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Physiol. Genomics 36: 140-148, 2009. First published November 25, 2008; doi:10.1152/physiolgenomics.00022.2008
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Received 25 January 2008; accepted in final form 18 November 2008.
Physiological Genomics 36:140-148 (2009)
1094-8341/09 $8.00 © 2009 American Physiological Society

Identification of differential gene expression during porcine conceptus rapid trophoblastic elongation and attachment to uterine luminal epithelium

Jason W. Ross 1, Morgan D. Ashworth 1, Daniel R. Stein 1, Oliver P. Couture 2, Christopher K. Tuggle 2 and Rodney D. Geisert 1

1 Department of Animal Science, Oklahoma State University, Stillwater, Oklahoma
2 Department of Animal Science, Iowa State University, Ames, Iowa


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Early embryonic development in the pig is characterized by a rapid elongation of the conceptus trophectoderm on days 11–12 of gestation. Initially, the conceptus trophoblast is morphologically rearranged from a 10-mm sphere into a tubular shape, transitioning into a thin filamentous form >150 mm in length in 2–3 h, followed by continued expansion within the uterine lumen for several days. Conceptus elongation is critical for establishing adequate placental surface area needed for embryo and fetal survival throughout gestation. The objective of this study was to characterize conceptus gene expression during trophoblastic elongation and the early attachment to the uterine endometrium on days 11–14 of gestation with the GeneChip Porcine Genome Array. In all, 3,759 different probe sets were statistically different in at least one comparison [spherical vs. tubular, spherical vs. day 12 filamentous (D12F), spherical vs. day 14 filamentous (D14F), tubular vs. D12F, tubular vs. D14F, and D12F vs. D14F]. When restricted to the spherical vs. D12F and D12F vs. D14F comparisons, 482 and 232 genes, respectively, were statistically different with greater than twofold change in expression. Utilization of k-means clustering, in addition to the Database for Annotation, Visualization, and Integrated Discovery (DAVID), identified genes of interest. Quantitative RT-PCR expression profiles for interferon-{gamma} (IFNG), heat shock protein 27 kDa (HSPB1), angiomotin, B-cell linker (BLNK), chemokine ligand 14 (CXCL14), parathyroid hormone-like hormone (PTHLH), and maspin were supportive of the GeneChip Porcine Genome Array data.

pregnancy; implantation; uterus; pig


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
AS IN ANY MAMMALIAN SPECIES, successful establishment of pregnancy and embryonic development in the pig follows a specific pattern of temporal and spatial gene expression. It is estimated that successful preimplantation and early fetal development involve the appropriate expression pattern of ~10,000 genes (41). During early pregnancy in the litter-bearing pig, the peri-implantation period is one of the most critical stages of conceptus development. After ovulation and fertilization, prenatal mortality in the pig ranges from 20% to 46% by term (44), with the majority of the loss occurring during the peri-implantation stage of development, between days 12 and 18 of gestation (52). This significant period of elevated conceptus mortality during the peri-implantation stage of development coincides with conceptus rapid trophoblastic elongation (20); neurulation of the inner cell mass; transient synthesis and release of the maternal recognition of pregnancy signal, estrogen (20); and, finally, trophectoderm differentiation followed by adhesion and attachment to the uterine surface epithelium (9). Disruption of any of these critical biological processes can result in conceptus mortality and a reduction in litter size.

Conceptus trophoblast elongation, which occurs during days 11 and 12 of gestation, is characterized by transition through four distinct morphological stages (spherical, ovoid, tubular, and filamentous). The cellular remodeling and migration of the trophectoderm is initiated when a conceptus reaches a 9- to 10-mm spherical morphology and initiates transformation to an ovoid shape and then into a tubular morphology that rapidly expands into a long filamentous thread >150 mm in length within 2–3 h (20). Secretion of the conceptus-produced maternal recognition signal, estrogen (5, 23), and interleukin-1β (48) occurs concomitantly with elongation of the trophoblastic membrane. Trophoblastic elongation and expansion through the uterine horns maximizes nutrient exchange throughout gestation through increased placenta-uterine contact after the formation of a diffuse epitheliochorial type of placenta (52). The associated release of estrogen induces alterations in the endometrium, changing the uterine environment, which may be unfavorable for less developed littermates (22, 44). Litter variation in conceptus morphological stage of development around day 12 of gestation is not uncommon (3). Given that the amount of estrogen production by individual conceptuses is directly proportional to the morphological stage of development (45), it is plausible to conclude that variation within the onset of trophoblastic elongation and estrogen production between littermates can reduce survivability of the less developed conceptuses as the uterine microenvironment is altered. While conceptus trophoblastic elongation is a required phenomenon for the establishment of pregnancy in the pig, the subsequent adhesion of the trophectoderm to the uterine surface epithelium is also critical for continued conceptus development.

