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.

GABAergic input through GABAB receptors is necessary during a perinatal window to shape gene expression of factors critical to reproduction such as Kiss1.

Lack of GABAB receptors in GABAB1 knockout mice decreases neonatal ARC kisspeptin 1 (Kiss1) expression in the arcuate nucleus of the hypothalamus (ARC) in females, which show impaired reproduction as adults. Our aim was to selectively impair GABAB signaling during a short postnatal period to evaluate its impact on the reproductive system. Neonatal male and female mice were injected with the GABAB antagonist CGP 55845 (CGP, 1 mg/kg body wt sc) or saline from postnatal day 2 (PND2) to PND6, three times per day (8 AM, 1 PM, and 6 PM). One group was killed on PND6 for collection of blood samples (hormones by radioimmunoassay), brains for gene expression in the anteroventral periventricular nucleus-periventricular nucleus continuum (AVPV/PeN), and ARC micropunches [quantitative PCR (qPCR)] and gonads for qPCR, hormone contents, and histology. A second group of mice was injected with CGP (1 mg/kg body wt sc) or saline from PND2 to PND6, three times per day (8 AM, 1 PM, and 6 PM), and left to grow to adulthood. We measured body weight during development and parameters of sexual differentiation, puberty onset, and estrous cycles. Adult mice were killed, and trunk blood (hormones), brains for qPCR, and gonads for qPCR and hormone contents were obtained. Our most important findings on PND6 include the CGP-induced decrease in ARC Kiss1 and increase in neurokinin B (Tac2) in both sexes; the decrease in AVPV/PeN tyrosine hydroxylase (Th) only in females; the increase in gonad estradiol content in both sexes; and the increase in primordial follicles and decrease in primary and secondary follicles. Neonatally CGP-treated adults showed decreased ARC Kiss1 and ARC gonadotropin-releasing hormone (Gnrh1) and increased ARC glutamic acid decarboxylase 67 (Gad1) only in males; increased ARC GABAB receptor subunit 1 (Gabbr1) in both sexes; and decreased AVPV/PeN Th only in females. We demonstrate that ARC Kiss1 expression is chronically downregulated in males and that the normal sex difference in AVPV/PeN Th expression is abolished. In conclusion, neonatal GABAergic input through GABAB receptors shapes gene expression of factors critical to reproduction.

Unraveling the connection between GABA and kisspeptin in the control of reproduction.

Neuroendocrine control of reproduction involves the interplay of various factors that become active at some point along development. GnRH is the main neurohormone controlling reproduction and among the most important inputs modulating GnRH synthesis/secretion are GABA and kisspeptins. These interactions of GABA and kisspeptin in the control of GnRH secretion can take place by the presence of the receptors of both factors on the GnRH neuron or alternatively by the actions of GABA on kisspeptin neurons and/or the actions of kisspeptin on GABA neurons. Kisspeptin acts on the Kiss1R, a seven transmembrane domain, Gαq/11-coupled receptor that activates phospholipase C, although some Gαq/11-independent pathways in mediating part of the effects of Kiss1R activation have also been proposed. GABA acts through two kinds of receptors, ionotropic GABAA/C receptors involving a chloride channel and associated with fast inhibitory/stimulatory conductance and metabotropic GABAB receptors (GABABR) that are Gi/0 protein linked inducing late slow hyperpolarization. In this review, we aim to summarize the different ways in which these two actors, kisspeptin and GABA, interact to modulate GnRH secretion across the reproductive lifespan.

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.

Postnatal development of the endocrine pancreas in mice lacking functional GABAB receptors.

Adult mice lacking functional GABAB receptors (GABAB1KO) have glucose metabolism alterations. Since GABAB receptors (GABABRs) are expressed in progenitor cells, we evaluated islet development in GABAB1KO mice. Postnatal day 4 (PND4) and adult, male and female, GABAB1KO, and wild-type littermates (WT) were weighed and euthanized, and serum insulin and glucagon was measured. Pancreatic glucagon and insulin content were assessed, and pancreas insulin, glucagon, PCNA, and GAD65/67 were determined by immunohistochemistry. RNA from PND4 pancreata and adult isolated islets was obtained, and Ins1, Ins2, Gcg, Sst, Ppy, Nes, Pdx1, and Gad1 transcription levels were determined by quantitative PCR. The main results were as follows: 1) insulin content was increased in PND4 GABAB1KO females and in both sexes in adult GABAB1KOs; 2) GABAB1KO females had more clusters (<500 µm(2)) and less islets than WT females; 3) cluster proliferation was decreased at PND4 and increased in adult GABAB1KO mice; 4) increased ß-area at the expense of the α-cell area was present in GABAB1KO islets; 5) Ins2, Sst, and Ppy transcription were decreased in PND4 GABAB1KO pancreata, adult GABAB1KO female islets showed increased Ins1, Ins2, and Sst expression, Pdx1 was increased in male and female GABAB1KO islets; and 6) GAD65/67 was increased in adult GABAB1KO pancreata. We demonstrate that several islet parameters are altered in GABAB1KO mice, further pinpointing the importance of GABABRs in islet physiology. Some changes persist from neonatal ages to adulthood (e.g., insulin content in GABAB1KO females), whereas other features are differentially regulated according to age (e.g., Ins2 was reduced in PND4, whereas it was upregulated in adult GABAB1KO females).

