2006-Endocrinology-The Mouse Testis Is the Source of Various Serine Proteases and Serine Proteinase

The Mouse Testis Is the Source of Various Serine

Proteases and Serine Proteinase Inhibitors (SERPINs):Serine Proteases and SERPINs Identified in Leydig Cells Are under Gonadotropin Regulation

Fanny Odet,Ade ′lie Verot,and Brigitte Le Magueresse-Battistoni

Institut National de la Sante ′et de la Recherche Me ′dicale,Unite ′418,Institut National de Recherche et de Se ′curite ′Unite ′Mixte de Recherche 1245,and Universite ′Lyon 1,Hopital Debrousse,69322Lyon cedex 05,France

The occurrence of various serine proteinases and serine pro-teinases inhibitors (SERPINs)was investigated by RT-PCR in whole testes of 1-,3-,and 8-wk-old mice in crude and enriched germ cell fractions,mouse Leydig tumor cells (mLTC-1),and primary cultures of 3-and 8-wk-old enriched fractions of Ley-dig cells and 3-wk-old Sertoli cells.New members were iden-tified in the testis protease repertoire.Within the Leydig rep-ertoire,a PCR product was found for plasminogen activators urokinase plasminogen activator (uPA)and tissue plasmino-gen activator (8-wk-old cells),matriptase-2(mLTC-1),kal-likrein-21,SERPINA5,SERPINB2(primary cultures),and serine peptidase inhibitor Kunitz type 2(SPINT2).The go-nadotropin regulation was explored by semiquantitative RT-PCR,using steroidogenic acute regulatory protein (StAR)as a positive control.Matriptase-2,kallikrein-21,SPINT2,and SERPINA5were down-regulated,whereas uPA and its recep-tor were up-regulated by human chorionic gonadotropin (hCG)via cAMP in the mLTC-1cells.Positive effects were observed transiently after 1–8h of hCG exposure,and nega-tive effects,first evidenced after 6h,lasted 48h.The hCG-induced effects were confirmed in primary cultures.In addi-tion,SERPINB2was augmented by hCG in primary cultures.Addition of either trypsin or protease inhibitors did not alter the hCG-induced surge of StAR.Because hCG regulated pro-teases and SERPINs (whereas testosterone did not),it could alter the proteolytic balance of Leydig cells and consequently the metabolism of extracellular matrix components.There-fore,even though a direct interplay between the early hCG-induced surge of uPA and StAR is unlikely,our data to-gether with the literature suggest that extracellular matrix proteins alter Leydig cell steroidogenesis.(Endocrinology 147:4374–4383,2006)

A

NUMBER OF important processes that regulate the activity and fate of many proteins are strictly depen-dent on proteolytic processing events.The serine proteinase family is one of the oldest characterized and largest families of proteolytic genes (227members in the mouse)(1,2),which has well-characterized roles in diverse cellular activities in-cluding blood coagulation,platelet activation,fibrinolysis and thrombolysis,extracellular matrix (ECM)remodeling,and cancer invasion (3).The serine proteinases can be further subdivided into 16families,among them the plasminogen activators,transmembrane serine proteinases,and kal-likreins.Serine proteinase activity is regulated by serine pro-teinase inhibitors (SERPINs).The SERPINs belong to an ex-panding superfamily of structurally similar but functionally diverse proteins,and most of them share a substrate suicide mechanism irreversibly inactivating the SERPINs (4–6).

The testis is a reproductive organ that serves two decisive functions,the production of spermatozoa taking place in the seminiferous tubules and the synthesis and secretion of tes-tosterone by the Leydig cells located in the interstitium.Spermatogenesis is hormonally regulated,pituitary gonad-otropins positively regulating Leydig and Sertoli cell secre-tions through LH and FSH,respectively.Testosterone,pro-duced as a result of LH action on Leydig cells,acts via androgen receptors localized on Sertoli,peritubular,and Leydig cells.Sertoli cells play several key roles in spermat-ogenesis.They are targets for FSH and testosterone,these hormones being responsible for the initiation and mainte-nance of spermatogenesis.Moreover,together with the peri-tubular cells,they also form the cytoarchitectural scaffolding of the tubule,providing structural and nutritional support for the developing germinal cells (7–10).

In an attempt to elucidate the role of proteinases and proteinase inhibitors in testicular physiology,we examined the presence of various serine proteinases and SERPINs in the whole testes of mice as well as isolated testicular cells.For this purpose,we used total RNA recovered from whole testes of 1-,3-,and 8-wk-old mice,from crude and enriched germ cell fractions,primary cultures of 3-wk-old Sertoli cells,3-and 8-wk-old enriched preparations of Leydig cells,and mouse Leydig tumor cells (mLTC-1).Because Leydig cells were found to express several proteinases and SERPINs,we determined whether they were under gonadotropin regula-tion.We also examined the influence of testosterone.

First Published Online June 1,2006

Abbreviations:aPC,Activated protein C;ECM,extracellular matrix;hCG,human chorionic gonadotropin;HGFA,hepatocyte growth factor activator;HPRT,hypoxanthine phosphoribosyl transferase;mLTC,mouse Leydig tumor cell;PA,plasminogen activator;PC,protein C;PCI,protein C inhibitor;SERPIN,serine proteinase inhibitor;SPINT1or 2,serine peptidase inhibitor Kunitz type 1or 2;StAR,steroidogenic acute regulatory protein;tPA,tissue plasminogen activator;uPA,urokinase plasminogen activator;uPAR,uPA receptor.

Endocrinology is published monthly by The Endocrine Society (https://www.wendangku.net/doc/0612774404.html,),the foremost professional society serving the endocrine community.

0013-7227/06/$15.00/0Endocrinology 147(9):4374–4383

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Copyright ?2006by The Endocrine Society

doi:10.1210/en.2006-0484

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Materials and Methods

Animals,tissues,and cell preparations

CD-1mice were purchased from Elevage Janvier(Le Genest,France). Testes were collected from1-,3-,and8-wk-old mice.Enriched prepa-rations of pachytene spermatocytes and early spermatids(75–80%en-richment)were obtained after centrifugal elutriation of a crude germ cell preparation recovered from adult mice testes by trypsination(11).Once harvested,testes or cells were immediately stored at?70C and pro-cessed for RNA analysis.Experiments were conducted according to the Guide for the Care and Use of Laboratory Animals.

Leydig-enriched fractions and Sertoli cells were isolated from3-wk-old mice testes and cultured in Ham’s F12-DMEM(Life Technologies, Grand Island,NY)at32C in a humidified atmosphere of5%CO2as previously described(12),except that a hyaluronidase treatment(0.1%, 20min,32C)was introduced after the second collagenase digestion to reduce peritubular cell contamination in the Sertoli cell fraction(13). Leydig cells were recovered in the supernatant of the first collagenase digestion,and the resulting suspension was hypotonic treated to elim-inate the contaminating germ cells,as described recently(14).After several washes,the cell suspension was plated in12-well plates(50,000 cells/well).Enrichment of the cell suspension was higher than80%as judged by3?-hydroxysteroid dehydrogenase immunostaining(not shown).At the end of the enzymic procedure,the Sertoli cell suspension was washed with fresh culture medium,and cells were seeded in6-well plates at a ratio of1?106viable cells/well in Ham’s F12-DMEM.They were cultured for2d.At that time,the purity of the cultures was higher than90%,and contamination was mainly due to residual germ cells(an average of5–7%)(not shown).

Leydig-enriched fractions were also isolated from8-wk-old mice testes and cultured in Ham’s F12-DMEM at32C in a5%CO2incubator as described for the3-wk-old testes.They were recovered in the super-natant of the first collagenase digestion,and the resulting suspension was harvestly washed,filtered through a45-mm nylon mesh and hy-potonic treated.After several washes,the cell suspension was plated in 12-well plates(50,000cells/well).Enrichment of the cell suspension examined on the next day was higher than70%as judged by3?-hy-droxysteroid dehydrogenase immunostaining(not shown).

Cell line cultures

The immortalized Leydig cell mLTC-1line was kindly provided by D.M.Stocco(Texas Tech,Lubbock,TX).The mLTC-1cells were culti-vated at37C in a5%CO2incubator.Culture medium was a RPMI1640 medium(Sigma-Aldrich Corp.,St.Louis,MO)supplemented with10% fetal calf serum until cells reached subconfluency(70%),as described (15).Serum was then omitted,cells were rinsed abundantly,and fresh medium was replaced.

Cell treatment

In the relevant experiments,testosterone(0.1?m),human chorionic gonadotropin(hCG;from1to100ng/ml;Organon,Puteaux,France), bu2cAMP(1m m),actinomycin D(5?g/ml),cycloheximide(50?g/ml), trypsin(from1to1000ng/ml),or proteases inhibitors(a cocktail of aprotinin and leupeptin at100?m each)(all from Sigma)were added to the cultures at the doses and for the time periods indicated.

RNA extraction,RT-PCR,and semiquantitative RT-PCR Procedures for RNA extraction and RT-PCR have been described elsewhere(16).Specific primers were designed using the Gene-Jockey sequence processor(Biosoft,Cambridge,Cambridgeshire,UK),and the optimal temperature of annealing was defined for each couple of prim-ers(Table1).Negative controls contained water instead of cDNA.PCR with no reverse transcription gave no product,eliminating the possi-bility of a genomic DNA contamination in the RNA preparations.Am-plified cDNAs were visualized in a1.5%agarose gel stained with ethidium bromide.A DNA ladder(Promega,Charbonnie`res,France) was loaded on each gel,and18S was used to ensure equal loading.PCR products were sequenced by Biofidal(Lyon,France).Conditions for reliable semiquantitative RT-PCR were optimized for each series of primers in the presence of?-33P dATP(0.75?Ci;2500Ci/mmol;Am-ersham Pharmacia Biotech Europe GmbH,Orsay,France),as described elsewhere(17,18).The PCR products were separated on8%polyacryl-amide gel electrophoresis in1?Tris borate EDTA(TBE)buffer.Gels were transferred to filter paper,dried,and exposed to Kodak biomax MR1films(Sigma).The band densities were determined by scanning densitometric analysis(Scion image?,4.03;Scion Corp.).Densitometry data were normalized using hypoxanthine phosphoribosyl transferase (HPRT).

