Positive Feedback Action Of Estrogen Research Articles

Kobayashi Award 2019: The neuroendocrine regulation of the mammalian reproduction

Mammalian reproductive function is a complex system of many players orchestrated by the hypothalamus-pituitary–gonadal (HPG) axis. The hypothalamic gonadotropin-releasing hormone (GnRH) and the consequent pituitary gonadotropin release show two modes of secretory patterns, namely the surge and pulse modes. The surge mode is triggered by the positive feedback action of estrogen secreted from the mature ovarian follicle to induce ovulation in females of most mammalian species. The pulse mode of GnRH release is required for stimulating tonic gonadotropin secretion to drive folliculogenesis, spermatogenesis and steroidogenesis and is negatively fine-tuned by the sex steroids. Accumulating evidence suggests that hypothalamic kisspeptin neurons are the master regulator for animal reproduction to govern the HPG axis. Specifically, kisspeptin neurons located in the anterior hypothalamus, such as the anteroventral periventricular nucleus (AVPV) in rodents and preoptic nucleus (POA) in ruminants, primates and others, and the neurons located in the arcuate nucleus (ARC) in posterior hypothalamus in most mammals are considered to play a key role in generating the surge and pulse modes of GnRH release, respectively. The present article focuses on the role of AVPV (or POA) kisspeptin neurons as a center for GnRH surge generation and of the ARC kisspeptin neurons as a center for GnRH pulse generation to mediate estrogen positive and negative feedback mechanisms, respectively, and discusses how the estrogen epigenetically regulates kisspeptin gene expression in these two populations of neurons. This article also provides the mechanism how malnutrition and lactation suppress GnRH/gonadotropin pulses through an inhibition of the ARC kisspeptin neurons. Further, the article discusses the programming effect of estrogen on kisspeptin neurons in the developmental brain to uncover the mechanism underlying the sex difference in GnRH/gonadotropin release as well as an irreversible infertility induced by supra-physiological estrogen exposure in rodent models.

Oestrogen-induced activation of preoptic kisspeptin neurones may be involved in the luteinising hormone surge in male and female Japanese monkeys.

The oestrogen-induced luteinising hormone (LH) surge is evident in male primates, including humans, whereas male rodents never show the LH surge, even when treated with a preovulatory level of oestrogen. This suggests that the central mechanism governing reproductive hormones in primates is different from that in rodents. The present study aimed to investigate whether male Japanese monkeys conserve a brain mechanism mediating the oestrogen-induced LH surge via activation of kisspeptin neurones. Adult male and female Japanese monkeys were gonadectomised and then were treated with oestradiol-17β for 2 weeks followed by a bolus injection of oestradiol benzoate. Both male and female monkeys showed an oestrogen-induced LH surge. In gonadectomised monkeys sacrificed just before the anticipated time of the LH surge, oestrogen treatment significantly increased the number of KISS1-expressing cells in the preoptic area (POA) and enhanced the expression of c-fos in POA KISS1-positive cells of males and females. The oestrogen treatment failed to induce c-fos expression in the arcuate nucleus (ARC) kisspeptin neurones in both sexes just prior to LH surge onset. Thus, kisspeptin neurones in the POA but not in the ARC might be involved in the positive-feedback action of oestrogen that induces LH surge in male Japanese monkeys, as well as female monkeys. The present results indicate that oestrogen-induced activation of POA kisspeptin neurones may contribute to the LH surge generation in both sexes. The conservation of the LH surge generating system found in adult male primates, unlike rodents, could be a result of the capability of oestrogen to induce POA kisspeptin expression and activation.

