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1.
Cell Tissue Res ; 393(1): 47-62, 2023 Jul.
Article in English | MEDLINE | ID: mdl-37227506

ABSTRACT

The seahorse is one of the most unique teleost fishes in its morphology. The body is surrounded by bony plates and spines, and the male fish possess a brooding organ, called the brood pouch, on their tail. The surfaces of the brood pouch and the spines are surrounded by characteristic so-called flame cone cells. Based on our histological observations, flame cone cells are present in the seahorse Hippocampus abdominalis, but not in the barbed pipefish Urocampus nanus or the seaweed pipefish Syngnathus schlegeli, both of which belong to the same family as the seahorse. In the flame cone cells, we observed expression of an "orphan gene" lacking homologs in other lineages. This gene, which we named the proline-glycine rich (pgrich) gene, codes for an amino acid sequence composed of repetitive units. In situ hybridization and immunohistochemical analyses detected pgrich-positive signals from the flame cone cells. Based on a survey of the genome sequences of 15 teleost species, the pgrich gene is only found from some species of Syngnathiformes (namely, the genera Syngnathus and Hippocampus). The amino acid sequence of the seahorse PGrich is somewhat similar to the sequence deduced from the antisense strand of elastin. Furthermore, there are many transposable elements around the pgrich gene. These results suggest that the pgrich gene may have originated from the elastin gene with the involvement of transposable elements and obtained its novel function in the flame cone cells during the evolution of the seahorse.


Subject(s)
Smegmamorpha , Animals , Male , Smegmamorpha/genetics , Smegmamorpha/anatomy & histology , Elastin , DNA Transposable Elements , Fishes/genetics , Epithelium
2.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Article in English | MEDLINE | ID: mdl-35217609

ABSTRACT

Insects comprise over half of the described species, and the acquisition of metamorphosis must have contributed to their diversity and prosperity. The order Odonata (dragonflies and damselflies) is among the most-ancestral insects with drastic morphological changes upon metamorphosis, in which understanding of the molecular mechanisms will provide insight into the evolution of incomplete and complete metamorphosis in insects. In order to identify metamorphosis-related genes in Odonata, we performed comprehensive RNA-sequencing of the blue-tailed damselfly Ischnura senegalensis at different developmental stages. Comparative RNA-sequencing analyses between nymphs and adults identified eight nymph-specific and seven adult-specific transcripts. RNA interference (RNAi) of these candidate genes demonstrated that three transcription factors, Krüppel homolog 1 (Kr-h1), broad, and E93 play important roles in metamorphosis of both I. senegalensis and a phylogenetically distant dragonfly, Pseudothemis zonataE93 is essential for adult morphogenesis, and RNAi of Kr-h1 induced precocious metamorphosis in epidermis via up-regulation of E93 Precocious metamorphosis was also induced by RNAi of the juvenile hormone receptor Methoprene-tolerant (Met), confirming that the regulation of metamorphosis by the MEKRE93 (Met-Kr-h1-E93) pathway is conserved across diverse insects including the basal insect lineage Odonata. Notably, RNAi of broad produced unique grayish pigmentation on the nymphal abdominal epidermis. Survey of downstream genes for Kr-h1, broad, and E93 uncovered that unlike other insects, broad regulates a substantial number of nymph-specific and adult-specific genes independently of Kr-h1 and E93 These findings highlight the importance of functional changes and rewiring of the transcription factors Kr-h1, broad, and E93 in the evolution of insect metamorphosis.


Subject(s)
Biological Evolution , Metamorphosis, Biological/genetics , Odonata/growth & development , Wings, Animal , Animals , Female , Gene Expression Profiling , Genes, Insect , Male , Odonata/genetics , RNA Interference
3.
J Reprod Dev ; 68(1): 30-37, 2022 Feb 18.
Article in English | MEDLINE | ID: mdl-34719558

