Abstract
When removed from the follicles, during the 44 h process of in vitro maturation (IVM) fully grown porcine oocytes resume meiosis spontaneously from the late diplotene stage of the first meiotic prophase and proceed to the metaphase-II (MII) stage at which they remain arrested until fertilization. However, the spontaneous resumption may start at various times causing heterogeneity in the nuclear stage and also in cytoplasmic characteristics within a population. Those oocytes that reach the MII stage earlier than others undergo an aging process which is detrimental for further embryo development. The synchronization of nuclear progression of porcine oocytes can be achieved by a transient inhibition of meiotic resumption during the first 20–22 h of IVM by the elevation of intracellular levels of cyclic adenosine monophosphate (cAMP) using the cellular membrane-permeable analog of cAMP, dibutyryl cyclic AMP. A simple and efficient protocol for such treatment is described below.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Betthauser J, Forsberg E, Augenstein M, Childs L, Eilertsen K, Enos J et al (2000) Production of cloned pigs from in vitro systems. Nat Biotech 18:1055–1059
Motlik J, Fulka J (1976) Breakdown of the germinal vesicle in pig oocytes in vivo and in vitro. J Exp Zool 198:155–162
Pincus G, Enzmann EV (1935) The comparative behavior of mammalian eggs in vivo and in vitro. I. The activation of ovarian eggs. J Exp Med 62:665–675
Wehrend A, Meinecke B (2001) Kinetics of meiotic progression, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes: species specific differences in the length of the meiotic stages. Anim Reprod Sci 66:175–184
Funahashi H, Cantley TC, Day BN (1997) Preincubation of cumulus–oocyte complexes before exposure to gonadotropins improves the developmental competence of porcine embryos matured and fertilized in vitro. Theriogenology 47:679–686
Nagai T, Ebihara M, Onishi A, Kubo M (1997) Germinal vesicle stages in pig follicular oocytes collected by different methods. J Reprod Dev 43:339–343
Funahashi H, Cantley TC, Day BN (1997) Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization. Biol Reprod 57:49–53
Somfai T, Kikuchi K, Onishi A, Iwamoto M, Fuchimoto D, Papp AB et al (2004) Relationship between the morphological changes of somatic compartment and the kinetics of nuclear and cytoplasmic maturation of oocytes during in vitro maturation of porcine follicular oocytes. Mol Reprod Dev 68:484–491
Miao YL, Kikuchi K, Sun QY, Schatten H (2009) Oocyte aging: cellular and molecular changes, developmental potential and reversal possibility. Hum Reprod Update 15:573–585
Grupen CG, Nagashima H, Nottle MB (1997) Asynchronous meiotic progression in porcine oocytes matured in vitro: a cause of polyspermic fertilization? Reprod Fertil Dev 9:187–191
Grupen CG (2014) The evolution of porcine embryo in vitro production. Theriogenology 81:24–37
Alfonso J, García-Rosello E, García-Mengual E, Salvador I, Silvestre MA (2009) The use of R-roscovitine to fit the “time frame” on in vitro porcine embryo production by intracytoplasmic sperm injection. Zygote 17:63–70
Lazzari G, Galli C, Moor RM (1992) Centrifugal elutriation of porcine oocytes isolated from the ovaries of newborn piglets. Anal Biochem 200:31–35
Banfalvi G (2008) Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation. Nat Protoc 3:663–673
Yoshioka K, Suzuki C, Onishi A (2008) Defined system for in vitro production of porcine embryos using a single basic medium. J Reprod Dev 54:208–213
Mcgaughey RW, Montgomery DH, Richter JD (1979) Germinal vesicle configurations and patterns of polypeptide synthesis of porcine oocytes from antral follicles of different size as related to their competency for spontaneous maturation. J Exp Zool 209:239–254
Marchal R, Vigneron C, Perreau C, Bali-Papp Á, Mermillod P (2002) Effect of follicular size on meiotic and developmental competence of porcine oocytes. Theriogenology 57:1523–1532
Iwamoto M, Onishi A, Fuchimoto D, Somfai T, Takeda K, Tagami T et al (2005) Low oxygen tension during in vitro maturation of porcine follicular oocytes improves parthenogenetic activation and subsequent development to the blastocyst stage. Theriogenology 63:1277–1289
