Luteinizing hormone releasing factor in pituitary stalk plasma from long-term ovariectomized rats: effects of steroids

The concentration of LH releasing factor (LH-RF) was measured by radioimmunoassay in blood collected from the cut pituitary stalk of long-term ovariectomized rats anaesthetized with Althesin. Stalk plasma LH-RF concentrations were increased immediately after ovariectomy (carried out at oestrus) and low at 2 and 4 days after operation. The concentrations then began to increase to reach a level at 24-28 days which was significantly higher than the concentrations during the oestrous cycle except for the time of the ovulatory surge at pro-oestrus. This pattern was similar to that of the concentrations of LH in jugular venous plasma taken from the same animals before exposure of the pituitary stalk. Like peripheral plasma LH concentrations, the concentrations of LH-RF in stalk plasma fluctuated and fell significantly and rapidly after the intravenous injection of 1 microgram oestradiol-17 beta. The release of LH-RF in long-term ovariectomized rats, into which had been implanted an oestradiol-containing Silastic capsule, was similar to the diurnal pattern of LH release; the afternoon increase in stalk plasma LH-RF concentration could be blocked by sodium pentobarbitone administered at 13.00 h and augmented by administering this anaesthetic at 13.00 h of the preceding day. The stalk plasma LH-RF concentrations in animals injected with oestradiol benzoate (OB) followed 72 h later with either OB or progesterone were lower than the concentrations in animals injected only with oil. These data show that in the rat (1) ovarian steroids could moderate LH release ('negative feedback') by inhibiting LH-RF release, and that in long-term ovariectomized animals (2) the oestradiol-induced circadian pattern of LH release is due to a circadian pattern of LH-RF release, and (3) the surge of LH produced by administering OB followed by either OB or progesterone is probably due mainly to a massive increase in the responsiveness of the anterior pituitary gland to LH-RF.     

Central administration of antisense oligodeoxynucleotides to neuropeptide Y (NPY) mRNA reveals the critical role of newly synthesized NPY in regulation of LHRH release

Compelling evidence shows that the episodic and cyclic secretion of hypothalamic luteinizing hormone releasing hormone (LHRH), the primary stimulator of pituitary LH release, is subject to regulation by neuropeptide Y (NPY). We have reported earlier that sequential treatment of ovariectomized (ovx) rats with estrogen and progesterone to stimulate a preovulatory-type LH surge elevated the levels of both NPY and preproNPY mRNA levels in the hypothalamus concomitant with dynamic changes in LHRH activity. The present study was designed to determine whether these elevations in NPY content and gene expression represent new synthesis of NPY that is crucial to elicit LHRH discharge. Ovx, steroid-primed rats received intracerebroventricular injections of an unmodified 20-mer oligodeoxynucleotide (oligo) complementary to the NPY mRNA sequence. Control rats were injected similarly with either saline or the sense or missense oligos. Results showed that control rats displayed a characteristic surge-type elevation in plasma LH levels that was not affected by the administration of missense or sense oligos. However, in rats injected with the antisense oligo, the steroid-induced LH surge was completely blocked. In an additional experiment, NPY peptide levels were measured in microdissected hypothalamic sites following the injection of antisense or missense oligos. NPY antisense oligo administration blocked the significant increases in NPY levels in the median eminence-arcuate area, the medial preoptic area and lateral preoptic area seen in control rats. These results suggest that sequential ovarian steroid treatment augments NPY synthesis in the hypothalamus and this newly synthesized NPY is critical for induction of the LHRH and LH surge.     

