Cybrid Model Supports Mitochondrial Genetic Effect on Pig Litter Size

Abstract

In pigs, mitochondrial DNA (mtDNA) polymorphism and the correlation to reproductive performance across breeds and individuals have been largely reported, however, experimental proof has never been provided. In this study, we analyzed 807 sows for correlation of total number born (TNB) and mitotype, which presented the maximum of 1.73 piglets for mtDNA contribution. Cybrid models representing different mitotypes were generated for identification of the mtDNA effect. Results indicated significant differences on cellular and molecular characteristics among cybrids, including energy metabolic traits, mtDNA copy numbers and transcriptions, mRNA and protein expressions on mitochondrial biogenesis genes and reproduction-related genes. Referring to mitotypes, the cybrids with prolific mitotypes presented significantly higher oxygen consumption rate (OCR) productions, mtDNA transcriptions and copy numbers than those with common mitotypes, while both mRNA and protein expressions of PPARA, TFAM, ER1, ER2, and ESRRG in prolific cybrids were significantly higher than those with common mitotypes. Cybrid models reflected the mtDNA effect on pig litter size, suggesting the potential application of mtDNA polymorphism in pig selection and breeding practices.

Keywords: cybrid; genetic effect; litter size; mitochondrial DNA; pig.

FIGURE 1

Network analysis of mitotypes. 7 mitotypes clustered into two mitogroups, HG1 (H1, H4, and H5) and HG2 (H2, H3, H6, and H7).

FIGURE 2

Cell proliferation, oxygen consumption rate and ROS assays in different cell lines. (A) Cell proliferation in fibroblasts. (B) Cell proliferation in cybrids. (C) OCR assays for fibroblasts. (D) OCR assays for cybrids. (E) The relative ROS contents in fibroblasts. (F) The relative ROS contents in cybrids. Different capital or lowercase letters meant significant differences at P < 0.01 or P < 0.05, respectively. PH1-7, fibroblast with mitotype 1–7, respectively; IPH1-7, cybrid with mitotype 1–7, respectively.

FIGURE 3

Detections mitochondrial DNA copy number and transcription in fibroblasts and cybrids by RT-qPCR assays. (A) mtDNA copy numbers in fibroblasts. (B) mtDNA copy numbers in cybrids. (C) Transcription levels of mtDNAs in fibroblasts. (D) Transcription levels of mtDNAs in cybrids. Different letters on columns meant extremely significant differences at P < 0.01. PH1-7, fibroblast with mitotype 1–7, respectively, IPH1-7, cybrid with mitotype 1–7, respectively.

FIGURE 4

mRNA expressions of mitochondrial biogenesis-related genes in fibroblasts and cybrids. (A–C) Illustrated the expressions of TFAM, TFB1M, and PPARA in fibroblasts, (D–H) presented the expressions of TFAM, TFB2M, PPARA, NRF1, and PPARGC1A in cybrids. Capital or lowercase letters on columns meant significant differences at P < 0.01 or P < 0.05 levels, respectively. PH1-7, fibroblast with mitotype 1–7, respectively; IPH1-7, cybrid with mitotype 1–7, respectively.

FIGURE 5

Measurements of PPARA and TFAM in fibroblasts and cybrids by western blot assays. (A,D) Illustrations of western blotting images in fibroblasts and cybrids; (B,C) protein levels of TFAM and PPARA in fibroblasts; (E,F) protein contents of TFAM and PPARA in cybrids. Different letters on columns meant significant differences at P < 0.05 levels. PH1-7, fibroblast with mitotype 1–7, respectively; IPH1-7, cybrid with mitotype 1–7, respectively.

FIGURE 6

Expressions of reproduction-related genes in fibroblasts and cybrids. (A–C) Illustrated the mRNA expressions of ER1, ER2, and ESRRG in fibroblasts. (D–H) Presented the mRNA expressions of ER1, ER2, ESRRG, FSHR, and ESRRa in cybrids. (I,M) Illustrations of western blotting images in fibroblasts and cybrids. (J–L) Protein levels of ER1, ER2, and ESRRG in fibroblasts. (N–P) Protein contents of ER1, ER2, and ESRRG in cybrids. Different capital and lowercase letters on columns meant significant differences at P < 0.01 or P < 0.05, respectively. PH1-7, fibroblast with mitotype 1–7, respectively; IPH1-7, cybrid with mitotype 1–7, respectively.