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Endocrinology Vol. 145, No. 3 1410-1418
Copyright © 2004 by The Endocrine Society

Cochlin, a Secreted von Willebrand Factor Type A Domain-Containing Factor, Is Regulated by Leukemia Inhibitory Factor in the Uterus at the Time of Embryo Implantation

Clara I. Rodriguez, Jr-Gang Cheng, Linda Liu and Colin L. Stewart

Cancer and Developmental Biology Laboratory, National Cancer Institute, Division of Basic Science, National Cancer Institute at Frederick, Frederick, Maryland 21702

Address all correspondence and requests for reprints to: Colin L. Stewart, Building 539, Room 121, National Cancer Institute, Division of Basic Science, National Cancer Institute at Frederick, Frederick, Maryland 21702. E-mail: stewartc{at}ncifcrf.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Embryo implantation is a required step in the reproduction of all mammals. In mice, a transient rise in the uterine expression of leukemia inhibitory factor (LIF) occurs on d 4 of pregnancy and is essential for embryo implantation. However, which genes are regulated by LIF in the uterus at implantation has not been determined. We performed a subtractive hybridization assay between luminal epithelial (LE) mRNAs from d 3 and 4 of pregnancy to find genes up-regulated on d 4 and which would be potentially regulated by LIF. One candidate, Coch-5b2, was up-regulated on the day of implantation. Coch mRNA localized to the LE of wild-type mice and was not detected in uteri from Lif-deficient mice. Treatment of LE with LIF, both in vitro and in vivo, resulted in the up-regulation of Coch. Coch is also highly expressed in other tissues, including the spleen and inner ear, but only in the uterus is Coch expression regulated by LIF. Mice were derived in which Coch was either deleted or tagged with a LacZ reporter. In mice carrying the tagged Coch gene, expression of Coch was detected in the LE and also at the site of embryo implantation. However, mice in which the Coch gene was deleted were normal, showing no overt defects in their reproduction. Although loss of Coch expression is not essential to reproduction in mice, it may serve as a useful marker for assessing the state of uterine receptivity in response to LIF at the onset of implantation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MAMMALS, AFTER fertilization, as the embryo moves down the reproductive tract, it undergoes a series of cleavage divisions, resulting in the formation of a blastocyst. For embryonic development to continue, the blastocyst must come into physical contact with the maternal uterine tissue through the process of implantation. The function of implantation is to establish an effective union between the maternal tissue and the embryo, providing oxygen and nutrients to the embryo and removing its metabolic waste products, which is necessary for the embryo’s continued development (1).

A fundamental feature of mammalian reproduction is that development of the preimplantation embryo is synchronized with the differentiation of the uterus into a receptive state that will allow implantation. In mice, preparation of the uterus for implantation is primarily under control of the ovarian steroid hormones estrogen [estradiol (E2)] and progesterone (P4). In the mouse, after ovulation, ovarian P4 levels increase and are essential to the uterus becoming pre-receptive. By d 4 of pregnancy (day of plug {equiv} d 1 of pregnancy), the P4-primed uterus becomes fully receptive due to a transient rise in nidatory E2 levels (2). These hormones act both directly on the uterine tissues and indirectly by regulating the expression of locally produced growth factors and cytokines that also affect the uterine tissues (3, 4, 5, 6). The combined effects of all these factors are to regulate uterine cell proliferation and differentiation in preparation for implantation of the embryo. The onset of implantation is marked by a reciprocal series of interactions between the activated blastocyst and uterine luminal epithelium (LE) (7).

One of the effects of nidatory E2 is to up-regulate the expression of the cytokine leukemia inhibitory factor (LIF), which has an essential role in implantation (8, 9). In fact, LIF can replace nidatory estrogen, inducing both implantation and decidualization in ovariectomized mice (9). LIF is transiently expressed in the glandular epithelium (GE) of mice at ovulation and again on d 4, just before the onset of implantation (10, 11). Female mice with a null mutation in the LIF gene are sterile due to a failure of blastocyst implantation. However, normal pregnancies can be induced in LIF-null mice when they are injected with recombinant LIF (rLIF), demonstrating that maternal expression of LIF is essential for implantation in the mouse (9).

