WFIKKN2 may play a role in the regulation of muscle growth and development through its interaction with growth and differentiation factor 8 (GDF8) and growth and differentiation factor 11 (GDF11), but to date research into the function of the protein has been focused on mice, even though the WFIKKN2 gene (WFIKKN2) was first identified in humans in 2001. In this study two regions (intron 1 and the 3' UTR) of ovine WFIKKN2 were investigated, using Polymerase Chain Reaction-Single Stranded Conformational Polymorphism (PCR-SSCP). Two different PCR-SSCP patterns, representing two unique DNA sequences (designated a and b) were detected in a 399-bp amplicon derived from the 3' UTR, with sequence analysis revealing one single nucleotide polymorphism (SNP). In a 421-bp amplicon from intron 1, five different PCR-SSCP patterns (designated A-E) were observed and twelve SNPs were detected. Either one or two different sequences were detected in individual sheep and all the sequences identified shared homology with the WFIKKN2 sequences from cattle and other animal species, suggesting that these sequences represent variants of the ovine WFIKKN2 gene. In intron 1 of 487 sheep from eight breeds, variants B and C were the most common, followed by A, D and E. These results indicate that ovine WFIKKN2 is polymorphic and suggest that further analysis is required to see if variation in the gene is associated with variation in growth and muscle traits in sheep.

Although butterflies undergo a dramatic morphological transformation from larva to adult via a pupal stage (holometamorphosis), crickets undergo a metamorphosis from nymph to adult without formation of a pupa (hemimetamorphosis). Despite these differences, both processes are regulated by common mechanisms that involve 20-hydroxyecdysone (20E) and juvenile hormone (JH). JH regulates many aspects of insect physiology, such as development, reproduction, diapause, and metamorphosis. Consequently, strict regulation of JH levels is crucial throughout an insect's life cycle. However, it remains unclear how JH synthesis is regulated. Here, we report that in the corpora allata of the cricket, Gryllus bimaculatus, Myoglianin (Gb'Myo), a homolog of Drosophila Myoglianin/vertebrate GDF8/11, is involved in the down-regulation of JH production by suppressing the expression of a gene encoding JH acid O-methyltransferase, Gb'jhamt In contrast, JH production is up-regulated by Decapentaplegic (Gb'Dpp) and Glass-bottom boat/60A (Gb'Gbb) signaling that occurs as part of the transcriptional activation of Gb'jhamt Gb'Myo defines the nature of each developmental transition by regulating JH titer and the interactions between JH and 20E. When Gb'myo expression is suppressed, the activation of Gb'jhamt expression and secretion of 20E induce molting, thereby leading to the next instar before the last nymphal instar. Conversely, high Gb'myo expression induces metamorphosis during the last nymphal instar through the cessation of JH synthesis. Gb'myo also regulates final insect size. Because Myo/GDF8/11 and Dpp/bone morphogenetic protein (BMP)2/4-Gbb/BMP5-8 are conserved in both invertebrates and vertebrates, the present findings provide common regulatory mechanisms for endocrine control of animal development.

Myostatin, also known as growth differentiation factor 8 (GDF8), is a transforming growth factor-β (TGF-β) family member that functions to limit skeletal muscle growth. Accordingly, loss-of-function mutations in myostatin result in a dramatic increase in muscle mass in humans and various animals, while its overexpression leads to severe muscle atrophy. Myostatin also exerts a significant effect on bone metabolism, as demonstrated by enhanced bone mineral density and bone regeneration in myostatin null mice. The identification of myostatin as a negative regulator of muscle and bone mass has sparked an enormous interest in developing myostatin inhibitors as therapeutic agents for treating a variety of clinical conditions associated with musculoskeletal disorders. As a result, various myostatin-targeting strategies involving antibodies, myostatin propeptides, soluble receptors, and endogenous antagonists have been generated, and many of them have progressed to clinical trials. Importantly, most myostatin inhibitors also repress the activities of other closely related TGF-β family members including GDF11, activins, and bone morphogenetic proteins (BMPs), increasing the potential for unwanted side effects, such as vascular side effects through inhibition of BMP 9/10 and bone weakness induced by follistatin through antagonizing several TGF-β family members. Therefore, a careful distinction between targets that may enhance the efficacy of an agent and those that may cause adverse effects is required with the improvement of the target specificity. In this review, we discuss the current understanding of the endogenous function of myostatin, and provide an overview of clinical trial outcomes from different myostatin inhibitors.

