FOXO1 mediates hypoxia-induced G0/G1 arrest in ovarian somatic granulosa cells by activating the TP53INP1-p53-CDKN1A pathway.

The development of ovarian follicles constitutes the foundation of female reproduction. The proliferation of granulosa cells (GCs) is a basic process required to ensure normal follicular development. However, the mechanisms involved in controlling GC cell cycle are not fully understood. Here, by performing gene expression profiling in the domestic pig (Sus scrofa), we showed that cell cycle arrest at G0/G1 phase is highly correlated with pathways associated with hypoxic stress and FOXO signalling. Specifically, the elevated proportion of GCs at the arrested G0/G1 phase was accompanied by increased nuclear translocation of FOXO1 under conditions of hypoxia both in vivo and in vitro. Furthermore, phosphorylation of 14-3-3 by the JNK kinase is required for hypoxia-mediated FOXO1 activation and the resultant G0/G1 arrest. Notably, a FOXO1 mutant without DNA-binding activity failed to induce G0/G1 arrest of GCs during hypoxia. Importantly, we identified a new target gene of FOXO1, namely TP53INP1, which contributes to suppression of the G1-S cell cycle transition in response to hypoxia. Furthermore, we demonstrated that the inhibitory effect of the FOXO1-TP53INP1 axis on the GC cell cycle is mediated through a p53-CDKN1A-dependent mechanism. These findings could provide avenues for the clinical treatment of human infertility caused by impaired follicular development.

FSH mediates estradiol synthesis in hypoxic granulosa cells by activating glycolytic metabolism through the HIF-1α-AMPK-GLUT1 signaling pathway.

Owing to the avascular environment within ovarian follicles, granulosa cells (GCs) are believed to live in a hypoxic niche. Follicle-stimulating hormone (FSH)-mediated steroidogenesis is crucial for normal growth and maturation of ovarian follicles, but it remains unclear how FSH stimulates estradiol (E2) synthesis under hypoxic conditions. Here, we aimed to explore whether FSH affects the ATP production required for estrogen synthesis from the perspective of glucose metabolism. It was observed that the levels of both E2 and HIF-1α were markedly increased in a dose-dependent manner in mouse ovarian GCs after the injection of FSH in vivo, indicating that hypoxia/HIF-1α may be relevant to FSH-induced E2 synthesis. By treating hypoxic GCs with FSH in vitro, we further revealed that the activation of the AMP-activated protein kinase (AMPK)-GLUT1 pathway, which in turn stimulates ATP generation, may be essential for FSH-mediated E2 production during hypoxia. In contrast, inhibition of AMPK or GLUT1 with siRNAs/antagonist both repressed glycolysis, ATP production, and E2 synthesis despite FSH treatment. Moreover, blocking HIF-1α activity using siRNAs/PX-478 suppressed AMPK activation, GLUT1 expression, and E2 levels in FSH-treated GCs. Finally, the in vitro findings were verified in vivo, which showed markedly increased AMPK activity, GLUT1 expression, glycolytic flux, ATP levels, and E2 concentrations in ovarian GCs following FSH injection. Taken together, these findings uncovered a novel mechanism for FSH-regulating E2 synthesis in hypoxic GCs by activating glycolytic metabolism through the HIF-1α-AMPK-GLUT1 pathway.

Oocytes and hypoxanthine orchestrate the G2-M switch mechanism in ovarian granulosa cells.

In mammalian growing follicles, oocytes are arrested at the diplotene stage (which resembles the G2/M boundary in mitosis), while the granulosa cells (GCs) continue to proliferate during follicular development, reflecting a cell cycle asynchrony between oocytes and GCs. Hypoxanthine (Hx), a purine present in the follicular fluid, has been shown to induce oocytes meiotic arrest, although its role in GC proliferation remains ill-defined. Here, we demonstrate that Hx indiscriminately prevents G2-to-M phase transition in porcine GCs. However, oocyte-derived paracrine factors (ODPFs), particularly GDF9 and BMP15, maintain the proliferation of GCs, partly by activating the ERK1/2 signaling and enabling the G2/M transition that is suppressed by Hx. Interestingly, GCs with lower expression of GDF9/BMP15 receptors appear to be more sensitive to Hx-induced G2/M arrest and become easily detached from the follicular wall. Importantly, Hx-mediated inhibition of G2/M progression instigates GC apoptosis, which is ameliorated in the presence of GDF9 and/or BMP15. Therefore, our data indicate that the counterbalance of intrafollicular factors, particularly Hx and oocyte-derived GDF9/BMP15, fine-tunes the development of porcine follicles by regulating the cell cycle progression of GCs.

