DUB failed to ovulate during at least three consecutive cycles. The profiles of urinary total estrogen excretion in these seven subjects were consistent with regular follicular development, but the follicular phase was prolonged and the amount of estrogen excretion increased, as compared to controls. In four of these seven patients, the endometrium had previously shown cystic glandular hyperplasia. Although the release of LH and FSH after injection of 50 micrograms synthetic LRH was normal, the surge of LH induced in response to exogenous estrogen (200 micrograms ethinylestradiol/day for 3 days) was significantly (P less than 0.005) lower in the patients (16.2 +/- 3.7 mU/ml) than that of control women )35.0 +/- 5.5 mU/ml). It is concluded that the failure to ovulate in young women with anovulatory dysfunctional uterine bleeding is due to inadequate release of LH in response to estrogen. The results support the hypothesis that the basic defect in these women may be a decrease of hypothalamic sensitivity to positive feedback.

Cyclic and temporal differences in LH-RH-stimulated LH release in cultured rat pituitary cells

To establish whether the enhanced LH-RH responsiveness shown by pituitary gonadotrophs at proestrus in vivo could be maintained in vitro, rat anterior pituitary cells were investigated to determine differences in LH release in response to LH-RH through the estrous cycle and with time in primary culture. Pooled or individual anterior pituitary glands from each day of the cycle were dissociated with collagenase, hyaluronidase and Viokase® and cultured for from 1 to 4 days. Four-day cultures of proestrous cells did not show differences in LH-RH responsiveness when compared to estrous, diestrous I and diestrous II cells. In addition, proestrous cells did not show differences in LH-RH responsiveness when compared to diestrous II cells after 1,2, 3 or 4 days of culture; however, over the same 1–4 days of culture, proestrous cells contained higher amounts of LH and released greater quantities of LH into the growth medium than did diestrous II cells. It was also observed that both proestrous and diestrous II cells exhibited significantly greater LH-RH responsiveness after 3 or 4 days of culture than after 1–2 days of culture. These results suggest that the differential LH-RH responsiveness shown by pituitary gonadotrophs at proestrus in vivo is not maintained when pituitary cells are placed in primary culture.

LH release in response to GnRH during the postpartum period of dairy cows.

LH release in response to GnRH during the postpartum period of dairy cows.

Estradiol Potentiation of Hypothalamic Uptake of LH-RH from the CSF

Ovariectomized (Ovx) rats were treated (s.c.) with estradiol benzoate (E2B) for 7 days, and then 10, 25 or 100 ng LH-RH were microinjected into the 3rd ventricle. Intraventricular LH-RH elevated plasma LH at 10 and 30 min in a dose-response manner. In experiment 2, Ovx rats were treated with E2B or oil for 7 days and then intraventricularly injected with 25 ng LH-RH. Two days later, 100 ng LH-RH were systemically administered. E2B treatment resulted in a greater release of LH in response to the intraventricular administration of LH-RH but not to the systemic injection. A direct measure of median eminence (ME) uptake of LH-RH was used in experiment 3. Ovx-E2B or oil-treated rats were decapitated 10 min after intraventricular injection of 125I-LH-RH, 125I or 3H-glycine. The ME region of the hypothalamus, cortex, anterior pituitary (AP) and plasma were solubilized and their radioactivity determined. E2B increased the radioactivity in the ME following injection of 125I-LH-RH but not in other tissues. Tissue uptake of 125I and 3H-glycine were similar in E2B or oil-treated rats. These data indicate E2B facilitates the incorporation of LH-RH into the ME from the cerebrospinal fluid (CSF).

Pituitary-Ovarian Relationships in Polycystic Ovary Syndrome

The spontaneous pattern of pituitary gonadotropins and ovarian steroids and their response to dynamic tests were measured in 12 women with polycystic ovarian syndrome (PCO) and the results compared to those from 6 normal women during the early follicllar phase of the cycle (controls). As judged by serial measurements of urinary total estrogen and pregnanediol over a 12-week period, in PCO patients 75% of cycles were anovulatory (anovulatory PCO) as compared to 100% ovulatory in controls. The basal concentrations of LH, androstenedione and estrone were significantly higher and the concentration os FSH significantly lower in anovulatory PCO than in the controls (P less than .05). In PCO patients the concentration of LH was lower following an ovulatory cycle than that following a period of anovulation. Negative and positive feedback responses to an estrogen provocation test (200 microgram ethinyl estradiol per day for 3 days) were normal in anovulatory PCO although the LH peak occurred 24 h earlier than in the controls. The amplitude of the pulses of LH was significantly greater in anovulatory PCO than in the controls and was suppressed in both groups after ethinyl estradiol. The peak release of LH in response to 56 microgram LRF in ovulatory PCO was similar in controls but LH responses in anovulatory PCO were significantly greater. It is suggested that the abnormalities in gonadotropin secretion in PCO are secondary to excessive and prolonged extraglandular production of estrogen from androstenedione.