Keywords: Activins; Bones; Follistatin; GDF11; Muscles; Myostatin.

Breast adenocarcinoma continues to be the most frequently diagnosed tumor entity. Despite established therapy options, mortality for breast cancer remains to be as high as 40,000 patients in the US annually. Thus, a need to develop a patient-oriented, targeted therapy exists. In this study, we investigated the interaction of breast adenocarcinoma with the ubiquitously present protein Follistatin and subsequently the GDF8/11 pathway. We analyzed primary histological samples from adenocarcinoma patients for expression of Follistatin and GDF8/11. Furthermore, expression levels of Follistatin and GDF8/11 in MCF7 were compared with MCF10a cells. From the resulting data, GDF8 and Follistatin were used as chemotherapeutic agents in MCF7 cells and their migratory, proliferative behavior and viability were measured. From the experiments, we were able to detect a significantly increased expression of Follistatin and GDF8/11 in the low malignant breast adenocarcinoma (G1) as compared to benign breast fibroadenoma. Interestingly, a decrease was demonstrated in higher grade malignancies. These findings were accompanied by the clinical observation that increased expression of Follistatin and GDF8 is associated with a higher overall survival rate of breasts cancer patients. Substitution of GDF8 and Follistatin reduces the viability of the MCF7 cells and disrupts the migrative and proliferative potential. In summary, MCF7 cells show high chemosensitivity to Follistatin and especially GDF8 and both proteins might serve as targets to improve systemic treatment in breast cancer. In contrast to most established chemotherapy regimens Follistatin and GDF8 show no cytotoxicity to other organs.

Myostatin, which is also known as growth and differentiation factor 8 (GDF8), acts as a negative regulator of skeletal muscle growth. Variation in the myostatin gene (MSTN) has been associated with variation in muscularity in many animals including sheep. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis was used to investigate MSTN in a diverse range of sheep breeds including the New Zealand (NZ) Romney, Coopworth, Corriedale, Dorper, Perendale, Suffolk, Merino, Dorset Down, Poll Dorset, Texel and other NZ cross-bred sheep. A total of 28 nucleotide substitutions were identified from nucleotide c.-1199 in the promoter region to c.*1813 (based on NCBI GenBank accession number DQ530260) and including the well-described substitution c.*1232G>A (MSTN g+6223G>A). Of these 28 substitutions, 3 were located in the promoter region, 3 in the 5'UTR, 11 in intron 1, 5 in intron 2 and 5 in the 3'UTR. One substitution in exon 1 (c.101G>A) potentially results in an amino acid substitution of glutamic acid (Glu) with glycine (Gly) at codon 34. Ten of these substitutions have not been reported previously. The genetic variation revealed in this study suggests this gene is more variable than hitherto reported and provides a foundation for future research into how this variation affects muscle and growth traits.

One of the most studied and widely accepted conjectures of aging process is the oxidative stress theory. Current studies have generated disputes on the effects of GDF11 and GDF8, a closely related member of GDF11, on rejuvenation and anti-aging properties. In this study, we first demonstrated that when recombinant GDF8 (rGDF8) and GDF11 (rGDF11) of the fish Nothobranchius guentheri were injected into 20-month-old male mice, their serum GDF8 and GDF11 levels were clearly increased. We also showed that injection of rGDF8 and rGDF11 had little influences on the body weight and serological parameters of the mice, indicating their general condition and physiology were not affected. Based on these findings, we started to test the effects of administration of piscine rGDF11 and rGDF8 on the aging process of male mice and to explore the underlying mechanisms. It was found that rGDF11 was able to reduce the levels of AGEs, protein oxidation and lipid peroxidation, and to slow down the accumulation of age-related histological markers, while rGDF8 was not. Moreover, rGDF11 significantly prevented the decrease in CAT, GPX and SOD activities, but rGDF8 did not. Collectively, these results suggest that it is GDF11 but not GDF8 that can exert rejuvenation and anti-aging activities via the action of antioxidant system. It is also the first report that shows the activity of GDF11 is not species-specific, implicating potential usefulness of piscine GDF11 in prolonging the lifespan of the elderly.

Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF-β family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid-cleaved GDF8 pro-complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro-complexes reveals a V-shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring-like, cross-armed conformation of latent TGF-β1. Surprisingly, Tolloid-cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-β1 to the α1-helix, latency lasso, and β1-strand in the prodomain and to the β6'- and β7'-strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain.

Myostatin, also known as GDF8 (growth differentiation factor 8), belongs to one of the biggest groups of proteins, called transforming growth factors (TGF-β). The protein regulates embryonic development and maintains homeostasis of full-grown individuals. When the process of maturation is over, GDF8 becomes a negative regulator of skeletal muscle growth. The physiological role of myostatin is to prevent overgrowth of muscle tissue in various stages of organism development. It also inhibits the regeneration of skeletal muscles by weakening activation and proliferation of satellite cells and the migration of macrophages and myoblasts to the site of injury. Several mutations within the sequence of MSTN have been described; they may affect the activity of the protein in the tissues, and hence the level of the functional characteristics of the animals, which determine the value of breeding animals. Understanding the impact of individual mutations in the gene encoding the protein is particularly important in relation to production traits of livestock.

Myostatin, encoded by the MSTN gene (previously GDF8), is a member of the transforming growth factor-β superfamily, which normally acts to limit skeletal muscle mass by regulating the number and growth of muscle fibers. In this study, a total of 84 myostatin gene sequences with known complete coding regions (CDS) and corresponding amino acid sequences were analyzed from 17 species, and differentiation within and among species was studied using comparative genomics and bioinformatics. Characteristics of the nucleotide and amino acid sequences were also predicted. The results indicated that a total of 569 polymorphic sites, including 53 singleton variable sites and 516 parsimony informative sites, which could be sorted into 44 haplotypes, were detected from 17 species. Observed genetic diversity was higher among species than within species, and Vulpes lagopus was more polymorphic than other species. There was clear differentiation of the myostatin gene among species and the reconstructed phylogenetic tree was consistent with the NCBI taxonomy. The myostatin gene was 375-aa long in most species, except for Mus musculus (376 aa) and Danio rerio (373 aa). The amino acid sequences of myostatin were deemed hydrophilic, and had theoretical pI values of <7.0, mostly due to the acidic polypeptide. The instability index of the myostatin protein was 40.48-51.63, indicating that the polypeptide is not stable. The G+C content of the CDS nucleotide sequence in different species was 40.60-51.69%. The predicted promoter region of the Ovis aries myostatin gene was 150-220 bp upstream of the start codon.

Growth differentiation factor 11 (GDF11) is a TGF-β superfamily circulating factor that regulates cardiomyocyte size in rodents, sharing 90% amino acid sequence identity in the active domains with myostatin (GDF8)- the major determinant of skeletal muscle mass. Conflicting data on age-related changes in circulating levels have been reported mainly due to the lack of specific detection methods. More recently, liquid chromatography tandem mass spectrometry (LC-MS/MS)-based assay showed that the circulating levels of GDF11 do not change significantly throughout human lifespan, but GDF8 levels decrease with aging in men. Here we demonstrate a novel detection method based on parallel reaction monitoring (PRM) LC-MS/MS assay combined with immunoprecipitation to reliably distinguish GDF11 and GDF8 as well as determine their endogenous levels in mouse serum. Our data indicate that both GDF11 and GDF8 circulating levels significantly decline with aging in female mice. This article is protected by copyright. All rights reserved.