Comprehensive analysis of nonsurrounded nucleolus and surrounded nucleolus oocytes on chromatin accessibility using ATAC-seq.

Mouse germinal vesicle (GV) oocytes are divided into surrounded nucleolus (SN) and nonsurrounded nucleolus (NSN) oocytes based on chromatin morphology. NSN oocytes spontaneously transform into SN oocytes after accumulating enough maternal transcripts. SN oocytes show transcriptional silencing. When oocyte maturation is abnormal or takes place in vitro, NSN oocytes do not go through SN stage before proceeding to MII. Nontransitive oocytes show developmental retardation, a low fertilization rate, and arrest at the two-cell embryo stage in mice. Here, chromatin-binding ribonucleic acid polymerase II (RNAP II) activity, newly synthesized RNA, and chromatin accessibility in GV oocytes were examined. In SN oocytes, RNAP II did not bind to DNA, neo-RNA was not generated in nuclei, and the phosphorylation state of RNAP II did not affect the chromatin-binding activity. The number of accessible genes in SN oocytes was remarkably lower than that in NSN oocytes. The accessibility of different functional genes was also different between the two types of oocytes. Thus, low chromatin accessibility leads to transcriptional silencing in SN oocytes.

Albumin (ALB) and protein disulfide isomerase family A member 4 (PDIA4) are novel markers to predict sperm freezability of Erhualian boar.

The frozen semen of Erhualian pig can promote the continuous improvement of commercial pigs, but currently, frozen semen fails to satisfy the practical application requirement. Oxidative damage is one of the crucial factors affecting the quality of frozen semen; besides, there are individual differences in boar sperm freezability. Based on the previous analysis of the proteomic differences of Erhualian boar sperm with different freezability, two differentially abundant proteins (DAPs) in boar sperm, albumin (ALB) and protein disulfide isomerase family A member 4 (PDIA4), were selected as the research objects in the current study. It is assumed that redox-related proteins ALB and PDIA4 can be used as markers to predict Erhualian boar sperm freezability. We cryopreserved the semen of 14 Erhualian boars. According to the difference of frozen semen quality, boars with good and poor freezability ejaculates (GFE and PFE, n = 3) were selected respectively. The relative contents of ALB and PDIA4 in GFE and PFE were analyzed by Western blot, and the localization patterns of ALB and PDIA4 in pre-frozen and frozen-thawed sperm were detected by immunofluorescence. The results showed that the abundances of ALB and PDIA4 in GFE were significantly higher than PFE, and there was a significant correlation between the relative contents of ALB and PDIA4 and frozen-thawed sperm quality parameters. Additionally, the freezing process had no effect on the localization patterns of ALB and PDIA4 in spermatozoa. In conclusion, these results suggest that ALB and PDIA4 are related to boar sperm cryotolerance and may be used as novel freezability markers.

G6PD Deficiency Is Crucial for Insulin Signaling Activation in Skeletal Muscle.

Glucose 6-P dehydrogenase (G6PD) is the first rate-limiting enzyme in pentose phosphate pathway (PPP), and it is proverbial that G6PD is absent in skeletal muscle. However, how and why G6PD is down-regulated during skeletal muscle development is unclear. In this study, we confirmed the expression of G6PD was down-regulated during myogenesis in vitro and in vivo. G6PD was absolutely silent in adult skeletal muscle. Histone H3 acetylation and DNA methylation act together on the expression of G6PD. Neither knock-down of G6PD nor over-expression of G6PD affects myogenic differentiation. Knock-down of G6PD significantly promotes the sensitivity and response of skeletal muscle cells to insulin; over-expression of G6PD significantly injures the sensitivity and response of skeletal muscle cells to insulin. High-fat diet treatment impairs insulin signaling by up-regulating G6PD, and knock-down of G6PD rescues the impaired insulin signaling and glucose uptake caused by high-fat diet treatment. Taken together, this study explored the importance of G6PD deficiency during myogenic differentiation, which provides new sight to treat insulin resistance and type-2 diabetes.

