Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print September 12, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.063594
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
77/6/1027    most recent
biolreprod.107.063594v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lewis, S. K
Right arrow Articles by Spencer, T. E
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lewis, S. K
Right arrow Articles by Spencer, T. E
Agricola
Right arrow Articles by Lewis, S. K
Right arrow Articles by Spencer, T. E
BIOLOGY OF REPRODUCTION 77, 1027–1036 (2007)
DOI: 10.1095/biolreprod.107.063594
© 2007 by the Society for the Study of Reproduction, Inc.

Galectin 15 (LGALS15): A Gene Uniquely Expressed in the Uteri of Sheep and Goats that Functions in Trophoblast Attachment1

Shaye K Lewis 3 4, Jennifer L Farmer 3 4, Robert C Burghardt 5, Gary R Newton 6, Greg A Johnson 5, David L Adelson 4, Fuller W Bazer 4, and Thomas E Spencer 2 4

Center for Animal Biotechnology and Genomics, Departments of Animal Science4 and Veterinary Integrative Biosciences,5 Texas A&M University, College Station, Texas 77843 Cooperative Agricultural Research Center,6 Prairie View A&M University, Prairie View, Texas 77446

ABSTRACT

Galectins are a family of secreted animal lectins with biological roles in cell adhesion and migration. In sheep, galectin 15 (LGALS15) is expressed specifically in the endometrial luminal (LE) and superficial glandular (sGE) epithelia of the uterus in concert with blastocyst elongation during the peri-implantation period. The present study examined LGALS15 expression in the uterus of cattle, goats, and pigs. Although the bovine genome contains an LGALS15-like gene, expressed sequence tags encoding LGALS15 mRNA were found only for sheep, and full-length LGALS15 cDNAs were cloned only from endometrial total RNA isolated from pregnant sheep and goats, but not pregnant cattle or pigs. Ovine and caprine LGALS15 were highly homologous at the mRNA (95%) and protein (91%) levels, and all contained a conserved carbohydrate recognition domain and RGD recognition sequence for integrin binding. Endometrial LGALS15 mRNA levels increased after Day 11 of both the estrous cycle and pregnancy, and were considerably increased after Day 15 of pregnancy in goats. In situ hybridization detected abundant LGALS15 mRNA in endometrial LE and sGE of early pregnant goats, but not in cattle or pigs. Immunoreactive LGALS15 protein was present in endometrial epithelia and conceptus trophectoderm of goat uteri and detected within intracellular crystal structures in trophectoderm and LE. Recombinant ovine and caprine LGALS15 proteins elicited a dose-dependent increase in ovine trophectoderm cell attachment in vitro that was comparable to bovine fibronectin. These results support the hypothesis that LGALS15 is uniquely expressed in Caprinae endometria and functions as an attachment factor important for peri-implantation blastocyst elongation.

conceptus, implantation, pregnancy, trophoblast, uterus

INTRODUCTION

Maternal support of conceptus (embryo/fetus and associated membranes) growth and development is critical for pregnancy recognition signaling and implantation in domestic animals [1–3]. In ruminants, morula-stage embryos enter the uterus on Days 4–6 and then form a blastocyst that contains a blastocoele or central cavity surrounded by a monolayer of trophectoderm [4, 5]. After hatching from the zona pellucida, blastocysts develop into a tubular form and then elongate to 10 cm or more in length beginning on Day 12 in sheep and Day 15 in goats and cattle. Peri-implantation blastocyst growth and elongation is crucial for pregnancy recognition signaling, which involves production of interferon tau (IFNT) from mononuclear trophectoderm cells of the elongating blastocyst that inhibits luteolysis [6, 7]. Hatched blastocysts of ruminants will only elongate when transferred to uteri in domestic ruminants [8]. Thus, factors supporting and regulating growth of peri-implantation blastocysts and elongating conceptuses are thought to be derived primarily from secretions of the uterus or histotroph. This hypothesis is supported by studies of asynchronous uterine transfer of embryos and trophoblast vesicles [9, 10], progesterone regulation of blastocyst elongation [11–13], and the phenotype of uterine gland knockout (UGKO) ewes [14, 15].

UGKO ewes display recurrent early pregnancy loss due to inadequate histotroph from the endometrial luminal (LE) and glandular (GE) epithelia that is required for peri-implantation blastocyst survival and elongation [14, 15]. To better understand the peri-implantation pregnancy defect in UGKO ewes, a gene-expression-profiling project was conducted using an endometrial cDNA library from Day 14 pregnant ewes [16, 17]. Interestingly, approximately 1.4% of the expressed sequence tags (ESTs) sequenced from that cDNA library were highly similar to OVGAL11, a novel member of the galectin family of secreted animal lectins [18]. The sequence of OVGAL11 protein displayed the highest similarity to human LGALS10 (also known as Charcot-Leyden Crystal [CLC] protein) [19, 20] and human LGALS13 (also known as placental tissue protein 13 or PP13) [21]. Because OVGAL11 did not have a known orthologue, it was proposed to be a new family member and renamed galectin 15 (LGALS15). Galectins are proteins with a conserved carbohydrate recognition domain (CRD) that bind beta-galactosides, thereby cross-linking glycoproteins as well as glycolipid receptors on the surface of cells and initiating biologic responses [22–24] that include adhesion, chemoattraction, migration, growth, differentiation, and apoptosis [25, 26].

Ovine endometrial LGALS15 contains a predicted CRD as well as C-terminal LDV and RGD recognition sequences that allow proteins to interact with integrins and other components of the extracellular matrix [27]. The temporal and spatial alterations in LGALS15 mRNA and protein in the uterine endometrial LE and sGE and lumen during the peri-implantation period of early pregnancy in sheep, combined with known biological activities of other galectins, make LGALS15 a strong candidate mediator of conceptus-endometrial interactions during implantation [17, 28]. One proposed extracellular role for LGALS15 in the uterine lumen is to function as a heterotypic adhesion molecule bridging the conceptus trophectoderm and endometrial LE and stimulating biological responses within the trophoblast, such as attachment and migration, that are critical for successful blastocyst elongation [5, 29]. Indeed, advanced growth and elongation of blastocysts in sheep uteri can be elicited by early progesterone treatment that was associated with early induction of LGALS15 in endometrial LE and sGE [11].

Although blastocyst elongation occurs in most domestic animals (sheep, goats, cattle, and pigs), LGALS15 has only been investigated in sheep. Therefore, objectives of this study were to (1) determine if LGALS15 is expressed in uteri of other domestic ruminants (goat and cattle) as well as pigs and (2) investigate the attachment function of LGALS15 using ovine trophectoderm (oTr1) cells. Results indicate that the LGALS15 gene is present in sheep, goats, and cattle, but is uniquely expressed only in endometria of sheep and goats during the peri-implantation period of pregnancy. Both sheep and goat LGALS15 support in vitro attachment of oTr1 cells, thereby supporting a role for LGALS15 in peri-implantation blastocyst elongation in Caprinae, a subfamily of the family Bovidae.

MATERIALS AND METHODS

Animals and Experimental Design

All experimental and surgical procedures involving animals met the Guidelines for the Care and Use of Agricultural Animals in Agricultural Teaching and Research and were approved by the Institutional Animal Care and Use Committees of Texas A&M and Prairie View A&M Universities.

