Mediation by gonadal steroids of plasma beta-endorphin and LH in castrated female and male rats

Immunoreactive beta-endorphin (IR-BE) was significantly decreased and luteinizing hormone (LH) significantly increased in female rats castrated for four weeks. Forty eight hours after a single injection of estradiol benzoate (EB), IR-BE levels increased, and LH levels were reduced. On the afternoon following the administration of a second injection of EB given six hours earlier, IR-BE levels were reduced below control values, whereas LH levels were significantly elevated. There was no change in IR-BE levels during the remainder of that afternoon whereas LH levels decreased over time. Similar to female rats, IR-BE was diminished and LH increased in castrated male rats. IR-BE was increased significantly above those values observed in intact animals 24 hr after a single injection of TP and returned to control levels by 48 hr after administration of TP. Injection of TP reduced LH to levels observed prior to castration. These findings suggest that gonadal steroids exert a feedback on the release of IR-BE from the pituitary of female and male rats opposite to their feedback effect on the release of pituitary gonadotropins.     

Effects of neonatal gonadal steroids on adult CA3 pyramidal neuron dendritic morphology and spatial memory in rats

The hippocampus is implicated in spatial cognition, which is sexually dimorphic and developmentally sensitive to gonadal steroids. Previously we have shown a sex difference in CA3 pyramidal cell layer volume and neuronal soma size that was reversible with neonatal castration in males or prenatal treatment of females with either testosterone propionate (TP) or a nonaromatizable androgen, dihydrotestosterone propionate, but not estradiol benzoate, all of which correlated with adult water maze navigation. The present study further investigates developmental androgen sensitivity of CA3 pyramidal neurons by measuring dendritic morphology and its relation to adult spatial ability. Female rats were injected with TP on postnatal day (P) 3 and P5 or ovariectomized (OVX) on P2, and male rats were castrated on P2, with or without testosterone replacement (Cas+T). Sham surgery controls were also included. Animals were tested on a water maze in adulthood, sacrificed, and CA3 pyramidal neurons were Golgi-stained and reconstructed in three dimensions using a computer-interfaced morphometry system. High-androgen groups (control males, Cas+T, TP females) performed better in spatial navigation and exhibited CA3 neurons with longer dendrites, a larger number of dendritic branches, and volumes of influence compared to low-androgen groups (control females, castrated males, OVX). Collectively, these findings indicate that the critical time period for organizational effects of androgens on the CA3 pyramidal neurons includes both prenatal and postnatal life, during which time androgens regulate developmental events such as somal growth and neuronal differentiation, all of which significantly contribute to establishing the sex difference in adult spatial navigation.     

Effect of neonatal beta-endorphin imprinting on sexual behavior and brain serotonin level in adult rats

A single dose (3 microg) beta-endorphin was administered to newborn female and male rats (hormonal imprinting). In adult age (at 5 months) sexual behavior, steroid hormone binding capacity and brain serotonin content was studied. Females' sexual activity (lordosis quotient) significantly decreased and more animals protested against mounting (ratio of kicking and crying 21/24 vs. 8/24; p < 0.001). Males' sexual activity did not change, however more males were aggressive (4/10 vs. 1/10). Uterine estrogen receptor density significantly increased and affinity decreased. There was no change in the binding capacity of thymic glucocorticoid receptors. In the brain, five regions were studied for serotonin content. There was a gender difference in serotonin level and the intragroup differences were also high. In the endorphin treated males the serotonin level was significantly lower than in the controls. In the endorphin treated females the intragroup scattering has been significantly reduced. Nociceptin content of the cerebrospinal fluid was not changed. The experiments call attention to the possibility of adjustment of sexual and behavioral sphere by the individually different endorphin surge during labor.     

