Changes In Pituitary Sensitivity Research Articles

Exploring the Role of Pituitary Kisspeptin Receptor on Gonadotrophic Function In Vivo and In Vitro

The hypothalamic‐pituitary‐gonadal (HPG) axis is classically comprised of gonadotropin‐releasing hormone (GnRH) neurons in the hypothalamus that regulate the anterior pituitary gland, stimulating the gonadotrophs to secrete luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) into the bloodstream. LH and FSH after being synthesized and released by the pituitary gland go on to play essential roles in folliculogenesis, ovulation, and steroidogenesis. Kisspeptin (KISS1)/kisspeptin receptor (KISS1R) signaling in GnRH neurons has been shown by our group and others to play an essential role in HPG function. While the gonadotropes of the anterior pituitary express the Kiss1r gene, a physiological role for kisspeptin signaling at the level of the pituitary has not been demonstrated. We knocked out the Kiss1r gene specifically in pituitary gonadotropes by crossing the αGSUCre mouse with the floxed Kiss1r mouse, producing the PKiRKO mouse. Q‐RT‐PCR and immunohistochemistry were used to demonstrate a disruption in pituitary Kiss1r mRNA and KISS1R protein in PKiRKO mice relative to controls. Q‐RT‐PCR demonstrated a reduction in Kiss1r mRNA by 88% and 64% in the pituitary of male and female PKiRKO mice (n=8), respectively, compared with wild type (WT) mice (n=8). Immunostaining for KISS1R protein levels exhibited similar trends for protein knock down as observed for the relative mRNA levels. Our results revealed no difference in the age of puberty between WT and PKiRKO littermates, as assessed by the ages of vaginal opening, and first estrus for female mice, and preputial separation for male mice. Furthermore, we saw no difference in ovarian and testes weight respectively in female and male mice. While there were no differences in basal LH and FSH levels, upon performing a GnRH stimulation test in vivo, we observed a significant attenuation (P<0.05) in stimulated luteinizing hormone (LH) serum of PKiRKO male mice compared with WT male mice, while stimulated LH and FSH levels were no different between WT and PKiRKO female mice. These changes in pituitary sensitivity in males were associated with lower mRNA levels of GnRH receptor and estrogen receptor alpha. To directly study the effects of KISS1 on the pituitary gonadotroph, we sought to develop an in vitro primary culture system. To test the system, a GnRH dose response and time course study was performed on dispersed and adherent pituitary cells harvested from WT male and female mice. Groups of cells were treated with either 5nM, 30nM or 100nM GnRH, for a duration of 30 and 60 minutes. A significant increase in LH levels was observed in both male and female mice pituitary cells when treated with either 30nM or 100nM GnRH for 60 minutes. Female pituitary cells also showed a significant increase in LH secretion at the 30 minute time point. Therefore, a 30nM treatment for 60 minutes is suitable for GnRH stimulation and will be used for future studies. Included in those studies will be; WT and PKiRKO pituitary cells treatment with/without GnRH and with/without different doses of Kp10 (kisspeptin agonist). LH and FSH levels in the media will be measured after a 60 minute incubation. These studies will allow us to assess the direct effects of KISS1 on pituitary gonadotroph function that are mediated via the KISS1R. Overall this work will provide insight into the signaling of kisspeptin and Kiss1r on the gonadotroph neuron and how it contributes to both normal and abnormal reproductive physiology.Support or Funding InformationWe acknowledge support from the following grants 1.) U01HD066432 2.) P30DK079637 3.) R01068777 4.) R01DK101591

Pituitary gonadotropin-releasing hormone (GnRH) receptor: Structure, distribution and regulation of expression

Reproduction in mammals is controlled by interactions between the hypothalamus, anterior pituitary and gonads. Interaction of GnRH with its cognate receptor is essential to regulating reproduction. Characterization of the structure, distribution and expression of GnRH receptors (GnRH-R) has furthered our understanding of the physiological consequences of GnRH stimulation of pituitary gonadotropes. Based on the putative topology of the amino acid sequence of the GnRH-R and point mutation studies, key elements of the GnRH-R have been identified to play a role in ligand recognition and binding, G-protein activation and internalization. Normally, reproductive function is mediated by GnRH-R expressed only on the membranes of pituitary gonadotropes. The density of GnRH-R on gonadotropes determines their ability to respond to GnRH. This density is highest just prior to ovulation and likely is important for complete expression of the pre-ovulatory surge of LH. Therefore, knowledge regarding what regulates the density of GnRH-R is essential to understanding changes in pituitary sensitivity to GnRH and ultimately, to expression of the LH surge. Regulation of GnRH-R gene expression is influenced by a multitude of factors including gonadal steroid hormones, inhibin, activin and perhaps most importantly GnRH itself.

