Inactivation of Myostatin Delays Senescence via TREX1-SASP in Bovine Skeletal Muscle Cells.

The myostatin (MSTN) gene also regulates the developmental balance of skeletal muscle after birth, and has long been linked to age-related muscle wasting. Many rodent studies have shown a correlation between MSTN and age-related diseases. It is unclear how MSTN and age-associated muscle loss in other animals are related. In this study, we utilized MSTN gene-edited bovine skeletal muscle cells to investigate the mechanisms relating to MSTN and muscle cell senescence. The expression of MSTN was higher in older individuals than in younger individuals. We obtained consecutively passaged senescent cells and performed senescence index assays and transcriptome sequencing. We found that senescence hallmarks and the senescence-associated secretory phenotype (SASP) were decreased in long-term-cultured myostatin inactivated (MT-KO) bovine skeletal muscle cells (bSMCs). Using cell signaling profiling, MSTN was shown to regulate the SASP, predominantly through the cycle GMP-AMP synthase-stimulator of antiviral genes (cGAS-STING) pathway. An in-depth investigation by chromatin immunoprecipitation (ChIP) analysis revealed that MSTN influenced three prime repair exonuclease 1 (TREX1) expression through the SMAD2/3 complex. The downregulation of MSTN contributed to the activation of the MSTN-SMAD2/3-TREX1 signaling axis, influencing the secretion of SASP, and consequently delaying the senescence of bSMCs. This study provided valuable new insight into the role of MSTN in cell senescence in large animals.

High histone crotonylation modification in bovine fibroblasts promotes cell proliferation and the developmental efficiency of preimplantation nuclear transfer embryos.

Lysine crotonylation (Kcr) is a recently discovered histone acylation modification that is closely associated with gene expression, cell proliferation, and the maintenance of stem cell pluripotency and indicates the transcriptional activity of genes and the regulation of various biological processes. During cell culture, the introduction of exogenous croconic acid disodium salt (Nacr) has been shown to modulate intracellular Kcr levels. Although research on Kcr has increased, its role in cell growth and proliferation and its potential regulatory mechanisms remain unclear compared to those of histone methylation and acetylation. Our investigation demonstrated that the addition of 5 mM Nacr to cultured bovine fibroblasts increased the expression of genes associated with Kcr modification, ultimately promoting cell growth and stimulating cell proliferation. Somatic cell nuclear transfer of donor cells cultured in 5 mM Nacr resulted in 38.1% blastocyst development, which was significantly greater than that in the control group (25.2%). This research is important for elucidating the crotonylation modification mechanism in fibroblast proliferation to promote the efficacy of somatic cell nuclear transfer.

Effects of Different Generations and Sex on Physiological, Biochemical, and Growth Parameters of Crossbred Beef Cattle by Myostatin Gene-Edited Luxi Bulls and Simmental Cows.

(1) Background: Myostatin (MSTN) is a protein that regulates skeletal muscle development and plays a crucial role in maintaining animal body composition and muscle structure. The loss-of-function mutation of MSTN gene can induce the muscle hypertrophic phenotype. (2) Methods: Growth indexes and blood parameters of the cattle of different months were analyzed via multiple linear regression. (3) Results: Compared with the control group, the body shape parameters of F2 cattle were improved, especially the body weight, cross height, and hip height, representing significant development of hindquarters, and the coat color of the F2 generation returned to the yellow of Luxi cattle. As adults, MSTN gene-edited bulls have a tall, wide acromion and a deep, wide chest. Both the forequarters and hindquarters are double-muscled with clear muscle masses. The multiple linear regression demonstrates that MSTN gene-edited hybrid beef cattle gained weight due to the higher height of the hindquarters. Significant differences in blood glucose, calcium, and low-density lipoprotein. Serum insulin levels decreased significantly at 24 months of age. MSTN gene editing improves the adaptability of cattle. (4) Conclusions: Our findings suggest that breeding with MSTN gene-edited Luxi bulls can improve the growth and performance of hybrid cattle, with potential benefits for both farmers and consumers.

The Effect of MSTN Mutation on Bile Acid Metabolism and Lipid Metabolism in Cattle.

