Clustering of PCOS-like traits in naturally hyperandrogenic female rhesus monkeys.

Study question: Do naturally occurring, hyperandrogenic (≥1 SD of population mean testosterone, T) female rhesus monkeys exhibit traits typical of women with polycystic ovary syndrome (PCOS)? Summary answer: Hyperandrogenic female monkeys exhibited significantly increased serum levels of androstenedione (A4), 17-hydroxyprogesterone (17-OHP), estradiol (E2), LH, antimullerian hormone (AMH), cortisol, 11-deoxycortisol and corticosterone, as well as increased uterine endometrial thickness and evidence of reduced fertility, all traits associated with PCOS. What is known already: Progress in treating women with PCOS is limited by incomplete knowledge of its pathogenesis and the absence of naturally occurring PCOS in animal models. A female macaque monkey, however, with naturally occurring hyperandrogenism, anovulation and polyfollicular ovaries, accompanied by insulin resistance, increased adiposity and endometrial hyperplasia, suggests naturally occurring origins for PCOS in nonhuman primates. Study design, size, duration: As part of a larger study, circulating serum concentrations of selected pituitary, ovarian and adrenal hormones, together with fasted insulin and glucose levels, were determined in a single, morning blood sample obtained from 120 apparently healthy, ovary-intact, adult female rhesus monkeys (Macaca mulatta) while not pregnant or nursing. The monkeys were then sedated for somatometric and ultrasonographic measurements. Participants/materials, setting, methods: Female monkeys were of prime reproductive age (7.2 ± 0.1 years, mean ± SEM) and represented a typical spectrum of adult body weight (7.4 ± 0.2 kg; maximum 12.5, minimum 4.6 kg). Females were defined as having normal (n = 99) or high T levels (n = 21; ≥1 SD above the overall mean, 0.31 ng/ml). Electronic health records provided menstrual and fecundity histories. Steroid hormones were determined by tandem LC-MS-MS; AMH was measured by enzymeimmunoassay; LH, FSH and insulin were determined by radioimmunoassay; and glucose was read by glucose meter. Most analyses were limited to 80 females (60 normal T, 20 high T) in the follicular phase of a menstrual cycle or anovulatory period (serum progesterone <1 ng/ml). Main results and the role of chance: Of 80 monkeys, 15% (n = 12) exhibited classifiable PCOS-like phenotypes. High T females demonstrated elevations in serum levels of LH (P < 0.036), AMH (P < 0.021), A4 (P < 0.0001), 17-OHP (P < 0.008), E2 (P < 0.023), glucocorticoids (P < 0.02-0.0001), the serum T/E2 ratio (P < 0.03) and uterine endometrial thickness (P < 0.014) compared to normal T females. Within the high T group alone, anogenital distance, a biomarker for fetal T exposure, positively correlated (P < 0.015) with serum A4 levels, while clitoral volume, a biomarker for prior T exposure, positively correlated (P < 0.002) with postnatal age. Only high T females demonstrated positive correlations between serum LH, and both T and A4. Five of six (83%) high T females with serum T ≥2 SD above T mean (0.41 ng/ml) did not produce live offspring. Large scale data: N/A. Limitations, reasons for caution: This is an initial study of a single laboratory population in a single nonhuman primate species. While two biomarkers suggest lifelong hyperandrogenism, phenotypic expression during gestation, prepuberty, adolescence, mid-to-late reproductive years and postmenopause has yet to be determined. Wider implications of the findings: Characterizing adult female monkeys with naturally occurring hyperandrogenism has identified individuals with high LH and AMH combined with infertility, suggesting developmental linkage among traits with endemic origins beyond humans. PCOS may thus be an ancient phenotype, as previously proposed, with a definable pathogenic mechanism(s). Study funding/competing interest(s): Funded by competitive supplement to P51 OD011106 (PI: Mallick), by P50 HD028934 (PI: Marshall) and by P50 HD044405 (PI: Dunaif). The authors have no potential conflicts of interest.

Mycobacterium chelonae necrotizing pneumonia after allogeneic hematopoietic stem cell transplant: report of clinical response to treatment with tigecycline.

