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Endocrinology Vol. 147, No. 7 3478-3483
Copyright © 2006 by The Endocrine Society

Progesterone and Interferon-{tau} Regulate Cystatin C in the Endometrium

Gwonhwa Song, Thomas E. Spencer and Fuller W. Bazer

Center for Animal Biotechnology and Genomics and Department of Animal Science, Texas A&M University, College Station, Texas 77843

Address all correspondence and requests for reprints to: Fuller W. Bazer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, Texas 77843-2471. E-mail: fbazer{at}cvm.tamu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cystatin C (CST3) is a secreted inhibitor of lysosomal cysteine proteases cathepsins B (CTSB) and CTSL, which are abundant in the ovine endometrium and conceptus. In mice, cathepsins and cystatins play important roles in implantation and placentation. This study determined effects of the estrous cycle, pregnancy, progesterone (P4), and interferon-{tau} (IFNT) on CST3 in the ovine uterus. In cyclic ewes, CST3 mRNA was low on d 10, increased about 12-fold by d 12, and declined thereafter. In early pregnant ewes, CST3 mRNA was low on d 10 and increased about 130-fold from d 10 to d 20. CST3 mRNA and protein were abundant in the endometrial luminal epithelium (LE) and glandular epithelium and also in conceptus trophectoderm. In uterine flushes from pregnant ewes, CST3 protein was not detected on d 10 but was abundant on d 12, 14, and 16. In another study, treatment of ovariectomized, cyclic ewes with P4 induced a 14-fold increase in endometrial CST3 mRNA, and IFNT stimulated an additional 2-fold increase in CST3 mRNA in P4-treated ewes but not in ewes treated with P4 and the antiprogestin ZK 136,317. CST3 mRNA and protein were abundant in the endometrial luminal epithelium and superficial glandular epithelium of P4-treated ewes but were very low or not detectable in endometria of P4- and ZK-treated ewes. These results indicate that CST3 is a novel P4-induced and IFNT-stimulated gene expressed only in the epithelial cells of the ovine endometrium and implicate CST3 in regulation of uterine cathepsin activity during conceptus implantation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A VARIETY OF proteases, as well as their inhibitors, regulate endometrial remodeling and trophoblast invasion in many species (e.g. mouse, rat, cat, sheep, pig, and human) during conceptus (embryo/fetus and associated extraembryonic membranes) implantation and placentation (1, 2, 3, 4, 5). Cathepsins are a family of lysosomal proteinases that can degrade extracellular matrix molecules and influence catabolism of intracellular proteins and prohormone processing (6). Cystatin C (CST3) is a low-molecular-weight secretory protein that functions as an inhibitor of lysosomal cysteine proteinases, including cathepsins B (CTSB) and L (CTSL) (7, 8, 9, 10). In mice, CTSB and CTSL are necessary for normal embryo development and uterine decidualization, and the decidua coordinately expresses CST3 presumably to control cathepsin actions within the implantation site (11). We recently reported expression of CTSB, CTSD, CTSH, CTSK, CTSL, CTSS, and CTSZ in the endometrium and/or conceptus of sheep during early pregnancy (12). In that study, CTSL was the most abundant cathepsin expressed by the endometrial epithelia and conceptus trophectoderm during early pregnancy, and the CTSL gene was induced by progesterone (P4) and stimulated by interferon-{tau} (IFNT). However, expression of CST3 has not been investigated in the ovine uterus.

Trophoblast invasion in ruminants (sheep, cattle, and goats) is limited to fusion of migrating trophoblast giant binucleate cells with uterine luminal epithelium (13); however, considerable tissue remodeling and angiogenesis occurs within the endometrium at implantation that is associated with the cysteine and serine proteases and production of matrix metalloproteinases by the endometrium and conceptus (14, 15). Endometrial functions during this period of pregnancy are primarily regulated by P4 from the corpus luteum (CL) and hormones from the conceptus, including IFNT, placental lactogen, and placental GH (16, 17). IFNT is the signal for maternal recognition of pregnancy in ruminants (18, 19) and is produced between d 10 and 21–25 of pregnancy in sheep by the mononuclear trophoblast cells of the conceptus (20). In sheep, IFNT acts in a paracrine manner on endometrial luminal epithelium (LE) and superficial glandular epithelium (sGE) to inhibit transcription of the estrogen receptor-{alpha} gene (21), thereby preventing induction of the oxytocin receptor gene and endometrial release of luteolytic pulses of prostaglandin F2{alpha} (18, 22). The antiluteolytic actions of IFNT are required for maintenance of a functional CL and secretion of P4, the essential hormone of pregnancy. IFNT also induces or stimulates expression of a number of genes, termed IFNT-stimulated genes (ISGs), in the endometrium that are hypothesized to play important biological roles in uterine receptivity and conceptus implantation (23). In the ovine uterus, most ISGs are induced or increased in the endometrial stroma and middle to deep GE. Indeed, LGALS15 (galectin-15) (24), WNT7A (wingless-type mouse mammary tumor virus integration site family, member 7A) (25), and CTSL (12) are the only genes known to be induced or increased by IFNT in endometrial LE and sGE.