Myostatin (MSTN) is a negative regulator of skeletal muscle genesis during development. MSTN mutation leads to increased lean meat production and reduced fat deposition in livestock. However, the mechanism by which MSTN promotes myogenesis by regulating metabolism is not clear. In this study, we compared the metabolomics of the livers of wild-type (WT) and MSTN mutation cattle (MT), and found changes in the content and proportion of fatty acids and bile acids in MT cattle. The differential metabolites were enriched in sterol synthesis and primary bile acid synthesis. We further analyzed the expression of genes involved in the regulation of lipid and bile acid metabolism, and found that the loss of MSTN may alter lipid synthesis and bile acid metabolism. This study provides new basic data for MSTN mutations in beef cattle breeding.

Combined Transcriptome and Metabolome Analysis of Smooth Muscle of Myostatin Knockout Cattle.

Myostatin (MSTN), a growth and differentiation factor, plays an important role in regulating skeletal muscle growth and development. MSTN knockout (MSTN-KO) leads to skeletal muscle hypertrophy and regulates metabolic homeostasis. Moreover, MSTN is also detected in smooth muscle. However, the effect of MSTN-KO on smooth muscle has not yet been reported. In this study, combined metabolome and transcriptome analyses were performed to investigate the metabolic and transcriptional profiling in esophageal smooth muscles of MSTN-KO Chinese Luxi Yellow cattle (n = 5, 24 months, average body weight 608.5 ± 17.62 kg) and wild-type (WT) Chinese Luxi Yellow cattle (n = 5, 24 months, average body weight 528.25 ± 11.03 kg). The transcriptome was sequenced using the Illumina Novaseq™ 6000 sequence platform. In total, 337 significantly up- and 129 significantly down-regulated genes were detected in the MSTN-KO cattle compared with the WT Chinese Luxi Yellow cattle. Functional enrichment analysis indicated that the DEGs were mainly enriched in 67 signaling pathways, including cell adhesion molecules, tight junction, and the cGMP-PKG signaling pathway. Metabolomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 130 differential metabolites between the groups, with 56 up-regulated and 74 down-regulated in MSTN knockout cattle compared with WT cattle. Differential metabolites were significantly enriched in 31 pathways, including glycerophospholipid metabolism, histidine metabolism, glutathione metabolism, and purine metabolism. Transcriptome and metabolome were combined to analyze the significant enrichment pathways, and there were three metabolically related pathways, including histidine metabolism, purine metabolism, and arginine and proline metabolism. These results provide important references for in-depth research on the effect of MSTN knockout on smooth muscle.

Loss of Myostatin Alters Mitochondrial Oxidative Phosphorylation, TCA Cycle Activity, and ATP Production in Skeletal Muscle.

Myostatin (MSTN) is an important negative regulator of skeletal muscle growth in animals. A lack of MSTN promotes lipolysis and glucose metabolism but inhibits oxidative phosphorylation (OXPHOS). Here, we aimed to investigate the possible mechanism of MSTN regulating the mitochondrial energy homeostasis of skeletal muscle. To this end, MSTN knockout mice were generated by the CRISPR/Cas9 technique. Expectedly, the MSTN null (Mstn-/-) mouse has a hypermuscular phenotype. The muscle metabolism of the Mstn-/- mice was detected by an enzyme-linked immunosorbent assay, indirect calorimetry, ChIP-qPCR, and RT-qPCR. The resting metabolic rate and body temperature of the Mstn-/- mice were significantly reduced. The loss of MSTN not only significantly inhibited the production of ATP by OXPHOS and decreased the activity of respiratory chain complexes, but also inhibited key rate-limiting enzymes related to the TCA cycle and significantly reduced the ratio of NADH/NAD+ in the Mstn-/- mice, which then greatly reduced the total amount of ATP. Further ChIP-qPCR results confirmed that the lack of MSTN inhibited both the TCA cycle and OXPHOS, resulting in decreased ATP production. The reason may be that Smad2/3 is not sufficiently bound to the promoter region of the rate-limiting enzymes Idh2 and Idh3a of the TCA cycle, thus affecting their transcription.

