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The problem of male infertility is a global health crisis and poses a serious threat to the well-being of families. Under heat stress (HS), the reduction of Sertoli cells (SCs) inhibits energy transport and nutrient supply to germ cells, leading to spermatogenesis failure. DNA methylation of genes is a central epigenetic regulatory mechanism in mammalian reproduction. However, it remains unclear how DNA methylation regulates gene expression in heat-stressed SCs. In this study, we investigated whether the decrease in SC levels during HS could be related to epigenetic DNA modifications. The cells exposed to HS showed changes in differential methylation cytosines and regions (DMCs/DMRs) and differential expression genes (DEGs), but not in global DNA methylations. One of the most important biological processes affected by HS is cell apoptosis induced by the intrinsic apoptotic signaling pathway (GO: 2,001,244, P < 0.05) by enrichment in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The joint analysis showed that several gene expressions in RNA-seq and WGBS overlapped and the shortlisted genes BAX, HSPH1, HSF1B, and BAG were strongly correlated with stress response and apoptosis. Methylation-specific PCR (MSP) and flow cytometry (FCM) analyzes showed that reduced promoter methylation and enhanced gene expression of BAX with a consequence of apoptosis. The activity of BAX, as well as an increase in its expression, is likely to result in a reduction of SCs population which could further impair ATP supply and adversely affect membrane integrity. These findings provide novel insights into the molecular mechanisms through which stressors cause male reproductive dysfunction and a new molecular etiology of male infertility.
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Apoptose , Metilação de DNA , Resposta ao Choque Térmico , Células de Sertoli , Proteína X Associada a bcl-2 , Animais , Masculino , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Epigênese Genética , Resposta ao Choque Térmico/fisiologia , Resposta ao Choque Térmico/genética , Células de Sertoli/metabolismo , SuínosRESUMO
Selection of stable housekeeping genes (HKGs) is very important for accurate calculation of relative expression levels of target genes by quantitative real-time polymerase chain reaction (qRT-PCR). At present, the appropriate HKGs have not been identified in placental tissues throughout the pregnancy of the goat. In our study, 20 HKGs were tentatively selected from RNA-seq data and previous reports. The cycle threshold (Ct) of HKGs was determined by qRT-PCR in trophoblast membrane and cotyledon villus collected from 38 Dazu Black goats on gestation days of 20, 25, 30, 45, 60, 90, 120, and 150 (birth). The expression stability of the HKGs was analyzed by geNorm, Normfinder, Bestkeeper and Delta Ct algorithms, and comprehensively evaluated by ReFinder and ComprFinder. In addition, the optimal HKGs were further verified by placenta-specific genes (SPP1, VEGFA and PAG6). The 16 candidate HKGs (except POP4, TBP, RNF10, UBC) showed a qualified Ct value, less than 28. Among them, YWHAZ, EIF3K and PPIB showed the most stable expression in placental tissues during early, mid-late pregnancy and postpartum, but the least stable expression was B2M at early and mid-late stage, and PPIB at postpartum. After comprehensive analysis, RPLP0, EIF3K and YWHAZ were found to be the most stable placental HKGs throughout pregnancy. The classical HKGs, ACTB, GAPDH and 18S RNA have unstable expressions and even ranked at the bottom of the list from comprehensive index, suggesting an inappropriate for target gene normalization. Taken together, our study confirmed that YWHAZ, EIF3K, HMBS and RPLP0 may be the optimal HKGs in goat placenta at different stage of pregnancy, which provided a valuable reference of HKGs on functional gene expression detection for further research on placenta development and growth in ruminants.
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Perfilação da Expressão Gênica , Genes Essenciais , Animais , Feminino , Gravidez , Genes Essenciais/genética , Reação em Cadeia da Polimerase em Tempo Real , Cabras/genética , Placenta , Placentação , RNARESUMO
Cold stimulation dynamically remodels mitochondria in brown adipose tissue (BAT) to facilitate non-shivering thermogenesis in mammals, but what regulates mitochondrial plasticity is poorly understood. Comparing mitochondrial proteomes in response to cold revealed FAM210A as a cold-inducible mitochondrial inner membrane protein. An adipocyte-specific constitutive knockout of Fam210a (Fam210aAKO) disrupts mitochondrial cristae structure and diminishes the thermogenic activity of BAT, rendering the Fam210aAKO mice vulnerable to lethal hypothermia under acute cold exposure. Induced knockout of Fam210a in adult adipocytes (Fam210aiAKO) does not affect steady-state mitochondrial structure under thermoneutrality, but impairs cold-induced mitochondrial remodeling, leading to progressive loss of cristae and reduction of mitochondrial density. Proteomics reveals an association between FAM210A and OPA1, whose cleavage governs cristae dynamics and mitochondrial remodeling. Mechanistically, FAM210A interacts with mitochondrial protease YME1L and modulates its activity toward OMA1 and OPA1 cleavage. These data establish FAM210A as a key regulator of mitochondrial cristae remodeling in BAT and shed light on the mechanism underlying mitochondrial plasticity in response to cold.
