Effects Of Phenanthrene Stress On Germ Cell Development In Loach

Phenanthrene is the main PAHs pollutant in water,which has carcinogenic,teratogenic and mutagenic effects.Long-term pollution may cause abnormal expression of genes related to the development of fish germ cells and lead to structural abnormalities of gonads.Therefore,it is of great significance to study its effects on the gonads of fish.In this study,loach was used as the research object to analyze the growth performance,sperm nest aminotransferase and its genes,spermal nest sex steroid hormone and its receptor,gonadal sex steroid hormone receptor gene,gonadal development-related genes and gonadal changes under Phe stress.In order to explore the influence of Phe on the development of fish gonads,lay a foundation for future research in this field,so as to better detect and protect the aquatic environment and maintain the stability of biodiversity.The main conclusions are as follows:(1)The growth performance was determined by using different concentrations(0.15,1.33,1.77,2.36,3.13 mg/L)and combining different treatment times(15,30,45,60,75,90 d);The results showed that Phe had a toxic effect on male loach,and compared with the control group,Phe stress could lead to an increase in body weight growth and then decrease,and long-term stress significantly reduced the growth rate.Phe stress can significantly increase the fertility of male loach.Phe had an inhibitory effect on the body length and weight of male loach,and the inhibition of body length was more significant.(2)The sperm nest index of loach was determined,and the sperm nest aminotransferase activity was determined by Lai’s colorimetric method;qRT-PCR was used to detect the transcription level of transaminase genes(ast,alt)in loach sperm nests.The results showed that short-term Phe stress induced the increase of the sperm nest index of loach,and long-term Phe stress inhibited the nesting index.Phe stress had a significant effect on the AST and ALT activities of sperm nests,and the trend of AST activity was more obvious.The changes in AST and ALT activities had obvious lagging effects compared with the changes in ast and alt expressions.The AST and ALT activities and the transcription levels of ast and alt in loach sperm nest under Phe stress were not significantly correlated with the change of sperm nest index.(3)The contents of E2,T,ER and AR were determined by enzyme-linked immunosorbent method,and the transcription level of sex steroid receptor genes(era,erβ1,ar)in the sperm nest of loach was detected by qRT-PCR.The results showed that Phe stress could significantly reduce the contents of T and AR and significantly increase the contents of E2 and ER.Under short-term Phe stress,the expression of era and erβ1was induced,and under long-term Phe stress,the expression of era and erβ1 was inhibited,but the contribution of era to ER synthesis was greater.Phe exhibits induction and then inhibition of ar,which may be related to erβ1;The relationship between E2 and ER was enhanced under Phe coercion,while the relationship between T and AR was weakened.This suggests that Phe may regulate hormone synthesis and conversion by influencing steroid-producing pathways,resulting in strong estrogen-like effects on male loach.(4)The qRT-PCR method was used to detect the transcription level of sperm nest development related genes(amh,sox9 a,sox9b)in loach sperm nest,and paraffin tissue sectioning,HE staining and electron microscopy were used to observe the histological structure of loach sperm nest.The results showed that short-term Phe stress induced the rise of amh,sox9 a and sox9 b,and long-term high-concentration Phe stress inhibited the expression of amh,sox9 a and sox9 b.Under long-term stress,the structure of seminal vesicles was damaged,sperm cells and sperm counts decreased significantly,the number of spermatogonia,primary spermatoblasts and secondary spermatoblasts increased significantly,and a small number of cavities appeared in the seminal nest.3.13 mg/L Phe was stressed for 90 days,no sperm cells and spermatozoa were observed in the seminal nest,a small number of spermatogonia were observed,the number of spermatogonia was large,and the volume of a small number of spermatogonia increased significantly,and a small number of oogonia and oocytes were observed in the seminal nest.This indicates that Phe has a retarding effect on the development of loach sperm nest,and high-concentration long-term Phe stress can lead to the degradation or transformation of loach sperm nest into ovaries,and the decrease in the transcription level of amh,sox9 a and sox9 b may be caused by damage to the tissue structure of the sperm nest.(5)qRT-PCR was used to detect the transcription level of ovarian sex steroid receptor genes(era,erβ1,ar)and ovarian development-related genes(amh,sox9 a,sox9b)in loach ovaries,and the histological changes of loach ovary were observed by paraffin tissue section,HE staining and electron microscopy.The results showed that under Phe stress,erα,erβ1,ar and sox9 b were significantly induced in loach ovaries.Phe affects the normal development of loach ovaries,accelerates the development of ovoblasts to stage Ⅱ oocytes,and destroys the morphological structure of oocytes.In summary,Phe had obvious toxic effects on the growth and development of male loach and the maturation of sperm nest.AST and ast play the main detoxification role in the seminal nest,and the detoxifying amino acids have no significant effect on the development of the seminal nest,and the more serious damage to the development of the seminal nest may be related to the estrogenoid effect of Phe.The estrogenoid effect of Phe leads to an endocrine imbalance of spermatozonic sex steroid hormones and their receptors,a sharp increase in the level of estrogen and its receptors,and a sharp decrease in the level of androgens and their receptors;The increase in estrogen receptor levels may be mediated by erα,and the decrease in transcription levels of erβ1,ar,amh,sox9 a,and sox9 b may be an important factor in the damage to the sperm nest structure;Phe can significantly induce the transcription of erα,erβ1,ar and sox9 b in loach ovary,accelerate the development of ovogonia to stage Ⅱ oocytes,and destroy the morphological structure of oocytes.