Transient LIF expression in the uterus at the time of embryo implantation has been described in many mammalian species and may, therefore, be of central importance to regulating uterine receptivity (6). In the mouse uterus, LIF is expressed in the GE, from which it is secreted into the uterine lumen. The uterine LE, but not the stroma or GE, expresses LIF receptors and responds to LIF on d 4, primarily by activating the latent transcription factor signal transducer and activator of transcription 3 (STAT3) (12). However, the genes regulated by LIF in the LE are unknown and potentially would include genes whose expression is either up-regulated or down-regulated. Identification of such genes should further our understanding as to which factors are crucial for implantation. Their identification and interactions with each other would provide new opportunities to regulate fertility in mammals and help to gain a deeper understanding of potential causes of human infertility. Here, we describe an initial search for genes regulated by LIF in the LE using a suppressive subtractive hybridization procedure with mRNA isolated from purified LE before and at the time of implantation. Among the candidates, Coch-5b2 was identified and upregulated on the day of implantation. Coch expression is not detectable in the uterus of LIF null mice and is upregulated in the LE of mice treated with rLIF. We derived a Coch-LacZ reporter mouse that corroborated the up-regulation of Coch by LIF in the LE at implantation. However, Coch deficient mice have not shown any overt defects in implantation or in their reproduction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and preparation of the LE
LIF-deficient mice were maintained in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Research Council, Washington, DC). All wild-type mice were (C57BL6XC3H) F1s. Surgical procedures were performed under tribromoethanol (Avertin, Sigma-Aldrich, St. Louis, MO) anesthesia according to institutional guidelines (National Cancer Institute-Frederick Animal Care and Use Committee Guidelines and Policies). All mice used were between 6 and 10 wk old. To assess the time of pregnancy (day of plug {equiv} d 1 pregnancy), the uteri or oviducts were flushed to isolate embryos that were then developmentally staged by visual inspection. After ovariectomy on the morning of d 3, mice received a single 3-mg injection of Depo-Provera (P4; Pfizer, New York, NY) to establish a state of delayed implantation. The mice primed with P4 were rested for 3 d (to avoid any possible alterations due to inflammation) before they were killed, and RNA was isolated from uteri by the RNeasy protocol (Qiagen, Valencia, CA). Uterine LE from wild-type matings were purified by using mild enzymatic digestion and mechanical suction, slightly modified for mature mice (13). Sheets of LE were collected, washed three times in PBS, and RNA was isolated. Gravity sedimentation was used to separate large pieces of LE from contaminating blood cells. For LIF treatment, the collected LE were resuspended in serum-free optiMEM (Bio-Rad Laboratories, Hercules, CA), divided in two, and rLIF (200 ng/ml) was added to one sample. Epithelia were incubated at 37 C with gentle shaking for 4 h. Subsequently, RNAs were isolated from the rLIF-treated and untreated LE (RNeasy, Qiagen).

Subtractive hybridization
Total RNA from LE, early in the afternoon of d 3 and 4 of pregnancy, was isolated using the RNeasy procedure (Qiagen). One microgram of total RNA was converted to cDNA with SMART technology (Clontech, Palo Alto, CA). Suppressive subtractive hybridization was carried out using the PCR-Select cDNA subtraction kit (Clontech) according to the manufacturer’s protocol. Day 4 cDNA was used as tester, whereas d 3 cDNA served as a driver to detect genes highly expressed on late d 4, the day of implantation. Further screening of candidates was carried out by the Differential Screen procedure (Clontech). The subtracted cDNA pool was cloned into pCR-blunt-topo vector (Invitrogen, Carlsbad, CA). Individual clones were PCR amplified with common adapter primers and applied into duplicate 96-well dot blot membranes. One membrane was hybridized with d 3 cDNA, one membrane was hybridized with d 4 cDNA, and signals were integrated by phosphor imaging (Amersham Biosciences, Piscataway, NJ). The clones showing highly differential signals were subjected to sequencing and Blast analysis against GenBank.

Northern blotting
Total RNA from LE or different tissues of wild-type and/or LIF-deficient mice were obtained using the RNeasy system (Qiagen). Four to six mice were used to provide a particular tissue or to provide tissues from any particular time point. Ten micrograms of each sample were loaded per slot onto a formaldehyde1% agarose gel. The RNA was transferred to Hybond-N+ membranes (Amersham Pharmacia, Uppsala, Sweden) and hybridized with 32P-labeled cDNA probes using ExpressHyb Hybridization solution (Clontech) following the manufacturer’s instructions. The mouse Multiple Tissue Northern filters were obtained from Clontech. The mouse Conceptus Tissue and Placenta Full Stage Filters were from SeeGene USA (Del Mar, CA).