Changes in pituitary sensitivity to GnRH in estrogen-treated post-menopausal women: evidence that gonadotrophin surge attenuating factor plays a physiological role.

The purpose of this study was to investigate changes in pituitary response to GnRH in post-menopausal women during substitution treatment with exogenous estrogen and progesterone. Seven healthy post-menopausal women (group 1) were treated with various doses of E2 valerate for 43 days, so as the serum concentrations of E2 mimicked those of a follicular (FP-1), a luteal (LP) and a second follicular (FP-2) phase. During the LP, progesterone was also administered. The 30 min response of LH (DeltaLH) and FSH (DeltaFSH) to GnRH (10 microg i.v.) (pituitary sensitivity) was investigated every 24 h in group 1 and also in seven normally cycling women (group 2) during a spontaneous (control) follicular phase (FP). Based on the hormone profiles, day 32 in group 1 (FP-2) corresponded to day 2 in the spontaneous FP of group 2. Basal FSH concentrations were significantly higher in FP-2 than in the control FP (P < 0.05), while basal LH concentrations were similar in the two phases with higher values in FP-2 towards the end of the experiment (corresponding to days 10 and 11, P < 0.05). However, an LH surge was seen only in the control FP. DeltaFSH values remained stable in both phases and increased only in the control FP on days 12 and 13. DeltaLH values remained stable in the control FP and only increased on days 12 (P < 0.05) and 13 (P < 0.05), but in FP-2, DeltaLH values increased earlier (corresponding to day 7, P < 0.05). The present study demonstrates for the first time that in the absence of ovarian function, follicular phase E2 concentrations sensitize the pituitary to GnRH at an earlier stage (corresponding to the midfollicular phase) than in the normal menstrual cycle (late follicular phase). It is suggested that during the early to midfollicular phase the ovaries produce a gonadotrophin surge attenuating factor (GnSAF) that antagonizes the pituitary-sensitizing effect of E2 to GnRH.

Chapter 7 Hypothalamic and limbic expression of CRF and vasopressin during lactation: implications for the control of ACTH secretion and stress hyporesponsiveness

Lactation is associated with physiological and behavioral changes that optimize conditions for development of the offspring. Although neuroendocrine and emotional stress responses are blunted, the central mechanisms involved are unclear. In addition to a reduction in stimulatory noradrenergic inputs to paraventricular nucleus (PVN) neurons, we demonstrate that lactation induces: (1) unique phenotypic changes in neuropeptide expression by hypothalamic PVN neurons (reduced expression of corticotropin-releasing factor (CRF) mRNA and increased expression of vasopressin mRNA in parvocellular PVN neurons); and (2) changes in pituitary sensitivity to CRF (reduced) and vasopressin (increased) as a consequence of differential CRF/vasopressin secretion into the hypophysial portal blood. Neurons in the bed nucleus of the stria terminalis (BNST) and the central amygdala (CeA) that are implicated in the control of the hypothalamopituitary-adrenal axis also display changes in lactation: expression of CRF mRNA in the CeA is reduced, consistent with the diminished responsiveness to acoustic startle observed in nursing mothers. In contrast, expression of CRF mRNA is increased in the dorsolateral portion of the BNST, probably because of the tonic increases in endogenous glucocorticoid production during this period. Using immuno-targeted lesions of CRF or vasopressin in the PVN of virgin females, we have shown that CRF neurons of the PVN send inhibitory projections to the dorsolateral portion of the BNST and stimulatory inputs to CRF neurons in the CeA. Thus, it is possible that lactation-induced changes in the activity of parvocellular PVN neurons might also modulate the expression of neuropeptides and neurotransmitters in the BNST and the amygdala.

