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BOR - Papers in Press, published online ahead of print July 30, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.019307
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BIOLOGY OF REPRODUCTION 69, 1697–1706 (2003)
DOI: 10.1095/biolreprod.103.019307
© 2003 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Isolation of Differentially Expressed Genes in Conceptuses and Endometrial Tissue of Sows in Early Gestation1

Maud Vallée3, Danièle Beaudry4, Claude Roberge4, J. Jacques Matte4, Richard Blouin3, and Marie-France Palin2,4

Département de Biologie,3 Université de Sherbrooke, Sherbrooke, Québec, Canada J1K 2R1 Dairy and Swine Research and Development Center,4 Agriculture and Agri-Food Canada, Lennoxville, Québec, Canada J1M 1Z3


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The implantation period is a critical time for embryonic survival in pigs. During this period, numerous growth factors are secreted by the conceptuses and the uterine endometrium in order to establish pregnancy and to provide a proper environment for embryonic development. It is well known that the Chinese Meishan sows have a larger litter size when compared with occidental sows mainly because of a superior embryonic survival rate. As a further step toward understanding the mechanisms involved in embryonic survival, we used a suppression subtractive hybridization technique to identify genes that were differentially expressed in Meishan-Landrace conceptuses and endometrial tissue at Day 15 of gestation when compared with conventional Landrace sows. Of the 1000 subtractive clones isolated from each library, 137 endometrial and 166 conceptus-enriched cDNAs were single-pass sequenced and examined by BLAST analysis for identification. Sixty-two percent of the clones found in the endometrial library and 78% of the clones found in the conceptus library showed homology with known genes. Among these genes, the 20 most relevant to embryonic survival based on the available literature were validated through real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. Our results show that suppression subtractive hybridization is a powerful method applicable in identifying putative candidate genes that might be used for selection of high litter-size breeds.

conceptus, early development, implantation, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The second week of pregnancy is a critical period for embryonic survival in pigs, and about 30% of conceptuses are lost between Days 12 and 15 of gestation [1, 2]. During the implantation period, the developing conceptuses secrete numerous growth factors, hormones, and proteins in order to develop properly [3–6]. The uterine environment also provides an optimal and essential biochemical environment for proper conceptus development [4, 7–10]. Porcine conceptuses begin producing estradiol-17ß (E2) on Days 11–12 of gestation during the period of rapid trophoblast elongation [11, 12]. This increase in E2 production by conceptuses results in an increase in uterine luminal secretions of endometrial proteins [13]. It has been hypothesized that the changes in the uterine milieu induced by E2 secreted by the larger and more advanced conceptuses of a litter may lead to the loss of lesser developed littermates and thus results in a smaller litter size [14].

It is well documented that litter size at farrowing is greater in multiparous Chinese Meishan sows and is due, in part, to a greater ovulation rate, greater uterine capacity, and greater embryonic survival [15–18]. Meishan conceptuses secrete less E2 into uterine luminal fluids, causing more gradual changes in the uterine environment and potentially allowing the lesser developed conceptuses to survive as compared with occidental breeds. Furthermore, the Meishan conceptuses elongate to a reduced length when compared with occidental pig (e.g., Landrace) conceptuses at a similar stage, and this is due to a maternal effect of the Meishan uterus [17, 19]. The protein content found in the uterine flushings of Meishans is also lower than that of occidental breeds [20]. All of these factors contribute to the potential mechanisms by which embryonic survival is improved in the Meishan pig.

Previous studies comparing the mechanisms of embryonic survival between Meishan and occidental breeds have concentrated on the relative spatial and temporal expression of genes and proteins known to be present or have a function in the developing conceptus and/or uterus [13, 19, 21–25]. However, the Meishan pig may have an improved embryonic survival rate due to the expression of various genes, and genes not already considered to be implicated in embryonic survival. The use of suppressive subtractive hybridization (SSH) as a technique to identify genes that are differentially expressed in conceptuses and the uterine endometrium between Meishan and Landrace breeds presents a powerful method to identify putative candidate genes implicated in embryonic survival. Recently, Ross et al. [26] demonstrated the potential of the SSH technique to identify genes that were temporally expressed during rapid trophoblastic elongation in the pig. Therefore, the present study was undertaken to identify genes that could be associated with the high embryonic survival rate in the Meishan pig. To achieve this objective, two enriched cDNA libraries were constructed using SSH prepared from conceptuses and endometrial tissue of Meishan-Landrace and Landrace breeds taken during the implantation period [27]. Among the different clones that were analyzed from each library, we found several sequences related to embryonic development and survival in different animal species.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments

Three multiparous Meishan-Landrace sows (ML; 6 parities, 16.8 piglets/litter) and 3 multiparous Landrace sows (LL; 7 parities, 11.2 piglets/litter), generously provided by Genetiporc (St-Bernard, QC), were selected to perform SSH and real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis. Three multiparous ML cyclic sows (5 parities, 14.3 piglets/litter) and 3 multiparous LL cyclic sows (5 parities, 8.8 piglets/litter) were also selected on which to perform real-time RT-PCR analysis. Despite the fact that we used sows that were not pure Meishan but crossbred animals, we believe that we will still be able to identify genes associated with embryonic survival because the crossbreeding was made in order to achieve a more prolific line of sows. All sows received 2.5 kg daily of a basal diet (50% [w/w] corn, 20% barley, 20% wheat bran, 5% soybean, and 15 ppm folic acid meal). Heat detection was performed twice a day, between 0800 and 0900 h and between 1600 and 1700 h by introducing a boar into the pen. Sows were inseminated twice with a Landrace mix semen (pooled semen from three Landrace boars of proven fertility: CIPQ, Inc., St-Lambert, QC, Canada), 12 and 24 h after estrus detection. Landrace semen was used for both breeds of sows because it was previously reported that the Meishan's high prolificacy is due to the mother's genotype and not to the litter's genotype [28, 29]. The time of the first insemination was considered as Day 0.

Tissue Collection

Sows were slaughtered on Day 15 of gestation or Day 15 of the estrous cycle (control). This time frame was chosen to ensure that uterine epithelium samples were taken from implantation sites, and immediately after implantation had successfully occurred. Furthermore, there is a greater potential to identify genes involved in embryonic survival when the library is made with conceptuses that have all successfully implanted in the uterus. The reproductive tract was collected and the uterine horns were dissected from the mesometrium. The ovaries, oviducts, and cervix were also removed. Both horns were flushed with 20 ml of PBS (135 mM NaCl, 2.5 mM KCl, 10 mM Na2HPO4, 1.5 mM KH2PO4), the fluids collected, and centrifuged at 1000 x g for 5 min at 4°C to precipitate the filamentous conceptuses. The pellet containing the conceptuses was washed twice with PBS. Randomly chosen strips of epithelial endometrium (1 x 5 cm) were collected at five different sites of implantation, and this protocol was repeated for each horn. Samples of endometrial tissue were also collected for the cyclic sows. All tissue samples were immediately frozen in liquid nitrogen. The whole procedure was completed within 20 min of slaughter. Animals were cared for according to the recommended code of practice [30] and killed using a penetrating bolt pistol as an acceptable method approved by the local Animal Care Committee following the guideline of the Canadian Council on Animal Care [30].

RNA Extraction and mRNA Purification

Total RNA was extracted from gestating sow endometrial tissue and conceptuses. Briefly, 2 g of tissue was extracted with 20 ml of Trizol reagent (Gibco BRL, Burlington, ON, Canada) according to the manufacturer's instructions. The extracted RNA was dissolved in water and quantified spectrophotometrically at 260 nm. An RNA aliquot was electrophoresed in a 1% agarose gel to verify its integrity. Polyadenylated RNA was isolated from total RNA of endometrial tissue and conceptuses using the mRNA Separator Kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions.

Suppressive Subtractive Hybridization

SSH was performed with the PCR-Select cDNA Subtraction Kit (Clontech) as described by the manufacturers. Briefly, pools of mRNA ML endometrial tissue and of LL endometrial tissue were used for cDNA production. The same procedure was repeated with conceptuses. The pools consisted of mRNA (2 µg) from three different ML sows for the tester and three different LL sows for the driver. The endometrial tissue consisted of tissue pooled from 10 different implantation sites, 5 on each horn. Embryonic tissue came from pooled conceptuses from both horns. Polymerase chain reaction (PCR) was performed according to a standard protocol (PCR-Select cDNA Subtraction Kit; Clontech) in a PTC-200 Programmable Thermal Controller (MJ Research, Foster City, CA). The subtracted PCR products generated by SSH were cloned into the pT-Adv vector (Advantage PCR cloning kit; Clontech) and transformed into DH5{alpha} Escherichia coli cells. An average of 1000 clones was obtained from each subtracted library.

