Figure 1.
Long chain fatty acid activation, entry into mitochondria and metabolism via fatty acid oxidation.
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.
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.
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.
Acetyl-CoA is able to enter the tricarboxylic acid (TCA) cycle which with fatty acid oxidation generates electrons forming NADH and FADH2 which donate electrons to the electron transport chain (ETC) required for ATP synthesis
Genes involved in fatty acid oxidation were measured in cumulus oocyte complexes during in vivo maturation in response to hCG. A summary of their expression pattern is depicted with blue, red and black coloured genes representing significantly up-regulated, down-regulated and unchanged respectively, as demonstrated in Figure 2.
Figure 2.
Modulation of genes involved in fatty acid oxidation in the cumulus oocyte complex during oocyte maturation in vivo.
Analysis of Acsbg2, Acsl1, Acsl4, Acsl5, Acsl6, Acsm3, Cpt1a, Acad10, Acad11, Acadl, Acadm, Acadsb, and Acaa2 (A–M, respectively) expression in cumulus oocyte complexes at 0, 6, 10, and 16 h post-hCG administration is shown. mRNA expression was normalized to the geometric mean of Gusb, Hprt, Actb, Gapdh and Hsp90ab1 and presented as mean ± SEM (n = 3 experimental replicates, different superscripts signify statistical difference of P<0.05 by one-way ANOVA with Tukey post hoc test).
Figure 3.
Gene transcripts involved in fatty acid oxidation are dysregulated in cumulus oocyte complexes matured in vitro (IVM).
Analysis of Ascbg1, Acsbg2, Acsl1, Acsl4, Acsl5, Acsl6, Acsm3, Acsm4, Cpt1b, Acad10, Acad11, Acadm, Acadsb, Acadvl, and Acaa2 (A-O, respectively) expression in cumulus oocyte complexes following 10 h of in vivo or in vitro maturation (IVM). mRNA expression was normalized to the geometric mean of Gapdh and Hsp90ab1 and presented as mean ± SEM (n = 3 experimental replicates, * P<0.05, ** P<0.01, *** P<0.001 by unpaired t test).
Figure 4.
FAO is reduced in cumulus oocyte complexes matured in vitro and is modulated by rosiglitazone but not bezafibrate.
Cumulus oocytes complexes (COC) were matured for 10 h in vitro (IVM) or collected 10 h following the administration of hCG (in vivo matured). β-oxidation was measured over 4 h of culture and expressed as pmol palmitic acid metabolized per COC per hour (A). Data presented as mean ± SEM, n = 7 per treatment from 3 independent experiments, representative of 280 COCs per treatment. ***P<0.001 by unpaired t test. The effect of PPAR agonists bezafibrate and rosiglitazone on β-oxidation was measured in COCs maturing in vitro over 20 h in the presence of increasing doses of bezafibrate (B) or rosiglitazone (C). Data presented as mean ± SEM, n = 5 independent experiments, representative of 100 COCs per group. Data analyzed by one-way ANOVA and Tukey post hoc test, different letters signifying statistical difference (P<0.05).
Figure 5.
Rosiglitazone treatment of COCs during in vitro maturation significantly modulates expression of genes involved in fatty acid oxidation.
Analysis of Acsl1 (A), Cpt1b (B), Cpt1c (C), Cpt2 (D) and Acaa2 (E) expression in cumulus-oocyte complexes following 10 h of in vitro maturation (IVM) in the absence (0) or presence of rosiglitazone (20 µM). mRNA expression was normalized to the geometric mean of Gapdh and Hsp90ab1 and presented as mean ± SEM (n = 3 experimental replicates, * P<0.05, ** P<0.01 by unpaired t test).
Figure 6.
Mitochondrial membrane potential is increased in oocytes following maturation of cumulus oocyte complexes in vitro in the presence of rosiglitazone.
Mitochondrial membrane potential was assessed by JC-1 staining of oocytes following in vitro maturation of cumulus oocyte complexes in the absence (A–C) or presence of 20 µM rosiglitazone (D–F). Red (G), green (H) and the ratio of red:green (I) fluorescence within oocytes was quantified and presented as mean ± SEM, representative of 30–35 COCs per group, from 3 independent experiments. Data analyzed by unpaired t test on untransformed (H) or Log10 transformed (G and I) data, * P<0.05; *** P<0.001.
Figure 7.
Rosiglitazone treatment during in vitro maturation of cumulus-oocyte complexes negatively affects oocyte developmental competence.
Oocyte developmental competence was assessed following in vitro fertilization of cumulus-oocyte complexes matured in vitro in control conditions or in the presence of rosiglitazone (20 µM). Embryo development was assessed on days, 2, 3, 4 and 5, with day 1 designated as the day of fertilization (A and B). Development on day 5 was assessed as blastocyst or hatching blastocyst (B). Data presented as mean ± SEM, n = 7 independent experiments, representative of 132–138 oocytes per treatment group. Arcsine transformed data were analyzed by either repeated measures two-way ANOVA (A) or regular two-way ANOVA with Bonferroni post hoc test (B). Asterix indicates statistical difference (P<0.05) within developmental stage.