Protein extraction and analysis

Gonadal proteins were extracted in PBS containing1%Nonidet P-40 and5m m EDTA,as previously described(16).For plasminogen zy-mography,10-fold concentrated proteins were electrophoresed at4C on 10%PAGE in the absence of any reducing agent.After electrophoresis, sodium dodecyl sulfate was removed from the gel by exchange in Triton X-100(two washes,30min each,at room temperature in2.5%Triton X-100,followed by three washes,30min each,in distilled water).The gel was subsequently placed on a casein-agar-plasminogen underlay as

TABLE1.Nomenclature and selected information on the serine proteinases and SERPINs hereby studied(1)

Nomenclature Selected information Ref. Serine proteinases

HGFA Activates the conversion of proHGF into active HGF20 Hepsin Transmembrane serine proteinase which may also activate proHGF21 Kallikrein-21Specifically expressed in Leydig cells in testis where it degrades at least fibronectin22 Matriptase-2Transmembrane serine proteinase showing degrading activity against

type I collagen and fibronectin

23 PA

tPA uPA Catalyse the conversion of plasminogen into plasmin,a serine proteinase with a

large spectrum of activity toward latent proteinases and ECM components

3

aPC Anticoagulant previously identified in the testis24

Targeted proteinases among those studied

SERPINs

HGFA inhibitor(HAI)

SPINT1(HAI-1)HGFA,matriptase-2,hepsin20–23 SPINT2(HAI-2)HGFA,kallikrein,hepsin and plasmin,but not uPA25–27 PA inhibitor(PAI)

SERPINE1(PAI-1)uPA,aPC,tPA,plasmin

SERPINB2(PAI-2)uPA,tPA(poor inhibitor)

SERPINA5(PAI-3or PCI)aPC,uPA,tPA,kallikrein3–6 SERPINE2(Protease nexin1,or PN-1)uPA,tPA,plasmin

Odet et al.?Serine Proteinases,Serpins,and Mouse Testis Endocrinology,September2006,147(9):4374–43834375

previously described(18,19).Plasminogen-degrading activity was vi-sualized after incubation at37C for12h,and gels were scanned. SDS-PAGE(10%)and Western blotting were carried out as described elsewhere(16–18),using the concentrated culture media also assayed in plasminogen zymography.A rabbit polyclonal antibody was used against recombinant mouse protein C inhibitor(PCI or SERPINA5;di-lution1:1000;a gift from M.Geiger,Department of Vascular Biology and Thrombosis Research,University of Vienna,Vienna,Austria)and was detected by an antirabbit IgG(dilution1:10,000)conjugated to peroxy-dase(Jackson ImmunoResearch,Baltimore,MD).Precision protein stan-dards(Bio-Rad Laboratories,Hercules,CA)were loaded for estimation of the molecular masses of the bands,which were revealed using an ECL?chemiluminescent detection system(Amersham Pharmacia Biotech).

Data analysis

The densitometric data are given as the mean?sem(n?3–4),and experiments were repeated at least three times(twice for the8-wk-old preparations of Leydig cells).The significance of the results was deter-mined by ANOVA followed by a Mann-Whitney U test.Differences were considered significant at P?0.05.

Results

RT-PCR screening of serine proteinases and SERPINs Selected information and nomenclature for the seven serine proteinases and six SERPINs studied are summarized in Table1,and the list and sequences of the designed specific primers for PCR studies are described in Table2.Total RNA was recovered from1-,3-,or8-wk-old testes from total germ cells or enriched fractions in pachytene spermatocytes or early spermatids;the mLTC-1;Leydig-enriched fractions re-covered from3-and8-wk-old mice testes;and3-wk-old Sertoli cells,which had been cultured for2d in basal con-ditions.A PCR product of the right size and sequence(not shown)was detected for each of the serine proteinases and SERPINs(Fig.1).Plasminogen activators(PAs)were among the most abundant serine proteases within the testis at the three ages investigated,and tissue plasminogen activator (tPA)was more abundant than urokinase plasminogen ac-tivator(uPA).SERPINA5,serine peptidase inhibitor Kunitz type1(SPINT1),and SPINT2were the most abundant SERPINs within the testis.Germ cells had the smallest rep-ertoire with hepatocyte growth factor activator(HGFA),ac-tivated protein C(PC),SERPINA5,SPINT1,and the longest form of SPINT2.The weak bands seen for tPA and SERPINE2 in the nonelutriated fraction probably resulted from a so-matic contamination because they were not detected in the enriched fractions(Fig.1).These data extend previous results showing activated PC(aPC)in mouse germ cells(24)and SPINT2in human germ cells(27).

Primary cultures of Leydig-enriched fractions as well as mLTC-1cells exhibited a PCR product for six of the13serine proteases and SERPINs investigated,including the two plas-minogen activators,matriptase-2,kallikrein-21,SERPINB2, SERPINA5,and SPINT2.No signals were seen with primers designed against activated protein C,HGFA,or hepsin(Fig.

1).Interestingly,the PCR band for matriptase-2was detected in mLTC-1but not Leydig-enriched fractions,Sertoli cells,or germ cells.Thus,the origin of the weak band in whole testes is yet unknown.Inversely,a PCR band for SERPINB2was found in Leydig-enriched fractions but not mLTC-1cells. Finally,a PCR band for tPA was found in8-wk-old enriched fractions of Leydig cells(Fig.1).All these data on Leydig cells are original except for tPA(28)and kallikrein-21(22).Sertoli cells exhibited stronger PCR products corresponding to uPA and tPA than Leydig cells.A weak PCR product correspond-ing to hepsin was also found in Sertoli cells.Regarding the SERPINs,Sertoli cells exhibited a PCR product for each of them.In addition,PCR products were stronger in Sertoli cells than Leydig cells,with the exception of SERPINB2(Fig.1). Addition of hCG altered the expression of proteinases and SERPINs in the mLTC-1cells

Leydig mLTC-1cells were cultured to80%confluence, serum starved,and then treated with hCG at1,10,or100 ng/ml for6h.Cells were then scraped,total RNA was extracted,and RT-PCR experiments performed.Primers

TABLE2.List and sequence of the designed specific primers for PCR studies

Accession no.

Primers(5?–3?)

Size(bp)T Opt(C) Sense Antisense

HPRT J00423CCTGCTGGATTACATTAAAGCACTG GTCAAGGGCATATCCAACAACAAAC35465 18S X00686GGAATAATGGAATAGGACCG TCTGTCAATCCTGTCCGTGTCC43561 StAR L36062GCATACTCAACAACCAGGAAGG CTGGTTGATGATTGTCTTCGGC51166 AR NM_013476TACATGTGGTCAAGTGGG TGTGTGGAAAATAGATGGG51166 Serine proteinases and receptor for uPA

uPA NM_008873TGTCAGAACGGAGGTGTATGCG TGTTCTTCTGGTAGATGGCTGC50765 uPAR X62700GAATGGCAAGATGATAGAGAGC AACACTGGAAGCCATTCGGTGG48969 tPA J03520TATTGTCGGAATCCAGATGG AACGAACCTGACACTGGAGTCG46163 Matriptase-2AY240929CTCTCTGGACTACGGCTTGGCGCTC TGGAGGCCACAGTCACAGTGTTGC59665 Hepsin NM_008281TCATCTCTGTATGTGACTGTCC TAGAGAGGTGGACCAAGGCAA39565 HGFA AF224724ACTTCTTCAACCGCACCACG CTACTACGGTATGTCCATTCGG45766 Kallikrein-21NM_010642GAACCTGGACACCTGTTACC CGTAGTAACATCAGGTCATTGC39666 aPC NM_008934CCAAGGATAGTCAACGGAACGC CTGCCCATCCTTTGATTCTGTCGC43366 SERPINs

E1M33960CCAACAAGAGCCAATCACAAGG GAAAGGTGGAGACTCTTC50363 B2NM_011111TCCTTGAATGTGCTGAAGAAGC GACCACAACATCATCTTCATCC45169 A5AH006766ACAACTATGTAGCCAAGCAGACC TCCTCCACCTGCTTCATCTTGC32465 E2NM_009255TGAAGTCAGTGAAGATGGAACC CCTGCTCATCCTTCACTACAGC46458 SPINT1AF099018TGACTATCACTGTGCTAACTGC TAGGAAGAACCTCTACGGTGCC59265 SPINT2AF099016AATGGAGCCGACTCTTCTGTCC CTACTCCGTCTGGTATCTCC35665

The optimal temperature(T

Opt )for annealing,the size of the expected PCR fragments,and the source for the design of the primers are

reported.The number of cycles was determined for each set of primers.It ranged from19to26.

4376Endocrinology,September2006,147(9):4374–4383Odet et al.?Serine Proteinases,Serpins,and Mouse Testis

used included those specific for uPA,kallikrein-21,matriptase-2,SPINT2,SERPINA5,HPRT as a house keeping gene,and the steroidogenic acute regulatory protein (StAR)to ensure that Leydig cells responded to hCG under our experimental conditions.Addition of hCG resulted in a sig-nificant enhancement of StAR at the three doses,and 100ng/ml induced a 2.5-fold increase (P ?0.05).Interestingly,hCG positively regulated the expression levels of uPA and down-regulated the expression levels of matriptase-2,kal-likrein-21,SERPINA5,and SPINT2(Fig.2).These effects depended on the doses of hCG,and a significant (P ?0.05)effect was obtained with the dose of 100ng/ml.Indeed,uPA levels increased 4.2-fold,whereas levels of kallikrein-21,matriptase-2,SPINT2,and SERPINA5were halved in the hCG-treated cultures,compared with the untreated cultures.

Gonadotropins positively regulate the expression of uPA,its receptor (uPAR),and the uPA enzymatic activity via cAMP

The addition of hCG at a dose of 100ng/ml for 2h,which significantly increased (2.5-fold increase;P ?0.05)the ex-pression of StAR,also significantly enhanced (a 5.2-fold in-crease;P ?0.05)the expression of uPA (Fig.3).The hCG-induced expression of uPA was maintained after 8h (a 2-fold increase;P ?0.05),but it was no longer observed after 24h of stimulation.The time-course effects of hCG were mirrored by the addition of bu 2cAMP 1m m ,indicating the involve-ment of the protein kinase A (Fig.3A).We next examined the expression levels of the uPAR because uPAR dictates the site and extent of proteolysis of uPA (3).Addition of hCG or bu 2cAMP was found to exert a significant but transient stim-ulatory effect on the expression of uPAR.The effect was observed after 2h (a 3-and 2.9-fold increase,respectively;P ?0.05)but not after 8or 24h of stimulation (Fig.3A).The role of transcription and ongoing protein synthesis in the hCG-induced uPA mRNA levels was evaluated.For this purpose,mLTC-1cells were pretreated for 30min with ac-tinomycin-D or cycloheximide and stimulated with hCG 100ng/ml for 6h.The results indicate that the presence of the inhibitor of either RNA or protein synthesis abolishes the hCG-induced positive regulation of uPA and StAR (Fig.3B).The biological activity of uPA was examined by zymog-raphy in culture media from mLTC-1treated for 24h with hCG or bu 2cAMP.We observed a strong enhancement of the uPA lytic band in the stimulated cells.No tPA lytic band was detectable (Fig.3C).

Gonadotropins negatively regulate the expression of matriptase-2,kallikrein-21,SERPINA5,and SPINT2via cAMP

Matriptase-2,kallikrein-21,SERPINA5,and SPINT2were decreased in hCG-treated cells after a kinetics different from that described for uPA and uPAR (Fig.4A),and no

effects

F I

G .1.RT-PCR analysis of various serine proteinases and SERPINs in 1-,3-,8-wk-old mice testes;germ cells either from total germinal cells (total)or fractions enriched in pachytene spermatocytes (scytes)or early spermatids (stids);mLTC-1cells;Leydig-enriched fractions recovered from 3-and 8-wk-old mice testes;or Sertoli cells recovered from 3-wk-old mice testes (SC 3-wk),which had been cultured for 2d in basal conditions.The set of primers used is listed in Table 2.RT-PCR studies were also conducted using primers directed against 18S to ensure that equal amounts of material were used.A DNA ladder was included in each gel for accurate determination of the size of the PCR product (not shown).Mat.-2,Matriptase-2;kall.-21,kal-

likrein-21.

F I

G .2.Regulation of StAR,uPA,kallikrein-21,matriptase-2,SPINT2,and SERPINA5in the mLTC-1cells by hCG.Cells were cultured for 6h in the absence or presence of three different doses of hCG (1,10,and 100ng/ml,respectively).Three experiments were performed and a representative autoradiograph is shown.HPRT was used as a control in the RT-PCR studies.