Differential Screening

The cDNA inserts obtained from the different clones were amplified by PCR. Briefly, a bacterial colony was picked and resuspended in 300 µl Luria broth (LB) + ampicillin (50 µg/ml) and grown for 2 h at 37°C. PCR amplifications were carried out from 1 µl of bacterial suspension with the Advantage PCR polymerase mix (Clontech) according to the manufacturer's instructions. The remaining bacterial suspension was kept in 20% glycerol at -80°C for later identification. Five microliters from each PCR reaction was electrophoresed on a 2% agarose gel to identify insert-containing clones. PCR products from positive clones were dotted onto eight identical Hybond N+ membranes (Amersham Pharmacia Biotech., Baie d'Urfée, QC, Canada) according to a standard protocol. Forward and reverse subtracted PCR products as well as nonsubtracted cDNA from testers and drivers were used as probes to hybridize one of each identical dot blot membrane containing the PCR-amplified cDNA inserts. This last procedure was performed in duplicate. Probes were 32P-labeled using PCR-Select Subtraction Hybridization Screening Kit (Clontech) in the presence of [{alpha}-32P]-dCTP (Amersham). The labeled probes were then purified with CHROMA SPIN-100 Columns (Clontech). Membranes were prehybridized with ExpressHyb Hybridation Solution (Clontech) for 2 h at 72°C with continuous agitation. Membranes were then hybridized with equivalent amounts of labeled probes, each with approximately equal specific activity, and then washed according to standard protocols. Each membrane was then subjected to autoradiography (BIOMAX MR autoradiographic film; Interscience, Markham, ON, Canada) for 3, 6, 12 h and 3 days. Hybridization signals were analyzed using a densitometer (BIO-RAD Imaging Densitometer Model GS-670; Bio-Rad Laboratories Ltd., Mississauga, ON, Canada) and the Molecular Analyst Software (version 1.5) (Bio-Rad Laboratories Ltd.).

DNA Sequencing and Analysis

DNA sequencing was performed using an automated ABI 377 DNA Sequencer (PE Applied BioSystems, Foster City, CA). Sequence reactions were carried out with the Big Dye Terminator Cycle Sequencing Ready Reactions Sequencer (PE Applied BioSystems) and nested primer 2R (Clontech). The resulting sequences were compared against the GenBank database using the online computer BLAST program (http://www.ncbi.nlm.gov/BLAST/).

Real-Time RT-PCR

Confirmation of differentially expressed cDNA was made by real-time RT-PCR. Total RNA from the same tissue samples used previously and from endometrial tissue of six cyclic sows (n = 3 ML and n = 3 LL) was extracted as described above and reverse transcribed to cDNA as described previously [24]. The cDNA samples were used individually and not pooled, for a total of six pig samples for the embryonic tissue and 12 pig samples for the endometial tissue (n = 6 gestating and n = 6 cyclic sows). Briefly, first strand cDNA was synthesized with SuperScript II (Gibco BRL) from 5 µg of total RNA. The primers were designed based on the sequence of the cloned cDNA segments (Table 1) and were selected using the Primer Express Software (PE Applied BioSystems). Real-time PCR amplification was performed in 25-µl reaction volume consisting of 300 nM of each primer, 12.5 µl of 2x SYBRGreen Master Mix (PE Applied BioSystems), and 1 µl of cDNA. Cycling conditions were 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. Amplification, detection, and data analysis were performed with an ABI 7700 Prism Sequence Detector (PE Applied BioSystems). Samples were normalized using the housekeeping genes GAPDH (primer sequence; 5'-CAG CAA TGC CTC CTG TAC CA-3' and 5'-GAT GCC GAA GTT GTC ATG GA-3', GenBank accession no. AF017079) and Cyclophilin (primer sequence; 5'-GCA CTG GTG GCA AGT CCA T-3' and 5'-AGG ACC CGT ATG CTT CAG GA-3', GenBank accession no. AY008846); each amplification was performed in triplicate. Standard curves were prepared for both the target SSH clone and the endogenous reference (GAPDH or Cyclophilin). For each experimental sample, the amount of target SSH clone and endogenous reference were determined from the appropriate standard curve. Then the amount of the target SSH clone was divided by the endogenous reference amount to obtain a normalized target value. Specificity of the amplified products was verified on 3% agarose gel and with the melting curve analysis.


