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The Mechanism of Active Immunization Against GnRH in Rats and Its Reversibility

Author: CaoXiaoHan
Tutor: ZengXianZuo
School: Sichuan Agricultural University
Course: Clinical Veterinary Medicine
Keywords: GnRH active immunization rat mechanism reversibility
CLC: S814.1
Type: PhD thesis
Year: 2013
Downloads: 78
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Abstract

Gonadotropin-releasing hormone (GnRH) is a central regulator of reproductive functions. It is released from hypothalamic neurons in a pulsatile manner, then pulsatile releases into the hypophyseal portal blood vessels and then gose in anterior pituitary, The peptide binds to specific GnRH receptors on pituitary gonadotrophs, regulates the secretion of Luteinizing and Follicle-Stimulating Hormones which together orchestrate the secretion of gonadal hormones and gametogenesis. In animals, active immunization against GnRH degrads and even ends the gonadals development, lowers serum FSH and LH levels, suppression of reproductive function. Active immunization against GnRH is a new method that avoids the problem of surgical castration-induced stress, infection, drug residues without an influence on the performance of animal production. Thus, it is more in line with the requirements of the Animal Welfare and has been a good alternative o surgical castration in mammals.KiSS-1gene is one of the isoforms of KiSS gene, express in central nervous system and placenta. The kisspeptins was a group of peptides encoded by kiSS-1gene, whom called GPR54(G protein-coupled receptor54), was the natural receptor of kisspeptin. The pivotal role of the KiSS-1/GPR54system is in the control of reproduction system development. It was shown that kisspeptins regulate the GnRH neurons directly, the pulsatile release of FSH and LH from the pituitary gland is dependent on GnRH stimulation by combining of kisspeptins and GPR54.Active immunization against GnRH causes infertility in both males and females, but was is the mechanisms? Why there were differences among different studies about the castrated period? In view of these questions, the experiments in present study prepared GnRH vaccine against which active immunization SD rats, detected the antibody titer, concentration of serum hormones, mRNA expression of reproductive-relation and their receptor genes, investigate the regulatory relations between KiSS-1/GPR54and hormones, as well as their receptor in hypothalamus-pituitary-gonads axis after active immunization against GnRH, elucidate the mechanism of active immunization against GnRH; furthermore, do researches on the reversibility of active immunization against GnRH. The studies and results of present research are summarized as following:1. Synthesis of GnRH-Tandem and GnRH-Tandem-Dimer (GnRH-TD), conjugation the GnRH-TD to OVA via N-ethyl-N’-(3’-dimethylaminopropyl) carbodiimide hydrochloride (EDC), made it to GnRH-TDO, which was identified by SDS-PAGE; made specol adjuvant with mineral oil, Tween-85and span-85, emulsified the GnRH-TDO and specol as the vaccine. The physical property and stability of the vaccine were analysis and confirm as qualified vaccine.2.72sexually mature male SD rats were slected and equally divided into male immuzed group (mIG), male surgical group (mSG) and male control group (mCG),(the24rats in every group equally divided into3groups again, n=8). All of the24rats in mIG were injected i.m. with a total of1mL GnRH-TDO (contains GnRH-TD50μg) emulsified in specol adjuvant at12week of age, an equal dose booster was administered4weeks later; mSG were surgically castrated at11week of age while mCG without any treatments. The day of first immunization denoted as0weeks post vaccination (0wpv). Blood samples were collected from each rat at day0(0wpv) and every2weeks thereafter, until just before sacrificed (8wpv,12wpv, and16wpv, respectively.). Sera were prepared. Titers of anbodies against GnRH were determind using ELISA, and through out the RIA to assess the serum levels of T, FSH and LH. At sacrificed, hypothalamus and pituitary were removed, then the mRNA expressions for reproduction-related genes were determined by real-time PCR; Testicles of mIG and mCG were excised, epididymes were removed and testes weight was recorded. then the mRNA expressions for reproduction-related genes were determined by real-time PCR.After immunization twice of male SD rats against GnRH, serum titers of GnRH antibodies rose, and caused shrinking and a continued decline in testicular weight, and the testis involuted to55.22%,54.31%and42.55%of the mCG weight at8wpv,12wpv and16wpv, respectively, which significantly lower in mIG rats than in mCG (P＜0.05). Dramatic decreases in serum concentrations of T, LH and FSH were observed,the concentrations of T, LH and FSH were decreased in mIG rats than those in mCG significantly(P＜0.05). In