The Effects And Mechanism Of KISS1 Gene Immunization On Castration

Surgical castration of thousands of years has been challenging for the standardized scale livestock production. Gene immunocastration as a new, most promising castration technology can replace surgical castration. Kisspeptin encoded by KISS1 gene, which is secreted by hypothalamus from the most upstream of reproductive axis, plays a decisive role in reproduction switch by controlling the secretion of Gn RH,meanwhile, it is also critical for onset of puberty and control of adult fertility. This study selects KISS1 gene in the topmost of reproductive axis as a target gene to construct eukaryotic expression plasmid p KS-asd by a series of techniques, such as gene cloning, RT-PCR, Western blot, ELISA, IHC, QPCR and histological HE staining. When the expression of fusion antigen KS is detected correctly, the ram lambs are immunized with p KS-asd plasmid, aims to,explore the effects,mechanism,impact on growth, reversibility and safety of KISS1 gene immunocastration. This study provides a theoretical basis and technical support for the development of high efficiency and low cost of new technology and new method of immunocastration.1. The construction and identification of p KS-asd eukaryotic expression plasmid without antibiotic resistance geneChemically-synthesized KISS1 gene was subcloned into p VAX1 vector to construct p VAX1-KISS1 plasmid and then HBs Ag-S gene was subcloned into the p VAX-KISS1 plasmid to construct p KS plasmid. Finally, antibiotic resistance gene kan of the p VAX-asd plasmid was replaced by nutritional selected marker asd gene to construct p KS-asd eukaryotic expression plasmid without antibiotic resistance gene. The sequence, insertion site and direction of the fusion gene KS were identified correctly by restriction endonuclease digestion and sequencing. The KS fusion gene was detected by RT-PCR and KS fusion protein was detected by Western blot when the He La cells were transfected for 24 h and for 48 h, respectively.2. The effects of KISS1 gene immunization on castrationSix 8 week old ram lambs were randomly divided into two equal groups. Vaccinated group were injected intramuscularly with p KS-asd while the control group animals were treated with p VAX-asd, and the same treatments were repeated at weeks 3 and 6 after the primary immunization.The lambs were slaughtered at week 14 after the primary immunization. Anti-kisspeptin-antibodies were detected in vaccinated group ram lambs at weeks 4-14 after the primary immunization, meanwhile, serum testosterone level in vaccinated group ram lambs was showed significantly lower than that in control animals(P < 0.05). Scrotal circumference in vaccinated group ram lambs was significantly lower than that control animals at weeks 4-14 after the primary immunization(P < 0.05), likewise testes weight and length in vaccinated group was significantly lower than that control animals(P < 0.05). The seminiferous tubules of vaccinated animals contained low densities of spermatogonium, spermatocytes and spermatids compared to control group animals. Sexual behaviours of vaccinated animals were significantly lower than controls(P < 0.05). In conclusion, immunization of KISS1 gene vaccine induced a strong antibody response and resulted in the suppression of gonadal function and sexual behaviour in animals, achieved the purpose of the castration, demonstrated as KISS1 can be used as a novel target for developing a DNA immunocastration vaccine.3. The mechanism of KISS1 gene immunocastration①Immunoneutralization of anti-kisspeptin-antibodies on the endogenic kisspeptin in peripheral bloodAnti-kisspeptin-antibodies were detected in vaccinated group ram lambs at weeks 4-14 after the primary immunization, meanwhile, serum testosterone level in vaccinated group animal was significantly lower than control animals at weeks 6-14 after the primary immunization(P < 0.05). The results demonstrated that anti-kisspeptin-antibodies from KISS1 gene immunization could neutralize the endogenic kisspeptin in peripheral blood, thus leading to a sharp drop in concentration of kisspeptin in serum and achieving the purpose of castration.②Immunoneutralization of anti-kisspeptin-antibodies crossing blood-brain barrier or blood-testis barrierThe anti-kisspeptin-antibodies were undetectable in the brain and testis tissue. The results demonstrated that anti-kisspeptin-antibodies from KISS1 gene immunization could not cross blood-brain barrier or blood-testis barrier and may play a role of immunoneutralization directly in the brain or testis.