Functional capacity of fetal zone cells of the baboon fetal adrenal gland: a major source of alpha-inhibin.

We have shown that ACTH receptor mRNA expression and steroidogenesis were increased in the transitional zone and decreased in the fetal zone of the baboon fetal adrenal in the second half of gestation. Thus, we proposed that there is a divergence in ACTH receptor-mediated zone-specific steroidogenesis within the fetal adrenal during mid to late gestation. We have also demonstrated that fetal serum alpha-inhibin levels decline with advancing development. It is possible, therefore, that the alpha subunit of inhibin provides a good marker of fetal zone cellular function and that the changes in circulating fetal alpha-inhibin with advancing pregnancy reflect ontogenetic changes in fetal adrenal cortical zone-specific cell function. However, it remains to be determined whether the fetal adrenal is a major source of circulating alpha-inhibin in the fetus and whether alpha-inhibin is expressed in the fetal, definitive, and/or transitional zones. Therefore, the current study compared fetal serum alpha-inhibin levels with immunocytochemical localization of alpha-inhibin in baboon fetal adrenals obtained on Days 60 (early), 100 (mid), and 165 or 182 (late) of gestation (term averages Day 184) from animals untreated or treated with betamethasone, which we previously demonstrated suppressed fetal pituitary ACTH and adrenal weight. Fetal serum alpha-inhibin levels (mean +/- SE) were greater (p < 0.05) at mid (5863 +/- 730 microliter eq/ml) than at late (3246 +/- 379) gestation and were reduced (p < 0. 05) by betamethasone. The inhibin alpha subunit was expressed in abundant quantities in the fetal adrenal cortex, but not in medulla, throughout gestation. At mid and late gestation, alpha-inhibin was expressed throughout the fetal adrenal cortex but most intensely in the innermost area of fetal zone cells. By late gestation, the fetal adrenal exhibited a gradient of alpha-inhibin expression. Thus, the outermost definitive zone cells were devoid of alpha-inhibin, the transitional zone exhibited a relatively low alpha-inhibin content, and fetal zone cells continued to exhibit extensive expression of alpha-inhibin. Betamethasone diminished the intensity of alpha-inhibin expression throughout the fetal adrenal cortex. These results indicate that the fetal adrenal fetal zone is a significant source of circulating alpha-inhibin in the baboon fetus and that alpha-inhibin provides a good marker to study the developmental regulation of fetal zone-specific adrenocortical function.

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.

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.

Identification of immunoreactive inhibin in human and baboon fetal serum at term as free alpha-subunit(s).

Immunoreactive inhibin (i-inhibin) in the serum of the baboon fetus is high at midgestation and decreases toward term. Human cord serum also contains immunoreactive inhibin, but bioactive inhibin is nondetectable or very low. In the present study we report that baboon fetal serum is also inactive in the sheep anterior pituitary FSH bioassay. Furthermore, both fetal baboon serum and term human cord serum are inactive in two enzyme-linked immunosorbent assays (ELISAs) that use a monoclonal antibody (R-I) against the alpha-subunit of human inhibin as the capturing agent and a monoclonal antibody (E-4) against the beta A-subunit as a detection/quantification agent (ELISA-A), or the E-4 antibody as the capturing agent and a R-1 F(ab) fragment as the detection/quantification agent (ELISA-B). Recombinant human inhibin A was reactive in both ELISAs. Human cord serum inhibited recombinant human inhibin activity in the ELISA-A, but not in the ELISA-B. An immunoaffinity column to which the R-1 antibody had been coupled extracted i-inhibin activity from both baboon fetal heart serum and human cord serum. Together, these results suggest that the i-inhibin in fetal serum is the free alpha-subunit, with little, if any, dimeric inhibin present. Western blot analysis confirmed that the i-inhibin activity extracted by the R-1 immunoaffinity column from baboon fetal serum can be attributed to a free alpha-subunit(s).

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

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), postpubertal (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.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of age and long-term ovariectomy on the estrogen-receptor containing subpopulations of beta-endorphin-immunoreactive neurons in the arcuate nucleus of female C57BL/6J mice.