Growth differentiation factor 11 (GDF11 or bone morphogenetic protein 11, BMP11) belongs to the transforming growth factor-β superfamily and is closely related to other family member-myostatin (also known as GDF8). GDF11 was firstly identified in 2004 due to its ability to rejuvenate the function of multiple organs in old mice. However, in the past few years, the heralded rejuvenating effects of GDF11 have been seriously questioned by many studies that do not support the idea that restoring levels of GDF11 in aging improves overall organ structure and function. Moreover, with increasing controversies, several other studies described the involvement of GDF11 in fibrotic processes in various organ setups. This review paper focuses on the GDF11 and its pro- or anti-fibrotic actions in major organs and tissues, with the goal to summarize our knowledge on its emerging role in regulating the progression of fibrosis in different pathological conditions, and to guide upcoming research efforts.

Keywords: BMP11; Fibrosis; GDF11; Growth differentiation factor 11.

Myostatin, also known as growth differentiation factor 8 (GDF8), belongs to the TGF-β superfamily of proteins. MSTN is a highly conserved protein that acts as a negative regulator of skeletal muscle growth. Loss of myostatin functionality causes the phenotype to appear in the form of 'double musculature', among others in cattle, sheep, and house mice. The presented results of the research were carried out on two geese breeds-Landes and Kielecka. The aim of the study was to identify mutations in the MSTN gene and study their impact on body weight in both geese breeds in different periods of life. Analysis of the obtained results showed the existence of polymorphism in exon 3 (c.1231C>T) and suggested a possible association (p < 0.05) between BW and genotype in 12 weeks of life in male Kielecka geese breed. The identified polymorphism may be one of the factors important for improving body weight in the studied Kielecka breed, therefore, it is necessary to conduct further research on a larger population of geese breeds in order to more accurately estimate the effect of the identified SNP c.1231C>T on BW in geese.

UNASSIGNED

Growth/differentiation factors 8 (GDF8) and 11 (GDF11) have attracted attention as targets for rejuvenating interventions. The biological activity of these proteins may be affected by circulating antagonists such as their respective prodomains, follistatin (FST315), WFIKKN1, and WFIKKN2. Reports of the relationship of GDF8 and GDF11 and their antagonists with aging and aging phenotypes such as skeletal muscle strength have been conflicting possibly because of difficulties in measuring these proteins and polypeptides.

UNASSIGNED

Plasma GDF8 and GDF11 and their antagonists were measured using a multiplexed selected reaction monitoring (SRM) assay and liquid chromatography-tandem mass spectrometry in 160 healthy adults aged 22 to 93 years. Quadriceps strength was measured by knee extensor torque using isokinetic dynamometry.

UNASSIGNED

Spearman correlations with age were: GDF11 prodomain (r=0.30, P=0.001), GDF11 mature protein (r=0.23, P=0.004), FST315 (r=0.32, P<0.0001), WFIKKN1 (r=-0.21, P=0.008), and WFIKKN2 (r=0.18, P=0.02). Independent of age, FST315 and WFIKKN1 were negatively associated with knee strength (P=0.02, P=0.03, respectively) in a multivariable model that included both GDF8 and GDF11 mature proteins.

UNASSIGNED

When measured by an antibody-free SRM assay, GDF8, GDF11, and their antagonists are found in the circulation in the ng/mL range. In healthy adults, plasma GDF11 and antagonists FST315, WFIKKN1, and WFIKKN2 differed by age. Antagonists of GDF8 and GDF11, but not GDF8 and GDF11, were independently associated with skeletal muscle strength. Further work is needed to characterize the relationship of these protein and polypeptides with sarcopenia-related phenotypes such as physical function and walking disability.

The present study was undertaken to identify polymorphism in the coding sequence of GDF8gene across indigenous meat type sheep breeds. A 1647 bp sequence was generated, encompassing 208 bp of the 5'UTR, 1128 bp of coding region (exon1, 2 and 3) as well as 311 bp of 3'UTR. The sheep and goat GDF8 gene sequences were observed to be highly conserved as compared to cattle, buffalo, horse and pig. Several nucleotide variations were observed across coding sequence of GDF8 gene in Indian sheep. Three polymorphic sites were identified in the 5'UTR, one in exon 1 and one in the exon 2 regions. Both SNPs in the exonic region were found to be non-synonymous. The mutations c.539T>> G and c.821T>> A discovered in this study in the exon 1 and exon 2, respectively, have not been previously reported. The information generated provides preliminary indication of the functional diversity present in Indian sheep at the coding region of GDF8gene. The novel as well as the previously reported SNPs discovered in the Indian sheep warrant further analysis to see whether they affect the phenotype. Future studies will need to establish the affect of reported SNPs in the expression of the GDF8 gene in Indian sheep population.