Therefore, these studies were conducted to determine whether the CST3 gene is expressed in the ovine uterus and to determine effects of the estrous cycle, pregnancy, P4, and IFNT on CST3 gene expression in the endometrium and conceptus. The results indicate that CST3 is expressed coordinately with CTSL in the endometrial LE and GE and conceptus during the periimplantation period of pregnancy. Furthermore, CST3 is a novel P4-induced and IFNT-stimulated gene in the endometrial LE and sGE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Mature crossbred Suffolk ewes (Ovis aries) were observed daily for estrus in the presence of vasectomized rams and used in experiments only after they had exhibited at least two estrous cycles of normal duration (16–18 d). All experimental and surgical procedures were in compliance with the Guide for the Care and Use of Agriculture Animals and approved by the Institutional Animal Care and Use Committee of Texas A&M University.

Experimental design
Study 1.
At estrus (d 0), ewes were mated to either an intact or vasectomized ram and then hysterectomized (n = 5 ewes/d) on d 10, 12, 14, or 16 of the estrous cycle or d 10, 12, 14, 16, 18, or 20 of pregnancy as described previously (26). At hysterectomy, the uterus was flushed with 20 ml sterile saline. Pregnancy was confirmed on d 10–16 post mating by the presence of a morphologically normal conceptus in the uterine flush. It was not possible to obtain uterine flushes on either d 18 or 20 of pregnancy, because the conceptus is firmly adhered to the endometrial LE and basal lamina. At hysterectomy, several sections (~0.5 cm) from the midportion of each uterine horn ipsilateral to the CL were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2). After 24 h, fixed tissues were changed to 70% ethanol for 24 h, dehydrated through a graded series of alcohol to xylene, and then embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO). Several sections (1–1.5 cm) from the middle of each uterine horn were embedded in Tissue-Tek OCT compound (Miles, Oneonta, NY), frozen in liquid nitrogen vapor, and stored at –80 C. The remaining endometrium was physically dissected from myometrium, frozen in liquid nitrogen, and stored at –80 C for subsequent RNA or protein extraction. In monovulatory pregnant ewes, uterine tissue samples were marked as either contralateral or ipsilateral to the ovary bearing the CL, and only tissues from the ipsilateral uterine horn were used in subsequent analyses. Uterine flushes were clarified by centrifugation (3000 x g for 30 min at 4 C) and frozen at –80 C for Western blot analysis.

Study 2.
In study 2, cyclic ewes (n = 20) were checked daily for estrus and then ovariectomized and fitted with indwelling uterine catheters on d 5 as described previously (27). Ewes were then assigned randomly (n = 5 per treatment) to receive daily im injections of P4 and/or a P4 receptor (PGR) antagonist (ZK 136,317; Schering AG, Berlin, Germany) and intrauterine infusions of control serum proteins and/or recombinant ovine IFNT protein as follows: 1) 50 mg P4 (d 5–16) and 200 µg control (CX) serum proteins (d 11–16) (P4+CX); 2) P4 and 75 mg ZK 136,317 (d 11–16) and CX proteins (P4+ZK+CX); 3) P4 and IFNT (2 x 107 antiviral units, d 11–16) (P4+IFN); or 4) P4 and ZK and IFNT (P4+ZK+IFN). Steroids were administered daily in corn oil vehicle. Both uterine horns of each ewe received twice-daily injections of either CX proteins (50 µg/horn per injection) or recombinant ovine (ro)IFNT (5 x 106 antiviral units/horn per injection). The roIFNT was produced in Pichia pastoris and purified as described previously (28). Proteins were prepared for intrauterine injection as described previously (27). This regimen of P4 and roIFNT mimics the effects of P4 and the conceptus on endometrial expression of hormone receptors and IFNT-stimulated genes during early pregnancy in ewes (25, 29, 30, 31). All ewes were hysterectomized on d 17 and uteri and endometria processed as described for study 1.

