Cloning and expression analysis of wpHSP90 gene from Whitmania pigra at different temperatures / 中国中药杂志

HSP90 is a widely distributed molecular chaperone that participates in a variety of cellular processes and plays an important role in the meiosis of germ cells. However, its role in the gonadal development of hermaphroditic Whitmania pigra is not yet clear. To explore the effect of HSP90 on the germ cell development of Wh. Pigra, this study cloned the wpHSP90 gene, performed bioinformatics analysis, and measured its expression levels. The results showed that the cloned wpHSP90 was 2 592 bp in length, with an open reading frame(ORF) of 2 373 bp, encoding 790 amino acids. Prediction analysis revealed 85 phosphorylation modification sites on serine, threonine, and tyrosine residues of the wpHSP90 protein. Structural domain prediction and multiple sequence alignment results showed that wpHSP90 contained two conserved domains of HSP90 and exhibited the highest homology with Helobdella robusta, with a sequence similarity of 80.72%. RT-qPCR results showed that the relative expression level of wpHSP90 in the gonads of 5-month-old Wh. pigra was positively correlated with temperature within the range of 12 ℃ to 28 ℃. The expression level in the female gonads was significantly higher than in the male gonads and correlated with the trend of germ cell development in the ovaries and testes. In conclusion, wpHSP90 may be involved in regulating the development of germ cells, particularly oocytes, in Wh. pigra. This study provides a reference for further research on the gonadal development mechanism in Wh. pigra.

Cloning and expression analysis of protein kinase C gene from Whitmania pigra / 中国中药杂志

Protein kinase C(PKC) is a type of protein kinase widely involved in cell proliferation and development, but the developmental mechanism in the gonads of androgynous animals is still unclear. In order to explore the role of protein kinase C in the development of Whitmania pigra germ cells, the Wh. pigra PKC(Wp-PKC) gene was cloned, bioinformatics analysis was conducted, and fluorescent quantitative PCR was used to analyze the expression of female and male gonads. The results showed that:(1)The cloned Wp-PKC had a full length of 2 580 bp, a relative molecular weight of 76 555.19, and contains an open reading frame encoding 670 amino acids, Wp-PKC was closely related to Danio rerio PKC-α and rat PKC-γ. The similarity of amino acid sequence was 55% and 58%.(2)The protein encoded by Wp-PKC had no signal peptide and was a hydrophilic protein. The secondary structure is mainly composed of random coils, α-helices, extended chains, folds and folds, with the largest proportion of random coils and α-helices. Wp-PKC protein does not contain a transmembrane domain. Multiple sequence alignment and domain prediction analysis show that Wp-PKC contains 4 conserved domains of classical protein kinase C.(3)Fluorescence quantitative results showed that the expression of Wp-PKC in Wh. pigra gonads was positively correlated with the development of germ cells, and the expression in male gonads was significantly higher than that in female gonads. In summary, Wp-PKC is a classic PKC, and Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads, and provide references for further research on the developmental mechanisms of Wh. pigra.

Effects of Wp-PKC on gonadal development of Whitmania pigra / 中国中药杂志

Protein kinase C(PKC) is a kind of kinase which is widely involved in cell proliferation and development. PKC(Wp-PKC) in Whitmania pigra body belongs to classic PKC. In order to investigate the effect of Wp-PKC on the development of Wh. pigra germ cells, 17β-estradiol(17β-E2)(100 ng·mL~(-1)) and methyltestosterone(MT)(150 μg·L~(-1)), 150 μg·L~(-1)(MT)+0.5 mg·L~(-1) PKC, 0.5 mg·L~(-1) PKC inhibitor were added to Wh. pigra culture water, and no addition group(control group) was added, and the effects on the development of Wh. pigra germ cells and the expression of Wp-PKC were observed. The results showed that: Wp-PKC in male gonads was always higher than that in female gonads; MT promoted the development of male gonads in Wh. pigra, while the expression of Wp-PKC was significantly higher than that in the control; 17β-E2 promoted the development of female gonads in Wh. pigra and Wp-PKC expression significantly lower than that of the control; while the development of the female and male gonads in the PKC inhibitor group was inhibited, the expression of Wp-PKC was significantly lower than that of the control. In summary, Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads.

