BACKGROUND
the ratio of birth weight to placental weight can be used as a measure of placental efficiency  <cit> .
it is determined by many factors, including the thickness and surface area of the placenta, its vascular density, and the number and activity of transporters  <cit> .
in eutherian mammals, intrauterine growth shows a balance between fetal growth and the placental supply of nutrients and oxygen  <cit> .
fetal body weight is correlated positively with placental weight.
in fact, birth size affects the long-term health of an individual, and is critical in determining life expectancy.
in humans, smaller neonates are less likely to survive at birth and have a greater susceptibility to disease  <cit> .
thus, exploration of the genetic factors that regulate placental efficiency is an important research area.
the porcine placenta is chorioallantoic; it originates from the trophoblast and inner cell mass with no trophoblastic invasion of uterine vessels  <cit> .
it is responsible for the exchange of respiratory gases, nutrients, and waste products between the maternal and the fetal systems.
in fact, prenatal fetal organs do not participate in any nutrient metabolic pathways; all their metabolic demands are supplied by transplacental exchange from mother to fetus  <cit> .
the placenta is a provisional organ, which only emerges during gestation.
it secretes a variety of steroid and protein hormones that act in a paracrine manner on the endometrium and fetus to elicit changes in gene expression that support the growth and development of the conceptus.
high placental efficiency is thought to allow smaller placentas to maintain relatively larger fetuses, thereby contributing to higher uterine capacity and litter size  <cit> .
in fact, placental insufficiency is the primary mechanism through which intrauterine crowding of fetuses decreases fetal survival  <cit> .
in the pig, the period from gestation day  <dig> to gestation day  <dig> is an important stage for placental and fetal development, during which the fetuses grow rapidly and need adequate nutrition.
vonnahme et al.
<cit>  found that if one of two adjacent fetuses dies in the uterus, in the meishan pig the other conceptus fails to increase its placental weight or surface area.
in contrast, the adjacent conceptus of a large white pig accelerates its placental growth.
this indicates that meishan and large white conceptuses use different strategies in the competition for nutrients during gestation.
the meishan increases the vascular density of the placenta in order to obtain adequate nutrition, while the large white conceptus accelerates its placental growth instead of enhancing placental efficiency  <cit> .
previous studies that involved translocation of embryos from meishan to large white sows indicated that the number of conceptuses transferred at gestation day  <dig> is generally reduced during late gestation as a result of the limited ability of the uterus to maintain a large number of fetuses to term  <cit> .
it will be of great interest to understand the molecular mechanism underlying the differences in placental gene expression between chinese indigenous and western breeds of pig.
in this study, we investigated differences in the expression profiles of placental genes between erhualian and large white pigs in late gestation by use of the affymetrix genechip.
the erhualian is a type of taihu breed, and has larger litters than the meishan.
bioinformatics analysis revealed that the differentially expressed genes are involved in important biological processes such as angiogenesis and embryonic development in utero.
real-time rt-pcr was used to confirm the differential expression of various genes.
significant differential expression of five genes in the vegf pathway was also detected between large white and erhualian pigs using real-time rt-pcr.
the differentially expressed genes were also mapped to qtl regions related to reproduction traits such as number born alive, fully formed piglets, number of stillborn piglets, etc.
differential expression of candidate imprinted genes was also detected, and the imprinting status of three genes was determined.
our results offer new insight towards an understanding of the molecular basis of placenta efficiency.
RESULTS
transcriptomes in the placenta
there are  <dig>  probesets, which represent  <dig>  transcripts and  <dig> controls, on the affymetrix porcine genechip.
the transcriptome analysis indicated that  <dig>  probesets were expressed in the placenta .
through blast sequence similarity analyses,  <dig> transcripts were matched to human refseq entries.
go annotation was used to classify these genes into groups representing different biological processes.
the results revealed that these genes were involved in metabolic processes, transport and developmental process, etc.
.
it is interesting that many genes had high and stable expression in e <dig>  e <dig>  l <dig> and l <dig>  the strong expression of these genes in the placenta suggests that they may have important roles in placental development or nutrient transportation.
we further checked the relative expression of some homologous genes in data obtained from human .
some genes that are specifically expressed in the placenta , or are specifically expressed in a few tissues including the placenta, in other species  <cit> , such as foxf <dig>  col1a <dig>  col3a <dig>  perp, spp <dig> and pappa, were detected to have high expression in our study.
identification of differentially expressed genes
we identified  <dig>  transcripts that were differentially expressed between e <dig> and l <dig> .
among these genes,  <dig> transcripts were matched to human refseq entries through blast sequence similarity analyses, while the other 29% of the transcripts were non-annotated.
of the differentially expressed genes,  <dig> transcripts were expressed at higher levels in e <dig>  while  <dig> transcripts were expressed more abundantly in l <dig>
between e <dig> and l <dig>   <dig>  differentially expressed transcripts were found .
