Indian Journal of Experimental Biology Vol. 4 1, Ju ly 2003, pp. 756-763

Embryo-endometrial proteases during early mammalian development

P B Seshagiri *, H S Lalitha , A Mi shra & G V Sireesha

Department or Molecular Reproduction. Development and Genetics

Indian InstitUle or Sc ience, Bangalore 56() 0 12, India

In mamm;i1 s. extensive n:modeling or uterine endomet ri al mat ri x 0 curs duri ng reproduct i ve cyc le and blastocyst implanta ti on. Th is is regul atcd hy a varicty or Illolecules sueh as hormones, growlh ractors, cy tokines and proteases. In thi s arti cle , we rev iew the current state or know ledge avaibble on vari ous prot cases and their inhibitors runcti onall y invo lvcd in the cnlbryo-cndomet ri al ti ss ucs and prcsent sume data un endometri al pro teascs in hamste rs and rats during est rous cyc le and carly pregnancy. We ciemonstrate the presence or at Icast rour gelat inolyt ic acti vi tics in cndomet ri al samples, bc longing to ge latin ase-A and -B categori es and their dependence on ealciumlz inc ions ror enzyme ac ti vi ty and, their intcr­rela tionships hc!\Vccn 7.ymogcn and acti ve rorms. We be lieve th at th e embryo-endometrial proteases are esscnt ial for hatchi ng or blastocysts and 1'0 1' the dynamic remode ling or endometri al ti ssue" occurri ng duri ng the criti cal peri ­implanta tion period .

Keywords : Embryo implantati on, Endomctrium, Prot cases. ZYlllography

Hatching o f blastocyst and its implantati on into uter­ine endometrium is a criti cal step in the es tabli shment of earl y pregnancy. These events are thought to be under a coordinated control o f vari ous bioregulatory molecule. such as hormones, growth ractors , cyto­kines, adhes ion molecules and protcases t

.6 Timely

sec reti on of embryo-endometrial prOleases is respon­sible for hatching or blastocy sts and in vasion of im­planting embryos into uterine endometri al matri x.

The uterine endometrium is a dynamic tissue, which undergoes rapid and progressive, morphological and functi onal modifi cati ons, including ex tensive pro­li fe rati on and di f ferenti ati on, during reproducti ve cy­cle and earl y pregnancy. Ovarian steroid hormones are known to influence endometrial functi on by either direct ly inducing the ex press ion o f proteases and/or their inhibitors or indirec tl y through growth factorsU

and cy tok i nes).7 . Several cl asses of proteases like ser­ine proteases, cys teine proteases and the matri x mctal­loprO(eases (MMPs) are ex pressed during dirferent stages of reproducti ve cyc l e~ and embryo implanta-. ')to T I MMP' t- . li on ' . l e s are Important groups 0 Z II1C-

containing enzy mes, impli cated in endometrial ti ssue rcmodcl ing and organomorphogencs is tt . The process

" For COITc, pllnli<.: ncc: Phone: (OXO)2 <J3-2(lg7 I"ax: (tl,'Or,ClO-O<J<J<J!:\(1()-()6g3 le- Illail: poian i@l11 rdg. i isc.ernel. ill

o f remodeling requires a balance between the synthe­sis and degradati on of extra cellular matri x (ECM ) components, which is contro lled by oppos ing ac ti ons o f MMPs and their inhibitors viz., ti ssue inhibitors o f metall oproteases: TIMPs4

.x. There is a need for

knowledge on protease in vo lvement In peri ­impl antation development and their regulati on. In thi s arti cle, we rev iew the current status of know ledge on the mammal ian embryo-endometri al proteases and prov ide some data pertaining to hamsters and rats.

Materials and Methods Allill/ols -Golden hamsters and albino rats (3 -6

month old) were housed in controlled environment

(25°C, 14L- IOD) and were prov ided with pelleteci feed and water ad libitum. Females were paired w ith fe rtil e males and checked for the presence of sperm in vaginal smears. The day on which sperm were found in the smear was des ignated day I of pregnancy. Dif­ferent stages o f estrous cyc le were determined by ex­amining the vaginal smears, whi ch characteri ze spe­cifi c stages. Procedures for handling and ex perimenta­ti on foll owed were according to the Guidelines on the Use of Animal s in Scientifi c Research (I NSA, New Delhi , 1992).

