GABAB Receptor Antagonism from Birth to Weaning Permanently Modifies Kiss1 Expression in the Hypothalamus and Gonads in Mice.

INTRODUCTION: The kisspeptin gene Kiss1 is expressed in two hypothalamic areas: anteroventral periventricular nucleus/periventricular nucleus (AVPV/PeN) and arcuate nucleus (ARC), and also in gonads. Several pieces of evidence suggests that gamma-amino butyric acid B receptors (GABAB) signaling can regulate Kiss1 expression. Here, we inhibited GABAB signaling from PND2 to PND21 and evaluated the hypothalamic-pituitary-gonadal (HPG) axis. METHODS: BALB/c mice were treated on postnatal days 2-21 (PND2-PND21) with CGP55845 (GABAB antagonist) and evaluated in PND21 and adulthood: gene expression (qPCR) in the hypothalamus and gonads, hormones by radioimmunoassay, gonad histochemistry (H&E), puberty onset, and estrous cycles. RESULTS: At PND21, CGP inhibited Kiss1 and Tac2 and increased Pdyn and Gabbr1 in the ARC of both sexes and decreased Th only in female AVPV/PeN. Serum follicle-stimulating hormone (FSH) and testis weight were decreased in CGP-males, and puberty onset was delayed. In adults, Kiss1, Tac2, Pdyn, Pgr, Cyp19a1, and Gad1 were downregulated, while Gabbr1 was upregulated in the ARC of both sexes. In the AVPV/PeN, Kiss1, Th, Cyp19a1, and Pgr were decreased while Gad1 was increased in CGP-females, whereas Cyp19a1 was increased in CGP-males. Serum FSH was increased in CGP-males while prolactin was increased in CGP-females. Testosterone and progesterone were increased in ovaries from CGP-females, in which Kiss1, Cyp19a1, and Esr1 were downregulated while Hsd3b2 was upregulated, together with increased atretic and decreased ovulatory follicles. Testes from CGP-males showed decreased progesterone, increased Gabbr1, Kiss1, Kiss1r, and Esr2 and decreased Cyp19a1, and clear signs of seminiferous tubules atrophy. CONCLUSION: These results demonstrate that appropriate GABAB signaling during this critical prepubertal period is necessary for the normal development of the HPG axis.

Proposed mechanisms for oligonucleotide IMT504 induced diabetes reversion in a mouse model of immunodependent diabetes.

Type 1 diabetes (T1D) originates from autoimmune ß-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 ß-cell recovery, and impaired islet infiltration. IMT504 induced early blood glucose decrease and infiltration inhibition, increased ß-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 ß-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.

Lack of functional GABAB receptors alters Kiss1 , Gnrh1 and Gad1 mRNA expression in the medial basal hypothalamus at postnatal day 4.

BACKGROUND/AIMS: Adult mice lacking functional GABAB receptors (GABAB1KO) show altered Gnrh1 and Gad1 expressions in the preoptic area-anterior hypothalamus (POA-AH) and females display disruption of cyclicity and fertility. Here we addressed whether sexual differentiation of the brain and the proper wiring of the GnRH and kisspeptin systems were already disturbed in postnatal day 4 (PND4) GABAB1KO mice. METHODS: PND4 wild-type (WT) and GABAB1KO mice of both sexes were sacrificed; tissues were collected to determine mRNA expression (qPCR), amino acids (HPLC), and hormones (RIA and/or IHC). RESULTS: GnRH neuron number (IHC) did not differ among groups in olfactory bulbs or OVLT-POA. Gnrh1 mRNA (qPCR) in POA-AH was similar among groups. Gnrh1 mRNA in medial basal hypothalamus (MBH) was similar in WTs but was increased in GABAB1KO females compared to GABAB1KO males. Hypothalamic GnRH (RIA) was sexually different in WTs (males > females), but this sex difference was lost in GABAB1KOs; the same pattern was observed when analyzing only the MBH, but not in the POA-AH. Arcuate nucleus Kiss1 mRNA (micropunch-qPCR) was higher in WT females than in WT males and GABAB1KO females. Gad1 mRNA in MBH was increased in GABAB1KO females compared to GABAB1KO males. Serum LH and gonadal estradiol content were also increased in GABAB1KOs. CONCLUSION: We demonstrate that GABABRs participate in the sexual differentiation of the ARC/MBH, because sex differences in several reproductive genes, such as Gad1, Kiss1 and Gnrh1, are critically disturbed in GABAB1KO mice at PND4, probably altering the organization and development of neural circuits governing the reproductive axis.