N-cadherin is regulated by gonadal steroids in adult sexually dimorphic spinal motoneurons

Gonadal steroids influence the morphology and function of neurons in the adult spinal cord through cellular and molecular mechanisms that are largely unknown. The cadherins are cell adhesion molecules that participate in the formation and organization of the CNS during embryonic development, and recent evidence suggests that the cadherins continue to regulate neural structure and function in adulthood. Using degenerate oligonucleotides coding conserved regions of the catenin-binding domain of classical cadherins in a RT-PCR cloning strategy, we identified several cadherin subtypes, the most frequently cloned being N-, E-, and R-cadherin, suggesting that these are the major classical cadherin subtypes present in the adult male rat lumbosacral spinal cord. We then examined cadherin expression levels of these cadherin subtypes under steroid conditions known to induce plastic changes in spinal motoneurons. Semiquantitative PCR revealed that mRNA levels of N-cadherin, but not E-cadherin or R-cadherin, are elevated in castrated rats treated with testosterone, 17 beta-estradiol, or dihydrotestosterone relative to castrate rats not treated with steroids. Immunolocalization of N-cadherin revealed that steroid treatment increased N-cadherin expression levels in functionally related neural populations whose morphology and function are regulated by steroids. These results suggest a role for N-cadherin in steroid-induced neuroplastic change in the adult lumbar spinal cord.     

Neurotransmitters and estrogen interact to affect beta-endorphin levels in castrated female rats

The possibility of an interaction between neurotransmitter systems and estrogen in affecting levels of immunoreactive beta-endorphin (IR-BE) in the anterior pituitary (AP), the neurointermediate lobe of the pituitary (NIL) and the hypothalamus was investigated in ovariectomized (OVX) female rats. Chronic administration of the dopamine antagonist, haloperidol (HALO), had no effect on IR-BE levels in the AP. By contrast, the content of IR-BE in the NIL was increased and the content of IR-BE in the hypothalamus was decreased by HALO. Chronic treatment with estradiol benzoate (EB) produced a decrease in IR-BE in all three tissues. The effect of EB on IR-BE levels in the AP and NIL was reversed by administration of HALO, while EB and HALO appeared to act independently on the hypothalamus. Gel chromatography indicated that alterations in IR-BE in the AP corresponded to similar changes in beta-endorphin (BE) and beta-lipotropin (LPH) and that BE alone comprised the immunoreactivity detected in the NIL and hypothalamus regardless of treatment. Chronic treatment with the alpha-adrenergic agonist, clonidine (CLON), increased, whereas treatment with EB decreased, IR-BE levels in the AP, NIL and hypothalamus. EB attenuated the effect of CLON on IR-BE levels in the AP and hypothalamus. Chronic treatment with CLON appeared to promote the formation of BE in the AP, whereas the proportions of BE and LPH were similar in the AP of controls and animals treated with EB or EB and CLON. BE alone was detected in the NIL and hypothalamus of treated and control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple mechanisms of ethanol-induced gonadal toxicity to adult male rats.

In an attempt to elucidate the mechanism(s) underlying the alcohol-induced pathogenesis of testis, acute as well as chronic studies were undertaken in adult male rats. Ethanol reduced significantly the plasma and testicular testosterone contents in treated rats even at moderate dose levels. The alterations in pituitary gonadotrophins, LH and FSH, demonstrated a central defect in the hypothalamo-hypophyseal-gonadal axis. Major microsomal enzymes involved in the biosynthesis of testosterone, viz. 3 beta-hydroxysteroid dehydrogenase and steroidogenic mixed function oxidases were markedly inhibited in a dose and duration dependent manner. The terminal enzyme 17 beta-hydroxysteroid dehydrogenase was, however, unaffected by ethanol treatments except at a higher dose level of 6 g/kg body wt. Although, the activity of testicular alcohol dehydrogenase was relatively unchanged, a marked induction in the activity of cytosolic conjugation enzyme, GSH-s-transferase was noticed. The present study demonstrates the major role of the metabolism of ethanol in the underlying cause for in vivo toxicity of ethanol and warrants its further consideration.     

Platelet serotonin levels and gonadal hormones in rats

The role of gonadal hormones in the control of platelet serotonin levels was studied by evaluating the effect of sexual maturation in rats of both sexes and the time-course of changes following gonadectomy performed either prepubertally or on sexually mature animals. In males, platelet serotonin levels remained fairly stable during sexual maturation as well as during the whole postgonadectomy period monitored (four months). In females, somewhat higher values of platelet serotonin levels in adult than in sexually immature animals were found (9%, p less than 0.001, N = 34). A slight decrease of platelet serotonin (10-18%, p less than 0.05) was observed following ovariectomy of sexually mature females, but it was of transient nature. When females were ovariectomized prepubertally a tendency towards permanently lower platelet serotonin levels was noticed. These results suggest that gonadal hormones have no major role in the control of platelet serotonin levels in rats, although a subtle hormonal modulation of this platelet variable in females may exist.     

