Characterization of the rat pituitary capsule: Evidence that the cerebrospinal fluid filled the pituitary cleft and the inner side of the capsule.

In humans, the pituitary gland is covered by a fibrous capsule and is considered a continuation of the meningeal sheath. However, in rodents some studies concluded that only the pars tuberalis (PT) and pars nervosa (PN) are enwrapped by the pia mater, while others showed that the whole gland is covered by this sheath. At PT the median eminence subarachnoid drains cerebrospinal fluid (CSF) to its cisternal system representing a pathway to the hypothalamus. In the present study we examined the rat pituitary capsule to elucidate its configuration, its physical interaction with the pituitary border and its relationship with the CSF. Furthermore, we also revisited the histology of the pituitary cleft and looked whether CSF drained in it. To answer such questions, we used scanning and transmission electron microscopy, intracerebroventricular infusion of Evan´s blue, fluorescent beads, and sodium fluorescein. The latter was measured in the pars distalis (PD) and various intracranial tissues. We found a pituitary capsule resembling leptomeninges, thick at the dorsal side of the pars intermedia (PI) and PD, thicker at the level of PI in contiguity with the PN and thinner at the rostro-ventral side as a thin membrane of fibroblast-like cells embedded in a fibrous layer. The capsule has abundant capillaries on all sides. Our results showed that the CSFs bathe between the capsule and the surface of the whole gland, and ciliate cells are present in the pituitary border. Our data suggest that the pituitary gland intercommunicates with the central nervous system (CNS) through the CSF.

Impact of Hydrocortisone and of CRH Infusion on the Hypothalamus-Pituitary-Adrenocortical Axis of Septic Male Mice.

PURPOSE: Sepsis is hallmarked by high plasma cortisol/corticosterone (CORT), low adrenocorticotropic hormone (ACTH), and high pro-opiomelanocortin (POMC). While corticotropin-releasing hormone-(CRH) and arginine-vasopressin (AVP)-driven pituitary POMC expression remains active, POMC processing into ACTH becomes impaired. Low ACTH is accompanied by loss of adrenocortical structure, although steroidogenic enzymes remain expressed. We hypothesized that treatment of sepsis with hydrocortisone (HC) aggravates this phenotype whereas CRH infusion safeguards ACTH-driven adrenocortical structure. METHODS: In a fluid-resuscitated, antibiotics-treated mouse model of prolonged sepsis, we compared the effects of HC and CRH infusion with placebo on plasma ACTH, POMC, and CORT; on markers of hypothalamic CRH and AVP signaling and pituitary POMC processing; and on the adrenocortical structure and markers of steroidogenesis. In adrenal explants, we studied the steroidogenic capacity of POMC. RESULTS: During sepsis, HC further suppressed plasma ACTH, but not POMC, predominantly by suppressing sepsis-activated CRH/AVP-signaling pathways. In contrast, in CRH-treated sepsis, plasma ACTH was normalized following restoration of pituitary POMC processing. The sepsis-induced rise in markers of adrenocortical steroidogenesis was unaltered by CRH and suppressed partially by HC, which also increased adrenal markers of inflammation. Ex vivo stimulation of adrenal explants with POMC increased CORT as effectively as an equimolar dose of ACTH. CONCLUSIONS: Treatment of sepsis with HC impaired integrity and function of the hypothalamic-pituitary-adrenal axis at the level of the pituitary and the adrenal cortex while CRH restored pituitary POMC processing without affecting the adrenal cortex. Sepsis-induced high-circulating POMC may be responsible for ongoing adrenocortical steroidogenesis despite low ACTH.

The Type of Fat in the Diet Influences Regulatory Aminopeptidases of the Renin-Angiotensin System and Stress in the Hypothalamic-Pituitary-Adrenal Axis in Adult Wistar Rats.

