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Biology of Reproduction 66, 1006-1015 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Stage-Specific Expression of the Intermediate Filament Protein Cytokeratin 13 in Luminal Epithelial Cells of Secretory Phase Human Endometrium and Peri-Implantation Stage Rabbit Endometrium1

Gary E. Olson2,a, Virginia P. Winfreya, Gareth L. Blaeuera, John R. Palisano3,a, and Subir K. NagDasa

a Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37232


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In preparation for blastocyst implantation, uterine luminal epithelial cells express new cell adhesion molecules on their apical plasma membrane. Since one mechanism epithelial cells employ to regulate membrane polarity is the establishment of specific membrane-cytoskeletal interactions, this study was undertaken to determine if new cytokeratin (CK) intermediate filament assemblies are expressed in endometrial epithelial cells during developmental stages related to blastocyst implantation. Type-specific CK antibodies were used for immunocytochemical and immunoblot analyses of 1) intermediate filament networks of the endometrial epithelium during embryo implantation in rabbits and 2) proliferative and secretory phases of the human menstrual cycle. CK18, a type I CK found in most simple epithelia, was expressed in all luminal and glandular epithelial cells of both the human and rabbit endometrium at all developmental stages analyzed; it was also strongly expressed in trophectoderm of the implanting rabbit blastocyst. In contrast, CK13, another type I cytokeratin, exhibited a regulated expression pattern in luminal, but not glandular, epithelial cells of secretory phase human and peri-implantation stage rabbit endometrium. Furthermore, in the rabbit implantation chambers, CK13 was predominately localized at the cell apex of luminal epithelial cells, where it assembled into a dense filamentous network. These data suggest that the stage-specific expression of CK13 and a reorganization of the apical intermediate filament cytoskeleton of uterine luminal epithelial cells may play important functions in preparation for the implantation process.

female reproductive tract, implantation, pregnancy, trophoblast, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Early steps in the implantation process include the apposition and subsequent adhesion of the blastocyst trophectoderm to the uterine luminal epithelium [1–4]. Specific cell adhesion molecules localized to the apical surfaces of these interacting epithelia mediate this binding [4, 5]. Uterine epithelial cells are receptive to the implanting blastocyst for a restricted time, the implantation window [6], indicating that their apical plasma membrane undergoes essential differentiation during preimplantation development [2, 4, 7]. After blastocyst adhesion to the uterine epithelium, implantation processes vary among species [1, 3, 8]. For example, the mouse employs a displacement type of implantation, in which uterine luminal epithelial cells undergo apoptosis and sloughing to facilitate blastocyst penetration of the endometrial stroma [1]. The rabbit uses a fusion type of implantation, in which syncytial trophoblastic knobs fuse with uterine luminal epithelial cells that then express an invasive phenotype and penetrate into the endometrial stroma [9–11]. The human apparently uses the intrusive type of implantation in which the blastocyst trophectoderm extends invasive processes that penetrate between adjacent uterine epithelial cells to access the stroma [1, 5]. Each of these implantation strategies requires altered cell-cell and/or cell-extracellular matrix interactions of cells comprising the uterine luminal epithelium.

Epithelial cells exhibit a polarized architecture and express specific adhesion molecules on restricted surface domains in vivo [12]. For example, the cadherins, which regulate cell-cell adhesion, localize to the lateral surface domain [13, 14], whereas the integrins, which function in cell-substrate binding, localize primarily to the basal surface domain [15, 16]. Several mechanisms maintain the domain-specific localization of cell adhesion molecules, including the binding of their extracellular domain to ligands on the adjacent cell or extracellular matrix, the anchoring of their cytoplasmic domain to specific cytoskeletal elements, and the presence of membrane diffusion barriers such as the zonula occludens of the junctional complex that prevent protein diffusion between domains [12, 13, 16].