Melatonin promotes male reproductive performance and increases testosterone synthesis in mammalian Leydig cells†.

Leydig cells play a critical role in male reproductive physiology, and their dysfunction is usually associated with male infertility. Melatonin has an important protective and regulatory role in these cells. However, the lack of suitable animal models impedes us from addressing the impact of endogenous melatonin on these cells. In the current study, by using arylalkylamine N-acetyltransferase (AANAT) overexpression transgenic sheep and AANAT knockout mice, we confirmed the regulatory effects of endogenously occurring melatonin on Leydig cells as well as its beneficial effects on male reproductive performance. The results showed that the endogenously elevated melatonin level was correlated with decreased Leydig cell apoptosis, increased testosterone production, and improved quality of sperm in melatonin-enriched transgenic mammals. Signal transduction analysis indicated that melatonin targeted the mitochondrial apoptotic Bax/Bcl2 pathway and thus suppressed Leydig cell apoptosis. In addition, melatonin upregulated the expression of testosterone synthesis-related genes of Steroidogenic Acute Regulatory Protein (StAR), Steroidogenic factor 1 (SF1), and Transcription factor GATA-4 (Gata4) in Leydig cells. This action was primarily mediated by the melatonin nuclear receptor RAR-related orphan receptor alpha (RORα) since blockade of this receptor suppressed the effect of melatonin on testosterone synthesis. All of these actions of melatonin cause Leydig cells to generate more testosterone, which is necessary for spermatogenesis in mammals. In contrast, AANAT knockout animals have dysfunctional Leydig cells and reduced reproductive performance.

FSH prevents porcine granulosa cells from hypoxia-induced apoptosis via activating mitophagy through the HIF-1α-PINK1-Parkin pathway.

In developing follicles, the granulosa cells (GCs) live in a hypoxic environment due to the devoid of blood supply. Upon hypoxic conditions, several types of mammalian cells have been reported to undergo apoptosis. Follicle-stimulating hormone (FSH) is known as the primary survival factor for antral follicles by preventing GCs apoptosis. Mitophagy is a type of organelle-specific autophagy that removes damaged or stressed mitochondria to maintain cellular health. This study provides the first evidence suggesting that FSH-mediated mitophagy protected porcine GCs from hypoxia-induced apoptosis. Our data showed that the GCs apoptosis caused by mitochondrial pathway upon hypoxia stress was markedly attenuated after FSH treatment, which was correlated with enhanced activation of mitophagy. Interestingly, FSH also stimulated mitochondrial biogenesis as suggested by increased expression of mitochondrial transcription factor A and nuclear respiratory factor 1 during hypoxia exposure. Notably, the protein level of hypoxia inducible factor-1α (HIF-1α) was significantly increased in hypoxic GCs following FSH treatment, accompanied by elevated mitophagic activity and dampened apoptotic signaling. Blocking HIF-1α inhibited mitophagy and restored hypoxia-induced apoptosis despite FSH treatment. Importantly, FSH promoted the expression of serine/threonine kinase PTEN induced putative kinase 1 (PINK1) and the E3 ligase Parkin during hypoxia stress through a HIF-1α dependent manner. This induced the mitophagic clearance of damaged mitochondria, hence inhibiting apoptosis by reducing cytochrome c releasing. The inhibition of HIF-1α and/or PINK1 using inhibitor or RNAi further confirmed the role of the FSH-HIF-1α-PINK1-Parkin-mitophagy axis in suppressing GC apoptosis under hypoxic conditions. These findings highlight a novel function of FSH in preserving GCs viability against hypoxic damage by activating HIF-1α-PINK1-Parkin-mediated mitophagy.

Effect of Exogenous Melatonin on the Development of Mice Ovarian Follicles and Follicular Angiogenesis.