Numerous conceptus products have been hypothesized to be important in porcine conceptus development since detection of its mRNA expression in the various conceptus morphological stages with techniques such as semiquantitative RT-PCR (25, 32, 59, 60), differential display RT-PCR (58), suppression subtractive hybridization (SSH) (47), expressed sequence tag (EST) library construction and analysis (50), utilization of embryonic based cDNA array (35), and serial analysis of gene expression (SAGE) (8). While these techniques have been complementary and consistent in identifying genes and pathways potentially regulating trophoblastic elongation, most studies have only compared the expression of day 12 filamentous conceptuses to those of earlier morphological stages of development and not postelongated conceptuses, which are initiating attachment to the uterine epithelial surface. The objective of the present investigation was to utilize the GeneChip Porcine Genome Array from Affymetrix possessing 20,201 unique probe sets to identify differentially expressed genes during rapid trophoblastic elongation and attachment to the uterine surface in the pig. Identification and characterization of conceptus gene expression patterns during rapid trophoblastic elongation and attachment in the pig will provide a better understanding of the events required for successful attachment and embryonic survival.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Conceptus Collection
Research was conducted in accordance with the American Physiological Society's "Guiding Principles for Research Involving Animals and Human Beings," and protocols were approved by the Institutional Animal Care and Use Committee at Oklahoma State University. Twenty crossbred, cyclic gilts were checked for expression of estrus twice daily in the presence of an intact boar and naturally mated at the onset of the second estrus and again 24 h later. Gilts were hysterectomized on days 11–12 of gestation to collect spherical (8–10 mm) and tubular (20–80 mm) conceptuses, while filamentous (>100 mm) conceptuses were collected on days 12 and 14 of gestation. Hysterectomies were conducted as previously described for our laboratory (26). After removal of the uterine horns, conceptuses from each uterine horn were flushed into a sterile petri dish with 20 ml of physiological saline. Because of the limited time frame during which conceptuses are in tubular transitional development (2–3 h) and the difficulty in determining when tubular conceptuses are in the uterus after mating, one uterine horn was removed on day 11.5 of gestation in a subset of gilts as previously described (47). Conceptuses were evaluated to determine an appropriate time-delayed removal of the second horn corresponding to a predicted time that conceptuses would be in a tubular morphology according to the rate of development described by Geisert et al. (21). After collection from the uterine horns, conceptuses of identical morphologies were transferred to cryogenic vials, snap-frozen in liquid nitrogen, and stored at –80°C until RNA was extracted.

RNA Isolation
Total RNA was isolated from conceptus pools, each representing multiple individuals (4–8) of identical morphologies. Some uterine flushes produced an insufficient number of conceptuses at the appropriate morphology to provide adequate total RNA for downstream applications. For spherical and tubular morphologies, when fewer than four conceptuses were collected from a single gilt embryos of identical morphology from a second gilt were combined so that each pool represented four to eight conceptus pools from one or two gilts. Conceptus pools for day 12 filamentous (D12F) and day 14 filamentous (D14F) embryos are representative of one gilt per pool. Total RNA was isolated with RNAwiz (Ambion, Austin, TX) according to the manufacturer's recommendations. RNA concentrations were calculated based on absorbance at the 260-nm wavelength. Purity and integrity of the RNA were determined from the 260-to-280 ratio and visualization after agarose gel electrophoresis.

Microarray Analysis
Affymetrix porcine chip.
The GeneChip Porcine Genome Array (Affymetrix, Santa Clara, CA) contains 23,937 probe sets interrogating 23,256 transcripts, representing 20,201 genes. Four chips were used for each morphological stage of development (spherical, tubular, D12F, and D14F). RNA utilized for each chip represented a unique pool of conceptus total RNA for the respective morphological stage of development. Before target labeling, RNA was further purified (RNeasy MinElute Cleanup, Qiagen, Valencia, CA). Target labeling, GeneChip hybridization, scanning, and quantification were conducted by the University of Tulsa Microarray Core Facility. Affymetrix GeneChip Operating Software (GCOS version 1.1.1, Affymetrix) was used to quantitate each GeneChip. The summary intensities for each probe were loaded into DNA-Chip Analyzer (dChip) version 1.3 for normalization, standardization, and analysis. All microarray data have been deposited into the NCBI Gene Expression Omnibus (GEO accession no. GSE12705).