We present a case of progressive Mycobacterium chelonae ssp. chelonae necrotizing pneumonia after hematopoietic stem cell transplantation (HSCT) in the presence of chronic graft-versus-host disease. The patient failed to respond to standard combination therapy with multiple agents and developed resistance to most drugs over the course of treatment. Tigecycline, a new glycylcycline antimicrobial agent with in vitro activity against M. chelonae, was then used with a clinical response to treatment. To our knowledge, this is the first reported case demonstrating tigecycline to have a degree of clinical effectiveness to treat refractory pulmonary infection with M. chelonae in an HSCT recipient.

Estrogen receptor alpha signaling pathways differentially regulate gonadotropin subunit gene expression and serum follicle-stimulating hormone in the female mouse.

Estrogen, acting via estrogen receptor (ER)alpha, regulates serum gonadotropin levels and pituitary gonadotropin subunit expression. However, the cellular pathways mediating this regulation are unknown. ERalpha signals through classical estrogen response element (ERE)-dependent genomic as well as nonclassical ERE-independent genomic and nongenomic pathways. Using targeted mutagenesis in mice to disrupt ERalpha DNA binding activity, we previously demonstrated that ERE-independent signaling is sufficient to suppress serum LH levels. In this study, we examined the relative roles of ERE-dependent and -independent estrogen signaling in estrogen regulation of LH, FSH, prolactin, and activin/inhibin subunit gene expression, pituitary LH and FSH protein content, and serum FSH levels. ERE-independent signaling was not sufficient for estrogen to induce pituitary prolactin mRNA or suppress pituitary LHbeta mRNA, LH content, or serum FSH in estrogen-treated ovariectomized mice. However, ERE-independent signaling was sufficient to reduce pituitary glycoprotein hormone alpha-subunit, FSHbeta, and activin-betaB mRNA expression. Together with previous serum LH results, these findings suggest ERE-independent ERalpha signaling suppresses serum LH via reduced secretion, not synthesis. Additionally, ERE-dependent and ERE-independent ERalpha pathways may distinctly regulate steps involved in the synthesis and secretion of FSH.

Donor leukocyte infusions to treat hematologic malignancy relapse following allo-SCT in a pediatric population.

Donor leukocyte infusions (DLI) can reverse relapse of hematologic malignancy following allogeneic hematopoietic stem cell transplant (HSCT) in some cases. Little is known regarding the effectiveness of DLI in children who relapse after HSCT. We report outcomes of 49 children who received DLI for relapse after allogeneic transplant. Prognosis was particularly poor (0/14 responses) for patients relapsing within 6 months from transplant. DLI rarely induced remission when given as sole therapy for marrow relapse. One-year disease-free survival was 30% (6/20) in patients who received DLI as consolidation following chemotherapy. The development of GVHD grades 1-2 was associated with superior 3-year survival than patients who developed GVHD grades 3-4 (P<0.002). To determine the benefit of DLI, 45 children who received DLI for relapse (four children without matches were excluded) were compared to 1229 children with similar characteristics whose relapse was not treated with DLI. There was no difference in survival (P=0.30) once adjustments were made to account for the time from relapse to DLI. Although a few children achieved durable remissions when DLI was used as part of a post-relapse treatment strategy, DLI was unsuccessful in the majority of cases. Strategies may be better directed at preempting post transplant relapse.

Low-dose cidofovir treatment of BK virus-associated hemorrhagic cystitis in recipients of hematopoietic stem cell transplant.

In recipients of hematopoietic stem cell transplants (HSCTs), BK virus (BKV) has been associated with late-onset hemorrhagic cystitis (HC). In our institution, HSCT recipients with BKV-associated HC are treated with 1 mg/kg of cidofovir weekly. We identified HSCT recipients with BKV-associated HC, treated with weekly cidofovir. Microbiological response was defined as at least a one log reduction in urinary BKV viral load; clinical response was defined as improvement in symptoms and stability or reduction in the grade of cystitis. Nineteen allogeneic HSCT patients received a mean of 4.5 weekly doses of cidofovir. HC occurred at a mean of 68.7 days after transplant. A clinical response was detected in 16/19 (84%) patients, and 9/19 (47%) had a measurable microbiological response (8/10 nonresponders had a BKV viral load above the upper limit of the assay before treatment). Fourteen out of nineteen (74%) patients had no significant increase in serum creatinine. Five patients with renal dysfunction resolved after completion of the therapy and removal of other nephrotoxic agents. We conclude that weekly low-dose cidofovir appears to be a safe treatment option for BKV-associated HC. Although the efficacy of low-dose cidofovir is not proven, a prospective trial is warranted.

