The potential role of periovarian fat and leptin in initiation of puberty in the immature rat.

Twenty-four-day old female rats were injected with 25 microg recombinant leptin twice daily for 24, 48, 72, or 93 h. Periovarian fat contained significantly greater amounts of aromatase mRNA at 72 and 93 h and estradiol at 72 h after leptin treatment as compared to the ovary. On the other hand, there was an increase in testosterone at 72 h and progesterone at 72 and 93 h after leptin in the ovary as compared to periovarian fat. Cholesterol side chain cleavage and 17alpha-hydroxylase mRNA were found only in the ovary and not periovarian fat. Thus, periovarian fat has a greater capacity to synthesize estrogen than the ovary in the immature rat and adipose tissue may serve as the primary source of estrogen in the prepubertal period which results in the onset of puberty.

Estrogen-astrocyte-luteinizing hormone-releasing hormone signaling: a role for transforming growth factor-beta(1).

The purpose of this study was to identify factors from astrocytes that can regulate LHRH neurosecretion. Exposure of LHRH-secreting (GT1-7) cells to conditioned media (CM) from C6 glial cells and hypothalamic astrocytes (HA) stimulated LHRH release. Assays of C6 and HA CM revealed that transforming growth factor-beta(1) (TGF-beta(1)) and 3alpha-hydroxy-5alpha-pregnane-20-one (3alpha, 5alpha-THP), both known LHRH secretagogues, were present in CM and their levels increased in parallel to the LHRH-releasing activity of CM. In contrast, TGF-alpha was undetectable in C6 or HA CM. Ultrafiltration to remove peptides with molecular weights >10 kDa virtually abolished the LHRH-releasing ability of the HA CM. Furthermore, immunoneutralization with a panspecific THF-beta antibody dose-dependently attenuated the LHRH-releasing activity of the CM. Rat hypothalamus and GT1-7 cells were demonstrated to express TGF-beta receptors as well as furin, an enzyme that converts latent TGF-beta(1) to active TGF-beta(1). Estrogen receptor-alpha and ER-beta mRNA and protein were also demonstrated in HAs by reverse transcription-polymerase chain reaction and double immunofluorescence, and treatment with 17beta-estradiol (17beta-E(2)) increased both active and latent TGF-beta(1) levels in HA CM. The effect of 17beta-E(2) was completely blocked by the ER antagonist ICI8280. As a whole, these studies provide evidence of a previously undescribed 17beta-E(2)-TGF-beta(1)-LHRH signaling pathway.

Evidence that neuronal nitric oxide synthase but not heme oxygenase increases in the hypothalamus on proestrus afternoon.

Nitric oxide (NO) has been implicated in the control of the proestrus luteinizing hormone (LH) surge in the rat but to date no studies have attempted to measure neuronal nitric oxide synthase (nNOS) or NO production on proestrus in the hypothalamus in order to determine if endogenous NO is increased on proestrus afternoon to activate gonadotropin-releasing hormone (GnRH) neurons. To address this deficit in our knowledge, we measured nNOS mRNA and protein levels as well as NOS activity levels in rat preoptic area (POA) and medial basal hypothalamus (MBH) fragments at 12.00, 14.00, 16.00, and 18.00 h of proestrus. Serum LH levels were also assessed to determine whether NOS changes correlate to the LH surge. To determine the specificity of observed changes we also measured mRNA levels for the enzyme heme oxygenase-2, which is responsible for production of another putative gaseous transmitter, carbon monoxide. In all studies a metestrus 12.00 h control group was included since steroid and LH levels would be basal at this time as compared to proestrus. The results revealed that nNOS mRNA and protein levels, as well as NOS activity did not change significantly in the MBH on proestrus. In contrast, nNOS mRNA levels were significantly elevated in the POA at proestrus 12.00 and 14.00 h, as compared to metestrus 12.00 h. Likewise, at the protein and activity level, nNOS protein levels in the POA were significantly elevated on proestrus at 14.00 and 16.00 h, with NOS activity significantly increased at 16.00 h on proestrus. The elevation of nNOS protein and activity levels in the POA occurred at the time of initiation of the LH surge. The elevation of nNOS was specific as mRNA levels for the CO-synthetic enzyme heme oxygenase-2 did not change significantly on proestrus in the POA or MBH. As a whole, the current studies provide new evidence that nNOS is elevated in the POA on proestrus, and thus could play a role in the activation of GnRH neurons to produce the preovulatory LH surge.

