Deletion of GABAB receptors from Kiss1 cells affects glucose homeostasis without altering reproduction in male mice.

Kisspeptin and γ-amino butyric acid (GABA), synthesized in the central nervous system, are critical for reproduction. Both are also expressed in peripheral organs/tissues critical to metabolic control (liver/pancreas/adipose). Many kisspeptin neurons coexpress GABAB receptors (GABABR) and GABA controls kisspeptin expression and secretion. We developed a unique mouse lacking GABABR exclusively from kisspeptin cells/neurons (Kiss1-GABAB1KO) to evaluate the impact on metabolism/reproduction. We confirmed selective deletion of GABABR from Kiss1 cells in the anteroventral periventricular nucleus/periventricular nucleus continuum (AVPV/PeN; immunofluorescence and PCR) and arcuate nucleus (ARC), medial amygdala (MeA), pituitary, liver, and testes (PCR). Young Kiss1-GABAB1KO males were fertile, with normal LH and testosterone. Kiss1 expression was similar between genotypes in AVPV/PeN, ARC, MeA, bed nucleus of the stria terminalis (BNST), and peripheral organs (testis, liver, pituitary). Kiss1-GABAB1KO males presented higher fasted glycemia and insulin levels, an impaired response to a glucose overload, reduced insulin sensitivity, and marked insulin resistance. Interestingly, when Kiss1-GABAB1KO males got older (9 mo old) their body weight (BW) increased, in part due to an increase in white adipose tissue (WAT). Old Kiss1-GABAB1KO males showed higher fasted insulin, increased pancreatic insulin content, insulin resistance, and significantly decreased pancreatic kisspeptin levels. In sum, lack of GABABR specifically in Kiss1 cells severely impacts glucose homeostasis in male mice, reinforcing kisspeptin involvement in metabolic regulation. These alterations in glucose homeostasis worsened with aging. We highlight the impact of GABA through GABABR in the regulation of the pancreas kisspeptin system in contrast to liver kisspeptin that was not affected.NEW & NOTEWORTHY We developed a unique mouse lacking GABAB receptors specifically in Kiss1 cells to evaluate the impact on reproduction and metabolism. Knockout males showed a severe impact on glucose homeostasis, which worsened with aging. These results reinforce the proposed kisspeptin involvement in metabolic regulation and highlight the impact of GABA through GABABR in the regulation of the peripheral pancreas kisspeptin system.

Persistently expressed human chorionic gonadotropin induces premature luteinization and progressive alterations on the reproductive axis in female mice.

The hypothalamic-pituitary-gonadal axis plays a fundamental role in the endocrine regulation of the reproductive function in mammals. Any change in the function of the participating hormones or their receptors can lead to alterations in sexual differentiation, the onset of puberty, infertility, cancer development, and other dysfunctions. In this study, we analyzed the influence of persistently elevated levels of the human chorionic gonadotropin hormone (hCG), a powerful agonist of pituitary luteinizing hormone (LH), on the reproductive axis of female mice. As a consequence of chronic hCG hypersecretion through a global expression of the hCGbeta-subunit in transgenic (TG) female mice, a series of events perturbed the prepubertal to juvenile transition. The imbalance in gonadotropin action was first manifested by precocious puberty and alterations in gonadal hormone production, with the consequent ovarian function disruption and infertility in adulthood. The expansion of cumulus cells in vivo and in vitro, ovulatory capacity, and gene expression of ovulation-related marker genes after hormone stimulation were normal in 3-week-old TG females. However, the expression of genes related to steroidogenesis and luteinization such as Lhcgr, Prlr, and the steroidogenic enzymes Cyp11a1, Cyp17a1, and Cyp19a1 were significantly elevated in the TG females. This study demonstrates that the excessive secretion of hCG in concert with high prolactin, induced premature luteinization, and enhanced ovarian steroidogenesis, as was shown by the up-regulation of luteal cell markers and progesterone synthesis in the TG mice. Furthermore, progressively impaired reproductive function of the TG females occurred from the peripubertal stage to adulthood, thus culminating in infertility.

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.

Participation of TRPV1 in the activity of the GnRH system in male rats.