Uterine tissues from sheep, goats, cattle, and pigs were obtained during the estrous cycle and/or pregnancy and processed for analysis by in situ hybridization and immunohistochemistry. Uteri from Spanish crossbred female goats or does (Capra hircus) were obtained (Day 0 = estrus/mating) on Days 5, 11, 13, 15, 17, and 19 of the estrous cycle and pregnancy (n = 5/day per status) and Day 25 of pregnancy (n = 5). Uteri from Angus crossbred cattle (Bos Taurus) were obtained at estrus (n = 2) and on Days 16, 16.5, 17, 17.5, 18, and 19 of the estrous cycle and pregnancy (n = 3/day per status) and Days 22 (n = 2) and 23 (n = 3) of pregnancy. Uteri from Large White crossbred gilts (Sus scrofa) were obtained on Days 5, 9, 12, and 15 of the estrous cycle and Days 9, 10, 12, 13, 14, 15, and 20 of pregnancy (n = 3/day per status). Uteri from Suffolk crossbred ewes (Ovis aries) were obtained from Days 16 and 18 of pregnancy (n = 4/day) as a positive control. Uteri were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2) and embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO) for histology. Samples of endometria were also frozen in liquid nitrogen and stored at –80°C for RNA extraction.

Slot Blot Hybridization Analysis

Steady-state levels of LGALS15 mRNA in goat endometria were assessed by slot blot hybridization using methods described previously [30]. Radiolabeled antisense LGALS15 cRNA probes were generated by in vitro transcription with [{alpha}-32P]-UTP using linearized full-length O. aries LGALS15 cDNA as the template [17] and RNA polymerase. Denatured total endometrial RNA (20 µg) from each goat was hybridized with radiolabeled antisense LGALS15 cRNA. To correct for variation in total RNA loading, a duplicate RNA slot membrane was hybridized with radiolabeled antisense 18S cRNA (pT718S; Ambion, Austin, TX). Following washing, the blots were digested with ribonuclease A and radioactivity associated with slots quantified using a Typhoon 8600 MultiImager (Molecular Dynamics, Piscataway, NJ).

In Situ Hybridization Analysis

Location of LGALS15 mRNAs in uterine tissues was determined by radioactive in situ hybridization analysis as described previously [30]. Radiolabeled antisense and sense cRNA probes were generated by in vitro transcription using linearized full-length O. aries LGALS15 cDNA [17], RNA polymerases, and [{alpha}-35S]-UTP. Deparaffinized, rehydrated, and deproteinated uterine tissue sections were hybridized with radiolabeled antisense or sense cRNA probes. After hybridization, washing, and ribonuclease A digestion, slides were dipped in Kodak NTB-2 liquid photographic emulsion and exposed at 4°C for 3 days. Slides were developed in Kodak D-19 developer, counterstained with Gill's hematoxylin (Fisher Scientific, Fairlawn, NJ), and then dehydrated through a graded series of alcohol to xylene. Coverslips were then affixed with Permount (Fisher Scientific). Images of representative fields were recorded under brightfield or darkfield illumination using an Eclipse 1000 photomicroscope (Nikon Instruments, Inc., Lewisville, TX) fitted with a Nikon DXM1200 digital camera.

RT-PCR Analysis

Expression of LGALS15 mRNA in endometrial samples was determined by RT-PCR as described previously [31]. Total cellular RNA was isolated from endometria of cyclic and pregnant sheep, goats, cattle, and pigs using Trizol (Gibco-BRL, Bethesda, MD) according to the manufacturer's recommendations. The quantity of RNA was assessed spectrophotometrically, and the integrity of RNA was examined by gel electrophoresis in a denaturing 1% agarose gel. Briefly, cDNA was synthesized from total endometrial RNA (5 µg) using random and oligo-dT primers and SuperScript II Reverse Transcriptase (Life Technologies, Gaithersburg, MD). Newly synthesized cDNA was acid-ethanol precipitated, resuspended in 20 µl sterile water, and stored at –20°C. The cDNAs were diluted (1:10) with sterile water prior to use in PCR reactions. The PCR reactions were performed using Ex Taq DNA polymerase (2.5 U) and 10X Ex Taq buffer (Takara Bio, Carlsbad, CA) according to the manufacturer's recommendations.

The forward (5'-ACA CAG TTT CAA CAG GGA AG-3') and reverse (5'-CCG CCC CTT ATA ACG TA-3') primers amplified a cDNA of 443 bp that contained the entire coding sequence of the ovine LGALS15 mRNA. PCR amplifications were conducted as follows: 34 cycles of 95°C for 30 sec, 47°C for 1 min, and 72°C for 1 min. As a positive control, ACTB (beta actin) primers (forward: 5'-ATG AAG ATC CTC ACG GAA CG-3'; reverse: 5'-GAA GGT GGT CTC GTG AAT GC-3') were used to amplify a cDNA of 270 bp. PCR products were separated on a 1.5% agarose gel, visualized by ethidium bromide staining, cloned into pCR2 (Invitrogen, Carlsbad, CA), and sequenced in both directions. A minimum of five clones from five individual sheep and goats were sequenced, and representative clones were deposited in GenBank (accession numbers EU009323, EU009324, EU009325, and EU009326).

Multiple alignments of translated protein sequences were carried out using MUSCLE v.3.6 [32, 33] with the –maxiters flag set to 4. Phylogenetic trees were constructed by generating tree files from the MUSCLE alignments using ClustalW [34] and plotted using TreeView X [35].

Production of Recombinant LGALS15 Proteins

The entire coding sequence for the ovine and caprine endometrial LGALS15 mRNAs with either the LDVRGD or LVVRGD sequence polymorphism at the C-terminus was used to produce recombinant ovine and caprine LGALS15 in bacteria. PCR reactions (50 µl) were conducted in Optimized Buffer F (Invitrogen) and contained 10 ng of the appropriate ovine or caprine LGALS15 cDNA, 0.5 mg/ml forward primer (5'-AGA TGA AGC CAT GGA CTC CTT GCC GAA CCC CTA CC-3'), 0.5 mg/ml reverse primer (5'-AGA GTA AGC TTT GAT AAC GTA TCC ACT GAA GTC AGC-3'), and 1 U Ex Taq polymerase using an Eppendorf Mastercycler thermocycler (Eppendorf, Westbury, NY) with conditions of: 1) 95°C for 2 min; 2) 95°C for 30 sec, 54°C for 1 min, and 72°C for 1 min for 35 cycles; and 3) 72°C for 7 min. The amplified LGALS15 cDNA was restricted with NcoI and HindIII enzymes and then directionally subcloned into the pET-28b (+) vector (Novagen, Madison, WI). This cloning strategy mutated the stop codon of LGALS15 and placed a His•Tag sequence at the C-terminus. The resulting plasmid was sequenced in both directions to ensure that no mutations were present in the LGALS15 sequence.

