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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 7 2526-2536
Copyright © 2000 by The Endocrine Society

Original Studies

Dysregulated Expression of ebaf, a Novel Molecular Defect in the Endometria of Patients with Infertility1

Siamak Tabibzadeh, James M. Mason, Wendy Shea, Yiqiang Cai, Michael J. Murray and Bruce Lessey

Department of Pathology (S.T., Y.C.) and Viral Vector Laboratory (J.M.M.), Department of Research, North Shore University Hospital, Biomedical Research Center, Manhasset, New York 11030; and Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, University of North Carolina (M.J.M., B.L.), Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: S. Tabibzadeh, M.D., Department of Pathology, North Shore University Hospital, Biomedical Research Center, 350 Community Drive, Manhasset, New York 11030. E-mail: tabibzad{at}nshs.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently described the expression of ebaf, a novel member of the transforming growth factor-ß superfamily in human endometrium. ebaf messenger ribonucleic acid was expressed in late secretory and menstrual endometria. Here, we show that ebaf is secreted as 42-, 34-, 28-, and 14-kDa proteins into the conditioned medium of transfected cells, endometrial fluid, and serum. The amount of secreted proteins was markedly reduced during the implantation window in the endometria and sera of normal fertile subjects. The expression of ebaf was dysregulated in the endometria of a subset of women with infertility during the receptive phase of the menstrual cycle. Abundant secreted protein was present in the endometria of these women during the implantation window. During the critical period of endometrial receptivity, ebaf protein was more abundant in patients with endometriosis who did not conceive than in patients who became pregnant. These findings show that ebaf is a secreted product and is released into body fluids. Some types of infertility are associated with dysregulated expression of ebaf in human endometrium, suggesting that a molecular defect in uterine receptivity may be identified using such a marker protein.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HUMAN ENDOMETRIUM is a unique tissue that undergoes sequential phases of proliferation, secretory changes, tissue shedding, and bleeding during menstruation. After ovulation, during a defined period designated the period of endometrial receptivity or implantation window, a number of sequential changes at the structural and molecular levels make human endometrium susceptible to implantation (1). In humans, the ovum is fertilized in the Fallopian tube. The fertilized ovum starts to divide, migrates through the Fallopian tube, and enters the uterine cavity around the third to fourth day after ovulation. The blastocyst remains free floating within the endometrial cavity for a day and initiates implantation on days 5–10 after ovulation (2, 3, 4, 5, 6). This time frame coincides with the period that the endometrium becomes susceptible to implantation, the so-called endometrial receptivity period or implantation window. The identity of the members of the molecular repertoire that make endometrium receptive to the implantation process remains largely unknown, but it is thought to consist of adhesion molecules, cytokines, and heat shock proteins (1). If implantation does not occur, however, a second series of changes leads to refractoriness of the endometrium to implantation and to its menstrual shedding. We recently identified a member of this premenstrual molecular repertoire (7, 8). The maximal expression of this novel gene was found in endometrium immediately before and during menstrual bleeding, and hence, it was designated endometrial bleeding-associated factor (ebaf) (7). In fact, consistent with its intimate relation with endometrial bleeding, expression of the gene was found in endometrium during abnormal uterine bleeding regardless of the phase of the menstrual cycle (7).

The deduced amino acid sequence of ebaf showed a great amount of identity and similarity with the known members of the transforming growth factor-ß (TGFß) superfamily. A motif search revealed that the predicted ebaf protein contains most of the conserved cysteine residues of the TGFß-related proteins (7) that are necessary for the formation of the cysteine knot structure (9, 10). The ebaf sequence contains an additional cysteine residue, 12 amino acids upstream from the first conserved cysteine residue. The only other TGFß superfamily members known to contain an additional cysteine residue are TGFßs, inhibins, and growth and differentiation factor (GDF)-3 (9, 11). ebaf, similar to lefty-1, GDF-3/Vgr2, and GDF-9, lacks the cysteine residue necessary for the formation of intermolecular disulfide bond (11, 12, 13). Therefore, ebaf appears to be an additional member of the TGFß superfamily with an unpaired cysteine residue that may not exist as a dimer (8). The carboxyl-terminus of the TGFß family is usually CX1CX1, and ebaf has the C-terminal sequence, CX1CX13 (14). A gene called lefty-1/stra3 of the TGFß superfamily is expressed during development in the left side of the mouse embryo in the mesenchyme (14, 15). The deduced amino acid sequence of ebaf protein is 77% identical and 83% similar to lefty-1 (14). Therefore, lefty-1 is the mouse homolog of ebaf or a closely related protein (7, 14).

