Hypothalamic areas involved in prostaglandin (PG)-induced gonadotropin release. I: effects of PGE2 and PGF2alpha implants on luteinizing hormone release.

Ovariectomized rats had a cannula inserted unilaterally within various hypothalamic areas. Several days later they were primed with a sc dose of 10 microng of estradiol benzoate (Eb). Two days after priming they were etherized and an initial blood sample was drawn from the external jugular vein. An inner cannula containing PGE2 or PGF2alpha at its tip was inserted into the previously implanted outer cannula. Blood samples were drawn at 20, 40, 60, and 120 min following the implantation. PGE2 induced a 4-5-fold increase in plasma LH 40 to 60 min following its implantation in the arcuate nucleus-median eminence region (ARH-ME). Levels were already significantly elevated at 20 min. When PGE2 was placed slightly more dorsally, close to the ventromedial nucleus (VMH), LH titers rose to comparable levels but only after a delay of 120 min. PGE2 implanted in the caudal portion of the ARH-ME or dorsally in the VMH-dorsomedial nuclei, barely increased plasma LH, whereas its placement in the anterior portion of the ARH-ME clearly elevated LH titers. PGE2 implants located more than 1 mm lateral from the midline or outside the hypothalamus were ineffective. When PGE2 was placed in the preoptic area (POA) or anterior ventral portion of the anterior hypothalamic area (AHA), plasma LH levels rose strikingly, the first significant increase being observed at 20 min. PGE2 implants located in the vicinity of the paraventricular nucleus-dorsal portion of AHA were much less effective. PGF2alpha implanted in the ARH-ME or POA induced a small increase in plasma LH and the implantation of empty cannulae in the same areas was ineffective. Intrapituitary implants of PGE2 failed to alter plasma LH significantly. The results indicate that PGE2 acts at the ARH-ME region to induce LH release and that an even more effective site of action seems to be located in the POA-AHA. Since these are areas which contain LHRH, the results support the view that PGs can activated LHRH-secreting neurons in these regions.

Hypothalamic areas involved in prostaglandin (PG)-induced gonadotropin release. II: effect of PGE2 and PGF2alpha implants on follicle stimulating hormone release.

The effect of unilateral intrahypothalamic implants of PGE2 or PGF2alpha on FSH release in ovariectomized, estrogen-treated rats was studied. Both PGs induced an equally small increase in plasma FSH following their implantation in the body of the arcuate nucleus-median eminence region (BARH-ME). Levels were maximally elevated 120 min post-implantation. PGE2 implanted in the caudal portion of the ARH-ME (TARH-post-ME) also induced a similar increase in plasma FSH, whereas its placement in the region of the ventromedial nucleus (VMH)-dorsomedial nucleus (DMH) did not significantly alter FSH titers. By contrast, PGE2 placed in the post-chiasmatic region (HARH-ME) clearly elevated plasma FSH as early as 40 min after implantation. Implants of PGF2alpha in this area were ineffective. PGE2 implants located more than 1 mm lateral from the midline or outside the hypothalamus were also ineffective. When PGE2 was placed in the preoptic area (POA) or anterior ventral portion of the anterior hypothalamic area (AHA), plasma FSH rose markedly showing the first significant increase between 40 and 60 min after implantation. PGE2 implants located in the dorsal portion of the AHA-paraventricular nucleus (DAHA-PVH) were also clearly more effective than implants in BARH-ME, but less effective than implants in the POA. PGF2 implanted in the POA evoked a smaller increase in plasma FSH than that induced by PGE2 implants in this region. Implantation of empty cannulae in the ARH-ME region or POA failed to increase plasma FSH levels significantly. Intrapituitary implants of PGE2 did not alter plasma FSH. The results indicate that PGE2 acts mainly on the POA-AHA region to induce FSH release and that PGF2alpha, although less effective, also stimulates FSH release in this area. The ARH-ME appears to be a less important site. The greater effectiveness in the POA-AHA region than in the ARH-ME may indicate that PGE2 is more effective in stimulating cell bodies of LHRH neurons than their axons. Although most of the sites at which PGE2 released FSH coincide with the distribution of LHRH, it is noteworthy that a relatively enhanced release of FSH took place from certain sites in the dorsal AHA-PVH and from sites in the post-ME, which suggests that PGE2 may act on neurons which secrete a specific FSH-RF as well as on neurons which release LHRH.