Recombinant LGALS15 protein was produced in BL21 Star (DE3) One Shot Escherichia coli (Invitrogen) according to the manufacturer's suggestions. Expression of the LGALS15 fusion protein was induced with 5 mM isopropyl-beta-D-thiogalactopyranoside (IPTG; Sigma, St. Louis, MO). Bacteria were lysed with Bugbuster (Invitrogen) supplemented with recombinant lysozyme and benzonase. Recombinant LGALS15 protein was isolated by affinity chromatography using a Ni-NTA His•Bind Resin purification kit (Invitrogen). Elutions from the column were analyzed by 1D-SDS-PAGE followed by silver staining and Western blot analysis with rabbit anti-ovine LGALS15 IgG. Recombinant protein was dialyzed overnight in PBS (pH 7.2) at 4°C and then concentrated in a spin column with a 3500 MWCO (Vivaspin, Stonehouse, UK). Protein concentrations were determined using an RC/DC Protein Assay (Bio-Rad Laboratories, Hercules, CA) with BSA as the standard.

Production of Rabbit Antibodies to Ovine LGALS15

Purified recombinant ovine LGALS15 was used to immunize rabbits using a commercial service. Serum from high-titer rabbits was collected by terminal bleed, and anti-ovine LGALS15 IgG was purified from antiserum using an ImmunoPure (A/G) IgG Purification kit (Pierce, Rockford, IL). The antibody recognized a 15-kDa protein of the appropriate size in Western blot analysis of ovine uterine flush proteins and recombinant ovine and caprine LGALS15 protein produced in bacteria.

Immunohistochemistry

Immunocytochemical localization of LGALS15 protein in the uterus was performed using methods described previously [17]. Immunoreactive LGALS15 protein was detected using purified rabbit anti-ovine LGALS15 IgG at a final dilution of 1:5000 and a Vectastain ABC anti-rabbit kit (Vectastain Laboratories, Burlingame, CA). Antigen retrieval was performed using boiling citrate buffer as described previously [36]. Negative controls included substitution of the primary antibody with non-immune rabbit IgG. Immunoreactive protein was visualized using diaminobenzidine tetrahydrochloride (Sigma) as the chromagen. Sections were dehydrated and coverslips affixed with Permount.

Photomicroscopy

Photomicrographs of in situ hybridization and immunohistochemistry slides were taken using a Nikon Eclipse E1000 photomicroscope (Nikon Instruments, Melville, NY). Digital images were captured using a Nikon DXM 1200 digital camera (Nikon) and assembled using Adobe Photoshop 7.0 (Adobe Systems, Seattle, WA).

Trophectoderm Attachment Assays

Attachment assays were adapted from Liaw et al. [37] and Ochieng et al. [38]. Greiner Multiwell Tissue Culture Plates (24-well) for suspension cultures (PGC Scientific Co., Monroe, NC) were coated with either BSA Fraction V (Pierce) as a negative control, bovine fibronectin (bFN from bovine plasma; Sigma) as a positive control, and recombinant ovine or caprine LGALS15 proteins at the indicated amount in triplicate and allowed to dry overnight in a sterile hood at room temperature. Wells were then blocked with 1 ml per well of BSA (10 mg/ml) in PBS for 1 h and then rinsed three times with 1 ml per well of serum- and insulin-free medium. Derivation and culture of mononuclear oTr1 cells have been described previously [39]. The oTr1 cells were seeded into each well, and plates were incubated for 1.5 h. Wells were washed three times with 1 ml per well of serum- and insulin-free medium to remove nonattached cells. Cell number was then determined using a Janus Green assay [40] as described previously for oTr1 cells [39].

Statistical Analyses

All quantitative data were subjected to least-squares analysis of variance (ANOVA) using the General Linear Models procedures of the Statistical Analysis System (SAS Institute, Cary, NC). Slot blot hybridization data were corrected for differences in sample loading using the 18S rRNA data as a covariate. Slot blot data were analyzed for effects of day, pregnancy status (cyclic or pregnant), and their interaction. Next, least-squares regression ANOVA was conducted within pregnancy status. Tests of significance were performed using the appropriate error terms according to the expectation of the mean squares for error. A P-value of 0.05 or less was considered significant. Data are presented as least-square means (LSM) with SEM.

RESULTS

LGALS15 Is Present in Ruminants, but Only Expressed in the Uterus of Sheep and Goats

The coding sequence of ovine LGALS15 mRNA (GenBank AF252548) and the inferred LGALS15 protein sequence (GenBank AAF64320) were used to interrogate available databases. Multiple BLAST searches found evidence for LGALS15 mRNA only in sheep and an LGALS15-related sequence in bovine (GenBank XM_593263) with 86% and 77% identity to the ovine LGALS15 mRNA and protein, respectively. In sheep, ESTs for LGALS15 were found in several different tissues including endometrium, gall bladder, small intestine, Peyer's patches, skin, spleen/brain, dendritic cells, and mammary glands. Interestingly, LGALS15 ESTs were highly represented in an endometrial cDNA library from Day 14 pregnant ewes (1.8%), gall bladder (1.1 %), and small intestine (1.1%). In cattle, only 5 ESTs for the B. taurus mRNA similar to ovine LGALS15 were found out of 1.3 million bovine ESTs. Of these five sequences, two were full length, forward and reverse from one clone from a male Holstein (BARC 9 library). This full-length sequence aligned to a region of bovine chromosome 18 and spanned four predicted exons. The other three sequences were from the placenta, but two of those sequences appeared to be chimeric ribonucleoprotein/LGALS15, suggesting that they were most likely cloning artifacts. The remaining placental sequence spanned three of the four predicted exons for LGALS15.

Primers were developed to amplify the entire coding sequence of LGALS15 and used for RT-PCR analyses of total RNA isolated from endometria of cyclic and pregnant sheep, goats, cattle, and pigs. PCR products were generated in endometria from sheep and goats, but not cattle or pigs. Sheep and goat LGALS15 were highly homologous at the mRNA (95%) and protein (91%) levels (Fig. 1). Similarly, the bovine LGALS15-like sequence shared 86% and 77% identity to the ovine LGALS15 mRNA and protein, respectively.


Figure 01
View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1 MUSCLE alignment of the amino acid sequences of LGALS15 from ovine and caprine endometria compared to a predicted bovine protein with high similarity to LGALS15. The arrowheads denote the conserved residues forming the CRD in prototypical galectin family members. The black circle denotes a conserved C residue critical for mannose binding in LGALS10. The underlined residues denote the conserved LDV and RGD recognition sequences for integrin binding near the C-terminus in ovine and caprine LGALS15 and closely related LKV and KGD sequences in the predicted bovine LGALS15. The asterisks, colons, and single dots indicate the amino acid identity, conserved substitutions, and semiconserved substitutions, respectively.

A search of the amino acid sequence of the LGALS15 proteins revealed the presence of a CRD characteristic of galectins [41]. The CRD is a consensus motif consisting of 13 amino acids [42], eight of which (H.N.R.V.N.W.E.R) play a critical role in binding sugars [43, 44]. As illustrated in Figure 1, comparison of the putative CRDs of ovine and caprine LGALS15 with the conserved CRD of other galectins indicated that four residues are identical (V62, N64, W71, E74) and three are conservatively substituted (R54, W56, K76). Similarly, comparison of the predicted CRD of bovine LGALS15 with the conserved CRD of other galectins found that five residues were identical (V63, N65, W72, E75, R77) and two were conservatively substituted (H54, R56). However, ovine and caprine LGALS15 substituted a P52, and the bovine LGALS15 an A52, for the first H residue of the consensus CRD. The C57 in ovine and caprine LGALS15 is different from prototypical galectins, but appears to allow for binding of mannose in LGALS10 [45]. However, a C residue at position 58 was not found in bovine LGALS15.

