Activated Leukocyte Cell Adhesion Molecule : Expression in the Uterine Endometrium during the Estrous Cycle and Pregnancy in Pigs *

The pig exhibits true epitheliochorial placentation, where the fetal membrane maintains attachment throughout pregnancy but does not invade into the maternal uterine endometrium. Accordingly, the expression and function of cell adhesion molecules are very important for embryo implantation and the establishment of pregnancy. In our recent microarray analysis, we found that activated leukocyte cell adhesion molecule (ALCAM) was expressed in the uterine endometrium during pregnancy in pigs. To better understand the roles of ALCAM in the establishment and maintenance of pregnancy, we examined ALCAM expression in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that ALCAM was differentially expressed in the uterine endometrium during the estrous cycle and pregnancy, with the highest levels on D12 of pregnancy. ALCAM mRNA was localized to the luminal and glandular epithelial cells and to the trophectoderm of conceptuses during early pregnancy. The steroid hormones estrogen and progesterone had no effect on ALCAM expression in an endometrial explant culture study. Further, we found that ALCAM expression in the uterine endometrium from gilts with somatic cell nuclear transfer-derived embryos was not different from that in gilts with embryos from natural mating. ALCAM was expressed in a pregnancy stageand cell type-specific manner in the uterine endometrium and conceptuses during pregnancy. These findings suggest that ALCAM may play a role in the establishment of pregnancy. Further analysis of ALCAM will provide insight into the implantation process and establishment of pregnancy in pigs. (


