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Biomedical subjects

J E Levine

Publications and source records attributed to J E Levine.

At least 19 recordsLinked to original sources
PubMed2001

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.

Adolescent↗
PubMed2001

Progesterone receptors as neuroendocrine integrators.

Intracellular progesterone receptors (PRs) are ligand-inducible transcription factors that mediate the majority of the effects of progesterone (P) on neuroendocrine functions. During the past decade, evidence has accumulated which suggest that PRs can also be activated independently of P, by signals propagated through membrane-bound receptors to the interior of cells. The activation of PRs by this type of "cross-talk" mechanism has been implicated in the physiological regulation of several important neuroendocrine processes, including estrous behavior and periovulatory hormone secretions. We review evidence that both ligand-dependent and ligand-independent activation of PRs occurs in central neurons and in anterior pituitary cells and that the convergence and summation of these signals at the PR serves to integrate neural and endocrine signals which direct several critically important neuroendocrine processes. An integrative function for PRs is reviewed in several physiological contexts, including the display of lordosis behavior in female rodents, the neurosecretion of gonadotropin-releasing hormone surges, secretion of preovulatory gonadotropin surges, and release of periovulatory follicle stimulating hormone surges. The weight of evidence indicates that cross talk at the intracellular PR is an essential component of the integrative mechanisms that direct each of these neuroendocrine events. The recurrence of PR's integrative actions in several different physiological contexts suggests that other intracellular steroid receptors similarly function as integrators of neural and endocrine signals in other neuroendocrine processes.

Animals↗
PubMed2001

In vivo gonadotropin-releasing hormone secretion in female rats during peripubertal development and on proestrus.

Pubertal development in female rats is characterized by increased LH levels and the appearance of estrogen-dependent afternoon LH mini-surges. In these studies we performed the first analysis of GnRH patterns in peripubertal rats to determine whether there are similar changes in pulsatile GnRH release. Microdialysis samples were collected at 5-min intervals throughout a 5-h afternoon period from 22 rats sampled on a single day between 30-47 days of age. Adult female rats were sampled on proestrus for comparison. In 30- to 33-day-old rats, GnRH release was infrequent (2.7 pulses/5 h; n = 3), whereas intermediate pulse frequencies were observed in 34- to 37-day-old rats (6.4 pulses/5 h; n = 9) and 38- to 42-day-old (5.0 pulses/5 h; n = 5) rats. The highest GnRH pulse frequencies were observed in 43- to 47-day-old rats (9.4 pulses/5 h; n = 5). Mean GnRH pulse amplitude did not vary significantly with age. Animals sampled before vaginal opening (VO) exhibited significantly slower GnRH pulse frequencies than those sampled after vaginal opening (1.3 pulses/5 h pre-VO vs. 7.6 pulses/5 h post-VO; P = 0.01). An afternoon increase in GnRH secretion, defined operationally as a greater than 25% increase in mean GnRH levels in the last half of the sampling period and tentatively termed a mini-surge, was observed in 0%, 33%, 40%, and 60% of 30- to 33-, 34- to 37-, 38- to 42-, and 43- to 47-day-old rats, respectively. An overall increase in GnRH pulse frequency was observed in females displaying a mini-surge (9.0 pulses/5 h with mini-surge compared with 4.7 pulses/5 h with no mini-surge). The mini-surge itself, however, was associated with a late afternoon increase in GnRH pulse amplitude and not in pulse frequency. In adult proestrous rats, peak levels during the GnRH surge were an order of magnitude greater than those reached in pubertal animals. Our findings demonstrate that pubertal maturation in the female rat is associated with an acceleration of GnRH pulse generator activity and that later stages of pubertal maturation are characterized by the appearance of afternoon increases in GnRH release that may underlie previously reported mini-surges in LH.

Aging↗
PubMed2000

Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.