Vaccination of pediatric solid tumor patients with tumor lysate-pulsed dendritic cells can expand specific T cells and mediate tumor regression.

Dendritic cells (DCs) have been shown to be a promising adjuvant for inducing immunity to cancer. We evaluated tumor lysate-pulsed DC in a Phase I trial of pediatric patients with solid tumors. Children with relapsed solid malignancies who had failed standard therapies were eligible. The vaccine used immature DC (CD14-, CD80+, CD86+, CD83-, and HLA-DR+) generated from peripheral blood monocytes in the presence of granulocyte/monocyte colony-stimulating factor and interleukin-4. These DC were then pulsed separately with tumor cell lysates and the immunogenic protein keyhole limpet hemocyanin (KLH) for 24 h and then combined. A total of 1 x 10(6) to 1 x 10(7) DC are administered intradermally every 2 weeks for a total of three vaccinations. Fifteen patients (ages 3-17 years) were enrolled with 10 patients completing all vaccinations. Leukapheresis yields averaged 2.8 x 10(8) peripheral blood mononuclear cells (PBMC)/kg, and DC yields averaged 10.9% of starting PBMC. Patients with neuroblastoma, sarcoma, and renal malignancies were treated without obvious toxicity. Delayed-type hypersensitivity (DTH) response was detected in 7 of 10 patients for KLH and 3 of 6 patients for tumor lysates. Priming of T cells to KLH was seen in 6 of 10 patients and to tumor in 3 of 7 patients as demonstrated by specific IFN-gamma-secreting T cells in unstimulated PBMCs. Significant regression of multiple metastatic sites was seen in 1 patient. Five patients showed stable disease, including 3 who had minimal disease at time of vaccine therapy and remain free of tumor with 16-30 months follow-up. Our results demonstrate that it is feasible to generate large numbers of functional DC from pediatric patients even in those highly pretreated and with a large tumor burden. The DC can be administered in an outpatient setting without any observable toxicity. Most importantly, we have demonstrated the ability of the tumor lysate/KLH-pulsed DC to generate specific T-cell responses and to elicit regression of metastatic disease.

Safety and efficacy of allogeneic PBSC collection in normal pediatric donors: the pediatric blood and marrow transplant consortium experience (PBMTC) 1996-2003.

The use of peripheral blood stem cells (PBSC) for allogeneic transplants in adults has greatly increased. This trend is reflected in pediatrics, where healthy children increasingly are donating PBSC or donor lymphocyte infusion (DLI) via apheresis for use by ill siblings. There is a potential concern that the risks of PBSC collection may differ for pediatric donors. However, no large studies have assessed safety issues in this population. To address this need, we reviewed 218 (213 PBSC, five DLI) collections in 201 normal pediatric donors (8 months to 17 years, median 11.8 years) at 22 institutions in the Pediatric Blood and Marrow Transplant Consortium. Donors received a median of 4 days of growth factor, and mean collection yield was 9.1 x 10(6) CD34+ cells/kg recipient weight. Younger age, days of apheresis, and male gender predicted increased yield of CD34+ cells/kg donor weight. Growth factor-induced pain was mild and reported in less than 15% of patients. Most donors <20 kg (23/25, 92%) required PRBC priming of the apheresis machine. This experience with over 200 collections demonstrates that PBSC collection is safe in normal pediatric donors and desired CD34 cell yields are easily achieved. Younger children utilize more medical resources and children <20 kg usually require a single blood product exposure.

Stem cell transplantation in patients with severe congenital neutropenia with evidence of leukemic transformation.

Severe congenital neutropenia (SCN) is a hematologic condition characterized by arrested maturation of myelopoiesis at the promyelocyte stage of development. With appropriate treatment using recombinant human granulocyte-colony-stimulating factor (r-HuG-CSF), SCN patients are now surviving longer, but are at increased risk of developing myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). Hematopoietic stem cell transplantation (HSCT) is the only curative option for these patients, but transplantation outcomes after malignant transformation are not well established. We report results for six patients with SCN who underwent HSCT for MDS or AML between 1997 and 2001 at two transplant centers. Two patients transplanted for MDS survived. Both of these patients were transplanted without being given induction chemotherapy. Four patients, who all received induction chemotherapy for AML prior to HSCT, died. Administering induction chemotherapy prior to HSCT resulted in significant morbidity. Rapid transplantation should be the goal for the SCN patient once the diagnosis of MDS/AML is established. SCN patients should be monitored carefully for progression to MDS in order to be treated with HSCT as soon as they have progressed and before developing AML. For SCN patients who progress to AML, HSCT should still be considered, even though the risks appear to be greater.