INTRODUCTION
The establishment and maintenance of pregnancy requires complex and well-organized interactions between the implanting embryos and the maternal uterine endometrium.In pigs, embryo implantation begins around day (D) 12 of pregnancy.During the implantation period, trophoblastic elongation, maternal recognition of pregnancy, and embryo attachment occur at the fetal-maternal interface (Bazer et al., 2009).Pigs exhibit true epitheliochorial placentation, where the fetal membrane does not invade into the uterine endometrium but superficially interdigitates with the luminal epithelial layer.Thus, cell-cell adhesion between the conceptus trophectoderm and endometrial luminal epithelial cells is the final step for placentation, and various cell adhesion molecules are involved in this process (Burghardt et al., 1997;Burghardt et al., 2002).For example, SPP1 (secreted phosphoprotein 1, also called osteopontin) promotes trophectodermal cell migration and adhesion with luminal epithelial cells by interaction with integrin (Erikson et al., 2009).Integrin subunits, receptors for extracellular matrix (ECM) proteins such as SPP1, fibronectin, and vitronectin, are expressed in the conceptuses and luminal epithelial cells for cell-to-cell attachment during the implantation period (Bowen et al., 1996;Burghardt et al., 1997).Down-regulated mucin 1 (MUC1) expression exposes integrins localized in the apical surface of the uterine luminal epithelial cells and facilitates binding of integrins to ECM proteins at the fetal-maternal interface (Burghardt et al., 2002).Failures of these serial events and subsequent abnormal maternal-fetal interactions lead to early embryonic loss in pigs (Pope, 1994;Bazer et al., 2009;Kim et al., 2009), but the process of initial cell-tocell attachment and maintenance of cell adhesion during pregnancy is not fully understood.
Activated leukocyte cell adhesion molecule (ALCAM; also known as CD166) is a glycoprotein that acts as a cell surface ligand for CD6 (Ohneda et al., 2001).ALCAM interacts not only with CD6 in a heterotypic manner, but also homotypically with itself (Bowen et al., 1996;Cayrol et al., 2008).ALCAM is expressed in a variety of cells, including fibroblasts (Singer et al., 1997), pancreatic acinar and islet cells (Stephan et al., 1999), mesenchymal stem cells (Bruder et al., 1998;Arai et al., 2002), neurons (Konno et al., 2001), and trophectodermal cells (Fujiwara et al., 2003).Expression of ALCAM in trophectodermal cells is particularly critical for initial attachment of human embryos (Fujiwara et al., 2003), because ALCAM-ALCAM interactions form a tight ALCAM network between cells (Bowen et al., 1995).ALCAM expression is induced by cytokines such as interleukin-3 (IL3), macrophage-colony stimulating factor (M-CSF), granulocyte macrophagecolony stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, and interferon-γ (IFNG) (Levesque et al., 1998;Cayrol et al., 2008).In pigs, the uterine endometrial gene expression is influenced by various factors such as progesterone of ovarian origin and estrogen, interleukin-1β (IL1B), and IFNG of conceptus origin during early pregnancy (Lefevre et al., 1990;Burghardt et al., 1997;Geisert and Yelich, 1997;Ross et al., 2003;Bazer et al., 2009).Using microarray analysis, we have found that ALCAM is expressed in the uterine endometrium during pregnancy (Kim and Ka, manuscript in preparation), but the expression and functions of ALCAM in the uterine endometrium during the estrous cycle and pregnancy in pigs are not well understood.In addition, regulatory mechanism of ALCAM expression in the uterine endometrium needs to be elucidated.
The efficiency to produce viable offspring using somatic cell nuclear transfer (SCNT) technique is extremely low in pigs (Keefer, 2008), and is influenced by several factors, including quality of recipient oocytes, epigenetic reprogramming status of donor nucleus, and SCNT procedures (Humpherys et al., 2001;Campbell et al., 2005;Farin et al., 2006).Appropriate interaction between the conceptus and the uterine endometrium is required for normal uterine function to support fetal development (Bazer et al., 2009;Ashworth et al., 2010), but the interaction between the SCNT embryo and the maternal uterus is impaired in pigs carrying SCNT embryos (Chae et al., 2008;Bauersachs et al., 2009;Kim et al., 2009).Our recent studies have shown that many uterine endometrial genes related to cell-to-cell interaction are dysregulated in the uterine endometrium with SCNT embryos on D30 (Ka et al., 2008) and term pregnancy (Kim et al., 2009).Accordingly, it would be interesting to know whether endometrial ALCAM is appropriately expressed in the uterus with SCNT embryo on D12 of pregnancy.
Therefore, to better understand the role of ALCAM at the fetal-maternal interface in pigs during pregnancy, we determined i) the pattern of ALCAM expression in the uterine endometrium during the estrous cycle and pregnancy; ii) localization of ALCAM mRNA in the uterine endometrium; iii) effects of steroid hormones on ALCAM mRNA expression; and iv) ALCAM expression levels in the uterine endometrium from gilts with embryos derived from SCNT compared with those from gilts with embryos from natural mating.

Recovery and in vitro maturation of oocytes
Recovery and in vitro maturation of oocytes, SCNT, and embryo transfer were conducted as described in Ka et al. (2008).In brief, cumulus-oocyte complexes (COCs) were collected by aspiration from ovarian antral follicles of 3.0-6.0mm in diameter from ovaries collected at a local slaughterhouse.COCs having at least three layers of compacted cumulus cells were selected and cultured in tissue culture medium (TCM)-199 (Invitrogen, Carlsbad, CA) supplemented with 0.6 mM cysteine, 10 ng/ml epidermal growth factor, 4 IU/ml equine chorionic gonadotropin (eCG; Intervet, Boxmeer, The Netherlands) and human chorionic gonadotropin (hCG; Intervet), and 10% (v/v) porcine follicular fluid.The COCs were cultured for 22 h with hormones and then for 22 h without hormones in a humidified atmosphere of 5% CO 2 and 95% air at 38.5°C.

