cAMP modulation during sheep in vitro oocyte maturation delays progression of meiosis without affecting oocyte parthenogenetic developmental competence

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cAMP modulation during sheep in vitro oocyte maturation delays progression of meiosis without affecting oocyte parthenogenetic developmental competence Margaret Buell, James L. Chitwood, Pablo J. Ross ∗ Department of Animal Science, University of California, Davis, CA, United States

a r t i c l e

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Article history: Received 27 August 2014 Received in revised form 3 December 2014 Accepted 14 December 2014 Available online xxx Keywords: Oocyte maturation cAMP IBMX Forskolin Cilostamide GVBD

a b s t r a c t Removal of oocytes from their natural inhibitory follicular environment results in spontaneous resumption of meiosis independent of normal signaling events that occur in vivo. Controlling the onset of meiotic resumption via maintenance of elevated oocyte cAMP levels with adenylyl cyclase (AC) activation and phosphodiesterase (PDE) inhibition, and subsequent hormone stimulation with follicle FSH has been shown to dramatically improve developmental competence of bovine and murine IVM oocytes. This study evaluated the effect of cAMP modulation during IVM of sheep oocytes on meiotic progression and development to blastocyst after parthenogenetic activation. Changes in oocyte cAMP levels were quantified during the first 2 h of in vitro maturation in control or cAMP-modulating medium. No significant changes in intra-oocyte cAMP were observed under control conditions, though a slight and transient drop was noticed at 15 min of maturation. Addition of the AC stimulator Forskolin and the PDE inhibitors IBMX altered the cAMP profile, resulting in 10-fold elevation of cAMP by 15 min and sustained >3-fold elevated levels from 30 to 120 min. The effect of cAMP elevation on meiotic resumption was measured by completion of germinal vesicle breakdown. Modulated oocytes were significantly delayed when compared to control media oocytes. Also, progression to MII was significantly delayed in modulated versus control oocytes at 20 and 24 h, though no differences persisted to 28 h. Lastly, when control and modulated oocytes were parthenogenetically activated, no differences in blastocyst formation were observed. Thus, while cAMP modulation delayed meiotic progression, it did not improve developmental competence of sheep IVM oocytes. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Successful oocyte in vitro maturation (IVM) can facilitate the production of large numbers of embryos in a precisely controlled environment for research involving

∗ Corresponding author at: One Shields Avenue, Davis, CA 95616, United States. Tel.: +1 530 771 7225. E-mail address: [email protected] (P.J. Ross).

transgenics, cloning, and stem cells, as well as agricultural breeding programs and assisted fertility treatments. However, IVM oocytes are significantly less viable than those matured in vivo, as demonstrated by lower blastocyst yield, and implantation and live birth rates. This is, in part, because the mechanics of spontaneous maturation as they normally occur in vitro differ significantly from naturally induced maturational progression, resulting in significant abnormalities in the structure, composition and physiology between in vivo-derived (IVD) versus IVM

http://dx.doi.org/10.1016/j.anireprosci.2014.12.012 0378-4320/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Buell, M., et al., cAMP modulation during sheep in vitro oocyte maturation delays progression of meiosis without affecting oocyte parthenogenetic developmental competence. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2014.12.012

