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Gellan gum (GG) is a soft, tractable,ãand natural polysaccharide substrate used for cell incubation. In this study, we examined the effects of GG on porcine oocyte maturation. Cumulus cells and oocyte complexes (COCs) were collected from slaughterhouse-derived porcine ovaries and cultured on plastic plates containing 0.05% or 0.1% GG gels. The 0.1% GG gel improved the maturation rate and quality of blastocysts, as determined by the total cell number and the rate of abnormally condensed nuclei. GG gels have antioxidant abilities and oocytes cultured on GG gels (0.05% and 0.1%) have reduced reactive oxygen species (ROS) content. Furthermore, GG gels (0.05% and 0.1%) increased F-actin formation, whereas treatment of oocytes with H2O2 reduced F-actin levels. GG gels increased the ATP content in oocytes but did not affect the mitochondrial DNA copy number or mitochondrial membrane potential. In addition, the medium cultured on 0.05% GG increased the glucose consumption of COCs. In conclusion, GG gel reduced ROS content, increased energy content, and improved subsequent embryonic development in pigs.
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The present study examined whether male resveratrol intake affected mitochondrial DNA copy number (mt-cn) and telomere length (TL) in blastocysts fathered by young and aged male mice. C57BL/6N male mice supplied with water or water containing 0.1 mM resveratrol were used for embryo production at 14-23 and 48-58 weeks of age. Two-cell-stage embryos were collected from the oviducts of superovulated female mice (8-15 weeks old) and cultured for 3 days until the blastocyst stage. Mt-cn and TL levels were measured by real-time polymerase chain reaction. Resveratrol intake did not affect body weight or water consumption. Resveratrol intake increased the expression levels of SIRT1 in the liver, the antioxidative ability of serum, and extended TL in the heart, whereas there was no significant difference in mt-cn in the heart or TL in sperm. The rate of blastocyst development was significantly lower in aged male mice than in younger mice, and resveratrol intake increased the total number of blastocysts derived from both young and aged males. Resveratrol intake did not affect mt-cn or TL in blastomeres of blastocyst-stage embryos derived from young mice, but significantly increased both mt-cn and TL in blastomeres of blastocysts derived from aged fathers. In conclusion, resveratrol intake increased mt-cn and TL levels in blastocysts derived from aged male mice.
Assuntos
Blastocisto , DNA Mitocondrial , Camundongos Endogâmicos C57BL , Resveratrol , Telômero , Animais , Resveratrol/farmacologia , Masculino , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Feminino , Camundongos , DNA Mitocondrial/metabolismo , Telômero/efeitos dos fármacos , Telômero/metabolismo , Homeostase do Telômero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Sirtuína 1/metabolismo , Sirtuína 1/genética , Variações do Número de Cópias de DNA/efeitos dos fármacos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Envelhecimento , Estilbenos/farmacologia , Idade PaternaRESUMO
Global methylation levels differ in in vitro- and in vivo-developed embryos. Follicular fluid (FF) contains extracellular vesicles (EVs) containing miRNAs that affect embryonic development. Here, we examined our hypothesis that components in FF affect global DNA methylation and embryonic development. Oocytes and FF were collected from bovine ovaries. Treatment of zygotes with a low concentration of FF induced global DNA demethylation, improved embryonic development, and reduced DNMT1/3A levels. We show that embryos take up EVs containing labeled miRNA secreted from granulosa cells and the treatment of zygotes with EVs derived from FF reduces global DNA methylation in embryos. Furthermore, the methylation levels of in vitro-developed blastocysts were higher than those of in their vivo counterparts. Based on small RNA-sequencing and in silico analysis, we predicted miR-29b, -199a-3p, and -148a to target DNMTs and to induce DNA demethylation, thereby improving embryonic development. Moreover, among FF from 30 cows, FF with a high content of these miRNAs demethylated more DNA in the embryos than FF with a lower miRNA content. Thus, miRNAs in FF play a role in early embryonic development.
