Molecular Mechanisms Of Active Immunization Against GnRh On Reproduction And Performance In Mammals

Hypothalamic gonadotropin-releasing hormone(Gn RH)tightly controls reproductive function and reproductive behavior in mammals through hypothalamic-pituitary-gonadal(HPG)axis.At the same time,reproductive neuroendocrine system has tight linkages with productive system,immune system as well as the formation of boart taint in intact boars.Active immunization against Gn RH(also called immunocastration)effectively inhibits sex hormone production and gamatogenesis throught the immunoneutralization of endogenous Gn RH,finally resulting in infertility in both males and femals,which has been the most promising animal-fridendly alternative to surgical castration.However,there still exist some problems,which prevent the further application of Gn RH immunocastration: 1)a few part of animals are not responded well after anti-Gn RH vaccination;2)in order to achieve a good immunocastrative effect,the injected dosage of Gn RH antigen as well as vaccination times is usually high;3)the mechanism underlying immunocastration is unknow until now,which further prevents the the solution for the problem mentioned above;4)the interactions between reproductive axis and productive axis,immune system and boar taint compound metabolism,are largely unknown.Therefore,the present study,with five experiments,was aimed to explore the designing strategies for new Gn RH-like antigens,and the effects as well as the underlying mechanisms of active immunization against Gn RH on reproductive endocrine system,productive endocrine system,immune system as well as the metabolic metabolism of boar taint compounds.Our final data will provide suggestions to making highly effective anti-Gn RH vaccines and expanding the large-scale applications of Gn RH vaccine in animal hunsbandry.Exp.1 The designing strategies of new Gn RH-like constructs and comparative effects of three new different Gn RH-like peptide constructs in male SD ratsThe designing strategies for the new Gn RH-like peptide constructs are based on 1)increasing the number of Gn RH inserts;2)increasing the molecular weight of Gn RH antigen.The new Gn RH-like peptide constructs were designed based on a Gn RH-tandem peptide,which was modified by relacing Gly at position 6 of the decapeptide with D-Lys(G6KT)to allow conjugation of the Gn RH peptide to ovalbumin(OVA).The first new Gn RH-like construct was prepared by the dimerization of G6KT(G6KTD);the second and third one were prepared by conjugation G6 KT and G6 KTD to OVA,respectively.Sixty male Sprague-Dawlry(SD)rats at the age of 6 weeks,were randomly allocated to five groups(n=12):intact controls(no treatment),surgically castrated or vaccinated against 100 ug peptide equivalent of G6 KTD,G6KT-OVA and G6KTD-OVA in Specol adjuvant at 6 weeks of age(with a booster 8 wk later).All rats were decapitated at 4 weeks after booster vaccination,blood samples for antibody titter and hormone assays were collected at 0 week post vaccination(wpv),every 4 weeks thereafter until decapitation.At decapitation,testes weight and volume were recorded.Pituitary and testis were collected to detect the m RNA expressions of reproduction-related genes with q PCR,and testes were excised for morphological observation too.The results shown that,1)all of three different new Gn RH-like constructs can intrigue good antibody responses in male rats;2)active immunization against G6KT-OVA and G6KTD-OVA reduced(P < 0.05)serum LH,FSH,testosterone and inhibin B concentrations 4 weeks after the first injection and then further decreased after the booster vaccination,and testes at decapitation were reduced to lower than 50% of the weight and volume of testes in intact controls and spermatogenesis was completely stopped at decapitation;3)while the decrease of serum concentrations of LH,FSH,testosterone and inhibin B in G6KTD-immunized male rats only occurred after the booster injection.Among 12 of G6KTD-immunized male rats,9 rats were responded well to the vaccination(with significantly decreased serum reproductive hormones and atrophy of testes),whereas the remaining 3 were not responded well without effects on serum reproductive hormones and testes parameters.Based on those,we concluded that both G6KT-OVA and G6KTD-OVA were highly efficacious in two vaccinations at low dose,which could be possibly used as one-shot vaccine in the furthure,whereas G6 KTD itself was also effective in two vaccinations,which may be used as Gn RH antigen in relative high dose and so to avoid the drawbacks derived from the conjugation and purification processes of Gn RH-carrier conjugates.Exp.2 Active immunization against GnRH on the functional integrity of hypothalamic-pituitary-testicualr axis in male SD rats,sheep and boarsAll the three animal species were randomly and equally allocated into three groups: control(no treatment),surgically castrated,or immunized against G6KT-OVA for rats or G6 k TD-OVA for sheep and boars in Specol adjuvant(with a booster 8 wk later).Blood samples(for antibody titers and hormone concentrations)were collected.Compared to intact controls,anti-Gn RH immunization reduced(P < 0.05)serum concentrations of testosterone,LH and FSH,and the Gn RH content in the median eminence,reduced the weight of the hypohysis(P < 0.05),and induced testicular atrophy(suppression of spermatogenesis).Furthermore,m RNA expression of Gn RH in the hypothalamus arcuate nucleus(Arc)and anteroventral