María S. Bianchi

Neonatal exposure to bisphenol a alters reproductive parameters and gonadotropin releasing hormone signaling in female rats.

Background Bisphenol A (BPA) is a component of polycarbonate plastics, epoxy resins, and polystyrene and is found in many products. Several reports have revealed potent in vivo effects, because BPA acts as an estrogen agonist and/or antagonist and as an androgen and thyroid hormone antagonist. Objectives We analyzed the effects of neonatal exposure to BPA on the reproductive axis of female Sprague-Dawley rats. Methods Female rats were injected subcutaneusly, daily, from postnatal day 1 (PND1) to PND10 with BPA [500 μg/50 μL (high) or 50 μg/50 μL (low)] in castor oil or with castor oil vehicle alone. We studied body weight and age at vaginal opening, estrous cycles, and pituitary hormone release in vivo and in vitro, as well as gonadotropin-releasing hormone (GnRH) pulsatility at PND13 and in adults. We also analyzed two GnRH-activated signaling pathways in the adults: inositol-triphosphate (IP3), and extracellular signal-regulated kinase1/2 (ERK1/2). Results Exposure to BPA altered pituitary function in infantile rats, lowering basal and GnRH-induced luteinizing hormone (LH) and increasing GnRH pulsatility. BPA dose-dependently accelerated puberty onset and altered estrous cyclicity, with the high dose causing permanent estrus. In adults treated neonatally with BPA, GnRH-induced LH secretion in vivo was decreased and GnRH pulsatility remained disrupted. In vitro, pituitary cells from animals treated with BPA showed lower basal LH and dose-dependently affected GnRH-induced IP3 formation; the high dose also impaired GnRH-induced LH secretion. Both doses altered ERK1/2 activation. Conclusions Neonatal exposure to BPA altered reproductive parameters and hypothalamic–pituitary function in female rats. To our knowledge, these results demonstrate for the first time that neonatal in vivo BPA permanently affects GnRH pulsatility and pituitary GnRH signaling.

Guinea Pig Gonadotropin-Releasing Hormone: Expression Pattern, Characterization and Biological Activity in Rodents

Gonadotropin-releasing hormone (GnRH) is a decapeptide widely known for its role in regulating vertebrate reproduction by serving as a signal from the hypothalamus to pituitary gonadotropes. The first form of GnRH to be identified was isolated from mammals (mGnRH) and the same form has been reported for all mammals studied, which includes marsupials and placental mammals. Later, another variant, chicken GnRH-II (cGnRH-II) was shown to be expressed together with mGnRH in the brains of all jawed vertebrates, including mammals such as rats, monkeys and humans. Our objective was to characterize a third form of GnRH that was isolated previously as mRNA from guinea pigs (gpGnRH), but has not been reported for any other mammal to date. Furthermore, the gonadotropic activity of gpGnRH has not been fully characterized. Our results, using chromatographical and immunological methods, show for the first time that gpGnRH is expressed together with mGnRH in some rodents (wild guinea pig and capybara), but not in others (mouse and hamster). Also, the gonadotropic activity of gpGnRH and mGnRH was tested in two different rat cell culture systems. Although there have been reports that the salmon(s) form of GnRH is present in mammals, we did not detect sGnRH in capybara, wild guinea pigs, hamsters, rats or mice. Taken together with previous reports, the present results support the idea that the expression of multiple GnRH variants in a single species is a common pattern in most vertebrate groups.