(1) Background: Prolonged feeding with a high-fat diet (HFD) acts as a stressor by activating the functions of the hypothalamic-pituitary-adrenal gland (HPA) stress axis, accompanied of hypertension by inducing the renin-angiotensin-aldosterone system. Angiotensinases enzymes are regulatory aminopeptidases of angiotensin metabolism, which together with the dipeptidyl peptidase IV (DPP-IV), pyroglutamyl- and tyrosyl-aminopeptidase (pGluAP, TyrAP), participate in cognitive, stress, metabolic and cardiovascular functions. These functions appear to be modulated by the type of fat used in the diet. (2) Methods: To analyze a possible coordinated response of aminopeptidases, their activities were simultaneously determined in the hypothalamus, adenohypophysis and adrenal gland of adult male rats fed diets enriched with monounsaturated (standard diet (S diet) supplemented with 20% virgin olive oil; VOO diet) or saturated fatty acids (diet S supplemented with 20% butter and 0.1% cholesterol; Bch diet). Aminopeptidase activities were measured by fluorimetry using 2-Naphthylamine as substrates. (3) Results: the hypothalamus did not show differences in any of the experimental diets. In the pituitary, the Bch diet stimulated the renin-angiotensin system (RAS) by increasing certain angiotensinase activities (alanyl-, arginyl- and cystinyl-aminopeptidase) with respect to the S and VOO diets. DPP-IV activity was increased with the Bch diet, and TyrAP activity decrease with the VOO diet, having both a crucial role on stress and eating behavior. In the adrenal gland, both HFDs showed an increase in angiotensinase aspartyl-aminopeptidase. The interrelation of angiotensinases activities in the tissues were depending on the type of diet. In addition, correlations were shown between angiotensinases and aminopeptidases that regulate stress and eating behavior. (4) Conclusions: Taken together, these results support that the source of fat in the diet affects several peptidases activities in the HPA axis, which could be related to alterations in RAS, stress and feeding behavior.

Lithocholic acid inhibits P2X2 and potentiates P2X4 receptor channel gating.

The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes.

Anterior pituitary gland synthesises dopamine from l-3,4-dihydroxyphenylalanine (l-dopa).

Prolactin (PRL) is a hormone principally secreted by lactotrophs of the anterior pituitary gland. Although the synthesis and exocytosis of this hormone are mainly under the regulation of hypothalamic dopamine (DA), the possibility that the anterior pituitary synthesises this catecholamine remains unclear. The present study aimed to determine if the anterior pituitary produces DA from the precursor l-3,4-dihydroxyphenylalanine (l-dopa). Accordingly, we investigated the expression of aromatic l-amino acid decarboxylase (AADC) enzyme and the transporter vesicular monoamine transporter 2 (VMAT2) in the anterior pituitary, AtT20 and GH3 cells by immunofluorescence and western blotting. Moreover, we investigated the production of DA from l-dopa and its release in vitro. Then, we explored the effects of l-dopa with respect to the secretion of PRL from anterior pituitary fragments. We observed that the anterior pituitary, AtT20 and GH3 cells express both AADC and VMAT2. Next, we detected an increase in DA content after anterior pituitary fragments were incubated with l-dopa. Also, the presence of l-dopa increased DA levels in incubation media and reduced PRL secretion. Likewise, the content of cellular DA increased after AtT20 cells were incubated with l-dopa. In addition, l-dopa reduced corticotrophin-releasing hormone-stimulated adrenocorticotrophic hormone release from these cells after AADC activity was inhibited by NSD-1015. Moreover, DA formation from l-dopa increased apoptosis and decreased proliferation. However, in the presence of NSD-1015, l-dopa decreased apoptosis and increased proliferation rates. These results suggest that the anterior pituitary synthesises DA from l-dopa by AADC and this catecholamine can be released from this gland contributing to the control of PRL secretion. In addition, our results suggest that l-dopa exerts direct actions independently from its metabolisation to DA.

Pregnancy-induced changes in the transcript levels of prolactin receptor and its suppressor in the ovine hypothalamus and adenohypophysis.

