Diverse actions of estradiol on anorexigenic and orexigenic hypothalamic arcuate neurons.

Contribution to Special Issue on Fast effects of steroids. There is now compelling evidence for membrane-associated estrogen receptors in hypothalamic neurons that are critical for the hypothalamic control of homeostatic functions. It has been known for some time that estradiol (E2) can rapidly alter hypothalamic neuronal activity within seconds, indicating that some cellular effects can occur via membrane initiated events.

The Effects of Muscle Mass on Homocyst(e)ine Levels in Plasma and Urine.

The present study was designed to examine the relationship between homocyst(e)ine (H[e]) levels and muscle mass. Two experimental groups each of 24 Caucasian males, one consisting of higher-muscle mass subjects (HMM) and the other of lower-muscle mass subjects (LMM) participated in this study. Muscle mass was estimated from 24-hour urine collections of creatinine (Crt).

Age-related effects of estrogen on the expression of estrogen receptor alpha and beta mRNA in the ovariectomized monkey hypothalamus.

In the present study, we reported distribution of ER alpha and ER beta mRNAs in the hypothalamus of young and old ovariectomized (OVX) rhesus macaques. The ER alpha were detected in all six major vestiblular nuclei which included arcuate nucleus (ARC) , paraventricularis nucleus (PVN) , periventricular nucleus (PeriV) , supraoptic nucleus (SON) , medial prioptic nucleus (MPN) and lateral hypothalamus area (LHA). However, the ER beta mRNA can also detected in those nuclei excerpt SON, but the signals of ER beta mRNA were weaker than those of ER alpha mRNA.

Transgenic animals: current and alternative strategies.

Transgenic animal technology is one of the most fascinating technologies developed in the last two decades. It allows us to address questions in life sciences that no other methods have achieved. The impact on biomedical and biological research, as well as commercial interests are overwhelming.

Optimization strategies for production of mammalian embryos by nuclear transfer.

In order to optimize each of the individual steps in the nuclear transfer procedure, we report alternative protocols useful for producing recipient cytoplasts and for improving the success rate of nuclear transfer embryos in cattle, rhesus monkey, and hamster. Vital labeling of maternal chromatin/spindle is accomplished by long wavelength fluorochromes Sybr14 and rhodamine labeled tubulin allowing constant monitoring and verification during enucleation. The use of Chinese hamster ovary (CHO) donor cells expressing the viral influenza hemagglutinin fusion protein (HA-300a+), to adhere and induce fusion between the donor cells and enucleated cow, rhesus and hamster oocytes was examined.

Expression of the insulin receptor-related receptor is induced by the preovulatory surge of luteinizing hormone in thecal-interstitial cells of the rat ovary.

The insulin receptor-related receptor (IRR) is a member of the insulin receptor family that, on its own, recognizes neither insulin nor any of the identified insulin-related peptides. In both the nervous system and peripheral tissues, IRR mRNA is detected in cells that also express trkA, the nerve growth factor tyrosine kinase receptor. In the ovary, the trkA gene is transiently activated in thecal-interstitial cells of large antral follicles at the time of the preovulatory surge of gonadotropins.

High-dose estrogen and clinical selective estrogen receptor modulators induce growth arrest, p21, and p53 in primate ovarian surface epithelial cells.

Ovarian cancer is the most lethal gynecological cancer affecting women. Hormone-based therapies are variably successful in treating ovarian cancer, but the reasoning behind these therapies is paradoxical. Clinical reagents such as tamoxifen are considered to inhibit or reverse tumor growth by competitive inhibition of the estrogen receptor (ER); however, high-dose estrogen is as clinically effective as tamoxifen, and it is unlikely that estrogen is acting by blocking ER activity; however, it may be activating a unique function of the ER that is nonmitogenic.

Transcervical polidocanol as a nonsurgical method of female sterilization: a pilot study.

We have studied the effects of transcervically administered polidocanol on uterine and fallopian tube morphology in Wistar rats and Rhesus monkeys. Polidocanol is a synthetic, long-chain fatty acid that is widely used as a sclerosing agent in Europe. The goal of the study was to determine whether polidocanol would safely cause tubal occlusion in an animal model.

Estrogen inhibits cell cycle progression and retinoblastoma phosphorylation in rhesus ovarian surface epithelial cell culture.

Estrogen promotes the growth of some ovarian cancer cells at nanomolar concentrations, but has been shown to inhibit growth of normal ovarian surface epithelial (OSE) cells at micromolar concentrations (1 microg/ml). OSE cells express the estrogen receptor (ER)-alpha, and are the source of 90% of ovarian cancers. The potential sensitivity of OSE cells to estrogen stresses the importance of understanding the estrogen-dependent mechanisms at play in OSE proliferation and transformation, as well as in anticancer treatment.

