X-linked gene transcription patterns in female and male in vivo, in vitro and cloned porcine individual blastocysts

Abstract

To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF), and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average) were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes) but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT) process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process, which may eventually lead to problems with embryonic or placental defects.

Figure 1. XIST mRNA expression of individual in vivo blastocysts.

Each value derived from transcripts of the XIST gene in in vivo blastocysts derived from commercial (n = 15) and Yucatan miniature pig (n = 5), after normalization relative to ACTB and 18S (internal control) genes, were compared with that of one of 26 in vivo blastocysts defined as 1. An underlined Y above bars indicates the blastocysts derived from Yucatan miniature pigs.

Figure 2. X-linked gene transcription patterns of in vivo porcine blastocysts.

Aligned dot plots represent mRNA transcript levels for X-linked genes in female and male in vivo individual blastocysts. The expression values in female and male individuals are represented by red circle and blue square, respectively. A relative fold change of mRNA levels in in vivo male blastocysts compared with that of the in vivo female ones defined as 1. This data is presented as mean ± SEM. Asterisk indicates significant difference between the sexes by Student t test (P<0.05).

Figure 3. Imprinted gene transcription patterns of in vivo porcine blastocysts.

The dot plots represent mRNA transcript levels for imprinted genes in female and male in vivo blastocysts. There were no differences in gene expression between the sexes of blastocysts.

Figure 4. IVF embryo sexing by PCR.

a representative PCR result for embryo sexing is shown. M is a 100-bp ladder as a DNA size marker. Male and female indicate M¹ and F¹ fetal fibroblast cell lines, respectively. 1–12 lanes are shown for IVF embryos. A single PCR product (250-bp and 105-bp) was detected for the SRY and the ACTB, respectively.

Figure 5. X-linked gene transcription patterns of IVF blastocysts.

The dot plots of mRNA transcript levels for X-linked in female and male in vivo and in vitro fertilized (IVF) blastocysts. Asterisks indicate significant difference between in vivo and IVF embryos of each sex as well as between female and male IVF embryos. A relative fold change of mRNA levels of female and male IVF blastocysts compared with that of the in vivo female ones defined as 1. Asterisks indicate significant difference between in vivo and IVF blastocysts of each sex as well as between female and male IVF embryos (P<0.05).

Figure 6. X-linked gene transcription patterns of cloned blastocysts.

The dot plots of mRNA transcript levels for X-linked in female and male in vivo and cloned blastocysts. Other details are as described in the legends to Figure 5 .

Figure 7. X-linked gene transcription patterns of pFF cell lines.

Asterisks indicate significant difference between the different cell lines (P<0.01).

Figure 8. X-linked gene transcription patterns of female and male porcine cloned blastocysts by treatment of Scriptaid, a HDACi after SCNT.

A relative fold change of mRNA levels of female (Left panel) and male (right panel) cloned blastocysts compared with that of the in vivo female ones defined as 1. Asterisks indicate significant difference between in vivo and cloned groups (* P<0.05; ** P<0.01; *** P<0.001).

Figure 9. Calculation of

amplification efficiencies. qPCR efficiencies of reference (ACTB and RN18S) and target genes (BEX1, G6GD, HPRT1, PGK1, XIST and ZXDA). The C_q was plotted against the log of the initial quantity of template for each dilution of cDNA (50 ng–16 pg, n = 3).