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,1
* Center for Animal Biotechnology and Genomics and Department of Animal Science;
Image Analysis Laboratory and Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas, USA; and
Department of Animal Sciences, University of Florida, Gainesville, Florida, USA
2 Correspondence: Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, TX 77843-2471, USA. E-mail: tspencer{at}tamu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: trophoblast • apoptosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. In sheep, morula-stage embryos enter the uterus on day 4 and then form a blastocyst by day 6 that contains a blastocoele or central cavity surrounded by a monolayer of trophectoderm (3
, 4)
. After hatching from the zona pellucida on day 8, the blastocyst develops into a tubular form by day 11 and then elongates between days 12 and 16 to form a filamentous conceptus of 10 cm or more in length. Blastocyst growth and elongation is crucial for pregnancy recognition signaling, which involves synthesis and secretion of interferon tau (IFNT) from mononuclear trophectoderm cells of elongating blastocyst that inhibits luteolysis (5
, 6)
. The factors supporting growth of peri-implantation blastocysts and elongating conceptuses are thought to be derived primarily from secretions of the uterus, collectively referred to as histotroph (7
, 8)
. This hypothesis is supported by failure of conceptus development in the uterine gland knockout ewe model (9
, 10)
.
The uterine gland knockout ewe experiences recurrent early pregnancy loss between days 12 and 14 that is most likely due to inadequate histotroph from the endometrial lumenal (LE) and glandular (GE) epithelia (9
, 10)
. To understand the peri-implantation pregnancy defect, the UGKO ewe model was used in a gene expression profiling project based on an endometrial cDNA library from uteri of day 14 pregnant ewes (11
, 12)
. Interestingly, 
1.4% of the expression sequence tags sequenced from the cDNA library was highly similar to OVGAL11, a novel member of the galectin family of secreted animal lectins (13)
. OVGAL11 was originally shown to be induced in gastrointestinal tissue and secreted into the intestinal lumen in response to inflammation and eosinophil infiltration after infection of sheep with the helminth, Hemonchus contortus (13)
. The sequence of OVGAL11 protein displayed highest similarity to human LGALS10 (also known as Charcot-Leyden Crystal protein; refs. 14
, 15
) and human LGALS13 (also known as placental tissue protein 13 or PP13; ref. 16
). Since OVGAL11 does not have a known orthologue, it was designated a new member of the galectin superfamily and renamed galectin 15 (LGALS15). Galectins are proteins with a conserved carbohydrate recognition domain (CRD) that bind beta-galactoside sugars, thereby cross-linking glycoproteins as well as glycolipid receptors on the surface of cells and initiating biological responses (17
18
19)
. Functional studies of the extracellular and intracellular roles of galectins implicate them in cell adhesion, chemoattraction, and migration as well as cell growth, differentiation, and apoptosis (20
, 21)
. All of those biological roles are important for peri-implantation blastocyst growth and differentiation (3
, 4)
.
In the ovine uterus, LGALS15 mRNA is detected only after day 10 of pregnancy in the endometrial LE and superficial ductal GE (SGE), and LGALS15 is induced by ovarian progesterone and further stimulated by conceptus IFNT (12
, 22)
. In the endometrium, LGALS15 protein has a nucleocytoplasmic distribution within the LE and sGE and is also concentrated near and on the apical surface. Secreted LGALS15 protein is abundant in histotroph recovered from the uterine lumen where it exists in multimeric forms and is localized to the apical surface of conceptus trophectoderm and within intracellular crystals (12
, 23)
. The ovine LGALS15 protein contains predicted CRD, LDV, and RGD recognition sequences, which can bind and activate integrins (24)
. The temporal and spatial alterations in LGALS15 mRNA and protein in the uterine endometrial epithelia and lumen during the peri-implantation period of early pregnancy, combined with the known biological activities of other galectins, make it a candidate mediator of conceptus-endometrial interactions during implantation (12
, 23)
. Recently, advanced growth and development of blastocysts in response to early progesterone treatment of ewes was associated with induction of LGALS15 in the endometrial epithelia (22)
.
The working hypothesis for the present study was that LGALS15, synthesized and secreted by endometrial LE and sGE into the uterine lumen, functionally binds and crosslinks beta-galactosides on glycoproteins and glycolipids using the CRD and integrins through the LDV and RGD recognition sequences to allow LGALS15 to function as a heterotypic adhesion molecule bridging conceptus trophectoderm and endometrial LE, as well as stimulates migration and proliferation of trophectoderm that are critical for successful blastocyst elongation and conceptus implantation (4
, 25)
. Experiments to test this working hypothesis, using recombinant ovine LGALS15, bovine blastocysts, and ovine mononuclear trophectoderm cells in functional assays, indicated that LGALS15 primarily stimulates trophectoderm migration and attachment and has secondary effects on proliferation and inhibition of apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 12)
was used to produce recombinant ovine LGALS15 in a bacterial expression system to be described later. Polymerase chain reactions (PCR; 50 µl) were conducted in Optimized Buffer F (Invitrogen, Carlsbad, CA, USA) that contained 10 ng ovine LGALS15 cDNA from day 14 pregnant endometrium (11)
, 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 ExTaq polymerase (Takara Bio, Beijing, Japan, USA) using an Eppendorf Mastercycler thermocycler with conditions of: 95°C for 2 min; 95°C for 30 s, 54°C for 1 min, and 72°C for 1 min for 35 cycles; and 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, USA). This cloning strategy mutated the stop codon of LGALS15 and placed a His · Tag sequence at the C terminus for affinity purification (see Fig. 1
). The resulting plasmid was sequenced to ensure that no mutations were present in the LGALS15 sequence.