Changes in pituitary, ovarian, and testicular activity in harbour seals (Phoca vitulina) in relation to season and sexual maturity

This study examined pituitary sensitivity to exogenous gonadotrophin-releasing hormone (GnRH) in relation to time of year and changes in plasma progesterone, testosterone, luteinising hormone (LH), and follicle-stimulating hormone (FSH) concentrations during the annual cycle of adult and juvenile harbour seals (Phoca vitulina vitulina). There was no significant difference in seasonal changes in reproductive hormone levels between six captive individuals and seals caught from the wild (n = 80). Based on results from both the captive and the wild individuals, the plasma progesterone concentration was elevated in late gestation, whereas it declined at the equivalent time in nonpregnant females. The progesterone concentration was also elevated during the phase of delayed implantation, but there was no difference between pregnant and nonpregnant seals. In captive seals given exogenous GnRH, the LH concentration peaked 10-20 min after injection of GnRH, and the magnitude of the LH response depended upon the dose up to ~120 ng·kg-1. FSH did not respond to GnRH in the time course of the experiment. Pituitary sensitivity to GnRH was greatest in the summer and autumn and lowest in the winter and spring. Seasonal changes in pituitary sensitivity were apparent in both adults and juveniles. There was evidence that three of the four juvenile seals attained puberty during the study. We conclude that the seasonal patterns of pituitary sensitivity and plasma hormone concentration in harbour seals are consistent with a reproductive cycle under photoperiodic control and with the general mechanisms underlying photoperiodic control of seasonal reproduction in higher vertebrates.

Premature elevation in serum insulin-like growth factor-I advances first ovulation in rhesus monkeys.

Previous data suggest that developmental increases in peripheral concentrations of insulin-like growth factor-I (IGF-I) may be one of several neuroendocrine signals that regulate the timing of puberty. In order to test this hypothesis further, normal juvenile female rhesus monkeys (Con; n = 6) were compared with age-matched animals (Igf; n = 4) which received a constant subcutaneous infusion of recombinant human IGF-I (110 micrograms/kg/day) from 18 through 36 months of age. Menstrual bleeding was monitored and ovulation was inferred from a sustained rise in serum progesterone. In order to assess the sensitivity of luteinizing hormone-releasing hormone (LHRH) neurons to excitation, the response of serum LH to the acute administration of the glutamate receptor agonist N-methyl-D, L-aspartic acid (NMDA) was assessed prior to menarche, 2 months following menarche, and during the follicular phase of a female's third ovulation or 50 days after a female's first ovulation. In addition, the pituitary response of LH secretion to an LHRH agonist was assessed during the follicular phase of a female's fourth ovulation or 75 days following her first ovulation. IGF-I treatment effectively elevated serum concentrations by more than 86% of the values observed in Con animals. Although the treatment also enhanced the developmental increase in IGF binding protein-3 (IGFBP-3), IGF-I was increased proportionately more, resulting in a significantly higher molar ratio of IGF-I:IGFBP-3 in treated females throughout the course of the study. Treatment with IGF-I did not affect age at menarche but did significantly advance the age of first ovulation. Consequently, the interval between menarche and first ovulation was significantly shorter in Igf compared with Con females. Although the total number of ovulations exhibited by Igf (3.8 +/- 0.3) and Con females (3.0 +/- 0.5) in the 12 months following menarche was similar, significantly more of these were characterized by normal luteal phase progesterone secretion in Igf (100% +/- 0) compared with Con females (64% +/- 17). An analysis of cycles with a normal luteal phase revealed that serum estradiol during the luteal phase was significantly higher in Igf compared with Con females. Finally, IGF-treated females responded to NMDA treatment with a significantly greater increase in serum LH following menarche but not prior to menarche. In contrast, the response of serum LH to an LHRH agonist did not differ between Igf and Con females. These data suggest that the premature elevation in IGF-I levels, and consequently the ratio of IGF-I:IGFBP-3, accelerates the tempo of the final stages of puberty in rhesus monkeys. This action of IGF-I is probably the result of an increase in LHRH neuronal activity and is not due to a change in pituitary sensitivity to LHRH stimulation. In addition, ovarian sensitivity to LH stimulation during the luteal phase is also increased by IGF-I. Taken together, these data suggest that developmental increases in peripheral IGF-I secretion participate in the neuroendocrine regulation of puberty in female primates.

Developmental and physiological aspects of Ca2+ signaling in agonist-stimulated pituitary gonadotrophs.

