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Biology of Reproduction 67, 1259-1267 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Effects of Breed, Parity, and Folic Acid Supplement on the Expression of Folate Metabolism Genes in Endometrial and Embryonic Tissues from Sows in Early Pregnancy1

Maud Valléea, Frédéric Guayb, Danièle Beaudryc, Jacques Mattec, Richard Blouina, Jean-Paul Laforestb, Martin Lessardc, and Marie-France Palin2,c

a Département de Biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada J1K 2R1 b Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales Université Laval, Sainte-Foy, Québec, Canada G1K 7P4 c Dairy and Swine Research and Development Center, Agriculture and Agri-Food Canada, Lennoxville, Québec, Canada J1M 1Z3


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Folic acid and glycine are factors of great importance in early gestation. In sows, folic acid supplement can increase litter size through a decrease in embryonic mortality, while glycine, the most abundant amino acid in the sow oviduct, uterine, and allantoic fluids, is reported to act as an organic osmoregulator. In this study, we report the characterization of cytoplasmic serine hydroxymethyltransferase (cSHMT), T-protein, and vT-protein (variant T-protein) mRNA expression levels in endometrial and embryonic tissues in gestating sows on Day 25 of gestation according to the breed, parity, and folic acid + glycine supplementation. Expression levels of cSHMT, T-protein, and vT-protein mRNA in endometrial and embryonic tissues were performed using semiquantitative reverse transcription-polymerase chain reaction. We also report, for the first time, an alternative splicing event in the porcine T-protein gene. Results showed that a T-protein splice variant, vT-protein, is present in all the tested sow populations. Further characterizations revealed that this T-protein splice variant contains a coding intron that can adopt a secondary structure. Results demonstrated that cSHMT mRNA expression levels were significantly higher in sows receiving the folic acid + glycine supplementation, independently of the breed or parity and in both endometrial and embryonic tissues. Upon receiving the same treatment, the vT-protein and T-protein mRNA expression levels were significantly reduced in the endometrial tissue of Yorkshire-Landrace sows only. These results indicate that modulation of specific gene expression levels in endometrial and embryonic tissues of sows in early gestation could be one of the mechanism involved with the role of folic acid on improving swine reproduction traits.

early development, embryo, female reproductive tract, pregnancy, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Folic acid (B9) is now recognized as a factor of great importance in the control of sows' prolificacy [1]. In sows' early gestation, a critical period for recognition of pregnancy and embryonic survival, there is a decrease of serum folates, suggesting a temporary folate deficiency [2]. It is now hypothesized that these folates are quickly used by the conceptuses to sustain the rapid cellular division since B9 plays an essential role in DNA and RNA synthesis [3]. Matte et al. [1] showed that B9 supplements, given to multiparous gestating sows, can increase litter size by 10% through a decrease in embryonic mortality during early pregnancy [4]. However, a B9 supplement affects litter size of nulliparous sows to a lesser extent than multiparous sows, suggesting differences in the metabolism of folate in early gestation according to the parity of the sows [5, 6].

Metabolic provision of glycine to the uterus is also an important factor in sows' prolificacy. In sows, it has been reported that glycine is the most abundant amino acid in oviductal, uterine [7], and allantoic fluids [8]. Moreover, Wu et al. [9] showed that the glycine contribution to amino acid composition in fetal pigs increases during gestation. Although the role of glycine in early gestation has not been clearly elucidated yet, there is accumulating evidence that it may act as an organic osmoregulator both in mouse [10] and bovine embryos [11]. Because glycine catabolism provides a methyl group to B9 for DNA synthesis and for methylation of homocysteine [12], glycine levels in the uterus should also be adequate for an optimal folate response.

To better understand the possible role of B9 and glycine in early pregnancy, two enzymes, involved in folate metabolism, have been studied (Fig. 1). The first one is the T-protein, a component of the glycine cleavage enzyme system (GCS), which catalyzes the formation of ammonia and 5,10-methylenetetrahydrofolate (5,10-methylene-THF) from glycine and tetrahydrofolate [13]. The glycine cleavage system represents the major pathway of glycine catabolism in vertebrates, which consists of glycine decarboxylase (P-protein), H-protein, T-protein, and poamide dehydrogenase [14–16]. The second enzyme studied is the cytoplasmic serine hydroxymethyltransferase (cSHMT), an enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methylene-THF [17]. Aside from its well-known involvement in folate metabolism, the rationale for studying cSHMT mRNA expression levels was based on previous observations that sheep cSHMT displays tissue- and stage-specific expression, varying throughout gestation and rising after birth [18]. The investigation of T-protein mRNA profiles according to the parity or genotype of the sows was chosen mainly because its tissue-specific expression matches the cSHMT expression profile, suggesting that these two enzymes may work in concert [19].



