Charles A. Barraclough

Suppression of spontaneous LH surges in estrogen-treated ovariectomized rats by microimplants of antiestrogens into the preoptic brain

Studies by others have shown that parenteral administration of antiestrogens blocks the positive feedback effect of estrogen on the luteinizing hormone (LH) surge mechanism. Since all estrogen-accumulating cells could be affected by this treatment, it is difficult to identify the site(s) at which this steroid acts to affect LH surges. In the present study we attempted to deprive specific hypothalamic neurons of estrogen by stereotaxically implanting antiestrogen-containing microcannulae into the brains of ovariectomized (OVX) rats which, otherwise, were completely estrogenized. The animal model used in these studies was the 14-day OVX rat into which 2 estradiol-containing Silastic capsules were inserted s.c. on day 14 (day 0). Microcannulae were placed into either the medial or lateral preoptic nuclei (MPN, LPN) on day 0 and the effects on LH release were examined 2 days later (day 2). When empty cannulae were placed into the MPN or LPN, 6 of 7 and 8 of 8 rats, respectively, had normal spontaneous LH surges. In contrast, when cannulae containing either CI-628, LY 10074 or Keoxifene were implanted into MPN only 33.3, 0, and 14.3% of the rats, respectively, had LH surges by 16.00 h on day 2 (time of LH peak). When antiestrogen-containing cannulae were placed into the LPN, all rats displayed normal LH patterns of release and concentrations. The antiestrogens did not prevent estrogen from suppressing elevated high post-ovariectomy plasma LH concentrations (negative feedback). To evaluate whether Keoxifene affected releasable luteinizing hormone-releasing hormone (LH-RH), we examined the effects of MPN-Keoxifene implants on LH secretion evoked by electrochemical stimulation (ECS) of the MPN or the medial basal hypothalamus (MBH). In ketamine-anesthetized rats with empty cannulae, plasma LH increased significantly to reach peak concentrations 30-45 min after ECS. Similar LH concentrations and release patterns occurred in rats with the antiestrogen implant. Other studies examined the effects of MPN-Keoxifene implants on norepinephrine (NE) concentrations and rate constants following administration of alpha-methyl-p-tyrosine. NE concentrations and rate constants in the MPN and median eminence did not differ significantly in rats which had received empty versus Keoxifene-containing microcannulae. In the final series of studies we examined the response of LH-RH neurons to an intracerebroventricular (i.c.v.) infusion of norepinephrine (20 micrograms). Plasma LH peaked within 10 min after i.c.v. NE and, thereafter, declined towards baseline. Keoxifene did not affect LH-RH neuronal responsiveness to i.c.v. NE.(ABSTRACT TRUNCATED AT 400 WORDS)

A role for hypothalamic catecholamines in the regulation of gonadotropin secretion

Publisher Summary This chapter discusses the role of the hypothalamic catecholaminergic system in regulating Luteinizing-hormone-releasing hormone (LHRH) secretion. The functional activity of the LHRH neuron can be affected by numerous putative neurotransmitters that stimulate or inhibit LHRH release. Some neurotransmitters may also activate or inhibit other primary inputs to affect the activity of the LHRH neuron. Coupled with these complex controls are modulatory influences exerted within the brain by changing serum levels of sex steroids on input circuitry, on genomic functions, and on ionic transport properties of the LHRH neuronal membrane. The entire sequence of neuroendocrine events that ultimately leads to ovulation depends on the maturation of ovarian follicles which, under the influence of basal concentrations of LH and follicle-stimulating hormone FSH, increase their secretion of estrogen into blood during the second day of diestrous and morning of proestrous. Estrogen may not only increase the releasable pool size of median eminence but it also is essential for the increased release of norepinephrine during the early afternoon of proestrus.