In a substantial number of women, implantation fails to occur, and these women do not become pregnant. As shown in the classic Guttmacher’s table, about 7% of couples can be considered infertile after they have tried for 2 yr to attain pregnancy (16). In the U.S. in 1982, nearly one in five married women of reproductive age reported that during her lifetime, she had sought professional help for infertility (17), and in 1988, 8.4% (a total of 4.9 million) of women, aged 15–44 yr, reported impaired fecundity (18). After all of the standard clinical investigations are completed, and known causes of infertility attributable to tubal and pelvic pathologies, male factor, ovulatory dysfunction, and unusual problems are ruled out, a substantial number (10%) of infertility cases remain of unknown etiology. These cases are designated unexplained infertility (19). Regardless of the cause, however, infertility may be associated with the development of lesions within the molecular repertoire of endometrium during the critical period of endometrial receptivity. For example, it was shown that infertility is associated with aberrant expression of {alpha}vß3, which is normally present in endometrium during the receptive phase of the menstrual cycle (20, 21). Such a defect may be due to the lack of endometrial receptivity or, alternatively, to dysregulated expression of the premenstrual molecular repertoire, which leads to menstrual shedding and bleeding (22, 23). ebaf messenger ribonucleic acid (mRNA) is normally expressed in endometrium during the critical period when endometrium is destined to be shed, and it becomes refractory to the implantation (7). This association suggests that ebaf may be regarded as a member of a premenstrual molecular repertoire and a marker for a nonreceptive endometrium. To further test this hypothesis, we examined the temporal expression of ebaf in normal human endometria during the menstrual cycle and in the endometria of women with diverse forms of infertility during the implantation window.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

The full-length, 1.961-kb, ebaf complementary DNA (cDNA) was derived from a human placental cDNA library (8). A 1.1-kb cDNA fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). Other materials included, [{alpha}-32P]deoxy-CTP (3000 Ci/mmol; NEN Life Science Products, Boston, MA), Prime-a-Gene labeling kit (Promega Corp., Madison, WI), RNA STAT-60 (Tell-Test, Inc., Friendswood, TX), nick column (Pharmacia Biotech, Piscataway, NJ), TRIzol (Life Technologies, Inc., Gaithersburg, MD), Hybond nylon membrane and enhanced chemiluminescence system (Amersham Pharmacia Biotech, Arlington Heights, IL), Kodak-OMAT films (Sigma, St. Louis, MO), Coomassie Plus Protein Assay Reagent (Pierce Chemical Co., Rockford, IL), nitrocellulose membrane (MSI, Westborough, MA), biotin-labeled goat anti-rabbit antiserum and avidin-biotin complex (ABC) reagent (Vector Laboratories, Inc., Burlingame, CA), and Protein G Plus Agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). All other chemicals were from either Sigma or Fisher Scientific (Pittsburgh, PA).

Processing of endometria and sera

Tissues and sera were obtained after approval of the internal review board of the institute. Informed consent was obtained from each patient. A set of endometrial tissues was obtained as biopsy or curettings and from hysterectomy specimens of normal fertile women who underwent these procedures for diagnosis or treatment of nonendometrial abnormalities such as ovarian or cervical lesions (Tables 1Go and 2Go). Hysterectomy specimens and each endometrial biopsy sample were rapidly processed. In some cases the endometrial tissue was spun at 900 x g, and the cell-free fluid (endometrial fluid) was collected and stored at -80 C until use. The date of endometrium was determined based on the morphological evaluation of hematoxylin- and eosin-stained endometrial sections using established criteria of Noyes and Hertig (24). Each endometrial sample was aliquoted as required. Each sample was processed for paraffin sectioning and morphological examination, and some of the tissue was flash-frozen in a dry ice/ethanol bath for isolation of RNA and/or protein. The first set of serum samples was obtained from normal fertile subjects from whom endometrial samples were obtained and processed for paraffin sectioning and morphological examination for tissue dating. The second set of serum samples was obtained from normal fertile subjects after the LH surge. Sera were also obtained from five healthy men between the ages of 25–35 yr.


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  		Table 1. Cases used in the Northern and Western blot analysis of ebaf in the endometria of normal and infertile women

 

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  		Table 2. Cases used for the immunohistochemical localization of ebaf in endometria

 
Isolation of RNA and Northern blotting

The RNA was extracted using the acid guanidinium thiocyanate-phenol-chloroform extraction method as previously described (25). Briefly, the tissues were homogenized in RNA STAT-60. Each 50–100 mg tissue were homogenized in 1 mL RNA STAT-60 in a glass or Teflon Dounce homogenizer (Kontes Co., Vineland, NJ). Each homogenate was stored for 5 min at room temperature to permit the complete dissociation of nucleoprotein complexes. Then, 0.2 mL chloroform was added for each 1 mL RNA STAT-60 used. Each sample was covered and shaken vigorously for 15 s, then allowed to stand at room temperature for 2–3 min. After centrifugation at 12,000 x g for 15 min at 4 C, each homogenate was separated into a lower phenol/chloroform phase and an upper aqueous phase. RNA in the upper aqueous phase was transferred to fresh tubes and mixed with isopropanol to precipitate the total RNA. After centrifugation and drying, the precipitated RNA was dissolved in diethylpyrocarbonate-treated water by vigorous pipetting and gentle heating at 55–60 C. The amount of RNA in each sample was determined spectrophotometrically, and its quality was evaluated by the integrity of ribosomal RNA by electrophoresis of 20 µg total RNA in 1% formaldehyde-agarose gel in the presence of ethidium bromide. Northern blotting was performed as previously described (26). Briefly, 20 µg total RNA from each sample were denatured at 65 C in a RNA loading buffer, electrophoresed in 1% agarose containing 2.2 mol/L formaldehyde gel, and blotted onto a Hybond nylon membrane using a positive pressure transfer apparatus (Posiblot, Stratagene, La Jolla, CA). The RNA was fixed to the membrane by UV cross-linking. Using the Prime-a-Gene kit, cDNA was labeled with 32P to a high specific activity and purified by nick columns. Membranes were prehybridized in 50% formamide, 10 x Denhardt’s solution, 4% SSC (saline sodium citrate), 0.05 mol/L sodium pyrophosphate, and 0.1 mg/mL denatured herring sperm DNA at 42 C for 2–4 h and hybridized for 16 h at 42 C with 106 cpm/mL heat-denatured probe in the same buffer containing 10% dextran sulfate. Then, membranes were sequentially washed three times in 4 x SSC, once in 0.5 x SSC, and once in 0.1 x SSC. All washes contained 0.1% SDS and were performed at 65 C for 20 min each. The membranes were subjected to autoradiography at -70 C with intensifying screens. The same blot was stripped and reprobed for GAPDH. To reprobe a blot, the probe was stripped from the membrane in 75% formamide, 0.1 x SSPE (saline, sodium phosphate, and ethylenediamine tetraacetate), and 0.2% SDS at 50 C for 1 h.