Consistent with other galectins, none of the LGALS15 had apparent or predicted signal peptide, transmembrane domain, or glycosylation sites. A PROSITE search revealed two putative cell attachment sequences at positions 123 (LDV) and 126 (RGD) in ovine and caprine LGALS15 that are recognition sequences for integrin binding [27]. The putative CRD and LDVRGD recognition sequences were conserved in all cDNAs amplified from sheep and goat endometrium (GenBank EU009324 and EU009325). However, approximately 50% of the sheep and goat LGALS15 from each individual contained an LVV polymorphism next to the RGD sequence in the C-terminus (GenBank EU009323 and EU009326). As shown in Figure 1, the bovine LGALS15-like protein contained LKVKGD sequences at position 124 instead of the LDVRGD or LVVRGD sequence found in ovine and caprine LGALS15. The KGD recognition sequence binds integrins similar to the RGD sequence [27].

All galectin protein sequences present in UniProt [46] were downloaded and aligned with the translated ovine, caprine, and bovine LGALS15 sequences (data not shown). It was clear from this alignment and the resulting phylogeny that LGALS15, CLC/LGALS10, and CLC2/LGALS14 were most closely related and most similar to LGALS4. This relationship is illustrated in Figure 2, where only the LGALS4 node from the comprehensive tree is displayed. Based on this result, the LGALS15 genes are likely specific to the subfamily Caprinae of the family Bovinae. Further, the LGALS15 genes and the primate CLC/LGALS10, CLC2/LGALS14 and LGALS13 genes arose from an ancestral duplication of LGALS4. Specifically, the predicted protein sequence from the B. taurus LGALS15-like mRNA shares significant similarity to LGALS10, LGALS13, and LGALS14 from several species with no gaps and to ovine LGALS15 with one gap.


Figure 02
View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2 Phylogenetic tree of LGALS15 relationships to the other most closely related galectin superfamily members based on the Neighbor Joining method using the tree generated from the MUSCLE alignment. The branch lengths are proportional to an estimate of evolutionary change. The scale bar at the bottom denotes relative estimate of evolutionary distance.

LGALS15 mRNA and Protein Are Present in Endometria of Sheep and Goats, but Not Cattle or Pigs

Steady-state levels of LGALS15 mRNAs in endometria from cyclic and pregnant goats were determined by slot blot hybridization analyses (Fig. 3) and found to be affected (P < 0.01) by day, status, and their interaction. In cyclic goats, endometrial LGALS15 mRNA was low to undetectable before Day 13, increased (cubic effect of day, P < 0.01) about 88-fold from Days 13 to 17, and then declined to Day 19. In pregnant goats, LGALS15 mRNA levels were also low to undetectable before Day 13, increased between Days 13 and 17, and declined between Days 19 and 25 (cubic effect of day, P < 0.01). Between Days 13 and 17, endometrial LGALS15 mRNA levels increased about 88-fold in cyclic goats, compared to a 292-fold increase in pregnant goats (day x status, P < 0.0001). Thus, endometrial LGALS15 mRNA levels were not different between cyclic and pregnant goats between Days 11 and 15, but increased in pregnant over cyclic goats between Days 15 and 19, which correlates to the onset of definitive attachment of the trophectoderm to the endometrial LE in goats [47].


Figure 03
View larger version (9K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3 Steady-state levels of LGALS15 mRNA in endometria from cyclic and early pregnant goats determined by slot blot hybridization analysis. In cyclic goats, LGALS15 mRNA was low to undetectable from Days 5 to 11, increased about 88-fold from Days 11 to 17, and decreased to Day 19 (cubic effect of day, P < 0.01). In pregnant goats, LGALS15 mRNA was low to undetectable from Days 5 and 11, increased about 292-fold between Days 13 and 17, and declined somewhat to Day 25 (cubic effect of day, P < 0.01). Endometrial LGALS15 mRNA was higher in pregnant than cyclic goats on Days 17 and 19 (day x status, P < 0.0001). Data are expressed as LSM relative units with SEM.

In situ hybridization analyses found abundant LGALS15 mRNA in endometrial LE and sGE of cyclic and pregnant uteri from goats and sheep (Fig. 4). In contrast, no hybridization signal for LGALS15 mRNA was detected in uteri of cyclic or pregnant cattle and pigs. In goats, LGALS15 mRNA was first observed at low levels in LE, sGE, and upper glands of the endometrium on Day 13 of both the estrous cycle and pregnancy. In cyclic goats, LGALS15 mRNA was most abundant on Day 17 and then declined substantially on Day 19, whereas LGALS15 mRNA in pregnant goats increased from Days 15 to 17 and remained abundant thereafter. LGALS15 mRNA was not detected in conceptus trophectoderm. Thus, the presence of a conceptus increases LGALS15 mRNA levels in caprine endometrium.


Figure 04
View larger version (74K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4 In situ localization of LGALS15 mRNA in the endometrium. Cross-sections of the uterine wall from cyclic (C) and pregnant (P) goats were hybridized with radiolabeled antisense or sense ovine LGALS15 cRNA probes. LGALS15 mRNA was detected only in endometrial LE and sGE of goats and sheep, but was not detected in either cattle or pigs. All representative photomicrographs are shown in brightfield (left) and darkfield (right) illumination at the same width of field (420 µm). S, stroma; Tr, trophectoderm. Numbers in panels indicate days.

Overall changes in immunoreactive LGALS15 protein abundance in endometrial LE and sGE of goats paralleled changes in LGALS15 mRNA in cyclic and pregnant goats (Fig. 5). In both cyclic and pregnant goats, LGALS15 protein was localized primarily in the cytoplasm of endometrial LE and sGE. Consistent with an increase in LGALS15 mRNA, the abundance of LGALS15 protein increased in LE and sGE after Day 13 and was readily apparent near and on the apical surface of endometrial LE by Day 17 of the estrous cycle and pregnancy. Although LGALS15 mRNA was not present in the conceptus, LGALS15 protein was detected in crystal structures within conceptus trophectoderm as well as endometrial LE and sGE. Consistent with the lack of detectable LGALS15 mRNA, no immunoreactive LGALS15 protein was detectable in bovine or porcine uteri.


Figure 05
View larger version (93K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5 Immunolocalization of LGALS15 protein in the endometrium and conceptus in pregnant (P) and cyclic (C) goats. Note the presence of immunoreactive LGALS15 protein in the endometrial epithelia and conceptus trophectoderm in pregnant goats and sheep, but not cattle or pigs. For the IgG control, normal rabbit IgG was substituted for the primary antibody. Representative photomicrographs are shown at the same width of field (420 µm) with the exception of the higher magnifications of the caprine endometrium (right bottom) that is a width of field of 630 µm. Sections were not counterstained. S, stroma; Tr, trophectoderm. Numbers in panels indicate days.

