| Objective:The effects of gonadotropin releasing hormone analogue (GnRHa) on the regulatory role and mechanism of animal reproductive immunology were investigated through a series of studies on female sheep reproductive immune regulation by GnRHa, which provided the basis for animal clinical and production.Methods:(1)Arbodiimide hydrochloride(EDC.HCL)used as the coupling agent, GnRHa (agonist,Alarelin) was combined with bovine serum albumin(BSA) to make a compound, then incomplete Freund’s adjuvant was added into the compund for preparing GnRHa antigen. After the aseptic inspection, security and physical properties were tested according to Veterinary biological product quality inspection.(2)Fourty-two ewes (Ovis aries), five-to-six months old and24.21±2.51kg, were randomly divided into six groups (each of7eves), namely experiment group I (EG-I), experiment group II (EG-II), experiment group III (EG-III), experiment group Ⅳ(EG-Ⅳ), experiment group V(EG-V) and control group (CG). Animals were subcutaneously injected with different dose of GnRH agonist (alarelin) antigen emulsion.5mL blood was collected from lateral vein of hind limbs on the d0,7,14,21,28,35,45,60and70after antigen injection. The blood samples were immediately centrifuged at2000-2500r/min for10min. Serum was harvested and stored at-20℃. Concentration of GnRH antibody and GnRH were measured by enzyme-linked immunosorbent assay (ELISA).(3) The samples of uterus and ovaries were collected aseptically on d7. Immunohistochemistry SP (Streptomyces avidin-peroxidase) method and image analysis were used to locate and analyze GnRH receptor (GnRHR) distribution in uterus and ovaries.(4)The samples of pituitaries in each ewe were collected aseptically on d7. Total RNA were extracted, GnRHR were amplified with RT-PCR and sequencd. Gene expressions of GnRHR mRNA in pituitary were analyzed by RT-PCR.(5)Physicochemical properties, membrane structure, signal peptide, cellular localization, secondary and tertiary structure of GnRHR sequence or proteinwere were assayed and predicted by bioinformatics softwares and online tools.Results:(1)The alarelin antigen was security and stable.(2) Concentrations of GnRH antibody had a regular expression, and they increased firstly and then decreased in the experimental groups. While the EG-1, EG-II and EG-Ⅲ reached the peak on d21,EG-Ⅳand EG-Ⅴ on day35. The concentration of GnRH continued to decline on day45after immunization. GnRH concentration of EG-Ⅰ, EG-Ⅱ and EG-Ⅲ reached the bottom values on28d, GnRH concentrations of EG-Ⅳ and EG-Ⅴ also reached the bottom on d45.(3) GnRHR positive cells were distributed in the ovaries and uteri. They mainly were found in oocytes, follicle cells, endometrial epithelial cells and glandular epithelial cells. When GnRHa was injected at the different doses, positive cells had the different immunohistostaining intensity, which indicated that the location and expression of GnRHR were varied. However, both the number and location of distribution were not uniform. GnHRa may increased the distribution of GnRHR in the ovaries and uteri.(4) The GnRHR2-(ΔΔCt) values of experimental groups were lower than the control group. The2-(ΔΔCt) values in EG-Ⅳ and EG-V were lower than that in EG-Ⅰ and EG-Ⅱ (P<0.05)(5) The result showed that GnRHR gene amplification product was2027bp, and GnRHR gene coding product was a Hydrophobicity transmembrane protein, with out obvious signal peptide. The secondary structure was primarily composed of alpha helix, and the biological effects of GnRHR was performed in the endoplasmic reticulum. And the transmembrane Segments composed of extracellular domain, transmembrane domain and intracellular domain. Sequence analysis indicated that this gene coding product might involve in transporter, and play important roles in ion transmembrane and signal transduction, and influence the hormonal regulation of ewes (Ovis aries).Conclusions:(1) GnRHa (Alarelin) antigen had good immunogenicity, in order to last longer peak leve of antibody, it was necessary to strengthen immunization.(2)GnRH antibody concentration improved and GnRH concentration reduced after an active immunization of GnRHa.(3) Ovary and uterus had GnRHR positive cells, GnRHa active immunization enhanceed the distribution of GnRHR in the ovary and uterus in ewes.(4)Alarelin immunization can significantly decrease gene expressions of GnRHR, and increased doses and number of injections mske it more obvious.(5)GnRHR was an unstable Hydrophobicity transmembrane protein sequence, had obvious bioinformatics characteristics. |
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