among these genes,  <dig> transcripts were matched to human refseq entries through blast sequence similarity analyses, while the other 25% of transcripts were non-annotated.
in total,  <dig> transcripts were expressed at higher levels in e <dig>  and  <dig> transcripts were expressed more abundantly in l <dig>  a total of  <dig> transcripts were detected to have differential expression both in e <dig> vs l <dig> and in e <dig> vs l <dig>  but only  <dig> transcripts had the same expression trends in pigs of the two gestational ages.
real-time rt-pcr verification of differentially expressed genes
thirteen genes  were selected to confirm the expression by the use of real-time rt-pcr.
among these genes, eight genes were used to validate the microarray data.
the results indicated that expression patterns of these genes were consistent with the microarray .
the other five genes play important roles in placental angiogenesis and vascular permeability, so we also detected their expression by real-time rt-pcr.
four genes showed more than two-fold differences between breeds in the microarray analysis, but were not statistically significant.
gene β-arrestin <dig> is another important gene in the vegf pathway but is not on the microarray.
expression of vegfr- <dig> was significantly lower in e <dig> than l <dig>  but significantly higher in e <dig> than l <dig>  vegfr- <dig> showed significantly higher expression in e <dig> than in l <dig>  the expression levels of the ve-cadherin and β-arrestin <dig> genes were significantly lower in l <dig> than l <dig>
cluster analysis
to gain insight into similarities at the transcriptome scale among placentas from conceptuses of two breeds and two ages, data from all the differentially expressed genes in the placentas were used in a systematic cluster analysis.
the results showed that l <dig> and l <dig> were initially clustered together because their expression profiles were similar; e <dig> and e <dig> were clustered in another class .
go analysis
go classification on the basis of biological process was used to categorize  <dig> genes that were differentially expressed between e <dig> and l <dig>  the results indicated that the proteins encoded by these genes are associated with metabolic process , biological regulation , developmental process  and electron transport  .
the go biological process classification of  <dig> genes that were differentially expressed between e <dig> and l <dig> indicated that more genes encoded proteins associated with cellular process , metabolic process , developmental process  and transport  .
expression analysis of candidate imprinted genes
imprinted genes, which are expressed in a monoallelic fashion depending on their parental origin, play important roles in mammalian fetal development, growth and behavior.
they also influence the growth of the placenta.
a homolog search using the available sequences of imprinted genes in the human and mouse genomes  identified  <dig> candidate imprinted genes on the porcine affymetrix genechip .
eight candidate imprinted genes showed differential expression between the two breeds in the microarray analysis.
one genes  had significantly higher expression in e <dig> than in l <dig>  and seven genes  showed significant differential expression between e <dig> and l <dig>
to further investigate the allelic expression of candidate imprinted genes in the porcine placenta, cdna sequences of from both breeds were compared.
in the plagl <dig> gene, one single nucleotide polymorphism   was detected at position  <dig> .
in the slc38a <dig> gene, an snp  was detected at position  <dig> , and in the diras <dig> gene, one snp  was revealed at position  <dig> .
restriction enzyme digestion  were used to digest the genomic dna and cdna of placentas.
at least three heterozygotes were used to confirm the expression of imprinted genes, and the results showed that plagl <dig> and slc38a <dig> were paternal expressed.
unfortunately, we could not detect the parental source of diras <dig> gene, because that maternal genetype also was heterozygotes .
no csnps were detected in other candidate imprinted genes, thus the imprinting status was not determined for the remaining genes.
in silico mapping of differentially expressed genes to reproduction qtl regions
through in silico analysis,  <dig> differentially expressed transcripts were found in porcine reproduction qtl chromosomal regions .
the traits associated with these qtl regions included ovulation rate , age at puberty , and uterine capacity   .
discussion
during late gestation, erhualian and large white sows show different uterine responses to the rapid growth of the fetuses.
for example, the placenta of the erhualian pig stops growing, whereas large white pigs continue to increase the surface area of the placenta  <cit> .
this indicates indirectly that there may be many genes involved in the differences in placental development between erhualian and large white pigs .
the "ref" is the number of the paper in the reference.
genes involved in vascular development and permeability pathway
placental vascular development and permeability are important in transportation of nutrients, respiratory gases, and waste products between the maternal and fetal circulations.
in many cases, to keep pace with fetal growth, uterine and umbilical blood flow increases markedly during late gestation  <cit> .
insufficient blood flow leads to nutritional deficiency, and compromises fetal growth, which affects not only the neonate but also the health and productivity of the resulting adult throughout life  <cit> .
thus it is important to investigate the expression of genes in the vegf pathway in order to understand placental efficiency.
the results of this study showed that the expression of these genes was significantly decreased from l <dig> to l <dig>  while no significant changes were found between e <dig> and e <dig> .
this indicates that vascular development and permeability may be stable in e <dig> and e <dig>  but that the large white showed notable variation accompanying further development of the placenta during this period.
