Developmental expression of lineage specific genes in porcine embryos of different origins

Abstract

Purpose: This study compared the expression of genes involved in pluripotency, segregation of inner cell mass (ICM) and trophectoderm (TE), and primitive endoderm (PE) formation in porcine embryos produced by in vitro fertilization (IVF), parthenogenetic activation (PA), and nuclear transfer (NT) using either fetal fibroblasts (FF-NT) or mesenchymal stem cells (MSC-NT).

Methods: Blastocyst formation and total cell number were analyzed. The expression patterns of transcripts, including SRY-related HMG-box gene 2 (SOX2), reduced expression gene 1 (REX1/ZFP42), LIN28, caudal type homeobox 2 (CDX2), TEA domain family member 4 (TEAD4), integrin beta 1 (ITGB1) and GATA6 were assessed at the 4-8 cell and blastocyst stage embryos by real-time PCR.

Results: Developmental rates to blastocyst stage and total cell number were higher in IVF and PA embryos than in NT embryos. But MSC-NT embryos had increased blastocyst formation and higher total cell number compared to FF-NT embryos. The relative expressions of transcripts were higher in blastocysts than in 4-8 cell stage embryos. The mRNA expression levels of SOX2 and REX1 were largely similar in embryos of different origins. However, the genes such as LIN28, CDX2, TEAD4, ITGB1 and GATA6 showed the differential expression pattern in PA and NT embryos compared to IVF embryos. Importantly, the transcript levels in MSC-NT embryos were relatively less variable to IVF than those in FF-NT embryos.

Conclusion: MSCs seem to be better donors for porcine NT as they improved the developmental competency, and influenced the expression pattern of genes quite similar with IVF embryos than that of FFs.

Fig. 1

Characterization of porcine bone marrow derived MSCs. a. Representative images showing flow cytometric analysis of cell surface markers CD29, CD44, CD90 and CD45. Filled histograms represent staining with specific antibodies and open histograms correspond to matched isotypes. b. Adipogenic differentiation was indicated by intracellular lipid accumulation with Oil red O staining (I). Osteogenic induction was visualized by detection of dark calcium deposits after von Kossa staining (II). Untreated MSCs remained as fibroblast-like cells. Scale bar = 50 μM (I), 100 μM (II), 250 μM (III)

Fig. 2

Analysis of total cell number in porcine embryos of different origins. a. Representative images showing the total cell number in IVF (I), PA (II), FF-NT (III) and MSC-NT (IV) blastocysts. Nuclei were labeled with DAPI (blue) for counting of cell number. Scale bar = 50 μM. b. A bar graph showing the mean total cell number in IVF, PA, FF-NT and MSC-NT blastocysts. Different superscripts (a, b, c) indicate significant differences between the groups (P < 0.05)

Fig. 3

RT-PCR and real-time PCR analysis showing gene expression profile of porcine donor cells, and embryos derived from IVF, PA, FF-NT and MSC-NT. a. Expression of SRY-related HMG-box gene 2 (SOX2), reduced expression 1 (REX1/ZFP42), LIN28, caudal type homeobox 2 (CDX2), TEA domain family member 4 (TEAD4), integrin beta 1 (ITGB1) and GATA6 transcripts in donor cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. b. Relative expression of selected genes at 4–8 cell and blastocyst stages of embryos. GAPDH was employed as a housekeeping gene for normalization. Data is the mean ± SD. Different superscripts (a, b, c) represent significant differences in relative expression between IVF, PA, FF-NT and MSC-NT embryos at a given stage of development (P < 0.05, ANOVA, followed by Bonferroni or Tukey test for post-hoc comparisons). Representative RT-PCR gel photographs of the respective gene transcript in IVF, PA, FF-NT and MSC-NT embryos at 4–8 cell and blastocyst stages are presented at the bottom