In mammalian, the periodic growth and development of ovarian follicles constitutes the physiological basis of female estrus and ovulation. Concomitantly, follicular angiogenesis exerts a pivotal role in the growth of ovarian follicles. Melatonin (N-acetyl-5-methoxytryptamine, Mel), exists in follicle fluid, was suggested to affect the development of follicles and angiogenesis. This research was conducted to investigate the effects and mechanisms of Mel on the development of ovarian follicles and its angiogenesis. In total, 40 ICR mice at age of 3 weeks were allocated into four groups at liberty: control, Mel, FSH and FSH + Mel for a 12-day trial. Ovaries were collected at 8:00 a.m. on Day 13 for detecting the development of ovarian follicles and angiogenesis. Results indicated that Mel promoted the development of ovarian follicles of 50-250 µm (secondary follicles) and periphery angiogenesis, while FSH remarkably increased the number of antral follicles and periphery angiogenesis. Mechanically, Mel and FSH may regulate the expression of VEGF and antioxidant enzymes in different follicular stages. In conclusion, Mel primarily acted on the secondary follicles, while FSH mainly promoted the development of antral follicles. They both conduced to related periphery angiogenesis by increasing the expression of VEGF. These findings may provide new targets for the regulating of follicular development.

Insulin-like growth factor-I prevents hypoxia-inducible factor-1 alpha-dependent G1/S arrest by activating cyclin E/cyclin-dependent kinase2 via the phoshatidylinositol-3 kinase/AKT/forkhead box O1/Cdkn1b pathway in porcine granulosa cells†.

As the follicle develops, the thickening of the granulosa compartment leads to progressively deficient supply of oxygen in granulosa cells (GCs) due to the growing distances from the follicular vessels. These conditions are believed to cause hypoxia in GCs during folliculogenesis. Upon hypoxic conditions, several types of mammalian cells have been reported to undergo cell cycle arrest. However, it remains unclear whether hypoxia exerts any impact on cell cycle progression of GCs. On the other hand, although the GCs may live in a hypoxic environment, their mitotic capability appears to be unaffected in growing follicles. It thus raises the question whether there are certain intraovarian factors that might overcome the inhibitory effects of hypoxia. The present study provides the first evidence suggesting that cobalt chloride (CoCl2)-mimicked hypoxia prevented G1-to-S cell cycle progression in porcine GCs. In addition, we demonstrated that the inhibitory effects of CoCl2 on GCs cell cycle are mediated through hypoxia-inducible factor-1 alpha/FOXO1/Cdkn1b pathway. Moreover, we identified insulin-like growth factor-I (IGF-I) as an intrafollicular factor required for cell cycle recovery by binding to IGF-I receptor in GCs suffering CoCl2 stimulation. Further investigations confirmed a role of IGF-I in preserving G1/S progression of CoCl2-treated GCs via activating the cyclin E/cyclin-dependent kinase2 complex through the phoshatidylinositol-3 kinase/protein kinase B (AKT)/FOXO1/Cdkn1b axis. Although the present findings were based on a hypoxia mimicking model by using CoCl2, our study might shed new light on the regulatory mechanism of GCs cell cycle upon hypoxic stimulation.

NLRP7 is expressed in the ovine ovary and associated with in vitro pre-implantation embryo development.

NLRP (NACHT, LRR and PYD domain-containing proteins) family plays pivotal roles in mammalian reproduction. Mutation of NLRP7 is often associated with human recurrent hydatidiform moles. Few studies regarding the functions of NLRP7 have been performed in other mammalian species rather than humans. In the current study, for the first time, the function of NLRP7 has been explored in ovine ovary. NLRP7 protein was mainly located in ovarian follicles and in in vitro pre-implantation embryos. To identify its origin, 763 bp partial CDS of NLRP7 deriving from sheep cumulus oocyte complexes (COCs) was cloned, it showed a great homology with Homo sapiens. The high levels of mRNA and protein of NLRP7 were steadily expressed in oocytes, parthenogenetic embryos or IVF embryos. NLRP7 knockdown by the combination of siRNA and shRNA jeopardized both the parthenogenetic and IVF embryo development. These results strongly suggest that NLRP7 plays an important role in ovine reproduction. The potential mechanisms of NLRP7 will be fully investigated in the future.

Responses of Transgenic Melatonin-Enriched Goats on LPS Stimulation and the Proteogenomic Profiles of Their PBMCs.

