| Meiosis is the most important biological feature that distinguishes germ cells from somatic cells,and it is also a key biological process of gonadal development and gametogenesis in sexual reproductive organisms,which is precisely regulated by endogenous and exogenous factors.Its imbalance will cause tumors or reproductive disorders,which is an important research topic in the field of life science.In mammals,retinoic acid(RA)is a key factor in regulating the initiation of meiosis,mainly by inducing the expression of Stra8(stimulated by retinoic acid gene 8)in germ cells.In non-mammals such as birds,amphibians and fish,a large number of studies have confirmed that RA also plays an important role in the initiation of meiosis.Studies in our laboratory have shown that RA plays an important role in the initiation of meiosis in fish such as Southern catfish(Silurus meridionalis)and Nile tilapia(Oreochromis Niloticus).However,Stra8 exists only in a few fish such as Southern catfish,and no Stra8 homologue is found in many fish such as tilapia,medaka(Oryzias latipes)and zebrafish(Danio rerio).Therefore,how RA mediates the initiation of meiosis in fish without Stra8 is still a mystery.Meiotic recombination protein 8(Rec8)is a meiosis-specific adhesion complex protein,which exists in all meiotic organisms from yeast to human.It plays an important XII role in homologous chromosome pairing,sister chromatid adhesion and dissociation.However,what about the expression,regulation and function of Rec8 in fish? At present,there is a lack of related research.In view of this,this study studied the expression patterns,transcriptional and post-transcriptional regulation of two rec8 homologues(named Onrec8 a and Onrec8 b,respectively)in tilapia,and preliminarily explored their functions by overexpression.The details are as follows:1 cDNA cloning and sequence analysisTwo replication genes(Onrec8a and Onrec8b)of tilapia rec8,were cloned and analyzed.On Rec8 a and On Rec8 b are composed of 589 and 577 amino acid residues respectively,and the identity of amino acid sequences with other species is more than30%.The identity between On Rec8 b and On Rec8 a is 36%.Phylogenetic analysis showed that both On Rec8 a and On Rec8 b are homologous molecules of Rec8.Most fishes(without Stra8)had two replicative genes,but a few fishes(with Stra8)selectively lost one of them.2 Expression patternsThe onset time of meiosis of tilapia has obvious male and female dimorphism,which is about 30 dah(days after hatching)in female fish and 75 dah in male fish.Real-time PCR results showed that Onrec8 a and Onrec8 b were mainly expressed in gonads.In testis,both of them were significantly up-regulated at 80 dah.In ovary,Onrec8 a was significantly up-regulated at 30 dah,while the expression level of Onrec8 b was significantly lower than that of Onrec8 a,and up-regulated at 40 dah.The results of in situ hybridization showed that Onrec8 a and Onrec8 b were mainly expressed in differentiated germ cells such as spermatocytes and spermatids.3 Expression regulation3.1 Regulation of Onrec8a/b expression in testicular tissue of tilapia by RAAM580 is a selective agonist of RA receptor α,while BMS493 is a pan-inhibitor of RA receptor α,β and γ.The testicular tissues of 40 dah tilapia cultured in vitro were treated with AM580 and BMS493.The results showed that AM580 could significantly up-regulate the expression of Onrec8 a and Onrec8 b,while BMS493 could inhibit the expression of Onrec8 a and Onrec8 b.It is suggested that RA can promote the expression of Onrec8 a and Onrec8 b,suggesting that both Onrec8 a and Onrec8 b may play a role in the initiation of meiosis mediated by RA.3.2 Expression regulation at transcriptional level(1)Cloning of 5’-upstream sequence and construction of luciferase reporter vector and transcriptional activity analysis3005 bp and 2603 bp upstream sequences of translation initiation site of Onrec8 a and Onrec8 b were isolated and cloned from tilapia genome,and five luciferase reporter vectors with different lengths were constructed,respectively.The results of luciferase activity detection showed that the luciferase reporter vectors with different length of 5’upstream sequence had transcriptional activity,but their transcriptional activity levels were obviously different.For Onrec8 a,there are positive regulatory elements between-283 and-2571,and negative regulatory elements between-2571 and-3005.For Onrec8 b,there are positive regulatory elements in the region between-261 and-756 bp and negative regulatory elements in the region between-756 and-2603.(2)Transcriptional regulation of RAFive luciferase reporter vectors with different length of 5 ’upstream sequence of Onrec8 a and Onrec8 b were treated with RA,respectively.The results showed that RA can significantly up-regulate the luciferase activity except pr8a-3005 luc,pr8a-2571 luc and pr8b-261 luc.It is suggested that RA can up-regulate the expression of Onrec8 a and Onrec8 b through direct transcriptional regulation.(3)Transcriptional regulation of pluripotent factors and estrogensCells transfected with pr8a-3005 luc or pr8b-2603 luc were treated with different conditions.The results of luciferase activity detection showed that pluripotent molecules Sox2,Pou5f3 and estrogen significantly decreased their luciferase activity,indicating that pluripotent factors and estrogen could down-regulate the expression of Onrec8 a and Onrec8 b.3.3 Expression regulation at post-transcriptional levelThe 3’UTR(3’downstream untranslated region)of Onrec8 a and Onrec8 b were cloned from tilapia c DNA by 3’RACE,and construct into the luciferase expression vectors psi CHECKTM-2,which was used for post-transcriptional regulation analysis,and then named psi8a-luc and psi8b-luc,respectively.Luciferase activity analysis showed that tilapia germ cell-specific RNA binding molecules Dazl and Dnd could significantly enhance luciferase activity.It is suggested that Dazl and Dnd can regulate the expression of Onrec8 a and Onrec8 b at the post-transcriptional level.4.Effect of overexpression of Onrec8a/b on cell cycleOnrec8a and Onrec8 b were constructed into eukaryotic expression vectors driven by CMV,named p CMV-Onrec8a-Dsred and p CMV-Onrec8b-GFP,respectively,and transfected into cells.Fluorescence microscopic observation showed that Onrec8 a and Onrec8 b were mainly located in the nucleus,and the effect of overexpression of Onrec8a/b on cell cycle was detected by flow cytometry.The preliminary results showed that Onrec8 a and Onrec8 b can affect the cell cycle and block the cells in G0/G1 phase.In summary,this study isolated and identified two replication genes in tilapia rec8.The results of sequence analysis,spatio-temporal expression pattern,transcriptional and post-transcriptional expression regulation showed that the expression of Onrec8 a and Onrec8 b were related to meiosis and were regulated by RA,the key factor of meiosis initiation,and many other factors.This study provides scientific data and reference for the study of the initiation of meiosis in fish. |
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