Rho GTPases control a variety of cellular processes, including actin polymerization, integrin complex formation, cell adhesion, gene transcription, cell cycle progression, and cell proliferation. A patient is described who has recurrent infections and defective neutrophil cellular functions similar to those found in Rac2-deficient mice. Molecular methods were used to clone the expressed Rac2 cDNA from this patient, and a single base pair change (G-->A at nucleotide 169) in the coding sequence was identified. This results in an asparagine for aspartic acid mutation at amino acid 57 (D57N), a residue that is involved in nucleotide binding and is conserved in all mammalian Rho GTPases. The cloned cDNA was then introduced into normal bone marrow cells through retrovirus vectors, and neutrophils expressing this mutant exhibited decreased cell movement and production of superoxide in response to fMLP. The expressed recombinant protein was also analyzed biochemically and exhibited defective binding to GTP. Functional studies demonstrated that the D57N mutant behaves in a dominant-negative fashion at the cellular level. The syndrome of Rac2 dysfunction represents a human condition associated with mutation of a Rho GTPase and is another example of human disease associated with abnormalities of small G protein signaling pathways. (Blood. 2000;96:1646-1654)

3T3 Cells↗
PubMed2000

Stem cell transplantation in patients with severe congenital neutropenia without evidence of leukemic transformation.

Severe congenital neutropenia (CN) (Kostmann syndrome) is a hematologic disorder characterized by a maturation arrest of myelopoiesis at the promyelocyte/myelocyte stage of development. This arrest results in severe neutropenia leading to absolute neutrophil counts (ANC) below 0.2 x 10(9)/L associated with severe bacterial infections from early infancy. Data on over 300 patients with CN collected by the Severe Chronic Neutropenia International Registry (SCNIR) beginning in 1994 indicate that more than 90% of these patients respond to recombinant human granulocyte-colony stimulating factor (r-HuG-CSF) treatment with an ANC greater than 1. 0 x 10(9)/L. For patients who are refractory to r-HuG-CSF treatment and continue to have severe and often life-threatening bacterial infections, hematopoietic stem cell transplantation is the only currently available treatment. We report on a total of 11 patients with CN reported to the SCNIR who underwent transplantation for reasons other than malignant transformation between 1976 and 1998. Of these patients, 8 were nonresponders or showed only partial response to r-HuG-CSF treatment with ongoing infections. Results from these patients suggest that transplantation of stem cells from an HLA-identical sibling is beneficial for patients refractory to r-HuG-CSF. (Blood. 2000;95:1195-1198)

Adolescent↗
PubMed2000

Cytokine-mobilized allogeneic peripheral blood stem cell transplants in children result in rapid engraftment and a high incidence of chronic GVHD.

Between October 1995 and October 1998, 24 children aged 9 months to 17 years (median 11 years) underwent cytokine-mobilized allogeneic peripheral blood stem cell (PBSC) transplantation for treatment of hematological disorders. All of the transplants were the first allogeneic transplant for the recipient. Twenty patients were transplanted for hematological malignancies (ALL = 8, AML = 6, CML = 4, MDS = 2) and four patients were transplanted for non-malignant disease (thalassemia major = 2, Wiskott-Aldrich syndrome = 1, Kostmann's syndrome = 1). Nineteen donors were HLA-identical siblings, four were HLA-matched or single antigen mismatched parents, and one was a syngeneic transplant. Donors aged 8 to 38 years (median 15 years, 14 donors <18 years) received G-CSF 10 microg/kg/day subcutaneously beginning 4 days before PBSC collection and were submitted to one to three leukapheresis collections. The median CD34+ cell yield was 7.8 x 106 cells/kg recipient body weight. All patients achieved an ANC >0.5 x 109/l after a median of 13 days (range 10-21). Twenty-three patients eventually achieved platelet transfusion independence. One patient died on day 63 without ever achieving platelet transfusion independence. Four patients received platelet transfusions to maintain a platelet count well above 20 x 109/l due to bleeding complications. Of the 19 evaluable patients, the median time to a non-transfused platelet count of 20 x 109/l was 12 days (range 0-44). Ten of 23 at-risk patients developed acute GVHD grades II to IV, with grades III to IV in four patients. Twelve of 19 patients followed for at least 100 days have developed chronic GVHD (extensive = 2, limited = 10) with an actuarial risk of chronic GVHD of 75% at 1 year. The Kaplan-Meier estimate of event-free survival is 65% at 2 years. Four patients died (GVHD = 3, VOD = 1), three patients relapsed, and one patient with thalassemia major had a late graft failure with autologous recovery. Based upon our experience, allogeneic PBSCT is safe for both pediatric donors and recipients and engraftment of neutrophils and platelets is rapid. Bone Marrow Transplantation (2000) 25, 13-18.