vascular endothelial growth factor  is produced and secreted in the placenta; it serves as an angiogenic and permeability enhancing factor in several species, including the pig  <cit> .
it has two specific receptors .
the interaction of vegf and vegfr- <dig> could improve the mitogenic ability of vegf, and lead to survival, migration, and differentiation of endothelial cells, and mediation of vascular permeability  <cit> .
compared with vegfr- <dig>  vegfr- <dig> plays a major role in the biological activity of vegf  <cit> .
a number of signal transduction pathways downstream of vegfr- <dig> play important roles, especially in vasculogenesis.
in the results of our real-time rt-pcr, the expression of vegfr- <dig> had no significant difference between e <dig> and l <dig>  but was significantly higher in e <dig> than in l <dig>  this indicates that the erhualian placenta maybe have higher blood vessel density comparing with the large white placenta.
thus erhualian pigs can provide sufficient nutrients despite a markedly smaller placenta.
of interest, the expression of vegfr- <dig> in the e <dig> placenta was lower than that in the l <dig>  but at gestation day  <dig> in the large white placenta, this gene was down-regulated and showed no significant difference from that of the erhualian.
vegfr- <dig> may decrease the binding of vegf to vegfr- <dig> by sequestration of vegf, thereby preventing its binding to vegfr- <dig>  <cit> .
of course, vegfr- <dig> and - <dig> may also form heterodimers whose signal efficiency is equal to or even greater than that of the vegfr- <dig> homodimer  <cit> .
even so, recent data indicate that vegfr- <dig> plays a positive regulatory role in vascular permeability  <cit> .
ve-cadherin <dig>  a cell-cell adhesion molecule specific to the endothelium, also interacts with vegfr- <dig>  this complex may promote cell internalization, lead to decomposition of intercellular junctions, and promote endothelial permeability  <cit> .
β-arrestin  <dig> also aids endocytosis of ve-cadherin.
a knockdown experiment indicated that β-arrestin 2-/- inhibits the effect of vegf on both endocytosis of ve-cadherin and endothelial permeability  <cit> .
the expression of β-arrestin  <dig> and ve-cadherin in placentas from erhualian pigs was lower than in large white placentas on day  <dig> of gestation, but there was no significant difference on day  <dig>
various genes related to vascular development were also detected in the microarray analysis.
hoxa <dig>  which is essential for placental vascular patterning and labyrinth endothelial specification, showed no differential expression between breeds at gestation day  <dig>  but had higher expression in e <dig> than in l <dig>  <cit> .
tsp- <dig>  which is a negative regulator of angiogenesis, showed higher expression in l <dig> than in e <dig>  <cit> .
hand <dig> is required for vascular development and regulation of angiogenesis  <cit> .
it was expressed at a higher level in the erhualian than the large white.
all these results indicate that the vascular development and permeability of the placenta is different in the erhualian and large white.
during placental development in the large white, this difference increases.
this may explain the different response of the placenta of erhualian and large white pigs to rapid fetal development in late gestation.
hormone related genes
the placenta is an endocrine organ which synthesizes many kinds of hormones that regulate its development and alter maternal physiology to support gestation.
it can influence and regulate the expression of the fetal hormone genes  <cit> .
regulation of endocrine signals in the placenta, especially in late gestation, may over-ride maternal signals, owing to the commencement of accelerated growth of the fetus.
this parasecretion may lead to the death of both mother and offspring  <cit> .
in our results, some specific hormone related genes were shown to have differential expression between the two breeds of pig.
glucocorticoids are steroid hormones that play important roles in metabolic, immunological and development, in particular in stimulation of several processes that collectively serve to increase and maintain concentrations of glucose in blood  <cit> .
they exert their actions by binding to the complexes which is composed with hormone-receptor and glucocorticoid responsive elements complexes  <cit> .
in our experiment, we found that the level of expression of gr  was higher expression in e <dig> than l <dig>  the ptgs <dig> gene, which is a glucocorticoid responsive element, also could regulate angiogenesis in endothelial cells  <cit> .
its expression level was both higher in e <dig> and e <dig> than l <dig> and l <dig>  med <dig> , which is the gr coactivators, also showed higher expression in e <dig> than in l <dig>  <cit> .
all these results showed that the erhualian placenta may be more efficiently to able obtain glucose from the uterus than that of the large white.
moreover, the level of expression of the.
estrogen receptor  <dig>  mineralocorticoid receptor, growth hormone regulated tbc protein  <dig>  thyroid hormone receptor associated protein  <dig> and thyroid hormone receptor associated protein  <dig> all were significantly higher in the erhualian than in the large white at the same stage of gestation.
all these differentially expressed genes are major regulators of the function of hormone gene.
therefore, we presume that the erhualian placenta can provide an environment that supports optimal absorption of nutrients.
the placenta of this breed may be able to extract nutrition from the uterus more efficiently than that of the large white.
candidate genes for placental development and nutrient transportation
a reduction in placental size may mediate negative regulation of maternal nutrition on the birth weight and the number of secondary muscle fibers in the offspring  <cit> .
during late gestation, the large white placenta increases in size and surface area.
in contrast, the erhualian placenta remains unchanged in size and weight  <cit> .
the genes that are involved with placental development are poorly understood.
in our study, the expression of the gene chd <dig> was higher in the large white than in the erhualian on gestation day  <dig> and  <dig>  in the mouse, nishiyama et al.