LGALS15 Promotes Attachment of oTr1 Cells

Ovine trophectoderm cells isolated from Day 15 conceptuses were predominantly mononuclear and expressed IFNT by RT-PCR (data not shown). A dose-dependent increase (P < 0.01) in oTr1 cell attachment was induced in wells coated with increasing amounts of LGALS15 and bFN, but not BSA (Fig. 6). An increase in oTr1 cell attachment also occurred in response to bFN as well as in response to all forms of ovine and caprine LGALS15, and LGALS15 and bFN induced similar increases in oTr1 cell attachment.


Figure 06
View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 6 Attachment function assays of ovine and caprine LGALS15 using oTr1 cells. Wells of suspension culture plates were coated with increasing amounts (0.1, 1, or 10 µg) of recombinant ovine and caprine LGALS15 or purified bovine fibronectin, but not BSA. Freshly prepared oTr1 (labeled oTr) cells were seeded into each well and allowed to attach for 1.5 h. Unattached cells were washed off, and cell number in each well determined. Data are expressed as percentage of attached oTr1 cells relative to BSA. The entire experiment was independently repeated at least three times with similar results.

DISCUSSION

Results of bioinformatic and RT-PCR analyses indicate that LGALS15 is a unique member of the galectin family that is present in the genome of sheep and goats (Subfamily Caprinae) and cattle (Subfamily Bovinae), but not in pigs (Suborder Suina), which are Artiodactyls. Outside of the Artiodactyls, LGALS15 was not detected in human, nonhuman primate, mouse, chicken, dog, or any other species with a sequenced genome. These results suggest that LGALS15 is a unique gene in sheep, goats, and cattle. Given the lack of expression of LGALS15 in the bovine uterus and very rare abundance in other tissues based on EST analysis, the bovine LGALS15 gene may be a pseudogene, which can be defined as a defunct relative of a known gene that is no longer expressed in the cell [48]. Phylogenetic analyses of available galectins from a number of species (human, mouse, rat, dog, and cow) suggest that LGALS15 is likely a paralog derived from another closely related galectin family member such as LGALS10/CLC, LGALS11, LGALS13, or LGALS14/CLC2. Paralogs are genes related by duplication within a genome that evolve new functions, even if these are related to the original gene. Indeed, the LDV and RGD recognition sequences in LGALS15 of sheep and goats are conservatively substituted in cattle, but not present in the C-terminus of any other galectins. Interestingly, LGALS13, originally known as PP13, was originally cloned from human placenta [49] and is a homologue of the human eosinophil CLC protein that is known as LGALS10. Moreover, ovine, caprine, and bovine LGALS15 display the highest similarity to human CLC [19, 20] and LGALS13 [21]. Thus, LGALS15 most likely evolved as a paralog of LGALS13 or LGALS10/CLC in sheep, goats, and cattle. Functional studies of other galectins have implicated these proteins in cell adhesion, chemoattraction, and migration, as well as cell growth, differentiation, and apoptosis [22, 25, 50]. All of these biological activities are proposed to be important for ruminant blastocyst growth and elongation during the peri-implantation period of pregnancy [4, 5, 51].

The temporal changes in expression of endometrial LGALS15 mRNA support the hypothesis that ovarian progesterone and conceptus IFNT regulate transcription of the LGALS15 gene in endometrial epithelia of goat uteri as found in sheep [16, 17]. IFNT is the pregnancy recognition hormone in ruminants that acts on the endometrium to prevent development of the luteolytic mechanism, thereby maintaining the corpus luteum for production of progesterone [52]. The enhanced levels of LGALS15 mRNA in endometria of pregnant goats on Days 15–19 of pregnancy as compared to cyclic goats parallels the increase in production of IFNT by the conceptus, which is produced from Days 16 to 21 and is maximal between Days 16 and 18 in goats [53, 54]. Intrauterine administration of IFNT increases LGALS15 mRNA, but only in progesterone-treated ewes [17]. Indeed, several genes have been identified to be progesterone-induced and IFNT-stimulated specifically in endometrial LE/sGE in the ovine uterus, including cathepsin L and cystatin C [55, 56]. Thus, progesterone and IFNT act in concert to stimulate a number of genes apparently important for conceptus survival, growth, and implantation [5, 16, 29, 57].

Prototypical members of the galectin superfamily (LGALS1, LGALS2, LGALS5, LGALS7, LGALS10, LGALS11, LGALS13, LGALS14) have one conserved CRD. Interestingly, results of phylogenetic analyses suggest that LGALS10, LGALS13, and LGALS15 were derived from LGALS4, which is a tandem repeat galectin with two CRDs [58]. Although the CRD of LGALS15 differs slightly from that in the prototypical galectins, it does possess the "jellyroll" structural fold similar to that found in LGALS10 and LGALS13 [59]. Galectins bind beta-galactosides via the CRD, but the carbohydrate binding specificity for each galectin appears to be different [60]. In addition to the CRD, sheep and goat LGALS15 also contains predicted cell attachment sequences (LDV and RGD) that could mediate binding to integrins in extracellular matrix proteins [27]. Galectins can also bind and activate integrins via their CRD [25]. In the present study, all forms of recombinant ovine and caprine LGALS15 increased attachment of mononuclear oTr1 cells to a similar extent as bFN. Although the LDV sequence next to the RGD sequence is an integrin binding site [27], there were no detectable differences in cell attachment function among the different polymorphic variants of ovine or caprine LGALS15 that contained the LVVRGD sequence instead of the LDVRGD sequence in the C-terminus. The temporal and spatial alterations in LGALS15 mRNA and protein in the ovine uterus [17] and caprine uterus during pregnancy, combined with the in vitro attachment of oTr1 cells to recombinant ovine and caprine LGALS15, support the hypothesis that LGALS15 functions as a heterotypic cell adhesion molecule bridging endometrial LE and conceptus trophectoderm, which is required for the blastocyst growth, elongation, and attachment phases of implantation.

Both the RGD recognition sequence and perhaps the CRD of LGALS15 may be involved in cell attachment and adhesion via integrin binding and activation. Indeed, integrins are proposed to be the dominant glycoproteins that regulate trophectoderm adhesion to endometrial LE during implantation [61, 62]. During the peri-implantation period of pregnancy in sheep, integrin subunits {alpha}v, {alpha}4, {alpha}5, β1, β3, and β5 are constitutively expressed on conceptus trophectoderm and the apical surface of endometrial LE [63]. In goats, integrin subunits {alpha}v, {alpha}4, {alpha}5, β1, and β3 are expressed on conceptus trophectoderm and endometrial LE on Days 21 and 23 of pregnancy [64]. Thus, conceptus implantation in sheep and goats does not appear to involve changes in temporal or spatial patterns of integrin expression [63, 64], but appears to depend primarily on changes in expression of integrin ligands, such as LGALS15 and secreted phosphoprotein one (SPP1/osteopontin) [5, 65, 66]. Other galectins bind integrins, FN, and laminin, because these extracellular matrix proteins are modified with beta-galactoside sugars [22, 23]. Indeed, FN and vitronectin are also expressed on conceptus trophectoderm and endometrial LE on Days 21 and 23 of pregnancy in goats [64]. In the goat, close contact between the conceptus trophectoderm and endometrial LE occurs between Days 17 and 18, with firm adhesion developing between Days 19 and 23 [47, 67]. This time period coincides with rapid elongation of the goat blastocyst to form a filamentous conceptus [67]. Blastocyst elongation has not been achieved in vitro, suggesting that a factor present in the uterine lumen, perhaps a secreted protein like LGALS15 or SPP1, is required for blastocyst development into a filamentous conceptus. The idea that factors in uterine histotroph are required to promote blastocyst growth and elongation in ruminants is supported by studies of asynchronous uterine transfer of embryos and trophoblast vesicles [9, 10], progesterone regulation of blastocyst elongation [11–13], and failure of conceptus development in UGKO ewes [14, 15]. In fact, blastocyst elongation has been hypothesized to require transient attachment, detachment, and reattachment as the trophectoderm elongates from each side of the centrally located embryonic disc [4]. Thus, available evidence suggests that LGALS15 secreted by endometrial LE functions to promote blastocyst growth and elongation in sheep and goats by moderating adhesion of trophectoderm to endometrial LE via integrin binding.