Effects of MT on growth and gonad development of natural overwintering Whitmania pigra / 中国中药杂志

The objectives of study were to explore the effects of exogenous methyltestosterone( MT) on the growth and gonadal development of overwintering Whitmania pigra. Before overwintering,0. 1,1. 0,10. 0,100. 0,150. 0 μg·L-1 of MT were added to the aquaculture water for 6 weeks. The changes of growth performance,gonad index,endogenous steroid hormones level and internal quality were measured after hibernate for 60 days. Then the tissue slice technique was used to observe the spermary( ovary) of Wh. pigra.The results showed that the body weight,survival rate and gonadal index increased first and then decreased with the increase of exogenous MT concentration; the male gland index was found the highest at the concentration of MT 10. 0 μg·L-1 and the female gland index was the highest at the concentration of MT 1. 0 μg·L-1. The survival rate of Wh. pigra peaked at the concentration of MT 10. 0 μg·L-1.The weight reaches a peak at a concentration of MT 100. 0 μg·L-1( P<0. 05). The number of primary spermatocytes in the testis was negatively correlated with the concentration of exogenous MT. The number of secondary spermatocytes and sperm cells increased first and then decreased. The concentration of secondary spermatocytes was the highest when the concentration of MT was 100. 0 μg·L-1.The number and volume of oocytes in the ovary and the yolk granules increased first and then decreased with the increase of exogenous MT concentration,and the highest was observed at the MT concentration of 100. 0 μg·L-1. The endogenous steroid hormone of Wh.pigra increased first and then decreased with the increase of exogenous MT concentration. The concentration of androgen and progesterone was the highest in MT 100. 0 μg·L-1 treatment( P<0. 05),and the concentration of estrogen was found the highest in MT 10. 0 μg·L-1 treatment( P<0. 05). After adding exogenous MT,Wh. pigra moisture content,acid-insoluble ash content,p H and anti-thrombin activity met the quality criterion of medicinal Wh. pigra in Chinese Pharmacopoeia( 2015 edition). In conclusion,the short-term addition of 1. 0-100. 0 μg·L-1 exogenous MT before hibernation can promote the growth,the development of sperm cells and the antithrombin activity of Wh. pigra.

Effects of 17β-E2 on growth and gonad development of overwintering Whitmania pigra / 中国中药杂志

The present study was conducted to explores the effects of short-term addition of 17β-E2 on the growth,gonad development and internal quality of overwintering Whitmania pigra. Before overwintering,0. 0,1. 0,10. 0,25. 0,50. 0,100. 0 μg·L~(-1) of 17β-E2 were added to the aquaculture water for 6 weeks and then hibernated for 60 days. The changes of growth performance,gonad index,morphological structure of spermary( ovary),endogenous steroid hormones level and internal quality were measured. The results showed that the body weight,weight gain rate,specific growth rate,female gonad index,oocyte development and endogenous estrogen level of the leech increased first and then decreased with the increase of the concentration of exogenous 17β-E2,which were higher than those of the control group. The body weight,weight gain rate and specific growth rate of the leech at the concentration of 25 μg·L~(-1)17β-E2 were significantly higher than those of the other groups( P<0. 05),oocyte development and endogenous estrogen levels were significantly higher than those of other groups at the concentration of 50 μg·L~(-1)( P<0. 05). When the concentration of exogenous 17β-E2 was higher than 50 μg·L~(-1),the levels of male gonad index,spermatocyte development,endogenous androgen and progesterone were significantly inhibited( P< 0. 05). There was no significant difference in endogenous corticosteroid levels among the groups. In conclusion,short-term addition of exogenous 17β-E2 of 10-25 μg·L~(-1) could promote the growth of overwintering leeches,oocyte development and antithrombin activity without inhibiting the development of male gonads.