Uterine epithelial cells exhibit an altered polarized phenotype as they differentiate toward the receptive state for blastocyst implantation [2, 7]. New integrin family members are expressed in a stage-specific pattern, and specific integrins become localized to the apical plasma membrane domain, where they appear to function in the attachment to the embryo [17]. This apical localization contrasts dramatically with typical integrin localization to the basal plasma membrane domain of most polarized epithelial cells [16]. In addition, E-cadherin, which is typically localized to the lateral plasma membrane and functions in cell-cell adhesion, becomes localized to the basal plasma membrane domain of mouse and rabbit endometrial luminal epithelial cells during the peri-implantation period [2, 18, 19]. The cytoplasmic domains of integrins and cadherins interact with specific linker proteins that attach to the actin and/or intermediate filament cytoskeletons [13, 20], suggesting that alterations of the uterine epithelial cell cytoskeleton may function in localizing cell adhesion molecules to the apical surface.

In epithelial cells, cytokeratins (CKs) assemble into a flexible dynamic network of intermediate filaments that are a primary determinant of cell shape. Over 20 distinct proteins comprise the CK family [21–23], and CK-containing intermediate filaments assemble from heterodimers composed of a specific type I keratin paired to a specific type II keratin. Pairs of type I and type II CKs exhibit epithelial cell type-specific expression patterns, and changes in CK type expression are seen during epithelial cell differentiation in several tissues [21]. Human uterine epithelial cells express the CK pair CK8 and CK18, typically found in simple epithelial cells [24]. In the goat, it has been shown that a down-regulation of uterine epithelial CK18 occurs during the peri-implantation period [25], but in the rabbit, endometrial epithelium immunostaining with a pan-CK antibody revealed no changes in CK expression throughout the peri-implantation period [26]. The identification of new CK networks in uterine epithelial cells related to the process of blastocyst implantation has not been reported. This study was undertaken to determine if developmentally regulated alterations in CK expression and localization in endometrial epithelium of the human and rabbit are potentially related to the process of blastocyst implantation. Evidence is presented that the implantation period correlates with the expression of new CK types by luminal but not glandular epithelial cells. In the rabbit luminal endometrial epithelium, the new CK-containing intermediate filament network assembles into a dense layer localized at the cell cortex, suggesting a role in interaction with the implanting embryo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Animal protocols conformed to National Institutes of Health guidelines for humane animal care and use in research and were approved by the institutional Animal Care and Use Committee. Rabbits were housed in the university animal care facility and given free access to food and water. Timed pregnant animals were purchased from the supplier, or females were mated with an adult male and then injected with 50 IU of hCG (Ayerst, New York, NY) to ensure ovulation. Time 0 of pregnancy (PG) corresponded to the time of mating. Pseudopregnant (PsPG) animals were induced with a single injection of 50 IU of hCG; ovaries of PsPG animals were observed at the time the animals were killed to verify ovulation. The stages studied were estrus, Day 63/4 and Day 8 PG, and Day 8 PsPG. Animals were killed with an intravascular injection of sodium pentobarbital, and the uterus was removed and placed in ice-cold Hanks solution. Nonimplant and implant sites of pregnant animals were separated before tissue preparation for histologic analyses or SDS-PAGE.

Human Endometrial Biopsies

The use of human tissues was approved by the Vanderbilt University Institutional Review Board and Committee for the Protection of Human Subjects. Endometrial biopsies were obtained on specific cycle days from healthy human volunteer donors aged 21–45 yr exhibiting regular menstrual cycles. Serum progesterone was determined by the IMMULITE automated immunoassay (Diagnostics Products Corporation, Los Angeles, CA); progesterone values less than 1.5 ng/ml defined the proliferative phase, whereas values greater than 2.5 ng/ml were representative of the secretory phase. Four proliferative phase biopsies, from cycle Days 11 or 12, and four secretory phase biopsies, from cycle Days 18 to 21, were used in this study. Each proliferative phase biopsy had progesterone levels less than 0.5 ng/ml, and the serum progesterone levels of individuals in the secretory phase ranged between 8.2 and 17.5 ng/ml. Tissue samples were rinsed in cold Hanks solution and then immediately prepared for cryosectioning or SDS-PAGE.

