| Objective: To investigate deeply the effects of gonadotropin releasing hormone analogue (GnRH-A) on the reproductive functions and Molecular mechanism of immunological regulation.Methods: (1) Arbodiimide hydrochloride (EDC.HCL) used as the coupling agent, GnRH-A (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 GnRH-A antigen. After the aseptic inspection, security and physical properties were tested according to Veterinary biological product quality inspection. (2) 16 male rabbits(Oryctolagus cuniculus), 3-month-old, were randomly divided into 4 groups, three experimental groups were injected with GnRH-A antigen 1.0mL at the concentrations 50μg/mL,100μg/mL and 150μg/ml respectively. The control group rabbit was done with saline. 5 mL blood was collected from lateral vein of hind limbs on the d 0, 7, 14, 21, 28, 35 and 42 after antigen injection. The blood samples were immediately centrifuged at 2000～2500 r/min for 10 min. Serum was harvested and stored at -20?C. GnRH antibody titer were measured by enzyme-linked immunosorbent assay(ELISA). (3) 30 male rabbits, 3-month-old, were randomly divided into experiment group 1(EG-I), experiment group 2 (EG-II) and control group(CG). Rabbits in EG-I and EG-II were injected subcutaneously with 1.0 mL GnRH-A antigen at concentrations of 100μg/mL. EG-II were re-injected with the above doses three weeks later. CG was as blank without any treatment. 5 mL blood was collected on 7, 14, 28, 49, 70, 91 and 102 d. The testis length, testis weight and body weight of the immunized rabbits were measured, GnRH antibody titer and testosterone concentration were detected by ELISA. (4) 15 hematocytes parameters were checked by using the automatic hematology analyzer, 10 blood gas indexes were assayed with i-STAT blood analyzer. (5) The pituitary were collected on 102 d. Total RNA were extracted, GnRHR were amplified with RT-PCR and sequencd. Gene expressions of GnRHR, FSH-βand LH-βmRNA in pituitary were analyzed by real time PCR. GnRHR sequence and protein physicochemical properties, membrane structure, signal peptide, cellular localization, secondary and tertiary structure were assayed and predicted with bioinformatics softwares and online tools. (6) 24 rabbits were divided randomly into experiment group 1(EG-I), experiment group 2(EG-II), experiment group 3(EG-III) and control group(CG). and the experimental groups were injected respectively with GnRH-A antigen 1.0mL at the different doses(100μg/mL, 100μg/mL and 50μg/mL), EG-II and EG-III rabbits were re-injected at the same dose on the third week. Serum GnRH antibody titre, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) concentrations were determined with ELISA. (7) he samples of ovary and uterus were collected, which were fixed with the 10% formaldehyde and 3% glutaric dialdehyde.The slices were observed under carry on optical and electron microscope. (8)Immunohistochemistry SP method(Streptomyces avidin-peroxidase method) and image analysis techniques were used to locate and analyse in the experiment.Results: (1) The alarelin antigen was safity and stable. (2)There was a significant difference between the testis length in both EG-I and EG-II (P <0.01), antibody level of EG-II was higher obviously than EG-I; serum testosterone concentration of EG-I and EG-II differed remarkably from 7 to 16 weeks (P <0.05 ), testosterone concentration of EG-II was lower than control group(CG) at 102 d(P <0.01) and EG-I at 28 d(P <0.05) after the injecting GnRH-A antigen. Body weight and average gain of EG-II were the biggest and significantly higher than EG-I and CG (P <0.05). (2)There was a significant difference between the testis length in both EG-I and EG-II (P <0.01), antibody level of EG-II was higher obviously than EG-I; serum testosterone concentration of EG-I and EG-II differed remarkably from 7 to 16 weeks (P <0.05 ), testosterone concentration of EG-II was lower than control group(CG) at 102 d(P <0.01) and EG-I at 28 d(P <0.05)after the