Oligonucleotide IMT504 Improves Glucose Metabolism and Controls Immune Cell Mediators in Female Diabetic NOD Mice.

Type 1 diabetes occurs as a consequence of progressive autoimmune destruction of beta cells. A potential treatment for this disease should address the immune attack on beta cells and their preservation/regeneration. The objective of this study was to elucidate whether the immunomodulatory synthetic oligonucleotide IMT504 was able to ameliorate diabetes in NOD mice and to provide further understanding of its mechanism of action. We found that IMT504 restores glucose homeostasis in a diabetes mouse model similar to human type 1 diabetes, by regulating expression of immune modulatory factors and improving beta cell function. IMT504 treatment markedly improved fasting glycemia, insulinemia, and homeostatic model assessment of beta cell function (HOMA-Beta cell) index. Moreover, this treatment increased islet number and decreased apoptosis, insulitis, and CD45+ pancreas-infiltrating leukocytes. In a long-term treatment, we observed improvement of glucose metabolism up to 9 days after IMT504 cessation and increased survival after 15 days of the last IMT504 injection. We postulate that interleukin (IL)-12B (p40), possibly acting as a homodimer, and Galectin-3 (Gal-3) may function as mediators of this immunomodulatory action. Overall, these results validate the therapeutic activity of IMT504 as a promising drug for type 1 diabetes and suggest possible downstream mediators of its immunomodulatory effect.

Lack of functional GABA(B) receptors alters GnRH physiology and sexual dimorphic expression of GnRH and GAD-67 in the brain.

GABA, the main inhibitory neurotransmitter, acts through GABA(A/C) and GABA(B) receptors (GABA(B)Rs); it is critical for gonadotropin regulation. We studied whether the lack of functional GABA(B)Rs in GABA(B1) knockout (GABA(B1)KO) mice affected the gonadotropin axis physiology. Adult male and female GABA(B1)KO and wild-type (WT) mice were killed to collect blood and tissue samples. Gonadotropin-releasing hormone (GnRH) content in whole hypothalami (HT), olfactory bulbs (OB), and frontoparietal cortexes (CT) were determined (RIA). GnRH expression by quantitative real-time PCR (qRT-PCR) was evaluated in preoptic area-anterior hypothalamus (POA-AH), medial basal-posterior hypothalamus (MBH-PH), OB, and CT. Pulsatile GnRH secretion from hypothalamic explants was measured by RIA. GABA, glutamate, and taurine contents in HT and CT were determined by HPLC. Glutamic acid decarboxylase-67 (GAD-67) mRNA was measured by qRT-PCR in POA-AH, MBH-PH, and CT. Gonadotropin content, serum levels, and secretion from adenohypophyseal cell cultures (ACC) were measured by RIA. GnRH mRNA expression was increased in POA-AH of WT males compared with females; this pattern of expression was inversed in GABA(B1)KO mice. MBH-PH, OB, and CT did not follow this pattern. In GABA(B1)KO females, GnRH pulse frequency was increased and GABA and glutamate contents were augmented. POA-AH GAD-67 mRNA showed the same expression pattern as GnRH mRNA in this area. Gonadotropin pituitary contents and serum levels showed no differences between genotypes. Increased basal LH secretion and decreased GnRH-stimulated gonadotropin response were observed in GABA(B1)KO female ACCs. These results support the hypothesis that the absence of functional GABA(B)Rs alters GnRH physiology and critically affects sexual dimorphic expression of GnRH and GAD-67 in POA-AH.

The key action of estradiol and progesterone enables GnRH delivery during gestation in the South American plains vizcacha, Lagostomus maximus.