GnRH-induced Ca2+ signaling was analyzed in gonadotrophs from pituitary glands of neonatal, prepubertal, peripubertal, cycling, and ovariectomized female and adult male rats. In all cases, single cell studies showed three types of Ca2+ responses to increasing GnRH concentrations: subthreshold, baseline oscillatory, and biphasic (an early transient followed by sustained oscillations or a plateau). An increase in the frequency, but not the amplitude, of spiking, and the presence of both baseline oscillatory and biphasic Ca2+ responses were observed in single cells from all groups exposed to low and subsequently to higher GnRH concentrations. Thus, modulation of the frequency of Ca2+ spiking at low to medium GnRH concentrations and suppression of the oscillatory response at high agonist concentrations are characteristics of the gonadotroph that are not affected by age, sex, stage of the estrous cycle, or gonadectomy. Ca2+ influx through plasma membrane channels contributed to both baseline oscillatory and biphasic responses, and both types of Ca2+ signaling were associated with LH release. The sensitivity of gonadotrophs to GnRH stimulation was similar in cells from single and multigland preparations from the same group of donors, but varied according to age and physiological status. Thus, sensitivity was low in neonatal gonadotrophs and progressively increased in postnatal cells. In cycling females, gonadotrophs from proestrous animals were more sensitive than those from estrous and diestrous animals. Ca2+ signaling was more uniform in gonadotrophs from ovariectomized than in those from normal females. During prolonged culture there was an increase in the frequency of Ca2+ spiking in response to 1 nM GnRH over the first 3 days, followed by a gradual decrease over 7-10 days and loss of the response in older cultures. These changes in Ca2+ signaling were accompanied by similar changes in the GnRH-induced secretory responses. These data indicate that frequency-controlled spiking, with suppression of the oscillatory mechanism at high agonist concentrations, is a characteristic of Ca2+ signaling in rat gonadotrophs. In addition, developmental, physiological, and in vitro changes in pituitary sensitivity to GnRH are expressed through modulation of the Ca2+ signal and the consequent secretory response.

Induced moulting in cockerels: Effects on sperm production, plasma concentrations of luteinising hormone, testosterone and thyroxine, and on pituitary sensitivity to luteinising hormone‐releasing hormone

1. The ability of a moult-inducing procedure to restore high levels of sperm production was assessed, in two experiments, using cockerels with reduced sperm production. The moulting procedure consisted of a period of food and light restriction for 6 weeks. The moulted birds were compared with control birds for 20 weeks. 2. Moult induction resulted in decreased daily sperm output (DSO) and plasma testosterone concentration, from weeks 3 to 7. In the first experiment, plasma luteinising hormone (LH) concentration in moulted birds was reduced on week 5. 3. No change in pituitary sensitivity to chicken luteinising hormone-releasing hormone-I (cLHRH-I) was detected at week 3 in moulted cockerels. In moulted birds, a transient increase in plasma thyroxine concentration was detected. 4. After the end of moult induction, testosterone concentrations increased, plasma LH showed a rebound at week 7 and the pituitary sensitivity to LHRH was increased at week 9. 5. This increased activity of the pituitary-testicular axis resulted for a short time in an increase in DSO of moulted birds compared with that of controls. Although amelioration was moderate, this result indicates the possibility of improving sperm production in the cockerel by using an induced moulting procedure.

Destruction of testicular leydig cells reveals a role of endogenous inhibin in regulating follicle-stimulating hormone secretion in the adult male rat