As observed in sheep [17, 28], LGALS15 is detectable on the surface of the trophectoderm and within intracellular crystal structures of trophectoderm and the endometrial LE of the goat. LGALS10 was initially known as a CLC protein because it formed distinctive hexagonal bipyramidal crystals in eosinophils that accounted for nearly 10% of the total cellular protein [19, 68]. Further, LGALS15 was immunologically identical to the novel 14K progesterone-modulated protein from the sheep uterus associated with crystalline inclusion bodies in endometrial LE and conceptus trophectoderm [69]. Subsequent immunogold electron microscopy analysis revealed the 14K protein was localized to large, membrane-bound rhomboidal or needle-shaped crystal structures, but not in the endoplasmic reticulum and Golgi body. Therefore, Kazemi et al. [69] suggested that the protein was secreted by the endometrial epithelia and taken up by the conceptus from uterine histotroph. Indeed, needle-shaped crystalline structures have also been described in caprine endometrial epithelial and trophectoderm cells [67], and their development correlates with the synthesis and secretion of an unidentified 15-kDa protein with a pI of ~6.0 from explant cultures in response to the conceptus [70]. Interestingly, development of in vitro-produced bovine blastocysts transferred into sheep uteri resulted in the presence of crystals in trophectoderm cells [71, 72]. However, crystal-like structures were not observed in the trophectoderm of Day 15 or Day 19 bovine blastocysts produced by in vivo development in cattle [72]. Therefore, it is not surprising that the LGALS15 gene is expressed in endometria of ovine and caprine uteri, but not bovine uteri. The present results strongly indicate that LGALS15 is expressed by endometrial LE and sGE of the ovine and caprine uterus, secreted into the uterine lumen, and then adsorbed to the surface of or absorbed into conceptus trophectoderm where it forms crystals. Although the biological role(s) of LGALS15 crystals in the conceptus is not known, the intracellular roles of other galectins include modulation of cell growth, differentiation, and apoptosis through functioning as pre-mRNA splicing factors and interacting with specific intracellular ligands such as RAS and BCL2 [73, 74]. Similar to LGALS15, a number of galectins are present in the cytoplasm and nuclei of cells, including LGALS1, LGALS3, LGALS7, and LGALS12 [26].

Collectively, available data support the hypothesis that LGALS15 is uniquely expressed in uterine endometria of ruminants in the subfamily Caprinae (O. aries and C. hircus) and secreted into the uterine lumen where it functions as an attachment factor important for pre-implantation blastocyst growth, elongation, and attachment phase of implantation. Future work will focus on the extracellular and intracellular roles of LGALS15 in endometria and conceptuses of sheep and goats and will determine if other galectin family members are expressed in uteri of other mammals.

ACKNOWLEDGMENTS

The authors thank Dr. Thomas R. Hansen of Colorado State University for providing bovine uterine tissue.

FOOTNOTES

1Supported by the National Research Initiative Competitive Grant 2005-35203-16252 from the USDA Cooperative State Research, Education, and Extension Service and NIH Grant 5 P30 ES09106. Back

Correspondence: 2Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, Texas 77843-2471. FAX: 979 862 2662; e-mail: tspencer{at}tamu.edu

3These authors contributed equally to this work. Back

Received: 22 June 2007.

First decision: 22 July 2007.

Accepted: 17 August 2007.