Assuntos
Desenvolvimento Embrionário , Vesículas Extracelulares , Líquido Folicular , MicroRNAs , Animais , Feminino , MicroRNAs/genética , MicroRNAs/metabolismo , Bovinos , Líquido Folicular/metabolismo , Vesículas Extracelulares/metabolismo , Desenvolvimento Embrionário/genética , Metilação de DNA , Desmetilação do DNA , Oócitos/metabolismo , Blastocisto/metabolismo , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Zigoto/metabolismoRESUMO
Improvement in vitro maturation culture conditions has been achieved by mimicking in vivo culture environments such as the follicular fluid. Acetic acid is an energy substrate that is abundantly present in the follicular fluid but has not been considered in vitro maturation. This study examined the effects of acetic acid on oocyte quality during nuclear maturation. Cumulus cells and oocyte complexes were collected from the porcine antral follicles of gilt ovaries and matured with 0, 0.1 or 1 mmol/L of acetic acid. After 44 h of in vitro maturation, the energy status, mitochondrial quality and function and embryonic developmental rate following parthenogenetic activation were determined. RNA-sequencing and protein expression analyses were conducted to predict the effects of acetic acid. Supplementation of the in vitro maturation medium with acetic acid (1 mmol/L) improved embryonic development. Oocytes matured with acetic acid had low adenosine triphosphate and lipid contents, mitochondrial membrane potential and reactive oxygen species levels. RNA-sequencing revealed differential expression of genes associated with the adenosine monophosphate-activated protein kinase signalling pathway. Immunostaining revealed that acetic acid increased the levels of phospho-adenosine monophosphate-activated protein kinase, phospho-acetyl-coenzyme A carboxylase, and sirtuin 1 and decreased those of fatty acid synthase and acetyl-coenzyme A synthetase 1. In summary, the use of acetic acid during oocyte maturation improved oocyte developmental ability and metabolism by altering mitochondrial activity and lipid metabolism.
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Ácido Acético , Técnicas de Maturação in Vitro de Oócitos , Oócitos , Animais , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Suínos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Ácido Acético/farmacologia , Feminino , Desenvolvimento Embrionário/efeitos dos fármacosRESUMO
Ethanol affects pre-conceptional oocyte quality in women. In this study, we examined the effect of low ethanol concentrations on mouse oocytes. Oocytes were collected from the ovaries of 9-10 week old mice and allowed to mature in vitro in the presence of low concentrations of ethanol (0.1% and 0.2% v/v) for 24 h. Treatment of oocytes with ethanol (0.2%) during maturation decreased the mitochondrial DNA content and membrane potential compared to that in untreated ones, whereas the ATP content did not differ between the groups. Both 0.1% and 0.2% ethanol reduced the lipid content in the oocytes. In addition, immunostaining revealed that oocytes cultured in maturation medium containing ethanol (0.2%) had reduced levels of global DNA methylation and DNMT3A compared with untreated oocytes, and decreased rate of blastocyst development with low mitochondrial protein levels (TOMM40) in embryo. RNA-sequencing of the ethanol-treated (0.2%) and untreated oocytes revealed that mitochondria were a major target of ethanol. In conclusion, treatment of oocytes with low concentration of ethanol reduces the developmental rate to the blastocyst stage, with a lower total cell number and global DNA methylation. In addition, ethanol affected mitochondrial function and mitochondria-related gene expression.
Assuntos
Metilação de DNA , Etanol , Técnicas de Maturação in Vitro de Oócitos , Mitocôndrias , Oócitos , Animais , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Etanol/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Feminino , Metilação de DNA/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Meios de Cultura/química , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , DNA Mitocondrial/metabolismo , Transcriptoma/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacosRESUMO
Telomere length (TL) and mitochondrial DNA copy number (mt-cn) are associated with embryonic development. Here, we investigated the correlation between TL and mt-cn in bovine embryos to determine whether TL regulates mt-cn. TL and mt-cn were closely correlated in embryos derived from six bulls. Treatment of embryos with a telomerase inhibitor (TMPyP) and siTERT shortened the TL and reduced mt-cn in blastocysts. RNA-sequencing of blastocysts developed with TMPyP revealed differentially expressed genes associated with transforming growth factor-ß1 signaling and inflammation. In conclusion, TL regulates mt-cn in embryos.