RNA isolation
Total cellular RNA was isolated from frozen endometrium from the ipsilateral uterine horn (studies 1 and 2) using Trizol reagent (Life Technologies, Inc.-BRL, Bethesda, MD) according to the manufacturer’s recommendations. The quantity and quality of total RNA were determined by spectrometry and denaturing agarose gel electrophoresis, respectively.

Cloning of partial cDNA for ovine CST3
Partial cDNA for ovine CST3 mRNA was amplified by RT-PCR using total RNA from d-18 pregnant ovine endometrial tissues by specific primers based on the bovine CST3 mRNA (GenBank accession no. NM_174029; forward, 5'-CTG TCC TTT GCG GTC AGC-3'; reverse, 5'-CCT GGC AGC TAA ACT TCA CC-3'). PCR amplification was conducted as follows for ovine CST3: 1) 95 C for 5 min; 2) 95 C for 45 sec, 56.5 C for 1 min, and 72 C for 1 min for 35 cycles; and 3) 72 C for 10 min. The partial cDNAs for CST3 were cloned into pCRII using a T/A Cloning Kit (Invitrogen, Carlsbad, CA) and sequence verified using an ABI PRISM Dye Terminator Cycle Sequencing Kit and ABI PRISM automated DNA sequencer (PerkinElmer Applied Biosystems, Foster City, CA).

Slot blot hybridization analyses
Steady-state levels of mRNA in ovine endometria were assessed by slot blot hybridization as described previously (32). Antisense CST3 cRNA probes were generated by linearizing the pCR II-CST3 plasmid with BamHI, and in vitro transcription with T7 RNA polymerase and sense cRNA probes were generated using XbaI and SP6 RNA polymerase. And then, radiolabeled antisense and sense cRNA probes were generated by in vitro transcription with [{alpha}-32P]UTP. Denatured total endometrial RNA (20 µg) from each ewe was hybridized with radiolabeled cRNA probes. To correct for variation in total RNA loading, a duplicate RNA slot membrane was hybridized with radiolabeled antisense 18S cRNA (pT718S; Ambion, Austin, TX). After washing, the blots were digested with ribonuclease A, and radioactivity associated with slots was quantified using a Typhoon 8600 MultiImager (Molecular Dynamics, Piscataway, NJ).

In situ hybridization analyses
Location of mRNA expression in sections (5 µm) of the ovine uterine endometrium was determined by radioactive in situ hybridization analysis as described previously (32). Briefly, deparaffinized, rehydrated, and deproteinated uterine tissue sections were hybridized with radiolabeled antisense or sense cRNA probes generated from linearized ovine CST3 partial cDNA using in vitro transcription with [{alpha}-35S]UTP. After hybridization, washing, and ribonuclease A digestion, slides were then dipped in NTB-2 liquid photographic emulsion (Kodak, Rochester, NY) and exposed at 4 C for 1 wk. Slides were developed in Kodak D-19 developer, counterstained with Gill’s hematoxylin (Fisher Scientific, Fairlawn, NJ), and dehydrated through a graded series of alcohol to xylene, and coverslips were affixed with Permount (Fisher). Images of representative fields were recorded under bright-field or dark-field illumination using a Nikon Eclipse 1000 photomicroscope (Nikon Instruments Inc., Lewisville, TX) fitted with a Nikon DXM1200 digital camera.

Immunohistochemistry
Immunocytochemical localization of CST3 protein in the ovine uterus was performed as described previously (26) using antihuman CST3 polyclonal antibody (catalog no. 06-458; Upstate Biotechnology, Lake Placid, NY) at a 1:2000 dilution (0.5 µg/ml). Antigen retrieval was performed by using Pronase E digestion, and negative controls included substitution of the primary antibody with purified rabbit IgG at the same final concentration.

Western blot analyses
Uterine flushes from study 1 were concentrated using Centricon-3 columns (Amicon, Beverly, MA), and protein content was determined using the Bradford protein assay (Bio-Rad, Hercules, CA) with BSA as the standard. Proteins were denatured and separated by 15% SDS-PAGE, and Western blot analyses were conducted as described previously (26) using enhanced chemiluminescence detection (SuperSignal West Pico; Pierce, Rockford, IL) and X-OMAT AR x-ray film (Kodak) according to the manufacturer’s recommendations. Immunoreactive CST3 protein was detected by using the rabbit antihuman CST3 polyclonal antibody (Upstate) at a 1:10,000 (0.1 µg/ml) dilution. Negative control blots were performed by replacing the primary antibody with rabbit IgG at the same concentration.