Role of a Transbilayer pH Gradient in the Membrane Fusion Activity of the Influenza Virus Hemagglutinin: Use of the R18 Assay to Monitor Membrane Merging.

It had been suggested that influenza virus-mediated membrane fusion might be dependent on a pH gradient across a target membrane. We have designed experiments in which this issue could be addressed. Two populations of liposomes were prepared, both simulating the plasma membrane of target cells, but with the pH of the internal aqueous medium buffered either at pH 7.

Dexamethasone or interleukin-10 blocks interleukin-1beta-induced uterine contractions in pregnant rhesus monkeys.

Objective: The purpose of this study was to determine whether treatment with the immune modulators dexamethasone or interleukin-10 prevents interleukin-1beta-induced uterine contractions in a nonhuman primate model.

Study Design: Thirteen chronically instrumented rhesus monkeys at 135 +/- 1 days of gestation (term, 167 days) received one of three interventions: (1) intra-amniotic interleukin-1beta (10 microg) infusion with maternal dexamethasone (1 mg/kg) intravenously every 6 hours for 1 day before interleukin-1beta and for 2 days thereafter (n = 4), (2) intra-amniotic interleukin-1beta infusion with maternal interleukin-10 (25 microg/kg) given intravenously and 100 microg interleukin-10 given intra-amniotically before the interleukin-1beta and continued every 8 hours for 3 days (n = 5), and (3) intra-amniotic interleukin-1beta administered alone (n = 5). Uterine activity was monitored continuously and quantified as the hourly contraction area (millimeters of mercury times seconds per hour) in all groups until delivery.

Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes.

The initiation of mammalian puberty requires the activation of hypothalamic neurons secreting the neuropeptide luteinizing hormone-releasing hormone (LHRH). It is thought that this activation is caused by changes in trans-synaptic input to LHRH neurons. More recently, it has been postulated that the pubertal increase in LHRH secretion in female animals also requires neuron-glia signaling mediated by growth factors of the epidermal growth factor (EGF) family and their astrocytic erbB receptors.

Neurotrophic control of ovarian development.

Substantial evidence now exists indicating that the neurotrophins, a family of growth factors required for the survival, development, and differentiation of various neuronal populations of the nervous system, are also important for the development of nonneuronal tissues. Such a function was first suggested by studies showing the presence of high-affinity neurotrophin receptors in a variety of nonneuronal tissues including those of the cardiovascular, endocrine, immune, and reproductive systems. Within the latter, the gonads appear to be a preferential site of neurotrophin action as suggested by the presence in the mammalian ovary of at least four of the five known neurotrophins and all of the neurotrophin receptors thus far identified.

Measurement of changes in fluorescence resonance energy transfer between gonadotropin-releasing hormone receptors in response to agonists.

Oligomerization of membrane-bound G-protein-coupled receptors has recently emerged as an important step in cellular signaling. Fluorescence resonance energy transfer (FRET) has undergone a revival as the method of choice for demonstrating in vivo protein-protein interactions and receptor dimerization. We have used chimeras of gonadotropin-releasing harmone (GnRH) receptors and various fluorescent proteins to investigate receptor dimerization in relation to receptor activation.

Male contraception.

From a public health perspective, the need for contraception has never been greater. Although the existing male-specific methods (withdrawal, condoms, and vasectomy) are safe and effective, increasing male options for fertility control could improve family planning. For new male contraceptive methods to have an impact, they must be acceptable to both men and women, as well as effective.

Phosphodiesterase 3 inhibitors selectively block the spontaneous resumption of meiosis by macaque oocytes in vitro.

Background: The purpose of this study was to determine whether phosphodiesterase (PDE) 3 inhibitors selectively prevent the resumption of meiosis in primates.

Methods: Immature oocytes (intact germinal vesicles) obtained from large pre-ovulatory follicles following ovarian stimulation in rhesus macaques were incubated with or without various doses of the PDE3 inhibitors, Cilostamide, Milrinone or ORG 9935, or a selective PDE4 inhibitor, Rolipram. Oocytes were observed for germinal vesicle breakdown (GVBD) as an indicator of resumption of meiosis.

Analysis of DNA fragmentation of in vitro cultured bovine blastocysts using TUNEL.

Programmed cell death (apoptosis) characteristically affects the single cells of blastocysts whereas necrosis affects cluster of cells in both the inner cell mass (ICM) and the trophectoderm (TE). This study uses the trophectodermrminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) assay as a way of evaluating the proportion of apoptotic cells and, thus, bovine blastocyst quality during in vitro culture at Days 6,7, and 8. Furthermore, parthenogenetic blastocysts were compared to in vitro fertilized blastocysts at Day 7.

Nuclear transfer and cloning.