Multidimensional screening and design of pharmaceuticals by using endocrine pharmacophores.

A novel computational technology derived from gene structure has been developed for screening, selecting, and designing pharmaceutical candidates. Pharmacophores, or three-dimensional molecular blueprints, were created by docking known active structures into specific sites in partially unwound DNA. The pharmacophores are composites of the van der Waals surfaces and hydrogen bonding functional groups of active molecules. Once created, molecules can be inserted into the pharmacophores and degree of fit quantitated by the volume of the molecule that fits within the composite surface and the magnitude of electrostatic interactions with charged atoms on the pharmacophore. Here, we describe endocrine pharmacophores and in particular the estrogen pharmacophore derived by docking active ligands into partially unwound DNA. Fit of candidate structures into the estrogen pharmacophore correlated with estrogenic (uterotropic) activity. For example, the super active estrogens moxestrol and 11beta-acetoxyestradiol fit better within the site than estradiol. Bisphenol A, a putative endocrine disrupter with suspected estrogenic activity, was a poor fit in the pharmacophore. Consistent with this prediction, bisphenol A was recently shown to lack uterotropic activity. The capacity of the endocrine pharmacophores to predict certain nontarget activities was demonstrated by using the antiandrogen cyproterone acetate that did not fit the estrogen or thyroid pharmacophores but fit partially into the progestin and glucocorticoid pharmacophores. Cyproterone acetate has been reported to have weak progestational and glucocorticoid activities. The pharmacophores provide for the first time a multidimensional computational method that can simultaneously predict multiple activities of diverse molecular structures.

Autoregulation of the androgen receptor at the translational level: testosterone induces accumulation of androgen receptor mRNA in the rat ventral prostate polyribosomes.

Several studies have documented that androgens have the ability to autoregulate their own receptor levels; however, the mechanism of such autoregulation remains poorly understood. Along these lines, our laboratory has shown that testosterone increased androgen receptor (AR) protein levels and binding in the castrated rat ventral prostate within 1 h. Ongoing protein synthesis was required for the testosterone effect, as the protein synthesis inhibitor cycloheximide blocked this effect. Testosterone and/or actinomycin D, an mRNA synthesis inhibitor, did not affect the steady-state AR mRNA levels. Therefore, we suggest that the early events induced by testosterone are posttranscriptional and that protein synthesis is required for the maintenance of AR protein and AR mRNA levels. In addition, we hypothesize that the testosterone posttranscriptional effect is primarily through the sequestering of AR mRNA in the prostate polyribosomes. To test this hypothesis, total RNA was isolated from prostate polyribosomes of controls and testosterone-treated rats and AR mRNA levels were quantitated by competitive reverse transcription-polymerase chain reaction. Polyribosomes profiles on linear sucrose gradients showed no difference in the sedimentation characteristics of ribosomal particles from the vehicle-treated control or testosterone-treated animals. Furthermore, because both polyribosomal preparations can direct protein synthesis to the same extent in a cell-free system, testosterone does not increase the efficiency of translation. However, competitive reverse transcription-polymerase chain reaction revealed that testosterone increases AR mRNA associated with polyribosomes by threefold after 1 h of treatment compared with control. These data suggest a rapid testosterone-mediated posttranscriptional mechanism, in which testosterone regulates the stability of the AR mRNA by sequestering it in polyribosomes, and consequently increasing its translation.

Progesterone and regulation of the follicle-stimulating hormone (FSH-beta) gene.