ABSTRACT

Although hormonal induction of parturition in cattle results in the successful delivery of healthy calves, the risk of retained fetal membrane is significantly increased. In a previous study, a combination of the long-acting glucocorticoid, triamcinolone acetonide, with a high dose of betamethasone partially normalized the placentomal gene expression during parturition; however, the incidence of retained fetal membrane remained high. This study further explored placentomal dysfunction and aimed to elucidate the mechanism of retained fetal membrane in parturition-induced cows. In this study, transcriptome analysis revealed that enhanced glucocorticoid exposure normalized the expression of a substantial fraction of genes in the cotyledons. In contrast, a significant reduction in the multiple signaling pathway activities, including interferon signaling, was found in the caruncles during induced parturition. Real-time PCR showed that the expression of interferon-tau in the caruncles, but not interferon-alpha or interferon-gamma, was significantly lower in induced parturition than spontaneous parturition. Interferon-stimulated gene expression was also significantly decreased in the caruncles during induced parturition. These results indicate that interferon signaling could be important for immunological control in placentomes during parturition. Additionally, this suggests that interferon-tau might be a pivotal ligand for interferon receptors in the caruncles. This study revealed that peripheral blood leukocytes in prepartum cows transcribed interferon-tau. Macrophage infiltration in the placentome is known to participate in the detachment of the fetal membrane from the caruncle. Thus, this study raised the possibility that immune cells migrating into the caruncles at parturition may act as a source of ligands that activate interferon signaling.


Subject(s)
Cattle Diseases , Placenta, Retained , Animals , Cattle , Cattle Diseases/metabolism , Extraembryonic Membranes/metabolism , Female , Gene Expression Profiling , Parturition , Placenta/metabolism , Placenta, Retained/metabolism , Placenta, Retained/veterinary , Pregnancy
4.
Sci Rep ; 11(1): 22485, 2021 11 18.
Article in English | MEDLINE | ID: mdl-34795357

ABSTRACT

Asexual vertebrates are rare and at risk of extinction due to their restricted adaptability through the loss of genetic recombination. We explore the mechanisms behind the generation and maintenance of genetic diversity in triploid asexual (gynogenetic) Carassius auratus fish, which is widespread in East Asian fresh waters and exhibits one of the most extensive distribution among asexual vertebrates despite its dependence on host sperm. Our analyses of genetic composition using dozens of genetic markers and genome-wide transcriptome sequencing uncover admixed genetic composition of Japanese asexual triploid Carassius consisting of both the diverged Japanese and Eurasian alleles, suggesting the involvement of Eurasian lineages in its origin. However, coexisting sexual diploid relatives and asexual triploids in Japan show regional genetic similarity in both mitochondrial and nuclear markers. These results are attributed to a unique unidirectional gene flow from diploids to sympatric triploids, with the involvement of occasional sexual reproduction. Additionally, the asexual triploid shows a weaker population structure than the sexual diploid, and multiple triploid lineages coexist in most Japanese rivers. The generated diversity via repeated interploidy gene flow as well as an increased establishment of immigrants is assumed to offset the cost of asexual reproduction and might contribute to the successful broad distribution of this asexual vertebrate.


Subject(s)
Gene Flow , Goldfish/genetics , Goldfish/physiology , Alleles , Animals , Asia , Biological Evolution , Cell Nucleus/metabolism , DNA, Mitochondrial/genetics , Diploidy , Europe , Genetic Markers , Genetics, Population , Genotype , Geography , Heterozygote , Homozygote , Japan/epidemiology , Male , Mitochondria/metabolism , Models, Genetic , Polymorphism, Single Nucleotide , Principal Component Analysis , Rivers , Species Specificity , Spermatozoa/physiology , Transcriptome , Triploidy
5.
FASEB J ; 35(10): e21904, 2021 10.
Article in English | MEDLINE | ID: mdl-34569650

ABSTRACT

Blastocyst formation gives rise to the inner cell mass (ICM) and trophectoderm (TE) and is followed by the differentiation of the epiblast (Epi) and primitive endoderm (PrE) within the ICM. Although these two-round cell lineage differentiations underpin proper embryogenesis in every mammal, their spatiotemporal dynamics are quite diverse among species. Here, molecular details of the blastocyst stage in cattle were dissected using an optimized in vitro culture method. Blastocyst embryos were placed on agarose gel filled with nutrient-rich media to expose embryos to both gaseous and liquid phases. Embryos derived from this "on-gel" culture were transferred to surrogate mothers on day (D) 10 after fertilization and successfully implanted. Immunofluorescent studies using on-gel-cultured embryos revealed that the proportion of TE cells expressing the pluripotent ICM marker, OCT4, which was beyond 80% on D8, was rapidly reduced after D9 and reached 0% on D9.5. This first lineage segregation process was temporally parallel with the second one, identified by the spatial separation of Epi cells expressing SOX2 and PrE cells expressing SOX17. RNA-seq comparison of TE cells from D8 in vitro fertilized embryos and D14 in vivo embryos revealed that besides drastic reduction of pluripotency-related genes, TE cells highly expressed Wnt, FGF, and VEGF signaling pathways-related genes to facilitate the functional maturation required for feto-maternal interaction. Quantitative PCR analysis of TE cells derived from on-gel culture further confirmed time-dependent increments in the expression of key TE markers. Altogether, the present study provides platforms to understand species-specific strategies for mammalian preimplantation development.