Normalization and standardization.
Normalization was conducted to reduce technical variation between chips that can occur in part because of labeling and hybridization efficiencies, washing stringency, and scanning. For normalization, we used dChip's method of invariant set normalization, designed for oligo-based arrays (37), in which the chip with the median intensity value was used as the baseline against which the brightness of the remaining chips was adjusted in order to be of a comparable level. To reduce variance of expression level estimates by accounting for probe differences, standardization was conducted by calculating model-based expression indexes (MBEI) using dChip's Perfect-Match (PM)-only model.

Log transformation and statistical analysis.
The MBEI were log base 2 transformed to approximate a normal distribution for each gene and provide measures by which to conduct the statistical analysis. Unpaired t-tests were calculated with dChip to evaluate differences between two groups. Analysis of gene expression was done to compare expression changes between all morphological stages of development utilized in the study. Restriction of the list of candidate genes was accomplished by selecting those with a P value <0.001 as determined by the unpaired t-test and a numerical change in expression of at least twofold for each morphological comparison evaluated. While it should not be assumed that genes whose expression changes are less than twofold do not have a biological effect, production of a differentially expressed gene list based solely on statistical analysis can be irreproducible and this can be overcome by utilizing additional restrictions such as a minimum of twofold change (27, 51).

GeneChip Porcine Genome Array Reannotation
The annotation was updated by utilizing the provided sequence from Affymetrix that was utilized in probe development. Through the utilization of BLAST (2), a human accession number, based on homology, was assigned to each Affymetrix ID already correlated to each probe on the chip (Couture and Tuggle, manuscript in preparation). Briefly, an in-house assembly of 1.7 million public porcine sequences was used to produce consensus sequences. Affymetrix target sequences were then aligned with the porcine consensus sequences, setting a cutoff of an E value < 1e-5, thereby providing longer, more complete sequences than the consensus sequence produced by Affymetrix during the design of the array. The porcine consensus sequences were aligned against RefSeq, setting a stricter cutoff of an E value < 1e-10, because it is cross species. The highest rankings of each alignment, the Affymetrix to porcine consensus and the porcine consensus to RefSeq, were used to map the Affymetrix probe to RefSeq. The assignment of a human accession number allowed a more elaborate analysis of the biological processes being regulated during conceptus elongation and attachment, by enabling the more effective use of software such as the Database for Annotation, Visualization, and Integrated Discovery (DAVID), which cannot use porcine gene information.

Clustering Analysis
To provide a better biological understanding of the data, we conducted k-means clustering to group genes with similar expression patterns together and potentially indicate multiple genes associated with a single biological transition during conceptus development. Clustering genes based on expression profiles also provides the ability to predict genes that may be regulated by the same transcription factors based on similar expression patterns. All genes determined to be differentially expressed based on the criteria indicated for at least one comparison between morphological stages of development (n = 1,473) were utilized for analysis in Cluster 3.0 (12, 15). We sorted the 1,473 genes that were both statistically (P < 0.001) and numerically (±2-fold change) different into 25 clusters. The 25 clusters were identified with the k-means learning algorithm following 1,000 iterations within Cluster 3.0.

Database for Annotation, Visualization, and Integrated Discovery
DAVID version 2.0 (http://david.niaid.nih.gov/david/version2/index.htm) is a program that enables the utilization of microarray gene lists to generate specific functional annotations of the biological processes affected by the treatment as determined through microarray experiments (24). DAVID was utilized to annotate biological themes occurring during the two major developmental transitions during pig conceptus development: spherical to D12F and D12F to D14F. These two transitional stages are characteristic of trophoblastic elongation induction and initial conceptus-uterine attachment. All genes identified to be both significantly (P < 0.001) and numerically (±2-fold change) different for the spherical vs. D12F and D12F vs. D14F comparisons, and also successfully assigned a human accession number, were used in the analysis via DAVID. The background list utilized in the program included all genes assigned a human accession number that were identified as present on at least one of the eight chips representing a morphological comparison. With Gene Ontology (GO) terms as identified through biological process, cellular component, and molecular function as well as protein domain and biochemical pathway membership, biological themes were generated through DAVID by grouping like terms, thereby creating functional annotation clusters associated with each developmental transition.