The South American plains vizcacha, Lagostomus maximus, is the only mammal described so far that shows expression of estrogen receptors (ERs) and progesterone receptors (PRs) in gonadotropin-releasing hormone (GnRH) neurons. This animal therefore constitutes an exceptional model for the study of the effect of steroid hormones on the modulation of the hypothalamic-pituitary-ovarian (HPO) axis. By using both in vivo and ex vivo approaches, we have found that pharmacological doses of progesterone (P4) and estradiol (E2) produced an inhibition in the expression of hypothalamic GnRH, while physiological doses produced a differential effect on the pulsatile release frequency or genomic expression of GnRH. Our ex vivo experiment indicates that a short-term effect of E2 modulates the frequency of GnRH release pattern that would be associated with membrane ERs. On the other hand, our in vivo approach suggests that a long-term effect of E2, acting through the classical nuclear ERs-PRs pathway, would produce the modification of GnRH mRNA expression during the GnRH pre-ovulatory surge. Particularly, P4 induced a rise in GnRH mRNA expression and protein release with a decrease in its release frequency. These results suggest different levels of action of steroid hormones on GnRH modulation. We conclude that the fine action of E2 and P4 constitute the key factor to enable the hypothalamic activity during the pregnancy of this mammal.

Perinatal programming of the orexinergic (hypocretinergic) system in hypothalamus and anterior pituitary by testosterone.

Orexins A/B derived from hypothalamic prepro-orexin (PPO) are agonists for orexin receptors 1 (OX1) and 2 (OX2). Previously, we showed clear sex differences in the hypothalamic-pituitary-gonadal orexinergic system in adult rodents. Here, we studied the effect of sexual brain differentiation on the orexinergic system in neuroendocrine structures regulating reproduction. We evaluated: a: proestrous and neonatally androgenized female rats; b: adult males, untreated or gonadectomized in adulthood and injected with oil or estradiol and progesterone (E2/P4); c: control and demasculinized males (perinatally treated with flutamide and later castration) injected either with oil or E2/P4 in adulthood. Rats were sacrificed at 12:00 and 18:00h; blood samples and brains were collected. Hormones were measured using radioimmunoassay. PPO, OX1 and OX2 mRNAs were quantified by qPCR in medial basal hypothalamus, anterior hypothalamus, adenohypophysis, and cortex. Western blots for OX1 were done in the same structures. In normal females, gonadotropins surged at 18:00h coinciding with significant elevations of PPO, OX1 and OX2 mRNAs and OX1 protein in hypothalamus and pituitary; no increases were observed at noon. Afternoon changes were absent in masculinized females. Demasculinized males when treated with E2/P4 showed high PPO, OX1 and OX2 mRNAs and OX1 protein expression in hypothalamus and pituitary at 12:00 and 18:00h compared vehicle-treated controls. The same steroid treatment was ineffective in males with normal brain masculinization. Here we show that neonatal testosterone shapes the sexual differences in the hypothalamic-pituitary orexinergic system in synchronicity to establishing the brain sex differences of the reproductive axis. The female brain controls gonadotropin surges and concurrent elevations of all studied components of the orexinergic system, suggesting its participation as a possible link between food intake, behavior and hormonal control of reproduction.

Estradiol-Dependent and -Independent Stimulation of Kiss1 Expression in the Amygdala, BNST, and Lateral Septum of Mice.