SCNT
After in vitro maturation, cumulus cells were removed by repeatedly pippetting for 1 min in a tyrode-lactate-HEPES (TL-HEPES) medium supplemented with 0.1% (w/v) hyaluronidase (Hagen et al., 1991).A cumulus-free oocyte was held with a holding micropipette and the zona pellucida was partially incised with a fine glass needle to make a slit near the first polar body.The first polar body and adjacent cytoplasm of the oocyte were extruded by squeezing with the same needle (Hyun et al., 2003).Oocytes were enucleated in TL-HEPES supplemented with 0.3% BSA and 7.5 mg/ml cytochalasin B. The cultured porcine embryonic fibroblast cells as nuclear donors were used after cell cycle synchronization by serum starvation for 48 h.A donor cell was inserted into the perivitelline space of an enucleated oocyte.Donor cell-oocyte complexes were placed in a 280 mM mannitol solution containing 0.5 mM HEPES, 0.1 mM CaCl 2 and 0.1 mM MgCl 2 for 2 min and transferred to a chamber consisting of two electrodes overlaid.They were fused and activated simultaneously with a DC pulse (2.0 kV/cm 30 μs×1) using the BTX Electro-cell Manipulator 2001 (BTX, San Diego, CA).The reconstructed oocytes were then cultured in modified NCSU23 (Petters and Wells, 1993) supplemented with 4 mg/ml BSA in a humidified atmosphere of 5% CO 2 , 5% O 2 and 90% N 2 at 38.5°C.

Animals, embryo transfer, and tissue collection
All experimental procedures involving animals were conducted in accordance with the Guide for the Care and Use of Research Animals in Teaching and Research and approved by the Institutional Animal Care and Use Committee of Yonsei University.Sexually mature crossbred female pigs were assigned randomly to either cyclic or pregnant status.Twenty-four gilts were hysterectomized on D12 and D15 of the estrous cycle and D12, D15, D30, D60, D90, or D114 of pregnancy (n = 3 gilts/day/status).Pregnancy was confirmed by the presence of apparently normal conceptuses with filamentous morphology in uterine flushings or fetuses.Endometrium dissected from the myometrium was collected from two different areas of the middle portion of each uterine horn.
Endometrial tissue samples from gilts with embryos generated by SCNT on D12 of pregnancy were obtained as described in Ka et al. (2008).Crossbred prepubertal gilt weighing between 100 and 105 kg was used as the recipient of the embryos.To induce estrus in the gilt, 1,000 IU of eCG were injected intramuscularly, followed by an injection of 1,500 IU hCG 72 h later.The embryos produced by SCNT were cultured for 1 or 2 days.A total of 150 embryos that were morphologically normal at the 1-cell or 2-4-cell stages were selected for transfer.Embryos were then transferred into the oviducts of the recipient gilt approximately 48 h after hCG injection.Uterine endometrial tissues on D12 of pregnancy were obtained from four gilts with SCNT embryos and three gilts with embryos by natural mating (Non-NT).Endometrial tissues were snap-frozen in liquid nitrogen and stored at -80°C for RNA and protein extraction.For in situ hybridization and immunohistochemistry, cross-sections of endometrium and conceptuses were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 24 h and then embedded in paraffin, as previously described (Ka et al., 2000).

RNA isolation
Total RNA from endometrial tissues was extracted using TRIzol (Invitrogen, Carlsbad, CA) and the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.The purity and integrity of the total RNA were checked using a NanoDrop (NanoDrop Technologies, Wilmington, DE) and Experion (Bio-rad, Hercules, CA), respectively.