Adolescent↗
PubMed2000

Tacrolimus (FK506) and methotrexate as prophylaxis for acute graft-versus-host disease in pediatric allogeneic stem cell transplantation.

Currently, limited data exist on the role of tacrolimus (FK506) in pediatric allogeneic marrow transplantation. Forty-one patients who received tacrolimus as prophylaxis were reviewed, with a median age of 9 years (range 0.2-16 years). Twenty-one patients underwent related donor transplants and 20 underwent unrelated donor transplants. All patients received tacrolimus beginning the day prior to transplant at a dose of 0.03 mg/kg/day by continuous i.v. infusion. When clinically possible, patients were switched to oral therapy in two divided doses, at four times the intravenous dose. Tacrolimus levels were monitored twice a week, and dosages adjusted to maintain serum levels 5-15 ng/ml. Common adverse effects included hypomagnesemia (98%), hypertension (49%), nephrotoxicity (34%), and tremors (32%). Less common side-effects (<10% cases) included seizures and hyperglycemia. The median time to ANC recovery (ANC >500 x 106/l) was 15 days. For the related donor group, the incidence of grade II-IV acute GVHD was 33%, and grade III-IV GVHD 19%. For the unrelated donor group, the incidence of grade II-IV acute GVHD was 55%, and grade III-IV GVHD 30%. Overall, tacrolimus therapy was well tolerated as prophylaxis for acute GVHD in pediatric patients undergoing allogeneic transplantation.

Actuarial Analysis↗
PubMed2000

Donor leukocyte infusions for multiple myeloma.

Donor leukocyte infusion (DLI) has well-documented activity in CML, but the role of DLI in other diseases is less well defined. To evaluate the strategy in multiple myeloma (MM) we evaluated 25 MM patients from 15 centers who were treated with DLI. Patients with persistent or recurrent disease after allogeneic BMT received DLI from the original marrow donor (23 matched related, one mismatched family, and one matched unrelated). Chemotherapy was given before DLI in three patients. Two of 22 patients responded completely to DLI alone and three patients responded to the combination of DLI and chemotherapy. Nine patients who had not had sufficient disease control after DLI were given additional DLIs; five of these patients had either complete (two) or partial (three) responses. Thirteen of 25 evaluable patients developed acute GVHD and 11 of 21 evaluable patients developed chronic GVHD; all responders developed GVHD. No patients developed post-DLI pancytopenia. Four patients had responses which lasted >1 year after DLI, three patients had responses which lasted <1 year, and three patients had ongoing responses but with follow-up <1 year. In conclusion, DLI has anti-myeloma activity but the strategy is limited by no response or short duration of response in a significant percentage of patients and by significant GVHD in the majority of the responders.

Adult↗
PubMed2000

Successful bone marrow transplantation in a patient with Schimke immuno-osseous dysplasia.

Early death in Schimke immuno-osseous dysplasia often results from renal failure and/or cell-mediated immunodeficiency. Kidney transplants have improved renal function, but effective therapy for the immunodeficiency has not yet been reported. We describe markedly improved marrow function 2 years after bone marrow transplantation in a boy with Schimke immunoosseous dysplasia.

Antigens, CD↗
PubMed2000

Attenuation of luteinizing hormone surges in neuropeptide Y knockout mice.