We have reported a decrease in the number of arcuate nucleus (ARC)-immunoreactive beta-endorphin neurons in old (24 months) female C57BL/6J mice versus young (5 months) mice. Here, we have tested by immunocytochemistry whether age-related changes in beta-endorphin neuron numbers are selective for beta-endorphin neurons which do or do not contain estrogen receptors (E2R). We also compared beta-endorphin neuron number in mice with short- (S) and long-duration (L) ovariectomy (OVX), since the latter may protect against neuroendocrine aging. Mice were studied at 5 (young), 12 (middle-aged), or 23-24 months (old). When the mean number of neurons per tissue section (15 sections per animal) was examined, there were no significant differences between young and middle-aged S-OVX females for either beta-endorphin, E2R, or beta-endorphin/E2R neuron number. However, there were significant decreases in beta-endorphin-containing neurons in the oldest age group versus young females (young S-OVX: 74.4 +/- 11 (+/- SD) immunopositive neurons per tissue section, n = 10 mice; young L-OVX: 61.6 +/- 6.9, n = 6; old S-OVX: 45.7 +/- 9.9, n = 7; and old L-OVX: 37.5 +/- 7.3, n = 7). There were also decreases in beta-endorphin neurons which contained E2R in the oldest animals (young S-OVX: 16.6 +/- 6.4; young L-OVX: 13.7 +/- 1.3; old S-OVX: 9.2 +/- 1.8; L-OVX: 6.0 +/- 1.5) (p < 0.05 ANOVA). Both age (p < or = 0.001, two-way ANOVA) and ovarian status (p < or = 0.05) independently affected neuron number for both the beta-endorphin and beta-endorphin/E2R populations versus young mice. We tested whether the observed age and/or ovarian-related decreases were proportionally greater in the subpopulation of beta-endorphin neurons which contained E2R compared to the total beta-endorphin neuron population. In the oldest age group, there was no significant difference in the decrease with age in the population of beta-endorphin neurons which contained E2R and the total beta-endorphin population (p = 0.208). When we examined the E2R neuron population as compared to the beta-endorphin neuron populations, age-related decreases in the beta-endorphin neuronal population tended to be greater than the decreases seen in the E2R neuron population (p = 0.054 repeated measures ANOVA). The tyrosine hydroxylase (TH) neuron population was studied to test whether there were changes in another ARC neuron population.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of biologically active inhibin in the peritoneal fluid of women.

PURPOSE: Immunoreactive inhibin (i-inhibin) has been reported to be present in the peritoneal fluid of women. The radioimmunoassay employed measures free, biologically inactive alpha-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. METHODS: Peritoneal 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 alpha-subunit (ELISA-A) or the beta-subunit (ELISA-B) of inhibin and subsequent quantification of dimeric inhibin-A. RESULTS: There 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. CONCLUSION: A 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.

Gonadotropin hormone-releasing hormone induced luteinizing hormone responses in young and old female C57BL/6J mice.

The estrogen-induced luteinizing hormone (LH) surge is markedly attenuated in old (24 months) female mice compared with young (6 months) females. To test whether or not this attenuated LH response is due to a diminished capacity of the pituitary to respond to hypothalamic gonadotropin hormone-releasing hormone (GnRH), the pituitary response to exogenous GnRH was measured in young (5-6 months), normally cycling (n = 6) and old (24 months), acyclic constant diestrus (n = 6) C57BL/6J female mice. Mice were ovariectomized and estrogen-treated for 7 days. After intrajugular catheterization on Day 6, serial blood samples were taken at 15-min intervals for 165 min on Day 7. Serum LH was measured in samples obtained before and after infusion of either saline or GnRH (5 micrograms/5 microliters saline/kg body wt) and 15 micrograms/15 microliters saline/kg body wt 1 hr apart). Saline-treated animals demonstrated no LH response in either young (0.09 +/- 0.02 ng/ml baseline) or old (0.11 +/- 0.01 ng/ml baseline) females. However, a significant release of LH was obtained in response to each challenge of GnRH in both young (0.3 +/- 0.04 ng/ml first challenge, 0.69 +/- 0.1 ng/ml second challenge) and old (0.78 +/- 0.1 ng/ml first challenge, 1.76 +/- 0.2 ng/ml second challenge) mice. The LH response in the aged group was significantly greater (P < 0.05, analysis of variance) than in the young group. These results show that pituitaries of old female mice were at least as capable of responding to exogenous stimulation by GnRH as those of young. We conclude that alteration in the capacity of the pituitary to respond to GnRH is not likely to be a factor contributing to altered LH secretion with age in C57BL/6J mice.

