Developmental Changes In Concentrations Of Steroid Hormones And Yolk During Ovarian Development Of Chinese Mitten Crab (Eriocheir Sinensis)

Steroid hormones play an extremely important role in the reproduction and development of animals,however,there is still controversy about the presence of steroid hormones in crustaceans and their regulation of ovarian development.Therefore,this study establishing liquid chromatography–tandem mass spectrometry(LC-MS/MS)for the isolation and determination of steroid hormones in the Chinese mitten crab(Eriocheir sinensis).The changes of steroid hormones in the ovary,hepatopancreas and hemolymph were determined by optimized LC-MS/MS;and the Vitellogenin/Vitellin(Vg/Vn)levels of ovary,hepatopancreas and hemolymph during ovarian development of Chinese Mitten Crab were further studied.The purpose of this study is providing a reference information for further understanding the mechanism of steroid hormones in the breeding cycle of female crabs,as well as evaluating and controling the breeding process of crabs.The full text can be divided into the following contents:Exp.1.The development of liquid chromatography–tandem mass spectrometry(LC-MS/MS)method for the determination of steroid hormones in Eriocheir sinensisA simple,rapid and highly sensitive of LC-MS/MS method for the determination of steroid hormones in Chinese Mitten Crab was developed.Samples were extracted,purified and concentrated to eliminate matrix effects.In addition,other experimental influencing factors(such as solvent extraction,chromatographic conditions and derivatization process)were examined and optimized.The results showed that the calibration curves of 9 steroids were linear in the certain concentration range with correlation coefficients(r)more than 0.992.The average recoveries of steroids at three spiked levels ranged from 63.8%to 119.3%with relative standard deviations(RSDs)of2.8%-13.1%.The limits of quantitation(LOQ)were in the range of 0.05-10ng/mL.The method was successfully applied to the determination of steroid hormones during ovarian development of Chinese Mitten Crab.It was suitable for rapid detection of large batches and small volumes.Exp.2.Comparative study on the steroid hormones of three tissues during ovarian development of Eriocheir sinensisThe changes of steroid hormones in the ovary,hepatopancreas and hemolymph were determined by optimized LC-MS/MS,in addition,the correlation between steroid hormones and ovarian development was discussed.The results showed that:(1)During the period of ovary development,the progesterone levels in the hemolymph increased significantly,especially in stages I-II,then declines gradually.The change trends of estrone and estradiol were consistent and showed a trend of first increase and then decline.(2)The levels of estradiol and 17α-hydroxyprogesterone in the hepatopancreas was lower in ovarian stage I,and the levels of two hormones reach their peaks in ovarian stage III and IV,respectively,and after that both were decreasing.(3)There was a significant negative correlation between GSI and estrone as well as estradiol levels in ovary,and the levels of two hormones were decreasing along with the growth of GSI.Exp.3.Comparative study on the vitellogenin/vitellin(Vg/Vn)levels during ovarian development of Eriocheir sinensisIn this study,the levels of Vg/Vn in the hemolymph,ovary,and hepatopancreas was determined by Enzyme-Linked Immunosorbent Assay(ELISA)during the ovarian development of Eriocheir sinensis,and further explored the correlation between Vg/Vn levels and ovarian development.The results showed as followings:(1)During ovarian development,the Vn level in the hemolymph continued to increase,reaching the highest value in the ovarian stage V,which was 1233.87μg/mL;The Vn level of ovary increased significantly,and reached its peak in the ovarian stage V was 359.87 mg/g;In hepatopancreas,Vg was not detected at ovarian stage I and II,and the Vg level increased significantly were observed during ovarian stage III to ovarian stage V.(2)Vn of ovary and Vg of hepatopancreas were positively correlated with GSI,the equations were Y=33.85X+42.325(R~2=0.9155)and Y=1.4167X+0.3633(R~2=0.7886),respectively.