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Adipócitos Marrons , Hipotermia , Proteínas Mitocondriais , Animais , Camundongos , Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/metabolismo , Temperatura Baixa , Hipotermia/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Termogênese , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismoRESUMO
The uterus is a critical pregnancy organ for mammals. The normal growth and development of ruminant uterus caruncles are crucial to maintain gestation and fetal health in goats. Quantitative real-time polymerase chain reaction (qRT-PCR) is a reliable tool to study gene expression profiling for exploring the intrinsic mechanism underlying the conversion process of uterus caruncle tissue. However, the candidate housekeeping genes (HKGs) are required for normalizing the expression of function genes. In our study, 22 HKGs were selected from analyzing transcriptome data at non-pregnancy and pregnancy processes and previous reports about HKGs in goat tissues. We assessed them for expression suitability in 24 samples from uterus tissues at 15 non-pregnant days (Stage 1), early (Stage 2), and medium-later pregnant days (Stage 3). The expression stability of these genes was evaluated by using geNorm, Normfinder, Bestkeeper, and Delta Ct algorithms and, comprehensively, by ReFinder. In addition, the most and least stable HKGs were used to normalize the target genes expression of SPP1, VEGFA, and PAG8. It was found that traditional reference genes, such as ACTB and GAPDH, were not suitable for target gene normalization. In contrast, PPIB selected from RNA sequencing data and EIF3K selected from previous references showed the least variation and were recommended as the best HKGs during the nonpregnant stage and the whole stages of goat uterus caruncle tissue, respectively. It is the first time the HKGs genes in uterus during the non-pregnant day and throughout the total pregnancy have been explored. These findings found suitable HKGs in uterus caruncle tissues at various stages of non-pregnancy and pregnancy; these can be useful for gene expression studies to reveal the molecular mechanisms of uterus development in goats.
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The death of Sertoli cells (SCs) under condition of heat stress (HS) affects spermatogenesis and is associated with impaired function of the blood-testis barrier (BTB). The fatty acid arachidonic acid (AA) is essential for the maintenance of cellular function. However, excessive release of AA during HS may adversely affect the reproductive function. The molecular mechanisms through which AA modulates the BTB in SCs are unclear. In this study, we found that 100 µM AA damaged testicular morphology and accelerated SC apoptosis during HS, reducing the stability of tight junction proteins (TJPs), shown by measurement of the levels of Claudin 11, 5, Occludin, and trans-epithelial electrical resistance (TEER). It was also found that AA adversely affected TJPs by increasing the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), activating p38 mitogen-activated protein kinases (P38 MAPK) and reducing mitochondria DNA (mtDNA) and the expression of mitochondrial complexes I and III. In contrast, pretreatment with SB203508 (a P38 MAPK inhibitor), Rotenone (an inhibitor of complex I) and Antimycin A1 (an inhibitor of complex III) reversed TJPs degradation induced by AA. Interestingly, pretreatment of cells with 10 µM Baicalein, a 12/15 lipoxygenase (12/15-LOX) -dependent inhibitor of AA production, protected against AA-induced TJPs degradation, restored mitochondrial function, and reduced apoptosis. These results suggested an intriguing link between the induction of TJPs degradation induced by AA overload and mitochondrial antioxidant function during HS, which was found to be regulated by the mitochondrial complex-ROS-P38 MAPK axis.
