Animal Reproduction (AR)
Animal Reproduction (AR)

Effects of different stimulators of cGMP synthesis on lipid content in bovine oocytes matured in vitro

Letícia Schefer; Kátia Regina Lancelloti Schwarz; Daniela Martins Paschoal; Fernanda Cavallari de Castro; Hugo Fernandes; Ramon César Botigelli; Cláudia Lima Verde Leal

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Bovine oocytes and blastocysts produced in vitro are frequently of lower quality and less cryotolerant than those produced in vivo, and greater accumulation of lipids in the cytoplasm has been pointed out as one of the reasons. In human adipocytes cGMP signaling through the activation of PKG appears to be involved in lipid metabolism, and components of this pathway have been detected in bovine cumulus-oocyte complexes (COCs). The aim of this study was to investigate the influence of this pathway on the lipid content in oocytes and expression of PLIN2 (a lipid metabolism-related gene) in cumulus cells. COCs were matured in vitro for 24 h with different stimulators of cGMP synthesis. The activation of soluble guanylyl cyclase (sGC) by Protoporphyrin IX reduced lipid content (22.7 FI) compared to control oocytes (36.45 FI; P <0.05). Stimulation of membrane guanylyl cyclase (mGC) with natriuretic peptides precursors A and C (NPPA and NPPC) had no effect (36.5 FI; P>0.05). When the PKG inhibitor KT5823 was associated with Protoporphyrin IX, its effect was reversed and lipid contents increased (52.71 FI; P<0.05). None of the stimulators of cGMP synthesis affected the expression of PLIN2 in cumulus cells. In conclusion, stimulation of sGC for cGMP synthesis promotes lipolytic activities in bovine oocytes matured in vitro and such effect is mediated by PKG. However, such effect may vary depending on the stimulus received and/or which synthesis enzyme was activated, as stimulation of mGC had no effects.


natriuretic peptides, NPR1, guanylate cyclase, lipid droplets, Nile red


Ambruosi B, Lacalandra GM, Iorga AI, De Santis T, Mugnier S, Matarrese R, Goudet G, Dell’aquila ME. Cytoplasmic lipid droplets and mitochondrial distribution in equine oocytes : implications on oocyte maturation, fertilization and developmental competence after ICSI. Theriogenology. 2009;71(7):1093-104. PMid:19167745.

Asada M, Ishibashi S, Ikumi S, Fukui Y. Effect of polyvinyl alcohol (PVA) concentration during vitrification of in vitro matured bovine oocytes. Theriogenology. 2002;58(6):1199-208. PMid:12240922.

Auclair S, Uzbekov R, Elis S, Sanchez L, Kireev I, Lardic L, Dalbies-Tran R, Uzbekova S. Absence of cumulus cells during in vitro maturation affects lipid metabolism in bovine oocytes. Am J Physiol Endocrinol Metab. 2013;304(6):E599-613. PMid:23321473.

Barceló-Fimbres M, Seidel GE Jr. Cross-validation of techniques for measuring lipid content of bovine oocytes and blastocysts. Theriogenology. 2011;75(3):434-44. PMid:21111465.

Bickel PE, Tansey JT, Welte MA. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores. Biochim Biophys Acta. 2009;1791(6):419-40. PMid:19375517.

Bilodeau-Goeseels S. Effects of manipulating the nitric oxide / cyclic GMP pathway on bovine oocyte meiotic resumption in vitro. Theriogenology. 2007;68(5):693-701. PMid:17604095.

Cesaro MPD, Macedo MP, Santos JT, Rosa PRA, Ludke CA, Rissi VB, Gasperin BG, Gonçalves PBD. Natriuretic peptides stimulate oocyte meiotic resumption in bovine. Anim Reprod Sci. 2015;159:52-9. PMid:26051611.

De Loos FAM, Bevers MM, Dieleman SJ, Kruip TAM. Follicular and oocyte maturation in cows treated for superovulation. Theriogenology. 1991;35(3):537-46. PMid:16726923.

Del Collado M, Saraiva NZ, Lopes FL, Cruz MH, Gaspar RC, Oliveira CS, Perecin F, Garcia JM. Efeitos da redução ou substituição do soro fetal bovino por outros compostos na maturação in vitro de oócitos bovinos. Pesq Vet Bras. 2014;34(7):689-94.

Del Collado M, Saraiva NZ, Lopes FL, Gaspar RC, Padilha LC, Costa RR, Rossi GF, Vantini R, Garcia JM. Influence of bovine serum albumin and fetal bovine serum supplementation during in vitro maturation on lipid and mitochondrial behaviour in oocytes and lipid accumulation in bovine embryos. Reprod Fertil Dev. 2016;28(11):1721-32. PMid:25986410.

Del Collado M, da Silveira JC, Sangalli JR, Andrade GM, Souza LRS, Silva LA, Meirelles FV, Perecin F. Fatty acid binding protein 3 and transzonal projections are involved in lipid accumulation during in vitro maturation of bovine oocytes. Sci Rep. 2017;7:2645.