mSG, the concentrations of T in serum were decreased than those in mCG (P＜0.05), while the concentrations of FSH and LH in serum were increased significantly than those in mCG (P＜0.05). Furthermore, compared with mCG, active immunization against GnRH significantly down-regulated the mRNA levels of GnRH, GPR54, KiSS-land AR in the hypothalamus (p＜0.05), and GnRH-R, FSH-β and LH-β in the pituitary (p＜0.05),as well as FSHR, LHR and AR in testis (p＜0.05); and in mSG, surgical operation significantly down-regulated the mRNA levels of GnRH, GPR54, KiSS-1and AR in the hypothalamus (p＜0.05), and FSHR, LHR and AR in testis (p＜0.05),but up-regulated the mRNA levels of GnRH-R, FSH-β and LH-β in the pituitary (p＜0.05).3.72sexually mature female SD rats were slected and equally divided into female immuzed group (fIG), female surgical group (fSG) and female control group (fCG),(24SD rats in every group equally divided into3groups again). All of the24rats in fIG were injected i.m. with a total of1mL GnRH-TDO (contains GnRH-TD50μg) emulsified in specol adjuvant at12week of age, an equal dose booster was administered4weeks later. Rats in fSG were surgically castrated at11week of age while fCG without any treatment. The day of first immunization denoted as0wpv. Blood samples were collected from each rat at day0(0wpv) and every2weeks thereafter, until just before sacrificed (8wpv,12wpv, and16wpv, respectively.). Sera were prepared, titers of anbodies against GnRH were determind using ELISA, and through out the RIA to assess the serum levels of FSH, LH, P4and E2. After sacrificed, hypothalamus and pituitary were removed, then the mRNA expressions for reproduction-related genes were determined by real-time PCR; Ovarians of fIG and fCG were excised, adipose tissues were removed and ovaries weight was recorded. then the mRNA expressions for reproduction-related genes were determined by real-time PCR.The generation of serum GnRH antibodies after immunization caused a continued decline in the weight of ovary, and which was reduced to63.63%,38.46%and21.43%of the weight in fCG at8wpv,12wpv and16wpv, respectively. While the concentrations of E2, P4, FSH and LH in fIG were increased after active immunization against GnRH, which significantly lower than in fCG (p＜0.05). On the other hand, surgical castration increased the serum concentrations of E2, P4to the detection limit of rats in fSG, which were significantly lower than that in fCG (p＜0.05), while the serum concentrations of FSH and LH were significantly higher than that in fCG (p＜0.05). Moreover, active immunization against GnRH significantly down-regulated the mRNA levels of GnRH, GPR54, KiSS1and ER-a in the hypothalamus in fIG, and GnRH-R, FSH-β and LH-β in the pituitary (p＜0.05),as well as FSHR, LHR and ER-a in ovaries (p＜0.05); In fSG, surgical castration significantly down-regulated the mRNA levels of GnRH, GPR54, KiSS1and ER-a in the hypothalamus in fIG, and FSHR, LHR and ER-a in ovaries (p＜0.05);but opposite to fIG, the mRNA levels of GnRH-R, FSH-β and LH-β in the pituitary were significantly up-regulated in fSG (p＜0.05).4.24mature male SD rats were slected and equally divided into mIG, mSG and mCG. All of the8rats in mIG were injected i.m. with a total of1mL GnRH-TDO (contains GnRH-TD50μg) emulsified in specol adjuvant at12week of age, an equal dose booster was administered4weeks later. Rats in mSG were surgically castrated at11week of age while mCG without any treatment. The day of first immunization denoted as0wpv. Blood samples were collected from each rat at day0(0wpv) and every2weeks thereafter, until just before sacrificed at56wpv. Sera were prepared, titers of anbodies against GnRH were determind using ELISA, and through out the RIA to assess the serum levels of FSH, LH and T. After sacrificed, hypothalamus and pituitary were removed, the mRNA expressions for reproduction-related genes were determined by real-time PCR; testicles of mIG and mCG were excised, epididymes were removed and testes weight was recorded, then the mRNA expressions for reproduction-related genes were determined by real-time PCR.After immunization twice of male SD rats against GnRH, serum titers of GnRH antibodies rose and a peak was observed in12wpv and last a long period; the antibody titer didn’t decrease to the level the same as intact controls until40wpv, and when sacrificed at56wpv, the antibody titer was nearly absence. The weight of testes in mIG was significantly lower than that in mCG (p＜0.05) and they still shown atropy without elasticity at56wpv. While the concentrations of T, FSH and LH in mIG were increased after active immunization against GnRH, which significantly lower than in fCG (p< 0.05);and when the mIG rats were sacrificed at56wpv, the value of serum T level shown a slightly decreased but remain significantly lower than that in mCG (p＜0.05). On the other hand, surgical castration