GnRH主动免疫动物去势机制及其可逆性研究
摘要
促性腺激素释放激素(Gonadotropin-releasing hormone, GnRH)是动物体内重要生殖激素之一,其主要作用是调控动物生殖过程。GnRH由丘脑下部的GnRH神经元合成,以脉冲方式释放,通过垂体门脉循环进入垂体前叶,与垂体上的GnRH受体结合,调节促黄体激素(Luteinizing hormone, LH)和促卵泡激素(Follicle stimulating hormone, FSH)的合成和分泌,继而通过血液循环调节性腺类固醇激素的产生和雌雄配子的发生。GnRH主动免疫动物能降低动物体内FSH、LH含量,使性腺退化、萎缩甚至停止发育,影响其生殖功能从而达到去势的目的。利用GnRH主动免疫可以避免外科手术或药物去势对动物造成的应激、感染、药物残留等问题,并且不影响动物生产性能,也更符合动物福利的要求,是一种有望替代传统外科手术给动物去势的新方法。
KiSS-1基因是KiSS基因的亚型,主要在中枢神经系统和胎盘表达,该基因编码的一组肽产物称为kisspeptins, GPR54(G protein-coupled receptor54)是kisspeptin的天然受体。KiSS-1/GPR54系统与生殖系统的发育有密切关系：研究表明,GnRH神经元是kisspeptins的直接调节对象；目前普遍认为的作用机制是kisspeptins与GPR54结合,激活GnRH神经元,刺激GnRH的释放,进而调节FSH和LH的释放。
GnRH主动免疫动物能使其达到去势效果,但去势机制是怎样的?去势效果维持时间的长短在不同研究中为何有较大差别?关于这些问题目前尚无统一定论。鉴于此,本研究制备了GnRH免疫去势疫苗,主动免疫SD大鼠,结合大鼠血清抗体、血清激素、生殖相关基因及其受体mRNA表达变化情况,探讨主动免疫后KiSS-1/GPR54系统和下丘脑-垂体-性腺轴各级激素、受体和配体的调控关系,以期阐明GnRH主动免疫去势的作用机理；此外,还对GnRH免疫去势的可逆性进行了研究。本文开展的主要研究工作和取得的结果如下：
1.以组成GnRH的9种氨基酸为原料合成GnRH并列体(GnRH-Tandem)和GnRH并列体二聚物(GnRH-Tandem-Dimer, GnRH-TD),使用碳二亚胺法将GnRH-TD与卵清蛋白(OVA)偶联,制成GnRH-TDO免疫原,经SDS-PAGE分析,在51.5KDa处出现了预期条带,得到合格的免疫原；以矿物油、‘Tween-85和span-85为原料制作specol佐剂,将GnRH-TDO免疫原与specol佐剂充分乳化制成GnRH-TDO去势疫苗,检验去势疫苗的物理特性和稳定性,均符合要求。制得合格的GnRH-TDO免疫去势疫苗。
2.选取健康性成熟雄性SD大鼠72只,随机分为GnRH免疫去势组(mIG)、手术,去势组(mSG)和空白组(mCG),每组24只(各组再随机分为3个小组,n=8)。mIG于12周龄腿部肌肉注射1mL疫苗(含GnRH-TD50μg),4周后加免,加免时注射剂量及方法同初次免疫；mSG于11周龄进行手术,摘除睾丸；mCG不做任何处理。初次免疫当天记为Owpv (weeks post vaccination)。初免当天起每2周空腹尾尖采血1次,直至处死。所有血样分离血清,使用ELISA法测定血清抗体含量,RIA法测定血清激素T、FSH和LH含量。各小组雄鼠分别于8wpv、12wpv、16wpv处死,分离下丘脑、垂体,提取总RNA,使用荧光定量PCR测定各基因]mRNA表达情况；mIG和mCG分离双侧睾丸,去除附睾后精确称重,提取总RNA,使用荧光定量PCR测定各基因]nRNA表达情况。
