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Introduction: Male sexual potency and vigor are a complex neuroendocrine process and an important component of well-being. Psychological stress is one of the leading causes of male impotence worldwide. Therefore, to better understand the effects of psychological stress on male sexual potency, vigor, and the physiology of erection, we used the rat restraint stress (RS) model, which can most aptly simulate psychological stress. Methods: Adult male SD rats were exposed to RS for 1.5 or 3 h/day for 30 days. Neuromodulators and hormones of sexual potency and penile erection were quantified using ELISA kit. The histoarchitecture of the penis was examined using Masson trichrome staining. Immunoblotting and immunofluorescence were used to assess the expression and immunolocalization patterns of penile erection markers. To assess sexual potency and vigor, a noncontact erection and a copulatory test were performed. Results: RS exposure decreased the circulatory levels of gonadotropins and testosterone while increasing the serum corticosterone level. RS exposure altered the histomorphology of the penis by decreasing the smooth muscle/collagen ratio and increasing oxidative stress in penile tissue. Furthermore, RS adversely affected NO availability for penile erection by decreasing the neurotransmitter acetylcholine and other erection facilitatory markers such as p-Akt, nNOS, eNOS, and cGMP, while increasing the inhibitory marker PDE5α in the penis. RS exposure significantly reduced the frequencies of mount, intromission, and ejaculation, whereas it prolonged sexual exhaustion by increasing latencies of postejaculatory mount, intromission, and ejaculation. Conclusion: The current findings suggest that psychological stressors, such as RS, cause erectile dysfunction in adult male rats by modulating the hypothalamic-pituitary-testicular axis, oxidative balance, penile fibrosis, and the NO/cGMP/PDE5α pathway of penile erection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background/Aims: Ovarian theca cell hyperandrogenism in women with polycystic ovary syndrome (PCOS) is compounded by androgen receptor-mediated impairment of estradiol and progesterone negative feedback regulation of episodic luteinizing hormone (LH) release. The resultant LH hypersecretion, likely the product of accelerated episodic release of gonadotropin-releasing hormone (GnRH) from the median eminence of the hypothalamus, hyperstimulates ovarian theca cell steroidogenesis, enabling testosterone (T) and androstenedione excess. Prenatally androgenized (PA) female monkeys exposed to fetal male levels of T during early-to-mid gestation, when adult, demonstrate PCOS-like traits, including high T and LH levels. This study tests the hypothesis that progesterone resistance-associated acceleration in episodic LH release contributes to PA monkey LH excess. Methods: A total of 4 PA and 3 regularly cycling, healthy control adult female rhesus monkeys of comparable age and body mass index underwent (1) a 10 h, frequent intravenous sampling assessment for LH episodic release, immediately followed by (2) IV infusion of exogenous GnRH to quantify continuing pituitary LH responsiveness, and subsequently (3) an SC injection of a progesterone receptor antagonist, mifepristone, to examine LH responses to blockade of progesterone-mediated action. Results: Compared to controls, the relatively hyperandrogenic PA females exhibited ~100% increase (p = 0.037) in LH pulse frequency, positive correlation of LH pulse amplitude (p = 0.017) with androstenedione, ~100% greater increase (p = 0.034) in acute (0–10 min) LH responses to exogenous GnRH, and an absence (p = 0.008) of modest LH elevation following acute progesterone receptor blockade suggestive of diminished progesterone negative feedback. Conclusion: Such dysregulation of LH release in PCOS-like monkeys implicates impaired feedback control of episodic release of hypothalamic GnRH reminiscent of PCOS neuroendocrinopathy. [ABSTRACT FROM AUTHOR]

Background: Male testosterone levels decline by 1% per year from the age of 40 years. Whilst a primary testicular deficit occurs, hypothalamic or pituitary dysregulation may also coexist. This study aimed to compare the hypothalamic response to kisspeptin-54 and the pituitary response to gonadotropin-releasing hormone (GnRH) of older men with those of young men. Methods: Following 1 h of baseline sampling, healthy older men (n = 5, mean age 59.3 ± 2.9 years) received a 3-h intravenous infusion of either vehicle, kisspeptin-54 0.1, 0.3, or 1.0 nmol/kg/h or GnRH 0.1 nmol/kg/h, on five different study days. Serum gonadotropins and total testosterone were measured every 10 min and compared to those of young men (n = 5/group) (mean age 28.9 ± 2.0 years) with a similar body mass index (24 kg/m2) who underwent the same protocol. Results: Kisspeptin-54 and GnRH significantly stimulated serum gonadotropin release in older men compared to vehicle (p < 0.001 for all groups). Gonadotropin response to kisspeptin-54 was at least preserved in older men when compared to young men. At the highest dose of kisspeptin-54 (1.0 nmol/kg/h), a significantly greater luteinising hormone (LH) (p = 0.003) response was observed in older men. The follicle-stimulating hormone (FSH) response to GnRH was increased in older men (p = 0.002), but the LH response was similar (p = 0.38). Serum testosterone rises following all doses of kisspeptin-54 (p ≤ 0.009) were reduced in older men. Conclusions: Our data suggest that healthy older men without late-onset hypo­gonadism (LOH) have preserved hypothalamic response to kisspeptin-54 and pituitary response to GnRH, but impaired testicular response. Further work is required to investigate the use of kisspeptin-54 to identify hypothalamic deficits in men with LOH. [ABSTRACT FROM AUTHOR]

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. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Reproductive function in mammals, defined as the capacity to generate viable male and female gametes, and to support pregnancy and lactation selectively in the female, is sensitive to the metabolic state of the organism. This contention, long assumed on the basis of intuitive knowledge, became formulated on a scientific basis only recently, with the identification of a number of neuroendocrine signals which crucially participate in the joint control of energy balance and reproduction. A paradigmatic example in this context is the adipocyte-derived hormone, leptin; a satiety factor which signals the amount of body energy (fat) stores not only to the circuits controlling food intake but also to a number of neuroendocrine axes, including the reproductive system. More recently, the reproductive dimension of another metabolic hormone, namely the orexigenic stomach-secreted peptide, ghrelin, has been disclosed by observations on its putative roles in the control of gonadal function and gonadotropin secretion. Of note, leptin and ghrelin have been proposed to act as reciprocal regulators of energy homeostasis. However, their potential interplay in the control of reproduction remains largely unexplored. Based on the comparison of the biological actions of leptin and ghrelin at different levels of the hypothalamic-pituitary-gonadal axis, reviewed in detail herein, we propose that, through concurrent or antagonistic actions, the leptin-ghrelin pair is likely to operate also as modulator of different reproductive functions, thereby contributing to the physiological integration of reproduction and energy balance. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Unraveling of the master role of kisspeptins, the products of the KiSS-1 gene, and their receptor, GPR54, in the control of reproduction has been a major breakthrough in contemporary neuroendocrinology. Indeed, since the disclosure of their reproductive dimension in late 2003, an ever-growing number of genetic, molecular, physiologic and pharmacological studies have defined the crucial role of KiSS-1 neurons as central processors for the dynamic regulation of the gonadotropic axis and its full activation at puberty. Yet, the potential role of the hypothalamic KiSS-1 system as an intermediary factor for the well-known interplay between energy status and reproduction initially received little attention. Recent data, however, strongly suggest a prominent role of KiSS-1 in the metabolic control of fertility, as expression of KiSS-1 gene at the hypothalamus is down-regulated in conditions of negative energy balance and kisspeptin administration is capable of overcoming the hypogonadotropic state observed in undernutrition and disturbed metabolic conditions. Leptin, the adipocyte hormone signaling the size of body energy stores, is likely to play a pivotal role in the metabolic control of the KiSS-1 system, since kisspeptin neurons express leptin receptors and leptin is able to normalize defective KiSS-1 gene expression in models of impaired gonadotropin secretion linked to hypoleptinemia, such as the ob/ob mouse and streptozotocin-induced diabetic rat. In sum, these data provide strong evidence for a central role of kisspeptins and GPR54 as molecular conduits for the metabolic regulation of reproductive function – a phenomenon with potential physiopathologic and therapeutic implications. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

