Regulation And Molecular Mechanisms Of GnIH On Murine Reproduction And Study Of Genetic Immunization Techniques Against GnIH

GnIH(gonadotropin-inhibitory hormone, GnIH)is novel hormone containing 12 amino acids, and it is also called RFRP(RF-amide related peptide,RFRP) in mammals. Presumably, GnIH can inhibit synthesis and release of gonadotropin similar to physiological functions of inhibin, and may directly participate in the regulation of ovarian function by paracrine or autocrine manner in gonad. In this study, male mice were injected with different dose of GnIH, and then mouse ovarian granulosa cells were cultured with different concentrations of GnIH in vitro. The effect and mechanism of GnIH on hypothalamus, pituitary, testis and ovarian granulosa cells were analyzed. To provethe reliability of the results, three novel gene vaccines were constructed based on inhibin gene vaccines conserved in our laboratory and the expressions of these vaccines were analyzed in HeLa cells. Finally, immune responses and litter size of mice were analyzed after immunization with these vaccines by electroporation; meanwhile, immune responses, hormone levels and lambing rate of sheep were analyzed after immunization with three vaccines by intramuscular injection, which would provide a foundation for optimizing gene vaccine increasing fertility.The main contents and results of this study are as follows:1.Molecular mechanism of GnIH on reproductive and reproductive endocrinology in male mice.32 mice was divided into four groups according to the age and weight(8 of each group).Mice were subcutaneously injected(twice daily)with 150 ul saline containing 0(control),1, 3and 6μg for 11 days, then blood samples were collected. After collecting serum, the levels of LH, T and INH B were determined with ELISA; Total RNA of hypothalamus, pituitary and testis and total protein of testis were extracted. Then the expression of GnRH I、Kiss-1、FSH β、LH β、GnRHR and HSF-2mRNA were separately detected with qPCR; meanwhile, the protein expressions of p450scc、StAR、3β-HSD、LHR and AR were identified by western bolt; Testicular tissue morphology was viewed by H.E staining; germ cell apoptosis was analyzed by TUNEL. Results showed that GnIH treatment markedly reduced the concentration of LH in the peripheral blood. Further synthesis indicated that GnIH treatment caused marked decreases in the expression of GnRH I mRNA and Kiss-1 mRNA in the hypothalamus and down-regulated mRNA levels of FSH β, LH β and the GnRH receptor(GnRHR) genes in the pituitary. We also found that GnIH treatment significantly decreased the level of testosterone by reducing expression of the steroidogenesis-related enzymes(P450scc, StAR and 3β-HSD). Furthermore, GnIH treatment down-regulated the expression of spermatogenesis related genes(LHR, AR, and HSF-2). More importantly, the concentration of inhibin B(a major indicator of spermatogenesis) and the level of INH βb mRNA remarkably declined following GnIH treatment. Moreover, GnIH treatment also led to the abnormal morphology of the germ cells and induced germ cells apoptosis. These results showed(1)GnIH likely suppressed the release of GnRH by inhibiting the expressions of GnRH I and Kiss-1mRNA or reduced the expression of GnRHR mRNA, resulting in the decrease of LH secretion;(2)GnIH inhibited testosterone synthesis by directly reducing the expression of p450 scc, StAR and 3β-HSD proteins in testis;(3)GnIH likely inhibited spermatogenesis by directly down-regulating the expression of LHR, AR and HSF-2 genes in the testis of mice.2.Effect of GnIH on steroidogenesis and it‘sregulation mechanismin granulosa cells of miceFirst, mouse ovarian granulosa cells was separated 48 h after PMSG injection, then total RNA and protein were extracted at 24 h, 48 h, 72 h, 96 h after culturingin vitro.The gene expression of GnIH receptor(GPR147)was determined by qPCR and western blot. After 24 h treatment with different concentrations of mouse GnIH(RFRP-3), total RNA and protein of primary ovarian granulosa cells were extracted and cell supernatants were collected. The gene expressions of steroidogenesis-related enzymes(p450scc、3β-HSD、StAR)and FSHR were determined by qPCR, meanwhile the protein expression of p-ERK1/2 was analyzed by wester blot; Granulosa cells apoptosis was detected by flow cytometry. The results showed that GPR147 was indeed expressed in granulosa cells of mice; moderate doses of GnIH can significantly decrease the expression of p450 scc, 3β-HSD, StAR,FSHR mRNA and induce apoptosis of granulosa cells(P<0.05);low and moderate dose of GnIH can significantly reduce synthesis of progesterone in granulosa cells(P<0.05)and decrease the expression of p-ERK1/2 protein. These results indicated that the expression of GPR147 was firstly detected in ovarian granulosa cells