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Long chain fatty acid activation, entry into mitochondria and metabolism via fatty acid oxidation. U Entry of long chain fatty acids into mitochondria requires activation by acyl-CoA synthetase enzymes which catalyze the transfer of CoA from CoA-SH to form fatty acyl-CoA. V Activated fatty acids enter mitochondria via enzymatic transfer of CoA for carnitine which is catalyzed by carnitine palmitoyl transferase I (CPTI). Fatty acyl-carnitine enters the mitochondrial matrix via carnitine acylcarnitine translocase where carnitine palmitoyl transferase II (CPTII) replaces carnitine with CoA. This is known as the carnitine shuttle. W Fatty acyl-CoA then enters the fatty acid oxidation spiral which has 4 steps catalyzed by 1) fatty acyl CoA dehydrogenase, 2) enoyl CoA hydratase, 3) hydroxyacyl CoA dehydrogenase and 4) acetyl-CoA transferase (also known as ketoacyl-CoA thiolase) and yields an acetyl-CoA molecule for each cycle. X Acetyl-CoA is able to enter the tricarboxylic acid (TCA) cycle which with fatty acid
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Fatty acid oxidation is an important energy source for the oocyte; however, little is known about how this metabolic pathway is regulated in cumulus-oocyte complexes. Analysis of genes involved in fatty acid oxidation showed that many are regulated by the luteinizing hormone surge during in vivo maturation, including acyl-CoA synthetases, carnitine...
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... Acsl5 were significantly induced during oocyte maturation in vivo while Acsl6 and Acsm3 were significantly down-regulated (Fig. 2A–F). The expression of Acsbg1 , Acsl3 , Acsm2 , Acsm4 and Acsm5 were not regulated during in vivo maturation (data not shown). Expression of carnitine transporters Cpt1a , Cpt1b , Cpt1c and Cpt2 which transport activated long chain fatty acids into the mitochondria revealed that Cpt1a was significantly induced (Fig. 2G) while the other genes remained unaltered (data not shown). Analysis of acyl-CoA dehydrogenase isoforms, which catalyze the first step in the FAO spiral, revealed that Acad10 and Acad11 were significantly altered post hCG demonstrating an initial down regulation at 6 h post hCG while at 16 h, post ovulation, expression was not significantly different from immature 0 h COCs (Fig. 2H and I). Acadl , Acadm and Acadsb were significantly induced in the COC during maturation in vivo (Fig. 2 J–L). The fourth and final step in the FAO spiral is performed by acetyl-CoA transferase and encoded by Acaa2 . Acaa2 expression was significantly up-regulated during oocyte maturation in vivo compared to immature 0 h COCs (Fig. 2M). These results show that a number of FAO genes are dynamically regulated in COCs in vivo in response to ovulatory hCG and that even distinct isoforms of similar enzymes exhibit differential expression patterns (see Fig. 1 for summary of changes). We next assessed whether COCs matured in vitro had altered expression of these dynamically regulated fatty acid oxidation genes. The time point of 10 h maturation was chosen for comparison since the majority of genes were significantly different at this time point in vivo compared to levels at 0 h (Fig. 2). There was no difference in cell number between COCs from the two maturation conditions (in vivo: 3040 6 205.5; IVM: 2457 6 377.3 cells/COC). Analysis of COCs matured for 10 h in vitro showed that Acyl-CoA synthetase expression was significantly altered in IVM COCs compared to in vivo matured COCs with Acsbg1 , Acsbg2 , Acsl1 , Acsl4 and Acsm3 expression significantly decreased in IVM COCs at 10 h while Acsl5 , Acsl6 and Acsm4 were significantly higher compared to in vivo matured COCs (Fig. 3 A–H). Acsl3 , Acsm2 , and Acsm3 were not different in COCs matured in vivo for 10 h compared to non-matured COCs (Fig. 2 and data not shown) nor compared to COCs matured in vitro (data not shown). Expression of the isoform Cpt1b was significantly reduced in IVM COCs (Fig. 3I) while Cpt1a , Cpt1c and Cpt2 were unaltered (data not shown). Genes encoding isoforms for acyl-CoA dehydrogenases which perform the first step in the FAO spiral showed disparate expression patterns with Acad10 , Acad11 and Acadvl significantly higher in IVM COCs while Acadm and Acadsb were significantly reduced compared to in vivo matured COCs (Fig. 3 J–N). Acadl , Acad9 and Acads expression levels were not different between maturation treatment groups (data not shown). The expression of the acetyl-CoA transferase Acaa2 , which catalyzes the final step in the FAO spiral, showed a significant decrease following IVM (Fig. 3O). We next compared functional rates of FAO in COCs that were matured in vitro for 10 h versus COCs isolated from follicles at 10 h post-hCG (i.e. matured in vivo). There was a significant 2.8- fold reduction in the level of FAO occurring in COCs matured in vitro (IVM) compared to those matured in vivo (Fig. 4A). Thus, IVM conditions result in COCs performing significantly less FAO than in vivo matured COCs at this timepoint. Because of the dramatically reduced level of FAO in IVM COCs, we next determined whether agonists of PPARs, specifically bezafibrate and rosiglitazone, would increase FAO in COCs during in vitro maturation. Real-time RT-PCR was used to confirm the presence of Ppar a , Pparg and Ppard mRNA transcripts in COCs prior to experiments examining the effects of the PPAR agonists (data not shown). Treatment of COCs with bezafibrate using doses ranging from 50–800 m M had no significant effect on FAO rate during the maturation period (Fig. 4B). At 100 m M bezafibrate did result in a 1.4-fold increase in FAO compared to control, however this dose also failed to have