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  		FIG. 1. Schematic representation of the cytoplasmic folate metabolism. Folate metabolism is required for the synthesis of purines, thymidylate, and methionine in the cytoplasm. cSHMT, Cytoplasmic serine hydroxymethyltransferase; GCS, glycine cleavage system (which includes T-protein); THF, tetrahydrofolate

With this study, we wanted to estimate the effects of B9 + glycine supplement on the expression levels of folate metabolism genes in endometrial and embryonic tissues from sows on Day 25 of gestation. With this objective in mind, we discovered a splice variant for the porcine T-protein gene. We report here the characterization of a T-protein splice variant (vT-protein). The effects of B9 + glycine supplement on the mRNA expression levels of cSHMT, T-protein, and vT-protein genes were then examined. The effect of sow genotype on cSHMT, T-protein, and vT-protein mRNA expression was also verified by comparing Chinese Meishan-Landrace genotype, known for their higher litter size and lower embryonic mortality [20], with occidental Yorkshire-Landrace sows. The parity effect on cSHMT, T-protein, and vT-protein mRNA expression was verified as well. This information should be useful for characterizing part of the folate pathway and for better understanding the possible role of folic acid and glycine in early gestation of pigs.


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

Twenty-two multiparous Meishan-Landrace sows (ML; 2–3 parities, 193 ± 3 kg), 22 multiparous Yorkshire-Landrace sows (YL; 4–5 parities, 212 ± 2 kg), and 22 nulliparous Yorkshire-Landrace gilts (GT; 124 ± 2 kg), generously provided by Genetiporc (St.-Bernard, QC, Canada), were selected. They were fed an experimental diet with or without supplement of B9 and glycine (15 ppm B9 and 0.6% glycine) as described previously [21]. Glycine was added in order to prevent any possible short-term suboptimal provision of this amino acid, especially for the local uterine metabolism pool [7, 22]. Sows were inseminated twice with pooled semen from Duroc boars of proven fertility (graciously provided by CIPQ, Inc.) 12 and 24 h after estrus detection, which was considered as Day 0 (d0). Sows were slaughtered on Day 25 of gestation, a time that allowed us to assess embryonic survival rate and litter size. The reproductive tract was removed and the embryos were counted and collected. Ovaries were dissected to count the number of corpora lutea CL. Samples of endometrial and embryonic tissues were collected and frozen in liquid nitrogen as previously described [21]. Animals were cared for according to the recommended code of practice [23]. They were killed using a penetrating bolt pistol as an acceptable method approved by the local Animal Care Committee following the guidelines of the Canadian Council on Animal Care [23].

RNA Extraction and Complementary DNA Preparation

Total RNA was extracted from the endometrial tissue of gestating sows and from homogenates of embryonic tissues. Briefly, 200 mg of tissue were extracted with 2 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.

Total RNA was reverse transcribed to cDNA in a PTC-200 Programmable Thermal Controller (MJ Research, Foster City, CA). For all tissue samples, 5 µg of RNA were treated with three units of Dnase I (amplification grade #8068-015; Gibco BRL) to remove contaminating genomic DNA. First-strand cDNA was synthesized using a SuperScriptTM II preamplification system (Gibco BRL) and 500 ng of oligo(dT)12-18 as primer (Pharmacia Biotech, Baie D'Urfé, QC, Canada) in a total volume reaction of 50 µl.

Polymerase Chain Reaction Amplification and Semiquantitative RT-PCR

Polymerase chain reaction (PCR) amplification of cSHMT, T-protein, and vT-protein cDNAs was performed with forward and reverse primers listed in Table 1 (cSHMT, 1-TP, and 2-TP). Locations of primers used for all T-protein amplification are given in Figure 2. A 2-µl aliquot of the reverse transcriptase product from endometrial and embryonic tissues was subjected to PCR amplification in a PTC-200 Programmable Thermal Controller (MJ Research). The 100-µl PCR reaction contained MgCl2 (1.0 or 1.5 mM), forward and reverse primers (15 or 30 pmol each), 200 µM dNTPs, and 2.5 units of Taq polymerase in 1x Taq polymerase buffer (Amersham Pharmacia Biotech). The PCR profile consisted of an initial denaturation step at 94°C for 2 min, followed by an optimal number of three-step cycles: denaturation at 94°C for 1 min, annealing at an optimal temperature for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 5 min. Optimal PCR conditions (listed in Table 1) were defined for each amplification as those that generated a single PCR product of the predicted base-pair length.


