Cybrid Model Supports Mitochondrial Genetic Effect on Pig Litter Size

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.


INTRODUCTION
The mitochondrion is indispensable to eukaryotes, which involves in bioenergy metabolism, biosynthesis, anti-oxidant defense, signal transduction, redox status, and apoptosis (Wallace, 2012;Aanen et al., 2014;Wang et al., 2017b), hence the mitochondrial dysfunction may result in the decline of individual motor skill or even disease (Amaral et al., 2013;Cardoso et al., 2017;Herst et al., 2017;van der Bliek et al., 2017). Mitochondrial genome (mtDNA) is necessary for components of the respiratory chain complexes, which is essential for ATP production. In vertebrates, mtDNAs are approximately 16.5 kb-circular molecules, of which encodes 37 canonical genes, including 2 rRNAs, 22 tRNAs, and 13 polypeptide genes for respiratory chain subunits. Mitochondrial genome has been widely used to study origin evolution (Xiang et al., 2014), diseases (Wallace, 1994), aging (Boengler et al., 2017), and production and reproduction traits of farm animals (Reicher et al., 2012;Liu et al., 2019a).
However, the mtDNA effect on pig reproduction traits lacks the experimental proof. In this study, we focused on the litter size, the core trait for reproduction performance, to investigate the relationship with mitotypes, and using transmitochondrial cell model (cybrid) to test the validity of mtDNA effects.

Ethics Statement
The guidelines of the experimental animal management of China Agricultural University (CAU) were followed throughout the study, and the experimental protocols were approved by the Experimental Animal Care and Use Committee of CAU.

Sample Collection and Association Analysis
The records of TNB were collected and amounted to 1,766 in 807 Large White sows from Beijing Liuma pig company. All pigs used in this study lived in consistent circumstances (including farm, feeding and management, etc.). Ear samples were collected, and genomic DNAs were extracted using a Tissue/Cell Genome DNA Extraction Kit (Aidlab, Beijing, China) according to manufacturer's instruction. Mitochondrial genome was amplified in a 50 µL volume containing 25 µL 2 × A9 LongHiFi PCR MasterMix (Aidlab, Beijing, China), 1 µL each forward and reverse primers (referred to NC_000845.1, Supplementary Table 1), 2 µL DNA and 21 µL H 2 O using the following program: 95 • C for 3 min, followed by 38 cycles of 95 • C for 10 s, 60 • C for 15 s and 72 • C for 3 min, and PCR products were directly sequenced by TsingKe Biotech (China).
Mitochondrial DNA polymorphic sites were detected through sequence alignment using MEGA7 software 1 . Mitotypes were sorted by FaBox online software 2 and analyzed through median joining method by Network 5 software 3 , and mitogroups were sorted according to mutated positions among mitotypes. Association analyses of mitotype/mitogroup and TNB were performed by the linear mixed model using ASReml software as follows: In the model, the effects of pig population mean (µ), farrowing year-season (ys), parity number (parity), mtDNA variation (including mitotypes and mitogroups), the polygenic effect (ID), the permanent environmental effect (EP), and the random residual (e) were included. The response variable was TNB. The polygenic effect corrected the genetic background by the additive genetic relationship matrix, i.e., the pedigree information. The permanent environmental effect dealt with the repeated measurement data. When a set of statistical inferences were simultaneously considered, multiple comparisons were conducted by the Bonferroni method, and the adjusted P < 0.05 was regarded as statistical significance.