Total RNA extraction and reverse transcriptionpolymerase chain reaction (RT-PCR) for ALCAM analysis
Total RNA was extracted from endometrial tissues and conceptuses using TRIzol reagent (Invitrogen Life Technology, Carlsbad, CA) according to manufacturer's recommendations.The quantity of RNA was assessed spectrophotometrically, and integrity of RNA was examined by gel electrophoresis in 1% agarose gels.
Two micrograms of total RNA were treated with DNase I (Promega, Madison, WI) and reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen) to obtain cDNAs.The cDNA templates were then diluted 1:5 with sterile water and amplified by PCR using Taq polymerase (Takara Bio, Shiga, Japan), and specific primers based on mRNA sequences of porcine ALCAM (GenBank accession number AJ311681; forward, 5'-CCT TCA GGT CCT CCA CAA AG -3'; reverse, 5'-ATG TGA TGT TGC CAT CTG GA-3') and porcine ribosomal protein L7 (RPL7), (GenBank accession number NM_001113217; forward, 5'-AAG CCA AGC ACT ATC ACA AGG AAT ACA-3'; reverse, 5'-TGC AAC ACC TTT CTG ACC TTT GG-3') were designed to amplify cDNA of 302 bp and 172 bp, respectively.PCR conditions for ALCAM were 35 cycles of 94°C for 15 s, 60°C for 20 s, and 72°C for 1 min.PCR products were separated on 2% agarose gels and visualized by ethidium bromide staining.The identity of each amplified PCR product was verified by sequence analysis after cloning into a pCRII vector (Invitrogen).

Quantitative real-time RT-PCR
To analyze levels of ALCAM mRNA in the uterine endometrium, real-time RT-PCR was performed using the Applied Biosystems StepOnePlus System (Applied Biosystems, Foster City, CA) and the SYBR Green method.Complementary DNAs were synthesized from 4 μg of total RNA isolated from different uterine endometrial tissues, and newly synthesized cDNAs (total volume of 21 μl) were diluted 1:4 with sterile water and then used for PCR.Specific primers based on porcine ALCAM (GenBank accession number AJ311681; forward, 5'-CCA GAA TAC AAA AAC AGA ATT GAG CC-3'; reverse, 5'-TAG ATG GTT GCT TGG GAC ACC TTG AC-3') and porcine RPL7 (GenBank accession number NM_001113217; forward, 5'-AAG CCA AGC ACT ATC ACA AGG AAT ACA-3'; reverse, 5'-TGC AAC ACC TTT CTG ACC TTT GG-3') were designed to amplify cDNA of 150 bp and 172 bp, respectively.Power SYBR Green PCR Master Mix (Applied Biosystems) was used for the PCR reaction.The final reaction volume of 20 μl included 2 μl of cDNA, 10 μl of 2X Master mix, 2 μl of each primer, and 4 μl of dH 2 O. PCR conditions were 95°C for 15 min followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s.The results were reported as the expression relative to the level detected on D12 of the estrous cycle after normalization of the transcript amount to the endogenous RPL7 control by the 2 -ΔΔCT method (Livak and Schmittgen, 2001).

Non-radioactive in situ hybridization
Non-radioactive in situ hybridization was performed as described previously with some modifications (Braissant and Wahli, 1998).Sections (5 μm thick) were rehydrated through successive baths of xylene, 100% ethanol, 95% ethanol, diethylpyrocarbonate (DEPC)-treated water, and DEPC-treated PBS.Tissue sections were permeabilized with DEPC-treated PBS containing 0.3% Triton X-100 for 15 min.After washing in DEPC-treated PBS, they were digested with 5 μg/ml Proteinase K (Sigma) in TE (100 mM Tris-HCl, 50 mM EDTA, pH 7.5) at 37°C.After postfixation in 4% paraformaldehyde, sections were incubated twice for 15 min each in PBS containing 0.1% active DEPC, and equilibrated for 15 min in 5X SSC.The sections were prehybridized for 2 h at 68°C in hybridization mix (50% formamide, 5X SSC, 500 μg/ml herring sperm DNA; 200 μl on each section).Sense and antisense ALCAM riboprobes labeled with DIG-UTP were denatured for 5 min at 80°C and added to the hybridization mix.The hybridization reaction was carried out at 68°C overnight.Prehybridization and hybridization were performed in a box saturated with a 5X SSC -50% formamide solution to avoid evaporation, and no coverslips were used.After hybridization, sections were washed for 30 min in 2X SSC at room temperature, 1 h in 2X SSC at 65°C, and 1 h in 0.1X SSC at 65°C.Probes bound to the section were immunologically detected using sheep anti-DIG Fab fragments covalently coupled to alkaline phosphatase and nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (toluidine salt) as a chromogenic substrate, according to the manufacturer's protocol (Roche, Indianapolis, IN).

