Implantation Of Silastic Capsules Research Articles

Bisphenol A (BPA) pharmacokinetics with daily oral bolus or continuous exposure via silastic capsules in pregnant rhesus monkeys: Relevance for human exposures

We measured serum dBPA in non-pregnant and pregnant female rhesus monkeys, fetuses and amniotic fluid. dBPA was administered by a daily oral bolus or sc implantation of Silastic capsules; both resulted in daily average serum unconjugated dBPA concentrations of <1ng/ml. We observed lower serum concentrations of unconjugated dBPA in pregnant females relative to pre-pregnancy values, and generally lower concentrations in fetal serum than in maternal serum. Differences in pharmacokinetics of dBPA were evident between pre-pregnancy, early and late pregnancy, likely reflecting changes in maternal, fetal and placental physiology. The serum ratio of conjugated to unconjugated dBPA after continuous sc release of dBPA was similar to values reported in human biomonitoring studies and markedly lower than with oral administration, suggesting oral bolus exposure is not an appropriate human exposure model. We report elsewhere that there were numerous adverse effects on fetuses exposed to very low serum dBPA in these studies.

Особливості статевої поведінки самиць щурів при моделюванні гіперандрогенії в пубертатний та постпубертатний періоди

The parameters of female and male sexual behavior in 3- and 6- month old female rats which were exposed to an androgen excess (subcutaneous implantation of Silastic capsules containing 5 mg of crystalline testosterone) from the beginning of pubertal period (at the age of 35 days), or within postpubertal period (at the age of 4 months). Hyperandrogenia in pubertal period had no effect on female sexual behavior formation, but it led to appearance of male behavior components in 100% of animals. In female rats which were implanted with testosterone capsules in postpubertal period, sexual disturbances were more pronounced and were characterized by masculinization and defeminization, which was due to a higher degree of androgenic saturation. The data obtained suggest a leading role of hyperandrogenemia in the pathogenesis of sexual behavior disturbances in female rats in different periods of individual development.

Comparing the relative amount of testosterone required to restore sexual arousal, motivation, and performance in male rats

It is well established that male rat reproductive behaviors including sexual arousal, motivation, and performance are dependent on circulating levels of testosterone (T). The present study was designed to (1) compare the relative amount of T required to restore these different aspects of behavior in castrated rats, and (2) create an animal model for clinical populations with sexual impairments. Twenty-nine male Long–Evans rats were tested before and after castration for sexual performance (copulation), motivation (partner preference), and arousal (50 kHz ultrasonic vocalizations; measured together with scent marking). Sexual arousal was also inferred from copulation data. Rats were then assigned to one of four groups, and T was re-introduced via Silastic capsule implants varying in length and content: No T (empty capsules), Low T (2 mm capsules), Medium T (5 mm capsules), or High T (two 10 mm capsules). The highest dose was intended to restore physiological levels. Results indicate that High T is required for 50 kHz vocalizations, while Medium T was sufficient for the restoration of copulation, partner preference, and scent marking. These data suggest that sexual arousal may be most sensitive to reductions in testosterone. The role of T levels in measures of generalized and specific (sexual) arousal is discussed in the context of other reproductive behaviors. Furthermore, because the Low T group showed impairments across all behaviors during post-implant tests, we propose that these animals may provide a good animal model for studying clinical conditions marked by reduced motivation and arousal, including Hypoactive Sexual Desire Disorder.