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Blastocisto , Variações do Número de Cópias de DNA , Animais , Bovinos , Blastocisto/metabolismo , Blastocisto/efeitos dos fármacos , Telômero/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Masculino , Feminino , Homeostase do Telômero/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genéticaRESUMO
Preeclampsia (PE) is characterized by hypertension, proteinuria, and fetal growth restriction during pregnancy, suggesting that the preeclamptic intrauterine environment may affect the growth and health of the offspring. This study aimed to how maternal hypertension affects male offspring growth, focusing on lipid metabolism and blood pressure in mice. Female mice were infused with angiotensin II (Ang II) on gestational day 12. Dysregulation and accumulation of lipid were observed in the placenta of Ang II-induced maternal hypertensive dams, associating with fetal growth restriction. Ang II-offspring showed lower birth weight than in the control-offspring. Isolated and differentiated adipocyte from neonatal mice of Ang II-dams showed higher Pparγ mRNA expression compared with the control group. Lower body weight tendency had continued in Ang II-offspring during long period, body weight of Ang II-offspring caught up the control-offspring at 16 weeks of age. The adipose tissue of Ang II-offspring in adult also showed higher Pparγ mRNA expression with the accumulation of neutrophils and inflammatory monocytes than in those control. In addition, Ang II-offspring had higher basal blood pressure and higher sensitivity to hypertensive stimuli than in the control-offspring. Taken together, maternal hypertension induced by Ang II changes placental function, causing a lower birth weight. These changes in the intrauterine environment may affect adipocyte function and blood pressure of offspring after growth.
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Hipertensão , Pré-Eclâmpsia , Humanos , Feminino , Gravidez , Masculino , Animais , Camundongos , Pressão Sanguínea/fisiologia , Retardo do Crescimento Fetal/etiologia , Peso ao Nascer , PPAR gama/genética , PPAR gama/metabolismo , Placenta/metabolismo , Sistema Renina-Angiotensina/fisiologia , Hipertensão/metabolismo , Angiotensina II/metabolismo , Pré-Eclâmpsia/metabolismo , Tecido Adiposo/metabolismo , RNA Mensageiro/metabolismoRESUMO
Preeclampsia (PE) is a serious complication, and soluble fms-like tyrosine kinase (sFLT1) released from the placenta is one of the causes of PE pathology. Trophoblasts are the primary source of sFLT1; however, monocytes/macrophages exist enough in the placenta can also secrete sFLT1. Sterile inflammatory responses, especially NLRP3 inflammasome and its downstream gasdermin D (GSDMD)-regulated pyroptosis, may be involved in the development of PE pathology. In this study, we investigated whether human monocyte/macrophage cell line THP-1 cells secrete sFLT1 depending on the NLRP3 inflammasome and GSDMD. To differentiate THP-1 monocytes into macrophages, treatment with phorbol 12-myristate 13-acetate (PMA) induced sFLT1 with interleukin (IL)- 1ß, but did not induce cell lytic death. IL-1ß secretion induced by PMA inhibited by deletion of NLRP3 and inhibitors of NLRP3 and caspase-1, but deletion of NLRP3 and these inhibitors did not affect sFLT1 secretion in THP-1 cells. Both gene deletion and inhibition of GSDMD dramatically decreased IL-1ß and sFLT1 secretion from THP-1 cells. Treatment with CA074-ME (a cathepsin B inhibitor) also reduced the secretion of both sFLT1 and IL-1ß in THP-1 cells. In conclusion, THP-1 macrophages release sFLT1 in a GSDMD-dependent manner, but not in the NLRP3 inflammasome-dependent manner, and this sFLT1 release may be associated with the non-lytic role of GSDMD. In addition, sFLT1 levels induced by PMA are associated with lysosomal cathepsin B in THP-1 macrophages. We suggest that sFLT1 synthesis regulated by GSDMD are involved in the pathology of PE.