To clarify the role of neuropeptide Y (NPY) in the regulation of the reproductive axis, these experiments evaluated the extent to which reproductive hormone secretions may be compromised in the absence of NPY expression. In NPY knockout (NPY-KO) and wild-type (WT) mice, hormone secretions were analyzed under conditions of basal release, following ovariectomy (OVX), in proestrus, after estrogen treatments which induce gonadotropin surges and after injection of gonadotropin-releasing hormone (GnRH). Radioimmunoassays of serum from metestrous females revealed that basal luteinizing hormone (LH), follicular-stimulating hormone (FSH), estrogen and progesterone levels, as well as hypothalamic GnRH tissue concentrations, were not different between the two genotypes. The LH and FSH levels and GnRH tissue concentrations were likewise similar in WT and NPY-KO mice 5 and 10 days following OVX. Significant differences in LH levels were observed however when animals were exposed to pheromone stimulation (male mouse urine) to induce preovulatory LH surges. In proestrous animals, mean LH levels at 18.30-19.00 h were reduced by about 66% in NPY-KO versus WT mice (4.33 +/- 1.12 ng/ml in the WT mice vs. 1.47 +/- 0.42 ng/ml in the NPY-KO mice, p = 0.028). Despite diminishment of LH surges in NPY-KO mice, corpora lutea were equally abundant in the ovaries of NPY-KO and WT mice. In an additional experiment, a surge-inducing regimen of estradiol-17-beta (E2) and estradiol benzoate (E2B) was administered to OVX animals. The LH surges in the NPY-KO animals treated in this manner were again diminished by approximately 50% compared to corresponding values in WT animals (WT mice 7.33 +/- 0.97 ng/ml, NPY-KO mice 3.58 +/- 0.74 ng/ml; p = 0.0063). To assess the contribution of altered pituitary responsiveness to the diminishment of LH surges, LH responses to a GnRH challenge (200 ng/kg subcutaneously) were determined; NPY-KO animals exhibited LH responses that were significantly reduced compared to values in WT mice (WT mice 4.88 +/- 0.56 ng/ml, NPY-KO mice 3.00 +/- 0.41 ng/ml; p = 0.013). Taken together, these observations do not support the idea that NPY plays a major role in the regulation of basal gonadotropin secretion or in mediating negative feedback actions of gonadal hormones. They demonstrate however that preovulatory NPY release is required for normal amplification of the LH surge that occurs on proestrus. Involvement of NPY in the generation of normal LH surges is partially mediated by the ability of the peptide to prime the anterior pituitary gland to GnRH stimulation.

Animals↗
PubMed2000

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.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

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.

Animals↗
PubMed2000

Regulation of hypothalamic neuropeptide Y Y1 receptor gene expression during the estrous cycle: role of progesterone receptors.

Neuropeptide Y (NPY) stimulates the release of GnRH in an estrogen (E2)-dependent manner, which is important in generating preovulatory GnRH surges. We tested the hypothesis that E2 up-regulates NPY's actions by stimulating NPY Y1 receptor (Y1r) gene expression through a mechanism mediated by E2's ability to induce progesterone (P) receptors (PRs). In initial experiments, a specific Y1r antagonist BIBP3226 was used to confirm the involvement of Y1r in the stimulatory effects of NPY on in vivo GnRH release. Hypothalamic Y1r messenger RNA (mRNA) levels were then measured using competitive RT-PCR and were found to be significantly increased at 1000, 1200, and 1400 h on proestrus compared with other times of the day or cycle stage. Ovariectomy eliminated these increases, and E2 treatment restored them. Additional P treatment produced even larger increases in Y1r mRNA levels. To assess the role of PRs in stimulating Y1r expression, proestrous rats were treated with PR antagonist or oil vehicle and killed at 1200 h. Treatment with PR antagonist completely blocked the proestrous rise in Y1r gene expression. In parallel experiments, the same in vivo PR antagonist treatments also blocked NPY stimulation of GnRH release in vitro. Together our findings reveal that 1) Y1r mRNA levels are increased during the late morning and afternoon of proestrus; 2) Y1r mRNA levels are similarly increased by E2, and to an even greater extent by additional P; and 3) PR antagonism blocks both increased Y1r mRNA and induction of GnRH responsiveness to NPY. These observations support the idea that E2 up-regulates GnRH neuronal responses to NPY through stimulation of Y1r gene expression, and that E2's actions are mediated by the induction and subsequent activation of PRs.

Animals↗
PubMed1999

Requirement of PI3-kinase activity for the nuclear transport of prolactin in cloned murine T lymphocytes.

The murine T-cell clone, L2, requires both IL2 and PRL to proliferate. Proliferation and selected IL2-driven gene expression are blocked by treatment with rapamycin. Since prolactin translocation to the nucleus is IL2 dependent and required for proliferation, experiments were performed to identify activation pathways that might be involved in nuclear transport and proliferation. L2 cells were stimulated with IL2 in the presence and absence of the mTOR inhibitor rapamycin, PI3-kinase inhibitors (wortmannin, LY294002), the p38 MAP kinase inhibitor SB203580 and the vitamin D analog calcipotriol. The immunosuppressant rapamycin markedly inhibited IL2-induced proliferation and prolactin translocation to the nucleus. Similarly, wortmannin and LY294002 inhibited IL2-induced proliferation and markedly decreased the amount of prolactin transported to the nucleus. SB203580 and calcipotriol partially inhibited IL2-induced proliferation but had no effect on prolactin translocation. None of the inhibitors affected Lucifer Yellow uptake indicating that rapamycin, wortmannin and LY294002 did not inhibit early endosomal formation but rather worked to inhibit prolactin translocation at a later point in the retrograde transport pathway.