<cit>  found that chd <dig> could inhibit p53-dependent apoptosis through binding to p <dig>  chd <dig> mice died early during embryogenesis.
it has strong expression in the placenta and other reproductive tissues.
therefore we presume that this gene may play roles in placental development.
in fact, there are some analogous genes among those investigated in our study.
for example, fut <dig> is associated with in-utero development of the embryo  <cit> .
it showed very high expression in the placenta and may potentially be the genes that influence placental development.
the prep <dig> gene regulates multiple aspects of embryonic development in the mouse through a pbx-meis network  <cit> .
it was also found to have higher expression in erhualian placentas than in those of large white pigs.
the go molecular function classification showed that the percentage of differentially expressed genes that encoded proteins associated with developmental process was  <dig> % on gestation day  <dig> and  <dig> % on gestation day  <dig>  all these results provide indications of potential genes that warrant further study in investigating the disparity in placental development between indigenous chinese and exotic pig breeds.
one of the primary regulators of maternofetal transfer of nutrition is the density of transporter proteins in the placenta.
in our results, we also detected some genes in the solute carrier family.
slc16a <dig>  which is a member of a family of plasma membrane amino acid transporters, mediates the na-independent transport of aromatic amino acids across the plasma membrane  <cit> .
it showed up-regulation in the erhualian placenta on both gestation day  <dig> and gestation day  <dig>  in comparison to that of the large white.
slc2a <dig> has been designated as a new member of the glucose transporter family in the mouse  <cit> .
slc25a <dig> plays important roles in the net uptake and efflux of adenine nucleotides  <cit> .
slc1a <dig> also participates in glutamate transport  <cit> .
these genes showed no difference in expression between e <dig> and l <dig>  but showed higher expression in e <dig> than in l <dig>  differential expression was also detected in genes of other solute carrier families.
the slc20a <dig> gene was expressed at higher levels in l <dig> than in e <dig>  but in placentas on gestation day  <dig>  it had higher expression in e <dig>  all these results suggest that erhualian placentas can supply more transporters and transport nutrients more efficiently.
imprinted genes
the genome of the fetus inherits two copies of most genes, one from the mother and the other from the father.
generally, the two single strands have similar transcription activities and equivalent function.
however, imprinted genes escape this rule, and preferentially express maternally or paternally derived alleles.
they have evolved in mammals because of the conflicting interests of maternal and paternal genes in relation to the transfer of nutrients from the mother to her offspring  <cit> .
they play central roles in fetal growth through controlling the fetal demand for, and the placental supply of, maternal nutrients  <cit> .
detecting the imprinting status of genes, especially in the placenta, is very important.
the information available is very useful in comparisons of mouse, human, cattle and pig genes because of the conservation of expression and function of imprinted genes  <cit> .
in this study, we detected the imprinting status of three potentially imprinted genes in the porcine placenta.
the slc38a <dig> gene is paternal expression in the pig.
this is the same as the mouse, but not cattle  <cit> .
the diras <dig> gene displays paternal-specific expression both in mice and humans  <cit> .
we found that it is also expressed in a monoallelic fashion in the porcine placenta.
the plagl <dig> gene was found to be imprinted in the porcine placenta.
in the human and mouse, these genes are paternally expressed  <cit> .
from our evaluation of the conservation of imprinting status among species, we assume that these genes have the same function and expression in the pig as in the other species.
in the mouse, diras <dig> inhibits growth, and transgenic expression of this gene produces individuals of small stature  <cit> .
the plagl <dig> gene encodes a growth suppressor protein that is related to developmental disorders such as growth retardation and transient neonatal diabetes mellitus   <cit> .
moreover, it regulates an imprinted gene network that is critically involved in the control of embryonic growth  <cit> .
in our study, diras <dig> had higher expression in the placentas of e <dig> than l <dig>  the expression of plagl <dig> increased from e <dig> to e <dig>  but decreased from l <dig> to l <dig>  the dcn gene, a strong candidate for function in placental development  <cit> , was found to have higher expression in the large white placenta.
these expression patterns may be related to the phenotype, in that the erhualian has a smaller fetus and placenta than the large white pig.