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  		TABLE 1. PCR primer sequences and optimal PCR conditions for GAPDH, cSHMT, T-protein, and vT-protein genes.a



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  		FIG. 2. Approximate positions of primers used to amplify and sequence porcine T-protein gene. Residue numbers are given according to the porcine vT-protein sequence (GenBank accession no. AF239168). Dotted lines represent the retained intron found in the vT-protein sequence. (-1-) 1-TP primers used for the semiquantitative RT-PCR of T-protein; (-2-) 2-TP primers used for the amplification of T-protein splice variants; (-3-) 3-TP primers used for the sequencing of vT-protein; (-4-) 4-TP primers used for the genomic sequencing of T-protein; (-5-) 5-TP primers used for the semiquantitative RT-PCR of vT-protein

Quantification of cSHMT, T-protein, and vT-protein mRNA levels in endometrial and embryonic tissues was performed by semiquantitative reverse transcription-PCR (RT-PCR). Quantification of pig glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels in both tissues was also performed as an internal control of amplification. The PCR primer sequences, optimal PCR conditions, and the expected size of amplified fragments of GAPDH, cSHMT, 1-TP, and 5-TP primers are listed in Table 1. A 2-µl aliquot of the reverse transcriptase product from endometrial and embryonic tissues was subjected to PCR amplification as previously described. The amplified PCR fragments were electrophoresed on a 2.0% agarose gel and stained with ethidium bromide. Pictures of the resulting gels were taken with Polaroid positive-negative film (#55). Negatives were then scanned using an Imaging Densitometer (Model GS-670; Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). Five aliquots (1, 2, 3, 4, and 5 µl) of the PCR products were amplified for a fixed number of cycles to ensure analysis of the products in the exponential range of amplification. The relative density of the transcripts was expressed as arbitrary optical units. A ratio of the optical density of the cSHMT, T-protein, and vT-protein transcripts standardized with the GAPDH transcript was calculated before statistical analyses were performed to correct for possible differences in cDNA synthesis. Each PCR amplification was repeated twice and the average values from the experiments were used in the final calculation. A control amplification of GAPDH, cSHMT, T-protein, and vT-protein mRNA that corresponded to a single animal was also carried out on each gel to correct for possible migration or staining variations.

Sequencing

For each gene, the nucleotide sequence of the amplified fragment was determined by cycle-sequencing five different PCR reactions in both directions with the same primers used for PCR amplification (Table 1). The amplified PCR fragments were electrophoresed on a 1% agarose gel, then excised from it and purified with a GENECLEAN II Kit (BIO 101 Inc., La Jolla, CA). Sequence determination was performed using the Big Dye Terminator Cycle Sequencing Ready Reactions (PE Applied BioSystem, Foster City, CA) according to the manufacturer's instructions and run on a Perkin Elmer Applied BioSystems automated 377 DNA sequencer.

T-Protein Splice Variants

For endometrial and embryonic tissues, a set of primers (2-TP; Table 1) was used to amplify the T-protein splice variants. A 2-µl aliquot of reverse transcriptase product from these tissues was subjected to PCR amplification as described previously. Optimal PCR conditions for the amplification of T-protein DNA fragments are listed in Table 1. The amplified PCR fragments were electrophoresed on a 1% agarose gel and stained with ethidium bromide. These same PCR fragments were also electrophoresed on denaturing condition on a 6% polyacrylamide/bis (29:1) gel containing 40% urea and visualized with ethidium bromide. PCR amplification of T-protein splice variants was also carried out on different tissues (ovaries, mammary gland, muscle, backfat, liver, kidney, heart, lung, and stomach) using the same primers and the same PCR conditions used for endometrial and embryo tissues (Table 1) and subsequently electrophoresed on a 1% agarose gel.

The amplified T-protein fragments (fragment a; Fig. 3A) and vT-protein PCR fragments (fragments b and c; Fig. 3A) from endometrial and embryonic tissues were excised separately from 1% agarose gel and purified with a GENECLEAN II Kit (BIO 101 Inc.). The nucleotide sequence of these fragments was determined by cycle-sequencing five different PCR reactions from Meishan-Landrace and Yorkshire-Landrace sows in both directions with 2-TP primers (Table 1) used previously for PCR amplifications of T-protein splice variants. Sequencing was performed as described previously. A second reaction of cycle sequencing for vT-protein (Fig. 3A; fragment b and c) was carried out for both tissues with another 3-TP set of primers (Table 1) that was designed from the T-protein sequence previously obtained with 2-TP primers. The T-protein mRNA sequences obtained were submitted to GenBank (Sus scrofa glycine cleavage system T-protein mRNA, accession no. AF239167 [fragment a; Fig. 3A] and Sus scrofa similar to glycine cleavage system T-protein mRNA sequence, accession no. AF239168 [fragments b and c; Fig. 3A]).



