| The guinea pig is an excellent animal model for studies on the reproductive system in human and most domestic animals. Unlike other laboratorial rodents, guinea pigs have an extended estrous cycle of16to19days, a full gestation of68days, and a small litter size of3.8on average. In this paper, techniques such as gene cloning, immune-neutralization and Reverse transcription-PCR were applied to investigate the influence of inhibin gene immunization on follicular development and LHR mRNA expression in uterus in guinea pig.1. Effect of inhibin gene immunization on the production of antibody against inhibin and follicular development in guinea pigIn order to study the effect of inhibin gene immunization on follicular development in guinea pig,19adult female guinea pigs were divided into two groups. The trial guinea pigs (n=15) were immunized with200μg/500μl pCISI, and four boosters were given at the dose of100μg/500μl with an interval of20days, the control guinea pigs (n=4) received an equivalent volume of saline. After the fifth immunization, the guinea pigs were anaesthetized and sacrificed at day8of estrous cycle and ovaries were collected. Blood samples were collected every10days throughout the experiment for assaying inhibin antibody titres by ELISA and ovaries were for histological analysis of ovarian morphology. The results showed that pCISI immunization resulted in the increment of positive sera ratio with inhibin antibody, but the concentration of inhibin antibody was not promoted significantly in the experiment (P>0.05).The value of P/N was1.90±0.14in the test group and1.45±0.09in the control group after twice immunization,1.56±0.17versus1.56±0.06after the third immunization, and0.93±0.64versus0.30±0.40after the fifth immunization. The differences of follicular number of different sizes and ovulation rate were not significant between two groups (P>0.05). These results showed that the pCISI plasmid gene immunization was failure to affect follicular development or induce superovulation in guinea pig.2. Study on the expression of LHR mRNA in guinea pig uterus Guinea pigs at DayO (the day of ovulation was estimated as Day0)、Day4、Day8、Day12and Day8(Imu)(8days after pCISI gene immunization) of estrous cycle were anaesthetized and sacrificed. Total RNA extracted from the uterus of guinea pig and primers designed according to the conservative sequence of pig, rat and mice were used for RT-PCR to amplify LHR fragment. Positive clones were identified and sequenced. BLAST and DNAman softwares were applied to analyze the homology. β-actin was used as an inner control and an optimal semi-quantitative RT-PCR method was established to analyze LHR mRNA expression levels in the uterus during4estrous cycle phases. Sequencing analysis indicated that there were two kinds of spliced variants:One was the686bp fragment and the other was611bp with a deletion of75bp in exon4. The686bp fragment was83.8%identical to the reported human LHR mRNA,84.6%to the cattle,84.2%to the pig,83.6%to the sheep,93.7%to the rat, and99.4%to the mice. The uterine LHR mRNA expression was different during4estrous cycle phases. The levels on day0(0.635±0.019) and day4(0.617±0.099) were both significantly lower than that on day8(P<0.05), then it rose to the highest on day12(1.218±0.049). pCISI immunization didn’t affect the expression level of LHR mRNA (0.766±0.080)(P>0.05). |
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