CONCLUSIONS
we have reported differential gene expression in placentas at gestation day  <dig> and  <dig> from pig breeds with different placental efficiency.
cluster analysis indicated that e <dig> has more similarity on the transcriptome scale with e <dig> than with l <dig> and l <dig>  this finding could indicate that from gestation day  <dig> to gestation day  <dig>  erhualian and large white pigs show a large variance in placental development.
we also identified some differentially expressed genes that are related to angiogenesis and vascular permeability, transport, fetal development and hormonal regulation.
the up-regulation of genes associated with the promotion of proliferation of blood vessels and transporter proteins in erhualian pigs may be responsible for providing sufficient nutrients to support fetal development.
the results of this study provide an opportunity to elucidate the genetic control of placental efficiency and improve our understanding of placental development.

 the ratio of birth weight to placental weight can be used as a measure of placental efficiency. it is determined by many factors, including the thickness and surface area of the placenta, its vascular density, and the number and activity of transporters. in eutherian mammals, intrauterine growth shows a balance between fetal growth and the placental supply of nutrients and oxygen. fetal body weight is correlated positively with placental weight. in fact, birth size affects the long-term health of an individual, and is critical in determining life expectancy. in humans, smaller neonates are less likely to survive at birth and have a greater susceptibility to disease. thus, exploration of the genetic factors that regulate placental efficiency is an important research area. the porcine placenta is chorioallantoic; it originates from the trophoblast and inner cell mass with no trophoblastic invasion of uterine vessels. it is responsible for the exchange of respiratory gases, nutrients, and waste products between the maternal and the fetal systems. in fact, prenatal fetal organs do not participate in any nutrient metabolic pathways; all their metabolic demands are supplied by transplacental exchange from mother to fetus. the placenta is a provisional organ, which only emerges during gestation. it secretes a variety of steroid and protein hormones that act in a paracrine manner on the endometrium and fetus to elicit changes in gene expression that support the growth and development of the conceptus. high placental efficiency is thought to allow smaller placentas to maintain relatively larger fetuses, thereby contributing to higher uterine capacity and litter size. in fact, placental insufficiency is the primary mechanism through which intrauterine crowding of fetuses decreases fetal survival. in the pig, the period from gestation day to gestation day is an important stage for placental and fetal development, during which the fetuses grow rapidly and need adequate nutrition. vonnahme et al. found that if one of two adjacent fetuses dies in the uterus, in the meishan pig the other conceptus fails to increase its placental weight or surface area. in contrast, the adjacent conceptus of a large white pig accelerates its placental growth. this indicates that meishan and large white conceptuses use different strategies in the competition for nutrients during gestation. the meishan increases the vascular density of the placenta in order to obtain adequate nutrition, while the large white conceptus accelerates its placental growth instead of enhancing placental efficiency. previous studies that involved translocation of embryos from meishan to large white sows indicated that the number of conceptuses transferred at gestation day is generally reduced during late gestation as a result of the limited ability of the uterus to maintain a large number of fetuses to term. it will be of great interest to understand the molecular mechanism underlying the differences in placental gene expression between chinese indigenous and western breeds of pig. in this study, we investigated differences in the expression profiles of placental genes between erhualian and large white pigs in late gestation by use of the affymetrix genechip. the erhualian is a type of taihu breed, and has larger litters than the meishan. bioinformatics analysis revealed that the differentially expressed genes are involved in important biological processes such as angiogenesis and embryonic development in utero. real-time rt-pcr was used to confirm the differential expression of various genes. significant differential expression of five genes in the vegf pathway was also detected between large white and erhualian pigs using real-time rt-pcr. the differentially expressed genes were also mapped to qtl regions related to reproduction traits such as number born alive, fully formed piglets, number of stillborn piglets, etc. differential expression of candidate imprinted genes was also detected, and the imprinting status of three genes was determined. our results offer new insight towards an understanding of the molecular basis of placenta efficiency.  transcriptomes in the placenta there are probesets, which represent transcripts and controls, on the affymetrix porcine genechip. the transcriptome analysis indicated that probesets were expressed in the placenta. through blast sequence similarity analyses, transcripts were matched to human refseq entries. go annotation was used to classify these genes into groups representing different biological processes. the results revealed that these genes were involved in metabolic processes, transport and developmental process, etc.. it is interesting that many genes had high and stable expression in e e l and l the strong expression of these genes in the placenta suggests that they may have important roles in placental development or nutrient transportation. we further checked the relative expression of some homologous genes in data obtained from human. some genes that are specifically expressed in the placenta, or are specifically expressed in a few tissues including the placenta, in other species, such as foxf col1a col3a perp, spp and pappa, were detected to have high expression in our study. identification of differentially expressed genes we identified transcripts that were differentially expressed between e and l. among these genes, transcripts were matched to human refseq entries through blast sequence similarity analyses, while the other 29% of the transcripts were non-annotated. of the differentially expressed genes, transcripts were expressed at higher levels in e while transcripts were expressed more abundantly in l between e and l differentially expressed transcripts were found. among these genes, transcripts were matched