Estrogen inhibits luteinizing hormone (LH), but not follicle-stimulating hormone secretion in hypophysectomized pituitary-grafted rats receiving pulsatile LH-releasing hormone infusions.

The differential feedback actions of estrogen (E2) on gonadotropin secretion were studied by means of an in vivo isolated pituitary paradigm. Adult female rats were hypophysectomized (hypox) and the next day received single anterior pituitary transplants (graft) under the kidney capsule. At the same time rats underwent bilateral ovariectomy. On the third day each animal was fitted with a catheter system which allowed for intermittent infusions of LHRH (250 ng/5 min.h) and chronic blood sampling. Rats received LHRH infusions for 7 days. On the sixth day of LHRH infusions blood samples were collected for 4 h 5, 15, 25, 35, 45 min after each hourly LHRH pulse. After 1 h of sampling, animals received sc injections of 2 micrograms estradiol benzoate (EB; n = 5) or oil vehicle (n = 5). Plasma LH, FSH, E2, and PRL levels in samples from all groups were determined by RIA. In hypox/graft rats LH release, but not FSH release, was pulsatile in response to the hourly LHRH infusions. Injection of EB in the hypox/graft rats significantly (P less than 0.05) suppressed LH release within 3 h by 57%, while FSH was unaffected. PRL levels were elevated by approximately 10-fold in the hypox/graft animals compared to those in pituitary-intact rats. These levels, however, were not changed as a function of steroid treatment and, therefore, could not account for the effects of EB on LH secretion. On the basis of these observations we conclude that 1) a major inhibitory effect of an acute injection of EB on LH secretion is exerted by a direct action on pituitary gonadotropes, and 2) E2 can differentially affect the release of LH and FSH by an intrapituitary mechanism. It is hoped that development of this model will allow for further investigation of the cellular mechanisms that mediate feedback actions of E2 on pituitary gonadotropes exposed to intermittent LHRH stimulation.

Simultaneous measurement of luteinizing hormone (LH)-releasing hormone, LH, and follicle-stimulating hormone release in intact and short-term castrate rats.

The temporal relationship between LHRH release and gonadotropin secretion as well as the effects of castration on LHRH release were investigated in conscious, freely moving male rats. LHRH release was measured in hypothalamic/median eminence perfusates, while levels of pituitary gonadotropins (LH, FSH) were determined in sequential blood samples obtained via atrial catheters. Twenty-four to 26 h before experiments, rats underwent sham surgery or castration. LHRH release in push-pull perfusates from both groups was pulsatile, and nearly all identified LH pulses (83.3%) were temporally associated with LHRH pulses. Of the fewer irregular FSH pulses that were observed, only 43.7% were temporally associated with LHRH pulses. Mean LHRH pulse amplitude and mean LHRH levels were not different in intact and castrate animals. The frequency of LHRH pulses was moderately increased in castrate rats (1.30 pulses/h) compared to that in intact animals (0.83 pulses/h), and this acceleration was accompanied by a significant increase in LH pulse frequency, pulse amplitude, and mean level. It was also noted that the number of silent LHRH pulses (those not associated with LH pulses) was dramatically reduced in castrate animals. Characteristics of gonadotropin release (pulse frequency, pulse amplitude, and mean level) were not significantly different in animals undergoing push-pull perfusion/bleeding procedures from those in rats not receiving push-pull cannula implants. We conclude from these studies that 1) LH pulses show a high concordance with LHRH pulses, providing evidence that the LHRH pulse generator operates as the neural determinant of LH pulses in male rats, 2) FSH secretion is not associated with LHRH release in an obvious and consistent manner, suggesting that LHRH/FSH relationships are not easily discerned in these animals or that a FSH-releasing factor distinct from the LHRH decapeptide may regulate FSH secretion, 3) a modest increase in LHRH pulse frequency occurs 24-30 h after castration, and 4) silent LHRH pulses occur with much greater regularity in intact than in castrate rats. The latter two observations suggest that both hypothalamic and intrapituitary sequelae of castration may be critically important in the development of postcastration increases in LH secretion and the negative feedback of gonadal steroids.

