The implantation of the mouse embryo requires the controlled invasion of the uterine stroma by the embryonic trophoblast. This event is dependent, in part, on the secretion of matrix metalloproteinases and serine proteinases for the extracellular degradation of the uterine matrix. Proteinase activity is controlled by stromal decidualization and specific proteinase inhibitors. This work adds to our understanding of implantation and placentation by reporting the expression and function of another class of proteinases/inhibitors closely related to invasive cell behavior. We focused on the cysteine proteinases, cathepsins B and L, and their inhibitor cystatin C. Northern blots showed that trophoblast expressed cathepsin B throughout the invasive period (days 5.5-10.5). Both cathepsin B message and cathepsin L protein were localized to the mature, invasive trophoblast giant cells. Substrate gel electrophoresis showed an increase in giant cell cathepsin activity with enzyme profiles changing at the end of the invasive period. Northern and western blotting showed that cystatin C, the main inhibitor of cathepsins, was a major product of the decidualizing stroma. Message levels first increased in peripheral decidualizing cells, with the protein localizing close to the embryo during implantation (days 5.5-8.5). With the regression of the decidua beginning on day 9.5, a coordinated upregulation of both cathepsin B and cystatin C was observed implying a role for controlled cathepsin expression during apoptosis. E-64, a synthetic inhibitor of cathepsins B and L, was injected into pregnant females at the stage of blastocyst attachment (days 4.5-5.5). High doses resulted in the complete failure of implantation while lower doses resulted in stunted embryos and a reduced decidual reaction. These results suggested that cathepsins B and L are necessary for normal embryo development and uterine decidualization, and that decidua contributes to their control by a coordinated expression of cystatin C within the implantation site.

REFERENCES

REFERENCES
Abrahamsohn
P. A.
(
1983
)
Ultrastructural study of the mouse antimesometrial decidua.
Anatomy Embryology
166
,
263
74
Abrahamsohn
P. A.
,
Zorn
T. M. T.
(
1993
)
Implantation and decidualization in rodents.
J. Exp. Zool
226
,
603
628
Abrahamson
M.
,
Barrett
A. J.
,
Salvensen
G.
,
Grubb
A.
(
1986
)
Isolation of six cysteine proteinase inhibitors from human urine.
J. Biol. Chem
261
,
11282
11289
Akcali
K. C.
,
Khan
S. A.
,
Moulton
B. C.
(
1996
)
Effect of decidualization on the expression of bax and bcl-2 in the rat uterine endometrium.
Endocrinology
137
,
3123
3131
Alexander
C. M.
,
Hansell
E. J.
,
Behrendtsen
O.
,
Flannery
M. L.
,
Kishnani
N. S.
,
Hawkes
S. P.
,
Werb
Z.
(
1996
)
Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation.
Development
122
,
1723
1736
Apte
A.
,
Hayashi
K.
,
Seldin
M. F.
,
Mattei
M. G.
,
Hayashi
M.
,
Olsen
B. J.
(
1994
)
Gene encoding a novel murine tissue inhibitor of metalloproteinases (TIMP), TIMP-3 is expressed in developing mouse epithelia, cartilage, and muscle, and is located on mouse chromosome 10.
Dev. Dynamics
200
,
177
197
Babiarz
B.
,
Romagnano
L.
,
Kurilla
G.
(
1992
)
The interaction of mouse ectoplacental cone trophoblast and uterine decidua in vitro.
In Vitro Cellular and Developmental Biol
28
,
500
508
Babiarz
B.
,
Romagnano
L.
,
Afonso
S.
,
Kurilla
G.
(
1996
)
Localization and expression of fibronectin during mouse decidualization invitro: Mechanisms of cell:matrix interactions.
Dev. Dynamics
206
,
330
342
Barrett
A. J.
,
Kembhavi
A. A.
,
Brown
M. A.
,
Kirschke
H.
,
Knight
C. G.
,
Tamai
M.
,
Hanada
K.
(
1982
)
L- trans -Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L.
