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Acetyl CoA carboxylase inactivation and meiotic maturation in mouse oocytes. Valsangkar DS et al. (2015) In mouse oocytes, meiotic induction by pharmacological activation of PRKA (adenosine monophosphate-activated protein kinase; formerly known as AMPK) or by hormones depends on stimulation of fatty acid oxidation (FAO). PRKA stimulates FAO by phosphorylating and inactivating acetyl CoA carboxylase (ACAC; formerly ACC), leading to decreased malonyl CoA levels and augmenting fatty-acid transport into mitochondria. We investigated a role for ACAC inactivation in meiotic resumption by testing the effect of two ACAC inhibitors, CP-640186 and Soraphen A, on mouse oocytes maintained in meiotic arrest in vitro. These inhibitors significantly stimulated the resumption of meiosis in arrested cumulus cell-enclosed oocytes, denuded oocytes, and follicle-enclosed oocytes. This stimulation was accompanied by an increase in FAO. Etomoxir, a malonyl CoA analogue, prevented meiotic resumption as well as the increase in FAO induced by ACAC inhibition. Citrate, an ACAC activator, and CBM-301106, an inhibitor of malonyl CoA decarboxylase, which converts malonyl CoA to acetyl CoA, suppressed both meiotic induction and FAO induced by follicle-stimulating hormone, presumably by maintaining elevated malonyl CoA levels. Mouse oocyte-cumulus cell complexes contain both isoforms of ACAC (ACACA and ACACB); when wild-type and Acacb(-/-) oocytes characteristics were compared, we found that these single-knockout oocytes showed a significantly higher FAO level and a reduced ability to maintain meiotic arrest, resulting in higher rates of germinal vesicle breakdown. Collectively, these data support the model that ACAC inactivation contributes to the maturation-promoting activity of PRKA through stimulation of FAO. Mol. Reprod. Dev. 2015. © 2015 Wiley Periodicals, Inc.//////////////////
175 EFFECT OF ACETYL-CoA CARBOXYLASE (ACC) INHIBITOR ON THE LIPID CONTENT AND NUCLEAR MATURATION OF CANINE OOCYTES. McGill J 2013 et al.
Compared with other domestic species, embryo technologies are least developed for the dog. This is mainly due to difficulties in producing mature oocytes in vitro. Canine oocytes contain exceptionally high amounts of lipid. High lipid content increases the chilling sensitivity of oocytes and embryos. Mechanical and chemical reductions of the lipid content have been used to improve the cryotolerance of oocytes. Additionally, chemical stimulation of lipid catabolism improved oocyte in vitro maturation (IVM) rates in other species (You et al. 2012 Theriogenology 78, 235-543). Acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme in de novo lipogenesis and its expression has been reported in oocytes and embryos. In somatic cells, inhibition of ACC reduces lipogenesis and enhances -oxidation. Our hypothesis is that treatment of oocytes with an inhibitor of ACC (CP640186, Pfizer Animal Health, New York, NY, USA) reduces lipid content and improves IVM rate of oocytes. Ovaries were collected from a spay clinic and sliced in HEPES-buffered TCM-199 to recover oocytes. In vitro maturation was conducted at 38.5C, 5% CO2, and high humidity in TCM-199 supplemented with 1% fetal bovine serum, glutamine, sodium pyruvate, -mercaptoethanol, oestradiol, epidermal growth factor, and antimicrobial agents (Songsasen et al. Mol. Reprod. Dev. 79, 186-196). During the first 19 to 21h, the IVM media contained 4 concentrations of the inhibitor (0+DMSO, 0.02, 0.1, and 0.5M, designated as treatments 1, 2, 3, and 4, respectively) and then oocytes were transferred to a medium without the inhibitor and cultured for an additional 27 to 29h. At the end of culture (total of 48h), oocytes were denuded of cumulus layers, washed, fixed, and stained with Nile red (lipid) and Hoechst-33342 (chromatin), and then mounted on a microscope slide. Lipid content and chromatin status were evaluated using fluorescent microscopy (TRITC and DAPI filters, respectively). The relative lipid content was measured by the corrected total cell fluorescence (CTCF) using ImageJ software (http://rsbweb.nih.gov/ij/). Data on CTCF and proportions of chromatin status of oocytes were analysed using one-way ANOVA (SigmaPlot 11.0). The mean CTCF for each treatment was 5.510(9) (n=51, 5.210(9) (n=44), 4.510(9) (n=31), and 4.810(9) (n=34), respectively (P=0.3; 4 replicates). At the highest dose, the agent induced relatively more cumulus cell layer expansion but inhibited their attachment to the dish; the latter effect was reversible because cumulus cells attached and proliferated after washing the oocytes of the agent. Metaphase II was rare (=3.1%); however, the proportion of oocytes developing to =GVBD stage (Trt 1 14%, n=37; Trt 2 41%, n=56; Trt 3 5%, n=22; Trt 4 11%, n=43) was affected by treatments. Our preliminary data indicate that a low concentration of ACC inhibitor has a positive effect on the nuclear maturation of canine oocytes but the effect on lipid content as estimated by using Nile red fluorescence intensity appears to be minimal.