A Medial Basal Hypothalamic Site of Synergistic Action of Estrogen and Progesterone on the Inhibition of Pituitary Luteinizing Hormone Release1

Plasma LH concentrations were measured by radioimmunoassay in blood withdrawn from indwelling atrial cannulas in long-term ovariectomized (OVX) rats. Subcutaneous injection of 50 μg of estradiol benzoate (EB) in oil depressed the plasma LH concentration to about one-third within 2 h. In other rats, sc injection of 25 mg of progesterone (P) in oil at the time of EB injection did not alter this response. Plasma LH remained suppressed at that level until at least 8 h after injection in both groups, but fell an additional 50% within 24 h after injection only in the rats treated with both EB and P. In additional rats, P, but not the oil vehicle alone, when administered 18 h after the injection of EB, caused plasma LH to decline by about 50% within 60 min. Under these latter conditions, 5, 20 or 100 ng of LHRH was injected through the cannula at 1 h after the injection of P or oil. Progesterone did not inhibit pituitary LH release in response to LHRH as determined by comparison of plasma LH concentrations and increments at 3, 10 and 30 min after LHRH in the two groups. In OVX rats which still exhibited pulsatile LH release after surgical interruption of all of the neural connections to their medial basal hypothalami, injection of EB followed 18 h later by injection of P produced results similar to those seen in control OVX rats. The results suggest that P acts synergistically with estrogen at the level of the medial basal hypothalamus in long-term OVX rats to lower plasma LH concentrations to levels which approach but do not reach basal plasma LH levels in cyclic rats and that, under the conditions of this study, P does not lower plasma LH concentration in EB-treated OVX rats until estrogen has exerted its effect for >8 h but ≤18 h.

The effects of prostaglandin and mating on release of LH in the female rabbit.

The release of LH in response to prostaglandin (PG) treatment of female rabbits in various reproductive states was compared with the surge following mating. Intracarotid infusion of PGE-2 or PGF-2alpha (0-3--900 microgram/h) into non-receptive and pseudopregnant does resulted in small, 2--4-fold elevations in jugular vein LH concentration. Similar doses of PGF-2alpha in oestrogen-pretreated, oestrous does stimulated a 13-fold increase in plasma LH levels. Mating resulted in a much larger release of LH, as plasma levels increased approximately 60-fold from 1-1 +/- 0-2 (S.E.M.) ng/ml to 67-8 +/- 10-5 ng/ml. These results indicate that PG can stimulate the hypothalamic-hypophysial axis to release LH in non-receptive, pseudopregnant and oestrogen-pretreated, oestrous rabbits.

Regulatory Role of Estradiol in Pituitary Responsiveness to Luteinizing Hormone-Releasing Hormone on Proestrus in the Rat

Pituitary LH release in response to LHRH was examined following acute ovariectomy (OVX) at 1300 h on proestrus in rats that had received Nembutal® to block the LH surge. When LHRH (250 ng iv) was administered 1 h after OVX, the plasma LH peak (374 ± 34 ng/ml) was significantly greater than that observed in intact (155 ± 13 ng/ml), sham-OVX (168 ± 25 ng/ml) or intact progesterone-treated (156 ± 19 ng/ml) rats. When the pulse of LHRH was delayed until 2 h after OVX, the plasma LH peak was 676 ± 69 ng/ml. In controls not treated with LHRH, the first significant increase over baseline LH was not detected until 3 h after OVX. In intact proestrous rats, serum estradiol-17/3 (E2) was 56.1 ± 4.95 pg/ml at 1300 h. One hour following OVX serum E2 had fallen to 10.2 ± 2.0 pg/ml; by 2 h the concentration was 6.6 ± 0.66 pg/ml which was not significantly different from that found in diestrus-1 rats. OVX of such diestrus-1 animals had no effect on pituitary LH response to LHRH. In order to duplicate the LHRH-induced pat...

Impairment of luteninsing-hormone release following oestrogen administration to hyperprolactinaemic ewes.