Subject(s)
Antigens, Differentiation/biosynthesis , Blastocyst/metabolism , Cell Lineage , Embryonic Development , Gene Expression Regulation, Developmental , Animals , Cattle
6.
Nat Commun ; 10(1): 5766, 2019 12 18.
Article in English | MEDLINE | ID: mdl-31852900

ABSTRACT

Cognitive performance in people varies according to time-of-day, with memory retrieval declining in the late afternoon-early evening. However, functional roles of local brain circadian clocks in memory performance remains unclear. Here, we show that hippocampal clock controlled by the circadian-dependent transcription factor BMAL1 regulates time-of-day retrieval profile. Inducible transgenic dominant negative BMAL1 (dnBMAL1) expression in mouse forebrain or hippocampus disrupted retrieval of hippocampal memories at Zeitgeber Time 8-12, independently of retention delay, encoding time and Zeitgeber entrainment cue. This altered retrieval profile was associated with downregulation of hippocampal Dopamine-cAMP signaling in dnBMAL1 mice. These changes included decreases in Dopamine Receptors (D1-R and D5-R) and GluA1-S845 phosphorylation by PKA. Consistently, pharmacological activation of cAMP-signals or D1/5Rs rescued impaired retrieval in dnBMAL1 mice. Importantly, GluA1 S845A knock-in mice showed similar retrieval deficits with dnBMAL1 mice. Our findings suggest mechanisms underlying regulation of retrieval by hippocampal clock through D1/5R-cAMP-PKA-mediated GluA1 phosphorylation.


Subject(s)
Circadian Clocks/physiology , Hippocampus/metabolism , Mental Recall/physiology , Receptors, AMPA/metabolism , ARNTL Transcription Factors/genetics , ARNTL Transcription Factors/metabolism , Animals , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Dopamine/metabolism , Female , Gene Knock-In Techniques , Male , Maze Learning , Mice , Mice, Transgenic , Models, Animal , Phosphorylation/physiology
7.
Sci Rep ; 9(1): 4050, 2019 03 11.
Article in English | MEDLINE | ID: mdl-30858410

ABSTRACT

The 28,000-year-old remains of a woolly mammoth, named 'Yuka', were found in Siberian permafrost. Here we recovered the less-damaged nucleus-like structures from the remains and visualised their dynamics in living mouse oocytes after nuclear transfer. Proteomic analyses demonstrated the presence of nuclear components in the remains. Nucleus-like structures found in the tissue homogenate were histone- and lamin-positive by immunostaining. In the reconstructed oocytes, the mammoth nuclei showed the spindle assembly, histone incorporation and partial nuclear formation; however, the full activation of nuclei for cleavage was not confirmed. DNA damage levels, which varied among the nuclei, were comparable to those of frozen-thawed mouse sperm and were reduced in some reconstructed oocytes. Our work provides a platform to evaluate the biological activities of nuclei in extinct animal species.


Subject(s)
Cell Nucleus/metabolism , Fossils/diagnostic imaging , Mammoths/metabolism , Proteomics , Animals , Cell Nucleus/chemistry , Female , Male , Mammoths/genetics , Mice , Nuclear Transfer Techniques , Oocytes/metabolism
8.
Sci Rep ; 9(1): 5167, 2019 03 26.
Article in English | MEDLINE | ID: mdl-30914704

ABSTRACT

Although in vitro maturation (IVM) of oocytes is important for assisted reproduction, the rate of development of embryos from IVM oocytes is lower than from their in vivo counterparts. It has been shown that an artificial increase of intracellular cAMP before culture significantly improves oocyte developmental competence in cattle and mice. Here, we revealed that forskolin and 3-isobutyl-1-methylxanthine treatment of prophase-stage oocytes induced the expression of genes required for glycolysis, fatty acid degradation, and the mitochondrial electron transport system and improved mitochondrial functions and ATP levels in oocytes without involving nuclear maturation. We propose the existence of a comprehensive energy-supply system in oocytes under follicle-stimulating hormone stimulation as a potential explanation of how oocytes acquire developmental competence.