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Mutation of the LDVRGD recognition sequence in LGALS15 to LAVRAD was achieved using PCR amplification of the ovine endometrial LGALS15 cDNA described previously but with a different reverse primer (5'-CAG CAC GAT ATC TGC CCT CAC AGC CAG CAT TT-3') and the following modifications. PCR reactions were conducted with VENT polymerase buffer (New England BioLabs, Beverly, MA, USA) and 1 U VENT polymerase with conditions of: 95°C for 2 min; 95°C for 30 s, 60°C for 1 min, and 72°C for 1 min for 35 cycles; and 72°C for 7 min. The amplified LGALS15 cDNA was restricted with NcoI and EcoRV enzymes and then directionally subcloned into the pET-28b(+) vector (Novagen). The resulting plasmid was sequenced to ensure that the targeted mutations translated into a LAVRAD mutant of the LDVRGD sequences in the C terminus of wild-type LGALS15 (see Fig. 1
).
Mutation of the predicted CRD in ovine endometrial LGALS15 was achieved by using two sets of nested internal primers for PCR amplification. Set 1 mutated the first half of the CRD (forward 5'-CCA TTC GCT TTC GCG TAC GCC GAT GGC ATC GTG GCT ATG GCC ACT TTA AAG-3' and reverse 5'-CTT TAA AGT GGC CAT AGC CAC GAT GCC ATC GGC GTA CGC GAA AGC GAA TGG-3'). Set 2 mutated the second half of the CRD (forward 5'-GGG AGT GCG GGG AAG GCA CAG GCA CTG CAT ACT GAG GC-3' and reverse 5'-GCC TCA GTA TGC AGT GCC TGT GCC TTC CCC GCA CTC CC-3'). PCR reactions were conducted as described previously to generate the LGALS15 LAVRAD mutant. Partial cDNAs were gel purified and then used in a PCR reaction with primers to amplify the full coding sequence. This cloning strategy mutated the predicted amino acids in the CRD to alanine in LGALS15 and placed a His · Tag sequence at the C terminus (see Fig. 1
). The insert of the resulting plasmid was sequenced to ensure that targeted mutations were present in the CRD of the LGALS15 sequence.
Wild-type and mutant forms of ovine endometrial LGALS15 protein were produced in BL21 bacteria according to the manufacturer’s suggestions. Expression of LGALS15 fusion protein in bacteria was induced with 5 mM isopropyl-beta-D-thiogalactopyranoside (IPTG, Sigma, St. Louis, MO, USA). Bacteria were lysed with Bugbuster (Invitrogen) supplemented with recombinant lysozyme and benzonase (Invitrogen). 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 blotting using a rabbit anti-ovine LGALS15 antibody (12)
. Fractions containing recombinant LGALS15 were dialyzed overnight in PBS (pH 7.2) at 4°C, concentrated using a spin column with a 3,500 MWCO (Vivaspin, Stonehouse, UK), and frozen in aliquots at –80°C. Protein concentration was determined using a RC/DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin (BSA) as the standard.
In vitro blastocyst development assays
The HEPES-Tyrodes Lactate (TL), IVF-TL, and Sperm-TL media were purchased from Cell and Molecular Technologies Inc. (Lavallete, NJ, USA) or Caisson Laboratories (Sugar City, ID, USA) and used to prepare HEPES-Tyrodes albumin lactate pyruvate (TALP), IVF-TALP, and Sperm-TALP as described previously (26)
. Oocyte collection medium (OCM) was Tissue Culture Medium-199 (TCM-199) with Hank’s salts without phenol red (Atlanta Biologicals, Norcross, GA, USA) supplemented with 2% (v/v) bovine steer serum (Pel-Freez, Rogers, AR, USA) containing 2 U/ml heparin, 100 U/ml penicillin-G, 0.1 mg/ml streptomycin, and 1 mM glutamine. Oocyte maturation medium (OMM) was TCM-199 (Bio-Whittaker, Walkersville, MD, USA) with Earle’s salts supplemented with 10% (v/v) bovine steer serum, 2 µg/ml estradiol 17-β, 20 µg/ml bovine FSH (Folltropin-V; Vetrepharm Canada, London, ON), 22 µg/ml sodium pyruvate, 50 µg/ml gentamicin sulfate, and 1 mM glutamine. Percoll was from Amersham Pharmacia Biotech (Uppsala, Sweden). Frozen semen from various bulls was donated by Southeastern Semen Services (Wellborn, FL, USA). Potassium simplex optimized medium (KSOM) containing 1 mg/ml BSA was obtained from Caisson Laboratories (North Logan, UT, USA). Essentially fatty-acid free (EFAF) BSA was from Sigma. On the day of use, KSOM was modified for bovine embryos to produce KSOM-BE2 as described elsewhere (27)
.