increased the serum concentrations of T to the detection limit of rats in mSG, which were significantly lower than that in mCG (p＜0.05), while the serum concentrations of FSH and LH were significantly higher than that in mCG (p＜0.05). Moreover, active immunization against GnRH significantly down-regulated the mRNA levels of GnRH, GPR54, KiSS1and AR in the hypothalamus in mIG, and GnRH-R, FSH-β and LH-β in the pituitary (p＜0.05),as well as FSHR, LHR and AR in testes (p＜0.05); In mSG, surgical castration significantly down-regulated the mRNA levels of GnRH, GPR54, KiSS1and AR in the hypothalamus, and FSHR, LHR and AR in testes (p＜0.05);but opposite to mIG, the mRNA levels of GnRH-R, FSH-β and LH-β in the pituitary were significantly up-regulated in mSG (p＜0.05).5.24mature female SD rats were slected and equally divided into fIG, fSG and fCG All of the8rats in fIG were injected i.m. with a total of1mL GnRH-TDO (contains GnRH-TD50μg) emulsified in specol adjuvant at12week of age,4weeks later an equal dose booster was administered. Rats in fSG were surgically castrated at11week of age while mCG without any treatment. The day of first immunization denoted as0wpv. Blood samples were collected from each rat at day0(0wpv) and every2weeks thereafter, until just before sacrificed at56wpv. Sera were prepared, titers of anbodies against GnRH were determind using ELISA, and through out the RIA to assess the serum levels of FSH, LH E2and P4. After sacrificed, hypothalamus and pituitary were removed, the mRNA expressions for reproduction-related genes were determined by real-time PCR; Ovarians of fIG and fCG were excised, adipose tissues were removed and ovaries weight was recorde, then the mRNA expressions for reproduction-related genes were determined by real-time PCR.After immunization twice of female SD rats against GnRH, serum titers of GnRH antibodies rose and a peak was observed in16wpv and last a long period; the antibody titer didn’t decrease to the level the same as intact controls until40wpv, and when sacrificed at56wpv, the antibody titer was nearly absence.The ovary and uterus still remained a castrative status after56-weeks vaccination, and the weight of ovary in fIG was significantly lower than that in fCG (p＜0.05). Meanwhile, the concentrations of E2, P4, FSH and LH in fIG were increased after active immunization against GnRH, which significantly lower than that in fCG (p＜0.05); and when the fIG rats were sacrificed at56wpv, the value of serum E2and LH level shown a slightly decreased but remain significantly lower than that in mCG (p＜0.05). On the other hand, surgical castration increased the serum concentrations of E2and P4to the detection limit of rats in fSG, which were significantly lower than that in fCG (p＜0.05), while the serum concentrations of FSH and LH were significantly higher than that in fCG (p＜0.05). Moreover, active immunization against GnRH significantly down-regulated the mRNA levels of GnRH, GPR54, KiSS1and ER-a in the hypothalamus in fIG, and GnRH-R, FSH-β and LH-β in the pituitary (p＜0.05),as well as FSHR, LHR and ER-a in ovaries (p＜0.05); In fSG, surgical castration significantly down-regulated the mRNA levels of GnRH, GPR54, KiSS1and ER-a in the hypothalamus, and FSHR, LHR and ER-α in ovaries (p＜0.05); but opposite to fIG, the mRNA levels of GnRH-R, FSH-β and LH-β in the pituitary were significantly up-regulated in fSG (p＜0.05).conculusion:Results of this study indicate GnRH-TDO has excellent immunogenic and was a good alternative of surgical castration. This study has demonstrated the potent inhibitory effects of active immunization against GnRH on the gonadals development of SD rats, dramatic decreases in serum concentrations of FSH, LH and sex hormones (T in male rats and E2, P4in female rats) were observed. Active immunization against GnRH-TDO significantly down-regulated the mRNA expressions of GnRH, GPR54, KiSS-1and AR/ER-α in the hypothalamus, and GnRH-R, FSH-P and LH-β in the pituitary,as well as FSHR, LHR and ARIER-a in testes and ovaries. The mechanism of immune castration is:Active immunization against GnRH-TDO reduced the serum concentration of gonadal hormone, down-regulated the expression of GnRH mRNA in hypothalamus by the feedback loop of AR/ER-α-GPR54/KiSS-1, cause the decreacing of serum LH and FSH level, thus resulting in infertility of SD male and female rats. The castrative status could be sustained at least for56weeks after the boster vaccination, but the values of serum T in male rats and serum E2, LH in female rats were observed a trend of rose up till the last stage of this research, which shown that though active immunization against GnRH-TDO cause animals in infertility, the long-term infertility or sterility was not permanent, the fertility of immunocastrated rats show a tendancy to recover and gender difference.

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