| Whitmania pigra Whitman,also known as Leech,is the main basic protozoan of leech in Chinese Pharmacopoeia(2015 edition).It has good effect of activating blood circulation to dissipate blood stasis.Because of overfishing,environmental pollution and other reasons,the amount of wild W.pigra resources declined sharply.In recent years,a large number of studies have been carried out on the biological characteristics,digestive physiology,respiratory physiology and breeding density of W.pigra,and the artificial breeding of W.pigra has achieved initial success.Research shows that the W.pigra is a hermaphrodite.Under natural conditions,the male gland develops prior to the female gland,and then matures together gradually after overwintering to reproduce.It is reported that the gonad development of aquatic animals is not only controlled by genetic factors,but also affected by temperature and exogenous hormones in varying degrees.Therefore,this experiment studied the effects of exogenous addition of 17β-estradiol at different concentrations on the gonad development of W.pigra,providing theoretical basis for the study of the mechanism of the gonad development of W.pigra,and providing a reference for the indoor culture of W.pigra.In the experiment,healthy Wpigra aged 1.5 months and 5.5 months were selected,and the concentrations of 17β-E2 in the aquaculture water were 0.0,1.0,10.0,25.0,50.0 and 100.0 ng.mL-1 respectively.After six weeks of culture,the W.pigra in the second stage were recorded as experimental phase Ⅰ and Ⅱ respectively.After 60 days of natural hibernation,the W.pigra in the second stage were recorded as experimental phase Ⅲ,and the related indexes of growth and gonad development were determined.The results of this experiment are as follows:(1)With the increase of exogenous 17β-E2 concentration,the body weight,weight gain rate and specific growth rate of stage Ⅰ W.pigra increased gradually.The body weight,weight gain rate and specific growth rate of stage Ⅱ and Ⅲ W.pigra were significantly higher than those of other groups(P<0.05)when the concentration was 100 ng·mL-1.The body weight,weight gain rate and specific growth rate of stage Ⅱ and Ⅲ W.pigra increased first and then decreased with the increase of exogenous 17β-E2 concentration.Among them,the body weight,weight gain rate and specific growth rate at 25 ng mL-1 concentration were significantly higher than those of other groups(P<0.05).There was no significant difference in water,ash,acid-insoluble ash and acidity-basicity among the three groups of dried W.pigra(P>0.05).Antithrombin activity increased first and then decreased with the increase of exogenous 17β-E2 concentration.Antithrombin activity was the highest when the concentration was 25 ng·mL-1.After adding exogenous 17β-E2,the female gland index of the W.pigra was significantly higher than that of the control group(P<0.05).The female gland index of stage Ⅰ and Ⅱ increased at first and then decreased.The highest index of stage Ⅰ was at 50 ng·mL-1,the highest index of stage Ⅱ was at 10 ng·mL-1,and there was no significant difference between stage Ⅲ groups(P>0.05).The male gland index of stage Ⅲ was increased with the increase of exogenous 17β-E2 concentration.When the concentration of exogenous 17β-E2 was higher than 25.0 ng·mL-1,the index of male gland was significantly lower than that of the control group(P<0.05);exogenous 17β-E2 could promote the development of ovaries and female germ cells,and the development of ovaries in estrogen group was higher than that in control group at all stages,and the number of oocytes in estrogen group Ⅰ increased significantly.The proportion of yolk-phase cells increased significantly,and the proportion of yolk-phase cells and mature oocytes in ovaries of stage Ⅱ and Ⅲ estrogen group was significantly higher than that of control group.The development of testis and male germ cells was significantly inhibited by high concentration of 17β-E2.When the concentration of exogenous 17β-E2 was more than 25.0 ng·mL-1,the number of germ cells in the testis of W.pigra decreased significantly.