A significant role of leptin in the generation of steroid-induced luteinizing hormone and prolactin surges in female rats

Recent evidence suggests that leptin, the product of obese (ob) gene, may play an important role in the regulation of reproductive function. However, a possible role of leptin in the preovulatory surges of luteinizing hormone (LH) and prolactin (PRL) in rodents has yet to be explored, and thus examined in this study. Experiments were performed on both normally fed and 3-day starved rats, which were ovariectomized and primed with estradiol and progesterone. At 11:00 h on the day of the experiments, normally fed rats received an intracerebroventricular injection of artificial cerebrospinal fluid, anti-leptin serum, or normal rabbit serum. Three-day starved rats were given artificial cerebrospinal fluid or recombinant human leptin (2.5 microgram) via the same route. From 11:00 to 18:00 h, blood was collected every 30 min to measure LH and PRL. The 3-day starvation completely abolished both LH and PRL surges, but leptin resumed these hormonal surges to the levels of normally fed rats. In addition, anti-leptin serum given to normally fed rats significantly depressed LH surge and delayed the onset of PRL surge. This study is the first to demonstrate that leptin plays a physiologically important role in the generation of steroid-induced LH and PRL surges in female rats.     

The dithiocarbamate fungicide thiram disrupts the hormonal control of ovulation in the female rat

Thiram has been reported to inhibit dopamine-beta-hydroxylase (D beta H), thereby affecting norepinephrine (NE) synthesis. Because NE is a neurotransmitter that is known to play an important role in the hypothalamic regulation of pituitary function, the acute effects of the thiram on the hormonal control of ovulation in the rat were investigated. Ovariectomized, estrogen-primed female rats were given a single injection of thiram (0, 6, 12, 25, 50, and 100 mg/kg, i.p.) at 1100 h and serum LH was measured in serial bleeds. Thiram at 100 and 50 mg/kg completely blocked the LH surge in all rats tested, while 12 and 25 mg/kg blocked the surge in 40 and 75% of the treated animals, respectively. Six mg/kg had no effect. Ovulation was then assessed in intact, proestrous females in response to thiram administration (0, 12, 25, or 50 mg/kg) at 0900, 1100, 1300, or 1800 h. Ovulation was blocked by 25 and 50 mg/kg at 1300 h in all rats, but when injected at 1100 h only the 50 mg/kg dose was effective. No such blockade was found with 50 mg/kg injected at 0900 and 1800 h. To assess the influence of thiram on the LH surge in intact rats, additional females were dosed at 1300 h on the day of proestrus and blood collected over that same day. Thiram at 50 mg/kg blocked the LH surge in all rats, while 25 mg/kg blocked the surge in 60% of the females tested. No effect occurred with 12 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of prolactin secretion delays extinction of circadian LH surges in ovariectomized rats bearing estradiol implants

Ovariectomized rats bearing Silastic capsules containing estradiol exhibit a daily afternoon surge of luteinizing hormone (LH) which decreases with time until it is undetectable by Day 10 after implantation of estradiol. Increases in basal prolactin levels as well as afternoon surges are also observed. To determine if increased prolactin secretion contributed to the extinction of the circadian LH surges, we examined the patterns of LH and prolactin secretion in rats in which prolactin was suppressed by bromocriptine treatment. In vehicle-treated control rats, the magnitude of the LH surges decreased with time. Large LH surges were observed on Days 2 and 4. A significant decrease in the surge occurred on Day 6, and it disappeared by Day 10. Animals treated with bromocriptine also exhibited large LH surges on Days 2 and 4, and in addition, secreted a greater amount of LH than the control group on Days 6, 8, and 10. In ovariectomized rats bearing estradiol implants, large afternoon surges in prolactin were observed and by Day 6, basal prolactin levels were also elevated. Bromocriptine treatment completely suppressed prolactin secretion through Day 6, but a small afternoon rise was observed on Days 8 and 10. These findings suggest that elevated prolactin secretion may be one factor contributing to the extinction of circadian LH surges in the estrogen-treated rat.     