Modulation of hypothalamic mu-opioid receptor density by estrogen: a quantitative autoradiographic study of the female C57BL/6J mouse.

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.

Regulation of immunoreactive inhibin patterns in baboon pregnancy: maternal, placental, and fetal considerations.

To better understand the sources and regulation of circulating inhibin during primate pregnancy, immunoreactive inhibin was measured in sera obtained from the maternal saphenous vein, uterine vein, and the fetus at varying times of baboon pregnancy. In both intact and fetectomized (fetus removed on day 100 of gestation; term = 184 days) animals, maternal serum inhibin concentrations were relatively constant between day 80 (first sampling day) and day 110 of gestation, after which they then steadily increased until days 155-165 (end of sampling). The increase in inhibin concentrations was significantly less in the fetectomized animals than in the intact baboons. Restoration of estrogen levels in the fetectomized animals did not significantly alter the circulating inhibin concentrations. Similarly, administration of the estrogen antagonist MER-25 to intact animals in the last trimester had no effect on maternal serum inhibin concentrations. Inhibin concentrations in uterine venous blood collected on day 100 of gestation were not significantly different from those in the maternal saphenous vein. However, the inhibin concentrations of uterine venous blood collected late in gestation (days 155-165) in either intact or fetectomized animals were significantly higher than the corresponding maternal venous concentrations, suggesting that the uteroplacental tissue becomes a source of circulating inhibin during the third trimester of pregnancy. Consistent with this suggestion was the detection of inhibin alpha-subunit mRNA in the placentae of intact or fetectomized animals obtained late in pregnancy, but its absence at midgestation. Immunoreactive inhibin concentrations were about 16 times higher (6500 +/- 831 mu Leq/mL) in fetal blood than in maternal blood (411 +/- 23 mu Leq/mL) at midgestation. The fetal blood concentrations significantly decreased to about 2800 mu Leq/mL by days 160-165 of gestation, but were still greater than those in the mother (approximately 1000 mu Leq/mL). The umbilical arterial and venous concentrations were the same as the fetal blood concentration of inhibin. The role of the baboon fetal adrenal in inhibin production was studied. Fetal adrenals collected from days 59, 135, and 167 of gestation contained the mRNA for the inhibin alpha-subunit in relatively high abundance. The in utero administration of ACTH for 30 min to five fetuses at midgestation (days 100-110) apparently did not alter the fetal concentration of immunoreactive inhibin. In summary, maternal serum inhibin levels increase during the last trimester of baboon pregnancy. This is suggested to be due to an increasing contribution of placental inhibin secretion, which is regulated not by placental estrogen production but, perhaps, by placental growth.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for the presence of the estrogen receptor in the ovary of the baboon (Papio anubis).

Although intraovarian estrogen has been firmly established as an important factor in the regulation of ovarian follicular development and function in the rat, an autocrine-paracrine role for estrogen in the primate ovary is not yet established. Immunocytochemical identification of an estrogen receptor in the monkey follicle was negative, but it was positive for the granulosa cells of antral follicles of the human ovary. In the present study baboon ovaries obtained during the follicular phase were examined for the presence of estrogen receptor by immunocytochemical analysis of frozen sections and Northern blot analysis of RNA extracts of the ovaries. Immunocytochemistry identified the estrogen receptor in the granulosa cells of healthy appearing and atretic or cystic-like antral follicles and in occasional, but rare, thecal cells. The ovaries contained a prominent mRNA species for the estrogen receptor, approximately 7 kilobases in size, which was present in relatively low abundance compared to that in the nonpregnant baboon uterus, but in a similar abundance to the estrogen receptor mRNA content of the pregnant endometrium. These studies are the first to report the presence of estrogen receptor mRNA in the ovary of a primate. These results in conjunction with the immunocytochemical studies firmly establish the presence of the estrogen receptor in the primate ovary and suggest an autocrine-paracrine role for intraovarian estrogen in primate ovarian physiology.