GnRH主动免疫雄鼠后,血清抗体滴度升高,睾丸重量减小,睾丸严重皱缩。在8wpv、12wpv和16wpv采样时,mIG睾丸平均重量分别为mCG睾丸平均重量的55.22%、54.31%和42.55%,均显著低于mCG (p＜0.05)。GnRH主动免疫使mIG大鼠血清T、FSH和LH浓度显著下降,均显著低于mCG(p<0.05)。而外科手术去势使血清T含量显著下降至检测限附近,而使血清FSH和LH含量上升,二者浓度均显著高于空白组(p<0.05)。与mCG相比,GnRH主动免疫显著下调下丘脑GnRH, GPR54、KiSS-1和AR mRNA,垂体GnRH-R、FSH-β和LH-βmRNA以及睾丸FSHR、LHR和AR mRNA表达水平(p<0.05)；而外科手术显著下调下丘脑下丘脑GnRH、GPR54、KiSS-1和AR mRNA、睾丸FSHR、LHR和AR mRNA表达水平(p<0.05)的同时,对垂体GnRH-R、 FSH-β和LH-βmRNA表达水平显著上调(p<0.05)。
3.选取健康性成熟雌性SD大鼠72只,随机分为GnRH免疫去势组(fIG)、手术去势组(fSG)和空白组(fCG),每组24只(各组再随机分为3个小组,n=8)。fIG于12周龄腿部肌肉注射1mL疫苗(含GnRH-TD50μg),4周后加免,加免时注射剂量及方法同初次免疫；fSG于11周龄进行手术,摘除卵巢；fCG不做任何处理。初次免疫当天记为0wpv。初免当天起每2周空腹尾尖采血1次,直至处死。所有血样分离血清,使用ELISA法测定血清抗体含量,RIA法测定血清激素E2、P4、FSH和LH含量。各小组雌鼠分别于8wpv、12wpv、16wpv处死,分离下丘脑、垂体,提取总RNA,使用荧光定量PCR测定各基因mRNA表达情况；fIG和fCG分离双侧卵巢,去除脂肪组织后精确称重,提取总RNA,使用荧光定量PCR测定各基因mRNA表达情况。
GnRH主动免疫雌鼠后,血清抗体滴度升高,卵巢重量减小,卵巢和子宫萎缩。8wpv、12wpv和16wpv采样时fIG卵巢平均重量分别为fCG平均重量的63.63%、38.46%和21.43%,均显著低于fCG (p＜0.05)。GnRH主动免疫使fIG大鼠血清E2、P4、FSH和LH浓度显著下降,均显著低于fCG (p＜0.05)。而外科手术去势使fSG大鼠血清E2、P4含量显著下降至检测限附近；而使其血清FSH和LH含量上升,二者浓度均显著高于空白组(p＜0.05)。GnRH主动免疫明显下调雌鼠下丘脑GPR54、KiSS-1、GnRH和ER-α mRNA、垂体GnRH-R、FSH-β和LH-β mRNA以及卵巢FSHR、LHR和ER-α mRNA的表达水平(p<0.05)。而外科去势对雌鼠生殖相关基因]mRNA的表达调控有不同影响：显著下调下丘脑GPR54、KiSS-1、GnRH和ER-α mRNA,以及卵巢FSHR、LHR和ER-α mRNA的表达水平；与此同时,显著上调垂体GnRH-R、FSH-β和LH-β mRNA表达水平。
4.选取健康性成熟雄性SD大鼠24只,随机分为免疫组(mIG)、手术组(mSG)和空白组(mCG),每组8只(n=8)。mIG于12周龄腿部肌肉注射1mL疫苗(含GnRH-TD50μg),4周后加免,加免时注射剂量及方法同初次免疫；mSG于11周龄手术摘除睾丸；mCG不做任何处理。初次免疫当天记为0wpv。初免当天至8wpv每2周、8wpv至56wpv每4周空腹尾尖采血1次,直至处死(56wpv)。所有血样分离血清,使用ELISA法测定血清抗体含量,使用RIA法测定血清激素T、FSH和LH含量。各组大鼠于56wpv处死,迅速分离下丘脑、垂体,提取总RNA,使用荧光定量PCR测定各基因mRNA表达情况；mIG和mCG分离双侧睾丸,精确称重,提取总RNA,使用荧光定量PCR测定各基因mRNA表达情况。