Production of the polyclonal antibody

Rabbit antiserum was prepared according to established protocol against the peptide CASDGALVPRRLQHRP-amide, which resides at the carboxyl end of the ebaf molecule (7). Keyhole limpet hemocyanin was used as the carrier protein. The coupled peptide suspended in phosphate-buffered saline (PBS) at 1 mg/mL was mixed with an equal volume of complete Freund’s adjuvant. This material was mixed until it formed an emulsion, and thenit was injected at six sites sc. A total of 300 µg peptide was injected. Additional injections of the coupled peptide with incomplete Freund’s adjuvant were performed on days 14, 28, 35, and 70, and the production bleeds were performed on days 85 and 90 after the initial injection. The titers of the antiserum, compared to those of samples of the preimmune serum, were determined by enzyme-linked immunosorbent assay. Then, the antiserum was affinity purified using a SulfoLink Affinity column (Pierce Chemical Co.) containing the peptide. The affinity column was made by first washing the column with PBS according to the manufacturer’s instructions, followed by the addition of 1.2 mg peptide/mL resin. After allowing the gel and the peptide to react, the gel was washed extensively, and a solution of 50 mmol/L cysteine was incubated with the gel to react with any remaining functional groups. The column was then washed again before exposure to the immune serum and exposed to 20 mL serum and 20 mL PBS. After incubation for 3 h with shaking, the serum and the gel were poured back into the Econo column (Watrex, Pittsford, NY), and the serum flow-through was collected. The column was then washed with phosphate buffer containing 250 mmol/L NaCl until no protein could be eluted. Then, the column was exposed to 100 mmol/L glycine buffer (pH 2.5), and 1-mL fractions were collected into tubes containing 50 mL 1 mol/L Tris-HCl (pH 9.5) to neutralize the pH and protect the integrity of the antibody. The fractions containing proteins were pooled and dialyzed in 4 L of 5 mmol/L phosphate buffer (pH 7.4); the buffer was changed every few hours. This material was aliquoted, frozen, and kept at -70 C until used.

Transfection

Lipid-mediated transient transfection was carried out in NIH-3T3, 293T, and COS-7 cell lines using SuperFect (QIAGEN, Valencia, CA) as described by the manufacturer. Briefly, the required amount of SuperFect reagent was added to the DNA solution, mixed, and incubated for 5–10 min, and the SuperFect-DNA complexes were incubated with the cells. After a 2- to 3-h incubation, a medium change was performed, and the cells were incubated for gene expression. The cells and their conditioned media were examined for gene expression 24–72 h after transfection. NIH-3T3 and 293T cells were stably transfected with a mammalian expression vector containing a neomycin resistance gene and ebaf cDNA. These cells were selected in the presence of the neomycin analogue, G418, and maintained in the presence of this drug.

Mammalian expression plasmid construction

The sense and antisense orientations of the ebaf cDNA were constructed using plasmid pAdCMV5 (Quantum Biotechnologies, Inc., Montréal, Canada) in which ebaf gene expression is regulated by the cytomegalovirus immediate early promoter. A 1.2-kb BamHI/AflIII ebaf cDNA fragment containing minimal 5'- and 3'-untranslated regions from plasmid pBluescript2SK-ebaf was filled with T4 DNA polymerase and cloned into PmeI-digested pAdCMV5. Restriction mapping and flanking DNA sequencing confirmed the orientation of the resulting ebaf expression plasmids.