Kisspeptin and the Preovulatory Gonadotrophin‐Releasing Hormone/Luteinising Hormone Surge in the Ewe: Basic Aspects and Potential Applications in the Control of Ovulation

The identification of the neural mechanisms controlling ovulation in mammals has long been a 'holy grail' over recent decades, although the recent discovery of the kisspeptin systems has totally changed our views on this subject. Kisspeptin cells are the major link between gonadal steroids and gonadotrophin-releasing hormone (GnRH) neurones. In the female rodent, kisspeptin cells of the preoptic area are involved in the positive-feedback action of oestrogen on GnRH secretion, although the picture appears more complicated in the ewe. As in rodents, activation of preoptic kisspeptin neurones accompanies the GnRH surge in the ewe but an active role for arcuate kisspeptin neurones has also been proposed. Experimentally, kisspeptin is able to restore reproductive function when the hypothalamic-hypophyseal ovarian axis is quiescent. For example, i.v. infusion of a low dose of peptide in anoestrous ewes induces an immediate and sustained release of gonadotrophin, which subsides and then provokes a luteinising hormone (LH) surge a few hours later. This pharmacological intervention induces the same hormonal changes normally observed during the follicular phase of the oestrous cycle, including the secretion of oestrogen and its negative- and positive-feedback actions on the secretion of LH and follicle-stimulating hormone. Accordingly, a high percentage of kisspeptin-infused animals ovulated. Although the multiple facets of how the kisspeptin systems modulate GnRH secretion are not totally understood, the demonstration that exogenous kisspeptin administration can induce ovulation in anovulatory animals paves the way for future therapeutic applications aiming to control reproduction.

Involvement of Central Metastin in the Regulation of Preovulatory Luteinizing Hormone Surge and Estrous Cyclicity in Female Rats

Ovulation is caused by a sequence of neuroendocrine events: GnRH and LH surges that are induced by positive feedback action of estrogen secreted by the mature ovarian follicles. The central mechanism of positive feedback action of estrogen on GnRH/LH secretion, however, is not fully understood yet. The present study examined whether metastin, the product of metastasis suppressor gene KiSS-1, is a central neuropeptide regulating GnRH/LH surge and then estrous cyclicity in the female rat. Metastin had a profound stimulation on LH secretion by acting on the preoptic area (POA), where most GnRH neurons projecting to the median eminence are located, because injection of metastin into the third ventricle or POA increased plasma LH concentrations in estrogen-primed ovariectomized rats. Metastin neurons were immunohistochemically found in the arcuate nucleus (ARC) to be colocalized with estrogen receptors with some fibers in the preoptic area (POA) in close apposition with GnRH neuronal cell bodies or fibers. Quantitative RT-PCR has revealed that KiSS-1 and GPR54 mRNAs were expressed in the ARC and POA, respectively. The blockade of local metastin action in the POA with a specific monoclonal antibody to rat metastin completely abolished proestrous LH surge and inhibited estrous cyclicity. Metastin-immunoreactive cell bodies in the ARC showed a marked increase and c-Fos expression in the early proestrus afternoon compared with the day of diestrus. Thus, metastin released in the POA is involved in inducing the preovulatory LH surge and regulating estrous cyclicity.

Effects of Changing Gonadotropin-Releasing Hormone Pulse Frequency and Estrogen Treatment on Levels of Estradiol Receptor-α and Induction of Fos and Phosphorylated Cyclic Adenosine Monophosphate Response Element Binding Protein in Pituitary Gonadotropes: Studies in Hypothalamo-Pituitary Disconnected Ewes

Estrogen receptor-alpha (ER alpha) levels in gonadotropes are increased during the follicular phase of the ovine estrous cycle, a time of increased frequency of pulsatile secretion of GnRH and elevated plasma estrogen levels. In the present study, our first aim was to determine which of these factors causes the rise in the number of gonadotropes with ER alpha. Ovariectomized hypothalamo-pituitary disconnected ewes (n = 4-6) received the following treatments: 1) no treatment, 2) injection (im) of 50 microg estradiol benzoate (EB), 3) pulses (300 ng iv) of GnRH every 3 h, 4) GnRH treatment as in group 3 and EB treatment as in group 2, 5) increased frequency of GnRH pulses commencing 20 h before termination, and 6) GnRH treatment as in group 5 with EB treatment. These treatments had predictable effects on plasma LH levels. The number of gonadotropes in which ER alpha was present (by immunohistochemistry) was increased by either GnRH treatment or EB injection, but combined treatment had the greatest effect. Immunohistochemistry was also performed to detect phosphorylated cAMP response element binding protein (pCREB) and Fos protein in gonadotropes. The number of gonadotropes with Fos and with pCREB was increased only in group 6. We conclude that either estrogen or GnRH can up-regulate ER alpha in pituitary gonadotropes. On the other hand, during the period of positive feedback action of estrogen, the appearance of pCREB and Fos in gonadotropes requires the combined action of estrogen and increased frequency of GnRH input. This suggests convergence of signaling for GnRH and estrogen.