to human refseq entries through blast sequence similarity analyses, while the other 25% of transcripts were non-annotated. in total, transcripts were expressed at higher levels in e and transcripts were expressed more abundantly in l a total of transcripts were detected to have differential expression both in e vs l and in e vs l but only transcripts had the same expression trends in pigs of the two gestational ages. real-time rt-pcr verification of differentially expressed genes thirteen genes were selected to confirm the expression by the use of real-time rt-pcr. among these genes, eight genes were used to validate the microarray data. the results indicated that expression patterns of these genes were consistent with the microarray. the other five genes play important roles in placental angiogenesis and vascular permeability, so we also detected their expression by real-time rt-pcr. four genes showed more than two-fold differences between breeds in the microarray analysis, but were not statistically significant. gene β-arrestin is another important gene in the vegf pathway but is not on the microarray. expression of vegfr- was significantly lower in e than l but significantly higher in e than l vegfr- showed significantly higher expression in e than in l the expression levels of the ve-cadherin and β-arrestin genes were significantly lower in l than l cluster analysis to gain insight into similarities at the transcriptome scale among placentas from conceptuses of two breeds and two ages, data from all the differentially expressed genes in the placentas were used in a systematic cluster analysis. the results showed that l and l were initially clustered together because their expression profiles were similar; e and e were clustered in another class. go analysis go classification on the basis of biological process was used to categorize genes that were differentially expressed between e and l the results indicated that the proteins encoded by these genes are associated with metabolic process, biological regulation, developmental process and electron transport. the go biological process classification of genes that were differentially expressed between e and l indicated that more genes encoded proteins associated with cellular process, metabolic process, developmental process and transport. expression analysis of candidate imprinted genes imprinted genes, which are expressed in a monoallelic fashion depending on their parental origin, play important roles in mammalian fetal development, growth and behavior. they also influence the growth of the placenta. a homolog search using the available sequences of imprinted genes in the human and mouse genomes identified candidate imprinted genes on the porcine affymetrix genechip. eight candidate imprinted genes showed differential expression between the two breeds in the microarray analysis. one genes had significantly higher expression in e than in l and seven genes showed significant differential expression between e and l to further investigate the allelic expression of candidate imprinted genes in the porcine placenta, cdna sequences of from both breeds were compared. in the plagl gene, one single nucleotide polymorphism was detected at position. in the slc38a gene, an snp was detected at position, and in the diras gene, one snp was revealed at position. restriction enzyme digestion were used to digest the genomic dna and cdna of placentas. at least three heterozygotes were used to confirm the expression of imprinted genes, and the results showed that plagl and slc38a were paternal expressed. unfortunately, we could not detect the parental source of diras gene, because that maternal genetype also was heterozygotes. no csnps were detected in other candidate imprinted genes, thus the imprinting status was not determined for the remaining genes. in silico mapping of differentially expressed genes to reproduction qtl regions through in silico analysis, differentially expressed transcripts were found in porcine reproduction qtl chromosomal regions. the traits associated with these qtl regions included ovulation rate, age at puberty, and uterine capacity. discussion during late gestation, erhualian and large white sows show different uterine responses to the rapid growth of the fetuses. for example, the placenta of the erhualian pig stops growing, whereas large white pigs continue to increase the surface area of the placenta. this indicates indirectly that there may be many genes involved in the differences in placental development between erhualian and large white pigs. the "ref" is the number of the paper in the reference. genes involved in vascular development and permeability pathway placental vascular development and permeability are important in transportation of nutrients, respiratory gases, and waste products between the maternal and fetal circulations. in many cases, to keep pace with fetal growth, uterine and umbilical blood flow increases markedly during late gestation. insufficient blood flow leads to nutritional deficiency, and compromises fetal growth, which affects not only the neonate but also the health and productivity of the resulting adult throughout life. thus it is important to investigate the expression of genes in the vegf pathway in order to understand placental efficiency. the results of this study showed that the expression of these genes was significantly decreased from l to l while no significant changes were found between e and e. this indicates that vascular development and permeability may be stable in e and e but that the large white showed notable variation accompanying further development of the placenta during this period. vascular endothelial growth factor is produced and secreted in the placenta; it serves as an angiogenic and permeability enhancing factor in several species, including the pig. it has two specific receptors. the interaction of vegf and vegfr- could improve the mitogenic ability of vegf, and lead to survival, migration, and differentiation of endothelial cells, and mediation of vascular permeability. compared with vegfr- vegfr- plays a major role in the biological activity of vegf. a number of signal transduction pathways downstream of vegfr- play important roles, especially in vasculogenesis. in the results of our real-time rt-pcr, the expression of vegfr- had no significant difference between e and l but was significantly higher in e than in l this indicates that the erhualian placenta maybe have higher blood vessel density comparing with the large white placenta. thus erhualian pigs can provide sufficient nutrients despite a markedly smaller placenta. of interest, the expression of vegfr- in the e placenta was lower than that in the l but at gestation day in the large white placenta, this gene was