Prostaglandin E2 (PGE2)-induced growth hormone (GH) release: effect of intrahypothalamic and intrapituitary implants.

Ovariectomized rats were unilaterally implanted with a 23-gauge stainless steel cannula in different hypothalamic areas or in the pituitary gland and subsequently were treated with estrogen (sc, 10 micron g estradiol benzoate, Eb). Two days after the estrogen injection, an inner cannula containing PGE2 or PGF2alpha at its tip was inserted into the cannula. Other animals were implanted with an empty inner cannula. Plasma GH concentrations were measured by RIA in blood samples drawn from the jugular vein while the animals were lightly etherized before (-2) and at 20, 40, 60 and 120 min following the implantation. Plasma GH levels in control animals bearing an empty cannula in the body of the arcuate nucleus-median eminence region (BARH-ME) were significantly depressed by the ether stress. The implantation of PGF2alpha in this area was completely ineffective in preventing ether stress-induced decline in plasma GH. By contrast, PGE2 implanted in BARH-ME or the post-chiasmatic region of the hypothalamus (HARH-ME) elevated plasma GH 20 min following its implantation and partially prevented the subsequent decrease in GH levels induced by ether stress. PGE2 implants located in several other hypothalamic areas failed to induce GH release or to prevent the decline in GH levels induced by ether stress. However, PGE2 implanted in the pituitary gland elicited a marked increase in plasma GH at 20 min and completely prevented the subsequent ether stress-induced decline in GH levels. The results suggest that PGE2 can act at both hypothalamic (ARH-ME) and pituitary levels to stimulate GH release. At the hypothalamus, PGE2 may inhibit GH-inhibiting factor (GIF) release or induce release of GH releasing factor (GHRF).

Developmental patterns of plasma and pituitary growth hormone (GH) in the female rat.

Circulating GH levels measured by RIA in developing female rats were elevated between day 5 and 12. Titers declined by day 15, reaching a nadir at day 20 and by day 25 they began to increase again. During the pubertal period (days 32-38), plasma GH was elevated in the anestrous phase, reaching peak values in the afternoon of the early proestrous phase. Levels were also elevated the day of the first preovulatory discharge of gonadotropins, showing peak values in the afternoon of that day. Thereafter, plasma GH declined, reaching a nadir in the morning of diestrus 1 (day 2 after vaginal opening) and remained slightly more elevated during the afternoon of that day and throughout diestrus 2. Plasma GH levels at this time were similar to those of adult diestrus females. Pituitary GH concentration was low between days 5 and 15 and increased markedly thereafter, reaching adult concentrations by day 25. Body weight increased linearly between day 5 and 25. At this time, the slope of the curve of body weight gain increased significantly. Although the majority of animals revealed open vaginae only after they had reached a body weight of 100 g, a critical weight for vaginal opening could not be established. The pronounced changes in circulating GH levels which preceded by several days the surge in gonadotropin release that normally accompanies the onset of puberty in the rat suggest that GH may play a physiological role in the control of the pubertal process in this species.

Prolactin release in response to blockade of dopaminergic receptors and to TRH injection in developing and adult rats: role of estrogen in determining sex differences.