Myostatin (MSTN), a protein encoded by growth differentiation factor 8 (GDF8), is primarily expressed in skeletal muscle and negatively regulates the development and regeneration of muscle. Accordingly, myostatin-deficient animals exhibit a double-muscling phenotype. The CRISPR/Cas9 system has proven to be an efficient genome-editing tool and has been applied to gene modification in cells from many model organisms such as Drosophila melanogaster, zebrafish, mouse, rat, sheep, and human. Here, we edited the GDF8 gene in fibroblasts and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system. The CRISPR/Cas9-mediated mutation efficiency in fibroblasts was as high as 87.5% in pig and 78.9% in buffalo. We then obtained single-cell clones with mutations at the specific sites of the GDF8 gene by screening with G418 in fibroblasts of pig and buffalo. In addition, the frequencies of Cas9/gRNA-mediated mutations were at 36 and 25% in the intracytoplasmic sperm injection embryos of pig and in vitro fertilization embryos of buffalo, respectively. Our work demonstrates that the Cas9/gRNA system is a highly efficient and fast tool for genome editing in cultured cells and embryos of Debao pig and swamp buffalo. These results can be helpful for the establishment of a new animal strain that can generate more meat.

BACKGROUND

Adolescents with anorexia nervosa (AN) have low body mass and low bone mineral density (BMD). Growth differentiation factor 8 (Myostatin, GDF8) and its homologue growth differentiation factor 11 (GDF11), members of the TGF-β super-family, play an important role in muscle regeneration and bone metabolism in healthy individuals. However, their association with BMD in AN is unknown. The present study was undertaken to investigate the relationship between GDF8, GDF11 and BMD in adolescent girls with AN.

METHODS

Serum GDF8, GDF11 and BMD were determined in 25 girls (12-16 years old) with AN and 31 healthy girls (12-16 years old).

RESULTS

Growth differentiation factor 8 levels were lower in AN subjects. On the contrary, GDF11 levels were higher in AN subjects than controls. There was no relationship between GDF8 and BMD. A significant negative correlation between GDF11 and BMD was found. In multiple linear stepwise regression analysis, BMI, 25-hydroxyvitamin D, GDF11, or lean mass, but not fat mass and GDF8, were independent predictors of BMD in the AN and control groups separately.

CONCLUSIONS

Growth differentiation factor 11 was independent predictor of BMD in girls with AN. It suggested that GDF11 exerts a negative effect on bone mass.

The expression of inflammatory cytokines and growth factors in surgically repaired lacerated muscles over a 12-week recovery phase was investigated. We hypothesized that these expression levels are influenced by both neural and muscular damage within lacerated muscles. Microarrays were confirmed with reverse transcription-polymerase chain reaction assays and histology of biopsies at the lesion of three simulated lacerated muscle models in 130 adult rats. The lacerated medial gastrocnemius with the main intramuscular nerve branch either cut (DN), crushed but leaving an intact nerve sheath (RN); or preserved intact (PN) were compared. At 4 weeks, DN had a higher number of interleukins up-regulated. DN and RN also had a set of Bmp genes significantly expressed between 2 and 8 weeks (P ≤ 0.05). By 12 weeks, DN had a poorer and slower myogenic recovery and greater fibrosis formation correlating with an up-regulation of the Tgf-β gene family. DN also showed poorer re-innervation with higher mRNA expression levels of nerve growth factor (Ngf) and brain-derived neurotrophin growth factor (Bdnf) over RN and PN. This study demonstrates that the inflammatory response over 12 weeks in lacerated muscles may be directed by the type of intramuscular nerve damage, which can influence the recovery at the lesion site. Inflammatory-related genes associated to the type of intramuscular nerve damage include Gas-6, Artemin, Fgf10, Gdf8, Cntf, Lif, and Igf-2. qPCR also found up-regulation of Bdnf (1-week), neurotrophin-3 (2w), Lif (4w), and Ngf (4w, 8w) mRNA expressions in DN, making them possible candidates for therapeutic treatment to arrest the poor recovery in muscle lacerations (250).