Changes in tyrosine hydroxylase mRNA levels in medullary A1 and A2 neurons and locus coeruleus following castration and estrogen replacement in rats

Temporal changes in tyrosine hydroxylase (TH) mRNA levels in medullary A1 and A2 neurons and locus coeruleus (LC) cells were studied 6, 12 and 24 h following orchidectomy in rats. Brains from intact controls and sham castrated rats also were evaluated at these same time periods. In situ hybridization histochemistry and quantitative image analysis techniques were used to quantitate levels of cytoplasmic TH mRNA. Neither the time of day nor the stress of sham castration affected TH mRNA levels in A1, A2 and LC neurons. In contrast, 6 h following castration, TH mRNA levels in A1 neurons had declined significantly. Thereafter, there was a linear increase in A1 message levels such that, by 24 h, TH mRNA values were significantly higher than those obtained in intact controls. Placement of Silastic estrogen capsules immediately after castration prevented the 6 h decline in A1 message levels. At 12 h, TH mRNA levels in A1 neurons were significantly higher in estrogen-treated rats compared to those of the castrate or intact control groups. By 24 h, message levels in A1 neurons of steroid-treated rats were comparable to the intact control. Neither castration nor estrogen treatment altered TH mRNA levels in A2 neurons. TH mRNA levels in LC neurons increased significantly 6 h after castration and estrogen produced a further significant increase in message levels. Six hours later (12 h), TH mRNA values were still higher than controls but, in the estrogen-treated group, these levels had declined to those observed in the 12 h castrate group.(ABSTRACT TRUNCATED AT 250 WORDS)

Medial Preoptic Microimplants of the Antiestrogen, Keoxifene, Affect Luteinizing Hormone‐Releasing Hormone mRNA Levels, Median Eminence Luteinizing Hormone‐Releasing Hormone Concentrations and Luteinizing Hormone Release in Ovariectomized, Estrogen‐Treated Rats

We examined the temporal changes in plasma luteinizing hormone (LH) levels, median eminence luteinizing hormone‐releasing hormone (LHRH) concentrations and LHRH mRNA levels in estrogen‐treated, ovariectomized rats with empty or antiestrogen‐ containing microcannulae stereotaxically implanted into the medial preoptic area. Neither treatment disrupted the negative feedback effects of estrogen on LH secretion, but antiestrogen (Keoxifene) blocked the afternoon LH surges. In rats exhibiting LH surges, median eminence LHRH concentrations were similar at 0800, 1200 and 1600 h, but they were significantly elevated by 2000 h. In contrast, no alterations in LHRH concentrations occurred in the Keoxifene‐treated group. LHRH mRNA levels in control rats were significantly elevated at 1200, 1600 and 2000 h compared with 0800 h, but LHRH mRNA levels in Keoxifene‐treated rats did not change significantly over the time period examined. When we compared treatment effects over time we saw that serum LH levels were significantly higher in control than Keoxifene‐treated rats only at 1600 and 2000 h. Median eminence LHRH concentrations did not differ between treatment groups until 2000 h when control animals had significantly higher levels than those of Keoxifene‐treated animals. LHRH mRNA levels in Keoxifene‐treated rats were significantly higher than those of controls at 0800 hand significantly lower at 1600 h. No differences in LHRH mRNA levels were detected between groups at either 1200 h or 2000 h. In summary, although it was not clear on which neuronal system estrogen acted, depriving medial preoptic neurons of this steroid in systemically estrogenized rats certainly disrupted the neural mechanisms involved in surge, but not basal LH release. In addition, neither LHRH mRNA levels nor median eminence LHRH concentrations showed variations within the period studied when the estrogen‐sensitive mechanisms involved in LH release were disrupted. Therefore, the changes in LHRH mRNA levels and LHRH concentrations in the median eminence seen in surging animals probably resulted from the same neural events which triggered LH release.