Site-specific decrease of progesterone receptor mRNA expression in the hypothalamus of middle-aged persistently estrus rats

Middle-aged females gradually become acyclic and spontaneously develop a persistently estrus (PE) state. PE rats, acyclic for 30 days (early PE), are unresponsive to the positive feedback action of estrogen, but respond to a progesterone challenge with a luteinizing hormone (LH) surge and ovulation; unlike long-term PE rats, acyclic for 90 days, neither estrogen nor estrogen plus progesterone will elicit an LH surge [10th International Congress of Endocrinology, San Francisco, P3 (1996) 1061]. We hypothesize that the PE state may develop due to a diminished level of estrogen-induced progesterone receptor (PR) expression in the hypothalamus that prevents progesterone from stimulating LH regulating circuits. To test this hypothesis, PR mRNA levels were measured in hypothalamic regions of young, proestrus (2–3 months of age), early PE (10–12 months) and long-term PE (13–15 months) rats. The anteroventral periventricular nucleus (AVPV), an important regulatory site of the LH surge, had decreased PR mRNA levels in early and long-term PE rats compared with proestrus rats. However, PR mRNA levels were reduced only in long-term PE rats in the ventromedial nucleus (VMH) and arcuate nucleus (ARH). In the medial preoptic nucleus (MPN), levels of PR mRNA did not change. A previous report showed that exogenous progesterone stimulates an LH surge in young and early PE animals, indicating that the expression of PR mRNA demonstrated in this study is sufficient to mediate progesterone facilitation of the LH surge in early PE rats. In acyclic, long-term PE rats, diminished estrogen-induced expression of progesterone receptors is correlated with a previously shown inability to respond to exogenous progesterone.

Luteinizing hormone secretion as affected by hypophyseal stalk transection and estradiol-17β in ovariectomized gilts

The objectives were to determine hypothalamic regulation of pulsatile luteinizing hormone (LH) secretion in female pigs and the biphasic feedback actions of estradiol-17β (E 2-17β). In the first study, the minimum effective dosage of E 2-17β that would induce estrus in ovariectomized gilts was determined to be 20 μg/kg body weight. In the second study, ovariectomized gilts were assigned randomly on day 0 to treatments: (a) hypophyseal stalk transection (HST), (b) cranial sham-operated control (SOC), and (c) unoperated control (UOC). On day 3, gilts from each group received a single i.m. injection of either E 2-17β (20 μg/kg body weight) or sesame oil. Blood was collected from an indwelling jugular cannula at 15 min intervals for 3 h before (day −2) and after treatment (day 2) from HST, SOC and UOC gilts. On day 3, blood was collected at 2 h intervals for 12 h after E 2-17β or sesame oil injection and at 4 h intervals thereafter for 108 h. Pulsatile LH secretion in all gilts 2 days after ovariectomy exhibited a frequency of 0.9±0.06 peaks/h, amplitude of 1.3±0.13 ng/ml, baseline of 0.8±0.07. Serum LH concentrations from SOC and UOC gilts were similar on day 2 and profiles did not differ from those on day −2. In HST gilts pulsatile LH release was abolished and mean LH concentration decreased compared with controls (0 versus 0.9±0.06 peaks/h and 0.77±0.03 versus 1.07±0.07 ng/ml, respectively; P<0.05). E 2-17β or sesame oil did not affect serum LH concentration in HST gilts, and LH remained constant throughout 120 h (0.7±0.07 ng/ml). In SOC and UOC control gilts, E 2-17β induced a 60% decrease ( P<0.05) in LH concentration within 12 h, and LH remained low until 48 h, then increased to peak values ( P<0.05) by 72 h, followed by a gradual decline to 120 h. Although pituitary weight decreased 31% in HST gilts compared with controls (228 versus 332 mg, P<0.05), an abundance of normal basophils was evident in coronal sections of the adenohypophysis of HST comparable to that seen in control gilts. The third and fourth studies determined that hourly i.v. infusions of LHRH (2 μg) and a second injection of E 2-17β 48 h after the first had no effect on the positive feedback action of estrogen in UOC. However, in HST gilts that received LHRH hourly, the first injection of E 2-17β decreased ( P<0.05) plasma LH concentrations while the second injection of E 2-17β failed to induce a positive response to estrogen. These results indicate that both pulsatile LH secretion and the biphasic feedback action of E 2-17β on LH secretion depend on hypothalamic regulatory mechanisms in the gilts. The isolated pituitary of HST gilts is capable of autonomous secretion of LH; E 2-17β will elicit direct negative feedback action on the isolated pituitary gland if the gonadotropes are supported by exogenous LHRH, but E 2-17β at high concentrations will not induce positive feedback in isolated pituitaries. Thus, the direct effect of E 2-17β on the pituitary of monkeys cannot be mimicked in pigs.

