Trypsin Is a Multifunctional Factor in Spermatogenesis Author(s): Chiemi Miura, Takashi Ohta, Yuichi Ozaki, Hideki Tanaka, Takeshi Miura

and Ryuzo Yanagimachi Source: Proceedings of the National Academy of Sciences of the United States of America, Vol. 106, No. 49 (Dec. 8, 2009), pp. 20972-20977 Published by: National Academy of Sciences Stable URL: http://www.jstor.org/stable/40536106 . Accessed: 13/11/2013 11:52
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is a multifunctional factorin spermatogenesis Trypsin

Chiemi Miuraa, Takashi Ohtaa, Yuichi Ozaki3, Hideki Tanakab, and Takeshi Miura3-1
aResearch Group for Reproductive Physiology, Southern Ehime Fisheries Research Center, Ehime University, 1289-1 Funakoshi, Ainan, Ehime 798-4292, Nansei, Mie 516-0193, Japan Japan; and bNational Research Institute of Aquaculture, Fisheries Research Agency, 422-1 Nakatsuhamaura, Edited by Ryuzo Yanagimachi, University of Hawaii, Honolulu, HI, and approved October 16, 2009 (received for review July 10, 2009)

Trypsin is well known as a pancreatic enzyme that is typically secreted into the intestine to digest proteins. We show in our current study, however, that trypsin is also a key factor in the control of spermatogenesis. A progestin in teleost fish, 17a, 20/3(DHP), is an essential component of dihydroxy-4-pregnen-3-one the spermatogenesis pathway, particularly during the initiationof the firstmeiotic division. In the course of our investigations into the mechanisms underlyingprogestin-stimulatedspermatogenesis, we identifiedthat eel trypsinogen is upregulated in eel testis by DHP treatment. Trypsinogen is expressed in the Sertoli cells surroundingspermatogonia and in the membranes of spermatids and spermatozoa. Usingan in vitroeel testicularculturesystem,we furtheranalyzed the roles of trypsin in spermatogenesis. The inhibitionof trypsinusing specific antibodies or serine protease inhibitorswas found to compromise DHP-induced spermatogeneinduces DNA synthesisand the expression sis. A low dose of trypsin of Spoil, a molecular marker of meiosis, in germ cells. By comparison, a higherdose of trypsinpartiallyinduced spermiogenesis. Furthermore,trypsin was detectable in the membranes of the spermatozoa and found to be associated with fertilizationin fish. Our results thus demonstrate that trypsin and/or a trypsin-like protease is an essential and multifunctionalfactor in spermatogenesis.

ationofspermatogonial is regulated byan androproliferation of gen,11-ketotestosterone (11KT) (3), and thattheinitiation andsperm meiosis maturation areregulated 17a, bya progestin, ac20-dihydroxy4-pregnen-3-one (DHP) (6). It is generally is medithat thebiological ofsteroid hormones cepted activity intheexpression In the atedvia changes ofsteroid target genes. is mediated stem cell renewal eel,E2 activity byspermatogonial and 11-KTis mediated substance factor byMllerian inhibiting we first focused on the progestinstudy, (7). In our present theinitiation of meicontrol mechanisms governing regulated osis.During these we identified as a multifuncanalyses, trypsin We thus tionalfactor the male reproductive during process. and discussthe function of trypsin vertebrate report during and fertilization. spermatogenesis
Results

