Reinhart B. Billiar

Immunocytochemical identification of the oestrogen receptor in the nuclei of cultured human placental syncytiotrophoblasts

We have shown that oestrogen has a central integrative role in regulating key components of the progesterone biosynthetic and corticosteroid metabolic pathways within syncytiotrophoblasts that govern placental function and maturation of the fetal pituitary-adrenocortical axis. Studies utilizing classic binding procedures and RNAse protection have demonstrated that human placental villous tissue exhibits specific high affinity oestrogen binding and expresses the mRNA for the oestrogen receptor. However, it is not known whether the oestrogen receptor is expressed specifically in syncytiotrophoblasts. Therefore, the present study determined whether the oestrogen receptor protein was detectable by immunocytochemistry in cultured human syncytiotrophoblast maintained in a low oestrogen/progestin environment. Cytotrophoblasts were isolated from human term placentae by trypsin dispersion and Percoll gradient centrifugation and cultured for 5, 7 or 10 days. Incubation of syncytiotrophoblast with 5-10 micrograms/ml of the anti-oestrogen receptor rat monoclonal antibody D-75, which is specific for the primate oestrogen receptor, resulted in identification of the oestrogen receptor in the nuclei of these cells. In contrast, there was no reactivity of the trophoblasts to either rat IgG or an irrelevant rat monoclonal antibody IgG2a against mouse common leukocyte antigen T200. Collectively, these findings indicate that oestrogen receptor is expressed in the nuclei of human placental syncytiotrophoblasts and support the suggestion that the syncytiotrophoblast is an oestrogen-responsive tissue.

Modulation of hypothalamic mu-opioid receptor density by estrogen: A quantitative autoradiographic study of the female

The labelling of hypothalamic binding sites by [125I]-FK, a specific mu-opioid receptor ligand, was studied in female C57BL/6J mice to test whether removal of ovarian steroids affected the density of distribution of receptor binding. Labelling densities in the forebrain of normally cycling (intact) females (N = 12), were compared to those in mice that had been ovariectomized (OVX) for 6 weeks (n = 8) and in mice that had been OVX and implanted with an estradiol (E2) capsule (OVX+E2) for 6 weeks (n = 11). Frozen sections from the rostral forebrain were incubated with 1 nM [125I]-FK and processed for light microscopic autoradiography. The diagonal band of Broca (DBB), organum vasculosum lamina terminalis (OVLT), preoptic area (POA), septum, parietal cortex, and striatum were analyzed using computerized image analysis. The distribution of labelling was similar in all three experimental groups in all the regions; however, labelling was significantly reduced in the ventrolateral POA of OVX animals compared to intact females. Labelling densities in the OVX animals replaced with the gonadal steroid estradiol were not significantly different from those in normally cycling mice. This study demonstrates a region-specific loss of mu-opiate receptor labelling following long-term deprivation of gonadal steroids, and supports the hypothesis that estrogen directly or indirectly influences the density of mu-opioid receptors in the rostral forebrain of female mice.

Neosurugatoxin: CNS acetylcholine receptors and luteinizing hormone secretion in ovariectomized rats

Neosurugatoxin, a neurotoxin isolated from the Japanese ivory shell, inhibits ganglionic nicotinic acetylcholine receptors but not skeletal muscle nicotinic acetylcholine receptors. It has also been reported to inhibit (3H) L-nicotine binding to high-affinity agonist acetylcholine receptors in rat brain membrane preparations. In the present study, 10(-5) M neosurugatoxin inhibited the in vitro binding of (3H) L-nicotine to the medial habenular nucleus of frozen, coronal sections of rat brain as did 10(-5) M cytisine or nicotine and 10(-4) M dihydro-beta-erythroidine. Neosurugatoxin did not inhibit (125I) alpha-bungarotoxin binding to hypothalamic synaptosomal preparations or to frozen, coronal sections of rat brain. Injection of neosurugatoxin into the third ventricles of ovariectomized rats resulted in a significant decrease in the frequency of pulses of luteinizing hormone (LH) secretion but had no effect on the amplitude of pulses. A low dose (1 microgram/injection) of the nicotinic acetylcholine agent cytisine injected into the third ventricle had no significant effect on pulsatile LH secretion. Coadministration of cytisine could block the inhibitory effect of neosurugatoxin on LH secretion. It is suggested that neosurugatoxin is a useful antagonist to study the biological roles of a specific subclass of nicotinic acetylcholine receptors in mammalian brain and reproductive neuroendocrine functions.

