Effects Of Active Immunization Against GnRH On The Hypothalamic-Pituitary-Testicular Axis And Reproductive Feedback System In Male Rats

In late1960s, hormone immuno-nutralization techniques offered a new regulative method in animal reproduction. Active immunization against GnRH results in prolonged antibodies, which specifically neutralize endogenous GnRH and block the reproductive hormone cascade, disrupts the hypothalamic-pituitary-gonadal axis, inhibits the synthesis and secretion of LH and FSH, and results in sex steroids deprivation and arrest of spermatogenesis, thus causing infertility in males and females. Recently, active immunization against GnRH which avoids disadvantages of surgical castration, has successfully applied as a promising alternative to surgical castration. It is generally considered that hormone-immunoneutralization hypothesis is the "true" reason responsible for infertility caused by immunizing against GnRH, while the exact mechanism remains unclear so far. Furthermore, active immunization against GnRH disrupts the balance status of the reproductive axis as long as antibodies elevated, whether this has a detrimental effect on the reproductive axis is largely unknown. Therefore, the present study, using sexually mature male SD rat as a model, is aimed to investigate the molecular mechanisms of active immunization against GnRH by focusing on effects of active immunization against GnRH on the mRNA expressions of hypothalamic AR, ERα,kiss-1/GPR54、GnRH, pituitary receptor GnRHR, gonadotropin subunit genes and gonadotropin receptors in testes.Thirty-six adult male Sprague-Dawley (SD) rats (BW275-311) at the age of10-11weeks, were randomly allocated to one of three treatments (n=12) as follows. Briefly,12rats were immunized against50ug D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin (TDK-OVA) in Specol adjuvant at12weeks of age with a booster vaccination8weeks later.12intact males and12castrated rats (surgically castrated at age of11weeks) were not administrated and served as controls. All rats were decapitated at4weeks after booster vaccination, blood samples for antibody titter and hormone assays were collected at0week post vaccination (wpv), every2weeks thereafter, and the day of decapitation. At decapitation, testes weight and volume and pituitary weight were recorded. Hypothalamus, pituitary and testis were collected to detect the mRNA expressions of reproduction-related genes with real-time fluorescence quantitative PCR, and the hypothalamus and testes were excised for tissue GnRH detection and morphological observation, respectively. Results indicated that eleven of twelve immunized rats responded well to the immunization (immunocastrates), while the remaining one rat responded poorly (non-responder). Compared to intact controls, immunocastration significantly decreased the levels of gonadotropins (LH and FSH) and testosterone (T) levels (p<0.05) in sera, and the GnRH content in hypothalamic eminence and the pituitary weight at decapitation (p<0.01). Testes in immunocastrates were reduce to20%of both the average weight and volume of intact controls at decapitation (p<0.01). Testis histologic evaluation displayed severe damage of leydig-cell interstitial tissue and absence of spermatids. While the levels of serum LH and FSH, and the weight of the pituitary of surgical castrates were significantly increased after castration as compared to intact controls (p<0.05). By quantitative PCR analysis, the mRNA expression levels of androgen-receptor (AR), estrogen receptor alpha(ERa), kiss-1, GPR54and GnRH in the hypothalaus, and GnRH receptor (GnRH-R), LH-β, FSH-β in the pituitary as well as LH receptor (LH-R) and FSH receptor (FSH-R) in the testes of immunocastrates were significantly down-regulated after immunization as compared to intact controls (p<0.05). Similarly, surgical castration also reduced the amount of GnRH in the median eminence as well as mRNA expression levels of GnRH, AR, Kiss-1and GPR54in the hypothalamus (P<0.05).These results firstly suggest that immunization against GnRH1) reduces the synthesis of hypothalamic GnRH as sex steroids are necessary to maintain the synthesis of hypothalamic GnRH;2) causes dysfunction of the pituitary-testicular axis. Both the loss of the synthesis of GnRH and dysfunction of pituitary-testicular axis seems to be the true mechanisms underlying immunocastration.