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AMPK regulates progesterone secretion in rat granulosa cells Tosca L, et al .
The AMP-activated protein kinase (AMPK) is a major regulator of energy metabolism involved in fatty acid and cholesterol synthesis. In the ovary, cholesterol plays a key role in steroid production. We report the presence of AMPK in rat ovaries and we have investigated its role in granulosa cells. We show using RT-PCR and western-blot that the mRNAs for the alpha1/2 and beta1/2 subunits and the proteins are found in the ovaries. Immunohistochemistry localized the alpha1 AMPK subunit in granulosa cells, corpus luteum, oocyte, and less abundantly in theca cells. Treatment with AICAR (5-amino-imidazole-4-carboxyamide-1-beta-D-ribofuranoside, 1 mM), an activator of AMPK, increased dose-dependent and time-dependent phosphorylation of AMPKalpha1 on Thr 172 in primary granulosa cells. Simultaneously, phosphorylation of Acetyl-CoA Carboxylase at Ser-79 was also increased. AICAR treatment for 48 h halved progesterone secretion, 3beta-HSD protein and mRNA levels and phosphorylation of both basal MAPK ERK1/2 and p38 and in response to IGF-1 and/or FSH in granulosa cells. AICAR treatment (1 mM) had no detectable effect on basal and FSH- and/or IGF-1-induced estradiol production and on granulosa cell proliferation or viability. Adenovirus-mediated expression of dominant negative AMPK totally abolished the effects of AICAR on progesterone secretion, 3beta-HSD protein production, and MAPK ERK1/2 and p38 phosphorylation. Moreover, we showed using specific inhibitors of ERK1/2 and p38 MAPK that the MAPK ERK1/2 and not p38 is involved in progesterone secretion and 3beta-HSD expression, strongly suggesting that the activation of AMPK in response to AICAR reduces progesterone production through the MAPK ERK1/2 signaling pathway in rat granulosa cells.
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cAMP pulsing of denuded mouse oocytes increases meiotic resumption via activaton of AMP-activated protein kinase. Chen J et al. cAMP plays a critical role in the control of oocyte maturation, as a high level of cAMP maintains oocyte arrest at the first meiotic prophase. Yet this study shows that pulsing meiotically arrested denuded oocytes (DO) with cAMP induces oocyte maturation through the activation of AMP-activated protein kinase (PRKA). Short term (3 h) pulsing of meiotically arrested oocytes with forskolin, an adenyl cyclase (AC) activator, increased oocyte cAMP, led to elevated AMP and induced oocyte meiotic resumption compared to oocytes continuously cultured in the control medium with or without forskolin. Western analysis showed that GV-stage oocytes after forskolin pulsing contained increased levels of phospho-acetyl CoA carboxylase (pACACA), a primary substrate of PRKA. Pulsing oocytes with the PDE-sensitive cAMP analog, 8-Bromo-cAMP (8-Br-cAMP), also increased pACACA and pPRKA levels in GV-stage oocytes and induced oocyte meiotic resumption. Moreover, the PRKA inhibitors, compound C and araA, prevented 8-Br-cAMP pulsing-induced maturation. The lack of effect on meiotic induction and PRKA activation when oocytes were pulsed with the PDE-resistant activators of cAMP-dependent protein kinase, Sp-cAMP-AM and Sp-5,6-DCI-cBIMPS, suggests that cAMP degradation is required for pulsing-induced maturation. Pulsing oocytes with the Epac (exchange protein directly activated by cAMP)-specific activator, 8-CPT-2-O-Me-cAMP, had no stimulatory effect on oocyte maturation, suggesting Epac is not involved in the pulsing-induced maturation. Taken together, these data support the idea that a transient increase in oocyte cAMP can induce meiotic resumption via activation of PRKA.
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MALDI Mass Spectrometry Imaging of Lipids and Gene Expression Reveals Differences in Fatty Acid Metabolism between Follicular Compartments in Porcine Ovaries. Uzbekova S et al. (2015) In mammals, oocytes develop inside the ovarian follicles; this process is strongly supported by the surrounding follicular environment consisting of cumulus, granulosa and theca cells, and follicular fluid. In the antral follicle, the final stages of oogenesis require large amounts of energy that is produced by follicular cells from substrates including glucose, amino acids and fatty acids (FAs). Since lipid metabolism plays an important role in acquiring oocyte developmental competence, the aim of this study was to investigate site-specificity of lipid metabolism in ovaries by comparing lipid profiles and expression of FA metabolism-related genes in different ovarian compartments. Using MALDI Mass Spectrometry Imaging, images of porcine ovary sections were reconstructed from lipid ion signals for the first time. Cluster analysis of ion spectra revealed differences in spatial distribution of lipid species among ovarian compartments, notably between the follicles and interstitial tissue. Inside the follicles analysis differentiated follicular fluid, granulosa, theca and the oocyte-cumulus complex. Moreover, by transcript quantification using real time PCR, we showed that expression of five key genes (including ACACA) in FA metabolism significantly varied between somatic follicular cells (theca, granulosa and cumulus) and the oocyte. In conclusion, lipid metabolism differs between ovarian and follicular compartments.//////////////////
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