Tissue Fixation and Embedding

Tissues were fixed in 4% formaldehyde in 0.1 M sodium phosphate buffer, pH 7.4; rinsed in buffer; dehydrated in ethanol and xylene; and embedded in paraffin. Some samples were fixed in glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, followed by postfixation in 1% OsO4; the tissues were then dehydrated in ethanol, equilibrated in propylene oxide, and embedded in epoxy resin. Paraffin sections were stained with hematoxylin and eosin, whereas 1- to 2-µm plastic sections were stained with 1% toluidine blue.

Immunocytochemistry

Uterine segments were submerged in OCT embedding medium (Fisher Scientific, Atlanta, GA), frozen in liquid nitrogen, and stored at -75°C. Cryosections 4–6 µm in thickness were fixed with either 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2, at 4°C for 30 min or in absolute acetone at -20°C for 10 min. Sections were rinsed in Tris-buffered saline (TN: 20 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.05% Tween 20) and then blocked in TN containing 2.5% BSA (Fisher Scientific) and 5% normal donkey serum. Sections were then sequentially incubated in primary and secondary antibodies diluted in blocking solution with at least three washes in TN after each antibody incubation step. A set of well-characterized, type-specific anti-CK mouse monoclonal antibodies were used. These included antibodies from clone KS-1A3 (Sigma Chemical Co., St. Louis, MO) and clone AE8 (ICN Biomedicals Inc., Aurora, OH), which are specific to CK13, a polypeptide of Mr 54 000 [27, 28]. Two monoclonal antibodies specific to CK18, an Mr 44 000 polypeptide, clones CY-90 and CK5 [29], were obtained from Sigma, and a monoclonal antibody to CK19 [30], clone A53-B/A2.29 (also termed clone Ks19.1), was obtained from Neo Markers (Union City, CA). For primary antibody controls, parallel tissue sections were incubated in identical dilution of mouse ascites fluid or identical levels of affinity-purified nonimmune mouse immunoglobulin (Ig) G. Secondary antibody was an affinity-purified Cy3 donkey anti-mouse IgG from Jackson ImmunoResearch (West Grove, PA). Nuclei were stained by adding 0.2 µg/ml Hoechst 33258 (Molecular Probes, Inc., Eugene, OR) to the secondary antibody solution. Immunostaining was performed on cryosections prepared from eight estrous, four Day 63/4 PG, ten Day 8 PG, and two Day 8 PsPG rabbits and from four proliferative phase and four secretory phase human endometrial biopsies. The immunostaining patterns reported were consistent for all samples within each group. The results of staining were negative for all sections immunostained with primary antibody controls.

SDS-PAGE and Immunoblot Analyses

Rabbit endometrium was dissected from the underlying myometrium of fresh uterine samples and frozen on dry ice or in liquid nitrogen for storage at -75°C. In some cases, endometrium from the antimesometrial and mesometrial surfaces of implantation chambers were dissected separately. Frozen tissue samples were used to prepare either total tissue lysates or cytoskeleton-enriched fractions. To prepare a cytoskeletal fraction, endometrium was homogenized (50 mg tissue per milliliter) in high-salt extraction buffer (composed of 0.5% Triton X-100, 0.15 M NaCl, 0.5 M KCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM NaVO4, 0.5 mM PMSF, and 25 mM Tris-HCl, pH 7.5) and incubated on ice for 1 h. The homogenate was briefly sonicated at medium power using a microtip probe and then centrifuged at 14 000 x g for 30 min to obtain a cytoskeleton-enriched pellet. For SDS-PAGE, frozen tissues or cytoskeletal preparations were incubated in sample buffer at 95°C for 5 min and centrifuged at 13 000 x g for 5 min to remove insoluble material. Lysates were fractionated on 12% acrylamide gels [31], and polypeptides were transferred to polyvinylidene fluoride membranes [32]. Blots were blocked at least 1 h in Tris-buffered saline (TBS: 150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% Tween 20) containing 1% BSA and then incubated in primary antibody in blocking buffer; control blots were incubated with identical levels of affinity-purified nonimmune mouse IgG. Blots were rinsed several times in TBS and then incubated in affinity-purified, peroxidase-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch) diluted in TBS containing 5% nonfat dry milk. Immunoreactive bands were visualized either by enhanced chemiluminescence (SuperSignal; Pierce Chemical, Rockford, IL) on Kodak BioMax film or by color development with diaminobenzidine and H2O2.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Stage-Specific CK Expression in Human Endometrium

