Phenotypic and Gene Profiling Characteristics of Day 30 BP and PRT Fetuses
Of 352 PRT embryos transferred into seven recipients, four became pregnant. From the four pregnancies, we were able to collect 52 viable fetuses at Days 28–30 of gestation (15%; based on fetal heart beats), and fetal and placental weights were collected from 32 fetuses. The additional fetuses were used for experiments unrelated to the present study. Fetal and placental weights were compared between PRT and BP fetuses and, as predicted by the parental conflict hypothesis, both were significantly reduced in the PRT fetuses (). Histological analysis of PRT and control placentas at Day 28 revealed no significant differences between these placentae. By Day 30, however, there were placental differences, with the PRT samples having a reduction of branched structures or interdigitation, reduced number of chorionic protrusions or simple villus (P < 0.0136), and reduced chorionic surface area (P < 0.0175; ). In addition, maternal-fetal crosstalk seemed to be impaired, because uterine epithelium showed a trend (P < 0.129) toward reduction of the total number of maternal blood vessels at Day 30.
FIG. 1. A) Fetal and placental weight comparisons between BP and PRT concepti at Day 30 of gestation. Bars indicate mean ± SEM; n indicates the number of observations. B–D) Gross morphology of placentae from Day 30 (D30) naturally mated (B) and (more ...)
Additionally, the prediction that expression profiling of uniparental pregnancies could be used to identify conserved imprinted genes was evaluated. To reduce the dimensionality of the transcriptome data into clusters of similar arrays, and as a quality control to determine the quality of the hybridization and identify arrays that did not meet required quality controls, we performed a principal component analysis to clarify which of the four tissue-specific arrays clustered together (Supplemental Fig. S1 illustrates the data after mixed-model normalization [45
] and principal components analysis). The first three principal components were used because they explained 86%, 5%, and 5% of the total variation, respectively. Two arrays (LG2 and BG3; liver PRT 2 and brain PRT 3, respectively) fell outside the 95% concentration ellipse and were excluded from downstream analysis (Supplemental Fig. S1).
Because our experimental focus was the study of conservation of the imprinted gene family, we extracted from the microarray data informative probes that detected known or putative imprinted genes (Catalogue of Imprinted Genes). Of the 49 genes analyzed in this manner, eight were identified as not expressed at P < 0.001 in any tissue tested; they included CALCR, DIO3, GABRA5, HTR2A, INS, OSBPL5, SLC22A2, and WT1.
To examine in more detail the remaining expressed genes, we mapped each Affymetrix probe sequence to the known porcine transcript, or in its absence to the human transcript, and examined each gene individually. This identified the probe sets for GNAS, INPP5F, KCNQ1, and PPP1R9A as noninformative because of their inability to discriminate known imprinted and nonimprinted isoforms. To clarify the expression status of these genes, we attempted to design isoform-specific RT-PCR. Unfortunately, we were unable to do so for GNAS or KCNQ1. However, a semiquantitative RT-PCR assay for INPP5F variant 2 (INPP5F_V2) and PPP1R9A were successfully designed. Results shown in A indicate that INNP5F_V2 is preferentially expressed in carcass and liver BP tissues but not in brain and placental samples. Similarly, for PPP1R9A, results from the semiquantitative RT-PCR indicated that expressions from BP and PRT samples were similar in brain, fibroblasts, and liver (PRT:BP ratios not different from 1). In contrast, in the placental sample, expression from the PRT sample was higher than the BP sample, with a PRT:BP ratio of 1.7 (E).
FIG. 2. Fine structure of fetal-maternal interface from stage-matched, naturally mated controls (Day 28 [A and B] and Day 30 [E and F]) and swine PRTs (Day 28 [C and D] and Day 30 [G and H]) stained with hematoxylin and eosin. Defects become apparent in Day 30 (more ...)
