caspase3,9- DHT
Journal of Steroid Biochemistry &Molecular Biology 135 (2013) 15–23
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Journal of Steroid Biochemistry and Molecular
Biology
j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j s b m
b
Regulation of apoptotic signaling pathways by 5?-dihydrotestosterone and 17?-estradiol in immature rat Sertoli cells
V.L.Sim?es,M.G.Alves,A.D.Martins,T.R.Dias,L.Rato,S.Socorro,P.F.Oliveira ?
CICS –UBI –Health Sciences Research Centre,University of Beira Interior,6200-506Covilh?,Portugal
a r t i c l e
i n f o
Article history:
Received 10August 2012Received in revised form 14November 2012
Accepted 28November 2012
Keywords:Sertoli cells Apoptosis Androgens Estrogens Caspases
a b s t r a c t
Apoptosis is an important regulatory event in testicular homeostasis and optimization of sperm produc-tion.Sertoli cells (SCs)form the blood–testis barrier creating a special microenvironment where germ cells develop and are under strict hormonal control.Estrogens and androgens are known to play critical roles in SCs functioning,improving their in vitro survival by preventing apoptotic progression.Herein,we studied the in?uence of 17?-estradiol (E2)and 5?-dihydrotestosterone (DHT)on the apoptotic signaling pathways of immature rat cultured SCs.For that we chose key points of the apoptotic pathway that inter-act with the mitochondria and evaluated the mRNA expression and/or protein levels of several apoptotic markers such as p53,the anti-apoptotic protein Bcl2,the pro-apoptotic Bcl2family member Bax,the apoptosis-inducing factor (AIF)and caspase-3and 9.Caspase-3activity and DNA fragmentation were also evaluated as endpoint markers of apoptosis.E2and DHT down-regulated the mRNA transcript levels of p53,Bax,caspase-9and caspase-3.The protein levels of AIF were reduced after DHT treatment while E2-treated cells presented decreased levels of cleaved caspase-9protein.Moreover,Bax/Bcl2ratio was signi?cantly decreased in E2-treated cells.The apoptotic endpoints caspase-3activity and DNA fragmen-tation presented signi?cant decreased levels after hormonal treatment.Taken together,these results show that E2and DHT act as apoptotic signaling modulators in in vitro immature rat SCs suggesting that androgens and estrogens may be capable of modulating independent pathways of the apoptotic event by regulating different pro-apoptotic factors.
? 2012 Elsevier Ltd. All rights reserved.
1.Introduction
The whole spermatogenesis process is dependent on the deliv-ery of speci?c products that are produced by Sertoli cells (SCs)and are necessary for germ cell survival.SCs are responsible for creating a unique and essential environment in the adluminal compartment,in order to provide structural support,nutrition and immunolog-ical protection to the progression of spermatogenesis [1–3].The
Abbreviations:AIF,apoptosis-inducing factor;Apaf-1,apoptotic protease activating factor 1;AN,accession number;?2MG,?-2-microglobulin;BSA,bovine serum albumin;E2,17?-estradiol;DHT,5?-dihydrotestosterone;cCasp9,cleaved caspase 9;DAB,3,3 -diaminobenzidine hydrochloride;DMEM:Ham’s F12,Dulbecco’s modi?ed Eagle’s medium:Ham’s nutrient mixture F12;dNTPs,deoxynu-cleotide triphosphates;EDTA,ethylene diamine tetra acetic acid;FBS,fetal bovine serum;GPER,G protein coupled estrogen receptor;HBSS,Hank’s balanced salts solution;ITS,insulin–transferrin–sodium selenite;M-MLV RT,moloney murine leukemia virus reverse transcriptase;RT-PCR,reverse transcriptase polymerase chain reaction;SCs,Sertoli cells;TdT,terminal deoxynucleotidyl transferase;TUNEL,TdT-mediated dUTP nick end labeling.
?Corresponding author at:Health Sciences Research Centre,Faculty of Health Sciences,University of Beira Interior,Av.Infante D.Henrique,6201-506Covilh?,Portugal.Tel.:+351275329077;fax:+351275329099.
E-mail address:pfobox@https://www.wendangku.net/doc/e818364961.html, (P.F.Oliveira).
number of germ cells that are produced during spermatogenesis is directly related to the number of functional SCs [4],as one single SC can only support 30–50germ cells at different stages [5].
Although it is commonly accepted that SCs regulate apopto-sis of developing germ cells in a complex process controlled by a network of endocrine and other regulatory factors,particularly through the FasL/Fas signal transduction system [6],the mech-anisms by which SCs proliferation and death is controlled have been poorly investigated.It has been reported that SCs may have protective mechanisms that render them resistant to apoptosis derived from a variety of insults when compared to germ cells [7–9].Nevertheless,studies on infertile men demonstrated signif-icantly increased apoptotic levels in SCs of those patients [10].It has been reported that a combination of peptide,steroid hormone and growth factors are able to modulate SCs apoptosis and survival [11]but the mechanisms have not been explored.Furthermore,it is known that androgens and estrogens play an important role in the regulation of growth,development,homeostasis and programmed cell death in testicular cells [12–14].
The process of apoptosis is a fundamental biochemical cell death pathway characterized by several morphologic changes [15]and plays a fundamental role in the maintenance of tissue homeosta-sis in the adult organism,turned on in response to environment
0960-0760/$–see front matter ? 2012 Elsevier Ltd. All rights reserved.https://www.wendangku.net/doc/e818364961.html,/10.1016/j.jsbmb.2012.11.019
16V.L.Sim?es et al./Journal of Steroid Biochemistry&Molecular Biology135 (2013) 15–23
signals or triggered by intrinsic factors[16].These signals can be observed in several situations during cell development,homeo-static regulation of cell populations and aging[17].There are two main pathways involved in the apoptotic process:the extrinsic or death receptor pathway,occurring in response to activated death receptors present in the cell surface,and the intrinsic or mito-chondrial pathway occurring in response to signals originated from inside the cell[16].The intrinsic pathway can be triggered due to various signals as DNA damage,cytotoxic stress and growth factors deprivation[18]and involves the action of mitochondria,which stimulates and transduce signals to execute apoptosis via a dis-tinctive set of molecules[17–19].The Bcl-2family consists of anti-and pro-apoptotic members such as Bcl-2and Bax respectively and the Bax/Bcl-2ratio is often used as biomarker for apoptosis unbalance in several pathological conditions[20].Moreover,the up-regulation or activation of pro-apoptotic molecules,such as Bax, leads to its translocation to the outer membrane of mitochondria [21]and consequent permeabilization of the outer mitochondrial membrane,which is followed by the release of many apoptosis pro-moting proteins that reside in the mitochondrial intermembrane space,including cytochrome c,apoptosis-inducing factor(AIF)and some pro-caspases[21].This permeabilization can lead to sequen-tial activation of effector caspases(such as caspase-3)and when the caspases cascade is initiated,the process of cell death cannot be reversed[17].Additionally,it can also lead to the release of other pro-apoptotic factors(such as the?avoprotein AIF)that,ensuing translocation to the nucleus,contribute to chromatin condensation and chromatinolysis,resulting in apoptosis[22].
Several pathological conditions known to cause subfertility and infertility are characterized by severe hormonal deregulation [23,24].We have previously shown that17?-estradiol(E2)and 5?-dihydrotestosterone(DHT)are modulators of SCs metabolism [25,26]and there is a close relationship between metabolism and apoptosis,being the mitochondria the central organelle[27],thus we hypothesized that sex steroid hormones could have a role on the regulation of mitochondria related pro-apoptotic factors.So, the aim of our work was to study the in?uence of E2and DHT on the apoptotic signaling pathways in immature rat SCs.Therefore, we chose key points of the apoptotic pathway that interact with the mitochondria and evaluated mRNA expression of p53,the pro-apoptotic Bcl2family member Bax,caspase-3and caspase-9.The protein expression of caspase-9,Bax,the anti-apoptotic Bcl2,and AIF was also determined.Finally,Caspase-3activity and DNA frag-mentation(TUNEL assay)were evaluated as endpoint markers of apoptosis.
2.Materials and methods
2.1.Chemicals
Hank’s balanced salts solution(HBSS),Dulbecco’s modi-?ed Eagle’s medium:Ham’s nutrient mixture F12(DMEM:Ham’s F12),ethylene diamine tetra acetic(EDTA)acid,soybean trypsin inhibitor,DNAse,Collagenase type I,17?-estradiol (E2),5?-dihydrotestosterone(DHT),bovine serum albumin (BSA),ExtrAvidin-Peroxidase Staining Kit,3,3 -diaminobenzidine hydrochloride(DAB),trypsin–EDTA,insulin–transferrin–sodium selenite supplement(ITS supplement),TRI reagent and other drugs were obtained from Sigma Aldrich.(St.Louis,MO,USA).Fetal Bovine Serum(FBS)was obtained from Biochrom AG(Germany). Moloney murine leukemia virus reverse transcriptase(M-MLV RT) and random hexamer primers were obtained from Invitrogen(CA, USA).dNTPs were obtained from GE Healthcare(Buckinghamshire, UK).1×Buffer and Taq DNA polymerase were obtained from Fer-mentas Life Sciences(Ontario,Canada).Polyclonal antibodies were obtained from Santa Cruz Biotechnology(Heidelberg,Germany) and Cell Signaling(Massachusetts,USA).TdT-FragEL DNA fragmen-tation Detection kit was obtained from Calbiochem(Darmstadt, Germany).
2.2.Animals
Wistar male rats(Rattus norvegicus)were obtained from Charles River(Barcelona,Spain)and housed under a12h light-12h dark-ness cycle,with food and water available ad libitum.Housing, maintenance and handling of animals comply with the“Guide for the Care and Use of Laboratory Animals”;published by the US National Institutes of Health(NIH Publication No.85-23,revised 1996)and the rules for the care and handling of laboratory animals (Directive86/609/EEC).
2.3.Sertoli cell culture
Ten male Wistar rats(20-day old)were sacri?ced by cervical dislocation,the testis were immediately excised in aseptic con-ditions and washed two times in a50mL conical tube in30mL of ice cold HBSS containing10,000U/mL of penicillin,10mg/mL streptomycin and25?g/ml amphotericin B(pH7.4).SCs were isolated and cultured(in phenol-red free media)using a previ-ously described method by Meroni and collaborators[28],adapted by Oliveira and collaborators[29].Brie?y,tissue from decapsu-lated testes was placed in a Petri dish containing glycine medium (HBSS plus1M glycine,2mM EDTA,0.002%(w/v)soybean trypsin inhibitor;pH7.2).The tubular pellet was digested with Collagen-ase type I and DNAse in HBSS for15–20min at room temperature. The sertoli cell suspension,was collected by centrifugation(300×g for3min),washed in HBSS and resuspended in Sertoli culture medium which consisted of a1:1mixture of DMEM:Ham’s F12(pH 7.2–7.4)supplemented with15mM HEPES,50U/mL penicillin and 50mg/mL streptomycin sulfate,0.5mg/mL fungizone,50?g/mL gentamicin and10%heat inactivated FBS.This cellular suspension was then forced through a20G needle,in order to disaggregate large Sertoli clusters.The cellular suspension was plated on culture ?asks(Cell+;Sarstedt),and incubated at33?C in an atmosphere of5%CO2,95%O2in Sertoli culture medium.SC culture purity was assessed by the immunoperoxidase detection of speci?c mark-ers,anti-Mullerian hormone and Vimentin as described elsewhere [30].Brie?y,cells were grown on6well culture plates,incubated overnight at4?C with primary polyclonal antibody and labeled streptavidin–biotin method using an ExtrAvidin-Peroxidase Stain-ing Kit,giving a brown coloration to the SCs after reaction with diaminobenzidine.The cell nucleus was then stained with haema-toxylin.Negative-control incubations were executed using PBS instead of primary antibody.Cultures were examined by phase con-trast microscopy and selected if cells contaminants were below5% after96h.
2.4.Experimental groups
SCs were allowed to grow until reach90–95%con?uence,and then washed thoroughly and the medium replaced by serum and phenol-red free media(DMEM:F12,1:1,with ITS supplement, pH7.4).To evaluate the effects of sex hormones on mRNA and protein expression,SCs were treated during50h with100nM of E2or100nM of DHT.DHT was chosen as a androgen fam-ily representative because it is not conversable to E2by the cells [31].The concentrations of the sex steroid hormones were cho-sen based on available data which reported that intratesticular interstitial?uid concentrations of those hormones are particu-larly higher than those of circulating plasma,reaching values up to200nanomolar[32,33].Working stock solutions of DHT
V.L.Sim?es et al./Journal of Steroid Biochemistry&Molecular Biology135 (2013) 15–2317 Table1
Oligonucleotides and cycling conditions for PCR ampli?cation of p53,Bax,caspase-9,caspase-3,18S and?-2-microglobulin.