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  		FIG. 3. Tissue distribution of T-protein splice variants. A) Fragments a, b, and c, amplified with primers 2-TP (F) and (R). B) Relative mRNA expression of T-protein (both variants are amplified without distinction with forward and reverse 1-TP primers). C) Amplification of GAPDH used an internal control to determine the quality of total RNA

Genomic DNA was extracted from whole blood Meishan-Landrace pigs using a Genomic DNA Purification Wizard Kit (Promega, Madison, WI) according to the manufacturer's instructions. An additional step of RNase treatment was performed to ensure that no RNA contamination was present. Genomic DNA amplification of T-protein was carried out with the 4-TP primers located in exon 5 and exon 6 (Table 1). These primers were designed from the vT-protein sequence (GenBank accession no. AF239168). Fifty nanograms of genomic DNA were subjected to PCR amplification as described previously (sequencing of RT-PCR product). Optimal PCR conditions are listed in Table 1. The amplified PCR fragment was excised from 1% agarose gel and purified with a GENECLEAN II Kit (BIO 101 Inc.). The nucleotide sequence of this fragment was determined by cycle sequencing five different PCR reactions in both directions as described previously with the same primers used for PCR amplification (4-TP; Table 1).

Statistical Analysis

Data were analyzed using the mixed procedure of the SAS [24] according to a 3 x 2 factorial arrangement in a random design with type of sow (YL vs. GT, parity effect; YL vs. ML, genotype effect) and B9 + glycine supplement as the two main independent factors. For expression of endometrial and embryonic cSHMT, T-protein, and vT-protein genes, a logarithmic transformation was done to normalize experimental errors. Statistical analyses were considered to be significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sequencing of RT-PCR Products

Using RT-PCR on total RNA isolated from embryonic and endometrial tissues, partial cDNA coding sequences for cSHMT (GenBank accession no. AF239165) and T-protein (GenBank accession no. AF239167) genes were obtained. Similarity analysis showed that the PCR product for porcine cSHMT is 88% identical to the sheep [17], human [25], and rabbit [26] nucleotide sequences. The deduced amino acid sequence showed identities of 96%, 95%, and 94% between porcine cSHMT and cSHMT from sheep [17], human [25], and rabbit [26], respectively. Analysis of the T-protein nucleotide sequence (fragment a; Fig. 3A) showed that the porcine PCR product is 89% and 92% identical to the human [27] and bovine [13] sequences, respectively. Analysis of T-protein-deduced amino acid sequence showed a 92% and 94% identity when compared with the same species [13, 27].

T-Protein Splice Variants

Unexpectedly, three PCR products (fragments a, b, and c; Fig. 3A) resulted from the amplification of T-protein with 2-TP primers (Table 1). The characterization of these three products was then performed. The PCR product with the strongest intensity (fragment a) is the product with the expected length, ~600 base pairs (bp). The nucleotide sequence of these three fragments was determined by cycle sequencing and revealed only two different sequences. The T-protein sequence obtained from fragment a showed 89% similarity to the human T-protein nucleotide sequence. The second T-protein nucleotide sequence revealed that fragments b and c shared the same sequence and both showed a 288-bp insertion (Fig. 4; vT-protein). Splice site acceptor and donor consensus sequences are present in the 5' (AG: GUAAGC) and 3' (CCAG: GUCCC) regions of this 288-bp insertion (Fig. 4). Sequencing of porcine genomic DNA with 4-TP primers (Table 1) revealed that this 288-bp insertion, in which three premature stop codons are present, corresponds to a retained intron located between exons 5 and 6 of the T-protein gene. Exon numerical assignments for the porcine T-protein gene correspond to the previously reported human T-protein gene assignments (GenBank accession no. D14681).



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  		FIG. 4. Alignment of T-protein splice variant sequences. Splice site acceptor and donor consensus sequences in 5' and 3' are given in bold and the three premature stop codons are underlined. Residue numbers are indicated on the right according to the T-protein sequence (fragment a) (GenBank accession no. AF239167) and similar to T-protein sequences (fragments b, c) (GenBank accession no. AF239168)

The vT-protein sequence that includes the 288-bp insertion can adopt a secondary structure that accelerates its migration on nondenaturing agarose gel, leading this sequence to migrate at two different heights, whether it adopts the secondary structure (fragment b) or not (fragment c). When fragments b and c are electrophoresed on denaturing acrylamide gel, only one PCR product is observed (fragment c) (data not shown).