GABAB Receptors in Anterior Pituitary Cells

Fil: Lux, Victoria Adela R.. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Biologia y Medicina Experimental. Fundacion de Instituto de Biologia y Medicina Experimental. Instituto de Biologia y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaThe activation of pituitary GABA_B receptors by the specific agonist baclofen inhibits pituitary hormone secretion in vitro. Here we studied the mechanism of action of GABA_B receptors in rat adenohypophysis. Anterior pituitary cells were obtained by trypsinization and were either plated for hormonal studies and cAMP determination or incubated in FURA 2AM for calcium measurements. Baclofen (BACL: 1·10^–5 M) significantly inhibited basal and thyrotropic releasing hormone (TRH)-stimulated (1·10^–7 M) PRL secretion in anterior pituitary cells from proestrous rats. In the presence of pertussis toxin (PTX: 150 ng/ml, 20 h), which leads to the uncoupling of the G_i/o-protein from the receptor, both effects of BACL were abolished while the effect of dopamine (DA: 1·10^–8 M), used as an inhibitory control, was reduced from 70 to 25%. PTX also reversed BACL-induced inhibition of gonadotropin-releasing hormone (GnRH)-elicited luteinizing hormone (LH) secretion in anterior pituitary cells from 15-day-old female rats. In addition, though working in a pituitary mixed cell population, in which only some cell types possess GABA_B receptors, BACL (1·10^–5 M) attenuated the forskolin-induced (0.5 µM) increase in cAMP. This effect was prevented by co-incubation with the antagonist 2 hydroxysaclofen and by preincubation with PTX. BACL (5· 10^–5 M) and DA (5·10^–7 M) inhibited basal intracellular calcium concentrations ([Ca²⁺]_i) in pituitary cells and the effect of the latter was significantly stronger. The effect of BACL on [Ca²⁺]_i was abolished after preincubation with PTX. In the presence of the potassium channel blocking agents barium (200 µM and 1 mM) and tetraethylammonium (10 mM), BACL was still able to inhibit [Ca²⁺]_i. Blockade of voltage-sensitive calcium channels (VSCC) with either verapamil (5·10^–6 M) or nifedipine (1·10^–6 M) completely abolished the effect of BACL on [Ca²⁺]_i. In the presence of 12.5 mM potassium concentration baclofen significantly inhibited [Ca²⁺]_i. In conclusion, our results describe the negative coupling of adenohypophyseal GABA_B receptors to VSCC through PTX-sensitive G-proteins. These characteristics suggest a resemblance of these receptors to the typical presynaptic GABA_B sites described in the central nervous system.

Metformin regulates ovarian angiogenesis and follicular development in a female polycystic ovary syndrome rat model.

Polycystic ovary syndrome (PCOS) is a frequent pathology that affects more than 5% of women of reproductive age. Among other heterogeneous symptoms, PCOS is characterized by abnormalities in angiogenesis. Metformin has been introduced in the treatment of PCOS to manage insulin resistance and hyperglycemia. Besides its metabolic effects, metformin has been shown to improve ovulation, pregnancy and live birth rates in PCOS patients. In the present study, we used a dehydroepiandrosterone-induced PCOS rat model to analyze the effect of metformin administration on ovarian angiogenesis. We found that metformin was able to restore the increased levels of vascular endothelial growth factor, angiopoietin (ANGPT)1, and ANGPT1/ANGPT2 ratio and the decreased levels of platelet-derived growth factor B and platelet-derived growth factor D observed in the dehydroepiandrosterone-treated rats. These effects could take place, at least in part, through a decrease in the levels of serum insulin. We also found an improvement in follicular development, with a lower percentage of small follicles and cysts and a higher percentage of antral follicles and corpora lutea after metformin administration. The improvement in ovarian angiogenesis is likely to restore the accumulation of small follicles observed in PCOS rats and to reduce cyst formation, thus improving follicular development and the percentage of corpora lutea. These results open new insights into the study of metformin action not only in glucose metabolism but also in ovarian dysfunction in PCOS women.

Effect of Androgens on Sexual Differentiation of Pituitary Gamma-Aminobutyric Acid Receptor Subunit GABAB Expression

Previous work demonstrated a sexually dimorphic ontogenic expression of γ-aminobutyric acid receptors (GABABR) in rat pituitary. As sex steroids determine sex-specific expression patterns, we now studied the effect of sex hormones on pituitary GABABR expression. GABABR subunits, measured by Western blot and by semi-quantitative RT-PCR and luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone measured by RIA were determined in two experimental designs: First experimental design: 8- and 15-day-old females (8F, 15F); 8F and 15F treated with 100 µg testosterone propionate (TP) on day 1 of life (8F100TP, 15F100TP), 8- and 15-day-old males (8M, 15M) and 8M and 15M castrated on day 1 (8MC, 15MC). Second experimental design: 8-day-old female and male animals: 8F, 8F100TP, 8F treated with 1 µg/day TP on days 1–4 (8F1TP), 8F treated with the androgen antagonist Flutamide (Flut: 2.5 mg/100 g BW of pregnant mother on days E17-E23) (8F-Flut), 8M, 8MC, 8M treated with Flut as above (8M-Flut) and 8MC-Flut. In these animals, in addition, GABA, glutamate, aspartate and taurine were measured by HPLC in hypothalami and cortex. In the first set of experiments, GABAB1R mRNA/protein expression was higher in 8F than in 15F, 8M or 15M. In 8F100TP, GABAB1R mRNA/protein decreased to male levels. TP treatment did not alter GABAB1R expression in 15F. There was no difference in GABAB1R expression between 8M and 15M and neonatal castration did not modify its expression. In the second set of experiments, TP (1 µg) or Flut did not modify GABAB1R in 8F, while 100 µg TP continued to decrease GABAB1R expression. In 8M, Flut, alone or with castration, increased GABAB1R mRNA/protein expression to 8F. Hypothalamic GABA content followed the same pattern as pituitary GABABR expression in 8-day-old animals, suggesting a cross-regulation. With regard to hormonal levels, 100 µg, but not 1 µg TP altered gonadotropins at 8 days, although both treatments effectively androgenized females as evidenced by lack of cycling. We conclude that androgens, acting pre- and postnatally, decrease pituitary GABABR subunit expression.