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  		TABLE 1. Real-time reverse transcription-polymerase chain reaction primer sequences used for the validation of differentially expressed cDNA

Statistical Analysis

Data from quantitative real-time PCR were analyzed using the GLM Procedure of SAS [31]. For the endometrial tissue, the effect of breed (ML and LL) and gestational stage (cyclic vs. gestating) on mRNA levels from selected SSH clones were analyzed separately. For the endometrial tissue and conceptuses, a second analysis was conducted to validate our SSH clones and to assess the effect of breed on mRNA levels from selected SSH clones in gestating sows. Results are presented as least square means ± SEM and were considered to be significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SSH to Identify Genes Differentially Expressed in Meishan-Landrace Conceptuses and Endometrial Tissue at Day 15 of Gestation

The subtraction efficiency was estimated using the housekeeping gene GAPDH. In both tissues, the amount of GAPDH transcript was reduced over 100-fold after subtraction (data not shown). After cloning and transforming the subtracted cDNA, over 1000 clones from each subtracted library were picked and conserved in glycerol for further analyses. For both tissues, cDNA inserts from over 400 clones were amplified by PCR. This procedure revealed that 86% of the clones from endometrial tissue and 93% of the clones from conceptuses were positive insert-containing clones. The PCR products of 282 positives clones from each tissue were transferred onto eight identical nylon membranes (94 clones and 2 negative controls per membrane). Typical results from differential screening are shown in Figure 1. Dot blot analyses revealed a total of 137 clones (49%) for the endometrial and 166 clones (59%) for the conceptus libraries, which had a hybridization signal difference higher than 3 between ML and LL sows. All of these differentially expressed clones were then analyzed by DNA sequencing. The clone insert sizes ranged from 0.2 kilobase (kb) to 2 kb, and the average fragment insert size was ~500 base pairs (bp), which is in close agreement with that statistically predicted by RsaI digestion (~600 bp).



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  		FIG. 1. An example of colony hybridization for screening differentially expressed clones. The duplicate membranes with 96 clones were hybridized separately with the probes of (A) Meishan-Landrace cDNA fragments and (B) Landrace cDNA fragments. Example of clones detected as overexpressed in Meishan-Landrace sows when compared with Landrace sows are indicated with an arrow. Membranes hybridized with the nonsubtracted probes served as negative controls and are not shown here

We classified the cDNA sequences into different categories, according to their sequence identity with the GenBank database (Fig. 2). For the endometrial library (Fig. 2A), 66 clones had >=90% identity with known gene sequences or with apparent orthologs of genes and were considered as identified genes, and 16 clones had low homology (80–89%) with known gene sequences. Also, there were 52 novel sequences with no homology to any database entries, and 3 clones showed high homology with Sus scrofa 16S ribosomal DNA. For the conceptus library (Fig. 2B), 62 clones had >=90% identity with known gene sequences or with apparent orthologs of genes, and 26 clones had low homology (80–89%) with known gene sequences. Thirty-seven clones were novel sequences with no homology to any database entries, and 41 clones showed high homology with Sus scrofa 16S ribosomal DNA. Clones that were classified as novel genes were compared against one another to detect multiple occurrences. Five novel genes were present twice and three other novel genes were present three times. Overall, in both libraries, 51% of the cDNA contained untranslated region (UTR) sequences (5' or 3') either the partial (23%) or the complete (28%) UTR region. However, the majority of these clones had a 3' UTR region (45%), and a small number had a 5'UTR region (6%).



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  		FIG. 2. Pie diagram of the cDNA sequences found in the subtracted libraries for (A) endometrial tissue and (B) conceptuses. Identified genes, sequence with at least 90% homology to known genes (apparent orthologs of genes); low homology, sequence with 80–89% identity to known genes; novel genes, sequence having no identity to any GenBank entries or corresponding to hypothetical proteins, and repetitive DNA elements; mt 16S rRNA, mitochondrial 16S ribosomal RNA

Validation of the SSH Technique by Real-Time RT-PCR

Among all the cDNA identified by SSH, we decided to narrow the search for candidate genes to 10 specific cDNAs in each tissue. These candidate genes were chosen according to the following specific selection criteria: 1) having involvement in different pathways known or suspected to be critical for embryonic development and survival, such as apoptosis, tissue remodeling, embryonic patterning, iron transport, and lipid metabolism; 2) having the highest hybridization signal difference on dot blots between ML and LL sows. These candidate genes are listed in Table 2 (endometrial library) and Table 3 (conceptus library).


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  		TABLE 2. Differentially expressed clones in endometrial tissue when comparing Landrace and Meishan-Landrace gestating sows


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  		TABLE 3. Differentially expressed clones in conceptuses when comparing Landrace and Meishan-Landrace gestating sows

Effect of Breed on mRNA Levels of Selected SSH Clones

Real-time RT-PCR analysis was performed on both endometrial tissue and conceptuses to confirm the differential expression of the selected cDNA fragments and to assess the effect of breed on mRNA levels of selected SSH clones. The normalization performed with our two different housekeeping genes, GAPDH and Cyclophilin, gave identical results. Among the 20 clones that were validated for both tissues, 6 clones (45, 81, 163, 243, and 258 in endometrial tissue and 201 in embryonic tissue) showed higher (P < 0.05) mRNA levels in LL sows and 10 clones (183, 209, 238, and 283 in endometrial tissue and 27, 169, 277, 291, 297, and 348 in embryonic tissue) had higher (P < 0.05) mRNA levels in ML sows. Clone 17 gave no amplification signal, which might reflect its very low expression level in the conceptuses. Clones 275 in endometrial tissue and 209 in embryonic tissue showed a tendency for higher (P < 0.1) mRNA levels in the ML than in the LL sows. Among the 20 clones validated in both tissues by real-time RT-PCR, 1 clone (165 in embryonic tissue) showed no difference in mRNA levels between ML and LL sows (Table 4) and was thus considered as a false-positive clone.