In vitro embryo production was performed as described previously (28
, 29)
except for the culture medium described. Briefly, cumulus oocyte complexes (COCs) were obtained by slicing 2- to 10-mm follicles on the surface of ovaries (a mixture of beef and dairy cattle) obtained from Central Beef Packing (Center Hill, FL, USA). Those COCs with at least one complete layer of compact cumulus cells were washed two times in OCM and used for subsequent steps. Groups of 10 COCs were placed in 50 µl drops of OMM overlaid with mineral oil and matured for 20–22 h at 38.5°C in an atmosphere of 5% (v/v) CO2 in humidified air. Matured COCs were then washed once in HEPES-TALP and transferred in groups of 30 to 4-well plates containing 600 µl IVF-TALP and 25 µl PHE [0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 µM epinephrine in 0.9% (w/v) NaCl] per well and fertilized with 1 x 106 Percoll-purified spermatozoa from a pool of frozen-thawed semen from three bulls. After 20–22 h at 38.5°C in an atmosphere of 5% (v/v) CO2 in humidified air, putative zygotes were removed from fertilization wells, denuded of cumulus cells by vortex mixing in 1 ml of 1000 U/ml hyaluronidase in HEPES-TALP, and placed in groups of 30 in 50-µl drops of KSOM-BE2.
Four replicates of this experiment were conducted using a total of 363 putative zygotes. Approximately one-third of the putative zygotes were cultured in KSOM-BE2 containing 100 ng/ml recombinant LGALS15 for the entire culture period, one-third of the zygotes were cultured in KSOM-BE2 containing 1 µg/ml LGALS15, and the final third of zygotes were cultured in an equivalent amount of PBS with no LGALS15. All drops of culture medium containing embryos were overlaid with mineral oil and cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 (v/v) in humidified air. Rates of embryo cleavage and blastocyst formation by putative zygotes were assessed on day 3 and day 8 after fertilization, respectively.
Isolation and culture of mononuclear ovine trophectoderm (oTr) cells
All animal experiments were approved by the Institutional Animal Care and Use Committee of Texas A&M University. Mature Suffolk-type ewes (Ovis aries) were observed for estrus (designated as day 0) in the presence of a vasectomized ram and used in experiments only after exhibiting at least two estrous cycles of normal duration (16–18 days). At estrus, ewes were mated to intact rams. As described previously (30
, 31)
, elongating conceptuses were recovered on day 15 postmating by flushing the uterine lumen with 20 ml sterile PBS (pH 7.2) supplemented with 100 U penicillin and 100 µg streptomycin per liter. The inner cell mass was removed from conceptuses, and the remaining tissues were carefully minced, pooled, and placed in trophoblast growth medium [DMEM/F-12 supplemented with 10% fetal bovine serum, glutamine (2 mM), insulin (700 nM), pyruvate (1.0 mM), nonessential amino acids (0.1 mM), and antibiotics (50 U penicillin, 50 µg streptomycin)] and maintained in a 5% CO2 environment at 37°C. Two different ovine trophectoderm cell lines were established and designated oTr1 and oTrF. The oTr1 cell line was established in tissue-culture-treated plastic dishes, whereas the oTrF cell line was established on collagen-coated plastic dishes (Cohesion, Palo Alto, CA, USA). Fluid-filled trophoblastic vesicles, which spontaneously developed in culture, were physically ruptured with a sterile 28-gauge needle to enhance generation of a cellular monolayer. This primary culture was propagated on the same support after serial trypsinizations and cell passage.
Migration assay
Migration assays were conducted with both oTr1 and oTrF cells, hereafter referred to as oTr cells, as described previously (31)
with minor modifications. Briefly, oTr cells (50,000 cells per 100 µl serum and insulin-free DMEM) were seeded on 8 µm pore Transwell inserts (Corning Costar #3422, Corning, NY, USA). Treatments were then added to each well (n=3 wells per treatment) that included combinations of the following: 1) serum and insulin-free DMEM medium (600 µl); 2) recombinant ovine LGALS15 at 100 ng, 1 µg, or 10 µg; 3) 1 µg/ml recombinant ovine LGALS15 with 10, 50 or 100 µM 264 Y27632 [Rho-kinase inhibitor (ROCK) inhibitor, Calbiochem, San Diego, CA, USA]; 4) 1 µg/ml recombinant ovine LGALS15 with 10, 50, or 100 µM cell permeable c-Jun N-terminal kinase (JNK) inhibitor (JNKI1, Alexis, San Diego, CA, USA); or 5) trophoblast growth medium including serum and insulin as a positive control. After 12 h, cells on the upper side of the inserts were removed with a cotton swab. For evaluation of cells that migrated onto the lower surface, inserts were fixed in 50% ethanol for 5 min. The Transwell membranes were then removed, placed on a glass slide with the side containing cells facing up, overlaid with Prolong antifade mounting reagent with DAPI, and overlaid with a coverslip (Invitrogen-Molecular Probes, Eugene, OR, USA). The migrated cells were systematically counted using a Zeiss Axioplan 2 fluorescence microscope with Axiocam HR digital camera and Axiovision 4.3 software (Carl Zeiss Microimaging, Thornwood, NY, USA). The entire experiment was repeated at least three times with different passages of oTr cells.