It has previously been demonstrated that passive immunoneutralization of endogenous inhibin results in a dramatic elevation in follicle-stimulating hormone (FSH) secretion in the adult female rat but not in the adult male. The purpose of the present study was to investigate whether the effects of immunoneutralizing endogenous inhibin on FSH secretion in the adult male rat might be masked by the presence of additional, compensating, FSH-suppressing factors. This was determined by examining the individual and combined effects of removing the testicular influences provided by the Leydig cells using the selective toxicant, ethane dimethane sulfonate (EDS), and passive immunoneutralization of endogenous inhibin. Within 24 h of a single i.p. injection of EDS, plasma testosterone levels were lowered to near assay limits and by 3 days were undetectable. Plasma FSH levels were significantly elevated 3 and 7 days after EDS treatment, but not to the levels observed in rats castrated for similar periods of time. Castration of rats, treated 3 days earlier with EDS, resulted in a further significant increase in FSH secretion as compared with EDS-treated, sham-operated controls, indicating that the testes were providing an additional FSH-suppressing factor(s) other than those originating in the Leydig cells. Injection of anti-inhibin serum, into rats treated 3 or 7 days earlier with EDS, induced a further significant increase in FSH secretion that raised plasma FSH to a level comparable to that observed in male rats castrated for similar periods of time. Plasma LH secretion was also dramatically elevated by EDS treatment to levels that equaled or exceeded those observed in similarly timed castrates. Pituitary sensitivity, as tested by the injection of an exogenous challenge of luteinizing hormone-releasing hormone (LHRH), was significantly increased 3 or 7 days after either EDS treatment or castration in terms of LHRH-stimulated LH release, but not in terms of LHRH-stimulated FSH release. Immunoneutralization of endogenous inhibin induced no further observable changes in pituitary sensitivity to LHRH. These results demonstrate that in the absence of the Leydig cells a secondary role is revealed for endogenous inhibin in suppressing FSH secretion that, in combination with the Leydig cell influence(s), accounts for the postcastration increase in FSH. The need to remove the Leydig cell influence(s) to reveal an effect of endogenous inhibin on FSH secretion in the adult male rat may suggest that the inhibin effect is normally masked by the presence of the comparatively larger

Paradoxical effect of corticosteroids on pituitary ACTH/β-endorphin release in stressed animals

We have previously demonstrated a number of changes in the anterior lobe proopiomelanocortin (POMC) system in chronically stressed rats. The purpose of the present experiments was to investigate whether chronically stressed rats demonstrate changes in pituitary sensitivity to glucocorticoid negative feedback. To study this question we compared the effects of glucocorticoids on ovine corticotropin releasing factor (oCRF)-stimulated ACTH and β-endorphin release from cell suspensions prepared from naive unhandled rats versus chronically stressed rats. After dexamethasone, there was a 50% decrease in oCRF-stimulated hormone release in control rats but no inhibition of oCRF-stimulated hormone release in anterior lobe suspension from chronically stressed rats. Rather, the chronically stressed group exhibited a 50% hormone increase above the oCRF-stimulated baseline. The same pattern was observed after the addition of corticosterone to the medium. These findings suggest that there may be a positive feedback effect of glucocorticoids at the pituitary level under some conditions of chronic stress.

Continuous infusion of GnRH reduces the LH response to an intravenous GnRH injection but does not inhibit endogenous LH secretion in cows.

Plasma LH concentrations were monitored in 6 Hereford X Friesian suckled cows at about 80 days post partum, before and during a 14-day period of continuous s.c. infusion of GnRH (20 micrograms/h). Blood samples were collected at 10-min intervals on Days -2, -1, 1, 2, 3, 4, 7, 10, 13 and 14 (Day 1 = start of infusion). Plasma LH concentrations rose from mean pretreatment levels of 1.3 +/- 0.20 ng/ml to a maximum of 17.1 +/- 3.09 ng/ml within the first 8 h of GnRH infusion, but returned to pretreatment levels by Day 2 or 3. In 4/6 animals, the initial increase was of a magnitude characteristic of the preovulatory LH surge. In all animals, an i.v. injection of 10 micrograms GnRH, given before the start and again on the 14th day of continuous infusion, induced an increase in LH concentrations but the increase to the second injection was significantly (P less than 0.01) less (mean max. conc. 6.4 +/- 0.76 and 2.3 +/- 0.19 ng/ml). Mean LH concentrations (1.0 +/- 0.08, 1.1 +/- 0.08 and 0.9 +/- 0.06 ng/ml) and LH episode frequencies (3.3,4.3 and 3.2 episodes/6 h) did not differ significantly on Days -2,7 and 13. However, the mean amplitude of LH episodes was significantly lower (P less than 0.05) on Day 13 (1.3 +/- 0.10 ng/ml) than on Day -2 (1.8 +/- 0.16 ng/ml). Therefore, although the elevation in plasma LH concentrations that occurs in response to continuous administration of GnRH is short-lived and LH levels return to pre-infusion values within 48 h of the start of infusion, these results show that the pituitary is still capable of responding to exogenous GnRH, although the LH response to an i.v. bolus injection of GnRH is reduced. In addition, this change in pituitary sensitivity is not fully reflected in endogenous patterns of episodic LH secretion.