Rec{Jllerv of' uterill e tisslles- Uterine ti ssues were recove red dail y at lOAM during different days of estrous cycle alld days 1-6 o f pregnancy . Uterine horn s were excised longitudinall y anJ endometri al

y

-,

)0 .

SESHAGIRI el al.: EMBRYO-E DOM ETRI AL PROTEASES 757

ti ssues were scraped from the remaining myometrium and homogeni zed in 50 mM Tri s- HCI buffer (pH 7.5 ), containing 0.25% Triton X-IOO. The ti ssue homoge­nate was spun ( 12,000 rpm) for 30 min at 4°C. Super­natant was transferred to a fresh tube and protein con­centration quant ified u ing Bradford's method. Pro­tein lysate was stored at - 70°C till further use.

ZVlI10graphic analysis - This was carried out ac­cording to the published protocol12

. Briefl y. 8% poly­acrylamide gel was impregnated with gelatin at a con­centrati on of I mg/ml. About 100 ~g of tOlal protein was loaded on the ge l under non-denaturing condi­ti ons and electrophoresed at 100 V for 3 hI' at 4°C. The gel was then soaked in 2.5% Triton X- IOO for I hI' with gCllll e shak ing. It was then incubated in 50 mM Tri s- HCI buffer (p H 7.5), containing 10 mM CaCl 2 and I pM ZnCl 2 for 16 hI' at 37°C. The gel was stained with 0.5 % Coomassie brilliant blue Rand de­stained in 40% methanol: 10% acetic ac id : 50% wa­ter. Clearing of gel-trapped gelatin by proteases was revealed as clear bands.

To study the dependency of metal ions for these proteases, metal chelator (EGT A) or spec i fic metall o­protease inh ibitor ( I, IO-phenanthroline) was used in the incubati on buffer. To test enzy me spec ifi city, ser­ine protease inhibitor (S BTI) was used. To assess the latent protease activi ty, total protein was pre­incubated either with 2 mM 2-amino phenyl mercuric acetate (A PMA) or tryps in (0. 1- 1 00 ~g) and in acti­vated by adding a three- fold excess of SBT!. Samples "vere then electrophoresed as desc ribed above.

Results and Discussion During the reproductive cycle (estrous/menstrual

cyc le) and embryo implantation, rapid ti ssue remodel­ing occurs in the endometrial matri x and also a loca l breakdown of ECM4

.8

.13

. Prior to implantation, blas to­cyst undergoes hatching, mediated by em­bryo/endometri al proteases I . The ro le of embryo­derived proteases in the process of blastocyst hatching and trophoblast in vasion remains to be understood clearl y. It is know n that the process of embryo hatch­ing varies depending on the species and it would be of interest to study embryo-deri ved proteases. The hatch­ing and impl antat ion of blastocys ts are believed to be governed by a va ri ety of different classes of proteases of embryo-endometri al origin , whi ch include serine proteases, cysteine proteases and MMPs (Tables 1,2). Ex pectedl y, the ac tiviti es of these proteases are tightl y regulated during the reproducti ve cyc le4

.8 and early

es tabli shment of pregnancy l.3 . The balance between the producti on of these enzy mes and that of thei I' spe­cifi c inhibitors and ac tivators plays a crucial role In the remodeling of uterine endometrial matri x.

