Effects of caffeine, cumulus cell removal and aging on polyspermy and embryo development on in vitro matured and fertilized ovine oocytes
Introduction
In the majority of mammals, oocytes are ovulated at metaphase of the second meiotic division (MII). The ovulated, matured oocyte or unfertilized egg then remains at MII until fertilization occurs and development begins. Alternatively, oocytes can be activated artificially by a range of physical or chemical stimuli including electric shock, ethanol, Ca2+ ionophore, or Sr2+, these treatments can be applied individually or in combination with the protein synthesis inhibitor cycloheximide or the serine threonine kinase inhibitor di-methyl-amino-purine (DMAP) [1]. The maturation of oocytes from the germinal vesicle (GV) stage to MII is a dynamic process that requires coordination of both nuclear and cytoplasmic processes [2]. The control of nuclear maturation is intrinsically linked to the levels of two cytoplasmic protein kinases, maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) [3]. MPF is a cyclin dependent serine/threonine protein kinase, its activation occurs in late G2 by de-phosphorylation of T14 and Y15 by cdc25 phosphatase [4]. Active MPF phosphorylates a range of proteins initiating entry into M-phase resulting in nuclear envelope breakdown, chromatin condensation and microtubular reorganization [5], [6], [7]. MAPKs are serine/threonine kinases that require phosphorylation on threonine and tyrosine residues to become activated [8], [9], this involves a cascade of upstream kinases [9]. The increases in the activities of both kinases are responsible for the onset of germinal vesicle breakdown (GVBD) and required for the arrest of oocytes at metaphase of second meiotic division (MII) [10], [11], [12], [13], [14], MII arrest is then maintained by continued high activities of both kinases [12], [14], [15].
The matured (MII) oocyte acquires fertilization competence, however, the lifespan window for fertilization varies between different species [16]. If oocytes are not fertilized during this optimal time frame, then they consequently age. Aging is associated with a range of changes including; alteration of intracellular Ca2+ dynamics [17], decreases in the activities of both MPF and MAP kinases [18], an increase in activation sensitivity [19], alteration of cortical granule release and increased risk of polyspermy [20]. In addition a deterioration of the spindle can result in the loss of attachment of kinetochores to the spindle fibres and displacement of the chromosomes from the spindle equator [21], [22], [23]. Furthermore, an increased frequency of fragmentation, with decreased frequencies of cleavage and development to the blastocyst stage have been reported in a variety of species [16], [20], [24], [25].
MPF activity is controlled by association of cdc2 with cyclin B and phosphorylation of cdc2 at T14 and Y15. Caffeine, a phosphodiesterase inhibitor has been reported to artificially increase the activity of MPF by inducing the dephosphorylation of cdc2 at T14 and Y15 in pig oocytes [26], [27], cultured mammalian cells [28] and Xenopus oocytes [29]. However, it cannot restore loss of MPF activity caused by degradation of cyclin B, which occurs on aging in pig oocytes [26]. We have previously reported that treatment of in vitro matured ovine oocytes with caffeine increases activities of both MPF and MAPK and prevents the decline in kinase activities associated with aging. Furthermore, maintaining the levels of both kinases in aging oocytes prevented the acquisition of activation sensitivity [30]. In addition, caffeine treated ovine oocytes used for nuclear transfer resulted in an increased occurrence of nuclear envelope breakdown (NEBD) in the transferred nuclei, and in blastocysts with a significantly higher cell number than control groups, however, there was no improvement in the frequency of development to the blastocyst stage [31]. In this manuscript, the effects of caffeine on the incidence of polyspermy, frequency of embryo cleavage and development to blastocyst of ovine oocytes aged and then fertilized in vitro are reported and discussed.
Section snippets
Materials and methods
All chemicals and reagents were purchased from Sigma–Aldrich, Dorset, UK, unless otherwise stated.