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Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Catepsina B/metabolismo , Gasderminas , Macrófagos/metabolismoRESUMO
Purpose: Oocyte and embryo quality differs significantly among individuals. Follicular fluid (FF) is a solo environment of oocyte maturation and may flux into the oviduct. Supplementation of in vitro maturation (IVM) and culture (IVC) medium with extracellular vesicles of FFs supports oocyte maturation and embryonic development. We addressed a hypothesis that miRNA profiles in FFs are crucial background of oocyte maturation and embryonic development. Methods: FFs were collected from the ovaries of individual cows, and the FFs were classified into Good or Poor FF based on the developmental rate to the blastocyst stage of enclosed oocytes. miRNAs associated with the Good FFs were explored using small RNA sequencing. In addition, FFs were classified using the concentration of Good-FF-associated miRNAs. These classified FFs or miRNA were added to the IVM or IVC mediums. Results: Supplementation of IVM and IVC medium with Good FF improved embryonic development. Good FFs contained miR-151-3p and miR-425-5p at a high concentration compared with those in Poor FFs. FFs selected by the concentration of miR-151-3p and miR-425-5p improved oocyte maturation and embryonic development. Supplementation of IVM or IVC medium with either miR-151-3p or miR-425-5p improved embryonic development to the blastocyst stage. Conclusion: miRNAs were associated with the Good FFs determined oocyte maturation and embryonic development.
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In this study, we examined the effects of paternal aging on the mitochondrial DNA copy number (mt-cn), telomere length (TL), and gene expression in mouse embryos. The effects of vitrification on the mt-cn and TL of the embryos derived from young and aged male parents (YF and AF, respectively) were examined. C57BL/6N male mice were used for embryo production at 13-23 and 50-55 weeks of age. Two-cell stage embryos were collected from the oviducts of superovulated female mice (8-15 weeks old) and cultured for 24 h until the 8-cell stage, followed by embryo vitrification. Fresh and vitrified-warmed embryos were incubated for 2 days until the blastocyst stage, and mt-cn and TL were investigated. The cell-free mitochondrial DNA copy number (cf-mt-cn) in the spent culture medium (SCM) of the embryos was then investigated. RNA sequencing of blastocysts revealed that metabolic pathways, including oxidative phosphorylation and mTOR pathways, were enriched in differentially expressed genes. The mt-cn and TL of AF-derived blastocysts were lower and shorter, respectively, than those of YF-derived blastocysts. Paternal aging did not affect the blastocyst rate after vitrification. Vitrification of the 8-cell stage embryos did not affect the mt-cn of the blastocysts. However, it increased the cf-mt-cn (cell-free mt-cn) in the SCM of both YF- and AF-derived embryos. Vitrification did not affect the TL of either YF- or AF-derived embryos. Thus, paternal aging affected the mt-cn and TL of the embryos, but vitrification did not affect these parameters in either age groups.