Glucose availability modulates the timing of the luteinizing hormone surge in the ewe.

To determine if glucose availability modulates the timing of the positive feedback action of oestrogen on gonadotropin secretion, we monitored the estradiol-induced luteinizing hormone (LH) surge in sheep (n = 5/group) made transiently hypoglycemic by insulin. Experiment 1 determined an effective insulin treatment, one which would depress tonic LH secretion. Two injections of insulin (5 IU/kg iv) 4 h apart were found to induce extended hypoglycemia (10-13 h) and to decrease the LH pulse frequency for 8 h (5.0 +/-0.32 pulses/4 h before versus 2.5+/-0.34 pulses/4 h after insulin; P<0.05; mean +/- SEM). Using this same paradigm, experiment 2 determined the influence of the transient hypoglycemia on the LH surge mechanism. In control sheep, estradiol (subcutaneous implants at hour 0) evoked an LH surge with a latency period of 12.4+/-0.5 h. When insulin was administered either before (hours -4 and 0) or after the estradiol stimulus (hours 4 and 8, or 12 and 16), the onset of the LH surge was delayed to 29.0+/-2.4 h (average of all three time groups, P <0.05). Infusion of glucose from hours 12-30, along with insulin, prevented hypoglycemia and restored the normal timing of the oestrogen-induced LH surge to that of controls (15.4+/-0.93 h, P>0.05). These findings suggest that not only is the tonic mode of LH secretion sensitive to metabolic fuel availability, but the surge mode of LH secretion is as well.

The positive feedback action of estrogen mobilizes LH-containing, but not FSH-containing secretory granules in ovine gonadotropes.

It has previously been shown in the ewe that luteinizing hormone (LH)-containing secretory granules become polarized to the side of the gonadotrope nearest to a vascular sinusoid during the preovulatory period. We have used laser scanning confocal microscopy to monitor the migration of LH and follicle stimulating hormone (FSH)-containing secretory granules in the gonadotrope of the ewe. Ovariectomized (OVX) ewes (n = 4/group) were given either 50 micrograms of estradiol benzoate (EB) or oil and were killed 16 h later for collection of the pituitary glands for immunohistochemistry and confocal microscopy. Pituitary sections were simultaneously immunolabelled for LH and FSH and were visualized on the same sections using fluorescent markers. All cells that contained LH also contained FSH and vice versa. The results showed that LH-containing granules were uniformly distributed throughout the cytoplasm in 83% of gonadotropes from controls, while in the remaining 17% the granules were predominantly located adjacent to the plasma membrane. Following EB treatment, LH-containing secretory granules were distributed around the plasma membrane in 84% of immunopositive gonadotropes; FSH remained uniformly distributed throughout the cytoplasm. We conclude that estrogen causes the movement of LH-containing granules to the plasma membrane in gonadotropes but does not influence the distribution of FSH-containing granules. Rather than being polarized to one side of the cell, the LH-containing granules were distributed around the periphery of the cell.