Proliferative phase and secretory phase human endometrial biopsies were examined for expression patterns of the type I cytokeratins CK13, CK18, and CK19. Only CK13 demonstrated a stage- and region-dependent expression pattern. In secretory phase biopsies, most luminal epithelial cells stained intensely with anti-CK13 (Fig. 1A). Although CK13 was distributed throughout the epithelial cell cytoplasm, some cells displayed an apical accumulation of CK13 (Fig. 1A). Glandular epithelium of secretory phase biopsies exhibited no detectable anti-CK13 staining (Fig. 1A). In contrast to secretory stage endometrium, endometrium from proliferative phase biopsies displayed little detectable CK13 expression. Most luminal epithelial cells exhibited no staining with anti-CK13, although occasional luminal epithelial cells were weakly positive (Fig. 1B). No detectable staining of glandular epithelial cells with anti-CK13 was noted in proliferative phase endometrial biopsies (not shown). Identical stage-specific CK13 expression patterns were noted using the two different monoclonal antibodies to CK13.



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  		FIG. 1. A, A') Matched phase contrast and fluorescence images of cycle Day 18 human secretory endometrial biopsy immunostained with antibody to CK13. Note that the luminal epithelium (le) displays intense fluorescence, whereas the glandular epithelium (ge) and stroma (st) exhibit no detectable staining. B, B') Matched phase contrast and fluorescence images of cycle Day 11 human proliferative endometrial biopsy immunostained with anti-CK13. Only an occasional cell in the luminal epithelium (le) exhibits low levels of detectable staining. C, C', and D, D') Matched phase contrast and fluorescence images of cycle Day 18 human secretory endometrium immunostained with anti-CK18. Both the luminal epithelium (le) and the glandular epithelium (ge) exhibit intense staining. st, Stroma

To determine if other type I CKs demonstrated temporal and/or spatial stage-specific expression patterns, endometrial biopsies were also immunostained with monoclonal antibodies to CK18 and CK19. Both luminal and glandular endometrial epithelial cells expressed CK18 in all biopsies examined, consistent with a constitutive expression pattern (Fig. 1, C and D); identical staining patterns were noted using two different monoclonal antibodies to CK18. CK19 was also constitutively expressed in both glandular and luminal epithelium of proliferative and secretory biopsies, and no stage-dependent expression changes were detected (data not shown). These data indicate that CK13 expression is upregulated in human luminal endometrial epithelial cells during the secretory phase of the menstrual cycle, suggesting a potential role in preparation for the implantation process.

Expression of CK13 in Peri-Implantation Stage Rabbit Endometrium

To define uterine endometrial epithelial CK expression and/or distribution patterns during the peri-implantation period, as well as potential modulation of CK expression by the implanting blastocyst, rabbit uteri were obtained from estrous animals and from animals on Day 63/4 and Day 8 of pregnancy. In the rabbit, Day 63/4 of pregnancy represents a time point approximately 6 h before the attachment between the blastocyst and the endometrium, which occurs initially at the antimesometrial pole of the implantation chamber; blastocyst attachment to the mesometrial endometrium does not occur until Day 8 of pregnancy [11]. The endometrium undergoes extensive differentiation between estrus and Day 8 of pregnancy [9, 11]. In the Day 8 implantation chamber, the mesometrial endometrium consists of two prominent placental folds (Fig. 2A); the luminal epithelium of these folds is composed of multinucleated cells that possess a prominent cell cortex (Fig. 2B). Each placental fold is flanked by a smaller paraplacental fold, and the antimesometrial endometrium is attenuated and lined by a symplasm of fused luminal epithelial cells (Fig. 2A).



