Mammalian spermatozoa must undergo capacitation, before becoming competent for fertilization. Despite its importance, the fundamental molecular mechanisms of capacitation are poorly understood. Therefore, in this study, we applied a proteomic approach for identifying capacitation-related proteins in boar spermatozoa in order to elucidate the events more precisely. 2-DE gels were generated from spermatozoa samples in before- and after-capacitation. To validate the 2-DE results, Western blotting and immunocytochemistry were performed with 2 commercially available antibodies. Additionally, the protein-related signaling pathways among identified proteins were detected using Pathway Studio 9.0. We identified Ras-related protein Rab-2, Phospholipid hydroperoxide glutathione peroxidase (PHGPx) and Mitochondrial pyruvate dehydrogenase E1 component subunit beta (PDHB) that were enriched before-capacitation, and NADH dehydrogenase 1 beta subcomplex 6, Mitochondrial peroxiredoxin-5, (PRDX5), Apolipoprotein A-I (APOA1), Mitochondrial Succinyl-CoA ligase [ADP-forming] subunit beta (SUCLA2), Acrosin-binding protein, Ropporin-1A, and Spermadhesin AWN that were enriched after-capacitation (>3-fold) by 2-DE and ESI-MS/MS. SUCLA2 and PDHB are involved in the tricarboxylic acid cycle, whereas PHGPx and PRDX5 are involved in glutathione metabolism. SUCLA2, APOA1 and PDHB mediate adipocytokine signaling and insulin action. The differentially expressed proteins following capacitation are putatively related to sperm functions, such as ROS and energy metabolism, motility, hyperactivation, the acrosome reaction, and sperm-egg interaction. The results from this study elucidate the proteins involved in capacitation, which may aid in the design of biomarkers that can be used to predict boar sperm quality.

Muscle development and lipid accumulation in muscle critically affect meat quality of livestock. However, the genetic factors underlying myofiber-type specification and intramuscular fat (IMF) accumulation remain to be elucidated. Using two independent intercrosses between Western commercial breeds and Korean native pigs (KNPs) and a joint linkage-linkage disequilibrium analysis, we identified a 488.1-kb region on porcine chromosome 12 that affects both reddish meat color (a*) and IMF. In this critical region, only the MYH3 gene, encoding myosin heavy chain 3, was found to be preferentially overexpressed in the skeletal muscle of KNPs. Subsequently, MYH3-transgenic mice demonstrated that this gene controls both myofiber-type specification and adipogenesis in skeletal muscle. We discovered a structural variant in the promotor/regulatory region of MYH3 for which Q allele carriers exhibited significantly higher values of a* and IMF than q allele carriers. Furthermore, chromatin immunoprecipitation and cotransfection assays showed that the structural variant in the 5′-flanking region of MYH3 abrogated the binding of the myogenic regulatory factors (MYF5, MYOD, MYOG, and MRF4). The allele distribution of MYH3 among pig populations worldwide indicated that the MYH3 Q allele is of Asian origin and likely predates domestication. In conclusion, we identified a functional regulatory sequence variant in porcine MYH3 that provides novel insights into the genetic basis of the regulation of myofiber type ratios and associated changes in IMF in pigs. The MYH3 variant can play an important role in improving pork quality in current breeding programs. Local native livestock breeds, such as pigs and cattle, are important reservoirs of genetic variation, because they have adapted to local conditions and have not been as strongly selected as commercial livestock breeds. Therefore, the study of local native breeds is of utmost interest to identify causative mutations for traits in which large differences exist between local native breeds and breeds used internationally for meat production. Herein, we apply these principles to meat quality related traits in pigs. A combined linkage-linkage disequilibrium analysis identified the MYH3 gene on pig chromosome 12, which encodes myosin heavy chain 3 protein. Subsequently, we discovered a regulatory mutation in MYH3, which occurs at high frequency in Korean Native Pigs compared to the frequency at which it occurs in Western pig breeds. We confirmed the effect of MYH3 on muscle fiber-type composition and intramuscular fat content using transgenic mice and transfection assays in porcine fibroblast cells. This is the first causal mutation identified in livestock for these traits and the result is immediately applicable for the genetic improvement of pork quality. These results also enhance our understanding of the genetic basis regarding the regulation of myofiber type ratios and associated changes in intramuscular fat contents in pigs and mammals in general.