A reproductive facet of ghrelin, a stomach-derived orexigenic peptide involved in energy homeostasis, has been recently suggested, and predominantly inhibitory effects of ghrelin upon luteinizing hormone (LH) secretion have been demonstrated in rat models. Yet, the modulatory actions of ghrelin on the gonadotropic axis remain scarcely evaluated. We report herein a detailed analysis of the effects of ghrelin upon LH and follicle-stimulating hormone (FSH) secretion in the female rat, using a combination of in vivo and in vitro approaches. Intracerebroventricular administration of ghrelin (3 nmol/rat) evoked a significant inhibition of LH secretion in cyclic female rats throughout the estrous cycle (proestrus afternoon, estrus, metestrus), as well as in ovariectomized females. In good agreement, gonadotropin-releasing hormone (GnRH) secretion by hypothalamic fragments from ovariectomized females was significantly inhibited by ghrelin. In contrast, ghrelin dose-dependently stimulated basal LH and FSH secretion by pituitary tissue in vitro; a phenomenon that was proven dependent on the phase of estrous cycle, as it was neither detected at estrus nor observed after ovariectomy. Conversely, GnRH-stimulated LH secretion in vitro was persistently inhibited by ghrelin regardless of the stage of the cycle, whereas stimulated FSH secretion was only inhibited by ghrelin at estrus. In addition, cyclic fluctuations in mRNA levels of growth hormone secretagogue receptor (GHS-R)1a, i.e. the functional ghrelin receptor, were observed in the pituitary, with low values at estrus and metestrus. GHS-R1a mRNA levels, however, remained unchanged after ovariectomy. In summary, our data illustrate a complex mode of action of ghrelin upon the gonadotropic axis, with predominant inhibitory effects at central (hypothalamic) levels and upon GnRH-induced gonadotropin secretion, but direct stimulatory actions on basal LH and FSH secretion. Overall, our results further document the reproductive role of ghrelin, which might be relevant for the integrated control of energy balance and reproduction. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid (GABA) has been implicated in the control of hypophyseal functions. We evaluated whether the constitutive loss of functional GABAB receptors in GABAB1 knockout (GABAB1–/–) mice alters hormonal levels, under basal and stimulated conditions, and reproductive function. The serum hormone levels were measured by radioimmunoassay, the estrous cyclicity was evaluated by vaginal lavages, and the mating behavior was determined by the presence of vaginal plugs. A moderate hyperprolactinemic condition was observed, in which prolactin increase and thyroid-stimulating hormone decrease were similar between genotypes. Basal luteinizing hormone (LH), follicle-stimulating hormone, thyroid-stimulating hormone, and growth hormone levels were similar between genotypes in each sex. Analysis of the gonadotropin axis revealed no differences in puberty onset between female genotypes. In con trast, the estrous cyclicity was significantly disrupted in GABAB1–/– female mice, showing significantly extended periods in estrus and shortened periods in proestrus. Reproduction was significantly compromised in GABAB1–/– females, with a significantly lower proportion of mice (37.5%) getting pregnant during the first 30 days of mating as compared with wild-type controls (87.5%). Moreover, only 14% of vaginal plug positive GABAB1–/– females had successful pregnancies as compared with 75% in the controls. In addition, the postovariectomy LH rise was significantly advanced in GABAB1–/– mice, while the response to estradiol feedback was similar in both genotypes. In conclusion, our endocrine analysis of GABAB1–/– mice reveals that GABAB receptors are involved in the regulation of basal prolactin titers. Moreover, the hypothalamic-hypophyseal-ovarian axis is seriously disturbed, with alterations in cyclicity, postcastration LH increase, and fertility indexes. The molecular mechanism underlying these hormonal disturbances remains to be addressed. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Silvering (transition from yellow to silver eel) has been traditionally considered as a metamorphosis in view of the numerous morphological, physiological and behavioral changes preparing the eel for the oceanic migration. However, some changes, such as increases in gonad weight and steroidogenesis, suggest that silvering could also be considered as a pubertal event. In order to assess which endocrine axis may be involved in the induction of silvering, we compared the profiles of pituitary and peripheral hormones during the transition from yellow to silver female eels. A strong activation of the gonadotropic axis was shown during silvering. Follicle-stimulating hormone (FSH) mRNA levels increased during the early stages of silvering, followed by a later increase in luteinizing hormone (protein and mRNA) levels. In addition, plasma levels of sexual steroids (estradiol, E2; testosterone, T, and 11-ketotestosterone) and of vitellogenin significantly increased. In contrast, thyrotropin mRNA levels did not change and no or weak variations in plasma thyroid hormones were observed, indicating no or moderate change of the thyrotropic axis during silvering. Similarly, the somatotropic axis was not activated, as shown by pituitary growth hormone expression (protein and mRNA) and plasma levels. In addition, we studied the effects of chronic treatments of female yellow eels with thyroid hormone (thyroxine, T4) and sex steroids (T and E2) on biometrical parameters characteristics of silvering. T induced an increase in eye size and a reduction of digestive tract, whereas T4 and E2 had no effect. These hormonal profiles and experimental data lead to the conclusion that eel silvering should be considered as an onset of puberty rather than a ‘genuine’ metamorphosis. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The excitatory transmitter, glutamate has been implicated in the control of reproduction, hormone secretion and neuroendocrine regulation. The present study examined whether the hypothalamic expression of three key ionotropic glutamate receptor subunits (NMDAR1, GluR1 and GluR6) fluctuates significantly on proestrus in the rat, and whether treatment with the antiprogestin, RU486 affected glutamate receptor subunit expression. The studies revealed that NMDAR1, GluR1 and GluR6 mRNA levels in the mediobasal hypothalamus (MBH) and preoptic area (POA) fluctuate little throughout the day of proestrus. However, treatment with the antiprogestin, RU486 induced a significant elevation of GluR6 mRNA levels at 14.00 and 16.00 h on proestrus in the MBH, suggesting that endogenous progesterone (P4) may act to inhibit hypothalamic GluR6 levels. In support of this suggestion, exogenous P4 treatment to estrogen (E2)-primed ovariectomized (ovx) rats significantly suppressed GluR6 mRNA levels in the afternoon (12.00–16.00 h) in the MBH, and at 12.00 h in the POA, which preceded LH surge induction. Likewise, temporal examination of hypothalamic GluR6 protein levels in E2 + P4-treated young and middle-aged ovx rats revealed an early elevation from 12.00 to 14.00 h, which was followed by a fall from 16.00 to 20.00 h. The early elevation of GluR6 protein levels was most pronounced in the POA of the young rat, and this elevation was markedly attenuated in the middle-aged rat. As a whole, the studies suggest that glutamate receptor expression fluctuates little on proestrus in the hypothalamus, but that expression of the kainate GluR6 receptor subunit may be modulated by progesterone and aging. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are, in teleosts as in mammals, under the control of hypothalamic factors and steroid feedbacks. In teleosts, feedback regulations largely vary depending on species and physiological stage. In the present study the regulation of FSH and LH expression was investigated in the European eel, a fish of biological and phylogenetical interest as a representative of an early group of teleosts. The eel FSHβ subunit was cloned, sequenced and together with earlier isolated eel LHβ and glycoprotein hormone α (GPα) subunits used to study the differential regulation of LH and FSH. In situ hybridization indicated that FSHβ and LHβ are expressed by separate cells of the proximal pars distalis of the adenohypophysis, differently from the situation in mammals. The profiles of LHβ and FSHβ subunit expression were compared during experimental ovarian maturation, using dot-blot assays. Expression levels for LHβ and GPα increased throughout ovarian development with a positive correlation between these two subunits. Conversely, FSHβ mRNA levels decreased. To understand the role of sex steroids in these opposite variations, immature eels were treated with estradiol (E2)and testosterone (T), both steroids being produced in eel ovaries during gonadal development. E2 treatment induced increases in both LHβ and GPα mRNA levels, without any significant effect on FSHβ. In contrast, T treatment induced a decrease in FSHβ mRNA levels, without any significant effect on the other subunits. These data demonstrate that steroids exert a differential feedback on eel gonadotropin expression, with an E2-specific positive feedback on LH and a T-specific negative feedback on FSH, leading to an opposite regulation of LH and FSH during ovarian development. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We utilized mass spectrometry (MS) and bioinformatics to investigate the proteome of the anterior pituitary gland (AP). Subcellular fractions of APs from 2-month-old male Golden Syrian hamsters were prepared for protein denaturation, treatment with trypsin and analyses utilizing micro liquid chromatography MS/MS and the database search software SEQUEST. In the nuclear, non-nuclear 100,000 × g and cytosolic fractions we identified 76, 52 and 52 different proteins, respectively. A total of 145 distinct proteins were detected. We identified growth hormone, prolactin, pro-opiomelanocortin, the α-subunit for the glycoprotein hormones, luteinizing hormone-β and follicle-stimulating hormone-β. Groups of other identified proteins included hormone processing, secretion granule associated, non-hormonal endoplasmic reticulum associated, calcium binding, protein kinase C associated histone and non-histone chromosomal material, other RNA-binding, splicing factors, heterogeneous nuclear ribonucleoproteins, helicases, lamins, microfilament associated, microtubule associated, adenosine triphosphate and guanosine diphosphate associated, keratins, lysosomal, ribosomal, enzymes in glycolysis and the tricarboxylic and pentose phosphate paths, glutathione associated, transmethylation, catabolic and unknown protein products as well as blood hemoglobins. Proteins previously not reported in the AP, such as fertility protein SP22, were identified. The proteins identified in the present study form a foundation for defining the proteome in normal adult male AP. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The neurochemical mechanisms underlying hindbrain glucoprivic suppression of the luteinizing hormone (LH) surge are not known. A body of experimental evidence supports the view that gonadal steroid positive-feedback action on the reproductive neuroendocrine axis relieves tonic GABAergic inhibition of gonadotropin-releasing hormone neurons by diminishing preoptic release of this neurotransmitter. The present studies evaluated the hypothesis that hindbrain glucoprivic attenuation of the LH surge may be correlated with site-specific modifications in gonadal steroid suppression of γ-aminobutyric acid release in this region of the brain. Individual septopreoptic loci were microdissected from the brains of estrogen, progesterone-primed ovariectomized female rats injected with the glucose antimetabolite, 5-thioglucose (5-TG), or vehicle into the caudal fourth ventricle during the ascending phase of the surge, and analyzed by high-performance liquid chromatography. The data show that 5- TG administration increased GABA release within the rostral preoptic area (rPO), anteroventral periventricular nucleus (AVPV), and median preoptic nucleus (MEPO), relative to the vehicle-treated controls, but did not alter neurotransmitter release in other structures evaluated. The rate of GABA turnover in each brain site was equivalent between animals injected with the μ opioid receptor antagonist CTOP and 5-TG versus their vehicle-treated controls. These results constitute novel evidence for site-specific modulation of steroid positive-feedback suppression of this inhibitory neurotransmitter by caudal hindbrain signaling of glucose insufficiency, and support the need for neurochemical characterization of glucoprivic-sensitive afferent input to GABAergic neurons terminating within the rPO, AVPV, and MEPO, as well as the relevance of enhanced local GABA release for reproductive neuroendocrine function. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Previous work demonstrated a sexually dimorphic ontogenic expression of γ-aminobutyric acid receptors (GABABR) in rat pituitary. As sex steroids determine sex-specific expression patterns, we now studied the effect of sex hormones on pituitary GABABR expression. GABABR subunits, measured by Western blot and by semi-quantitative RT-PCR and luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone measured by RIA were determined in two experimental designs: First experimental design: 8- and 15-day-old females (8F, 15F); 8F and 15F treated with 100 μg testosterone propionate (TP) on day 1 of life (8F100TP, 15F100TP), 8- and 15-day-old males (8M, 15M) and 8M and 15M castrated on day 1 (8MC, 15MC). Second experimental design: 8-day-old female and male animals: 8F, 8F100TP, 8F treated with 1 μg/day TP on days 1–4 (8F1TP), 8F treated with the androgen antagonist Flutamide (Flut: 2.5 mg/100 g BW of pregnant mother on days E17-E23) (8F-Flut), 8M, 8MC, 8M treated with Flut as above (8M-Flut) and 8MC-Flut. In these animals, in addition, GABA, glutamate, aspartate and taurine were measured by HPLC in hypothalami and cortex. In the first set of experiments, GABAB1R mRNA/protein expression was higher in 8F than in 15F, 8M or 15M. In 8F100TP, GABAB1R mRNA/protein decreased to male levels. TP treatment did not alter GABAB1R expression in 15F. There was no difference in GABAB1R expression between 8M and 15M and neonatal castration did not modify its expression. In the second set of experiments, TP (1 μg) or Flut did not modify GABAB1R in 8F, while 100 μg TP continued to decrease GABAB1R expression. In 8M, Flut, alone or with castration, increased GABAB1R mRNA/protein expression to 8F. Hypothalamic GABA content followed the same pattern as pituitary GABABR expression in 8-day-old animals, suggesting a cross-regulation. With regard to hormonal levels, 100 μg, but not 1 μg TP altered gonadotropins at 8 days, although both treatments effectively androgenized females as evidenced by lack of cycling. We conclude that androgens, acting pre- and postnatally, decrease pituitary GABABR subunit expression. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In this study we have isolated the follicle-stimulating hormone β subunit gene from the Chinook salmon (csFSHβ). This gene encodes for a protein that is highly similar to those isolated from other salmonids and shares all of the structural constraints seen in mammalian gonadotropins, including twelve conserved cysteines and a putative N-linked glycosylation site. The organization of the gene follows the conserved pattern regarding the numbers and positions of the introns, although the csFSHβ gene contains a particularly large 6.2-kb first intron due to the inclusion of several transposon-like elements. Isolation of 1.2 kb of the 5′ flanking region of the csFSHβ gene and subsequent analysis in silico have revealed a number of putative elements which appear highly conserved in teleost FSHβ gene promoters and are thus likely involved in basal and hormone-induced transcriptional regulation. The functionality of this 1.2-kb fragment in driving expression of a reporter gene and its response to GnRH was shown in gonadotropes, while the overexpression of AP-1 factors, Sf-1, estrogen receptor or Smad1 revealed that the promoter is responsive to these transcription factors. Our current study has opened the way for future analysis to verify the role of these factors in mediating hormonally induced transcription of this gene. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The relationship between the brain and the endocrine system is now seen to extend far beyond the regulation of somatic hormone production by the hypothalamus and pituitary: the brain itself can be considered both as an endocrine organ, producing hormones that act both within and outside the central nervous system, and as a target for hormones. The current extent of this concept with respect to the gonadal hormones was explored at a recent meeting (‘Hormones and the Brain’, Third Endocrinology Colloquium of the Fondation Ipsen, Paris, December 8, 2003). The discussion, reviewed in this article, ranged from intracellular signalling pathways and intercellular networks regulating hormone production and action in the central nervous system to hormone involvement in the generation of sexual behaviour and in development, plasticity, neuroprotection and repair. The hormonal contribution to psychiatric and neurodegenerative illnesses was also examined. The picture presented is complex, with layers of controls and with hormones that have diverse actions at different sites in the central nervous system. This richness of actions and functions is providing some interesting leads for developing new therapeutics. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Estrogen (E) is a key regulator of the synthesis and secretion of pituitary reproductive hormones [luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL)]. Until recently, it was thought that all biological actions of E at the pituitary were manifested through a single E receptor (R). The pituitary, like many other reproductive tissues, expresses two isoforms of ER, α and β, both activated by E. The relative contribution of α and β forms in E regulatory actions is largely unknown. To this end, 2-week-old ovariectomized (OVX) rats were injected over 3 days with 25 μg estradiol benzoate (EB), 1.5 mg of propylpyrazole triol (PPT), a selective ERα agonist, 1.5 mg of the selective ERβ agonist diarylpropionitrile (DPN) or a combination of PPT and DPN. Controls were injected with 0.2 ml oil. At 10:00 h on the day after treatment, trunk blood was collected to determine serum concentration of LH, FSH and PRL, and pituitaries were processed for RT-PCR analysis of total (A+B) progesterone receptor (PR) mRNA, immunocytochemistry of PR and incubation. Pituitaries from each of the five groups were incubated in DMEM, with or without 20 nM of the antiprogestin at the receptor ZK299, for 3 h with: 10–8M 17β-estradiol, 10–6M PPT, 10–6M DPN, PPT+DPN or medium alone, respectively, to determine LH, FSH and PRL secretion, and, when challenged with two pulses of 15 min 1 h apart of 10–8M gonadotropin-releasing hormone (GnRH) (GnRH self-priming). EB, PPT and PPT+DPN treatments increased PR mRNA and the number and intensity of nuclei immunoreactive (IR) for PR in gonadotropes, and reduced the number of gonadectomy cells. Like E, PPT alone or in combination with DPN stimulated PRL secretion, increased basal and GnRH-stimulated LH and FSH secretion and induced GnRH self-priming in the absence of ZK299 in the incubation medium. DPN alone had only a significant E-like effect on gonadectomy cells and IR-PR, but not on GnRH self-priming. In addition, while DPN lacked an agonistic action on peripheral tissue and serum pituitary reproductive hormones concentration, EB, PPT and PPT+DPN induced similar uterine ballooning and vaginal cornification, and increased and decreased, respectively, serum concentrations of PRL and gonadotropins. Overall, these results indicate that most of these E actions on the pituitary are exerted through the ERα isoform. The finding that activation of ERβ with its selective DPN agonist had an estrogenic effect on IR-PR nuclei, but not on GnRH self-priming, a characteristic ERα-mediated effect of E, suggests that the biological action of E at the pituitary may involve both isoforms of ER. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Galanin-like peptide (GALP) shares partial sequence identity with galanin and exhibits agonistic activity at two of the galanin receptor subtypes (GALR1 and GALR2) in vitro. The goal of these experiments was to determine whether galanin receptors mediate the effects of central GALP administration on food intake, body weight, and luteinizing hormone (LH) secretion in the mouse. We first evaluated the effects of intracerebroventricular injections of GALP or its vehicle alone in GALR1 knockout mice, GALR2 knockout mice, and their respective wild-type controls. GALP reduced food intake and body weight after 24 h to a similar degree in wild-type, GALR1 knockout, and GALR2 knockout mice. The wild-type, GALR1 knockout, and GALR2 knockout mice also exhibited significant increases in serum levels of LH following the GALP injections. To help delineate the biologically active moiety of the GALP molecule, we injected wild-type mice with shorter fragments of the full-length GALP peptide. Neither GALP(1–21) (the fragment containing the galanin-homologous sequence) nor GALP(22–60) (the C-terminal portion of the GALP molecule lacking sequence identity with galanin) had any discernable effect on food intake, body weight or circulating LH. These observations demonstrate that neither GALR1 nor GALR2 are essential for mediating the effects of GALP on feeding, body weight or LH secretion. Furthermore, the galanin-homologous region of the GALP molecule is not sufficient to mimic the effects of full-length GALP. Together, these findings argue against the hypothesis that GALP signals solely through galanin receptors in vivoand suggest the existence of a yet-to-be-identified GALP-specific receptor. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