Progesterone (P) biphasically modulates follicle-stimulating hormone (FSH) secretion in the rat both in vivo and in vitro with the duration of estrogen priming determining the biphasic nature of the P action, probably through estrogen up-regulation of the anterior pituitary progesterone receptor (PR) levels. P has been also shown to regulate anterior pituitary levels of FSH-beta mRNA in the rat. Although the mechanism of this action has not been determined, steroids may regulate gene expression through the binding of liganded receptors to gene sequences known as hormone response elements (HRE); however, it is not known whether HRE's exist on the rat FSH-beta gene. We have localized a series of progesterone response elements (PRE)-like sequences on the rat FSH-beta gene and have begun testing the hypothesis that P modulates the expression of the rat FSH-beta gene through the direct binding of the P/PR complex to these PRE-like sequences. Electromobility shift assays indicate that these PRE-like sequences bind PR with high affinity and specificity. In addition, when a 361-base pair sequence, which contains the three PRE-like sequences localized in the upstream region of the gene, was cloned into a luciferase expression vector driven by a heterologous promoter and transiently transfected into anterior pituitary cell cultures, progestin stimulation elicited increased luciferase expression. These results indicated that the 361-base pair sequence conferred P-responsiveness to a heterologous promoter. The data further suggest that FSH synthesis in the rat is modulated by direct binding of PR to PRE-like sequences.

Regulation of leptin gene expression and secretion by steroid hormones.

Previous work has shown that 17 beta-estradiol is the primary ovarian signal regulating body weight and adiposity, although its mechanisms of action remain unclear. We hypothesized that 17 beta-estradiol could enhance leptin levels as a mechanism of its anorectic effects. Administration of 5 microg 17 beta-estradiol subcutaneously (s.c.) for 2 days significantly elevated leptin mRNA levels in adipose tissue as compared to vehicle controls (P < 0.003). A time-course administration of estrogen showed increased mRNA levels in adipose tissue between 6 and 12 h after estrogen injection as compared to vehicle controls (P < 0.03). Corresponding to the increased leptin mRNA levels at 6 and 12 h, elevated plasma leptin levels were observed at 12 h after estrogen administration as compared to controls (P < 0.05). Administration of progesterone (1 mg/rat) after estradiol injection did not enhance the elevated leptin mRNA levels in adipose tissue. Serum leptin levels from cycling rats did not differ significantly between metestrous and proestrous animals. In conclusion, the present studies demonstrate that 17 beta-estradiol can regulate leptin gene expression and secretion in the female rat, thus providing a better understanding of the possible anorectic effect of estrogens.

Bradykinin receptor localization and cell signaling pathways used by bradykinin in the regulation of gonadotropin-releasing hormone secretion.

In a previous publication we provided evidence of a novel neuronal pathway for the control of GnRH secretion by bradykinin. The action of bradykinin appeared to be exerted through the bradykinin B2 receptor. In this study we demonstrated that the bradykinin B2 receptor is densely localized in the arcuate nucleus, median eminence, organum vasculosum of the lamina terminalis, and preoptic area, regions known to be critical for the control of GnRH secretion. To determine the mechanism of action of bradykinin in stimulating GnRH release, we used immortalized GnRH (GT1-7) cells in vitro. Bradykinin stimulation of GnRH secretion from GT1-7 cells appears to involve activation of the phospholipase C signaling pathway and mobilization of extracellular and intracellular calcium stores. Evidence to support this contention was derived from the observations that incubation of the phospholipase C inhibitor, U-73122 with bradykinin, blocked the ability of bradykinin to stimulate release from GT1-7 cells. This effect was specific, as a nitric oxide synthase inhibitor and a cyclooxygenase inhibitor were found to have no effect on bradykinin-induced GnRH secretion, suggesting that nitric oxide and PGs do not mediate bradykinin effects. Pertussis toxin also had no effect on bradykinin action. This suggests that the bradykinin B2 receptor may be coupled to a pertussis toxin-insensitive G protein in GT1-7 cells. With respect to calcium involvement in bradykinin action, fura-2 calcium indicator studies revealed that bradykinin can rapidly increase intracellular Ca2+ levels in GT1-7 cells. A role for intracellular Ca2+ in bradykinin action was further suggested by the finding that an intracellular calcium chelator, 1,2-bis(O-aminophenoxy)]ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, significantly attenuated the effects of bradykinin on GnRH release. The elevation of intracellular calcium by bradykinin appears to be due to mobilization of calcium from the endoplasmic reticulum, as incubation of the Ca2+-adenosine triphosphatase inhibitor thapsigarin, which depletes endoplasmic reticulum Ca2+ stores, significantly attenuated bradykinin action on GnRH release. Extracellular calcium may also be involved in bradykinin action, as the L-type Ca2+ channel blockers verapamil and nifedipine had no effect on bradykinin-induced GnRH release, whereas the nonselective Ca2+ channel blocker, nickel chloride, attenuated bradykinin-induced GnRH release. Taken as a whole, these studies demonstrate that the bradykinin B2 receptor is densely localized in key hypothalamic nuclei responsible for regulation of GnRH release, and that the mechanism of bradykinin stimulation of GnRH secretion involves activation of the phospholipase C signaling pathway, with a critical role implicated for calcium in bradykinin action in GT1-7 cells.