Estrogen and aging affect the synaptic distribution of estrogen receptor beta-immunoreactivity in the CA1 region of female rat hippocampus

Estradiol (E) mediates increased synaptogenesis in the hippocampal CA1 stratum radiatum (sr) and enhances memory in young and some aged female rats, depending on dose and age. Young female rats express more estrogen receptor α (ERα) immunolabeling in CA1sr spine synapse complexes than aged rats and ERα regulation is E sensitive in young but not aged rats. The current study examined whether estrogen receptor β (ERβ) expression in spine synapse complexes may be altered by age or E treatment. Young (3–4 months) and aged (22–23 months) female rats were ovariectomized 7 days prior to implantation of silastic capsules containing either vehicle (cholesterol) or E (10% in cholesterol) for 2 days. ERβ immunoreactivity (ir) in CA1sr was quantitatively analyzed using post-embedding electron microscopy. ERβ-ir was more prominent post-synaptically than pre-synaptically and both age and E treatment affected its synaptic distribution. While age decreased the spine synaptic complex localization of ERβ-ir (i.e., within 60 nm of the pre- and post-synaptic membranes), E treatment increased synaptic ERβ in both young and aged rats. In addition, the E treatment, but not age, increased dendritic shaft labeling. This data demonstrates that like ERα the levels of ERβ-ir decrease in CA1 axospinous synapses with age, however, unlike ERα the levels of ERβ-ir increase in these synapses in both young and aged rats in response to E. This suggests that synaptic ERβ may be a more responsive target to E, particularly in aged females.

Resveratrol Inhibits Development of Experimental Endometriosis In Vivo and Reduces Endometrial Stromal Cell Invasiveness In Vitro1

Endometriosis is a common gynecologic disorder characterized by ectopic attachment and growth of endometrial tissues. Resveratrol is a natural polyphenol with antiproliferative and anti-inflammatory properties. Our objective was to study the effects of resveratrol on human endometriotic implants in a nude mouse model and to examine its impact on human endometrial stromal (HES) cell invasiveness in vitro. Human endometrial tissues were obtained from healthy donors. Endometriosis was established in oophorectomized nude mice by intraperitoneal injection of endometrial tissues. Mice were treated with 17β-estradiol (8 mg, silastic capsule implants) alone (n = 16) or with resveratrol (6 mg/mouse; n = 20) for 10-12 and 18-20 days beginning 1 day after tissue injection. Mice were killed and endometrial implants were evaluated. A Matrigel invasion assay was used to examine the effects of resveratrol on HES cells. We assessed number and size of endometriotic implants in vivo and Matrigel invasion in vitro. Resveratrol decreased the number of endometrial implants per mouse by 60% (P < 0.001) and the total volume of lesions per mouse by 80% (P < 0.001). Resveratrol (10-30 μM) also induced a concentration-dependent reduction of invasiveness of HES by up to 78% (P < 0.0001). Resveratrol inhibits development of endometriosis in the nude mouse and reduces invasiveness of HES cells. These observations may aid in the development of novel treatments of endometriosis.

Abstract 5361: Progesterone alters cell survival and cell death pathways in estrogen-induced mammary carcinogenesis

Abstract Breast cancer is one of the most frequent and common malignancy in women. The incidence of breast cancer is high in postmenopausal women and majority of these cancers are estrogen receptor positive. Epidemiological data suggests that estrogen plus progesterone hormone replacement therapy increases the risk of breast cancer in postmenopausal women. Further, estrogen alone therapy did not increase the risk instead decreased the risk of breast cancer in postmenopausal women. Till date the role of progesterone in breast cancer is not well understood. Our earlier studies using ACI rats have clearly demonstrated the significant role progesterone plays in the estrogen-induced mammary carcinogenesis. We now, aim to investigate the mechanisms by which progesterone promotes estrogen-induced mammary carcinogenesis in ACI rats. We chose to use the ACI rats as it is a good model to study hormone-induced mammary carcinogenesis. In brief, seven weeks old female ACI rats were divided into the following groups: 1) untreated control, 2) rats treated with 30 mg estradiol, 3) rats treated with 30 mg progesterone, 4) rats treated with 30 mg estradiol plus 30 mg of mifepristone (a progesterone inhibitor). All hormone treatments were given for 30 days by subcutaneous implants of silastic capsules. At the end of the treatment, mammary glands were excised and used for molecular analysis. Cell survival, cell death pathways, and hormone receptor status were analyzed using different molecular techniques. Expression patterns of mRNA and proteins involved in major cell survival and apoptotic pathways were significantly altered in response to progesterone inhibition. Our data demonstrates that progesterone plays a prominent role in regulating key regulatory pathways in estrogen-induced mammary carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5361.