Odet et al.?Serine Proteinases,Serpins,and Mouse Testis Endocrinology,September 2006,147(9):4374–43834377

were observed after 2h of stimulation.However,in cells treated for 8h with hCG,matriptase-2,kallikrein-21,SERPINA5,and SPINT2were significantly decreased (P ?0.05),and their levels represented 33,40,36,and 50%of their respective control.After 24h of hCG exposure,the inhibition was stronger than that after 8h (except for SPINT2),with levels of matriptase-2,kallikrein-21,and SERPINA5de-creased to baseline levels.The kinetics of the effects induced by hCG were mimicked by the addition of bu 2cAMP,indi-cating the involvement of the protein kinase A (Fig.4).To determine the reversibility of the hCG-induced inhi-bition,mLTC-1cells were treated for 16h with hCG 100ng/ml,after which cells were washed and fresh culture me-dium was added.After 24h,cells were scraped and total RNA was extracted for RT-PCR studies.A densitometry analysis indicated a full recovery of the expression levels of matriptase-2and kallikrein-21,whereas the expression levels of SERPINA5and SPINT2had only partially recovered (Fig.4B).After 48h,half and full recovery of the expression levels of SERPINA5and SPINT2were observed,respectively (not shown).

By Western blotting,a single protein,comigrating at 46kDa with proteins extracted from seminal vesicles rich in PCI (24),was detected in testis extracts as well as the culture media of mLTC-1.Addition of hCG 100ng/ml or bu 2cAMP 1m m for 24h resulted in a strong decrease in the 46-kDa migrating band corresponding to PCI (Fig.4C).

Testosterone did not influence the expression of proteases and SERPINs in the mLTC-1cells

We also investigated a possible influence of testosterone 0.1?m added for 2,8,or 24h.Androgen receptors were

first evidenced by RT-PCR (not shown).No effects of tes-tosterone were observed on uPA,matriptase-2,kallikrein-21,SERPINA5,or SPINT2(not shown).

Gonadotropins regulate serine proteases and SERPINs expressed in primary cultures

Because mLTC-1cells were tumor cells,we wished to examine whether hCG and testosterone could regulate serine proteases and SERPINs expressed in normal Leydig cells.For this purpose,enriched fractions of Leydig cells were isolated from 3-and 8-wk-old mouse testes,cultured for 2d,and stimulated with hCG 100ng/ml or testosterone 0.1?m for 2,24,or 48h (Fig.5).Androgen receptors were first evidenced by RT-PCR (not shown).The serine proteases and SERPINs examined include uPA,kallikrein-21,SERPINA5,SPINT2,and SERPINB2.tPA was also studied using the 8-wk-old preparations,but levels in the culture were too weak to be quantified (not shown).The data presented indicate that hCG significantly enhanced uPA and SERPINB2and that kallikrein-21,SERPINA5,and SPINT2were down-regulated by hCG in both types of primary cultures,i.e.from the 3-and 8-wk-old preparations (Fig.5).After 2h of stimulation,fold increases of uPA were 2.5and 4.9(P ?0.05),respectively for the 3-and 8-wk-old preparations in primary cultures.No effects of hCG were observed in long-term cultures (24or 48h)(Fig.5A).The addition of hCG significantly increased SERPINB2levels after 24h of stimulation (a 4.1-fold increase;P ?0.05),using the 8-wk-old preparations.The fold increase was 1.6(P ?0.05)after 48h of stimulation using the 3-wk-old preparations.No significant effects were observed when the cells were treated for 2h with hCG (Fig.5A).The effects induced by hCG in primary cultures from the 8-wk-old

prep-

F I

G .3.Effects of hCG and bu 2cAMP on the expression levels of StAR,uPA,uPAR (RT-PCR),and uPA biological ac-tivity (plasminogen zymography)in mLTC-1cells.A,mLTC-1cells were stimulated for 2,8,or 24h in the pres-ence of hCG 100ng/ml or bu 2cAMP 1m M .C,Control,i.e.untreated cells.B,mLTC-1cells were pretreated for 30min with actinomycin D (Act)or cyclohexi-mide (CHX)and stimulated with hCG for 6h.Representative autoradiographs are shown in A and B.Autoradiographs were scanned and the expression was normal-ized to the HPRT signal.Values are the mean ?SEM of n ?3;*,P ?0.05,com-pared with untreated dishes.C,Zymo-graphic analysis of the culture media of mLTC-1cells stimulated for 24h in the presence or absence of 100ng/ml hCG or bu 2cAMP 1m M .A representative zymo-gram of four experiments performed is shown.The uPA lytic band is noted on the left.Cont,Control.

4378Endocrinology,September 2006,147(9):4374–4383Odet et al.?Serine Proteinases,Serpins,and Mouse Testis

arations were mimicked by bu 2cAMP (not shown).Primary cultures from the 3-wk-old preparations were not examined.The expression levels of kallikrein-21,SERPINA5,and SPINT2were not modified by a 2-h exposure with hCG or testosterone (not shown).However,their levels were signif-icantly (P ?0.05)halved in primary cultures treated for 24or 48h with hCG (Fig.5B)or bu 2cAMP (not shown).Tes-tosterone had no effect (Fig.5B).

Effects of the exogenous addition of protease inhibitors or of trypsin

We explored the hypothesis that the enhancement of uPA and StAR may be interdependent events.For this purpose,we carefully examined the kinetics of the hCG-induced stim-ulatory effects during the first hours of treatment to deter-mine which of uPA or StAR was first induced by hCG.Our data show that the hCG-induced enhancement of StAR and uPA followed closely the same pattern of expression up to 4h of stimulation,after which uPA levels decreased,whereas StAR levels were maintained at a significantly higher level than in the controls,in mLTC-1cells (Fig.6A).We also examined SERPINB2expression levels in the hCG-stimu-

lated mLTC-1cells.We observed the appearance of a PCR product in the hCG-treated mLTC-1cells,whereas no PCR product was detected in untreated cells.However,the signal was too weak to be quantified (not shown).The expression levels of StAR were also significantly increased in primary cultures after a 2-h treatment with hCG (Fig.6B).

We then explored whether the hCG-induced early peak of StAR could be mimicked by the addition of serine proteases.If so,the addition of serine protease inhibitors would de-crease the hCG-induced increase of StAR.Cells were there-fore pretreated with protease inhibitors or trypsin and then exposed to hCG for 2h and the expression of StAR was examined.The addition of serine protease inhibitors (in both normal and tumor cells)or trypsin 1,100,or 1000ng/ml (performed in the mLTC-1cells)had no effect on the hCG-or bu 2cAMP-induced StAR (Fig.6,B and C).Higher doses of trypsin resulted in cell detachment,and thus,their effect on StAR could not be examined (not shown).

Discussion

In the present study,we demonstrate that the mouse testis is the source of several serine proteinases and inhibitors

and

F I

G .4.Effects of hCG and bu 2cAMP on the expression levels of matriptase-2,kallikrein-21,SERPINA5,and SPINT2assessed by RT-PCR in mLTC-1cells.A,mLTC-1cells were stimulated for 2,8,or 24h with hCG 100ng/ml or bu 2cAMP 1m M .C,Control cells,i.e.untreated cells.Representative autoradiographs are shown.mLTC-1cells were cultured with (B)(?hCG)or without (C)hCG 100ng/ml for 16h.Cells were washed and fresh hCG-free medium was added for 24h.In A and B,autoradiographs were scanned and the expression was normalized to the HPRT signal (the same as in Fig.3for A).Values are the mean ?SEM of n ?3.*,P ?0.05,compared with untreated dishes in A and the time-matched controls in B.C,Western blot analysis of culture media of mLTC-1cells cultured for 24h with or without (C)100ng/ml hCG or bu 2cAMP 1m M (same culture media as in Fig.3C).Thirty micrograms of proteins were loaded per lane.Seminal vesicles (SV)and testis from adult mice (testis)were used as a positive control.The arrow points to the 46-kDa migrating band corresponding to SERPINA5.A representative gel of the three experiments performed is presented.

Odet et al.?Serine Proteinases,Serpins,and Mouse Testis Endocrinology,September 2006,147(9):4374–43834379

that not only Sertoli cells and germ cells but also Leydig cells (enriched fractions from 3-or 8-wk-old mice testes or mLTC-1cells)contribute to the protease repertoire.In ad-dition,serine proteases and inhibitors detected in Leydig cells were regulated by hCG in both normal and tumor cells.The physiological significance of the data is discussed,taking into account the predicted role of proteinases and inhibitors in degrading the ECM components as well as data from the literature showing that ECM proteins alter Leydig cell steroidogenesis.

In a first series of experiments using a RT-PCR procedure,we show the presence of mRNAs encoding several protein-ases and inhibitors in the mouse testis of various ages,en-riched germ cell fractions,3-wk-old mouse Sertoli cells,Ley-dig-enriched fractions from 3-and 8-wk-old mice testes,and the mLTC-1Leydig cells.The purpose of using these differ-ent fractions was to evidence potential cross-contaminations because enriched Leydig fractions but not pure Leydig cells could be isolated and mLTC-1cells are tumor cells.In ad-dition,because of the differential proportion of somatic and germinal cells during testicular development,an age-devel-opmental decrease of a PCR product suggests a somatic origin of the corresponding transcript,whereas an age-developmental enhancement of a PCR product suggests a germ cell origin.New members were identified in the mouse testis protease repertoire including HGFA in germ cells,hep-sin in Sertoli cells,SPINT1in Sertoli cells,and SPINT2in the different fractions examined.Because all these molecules converge on the HGF signaling pathway and considering the importance of HGF in testicular physiology (29),our data support the hypothesis that in addition to the standard tran-scriptional level,the regulation of this pathway could in-volve a proteolytic level of regulation.This expression screening also demonstrates that Leydig cells exhibit a PCR product corresponding to six of the 13proteases and inhib-itors investigated.In addition to SERPINA5(18),kal-likrein-21(22)and tPA weakly present in adult Leydig cells (28);the somatic form of SPINT2was identified in Leydig cells.Interestingly,SERPINB2was found in primary cultures of Leydig cells but not the mLTC-1cells,and matriptase-2was found in the mLTC-1cells but not the primary cultures.In the second part of this study,we show that hCG

reg-

F I

G .5.Effects of hCG and testosterone on primary cultures of Leydig cells isolated from 3-and 8-wk-old testes.Leydig cells were cultured for 2,24,or 48h with or without hCG 100ng/ml or testosterone 0.1?M ,as indicated.RT-PCR studies were performed using uPA and SERPINB2(A)and kallikrein-21,SPINT2,and SERPINA5(B).In A,the 3-wk-old Leydig cells were stimulated for 2and 48h,and the 8-wk-old Leydig cells were stimulated for 2and 24h;in B,the 3-wk-old Leydig cells were stimulated for 48h,and the 8-wk-old Leydig cells were stimulated for 24h.Representative autoradiographs are shown for serine proteases and SERPINs but not HPRT.Autoradiographs were scanned and expression was normalized to the HPRT signal.The values correspond to the mean ?SEM of n ?4(3wk old Leydig cells)and n ?3(8wk old Leydig cells).*,P ?0.05,compared with untreated cells of the time-matched control.