Characterization of ionotropic glutamate receptors in rat hypothalamus, pituitary and immortalized gonadotropin-releasing hormone (GnRH) neurons (GT1-7 cells).

Evidence from various sources suggested that the Gonadotropin-Releasing Hormone (GnRH) neuron does not contain glutamate receptors. Northern analysis of the hypothalamus showed the presence of NMDAR1, GluR1, GluR4 and GluR6 mRNA, while the pituitary showed the presence of NMDAR1, GluR1 and GluR6 mRNA. Western blot analysis also showed the presence of NMDAR1 and GluR1 protein. Since there are relatively few GnRH neurons in the hypothalamus, and GT1-7 cells have been considered to be a GnRH neuronal cell line, GT1-7 cells were studied in detail. GT1-7 cells contained NMDAR1 mRNA levels as shown by Northern analysis but did not contain GluR1, GluR4, or GluR6 mRNA. They did not show the presence of NMDAR1 and GluR1 protein by Western analysis. In addition, GT1-7 cells showed no NMDA receptor binding using the competitive inhibitor CGP-39563 and the noncompetitive inhibitor MK-801. Likewise, no binding was detected for kainate receptors. However, a small amount of binding for AMPA receptors was found in GT1-7 cells. GT1-7 cells did not exhibit glutamate toxicity and NMDA failed to elicit inward currents using patch-clamp techniques, although GABA did induce currents in the cells. As a whole, these studies suggest that GT1-7 cells lack or possess only low levels of ionotropic glutamate receptors.

Expression of mRNA and proteins for testicular steroidogenic enzymes and brain and pituitary mRNA for glutamate receptors in rats exposed to immobilization stress.

The objectives of this study were to determine whether stress attenuates the pituitary LH response to excitatory amino acids by altering expression of glutamate receptor 1 (GluR1) and N-methyl-D-aspartic acid (NMDA) receptor mRNA levels in the hypothalamus or pituitary, and assess whether stress influences testicular levels of mRNA or protein for steroidogenic enzymes. Three hours (h) of immobilization stress was associated with a greater than 7-fold increase in serum corticosterone, and a marked reduction in serum testosterone (T) concentrations. Stress did not significantly alter hypothalamic or pituitary GluR1 and NMDA receptor mRNA levels. Although transcript levels for P450SCC and P45017alpha mRNA in the testis were unchanged in stressed rats, western blotting of testicular fractions revealed reduced amounts of P450SCC and 3beta-HSD, but not P45017alpha. The data suggest that immobilization stress reduces T production by suppressing the translation of transcripts for P450SCC and 3beta-HSD, but the attenuated LH response of stressed animals to NMDA is not mediated by altered hypothalamic or pituitary expression of GluR1 and NMDA receptor levels.

Regulation of the preovulatory gonadotropin surge by endogenous steroids.

Estradiol secreted by growing ovarian follicle(s) has been considered classically to be the neural trigger for the preovulatory surge of gonadotropins. The observation that the estradiol-induced gonadotropin surge in ovariectomized rats is of lesser magnitude and duration than that found in the cycling rat at proestrus has resulted in a search for other steroid regulators. Progesterone is a major regulator of the preovulatory gonadotropin surge. It can only act in the presence of an estrogen background, which is necessary for the synthesis of progesterone receptors. In the estrogen-primed ovariectomized rat, progesterone is able to initiate and enhance the gonadotropin surge to the magnitude observed on the day of proestrus and limit it to 1 day. The physiological role of progresterone in the induction of the preovulatory gonadotropin surge has been demonstrated by the attenuation of the progesterone-induced surge and the endogenous proestrus surge by progesterone receptor antagonist RU486 and the progesterone synthesis inhibitor trilostane. The promoter region of the follicle-stimulating hormone (FHS)-beta gene contains multiple progesterone response elements and progesterone brings about FSH release as well. The reduction of progesterone in the 5 alpha-position appears to be important for the regulation of progesterone secretion. Corticosteroids appear to play a significant role in the secondary FSH surge on late proestrus and early estrus.