The pituitary response to luteinzing hormone-releasing hormone (LH-RH) was studied in lactating and nonlactating spayed ewes. During the 1st 4 weeks postpartum the release of LH in response to LH-RH increased progressivily to a constant pattern in lactating animals. However as the pattern of LH release was indistinguishable from controls it was concluded that prolactin does not alter the ability of the pituitary to release LH in response to LH-RH. Similarly the LH response to LH-RH in nonlactating spayed ewes was not modified by hyperprolactinemia. Another experiment was performed to determine whether the positive feedback effect of estradiol on the hypothalamus was impaired by high prolactin levels. The most effective dose of estra diol-17beta was rendered ineffective by thyrotopin-releasing hormone induction of hyperprolactinemia. The results suggest that high prolactin levels are involed in the suppression of ovulation during lactation.

Effect of melatonin on the luteinizing hormone release induced by clomiphene and luteinizing hormone-releasing hormone.

The effect of melatonin on the LH-release response after the administration of synthetic LH-RH and clomiphene citrate was investigated in adult male rats. The sc administration of melatonin (1 mg/day) for 6 days produced a significant decrease in serum LH levels and in seminal vesicles and ventral prostate weights. On the other hand, the daily injection of 0.01 mg/100 g of body weight of clomiphene citrate during 6 days significantly stimulated LH levels and the weights of the accessory sex glands. Simultaneous treatment with melatonin and clomiphene produced an inhibitory effect similar to that obtained with melatonin alone. Neither pretreatment with melatonin alone. Neither pretreatment with melatonin (1 mg/day, sc for 6 days) nor its simultaneous iv administration (500 mug) with 75 ng of LH-RH modified the LH-release in response to the hypothalamic hormone. The fact that melatonin was able to suppress the effect of clomiphene, which is probably exerted at the hypothalamic level, but not the actiion of LH-RH on the pituitary, appears to indicate that the hypothalamus may be the site involved in the inhibitory effect of the pineal principle on the reproductive function.

Effects of intravenous infusion of catecholamines on rat plasma luteinizing hormone and prolactin concentrations.

Catecholamines were infused through an atrial cannula in unanesthetized rats on the afternoon of proestrus and blood was withdrawn through a second cannula for radioimmunoassay of LH and prolactin. Infusion of epinephrine, but not of norepinephrine or dopamine, blocked spontaneous pituitary LH release and ovulation. Ultimately, this effect appears to be exerted on the brain and not on the pituitary or through changes in pituitary blood flow. Pituitary LH release in response to exogenous LHRH, when administered in an amount that simulated the proestrous LH surge in phenobarital-treated rats, was unaltered by epinephrine infusion. In addition, epinephrine infusion did not alter the timing of the rise in plasma prolactin. Infusion of dopamine blocked the spontaneous rise in plasma prolactin and depressed basal prolactin levels. After the end of infusion, plasma prolactin rose rapidly. Infusion of norepinephrine or epinephrine partially suppressed the prolactin rise but only after 2 h of infusion. 1) point out the possibility that chronic release of epinephrine from the adrenal medulla may be involved in phenomena in which "stress" inhibits reproductive function; and 2) are consistent with the view that dopamine, but not norepinephrine, may be PIF.

Effect of inhibitors of prostaglandin synthesis on gonadotropin release in the rat.

To study the effect of blockade of prostaglandin (PG) synthesis on gonadotropin release in the rat, inhibitors of PG synthesis were injected by various routes in various experimental conditions. The injection of 5-, 8-, 11-, 14-eicosatetraynoic acid (TYA) into the third ventricle (3rd V) significantly decreased plasma LH of ovariectomized (OVX) rats 1, 2, and 4 h following its injection; however, TYA failed to alter plasma LH in OVX rats when administered as a single sc injection and also failed to prevent the post-castration rise in plasma LH when administered sc once daily for 4 days to short-term OVX rats. None of these treatments altered plasma FSH concentrations. Indomethacin (Id) injected into the 3rd V or implanted into the medial basal hypothalamus (MBH) of OVX rats depressed plasma LH 1--6 h later. This effect was no longer observed 24--72 h following its implantation in the MBH. When different doses of Id were administered as single sc injections to OVX rats, plasma LH titers were depressed 24--32 h later, whereas plasma FSH remained either unaltered or was slightly increased. Similarly, the post-castration rise of plasma LH but not that of FSH in male rats was suppressed by a single sc injection of Id given 6 h before orchidectomy. Id administered acutely iv failed to modify the pulsatile release of LH in OVX rats, but it effectively inhibited this release when injected sc 20--30 h before the initiation of blood collection. Moreover, Id blocked the progesterone-induced LH and FSH release in OVX estrogen-primed rats when given sc 24 h before progesterone, but not when it was injected either sc or iv shortly (2 h) before or shortly after (1--3 h) progesterone treatment. Rats treated with Id showed a decrease in BW 24--32 h afters its sc injection. However, the effects of Id on LH release could not be explained by lack of food intake since fasted controls showed LH titers similar to fed rats. Id did not significantly inhibit the LH release in response to synthetic LH-releasing hormone (LHRH) in OVX rats, but partially blocked the response in OVX estrogen, progesterone-treated (OEP) rats. Surprisingly, in OEP rats, Id appeared to potentiate the FSH release in response to LHRH. The results of this study indicate that inhibitors of PG synthesis administered at high doses can inhibit LH release in the rat and that this effect is mainly due to a direct effect of the drug or drugs on the central nervous systen. Consequently, the results of this study give further support to the hypothesis that PGs play a physiological role in the control of gonadotropin secretion.

