Ovarian steroids but not the locus coeruleus regulate stress-induced prolactin secretion in female rats

Stress has been proposed to stimulate prolactin release if its prestress levels are low, or to inhibit it if they are elevated, but the role of ovarian-steroid fluctuations in the prolactin stress response is not yet clearly understood. Because the noradrenergic nucleus locus coeruleus has been implicated in stress responses and generation of prolactin surges in female rats, the present study aimed to evaluate stress-induced prolactin secretion under different hormonal conditions, determining the effect of locus coeruleus lesion on each response. Blood samples were withdrawn from a jugular vein catheter 5 and 2 min before and 2, 5, 10, 15 and 30 min after ether stress in male rats, female rats during the oestrous cycle and ovariectomised rats treated with oil (OVX), oestradiol (OVE) or oestradiol plus progesterone (OVEP). Increased Fos immunoreactivity demonstrated locus coeruleus activation following ether stress. Ether stress increased both low (at 16.00 h in males, in OVX and on dioestrous and at 11.00 h on pro-oestrous and oestrous) and high plasma prolactin (at 16.00 h on oestrous and in OVE), but it decreased elevated prolactin levels during the afternoon on pro-oestrous and in OVEP. Locus coeruleus lesion prevented prolactin surges during the afternoon on pro-oestrous, oestrous, OVE and OVEP but did not modify either pattern (i.e. increase or decrease) or degree of prolactin stress response under any condition studied. The present data therefore suggest that oestradiol and progesterone modulate stress-induced prolactin secretion, regardless of its prestress levels. Moreover, the locus coeruleus is probably not involved in prolactin response to stress and most likely has a specific role in prolactin surges induced by ovarian steroids.     

Luteinizing hormone and luteinizing hormone-releasing hormone secretion is under locus coeruleus control in female rats

It has been suggested that norepinephrine (NE) from the locus coeruleus (LC) plays an important role in triggering the preovulatory surge of gonadotropins. This work intended to study the role of LC in luteinizing hormone (LH) secretion during the estrous cycle and in ovariectomized rats treated with estradiol and progesterone (OVXE(2)P) and to correlate it with LH releasing hormone (LHRH) content in the medial preoptic area (MPOA) and median eminence (ME). Female rats on each day of the estrous cycle and OVXE(2)P were submitted to jugular cannulation and LC electrolytic lesion or sham-operation, at 09:00 h. Blood samples were collected hourly from 11:00 to 18:00 h, when animals were decapitated and their brains removed to analyze LC lesion and punch out the MPOA and ME. Plasma LH levels and LHRH content of MPOA and ME were determined by radioimmunoassay. During metestrus, diestrus and estrus, LC lesion did not modify either LH plasma concentrations or LHRH content, but completely abolished the preovulatory LH surge during proestrus and the surge of OVXE(2)P. These blockades were accompanied by an increased content of LHRH in the MPOA and ME. The results suggest that: (1). LC does not participate in the control of basal LH secretion but its activation is essential to trigger spontaneous or induced LH surges, and (2). the increased content of LHRH in the MPOA and ME may be due to a decreased NE input to these areas. Thus, LC activation may be required for depolarization of LHRH neurons and consequent LH surges.     

Evidence in support of nitric oxide (NO) involvement in the cyclic release of prolactin and LH surges

Studies were undertaken to determine whether nitric oxide (NO) is involved in induction of the prolactin surge on proestrus and in that induced by ovarian steroids in ovariectomized (ovx) rats, by using inhibitors of NO synthase, the enzyme that generates NO. Two week-ovariectomized rats were treated either with estradiol benzoate (EB, 30 μg/rat, s.c.) alone, or with EB and 2 days later with progesterone (P, 2 mg/rat, s.c.) to evoke a prolactin surge in the afternoon. Injections of the NO synthase inhibitor methyl ester (40 mg/kg, s.c.) at 10.00, 12.00, and 14.00 h completely suppressed the steroid-induced prolactin surge in the afternoon. Similarly, another NO synthase inhibitor, (100 mg/kg, s.c.), injected at 1000, 1200, and 1400 h on proestures suppressed prolactin and luteinizing hormone (LH) surges, but failed to alter the daily increase of corticosterone. These studies confirm our earlier report and show that NO is involved in the complex processes that initiate the afternoon prolactin and LH surges of proestrus.     

