1 Department of Obstetrics and Gynecology, Stony Brook University, Stony Brook, NY and 2 Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA
3 To whom correspondence should be addressed at: Frontiers in Bioscience, P.O.Box 160, Searingtown, NY 11507, USA. Email: tabibzadeh{at}bioscience.org
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Abstract |
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Key words: Ebaf/endometrium/estrous cycle/lefty/mouse
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Introduction |
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A prevailing hypothesis is that steroid hormones control the homeostasis of endometrial ECM, but the molecular basis of this control has not been thoroughly worked out (Bruner et al., 1999). This remains a critical gap in our knowledge. We identified a protein that appears to be involved in tissue remodelling required for embryo implantation and menstrual shedding. The expression of this endometrial bleeding-associated factor (ebaf/lefty) in humans was minimal during the proliferative, early and mid-secretory phases, but was increased during peri-menstrual and menstrual phases (Kothapalli et al., 1997
). Lefty is a member of the transforming growth factor (TGF)-
family of molecules (Kothapalli et al., 1997
). Because lefty does not form dimers, it acts as an inhibitor of other members of the TGF-
family such as nodal and activin. Expression of lefty was increased up to
100-fold during menstruation (Cornet et al., 2002
). These findings suggest that lefty might be involved in ECM remodelling, a requisite for menstrual tissue shedding. We recently showed that lefty induced tissue remodelling by induction of collagenolysis by MMPs (Mason et al., 2002
). Lefty induced MMP-3 and MMP-7 in human endometrial explants maintained ex vivo (Cornet et al., 2002
). Lefty also induced collagenolytic activity in vivo by cells forced to express lefty (Ulloa et al., 2001
; Mason et al., 2002
). We were interested in extending these observations to a model system, more amenable to experimental manipulation in vivo. Therefore, the current study was carried out to precisely define the temporal expression and spatial distribution profile of lefty and to characterize the tissue remodelling events in mouse endometrium during the estrous cycle and implantation. The results showed that lefty was expressed in mouse endometrium during estrous cycle and pregnancy in a unique spatial distribution pattern consistent with its role in tissue remodelling in endometrium.
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Materials and methods |
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Approvals
All experiments in animals were conducted in accord with accepted standards of humane animal care and in accord with NIH guidelines. Approval of institutional IACUC was obtained for carrying out the animal procedures used in this study.
Sample preparation
Lefty, MMP and collagen were analysed in the endometria of virgin outbred CF-1 mice throughout the estrous cycle and during the first 9 days of pregnancy. The day of the estrous cycle was determined by vaginal smears as described by Snell et al. (1966) and Rugh (1968)
. The day when the vaginal plug was detected was designated as day 1 of pregnancy. The expression of lefty and MMP was assessed in endometrial tissues obtained from six mice, for each day of estrous cycle and on days 1, 3, 5 and 9 of pregnancy. Each experiment was repeated at least twice to confirm reproducibility of data.
Northern blot analysis
For Northern blot analysis, total RNA and poly-A RNA were extracted and pooled from 10 uterine horns of virgin CD1 mice. Mouse uterine horns and segments of colon were immediately placed in RNAlaterTM RNA Stablization Reagent and stored at 80 °C. Total RNA was isolated from tissues using the RNeasy Mini Kit. PolyA RNA was isolated on the oligo(dT) cellulose from total RNA using the Poly(A)PuristTM mRNA purification Kit following the manufacturer's protocol. The 32P-labelled cRNA lefty and RNA marker probes were made by in vitro transcription reaction using MAXIscriptTM in vitro transcription kit. The template used for generation of cRNA lefty probe was the pSTblue-mouse lefty 1 linearized by Sph I. The template was made by inserting coding region of mouse lefty 1 into the EcorV site of the pSTblue-1 cloning vector. The template construct was subjected to DNA sequencing to ensure insertion and orientation. Millenium RNA MarkerTM Probe Template cDNA was used for generation of probe for RNA marker. The cRNA probes were precipitated by NH4Ac/ethanol, rinsed once in 70% ethanol and resuspended in 50 µl of nuclease-free water. Northern blot analysis was performed using the NorthernMaxTM Formaldehyde-based system following the manufacturer's instructions.
