Our previous study found that CLOCK knockdown in the testes of male mice led to a reduced fertility, which might be associated with the lower acrosin activity. In this present study, we examined the differential expression in proteins of CLOCK knockdown sperm. Clock gene expression was knocked down in cells to confirm those differentially expressions and serine protease inhibitor SERPINA3K was identified as a potential target. The up-regulated SERPINA3K revealed an inverse relationship with Clock knockdown. Direct treatment of normal sperm with recombinant SERPINA3K protein inhibited the acrosin activity and reduced in vitro fertilization rate. The luciferase reporter gene assay showed that the down-regulated of Clock gene could activate the Serpina3k promoter, but this activation was not affected by the mutation of E-box core sequence. Co-IP demonstrated a natural interaction between SERPIAN3K and RORs (α and β). Taken together, these results demonstrated that SERPINA3K is involved in the Clock gene-mediated male fertility by regulating acrosin activity and provide the first evidence that SERPINA3K could be regulated by Clock gene via retinoic acid-related orphan receptor response elements.
LimFLCurrieRAOrphanidesGMoggsJG. Emerging evidence for the interrelationship of xenobiotic exposure and circadian rhythms: a review. Xenobiotica2006; 36: 1140–51.
2.
ReppertSMWeaverDR. Molecular analysis of mammalian circadian rhythms. Annu Rev Physiol2001; 63: 647–76.
3.
BuhrEDTakahashiJS. Molecular components of the Mammalian circadian clock. Handb Exp Pharmacol2013; 217: 3–27.
RoennebergTMerrowM. The network of time: understanding the molecular circadian system. Curr Biol2003; 13: R198–207.
6.
KawamotoTNoshiroMSatoFMaemuraKTakedaNNagaiRIwataTFujimotoKFurukawaMMiyazakiKHonmaSHonmaKKatoY. A novel autofeedback loop of Dec1 transcription involved in circadian rhythm regulation. Biochem Biophys Res Commun2004; 313: 117–24.
7.
EtchegarayJPLeeCWadePAReppertSM. Rhythmic histone acetylation underlies transcription in the mammalian circadian clock. Nature2003; 421: 177–82.
8.
MunozEBalerR. The circadian E-box: when perfect is not good enough. Chronobiol Int2003; 20: 371–88.
9.
KyriacouCPRosatoE. Squaring up the E-box. J Biol Rhythms2000; 15: 483–90.
10.
GekakisNStaknisDNguyenHBDavisFCWilsbacherLDKingDPTakahashiJSWeitzCJ. Role of the CLOCK protein in the mammalian circadian mechanism. Science1998; 280: 1564–9.
11.
OishiKShiraiHIshidaN. Identification of the circadian clock-regulated E-box element in the mouse plasminogen activator inhibitor-1 gene. J Thromb Haemost2007; 5: 428–31.
12.
KennedyWPKaminskiJMVan der VenHHJeyendranRSReidDSBlackwellJBielfeldPZaneveldLJ. A simple, clinical assay to evaluate the acrosin activity of human spermatozoa. J Androl1989; 10: 221–31.
13.
GuillaumondFDardenteHGiguereVCermakianN. Differential control of Bmal1 circadian transcription by REV-ERB and ROR nuclear receptors. J Biol Rhythms2005; 20: 391–403.
14.
YinLJoshiSWuNTongXLazarMA. E3 ligases Arf-bp1 and Pam mediate lithium-stimulated degradation of the circadian heme receptor Rev-erb alpha. Proc Natl Acad Sci U S A2010; 107: 11614–9.
15.
GiguereV. Orphan nuclear receptors: from gene to function. Endocr Rev1999; 20: 689–725.
16.
MengQJMcMasterABeesleySLuWQGibbsJParksDCollinsJFarrowSDonnRRayDLoudonA. Ligand modulation of REV-ERBalpha function resets the peripheral circadian clock in a phasic manner. J Cell Sci2008; 121: 3629–35.
17.
MazzoccoliGCaiYLiuSFrancavillaMGiulianiFPiepoliAPazienzaVVinciguerraMYamamotoTTakumiT. REV-ERBalpha and the clock gene machinery in mouse peripheral tissues: a possible role as a synchronizing hinge. J Biol Regul Homeost Agents2012; 26: 265–76.
18.
SatoTKPandaSMiragliaLJReyesTMRudicRDMcNamaraPNaikKAFitzGeraldGAKaySAHogeneschJB. A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron2004; 43: 527–37.
19.
TeiHOkamuraHShigeyoshiYFukuharaCOzawaRHiroseMSakakiY. Circadian oscillation of a mammalian homologue of the Drosophila period gene. Nature1997; 389: 512–6.
20.
AlvarezJDChenDStorerESehgalA. Non-cyclic and developmental stage-specific expression of circadian clock proteins during murine spermatogenesis. Biol Reprod2003; 69: 81–91.
21.
HodzicARistanovicMZornBTulicCMaverANovakovicIPeterlinB. Genetic variation in circadian rhythm genes CLOCK and ARNTL as risk factor for male infertility. PLoS One2013; 8: e59220–e59220.
22.
LiangXChengSJiangXHeXWangYJiangZHouWLiSLiuYWangZ. The noncircadian function of the circadian Clock gene in the regulation of male fertility. J Biol Rhythms2013; 28: 208–17.
23.
Huang daWShermanBTLempickiRA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc2009; 4: 44–57.
24.
Huang daWShermanBTLempickiRA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res2009; 37: 1–13.