Stimulation of gonadotropin-releasing hormone surges by estrogen. I. Role of hypothalamic progesterone receptors.

Estrogen (E2) stimulates GnRH surges by coupling a daily neural signal to neuronal circuitries governing GnRH release. We have hypothesized that E2 promotes this coupling process by inducing expression of neuronal transcription factors, which are subsequently activated by neurotransmitter-mediated mechanisms representing the daily neural signal. These experiments tested the specific hypothesis that the progesterone receptor (PR) functions in this manner, viz. as an E2-induced factor whose activation is necessary for the stimulation of GnRH surges. Two complimentary experiments were performed to determine whether activation of hypothalamic PRs is obligatory for the stimulation of GnRH surges by E2. In the first, the effects of a PR antagonist on GnRH and LH surges were assessed in ovariectomized (OVX), E2-primed rats. Rats were OVX on diestrous day 2, treated with 30 microg estradiol benzoate or oil vehicle, sc, and then administered either oil vehicle or the type I antiprogestin, ZK98299 at 0900 h on proestrus. GnRH release rates and plasma LH levels were determined in each animal by microdialysis of median eminence and atrial blood sampling, respectively. Estrogen, but not oil vehicle, treatment evoked robust and contemporaneous GnRH and LH surges in animals that received no PR antagonist on proestrus. Additional treatment with ZK98299, however, completely blocked both GnRH and LH surges. In a second experiment, specific involvement of anteroventral periventricular (AVPV) PRs in E2-induced GnRH surges was assessed. Additional groups of OVX, E2-primed rats were fitted with intracerebroventricular cannulas, and PR antisense oligonucleotides were infused into the third ventricle adjacent to the AVPV to prevent expression of PR in this periventricular region. Animals infused with PR antisense oligos did not exhibit any LH surges, whereas surges were observed in saline-, missense-, and sense oligo-treated controls. Immunohistochemistry confirmed the effectiveness of PR antisense oligonucleotides in blocking PR expression. These findings provide direct support for the hypothesis that activation of PRs, specifically those in hypothalamic regions including the AVPV, is an obligatory event in the stimulation of GnRH surges by E2.

Acute increase in responsiveness of luteinizing hormone (LH)-releasing hormone nerve terminals to neuropeptide-Y stimulation before the preovulatory LH surge.

Neuropeptide-Y (NPY) neurons regulate LH secretion in part through facilitation of LHRH release. We tested the hypothesis that responsiveness of LHRH neurons to NPY's facilitatory actions is physiologically regulated during the estrous cycle, and specifically, that it may be increased as a component of the gonadotropin surge-generating process. A dynamic superfusion paradigm was used to examine the role of cycle stage and time of day on LHRH responsiveness to NPY stimulation, using median eminence tissue from animals killed at 0900, 1400, and 1800 h on metestrus and proestrus. Tissue obtained at 0900 and 1800 h on metestrus did not exhibit significant LHRH responses to 10(-7) M NPY, and only moderate responses were seen at 1400 h on metestrus and 0900 h on proestrus. At 1400 h on proestrus, however, median eminence responsiveness to the same concentration of NPY was significantly increased, with LHRH responses to NPY being 2- to 5-fold greater than those at 0900 (P < 0.01), 1400 (P < 0.05), and 1800 h on metestrus (P < 0.01) and at 0900 h on proestrus (P < 0.05). Neither cycle-related changes in basal LHRH release nor changes in the releasability of LHRH in response to depolarization could account for the accentuated responses in the 1400 h proestrous group. These data clearly demonstrate that the responsiveness of LHRH terminals and/or their afferents to the actions of NPY is acutely enhanced during a brief window of time on proestrus, viz. immediately before generation of gonadotropin surges. Our findings are consistent with the hypothesis that the preovulatory endocrine milieu permits an acute increase in the responsiveness of LHRH nerve terminals to the actions of NPY, perhaps by prompting increases in the number and/or affinity of NPY receptors.

Role of protein kinase C in facilitation of luteinizing hormone (LH)-releasing hormone-induced LH surges by neuropeptide Y.