Statistical analysis
Data from real-time RT-PCR for ALCAM expression were subjected to least squares ANOVA using the General Linear Models procedures of SAS (Cary, NC).As sources of variation, the model included day, pregnancy status (cyclic or pregnant), and their interactions to evaluate the steadystate level of ALCAM mRNA, and treatment and animal to evaluate the effect of steroid hormones on ALCAM mRNA.Preplanned contrasts (control vs. E 2 ; control vs. P 4 ; E 2 vs. E 2 +P 4 ; E 2 +P 4 vs.E 2 +P 4 +ICI) for ALCAM expression were applied to test for effects of treatments in the explant cultures.Data are presented as least squares means with SE (standard error).Data from real-time RT-PCR analysis for comparison of ALCAM levels in the endometrium with SCNT and Non-NT embryos were subjected to t tests, and are presented as means with SE.

Expression and regulation of ALCAM mRNA in the uterine endometrium during the estrous cycle and pregnancy in pigs
To determine ALCAM mRNA levels in the uterine endometrium during the estrous cycle and pregnancy in pigs, we conducted real-time RT-PCR analysis.As shown in Figure 1, relative levels of ALCAM mRNA were high during early pregnancy in the uterine endometrium and decreased after D15 of pregnancy (day, p<0.05).ALCAM mRNA levels in the uterine endometrium on D12 of pregnancy were higher than those on D12 of the estrous cycle (day x status, p<0.05).

Localization of ALCAM mRNA in the uterine endometrium during the estrous cycle and pregnancy
To determine which cell types express ALCAM mRNA in the uterine endometrium during the estrous cycle and pregnancy and in conceptuses, we performed in situ hybridization analysis using uterine endometrial tissues from D12 and D15 of the estrous cycle and pregnancy and conceptuses from D12 of pregnancy.ALCAM mRNA was mainly localized in the luminal and glandular epithelial cells in the uterine endometrium during the estrous cycle and pregnancy (Figure 2).The signal intensity of ALCAM mRNA was stronger in the uterine endometrium on D12 of pregnancy than that on D12 of the estrous cycle, which was consistent with real-time RT-PCR results.In addition, the  signal intensity of ALCAM mRNA seemed to be slightly stronger in luminal compared to glandular epithelial cells.ALCAM mRNA was also detected in the trophectoderm of conceptuses on D12 of pregnancy.

Expression of ALCAM in the conceptus during early pregnancy
To determine whether conceptuses express ALCAM mRNA during the implantation period, we analyzed ALCAM expression in conceptuses from D12 and D15 of pregnancy by RT-PCR and found ALCAM mRNA expression on both D12 and D15 of pregnancy (Figure 3).

Effect of steroid hormones on ALCAM expression
Because ALCAM expression was highest on D12 of pregnancy in the uterine endometrium, we hypothesized that ALCAM expression might be regulated by estrogen secreted by elongating conceptuses and progesterone of ovarian origin.To determine the effect of steroid hormones on ALCAM expression in the uterine endometrium, we treated explant cultures of uterine endometrial tissues from gilts on D12 of the estrous cycle with steroid hormones and inhibitors of those hormones receptors.Estrogen and progesterone did not affect ALCAM mRNA expression in the uterine endometrium (Figure 4).

Comparison of ALCAM expression levels in the uterine endometrium in pigs carrying Non-NT and NT-derived embryos
To determine influence of ALCAM expression on the establishment of pregnancy in recipients with SCNT embryos, we compared ALCAM mRNA expression levels in the uterine endometrium of pigs carrying Non-NT and SCNT-derived embryos by real-time RT-PCR.ALCAM levels were not significantly different depending on embryo type.