Assuntos
Criopreservação , Vitrificação , Masculino , Feminino , Animais , Camundongos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Variações do Número de Cópias de DNA , Camundongos Endogâmicos C57BL , Blastocisto/metabolismo , TelômeroRESUMO
Vitrification is an important assisted reproductive technology, although it induces mitochondrial dysfunction in embryos. Herein, we aimed to investigate whether age-associated accumulation of advanced glycation end-products (AGEs) in oocytes impairs the recovery of embryos from cryopreservation-induced mitochondrial dysfunction/damage. Mouse eight-cell stage embryos developed in vitro were vitrified and warmed and incubated up to the blastocyst stage. AGE levels in oocytes were higher in both aged mice and AGE accumulation mouse models (MGO-mice) than those in young and control mice. In addition, the level of SIRT1 upregulation was lower for embryos of aged and MGO-mice than that for embryos of young and control mice. The highest mitochondrial DNA (mtDNA) content was detected in blastocysts derived from vitrified embryos of aged and MGO-mice. The spent culture medium of blastocysts derived from both aged and MGO-mice contained higher mtDNA content than that of the blastocysts derived from young and control mice. EX527 increased mtDNA content in the spent culture medium of vitrified embryos derived from young mice. In addition, p62 aggregate levels were higher in vitrified embryos of control mice than those in vitrified embryos of MGO-mice. The SIRT1 activator, resveratrol, increased p62 aggregation levels in vitrified embryos derived from young and aged mice, whereas vitrification did not affect p62 aggregation levels in embryos from aged mice. Therefore, age-associated AGE accumulation induces decreased responsive SIRT1 upregulation following vitrified-warmed treatment and impairs mitochondrial quality control activity in vitrified embryos.
Assuntos
Sirtuína 1 , Vitrificação , Animais , Camundongos , Sirtuína 1/genética , Sirtuína 1/metabolismo , Óxido de Magnésio/metabolismo , Reação de Maillard , Desenvolvimento Embrionário/fisiologia , Criopreservação , Blastocisto/metabolismo , Mitocôndrias , DNA Mitocondrial/metabolismoRESUMO
The present study investigated the effects of low ethanol exposure on bovine oocytes. Cumulus-oocyte complexes (COCs) were aspirated for the antral follicles of slaughterhouse-derived ovaries. These COCs were incubated in maturation medium containing 0, 0.1, and 0.2% ethanol for 21 h and subjected to fertilization and in vitro development, and then the rates of nuclear maturation, mitochondrial DNA copy number (Mt-cn) and protein (TOMM40), ATP content and lipid content in oocyte, fertilization, and blastulation were examined. Furthermore, COCs were incubated with 0 or 0.1% ethanol and then mitochondrial membrane potential (MMP) and the glucose consumption of COCs was determined. In addition, gene expression in oocytes was examined by RNA sequencing. Ethanol (0.1 and 0.2%) increased Mt-cn and Mt-protein levels whereas 0.2% ethanol increased the blastulation rate and ATP content in oocytes and decreased lipid content in oocytes. Ethanol (0.1%) increased MMP in oocytes and decreased glucose consumption of COCs. Eight stage embryos derived from 0.1% ethanol treated oocytes had higher levels of trimethyl-H3K9 compared with that of nontreated counterpart. RNA sequencing revealed that differentially expressed genes were associated with glycolysis/gluconeogenesis, carbon metabolism, sphingolipid metabolism, amino acid metabolism, and fatty acid degradation pathways. In conclusion, even 0.1% concentrations of ethanol during in vitro maturation considerably affects oocyte metabolism and histone configuration of embryos.
Assuntos
DNA Mitocondrial , Oócitos , Bovinos , Animais , Feminino , Desenvolvimento Embrionário , Etanol/farmacologia , Glucose/farmacologia , Lipídeos , Trifosfato de Adenosina , Técnicas de Maturação in Vitro de Oócitos/veterinária , Células do CúmuloRESUMO
The present study examined the effect of polysaccharides gels made of xanthan gum and locust bean gum (gel culture system) on oocyte maturation and explored the molecules causing the beneficial effect of the gel culture system. Oocytes and cumulus cells complexes were collected from slaughterhouse-derived ovaries and cultured on a plastic plate or gel. The gel culture system improved the rate of development to the blastocyst stage. The oocytes that matured on the gel contained high lipid contents and F-actin formation, and the resultant 8-cell stage embryos had low DNA methylation levels compared to their plate counterparts. RNA sequencing of the oocytes and embryos revealed the differentially expressed genes between the gel and plate culture systems, and upstream regulator analysis revealed estradiol and TGFB1 as top activated upstream molecules. The medium of the gel culture system contained higher concentrations of estradiol and TGFB1 than that of the plate cultures system. Supplementation of the maturation medium with either estradiol or TGFB1 resulted in high lipid content in oocytes. In addition, TGFB1 improved the developmental ability of the oocytes and increased F-actin content while reducing DNA methylation levels in the 8-cell stage embryos. In conclusion, the gel culture system is useful for embryo production, potentially through the upregulation of TGFB1.