Western blot analysis

Proteins were isolated from human endometrium or serum using TRIzol reagent according to the manufacturer’s recommendations. Briefly, after RNA and DNA were extracted from the sample, the proteins were precipitated with isopropanol and washed with 0.3 mol/L guanidine-hydrochloride in 95% (vol/vol) ethanol. Vacuum-dried protein pellets were solubilized in 10 mol/L urea containing 50 mmol/L dithiothreitol for 1 h, boiled, dissolved and diluted with 4-fold concentrated Laemmli sample buffer, and boiled again for 5 min before loading onto the gels. The protein concentration was determined by the Coomassie Plus protein assay reagent. Fifteen micrograms of total protein were electrophoresed on 15% polyacrylamide-SDS gels according to the method of Laemmli (27), transferred to nitrocellulose membrane, and blocked for 2 h at 25 C with 5% nonfat dried milk powder in TBS buffer (150 mmol/L NaCl and 10 mmol/L Tris, pH 7.4). Membranes were washed in TBS containing 5% nonfat dried milk powder and 0.1% (vol/vol) Tween-20 and were incubated overnight at 4 C with rabbit polyclonal affinity-purified antiserum against ebaf (1:250 dilution of 0.8 mg protein/mL) in TBS containing 1% (wt/vol) BSA. Membranes were washed and then incubated with biotin-labeled goat antirabbit antiserum (1:2000 dilution) for 90 minat 25 C. The membranes were washed and incubated for 30 min at 25 C with the ABC reagent (1:300 dilution) and developed using the enhanced chemiluminescence system. The optical density of each band was determined by laser scanning densitometry. Kruskal-Wallis and Mann-Whitney tests were used for statistical evaluation. Significance was established at the P < 0.05 level.

Immunoprecipitation of proteins for Western blot analysis

Two hundred and fifty micrograms of TRIzol-extracted protein were preincubated with 1 µg normal rabbit IgG and 20 µl protein G plus agarose for 30 min at 4 C. The sample was centrifuged at 500 x g for 5 min at 4 C, the pellet was discarded, and then 2.5 µg affinity-purified anti-ebaf antiserum were added to the supernatant. This preparation was incubated for 2 h at 4 C. Twenty microliters of protein G plus agarose were then added, and the incubation was continued for an additional hour. The mixture was centrifuged at 500 x g. The pellet was washed four times with cold PBS, and an equal volume of 2-fold concentrated Laemmli sample buffer was added to the pellet. The pellet was boiled for 5 min and centrifuged at 500 x g, and the supernatant was loaded directly onto a 15% polyacrylamide-SDS gel.

Immunohistochemical staining

Immunostaining was performed according to the ABC procedure as described previously (28). Briefly, the staining consisted of fixation of the sections in 10% buffered formalin, followed by a 5-min wash in PBS (0.1 mol/L; pH 7.4). The slides were then incubated with the appropriate concentration of the antiserum, biotinylated goat antirabbit IgG, and avidin-peroxidase complex. Each incubation was performed for 30 min at room temperature followed by a 5-min wash in PBS. The slides were developed in the Vector VIP peroxidase kit (SK-4600, Vector Laboratories, Inc.). The proper concentration of the antibody was determined by serial dilutions of the antiserum. Two sets of controls were used. In one set of experiments, the primary antibody was omitted. In the second set of experiments, the antibody was preincubated with various concentrations of the peptide for 30 min at 37 C before application of the antiserum to the slide. Sections were viewed, evaluated, and photographed at the light microscopic level without a counterstain. The immunoreactivity was scored as: 0, negative; 1+, weak; 2++, moderate; and 3+++, strong.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of mRNA expression in the endometria of patients with infertility

We previously carried out Northern blot analysis for analysis of the expression level of ebaf mRNA in endometrium throughout the menstrual cycle (7). ebaf mRNA was expressed at a detectable level in human endometrial tissue during the premenstrual and menstrual phases (7). In the menstrual endometrium, up to three mRNA species of 2.5, 2.1, and 1.5 kb hybridized with the full-length ebaf cDNA (Fig. 1Go). The distinct temporal pattern of ebaf mRNA expression in human endometrium suggested that ebaf belongs to a premenstrual molecular repertoire that marks the closure of the implantation window. Therefore, we tested the hypothesis that infertility is associated with dysregulated expression of ebaf mRNA during the implantation window. Northern blot analysis was carried out on endometria of several normal controls and during the implantation window on the endometria of patients with various types of infertility (Table 1Go). As reported previously (7), in two normal women undergoing tubal ligation, the menstrual endometrium exhibited a significant level of ebaf mRNA (Fig. 2Go, lanes 25 and 26). Consistent with our previous findings (7), endometrium obtained during the implantation window from a normal fertile woman who donated her egg (egg donor), exhibited a low level of mRNA expression (Fig. 2Go, lane 21). The HL60 cell line, used as a negative control, did not express any detectable mRNA (Fig. 2Go, lane 15). In more than 50% of infertile patients, a mRNA that hybridized to the full-length ebaf cDNA was detectable in the endometria of women on postovulatory days 6–10 (Fig. 2Go). This was common in women with endometriosis and unexplained infertility (Table 1Go and Fig. 2Go). The mRNA detected was primarily the 2.1-kb species. Additional smaller bands were also detected in a smaller number of patients (Fig. 2Go).