The Positive Feedback Action of Estrogen Mobilizes LH-Containing, but not FSH-Containing Secretory Granules in Ovine Gonadotropes

The Positive Feedback Action of Estrogen Mobilizes LH-Containing, but not FSH-Containing Secretory Granules in Ovine Gonadotropes

Evidence that the arcuate nucleus is one of the site for positive feedback action of estrogen on LHRH release

Evidence that the arcuate nucleus is one of the site for positive feedback action of estrogen on LHRH release

The neuroendocrine control of ovulation.

In all species studied to date, follicular development is the consequence of pulsatile gonadotrophin stimulation occasioned by a GnRH pulse generator located in the mediobasal hypothalamus. This pulse generator operates within a narrow frequency range established by a neuronal system which, in the higher primates including man, discharges with a period of approximately 1 h. This frequency does not appear to change significantly as the follicular phase of the menstrual cycle progresses and plasma oestradiol concentrations rise. When these exceed a defined threshold for approximately 36 h, the pre-ovulating gonadotrophin surge is initiated and ovulation ensues some 24 h later, the so-called positive feedback action of oestrogen. This sequence of events can be replicated experimentally in subjects with various hypothalamic lesions which abolish endogenous GnRH production, simply by the administration of exogenous GnRH at an unchanging physiological frequency. This implies that the site of the positive feedback action of oestradiol is the pituitary gland and that GnRH plays but a permissive role in the initiation of the mid-cycle gonadotrophin surge. This process can be disrupted by a number of inputs which perturb the proper functioning of the hypothalamic GnRH pulse generator.

Possible site of negative and positive feedback action of oestrogen on gonadotrophin secretion in normal women.

For determination of the site of action of oestrogen (E) during the negative and positive feedback phases of gonadotrophin secretions, studies were made on the pituitary response to a small amount of LRH and the pulsatility of gonadotrophins after E administration in normal cycling women in the mid-follicular phase. The pituitary responses to an iv bolus of 2.5 micrograms of synthetic LRH were evaluated by measuring serum LH and FSH 2 h before and 8 h after administration of 20 mg of conjugated E (Premarin). In the next cycle, the pituitary responses to a same dose of LHRH were also observed 2 h before and 56 h after E injection. The mean levels of serum LH and FSH and the peak responses to LRH were significantly (P less than 0.05) decreased 8 h after E injection, but were significantly (P less than 0.05) increased 56 h after E administration. In the third cycle, the pulsatility of gonadotrophins was evaluated by measuring serum LH and FSH every 15 min for 180 min before and 8 h and 56 h after E injection. The pulse frequencies of gonadotrophins were not significantly different before and 8 h and 56 h after E injection. The amplitudes of pulses 56 h after Premarin injection were significantly higher than those before the injection. These findings suggest that the negative and positive feedback effects of E on gonadotrophin secretion may be caused, in part, by its direct action on the pituitary response to LRH.

Drug-induced hyperprolactinaemia and discharges of luteinizing hormone evoked by oestrogen in ovariectomized rhesus monkeys.

Ovulatory failure associated with hyperprolactinaemia has been attributed to the inability of oestrogen to evoke a preovulatory discharge of gonadotrophins. This hypothesis was tested in ovariectomized rhesus monkeys rendered hyperprolactinaemic by treatment with two dopamine antagonists, domperidone, a compound reported not to cross the blood-brain barrier in rats, and sulpiride, a drug with known actions on both pituitary and central dopaminergic receptors. An artificial surge of oestrogen similar to that observed at mid-cycle in intact monkeys was obtained with subcutaneous capsules filled with oestradiol-17 beta. Treatment with either drug increased prolactin concentrations to 1.0 i.u./l or more compared with control values of 0.1-0.3 i.u./1. In the presence of these greatly increased prolactin levels, a normal discharge of LH, indistinguishable from that observed in control animals, was provoked by oestrogens in all but one drug-treated monkey. Furthermore, neither the basal (pre-oestrogen) level of LH nor the negative feedback action of oestrogen was consistently altered in hyperprolactinaemic monkeys. Administration of progesterone or 17 alpha-hydroxyprogesterone to simulate more closely the conditions observed in intact animals during the periovulatory period did not alter the LH discharge induced by oestrogen in animals treated with domperidone. The present results suggest that the high levels of prolactin brought about by dopaminergic blockade do not affect the positive feedback action of oestrogen. Furthermore, low levels of progestins do not sensitize the hypothalamic-pituitary axis towards an inhibitory action of prolactin on oestrogen-induced LH release. The rhesus monkey thus differs from other species, including man, in which artificially induced hyperprolactinaemia may impair or abolish the positive feedback action of oestrogen.