GnRH主动免疫雄性大鼠,产生抗GnRH抗体,在12wpv时达到高峰并维持较长时间,20wpv后开始下降,至40wpv时抗体滴度下降至免疫前水平,至56wpv处死时几乎已无抗体存在。56wpv采样时,大鼠睾丸表面仍然皱缩,轻按可以感觉到并无弹性,且重量显著低于mCG (p＜0.05);主动免疫显著降低mIG大鼠血清T、FSH和LH含量,至56wpv处死时除T含量有微弱上升,FSH和LH含量维持较低水平。三种激素含量显著低于mCG (p＜0.05)。外科手术去势使mSG大鼠血清T含量显著下降至检测限附近；而使其血清FSH和LH含量上升,二者浓度均显著高于空白组(p<0.05)。GnRH主动免疫56wpv后,下丘脑GPR54、KiSS-1、GnRH和AR mRNA,垂体GnRH-R、FSH-β和LH-βmRNA以及睾丸FSHR、LHR和AR mRNA的表达水平均显著低于mCG(p<0.05)；对于mSG,56wpv采样时,雄鼠下丘脑GPR54、KiSS-1、 GnRH和AR mRNA,以及睾丸FSHR、LHR和AR mRNA的表达水平均显著低于mCG,(p<0.05),而垂体GnRH-R, FSH-β和LH-β mRNA表达量显著高于mCG(p＜0.05)。
5.选取健康性成熟雌性SD大鼠24只,随机分为免疫组(fIG)、手术组(fSG)和空白组(fCG),每组8只(n=8),fIG于12周龄腿部肌肉注射1mL疫苗(含GnRH-TD50μg),4周后加免,加免注射剂量及方法同初次免疫；fSG于11周龄进行手术,摘除卵巢；fCG不做任何处理。初次免疫当天记为0wpv。初免当天至8wpv每2周、8wpv至56wpv每4周空腹尾尖采血1次,直至处死。所有血样分离血清,使用ELISA法测定血清抗体含量,RIA法测定血清激素E2、P4、FSH和LH含量。各组大鼠于56wpv处死,分离下丘脑、垂体,提取总RNA,使用荧光定量PCR测定各基因mRNA表达情况；fIG和fCG分离双侧卵巢,去除脂肪组织后精确称重,提取总RNA,使用荧光定量PCR测定各基因mRNA表达情况。
GnRH主动免疫雌性大鼠，产生抗GnRH抗体,在12wpv时达到高峰并维持较长时间,20wpv后开始缓慢下降,40wpv时抗体滴度下降至免疫前水平,至56wpv处死时几乎已无抗体存在。GnRH主动免疫56周后,卵巢和子宫仍然保持较好的去势状态,fIG卵巢重量显著低于fCG (p＜0.05)。主动免疫显著降低fIG大鼠血清E2、P4、FSH和LH含量,至56wpv处死时除E2和LH含量有微弱上升,P4和FSH含量维持较低水平。四种激素含量显著低于mCG (p＜0.05)。而外科手术去势使fSG大鼠血清E2、P4含量显著下降至检测限附近,血清FSH和LH含量上升,二者浓度均显著高于空白组(p<0.05)。GnRH主动免疫56wpv后,雌鼠下丘脑GPR54、KiSS-1、 GnRH和ER-α mRNA,垂体GnRH-R、FSH-β和LH-β mRNA以及卵巢FSHR、LHR和ER-α mRNA的表达水平均显著低于mCG (p＜0.05);对于fSG,56wpv采样时,下丘脑GPR54、KiSS-1、GnRH和ER-α mRNA,以及卵巢FSHR、LHR和ER-a mRNA的表达水平均显著低于mCG (p＜0.05),而垂体GnRH-R、FSH-β和LH-β mRNA表达量显著高于mCG。
综上所述：GnRH-TDO具有良好的免疫原性。GnRH-TDO主动免疫能显著抑制SD大鼠性腺发育,显著降低血清FSH、LH以及性激素(雄性血清T,雌性血清E2和P4)的含量；GnRH-TDO主动免疫显著下调雄性和雌性SD大鼠下丘脑GnRH、 GPR54、KiSS-1和AR mRNA/ER-a mRNA,垂体GnRH-R、FSH-β和LH-βmRNA以及性腺FSHR、LHR和AR mRNA/ER-α mRNA表达水平。主动免疫降低血清性激素含量,通过下丘脑AR mRNA/ER-a mRNA-GPR54/KiSS-1通路,下调GnRH mRNA表达,从而降低血清FSH、LH含量,最终使动物去势。经GnRH-TDO主动免疫56w后的SD大鼠仍处于去势状态,雄性大鼠血清T含量和雌性大鼠血清LH含量在试验末期出现上升趋势,揭示GnRH主动免疫造成的动物去势存在恢复的可能,且动物去势状态维持时限可能存在性别差异。
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.
