Effects Of A New Inhibin-like Peptide Construct Vaccine On Reproductive Performance Of Female SD Rats

Inhibin is an important reproductive hormone produced by the ovary,which can bind to its co-receptor Betaglycan to inhibit pituitary secretion of FSH.Vaccination with inhibin result in increasing serum FSH level and ovulation rate in animals,which has a good application prospect.In earlier studies,the inhibin immunogen was prepared in the following several methods: a)extraction from gonadal;b)inhibin subunit preparation by recombinant techniques;c)peptide synthetization using different amino acid series among inhibin α subunit N-terminal 1-30 amino acid.However,all of the above-mentioned immunogen preparation methods are unsuitable due to the low Inhibin content and purification difficult(a),complex preparation process(b),insufficient efficacy(c).Recently,it has been reported that the N-terminal 108-120 amino acid sequence of human inhibin αsubunit is the epitope of binding of inhibin to Betaglycan.We speculate that the vaccine based on this sequence can block the binding of inhibin to Betaglycan and consequently increase FSH production,thereby promoting fertility in females.Therefore,the current study will use the N-terminal 108-120 amino acid sequence of human inhibin α subunit(VRTTSDGGYSFKY)tandem and conjugate to OVA,to investigate the effects of the new inhibin-like construct vaccine on the reproductive function of female SD rats.The experiments carried out,and results obtained are as follows:Experiment Ⅰ: Preparation of new inhibin-like peptide construct vaccineThe homology analysis and spatial structure prediction of N-terminal 108-120 amino acid sequence of human inhibin α subunit were carried out by sequence alignment and model prediction.The results showed that the sequence was the epitope of inhibin binding to Betaglycan and was highly conserved among species.The spatial structure predicts that the sequence is exposed to the periphery of the spatial structure of inhibin α subunit,so that the antibody can bind to it efficiently.The N-terminal 108-120 region of the human inhibin alpha subunit tandem(INH13AA-Tandem:INH13AA-T)was synthesized.The molecular weight of INH13AA-T was 3046.33 Da with the purity of 91.36%.Subsequently,INH13AA-T was coupled with ovalbumin(Ovalbumin,OVA)and homogenized with Specol adjuvant to form a new inhibin-like peptide construct vaccine.Experiment Ⅱ: Effects of active immunization against INH13AA-T on blood antibody titer,hormone content and fertility in female rats.Ninety female rats with similar body weight and 8weeks of age were randomly assigned to control group,and INH13AA-T immunized group,each group consist of 45 animals.The female rats in the INH13AA-T immunized group received 0.5 m L emulsion(including300μg INH13AA-T)intramuscular in the leg at the age of 8 weeks.Booster immunization was performed at the 4th and 8th week after the first immunization(dose as the first immunization),respectively.The female rats in the control group were each similarly treated with 0.5 m L adjuvant on the three corresponding occasions.Blood for serum anti-inhibin antibody and reproductive hormone concentrations was sampled every 4 weeks from the tail tips.The estrous cyclicity of female rats were assessed for 14 consecutive days,starting 2weeks after the second booster.At 20 weeks of age,namely 4 weeks after the second booster vaccination,rats in each group were randomly allocated into three subgroups(subgroup I,subgroup II and subgroup Ⅲ,respectively).Subgroup I were anaesthetized with ether and then decapitated at estrus.After decapitation,various organs including liver,inguinal white adipose tissue,periovarian white adipose tissue,kidney,adrenal glands,thymus,spleen,pituitary,ovaries and uteri were collected and weight were recorded.Gene expression and histological evaluation were carried out for pituitary and ovary.The subgroup II were paired with male SD rats for the detection of ovulation and were euthanized immediately after the occurrence of vaginal plug.The subgroup Ⅲ were caged together with one male rat for 2weeks,and then the male rat was removed.The litter size,litter weight were recorded.Results show that,INH13AA-T immunization induced a good antibody response,especially after the booster immunizations.In response to the sharp increase of serum inhibin-specific antibodies,serum concentrations of FSH at diestrus,proestrus and estrus,and estradiol at proestrus and estrus were increased in INH13AA-T immunized rats compared to placebo immunized controls at decapitation.However,serum LH concentrations were similar between INH13AA-T-immunized and placebo-immunized group.INH13AA-T immunization shortened the duration of metestrus/diestrus phases,but prolonged estrus phases.However,the estrous cycle length was not changed by INH13AA-T immunization.INH13AA-T immunization increased the thymus weight and index but without effects on other organs in female rats.The expression of FSH β,Foxl2,and Gn RHR m RNA in the INH13AA-T immunized group were markedly upregulated by INH13AA-T immunization.While,m RNA expression of Smad4 was down-regulated by INH13AA-T immunization.Compared with the control group,there was a clear increase in the number of antral follicles and corpora lutea in female rats following INH13AA-T immunization,and the expressions of folliculogenesis-associated genes Creb,Ccnd2,Inhα and steroidogenesis-associated genes Hsd-3β1,Cyp11a1 and Cyp191a1 m RNA were significantly increased.Consequently,compared to placebo-immunized controls,INH13AA-T immunized group ovulated more eggs.INH13AA-T immunized group had average 3.8 more pups per litter than the placeboimmunized controls.However,litter weight of pups was similar between INH13AA-T and placebo-immunized,with a significant decrease in individual body weight of newborn pups of INH13AA-T immunized females than that of placebo-immunized controls.In summary,the present study was the first to show that,using the female rat as a model,active immunization against the tandem form of conserved Betaglycan-binding epitope(VRTTSDGGYSFKY)on inhibin α subunit led to increased pituitary FSH synthesis and secretion,and,in turn,improved folliculogenesis and the fertility without pathological effects on the length of the estrus cycle,as well as the weight and index of various organs.Our findings demonstrate that active immunization against INH13AA-T could be a useful method for improving the ovulation rate and fertility in females.