Extraocular muscles (EOMs) are a highly specialized type of tissue with a wide range of unique properties, including characteristic innervation, development, and structural proteins. Even though EOMs are frequently and prominently affected by thyroid-associated diseases, little is known about the direct effects of thyroid hormone on these muscles. To create a comprehensive profile of changes in gene expression levels in EOMs induced by thyroid hormone, hyperthyroid conditions were simulated by treating adult Sprague-Dawley rats with intraperitoneal injections of the thyroid hormone 3,3',5-triiodo-L-thyronine (T(3)); subsequently, microarray analysis was used to determine changes in mRNA levels in EOMs from T(3)-treated animals relative to untreated control animals. The expression of 468 transcripts was found to be significantly altered, with 466 of these transcripts downregulated in EOMs from T(3)-treated animals. The biological processes into which the affected genes could be grouped included cellular metabolism, transport, biosynthesis, protein localization, and cell homeostasis. Moreover, 15 distinct biochemical canonical pathways were represented among the genes with altered transcription levels. Strikingly, myostatin (Gdf8), a potent negative regulator of muscle growth, was found to be strongly downregulated in EOMs from T(3)-treated animals. Together, these findings suggest that pathological concentrations of thyroid hormone have a unique effect on gene expression in EOMs, which is likely to play a hitherto neglected role in thyroid-associated ophthalmopathies.

Insulin resistance is associated with aging in mice and humans. We have previously shown that administration of recombinant GDF11 (rGDF11) to aged mice alters aging phenotypes in the brain, skeletal muscle, and heart. While the closely related protein GDF8 has a role in metabolism, limited data are available on the potential metabolic effects of GDF11 or GDF8 in aging. To determine the metabolic effects of these two ligands, we administered rGDF11 or rGDF8 protein to young or aged mice fed a standard chow diet, short-term high-fat diet (HFD), or long-term HFD. Under nearly all of these diet conditions, administration of exogenous rGDF11 reduced body weight by 3-17% and significantly improved glucose tolerance in aged mice fed a chow (~30% vs. saline) or HF (~50% vs. saline) diet and young mice fed a HFD (~30%). On the other hand, exogenous rGDF8 showed signifcantly lesser effect or no effect at all on glucose tolerance compared to rGDF11, consistent with data demonstrating that GFD11 is a more potent signaling ligand than GDF8. Collectively, our results show that administration of exogenous rGDF11, but not rGDF8, can reduce diet-induced weight gain and improve metabolic homeostasis.

Administration of active growth differentiation factor 11 (GDF11) to aged mice can reduce cardiac hypertrophy, and low serum levels of GDF11 measured together with the related protein, myostatin (also known as GDF8), predict future morbidity and mortality in coronary heart patients. Using mice with a loxP-flanked ("floxed") allele of Gdf11 and Myh6-driven expression of Cre recombinase to delete Gdf11 in cardiomyocytes, we tested the hypothesis that cardiac-specific Gdf11 deficiency might lead to cardiac hypertrophy in young adulthood. We observed that targeted deletion of Gdf11 in cardiomyocytes does not cause cardiac hypertrophy but rather leads to left ventricular dilation when compared with control mice carrying only the Myh6-cre or Gdf11-floxed alleles, suggesting a possible etiology for dilated cardiomyopathy. However, the mechanism underlying this finding remains unclear because of multiple confounding effects associated with the selected model. First, whole heart Gdf11 expression did not decrease in Myh6-cre; Gdf11-floxed mice, possibly because of upregulation of Gdf11 in noncardiomyocytes in the heart. Second, we observed Cre-associated toxicity, with lower body weights and increased global fibrosis, in Cre-only control male mice compared with flox-only controls, making it challenging to infer which changes in Myh6-cre;Gdf11-floxed mice were the result of Cre toxicity versus deletion of Gdf11. Third, we observed differential expression of cre mRNA in Cre-only controls compared with the cardiomyocyte-specific knockout mice, also making comparison between these two groups difficult. Thus, targeted Gdf11 deletion in cardiomyocytes may lead to left ventricular dilation without hypertrophy, but alternative animal models are necessary to understand the mechanism for these findings. NEW & NOTEWORTHY We observed that targeted deletion of growth differentiation factor 11 in cardiomyocytes does not cause cardiac hypertrophy but rather leads to left ventricular dilation compared with control mice carrying only the Myh6-cre or growth differentiation factor 11-floxed alleles. However, the mechanism underlying this finding remains unclear because of multiple confounding effects associated with the selected mouse model.

