Abstract
Increasing attention has been focused on the role of microRNAs in post-transcription regulation during spermatogenesis. Recently, the miR-34 family has been shown to be involved in the spermatogenesis, but the clear function of the miR-34 family in spermatogenesis is still obscure. Here we analyzed the function of miR-34a, a member of the miR-34 family, during spermatogenesis using miR-34a knockout zebrafish generated by the clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) system. miR-34a knockout zebrafish showed no obvious defects on testis morphology and sperm quantity. However, we found a significant increase in progressive sperm motility that is one of the pivotal factors influencing in vitro fertilization rates, in the knockout zebrafish. Moreover, breeding experiments showed that, when miR-34a-knockout male zebrafish mated with the wide-type females, they had a higher fertilization rate than did the wide-type males. Glycogen synthase kinase-3a (gsk3a), a potential sperm motility regulatory gene was predicted to be targeted by miR-34a, which was further supported by luciferase reporter assays, since a significant decrease of luciferase activity was detected upon ectopic overexpression of miR-34a. Our findings suggest that miR-34a downregulates gsk3a by targeting its 3′ untranslated region, and miR-34a/gsk3a interaction modulates sperm motility in zebrafish. This study will help in understanding in the role of the miR-34 family during spermatogenesis and will set paths for further studies.
Highlights
Spermatogenesis is a complicated and highly regulated process in which diploid spermatogonia proliferate and differentiate into mature spermatozoa through three distinct phases: mitosis, meiosis, and spermiogenesis [1,2,3]
Discussion miRNAs were first discovered in C. elegans as early as the 1990s [37], but the biological functions have not attracted considerable attention until a number of endogenous miRNAs were identified in worms, flies, and mammals
Zebrafish was used as a model in the current study to observe the functions of miR-34a is in spermatogenesis and sperm motility
Summary
Spermatogenesis is a complicated and highly regulated process in which diploid spermatogonia proliferate and differentiate into mature spermatozoa through three distinct phases: mitosis, meiosis, and spermiogenesis [1,2,3]. Noncoding RNAs (ncRNAs) including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and long ncRNAs (lncRNAs) have been considered to have crucial roles in spermatogenesis [15]. These molecules are gradually becoming the focus of studies in the field of gametogenesis. ETssh.eTrhoeleroolfe ospf escpieficcifimc imRNiRANsAssuscuhchasasmmiRiR-1-51a5,a,mmiRiR-1-18844, ,aanndd mmiiRR--338844 iiss ddeemmoonnssttrraatteedd iinn tthhee pprroocceessss ooff ssppeerrmmaattooggeenneessiiss. MMoorreeoovveerr,, mmiiRR--1155aa aanndd iittss ttaarrggeett ggeennee ccyycclliinn TT22 ((CCccnntt22)) aassssoocciiaatteess iinn eeaarrllyy ssppeerrmmaattooggeenneessiiss [[2233]]. IInn rreecceenntt ddeeccaaddeess,, sseevveerraall ssppeerrmm mmoottiilliittyyrreegguullaattoorryy ggeenneess hhaavveebbeeeennddiissccoovveerreedd,, ssuucchh aass ccaattiioonn cchhaannnneell ooff ssppeerrmm ((CCaattSSppeerr)) [[2255]],, rraabb--lliikkee 22 ((RRAABBLL22)) [[2266]],, ggsskk33aa [[2277]],, aanndd ccyyssttiicc fifibbrroossiiss ttrraannssmmeemmbbrraannee ccoonndduuccttaanncceerreegguulalatotorr(C(CFFTTRR) )[2[82]8.]R. Tmhaenms [iR30-3].4 TfahmeimlyiiRs-h3i4ghfalmy eilvyoilsuthioignhalryilyevcoolnusteirovneadriilny cvoenrtseebrvraetdesinanvdercteobnrtaatienssatnhdreceocnlotasienlsytrherleaetecdlomseelmy rbeelarste: dmmiRe-m34bae/brs/:cm[3i1R]-.3E4aar/libe/rcst[u31d]i.eEs ahraliveer pstruodvieeds hthaavtethperomveiRd-t3h4aftatmheilmy aiRct-s34asfarmegiluylaatcotrssaosfrgegenuelaetoxrpsreosfsgioenneinexcpelrlecssyicolne pinrocgelrlecsysicolenp[3ro2g],rseesnsieosnce[n32ce], s[3e3n]e,sacnendceap[3o3p]t,oasnisd[a3p4o].pOtotshiesr[3e4v]i.dOenthceers esvuigdgeensctesthsautgtgheestmthiRat-3th4efammiRil-y34alfsaomhilaysaalscorhuacisaal rcoruleciianl rsopleerminatsopgeernmeastisogaenndessipsearnmd mspoetrilmitym[3o5ti,l3i6ty]. [A35t,3p6r]e.seAntt, phroewseenvte, rh, otwheevperre,ctihsee rporleeciosfe trhoelemoifRt-h34e mfamiRi-l3y4dfuarminilgysdpuerrminagtospgeenrmesaistoisgesntiellspisoiosrslytilulnpdoeorrsltyooudndinervsitvooo.dIninthviisvsot.udIny,thwies ssttaurdteyd, wweitshtamrtieRdw34iath tmo iRex-3p4laorteo etxhpelofruentchtieofnuanlctrioolneasl roofletshoef mthieRm-3i4R-f3a4mfailmy iliyninspsepremrmataotgoegneenseissisuussiningg mmiiRR--3344aa hhoommoozzyyggoouuss kknnoocckkoouutt zzeebbrraaffiisshh pprroodduucceedd bbyy tthhee CCRRIISSPPRR//CCaass ssyysstteemm,, aanndd ttrriieedd ttoo iiddeennttiiffyy tthhee ttaarrggeetteedd ggeennee ooff mmiiRR--3344aa uussiinngg tthhee bbiiooiinnffoorrmmaattiiccss aanndd lluucciiffeerraassee rreeppoorrtteerraassssaayy
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