Subject(s)
Cell Differentiation , Cyclic AMP/metabolism , Mitochondria/metabolism , Oocytes/metabolism , 1-Methyl-3-isobutylxanthine/pharmacology , Animals , Blastocyst/cytology , Blastocyst/drug effects , Blastocyst/metabolism , Cattle , Cell Differentiation/drug effects , Cell Differentiation/genetics , Colforsin/pharmacology , Fatty Acids/metabolism , Female , Gene Expression Regulation/drug effects , Glucose Transport Proteins, Facilitative/metabolism , Glycolysis/drug effects , Granulosa Cells/drug effects , Granulosa Cells/metabolism , Meiosis/drug effects , Meiosis/genetics , Methyltransferases/metabolism , Mitochondria/drug effects , Oocytes/cytology , Oocytes/drug effects , Oxidation-Reduction , Transcriptome/drug effects , Transcriptome/genetics
9.
Elife ; 82019 01 15.
Article in English | MEDLINE | ID: mdl-30642432

ABSTRACT

Many animals change their body color for visual signaling and environmental adaptation. Some dragonflies show wax-based color change and ultraviolet (UV) reflection, but the biochemical properties underlying the phenomena are totally unknown. Here we investigated the UV-reflective abdominal wax of dragonflies, thereby identifying very long-chain methyl ketones and aldehydes as unique and major wax components. Little wax was detected on young adults, but dense wax secretion was found mainly on the dorsal abdomen of mature males of Orthetrum albistylum and O. melania, and pruinose wax secretion was identified on the ventral abdomen of mature females of O. albistylum and Sympetrum darwinianum. Comparative transcriptomics demonstrated drastic upregulation of the ELOVL17 gene, a member of the fatty acid elongase gene family, whose expression reflected the distribution of very long-chain methyl ketones. Synthetic 2-pentacosanone, the major component of dragonfly's wax, spontaneously formed light-scattering scale-like fine structures with strong UV reflection, suggesting its potential utility for biomimetics.


Subject(s)
Odonata/radiation effects , Pigmentation/radiation effects , Ultraviolet Rays , Waxes/chemistry , Abdomen/anatomy & histology , Animals , Color , Epidermis/radiation effects , Epidermis/ultrastructure , Female , Genes, Insect , Male , Odonata/anatomy & histology , Odonata/genetics , Odonata/ultrastructure , Phylogeny , Solubility , Transcriptome/genetics , Up-Regulation/genetics , Wettability
10.
Int J Mol Sci ; 19(8)2018 Aug 08.
Article in English | MEDLINE | ID: mdl-30096769

ABSTRACT

BACKGROUND: Three-dimensional (3D) culture changes cell characteristics and function, suggesting that 3D culture provides a more physiologically relevant environment for cells compared with 2D culture. We investigated the differences in cell functions depending on the culture model in human trophoblast cells (Sw.71). METHODS: Sw.71 cells were incubated in 2D monolayers or simple 3D spheroids. After incubation, cells were corrected to assess RNA-seq transcriptome or protein expression, and culture medium were corrected to detect cytokines. To clarify the role of actin cytoskeleton, spheroid Sw.71 cells were treated mycalolide B (inhibitor of actin polymerization) in a 3D culture. RESULTS: RNA-seq transcriptome analysis, results revealed that 3D-cultured cells had a different transcriptional profile compared with 2D-cultured cells, especially regarding inflammation-related molecules. Although interleukin-6 (IL-6) mRNA level was higher in 3D-culured cells, its secretion levels were higher in 2D-cultured cells. In addition, the levels of mRNA and protein expression of regnase-1, regulatory RNase of inflammatory cytokine, significantly increased in 3D culture, suggesting post-translational modification of IL-6 mRNA via regnase-1. Treatment with mycalolide B reduced cell-to-cell contact to build 3D formation and increased expression of actin cytoskeleton, resulting in increased IL-6 secretin. CONCLUSION: Cell dimensionality plays an essential role in governing the spatiotemporal cellular outcomes, including inflammatory cytokine production and its negative regulation associated with regnase-1.


Subject(s)
Cell Culture Techniques , Interleukin-6/genetics , Ribonucleases/genetics , Trophoblasts/cytology , Actin Cytoskeleton/metabolism , Base Sequence/genetics , Gene Expression Regulation/genetics , Humans , Microtubules/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Spheroids, Cellular/cytology , Spheroids, Cellular/metabolism , Transcriptome/genetics , Trophoblasts/metabolism
11.
J Anim Sci ; 96(6): 2226-2237, 2018 Jun 04.
Article in English | MEDLINE | ID: mdl-29762736