Caffeine decreases the frequency of polyspermy in denuded oocytes aged in vitro prior to in vitro fertilization
After 24 h of in vitro maturation, 85–95% of ovine oocytes reached metaphase of the second meiotic division (MII) as confirmed by the presence of the first polar body (PBI). The results from the development of cumulus oocyte complexes (COC's) fertilized at 24 hpm (control; Group A) were used as a reference control for the treatment groups. The percentage of polyspermy varied between 3 and 21% dependent on the protocol (in the presence or absence of cumulus cells) or on the treatment (in the
Discussion
This manuscript reports the effects of caffeine on aging of in vitro cultured ovine oocytes before fertilization. To our knowledge, this is the first paper reporting the benefits of caffeine on fertilization of ageing oocytes. As described earlier, elevated levels of maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) activities maintain the oocytes arrested at metaphase of the second meiotic division (MII). In most mammalian species, the level of activities of both
Acknowledgement
This work was supported by the University of Nottingham.
References (51)
- et al.
In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase
Cell
(1990) - et al.
Okadaic acid accelerates germinal vesicle breakdown and overcomes cycloheximide- and 6-dimethylaminopurine block in cattle and pig oocytes
Dev Biol
(1993) - et al.
Mitogen-activated protein kinase kinase is required for the mos-induced metaphase arrest
J Biol Chem
(1994) - et al.
Fertilization and early cleavage in vitro of ageing bovine oocytes after maturation in culture
Therio
(1992) - et al.
Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34cdc2
Cell
(1992) - et al.
High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins
Theriogenology
(1999) - et al.
Effects of caffeine on in vivo and in vitro oocyte maturation in mice
Theriogenology
(2007) - et al.
Caffeine effects on meiotic maturation in hamster oocytes in vitro
Reprod Toxicol
(1992) - et al.
Acquisition of meiotic competence by denuded mouse oocytes: participation of somatic-cell product(s) and cAMP
Dev Biol
(1994) - et al.
Phosphatase inhibition leads to histone deacetylases 1 and 2 phosphorylation and disruption of corepressor interactions
J Biol Chem
(2002)
MAP kinase-mediated phosphoacetylation of histone H3 and inducible gene regulation
FEBS Lett
Inhibition of histone deacetylases alters allelic chromatin conformation at the imprinted U2af1-rs1 locus in mouse embryonic stem cells
J Biol Chem
Mammalian oocyte activation: lessons from the sperm and implications for nuclear transfer
Int J Dev Biol
Morphodynamics of nuclear and cytoplasmic reorganization during the resumption of arrested meiosis in the mouse oocyte
Prog Clin Biol Res
Control of the cell cycle in early embryos
On cyclins, oocytes, and eggs
Mol Reprod Dev
Mitogen-activated protein kinases phosphorylate nuclear lamins and display sequence specificity overlapping that of mitotic protein kinase p34cdc2
Eur J Biochem
Regulation of microtubule dynamics by CDC2 protein-kinase in cell-free extracts of Xenopus eggs
Nature
Requirements for phosphorylation of MAP kinase during meiosis in Xenopus oocytes
Science
The MAPK signaling cascade
FASEB J
Universal control mechanism regulating the onset of M-phase
Nature
Potential role of mitogen-activated protein kinase during meiosis resumption in bovine oocytes
Biol Reprod
Cytoplasmic control of nuclear behaviour during meiotic maturation of frog oocytes
J Exp Zool
Cell cycle dynamics of maturation-promoting factor during mouse oocyte maturation
Tokai J Exp Clin Med
Mechanisms underlying oocyte activation and postovulatory ageing
Reproduction
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- 1
Present address: University of Edinburgh W3.33 Centre for CVS, QMRI, 47 Litlle France Crecent, Edinburgh EH16 4TJ, UK.
- 2
Present address: Animal Development and Biotechnology Group, Division of Applied Life Science, College of Agriculture and Life Science, Gyeongsang National University, Jinju, Gyeongnam 660-701, South Korea.