Regulation of neural oxytocin gene expression by gonadal steroids in pubertal rats

We have previously demonstrated that neuronal oxytocin mRNA increases during the pubertal development of female rats. In this paper we have examined the factors that regulate this developmental increase in both male and female rats. Northern blot analysis demonstrated that neural oxytocin mRNA increased 5- to 10-fold from postnatal day 20 (P20) to P60 in animals of both sexes, coincident with puberty. Mature male rats and females at all stages of the estrous cycle expressed similar levels of neural oxytocin mRNA. Pubertal up-regulation of oxytocin mRNA was largely, but not completely, inhibited by prepubescent gonadectomy, indicating a requirement for intact gonads as well as some other as yet undefined factor(s). Pubertal treatment of gonadectomized animals with estradiol or testosterone abolished the effects of gonadectomy; treated animals expressed levels of neural oxytocin mRNA similar to those in controls. However, treatment of prepubertal animals with estradiol or testosterone from P10 to P20 had no effect on oxytocin mRNA levels, suggesting that neural maturation or other factors are necessary requisites for steroid sensitivity. To determine whether neural activin played any role in regulating oxytocin mRNA during puberty, we examined levels of inhibin/activin beta A-chain mRNA. This mRNA was expressed at similar levels in all brain regions and did not vary as a function of gonadectomy or steroid treatment, making it unlikely that activin mediates the observed changes. Together, these data indicate that neural oxytocin mRNA is induced by gonadal steroids during puberty, and suggest a mechanism for coordinating development of reproductive functions with other pubertal changes.     

Single neonatal treatment with beta-endorphin (hormonal imprinting) extremely enhances nocistatin level of cerebrospinal fluid in adult rats

In earlier experiments endorphin treatment of newborn rats caused the decrease of brain serotonin content, increasing aggressivity, enhanced sexual activity of females and changes in the binding capacity of uterine estrogen receptors at adult age, however nociceptin content of the cerebrospinal fluid was not changed. In the present experiment neonatal treatment of male and female rats was done with a single dose of 3 microg beta-endorphin and in five months old rats the level of nociceptin antagonist nocistatin was determined by radioimmunoassay in the cerebrospinal fluid. In both genders the amount of nocistatin was one magnitude higher in the endorphin treated groups. There was also a significant difference between the male and female nocistatin level in the treated and non-treated groups alike, with the advantage of females. The results call attention to the possibility of influencing pain-tolerance for life, by the pain-provoked endorphin levels during delivery.     

Cimetidine and plasma levels of gonadotropins,prolactin and gonadal steroids in women.

The endocrine effects of cimetidine (Tagamet) during the menstrual cycle were investigated in seven healthy female volunteers. The subjects were studied for six menstrual cycles divided into the pretreatment phase, a phase of therapy with 1.2 g of orally administered cimetidine daily for two cycles, and a post-treatment phase. Cimetidine therapy induced a significant increase in the mean plasma level of follicle-stimulating hormone during the periovulatory period, followed by modest but sustained hyperprolactinemia throughout the luteal phase of each cycle. No significant changes were found in the mean plasma levels of luteinizing hormone and progesterone, and the mean plasma estradiol level was significantly decreased only in the midproliferative phase of each cycle. The mean plasma prolactin levels after a bolus injection of thyrotropin-releasing hormone in the midluteal phase during cimetidine administration did not differ from the mean control levels, which indicates that cimetidine modulates the release of prolactin at the suprapituitary locus. However, the significance of the endocrine changes remains to be established.     