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  		TABLE 4. Analysis of differentially expressed clones isolated from the SSH libraries by real-time reverse transcription-polymerase chain reaction

Effect of Pregnancy Status on mRNA Levels of SSH Clones in Endometrial Tissue

Real-time RT-PCR analyses were conducted to assess the effect of pregnancy status on mRNA levels of SSH selected clones in the endometrial tissue (Table 5). Among the 10 clones that were analyzed, clones 45, 81, 163, and 183 showed higher (P < 0.05) mRNA levels in both ML and LL pregnant sows when compared with the cyclic sows. Clones 209, 238, and 283 showed lower (P < 0.05) mRNA levels for the LL pregnant sows while their mRNA levels were not affected (P > 0.05) by the pregnancy status in the ML sows. Clone 243 showed lower (P < 0.01) mRNA levels in the pregnant ML sows, while the opposite was observed for the LL sows where the mRNA level increased (P < 0.01) with pregnancy. Clone 258 showed a higher (P < 0.01) mRNA level in the LL pregnant sows, while it remained unchanged (P > 0.05) in the ML sows. Finally, the mRNA level for clone 275 was higher (P < 0.01) for the ML pregnant sows compared with the cyclic sows, while it was not affected (P > 0.05) by pregnancy status in the LL sows.


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  		TABLE 5. Real-time reverse transcription-polymerase chain reaction analysis of gene expression in endometrial tissue from cyclic and pregnant sows


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This project was aimed at identifying 1) differentially expressed genes between highly prolific Meishan-Landrace and more conventional Landrace sows during the implantation period in conceptuses and endometrial tissue and 2) putative candidate marker genes associated with pig embryonic development and survival rate. In the present experiment, we focused on comparing prolific sows (ML) to sows that are considered to be more conventional (LL).

Sequence Analysis of Differentially Expressed Clones

Sequence analyses revealed that some of the cDNA clones expressed in conceptuses and endometrial tissue displayed significant similarity to existing sequences, whereas some clones showed no similarity with known GenBank database entries. Clones that corresponded to hypothetical proteins, repetitive DNA elements, and those with no identity to GenBank entries were considered as novel. These clones were considered novel because we could not relate them to any known functions or pathways. In the present study, it is surprising to see that such a large number of cDNA clones remains unrecognized because the number of genes in the database has grown exponentially during recent years. The most likely explanation is that a different part of the gene in question could be present in GenBank because of the frequency of partial sequences and the presence of non-full-length cDNAs in the databases. The novel cDNA may also represent unknown gene families or uncharacterized branches of known families that have not been previously identified because of their low occurrence. Alternatively, the genes in question could be subjected to alternative splicing events, which may be species-, tissue-, or stage-specific regulated. Inaccurate sequences are also a likely explanation because most Expressed Sequence Tags (ESTs) are generated by single pass sequencing of one strand of the cDNA and are therefore prone to error. Nevertheless, the percentages of novel cDNA obtained in our study are in close agreement with those that have been reported in other studies [32].

Our study revealed a high percentage of clones that corresponded to untranslated region. The 3'UTR regions are involved in the control of mRNA stability and degradation as well as in translation regulation [33]. Several studies have shown that mRNAs with a long poly(A) tail are translated with greater efficiency than those lacking poly(A) [33]. During the implantation period, there is intensive tissue remodeling in conceptuses and endometrial tissue [1, 6, 34]. Thus, mRNA must be highly available for translation in order to ensure a maximum protein synthesis. We believe that the high number of cDNA clones corresponding to mRNA untranslated sequences in conceptus and endometrial libraries may reflect these last observations.