The aim of this study was to analyse changes in the abundance of prolactin (PRL) receptor (PRLR) and suppressor of cytokine signalling-3 (SOCS-3) mRNA in the ventro-/dorsomedial nucleus (VMH/DMH) and arcuate nucleus (ARC) of the hypothalamus as well as in the median eminence (ME) and adenohypophysis (AP) in sheep at 30, 60, 90 and 120 d of pregnancy compared to non-pregnant animals. In the VMH/DMH, PRLR transcripts were detected only in non-pregnant ewes. In the ARC, the abundances of PRLR mRNA were higher in pregnant sheep on days 30 (p < .01), 90 (p < .01) and 120 (p < .05) than in non-pregnant sheep. In contrast, the expression of PRLR mRNA in the ME was lower (p < .01) in pregnant ewes at days 30 and 60 than in non-pregnant ewes and was undetectable at later stages of gestation. In all studied stages of pregnancy except day 60, the abundance of PRLR mRNA was higher (p < .01) in the ARC than in the AP, while in non-pregnant sheep, there were no differences (p ≥ .05) in the transcript levels between these two tissues. In non-pregnant ewes, the abundance of SOCS-3 mRNA in the AP was lower than that in any other studied tissue (p < .05-p < .01). In conclusion, the observed changes in PRLR and SOCS-3 mRNA abundance in the hypothalamus and AP during pregnancy may be important components of the mechanisms regulating the action of PRL in energy homeostasis and neuroendocrine interactions within the hypothalamic-pituitary axis.

Bioactive growth hormone in humans: Controversies, complexities and concepts.

OBJECTIVE: To revisit a finding, first described in 1978, which documented existence of a pituitary growth factor that escaped detection by immunoassay, but which was active in the established rat tibia GH bioassay. METHODS: We present a narrative review of the evolution of growth hormone complexity, and its bio-detectability, from a historical perspective. RESULTS: In humans under the age of 60, physical training (i.e. aerobic endurance and resistance training) are stressors which preferentially stimulate release of bioactive GH (bGH) into the blood. Neuroanatomical studies indicate a) that nerve fibers directly innervate the human anterior pituitary and b) that hind limb muscle afferents, in both humans and rats, also modulate plasma bGH. In the pituitary gland itself, molecular variants of GH, somatotroph heterogeneity and cell plasticity all appear to play a role in regulation of this growth factor. CONCLUSION: This review considers more recent findings on this often forgotten/neglected subject. Comparison testing of a) human plasma samples, b) sub-populations of separated rat pituitary somatotrophs or c) purified human pituitary peptides by GH bioassay vs immunoassay consistently yield conflicting results.

Hypothalamic-Pituitary and Adipose Tissue Responses to the Effect of Resistin in Sheep: The Integration of Leptin and Resistin Signaling Involving a Suppressor of Cytokine Signaling 3 and the Long Form of the Leptin Receptor.

We hypothesized that resistin is engaged in the development of leptin central insensitivity/resistance in sheep, which is a unique animal model to explore reversible leptin resistance. Thirty Polish Longwool ewes, which were ovariectomized with estrogen replacement, were used. Treatments consisted of the intravenous injection of control (saline) or recombinant bovine resistin (rbresistin): control (Control; n = 10), a low dose of rbresistin (R1; 1.0 µg/kg body weight (BW); n = 10), and a high dose of rbresistin (R2; 10.0 µg/kg BW; n = 10). The studies were performed during short-day (SD) and long-day (LD) photoperiods. Leptin and resistin concentrations were determined. Expression levels of a suppressor of cytokine signaling (SOCS)-3 and the long form of the leptin receptor (LeptRb) were determined in selected brain regions, including in the anterior pituitary (AP), hypothalamic arcuate nucleus (ARC), preoptic area (POA), and ventro- and dorsomedial nuclei (VMH/DMH). The results indicate that resistin induced a consistent decrease in LeptRb (except in POA) and an increase in SOCS-3 expression during the LD photoperiod in all selected brain regions. In conclusion, the results demonstrate that the action of resistin appears to be strongly associated with photoperiod-driven changes in the leptin signaling pathway, which may underlie the phenomenon of central leptin resistance.

Selenium protection against mercury neurotoxicity: Modulation of apoptosis and autophagy in the anterior pituitary.