Myostatin Knockout Affects Mitochondrial Function by Inhibiting the AMPK/SIRT1/PGC1α Pathway in Skeletal Muscle.

Myostatin (Mstn) is a major negative regulator of skeletal muscle mass and initiates multiple metabolic changes. The deletion of the Mstn gene in mice leads to reduced mitochondrial functions. However, the underlying regulatory mechanisms remain unclear. In this study, we used CRISPR/Cas9 to generate myostatin-knockout (Mstn-KO) mice via pronuclear microinjection. Mstn-KO mice exhibited significantly larger skeletal muscles. Meanwhile, Mstn knockout regulated the organ weights of mice. Moreover, we found that Mstn knockout reduced the basal metabolic rate, muscle adenosine triphosphate (ATP) synthesis, activities of mitochondrial respiration chain complexes, tricarboxylic acid cycle (TCA) cycle, and thermogenesis. Mechanistically, expressions of silent information regulator 1 (SIRT1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were down-regulated, while peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) acetylation modification increased in the Mstn-KO mice. Skeletal muscle cells from Mstn-KO and WT were treated with AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR), and the AMPK inhibitor Compound C, respectively. Compared with the wild-type (WT) group, Compound C treatment further down-regulated the expression or activity of pAMPK, SIRT1, citrate synthase (CS), isocitrate dehydrogenase (ICDHm), and α-ketoglutarate acid dehydrogenase (α-KGDH) in Mstn-KO mice, while Mstn knockout inhibited the AICAR activation effect. Therefore, Mstn knockout affects mitochondrial function by inhibiting the AMPK/SIRT1/PGC1α signaling pathway. The present study reveals a new mechanism for Mstn knockout in regulating energy homeostasis.

Low Expression of Mitofusin 1 Gene Leads to Mitochondrial Dysfunction and Embryonic Genome Activation Failure in Ovine-Bovine Inter-Species Cloned Embryos.

Inter-species somatic cell nuclear transfer (iSCNT) is significant in the study of biological problems such as embryonic genome activation and the mitochondrial function of embryos. Here, we used iSCNT as a model to determine whether abnormal embryo genome activation was caused by mitochondrial dysfunction. First, we found the ovine-bovine iSCNT embryos were developmentally blocked at the 8-cell stage. The reactive oxygen species level, mitochondrial membrane potential, and ATP level in ovine-bovine cloned embryos were significantly different from both bovine-bovine and IVF 8-cell stage embryos. RNA sequencing and q-PCR analysis revealed that mitochondrial transport, mitochondrial translational initiation, mitochondrial large ribosomal subunit, and mitochondrial outer membrane genes were abnormally expressed in the ovine-bovine embryos, and the mitochondrial outer membrane and mitochondrial ribosome large subunit genes, mitochondrial fusion gene 1, and ATPase Na+/K+ transporting subunit beta 3 gene were expressed at lower levels in the ovine-bovine cloned embryos. Furthermore, we found that overexpression and knockdown of Mfn1 significantly affected mitochondrial fusion and subsequent biological functions such as production of ATP, mitochondrial membrane potential, reactive oxygen species and gene expressions in cloned embryos. These findings enhance our understanding of the mechanism by which the Mfn1 gene regulates embryonic development and embryonic genome activation events.

Myostatin Deficiency Enhances Antioxidant Capacity of Bovine Muscle via the SMAD-AMPK-G6PD Pathway.

During exercise, the body's organs and skeletal muscles produce reactive oxygen species (ROS). Excessive ROS can destroy cellular lipids, sugars, proteins, and nucleotides and lead to cancer. The production of nicotinamide adenine dinucleotide phosphate (NADPH) by the pentose phosphate pathway (PPP) is an auxiliary process of the cellular antioxidant system that supplements the reducing power of glutathione (GSH) to eliminate ROS in the cell. Myostatin (MSTN) is mainly expressed in skeletal muscle and participates in the regulation of skeletal muscle growth and development. Loss of MSTN leads to muscular hypertrophy, and MSTN deficiency upregulates glycolysis. However, the effect of MSTN on the PPP has not been reported. This study investigated the effect of MSTN on muscle antioxidant capacity from a metabolic perspective. We found that reducing MSTN modulates AMP-activated protein kinase (AMPK), a key molecule in cellular energy metabolism that directly regulates glucose metabolism through phosphorylation. Downregulation of MSTN promotes tyrosine modification of glucose-6-phosphate-dehydrogenase (G6PD) by AMPK and is regulated by the Smad signaling pathway. The Smad2/3 complex acts as a transcription factor to inhibit the AMPK expression. These results suggest that reduced MSTN expression inhibits the Smad signaling pathway, promotes AMPK expression, enhances the activity of G6PD enzyme, and enhances the antioxidant capacity of nonenzymatic GSH.