RTPCR and real-time RTPCR
For RTPCR and real-time PCR, uterine horns were opened longitudinally and the endometrium was scraped from myometrium using a sterile scalpel. On day 9 of pregnancy, the embryo was removed and the decidua underlying the embryo was used for RNA isolation. Tissues were suspended in phosphate-buffered saline (PBS) and pelleted by centrifugation. The pellets were frozen on dry ice and stored at 80 °C. Scraped endometrial tissues were homogenized in a guanidinium thiocyanateacid phenol solution, and total RNA was extracted as described by Chomczynski and Sacchi (1987). RNA were treated with DNAse to remove any potential contaminating DNA. RTPCR for lefty was performed using primer sets shown in Table I. This was performed on 5 µg of RNA which was reverse-transcribed with Superscript II (InVitrogen) in 20 µl volume. The cDNA was diluted to 300 µl with TE. Aliquots (1 µl) of the RT products were subjected to PCR in a total volume of 20 µl PCR mix, containing 22 mmol/l TrisHCl pH 8.4, 55 mmol/l KCl, 1.65 mmol/l MgCl2, 220 µmol/l dNTP, 22 IU/ml Taq DNA polymerase, with 0.5 µmol/l of adequate paired primers (Table I). A control PCR with actin template DNA was performed in each experiment. After an initial denaturation at 95 °C for 5 min, the DNA was amplified through 30 cycles of 30 s at 95 °C, 30 s at the optimal annealing temperature of the primer pair and 30 s at 72 °C. The reaction was terminated at 72 °C for 10 min. PCR products were stored at 4 °C until use. Real-time PCR was performed using QuantiTect SYBR Green PCR Kit (Qiagen, USA) and DNA Engine Opticon system (MJ Research, USA).
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Immunohistochemcial staining
For immunohistochemical staining, uterine horns were placed in OCT medium and frozen in liquid nitrogen. Lefty was localized by immunostaining according to the ABC procedure as described previously (Hsu et al., 1981; Tabibzadeh and Gerber, 1985
; Tabibzadeh et al., 1987
). Briefly, sections or cells were fixed in 10% buffered formalin (v/v) for 5 min and then washed in PBS. These were incubated with primary and then with secondary antibody at a concentration of 12 µg/ml for 30 min at room temperature. This was followed by incubation in ABC complex. Each incubation was carried out at 37 °C and was followed by two washes in 0.1 mol/l PBS (pH 7.4). Immunoreactive cells were visualized by incubation of sections in a mixture of 3,3'-diaminobenzidine tetrahydrochloride (DAB)H2O2 which formed a brown precipitate in stained cells.
Neutralization of lefty antibody by peptide
In immunohistochemical and western blot analyses, the goat polyclonal antibody to lefty peptide (M-20) was neutralized using the peptide used for immunization of the animals. The neutralization was done according to the manufacturer's recommendation. Briefly, the antibody was incubated with 5-fold excess of the peptide (20 ng/ml) in 500 µl of PBS overnight at 4 °C. The mixture was then used without dilution.
Histochemical staining and morphometric analysis
To quantify the amount of collagen in tissues, sections of endometria were stained with Mason-Trichrome, which in view of its affinity, casts a blue colour onto collagen fibres. Collagen fibres appeared as cord- or tape-shaped wavy fibres. The abundance of collagen fibres in stained sections was assessed by morphometric analysis as described previously (Mason et al., 2002). The mean total area/pixel density of the trichrome-positive areas was measured per microscopic field, at x4 magnification. Three separate, randomly selected fields for each sample were examined.
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Results |
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Discussion |
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We identified a protein, lefty/ebaf, which caused collagenolysis by activation of MMP in human endometrial explants, suggesting that it might be the mediator of endometrial ECM lysis around the time of menstruation (Tabibzadeh, 2002). In this report, we showed that lefty was expressed and that lefty proteins were present in mouse uterine horns during estrous cycle. Lefty proteins were found in the ECM, in the basal lamina around glands, in the interstitium and around vessels. Thus, lefty is strategically positioned and intimately associated with structures in endometrium that are most prone to lysis. Despite its presence, lefty was expressed in mouse uterine horns at a significantly lower level as compared to that expressed in human uterus (Kothapalli et al., 1997
). Also, the magnitude of changes which occurred in lefty expression in mouse uterine horn during estrus cycle was much lower than that which occurred in human endometrium during menstrual cycle (Kothapalli et al., 1997
; Cornet et al., 2002
). We showed that lefty was increased up to 100-fold in human endometrium around the time menstruation whereas expression of lefty was only increased <5-fold in mouse uterine horn from estrus to metestrus, a catabolic stage of the estrous cycle characterized by degenerative changes in the genital tract (Kothapalli et al., 1997
; Cornet et al., 2002
). In human endometrium, the increase in lefty expression occurred at the same time and at a magnitude similar to that of MMP (Goffin et al., 2003
). The level of expression of MMP-3, -7 and MMP-12 increased >100-fold in human endometrium during menstruation (Goffin et al., 2003
). In marked contrast, in mouse uterine horns, the magnitude of increase in MMP expression was <6-fold during estrous cycle. The peak of expression of all MMPs, with the exception of MMP-12, was at metestrus (MMP-7) or at diestrus (MMP-2, -3 and -14).