KiSS-1基因是KiSS基因的亚型,主要在中枢神经系统和胎盘表达,该基因编码的一组肽产物称为kisspeptins, GPR54(G protein-coupled receptor54)是kisspeptin的天然受体。KiSS-1/GPR54系统与生殖系统的发育有密切关系：研究表明,GnRH神经元是kisspeptins的直接调节对象；目前普遍认为的作用机制是kisspeptins与GPR54结合,激活GnRH神经元,刺激GnRH的释放,进而调节FSH和LH的释放。
GnRH主动免疫动物能使其达到去势效果,但去势机制是怎样的?去势效果维持时间的长短在不同研究中为何有较大差别?关于这些问题目前尚无统一定论。鉴于此,本研究制备了GnRH免疫去势疫苗,主动免疫SD大鼠,结合大鼠血清抗体、血清激素、生殖相关基因及其受体mRNA表达变化情况,探讨主动免疫后KiSS-1/GPR54系统和下丘脑-垂体-性腺轴各级激素、受体和配体的调控关系,以期阐明GnRH主动免疫去势的作用机理；此外,还对GnRH免疫去势的可逆性进行了研究。本文开展的主要研究工作和取得的结果如下：
1.以组成GnRH的9种氨基酸为原料合成GnRH并列体(GnRH-Tandem)和GnRH并列体二聚物(GnRH-Tandem-Dimer, GnRH-TD),使用碳二亚胺法将GnRH-TD与卵清蛋白(OVA)偶联,制成GnRH-TDO免疫原,经SDS-PAGE分析,在51.5KDa处出现了预期条带,得到合格的免疫原；以矿物油、‘Tween-85和span-85为原料制作specol佐剂,将GnRH-TDO免疫原与specol佐剂充分乳化制成GnRH-TDO去势疫苗,检验去势疫苗的物理特性和稳定性,均符合要求。制得合格的GnRH-TDO免疫去势疫苗。
2.选取健康性成熟雄性SD大鼠72只,随机分为GnRH免疫去势组(mIG)、手术,去势组(mSG)和空白组(mCG),每组24只(各组再随机分为3个小组,n=8)。mIG于12周龄腿部肌肉注射1mL疫苗(含GnRH-TD50μg),4周后加免,加免时注射剂量及方法同初次免疫；mSG于11周龄进行手术,摘除睾丸；mCG不做任何处理。初次免疫当天记为Owpv (weeks post vaccination)。初免当天起每2周空腹尾尖采血1次,直至处死。所有血样分离血清,使用ELISA法测定血清抗体含量,RIA法测定血清激素T、FSH和LH含量。各小组雄鼠分别于8wpv、12wpv、16wpv处死,分离下丘脑、垂体,提取总RNA,使用荧光定量PCR测定各基因]mRNA表达情况；mIG和mCG分离双侧睾丸,去除附睾后精确称重,提取总RNA,使用荧光定量PCR测定各基因]nRNA表达情况。
GnRH主动免疫雄鼠后,血清抗体滴度升高,睾丸重量减小,睾丸严重皱缩。在8wpv、12wpv和16wpv采样时,mIG睾丸平均重量分别为mCG睾丸平均重量的55.22%、54.31%和42.55%,均显著低于mCG (p＜0.05)。GnRH主动免疫使mIG大鼠血清T、FSH和LH浓度显著下降,均显著低于mCG(p<0.05)。而外科手术去势使血清T含量显著下降至检测限附近,而使血清FSH和LH含量上升,二者浓度均显著高于空白组(p<0.05)。与mCG相比,GnRH主动免疫显著下调下丘脑GnRH, GPR54、KiSS-1和AR mRNA,垂体GnRH-R、FSH-β和LH-βmRNA以及睾丸FSHR、LHR和AR mRNA表达水平(p<0.05)；而外科手术显著下调下丘脑下丘脑GnRH、GPR54、KiSS-1和AR mRNA、睾丸FSHR、LHR和AR mRNA表达水平(p<0.05)的同时,对垂体GnRH-R、 FSH-β和LH-βmRNA表达水平显著上调(p<0.05)。