GnRH neuron activity is under the influence of multiple stimuli, including those coming from the endocannabinoid and the immune systems. Since it has been previously suggested that some of the main elements controlling the GnRH pulse generator possess the TRPV1 receptor, the aim of the present study was to evaluate the participation of the hypothalamic TRPV1, through its pharmacological blockade, in the activity of the hypothalamic-pituitary-testicular axis in male rats under basal or acute inflammatory conditions. Our hypothesis was based on the idea that the hypothalamic TRPV1 participates in the synthesis of the main neuromodulatory signals controlling GnRH, and therefore the reproductive axis. Our results showed that the hypothalamic TRPV1 blockade induced pro-inflammatory effects by increasing Tnfα and Il-1ß mRNA hypothalamic levels and inhibited the reproductive axis by affecting Gnrh, Kiss1 and Rfrp3 mRNA levels and decreasing plasma levels of luteinizing hormone and testosterone under basal conditions, without significant additive effects in rats exposed to systemic LPS. Altogether, these results suggest that the hypothalamic TRPV1 receptor participates in the regulation of the GnRH system, probably by modulating immune-dependent mechanisms.

Mammary gland-specific regulation of GNRH and GNRH-receptor gene expression is likely part of a local autoregulatory system in female vizcachas (Rodentia: Chinchillidae).

In addition to key mammotrophic hormones such as the pituitary prolactin (PRL) and the ovarian steroids progesterone and estradiol, there are local factors that modulate the tissue dynamics of the mammary glands during pregnancy and lactation. By immunohistochemistry and RT-PCR, we found local transcription and translation of gonadotropin-releasing hormone (GNRH), GNRH receptor (GNRHR), PRL and PRL receptor (PRLR) in mammary glands of adult vizcachas during pregnancy and lactation. Both GNRH and GNRHR showed a lag between protein expression and gene transcription throughout the gestational period: while the highest transcription levels of these genes were recorded at early-pregnancy, the epithelial immunoexpressions of both showed their maximum during lactation. RIA results corroborated the presence of GNRH in mammary glands at all the analyzed stages and confirmed the maximum amount of this peptide in the lactating group. Significant amounts of GNRH were detected in milk samples as well. Conversely, PRL and PRLR shared similar protein and gene expression profiles, all exhibiting maximum values during lactation. GNRH peptide content in mammary glands of females with sulpiride-induced hyperprolactinemia (HP) was significantly lower than that of control females (CT). Although PRL mRNA levels remained unchanged, there was a marked increase in theα-lactalbumin (LALBA) transcription in mammary glands of HP- vs CT-females. These results suggest that after targeting mammary glands, PRL stimulates the expression of milk protein genes, but also, tempers the local expression of GNRH. Mammary gland-explantssupplemented with a GNRH analogue (GN-explants) had no differences in terms of PRLR orLALBA transcription levels compared to CT-explants, so the mammary PRLR signaling would not appear to be modulated by GNRH. Yet, mRNA expression levels of both GNRH and the GNRHR-downstream factor, EGR1, were significantly higher in GN-explants compared to that of CT which would point to a GNRH-positive feedback mechanism. In summary, the local coupled expression of GNRH, GNRHR and EGR1 in the mammary gland throughout pregnancy of vizcachas, the PRL-dependent mammary GNRH secretion as well as the GNRH positive feedback on its own transcription suggest an autocrine-paracrine regulatory mechanism and propose an active role for GNRH in mammary gland tissue remodeling.

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.

Pituitary estrogen receptor alpha is involved in luteinizing hormone pulsatility at mid-gestation in the South American plains vizcacha, Lagostomus maximus (Rodentia, Caviomorpha).