Possible Role of Neurokinin A in the Control of Prolactin Secretion in Rats and Hamsters

The possible role of neurokinin A (NKA) in the control of prolactin secretion was studied in vivo, by injecting anti-NKA serum to ovariectomized rats treated with estrogens and to proestrous rats and hamsters. Injections of an anti-NKA serum to ovariectomized rats treated with two doses of 80 μg 17ß-estradiol 24 h apart, or treated chronically with estradiol implants induced a significant decrease of serum prolactin levels as compared with those of similarly treated rats injected with normal rabbit serum. In proestrous rats, the anti-NKA serum did not modify the afternoon surge of prolactin or luteinizing hormone, but when the antiserum was injected the day before, on diestrus II, it significantly reduced the prolactin surge during the afternoon of proestrus. As in these results obtained in the rat, injections of anti-NKA serum to golden hamsters on diestrus II also significantly decreased the prolactin surge in the afternoon of proestrus. These results suggest a possible physiological role of NKA on prolactin secretion, exerting a stimulatory influence on the release of this hormone.     

LHRH release depends on Locus Coeruleus noradrenergic inputs to the medial preoptic area and median eminence

We tested the hypothesis that Locus Coeruleus (LC) inputs to the medial preoptic area (MPOA) and median eminence (ME) are essential for gonadotropin release. Proestrus and ovariectomized (OVX) rats were decapitated at 16:00 h. LC electrolytic lesion was performed at 11:00 h during proestrus and 24h before decapitation in OVX rats. Plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured and MPOA and ME were microdissected for LHRH content measurement. In addition, FOS protein in LC and MPOA were studied in proestrus and OVX rats at 12:00, 15:00, and 17:00 h. On proestrus, LC lesion blocked the LH surge and only decreased plasma FSH; in OVX rats the lesion induced only a slight decrease on plasma LH without affecting FSH secretion. An increased content of LHRH in the MPOA and ME of both groups accompanied the decreases of plasma LH. In proestrus, the number of FOS-immunoreactive (FOS-ir) neurons increased from 12:00 to 17:00 h in the LC and MPOA. In OVX rats, there was an increase at 15:00 h in the LC and a decrease at 17:00 h in both areas. The number of FOS-ir neurons was lower in OVX than in proestrus animals. Thus, LC (1) is responsible, at least in part, for gonadotropin release through the activation of LHRH neurons, (2) is more closely related to the positive than the negative feedback, and (3) seems to show an intrinsic cyclic activity which is amplified by ovarian steroids.     

Locus coeruleus norepinephrine regulates the surge of prolactin during oestrus

A secondary surge of prolactin has been recently characterised on the afternoon of oestrus. Because the noradrenergic nucleus locus coeruleus participates in the genesis of the pro-oestrous and steroid-induced surges of prolactin, the aim of the present study was to investigate the importance of locus coeruleus norepinephrine in the generation of the prolactin surge of oestrus. For this purpose, we initially re-evaluated the profile of prolactin secretion during the oestrous cycle to verify whether this surge of prolactin was physiological and specific to the day of oestrus. Thereafter, the following were evaluated: (i) the effect of locus coeruleus lesion on the secondary surge of prolactin and on norepinephrine concentration in the medial preoptic area (MPOA), medial basal hypothalamus (MBH) and paraventricular nucleus (PVN) during the day of oestrus and (ii) locus coeruleus neurones activity during the same day by Fos immunoreactivity. Locus coeruleus lesion completely blocked the prolactin surge of oestrus in all rats studied and also significantly reduced norepinephrine concentration in the MPOA, MBH and PVN during the day of oestrus. The number of double-labelled tyrosine hydroxylase/Fos immunoreactive neurones in locus coeruleus was significantly higher at 14.00 h of oestrus, suggesting an increase in its activity preceding the prolactin surge that generally occurs at 15.00 h. Therefore, the increase in locus coeruleus activity on the afternoon of oestrus supports the data obtained with bilateral lesion of this nucleus, suggesting a stimulatory role of locus coeruleus norepinephrine in the genesis of the secondary surge of prolactin.     