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  		FIG. 2. A) Cross section of rabbit Day 8 implantation chamber showing regional differentiation of the endometrium. The prominent placental folds (pf) at the mesometrial (m) pole are laterally flanked by the smaller paraplacental folds (ppf), and over the antimesometrial pole (am), the endometrium is highly attenuated. E, Embryo. B) Photomicrograph of a plastic section showing the prominent lucent-staining cortex (arrows) of the multinucleated luminal epithelial cells (le) on the placental fold of the rabbit Day 8 implantation chamber. st, Stroma

Expression of CK18 was evident in all endometrial epithelial cells at all developmental stages examined (data not shown), whereas CK13 displayed a regulated temporal and cell-specific expression pattern in the peri-implantation stage endometrium. In estrous and Day 63/4 PG animals, neither luminal nor glandular endometrial epithelial cells expressed detectable levels of CK13 (Fig. 3, A and B). Some multinucleated luminal epithelial cells were noted in the placental folds of Day 63/4 implant sites, indicating the occurrence of cell fusion, but these fused cells were not stained with anti-CK13. However, in Day 8 implantation chambers, multinucleated luminal epithelial cells of the placental folds exhibited intense staining with anti-CK13. The staining pattern was polarized within the multinucleated cells, with a prominent accumulation of CK13 at the cell apex and a positive, but less intense, staining at the basal and lateral surfaces (Fig. 3C). When viewed in frontal sections, the CK13 staining comprised a filamentous network extending over the entire apical surface (Fig. 4A). Expression of CK13 terminated abruptly at the junction of the luminal epithelium with the glandular epithelium (Fig. 4B). CK13 was also expressed in luminal epithelial cells of the paraplacental folds and in the antimesometrial symplasm (Fig. 4C); glandular epithelium in both of these regions was also negative for CK13 expression (data not shown). No detectable staining of the blastocyst was noted with anti-CK13 (Fig. 4C).



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  		FIG. 3. Matched phase contrast (A, B, C) and fluorescence (A', B', C') images of estrous (A, A'), Day 63/4 PG implantation chamber placental fold (B, B'), and Day 8 implantation chamber placental fold (C, C') rabbit endometrium immunostained with anti-CK13. No detectable staining is seen in estrous (A') or Day 63/4 (B') samples. However, at Day 8 (C'), the multinucleated luminal epithelial cells exhibit intense staining. Note that the epithelial staining is most intense at the cell cortex. le, Luminal epithelial cells; st, stroma; lu, uterine lumen



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  		FIG. 4. A) Fluorescence image showing frontal view of luminal epithelial cells on placental fold of a Day 8 implantation site immunostained with anti-CK13. Note that CK13 forms an extensive filamentous network over the cell apex. B) Fluorescence image showing CK13 expression at the transition of luminal epithelium (le) to glandular epithelium (ge) on placental fold of a Day 8 implantation site. Note that the luminal epithelium exhibits prominent CK13 expression at the cell apex, but the glandular epithelium shows no CK13 expression. Nuclei (n) are counterstained with Hoechst. St, Stroma. C, C') Matched phase contrast and fluorescence images showing CK13 expression in antimesometrial symplasm (s) of a Day 8 implantation chamber. Note that the trophoblastic knob (tk) of the implanting embryo exhibits no detectable CK13 expression

To determine if the presence of an adjacent embryo affected CK13 expression and/or localization patterns in luminal epithelial cells, both nonimplant site endometrium of Day 8 PG animals and endometrium of Day 8 PsPG animals were compared with those of the Day 8 implantation chamber. Luminal epithelium of Day 8 nonimplant endometrium expressed CK13 (Fig. 5A); however, in comparison with luminal epithelium of implantation chambers, the staining pattern was weaker and not apically polarized. Endometrium of Day 8 PsPG animals also exhibited CK13-positive luminal epithelial cells; however, the staining pattern was variable, and some cells exhibited no detectable CK13 expression (Fig. 5B). All endometrial epithelial cells of Day 8 PsPG animals exhibited positive staining with anti-CK18 (Fig. 5C).