Biochem. J
201
,
189
198
Barrett
A. J.
,
Davies
M. E.
,
Grubb
A.
(
1984
)
The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors.
Biochem. Biophysical Res. Commun
120
,
631
636
Bevilacqua
E. M.
,
Abrahamsohn
P. A.
(
1989
)
Trophoblast invasion during implantation of the mouse embryo.
Archivos de Biologia y Medicina Experimentales
22
,
107
118
Burleigh
M. C.
,
Barrett
A. J.
,
Lazarus
G. S.
(
1974
)
Cathepsin B1. A lysosomal enzyme that degrades native collagen.
Biochem. J
137
,
387
394
Chambers
A. F.
,
Colella
R.
,
Denhardt
D. T.
,
Wilson
S. M.
(
1992
)
Increased expression of cathepsins L and B and decreased activity of their inhibitors in metastatic, ras-transformed NIH 3T3 cells.
Molecular Carcinogenesis
5
,
238
245
Chan
S. J.
,
San Segundo
B.
,
McCormack
M.
,
Steiner
D. F.
(
1986
)
Nucleotide and predicted amino acid sequences of cloned human and mouse preprocathepsin B cDNAs.
Proc. Natl. Acad. Sci. USA
83
,
7721
7725
Cross
J. C.
,
Werb
Z.
,
Fisher
S. J.
(
1994
)
Implantation and the placenta: key pieces of the developmental puzzle.
Science
266
,
1508
1518
Daston
G. P.
,
Baines
D.
,
Yonker
J. E.
,
Lehman-McKeeman
L.
(
1991
)
Effects of lysosomal proteinase inhibition on the development of the rat embryo in vitro.
Teratology
43
,
253
261
Dowd
A.
,
Dalton
J. P.
,
Loukas
A.
,
Prociv
P.
,
Brindley
P.
(
1994
)
Secretion of cysteine proteinase activity by the zoonotic hookworm Ancylostomacaninum.
Am. J. Trop. Med. Hyg
51
,
341
347
Everts
V.
,
van der Zee
E.
,
Creemers
L.
,
Beertsen
W.
(
1996
)
Phagocytosis and intracellular digestion of collagen, its role in turnover and remodeling.
Histochemical J
28
,
229
245
Farrar
J. D.
,
Carson
D. D.
(
1992
)
Differential temporal and spatial expression of mRNA encoding extracellular matrix components in decidua during the peri-implantation period.
Biol. Reprod
46
,
1095
1108
Glasser
S.
,
Lampelo
S.
,
Munir
M.
,
Julian
J.
(
1987
)
Expression of desmin, laminin and fibronectin during in situ differentiation (decidualization) of rat uterine stromal cells.
Differentiation
35
,
132
142
Gong
Q.
,
Chan
S. J.
,
Bajkowski
A. S.
,
Steiner
D. F.
,
Frankfater
A.
(
1993
)
Characterization of the cathepsin B gene and multiple mRNAs in human tissues: evidence for alternative splicing of cathepsin B pre-mRNA.
DNA and Cell Biology
12
,
299
309
Goretzki
L.
,
Schmitt
M.
,
Mann
K.
,
Calvete
J.
,
Chucholowski
N.
,
Kramer
M.
,
Gunzler
W.
,
Janicke
F.
,
Graeff
H.
(
1992
)
Effective activation of the proenzyme form of the urokinase-type plasminogen activator (pro-uPA) by the cysteine protease cathepsin L.
FEBS
297
,
112
118
Gottesman
M. M.
(
1990
)
Do proteases play a role in cancer?.
Seminars in Cancer Biol
1
,
97
99
Grubb
J. D.
,
Koszalk
T. R.
,
Drabick
J. J.
,
Metrione
R. M.
(
1991
)
The activities of thiol proteases in the rat visceral yolk sac increase during late gestation.
Placenta
12
,
143
151
Gu
Y.
,
Jow
G. M.
,
Moulton
B. C.
,
Lee
C.