Alterations during the estrous cycle in the responsiveness of the pituitary to subcutaneous administration of synthetic LH-releasing hormone (LHRH).

To further evaluate the alterations in responsiveness of the pituitary to synthetic LHRH during the proestrous discharge of gonadotropins, LHRH was given SC in the hope of producing a release of FSH as well as LH, and blood samples were removed prior to and at various intervals after the injections of the neurohormone while the rats were anesthetized with tribromoethanol. The procedure of anesthesia and blood sampling produced small declines in both FSH and LH, but these only achieved significance in the case of FSH on the morning of estrus and diestrus day 2. An increase in plasma LH occurred in response to LHRH at all stages of the estrous cycle but was minimal during diestrus. The increment in plasma LH was already increased by 9 AM on proestrus and the titer remained elevated for 2 h. A further increase in response to LHRH occured at 2 PM associated with an increase in initial plasma LH. The maximum response occurred at 5 PM together with maximal initial plasma LH, and the characteristics of the response changed such that there was a much larger increase in plasma LH at 20 min and a rapid decline thereafter, so that the response became pulselike. Initial plasma LH had already declined significantly by 9 PM, and this was associated with a dimished pulselike release of LH in response to LHRH. By the morning of estrus, an even smaller pulse-like release occurred. Significant FSH release in response to LHRH occurred at all stages of the cycle as indicated by the increments in plasma FSH. The relative increase in plasma titers was much less than that for LH and responsiveness to LHRH did not increase until 5 PM on proestrus. Responsiveness had already begun to decline by 9 PM on proestrus and by the morning of estrus was similar to that obtained on diestrus. Initial plasma FSH titers were first elevated by 2 PM on proestrus and they remained elevated on the morning of estrus. The results indicate that maximal responsiveness to LHRH in terms of LH release is associated with the proestrous discharge of LH, but that the maximal responsiveness in terms of FSH release terminates long before the endogenous release of hormone ceases sometime on estrus. The increased responsiveness in terms of LH release may be related to prior estrogen secretion and to the priming action of endogenous LHRH. The continued release of FSH in the face of a return of responsiveness to LHRH to low levels indicates that release of FSH on late proestrus and early estrus is not caused by endogenous release to LHRH.

Studies on the Site(s) of Blockade by Actinomycin-D of Estrogen-Induced LH Release

Ovariectomized rats were injected with estradiol benzoate (EB) on day 1. The rats received propylene glycol alone (control) or with Actinomycin-D (Act-D) at 08.00 h, followed by EB at 12.00 h on day 3. Plasma LH levels were markedly elevated at 18.00 h on days 3 and 4 in control rats; Act-D treatment failed to modify the surge of LH on day 3, but abolished it on day 4. The site of Act-D's blockade of estrogen-induced LH release was investigated. Act-D treatment suppressed serum levels of LRF while it failed to modify either hypothalamic LRF content or noon levels of pituitary and serum LH on day 4. Act-D treatment consistently reduced the weight and the ribonucleic acid (RNA) content of the pituitary; a similar effect on RNA in the medial basal hypothalamus was not detected on day 4. On the other hand, electrochemical stimulation of the medial preoptic area of Act-D-treated rats on day 4 raised plasma LH significantly. However, the elevations at 45 min after stimulation were less in Act-D-treated than in control rats. Similarly, a significantly smaller response of LH release was observed at 15 min following intravenous injections of LRF in Act-D rats than in controls. These studies suggest that Act-D-sensitive steps exist at more than one site on the preopticotuberal pituitary axis involved in the stimulatory feedback action of estrogen on LH release.