3.选取健康性成熟雌性SD大鼠72只,随机分为GnRH免疫去势组(fIG)、手术去势组(fSG)和空白组(fCG),每组24只(各组再随机分为3个小组,n=8)。fIG于12周龄腿部肌肉注射1mL疫苗(含GnRH-TD50μg),4周后加免,加免时注射剂量及方法同初次免疫；fSG于11周龄进行手术,摘除卵巢；fCG不做任何处理。初次免疫当天记为0wpv。初免当天起每2周空腹尾尖采血1次,直至处死。所有血样分离血清,使用ELISA法测定血清抗体含量,RIA法测定血清激素E2、P4、FSH和LH含量。各小组雌鼠分别于8wpv、12wpv、16wpv处死,分离下丘脑、垂体,提取总RNA,使用荧光定量PCR测定各基因mRNA表达情况；fIG和fCG分离双侧卵巢,去除脂肪组织后精确称重,提取总RNA,使用荧光定量PCR测定各基因mRNA表达情况。
GnRH主动免疫雌鼠后,血清抗体滴度升高,卵巢重量减小,卵巢和子宫萎缩。8wpv、12wpv和16wpv采样时fIG卵巢平均重量分别为fCG平均重量的63.63%、38.46%和21.43%,均显著低于fCG (p＜0.05)。GnRH主动免疫使fIG大鼠血清E2、P4、FSH和LH浓度显著下降,均显著低于fCG (p＜0.05)。而外科手术去势使fSG大鼠血清E2、P4含量显著下降至检测限附近；而使其血清FSH和LH含量上升,二者浓度均显著高于空白组(p＜0.05)。GnRH主动免疫明显下调雌鼠下丘脑GPR54、KiSS-1、GnRH和ER-α mRNA、垂体GnRH-R、FSH-β和LH-β mRNA以及卵巢FSHR、LHR和ER-α mRNA的表达水平(p<0.05)。而外科去势对雌鼠生殖相关基因]mRNA的表达调控有不同影响：显著下调下丘脑GPR54、KiSS-1、GnRH和ER-α mRNA,以及卵巢FSHR、LHR和ER-α mRNA的表达水平；与此同时,显著上调垂体GnRH-R、FSH-β和LH-β mRNA表达水平。
4.选取健康性成熟雄性SD大鼠24只,随机分为免疫组(mIG)、手术组(mSG)和空白组(mCG),每组8只(n=8)。mIG于12周龄腿部肌肉注射1mL疫苗(含GnRH-TD50μg),4周后加免,加免时注射剂量及方法同初次免疫；mSG于11周龄手术摘除睾丸；mCG不做任何处理。初次免疫当天记为0wpv。初免当天至8wpv每2周、8wpv至56wpv每4周空腹尾尖采血1次,直至处死(56wpv)。所有血样分离血清,使用ELISA法测定血清抗体含量,使用RIA法测定血清激素T、FSH和LH含量。各组大鼠于56wpv处死,迅速分离下丘脑、垂体,提取总RNA,使用荧光定量PCR测定各基因mRNA表达情况；mIG和mCG分离双侧睾丸,精确称重,提取总RNA,使用荧光定量PCR测定各基因mRNA表达情况。
GnRH主动免疫雄性大鼠,产生抗GnRH抗体,在12wpv时达到高峰并维持较长时间,20wpv后开始下降,至40wpv时抗体滴度下降至免疫前水平,至56wpv处死时几乎已无抗体存在。56wpv采样时,大鼠睾丸表面仍然皱缩,轻按可以感觉到并无弹性,且重量显著低于mCG (p＜0.05);主动免疫显著降低mIG大鼠血清T、FSH和LH含量,至56wpv处死时除T含量有微弱上升,FSH和LH含量维持较低水平。三种激素含量显著低于mCG (p＜0.05)。外科手术去势使mSG大鼠血清T含量显著下降至检测限附近；而使其血清FSH和LH含量上升,二者浓度均显著高于空白组(p<0.05)。GnRH主动免疫56wpv后,下丘脑GPR54、KiSS-1、GnRH和AR mRNA,垂体GnRH-R、FSH-β和LH-βmRNA以及睾丸FSHR、LHR和AR mRNA的表达水平均显著低于mCG(p<0.05)；对于mSG,56wpv采样时,雄鼠下丘脑GPR54、KiSS-1、 GnRH和AR mRNA,以及睾丸FSHR、LHR和AR mRNA的表达水平均显著低于mCG,(p<0.05),而垂体GnRH-R, FSH-β和LH-β mRNA表达量显著高于mCG(p＜0.05)。