,
Sensibar
J. A.
,
Park-Sarge
O. K.
,
Chen
T. J.
,
Gibori
G.
(
1994
)
Apoptosis in decidual tissue regression and reorganization.
Endocrinology
135
,
1272
9
Guenette
R. S.
,
Mooibroek
M.
,
Wong
K.
,
Wong
P.
,
Tenniswood
M.
(
1994
)
Cathepsin B, a cysteine protease implicated in metastatic progression, is also expressed during regression of the rat prostate and mammary glands.
European J. Biochem
226
,
311
321
Hamilton
R. T.
,
Bruns
K. A.
,
Shim
J. K.
,
Delgado
M. A.
,
Fang
Y.
,
Nilson-Hamilton
M.
(
1990
)
Developmental expression of cathepsin L and c-rasin the mouse placenta.
J. Cell Biol
111
,
357
–.
Hamilton
R. T.
,
Bruns
K. A.
,
Delgado
M. A.
,
Shim
J.
,
Fang
Y.
,
Denhardt
D. T.
,
Nilsen-Hamilton
M.
(
1991
)
Developmental expression of cathepsin L and c-rasHain the mouse placenta.
Molec. Reprod. Dev
30
,
285
292
Harvey
M. B.
,
Leco
K. J.
,
Arcellana-Panlilio
M. Y.
,
Zhang
X.
,
Edwards
D. R.
,
Schulta
G. A.
(
1995
)
Proteinase expression in early mouse embryos is regulated by leukemia inhibitory factor and epidermal growth factor.
Development
121
,
1005
1014
Henskens
Y. M.
,
Veerman
E. C.
,
Mantel
M. S.
,
Van der Veldem
U.
,
Nieuw-Amerongen
A. V.
(
1994
)
Cystatins S and C in human whole saliva and in glandular salivas in periodontal health and disease.
J. Dental Res
73
,
1606
1614
Huh
C.
,
Nagle
J.
,
Kozak
C.
,
Abrahamson
M.
,
Karlsson
S.
(
1995
)
Structural organization, expression and chromosomal mapping of mouse cystatin C encoding gene (Cyst 3).
Gene
152
,
221
226
Ichikawa
S.
,
Shibata
T.
,
Takehara
Y.
,
Tamada
H.
,
Oda
K.
,
Murao
S.
(
1985
)
Effects of proteinase inhibitors on preimplantation embryos in the rat.
J. Reprod. Fertil
73
,
385
390
Lah
T. T.
,
Buck
M. R.
,
Honn
K. V.
,
Crissman
J. D.
,
Rao
N. C.
,
Liotta
A. L.
,
Sloane
B. F.
(
1989
)
Degradation of laminin by human tumor cathepsin B.
Clin. Exp. Metastasis
7
,
461
469
Lah
T. T.
,
Babnik
J.
,
Schiffman
E.
,
Turk
V.
,
Skaleric
U.
(
1993
)
Cysteine proteinases and inhibitors in inflammation: their role in periodontal disease.
J. Periodontal
64
,
485
491
Lefebvre
O.
,
Regnier
C.
,
Chenard
M. P.
,
Wendling
C.
,
Chambon
P.
,
Basset
P.
,
Rio
M. C.
(
1995
)
Developmental expression of mouse stromelysin-3 mRNA.
Development
121
,
947
955
Lerner
U. H.
,
Grubb
A.
(
1992
)
Human cystatin C, a cysteine proteinase inhibitor, inhibits bone resorption in vitro stimulated by parathyroid hormone and parathyroid hormone-related peptide malignancy.
J. Bone Miner. Res
7
,
433
440
Mach
L.
,
Schwihla
H.
,
Stuwe
K.
,
Rowan
A. D.
,
Mort
J. S.
,
Glossl
J.
(
1993
)
Activation of procathepsin B in human hepatoma cells: the conversion into the mature enzyme relies on the action of cathepsin B itself.
Biochem. J
293
,
437
442
Mason
R.
,
Johnson
D.