There is evidence that α-melanocyte-stimulating hormone (α-MSH) has immunomodulatory and anti-inflammatory actions within the brain. In this study, we tested whether these actions are due to inhibition of the synthesis of nitric oxide (NO) and prostaglandins induced by lipopolysaccharide (LPS). Since melanocortin subtype MC4 receptor has been detected in the hypothalamus, we investigated the effect of central administration of α-MSH and HS024 (a selective MC4 receptor antagonist) on the gene expression of inducible, neuronal and endothelial NO synthase (iNOS, nNOS and eNOS) and on cyclooxygenase (COX-1 and COX-2) expression in the mediobasal hypothalamus (MBH) of LPS-treated male Wistar rats. Peripheral administration of LPS (250 μg/rat, 3 h) induced iNOS and COX-2 gene expression in the MBH. This stimulatory effect was reduced by α-MSH (3 nmol/rat) injected 30 min before LPS. α-MSH and HS024 (1 nmol/rat) alone had no effect on iNOS and COX-2 expression. The action of α-MSH on LPS-induced iNOS and COX-2 mRNA levels was not observed in the presence of HS024, suggesting that MC4-R may be involved in the modulatory effect of α-MSH. None of these treatments produced any modifications in nNOS, eNOS and COX-1 expression in MBH. The increase in serum corticosterone levels induced by LPS was attenuated by α-MSH. Both LPS and α-MSH decreased serum LH and prolactin levels. HS024 failed to modify the inhibitory effects of LPS and α-MSH on prolactin release but reverted the effect of LPS on LH secretion, indicating that MC4-R activation may be involved in the effects of α-MSH on LH secretion in male rats. When we examined the in vitro effect of LPS (10 μg/ml) and LPS plus interferon-γ (IFN-γ, 100 ng/ml) on iNOS expression in MBH, an increase in iNOS mRNA levels was observed only in the presence of LPS + IFN-γ. This stimulatory effect was attenuated in the presence of α-MSH (5 μM), which by itself had no effect. No changes were found in nNOS, eNOS, COX-1 or COX-2 expression. These results indicate that α-MSH reduces the induction of iNOS and COX-2 gene expression at the hypothalamic level during endotoxemia and suggest that endogenous α-MSH may exert an inhibitory tone on iNOS and COX-2 transcription via MC4 receptors acting as a local anti-inflammatory agent within the hypothalamus. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Through binding with estrogen receptors, phytoestrogens, plant-derived estrogen-like compounds, affect numerous reproductive functions. It is not known whether these compounds are capable of evoking effective changes in luteinizing hormone (LH) and prolactin (PRL) secretion in ewes by acting directly within the central nervous system (CNS). The hypothesis studied was that genistein, infused for several hours into the third ventricle, could immediately affect LH and PRL secretion in ovariectomized (OVX) ewes during seasonal anestrus. Two doses of genistein, 1 μg/100 μl/h (total 4 μg, n = 7) and 10 μg/100 μl/h (total 40 μg, n = 7), were infused intracerebroventricularly from 12.00 to 16.00 h and blood samples were collected from 8.00 to 20.00 h at 10-min intervals. Randomly selected ewes were infused with a vehicle (control, n = 5). The mean plasma LH concentration in control ewes was significantly (p < 0.01) higher during infusion of the vehicle than before the infusion. It remained on an insignificantly changed level after the infusion. The frequency of LH pulses in control ewes did not differ significantly before, during, or after vehicle infusion. In ewes infused with a lower dose of genistein, plasma LH concentrations decreased significantly (p < 0.001) after the infusion, as compared with the values noted before and during genistein infusion. Only a tendency towards a decrease in LH pulse frequency occurred after infusion of a lower dose of genistein. In ewes infused with a higher dose of genistein, the plasma LH concentration decreased significantly (p < 0.01) after phytoestrogen administration as compared with the values noted before and during infusion. The frequency of LH pulses was also significantly (p < 0.01) lower after genistein administration. Because the changes in PRL secretion were more dynamic in response to genistein infusion, the statistical analysis included 2-hour periods. The mean plasma PRL concentration in control animals was significantly enhanced (p < 0.01) only during the first 2-hour period of sampling. After that it decreased and remained on an unchanged level up to the end of sampling. Similar changes in PRL secretion were observed in both experimental groups before genistein infusion. In contrast, significant (p < 0.01 to p < 0.001) increases in PRL concentration were noted regularly during and shortly after the genistein infusion in either low-dose or high-dose genistein-infused ewes, compared with the concentrations noted before genistein treatment. Plasma PRL concentrations during and after genistein infusion in both experimental groups were also significantly higher than the control (p < 0.01 to p < 0.001). The presented data demonstrate that genistein, a phytoestrogen, may effectively modulate LH and PRL secretion in OVX ewes by acting within the CNS. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Genetic targeting of the cAMP-specific phosphodiesterase 4D1 (PDE4D1) to gonadotropin-releasing hormone (GnRH) neurons in the GPR-4 transgenic rat resulted in decreased luteinizing hormone (LH) pulse frequency in castrated female and male rats. A similar decrease in the intrinsic GnRH pulse frequency was observed in GT1 GnRH cells expressing the PDE4D1 phosphodiesterase. We have extended these findings in ovariectomized (OVX) GPR-4 rats by asking what effect transgene expression had on pulsatile LH and follicle-stimulating hormone (FSH) secretion, plasma and pituitary levels of LH and FSH, and levels of the α-glycoprotein hormone subunit (α-GSU), LH-β and FSH-β subunit mRNAs. In OVX GPR-4 rats the LH pulse frequency but not pulse amplitude was decreased by 50% compared to wild-type littermate controls. Assaying the same samples for FSH, the FSH pulse frequency and amplitude were unchanged. The plasma and anterior pituitary levels of LH in the GPR-4 rats were significantly decreased by approximately 45%, while the plasma but not anterior pituitary level of FSH was significantly decreased by 25%. As measured by real-time RT-PCR, the mRNA levels for the α-GSU in the GPR-4 rats were significantly decreased by 41%, the LH-β subunit by 38% and the FSH-β subunit by 28%. We conclude that in the castrated female GPR-4 rats the decreased GnRH pulse frequency results in decreased levels of LH and FSH and in the α- and β-subunit mRNA levels. Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The effect of androgens on the oligosaccharide structure of follicle-stimulating hormone (FSH) isoforms was studied in adult male rats treated with a potent gonadotropin-releasing hormone (GnRH) antagonist. Animals were castrated (Cx), and 24 h later were treated with physiological doses of either testosterone propionate (T) or dihydrotestosterone propionate (DHT) (0.25 mg/rat in corn oil, daily, s.c.) for 7 days. The antiandrogen flutamide (F; 5 mg/day/rat, twice a day, s.c.) was administered to either Cx-T or Cx-DHT rats to block androgen action. The GnRH antagonist Org 30276 (Ant; 1 mg/kg/day, s.c.) was injected to both Cx- and Cx-DHT treated rats. FSH serum levels reached normal values in Cx rats treated with either T or DHT, whereas those treated with F retained Cx conditions. Both Cx-Ant and Cx-Ant-DHT treated animals presented normal serum FSH levels. Concanavalin A affinity chromatography was used to isolate pituitary FSH isoforms according to their carbohydrate inner structure. An increased proportion of FSH isoforms bearing complex-type oligosaccharides was observed in Cx rats treated with T or DHT, whereas the proportion of these isoforms was reduced in Cx and Cx-T-F or Cx-DHT-F rats. These results demonstrate that functional androgens are needed to complete the oligosaccharide processing of FSH. In addition, the proportion of the different pituitary FSH isoforms after DHT replacement was equivalent to that found when GnRH action was blocked, indicating that androgens are involved in the regulation of carbohydrate incorporation to the FSH molecule by acting mainly at the pituitary level, independently of GnRH action.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Estrogen exerts an important regulatory influence upon the functioning of the gonadotropin-releasing hormone (GnRH) neurons. Whether this is mediated by estrogen receptor α (ERα) or ERβ or both ERs is presently unclear. Using female mice with targeted disruptions of ERα and ERβ (αERKO and βERKO, respectively) we have investigated the in vivo role of the two ERs in the negative feedback influence of estrogen upon GnRH mRNA expression. Compared with intact wild-type mice, plasma luteinizing hormone (LH) levels were substantially (p < 0.01) higher in intact αERKO females and increased modestly (p < 0.05) in intact βERKO mice. Three weeks after ovariectomy, LH concentrations were elevated significantly in wild-type (p < 0.01) and βERKO (p < 0.05) mice but not changed in αERKO females. Quantitative analysis of GnRH mRNA expression using in situ hybridization revealed that cellular GnRH mRNA content was greater (p < 0.05) in intact αERKO mice compared with intact wild-type and βERKO mice. Following ovariectomy, GnRH mRNA expression was elevated in wild-type (p = 0.06) and βERKO (p < 0.05) females but not αERKO mice. These data demonstrate that both ERα and ERβ are involved in inhibiting LH levels at times of estrogen-negative feedback in vivo. However, only ERα appears to be critical for the estrogen-negative feedback suppression of GnRH mRNA expression in the female mouse.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

As circulating estrogen levels rise on the afternoon of proestrus, they stimulate the hypothalamo-pituitary axis. This estrogen positive feedback is pivotal to stimulate the luteinizing hormone (LH) surge required for ovulation and luteinization of ovarian follicles. In addition to estrogen, pre-LH surge progesterone is critical for an LH surge as was demonstrated by blocking progesterone synthesis. In ovariectomized (OVX) rats treated with trilostane, a blocker of the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) that catalyzes the conversion of pregnenolone to progesterone, estrogen did not induce an LH surge. Further, estrogen induced an LH surge in OVX and adrenalectomized (ADX) rats, indicating that the source of progesterone was neither the ovary nor adrenal gland. This estrogen-only LH surge was inhibited by pretreatment with trilostane, indicating that although the adrenal gland and ovary were not necessary for positive feedback, progesterone synthesis was critical for estrogen-induced positive feedback in an OVX/ADX rat. This suggested that the LH surge is dependent on the pre-LH surge synthesis of progesterone. Estrogen-induced progesterone receptors in the hypothalamus are vital for the LH surge, so a potential location for progesterone synthesis is the hypothalamus. OVX/ADX female rats were treated with 17β-estradiol (50 μg) and progesterone levels were assayed by RIA. Progesterone levels were elevated in hypothalamic tissue following estrogen treatment. No increases in tissue progesterone levels were found in parietal cortex, cerebellum, medulla, pituitary or plasma. Additionally, male rats that do not have an estrogen positive feedback-induced LH surge were examined. Castrated/ADX male rats had no increase in hypothalamic progesterone levels after estrogen treatment. Together, these data strongly suggest that estrogen enhances neuroprogesterone synthesis in the hypothalamus that is involved in the positive feedback regulating the LH surge.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