are regulated outgeneexpression byDHP, we carried cloning eel testicular that werecultured with using fragments (+D) or DHP for 6 days. After without cultivation, poly (-D) 100ng/mL from these testicular and fragments (A)+ RNAswereextracted +D and was then constructed from a subtracted cDNA library via the RDA procedure. -D cDNA enriched One thousand weresubsequently screened clones from eachoftheselibraries fish | germ cell | in vitroculture | teleost | testis cDNA prepaeachenriched bydifferential hybridization using We thereby screened 25 non-cross-hybridizing ration as a probe. is a complex DHP treatment. cDNA fragments after developmental processthat andupregulated Among and these,a cDNA fragment with themitotic ofspermatogonia Spermatogenesis to trypsinogen was begins proliferation corresponding that convert identified extensive through morphological changes proceeds (GenBankAce. No. AB519643).Database searches intofunctional Sincethepro- showed thatthededucedaminoacid sequenceof thiscloneis spermatozoa. haploid spermatids is similar similar of the cellularstagesof spermatogenesis to eel pancreatic gression (Fig. SI). Eel testicular trypsinogen itis very that common trypsinogen thevertebrate across withthe aminoacid sesharesa 92% similarity kingdom, possible there are quenceofeel pancreatic mechanisms existin thesespecies. control However, andboth contain trypsinogen, proteins in thisprocess between each classofverte- twoconserved known differences sites.The serine active family protease trypsin brate. For example, ofeel testicular was aminoacidsequence androgens playa majorrolein spermato- deduced trypsinogen in fish, but in mammals, the spermatogonia phylogenetically that vertebrates inferred from ofother gonialpathways (Fig.S2). unaffected remain by a loss of androgen production. Ouranalysis that eel testicular largely shows trypsinogen belongs clearly verte- to thetrypsinogen also differ thestructures ofthetestes Moreover, among cluster. tubules contain several In mammals, theseminiferous brates. betweentesticular To further elucidatethe relationship a cystic trypsinogen ofgerm whereas fish exhibit successive cells, generations suchas DHP thatare and sexsteroids expression ofspermatogenesis. type with ofspermatogenesis, we examines associated theregulation in theeffects andgametogenic exist Various styles patterns reproductive mRNA E2 andDHP uponthetrypsinogen of11-KT, has a levelsin the different teleost The Japanese eel, forexample, species. were eel testis. Eel testicular fragments Japanese culture cultured andunder fresh-water spermatogenetic pattern specific with three andtheexpression 6 days these for steroids, testes of the males of thisspeciesshow immature conditions, blotanalysis. The wasthen analyzed byNorthern trypsinogen onlynon-proliferating typeA and earlytype results containing is induced showed thattrypsinogen only by expression thatinjection DHP stimulation It has been reported, however, spermatogonia. (Fig. 1). all stages with human chorionic gonadotropin (hCG) caninduce inthetestis, we oftrypsinogen To determine thedistribution intheJapanese eel invivo(1). Furthermore, ofspermatogenesis anan eel trypsinogen immunohistochemistry using performed inwhich animal theJapanese eel is theonly spermatcomplete an in treatments hasbeeninduced using byhormonal ogenesis anda germ cellcoculture Author contributions:CM., T.O., and T.M. designed research; CM., T.O., Y.O., H.T., and vitro culture cell/somatic organ system modelsystem T.M. performedresearch;CM., T.O., and T.M. analyzed data; and CM. and T.M. wrote the is an excellent system (2-4). Hence,theeel testis paper. forstudying theregulation ofspermatogenesis. we have The authors declare no conflictof interest. Usingthe eel modelboth in vivo and in vitro, in a number studies thatspermato- This article is a PNAS Direct Submission. of previous demonstrated in fish. 1To whom correspondence should be addressed. E-mail:miutake@agr.ehime-u.ac.jp. hormones sexsteroid is controlled bydifferent genesis is This article contains supporting information online at www.pnas.org/cgi/content/full/ cellrenewal for that stem We found example spermatogonial theiniti- 0907631 106/DCSupplemental. estradiol-17 regulated byan estrogen, (E2) (5), that
20972-20977 | PNAS | December 8f 2009 | vol.106 | no. 49

from ofTrypsinogen theEelTestis. To identify factors that Cloning

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Fig. 1. Trypsinogenexpression in the Japanese eel testis determined by Northern blot analysis of cultured testicular fragments. Pooled testicular fragments from 10 eels were cultured without (Q or with 10 ng/mLof 11-ketotestosterone(KT),estradiol-17 (2) or 17a,20/3-dihydroxy-4-pregnen3-one (DHP) for 6 days. Lane IC shows the initial control before culturing. Northernblot analysis of EF-1,which serves as reference,is also shown.