In female rats, neuropeptide Y (NPY) facilitates LHRH-induced LH surges without affecting basal LH release. The signal transduction mechanisms mediating this facilitation are unknown. Here, the involvement of PKC in this process was investigated. Anterior pituitaries (APs) were removed from rats at 1400 h proestrus and perifused in vitro with M199 for 5 h. After an equilibration and baseline period, tissue received hourly 5-minute pulses of the PKC inhibitor GF109203X (GFX), 2.5 microM, followed 15 min later by a 5-minute pulse of LHRH (10(-8) M), NPY (10(-6) M), or phorbol 12-myristate 13-acetate (PMA, 50 nM), or some combination. This regimen was repeated hourly for 3 h. As shown previously, NPY had no effect on basal LH release but greatly facilitated LHRH-induced LH release. Treatment with PMA also facilitated LHRH-induced LH release, to approximately the same degree as NPY. Inhibition of PKC activity with GFX completely prevented NPY's and PMA's facilitation of LH release but did not inhibit LH release stimulated by LHRH alone. Because previous work suggested involvement of both NPY and PKC in alterations of LHRH receptor affinity or number, the in vivo effects of NPY on LHRH binding characteristics were also investigated. Although NPY treatment reliably enhanced LHRH-induced LH and FSH surges in proestrous rats, this action was not accompanied by any detectable change in the affinity or concentration of LHRH receptors in anterior pituitary cell membranes. In summary, we have found that NPY's actions are blocked by PKC inhibition, mimicked by PKC stimulation, and not associated with any overt alterations in LHRH receptor affinity or number. We conclude that PKC activation is required for NPY's facilitation of LHRH-induced LH surges, and that this mechanism likely involves PKC targets other than those which may alter LHRH receptor number or affinity.

Stimulation of gonadotropin-releasing hormone surges by estrogen. II. Role of cyclic adenosine 3'5'-monophosphate.

Release of GnRH surges in female rats is directed by a daily neural signal and occurs only after exposure of the hypothalamus to sustained, elevated estrogen (E2) levels in serum. We have proposed that preovulatory E2 couples the daily neural signal to the circuitry governing GnRH release by a two-step process, which includes stimulation of neuronal progesterone receptors (PRs) by E2 and subsequent activation of PRs by the daily neural signal. In the preceding report we documented that PR activation is obligatory for the stimulation of GnRH surges by E2. In these studies we assess the validity of a second essential feature of this model, that neural signals can activate PRs and thereby prompt the release of GnRH and LH surges. Our efforts specifically focused on the role of cAMP in mediating neural PR trans-activation leading to GnRH surges. To assess whether cAMP may function as a daily neural signal, cAMP levels were examined via a competitive binding assay in anteroventral periventricular nucleus (AVPV) homogenates obtained at 0900, 1200, 1500, 1800, and 2100 h on all days of the estrous cycle. A significant rise in cAMP concentrations was observed at 1500 h on all estrous cycle days. A similar rise at the same time was observed in AVPV tissues of ovariectomized (OVX) rats regardless of steroid treatment. No significant increase in cAMP levels was observed at any time point in homogenates of ventromedial nucleus or cerebral cortex. In a second experiment, female rats were OVX on the afternoon of diestrous day 2 and simultaneously administered 30 microg estradiol benzoate or oil vehicle. On the following day of presumptive proestrus, rats received intracerebroventricular infusions of the cAMP analog, 8-bromo-cAMP, or saline vehicle at 0900 h. Rats treated with 8-bromo-cAMP exhibited LH surges that were advanced by 3 h compared with those in saline-treated controls. This advance did not occur in 8-bromo-cAMP-treated rats not primed with E2, or in E2-treated rats given the antiprogestin RU486. In a third experiment, OVX, estradiol benzoate-primed rats received intracerebroventricular infusions of saline vehicle or the adenylyl cyclase inhibitor SQ22536; although saline-treated rats exhibited normal LH surges, no surges were observed in the rats receiving SQ22536. In additional SQ22536-treated animals, however, LH surge release was rescued and greatly augmented by a pharmacological dose of progesterone. These results demonstrate that 1) cAMP levels in the AVPV are significantly elevated at 1500 h on a daily basis; 2) cAMP elevations in the AVPV can prematurely evoke LH surges by a mechanism that requires PR activation; 3) inhibition of adenylyl cyclase activity in the AVPV blocks LH surges, an action that can be reversed by progesterone; and 4) cAMP generation leads to PR transactivation in the AVPV. Our observations thus provide support for the hypothesis that an increase in intracellular cAMP in the AVPV acts as a component of the daily neural signal required to initiate GnRH and subsequent LH surges, and that transmission of this signal is mediated by cAMP-induced PR trans-activation in the AVPV.