Resveratrol inhibits development of experimental endometriosis and induces apoptosis of endometrial stroma

OBJECTIVE: Resveratrol (RES), a polyphenol abundant in red grapes, has antioxidant, anti-proliferative and anti-inflammatory properties. Pathophysiology of endometriosis involves ectopic attachment and proliferation of endometrial tissues, oxidative stress and inflammation. This study evaluated effects of RES on a nude mouse model of endometriosis and on cultures of human endometrial stromal cells (HEC). DESIGN: In vitro and in vivo experiments. MATERIALS AND METHODS: Human proliferative phase endometrial biopsies were established as organ cultures or used for HEC isolation. To establish endometriosis in the ovariectomized nude mouse, endometrial tissues were maintained in 1 nM estradiol (E) for 24 hrs and subsequently injected intraperitoneally. Mice (N=12) were randomly assigned to RES Group or Control Group. RES Group received E (8 μg, silastic capsule implants) plus RES (5.9 mg/kg per day) by gavage for 10 days. Control mice received E+vehicle. The animals were then sacrificed and endometrial implants were measured and stained (TUNEL) for apoptosis. Studies of HEC evaluated effects of RES (30-100 μM) on apoptosis by detection of caspases 3/7. RESULTS: Mice treated with RES had significantly lower number and volume of endometrial implants: Control Group had on average 2.5±0.3 lesions/animal while mice treated with RES had 74% fewer lesions (0.6±0.2/animal; P<0.001). Average volume of implants was 11.5±1.5 mm3/animal in the Control Group and was 88% lower in RES Group (1.4±1.1 mm3; P=0.0003). TUNEL staining of implants from RES Group was significantly greater than that from Control mice. In cultures of HEC, RES induced a concentration dependent wave of apoptosis with maximum expression of caspases 3/7 at 48 hours increased 18-fold above control (P<0.001). CONCLUSIONS: RES significantly reduced the number and size of endometrial implants in the nude mouse model of endometriosis as well as induced HEC apoptosis. The present findings may lead to the development of novel treatments of endometriosis involving RES.

Simvastatin Protects against the Development of Endometriosis in a Nude Mouse Model

Endometriosis is a common condition associated with infertility and pelvic pain in women. Recent in vitro studies have shown that statins decrease proliferation of endometrial stroma (ES) and inhibit angiogenesis. The aim was to evaluate effects of simvastatin on development of endometriosis in a nude mouse model. Proliferative phase human endometrial biopsies were obtained from healthy donors and established as organ cultures or used to isolate ES cells. To establish endometriosis in the nude mouse, endometrial tissues were maintained in 1 nm estradiol (E) for 24 h and subsequently injected into ovariectomized nude mice. Mice (n = 37) were treated with E (8 mg, SILASTIC capsule implants; made in author laboratory) alone or with E plus simvastatin (5 or 25 mg/kg x d) for 10 d beginning 1 d after tissue injection (from three donors). Mice were killed and examined for disease. Effects of simvastatin on matrix metalloproteinase-3 (MMP-3) were evaluated in cultures of ES cells. The number and size of endometriotic implants were measured. Simvastatin induced a dose-dependent decrease of the number and size of endometrial implants in mice. At the highest dose of simvastatin, the number of endometrial implants decreased by 87%, and the volume by 98%. Simvastatin also induced a concentration-dependent decrease in MMP-3 in the absence and presence of inflammatory challenge (using IL-1alpha). Simvastatin exerted a potent inhibitory effect on the development of endometriosis in the nude mouse. Mechanisms of action of simvastatin may include inhibition of MMP-3. The present findings may lead to the development of novel treatments of endometriosis involving statins.