The T-protein splice variants were amplified from sows' endometrial and embryonic tissues of different breeds and parities (data not shown) and from different tissues (Fig. 3A). In all cases, fragments a, b, and c were amplified and showed the same migration pattern. The only difference was within the ratio of the three PCR products. For example, in the stomach, the intensity of the three fragments seems equivalent, while in the mammary gland, fragment a is much more intense than fragments b and c (Fig. 3A). A PCR reaction performed with (F) and (R) primers 1-TP (Table 1) enabled us to amplify both splice variants simultaneously in order to see overall T-protein expression in different tissues (Fig. 3B).

Expression of cSHMT mRNA in Porcine Endometrial and Embryonic Tissues

Expression levels of cSHMT mRNA in endometrial (Table 2, Fig. 5) and embryonic (Table 2, Fig. 6) tissues were evaluated in ML, YL, and GT sows in early pregnancy receiving different experimental diets. Independent of breed and parity, the B9 + glycine supplementation significantly increased cSHMT mRNA expression levels in endometrial tissue (treatment, P < 0.0001) (Table 2, Fig. 5) as well as in embryonic tissue (treatment, P < 0.0001) (Table 2, Fig. 6). The cSHMT expression levels was also found to be higher in YL than in ML, and this difference was significant in endometrial tissue (breed, P < 0.05) (Table 2, Fig. 5, A and B).


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  		TABLE 2. Effect of parity, breed, and dietary supplementation of folic acid + glycine on expression levels of cSHMT, T-protein, and vT-protein mRNA in endometrial and embryonic tissues.a,b



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  		FIG. 5. Effect of parity, breed, and dietary supplementation of folic acid + glycine on expression levels of cSHMT, T-protein, and vT-protein mRNA in endometrial tissues. A) Multiparous Meishan-Landrace sows (ML). B) Multiparous Yorkshire-Landrace sows (YL). C) Nulliparous Yorkshire-Landrace sows (GT). Each column represents the average value for a specific group of sows (n = 11). The highest mRNA expression value for each gene was considered as 100% and transcription levels of all pigs were adjusted relative to this pig. The relative density of the transcripts was expressed as arbitrary optical units. A ratio of the optical density of the cSHMT, T-protein, and vT-protein transcripts standardized with the GAPDH transcript was calculated before statistical analyses were performed to correct for possible differences in cDNA synthesis. Each PCR amplification was repeated twice and the average value from each individual experiment was used in the final calculation. A control amplification of GAPDH, cSHMT, T-protein, and vT-protein mRNA that corresponded to a single animal was also carried out on each gel to correct for possible migration or staining variations. P values corresponding to these data are given in Table 2. cSHMT, Cytosolic serine hydroxymethyltransferase; T-protein, T-protein fragments a, b, and c; vT-protein, T-protein fragments b and c. Animals were fed an experimental diet with or without (0) a supplement of B9 + glycine (15 ppm B9 and 0.6% glycine). Least-squares mean ± SEM



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  		FIG. 6. Effect of parity, breed, and dietary supplementation of folic acid + glycine on expression levels of cSHMT, T-protein, and vT-protein mRNA in embryonic tissues. A) Multiparous Meishan-Landrace sows (ML). B) Multiparous Yorkshire-Landrace sows (YL). C) Nulliparous Yorkshire-Landrace sows (GT). Each column represents the average value for a specific group of sows (n = 11). The highest mRNA expression value for each gene was considered as 100% and transcription levels of all pigs were adjusted relative to this pig. The relative density of the transcripts was expressed as arbitrary optical units. A ratio of the optical density of the cSHMT, T-protein, and vT-protein transcripts standardized with the GAPDH transcript was calculated before statistical analyses were performed to correct for possible differences in cDNA synthesis. Each PCR amplification was repeated twice and the average value from each individual experiment was used in the final calculation. A control amplification of GAPDH, cSHMT, T-protein, and vT-protein mRNA that corresponded to a single animal was also carried out on each gel to correct for possible migration or staining variations. P values corresponding to these data are given in Table 2. cSHMT, Cytosolic serine hydroxymethyltransferase; T-protein, T-protein fragments a, b, and c; vT-protein, T-protein fragments b and c. Animals were fed an experimental diet with or without supplement of B9 + glycine (15 ppm B9 and 0.6% glycine). Least-squares mean ± SEM

Expression of T-Protein and vT-Protein mRNA in Porcine Endometrial and Embryonic Tissues