Termination at midpregnancy of the two daily surges of plasma prolactin initiated by mating in the rat

The pattern of plasma prolactin following uterine cervical stimulation consists of two surges each day, one nocturnal, occurring between 0100-0900 h (lights on 0600-1800 h), and one diurnal, occurring between 1500-2100 h. This pattern of prolactin continued throughout pseudopregnancy; the last surge was observed on the morning of day 11 (day 1 was taken as the first day of diestrus of pregnancy or pseudopregnancy). Prolactin levels remained low thereafter until the spontaneous proestrous surge on the afternoon of day 12, signalling the onset of a new estrous cycle. In contrast, the two daily prolactin surges did not continue throughout pregnancy, and in fact, were terminated sooner in pregnant animals than in pseudopregnant animals. The last diurnal surge was observed on day 8 while the last nocturnal surge was observed on day 10. The early termination of prolactin surges during pregnancy correlated with the increased secretion of rat placental lactogen. However, placental extracts obtained from day 11 of pregnancy and injected in large doses failed to inhibit prolactin surges in pseudopregnant animals. Prolactin surges also continued for a longer period of time in pseudopregnant rats bearing decidualized uteri than in pregnant animals. Thus, the two major components of pregnancy that differed from pseudopregnancy, that is, the presence of rat placental lactogen or decidual tissue, did not appear to account for the early termination of prolactin surges during pregnancy.     

Unimpaired postreceptor regulation of luteinizing hormone secretion by gonadotropin-releasing hormone and estrogen in aged rat anterior pituitary cells

Delayed, attenuated, or absence of the proestrous LH surge occurs in aging rats. To assess how aging affects the positive feedback action of 17 beta-estradiol (E2) on the pituitary, we determined the responsiveness of rat pituitary cells to GnRH and the secretagogues affecting intracellular signal transduction mechanisms in the presence or absence of E2. We also correlated the LH response to pituitary LH content. Anterior pituitaries excised from ovariectomized Sprague-Dawley rats, either young (3-4 months) or old (19-20 months), were enzymatically dispersed and then pretreated with or without E2 (0.6 nM) for 48 h, followed by incubation for 3 h with or without various secretagogues. The secretagogues included GnRH (1 and 10 nM), veratridine (increases Ca2+ influx; 5 and 10 microM), and phorbol 12-myristate 13-acetate (a protein kinase-C activator; 10 and 100 nM). LH in media and cells were measured by RIA and expressed on the basis of cellular DNA. GnRH, veratridine, and phorbol 12-myristate 13-acetate at all doses stimulated (P < 0.01) LH release in cells from both young and old rats. E2 stimulated (P < 0.05 to P < 0.01) all secretagogue-induced LH release in cells from both young and old rats, but only basal LH release (P < 0.05) in cells from young rats. The magnitude of both basal and secretagogue-induced LH release in either the presence or absence of E2 was smaller (P < 0.01) in cells from old than in those from young rats. The initial cellular LH was lower (P < 0.01) in cells from old than in those from young rats. The LH-releasing ability (expressed as a percentage of total cellular LH) of cells from old rats was identical (P > 0.05) to that of cells from young rats under all conditions studied. These results suggest that the reduced magnitude of LH release by cells from old rats may be attributed to reduced cellular LH, rather than to impaired estrogen feedback or impaired signal transduction mechanisms. It remains to be determined whether LH biosynthesis per cell and/or the number of gonadotropes decrease with age.     

Effect of restraint stress on the preovulatory luteinizing hormone profile and ovulation in the rat

Plasma profiles of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured during restraint stress on the day of pro-oestrus; these profiles were considered in relation to ovulation rate on the next day. Rats bearing a permanent jugular vein cannula were subjected to restraint, which was started 0, 1 or 2 h before the presumed onset of the LH surge and ended just before the beginning of the dark period. Exposure to restraint resulted in a suppression of the secretion of both gonadotrophins on the day of pro-oestrus. Suppression of the LH surge was virtually complete (plasma LH < or = 0.2 ng/ml) in 15 out of 32 stressed rats, and the ovaries of these rats contained graafian follicles with oocytes in germinal vesicle stage. In these rats, the LH surge did not occur 24 h later. In the remaining 17 rats, restraint resulted in a considerable suppression of the LH surge. Of these rats, five had an ovulation rate of 100% and four ovulated partially. In unruptured follicles of the latter, the oocyte had not resumed meiosis and the follicle wall was not luteinized. In the remaining eight rats with a reduced LH surge, ovulations had not occurred and graafian follicles were unaffected. The results of this study indicate that during pro-oestrus restraint stress suppresses and does not delay the release of preovulatory gonadotrophins. Partial suppression of LH by restraint does not result in induction of meiotic resumption without subsequent ovulation or in luteinized unruptured follicles.     