AIMS: The aim of the present study is to shed light on the modulating action of selenium on two of the most crucial cellular pathways; apoptosis and autophagy and the possible interplay between them in determining the pituitary fate in the context of mercury intoxication through demonstration of the molecular, histopathological, immunohistochemical, and ultrastructural features of selenium mercury-treated adenohypophysis. METHODS: Thirty adult Sprague Dawley male albino rats were assigned into control group, mercury-treated group and mercury­selenium concomitantly-treated group. The adenohypophysis was subjected to structural, molecular and protein expression assessment of autophagy and apoptotic markers and western blotted analysis of Beclin 1 as a key cross-regulator of autophagy and apoptosis. KEY FINDINGS: Selenium treatment ameliorated the mercury-induced apoptosis detected by improvement in PCR and immunohistochemical expression of the apoptotic markers Bax, Bcl-2 and Caspase-3. Selenium also improved mercury-induced autophagic dysfunction with statistically significant improvement in western blotted levels of the autophagy markers LC3I, LC3II and Beclin1. The histopathological and ultrastructural studies strongly confirmed those findings. SIGNIFICANCE: The crosstalk between the apoptotic Bcl-2 family of proteins and the autophagic Beclin-1LC3 pathway in the context of mercury intoxication paves the way for developing novel effective treatment strategies for several mercury-induced pituitary diseases.

Dysregulation of hypothalamic-pituitary estrogen receptor α-mediated signaling causes episodic LH secretion and cystic ovary.

Polycystic ovary syndrome (PCOS) is a hypothalamic-pituitary-gonadal (HPG) axis disorder. PCOS symptoms most likely result from a disturbance in the complex feedback regulation system of the HPG axis, which involves gonadotrophic hormones and ovarian steroid hormones. However, the nature of this complex and interconnecting feedback regulation makes it difficult to dissect the molecular mechanisms responsible for PCOS phenotypes. Global estrogen receptor α (ERα) knockout (KO) mice exhibit a disruption of the HPG axis, resulting in hormonal dysregulation in which female ERα KO mice have elevated levels of serum estradiol (E2), testosterone, and LH. The ERα KO females are anovulatory and develop cystic hemorrhagic ovaries that are thought to be due to persistently high circulating levels of LH from the pituitary. However, the role of ERα in the pituitary is still controversial because of the varied phenotypes reported in pituitary-specific ERα KO mouse models. Therefore, we developed a mouse model where ERα is reintroduced to be exclusively expressed in the pituitary on the background of a global ERα-null (PitERtgKO) mouse. Serum E2 and LH levels were normalized in PitERtgKO females and were comparable to wild-type serum levels. However, the ovaries of PitERtgKO adult mice displayed a more overt cystic and hemorrhagic phenotype when compared with ERα KO littermates. We determined that anomalous sporadic LH secretion caused the severe ovarian phenotype of PitERtgKO females. Our observations suggest that pituitary ERα is involved in the estrogen negative feedback regulation, whereas hypothalamic ERα is necessary for the precise control of LH secretion. Uncontrolled, irregular LH secretion may be the root cause of the cystic ovarian phenotype with similarities to PCOS.-Arao, Y., Hamilton, K. J., Wu, S.-P., Tsai, M.-J., DeMayo, F. J., Korach, K. S. Dysregulation of hypothalamic-pituitary estrogen receptor α-mediated signaling causes episodic LH secretion and cystic ovary.

Energy balance affects pulsatile secretion of luteinizing hormone from the adenohypophesis and expression of neurokinin B in the hypothalamus of ovariectomized gilts.