any significant effect over control conditions in subsequent assays investigating oocyte quality (data not shown). Conversely, treatment of COCs with rosiglitazone during IVM significantly reduced FAO in COCs in a dose dependant manner (Fig. 4C). Interestingly, despite rosiglitazone treatment (20 m M) significantly inhibiting FAO in COCs during IVM this dose caused a significant increase in the expression of genes involved in fatty acid activation ( Acsl1 ), carnitine mediated transport ( Cpt1b and Cpt2 ) and the FAO spiral ( Acaa2 ) compared to untreated controls (Fig. 5). In contrast, expression of Cpt1c was significantly decreased in the presence of rosiglitazone (Fig. 5C). The remaining 19 genes measured during in vivo maturation described above (or see Fig. 1) were also assessed but did not show significant modulation by rosiglitazone. Rosiglitazone treatment (20 m M) also significantly increased the degree of cumulus expansion compared to control (cumulus expansion index: control: 3.42 6 0.05 vs. rosiglitazone: 3.58 6 0.05, P , 0.05, Mann Whitney t test due to non-normal distribution of data, n = 7 experimental replicates, representative of $ 155 COCs per treatment). These results show that rosiglitazone significantly inhibits FAO in the COC during IVM despite upregulating a subset of genes involved in the FAO metabolic pathway. Oocyte mitochondrial membrane potential analyzed by JC-1 staining was altered by rosiglitazone (20 m M) during in vitro maturation of COCs (Fig. 6 A–F). Quantification of fluorescence confirmed that rosiglitazone treatment of COCs resulted in oocytes with a higher red:green fluorescence ratio indicating increased mitochondrial activity and that this was due to significantly higher levels of both red and green fluorescence(Fig. 6 G–I). Further, the distribution of green fluorescence appeared altered in rosiglitazone treated COCs with more of these mitochondria localised in the center of the oocyte (Fig. 6E) compared to a more homogeneous distribution observed in the oocytes matured in control conditions (Fig. 6B). COCs matured in vitro in the presence of rosiglitazone (20 m M) did not differ significantly in their ability to be fertilized and develop into a 2-cell embryo compared to COCs matured under control conditions (Fig. 7A). However, rosiglitazone treated COCs resulted in significantly fewer morula embryos on day 4 of embryo culture (Fig. 7A) and significantly fewer hatching blastocyst embryos on day 5 compared to control (Fig. 7B). Together these data show that despite rosiglitazone treatment of COCs increasing oocyte mitochondrial membrane potential, indicative of mitochondrial hyperpolarization, this was associated with decreased oocyte developmental competence with fewer fertilized oocytes capable of developing to the blastocyst stage of preimplantation development compared to oocytes from COCs matured in control conditions. Little is known about the regulation of FAO in the COC during maturation and how it impacts subsequent embryo development. In the current study we have shown that genes involved in FAO are hormonally regulated in the COC during maturation in vivo and are dysregulated during in vitro maturation. Since mature oocytes are transcriptionally inactive and oocyte mRNA would represent a small fraction of the mRNA in the COC, these dynamic changes in expression levels are attributed to changes in cumulus cells. This supports previous reports demonstrating that b -oxidation by the oocyte represents a small fraction of that occurring in the whole COC [16,17]. Further, using a functional assay we show that FAO is decreased in COCs matured in vitro compared to those that mature in vivo within the follicular environment. Whether decreased FAO persists throughout maturation in vitro remains to be determined, as does whether this reduction is directly due to the identified alterations in gene expression or deficiencies in other factors, such as growth factors and specific metabolites that are lacking in in vitro systems. Lastly, we show that treatment with the PPAR c agonist, rosiglitazone, significantly inhibits FAO and results in poor developmental competence. The significant upregulation of acyl-CoA synthetases Acsbg2 , Acsl1 , Acsl4 , and Acsl5 in the COC during maturation in vivo indicate that the COC is preferentially utilizing fatty acids with chain lengths of C10-C20 [42–45]. This correlates well with the predominant forms of fatty acids present in human and bovine follicular fluid; palmitic, oleic, linoleic and arachidonic acid [46– 49] for which Acsl1, Acsl5 and Acsl4 have high affinity [43–45]. Significant 5-fold induction of Cpt1a during maturation in vivo indicates that there is increased capacity for carnitine-mediated transport of fatty acids into mitochondria which is required for the oxidation of long chain fatty acids. We have previously shown significant upregulation of the isoform Cpt1b during COC maturation in vivo [17] here we again saw a 2-fold increase in Cpt1b at 10 h post-hCG though this was not statistically significant (data not shown). However, the Cpt isoforms, Cpt1a, b and c do not differ in their specificity for different activated fatty acids only in their tissue localization. Thus both our previous and current study illustrate increased Cpt1 activity indicating increased capacity for carnitine-mediated transport of activated long chain fatty acids into mitochondria during maturation in vivo. Acyl-CoA dehydrogenases catalyze the first step in FAO and those encoded by Acadl , Acadm and Acadsb have substrate optima of 16, 8, and 4 carbon chains, respectively [50,51]. Expression of these isoforms indicate that the COC is capable of metabolizing fatty acids of 4– 16 carbons long ...