Quantitative Two-Step RT-PCR
Quantitative RT-PCR analysis of transcripts of interest was conducted as previously described by our laboratory (4). RNA from the same conceptus pools utilized for Affymetrix analysis was aliquoted to be used for PCR analysis. Genomic DNA removal and synthesis of cDNA to be used for quantitative analysis were done with the QuantiTect Reverse Transcription kit according to manufacturer's recommendations (Qiagen). PCR amplification was conducted with the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). All primers and probes utilized for quantitative analysis for each target gene are presented in Table 1. One hundred nanograms of synthesized cDNA was assayed for each sample in duplicate. Thermal cycling conditions with the dual-labeled probes were 50°C for 30 min, 95°C for 15 min, followed by 40 repetitive cycles of 95°C for 15 s and a combined annealing/extension stage, 59°C for 1 min. Fluorescent data acquisition was done during the annealing/extension phase when using the dual-labeled probes. Cycling conditions for SYBR Green detection were 50°C for 30 min and 95°C for 15 min, followed by 40 repetitive cycles of 95°C for 15 s and variable annealing temperature for 30 s, 72°C for 33 s, and variable temperature during fluorescent data acquisition for 33 s. 18S ribosomal RNA was assayed as a normalization control to correct for loading discrepancies. Relative quantification of target gene expression was evaluated with the comparative cycle threshold (CT) method as previously described (4, 30). The {Delta}CT value was determined by subtracting the target CT of each sample from its respective ribosomal 18S CT value. Calculation of {Delta}{Delta}CT involves using the single greatest sample {Delta}CT value (the sample with the lowest expression) as an arbitrary constant to subtract from all other sample {Delta}CT values. Relative mRNA units for each sample were calculated assuming an amplification efficiency of 2 during the geometric region of amplification and applying the equation 2Formula. To compare the expression patterns determined through quantitative RT-PCR with that determined with microarray analysis, the mean relative mRNA units for tubular, D12F, and D14F morphologies, as determined for each gene, were divided by the mean relative mRNA units for the spherical morphology to produce the fold differences presented in Table 4.


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  		Table 1. Primer and probe sequence information for quantitative amplification of each target gene

 
Statistical analysis.
Normalized quantitative RT-PCR {Delta}CT values were analyzed with PROC MIXED of the Statistical Analysis System. The statistical model used in the analysis tested the fixed effect of morphology (spherical, tubular, D12F, and D14F). Significance (P < 0.05) was determined by probability differences of least-squares means between morphological stages of development on normalized gene expression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Affymetrix Analysis
Chips with >5% of probe sets flagged as array outliers are of suspect quality. dChip did not flag any of the arrays as an outlier (when fitted expression for the entire probe set has a standard error >3 standard deviations from the mean compared with other chips). Accordingly, no tissue was rehybridized to a new array, nor was any array dropped from analysis. Single outliers are lone probes of unusual intensity within a chip. In this set of samples, outlier percentages ranged from 0.02% to 0.13%. Single outliers were treated as missing values in subsequent analyses. The percentage of genes called "present" by the GCOS software ranged from 65.49% to 70.37%. Table 2 lists the intensities, present call percentages, and outlier percentages for each gene chip as produced by dChip. When the results from the six comparisons were combined, there were 3,850 significantly altered probe sets, of which 3,759 remained after deletion of those with an "absent" detection call across all chips in both comparison groups. When the results were restricted to unique GenBank accession numbers, 3,736 were found to differ significantly in their expression in one or more of the six comparisons (spherical vs. tubular, spherical vs. D12F, spherical vs. D14F, tubular vs. D12F, tubular vs. D14F, D12F vs. D14F). The number of genes statistically different, in addition to statistically and greater than twofold different for each comparison between morphologies, is presented in Table 3.