Chronic hyperestrogenemia: lack of positive feedback action on gonadotropin-releasing hormone-induced luteinizing hormone release and dual site of negative feedback action.

To test whether sustained midfollicular estrogen concentrations sensitize the pituitary response to GnRH in the continued presence of a GnRH stimulus, six female monkeys with regular menstrual cycles were administered hourly pulses of GnRH in the presence or absence of an sc estrone implant. Three were studied in a sequence of 2- to 8-week blocks of 1) control, 2) hourly pulses of exogenous GnRH (6 micrograms/1 min), 3) hourly GnRH pulses plus an estrone (E1) implant, and 4) the E1 alone. In the other three animals the sequence was 1) control, 2) E1, 3) E1 implant plus hourly GnRH pulses, and 4) GnRH pulses only. E1 increased mean estradiol concentrations from 55 pg/ml to 100 pg/ml and the corresponding E1 concentrations from 95 pg/ml to 160 pg/ml. LH concentrations, excluding midcycle surges, were 10.9 +/- 2.2 (SEM) ng/ml, 12.6 +/- 1.5 ng/ml, 11.7 +/- 1.5 ng/ml, and undetectable (less than 6 ng/ml) for the control, GnRH, GnRH plus E1, and E1-treatment periods, respectively. Of note was the suppression of LH concentrations to undetectable levels by midfollicular concentrations of estrogen during the E1-alone treatment period, and the return of LH concentrations to normal follicular phase levels by the application of exogenous GnRH support. This observation suggested that an estrogen negative feedback signal can suppress endogenous GnRH. To further examine this hypothesis we applied the same protocol to two hypogonadal female monkeys. E1 capsule placement increased the mean estradiol concentration from 22 to 61 pg/ml and suppressed LH and FSH to undetectable levels. When hourly pulses of GnRH (6 micrograms/1 min) were supplied, mean LH and FSH increased to 29.8 and 14.9 ng/ml, respectively. These studies demonstrate that elevation of estrogen concentrations to midfollicular levels does not sensitize the pituitary to GnRH stimulation, and pituitary sensitization is therefore unlikely to be important as a cause of elevated LH secretion in anovulatory states, such as the polycystic ovaries syndrome. In the hypogonadal monkeys, a 5-fold decrease in gonadotropin concentrations occurred in spite of full exogenons GnRH support, consistent with a hypophyseal site of estrogen negative feedback action. However, the GnRH clamp did prevent the complete suppression of LH and FSH noted when only estrogen was applied, consistent with an additional negative feedback effect on the hypothalamus. Although this same phenomenon is observed in the eugonadal monkeys, it appears unlikely that a hypothalamic site of estrogen inhibition plays a significant role during the menstrual cycle, otherwise the progressive rise in follicular phase estrogen concentrations would, by arresting GnRH secretion, abort folliculogenesis.

Reversed-phase liquid chromatographic purification and isolation of a radio-iodinated selective probe for mu opioid receptors in the brain.