The pubertal transition of gonadotropin secretion in pigs is metabolically gated. Kisspeptin (KISS1) and neurokinin B (NKB) are coexpressed in neurons within the arcuate nucleus of the hypothalamus (ARC) and are thought to play an important role in the integration of nutrition and metabolic state with the reproductive neuroendocrine axis. The hypothesis that circulating concentrations of luteinizing hormone (LH) and expression of KISS1 and tachykinin 3(TAC3, encodes NKB) in the ARC of female pigs are reduced with negative energy balance was tested using ovariectomized, prepubertal gilts fed to either gain or lose body weight. Restricted feeding of ovariectomized gilts caused a rapid and sustained metabolic response characterized by reduced concentrations of plasma urea nitrogen, insulin, leptin, and insulin-like growth factor-1 and elevated concentrations of free fatty acids. The secretory pattern of LH shifted from one of low amplitude to one of high amplitude, which caused overall circulating concentrations of LH to be greater in restricted gilts. Nutrient-restricted gilts had greater expression of follicle-stimulating hormone and gonadotropin-releasing hormone receptor, but not LH in the anterior pituitary gland. Expression of KISS1 in the ARC was not affected by dietary treatment, but expression of TAC3 was greater in restricted gilts. These data are consistent with the idea that hypothalamic expression of KISS1 is correlated with the number of LH pulse in pig, and further indicate that amplitude of LH pulses may be regulated by NKB in the gilt.

Diet-Induced Growth Is Regulated via Acquired Leptin Resistance and Engages a Pomc-Somatostatin-Growth Hormone Circuit.

Anorexigenic pro-opiomelanocortin (Pomc)/alpha-melanocyte stimulating hormone (αMSH) neurons of the hypothalamic melanocortin system function as key regulators of energy homeostasis, also controlling somatic growth across different species. However, the mechanisms of melanocortin-dependent growth control still remain ill-defined. Here, we reveal a thus-far-unrecognized structural and functional connection between Pomc neurons and the somatotropic hypothalamo-pituitary axis. Excessive feeding of larval zebrafish causes leptin resistance and reduced levels of the hypothalamic satiety mediator pomca. In turn, this leads to reduced activation of hypophysiotropic somatostatin (Sst)-neurons that express the melanocortin receptor Mc4r, elevated growth hormone (GH) expression in the pituitary, and enhanced somatic growth. Mc4r expression and αMSH responsiveness are conserved in Sst-expressing hypothalamic neurons of mice. Thus, acquired leptin resistance and attenuation of pomca transcription in response to excessive caloric intake may represent an ancient mechanism to promote somatic growth when food resources are plentiful.

Autocrine actions of prolactin contribute to the regulation of lactotroph function in vivo.

Prolactin (PRL), whose principal role is regulation of lactation, is mainly synthesized and secreted by lactotroph anterior pituitary cells. Its signaling is exerted via a transmembrane PRL receptor (PRLR) expressed in a wide variety of tissues, including the anterior pituitary. Dopamine, which is secreted by tuberoinfundibular hypothalamic neurons, is the major inhibitory regulator of prolactin secretion. Although PRL is well established to stimulate hypothalamic dopamine secretion, thereby exerting a negative feedback regulation on its own release, autocrine or paracrine actions of PRL on lactotroph cells have also been suggested. Within the pituitary, PRL may inhibit both lactotroph proliferation and secretion, but in vivo evaluation of these putative functions is limited. To determine whether the autocrine actions of prolactin have a significant role in the physiologic function of lactotrophs in vivo, we examined the consequences of conditional deletion of Prlr in lactotroph cells using a novel mouse line with loxP sites flanking the Prlr gene ( Prlrlox/lox) and Cre-recombinase (Cre) expressed under the control of the pituitary-specific Prl promoter. Prlrlox/lox/Prl-Cre mice have normal PRL levels and did not develop any pituitary lactotroph adenoma, even at 20 mo of age. Nevertheless, Prlrlox/lox/Prl-Cre mice displayed an increased dopaminergic inhibitory tone compared with control Prlrlox/lox mice. These results elegantly confirm an autocrine/paracrine feedback of PRL on lactotroph cells in vivo, which can be fully compensated by an intact hypothalamic feedback system.-Bernard, V., Lamothe, S., Beau, I., Guillou, A., Martin, A., Le Tissier, P., Grattan, D., Young, J., Binart, N. Autocrine actions of prolactin contribute to the regulation of lactotroph function in vivo.