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Actinas , Técnicas de Maturação in Vitro de Oócitos , Animais , Bovinos , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos , Polissacarídeos Bacterianos/farmacologia , Estradiol/farmacologia , Géis/farmacologia , Lipídeos/farmacologia , BlastocistoRESUMO
To investigate possible causes of reproductive failure, we conducted global endometrial gene expression analyses in fertile and subfertile cows. Ingenuity pathway analysis showed that RICTOR and SIRT3 are significant upstream regulators for highly expressed genes in fertile cows, and are predicted to be activated upstream regulators of normal mitochondrial respiration. Canonical pathway analysis revealed that these highly expressed genes are involved in the activation of mitochondrial oxidative phosphorylation. Therefore, in subfertile cows, the inactivation of RICTOR and SIRT3 may correlate with decreased capacity of mitochondrial respiration. Furthermore, the expression levels of most mitochondrial DNA genes and nuclear genes encoding mitochondrial proteins were higher in subfertile cows. The mitochondrial DNA copy number was significantly higher in the endometrium of subfertile cows, whereas the ATP content did not differ between fertile and subfertile cows. Quantitative reverse transcription-PCR analysis demonstrated that the expression of PGC1a, TFAM, MFN1, FIS1, and BCL2L13 were significantly lower in subfertile cows. In addition, transmission electron microscopy images showed mitochondrial swelling in the endometrial cells of the subfertile cow. These results suggest that poor-quality mitochondria accumulate in the endometrium owing to a reduced capacity for mitochondrial biogenesis, fusion, fission, and degradation in subfertile cows, and may contribute to infertility.
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Infertilidade , Sirtuína 3 , Feminino , Bovinos , Animais , Biogênese de Organelas , Sirtuína 3/metabolismo , Endométrio/metabolismo , Infertilidade/metabolismo , DNA Mitocondrial/genética , Fatores de Transcrição/metabolismoRESUMO
The developmental origins of health and disease (DOHaD) hypothesis is that future lifestyle diseases in offspring are associated with intrauterine origins in the mother; stress during pregnancy is a risk factor for these diseases in offspring. This study aimed to clarify association of maternal stress with placental dysfunction and offspring development in mice. We applied water stress for 24 h during late pregnancy to explore the metabolic response of offspring to a normal diet (ND) and high-fat diet (HFD). Placental functions were altered by maternal stress, reducing the birth weight of the offspring. In the later life of offspring fed with ND, maternal stress impaired systemic glucose tolerance and altered adipokine secretion in adipose tissue and/or liver. The female offspring of stress-induced dams were light in body weight with lower adipose tissue and smaller adipocytes in both the ND and HFD groups. Abnormal situations, such as dysregulation of plasma glucose levels and fatty liver despite and lower increases in body weight, were observed in the female offspring of stress-induced dams, especially in the HFD-treated group. These findings suggest that long-lasting abnormal conditions and responses to metabolic challenges in maternal stress-induced offspring are linked to placental dysregulation and fetal programming.
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Fígado Gorduroso , Efeitos Tardios da Exposição Pré-Natal , Animais , Camundongos , Gravidez , Feminino , Humanos , Obesidade/metabolismo , Placenta/metabolismo , Dieta Hiperlipídica/efeitos adversos , Tecido Adiposo/metabolismo , Fígado Gorduroso/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismoRESUMO
Mitochondrial DNA (mtDNA) copy number and telomere length (TL) in blastocysts derived from the same male mice at young (10-19-week-old) and aged (40-49-week-old) time points and mtDNA and TL in the hearts of offspring derived from young and aged male mice were examined. Paternal aging correlated with reduced mtDNA and TL in blastocysts. mtDNA and TL were significantly correlated, which was also observed in bovine blastocysts. Moreover, mtDNA in the heart of offspring was reduced in male mice with paternal aging. In conclusion, paternal aging affects embryonic mtDNA and TL, potentially impacting their offspring.
