Effects Of Passive Immunization Against Inhibin On Hormone Profile Of Ewes And Apoptosis Of Follicular Granulosa Cell In Vitro

The advantages of passive immunization against inhibin of ewes were economic, security and good effect.However,it is difficult for us to gain a great deal of anti-inhibin antibody,which hinders implication of passive immunization against inhibin in animal husbandry.The objectives of the present study were to get a highly effective method to obtain anti-inhibin antibody through hens as a bioreactor using a synthetic peptide of human inhibin a subunit,and to investigate the effect of immunoneutralization against anti-inhibin eIgY on reproductive hormone secretion in goats and apoptosis of follicular cell in vitro.32 local hens with the age of 20 weeks were randomly divided into two groups,16 hens of each group.One group was injected with 600ug of human inhibinα-subunit fragment(1-26) conjugated with keyhole limpet hemocyanin in Freund's complete adjuvant.Five booster injections(contain 300ug of human inhibinα-subunit fragment (1-26) in Freund's incomplete adjuvant) were given at the interval of 21-days.Another group was mocked with adjuvant only and treated as control.Eggs were collected once 10 days after every immunization,the titer and specificity of the harvested yolk antibody(IgY) were determined with enzyme labeling linked immunosorbent assay(ELISA),put eggs yolk together while the titer of IgY reached 1:1000 is high(binding%＞9),keep purified and freeze-dried eIgY in low sub-zero temperatures.The antibody titer of purification eIgY, lyophilized eIgY and its cryopreservation after one month were determined with ELISA, the result showed that antibody titer of eIgY cryopreservation was not decreased significantly(P＞0.05).Specificity of anti-inhibin eIgY is high,but no cross reaction with FSH,with blocking immunosuppression.To determine bioactivity of eIgY,thirty Wistar Rats(45 days old;weighing 100 to 115g) were randomly assigned to six groups of five animals per treatment,group 1 to 6 were injected with 100,200,400,800ul purification eIgY,10IU FSH and 800ul negative yolk liquid per day,respectively,three consecutive times.Weights of uteruses were measured on the fifth day after the primary injection,compared with control(1.22±0.31), uterus weight gain of treatments with 200(3.15±0.33),400(3.43±0.55),800ul(3.52±0.46) eIgY and FSH(2.77±0.99) is significantly(P＜0.05),and uterus weight gains an improving trend in 100ul(1.47±0.28) group;there is no significant difference(P＜0.05) and dose effect on uterus weight gain in groups treated with200,400 and 800ul eIgY.To determine effects of passive Immunization against Inhibin on hormone profile of goats,twenty Boer goats(45 days old;weighing 100 to 115g) were randomly assigned to tow groups of six animals per treatment,Oestrous cycles were synchronized with injection of 2mg of a synthetic analogue of prostaglandin F2a(PGF2a).On day 0 of the oestrous cycle,animals were intravenous injection with 2mg anti-inhibin eIgY or physiological saline(control;n=6).Blood samples were collected at 0,6,12,24,48,72,96 and 120hours after immunization,FSH,LH,E₂ and P levels in serum were determined using RIA.The result show that blood FSH levels rapidly increased in treatment group,to peak at 12h after immunization,levels in treatment were significantly higher than control group in 96h; Blood LH levels in treatment was flat,lower than control group;Blood E₂ levels was consistent with control group in 12 hours after immunization,then increased rapidly,to peak at 120h,was higher than control;Blood P levels in treatment is higher than control during 12h to 96h after immunization.To determine effects of anti-inhibin antibody on apoptosis cell,12-well cell culture plates with culturing Single layer of follicular granulosa cells are allocated four groups of three wells per treatment with adding inhibin(20IU/ml),anti-inhibin eIgY(diluted 100 times),FSH(10ng/ml) and blank,respectively.Samples were collected at 1,4,8,12,16, 20,24 and 48 hours after treatment,apoptosis rate were calculated using fluorescent staining method.The result indicated that anti-inhibin eIgY and FSH group both can inhibited significantly apoptosis of follicular granulosa cells(P＜0.01).The results of study show that we obtained a great deal of anti-inhibin antibody which is high titer and specific through hens as a bioreactor using a synthetic peptide of human inhibinαsubunit.Increased blood FSH,E₂,P levels and declined blood LH levels in goats with immunization against purification eIgY.Under primary culture condition,the anti-inhibin eIgY can inhibit apoptosis of follicular granulosa cells.The present study is to provide data for studying the effect of passive immunization of inhibin on animal,made a foundation for the further study effects of passive immunization against inhibin on reproduction of goats.