latetype Positive oftheSertoli cellssurrounding tibody. staining spermatogonia andsperma(Fig.2 A andB), andspermatids No staining wasobtained tozoa(Fig.2 C andD) wasobserved. as a negative control. serum using preimmune To investigate the relationship between Spermatogenesis. andspermatogenesis, cellpellets cell/somatic trypsinogen germ werecultured with orwithout an anti-eel antibody trypsinogen To assay Fig. 3. Effectsof trypsinon spermatogenesis in the Japanese eel in vitro.(A) DHP (10ng/mL) or11-KT for 6 days. and/or (10ng/mL) treatmenton DHP-induced DNA antibody (Anti-TN) DNA synthesis, we monitored BrdUincorpora- Effectof anti-trypsinogen spermatogonial cell coculture system.IC, Initial replicationof germ cells in a germ cell/somatic with 11-KT DHP alone tion. As positive treatment or controls, control; KT, 10 ng/mL; DHP, 10 ng/mL;+, with anti-TN;-, without anti-TN.(B) stimulated DNA synthesis in spermatogonial cells. Effectof serine protease inhibitortreatmenton DHP-induced DNA replication significantly The addition of an anti-eel antibody significantlyof germ cells in a germ cell/somaticcell coculture system.PM, Phenylmethyltrypsinogen butnot11-KT-induced, reduced DHP-induced, spermatogonial sulfonyl fluoride (PMSF); AEB, 4-(2-Aminoethyl)-benzenesulfonylfluoride DNA synthesis (AEBSF). (O Effect of various concentrations of porcine trypsinon germ (Fig.14). cell/somatic cell cocultures. is a serine and we therefore the Trypsin protease investigated effects of the serineproteaseinhibitors, 4-(2-aminoethyl)fluoride benzenesulfonyl (AEBSF) and phenylmethylsulfonyl cell coculture system (Fig. 3C). Germcell/somatic DNA synthesis fluoride stimulated cell/somatic (PMSF) on spermatogonial cell pelletswere cultured withincreasing concentrations of with DHP. Germ cell were cultured or cell/somatic by pellets 10 1 100 10 and 100 nM, nM, nM, , , pig-trypsin (1 ) DHP (10 ng/mL) without these inhibitors andwith or 11-KT(10 11-KTor 10 ng/mL DHP as positive controls DNA orwith10 ng/mL for 6 days butonly DHP-induced spermatogonial ng/mL) 2 for We then monitored DNA the of days. synthesis spermatowas reduced synthesis (Fig.3B). thepellets toBrdU.Atthestart ofcultivation, goniabyexposing all ofthegerm cellsinpellets areundifferentiated spermatogoDirectEffects of Trypsin on Eel Spermatogenesis.The direct effects a BrdUindex of15.2 2.42%.The addition oftrypsin oftrypsin aneel germ niawith onspermatogenesis were monitored using to the culture medium DNA replication induces witha peak in thecontrols without (56.6 2.75%) at 10 nM. Significantly, theBrdUindex didnotchange from itsinitial anysupplement, value. weexamined theeffects oftrypsin onthe immunohistochemistry of the meiosis-specific marker, expression Spoil, in a germ cellcoculture Before there was cell/somatic cultivation, system. no detectable cellswithin thepellets. 2 After Spoil inthegerm the trypsin treatment was found to however, daysin culture, induceSpoil expression in thesegermcells witha peak of at 10 nM (Fig.4A-C). activity were cultured withvariousconcentrations of spermatogonia 15days andtheappearance ofthecells trypsin (0.1-100) for was thenevaluated. Before A spermatogonia cultivation, type rounded butafter 3 daysin culture with100 of (Fig. 5/4), these cellsadopta spindle-shaped After 15 trypsin, morphology. with100 oftrypsin, a flagelliform strucdaysoftreatment ture is detectable on one sideofthese cells spindle-shaped germ without orwith 0.1,1,and (Fig.5 C andD). In cultures trypsin 10 trypsin, no morphological wereevident in the changes cells(Fig.5B). Usingflow-cytometry, thenuclear germ phases ofthese cellswererecorded. The resulting spindle-shaped germ
PNAS | December8, 2009 | vol.106 | no. 49 | 20973 Effects of Trypsin on the Expression of Meiosis-Specific Markers. By

Effects of a Trypsinogen and Serine Protease Inhibitors upon Antibody

Effects of Trypsin on the Spermatogonial Morphology. Type A

Fig.2. Cellularlocalization of eel trypsinogeninthe testis.(A and B) Eel testis at 12 days post-hCGinjection.(CandD) Eel testisat 18dayspost-hCG injection. (A and were assessed byhistochemistry using a specificanti-eel trypsinogen stained with antibody,( and D) show serial sections of A and C, respectively, and eosin. TypeA and earlytype spermatogonia (GA), late type hematoxylin spermatogonia (GB), spermatocytes(SC) spermatids(ST), spermatozoa (SZ), and enlarged Sertoli cells (arrowheads) are also shown. (Scale bar, 50 .) Miuraetal.