Gene Sequence(5 –3 )AT(?C)Ampli?con size(bp)C
P53
AN:NM030989.3Sense:CTG CCC ACC ACA GCG ACA GG5947135 Antisense:AGG AGC CAG GCC GTC ACC AT
Bax
AN:NM017059.2Sense:CGC GTG GTT GCC CTC TTC TAC TTT5912435 Antisense:CAA GCA GCC GCT CAC GGA GGA
Casp9
AN:NM031632.1Sense:TGC AGG GTA CGC CTT GTG CG6113035 Antisense:CCT GAT CCC GCC GAG ACC CA
Casp3
AN:NM012922.2Sense:AGG CCT GCC GAG GTA CAG AGC6025535 Antisense:CCG TGG CCA CCT TCC GCT TA
18S
AN:NR046237.1Sense:AAG ACG AAC CAG AGC GAA AG5614925 Antisense:GGC GGG TCA TGG GAA TAA
?2MG
AN:NM012512.2Sense:GCG TGG GAG GAG CAT CAG GG5926425 Antisense:CTC ATC ACC ACC CCG GGG ACT
Abbreviations:AT,annealing temperature;C,number of cycles during exponential phase of ampli?cation;AN,accession number.
and E2were prepared in ethanol(EtOH).The Control groups were treated with same amount of solvent/vehicle(EtOH)used in DHT and E2groups(<0.025%,v/v).After the50h incubation period,the detached cells were collected and the attached cells were detached with a trypsin–EDTA solution.A viability test was executed on all cells of the different experimental groups using the Trypan Blue Exclusion Test[34].Viability averaged85–95%, always with values higher than85%.At the end of the treat-ment,the total number of cells per?ask was determined with a neubauer chamber and cells were collected for RNA or protein extraction.
2.5.RT-PCR
Total ribonucleic acid(RNAt)was extracted from isolated SCs using TRI reagent according to the manufacturer’s instruc-tions.RNA concentration and absorbance ratios(A260/A280)were spectrophotometricaly determined(Nanophotometer TM,Implen, Germany).RNAt(1?g)was reversely transcribed in a?nal volume of20?L with200U of M-MLV RT according to the manufacturer’s protocol,using250ng of random hexamer primers and0.5mM of each dNTP.The resulting cDNA was used to amplify Bax,caspase-3,caspase-9and p53cDNA fragments using exon–exon spanning primer sets(Table1).cDNA(1?L)was ampli?ed in a?nal volume of25?L,containing reaction buffer(2mM MgCl2,0.2mM dNTP), 0.2?M of each primer and0.625U Taq DNA polymerase.Opti-mal annealing temperature and the number of cycles needed for ampli?cation phase of fragments are shown in Table1.Expression of Bax,caspase-3,caspase-9and p53mRNA was normalized with 18S gene and?-2-microglobulin expression levels.Densities from each band were obtained with BIO-PROFIL Bio-1D Software from Quantity One(Vilber Lourmat,Marne-la-Vallée,France)accord-ing to standard methods.The band density acquired for each studied gene was then divided by the respective18S gene and?-2-microglobulin band densities and the results are expressed as the mean fold variation(induction/reduction)versus the control group.
2.6.Western blot
Western blot procedure was performed as previously described [35].In brief,total proteins were isolated from rat SCs using RIPAS buffer supplemented with protease and phosphatase inhibitors(1×PBS,1%NP-40,0.5%sodium deoxycholate,0.1%SDS,1mM PMSF, supplemented with1%protease inhibitor cocktail and100mM sodium orthovanadate).Protein concentration was determined by the Bradford micro assay.Proteins samples were fractionated in polyacrylamide gels and transferred to polyvinylidene di?uoride membranes.The membranes were then incubated overnight at 4?C with rabbit anti-Bax(1:5000,ref.2772,Cell Signaling Tech-nology),rabbit anti-Bcl2(50E3)(1:1000,ref.2870,Cell Signaling Technology),rabbit anti-cleaved caspase-9(Asp353)(1:1000,ref. 9507,Cell Signaling Technology)and mouse anti-AIF(1:1000,ref. SC-13116,Santa Cruz Biotechnology)primary antibodies.Mouse anti-actin primary antibody(1:1000,ref.A-5441,Sigma–Aldrich, Roedermark,Germany)was used as protein loading control in different experimental conditions.The immune-reactive proteins were detected with goat anti-rabbit IgG-AP(1:5000,ref.SC-2007, Santa Cruz Biotechnology Heidelberg,Germany,)or goat anti-mouse IgG-AP(1:5000,ref.SC-2008,Santa Cruz Biotechnology Heidelberg,Germany).Membranes were reacted with ECF detec-tion system(GE,Healthcare,We?ling,Germany)and read with the BioRad FX-Pro-plus(Bio-Rad,Hemel Hempstead,UK).Using the Quantity One Software(Bio-Rad,Hemel Hempstead,UK)the densities from each band were obtained,according to standard methods.The obtained band density was divided by the respec-tive actin band density and then normalized as percentage of the respective control.
2.7.Caspase-3activity assay
Caspase-3activity was spectrophotometrically assessed by determining the cleavage of the respective colorimetric substrate as previously described[35].In brief,25?g of proteins were incubated in assay buffer(25mM HEPES,pH7.5,0.1%CHAPS, 10%sucrose and10mM DTT)with100?M of caspase-3sub-strate(Ac-DEVD-pNA)for2h at37?C.The caspase-3activity was determined by detection of the chromophore p-nitroanilide, measured at405nm in a spectrophotometer.The method was calibrated with known concentrations of p-nitroanilide.The attained activities were expressed in percentage versus the control group.
2.8.TUNEL assay
DNA fragmentation was determined by evaluating TdT-mediated dUTP nick end labeling(TUNEL)staining.TdT-FragEL DNA Fragmentation Detection Kit(Calbiochem,Germany)was used, according to the manufacturer’s instructions,to identify apoptotic cells in the different experimental groups.Each sample was ana-lyzed by counting the total number of cells and the number of TUNEL-positive stained SCs(displaying dark-brown precipitates in
18V.L.Sim?es et al./Journal of Steroid Biochemistry&Molecular Biology135 (2013) 15–23
their nuclei)in5random?elds in a“blinded”manner.Positive controls were carried out by treating SCs with DNAse previously to the TUNEL assay.Negative controls were obtained by replacing TdT enzyme by dH2O in the TUNEL assay.
2.9.Statistical analysis
The statistical signi?cance of mRNA and protein expression,as well as caspase-3activity among the experimental groups was assessed by two-way ANOVA,followed by Bonferroni post-test.All experimental data are shown as mean±SEM(n=5for each con-dition).Statistical analysis was performed using GraphPad Prism5 (GraphPad Software,San Diego,CA).P<0.05was considered signif-icant.
3.Results
3.1.E2down-regulates mRNA transcript levels of pro-apoptotic signaling markers
The treatment with E2was followed by gene expression analysis of apoptotic signaling markers such as p53,which is a key apoptosis regulator that can be translocated to the mitochondria,inducing apoptosis,or can be directly localized in the sites of DNA damage and promote its proper repair[36].For this reason we evaluated the effect of E2on the transcription of p53in cultured rat SCs.The mRNA expression levels of p53in E2-treated cells were signi?cantly reduced.We observed a0.71±0.05fold variation of p53mRNA levels in E2-treated cells when compared with the control group (Fig.1).
A crucial event for initiating the intrinsic apoptotic pathway is the opening of a permeability transition pore that requires the activation of pro-apoptotic members,such as Bax,in a process mediated by p53[27].Thus,we evaluated the mRNA levels of this pro-apoptotic protein and we also detected a signi?cant decrease in E2-treated cells(0.78±0.06fold variation to control)(Fig.1).
After Bax activation and its translocation to the outer membrane of mitochondria,the release of pro-apoptotic factors occurs,lead-ing to the cleavage of caspase-9followed by sequential activation of the effector caspase-3.Thereby,we assessed the mRNA levels of caspase-9and caspase-3that were also signi?cantly reduced (0.79±0.05and0.78±0.05fold variation,respectively)in E2-treated cells(Fig.1).3.2.mRNA transcript levels of pro-apoptotic markers are downregulated after DHT treatment
Following the treatment of SCs with DHT,we analyzed the effect of this androgen in the transcript levels of signaling apoptotic mark-ers.Starting with p53,we observed a pronounced decrease for the mRNA levels(0.59±0.07fold variation)(Fig.2).Bax activation is a key point of apoptosis as it leads to the release of pro-apoptotic factors.So we analyzed the effect of DHT in the gene transcript lev-els of this pro-apoptotic protein.The results showed a signi?cant 0.82±0.06fold variation in the mRNA expression levels of Bax in DHT-treated cells(Fig.2).
The apoptotic markers,caspase-9and caspase-3,represent an endpoint in this process.We observed a signi?cant decrease in mRNA levels of both caspase-9and caspase-3(0.74±0.03and 0.75±0.06fold variation,respectively)(Fig.2)in DHT-treated cells.
3.3.E2increases Bcl2protein levels and decreases Bax/Bcl2ratio
To determine if estrogens differentially modulated the balance between pro-and antiapoptotic markers,we measured the protein levels of the antiapoptotic Bcl-2and the pro-apoptotic Bax using Western blot after treating SCs with E2during50hours.E2signif-icantly increased Bcl-2levels by1.45±0.09fold as compared to control(Fig.3).Although we observed a signi?cant decrease in the mRNA expression levels of Bax,these results were not followed by a signi?cant decrease in the protein levels,as Bax protein levels in E2-treated cells did not present signi?cant differences relatively to the control group(Fig.3).Overall,treatment of SCs for50h sig-ni?cantly decreased the Bax/Bcl-2ratio by0.74±0.09fold when compared to control(Fig.3).
3.4.E2decrease cleaved caspase-9protein levels
The intrinsic apoptotic pathway also involves the release of caspase-independent death effectors such as AIF[22].Its release represents an important stage in apoptosis but we found no differ-ences regarding the protein expression of AIF in E2-treated when compared to the control SCs(Fig.3).
Caspase-9triggers a caspase-signaling cascade to induce apo-ptosis[37].The binding of pro-caspase-9to Apaf-1leads to the autolytic cleavage of pro-caspase-9[37],which allows the initi-ation of the caspases cascade[38].Following the noted decrease on caspase-9mRNA levels,in E2-treated cells,we also evaluated the protein levels that were consistent with those obtained for
the Fig.1.Effect of E2on p53,Bax,caspase-9and caspase-3mRNA levels in rat Sertoli cells.Panel A shows representative agarose gel electrophoresis.Panel B shows pooled data of independent experiments,indicating the fold variation of mRNA levels found in cultures with100nM E2when compared with cultures on control condition(dashed line). Results are expressed as mean±SEM(n=5for each condition).The statistical signi?cance was assessed by a two-way ANOVA,followed by Bonferroni post-test.Signi?cantly differently results(p<0.05)are indicated:*relatively to control.
V.L.Sim?es et al./Journal of Steroid Biochemistry&Molecular Biology135 (2013) 15–23
19
Fig.2.Effect of DHT on p53,Bax,caspase-9and caspase-3mRNA levels in rat Sertoli cells.Panel A shows representative agarose gel electrophoresis.Panel B shows pooled data of independent experiments,indicating the fold variation of mRNA found in cultures with100nM DHT when compared with cultures on control condition(dashed line). Results are expressed as mean±SEM(n=5for each condition).The statistical signi?cance was assessed by a two-way ANOVA,followed by Bonferroni post-test.Signi?cantly differently results(p<0.05)are indicated:*relatively to control.
mRNA levels,as we observed a signi?cant0.81±0.06fold variation to the control SCs(Fig.3).
3.5.DHT increases Bcl2protein levels and decreases Bax/Bcl2
ratio
After assessing the effect of DHT on Bax mRNA levels,we evalu-ated if whether DHT treatment resulted on altered expression of the pro-or anti-apoptotic proteins,Bax and Bcl-2,respectively. We observed that DHT treatment did not cause a signi?cant alter-ation in Bax protein levels(Fig.4).While there were no signi?cant changes in Bax expression,Bcl-2protein levels were signi?cantly increased(1.30±0.06fold variation to control)and the Bax/Bcl-2 ratio was found to be decreased,although not signi?cantly,when cells were treated with DHT for50h(Fig.4).