,
Chapman
H.
(
1986
)
Elastolytic activity of human cathepsin L.
Biochem. J
233
,
925
927
Murphy
G.
,
Ward
R.
,
Gavrilovic
J.
,
Atkinson
S.
(
1992
)
Physiological mechanisms for metalloproteinase activation.
Matrix Supplement
1
,
224
230
Ono
H.
,
Ide
C.
,
Nishya
I.
(
1989
)
Electron microscopic study of early decidualization of the endometrium of pregnant mice, with special reference to gap junctions.
Placenta
10
,
247
261
O'Shea
J.
,
Kleinfeld
R.
,
Morrow
H.
(
1983
)
Ultrastructure of decidualization in the pseudopregnant rat.
Am. J. Anat
166
,
271
298
Palis
J.
,
Kingsley
P. D.
(
1995
)
Differential gene expression during murine yolk sac development.
Molec. Reprod. Dev
42
,
19
27
Parr
E. L.
,
Tung
H. N.
,
Parr
M. B.
(
1987
)
Apoptosis as the mode of uterine epithelial cell death during embryo implantation in mice and rats.
Biol. Reprod
36
,
211
225
Qian
F.
,
Bajkowski
A. S.
,
Steiner
D. F.
,
Chan
S. J.
,
Frankfater
A.
(
1989
)
Expression of five cathepsins in murine melanomas of varying metastatic potential and normal tissues.
Cancer Res
49
,
4870
4875
Romagnano
L.
,
Babiarz
B.
(
1990
)
The role of murine cell surface galactosyltransferase in trophoblast: laminin interactions in vitro.
Dev. Biol
141
,
254
261
Romagnano
L.
,
Afonso
S.
,
Babiarz
B.
(
1996
)
An in vitro system for the study of matrix metalloproteases during decidualization in the mouse.
Biochem. Cell Biol
74
,
911
919
Ryan
R. E.
,
Sloane
B. F.
,
Sameni
M.
,
Wood
P. L.
(
1995
)
Microglial cathepsin B: an immunological examination of cellular and secreted species.
J. Neurochem
65
,
1035
1045
Schaeren-Wiemers
N.
,
Gerfin-Moser
A.
(
1993
)
A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labeled cRNA probes.
Histochemistry
100
,
431
440
Sloane
B. F.
,
Moin
K.
,
Krepela
E.
,
Rozhin
J.
(
1990
)
Cathepsin B and its endogenous inhibitors: the role in tumor malignancy.
Cancer Metastasis Rev
9
,
333
352
Tachikura
T.
(
1990
)
The teratogenic effects of E-64 on rat embryogenesis.
Acta Paediatrica Japonica
32
,
495
501
Teesalu
T.
,
Blasi
F.
,
Talarico
D.
(
1996
)
Embryo implantation in mouse: fetomaternal coordination in the pattern of expression of uPA, uPAR, PAI-1 and alpha 2MR/LRP genes.
Mechan. Dev
56
,
103
116
Waterhouse
P.
,
Denhardt
D. T.
,
Khoka
R.
(
1993
)
Temporal expression of tissue inhibitors of metalloproteinases (TIMPS) in mouse reproductive tissues during gestation.
Mol. Reprod. Dev
35
,
219
226
Wewer
U. M.
,
Damjanov
A.
,
Weiss
J.
,
Liotta
L. A.
,
Damjanov
I.
(
1986
)
Mouse endometrial stromal cells produce basement-membrane components.
Differentiation
32
,
49
58
Zhang
X.
,
Shu
M. A.
,
Harvey
M. B.
,
Schultz
G. A.
(
1996
)
Regulation of urokinase plasminogen activator production in implanting mouse embryo: effect of embryo interaction with extracellular matrix.
Biol. Reprod
54
,
1052
1058
Zorn
T.
,
Bevilacqua
E.
,
Abrahamsohn
P. A.
(
1986
)
Collagen remodeling during decidualization in the mouse.
Cell Tissue Res
244
,
443
448
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