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  		Figure 1. Northern blot analysis of ebaf mRNA expression in a menstrual endometrium. Total RNA of a menstrual endometrium was subjected to Northern blot analysis for detection of the level of expression of ebaf mRNA. The integrity of RNA was verified by staining the 18S and 28S ribosomal RNA (not shown) and hybridization of the blot with a cDNA probe to GAPDH (lower panel). The tissues in the menstrual endometrium are degenerating; thus, some degradation of RNA is unavoidable. The figure is representative of the data (n = 3). ebaf cDNA hybridizes to at least three mRNA species with sizes of 2.5, 2.1, and 1.5 kb.

 


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  		Figure 2. Northern blot analysis of ebaf mRNA expression in the endometria of fertile and infertile women. Upper panel, Twenty micrograms of total RNA from endometria of fertile and infertile women and a negative control (RNA from HL60 cell line) were subjected to the Northern blot analysis using the full-length ebaf cDNA as the probe. For lane legends, see Table 1Go. The integrity of RNA was verified by staining the 18S and 28S ribosomal RNA (not shown) and hybridization of the blot with a cDNA probe to GAPDH (lower panel). Some unavoidable degradation of RNA is detectable in the RNA of menstrual endometria. The optical densities of the bands were determined by laser scanning densitometry and expressed as relative optical density.

 
ebaf is a secreted protein

Based on the existence of a signal peptide in the ebaf sequence, we reported that ebaf is a secreted protein (7). The predicted size of ebaf precursor protein is 42 kDa (Fig. 3AGo). ebaf protein contains three potential RXXR cleavage sites that conform to the minimal requirement for efficient processing by convertases (29). To prove secretion of ebaf and to determine the sizes of the secreted products, several cell lines (NIH-3T3, 293T, and COS-7) were transfected with an ebaf expression plasmid. The culture media and the cell lysates of the transfected cells were subjected to Western blot analysis within 24–72 h after transfection. To detect ebaf, we raised a rabbit antiserum against a peptide at the COOH-terminal end of the protein. The antibody reacted in an enzyme-linked immunosorbent assay with the peptide, and its reactivity could be inhibited by preincubation of the antibody with an excess of the peptide (data not shown). The antibody reacted with a recombinant ebaf protein generated in Escherichia coli (Fig. 3Go). This reactivity was similar to that observed with a mouse monoclonal antibody to ebaf (Fig. 3Go). The blots from cell lysates and conditioned media of transfected cells were probed using the affinity-purified polyclonal rabbit antiserum to ebaf. The cell lysates of all three cell lines, including the cell lysate of 293T cells, contained a 42-kDa protein, consistent with the size of the precursor protein (Fig. 3Go). The conditioned media of all three cells lines, including those of NIH-3T3 cells, contained three secreted products (Fig. 3Go). The experiments were repeated with the cell lysates and conditioned media of NIH-3T3 and 293T cells stably transfected with an ebaf expression plasmid, and similar results were obtained (data not shown).



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  		Figure 3. A, The processing sites in the ebaf. Three potential RXXR processing sites are present in ebaf, leading to three secreted products (not drawn to scale). B, Western blot analysis of ebaf in the cell lysates and conditioned media of 293 cells transfected with expression plasmids containing ebaf cDNAs. Lanes 1 and 2, 293 cells transfected with an antisense ebaf cDNA expression plasmid. Lanes 3 and 4, 293 cells transfected with the sense ebaf cDNA expression plasmid. Lanes 5 and 6, Recombinant E. coli-produced ebaf. The conditioned media of transfected cells (lanes 1 and 3), their cell lysates (lanes 2 and 4), and a recombinant E. coli (26 kDa)-produced ebaf (5 ng; lanes 5 and 6) were subjected to Western blot analysis and probed with an affinity-purified rabbit polyclonal antiserum to ebaf (lanes 1–5) and a mouse monoclonal antibody to ebaf (lane 6). Size is shown in kilodaltons.

 
To show the specificity of the antibody, endometrial proteins extracted from a day 1 menstrual endometrium were resolved in a 15% gel by SDS-PAGE and subjected to Western blot analysis. The blot was probed with the antiserum (1:250 dilution of a solution containing 0.8 mg protein/mL) alone (Fig. 4Go, lane 1) and with the antiserum preincubated with the CASDGALVPRRLQHRP-amide (0.32 mg/mL; Fig. 4Go, lane 2). Several immunoreactive bands (~55/60, 42, 34, and 28 kDa) were detected by the antiserum (Fig. 4Go, lane 1). The immunoreactivity of the antiserum was markedly reduced by preincubation of the antibody with the peptide (Fig. 4Go, lane 2).



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  		Figure 4. Demonstration of specificity of the rabbit antiserum to ebaf by Western blot analysis. A, Endometrial proteins extracted from a day 1 menstrual endometrium were resolved in SDS-PAGE and Western blotting. The blot was probed with the antiserum alone (lane 1) and with the antiserum preincubated with CASDGALVPRRLQHRP-amide (0.32 mg/mL). Protein size is shown in kilodaltons. B, Total protein (250 µg) from a menstrual day 1 endometrium was immunoprecipitated using the rabbit antiserum to ebaf.