The anti-inflammatory activity of melatonin (MT) has been well documented; however, little is known regarding endogenously occurring MT in this respect, especially for large animals. In the current study, we created a MT-enriched animal model (goats) overexpressing the MT synthetase gene Aanat. The responses of these animals to lipopolysaccharide (LPS) stimulation were systematically studied. It was found that LPS treatment exacerbated the inflammatory response in wild-type (WT) goats and increased their temperature to 40 °C. In addition, their granulocyte counts were also significantly elevated. In contrast, these symptoms were not observed in transgenic goats with LPS treatment. The rescue study with MT injection into WT goats who were treated with LPS confirmed that the protective effects in transgenic goats against LPS were attributed to a high level of endogenously produced MT. The proteomic analysis in the peripheral blood mononuclear cells (PBMCs) isolated from the transgenic animals uncovered several potential mechanisms. MT suppressed the lysosome formation as well as its function by downregulation of the lysosome-associated genes Lysosome-associated membrane protein 2 (LAMP2), Insulin-like growth factor 2 receptor (IGF2R), and Arylsulfatase B (ARSB). A high level of MT enhanced the antioxidant capacity of these cells to reduce the cell apoptosis induced by the LPS. In addition, the results also uncovered previously unknown information that showed that MT may have protective effects on some human diseases, including tuberculosis, bladder cancer, and rheumatoid arthritis, by downregulation of these disease-associated genes. All these observations warranted further investigations.

An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving as the mammary gland bioreactor to produce melatonin-enriched milk in sheep.

Melatonin as a potent antioxidant exhibits important nutritional and medicinal values. To produce melatonin-enriched milk will benefit the consumers. In this study, a sheep bioreactor which generates melatonin-enriched milk has been successfully developed by the technology that combined CRISPR/Cas9 system and microinjection. The AANAT and ASMT were cloned from pineal gland of Dorper sheep (Ovis aries). The in vitro studies found that AANAT and ASMT were successfully transferred to the mammary epithelial cell lines and significantly increased melatonin production in the culture medium compared to the nontransgenic cell lines. In addition, the Cas9 mRNA, sgRNA, and the linearized vectors pBC1-AANAT and pBC1-ASMT were co-injected into the cytoplasm of pronuclear embryos which were implanted into ewes by oviducts transferring. Thirty-four transgenic sheep were generated with the transgenic positive rate being roughly 35% which were identified by Southern blot and sequencing. Seven carried transgenic AANAT, two carried ASMT, and 25 carried both of AANAT and ASMT genes. RT-PCR and Western blot demonstrated that the lambs expressed these genes in their mammary epithelial cells and these animals produced melatonin-enriched milk. This is the first report to show a functional AANAT and ASMT transgenic animal model which produce significantly high levels of melatonin milk compared to their wild-type counterparts. The advanced technologies used in the study laid a foundation for generating large transgenic livestock, for example, the cows, which can produce high level of melatonin milk.

Melatonin Improves the Quality of Inferior Bovine Oocytes and Promoted Their Subsequent IVF Embryo Development: Mechanisms and Results.

The inferior oocytes (IOs), which are not suitable for embryo development, occupy roughly one-third or more of the collected immature bovine oocytes. The IOs are usually discarded from the in vitro bovine embryo production process. Improving the quality of the inferior oocytes (IOs) and make them available in in vitro embryo production would have important biological, as well as commercial, value. This study was designed to investigate whether melatonin could improve the quality of IOs and make them usable in the in vitro maturation (IVM) and subsequent (in vitro fertilization) IVF embryo development. The results indicated that: the maturation rate of IOs and their subsequent IVF embryo developments were impaired compared to cumulus-oocyte complexes and melatonin treatment significantly improved the quality of IOs, as well as their IVF and embryo developments. The potential mechanisms are that: (1) melatonin reduced reactive oxygen species (ROS) and enhanced glutathione (GSH) levels in the IOs, thereby protecting them from oxidative stress; (2) melatonin improved mitochondrial normal distribution and function to increase ATP level in IOs; and (3) melatonin upregulated the expression of ATPase 6, BMP-15, GDF-9, SOD-1, Gpx-4, and Bcl-2, which are critical genes for oocyte maturation and embryo development and downregulated apoptotic gene expression of caspase-3.

Autophagy-Lysosome Pathway in Renal Tubular Epithelial Cells Is Disrupted by Advanced Glycation End Products in Diabetic Nephropathy.