ABSTRACT

The length and density of rumen papillae starts to increase during weaning and growth of ruminants. This significant development increases the intraruminal surface area and the efficiency of VFA (acetate, propionate, butyrate, etc.) uptake. Thus, it is important to investigate the factors controlling the growth and development of rumen papillae during weaning. This study aimed to compare the transcriptomes of rumen papillae in suckling and weaned calves. Total RNA was extracted from the rumen papillae of 10 male Japanese Black calves (5 suckling calves, 5 wk old; 5 weaned calves, 15 wk old) and used in RNA-sequencing. Transcript abundance was estimated and differentially expressed genes were identified and these data were then used in Ingenuity Pathway Analysis (IPA) to predict the major canonical pathways and upstream regulators. Among the 871 differentially expressed genes screened by IPA, 466 genes were upregulated and 405 were downregulated in the weaned group. Canonical pathway analysis showed that "atherosclerosis" was the most significant pathway, and "tretinoin," a derivative of vitamin A, was predicted as the most active upstream regulator during weaning. Analyses also predicted IgG, lipopolysaccharides, and tumor-necrosis factor-α as regulators of the microbe-epithelium interaction that activates rumen-related immune responses. The functional category and the up-regulators found in this study provide a valuable resource for studying new candidate genes related to the proliferation and development of rumen papillae from suckling to weaning Japanese Black calves.


Subject(s)
Cattle/genetics , Rumen/growth & development , Transcriptome , Age Factors , Animals , Cattle/growth & development , Epithelium/growth & development , Gene Expression Profiling/veterinary , Male , Sequence Analysis, RNA/veterinary , Weaning
12.
Sci Rep ; 8(1): 2158, 2018 02 01.
Article in English | MEDLINE | ID: mdl-29391571

ABSTRACT

Maternal immune activation (MIA) contributes to behavioral abnormalities relevant to schizophrenia in adult offspring, although the molecular mechanisms underlying MIA-induced behavioral changes remain unclear. Here we demonstrated that dietary intake of glucoraphanin (GF), the precursor of a natural antioxidant sulforaphane, during juvenile and adolescent stages prevented cognitive deficits and loss of parvalbumin (PV) immunoreactivity in the medial prefrontal cortex (mPFC) of adult offspring after MIA. Gene set enrichment analysis by RNA sequencing showed that MIA caused abnormal expression of centrosome-related genes in the PFC and hippocampus of adult offspring, and that dietary intake of GF improved these abnormal gene expressions. Particularly, MIA increased the expression of suppressor of fermentation-induced loss of stress resistance protein 1 (Sfi1) mRNA in the PFC and hippocampus of adult offspring, and dietary intake of GF prevented the expression of Sfi1 mRNA in these regions. Interestingly, we found altered expression of SFI1 in the postmortem brains and SFI1 mRNA in hair follicle cells from patients with schizophrenia compared with controls. Overall, these data suggest that centrosome-related genes may play a role in the onset of psychosis in offspring after MIA. Therefore, dietary intake of GF-rich vegetables in high-risk psychosis subjects may prevent the transition to psychosis in young adulthood.


Subject(s)
Brain/immunology , Diet , Glucosinolates/administration & dosage , Imidoesters/administration & dosage , Prenatal Exposure Delayed Effects/immunology , Psychotic Disorders/prevention & control , Schizophrenia/complications , Adult , Animals , Brain/drug effects , Brain/pathology , Disease Models, Animal , Female , Humans , Male , Middle Aged , Oximes , Pregnancy , Prenatal Exposure Delayed Effects/diet therapy , Prenatal Exposure Delayed Effects/physiopathology , Psychotic Disorders/etiology , Psychotic Disorders/pathology , Sulfoxides
13.
J Interferon Cytokine Res ; 37(10): 456-466, 2017 10.
Article in English | MEDLINE | ID: mdl-29028431

ABSTRACT

Type I interferons (IFN), including IFN-beta (IFNB), activate multiple STAT signaling to drive various biological responses. Another type I IFN, IFN-tau (IFNT), secreted by ruminant embryonic trophoblast cells, has multiple functions with low cytotoxicity. Here, we examined the effects of IFNT on human trophoblast cell functions. First, we performed next-generation sequencing and demonstrated that IFNT-dependent changes in the human Sw.71 trophoblast cell line are partly mediated by proinflammatory as well as IFN signaling. Next, we validated candidate genes, and data confirmed that IFNT stimulated interleukin-6 (IL-6) and IL-8 mRNA expression and secretion. However, human IFNB did not affect IL-6 and IL-8 mRNA expression and secretion. IFNT-induced cytokine secretion was dependent on STAT3 signaling, but not STAT1 signaling. In addition, treatment with IFNT, IL-6, or IL-8 increased cell proliferation, and IFNT also stimulated cell migration in human trophoblast cells. Although IFNT did not affect superoxide dismutase (SOD) 1 mRNA expression, it clearly increased mitochondrial SOD2 mRNA expression, resulting in the acceleration of SOD activity. We demonstrated that in addition to IFN signaling, IFNT also regulated inflammation-related signaling as well as cell proliferation, migration, and redox signaling in human trophoblast cells.