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  		Table 2. Intensities, percent present, and outliers for each AffyChip utilized during microarray analysis

 

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  		Table 3. Numbers of statistically different mRNA abundance for genes identified between morphological comparisons

 
GeneChip Porcine Genome Array Reannotation
Two comparisons, spherical vs. D12F and D12F vs. D14F, were further analyzed because of their biological importance with respect to this stage of pregnancy. For the specific biological comparisons of interest, spherical vs. D12F and D12F vs. D14F, 280 of 482 and 157 of 232 genes, respectively, with an Affymetrix identification number were successfully assigned a GenBank accession number for a homologous human gene. Genes with an appropriately assigned GenBank accession number with homology to human transcripts were further utilized in functional annotation clustering through DAVID. The human accession numbers assigned to each Affymetrix ID representing a gene with statistical and greater than twofold difference are available in Supplemental Table S1.1

Database for Annotation, Visualization, and Integrated Discovery
Analysis through DAVID was conducted to allow detection of biological processes affected during the two major transitions: spherical vs. D12F and D12F vs. D14F. Several processes were consistent throughout this overall period of development, but distinct differences were also notable. During the transition from spherical to D12F morphology, genes associated with lipid metabolism, phospholipid metabolism, localization/transport, locomotion/cell motility, ATP activity and binding, regulation of cell growth, amino acid utilization/metabolism, metal ion binding, and regulation of apoptosis were detected (Supplemental Table S2). Transition from D12F to D14F morphology revealed that several biological themes were still persistent from the spherical to D12F transition. Regulation of apoptosis was one of the most prominent biological themes during the transition from D12F to D14F, because three annotation clusters represented terms associated with apoptosis. Other critical processes appear to be those involved with protein kinase activity, regulation of cell cycle progression and other cell physiological processes, and chromosome organization (Supplemental Table S3). The processes that appeared to be involved in both developmental transitions did differ in their significance ranking as determined through DAVID. While cell motility/locomotion ranked high and apoptosis regulation ranked low during the spherical to D12F transition, apoptosis was of the most prominent themes during the D12F to D14F transition and cell motility/locomotion was much less distinguished (Supplemental Tables S2 and S3).

Clustering Analysis
The 25 clusters generated with the k-means clustering algorithm were distinguishable by their expression patterns and represented the 1,473 genes meeting the probability and fold difference threshold for one or more of the comparisons between morphological stages of development. The smallest cluster contained 3 genes, while the largest contained 364 genes. The mean cluster size was nearly 59 genes; however, the median cluster size was 19 genes, suggesting that most of the clusters represented unique expression patterns and a few larger clusters represented general trends (Supplemental Table S4). Expression patterns that displayed a transient shift in expression during the spherical to D12F transition are represented by clusters 4, 7, 14, 21, and 24. Clusters 4, 14, 21, and 24 represent 17, 10, 3, and 76 genes, respectively, whose expression was greatest in the elongating D12F conceptuses, while cluster 7 represents 88 genes whose expression was at a transient nadir in D12F conceptuses. The Affymetrix ID, associated human GenBank accession number, and putative identity for each gene and the expression profile for each gene that was significantly (P < 0.001) and numerically (>2-fold change) different between any two developmental stages are listed in association with their k-means cluster in Supplemental Table S4.

Quantitative Two-Step RT-PCR
On the basis of the evaluation through DAVID and k-means clustering, differences in mRNA abundances between the four conceptus developmental stages were further quantified with the two-step method for quantitative RT-PCR. Messenger RNAs that were evaluated with this method are listed in association with the primer/probe sequences used for detection in Table 1. The transcripts chosen to validate the results of the array were chosen so that further analysis would provide novel data with regard to conceptus development in the pig and would be physiologically relevant based on their putative expression profile as indicated in the k-means analysis and their association with biological themes as determined through DAVID. Quantitative RT-PCR was done to analyze changes in mRNA expression of interferon-{gamma} (IFNG), heat shock protein 27 kDa (HSPB1), angiomotin, epidermal growth factor receptor (EGFR), actinin {alpha}4 (ACTN4), B-cell linker (BLNK), chemokine ligand 14 (CXCL14), parathyroid hormone-like hormone (PTHLH), and maspin. The expression patterns for IFNG, HSPB1, angiomotin, BLNK, CXCL14, PTHLH, and maspin mRNA, as determined by quantitative RT-PCR, were similar to those determined by microarray analysis, while expression patterns for EGFR and ACTN4 differed slightly between the two techniques (Table 4).