Androstadienes↗
PubMed1999

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.

Animals↗
PubMed1999

Absence of gonadotropin surges and gonadotropin-releasing hormone self-priming in ovariectomized (OVX), estrogen (E2)-treated, progesterone receptor knockout (PRKO) mice.

It is well known that estrogen (E2) stimulates expression of progesterone receptors (PRs), thereby inducing responsiveness of several tissues to the actions of progesterone (P). Recent studies have also suggested, however, that biological actions previously ascribed to E2 alone may also be mediated by activation of E2-induced PRs, even independently of signal changes in P concentrations. In the present experiments, the progesterone receptor knockout (PRKO) mice were used to assess the role of PR activation in the positive feedback actions of E2 on gonadotropin release. Ovariectomized (OVX) PRKO mice were tested for their capacity to mount primary gonadotropin surges in response to exogenous E2, and to exhibit a GnRH self-priming effect in response to sequential injections of the decapeptide. Wild-type (WT) and PRKO mice were OVX, treated with both 17beta-estradiol and estradiol benzoate (EB), and then killed at 1900 h on day 7 postOVX. Plasma LH RIA revealed that WT mice exhibited surges in response to the E2 treatment; the PRKO mice, however, showed no elevation in plasma LH above untreated controls. Instead, plasma LH levels in E2-treated, OVX PRKO mice decreased significantly in comparison to untreated OVX PRKO mice, suggesting that E2 can exert a negative feedback influence on LH release in PRKO mice, despite the absence of positive feedback effects. A slight but significant rise in plasma FSH was observed in E2-treated OVX WT mice in comparison to untreated controls: an effect not seen in E2-treated OVX PRKO mice, reinforcing the observation that estrogen's positive feedback effects are compromised in PRKO mice. In a second experiment, E2-treated OVX WT and PRKO mice were given either one or two pulses of GnRH 60 min apart, and killed 10 min later. The WT mice were found to exhibit a robust GnRH self-priming effect, as WT mice receiving two GnRH pulses displayed LH responses approximately 2-fold greater than those receiving only one pulse. By contrast, PRKO mice receiving two GnRH pulses exhibited no additional increase in plasma LH levels. We conclude that PR activation is obligatory for expression of the GnRH self-priming effect as well as for generation of E2-induced LH and FSH surges. The extent to which failure of LH surge secretion in PRKO mice is due to the absence of GnRH self-priming, lack of hypothalamic GnRH surges, and/or defects in other processes remains to be determined. These observations clearly demonstrate, however, that the presence of PR is an absolute requirement for the transmission of E2-induced signals leading to gonadotropin surges.

Animals↗
PubMed1998

Oocytes are a source of catecholamines in the primate ovary: evidence for a cell-cell regulatory loop.

Catecholamines, thought to derive from the extrinsic innervation of the ovary, participate in the regulation of ovarian development and mature gonadal function. Recently, intraovarian neurons containing tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, were described in the ovary of nonhuman primates. We now show that the primate ovary expresses both the genes encoding TH and dopamine beta-hydroxylase (DBH), the key enzymes in norepinephrine (NE) biosynthesis. Ovarian neurons were identified as a site of TH and DBH gene expression, and surprisingly, oocytes were identified as an exclusive site of DBH synthesis. Oocytes contain neither TH mRNA nor protein, indicating that they are unable to synthesize dopamine (DA). They did, however, express a DA transporter gene identical to that found in human brain. The physiological relevance of this transporter system and DBH in oocytes was indicated by the ability of isolated oocytes to metabolize exogenous DA into NE. Isolated follicles containing oocytes-but not those from which the oocytes had been removed-responded to DA with an elevation in cAMP levels; this elevation was prevented by propranolol, a beta-adrenoreceptor antagonist. The results suggest that oocytes and somatic cells are linked by a neuroendocrine loop consisting of NE synthesized in oocytes from actively transported DA and cAMP produced by somatic follicular cells in response to NE-induced beta-adrenoreceptor activation.

Amino Acid Sequence↗