Subject(s)
Interferon Type I/biosynthesis , Pregnancy Proteins/biosynthesis , Trophoblasts/cytology , Trophoblasts/metabolism , Cell Line , Cell Movement , Cell Proliferation , Cells, Cultured , Cytokines/metabolism , Female , Gene Expression Profiling , Humans , Inflammation Mediators/metabolism , Interferon Type I/metabolism , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Signal Transduction , Superoxide Dismutase/metabolism , Ubiquitins/metabolism
14.
Theriogenology ; 103: 98-103, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28779614

ABSTRACT

Oocytes and granulosa cells rely primarily on mitochondrial respiration and glycolysis for energy production, respectively. The present study examined the effect of mitochondrial inhibitors on the ATP contents of oocytes and granulosa cells. Cumulus cell-oocyte complexes (COCs) and granulosa cells (GCs) were collected from the antral follicles of porcine ovaries. Treatment of denuded oocytes with either carbonyl cyanide m-chlorophenyl hydrazine (CCCP), antimycin, or oligomycin significantly reduced ATP content to very low levels (CCCP, 0.12 pM; antimycin, 0.07 pM; and oligomycin, 0.25 pM; P < 0.05), whereas treatment with a glycolysis inhibitor (bromopyruvic acid, BA) had no effect. Conversely, the ATP content of granulosa cells was significantly reduced by treatment with the glycolysis inhibitor but was not affected by the mitochondrial inhibitors (ATP/10,000 cells; control, 1.78 pM and BA, 0.32 pM; P < 0.05). Reactive oxygen species (ROS) generation after CCCP treatment was greater in oocytes (1.6-fold) than that seen in granulosa cells (1.08-fold). Oocytes surrounded by granulosa cells had higher ATP levels than denuded oocytes. Treatment of COCs with CCCP reduced, but did not completely abolish, ATP content in oocytes (control, 3.15 pM and CCCP, 0.52 pM; P < 0.05), whereas treatment with CCCP plus a gap junction inhibitor, 18α-glycyrrhetinic acid, and CCCP decreased the ATP content to even lower levels (0.29 pM; P < 0.05). These results suggest that granulosa cells are dependent on glycolysis and provide energy to oocytes through gap junctions, even after treatment with CCCP.


Subject(s)
Granulosa Cells/drug effects , Mitochondria/drug effects , Oocytes/drug effects , Swine , Adenosine Triphosphate/metabolism , Animals , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/pharmacology , Antimycin A/administration & dosage , Antimycin A/analogs & derivatives , Antimycin A/pharmacology , Carbonyl Cyanide m-Chlorophenyl Hydrazone/administration & dosage , Carbonyl Cyanide m-Chlorophenyl Hydrazone/analogs & derivatives , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Cells, Cultured , Female , Granulosa Cells/physiology , Oligomycins/administration & dosage , Oligomycins/pharmacology , Oocytes/physiology , Proton Ionophores/administration & dosage , Proton Ionophores/pharmacology , Reactive Oxygen Species , Uncoupling Agents/administration & dosage , Uncoupling Agents/pharmacology
15.
Reprod Biol Endocrinol ; 15(1): 63, 2017 Aug 14.
Article in English | MEDLINE | ID: mdl-28806906