Influence of suckling on gonadotropin secretion in the female rhesus monkey (Macaca mulatta).

The ability of exogenous estrogen to elicit gonadotropin surges was determined at approximately monthly intervals in postpartum female rhesus monkeys nursing their infants, in postpartum females whose infants were weaned at birth, and in pregestational females that served as foster mothers to these weaned infants. The stimulatory action of estrogen on gonadotropin secretion was abolished or severely inhibited for the first 6 months of nursing in both natural and foster mothers. In contrast, the administration of estrogen to postpartum females, whose infants were weaned at birth, elicited luteinizing hormone (LH) and follicle stimulating hormone (FSH) surges on the first trial 30 days after delivery. The abolition of the positive feedback action of estrogen in nursing mothers was associated with a suppression of basal serum gonadotropin levels. These findings demonstrate that the protracted inhibition of gonadotropin secretion in lactating female rhesus monkeys is occasioned by the presence of a suckling infant and is independent of antecedent events associated with gestation and parturition.

Influence of ovarian steroids on pituitary sensitivity to luteinizing hormone-releasing hormone in the ovariectomized guinea pig.

To determine the sites of positive feedback action of estrogen and progesterone (P), pituitary responsiveness to LHRH under estradiol benzoate (EB) and P influence was examined in the ovariectomized guinea pig. Two to 3 days before the experiment, animals received an implantation of an indwelling venous catheter for blood sampling (drawn every 15 min) and for LHRH administration. Plasma LH was measured by RIA. In the fust experiment, a single dose of LHRH (1.0μg) was given to five groups of animals: non-EB-treated; 12 h, 30 h, and 36 h after 1.5 μg EB; and 30 h after 1.5 μg EB plus 2 h after 0.5 μg P. Plasma LH levels were significantly elevated 15 min after LHRH injection in all treatments. In the subsequent 60-90 min, LH levels decreased exponentially. The peak value was highest in the group of non-EB-treated animals and diminished as time elapsed after EB treatment. P plus EB priming did not facilitate an LH response significantly greater than priming with EB alone. In the second experiment, LHRH was g...

Anovulation after pregnancy termination: ovarian versus hypothalamic-pituitary factors.

28 female rhesus monkeys were studied after induced abortion or after spontaneous delivery at term to differentiate ovarian from hypothalamic-pituitary factors responsible for suppression of ovulation. Human menopausal gonadotropin (HMG) was administered to stimulate follicular maturation, and estradiol benzoate was injected to test induction of gonadotropin surge. Spontaneous ovulation was delayed until about 39 days after abortion. The presence of multiple preovulatory follicles on both ovaries confirmed that folliculogenesis had been stimulated by HMG, which also caused the maturation and rupture of these follicles. These findings show that pregnancy does not hinder gonadotropin stimulation on the ovaries, but that it suppresses hypothalamic-pituitary responsivity to the positive feedback action of estrogen on FSH and on LH surges. It is possible that tonic gonadotropin secretion is inadequate to initiate follicular maturation until late in the puerperium.

Resumption of estrogen-induced gonadotropin surges in postpartum monkeys.

