| Steroid hormone is a class of tetracyclic aliphatic hydrocarbons,which catalyzed by a series of steroid synthetases,takes cholesterol as a precursor and plays an important role in many aspects of animal sex differentiation and gonad development.The translocation of cholesterol from cytoplasmic matrix to mitochondrial intima is the first rate-limiting step of steroid hormone synthesis,which depends on the participation of Steroidogenic acute regulatory protein(StAR).3 beta Hydroxy Steroid Dehydrogenase can catalyze the generation of different types of steroid hormones,and the fushi-tarazu factor 1 is an important regulatory factor in steroid hormone synthesis,which can regulate the expression of downstream target genes.Environmental factor hypoxia has been proven to be an endocrine disruptor.Hypoxia inhibits gonadal development and reproductive function by reducing the production of steroid hormones in vertebrates.However,there are few reports on the synthetic pathway and molecular mechanism of steroid hormones in crustaceans under hypoxia stress.Macrobrachium nipponense,commonly known as green shrimp,is one of the most important freshwater shrimp in China.In recent years,with the continuous expansion of the scale and intensification of M.nipponense culture,the hypoxic biological characteristics of M.nipponense seriously restrict the green and healthy development of the culture industry.Therefore,it is urgent to investigate the effects of hypoxia on steroid hormone levels of M.nipponense and its regulatory mechanism.In this study,we cloned the full-length cDNA sequences of StAR,3β-HSD and FTZ-F1 of M.nipponense,and detected the m RNA expression patterns in different tissues and at different hypoxia stages.The prokaryotic expression of StAR and3β-HSD and the preparation of polyclonal antibodies were conducted to investigate the expression and localization of StAR and 3β-HSD in testis tissues under hypoxia stress.The influence of hypoxia on the steroid hormones Estradiol(E2)and Testosterone(T)content in testis tissue was investigated by enzyme-linked immunosorbent assay(ELISA).The contents and main results of this study are as follows:1.The full length of StAR gene cDNA of M.nipponense was 3,361 bp(NCBI entry number: MW263908),including 5 ’-UTR 323 bp,3’-UTR 2,129 bp,open reading frame909 bp,encoding 302 amino acids.The results of amino acid sequence alignment and phylogenetic analysis showed that the StAR gene of M.nipponense had the highest similarity with that of Portellus tritatus,and was clustered into a genus with crustaceans,which was in line with its species taxonomic status.The results of semi-quantitative RTPCR showed that StAR gene was expressed in different tissues of M.nipponense,with the lowest expression level in hepatopancreas and heart tissues,and the highest expression level in testis tissues.Real-time quantitative PCR was used to detect the temporal and spatial expression of StAR gene in the testis of M.nipponense under hypoxia stress.The results showed that,compared with the control group,the expression of StAR gene in the testis of hypoxia treatment group was significantly up-regulated from1 to 48 h,but significantly decreased at 96 h,showing a downward trend overall.In addition,the expression abundance of StAR protein was basically similar to the gene expression pattern by Western blot.Immunohistochemical results showed that StAR protein was expressed in both sperm cells and stromal cells.This study showed that hypoxia stress inhibited the expression of StAR gene in M.nipponense,indicating that it may affect spermatogenesis and gonadal development of M.nipponense,providing data support for subsequent research on steroid synthesis mechanism of M.nipponense under hypoxia stress.2.The open reading frame of 3β-HSD gene of M.nipponense was 1179 bp(NCBI entry number: ON382563),encoding 302 amino acids.The results of amino acid sequence alignment and phylogenetic analysis showed that the 3β-HSD gene of M.nipponense had the highest similarity with that of Procambarus clari,and was clustered into a genus with crustaceans,which was in line with its taxonomic status.The results of semi-quantitative RT-PCR showed that 3β-HSD gene was expressed in different tissues of M.nipponense,with higher expression in hepatopancreas and gonadal tissues.Realtime quantitative PCR was used to detect the spatial and temporal expression of 3β-HSD gene in the testis of M.nipponense under hypoxia stress.The results showed that compared with the control group,The expression of 3β-HSD gene in the testis of hypoxic treatment group was significantly up-regulated from 1 to 48 h,while it was significantly decreased at 96 h in hypoxic treatment group,showing a downward trend overall.In addition,the expression level of 3β-HSD protein was basically similar to the gene expression pattern by Western blot analysis.Immunohistochemical results showed that3β-HSD protein was expressed in both sperm cells and stromal cells.This study showed that hypoxia stress inhibited the expression of 3β-HSD gene in M.nipponense,which laid the foundation for subsequent functional analysis of 3β-HSD gene under hypoxia stress.3.The full-length cDNA of FTZ-F1 gene of M.nipponense was 2,032 bp(NCBI entry number: MW480556),including 5 ’-UTR 59 bp,3’-UTR 152 bp,open reading frame 1821 bp,encoding 306 amino acids.Amino acid sequence alignment and phylogenetic analysis showed that the FTZ-F1 gene of M.nipponense had the highest similarity with that of Procambarus clarkii,and was clustered into a genus with crustaceans,which was in line with its taxonomic status.Semi-quantitative RT-PCR test results showed that FTZ-F1 gene was expressed in different tissues of M.nipponense,and the expression level of FTZ-F1 gene was higher in other tissues except intestines.The results showed that compared with the control group,the expression level of FTZF1 gene in the testis of hypoxia treatment group was significantly up-regulated from 1 to48 h.In the hypoxic treatment group,96 h decreased significantly,showing a downward trend overall.ELISA test showed that the estradiol and testosterone contents decreased with the increase of hypoxia stress time,suggesting that hypoxia may inhibit steroid hormone synthesis in M.nipponense in the form of endocrine disruptors.This study showed that hypoxia stress inhibited the expression of FTZ-F1 gene in M.nipponense,suggesting that it may also play a role in regulating steroid hormone synthesis in crustacea,laying a foundation for subsequent studies on the expression of FTZ-F1 gene in M.nipponense under hypoxia stress.The results of this study are expected to provide new insights into the steroid synthesis pathway of M.nipponense under hypoxia stress. |
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