Kisspeptin, encoded by Kiss1, activates reproduction by stimulating GnRH neurons. Although most Kiss1 neurons are located in the hypothalamus, smaller Kiss1 populations also reside in the medial amygdala (MeA), bed nucleus of the stria terminalis (BnST), and lateral septum (LS). However, very little is known about the regulation and function of these extra-hypothalamic Kiss1 neurons. This study focused on the roles and interactions of two signaling factors, estradiol (E2) and GABA, known to stimulate and inhibit, respectively, extra-hypothalamic Kiss1 expression. First, using estrogen receptor (ER)α knockout (KO) and ßERKO mice, we demonstrated that Kiss1 in both the BnST and LS is stimulated by E2, as occurs in the MeA, and that this E2 upregulation occurs via ERα, but not ERß. Second, using GABABR KO and wild-type mice, we determined that whereas E2 normally increases extra-hypothalamic Kiss1 levels, such upregulation by E2 is further enhanced by the concurrent absence of GABABR signaling in the MeA and LS, but not the BnST. Third, we demonstrated that when GABABR signaling is absent, the additional removal of gonadal sex steroids does not abolish Kiss1 expression in the MeA and BnST, and in some cases the LS. Thus, Kiss1 expression in these extra-hypothalamic regions is not solely dependent on E2 stimulation. Finally, we demonstrated a significant positive correlation between Kiss1 levels in the MeA, BnST, and LS, but not between these regions and the hypothalamus (anteroventral periventricular nucleus/periventricular nucleus). Collectively, our findings indicate that both E2 and GABA independently regulate all three extra-hypothalamic Kiss1 populations, but their regulatory interactions may vary by brain region and additional yet-to-be-identified factors are likely involved.

Immunomodulatory oligonucleotide IMT504: Effects on mesenchymal stem cells as a first-in-class immunoprotective/immunoregenerative therapy.

The immune responses of humans and animals to insults (i.e., infections, traumas, tumoral transformation and radiation) are based on an intricate network of cells and chemical messengers. Abnormally high inflammation immediately after insult or abnormally prolonged pro-inflammatory stimuli bringing about chronic inflammation can lead to life-threatening or severely debilitating diseases. Mesenchymal stem cell (MSC) transplant has proved to be an effective therapy in preclinical studies which evaluated a vast diversity of inflammatory conditions. MSCs lead to resolution of inflammation, preparation for regeneration and actual regeneration, and then ultimate return to normal baseline or homeostasis. However, in clinical trials of transplanted MSCs, the expectations of great medical benefit have not yet been fulfilled. As a practical alternative to MSC transplant, a synthetic drug with the capacity to boost endogenous MSC expansion and/or activation may also be effective. Regarding this, IMT504, the prototype of a major class of immunomodulatory oligonucleotides, induces in vivo expansion of MSCs, resulting in a marked improvement in preclinical models of neuropathic pain, osteoporosis, diabetes and sepsis. IMT504 is easily manufactured and has an excellent preclinical safety record. In the small number of patients studied thus far, IMT504 has been well-tolerated, even at very high dosage. Further clinical investigation is necessary to demonstrate the utility of IMT504 for resolution of inflammation and regeneration in a broad array of human diseases that would likely benefit from an immunoprotective/immunoregenerative therapy.

Adenohypophyseal and hypothalamic GABA B receptor subunits are downregulated by estradiol in adult female rats.

Gamma-aminobutyric acid (GABA) participates in neuroendocrine regulation. Since steroid hormones have been shown to modulate the GABAergic system, here we evaluated the effect of chronic in vivo estradiol administration on GABA B receptor (GABA(B)R) expression. GABA(B1) and GABA(B2) subunits were analyzed by Western Blot and RT-PCR, in hypothalami and anterior pituitaries of adult female rats: a) treated for 1 week with estradiol-valerate (a single dose of 100 mug /kg: E1), b) implanted with a 10 mg pellet of estradiol-benzoate for 5 weeks (E5) or c) on proestrous (P), d) ovariectomized (OVX). Pituitary GABA(B)R levels were correlated to a biological effect: baclofen, a GABA(B)R agonist, action on intracellular calcium titers ([Ca(2+)](i)) in pituitary cells. E5 pituitaries showed a significant decrease in the expression of GABA(B1) and GABA(B2) mRNAs compared to P. The GABA(B1a) splice variant of GABA(B1) was always more abundant than GABA(B1b) in this tissue. Similar to the pituitary, hypothalamic GABA(B1) and GABA(B2) mRNAs decreased in E5; this was confirmed at the protein level. In the hypothalamus GABA(B1b) was the main variant expressed in P rats, and was the one significantly sensitive to estradiol-induced decrease, as determined by Western Blots. Castration did not modify GABA(B)R expression with regards to P in either tissue. In P pituitary cells baclofen induced a decrease in [Ca(2+)](i), in contrast this effect was lost in E5 cells. We conclude that chronic estradiol treatment negatively regulates the expression of the GABA(B)R subunits in the pituitary and the hypothalamus. This effect is coupled to a loss of baclofen action on intracellular calcium in pituitary cells.