5.选取健康性成熟雌性SD大鼠24只,随机分为免疫组(fIG)、手术组(fSG)和空白组(fCG),每组8只(n=8),fIG于12周龄腿部肌肉注射1mL疫苗(含GnRH-TD50μg),4周后加免,加免注射剂量及方法同初次免疫；fSG于11周龄进行手术,摘除卵巢；fCG不做任何处理。初次免疫当天记为0wpv。初免当天至8wpv每2周、8wpv至56wpv每4周空腹尾尖采血1次,直至处死。所有血样分离血清,使用ELISA法测定血清抗体含量,RIA法测定血清激素E2、P4、FSH和LH含量。各组大鼠于56wpv处死,分离下丘脑、垂体,提取总RNA,使用荧光定量PCR测定各基因mRNA表达情况；fIG和fCG分离双侧卵巢,去除脂肪组织后精确称重,提取总RNA,使用荧光定量PCR测定各基因mRNA表达情况。
GnRH主动免疫雌性大鼠，产生抗GnRH抗体,在12wpv时达到高峰并维持较长时间,20wpv后开始缓慢下降,40wpv时抗体滴度下降至免疫前水平,至56wpv处死时几乎已无抗体存在。GnRH主动免疫56周后,卵巢和子宫仍然保持较好的去势状态,fIG卵巢重量显著低于fCG (p＜0.05)。主动免疫显著降低fIG大鼠血清E2、P4、FSH和LH含量,至56wpv处死时除E2和LH含量有微弱上升,P4和FSH含量维持较低水平。四种激素含量显著低于mCG (p＜0.05)。而外科手术去势使fSG大鼠血清E2、P4含量显著下降至检测限附近,血清FSH和LH含量上升,二者浓度均显著高于空白组(p<0.05)。GnRH主动免疫56wpv后,雌鼠下丘脑GPR54、KiSS-1、 GnRH和ER-α mRNA,垂体GnRH-R、FSH-β和LH-β mRNA以及卵巢FSHR、LHR和ER-α mRNA的表达水平均显著低于mCG (p＜0.05);对于fSG,56wpv采样时,下丘脑GPR54、KiSS-1、GnRH和ER-α mRNA,以及卵巢FSHR、LHR和ER-a mRNA的表达水平均显著低于mCG (p＜0.05),而垂体GnRH-R、FSH-β和LH-β mRNA表达量显著高于mCG。
综上所述：GnRH-TDO具有良好的免疫原性。GnRH-TDO主动免疫能显著抑制SD大鼠性腺发育,显著降低血清FSH、LH以及性激素(雄性血清T,雌性血清E2和P4)的含量；GnRH-TDO主动免疫显著下调雄性和雌性SD大鼠下丘脑GnRH、 GPR54、KiSS-1和AR mRNA/ER-a mRNA,垂体GnRH-R、FSH-β和LH-βmRNA以及性腺FSHR、LHR和AR mRNA/ER-α mRNA表达水平。主动免疫降低血清性激素含量,通过下丘脑AR mRNA/ER-a mRNA-GPR54/KiSS-1通路,下调GnRH mRNA表达,从而降低血清FSH、LH含量,最终使动物去势。经GnRH-TDO主动免疫56w后的SD大鼠仍处于去势状态,雄性大鼠血清T含量和雌性大鼠血清LH含量在试验末期出现上升趋势,揭示GnRH主动免疫造成的动物去势存在恢复的可能,且动物去势状态维持时限可能存在性别差异。
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.
引文
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