25.
SgourasDDuncanR. Methods for the evaluation of biocompatibility of soluble synthetic polymers which have potential for biomedical use: 1 — use of the tetrazolium-based colorimetric assay (MTT) as a preliminary screen for evaluation ofin vitro cytotoxicity. J Mater Sci: Mater Med1990; 1: 61–8.
26.
ToshimoriKSaxenaDKTaniiIYoshinagaK. An MN9 antigenic molecule, equatorin, is required for successful sperm-oocyte fusion in mice. Biol Reprod1998; 59: 22–9.
27.
SaxenaDKTaniiIYoshinagaKToshimoriK. Role of intra-acrosomal antigenic molecules acrin 1 (MN7) and acrin 2 (MC41) in penetration of the zona pellucida in fertilization in mice. J Reprod Fertil1999; 117: 17–25.
28.
BalakierHCasperRF. A morphologic study of unfertilized oocytes and abnormal embryos in human in vitro fertilization. J In Vitro Fertil Embryo Transf1991; 8: 73–9.
29.
HuangFJChangSYTsaiMYLinYCKungFTWuJFLuYJ. Relationship of the human cumulus-free oocyte maturational profile with in vitro outcome parameters after intracytoplasmic sperm injection. J Assist Reprod Genet1999; 16: 483–7.
30.
JinXShearmanLPWeaverDRZylkaMJde VriesGJReppertSM. A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock. Cell1999; 96: 57–68.
31.
OishiKShiraiHIshidaN. CLOCK is involved in the circadian transactivation of peroxisome-proliferator-activated receptor alpha (PPARalpha) in mice. Biochem J2005; 386: 575–81.
32.
PreitnerNDamiolaFLuis LopezMZakanyJDubouleDAlbrechtUSchiblerU. The orphan nuclear receptor REV-ERB ± controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell2002; 110: 251–60.
33.
YooSHYamazakiSLowreyPLShimomuraKKoCHBuhrEDSiepkaSMHongHKOhWJYooOJMenakerMTakahashiJS. PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci U S A2004; 101: 5339–46.
34.
RippergerJAShearmanLPReppertSMSchiblerU. CLOCK, an essential pacemaker component, controls expression of the circadian transcription factor DBP. Genes Dev2000; 14: 679–89.
35.
ChaoJTillmanDMWangMYMargoliusHSChaoL. Identification of a new tissue-kallikrein-binding protein. Biochem J1986; 239: 325–31.
36.
SchraderMDanielssonCWiesenbergICarlbergC. Identification of natural monomeric response elements of the nuclear receptor RZR/ROR. They also bind COUP-TF homodimers. J Biol Chem1996; 271: 19732–6.
KeimeSAdhamIMEngelW. Nucleotide sequence and exon-intron organization of the human proacrosin gene. Eur J Biochem1990; 190: 195–200.
39.
SilvermanGAWhisstockJCBottomleySPHuntingtonJAKaisermanDLukeCJPakSCReichhartJMBirdPI. Serpins flex their muscle: I. Putting the clamps on proteolysis in diverse biological systems. J Biol Chem2010; 285: 24299–305.
40.
WhisstockJCSilvermanGABirdPIBottomleySPKaisermanDLukeCJPakSCReichhartJMHuntingtonJA. Serpins flex their muscle: II. Structural insights into target peptidase recognition, polymerization, and transport functions. J Biol Chem2010; 285: 24307–12.
41.
UhrinPDewerchinMHilpertMChrenekPSchoferCZechmeister-MachhartMKronkeGValesACarmelietPBinderBRGeigerM. Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility. J Clin Invest2000; 106: 1531–9.
42.
LinM-HLeeRK-KHwuY-MLuC-HChuS-LChenY-JChangW-CLiS-H. SPINKL, a Kazal-type serine protease inhibitor-like protein purified from mouse seminal vesicle fluid, is able to inhibit sperm capacitation. Reproduction2008; 136: 559–71.
43.
YamagataKMurayamaKOkabeMToshimoriKNakanishiTKashiwabaraSBabaT. Acrosin accelerates the dispersal of sperm acrosomal proteins during acrosome reaction. J Biol Chem1998; 273: 10470–4.
44.
GiguereVTiniMFlockGOngEEvansRMOtulakowskiG. Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR alpha, a novel family of orphan hormone nuclear receptors. Genes Dev1994; 8: 538–53.
45.
YamamotoTNakahataYSomaHAkashiMMamineTTakumiT. Transcriptional oscillation of canonical clock genes in mouse peripheral tissues. BMC Mol Biol2004; 5: 18–18.
46.
CrumbleyCWangYKojetinDJBurrisTP. Characterization of the core mammalian clock component, NPAS2, as a REV-ERBalpha/RORalpha target gene. J Biol Chem2010; 285: 35386–92.
47.
YinLWuNCurtinJCQatananiMSzwergoldNRReidRAWaittGMParksDJPearceKHWiselyGBLazarMA. Rev-erbα, a heme sensor that coordinates metabolic and circadian pathways. Science2007; 318: 1786–9.
48.
KornmannBSchaadOBujardHTakahashiJSSchiblerU. System-driven and oscillator-dependent circadian transcription in mice with a conditionally active liver clock. PLoS Biol2007; 5: e34–e34.
49.
DebruyneJPNotonELambertCMMaywoodESWeaverDRReppertSM. A clock shock: mouse CLOCK is not required for circadian oscillator function. Neuron2006; 50: 465–77.