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of trypsinupon the induction of meiosis in the Japanese eel Fig. 4. Effects invitro.(A and B) Microphotographsshowing anti-eel Spo1 1 immunoreactive cell pellets without (A) or with (B) 0.01 of material in germ cell/somatic ( Percentages of Spo1 1-positivegerm cells. The (Scale bar, 10 .) trypsin. numberof immunoreactive germcells is expressed as a percentage of the total numberof germcells. Resultsare given as means SEM calculated foreach of different lettersare significantly sixsamples. Values with different (P < 0.05).

Fig. 6. Flagella and the manchette-likestructuresof sperm-likecells are treatment.(A) Electronmicrophotographof a crosssection induced bytrypsin of a flagellum of an eel spermatozoon, showing the 9 + 0 axonemal structure. (Scale bar, 0.1 .) () Electron microphotograph of cross section of an eel sperm head. Arrowheads indicate the nine sets of microtubules(manchette structure). (Scale bar, 1 .)( Localization of a-tubulin in a normal eel of whole cells using an spermatozoon assessed by immunohistochemistry anti-a-tubulin antibody, a-tubulin exists in the manchette structureof the (D) Site localization of sperm head and in the flagellum. (Scale bar, 5 .) assessed by immua-tubulin in spermatogonia cultured with 100 trypsin of whole cells using an anti-a-tubulinantibody. (Scale bar, nohistochemistry 5 .)

showed 2C and 4C peaks,thatis, histograms flow-cytometric division. a normal meiotic these cellsdidnotundergo of the elongated The morphology germcells exposedto withnormal eel spermatozoa was compared byhistotrypsin oftheeel sperm observation (Fig.6A-C). The flagella logical structure a 9 + 0 axonemal cellsexhibit (Fig.6A) andninepairs intofour andfive ineachflagellum aredivided ofmicrotubules where on the head they sperm (Fig. 6B) pairs,respectively, and the to the caputend. Both sets of microtubules extend a-tubucanbe detected using byimmunocytochemistry flagella an In sperm-like cells treatedwithtrypsin, lin antibodies.

in theflagelliform could notbe detected axonemal structure strucwhereas structure electron microscopy, flagelliform using found to atthecellsurface were andmicrotubule-like lines tures antibodies stained be specifically (Fig. 6D). bya-tubulin intheEelSperm Head. The trypsinogen Trypsin expression profile a specific wasnext ineel spermatozoa antibody, analyzed. Using in thesperm membranes was detected (Fig.1A) andits trypsin In thisanalysis, was assayed using gelatin zymography. activity and weredetected 80-and95-kDabandswith activity protease could be reducedby PMSF treatment. these two products toanti-trypsinogen two bands these Furthermore, corresponded blotanalysis, bandsdetected respectively byWestern positive (Fig.IB). was and sperm head trypsin between fertilization relationship in intheeel. Ejaculated eel sperm wereincubated also analyzed serine with the seminal artificial specific plasmasupplemented inhibitors PMSF or AEBSF, or withanti-eel trypsin protease werethenperformed for3 h. Inseminations antibodies, using eel eggs.Fourhoursafter and normal theseincubated sperm fertilized insemination, eggs were countedand the rate of that both Theresults showed calculated. fertilization wasthereby antibodies the trypsin serineproteaseinhibitors significantly rate(Fig.8). thefertilization reduced screencDNAswerecloned Eel trypsinogen bygeneexpression of factors that areregulated bytheDHP initiator ingto identify in theJapanese eel. Trypsinomeiosis during spermatogenesis a member of theserine of trypsin, protease genis a precursor in thepancreas as a digestive thatis mainly family produced are that serine ithasbeenreported proteases enzyme. Although
Miuraet al. Relationship Between Sperm Trypsin and Fertilization Pathways. The

Discussion

on spermiogenesis. Spermatogonia before culture of trypsin Fig. 5. Effects {A), and spermatogonia culturedwithout () or with (C and D) 100 trypsin in a spermatogonial germ cell culturefor 15 days are shown. D shows a higher (Scale bar 'nA-D, image of spermatogonia treated withtrypsin. magnification 20 .) 20974 | www.pnas.org/cgi/doi/10.1073/pnas.0907631106

