The Study On The Mechanism Of Orexin On The Hypothalamus-pituitary-gonad Axis In Suzhong Swine In Vivo And Vitro

Orexin,a recently discovered neuropeptide,are divided as orexin A and orexin B. Orexin roles are mediated by it's two different subtypes receptor(OXR)(OX1R and OX2R).Orexin has been implicated in the regulation of food intake,arousal states, energy metabolism,activity of visceral organs,neuroendocrine and reproduction.The datas that orexin involved in the regulation of reproduction were got mostly from rat and sheep,and very limited.Moreover,the distributions of orexin B and regulation of reproduction by orexin in swine were not reported in present literatures.Therefore, the distribution of orexin B in Suzhong swine,the relationship with GnRH and OB-Rb in the forebrain,expression of reproduction related neuropeptide and hormone mRNA in the hypothalamus and pituitary and PRL,FSH,leptin and insulin levels in serum after orexin A injected into lateral ventricle in ovariectomized swine,and effect of orexin on hypothalamus-pituitary-ovary in vitro were studied in present study with immunohistochemistry,double-labelled immunofluorescence combined laser-scanning confocal microscopy,real time-PCR and radioimmunoassay(RIA),in order to provide the morphological and physiological data for orexin role in regulating reproduction of swine.1 Localizatin of orexin B immunoreactive cells in swine10 Suzhong swines,1-2 months old,were used in this study in order to investigate localization of orxin B in the brain,spinal cord,some visceral organs and endocrine glands of swine with immunohistochemical method.The results were as follows:(1) Orexin B immunoreactive neurons were observed in the forebrain,brainstem, spinal cord,some visceral organs and endocrine glands in swine.In the forebrain, orexin B immunoreactive neurons mainly localized in the preoptic area,anterior hypothalamic area,periventricular and paraventricular nuclei,posterior hypothalamic area,dorsomedial hypothalamic nucleus,lateral hypothalamic area, perifornical area,zona incerta,mammillary area and so on.In the brainstem,orexin B immunoreactive neurons distributed in many nuclei,including the vestibular nuclus, locus coeruleus,solitary tract nucleus,inferior olivary nucleus and raphe pallidus.In the spinal cord,orexin B immunoreactive neurons only presented in the dorsal horn. In visceral organs,orexin B immunoreactive cell were observed mostly in the ovary, testicle,epididymis,stomach,duodenum,jejunum,ileum,colon,pancreas and spleen. In endocrine glands,orexin B immunoreactive cells were distributed in the pituitary and thyroid gland.(2) Orexin B immunoreactive fibers projected extensively in the the central nervous system.In the telencephalon,orexin B immunoreactive fibers projected to the cortex, septal area,bed nucleus of stria terminalis,caudate nucleus,amygdaloid nucleus and diagonal band.In the diencephalons,orexin B immunoreactive fibers densely projected to the habenular nucleus,dorsomedial hypothalamic nucleus,periventricular nucleus,supraoptic and suprachiasmatic nuclei.There were moderately dense orexin B immunoreactive fibers in the supramammillary nucleus,ventromedial nucleus, anterior hypothalamic area,posterior hypothalamic area,arcuate nucleus/median eminence.In the brain stem,there were very dense orexin B immunoreactive fibers in the superior colliculus,inferior colliculus,interpeduncular nucleus,dorsal raphe nucleus and central gray.Abundant orexin B immunoreactive fibers also projected to the pontine nucleus,locus coeruleus and the dorsal tegmental nucleus.Moderate numbers of orexn B fibers were observed in,area postrema,fourth ventricle and parabrachial nucleus.In addition,Small numbers of orexn B fibers were observed in the cerebellum,pituitary and spinal cord.2 The relationship of orexin B with GnRH and OB-Rb in the pig forebrain10 Suzhong swines,1-2 months old were used in this study in order to investigate the morphological relationship between orexin B and GnRH,orexin B and OB-Rb using double-labelled immunofluorescence combined laser-scanning confocal microscopy.The results showed that approximately 30%of GnRH fibres and neurons were contacted by orexin B fibres from the septal area and OVLT to the arcuate nucleus. Approximately 6%GnRH perikarya overlapped with orexin B perikarya The overlapped neurons were mainly concentrated in the supraoptic nucleus.In the anterior hypothalamic area,periventricular,paraventricular nucleus,zona