Proliferation assay
Trophectoderm proliferation assays were conducted as described previously (30)
with minor modifications. Briefly, oTr cells were subcultured into 12-well plates (Corning Costar #3513) to 50% confluency in trophoblast growth medium for 6 to 8 h and then switched to serum and insulin-free DMEM for 24 h. After 24 h, the wells (n=4 per treatment) were treated with either increasing amounts of recombinant LGALS15 (10 ng, 100 ng, 1 µg, or 10 µg) in serum and insulin-free DMEM, trophoblast growth medium containing serum and insulin as a positive control, or DMEM alone as a negative control. After 48 h of culture, cell numbers were determined as described previously (32)
. Briefly, DMEM was removed from cells by vacuum aspiration and cells were fixed in 50% (v/v) ethanol for 30 min followed by vacuum aspiration of the fixative. Fixed cells were stained with Janus Green B in PBS [0.2% (w/v)] for 3 min at room temperature. The stain was removed using a vacuum aspirator, and the whole plate was sequentially dipped into water and destained by gentle shaking. The remaining water was removed by shaking, and stained cells were immediately lysed in 0.5 N HCl and absorbance readings taken at 595 nm using a microplate reader. As described previously (32)
, cell numbers were calculated from absorbance readings using the formula [cell number=(absorbance-0.00462)/0.00006926]. The entire experiment was repeated at least three times with different passages of oTr cells.
Apoptosis assays
Four-well chamber slides (Nunc, Rochester, NY, USA) were seeded in triplicate with oTr cells at 60% confluency in serum and insulin-free oTr medium and incubated for 24 h. Fresh medium containing recombinant ovine LGALS15 (100 ng, 1 µg, or 10 µg) was added to each well and cultured for another 24 h. After 24 h, the medium was removed and fresh medium containing staurosporine and LGALS15 was added to each well (LC Laboratories, Woburn, MA, USA) and cultured for another 24 h. Cells were then fixed in 4% (w/v) paraformaldehyde in PBS and analyzed for apoptosis using In Situ Cell Death Detection Kit (Roche, Nutley, NJ, USA). Apoptotic nuclei were quantified using a Zeiss Axioplan 2 fluorescence microscope with Axiocam HR digital camera and Axiovision 4.3 software (Carl Zeiss Microimaging, Thornwood, NY, USA). The entire experiment was repeated at least three times with different passages of oTr cells.
Attachment assay
Attachment assays were adapted from published procedures (33
, 34)
. Cell suspension plates with 24 wells (Greiner Multiwell Tissue Culture Plates, PGC Scientific Co, Monroe, NC, USA) were coated with either BSA (Bovine Serum Albumin Fraction V, Pierce, Rockford, IL, USA) as a negative control, bovine FN [fibronectin 0.1% (w/v) solution from bovine plasma] as a positive control, or recombinant ovine LGALS15 protein (wild type or mutants) in triplicate and allowed to dry overnight in a sterile hood at room temperature. Wells were then blocked with 1 ml BSA (10 mg/ml) in PBS for 1 h and rinsed three times with 1 ml per well serum and insulin-free DMEM. Equal numbers of freshly trypsinized oTr cells were seeded into each well, and plates were incubated for 1.5 h. In some experiments, a cyclic blocking peptide (GRDGS; Peptides International, Inc., Louisville, KY, USA) or the cyclic control peptide (GRADS) was added to the wells. Wells were washed three times with 1 ml serum- and insulin-free medium to remove unattached cells. Cell numbers were determined using a Janus Green assay as described above for cell proliferation assays. The entire experiment was repeated at least three times with different passages of oTr cells.
Focal adhesion formation assays
The oTr cells were seeded into four-well Lab-Tek glass chamber slides (Nunc), which were coated with either recombinant LGALS15 (1, 5, 10, or 20 µg per well), bovine FN as a positive control, and poly-L-lysine or nothing as negative controls. After 1.5 h, cells were fixed in cold methanol (–20°C) for 10 min and air dried. Fixed cells were rehydrated at room temperature with 0.3% (v/v) Tween 20 in 0.02 M PBS (rinse solution), blocked in antibody dilution buffer [2 parts 0.02 M PBS, 1.0% (w/v) BSA, 0.3% (v/v) Tween 20 (pH 8.0) and one part glycerol] containing 5% normal goat serum (v/v) for 1 h at room temperature, and incubated overnight at 4°C with a mouse monoclonal anti-talin antibody (1:1000) or mouse serum (1:1000) (Sigma T3287 Clone 8d4). Immunoreactive protein was then detected using an Alexa Fluor 488-conjugated secondary antibody for 1 h at room temperature. Slides were overlaid with Prolong antifade mounting reagent with DAPI (Invitrogen-Molecular Probes) and affixed with a coverslip. The entire experiment was repeated at least three times with different passages of oTr cells.
Statistical analyses
All quantitative data were subjected to least-squares ANOVA using the General Linear Models (GLM) procedures of the Statistical Analysis System (SAS Institute, Cary, NC, USA). Tests of significance were performed using the appropriate error terms according to the expectation of the mean squares for error. Orthogonal contrasts were used to elucidate effects of treatment. Data on rates of embryo cleavage and blastocyst formation were calculated for each replicate. Percentage data were transformed by arcsin transformation before analysis. Independent variables included LGALS15 treatments and replicate. Probability values for percentage data are based on analysis of arcsin-transformed data, while least-squares means are from analysis of untransformed data. A P value of 0.05 or less was considered significant. Data are presented as least-square means (LSM) with SE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Therefore, the objective of this study was to determine effects of LGALS15 on in vitro preimplantation growth and development of bovine embryos. Bovine embryos were produced in vitro by standard conditions, and putative zygotes were cultured in the presence of increasing amounts of recombinant ovine LGALS15 protein (0, 100, or 1000 ng/ml). Rates of embryo cleavage on day 3 (82.5±1.7%) and blastocyst formation on day 8 (39.8±4.1%) were not affected (P>0.10) by treatment of zygotes with LGALS15.