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  		Table 4. Results from quantitative RT-PCR in comparison with Affymetrix results

 
Morphological stage of development significantly affected the mRNA abundance of IFNG (P < 0.001), HSPB1 (P < 0.002), angiomotin (P < 0.001), EGFR (P < 0.026), ACTN4 (P < 0.045), BLNK (P < 0.002), CXCL14 (P < 0.001), PTHLH (P < 0.001), and maspin (P < 0.004) as determined through quantitative RT-PCR. The mRNA abundance of IFNG, HSPB1, angiomotin, CXCL14, and PTHLH all increased significantly as conceptus development proceeded after the initiation of trophoblastic elongation, with transcript abundance being greater in D12F and D14F conceptuses compared with spherical or tubular conceptuses. BLNK and maspin mRNA abundance was significantly lower after the initiation of trophoblastic elongation and initial attachment to the uterine epithelium compared with spherical and tubular conceptuses.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Conceptus and endometrial changes during the peri-implantation development in the pig are critical for successfully establishing pregnancy and minimizing prenatal mortality throughout the gestation period (44). The extent of initial trophoblastic elongation is one factor that influences nutrient flow between the dam and the conceptus during later gestation, which impacts conceptus survivability.

Utilization of the Affymetrix GeneChip Porcine Genome Array allowed the identification of numerous factors that are differentially expressed during the transition through the multiple developmental stages in early pregnancy in the pig. Interestingly, as determined through the statistical analysis, no genes were detected during the transition from spherical to tubular morphology (Table 3) that met the probability and fold difference thresholds, whereas targets for 482 probes had different abundance levels between spherical and D12F morphologies. While differential expression of a couple of genes was detected by SSH (47) between the spherical and tubular morphologies, it is difficult to compare the results of the present study to the results of other somewhat similar comparisons utilizing SAGE (7, 8). While this study utilized 8- to 10-mm conceptuses (just before trophoblastic elongation) to represent the spherical morphology, the first experiment utilizing SAGE (8) compared 6- to 10-mm spherical to filamentous conceptuses and the second SAGE analysis, including the transitional tubular morphology, utilized 4- to 10-mm spherical conceptuses (7). The variation of spherical conceptus morphologies used complicates the comparison between these studies, as the SAGE experiments may be somewhat confounded by morphologies utilized as spherical because differential gene expression between spherical conceptuses of large (8–10 mm) and small (4–6 mm) diameter has been documented and can be dramatically different in these embryos that are all spherical (60).

The lack of significant gene expression changes detected during the spherical to tubular transition while a large transcriptional change is present between D12F vs. spherical conceptuses suggests that the initial trophectodermal remodeling is not dependent on large transcriptional changes. Additionally, the fact that porcine conceptus elongation has not been successful in vitro is suggestive that the uterine environment plays a critical role in the initiation of trophoblastic elongation and that elongation may not be exclusively controlled by conceptus transcriptional changes. While D14F conceptuses are still elongating to some degree, elongation is not as rapid as the initial several hundred millimeters of the spherical to D12F transition. However, the D14F conceptuses represent the stage of conceptus development in which trophoblast attachment to the uterine luminal epithelia occurs. During this initial attachment to the uterine endometrium, 232 genes had at least a twofold change in mRNA abundance. These genes likely represent factors associated with reduced trophoblastic elongation, placental differentiation, and conceptus-uterine cross talk.

Because no differences between spherical and tubular morphologies were detected, the transitions from spherical to D12F and from D12F to D14F were the two developmentally critical comparisons whose gene lists were utilized for biological annotation in DAVID. The association with cell motility and locomotion is plausible in that conceptuses collected in early stages of elongation are undergoing structural remodeling (21). The alterations in genes during the period also suggest the involvement of significant ATP utilization, cell growth factors, amino acid transport, and regulation of apoptosis (Supplemental Table S4). It is logical that the dramatic increase in conceptus mass from spherical to D12F is a result of energy catabolism, and several cell growth factors contribute to the morphological change. Interestingly, as DAVID ranks the significance of each cluster, apoptosis-related genes were ranked low in the spherical vs. D12F transition but apoptosis represented clusters 1, 8, and 9 in the D12F to D14F transition, suggesting the critical role apoptosis may play regarding conceptus development during attachment to the uterine epithelium. Differential expression of apoptosis-related transcripts during conceptus elongation and attachment is suggestive of the critical impact cell death can or does confer during this stage of pig conceptus development.