ABSTRACT

BACKGROUND: Endometrial cells secrete various cytokines and the dysfunction of endometrial cells may directly lead to infertility. Interferon tau (IFNT) secreted by trophoblast cells, a well-known pregnancy recognition signal in ruminants, acts on the uterus to prepare for pregnancy. Aging causes cellular and organ dysfunction, and advanced maternal age is associated with reduced fertility. However, few studies have investigated age-dependent changes in the uterus. METHODS: Using next generation sequencing and real-time PCR, we examined mRNA expression in bovine endometrial cells in vitro obtained from young (mean 45.2 months) and aged (mean 173.5 months) animals and the effects of IFNT depending on the age. RESULTS: We showed that inflammation-related (predicted molecules are IL1A, C1Qs, DDX58, NFKB, and CCL5) and interferon-signaling (predicted molecules are IRFs, IFITs, STATs, and IFNs) pathways were activated in endometrial cells obtained from aged compared to young cows. Also, the activation of "DNA damage checkpoint regulation" and the inhibition of "mitotic mechanisms" in endometrial cells obtained from aged cows were evident. Moreover, we showed lower cell viability levels in endometrial cells obtained from aged compared to young cows. Although treatment with IFNT upregulated various types of interferon stimulated genes both in endometrial cells obtained from young and aged cows, the rate of increase by IFNT stimulus was obviously lower in endometrial cells obtained from aged compared to young cows. CONCLUSIONS: Endometrial cells obtained from aged cows exhibited higher levels of inflammatory- and IFN-signaling, and dysfunction of cell division compared with young cows. In addition, a high basal level of IFN-related genes in endometrial cells of aged cows is suggested a concept of "inflammaging".


Subject(s)
Endometrium/metabolism , RNA, Messenger/metabolism , Age Factors , Animals , Cattle , Cell Division , Cells, Cultured , Female , Gene Expression Profiling , Interferon Type I/metabolism , Maternal Age , Pregnancy Proteins/metabolism , Signal Transduction
16.
J Reprod Dev ; 63(5): 455-461, 2017 Oct 18.
Article in English | MEDLINE | ID: mdl-28603221

ABSTRACT

Resveratrol is a potent activator of NAD-dependent deacetyltransferase sirtuin-1 (SIRT1) and affects lipid metabolism and ATP generation in somatic cells. In the present study, the effects of supplementing culture medium with resveratrol on lipid metabolism, ATP generation, and cryosensitivity of bovine in vitro produced embryos were investigated. Bovine early cleaved-stage embryos were cultured in medium containing 0 or 0.5 µM resveratrol for 1 or 5 days. Resveratrol treatment for both 1 day and 5 days increased the expression levels of SIRT1 and phosphorylated AMP-activated protein kinase (pAMPK) in the embryos. Furthermore, resveratrol treatment was effective to increase ATP generation and reduce lipid content of the embryos. The effects of resveratrol treatment were diminished by the SIRT1 inhibitor "EX527", and the reduced lipid content was reversed by treatment with etomoxir (a potent inhibitor of beta-oxidation). Blastocysts developed after resveratrol treatment showed low levels reactive oxygen species and increased cryotolerance. These results demonstrate that resveratrol improves in vitro development of bovine embryos, while reducing cytoplasmic lipid content through activation of beta-oxidation, thereby effective for production of bovine blastocysts with enhanced cryotolerance.


Subject(s)
Cryopreservation , Cytoplasm/drug effects , Embryo, Mammalian/drug effects , Lipid Metabolism/drug effects , Mitochondria/drug effects , Stilbenes/pharmacology , Animals , Blastocyst/cytology , Blastocyst/drug effects , Blastocyst/metabolism , Cattle , Cell Survival/drug effects , Cells, Cultured , Culture Media , Cytoplasm/chemistry , Cytoplasm/metabolism , Embryo Culture Techniques/veterinary , Embryo, Mammalian/physiology , Embryonic Development/drug effects , Female , Fertilization in Vitro/veterinary , Lipids/analysis , Male , Mitochondria/physiology , Resveratrol
17.
Sci Rep ; 7(1): 3800, 2017 06 19.
Article in English | MEDLINE | ID: mdl-28630431

ABSTRACT

Methionine adenosyltransferase (MAT) is involved in folate-mediated one-carbon metabolism, which is essential for preimplantation embryos in terms of both short-term periconceptional development and long-term phenotypic programming beyond the periconceptional period. Here, our immunofluorescence analysis of bovine oocytes and preimplantation embryos revealed the consistent expression of MAT2A (the catalytic subunit of the ubiquitously expressed-type of MAT isozyme) during this period. Addition of the MAT2A inhibitor FIDAS to the culture media of bovine preimplantation embryos reduced their blastocyst development, revealing the particular importance of MAT2A in successful blastocyst development. Exploration of MAT2A-associated genomic regions in bovine blastocysts using chromatin immunoprecipitation and sequencing (ChIP-seq) identified candidate MAT2A-associated genes implicated not only in short-term periconceptional embryo development, but also in long-term phenotypic programming during this period in terms of growth, metabolism, and immune functions. These results suggest the critical involvement of MAT2A in the periconceptional period in life-long programming of health and disease as well as successful preimplantation development.