Emhryonic pro (eases and (h eir role ill zona lysis Hatching of blastocysts is thought to occur by the

in vo lvement of protease- like hatching factor(s) of embryoni c ori gin , ass isting in the rupture of zona ill vi(ro. A variety of proteases of different spec ificities has been shown to be produced by preimplanlat ion embryos and embryo-deri ved trophoblasts (Table I). TI . I d h ' loll 5 I . I . lese Inc u e cat epsll1 s . , p asmln-p asmll10gen ac ti vators2 and MMPs I6. Even, ea rl y cleavage-stage embryos have been shown to produce proteases 17. 1

Tab le 1- SU lllmary of va ri ous protcascs of cmbryonic orig in

Spccics

Mouse

Rat

Hamster

Human

Protcase

Cathcpsin L Cat hcpsi n B uPA . tPA MMP-9 MTI-MMP Trypsi n Ii kc prot casc StrypsinliSP I

uPA tPA

Cys tc inc protcasc

uPA MMP-2 MMP-9

Locati on Rcfcrenccs

Trophob last 14.15 Trophob last I-US Trophob last 1.2 Trophoblast 1.2 Trophoblast 16 Hatchin g blastocysts 2 1 Rlastocysts 2 1

Prc- implan tali on cmbryos IH 2-cc ll cJllbryos . oocy tcs 18

Blastocyst 25

Cytlltrophoblast 4H Trophoblast 49 Trophoblast 49

Abbrcv iations uscd: lIPA. urokinase- type plasminogen acti vator: tPA. tissue-typc plasminogen acti vator: MMP, matri x Illctall o protc in ase: MT. mClll br;1I1C type. lSI'. implantation scri nc prot ease

758 I DIAN J EXP BIOL. JUL Y 2003

In the mouse, erine protease, in parti cul ar, trypsi n like enzyme (strypsin/heps in ) has been reported to be . I d' h f I h' 1 7 19-? 1 S' '1 . ll1 VO ve In t e process 0 latc Ing . -. IITIl ar IS the observati on with other mammalian species such as

" bb' 1, d '4 Of . I' h ' rat--, ra It-· an cow-. parllcu ar ll1terest, ere, IS the lack of in vo lvement of trypsin-like ac tivity in hamster zona lys is2S. Our studies showed that hamster blastocysts are self-sufficient to undergo hatching with concomitant and complete lys is of zonae, by their intrinsic ab ility to produce zona lyt ic factor with cysteine protease- like act ivity. lncidentall y, ours is the first report to implicate cysteine protease-like ac­ti vity in the phenomenon of hatching of mammalian blastocysts25 . Our unpublished data show that cys­tatin-inh ibitable, temporally-produced , proteolytic fac tor is in volved in hamster blastocyst hatching26

.

We believe that the uterine zona lys in appears unlikely to be in volved in the phenomenon of zona escape of hamster blastocysts in vivo25

. Blastocyst hatching in vivo by uterine zona lys in has however,

Table 2 - Summary of various protcascs of cndomctria l orig in

Spccics Protcasc Refcrcnces

Mousc MMP-2 38 MMP-9 50 MMP-3 51 MMP-7 5 1 Stromclysin l, :1 33 uPA 52 ISP-2 21

Rat Cathcpsin-D 53 MMP-2 4 1

32 MMP-9 4 1, 54 uPA 55 Matril ys in 56 Stromelys in 3 1 Col lagenase 40

Hamster Gclatinolytic 57 (Mr 70, 60 x 10-' )

Human MMP-2 13.49 MMP-9 13,49 uPA 48 Enkcphal in ase 58 Matril ys in 44 Cul lagcnase 43 Cathcpsins 59

Abbreviations used : uPA, urokinasc type plasminoge n ac ti va tor: MMP, matrix Illcta ll o protcinasc. ISP, implanta tion scrine proteasc

been impl icated fo r mouse27, rat28.29 and rabbit30. It is

important to study the three classes of proteases (serine, cysteine, and metalloproteases) In the embryo-endometri al tissues during the peri­implantation period. A variety of proteases produced both by the embryo (Tab le I ) and the uterine endometrium (Tab le 2) are extensively described. Seri ne proteases are normally known to be in volved in blastocyst hatch ing2.' 9.2o However, all the three fami lies of protease are involved in the ECM degradation, associated with endometrial shedding during menstruation and tissue remodeling during

b . I . 4 8 lO TI d h . em ryo Imp antatlon " . lese prot eases an t elr inhibitors regulate the ex tent to which the embryo can in vade the endometrium, wh ich varies considerably among mammals.

