Cyclophosphamide reduces gene transcriptional activity and embryo in vitro development by inhibiting NF-κB expression through decreasing AcH4K12.

Cyclophosphamide (CTX), a widely used chemotherapeutic agent for cancer treatment, has been associated with long-term toxicity and detrimental effects on oocytes and ovaries, resulting in female reproductive dysfunction. This study aimed to investigate the potential impact of CTX on in vitro maturation (IVM) injury of porcine oocytes and subsequent embryonic development, as well as its effects on epigenetic modification and gene activation during early embryonic development. The results demonstrated that CTX treatment caused aberrant spindle structure and mitochondrial dysfunction during oocyte maturation, inducing DNA damage and early apoptosis, which consequently disrupted meiotic maturation. Indeed, CTX significantly reduced the in vitro developmental capacity of porcine embryos, and induced DNA damage and apoptosis in in vitro fertilization (IVF) blastocysts. Importantly, CTX induced abnormal histone modification of AcH4K12 in early porcine embryos. Moreover, addition of LBH589 before zygotic genome activation (ZGA) effectively increased AcH4K12 levels and restored the protein expression of NF-κB, which can effectively enhance the in vitro developmental potential of IVF embryos. The DNA damage and apoptosis induced by CTX compromised the quality of the blastocysts, which were recovered by supplementation with LBH589. This restoration was accompanied by down-regulation of BAX mRNA expression and up-regulation of BCL2, POU5F1, SOX2 and SOD1 mRNA expression. These findings indicated that CTX caused abnormal histone modification of AcH4K12 in early porcine embryos and reduced the protein expression of NF-κB, a key regulator of early embryo development, which may block subsequent ZGA processes.

Application of PCR-RFLP for quick identification of MSTN mutants in MSTN mutant pig breeding.

Knockout of the MSTN gene is linked to the enlarged tongue, and it causes suckling difficulty in animals. The suckling difficulty has a severe effect on animal mortality. Thus, special care was required to ensure their survivability. Here, it is critical to promptly ascertain the genotype of all pigs after birth. The main objective of the present study was to develop the restriction enzyme-mediated PCR-RFLP assay for MSTN mutant pig genotyping. To accomplish this, conserved oligonucleotide primer and restriction site were deduced according to the mutated sequence of the MSTN mutant pigs. PCR amplification yielded a 176 bp band for all homozygous MSTN mutant (MSTN-/-), heterozygous MSTN mutant (MSTN+/-) and wild-type (WT) pigs. However, MSTN+/- samples produced two fragments with 176 and 87 bp, and WT samples produced one fragment with 87 bp after being digested by BstNI. MSTN-/- samples were not digested by BstNI and yielded a 176 bp band. Thus, we were able to determine the genotype of all pigs using BstNI restriction enzyme-mediated PCR-RFLP method. Overall, the present study reported a simple and fast PCR-RFLP genotyping method for MSTN mutant pig breeding. The present study may contribute to the establishment of commercial breeding systems and the production of double muscle pigs.

Effects of myostatin gene knockout on porcine extraocular muscles.

Myostatin (MSTN), a negative regulator of skeletal muscle mass, is not well known in extraocular muscles (EOMs). EOMs are specialized skeletal muscles. Hence, in this study, the effect of MSTN on the superior rectus (SR) and superior oblique (SO) of 2-month-old MSTN knockout (MSTN-/-) and wild-type (WT) pigs of the same genotype was investigated. SR (P < 0.01) and SO (P < 0.001) fiber cross-sectional areas of MSTN-/- pigs were significantly larger than those of WT pigs. Compared with WT pigs, MSTN-/- SO displayed a decrease in type I fibers (WT: 27.24%, MSTN-/-: 10.32%, P < 0.001). Type IIb fibers were higher in MSTN-/- pigs than in WT pigs (WT: 30.38%, MSTN-/-: 62.24%, P < 0.001). The trend in SR was the same as that in SO, although the trend in SO was greater than that in SR. The expression of myogenic differentiation factor (MyoD) and myogenic (MyoG) showed a significant increase in MSTN-/- SO (about 2.5-fold and 2-fold, respectively at the gene expression level, about 1.5-fold at the protein level) compared with WT pigs. MSTN plays an important role in the development of EOMs and regulates the muscle fiber type by modulating the gene expression of MyoD and MyoG in pigs.

Effect of myostatin gene mutation on erythrocyte osmotic fragility, hematological parameters and fatty acid composition of serum and erythrocyte membranes in piglets.