Subject(s)
Blastocyst/enzymology , Embryonic Development/physiology , Gene Expression Regulation, Developmental/physiology , Gene Expression Regulation, Enzymologic/physiology , Methionine Adenosyltransferase , Animals , Blastocyst/cytology , Cattle , Embryonic Development/drug effects , Enzyme Inhibitors/pharmacology , Female , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Methionine Adenosyltransferase/antagonists & inhibitors , Methionine Adenosyltransferase/biosynthesis , Methionine Adenosyltransferase/genetics
18.
Biochem Biophys Res Commun ; 486(3): 796-803, 2017 05 06.
Article in English | MEDLINE | ID: mdl-28347822

ABSTRACT

Jasmonic acid (JA) plays central roles in various events in plants, especially defence against pathogens and insects. The basic helix-loop-helix (bHLH) transcription factor MYC2 has attracted attention as a master regulator of JA signalling in dicotyledonous plants. However, how MYC2 functions in monocotyledonous plants, including agriculturally important crops such as cultivated rice, has been poorly understood. To elucidate the comprehensive effects of rice MYC2 (OsMYC2) on the JA-inducible transcriptional modifications, we performed RNA-sequencing by using OsMYC2-knockdown plants (osmyc2RNAi). In osmyc2RNAi, JA-inducible expression of many defence-related genes, for example chitinases and proteinase inhibitors, was compromised. Decrease in JA-dependent activation of the biosynthetic pathways of specialised metabolites, especially defence compounds, was also evident in the osmyc2RNAi line. Furthermore, a substantial change was noted in the expression of distinct types of transcription factors, such as MYB-type factors, likely depicting the importance of OsMYC2 in not only defence responses but also other morphogenetic events. Our findings provide fundamental information to understand the overall functions of MYC2 in JA signalling in monocotyledonous plants, which might yield agricultural benefits.


Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Cyclopentanes/metabolism , Gene Expression Regulation, Plant , Oryza/genetics , Oxylipins/metabolism , Plant Proteins/genetics , Trans-Activators/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/antagonists & inhibitors , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Cyclopentanes/pharmacology , Disease Resistance/genetics , Gene Ontology , Gene Silencing , High-Throughput Nucleotide Sequencing , Metabolic Networks and Pathways/drug effects , Molecular Sequence Annotation , Oryza/drug effects , Oryza/metabolism , Oxylipins/pharmacology , Plant Growth Regulators/pharmacology , Plant Proteins/antagonists & inhibitors , Plant Proteins/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Trans-Activators/antagonists & inhibitors , Trans-Activators/metabolism , Transcription, Genetic
19.
Mol Reprod Dev ; 84(1): 44-54, 2017 01.
Article in English | MEDLINE | ID: mdl-27864905

ABSTRACT

A major difference between in vivo and in vitro follicle culture is the stiffness of the substrate in which the follicles grow. In this study, we examined the effect of polyacrylamide gel (PAG), as a culture substrate, on the development of porcine oocytes derived from early antral follicles. Oocyte-granulosa cell complexes (OGCs) were collected from the early antral follicles of gilts, and incubated individually for 14 days in a 96-well culture plate without or with PAG. We then evaluated the number of granulosa cells present in OGCs as well as the abundance of ATP, lipid, and acetylated lysine in oocytes. OGCs cultured on PAG showed significantly greater antrum formation and granulosa cell proliferation than controls cultured on standard plastic. Oocytes grown on PAG also possessed significantly larger diameter, ATP and lipid content, and lysine acetylation, as well as competence to develop to the blastocyst stage. Transcriptome analysis of the granulosa cells revealed that genes involved in follicular development and mechanosensing are up-regulated under PAG culturing conditions. Thus, in vitro culturing OGCs on PAG profoundly induced granulosa cell proliferation, resulting in improved developmental competence of the oocytes. Mol. Reprod. Dev. 84: 44-54, 2017. © 2016 Wiley Periodicals, Inc.


Subject(s)
Acrylic Resins/chemistry , Cell Culture Techniques/methods , Granulosa Cells/metabolism , Oocytes/metabolism , Transcriptome , Animals , Cells, Cultured , Female , Granulosa Cells/cytology , Oocytes/cytology , Swine
20.
Proc Natl Acad Sci U S A ; 113(32): 9021-6, 2016 08 09.
Article in English | MEDLINE | ID: mdl-27457928

ABSTRACT

Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogen-receptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells.


Subject(s)
Oogenesis/physiology , Animals , Cryopreservation , Female , Male , Meiosis , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Oocytes/physiology , Ovarian Follicle/physiology
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