Assuntos
Células de Sertoli , Proteínas Quinases p38 Ativadas por Mitógeno , Masculino , Humanos , Células de Sertoli/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ácido Araquidônico/farmacologia , Ácido Araquidônico/metabolismo , Barreira Hematotesticular/metabolismo , Mitocôndrias/metabolismo , Proteínas de Junções Íntimas/metabolismoRESUMO
Embryo implantation is a critical step in the establishment of pregnancy. However, the mechanisms of embryo implantation during early pregnancy in goats remain unclear due to the lack of published studies examining the genes involved in embryo implantation. As a popular goat breed in southwest China, Dazu black goats (DBGs) are highly adaptable and exhibit high fertility, making this breed a good model in which to study reproductive performance of goats. Here, morphological analysis showed that compared with the non-pregnant (NP) groups, the endometrial thickness of the goats in the P15 and P19 groups (15 and 19-day pregnant groups, respectively) were increased (P < 0.01). Proliferating Cell Nuclear Antigen (PCNA) staining showed that PCNA was expressed in the NP, P15, and P19 groups. Transcriptome analysis was then conducted to identify gene expression patterns in uterine tissue during DBG embryo implantation. By comparing uterine tissue at different stages of embryonic implantation, 48 in NP_vs._P15, 318 in NP_vs._P19, and 1439 in P15_vs._P19, differentially expressed mRNAs were identified. Gene Ontology (GO) enrichments of the differentially expressed genes were enriched in the extracellular region, extracellular space, transporter activity, extracellular region, immune system process, immune response, and defense response etc. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the biological metabolic pathways with which the differentially expressed genes are associated were explored. Through KEGG analysis, the DBGs were associated with oxidative phosphorylation, complement and coagulation cascades, arginine and proline metabolism, metabolic pathways, arachidonic acid metabolism, and ECM-receptor interaction. These candidate genes (CSF1, C1S, CST6, SLC24A4, HOXA10, HOXA11, MMP9, and ITGA11) and enriched signaling pathways could be valuable references for exploring the molecular mechanisms underlying goat embryo implantation.
Mammalian embryo implantation refers to the process that the embryo normally develops to the blastocyst stage, contacts the maternal endometrium, and establishes one kind structural connection. This intimate connection allows for the process of maternalfetal material exchange, which is one of the key steps in the successful pregnancy. The success of embryo implantation depends on two aspects of the endometrium and the embryo, 1) the maternal endometrium is in a receptive state, and 2) the embryo develops normally, both of which are indispensable. In this stage, the mechanism of embryo implantation early in goat pregnancy is not clear, as only few limited studies have been conducted into gene expression in the uterus during embryo implantation. In this study, goat uterine tissue was systematically collected during the periods of non-pregnancy, pregnancy recognition, and embryo adhesion. And the morphological changes of the uterus in the different gestational stages were also observed, and gene expression associated with embryo implantation was further analyzed by RNA-seq method. This study provides a preliminary dataset for analyzing the molecular mechanisms regulating goat embryo implantation.
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Cabras , Transcriptoma , Gravidez , Feminino , Animais , Antígeno Nuclear de Célula em Proliferação/metabolismo , Cabras/genética , Cabras/metabolismo , Endométrio/metabolismo , Perfilação da Expressão Gênica/veterinária , Implantação do Embrião/genéticaRESUMO
Family with sequence similarity 83 A (FAM83A) is a newly discovered proto-oncogene that has been shown to play key roles in various cancers. However, the function of FAM83A in other physiological processes is not well known. Here, we report a novel function of FAM83A in adipocyte differentiation. We used an adipocyte-targeting fusion oligopeptide (FITC-ATS-9R) to deliver a FAM83A-sgRNA/Cas9 plasmid to knockdown Fam83a (ATS/sg-FAM83A) in white adipose tissue in mice, which resulted in reduced white adipose tissue mass, smaller adipocytes, and mitochondrial damage that was aggravated by a high-fat diet. In cultured 3T3-L1 adipocytes, we found loss or knockdown of Fam83a significantly repressed lipid droplet formation and downregulated the expression of lipogenic genes and proteins. Furthermore, inhibition of Fam83a decreased mitochondrial ATP production through blockage of the electron transport chain, associated with enhanced apoptosis. Mechanistically, we demonstrate FAM83A interacts with casein kinase 1 (CK1) and promotes the permeability of the mitochondrial outer membrane. Furthermore, loss of Fam83a in adipocytes hampered the formation of the TOM40 complex and impeded CK1-driven lipogenesis. Taken together, these results establish FAM83A as a critical regulator of mitochondria maintenance during adipogenesis.