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... cultured at 37 C in an atmosphere of 6% CO 2 and 94% air. Fertilization rate was scored 24 hours post-insemination (day 2) based on the division of the oocyte into a two-cell embryo. On day three of embryo culture (48 h post-insemination), the embryos were moved to a new 20 m l culture drop of ‘‘vitro cleave’’ medium (A905969, Cook Medical). Embryo development was assessed on day 3 (48 h post-insemination), 4 (78 h post-insemination) and day 5 (96 h post-insemination) of embryo culture for development to the 4–8 cell, $ morula and blastocyst stages of development, respectively. Results are presented as the mean 6 SEM. Statistical analyses were performed as indicated in figure legends using GraphPad Prism Version 5.1 for Windows (GraphPad Software, Inc.). All data were checked for normality and transformed where necessary as indicated in the figure legends. One-way ANOVA, two-way ANOVA, and t tests were used as described in the figure legends and statistical significance was considered at a P value of , 0.05. Proportional data (embryo development) were arcsine transformed prior to statistical analysis. We first determined whether genes involved in the fatty acid oxidation (FAO) metabolic pathway (Fig. 1) are regulated in the COC following an ovulatory dose of hCG to induce oocyte maturation in vivo. We analysed the expression of several isoforms of acyl-CoA synthetases, which activate long chain fatty acids by catalyzing their conversion to acyl CoA derivatives allowing transport into mitochondria, and found that Acsbg2 , Acsl1 , Acsl4 and Acsl5 were significantly induced during oocyte maturation in vivo while Acsl6 and Acsm3 were significantly down-regulated (Fig. 2A–F). The expression of Acsbg1 , Acsl3 , Acsm2 , Acsm4 and Acsm5 were not regulated during in vivo maturation (data not shown). Expression of carnitine transporters Cpt1a , Cpt1b , Cpt1c and Cpt2 which transport activated long chain fatty acids into the mitochondria revealed that Cpt1a was significantly induced (Fig. 2G) while the other genes remained unaltered (data not shown). Analysis of acyl-CoA dehydrogenase isoforms, which catalyze the first step in the FAO spiral, revealed that Acad10 and Acad11 were significantly altered post hCG demonstrating an initial down regulation at 6 h post hCG while at 16 h, post ovulation, expression was not significantly different from immature 0 h COCs (Fig. 2H and I). Acadl , Acadm and Acadsb were significantly induced in the COC during maturation in vivo (Fig. 2 J–L). The fourth and final step in the FAO spiral is performed by acetyl-CoA transferase and encoded by Acaa2 . Acaa2 expression was significantly up-regulated during oocyte maturation in vivo compared to immature 0 h COCs (Fig. 2M). These results show that a number of FAO genes are dynamically regulated in COCs in vivo in response to ovulatory hCG and that even distinct isoforms of similar enzymes exhibit differential expression patterns (see Fig. 1 for summary of changes). We next assessed whether COCs matured in vitro had altered expression of these dynamically regulated fatty acid oxidation genes. The time point of 10 h maturation was chosen for comparison since the majority of genes were significantly different at this time point in vivo compared to levels at 0 h (Fig. 2). There was no difference in cell number between COCs from the two maturation conditions (in vivo: 3040 6 205.5; IVM: 2457 6 377.3 cells/COC). Analysis of COCs matured for 10 h in vitro showed that Acyl-CoA synthetase expression was significantly altered in IVM COCs compared to in vivo matured COCs with Acsbg1 , Acsbg2 , Acsl1 , Acsl4 and Acsm3 expression significantly decreased in IVM COCs at 10 h while Acsl5 , Acsl6 and Acsm4 were significantly higher compared to in vivo matured COCs (Fig. 3 A–H). Acsl3 , Acsm2 , and Acsm3 were not different in COCs matured in vivo for 10 h compared to non-matured COCs (Fig. 2 and data not shown) nor compared to COCs matured in vitro (data not shown). Expression of the isoform Cpt1b was significantly reduced in IVM COCs (Fig. 3I) while Cpt1a , Cpt1c and Cpt2 were unaltered (data not shown). Genes encoding isoforms for acyl-CoA dehydrogenases which perform the first step in the FAO spiral showed disparate expression patterns with Acad10 , Acad11 and Acadvl significantly higher in IVM COCs while Acadm and Acadsb were significantly reduced compared to in vivo matured COCs (Fig. 3 J–N). Acadl , Acad9 and Acads expression levels were not different between maturation treatment groups (data not shown). The expression of the acetyl-CoA transferase Acaa2 , which catalyzes the final step in the FAO spiral, showed a significant decrease following IVM (Fig. 3O). We next compared functional rates of FAO in COCs that were matured in vitro for 10 h versus COCs isolated from follicles at 10 h post-hCG (i.e. matured in vivo). There was a significant 2.8- fold reduction in the level of FAO occurring in COCs matured in vitro (IVM) compared to those matured in vivo (Fig. 4A). Thus, IVM conditions result in COCs performing significantly less FAO than in vivo matured COCs at this timepoint. Because of the dramatically reduced level of FAO in IVM COCs, we next determined whether agonists of PPARs, specifically bezafibrate and rosiglitazone, would increase FAO in COCs during in vitro maturation. Real-time RT-PCR was used to confirm the presence of Ppar a , Pparg and Ppard mRNA transcripts in COCs prior to experiments examining the effects of the PPAR agonists (data not shown). Treatment of COCs with bezafibrate using doses ranging from 50–800 m M had no significant effect on FAO rate during the maturation period (Fig. 4B). At 100 m M bezafibrate did