Statistical analyses
All quantitative data were subjected to least-squares ANOVA using the general linear models procedures of the Statistical Analysis System (SAS Institute, Cary, NC). Slot blot hybridization data were corrected for differences in sample loading using the 18S rRNA data as a covariate. Data from study 1 were analyzed for effects of day, pregnancy status (cyclic or pregnant), and their interaction. Next, least-squares regression ANOVA was conducted within pregnancy status. Orthogonal contrasts were used to determine effects of treatment in study 2. All tests of significance were performed using the appropriate error terms according to the expectation of the mean squares for error. A P value of 0.10 or less was considered significant. Data are presented as least-square means with SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of estrous cycle and early pregnancy on expression of CST3 mRNAs in the ovine endometrium (study 1)
Steady-state levels of ovine CST3 mRNAs in endometria from cyclic and pregnant ewes were determined by slot blot hybridization analyses (Fig. 1Go) and found to be affected (P < 0.05) by day, status, and their interaction. In cyclic ewes, endometrial CST3 mRNA was low to undetectable on d 10, increased (quadratic effect of day, P < 0.05) about 12-fold from d 10 to d 12, and then declined to d 16. In pregnant ewes, CST3 mRNA levels were also low to undetectable on d 10 but then increased (linear effect of day, P < 0.01) about 130-fold between d 10 and 20.


Figure 1
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  		FIG. 1. Steady-state levels of CST3 mRNAs in endometria from cyclic and early pregnant ewes as determined by slot blot analysis. In cyclic ewes, CST3 mRNA was low on d 10, increased to d 12, and decreased thereafter (quadratic effect of day, P < 0.05). In pregnant ewes, CST3 mRNA was lowest on d 10 and increased 130-fold between d 10 and 20 (linear effect of day, P < 0.01). Data are expressed as least-square mean relative units (RU) with SE.

 
In situ hybridization analyses to determine the location of CST3 mRNA in the uterus (Fig. 2Go) revealed that it is present only in LE and GE of the endometrium. No hybridization signal was detected in endometrial stroma, myometrium, blood vessels, or immune cells. In cyclic ewes, CST3 mRNA appeared in LE and sGE of the endometrium between d 10 and 12 but decreased thereafter. In pregnant ewes, CST3 mRNA was also detected in endometrial LE and sGE between d 10 and 12. Between d 12 and 20 of pregnancy, CST3 mRNA was abundant in the endometrial LE and sGE and also increased in the middle to deep GE by d 20. In addition, CST3 mRNA was abundant in the conceptus trophectoderm on d 18 and 20 of pregnancy.


Figure 2
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  		FIG. 2. In situ hybridization analyses of CST3 mRNAs in uteri of cyclic and early pregnant ewes. Cross-sections of the uterine wall from cyclic (C) and pregnant (P) ewes were hybridized with radiolabeled antisense or sense ovine CST3 cRNA probes. CST3 mRNA was detected only in endometrial LE and GE as well as trophectoderm of the conceptus. S, Stroma; Tr, trophectoderm. Scale bar, 10 µm.

 
Consistent with results from in situ hybridization analyses, immunoreactive CST3 protein was detected in endometrial LE, sGE, and conceptus trophectoderm (Fig. 3AGo). In pregnant ewes, CST3 protein increased after d 10 and was concentrated near or on the apical surface of LE and sGE as well as in secretions of the uterine glands (Fig. 3BGo). The rabbit antihuman CST3 polyclonal antibody used in these studies detected a single immunoreactive protein of approximately 14 kDa in Western blot analyses of proteins in uterine extracts from cyclic and pregnant ewes (Fig. 3CGo). Consistent with immunohistochemical studies, CST3 protein was detected predominantly in uterine flushes from d 12 cyclic ewes. In uterine flushes of pregnant ewes, CST3 protein was not detected on d 10 but was abundant thereafter. Collectively, the temporal and spatial alterations in CST3 mRNA and protein expression during the estrous cycle and early pregnancy are consistent with its regulation by ovarian P4 and the conceptus.