Development of an experimental ovarian tumor over a year in the rat

Tumor growth, possible malignant transformation or metastatic propagation and hormonal patterns were evaluated over a year in luteoma induced by introducing an ovary into the spleen of ovariectomized 60 day-old rats. Sham castrated animals had a piece of muscle inserted into the spleen. Jugular blood samples were taken monthly. After a year animals were cycled and decapitated. Troncal blood was collected, autopsies were performed and luteoma were measured and fixed in 10% buffered formalin. Serum LH, FSH, PRL, estradiol and progesterone were measured. Serum inhibin content was determined in one month-old tumors-bearing animals and estrous rats as controls. After one year no external changes in tumor-bearing rats were observed, nor differences in body weight or mortality rates compared to Sham animals. Metastatic propagation was absent. Routine histological examination showed two types of tumors according to either granulosa or luteal cell predomination, tumor type did not determine hormonal patterns. However, a clear relationship between gonadotropin levels and tumor size was established. Low gonadotropins: Small tumors, 18.7% of cases and high gonadotropins: Large tumors, 81.3%. In Sham animals gonadotropins attained castrate levels and remained elevated until the end of the experiment. In the Small group no increases in gonadotropins or estradiol were detected, progesterone and PRL fluctuated. In the Large tumor group LH increased to Sham titers until month 7, then fell to initial levels, FSH augmented significantly as from month three and remained high up to month 5. No variations in either estradiol, progesterone or PRL were observed. Serum inhibin of one month-old tumor-bearing rats was significantly elevated, justifying the lack of FSH increase at this time point. We conclude that these luteoma do not suffer malignant transformation or induce metastases. They appear in two histological types. Tumor size depends on hormonal patterns. The delay in the initial increase and the sharp decrease observed in FSH in animals bearing Large tumors suggest a possible role for inhibin in this regulation.

GABAB Receptors in Neuroendocrine Regulation

Gamma-amino butyric acid (GABA), in addition to being a metabolic intermediate and the main inhibitory neurotransmitter in the synaptic cleft, is postulated as a neurohormone, a paracrine signaling molecule, and a trophic factor. It acts through pre- and post-synaptic receptors, named GABAA and GABAC (ionotropic receptors) and GABAB (metabotropic receptor). Here we reviewed the participation of GABAB receptors in the regulation of the hypothalamic-pituitary-gonadal axis, using physiological, biochemical, and pharmacological approaches in rats, as well as in GABAB1 knock-out mice, that lack functional GABAB receptors. Our general conclusion indicates that GABAB receptors participate in the regulation of pituitary hormone secretion acting both in the central nervous system and directly on the gland. PRL and gonadotropin axes are affected by GABAB receptor activation, as demonstrated in the rat and also in the GABAB1 knock-out mouse. In addition, hypothalamic and pituitary GABAB receptor expression is modulated by steroid hormones. GABA participation in the brain control of pituitary secretion through GABAB receptors depends on physiological conditions, being age and sex critical factors.These results indicate that patients receiving GABAB agonists/antagonists should be monitored for possible endocrine side effects.

In vivo xenoestrogenic actions of cadmium and arsenic in anterior pituitary and uterus

Cadmium (Cd) and arsenic (iAs) are toxic metals ubiquitously present in the environment. Both pollutants exert nonmonotonic dose responses, being mostly cytotoxic at high concentrations but mimicking estrogen (E2) effects at low doses. Xenoestrogenic activity of Cd and iAs has been demonstrated in different hormone-dependent tumor cell lines; however, their actions in vivo remain largely unknown. Here, we investigated whether in vivo administration of low doses of Cd and iAs through drinking water would display xenoestrogenic effects in the anterior pituitary gland and uterus of ovariectomized rats. Cd (1ppm) and iAs (0.1ppm) exposure increased the wet weight of anterior pituitary gland and uterus and induced proestrus- and estrus-like vaginal smears. Both metals stimulate cell proliferation of these tissues as they increased the expression of proliferation markers. More importantly, they augmented soluble guanylyl cyclase α1 subunit expression, which has been linked to hormone-dependent tumor progression. Also, Cd and iAs modified protein levels of full-length estrogen receptor α and its truncated variants in an E2-like manner. Anterior pituitary hormone secretion was differentially affected by both metals. Luteinizing hormone synthesis and release were strongly diminished after Cd exposure and only mildly reduced by iAs. Both metals were able to increase prolactin synthesis, although only iAs augmented serum prolactin levels. This study shows for the first time that Cd and iAs exert strong xenoestrogenic effects on anterior pituitary gland at low doses. The differences between Cd and iAs E2-like behavior indicate that other Cd- and iAs-specific mechanisms could be involved. Altogether, these results contribute to the knowledge of reproductive disorders associated with Cd and iAs environmental contamination.