result in a 1.4-fold increase in FAO compared to control, however this dose also failed to have any significant effect over control conditions in subsequent assays investigating oocyte quality (data not shown). Conversely, treatment of COCs with rosiglitazone during IVM significantly reduced FAO in COCs in a dose dependant manner (Fig. 4C). Interestingly, despite rosiglitazone treatment (20 m M) significantly inhibiting FAO in COCs during IVM this dose caused a significant increase in the expression of genes involved in fatty acid activation ( Acsl1 ), carnitine mediated transport ( Cpt1b and Cpt2 ) and the FAO spiral ( Acaa2 ) compared to untreated controls (Fig. 5). In contrast, expression of Cpt1c was significantly decreased in the presence of rosiglitazone (Fig. 5C). The remaining 19 genes measured during in vivo maturation described above (or see Fig. 1) were also assessed but did not show significant modulation by rosiglitazone. Rosiglitazone treatment (20 m M) also significantly increased the degree of cumulus expansion compared to control (cumulus expansion index: control: 3.42 6 0.05 vs. rosiglitazone: 3.58 6 0.05, P , 0.05, Mann Whitney t test due to non-normal distribution of data, n = 7 experimental replicates, representative of $ 155 COCs per treatment). These results show that rosiglitazone significantly inhibits FAO in the COC during IVM despite upregulating a subset of genes involved in the FAO metabolic pathway. Oocyte mitochondrial membrane potential analyzed by JC-1 staining was altered by rosiglitazone (20 m M) during in vitro maturation of COCs (Fig. 6 A–F). Quantification of fluorescence confirmed that rosiglitazone treatment of COCs resulted in oocytes with a higher red:green fluorescence ratio indicating increased mitochondrial activity and that this was due to significantly higher levels of both red and green fluorescence(Fig. 6 G–I). Further, the distribution of green fluorescence appeared altered in rosiglitazone treated COCs with more of these mitochondria localised in the center of the oocyte (Fig. 6E) compared to a more homogeneous distribution observed in the oocytes matured in control conditions (Fig. 6B). COCs matured in vitro in the presence of rosiglitazone (20 m M) did not ...
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... cultured at 37 C in an atmosphere of 6% CO 2 and 94% air. Fertilization rate was scored 24 hours post-insemination (day 2) based on the division of the oocyte into a two-cell embryo. On day three of embryo culture (48 h post-insemination), the embryos were moved to a new 20 m l culture drop of ‘‘vitro cleave’’ medium (A905969, Cook Medical). Embryo development was assessed on day 3 (48 h post-insemination), 4 (78 h post-insemination) and day 5 (96 h post-insemination) of embryo culture for development to the 4–8 cell, $ morula and blastocyst stages of development, respectively. Results are presented as the mean 6 SEM. Statistical analyses were performed as indicated in figure legends using GraphPad Prism Version 5.1 for Windows (GraphPad Software, Inc.). All data were checked for normality and transformed where necessary as indicated in the figure legends. One-way ANOVA, two-way ANOVA, and t tests were used as described in the figure legends and statistical significance was considered at a P value of , 0.05. Proportional data (embryo development) were arcsine transformed prior to statistical analysis. We first determined whether genes involved in the fatty acid oxidation (FAO) metabolic pathway (Fig. 1) are regulated in the COC following an ovulatory dose of hCG to induce oocyte maturation in vivo. We analysed the expression of several isoforms of acyl-CoA synthetases, which activate long chain fatty acids by catalyzing their conversion to acyl CoA derivatives allowing transport into mitochondria, and found that Acsbg2 , Acsl1 , Acsl4 and Acsl5 were significantly induced during oocyte maturation in vivo while Acsl6 and Acsm3 were significantly down-regulated (Fig. 2A–F). The expression of Acsbg1 , Acsl3 , Acsm2 , Acsm4 and Acsm5 were not regulated during in vivo maturation (data not shown). Expression of carnitine transporters Cpt1a , Cpt1b , Cpt1c and Cpt2 which transport activated long chain fatty acids into the mitochondria revealed that Cpt1a was significantly induced (Fig. 2G) while the other genes remained unaltered (data not shown). Analysis of acyl-CoA dehydrogenase isoforms, which catalyze the first step in the FAO spiral, revealed that Acad10 and Acad11 were significantly altered post hCG demonstrating an initial down regulation at 6 h post hCG while at 16 h, post ovulation, expression was not significantly different from immature 0 h COCs (Fig. 2H and I). Acadl , Acadm and Acadsb were significantly induced in the COC during maturation in vivo (Fig. 2 J–L). The fourth and final step in the FAO spiral is performed by acetyl-CoA transferase and encoded by Acaa2 . Acaa2 expression was significantly up-regulated during oocyte maturation in vivo compared to immature 0 h COCs (Fig. 2M). These results show that a number of FAO genes are dynamically regulated in COCs in vivo in response to ovulatory hCG and that even distinct isoforms of similar enzymes exhibit differential expression patterns (see Fig. 1 for summary of changes). We next assessed whether COCs matured in vitro had altered expression of these dynamically regulated fatty acid oxidation genes. The time point of 10 h maturation was chosen for comparison since the majority of genes were significantly different at this time point in vivo compared to levels at 0 h (Fig. 2). There was no difference in cell number between COCs from the two maturation conditions (in vivo: 3040 6 205.5; IVM: 2457 6 377.3 cells/COC). Analysis