LGALS15 has moderate effects on trophectoderm proliferation
Ovine trophectoderm (oTr1 and oTrF) cell lines isolated from day 15 conceptuses were predominantly mononuclear and expressed IFNT by RT-PCR (data not shown). Several members of the galectin family affect proliferation of cells (17)
, and proliferation of trophectoderm cells is involved in elongation and differentiation of peri-implantation ruminant conceptuses (3)
. This study determined effects of LGALS15 on oTr1 and oTrF cell proliferation in serum- and insulin-free media (Fig. 2
). Recombinant ovine LGALS15 did not affect (P>0.10) proliferation of oTr1 cells, but did stimulate oTrF cell proliferation. A 24% increase in oTrF cell numbers was detected at 100 ng LGALS15, but not at other amounts (cubic effect, P<0.03). In both types of oTr cells, BSA did not affect (P>0.10) cell number, whereas there was a 215 and 368% increase (P<0.01) in oTr1 and oTrF cell numbers, respectively, in response to serum- and insulin-containing trophoblast growth medium (data not shown). These results suggest that LGALS15 moderately increases trophectoderm cell proliferation in a dose- and cell-dependent manner.
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LGALS15 does not induce apoptosis but reduces staurosporine-induced apoptosis in trophectoderm cells
Several members of the galectin family have either negative or positive effects on cell apoptosis (17)
. This study examined effects of LGALS15 on apoptosis in oTr1 cells and determined that LGALS15 alone had no effect (P>0.10) on oTr1 cell apoptosis regardless of dose (data not shown). Next, staurosporine (Streptomyces staurospores), a relatively nonselective protein kinase inhibitor, was used to induce apoptosis (35)
. Staurosporine induced apoptosis of oTr1 cells in a dose-dependent manner (Fig. 3
). Almost 100% of oTr1 cells were apoptotic at 3 x 10–4 M and 85% of oTr cells were apoptotic at 3 x 10–5 M, whereas only 60% were apoptotic at 3 x 10–6 M staurosporine. At high levels of staurosporine-induced apoptosis, preincubation of oTr1 cells with recombinant ovine LGALS15 had no effect (P>0.10). In contrast, 10 µg LGALS15 decreased (P<0.03) the level of apoptosis by 30% in oTr1 cells incubated with 3 x 10–6 M staurosporine. Lower amounts of LGALS15 (100 ng or 1 µg) had no effect (P>0.10) on staurosporine-induced apoptosis. These results suggest that LGALS15 alone does not cause apoptosis but that LGALS15 can inhibit trophectoderm cell apoptosis induced by staurosporine in a dose-dependent manner.
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LGALS15 increases migration of trophectoderm cells via JNK
Several members of the galectin family stimulate migration of cells (17)
, and trophectoderm migration occurs during elongation of ruminant conceptuses (3)
. Therefore, effects of LGALS15 on migration of oTr1 and oTrf cells were determined. As illustrated in Fig. 4
A, B, recombinant ovine LGALS15 dose dependently increased (P<0.001) migration of both oTr1 and oTrF cells in serum- and insulin-free media. Cell movement and migration can be stimulated by the planar cell polarity pathway involving activation of Rho-ROCK and JNK-JUN pathways (36)
. Treatment of oTr cells with a ROCK (Rho-kinase) inhibitor, and JNK (JUN N-terminal kinase) inhibitor did not (P>0.10) affect basal rates of oTr1 or oTrF cell migration in the absence of LGALS15 (data not shown). However, the JNK inhibitor, but not the ROCK inhibitor, reduced (P<0.001) LGALS15-stimulated oTr1 and oTrF cell migration in a dose-dependent manner (Fig. 4)
. These results support the hypothesis that LGALS15 from the endometrium acts in a paracrine manner on ovine conceptus to stimulate trophectoderm cell migration and movement via activation of a signaling pathway involving JNK.
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LGALS15 mediates attachment of ovine trophectoderm cells that is RGD dependent
Several members of the galectin family are involved in heterotypic cell adhesion mediated by CRD binding of beta-galactosides on glycoproteins that include integrins (20
, 34)
. In addition to a predicted CRD, LGALS15 also contains predicted LDV and RGD integrin recognition sequences in the C terminus (Fig. 1)
. The RGD sequence is the cell attachment site present in many adhesive extracellular matrix (ECM), blood, and cell surface proteins and nearly one-half of over 20 known integrins recognize this sequence in their adhesion protein ligands (24)
. Integrins are heterodimeric transmembrane cell surface receptors that mediate adhesion between cells and the ECM by binding to ligands with an exposed RGD sequence. These receptors also stimulate intracellular signaling and gene expression involved in cell growth, migration, and survival. Integrin binding and activation is an essential element of conceptus-endometrial interactions, blastocyst implantation, and trophoblast differentiation in many species (37
, 38)
. Therefore, a series of studies were conducted to test the hypothesis that LGALS15 affects attachment functions of ovine trophectoderm cells.
Recombinant ovine LGALS15 mediated attachment of oTr1 and oTrF cells in a dose-dependent manner (Figs. 5
A, B). Relative to wells coated with BSA, there was an increase (P<0.01) in oTr1 and oTrF cell attachment in wells of nonadherent suspension plates coated with either 100 ng, 1 µg, or 10 µg recombinant ovine LGALS15. Moreover, the attachment function of LGALS15 was similar to that for bFN (bovine FN). These results indicate that LGALS15 contains an intrinsic attachment function and supports the hypothesis that it mediates heterotypic interactions between the conceptus trophectoderm and endometrial LE.