Hyperprolactinemia induced by hCG leads to metabolic disturbances in female mice.

The metabolic syndrome is a growing epidemic; it increases the risk for diabetes, cardiovascular disease, fatty liver, and several cancers. Several reports have indicated a link between hormonal imbalances and insulin resistance or obesity. Transgenic (TG) female mice overexpressing the human chorionic gonadotropin ß-subunit (hCGß+ mice) exhibit constitutively elevated levels of hCG, increased production of testosterone, progesterone and prolactin, and obesity. The objective of this study was to investigate the influence of hCG hypersecretion on possible alterations in the glucose and lipid metabolism of adult TG females. We evaluated fasting serum insulin, glucose, and triglyceride levels in adult hCGß+ females and conducted intraperitoneal glucose and insulin tolerance tests at different ages. TG female mice showed hyperinsulinemia, hypertriglyceridemia, and dyslipidemia, as well as glucose intolerance and insulin resistance at 6 months of age. A 1-week treatment with the dopamine agonist cabergoline applied on 5-week-old hCGß+ mice, which corrected hyperprolactinemia, hyperandrogenism, and hyperprogesteronemia, effectively prevented the metabolic alterations. These data indicate a key role of the hyperprolactinemia-induced gonadal dysfunction in the metabolic disturbances of hCGß+ female mice. The findings prompt further studies on the involvement of gonadotropins and prolactin on metabolic disorders and might pave the way for the development of new therapeutic strategies.

Expression of gamma-aminobutyric acid B receptor subunits in hypothalamus of male and female developing rats.

GABA and its receptors show particular ontogenic distributions in different rat brain areas. Recently, GABAB receptors (GBR) have been described to assemble as heterodimers formed by a GBR1a/b and a GBR2 subunit. Here, the ontogeny of rat GBRs and the pattern of subunit expression in both sexes were determined in the hypothalamus, a critical area for homeostatic regulation. Male and female rats were sacrificed at 1, 4, 12, 20, 28, 38 days of life and at adulthood and hypothalami were removed and frozen. Western blots analysis for GBR1 and GBR2 subunits showed that both were expressed in male and female hypothalamic membranes from day 1 to adulthood. In females, both GBR1a and GBR1b were maximally expressed in newborns and decreased towards adulthood. At birth, expression of GBR1a was significantly higher than GBR1b, while at 38 days, GBR1b was more abundant. In males, GBR1a and GBR1b expression was higher in young animals and decreased gradually showing adult levels between the second and third weeks of age without differences between isoforms. Comparing GBR1 variants levels in hypothalamus between sexes, GBR1a was significantly more abundant in females at birth while at 38 days its expression was higher in males; GBR1b showed no sex differences along development. GBR2 was detected in hypothalami of females and males at all ages; maximum levels were observed at 12 days and adult levels were attained at 38 days, without sex differences. This is the first report on the ontogeny of hypothalamic GABAB receptors in male and female rats, with a particular developmental pattern of subunit and isoform expression and presenting some sex differences.

Gonadotropin-releasing hormone signaling pathways in an experimental ovarian tumor.