4380Endocrinology,September 2006,147(9):4374–4383Odet et al.?Serine Proteinases,Serpins,and Mouse Testis

ulates the expression of serine proteinases and SERPINs identified in the mLTC-1cells and the primary cultures.Furthermore,a positive regulation was highlighted for uPA (which depended on ongoing RNA and protein synthesis;studied in the mLTC-1cells)and SERPINB2(shown in nor-mal cells),whereas SPINT2,kallikrein-21,SERPINA5,and matriptase-2(shown in tumor cells)were negatively regu-lated by hCG.The hCG concentrations effective in regulating proteinases and SERPINs were identical with those required to stimulate the expression of StAR in the mLTC-1cells (15).The striking difference was the kinetics of the hCG-induced effects.Indeed,we essentially observed an early and tran-sient positive effect or a delayed (not evidenced before 6to 8h of stimulation)negative effect,which lasted 48h.Inter-estingly,the hCG-induced enhancement of uPA paralleled the hCG-induced StAR during the first 4h,after which uPA but not StAR levels decreased.Given that SERPINB2may oppose PA activity (3–6,30),the hCG-induced enhancement of SERPINB2observed in primary cultures might contribute to restrict excessive proteolysis of uPA.Therefore,the pres-ence of matriptase-2(not found in primary cultures)together with the very low level of SERPINB2detected on hCG stim-ulation could account for the specific characteristics of the mLTC-1cells.For instance,if the role of SERPINB2is to oppose uPA within the Leydig cells,the lack of inhibition of uPA by SERPINB2in mLTC-1cells could explain a high uPA activity at a time (24h of hCG stimulation)when uPA and uPAR mRNA levels return to their basal levels.Future ex-periments are required to determine the precise kinetics of the hCG-induced enhancement of SERPINB2in normal Ley-dig cells and test whether SERPINB2could degrade the pro-tein uPA and its receptor in the primary Leydig cell cultures,keeping in mind that uPA and SERPINB2actually perform many additional and unrelated functions (30).

We also explored the hypothesis that the hCG-induced enhancement of StAR and of uPA may be interdependent events.For this purpose,we examined the effect of a pre-treatment with either serine protease inhibitors or trypsin on the expression of StAR in basal and hCG-treated cultures.We did not observe any effect.Because hCG can induce testos-terone formation as early as 30min in normal Leydig cells (9,31–33),we also examined whether androgens could stimu-late uPA.We did not observe any effect of testosterone on the proteases or SERPINs analyzed in the mLTC-1cells or the Leydig cell primary cultures.We could not explain why kallikrein-21was unresponsive to testosterone in our hands,whereas a positive effect had previously been reported (22).In addition,using the same batch of testosterone and mouse Sertoli cells from 3-wk-old mice testes,we observed that testosterone could induce the expression of SERPINA5(Odet,F.,A.Verot,and B.Le Magueresse-Battistoni,unpub-lished data),extending previous findings (10,14).Collec-tively,our data could be interpreted as a lack of a direct interaction between the hCG-induced surge in StAR and uPA.

However,considering that plasmin generated by uPA

is

F I

G .6.Kinetics of the early effects of hCG on StAR and uPA in the mLTC-1cells (A).Cells were stimulated with hCG 100ng/ml for 15min,30min,60min,2h,4h,and 8h.Autoradiographs of the PCR products were scanned,the expression was normalized to the HPRT signal,and levels of uPA and StAR at the beginning of the experiment were arbitrarily fixed to 1.The values are the mean ?SEM of n ?3;*,P ?0.05.B and C,Effects of a cocktail of protease inhibitors and trypsin on the hCG-or bu 2cAMP-induced StAR expression in mLTC-1cells (B and C)and primary cultures of Leydig cells prepared from 3-and 8-wk-old testes (B).Cells were pretreated with a cocktail of inhibitors or trypsin (100or 1000ng/ml)and stimulated for 2h with hCG 100ng/ml (B)or bu 2cAMP 1m M (C).Autoradiographs of the PCR products were scanned and expression was normalized to the HPRT signal.The values are the mean ?SEM of n ?4.*,P ?0.05,compared with untreated cells of the time-matched control.In C,the number of PCR cycles was lowered to 19instead of 22for the bu 2cAMP samples to avoid saturation of the signal.This explains why the intensity of the PCR bands between the treated and control samples are of the same amplitude.

Odet et al.?Serine Proteinases,Serpins,and Mouse Testis Endocrinology,September 2006,147(9):4374–43834381

highly active in degrading ECM components and initiating

a proteolytic cascade through activation of other proteases(3,

34),indirect interactions are likely.A similar situation has been described in the ovary in which plasmin action takes place early after the LH peak to ensure follicular rupture and ovulation(35,36).In addition,ECM serves as a reservoir for a variety of biologically active molecules,which may inter-fere with Leydig cell steroidogenesis ability(31,33,37)when released and/or activated by proteases(3,34).Thus,it could be suggested that uPA is involved in the metabolism of ECM components in the interstitial tissue surrounding the Leydig cells in the testis.In this context,it should be mentioned that adhesion,shape,proliferation,and gene expression of mouse Leydig cells are influenced by ECM in vitro(38),that rat Leydig cells are able to synthesize ECM proteins in vitro(39), and that fibronectin and type IV collagen induce the down-regulation of steroidogenic response to gonadotropins in adult rat Leydig cells(40,41).It would be of interest to determine whether hCG modulates the nature of the ECM components synthesized in vitro by Leydig cells.

The second set of serine proteases and SERPINs expressed in Leydig cells,i.e.matriptase-2(in tumor cells),kallikrein-21, SERPINA5,and SPINT2,was negatively regulated by hCG. Taking into account that serine proteinases exhibit degrading activity against ECM(3,34),whereas inhibitors oppose this activity,our data suggest that,in contrast to its early-induced effects,the hCG-induced long-term effects may stabilize the ECM proteins in the interstitial area surrounding the Leydig cells in the mouse testis.However,the situation is far from clear regarding SERPINA5.Indeed,we previously reported that SERPINA5is expressed in Leydig cells throughout de-velopment in which it could oppose a uPA activity(18).The present findings on the kinetics of the hCG-induced effects on uPA and SERPINA5do not support our previous hy-pothesis,and SERPINB2is a better candidate for opposing uPA activity in Leydig cells.SERPINA5is unique in that serpina5?/?male mice are sterile because of increased or unopposed proteolytic activity,which might be responsible for premature release and degeneration of developing germ cells in the lumen of the tubules(42).The recent findings that SERPINA5is also a Sertoli cell product and that its expres-sion is positively controlled by testosterone(Refs.10and14 and our unpublished data)may explain the phenotype of the transgenic mice.In this context,the relationship between the hCG down-regulation of SERPINA5and the phenotype of the serpina5?/?male mice is still unclear.Future studies will be useful to clarify this point.

In conclusion,we demonstrate that gonadotropins sub-stantially modify the proteolytic balance in Leydig cells and probably the metabolism of ECM proteins.Therefore,even though a direct interplay between the early hCG-induced surge of uPA and StAR is unlikely,our data should be con-sidered together with the described influence of ECM pro-teins on Leydig cell steroidogenesis.Future studies will aim at clarifying whether the molecules studied here interact or display independent proteolytic roles within Leydig cells.It would also be of interest to determine whether common transcription factors drive their expression in Leydig cells.

Acknowledgments

We are indebted to Douglas M.Stocco(Texas University,Lubbock, TX),and Margarethe Geiger(Department of Vascular Biology and Thrombosis Research,University of Vienna,Vienna,Austria)for pro-viding us with the MLTC-1cells and the anti-PCI antibody,respectively. Thierry Blache`re and Annick Lefe`vre are thanked for providing us with the enriched fractions of germ cells from mouse testes.We are grateful to Maguelone G.Forest(Hopital Debrousse,Lyon,France)and Sophie Rousseaux(Institut National de la Sante′et de la Recherche Me′dicale U309,La Tronche,France)for their contribution in careful reading of the manuscript.

Received April13,2006.Accepted May19,2006.

Address all correspondence and requests for reprints to:Dr.B.Le Magueresse-Battistoni,Institut National de la Sante′et de la Recherche Me′dicale Unite′418/Institut National de la Recherche Agronomique, Unite′Mixte de Recherche1245,University of Lyon Claude Bernard, Hopital Debrousse,29rue soeur Bouvier,69322Lyon cedex05,France. E-mail:lemagueresse@lyon.inserm.fr.

This work was supported by Institut National de la Sante′et de la Recherche Me′dicale,Institut National de la Recherche Agronomique, and the University of Lyon Claude Bernard.F.O.is funded by the Ministe`re de la Recherche et de la Technologie and the Fondation pour la Recherche Me′dicale.A.V.is funded by Organon(Azko,Nobel).

Author disclosure summary:F.O.,A.V.,B.L.M.-B.have nothing to declare.

References

1.Puente XS,Lopez-Otin C2004A genomic analysis of rat proteases and pro-

tease inhibitors.Genome Res14:609–622

2.Puente XS,Sanchez LM,Overall CM,Lopez-Otin C2003Human and mouse

proteases:a comparative genomic approach.Nat Rev Genet4:544–558

3.Dano K,Behrendt N,Hoyer-Hansen G,Johnsen M,Lund LR,Ploug M,

Romer J2005Plasminogen activation and cancer.Thromb Haemost93:678–681

4.Silverman GA,Bird PI,Carrell RW,Church FC,Coughlin PB,Gettins PGW,

Irving JA,Lomas DA,Luke CJ,Moyer RW,Pemberton PA,Remold-O’Donnell E,Salvesen GS,Travis J,Whisstock JC2001The Serpins are an expanding superfamily of structurally similar but functionally diverse pro-teins.J Biol Chem276:33293–33296

5.Gettins PGW2002Serpin structure,mechanism and function.Chem Rev

102:4751–4803

6.Pike RN,Buckle AM,Le Bonniec BF,Church FC2005Control of the coag-

ulation system by serpins.FEBS J272:4842–4851

7.De Kretser DM,Loveland KL,Meinhardt A,Simorangkir D,Wreford N1998

Spermatogenesis.Hum Reprod13(Suppl1):1–8

8.Griswold MD1998The central role of Sertoli cells in spermatogenesis.Sem

Cell Dev Biol9:411–416

9.Huhtaniemi I2003Gonadotrophin actions on the testis—genotypes and phe-

notypes of gonadotrophin and gonadotrophin receptor mutations.Endocr Dev 5:81–103

10.Denolet E,De Gendt K,Allemeerch J,Engelen K,Marchal K,Van Hum-

melen P,Tan KAL,Sharpe RM,Saunders PTK,Swinnen J,Verhoeven G2006 The effect of a Sertoli cell-selective knockout of the androgen receptor on testicular gene expression in prepubertal mice.Mol Endocrinol20:321–334 11.Meistrich MC1977Separation of spermatogenic cells and nuclei from rodent

testes.Methods Cell Biol15:15–54

12.Mather JP,Phillips DM1984Primary culture of testicular somatic cells.In:

Barnes DW,Sirbasku DA,Sato GH,eds.Methods for serum-free culture of cells of the endocrine system.New York:Liss;29–45

13.Le Magueresse-Battistoni B,Pernod G,Sigillo F,Kolodie′L,Benahmed M

1998Plasminogen activator inhibitor-1is expressed in cultured rat Sertoli cells.

Biol Reprod59:591–598

14.Anway MD,Show MD,Zirkin BR2005Protein C inhibitor expression by

adult rat Sertoli cells:effects of testosterone withdrawal and replacement.J Androl26:578–585

15.Manna PR,Huhtaniemi IT,Stocco DM2004Detection of hCG responsive

expression of the steroidogenic acute regulatory protein in mouse Leydig cells.