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The integrin-binding activity of adhesion proteins can be reproduced by short synthetic peptides containing the RGD sequence. Such peptides promote cell adhesion when bound to the cell surface but inhibit adhesion when presented to cells in solution (24)
. As illustrated in Fig. 5C, D
, inclusion of a cyclic GRGDS peptide inhibited (P<0.01) LGALS15-mediated oTr cell attachment, whereas the control peptide (GRADS), which contains the conservative substitution of alanine for glycine, had no detectable inhibitory activity. Thus, trophectoderm cells adhere to LGALS15 via receptors, such as integrins, that recognize a RGD sequence.
In contrast to other galectin family members, ovine LGALS15 contains predicted LDV and RGD sequences near the C terminus (12)
that serve as recognition sites for binding to integrins and other cell adhesion proteins such as SPP1 (secreted phosphoprotein 1 or osteopontin; refs. 24
, 39
). Therefore, the LDV and RGD recognition sequences in ovine LGALS15 were mutated to LAV and RAD using a PCR-based mutagenesis strategy, and the recombinant protein was used in oTr cell attachment assays. Wild-type LGALS15 increased attachment of oTr1 and oTrF cells in a dose-dependent manner (Fig. 6
A, B). There were no differences in attachment function of LGALS15 and the LGALS15 LAVRAD mutant in wells precoated with either 100 ng or 1 µg of protein, but there was a decrease (P<0.01) in oTr cell attachment in wells precoated with 10 µg LGALS15 LAVRAD mutant compared to 10 µg wild-type LGALS15. Collectively, results support the hypothesis that cell attachment function of LGALS15 is not entirely dependent on the RGD sequence in the C terminus and that another integrin recognition sequence may exist within the protein that recognizes a sequence similar to the RGD recognition sequence.
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Other galectin family members, which all lack a RGD recognition sequence, can modulate cell adhesion by binding to integrins via their CRD (20)
. Ovine LGALS15 has a predicted CRD based on information from studies of other galectins (12
, 40)
. The CRD is a consensus motif that consists of 13 amino acids (41)
, of which 8 (H.N.R.V.N.W.E.R) play a critical role in sugar binding (42
, 43)
. As compared to the conserved CRD of other galectins depicted in Fig. 1
, ovine endometrial LGALS15 has four residues that are identical (V62, N64, W71, E74) and three that are conservatively substituted (R54, W56, K76). The C57 in ovine LGALS15 is different from prototypical galectins but appears to allow binding of mannose in LGALS10 (44)
. To determine if the CRD of ovine LGALS15 plays a role in cell attachment function, each of the eight predicted residues forming the putative CRD were mutated to alanine using a PCR-based mutagenesis strategy, and the recombinant protein was used in oTr cell attachment assays. Compared to equivalent doses of native LGALS15, oTr cell attachment was greater (P<0.05) in wells precoated with 100 ng of the LGALS15 CRD mutant but less (P<0.05) in wells precoated with 10 µg of the LGALS15 CRD mutant (Fig. 7
A, B). To test whether the attachment function of the LGALS15 CRD mutant was dependent on integrin binding, competitive inhibition assays were conducted with a cyclic GRGDS peptide. As depicted in Fig. 7C, D
, the GRGDS peptide inhibited (P<0.01) the ability of both the wild-type LGALS15 and the LGALS15 CRD mutant to mediate oTr cell attachment, whereas the control peptide (GRADS) was not (P>0.10) inhibitory to cell attachment. These results implicate integrin binding via the RGD recognition sequence in the attachment function of LGALS15 for adhesion of trophectoderm cells and suggest that the sugar binding activity of LGALS15 CRD is not a primary determinant of its cell attachment function.
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LGALS15-mediated trophectoderm cell attachment involves formation of focal adhesions
Results of the present studies indicated that LGALS15 possesses an intrinsic cell attachment function involving RGD-dependent binding of integrins on the trophectoderm. Activation of integrins in the trophectoderm by cell adhesion molecules with a RGD integrin recognition sequence, such as SPP1 and FN, elicits formation of focal adhesions (45)
. As shown in Fig. 8
A, there was an increase in oTr1 cell attachment in glass slides coated with recombinant ovine LGALS15, whereas no difference in oTr1 cell attachment occurred between glass slides precoated with nothing or BSA as a control. Further, there was an increase in oTr1 cell attachment and spreading on slides precoated with 10 µg compared to 1 µg LGALS15.
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Next, the formation of focal adhesions was studied by visualizing talin, a focal adhesion protein that aggregrates in response to integrin binding and activation that is essential for the stable linkage of aggregating integrins to the actin cytoskeleton, the organization of actin and the contractile apparatus, and integrin signaling (46)
. Focal adhesions, visualized by punctate aggregates of talin protein, were observed in oTr1 cells that attached to glass slides precoated with LGALS15 or bovine FN in a dose-dependent manner (Fig. 8B
). Although some oTr1 cells attached on glass slides precoated with the LGALS15 LADRAD mutant, they exhibited reduced spreading on the substrate and very few focal adhesions. Focal adhesions were not observed for attached oTr1 cells on glass slides that were uncoated or coated with poly-L-lysine as negative controls. The accumulation of talin indicates functional integrin activation as well as cytoskeletal reorganization in response to attachment of oTr1 cells to LGALS15 and recognition of the RGD sequence.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In the present study, LGALS15 did not affect development of bovine embryos in vitro with respect to rates of cleavage or blastocyst formation. Further, effects of LGALS15 on proliferation of mononuclear ovine trophectoderm cells were moderate and oTr cell line-dependent, and LGALS15 alone did not inhibit apoptosis of these cells. These results suggest that LGALS15 would not be particularly effective to increase in vitro rates of development in cleavage-stage embryos or be useful for improving yield of transferable embryos from in vitro ruminant embryo production systems. Thus, the increase in blastocyst size observed in early progesterone-treated ewes (22)
may be due to other progesterone-regulated factors present in uterine histotroph of both endometrial origin and/or selective components of serum transudate (7
, 8
, 25
, 47
48
49
50)
.