Elldol1le/ria l pro/eases during es/rous cycle and early pregnallcy

Among the endometri al proteases, MMPs are pri­marily responsible for degradation of EC M compo­nents, which are produced under va rious physiologi­cal conditions (Tab le 2) and are synthesized as zymo­gens4

.R

. They are grouped loosely by their substrate specificity and include collagenases, ge latinases, stromelysins and membrane-bou nd MMPs. Following activation, they degrade a broad range of substrates such as proteoglycans, glycoprotei ns, lam inins, fibronectins, elastins and denatured cOllagens8

. The role of MMPs in ti ssue remodeling occurring during reproduct ive cycle and implantati on has been de­scribed in rats3 1

.32

, mice3D4, rabbits35 and humans '3.36.

However, the involvement of a network of proteases and their inhibitors in a spatial and temporal manner duri ng various physiological states is not clear.

To obtain comparati ve data , we analyzed gelatinolyti c protease actIvities for endometri al samples fro m hamsters and rats by zymography. Interestingly, gross similarities exis t between endometrial protease profiles of both spec ies. In the rat, three major act ivity bands (Mr 5EK. 65 K and 70K) were identifi ed during all stages of estrous cyc le and among these, the 65K band was most prominent (Fig. 1 A). Intensiti es of these bands were however, different and were more intense during the diestrous and proestrous, followed by estrous and metestrous stages. Moreover, we also observed two hi gher molecular weight activity bands (80K and 87K) and thei r intensities showed variations during estrous cycle. In all stages of estrou cycle, an ac tivity band of Mr 20K was also observed (Fig. I A). In the

SES HAG IRI el 01.: EMBRYO-ENDOM ETRI A L PROTEASES 759

hamster, we observed two distinct enzyme actI vIty bands with approx imate Mr of 65K and 70K in the hamster endometrium during all stages of estrous cycle (Fig. I B). Two additional bands of Mr 58 K and 80K were observed in diestrous and metestrous stages, which were either absent or less intense in proestrous and estrous stages. intensities of bands detected were similar in all samples (Fig. I B). Qualitati ve changes in relati ve intensities of bands aml appearance of additional bands may indicate that the expression and acti viti es of these proteins are tightl y regul ated by local hormonal and non-hormonal fac tors.

During earl y pregnancy, the protease profi Ie, ob­served with hamster samples (Fi g. 2A ), was similar to

2 3 4

that observed with sampl es of estrous cycle (Fi g. IB ). Bands observed with days 1 -6 samples were corre­sponding to Mr 20K, 65K and 70K. But a 58K band was detected onl y during days 1-2 of pregnancy (Fi g. 2A). With rat endometri al samples, four major bands (Mr 55K, 65K, 70 K and 87 K) were identified during days 1-6 of pregnancy (Fig 2B). An addi tional band at Mr lOO K was also observed in all samples, whose intensiti es decreased by days 5-6 of pregnancy. The intensiti es of other bands were more or less simi ­lar. The enzy me acti vity observed at Mr 65 K was more intense than the other bands. Two low molecul ar weight bands (40 K and 30K) were also seen, but their ac ti viti es appeared to be very minimal.

2 3 4 Mr X 10-3

A 87

~ 80-~70-'\~F

-20

B

Fig. I - Protease activity profil e during estrous cyc le in the rat (A) and hamster (B). Lanes I, 2. 3 and 4 in both panels correspond to .:ndometri al sa mples from di .:strous. metes trous , es trous and proestrous stages , respectively. In each lane. approximately 100 ~lg of total protein was loaded.

1 2 3 4 5 6

Mr X 10-3

lOu 8, 7fJ 6)

4u 3u

1 2 3 4 5 6

Fig. 2 - Protcase acti vity profi le during early preg nancy in the hamster (A) and rat ( B). Lanes 1-6 corrcspond to endomct rial sa mples fro m days 1-6 of pregnancy. res pccti vely. In each la ne, approximately 100 ~g of total protein was loaded.