Bu-shen-zhu-yun decoction promotes synthesis and secretion of FSHß and LHß in anterior pituitary cells in vitro.

Luteal phase defects (LPD) are an important etiology of infertility which has increased in recent years. Studies have shown that bu-shen-zhu-yun decoction (BSZY-D) can lower the expression of estrogen receptor and progesterone receptor, in rats endometrium of embryonic implantation period, which upregulated by mifepristone, and improve uterine receptivity. The aim of present study was to determine the effect of BSZY-D on the synthesis and secretion of gonadotropic hormones in the anterior pituitary cells of rats. Rats were treated with saline (control) or BSZY-D two times/day for three estrous cycles by gavage. The cerebrospinal fluid (CSF) were collected for further cell treatment. The components in BSZY-D, serum and CSF were analysed by High Performance Liquid Chromatography (HPLC). Cells were either pretreated with normal CSF or BSZY-D/CSF before being stimulated with or without cetrorelix. The mRNA and proteins levels of receptors, hormones, and transcription factors were detected by RT-PCR, western blot analysis and immunostaining. We show that non-toxic concentrations of cetrorelix, a GnRH antagonist, can reduce the mRNA and protein levels of GnRHR, LH, and FSH. This effect could be reversed by the addition of BSZY-D/CSF. We also show decreased mRNA and protein expression of transcription factors, such as CREB, and Egr-1 and secretory vescicles, including SNAP-25 and Munc-18 upon treatment with cetrorelix could be reversed post co-treatment with BSZY-D/CSF. These results indicate that BSZY-D/CSF treatment led to increased levels of GnRHR, transcription factors, and secretory vesicles leading to increased secretion of FSH and LH. Thus, BSZY-D presents a promising candidate to treat luteal phase defects and infertility.

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

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

The influence of dopaminergic system inhibition on biosynthesis of gonadotrophin-releasing hormone (GnRH) and GnRH receptor in anoestrous sheep; hierarchical role of kisspeptin and RFamide-related peptide-3 (RFRP-3).

This study aimed to explain how prolonged inhibition of central dopaminergic activity affects the cellular processes governing gonadotrophin-releasing hormone (GnRH) and LH secretion in anoestrous sheep. For this purpose, the study included two experimental approaches: first, we investigated the effect of infusion of sulpiride, a dopaminergic D2 receptor antagonist (D2R), on GnRH and GnRH receptor (GnRHR) biosynthesis in the hypothalamus and on GnRHR in the anterior pituitary using an immunoassay. This analysis was supplemented by analysis of plasma LH levels by radioimmunoassay. Second, we used real-time polymerase chain reaction to analyse the influence of sulpiride on the levels of kisspeptin (Kiss1) mRNA in the preoptic area and ventromedial hypothalamus including arcuate nucleus (VMH/ARC), and RFamide-related peptide-3 (RFRP-3) mRNA in the paraventricular nucleus (PVN) and dorsomedial hypothalamic nucleus. Sulpiride significantly increased plasma LH concentration and the levels of GnRH and GnRHR in the hypothalamic-pituitary unit. The abolition of dopaminergic activity resulted in a significant increase in transcript level of Kiss1 in VMH/ARC and a decrease of RFRP-3 in PVN. The study demonstrates that dopaminergic neurotransmission through D2R is involved in the regulatory pathways of GnRH and GnRHR biosynthesis in the hypothalamic-pituitary unit of anoestrous sheep, conceivably via mechanisms in which Kiss1 and RFRP-3 participate.

Intrauterine Zn Deficiency Favors Thyrotropin-Releasing Hormone-Increasing Effects on Thyrotropin Serum Levels and Induces Subclinical Hypothyroidism in Weaned Rats.