Ruminant blastocysts will not fully elongate in culture but will elongate if transferred to the uterus in domestic animals (51)
. Parallel increases in proliferation, migration and attachment of trophectoderm cells are presumed requirements for blastocyst elongation in uteri of ruminants (3
, 52
53
54)
. The onset of blastocyst elongation on day 12 in sheep is correlated with the induction of LGALS15 in the endometrium by progesterone between days 10 and 12, and the onset of conceptus implantation is correlated with further increases in LGALS15 by trophectoderm-derived IFNT and its presence within the uterine lumen between days 14 and 16 (12)
. Indeed, the total amount of LGALS15 protein recovered from the uterine lumen on days 12 to 16 of pregnancy in sheep ranges from 1 to 20 µg (J.L. Farmer and T.E. Spencer, unpublished observations). Results of the present study strongly support the hypothesis that LGALS15 possesses an intrinsic ability to bind and activate integrins on trophectoderm cells that, in turn, stimulates their migration and attachment and may reduce cell death due to apoptosis. Further, the biological activities of LGALS15 are not mediated by the CRD, but by integrin recognition sequences such as RGD in the C terminus.
Several members of the galectin family have either negative or positive effects on apoptosis (17)
. Of particular note, LGALS15 alone did not stimulate apoptosis of trophectoderm cells. Indeed, apoptosis is a common feature of conceptus development that may be required for cell remodeling or removal of genetically deficient cells (55)
. However, LGALS15 did partially inhibit apoptosis of trophectoderm cells that was elicited by staurosporine. Interestingly, focal adhesion kinase (FAK) dephosphorylation and focal adhesion disassembly is a very early event mediating the onset of staurosporine-induced endothelial cell apoptosis (35)
. Thus, the ability of LGALS15 to counteract staurosporine-induced apoptosis may be mediated by its ability to bind and activate integrins that result in formation of focal adhesions.
Results of the present studies revealed that LGALS15 potently stimulated migration of mononuclear trophectoderm cells in a manner dependent on activation of the JNK-JUN cell signaling pathway involved in a number of cellular processes including epithelial sheet migration (56)
. Indeed, JNK has been implicated as a downstream target of integrin activation (57)
and human trophoblast responses to placental growth factor (58)
. Both phosphorylated JNK and JUN proteins are present in trophectoderm cells of the days 16–20 ovine conceptus (K. Hayashi and T. E. Spencer, unpublished observations). These results support the hypothesis that endometrial-derived LGALS15 acts in a paracrine manner on trophectoderm cells of the conceptus to stimulate their motility and migration. In contrast to humans and rodents, blastocysts of domestic ruminants must elongate before implantation (3
, 4)
. Trophectoderm elongation involves cell migration and proliferation required for formation of the conceptus and developmentally regulated production of IFNT for pregnancy recognition (3
, 59)
. In sheep, the spherical blastocyst on day 7 begins elongation on day 12 to form a filamentous conceptus by day 18 that is in contact with the entire endometrial LE of the uterine horn ipsilateral to the corpus luteum and extends through the common uterine body into the contralateral uterine horn. Thus, blastocyst elongation undoubtedly requires an extraordinary amount of trophectoderm cell motility and migration. Blastocyst elongation is compromised in the uterine gland knockout ewe model which lacks endometrial glands and has a reduced amount of LE and consequently LGALS15 (9
, 11
, 12)
. Collectively, available results link the induction and increase in LGALS15 in the uterine lumen, which is synthesized and secreted by the endometrial LE and sGE, to stimulation of trophectoderm migration required for peri-implantation blastocyst elongation, formation of a filamentous conceptus and implantation
In the present studies, LGALS15 was found to mediate attachment of trophectoderm cells and formation of focal adhesions via binding and activation of integrins, which is an essential element of blastocyst implantation and trophoblast differentiation in many species (37
, 38)
. Indeed, integrins are proposed to be the dominant glycoproteins that regulate trophectoderm adhesion to endometrial LE during implantation in mammals (38
, 60)
. During the peri-implantation period of pregnancy in sheep, integrin subunits 
v, 4, 5, β1, β3, and β5 are constitutively expressed on apical surfaces of the conceptus trophectoderm and endometrial LE (45)
. Thus, conceptus implantation in sheep does not appear to involve temporal or spatial changes in patterns of integrin expression (45)
, but may depend primarily on changes in expression of secreted integrin ligands, such as LGALS15 and SPP1/osteopontin (4
, 39
, 61)
. Adhesive LE ligands, normally masked by mucins, become exposed during the receptive period, and various adhesion molecules then function sequentially, or in parallel, to stabilize adhesion of the trophectoderm to the endometrial LE (37
, 39
, 45)
.