Slow-release and injected progesterone treatments enhance acute recovery after traumatic brain injury

The benefits of continuous progesterone release via subcutaneous silastic capsule implants were compared to daily subcutaneous injections in a rat model of traumatic brain injury (TBI). Adult male Sprague–Dawley rats received either bilateral frontal cortex contusions or sham surgery. Rats were injected with progesterone or vehicle at 1 and 6 h post-injury, then once every 24 h for six days with tapering of the dose over the final two treatments. Progesterone-packed silastic capsules were implanted post-injury while the animals were anesthetized. Behavioral assays for anxiety and locomotor activity were evaluated pre- and post-TBI. Brains were extracted eight days post-TBI and prepared for molecular assays. Decreased GABAA-4 levels complemented a decrease in anxiety behaviors on the Elevated Plus Maze for capsule compared to progesterone-injected animals prior to daily injections. All groups with implanted capsules increased locomotor activity compared to those given progesterone injections. In conclusion, steady-state progesterone treatment after TBI decreases edema and anxiety and increases activity, thus enhancing behavioral recovery. A continuous mode of pharmacological administration may prove to be more beneficial in translational and clinical testing than bolus injections over the same period of time.

Testosterone implants alter the frequency range of zebra finch songs

To investigate whether changes in testosterone alter the frequency range in which a zebra finch produces song, we assigned male zebra finches to two groups, one of which received testosterone implants and the other empty silastic capsule implants. We then recorded songs up to 52 weeks after the surgery and measured frequency changes in the fundamental frequencies of arbitrarily chosen harmonic stacks in the songs of birds in either group. We found statistically significant decreases after 5 weeks in the songs of the testosterone-treated birds. No changes were found in the fundamental frequencies of the control group. The frequency change remained after the apparent effects of the testosterone implants ended. These data show that high levels of testosterone can lower the frequencies of elements in zebra finch songs.

Preservation of spermatogenesis in spinal cord injured rats with exogenous testosterone. Relationship with serum testosterone levels and cellular localization of cAMP responsive element modulator.

The current experiment examined the effects of exogenous testosterone (T) on spermatogenesis in rats with spinal cord injury (SCI) and their relationship with the cellular distribution of a cyclic AMP-responsive element modulator (CREM) in testicular cells. Implantation of T-filled Silastic capsules (TCs, 1-20 cm) resulted in dose-dependent, biphasic changes in testicular T levels and spermatogenesis in SCI rats. However, dose responsiveness of spermatogenesis to exogenous T in SCI rats differed from that in sham control rats. Specifically, implantation of 2-cm TCs enhanced the effects of SCI on spermatogenesis, resulting in total regression of the seminiferous epithelium. Although 3-cm TCs maintained complete spermatogenesis in sham control rats, this regimen failed to support complete spermatogenesis in SCI rats. Although complete spermatogenesis was maintained in SCI rats given 5-20-cm TC implants, various abnormalities persisted. Cellular distribution of CREM remained normal in SCI rats but was altered in those SCI rats that received 3- or 5-cm TC implants. Such effects were associated with reduced CREM proteins in testicular tissues. These results were consistent with altered cAMP signaling and its regulation in testicular cells after SCI and provided possible mechanistic explanations for the effects of SCI on spermatogenesis. SCI resulted in changes in the responsiveness of spermatogenesis to exogenous T. These effects were associated with altered cAMP/CREM signaling in testicular cells. Further studies, including a study of the relationship between serum T levels and normalcy of sperm functions and the role of neural-endocrine interactions in mediating the effects of SCI on spermatogenesis and sperm function, are needed so that therapeutic regimens can be designed for clinical use.