The South American plains vizcacha, Lagostomus maximus, is a caviomorph rodent native from Argentina, Bolivia and Paraguay. It shows peculiar reproductive features like pre-ovulatory follicle recruitment during pregnancy with an ovulatory process at around mid-gestation. We have described the activation of the hypothalamic - pituitary - ovarian (HPO) axis during pregnancy. A progressive decrease of progesterone (P4) at mid-pregnancy elicits the delivery of gonadotropin-releasing hormone (GnRH) with the consequent secretion of follicle stimulating hormone (FSH) and estradiol (E2) followed by luteinizing hormone (LH) release resulting in follicular luteinization and the P4 concentration recover. Pituitary gland is the central regulator of the HPO axis being E2 a key hormone involved in the regulation of its activity. In this work we analyzed the action of E2 on the pituitary response to the GnRH wave as well as its involvement on LH secretion at mid-gestation in L. maximus. The expression of GnRHR at the pituitary pars distalis showed a significant decrease at mid-pregnancy compared to early- and term-gestating females. ERα showed a significant increment from mid-gestation whereas ERß did not show variations throughout pregnancy; whereas the LH expression in the pituitary pars distalis showed a significant increase at mid-gestation, concordantly with serum LH, which was followed by a decrease at term-gestation with similar values than at early-pregnancy. The number of cells with co-localization of ERα and GnRHR showed a decline at mid-pregnancy related to early- and term-gestation, whereas the cells with co-localization of ERα and LH increased at mid- and term-pregnancy. On the other hand, ex vivo measuring of LH pulsatility showed a significant increment in the total mass of LH delivered at mid-pregnancy followed by a decrease at term-gestation. The stimulation of ERα with the PPT specific agonist induced a significant increment in the total mass of LH released, whereas no changes were determined when ERß was stimulated with its specific agonist MPP. These results suggest that LH pulsatility rise at mid-pregnancy would be enabled by the increase of E2 acting through ERα.

Sequence analysis, tissue distribution and molecular physiology of the GnRH preprogonadotrophin in the South American plains vizcacha (Lagostomus maximus).

Gonadotropin-releasing hormone (GnRH) is the regulator of the hypothalamic-hypophyseal-gonadal (HHG) axis. GnRH and GAP (GnRH-associated protein) are both encoded by a single preprohormone. Different variants of GnRH have been described. In most mammals, GnRH is secreted in a pulsatile manner that stimulates the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The South-American plains vizcacha, Lagostomus maximus, is a rodent with peculiar reproductive features including natural poly-ovulation up to 800 oocytes per estrous cycle, pre-ovulatory follicle formation throughout pregnancy and an ovulatory process which takes place at mid-gestation and adds a considerable number of secondary corpora lutea. Such features should occur under a special modulation of the HHG axis, guided by GnRH. The aim of this study was to sequence hypothalamic GnRH preprogonadotrophin mRNA in the vizcacha, to compare it with evolutionarily related species and to identify its expression, distribution and pulsatile pattern of secretion. The GnRH1variant was detected and showed the highest homology with that of chinchilla, its closest evolutionarily related species. Two isoforms of transcripts were identified, carrying the same coding sequence, but different 5' untranslated regions. This suggests a sensitive equilibrium between RNA stability and translational efficiency. A predominant hypothalamic localization and a pulsatile secretion pattern of one pulse of GnRH every hour were found. The lower homology found for GAP, also among evolutionarily related species, depicts a potentially different bioactivity.

Variation in progesterone receptors and GnRH expression in the hypothalamus of the pregnant South American plains vizcacha, Lagostomus maximus (Mammalia, Rodentia).

In mammals, elevated levels of progesterone (P4) throughout gestation maintain a negative feedback over the hypothalamic-hypophyseal-gonadal (H-H-G) axis, avoiding preovulatory follicular growth and preventing ovulation. Recent studies showed that in the South American plains vizcacha (Lagostomus maximus) folliculogenesis progresses to preovulatory stages during gestation, and an ovulatory process seems to occur at midgestation. The aim of this work was to analyze hypothalamic gonadotropin-releasing hormone (GnRH) and P4 receptors (PR) expression and luteinizing hormone (LH) secretion and correlate these with the functional state of the ovary in nonovulating and ovulating females and gestating females with special emphasis in the supposedly ovulating females at midgestation. We investigated P4 and LH serum levels as well as the distribution, localization, and expression of PR and GnRH in the hypothalamus of L. maximus at different time points during gestation and in nongestating, ovulating and nonovulating, females. A significant increment in GnRH, P4, and LH was detected in midpregnant vizcachas with respect to early-pregnant and to ovulating females. PR was also significantly increased in midpregnant animals. PR was detected in neurons of the preoptic and hypothalamic areas. Coexistence of both PR and GnRH in neurons of medial preoptic area and supraoptic nucleus was detected. Midpregnant animals showed increased number of PR immunoreactive cells at median eminence, localized adjacently to GnRH immunoreactive fibers. High expression of hypothalamic GnRH and PR, despite an increased level of P4, was correlated with the presence of antral, preovulatory follicles, and luteinized unruptured follicles at midgestation that suggest a possible role of the H-H-G axis in the modulation of ovulation during gestation in L. maximus.