down-regulated and showed no significant difference from that of the erhualian. vegfr- may decrease the binding of vegf to vegfr- by sequestration of vegf, thereby preventing its binding to vegfr-. of course, vegfr- and - may also form heterodimers whose signal efficiency is equal to or even greater than that of the vegfr- homodimer. even so, recent data indicate that vegfr- plays a positive regulatory role in vascular permeability. ve-cadherin a cell-cell adhesion molecule specific to the endothelium, also interacts with vegfr- this complex may promote cell internalization, lead to decomposition of intercellular junctions, and promote endothelial permeability. β-arrestin also aids endocytosis of ve-cadherin. a knockdown experiment indicated that β-arrestin 2-/- inhibits the effect of vegf on both endocytosis of ve-cadherin and endothelial permeability. the expression of β-arrestin and ve-cadherin in placentas from erhualian pigs was lower than in large white placentas on day of gestation, but there was no significant difference on day various genes related to vascular development were also detected in the microarray analysis. hoxa which is essential for placental vascular patterning and labyrinth endothelial specification, showed no differential expression between breeds at gestation day but had higher expression in e than in l. tsp- which is a negative regulator of angiogenesis, showed higher expression in l than in e. hand is required for vascular development and regulation of angiogenesis. it was expressed at a higher level in the erhualian than the large white. all these results indicate that the vascular development and permeability of the placenta is different in the erhualian and large white. during placental development in the large white, this difference increases. this may explain the different response of the placenta of erhualian and large white pigs to rapid fetal development in late gestation. hormone related genes the placenta is an endocrine organ which synthesizes many kinds of hormones that regulate its development and alter maternal physiology to support gestation. it can influence and regulate the expression of the fetal hormone genes. regulation of endocrine signals in the placenta, especially in late gestation, may over-ride maternal signals, owing to the commencement of accelerated growth of the fetus. this parasecretion may lead to the death of both mother and offspring. in our results, some specific hormone related genes were shown to have differential expression between the two breeds of pig. glucocorticoids are steroid hormones that play important roles in metabolic, immunological and development, in particular in stimulation of several processes that collectively serve to increase and maintain concentrations of glucose in blood. they exert their actions by binding to the complexes which is composed with hormone-receptor and glucocorticoid responsive elements complexes. in our experiment, we found that the level of expression of gr was higher expression in e than l the ptgs gene, which is a glucocorticoid responsive element, also could regulate angiogenesis in endothelial cells. its expression level was both higher in e and e than l and l med, which is the gr coactivators, also showed higher expression in e than in l. all these results showed that the erhualian placenta may be more efficiently to able obtain glucose from the uterus than that of the large white. moreover, the level of expression of the. estrogen receptor mineralocorticoid receptor, growth hormone regulated tbc protein thyroid hormone receptor associated protein and thyroid hormone receptor associated protein all were significantly higher in the erhualian than in the large white at the same stage of gestation. all these differentially expressed genes are major regulators of the function of hormone gene. therefore, we presume that the erhualian placenta can provide an environment that supports optimal absorption of nutrients. the placenta of this breed may be able to extract nutrition from the uterus more efficiently than that of the large white. candidate genes for placental development and nutrient transportation a reduction in placental size may mediate negative regulation of maternal nutrition on the birth weight and the number of secondary muscle fibers in the offspring. during late gestation, the large white placenta increases in size and surface area. in contrast, the erhualian placenta remains unchanged in size and weight. the genes that are involved with placental development are poorly understood. in our study, the expression of the gene chd was higher in the large white than in the erhualian on gestation day and in the mouse, nishiyama et al. found that chd could inhibit p53-dependent apoptosis through binding to p chd mice died early during embryogenesis. it has strong expression in the placenta and other reproductive tissues. therefore we presume that this gene may play roles in placental development. in fact, there are some analogous genes among those investigated in our study. for example, fut is associated with in-utero development of the embryo. it showed very high expression in the placenta and may potentially be the genes that influence placental development. the prep gene regulates multiple aspects of embryonic development in the mouse through a pbx-meis network. it was also found to have higher expression in erhualian placentas than in those of large white pigs. the go molecular function classification showed that the percentage of differentially expressed genes that encoded proteins associated with developmental process was % on gestation day and % on gestation day all these results provide indications of potential genes that warrant further study in investigating the disparity in placental development between indigenous chinese and exotic pig breeds. one of the primary regulators of maternofetal transfer of nutrition is the density of transporter proteins in the placenta. in our results, we also detected some genes in the solute carrier family. slc16a which is a member of a family of plasma membrane amino acid transporters, mediates the na-independent transport of aromatic amino acids across the plasma membrane. it showed up-regulation in the erhualian placenta on both gestation day and gestation day in comparison to that of the large white. slc2a has been designated as a new member of the glucose transporter family in the mouse. slc25a plays important roles in the net uptake and efflux of adenine nucleotides. slc1a also participates in glutamate transport. these genes showed no difference in expression between e and l but showed higher expression in e than in l differential expression was also detected in genes of other solute carrier families. the slc20a gene was expressed at