| The dryness of Whitmania pigra Whitman is the Chinese herbal medicine leeches of Chinese pharmacopoeia（2015）,with the effect of removing blood stasis and resolving static blood.W.pigra is a synchronous hermaphroditic animal.The testis precedes the development of the ovary,testis and ovary develop in an orderly manner but mature synchronously.The proliferation,formation and maturation of germ cells are mainly related to the involvement of androgen and estrogen in the testis and ovary.Methyltestosterone（MT）is an important androgen that not only affects gonadal development,but also promotes animal growth.Therefore,we add different concentrations of MT to the culture water of W.pigra during the testis proliferative phase（ovary formation phase）and testis dormancy（ovarian growth stage）,and study the effect of MT on the growth and gonadal development of W.pigra from tissue,physiological and molecular levels.The results can provide a theoretical basis for the study of the reproductive and developmental mechanism.of W.pigra,and provide guidance for the artificial breeding of W.pigra.The research results are as follows:（1）The effects of exogenous MT on the growth and gonadal development of W.pigra at different stages were studied.Five concentrations of MT（0.1 μg·L-1、1.0 μg·L-1、10.0μg·L-1、100.0 μg·L-1、150.0 μg·L-1）were used to treat W.pigra in water bath for 6 weeks at the age of 1.5 months and 5.5 months,respectively.At the same time,some 5.5-month-old Wpigra were hibernated for 60 days with MT for 6 weeks.The body weight,gonadal index and intrinsic quality of W.pigra in these three periods were determined by weighing method.Tissue sections and HE staining technique were used to observe the development of W.pigra sperm ovum.The results showed that exogenous MT could promote the growth and gonadal development of W.pigra in different degrees at 0.1～150.0μg·L-1,and the degree of promotion first increased and then decreased with the increase of MT concentration.Among them,1.5-month-old W.pigra added MT100.0 μg·L-1 had the highest specific growth rate,and the highest gonadal index was found the highest at the concentration of MT10.0 μg·L-1.5.5-month-old W.pigra added with MT6 10.0 μg·L-1 had the highest specific growth rate,and the gonadal index was found the highest at the concentration of MT100.0 μg·L-1.For W.pigra after natural wintering,the weigh was found the highest at the concentration of MT100.0 μg·L-1,the male gonadal index was found the highest at the concentration of MT10.0 μg·L-l,and the female gonadal index was found the highest at the concentration of MT1.0 μg·L-1.The quality of this kind of W.pigra meets the standard of Chinese Pharmacopoeia（2015 edition）,and antithrombin activity was higher than that of control group.When the concentration of exogenous MT increased to 10.0μg·L-1,primary and secondary spermatocytes appeared in the testis of 3 months W.pigra,and oogonia cells appeared in the ovary.When the concentration of MT was 100.0 μg·L-1,the number of gonadal spermatocytes,spermatocytes and oocytes in the 7 and 9-month-old groups were significantly higher than those in the other groups.（2）The effects of exogenous MT on endogenous steroid hormones in W.pigra at different stages were studied.The results showed that endogenous testosterone（T）,estriol（E3）and progesterone（P）increased first and then decreased with the increase of exogenous MT concentration in W.pigra for 1.5 months,and T,E3 and P was highest at concentrations of 100.0 ug.L-1,1.0 μg·L-1,1.0μg·L-1（P<0.05）;Methyl testosterone（MT）,estrone（E1）was positively correlated with exogenous MT concentration.After adding MT6 weeks to 5.5 months old W.pigra,endogenous T,E1 and E3 were positively correlated with exogenous MT concentration;MT and P increased first and then decreased with the increase of exogenous MT concentration,and reached the highest at 100.0 ug.L-1（P<0.05）.The hormones of natural wintering W.pigra increased first and then decreased.T,MT and P reached the highest at 100.0 ug.L-1,E1 at 1.0 ug.L-1,E3 at 10.0 ug.L-1.Endogenous T/E3 and T/E1 were lower than those of the control group after exogenous MT treatment.（3）The effects of exogenous MT on the expression of endogenous steroid hormone-related genes in W.pigra at different stages were studied.The results showed that with the increase of exogenous MT concentration,the growth hormone gene（GH）,insulin-like growth factor-1（IGF-1）,aromatasα（CYP19α）、3β-hydroxysteroid dehydrogenase（3β-HSD）and FOXL2 gene increased at first and then decreased,and were up-regulated in the concentration range of 10.0～100.0 μg·L-1（P<0.05）.SRY gene was up-regulated in male gonad（P<0.05）,but no significant expression in female gonads（P>0.05）;WNT4 gene was not significantly expressed in male gonad（P>0.05）,but significantly up-regulated in female gonad（P<0.05）.In male gland,with the increase of exogenous MT concentration,SRY/WNT4 was higher than that of CK group and increased first and then decreased.The W.pigra at 1.5 and 5.5 months old were highest at 10 μg·L-1,2.06 and 4.29 respectively,the natural wintering W.pigra was the highest at 100μg·L-1,at 2.84;In female gonad,with the increase of exogenous MT concentration,SRY/WNT4 was lower than the CK group and decreased first and then increased.The 1.5 and 5.5 month old W.pigra had the lowest concentration of 10 μg·L-1,respectively 0.15 and 0.32,and the natural wintering had the lowest concentration of 0.25 at a concentration of 100μg·L-1. |
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