A Guard-PAK precolumn system was used for the reversed-phase liquid chromatography purification of a small, synthetic radiolabeled opioid peptide, FK 33-824 (D-Ala2, methyl-phe4, Met (O)ol5 enkephalin) (FK). This procedure involves trace enrichment of iodinated peptide onto the precolumn while iodination reagents are not retained. Radioactive contamination of high-performance liquid chromatography columns and injectors is thus avoided. Precolumn chromatography has sufficient resolving power to separate not only labeled from unlabeled peptide but also mono- from di-iodinated peptide. Purified 125I-labeled FK (estimated specific activity 85.9-153.7 Ci/mmol) showed high specific binding to mouse corpus striatum, neocortex, cingulate cortex, nucleus accumbens septi, diagonal band of Broca, nucleus medialis septi, area preopticus magnocellularis, and the nucleus of the caudate/putamen. Radioligand binding was inhibited by both antagonists (naloxone and naltrexone); and agonists D-Ala2, N-methyl-phe4, gly-ol5-enkephalin [DAGO]; FK; and beta-endorphin at all concentrations tested (1 x 10(-8) to 1 x 10(-4) M). Adrenocorticotropin hormone (ACTH) did not block ligand binding at any concentration tested. Distribution of mu opioid receptors was analyzed by light microscopic autoradiography. Sections incubated with 125I-labeled FK in the presence of agonists and antagonists demonstrated decreasing ligand binding with increasing doses of competitor. ACTH did not block ligand binding at any concentration tested. HPLC analyses of ligand which had been iodinated 1.5 half lives before the date of the experiment demonstrated a single peak similar to that of freshly iodinated ligand. Similar binding kinetics and autoradiographic labeling patterns were observed as compared to those obtained with freshly iodinated peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced serum inhibin concentrations during ovulatory cycles of estrogen-treated rhesus monkeys: an indicator of FSH bioactivity.

Female rhesus monkeys treated with exogenous estrone initially were anovulatory. Although estrone and estradiol concentrations were maintained 1.5- to 2.5-fold elevated, i.e. in the midfollicular range, ovulatory cycles resumed in three of four animals after 6-15 months of anovulation. During the ovulatory cycles the serum bio LH concentrations were the same in estrone-treated animals as during ovulatory cycles of control monkeys, but the daily basal serum FSH concentrations detectable by RIA were significantly reduced during the ovulatory cycles of the estrone-treated animals compared to the cycles of the controls. In the present study serum inhibin concentrations were measured to determine whether or not they were increased and the cause of the selective decrease in FSH concentrations in the estrogen-treated monkeys. Serum LH, FSH, progesterone, and inhibin concentrations were measured by RIA in blood samples collected during the third year of continuous estrogen treatment. The lack of an effect of elevated estrogen on LH concentrations and a significant estrogen-induced decrease in serum FSH concentrations during ovulatory cycles was confirmed (FSH, control: 5.6 +/- 0.68 ng/ml; estrogen-treated: 2.5 +/- 0.09 ng/ml; P = 0.01). There was also a significant decrease in the serum inhibin concentrations detectable by RIA during the follicular phase of the estrone-treated monkeys compared to the follicular phase of the control animals (119 +/- 17 vs. 462 +/- 105 microliter eq/ml; P = 0.04). These results indicate that the lower serum concentrations of FSH in the estrogen-treated monkeys were not a result of an increase in ovarian secretion of inhibin. The lower inhibin concentrations suggest that FSH bioactivity, as well as immunoreactive FSH, is significantly reduced during the ovulatory cycles of the estrone-treated monkeys. Even though the estrogen treatment decreased the FSH bioactivity, sufficient FSH secretion occurs in the presence of the elevated estrone and estradiol concentrations to induce and support apparently normal ovulatory cycles.

Loss during aging of beta-endorphinergic neurons in the hypothalamus of female C57BL/6J mice.

Beta-endorphin (B-EP) content is often reduced in hypothalami of aging rodents. The objective of this study was to determine whether reduced B-EP content is associated with a reduced number of B-EP immunoreactive neurons. Serial coronal sections extending from the caudal hypothalamus through the retrochiasmatic area were examined by quantitative light microscopy in mature (5-6 month) and senescent (24-28 month) mice that had been ovariectomized 1 week earlier and injected with colchicine 24-48 h before sacrifice. Old mice were acyclic. As expected, B-EP immunoreactive cell bodies were restricted to the region of the arcuate nucleus. There was a 35% loss of B-EP immunopositive neurons in old, macroscopically disease-free animals. By contrast, some old animals with pituitary tumors had no loss of B-EP neurons. These results suggest that a subpopulation of B-EP neurons either die or stop synthesizing detectable concentrations of B-EP in aged mice. The basis for the absence of reduced B-EP neurons in some mice with pituitary tumors is unclear, but this observation underscores the importance of distinguishing age-related changes associated with diseases of aging from those that are independent of such diseases.