Diminished responsiveness of the hypothalamic-pituitary-adrenal axis of the rat during exposure to prolonged stress: a pituitary-mediated mechanism.

Intact rats exposed to low or moderate intensity electroshocks for 3-5 h showed a marked increase in plasma ACTH levels 10 min after the beginning of the stress, followed by a decline despite continuous exposure to the stimulus. We have explored the role of steroid feedback, desensitization of the pituitary response to CRF, or changes in pituitary ACTH content in mediating this phenomenon. The following results were obtained. Exposure of adrenalectomized rats to shocks showed that removal of steroid feedback did not restore the ability of the animals to maintain elevated levels of circulating ACTH during electroshocks. To determine whether prolonged stress caused changes in pituitary sensitivity to CRF, intact rats received CRF, epinephrine, vasopressin, or phorbol ester at the end of the 3-h shock session; all secretagogues caused a significantly smaller increase in the plasma ACTH levels in intact rats subjected to low or moderate intensity shocks compared to that of control animals, which suggested that there was no specific desensitization to CRF. By contrast, pituitary responsiveness to CRF was not significantly altered in adrenalectomized rats submitted to low intensity shocks for 1-3 h; however, when moderate intensity shocks were used, adrenalectomized rats showed a blunting of the response to CRF comparable to that in intact animals. Finally, we observed a comparable decrease in the pituitary ACTH content of intact or adrenalectomized rats exposed to electroshocks; this decrease was proportional to the length and intensity of the shocks. We conclude that the inability of continuously stressed rats to maintain elevated plasma ACTH levels appears to be mediated through both the temporary decrease in a readily releasable pituitary ACTH pool and the negative feedback exerted by corticosterone.

Luteinizing hormone secretion from the quail pituitary in vitro.

An enzymatically dispersed pituitary preparation from Japanese quail (Coturnix coturnix) was used to study the dynamics of gonadotropin release. After an 18-h incubation, the cells were challenged with different luteinizing hormone-releasing hormones (LHRH) for 90 min. Using pituitary cells from mature males, mammalian and chicken LHRH I (Gln8-LHRH) had approximately equal luteinizing hormone (LH)-releasing activity whereas chicken LHRH II (His5, Trp7, Tyr8-LHRH) was 8-9 times more potent. The LHRH agonist (Trp6, Pro9-NEt-LHRH) had 15 times greater potency than chicken LHRH I. Pre-incubation with an LHRH antagonist (D-Phe2, D-Trp6-LHRH) significantly suppressed LH release. Acid extracts of median eminence released LH from pituitary cells, extracts from short-day and long-day males had equal activity, while tissue extracts from castrated males had significantly greater LH-releasing activity. Pituitary cells from sexually immature males released LH in response to chicken LHRH I in a similar profile to cells from mature males. These data indicate that the quail LHRH receptor in the male recognizes several different molecular species of LHRH and the response to LHRH is comparable between short- and long-day males. Pituitary cells from ovulating females were variably sensitive to LHRH peptides, possibly due to changes in pituitary sensitivity during the ovulatory cycle. Pituitary cells from immature females did not release LH in response to chicken LHRH I. However, pituitary cells from immature females photostimulated for 1 wk displayed a response to chicken LHRH I and II similar to that of pituitary cells from males.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperprolactinemia alters the frequency and amplitude of pulsatile luteinizing hormone secretion in the ovariectomized rat.

Studies were undertaken to examine the effects of hyperprolactinemia on the frequency and amplitude of pulses of LH, and determine if changes in pituitary sensitivity to LHRH were involved in the prolactin-induced suppression of LH secretion. Rats were bilaterally ovariectomized (day 0). Ovine prolactin (4 mg/kg body weight, subcutaneously) or vehicle was administered every 8 h beginning at 09.00 h on day 4 after ovariectomy and continuing until 09.00 h on day 6. On day 6, between 07.00 and 09.00 h all animals received a right atrial cannula, using ether anesthesia. In experiment I blood samples were taken at 10-min intervals beginning at 12.00 h on day 6, for a total of 180 min. To test the effect of hyperprolactinemia on pituitary responsiveness (experiment II) animals received an intravenous injection of LHRH (25 ng/100 g body weight) after the 180-min and again after the 240-min sample. Blood was drawn every 10 min for a total of 300 min. Serum was assayed for LH. Hyperprolactinemia altered the pattern of pulsatile secretion of LH. Treatment with ovine prolactin produced a decrease in both the frequency and amplitude of the LH pulses compared to values found in control animals. However, no differences in pituitary responsiveness between hyperprolactinemic and control animals were found at the dose of LHRH given. Thus, the prolactin-induced suppression of pulsatile secretion of LH was not apparently a result of alterations in the sensitivity of the pituitary to LHRH. From these studies we suggest that hyperprolactinemia directly affects a hypothalamic site which ultimately alters the LHRH pulse generator, thereby changing the secretion of LHRH.