We also found in this study a high number of clones corresponding to 16S ribosomal RNA (41 clones) in the conceptus library compared with only three clones in the endometrial library (Fig. 2). The unexpected annealing of 16S rRNA to oligo dT primers may be explained by the presence of long stretches of internal A sequences in the pig 16S rRNA transcripts [35]. On the other hand, a prior study provided evidence for polyadenylation of some human mitochondrial 16S ribosomal RNA (mt 16S rRNA) transcripts [36]. We believe that the presence of internal stretches of A in the pig 16S rRNA sequence is probably not responsible for the expression-level differences observed between Meishan-Landrace and Landrace conceptuses. A more likely explanation would be that Meishan-Landrace and Landrace conceptuses really differed from one another with respect to the abundance of 16S rRNA. The Meishan conceptuses elongate to a reduced length when compared with occidental pig conceptuses at a similar stage [37]. These development differences between Meishan and Landrace conceptuses may be responsible for the observed variation of 16S rRNA abundance. On the other hand, expression of mitochondrial 16S rRNA may be upregulated in the ML sows by a yet unknown factor that could be involved in 16S rRNA regulation. Recently, Ibrahim [35] reported that p53 can have such a regulatory effect on 16S rRNA.

Differentially Expressed SSH Clones in the Endometrial Tissue

It has been suggested that successful pregnancy in pigs is associated with intrauterine immunosuppression [38]. During the implantation period, the pig conceptus is known to secrete a high amount of interferon-{gamma} (IFN{gamma}), which seems to play an active role in helping to establish the noninvasive epitheliochorial placenta [39, 40]. Interestingly, INF{gamma} has the ability to regulate MHC class II molecules on a variety of immunologically important cells like macrophages, monocytes, and endothelial and epithelial cells [41]. Among the SSH clones that were differentially expressed between ML and LL sows, three of them (Major Histocompatibility Complex [MHC] class II SLA-DQ, STAT1, and type III collagen) seem to be related to INF{gamma} activity during the implantation period.

At Day 15 of gestation, we observed that MHC class II mRNA is higher in ML sows when compared with the LL sows, although its mRNA level is upregulated for both ML and LL sows at this stage of gestation when compared with the cyclic sows. In gestating sows, it was recently reported that the number of MHC class II-expressing cells were reduced at Days 11 and 19 of gestation in the surface epithelium [42]. These authors suggested that the porcine embryos at this stage might initiate some processes to suppress the immune response in the surface epithelium. Thus, we believe that the lower mRNA levels found for the MHC class II gene in ML sows might reflect a higher capability of suppressing the immune response during the implantation period that would prevent embryo rejection.

Our results also showed that STAT1 mRNA level is upregulated in the endometrial tissue during pregnancy (Day 15) for both ML and LL sows. Biochemical and genetic analysis has demonstrated that STAT1 is essential for gene activation in response to INF{gamma} stimulation [43, 44]. Thus, we believe that the upregulation of STAT1 mRNA observed in our study could be associated with the higher secretion of INF{gamma} by the pig conceptus during the implantation period. At Day 15 of gestation, we observed that the higher MHC class II mRNA level also accompanies higher mRNA levels for STAT1 gene. This may reflect findings reported by Muhlethaler-Mottet et al. [45], which stated that INF{gamma} activation of MHC class II expression requires the STAT1 trans acting factor. Although the differential mRNA expression level of the protein inhibitor of activated STAT1 (PIAS1) could not be confirmed by real-time PCR analyses (P = 0.085), it is interesting to mention that PIAS1 can inhibit STAT1-mediated gene activation in response to interferon [43, 46].

Our results also showed that the type III collagen mRNA level is downregulated in the endometrial tissue of Day 15 pregnant ML sows while it is upregulated in the LL sows. Shortly after the initiation of implantation (Days 7 and 8), the rat uterus responds to the presence of the implanting embryo by decreasing the concentration of type III collagen [47]. In humans, it has been reported that INF{gamma} can downregulate the synthesis of type III collagen in lung fibroblast and synovial cells [48, 49]. In the human cartilage extracellular matrix, it has been reported that INF{gamma} can suppress the expression of type II collagen gene at the transcriptional level and that this suppression requires the STAT1 protein. Based on these previous findings, we could hypothesize that the lower type III collagen mRNA level observed in endometrial tissue of ML sows may reflect higher secretion levels of INF{gamma} by the ML conceptus at Day 15 of gestation.

During pregnancy, the porcine uterus secretes several proteins into the uterine lumen that can be used to promote conceptus development [14–16, 18]. Thus, it is not surprising to identify SSH clones from the endometrial tissue whose protein can be found in uterine secretions (SERPINB7) or that is known to have specific roles in the secretory machinery (adseverin).