Third Ventricular Injection of Alpha-Bungarotoxin Decreases Pulsatile Luteinizing Hormone Secretion in the Ovariectomized Rat

The injection of purified alpha-bungarotoxin (alpha-BTX), an antagonist of the muscle nicotinic cholinergic receptors, into the third ventricle (3rd V) of ovariectomized rats decreased the frequency of pulses of luteinizing hormone (LH) but had no significant effect on the amplitude or nadir of LH secretion. Approximately 24 h after the 3rd V injection of alpha-BTX, the pulsatile secretion rate of LH was increased and thus the alpha-BTX effect was reversible. The 3rd V injection of the two nicotinic cholinergic agents, cytisine (10 micrograms) and dihydro-beta-erythroidine, also decreased the frequency of pulses of LH. Cytisine and dihydro-beta-erythroidine inhibited the binding of (125I)alpha-BTX to rat hypothalamic synaptosomes (P2B) and also to frozen coronal sections of the hypothalamus as studied by film autoradiography. Injection of a lower dose of cytisine (1 micrograms) had no effect on LH secretion, but when administered with alpha-BTX into the 3rd V, cytisine significantly reduced the alpha-BTX-induced decreased LH secretion. The injection of cytisine with (125I)alpha-BTX into the 3rd V also decreased in vivo labeling of the (125I)alpha-BTX-binding sites in the hypothalamus of ovariectomized rats. The results suggest that alpha-BTX can inhibit the pulsatile secretion of LH in ovariectomized rats and this inhibition may be mediated by specific subclass of a CNS (central nervous system) nicotinic acetylcholine receptor.

Luteinizing hormone response to N-methyl-D, L-aspartic acid in the presence of physiological estradiol concentrations: influence of age and the ovary.

Abstract We have previously reported that the pituitary of Intra-atrially cannulated old female C57BL/6J mice is as capable of responding to a GnRH challenge as is that of young females (10). We have observed elevated luteinizing hormone (LH) levels in ovariectomized (OVX) intra-atrially cannulated mice. Sustained physiologic levels of estradiol (E2) for 6 days suppressed circulating LH to intact levels. However, in that model, a bolus of E2 following E2 priming was unable to elicit an LH surge (Joshi et al., unpublished findings). The present studies were designed to examine: first, whether GnRH neurons are competent to release GnRH in the presence of tonic physiologic levels of E2 and, second, whether either age or the ovary can influence GnRH neuronal responsiveness. The N-methyl-D, L-aspartic acid (NMA)-evoked GnRH response was assessed indirectly by measuring LH in two groups of OVX C57BL/6J mice: short-term OVX (S-OVX) (1 week) mice were either prepubertal (5 weeks), postpuberal (10 weeks), young (5 months), middle aged (12 months), or old (24 months). Long-term OVX (L-OVX) mice were either young (5 months), or old (24 months) and OVX at puberty; middle-aged L-OVX mice were OVX at 8 months and examined at 12 months of age. Animals were administered physiologic levels of E2 by subcutaneous silastic capsule for 1 week before testing. LH secretion was Inhibited by E2 in S-OVX mice of all ages. In no case did NMA overcome this inhibition in E2 primed S-OVX females. E2 also inhibited LH secretion in L-OVX mice of all ages, but NMA was able to overcome the E2 inhibition of LH secretion in L-OVX mice (young: 0.5 ± 0.1, 0.84 ± 0.19 ng/ml, first and second challenge, respectively; middle-aged: 0.46 ± 0.1, 1.08 ± 0.16 ng/ml; and old: 1.44 ± 0.19, 0.99 ± 0.27 ng/ml). This last effect was independent of animal maturity at the time of OVX or animal age at the time of experiment. These findings suggest that although the ovaries in the 24-month-old S-OVX mice had not produced enough E2 to alter the vaginal cytology for 2 ± 0.5 months before the experiment, the ovarian modulation of the inhibitory effect of E2 on NMA-induced LH secretion was still present. The nature of the ovarian factor(s) modulating this effect is unknown. These results demonstrate that in the intra-atrially cannulated female C57BL/6J mouse, the negative feedback effect of E2 on hypothalamic GnRH release predominates and prevents the induction of an LH surge by a bolus of E2. The ability of E2 to inhibit the NMA response is mediated by the length of time between removal of the ovary and initiation of estrogen replacement, and this effect is independent of age.