In situ hybridization
In situ hybridization was performed as described previously (14), with some modifications. Briefly, sense and antisense probes of Coch (1364–1837 bp) were labeled using an RNA labeling kit (Amersham Pharmacia). Uteri from different days of pregnancy were embedded and frozen in Tissue-Tek O.C.T. compound (Sakura, Torrance, CA). Frozen sections (8–10 µm) were mounted onto Superfrost Plus slides (VWR, West Chester, PA) and fixed for 1 h in 4% paraformaldehyde solution in PBS (pH 9.5). Slides were treated with proteinase K (0.1 µg/ml) for 10 min at 37 C, washed in diethylpyrocarbonate-treated water, acetylated, dehydrated using 50–100% graded alcohol washes, and air-dried. The hybridization was carried on at 58 C for 16 h using hybridization buffer containing the 33P-labeled Coch RNA probes. After hybridization, the sections were incubated with RNase A (40 µg/ml) for 45 min. RNase A-resistant hybrids were detected by autoradiography using NTB-2 liquid emulsion (Kodak, Rochester, NY).

Generation of embryonic stem (ES) cells and mice
The Coch cDNA fragment corresponding to 1364–1837 bp from the Coch cDNA was used to screen a 129/SvJ mouse phage library (Clontech). One of these phages contained the genomic sequence of Coch matching exon 4 through 4 kb past the last exon and was used to produce a replacement targeting vector containing PGKneo and TK cassettes (for positive and negative selection, respectively) as well as LacZ reporter cassette. Targeting vectors were derived by the phage-based Escherichia Coli homologous recombination system (recombineering) (15, 16). The Coch clone was inserted in pBR322. A neo cassette flanked by loxp sites was inserted in the StuI site of the PvuII Coch fragment (See Fig. 4AGo). After the neo cassette was inserted into pBR322 Coch clone by homologous recombination in E. coli, the construct was subjected to Cre recombinase to generate the orphan loxp site. The IRESLacZloxpPGKneo cassette was cloned into the NsiI site of an XbaI/BglII Coch fragment, and the cassette was similarly inserted into the pBR322 Coch clone by homologous recombination in E. coli. The TK cassette was introduced in the NotI site of the NheI fragment. This final construct was used for the electroporation of the W9.5 ES cells (17). ES clones heterozygous for the mutated allele were derived, and two independent lines of mice (no. 17 and no. 20) were derived by C57Bl/6 blastocyst injection. Both lines showed the same expression characteristics. Coch-deficient mice were derived from crosses between the Coch-LacZ mice and mice transgenic for the ubiquitously expressed ß-actin-Cre (18).



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  		FIG. 4. Generation of Coch-LacZ reporter mice. A, Top, a partial restriction map of the mouse Coch locus. Middle, the construct used to insert the different cassettes and three loxP sites into Coch. Bottom, the Coch locus after a homologous recombination event showing the insertion and exons of Coch. B, PCR using primers flanking the orphan loxP site. Wild-type and mutant alleles yield 154- and 204-bp fragments, respectively. C, Southern blot with probe (black rectangle in top of A) upstream of exon 4, revealing 18-kb wild-type and 24-kb mutant ScaI fragments. D, PCR amplification of a 400-bp fragment from the neo cassette in the mutant allele. E, LacZ staining of uterine stroma and LE from LacZ-Coch reporter mouse on d 3 of pregnancy. F, LacZ staining of uterine stroma and LE from LacZ-Coch reporter mouse on d 4 of pregnancy showing clear localization of LacZ activity to the LE. G, LacZ staining of uterus from LacZ-Coch reporter mice on d 5 of pregnancy. Note the expression of Coch around the implantation site. H, LacZ staining of uterine stroma and LE from LacZ-Coch reporter mouse on d 5 of pregnancy where no embryo implantation is occurring. LacZ activity is absent from the LE.

 
ß-Galactosidase staining
Uteri were immersed for 20 min in fixative solution (37% formaldehyde, 25% glutaraldehyde, and 10% Nonidet P-40 in PBS), washed two times for 5 min with detergent rinse (2 mM MgCl2, 0.02% Nonidet P-40 in PBS), and incubated overnight at 30 C in staining solution (2 mM MgCl2, 5mM potassium ferricyanide, 5 mM potassium ferrocyanide, and X-gal 5-bromo-4-chloro-3-indolyl-ß-D-galactosidase 1 mg/ml). The uteri were then embedded in paraffin, sectioned at 8–10 µm, and counterstained with neutral red.