Previous results showed that GnRH signaling is altered in cells from rat luteinized ovarian tumors (tumor group) because it did not activate the phospholipase C pathway, in contrast to control ovarian cells from superovulated prepubertal rats (SPO). In the present work, alternate GnRH-induced second messengers such as phospholipase A(2) and phospholipase D activation, cAMP production, ERK1/2 phosphorylation, and the presence of G proteins were evaluated to determine GnRH mechanism of action in tumor cells. G proteins examined were present in both cell types. Buserelin, a GnRH agonist, (1, 10, and 100 ng/ml) increased phosphatidylethanol in SPO, indicating phospholipase D activation. Only 100 ng/ml buserelin induced a significant response in the tumor group. Buserelin (100 ng/ml) increased (3)H-arachidonic acid in culture media in SPO, indicating phospholipase A(2) activation; no effect was observed in the tumor group. Buserelin (100 and 1000 ng/ml) induced pertussis toxin-insensitive cAMP increases in both cell types, with similar potencies. In the tumor group, buserelin (100 ng/ml) inhibited human chorionic gonadotropin-induced cAMP and progesterone; this effect was protein kinase C (PKC) dependent (inhibited by GF109203X, a PKC inhibitor). Buserelin (100 and 1000 ng/ml) induced ERK1/2 phosphorylation in both cell kinds. Buserelin-induced ERK1/2 activation was G(i/0) independent and PKC dependent. Only in the tumor group, buserelin-induced ERK1/2 activation was cAMP dependent (abolished by SQ 22536, the adenylyl cyclase inhibitor). Furthermore, dibutyryl cAMP-induced ERK1/2 activation in the tumor group was PKC dependent (inhibited by GF109203X). In conclusion, activation of phospholipases in tumor cells does not seem to mediate GnRH effects. GnRH signaling seems to involve adenylyl cyclase activation, PKC stimulation, and ERK1/2 phosphorylation.

Effects of polyamines on the release of gonadotropin-releasing hormone and gonadotropins in developing female rats.

Polyamines, putrescine (PUT), spermidine (SPD), spermine (SPM), and agmatine (AGM), are polycationic amines related to multiple cell functions found in high concentrations during the development of hypothalamus and pituitary. In previous works, we demonstrated that alpha-difluoromethylornithine (DFMO), an inhibitor of polyamines biosynthesis, induced a delay in puberty of female rats, accompanied by high, sustained follicle-stimulating hormone (FSH) levels during the infantile period. Also, DFMO treatment induced changes in polyamine concentration both in hypothalamus and pituitary of rats, mainly a decrease of PUT and SPD, an increase in SPM, and no change in AGM. In the present work, we investigated the direct effects of polyamines on the secretion of hypothalamic GnRH and pituitary gonadotropins in 6- and 15-day-old female rats. In 6-day-old animals, in vitro incubations with PUT, SPD, and AGM of hypothalami or anterior pituitaries were able to inhibit GnRH, FSH, and leutinizing hormone (LH) secretion, respectively. SPM showed a nonspecific transient inhibitory effect on FSH. When challenged with either high K(+) (hypothami) or GnRH (pituitaries), the tissues incubated in the presence of polyamines showed no differences when compared with their controls. No effects of polyamines in 15-day-old rats in either tissue were observed. Pituitary cell cultures of 6-day-old animals incubated with DFMO for 4 days showed a significant increase in FSH, but not in LH. We conclude that high PUT, SPD, and AGM levels during the first 10 days of life are important for the development of the hypothalamic-hypophyseal unit, probably related to an inhibitory effect on GnRH and gonadotropins. Therefore, polyamine participation, especially PUT and SPD, is of importance in the regulation of GnRH and gonadotropin secretion in the neonatal and infantile periods, critical stages in the establishment of sexual differentiation.

Impaired GABAB receptor signaling dramatically up-regulates Kiss1 expression selectively in nonhypothalamic brain regions of adult but not prepubertal mice.