3.6.AIF protein levels are decreased after DHT-treatment
As said,the apoptotic cascade occurs with the release of poten-tially lethal proteins from the mitochondrial intermembrane space, including caspase-independent apoptosis effectors such as AIF[22]. This apoptotic marker has the nucleus of the cells as a target,where it participates in the degradation of DNA[39].The protein lev-els of AIF remained unaltered when treated with E2(Fig.3),but DHT-treated cells presented a signi?cant decrease(0.82±0.06fold variation)in AIF protein expression levels(Fig.4).Finally,we ana-lyzed the protein expression of cleaved caspase-9in DHT-treated cells,concluding that cleaved caspase-9protein levels were not signi?cantly different from the control group(Fig.4).
3.7.Caspase-3activity is highly decreased after hormonal treatment
Caspases are critical mediators of the apoptotic process.Activa-tion of caspase-3requires the proteolytic cleavage of its inactive precursor enzyme,pro-caspase-3[37].The activation of caspase-3 turns the apoptotic pathway irreversible thus,following the alter-ation on the mRNA or protein expression of caspase-3and9,we measured caspase-3activity in SCs subject to hormonal treatment (DHT and E2-treated groups)by measuring the cleavage of a spe-ci?c substrate and the release of a chromophore(p-nitroanilide). We observed that caspase-3activity was decreased in both E2and DHT-treated cells(Fig.5).The results obtained for E2-treated cells showed a more striking effect,as we observed a decrease to47±9% of caspase-3activity relatively to the control group(Fig.5).DHT-treated cells also showed a signi?cant decrease in the activity of caspase-3to59±12%(Fig.5).These results are in agreement with and emphasize the alterations observed in caspase-3mRNA
levels. Fig.3.Effect of E2on Bax,Bcl2,AIF,cleaved caspase-9protein levels and Bax/Bcl2protein ratio in rat Sertoli cells.Panel A shows representative Western blot experiment. Panel B shows pooled data of independent experiments,indicating the fold variation of protein levels found in cultures with100nM E2when compared with cultures on control condition(dashed line).Results are expressed as mean±SEM(n=5for each condition).The statistical signi?cance was assessed by a two-way ANOVA,followed by Bonferroni post-test.Signi?cantly differently results(p<0.05)are indicated:*relatively to control.
20V.L.Sim?es et al./Journal of Steroid Biochemistry &Molecular Biology 135 (2013) 15–
23
Fig.4.Effect of DHT on Bax,Bcl2,AIF,cleaved caspase-9protein levels and Bax–Bcl2protein ratio in rat Sertoli cells.Panel A shows representative Western blot experiment.Panel B shows pooled data of independent experiments,indicating the fold variation of protein levels found in cultures with 100nM DHT when compared with cultures on control condition (dashed line).Results are expressed as mean ±SEM (n =5for each condition).The statistical signi?cance was assessed by a two-way ANOVA,followed by Bonferroni post-test.Signi?cantly differently results (p <0.05)are indicated:*relatively to
control.
Fig.5.Effects of E2and DHT on caspase-3activity in rat Sertoli cells.Results are presented as pooled data of independent experiments,indicating the percentage variation of activity levels found in cultures with 100nM E2(E2)or 100nM DHT (DHT)when compared with cultures on control condition (dashed line).Results are expressed as mean ±SEM (n =5for each condition).The statistical signi?cance was assessed by a two-way ANOVA,followed by Bonferroni post-test.Signi?cantly differently results (p <0.05)are indicated:*relatively to control.
3.8.DNA fragmentation is decreased after hormonal treatment
During apoptosis,DNA fragmentation occurs via the activation of nucleases [40].In order to estimate DNA fragmentation in SCs treated with E2or DHT for 50h,cells were ?xed and subjected to the TUNEL assay.When we assessed the DNA
fragmentation
Fig.6.Effects of E2and DHT on DNA fragmentation levels in rat Sertoli cells.Results are presented as pooled data of independent experiments,indicating the percentage of TUNEL-positive stained cells found in cultures Control conditions,with 100nM E2(E2)or 100nM DHT (DHT).TUNEL-positive cells were counted in 5random ?elds of each sample in a “blinded”manner.Results are expressed as mean ±SEM (n =5for each condition).The statistical signi?cance was assessed by a two-way ANOVA,followed by Bonferroni post-test.Signi?cantly differently results (p <0.05)are indicated:*relatively to control.
levels of hormone-treated SCs,we observed a signi?cant decrease in both E2and DHT-treated cells (Fig.6).Similarly to what was determined in caspase-3activity,in E2-treated cells the results obtained for DNA fragmentation showed a more intense effect,as we observed a decrease to 72%relatively to that of the control group (Fig.6).DHT-treated cells also showed a signi?cant decrease of the number of TUNEL positive cells (DNA fragmentation)of approximately 59%(Fig.6).These results are in agreement with and emphasize the observed alterations in caspase-3mRNA and activity levels.
4.Discussion
The role of SCs in the establishment of a successful spermato-genic event has been widely investigated but many questions are still unanswered.During their functioning,these cells suffer the in?uence of various endocrine and exocrine regulators [41,42].Sex steroid hormones are known as the key regulators of sper-matogenesis and SCs mediate their action [43,44].In fact,within the seminiferous tubules,SCs are known to express receptors for androgens and estrogens [45,46],rendering them the prime targets for the hormonal signaling that regulates spermatogenesis [47,48].Estrogens and androgens are known to play a critical role in pre-venting apoptosis in wide range of mammalian cells,namely in cells from the male reproductive tract [13,14,49–51].Additionally,apoptosis is a cellular event essential for the occurrence of a nor-mal spermatogenesis as it permits the control of germ cells number [12].Nevertheless,as SCs can only accommodate the differentiation of a ?nite number of germ cells [4],any agent that impairs the via-bility of SCs may cause deleterious effects on spermatogenesis,and may result in lower fertility or even in infertility.So the enlighten-ment of factors that can in?uence and modulate SCs survival and/or apoptosis is of extreme relevance.
Several pathological conditions that end-up in subfertility or infertility are characterized by severe hormonal deregula-tion [23,24].Recently,it was reported that E2and DHT are cell metabolism modulators in rat and human SCs [25,26,52]and it is known that alterations of cell metabolism,particularly in glucose metabolism,are closely related with apoptosis [27].Decreases in intracellular ATP or increases in oxidative stress due to reduced glycolysis can lead to increased apoptosis [53]and mitochondria seem to be the link between metabolic and apoptotic signaling and are the source of many of the apoptotic proteins.Hence,fol-lowing previously reported results on the effect of E2and DHT in SCs metabolism,we hypothesized that sex steroid hormones could have an active participation on the regulation of these mitochondria related pro-apoptotic proteins.
V.L.Sim?es et al./Journal of Steroid Biochemistry&Molecular Biology135 (2013) 15–2321
The apoptotic pathway can be triggered due to several signals. When a death signal occurs and the p53protein is activated,a cas-cade of events is initiated resulting in apoptosis[54].p53protein is known to induce the expression of pro-apoptotic members of the of Bcl-2protein family,such as Bax[55]that can be counteracted by the release of antiapoptotic proteins of Bcl-2family such as Bcl-2. When we assessed the effect of E2or DHT on the expression of the apoptotic markers p53and Bax in rat SCs we could observe a sig-ni?cant down-regulation.Indeed,the results obtained showed that both sex steroid hormones were able to signi?cantly decrease the mRNA levels of p53and Bax,which is in agreement with previous results reporting that both estrogens and androgens are capable of preventing apoptosis[56,57].
The Bax activation causes mitochondria permeabilization, which leads to the release of several apoptotic factors from the intermembrane space and can result on the activation of the caspase-dependent death pathway(via activation of caspase-9,fol-lowed by sequential activation of the effector caspase-3)[58]or by the activation of caspase-independent death effectors such as AIF (which translocates to the nucleus and contributes to chromatin condensation and chromatinolysis)[22].As could be expected,after observing a decrease in Bax mRNA levels,when we evaluated the effect of E2or DHT on the caspase-9and caspase-3mRNA levels we could also observe a signi?cant down-regulation.Furthermore, we also demonstrated that DHT treatment had a down-regulatory effect on AIF protein levels.This is in clear agreement with works by others that reported a decrease in SCs apoptosis,through a decrease in DNA fragmentation,induced by androgens[56].It has also been reported that testosterone improved in vitro SC survival through the reduction of DNA fragmentation,preventing the apoptotic pathway [12]and DHT is capable of up-regulating in vitro glucose consump-tion by SCs[25,26].In fact,the TUNEL assay showed that E2and DHT treatment highly decreased the percentage of TUNEL positive cells per?eld indicating that DNA fragmentation is decreased in E2and DHT-treated cells.Moreover,an increase in glucose metabolism can sustain the mitochondria membrane potential and slow or even prevent the apoptotic process[53].Thus,the results described for AIF protein levels,after DHT treatment,are in full agreement with this hypothesis and the increased glucose consumption observed in DHT-treated cells could be associated with this decreased apoptotic signaling and consequently with the decreased DNA fragmentation attained.
In our experiments,E2was also able to modulate the protein levels of cleaved caspase-9.The cleavage of caspase-9leads to the activation of the caspase cascade that culminates in the activa-tion of effector caspases[59].As previously said,the anti-apoptotic effect of estrogens has been widely reported for a variety of cel-lular systems.In fact,estrogens are capable of protecting SCs even from the action of exogenous apoptotic promoting substances[60]. So,the observed decrease in the cleaved caspase-9protein lev-els for E2-treated SCs is clearly in agreement with those reports.
A decrease in the active form of caspase-9will certainly trans-late into a reduction of the cellular apoptotic levels.Moreover,it has also been reported that E2-treatment increases the expres-sion of the anti-apoptotic proteins Bcl2and Bcl2l2in immature rat SCs,through MAPK and PIK3pathway[57,61].The interaction of E2-GPER modulates this CREB-mediated transcription of anti-apoptotic genes of the Bcl2gene family[57].Bcl2has been shown to prevent apoptosis by antagonizing the pore forming activity and the release of mitochondrial pro-apoptotic factors[35]leading to a decrease in the activation of caspase-9,as we report here.In fact, our results show that the Bax/Bcl-2ratio is signi?cantly decreased after E2treatment thus evidencing that there is an unbalance in the pro-and antiapoptotic proteins favoring the last.Consequently these results suggest that apoptotic signaling is decreased in E2-treated cells.
After observing a decrease on the mRNA levels and also on the protein levels of key pro-apoptotic factors in DHT and E2-treated cells,we chose the caspase-3activity and DNA fragmentation as endpoint markers for apoptosis.These endpoints allow a roughly quantitative assessment of cellular apoptosis levels,being that from these points the apoptotic process is irreversible[62].We observed that caspase-3activity in both DHT and E2-treated SCs was signi?cantly decreased.This decrease in caspase-3activity is in agreement with what was observed for caspase-3mRNA levels, and other pro-apoptotic factors.Moreover,DNA fragmentation as measured by TUNEL assay clearly indicates that both E2and DHT prevent DNA fragmentation.These results con?rm an effect of these hormones on reducing the apoptotic levels in cultured SCs.
5.Conclusion
In conclusion,our results show that E2and DHT act as apoptotic signaling modulators in in vitro immature rat SCs.DHT and E2are both capable of down-regulating p53,Bax,caspase-9and caspase-3mRNA levels.DHT alone is capable of decreasing the protein levels of AIF(a caspase-independent death effector released from mitochondria)[63],and E2alone is capable of down-regulating cleaved caspase-9protein levels(regulated by the release of cytochrome c from the mitochondria and its binding to and acti-vation of Apaf-1to form the apoptosome complex)[37],which may suggest that androgens and estrogens are capable of modu-lating independent pathways of the apoptotic event,regulated by different pro-apoptotic factors.Further studies will be needed to full elucidate the mechanisms behind this phenomenon.Never-theless,both DHT and E2were able to reduce caspase-3activity and the DNA fragmentation,which are endpoint markers for apo-ptosis,clearly con?rming the anti-apoptotic action of DHT and E2. Our?ndings provide an important contribution for the enlighten-ment of androgens and estrogens role in the SCs functioning and homeostasis and may contribute to better understand some male infertility causes,especially because several pathological condi-tions that end-up in subfertility or infertility are characterized by severe hormonal deregulation.