Western blot analysis
Tissues were lysed with 7 M urea, 2 M thiourea, 2% Triton X-100, and 100 mM dithiothreitol, and with complete protease inhibitor cocktail (Roche, Basel, Switzerland), and separated on 12.5% denaturing acrylamide gels. Proteins were transferred to polyvinyl difluoride membranes. A rabbit antibody to the carboxy terminus was raised against a specific peptide of Coch (aa529–544) conjugated to keyhole limpet hemocyanin. Proteins were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). For the peptide competition assay, the rabbit antibody to COCH was absorbed with the peptide (aa529–544) for 30 min before hybridization to the filter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The search for LIF-induced genes
In this initial attempt to identify genes regulated by LIF in the LE, mRNAs from the LE on d 3 and 4 of pregnancy were reverse transcribed and subjected to suppressive subtractive hybridization (19). mRNAs showing differences in expression between d 3 and 4 of pregnancy would be potential candidates of regulation by LIF because the LE is responsive to LIF on d 4 of pregnancy (12). Twenty-two candidates were identified, three of which showed increased expression on d 4 compared with d 3 by virtual Northern analysis (data not shown). All three candidates were further screened by Northern analysis using total uterine RNA from d 1–5 of pregnancy. One candidate, EST1, had homology to several retroviral-like sequences. The second candidate, EST2, was 99% identical to a Mus musculus cDNA (BC003974) encoding a 98-amino acid peptide of unknown function. EST1 showed no evidence for up-regulation on d 4, whereas EST2 showed some up-regulation on d 4, but because it was expressed throughout the preimplantation period, it was unlikely to be directly regulated by LIF on d 4 (Fig. 1AGo). Both ESTs were not further characterized. The third candidate corresponded to a previously identified gene (20), Coch (AF006741; also known as Coch-5b2), which is associated with a human deafness disorder, DFNA9. The predicted protein encoded by Coch (cochlin) has several motifs including a secretory signal peptide, an LCCL structural domain, and two von Willebrand factor type A domains (VWA; Fig. 1BGo). Coch expression was increased at least 10-fold on d 4 in the uterus, suggesting that it may be up-regulated by LIF.



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  		FIG. 1. Suppressive subtractive hybridization results. A, Northern analysis of total uterus RNA from d 1–5 of pregnancy. The same filter was probed with the three candidates obtained from the suppressive subtractive hybridization assay. Ribosomal protein L19 (RPL19; accession no. BC010710) expression serves as a loading control. The bar graphs quantified levels of expression. B, Schematic representation of Coch gene product. The cochlin protein contains a predicted secretory signal peptide (SP), followed by an LCCL domain. The asterisks represent LCCL domain missense mutations, which are associated with the human deafness disorder DFNA9. In the carboxy terminus of cochlin, there are two VWA-like domains.

 
Coch is up-regulated by LIF in the LE in vitro and in vivo
Because Coch was up-regulated in the uterus on the day of implantation, we determined whether Coch was specifically expressed in the LE on d 4. We compared Coch expression by Northern analysis of RNA isolated from purified LE between d 3 and 4 of pregnancy (Fig. 2AGo) and confirmed that Coch was up-regulated in the LE of wild-type mice on d 4. However, in Lif-deficient mice, Coch was not up-regulated on d 4 (Fig. 2BGo). Coch mRNA was detected only in the LE of wild-type mice by in situ hybridization and not in the Lif-deficient mice on d 4 of pregnancy. Therefore, Coch mRNA is specifically expressed in the uterine LE, suggesting that Coch is regulated by LIF (Fig. 2CGo).



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  		FIG. 2. Coch expression in the LE is induced by LIF. A, Northern analysis of LE RNA from wild-type mice in d 3 and 4 of pregnancy. As a loading control, the filter was reprobed with RPL19. B, Northern analysis of LE RNA from d 4 pregnant LIF+/+ and LIF-/- mice. RPL19 was used as a loading control. C, In situ hybridization with an antisense probe to Coch in the uterus from d 4 pregnant LIF+/+ and LIF-/- mice. Arrows indicate the LE and shows a strong Coch signal in the LE of +/+ mice. D, Northern analysis of LE from wild-type animals on d 3 of pregnancy with and without treatment with rLIF for 6 h in vitro. Treatment with LIF up-regulates Coch expression. RPL19 was used as a loading control. E, Northern analysis of LE from LIF-deficient animals injected with rLIF or saline control on d 4 showing up-regulation of Coch expression in the mice that received LIF.

 
To determine whether expression in LE is directly regulated by LIF, we analyzed Coch expression in vitro. Mice were ovariectomized, treated with P4, and allowed to recover for 3 d to avoid possible side effects from the surgical procedure. The LE was then isolated and incubated with or without rLIF. In the LIF-treated LE (Fig. 2DGo), Coch was up-regulated compared with the untreated LE.