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this acts surface from that (13). Itwassuggested finding trypsin to on intact cellsvia theinsulin as it has beenshown receptor stimulate thephosphorylation ofthe-subunit ofthis receptor. the membrane Moreover, may also directly trypsin modify whichformthe Ca2+ channel(14, 15). Although proteins further areneeded, itmay be possible that investigations trypsin inspermatogenesis function via someofand/or all ofthese may pathways. In theJapanese is regulated sex eel,spermatogenesis bythree steroids; E2, 11-KT,and DHP. E2 regulates spermatogonial renewal proliferation (5, 16), 11-KTregulates spermatogonial theinitiation ofmeiosis proliferation (3), andDHP regulates (6). Each of thesesteroids can altertheexpression of trypsinogen andthus havea roleateachstage ofspermatogenesis. Inour may Northern blotanalysis was used to examine the present study, effects ofthese three steroids on thetrypsinogen mRNAlevels in eel sperm. (>A)ImmunohistochemicalanalFig. 7. Localization of trypsin in an eel testicular culture The results show that organ system. localizes at ysisof whole cells using an anti-eel trypsinogenantibody. Trypsin these to DHP stimulation thus transcripts only respond suggestthe sperm membrane and within the mitochondria in the caput end of the functions ing that trypsinogen duringthe meioticsteps in sperm head. (Scale bar, 10 .) () Gelatin zymographyassay and Western we Furthermore, blot analysisof the eel sperm membrane with an anti-trypsinogen spermatogenesis. bytesticular histochemistry antibody is expressed in the Sertolicells (Anti-TN).PMSF - and + indicate with or without Phenylmethylsulfonyl observedthat trypsinogen Arrowheads indicate molecular markers(Left),trypsin thelatetype spermatogonia, butnotintheSertoli fluoride,respectively. surrounding cellsof thespermatocytes. was activity assayed by zymography,or signal detection by Western blot analysis Strong trypsinogen expression using the anti-eel trypsinogenantibody. inearlier alsoobserved spermatogonial latetype generations. In general, thenumber ofmitotic divisions that spermatogonia before is species-specific meiosis undergo entering (17). Previin testicular their functions have not been tissue, present yet weproposed that this number is related toa mediator that ously, that clarified we show study, (8, 9). In ourcurrent trypsin plays regulates ofJapanese eel spermatogonia into meiosis entry (18). an important roleintheregulation ofthree events The results reproductive of our histochemical the analysesalso support inthemaleeel; that ofmeiosis, is,theinitiation spermiogenesis,contention that a roleinmeiosis inearly trypsinogen plays stages andfertilization. oflatetype spermatogonial wespeculate Hence, development. Previous studies haveshown that is expressed in thatthemechanism trypsinogen theentry to meiosis is an regulating point several tumors andcancer celltypes (10). It has beenreported early process involving trypsinogen. that affects theproliferation, andmetastasis of invasion, The addition ofanti-trypsinogen trypsin antibodies intothemedium breast cancer cells(11). Thematrix cellcocultures DHPmetalloproteinases (MMPs) ofgerm cell/somatic significantly abrogates andproteinase-activated 2 (PAR-2) are stimulated butnot11-KT-induced, DNA synthereceptor by induced, spermatogonial andboth MAPK-ERKpath- sis.The neutralization activate themitogenic of trysinogen thusblocks DHP-induced trypsin may via theinduction ofepidermal factor its essential role in thisprocess. way growth receptor (11). spermatogenesis indicating Activation ofthis in many Moreover, also promotes cell division sinceinhibitors of serineproteases DHPpathway prevent induced in a celltypes. similar another has the we conclude that Moreover, pathways study investigated expresway, trypsinogen its functional role in spermatogenesis as trypsin, the sionof PAR-2,a G-protein-coupled and theroleof exerts receptor, active form ofthismolecule. incellproliferation inhuman coloncancer celllines biologically trypsin (12). mature, theeelgerm cellcoculture wecould cell/somatic Theresults ofthese indicate that shows Using system, previous reports trypsin effects of trypsinogen of as a growth factor andunravel a mechanism upon the initiation properties whereby assaythe direct meiosis. The addition of into the medium of these serine control the of colon tumors. In trypsin proteases proliferation yet stimulated DNA replication ingerm which significantly another ithasbeendemonstrated that initiates its cultures report, trypsin occurs in both mitosis and meiosis. cells, Furthermore, during intracellular role in growth on the cell by acting promotion was also found to significantly induce theexpression of trypsin cells.Spoil is involved intheformation ofDNA Spoil ingerm double-strand in eukaryotic breaks speciesduring homologous in meioticprophase(19, 20) and has been recombination as a molecular marker oftheinitiation in ofmeiosis recognized cells. Thesedatathus indicate that hasanimportant germ trypsin rolein theinitiation ofmeiosis uponDHP stimulation. In our histochemical antibodies, analyses usingtrypsinogen thespermatids andspermatozoa werealsopositively stained and we therefore therelationship between and investigated trypsin latestagespermatogenesis. We found that theshapeoftype A became "sperm-like"; thatis, spindle-shaped spermatogonia witha flagelliform aftertreatment with100 structure, of thesecellsrevealed 2C trypsin. Flow-cytometric histograms and4C peaks, that cells"didnotundergo is,these"sperm-like meiotic division and exhibited a spermatogonia-type nucleus. In theJapanese in as other the eel, anguillidae (21), flagella 8. Functions of the in membrane bound eel The effects Fig. sperm. trypsin of the spermcells exhibit a 9 + 0 axonemal structure. The of serine protease inhibitorsor an anti-eel trypsinogenantibody (Anti-TN) in each flagellum microtubules divide intofour and five pairs, upon the fertilizationrate of eel eggs were evaluated. Results are given as on thesperm extend to thecaput means SEM calculated for each of six samples. AEBSF, 4-(2-aminoethyl)head,where respectively, they fluoride.*, significantly different fromthe control(P < 0.05). end.Thismicrotubule ineel sperm structure headsis equivalent benzenesulfonyl
Miuraetal. PNAS | December8, 2009 | vol.106 | no. 49 | 20975