of COCs matured for 10 h in vitro showed that Acyl-CoA synthetase expression was significantly altered in IVM COCs compared to in vivo matured COCs with Acsbg1 , Acsbg2 , Acsl1 , Acsl4 and Acsm3 expression significantly decreased in IVM COCs at 10 h while Acsl5 , Acsl6 and Acsm4 were significantly higher compared to in vivo matured COCs (Fig. 3 A–H). Acsl3 , Acsm2 , and Acsm3 were not different in COCs matured in vivo for 10 h compared to non-matured COCs (Fig. 2 and data not shown) nor compared to COCs matured in vitro (data not shown). Expression of the isoform Cpt1b was significantly reduced in IVM COCs (Fig. 3I) while Cpt1a , Cpt1c and Cpt2 were unaltered (data not shown). Genes encoding isoforms for acyl-CoA dehydrogenases which perform the first step in the FAO spiral showed disparate expression patterns with Acad10 , Acad11 and Acadvl significantly higher in IVM COCs while Acadm and Acadsb were significantly reduced compared to in vivo matured COCs (Fig. 3 J–N). Acadl , Acad9 and Acads expression levels were not different between maturation treatment groups (data not shown). The expression of the acetyl-CoA transferase Acaa2 , which catalyzes the final step in the FAO spiral, showed a significant decrease following IVM (Fig. 3O). We next compared functional rates of FAO in COCs that were matured in vitro for 10 h versus COCs isolated from follicles at 10 h post-hCG (i.e. matured in vivo). There was a significant 2.8- fold reduction in the level of FAO occurring in COCs matured in vitro (IVM) compared to those matured in vivo (Fig. 4A). Thus, IVM conditions result in COCs performing significantly less FAO than in vivo matured COCs at this timepoint. Because of the dramatically reduced level of FAO in IVM COCs, we next determined whether agonists of PPARs, specifically bezafibrate and rosiglitazone, would increase FAO in COCs during in vitro maturation. Real-time RT-PCR was used to confirm the presence of Ppar a , Pparg and Ppard mRNA transcripts in COCs prior to experiments examining the effects of the PPAR agonists (data not shown). Treatment of COCs with bezafibrate using doses ranging from 50–800 m M had no significant effect on FAO rate during the maturation period (Fig. 4B). At 100 m M bezafibrate did result in a 1.4-fold increase in FAO compared to control, however this dose also failed to have any significant effect over control conditions in subsequent assays investigating oocyte quality (data not shown). Conversely, treatment of COCs with rosiglitazone during IVM significantly reduced FAO in COCs in a dose dependant manner (Fig. 4C). Interestingly, despite rosiglitazone treatment (20 m M) significantly inhibiting FAO in COCs during IVM this dose caused a significant increase in the expression of ...
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... On the other hand, top downregulated GO KEGG analysis in Cbfβ-deficient cartilage also shows downregulated PPAR signaling, in line with decreased Plin5 expression shown in the volcano plot ( Figure 4A and E). As mentioned previously, PPAR signaling is crucial for cell differentiation and lipid metabolism (Dunning et al., 2014;Feige et al., 2006). Upregulated GO BP and GO KEGG analysis results further elucidated the regulatory mechanism of Cbfβ in mice articular cartilage ( Figure 4). ...
As the most common degenerative joint disease, osteoarthritis (OA) contributes significantly to pain and disability during aging. Several genes of interest involved in articular cartilage damage in OA have been identified. However, the direct causes of OA are poorly understood. Evaluating the public human RNA-seq dataset showed that CBFB (subunit of a heterodimeric Cbfβ/Runx1, Runx2, or Runx3 complex) expression is decreased in the cartilage of patients with OA. Here, we found that the chondrocyte-specific deletion of Cbfb in tamoxifen-induced Cbfb f/f ;Col2a1-CreER T mice caused a spontaneous OA phenotype, worn articular cartilage, increased inflammation, and osteophytes. RNA-sequencing analysis showed that Cbfβ deficiency in articular cartilage resulted in reduced cartilage regeneration, increased canonical Wnt signaling and inflammatory response, and decreased Hippo/Yap signaling and Tgfβ signaling. Immunostaining and western blot validated these RNA-seq analysis results. ACLT surgery-induced OA decreased Cbfβ and Yap expression and increased active β-catenin expression in articular cartilage, while local AAV-mediated Cbfb overexpression promoted Yap expression and diminished active β-catenin expression in OA lesions. Remarkably, AAV-mediated Cbfb overexpression in knee joints of mice with OA showed the significant protective effect of Cbfβ on articular cartilage in the ACLT OA mouse model. Overall, this study, using loss-of-function and gain-of-function approaches, uncovered that low expression of Cbfβ may be the cause of OA. Moreover, Local admission of Cbfb may rescue and protect OA through decreasing Wnt/β-catenin signaling, and increasing Hippo/Yap signaling and Tgfβ/Smad2/3 signaling in OA articular cartilage, indicating that local Cbfb overexpression could be an effective strategy for treatment of OA.
... Real-time PCR analysis was followed to determine the role of trifuhalol A in key adipogenesis-related genes. Peroxisome proliferatoractivated receptor-gamma (PPAR-γ) and CCAAT/enhancer-binding protein-alpha (C/EBP-α) are key transcription factors of adipogenesis and activate expressions of down-stream genes involved in adipogenesis (Dunning et al., 2014;Ye et al., 2017;Zhang et al., 2018). Treating cells with trifuhalol A at 22 and 44 μM was able to suppress the expression of PPAR-γ by 18% and 26%, and C/EBP-α by 21% and 31% compared to the controls, respectively (Fig. 5A and B). ...