higher levels in l than in e but in placentas on gestation day it had higher expression in e all these results suggest that erhualian placentas can supply more transporters and transport nutrients more efficiently. imprinted genes the genome of the fetus inherits two copies of most genes, one from the mother and the other from the father. generally, the two single strands have similar transcription activities and equivalent function. however, imprinted genes escape this rule, and preferentially express maternally or paternally derived alleles. they have evolved in mammals because of the conflicting interests of maternal and paternal genes in relation to the transfer of nutrients from the mother to her offspring. they play central roles in fetal growth through controlling the fetal demand for, and the placental supply of, maternal nutrients. detecting the imprinting status of genes, especially in the placenta, is very important. the information available is very useful in comparisons of mouse, human, cattle and pig genes because of the conservation of expression and function of imprinted genes. in this study, we detected the imprinting status of three potentially imprinted genes in the porcine placenta. the slc38a gene is paternal expression in the pig. this is the same as the mouse, but not cattle. the diras gene displays paternal-specific expression both in mice and humans. we found that it is also expressed in a monoallelic fashion in the porcine placenta. the plagl gene was found to be imprinted in the porcine placenta. in the human and mouse, these genes are paternally expressed. from our evaluation of the conservation of imprinting status among species, we assume that these genes have the same function and expression in the pig as in the other species. in the mouse, diras inhibits growth, and transgenic expression of this gene produces individuals of small stature. the plagl gene encodes a growth suppressor protein that is related to developmental disorders such as growth retardation and transient neonatal diabetes mellitus. moreover, it regulates an imprinted gene network that is critically involved in the control of embryonic growth. in our study, diras had higher expression in the placentas of e than l the expression of plagl increased from e to e but decreased from l to l the dcn gene, a strong candidate for function in placental development, was found to have higher expression in the large white placenta. these expression patterns may be related to the phenotype, in that the erhualian has a smaller fetus and placenta than the large white pig.  we have reported differential gene expression in placentas at gestation day and from pig breeds with different placental efficiency. cluster analysis indicated that e has more similarity on the transcriptome scale with e than with l and l this finding could indicate that from gestation day to gestation day erhualian and large white pigs show a large variance in placental development. we also identified some differentially expressed genes that are related to angiogenesis and vascular permeability, transport, fetal development and hormonal regulation. the up-regulation of genes associated with the promotion of proliferation of blood vessels and transporter proteins in erhualian pigs may be responsible for providing sufficient nutrients to support fetal development. the results of this study provide an opportunity to elucidate the genetic control of placental efficiency and improve our understanding of placental development. 
to understand the molecular basis of placental development during late gestation in chinese indigenous and western breeds with different placental efficiency, female placental samples were collected from six pregnant erhualian gilts at gestation day and day and from six pregnant large white gilts at gestation day and day. these findings help to elucidate the genetic control of placental efficiency and improve the understanding of placental development. twelve pooled samples were hybridized to the porcine affymetrix genechip. significant differential expression of five genes in the vegf pathway was also detected between the breeds. together with its rapid growth during late gestation, the large white pig breed shows a significant increase in placental size and weight, but this does not occur in the highly prolific chinese pig breeds. real-time rt-pcr confirmed the differential expression of eight selected genes. a search of chromosomal location revealed that differentially expressed genes located to qtl regions related to reproduction. three of the six genes showed monoallelic expression in the porcine placenta. placental efficiency is strongly associated with litter size, fetal weight and prenatal mortality. differential expression of six candidate imprinted genes was also confirmed. our study detected many genes that showed differential expression between placentas of two divergent breed of pigs, and confirmed the imprinting of three genes.
 placental efficiency is strongly associated with litter size, fetal weight and prenatal mortality. together with its rapid growth during late gestation, the large white pig breed shows a significant increase in placental size and weight, but this does not occur in the highly prolific chinese pig breeds. to understand the molecular basis of placental development during late gestation in chinese indigenous and western breeds with different placental efficiency, female placental samples were collected from six pregnant erhualian gilts at gestation day and day and from six pregnant large white gilts at gestation day and day. two female placentas from one sow were used to extract rna and then pooled in equal volumes. twelve pooled samples were hybridized to the porcine affymetrix genechip.  a total of and transcripts were detected that were differentially expressed between e and l and between e and l, respectively. gene ontology analysis revealed that these genes belong to the class of genes that participate in angiogenesis and development. real-time rt-pcr confirmed the differential expression of eight selected genes. significant differential expression of five genes in the vegf pathway was also detected between the breeds. a search of chromosomal location revealed that differentially expressed genes located to qtl regions related to reproduction. differential expression of six candidate imprinted genes was also confirmed. three of the six genes showed monoallelic expression in the porcine placenta.  our study detected many genes that showed differential expression between placentas of two divergent breed of pigs, and confirmed the imprinting of three genes. these findings help to elucidate the genetic control of placental efficiency and improve the understanding of placental development. 