Assuntos
Adipócitos Brancos , Adipogenia , Caseína Quinase I , Mitocôndrias , Proteínas de Neoplasias , Proto-Oncogenes , Animais , Camundongos , Células 3T3-L1 , Adipócitos Brancos/citologia , Adipócitos Brancos/metabolismo , Adipogenia/genética , Caseína Quinase I/metabolismo , Diferenciação Celular , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMO
The Chchd10 gene encodes a coiled-coil-helix-coiled-coil-helix-domain containing protein predicted to function in the mitochondrion and nucleus. Mutations of Chchd10 are associated with ALS, dementia and myopathy in humans and animal models, but how knockout of Chchd10 (Chchd10KO) affects various tissues especially skeletal muscle and adipose tissues remains unclear. Here we show that Chchd10 expression increases as myoblasts and preadipocytes differentiate. During myogenesis, CHCHD10 interacts with TAR DNA binding protein 43 (TDP-43) in regenerating myofibers in vivo and in newly differentiated myotubes ex vivo. Surprisingly, Chchd10KO mice had normal skeletal muscle development, growth and regeneration, with moderate defects in grip strength and motor performance. Chchd10KO similarly had no effects on development of brown and white adipose tissues (WAT). However, Chchd10KO mice had blunted response to acute cold and attenuated cold-induced browning of WAT, with markedly reduced UCP1 levels. Together, these results demonstrate that Chchd10 is dispensable for normal myogenesis and adipogenesis but is required for normal motility and cold-induced, mitochondrion-dependent browning of adipocytes. The data also suggest that human CHCHD10 mutations cause myopathy through a gain-of-function mechanism.
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Occurrence of low birth weight (LBW) is a major concern in livestock production, resulting in poor postnatal growth, lowered efficiency of feed utilization, and impaired metabolic health in adult life. In the southwest region of China, birth weight of indigenous strains of goats varies seasonally with lower weights in summer and winter, but the metabolic regulation of the LBW offspring is still unknown. In this study, by comparing LBW goats to normal birth weight group, we examined hepatic lipid content in association with regulatory mechanisms. Histological studies showed higher microvesicular morphology in the liver of LBW goats in accompany with a significantly higher level of hepatic free fatty acids, total triglycerides, and cholesterols. Lipid metabolism impairment, increased oxidative stress, and inflammation were observed by transcriptome analysis. Meanwhile, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation further demonstrated lipid peroxidation, antioxidant pathway, and pro-inflammatory response involved in the hepatic lipid dysregulation from LBW group. Therefore, dysregulations of hepatic lipid metabolism, including fatty acid biosynthesis and degradation, lipid transportation, and oxidative stress, played important roles to contribute the lipid accumulation in LBW goats. Moreover, due to impaired antioxidant capacity, the oxidative damage could interact with persisting pro-inflammatory responses, leading to a higher risk of liver injury and metabolic syndromes in their adult life.
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Propionate is a gut microbial metabolite that has been reported to have controversial effects on metabolic health. Here we show that propionate is activated by acyl-CoA synthetase short-chain family member 3 (ACSS3), located on the mitochondrial inner membrane in brown adipocytes. Knockout of Acss3 gene (Acss3-/- ) in mice reduces brown adipose tissue (BAT) mass but increases white adipose tissue (WAT) mass, leading to glucose intolerance and insulin resistance that are exacerbated by high-fat diet (HFD). Intriguingly, Acss3-/- or HFD feeding significantly elevates propionate levels in BAT and serum, and propionate supplementation induces autophagy in cultured brown and white adipocytes. The elevated levels of propionate in Acss3-/- mice similarly drive adipocyte autophagy, and pharmacological inhibition of autophagy using hydroxychloroquine ameliorates obesity, hepatic steatosis and insulin resistance of the Acss3-/- mice. These results establish ACSS3 as the key enzyme for propionate metabolism and demonstrate that accumulation of propionate promotes obesity and Type 2 diabetes through triggering adipocyte autophagy.