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  		FIG. 5. Matched phase contrast and fluorescence images of Day 8 PG nonimplant endometrium (A, A') and Day 8 PsPG endometrium (B, B', C, C') immunostained with anti-CK13 (A', B') and anti-CK18 (C'). Note the expression of CK13 in luminal epithelium of both Day 8 nonimplant (A') and PsPG (B') endometrium. Compared with Day 8 PG specimens, the luminal epithelial cells of PsPG samples exhibit lower levels of CK13 expression, and many cells appear negative. C') Anti-CK18 stains all endometrial epithelial cells of Day 8 PsPG animals. le, Luminal epithelium; st, stroma; lu, uterine lumen

Immunoblot Analysis of CK Expression in Rabbit Endometrium

Cytoskeletal fractions prepared from endometrium of estrous uteri and Day 8 PG implantation chambers were fractionated by SDS-PAGE for immunoblot analysis (Fig. 6). Anti-CK18 recognized a band of ~44 kDa in both estrous and Day 8 PG endometrial cytoskeletal preparations (Fig. 6, lanes 1 and 2). No bands were detected in control lanes probed with identical levels of nonimmune mouse IgG (Fig. 6, lanes 3 and 4). Cytoskeletal preparations of estrous endometrium exhibited no detectable immunoreactive bands with anti-CK13 (Fig. 6, lane 5), whereas the Day 8 PG endometrial cytoskeletal fraction exhibited a prominent immunoreactive band of ~54 kDa (Fig. 6, lane 6). These data demonstrate the implantation stage-specific expression of CK13 and its assembly into an insoluble cytoskeletal fraction.



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  		FIG. 6. Immunoblot analysis of CK expression in estrous (lanes 1, 3, 5) and Day 8 implant site (lanes 2, 4, 6) endometrial cytoskeletal fractions. Lanes 1–4 were loaded with cytoskeletal protein from 2 mg of total endometrial tissue, and lanes 5 and 6 were loaded with cytoskeletal protein from 8 mg of total endometrial tissue. Lanes 1 and 2 were immunostained with anti-CK18, and both estrous and Day 8 PG samples exhibit a prominent band of ~44 kDa. Lanes 3 and 4 represent controls immunostained with a nonimmune mouse IgG and exhibit no detectable bands. Lanes 5 and 6 were immunostained with anti-CK13; no band was detected in the cytoskeletal fractions of estrous endometrium, but a prominent band of ~54 kDa was present in Day 8 implant site endometrium


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented in this study demonstrate that in two species that employ different modes of implantation [1], the human and the rabbit, a common feature of the secretory phase and peri-implantation stage, respectively, is the up-regulated expression and assembly of a CK13-containing intermediate filament network in luminal but not glandular epithelial cells. In the rabbit, CK13 exhibits a striking polarized distribution within luminal epithelial cells at implantation sites, with concentration at the cell apex, which represents the surface domain that directly interacts with the trophectoderm of the blastocyst [2, 11]. The data further indicate that initiation of CK13 expression in both the human and the rabbit is not dependent on an implanting embryo since in the human CK13 expression is cycle stage dependent and appears during the secretory phase. In the human, the window of receptivity for blastocyst implantation is approximately Days 20–24 of the cycle [5, 17]. Our data suggest that CK13 expression may correlate with this time frame; however, the precise time at which CK13 expression is initiated in the human endometrium remains to be established. In the rabbit, CK13 is expressed in Day 8 PsPG endometrium and in both nonimplant as well as implant endometrium at Day 8 of pregnancy; this finding also suggests that the presence of a blastocyst is not required for initiating CK13 expression. However, the immunocytochemical data do indicate that CK13 expression is significantly greater in both nonimplant and implant endometrium of Day 8 PG animals than in the endometrium of Day 8 PsPG animals. Moreover, in Day 8 PG animals, a striking difference in CK13 distribution is noted between luminal epithelial cells of nonimplant and implant sites, with implantation chamber epithelium exhibiting a polarized apical accumulation of CK13. These results suggest a relationship between the intracellular organization of the intermediate filament cytoskeleton of the endometrial epithelium and the presence of an implanting blastocyst.

Expression of CK13 appears stringently regulated in the peri-implantation rabbit endometrium. Epithelial CK13 is not detectable at implant sites at Day 63/4 of pregnancy, which is approximately 6 h before blastocyst attachment begins [11], whereas at Day 8, which is when blastocyst attachment is beginning on the mesometrial placental folds [9–11], prominent CK13 expression is detected in luminal epithelial cells around the entire perimeter of the implantation chamber. Thus, over a time frame of approximately 24 h, coincident with early phases of embryo implantation, luminal epithelial cells extensively transform their intermediate filament cytoskeleton.