Effects Of Exogenous Testosterone On Testicular Function During The Chronic Phase Of Spinal Cord Injury: Dose Effects On Spermatogenesis And Sertoli Cell And Sperm Function

lntroduction: Exogenaustestosterone has been shown to attenuate spinal cord injury (SCI)-related regression of spermatogenesis in the rat The current experiment investigated the effects of exogenaus testosterone in testicular and sperm functions in the rat du ring the chronic phase of SCI.Methods: Chronic SCI rats were given subcutaneous implants of testosterone-filled silastic capsules (TC). Northern blot cDNA hybridization was used to measure testicular Ieveis of Sertoli cell- and germ cell-specific transcripts. Western blot and immunohistochemistry were used to determine protein Ievei and cellular localization , respectively, of cyclic adenosine monophosphate-responsive element modulator (CREM) in the testes. Flow cytometry was used to determine sperm viability and mitochondrial potential.Results: Spontaneaus restoration of spermatogenesis occurred in 7 of the 8 untreated SCI rats. Although exogenaus testosterone restored complete spermatogenesis in all SCI rats, regressed seminiferous epithelium remained in 30% to 70% of tubular cross sections in these rats. These effects were associated with altered responses of germ cell-specific mRNA transcripts to exogenaus testosterone, and abnormal cellular distribution of CREM. Sperm of untreated SCI rats exhibited lowered motility, viability, and mitochondrial potential. Implantation of 1 0 cm of TC worsened sperm motility in sham control and SCI rats, but restored sperm viability and mitochondrial potential in SCI rats.Conclusion: Administration of exogenaus testosterone to SCI rats during the chronic phase of injury failed to facilitate spermatogenic restoration over that achieved in untreated SCI rats. Abnormalities in postmeiotic spermatogenic differentiation could contribute to these effects, and perhaps the production of sperm with abnormal morphology and/or functions during the chronic phase of SCI.

Prolactin and oxytocin interaction in the paraventricular and supraoptic nuclei: effects on oxytocin mRNA and nitric oxide synthase.

We investigated the contribution of prolactin and oxytocin to the increase in staining for NADPH-d and oxytocin mRNA in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) observed at the end of pregnancy, or following a steroid-priming regimen that mimics the hormonal profile of late pregnant females. Ovariectomized rats received chronic implants of silastic capsules containing oestrogen and progesterone followed by progesterone removal. In experiment 1, oxytocin antagonist (OTA) was administered to rats to investigate whether intranuclear oxytocin release was necessary for NADPH-d staining. In experiments 2a and b, rats received concurrent treatment with bromocryptine (0.5 mg/day) to suppress endogenous prolactin release, and either systemic prolactin (0.5 mg once daily), or prolactin (2 micro g/ micro l), or vehicle infused twice a day into the third ventricle, or chronic oxytocin infusion (24 ng/day) for 3 days following progesterone removal. Brains were then processed for NADPH-d histochemistry. In experiment 3, the interaction of prolactin and oxytocin on oxytocin mRNA within the SON and PVN was examined. NADPH-d staining in the SON and PVN was reduced by the highest dose of the OTA, and by bromocryptine treatment. Central prolactin and oxytocin replacement completely restored NADPH-d staining in bromocryptine-treated rats. Finally, both bromocryptine and the OTA suppressed oxytocin mRNA expression and prolactin replacement restored expression levels to that of controls. Together, these data suggest that the increased capacity to produce nitric oxide in the SON and PVN during late pregnancy is dependent on prolactin stimulating oxytocin gene mRNA and hence intranuclear oxytocin release.

Different effects of subnormal levels of progesterone on the pulsatile and surge mode secretion of luteinizing hormone in ovariectomized goats.