DISCUSSION
In the present study, we determined in pigs that i) ALCAM expression in the uterine endometrium changed during the estrous cycle and pregnancy with the highest levels on D12 of pregnancy; ii) ALCAM mRNA was localized in the luminal and glandular epithelial cells in the endometrium; iii) ALCAM expression was detected in conceptuses on D12 and D15 of pregnancy, localized to the conceptus trophectoderm; iv) steroid hormones (estrogen and progesterone) did not regulate ALCAM expression in the uterine endometrium; and v) ALCAM expression levels in the uterine endometrium were not different between pigs carrying SCNT and Non-NT-derived embryos.
We observed that ALCAM expression was differentially regulated in the uterine endometrium during pregnancy in pigs.ALCAM expression was higher in the uterine endometrium during early pregnancy than in the mid-to late stages of pregnancy.In particular, ALCAM expression levels were highest on D12 of pregnancy, which is the period of maternal recognition of pregnancy in pigs (Bazer Endometrial explants from gilts on D12 of the estrous cycle were cultured in DMEM/F-12 in the presence of control (C), E 2 (E; 50 ng/ml), P 4 (P; 3 ng/ml), E 2 +P 4 , E 2 +P 4 +ICI (I; 200 ng/ml, an estrogen receptor antagonist), or E 2 +P 4 +RU (R; 30 ng/ml, a progesterone receptor antagonist), at 37°C for 24 h.For each treatment, all experiments were repeated in triplicate with endometrium from each of three gilts.Data are presented as least squares means relative units with standard error.et al., 2009).Conceptuses during this period elongate to become filamentous forms and begin contact with the uterine endometrium for the establishment of pregnancy (Dantzer, 1985;Bazer et al., 2009).Cell-to-cell attachment between the trophectoderm of conceptuses and uterine epithelial cells is critical for the establishment of pregnancy (Dantzer, 1985), and a variety of cell adhesion molecules are dynamically regulated during the implantation period in the uterine endometrium (Burghardt et al., 1997;Geisert and Yelich, 1997;Fernando et al., 2007;Song et al., 2010).Among them, integrins, selectins, galectins, heparan sulfate proteoglycans, heparin-binding EGF-like growth factors, cadherins, CD44, and ECM molecules are known to be associated with initial attachment of conceptuses to the endometrial epithelial cells (Burghardt et al., 1997;Bazer et al., 2009).Because ALCAM, a cell adhesion molecule, is also involved in cell-to-cell attachment by interacting with CD6 heterotypically or with ALCAM homotypically (Bowen et al., 1995;Ohneda et al., 2001) and its expression is high at the time of conceptus implantation, our result suggests that ALCAM may also be critical for cell-to-cell adhesion between the conceptus trophectoderm and the uterine endometrium during the implantation process in pigs.
ALCAM was localized to the luminal and glandular epithelial cells and the trophectoderm of conceptuses during early pregnancy.ALCAM has been detected in brain, heart, guts, bone marrow, thymus, and hematopoietic sites in humans (Patel et al., 1995;Uchida et al., 1997;Bruder et al., 1998;Cortes et al., 1999;Stephan et al., 1999;Konno et al., 2001;Gessert et al., 2008), as well as luminal epithelial cells during the secretory phase in the human placenta and in blastocysts (Fujiwara et al., 2003).In our study, localization of ALCAM expression in the luminal epithelial cells and conceptus trophectoderm suggest the possibility of homotypic interactions of ALCAM between the uterus and conceptuses.Localization of ALCAM mRNA in the glandular epithelial cells indicates that ALCAM may be secreted from glandular epithelial cells into the uterine lumen, since the soluble isoform of ALCAM is produced by lung microvascular endothelial cells in humans (Ikeda and Quertermous, 2004).Further study to determine if soluble ALCAM is present in the uterine lumen is needed.
ALCAM expression is regulated by different hormones and cytokines in various cell types.In MCF7 cells, estrogen increases ALCAM expression (Jezierska et al., 2006), and human chorionic gonadotrophin induces ALCAM expression in the cumulus-oocyte complex of the human ovary (Hernandez-Gonzalez et al., 2006).GM-CSF induces ALCAM expression in CD14+ monocytes (Levesque et al., 1998).Co-treatment with TNF and IFNG induce ALCAM expression in human endothelial cells of the blood-brain barrier (Cayrol et al., 2008).We hypothesized that estrogen of conceptus origin induces ALCAM expression in the uterine endometrium for a number of reasons.First, ALCAM expression is induced by estrogen in MCF7 cells (Jezierska et al., 2006).In addition, estrogen, a signal for the maternal recognition of pregnancy in pigs, is secreted