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DNA Mitocondrial , Telômero , Masculino , Animais , Bovinos , Camundongos , DNA Mitocondrial/genética , Telômero/genética , Mitocôndrias/genética , Envelhecimento/genética , BlastocistoRESUMO
Embryo-maternal reproductive tract interactions are pivotal for successful pregnancy. The present study predicted the molecules modulating embryo-uterine communication by comparing two sets of differentially expressed genes (DEGs): DEGs in uterine epithelial cells (UECs) collected from the uterus with and without blastocysts and DEGs between blastocysts developed in vivo and in vitro. Cows were subjected to super ovulation (SOV), followed by insemination or non-insemination at estrus (SOV + AI and SOV cows). Seven days after estrus, the uterus was flushed to collect UECs, and the presence of blastocysts in the uterus was confirmed. UECs were subjected to RNA-Sequencing (RNA-Seq) to identify DEGs. Publicly available RNA-Seq data of in vivo and in vitro developed bovine blastocysts were used to determine DEGs. Then, using ingenuity pathway analysis, activated- and inhibited-upstream regulators (USRs) for UECs in blastocysts were compared with those for blastocysts developed in vivo. RNA-Seq of UECs revealed that the DEGs were associated with immune response and cell adhesion pathways. The activated and inhibited USRs of UECs derived from SOV+ AI cows overlapped with the activated and inhibited USRs of blastocysts developed in vivo. Overlapping activated USRs include leukemia inhibitory factor, interleukin 6, fibroblast growth factor-2, transforming growth factor beta-1, and epidermal growth factor. In conclusion, the present study predicted the molecules that potentially mediate communication between the developing embryo and the uterus in vivo and prepare the uterus for pregnancy.
Assuntos
Fator 2 de Crescimento de Fibroblastos , Interleucina-6 , Animais , Blastocisto/metabolismo , Bovinos , Família de Proteínas EGF/metabolismo , Células Epiteliais , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Interleucina-6/metabolismo , Fator Inibidor de Leucemia/metabolismo , Gravidez , RNA/metabolismo , Fator de Crescimento Transformador beta/metabolismo , ÚteroRESUMO
This study identified microRNAs (miRNAs) in bovine oviductal fluids (OFs) and examined the effect of miR-17-5p in OFs on embryonic development to the blastocyst stage. Small RNA-seq of extracellular vesicles of OFs revealed 242 miRNAs. Additionally, analyzing expressions of randomly selected OF-miRNAs with RT-qPCR in the culture medium of oviductal epithelial cells indicated that the abundance of miRNAs in OFs increased during the luteal phase. miR-17-5p mimic-treated eight-cell-stage zona pellucida-free embryos showed improved embryonic development to the blastocyst stage. The effect of the miR-17-5p mimic was confirmed using a dual-luciferase assay and immunostaining. In addition, RNA-seq of the miR-17-5p mimic- or control-treated embryos revealed differentially expressed genes (DEGs), suggesting possible pathways that overlapped with the in silico-predicted pathways for miR-17-5p targeting genes. Furthermore, ingenuity pathway analysis of DEG predicted miR-17 to be a significant upstream regulator. Our results suggest that miR-17-5p in OFs regulates embryonic development in bovines.