REFERENCES

  1. Gray C, Bartol FF, Taylor KM, Wiley AA, Ramsey WS, Ott TL, Bazer FW, Spencer TE. Ovine uterine gland knock-out model: effects of gland ablation on the estrous cycle Biol Reprod 2000 62448–456[Abstract/Free Full Text]
  2. Ashworth CJ, Bazer FW. Changes in ovine conceptus and endometrial function following asynchronous embryo transfer or administration of progesterone Biol Reprod 1989 40425–433[Abstract]
  3. Geisert RD, Ross JW, Ashworth MD, White FJ, Johnson GA, DeSilva U. Maternal recognition of pregnancy signal or endocrine disruptor: the two faces of oestrogen during establishment of pregnancy in the pig Soc Reprod Fertil Suppl 2006 62131–145[Medline]
  4. Guillomot M. Cellular interactions during implantation in domestic ruminants J Reprod Fertil Suppl 1995 4939–51[Medline]
  5. Spencer TE, Johnson GA, Bazer FW, Burghardt RC. Implantation mechanisms: insights from the sheep Reproduction 2004 128657–668[Abstract/Free Full Text]
  6. Bazer FW. Mediators of maternal recognition of pregnancy in mammals Proc Soc Exp Biol Med 1992 199373–384[CrossRef][Medline]
  7. Thatcher WW, Meyer MD, Danet-Desnoyers G. Maternal recognition of pregnancy J Reprod Fertil Suppl 1995 4915–28[Medline]
  8. Maddox-Hyttell P, Gjorret JO, Vajta G, Alexopoulos NI, Lewis I, Trounson A, Viuff D, Laurincik J, Muller M, Tveden-Nyborg P, Thomsen PD. Morphological assessment of preimplantation embryo quality in cattle Reprod Suppl 2003 61103–116[Medline]
  9. Flechon JE, Guillomot M, Charlier M, Flechon B, Martal J. Experimental studies on the elongation of the ewe blastocyst Reprod Nutr Dev 1986 261017–1024[CrossRef][Medline]
  10. Lawson RA, Parr RA, Cahill LP. Evidence for maternal control of blastocyst growth after asynchronous transfer of embryos to the uterus of the ewe J Reprod Fertil 1983 67477–483[Abstract/Free Full Text]
  11. Satterfield MC, Bazer FW, Spencer TE. Progesterone regulation of preimplantation conceptus growth and galectin 15 (LGALS15) in the ovine uterus Biol Reprod 2006 75289–296[Abstract/Free Full Text]
  12. Geisert RD, Fox TC, Morgan GL, Wells ME, Wettemann RP, Zavy MT. Survival of bovine embryos transferred to progesterone-treated asynchronous recipients J Reprod Fertil 1991 92475–482[Abstract/Free Full Text]
  13. Garrett JE, Geisert RD, Zavy MT, Morgan GL. Evidence for maternal regulation of early conceptus growth and development in beef cattle J Reprod Fertil 1988 84437–446[Abstract/Free Full Text]
  14. Gray CA, Burghardt RC, Johnson GA, Bazer FW, Spencer TE. Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongation Reproduction 2002 124289–300[Abstract]
  15. Gray CA, Taylor KM, Ramsey WS, Hill JR, Bazer FW, Bartol FF, Spencer TE. Endometrial glands are required for preimplantation conceptus elongation and survival Biol Reprod 2001 641608–1613[Abstract/Free Full Text]
  16. Gray CA, Abbey CA, Beremand PD, Choi Y, Farmer JL, Adelson DL, Thomas TL, Bazer FW, Spencer TE. Identification of endometrial genes regulated by early pregnancy, progesterone, and interferon tau in the ovine uterus Biol Reprod 2006 74383–394[Abstract/Free Full Text]
  17. Gray CA, Adelson DL, Bazer FW, Burghardt RC, Meeusen EN, Spencer TE. Discovery and characterization of an epithelial-specific galectin in the endometrium that forms crystals in the trophectoderm Proc Natl Acad Sci U S A 2004 1017982–7987[Abstract/Free Full Text]
  18. Dunphy JL, Balic A, Barcham GJ, Horvath AJ, Nash AD, Meeusen EN. Isolation and characterization of a novel inducible mammalian galectin J Biol Chem 2000 27532106–32113[Abstract/Free Full Text]
  19. Weller PF, Bach DS, Austen KF. Biochemical characterization of human eosinophil Charcot-Leyden crystal protein (lysophospholipase) J Biol Chem 1984 25915100–15105[Abstract/Free Full Text]
  20. Dvorak AM. Human basophil recovery from secretion. A review emphasizing the distribution of Charcot-Leyden crystal protein in cells stained with the postfixation electron-dense tracer, cationized ferritin Histol Histopathol 1996 11711–728[Medline]
  21. Bohn H, Kraus W, Winckler W. Purification and characterization of two new soluble placental tissue proteins (PP13 and PP17) Oncodev Biol Med 1983 4343–350[Medline]
  22. Yang RY, Liu FT. Galectins in cell growth and apoptosis Cell Mol Life Sci 2003 60267–276[CrossRef][Medline]
  23. Cooper DN. Galectinomics: finding themes in complexity Biochim Biophys Acta 2002 1572209–231[Medline]
  24. Liu FT, Rabinovich GA. Galectins as modulators of tumour progression Nat Rev Cancer 2005 529–41[CrossRef][Medline]
  25. Hughes RC. Galectins as modulators of cell adhesion Biochimie 2001 83667–676[Medline]
  26. Wang JL, Gray RM, Haudek KC, Patterson RJ. Nucleocytoplasmic lectins Biochim Biophys Acta 2004 167375–93[Medline]
  27. Ruoslahti E. RGD and other recognition sequences for integrins Annu Rev Cell Dev Biol 1996 12697–715[CrossRef][Medline]
  28. Gray CA, Dunlap KA, Burghardt RC, Spencer TE. Galectin-15 in ovine uteroplacental tissues Reproduction 2005 130231–240[Abstract/Free Full Text]
  29. Spencer TE, Johnson GA, Bazer FW, Burghardt RC, Palmarini M. Pregnancy recognition and conceptus implantation in domestic ruminants: roles of progesterone, interferons and endogenous retroviruses Reprod Fertil Dev 2007 1965–78[CrossRef][Medline]
  30. Spencer TE, Stagg AG, Joyce MM, Jenster G, Wood CG, Bazer FW, Wiley AA, Bartol FF. Discovery and characterization of endometrial epithelial messenger ribonucleic acids using the ovine uterine gland knockout model Endocrinology 1999 1404070–4080[Abstract/Free Full Text]
  31. Stewart MD, Johnson GA, Gray CA, Burghardt RC, Schuler LA, Joyce MM, Bazer FW, Spencer TE. Prolactin receptor and uterine milk protein expression in the ovine endometrium during the estrous cycle and pregnancy Biol Reprod 2000 621779–1789[Abstract/Free Full Text]
  32. Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time and space complexity BMC Bioinformatics 2004 5113[CrossRef][Medline]
  33. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput Nucleic Acids Res 2004 321792–1797[Abstract/Free Full Text]
  34. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 1994 224673–4680[Abstract/Free Full Text]
  35. Page RD. TreeView: an application to display phylogenetic trees on personal computers Comput Appl Biosci 1996 12357–358[Free Full Text]
  36. Taylor KM, Gray CA, Joyce MM, Stewart MD, Bazer FW, Spencer TE. Neonatal ovine uterine development involves alterations in expression of receptors for estrogen, progesterone, and prolactin Biol Reprod 2000 631192–1204[Abstract/Free Full Text]
  37. Liaw L, Almeida M, Hart CE, Schwartz SM, Giachelli CM. Osteopontin promotes vascular cell adhesion and spreading and is chemotactic for smooth muscle cells in vitro Circ Res 1994 74214–224[Abstract/Free Full Text]
  38. Ochieng J, Leite-Browning ML, Warfield P. Regulation of cellular adhesion to extracellular matrix proteins by galectin-3 Biochem Biophys Res Commun 1998 246788–791[CrossRef][Medline]
  39. Dunlap KA, Palmarini M, Varela M, Burghardt RC, Hayashi K, Farmer JL, Spencer TE. Endogenous retroviruses regulate periimplantation placental growth and differentiation Proc Natl Acad Sci U S A 2006 10314390–14395[Abstract/Free Full Text]
  40. Raspotnig G, Fauler G, Jantscher A, Windischhofer W, Schachl K, Leis HJ. Colorimetric determination of cell numbers by Janus green staining Anal Biochem 1999 27574–83[CrossRef][Medline]
  41. Hirabayashi J, Hashidate T, Nishi N, Nakamura T, Hirashima M, Urashima T, Oka T, Futai M, Muller WE, Yagi F, Kasai K. How galectins have evolved oligosaccharide specificity Biochim Biophys Acta 2002 1572232–254[Medline]