Female rats released more prolactin in response to blockade of dopaminergic receptors with Pimozide (P) than males at all ages studied (from day 15 to day 75). Testosterone propionate (Tp) administered during the neonatal period did not change the female response to P. The male response to P was not faciliated by neonatal orchidectomy, orchidectomy at day 21 or "feminization" of male fetuses, but it was enhanced by an injection of estradiol benzoate (Eb; 5 mug, SC, 48 h before P). Ovariectomy of adult androgenized or normal female rats blunted the prolactin response to P and an estrogen injection facilitated it. Pituitary prolactin reserve was evaluated by injecting TRH and measuring the resulting plasma prolactin levels. Adult males released less prolactin in response to TRH than females on proestrus or on diestrus day 1. The response of adult females to TRH varied during the cycle, being maximal on proestrus and minimal on diestrus day 2. In both sexes, castration suppressed the prolactin response to TRH and a single Eb injection restored it. Pituitary prolactin content was lower in males than in females and decreased following castration in both sexes. Eb partially restored it. The results indicate that a) the prolactin response to blockade of DA receptors is more pronounced in females than in males, b) this difference is not determined by a process of neonatal or fetal hypothalamic sexual differentiation, but rather is a consequence of a modulating action of estrogen at the hypothalamic-hypophyseal level during the coruse of sexual development, and c) the main site of action of estrogen in determining this sex difference appears to be the pituitary gland.

Developmental changes in pituitary responsiveness to luteinizing hormone-releasing hormone (LHRH) in the female rat: ovarian-adrenal influence during the infantile period.

Pituitary LH and FSH repsonses to synthetic LHRH as estimated by increases in plasma FSH and LH 15 and 45 min following its iv injection were enhanced during the first 2 weeks of life, reaching a maximum around day 10-15 and declining thereafter. No AM.-PM. variations in pituitary responsiveness were observed at any age studied. The increased pituitary response found in infantile rats did not appear to be caused by a slower rate of disappearance of LHRH in blood of the younger animals. Ovariectomy-adrenalectomy. (Ovx-Adrx) or Ovx at day 10, but not Adrx alone, resulted in elevated LH and FSH levels 5 days later and almost complete obliteration of the FSH response to LHRH. The LH response was not altered. Treatment with 5alha-dihydrotestosterone (DHT) but not with estradiol benzoate (EB) or testosterone propionate (TP) suppressed the post-Ovx-Adrx rise in plasma LH and FSH. Progesterone (P) potentiated the effect of DHT. Restoration of basal plasma LH and FSH levels (by DHT and/or P) restored FSH responsiveness to exogenous LHRH. EB and TP were ineffective. The LH response was slightly depressed by EB + DHT. It is concluded that the elevated plasma FSH levels in the infantile female rat may be due at least in part to a high degree of pituitary responsiveness to LHRH and/or FSH-RF brought about by steroidal signal of ovarian origin. DHT and P appear to be the steroids responsible for such a stimulatory action.

On the role of the central noradrenergic and dopaminergic systems in the regulation of TSH secretion in the rat.

Systemic administration of drugs affecting central noradrenergic and dopaminergic systems was used to evaluate their role in the regulation of TSH secretion in the rat. Alpha-methyl-p-tyrosine (alpha-MT) caused a depletion of brain norepinephrine and dopamine and a gradual decrease of serum TSH levels. Specific inhibitors of dopamine-beta-hydroxylase, diethyldithiocarbamate (DDC) and FLA 63, depleted central norepinephrine only and led to a simultaneous striking decrease of serum TSH. Blockade of alpha adrenergic receptors with phenoxybenzamine, but not with phentolamine, also depressed serum TSH. Blockade of beta receptors with propranolol had no effect. In contrast, the centrally and peripherally acting alpha receptor agonist, clonidine, increased serum TSH, whereas the peripherally acting methoxamine caused a decrease, probably due to non specific stress effect. A dose-related rapid inhibition of TSH secretion was observed following stimulation of dopamine receptors with apomorphine. Injection of L-Dopa had a similar effect. Blockade of the dopamine receptors with pimozide did not alter serum TSH, while blockade with spiroperidol led to a slight increase. The cold-induced surgeof TSH was abolished by pretreatment with DDC or phenoxybenzamine, reduced by apomorphine, but unaffected by pimozide or propranolol. The pituitary responsiveness to exogenous TRH was unaffected by administration of DDC or apomorphine. On the basis of these results, it is assumed that the central noradrenergic system has a stimulatory effect on the release of TRH from the hypothalamus, reflected in our experiments by the changes of serum TSH levels. It probably provides the drive for the tonic release of TRH in resting conditions and stimuli for the enhanced secretion during cold exposure. The effect is probably mediated by a central alpha-adrenergic mechanism. Activation of the dopaminergic system is inhibitory, but the physiological role of this effect remains to be established.