Trifuhalol A, a fucol-type phlorotannin, was extracted and identified from the brown algae Agarum cribrosum. The total yield and purity of trifuhalol A from A. cribrosum were 0.98% and 86%, respectively. Trifuhalol A at 22 and 44 μM inhibited lipid accumulation in human primary adipocytes. Consistently trifuhalol A suppressed the expression of adipogenesis-related genes, such as proliferator-activated receptor-gamma (PPAR-γ), CCAAT/enhancer-binding protein-alpha (C/EBP-α), fatty acid synthase (FAS), and sterol regulatory element-binding protein-1 (SREBP-1), in a dose-dependent manner. Trifuhalol A increased the level of proteins such as wingless/integrated (Wnt)10b, nuclear-β-catenin, total-β-catenin, phospho-AMP-activated protein kinase (pAMPK), and phospho-liver kinase B1 (pLKB1) as well as the expression of genes such as Wnt10b, Frizzled 1, and low-density lipoprotein receptor-related protein 6 (LRP6). Additionally, trifuhalol A decreased the expression of the glycogen synthase kinase-3beta (GSK3β) gene. These results suggest that trifuhalol A reduces fat accumulation in human adipocytes via the Wnt/β-catenin- and AMPK-dependent pathways.
... In this sense, in vitro embryo production (IVEP) and cryopreservation represent valuable tools [1,2], however, despite many advances achieved in IVEP, the efficiency of the oocyte in vitro maturation (IVM) still limits the application of this biotechnology [3]. Intracellular lipids provide energy for oocyte maturation and the subsequent early embryonic development [4] and exhibit a variety of important cellular functions, including membrane composition, energy storage, and cell signaling [5]. Some species have physiologically a greater amount of lipids in their oocytes, such as pigs, cattle, and cats [6,7] and despite the physiological amount and their use for metabolic functions, there are studies in cattle indicating that lipid content increases abnormally during IVM [8,9]. ...
... Forskolin (FSK), a cAMP modulator widely used in IVM of several species, can enhance lipolytic activity and induce the activation of intracellular lipases [3]. L-carnitine (LC) has antioxidant properties and stimulates the metabolism of intracellular lipid stores through the ability to upregulate β-oxidation [5]. Conjugated linoleic acid t10, c12 (CLA) can induce linoleic acid incorporation and triacylglycerols and phospholipids production which improves the lipid cellular lipid profile [14] (Fig. 1). ...
... In turn, no difference was found regarding mitochondrial activity between groups. The use of oocyte intracellular stores for energy production during IVM is relevant in species with high levels of stored lipids [5,34]. β-oxidation during IVM seems essential for subsequent developmental competence and LC increases beta-oxidation, taking fatty acids from the cytosol into mitochondria [34], however, the mitochondrial activity in the MIX group was not different from CONTROL. ...
This study investigated the time course of lipid accumulation during IVM and assessed the role of lipid modulators added during IVM on lipid content, nuclear maturation, oxidative stress, mitochondrial activity, gene expression, and cryosurvival of cat oocytes. First, the lipid content of immature COCs was compared to those subjected to different IVM duration times (24, 28, and 32 h). Then, the lipid content was investigated after the use of different lipid modulators [conjugated linoleic acid (CLA), forskolin (FK), L-carnitine (LC), and linolenic acid (LA)]. Subsequently, both the CONTROL group and MIX (CLA+FK+LC) were compared regarding nuclear maturation, mitochondrial activity, reactive oxygen species (ROS), and glutathione (GSH) levels, to the expression of SDHA, GDF9, BMP15, ZAR-1, PRDX1, SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes); and to vitrification and post-warming viability assessment. When not using any lipid modulator, an increase (P<0.05) in lipid content could be observed after 28 h of IVM. The MIX group showed the greatest (P<0.05) reduction in oocyte lipid content after 28 h of IVM. No difference (P>0.05) was observed in the MII rate in the CONTROL (45%) and MIX (41%) groups and in mitochondrial activity ((1.00 ± 0.35 A.U vs 1.19 ± 0.14 A.U). Although ROS and GSH levels were higher (P<0.05) in MIX than in CONTROL, the redox balance (ROS/GSH) was greater (P<0.05) in the latter (C:1.00±0.20b vs M:0.26±0.06 a A.U). The GDF9, HSP70, PRDX1, and SIRT1 transcripts were downregulated (P<0.05) in MIX-oocytes, compared to the CONTROL. After vitrification, MIX (74%) presented a higher (P<0.05) viability compared to control (53%). In conclusion, MIX can reduce the total lipid content and improve viability after cryopreservation, however, it seems to affect the oocyte metabolism in a way that still needs to be better understood in the cat biological model.
... From ou experiments, Cpt1a is expressed in mouse oocytes at the GV stage and becomes unde tectable at the MII stage. These findings are consistent with previous studies showing th expression of this isoform in mammalian oocytes from ovarian follicles [61][62][63]. In con trast to Cpt1a, Cpt1c is known to have low catalytic activity and is mainly localized in th ...
... From our experiments, Cpt1a is expressed in mouse oocytes at the GV stage and becomes undetectable at the MII stage. These findings are consistent with previous studies showing the expression of this isoform in mammalian oocytes from ovarian follicles [61][62][63]. In contrast to Cpt1a, Cpt1c is known to have low catalytic activity and is mainly localized in the endoplasmic reticulum (ER) rather than in mitochondria [64][65][66]. ...