③ The effect of KISS1 gene immunization on expression of KISS1 gene in hypothalamic-pituitary-gonadal axis(HPG axis)There was no difference in KISS1 m RNA level of hypothalamus, pituitary and testis tissue between vaccinated group animals and control animals. The results demonstrated that the pathway may not be the mechanism of KISS1 gene immunocastration by influencing the synthesis of KISS1 gene in HPG axis.④The effect of KISS1 gene immunization on expression of GPR54 gene in HPG axisThe GPR54 m RNA level of hypothalamus, pituitary and testis tissue between vaccinated group animals and control animals did not show significant difference. The results demonstrated that the pathway may not be the mechanism of KISS1 gene immunocastration by influencing the synthesis of GPR54 gene in HPG axis.In conclusion, KISS1 gene immunocastration achieved the purpose of the castration through immunoneutralization of anti-kisspeptin-antibodies on the endogenic kisspeptin in peripheral blood.4. The effect of KISS1 gene immunocastration on growthThe serum testosterone concentration in vaccinated group animals was significantly lower than that control animals at weeks 6-14 after the primary immunization(P < 0.05), however, there was no significant difference between serum estradiol, IGF-1 and GH concentration of vaccinated group animals and those of control animals; The KISS1 m RNA level of pancreas and tongue in the vaccinated group animal was significantly lower than in control animals(P < 0.05) and the GPR54 m RNA level of pancreas in the vaccinated group animal was significantly lower than that in control animals(P < 0.05). There was no significant difference between body weight of vaccinated group animals and those of control animals. In conclusion, KISS1 gene immunocastration did not affect the growth of animals.5. The reversibility of KISS1 gene immunization on castrationSix 8 week old ram lambs were randomly divided into two equal groups. The vaccinated group was injected intramuscularly with p KS-asd and the control group animals were treated with p VAX-asd. The same treatments were repeated at weeks 3 and 6 after the primary immunization, subjected lambs were slaughtered at 30 weeks after the primary immunization. The anti-kisspeptin-antibodies were detected in vaccinated group ram lambs at weeks 4-14 after the primary immunization, meanwhile, serum testosterone level in vaccinated group ram lambs was significantly lower than that in control animals(P < 0.05), however, as time goes on, anti-kisspeptin-antibodies levels decreased while serum testosterone level increased. Scrotal circumference in vaccinated group was significantly lower than control animals at weeks 6-22 after the primary immunization(P < 0.05), however, there were no significant difference between scrotal circumference in vaccinated group animals and that in control animals at week 30 after the primary immunization, meanwhile, there was no significantly difference between testes weight, length and width in vaccinated group animals and in control animals at week 30 after the primary immunization. There was no difference between densities of sperms, spermatids, spermatocytes, and spermatogonium of seminiferous tubules of vaccinated group animals and that of control animals at week 30 after the primary immunization. Therefore, this results revealed that the inhibitory effect of KISS1 gene immunization on testicular growth and development was reversible.6. The safety of KISS1 gene immunocastrationHypothalamus, testis, heart, liver, lung, spleen, kidney, muscle and small intestine in vaccinated and control group ram lambs were collected from the animals of reversible experimental. Results of HE staining showed that every tissues and organs were normal in vaccinated group and control group ram lambs. There was no obvious pathological changes in vaccinated group and control group ram lambs, which indicated that KISS1 gene vaccine p KS-asd was not toxic. Genomic DNA was extracted to detect the integration between p KS-asd and genomic DNA by sensitive PCR method. The p KS-asd gene was not integrated into genomic DNA. All the results showed that KISS1 gene immunocastration was safe.