Con?icts of interest
None to declare.
Acknowledgements
This work was supported by the Portuguese“Fundac??o para a Ciência e a Tecnologia”–FCT(PTDC/QUI-BIQ/121446/2010) co-funded by FEDER via Programa Operacional Factores de Com-petitividade–COMPETE/QREN.L.Rato(SFRH/BD/72733/2010)and M.G.Alves(SFRH/BPD/80451/2011)were?nanced by FCT.P.F. Oliveira was?nanced by FCT through FSE and POPH funds(Pro-grama Ciência2008).
References
[1]M.D.Griswold,Interactions between germ cells and sertoli cells in the testis,
Biology of Reproduction52(1995)211–216.
[2]L.Johnson,D.L.Thompson Jr.,D.D.Varner,Role of sertoli cell number and func-
tion on regulation of spermatogenesis,Animal Reproduction Science105(1–2) (2008)23–51.
[3]L.Rato,S.Socorro,J.E.B.Cavaco,P.F.Oliveira,Tubular?uid secretion in the sem-
iniferous epithelium:ion transporters and aquaporins in sertoli cells,Journal of Membrane Biology236(2)(2010)215–224.
[4]J.M.Orth,G.L.Gunsalus,https://www.wendangku.net/doc/e818364961.html,mperti,Evidence from sertoli cell-depleted
rats indicates that spermatid number in adults depends on numbers of sertoli cells produced during perinatal development,Endocrinology122(3)(1988) 787–794.
22V.L.Sim?es et al./Journal of Steroid Biochemistry&Molecular Biology135 (2013) 15–23
[5]C.Y.Cheng,E.W.Wong,H.H.Yan,D.D.Mruk,Regulation of spermatogenesis
in the microenvironment of the seminiferous epithelium:new insights and advances,Molecular and Cellular Endocrinology315(1–2)(2010)49–56. [6]K.K.Shukla,A.A.Mahdi,S.Rajender,Apoptosis,spermatogenesis and male
infertility,Frontiers in Bioscience4(2012)746–754.
[7]N.Eid,Y.Ito,Y.Otsuki,Enhanced mitophagy in sertoli cells of ethanol-treated
rats:morphological evidence and clinical relevance,Journal of Molecular His-tology(2012)1–10.
[8]A.Bozec,F.Chuzel,S.Chater,C.Paulin,R.Bars,M.Benahmed,C.Mauduit,
The mitochondrial-dependent pathway is chronically affected in testicular germ cell death in adult rats exposed in utero to anti-androgens,Journal of Endocrinology183(1)(2004)79–90.
[9]Y.J.Kim,J.Y.Chung,S.G.Lee,J.Y.Kim,J.E.Park,W.R.Kim,B.S.Joo,S.H.Han,
K.S.Yoo,Y.H.Yoo,Arsenic trioxide-induced apoptosis in TM4sertoli cells:The potential involvement of p21expression and p53phosphorylation,Toxicology 285(3)(2011)142–151.
[10]S.K.Kim,Y.D.Yoon,Y.S.Park,J.T.Seo,J.H.Kim,Involvement of the Fas–Fas
ligand system and active caspase-3in abnormal apoptosis in human testes with maturation arrest and sertoli cell-only syndrome,Fertility and Sterility 87(3)(2007)547–553.
[11]G.Dirami,N.Ravindranath,H.Kleinman,M.Dym,Evidence that basement
membrane prevents apoptosis of sertoli cells in vitro in the absence of known regulators of sertoli cell function,Endocrinology136(10)(1995)4439–4447.
[12]J.Tesarik,M.Guido,C.Mendoza,E.Greco,Human spermatogenesis in vitro:
respective effects of follicle-stimulating hormone and testosterone on meiosis, spermiogenesis,and sertoli cell apoptosis,Journal of Clinical Endocrinology and Metabolism83(12)(1998)4467–4473.
[13]V.Pentik?inen,K.Erkkil?,L.Suomalainen,M.Parvinen,L.Dunkel,Estradiol,
Acts as a germ cell survival factor in the human testis in vitro,Journal of Clinical Endocrinology and Metabolism85(5)(2000)2057–2067.
[14]https://www.wendangku.net/doc/e818364961.html,urentino,J.Gonc?alves,J.E.Cavaco,P.F.Oliveira,M.G.Alves,M.Sousa,A.
Barros,S.Socorro,Apoptosis-inhibitor Aven is downregulated in defective sper-matogenesis and a novel estrogen target gene in mammalian testis,Fertility and Sterility96(3)(2011)745–750.
[15]J.Skommer,D.Wlodkowic,A.Deptala,Larger than life:mitochondria and the
Bcl-2family,Leukemia Research31(2006)277–286.
[16]S.Elmore,Apoptosis:a review of programmed cell death,Toxicologic Pathology
35(4)(2007)495–516.
[17]P.Saikumar,Z.Dong,V.Mikhailov,M.Denton,J.M.Weimberg,M.A.Venkatacha-
lam,Apoptosis:de?nition,mechanisms,and relevance to disease,American Journal of Medicine107(5)(1999)489–506.
[18]B.Fadeel,S.Orrenius,Apoptosis:a basic biological phenomenon with wide-
ranging implications in human disease,Journal of Internal Medicine258(6) (2005)479–517.
[19]A.Jourdain,J.C.Martinou,Mitochondrial outer-membrane permeabilization
and remodelling in apoptosis,International Journal of Biochemistry and Cell Biology41(10)(2009)1884–1889.
[20]R.Zhang,Y.He,X.Zhang,B.Xing,Y.Sheng,H.Lu,Z.Wei,Estrogen receptor-
regulated microRNAs contribute to the Bcl2/Bax imbalance in endometrial adenocarcinoma and precancerous lesions,Cancer Letters314(2)(2011) 155–165.
[21]M.H.Harris,C.B.Thompson,The role of the Bcl-2family in the regulation of
outer mitochondrial membrane permeability,Cell Death and Differentiation7
(12)(2000)1182–1191.
[22]C.Artus,H.Boujrad,A.Bouharrour,M.N.Brunelle,S.Hoos,V.J.Yuste,P.Lenor-
mand,J.C.Rousselle,A.Namane,P.England,AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX, EMBO Journal29(9)(2010)1585–1599.
[23]S.S.Du Plessis,S.Cabler,D.A.McAlister,E.Sabanegh,A.Agarwal,The effect of
obesity on sperm disorders and male infertility,Nature Reviews Urology7(3) (2010)153–161.
[24]D.A.Paduch,A.Bolyakov,P.Cohen,A.Travis,Reproduction in men with Kline-
felter syndrome:the past,the present,and the future,Seminars in Reproductive Medicine27(2)(2009)137–148.
[25]P.F.Oliveira,M.G.Alves,L.Rato,J.Silva,R.Sa,A.Barros,M.Sousa,R.A.Carvalho,
J.E.Cavaco,S.Socorro,In?uence of5alpha-dihydrotestosterone and17beta-estradiol on human sertoli cells metabolism,International Journal of Andrology 34(6Pt2)(2011)e612–e620.
[26]L.Rato,M.G.Alves,S.Socorro,R.A.Carvalho,J.E.Cavaco,P.F.Oliveira,Metabolic
modulation induced by oestradiol and DHT in immature rat sertoli cells cul-tured in vitro,Bioscience Reports32(1)(2012)61–69.
[27]B.S.Majors,M.J.Betenbaugh,G.G.Chiang,Links between metabolism and
apoptosis in mammalian cells:applications for anti-apoptosis engineering, Metabolic Engineering9(4)(2007)317–326.
[28]S.Meroni,M.Riera,E.Pellizzari,S.Cigorraga,Regulation of rat sertoli cell func-
tion by FSH:possible role of phosphatidylinositol3-kinase/protein kinase B pathway,Journal of Endocrinology174(2)(2002)195–204.
[29]P.F.Oliveira,M.G.Alves,L.Rato,https://www.wendangku.net/doc/e818364961.html,urentino,J.Silva,R.Sa,A.Barros,M.Sousa,
R.A.Carvalho,J.E.Cavaco,S.Socorro,Effect of insulin deprivation on metabolism and metabolism-associated gene transcript levels of in vitro cultured human sertoli cells,Biochimica et Biophysica Acta1820(2)(2012)84–89.
[30]K.Steger,R.Rey,S.Kliesch,F.Louis,G.Schleicher,M.Bergmann,Immunohis-
tochemical detection of immature sertoli cell markers in testicular tissue of infertile adult men:a preliminary study,International Journal of Andrology19
(2)(1996)122–128.[31]V.B.Mahesh,T.G.Muldoon,J.C.Eldridge,K.S.Korach,The role of steroid
hormones in the regulation of gonadotropin secretion,Journal of Steroid Bio-chemistry6(6)(1975)1025–1036.
[32]T.T.Turner,C.E.Jones,S.S.Howards,L.L.Ewing,B.Zegeye,G.L.Gunsalus,On the
androgen microenvironment of maturing spermatozoa,Endocrinology115(5) (1984)1925–1932.
[33]B.P.Setchell,Hormones:what the testis really sees,Reproduction,Fertility,and
Development16(5)(2004)535–545.
[34]W.Strober,Appendix3B Trypan Blue Exclusion Test of cell viability,Current
Protocols in Immunology(2001).
[35]M.G.Alves,N.G.Machado,V.A.Sardao,R.A.Carvalho,P.J.Oliveira,Anti-
apoptotic protection afforded by cardioplegic celsior and histidine buffer solutions to hearts subjected to ischemia and ischemia/reperfusion,Journal of Cellular Biochemistry112(12)(2011)3872–3881.
[36]D.Speidel,Transcription-independent p53apoptosis:an alternative route to
death,Trends in Cell Biology20(1)(2010)14–24.
[37]H.Zou,R.Yang,J.Hao,J.Wang,C.Sun,S.W.Fesik,J.C.Wu,K.J.Tomaselli,R.C.
Armstrong,Regulation of the Apaf-1/caspase-9apoptosome by caspase-3and XIAP,Journal of Biological Chemistry278(10)(2003)8091–8098.
[38]D.Twiddy,K.Cain,Caspase-9cleavage,do you need it?Biochemical Journal
405(1)(2007)e1–e2.
[39]C.Cande,N.Vahsen,D.Metivier,H.Tourriere,K.Chebli,C.Garrido,J.Tazi,
G.Kroemer,Regulation of cytoplasmic stress granules by apoptosis-inducing
factor,Journal of Cell Science117(Pt19)(2004)4461–4468.
[40]P.R.Walker,M.Sikorska,New aspects of the mechanism of DNA fragmentation
in apoptosis,Biochemistry and Cell Biology75(4)(1997)287–299.
[41]N.So?kitis,N.Giotitsas,P.Tsounapi, D.Baltogiannis, D.Giannakis,N.
Pardalidis,Hormonal regulation of spermatogenesis and spermiogenesis, Journal of Steroid Biochemistry and Molecular Biology109(3)(2008) 323–330.
[42]M.D.Griswold,The central role of sertoli cells in spermatogenesis,Seminars in
Cell and Developmental Biology9(4)(1998)411–416.
[43]W.H.Walker,Non-classical actions of testosterone and spermatogenesis,Philo-
sophical Transactions of the Royal Society of London,Series B:Biological Sciences365(1546)(2010)1557–1569.
[44]S.Carreau,D.Silandre,C.Bois,H.Bouraima,I.Galeraud-Denis,C.Delalande,
Estrogens:a new player in spermatogenesis,Folia Histochemica et Cytobiolog-ica45(Suppl.1)(2007)4–5.
[45]J.E.B.Cavaco,https://www.wendangku.net/doc/e818364961.html,urentino,A.Barros,M.Sousa,S.Socorro,Estrogen,Receptors
alfa and beta in human testis:both isoforms are expressed,Systems Biology in Reproductive Medicine55(0)(2009)137–144.
[46]G.Verhoeven,J.Cailleau,Follicle-stimulating hormone and androgens increase
the concentration of the androgen receptor in sertoli cells,Endocrinology122
(4)(1988)1541–1550.
[47]L.Rato,M.G.Alves,S.Socorro,A.I.Duarte,J.E.Cavaco,P.F.Oliveira,Metabolic
regulation is important for spermatogenesis,Nature Reviews Urology9(2012) 330–338.