It has been reported that the impaired reproductive function found in endotoxemia is mediated by proinflammatory cytokines, prostaglandins, and opioid peptides in the brain and that the synthesis of all these molecules is stimulated by endotoxins. The role of glucocorticoids in the endotoxin-induced hypogonadism may also be important, because endotoxins are potent stimulators of the hypothalamo-pituitary-adrenal axis, and glucocorticoids are inhibitory to the reproductive system. However, no previous study examined directly whether glucocorticoids contribute to the endotoxin-induced suppression of the reproductive competence until a very recent study performed in sheep. To examine directly such a role of glucocorticoids in rats, we compared the effects of lipopolysaccharide (LPS) on the pulsatile luteinizing hormone (LH) release between adrenal-intact orchidectomized rats and adrenalectomized plus orchidectomized rats. The latter group received a constant subcutaneous infusion of corticosterone to maintain physiological plasma levels of the steroid. An intravenous injection of LPS promptly decreased both amplitude and frequency of the LH pulses in the orchidectomized rats, and the LPS effects were very similar in the double endocrinectomy group which did not show an increased corticosterone release after LPS. These results strongly suggest that glucocorticoids do not have a significant role in mediating the LPS-induced acute suppression of pulsatile LH secretion in orchidectomized rats.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Tamoxifen (TX) is an antiestrogen with varying levels of antagonist/agonist activity on the reproductive axis of the rat. It has been reported that TX, in contrast to other selective estrogen receptor modulators (SERMs), increases the content of cytosolic estrogen receptors (ER) in the gonadotrope and induces gonadotropin releasing hormone (GnRH) self-priming in the absence of E. GnRH priming is believed to be a consequence of E-dependent progesterone receptor (PR) activation. The purpose of this study was to determine whether TX induces PR expression in the gonadotrope in an E-dependent manner, and whether the blockade of PR activation affects TX-dependent GnRH self-priming in ovariectomized (OVX) rats. Chronic OVX rats were injected (sc) over 3 days with 25 μg estradiol benzoate (EB), 3 mg TX, 0.5 mg RU58668, a ‘pure’ anti-E (aE), 2 mg RU38486, an anti-P at the receptor (aP), TX+aE and TX+aP. Controls were given 0.2 ml oil. While EB and TX increased mRNA for both PR A+B and PR B expression and the number and intensity of nuclei immunoreactive (IR) for PR in the gonadotrope, the aE and aP given alone had no effect on either PR mRNA levels or nuclear PR-IR. The aE reduced the effect of TX on PR expression (mRNA and nuclear IR) while the aP slightly reduced nuclear PR-IR only. In addition, pituitaries from each of the seven groups were incubated with: 10[sup –8] M E[sub 2] , 10[sup –7] M TX, 10[sup –8] M aE, 10[sup –8] M aP, TX+aE, TX+aP or medium alone, respectively. Pituitaries were tested for GnRH self-priming (two pulses of 15 min 1 h apart) and the secretion of LH and PRL determined by specific RIAs. Pituitaries from rats treated with EB and incubated with E[sub 2] had increased basal and GnRH-stimulated luteinizing hormone (LH) and prolactin (PRL) secretion and GnRH self-priming. TX reduced basal and stimulated LH secretion, increased PRL secretion and induced a robust GnRH self-priming. All these effects of TX were blocked by the aE, while the aP blocked GnRH self-priming only. In conclusion, tamoxifen induced PR expression (mRNA and nuclear IR) in the gonadotrope in an E-dependent manner, while activation of these PR through intracellular signaling of GnRH induced GnRH self-priming.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

This study was designed to evaluate the effects of glucocorticoids and gonadal steroids on the expression of inhibin/activin subunits and follistatin of the anterior pituitary and test the hypothesis that resulting changes in the local activin/inhibin/follistatin tone contribute to steroid effects on follicle stimulating hormone (FSH) production from gonadotropes. In primary cell cultures of male rat anterior pituitaries, dexamethasone (DEX) or testosterone (T) stimulated FSH secretion and FSHβ mRNA and their effects were additive with activin-A. Follistatin (FS288) and inhibin-A antagonized the rise in FSH secretion both in the absence and presence of exogenous activin-A. Despite the similarity in their action on FSH production, DEX and T had opposite effects on follistatin mRNA levels. Follistatin mRNA levels of cultured rat anterior pituitary cells were elevated upon the addition of DEX but attenuated by T. On the other hand, both DEX and T suppressed inhibin/activin βB mRNA levels while only DEX affected βA mRNA. In these cells, activin-A stimulated follistatin and inhibin/activin βB mRNA levels but had no effect on βA. Together, DEX and activin-A caused a further increase in follistatin mRNA levels while T attenuated the effect of activin-A alone. Both steroids attenuated the effect of activin-A on βB mRNA accumulation. These results support the possibility that DEX and T, possibly acting on different subsets of anterior pituitary cells, use distinct mechanisms to modify the local activin/inhibin/follistatin circuitry and thereby upregulate FSH production from the anterior pituitary gonadotropes.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The median eminence (ME) of the hypothalamus is known to be an important brain site where hypophysiotropic release might be regulated by excitatory and inhibitory signals impinging on their neuronal terminals. Since a role for neuropeptide Y (NPY) on preovulatory luteinizing hormone (LH) release has been suggested, we hypothesized that NPY might act at the ME to control preovulatory gonadotropin-releasing hormone (GnRH) release and thus the onset of the preovulatory surge of LH. To examine this possibility, we used the ewe as an animal model to determine: (a) immunocytochemical distribution of GnRH and NPY in the ewe ME; (b) changes in in vivo release of NPY and GnRH using ME push-pull cannula (PPC) perfusate samples, as well as in plasma LH, during the luteal, follicular and preovulatory phases of a synchronized estrous cycle, and (c) effects of ME perfusion of NPY or a Y1-NPY antagonist, or an NPY antiserum on in vivo release of ME-GnRH and plasma LH during a synchronized follicular phase. Immunolocalization reveals a dense plexus of beaded GnRH-containing neurites in the arcuate nucleus and in its vicinity, the pituitary stalk and the palisade. In contrast, a dense plexus of NPY-containing neurites occurs in the internal layer, with occasional fibers found in the intermediate and lateral external zone of the ME. In the area between the lateral internal and lateral external layers, both NPY and GnRH-containing processes were found, thus providing opportunities for synaptic and/or paracrine interactions between NPY- and GnRH-containing neurons. Hormonal analysis indicated that a synchronized preovulatory surge of LH is elicited within a 2-hour window by the sequential implantation and removal of silastic-encased estradiol (E2) or progesterone (P4) implants. In this paradigm, there was a parallel increase in ME release of both NPY and GnRH preceding the synchronized LH surge. The onset of this synchronized LH surge was advanced by ME perfusion of exogenous NPY and was both delayed and blunted by ME perfusion with the NPY antagonist (both were perfused through the PPC probe for 2 h, starting 2–3 h before the expected onset of the LH surge). In addition, NPY perfusion in the ME increases, while perfusion of the Y1-NPY antagonist or of the NPY antiserum decreases ME-PPC GnRH content and plasma levels of LH in early follicular ewes. Finally, perfusion of NPY antiserum during an ongoing LH surge disrupted LH release. These results suggest that interactions between NPY and GnRH neurons are important in controlling the timing, magnitude and maintenance of the preovulatory LH surge.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Previous work by our laboratory demonstrated that activation of the progesterone receptor through exogenous administration of progesterone suppressed glutamic acid decarboxylase-67 (GAD[sub 67] ) mRNA in the hypothalamus of the estrogen-primed ovariectomized rat. Since GAD[sub 67] is the major synthetic enzyme for the inhibitory transmitter, γ-aminobutyric acid, the finding raised the possibility that the endogenous activation of the progesterone receptor may act to restrain GAD[sub 67] expression during the natural preovulatory gonadotropin surge during proestrus in the rat, thereby allowing GnRH secretion and the resultant LH surge. To test this hypothesis, the progesterone receptor antagonist, RU486, was administered to regularly cycling proestrous rats and the effect on GAD[sub 67] and GAD[sub 65] mRNA levels in the preoptic area (POA) and medial basal hypothalamus (MBH) was examined. Serum luteinizing hormone (LH) levels were also examined in order to identify correlations between changes in POA and MBH GAD levels and production of the LH surge. GAD[sub 67] mRNA levels in the POA were increased in the cycling rat during proestrus at 18.00 h at the peak and just preceding the termination of the LH surge. There was no change in GAD[sub 67] mRNA levels in the MBH, and GAD[sub 65] expression was also unchanged during proestrus in the POA and MBH. Treatment with the antiprogestin RU486 resulted in an increase in GAD[sub 67] mRNA levels at 12.00 and 14.00 h in the POA, and in the MBH at 14.00, 16.00, and 18.00 h during proestrus, effects which preceded and correlated with the attenuated LH surge in RU486-treated rats at 18.00 h. GAD[sub 65] mRNA levels were also elevated by RU486 at 14.00 and 16.00 h in the POA, and at 14.00 h in the MBH during proestrus. These findings suggest that the progesterone receptor plays a role in restraining GAD expression in the hypothalamus during proestrus, and that this effect may be important for the production of the GnRH and LH surge.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We have suggested that gamma-aminobutyric acid (GABA) in the hypothalamus plays a tonic inhibitory role in the control of the luteinizing hormone (LH) release in intact male rats. To assess whether feedback from the testis alters the inhibitory GABAergic tone in the medial preoptic area (MPO) of male rats, an in vivo microdialysis study was performed in gonadally intact (n = 10), castrated (n = 12) and castrated testosterone-primed (n = 10) male rats. The microdialysis samples were collected and sequential blood samples were also obtained at 1-hour intervals. GABA in the dialysate was determined by high-performance liquid chromatography system and serum LH concentration was determined by radioimmunoassay. Episodic GABA release in the MPO was observed in all three groups of male rats, although castrated male rats showed lower GABA release (2.3 ± 0.3 ng/h) than intact and castrated testosterone-primed male rats (4.0 ± 0.5 and 4.6 ± 1.0 ng/h, respectively). Conversely, castrated male rats showed higher serum LH concentration (7.31 ± 0.46 ng/ml) than intact and castrated testosterone-primed male rats (0.71 ± 0.04 and 0.53 ± 0.07 ng/ml, respectively). In addition, intravenous infusion of bicuculline significantly increased serum LH in intact male rats, whereas bicuculline did not alter serum LH concentrations in castrated male rats. These results are consistent with the hypothesis that the feedback of testosterone stimulates GABA release in the region of the GnRH cell bodies and dendrites in male rats.