Carnitines play a key physiological role in oocyte metabolism and redox homeostasis. In clinical and animal studies, carnitine administration alleviated metabolic and reproductive dysfunction associated with polycystic ovarian syndrome (PCOS). Oxidative stress (OS) at systemic, intraovarian, and intrafollicular levels is one of the main factors involved in the pathogenesis of PCOS. We investigated the ability of different acyl-carnitines to act at the oocyte level by counteracting the effects of OS on carnitine shuttle system and mitochondrial activity in mouse oocytes. Germinal vesicle (GV) oocytes were exposed to hydrogen peroxide and propionyl-l-carnitine (PLC) alone or in association with l-carnitine (LC) and acetyl-l-carnitine (ALC) under different conditions. Expression of carnitine palmitoyltransferase-1 (Cpt1) was monitored by RT-PCR. In in vitro matured oocytes, metaphase II (MII) apparatus was assessed by immunofluorescence. Oocyte mitochondrial respiration was evaluated by Seahorse Cell Mito Stress Test. We found that Cpt1a and Cpt1c isoforms increased under prooxidant conditions. PLC alone significantly improved meiosis completion and oocyte quality with a synergistic effect when combined with LC + ALC. Acyl-carnitines prevented Cpt1c increased expression, modifications of oocyte respiration, and ATP production observed upon OS. Specific effects of PLC on spare respiratory capacity were observed. Therefore, carnitine supplementation modulated the intramitochondrial transfer of fatty acids with positive effects on mitochondrial activity under OS. This knowledge contributes to defining molecular mechanism underlying carnitine efficacy on PCOS.
... This is because the PPAR signaling pathway is a type of gene expression pattern that transforms nutritional signals into specific genes and controls cellular energy metabolism [33]. In this study, the PPAR signaling pathway was enriched in yak oocytes, which may be related to the regulation of oocyte development by granulosa cells, suggesting that the PPAR signaling pathway plays an important role in maintaining oocyte meiosis [34]. Previous studies have demonstrated that the Hippo pathway plays an important regulatory role in follicular growth and early embryonic development. ...
The aim of this study was to investigate protein regulation at different time points during the in vitro maturation of yak oocytes. Yak oocytes at GV, MI, and MII stages were collected during in vitro maturation, and differential proteomics sequencing was performed using iTRAQ technology. GO functional classification indicated that the differential proteins were closely associated with biological processes such as “metabolic processes”, and molecular events such as “binding” molecular-function-related categories were active. KOG analysis showed that energy-metabolism-related activities were vigorous during oocyte development from the GV phase to MI phase, and genetic material preparation activities were more active when oocytes developed from the MI stage to MII stage. KEGG pathway analysis showed that the PPAR metabolic pathway, Hippo signaling pathway, and ECM–receptor interaction and metabolic pathway were enriched from the GV to the MI stages. The PI3K-Akt, TGF-β, and phagosome pathways were enriched from the MI stage to the MII stage. These results indicate that transient dynamic changes occurred in the proteome during the maturation of yak oocytes, and the physiological functions mediated by these were also different. The accurate identification of the differential proteins in the three stages of GV, MI, and MII was helpful in further analyzing the molecular regulatory mechanism of yak oocyte maturation.
... Fatty acid oxidation is a metabolic process responsible for the production of ATP, which is essential for cell proliferation event in pre-implantation embryos [48]. Surprisingly, the imbalance in the redox state can impair lipid metabolism, leading to accumulation of intracellular lipids [49]. ...
Vanillic acid (VA) has shown antioxidant and anti-inflammatory activities in different cell types, but its biological effects in the context of early embryo development have not yet been clarified. In the current study, the impact of VA supplementation during in vitro maturation (IVM) and/or post-fertilization (in vitro culture; IVC) on redox homeostasis, mitochondrial function, AKT signaling, developmental competence, and the quality of bovine pre-implantation embryos was investigated. The results showed that dual exposure to VA during IVM and late embryo culture (IVC3) significantly improved the blastocyst development rate, reduced oxidative stress, and promoted fatty acid oxidation as well as mitochondrial activity. Additionally, the total numbers of cells and trophectoderm cells per blastocyst were higher in the VA-treated group compared to control (p < 0.05). The RT-qPCR results showed down-regulation of the mRNA of the apoptosis-specific markers and up-regulation of AKT2 and the redox homeostasis-related gene TXN in the treated group. Additionally, the immunofluorescence analysis showed high levels of pAKT-Ser473 and the fatty acid metabolism marker CPT1A in embryos developed following VA treatment. In conclusion, the study reports, for the first time, the embryotrophic effects of VA, and the potential linkage to AKT signaling pathway that could be used as an efficacious protocol in assisted reproductive technologies (ART) to improve human fertility.
... In addition to the synergistic effects of PPARd and b-catenin on proliferation and lineage specification, the coordination of Wnt-b-catenin with PPARd signaling regulates lipid metabolism, enhancing the blastocyst development and implantation potential (21,89,90). Lipid metabolism and ATP generated through fatty acid oxidation is an important energy source for early embryonic development (95,96). When Wnt stimulation induces PPARd activity, there is a significant reduction in lipid droplet content, indicative of a high fatty acid oxidation metabolism (89). ...
Cell-cell junctions form strong intercellular connections and mediate communication between blastomeres during preimplantation embryonic development and thus are crucial for cell integrity, polarity, cell fate specification and morphogenesis. Together with cell adhesion molecules and cytoskeletal elements, intercellular junctions orchestrate mechanotransduction, morphokinetics and signaling networks during the development of early embryos. This review focuses on the structure, organization, function and expressional pattern of the cell–cell junction complexes during early embryonic development. Understanding the importance of dynamic junction formation and maturation processes will shed light on the molecular mechanism behind developmental abnormalities of early embryos during the preimplantation period.