Because implantation failure in Lif-null females is rescued by an ip injection of LIF (9), we tested Coch expression in Lif-null mice treated with rLIF. On the morning of d 4 of pregnancy, Lif-null mice were either administered a bolus injection of 20 µg rLIF or an injection of saline as a control. Six hours later, the animals were killed, and uterine mRNAs were isolated. Coch expression was up-regulated in the uteri of Lif-null mice treated with rLIF (Fig. 2EGo), further supporting a role for LIF in regulating Coch transcription.

Coch is up-regulated by LIF in a tissue-specific manner
In adult tissues of the mouse, Coch mRNA is primarily expressed in the spleen and brain, with weak expression in kidney and testis. No expression was detected in liver, muscle, or lung (Fig. 3AGo). During postimplantation development, strong expression of Coch was detected in the placenta, and weak expression was detected in the head of embryonic d (E) 17.5 embryos (Fig. 3CGo). Subsequent analysis revealed Coch to be expressed in the placentas from E10.5–E18.5 (Fig. 3DGo).



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  		FIG. 3. Coch expression is regulated by LIF in the LE but not in other tissues. A, Coch expression in a panel of mouse adult tissues revealing high levels of expression in spleen and in total brain. Sk, Skeletal. B, Coch levels were compared in different tissues of wild-type and LIF-deficient mice by Northern blot analysis, which showed that absence of LIF did not affect Coch expression in spleen and brain. RPL19 was used as a loading control. C, Coch expression in postimplantation embryos at E17.5, revealed strong expression in the placenta and weaker expression in the head. D, Coch is expressed in the placenta throughout postimplantation development.

 
The expression of Coch in several adult tissues raised the question of whether LIF could be regulating Coch expression in all the tissues in which it was expressed. To study this, we investigated the expression pattern of Coch in Lif-deficient animals. Coch expression in brain, kidney, and spleen of Lif-deficient mice showed no differences when compared with wild-type tissues. Thus, the differences in the LE of wild-type and Lif-deficient mice at implantation was not recapitulated in other tissues. These data show that Lif only regulates Coch in the uterus (Fig. 3BGo) but not in other tissues where Coch is expressed.

Coch-LacZ reporter mice
To facilitate the study of Coch regulation during embryo implantation, we generated Coch-LacZ reporter mice by targeting a LacZ reporter gene to the Coch locus by homologous recombination in ES cells. A targeting vector was used in which an IRESLacZ cassette, followed by a floxed PGKneo cassette, was introduced after the Coch stop codon and before the poly A in the 3' untranslated region. An orphan loxP site was introduced between exons 6 and 7 (Fig. 4AGo). Mice carrying the LacZ gene targeted to the Coch gene were derived (Figs 4Go, B–D). Analysis of uteri from Coch-LacZ mice on d 4–5 of pregnancy showed that ß-gal expression was restricted to the LE on d 4 of pregnancy (Fig. 4FGo). On d 5 of pregnancy, Coch-directed ß-gal expression was specific to the site of implantation (Fig. 4GGo), with no expression being detected in d 3 or in d 5 LE in the absence of embryo implantation (Fig. 4Go, E and H). These results were consistent with the Northern analysis and in situ hybridization data, further demonstrating the specific localization of Coch expression to the uterine LE during embryonic implantation.

Derivation of mice lacking Coch by Cre recombination
Because of the tissue-specific expression of Coch and its up-regulation by LIF in the uterus, Coch may play a potential role in embryo implantation. To delete the Coch gene, we crossed the floxed-LacZCoch mice with a general Cre deletor line of mice in which Cre was expressed under control of the ß-actin promoter. After recombination, the exons (7, 8, 9, 10, 11) coding for amino acids 147–552 were eliminated, removing the two VWA domains as well as the IRESLacZ and PGKneo cassettes. Coch coding sequences for the secretory signal peptide and LCCL domain remained intact (Fig. 5Go).



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  		FIG. 5. Derivation of Coch-deficient mice. A, Top, Coch locus of Coch-LacZ reporter mice. Bottom, final configuration of the Coch-deleted allele after in vivo Cre-driven recombination, where exons 7 through half of 12, as well as the IRESLacZ and neo cassettes, are excised. B, Southern blot revealing 24-kb wild-type and 12-kb mutant ScaI fragments. C, PCR using the primers that flanked the orphan loxP site. In the mutant mice, the amplification does not occur because one of the primer binding sites has been deleted after Cre excision. D, PCR amplification of a 400-bp fragment from the neo cassette in the wild-type allele. E, PCR amplification of a 342-bp fragment only when the excision driven by Cre has been completed.