The effect of blockade of kappa-opioid receptors in the medial preoptic area on the luteinizing hormone surge in the proestrous rat

The present study examined whether blockade of kappa-opioid receptors in the medial preoptic area (MPOA) prior to the critical period on the afternoon of proestrus could prematurely evoke an ovulatory luteinizing hormone (LH) surge, and if so, whether norepinephrine (NE) is involved in mediating this response. In the first experiment, push-pull perfusion of the MPOA with nor-binaltorphimine (nor-BNI), a specific kappa-opioid receptor antagonist, was done in rats between 10.30 and 13.50 h on proestrus. To determine whether any resulting ovulation was due to a nor-BNI-induced increase in LH release, rats were injected with pentobarbital at 13.55 h to block the afternoon LH surge. In 7 of 10 rats, nor-BNI in the MPOA produced a large increase in LH release beginning between 12.30 and 13.30 h, and 5 of 7 ovulated. During MPOA perfusion with cerebrospinal fluid in our normal colony between 14.00 and 17.00 h, surges of LH release began in the majority of rats between 15.30 and 16.30 h. Thus blockade of MPOA kappa-opioid receptors advanced the LH surge by 3 h. The next experiment examined the effect of NE synthesis inhibition with bis(4-methyl-1-homopiperazinylthiocarbonyl) disulfide (FLA-63), or alpha-adrenergic receptor blockade with phenoxybenzamine (PBZ), on the nor-BNI-induced LH response. In 5 of 6 vehicle-treated rats, blockade of MPOA kappa-opioid receptors elicited a large increase in LH release and all 5 ovulated. In contrast, only 3 of 8 rats pretreated with FLA-63 had a large increase in LH release and ovulated, and PBZ prevented the nor-BNI-induced LH increase and ovulation in 4 of 4 rats. PBZ also prevented the afternoon LH surge and ovulation in 4 of 4 rats in our normal colony. Finally, HPLC measurement of NE levels in MPOA push-pull perfusate indicated no increase in NE release during the nor-BNI-induced or normal afternoon LH surges. These results indicate that antagonism of kappa-opioid receptors in the MPOA can prematurely evoke an ovulatory LH surge prior to the critical period on the afternoon of proestrus. Furthermore, the nor-BNI-induced as well as the normal afternoon LH surges are dependent on the proper functioning of central noradrenergic neurons, but do not involve increased NE release within the MPOA.     

Short-term regulation of gonadotropin subunit mRNA levels by estrogen: studies in the hypothalamo-pituitary intact and hypothalamo-pituitary disconnected ewe

In this study the levels of mRNA for the pituitary gonadotropin hormone subunits luteinizing hormone beta (LHbeta), follicle stimulating hormone beta (FSHbeta) and the common alpha-subunit were assessed during the acute feedback stages of estradiol benzoate (EB) action in ovariectomized (OVX) ewes with and without hypothalamo-pituitary disconnection (HPD). In OVX/HPD ewes maintained on hourly pulses of 250 micrograms of gonadotropin-releasing hormone (GnRH) a single i.m. injection of EB in oil caused a biphasic (decrease and then increase) change in plasma LH levels and a monophasic decrease in FSH levels. There was a decrease in pituitary alpha-subunit and FSHbeta mRNA levels during the acute negative (8 h post EB) and through the positive feedback (20 h post EB) stages of the response. No significant change was seen in LHbeta mRNA levels following treatment with EB. In hypothalamic-pituitary intact OVX ewes the same EB treatment as above caused a biphasic change in LH secretion with the positive feedback component being much greater than in GnRH-pulsed OVX-HPD ewes. The levels of mRNA for all three gonadotropin subunits were reduced by 8 h after EB injections and remained low throughout the positive feedback period. These data suggest that the LH surge in this experimental model does not require an increase in LHB mRNA levels. Furthermore, the fall in LHbeta subunit mRNA seen after estrogen injection of OVX ewes is most likely due to an effect of estrogen to decrease GnRH secretion, since pulsatile GnRH replacement prevents this effect. These data also show that estrogen feedback can effect rapid alterations in pituitary gonadotropin subunit mRNA levels. Short-term changes in FSHbeta mRNA are reflected in changes in FSH secretion; the same is not true for LH.     