It is difficult to ascertain whether gene expression changes are specific to the trophectoderm, the inner cell mass, or both. Overall, the processes identified in the transition of D12F to D14F compared with spherical to D12F involved more complicated processes, which is expected because conceptus complexity is greatly increased during these 2 days of development, such as attachment to the uterine epithelium, neurulation of the inner cell mass, as well as cell lineage branching in preparation for early organ development of the inner cell mass and the differentiation of placental layers from the single trophectoderm.

Clustering analysis is critical to the ability to identify expression patterns for specific genes allowing the identification of associations between genes that may be regulated by a similar mechanism. This transient expression pattern associated with the rapid stage of trophectoderm remodeling (clusters 4, 7, 14, 21, and 24; Supplemental Table S4) would indicate those genes that could be considered as putative markers of trophoblastic elongation. Thus these markers of trophoblastic elongation are targets for exploration regarding biological function during this stage of conceptus development to determine their effect during the establishment of pregnancy.

A few genes, PTGS2 (57), steroidogenic acute regulatory factor (6), transforming growth factor β (28), epidermal growth factor receptor (56), interferon-{gamma} (34), and retinol binding protein (59), are among those previously identified to be differentially regulated during early peri-implantation conceptus development in the pig and were also identified to have a similar differential expression pattern during or after trophoblastic elongation as presented in this communication (Supplemental Table S1).

Methodological differences were evident in that statistical significance appeared to be greater based on the quantitative RT-PCR data than the array data, particularly for differences between spherical and tubular conceptuses; however, the expression patterns indicated by both the array and the quantitative RT-PCR were quite similar. Not only are the expression patterns based on the microarray data largely confirmed by the quantitative RT-PCR results, but the physiological relevance that the products of the differentially expressed genes have to conceptus elongation and attachment to the uterine epithelium also confers validation to the microarray data presented in this article.

Interferon-{gamma} is a significant component of pregnancy in many species and is generally known for its immune-related function in non-pregnancy-related biology. In certain B-cell lymphoma cell lines, IFNG is capable of reducing cell growth through apoptosis sensitivity (42). Generally, IFNG largely elicits its effects through the activation of either signal transducer and activator of transcription (STAT)-1 or -3 JAK-STAT activation pathways leading to transcription through the activation of interferon regulatory factors (11). STAT-3 upregulation (Supplemental Table S4, cluster 11) temporally associated with increased IFNG gene expression is suggestive that this mechanism of regulation is present during peri-implantation conceptus development in the pig.

With yeast two-hybrid screening, HSPB1 has been shown to strongly associate with estrogen receptor (ER)β, but not ER{alpha}, suggesting a role in estrogen-modulated signaling (40). Interestingly, pig conceptuses also express ERβ during trophoblastic elongation (27), while lacking detectable ER{alpha} gene expression (60). It is possible that HSPB1 may also regulate the ability of conceptus-secreted estrogen to function in an autocrine fashion. HSPB1 has also been shown to closely associate with F-actin, which may contribute to the maintenance of cell architecture during ATP depletion, as in renal epithelial cells (55). It is clear that actin stabilization/remodeling is tightly regulated based on the data presented within this article that many of the genes differentially expressed during trophoblastic elongation have the ability to attach to actin, particularly those with PDZ-LIM domains.

Angiomotin has been observed to promote angiogenesis via endothelial cell migration through the ability to bind and block the inhibitory affects of angiostatin (53). While deletion of the COOH-terminal PDZ binding motif of angiomotin in mouse endothelial cells blocked chemotactic capacity of those cells, the mutant protein was still localized to the lamellipodia of cells, as in migrating cells that contain PDZ-intact angiomotin (36). More recently, the report of two alternative splice isoforms of angiomotin working coordinately suggests that an 80-kDa (p80-angiomotin) form is primarily responsible for cell migration and a 130-kDa isoform (p130-angiomotin) colocalizes with F-actin regulating cell shape. The finding that both isoforms are expressed in mouse placental tissue (17) is suggestive that angiomotin could be involved with trophectoderm cell migration in multiple species. Localization to the lamellipodia suggests an association with cellular homing in addition to an ability to interact with F-actin, which has previously been suggested to be rearranged during rapid trophectoderm changes in the pig (39).