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oocytes (Gilchrist and Thompson, 2007; Hyttel and Fair, 1997; Hyttel et al., 1986, 1988, 1989; Martino et al., 1996; Pollard and Leibo, 1994; Rizos et al., 2002a). A complex interplay of autocrine and paracrine signaling events between the oocyte and its surrounding cumulus and granulosa cells is responsible for maintaining the highly regulated synchrony of in vivo maturation, and is critical for proper capacitation and acquisition of developmental competence. High levels of intra-oocyte 3 -5 -cyclic AMP (cAMP) are directly responsible for maintaining meiotic arrest until shortly prior to ovulation, when the LH-surge overrides this inhibition. High intra-oocyte cAMP levels are achieved by a mechanism that involves continuous production of cAMP by adenylyl cyclase (AC) and inhibition of phosphodiesterase (PDE) catalyzed cAMP hydrolysis. In vivo, the spike in LH at the pre-ovulatory gonadotropin surge leads to an indirect signaling cascade that overrides inhibitory cAMP signaling, leading to a drop in cAMP in the oocyte and its surrounding cumulus cells, inducing the synchronous, final maturation of the oocyte nucleus and ooplasm (Bilodeau-Goeseels, 2011; Liang et al., 2007; Sasseville et al., 2007; Zhang et al., 2007). Maturation of the ooplasm (cytoplasmic maturation) is not readily tangible, broadly referring to the ultra-structural and molecular changes that occur within the ooplasm that capacitate the oocyte to undergo fertilization and support embryonic and fetal development (Bevers and Dieleman, 1996). These changes include remodeling and redistribution of organelles, alterations in gene expression, accumulation of mRNA, alterations in protein expression and post-translational modifications (Rizos et al., 2002b; Sirard et al., 2006). When the immature oocyte is removed from the follicular environment, as in IVM, regulatory pathways are disrupted, resulting in asynchrony between the two critical maturational processes, nuclear and cytoplasmic (Albuz et al., 2010; Mehlmann, 2005). Premature nuclear maturation leads to inadequate cytoplasmic maturation at the time of fertilization, resulting in diminished developmental competence and reduced embryo yields. Spontaneous maturation has been linked to the critical secondary messenger cAMP; as removal of COCs from the follicular environment, and thus isolation from the mural granulosa cells, results in precipitous drop in intra-oocyte and cumulus cAMP levels (Albuz et al., 2010; Schultz et al., 1983; Vivarelli et al., 1983). Elevation of cAMP during IVM has been investigated for decades, and can be achieved through membrane permeant cAMP and cGMP analogues, or through direct stimulation of AC and inhibition of PDE (Aktas et al., 1995a; BilodeauGoeseels, 2011; Cho et al., 1974; Conti et al., 2002; Crosby et al., 1985). However, until recently, such modulation of cAMP during IVM had not resulted in improved oocyte developmental competence. Recently, a novel system, based on maintenance of elevated cAMP levels during IVM and delayed meiotic maturation has been shown to improve developmental competence of murine and bovine oocytes, presumably through recapitulation of in vivo signaling pathways (Aktas et al., 1995a,b; Albuz et al., 2010). This system, termed

Simulated Physiological Oocyte Maturation (SPOM), consists of a 2 h pre-maturation period in which cumulus oocyte complexes (COCs) are exposed to the general AC stimulator Forskolin and PDE inhibitor IBMX, followed by an extended maturation in medium supplemented with high levels of FSH and cilostamide, an inhibitor of the oocyte-specific PDE3A. The aim of the present study was to evaluate the effectiveness of this protocol in the sheep. It has been previously suggested that significant species-specific differences exist between bovine and ovine with regard to in vitro oocyte developmental parameters (Rizos et al., 2002a). Initially, we quantified changes in intra-oocyte cAMP concentration under control and cAMP-modulating conditions to evaluate the effectiveness of the treatment to elevate intra-oocyte cAMP. Next, we evaluated the effect of elevated cAMP on meiotic progression to GVBD and MII in order to elucidate any changes in the rate of maturational progression, and evaluate whether modulation produces a maturational delay. Lastly, we evaluated the effects of cAMP modulation during IVM on parthenogenetic blastocyst formation to evaluate oocyte developmental competence. 2. Materials and methods Chemicals were purchased from Sigma–Aldrich Co. (St Louis, MO) unless otherwise indicated. 2.1. Ovary collection and oocyte aspiration Ovine ovaries were collected from sheep of unknown reproductive status from a local abattoir (Superior Farms; Dixon, CA), and transported to the laboratory in sterile, pre-warmed saline solution. All ovaries were processed within 2 h of collection. COCs from mature follicles approximately 1–5 mm in diameter were aspirated directly into follicular fluid using an 18-gauge needle and negative pressure delivered through a vacuum pump at an adjusted fill rate of 9–11 mL/min. COCs with multiple compact cumulus cell layers and an even oocyte cytoplasm were selected, washed through HEPES-buffered synthetic oviductal fluid (hSOF) and transferred into maturation medium. 2.2. Oocyte maturation Groups of 20 washed COCs were matured at 38.5 ◦ C and 5% CO2 in 60 ␮L drops of equilibrated maturation media covered with mineral oil. Base maturation medium consisted of TCM-199 supplemented with 0.2 mM Sodium Pyruvate, 0.105 mM Gentamicin (Life Technologies, Carlsbad, CA), and 0.1 ␮M Cysteamine. Standard Maturation Medium (STD) consisted of the base media supplemented with 50 ng/mL ovine FSH (oFSH; AFP7558C; National Hormone and Peptide Program, Los Angeles, CA). Modulated Pre-Maturation Medium (PRE) consisted of base medium supplemented with 500 ␮M 3-isobutyl-1methylxanthine (IBMX) and 100 ␮M Forskolin. Modulated Extended Maturation Medium (EXT) consisted of base medium supplemented with 250 ng/mL oFSH and 200 ␮M

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Cilostamide. All media were prepared fresh for each collection, and equilibrated for at least 4 h prior to use. IBMX, Forskolin, Cilostamide and Cysteamine were added with washed COCs at collection to prevent degradation over the equilibration period. Forskolin and Cilostamide containing media were protected from light with foil to prevent lightmediated degradation. For STD maturation, COCs were matured in STD media for the entire maturation period. For modulated maturation, COCs were matured for 2 h in PRE media, washed through hSOF, and transferred to EXT media for the remainder of maturation. 2.3. Characterization of oocyte cAMP Intra-oocyte cAMP concentration was measured using a chemiluminescent immunoassay (CLIA; DetectX© High Sensitivity Direct Cyclic AMP Kit Acetylated Format; Arbor Assays, Ann Arbor, MI). Measurements were performed on groups of ten oocytes and repeated for three replicates per treatment and timepoint. COCs were manually aspirated from mature follicles directly into follicular fluid using a 1 cc syringe and 18-gauge needle. For Time 0 (T0), 10 COCs were quickly aspirated and placed immediately into hyaluronidase supplemented with 500 ␮M IBMX to prevent cAMP degradation. COCs were then vortexed for 5 min to release cumulus cells. Denuded oocytes were washed through hSOF with 500 ␮M IBMX to wash away remaining cumulus cells before a final wash through D-PBS (Life Technologies) supplemented with 1 mg/mL polyvinyl alcohol (PVA) and 500 ␮M IBMX. Oocytes were transferred into a 1.5 mL tube with minimal medium, snap-frozen in liquid nitrogen and stored at −80 ◦ C until used. For remaining time-points, COCs were manually aspirated in groups of ten, and matured in either STD or PRE media for 15, 30, 60 or 120 min before stripping, washing and fixing identically to the T0 oocytes. CLIA was carried out according to the manufacturer’s protocol. Reactions were read on a chemiluminescent plate reader (Tecan Spectrafluor, San Jose, CA) at 0.1 s read time per well. cAMP concentrations were then calculated by interpolating luminescence units into a standard curve generated from known amounts of cAMP. 2.4. Measurement of progression to germinal vesicle breakdown (GVBD) via Lamin A/C immunostaining Groups of 20 COCs (3–4 replicates per treatment and timepoint) were matured in CON or MOD media for 6, 8 or 12 h. COCs were stripped of cumulus cells with hyaluronidase and vortexing, and washed through hSOF to remove any remaining cumulus cells. Denuded oocytes were fixed for 10 min in 4% paraformaldehyde, washed through PBS–PVA and stored at 4 ◦ C in PBS–PVA until processing. At staining, groups of oocytes were washed through three successive 10 min washes at room temperature in Washing Buffer (WB; 0.1% Triton X-100 in D-PBS). Oocytes were then incubated for 30 min RT in Permeabilization Buffer (1% Triton X-100 in D-PBS) and washed 1× 10 min RT in WB. Samples were incubated for 2 h RT in Blocking Buffer (1% BSA and 10% Normal Goat Serum in WB) and then in 500 ␮L of Antibody Buffer Solution (ABS;

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WB supplemented with 1% BSA) with primary antibody (1:100, SC 7292, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 ◦ C. Oocytes were then washed 3× for 10 min and 3× for 20 min in WB at RT. Oocytes were then incubated with the secondary antibody (1:400, goat anti-mouse AlexaFlour 488; Life Technologies) in ABS for 1 h RT. Following incubation, oocytes were washed 3× 10 min and 3× 20 min RT in WB. Hoechst 33342 was added to the WB for the final 10 min of the last wash for DNA labeling. Samples were then mounted to slides in antifade reagent (Life Technologies), and observed with a fluorescent microscope. Oocytes were categorized as GV (Germinal Vesicle; intact nuclear lamina), TRANS (Transitioning; shrunken nuclear lamina), or GVBD (Germinal Vesicle Breakdown; dissipated nuclear lamina). 2.5. Measurement of progression to MII via DNA staining Groups of 20 COCs (3–4 replicates per treatment and timepoint) were matured in CON media or MOD media for 20, 24 or 28 h. COCs were then stripped of cumulus cells via hyaluronidase and vortexing and fixed as described above. Oocytes were incubated for 10 min in 10 ␮g/mL Hoechst 33342, washed through PBS–PVA and mounted to slides as above. Oocytes were categorized as GV, MI or MII under fluorescence microscopy. 2.6. Parthenogenetic activation and culture to blastocyst Groups of 20 COCs (3–4 replicates) were matured for 24 or 28 h in CON or MOD media before they were stripped of cumulus cells. Denuded oocytes were washed through hSOF and incubated for 4 min in ionomycin (5 ␮M in hSOF). Oocytes were then washed through hSOF and transferred in groups of 15–50 ␮L drops of 2 mM 6-(Dimethylamino) purine (DMAP) in potassium simplex optimized medium (KSOM, MR-121-D, Millipore, Billerica, MA) covered in mineral oil for 4 h for activation. Following activation, oocytes were washed through hSOF and transferred in groups of 20–60 ␮L drops of equilibrated KSOM culture medium supplemented with 4 mg/mL BSA and covered in mineral oil. Media was supplemented at 72 h postactivation with 5% fetal bovine serum (FBS) and blastocysts were counted on day 8. 2.7. Statistical analyses The levels of cAMP per oocyte were analyzed as a continuous variable using one-way ANOVA implemented by the PROC MIXED procedure of SAS. First, the effect of time was compared for oocytes cultured in control medium using replicate as a random variable. Then, the effect of time, treatment and their interaction was contrasted between oocytes cultured in control and modulated maturation medium. Replicate was included as a random variable. The proportion of oocytes at different stages of meiosis at different timepoints was evaluated using a logit mixed model implemented in the PROC GLIMMIX of SAS. Replicate was included as a random variable and the treatments and time were included as fixed variables. Blastocyst rates were

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compared among treatments using the GLIMMIX procedure of SAS.

3. Results 3.1. Changes in intra-oocyte cAMP concentration across the first 2 h of maturation with control (CON) and modulated maturation (MOD) media At the time of collection (T0), denuded oocytes contained an average of 6.1 ± 0.5 fmol of cAMP. Under CON conditions, the cAMP level transiently dropped to 3.5 fmol/oocyte after 15 min, before increasing to between 7 and 8 fmol/oocyte for the remainder of the 120 min of maturation time (Fig. 1). Under MOD maturation, cAMP levels increased (p < 0.05) from 6.1 ± 0.5 fmol/oocyte at T0 to 37.9 ± 10.4 fmol/oocyte after 15 min and 41.15 ± 11.09 fmol/oocyte after 30 min of maturation. Then, cAMP levels significantly decreased (p < 0.05) to 31.3 ± 7.7 fmol/oocyte by 60 min and to 26.9 ± 2.7 fmol/oocyte by 120 min of maturation. The cAMP concentration was significantly higher (p < 0.0001) in MOD oocytes compared to control oocytes, at each timepoint analyzed (Fig. 1). Furthermore, the increase in intra-oocyte cAMP levels induced by the presence of modulators was a concentration-dependent effect, with higher concentrations of modulators resulting in higher levels of cAMP (Supplementary Figure S1).

3.2. Effects of AC stimulation and PDE inhibition on resumption of meiosis Oocytes matured in control (CON) and cAMPmodulating media (MOD) were immunoflourescently stained for Lamin A/C to evaluate meiotic progression from 6 to 12 h of maturation (Fig. 2A ). There was a significant effect of time and treatment on oocyte meiotic stage (p < 0.05). By examining change within each treatment over time, a significant decrease in the prevalence of GV oocytes (p < 0.01) and a significant increase in GVBD oocytes (p < 0.01) was observed from 6 to 8 h in MOD and CON conditions. At these timepoints, the prevalence of oocyte in transitionary state (TRAN) did not significantly differ within treatments (p > 0.10; Fig. 2B). From 8 to 12 h, the progression towards GVBD continued in both treatment groups. There was no significant change in proportion of GV oocytes for CON (p = 0.72) or MOD (p = 0.25), indicating that in both groups the transition out of GV stage was mostly completed by 8 h. On the other hand, the proportion of GVBD oocytes significantly increased (p < 0.05) from 8 to 12 h, mostly at the expense of a decrease in the proportion of oocytes in TRAN state (p < 0.05; Fig. 2B). Comparing treatments within the same timepoint, we observed significant differences at each of the stages, indicating a significant effect of cAMP modulation on meiotic progression. At 6 h, there was a higher prevalence at GV oocytes in MOD versus CON (65.8% versus 29.5%; p < 0.01).

Fig. 1. Level of cAMP per ovine oocyte (fmol) during in vitro maturation in control (CON) and modulated (MOD) conditions from time of collection to 120 min of maturation. Each data point represents 3–6 replicates of 10 oocytes each. a,b,c : Different letters denote significant differences (p < 0.05).

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Fig. 2. Germinal vesicle break down is delayed by cAMP modulation. (A) Nuclear status of ovine oocytes determined by Lamin A/C immunostaining and stained Hoechst DNA staining (GV: germinal vesicle, intact nuclear lamina and uncondensed DNA; TRAN: transition state, shrunken nuclear lamina and condensing DNA; GVBD: germinal vesicle break-down, dissipated nuclear lamina and condensed DNA. (B) Percent of oocytes at each nuclear stage after 6, 8 or 12 h under control (CON) or cAMP modulated (MOD) conditions. Each data point represents 3–4 replicates of 10–30 oocytes each.

There were also fewer MOD oocytes having completed GVBD than CON (8.9% versus 38.2%; p < 0.01), while there was no difference in TRAN oocytes (p = 0.34; Fig. 2B). At 8 h of maturation more MOD oocytes remained at GV than CON (37.8% versus 7.5%; p < 0.001) and fewer MOD oocytes had completed GVBD than CON (30.2% versus 80.1%, p < 0.001). Also, more MOD oocytes remained in an intermediate TRAN phase than CON (31.9% versus 12.3%; p < 0.05). By 12 h of maturation, more MOD oocytes were still in GV than CON (18.8% versus 3.7%; p < 0.05) and fewer MOD oocytes had reached GVBD than CON (73.5% versus 92.8%, p = 0.01). But again, there was no significant difference in proportion of oocytes at TRAN state (p = 0.30) between MOD (7.7% TRAN) and CON (3.4% TRAN). Overall, cAMP modulation treatment transiently delayed progression through meiosis from GV, through a transition state (TRANS), and eventually to GVBD when compared to control. Control oocytes had largely reached GVBD by 8 h, and thus the majority of maturation occurred during the 6–8 h period, with a small amount continuing up to 12 h when essentially all had reached GVBD. Conversely, modulated oocytes were just beginning to transition from GV to GVBD from 6 to 8 h, and continued to exit GV stage through the full 12 h period. It is interesting to note the similarity in relative prevalence of GV, TRANS and GVBD in control oocytes at 6 h and modulated oocytes at 8 h. In fact, no significant difference between 6 h CON

and 8 h MOD exist in %GV (p = 0.60), %TRANS (p = 0.78) or %GVBD (p = 0.37). 3.3. Effects of AC stimulation and PDE inhibition on nuclear maturation to MII stage Both CON and MOD oocytes were categorized as GV, MI or MII after 20, 24 and 28 h of maturation via examination of nuclear material after Hoechst staining (Fig. 3A ). No differences in proportion of GV oocytes were detected between any of the conditions at any timepoint (p = 0.64) or between timepoints regardless of treatment (p = 0.95). An increasing proportion (p < 0.05) of MII oocytes and decreasing proportion of MI oocytes were observed between 20 and 24 h in CON conditions and between 20 & 24 h and 24 & 28 h in MOD conditions (Fig. 3B). Additionally, the proportion of MII oocytes was higher (p < 0.05) in CON than in MOD conditions at 20 and 24 h of maturation, mainly due to differences in MI oocyte proportions; while no differences were observed between treatments at 28 h of maturation (p = 0.77; Fig. 3B). In summary, control oocytes have largely completed maturation by 24 h, with no significant change over the subsequent 4 h from 24 to 28 h. Conversely, modulated oocytes progressed over the entire experimental window from 20 to 28 h, reaching a similar level of maturation to that of controls.

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Fig. 3. Nuclear maturation is delayed by cAMP modulation. (A) Classification of oocytes based on state of nuclear maturation. GV: germinal vesicle, MI: metaphase I, MII: metaphase II. (B) Percent of oocytes at different nuclear maturation stages after 20, 24 or 28 h under control (CON) or cAMP modulated (MOD) conditions. Each data point represents 4–6 replicates of 10–50 oocytes each.

3.4. Effects of increased cAMP on D8 blastocyst formation To evaluate developmental potential and cytoplasmic maturation, COCs matured for 24 or 28 h in each maturation condition (CON and MOD) were parthenogenetically activated and blastocyst formation scored. No effect of MOD maturation on parthenogenetic blastocyst formation was observed between any conditions (p > 0.05; Fig. 4). 4. Discussion A new approach to modulate the onset of oocyte maturation during in vitro culture conditions has been recently proposed (Aktas et al., 1995a,b; Albuz et al., 2010). Under these conditions, spontaneous initiation of oocyte maturation is prevented by increasing oocyte cAMP levels and then induced by FSH stimulation. In this study we characterized the early dynamics of cAMP levels in control and modulated conditions. Also, the effect of cAMP modulation on GVBD and MII formation kinetics was evaluated, as well as the developmental competence of the oocytes based on a parthenogenetic activation model. At the time of extraction from the follicle, ovine oocytes have a basal cAMP concentration of 6.09 ± 0.46 fmol/ oocyte, which is in agreement with previously reported concentrations of 6.3 ± 0.66 fmol/oocyte (Moor et al., 1981). This level was rather stable across the first 2 h

of in vitro culture, with only a slight decrease at 15 min when levels diminished to roughly half of the initial ones, although this was not statistically significant. Similarly, Crosby et al. (1985) reported no significant difference between T0 oocytes and those incubated in media with hormonal supplements for 1 h. In mice, upon removal of COCs from the follicle, a drop in cAMP is thought to be caused by the elimination of the uncharacterized ligand to GPR3/12, resulting in loss of AC stimulation (Mehlmann, 2005). Also, loss of the granulosa ligand NPPC would exacerbate the reduction in cAMP production through reduced cGMP production, and thus increased PDE3A cAMP hydrolyzing activity (Kawamura et al., 2011; Zhang et al., 2010). It is not clear if these systems are functional in ovine oocytes, but based on findings of this and others studies (Crosby et al., 1985) the drop in cAMP does not seem to be as dramatic as in other species. cAMP elevating agents have been shown to prevent spontaneous maturation in mice (Schultz et al., 1983) and rats (Aberdam et al., 1987), and transiently delay maturation in cattle (Homa, 1988; Sirard and First, 1988). As previously shown, treatment of ovine oocytes with Forskolin and IBMX resulted in a significant and concentration dependent increase in cAMP levels (Albuz et al., 2010). These results confirm that ovine COCs are capable of synthesizing significant amounts of endogenous cAMP, enough to elevate 5 to 10-fold over basal levels during the

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Fig. 4. Percent blastocyst formation from oocytes matured under control (CON) or cAMP modulated (MOD) conditions and activated parthenogenetically at 24 or 28 h of maturation. Each bar represents 3–6 replicates of 20–50 oocytes.

first 2 h of maturation, and prevent any transient drop in cAMP that could be associated with removal from the follicular environment and in vitro culture conditions. As previously suggested, GVBD in the sheep typically occurs at 8 h of maturation (Gharibi et al., 2013; Moor and Crosby, 1986). Nuclear progression following cAMP elevation in sheep has been examined previously. One study concluded that elevation of cAMP to 4× basal levels with cholera toxin (CT; 1 ␮g/mL) decreased the proportion GVBD by 18 h from approximately 90% in controls to approximately 67% in treated oocytes (Crosby et al., 1985). However, this decrease in GVBD appeared to be due to increased incidence of an abnormal phenotype, characterized by condensation of chromosomes with an intact nuclear lamina, which was suggested to potentially be due to inhibition of protein phosphorylation that accompanies GVBD due to terminally high CT levels. Thus, it was tentatively concluded that evidence at that time suggested that cAMP had little, if any, effect on the onset of GVBD in the sheep, and that changes in oocyte cAMP concentration did not act as a primary regulator controlling maturation in the sheep. Elevation of cAMP to 10× basal levels via AC stimulation and PDE inhibition, followed by gonadotropin stimulation of maturation to recapitulate in vivo hormonal signaling, was successful at transiently delaying maturation without inducing gross morphologic effects. Perhaps most interestingly, we too observed a phenotype characterized by partially condensed chromosomes with an intact nuclear lamina, similar to that described by Crosby et al. However, we determined that under our experimental conditions this was not an abnormal phenotype, but merely a transitional stage as chromatin condensation progressed

during the shift from GV to MI. Oocytes in our study did not become terminally arrested and progressed to GVBD in both control and modulated conditions in similar numbers, though with significantly delayed kinetics under cAMP modulation. A recent study utilizing an ovine-SPOM protocol, very similar to the one described presently, modulated cAMP levels during maturation and showed no deleterious effects on nuclear progression through 27 h of maturation (Rose et al., 2013). These, together with our results suggest that cAMP is in fact an important regulator of ovine oocyte maturation. In addition to measuring the effect at early time-points for GVBD, we also measured progression to MII at 20, 24 and 28 h to determine if cAMP modulation had any effect on kinetics of MII formation. As with GVBD, differences were present at early time-points with modulated oocytes significantly less mature. Approximately 20% more control oocytes had reached MII by 20 h than modulated (72.26% versus 55.15%), which was reduced to a difference of approximately 10% by 24 h (83.26% versus 73.47%). By 28 h, no significant differences remained, once again affirming that the delay induced by the experimental conditions in this study resulted in a transient, not terminal, meiotic delay. Combined, these findings reinforce that transient cAMP elevation through modulation does not have an injurious effect on the completion of oocyte maturation, as the same number of oocytes eventually reach MII in both control and modulated condition. To evaluate if transiently delaying nuclear maturation would affect oocyte developmental competence, development to blastocyst after parthenogenetic activation was analyzed. We chose parthenogenetic activation versus

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IVF in order to decrease potential variability inherent to natural variation in quality and viability of sperm. Diploid parthenogenetic embryos have shown to develop appropriately until at least the blastocyst stage in mice (Cuthbertson, 1983), cattle (Gómez et al., 2009), and sheep (Loi et al., 1998). Therefore, parthenogenetic activation has been used to evaluate oocytes competence in a number of mammalian species, including human (McElroy et al., 2010), pigs (Mizobe et al., 2010; Silvestre et al., 2007), and sheep (Ptak et al., 2006), among others. Under our conditions, cAMP modulation did not result in higher developmental competence. Similarly, blastocyst development after IVF was not improved by SPOM treatment of oocyte derived from peripubertal animals (Rose et al., 2013). Overall, these results suggest that if SPOM has beneficial effects on ovine oocyte developmental competence they may be minimal, as suggested by an increased number of cells in IVF blastocysts (Rose et al., 2013). We conclude that despite successful maturational delay via cAMP elevation, ovine oocyte developmental competence is neither positively nor negatively affected by this system, as evidenced by similar embryo yields under standard and modulated conditions. Further optimization of modulation and hormonal milieus are necessary to enable this type of system to be successfully utilized over standard methods in sheep for high efficiency embryo production. Conflict of interest We declare no conflict of interests that could influence the outcome of the study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.anireprosci.2014.12.012. References Aberdam, E., Hanski, E., Dekel, N., 1987. Maintenance of meiotic arrest in isolated rat oocytes by the invasive adenylate cyclase of Bordetella pertussis. Biol. Reprod. 36, 530–535. Aktas, H., Wheeler, M.B., First, N.L., Leibfried-Rutledge, M.L., 1995a. Maintenance of meiotic arrest by increasing [cAMP]i may have physiological relevance in bovine oocytes. J. Reprod. Fertil. 105, 237–245. Aktas, H., Wheeler, M.B., Rosenkrans Jr., C.F., First, N.L., Leibfried-Rutledge, M.L., 1995b. Maintenance of bovine oocytes in prophase of meiosis I by high [cAMP]i. J. Reprod. Fertil. 105, 227–235. Albuz, F.K., Sasseville, M., Lane, M., Armstrong, D.T., Thompson, J.G., Gilchrist, R.B., 2010. Simulated physiological oocyte maturation (SPOM): a novel in vitro maturation system that substantially improves embryo yield and pregnancy outcomes. Hum. Reprod. 25, 2999–3011. Bevers, M.M., Dieleman, S.J., 1996. Regulation and modulation of oocyte maturation in the bovine. Theriogenology 47, 13–22. Bilodeau-Goeseels, S., 2011. Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes. Mol. Reprod. Develop. 78, 734–743. Cho, W.K., Stern, S., Biggers, J.D., 1974. Inhibitory effect of dibutyryl cAMP on mouse oocyte maturation in vitro. J. Exp. Zool. 187, 383–386. Conti, M., Andersen, C.B., Richard, F., Mehats, C., Chun, S.Y., Horner, K., Jin, C., Tsafriri, A., 2002. Role of cyclic nucleotide signaling in oocyte maturation. Mol. Cell. Endocrinol. 187, 153–159. Crosby, I.M., Moor, R.M., Heslop, J.P., Osborn, J.C., 1985. cAMP in ovine oocytes: localization of synthesis and its action on

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Please cite this article in press as: Buell, M., et al., cAMP modulation during sheep in vitro oocyte maturation delays progression of meiosis without affecting oocyte parthenogenetic developmental competence. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2014.12.012