It has been suggested that autophagy protects renal tubular epithelial cells (TECs) from injury in diabetic nephropathy (DN). However, the manner in which the autophagy-lysosome pathway is changed in this state remains unclear. In this study of DN, we investigated the autophagic activity and lysosomal alterations in vivo and in vitro. We found that autophagic vacuoles and SQSTM1-positive proteins accumulated in TECs from patients with DN and in human renal tubular epithelial cell line (HK-2 cells) treated with advanced glycation end products (AGEs), the important factors that involved in the pathogenesis of DN. In HK-2 cells, exposure to AGEs caused a significant increase in autophagosomes but a marked decrease in autolysosomes, and the lysosomal turnover of LC3-II was not observed, although LC3-II puncta were co-localized with the irregular lysosomal-associated membrane protein1 granules after AGEs treatment. Furthermore, lysosomal membrane permeabilization was triggered by AGEs, which likely resulted in a decrease in the enzymatic activities of cathepsin B and cathepsin L, the defective acidification of lysosomes, and suppression of the lysosomal degradation of DQ-ovalbumin. Oxidative stress evoked by AGEs-receptor for AGE interaction likely played an important role in the lysosomal dysfunction. Additionally, ubiquitinated proteins were co-localized with SQSTM1-positive puncta and accumulated in HK-2 cells after exposure to AGEs, indicating blocked degradation of SQSTM1-positive and ubiquitinated aggregates. Taken together, the results show that lysosomal membrane permeabilization and lysosomal dysfunction are triggered by AGEs, which induce autophagic inactivation in TECs from patients with DN. Disruption of the autophagy-lysosome pathway should be focused when studying the mechanisms underlying DN.

Mitochondria Synthesize Melatonin to Ameliorate Its Function and Improve Mice Oocyte's Quality under in Vitro Conditions.

The physiology of oocyte in vitro maturation remains elusive. Generally, the oocytes have a very low maturation rate under in vitro conditions. In the current study, we found that melatonin promotes the maturation of oocytes in which mitochondria play a pivotal role. It was identified that; (1) mitochondria are the major sites for melatonin synthesis in oocytes and they synthesize large amounts of melatonin during their maturation; (2) melatonin improves mitochondrial function by increased mtDNA copy, mitochondrial membrane potential (ΔΨm) and mitochondrial distribution and ATP production in oocytes; (3) the meiotic spindle assembly is enhanced; (4) melatonin reduces ROS production and inhibits 8-oxodG formation, thereby protecting potential DNA mutation from oxidative damage. As a result, melatonin improves the quality of oocytes, significantly accelerates the developmental ability of IVF embryo. The results provide novel knowledge on the physiology of oocyte's maturation, especially under in vitro conditions.

Melatonin protects porcine oocyte in vitro maturation from heat stress.

Melatonin is a pleiotropic molecule which plays an important role in animal reproductive activities. Because of the increased global warming, the impact of heat stress (HS) on stockbreeding has become an inevitable issue to be solved. To investigate the potential effects of melatonin on the in vitro maturation of porcine oocyte under the HS, a HS model for porcine oocyte maturation has been used in this study and the different concentrations of melatonin (10(-6) -10(-9)  m) were also tested for their protective effects on oocytes. The polar body rate, the index of the nuclear maturation of the oocytes, and the cleavage rate as well as the blastocyst rate were measured to evaluate the developmental competence of the oocytes after parthenogenetic activation (PA). The results showed that HS [in vitro maturation (IVM) 20-24 hr, 42°C] significantly reduced the polar body rate of oocytes and the blastocyte rate of porcine PA embryos, while melatonin (10(-7)  m) application not only improved polar body rate and blastocyte rate, but also preserved the normal levels of steroid hormone which is disrupted by HS. The presence of melatonin (10(-7)  m) during the oocyte maturation under the HS reduced reactive oxygen species (ROS) formation, enhanced glutathione (GSH) production, inhibited cell apoptosis, and increased the gene expressions of SIRT1, AKT2, and Polg2. Importantly, the endogenously occurring melatonin of cumulus-oocyte complexes was significantly induced by HS. The results indicated that melatonin application effectively protected the oocytes from HS. These observations warranted the further studies in vivo regarding to improve the reproductive activities of animals under the global warming environment.

Lactate promotes myogenesis via activating H3K9 lactylation-dependent up-regulation of Neu2 expression.