Selective release of follicle-stimulating hormone by 5 alpha-dihydroprogesterone in immature ovariectomized estrogen-primed rats.

The effect of 5 alpha-dihydroprogesterone (5 alpha-DHP) on gonadotropin release was examined in the immature acutely ovariectomized (OVX) rat primed with a low dose of estradiol (E2). Treatment with various doses of 5 alpha-DHP given in combination with E2 increased levels of follicle-stimulating hormone (FSH) but had no effect on serum luteinizing hormone (LH). A single injection of a maximally stimulating dose of 5 alpha-DHP (0.4 mg/kg) stimulated increases in serum FSH at 1200 h and, 6 h later, at 1800 h. Pituitary LH and FSH content was dramatically enhanced by 1600 h and levels remained elevated at 1800 h. The administration of pentobarbital at 1200 h, versus 1400 h or 1600 h, prevented the increase in basal serum FSH levels at 1800 h, implying that the release of hypothalamic LH releasing hormone (LHRH) is modulated by 5 alpha-DHP. In addition, changes in pituitary sensitivity to LHRH as a result of 5 alpha-DHP were measured and a significant increase in the magnitude of FSH release was observed at 1200 h and 1800 h. Although the LH response to LHRH in 5 alpha-DHP-treated rats was not different from controls, the duration of LH release was lengthened. These results suggest that 5 alpha-DHP may stimulate FSH release by a direct action at the pituitary level. Together, these observations support the theory that 5 alpha-DHP mediates the facilitative effect of progesterone on FSH secretion and further suggests an action of 5 alpha-DHP in this phenomenon at both pituitary and hypothalamic sites.

The absence of an age-related change in the pituitary response to growth hormone-releasing factor in rats.

The capability of the anterior pituitary gland to secrete growth hormone (GH) in response to an intravenous injection of growth hormone-releasing factor, hpGRF-44, was evaluated in male rats ranging from 22 days to 24 months of age. The increase in GH concentrations following a submaximal dose of hpGRF-44 (0.5 microgram/kg) was similar among the different aged rats suggesting there is no age-related change in pituitary sensitivity to hpGRF-44. Likewise, the response to a maximal dose of this peptide (25 micrograms/kg) was not different in young and old animals indicating there is no decrease in the readily releasable pool of GH. Thus, the age-related changes in GH secretion observed in rats do not appear to be due to a change in the pituitary response to the hypothalamic growth hormone-releasing factor.

Desensitization to Gonadotropin-Releasing Hormone Observed in Superfused Pituitary Cells on Cytodex Beads*

Chronic exposure to gonadotropin-releasing hormone (GnRH) in vivo eventually results in a failure to maintain maximal gonadotropin secretion, suggesting a loss of pituitary responsiveness. To examine changes in pituitary sensitivity in vitro, we have developed a superfusion technique in which dissociated rat anterior pituitary cells are attached to Cytodex beads. In this system, continuous administration of 30 nM GnRH caused an initial 20-fold increase in LH and FSH secretion rates. However, hormone secretion declined rapidly, reaching basal levels within 12 h. Both the calcium ionophore A23187 and the cocarcinogen phorbol myristate acetate were effective in releasing additional LH and FSH from cells made refractory to 30 nM GnRH, whereas higher doses of GnRH and 8-bromo-cAMP were minimally active. A rapid loss of response was also seen with a superactive GnRH agonist but was not observed with a GnRH antagonist. Desensitization to prolonged superfusion with phorbol myristate acetate was also observed, although these cells remained sensitive to GnRH. In contrast to the development of tolerance produced by continuous exposure to GnRH, repeated delivery of GnRH in a pulsatile fashion maintained pituitary responsiveness for more than 24 h. A priming effect was observed for both LH and FSH. Such results demonstrate that continuous, but not intermittent, exposure to GnRH is capable of producing desensitization in superfused anterior pituitary cells.