Biol Proceed Online6:89–93

16.Longin J,Guillaumot P,Chauvin MA,Morera AM,Le Magueresse-Battis-

toni B2001MT1-MMP in rat testicular development and the control of Sertoli cell proMMP-2activation.J Cell Sci114:2125–2134

17.Guyot R,Magre S,Leduque P,Le Magueresse-Battistoni B2003Differential

expression of tissue inhibitor of metalloproteinases type1(TIMP-1)during mouse gonad development.Dev Dyn227:357–366

18.Odet F,Guyot R,Leduque P,Le Magueresse-Battistoni B2004Evidence for

4382Endocrinology,September2006,147(9):4374–4383Odet et al.?Serine Proteinases,Serpins,and Mouse Testis

similar expression of protein C inhibitor and the urokinase-type plasminogen activator system during mouse testis development.Endocrinology145:1481–1489

19.Tolli R,Monaco L,DiBonito P,Canipari R1995Hormonal regulation of

urokinase-and tissue-type plasminogen activator in rat Sertoli cells.Biol Re-prod53:193–200

20.Itoh H,Hanasuna R,Kataoka H,Yamauchi M,Akiyama Y,Miyazawa K,

Kitamura N2000Mouse hepatocyte growth factor activator gene:its expres-sion not only in the liver but also in the gastrointestinal tract.Biochim Biophys Acta1491:295–302

21.Kirchhofer D,Peek M,Lipari MT,Billeci K,Fan B,Moran P2005Hepsin

activates pro-hepatocyte growth factor and is inhibited by hepatocyte growth factor inhibitor-1B(HAI-1B)and HAI-2.FEBS Lett579:1945–1950

22.Matsui H,Takahashi T2001Mouse testicular Leydig cells express Klk21,a

tissue kallikrein that cleaves fibronectin and IGF binding protein-3.Endocri-nology142:4918–4929

23.Hooper JD,Campagnolo L,Goodarzi G,Truong TN,Stuhlmann H,Quigley

JP2003Mouse matriptase-2:identification,characterization and comparative mRNA expression analysis with mouse hepsin in adult and embryonic tissues.

Biochem J373:689–702

24.Yamamoto K,Loskutoff DJ1998Extrahepatic expression and regulation of

protein C in the mouse.Am J Pathol153:547–555

25.Delaria KA,Muller DK,Marlor CW,Brown JE,Das RC,Roczniak SO,

Tamburini PP1997Characterization of placental bikunin,a novel human serine protease inhibitor.J Biol Chem272:12209–12214

26.Kataoka H,Itoh H,Nuki Y,Hamasuna R,Nagamuna S,Kitamura N,Shi-

momura T2002Mouse hepatocyte growth factor(HGF)activator inhibitor type2lacking the first kunitz domain potently inhibits the HGF activator.

Biochem Biophys Res Commun290:1096–1100

27.Yamauchi M,Itoh H,Nagamuna S,Koono M,Hasui Y,Osada Y,Kataoka H

2002Expression of hepatocyte growth factor activator inhibitor type2(HAI-2) in human testis:identification of a distinct transcription start site for the HAI-2 gene in testis.Biol Chem383:1953–1957

28.Matsui H,Takano N,Moriyama A,Takahashi T2005Single-chain tissue-type

plasminogen activator is a substrate of mouse glandular kallikrein24.Zoolog Sci22:1105–1111

29.Catizone A,Ricci G,Galdieri M2001Expression and functional role of

hepatocyte growth factor receptor(c-Met)during postnatal rat testis devel-opment.Endocrinology141:1828–183430.Medcalf RL,Stasinopoulos SJ2005The undecided serpin.The ins and outs

of plasminogen activator inhibitor type2.FEBS J272:4858–4867

31.Saez JM1994Leydig cells:endocrine,paracrine and autocrine regulation.

Endocr Rev15:574–626

32.Hauet T,Liu J,Li H,Gazouli M,Culty M,Papadopoulos V2002PBR,StAR,

and PKA:partners in cholesterol transport in steroidogenic cells.Endocr Res 28:395–401

33.Stocco DM,Wang X Jo Y,Manna PR2005Multiple signaling pathways

regulating steroidogenesis and steroidogenic acute regulatory protein expres-sion more complicated than we thought.Mol Endocrinol19:2647–2659 34.Vu TH,Werb Z2000Matrix metalloproteinases:effectors of development and

normal physiology.Genes Dev14:2123–2133

35.Macchione E,Epifano O,Stefanini M,Belin D,Canipari R2000Urokinase

redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles.Biol Reprod62:895–903

36.Ny T,Wahlberg P,Bra¨ndstro¨m IJM2002Matrix remodeling in the ovary:

regulation and functional role of the plasminogen activator and matrix met-alloproteinase systems.Mol Cell Endocrinol187:29–38

37.Gnessi L,Fabbri A,Spera G1997Gonadal peptides as mediators of devel-

opment and functional control of the testis:an integrated system with hor-mones and local environment.Endocr Rev18:541–609

38.Vernon RB,Lane TF,Angello JC,Sage H1991Adhesion,shape,proliferation,

and gene expression of mouse Leydig cells are influenced by extracellular matrix in vitro.Biol Reprod44:157–170

39.Denduchis B,Schteingart H,Cigorraga S,Vianello SE Casanova MB,Lustig

L1996Immunodetection of cell adhesion molecules and extracellular matrix proteins in rat Leydig cel cultures.Int J Androl19:33–361

40.Diaz ES,Pellizzari E,Meroni S,Cigorraga S,Lustig L,Denduchis B2002

Effect of extracellular matrix proteins on in vitro testosterone production by rat Leydig cells.Mol Reprod Dev61:493–503

41.Diaz ES,Pellizari E,Casanova M,Cigorraga SB,Denduchis B2005Type IV

collagen induces down-regulation of steroidogenic response to gonadotropins in adult rat Leydig cells involving mitogen-activated protein kinase.Mol Reprod Dev72:208–215

42.Uhrin P,Dewerchim M,Hilpert M,Chrenek P,Schofer C,Zechmeister-

Machhart M,Kronke G,Vales A,Carmeliet P,Binder BR,Geiger M2000 Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility.J Clin Invest106:1531–1539

Endocrinology is published monthly by The Endocrine Society(https://www.wendangku.net/doc/0612774404.html,),the foremost professional society serving the

endocrine community.

Odet et al.?Serine Proteinases,Serpins,and Mouse Testis Endocrinology,September2006,147(9):4374–43834383

部编七下《阿长与山海经》教学设计

For personal use only in study and research; not for commercial use 《阿长与<山海经>》教学设计 鲁迅先生的《阿长与〈山海经〉》所追忆的保姆，她是一个粗人，没有文化、粗俗、 好事，是一个很不幸的人，但她又是一个热望一生平安的劳动妇女，质朴善良，热心帮助孩 子解决疑难。鲁迅先生深情地抒写了对她的真挚的怀念。教读这篇文章，应引导学生学习本文选取典型事例表现人物性格的方法，从而更深地感悟鲁迅对阿长的深厚怀念之情，领会阿长性格中的纯真美。 教学设想： 本单元的课文都是关于“小人物”的故事，平凡又普通，人物也不尽善尽美，但却给 人留下深刻的印象，阿长这个人物初看令人无奈，甚至厌烦，细读却让人回味无穷，通过此篇教学，力图让学生明白人都是复杂的，性格有多面，通过指导，力图使学生戒绝对人物的线性分析，要树立一个意识：不能用简单的好人坏人来评判人物，应从文本出发，细品人物形象，得出结论。 像《阿长与〈山海经〉》这样难度较大的作品，学生活动往往难以设计，因为篇幅长， 头绪多，可资学习供鉴的东西很多，很容易搞成满堂灌，在教学实践中发现采用主问题讨论 法比较可行。针对此篇的教学，基本上是扣住感情的变化，抓住四个视角，设计主问题讨论法来进行!从不太佩服——不耐烦——一时的空前的敬意——憎恶——新的敬意——怀 念，这是对阿长感情的变化，这时就有“少年鲁迅眼中的阿长”“中年鲁迅眼中的阿长”两个视角，在解读中，还有“学生眼中的阿长”“老师眼中的阿长”这些角度的衍生，有了这 些角度才有了自我的阅读，于是设计了主问题：“阿长买《山海经》为什么会让鲁迅铭记终 身呢?”之后又把这个问题分解为几个子问题： 1.你眼中的阿长是个什么样的人? 2.鲁迅对阿长的感情发生了什么变化?3.买回《山海经》为什么会让少年震惊?4.少年鲁迅与中年的鲁迅对阿长的感情有无变化?此外，课前的充分预习也相当重要，收集学生问题，进行归类整理，在查资料的基础上把课堂交给学生。 教学目标： 1.整体感知课文，了解阿长这个人物形象及作者的感情。 2.重点研读买《山海经》的部分，体会语句的深层含义。

《阿长与山海经》教学设计

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（）、（）。 2、本文选自（）。这是鲁迅唯一的一本（）集。 3、简介《山海经》 《山海经》是中国先秦重要古籍，也是一部富于神话传说的最古老的奇书。 《山海经》传世版本共计18卷，包括《山经》5卷，《海经》13卷。 山海经内容主要是民间传说中的地理知识，包括山川、地理、民族、物产、药物、祭祀、巫医等。保存了包括夸父逐日、精卫填海、大禹治水等不少脍炙人口的远古神话传说和寓言故事。 《山海经》具有非凡的文献价值，对中国古代历史、地理、文化、中外交通、民俗、神话等的研究，均有参考。其中的矿物记录，更是世界上最早的有关文献。 4、注意加点字词的读音。 骇． ( ) 惶．急( ) 疮疤 ．．( ) ( ) 掳． ( ) 渴慕．( ) 霹．雳( ) 悚．( ) 诘．问( ) 惧惮．( ) 孤孀．( ) 5、解释词语： 莫名其妙：深不可测：情有可原： 惶急：诘问：惧惮：震悚： 合作探究 1、本文主要刻画了哪个人物？围绕着这个人物写了哪几件事？ 阿长，共写了七件事： （1）切切察察的毛病。（2）摆成“大”字的睡相。 （3）令人讨厌的种种规矩。（4）一肚子烦琐的道理。 （5）“长毛”的故事。（6）谋害我的隐鼠。 （7）吃福橘。（8）给我买《山海经》。 2、文章写长妈妈的事情哪些事是详写？哪些事是略写？为什么要这样 安排？ 略写了阿长的称呼的来历、“切切察察”的毛病、摆成“大”字的睡相、令人讨厌的种种规矩、“长毛”的故事，谋死隐鼠，吃福橘。略写的那些内容能使人对长妈妈有个初步了解，她的外形特征，真实地反映长妈妈的一些毛病，但这些并不能削弱对阿长妈妈的敬意。 详写了为“我”买《山海经》一事。详写买《山海经》一事，却令人对长妈妈刮目相看，在作者看来“别人不肯做，或不能做的事情，她却能够做成功”，我们可以想到幼小的鲁迅对长妈妈的敬佩和感激之情。

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《阿长与山海经》教学设计 教学目标 一、知识目标 1、详略得当。 2．学习细节描写对刻画人物的作用。 二.能力目标 1.培养学生观察生活，感悟生活的能力。 2.掌握文中出现的陌生字词，达到会读、会写、会用。 3.能自主发现问题，提出问题，合作解决文章理解上的疑难，培养学生问题能力。 三.情感态度与价值观 深入领会作者对阿长的感情，建立朴素的阶段意识，热爱劳动人民。 教学重点 学习鲁迅通过人物动作、语言描写来塑造人物的写法，即人物的语言、动作描写。 教学难点： 学生对文章主题的领会。 教学方法 1.阅读领悟法 2.互动研讨法 教学过程： 第一课时 课型：新授。 方法：自学指导。 过程： 一、导入。 同学们，在初一时我们学过鲁迅先生的回忆性散文《从百草园到三味书屋》，文中美女蛇的故事给美丽的百草园蒙上了一层神秘的面纱。而故事的讲述者长妈妈，在鲁迅先生的作品中几次都提起，可见她是一个对自己很有影响的人物．那么她到底是个什么样的人呢？今天就走进鲁迅先生为她专门写的《阿长与<山海经>》。