Loss of LH-RH neurons in the rostral forebrain of old female C57BL/6J mice.

This study reports the quantitative immunohistochemical distribution of luteinizing hormone-releasing hormone (LH-RH) neurons within the rostral forebrain of young (5-6 month) and old (26-28 month) C57BL/6J mouse. Old mice demonstrated a significant (18%) loss of LH-RH-containing neurons (p less than 0.001, ANOVA). The most striking losses involved the preoptic area (24%) and more rostral regions (26%). The presence of pituitary or mesenteric tumors in older mice did not affect the density of LH-RH neurons. These observations indicate that LH-RH neurons comprise part of the neuronal population previously reported to be reduced in the preoptic area of older mice (4).

Escape from chronic estrogenic suppression of ovarian function in the adult rhesus monkey: evidence for changing sensitivity of gonadotropin secretion to estrogen inhibition.

Regularly menstruating female rhesus monkeys were implanted sc with Silastic tubing packed with estrone. The implants were replaced every 6-8 months to maintain the serum estrone and estradiol concentrations constantly elevated 1.5-2.5-fold, i.e. in the midfollicular phase range, for 4 yr. Increasing the estrone and estradiol concentrations initially led to anovulation which persisted for 6 to 15 months after which three of four of the estrogen-treated animals resumed ovulatory cycles. These animals then waxed and waned between anovulation and ovulatory cycles for the remainder of the study period. The resumption of ovulatory cycles were attributed to an escape of the central nervous system-pituitary axis from the suppressive effect of the elevated estrogen concentrations, since serum estradiol concentrations did not change. During anovulatory and ovulatory months the basal LH concentration was not significantly increased in the estrogen-treated monkeys compared to the control animals, but it was significantly reduced during anovulatory months compared to ovulatory months. Furthermore, LH secretion in response to a bolus of GnRH was attenuated in the monkeys chronically exposed to the acyclic elevation of blood estrogen levels. As the sensitivity to estrogen decreased, sufficient basal concentrations of FSH and LH were achieved to support ovulatory cycles with associated midcycle surges of LH and FSH secretion and apparently normal luteal patterns of progesterone secretion. Since the daily FSH concentrations of these ovulatory cycles of the estrone-treated animals were significantly lower than that of ovulatory cycles of the control monkeys, the ovulatory cycles of the estrogen animals may not have been normal, but this was not directly documented. These studies suggest that elevated basal estrogen levels do not lead to elevated basal LH secretion in the female primate; and also in the intact adult primate the central nervous system-pituitary axis has the potential to change its sensitivity to the negative feedback effects of estrogen on gonadotropin secretion.

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.

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.

Effect of chronically elevated androgen or estrogen on the glucose tolerance test and insulin response in female rhesus monkeys.

Insulin concentrations, in response to an intravenous glucose bolus after a 24-hour fast, have been studied in female rhesus monkeys in which the circulating levels of androstenedione and testosterone or estrone and estradiol have been increased for as long as 4 1/2 years. No significant differences were observed in the basal insulin or C-peptide concentrations in the androgen or estrogen-treated animals compared with each other or with normal cycling, nontreated control animals. The insulin and C-peptide responses to intravenous glucose were similar in control and androgen-treated monkeys. Compared with both the control and androgen-treated monkeys, the responses of the estrogen-treated monkeys tended to be lower but were not significantly different. The glucose disappearance rate after the intravenous glucose bolus was not significantly different in androgen and control monkeys but was significantly slower during the initial 30 minutes in the estrogen-treated monkeys compared with both the control and androgen-treated monkeys. These studies suggest that chronically elevated androgen levels in the mature female subhuman primate do not lead to insulin resistance or overt glucose intolerance.