Catecholamine synthesizing enzymes in the hypothalamus during the estrous cycle

The activities of tyrosine hydroxylase and dopamine-beta-hydroxylase were measured in the medial basal hypothalamus and remaining hyothalamic tissue of female rats at various times during diestrus 2, proestrus and estrus. Tyrosine hydroxylase activity in the medial basal hypothalamus was significantly lower at 12.00 h compared with other times on proestrus. This decrease preceded the elevation of serum prolactin and LH during the afternoon of proestrus. Tyrosine hydroxylase activity did not change significantly during diestrus 2 or estrus nor was it altered at any time in the remainder of the hypothalamus. Dopamine-beta-hydroxylase activity in the basal medial hypothalamus was significantly elevated at 12.00 h on proestrus and at 14.00 h on diestrus. The results provide further evidence for a decrease in dopaminergic neuron activity in the medial basal hypothalamus which may precipitate the series of events leading to the LH surge during proestrus. The increase in dopamine-beta-hydroxylase activity suggests that an increase in noradrenergic neuron activity may also be involved in triggering the release of LH.     

A method to study preovulatory surges of gonadotropins

The aim of this study was to describe and validate a method to evaluate the preovulatory surges of gonadotropins in rats submitted to anesthesia and implantation of a jugular vein cannula in the morning of proestrus and to withdrawal of serial blood samples in the afternoon of the same day. In experiment I, to choose an adequate anesthetic, cycling female rats were anesthetized in the morning of proestrus (10:00-11:00 h) with tribromoethanol, ketamine/xylazine or tiletamine/zolazepam and a Silastic cannula was implanted into the jugular vein. Blood samples (0.6 ml) were withdrawn hourly between 12:00 and 18:00 h of the same day and, on estrus morning, the rats were decapitated and the number of ova was counted. The preovulatory gonadotropin surges as well as ovulation occurred in rats anesthetized with tribromoethanol, while they were prevented by ketamine/xylazine or tiletamine/zolazepam. To investigate if the jugular cannulation under tribromoethanol anesthesia and serial blood sampling performed in experiment I altered the magnitude of the gonadotropin surges and the number of ova, intact rats (control) or rats anesthetized with tribromoethanol followed or not by jugular vein cannulation were decapitated at 16:00 h of proestrus and in the morning of estrus. The magnitude of preovulatory gonadotropin surges and the number of ova were not different among groups. Thus, since neither tribromoethanol nor surgical procedures or serial blood sampling altered the preovulatory gonadotropin surges or the ovulation process, this method seems to be suitable for this sort of study in rats.     

Prolactin secretory surge during estrus coincides with increased dopamine activity in the hypothalamus and preoptic area and is not altered by ovariectomy on proestrus

Prolactin (PRL) secretory surges have been reported on the afternoons of both proestrus and estrous in cycling rats. As neuroendocrine regulation of estrous PRL surge is poorly understood, the present study aimed to investigate the involvement of hypothalamic dopamine and serotonin as well as of plasma ovarian steroids in this hormonal surge generation. For that, we determined the concentrations of dopamine, serotonin and their respective metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole-3-acetic acid (5-HIAA) in the mediobasal hypothalamus (MBH) and medial preoptic area (MPOA) throughout the day of estrus and correlated them with plasma PRL levels. In a second study, we evaluated the effect of ovariectomy on the morning of proestrus on PRL surges of both proestrus and estrus. Dopamine turnover, as determined by DOPAC/dopamine ratio, increased in both the MBH and MPOA coinciding with the afternoon PRL surge on estrus. In contrast, both the concentration and turnover (5-HIAA/serotonin) of serotonin within these areas were unaltered during estrus. In addition, ovariectomy reduced plasma estradiol and progesterone levels but did not alter the PRL surges on proestrus and estrus. Considering that dopamine is the main inhibitor of PRL release and that PRL auto-regulates its secretion through a short-loop feedback mechanism, our present results suggest that PRL may suppress its own secretion during the estrus surge through the activation of the dopaminergic neurons in the MBH and MPOA. In addition, the PRL surge on estrus seems do not depend on either the activity of hypothalamic serotonin or the increased secretion of ovarian steroids on proestrus.     