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.

Multiple failures in the lutenising hormone surge generating system in GABAB1KO female mice.

Female mice lacking GABAB receptors, GABAB1KO, show disrupted oestrous cycles, reduced pregnancies and increased hypothalamic Gnrh1 mRNA expression, whereas anteroventral periventricular/periventricular preoptic nucleus (AVPV/PeN) Kiss1 mRNA was not affected. In the present study, we characterise the important components of the gonadotrophic preovulatory surge, aiming to unravel the origin of this reproductive impairment. In GABAB1KO and wild-type (WT) females, we determined: (i) hypothalamic oestrogen receptor (ER)α and ß and aromatase mRNA and protein expression; (ii) ovulation index and oestrus serum follicle-stimulating hormone (FSH) and pituitary Gnrh1r expression; (iii) in ovariectomised-oestradiol valerate-treated mice, we evaluated ex vivo hypothalamic gonadotrophin-releasing hormone (GnRH) pulsatility in the presence/absence of kisspeptin (Kiss-10, constant or pulsatile) and oestradiol (constant); and (iv) in ovariectomised-oestradiol silastic capsule-treated mice (proestrous-like environment), we evaluated morning and evening kisspeptin neurone activation (c-Fos+) and serum luteinising homrone (LH). In the medial basal hypothalamus of oestrus GABAB1KOs, aromatase and ERα mRNA and protein were increased, whereas ERß was decreased. In GABAB1KOs, the ovulation index was decreased together with decreased first oestrus serum FSH and increased pituitary Gnrh1r mRNA. Under constant Kiss-10 stimulation, hypothalamic GnRH pulse frequency did not vary, although GnRH mass/pulse was increased in GABAB1KOs. In WTs, pulsatile Kiss-10 together with constant oestradiol significantly increased GnRH pulsatility, whereas, in GABAB1KOs, oestradiol alone increased GnRH pulsatility and this was reversed by pulsatile Kiss-10 addition. In GABAB1KOs AVPV/PeN kisspeptin neurones were similarly activated (c-Fos+) in the morning and evening, whereas WTs showed the expected, marked evening stimulation. LH correlated with activated kisspeptin cells in WT mice, whereas GABAB1KO mice showed high, similar LH levels both in the morning and evening. Taken together, all of these alterations point to impairment in the trigger of the preovulatory GnRH surge that entails the reproductive alterations described.

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.

Neonatal exposure to bisphenol A alters the hypothalamic-pituitary-thyroid axis in female rats.

Bisphenol A (BPA) is a component of polycarbonate plastics, epoxy resins and polystyrene found in many common products. Several reports revealed potent in vivo and in vitro effects. In this study we analyzed the effects of the exposure to BPA in the hypothalamic-pituitary-thyroid axis in female rats, both in vivo and in vitro. Female Sprague-Dawley rats were injected sc from postnatal day 1 (PND1) to PND10 with BPA: 500 µg 50 µl-1 oil (B500), or 50 µg 50 µl-1 (B50), or 5 µg 50 µl-1 (B5). Controls were injected with 50 µl vehicle during the same period. Neonatal exposure to BPA did not modify TSH levels in PND13 females, but it increased them in adults in estrus. Serum T4 was lower in B5 and B500 with regards to Control, whereas no difference was seen in T3. No significant differences were observed in TRH, TSHß and TRH receptor expression between groups. TSH release from PPC obtained from adults in estrus was also higher in B50 with regard to Control. In vitro 24 h pre-treatment with BPA or E2 increased basal TSH as well as prolactin release. On the other hand, both BPA and E2 lowered the response to TRH. The results presented here show that the neonatal exposure to BPA alters the hypothalamic pituitary-thyroid axis in adult rats in estrus, possibly with effects on the pituitary and thyroid. They also show that BPA alters TSH release from rat PPC through direct actions on the pituitary.

Evaluation of sodium arsenite exposure on reproductive competence in pregnant and postlactational dams and their offspring.