二、检查预习 （一）注音： 掳（lu ）去震悚（song ）惶（huang ）急疮（chuang ）疤 诘（jie ）问惧惮（dan ）霹雳{pi li } （二）为下列词语中加括号的字选择正确的解释，将序号填在括号内。 １.莫（名）其妙（）Ａ名字Ｂ说出Ｃ名 誉Ｄ有名的 ２.（念）念不忘（）Ａ惦记、常常地想Ｂ念头Ｃ读Ｄ“廿”的大写 ３.（疏）懒（）Ａ分散Ｂ不亲密Ｃ粗心Ｄ不熟悉 ４.烦琐之（至（）Ａ极Ｂ以至Ｃ到Ｄ至于 三、查工具书，解释下列词语： 惶急：恐惧着急。 诘问：追问、责问。 惧惮：惧怕。惮，害怕。 渴慕：迫切地羡慕。 霹雳：云和地面之间发出的一种强烈的雷电现象。 烦琐：繁杂琐碎。 疮疤：疮口表面所结的痂。 毫不相干：一点关系也没有。 情有可原：这种情况有可以原谅之处。 深不可测：深度大，难以测量。形容深奥难以理解。 莫名其妙：没有人能说明它的奥妙{道理}。名，说出。 四、品味细节 1．读课文，要求找出作者感情变化的原因。 教师提示：我们知道，“没有无缘无故的爱，也没有无缘无故的恨”作者的感情变化是因为发生了一些事情。也就是说，这些事情就是我感情变化的原因。

《阿长与山海经》优秀教案设计一3篇

《阿长与山海经》优秀教案设计一3篇 《阿长与山海经》优秀教案设计一【教学内容】 选自义务教育课程标准实验教科书《语文》八年级上册第二单元 【教材分析】 鲁迅先生的《阿长与山海经》所追忆的保姆，她是一个粗人，没有文化、粗俗、好事，是一个很不幸的人。但她又是一个热望一生平安的劳动妇女，质朴善良、热心帮助孩子解决疑难。鲁迅先生深情地抒写了对她的真挚的怀念。教读这篇文章，应引导学生学习本文选取典型事例表现人物主要性格以及详写与略写相结合的写法。了解叙述、描写等表达方式，体会描写的作用和方法，从而更深地感悟鲁迅对阿长的深厚怀念之情，领会阿长性格中的纯真美。 【教学目标】 1、整体感知课文，了解阿长这个人物形象及作者的感情 2、重点研读买《山海经》的部分，体会语句的深层含义。 3、感悟鲁迅对阿长的深厚怀念之情，领会阿长性格中的纯真美 【重点难点】 1、学习本文选取典型事例表现人物主要性格的写法。 2、领悟这篇回忆性散文的用双重眼光看待人物和欲扬先抑的

写法。 【教具准备】录音机、配乐磁带、幻灯片 【课时安排】2课时 第一课时 【教学过程】 一、谈话导入： 1、说起鲁迅，我想大家应该都不陌生吧!谁来说说鲁迅? 2、在鲁迅的童年生活中，长妈妈是个很有影响的、特殊的人物。鲁迅没有专门写过回忆母亲的文章，却在他四十五岁那年，写了这篇回忆性散文——《阿长与》。以纪念自己童年时期的一个保姆，可见对她的感情之深。(背景音乐) 让我们满怀深情地读一读课题。 3、读了这个题目，你有什么疑问? (生质疑) 二、整体感知： 1、阿长究竟是怎样一个人，她与《山海经》又有什么关系，让鲁迅对他有这样深的感情，让我们带着疑问速读课文。 2、说说阿长在你眼中是个怎样的人? (生预设：无知、淳朴、善良、粗鲁……) 3、师小结： 总之，阿长是个好人，但又不是十全十美的人，她是一个复杂的人。 三、拎出线索： 1、那么这样一个人，作者对她的感情怎样呢?

阿长与山海经教案

阿长与《山海经》教学设计 54杨丽娟 一、教学目标 1、知识目标 掌握以下字词： 骇掳悚惶急疮疤诘问渴慕霹雳疏懒惧惮孤孀 2、能力目标 （一）学习本文抓住特征刻画人物的方法———详略得当。 （二）学习本文欲扬先抑的写法。 （三）了解阿长的性格特点，并能够用简单的语言概括。 3、人文目标 把握文中作者情感变化的过程，体会鲁迅先生对长妈妈的怀念、同情和赞美之情。 二、教学难点 文章围绕阿长写了哪些事，重点写了什么，从这些事情中可以看出阿长什么样的性格特征。找出表现作者对待长妈妈情感态度变化的语句，学习欲扬先抑的表现手法，体会“抑”中的“扬”。

三、教学重点 教学中要引导学生通过反复的朗读加深对课文内容的理解，分析课文详略得当的写作特点。 四、学时安排2课时 五、教学过程 （一）第一学时 1、本学时教学目标 整体感知全文，学习欲扬先抑的表现手法，把握长妈妈的性格特征。 2、教学过程 ⑴导入语 同学们，在上个学期，我们已经学过鲁迅的一篇散文叫《从白草园到三味书屋》，其中作者写到了美女蛇故事的惊险、有趣、生动。大家还有印象吗同时，在文章中鲁迅还提到了一个人，谁没错！就是长妈妈。长妈妈是鲁迅儿时的保姆。从文章中我们可以看出鲁迅与长妈妈的关系是非常密切的，可以说长妈妈对鲁迅今后的写作有着一定的影响，在鲁迅的许多文章中，我们都可以找到阿长的影子。那么，阿长到底是个什么样的人呢今天，我们就一起来看鲁迅先生的《阿长与〈山海经〉》。 ⑵作者简介 鲁迅（1881年9月25日～1936年10月19日）原名周樟寿,后改

名周树人，浙江绍兴人。伟大的无产阶级文学家、思想家和革命家。中国现代文学的奠基人。“鲁迅”是他在1918年5月发表《狂人日记》时用的笔名。而《狂人日记》也是中国文学史上第一篇白话小说。鲁迅的小说集还有《呐喊》、《彷徨》、《故事新编》等。散文集有《朝花夕拾》等。 ⑶写作背景 1926年3月18日，北洋军阀政府枪杀进步学生，鲁迅受反动政府通缉，不得不到厦门大学任教，后又受守旧势力的排挤。在那种境况下，作者不愿想到目前，只能回忆少年生活写点文章聊以自慰。于是，写出了回忆散文集《朝花夕拾》，本文就选自《朝花夕拾》的第二篇。 ⑷请同学们仔细阅读课文，并思考下列问题（在预习的前提下进行，并用铅笔在文中相应的地方勾画出答案） ①文章写了阿长的哪些事情重点写了哪件事情 明确： 文章介绍了人们对长妈妈的称呼，称呼的由来和她外形的特点，以及她的一些不好的习惯。如写她喜欢“切切察察”（3段），喜欢“告状”，睡觉爱摆“大”字等；接着写她懂得的许多“我听得不耐烦”的规矩。比如元旦、除夕吃福橘，人死了要说“老掉了”等（6-12）；最后写长妈妈为“我”买《山海经》的事，给“我”买《山海经》一事为重点。 ②归纳每件事情刻画出阿长的性格特征，总结归纳阿长的形象。 明确： 长妈妈名称的来历（1-2）------地位低下 切切察察，竖起第二个手指，在空中上下摆动，或者点着对方或自

(完整版)阿长与山海经-优秀教案

6 阿长与《山海经》 一、教学目标 1．知识和能力目标： 诵读感知课文，理清文章的思路，理解文中带有感情色彩的词句，把握作者情感变化的过程。 2．过程和方法目标： 体会先抑后扬的表现手法；学习详略得当来使主题更鲜明，人物更突出的写法。 3．情感态度和价值观目标： 体会作者对长妈妈的怀念、同情和赞美之情。 二、教学重难点 重点：理解文中带有感情色彩的词句，把握作者情感变化的过程。 难点：体会先抑后扬的表现手法；学习详略得当来使主题更鲜明，人物更突出的写法。 三、教学课时 2课时 四、教学方法讨论法、点拨法、讲授法相结合 五、教学过程 第一课时： 教学内容：整体感知文章，根据对文章描写人物的语句进行分析，初步掌握人物形象 （一）导入新课 1、同学们，提到鲁迅，大家并不陌生，谁来说说你了解的鲁迅的相关知识？（鲁迅，原名周树人，字豫才，浙江绍兴人。我国著名思想家、文学家、革命家。1918年5月，首次用“鲁迅”的笔名，发表中国现代文学史上第一篇白话小说《狂人日记》，奠定了新文学运动的基石。） 2、那我们学过鲁迅先生的哪些作品呢？（《风筝》《从百草园到三味书屋》《社戏》）在这些文章中，你们觉得鲁迅先生对他的家乡，家乡的亲人怀有一种什么样的情感？（深深的眷恋、无尽的思念） 3、鲁迅先生的文章在过去被誉为“匕首”、“投枪”，但在这些作品中，尤其是在《从百草园到三味书屋》和《社戏》中，我们看到的已不再是一个“横眉冷对千夫指”的战士，而是一个浪迹天涯的游子，心中充满了对家乡的人和事的眷念。在他的记忆中，家乡几乎已经幻化成了一个梦境般的美好世界。 4、大家还记得《从百草园到三味书屋》中那个神秘莫测的美好蛇的故事吗？（指名复述故事）它给百草园蒙上了一层面纱。讲这故事的人是谁呢？（长妈妈）长妈妈是作者儿时的保姆，长妈妈知道许多事情，懂得许多道理，可以说，她是民间文化的载体，对于鲁迅来说，她可是一个有影响力的人物。今天，就让我们一起走进课文，去感受作者对长妈妈的深情。 5、板书课题，作者。 （二）《朝花夕拾》及《山海经》简介： 《朝花夕拾》，鲁迅散文集。在《莽原》上发表示题为《旧事重提》，编辑出版时改用现名。共收入1926年创作的回忆性散文10篇。本散文集以一个侧面表现了鲁迅说处的时代的社会风貌。即《阿长与<山海经>》《藤野先生》《范爱农》。《藤野先生》记师恩，《范爱农》记友情，《阿长与<山海经>》则记作者儿时的保姆。