This study tested the hypothesis that endocrinological threshold levels of progesterone that induce negative feedback effects on the pulsatile and surge modes of LH secretion are different. Our approach was to examine the effects of subnormal progesterone concentrations on LH secretion. Long-term ovariectomized Shiba goats that had received implants of silastic capsules containing estradiol were divided into three groups. The high progesterone (high P) group received a subcutaneous implant of a silastic packet (50 x 70 mm) containing progesterone, and the low progesterone (low P) group received a similar implant of a small packet (25 x 40 mm) containing progesterone. The control (non-P) group received no treatment with exogenous progesterone. Blood samples were collected daily throughout the experiment for the analysis of gonadal steroid hormone levels and at 10-min intervals for 8 h on Days 0, 3, and 7 (Day 0: just before progesterone treatment) for analysis of the pulsatile frequency of LH secretion. Then estradiol was infused into the jugular vein of all animals at a rate of 3 microg/h for 16 h on Day 8 to determine whether an LH surge was induced. Blood samples were collected every 2 h from 4 h before the start of the estradiol infusion until 48 h after the start of the infusion. In each group, the mean +/- SEM concentration after progesterone implant treatment was 3.3 +/- 0.1 ng/ml for the high P group, 1.1 +/- 0.1 ng/ml for the low P group, and <0.1 ng/ml for the non-P group, concentrations similar to the luteal levels, subluteal levels, and follicular phase levels of the normal estrous cycle, respectively. The estradiol concentration ranged from 4 to 8 pg/ml after estradiol capsule implants in all groups. The LH pulse frequency was significantly (P < 0.05) suppressed on Day 3 (6.2 +/- 0.5 pulses/8 h) and on Day 7 (2.6 +/- 0.9 pulses/8 h) relative to Day 0 (9.0 +/- 0.5 pulses/8 h) in the high P group. In both the low P and non-P groups, however, the changes of pulsatile frequency of LH were not significantly different, and high pulses (7-9 pulses/8 h) were maintained on each of the 3 days they were tested. An LH surge (peak concentration, 100.3 +/- 11.0 ng/ml) occurred in all goats in the non-P group, whereas there was no surge mode secretion of LH in either the high P or the low P group. The results of this study support our hypothesis that the threshold levels of progesterone that regulate negative feedback action on the LH pulse and the LH surge are different. Low levels of progesterone, around 1 ng/ml, completely suppressed the LH surge but did not affect the pulsatile frequency of LH secretion.

Neuroendocrine mechanisms responsible for experimental anovulation of hyperandrogenie origin

An experimental model of anovulatory infertility of hyperandrogenic origin (subcutaneous implantation of silastic capsules with testosterone to mature female rats) was used to study morphological, hormonal, and biochemical indices that characterize the hypothalamic-pituitary-ovarian system. Disorders found in the functional metabolism of androgens in the hypothalamus, decreased pituitary response to hypothalamic hormone (HH), changes in the pattern of estrous cycles, ovarian morphological changes suggest that there are neuroendocrine disturbances ovulation regulation. The use of flutamide as a drug of experimental antiandrogenic therapy led to a partial normalization of hormonal, biochemical, and morphological characteristics and to recovery of fertility in female rats with anovulatory fertility.

Effects of chronic restraint stress and estradiol on open field activity, spatial memory, and monoaminergic neurotransmitters in ovariectomized rats

Twenty-one days of chronic restraint stress impairs male rat performance on the radial arm maze [Luine et al. (1994) Brain Res. 639, 167–170], but enhances female rat performance [Bowman et al. (2001) Brain Res. 904, 279–289]. To assess possible ovarian hormone mechanisms underlying this sexually dimorphic response to stress, we examined chronic stress effects in ovariectomized rats. Ovariectomized rats received Silastic capsule implants containing cholesterol or estradiol and were assigned to a daily restraint stress (21 days, 6 h/day) or non-stress group. Following the stress period, subjects were tested for open field activity and radial arm maze performance. Stress and estradiol treatment affected open field activity. All stressed animals, with or without estradiol treatment, made fewer total outer sector crossings. In contrast, estradiol-treated animals, with or without stress, made more inner sector visits, an indication that estradiol decreased anxious behavior on the open field across time. As measured by the total number of visits required to complete the task, stress did not affect radial arm maze performance in ovariectomized rats, but estradiol-treated animals, with or without stress, performed better than non-treated animals on the radial arm maze. Stressed subjects receiving estradiol showed the best radial arm maze performance. Following killing, tissue samples were obtained from various brain regions known to contribute to learning and memory, and monoamine and metabolite levels were measured. Several changes were observed in response to both stress and estradiol. Most noteworthy, stress treatment decreased homovanillic acid levels in the prefrontal cortex, an effect not previously observed in stressed intact females. Estradiol treatment increased norepinephrine levels in CA3 region of the hippocampus, mitigating stress-dependent changes. Both stress and estradiol decreased dentate gyrus levels of 5-hydroxyindole acetic acid. In summary, the current study provides novel information showing that estradiol alters behavioral and neurochemical responses to stress in ovariectomized rats. Estradiol treatment decreased anxious behavior on the open field and stressed animals receiving estradiol had enhanced radial arm maze performance. In relation to interactions between stress and estradiol on cognition and anxiety, changes in the prefrontal cortex dopaminergic system, dentate gyrus serotonergic system, and norepinephrine levels in the CA3 region appear important. Results show that estradiol may moderate stress effects on cognition and anxiety through both organizational and activation effects.