Inhibition of LH and prolactin release in the cycling rat following inhibition of dopamine-ß-hydroxylase

The effect of fusaric acid, an inhibitor of dopamine-ß-hydroxylase (DBH), on luteinizing hormone (LH) and prolactin levels during the estrous cycle was determined.Fusaric acid was found to cause a selective dose- and time-dependent inhibition of DBH activity in the medial basal hypothalamus without altering tyrosine hydroxylase activity. When DBH was inhibited during the afternoon of diestrus, the proestrous surges of both LH and, to a lesser extent, prolactin were inhibited. These results suggest that noradrenergic neuronal activity in the mediobasal hypothalamus is required during afternoon preceding proestrus in order for the LH and prolactin surges to occur. It is possible that the rise in serum estrogen during late diestrus 2 is blocked by fusaric acid treatment. This estrogen increase is necessary for LH and prolactin surges to occur during proestrus. When DBH was inhibited during the afternoon of proestrus, the LH and prolactin surges were completely eliminated. This indicates that noradrenergic neuronal activity in the mediobasal hypothalamus during the afternoon of proestrus is important for both the LH and prolactin surges to occur.     

Normalization of circulating leptin levels by fasting improves the reproductive function in obese OLETF female rats

In order to examine a possible detrimental effect of hyperleptinemia on the reproductive system, we examined whether a decrease in circulating leptin levels by fasting affects the estradiol/progesterone-induced luteinizing hormone (LH) and prolactin (PRL) surges in genetically obese OLETF (Otsuka-Long-Evans-Tokushima-Fatty) rats. Experiments were performed on both normally fed and 3-day starved groups from ovariectomized OLETF rats and their controls LETO (Long-Evans-Tokushima-Otsuka). Starved LETO rats, whose leptin levels were less than 0.5 ng/ml, did not show a significant surge of either LH or PRL. Normally fed OLETF rats, whose leptin levels were 9.7 +/- 1.8 ng/ml, showed a significant but small surge for both LH and PRL. Interestingly, starved OLETF rats, whose leptin levels (4.1 +/- 0.7 ng/ml) were similar to those in normally fed LETO rats (3.3 +/- 0.4 ng/ml), had significantly greater surges of both hormones than normally fed OLETF group. This study demonstrates for the first time that the normalization of circulating leptin levels in female OLETF rats augments the steroid-induced LH and PRL surges, and also suggests a deleterious effect of hyperleptinemia on the reproductive axis.     

Inhibition of LH and prolactin release in the cycling rat following inhibition of dopamine-beta-hydroxylase

The effect of fusaric acid, an inhibitor of dopamine-beta-hydroxylase (DBH), on luteinizing hormone (LH) and prolactin levels during the estrous cycle was determined. Fusaric acid was found to cause a selective dose- and time-dependent inhibition of DBH activity in the medial basal hypothalamus without altering tyrosine hydroxylase activity. When DBH was inhibited during the afternoon of diestrus, the proestrous surges of both LH and, to a lesser extent, prolactin were inhibited. These results suggest that noradrenergic neuronal activity in the mediobasal hypothalamus is required during the afternoon preceding proestrus in order for the LH and prolactin surges to occur. It is possible that the rise in serum estrogen during late diestrus 2 is blocked by fusaric acid treatment. This estrogen increase is necessary for LH and prolactin surges to occur during proestrus. When DBH was inhibited during the afternoon of proestrus, the LH and prolactin surges were completely eliminated. This indicates that noradrenergic neuronal activity in the mediobasal hypothalamus during the afternoon of proestrus is important for both the LH and prolactin surges to occur.     

Gonadotropin responses to naloxone may depend upon spontaneous activity in noradrenergic neurons at the time of treatment