阿长与山海经教学设计

《阿长与<山海经>》教学设计 【教学目标】 1．学习详略得当来使主题更鲜明,人物更突出得写法. 2。学习如何才能让所写得人物真实、感人得写法。 3．体会文中充满情感语言得深意. 【教学重难点】 文章围绕阿长写了哪些事,重点写了什么,从这些事中可以瞧出阿长什么样得性格特征。找出表现作者对待长妈妈情感态度变化得语句，学习欲扬先抑得表现手法,体会“抑”中得“扬”。 【教学时数】2课时 【教学过程】 一、导入新课 同学们，还记得《从百草园到三味书屋》中那个神秘莫测得美女蛇得故事吗?它给百草园蒙上了一层神秘得面纱.讲这故事得人就是谁呢?——对，她就就是长妈妈.长妈妈就是作者儿时得保姆，长妈妈知道许多事情，懂得许多道理,可以说，她就是民间文化得载体, 对于鲁迅来说，她可就是一个有影响力得人物.可就是在《阿长与《山海经》》里,鲁迅却将长妈妈称为“阿长”,这就是为什么呢？作者究竟就是怀着怎样得一种感情来写长妈妈得呢？今天就让我们一起来走进课文,去感悟作者对长妈妈得深切感情。本文选自《朝花夕拾》。 二、作者与《朝花夕拾》简介： 鲁迅(1881～19３６）,中国文学家、思想家、革命家与教育家.原名周树人,字豫才，浙江绍兴人， 《朝花夕拾》就是鲁迅得回忆性散文集，写于1926年２月至11月间,共10篇。前５篇写于北京,后5篇写于厦门大学.这１0篇散文，曾以“旧事重提”为总题，陆续发表在《莽原》杂志上。１９27年广州“七·一五"政变之后,鲁迅因营救被捕学生无效，愤而辞去中山大学教职,在白色恐怖中又将这10篇回忆散文汇拢,加写了《小引》与《后记》，编成一集，改了一个更好听得名称：《朝花夕拾》本散文集以一个侧面表现了鲁迅所处得时代得社会风貌。即《阿长与〈山海经>》《藤野先生》《范爱农》。《藤野先生》记师恩，《范爱农》记友情,《阿长与<山海经＞》则记作者儿时得保姆. 三、整体感知 (一）仔细读课文, 思考下列问题，小组自由讨论. 1、为阿长设计“个人简历”认识阿长。 明确：

《阿长与山海经》 教案教学设计

《阿长与山海经》教案教学设计 鲁迅 教学目标 一.知识目标 1．围绕中心选取材料；选取材料详略得当。 2．学习细节描写对刻画人物的作用。 二.能力目标 1.培养学生观察生活，感悟生活的能力。 2.掌握文中出现的陌生字词，达到会读、会写、会用。 3.能自主发现问题，提出问题，合作解决文章理解上的疑难，培养学生问题能力。 三.情感态度与价值观 深入领会作者对阿长的感情，建立朴素的阶段意识，热爱劳动人民。 教学重点 学习鲁迅通过人物动作、语言描写来塑造人物的写法，即人物的语言、动作描写。 教学难点： 学生对文章主题的领会。 教学方法 1.阅读领悟法 2.互动研讨法 教学过程： 第一课时 一、导入。 同学们，在初一时我们学过鲁迅先生的回忆性散文《从百草园到三味书屋》，文中美女蛇的故事给美丽的百草园蒙上了一层神秘的面纱。而故事的讲述者长妈妈，在鲁迅先生的作品中几次都提起，可见她是一个对自己很有影响的人物．那么她到底是个什么样的人呢？今天就走进鲁迅先生为她专门写的《阿长与<山海经>》。 二、检查预习 （一）注音： 掳（ lu ）去震悚（ song ）惶（huang ）急疮（ chuang ）疤 诘（ jie ）问惧惮（dan ）霹雳{pi li } （二）为下列词语中加括号的字选择正确的解释，将序号填在括号内。 １.莫（名）其妙（）Ａ名字Ｂ说出Ｃ名誉Ｄ有名的 ２.（念）念不忘（）Ａ惦记、常常地想Ｂ念头Ｃ读Ｄ“廿”的大写 ３.（疏）懒（）Ａ分散Ｂ不亲密Ｃ粗心Ｄ不熟悉

４.烦琐之（至（）Ａ极Ｂ以至Ｃ到Ｄ至于 三、查工具书，解释下列词语： 惶急：恐惧着急。 诘问：追问、责问。 惧惮：惧怕。惮，害怕。 渴慕：迫切地羡慕。 霹雳：云和地面之间发出的一种强烈的雷电现象。 烦琐：繁杂琐碎。 疮疤：疮口表面所结的痂。 毫不相干：一点关系也没有。 情有可原：这种情况有可以原谅之处。 深不可测：深度大，难以测量。形容深奥难以理解。 莫名其妙：没有人能说明它的奥妙{道理}。名，说出。 四、品味细节 1．读课文，要求找出作者感情变化的原因。 教师提示：我们知道，“没有无缘无故的爱，也没有无缘无故的恨”作者的感情变化是因为发生了一些事情。也就是说，这些事情就是我感情变化的原因。下面请同学们分小组联读课文，用简要的话概括出这些事情。 明确：一共写了七件事。 ①常喜欢切切察察 ②限制我的行动 ③睡觉时挤得我无法翻身 ④懂得许多规矩和麻烦的礼节 ⑤讲长毛攻城时护城 ⑥谋害我的隐鼠 ⑦为我买《山海经》 2、这些事中哪些地方写得最细，最精彩。请找出来，用“ .......写得好，它写出了......”的句式进行品析并说话。 五.小结 这节课我们理出了事件，认识了人物。 六.布置作业 研讨与练习一.二。

《阿长与山海经》教案 (人教版七年级下册)

《阿长与山海经》教案 (人教版七年级下 册) 学习目标： 1.知识与能力： 1、积累有关字词了解文学常识。 2、了解叙述、描写等表达方式，体会人物描写方法。 3、整体把握课文，深入了解阿长的人物形象以及作者 深厚感情。 2.过程与方法：自主合作探究的方法。 3.情感态度与价值观：感悟鲁迅对阿长的深厚怀念之情，领会“阿长”性格中人性美。 4.教学重点：透过文章平和含蓄的语言，文章深刻隽永 的意蕴。 5.教学难点：通过文章欲扬先抑的写法深入了解长妈妈 的人物形象，体会作者所表达的思想感情。 课时安排：2课时 教与学互动设计： 一、导入语： 同学们，在上个学期，我们已经学过鲁迅的一篇散文叫《从白草园到三味书屋》，其中作者写到了美女蛇故事的惊险、有趣、生动。大家还有印象吗？同时，在文章中鲁迅还 提到了一个人，谁？没错！就是长妈妈。长妈妈是鲁迅儿时

的保姆。从文章中我们可以看出鲁迅与长妈妈的关系是非常 密切的，可以说长妈妈对鲁迅今后的写作有着一定的影响， 在鲁迅的许多文章中，我们都可以找到阿长的影子。那么， 阿长到底是个什么样的人呢？今天，我们就一起来看鲁迅先 生的《阿长与〈山海经〉》。 二、预习交流 1、走近鲁迅及《朝花夕拾》 2、关于《山海经》有 3、4个同学提出 3、关于字词 4、简单的课文内容感知（集中2个问题）学贵质疑，在预习时，同学们对课文内容提出许多问题，我整理了一下， 有两个最典型的问题：第一个是有关作者思想感情的，正如 很多同学所述“作者对阿长的感情究竟是怎样的？”这类问 题提的很有深度，抓住了文章的要点、把握住了文章的主旨。第二类是有关题目的，正如部分同学所述“本文主要写的是 阿长，但题目为什么是《阿长与山海经》，能不能改成阿长？”这类问题提得很有价值，说明这些同学对文章的内容有整合 的过程，是善于动脑、善于思考的人。 三、探讨问题 1.请同学们浏览文章后，把预习中勾画的表现作者对阿 长感情变化的词语交流一下。 （预习稿）

(完整版)《阿长与山海经》教学设计

《阿长和山海经》 【教学目标】 1. 会读会写本文的生字词；把握课文的内容，理解阿长的形象。 2．了解课文围绕中心选择材料、安排材料，详略得当的写作特点。 3.注意分析关键语句，体会作者词语运用之妙。 4.学习欲扬先抑的写作手法。 5．分析人物形象，把握文章中心，理解文章所表达的思想感情。 【教学重难点】 分析人物形象，把握文章中心，理解文章所表达的思想感情； 了解课文围绕中心选择材料、安排材料，详略得当的写作特点。 学习欲扬先抑的写作手法 【教学方法】朗读法、引导法、讨论法。 【课时安排】两课时 【教学过程】第一课时： 一、创设情境，激情导入 （一）导语 在鲁迅先生的散文《从百草园到三味书屋》里，鲁迅除写了自己的老师寿镜吾先生之外，还写到了一个人，这个人是谁呢？对，是长妈妈，她讲的美女蛇的故事真是神秘莫测，给百草园蒙上一层神秘的面纱。那么，长妈妈是万个什么样的人呢？我们一起来看课文。板书标题、作者， （二）作者介绍 指定学生简介作者。 （三）出示教学目标 （四）检查预习情况 学生借助工具书解决字词：骇掳悚煌急疮疤诘问渴慕霹雳烦琐惧惮 二、自学活动一：朗读课文，感知文章内容，理解阿长的形象。 1、认真阅读课文，看看文章围绕阿长写了哪些事? 明确：文章先介绍了人们对长妈妈的称呼，称呼的由来和她外形的特点，以及她的一些不好的习惯。如写她喜欢“切切察察”、喜欢“告状”、睡觉爱摆“大”字等；接着写她懂得的许多“我听不耐烦”的规矩。比如元旦、除夕吃福橘、人死了要说“老掉了”等；长妈妈给我讲“长毛”的故事，谋害我的隐鼠，最后，写了长妈妈为“我”买《山海经》的事。 2、思考：课文对哪些内容详写？哪些内容写得比较简单？从这些事情中，可以看出阿长是个什么样的人？为什么这样安排？ 明确：第一部分，介绍她的身份和称呼。第二部分，首先围绕阿长的日常言行，略写“喜欢切切察察”、对“我”过分看管，详写睡相粗俗；其次围绕阿长“满肚子是麻烦的礼节”，详写“元旦的古怪仪式”，略写给“我”灌输各种礼仪禁忌；再次，围绕阿长的迷信可笑，详写讲长毛故事赢得“我”“空前的敬意”，略写“谋害”隐鼠而失去“我”的敬意；第三部分，围绕阿长对“我”的真诚慈爱，详写阿长为“我”买《山海经》。 从这些事情中，可以看出阿长虽然地位卑微、身世不幸，却乐天安命；虽没有文化、粗俗、好事、迷信，却天性纯朴善良、仁厚慈爱。 略写的那些内容能使人对长妈妈有个初步了解，她的外形特征，真实地反映长妈妈的一些毛病，但这些并不能削弱对阿长妈妈的敬意，而详写买《山海经》一事，却令人对长妈妈刮目相看，在作者看来“别人不肯做，或不能做的事情，她却能够做成功”，我们可以想到幼小的鲁迅对长妈妈的敬佩和感激之情。

阿长与山海经教学设计

阿长与山海经教学设计 Company Document number：WTUT-WT88Y-W8BBGB-BWYTT-19998

《阿长和山海经》 【教学目标】 1.会读会写本文的生字词；把握课文的内容，理解阿长的形象。 2．了解课文围绕中心选择材料、安排材料，详略得当的写作特点。 3.注意分析关键语句，体会作者词语运用之妙。 4.学习欲扬先抑的写作手法。 5．分析人物形象，把握文章中心，理解文章所表达的思想感情。 【教学重难点】 分析人物形象，把握文章中心，理解文章所表达的思想感情； 了解课文围绕中心选择材料、安排材料，详略得当的写作特点。 学习欲扬先抑的写作手法 【教学方法】朗读法、引导法、讨论法。 【课时安排】两课时 【教学过程】第一课时： 一、创设情境，激情导入 （一）导语 在鲁迅先生的散文《从百草园到三味书屋》里，鲁迅除写了自己的老师寿镜吾先生之外，还写到了一个人，这个人是谁呢对，是长妈妈，她讲的美女蛇的故事真是神秘莫测，给百草园蒙上一层神秘的面纱。那么，长妈妈是万个什么样的人呢我们一起来看课文。板书标题、作者，