[48]M.G.Alves,L.Rato,R.A.Carvalho,P.I.Moreira,S.Socorro,P.F.Oliveira,Hormonal
control of sertoli cell metabolism regulates spermatogenesis,Cellular and Molecular Life Sciences(2012),https://www.wendangku.net/doc/e818364961.html,/10.1007/s00018-012-1079-1.
[49]M.Bialek,P.Zaremba,K.K.Borowicz,S.J.Czuczwar,Neuroprotective role of
testosterone in the nervous system,Polish Journal of Pharmacology56(2004) 509–518.
[50]K.Erkkil?,K.Henriksén,V.Hirvonen,S.Rannikko,J.Salo,M.Parvinen,L.
Dunkel,Testosterone regulates apoptosis in adult human seminiferous tubules in vitro,Journal of Clinical Endocrinology and Metabolism82(7)(1997) 2314–2321.
[51]T.Pelzer,M.Schumann,M.Neumann,T.deJager,M.Stimpel,E.Ser?ing,
L.Neyses,17[beta]-Estradiol prevents programmed cell death in cardiac myocytes,Biochemical and Biophysical Research Communications268(1) (2000)192–200.
[52]M.G.Alves,S.Socorro,J.Silva,A.Barros,M.Sousa,J.E.Cavaco,P.F.Oliveira,
In vitro cultured human sertoli cells secrete high amounts of acetate that is stimulated by17?-estradiol and suppressed by insulin deprivation,Biochimica et Biophysica Acta1823(8)(2012)1389–1394.
[53]K.Moley,M.Mueckler,Glucose transport and apoptosis,Apoptosis5(2)(2000)
99–105.
[54]D.Schwartz,V.Rotter,p53-Dependent cell cycle control:response to genotoxic
stress,Seminars in Cancer Biology8(5)(1998)325–336.
[55]O.Terrones, B.Antonsson,H.Yamaguchi,H.G.Wang,J.Liu,R.M.Lee, A.
Herrmann,G.Basanez,Lipidic pore formation by the concerted action of proapoptotic Bax and tBID,Journal of Biological Chemistry279(29)(2004) 30081–30091.
[56]J.Tesarik,F.Martinez,L.Rienzi,M.Iacobelli,F.Ubaldi,C.Mendoza,E.Greco,
In-vitro effects of FSH and testosterone withdrawal on caspase activation and DNA fragmentation in different cell types of human seminiferous epithelium, Human Reproduction17(7)(2002)1811–1819.
[57]C.Royer,T.F.Lucas,https://www.wendangku.net/doc/e818364961.html,zari,C.S.Porto,17?-Estradiol signaling and regula-
tion of proliferation and apoptosis of rat sertoli cells,Biology of Reproduction 86(4)(2012)1–13.
[58]I.Schmitz,S.Kirchhoff,P.H.Krammer,Regulation of death receptor-mediated
apoptosis pathways,International Journal of Biochemistry and Cell Biology32 (11–12)(2000)1123–1136.
[59]K.M.Boatright,G.S.Salvesen,Mechanisms of caspase activation,Current Opin-
ion in Cell Biology15(6)(2003)725–731.
V.L.Sim?es et al./Journal of Steroid Biochemistry&Molecular Biology135 (2013) 15–2323
[60]D.Le Goff,C.Viville,S.Carreau,Apoptotic effects of25-hydroxycholesterol
in immature rat sertoli cells:prevention by17beta-estradiol,Reproductive Toxicology21(3)(2006)329–334.
[61]T.F.G.Lucas,C.Royer,E.R.Siu,https://www.wendangku.net/doc/e818364961.html,zari,C.S.Porto,Expression and signaling
of G protein-coupled estrogen receptor1(GPER)in rat sertoli cells,Biology of Reproduction83(2)(2010)307–317.
[62]I.Petrache,T.R.Medler,A.T.Richter,K.Kamocki,U.Chukwueke,L.Zhen,Y.Gu,J.
Adamowicz,K.S.Schweitzer,W.C.Hubbard,E.V.Berdyshev,G.Lungarella,R.M.
Tuder,Superoxide dismutase protects against apoptosis and alveolar enlarge-ment induced by ceramide,American Journal of Physiology-Lung Cellular and Molecular Physiology295(1)(2008)L44–L53.
[63]C.Cande,I.Cohen, E.Daugas,L.Ravagnan,https://www.wendangku.net/doc/e818364961.html,rochette,N.Zamzami,
G.Kroemer,Apoptosis-inducing factor(AIF):a novel caspase-independent
death effector released from mitochondria,Biochimie84(2–3)(2002) 215–222.
LED数码管的识别与检测方法使用常识 LED数码管也称半导体数码管，它是将若干发光二极管按一定图形排列并封装在一起的最常用的数码显示器件之一。LED数码管具有发光显示清晰、响应速度快、耗电省、体积小、寿命长、耐冲击、易与各种驱动电路连接等优点，在各种数显仪器仪表、数字控制设备中得到广泛应用。 LED数码管种类很多，品种五花八门，这里仅向初学者介绍最常用的小型“8”字形LED数码管的识别与使用方法。 如何识别LED数码管 1.结构及特点 目前，常用的小型LED数码管多为“8”字形数码管，它内部由8个发光二极管组成，其中7个发光二极管（a～g）作为7段笔画组成“8”字结构（故也称7 段LED数码管），剩下的1个发光二极管（h或dp）组成小数点，如图1（a）所示。各发光二极管按照共阴极或共阳极的方法连接，即把所有发光二极管的负极（阴极）或正极（阳极）连接在一起，作为公共引脚；而每个发光二极管对应的正极或者负极分别作为独立引脚（称“笔段电极”），其引脚名称分别与图 1（a）中的发光二极管相对应，即a、b、c、d、e、f、g脚及h脚（小数点），如图1（b）所示。若按规定使某些笔段上的发光二极管发光，就能够显示出图1（c）所示的“0～9”10个数字和“A～F”6个字母，还能够显示小数点，可用于2进制、10进制以及16进制数字的显示，使用非常广泛。
（a）结构图
（b）电路图
（c）显示符 常用小型LED数码管是以印制电路板为基板焊固发光二极管，并装入带有显示窗口的塑料外壳，最后在底部引脚面用环氧树脂封装而成。由于LED数码管的笔段是由发光二极管组成的，所以其特性与发光二极管相同。LED数码管的主要特点：能在低电压、小电流条件下驱动发光，并能与CMOS、TTL电路兼容；它不仅发光响应时间极短（＜0.1μs）、高频特性好、单色性好、亮度高，而且体积小、重量轻、抗冲击性能好、使用寿命长（一般在10万小时以上，最高可达 100万小时）、成本低。 2.外形和种类 常用小型LED数码管的封装形式几乎全部采用了双列直插结构，并按照需要将1至多个“8”字形字符封装在一起，以组成显示位数不同的数码管。如果按照显示位数（即全部数字字符个数）划分，有1位、2位、3位、4位、5位、6位……数码管，如图2所示。如果按照内部发光二极管连接方式不同划分，有共阴极数码管和共阳极数码管两种；按字符颜色不同划分，有红色、绿色、黄色、橙色、蓝色、白色等数码管；按显示亮度不同划分，有普通亮度数码管和高亮度数码管；按显示字形不同，可分为数字管和符号管。
1.暗影步+闷棍 这个宏需要跃出近战范围，但它能有效地在猎人用闪光弹照出你之前把他闷棍，也能在目标移动的太远，超出范围之前闷了他。 #showtooltip /cast 暗影步 /cast 闷棍 2.暗影步+肾击 对于使用肾击，这个宏非常有效。为了使用这个宏，你得先跑出近战范围或者背对你的目标，以免在使用暗影步之前使用肾击 #showtooltip 肾击 /cast 暗影步 /cast 肾击 3.暗影之舞+匕首+伏击 注意：宏中你的匕首名称需改成你自己使用的匕首名称，才可正常使用！当你在暗影之舞时，装备匕首使用能够使用伏击。也可以使用其他起手/潜行技能，因为它们并不基于副手武器伤害。将暗影之舞放到你的第二动作条上。 #showtooltip 暗影之舞 /equipslot 16 你的匕首名称 /cast 暗影之舞 /swapactionbar 2 4.暗影之舞+匕首+伏击—换回原来的装备 注意：宏中你的武器名称需改成你自己使用的武器名称，才可正常使用！在暗影之舞结束后，用这个宏在你使用出血之前换回你的慢速主手武器 #showtooltip 出血(等级 4) /equipslot 16 你的武器名称 /cast 出血(等级 4) /startattack 5.出血+自动攻击 这个宏的意义在于，如果能量不足，将会自动开始自动攻击，而不是使用其他技能 #showtooltip 出血 /startattack /cast 出血 6.冷血剔骨 一键冷血剔骨 /cast 冷血 /cast 刺骨 7.闷棍+平砍 闷棍同时平砍目标，而且不会将对方打醒。因为会使你脱离潜行状态，所以你需要额外的10
内部的四个数码管共用a~dp这8根数据线，为人们的使用提供了方便，因为里面有四个数码管，所以它有四个公共端，加上a~dp，共有12个引脚，下面便是一个共阴的四位数码管的内部结构图（共阳的与之相反）。引脚排列依然是从左下角的那个脚（1脚）开始，以逆时针方向依次为1~12脚，下图中的数字与之一一对应。 数码管使用条件： a、段及小数点上加限流电阻 b、使用电压：段：根据发光颜色决定；小数点：根据发光颜色决定 c、使用电流：静态：总电流 80mA（每段 10mA）；动态：平均电流 4-5mA 峰值电流 100mA
上面这个只是七段数码管引脚图，其中共阳极数码管引脚图和共阴极的是一样的，4位数码管引脚图请在本站搜索我也提供了数码管使用注意事项说明： （１）数码管表面不要用手触摸，不要用手去弄引角； （２）焊接温度：２６０度；焊接时间：５Ｓ （３）表面有保护膜的产品,可以在使用前撕下来。 数码管测试方法与数字显示译码表