injecting GnRH-A antigen. Body weight and average gain of EG-II were the biggest and significantly higher than EG-I and CG (P <0.05). (3) There was no significant differences of 15 between experimental and control rabbits at the end of experiment; the values of 10 blood gas indexes were within normal limits. TCO2 and HCO3- increased significantly (P <0.05) during the first four weeks. (4) GnRHR nucleotide was 1179bp with homology 96%, the nucleotide contained A 338 (28.7%), C 293 (24.9%), G 224 (19.0%), T 324 (27.5%). molecular weight of ssDNA and dsDNA were 362.66 kDa and 726.78kDa respectively. The patterns and secondary structure diagram of GnRHR sequence obtained, the secondary structure consisted of 151 (40.27%) amino acidα-helixes, 15 (4.00%) amino acidsβ-sheets. GnRHR isoelectric point (pI) was 5.02, instability index (II) 58.08, aliphatic index 28.67, the average hydrophobicity (GRAVY ) 0.869, which indicated that GnRHR protein was hydrophobic unstable protein. The transmembrane (TM ) helix length of GnRHR protein was 17 - 23, and comprised of 34 TM helixes areas. Signal peptide probability of GnRHR protein was 0.999, max cleavage site probability were in 17 and 18 positions. (5) Serum GnRH antibody titers were detected in three experiment groups rabbits after 10d of injecting GnRH-A without detection in CG. EG-I, EG-II and EG-III reached respectively peak level on 30d and 40d～50d. The antibody titers of EG-II were higher than EG-I and EG-III(P<0.01)during 40d～70d. LH concentration of EG-II exceeded other groups at 30d-50d. FSH concentrations of EG-II and EG-III reached the peak on 40d, EG-II FSH were higher other group(P<0.05).There was no significant difference between EG-I and CG. (6) GnRH-A may increase the numbers of ovary primary follicle, enlarge vertical diameter and the transverse diameter of follicle, promotes growing and maturing of ovary and follicle, which was depedend the used dosage. However, re-injection seemed not to accelerate these effects. GnRH-A could obviously speed up oocyte development by enlarging nucleus and chondriosome, increasing quantities of mitochondrial cristae, cortical granule and secretions in cytoplasm.The zona pellucida and microvillus of oocyte were broadened and lengthened. (7) There existed positive cells of GnRHR and GnRH in the ovaries and uterine. GnRHR and GnRH positive cells were found in oocytes, follicle cells, endometrial epithelial cells and glandular epithelial cells. When GnRH-A were injected at the different dose, positive cells had the different staining intensity, which indicated that the location and expression of GnRHR and GnRH varied. However, both the number and location of distribution were not uniform. GnHR-A may increased the distribution of GnRH and GnRHR in the ovary and uterine.Conclusions: (1)GnRH-A (Alarelin, Alarelin) antigen has good immunogenicity, in order to last longer peak leve of antibody, it is necessary to strengthen immunization.(2)The testis development, serum GnRH antibody titer and testosterone of the experimental rabbits changed obviously after an active immunization of GnRH-A and adding another injection would strengthen castration effects.(3) Alarelin immunization can significantly decrease gene expressions of GnRHR, FSH-βand LH-βin rabbit pituitary. GnRHR is an unstable hydrophobic transmembrane protein containing signal-peptide sequence containing, has obvious bioinformatics characteristics.(4)Injecting GnRH-A for rabbits may obviously improve GnRH antibody titer, enhance LH and FSH synthesis and secretion, strengthening injection is more effective, which may be relative with the injecting dosage.(5)GnRH-A promoted the growth and maturation of ovary and follicle, and improved remarkably microstructure and ultrastructure of ovary and uterus in rbbits.(6) Ovary and uterus have GnRHR and GnRH positive cells, GnRH-A active immunization enhanced the distribution of GnRHR and GnRH in the ovary and uterus in female rabbits.
|
PDF Full Text Request