Growth Traits and Sperm Proteomics Analyses of Myostatin Gene-Edited Chinese Yellow Cattle.

Chinese Yellow Cattle, an ancient and domesticated breed for draft service, provide unique animal genetic resources with excellent genetic features, including crude feed tolerance, good stress resistance, strong adaptability, and tender meat quality; however, their production performance and meat yield are significantly inferior. Herein, the myostatin gene (MSTN), a negative regulator of skeletal muscle development, was knocked out by CRISPR/Cas9 technology. Eight MSTN gene-edited bull calves (MT) were born, and six of them are well-developed. Compared with the control cattle (WT), the growth trait indexes of MT cattle were generally increased, and the hindquarters especially were significantly improved. The biochemical indexes and the semen characteristics demonstrated that MT bulls were healthy and fertile. Consistent with our conjecture, the wobble and beating of MT bull spermatozoa were significantly higher than that of WT. Nine sperm motility-related proteins and nineteen mitochondrial-related proteins were identified by up-regulation in MT bull spermatozoa using FLQ proteomic technique and act to govern sperm flagellum assembly, organization, and beating and provide sufficient energy for sperm motility. The current study confirmed that the MSTN gene-edited Chinese Yellow cattle have improved growth traits and normal fertility, which can be used for beef cattle production and breeding.

N-3 Polyunsaturated Fatty Acid Dehydrogenase Fat-1 Regulates Mitochondrial Energy Metabolism by Altering DNA Methylation in Isolated Cells of Transgenic Cattle.

The fatty acid dehydrogenase fat-1 gene, derived from Caenorhabditis elegans, encodes n-3 polyunsaturated fatty acid dehydrogenase (Δ15 desaturase) and catalyzes the 18-20-carbon n-6 polyunsaturated fatty acids (n-6 PUFA) to generate corresponding n-3 polyunsaturated fatty acids (n-3 PUFA). Subsequently, fat-1 can influence the n-6: n-3 PUFA ratio in fat-1 transgenic cells. This study aimed to explore which processes of energy metabolism are affected exogenous fat-1 transgene and the relationship between these effects and DNA methylation. Compared with the wild-type group, the n-3 PUFA content in fat-1 transgenic bovine fetal fibroblasts was significantly increased, and the n-6 PUFA content and the n-6: n-3 PUFA ratio decreased. In the context of energy metabolism, the increase of exogenous fat-1 transgene decreased ATP synthesis by 39% and reduced the activity and expression of key rate-limiting enzymes in glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation, thus weakening the cells' capacity for ATP production. DNA methylation sequencing indicated that this inhibition of gene expression may be due to altered DNA methylation that regulates cell energy metabolism. Exogenous fat-1 transgenic cells showed changes in the degree of methylation in the promoter region of genes related to energy metabolism rate-limiting enzymes. We suggest that alters the balance of n-6/n-3 PUFA could regulate altered DNA methylation that affect mitochondrial energy metabolism.

Comparison of Microbial Community and Metabolites in Four Stomach Compartments of Myostatin-Gene-Edited and Non-edited Cattle.