We investigated arsenite exposure on the reproductive axis of dams (during pregnancy and at cyclicity resumption) and their offspring. Pregnant rats were exposed to 5 (A5) or 50ppm (A50) of sodium arsenite in drinking water from gestational day 1 (GD1) until sacrifice at GD18 or two months postpartum. Offspring were exposed to the same treatment as their mothers from weaning to adulthood. A50-pregnant rats gained less weight, showed increased testosterone and estradiol but pregnancy was unaffected. After lactation, arsenic-exposed dams presented compromised cyclicity, decreased estradiol, increased follicle-stimulating hormone (FSH), less preovulatory follicles and presence of ovarian cysts, suggesting impaired reproduction. A50-offspring presented lower body weight; A50-female-offspring showed elevated gonadotropin releasing hormone (GnRH), FSH and testosterone, while A50-males showed diminished GnRH/FSH, but normal testosterone. We conclude that arsenite at the present exposure levels did not compromise pregnancy outcome while it negatively affected reproductive physiology in postpartum dams and their offspring.

Arsenite in drinking water produces glucose intolerance in pregnant rats and their female offspring.

Drinking water is the main source of arsenic exposure. Chronic exposure has been associated with metabolic disorders. Here we studied the effects of arsenic on glucose metabolism, in pregnant and post-partum of dams and their offspring. We administered 5 (A5) or 50 (A50) mg/L of sodium arsenite in drinking water to rats from gestational day 1 (GD1) until two months postpartum (2MPP), and to their offspring from weaning until 8 weeks old. Liver arsenic dose-dependently increased in arsenite-treated rats to levels similar to exposed population. Pregnant A50 rats gained less weight than controls and recovered normal weight at 2MPP. Arsenite-treated pregnant animals showed glucose intolerance on GD16-17, with impaired insulin secretion but normal insulin sensitivity; they showed dose-dependent increased pancreas insulin on GD18. All alterations reverted at 2MPP. Offspring from A50-treated mothers showed lower body weight at birth, 4 and 8 weeks of age, and glucose intolerance in adult females, probably due to insulin secretion and sensitivity alterations. Arsenic alters glucose homeostasis during pregnancy by altering beta-cell function, increasing risk of developing gestational diabetes. In pups, it induces low body weight from birth to 8 weeks of age, and glucose intolerance in females, demonstrating a sex specific response.

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.

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.

Gonadotropins and inhibins along the development of a luteinized rat ovarian tumor.

Luteinized intrasplenic ovarian tumors develop in response to high circulating gonadotropins. The relationship between tumor development, gonadotropins and inhibins was studied. Tumor-bearing animals were sacrificed weekly along the first 6 weeks of development. Inhibins were measured by enzyme-linked immunosorbent assay (ELISA), serum gonadotropins, GH and IGF-1 by RIA. Inhibin subunit mRNAs were determined by Northern blot. Tumor histology was examined. Ovarian grafts grew significantly along development. LH increased ten-fold on week 1; a further significant increment was observed on week 3. FSH peaked on weeks 1 and 2 and fell significantly thereafter. Serum inhibins markedly increased on weeks 3-5. Tumor inhibin A content and mRNA levels for alpha and beta A subunits also increased on week 3. Inverse correlations between inhibins and FSH and direct correlations between inhibins and LH were observed. Tumor inhibin A and IGF-1 contents correlated significantly. Increasing levels of luteinization were observed along tumor development. These luteinized tumors develop mainly in response to LH, since growth continues under FSH inhibition. The active inhibin secretion and the positive correlation between inhibins and LH suggests that LH may be the main driving force behind this production, while growth factors produced by the gonads may also participate in their regulation.

Quantitation of polyamines in hypothalamus and pituitary of female and male developing rats.

The quantitation of four polyamines in hypothalamus and pituitary is studied in male and female developing rats using an improved high-performance liquid chromatography method. In the hypothalamus, putrescine (PUT) reaches the highest concentration (nmol/mg protein) on day 6. It shows the lowest value in comparison with any other polyamine. Spermidine (SPD) is high during the first postnatal days. Spermine (SPM) fluctuates, and agmatine (AGM) is highest during the first week. SPD, SPM and AGM are lower in females. In the pituitary, PUT, SPD and AGM are high during the first week. SPM remains constant and it is higher in males. AGM is higher in males only on day 1. PUT shows the lowest concentration of all. Concentrations of PUT, SPD and SPM are higher in the pituitary; AGM is higher in the hypothalamus. alpha-Difluoromethylornithine (a specific and irreversible inhibitor of ornithine decarboxylase) decreases PUT and SPD, increased SPM and AGM remain unchanged in the hypothalamus and pituitary. Thus, each polyamine has its own pattern in hypothalamus and in pituitary during development in males and females; these changes could be related to the hypothalamic control of pituitary secretion of hormones related to reproduction in mammals.

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.