Fatty acid composition of serum and erythrocyte membrane, erythrocyte osmotic fragility and hematological parameters were estimated with the objective of determining effects of the gene mutation in one-week-old MSTN homozygous mutant (KO, MSTN-/-), heterozygous mutant (MSTN-/+) and wild type (WT, MSTN+/+) piglets (n = 4 each). Erythrocyte osmotic fragility, complete blood count (CBC), and fatty acid composition of serum and erythrocyte membrane were determined by flow cytometric analysis, automated hematology analyzer system, and liquid chromatography, respectively. Mean of median corpuscular fragility (MCF) was lower (P < 0.05, 0.001) in KO than MSTN-/+ and WT piglets. KO piglets had decreased (P < 0.05) white blood cell (WBC) count, lymphocyte (LYM) count, platelet (PLT) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell distribution width-standard deviation (RDW-SD), red cell distribution width-coefficient volume (RDW-CV), plateletcrit (PCT), mean platelet volume (MPV), platelet distribution width (PDW), platelet-large cell ratio (P-LCR), and an increased red blood cell (RBC) count when compared with MSTN-/+ and WT piglets. The ratios of unsaturated fatty acid (UFA) to saturated fatty acid (SFA) concentrations in serum and erythrocyte membranes of MSTN KO piglets were 2-fold and 4-fold higher compared to WT piglets (P < 0.001), respectively. In conclusion, MSTN KO piglets had a decreased erythrocyte osmotic fragility, and altered hematological profile and fatty acid composition of serum and erythrocyte membranes, as characteristic phenotype.

Silencing myostatin increases area fraction of smooth muscle in the corpus cavernosum of pigs.

Myostatin (MSTN), an inhibitor of skeletal muscle growth, is also expressed in penile smooth muscle; however, it is unclear whether MSTN plays an inhibitory role in penile smooth muscle growth. We investigated the role of MSTN in the smooth muscle of the penile corpus cavernosum of pigs using MSTN homozygous mutant knockout (KO) and wild type (WT) pigs (n = 4 in each group). The mean of area fraction (%) of smooth muscle in the penile corpus cavernosum was 65.9 % ± 1.79 in the KO and approximately 41.7 % ± 5.39 in the WT (P < 0.001). KO pigs showed significantly increased expression of smooth muscle-specific genes, including smooth muscle protein 22 (TAGLN) (6.62-fold), smooth muscle myosin heavy chain (MYH11) (2.41-fold), myocardin (MYOCD) (3.05-fold), and serum response factor (SRF) (4.95-fold), and decreased expression of vimentin (VIM) (1.36-fold). Immunofluorescence staining and Western blotting showed smooth muscle-specific expression of α-smooth muscle actin (SMA) and calponin was higher in KO pigs (P < 0.05) than in WT pigs. KO pigs had less fat deposition inside the corpus cavernosum, and showed downregulation of adiponectin (ADIPOQ) and fatty acid synthase (FASN) (2.5-fold and 1.9-fold loss, respectively). In vitro experiments showed MSTN interference promoted corporal smooth muscle cell growth and expression of smooth muscle-specific markers, whereas it downregulated the expression of fat-specific genes, ADIPOQ and FASN. MSTN inhibition could promote smooth muscle growth and decrease fat deposition in the corpus cavernosum. MSTN, thus, could be a possible target for the treatment of smooth muscle dystrophy-related disorders such as erectile dysfunction.

Myostatin deficiency decreases cardiac extracellular matrix in pigs.

Myostatin (MSTN), a member of the TGF-ß superfamily, negatively regulates muscle growth. MSTN inhibition has been known to cause a double-muscled phenotype in skeletal muscle and fibrosis reduction in the heart. However, the role of MSTN in the cardiac extracellular matrix (ECM) needs more studies in various species of animal models to draw more objective conclusions. The main objective of the present study was to investigate whether loss of MSTN affects the cardiac extracellular matrix in pigs. Three MSTN knockouts (MSTN-/-) and three wild type (WT) male pigs were generated by crossing MSTN ± heterozygous gilts and boars. Cardiac ECM and underlying mechanisms were determined post-mortem. The role of MSTN on collagen expression was investigated by treating cardiac fibroblasts with active MSTN protein in vitro. MSTN protein was detected in WT hearts, while no expression was detected in MSTN-/- hearts. The heart-to-body weight ratio was significantly decreased in MSTN-/- pigs. The morphometric analyses, including picrosirius red staining, immunofluorescent staining, and ultra-structural thickness examination of the endomysium, revealed a significant reduction of connective tissue content in MSTN-/- hearts compared to WT. Hydroxyproline, type I collagen (Col1A), and p-Smad3/Smad3 levels were significantly lower in MSTN-/- hearts in vivo. On the contrary, cardiac fibroblasts treated with exogenous MSTN protein overexpressed Col1A and activated Smad and AKT signaling pathways in vitro. The present study suggests that inhibition of MSTN decreases cardiac extracellular matrix.

Positive growth of smooth muscle in uterine horns of myostatin homozygous mutant gilt.