Figure 3
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  		FIG. 3. CST3 protein in endometria and uterine flushings from cyclic and pregnant ewes from study 1. A, Immunoreactive CST3 protein was localized using a rabbit antihuman CST3 polyclonal antibody. For the IgG control, normal rabbit IgG was substituted for the primary antibody. Sections were not counterstained. S, stroma; Tr, trophectoderm. Scale bar, 5 µm. B, Immunoreactive CST3 protein was localized predominantly near the apical surface of endometrial LE and sGE and was detected in secretions in the lumen of the upper endometrial glands. Sections were not counterstained. Scale bar, 1.25 µm. C, Representative Western blot analysis of CST3 in uterine flushings. Proteins in uterine flushings were separated by 15% SDS-PAGE (10 µg/lane), and immunoreactive CST3 protein detected using rabbit antihuman CST3 polyclonal antibody. Positions of prestained molecular weight standards (x10–3) are indicated.

 
Progesterone and IFNT regulate endometrial CST3 expression (study 2)
To determine whether P4 and IFNT regulate endometrial CST3 gene expression, cyclic ewes were ovariectomized and fitted with indwelling uterine catheters on d 5 and then treated with P4 or P4 and ZK 136,317 antiprogestin (P4+ZK) and infused with control proteins (CX) or roIFNT (see Fig. 4AGo). Slot blot analyses of endometrium found that treatment of ewes with P4 induced a 14-fold increase in CST3 mRNA (P4+CX vs. P4+ZK+CX, P < 0.001; Fig. 4BGo). Moreover, intrauterine infusions of roIFNT stimulated an additional 2-fold increase in CST3 mRNA (P4+CX vs. P4+IFN, P < 0.01). However, roIFNT did not stimulate CST3 mRNA in endometria of ewes receiving the ZK antiprogestin (P4+IFN vs. P4+ZK+IFN, P > 0.10). In situ hybridization (Fig. 4CGo) and immunohistochemical (Fig. 4DGo) analyses detected CST3 mRNA and protein only in endometrial LE and sGE of ewes treated with P4 (P4+CX and P4+IFN).


Figure 4
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  		FIG. 4. Effects of P4 and IFNT on CST3 mRNA and protein in the ovine uterus (study 2). A, Experimental design. See Materials and Methods for complete description. CX, Control serum proteins; Hystx, hysterectomy; Ovx/Cath, ovariectomy and uterine catheterization; ZK, ZK 137,316 antiprogestin. B, Steady-state levels of CST3 mRNA in endometria as determined by slot blot hybridization analysis. Treatment of ewes with P4 increased CST3 mRNA in the endometrium (P4+CX vs. P4+ZK+CX, P < 0.001). Intrauterine infusion of IFNT stimulated CST3 mRNA in endometria of ewes treated with P4 (P4+CX vs. P4+IFN, P < 0.01) but not in ewes receiving P4 and the ZK antiprogestin (P4+IFN vs. P4+ZK+IFN, P > 0.10). C, In situ hybridization analysis of CST3 mRNA expression. Cross-sections of the uterine wall from treated ewes were hybridized with radiolabeled antisense or sense ovine CST3 cRNA probes. Scale bar, 10 µm. D, Immunoreactive CST3 protein in the uterus. Sections were not counterstained. S, Stroma. Scale bar, 5 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, temporal changes in expression of endometrial CST3 mRNA in cyclic and pregnant ewes supported the hypothesis that ovarian P4 is required to induce transcription of the CST3 gene in endometrial LE and sGE. The increase in CST3 mRNA in LE and sGE, between d 10 and 12 post estrus/mating is coincident with the disappearance of PGR mRNA and protein in these epithelia (33, 34). Similarly, the decrease in CST3 mRNA between d 14 and 16 of the cycle is coincident with the reappearance of PGR protein in endometrial LE as a result of the regression of the CL and loss of P4. In study 2, CST3 mRNA was detected in endometrial LE and sGE of ovariectomized ewes treated with P4 for 12 d, but this expression was prevented by administration of the PGR antagonist ZK 136,317. Continuous exposure of the sheep uterus to P4 for about 10 d down-regulates PGR expression in endometrial LE and sGE but not in stroma or myometrium (35). PGRs are present in endometrial epithelia of P4+ZK-treated sheep (35), because PGR antagonists prevent the inhibitory effects of P4 on expression of the PGR gene. Consequently, P4 modulation of CST3 mRNA may be attributed, at least in part, to down-regulation of PGR by P4 that occurs in LE and sGE between d 10 and 12 of the cycle and pregnancy (16, 17). Thus, PGR loss in endometrial epithelia may reprogram these cells, allowing them to increase expression of genes associated with implantation (16). Alternatively, P4 may act on PGR-positive stromal cells to induce growth factors or changes in the extracellular matrix that regulate expression of selected epithelial genes (16).