In Vivo and In Vitro Arsenic Exposition Induces Oxidative Stress in Anterior Pituitary Gland.

Inorganic arsenic (iAs) is at the top of toxic metalloids. Inorganic arsenic-contaminated water consumption is one of the greatest environmental health threats worldwide. Human iAs exposure has been associated with cancers of several organs, neurological disorders, and reproductive problems. Nevertheless, there are no reports describing how iAs affects the anterior pituitary gland. The aim of this study was to investigate the mechanisms involved in iAs-mediated anterior pituitary toxicity both in vivo and in vitro. We showed that iAs administration (from 5 to 100 ppm) to male rats through drinking water increased messenger RNA expression of several oxidative stress-responsive genes in the anterior pituitary gland. Serum prolactin levels diminished, whereas luteinizing hormone (LH) levels were only affected at the higher dose tested. In anterior pituitary cells in culture, 25 µmol/L iAs significantly decreased prolactin release in a time-dependent fashion, whereas LH levels remained unaltered. Cell viability was significantly reduced mainly by apoptosis evidenced by morphological and phosphatidylserine externalization studies. This process is characterized by early depolarization of mitochondrial membrane potential and increased levels of reactive oxygen species. Expression of some key oxidative stress-responsive genes, such as heme oxygenase-1 and metallothionein-1, was also stimulated by iAs exposure. The antioxidant N-acetyl cysteine prevented iAs-induced effects on the expression of oxidative stress markers, prolactin release, and apoptosis. In summary, the present work demonstrates for the first time that iAs reduces prolactin release both in vivo and in vitro and induces apoptosis in anterior pituitary cells, possibly resulting from imbalanced cellular redox status.

PROPOSED MECHANISMS FOR OLIGONUCLEOTIDE IMT504 INDUCED DIABETES REVERSION IN A MOUSE MODEL OF IMMUNODEPENDENT DIABETES

Type 1 diabetes (T1D) originates from autoimmune β-cell destruction. IMT504 is an immunomodulatory oligonucleotide that increases mesenchymal stem cell cloning capacity and reverts toxic diabetes in rats. Here, we evaluated long-term (20 doses) and short-term (2-6 doses) effects of IMT504 (20 mg·kg(-1)·day(-1) sc) in an immunodependent diabetes model: multiple low-dose streptozotocin-injected BALB/c mice (40 mg·kg(-1)·day(-1) ip for 5 consecutive days). We determined blood glucose, glucose tolerance, serum insulin, islet morphology, islet infiltration, serum cytokines, progenitor cell markers, immunomodulatory proteins, proliferation, apoptosis, and islet gene expression. IMT504 reduced glycemia, induced β-cell recovery, and impaired islet infiltration. IMT504 induced early blood glucose decrease and infiltration inhibition, increased β-cell proliferation and decreased apoptosis, increased islet indoleamine 2,3-dioxygenase (IDO) expression, and increased serum tumor necrosis factor and interleukin-6 (IL-6). IMT504 affected islet gene expression; preproinsulin-2, proglucagon, somatostatin, nestin, regenerating gene-1, and C-X-C motif ligand-1 cytokine (Cxcl1) increased in islets from diabetic mice and were decreased by IMT504. IMT504 downregulated platelet endothelial cell adhesion molecule-1 (Pecam1) in islets from control and diabetic mice, whereas it increased regenerating gene-2 (Reg2) in islets of diabetic mice. The IMT504-induced increase in IL-6 and islet IDO expression and decreased islet Pecam1 and Cxcl1 mRNA expression could participate in keeping leukocyte infiltration at bay, whereas upregulation of Reg2 may mediate β-cell regeneration. We conclude that IMT504 effectively reversed immunodependent diabetes in mice. Corroboration of these effects in a model of autoimmune diabetes more similar to human T1D could provide promising results for the treatment of this disease.