Two different experiments were conducted to analyze T-protein expression levels. In the first experiment, the RT-PCR amplification was performed using 1-TP primers (Table 1) to analyze expression levels of both T-protein fragments simultaneously. In the second experiment, the RT-PCR amplification was performed with 5-TP primers (Table 1) to analyze expression levels of vT-protein only. Concerning T-protein mRNA expression levels in endometrial tissue, there was an effect of B9 + glycine supplementation (treatment, P < 0.0001) (Table 2, Fig. 5), and this effect was also observed for vT-protein mRNA expression levels (treatment, P < 0.01) (Table 2, Fig. 5). The treatment effect varied according to breed and parity of sows (Table 2, Fig. 5). T-protein mRNA expression levels in endometrial tissue were lower when YL and ML sows received this treatment, and the reduction was more pronounced in YL sows than in ML sows (breed x B9 + glycine, P < 0.05) (Table 2, Fig. 5, A and B). The B9 + glycine effect was also dependent on the parity of sows; T-protein mRNA expression levels in endometrial tissues increased slightly in GT sows while they decreased significantly in YL sows (parity x B9 + glycine, P < 0.0001) (Table 2, Fig. 5, B and C). Variations in treatment effect according to the breed and parity of sows were also observed for vT-protein expression levels in endometrial tissue (Table 2, Fig. 5). The mRNA expression levels of T-protein and vT-protein were always affected in the same way by the B9 + glycine treatment, both in endometrial and embryonic tissues.

There were no significant parity or breed effects on T-protein and vT-protein mRNA expression levels in embryonic tissue. The B9+ glycine supplementation increased vT-protein expression levels in all groups of sows (treatment, P < 0.05) (Table 2), and this effect was more marked in ML sows than in both groups of Yorkshire-Landrace sows, i.e., YL and GT (Fig. 6).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, this article provides the first report on partial sequences of porcine cSHMT, T-protein, and vT-protein cDNAs. This study also reports the characterization of an alternative splicing event in the porcine T-protein mRNA.

The reported porcine T-protein and vT-protein mRNA sequences are identical except for the inclusion in the vT-protein sequence of a 288-bp coding intron located between exons 5 and 6. The divergent region of vT-protein cDNA was identified as an intronic sequence when compared with the genomic DNA sequence of porcine T-protein. Indeed, sequences homologous to donor-acceptor sites (GT-AG) were identified at the boundaries of this region, suggesting that this extra sequence corresponds to a retained intron. Retention of a coding intron, resulting in a unique isoform, has been previously described for numerous genes such as rat Id1 (inhibitor of DNA binding) [28], Id3 [29], and mouse KOR-3e (opioid receptor) [30]. It has been reported that translation of intron-containing RNAs could result in the production of a nonfunctional, dominant, negative loss or gain of function proteins [31]. This T-protein variant could also be a semiprocessed pseudogene, although few of these appear to be transcribed [32]. A possible case of a functioning pseudogene transcript has been described recently for neural nitric oxide synthase, where this pseudogene can interfere through RNA duplex formation with the expression of nitric oxide synthase [33].

The retained intron encodes a unique 96-amino acids sequence with three potential stop codons. Generally, the termination codon of most intron-containing genes resides within the last exon [34]; thus, termination codons located upstream of the last exon-exon junction are prone to mediate a reduction in mRNA abundance [31]. In support of this last statement, we observed that the mRNA expression levels of vT-protein were lower than the expression levels of fully spliced T-protein (fragment a) in most tissues. Alternatively, Pan et al. [30] suggested that the presence of an alternative start codon located upstream of a retained intron that contains a stop codon could result in a newly functional protein by changing the reading frame of the protein.

Results presented herein suggest that the T-protein splicing mechanism differs between pig and human, and, to our knowledge, no such report has been made for human T-protein gene. In this study, we observed that vT-protein transcripts can adopt a secondary structure, suggesting that a different conformation might have an impact on the splicing machinery and modulate the T-protein splicing event. It has been reported that the abolishment of a hairpin structure can affect the splicing machinery of the human dystrophin gene [35]. Libri et al. [36] also reported that, in the human ß-tropomyosin gene, regulation of splicing is due in part to a secondary structure of the primary transcript. The function of the alternatively spliced vT-protein variant, if any, remains unknown.

In the present study, the vT-protein mRNA is always detectable, no matter the breed, parity, or B9 + glycine supplementation. Differences were noted in the ratio between T-protein (fragment a) and vT-protein (fragments b and c) mRNAs among tissues, suggesting a possible tissue-specific regulation or differences in mRNA stability. Interestingly, Xia et al. [37] have studied the mRNA expression of porcine T-protein in oocytes, cumulus cells, granulosa cells, and oviductal epithelial cells using RT-PCR and have made no mention of the presence of a splice variant in oviductal epithelial cells, although two PCR products are clearly seen on their polyacrylamide gel.