For the remaining genes, we used the Affymetrix array data to determine the ratio of expression of the BP tissues to the PRT tissues and determined whether the ratios differed from 1, an indication of a shift from biallelic expression. As shown in , DIRAS3, MEST, NNAT, NAP1L5, NDN, PEG3, APEG3, PEG10, PLAGL1, PRIM2A, SGCE, and SNRPN had ratios greater than 1, indicating greater expression from the BP samples, a pattern expected of paternally expressed genes. For MEST, NNAT, NAP1L5, NDN, PEG3, APEG3, PEG10, and SNRPN, increased expression from the BP sample and lack of PRT expression were detected in all samples where the genes were expressed. Results of the semiquantitative RT-PCR for PEG3, PEG10, and SNRPN are shown in and confirm the microarray data. Biparental:PRT expression ratios for each of these three genes ranged from a low of 2.7 (PEG10, liver sample) to a high of 150 (PEG3, brain sample), but in all cases there were clear differences between BP and PRT samples. For PRIM2A, significant expression differences were observed in the paternal direction (greater than 1). However, the microarray data indicated transcript expression in PRTs, and hence do not support complete silencing of the maternal allele. For DIRAS3, PLAGL1, and SGCE, tissue-specific differences were observed. DIRAS3 (ARHI) expression between BP and PRT was significantly different in brain, fibroblasts, and liver but not in the placenta (). In the placenta, there was significant expression from the PRT sample, something not seen in any of the other tissues. Results in A show the detection of a transcript in the placental PRT sample but not in the other tissues. To confirm this expression pattern, an RT-PCR was performed. Results confirmed that the placenta had similar expression levels from the BP and PRT samples (BP:PRT = 1.3), whereas the other tissues showed greater differences between BP and PRT expression (BP:PRT ratios of 3.6, 5.6, and 48.2 for brain, fibroblast, and liver, respectively; ).
Gene expression in biparental (BP) and parthenogenetic (PRT) fetal tissues expressed as BP/PRT ratios (mean ± SE).a
FIG. 3. Semiquantitative PCR analysis of candidate imprinted genes. Samples were analyzed as described in the text. A) Expression of variant 1 and/or variant 2 for INPP5F. INNP5F_V2 has increased expression in BP compared with PRT samples in all tissues tested. (more ...)
FIG. 4. Tissue-specific differences in BP and PRT fetal tissues for DIRAS3. A) A comparison of expression of BP and PRT samples using a probe-by-probe analysis. This allows for the identification of tissue-specific differences. Each Affymetrix probe set contains (more ...)
Figure 5A shows the presence of PLAGL1 expression in PRT placental tissues; however, no expression was detected in PRT brain, fibroblast, or liver tissues. In comparison, the expression level was still significantly higher in the BP placenta than the PRT sample, which suggests either coexpression of an imprinted and nonimprinted isoform or partial relaxation of imprinting. A series of RT-PCRs amplifying different exons was developed, and results supported a complex pattern of tissue- and isoform-specific imprinting, with PLAGL1 exon 1–2, exon 1–4, and exon 3–4 showing slight expression from the PRT placenta but not other PRT tissues (). In contrast, PLAGL1 exon 1–7 had partial maternal expression in fibroblasts but lack of PRT expression in other tissues. PLAGL1 exon 1–8 was only detected in the BP brain.
FIG. 5. Analysis of expression at the PLAGL1 locus by isoform-specific semiquantitative PCR. A) PLAGL1 expression in two representative samples. Although pattern of expression in brain shows lack of expression in the PRT sample, in the placental sample there (more ...)
SGCE also had a tissue-specific pattern, but in this case it was the liver that showed expression from the PRT genome when compared to the other three tissues (C). An RT-PCR amplifying exon 7–9 and exon 7–11 confirmed expression from the PRT liver seen in the probe-by-probe analysis, but in addition it indicated SGCE exon 7–9 is also expressed, albeit at a low level compared with the BP, in all PRT samples (). However, BP:PRT ratios were lower in the liver and placenta (0.9 and 1.8, respectively) than in brain and fibroblast (7.8 and 4.0, respectively). The RT-PCR for exon 7–11 had a similar pattern, with respective BP:PRT ratios for liver and placenta of 0.9 and 1.0 versus 3.0 and 4.0 for brain and fibroblasts.
FIG. 6. Analysis of expression at the SGCE locus by isoform-specific semiquantitative PCR. A) Expression of SGCE in BP and PRT tissues. Although there was expression of SGCE in the PRT liver, no PRT expression could be detected in any other tissue, including (more ...)