The ligand insertion hypothesis in the genomic action of steroid hormones.

Gene regulation by steroids is tightly coupled to hormone concentration and stereochemistry. A key step is binding of hormones to receptors which interact with consensus DNA sequences known as hormone response elements (HREs). The specificity and strength of hormone binding do not correlate well with hormonal activity suggesting an additional step involving recognition of ligand by the gene. Stereospecific fit of hormones between base pairs and correlation of fit with hormonal activity led to the proposal that such recognition involves insertion of hormone into DNA. Here, the feasibility of insertion was investigated using computer models of the glucocorticoid receptor DNA binding domain bound to its HRE. The site reported to accommodate glucocorticoids was found in the HRE and was exposed to permit unwinding at this locus. The resulting cavity in the unwound DNA/receptor interface fit cortisol remarkably well; cortisol formed hydrogen bonds to both the receptor and DNA. Current experimental evidence is generally consistent with ligand binding domains of receptors undergoing a conformational change which facilitates transfer of the ligand into the unwound DNA/receptor interface. We propose this step is rate limiting and alterations in receptor, DNA or hormone which attenuate insertion impair hormonal regulation of gene function.

Neuroendocrine mechanisms underlying the control of gonadotropin secretion by steroids.

There is considerable evidence that although estradiol may trigger the preovulatory surge of gonadotropins, progesterone is required for its full magnitude and duration and that glucocorticoids bring about selective follicle-stimulating hormone release. The luteinizing hormone-releasing hormone (LHRH) neuron does not have steroid receptors and is regulated by excitatory amino acid neurotransmission. Steroids do not appear to modulate excitatory amino acid receptors directly but increase release of glutamate in the preoptic area. This may be due to the suppression by steroids of the enzyme glutamatic acid decarboxylase67 that converts glutamate into GABA. NMDA receptors colocalize with nitric oxide synthase-containing neurons that surround the LHRH neurons in the preoptic area and intersect the LHRH fibers in the median eminence. Other potential novel pathways of LHRH release that are currently being explored include carbon monoxide generated by the action of heme oxygenase-2 on heme molecules and bradykinin acting via bradykinin B2 receptors.

Excitatory amino acid receptors and puberty.

Glutamate is an important excitatory signal in the hypothalamus for the steroid-mediated preovulatory gonadotropin surge. Steroids may exert this action by regulating glutamate receptor levels or glutamate release, or both. Work in our laboratory found no changes in NMDA and kainate receptor binding in the hypothalamus of castrated or castrated plus steroid-replaced male and female rats. Likewise, we found that NMDA and kainate binding did not change over the onset of puberty in the female rat. A competitive quantitative RT-PCR assay using exogenous internal standards was used to measure NMDAR1, GluR1, and beta-actin mRNAs levels. NMDAR1 and GluR1 expression was examined in the preoptic hypothalamic area and in the medial basal hypothalamus at Postnatal Days 10, 15, 20, 25, 30, 32, 34, 36, 40, and 63. A transient increase in GluR1 mRNA levels in the preoptic hypothalamic area was observed on Day 20, with all other time points showing comparable levels. NMDAR1 levels in the POA and medial basal hypothalamus did not change significantly at any of the time points; in contrast, however, AMPA receptor binding levels were increased in the hypothalamus at the time of puberty in the female rat. Thus, in addition to the previously reported elevation of glutamate release rates in the hypothalamus at the time of puberty, AMPA receptors may also be elevated and play a role in mediating glutamate regulatory effects on the timing of puberty in the female rat.

Evidence for a role of bradykinin neurons in the control of gonadotropin-releasing hormone secretion.