Assuntos
Tecido Adiposo Marrom/efeitos dos fármacos , Coenzima A Ligases/efeitos adversos , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/metabolismo , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/metabolismo , Tecido Adiposo Marrom/crescimento & desenvolvimento , Animais , Coenzima A Ligases/farmacologia , Modelos Animais de Doenças , Camundongos , Camundongos Knockout/metabolismo , Propionatos/metabolismo , Propionatos/farmacologiaRESUMO
The placenta is a temporary maternal−fetal organ, and its maternal placenta (caruncle) is essential for fetal growth and development. The exchange function of the placenta requires vascular development (angiogenesis). However, the angiogenesis of the caruncle is poorly understood in goats during the early stage of pregnancy. Here, we investigated the vascular distribution, mRNA expression of major angiogenic factors, and the methylation levels of ANGPT2 in the goat caruncle. It showed that CAD (capillary area density), CSD (capillary surface density), and APC (area per capillary) increased gradually, while CND (capillary number density) showed an insignificant change, probably due to the variability between animals. The proportion of proliferating cells was observed to be very high (>26%) and increased (p < 0.002) approximately 2-fold from day 20 to 60 of pregnancy. Furthermore, the expression patterns of major angiogenic factors changed during the early stage of pregnancy. Interestingly, we discovered an absolute correlation between the mRNA for ANGPT2, TEK, FGF2, and vascular distribution. Subsequently, we evaluated the DNA methylation of ANGPT2, where we found that mean methylation was negatively correlated with CAD. The methylation at the CpG sites, such as CpG 4/18, CpG 9.10.11, and CpG 15, showed significant changes during the early stage of pregnancy. Thus, our findings suggest that the methylation of ANGPT2 may be involved in the regulation of caruncle angiogenesis during the early stage of pregnancy.
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This study explored the trophoblast transcriptome to understand potential functional genes involved in early placental development in goats and their enriched signaling pathways. Trophoblast samples were collected from nine Dazu Black goats on days 20, 25, and 30 of pregnancy (D20, D25, and D30). As the pregnancy progressed, the morphology and histological structures showed significant growth, adhesion, and angiogenesis. A total of 23,253 commonly expressed genes (CEGs) and 4439 differently expressed genes (DEGs) were detected by RNA sequencing. The common highly expressed genes (ChEGs) (the top 100 CEGs) with the highest FPKM percentage (29.9%) of all CEGs were annotated to the ribosome pathway and maintain pregnancy. DEGs were abundant in D30 vs. D20 (3715 DEGs). Besides, the DEGs were associated with the inhibition of oxidative phosphorylation and activation of PI3K-Akt, focal adhesion, ECM-receptor interaction, Rap1, and CAM signaling pathways. The RAP1 may be a central pathway since it coordinates with others to regulate the cell proliferation, invasion, migration, and fusion of trophoblasts. qRT-PCR and Western blot analysis confirmed the transcriptional expression in IGF1, VEGFC, RAPGEF3, PIK3CA, AKT3, ITGB3, ITGA11, SPP1, NOS1, and ATP6V0B genes and protein levels in VEGF, RAPGEF3, and Akt. This is the first study of transcriptome profiling in goat placenta and provides diverse genetic resources for further research on placenta development.
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Mitochondrial remodeling through fusion and fission is crucial for progenitor cell differentiation but its role in myogenesis is poorly understood. Here, we characterized the function of mitofusin 2 (Mfn2), a mitochondrial outer membrane protein critical for mitochondrial fusion, in muscle progenitor cells (myoblasts). Mfn2 expression is upregulated during myoblast differentiation in vitro and muscle regeneration in vivo. Targeted deletion of Mfn2 gene in myoblasts (Mfn2MKO ) increases oxygen-consumption rates (OCR) associated with the maximal respiration and spare respiratory capacity, and increased levels of reactive oxygen species (ROS). Skeletal muscles of Mfn2MKO mice exhibit robust mitochondrial swelling with normal mitochondrial DNA content. Additionally, mitochondria isolated from Mfn2MKO muscles have reduced OCR at basal state and for complex I respiration, associated with decreased levels of complex I proteins NDUFB8 (NADH ubiquinone oxidoreductase subunit B8) and NDUFS3 (NADH ubiquinone oxidoreductase subunit S3). However, Mfn2MKO has no obvious effects on myoblast differentiation, muscle development and function, and muscle regeneration. These results demonstrate a novel role of Mfn2 in regulating mitochondrial complex I protein abundance and respiratory functions in myogenic progenitors and myofibers.