The CK family members exhibit cell-specific expression patterns in various epithelia of the body [21, 22]. CK13, a type I CK of Mr 54 000, was initially identified as a component of stratified nonkeratinized epithelia such as the esophageal epithelium, in which CK13 is restricted to the suprabasal cell layers [33, 34]. A previous analysis of epithelia of the human female reproductive tract demonstrated that proliferative stage endometrium contains CKs 7, 8, 18, and 19 but not CK13, or its coordinately expressed type II CK partner, CK4 [24]. Our data agree with this finding and further indicate that human and rabbit endometrial CK13 expression is temporally linked to the secretory phase and peri-implantation stage differentiation of luminal epithelial cells, respectively. Retinoic acid regulates CK13 expression in vivo and in cultured cells [35–37], and the human CK13 promoter contains two AP-2 sites, three sites with homology to SP-1, and four half sites for retinoid action [38]. During decidualization, rat endometrium synthesizes retinoic acid [39], raising the possibility that locally produced retinoic acid may participate in regulating epithelial CK13 expression during the peri-implantation period. Moreover, if paracrine factors are key elements regulating CK13 expression, it will be of interest to define the basis for the differential response of the glandular epithelium, which expresses no detectable CK13, and the luminal epithelium, which exhibits intense CK13 expression.

A key question that remains is to define the function of the stage-specific apical CK13 network in secretory phase human and peri-implantation stage rabbit endometrial luminal epithelium. Intermediate filaments have long been recognized to play major roles in regulating the shape and mechanical stability of epithelial cells, but in recent years, it has become evident that extensive structural and functional interactions between intermediate filaments, actin filaments, and microtubules are responsible for maintaining a dynamic cellular architecture [40, 41]. In cultured MDCK cells, recent studies have identified an apical accumulation of CK19 proposed to function in the organization of the apical pole of these polarized epithelial cells [42, 43]. The importance of transformation of the apical surface domain of endometrial epithelial cells in relationship to the implantation process is widely recognized [2, 4, 5, 7]. It appears likely that the newly organized CK13 network at the apex of the endometrial luminal epithelial cells could function as one component of the cytoskeletal framework required to anchor cell adhesion molecules to the apical membrane domain. Prior immunocytochemical studies of peri-implantation stage rabbit endometrium has demonstrated that the desmosome-associated protein desmoplakin is localized principally to the subapical lateral membrane of epithelial cells in the prereceptive phase, but this polarity is lost at receptivity, and staining is present throughout the lateral membrane [26, 44], these data too are consistent with a reorganization of the intermediate filament cytoskeleton during the peri-implantation period. In the mouse, a down-regulation of desmosome content of the endometrial luminal epithelium occurs during the peri-implantation stage [45], and in the rat, a loss of terminal web components and filamentous actin occurs in the endometrial epithelium during the peri-implantation stage [46, 47]. The early implantation stages may entail remodeling of multiple cytoskeletal assemblies at the cell apex. It will be of interest to define interactions of the apical CK13 network with both F-actin and microtubule assemblies of the cytoskeleton and to establish the roles of the terminal web complex in regulating the apical plasma membrane of endometrial luminal epithelial cells during interactions with the implanting blastocyst.


   ACKNOWLEDGMENTS
 
We thank Drs. Esther Eisenberg and Kevin Osteen for providing staged endometrial biopsies and Drs. Mike Melner, Kevin Osteen, and Ben Danzo for stimulating discussions during the course of these studies.


   FOOTNOTES
 
First decision: 5 March 2001.

1 This research was supported by NICHD/NIH through cooperative agreement U54 HD37321 as part of the Specialized Cooperative Centers Program in Reproductive Research. Some of these data were presented in abstract form at the 33rd annual meeting of the Society for Reproduction (Biol Reprod 2000; 62[suppl 1]:514). Back

2 Correspondence. FAX: 615 343 4539; gary.olson{at}mcmail.vanderbilt.edu Back

3 Current address: Department of Biology, University of the South, Sewanee, TN 37383 Back

Accepted: November 5, 2001.

Received: February 14, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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