Kisspeptin, encoded by Kiss1, stimulates reproduction and is synthesized in the hypothalamic anteroventral periventricular and arcuate nuclei. Kiss1 is also expressed at lower levels in the medial amygdala (MeA) and bed nucleus of the stria terminalis (BNST), but the regulation and function of Kiss1 there is poorly understood. γ-Aminobutyric acid (GABA) also regulates reproduction, and female GABAB1 receptor knockout (KO) mice have compromised fertility. However, the interaction between GABAB receptors and Kiss1 neurons is unknown. Here, using double-label in situ hybridization, we first demonstrated that a majority of hypothalamic Kiss1 neurons coexpress GABAB1 subunit, a finding also confirmed for most MeA Kiss1 neurons. Yet, despite known reproductive impairments in GABAB1KO mice, Kiss1 expression in the anteroventral periventricular and arcuate nuclei, assessed by both in situ hybridization and real-time PCR, was identical between adult wild-type and GABAB1KO mice. Surprisingly, however, Kiss1 levels in the BNST and MeA, as well as the lateral septum (a region normally lacking Kiss1 expression), were dramatically increased in both GABAB1KO males and females. The increased Kiss1 levels in extrahypothalamic regions were not caused by elevated sex steroids (which can increase Kiss1 expression), because circulating estradiol and testosterone were equivalent between genotypes. Interestingly, increased Kiss1 expression was not detected in the MeA or BNST in prepubertal KO mice of either sex, indicating that the enhancements in extrahypothalamic Kiss1 levels initiate during/after puberty. These findings suggest that GABAB signaling may normally directly or indirectly inhibit Kiss1 expression, particularly in the BNST and MeA, and highlight the importance of studying kisspeptin populations outside the hypothalamus.

Elevated hypothalamic aromatization at the onset of precocious puberty in transgenic female mice hypersecreting human chorionic gonadotropin: effect of androgens.

Transgenic female mice overexpressing the α- and ß- subunits of human chorionic gonadotropin (hCGαß+) exhibited precocious puberty, as evidenced by early vaginal opening. Chronically elevated hCG in 21-day-old hCGαß+ females stimulated gonadal androgen production, which exerted negative feedback over the endogenous gonadotropin synthesis, and activated the hypothalamic GnRH pulsatility and gene expression. Transgenic females also exhibited elevated hypothalamic aromatization in the preoptic area (POA), which is the sexually-differentiated area that controls the LH surge in adulthood. Ovariectomy at 14 days of age was unable to rescue this phenotype. However, the blockade of androgen action by flutamide from postnatal day 6 onwards reduced the aromatase levels in the POA of hCGαß+ females. Our results suggest that early exposure of females to androgen action during a critical period between postnatal days 6-14 induces sex-specific organizational changes of the brain, which affect the aromatase expression in the POA at the onset of precocious puberty.

Effects of orexins A and B on expression of orexin receptors and progesterone release in luteal and granulosa ovarian cells.

Orexin-A and orexin-B are neuropeptides controlling sleep-wakefulness, feeding and neuroendocrine functions via their G protein-coupled receptors, orexin-1R and orexin-2R. They are synthesized in the lateral hypothalamus and project throughout the brain. Orexins and orexin receptors have also been described outside the brain. Previously we demonstrated the presence of both receptors in the ovary, their increased expression during proestrous afternoon and the dependence on the gonadotropins. Here we studied the effects of orexins on the mRNA expression of both receptors, by quantitative real-time PCR, on luteal cells from superovulated rat ovaries and granulosa cells from diethylstilbestrol-treated rat ovaries. Effects on progesterone secretion were also measured. In luteal cells, 1 nM of either orexin-A or orexin-B decreased progesterone secretion. Orexin-A treatment increased expression of both orexin-1R and orexin-2R mRNA. The effect on orexin-1R mRNA expression was abolished by an orexin-1R selective receptor antagonist SB-334867 and the effect on orexin-2R mRNA expression was abolished by a selective orexin-2R antagonist JNJ-10397049. Orexin-B did not modify orexin-1R mRNA expression, but increased orexin-2R mRNA expression. The effect of orexin-B on orexin-2R was abolished by a selective orexin-2R antagonist. Neither the expression of orexin receptors nor progesterone secretions by granulosa cells were affected by orexins. FSH, as positive control, increased both steroid hormones secretion, but did not induce the expression of OX receptors in granulosa cells isolated from late preantral/early antral follicles. Finally in ovaries obtained immediately after sacrifice, the expression of orexin-1R and orexin-2R was higher in superovulated rat ovaries compared to control or diethylstilbestrol treated rat ovaries. A selective presence and function of both orexinergic receptors in luteal and granulosa cells is described, suggesting that the orexinergic system may have a functional role in the ovary.