 
Northern analysis on total RNA from inner ear, spleen, and d-4 LE from wild-type and homozygous mutant animals using a probe against the carboxy terminus of Coch failed to detect any transcript in the homozygous mutant animals, as was expected because these animals lacked the carboxy terminus. In wild-type tissues, transcripts of 2.5 and 2 kb were detected, corresponding with the two different polyadenylated forms of Coch (21). To examine whether a truncated Coch transcript was produced, RNA from homozygous mutant inner ear, spleen, and LE were subjected to Northern analysis using a cDNA probe to Coch. Neither full-length nor truncated Coch transcripts were detected in inner ear and LE of the homozygous null mice using probes to both the 3' terminus and the complete cDNA coding sequence, demonstrating that Cre recombination resulted in the generation of a Coch deficiency in these tissues (Fig. 6Go, A and B). As was expected, both probes failed to detect the full-length 2.0- and 2.5-kb transcripts in the mutant animals. Furthermore, no truncated transcript was detected.



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  		FIG. 6. Coch-deficient mice have a complete knockout of Coch mRNA, and cochlin isoform regulation is tissue specific. A, Northern analysis of Coch with a carboxy 3'-terminal probe on inner ear, spleen, and LE RNA on d 4 of pregnancy from wild-type and mutant mice. B, Northern analysis with the full-length cDNA probe on inner ear, spleen, and LE on d 4 from wild-type and mutant mice. In both cases, the filters were reprobed with RPL19. C, Western blot analysis of cochlin products on different tissues from wild-type and mutant animals. The antibody detected four cochlin products in the inner ear of wild-type animals that were absent in the COCH knockout animal (lanes 2 and 3). The four Cochlin proteins were selectively competed out by preincubating the antibody against the peptide used in its generation. Wild-type spleen revealed two cochlin isoforms not detected in mutant animals (lanes 4 and 5). No cochlin isoform was detected in the uteri of wild-type animals, and the bands visible were background (lanes 6 and 7). The filters were stripped and reprobed with antibody against ß-actin.

 
A previously described antibody raised against the amino terminus of human COCH (cochlin) (22) recognizes two predominant bands of around 60 and 40 kDa in the human fetal cochlea. The same two bands of 60 and 40 kDa were detected in cochlear extracts in mice (data not shown). The larger band corresponds to the expected size of the full-length protein deduced from the cDNA, and the 40-kDa band corresponds to one of the cochlin isoforms (23). Cochlin is the most abundant protein in the bovine inner ear, and sequencing three bovine isoforms revealed that their N termini correspond to residues 25, 133, and 152 of full-length cochlin (63, 44, and 40 kDa) (23). The largest isoform of cochlin starts directly after the signal peptide, the midsize isoform starts 32 residues before the first VWA domain, and the smallest isoform starts 13 amino acids before the first VWA domain. We generated a polyclonal antibody to a 16-amino acid peptide at the carboxy terminus of mouse cochlin. This antibody recognizes four bands in extracts from the cochlea of wild-type mice (Fig. 6CGo, lane 2). The two largest bands (60 and 42 kDa) corresponded to the two larger isoforms recognized by the amino-terminus antibody (22), with the smaller peptides having a molecular mass of approximately 30 and 18 kDa, respectively. None of these peptides were observed in the cochlea of the Coch-null mice, and no truncated versions were detected (Fig. 6CGo, lane 3). All four bands disappeared when the antibody was preincubated with the peptide used in the generation of the antibody (Fig. 6CGo, lane 1). In the wild-type spleen, our antibody recognizes two specific bands (~40 and 45 kDa) (Fig. 6CGo, lane 4). These bands were absent in the knockout mice (Fig. 6CGo, lane 5) and were competed by the peptide used to raise the antibody (data not shown). The cochlin proteins detected in spleen extracts from the wild-type mice did not appear to correspond to the isoforms described in the cochlea (23) or to the bands we detected by the same antibody in the inner ear of the wild-type mice. Our carboxy-terminus antibody did not detect any specific cochlin product in the uterus of wild-type mice on the day of implantation. The antibody to the amino terminus (22) also did not detect any peptides in the spleen or in the uterus of the wild-type mice on the day of implantation (data not shown).