Ovarian regulation of postcoital gonadotropin release in the rabbit: reexamination of a functional role for 20 alphadihydroprogesterone

In the rabbit, it has proposed that an ovarian progestin, 20 alpha-dihydroprogesterone (20alphaP), released at mating, is essential for a normal postcoital LH surge. However, we measured plasma levels of LH and 20alphaP after mating in rabbits and observed that the frequency, magnitude and time-course of changes in circulating levels of 20alphaP seemed inappropiate to account for the rapid and major surge of LH secretion. This prompted us to re-evalute the role of the ovary in regulating postcoital LH secretion. In chronically ovariectomized (greater than 30 days) does pretreated with estrogen, mating induced a normal LH surge in only 1 of 10 animals, indicating that an ovarian product in addition to estrogen is required for a normal postcoital LH surge. However, when 20alphaP was injected soon after mating in chronically ovariectomized does pretreated with estrogen, only 2 of 9 displayed normal LH surges; this proportion is not different from that (1/10) observed with estrogen treatment alone. To demonstrate that the estrogen treatment, which produced supraphysiologic plasma estradiol levels, did not itself block LH release, 6 intact anestrus females were treated with the same estrogen regimen. Estrus was induced in 5 and each displayed a large post-coital LH surge and ovulated. As a final test of the 20alphaP hypothesis, 5 spontaneously estrous does were ovariectomized within 15 min post coitum to abolish acute increases in circulating ovarian hormones. Three animals released LH in amounts and temporal pattern indistinguishable from intact estrous does. A fourth released smaller amounts of LH. Two of 4 sham-operated does also had normal LH surges. These findings indicate that ovarian hormones are required before mating to support the capacity of the LH secretory mechanism to respond to coitus. Chronic alteration in the hormonal milieu by ovariectomy appears to produce a change in the hypothalamo-hypophysial complex that is not reversed by estrogen, alone. More importantly, these results demonstrate clearly that neither 20alphaP nor any other ovarian hormone is required post coitum, at least after 15 min, for normal LH release.     

Changes in mediobasal hypothalamic gonadotropin-releasing hormone messenger ribonucleic acid levels induced by mating or ovariectomy in a reflex ovulator, the ferret

The ferret is a reflex-ovulating species in which receipt of an intromission induces a prolonged (+/- 12 h) preovulatory LH surge in the estrous female. This LH surge is probably stimulated by a large release of GnRH from the mediobasal hypothalamus (MBH). In Exp 1 we asked whether GnRH messenger RNA (mRNA) levels increase in response to mating so as to replenish the MBH GnRH stores needed to sustain the preovulatory LH surge. Estrous females were killed 0, 0.25, 0.5, 1, 3, 6, 14, or 24 h after the onset of a 10-min intromission from a male. Coronal brain sections ranging from the rostral preoptic area caudally to the posterior hypothalamus were processed for in situ hybridization using a 35S-labeled oligoprobe complementary to the human GnRH-coding region. We found no evidence of increased MBH GnRH mRNA levels during the ferret's mating-induced preovulatory LH surge. Instead, the number of GnRH mRNA-expressing cells dropped significantly in the arcuate region beginning 6 h after onset of intromission and remained low thereafter. Furthermore, cellular GnRH mRNA levels decreased in the arcuate region toward the end of the preovulatory LH surge. In Exp 2 we asked whether ovarian hormones regulate MBH GnRH mRNA levels in the female ferret. Ovariectomy of estrous females significantly reduced the number of GnRH mRNA-expressing cells in the arcuate region. This decrease was probably not due to the absence of circulating estradiol. Gonadally intact anestrous females had levels of MBH GnRH mRNA similar to those in estrous females even though plasma estradiol levels were equally low in anestrous females and ovariectomized females. Ovarian hormones other than estradiol may stimulate MBH GnRH mRNA levels in anestrous and estrous females.     