Adrenal corticosteroids and immunoreactive ACTH in chronically cannulated ovine foetuses with bilateral adrenalectomy.

Abstract. The adrenal fails to develop in the hypophysectomised ovine foetus but adrenal growth, maturation and increased cortisol secretion occur in the intact foetus whilst plasma immunoreactive ACTH remains at a constant value. The presence of a feedback relationship between foetal adrenal and pituitary was examined in 8 ovine foetuses which were bilaterally adrenalectomised (adrx.) at the time of implantation of catheters in the carotid artery and jugular vein (90–127 days of gestation). Ten chronically cannulated foetuses served as controls. Gestation length in these sheep was 147 ± 5 days. Plasma immunoreactive (IR) ACTH (IR.ACTH) was measured with a Sorin Biomedica kit. The antibody cross-reacts with ACTH1–39, and ACTH1–24, but not with ACTH1–13, ACTH18–39, β-LPH or β-endorphin. Cortisol (F) and corticosterone (B) were measured by a double isotope dilution derivative assay. In intact foetuses the IR.ACTH was 69.6 ± 7.1 pg/ml (mean ± sem, n = 37) until the day before parturition when values increased to 326 ± 60 pg/ml (n = 6). In adrx. foetuses of 91–121. 122–135 and &gt; 136 days gestation the IR.ACTH values were 50.3 ± 4 (n = 14), 444 ± 46 (n = 26) and 1155 ± 175 (n = 6) pg/ml, respectively. The foetal F and B concentrations were 0.12 ± 0.027 μg/100 ml and 40 ± 6 ng/100 ml (n = 14), respectively, in adrx. foetuses. These values are not significantly different from those in intact foetuses younger than 135 days. The mean F and B concentrations in adrx. foetuses were 22 ± 4.3% (n = 14) and 49 ± 5.4% (n = 14) of the respective maternal values. In summary prior to 120 days the IR.ACTH, F and B concentrations are similar in intact and adrx. foetuses. After 122 days the IR.ACTH secretion by the foetal pituitary first begins to exhibit a feedback relationship with some adrenal secretion (?not F or B) and after 130–135 days some subtle change in pituitary sensitivity to F and/or B may operate. It is suggested that about 120 days a significant alteration in the foetal pituitary capacity for ACTH synthesis occurs.

The effect of LH-RH administration on LH release in the female rabbit

Intracarotid infusion of LH-RH to female rabbits stimulated a significant increase in plasma LH concentration in the jugular vein. This response varied with the reproductive state of the animal, with a greater release occurring in oestrous (spontaneous or oestrogen-induced) and non-receptive does than in pseudopregnant or ovariectomized animals. If ovariectomized rabbits were pretreated with oestrogen, the pituitary response to LH-RH was restored. These findings suggest that there is little change in pituitary sensitivity to LH-RH infusion between oestrous and non-receptive rabbits, although pseudopregnancy (high physiological levels of progesterone) or ovariectomy inhibit its ability to respond to a releasing-hormone stimulus.

Environmental influences on hormonally induced spermiation of the bullfrog, Rana catesbeiana

AbstractFrog pituitaries, human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH) and gonadotropinreleasing hormone (LH/FSH‐RH) were tested to determine their effectiveness in eliciting the spermiation response in laboratory‐reared male bullfrogs (Rana catesbeiana). LH/FSH‐RH gave the highest percentage of positive responses and the most rapid responses. Sperm acquired by cloacal sampling after LH/FSH‐RH injections were fertile.Day length (14L:10D; 8L:16D, 25°) had no measurable effect on reproductive condition, growth or sensitivity to injections of LH/FSH‐RH. Spermiation response to LH/FSH‐RH was depressed at low temperatures (15°, 20° C) and testicular recrudescence appeared to be inhibited at warm temperature (30°C). Optimum spermiation responses were obtained at 25°C.A marked daily rhythm (unimodal) of spermiation response to LH/FSH‐RH was observable. Injections given one hour before the onset and offset of a 12L:12D photoperiod (25°C) were respectively the most and least effective in stimulating spermiation. The peak and trough of the rhythm were attributed to changes in pituitary sensitivity to LH/FSH‐RH and not changes in testicular sensitivity to LH.Age and size of bullfrogs utilized in these tests had no effect on spermiation response.