of mice, and GnIH may inhibit steroidogenesis and viability of granulosa cells by down-regulating the expression of FHSR mRNA and p-ERK1/2 protein, and would indirectly affect follicular development in mice.3.Construction and evaluation of the novel gene vaccine harboring the inhibin α(1-32)and the RFamide related peptide-3 genes for improving fertility in miceSynthesized SINH(swine INHα(1-32)gene was ligated to the C-terminus of the small envelope protein of the hepatitis B virus(HBV-S)and SRFRP(cattle RFRP-3 was ligated to the C-terminus of HBV-S gene)fragments were inserted into multiple cloning site of pIRES vector to develop p-SINH/SRFRP expressing INH and RFRP-3 genes(called co-expression plasmid). Then synthesized tissue plasminogen activator(TPA)signal sequence was then inserted into N-terminus of target genes of p-SINH/SRFRP plasmid to construct p-TPA-SINH/TPA-SFRFP expressing INH and RFRP-3 and TPA genes(called signal peptide co-expression plasmid). Meanwhile, single SINH was inserted into the multiple cloning siteof pIRES to develop p-SINH(single expression plasmid, inhibin vaccine)and considered as positive control. Constructed plasmids were identified by double enzyme digestion and sequencing analysis, then the expression of target genes was detected in HeLa cells transfected with constructed plasmids. Forty Kunming mice were equally divided into four groups and respectively immunized by electroporation with p-SINH, p-SINH/SRFRP and p-TPA-SINH/TPA-SRFRP vaccine and saline as control(two booster immunization at 2 weeks interval). At 0 d, 14 d, 28 d, 42 d after primary immunization, blood samples were collected. The antibody levels of against INH and RFRP-3 were detected by ELISA. Female mice were mated with male mice after collecting blood, and litter size of three successive generations was recorded. The results of double enzyme digestion and sequencing analysis showed that the gene insertion point, direction and sequence were inserted correctly. Results of immunization showed that the average antibodies(P/N value)of anti-INH and anti-RFRP in mice inoculated with p-TPA-SINH/TPA-SFRFP were significantly higher(p<0.05)than those inoculated with p-SINH/SRFRP and the positive rates of mice were 100%(anti-INH)and 90%(anti-RFRP)respectively, at 2 weeks after the third immunization; Results of reproduction showed that litter size of mice immunized with the three recombinant plasmids was higher(p<0.05)than that of the control, and litter size of mice immunized with p-TPA-SINH/TPA-SRFRP significantly increased( p<0.05) compared with of p-SINH. These results suggested that p-SINH, p-SINH/SRFRP and p-TPA-SINH/TPA-SRFRP plasmids were successfully constructed, and three vaccines induced antibodies against INH and/or GnIH(RFRP-3)after immunization and increased litter size of mice. Signal peptide co-expression plasmid was best in immunogenicity and improving litter size of mice.4.Effect of the novel gene vaccine fusing inhibin α(1-32) and the RF-amide related peptide-3 genes on immune response, hormone levels and fertility in Tan sheepThirty-two female Tan sheep were divided into four groups(8 each of groups)according to the age and weight, and respectively immunized with 0.6 mg of signal peptide co-expression vaccine(group A), co-expression vaccine(group B), inhibin vaccine(group C) or 0.4 ml saline(group D).Two booster immunizations were finished with 20 d interval. All ewes were synchronized on the day after immunization. Blood samples were collected by jugular vein on 20, 30, 40, 60 d after primary immunization. Rams were introduced into flock followed by collecting blood samples and lambing rate of ewes was recorded after parturition. Antibody levels of against INH and RFRP-3 were separately detected by indirectly ELISA; the levels of FSH、LH、E2 and P in the peripheral blood were determined by RIA after estrous synchronization. Results of immunization showed that all vaccines elicited significant immune responses, and the antibody levels of anti-INH and anti-RFRP-3 in the vaccinated groups were significantly higher(p<0.05) than that of the control group at 20 d after primary immunization. Immunization with signal peptide co-expression vaccine induced higher antibodies against INH and RFRP-3. Results of reproduction showed that levels of FSH and LH in group A were significantly higher(p<0.05) than those in group C at 20 d after the third immunization. Additionally, twinning rate of ewes in group A, B, C and D were 37.5%, 37.5%, 12.5% and 0, respectively. These results suggested that novel DNA vaccine expressing INH α(1-32) and RFRP-3 genes successfully elicited humoral immune responses of ewes, and increased the levels of gonadotropin in the peripheral blood, and showed certain potency in improving twinning rate of ewes in the present study.