ARK SM410501K SM420501K 数码管引脚图判断 数码管识别 ARK SM410501K 共阳极数码管 ARK SM420501K 共阴极数码管 到百度搜索下，这两种数码管只有销售商，并无引脚图。 对于判断引脚，对于老手来说，很简单，可是对于新手来讲，这是件很难的事情，因为共阴、 共阳表示的含义可能还不太懂 ZG工作室只是将该数码管的引脚图给出，并让大家一起分享。 注：SM410501K 和SM420501K 的引脚排列是一模一样的。 这张图很明确给出该数码管的引脚排列。 数字一面朝向自己，小数点在下。左下方第一个引脚为1、右下方第二个引脚为5，右上方第一个引脚为6。见图所示。 其中PROTEL图中K 表示共阴、A表示共阳。 能显示字符的LED数码管(三) 常用LED数码管的引脚排列图和内部电路图 CPS05011AR(1位共阴/红色 0.5英寸)、SM420501K(红色 0.5英寸)、 SM620501(蓝色0.5英寸)、SM820501(绿色0.5英寸)
DHT11温湿度传感器与单片机之间的通信 一DHT11的简介： 1 接口说明 建议连接线长度短于20米时用5K上拉电阻,大于20米时根据实际情况使 用合适的上拉电阻 2数据帧的描述 DATA 用于微处理器与DHT11之间的通讯和同步,采用单总线数据格式,一次通讯时间4ms左右,数据分小数部分和整数部分,具体格式在下面说明,当前小数部分用于以后扩展,现读出为零.操作流程如下: 一次完整的数据传输为40bit,高位先出。 数据格式:8bit湿度整数数据+8bit湿度小数数据 +8bi温度整数数据+8bit温度小数数据 +8bit校验和 数据传送正确时校验和数据等于“8bit湿度整数数据+8bit湿度小数数据+8bi 温度整数数据+8bit温度小数数据”所得结果的末8位。 3时序描述 用户MCU发送一次开始信号后,DHT11从低功耗模式转换到高速模式,等待主机开始信号结束后,DHT11发送响应信号,送出40bit的数据,并触发一次信号采集,用户可选择读取部分数据.从模式下,DHT11接收到开始信号触发一次温湿度采集,如果没有接收到主机发送开始信号,DHT11不会主动进行温湿度采集.采集数据后转换到低速模式。 1.通讯过程如图1所示
图1 总线空闲状态为高电平,主机把总线拉低等待DHT11响应,主机把总线拉低必须大于18毫秒,保证DHT11能检测到起始信号。DHT11接收到主机的开始信号后,等待主机开始信号结束,然后发送80us低电平响应信号.主机发送开始信号结束后,延时等待20-40us后, 读取DHT11的响应信号,主机发送开始信号后,可以切换到输入模式,或者输出高电平均可, 总线由上拉电阻拉高。 图2 总线为低电平,说明DHT11发送响应信号,DHT11发送响应信号后,再把总线拉高80us,准备发送数据,每一bit数据都以50us低电平时隙开始,高电平的长短定了数据位是0还是1.格式见下面图示.如果读取响应信号为高电平,则DHT11没有响应,请检查线路是否连接正常.当最后一bit数据传送完毕后，DHT11拉低总线50us,随后总线由上拉电阻拉高进入空闲状态。 数字0信号表示方法如图4所示
玩家分享 WOW7.0火法爆发宏命令 第一次发帖，属性比较差50%暴都没有，感觉如果暴击高的话伤害会更高，打的是职业大厅里的木桩，伤害不高 天赋方面没有点反斩杀，因为这个宏直接起手就用，不用先攒顺发，之前用的起手爆发宏是先攒炎爆的。和攒好炎爆的爆发宏比少了一个炎爆，好处就是在平缓期可以打更多的炎爆，特别是暴击高了之后。动荡魔法换成活动炸弹感觉差距不大，AOE场合肯定炸弹更好。 再说说使用方面，需要2层火冲，3层凤凰，飞舞，法阵，燃烧全部可用。这个宏不止是起 手用，只要这些技能CD好了都可以用，所以再起手打完之后最多放1次法阵和飞舞，火冲看着3 层凤凰CD用，都准备好了可以再用一次来爆发。 也就是说爆发宏打完到下一次爆发期间一次凤凰都不用的。因为我点了凤凰减CD的大 特质，CD还是跟的上的。 一点见解清各位轻喷，指教。 这个宏只是为了省事，在技能CD都好的时候可以打一波爆发。平缓期也只是打火球攒小括 号然后火冲出大括号打瞬发炎爆。或者第一层天赋特效出的时候直接打瞬发炎爆。也许不是最高的输出方式，但也不会差太多。但省事很多，输出方式不会太复杂。在打本的时候有精力处理更多的事情。再说一次只是为了省事。 下面是宏 #showtooltip 燃烧 /castsequence reset=10 能量符文,燃烧,火焰冲击,凤凰烈焰,炎爆术,火焰冲击,炎爆术,烈焰 飞舞,火焰冲击,炎爆术,火焰冲击,炎爆术,凤凰烈焰,炎爆术,凤凰烈焰,炎爆术,火球术再一个是起手要攒顺发炎爆的宏 #showtooltip 燃烧 /castsequence reset=10 能量符文,燃烧,炎爆术,火焰冲击,炎爆术,火焰冲击,炎爆术,烈焰飞舞,火焰冲击,炎爆术,火焰冲击,炎爆术,凤凰烈焰,炎爆术,凤凰烈焰,炎爆术,火球术 百度攻略&265G 提供，更多精彩攻略访问https://www.wendangku.net/doc/e818364961.html, 1
LED数码管的结构及工作原理 LED数码管（LED Segment Displays）是由多个发光二极管封装在一起组成“8”字型的器件，引线已在内部连接完成，只需引出它们的各个笔划，公共电极。LED数码管常用段数一般为7段有的另加一个小数点，还有一种是类似于3位“+1”型。位数有半位，1，2，3，4，5，6，8，10位等等....，LED数码管根据LED的接法不同分为共阴和共阳两类，了解LED的这些特性，对编程是很重要的，因为不同类型的数码管，除了它们的硬件电路有差异外，编程方法也是不同的。图2是共阴和共阳极数码管的内部电路，它们的发光原理是一样的，只是它们的电源极性不同而已。颜色有红，绿，蓝，黄等几种。LED数码管广泛用于仪表，时钟，车站，家电等场合。选用时要注意产品尺寸颜色，功耗，亮度，波长等。下面将介绍常用LED数码管内部引脚图。 图1 这是一个7段两位带小数点10引脚的LED数码管 图2 引脚定义
每一笔划都是对应一个字母表示 DP 是小数点. 数码管分为共阳极的LED 数码管、共阴极的LED 数码管两种。下图例举的是共阳极的LED 数码管，共阳就是7段的显示字码共用一个电源的正。led 数码管原理图示意： 图3 引脚示意图 从上图可以看出，要是数码管显示数字，有两个条件：1、是要在VT 端（3/8脚）加正电源；2、要使（a,b,c,d,e,f,g,dp)端接低电平或“0”电平。这样才能显示的。 共阳极LED 数码管的内部结构原理图图4： 图4 共阳极LED 数码管的内部结构原理图 a b c d e f g dp
共阴极LED数码管的内部结构原理图： a b c d e f g dp 图5 共阴极LED数码管的内部结构原理图 表1.1 显示数字对应的二进制电平信号 LED数码管要正常显示，就要用驱动电路来驱动数码管的各个段码，从而显示出我们要的数位，因此根据LED数码管的驱动方式的不同，可以分为静态式和动态式两类。 A、静态显示驱动：
本教程没有收录所有的宏，意在教会新手制作属于自己的宏，得到更多游戏的快乐。 /cast [<第一组条件选项>] <第一个法术名称>; [<第二组条件选项>] <第二个法术名称>; [<第三组条件选项>] <第三个法术名称>;...” /castsequence [<条件选项>] reset=<#>/target/combat <法术1>, <法术2>, <法术3> 你可以在任何条件选项前加上“no”来得到反效果，比如，“nocombat”则在脱离战斗的情况下成立。 用逗号“,”来分隔条件选项作用和“and”一样，当条件选项同时成立时执行。 用斜杠“/”来分隔条件选项作用和“or”一样，当其中一个条件选项成立时执行。 2.0以前宏举例。 1、小D判断连击点数释放技能，也适用于盗贼： /script if ( GetComboPoints() >= 3 ) then CastSpellByName("凶猛撕咬(等 级 3)"); else CastSpellByName("爪击(等级 4)") end 2、常用的密语格式 /script SendChatMessage(“主动给钱或是由我抢劫二选一”,” say”,”通用语”,” YELL”) "SAY"：普通说话2."WHISPER"：密语 1."GUILD"：工会 2."PARTY"：小队 3."RAID"：组团 4."YELL"：大喊 /script UIErrorsFrame:Clear()可以隐藏并清除提示。Clear也可改为Hide。 3、如果目标生命大于20，释放抽取生命法术，否则使用灵魂抽取。 /script if (UnitHealth("target")>20) then CastSpellByName("抽取生命") else CastSpellByName("灵魂抽取") end 4、使用奥暴，当法力值不足400，用法力红宝石补充并提醒队友 /script if (UnitMana("player")>400) then CastSpellByName("魔爆术(等 级 6)") else UseContainerItem(4, 1);SendChatMessage(“魔法将要耗尽，大家小心！”,”yell”); end 2.0可用的条件选项有：[……] help - 检测目标是否为友善 harm - 检测目标是否为敌对 combat - 检测你是否在战斗中 stance或stance:0/1/2../n检测你是否在姿态中，或是否在某个特定的姿态中 stealth - 检测你是否潜行 equipped: - 检测某个物品是否被装备。可以是任何有效的装备槽，物品分类，或者物品子类target =player/pet/targettarget/Unit 它把当前目标改变为任何有效的单位 pet: <宠物名称或类型> 玩家当前宠物为某宠物Voidwalker，Boar，Imp，Wolf，pet为所有宠物类型 actionbar:1/…./6检测当前动作条是否为列出的那个 button:1/…/5/<虚拟按键号>检测某个特定的按钮被用来触发法术，默认为1即左键点击，2为右键点击，3为鼠标中间点击，4，5为鼠标特殊按键点击。 modifier或modifier:shift/ctrl/alt - 检测命令被执行时是否某个特定的键被按下,可以简写为mod以节省字节。 pet: - 检测宠物是否存在。可以接受宠物类型（枭，熊，小鬼）或者名字（Fluffy，我家坏坏）作为条件。不带条件则检测是否有任何宠物存在。 mounted，swimming，flying，flyable- 检测是否在坐骑上，游泳，或者飞行中，能够飞行状态 indoors，outdoors - 检测是在室内还是室外 exists 和dead - 检测是否目标已经死亡，或是否真的存在
L E D数码管结构及工作原理-标准化文件发布号：（9556-EUATWK-MWUB-WUNN-INNUL-DDQTY-KII
LED数码管的结构及工作原理 沈红卫 LED数码管（LED Segment Displays）是由多个发光二极管封装在一起组成“8”字型的器件，引线已在内部连接完成，只需引出它们的各个笔划，公共电极。LED数码管常用段数一般为7段有的另加一个小数点，还有一种是类似于3位“+1”型。位数有半位，1，2，3，4，5，6，8，10位等等....，LED数码管根据LED的接法不同分为共阴和共阳两类，了解LED的这些特性，对编程是很重要的，因为不同类型的数码管，除了它们的硬件电路有差异外，编程方法也是不同的。图2是共阴和共阳极数码管的内部电路，它们的发光原理是一样的，只是它们的电源极性不同而已。颜色有红，绿，蓝，黄等几种。LED数码管广泛用于仪表，时钟，车站，家电等场合。选用时要注意产品尺寸颜色，功耗，亮度，波长等。下面将介绍常用LED数码管内部引脚图。 图1 这是一个7段两位带小数点 10引脚的LED数码管 图2 引脚定义 每一笔划都是对应一个字母表示 DP是小数点. 数码管分为共阳极的LED数码管、共阴极的LED数码管两种。下图例举的是共阳极的LED数码管，共阳就是7段的显示字码共用一个电源的正。led 数码管原理图示意：
图3 引脚示意图 从上图可以看出，要是数码管显示数字，有两个条件：1、是要在VT端（3/8脚）加正电源；2、要使（a,b,c,d,e,f,g,dp)端接低电平或“0”电平。这样才能显示的。 共阳极LED数码管的内部结构原理图图4： 图4 共阳极LED数码管的内部结构原理图共阴极LED数码管的内部结构原理图： 图5 共阴极LED数码管的内部结构原理图
基于单片机的DHT11温湿度 传感器设计 姓名：史延林 指导老师：黄智伟 学院：电气工程学院 学号：20094470321 摘要： 温湿度是生活生产中的重要的参数。本设计为基于单片机的温湿度检测与控制系统，采用模块化、层次化设计。用新型的智能温湿度传感器DHT11主要实现对温度、湿度的检测，将温度湿度信号通过传感器进行信号的采集并转换成数字信号，再运用单片机STC89C52进行数据的分析和处理，为显示和报警电路提供信号，实现对温