The opiate system is thought to modulate gonadotropin secretion by its effect on catecholamine secretion. This action may be produced by opiates regulating the amount of catecholamine released from presynaptic terminals at a given frequency of depolarization and/or by increasing the rate of impulse traffic within catecholamine neurons. We examined the effects of naloxone, an opiate receptor antagonist, on luteinizing hormone (LH) and prolactin (Prl) secretion in 3 sex steroid-treated, gonadectomized rat models in which we have considerable information on the rates of turnover of norepinephrine (NE) and dopamine (DA) in the hypothalamus. In 7 day ovariectomized rats treated for 2 days with estradiol (E2), the injection of naloxone (10 mg/kg) at 09.15 h produced a small 3-fold rise in LH and a short-lived decline in Prl. In contrast, naloxone, given at 12.15 h, markedly amplified (10-fold) and advanced the time of the LH surge but did not affect afternoon Prl surges. Hypothalamic NE turnovers are low in the morning and high in the afternoon for such animals. Other ovariectomized (OVX) rats received E2 for 2 days and progesterone (P4) on day 2. Such treatment extinguishes the LH surges which normally occur the next day (day 3) but does not affect phasic Prl secretion. Naloxone, given at 09.15 h to E2P4-treated rats on day 3, did not affect basal LH levels but serum Prl declined for about 1 h. When given at 12.15 h, naloxone produced a small 3-fold rise in LH but did not affect phasic Prl release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Noradrenaline release in the medial preoptic area during the rat oestrous cycle: temporal relationship with plasma secretory surges of prolactin and luteinising hormone

During the rat oestrous cycle, the afternoon of pro-oestrous is characterised by preovulatory surges of luteinising hormone (LH) and prolactin. On the afternoon of oestrous, a secretory surge of prolactin has also been reported. Because the medial preoptic area (MPOA) is known to regulate prolactin and LH secretory surges and noradrenaline has been demonstrated to stimulate these hormones release, we evaluated whether noradrenaline release in the MPOA was temporally associated with plasma prolactin and LH surges in cycling rats. During the 4 days of oestrous cycle, noradrenaline concentrations were determined in microdialysates from the MPOA, collected at 30-min intervals from 10.30 h to 19.00 h. Plasma prolactin and LH levels were measured in blood samples withdrawn hourly from 14.00 h to 19.00 h on pro-oestrous and from 13.00 h to 18.00 h on the other days of the cycle. On the afternoons of both pro-oestrous and oestrous, noradrenaline levels increased at 14.00 h and remained elevated until 16.30 h. Conversely, they were low and constant throughout metoestrous and dioestrous. Correlating with noradrenaline release in the MPOA, plasma prolactin surges occurred during the afternoons of both pro-oestrous and oestrous. On pro-oestrous, the afternoon LH surge was also preceded by the increase in MPOA noradrenaline whereas, during oestrous, LH secretion was low and unaltered. A temporal association between noradrenaline release and prolactin secretion suggests that noradrenergic neurotransmission in the MPOA regulates prolactin surges in female rats. Moreover, our data also suggest that MPOA noradrenaline requires specific conditions to physiologically regulate LH secretion, which seems to occur during the afternoon of pro-oestrous.     

Ovarian Steroid-Independent Diurnal Rhythm in Cyclic GMP/Nitric Oxide Efflux in the Medial Preoptic Area: Possible Role in Preovulatory and Ovarian Steroid-Induced LH Surge*

The aim of this study was to evaluate the relationship between cyclic LH hypersecretion and nitric oxide (NO) release in the medial preoptic area (MPOA), the hypothalamic site implicated in induction of LH hypersecretion. The MPOA extracellular cyclic GMP (cGMP) efflux (an index of NO release), was monitored by microdialysis. Quite unexpectedly, we observed a daily afternoon rise in the MPOA cGMP efflux in cycling female rats on proestrus and diestrus II, in ovariectomized (ovx) rats and in ovx rats treated with ovarian steroids to induce the LH surge. The daily rise in cGMP efflux occurred earlier in diestrous and in estradiol benzoate (EB)-treated ovx rats than in ovx rats. Progesterone (P) injection to estrogen-primed ovx rats further advanced the onset of the rise close to the earliest time of rise as seen on proestrus. The afternoon increase in the cGMP efflux in proestrous rats was abolished by pentobarbital treatment that blocked the LH surge. Intracerebroventricular (icv) injection of 1H-[1,2,4]oxadiazo[4,3-a]quinoxalin-one (ODQ), a selective inhibitor of soluble guanylyl cyclase, suppressed the P-induced LH surge in EB-primed ovx rats, but not basal LH secretion in unprimed ovx rats. Analysis of brain NOS (bNOS) levels in the POA by Western blotting showed that the morning bNOS levels were higher in the POA of EB-treated rats than in unprimed ovx rats. Further, with the exception of ovx rats treated with sequential EB and P treatment, the POA bNOS levels rose significantly in the afternoon in unprimed ovx and EB-treated ovx rats. Collectively, these findings reveal a diurnal rhythm in the MPOA cGMP/NO efflux that is ovarian steroid-independent. Ovarian steroids apparently shift the timing of the afternoon rise in cGMP/NO efflux to synchronize with the activation of steroid-dependent neuronal systems responsible for the LH surge.     