Further studies on prostaglandin E2 (PGE2)-induced gonadotropin release: comparison with the effect of 15-methyl PGE2, PGAs, PGBs and prostaglandin endoperoxide analogs.

It is known that PGE2 is a potent stimulus of LH release. To determine if the effect of PGE2 could be enhanced and/or prolonged by retarding its metabolic degradation, a derivative, 15-methyl PGE2 (15-E2) which is more slowly degraded than the natural compound was injected intravenously (i.v.) at various dose levels or into the third ventricle (3rd V) of ether-anesthetized, ovariectomized, estrogen (OVX, Eb)-treated rats and its effect on gonadotropin release was compared with that of PGE2. Both PGs injected i.v. were equally effective in increasing plamsa LH and maintaining the elevated levels, although 15-E2 induced a larger and more sustained increase in plasma FSH than PGE2. By contrast, 3rd V PGE2 was clearly more effective than 3rd V 15-E2 in releasing LH and to a lesser extent, FSH. The effect of 15-E2 on LH was similar to that produced by 3rd V PGE1, injected at a similar dose. However, its effect on FSH was greater than that of PGE1. To evaluate the effect(s) of prostaglandins of the A and B series on gonadotropin release, PGA1, PGA2, PGB1 or PGB2 were injected intraventricularly in OVX, Eb-treated rats. PGBs were injected into conscious, free-moving rats. PGA2 or PGB2 increased plasma LH concentrations although much less effectively than PGE2. Third V PGA1 or PGB1 were ineffective. The 3rd V injection of two cyclic esters (U-44069 and U-46619), stable analogs of the PG endoperoxide PGG2 and PGH2, induced a small, transient increase in LH levels and did not alter plasma FSH in conscious, free-moving animals. PGE2 injected intraventricularly at a similar dose was demonstrated to be mcuh more potent than the analogs in stimulating LH and FSH release. The results indicate that: 1) 15-E2, in spite of its described long-lasting activity, does not appear to be more potent than the natural compound in releasing LH, although when injected i.v., it appeared to induce a more sustained increase in plasma FSH; 2) although PGA2 and PGB2 can also act centrally to stimulate LH release, their low potency suggests that this is a pharmacological effect; and 3) the two analogs of PG endoperoxides tested proved to be poor stimuli for gonadotropin release. The significance of these findings is discussed.

The onset of puberty in the female rat: changes in plasma prolactin, gonadotropins, luteinizing hormone-releasing hormone (LHRH), and hypothalamic LHRH content.