湿度的控制报警。报警系统根据设定报警的上下限值实现报警功能，显示部分采用LCD1602液晶显示所测温湿度值。系统电路简单、集成度高、工作稳定、调试方便、检测精度高，具有一定的实用价值。 关键词：单片机；DHT11温湿度传感器； LCD1602显示 第一章：课程构思 1.1课题背景 温湿度的检测与控制是工业生产过程中比较典型的应用之一，随着传感器在生产和生活中的更加广泛的应用。在生产中，温湿度的高低对产品的质量影响很大。由于温湿度的检测控制不当，可能使我们导致无法估计的经济损失。为保证日常工作的顺利进行，首要问题是加强生产车间内温度与湿度的监测工作，但传统的方法过于粗糙，通过人工进行检测，对不符合温度和湿度要求的库房进行通风、去湿和降温等工作。这种人工测试方法费时费力、效率低，且测试的温度及湿度误差大，随机性大。目前，在低温条件下(通常指100℃以下)，温湿度的测量已经相对成熟。利用新型单总线式数字温度传感器实现对温度的测试与控制得到更快的开发。但人们对它的要求越来越高，要为现代人工作、科研、学习、生活提供更好的更方便的设施就需要从数字单片机技术入手，一切向着数字化，智能化控制方向发展。 对于国内外对温湿度检测的研究，从复杂模拟量检测到现在的数字智能化检测越发的成熟，随着科技的进步，现在的对于温湿度研究，检测系统向着智能化、小型化、低功耗的方向发展。在发展过程中，以单片机为核心的温湿度控制系统发展为体积小、操作简单、量程宽、性能稳定、测量精度高，等诸多优点在生产生活的各个方面实现着至关重要的作用。 温湿度传感器除电阻式、电容式湿敏元件之外，还有电解质离子型湿敏元件、重量型湿敏元件（利用感湿膜重量的变化来改变振荡频率）、光强型湿敏元件、声表面波湿敏元件等。湿敏元件的线性度及抗污染性差，在检测环境湿度时，湿敏元件要长期暴露在待测环境中，很容易被污染而影响其测量精度及长期稳定性。1.2主要内容
LED数码管及引脚图资料LED数码管实际上是由七个发光管组成8字形构成的，加上小数点就是8个。这些段分别由字母a,b,c,d,e,f,g,dp来表示。当数码管特定的段加上电压后，这些特定的段就会发亮，以形成我们眼睛看到的 2个8数码管字样了。如：显示一个“2”字，那么应当是a亮b亮g亮e亮d亮f不亮c不亮dp不亮。LED数码管有一般亮和超亮等不同之分，也有0.5寸、1寸等不同的尺寸。小尺寸数码管的显示笔画常用一个发光二极管组成，而大尺寸的数码管由二个或多个发光二极管组成，一般情况下，单个发光二极管的管压降为1.8V左右，电流不超过30mA。发光二极管的阳极连接到一起连接到电源正极的称为共阳数码管，发光二极管的阴极连接到一起连接到电源负极的称为共阴数码管。常用LED数码管显示的数字和字符是0、1、2、3、4、5、6、7、8、9、A、B、C、D、E、F。 led数码管（LED Segment Displays）是由多个发光二极管封装在一起组成“8”字型的器件，引线已在内部连接完成，只需引出它们的各个笔划，公共电极。led数码管常用段数一般为7段有的另加一个小数点，还有一种是类似于3位“+1”型。位数有半位，1，2，3，4，5，6，8，10位等等....，led数码管根据LED的接法不同分为共阴和共阳两类，了解LED的这些特性，对编程是很重要的，因为不同类型的数码管，除了它们的硬件电路有差异外，编程方法也是不同的。图2是共阴和共阳极数码管的内部电路，它们的发光原理是一样的，只是它们的电源极性不同而已。颜色有红，绿，蓝，黄等几种。led数码管广泛用于仪表，时钟，车站，家电等场合。选用时要注意产品尺寸颜色，功耗，亮度，波长等。下面将介绍常用LED数码管内部引脚图片 10引脚的LED数码管 图1 这是一个7段两位带小数点 10引脚的LED数码管
数字温湿度传感器 DHT11 ?相对湿度和温度测量 ?全部校准，数字输出 ?卓越的长期稳定性 ?无需额外部件 ?超长的信号传输距离 ?超低能耗 ?4 引脚安装 ?完全互换 DHT11产品概述 DHT11数字温湿度传感器是一款含有已校准数字信号输出的温湿度复合 传感器。它应用专用的数字模块采集技术和温湿度传感技术，确保产品具有极 高的可靠性与卓越的长期稳定性。传感器包括一个电阻式感湿元件和一个NTC 测温元件，并与一个高性能8位单片机相连接。因此该产品具有品质卓越、超 快响应、抗干扰能力强、性价比极高等优点。每个DHT11传感器都在极为精确 的湿度校验室中进行校准。校准系数以程序的形式储存在OTP内存中，传感器 内部在检测信号的处理过程中要调用这些校准系数。单线制串行接口，使系统 集成变得简易快捷。超小的体积、极低的功耗，信号传输距离可达20米以上， 使其成为各类应用甚至最为苛刻的应用场合的最佳选则。产品为 4 针单排引 脚封装。连接方便，特殊封装形式可根据用户需求而提供。 应用领域 ?暖通空调?测试及检测设备 ?汽车?数据记录器 ?消费品?自动控制 ?气象站?家电 ?湿度调节器?医疗 ?除湿器 订货信息 型号测量范围测湿精度测温精度分辨力封装DHT11 20－90％RH 0－50℃±5％RH ±2℃ 1 4针单排直插
1、传感器性能说明 参数条件Min Typ Max 单位 湿度 分辨率 1 1 1 %RH 16 Bit 重复性±1 %RH 精度25℃±4 %RH 0－50℃±5 %RH 互换性可完全互换 量程范围0℃30 90 %RH 25℃20 90 %RH 50℃20 80 %RH 响应时间1/e(63%)25℃， 6 10 15 S 1m/s 空气 迟滞±1 %RH 长期稳定性典型值±1 %RH/yr 温度 分辨率 1 1 1 ℃ 16 16 16 Bit 重复性±1 ℃ 精度±1 ±2 ℃ 量程范围0 50 ℃ 响应时间1/e(63%) 6 30 S 2、接口说明 建议连接线长度短于20米时用5K上拉电阻,大于20米时根据实际情况使 用合适的上拉电阻 3、电源引脚 DHT11的供电电压为3－5.5V。传感器上电后，要等待1s 以越过不稳定状态在此期间无需发送任何指令。电源引脚（VDD，GND）之间可增加一个100nF 的电容，用以去耦滤波。
魔兽世界猎人国服3.35版常用宏 冰冻陷阱宏，避免刚冻上就被自己打醒，作用是放冰冻陷阱的同时，停止攻击。 #show冰冻陷阱 /consoleSound_EnableSFX0 /stopattack /cast冰冻陷阱 /consoleSound_EnableSFX1 /scriptUIErrorsFrame:Clear() 冰冻陷阱宏，避免刚冻上就被自己打醒，作用是放冰冻陷阱的同时，停止攻击。 #show冰冻陷阱 /console Sound_EnableSFX 0 /stopattack /cast冰冻陷阱 /console Sound_EnableSFX 1 /script UIErrorsFrame:Clear() 杀戮奥术宏,停止所有行动（主要是卡猛禽一击的时候），立刻释放奥术射击，奥术冷却中，则自动开始自动射击。 #show奥术射击 /stopcasting /startattack
/cast急速射击 /castrandom [target=pettarget,exists]杀戮命令 /cast奥术射击 /console Sound_EnableSFX 1 /script UIErrorsFrame:Clear() 威慑灵猴宏，起动威慑的同时切换到灵猴守护。 #show威慑 /console Sound_EnableSFX 0 /castsequence威慑,灵猴守护 /console Sound_EnableSFX 1 /script UIErrorsFrame:Clear() 毒蛇输出宏，同抽筋宏，无缝毒蛇钉刺、稳固、奥术。超过2秒不执行该宏，作用等同毒蛇钉刺。#show毒蛇钉刺 /console Sound_EnableSFX 0 /startattack /cast急速射击 /castrandom [target=pettarget,exists]杀戮命令 /castsequence reset=2/target毒蛇钉刺,稳固射击,!自动射击,稳固射击,!自动射击,稳固射击,奥术射击,稳固射击,!自动射击,稳固射击,!自动射击,稳固射击,奥术射击 /console Sound_EnableSFX 1 /script UIErrorsFrame:Clear()
LED数码管知识介绍 什么是led数码管 LED数码管（LED Segment Displays）是由多个发光二极管封在在一起组成“8”字型的器件，引线已在内部连接完成，只需引出它们的各个笔划，公共电极。LED数码管常用段数一般为7段有的另加一个小数点，还有一种是类似于3位“+1”型。位数有半位，1，2，3，4，5，6，8，10位等等....，LED数码管根据LED 的接法不同分为共阴和共阳两类，了解LED的这些特性，对编程是很重要的，因为不同类型的数码管，除了它们的硬件电路有差异外，编程方法也是不同的。右图是共阴和共阳极数码管的内部电路，它们的发光原理是一样的，只是它们的电源极性不同而已。颜色有红，绿，蓝，黄等几种。LED数码管广泛用于仪表，时钟，车站，家电等场合。选用时要注意产品尺寸颜色，功耗，亮度，波长等。下页将介绍常用LED数码管内部引脚图片 图1 这是一个7段两位带小数点 10引脚的LED数码管 图2 引脚定义每一笔划都是对应一个字母表示 DP是小数点 LED数码管要正常显示，就要用驱动电路来驱动数码管的各个段码，从而显示出我们要的数位，因此根据LED数码管的驱动方式的不同，可以分为静态式和动态式两类。 A、静态显示驱动： 静态驱动也称直流驱动。静态驱动是指每个数码管的每一个段码都由一个单片机的I/O埠进行驱动，或者使用如BCD码二
-十进位解码器解码进行驱动。静态驱动的优点是编程简单，显示亮度高，缺点是占用I/O埠多，如驱动5个数码管静态显示则需要5×8＝40根I/O埠来驱动，要知道一个89S51单片机可用的 I/O埠才32个呢。故实际应用时必须增加解码驱动器进行驱动，增加了硬体电路的复杂性。 B、动态显示驱动： 数码管动态显示介面是单片机中应用最为广泛的一种显示方式之一，动态驱动是将所有数码管的8个显示笔划 "a,b,c,d,e,f,g,dp "的同名端连在一起，另外为每个数码管的公共极COM增加位元选通控制电路，位元选通由各自独立的I/O线控制，当单片机输出字形码时，所有数码管都接收到相同的字形码，但究竟是那个数码管会显示出字形，取决于单片机对位元选通COM端电路的控制，所以我们只要将需要显示的数码管的选通控制打开，该位元就显示出字形，没有选通的数码管就不会亮。 透过分时轮流控制各个LED数码管的COM端，就使各个数码管轮流受控显示，这就是动态驱动。在轮流显示过程中，每位元数码管的点亮时间为1～2ms，由于人的视觉暂留现象及发光二极体的余辉效应，尽管实际上各位数码管并非同时点亮，但只要扫描的速度足够快，给人的印象就是一组稳定的显示资料，不会有闪烁感，动态显示的效果和静态显示是一样的，能够节省大量的I/O埠，而且功耗更低。 恒流驱动与非恒流驱动对LED数码管的影响主要有以下几点： 1、显示效果： 由于LED基本上属于电流敏感元件，其正向压降的分散性很大，并且还与温度有关，为了保证数码管具有良好的亮度均匀度，就需要使其具有恒定的工作电流，且不能受温度及其它因素的影响。另外，当温度变化时驱动晶片还要能够自动调节输出电流的大小以实现色差平衡温度补偿。 2、安全性： 即使是短时间的电流超载也可能对发光管造成永久性的损坏，采用恒流驱动电路后可防止由于电流故障所引起的数码管的大面积损坏。 另外，我们所采用的超大型积体电路还具有级联延时开关特性，可防止反向尖峰电压对发光二极体的损害。超大型积体电路还具有热保护功能，当任何一片的温度超过一定值时可自动关断，并且可在控制室内看到故障显示。 什么数码管亮度不均匀？ 有两个大的因素影响到亮度一致性。一是使用原材料晶片的选取，一是使用数码管时采取的控制方式。 1、原材料--LED晶粒的VF和亮度和波长是一个正态分布， 即使筛选过LED晶粒，VF和亮度和波长已在一个很小的范围了，生产出来的产品还是在一个范围内，结果就是亮度不一致。 2、要保证LED数码管亮度一样，在控制方式选取上也有差别 最好的办法是恒流控制，流过每一个发光二极体的电流都是相同的，这样发光二极体看起来亮度就是一样的了。如恒压控制，则导致VF不相同的发光二极体分到的电流不相同，所以亮度也不同。当然这两个条件是相辅相成的。 怎样测量数码管引脚，分共阴和共阳? 找公共共阴和公共共阳，首先，我们找个电源（3到5伏）和不同规格的电阻，VCC串接个电阻后和GND接在任意2个脚上，组合有很多，但总有一个LED 会发光的找到一个就够了，然后用GND不动，VCC（串电阻）逐个碰剩下的脚，如果有多个LED（一般是8个），那它就是共阴的了。相反用VCC不动，GND 逐个碰剩下的脚，如果有多个LED（一般是8个），那它就是共阳的。也可以直接用数位万用表，红表笔是电源的正极，黑表笔是电源的负极。