In addition to regulation by P4, results of the present studies indicate that CST3 expression is further regulated by IFNT. IFNT is the pregnancy recognition hormone in sheep that acts on the endometrium to prevent development of the luteolytic mechanism, thereby maintaining the CL and its production of P4 (18, 19). Of particular note, CST3 is a novel gene stimulated by IFNT in endometrial LE and sGE because expression between d 10 and 18 of early pregnancy parallels the increase in production of IFNT by the elongating conceptus, which is maximal on d 16 (36, 37). In study 2, intrauterine administration of IFNT increased CST3 mRNA, but only in P4-treated ewes. One hypothesis is that IFNT can only stimulate transcription of the CST3 gene in the absence of liganded PGR, i.e. after down-regulation of PGR by P4. Alternatively, the PGR-positive stroma may produce a progestamedin, e.g. fibroblast growth factor 7 or 10 or hepatocyte growth factor, that could be required for LE and sGE to respond to IFNT (16, 25, 38). The signaling pathway whereby IFNT regulates transcription of the CST3 gene is not known, but it clearly does not involve the classical Janus kinase/signal transducer and activator of transcription 1/IFN regulatory factor signaling pathway (39). The 5'-flanking promoter/enhancer region of the bovine CST3 gene (GenBank NW_928624) does not have any predicted transcription factor binding sites for classical ISGs, such as {gamma}-activation sequence elements for STAT1 binding, IFN-stimulated response elements for ISGF3, or IFN regulatory factor response elements; however, the region does have several predicted PGR response elements (data not shown). To date, CTSL, WNT7A, and LGALS15 are the only other genes identified in endometrial LE and sGE that are induced or stimulated by IFNT (12, 25, 40). Thus, the diverse actions of IFNT on the endometrium include repression of genes, including ESR1 (estrogen receptor {alpha}), to abrogate development of the endometrial luteolytic mechanism as well as stimulation of genes that are potentially critical to implantation, placentation, and conceptus growth and development (16). Knowledge of mechanisms whereby IFNT stimulates CST3 gene expression in endometrial LE and sGE is expected to help unravel a nonclassical signaling pathway for type I IFNs.

CST3 is an inhibitor of cysteine proteases, e.g. CTSB and CTSL, that have biological roles in the processing and catabolism of proteins (6). Results of the present studies of CST3 in the ovine uterus are similar to those for mice (11), in which expression of CTSL and CTSB by invasive trophoblast giant cells was balanced by coordinated expression of CST3 in the decidualizing stroma at the implantation site. Coordinated increases in CTSL and CTSB with CST3 occur in endometrial LE and sGE as well as in conceptus trophectoderm during early pregnancy (12). Thus, one biological role of CST3 may be to inhibit the actions of cysteine proteases produced by the conceptus and endometrial epithelia to limit the invasive activity of the trophoblast. These results support the general idea that proteases and their inhibitors expressed at the maternal-fetal interface are important for uterine receptivity, endometrial remodeling, and conceptus implantation during pregnancy in mammals (1, 2, 3, 4, 5). Interestingly, cathepsins and cystatins have recently been implicated in recurrent miscarriage in women (41) who had higher than normal decidual levels of CTSB and CTSH and lower than normal levels of serum CST3. Thus, increased knowledge of uterine proteases and their inhibitors is important for developing therapeutic strategies to prevent, treat, and diagnose infertility in humans and domestic animals.


   Acknowledgments
 
We thank all members of the Bazer/Spencer Laboratory for assistance and management of animals.


   Footnotes
 
This research was funded by National Institutes of Health Grants 5 R01 HD32534 and 5 P30 ES09106.

Disclosure of potential conflicts of interest: G.S., T.E.S, and F.W.B. have nothing to declare.

First Published Online March 23, 2006

Abbreviations: CL, Corpus luteum; CST3, cystatin C; CTSB, cathepsin B; IFNT, interferon-{tau}; ISG, IFNT-stimulated gene; LE, luminal epithelium; P4, progesterone; PGR, P4 receptor; ro, recombinant ovine; sGE superficial glandular epithelium.

Received January 30, 2006.

Accepted for publication March 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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