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Selective estrogen receptor modulators (SERMs) are compounds which may function as agonists or antagonists depending upon the target tissue. This study compares the actions of different SERMs on luteinizing hormone (LH) secretion, and on gonadotropin-releasing hormone (GnRH) self-priming in the rat. To do this, 4-day cyclic rats were injected twice, on day 2 (metestrus) and day 3 of the estrous cycle, with one of the following SERMs: 0.25 mg ICI 182,780, 3 mg tamoxifen (TX), LY139481-HCl or LY117018-HCl, or 0.5 mg RU58668. Control rats were given subcutaneous injections of 0.2 ml oil. On the morning of day 4 (proestrus in controls), rats from each group were either injected intraperitoneally with pentobarbital (40 mg/kg) for in vivo study or decapitated and their pituitaries collected for incubation (in vitro study). Additionally, pituitaries taken on each day of the estrous cycle from control rats as well as on day 4 from SERM-treated rats were processed for immunohistochemical determination of the estrogen receptor-α (ERα) gonadotrope. The plasma concentration or accumulation of LH in the medium was determined after 1 h (basal secretion). Thereafter, an intravenous bolus of GnRH (50 ng/0.5 ml/100 g BW) or 10[sup –8] M GnRH was injected or added to the medium, respectively. After 1 h of GnRH exposure, blood or medium were taken, and another challenge of GnRH was made. At the end of the 3rd h of the experiment, blood or medium samples were taken again and the LH plasma concentration or accumulation in the medium were determined. All SERM treatments reduced uterus weight and decreased basal and stimulated LH secretion. Also, on day 4, rats treated with any SERM other than TX showed vaginal smears infiltrated by leukocytes and a reduction in GnRH self-priming. TX-treated rats exhibited cornified vaginal smears and an estrogenic effect on GnRH self-priming. Moreover, 15-min exposure to two consecutive GnRH (10[sup –8] M) challenges 1 h apart in incubated pituitaries with estradiol (E[sub 2] , 10[sup –8] M), TX (10[sup –7] M), E[sub 2] + TX, or medium alone form ovariectomized rats injected for 3 days with estradiol benzoate (25 μg), TX (3 mg), estradiol benzoate + TX, or 0.2 ml oil, respectively, showed that TX increased GnRH self-priming, as did E[sub 2] , whereas it reduced the E[sub 2] -sensitizing effect on GnRH-stimulated LH secretion and cancelled the E[sub 2] -dependent GnRH self-priming. All SERMs prevented the physiological nucleocytoplasmic shuttling of ERα exhibited during proestrus in control rats, and TX, in addition, induced a significantly larger number of gonadotropes displaying strong cytosolic immunosignals corresponding to ERα than the rest of the experimental groups. Overall, data from this study indicated that, in contrast to the general antagonistic effect of the tested SERMs, TX seemed to display both selective agonist and antagonist activity at the gonadotrope level and on GnRH self-priming of LH secretion respectively.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The adage ‘we are what we eat’ is taking on a new meaning in our well-fed and increasingly sedentary culture, as many of us convert much of our excess food into body fat; in the USA, 60% of the population is now considered to be overweight. Obesity brings with it an increased risk of developing type II diabetes, hypertension and heart disease, so the mechanisms that control food intake and body weight are of considerable importance for public health and clinical medicine. The mass of body fat is now known to be regulated by several hormones and neuropeptides. Two of these, the circulating peptide hormones leptin and ghrelin have actions that include reciprocal effects on appetite-regulating neurons in the hypothalamus. This article reviews data discussed at a recent meeting[sup 1] , where an overview of recent developments in research into leptin and ghrelin was presented. Topics covered are the roles of leptin and ghrelin in the regulation of food intake and energy production; the integration of food intake with other energy-regulated processes, such as growth, sexual maturation and reproduction, sleep and the immune response; and pathological conditions, ranging from diabetes to psychiatric disorders.1This report summarizes conclusions of the meeting ‘Brain Somatic Cross-Talk and the Central Metabolism’ held in Paris on January 28, 2002.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

During reproductive aging, female rodents show impaired inducibility of the estradiol (E2)-induced (preovulatory) surge of luteinizing hormone (LH), which is associated with hypothalamic neuronal impairments of aging. To evaluate if astrocytes show comparable age changes, we analyzed the regulation of glial fibrillary acidic protein (GFAP) mRNA which is transiently increased in the arcuate nucleus in association with the proestrus LH surge in young rats. In aging (18-month-old) F344 rats in persistent estrus, the loss of the E2-induced LH surge was paralleled by the lack of increased GFAP mRNA in the arcuate nucleus (in situ hybridization with X-ray film). We then tested the hypothesis that restoration of the LH surge by chronic ovariectomy (OVX) in aging rats would restore or block GFAP mRNA induction, respectively. Despite restoration of the inducible LH surge in aging rats by chronic OVX, there was no induction of GFAP mRNA in the arcuate nucleus. Moreover, young rats given chronic E2 implants for 6 weeks as a model for persistent estrus, retained induction of arcuate nucleus GFAP mRNA, despite loss of the induced LH surge. The aging rats were highly sensitive to gross pituitary enlargement from chronic E2 with 50% mortality; thus, the F344 genotype is not optimum for studies of aging-E2 interactions that require prolonged E2 treatments. More detailed cellular level analysis of GFAP mRNA is needed to define the relationship of GFAP expression to synaptic reorganization during the LH surge. The aging rats also showed higher levels of GFAP mRNA in the arcuate nucleus and ventromedial nucleus, consistent with the general trend for elevated GFAP mRNA during aging in other brain regions and in both sexes.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Estradiol induces surges of prolactin (PRL) and luteinizing hormone (LH) secretion as well as lactotrope proliferation in female rats. We examined whether these hypothalamus-dependent events require the direct action of estradiol on the anterior pituitary gland by selective blockade of its peripheral actions, using ICI182,780 (ICI), an antiestrogen that cannot cross the blood-brain barrier. Injection of ICI into ovariectomized rats, at a dose of 250 μg/day for 4 days, almost completely inhibited estradiol-induced growth of the uterus, proliferation of lactotropes as determined by bromodeoxyuridine incorporation, and afternoon surges of LH secretion. However, ICI only partially inhibited estradiol-induced surges of PRL secretion and had no effect on estradiol-induced tonic inhibition of LH secretion even at the highest dose of 1,000 μg/day. The inhibitory effects of ICI found at 250 μg/day were attributable to its selective peripheral, but not central actions since ICI did not alter hypothalamic expression of progesterone receptors, an estradiol-dependent brain process. Estradiol-induced increases in the number of progesterone receptor-immunoreactive cells in the hypothalamic ventromedial nucleus and the medial preoptic area were not inhibited by this dose of ICI but were inhibited by 500 μg/day tamoxifen, an antiestrogen that can cross the blood-brain barrier. Treatment of cycling female rats with 250 μg/day ICI beginning from diestrus day 2 was also effective in blocking estrous lactotrope proliferation and preovulatory surges of LH secretion but not PRL secretion. Finally, in ovariectomized estradiol-treated pup-deprived lactating rats, ICI did not affect suckling-induced PRL secretion but completely blocked lactotrope proliferation. These results suggest that a direct estradiol action on the anterior pituitary gland is required for lactotrope proliferation and the positive feedback action on LH secretion but not for the secretory surges of PRL or for negative feedback.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Gonadotropin-releasing hormone (GnRH) is a decapeptide widely known for its role in regulating vertebrate reproduction by serving as a signal from the hypothalamus to pituitary gonadotropes. The first form of GnRH to be identified was isolated from mammals (mGnRH) and the same form has been reported for all mammals studied, which includes marsupials and placental mammals. Later, another variant, chicken GnRH-II (cGnRH-II) was shown to be expressed together with mGnRH in the brains of all jawed vertebrates, including mammals such as rats, monkeys and humans. Our objective was to characterize a third form of GnRH that was isolated previously as mRNA from guinea pigs (gpGnRH), but has not been reported for any other mammal to date. Furthermore, the gonadotropic activity of gpGnRH has not been fully characterized. Our results, using chromatographical and immunological methods, show for the first time that gpGnRH is expressed together with mGnRH in some rodents (wild guinea pig and capybara), but not in others (mouse and hamster). Also, the gonadotropic activity of gpGnRH and mGnRH was tested in two different rat cell culture systems. Although there have been reports that the salmon(s) form of GnRH is present in mammals, we did not detect sGnRH in capybara, wild guinea pigs, hamsters, rats or mice. Taken together with previous reports, the present results support the idea that the expression of multiple GnRH variants in a single species is a common pattern in most vertebrate groups.