 
Various forms of ebaf protein in the endometrium, endometrial fluid, and serum during the menstrual cycle

A number of endometrial proteins reacted with ebaf antiserum in Western blot analysis. These included 42-, 34-, 28, and 14-kDa protein bands (Fig. 4Go, lane 3). These bands were not detected when the antibody was omitted (data not shown). When adequately resolved, the 42-kDa protein appeared as a doublet (Fig. 4AGo). These findings show that the antibody reacts with ebaf. A 55/60-kDa protein was also detected. This protein band was not detected in the cell lysates or the conditioned medium of transfected cells (Fig. 3Go) and is probably a related or cross-hybridizing protein. This band was not observed when the primary antibody was omitted during the immunostaining of the blot (data not shown). The predicted 14-kDa protein was not detected by Western blot analysis in the endometrial proteins. To show that such protein exists in human endometrium, the endometrial proteins were immunoprecipitated by the antiserum. The immunoprecipitated proteins were subjected to SDS-PAGE and examined by Western blot analysis. In addition to the bands detected by Western blot analysis, the 14-kDa protein was detected as a weak band (Fig. 4BGo). To show the temporal pattern of synthesis and/or secretion of endometrial ebaf proteins throughout the menstrual cycle, we carried out Western blot analysis on a number of endometria obtained from various phases of the menstrual cycle (Fig. 5Go). The immunoreactive bands were detected during the menstrual cycle (Fig. 5Go). However, this immunoreactivity was greatly reduced during the implantation window (P < 0.05). As shown in Fig. 5Go, the immunoreactivity of the bands was markedly reduced in endometrial samples obtained on postovulatory days 4, 5, 8, and 9. In these samples, the disappearance of the smaller bands was pronounced, and only the 55/60-kDa band could be detected, presumably due to its excessive amount. To determine whether ebaf was secreted, the endometrial fluid and the sera of normal fertile subjects were subjected to Western blot analysis (Figs. 6Go and 7Go). Immunoreactive bands were detected in both the endometrial fluid (Fig. 6Go) and serum (Fig. 7Go). The 42-, 34-, and 28-kDa ebaf bands were relatively more abundant during the late secretory/menstrual phase in both the endometrial fluid (Fig. 6Go) and serum (Fig. 7Go). The amounts of these proteins were particularly low in the serum during the early and midsecretory phases (Fig. 7Go). To further validate these findings, sera were obtained from normal fertile women on different days after the LH surge. The amount of the immunoreactive ebaf bands were markedly reduced during days 5–9 after the LH surge and were elevated on days 10–14 after the LH surge (data not shown). In the male sera, the 55/60, 42- and 28-kDa proteins were detected; however, the 34-kDa form of ebaf protein was not found in these sera (Fig. 7Go).



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  		Figure 5. Immunoreactive ebaf bands in human endometria throughout the menstrual cycle. In each lane, 10 µg extracted endometrial proteins were resolved in a 15% gel by SDS-PAGE and then subjected to Western blot analysis. The blot was probed with the affinity-purified rabbit antiserum to ebaf. Similar results were obtained in three additional experiments using tissues obtained from endometria of normal subjects. The optical densities of the bands were determined by laser scanning densitometry and were expressed as relative optical density. MP, Midproliferative; LP, late proliferative; P, postovulatory day; D1, first day of menstruation. Protein size is shown in kilodaltons.

 


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  		Figure 6. Immunoreactive ebaf bands in the endometrial fluid during the secretory phase. In each lane, 10 µg extracted endometrial proteins were resolved in a 15% gel by SDS-PAGE and then subjected to Western blot analysis. The blot was probed with the affinity-purified rabbit antiserum to ebaf. MP, Midproliferative; LP, late proliferative; P, postovulatory day; D1, first day of menstruation. Protein size is shown in kilodaltons. The optical densities of the bands were determined by laser scanning densitometry and were expressed as relative optical density.

 


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  		Figure 7. Immunoreactive ebaf bands in human sera. In each lane, 10 µg extracted serum proteins were resolved in a 15% gel by SDS-PAGE and then subjected to Western blot analysis. The blot was probed with the affinity-purified rabbit antiserum to ebaf. Protein size is shown in kilodaltons. The optical densities of the bands were determined by laser scanning densitometry and were expressed as relative optical density. END, Endometrial proteins from a day 1 menstrual endometrium included for comparison; MP, midproliferative; LP, late proliferative; P, postovulatory day; D1, first day of menstruation.

 
ebaf protein in the endometria of patients with infertility

We next examined the presence of ebaf proteins in the endometria of infertile patients during the implantation window (Table 1Go and Fig. 8Go). In contrast to the normal controls, the immunoreactive ebaf bands were found in differing amounts in the endometria of infertile women during the implantation window. In some infertile women, the immunoreactive ebaf bands were as abundant as those found during menstruation (Table 1Go and Fig. 8Go). In different endometria, different forms of the protein were found to be the abundant species. In these endometria, all or a single species of ebaf were found to predominate (Table 1Go and Fig. 8Go).