For both studied breeds, we observed that SERPINB7 mRNA was upregulated at Day 15 of gestation compared with the cyclic sows. This observation may be explained by the higher progesterone levels found in pregnant sows at the peri-implantation period when compared with the cyclic ones. It has been recently reported that, during pregnancy, the endometrium of the ewe secretes a progesterone-induced member of the serpin superfamily (OvUS) of serine proteinase inhibitors [50]. It was also suggested that uterine serpin may mediate the immunosuppressive effects of progesterone and prevent immunological rejection of the fetus [50]. In the pig, it was also reported that uterine endometrium locally produces protease inhibitors in order to modulate peri-implantation events and embryo-maternal communication [51]. The lower SERPINB7 mRNA levels that we observed in ML sows may be explained by a recently published article, where it was reported that Meishan gilts undergo a more gradual increase in both uterine protein secretion and estrogen secretion when compared with White crossbred gilts [20, 52]. The decreased secretion of uterine proteins in the Meishan breed may partially explain the slower embryonic development that has been reported for this breed.

Adseverin is a component of the secretory machinery [53] and seems to be restricted to tissues with high secretory activity, which is the case for the endometrium [54]. Adseverin, also known as scinderin, is a Ca2+-dependent actin filament-serving protein that is presumed to have a regulatory function in exocystosis by affecting the organization of the microfilament network underneath the plasma membrane [55]. We observed that the adseverin mRNA level was downregulated in endometrial tissue from Day 15 gestating LL sows, while it remained unchanged in the ML sows. Because there are no previous reports regarding adseverin mRNA expression in pigs, it would be premature to conclude anything about this breed discrepancy.

Among the differentially expressed genes identified in the endometrial tissue, we found that stanniocalcin (STC1) is upregulated in both ML and LL sows at Day 15 of gestation. Stanniocalcin is a glycoprotein hormone that plays a role in calcium and phosphate homeostasis [56]. In pigs, the increase of conceptus estrogen secretion, at the time of trophoblast elongation, stimulates a rapid release of calcium into the uterine lumen [57]. It has been reported that STC1 is upregulated by an increase of ambient Ca2+ concentration [58]. Thus, we believe that the estrogen-dependent calcium release observed in the pig uterine lumen during the peri-implantation period may trigger an increase of STC1 mRNA levels. Moreover, the lower STC1 mRNA levels found in the ML sows may only reflect the fact that Meishan conceptuses secrete less E2 into uterine luminal fluids, causing more gradual changes in the uterine environment, including more gradual calcium release [59].

The splicing factor, arginine/serine-rich (SFRS3) is also differentially expressed in the endometrial tissue, with higher mRNA levels in the ML sows at Day 15 of gestation. Using a mouse model, another splicing factor named SC35 has been identified when using an RNA differential display technique to identify new genes involved in implantation [60]. The SC35 splicing factor belongs to the SR proteins (rich in serine and argenine residues), which is also the case for SFRS3 splicing factor. In the mouse, the mRNA level for SC35 splicing factor appears to be upregulated by the presence of an embryo, while our results rather showed a downregulation of SFRS3 mRNA level in Day 15 pregnant LL sows or no variations in the ML pregnant sows [61]. Although nothing has been reported on SFRS3 in pigs, we know that, in the uterus, many of the genes so far identified as being important for implantation require processing of their mRNA by splicing mechanisms. Thus, it can be predicted that correct regulation of splicing factors such as SFRS3 at implantation sites may also be pivotal.

Finally, two different protein kinases (CKS1B and PRKAR1) were identified in the endometrial SSH library. Because of the lack of information regarding the effect of protein kinase modulation during the gestation period, attempts to explain the observed discrepencies for CKS1B and PRKAR1 mRNA levels between ML and LL would only be speculative. Nevertheless, we can say that protein kinase, through phosphorylation of target proteins, controls many crucial biochemical events in the cell, including regulation of metabolism, ion transport, and gene transcription.

Differentially Expressed SSH Clones in Conceptuses

Meishan embryos contain fewer cells than the Yorkshire embryos at the blastocyst stage, and this lower number of cells is due entirely to fewer trophectoderm cells [62]. Moreover, it is believed that the lower trophectoderm mitotic rate associated with the Meishan breed may limit estrogen synthesis, a limitation that could lead to decreased conceptus mortality for the Meishan less developed littermates [19]. Thus, we believe that a large part of identified SSH clones isolated from the conceptus library may be associated with the Meishan's lower growth rate. For example, SSH clone 277 (translation elongation factor 1 alpha (Eef1a)) may reflect this growth discrepancy because it was previously identified as a gene for which the expression varies with pig conceptus elongation [63]. Aside from its role in delivery of aminoacyl-tRNA (aa-tRNA) to the elongating ribosome, Eef1a protein has also been demonstrated to bind actin [64] and to sever microtubules, a prerequisite for cytoskeletal rearrangements that occur during the cell cycle [65].