Current studies on myostatin (MSTN), a well-known negative regulator of skeletal muscle, studies mainly focus on the its effects on skeletal muscle.However, its effects on smooth muscle are less studied, especially in the uterine horns. To identify the role of MSTN in uterine horn smooth muscle, this study used 6-8-month-old homozygous MSTN mutant (MSTN-/-) gilts in anoestrum as animal models. Histochemical and immunofluorescence staining, western blotting, and RT-qPCR were performed. The results showed that the uteri of the MSTN-/- gilts were morphologically normal, and the uterine horn smooth muscle content was increased (MSTN-/-: 75.19%, Wild type: 51.52%, P < 0.01). In vivo immunofluorescence staining showed that the expression of the uterine horn smooth muscle-specific marker proteins, namely α-smooth muscle actin (ACTA2) and calponin, increased after MSTN knockout (1.41- and 1.21-fold, respectively, P < 0.05). Increased gene expression was also seen in MSTN-/- gilts in vivo for ACTA2 (approximately 2-fold), smooth muscle myosin heavy chain (7.14-fold), myocardin (9.32-fold), and serum response factor (2.17-fold). Protein expression of smooth muscle-specific markers was increased (1.51-fold for ACTA2, 1.57-fold for calponin, P<0.05). MSTN knockout promoted proliferation of the smooth muscle cell and the gene expression of c-kit, a peristaltic marker (2.43-fold, P < 0.05). The results of the in vitro experiments were consistent with those of the in vivo experiments. The present study indicates that MSTN knockout can increase the smooth muscle content of uterine horns, thus providing potential therapeutic targets for pregnancy disorders caused by increased smooth muscle content.

miR-455-3p Is Negatively Regulated by Myostatin in Skeletal Muscle and Promotes Myoblast Differentiation.

Myostatin (MSTN) is a growth and differentiation factor that regulates proliferation and differentiation of myoblasts, which in turn controls skeletal muscle growth. It may regulate myoblast differentiation by influencing miRNA expression, and the present study aimed to clarify its precise mechanism of action. Here, we found that MSTN-/- pigs showed an overgrowth of skeletal muscle and upregulated miR-455-3p level. Intervention of MSTN expression using siMSTN in C2C12 myoblasts also showed that siMSTN significantly increased the expression of miR-455-3p. It was found that miR-455-3p directly targeted the 3'-untranslated region of Smad2 by dual-luciferase assay. qRT-PCR, Western blotting, and immunofluorescence analyses indicated that miR-455-3p overexpression or Smad2 silencing in C2C12 myoblasts significantly promoted myoblast differentiation. Furthermore, siMSTN significantly increased the expression of GATA3. The levels of miR-455-3p were considerably reduced in C2C12 myoblasts following GATA3 knockdown. Consistently, GATA3 knockdown also reduced the enhanced miR-455-3p expression caused by siMSTN. Finally, we illustrated that GATA3 has a role in myoblast differentiation regulation. Taken together, we identified the expression profiles of miRNAs in MSTN-/- pigs and found that miR-455-3p positively regulates myoblast differentiation. In addition, we revealed that MSTN acts through the GATA3/miR-455-3p/Smad2 cascade to regulate muscle development.

Altered fibrinogen level and fibrin clot structure in myostatin homozygous mutant pig.

Obesity is associated with increased serum fibrinogen level. Myostatin (MSTN), a strong inhibitor of skeletal muscle growth, is recognized as a potential target for obesity. However, the effect of MSTN inhibition on fibrinogen is not largely known. The objective of the present study was to explore fibrinogen levels after MSTN inhibition. Fibrinogen levels and the fibrin clot structure of MSTN homozygous knockout (KO) and wild-type (WT) pigs (n = 4 in each group) were investigated. The protein expression of fibrinogen in the serum and liver of KO pigs decreased greatly (1.6-fold loss for serum and 2.5-fold loss for liver). KO pigs showed significantly decreased gene expression of fibrinogen chains: FGA (fibrinogen-α; 11-fold), FGB (fibrinogen-ß; 8-fold) and FGG (fibrinogen-γ; 7.4-fold). The basal transcriptional regulators of fibrinogen, HNF1 (hepatocyte nuclear factor 1) and CEBP-α (CCAAT/Enhancing-binding protein-alpha) were remarkably down-regulated after interruption of MSTN expression by siRNA (small interfering RNA) in cultured hepatocytes (about 2- and 4-fold, respectively). Compared with WT pigs, KO pigs displayed altered fibrin clot structure with thinner fibers, decreased turbidity and increased permeability. The findings indicate that the inhibition of MSTN could affect fibrinogen levels and the fibrin clot structure.

Association of myostatin deficiency with collagen related disease-umbilical hernia and tippy toe standing in pigs.