  42. Oda Y, Herrmann J, Gitt MA, Turck CW, Burlingame AL, Barondes SH, Leffler H. Soluble lactose-binding lectin from rat intestine with two different carbohydrate-binding domains in the same peptide chain J Biol Chem 1993 2685929–5939[Abstract/Free Full Text]
  43. Hirabayashi J, Kasai K. Further evidence by site-directed mutagenesis that conserved hydrophilic residues form a carbohydrate-binding site of human galectin-1 Glycoconj J 1994 11437–442[CrossRef][Medline]
  44. Dyer KD, Rosenberg HF. Eosinophil Charcot-Leyden crystal protein binds to beta-galactoside sugars Life Sci 1996 582073–2082[CrossRef][Medline]
  45. Swaminathan GJ, Leonidas DD, Savage MP, Ackerman SJ, Acharya KR. Selective recognition of mannose by the human eosinophil Charcot-Leyden crystal protein (galectin-10): a crystallographic study at 1.8 A resolution Biochemistry 1999 3813837–13843[CrossRef][Medline]
  46. The UniProt Consortium. The Universal Protein Resource (UniProt) Nucleic Acids Res 2007 35D193–D197[CrossRef][Medline]
  47. Dent J. Ultrastructural changes in the intercotyledonary placenta of the goat during early pregnancy J Anat 1973 114245–259[Medline]
  48. Vanin EF. Processed pseudogenes: characteristics and evolution Annu Rev Genet 1985 19253–272[CrossRef][Medline]
  49. Than NG, Sumegi B, Than GN, Berente Z, Bohn H. Isolation and sequence analysis of a cDNA encoding human placental tissue protein 13 (PP13), a new lysophospholipase, homologue of human eosinophil Charcot-Leyden Crystal protein Placenta 1999 20703–710[CrossRef][Medline]
  50. Barondes SH, Cooper DN, Gitt MA, Leffler H. Galectins. Structure and function of a large family of animal lectins J Biol Chem 1994 26920807–20810[Free Full Text]
  51. Guillomot M, Flechon JE, Leroy F. Blastocyst development and implantation In: Thibault C, Levasseur MC, Hunter RHF (eds.), Reproduction in Mammals and Man Paris Ellipses 1993387–411
  52. Bazer FW, Vallet JL, Harney JP, Gross TS, Thatcher WW. Comparative aspects of maternal recognition of pregnancy between sheep and pigs J Reprod Fertil Suppl 1989 3785–89[Medline]
  53. Guillomot M, Reinaud P, La Bonnardiere C, Charpigny G. Characterization of conceptus-produced goat interferon tau and analysis of its temporal and cellular distribution during early pregnancy J Reprod Fertil 1998 112149–156[Abstract/Free Full Text]
  54. Gnatek GG, Smith LD, Duby RT, Godkin JD. Maternal recognition of pregnancy in the goat: effects of conceptus removal on interestrus intervals and characterization of conceptus protein production during early pregnancy Biol Reprod 1989 41655–663[Abstract]
  55. Song G, Spencer TE, Bazer FW. Cathepsins in the ovine uterus: regulation by pregnancy, progesterone, and interferon tau Endocrinology 2005 1464825–4833[CrossRef][Medline]
  56. Song G, Spencer TE, Bazer FW. Progesterone and interferon-tau regulate cystatin C in the endometrium Endocrinology 2006 1473478–3483[CrossRef][Medline]
  57. Spencer TE, Johnson GA, Burghardt RC, Bazer FW. Progesterone and placental hormone actions on the uterus: insights from domestic animals Biol Reprod 2004 712–10[Abstract/Free Full Text]
  58. Hirabayashi J, Hashidate T, Arata Y, Nishi N, Nakamura T, Hirashima M, Urashima T, Oka T, Futai M, Muller WE, Yagi F, Kasai K. Oligosaccharide specificity of galectins: a search by frontal affinity chromatography Biochim Biophys Acta 2002 1572232–254[Medline]
  59. Barondes SH, Castronovo V, Cooper DN, Cummings RD, Drickamer K, Feizi T, Gitt MA, Hirabayashi J, Hughes C, Kasai K, Leffler H, Liu FT, et al. Galectins: a family of animal beta-galactoside-binding lectins Cell 1994 76597–598[CrossRef][Medline]
  60. Cho M, Cummings RD. Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells. I. Physical and chemical characterization J Biol Chem 1995 2705198–5206[Abstract/Free Full Text]
  61. Aplin JD. Adhesion molecules in implantation Rev Reprod 1997 284–93[Abstract]
  62. Armant DR. Blastocysts don't go it alone. Extrinsic signals fine-tune the intrinsic developmental program of trophoblast cells Dev Biol 2005 280260–280[CrossRef][Medline]
  63. Johnson GA, Bazer FW, Jaeger LA, Ka H, Garlow JE, Pfarrer C, Spencer TE, Burghardt RC. Muc-1, integrin, and osteopontin expression during the implantation cascade in sheep Biol Reprod 2001 65820–828[Abstract/Free Full Text]
  64. Garcia P, Nieto A, Sanchez MA, Pizarro M, Flores JM. Expression of alphav, alpha4, alpha5 and beta3 integrin subunits, fibronectin and vitronectin in goat peri-implantation Anim Reprod Sci 2004 8091–100[CrossRef][Medline]
  65. Joyce MM, Gonzalez JF, Lewis S, Woldesenbet S, Burghardt RC, Newton GR, Johnson GA. Caprine uterine and placental osteopontin expression is distinct among epitheliochorial implanting species Placenta 2005 26160–170[CrossRef][Medline]
  66. Johnson GA, Burghardt RC, Bazer FW, Spencer TE. Osteopontin: roles in implantation and placentation Biol Reprod 2003 691458–1471[Abstract/Free Full Text]
  67. Wango EO, Wooding FB, Heap RB. The role of trophoblast binucleate cells in implantation in the goat: a quantitative study Placenta 1990 11381–394[CrossRef][Medline]
  68. Ackerman SJ, Loegering DA, Gleich GJ. The human eosinophil Charcot-Leyden crystal protein: biochemical characteristics and measurement by radioimmunoassay J Immunol 1980 1252118–2126[Medline]
  69. Kazemi M, Amann JF, Keisler DH, Ing NH, Roberts RM, Morgan G, Wooding FB. A progesterone-modulated, low-molecular-weight protein from the uterus of the sheep is associated with crystalline inclusion bodies in uterine epithelium and embryonic trophectoderm Biol Reprod 1990 4380–96[Abstract]
  70. Weise DW, Newton GR, Emesih GC. Effects of day of the estrous cycle or pregnancy on protein secretion by caprine endometrial tissues Biol Reprod 1993 49522–527[Abstract]
  71. Rexroad CE Jr, Powell AM. The ovine uterus as a host for in vitro-produced bovine embryos Theriogenology 1999 52351–364[CrossRef][Medline]
  72. Talbot NC, Powell A, Garrett W, Edwards JL, Rexroad C Jr. Ultrastructural and karyotypic examination of in vitro produced bovine embryos developed in the sheep uterus Tissue Cell 2000 329–27[CrossRef][Medline]
  73. Hernandez JD, Baum LG. Ah, sweet mystery of death! Galectins and control of cell fate Glycobiology 2002 12127R–136R[Abstract/Free Full Text]
  74. Liu FT, Patterson RJ, Wang JL. Intracellular functions of galectins Biochim Biophys Acta 2002 1572263–273[Medline]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
N. G. Than, R. Romero, M. Goodman, A. Weckle, J. Xing, Z. Dong, Y. Xu, F. Tarquini, A. Szilagyi, P. Gal, et al.
A primate subfamily of galectins expressed at the maternal-fetal interface that promote immune cell death
PNAS, June 16, 2009; 106(24): 9731 - 9736.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
R. C Burghardt, J. R Burghardt, J. D Taylor II, A. T Reeder, B. T Nguen, T. E Spencer, K. J Bayless, and G. A Johnson
Enhanced focal adhesion assembly reflects increased mechanosensation and mechanotransduction at maternal-conceptus interface and uterine wall during ovine pregnancy
Reproduction, March 1, 2009; 137(3): 567 - 582.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
T. E Spencer, O. Sandra, and E. Wolf
Genes involved in conceptus-endometrial interactions in ruminants: insights from reductionism and thoughts on holistic approaches
Reproduction, February 1, 2008; 135(2): 165 - 179.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
77/6/1027    most recent
biolreprod.107.063594v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lewis, S. K
Right arrow Articles by Spencer, T. E
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lewis, S. K
Right arrow Articles by Spencer, T. E
Agricola
Right arrow Articles by Lewis, S. K
Right arrow Articles by Spencer, T. E

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS