Effects of caffeine, cumulus cell removal and aging on polyspermy and embryo development on in vitro matured and fertilized ovine oocytes

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Theriogenology 71 (2009) 1083–1092 www.theriojournal.com

Effects of caffeine, cumulus cell removal and aging on polyspermy and embryo development on in vitro matured and fertilized ovine oocytes W.E. Maalouf 1, J.-H. Lee 2, K.H.S. Campbell * Animal Development and Biotechnology Group, School of Biosciences, Division of Animal Physiology, School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough, Leicestershire LE12 5RD, UK Received 8 April 2008; received in revised form 25 November 2008; accepted 5 December 2008

Abstract The objectives of these studies were to determine the effects of cumulus cell removal and caffeine treatment on the development of in vitro matured ovine oocytes aged in vitro until until fertilization. Oocytes were denuded (DO) at 24 h post-onset of maturation (hpm), control cumulus oocyte complexes (COC’s) and DO groups were fertilized at 24 hpm or returned to culture in the presence or absence of 10 mM caffeine and fertilized at 30 hpm. Removal of cumulus cells and aging both increased polyspermy, caffeine reduced this increase, however, with the exception of DO’s (30 hpm) vs. COC’s (24 hpm) the differences were not statistically significant. Aging significantly decreased cleavage between COC groups at 24 hpm and 30 hpm and caffeine did not affect this (68.4%, 73.4%, 74.0% respectively). In contrast, the frequency of cleavage was significantly reduced in the DO (24 hpm) group as compared to COC controls (45.6% vs. 68.4% (P < 0.05)), however, cleavage increased in the DO group on aging (73.4%) and this was not affected by caffeine (73.0%). The percentage of COC’s and DO’s developing to the blastocyst stage significantly decreased on aging, caffeine treatment of DO’s prevented this (31.3%, 12.7% and 29.4% respectively (P < 0.05)) but had no effect on COC’s (4.2% vs. 3.9%). Total cell numbers in blastocysts were not statistically different (92.4  5.2, 84.7  3.7 and 80.4  5.8 (P > 0.05)). In summary caffeine treatment of aged COC’s had no significant effect on the frequency of development, however, in aged DO’s caffeine treatment statistically increased development to blastocyst and lowered the frequency of polyspermy. # 2009 Elsevier Inc. All rights reserved. Keywords: Ovine; Oocyte; Embryo; Caffeine; Polyspermy; MPF; MAPK

1. Introduction

* Corresponding author. Tel.: +44 115 951 6298 fax: +44 115 951 6302. E-mail address: [email protected] (K.H.S. Campbell). 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 660701, South Korea. 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.12.001

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

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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 [5–7]. 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) [10–14], 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 [21–23]. 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. 2. Materials and methods All chemicals and reagents were purchased from Sigma–Aldrich, Dorset, UK, unless otherwise stated. 2.1. Collection of oocytes Ovine ovaries were collected from a local slaughter house in warm (25 8C) phosphate buffered saline (PBS), transferred to the laboratory and processed within a maximum of 2 h after collection. In the laboratory, cumulus oocyte complexes (COC’s) were aspirated from follicles of 2–10 mm in diameter using a 10 mL syringe fitted with an 18-gauge needle. COC’s with a uniform cytoplasm and at least 3 layers of unexpanded cumulus cells were selected for subsequent maturation. 2.2. In vitro oocyte maturation Selected oocytes were washed three times in dissection medium (Medium 199 containing 10% FCS (Gibco Life Technologies Inc., Paisley, UK), and 1 M HEPES), and then once in maturation medium (Medium 199 containing 10% FCS (Gibco Life Technologies Inc., Paisley, UK), 5 mg/mL FSH (Vetropharm, Ireland), 5 mg/mL LH (Vetropharm, Ireland), 1 mg/mL oestradiol-17b and 50 mg/mL gentamicin). For maturation, groups of 40–45 oocytes were cultured in 500 mL of maturation medium overlaid with mineral oil in 4-well dishes (Nunclon, Rosklide, Denmark) and incubated at 39 8C in a humidified atmosphere of 5% CO2 for a period of 24 or 30 h depending on the experimental group.

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2.3. Removal of cumulus cells COC’s were denuded by vortexing in PBS containing 300 IU/mL of hyaluronidase and 1% polyvinylpyrollidine (PVP) for 4–5 min followed by gentle pipetting, they were then washed thee times in HEPES-buffered modified synthetic oviductal fluid medium (H-SOF; ref. [32]). Cumulus free (denuded oocytes) were selected on the basis of a uniform cytoplasm and presence of a polar body and termed (DO) thereafter. 2.4. In vitro fertilization of ovine oocytes and embryo culture In vitro fertilization of ovine oocytes Good quality c was carried out as previously described [33]. Briefly, cryopreserved semen pellets collected from one ejaculate of a Texel ram with an excellent post-thaw motility (Britbreed, UK) were thawed in a pre-warmed glass tube. The thawed semen was divided into two and each half layered on top of a double density layer of percoll solutions (1 mL of 45% over 1 mL of 90% v:v in sperm washing medium) in a 15 mL polystyrene conical tube (Bibby Sterilin, Stone, UK). The tubes were centrifuged at 1000  g for 7 min at 4 8C. The supernatants were discarded and the pellets mixed and suspended in 10 mL of sperm wash medium. The sperm suspension was then centrifuged at 600  g for 5 min. The supernatant was removed leaving 0.1–0.2 mL of media above the pellet, 0.8–0.9 mL of ovine fertilization medium was then added and the number of sperm counted using an improved Neubauer counting chamber. The concentration of the sperm suspension was adjusted to 2.0–4.0  106 mL live sperm by addition of ovine fertilization medium. Drops of 450 mL of the final sperm suspension were prepared in four-well dishes (Nunclon, Roskilde, Denmark), overlaid with equilibrated embryotested mineral oil, and kept in a humidified incubator at 398C, and 5% CO2 until the oocytes were prepared. COC’s or DOs from all groups were washed once in ovine fertilization medium, and then groups of 40–50 oocytes were transferred into the prepared sperm drops and cultured for 20 h at 39 8C in a humidified atmosphere of 5% CO2. After 20 h (day 1), all embryos were washed twice in H-SOF medium, and transferred into pre-equilibrated modified synthetic oviduct fluid medium containing 2% (v:v) BME-essential amino acids, 1% (v:v) MEM-nonessential amino acids, and 2.75 mM myo-inositol (C-SOF) supplemented with 3 mg/mL fraction V fatty acid free BSA (bovine serum albumin; C-SOF-BSA) for 48 h (day 2). On day 2, cleaved embryos were transferred into fresh C-SOF medium

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supplemented with 10% FCS (C-SOF-FCS). Embryo culture was carried out at 39 8C in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2. 2.5. Treatment of oocytes with caffeine An average of 65 oocytes for each treatment group were incubated in 500 mL drops of fresh pre-equilibrated maturation medium containing 10 mM caffeine at 39 8C in a humidified atmosphere of 5% CO2 in air. 2.6. Experimental design Six groups, including four controls and two treatment groups, were prepared as outlined below and in Fig. 1; Group A: COC’s were cultured in IVM media until fertilization at 24 hpm. Group B: COC’s were cultured in IVM media until 24 hpm. Cumulus cells were removed and then the denuded metaphase II oocytes (DO’s) were fertilized. Group C: COC’s were cultured in IVM media, cumulus cells were removed at 24 hpm. DO’s were cultured in fresh IVM medium and fertilized at 30 hpm. Group D: COC’s were cultured in IVM media for 24 h, cumulus cells were removed, DO’s were cultured in fresh IVM containing 10 mM caffeine for a further 6 h of culture and fertilized at 30 hpm. Group E: COC’s were cultured in IVM media for 24 h, transferred into fresh IVM medium and fertilized at 30 hpm. Group F: COC’s cultured in IVM media for 24 h, were transferred into fresh IVM medium containing 10 mM caffeine for a further 6 h of culture and fertilized at 30 hpm. 2.7. Determination of polyspermy At 20 h post-insemination (hpi; day 1), a number of putative zygotes were removed from each treatment group, washed thoroughly in PBS containing 0.1% PVA (PBS–PVA) and then fixed in 70% ethanol containing 50 mM glycine in a glass well at 20 8C for 20 min. After fixation, zygotes were washed well in PBS–PVA, then mounted on a glass slide in Vectashield medium containing DAPI (Vector Laboratories, USA) and covered with thin cover-slip (SLS, UK). Samples were examined using a fluorescence microscope (Leica DMR, Heidelberg, Germany) fitted with a digital camera (Hammamatsu, ORCA-er, Japan) and image analysis software (Simple PCI, Compix Inc., USA). The number of pronuclei in each fertilized zygote was counted; zygotes containing two pronuclei were considered normal, those containing three or more

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Fig. 1. Diagramatic representation of experimental design. Group A: COC’s were cultured in IVM media until fertilization at 24 hpm. Group B: COC’s were cultured in IVM media until 24 hpm. Cumulus cells were removed and then the denuded oocytes (DO’s) were fertilized. Group C: COC’s were cultured in IVM media, cumulus cells were removed at 24 hpm. DO’s were then cultured in fresh IVM medium and fertilized at 30 hpm. Group D: COC’s were cultured in IVM media for 24 h, cumulus cells were removed, DO’s were cultured in fresh IVM containing 10 mM caffeine for a further 6 h of culture and fertilized at 30 hpm. Group E: COC’s were cultured in IVM media for 24 h, transferred into fresh IVM medium and fertilized at 30 hpm. Group F: COC’s cultured in IVM media for 24 h, were transferred into fresh IVM medium containing 10 mM caffeine for a further 6 h of culture and fertilized at 30 hpm.

pronuclei were considered polyspermic. The number of unfertilized oocytes was also noted. 2.8. Determination of the frequency of cleavage On day 2 the number of cleaved embryos in each group was recorded. The presence or absence of fragmentation was also noted as a general indicator of embryo quality. 2.9. Determination of blastocyst development, total cell number and ratio of ICM:TE cells On day 7, the number of blastocyst stage embryos was counted and expressed as a percentage of the total number of cleaved embryos selected on day 2. All blastocysts were double-stained as previously described [34] to obtain total cell numbers as well as differentiate trophectoderm (TE) cells from inner cell mass (ICM) cells. In summary, the blastocysts were fixed and the TE cells were permeabilized to stain red with propidium iodide (PI), while the non-permeabilized ICM cells stained only blue with 40 ,6-diamidino-2-phenylindole (DAPI) (Fig. 2). Total cell numbers and the ratio of ICM:TE were recorded.

2.10. Statistical analysis At least three replicates were carried out for each experimental group with an average of 20 embryos per replicate. Two-tailed T-test for binomial proportions or Fisher’s exact test were used to compare the frequencies of polyspermy, cleavage and blastocyst development across all six treatments depending on whether the data passed the test for normality or not respectively. Twotailed T-test for means or Mann–Whitney test were used to compare the means or ratios depending if the data passed the test for normality or not respectively. Statistical significance was reported when P was <0.05. 3. Results 3.1. 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

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Fig. 2. Differerential staining of day 7 ovine blastocysts; ICM cells stained blue with DAPI and TE cells stained red with PI. (A) Hatched control blastocyst derived from control untreated oocytes and (B) non-hatched blastocyst derived from 6 h caffeine-treated oocytes. Arrow indicates location of the inner cell mass. Scale bar = 10 mM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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 presence or absence of caffeine) used. COC’s fertilized at 24 h hpm (Group A) had the lowest frequency of polyspermy (3.0  1.7%; Fig. 3), in contrast denuded oocytes fertilized at the

same time point (DO control; Group B) showed an increased frequency of polyspermy (8.1  4.4%; Fig. 3), but this was not statistically significant (P > 0.05; Fig. 3). Zygotes produced after denuding oocytes at 24 hpm and fertilizing at 30 hpm (aged control; Group C) had the highest frequency of polyspermy (20.6  7.3%) and this differed significantly from the control group (P < 0.05; Fig. 3).

Fig. 3. Effects of aging, cumulus removal and caffeine treatment of in vitro matured and fertilized ovine oocytes on the frequency of polyspermy, cleavage and blastocyst developmental rates in different ovine IVF treatment groups. COC: cumulus oocyte complex; D: denuded oocytes; Ctrl: controls in the absence of caffeine; Caff: caffeine treated groups. Statistically significant differences are marked by different superscripts (P < 0.05).

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Oocytes that were denuded at 24 hpm and treated with 10 mM caffeine until fertilization at 30 hpm (Group D) had a lower frequency of polyspermy (10.2  4.8%) than non caffeine treated controls, but there was no statistical significance (P > 0.05; Fig. 3). COC’s fertilized at 30 hpm after treatment (Group F) or no treatment (Group E) with 10 mM caffeine for 6 h resulted in similar frequencies of polyspermy (15.3  5.0% and 15.0  7.4% respectively), but were not statistically different from each other or from any other treatment group (P > 0.05; Fig. 3). 3.2. Caffeine did not affect the frequency of cleavage of in vitro fertilized normal or aged oocytes The frequency of cleavage of zygotes derived form COC’s that were fertilized at 24 hpm (Group A), and denuded oocytes that were fertilized at 30 hpm (Group C) or fertilized at 30 hpm but after 6 h treatment with 15 mM caffeine (DO control; Group D) varied between 65% and 75%, however, there were no statistical significant differences between groups (P > 0.05; Fig. 3). In contrast oocytes that were denuded and fertilized at 24 hpm (Group B) had a statistically significant lower frequency of cleavage (45.6  5.6%) than Groups A, C and D (P < 0.05; Fig. 3). COC’s that were fertilized without (aged COC control; Group E) or with caffeine (COC aged with caffeine; Group F) had significantly lower frequencies of cleavage than all other groups (23.3  3.8% and 26.0  5.0% respectively; P < 0.05; Fig. 3). 3.3. Caffeine improves developmental to blastocyst of aged oocytes denuded and in vitro fertilized The frequency of development to blastocyst was calculated as the number of blastocysts which developed from cleaved embryos present on day 2 postinsemination. COC’s that were fertilized at 24 hpm (Group A) and denuded oocytes treated with 10 mM caffeine and fertilized at 30 hpm (Group D) had the highest frequency of development to blastocyst (31.3  3.8% and 29.4  3.9% respectively) and were significantly higher than the frequency of blastocyst development in all other treatment groups (P < 0.05; Fig. 3). Denuded oocytes fertilized at 24 hpm (Group B) or at 30 hpm (Group C) did not differ statistically in the frequency of development to blastocyst (15.2  5.0% and 12.7  3.1% respectively (P > 0.0; Fig. 3)), however both had a significantly higher frequency of development to blastocyst than COC’s fertilized at

30 hpm without (Group E) or with caffeine (Group F) (4.2  1.8% and 3.9  2.2% respectively; P < 0.05; Fig. 3). The mean cell number (MCN) of day 7 blastocysts from COC’s that were fertilized at 24 hpm (Group A) was 85.3  4.2, this was significantly higher than the MCN of blastocysts obtained from COC’s denuded and fertilized at 24 hpm (Group B) and COC’s incubated with 10 mM caffeine between 24 hpm and fertilization at 30 hpm (Group F) with MCNs of 76.1  2.7 and 62.3  3.8 respectively (P < 0.05; Fig. 4). The MCN of day 7 blastocysts produced from denuded oocytes fertilized at 24 hpm (Group C) was 96.2  8.5, this was not significantly different from any of the other treatment groups (P > 0.05; Fig. 4). Denuded oocytes treated with 10 mM caffeine and fertilized at 30 hpm (Group D) had a MCN of day 7 blastocysts of 107.8  6.7, this was significantly increased as compared to the MCN of blastocysts from Group E (COC’s fertilized at 30 hpm) of 67.5  4.9 and Group F (62.3  3.8) (P < 0.05; Fig. 4). The ratio of inner cell mass (ICM) cells to trophectodermal (TE) cells (ICM:TE) in blastocyst stage embryos was statistically similar in all groups (P > 0.05; Fig. 5). 4. 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 kinases decrease in MII oocytes after an extended period of maturation in vitro [18,30,31]. In addition, in vitro aged then fertilized oocytes exhibit an increased frequency of polyspermy and fragmentation, a decrease in the frequency of cleavage and a decrease in frequency of development to the blastocyst stage [20,24,25]. Previous studies in our laboratory have reported that treatment of ovine oocytes with 10 mM caffeine from 18 to 24 hpm can increase the activities of both MPF and MAPK in MII oocytes. In addition treatment of MII oocytes from 24 to 30 hpm with 10 mM caffeine can prevent the decline in MPF and MAPK activities associated with aging and also prevent the acquisition of activation competence [30,31]. Following these observations, we hypothesised that by maintaining the activities of MPF and MAPK during

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Fig. 4. Effects of aging, denudation and caffeine treatment of in vitro matured and fertilized ovine oocytes on the on the mean cell numbers of day 7 blastocysts cultured in vitro. COC: cumulus oocyte complex; D: denuded oocytes; Ctrl: controls in the absence of caffeine; Caff: caffeine treated groups. Statistically significant differences is marked by different superscripts (P < 0.05).

oocyte aging, the window for optimal fertilization may be extended. Oocytes that were denuded at 24 hpm and fertilized at 30 hpm resulted in zygotes with a significantly higher frequency of polyspermy than control oocytes. This observation was expected since cumulus cells play a role in filtering out the subfertile spermatozoa [35] and aged oocytes fail to block polyspermy which could be

due to dysfunctional cortical granule (CG) exocytosis, or reduced CG density at the cytoplasmic membrane as reported in some mammalian oocytes [20]. COC’s that were aged and fertilized at 30 hpm did not result in a significant increase in polyspermy, suggesting that cumulus cells may play a major role in blocking polyspermy [35]. In the studies reported here, treatment of COC’s and cumulus stripped oocytes with caffeine

Fig. 5. Effects of aging, cumulus removal and caffeine treatment of in vitro matured and fertilized ovine oocytes on the ICM to TE ratios in day 7 blastocysts cultured in vitro. COC: cumulus oocyte complex; D: denuded oocytes; Ctrl: controls in the absence of caffeine; Caff: caffeine treated groups. Statistically significant differences are marked by different superscripts (P < 0.05).

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for a period of 6 h between 24 hpm and 30 hpm reduced the frequency of polyspermy but this was only of borderline significance. The mechanism/s through which caffeine acts upon cumulus oocyte complexes probably differ to those which act on denuded oocytes directly. In the mouse, caffeine was reported to reduce the frequency of maturation and to decrease survival of oocytes matured in vitro dependent upon concentration [36]. The reasons for the differences to our observations are probably many, however, we have previously reported that survival of ovine oocytes is dependent upon the concentration of caffeine and the duration of exposure [30]. In the studies reported in the mouse, COC’s were exposed throughout the maturation period. In our previous studies in sheep, denuded oocytes were not exposed until 18 h post-onset of maturation, when oocytes were at anaphase/telophase of the 1st meiotic division (AI/TI) or until 24 hpm when at MII. In both cases, exposure to 10 mM caffeine for a period over 6 h, or a concentration greater than 10 mM reduced oocyte viability. In the present studies oocytes were not treated with caffeine until having reached MII. The mechanism of action of caffeine may differ in COC’s and denuded oocytes. In COC’s caffeine, like other purine derivatives, possibly acts by blocking cAMP phosphodiesterase and cdc2 kinase, increasing cAMP levels and thus changing the signalling pathways [37–39]. In a number of mammalian oocytes, caffeine is known to increase and maintain the activities of MPF and MAPK in denuded oocytes possibly by inhibition of Myt1/Wee1 kinases [31,40], however, other mechaisms cannot be excluded. The reduction in the frequency of polyspermy following caffeine treatment may be the result of a number of mechanisms. Firstly, maintaining high activities of MPF and MAPK may prevent some of the changes associated with aging, for instance it was reported that increased activity of MPF reorganises the endoplasmic reticulum and increases the dynamics of calcium ions in maturing mouse [41] and marine worm [42] oocytes. The release of intracellular Ca2+ mediates a block to polyspermy [43]. In MII bovine oocytes, inhibition of MAPK with the inhibitor U2016, increased the incidence of polyspermy suggesting that an ERKlike cascade is part of a mechanism that controls cortical granule reaction [44]. Although, in the studies reported here caffeine was removed after fertilization, other studies have reported that caffeine treatment can decrease histone acetylation in aged mouse oocytes, suggesting that epigenetic changes occur during oocyte aging [45]. Caffeine may affect such changes through elevated kinase activities; in particular MAPK has been implicated in a number of pathways which may regulate

epigenetic state [45–49]. In addition inhibition of MAPK during the 1st cell cycle of bovine oocytes results in an increased incidence of aberrant spindle microtubule assembly and cell division [44]. The second parameter that was compared between treatments was the frequency of cleavage on day two after insemination. The physical effects of cumulus removal followed by immediate fertilization may explain the lower fertilization rate for Group B. In contrast, oocytes that were denuded at 24 hpm and then fertilized at 30 hpm had a significantly higher frequency of fertilization; one explanation for this is that these oocytes were allowed 6 h to recover from any physical stress due to vortexing used in removal of cumulus cells, prior to fertilization. The fertilization capability of the sperm was not reduced 6 h after preparation as demonstrated by the development of embryos from Group D (30 hpm DO with caffeine). The fertilization of COC’s at 30 hpm in the presence or absence of caffeine was significantly lower than in all other groups. The actual time of fertilization in the latter two groups is more likely to be around 34 hpm, and this is because spermatozoa require more time to penetrate between the cumulus cells before reaching the zona pellucida (ZP) [50]. In contrast, in denuded oocytes that were inseminated at 30 hpm, fertilization was almost complete within 1 h from the insemination time, and thus these oocytes had a significantly higher frequency of cleavage than their non-denuded counterparts. Caffeine treatment of denuded and non-denuded groups had no effect on the frequency of cleavage. However, the quality of two-cell embryos from the caffeine treated group appeared morphologically superior as less fragmentation was observed than in the appropriate control. The frequency of development to the blastocyst stage on day seven of aged denuded oocytes was significantly lower than the control group. In contrast, the supplementation of the oocyte maturation media with 10 mM caffeine for 6 h before insemination of denuded oocytes significantly improved frequency of development to levels comparable with Group A. However, caffeine did not have any effect on the frequency of development of COC’s; both treated and untreated groups of COC’s that were fertilized at 30 hpm resulted in a very low frequency of development to blastocyst. For the same reasons as suggested for differeces in cleavage, delayed fertilization due to the presence of cumulus cells [50] may explain the low frequency of development to blastocyst in these last two groups. The mean cell number (MCN) of blastocysts varied between treatments, but there was no significant difference

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observed in terms of caffeine effects. Only the last two groups demonstrated a lower MCN than all other groups irrespective of the presence or absence of caffeine. Although the ratio of ICM:TE varied between treatments, this was not significant. In summary, ovine denuded oocytes that were treated with caffeine had an improved rate of development to blastocyst but there were no effects on the quality of blastocysts produced in terms of mean cell number or ICM:TE ratios. Further studies are required to look into the quality and ploidy of the embryos produced after caffeine treatment. Thus, caffeine appears to extend the fertilization period of in vitro matured ovine oocytes by reducing polyspermy and retaining the ability to develop to the blastocyst. Since caffeine was recently reported to affect the in vitro development of nuclear transfer porcine embryos in relation to their nuclear reprogramming [51], effects in other species have to be studied before caffeine can be considered for improving human assisted reproductive technologies. Acknowledgement

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