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Orexins are hypothalamic neuropeptides which stimulate luteinizing hormone (LH) secretion in estrogen- and progesterone-treated ovariectomized (OVX) rats and suppress it in OVX rats not treated with estrogen, suggesting a modulation by estrogen of the response to orexins. We examined the effects of orexin A on pulsatile LH secretion in OVX rats treated with a very small dose of estrogen so as to maintain the pulsatile secretion of LH. The estrogen treatment was done 24 h before the blood sampling by subcutaneously implanting a silicone tube (id = 1.5 mm, od = 2.5 mm, length = 25 mm) containing 17β-estradiol (E[sub 2] ) dissolved in sesame oil at 20 μg/ml. In OVX rats treated with sesame oil as a control, the intracerebroventricular (icv) injection of orexin A (0.3 nmol, dissolved in 3 μl artificial cerebrospinal fluid) had no significant effect on the parameters of pulsatile LH secretion, i.e., pulse frequency and pulse amplitude, although it caused a small but statistically significant decrease in overall mean LH concentrations within 1 h. In OVX rats treated with E[sub 2] , the icv injection of orexin A significantly suppressed the pulsatile LH secretion; the frequency decreased for more than 2 h, inducing a rapid decline in overall mean LH concentrations. In view of the finding that a much higher dose of orexin A suppresses pulsatile LH secretion in OVX rats not treated with E[sub 2] , we suggest that the suppressive action of orexin A on pulsatile LH secretion is potentiated by estrogen.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The relationship between the hypothalamo-pituitary-gonadal (HPG) axis and the hypothalamo-pituitary-adrenal (HPA) axis has been well documented in the rat. In most cases, a negative coupling was observed and an inhibitory effect of the HPA axis upon the HPG was shown. In the female rat, a marked circadian rhythm of corticosterone plasma values is observed during each day of the estrous cycle, with maximal values around 08:00 p.m. The preovulatory luteinizing hormone (LH) surge also occurs at 08:00 p.m. on the day of proestrus. Here we measured circadian variations of plasma cortisol in humans in relation with the time of initiation of the preovulatory LH surge. Blood samples were taken at 08:00 a.m., 12:00 a.m., 04:00 p.m., 08:00 p.m., 12:00 p.m., and 04:00 a.m. from 19 subjects for 4 consecutive days, once 17β-estradiol (E[sub 2] ) values reached 125 pg/ml (days 7–10 of the menstrual cycle). Serum E[sub 2] and LH determinations were performed by microparticle enzyme immunoassays. Serum progesterone and plasma cortisol determinations were made using RIA methods. For plasma cortisol values, a marked circadian rhythm, with 2- to 3-fold higher values during the morning than during the afternoon, was almost identical before, during and after the LH surge. However, values were generally higher during the follicular phase than during the luteal phase. Maximum cortisol values occurred between 04:00 and 08:00 a.m. and minimal cortisol values between 04:00 and 08:00 p.m. Initiation of the LH surge (50% over the mean of previous values) occurred at 04:00 a.m. (20% of the cases) or at 08:00 a.m. (80% of the cases). There was a strong coupling between the onset of the surge and the acrophase of the cortisol circadian rhythm: maximal cortisol plasma values were seen at 04:00 a.m. when the LH preovulatory surge started at 04:00 a.m. and 08:00 a.m. when it started at 08:00 a.m. The present results show that the positive coupling documented in the female rat between the HPA and the HPG axis at the time of preovulatory LH surge is also present during the menstrual cycle in the human.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

There is ample information on the hypophysiotropic function of pituitary adenylate cyclase-activating polypeptide (PACAP) and neuropeptide Y (NPY) in fish as in mammals, although evidence as to their direct effects on gonadotropic cells is scarce. We have previously reported that NPY and PACAP38 augment gonadotropin-releasing hormone (GnRH)-induced expression of glycoprotein α (α) subunit gene in the teleost fish, tilapia. The aim of the present study was to elucidate possible direct effects of these peptides on gonadotropin subunit gene expression in culture of tilapia pituitary cells, as well as the transduction pathways involved. Both NPY and PACAP38 (0.001–10 nM) increased the level of phosphorylated extracellular signal-regulated kinase (pERK) dose-dependently, reaching a peak at 0.1 and 0.01 nM, respectively. Inhibition of protein kinase C (PKC) by GF109203X (GF; 0.01–10 nM) suppressed NPY-stimulated pERK levels and its effect on α and luteinizing hormone (LH) β subunit mRNA levels. However, NPY had no effect on follicle stimulating hormone (FSH) β mRNA levels. NPY-elevated α, LHβ mRNA and pERK levels were also attenuated by inhibition of protein kinase A (PKA) with H89 (0.01–10 nM). Exposure of the cells to the MAPK kinase (MEK) inhibitor (PD98059; PD 10, 25 and 50 μM) completely blocked NPY-induced ERK activity. In addition, this inhibitor abated the α and LHβ mRNA responses to NPY. Similar experiments conducted to elucidate PACAP38 signaling revealed that PACAP38 (0.01 nM) elevated all three-gonadotropin subunit gene expression via both PKC-ERK and PKA-ERK cascades. It is suggested that both NPY and PACAP38 act directly on gonadotropes to elevate gonadotropin subunit gene expression. Whereas the expression of α and LHβ subunit genes is regulated by both NPY and PACAP, the effect on the FSHβ transcript is elicited only by PACAP38. NPY and PACAP38 stimulatory actions are mediated via protein kinase C (PKC) and protein kinase A (PKA), converging at the MEK-ERK cascade. These findings represent one of the fine tuning levels that differentially regulates gonadotropin subunit gene expression.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Our previous studies on annexin 5, a member of the annexin family of proteins, have shown its expression in the anterior pituitary gland, its preferential distribution in gonadotropes, and its increase after ovariectomy. In the present study, we examined (1) whether annexin 5 is synthesized in gonadotropes, (2) whether its expression is under the control of gonadotropin-releasing hormone (GnRH), and (3) the effect of annexin 5 on gonadotropin release. Large cells, also called castration cells, appeared in anterior pituitary tissue 3 weeks after ovariectomy. These cells have been confirmed to be hyperfunctioning gonadotropes and are easily discriminated from other pituitary cells without immunostaining. Using in situ hybridization with a digoxigenin-labeled ribonucleic acid probe, enhanced expression of annexin 5 messenger ribonucleic acid (mRNA) in these gonadotropes was clearly demonstrated. Northern blot analysis showed an increase in the level of annexin 5 mRNA expression 3 weeks after ovariectomy. It was lessened 3 h after the injection of Cetrorelix (GnRH antagonist, 10 μg i.v.). Administration of a GnRH analog [GnRHa; Des-Gly 10 (Pro9) GnRH ethylamide, 0.2 ml of 2.5 μg/ml saline ten times intraperitoneally at 30-min intervals] significantly increased pituitary annexin 5 mRNA. In primary cultures of anterior pituitary cells, recombinant rat annexin 5 stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in a dose-dependent manner. Concomitant administration of annexin 5 (1 μg/ml) and GnRHa augmented the LH and FSH release induced by GnRHa. After a 1-hour incubation, cycloheximide (10 μg/ml) apparently inhibited the LH response to GnRHa, while annexin 5 (2 μg/ml) moderated this inhibition. Further, the antisense oligodeoxynucleotide to annexin 5 mRNA blunted the LH response to GnRHa. It is thus concluded that annexin 5 is synthesized in the gonadotropes under the effect of GnRH, and it is suggested that annexin 5 synthesis mediates at least partly GnRH receptor signaling to stimulate gonadotropin secretion.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Prolactin (PRL)-releasing peptide (PrRP) is a novel hypothalamic peptide reported to be a potent and specific stimulator of PRL secretion. This author recently reported that PrRP might play a significant role in mediating the steroid-induced PRL surge in the rat. In order to examine the secretory profile of PrRP in the rat hypothalamus before and during the luteinizing hormone (LH) and PRL surges, this study employed the push-pull perfusion technique and determined the in vivo release of PrRP and also of gonadotropin-releasing hormone (GnRH) in ovariectomized rats primed with estradiol and progesterone. In the medial preoptic area (MPOA) where the GnRH neuronal perikarya exist, GnRH release was increased prior to the initiation of the LH surge, and PrRP also started rising even earlier than GnRH. In the median eminence-arcuate nucleus complex (ME-ARC), where GnRH neuronal fibers terminate, GnRH secretion started increasing before the commencement of the LH surge, but the release of PrRP did not change significantly. These results suggest that PrRP may play a role in mediating the steroid-induced LH surge by activating GnRH neurons in the MPOA. A possible involvement of PrRP in the PRL surge was not suggested from the present data. The lack of a significant alteration in PrRP release in the ME-ARC may argue against a direct hypophysiotropic action of the peptide.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Ciliary neurotropic factor (CNTF) is a neuroregulatory cytokine belonging to the interleukin-6 type cytokine superfamily. Although a few studies have reported a facilitatory action of CNTF on the reproductive axis in rodents, information along this line is still very limited. In this study, we examined a possible role of CNTF in the generation of ovarian steroid-induced luteinizing hormone (LH) and prolactin (PRL) surges in the rat, a crucial physiological event in mammalian reproduction. Experiments were performed on both normally-fed and 3-day-fasted rats, ovariectomized and primed with estradiol and progesterone. Blood was collected every 30 min between 11:00 and 18:00 h, to measure LH and PRL. Drugs were given intracerebroventricularly at 11:00 h. Compared to control serum, undiluted as well as threefold dilutions of anti-CNTF serum caused partial but significant suppression of LH surges. Both concentrations of the antibody also delayed the onset of PRL surge to a comparable degree. Fasted rats did not exhibit significant surges of the hormones, while 0.3 and 1.0 nmol, but not 0.1 nmol, recombinant human CNTF led to a dose-dependent recovery of both LH and PRL surges. These results demonstrate for the first time a significant role of CNTF in the generation of preovulatory LH and PRL surges in the rat. CNTF may thus be another humoral signal linking nutrition and reproductive function.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