Myostatin (MSTN), a major negative regulator of skeletal muscle mass and an endocrine factor, can regulate the metabolism of various organisms. Inhibition of the MSTN gene can improve meat production from livestock. Rumen microorganisms are associated with production and health traits of cattle, but changes in the microbial composition and metabolome in the four stomach compartments of MSTN gene-edited cattle have not previously been studied. Our results indicated that microbial diversity and dominant bacteria in the four stomach compartments were very similar between MSTN gene-edited and wild-type (WT) cattle. The microbiota composition was significantly different between MSTN gene-edited and WT cattle. Our results show that the relative abundance of the phylum Proteobacteria in the reticulum of MSTN gene-edited cattle was lower than that of WT cattle, whereas the relative abundance of the genus Prevotella in the omasum of MSTN gene-edited cattle was significantly higher than that of WT cattle. Metabolomics analysis revealed that the intensity of L-proline and acetic acid was significantly different in the rumen, reticulum, and abomasum between the two types of cattle. Meanwhile, pathway topology analysis indicated that the differential metabolites were predominantly involved in arginine biosynthesis and glutamate metabolism in the rumen, reticulum, and omasum but were mainly involved in pyruvate metabolism and glycolysis/gluconeogenesis in the abomasum. Spearman correlation network analysis further demonstrated that there was a significant correlation between microflora composition and metabolic pathways. These findings provide clues for studying nutrient digestion and absorption ability of MSTN gene-edited cattle.

Myostatin Knockout Limits Exercise-Induced Reduction in Bovine Erythrocyte Oxidative Stress by Enhancing the Efficiency of the Pentose Phosphate Pathway.

Moderate exercise can strengthen the body, however, exhaustive exercise generates large amounts of reactive oxygen species (ROS). Although erythrocytes have antioxidant systems that quickly eliminate ROS, erythrocytes become overwhelmed by ROS when the body is under oxidative stress, such as during exhaustive exercise. Myostatin (MSTN) has important effects on muscle hair development. Individuals lacking myostatin (MSTN) exhibit increased muscle mass. The purpose of this study was to investigate the mechanism by which MSTN affects erythrocyte antioxidant changes after exhaustive exercise in cattle. Antioxidant and metabolite detection analysis, western blotting, immunofluorescence, and fatty acid methyl ester analysis were used to assess exercise-associated antioxidant changes in erythrocytes with or without MSTN. Knockdown of MSTN enhances Glucose-6-phosphate dehydrogenase (G6PD) activity after exhaustive exercise. MSTN and its receptors were present on the erythrocyte membrane, but their levels, especially that of TGF-ß RI, were significantly reduced in the absence of MSTN and following exhaustive exercise. Our results suggest that knockout of MSTN accelerates the pentose phosphate pathway (PPP), thereby enhancing the antioxidant capacity of erythrocytes. These results provide important insights into the role of MSTN in erythrocyte antioxidant regulation after exhaustive exercise.

Localized delivery of curcumin by thermosensitive hydrogels for promoting wound healing.

BACKGROUND: Curcumin can promote wound healing, but its drug delivery medium needs to be improved further. OBJECTIVES: A curcumin-loaded thermosensitive hydrogel was prepared, its characterization was evaluated, and its promoting effect on wound healing was observed. METHODS: Curcumin-loaded thermosensitive hydrogels were prepared with different percentages of poloxamer 188 and poloxamer 407. A small tube inversion assay was used to observe the sol-gel transition temperature, and a rotational rheometer was used to detect the sol viscosity, sol-gel phase transition temperature, and phase transition time. The microstructure of the gel was observed by scanning electron microscopy, and Fourier infrared spectroscopy was used to evaluate whether curcumin was successfully loaded. Finally, its promoting effect on wound healing was observed in vivo and in vitro. RESULTS: Poloxamer 407 24% and poloxamer 188 1% were selected to prepare curcumin-loaded thermosensitive hydrogels. After 60 ± 15 s at 32°C, the sol-gel transition process was completed, with certain elastic behavior and solid-like rheological properties. Scanning electron microscopy showed that the pores of the curcumin-P407/P188 thermosensitive hydrogel were interconnected, with an average pore size ranging from 5 to 10 µm. Hydrogels showed a higher swelling ratio. Fourier transform infrared spectroscopy showed that curcumin had been incorporated into the hydrogel. Live/dead cell assays suggested that the hydrogel was not toxic to fibroblasts. Curcumin-loaded thermosensitive hydrogels can promote an increase in S-phase fibroblasts and improve wound healing. CONCLUSIONS: Curcumin-loaded P407/P188 thermosensitive hydrogel improves wound healing. More in-depth research is needed in the future.