In order to study the sequence of hormonal changes that accompany the onset of puberty in the female rat, immature animals were sacrificed by decapitation between days 32 and 38, and plasma titers of gonadotropins, prolactin, and LHRH, and the hypothalamic content of LHRH were determined by specific radoimmunoassays (RIA). Animals were decapitated at 10:00 and 16:00 h throughout the pubertal period, the uterine weight was recorded, and the ovaries were inspected for signs of ovulation. Animals with the vagina closed were grouped according to the condition of the uterus as anestrus (A), early proestrus (EP) and late proetrus (LP), the uterus being unstimulated, dilated with some fluid, or ballooned, respectively. Vaginal opening was usually associated with ovulation and in most cases occurred at the end of the late proestrous phase. Animals were studied up to 3 days after vaginal opening and were grouped according to vaginal cytology. Uterine weight, taken as an index of estrogen secretion, was low during A, increased during EP, and reached a peak at LP, declining thereafter. Plasma LH and FSH were low from A until the afternoon of LP. At this time, uterine weight was maximal and both gonadotropins increased dramatically. The following morning (estrus), LH but not FSH, had returned to basal values. FSH returned to basal levels on the afternoon of estrus. Plasma prolactin was low in the morning during the entire period, but showed peaks on the afternoon, which reached a maximum at LP and declined thereafter following the pattern of changes in uterine weight. Plasma LHRH was uniformly low throughout the entire pubertal period, whereas hypothalamic LHRH content declined on the morning of estrus (day of vaginal opening). We suggest that the onset of puberty in the female rat is brought about by a gradual increase in estrogen secretion which, acting at the CNS-pituitary level, triggers a preovulatory proestrus-like surge of gonadotropins and prolactin.

Plasma prolactin levels in maturing intact and cryptorchid male rats: development of stress response.

In order to determine the possible role of the seminiferous tubules in the regulation of prolactin secretion during sexual development, male rats were rendered cryptorchid at 22 days of age, and thereafter different groups of animals were decapitated at 8-10 day intervals between Day 32 and Day 70. Cryptorchid rats showed destruction of the germinal epithelium accompanied by increased plasma FSH and, to a much lesser extent, increased plasma LH titers. Nevertheless, plasma prolactin levels were similar to those of intact controls throughout the entire period studied. Plasma prolactin titers in intact controls remained uniformly low from Day 20 to Day 70, contrasting with previous reports in which increasing prolactin levels have been observed during sexual development. To determine the reason for this apparent discrepancy, a longitudinal experiment was conducted in which intact and cryptorchid male rats were bled every 10 days from Day 30 to Day 70, following a 3-min period of exposure to ether fumes. The prolactin response to this stress increased markedly with age. A similar pattern of prolactin was observed in a cross-sectional study in which different groups of intact animals were bled following a 3-min period of ether exposure, at ages ranging from 20 to 70 daysmthe results indicate that unlike FSH secretion, prolactin secretion is not controlled by the seminiferous tubules. In addition, they suggest that the pattern of increasing plasma prolactin previously described in the developing male rat is at least in part caused by an age-dependent increase in responsiveness of prolactin to stress.

Developmental patterns of plasma and pituitary TSH and prolactin and hypothalamic TRH in the female rat.

In developing female rats, pituitary content and concentration of TSH and prolactin measured twice daily (1000 and 1600 h) were low during the first two weeks of life and increased markedly between day 15 and 30. AM-PM variations in pituitary levels of both hormones were apparent, particularly during this latter phase of development. Plasma TSH levels increased between days 5 and 12, showing peak values at this age. After day 15, levels declined gradually to reach a nadir shortly before puberty. At puberty, plasma TSH remained low, showing only minor fluctuations. No consistent AM-PM differences in plasma TSH were observed at any age studied. Plasma prolactin was low between day 5 and 15, increasing thereafter. Starting at day 10, AM-PM fluctuations in plasma levels were detected, titers being higher in the afternoon than in the mornings. It has already been reported (Endocrinology 98: 630, 1976) that during puberty this pattern becomes more evident, peak values being reached in the afternoon of the first proestrus. Hypothalamic TRH content increased between day 5 and 15, reaching a maximum at this age and declining thereafter to adult values. No qualitative changes in pituitary TSH during development were observed, as determined by exclusion chromatography. The existence of divergent patterns of plasma TSH and prolactin during female sexual maturation and the fact that hypothalamic TRH titers than with prolactin levels suggest that the mechanism(s) that stimulates pituitary release of these two hormones during sexual development is different.