Relationship of Gonadal Hormone Administration, Sex, Reproductive Status and Age to Monoamine Oxidase Activity Within the Hypothalamus

Activity of Type A monoamine oxidase (MAO) was measured in microdissected samples from preoptic-hypothalamic and hindbrain areas of young male and female rats, and aged female rats. Administration of estradiol and progesterone, in doses sufficient to facilitate lordosis behavior and induce a luteinizing hormone surge in ovariectomized, but not castrated rats, was associated with sexually dimorphic changes of MAO activity within the hypothalamus. Forty-two h following estradiol benzoate administration, increased MAO activity was measured in the ventromedial nucleus (VML) and midbrain central gray of females, while decreased MAO activity was measured in the VML and arcuate-median eminence (ArME) of males. Progesterone administration to estradiol benzoate-primed rats was associated with decreased MAO activity in the VML and medial preoptic nucleus (mPOA) of females and decreased activity in the dorsal raphe nucleus of males. Activity of MAO on diestrus, proestrus and estrus was assessed in ten preoptic-hypothalamic and hindbrain sites. Differences between days of the cycle were limited to the mPOA, ArME and VML. While activities were generally lowest at estrus, these areas exhibited different patterns of activity across the cycle. Activity was highest at proestrus in the mPOA and highest at diestrus in the VML and ArME. Activity of MAO in some areas of 25-month old, diestrus rats was altered as compared to young, cycling rats; however, ageing was not associated with widespread changes in MAO activity. In the suprachiasmatic nucleus, aged rats showed approximately 30% less activity than young rats. In the mPOA, VML and ArME, activity in aged females was different from some, but not all, days of the estrous cycle. These results show that MAO activity changes within specific hypothalamic sites when the neuroendocrine axis is altered. Since the changes are present in areas where activity of rnonoaminergic systems is critical for initiating gonadotrophin surges and inducing lordosis behavior, these results provide initial evidence that catabolism of monoamines by MAO may contribute to rnonoaminergic regulation of reproductive function.     

Changing patterns of Fos expression in brainstem catecholaminergic neurons during the rat oestrous cycle

Brainstem catecholaminergic neurons are believed to play an important role in the activation of luteinising hormone-releasing hormone (LHRH) neurons on the afternoon of proestrus which results in the luteinising hormone (LH) surge. To examine the respective roles of brainstem A1 and A2 neurons and the adjoining C1 and C2 adrenergic cells at this time, we have examined the patterns of Fos-immunoreactivity within tyrosine hydroxylase (TH) and phenylethanolamine-N-methyl transferase (PNMT) neurons during diestrus and proestrus. Initial studies demonstrated that the LH surge commenced at approximately 15:00 h in proestrous animals and that peak plasma levels of LH were observed between 16:00 and 17:00 h. Groups of cycling female rats (n = 6) were then perfused between 09:00 and 11:00 (diestrus early) and 18:00 to 19:30 h (diestrus late) on diestrus and at the same times on proestrus (proestrus early and proestrus late). Double-labelling immunocytochemistry revealed little Fos expressions by adrenergic neurons of the C1 or C2 cell groups and this did not change significantly between any of the experimental groups. Analysis of the A2 region was divided into rostral, middle and caudal divisions and all regions showed a significant (P < 0.01) increase in the number of Fos-expressing TH neurons (up to 35% of TH cells) in proestrus early animals compared with diestrus and proestrus late rats. In the A1 region, a significant increase in the number of TH neurons expressing Fos ( 33%) was detected in both proestrus early (P < 0.05) and diestrus early (P < 0.01) rats compared with animals perfused in the late afternoon ( 12%). These results indicate that TH-immunoreactive neurons in both A1 and A2 cell groups are activated on the morning of proestrus prior to the LH surge whilst the C1 and C2 adrenergic neurons express little Fos throughout. The morning increase in Fos-expression by TH neurons within the A1 region on both diestrus and proestrus indicates a circadian pattern of activation for A1 noradrenergic cells and suggests different roles for the A1 and A2 cell groups in regulating the activity of LHRH neurons on proestrus.     