from elongating conceptuses around D12 of pregnancy (Bazer et al., 2009).Finally, we observed that ALCAM expression was highest in the uterine endometrium on D12 of pregnancy.Because progesterone of ovarian origin is the major hormone affecting the uterine endometrium during pregnancy, we further postulated that progesterone affects ALCAM expression in the endometrium.We found, however, that estrogen and progesterone did not change ALCAM expression in the present study, indicating that factors other than estrogen and progesterone may be involved in ALCAM expression in the uterine endometrium in pigs.In the porcine uterus, elongating conceptuses secrete IL1B on D12 of pregnancy (Ross et al., 2003) and IFNG between D12 and D18 of pregnancy (Lefevre et al., 1990), and these cytokines may induce endometrial ALCAM expression.Another possibility is that the uterine endometrial tissues used in this study were from D12 of the estrous cycle and so had previous exposure to progesterone that minimized their response to in vitro treatment with estrogen and/or progesterone.Further study involving the influence of steroid hormones and cytokines on the regulation of ALCAM expression in the uterine endometrium of pigs during early pregnancy is still needed.
Early embryonic mortality reaches approximately 40% in pigs (Pope, 1994), and the efficiency of generating viable offspring using SCNT cloning is very low in pigs (Keefer, 2008).Unsynchronized interactions between the conceptuses and the maternal endometrium may lead to aberrant gene expression in the uterine endometrium, which consequently leads to early embryonic loss during early pregnancy (Bauersachs et al., 2009;Bazer et al., 2009;Kim et al., 2009).Premature estrogen administration results in decreased expression of ITIH2 in the uterine endometrium in pigs, which could be related to loss of uterine glycocalyx leading to degradation of conceptuses (Ashworth et al., 2010).Furthermore, it has been suggested that abnormal expression of cell adhesion molecules, including integrins and ECM proteins, and some growth factors in the uterine endometrium with SCNT embryos are related to early embryonic loss of cloned pig embryos (Chae et al., 2008;Kim et al., 2009).Our previous study showed that SPP1, retinol-binding protein, and fibroblast growth factor 7 were aberrantly expressed in the endometrium with SCNT embryos compared to embryos from natural mating at term (Kim et al., 2009).In this study, we also looked for differences in ALCAM expression in the endometrium with SCNT and Non-NT embryos and found no differences.These findings indicate that the SCNT procedure does not affect endometrial ALCAM expression in pigs carrying SCNT embryos and that ALCAM expression is not affected by the presence of the conceptuses for implantation initiation in pigs.Further study is needed to determine whether ALCAM expression is affected by the SCNT procedure during mid-to late-stage pregnancy and whether there is any crucial role for ALCAM in the maintenance of pregnancy for SCNT embryos after the implantation stage.
In the porcine uterus, the exact function of ALCAM has not yet been determined.Since ALCAM is involved in cellto-cell attachment to regulate migration of lymphocytes and cancer cells (Masedunskas et al., 2006;Cayrol et al., 2008), development of bone marrow (Arai et al., 2002), morphogenesis and maintenance of cardiac cells (Gessert et al., 2008), and initiation of the human embryo implantation (Fujiwara et al., 2003), we speculate that ALCAM may also play an important role in cell-to-cell attachment for embryo implantation at the maternal-fetal interface.Indeed, ALCAM expression was detected in both endometrial epithelial cells and conceptus trophectoderm in this study, and it is well known that ALCAM interacts with itself homotypically to trigger oligomerization of ALCAMs on the cell surface between the cells, enhancing a tight ALCAM network (Bowen et al., 1995).Expression of CD6, a heterophilic receptor for ALCAM expressed mainly in lymphocytes, has not been determined in the uterine endometrium or conceptuses in pigs so far, but it is possible that ALCAM may recruit and activate endometrial lymphocytes.
In conclusion, our results provide evidence that ALCAM is expressed in the luminal and glandular epithelial cells and trophectoderm of conceptuses during the implantation period in pigs.These findings suggest that ALCAM may be involved in the initial contact between the conceptuses and uterine endometrial epithelial cells for the establishment of implantation in pigs.These results provide valuable information on the mechanisms of the implantation process in the pig, which exhibits epitheliochorial placentation.