It has repeatedly been suggested that galanin acts within the anterior pituitary (AP) in an auto/paracrine manner to modulate luteinizing hormone (LH) release. Except for one recent report in the rat, evidence for this notion is absent. The purpose of this study was to investigate in the pig the effects of galanin on LH and growth hormone (GH) release and to evaluate putative local effects using various AP culture systems (monolayer, perifusion, reaggregates). Independent of age galanin dose dependently (0.05, 0.2, 1 μM) stimulated basal but not gonadotropin-releasing hormone (GnRH; ≥0.01 nM)-induced LH release. Neither basal nor GH-releasing hormone (GHRH)-stimulated GH release was affected at any age. Of 4 galanin receptor antagonists (0.2, 1 μM) tested C7 proved to have agonistic effects, whereas M40 and M15 (galantide) were ineffective in blocking galanin (0.2 μM)-induced LH secretion or affecting basal or GnRH-induced LH release. M32 [galanin (1–13) NPY (25–36) amide] inhibited (p ≤ 0.05) GnRH-induced LH release at doses of ≥2 μM, an effect which could be totally compensated by 1 μM galanin. However, the neuropeptide (NPY) antagonist BIBP 3226 (1 μM) partially overcame the effect of M32 (M32 is known to also bind to NPY receptors and NPY is inhibitory in the pig). In further studies using APs from preovulatory gilts a specific well-characterized galanin antiserum diluted 1:20 or 1:50 attenuated GnRH-induced LH release (p ≤ 0.05). However, an NPY antiserum (also affinity purified and at the same dilution) used as control unexpectedly inhibited GnRH (and galanin)-induced LH release as well, thus suggesting that attenuation of GnRH-induced LH release by galanin antiserum might be at least partly nonspecific. Furthermore 96-hour exposure of AP reaggregates to two types of porcine preprogalanin antisense oligodeoxynucleotides neither affected basal nor GnRH-induced LH release. In line with the failure to unequivocally prove paracrine effects of galanin, concentrations of galanin in AP cultures and AP culture medium were very low (≤2 pg galanin/10[sup 5] AP cells). In conclusion the present study provides some evidence to ascribe a hypophysiotropic role to galanin in regulating LH but not GH secretion in the pig. The study also points to the critical role of appropriate controls when trying to prove auto/paracrine control mechanisms within the anterior pituitary. Our findings do not provide convincing evidence to support the notion that intrapituitary galanin is involved in the fine tuning of LH secretion, at least in the preovulatory pig.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Rat and hamster brain tissues were used to investigate the possible existence of a follicle stimulating hormone (FSH)-releasing factor with similar characteristics to the lamprey gonadotropin-releasing hormone III (lGnRH-III) form proposed in previous reports. The present studies involved isolation and purification of the molecule by high-performance liquid chromatography (HPLC), identification by radioimmunoassay, sequence analysis by automated Edman degradation, mass spectrometry and examination of biological activity. Hypothalamic extracts from both species contained an HPLC fraction that was immunoreactive to GnRH and coeluted with lGnRH-III and 9-hydroxyproline mGnRH ([Hyp[sup 9] ]GnRH). Determination of primary structure from purified total brain material demonstrated that the isolated molecule was [Hyp[sup 9] ]GnRH. This is the first report showing the presence of the posttranslationally modified form already known as [Hyp[sup 9] ]GnRH by primary sequence analysis. The biological activity of distinct GnRH peptides was also tested in vitro for gonadotropin release using rat pituitary primary cell cultures. The results showed that [Hyp[sup 9] ]GnRH stimulated both luteinizing hormone and FSH release, as already reported, whereas lGnRH-III had no action on the secretion of either gonadotropin.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The study was designed to determine whether the ability of central oxytocin (OT) to stimulate gonadotropin-releasing hormone (GnRH) on the afternoon of proestrus (PE) in the cycling female rat is mediated at the level of GnRH terminals within the median eminence (ME), or at higher hypothalamic levels where GnRH cell bodies and axons are located. Determining the location of this OT effect in vivo has proven difficult. Therefore, an in vitro system utilizing ME or basal hypothalamic (BH) explants containing GnRH terminals, or GnRH neurons including the cell bodies, axons and terminals, respectively, were harvested from regular cycling female rats at 15:00 h on PE or diestrus (DI). The explants were allowed to preincubate in Krebs Ringer Bicarbonate Buffer containing glucose, ascorbic acid, calcium, and a metalloprotease inhibitor (KRBG) and enriched with 95% O[sub 2] /5% CO[sub 2] at 37°C until a stable baseline release of GnRH was achieved (30 min). The 0.05 level of probability was used as the minimum criterion of significance in all experiments. The ability of OT (10[sup –15] –10[sup –9] M) to stimulate the release of GnRH was determined in both ME and BH explants on PE and DI. The results demonstrated a sensitive, dose-dependent ability of OT to stimulate GnRH release from PE BH explants which was observed only in PE. Furthermore, OT failed to significantly stimulate GnRH release from ME explants on either PE or DI. The data indicate that the PE BH explant paradigm can be used to examine the manner and mechanisms by which OT influences GnRH release on the afternoon of PE. Furthermore, the results indicate for the first time that the stimulatory action of OT by itself on preovulatory GnRH release in cycling female rats is not mediated at the level of the GnRH terminals within the ME, but requires neuronal interactions and mechanisms within the BH explants.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Synaptic transmission at the vocal synapse, the laryngeal neuromuscular junction, of Xenopus laevis has been shown to be regulated by long-term changes in circulating estrogen. In females, high levels of circulating estrogen also accompany gonadotropin-induced ovulation and oviposition and the switch from sexually unreceptive to receptive states, including changes in vocal behaviors (ticking to rapping). Here we examine the effects of gonadotropin injection on laryngeal synaptic strength and call type. Gonadotropin acutely reduced quantal content values of laryngeal synapses in intact, adult females; the lowest values were attained by 12 h post-injection. Estrogen and progesterone levels increased following human chorionic gonadotropin (hCG) injection; the time course was similar to, but negatively correlated with, changes in synaptic strength. In ovariectomized frogs, exogenous estrogen, but not progesterone or hCG, mimicked the acute effects of hCG in weakening laryngeal synapses of intact frogs. hCG injection suppressed ticking and sometimes induced rapping. Females could tick with either strong or weakened laryngeal synapses while rapping was only produced during the weakening action of hCG. The normally strong synapses of females may enable vocal production even when laryngeal synapses are weakened by hormones that induce ovulation. In contrast to the acute effect of estrogen on weakening laryngeal synapses, juveniles required more than 2 weeks of estrogen treatment to strengthen laryngeal synapses while at least 4 weeks postovariectomy were required to weaken synapses in adult females. We conclude that acute (hours) increases in circulating levels of estrogen weaken synapses while chronic (weeks) increases strengthen laryngeal synapses.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Leptin is an adipocyte-derived hormone that has been implicated to serve as a metabolic signal to the reproductive axis. The role of leptin in pubertal maturation, however, has been a much-debated topic. We have previously reported that leptin serves as a permissive signal to the onset of puberty in the female rat. In an attempt to further understand the mechanics of leptin during pubertal maturation in rodent species, we had three experimental objectives: first, to describe the temporal relationship of leptin with development in the male and female rat; second, to seek evidence for an increase in responsiveness of the neuroendocrine axis to leptin by assessing for possible changes in leptin receptor expression during pubertal developmental in the female rat; and, third, to reevaluate the possible role of leptin as a permissive signal to the onset of puberty in the mouse. We found that serum leptin levels remain relatively constant during the prepubertal and postpubertal stages of both sexes. In addition, we could not detect any significant developmental changes in leptin receptor gene expression in the hypothalamus of the female rat. Lastly, we corroborated our findings in the female rat that leptin reversed the delay in pubertal maturation secondary to food restriction but did not advance the onset of puberty in female mice. Together, these results suggest that leptin is not a metabolic trigger for the onset of puberty in the rodent; instead, leptin is one of several permissive factors, whose presence may be necessary but alone is not sufficient to initiate sexual maturation in these species.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Photoperiod has profound effects upon the neuroendocrine axis underlying reproductive physiology in seasonally breeding mammals. For long-day (LD) breeders, such as the Siberian hamster, exposure to a short-day (SD) photoperiod results in declines in circulating levels of gonadal steroids, luteinizing hormone (LH), and prolactin (PRL). The current study sought to investigate the effects of photoperiod and steroid levels on norepinephrine (NE), one of the major neurochemical regulators of gonadotropin-releasing hormone (GnRH) function. Since NE release within the medial preoptic area (mPOA) has been shown to stimulate the activity of GnRH cells, it was hypothesized that exposure to a short photoperiod would decrease NE levels. Furthermore, since gonadal steroids show negative feedback on GnRH function, it was hypothesized that gonadectomy would result in increased levels of NE. Adult male and female Siberian hamsters were gonadectomized and implanted with silastic capsules containing either cholesterol (C) or a mixture of estradiol (E) or testosterone (T). Microdialysis sampling within the mPOA was conducted after 8 weeks of exposure to either an LD or an SD photoperiod. Blood samples were analyzed for LH and PRL, while dialysis samples were analyzed for NE and its major metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG). The results revealed significant suppression of LH and PRL by exposure to the SD photoperiod in both males and females. For LH, the steroid implants suppressed circulating hormone levels under both photoperiods, whereas for PRL, steroid treatment facilitated circulating levels. In contrast, there were no significant effects of photoperiod on NE or MHPG release for either males or females, but there was a significant decrease in extracellular levels of these neurochemicals in steroid-treated animals. These data suggest that photoperiodic modulation of GnRH neuronal function by NE is achieved largely through the indirect effects of photoperiod on circulating gonadal steroids.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]