Myosin heavy chain (MyHC) isoforms consist of Myh7, Myh2, Myh1, and Myh4, which are expressed in skeletal muscle tissues during postnatal development. These genes influence the contraction–relaxation activity in skeletal muscles and are involved in determining muscle composition such as the proportion of fast-to-slow and/or slow-to-fast fiber types. Among them, Myh1 is associated with skeletal muscle contraction and is involved in both slow-to-fast and fast-to-slow transition. However, the muscle transition mechanism is not well understood. For this study, we first produced porcine Myh1 transgenic (TG) mice to study whether the ectopic expressed porcine Myh1 gene had any effects on muscle composition, especially on slow-type muscle components. Our results showed that the factors associated with slow muscles, such as Myh7, Myoglobin, Troponin (slow-type units), and cytochrome C, were highly expressed in the quadriceps muscles of Myh1 transgenic mice. Furthermore, the ectopic porcine MYH1 protein was located only in the slow-type muscle fibers of the quadriceps muscles in Myh1 transgenic mice. In physical endurance tests, Myh1 transgenic mice ran longer and further on a treadmill than wild-type (WT) mice. These data fully supported our hypothesis that Myh1 is associated with slow muscle composition, with overexpression of Myh1 in muscle tissues possibly being a new key in modulating muscle fiber types. Our study provides a better understanding of muscle composition metabolism, physical mobility, and genetic factors in muscle fatigue.

Recent progress in genetic manipulation of pigs designated for xenotransplantation ha6s shown considerable promise on xenograft survival in primates. However, genetic modification of multiple genes in donor pigs by knock-out and knock-in technologies, aiming to enhance immunological tolerance against transplanted organs in the recipients, has not been evaluated for health issues of donor pigs. We produced transgenic Massachusetts General Hospital piglets by knocking-out the α-1,3-galactosyltransferase (GT) gene and by simultaneously knocking-in an expression cassette containing five different human genes including, DAF, CD39, TFPI, C1 inhibitor (C1-INH), and TNFAIP3 (A20) [GT^{−(DAF/CD39/TFPI/C1-INH/TNFAIP3)/+}] that are connected by 2A peptide cleavage sequences to release individual proteins from a single translational product. All five individual protein products were successfully produced as determined by western blotting of umbilical cords from the newborn transgenic pigs. Although gross observation and histological examination revealed no significant pathological abnormality in transgenic piglets, hematological examination found that the transgenic piglets had abnormally low numbers of platelets and WBCs, including neutrophils, eosinophils, basophils, and lymphocytes. However, transgenic piglets had similar numbers of RBC and values of parameters related to RBC compared to the control littermate piglets. These data suggest that transgenic expression of those human genes in pigs impaired hematopoiesis except for erythropoiesis. In conclusion, our data suggest that transgenic expression of up to five different genes can be efficiently achieved and provide the basis for determining optimal dosages of transgene expression and combinations of the transgenes to warrant production of transgenic donor pigs without health issues.

The generation and application of porcine induced pluripotent stem cells (iPSCs) may enable the testing for safety and efficacy of therapy in the field of human regenerative medicine. Here, the generation of iPSCs from the Massachusetts General Hospital miniature pig (MGH minipig) established for organ transplantation studies is reported. Fibroblasts were isolated from the skin of the ear of a 10-day-old MGH minipig and transduced with a cocktail of six human factors:POU5F1, NANOG, SOX2, C-MYC, KLF4,andLIN28. Two distinct types of iPSCs were generated that were positive for alkaline phosphatase activity, as well as the classical pluripotency markers:Oct4, Nanog, Sox2, and the surface marker Ssea-1. Only one of two porcine iPSC lines differentiated into three germ layers bothin vitroandin vivo. Western blot analysis showed that the porcine iPSCs were dependent on LIF or BMP-4 to sustain self-renewal and pluripotency. In conclusion, the results showed that human pluripotent factors could reprogram porcine ear fibroblasts into the pluripotent state. These cells may provide a useful source of cells that could be used for the treatment of degenerative and genetic diseases and agricultural research and application.