BLNK is a required component of B-cell development, because progenitor B cells in BLNK–/– mice fail transition into precursor B cells (43). BLNK may also regulate B-cell function through B-cell receptor-mediated signaling inhibition during conceptus attachment in the pig, because conceptus BLNK expression is so dramatically downregulated (28- and 17-fold in D12F and D14F, respectively, relative to spherical conceptuses; Table 4) during adhesion of the trophectoderm to the uterine endometrium. In pregnant mice, maternal B lymphocytes capable of responding to paternal major histocompatibility complex (MHC) antigens are partially deleted (1), suggesting the importance of B-cell regulation during pregnancy. Because BLNK is critical for B-cell function, it is likely that the significant reduction in BLNK expression during pig conceptus development involves the regulation of the maternal immune response to the developing conceptus.

CXCL14 promotes the migration of human cytotrophoblast cells while expression of the receptor for CXCL14 (CCR1) is localized in endometrium at the maternal-fetal interface during implantation (29). CCR1 is also expressed in human extravillous trophoblast cells but not in cytotrophoblast cells (49), suggestive of a role in trophoblast differentiation and invasion into the decidua. The significant upregulation of CXCL14 in D14F pig conceptuses may also be involved with regulating initial trophoblast differentiation that subsequently contributes to the formation of placental layers with unique function (18).

PTHLH is involved in the calcium mobilization and homeostasis (46) that is under significant regulation in the uterine lumen during the period of trophoblastic elongation and conceptus-uterine attachment in the pig (20). PTHLH has been shown to be involved with the rearrangement and cytoskeleton organization near the ectoplacental cone (EPC) in mice (16) and is expressed in human syncytiotrophoblast and avascular amnion cells, suggesting a role in regulation of migration and differentiation of trophectoderm cells (14). Expression of PTHLH and its receptor are spatially and temporally associated with the differentiation of uterine stromal cells into decidual cells during implantation in the rat (54).

Maspin expression during pig conceptus development is significantly downregulated in D12F and D14F conceptuses compared with those spherical in morphology. During early embryonic development in mice, maspin expression is critical to normal inner cell mass development and adequate cell growth rate (19), and maspin is also differentially expressed in human placenta throughout gestation (13). Maspin has demonstrated ability to function as a suppressor of tumor growth and metastasis (10), suggestive of a negative role in cell motility and attachment. Interestingly, conceptus downregulation of maspin is temporally associated with increased cell motility and attachment of the trophectoderm to the uterine endometrium, which is suggestive of a potential interaction between these biological processes during pig conceptus peri-implantation development and maspin expression.

The use of the Affymetrix GeneChip Porcine Genome Array effectively identified hundreds of porcine genes that are differentially expressed during the developmental period consisting of trophoblastic elongation and the initial attachment of the conceptus trophectoderm to the maternal uterine endometrium. While, based on the literature, mechanisms of action for the reported lists of genes can be hypothesized, it is also critical to determine what interactions do exist between these proteins and others during pig conceptus elongation and the exact regulatory functions these genes may elicit. Enhanced understanding of this process will allow a more comprehensive approach to selecting for effective conceptus development resulting in optimal degrees of placentation among conceptuses, promoting similar development between littermates and increased litter size in pigs.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Research Initiative Competitive Grants 2002-35203-12262 and 2004-35205-14202 from the US Department of Agriculture (USDA) Cooperative State Research, Education, and Extension Service to R. D. Geisert and C. K. Tuggle, respectively. O. P. Couture acknowledges support of USDA-Food and Agricultural Sciences Multidisciplinary Graduate Education and Training Grant 2001-52100-11506.


   ACKNOWLEDGMENTS
 
The authors thank the University of Tulsa Microarray Facility for conducting Affymetrix GeneChip hybridizations. We also thank Steve Welty for the maintenance and care of the animals used in this research study.

Present address of J. W. Ross: Animal Science Dept., College of Agriculture and Life Sciences, Iowa State University, Ames, IA 50011.


   FOOTNOTES
 
Address for reprint requests and other correspondence: R. D. Geisert, Animal Science Dept., Univ. of Missouri-Columbia, Coll. of Agriculture, Food, and Natural Resources, S108 Animal Science Research Ctr., Columbia, MO 65211 (e-mail: geisertr{at}missouri.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The online version of this article contains supplemental material. Back


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
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