| Onychostoma macrolepis(O.macrolepis),categorized as Onychostoma(Cypriniformes,Cyprinidae),commonly known as ?Qian Yu?,is one of the national key protected freshwater fish.O.macrolepis is rare atypical cave fish,and is called "living fossil".However,with the influence of climate change,environmental pollution,human activities etc,the wild population of the species is on the verge of extinction.In order to protect the germplasm resource of O.macrolepis,our research team has broken through the artificial breeding technology by a series of domestication experiments.Interestingly,we found the development of ovaries and eggs of O.macrolepis is asynchronous during reproduction.To exploit asynchronous development of eggs of O.macrolepis,the fatty acid composition of eggs and embryos was detected at different states by gas chromatography.Then the transcriptome of ovaries from adult O.macrolepis was analyzed in March and June.And further the genes relating to fatty acid metabolism was screened.Finally,the expression and localization of caveolin-1 was analyzed by q PCR and immunohistochemistry in ovary,liver and testes.The following research results were obtained:1.Characterization of breeding and spawning rules of O.macrolepisDuring the breeding period,broodstock of O.macrolepis were observed to harbor immature,mature and overripe eggs at the same time in the ovary with different component proportion in different individuals.After fertilization,their embryo survival rate seems to relate to this eggs composition.The results demonstrate that the spawning of O.macrolepis is batched and asynchronous.2.Determination and analysis of fatty acid component in eggs and embryos of O.macrolepisThe results from gas chromatography showed that the proportion of quinic acid(C10:0)in overripe eggs was lower than that of mature eggs,and no kulanic acid was detected in immature eggs.The proportion of normal embryonic fatty acids is identical with eggs,but quite different from sperm.Dead embryos incubated at 18 ± 1 ?,Kunic acid could not be detected in dead embryos even they were incubated in standard process(18 ± 1 ? for 72h).Furthermore,the mature eggs had higher proportion of unsaturated fatty acid and lower polyunsaturated fatty acid.These results indicate that kunoic acid(C10:0)should be an important marker for normal eggs and normal embryos.3.Transcriptome analysis of adult Onychostoma macrolepis ovary tissues in March and JuneThrough comparative analyzing the genes expression in the ovary between in March and June,the results showed that 3948 genes were significantly up-regulated(46.66%)and 4514 genes were significantly down-regulated(53.34%)in June compared with in March.GO enrichment analysis identified 28,745 genes,among which at least 7 of them are related to adipose tissue development.KEGG enrichment analysis revealed 59 genes relating to fatty acid metabolism.Based on these data,10 highly-expressed genes regulating fatty acid metabolism in June were screened,particularly,of which caveolin-1 is attractive among these 10 selected genes due to its involvement in lipid metabolism.4.Sequence alignment of caveolin-1 in O.macrolepis,and its expression and localization in liver,testis and ovary at different development stagesO.macrolepis caveolin-1 is highly conserved compared with other species,but relatively close to fish and far from mammals.The expression and location of caveolin-1 in the liver show seasonal differences,which seem to be positively correlated with the lipid content in the liver.All the fluorescence signal located in the membrane structure of liver cells,indicating caveolin-1 protein is membrane protein in O.macrolepis;In the adult O.macrolepis' s testis,caveolin-1 is highly expressed in June(breeding period)and is located on the surface of the sperm during this period;In the adult O.macrolepis' s ovary at different periods,the expression of caveolin-1 was highest in the ovary in June and was also the highest in mature eggs during reproduction,caveolin-1 was also observed in every oogenic cell line.In summary,this study provides further evidence that O.macrolepis spawn in batch and its ovaries develop asynchronously.Kunoic acid(C10:0)can be used as a molecular marker for mature normal eggs and healthy embryos;In liver,caveolin-1 expression level was positively correlated with fatty acid content.In gonads,expression and localization of caveolin-1 showed significant seasonal changes.It is speculated that caveolin-1 may regulate gonadal development by affecting fatty acid metabolism. |
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