Locus coeruleus (LC) stimulation augments LHRH release induced by medial preoptic stimulation. Evidence that the major LC stimulatory component enters contralaterally into the hypothalamus

Previous studies by others suggest that mid- and hindbrain noradrenergic projections to the hypothalamus may be stimulatory or inhibitory to luteinizing hormone releasing hormone (LHRH) release depending upon the steroid environment of the rat. In the present study we reevaluated the effects of electrical stimulation of the locus coeruleus (LC) in estrogen-primed ovariectomized (OVX) rats anesthetized with chloral hydrate. This anesthetic agent blocked the spontaneous luteinizing hormone (LH) surges which normally occur in estrogen-treated OVX rats. In such rats, bilateral LC electrical stimulation was ineffective in altering basal LH concentrations. Thereafter, we evaluated the effects of LC electrical stimulation on patterns and concentrations of plasma LH induced by electrochemical stimulation (ECS) of the medial preoptic nuclei (MPN). Bilateral MPN-ECS induced a significant rise in plasma LH. When the LC was bilaterally stimulated for 15 min beginning 30 min after MPN stimulation, peak LH concentrations were significantly augmented and remained elevated throughout the experiment. To learn more of how LC noradrenergic projections reach regions of the hypothalamus which contain LHRH neurons, the right MPN in a group of rats was unilaterally ECS and 30 min later the right LC was unilaterally stimulated for 15 min. Plasma LH levels increased after MPN activation but no further rise or fall in LH occurred after ipsilateral LC stimulation. In the final group of rats, the right MPN was unilaterally electrochemically stimulated and this was followed 30 min later by contralateral electrical stimulation of the left LC. In these animals, peak plasma LH concentrations were significantly elevated above those obtained after only unilateral MPN stimulation. These data demonstrated that LC stimulation augments LH release but only after preliminary depolarization of LHRH neurons by ECS. Moreover, the stimulatory noradrenergic projections from LC decussate to enter contralateral hypothalamic regions containing LHRH neurons.

Changes in Peripheral Plasma Progesterone During the Rat 4-Day Estrous Cycle: An Adrenal Diurnal Rhythm

Summary A diurnal rhythm exists in peripheral plasma progesterone concentrations during the 4-day estrous cycle of rats. Peak values in this rhythm were obtained during the early morning hours (0100-0500), whereas in the afternoon plasma concentrations of the steroid were at a nadir. Ovarian progesterone secretion rates within this same period remained unaltered, suggesting that the adrenal glands were responsible for these fluctuations.

Effects of morphine on luteinizing hormone secretion and catecholamine turnover in the hypothalamus of estrogen-treated rats

This study examined the effects of morphine sulfate and naloxone alone or in combination on phasic luteinizing hormone (LH) secretion in estrogen-treated ovariectomized rats. Thereafter, the effects of morphine on initial concentrations, rate constants and rates of turnovers of norepinephrine and dopamine were evaluated in untreated or morphine-injected, estrogen-primed rats. Morphine, when given at 12.30 h, completely suppressed the spontaneous LH surges which occur in steroid-treated rats. The opiate antagonist, naloxone, (12.15 h) markedly amplified and advanced the time of LH release and combination of morphine and naloxone produced peak afternoon LH values which were intermediate between those obtained in controls and in rats receiving only naloxone. Norepinephrine (NE) and dopamine (DA) turnover were calculated from data obtained in groups of rats sacrificed 0.45 or 90 min after administering 300 mg/kg b. wt. i.p. of alpha-methyl-p-tyrosine (alpha-MPT) at 10.00 or 15.00 h. In these experiments, the medial preoptic nucleus (MPN) and the median eminence (ME) were microdissected and analyzed for changes in NE and DA concentrations by a radioenzymatic procedure. In estrogen-treated rats, NE rate constants and turnover significantly increased at 15.00 vs 10.00 h in MPN and ME concomitant with increases in serum LH. Morphine blocked both increases in rate constants and turnover in the MPN and ME and also significantly reduced initial concentrations of NE in the MPN. None of the DA parameters measured in MPN or ME changed in estrogen-treated controls between morning and afternoon. Further, while morphine did not affect DA turnover in the MPN, DA turnover declined in the ME.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine hydroxylase and POMC mRNA in the arcuate region are increased by castration and hyperprolactinemia