Inhibition of the LH surge in cyclic rats by stress is not mediated by opioids

The effects of intravenous (iv) administration of the opioid antagonists naloxone and naltrexone on the restraint-induced suppression of the pro-estrous LH surge were studied in cyclic female rats. To minimize stress during repeated blood sampling, the rats were provided with a jugular vein cannula. Restraint stress for 6 hrs starting at t = -1 h (the onset of the LH surge being at t = 0 h) caused a suppression of LH levels (including peak height) during the period of the LH surge. Repeated naloxone injections, given 3 h (1 mg), 4 h (0.5 mg) and 5 h (0.5 mg) after the onset of the LH surge, did not affect the restraint-induced inhibition neither did pretreatment with 1 mg naloxone at t = -75 min (i.e. 15 min before application of restraint). Naltrexone (2 mg) administered at t = -15 min induced higher plasma LH levels at t = -6 min. When rats were subsequently subjected to restraint for 5 hrs starting at t = -5 min, the restraint-induced inhibition of surge levels of LH was not affected. The results indicate that withdrawal of opioid activity in cyclic female rats before the presumed onset of the LH surge results in a premature rise of LH levels. This is in accordance with the notion that LH levels prior to the surge are under tonic inhibition of endogenous opioid peptides (EOP). In addition, the data show that opioid receptor antagonism during or before application of restraint does not alter the restraint-induced suppression of the LH surge. It is therefore concluded that EOP do not mediate the inhibitory effect of restraint stress on the LH surge in cyclic rats.     

Neuropeptide Y regulation of LHRH release in the median eminence: immunocytochemical and physiological evidence in hens

It has been suggested that hypothalamic median eminence (ME) might be a control site for luteinizing hormone-releasing hormone (LHRH) release. Thus, stimulatory and/or inhibitory inputs acting at this site might be involved in regulating LHRH release from the ME and, therefore, luteinizing hormone (LH) release from the anterior pituitary. Since a role for neuropeptide Y (NPY) on LH release has been suggested, we have hypothesized that NPY might act in the ME to control preovulatory LHRH release in hens. To examine this possibility we have determined: (a) the immunocytochemical distribution of LHRH and NPY in the ME of the hen, (b) the basal and NPY-stimulated release of LHRH in vitro from the ME of hens undergoing a natural or a premature preovulatory surge of LH, and (c) the tissue content of LHRH and NPY in microdissected MEs, at various times before and during a natural or a premature preovulatory surge of LH. A potential role for NPY on LHRH release in the ME is suggested for the following reasons. (a) There are opportunities for synaptic interactions between NPY and LHRH-containing axons at this site. LHRH-containing cell bodies localized in the anterior hypothalamus/medial preoptic area project to the ME. NPY-containing perikarya, concentrated in the ventromedial aspect of the arcuate nucleus, might contact LHRH processes going to the ME and/or might themselves send axons to the ME, (b) Addition of NPY to the incubation media increases LHRH release from microdissected ME tissue of hens killed at the time of the natural preovulatory surge of LH, but not in hens killed 7 h before the occurrence of this surge. However, the stimulatory effect of NPY on LHRH release can be induced at this latter time when a premature LH surge is elicited. While the natural preovulatory surge of LH occurs 4 h before the second ovulation in a sequence (C2 ovulation), administration of progesterone (P4) 10-14 h before the expected natural C2 ovulation advances the natural LH surge by 7-8 h. Thus, NPY might act as a physiological stimulus of LHRH release at the ME during the preovulatory surge of LH. This is suggested since in vitro basal LHRH release from denervated ME tissue does not change before and during the natural or the premature LH surge. Therefore, preovulatory release of LHRH in vivo might be under a continuous drive from stimulatory inputs to the LHRH neuron and NPY might be one of these stimulating factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