LGALS15 is a candidate integrin bridging ligand in the uterine lumen during the peri-implantation period (11
, 12
, 23)
. Although trophectoderm cells do not express LGALS15 mRNA, LGALS15 protein accumulates in the uterine lumen and is present at the surfaces of these cells to act via integrin receptors (12
, 23)
. Within the uterine lumen, LGALS15 forms multimers on Days 14 and 16, which could increase bridging of integrins expressed on endometrial LE and conceptus trophectoderm (23)
. The cell attachment function of LGALS15 is due to sequences that mediate integrin recognition, such as the RGD, rather than the CRD. In the present studies, mutation of the CRD had little effect on the cell attachment function of LGALS15, which remained RGD-dependent in the CRD mutant. Other galectins, which do not display a conserved RGD recognition sequence, can bind and activate integrins via their CRD and bind fibronectin and laminin because these ECM proteins are modified with beta-galactoside sugars (17
, 18)
. Indeed, LGALS15 has little or no binding affinity for classical beta-galactosides (J. L. Farmer and T. E. Spencer, unpublished observations). Thus, the cell attachment function of LGALS15 is clearly most dependent on RGD recognition sequences, because the cyclic GRGDS peptide inhibited its cell attachment function. However, mutation of the LDVRGD recognition sequence in the C terminus to LAVRAD did not affect LGALS15 cell attachment function when wells were coated with low amounts of LGALS15 protein, but did reduce trophectoderm attachment to wells coated with high amounts of LGALS15 protein. Further, the LDV recognition sequence of LGALS15 is not likely important because natural polymorphic variants of LGALS15 with LVV instead of LDV sequences in the C terminus have been discovered in sheep and goat LGALS15 that do not alter their cell attachment function (62)
. One reasonable interpretation of results of the present studies is that LGALS15 has another integrin binding sequence(s) separate from the classical RGD sequence in the C terminus but recognizes the same site on integrins as the RGD sequence. This putative and unknown recognition sequence is functional in trophectoderm cell attachment assays using low amounts of LGALS15 LAVRAD mutant protein, but is inhibited when higher amounts of protein are used due to increased availability of the dysfunctional RAD sequence in the LGALS15 LAVRAD mutant. Indeed, the cyclic GRGDS peptide inhibited cell attachment function of the LGALS15 LAVRAD mutant, and cell attachment functions of both wild-type LGALS15 and LGALS15 CRD mutant were clearly inhibited by the GRGDS peptide but not by the GRADS peptide. One focus of future studies will be to identify the novel integrin recognition sequences of LGALS15. Although LGALS15 does not have any other obvious conserved integrin recognition sequence, many cell adhesion molecules, such as SPP1 and FN (39
, 63)
are known to have cryptic non-RGD integrin recognition sequences in addition to the conserved RGD recognition sequence (24)
.
Binding of integrins to ECM proteins promotes the aggregation of integrins and triggers a hierarchical response leading to transmembrane accumulation of cytoskeletal proteins and over 20 signal transduction molecules may be recruited to the β-integrin subunit cytoplasmic domain (64)
. The result is assembly of well-developed aggregates composed of ECM proteins, integrins, and cytoskeletal proteins known as focal adhesions (64
, 65)
. Attachment of the c-Src substrates, tensin, and focal adhesion kinase, can result from integrin aggregation alone, but aggregation of cytoskeletal proteins including talin, -actinin, vinculin, and F-actin requires ligand occupancy and integrin aggregation (64)
. Therefore, immunodetection of aggregated integrins, talin, or -actinin at focal adhesions can provide a sensitive functional index of integrin activation and outside-in signaling. The studies reported here exploited the ability of LGALS15 to induce focal adhesions by integrin-ECM interactions to demonstrate functional integrin activation and cytoskeletal reorganization in conceptus trophectoderm cells in response to LGALS15 binding. Accumulation of talin was detected at the interface between LGALS15-coated slides and ovine trophectoderm cells. The focal adhesions result from RGD-integrin interactions because mutation of the LGALS15 RGD sequence clearly eliminated cytoskeletal aggregation of talin, although the identity of activated integrins remains unknown. Interestingly, v and β3 integrin subunits that form the vβ3 receptor, which is capable of binding multiple matrix proteins, including SPP1, vitronectin, and fibronectin, aggregate at sites of cell anchorage to the substrate in both LE and trophectoderm cells, suggesting the presence of this versatile receptor at focal adhesion sites during the peri-implantation period (45)
. Therefore, it is reasonable to predict that in the pregnant ovine uterus, LGALS15 binding to integrin heterodimers induces focal adhesion sites that promote trophoblast elongation and stabilize attachment of trophectoderm to LE for implantation.
In summary, the temporal and spatial alterations in LGALS15 mRNA and protein in endometrial LE and lumen of the ovine uterus during pregnancy, combined with the functional aspects of LGALS15 discovered in the present studies, substantially support the hypothesis that LGALS15 functions as a heterotypic cell adhesion molecule bridging integrins in the endometrial LE and conceptus trophectoderm. These biological functions are undoubtedly required for ruminant blastocyst growth and elongation prior to implantation in utero. Of particular note, we recently determined that the LGALS15 gene is present in only ruminants (cattle, sheep, and goats), but is uniquely expressed in uterine endometria of ruminants in the subfamily Caprinae (sheep and goats; ref. 62
). However, other galectin family members are expressed in the endometria and placentae of other mammals where they may function in endometrial differentiation as well as blastocyst implantation and trophoblast differentiation (66
67
68)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication June 21, 2007. Accepted for publication August 16, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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