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to the"manchette" in mammalian structure heads(22, during spermatogenesis, these cultureswere exposed to porcine trypsin(Nasperm fluoride (PMSF; Nacalai Tesque), 4-(2and themanchette-like in mam- calai Tesque), Phenylmethylsulfonyl structures 23). The flagellum fluoride (AEBSF; Nacalai Tesque) or anti-eel Aminoethyl)-benzenesulfonyl malianspermalso comprise micro tubules(21) consisting of trypsinogenantibodies. heterodimers ofa and-tubulin can be detected Thus, (24). they an -tubulin intheeel. NorthernBlot byimmunocytochemistry using antibody Analysis.Total RNA was extracted using Sepasol RNA I super In particular, themanchette-like in (Nacalai structures can be observed Tesque) fromtesticularfragmentsculturedfor 6 days with 10 ng/mL twolineson sperm head. of 11-ketotestosterone (11-KT) or 17a, 20 -dihydroxy-4 -pregnen -3-one The flagelliform structures stimula- (DHP). Testicular fragments before culturingand testicular fragmentsculproduced bythetrypsin tionof sperm-like cellswerenotfound to exhibit the 9 + 0 tured without steroids were used as the initialcontrol and control, respecaxonemal structure observation. How- tively.One mg of poly (A) + RNA purifiedby Oligotex-dt30 (Takara Bio) was byelectron microscopic theflagelliform structures ofthesecells,stained ever, bya-tu- electrophoresed on a 1% agarose denatured gel and blotted onto nylon bulinantibodies, and manchette like structures of membranes. The cDNA fragments obtained from cDNA subtraction were composed using a DIG-dUTP PCR DIG probe synthesiskit (Roche) and served as a-tubulin were detectable at the cell surface. These results labeled The cDNA fragmentencoding Japanese eel elongation factori (EF1) probes. indicate that treatment butnotall aspects was also labeled for some, trypsin triggers use as an internal standard. After hybridization,the ofspermiogenesis; that which is,theprocesses by spermatogonia membrane was analyzed using a LAS-1000 mini(Fujifilm). becomespermatozoa and the induction to of spermatogonia intosperm-like cells. Our current data also Immunohistochemistry. was performedusing antiboddevelopdirectly Immunohistochemistry that a relatively lowconcentration oftrypsin is sufficient ies raised against eel trypsinogenand eel Spo1 1. Testiculartissue was fixed in suggest to induce while a high concentration oftrypsin induces Bouin's solution at 4 C for 18 h,embedded in paraffinwax, and cut into 5- meiosis, sections. Immunohistochemicalanalysis was performed using the Histofine partial spermiogenesis. In teleost forsomespecies suchas thesturgeon, SAB-AP kit(Nichirei). fish, except The anti-eel trypsinogenantibody was produced using bacterial recombido nothavean acrosome spermatozoa (25,26),andfertilization nant eel trypsinogen. cDNA fragmentsencoding the mature eel trypsinogen is facilitated via the egg's micropyle. Hence, an acrosome peptide were subcloned into a pQE-32 expressionvector (Qiagen) to create a is not required reaction the fertilization of fisheggs. 6x histidine tag on the expressed protein. After bacterial expression, the during there islittle information available However, currently regarding recombinantproteinwas purifiedand refolded.A rabbitwas then immunized thefactors ormechanisms that of with 1 mg recombinant eel trypsinogen.The IgG fractionwas purifiedfrom playa roleinthefertilization fish in have a the respective antiserum by anion exchange chromatography.Western blot or may eggs. Trypsin trypsin-like proteases sperm analysis of the testes at 12 days afterthe hCG injection was then carried out critical rolein this regard. usingthiseel trypsinogenantibodyand a single band of approximately27 kDa a immunoreactive material was Using trypsinogen antibody, was detected (Fig. S3). The productionof rabbit polyclonal antibodies against inthesperm detectable membrane that displayed trypsin activity eel Spoil was performedas described in ref.20. in a gelatin heads have zymography assay.Hence,eel sperm Using anti-eel trypsinogen and -eel Spoil antibodies at a dilution of inquestion differs from the 1:1,000, theprotein