Growth Differentiation Factor 8 (GDF8) is a member of the transforming growth factor-β (TGF-β) family and has been identified as a strong physiological regulator. This factor is expressed as a paracrine factor in mural granulosa cells. To investigate the effects of GDF8 on the in vitro maturation (IVM) of porcine oocytes, we assessed the quality of matured oocytes as well as the specific gene transcription and protein activation levels in oocytes and cumulus cells (CCs) after IVM and subsequent embryonic development after in vitro fertilization (IVF) and parthenogenetic activation (PA). Supplemental concentrations (0, 1, 10, and 100 ng/ml) of GDF8 were provided in IVM medium. Supplementation with GDF8 during IVM induced transcription of specific TGF-β receptor genes, such as ActRIIb and Alk4/5, and the recognition of the GDF8 by these receptors induced phosphorylation of p38 MAPK. Activated p38 MAPK signaling changed oocyte maturation and cumulus expansion-related gene transcription: Nrf2 and Bcl-2 in oocytes and PCNA, Nrf2, Has2, Ptx3, and TNFAIP6 in CCs. The altered gene expression pattern during IVM resulted in a 10% lower level of intracellular ROS in mature oocytes. The improved cytoplasmic maturation led to an increase in the fertilization efficiency and subsequent embryonic developmental competence. The embryonic development showed increases in the blastocyst formation rate and higher transcription levels of POU5F1 and BCL-2 in the blastocysts. The present study suggests that supplementation of GDF8 during IVM synergistically improved the developmental potential of IVF- and PA-derived porcine embryos by reducing the intracellular ROS level in oocytes by altering the transcription of specific genes and increasing the phosphorylation of p38 MAPK during IVM. In conclusion, for the first time, our results demonstrate that GDF8 can act as a paracrine factor to modulate oocyte maturation by regulating p38 MAPK phosphorylation and intracellular ROS level during porcine IVM.

Growth differentiation factor 8 (GDF8), also known as myostatin, is a member of the transforming growth factor-β (TGF-β) family and has been identified as a strong physiological regulator of muscle differentiation. Recently, the functional role of GDF8 in reproductive organs has received increased interest following its detection in the human placenta and uterus. To investigate the effects of GDF8 during porcine oocyte in vitro maturation (IVM), we assessed the quality of matured oocytes. Furthermore, we investigated the specific gene transcription and protein activation levels in oocytes and cumulus cells after IVM and subsequent embryonic development after in vitro fertilization and parthenogenetic activation. Prior to these experiments, the concentration of GDF8 in porcine follicular fluid was determined. During the entire IVM period, 1.3 ng/mL GDF8 and its signaling inhibitor SB431542 (SB) at 5 μM were added as control, SB, SB + GDF8, and GDF8 groups, respectively. Our results demonstrate that supplementation with GDF8 during porcine oocyte IVM enhanced both meiotic and cytoplasmic maturation, with altered transcriptional patterns, via activation of Sma- and Mad-related protein 2/3 (SMAD2/3). Using the pharmacological inhibitor SB431542, we demonstrated that inhibition of GDF8-induced Smad2/3 signaling reduces matured oocyte quality. In conclusion, for the first time, we demonstrated paracrine factor GDF8 in porcine follicular fluid in vivo. Furthermore, we showed that GDF8 supplementation improved mature oocyte quality by regulating p38 mitogen-activated protein kinase phosphorylation and intracellular glutathione and reactive oxygen species levels during porcine IVM.