| Orexins, a recently discovered neuropeptide, are divided as orexin A and orexin B. Orexin roles are mediated orexin receptor (OXR) of two different subtypes (OX1R and OX2R). Orexins have been implicated in the regulation of food intake, arousal states, energy metabolism, activity of visceral organs, neuroendocrine and reproduction. The distributions of orexins have been reported in human and some animals, such as rat, mice, sheep, rabbit and so on. The data that orexin involving in the regulation of reproduction was got mostly from rat, sheep and human, and very limited. Moreover, the distributions of OXR and regulation of reproduction by orexin in swine were not reported in present literatures. Therefore, the distribution of OXR in Suzhong swine, changes of expression of orexin and OXR mRNA in estrous cycle of sow, and expression of related neuropeptide mRNA in hypothalamus and pituitary and LH levels in serum after orexin A injected into lateral ventricle in ovariectomized sow were studied in present study with immunohistochemistry, relative quantity RT-PCR, radioimmunoassay (RIA), in order to provide the physiological and morphological data for orexin role in regulating reproduction of swine.1 Localizatin of OXR in swine12 Suzhong swines, 1-2 months old, were used in this study in order to investigate localization of OXR in the brain, spinal cord, some visceral organs and endocrine glands of swine with immunohistochemical method. The results were as follows:(1) Orexin 1 receptors (OX1R) were observed in the forebrain, brainstem, spinal cord, some visceral organs and endocrine glands in swine. In the forebrain, OX1R-like immunoreactive neurons were present mostly in the anterior hypothalamic area, supraoptic nucleus, paraventricular nucleus, periventricular nucleus, arcuate nucleus and tuberomammillary nucleus. In addition, sparse OX1R-like immunoreactive neurons were also found in the thalamic paraventricular nucleus and diagonal band. In the brainstem, OX1R-like immunoreactive cells were observed mainly in the nucleus raphe pallidus and locus caeruleus. In visceral organs, OX1R-like immunoreactive neurons were observed mostly in the ovary, testicle, epididymis, duodenum, jejunum, ileum, colon, pancreas and kidney. In endocrine glands, OX1R-like immunoreactive cells were distributed in the pituitary and thyroid gland.(2) Orexin 2 receptors (OX2R) were observed in the forebrain, brainstem, somevisceral organs and endocrine glands in swine. In the forebrain, OX2R-like immunoreactive neurons were present mostly in anterior hypothalamic area, preoptic area, supraoptic nucleus, suprachiasmatic nucleus, periventricular nucleus and paraventricular nucleus. Moreover, sparse OX2R-like immunoreactive neurons were also found in the thalamic anteromedial nucleus, olfactory tubercle and diagonal band. In the brainstem, OX2R-like immunoreactive cells were observed mainly in the hypoglossal nucleus and the dorsal motor nucleus of the vagus nerve. In visceral organs and endocrine glands, OX2R-like immunoreactive neurons were observed mostly in the lung, small intestine, pituitary and adrenal gland. So the difference of distribution of OX1R and OX2R in swine was present exactly.2 Expression of preproorexin and OX1R mRNA in sow hypothalamus-pituitary-ovary axes at different stages of the estrous cycleThe expression of preproorexin and OX1R mRNA in sow hypothalamus-pituitary-ovary axes at different stages of the estrous cycle (proestrus, oestrus, metestrus and diestrus, n=4) was studied with relative quantity RT-PCR in order to assess the relation of orexin and reproduction. The results were as follows:(1) Preproorexin mRNA and OX1R mRNA were expressed in the hypothalamus, pituitary and ovary of Suzhong sows and changed regularly with the stages of sow estrous cycle.(2) The expression of preproorexin mRNA in the hypothalamus, pituitary and ovary was the highest in the proestrus, and then decreased in the oestrus, reached the lowest in the metestrus and increased again in the diestrus.(3) The expression of OX1R mRNA in the hypothalamus, pituitary and ovary was the lowest in the proestrus, then increased in the oestrus, reached the highest in the metestrus and decreased in the diestrus at last.(4) The expression of preproorexin and OX1R mRNA showed a complementary relation, which implies that orexin roles are mediated via OX1R. 3 The effect of orexin A on serum LH level after injection of orexin A into the lateral ventricle in ovariectomized sowLH level 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 sows in order to study the effect of orexin A on LH secretion(blood sample interval each time was 10 min). The results were as follows:(1) Orexin A promoted the secretion and release of LH in short term after injection of it into the lateral ventricle.(2) LH concentration in serum began to increase 10 min after injection of orexin A into the lateral ventricle, was up to the peak 30 min after injection, and returned to the initial level 1.5 h after injection.4 The effect of orexin A on expression of relative neuropeptide mRNA in the hypothalamus and pituitary after injection of orexin A into the lateral ventricle in ovariectomized sowThe expression of GnRH, NPY and POMC mRNA in the hypothalamus and LH, FSH, POMC and ghrelin mRNA in the pituitary was studied with relative quantity RT-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) Orexin A stimulated expression of GnRH mRNA and decreased expression of NPY and POMC mRNA in the hypothalamus after injection of it into the lateral ventricle.(2) After injection of it into the lateral ventricle, orexin A promoted expression of LH and FSH mRNA in the pituitary, but the difference for FSH was not remarkable(p >0.05). Orexin A depressed expression of ghrelin and POMC mRNA in the pituitary, but the difference for POMC was not remarkable(p >0.05).The above studies showed that orexin receptors were observed in the hypothalamus, pituitary and ovary of swine, which provided the morphologic data for orexin role involving in regulation of pig reproduction. Regular expression of preproorexin and OX1R mRNA in the hypothalamus, pituitary and ovary of sow at different stages of the estrous cycle suggested that changes of orexin and its receptor be related to secretion of some reproductive hormones. After injection of orexin A into the lateral ventricle, it promoted the expression of GnRH mRNA in the hypothalamus and LH mRNA in the pituitary, increased LH level in serum, and affected the expression of NPY, POMC and ghrelin mRNA in the hypothalamus or the pituitary, which 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 regulating expression of NPY, POMC and ghrelin. These studies provided physiological data for orexin role in regulating reproductive activity.
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