Figure 1 .
Figure1.Analysis of ALCAM mRNA levels in the uterine endometrium during the estrous cycle and pregnancy in pigs by real-time RT-PCR analysis.Endometrial tissue samples from cyclic and pregnant gilts (n = 3 per day) were tested.Abundance of mRNA is presented as the expression relative to the level of ALCAM measured on D12 of the estrous cycle after normalization of the transcript amount to RPL7.ALCAM mRNA levels were highest on D12 during pregnancy (day, p<0.05), and the levels of ALCAM mRNA on D12 of pregnancy were significantly higher than those on D12 of the estrous cycle (day x status, p<0.05, indicated as asterisk).Data are presented as least squares means with standard error.

Figure 2 .
Figure 2. In situ hybridization analysis of ALCAM mRNA in the uterine endometrium during the estrous cycle and pregnancy in pigs.A section from D12 of pregnancy hybridized with a DIG-labeled sense ALCAM cRNA probe (Sense) is shown as a positive and a negative control, respectively.ALCAM mRNA was primarily localized to the luminal and glandular epithelial cells of the uterine endometrium and trophectoderm of conceptuses on D12 of pregnancy.D, day; C, estrous cycle; P, pregnancy; LE, luminal epithelium; St, stroma; Tr, trophectoderm, Con; conceptus.Arrow heads indicate ALCAM transcript-positive signals on luminal epithelial cells and conceptus trophectoderm cells.Scale bar = 100 μm and 200 μm in D12P Con.

Figure 4 .
Figure 4. Effects of steroid hormones on ALCAM mRNA levels in endometrial explant cultures by real-time RT-PCR analyses.Endometrial explants from gilts on D12 of the estrous cycle were cultured in DMEM/F-12 in the presence of control (C), E 2 (E; 50 ng/ml), P 4 (P; 3 ng/ml), E 2 +P 4 , E 2 +P 4 +ICI (I; 200 ng/ml, an estrogen receptor antagonist), or E 2 +P 4 +RU (R; 30 ng/ml, a progesterone receptor antagonist), at 37°C for 24 h.For each treatment, all experiments were repeated in triplicate with endometrium from each of three gilts.Data are presented as least squares means relative units with standard error.

Figure 5 .
Figure 5. Analysis of ALCAM mRNA levels in the uterine endometrium of pigs with embryos derived from SCNT (NT) compared to embryos from natural mating (Non-NT) on D12 by real-time RT-PCR.Abundance of mRNA is presented as the expression relative to the levels of ALCAM mRNA measured in the uterine endometrium from gilts with non-NT embryos after normalization of the transcript amount to RPL7 mRNA.Data are presented as means with standard error.