Cybrid Generation and Culture
Cybrids were generated by enucleation of mitochondria donor cells and fusion of the cytoplasts with ρ0 cells according to modified procedures of the previous study (Yu et al., 2015;Wang et al., 2017a). Ear primary fibroblasts were used as mitochondria donors, which were isolated from Large White sows using tissue culture method (Wang et al., 2017a). And IPEC-J2 cell line was used as nucleus donor (ρ0 cell), which was presented by Dr. Jianguo Zhao, Chinese Academy of Sciences, China. Briefly, IPEC-J2 was cultured in former medium (containing 10% FBS, 4 µg/ml rhodamine 6G, 50 µg/mL uridine, 1 mM sodium pyruvate and 1% penicillin-streptomycin; Gibco, United States) for 7 days with replacement of former medium at 24 h intervals to generate ρ0 cells. Before cytoplast fusion with fibroblasts, ρ0 cells were cultured in normal medium for 3 h. Primary fibroblasts were enucleated using ultracentrifugation (44,000 g) with cytochalasin B. Cybrids were created by the fusion of the ρ0 cells with the enucleated cells using polyethylene glycol (PEG), and positive cells were identified by mtDNA sequencing.

Cell Proliferation Assay
An Enhanced Cell Counting Beyotime,China) was used in the cell proliferation assay, wherein 3 × 10 3 fibroblasts/well or 2 × 10 3 cybrids/well were seeded in 96-well plates, respectively. After 1-7 days culture, cells were further incubated with 10 µL of CCK-8 solution for 2 h at 37 • C, respectively. Then, the absorbance at 450 nm was measured with reference wavelength at 650 nm using a microplate reader.

Analysis of Mitochondrial Respiration Ability
Approximately 1.8 × 10 4 cells for fibroblasts and 8 × 10 3 cells for cybrids were seeded in 96 wells of XF96 cell culture microplates (Seahorse Bioscience, United States), respectively. For respiratory analysis, cells were analyzed according to the procedures described in the Seahorse XF Cell Mito Stress Test kit (Agilent, United States). After baseline measurements, OCR values were measured after sequentially adding Oligomycin (2 µM final concentration), FCCP (carbonyl cyanide 4-trifluoromethoxyphenylhydrazone, 0.5 µM final concentration for fibroblasts and 1 µM for cybrids), and Rotenone plus Antimycin A (0.5 µM final concentration of each). Subtracting the non-mitochondrial OCR from the total OCR yields the mitochondrial OCR.

Quantification of mtDNA Copy Number
Genomic DNAs were extracted using a Tissue/Cell Genome DNA Extraction Kit (Aidlab, Beijing, China). The mtDNA specific primers were listed in Supplementary Table 1 were designed using GenBank sequence (NC_000845.1). Beta-globin gene was used as the internal standard (Liu et al., 2019b). The mtDNA copy number of each sample was compared by calculating the ratio of mitochondrial to nuclear DNA abundance (mtDNA/nDNA) (Wang et al., 2017a). The baseline adjustment method of the qPCRsoft software (ANALYTIKJENA, Germany) was used to determine the Ct of each reaction. The amplification efficiencies were close to 100%, and each RT-qPCR experiment was performed in triplicates.

The mRNA Expression Levels of Mitochondrial H Strand, Mitochondrial Biogenesis-Related Genes, and Reproduction-Related Genes
RNA samples of primary fibroblasts and cybrids were extracted by Tissue/Cell RNA Rapid Extraction Kit (Aidlab, China) according to the manufacturer's instruction, and the cDNA was synthesized using 1 µg RNA with TRUE script One Step RT-PCR Kit (Aidlab, China). The relative expressions of mitochondrial H strand, as well as 11 genes (NRF-1, PPARA, TFAM, TFB1M, TFB2M, PPARGC1A, ER1, ER2, ESRRA, ESRRG, and FSHR) were measured by RT-qPCR with specific primers (Supplementary Table 1). GAPDH was used as the internal control (Li et al., 2016b). The baseline adjustment method of the qPCR software (ANALYTIKJENA, Germany) was used to determine the Ct of each reaction. The amplification efficiencies were close to 100%, and all samples were amplified in triplicate. For data analysis, 2 − ct method was used to calculate the relative level of samples.

Reactive Oxygen Species (ROS) Detection
The ROS level was determined by ROS Assay Kit (Beyotime, China). Approximately 4 × 10 5 cells in a 6-well plate were incubated with DCFH-DA (10 mM) for 20 min at 37 • C and washed 3 times with DMEM. ROS production in cells was measured fluorometrically with excitation and emission settings at 488 and 525 nm, respectively, and expressed as arbitrary units.