... Upregulation of these genes is significant as they are involved in a multitude of different pathways that could potentially lead to cardiac remodeling effects. PPARα and ACADM are both involved in fatty acid oxidation and metabolism [29,30], expressions of both NPPA and NPPB encoding for ANP (atrial natriuretic protein) and BNP (brain natriuretic protein), respectively, have been shown to have blood pressure-lowering effects [31] as well as contribute to cardio-renal homeostasis [32,33], and TNFα is a potent paracrine and endocrine mediator of inflammatory and immune functions that is responsible for mediating signaling pathways that play an important role, both in homeostasis and pathophysiology [34,35]. The aforementioned genes show promising potential for research and are all subject to Content courtesy of Springer Nature, terms of use apply. ...
Sodium-glucose co-transporter 2 (SGLT2) inhibitors represent one type of new-generation type 2 diabetes (T2DM) drug treatment. The mechanism of action of an SGLT2 inhibitor (SGLT2i) in treating T2DM depends on lowering blood glucose levels effectively via increasing the glomerular excretion of glucose. A good number of randomized clinical trials revealed that SGLT2is significantly prevented heart failure (HF) and cardiovascular death in T2DM patients. Despite ongoing clinical trials in HF patients without T2DM, there have been a limited number of translational studies on the cardioprotective properties of SGLT2is. As the cellular mechanism behind the cardiac benefits of SGLT2is is still to be elucidated, animal models are used to better understand the pathways behind the cardioprotective mechanism of SGLT2i. In this review, we summarize the animal models constructed to study the cardioprotective mechanisms of SGLT2is to help deliver a more comprehensive understanding of the in vivo work that has been done in this field and to help select the most optimal animal model to use when studying the different cardioprotective effects of SGLT2is.
Graphical Abstract
... It has, therefore, been suggested that these compounds may act as agonists and, through their influence on PPARγ, they determine many of the body's functions that are regulated by this enzyme [67]. In addition, other studies have shown that the activation of PPARγ induces the expression of antioxidant enzymes, e.g., catalase and superoxide dismutase (SOD), i.e., two enzymes capable of alleviating oxidative stress and inhibiting NADPH oxidase [68][69][70]. ...
Citation: Tabęcka-Łonczyńska, A.; Skóra, B.; Kaleniuk, E.; Szychowski, K.A. Reprotoxic Effect of Tris(2,3-Dibromopropyl) Isocyanurate (TBC) on Spermatogenic Cells In Vitro. Molecules 2023, 28, 2337. https:// Abstract: Tris(2,3-dibromopropyl) isocyanurate (TBC) belongs to the class of novel brominated flame retardants (NFBRs) that are widely used in industry. It has commonly been found in the environment , and its presence has been discovered in living organisms as well. TBC is also described as an endocrine disruptor that is able to affect male reproductive processes through the estrogen receptors (ERs) engaged in the male reproductive processes. With the worsening problem of male infertility in humans, a mechanism is being sought to explain such reproductive difficulties. However, so far, little is known about the mechanism of action of TBC in male reproductive models in vitro. Therefore , the aim of the study was to evaluate the effect of TBC alone and in cotreatment with BHPI (estrogen receptor antagonist), 17β-estradiol (E 2), and letrozole on the basic metabolic parameters in mouse spermatogenic cells (GC-1 spg) in vitro, as well as the effect of TBC on mRNA expression (Ki67, p53, Pparγ, Ahr, and Esr1). The presented results show the cytotoxic and apoptotic effects of high micromolar concentrations of TBC on mouse spermatogenic cells. Moreover, an increase in Pparγ mRNA levels and a decrease in Ahr and Esr1 gene expression were observed in GS-1spg cells cotreated with E 2. These results suggest the significant involvement of TBC in the dysregulation of the steroid-based pathway in the male reproductive cell models in vitro and may be the cause of the currently observed deterioration of male fertility. However, more research is needed to reveal the full mechanism of TBC engagement in this phenomenon.
... Embryo development is a complex biological process known to require great energy amount. Besides, intracellular lipids can be considered a potential and more economical energy source for preimplantation embryos than others exogenous sources, such as carbohydrates [38,40]. However, a high lipid content in IVP embryos is also related to a lower embryonic development and pregnancy rate, which might indicate an inadequate culture conditions when comparing different in vitro culture systems or even in relation to the in vivo system [41]. ...
Supplementation of culture media with IGF-1 during in vitro culture of embryos has had controversial results over the years. In the present study, we show that differences previously observed in response to IGF addition might be related to intrinsic heterogeneity of the embryos. In other words, the effects exerted by IGF-1 are dependent on the characteristics of the embryos and their ability to modulate metabolism and overcome stressful conditions, such as the ones found in a non-optimized in vitro culture system. To test this hypothesis, in vitro produced bovine embryos with distinct morphokinetics (fast- and slow-cleavage) were submitted to treatment with IGF-1 and then evaluated for embryo production rates, total cell number, gene expression and lipid profile. Our results show that remarkable differences were found when fast and slow embryos treated with IGF-1 were compared. Fast embryos respond by upregulating genes related to mitochondrial function, stress response, and lipid metabolism, whereas slow embryos presented lower mitochondrial efficiency and lipid accumulation. We conclude that indeed the treatment with IGF-1 selectively affects embryonic metabolism according to early morphokinetics phenotypes, and this information is relevant for decision-making in the design of more appropriate in vitro culture systems.