The use of nuclear transfer in human reproductive and therapeutic cloning is reviewed with attention on the origins of this technology from its evolution to the present. The successes and limitations of mammalian reproductive cloning are itemized. A case is made against the use of human reproductive cloning to reproduce an existing person, based on the unacceptable risks to the embryo, fetus, or newborn.

Injection of antiangiogenic agents into the macaque preovulatory follicle: disruption of corpus luteum development and function.

Ovulation and conversion of the follicle into the corpus luteum involve remarkable changes in vascular permeability and neovascularization of the luteinizing granulosa layer. To evaluate the importance of these vascular events in follicle rupture and luteal development, sequential experiments were designed in which vehicle or angiogenic inhibitors (TNP-470 or angiostatin) were injected directly into the preovulatory follicle of rhesus monkeys during spontaneous menstrual cycles. After control injections, 13 of 14 animals exhibited serum levels of progesterone (P) during the subsequent luteal phase that were comparable to untreated animals in our colony.

Rescue of hypogonadotropic hypogonadism-causing and manufactured GnRH receptor mutants by a specific protein-folding template: misrouted proteins as a novel disease etiology and therapeutic target.

In the present study, we demonstrate pharmacological rescue (assessed by ligand binding and restoration of receptor coupling to effector) of five naturally occurring GnRH receptor (GnRHR) mutants (T(32)I, E(90)K, C(200)Y, C(279)Y, and L(266)R), identified from patients with hypogonadotropic hypogonadism, as well as rescue of other defective receptors intentionally manufactured with internal or terminal deletions or substitutions at sites expected to be involved in establishment of tertiary receptor structure. The pharmacological agent used is a small, membrane-permeant molecule, originally designed as an orally active, nonpeptide receptor antagonist, but is believed to function as a folding template, capable of correcting the structural defects caused by the mutations and thereby restoring function. After rescue, this agent can be demonstrably removed.

Ovarian steroid regulation of 5-HT1A receptor binding and G protein activation in female monkeys.

Serotonin 5-HT(1A) receptors play an important role in serotonin neurotransmission and mental health. We previously demonstrated that estradiol (E) and progesterone (P) decrease 5-HT(1A) autoreceptor mRNA levels in macaques. In this study, we questioned whether E and P regulate 5-HT(1A) binding and function and G(alpha) subunit protein expression.

Wired for reproduction: organization and development of sexually dimorphic circuits in the mammalian forebrain.

Mammalian reproduction depends on the coordinated expression of behavior with precisely timed physiological events that are fundamentally different in males and females. An improved understanding of the neuroanatomical relationships between sexually dimorphic parts of the forebrain has contributed to a significant paradigm shift in how functional neural systems are approached experimentally. This review focuses on the organization of interconnected limbic-hypothalamic pathways that participate in the neural control of reproduction and summarizes what is known about the developmental neurobiology of these pathways.

Progesterone receptor localization and isoforms in myometrium, decidua, and fetal membranes from rhesus macaques: evidence for functional progesterone withdrawal at parturition.

Objective: It is not known whether withdrawal of progesterone (P) action is a prerequisite for parturition in women or in nonhuman primates because concentrations of circulating progesterone or progesterone receptors (PR) in myometrium and decidua do not decrease before delivery. To examine this potentially important regulatory mechanism, we determined PR isoforms, PR localization, and mRNA in myometrium, decidua, and fetal membranes from rhesus monkeys during pregnancy and in spontaneous labor at term.

Methods: Gestational tissues were obtained midpregnancy (day 80-100), late pregnancy (day 130-145), and during spontaneous labor at term (day 161-167).

Photoperiod-dependent response to androgen in the medial amygdala of the Siberian hamster, Phodopus sungorus.

The medial nucleus of the amygdala (MeA) is a steroid-sensitive region that has been implicated in the expression of behaviors such as mating and aggression. The male Siberian hamster (Phodopus sungorus) uses light cues to regulate its reproductive neuroendocrine system, reducing androgen synthesis in the autumn and increasing it in the spring. There is also evidence that short photoperiods reduce the sensitivity of the brain to the behavioral effects of androgen.

Glial-neuronal-endothelial interactions are involved in the control of GnRH secretion.

In recent years compelling evidence has been provided that cell-cell interactions involving non-neuronal cells, such as glial and endothelial cells, are important in regulating the secretion of GnRH, the neuropeptide that controls both sexual development and adult reproductive function. Modification of the anatomical relationship that exist between GnRH nerve endings and glial cell processes in the external zone of the median eminence modulates the access of GnRH nerve terminals to the portal vasculature during the oestrous cycle. The establishment of direct neuro-haemal junctions between GnRH neuroendocrine terminals and the portal vasculature on the day of pro-oestrus may be critical for the transfer of GnRH upon its release into the fenestrated capillaries of the median eminence.