incerta, lateral hypothalamic area and ventromedial nucleus,the orexin B neurons expressed OB-Rb. 3.The effect of orexin A on expression of reproduction related neuropeptide and hormone mRNA in the hypothalamus and pituitary after injection of orexin A into the lateral ventricle in ovariectomized swineThe expression of PRL,leptin,OB-Rb and OXRS mRNA in the hypothalamus and pituitary was studied with real time PCR in 10 ovariectomized sows after injection of orexin A or physiological saline into the lateral ventricle in order to approach the pathway through which orexin A regulate pig reproduction.The results were as follows:(1) After injection of orexin A into the lateral ventricle,it depressed expression of PRL mRNA in the pituitary.(2) After injection of it into the lateral ventricle,orexin A depressed expression of leptin,OB-Rb mRNA,and promoted expression of OX1R mRNA in the hypothalamus and pituitary.4 The effect of orexin A on serum PRL,FSH,leptin and insulin level after injection of orexin A into the lateral ventricle in ovariectomized swineHormone levels in serum was analyzed with radioimmunoassay(RIA) from 1.5 h before injection to 3 h after injection of orexin A or physiological saline into the lateral ventricle in 10 ovariectomized swines in order to study the effect of orexin A on PRL,FSH,leptin and insulin secretion(blood sample interval was 10 min).The results were as follows:(1) Orexin A depressed the secretion and release of leptin after injection of orexin A into the lateral ventricle,and leptin concentration in serum began to descend at 10 min,was down to the lowest point at 30 min after injection,and maintained the low level for 80 min.(2) Orexin A promoted the secretion and release of insulin after injection of orexin A into the lateral ventricle.Insulin concentration in serum began to increase 10 min, was up to the peak 30 min after,and returned to the initial level at 110 min after injection.(3) Orexin A depressed the secretion and release of PRL after injection of orexin A into the lateral ventricle.PRL concentration in serum began to descend at 10 min, was down to the lowest 40 min after injection,maintained the low level for 80 min.(4) Orexin A had no significant influence on FSH level in plasm after injection of orexin A into the lateral ventricle.5.The effect of orexin on the hypothalamus-pituitary-ovary axis in vitro To study the action target of orexin,we studied hypothalamic explants anterior pituitary and ovary gransola cells in vitro.Our results were as follows: (1) After hypothalamic explants were challenged for 45 min with orexin A(1,10,100 and 1000 nm),the GnRH levels in the incubated aCSF were measured by radioimmunoassay(RIA).Orexin A(100 and 1000 nM) stimulated the release of GnRH from hypothalamic explants harvested from prepuberal swine.However,only 1000 nM orexin B could stimulated the release of GnRH from the hypothalamic explants harvested from prepuberal swine.(2) orexin A(10,100 and 1000 nm) could dose-dependent stimulate the release of LH from anterior pituitary cells.And orexin B(100,1000 nM) also could stimulate the release of LH from anterior pituitary cells.(3) Orexin A(10,100 and 1000 nM) could stimulate the release of E2 from ovary gransola cells.But,the 100 nM dose was more effective than 1000 nM.Orexin B(100 and 1000 nM) also could stimulate the release of E2 from ovary gransola cells.The above studies showed that orexin B was observed in the hypothalamus, pituitary and ovary of swine,which provided the morphologic data for orexin role involving in regulation of pig reproduction.Orexin B colocalized with GnRH and OB-Rb suggested that orexin could directly act to GnRH neuron or indirectly act to leptin participate in regulating reproduction.After injection of orexin A into the lateral ventricle,it depressed the expression of PRL,leptin and OB-Rb mRNA and promoted the expression of OX1R mRNA in the hypothalamus and pituitary, Meanwhile,it depressed PRL,leptin and increased insulin level in serum,and orexin could stimulate the release of GnRH,LH and E2 from hypothalamus plants,the anterior lobe of pituitary and ovary gransola cells.This investigation suggested that orexin be involved in regulation of reproduction either by stimulating directly release of GnRH and LH,or via affecting indirectly release of GnRH and LH by the way of leptin and insulin.These studies provided morphological and physiological data for orexin role in regulating reproductive activity.