We have examined the changes which occur in neuronal expression of tyrosine hydroxylase (TH) and proopiomelanocortin (POMC) mRNA in response to castration and hyperprolactinemia (HP) in male rats. Steady-state mRNA levels were determined by quantitative in situ hybridization histochemistry (ISHH) using 35S-labeled synthetic 48-base oligodeoxynucleotide probes. Castration produced a 27% increase in TH mRNA in the periventricular and arcuate nuclei. PRL-exposed rats exhibited a further 27% increase in the level of TH mRNA and a striking 48% increase in POMC mRNA in periarcuate region cell bodies. These results indicate that gonadal steroids and PRL are involved, either directly or indirectly, in regulating the biosynthesis of TH and POMC in the hypothalamus.

Stimulatory versus inhibitory effects of progesterone on estrogen-induced phasic LH and prolactin secretion correlated with estrogen nuclear and progestin cytosol receptor concentrations in brain and pituitary gland

The purpose of these studies was to determine the temporal requirements and time of day required for progesterone (P4) to amplify versus extinguish LH surges. The experimental animal model used in these studies was the 7-day ovariectomized rat (day 0) treated continuously with estradiol (E2) for 2-4 days (days 2, 3 or 4). In these studies we also examined the changes which occur in estrogen nuclear (ERn) and progestin cytosol (PRc) receptor concentrations in the preoptic area (POA), medial basal hypothalamus (MBH), corticomedial amygdala (CMA) and pituitary gland (PIT) associated with these physiological responses. When two P4 capsules (50 mg/ml) were placed subcutaneously at 09.00 h on day 2 and removed 3 h later (12.00 h), LH surges were amplified that afternoon. 24 and 48 h later (days 3, 4) phasic LH release reoccurred albeit in reduced concentrations compared to the LH values obtained in E2-treated rats on day 2. In contrast, if P4 capsules remained in situ for greater than 24 h, LH surges did not reappear on day 3. In a separate group of E2-treated rats, P4 capsules were placed subcutaneously at 15.00 h and removed at 21.00 h on day 2. Only minor increases in LH occurred on day 3 compared to E2-treated controls. Thus, both the duration of time and hour of day that the hypothalamo-pituitary unit is exposed to P4 seem important in determining which component of the biphasic LH response (amplification vs. inhibition) will occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of luteinizing hormone surges with estrogen nuclear and progestin cytosol receptors in the hypothalamus and pituitary gland. I. Estradiol dose response effects.

The present studies were designed to answer three questions: (1) how will a progressive increase in serum estradiol (E2) in ovariectomized (OVX) rats affect progesterone (P4)-induced luteinizing hormone (LH) surge concentrations? (2) Can steroid-induced LH surges be correlated with estrogen nuclear receptor (E2Rn) and progestin cytosol receptor (PRc) levels in brain regions known to regulate LH secretion, and (3) do differences in pituitary responsiveness to LHRH in E2- or E2P4-treated OVX rats parallel changes in E2Rn and PRc concentrations in this gland? 1 week after ovariectomy of adult cyclic rats (day 0), Silastic E2 capsules were placed subcutaneously at 09.00 h and produced serum E2 levels of 6-8 (low), 12-19 (medium) and 27-37 (high) pg/ml, respectively. 2 days later (day 2), some rats also received Silastic P4 capsules subcutaneously which elevated serum P4 concentrations to 10-12 ng/ml. In rats with low serum E2, P4 treatment induced peak serum LH levels of 913 ng/ml. When serum E2 was increased to the medium or relatively high physiologic range, P4 treatment resulted in LH surge levels of 4,686 and 5,030 ng/ml. OVX controls and E2-treated OVX rats were sacrificed at 10.00 h on day 2 and E2Rn and PRc were measured concurrently in the preoptic area (POA), mediobasal hypothalamus (MBH), corticomedial amygdala (CMA) and pituitary gland (PIT). Raising serum E2 from OVX levels to the low range significantly increased both E2Rn and PRc in MBH and PIT, but not in the POA or CMA.(ABSTRACT TRUNCATED AT 250 WORDS)