Statistical Analysis
The mean values shown in Figures 2-6 and used in the statistical analyses represented at least three independent trials. Differences between each group were evaluated by analysis of variance (ANOVA), followed by Duncan's multiple-range test using the General Linear Model procedure of SAS (SAS version 8.2; Cary, NC, United States). Results were expressed as the mean ± standard error of the mean (SEM), and the data used in the statistical analyses represented at least three independent trials. Results with P-values < 0.05 or < 0.01 were considered statistically significant or extremely significant when testing for differences among samples, respectively.

DISCUSSION
Due to porcine litter size is considered a trait with low heritability, DNA markers could be applied in molecular breeding for greater predictability on reproduction (Shields et al., 1985;King et al., 2003;Wang et al., 2018;Liu et al., 2019a). The potential role of mtDNA impacting pig reproduction traits was postulated for several decades (Sun et al., 2001;El Shourbagy et al., 2006;Wang et al., 2018), and the question remains open with some ambiguity in accumulated studies of mtDNA variations and associated reports.
to evaluate the mitochondrial genetic effects on animal traits directly. The transmitochondrial cybrid model is created by fusing cells devoid of mtDNA (ρ0 cells) with cytoplasts (enucleated cells) from different individuals, so the resultant cybrids have uniform nuclear background but different mitochondrial genome (Yu et al., 2015;Swerdlow et al., 2017), which are valid tools for dissecting mtDNA effect of dynamic biological function under the same background. Thus, cybrid model was utilized in this study to identify the correlations to cell metabolisms and gene expressions caused by mitotypes.
It is noteworthy that prolific mitotypes (H1, H4, and H5) exhibited higher levels of OCRs, mtDNA copy numbers and transcriptions, expressions of mitochondrial biogenesis-related genes and reproduction-related genes, and lower ROS contents compared to common mitotypes (H2, H3, H6, and H7), which were consistent with the effect on litter size (Figures 2-5 and Table 1).
Copy number and transcription of mitochondrial genome are dynamic and cell-specific biomarkers (Moraes, 2001), which depends on the regulation of mitochondrial biogenesis (Lee et al., 2015;Laubenthal et al., 2016) and ATP generation through oxidative phosphorylation (Wai et al., 2010). In the present research, cells with prolific mitotypes (H1, H4, and H5) contained larger mtDNA copy numbers and transcriptions than common mitotypes, which were consistent with OCR results. Previous studies reported that mitochondrial genome did not replicate at the early stage of embryonic development, and high-quality mtDNA copies were essential for cell division and energy supply (Sun et al., 2001;Babayev and Seli, 2015), which reflects the correlation of mtDNA copies, mitotypes and pig litter size.
The proliferation rate is a fundamental indicator for assessing cell states (Golias et al., 2004), which is a prerequisite for further experiments. In the current study, cell proliferation rates of fibroblasts or cybrids presented stable and similar growing states (Figures 2A,B), indicating the validity of verification experiments.

CONCLUSION
This is the first report, to our knowledge, that demonstrates mitotype effect on pig TNBs, which ultimately confers differences on mitochondrial energy metabolic traits and gene expressions involved in mitochondrial biogenesis and reproduction traits using transmitochondrial cell models. This study provides an insight into mitotype effect on reproduction traits of domestic animals. Therefore, mitotype can be considered as candidate markers for animal selection and breeding.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by the Experimental Animal Care and Use Committee of CAU.

AUTHOR CONTRIBUTIONS
XZo and HL conceived and designed the study. HL, JW, DW, and XZn performed the experiments. HL, DW, and CN analyzed the data. XZg, HL, JX, and MK collected the samples. XZo and HL interpreted the data and drafted the manuscript. All authors read and approved the final manuscript. Different letters on columns meant significant differences at P < 0.01. PH1-7, fibroblast with mitotype 1-7, respectively; IPH1-7, cybrid with mitotype 1-7, respectively.