760 INDI AN J EXP 1310L. JULY 2003

We also determined the nature of hamster endo­metrial protease. The protease acti vity was completely inhibited with the calcium ion chelator, EGTA (Fig. 3) but not with serin e protease inhibitor, SBT!. When the gel was incubated w ith either Ca~+ alone or a mi xture of C}+ and Zn 1+ or a combination of Ca~+, Zn~+ and EGT A, we found that the addition o f EGT A completely inhibited the Ca2+ and/or Zn ~+ -dependent protease activity. The enzy me activ ity was not de­tec ted when the sample "vas incubated w ith Zn 2+ alone. But, in the presence o f Ca"+ -conta ining buller. the protease ac ti vity was detec tabl e (Fig. 3) . More­over, when I , JO-phenanthro line (a general metallo­protease inhibitor) was used, the activity 'was com­pletely inhibited. These results clearl y indica te th at the observed protease acti v ity is a Cac+ -dependent. metalloprotease.

It is known that MMPs are produced as active pro­enzy mes (zy mogens) and when required , they are activated by several mechanisms. Ac ti va ti on and inhibiti on of these latent pro-MMPs arc brought about by a comp lex network of specific ac tivators and T IMPs. Zy mogens can be activated by serine proteases such as trypsin/p lasminx, or by cys teine proteases like cathepsin B1 4. and also by physical and chemica l (HOCI, organomercurial s, nitric ox ide) means·\7·YI Act ivat ion of pro-MMPs is thought to be

Mr X 10-3

87- 1 70-65- I r 58 ,

20-

Ca+2

EGTA

+ + +

+ + +

- + l-'i g . .1 - Relj uin.: nlcnl ur metal Iuns ror halllstcr endo lllct ri a l protca,c activity . Following clectrophoresis of approx imatel y 100 pg or IOtal prolcin. eac h lane was incubated unclcr dif'f'crcnl conditions as indi cated, Thcn. activ it y stain ing \\as ca rri cd out as detailed in thc tex t.

regulated by hormones. In the rat, whil e serotonin is an acr i vat i ng hormone, estrogen and progesterone

. " 1\ WI' d I I prevent actl vat Ion . . e c etenl1lne W l et l er or not the observed enzy me activit y o f hamster endometrial tissues was due to pro-form or ac ti ve form of protease by pre- incubating samples with 2 mM APMA. W e observed a decrease in the intensity or the 70K band w ith an appearance of a low molecular weight (60 K) band. indi ca tin g th at there is act iva ti on of 70 K band by A PM A (Fig. 4A). Interestingly. trypsin-activation o f endometrial sampl es did not show any major change in the protease ac ti vity profile of the 6S K-70 K proteins, However, a few minor bands of lower mol ecular weigh ts (40K-SO K ) were observed

.(Fig 4B). When rat samples were anal yzed si mi larl y,

(Fig. 4C), i t was Found th at, following acti va ti on of samples by 2 mM A PMA, there was a complete dis­appearance of high molecular we ight bands (80K and 97 K) and an increase in the intensity of 6SK band (Fig. 4C). T hesc results indica te the presence of high molecular weigh t latent protcases w ith res idual activ­ity, wh ich upon A PMA-induced activati on increases the ca tal y ti c act ivit y. Because we observed similar ac ti va ti on process w ith day 5 pregnant samp les. we believe that thi s ac ti va tion mechan ism could be occur­ring in Fi FO during ac ti ve ti ssue remodel ing in estrous cycle and embryo implantation.