Individuals who consume a diet deficient in zinc (Zn-deficient) develop alterations in hypothalamic-pituitary-thyroid axis function, i.e., a low metabolic rate and cold insensitivity. Although those disturbances are related to primary hypothyroidism, intrauterine or postnatal Zn-deficient adults have an increased thyrotropin (TSH) concentration, but unchanged thyroid hormone (TH) levels and decreased body weight. This does not support the view that the hypothyroidism develops due to a low Zn intake. In addition, intrauterine or postnatal Zn-deficiency in weaned and adult rats reduces the activity of pyroglutamyl aminopeptidase II (PPII) in the medial-basal hypothalamus (MBH). PPII is an enzyme that degrades thyrotropin-releasing hormone (TRH). This hypothalamic peptide stimulates its receptor in adenohypophysis, thereby increasing TSH release. We analyzed whether earlier low TH is responsible for the high TSH levels reported in adults, or if TRH release is enhanced by Zn deficiency at weaning. Dams were fed a 2 ppm Zn-deficient diet in the period from one week prior to gestation and up to three weeks after delivery. We found a high release of hypothalamic TRH, which along with reduced MBH PPII activity, increased TSH levels in Zn-deficient pups independently of changes in TH concentration. We found that primary hypothyroidism did not develop in intrauterine Zn-deficient weaned rats and we confirmed that metal deficiency enhances TSH levels since early-life, favoring subclinical hypothyroidism development which remains into adulthood.

Relationship of neuropeptide FF receptors with pubertal maturation of gilts.

Mechanisms governing the timing of puberty in pigs are poorly understood. A genome-wide association study for age at first estrus in pigs identified candidate genes including neuropeptide FF receptor 2 (NPFFR2), which is a putative receptor for RFamide-related peptides (RFRP). RFRP has been shown to negatively regulate secretion of reproductive hormones from hypothalamic and pituitary tissue of pigs in culture. Here, the porcine NPFFR2 gene was further screened and four potentially functional variants were identified to be associated with age at first estrus in pigs (1,288 gilts). The RFRP neurons in the porcine hypothalamus were localized in the paraventricular and dorsomedial nuclei with RFRP fibers in the lateral hypothalamic area. There were marked changes in expression of NPFF receptors in the anterior pituitary gland and hypothalamus of gilts beginning with the peripubertal period. The hypothesis that NPFF receptor function is related to secretion of luteinizing hormone (LH) in gilts was tested with various NPFF receptor ligands. The NPFF receptor antagonist RF9 stimulated a pulse-like release of LH in prepubertal gilts. The putative NPFF receptor agonist RFRP3 modestly suppressed LH pulses in ovariectomized (OVX) prepubertal gilts. A porcine-specific RFRP2 failed to have an effect on LH secretion in OVX prepubertal gilts despite its high degree of homology to avian gonadotropin-inhibitory hormone. Results indicate that an RFRP system is present in the pig and that NPFFR2 is important for pubertal onset in gilts. It is not clear if this regulation involves major control of LH secretion or another unknown mechanism.

Role of Nesfatin-1 in the Reproductive Axis of Male Rat.

Nesfatin-1 is an important molecule in the regulation of reproduction. However, its role in the reproductive axis in male animals remains to be understood. Here, we found that nesfatin-1 was mainly distributed in the arcuate nucleus (ARC), paraventricular nucleus (PVN), periventricular nucleus (PeN), and lateral hypothalamic area (LHA) of the hypothalamus; adenohypophysis and Leydig cells in male rats. Moreover, the concentrations of serum nesfatin-1 and its mRNA in hypothalamo-pituitary-gonadal axis (HPGA) vary with the age of the male rat. After intracerebroventricular injection of nesfatin-1, the hypothalamic genes for gonadotrophin releasing hormone (GnRH), kisspeptin (Kiss-1), pituitary genes for follicle-stimulate hormone ß(FSHß), luteinizing hormone ß(LHß), and genes for testicular steroidogenic acute regulatory (StAR) expression levels were decreased significantly. Nesfatin-1 significantly increased the expression of genes for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and cytochrome P450 cleavage (P450scc) in the testis of pubertal rats, but their levels decreased in adult rats (P < 0.05), along with the serum FSH, LH, and testosterone (T) concentrations. After nesfatin-1 addition in vitro, T concentrations of the supernatant were significantly higher than that in the control group. These results were suggestive of the role of nesfatin-1 in the regulation of the reproductive axis in male rats.