periventricular nucleus(AVPV),Gn RH receptor,LH? and FSH? in the pituitary,LH receptor and FSH receptor in the testes,and genes in Gn RH upstream signal pathways,including androgen receptor(AR),kisspeptin encoded gene(Kiss1)and kisspeptin receptor(GPR54)in the hypothalamus were decreased in immunocastrated rats compared to intact controls(P < 0.05).Similarly,surgical castration also reduced Gn RH in the median eminence as well as m RNA expression of Gn RH,AR,Kiss1 and GPR54 in the hypothalamus(P < 0.05).All these results were consistent among the three different animal species.Accordingly,we concluded that anti-Gn RH immunization reduced synthesis of hypothalamic Gn RH by decreasing androgen-androgen receptor-Kisspeptin-GPR54 signaling pathways,which should be one important reason causing immunocastration.Moreover,the downregulation of Gn RHR in pituitary and LHR and FSHR in testes after immunocastration disrupted the functional integrity of HPG axis,which is also partly responsible for immunocastration.Exp.3 Active immunization against GnH the function of GHRH/SS-GH-IGF1 in male rats and boarsThis study was conducted on the same rats and boars used in Exp.2.Blood samples were collected(for hormone and urea nitrogen concentrations)and growth performance was evaluated.Compared to intact controls,surgical castration reduced(P < 0.05)m RNA expressions of hypothalamic gonadotropin-inhibitoary hormone(Gn IH)and growth hormone-releasing hormone(GHRH),pituitary growth hormone(GH)and liver insulin-like growth factor-1(IGF1),reduced(P < 0.05)serum concentrations of GH and IGF-1 and increased(P < 0.05)serum concentrations of urea nitrogen.In contrast,immunocastration did not alter m RNA expressions of hypothalamic Gn IH,GHRH and pituitary GH,and the serum concentrations of GH(P > 0.05).Moreover,serum concentrations of IGF-1 and urea nitrogen in immunocastrates were substantially higher and lower than in surgical castrates,respectively(P < 0.05).Compared to surgical castrates,immuncastrates had superior feed conversion efficiency(P < 0.05).These results were consistent between SD male rats and boars.Accordingly,we concluded that surgical castration but not immunocastration is associated with reduced hypothalamic Gn IH and GHRH/GH/IGF-I axis function in male rats and boars.And Gn IH likely acts as the linkage between reproductive endocrine axis and productive endocrine axis.Exp.4 Active immunization against Gn RH in immune function of spleen in ramsThis study was conducted using the same animals used in Exp.2.Blood samples(for hormone and immune cytokine profiles)were collected at 4 weeks intervals until rams were slaughtered(10 mo).Compared to intact controls,anti-Gn RH immunization reduced(P < 0.05)serum concentrations of LH,FSH,and testosterone.Reduced testosterone abrogated its inhibitor feedback effect on the synthesis of Gn RH in spleen,as evidenced by increased(P < 0.05)protein content and m RNA expressions of Gn RH,and simultaneously decreased(P < 0.05)m RNA expressions of androgen receptor in spleen.In parallel with the increased Gn RH production in spleen,the m RNA expressions of interleukin(IL)-2,IL-4,IL-6 and tumor necrosis factor alpha(TNF-?)as well as lymphocyte marker CD4,CD8 and CD19 molecules were increased(P<0.05)in spleen.Consistently,serum concentrations of IL-2,IL-4,IL-6,TNF-? were increased(P<0.05)in rams following immunization.Similarly,deprivation of testosterone by surgical castration also increased(P < 0.05)Gn RH and thus immune cytokine expressions in spleen.Collectively,our data suggested that immunocastration increased Gn RH production in spleen by abrogating the inhibitory feedback effects from testosterone,consequently improving the immune markers of spleen and serum immune cytokines in rams.Exp.5 Active immunization against GnRH on boar taint compound metabolism in boarsThis study was conducted using the same animals as in Exp.2.Serum,fat,liver and testis tissues were collected for parameter analysis.Compared to intact controls,immunocastration substantially abolished testicular androstenone synthesis as evidenced by almost undetectable androstenone levels in serum and fat,and decreased m RNA and protein expressions of androstenone synthetic enzymes(CYP11A1,CYP17A1 and CYB5A)in testes(P < 0.05).Deprivation of testicular steroids accelerated androstenone metabolism in liver by increasing m RNA and protein expressions of hepatic androstenone metabolizing enzymes(3?-HSD,3?-HSD and SULT2A1)in both immunologically-and surgically-castrated boars.Likewise,immunocastration decreased(P < 0.01)levels of skatole in serum and fat in boars at slaughter.In liver,expressions of key skatole metabolizing enzymes including CYP2E1,CYP2A19,CYP2C49 and CYB5 A were increased(P < 0.05)by immunocastration,which persisted in surgical castrates.Moreover,androstenone,were more positively correlated with skatole levels in serum than testosterone and 17?-estradiol in intact males,suggesting androstenone exert a great suppressive effect on the hepatic skatole metabolism.Collectively,immunocastration could effectively abolish testicular androstenone as well as other steroid synthesis,which in turn accelerates hepatic skatole and androstenone clearance-metabolism,thereby ultimately eliminating boar taint accumulation in boars.It seems that testicular biosynthesis of androstenone is the primary determinant factor responsible for the deposition of both androstenone and skatole in adipose tissues in boars.