The major protease acti v ities of roden t endomctrial samples, v iz, the 65 K and 70 K bands that we ob­served be long to ge latinase- (MMP-2) class and the other spec ies. vi;:. SO K- 100K bands be long to ge lat i­nase-B (MMP-9) c las s~·4o This is based on the follow­ing observati ons: ( i ) molecular weights or ge lat i­noly ti c activ iti es of th e endometri al samp les during es trous cyc le and early pregnancy . ( ii ) dependence on di va lent ca ti ons for enzy me act i vi ti es, ( i i i ) response to va rious i nh i bitors and ( i v) rel ation shi p bet ween zy­mogens and active forms. These f indings arc consi s­tent with th ose observed with mice33

.3x

, rats41,40, mon-

k 4" I h 41~4 T h . b'I' ,. , eys - all( Ulllans " , e lila I lIy 0 tryps lIl prote-ases to acti vate the latent forms in the hamster endo­metriulll suggests the role or other protcascs li ke, cys­teine proteases, viz. ca theps in L, that can activate MMPs~ ·' . Of interest here is our unpubli shed data. wh ich show that cathespin L is detected in all days of es trous cycle and during ear ly days of pregnancy in the hamster endometrium"'h

For proper remodeling of endometrial matri x dur­i ng implan ta ti on. excess i ve or i nappropri atel y ti med proteo ly ti c events need to be res trained and hence

'.

J

X r-- Otr) 00 r--\O

SES HAG IRI (! I (II .: EMBRYO-ENDOM ETRI AL PROTEASlS 76 1

proteases have their respccti ve inhibitors which are ortcn w idely di stributed in the sa me ti ssues in which thc proteases are Ii kel y to ac t ~. Thi s molecu lar mechanism during il11plantati on appears to be con­served in all spec ies. regardl ess o f the ex tent of in va­sion. It is still not clear how enzymcs arc acti va tcd and their ac ti viti es regulated. It is also not clear whethcr stcroid hormones (es trogen/proges terone) arc rcquircd for thcir ex prcss ion and/or therc arc loca ll y produced non-cndocri ne factors (growth hormoncs/ cy tok incs) acting as major stimul ators for proteasc pro­duction5

.6

. Thc knockout phenotypcs of a few of the MMPs and thcir inhibitors however, show abnorl11al uterinc morphology and reduccd fe rtilit / 7 It is in tercst­ing to note that ill vim mutational studics involv ing various MMPs and TIMPs do not show major defccts in either ti ssue rcmodeling during estrous cycle or cmbryo ill1plantati o n~'x, suggesting that embryo-endomctri al pro­teascs may bc hav ing overlapping funct ions during this vital stage of l11ammalian dcvelopmcnt.

Conclusion Wc observcd exprcss ion of MMPs during estrous

cyc le and earl y pregnancy in rodent cndometrium . Thi s is consistent w ith publi shed observa tions that MMPs are abundantl y expressed during rcproducti vc cyc lc and ea rl y prcgnancy in humans. which are shown to be in vol ved in uterine shcdd ing and remod­eling of stroma. For cs tabli sh mcnt of p'·cgnL\ncy. thc two events viz .. hatching of blastocysts and it s inva­sion into utcrinc endomctrium arc ti ghtl y couplcd which are under the control or oppos ing ac ti ons of MMPs and TIMPs and other c lasses o f proteases . For thi s, a timcly synthesis and sccrct ion of thesc pmtc­ases- rcgulators by embryo-e ndomctr ial ti ss ucs is rc­quircd. We bclic vc that they arc undcr thc control of steroid hormones and/or growth factors. The complcx intcrplay of thcse factors in the proteasc-mediated blastocyst hatching and cndometri al ti ssuc rcmodel ing during mammali an peri - implan tation devclopment remains to be investigated in dctail.

Acknowlcdgemcnt The rin(lncial support from thc DST and ICMR.

Ncw Dclhi is gratefull y ack nowledgcd. We thank Ms. V idya Raja for hcr hclp in zymography. Our th anb arc al so duc to M.S. Padmavathi for hcr hclp during the preparation of this manusc ript.

Refcl'clll'CS 1 I br\'ey M H. Lew K J. Arccll;lIla -Panli lio M Y. Zhang X.

I'::dwards D R & Schu ll ": CI A. RoiL- of gn)\\ Ih raelnrs during pcri -illlpbnlalion cic\'c lofllllcni. 111111/ /?'proil. 1 () ( I lJlJ);J) 7 12.

762 INDIAN J EX P BIOL, JUL Y 2003

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