Autocrine regulation of prolactin secretion by endothelins: a permissive role for estradiol.

We have previously found that lactotrophs express and secrete endothelin-like peptides that influence prolactin (PRL) secretion in an autocrine fashion. We have also observed that the incidence of endothelin-immunoreactive lactotrophs is markedly affected by ovarian steroids. In this study, we examined how the ovarian steroid background determines the efficiency of the endothelin-mediated autocrine feedback regulation of PRL secretion. Ovariectomized adult female rats were used throughout these studies. Steroid replacements were made by sc implantation of Silastic capsules immediately following ovariectomy. Eight to 10 wk later, three animals from each treatment group (no steroid control, estradiol, progesterone, estradiol plus progesterone) were sacrificed by decapitation, and the anterior pituitary cells were enzymatically dispersed using collagenase and hyaluronidase. A PRL-specific reverse hemolytic plaque assay was used to measure PRL secretion at the single-cell level. BQ123, a synthetic cyclic pentapeptide with distinctive endothelin-A receptor antagonist quality, caused only a modest elevation of PRL secretion in the control group. Endothelin antagonism did not affect PRL secretion in cells obtained from progesterone-implanted animals. Endothelin antagonism did, however, increase overall PRL secretion in the estradiol and estradiol plus progesterone groups by five- and threefold, respectively. Frequency distribution of PRL plaques in these same two BQ123-treated groups revealed two subpopulations, indicating that lactotrophs differ in their response to endogenous endothelin feedback and that this difference is steroid dependent. These observations clearly suggest that the ovarian steroid milieu (estrogens in particular) can have a profound influence on the self-regulatory mechanisms of lactotrophs. Our results also emphasize that endogenous endothelins may play an important role in the negative feedback regulation of PRL secretion in female rats.

Regulation of Noncontact Erection in Rats by Gonadal Steroids

Male rats exhibit erections in the presence of inaccessible estrous females, and we investigated which gonadal steroids regulate these noncontact erections (NCEs). Sexually experienced Wistar males (n ≥ 8/group) were tested for NCE four times (every 3 days) before castration, after castration, and after receiving subcutaneous implants of 10-mm Silastic capsules that were empty or filled with crystalline testosterone propionate (TP), dihydrotestosterone (DHT), estradiol benzoate (EB), or DHT + EB (10 mm each). Before castration, males responded with NCE in approximately 50% of tests. No males had NCEs after castration, beginning 3 days after surgery. Also, no males responded after treatment with EB or empty capsules. After receiving implants of TP, DHT, or DHT + EB, 50% of males had NCEs, beginning with the first test 3 days after treatment. On every measure of NCE, males treated with DHT or DHT + EB were indistinguishable from each other and from TP-treated males. Among the sexual responses of male rats, NCE appears to be more sensitive than other behaviors to changes in gonadal condition. In its profile of response to gonadal steroids (testosterone+, dihydrotestosterone+, estradiol−), NCE is similar to reflexive erection, for which spinal systems are sufficient, and unlike copulation (T+, DHT−, E+), which depends on discrete areas of the brain. We nonetheless conclude that NCE depends on androgen-sensitive systems in the brain, but androgen-sensitive neurons in the lumbosacral spinal cord may also play a role.