Transcriptome Analysis Reveals that MAPK Signaling Pathway Mediates Salt Tolerance of YMR253C ORF in Saccharomyces cerevisiae.

The YMR253C open reading frame encodes a membrane protein that is highly expressed in NaCl-resistant Saccharomyces cerevisiae mutants. Whether it mediates NaCl tolerance is unclear. By knocking out YMR253C in S. cerevisiae, we found that the salt tolerance of yeast was reduced, the integrity of the cell wall was impaired, and cell death was induced; transcriptome analysis further revealed that YMR253C gene knockout mediates significant changes of 1291 genes, and YMR253C mediates the regulation of MAPK signal pathways. Therefore, the transmembrane protein YMR253C may regulate the MAPK signaling pathway to regulate the salt stress of S. cerevisiae.

Myostatin Knockout Regulates Bile Acid Metabolism by Promoting Bile Acid Synthesis in Cattle.

Myostatin (MSTN) is a major negative regulator of skeletal muscle mass and causes a variety of metabolic changes. However, the effect of MSTN knockout on bile acid metabolism has rarely been reported. In this study, the physiological and biochemical alterations of serum in MSTN+/- and wild type (WT) cattle were investigated. There were no significant changes in liver and kidney biochemical indexes. However, compared with the WT cattle, lactate dehydrogenase, total bile acid (TBA), cholesterol, and high-density lipoprotein (HDL) in the MSTN+/- cattle were significantly increased, and glucose, low-density lipoprotein (LDL), and triglycerides (TG) were significantly decreased, indicating that MSTN knockout affected glucose and lipid metabolism and total bile acids content. Targeted metabolomic analysis of the bile acids and their derivatives was performed on serum samples and found that bile acids were significantly increased in the MSTN+/- cattle compared with the WT cattle. As the only bile acid synthesis organ in the body, we performed metabolomic analysis on the liver to study the effect of MSTN knockout on hepatic metabolism. Metabolic pathway enrichment analysis of differential metabolites showed significant enrichment of the primary bile acid biosynthesis and bile secretion pathway in the MSTN+/- cattle. Targeted metabolomics data further showed that MSTN knockout significantly increased bile acid content in the liver, which may have resulted from enhanced bile acid synthesis due to the expression of bile acid synthesis genes, cholesterol 7 alpha-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1), and upregulation in the liver of the MSTN+/- cattle. These results indicate that MSTN knockout does not adversely affect bovine fitness but regulates bile acid metabolism via enhanced bile acid synthesis. This further suggests a role of MSTN in regulating metabolism.

C23 gene regulates the nucleolin structure and biosynthesis of ribosomes in bovine intraspecific and interspecific somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) can reprogram differentiated somatic cells to produce individual animals, thus having advantages in animal breeding and chromatin reprogramming. Interspecies SCNT (iSCNT) provides extreme cases of reprogramming failure that can be used to understand the basic biological mechanism of genome reprogramming. It is important to understand the possible mechanisms for the failure of zygotic genome activation (ZGA) in iSCNT embryos in order to improve the efficiency of SCNT embryos. In the present study, we compared the development of bovine-bovine (B-B), ovine-ovine (O-O) SCNT, and ovine-bovine (O-B) iSCNT embryos and found that a developmental block existed in the 8-cell stage in O-B iSCNT embryos. RNA sequencing and q-PCR analysis revealed that the large ribosomal subunit genes (RPL) or the small ribosomal subunit genes (RPS) were expressed at lower levels in the O-B iSCNT embryos. The nucleolin (C23) gene that regulates the ribosomal subunit generation was transcribed at a lower level during embryonic development in O-B iSCNT embryos. In addition, the nucleolin exhibited a clear circular-ring structure in B-B 8-cell stage embryos, whereas this was shell-like or dot-like in the O-B embryos. Furthermore, overexpression of C23 could increase the blastocyst rate of both SCNT and iSCNT embryos and partly rectify the ring-like nucleolin structure and the expression of ribosomal subunit related genes were upregulation, while knockdown of C23 increased the shell-like nucleolin-structure in B-B cloned embryos and downregulated the expression of ribosomal subunit related genes. These results implied that abnormal C23 and ribosome subunit gene expression would lead to the developmental block of iSCNT embryos and ZGA failure. Overexpression of the C23 gene could partly improve the blastocyst development and facilitate the nucleolin structure in bovine preimplantation SCNT embryos.