Engeli S, Birkenfeld AL, Badin P, Bourlier V, Louche K, Viguerie N, Thalamas C, Montastier E, Larrouy D, Harant I, de Glisezinski I, Lieske S, Reinke J, Beckmann B, Langin D, Jordan J, Moro C. Natriuretic peptides enhance the oxidative capacity of human skeletal muscle. J Clin Invest. 2012;122(12):4675-9. PMid:23114600.

Fair T, Lonergan P, Dinnyes A, Cottell DC, Hyttel P, Ward FA, Boland MP. Ultrastructure of bovine blastocysts following cryopreservation : effect of method of blastocyst production. Mol Reprod Dev. 2001;58(2):186-95.<186::AID-MRD8>3.0.CO;2-N. PMid:11139231.

Franciosi F, Coticchio G, Lodde V, Tessaro I, Modina SC, Fadini R, Dal Canto M, Renzini MM, Albertini DF, Luciano AM. Natriuretic peptide precursor C delays meiotic resumption and sustains gap junction-mediated communication in bovine cumulus-enclosed oocytes. Biol Reprod. 2014;91(3):61. PMid:25078681.

Francis SH, Blount MA, Corbin JD. Mammalian cyclic nucleotide phosphodiesterases : molecular mechanisms and physiological functions. Physiol Rev. 2011;91(2):651-90. PMid:21527734.

Fu XW, Wu GQ, Li JJ, Hou YP, Zhou G, Lun-Suo, Wang YP, Zhu SE. Positive effects of Forskolin (stimulator of lipolysis) treatment on cryosurvival of in vitro matured porcine oocytes. Theriogenology. 2011;75(2):268-75. PMid:21187280.

Genicot G, Leroy JL, Soom AV, Donnay I. The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes. Theriogenology. 2005;63(4):1181-94. PMid:15710202.

Greenspan P, Mayer EP, Fowler SD. Nile Red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol. 1985;100(3):965-73. PMid:3972906.

Haemmerle G, Lass A, Zimmermann R, Gorkiewicz G, Meyer C, Rozman J, Heldmaier G, Maier R, Theussl C, Eder S, Kratky D, Wagner EF, Klingenspor M, Hoefler G, Zechner R. Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase. Science. 2006;312(5774):734-7.

Huang GH, Chen G, Chen F. Rapid screening method for lipid production in alga based on Nile red fluorescence. Biomass Bioenergy. 2009;33(10):1386-92.

Huang HC, Ho YC, Lim JM, Chang TY, Ho CL, Chang TM. Investigation of the anti-melanogenic and antioxidant characteristics of eucalyptus camaldulensis flower essential oil and determination of its chemical composition. Int J Mol Sci. 2015;16(5):10470-90. PMid:25961954.

Kuhn M, Ng CKD, Su Y-H, Kilić A, Mitko D, Bien-Ly N, Kömüves LG, Yang RB. Identification of an orphan guanylate cyclase receptor selectively expressed in mouse testis. Biochem J. 2004;379(Pt 2):385-93. PMid:14713286.

Kuhn M. Structure, regulation, and function of mammalian membrane guanylyl cyclase receptors, with a focus on guanylyl cyclase-A. Circ Res. 2003;93(8):700-9. PMid:14563709.

Lafontan M, Moro C, Berlan M, Crampes F, Sengenes C, Galitzky J. Control of lipolysis by natriuretic peptides and cyclic GMP. Trends Endocrinol Metab. 2008;19(4):130-7. PMid:18337116.

Langin D. Control of fatty acid and glycerol release in adipose tissue lipolysis. C R Biol. 2006;329(8):598-607, discussion 653-5. PMid:16860278.

Listenberger LL, Studer AM, Brown DA, Wolins NE. Fluorescent dtection of lipid droplets and associated proteins. Curr Protoc Cell Biol. 2016;71:4.31.1-14. PMid:27245427.

Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real- Time Quantitative PCR and the 2∆∆ C T method. Methods. 2001;25(4):402-8. PMid:11846609.

Lohmann SM, Vaandrager AB, Smolenski A, Walter U, De Jonge HR. Distinct and specific functions of cGMP-dependent protein kinases. Trends Biochem Sci. 1997;22(8):307-12. PMid:9270304.

Lolicato F, Brouwers JF, de Lest CH, Wubbolts R, Aardema H, Priore P, Roelen BA, Helms JB, Gadella BM. The cumulus cell layer protects the bovine maturing oocyte against fatty acid-induced lipotoxicity. Biol Reprod. 2014;92(1):1-16. PMid:25297544.

Lonergan P, Fair T, Corcoran D, Evans ACO. Effect of culture environment on gene expression and developmental characteristics in IVF-derived embryos. Theriogenology. 2006;65(1):137-52. PMid:16289260.