activity trypsin although was also performed. As a negative control, immunohistochemistry mass immunohistochemicalanalysiswas performedusing preimmune normal rabtesticular that we havecloned, sincethemolecular trypsin of sperm head trypsin from thatof bit serum (NRS), which did not react with any cells. Using anti-a-tubulinand (80 and 95 kDa) differs of germ cells was also also anti-eel trypsin,whole mount immunohistochemistry testicular type trypsin (27 kDa). Sinceithasbeenreported Cells were attached to the polyL-lysine coated glass slides and performed. thatmembrane-type serine exists in elongated sperprotease inmammals matids isa possibility that head fixedwith 4% paraformaldehyde in 0.1 M phosphate buffer(pH 7.4) for 1 h. sperm (27,28),there Afterwashing with PBS (PBS), the cells were permeabilized for 30 min in 1% in in eel is similar to this serine mammals. protease trypsin TritonX-100 in PBS. The slides were then incubated for30 minin 5% skimmed also thepossibility that head milkin PBS to block We wished to investigate sperm nonspecificbinding sites before immersionfor 1 h in PBS is related to fertilization andevaluated therelationship containing antibodies trypsin against a-tubulin (mouse monoclonal, diluted 1: 100) or in the eel. The eel trypsin(diluted 1:1,000). After washing with PBS containing 0.025% between fertilization and sperm head trypsin serineproteaseinhibitors and the anti-trypsinogen antibody Tween 20 (TPBS), the slides were next incubated with FITC-conjugatedgoat reduced head anti-mouse IgG (diluted 1:100) oranti-rabbitIgG(diluted 1:100)for1 h. Finally, thefertilization rate;hence, sperm significantly the slides were washed with TPBS and then with PBS and mounted in 50% has an important rolein fertilization. indeed trypsin glycerolin PBS. In conclusion, we provide evidence that the testicular strong in thecontrol are important factors of meiosis, spertrypsins onlytype A spermatogonia In other andfertilization. is a multi- SpermatogonialCell Culture.Eel testes containing words, miogenesis, trypsin were digested by collagenase/dispase II for 2-3 h at room temperature. The in malereproduction. functional factor resultingcell suspension was passed successivelythrough meshes with a 75-,
Materials and Methods
cDNACloning.Testicularorgan cultureswere prepared as described in ref. 1. The testicularfragmentswere culturedfor 6 days with (+D) or without (-D) DHP at a concentration of 100 ng/mL.cDNA subtractions were performed - D culturedtesticularfragments using poly (A)+ RNAextractedfrom+Dand as described in ref. 7. Afterthe subtraction enrichment process, enriched upregulated +D cDNA fragmentswere packaged into Azapll phage particles. Enriched +D and -D cDNAs were then labeled using PCR DIG labeling MixP|us (Roche). Labeled +D and -D cDNA probes were hybridizedseparately to duplicate copies of the library. Onlythose clones that hybridizedto the + D probe were collected and purified,and these plasmids were then excision reduced. These partial cDNA fragmentswere then labeled with DIG-dUTP using PCR and used as probes to screen an intact cDNA libraryconstructed from oligo (dT) primed mRNA extracted from testes at 12 days after hCG injection.Positiveclones obtained fromthislibrary screeningwere sequenced by the dideoxy chain terminationmethod using the Dual CyDyeTerminator sequencing kit(Amersham Biosciences). Germ Cell/SomaticCell Pellet Cultures. Germ cell/somaticcell pellets were culturedas described in ref.4. To furtherunderstand the functionof trypsin 20976 | www.pnas.org/cgi/doi/10.1073/pnas.0907631106 45-, and 20- pore size. Afterwashing with eel Ringer'ssolution, the cells were layered on top of a 13.8% Nycodenz solution and centrifugedfor 10 min The resultingcells were then plated at 1,300 x g to remove the erythrocytes. on collagen-coated dishes to remove (adhering) somatic cells. Non-adhering germ cells (1 106 cells, mainlytype A spermatogonia) were then cultured in L-15 culture medium with or without various concentrations (0.1, 1, 10, and 100 ) of pig trypsinin non-coated culture dishes (3-cm diameter) at 28 C for 15 days.