Melatonin improves the quality of frozen bull semen and influences gene expression related to embryo genome activation.

The efficiency of animal artificial breeding in vitro is still low. Oxidative damage is an important obstacle for in vitro artificial breeding of animals. Melatonin can reduce the degree of oxidative damage to both gametes and embryos caused by the external environment. However, there is still some controversy concerning the effect of melatonin on frozen semen, especially in the processes of freezing semen, IVM, IVF and IVC. Here, the effects of melatonin on the whole processes of sperm cryopreservation, oocyte maturation, and embryonic development were studied. The results demonstrated that melatonin at 10-3 M concentration significantly improved progressive sperm viability, plasma membrane integrity, mitochondrial membrane integrity, and acrosome integrity; however, there were also individual differences between bulls, depending on the age of different individuals. The 10-3 M melatonin treatment reduced the reactive oxygen species (ROS) level by nearly 50% in sperm during IVF. Meanwhile, during IVM, the addition of 10-7 M melatonin significantly increased the maturation rate of oocytes and reduced the ROS levels by 58.8%. In addition, 10-7 M melatonin improved the total cell numbers of the IVF blastocysts. Notably, treatment of IVF embryos with melatonin significantly reduced the levels of ROS and influenced the expression levels of key regulatory genes associated with embryo genome activation. This study is of significance for understanding the function of melatonin in animal artificial breeding.

Single-cell RNA sequencing reveals atlas of dairy goat testis cells.

Single-cell RNA sequencing (scRNA-seq) is useful for exploring cell heterogeneity. For large animals, however, little is known regarding spermatogonial stem cell (SSC) self-renewal regulation, especially in dairy goats. In this study, we described a high-resolution scRNA-seq atlas derived from a dairy goat. We identified six somatic cell and five spermatogenic cell subtypes. During spermatogenesis, genes with significantly changed expression were mainly enriched in the Notch, TGF-ß, and Hippo signaling pathways as well as the signaling pathway involved in the regulation of stem cell pluripotency. We detected and screened specific candidate marker genes ( TKTL1 and AES) for spermatogonia. Our study provides new insights into goat spermatogenesis and the development of testicular somatic cells.

Eif2s3y Promotes the Proliferation of Spermatogonial Stem Cells by Activating ERK Signaling.

The future fertility of males with cancer may be irreversibly compromised by chemotherapy and/or radiotherapy. Spermatogonial stem cell transplantation is believed to be a way to restore fertility in men. However, the survival efficiency of transplanted cells is still low. Eukaryotic translation initiation factor 2 subunit 3 and structural gene Y-linked (Eif2s3y) located on the Y chromosome of male animals is a coding gene of eIF2γ which mainly functions in translation initiation. Recently, the emerging role of Eif2s3y in spermatogenesis has been emphasized in several studies. However, the underlying mechanism is still unclear. In addition, how Eif2s3y functions in large animals remains largely unknown. In this study, we obtained the CDS sequence of the Eif2s3y gene from the testis of dairy goats and found that this gene was highly expressed in the testis and was evolutionarily conserved among different species. Interestingly, overexpression of Eif2s3y promoted the proliferation of spermatogonial stem cells of dairy goats by activating the ERK signaling pathway. In animal experiments, overexpressing Eif2s3y promoted transplanted goat spermatogonial stem cells and produced more colonies after microinjection into the seminiferous tubules of infertile mice. In conclusion, our study highlights an undiscovered role of Eif2s3y in dairy goat reproduction. This finding may provide an important basis for future works regarding male spermatogenic cell restoration and represent a major advance toward surrogate sires becoming a tool for disseminating and regenerating germplasm in all mammals.