Coordinated with changes in lefty and MMP expression, the collagen fibres were slightly reduced in mouse uterine horns at metestrus and diestrus. Similar to these findings, it was reported that, during metestrus in cycling rats, the wet weight and the collagen content of endometrium decreased by 20% of their proestrus values (Yochim and Blahna, 1976). These findings indicate that, although ECM lysis is an essential feature of mouse endometrium in an undisturbed estrous cycle, the extent of this breakdown is sufficient only to cause regression and not tissue shedding (Marbaix et al., 1996
).
Given that mouse endometrium expresses the molecular repertoire required for tissue breakdown, the low level of ECM lysis in mouse endometrium might be related to the low level of lefty and MMP. Significantly higher levels of lefty and MMP might be required to cause tissue shedding in mouse endometrium similar to that which occurs in human endometrium during menstruation. Another factor might involve processing of lefty proteins. Most of lefty protein found in mouse uterine horn during estrous cycle was in a precursor form, suggesting that the precursor might not be processed or that its processed forms were rapidly secreted. Mouse endometrium, however, can be forced to show changes similar to those observed in human endometrium during menstruation and to exhibit tissue shedding. Recently, Brasted et al. (2003) showed that steroid hormone treatment followed by application of a deciduogenic stimulus made mouse endometrium ready to show changes that closely mimicked those which occur in human endometrium at menstruation. Following these treatments, progesterone withdrawal led to infiltration of endometrium by leukocytes, apoptosis and ultimately led to shedding of decidual zone.
Lefty and MMP were also coordinately expressed during pregnancy in mouse endometrium. Lefty was maximally expressed in mouse endometrium around the time of implantation and its expression subsided on day 9. Similar to these findings, lefty proteins were detectable at the time of implantation and disappeared by day 9 of pregnancy. Interestingly, on day 5 of pregnancy, most if not all of lefty protein was a processed form. MMP including MMP-3 and -7 were also expressed in mouse endometrium during pregnancy (Curry and Osteen, 2003). Expression of MMPs in mouse endometrium increased on days 3 and/or 5 of pregnancy and decreased by day 9. Consistent with these results, Das et al. showed that in mouse uterus, MMP-2 mRNA was highly expressed in the subepithelial stroma on days 35 and was primarily expressed in the secondary decidual zone on day 6. MMP-3 mRNA was first detected on day 5 in stromal cells located exclusively at the site of implantation (Das et al., 1997
). MMPs are critical to embryo implantation. Injection of peptide hydroxamate MMP inhibitor retarded decidual development. Similarly, development of decidua was inhibited in transgenic mice overexpressing TIMP-1 which inhibits MMP activity (Alexander et al., 1996
). MMPs appear to be required for remodelling of collagen network that takes place in response to decidualization and implantation. The process of decidualization is associated with a vanishing collagen type I, III, V and VI content in pregnant uteri (Mulholland et al., 1992
; Hurst et al., 1994
). Here, we confirmed these findings and showed that during pregnancy, collagen fibres were progressively lost in mouse endometrium from day 3, with relatively less fibers detected by day 9. Several studies showed that this loss of collagen occurred more around the implanting blastocyst (Alexander et al., 1996
; Das et al., 1997
; Curry and Osteen, 2003
). Collagen type I was virtually absent around rat embryos on day 5 of pregnancy, and on days 68 of pregnancy very little collagen was left at the primary and secondary decidualized zones (Clark et al., 1992
, 1993
). Similarly, in humans, collagen types I, III, IV and VI were all reduced during peri-implantation period and were no longer detected in first trimester decidua (Mylona et al., 1995
; Iwahashi et al., 1996
). Decreased collagen expression has also been described in human decidual tissues in spontaneous abortions (Iwahashi et al., 1996
).
In summary, lefty and MMPs are expressed in mouse uterine horns in a coordinated fashion. During estrous cycle, lefty is lower at estrus and higher at metestrus and diestrus. MMP (MMP-2, -3, -7 and -14) are also higher during metestrus and/or diestrus. During estrous cycle, the change in expression level of lefty and MMPs in mouse endometrium does not occur to the extent sufficient for tissue shedding. During pregnancy, expression of lefty and MMP (MMP-3, -7, -12 and -14) is jointly increased around the time of implantation while their expression is coordinately decreased following implantation on day 9 of pregnancy. Spatially, mouse endometrial lefty is found associated with structures that are prone to tissue remodelling, namely the ECM in stroma and vessel walls and basal lamina around glands. Unique spatial pattern of distribution of lefty in endometrium might be important to its biological effect on ECM, and significant when tissue undergoes remodelling.
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Acknowledgements |
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Submitted on May 28, 2004; resubmitted on October 7, 2004; accepted on December 7, 2004.
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