Neuropeptide Y Y1-receptor stimulation is required for physiological amplification of preovulatory luteinizing hormone surges.

Neuropeptide Y (NPY) has been shown to potentiate the actions of LHRH during the generation of preovulatory LH surges. It is not yet known, however, if activation of a specific subtype of NPY receptors in the anterior pituitary gland is an obligatory event in the stimulation of spontaneous LH surges. A battery of NPY receptor agonists, as well as the specific NPY Y1 receptor antagonist BIBP3226, were used to assess the role of Y1 receptors in the amplification of LH surges. In Exp 1, the potencies of a number of NPY agonists in facilitating LHRH-induced LH surges were assessed in pentobarbital (PB)-blocked, proestrous rats. The rank-ordered potencies of these compounds were determined to be PYY = [Leu31Pro34]NPY > NPY >> hPP = rPP = NPY(13-36), which most closely reproduces the known rank-ordered affinties of these compounds for the Y1 receptor. In Exp 2, a Y1 subtype- specific antagonist, BIBP3226, was administered to unanesthetized, proestrous rats to assess the involvement of the Y1 receptor in the stimulation of spontaneous LH surges. The BIBP3226 compound strongly attenuated the endogenous proestrous LH surge, reducing the integrated value of LH secretion during the proestrous surge by more than 70%. In Exp 3, we assessed the ability of the Y1 receptor antagonist to block exogenous NPY effects on LHRH-induced LH surges. Treatment with BIBP3226 was found to completely prevent NPY amplification of LHRH-induced LH surges in pentobarbital-blocked, proestrous rats, thus confirming a pituitary locus of action of the drug. Taken together, these data clearly demonstrate that activation of neuropeptide Y receptors of the Y1 subtype is required for the physiological amplification of the spontaneous preovulatory LH surge in rats.

Neuropeptide Y potentiates luteinizing hormone (LH)-releasing hormone-induced LH secretion only under conditions leading to preovulatory LH surges.

We recently demonstrated that neuropeptide Y (NPY) potentiates the ability of pulsatile LHRH infusions to restore LH surges in pentobarbital (PB)-blocked, proestrous rats. In the present study we determined if specific endocrine conditions are necessary for the expression of these direct pituitary effects of NPY. Facilitatory actions of NPY were examined in the absence of gonadal feedback [ovariectomy (OVX)], in the presence of negative gonadal feedback (metestrus), after estrogen priming of the pituitary gland [OVX plus 30 micrograms estradiol benzoate (EB) 2 days before experiments], and after treatments which evoke preovulatory-like LH surges (OVX plus EB and 5 mg progesterone or P the morning of experiments). Rats received jugular catheter implants the day before experiments. On the day of experiments, hourly blood samples were taken from 1100-2100 h. At 1330 h, rats received injections of PB to block endogenous LHRH release, or saline. Every 30 min from 1400-1800 h, PB-treated rats received iv pulses of LHRH (15 ng/pulse) or saline, along with concurrent pulses of NPY (1 or 5 micrograms/pulse) or saline. Plasma samples were analyzed by LH RIA. In all cases, pulsatile administration of 15 ng LHRH resulted in plasma LH levels that were significantly elevated above saline-treated, PB-blocked controls. Only in the case of EB+P-treated rats did coadministration of 5 micrograms NPY along with LHRH significantly enhance LHRH-stimulated LH secretion (P < 0.001). NPY had no effect on LHRH-stimulated LH secretion in OVX, OVX + EB-treated, or metestrous rats. Pulsatile administration of either dose of NPY alone did not stimulate LH release in any of the four groups examined. These results demonstrate that the facilitatory effects of NPY on LHRH-stimulated LH secretion can be manifest only under the endocrine conditions required to produce full, preovulatory-like LH surges, i.e. after estrogen and P treatment.

Neuropeptide Y gene expression in the arcuate nucleus is increased during preovulatory luteinizing hormone surges.