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Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Animais , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons , Feminino , GTP Fosfo-Hidrolases/genética , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias Musculares , Desenvolvimento Muscular , Músculo Esquelético , Estresse Oxidativo , Consumo de Oxigênio , Condicionamento Físico Animal , Espécies Reativas de Oxigênio , Células-TroncoRESUMO
The subtropical monsoon climate characterized by high or low temperature and humidity can induce cold and heat stress for newborn animals, which results in adverse effect on birth weight and even pre-weaning mortality. However, this early growth performance on indigenous goats is affected by cold and heat climatic environments and is still unclear in subtropical climate. In this study, we continuously measured (July 2011 to June 2016) the birth weight and mortality of an indigenous goat species (n = 530), and collected temperature, humidity, temperature-humidity index (THI) in original farming area, Chongqing, southwest China. As the result, the mean birth weights in cold months (January and February, mean temperature < 10 °C and THI < 56) and heat months (July and August, mean temperature > 29 °C and THI > 76) were significantly lower (P < 0.05) compared to the other months (June and October, mean temperature = 16~25 °C and THI = 61~75). Meanwhile, the birth weight was positively correlated (P < 0.01) with gestational THI from November to May, and was negatively correlated (P < 0.01) with those parameters from June to October, respectively. The maximum pre-weaning mortality, occurring in the 1st month after birth, is 16.17 ± 2.56%. However, when the birth weight was 20% lower than annual average (2.09 ± 0.54 kg), the mortality was significantly enhanced (P < 0.01) to 46%. In addition, cold and heat climates respectively enhanced mortality in the 1st month and 2nd~4th months after birth. In conclusion, annually chronic heat and cold climates could play important roles in lowering birth weight and their survival in subtropical monsoon region. Low birth weight and cold temperature play critical role to contribute the advent of higher mortality after birth. Our results potentially provide the appropriate ranges of temperature (16~26 °C) and THI (61~75) as pregnant goat and kids raising condition to avoid these negative influences.
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Peso ao Nascer , Cabras/fisiologia , Mortalidade , Tempo (Meteorologia) , Animais , Animais Recém-Nascidos , China , Temperatura Baixa , Feminino , Transtornos de Estresse por Calor/veterinária , Resposta ao Choque Térmico , Temperatura Alta , Umidade , Parto , Gravidez , Temperatura , DesmameRESUMO
Lactate produced by glycolysis in Sertoli cells (SCs) is the main energy substrate for developing germ cells and plays a vital role in spermatogenesis. MicroRNAs (miRNAs) function as posttranscriptional regulators of gene expression in biological processes. We have previously shown that hyperthermia (43°C, 30 min) promotes lactate secretion by inhibiting phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in cultured immature boar SCs. However, it is unclear whether miRNAs are involved in AMPK-modulated glycolysis in SCs. In the present study, we identified 349 miRNAs (227 upregulated and 122 downregulated) in hyperthermia-treated boar SCs by next-generation high-throughput RNA sequencing. MiR-8-3p, which was found to be a novel upregulated miRNA in hyperthermia-treated SCs, suppressed the expression of AMPK upstream genes (protein phosphatase 2 subunit B, PPP2R5B), and further downregulated the expression of p-AMPK. The miR-8-3p mimic upregulated expression of glucose transporter 3, lactate dehydrogenase A and monocarboxylate transporter 1, and increased lactic acid dehydrogenase activity, lactate secretion, and ATP depletion in SCs; the miR-8-3p inhibitor had the opposite effects on these parameters. Our findings indicate that miR-8-3p acts as a novel regulator of AMPK-modulated lactate secretion by targeting PPP2R5B in hyperthermic boar SCs.
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Resposta ao Choque Térmico , Ácido Láctico/metabolismo , MicroRNAs/metabolismo , Proteína Fosfatase 2/metabolismo , Células de Sertoli/metabolismo , Animais , Masculino , SuínosRESUMO
Goats (Capra hircus) are one of the oldest livestock domesticated species, and have been used for their milk, meat, hair and skins over much of the world. Detection of selection footprints in genomic regions can provide potential insights for understanding the genetic mechanism of specific phenotypic traits and better guide in animal breeding. The study presented here has generated 192.747G raw data and identified more than 5.03 million single-nucleotide polymorphisms (SNPs) and 334,151 Indels (insertions and deletions). In addition, we identified 155 and 294 candidate regions harboring 86 and 97 genes based on allele frequency differences in Dazu black goats (DBG) and Inner Mongolia cashmere goats (IMCG), respectively. Populations differentiation reflected by Fst values detected 368 putative selective sweep regions including 164 genes. The top 1% regions of both low heterozygosity and high genetic differentiation contained 239 (135 genes) and 176 (106 genes) candidate regions in DBG and IMCG, respectively. These genes were related to reproductive and productive traits, such as "neurohypophyseal hormone activity" and "adipocytokine signaling pathway". These findings may be conducive to molecular breeding and the long-term preservation of the valuable genetic resources for this species.