Expression Of Extragonadal Gonadotrophins Receptors In The Prepubertal Gilts

Two months old crossbred (Landrace×Large White) gilts were used to investigate: 1) the expression and location of the functional FSHR and LH/CGR in both uterus and oviducts. 2) the effects of dietary energy on hormones release in prepubertal gilts 3) the effects of dietary energy on the expression of gonadotropins receptors in extragonadal prepubertal gilts. The present study was to clarify the preliminary molecular biology mechanism of the regulating role of extragonadal gonadotropins in the extragonadal tissues , and to establish theoretical basis for feeding of prepubertal gilts as well.Firstly, we determined the localization of FHSR and LH/CGR in the uteri and oviducts of prepubertal gilts with immunohistochemical method. The uterus, oviducts and ovaries were collected from five prepubertal gilts. the ovaries were used as the positive control, and the negtive control was non-specific IgG instead of specific antibodies. The results showed that LH/CGR and FSHR were present in the uteri and oviducts of prepubertal gilts. In the uteri, FSHR was expressed in the endometrial epithelial cells, but not in the myometrium and perimetrium. LH/CGR was present in the endometrial epithelial cells and the myometrial vascular smooth muscle. However, there was no LH/CGR in the perimetrium. FSHR and LH/CGR were all expressed in the oviductal mucosal epithelium, but not in the myosalpinx and the serosa. The resluts confirmed that the ovaries was not the only target organ of gonadotropins receptors, and FSHR and LH/CGR were also located in the prepubertal extragonadal organs such as uterus and oviducts.Secondly, we investigated the expression of functional FHSR and LH/CGR in the prepubertal uterus and oviducts with radioactive immunoassay, and Western blotting. Fifteen growth-matched crossbred prepubertal gilts were randomly allocated to three groups after one week reared in the same room. One group animals (n=5) were performed ovariectomy. Two days later, the fasting vein blood was collected from all three groups. Then we carried out treatments according to the experimental design. The groupⅠgilts were injected with physiological saline (5 ml per gilt); the groupⅡgilts were injected, i.m., with eCG (300IU per gilt); the groupⅢgilts (ovariectomizd) were injected with eCG (300IU per gilt). Twenty four hours after eCG treatment, vein blood was collected from all three groups for serum preparation, uteri and oviducts were also removed for detection. The results showed that the concentrations of serum FSH and LH were significantly increased after eCG injection(p<0.01)in groupⅡand groupⅢ. The ovary excision removed the major source of in vivo estrogen synthesis and significantly decreased the concentration of serum estradiol compared with that of intact gilts(p<0.01). After eCG injection, the concentration of serum estradiol in the ovariectomized gilts was significantly lower than that of saline injection pig (p<0.01). Regardless of ovaries removed or not, eCG injection could promote the expression of FSHR and LH/CGR in the uterus and oviducts. The FSHR expression level of uterus and oviducts in intact gilts was significantly higher than that of ovariectomized gilts. The similar consequence was found in oviducal LH/CGR(p<0.05). These results showed that the FSHR and LH/CGR in prepubertal uterus and oviducts were active and functional, and the expression of LH/CGR and FSHR were regulated by FSH, LH, and the estrogen.Finally, we observed the effects of dietary energy on the hormones release and the expression of FSHR and LH/CGR on prepubertal gilts with radioactive immunoassay, fluorescence quantitative-PCR and Western blotting. Fifteen gilts were randomly allocated to three groups, the groupⅠfed with the moderate energy level (100%of NRC energy requirements), the groupⅡfed with the low energy level (90% of NRC energy requirements), the groupⅢfed with the diet of high energy level (100%of NRC energy requirements), for 12d. Then the fasting vein blood was collected from all three groups for serum preparation on d1, d5, d9, and d12.Then uterus and oviducts were collected at the slaughter on d12 The results showed that dietary energy could affect the concentrations of peripheral serum FSH, LH, GH, leptin, Ins, and IGF-Ⅰin prepuberty gilts. High energy level significantly enhanced the concentrations of serum FSH, LH, leptin, GH, insulin and IGF-Ⅰ, whereas the change of GH concentration was not significantly(p<0.01). Low energy level significantly reduced the concentrations of serum FSH, LH, GH, leptin, Ins and IGF-Ⅰ. Dietary energy level affected the expression of FSHR and LH/CGR in prepubertal gilts. High energy intake significantly enhanced the expresioon of uterine and oviducal FSHR and LH/CGR, whereas insufficient energy intake restricted FSHR and LH/CGR expression. In the uterus, the expression level of FSHR mRNA was the highest in follicular phase and LH/CGR mRNA expression level was the highest in luteal phase. In the oviducts, The expression level of FSHR mRNA and LH/CGR mRNA were all the highest in follicular phase .Overall, our results indicated that there were functional FSHR and LH/CGR in uterus and oviducts of prepubertal gilts, and confirmed for the first time that FSHR was present in the endometrium epithelial cells. FSH, LH and estrogens regulated the expression of FSHR and LHR in the uterus and oviducts. Gonadotropins could regulate the uterine and oviductal function not only indirectly through gonadal hormone secreted by ovaries. but also directly through gonadotrophins receptors in the uterus and oviducts. These results imply that dietary energy level could influence the expression of FSHR and LH/CGR in uterus and oviducts which might be mediated by insulin, IGF-Ⅰ, leptin and GH, These results provided the hypothesis that gonadotropins might play direct roles on the extragonadal organs and consummate the mechanism of gonadotropins in animal reproduction. Furthermore, we explored the effect and mechanism of energy on reproductive development of prepubertal gilts from aspects of reproductive endocrinology, gene and protein expression, which might provide a theoretical basis for feeding of the prepubertal gilts.