Site-Specific Induction of Fos Immunoreactivity in Preoptic and Hypothalamic NADPH-Positive Neurons during Glucoprivation

Neuronal nitric oxide synthase, e.g. NADPH diaphorase (NADPH-d), catalyzes formation of the free radical, nitric oxide (NO), and occurs within brain structures that have functional significance for energy fuel homeostasis. The following studies examined whether populations of NADPH-d-positive neurons in the hypothalamus and nearby preoptic area express immunoreactivity for the nuclear transcription factor, Fos, in response to glucose substrate imbalance. Eight days after bilateral ovariectomy (OVX) and subcutaneous implantation of silastic capsules containing 30 µg estradiol benzoate/ml, female rats were injected i.p. with the glucose antimetabolite, 2-deoxy-D-glucose (2DG; 400 mg/kg), or the vehicle, saline. The animals were sacrificed by transcardial perfusion 2 h after these treatments. Sections at 150-µm intervals throughout preoptic area and anterior and tuberal regions of the hypothalamus were processed for dual cytoplasmic NADPH-d enzyme activity and nuclear Fos-immunoreactivity (-ir). The glucose antimetabolite elicited expression of nuclear Fos-ir by NADPH-d-positive neurons in several neural structures, including the medial preoptic area, median preoptic nucleus, anterior commissural, periventricular magnocellular supraoptic nucleus, paraventricular nucleus, and medial part of the bed nucleus of the stria terminalis. In contrast, the extensive populations of NADPH-d-positive neurons in the ventromedial hypothalamic nucleus and lateral hypothalamic area showed very little immunolabeling for Fos in response to glucoprivation. This demonstration of nuclear immunoreactivity for Fos suggests that cellular glucopenia elicits the transcriptional activation, via AP-1 regulatory sites, of multiple populations of hypothalamic neurons characterized by the functional capacity to generate NO, and thus that this gaseous neurotransmitter may fulfill a role(s) in central neural mechanisms governing regulation of compensatory motor responses to metabolic imbalance.

Induction of Fos immunoreactivity by acute glucose deprivation in the rat caudal brainstem: relation to NADPH diaphorase localization.

Neuronal nitric oxide (NO) synthase, a NADPH diaphorase (NADPH-d) enzyme, catalyzes formation of the free radical neurotransmitter, NO, and is distributed within several caudal brainstem structures. The following studies investigated whether these neuron populations express immunoreactivity for the inducible nuclear transcription factor, Fos, in response to acute glucose deprivation. Eight days after bilateral ovariectomy and subcutaneous implantation of silastic capsules containing 30 microg estradiol benzoate/ml, adult female rats were injected i.p. with the glucose antimetabolite, 2-deoxy-D-glucose (2DG; 400 mg/kg), or the vehicle, saline, and killed by transcardial perfusion 2 h later. At 150-microm intervals through the midbrain, pons, and medulla 25-microm sections were taken and processed for dual cytoplasmic NADPH-d enzyme activity and nuclear Fos immunoreactivity (Fos-ir). Although NADPH-d-positive neurons were demonstrated in several neural structures, only those in the dorsal raphe nuclei, central subnucleus of the nucleus of the solitary tract, dorsal vagal motor nucleus, lateral paragigantocellular nucleus, nucleus ambiguus, reticular parvocellular nucleus, and medullary A5 noradrenergic cells were colabeled for nuclear Fos-ir following injection of 2DG. While NADPH-d neurons in the midbrain central gray and the latero- and posterodorsal tegmental, lateral parabrachial, motor trigeminal, and gigantocellular nuclei were not immunolabeled for Fos in the 2DG-treated animals, there was a close neuroanatomical proximity between neurons capable of generating NO and others expressing Fos-ir in these sites. These data reveal that only discrete populations of NADPH-d-containing neurons in the caudal brainstem are transcriptionally activated via the Fos stimulus-transcription cascade in response to glucose substrate imbalance, and suggest that NO and/or other neurotransmitters released by these neurons may function as neurochemical mediators of glucoprivic regulatory effects within this part of the brain.