WOW7.0狂暴战一键宏分享 狂暴战一键宏 狂暴也是有2套 第一套： #showtoplist /castsequence reset=5 怒击,嗜血,狂暴挥砍,怒击,嗜血,暴怒,怒击,嗜血,狂暴挥砍,怒击,嗜血,暴怒,怒击,嗜血,狂暴挥砍,怒击,嗜血,巨龙怒吼,暴怒 /castsequence reset=10 战吼 /cast 天神下凡 狂暴加点：233333333 这套宏经过测试860装等无橙1亿输出稳定26W秒伤，至于战吼哪里设了个进程宏是方便玩家打到软泥怪或是其他主动伤害饰品的话，可以写在战吼的后面,起手按宏打奥丁有神器的支持后战吼和奥丁的CD差不多同时结束，战吼时打奥丁。其他时候一直按宏就 行了。 第二套是翻页宏 宏一： #showtoplist /castsequence reset=5 怒击,嗜血,狂暴挥砍,怒击,嗜血,狂暴挥砍,怒击,嗜血,狂暴挥砍,怒击,嗜血,狂暴挥砍,怒击,嗜血,巨龙怒吼 /castsequence reset=10 战吼 /cast 天神下凡 /changeactionbar 2 宏二： #showtoplist /castsequence reset=1 暴怒 /changeactionbar 1 这套宏怒气够用时候可以第一时间打出暴怒，但是暴怒过后无法保证第一时间打怒击,出了橙头后感觉第二套要好些 狂暴战一键AOE宏
#showtooltip /castsequence reset=5 旋风斩,旋风斩,嗜血,旋风斩,旋风斩,嗜血,旋风斩,暴怒/castsequence reset=10 战吼 /cast 天神下凡 狂暴战一键翻页斩杀宏 宏一： #showtooltip 斩杀 /startattack /castsequence reset=5 嗜血,怒击 /castsequence reset=10 战吼 /cast 天神下凡 /changeactionbar 2 宏二： #showtooltip /castsequence reset=1 斩杀 /changeactionbar 1 翻页宏比较好，怒气不会溢出 https://www.wendangku.net/doc/e818364961.html,/read.php?&tid=10154339
电子综合设计实训 题目数码管动态显示 _ 姓名 专业 学号 指导教师 郑州科技学院电气工程学院
目录 摘要.................................................................................................. I 1背景. (1) 1.1介绍 (1) 1.2设计步骤 (2) 2 设计思路 (3) 2.1方案对比 (3) 3元件的选择 (6) 3.1单片机 (6) 3.2 显示元器件的选择 (6) 4 设计原理及功能说明 (8) 4.1 各部分功能说明 (8) 5 装配与调试 (14) 5.1装配 (14) 5.2调试 (14) 6 总结 (15) 附录 (17) 附录一：元件清单 (17) 附录二：电路源程序 (17)
数码管动态显示的设计 摘要 本文介绍了一种基于AT89C51单片机的8个数码管滚动显示单个数字的设计，让八位数码管滚动显示0、1、2、3、4、5、6、7，我们以液晶显示技术的发展为背景，选择了比较常用的液晶数码管显示模块，利用了单片机控制数码管模块的显示机理。研究学习AT89C51单片机其功能，对学习过的单片机，C语言课程进行巩固，设计一款在8只数码管上流动显示单个数字的程序，并用PROTEUS进行电路设计和实时仿真。该电路有两部分组成：AT89C51单片机和显示模块组成。AT89C51单片机具有超低功耗和CPU外围的高度整合性；显示模块数码管是由多个发光二极管封装在一起组成“8”字型的器件，引线已在内部连接完成，只需引出它们的各个笔划，公共电极，方便易用。实际应用中不需要外部任何元器件即可实现，具有接口电路简单、可靠，易于编程的特点，抗干扰性好等特点。 单片机技术使我们可以利用软硬件实现数码管准确显示各种数码。而且这种技术相对简单，性价比较高，在我们生活中应用很广泛，具有一定的发展前景。 关键词：AT89C51单片机；数码管；滚动显示
宏命令全表 本文WOWWiki共享维护Subelf译 有些命令需要参数，而部分命令的参数则是可以省略的。比如/join可以接受两个参数，一个是可选的，另一个则是必需的，我们用/join <频道名称> [密码]来表示。也就是说“<>”中的是必需的，“[]”中的则是可选的。 一、基本命令 协助[单位]，也就是选择[单位]的目标。如果没有指定[单位]，当前目标将作为 为自己装备<物品>。 自动跟随[玩家]。如果没有指定[玩家]，当前目标将作为参数。 掷骰子，获得一个[下限]到[上限]之间的随机数。如果只指定了一个参数值，那么范围就是从1到这个指定的值。如果没有给出任何参数，那么范围就是从1到100。如果你在小队或团队中，附近的队员可以看到你得到的随机数，范围和/say
显示服务器时间。 向[玩家]发起交易。如果没有指定[玩家]，当前目标将作为参数。 定[搜索内容]，那么会进行一个默认的搜索，并打开搜索对话框显示结果。 二、聊天命令 下面这些是用于玩家交流的命令，如果没有特别设定，聊天内容都是用当前的 表情的作用是向周围发送一个“玩家名<表情>”的信息，和/say类似。不过当敌对阵营玩家收到信息后只会显示“玩家名做了一些奇怪的动作”。 向战场团队频道发送[信息]。 说话，向周围发送<信息>，附近的玩家都可以收到。 仅向<玩家>发送<信息>。 三、在线状态 你“处在暂离状态”的自动回复。该回复也可以通过设置[信息]修改。
切换勿扰状态。玩家向你发送悄悄话时将在你名字左侧显示“勿扰”，并得到你“请勿打扰”的自动回复。该回复也可以通过设置[信息]修改。 四、好友列表 将[玩家]添加到忽略名单，如果没有指定[玩家]，则将你的当前目标作为参数。 为参数。 为参数。 五、小队、团队命令 邀请[玩家]加入队伍，如果没有指定[玩家]，则将你的当前目标作为参数。至 将[玩家]开除队伍，如果没有指定[玩家]，则将你的当前目标作为参数。至少需要助理权限。 队长权限 向团队发送[警告信息]，队员收到信息后将有警告音，并且屏幕中将会显示[警告信息]。至少需要助理权限。 向全团发送就位确认询问，所有队员需要在30秒内做“是”或“否”的选择。 之后将收到全团就位情况的统计结果。至少需要助理权限。 六、战利品分配
LED数码管的结构及工作原理 沈红卫 LED数码管(LED Segment Displays)就是由多个发光二极管封装在一起组成“8”字型的器件,引线已在内部连接完成,只需引出它们的各个笔划,公共电极。LED数码管常用段数一般为7段有的另加一个小数点,还有一种就是类似于3位“+1”型。位数有半位,1,2,3,4,5,6,8,10位等等、、、、,LED数码管根据LED的接法不同分为共阴与共阳两类,了解LED的这些特性,对编程就是很重要的,因为不同类型的数码管,除了它们的硬件电路有差异外,编程方法也就是不同的。图2就是共阴与共阳极数码管的内部电路,它们的发光原理就是一样的,只就是它们的电源极性不同而已。颜色有红,绿,蓝,黄等几种。LED数码管广泛用于仪表,时钟,车站,家电等场合。选用时要注意产品尺寸颜色,功耗,亮度,波长等。下面将介绍常用LED数码管内部引脚图。 图1 这就是一个7段两位带小数点 10引脚的LED数码管 图2 引脚定义 每一笔划都就是对应一个字母表示 DP就是小数点、 数码管分为共阳极的LED数码管、共阴极的LED数码管两种。下图例举的就是共阳极的LED数码管,共阳就就是7段的显示字码共用一个电源的正。led数码管原理图示意:
图3 引脚示意图 从上图可以瞧出,要就是数码管显示数字,有两个条件:1、就是要在VT端(3/8脚)加正电源;2、要使(a,b,c,d,e,f,g,dp)端接低电平或“0”电平。这样才能显示的。 共阳极LED数码管的内部结构原理图图4: 图4 共阳极LED数码管的内部结构原理图共阴极LED数码管的内部结构原理图: 图5 共阴极LED数码管的内部结构原理图
1.鼠标指向治疗 对你鼠标指向的目标施放快速治疗，不需要选中目标，改变当前目标。 /cast [target=mouseover] 快速治疗 2.暗牧伤害输出 注意：宏中饰品名字需改成你所要使用的饰品名字后，才可正常使用！暗牧输出宏，适用团队开嗜血英勇时候，配合自身饰品爆发用。/USE 后修改为个人饰品，已屏蔽系统红字提示。/castsequence [nochanneling] reset=combat 吸血鬼之触, 心灵震爆, 精神鞭笞, 暗言术：灭, 精神鞭笞, 心灵震爆, 精神鞭笞,精神鞭笞, 精神鞭笞 /cast 噬灵瘟疫 /script UIErrorsFrame:Clear() /use 饰品名字 3.暗牧一键输出 注意：宏中饰品名字需改成你所要使用的饰品名字后，才可正常使用！实现输出一键循环宏，按循环施放技能。技能循环中没有插入痛，需要手动上。 /castsequence [nochanneling] reset=6/combat 吸血鬼之触,心灵震爆,精神鞭笞,精神鞭笞,心灵震爆,精神鞭笞,吸血鬼之触,心灵震爆,精神鞭笞,精神鞭笞,心灵震爆,精神鞭笞 /use 饰品名字 /施放暗言术：灭 4.暗牧治疗 第一次点宏，取消暗影形态进行快速治疗，第二次点击，返回暗影形态 /castsequence 快速治疗, 暗影形态 5.暗影魔 施放暗影恶魔，并且将其调成攻击当前目标，这样暗影恶魔就不会乱跑了。 #showtooltip 暗影恶魔 /cast 暗影恶魔 /petattack /petdefensive 6.沉默焦点 点宏则停止当前法术，对焦点目标(前提当然是你设置了焦点)施放沉默，否则将会沉默你的当前目标 #showtooltip 沉默 /stopcasting /cast [target=focus,exists,harm,nodead] 沉默; 沉默 7.法力燃烧 当你的目标是你的队友时，点宏将会对你的焦点目标施放法力燃烧，如果你当前目标是敌人，则会直接施放法力燃烧。 #showtooltip 法力燃烧 /target [help] focus
数码管显示原理及应用实现 1.数码管显示原理 （1）数码管外形 图1 单位数码管图2 双位数码管图3 四位数码管 （2）数码管内部原理 图4 引脚图5 共阳极数码管 图6 共阴极数码管 （3）数码管工作电压和电流 红色和黄色的发光二极管的工作电压是2伏的，其他颜色的工作电压都是3伏；一般的发光二极管的工作电流是20毫安。可以使用电阻或者限流二极管来分压。 （4）数码管的检测 一、指针表： ①前提是你的万用表最好是用3V以上电池，因为1.5V不够点亮LED，特别是高亮超高亮的，点亮电压高。另外万用表在RX1档或最高档。 ②万用表笔随便一脚，假设红笔，搭在数码管上任一脚。黑笔在其它脚上扫过，如果不亮，有可能此管为共阴，可用3法再试。如有一段点亮。黑笔不动，移动红笔，在其它脚测。如果其它脚分别都能点亮，则可以说明黑笔接的是公共脚，此管共阳。（指针表的黑表笔是正电源）
③.表笔更换一下，黑笔先搭一脚，扫红笔。如有一段点亮，红笔不动，扫黑笔。如各段分别点亮，则红笔所接为公共 ，此管共阴。 4.如2、3两法均不亮，可能数码管额定电压较高，也可能数码管是坏的。这时，可用5V 电源串一500欧电阻继续测试。 二、数字表： 用二极管档（有个二极管符号的，也作通路档使用），方法同指针表。 不过，红表笔所对应的共阳共阴和指针表是相反的。因为数字表的红笔就是正电源。 （5）与单片机的接口 P0口8个LS TTL 门电路构成，P1-P3口由4个LS TTL 门电路构成。单个LS TTL 门电路输出电流约1.2mA ，输入电流20mA ，总的灌电路一般不超过50mA 。 a ．三极管驱动 图7 三极管驱动线路（图中有错误） b ．专用驱动芯片 E 1L 11 D02D13D24D35D46D57D68D7 9VCC 20GND 10Q019Q118Q217Q316Q415Q514Q613Q712 U174HC573E 1L 11D02D13D24D35D46D57D68D79VCC 20GND 10 Q019Q118Q217Q316Q415Q514Q613Q712 U274HC573 D0D1D2D3D4D5D6D7D0D1D2D3D4D5D6D7 DULA WELA A B C D E F G H WE1WE2WE3WE4WE5WE6C SAD VCC 40 P10/T 1P11/T 2P123P134P145P156P167P178 INT113INT012T115T014 EA/VP 31 X119 X218 R ES ET 9R D 17WR 16GND 20PSEN 29 ALE/P 30TXD 11R XD 10P0039P0138P0237P0336P0435P0534P0633P0732P2021P2122P2223P2324P2425P2526P2627P272889C5289C52 VCC DB1DB2DB3DB4DB5DB6DB7DB8INT1C SDA R S LCDEN R ST R D WR X1X2D0D1D2D3D4D5D6D7SDA SC L 18B20FM C SUSB DIOLA DULA WELA P3.0P3.1ALE VCC VDD VCC