BACKGROUND: Lactate, a glycolytic metabolite mainly produced in muscles, has been suggested to regulate myoblast differentiation, although the underlying mechanism remains elusive. Recently, lactate-mediated histone lactylation is identified as a novel epigenetic modification that promotes gene transcription. METHODS: We used mouse C2C12 cell line and 2-month-old male mice as in vitro and in vivo models, respectively. These models were treated with lactate to explore the biological function and latent mechanism of lactate-derived histone lactylation on myogenic differentiation by quantitative real-time PCR, western blotting, immunofluorescence staining, chromatin immunoprecipitation, cleavage under targets and tagmentation assay and RNA sequencing. RESULTS: Using immunofluorescence staining and western blotting, we proposed that lactylation might occur in the histones. Inhibition of lactate production or intake both impaired myoblast differentiation, accompanied by diminished lactylation in the histones. Using lactylation site-specific antibodies, we demonstrated that lactate preferentially increased H3K9 lactylation (H3K9la) during myoblast differentiation (CT VS 5, 10, 15, 20, 25 mM lactate treatment, P = 0.0012, P = 0.0007, and the rest of all P < 0.0001). Notably, inhibiting H3K9la using P300 antagonist could block lactate-induced myogenesis. Through combined omics analysis using cleavage under targets and tagmentation assay and RNA sequencing, we further identified Neu2 as a potential target gene of H3K9la. IGV software analysis (P = 0.0013) and chromatin immunoprecipitation-qPCR assay (H3K9la %Input, LA group = 9.0076, control group = 2.7184, IgG = 0.3209) confirmed that H3K9la is enriched in the promoter region of Neu2. Moreover, siRNAs or inhibitors against Neu2 both abrogated myoblast differentiation despite lactate treatment, suggesting that Neu2 is required for lactate-mediated myoblast differentiation. CONCLUSIONS: Our findings provide novel understanding of histone lysine lactylation, suggesting its role in myogenesis, and as potential therapeutic targets for muscle diseases.

Novel Pro-myogenic Factor Neoruscogenin Induces Muscle Fiber Hypertrophy by Inhibiting MSTN Maturation and Activating the Akt/mTOR Pathway.

Neoruscogenin is a plant-origin sapogenin that has the potential to modulate muscle growth among the small-molecule compounds that we previously predicted by artificial intelligence to target myostatin (MSTN). This study aimed to elucidate the biological role of neoruscogenin on muscle growth and its relationship with MSTN. Using molecular biological techniques, we found that neoruscogenin inhibited MSTN maturation, thereby repressing its signal transduction; further facilitated protein synthesis metabolism and reduced protein degradation metabolism, ultimately promoting the differentiation of myoblasts and hypertrophy of muscle fibers; and had the effect of repairing muscle injury. This study enriched the biological functions of neoruscogenin and provided a theoretical basis for the treatment of human myopathy and its application in the livestock industry.

Melatonin-Mediated Suppression of mtROS-JNK-FOXO1 Pathway Alleviates Hypoxia-Induced Apoptosis in Porcine Granulosa Cells.

Numerous studies have established that the hypoxic conditions within ovarian follicles induce apoptosis in granulosa cells (GCs), a pivotal hallmark of follicular atresia. Melatonin (N-acetyl-5-methoxytryptamine, MT), a versatile antioxidant naturally present in follicular fluid, acts as a safeguard for maintaining GCs' survival during stress exposure. In this study, we unveil an innovative protective mechanism of melatonin against hypoxia-triggered GC apoptosis by selectively inhibiting mitochondrial ROS (mtROS) generation. Specifically, under hypoxic conditions, a gradual accumulation of mitochondrial ROS occurred, consequently activating the JNK-FOXO1 pathway, and driving GCs toward apoptosis. The blocking of JNK or FOXO1 diminished hypoxia-induced GC apoptosis, but this effect was nullified in the presence of GSH, indicating that mtROS instigates apoptosis through the JNK-FOXO1 pathway. Consistent with this, hypoxic GCs treated with melatonin exhibited decreased levels of mtROS, reduced JNK-FOXO1 activation, and mitigated apoptosis. However, the protective capabilities of melatonin were attenuated upon inhibiting its receptor MTNR1B, accompanied by the decreased expression of antioxidant genes. Notably, SOD2, a key mitochondrial antioxidant gene modulated by the melatonin-MTNR1B axis, effectively inhibited the activation of mtROS-JNK-FOXO1 and subsequent apoptosis, whereas SOD2 knockdown abrogated the protective role of melatonin in hypoxic GCs. In conclusion, our study elucidates that melatonin, through MTNR1B activation, fosters SOD2 expression, effectively quelling mtROS-JNK-FOXO1-mediated apoptosis in follicular GCs under hypoxic stress.