To evaluate the functional integrity of the hypothalamic-pituitary (H-P) system in postgestational monkeys, peripheral serum concentrations of LH and FSH were determined in postpartum (nursing and nonnursing) and postabortion (midpregnancy) females challenged with estradiol benzoate (EB) at a dose known to be effective in eliciting gonadotropin surges in cycling monkeys. In a longitudinal study of nursing and nonnursing postpartum mothers and postabortion females, EB was administered serially during weeks 1, 3, 5, 7, 9, and 11 post delivery. Some nursing monkeys were challenged with EB up to 6 months post partum. In a cross-sectional study of nursing mothers, this response to EB was evaluated during weeks 1, 3, 5, 7, and 9 postpartum. H-P responsivity to EB was impaired in nursing monkeys through 180 days post partum, in that FSH or LH surges were not induced by estrogen therapy. In contrast, nonnursing postpartum mothers and postabortion females regained their sensitivity to this positive feedback action of estrogen within 35-49 days. Our findings indicate that during gestation a transient lesion develops in the H-P system and persists into the puerperium. It blocks the positive feedback response of surge gonadotropin secretion induced by estradiol. However, this H-P failure is not sufficient by itself to explain postpartum infertility in these primates, since in nonnursing monkeys, spontaneous resumption of ovulatory menstrual cycles seldom occurs until 2-4 months after delivery at term.

Factors influencing the positive feedback action of estrogen upon the luteinizing hormone surge in the ovariectomized guinea pig.

Factors influencing the positive feedback action of estrogen, especially those related to dose and the time of day of injection, were investigated in the ovariectomized female guinea pig Adult guinea pigs were housed in a light-controlled room (12 h light, 0600–1800 h) and ovariectomized 2 weeks before estradiol benzoate (EB) injections. EB doses of 1.5, 10, or 50 μg were administered sc at 1000 or 2200 h. Blood samples were obtained through a chronic indwelling venous catheter. Plasma LH was estimated by RIA. The mean latency from the time of EB injection to the occurrence of the LH surge was not influenced by the time of day of injection but was clearly related to the dose of estrogen administered. The mean latency to the LH surge from the time of injection decreased with increasing doses of estrogen but the mean dose-dependent latency was comparable in the same dose groups regardless of the time of day of injection. However, at each dose of estrogen, the temporal distributions of the individual LH surges were clearly different between morning and evening groups. Overall, a greater percentage of animals showed the LH surge during the dark phase than light phase. At all doses tested, 100% of the responders showed LH surges in the dark phase when the estrogen injection was timed so that the dosetypical latency was coincident with the midpoint of the dark phase of the cycle. When the dose-typical latency was coincident with the midpoint of the light phase, episodes of the LH surge were widely distributed and occurred in the dark as well as in the light phases of the cycle. This tendency for episodes of the LH surge to be shifted out of the light phase into the preceding and subsequent dark phases was inversely related to the dose of estrogen administered. Thus, the effect was most clearly observed in animals given 1.5 μg EB. These data indicate that the timing of the estrogen-induced LH surge in the ovariectomized guinea pig is regulated by two major factors: 1) the dose of estrogen given and 2) the lighting conditions (environmental light-dark cycle).

PITUITARY RESPONSIVENESS TO LUTEINIZING HORMONE‐RELEASING HORMONE DURING TREATMENT WITH SUBDERMAL D‐NORGESTREL IMPLANTS IN WOMEN

It has previously been reported that subdermal implants containing d-norgestrel inhibit ovulation through blockage of the positive feedback action of estrogen on luteinizing hormone (LH) release. In this study 100 micrograms of LH-releasing hormone (LRH) were injected intravenously to test the pituitary reserve capacity for gonadotrophin secretion in three women with three 40 mg d-norgestrel rods implanted subdermally for more than one year. The gonadotrophin release to LRH varied and seemed related to the ovarian steroid concentrations at the time of the LRH infusion in a manner similar to that seen during the normal menstrual cycle. It is concluded that low plasma levels of d-norgestreol do not inhibit the pituitary responsiveness to LRH. The results indicate that the blocking action of the gestagen on the positive feedback of estradiol on LH release occurs at the hypothalamus or higher CNS centers.