Herein, we investigate the high incidence of umbilical hernia and tippy-toe standing and their underlying changes in gene expression and proliferation in myostatin knockout (MSTN-/-) pigs. Thirty-six male MSTN-/- pigs were generated by somatic cell nuclear transfer (SCNT). These pigs presented a considerably high incidence of tippy-toe standing and umbilical hernia (69.4% and 61.1%, respectively). The tendon to body weight ratio was significantly lower than wild-type pigs (0.202 ± 0.017 vs 0.250 ± 0.004, respectively). The crimp length of the MSTN-/- tendon was significantly longer than that of wild-type pigs. The expression of MSTN and the activin type IIB (ACVR2B) was detected in the tendon and linea alba of MSTN-/- pigs. MSTN treatment significantly increased the phosphorylation of Smad2/3 in both tendon and linea alba fibroblasts. Type I collagen (Col1A) and Scleraxis (Scx) expression levels in the tendon and linea alba of MSTN-/- pigs were significantly lower than those in wild-type in vivo, whereas and cyclin-dependent kinase inhibitor 1 (p21) expression levels were higher. Treatment of tendon and linea alba fibroblasts with recombinant MSTN increased Col1A and Scx and decreased p21 expression in vivo. Moreover, there was a significant increase in fibroblast proliferation after treatment. The results indicated that MSTN regulates collagen expression and proliferation in tendon and linea alba fibroblasts; thus, MSTN deficiency causes collagen-related pathological features in MSTN-/- pigs. Hence, MSTN could be used as a therapeutic target for treating UH and tendon abnormalities.

Ginsenoside Rb1 protects porcine oocytes against methylglyoxal damage thus it improves the quality of parthenogenetic activation and in vitro fertilization embryos.

Panax ginseng, a functional food, has been widely used as an edible nourishment and medicinal supplement. Ginsenoside Rb1 is a major bioactive ingredient of ginseng, which shows very specific anti-apoptosis and anti-oxidant activities. Methylglyoxal (MGO) is one of intermediate products of glucose metabolism, which is absorbed easily from high sugar foods or carbonated beverages. It may involve in a variety of detrimental processes in vivo. However, it has not been fully explored the effects of ginsenoside Rb1 on MGO-induced oocytes damage. This study found that MGO-induced DNA damage and mitochondrial dysfunction result in the failure of porcine oocytes maturation and low in vitro development capacity of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos. Conversely, Rb1 supplementation recovered the rate of maturation, and improved in vitro development capacity of PA and IVF embryos. Rb1 also provided porcine oocytes a lower level of reactive oxygen species production, higher level of ATP content and mitochondrial membrane potential, and stimulated pluripotency gene expression in blastocysts. The findings of this study reveal ginsenoside Rb1 protects porcine oocyte from the cytotoxicity effects of methylglyoxal and provides novel perspectives for the protection of reproduction system by functional food of ginseng.

Reproduction traits of heterozygous myostatin knockout sows crossbred with homozygous myostatin knockout boars.

Few studies exist on homozygous myostatin gene mutant (MSTN-/- ) pigs, especially on their reproductive ability. We have previously shown that semen quality of homozygous MSTN-/- boars is comparable to that of wild type (WT). However, no data exist on the reproductive ability of heterozygous MSTN gene mutant (MSTN+/ - ) sows. The present study highlights showed that the heterozygous MSTN+/ - sows have delayed pubertal age than WT sows (255.80 ± 6.79 versus 191.10 ± 3.42, respectively). The number of services per pregnancy of heterozygous MSTN+/ - sows is significantly higher than that of WT sows (3.33 ± 0.43 versus 1.60 ± 0.25, respectively). Moreover, although heterozygous MSTN+/ - sows have natural reproduction ability, their litter size was significantly lower than that of WT sows (7.75 ± 0.44 versus 14.25 ± 0.60, respectively). Offsprings generated from heterozygous MSTN+/ - sow and homozygous MSTN-/- boar were genotyped with the PCR and sequencing method to detect myostatin mutation and to identify whether the piglets are homozygous MSTN-/- or heterozygous MSTN+/ - . The proportion of homozygous MSTN-/- piglets was significantly lower than that of heterozygous MSTN+/ - piglets (2.50 ± 0.35 versus 5.25 ± 0.60, respectively). Furthermore, none of the sows presented dystocia, and the phenotype of heterozygous MSTN+/ - piglets was normal. However, 10% homozygous MSTN-/- piglets died of dyspnoea within 2 hr after birth, 60% of homozygous MSTN-/- piglets showed large tongues, and 50% had umbilical hernias. In summary, this study for the first time reports the reproduction traits of heterozygous MSTN+/ - sows crossbred with homozygous MSTN-/- boars. This study will pave the way in a new direction for the breeding and development of super lean meat varieties in the future.