Fuchimoto D, Senbon S, Suzuki S, Onishi A (2011) Effects of preservation of porcine oocytes by dibutyryl cyclic AMP on in vitro maturation, fertilization and development. Jpn Agr Res Q 45:295–300
Somfai T, Kikuchi K, Onishi A, Iwamoto M, Fuchimoto D, Papp AB et al (2003) Meiotic arrest maintained by cAMP during the initiation of maturation enhances meiotic potential and developmental competence and reduces polyspermy of IVM/IVF porcine oocytes. Zygote 11:199–206
Kim JS, Cho YS, Song BS, Weec G, Park JS, Choo YK et al (2008) Exogenous dibutyryl cAMP affects meiotic maturation via protein kinase A activation; it stimulates further embryonic development including blastocyst quality in pigs. Theriogenology 69:290–301
Ozawa M, Nagai T, Somfai T, Nakai M, Maedomari N, Fahrudin M et al (2008) Comparison between effects of 3-isobutyl-1-methylxanthine and FSH on gap junctional communication, LH-receptor expression, and meiotic maturation of cumulus-oocyte complexes in pigs. Mol Reprod Dev 75:857–866
Bagg MA, Nottle MB, Grupen CG, Armstrong DT (2006) Effect of dibutyryl cAMP on the cAMP content, meiotic progression, and developmental potential of in vitro matured pre-pubertal and adult pig oocytes. Mol Reprod Dev 73:1326–1332
Racowsky C (1985) Effect of forskolin on maintenance of meiotic arrest and stimulation of cumulus expansion, progesterone and cyclic AMP production by pig oocyte-cumulus complexes. J Reprod Fertil 74:9–21
Shimada M, Nishibori M, Isobe N, Kawano N, Terada T (2003) Luteinizing hormone receptor formation in cumulus cells surrounding porcine oocytes, and its role during meiotic maturation of porcine oocytes. Biol Reprod 68:1149–1159
Wu GM, Sun QY, Mao J, Lai L, McCauley TC, Park KW et al (2002) High developmental competence of pig oocytes after meiotic inhibition with a specific M-phase promoting factor kinase inhibitor, butyrolactone I. Biol Reprod 67:170–177
Marchal R, Tomanek M, Terqui M, Mermillod P (2001) Effects of cell cycle dependent kinases inhibitor on nuclear and cytoplasmic maturation of porcine oocytes. Mol Reprod Dev 60:65–73
Schoevers EJ, Bevers MM, Roelen BA, Colenbrander B (2005) Nuclear and cytoplasmic maturation of sow oocytes are not synchronized by specific meiotic inhibition with roscovitine during in vitro maturation. Theriogenology 63:1111–1130
Coy P, Romar R, Ruiz S, Cánovas S, Gadea J, García Vázquez F et al (2005) Birth of piglets after transferring of in vitro-produced embryos pre-matured with R-roscovitine. Reproduction 129:747–755
Sugimura S, Ritter LJ, Rose RD, Thompson JG, Smitz J, Mottershead DG et al (2015) Promotion of EGF receptor signaling improves the quality of low developmental competence oocytes. Dev Biol 403:139–149
Kikuchi K, Onishi A, Kashiwazaki N, Iwamoto M, Noguchi J, Kaneko H et al (2002) Successful piglet production after transfer of blastocysts produced by a modified in vitro system. Biol Reprod 66:1033–1041
Hirao Y, Tsuji Y, Miyano T, Okano A, Miyake M, Kato S et al (1995) Association between p34cdc2 levels and meiotic arrest in pig oocytes during early growth. Zygote 3:325–332
Shimada M, Ito J, Yamashita Y, Okazaki T, Isobe N (2003) Phosphatidylinositol 3-kinase in cumulus cells is responsible for both suppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes. J Endocrinol 179:25–34
Maedomari N, Kikuchi K, Ozawa M, Noguchi J, Kaneko H, Ohnuma K et al (2007) Cytoplasmic glutathione regulated by cumulus cells during porcine oocyte maturation affects fertilization and embryonic development in vitro. Theriogenology 67:983–993
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Somfai, T., Hirao, Y. (2017). Synchronization of In Vitro Maturation in Porcine Oocytes. In: Banfalvi, G. (eds) Cell Cycle Synchronization. Methods in Molecular Biology, vol 1524. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6603-5_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6603-5_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6602-8
Online ISBN: 978-1-4939-6603-5
eBook Packages: Springer Protocols