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Vesículas Extracelulares , MicroRNAs , Animais , Bovinos , Desenvolvimento Embrionário/genética , Vesículas Extracelulares/metabolismo , Tubas Uterinas/metabolismo , Feminino , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Gravidez , RNA-SeqRESUMO
PROBLEM: Systemic inflammation induced by infection, which is associated with testicular inflammation, predisposes males to subfertility. Recently, the nucleotide-binding oligomerization domain, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome was identified as a key mediator of inflammation, and excessive activation of the NLRP3 inflammasome was shown to contribute to the pathogenesis of a wide variety of diseases. However, the mechanisms underlying infectious inflammation in the testis remain unclear. We investigated the effect of lipopolysaccharide (LPS)-induced systemic inflammation on the role of the NLRP3 inflammasome in murine testes. METHOD OF STUDY: We performed in vivo and in vitro studies using an LPS-induced model of NLRP3 inflammasome activation and testicular inflammation. RESULTS: Intraperitoneal administration of LPS significantly impaired sperm motility in the epididymis of wild type (WT) and NLRP3-knockout (KO) mice. LPS administration stimulated interleukin (IL)-1ß production and secretion in the testes of WT mice, and these adverse effects were improved in the testes of NLRP3-KO mice. LPS administration also stimulated neutrophil infiltration as well as its chemoattractant C-C motif chemokine ligand 2 (CCL2) in WT testes, which were suppressed in NLRP3-KO testes. In in vitro cell culture, treatment with LPS and NLRP3 inflammasome activation significantly induced IL-1ß and CCL2 secretion from WT but not NLRP3-KO testicular cells. CONCLUSIONS: Taken together, our results suggest that testicular cells have the potential to secrete IL-1ß and CCL2 in an NLRP3 inflammasome-dependent manner and that these cytokines from the testis may further exacerbate testicular function, resulting in subfertility during infectious diseases.
Assuntos
Infertilidade , Orquite , Animais , Humanos , Inflamassomos , Inflamação/induzido quimicamente , Interleucina-1beta , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Motilidade dos EspermatozoidesRESUMO
Repeat breeding, which is non-pregnancy following three or more breeding attempts, is a serious reproductive disorder in cattle. In the present study, metabolomic profiling was used to identify metabolites in the blood plasma of repeat breeder cows (RBCs) and non-RBCs. Metabolomic analysis showed that acetoacetate (AcAc), a ketone body, was detected in RBCs, but not in non-RBCs. In contrast, ß-hydroxybutyrate (BHB) was at similar levels in both RBCs and non-RBCs. We hypothesized that an imbalance of AcAc and BHB induces abnormal inflammatory conditions, especially the NLRP3 inflammasome, which regulates sterile inflammation to control interleukin (IL)-1ß secretion, and may be associated with repeat breeding in cattle. To investigate this hypothesis, blood samples were collected from both non-RBCs and RBCs on day 7 of the estrous cycle. The mRNA expression of IL1B in peripheral blood mononuclear cells (PBMCs) was observed to be higher in RBCs than in non-RBCs. To test the effects of AcAc and BHB on inflammatory responses, blood samples were collected from healthy cows and PBMCs were isolated. PBMCs were treated with AcAc and BHB to investigate the activation of the NLRP3 inflammasome (complex of NLRP3, ASC, and caspase-1) and IL-1ß secretion. AcAc treatment resulted in higher protein and/or mRNA expression of NLRP3 and IL-1ß in PBMCs. Moreover, AcAc increased the co-localization of NLRP3 and ASC and stimulated caspase-1 activation, indicating the formation of the platform of the NLRP3 inflammasome. Addition of specific NLRP3 inhibitor, MCC950, suppressed AcAc stimulation-induced IL-1ß secretion. Contrary to the effects of AcAc, BHB treatment suppressed the activation of NLRP3 inflammasome and IL-1ß secretion in response to AcAc and typical NLRP3 inflammasome triggers. These findings demonstrate that AcAc can potentially trigger NLRP3 inflammasome activation, resulting in IL-1ß secretion, and that these inflammatory responses are suppressed by BHB in bovine PBMCs. In addition, the imbalance between AcAc and BHB with higher levels of IL-1ß may be associated with repeat breeding in cattle.