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  		Figure 8. Immunoreactive ebaf bands in the endometria of infertile women. In each lane, 10 µg extracted endometrial proteins from the cases shown in Table 1Go were resolved in a 15% gel by SDS-PAGE and then subjected to Western blot analysis. The blots were probed with the affinity-purified rabbit antiserum to ebaf. Lane 1, Endometrial proteins from a normal menstrual day 1 endometrium included as a control. In this endometrium, all forms of ebaf are abundant. Lanes 2 and 3, Endometrial proteins from normal postovulatory day 6 endometria included as controls. Lanes 4–23, Endometrial proteins from infertile subjects. Protein size is shown in kilodaltons. The optical densities of the bands were determined by laser scanning densitometry and were expressed as relative optical densities.

 
To determine whether the immunoreactive ebaf bands can be used as a predictor of infertility, ebaf immunoreactivity was examined in the endometria of patients with endometriosis. In some women, the endometriosis is associated with infertility, whereas in others, its presence does not preclude pregnancy (30, 31). Immunoreactive protein bands were more abundant in the endometria of patients with endometriosis (Fig. 9Go, compare with normal control shown in Fig. 8Go). However, there was more immunoreactive protein in the endometria of infertile patients with endometriosis than in the endometria of patients with endometriosis who were fertile (Fig. 9Go). In these patients, the levels of the precursor and the secreted form of ebaf were strikingly greater than those in the fertile control (P < 0.05; Fig. 9Go).



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  		Figure 9. Immunoreactive ebaf bands in the endometria of patients with endometriosis. In each lane, 10 µg extracted endometrial proteins were resolved in a 15% gel by SDS-PAGE and then subjected to Western blot analysis. Lane 1, Postovulatory day 8; lane 2, postovulatory day 10; lane 3, postovulatory day 13; lane 4, postovulatory day 9; lane 5, postovulatory day 9; lane 6, postovulatory day 7; lane 7, postovulatory day 9; lane 8, postovulatory day 7; lane 9, postovulatory day 8. Protein size is shown in kilodaltons. Lines are drawn from the highest level of ebaf detected in fertile endometriosis patients for comparison with the levels detected in infertile endometriosis patients. The optical densities of the bands were determined by laser scanning densitometry and were expressed as relative optical density.

 
We recently showed that the immunoreactive ebaf protein can be detected in the stromal and endometrial epithelial cells in the late secretory endometria (32). The immunoreactive ebaf protein bands were not detectable in the early and midsecretory endometria (32). To localize the cells that expressed ebaf in the endometria of infertile patients, immunohistochemical staining was carried out on the frozen sections of endometria of infertile patients obtained from early to midsecretory phases (Table 2Go). In some endometria, both the endometrial glands and stroma strongly exhibited a positive immunoreactivity (Fig. 10AGo and Table 2Go). In some endometria, primarily a stromal or a glandular pattern of immunostaining emerged (Fig. 10Go, C and E). Yet, in some endometria, the immunoreactivity was not easily detectable (Fig. 10GGo). The sections that were immunostained in the absence of the primary antibody did not show any evidence of immunoreactivity (Fig. 10Go, B, D, F, and H). Based on the semiquantitative assessment of immunostaining, the infertile cases could be divided into four main groups (Fig. 11Go). In some infertile women, similar to normal control women, no staining in the gland or stroma was observed. In the second group of infertile patients, only stromal staining was detected. In the third group of infertile patients, both glandular and stromal cell staining were found, and in the fourth group, only glandular staining was present.



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  		Figure 10. Immunohistochemical staining of ebaf in the endometria of infertile patients during the implantation window. Sections of endometria of infertile patients were immunostained for ebaf using the affinity-purified rabbit antiserum to ebaf as described in the text (A, C, E, and G). In the control sections, the antibody was omitted (B, D, F, and H). Immunostaining was observed in the glands and stroma (A), in the gland alone (C and E), or in neither the gland nor the stroma (G). No staining was detected when the primary antibody was omitted (B, D, F, and H).

 