These results suggest that Coch is differentially processed at the transcriptional and/or posttranslational level, as previously suggested (23), but they also suggest that Coch regulation is tissue specific because different isoforms are found in different organs. However, here, the most important fact is that neither the amino- nor the carboxy-terminal antibodies were able to detect any isoform of Cochlin protein in the knockout tissues, whereas they detected several isoforms in wild-type organs. These data demonstrate that Cre recombination of the conditional Coch allele resulted in the generation of a complete Coch knockout in the inner ear, spleen, and uterus.

Homozygous Coch-/- mice were viable and indistinguishable from their wild-type littermates. The lifespan of the Coch-/- mice was comparable to the lifespan of their wild-type littermates. Coch-/- female mice were also implantation competent. We found no overt differences in implantation site morphology, implantation site distribution, or in the timing of decidualization in the uteri of null mice compared with wild-type littermates (Fig 7Go). Furthermore, histological analysis of the implantation sites in the Coch-/- mice on d 5–6 did not show any overt defects (data not shown). The total number of progeny produced from mating four pairs of homozygotes, compared with the numbers produced from four wild-type pairs, was slightly increased (234 vs. 204, respectively). However, this difference in numbers was not significant (Student’s t test, P > 0.5).



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  		FIG. 7. Coch knockout mice do not show any overt differences in implantation site formation or distribution. Uteri from Coch+/+ and Coch-/- mice were isolated at d 7 of pregnancy.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The initial events of embryo implantation, which include proliferation, differentiation, and apoptosis in the uterus, have been extensively described for more than three decades (24). However, the molecular basis to implantation, the interactions between embryonic and endometrial epithelial cells, and the initiation of implantation and its effects on the embryo, uterine stroma, and LE are largely unknown. Appropriate development and preparation of the LE is essential for normal implantation (25), and the blastocyst must be able to interact with the LE. In response to this interaction, both the embryo and uterus induce changes in the appropriate cellular adhesion molecules (24). Such a receptor-mediated model of implantation is based on the presence or absence of specific cell-adhesion receptors and their respective ligands. This model postulates a possible molecular mechanism for changes in adhesion, both between the LE and embryos and within cells of the LE and stroma (26).

In mice, the ovarian steroids E2 and P4 have a primary role in regulating uterine preparation for implantation. Their effects on cells of the uterus are both direct and indirect, with indirect effects being mediated by locally produced cytokines and growth factors. One such cytokine is LIF. In the mouse, LIF expression is induced by E2 and is essential for inducing a state of receptivity in the uterus, allowing implantation (9). LIF acts by binding to LIF receptors expressed in the uterine LE and activates the JAK-STAT3 pathway in the LE at the onset of embryo implantation (12). However, the genes regulated by LIF and their potential roles in embryo implantation are unknown.

The search for genes with potential roles in embryo implantation resulted in the identification of Coch, which has expression that is restricted to the LE on d 4 of pregnancy in mouse. COCH was originally isolated from a human fetal cochlear cDNA library (20) and is highly conserved between human (AF006740), mouse (AF006741), and chicken (AF012252). The human protein shares 94% and 79% amino acid identity to mouse and chicken orthologs, respectively (27). COCH maps to human chromosome 14q11.2–13 (21), within the locus for DFNA9 (28), a nonsyndromic, autosomal dominant hearing loss of vestibular malfunction with variable penetrance. Several point mutations in COCH have been described in unrelated families with vestibular dysfunction (27, 29, 30, 31, 32, 33, 34, 35, 36). The predicted protein encoded by COCH has several motifs including a secretory signal peptide, an LCCL structural domain, and two VWA domains (Fig. 3Go). The LCCL domain is found in Lg11 protein, cub-1-related proteins, Coch-5b2-related proteins, coagulation factor C of the horseshoe crab, and a predicted protein of Plasmodium falciparum. The function of the LCCL domain is unknown; however, all the DFNA9 mutations in COCH are found in the LCCL domain. Preliminary structural studies of COCH have shown that the mutations associated with hearing loss in humans affect residues critical for the proper folding of the LCCL domain (37, 38). Of the other domains in COCH, VWA domains have been identified in secreted proteins (with the exception of the integrins), including proteins involved in hemostasis, the complement system, cellular adhesion, and extracellular matrix proteins (39). Moreover, VWA domains bind to fibrillar collagens, glycoproteins, and proteoglycans (40, 41, 42).

In the process of embryo implantation, the remodeling of the extracellular matrix is indispensable for successful implantation in both humans and rodents (43, 44, 45). A promiscuous receptor that recognizes and binds several different extracellular matrix ligands is the integrin {alpha}Vß3. This integrin shows a temporal expression that is similar to that of LIF (46). The murine and human {alpha}Vß3 are expressed at the time of implantation on the subluminal border with the LE in the endometrium, as well as on the blastocyst. Furthermore, the blocking of the {alpha}Vß3 integrin using specific antibodies adversely affected implantation in mouse (47). The ß3 unit of this integrin can bind to VWA domains (48).