Presence of kappa-opioid tone at the onset of the ovulatory luteinizing hormone surge in the proestrous rat

A decrease in endogenous opioid peptide inhibitory tone on the afternoon of proestrus is one event underlying generation of the ovulatory luteinizing hormone (LH) surge. Whether this disinhibition involves a complete loss of opioid suppression at the time of the LH surge is controversial. The objective of the present study was to determine whether a total loss specifically of the kappa-opioid inhibitory component suppressing LH secretion occurs on proestrus at the onset of the LH surge. Proestrous rats were infused intraventricularly with either artificial cerebrospinal fluid (aCSF) or aCSF containing nor-binaltorphimine (nor-BNI), a selective kappa-opioid receptor antagonist, from 15:30 or 16:30 h (the approximate onset time of the spontaneous LH surge) to 18:50 h. The LH surge in rats treated with nor-BNI beginning at 15:30 h started 0.5 h earlier than the spontaneous surge in aCSF controls, and had significantly higher plasma LH levels from 16:30 to 17:30 h. Nor-BNI administration begun at 16:30 h also produced an LH surge with more elevated plasma LH levels at 17:30 and 18:00 h than in aCSF-treated controls. These results demonstrate that significant amounts of kappa-opioid tone are still present during the hours when the LH surge is initiated. Thus, a complete loss of kappa-opioid inhibition is not required for the onset of the LH surge on proestrus.     

Evidence that Hypothalamic Neuropeptide Y Gene Expression Increases Before the Onset of the Preovulatory LH Surge*

Neuropeptide Y (NPY), a 36 amino acid residue peptide, is involved in stimulation of LHRH and LH surges on proestrus and those induced by ovarian steroids in ovariectomized (ovx) rats. Recently, we observed that NPY gene expression in the medial basal hypothalamus (MBH) was increased before the onset of the LH surge in the ovarian steroid-primed ovx rats. Since the ovarian steroidal milieu during the estrous cycle is markedly different from that prevailing after ovarian steroid injections in ovx rats, we evaluated in cycling rats the temporal relationship between MBH preproNPY mRNA levels and the preovulatory LH surge on the day of proestrus and compared that with diestrus II, concomitant with basal LH levels. PreproNPY mRNA levels in the MBH were measured by solution hybridization/RNAse protection assay, using a cRNA probe. On the day of diestrus II, preproNPY mRNA levels changed little between 1000 and 1800 h. Quite unexpectedly, preproNPY mRNA levels at 1000 h on proestrus were similar to diestrus II levels, despite additional exposure to ovarian steroids during this interval. However, from these low levels at 1000 h, the preproNPY mRNA profile displayed a biphasic rise. During the first phase, preproNPY mRNA rose significantly at 1200 h and remained elevated at 1300 and 1400 h concomitant with basal serum LH levels. Thereafter, a second rise in preproNPY mRNA began at 1500 h, peaked rapidly at 1600 h and declined significantly at 1800 h. This secondary activation of NPY gene expression occurred with a slow, two-fold increase in serum LH at 1500 h, followed by a rapid ascension to peak levels at 1800 h and was associated with an increase at 1400 h of serum progesterone levels which reached their peak at 1800 h. These results demonstrate that a dynamic, biphasic augmentation in hypothalamic NPY gene expression occurs selectively on proestrus, and that the first incremental response is observed some time before the onset of preovulatory LH hypersecretion. Because preproNPY mRNA levels at 1000 h on proestrus were similar to the low levels seen on the preceding diestrous II phase, a neural timing mechanism, and not changes in ovarian hormone levels during this phase may be responsible for the increase in NPY gene expression after 1000 h of proestrus. Because of our previous observations that progesterone can rapidly augment preproNPY mRNA in the MBH and because a rise in serum progesterone occurs hours before the onset of the LH surge, we suggest that the secondary rise in preproNPY mRNA is facilitated by this antecedent increase in serum progesterone. Cumulatively, these results are in accord with the thesis that activation of hypothalamic NPY gene expression is one of the key early neural events initiated by the neural clock that times the preovulatory LHRH and LH surges.