The present study provides evidence of a novel neuronal pathway for the control of GnRH secretion involving bradykinin neurons. Bradykinin neurons were shown by immunohistochemistry to be densely localized in several regions of the brain including the cortex, hippocampus and supraoptic nucleus, as well as two regions critical in the control of GnRH secretion, the organum vasculosum of the lamina terminalis and arcuate nucleus. Bradykinin dose-dependently stimulated GnRH release from male and proestrous female rat hypothalami in vitro. Antagonist studies revealed that bradykinin effects are mediated by the bradykinin B2 receptor. The effect of bradykinin on GnRH release is not mediated by the classical major transmitter, glutamate, as glutamate antagonists had no effect on bradykinin stimulation of GnRH release. Rather, bradykinin appears to act directly on the GnRH neuron as bradykinin stimulated GnRH release directly from immortalized GnRH (GT1-7) neurons in vitro, and immunoblot studies revealed that the bradykinin B2 receptor is present in GT1-7 neurons. The bradykinin B2 receptor was also demonstrated in the rat hypothalamus and pituitary by immunoblotting. Bradykinin-induced exocytosis of GnRH appears to involve activation of the PKC signaling pathway, as a PKC inhibitor blocked bradykinin-induced GnRH release. Finally, bradykinin neurons appear to be important mediators of steroid signals in the hypothalamus to produce the LH surge, as central administration of a B2 antagonist, but not a B antagonist, significantly attenuated the steroid-induced LH surge in the ovariectomized female rat.

Regulation of the progesterone receptor and estrogen receptor in decidua basalis by progesterone and estradiol during pregnancy.

In this study we examined the roles of progesterone (P4) and estradiol-17beta (E2) in regulation of the P4 receptor (PR) and estrogen receptor (ER) in the decidua basalis (DB) during stromal cell proliferation and regression (Days 10 and 14 of pregnancy, respectively). Pregnant rats were ovariectomized (Ovx) on Day 8 or 12 and killed on Day 10 or 14, respectively, following treatment with P4, E2, or both. In some experiments, rats received pellets of the anti-progestin RU-486 on Day 9 and were killed 3, 6, 12, and 24 h later. Immunolocalization of PR and ER showed that both receptors decreased from Day 10 to Day 14. Histologic integrity of the placenta and DB were maintained only when P4 was present. Control and hormone-treated groups expressed established isoforms of PR and ER: PR-B, 110 kDa; PR-A, 80-90 kDa; PR-C, 64-60 kDa; ER-66, 66 kDa; and ER-49, 49 kDa. On Day 10, expression of PR-A, PR-B, and ER-66 decreased 50-99% (p < 0.05) after Ovx or RU-486 treatment but was restored to control levels after Ovx by exogenous P4. On Day 14, PR-B and ER-66 declined 66-75% (p < 0.05) after Ovx and could not be restored by P4 treatment. Estrogen could not substitute for P4, and co-administration of E2 with P4 did not enhance the response over P4 alone. In contrast, PR-C was abundantly expressed on Days 10 and 14 in all treatment groups after Ovx and RU-486. P4 maintained PR mRNA and ER mRNA after Ovx. Thus, regression of the DB may be initiated via changes in relative expression of PR isoforms, which result in impaired stromal cell response to P4 action.

Opioid-glutamate-nitric oxide connection in the regulation of luteinizing hormone secretion in the rat.

Opioid neurons are recognized to be an important component of the inhibitory "brake" in the CNS that restrains LHRH secretion. Opioid inhibition could be exerted directly on LHRH neurons, or it could be achieved via indirect mechanisms involving restrainment of excitatory "accelerator" neurons that facilitate LHRH release. The purpose of the present study was to explore the second hypothesis by investigating whether removal of opioid inhibition by administering the opioid antagonist, naloxone leads to enhanced activation of glutamate and nitric oxide (NO) neurons, which are known to be important excitatory "accelerator" components for the control of LHRH secretion. Naloxone administration (2.5 mg/kg) to adult male rats induced a significant elevation of serum LH levels at 20 min post injection. NOS activity in preoptic area (POA) and medial basal hypothalamic (MBH) fragments was demonstrated to be significantly elevated 20 min post naloxone injection. Administration of a glutamate (NMDA) receptor antagonist (MK-801, 0.2 mg/kg) abolished the naloxone-induced increase in NOS activity in the POA and MBH, with a corresponding block of the naloxone-induced LH release. Glutamate appears to only be involved in LH surge generation and not to regulate basal LH levels, as MK-801 had no effect on basal LH release. Because previous work by our laboratory and others have provided evidence that NO is a mediator of glutamate effects in the hypothalamus, these findings are interpreted to mean that opioid inhibition is mediated on glutamate neurons that are upstream of NO neurons. In support of this contention, we found that NMDA treatment enhanced NOS activity in the male rat POA and MBH fragments in vitro, an effect that was specific as it was completely blocked by the NMDA receptor antagonist, MK-801. Additionally, in vivo microdialysis studies revealed that naloxone treatment significantly enhances glutamate release in the preoptic area (POA) at 15 min post injection in conscious, unanesthetized, freely moving male rats. Release rates of the control amino acid, serine did not change significantly following naloxone injection. Taken as a whole, these findings provide evidence for an opioid-glutamate-NO pathway in the control of LHRH secretion, and they demonstrate the importance of "brake-accelerator" interactions in the control of LHRH and LH secretion.