Western Blot Analysis and Gelatin Zymography.Eel spermatozoa were collected from artificiallymatured, hCG-treated eels according to described methods in ref. 1. These spermatozoa were then suspended in solubilization buffer(10 mM Tris-HCI, pH 7.8, 0.15 M NaCI, 30 mM n-octyl-b-D-thioglucopyranoside, 1 mM PMSF, and 2 mM EDTA), shaken for 20 min at 4 C, and then centrifugedat 100,000 x g for 1 h at 4 C. The resultantsupernatants containing solubilized sperm were used as the sperm membrane fractions.SDS/ PAGE with gelatin was then performedas described by Gordon and Lilly (30). Gelatin was added to the separating gel to a final concentration of 0.2% The samples were mixedwith an equal volume of sample buffer[125 (wt/vol). mM Tris-HCI, 4% (wt/vol) SDS, 20% (vol/vol) glycerol, and 0.05% (wt/vol) bromophenol blue]. Afterelectrophoresis,the gels were equilibrated in 10 Miuraet al.

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mM Tris-HCI, pH 8.0, containing 2.5% (vol/vol)Triton X-100 for 1 h. After pH 8.0, the gels were incubated with 10 mM washing with 10 mM Tris-HCI, Tris-HCI pH 8.0 for 24 h at 37 C, stained with Coomassie brilliantblue and destained. of Japanese Eel. Male and female Insemination SpermIncubationand Artificial induced to reach maturityusing hormonal Japanese eels were artificially treatments(31, 32). Ejaculated miltfromfiveindividualmale eels was diluted seminal plasma forthe Japanese eel (33). The diluted (1 :10,000) withartificial miltwas then incubated at 4 C for 2 h with various concentrationsof either PMSF (1, 10, and 100 ), AEBSF (1, 10, and 100 ) or anti-eel trypsinogen antibody (0.5, 5, and 50 g/mL).the sperm remain immobilized under these conditions.The fertilization test was then performedas follows. Five hundred of ovulated eggs were inseminated with 500 _ diluted semen. micrograms
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From3-4 h afterfertilization, the fertilizationrate was estimated bycounting eggs displayingcleavage under a binocular microscope. Statistics.Resultsare expressed as the means SEM. Data analysiswas carried out using the Sceirer, Ray, and Hara extension of the Kruskal-Wallis test (a two-way ANOVA design for ranked data), followed by a post-hoc Bonferroni adjustment. ACKNOWLEDGMENTS. We thank Dr. R. W. Schulz (Utrecht University, The Netherlands) for his valuable comments on the manuscript.This work was supported by grants-in-aidfor scientificresearch and for Fellows fromthe Japan Society for the Promotion of Science, the Global Center of Excellence of Education, Culture, Sports, Science, and Techprogram, and the Ministry nology of the Japanese Government.
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Miura et al.

PNAS

December 8, 2009

vol.106

no. 49

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