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  		Figure 11. Semiquantitative assessment of immunostaining. The intensity of immunostaining in the glands and stroma of endometria of normal and infertile women was separately assessed semiquantitatively as described in the text and is shown on the y-axis. The case numbers on the x-axis are the same as those shown in Table 2Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We identified ebaf as a member of the premenstrual/menstrual molecular repertoire in human endometrium (7, 8). Northern blot analysis showed that ebaf mRNA was abundant in the late secretory and menstrual endometria (7). ebaf protein was detectable by Western blot analysis in the endometrium during the proliferative phase of the menstrual cycle, in the absence of a detectable ebaf mRNA. This is probably due to the low copy number of ebaf mRNA, which is undetectable by Northern blot analysis when total RNA is analyzed. A 42-kDa protein band that appeared as a doublet when adequately resolved was detected in the Western blot analysis of the endometrial proteins. The predicted size of the protein is smaller than 42 kDa, indicating that the larger size is due to posttranslational modification. Our analysis shows that ebaf is glycosylated. Such glycosylation adds approximately 2 kD to the size of protein. The cell lysates of the transiently transfected 293T, NIH-3T3, and COS-7 cells as well as stably transfected NIH-3T3 and 293T cells also contained this protein. The 293T cells transfected with lefty-1, the presumed mouse homolog of human ebaf, also contained a 42-kDa protein (14). The presence of a signal peptide suggested that ebaf is a secreted protein (7). Three potential cleavage sites exist within ebaf precursor. Western blot analysis of the conditioned media of transfected cells revealed proteins of 42, 34, 28, and 14 kDa. Similarly, transfection of BALB/3T3 cells with lefty-1 cDNA led to the secretion of 32- and 26-kDa proteins (14). Western blot analysis did not reveal the predicted 14-kDa ebaf protein. However, in the conditioned media of cells transfected with an ebaf expression plasmid and in the immunoprecipitated endometrial proteins, a 14-kDa protein band was detected. This protein is less abundant in both transfected cells and endometrium, making it difficult to detect by Western blot analysis. In addition to these bands, a 55/60-kDa protein band was detected by Western blot analysis in the endometrial proteins. This protein was not observed in the transfected cells. Therefore, it may represent a closely related or cross-hybridizing protein. Some of the proteins detected in the tissue lysates of endometrium may be secretory products that reside outside the cells and ultimately enter the peripheral circulation. Consistent with this, ebaf protein could be detected in the endometrial fluid. The presence of the 42-kDa precursor protein in serum is not unusual. It is interesting to note that serpins that inhibit furin (33) despite the lack of the typical cleavable N-terminal signal sequence have been found to reside extracellularly (34, 35, 36). ebaf protein was found in the male sera, indicating that sources other than endometrium exist in the body. By Northern blot hybridization, using total RNA for analysis, we previously showed that ebaf mRNA is expressed in other tissues at a low level (8). Northern and dot blot analyses, using polyadenylated RNA from various tissues, show that ebaf mRNA is expressed at a lower level in tissues other than endometrium (our unpublished data). These tissues are the likely source of immunoreactive ebaf in the serum in male subjects. The 28-kDa, but not the 34-kDa, protein band was detected in male sera, suggesting that only the 34-kDa protein may be uterine specific. Thus, the amount of this protein species in the serum may reflect the amount of ebaf synthesized by the endometrium.

We speculated that successful implantation occurs in the presence of a low level of ebaf protein in human endometrium, and that a high level of ebaf would be associated with infertility. Consistent with this, in over 50% of endometria from infertile patients, a mRNA that hybridized with full-length ebaf cDNA was up-regulated during the endometrial receptivity period. The infertility in these women was associated with endometriosis, polycystic ovary, bilateral tube occlusion, anovulatory cycle, luteal phase defect, premature ovarian failure, and habitual abortion. In some women, the underlying basis of infertility remained unknown (unexplained infertility). During this phase, an immunoreactive protein was also found in the endometria of patients with endometriosis, particularly those who were infertile. Women with endometriosis frequently experience abnormal uterine bleeding at times other than menstruation (37, 38). Abnormal expression of ebaf may be involved in such bleeding and may be a hallmark of imminent bleeding or abortion. In fact, it has been estimated that in up to 30% of normal fertile women, a subclinical pregnancy is ended with abortion (39, 40). Furthermore, nearly 50% of early pregnancy losses occur when implantation occurs after postovulatory day 10, when the ebaf protein is relatively abundant in endometrium (41). We have found that ebaf up-regulates the expression of matrix metalloprotease-3 and -7 and therefore may be involved in tissue shedding (unpublished data). A number of other molecular and biochemical alterations have been noted in the endometria of women with endometriosis. These include alterations in the expression of {alpha}vß3 integrin (21, 42, 43), complement (C3) (44), estrogen receptor splice variants (45), CA-125 (46), aromatase (47), metalloproteases (48), vascular endothelial growth factor (49), heat shock protein (50), soluble urokinase-type plasminogen activator (51), interleukin-6 (52), and, more recently, HoxA10 (53). Patients with endometriosis failed to show the expected midluteal rise in HOX gene expression, suggesting an altered development of the endometrium at the molecular level (53). HOXA10 and HOXA11 are homeobox genes that function as transcription factors and are essential to embryonic development and for implantation in the mouse (54). Therefore, it was suggested that abnormal expression of HoxA10 may contribute to the etiology of infertility in patients with endometriosis (53). With regard to ebaf, it seems that there are two abnormalities in the endometria of infertile patients. One involves a dysregulated expression of mRNA and protein, and a second defect relates to an abnormal processing of protein. In the endometria of some infertile patients, the 42-kDa form of ebaf was predominant, whereas in others the secreted forms were more abundant, and in yet another group both were present. Immunostaining shows that the protein is present in the glands, stroma, or both in such patients.

In summary, during a normal menstrual cycle in fertile women, ebaf is low during the implantation window and is abundantly expressed during the critical period of the menstrual cycle when implantation is unlikely to occur, such as proliferative or late secretory and menstrual phases. This regulation is disturbed in the endometria of infertile patients and is manifested as abnormal mRNA expression and processing of the protein.


   Footnotes
 
1 This work was supported by Grant CA-8466 from the NIH (to S.T.), and NIH Grants HD-35041 and HD-34824 and the National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation (to B.L.). Back

Received November 15, 1999.

Revised March 15, 2000.

Accepted March 29, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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