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.

Melatonin is involved in the modulation of the hypothalamic and pituitary activity in the South American plains vizcacha, Lagostomus maximus.

Melatonin, the key messenger of photoperiodic information, is synthesized in the pineal gland by arylalkylamine N-acetyltransferase enzyme (AANAT). It binds to specific receptors MT1 and MT2 located in the hypothalamus and pituitary gland. Melatonin can modulate the reproductive axis affecting the secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). The South American plains vizcacha, Lagostomus maximus, shows natural poliovulation of up to 800 oocytes per estrous cycle, a 154-day long pregnancy, and reactivation of the reproductive axis at mid-gestation with pre-ovulatory follicular recruitment, presence of active corpora lutea, and variations of the endocrine status. Here we analyzed the involvement of melatonin in the modulation of the hypothalamic and pituitary gland physiology of vizcacha thorough several approaches, including histological localization of melatoninergic system components, assessment of melatoninergic components expression throughout the reproductive cycle, and evaluation of the effect of melatonin on hypothalamic and pituitary activities during the follicular and luteal phases of the estrous cycle. AANAT and melatonin receptors were localized in the pineal gland and preoptic area of the hypothalamus. Increase in pineal AANAT and serum melatonin expression was observed as pregnancy progressed, with the lowest hypothalamic MT1 and MT2 levels at mid-pregnancy. Pulsatility assays demonstrated that melatonin induces GnRH and LH secretion at luteal phase. The melatoninergic system effects on hypothalamic and pituitary gland hormones secretion during pregnancy pinpoint to melatonin as a potential key factor underlying the reactivation of the reproductive axis activity at mid-gestation.

Neuroprotective Effects of Testosterone in Male Wobbler Mouse, a Model of Amyotrophic Lateral Sclerosis.

Patients suffering of amyotrophic lateral sclerosis (ALS) present motoneuron degeneration leading to muscle atrophy, dysphagia, and dysarthria. The Wobbler mouse, an animal model of ALS, shows a selective loss of motoneurons, astrocytosis, and microgliosis in the spinal cord. The incidence of ALS is greater in men; however, it increases in women after menopause, suggesting a role of sex steroids in ALS. Testosterone is a complex steroid that exerts its effects directly via androgen (AR) or Sigma-1 receptors and indirectly via estrogen receptors (ER) after aromatization into estradiol. Its reduced-metabolite 5α-dihydrotestosterone acts via AR. This study analyzed the effects of testosterone in male symptomatic Wobblers. Controls or Wobblers received empty or testosterone-filled silastic tubes for 2 months. The cervical spinal cord from testosterone-treated Wobblers showed (1) similar androgen levels to untreated control and (2) increased levels of testosterone, and its 5α-reduced metabolites, 5α- dihydrotestosterone, and 3ß-androstanediol, but (3) undetectable levels of estradiol compared to untreated Wobblers. Testosterone-treated controls showed comparable steroid concentrations to its untreated counterpart. In testosterone- treated Wobblers a reduction of AR, ERα, and aromatase and high levels of Sigma-1 receptor mRNAs was demonstrated. Testosterone treatment increased ChAT immunoreactivity and the antiinflammatory mediator TGFß, while it lessened vacuolated motoneurons, GFAP+ astrogliosis, the density of IBA1+ microgliosis, proinflammatory mediators, and oxidative/nitrosative stress. Clinically, testosterone treatment in Wobblers slowed the progression of paw atrophy and improved rotarod performance. Collectively, our findings indicate an antiinflammatory and protective effect of testosterone in the degenerating spinal cord. These results coincided with a high concentration of androgen-reduced derivatives after testosterone treatment suggesting that the steroid profile may have a beneficial role on disease progression.

Chronic exposure to low concentrations of chlorpyrifos affects normal cyclicity and histology of the uterus in female rats.