An interesting observation in this work was that, although the tissues we tested showed the same two transcripts (2.0 and 2.5 kb) corresponding to the two different polyadenylation signals of Coch (21), the same antibody (carboxy terminal) detected different products in different tissues. Moreover, antibodies against the carboxy and amino terminals detected different cochlin isoforms in the inner ear. In spleen, the antibody specific to the amino terminus did not detect any products, whereas the carboxy-terminus antibody detected two specific cochlin isoforms that were different from the products detected in the inner ear by the same antibody. These splenic variants did not correspond to any previously described cochlin isoforms (23). The 2.0- and 2.5-kb transcripts appeared in the LE on d 4, although neither the amino- nor the carboxy-terminal antibodies detected any cochlin isoforms in the uterus of the wild-type mice. These facts suggest that cochlin is processed at the posttranslational level and that this regulation is tissue specific. As previously mentioned, no antibody detected any specific cochlin isoforms in the wild-type LE at implantation. This doesn’t preclude the existence of a cochlin product in the LE because the posttranslational modification of cochlin in the LE could be different than that of the inner ear or spleen, rendering the protein unrecognizable by the available antibody reagents.

In addition to the extensive posttranslational regulation of cochlin, the tissue-specific transcriptional regulation of cochlin also appears to be complex. Coch expression in the cochlea and spleen is independent of LIF. We analyzed the sequence of the Coch gene up to10 kb upstream of the ATG start site and found one putative STAT3 consensus binding site at approximately 6.8 kb (data not shown). No consensus estrogen response or P4 response elements were identified within the 10-kb region, although the sequence for this region in the public National Center for Biotechnology Information database is still incomplete. We attempted to determine whether Coch transcription could be induced directly by LIF by transfecting the Coch promoter sequences linked to a reporter gene into cells that were then treated with rLIF. No up-regulation of Coch expression was detected in the transfected cells (data not shown). This indicates that Coch expression in the uterus, unlike the other tissues, including spleen, kidney, and brain, in which it is expressed, may be regulated by LIF/STAT3. However, regulation of Coch in the uterus by LIF may also be dependent on additional transcriptional regulators that are specific to the uterus.

Although the regulation of Coch expression by LIF during pregnancy suggests a role for Coch in embryo implantation, derivation of Coch-/- mice surprisingly showed no overt defects in implantation or changes in fertility. Embryos from mutant mothers developed normally and were indistinguishable from the progeny of wild-type mothers. It is puzzling that, despite the specific up-regulation of Coch by LIF in the LE, together with the complexity of the tissue-specific posttranslational modifications of cochlin, the loss of cochlin had no effect on implantation. This leaves open the question as to the function of cochlin. One possibility is that the local production of an LE-specific isoform of cochlin may act to regulate or limit vascular edema and bleeding at the site of implantation because cochlin is an extracellular matrix protein with VWA binding domains. The presence of the VWA domains suggests the possible function in helping create an extracellular matrix or clot to reduce any bleeding that may occur. Clearly cochlin is not essential to such clotting, possibly due to extensive redundancy that is built into such a vital process. Indeed, mice lacking the critical Factor IX in the clotting process die from excessive bleeding from any wound that is not cauterized, but female Factor IX nulls show no reproductive defects (49).

In summary, we have identified a secreted protein, cochlin, that is regulated by LIF in the LE on the day of implantation. Despite the function of cochlin in the uterus remaining obscure, the fact that it is tightly regulated by LIF may make it an important marker to further define the window of implantation. In particular, it may be useful in establishing that the uterus is responsive to LIF, a factor essential for implantation.


   Acknowledgments
 
We thank Cynthia Morton for kindly providing us with an anti-cochlin antibody, Lidia Hernandez for outstanding technical help, Lori Sewell for maintenance of the mouse colony, Richard Frederickson for preparation of the figures, and Leslie Mounkes for critical reading of the manuscript.


   Footnotes
 
Abbreviations: E, Embryonic day; E2, estradiol; ES, embryonic stem; GE, glandular epithelium; LE, luminal epithelium; LIF, leukemia inhibitory factor; P4, progesterone; rLIF, recombinant leukemia inhibitory factor; STAT3, signal transducer and activator of transcription 3; VWA, von Willebrand factor type A.

Received October 9, 2003.

Accepted for publication November 25, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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