（二）作者介绍 指定学生简介作者。 （三）出示教学目标 （四）检查预习情况 学生借助工具书解决字词：骇掳悚煌急疮疤诘问渴慕霹雳烦琐惧惮 二、自学活动一：朗读课文，感知文章内容，理解阿长的形象。 1、认真阅读课文，看看文章围绕阿长写了哪些事 明确：文章先介绍了人们对长妈妈的称呼，称呼的由来和她外形的特点，以及她的一些不好的习惯。如写她喜欢“切切察察”、喜欢“告状”、睡觉爱摆“大”字等；接着写她懂得的许多“我听不耐烦”的规矩。比如元旦、除夕吃福橘、人死了要说“老掉了”等；长妈妈给我讲“长毛”的故事，谋害我的隐鼠，最后，写了长妈妈为“我”买《山海经》的事。 2、思考：课文对哪些内容详写哪些内容写得比较简单从这些事情中，可以看出阿长是个什么样的人为什么这样安排？ 明确：第一部分，介绍她的身份和称呼。第二部分，首先围绕阿长的日常言行，略写“喜欢切切察察”、对“我”过分看管，详写睡相粗俗；其次围绕阿长“满肚子是麻烦的礼节”，详写“元旦的古怪仪式”，略写给“我”灌输各种礼仪禁忌；再次，围绕阿长的迷信可笑，详写讲长毛故事赢得“我”“空前的敬意”，略写“谋害”隐鼠而失去“我”的敬意；第三部分，围绕阿长对“我”的真诚慈爱，详写阿长为“我”买《山海经》。 从这些事情中，可以看出阿长虽然地位卑微、身世不幸，却乐天安命；虽没有文化、粗俗、好事、迷信，却天性纯朴善良、仁厚慈爱。

《阿长与山海经》说课教案

《阿长与山海经》说课教案 一、说课堂教学指导思想及课程标准教材 阿长与《山海经》一文出自初中语文课本第三册第二单元，体裁为叙事散文，根据素质教育要求：语文教学要注意“引导学生积极主动的学习，让学生在学习实践中得到听说读写能力的良好训练，，培养学生的各种学习能力，养成良好的学习习惯，最终全面提高学生的语文素质。”以及第三次全国教育工作会议的有关精神：教育要注重培养学生的创新精神和实践能力。因此，本文的课堂教学指导思想是：在教师的引导下，发挥学生在学习过程中的积极性、主动性，培养学生良好的学习习惯和科学的学习方法，使学生具有一定的语文自学能力；在合作讨论等活动中，体验合作与成功的喜悦。 本文体裁是叙事散文，语言平实自然、朴素亲切，其中所蕴含的感情需要学生通过听、读、说去分析去品味。对文章的阅读练习，《语文课程标准》就此提出如下要求：整体感知课文的大概内容，感受课文的语言所表达的思想感情，大体了解课文的思路和中心意思。 这篇课文主要通过写长妈妈的几件事,表达了对一个劳动妇女的深深的怀念之情。作者围绕中心选材，有详有略，通过对人物的刻画,很好的突出了文章的中心，并且记叙时有一定的顺序，这也是本文的重点；在写人记事的过程中，本篇运用了许多带有感情色彩的词语和句子，要注意引导学生，结合上下文，把握语句中蕴涵的感情。 综上所述，确定本文的教学目标有： 1、找出文章围绕阿长所写的事情，哪些详,哪些略。 2、分析本文刻画人物的方法。［重点］ 3、理解重要语句的深刻含义。 4、作文片段练习：针对本文所学,进行作文训练。 二、说教法 1、听说读写结合法。这是针对本文内容和语言上的特点，课堂教学中主要通 过听说读来调动学生的积极性，培养学生分析问题的能力和口头表达能力，以本文所学进行作文片段练习为手段，培养学生的写作能力。 2、提示法。学生在读书过程中遇到疑难问题，陷入迷茫时，教师在一旁给予 必要的引导、点拨，从而达到解决问题的目的。 3、赏识成功教育。学生在讨论合作，解决疑难问题过程中，适当的给予表扬 鼓励，让其体会到合作与成功的喜悦，能很好的调动学生的积极性，激发学生的兴趣。 三、说学法指导 1、圈注法。指导学生在对课文的分析中，用符号圈点出重点词、句，以助于 学生对文章内容的理解。养成圈点批画的良好习惯。 2、小组合作讨论法。通过探究、讨论，培养学生质疑解疑的能力。 本课课型：新授课 课时安排：2课时 教具：多媒体 四、说教学过程 第一课时 （一）导入新课：在鲁迅先生的散文《从百草园到三味书屋》里,鲁迅除了写自

部编七下《阿长与山海经》教学设计

《阿长与<山海经>》教学设计 鲁迅先生的《阿长与〈山海经〉》所追忆的保姆，她是一个粗人，没有文化、粗俗、好事，是一个很不幸的人，但她又是一个热望一生平安的劳动妇女，质朴善良，热心帮助孩子解决疑难。鲁迅先生深情地抒写了对她的真挚的怀念。教读这篇文章，应引导学生学习本文选取典型事例表现人物性格的方法，从而更深地感悟鲁迅对阿长的深厚怀念之情，领会阿长性格中的纯真美。 教学设想： 本单元的课文都是关于“小人物”的故事，平凡又普通，人物也不尽善尽美，但却给人留下深刻的印象，阿长这个人物初看令人无奈，甚至厌烦，细读却让人回味无穷，通过此篇教学，力图让学生明白人都是复杂的，性格有多面，通过指导，力图使学生戒绝对人物的线性分析，要树立一个意识：不能用简单的好人坏人来评判人物，应从文本出发，细品人物形象，得出结论。 像《阿长与〈山海经〉》这样难度较大的作品，学生活动往往难以设计，因为篇幅长，头绪多，可资学习供鉴的东西很多，很容易搞成满堂灌，在教学实践中发现采用主问题讨论法比较可行。针对此篇的教学，基本上是扣住感情的变化，抓住四个视角，设计主问题讨论法来进行!从不太佩服——不耐烦——一时的空前的敬意——憎恶——新的敬意——怀念，这是对阿长感情的变化，这时就有“少年鲁迅眼中的阿长”“中年鲁迅眼中的阿长”两个视角，在解读中，还有“学生眼中的阿长”“老师眼中的阿长”这些角度的衍生，有了这些角度才有了自我的阅读，于是设计了主问题：“阿长买《山海经》为什么会让鲁迅铭记终身呢?”之后又把这个问题分解为几个子问题：1.你眼中的阿长是个什么样的人? 2.鲁迅对阿长的感情发生了什么变化?3.买回《山海经》为什么会让少年震惊?4.少年鲁迅与中年的鲁迅对阿长的感情有无变化?此外，课前的充分预习也相当重要，收集学生问题，进行归类整理，在查资料的基础上把课堂交给学生。 教学目标： 1.整体感知课文，了解阿长这个人物形象及作者的感情。 2.重点研读买《山海经》的部分，体会语句的深层含义。 3.感悟鲁迅对阿长的深厚怀念之情，领会阿长性格中的纯真美 教学重点： 1.把握课文的主要内容，理解阿长的形象。 2.理解回忆性散文中，作者将写作时的回忆与童年的感受彼此交错转换的特点。 3.注意分析关键语句，体会作者词语运用之妙。

(完整版)阿长与山海经教学设计

《阿长与〈山海经〉》教学设计 保定二中分校于婧雯 【设计理念】 现代教育理念要求教学应当以学生为主体，教师为主导。在教学中，教师应该充分调动学生学习的主动性和积极性，用合作探究与交流的方式引导学生，让学生在理解文本的基础上，联系自身经验，对作品、作家进行了个性化解读，帮助学生树立正确的人生观价值观，培养学生的人文素养。 【教材分析】 本文是鲁迅先生散文集《朝花夕拾》中的名篇。作者在文中运用了儿童和成人两种视角对儿时与保姆阿长发生的故事进行了回忆。作者用儿童的口吻叙述，以儿童的视角来评价阿长的行为举动，体现了少年鲁迅的天真与幼稚。而在文章结尾处，作者又以一个成人的视角，用诗般的语言为已逝去的阿长进行祈祷，表现成年鲁迅对于阿长的深深的爱。阅读本文，会唤起学生的共鸣，学生会有很多切身的感受。学习这篇文章不仅要让学生理解鲁迅对于阿长的态度变化，体会人物特点，更要让学生走进作者的内心，并有感于现实生活，引导教育学生珍视生命中陪伴自己的每一个人。 【学情分析】 初一下学期，学生已对《朝花夕拾》有一定的认识和了解。教师要以引导为主，引导学生用已学知识和方法去探求新的理解，激发他们的参与意识，培养他们的合作精神和探究热情。同时，初一学生的理解力有限的，对于鲁迅先生文章的解读不够深入，需要老师多加指导，适当增加相关文章资料，帮助学生进行理解。 【教学目标】 1．根据文章内容，归纳少年鲁迅与中年鲁迅对阿长所持的不同态度。 2．结合拓展资料，理解鲁迅先生态度变化的原因。 3．引导学生联系实际生活，进行感恩教育。教育学生珍视父母，珍视陪伴在你身边的每一个人。【重点难点】 1．教学重点：根据文章内容，归纳少年鲁迅与中年鲁迅对阿长所持的不同态度。 2．教学难点：结合拓展资料，理解鲁迅先生态度变化的原因。 【教学方法】 合作讨论探究法朗读法 【教学手段】 采用多媒体教学。

阿长与《山海经》板书设计

阿长与《山海经》板书设计教学目标：1、整体把握文章，了解文章的内容。小编给大家提供阿长与《山海经》板书设计，欢迎参考！ 阿长与《山海经》板书设计【教学内容】 选自义务教育课程标准实验教科书《语文》八年级上册第二单元 【教材分析】 鲁迅先生的《阿长与山海经》所追忆的保姆，她是一个粗人，没有文化、粗俗、好事，是一个很不幸的人。但她又是一个热望一生平安的劳动妇女，质朴善良、热心帮助孩子解决疑难。鲁迅先生深情地抒写了对她的真挚的怀念。教读这篇文章，应引导学生学习本文选取典型事例表现人物主要性格以及详写与略写相结合的写法。了解叙述、描写等表达方式，体会描写的作用和方法，从而更深地感悟鲁迅对阿长的深厚怀念之情，领会阿长性格中的纯真美。 【教学目标】 1、整体感知课文，了解阿长这个人物形象及作者的感情 2、重点研读买《山海经》的部分，体会语句的深层含义。 3、感悟鲁迅对阿长的深厚怀念之情，领会阿长性格中的纯真美

【重点难点】 1、学习本文选取典型事例表现人物主要性格的写法。 2、领悟这篇回忆性散文的用双重眼光看待人物和欲扬先抑的写法。 【教具准备】录音机、配乐磁带、幻灯片 【课时安排】2课时 第一课时 【教学过程】 1、说起鲁迅，我想大家应该都不陌生吧！谁来说说鲁迅？ 2、在鲁迅的童年生活中，长妈妈是个很有影响的、特殊的人物。鲁迅没有专门写过回忆母亲的文章，却在他四十五岁那年，写了这篇回忆性散文——《阿长与》。以纪念自己童年时期的一个保姆，可见对她的感情之深。 让我们满怀深情地读一读课题。 3、读了这个题目，你有什么疑问？ 1、阿长究竟是怎样一个人，她与《山海经》又有什么关系，让鲁迅对他有这样深的感情，让我们带着疑问速读课文。 2、说说阿长在你眼中是个怎样的人？ 3、师小结： 总之，阿长是个好人，但又不是十全十美的人，她是一