Progesterone metabolite allopregnanolone in women with premenstrual syndrome.

OBJECTIVE: To evaluate the anxiolytic 3alpha-5alpha-reduced progesterone metabolite allopregnanolone in the luteal phase of the menstrual cycle in women with premenstrual syndrome (PMS) and controls. METHODS: Thirty-five women with prospectively documented PMS and 36 controls were evaluated. Serum progesterone and allopregnanolone levels were measured on days 19 and 26 of the cycle as determined by urinary LH detection kits. Analysis of variance and Student t tests were used to analyze the data. RESULTS: Allopregnanolone levels were significantly lower on day 26 in the PMS group than in controls (3.6 +/- 0.8 versus 7.5 +/- 1.3 ng/mL; P < .04). Significant differences in the ratio of the metabolite to progesterone also were noted, with a smaller ratio in the PMS subjects (0.9 +/- 0.3 versus 3.2 +/- 1.3 ng/mL; P < .05). There were no significant differences between the PMS and control groups with respect to serum progesterone levels. CONCLUSION: Subjects with PMS manifested lower levels of the anxiolytic metabolite allopregnanolone in the luteal phase when compared with controls. Diminished concentrations of allopregnanolone in women with PMS may lead to an inability to enhance gamma aminobutyric acid-mediated inhibition during states of altered central nervous system excitability, such as ovulation or physiologic or psychological stress. The lowered metabolite levels could contribute to the genesis of various mood symptoms of the disorder, such as anxiety, tension, and depression.

Stromal cell progesterone and estrogen receptors during proliferation and regression of the decidua basalis in the pregnant rat.

This study examines the distribution and abundance of progesterone receptors (PR) and estrogen receptors (ER) in the decidua basalis (DB) during proliferation (Days 8-12 of gestation) and regression (Days 14-21) in the rat. Stromal cells of the DB and metrial gland exhibited strong nuclear immunostaining for PR throughout gestation. Nuclear localization of ER was detectable only between Days 8-12. The heavily granulated natural killer cells were always negative for PR and ER. DB were dissected between Days 8 and 17 to measure progesterone (P4)-binding sites and receptor proteins by Western blotting. The latter revealed four specific PR isoforms: B (110 kDa), A1 (90 kDa), A2 (76-82 kDa), and C (60-64 kDa). Stromal cell nuclei contained more than 50% of P4-binding sites during DB proliferation but less than 22% during regression (p < 0.05). PR-A and PR-B expression was greatest at proliferative stages (p < 0.05). PR-C increased in relative abundance during DB regression. Two ER isoforms of 66 kDa and 49 kDa were revealed. The 66-kDa ER, the most abundant form, was maximally expressed during proliferation, declining 71% by Day 12 (p < 0.01), whereas the 49-kDa form accounted for up to 90% of ER during regression. Northern blot analysis revealed three prominent transcripts of approximately 11, 7, and 4 kilobases (kb) for PR mRNA, which declined markedly at Days 14 to 17 (p < 0.05), and one of 6.0 kb for ER mRNA, which declined markedly on Day 17 (p < 0.05). Our study establishes that the DB expresses heterogeneity of receptor message and proteins. We propose that preferential expression of receptor isoforms in late pregnancy limits P4 action and promotes DB regression in spite of invariant levels of serum P4, P4-binding sites, and total receptor protein.