The human p68 RNA-helicase is a nuclear RNA-dependant ATPase that belongs to a family of putative helicases known as the DEAD box proteins [66]. These proteins have been implicated in aspects of RNA function, including translation initiation, splicing, and ribosome assembly [67]. Interestingly, a DEAD box protein has also been identified in a porcine library prepared from elongating swine embryos [63]. Studies have shown that p68 is expressed in early development, is developmentally regulated, and appears to correlate with organ differentiation and maturation in the fetus [68, 69]. In human, it has also been reported that p68 acts as a transcriptional coactivator of estrogen receptor alpha [70].

Transcript for transferring (Tf) gene was also found to be differentially expressed in the conceptus library and retinol-binding protein (Rbp) tended (P = 0.091) to be differentially expressed between ML and LL sows. It was recently reported that the association of retinol-binding protein and transferrin in various maternal and conceptus compartments during the pregnancy protects maternal and fetal tissues from lipid peroxidation that is a possible consequence of iron transport via endometrial secretion [71]. This association would protect the developing conceptus from uncontrolled lipid peroxidation, which is known to result in cell damage and death.

Another interesting transcript identified in the conceptus library is the alpha1-acid glycoprotein (AGP1). AGP1 is one of the major acute-phase proteins in human, rats, mice, and other species [72]. In human, AGP serum concentration is found to increase in the developing fetus throughout gestation [73]. Interestingly, it has been reported that AGP gene expression can be modulated by interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFalpha), two cytokines that are present within the uterine luminal microenvironment and in the elongating porcine conceptuses [74, 75]. These findings support an anti-inflammatory and immunomodulating role for AGP protein.

Transcript for fibronectin 1 (FN1) was also identified as differentially expressed in the conceptus library. Human studies suggest the involvement of FN1 in mediating attachment, trophoblast spreading, and syncytial formation [76, 77]. Moreover, Tuo and Bazer reported the constitutive expression of fibronectin throughout gestation in the pig conceptus and uterus [78]. The presence of fibronectin at the fetal-maternal interface suggests a role in conceptus interactions and attachment to the endometrium.

Sterol carrier protein 2 (SCP2) was also differentially expressed in the conceptus library. SCP2 is a peroxisomal protein that stimulates various steps of cholesterol metabolism and function as a fatty acyl-CoAs carrier [79]. It has been reported that PGF2{alpha} can downregulate the SCP2 mRNA transcript in the ovary [80]. Because PGF2{alpha} is detected in the pig uterine lumen during early pregnancy [81], we could hypothesize that conceptus expression of SCP2 is regulated by PGF2{alpha} during the peri-implantation period.

Finally, NICE-5 protein was also identified in the conceptus library. This protein was recently characterized in human epidermal differentiation complex [82]. Because its function remains unknown, this protein could be considered as a novel uncharacterized protein. Nevertheless, all we know from Database searches is that NICE-5 is also expressed in many other tissues, such as placenta, blastocyst, embryonal carcinoma, pooled germ cell tumors, and many more.

In this study, we have isolated novel and known cDNA differentially expressed in conceptuses and endometrial tissue of prolific Meishan-Landrace and more conventional Landrace sows at Day 15 of gestation. Because the Meishan breed is recognized for its higher embryonic survival rate, these genes could become putative markers that may be resolved in further studies and eventually used in selection for high litter size breeds. Because this study was performed on a limited number of pigs, further confirmation will be needed on a much larger population and at various gestation stages to confirm these results. Also, further analysis of the expression pattern of these genes during the implantation period should facilitate the understanding of the molecular characteristics of this period, which is a critical time for embryonic survival.


   ACKNOWLEDGMENTS
 
We thank D. Morissette, F. Phaneuf, E. Bérubé, M. Turcotte, C. Mayrand, J. Boudreau, A. Marsh, and F. Champagne for animal care and sample collection; Steve Methot for statistical analysis; and Dr. Susan Novak for critical reading of the manuscript.


   FOOTNOTES
 
1 This work was supported by Genetiporc Inc. (St-Bernard, QC, Canada) and Agriculture and Agri-Food Canada. Lennoxville Dairy and Swine R&D Centre contribution 799. M.V. is supported by a FCAR fellowship. Back

2 Correspondence: Marie-France Palin, Agriculture and Agri-Food Canada, P.O. Box 90, 2000 Route 108 East, Lennoxville, Quebec, J1M 1Z3 Canada. FAX: 819 564 5507; palinmf{at}agr.gc.ca Back

Received: 12 May 2003.

First decision: 29 May 2003.

Accepted: 8 July 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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