The present study is also a first report on expression levels of cSHMT, T-protein, and vT-protein in endometrial and embryonic tissues of early pregnant sows. The higher levels of cSHMT mRNA both in endometrial and embryonic tissues in all groups of sows receiving the treatment suggest that a supplementation of B9 + glycine can influence cSHMT regulation at the transcriptional level. It was previously reported that availability of nutrients such as folic acid can regulate SHMT activity in chickens [38]. Currently, the metabolic role of the individual SHMT isoenzymes is not completely understood. Some results suggest that mitochondrial SHMT (mSHMT) is responsible for glycine synthesis, whereas the cytoplasmic SHMT is involved in thymidylate, purine, and methionine synthesis [39]. Our study focused on the cSHMT isoform because it seems to be a key regulator in folate metabolism [40] and may work in tandem with the glycine cleavage system [19]. The rationale for working with cSHMT is based on the observation that it appears to display tissue-specific expression, whereas mSHMT expression displays similar levels in all tissues [19]. Moreover, in fetal ovine liver, cSHMT RNA levels vary throughout gestation, rising after birth, while mSHMT levels are constant throughout gestation [18]. Furthermore, similar tissue-specific expressions of GCS enzymes and cSHMT suggest that these enzymes may be involved in a glycine metabolizing pathway [19].

As for protein activity in ovine liver over the gestational period, cSHMT accounts for the majority of SHMT activity [18]. The cSHMT may influence protein expression by directly modifying the translation of some RNA species, including its own mRNA, since it has been recently demonstrated that cSHMT is a mRNA-binding protein [41].

Our data showed that sows receiving the B9 + glycine supplementation had higher expression levels of cSHMT in the embryonic tissue, a result in agreement with the concomitant increase of folate status in sows and embryos, the decrease in embryo homocysteine, and the improved embryo development (DNA and protein in YL sows) [42]. It seems that cSHMT mRNA expression levels may have an impact on the embryonic homocysteine levels, thus favoring the conversion of homocysteine to methionine, an essential metabolism for conceptus integrity [43–45]. Folic acid alone is likely to be mainly responsible for the B9 + glycine effect on cSHMT. First, folic acid can cross the placental barrier in sows [46]. Second, the transfer of glycine in utero is likely to be small (if any occurs at all) because, as Wu et al. [22] reported, glycine does not cross the porcine placental barrier at times slightly later than in the present experiment, i.e., from Day 30 to Day 110 of pregnancy. Finally, if glycine were critical for the expression of GCS and cSHMT, the presence of exogenous glycine would have stimulated GCS (glycine catabolism) and inhibited cSHMT (glycine metabolic synthesis from serine). However, in contrast, GCS was not affected (or decreased in YL) by the B9 + glycine treatment while cSHMT was stimulated in all sows; this last effect on cSHMT is in agreement with the reduction of allantoic contents in serine (data not shown) [42].

The central role of SHMT isoenzymes is to produce one-carbon-substituted folate cofactors [47] used in thymidylate, methionine, lipid, and purine biosyntheses. These metabolisms are of great importance for the rapidly developing embryo. Evidence has been reported suggesting that SHMT plays a critical role in cell division and proliferation, as its activity is increased during the S phase of the cell cycle [48] and SHMT levels are found to be elevated in rapidly proliferating cell lines and tumors [49, 50].

The human proximal promoter region of the cSHMT gene contains a number of consensus regulatory motifs, including two potential steroid response elements, mammary and neuronal-specific elements, whose activity is influenced by retinoic acid and its related receptors [19]. The higher endometrial expression levels of cSHMT mRNA in YL compared with ML sows may be explained by the different hormonal status observed between Meishan and conventional breeds such as Yorkshire. Hypercortisolism was previously demonstrated, using plasma measurements, in Meishan pigs compared with a white breed [51–53]. There is also evidence that cSHMT is hormonally regulated because SHMT activity is elevated in the uterus after injection of 17ß-estradiol to ovariectomized rats, whereas testosterone increases the specific activity of SHMT in the prostate [54]. It was also previously demonstrated that concentrations of calcium, total protein, retinol-binding proteins, and IGF-1 in uterine flushings from Meishan sows were significantly lower compared with Yorkshire females on Day 12 of gestation [55, 56]. No significant breed effect was observed in embryonic tissue, although the high variation found in the ML group may account for this observation.