The development of the serum corticosterone rhythm in rats

The purpose of this study was to determine whether the sequence of changes that occur in the adrenal rhythm in maturing female rats (development of a peak, shift in acrophase and amplitude) requires experience with a photoperiodic stimulus or a change in ovarian status. The emergence of the serum corticosterone (CS) rhythm occured more quickly in adult rats placed in a 14 h light, 10 h dark (14:10) cycle at 50 days of age after rearing in constant light (LL) than in weanling rats placed in 14:10. Ovariectomy at weaning age did not alter the pattern of CS development in 14:10 although the amplitude of the peak was reduced even in 25-day-old rats. Adult rats reared in 14:10 held a population rhythm of CS longer after they were placed in LL than did weanling rats placed in LL. This difference was not dependent upon the presence of the ovaries since acutely and chronically ovariectomized (OVX) adult rats responded in a similar manner to adult controls. It can be concluded that the adrenal rhythm emerges as a function of age rather than as a result of a change in ovarian status. The capacity to synchronize serum CS to light-dark cycles develops in the absence of photoperiodic cues.     

Morphine amplifies norepinephrine (NE)-induced LH release but blocks NE-stimulated increases in LHRH mRNA levels: comparison of responses obtained in ovariectomized, estrogen-treated normal and androgen-sterilized rats

In these studies we examined the temporal effects of intracerebroventricular (i.c.v.) infusions of norepinephrine (NE) on plasma LH and on LHRH mRNA levels in the organum vasculosum of the lamina terminalis (OVLT) and in neurons located in the rostral (r), middle (m) and caudal (c) preoptic areas (POA) of ovariectomized, estrogen-treated rats. Thereafter, we compared these responses to those which occur in androgen-sterilized rats (ASR). NE infusions not only increased plasma LH concentrations but within 1 h after NE, LHRH mRNA levels also were increased significantly in the OVLT and rPOA but not in the mPOA or cPOA. By 4 h, these message levels still were elevated in the OVLT and rPOA and they now also were significantly higher than control values in the mPOA and cPOA. While NE also increased LH secretion in ASR, the plasma LH concentrations obtained were markedly blunted compared to control values. Moreover, NE infusions did not alter single cell levels of LHRH mRNA in any region of the rostral hypothalamus. Previously, we have reported that morphine (s.c.) markedly amplifies NE-induced LH release and questioned whether these responses are accompanied by concomitant augmented increases in LHRH mRNA levels. Morphine alone did not affect basal LHRH mRNA or plasma LH levels. However, when rats were pretreated with morphine (-15 min) and NE was infused i.c.v. at 0 time, significant amplification of LH release occurred but, unexpectedly, morphine completely blocked NE-induced increases in LHRH mRNA levels in all of the neurons we examined. Morphine also amplified LH release in ASR but these responses were significantly less than those obtained in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preoptic rather than mediobasal hypothalamic amino acid neurotransmitter release regulates GnRH secretion during the estrogen-induced LH surge in the ovariectomized rat