Effects of GABAB receptor agonists and antagonists on glycemia regulation in mice.

γ-Aminobutyric acid (GABA) inhibits insulin secretion through GABA(B) receptors in pancreatic ß-cells. We investigated whether GABA(B) receptors participated in the regulation of glucose homeostasis in vivo. BALB/c mice acutely pre-injected with the GABA(B) receptor agonist baclofen (7.5mg/kg, i.p.) presented glucose intolerance and diminished insulin secretion during a glucose tolerance test (GTT, 2g/kg body weight, i.p.). The GABA(B) receptor antagonist 2-hydroxysaclofen (15 mg/kg, i.p.) improved the GTT and reversed the baclofen effect. Also a slight increase in insulin secretion was observed with 2-hydroxysaclofen. In incubated islets 1.10(-5)M baclofen inhibited 20mM glucose-induced insulin secretion and this effect was reversed by coincubation with 1.10(-5)M 2-hydroxysaclofen. In chronically-treated animals (18 days) both the receptor agonist (5mg/kg/day i.p.) and the receptor antagonist (10mg/kg/day i.p.) induced impaired GTTs; the receptor antagonist, but not the agonist, also induced a decrease in insulin secretion. No alterations in insulin tolerance tests, body weight and food intake were observed with the treatments. In addition glucagon, insulin-like growth factor I, prolactin, corticosterone and growth hormone, other hormones involved in glucose metabolism regulation, were not affected by chronic baclofen or 2-hydroxysaclofen. In islets obtained from chronically injected animals with baclofen, 2-hydroxysaclofen or saline (as above), GABA(B2) mRNA expression was not altered. Results demonstrate that GABA(B) receptors are involved in the regulation of glucose homeostasis in vivo. Treatment with receptor agonists or antagonists, given acutely or chronically, altered glucose homeostasis and insulin secretion alerting to the need to evaluate glucose metabolism during the clinical use of these drugs.

Endogenously elevated androgens alter the developmental programming of the hypothalamic-pituitary axis in male mice.

Transgenic male mice that express human chorionic gonadotropin (hCG) α and ß subunits constitutively hypersecrete hCG and produce elevated levels of androgens. The aim of this study was to characterize the hypothalamic-pituitary function of these transgenic (hCGαß+) males by focusing on FSH regulation. Serum FSH levels and pituitary mRNA expression of Fshb, Lhb, Cga, Gnrhr and Esr1 were reduced, whereas Fst expression was increased in prepubertal hCGαß+ males as compared with wild-type. In the hypothalamus, Cyp19a1 expression, GnRH concentration and ex-vivo GnRH pulsatility were elevated in prepubertal hCGαß+ mice, whereas Kiss1 expression was decreased prepubertally and Gad67 expression was elevated neonatally. The effect of androgens on the developmental programming of the hypothalamic-pituitary axis of hCGαß+ males was evaluated by perinatal and prepubertal antiandrogen (flutamide) administration. Our studies identified a critical window between gestational day 18 and postnatal day 14, during which chronically elevated androgens and/or their locally produced metabolites activate the hypothalamus and concomitantly shut-down the gonadotropin axis.

GABA(B) 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 GABA(A) and GABA(C) (ionotropic receptors) and GABA(B) (metabotropic receptor). Here we reviewed the participation of GABA(B) receptors in the regulation of the hypothalamic-pituitary-gonadal axis, using physiological, biochemical, and pharmacological approaches in rats, as well as in GABA(B1) knock-out mice, that lack functional GABA(B) receptors. Our general conclusion indicates that GABA(B )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 GABA(B) receptor activation, as demonstrated in the rat and also in the GABA(B1) knock-out mouse. In addition, hypothalamic and pituitary GABA(B) receptor expression is modulated by steroid hormones. GABA participation in the brain control of pituitary secretion through GABA(B) receptors depends on physiological conditions, being age and sex critical factors.These results indicate that patients receiving GABA(B) agonists/antagonists should be monitored for possible endocrine side effects.