Estrogen, the ovary, and neutotransmitters: factors associated with aging

Our studies in the C57BL/6J mouse have been designed to examine the interactions of aging and the ovary, and their mutual effects on neuroendocrine function. In the pituitary, ovarian status and not age determines responsiveness to gonadotropin hormone releasing hormone (GnRH), but estrogen (E2) is an important mediator in CNS changes, and removal of the ovary (OVX) is deleterious to the neuroendocrine hypothalamus. OVX for just six days in young animals results in synaptic loss between noradrenergic terminals and gonadotropin hormone releasing hormone (GnRH) neurons. Long-term OVX, hypothesized to protect against neuroendocrine aging, fails to guard against any studied age-related changes. Some age-related changes occur as early as midlife. Although neuron number remains constant at middle age, opiatergic neurons undergo significant functional changes by producing opiate antagonist peptides. This change appears to be caused by alterations in the prohormone convertases, which cleave propeptide to peptide. Altered peptides may trigger the loss of reproductive capacity. The midlife shift in opiate peptide production is a component of natural developmental processes that begin in the neonate and continue through old age. In the cholinergic system, E2 mediates numbers of cholinergic receptors, cholinergic neurons, and cholinergic-modulated memory systems in both young and old animals. Regardless of age, ovarian steroids, if present at physiologic levels, are beneficial to the neuroendocrine CNS, and long-term deprivation from ovarian-produced factors is deleterious in the systems we have examined. Our studies have shown that deprivation from ovarian steroid hormones in the female appears to be a major factor in the health of the CNS and in events associated with aging.

Identification of biologically active inhibin in the peritoneal fluid of women

PurposeImmunoreactive inhibin (i-inhibin) has been reported to be present in the peritoneal fluid of women. The radioimmunoassay employed measures free, biologically inactive α-subunits(s) equally as well as dimeric, biologically active inhibin. The present study was designed to determine if biologically active, dimeric inhibin is present in the peritoneal fluid of women.MethodsPeritoneal fluid of four women was assayed by radioimmunoassay, a sheep pituitary bioassay, and two ELISA procedures which utilized specific monoclonal antibodies for the “capture” of the α-subunit (ELISA-A) or the β-subunit (ELISA-B) of inhibin and subsequent quantification of dimeric inhibin-A.ResultsThere was a good correlation between the values obtained by radioimmunoassay, bioassay, and both ELISAs; two samples (from the late follicular phase) with relatively high i-inhibin concentrations were positive in all four assays, whereas two samples (from the early follicular phase) with very low i-inhibin concentrations were negative in the bioassay and ELISAs.ConclusionA significant portion of the immunoreactive inhibin in the peritoneal fluid obtained during the late follicular phase of women is dimeric, biologically active inhibin. We speculate that this may have potential implications for oocyte maturation and early embryogenesis within the oviduct.