Inhibitory and excitatory amino acid neurotransmitters have been suggested to participate in the feedback actions of estradiol (E2) on LH secretion. In the rat estrogen-receptive neurons have been demonstrated in the preoptic/anterior hypothalamic area (POA) and mediobasal hypothalamus/median eminence (MBH) and many of these neurons utilize gamma-aminobutyric acid (GABA) as neurotransmitter. The actions of excitatory amino acids (EAA) differ in ovariectomized (ovx) and ovx E2-substituted rats indicating that EAAs also participate in the positive feedback action of E2 on LH release. However, little information is available as to whether in vivo these transmitters exert their effects in the POA, where most of the GnRH perikarya are located, or in the MBH, i.e. at the nerve terminals. Therefore we conducted push pull cannula perfusions to compare the release rates of GABA, aspartate (ASP) and glutamate (GLU) in the MBH and POA. A subcutaneous implant of a silastic tube containing E2 resulted in LH surges in the afternoon of all treated animals. Prior to and during this LH surge the MBH release rates of neither GABA nor ASP nor GLU were significantly altered. In contrast, a conspicuous drop in preoptic GABA release occurred prior to and during the time of estrogen-induced LH surges and this was accompanied by enhanced preoptic secretion of ASP and GLU. In conclusion, we present the first data about amino acid release in the MBH during the E2-induced LH surge. Since only in the POA the LH surge is associated with changes in amino acid release, it appears that both inhibitory and excitatory amino acids act at the level of the GnRH cell bodies and/or dendrites and not on GnRH nerve terminals to mediate the feedback mechanism of E2 on LH release.     

Relation between prolactin and gonadotrophin secretion in post-partum lactating rats

In post-partum lactating rats, sucking by the young was associated with high prolactin release and maintenance of lactation but severe inhibition of LH and FSH release and suspension of oestrous cycles. Shortly after the pups were removed on day 22 post partum LH and FSH release returned to normal and oestrous cycles resumed. Twice-daily injections of ergocornine methanesulphonate (ERG) into mothers beginning at 5 or 7 days post partum, resulted in sustained inhibition of prolactin release and diminished mild secretion. By frequent exchange of pups between control and ERG-treated mothers, it was possible to maintain vigorous sucking and almost normal pup growth despite low serum prolactin levels and diminished lactation. In these rats, serum levels of LH remained low during 11 or more days of treatment with ERG, but serum FSH was consistently higher than in untreated control mothers. After 11 or more days of ERG treatment, most rats showed a return to normal LH and FSH release and resumption of oestrous cycles. These results suggest (a) that the sucking stimulus rather than high prolactin levels in the circulation is mainly responsible for inhibition of LH and FSH release during the first 11 days post partum, (b) that the sucking stimulus acts to increase prolactin and inhibit LH release by separate hypothalamic mechanisms, and (c) that administration of ERG results in diminished prolactin release and lactation, and in increased release of FSH and subsequently of LH with earlier resumption of oestrous cycles.     

Effects of ovariectomy and hindlimb unloading on skeletal muscle

Female rats (7-8 mo old, n = 40) were randomly placed into the intact control (Int) and ovariectomized control (Ovx) groups. Two weeks after ovariectomy, animals were further divided into intact 2-wk hindlimb unloaded (Int-HU) and ovariectomized hindlimb unloaded (Ovx-HU). We hypothesized that there would be greater hindlimb unloading-related atrophy in Ovx than in Int rats. In situ contractile tests were performed on soleus (Sol), plantaris (Plan), peroneus longus (Per), and extensor digitorum longus (EDL) muscles. Body weight and Sol mass were approximately 22% larger in Ovx than in Int group and approximately 18% smaller in both HU groups than in Int rats (Ovx x HU interaction, P < 0.05), and there was a similar trend in Plan muscle (P < 0.07). There were main effects (P < 0.05) for both ovariectomy (growth) and hindlimb unloading (atrophy) on gastrocnemius mass. Mass of the Per and EDL muscles was unaffected by either ovariectomy or hindlimb unloading. Time to peak twitch tension for EDL and one-half relaxation times for Sol, Plan, Per, and EDL muscles were faster (P < 0.05) in Ovx than in Int animals. The results suggest that 1) ovariectomy led to similar increases of approximately 20% in body weight and plantar flexor mass; 2) hindlimb unloading may have prevented ovariectomy-related muscle growth; 3) greater atrophy may have occurred in Sol and Plan of Ovx animals compared with controls; and 4) removal of ovarian hormonal influence decreased skeletal muscle contraction times.