(2)The levels of endogenous estradiol(E2)and estriol(E3)increased first and then decreased with the increase of exogenous 17β-E2 concentration in different experimental periods.The levels of endogenous E2 and E3 in 25.0 ng·mL-1 group and 50.0 ng·mL-1 group were significantly higher than those in other groups(P<0.05).The levels of endogenous testosterone(T)and methyltestosterone(MT)in stage Ⅰ W.pigra decreased gradually with the increase of exogenous 17β-E2 concentration,and the levels of T and MT in 100.0 ng·mL-1 group were significantly lower than those in other groups(P<0.05);the levels of endogenous T and MT in stage Ⅱ and Ⅲ W,pigra increased first and then decreased with the increase of exogenous 17β-E2 concentration,when the concentration of 17β-E2 was higher than 100.0 ng·mL-1 group.MT level was significantly lower than that of control group(P<0.05).The endogenous progesterone(P)level in stage Ⅰ and Ⅲ decreased gradually with the increase of exogenous 17β-E2 concentration,while the endogenous P level in stage Ⅱ increased first and then decreased with the increase of exogenous 17β-E2 concentration.The highest P level was found when the concentration of 17β-E2 was 1.0 ng·mL-1,and significantly lower than that in the control group when the concentration of exogenous 17β-E2 was more than 25.0 ng·mL-1(P<0.05).The endogenous corticosterone(CORT)in stage I decreased gradually with the increase of exogenous 17β-E2 concentration,and was significantly lower than that in the control group(P<0.05).The CORT in stage Ⅱ increased first and then decreased with the increase of exogenous 17β-E2 concentration.The CORT level was the highest when the concentration of 17β-E2 was 1.0 ng·mL-1,and was significantly lower when the concentration of 17β-E2 was more than 25.0 ng·mL-1(P<0.05).There was no significant difference in endogenous CORT level among the W.pigra(P>0.05).When endogenous T/E2 was lower than 0.08,0.19 and 0.66 respectively,MT/E2 was less than 0.08,0.18 and 0.70,which significantly inhibited testicular development,while T/E2 could promote ovarian development at 0.02-0.13,0.10-0.97 and 0.29～1.33.In conclusion,when exogenous 17β-E2 is less than 25.0 ng·mL-1,estrogen level can be improved without affecting endogenous androgen level of the W.pigra.(3)The expression levels of GH and IGF-1 genes in the gonads of W.pigra increased first and then decreased with the increase of exogenous 17β-E2 concentration.The expression levels of WNT4 and FOXL2 genes in the gonads of W.pigra were significantly higher than those in the other groups(P<0.05).The expression levels of WNT4 and FOXL2 genes in the gonads of W.pigra increased first and then decreased with the increase of exogenous 17β-E2 concentration,when the concentration of exogenous 17β-E2 was greater than 25.0 ng·mL-1,the expression of WNT4 and FOXL2 was significantly lower than that of control group(P<0.05).WNT4 and FOXL2 genes increased first and then decreased with the increase of exogenous 17β-E2 concentration in female glands,and were significantly higher than that of control group(P<0.05).The expression of FOXL2 gene was the highest at 25.0 ng·mL-1,WNT4 gene was the highest at 50.0 ng·mL-1,and SR in male glands with the increase of exogenous 17β-E2 concentration.The expression of 17β-HSD gene in female and male glands at three stages increased first and then decreased,and were significantly higher than that in control group(P<0.05).The aromatase gene CYP19alpha in the gonads of cockroaches was significantly lower than that in control group(P<0.05).The expression of 17β-HSD gene in female and male glands of cockroaches was higher than that in control group(P<0.05).The concentration of Source 17β-E2 increased at first and then decreased.The expression level of Source 17β-E2 in female glands was higher than that in control group(P<0.05),while that in male glands was lower than that in control group(P<0.05).In conclusion,exogenous 17β-E2 promotes the growth and development of W.pigra by increasing the expression of GH and IGF-1 genes.17β-E2 promotes ovarian development by increasing the expression of FOXL2,WNT4,CYP19alpha and 17β-HSD genes,and inhibits the development of testis by reducing the expression of SRY genes in testis.Exogenous 17β-E2 may regulate the ratio of endogenous estrogen to androgen by affecting the expression of 17β-HSD gene,thus further regulating the development of female and male glands in W.pigra. |
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