Results from this study have shown that the B9 + glycine supplement significantly affected expression levels of T-protein and vT-protein mRNA in endometrial tissue. However, looking at each group of sows individually, we observed that the B9 + glycine supplement reduced T-protein and vT-protein mRNA expression only in the YL group. Because T-protein and vT-protein were similarly affected by the B9 + glycine supplement, the following comments on T-protein will stand for both T-protein and vT-protein. These different effects of the treatment on T-protein mRNA expression according to the breed or parity of sows could be explained by the different nutritional statuses observed between ML, YL, and GT sows. In a previous study, results obtained with the same sow populations showed higher levels of serum vitamin B12 in YL than in ML and GT sows [42]. Moreover, a breed effect was observed for vitamin B12 concentrations in allantoic fluid, with higher levels in YL than in ML sows [42]. The higher vitamin B12 status found in YL sows could possibly contribute to a more efficient use of vitamin B9. We also found that YL sows had lower levels of homocysteine in endometrial tissue than ML sows [42]. All these observations point toward a more efficient folate metabolism in the YL sows. This could, in part, explain an increased litter size through folic acid supplementation seen mostly in YL multiparous sows [1, 5, 46]. A more efficient folate metabolism can generate more S-adenosylmethionine (SAM), a molecule involved in DNA methylation [57]. The pattern of methylation of DNA is inheritable as well as being tissue and species specific [58]. Knowing that the DNA methylation pattern is critical for regulation of gene expression [59], we believe that the B9 + glycine treatment could modulate the T-protein expression levels according to the availability of SAM molecules.

The presence of glycine in the supplement might be responsible for the modulation of the expression levels of T-protein in the endometrial tissue by regulating its expression at the transcriptional level. It has been reported that in vivo administration of glycine is responsible for down-regulation of tumor necrosis factor-alpha (TNF-{alpha}) activity in rat hepatocytes [60]. Moreover, Spittler et al. [61] also reported that glycine reduced TNF-{alpha} and interleukin-1ß production in whole blood assay.

The B9 + glycine treatment did not affect the expression levels of T-protein mRNA in embryonic tissues. On the other hand, we observed that the treatment affected the mRNA expression levels of vT-protein despite the fact that both variants follow the same expression profile in this tissue. In sheep, there is relatively little transplacental transport of glycine across the epitheliochorial placenta; thus, the majority of the fetal glycine requirement must be met by endogenous fetal production [22]. In pigs, Wu et al. [22] reported that a high rate of glycine synthesis is likely to occur in the fetus because glycine concentrations are relatively low in the umbilical vein. Based on this information, results suggest that supplementation had no effect on the embryo GCS metabolism in our population.

In conclusion, the present article reports the characterization of an alternative splicing event in porcine T-protein gene that generates a splice variant containing a coding intron. The B9 + glycine supplement increased cSHMT mRNA expression levels in endometrial and embryonic tissues in all groups of sows. On the other hand, expression levels of T-protein and vT-protein mRNA in endometrial tissue were reduced in YL sows receiving the treatment. The results presented herein focused on cSHMT, T-protein, and vT-protein mRNA quantity only. Therefore, a role for the quality or stability of transcripts cannot be excluded as a possible explanation for the observed variations. Finally, results presented herein focused on mRNA expression levels only. Thus, it will be interesting to further understand the effects of B9 + glycine treatment on cSHMT, T-protein, and vT-protein activities as well as on their translational efficiencies. Studies at the protein level will be needed to confirm the present results.

In the present study, we demonstrated that parity and genotype are factors that can modulate gene expression. The study of the modulation of B9 + glycine supplementation on cSHMT, T-protein, and vT-protein gene expressions allowed us to better understand the possible roles of folic acid and glycine in swine during the early gestation period.


   ACKNOWLEDGMENTS
 
The authors are grateful to D. Morissette, F. Phaneuf, E. Bérubé, M. Turcotte, C. Mayrand, J. Boudreau, A. Marsh, and F. Champagne for animal care and sample collection and to S. Méthot for statistical analysis. This work was financially supported by Génétiporc (St.-Bernard, QC) and Agriculture and Agri-Food Canada. Lennoxville Dairy and Swine R&D Centre contribution no. 745.


   FOOTNOTES
 
1 M.V. is supported by a FCAR fellowship. This work was supported by Genetiporc Inc. (St.-Bernard, QC, Canada) and Agriculture and Agri-Food Canada. Back

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

Received: 8 February 2002.

First decision: 1 March 2002.

Accepted: 17 May 2002.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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M. Vallee, D. Beaudry, C. Roberge, J. J. Matte, R. Blouin, and M.-F. Palin
Isolation of Differentially Expressed Genes in Conceptuses and Endometrial Tissue of Sows in Early Gestation
Biol Reprod, November 1, 2003; 69(5): 1697 - 1706.
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