Recent studies have suggested that neuropeptide Y (NPY) plays an important role in the induction of the preovulatory LH surge. The present study was performed in order to determine if a change in NPY gene expression within arcuate nucleus NPY neurons is associated with the generation of the preovulatory LH surge. In Exp 1, in situ hybridization was used to measure NPY messenger RNA (mRNA) levels in the arcuate nucleus of female rats at 0900 h and every 2 h from 1400-2200 h on the day of proestrus (PRO). Comparisons between groups showed a clear, stepwise increase in NPY gene expression throughout the day of PRO. At 1600 h, when LH values were significantly greater than 0900 h values, NPY mRNA labeling intensities in the arcuate nucleus were significantly greater than 0900 h levels (P < 0.01). By 1800 h, the time at which the LH surge peaked, NPY mRNA levels also peaked and were nearly 3-fold greater than levels observed at 0900 h (P < 0.01). NPY mRNA levels at 2000 h and 2200 h remained elevated above 0900 h levels (P < 0.01) but by 2000 h had decreased significantly from 1800 h levels (P < 0.05). In Exp 2, NPY mRNA levels were measured once again at 0900 h and 1800 h on PRO, and then at 0900 h and 1800 h on metestrus (MET), in order to determine if the change in gene expression seen in Exp 1 was unique to the day of PRO, or if it simply reflected a daily rhythm of gene expression in the nucleus. Analysis of mRNA levels showed no difference in NPY mRNA levels between 0900-1800 h on MET. Also, NPY mRNA levels at 0900 h and 1800 h on MET were significantly less than levels at 1800 h on PRO (P < 0.01). These results are consistent with the hypothesis that NPY neurons participate in the generation of LH surges through increased production of NPY and subsequent potentiation of the release and/or actions of LHRH.

In vitro gonadotropin-releasing hormone release from hypothalamic tissues of ovariectomized estrogen-treated cynomolgus macaques.

The effects of in vivo 17 beta-estradiol (E2) treatment on in vitro GnRH release and serum LH levels were studied to determine the loci of E2 feedback actions and to examine the hypothalamic mechanisms by which this steroid may regulate LH secretion in monkeys. Ovariectomized cynomolgus macaques received sc Silastic capsule implants containing E2 and were killed 12, 36, 42, or 48 h later. At least one control (CTL) animal received a blank implant and was killed concurrently with each E2-treated monkey. Three untreated animals were used in validation experiments. Before death, each animal was anesthetized with ketamine (15 mg/kg, im), and blood samples were drawn for subsequent LH analysis by Leydig cell bioassay. A diencephalic tissue block was obtained at autopsy and immediately immersed in Krebs-Ringer-phosphate medium (KRP). Mediobasal hypothalamic (MBH) and anterior hypothalamic/preoptic (AH/POA) fragments were quickly dissected from the block and placed in separate superfusion chambers maintained at 37 C. Tissues were superfused at 50 microliter/min with KRP, and 10-min fractions were collected, acidified, and stored at -20 C for subsequent GnRH RIA. Basal immunoreactive GnRH (IR-GnRH) release was measurable from MBH (0.367 +/- 0.063 pg/min) and AH/POA (0.176 +/- 0.065 pg/min) fragments from CTL monkeys. In validation experiments, IR-GnRH release was increased 3- to 7-fold by superfusion with 60 mM K+-KRP only in the presence of Ca+2. Superfusate IR-GnRH coeluted with synthetic GnRH from a Sephadex G-25 chromatographic column, and superfusate and tissue extract GnRH showed appropriate LH-releasing capacities, as determined by rat pituitary cell culture assay. IR-GnRH release rates from MBH or AH/POA tissues varied as a function of in vivo estrogen treatment. GnRH release from both tissues was increased in the E2-treated group killed at 12 h when LH levels were suppressed. Thirty-six hours after E2 treatment, in vitro GnRH release was not significantly different from CTL values. GnRH release rates from MBH and AH/POA tissues obtained 42 h after E2 treatment were significantly greater than CTL release rates (P less than 0.01). This increased in vitro GnRH release at 42 h occurred during the apparent rising phase of the LH surge. Elevated GnRH release was not sustained at 48 h, when surge levels of LH were apparent.(ABSTRACT TRUNCATED AT 400 WORDS)