Chlorpyrifos (CPF), the most used insecticide in Argentina, can act as an endocrine disruptor at low doses. We previously demonstrated that chronic exposure to CPF induces hormonal imbalance in vivo. The aim of this work was to study the effects of low concentrations of CPF (0.01 and 1 mg/kg/day) on the reproductive system of virgin adult rats. In the ovary, we studied the effects of CPF on steroidogenesis by determining steroid hormone content by RIA and CYP11 and CYP19 enzyme expression by qRT-PCR. The estrous cycle was evaluated by microscopic observation of vaginal smear, as well as by changes in uterine histology. In endometrium, we determined the fractal dimension and expression of PCNA, ERα and PR by IHC. Our results showed that chronic exposure to CPF affects ovarian steroid synthesis, causing alterations in the normal cyclicity of animals. In addition, CPF induced proliferative changes in the uterus, suggesting that it could affect reproduction or act as a risk factor in the development of uterine proliferative pathologies.

Fetal Programming Effects of a Mild Food Restriction During Pregnancy in Mice: How Does It Compare to Intragestational Ghrelin Administration?

To explore in mice if a 15% food restriction protocol during pregnancy programs the offspring postnatal development, with emphasis on reproductive function, and to assess if ghrelin (Ghrl) administration to mouse dams exerts effects that mimic those obtained under mild caloric restriction. Mice were 15% food-restricted, injected with 4 nmol/animal/day of Ghrl, or injected with the vehicle (control) thorough pregnancy. After birth, the pups did not receive further treatment. Pups born from food-restricted dams (FR pups) were lighter than Ghrl pups at birth, but reached normal weight at adulthood. Ghrl pups were heavier at birth and gained more weight than control pups (C pups). This effect was not associated with plasma IGF-1. FR pups showed a delay in pinna detachment and eye opening, while an advance was observed in Ghrl pups. FR pups showed also impairment in the surface-righting reflex. In both female FR and Ghrl pups, there was an advance in vaginal opening and, in adulthood, FR pups showed a significant decrease in their own litter size and plasma progesterone, and an increase in embryo loss. A delay in testicular descent was evident in male Ghrl pups. Changes in puberty onset were not associated with differences in the expression of Kiss1 in hypothalamic nuclei. Finally, in adulthood, FR pups showed a significant decrease in sperm quality. In conclusion, a mild food restriction thorough gestation exerted programming effects on the offspring, affecting also their reproductive function in adulthood. These effects were not similar to those of intragestational Ghrl administration.

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.

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.

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.

Local production of neurostradiol affects gonadotropin-releasing hormone (GnRH) secretion at mid-gestation in Lagostomus maximus (Rodentia, Caviomorpha).

Females of the South American plains vizcacha, Lagostomus maximus, show peculiar reproductive features such as massive polyovulation up to 800 oocytes per estrous cycle and an ovulatory process around mid-gestation arising from the reactivation of the hypothalamic-hypophyseal-ovary (H.H.O.) axis. Estradiol (E2) regulates gonadotropin-releasing hormone (GnRH) expression. Biosynthesis of estrogens results from the aromatization of androgens by aromatase, which mainly occurs in the gonads, but has also been described in the hypothalamus. The recently described correlation between GnRH and ERα expression patterns in the hypothalamus of the vizcacha during pregnancy, with coexpression in the same neurons of the medial preoptic area, suggests that hypothalamic synthesis of E2 may affect GnRH neurons and contribute with systemic E2 to modulate GnRH delivery during the gestation. To elucidate this hypothesis, hypothalamic expression and the action of aromatase on GnRH release were evaluated in female vizcachas throughout pregnancy. Aromatase and GnRH expression was increased significantly in mid-pregnant and term-pregnant vizcachas compared to early-pregnant and nonpregnant females. In addition, aromatase and GnRH were colocalized in neurons of the medial preoptic area of the hypothalamus throughout gestation. The blockage of the negative feedback of E2 induced by the inhibition of aromatase resulted in a significant increment of GnRH-secreted mass by hypothalamic explants. E2 produced in the same neurons as GnRH may drive intracellular E2 to higher levels than those obtained from systemic circulation alone. This may trigger for a prompt GnRH availability enabling H.H.O. activity at mid-gestation with ovulation and formation of accessory corpora lutea with steroidogenic activity that produce the necessary progesterone to maintain gestation to term and guarantee the reproductive success.