Lonergan P, Gutierrez-adan A, Rizos D, Pintado B, de la Fuente J, Boland MP. Relative messenger RNA abundance in bovine oocytes collected In Vitro or In Vivo before and 20 hr after the preovulatory luteinizing hormone surge. Mol Reprod Dev. 2003;66(3):297-305. PMid:14502609.

Misono KS, Philo JS, Arakawa T, Ogata CM, Qiu Y, Ogawa H, Young HS. Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase. FEBS J. 2011;278(11):1818-29. PMid:21375693.

Miyoshi H, Perfield JW 2nd, Obin MS, Greenberg AS. Adipose Triglyceride lipase regulates basal lipolysis and lipid droplet size in adipocytes. J Cell Biochem. 2008;105(6):1430-6. PMid:18980248.

Moro C, Klimcakova E, Lafontan M, Berlan M, Galitzky J. Phosphodiesterase-5A and neutral endopeptidase activities in human adipocytes do not control atrial natriuretic peptide-mediated lipolysis. Br J Pharmacol. 2007;152(7):1102-10. PMid:17906676.

Moro C, Lafontan M. Natriuretic peptides and cGMP signaling control of energy homeostasis. Am J Physiol Heart Circ Physiol. 2013;304(3):H358-68. PMid:23203965.

Perry G. Statistics of embryo collection and transfer in domestic farm animals. Embryo Transf Newsl. 2015;33:14-26.

Pollard JW, Leibo SP. Chilling sensitivity of mammalian embryos. Theriogenology. 1994;41(1):101-6.

Potter LR, Yoder AR, Flora DR, Antos LK, Dickey DM. Natriuretic peptides: their structures, receptors, physiologic functions and therapeutic applications. Handb Exp Pharmacol. 2009;191(191):341-66. PMid:19089336.

Rettori V, McCann SM. Role of nitric oxide and alcohol on gonadotropin release in Vitro and in Vivo. Ann N Y Acad Sci. 1998;840(1):185-93. PMid:9629250.

Rizos D, Fair T, Papadopoulos S, Boland MP, Lonergan P. Developmental, qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro. Mol Reprod Dev. 2002;62(3):320-7. PMid:12112595.

Romek M, Gajda B, Krzysztofowicz E, Kepczynski M, Smorag Z. Lipid content in pig blastocysts cultured in the presence or absence of protein and vitamin E or phenazine ethosulfate. Folia Biol (Krakow). 2011;59(1-2):45-52. PMid:21614967.

Sasseville M, Côté N, Gagnon MC, Richard FJ. Up-regulation of 3′5′-cyclic guanosine monophosphate-specific phosphodiesterase in the porcine cumulus-oocyte complex affects steroidogenesis during in Vitro maturation. Endocrinology. 2008;149(11):5568-76. PMid:18669600.

Schwarz KRL, Botigelli RC, Del Collado M, Cavallari de Castro F, Fernandes H, Paschoal DM, Leal CLV. Effects of fetal calf serum on cGMP pathway and oocyte lipid metabolism in vitro. Reprod Fertil Dev. 2017;29(8):1593-601. PMid:27554265.

Schwarz KRL, de Castro FC, Schefer L, Botigelli RC, Paschoal DM, Fernandes H, Leal CLV. The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation. PLoS ONE. 2018;13(1):e0191023.

Schwarz KRL, Pires PRL, Mesquita LG, Chiaratti MR, Leal CLV. Effect of nitric oxide on the cyclic guanosine monophosphate (cGMP) pathway during meiosis resumption in bovine oocytes. Theriogenology. 2014;81(4):556-64. PMid:24331454.

Sengenès C, Berlan M, Glisezinski I, Lafontan M, Galitzky J. Natriuretic peptides: a new lipolytic pathway in human adipocytes. FASEB J. 2000;14(10):1345-51. PMid:10877827.

Sengenès C, Bouloumié A, Hauner H, Berlan M, Busse R, Lafontan M, Galitzky J. Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes. J Biol Chem. 2003;278(49):48617-26. PMid:12970365.

Sessa WC, Pritchard K, Seyedi N, Wang J, Hintze TH. Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circ Res. 1994;74(2):349-53. PMid:7507417.

Sudano MJ, Santos VG, Tata A, Ferreira CR, Paschoal DM, Machado R, Buratini J, Eberlin MN, Landim-Alvarenga FD. Phosphatidylcholine and Sphingomyelin Profiles Vary in Bos taurus indicus and Bos taurus taurus In Vitro- and In Vivo-Produced Blastocysts. Biol Reprod. 2012;87(6):130. PMid:23053436.

Törnell J, Billig H, Hillensjo T. Resumption of rat oocyte meiosis is paralleled by a decrease in guanosine 3′,5′ - cyclic monophosphate (cGMP) and is inhibited by microinjection of cGMP. Acta Physiol Scand. 1990;139(3):511-7. PMid:2173353.

Zhang M, Su Y-Q, Sugiura K, Xia G, Eppig JJ. Granulosa cell Ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes. Science. 2010;330(6002):366-9.

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