Elevation of plasma LH in response to systemic injection of beta-endorphin antiserum in adult male rats

A single injection of beta-endorphin antiserum into mature male rats produced approximately a 3-fold rise in plasma LH levels by 10 to 50 min, and declined by 90 min, but remained significantly elevated above pre-injection values. Control rats injected with normal rabbit serum showed no elevation in plasma LH values. These results indicate that beta-endorphin is one of the endogenous opiates that tonically depresses basal release of LH in mature male rats.     

Effect of beta-endorphin imprinting during late pregnancy on the brain serotonin and plasma nocistatin levels of adult male rats.

Female rats were treated with 10 microg of beta-endorphin on the 19th day of pregnancy. Offspring were studied when five months old. Serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content in four brain regions were determined by HPLC-EC and the nocistatin levels of blood plasma using RIA methods. In each brain region studied, the 5-HT levels were highly significantly reduced and that of 5-HIAA in three regions was highly significantly increased. When 5HIAA/5HT ratios, as a measure of serotonin turnover, were calculated, imprinted animals showed extremely high values. Plasma nocistatin level was also significantly elevated. The results call attention to the effect of perinatal endorphin imprinting and its long-term consequences (e.g., setting of aggressiveness, pain tolerance).     

The influence of beta-endorphin on testicular endocrine function in adult rats.

The role of beta-endorphin in testicular steroidogenesis is poorly understood. To address this issue, we treated adult hypophysectomized rats intratesticularly with either saline-50% polyvinylpyrrolidone (SAL-PVP) or human beta-endorphin (0.5 microgram/testis; a total of 1 microgram/rat/day) in SAL-PVP for 3 days. Testicular injections were made under ether anesthesia. On Day 3, rats also received injections (s.c.) of either SAL-PVP or 5 micrograms beta-endorphin in SAL-PVP to minimize the dilution of ether in the testis. One hour later, rats were treated (i.p.) with either saline or ovine LH (25 micrograms/rat). One hour after saline or LH injection, blood was obtained via heart puncture for determination of plasma progesterone (P), androstenedione (A-dione), and testosterone (T) levels. The effects of beta-endorphin (50 ng, equivalent to 13.9 pM; or 250 ng, equivalent to 69.6 pM) on P and androgen secretions in vitro were also examined. Intratesticular injections of beta-endorphin significantly (p less than 0.025) decreased the T response to LH treatment, but failed to affect plasma P and A-dione levels. Response of P to LH treatment was increased (p less than 0.005) in medium containing testicular fragments exposed to 250 ng (69.6 pM) beta-endorphin. However, beta-endorphin attenuated LH effects on A-dione and T production in vitro. These studies demonstrate that beta-endorphin inhibits T secretion, possibly because of its effect on the synthesis of T precursors. Thus, testicular beta-endorphin modulates the endocrine function of the testis in adult rats.     

Pulmonary vascular resistance in adult rats exposed to hypoxia in the neonatal period

Newborn rats were exposed to hypoxia (10% O2 + N2) from 24 h to day 6 of neonatal life and then returned to room air until 45 days of age (experimental). The rats were anaesthetized, heparinized, and exsanguinated. The chest was opened and the lungs were perfused with diluted autologous blood at a constant flow rate (Q). The pulmonary arterial pressure (Pa) and venous pressure (Pv) were monitored. The properties of the pulmonary vasculature were assessed by measuring baseline vascular resistance, PVR = (Pa-Pv)/Q, segmental pressure gradients (double occlusion technique), pressure-flow relationship, hypoxic pressor response (HPR, 3% O2), and the response to 0.5 microgram bolus of angiotensin II (AII). These were compared with similar measurements on age-matched control animals never exposed to hypoxia. The perfusate hematocrit and gases were not significantly different between the two groups. The PVR normalized to body weight was 30% higher in the experimental groups (p less than 0.005). The double occlusion results (obtained at a flow rate of 13 mL/min) revealed that this increase in resistance was primarily due to the increase in the postcapillary resistance. HPR was primarily in the upstream segment in both groups but was larger in the experimental group. In contrast, the response to AII occurred in both the upstream as well as in the downstream vascular segments and did not differ between the two groups. We conclude that adult rats exposed to hypoxia in the neonatal period have elevated pulmonary vascular resistance and increased vascular reactivity to hypoxia.