In addition, AMPD3
, and TFPI2
had ratios lower than 1 in at least one tissue type examined, indicating greater expression from the PRT than the BP samples, a pattern expected of maternally expressed genes (). In the case of SLC38A4
, the array was capable of detecting expression in liver with a higher level of expression in the PRT than the BP sample. In humans, transcription of SLC38A4
produces eight different mRNAs, six alternatively spliced variants, and two unspliced isoforms. From three alternative SLC38A4
], we designed a series of RT-PCRs for different regions of the gene and, as shown in B, a complex pattern of expression was seen. For the P1-Iso1 transcript, expression was greater in the PRT than the BP sample in all tissues except the liver, where the opposite was true. In contrast, for P1-Iso2, P2, and P1+P3, ratios of BP:PRT were lower than 1 (range, 0.8–0.03) in all tissues except the brain. In the brain, ratios were 1.4, 2.3, and 1.5 for P1-Iso2, P2, and P1+P3, respectively. For SLC22A3
, there was a trend () toward overexpression in the PRT placenta, which is suggestive of maternal imprinted gene expression. H19
had an unexpected result, with only the placenta showing a significant allelic imbalance. Consistent with the pattern of a maternally expressed imprinted gene, H19
showed higher expression in the PRT placental tissue. Unexpectedly, wide variation among replicates constrained the detection of significant maternal expression in other tissues (brain, fibroblast, liver) by microarray expression profiling, as would be predicted by the PRT samples. Fortunately, we were able to test imprinting of H19
by QUASEP and confirmed that H19
was imprinted in all tissues tested (). ASCL2
, and UBE3A-AS
were not differentially expressed between PRT and BP embryos in any tissue analyzed ().
FIG. 7. Analysis of expression at the SLC38A4 locus by isoform-specific semiquantitative PCR. A) Cartoon of SLC38A4 gene and mRNA isoforms that have been detected in swine and/or humans. Locations of PCR primers are designated by arrows. The heavy bar indicates (more ...)
Quantitative allelic pyrosequencing (QUASEP) analysis of reciprocal Meishan × White composite crosses (mean ± SE).a
Analysis of Imprinting by QUASEP
Although the expression profiling gave an overall view of the conservation of imprinted genes in swine, and it provided a unique set of observations with respect to imprinted gene expression, it was important to both validate the microarray data in a more direct way and to expand the analysis to imprinted genes not represented in the arrays. Thus, we developed hybrid crosses between purebred Meishans and WC (hybrid of Yorkshire, Landrace, Large White, and Chester White breeds) and used a pyrosequencing-based approach to examine monoallelic versus biallelic expression. Using methods described previously, tSNPs were identified in our reference population for all genes described in and .
FIG. 9. QUASEP results of Day 30 fetal samples obtained from reciprocal Meishan × WC matings. A representative set of pyrosequencing results for both (A and B) imprinted (SNORD107) and (C and D) nonimprinted (DCN) genes. Reciprocal matings (R) from WC (more ...)
The identified tSNPs were analyzed by QUASEP using DNA and cDNA collected from fetal tissues (brain, carcass, liver, and placenta) from both reciprocal interbreed crosses. Each of the 15 interbreed fetuses collected (seven fetuses from WC × MS and eight fetuses from MS × WC) were screened by QUASEP to identify heterozygotes. In general, three to six animals containing the informative polymorphisms were identified from reciprocal matings to clarify the imprinting status for each gene. These informative polymorphisms were identified in both reciprocal crosses, WC × MS and MS × WC, for all genes except ASB4, DLK1, IGF2AS, and NNAT; in these exceptions, tSNPs were identified in only one direction of the litter matings: WC × MS or MS × WC, but not both. A representative set of results is shown in depicting allelic quantification for DNA and cDNA. Analogous pyrograms were developed for each of the genes above and used to generate the results shown in .
As indicated previously, we define imprinting as a 1) significant allelic imbalance from 50:50 and 2) display of a parent-of-origin effect. In the current study, reciprocal crosses were used to clarify the parent-of-origin effects, and QUASEP was used to quantitate allelic imbalances, followed by a statistical test to determine significance. Although recent studies have identified genes that are expressed monoallelically, these genes are not expressed in a parent-of-origin nature. Taken together, QUASEP identified genes that are imprinted across all tissues tested in a tissue-specific manner or biallelically expressed genes (). Moreover, reciprocal crosses, if available, confirmed the parent-of-origin effect of the allelic bias as exemplified by SGCE (), with allelic shifts from 100 to 0 versus the expected biallelic 50:50. There were also two cases where the reciprocal crosses differed in the extent of allelic bias. For PHLDA2, the reciprocal crosses differed in the fibroblast and placenta, with one cross having a significant allelic bias and the other not having it. Similarly, for SGCE, the liver tissue showed complete bias in one direction (100% or 0%) but a 17% or 73% in the other direction (). For ASB4, CD81, and DCN, the allelic bias between genomic DNA and cDNA was not significantly different, indicating that these genes are not imprinted in swine.