Role of ataxia-telangiectasia mutated (ATM) in porcine oocyte in vitro maturation

Zi-Li Lin and Nam-Hyung Kim*

Abstract

Ataxia-telangiectasia mutated (ATM) is critical for the DNA damage response, cell cycle checkpoints, and apoptosis. Significant effort has focused on elucidating the relationship between ATM and other nuclear signal transducers; however, little is known about the connection between ATM and oocyte meiotic maturation. We investigated the function of ATM in porcine oocytes. ATM was expressed at all stages of oocyte maturation and localized predominantly in the nucleus. Furthermore, the ATM-specific inhibitor KU-55933 blocked porcine oocyte maturation, reducing the percentages of oocytes that underwent germinal vesicle breakdown (GVBD) and first polar body extrusion. KU-55933 also decreased the expression of DNA damage-related genes (breast cancer 1, budding uninhibited by benzimidazoles 1, and P53) and reduced the mRNA and protein levels of AKT and other cell cycle-regulated genes that are predominantly expressed during G2/M phase, including bone morphogenetic protein 15, growth differentiation factor 9, cell division cycle protein 2, cyclinB1, and AKT. KU-55933 treatment decreased the developmental potential of blastocysts following parthenogenetic activation and increased the level of apoptosis. Together, these data suggested that ATM influenced the meiotic and cytoplasmic maturation of porcine oocytes, potentially by decreasing their sensitivity to DNA strand breaks, stimulating the AKT pathway, and/or altering the expression of other maternal genes.

Keywords: ATM, Porcine oocytes, In vitro maturation

Introduction

In meiosis, a specialized type of cell division, double-strand breaks (DSBs) initiate recombination, following which homologous pairing and chromosome segregation occur; however, DNA DSBs can cause potentially lethal genomic lesions if they are not properly repaired (Bzymek et al., 2010). Mammalian oocyte maturation is blocked at the germinal vesicle (GV) stage. Following germinal vesicle breakdown (GVBD), meiosis I is completed, and oocytes arrest at metaphase (M) II until they are fertilized or parthenogenetically activated (Lin et al., 2014a). DNA DSBs occur during meiotic recombination in oocytes. DSBs are mainly sensed by ataxia-telangiectasia mutated (ATM) and repaired by the DNA damage checkpoint in cells (Shiloh, 2003; Valerie et al., 2007). Several studies have demonstrated functional interactions between ATM and growth factor-mediated signaling pathways in somatic cells with DNA DSBs. ATM influences the phosphoinositide 3-kinase/AKT pro-survival signaling pathway (Valerie et al., 2007; Sowd et al., 2013), resulting in increased proliferation, metastasis, invasion, and radioresistance (Valerie et al., 2007; McLendon et al., 2007; Viniegra et al., 2005). AKT is phosphorylated at Ser473 in response to DNA damage (Feng et al., 2004; Bozulic et al., 2008; Surucu et al., 2008); however, phosphorylation at both the Thr308 and Ser473 residues of AKT is required for its full activation (Alessi et al., 1996; Debabrata et al., 2013). The activity of AKT is increased at the G2/M transition of the cell cycle and is necessary for the timely progression of epithelial cells (Shtivelman et al., 2002) and
HEK293 cells (Katayama et al., 2005) through mitosis. However, the mechanism underlying the link between AKT and maturation progressionin oocytes is unclear. We hypothesize that ATM interacts with AKT to influence the resumption of growth factorinduced oocyte meiotic maturation. Many maternal transcripts important for mammalian oocyte maturation have been intensively investigated. CyclinB1, cell division cycle protein 2 (CDC2), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) are markers of female germ cells during meiotic maturation, and a proto-oncogene is a regulator of oocyte maturation in humans (Hashiba et al., 2001) and pigs (Ohashi et al., 2003; Marangos et al., 2012). Protein kinase A (PKA) and protein kinase C (PKC) also mediate oocyte maturation (Peyton and Thomas, 2011). ATM is activated in oocytes with DNA DSBs, following which it phosphorylates checkpoint kinase 1 (Chk1) and blocks oocytes at GV stage (Marangos et al., 2012). ATM may regulate DNA damage-induced pro-survival signaling and cell cycle arrest in oocytes via maternal transcripts. Thus, inhibition of ATM signaling is predicted to compromise pro-survival signaling, in addition to cell cycle checkpoints and DNA DSB repair targets, and influence the cell cycle and DNA DSB repair factors by phosphorylating downstream targets such as breast cancer 1 (BRCA1) (Cortez, et al., 1999), BRCA1/2-containing complex subunit (BRCC36) (Lukas, et al., 2011), budding uninhibited by benzimidazoles 1 (BUB1), mediator of DNA damage checkpoint protein 1 (MDC1) (Stewart et al., 2003), p53 (encoded by TP53), and DNA damage-inducible transcript 3 (DDIT3) (Yang et al., 2013).
In the present study, KU-55933, which is a potent and specific inhibitor of ATM and thereby regulates cell survival and cell cycle checkpoints that modulate apoptosis (Bozulic et al., 2008), was used to determine the effects of ATM on in vitro porcine oocyte meiotic maturation and embryo apoptosis. To elucidate the underlying molecular mechanism, the mRNA expression patterns and activities of several important maternal genes and the housekeeping gene GAPDH were determined by real-time PCR and Western blotting, respectively. Our results indicate that ATM plays an important role in oocyte meiotic maturation by regulating the expression of maternal genes.

Material and Methods

In vitro maturation (IVM) and parthenogenetic activation of porcine oocytes

Porcine cumulus-oocyte complexes (COCs) were collected as described previously (Lin et al., 2014a). COCs with intact and unexpanded cumulus cells were isolated and cultured in tissue culture medium-199 containing 0.1% polyvinyl alcohol (PVA, w/v), 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 ng/mL epidermal growth factor (Sigma, St. Louis, MO, USA), 10 IU/mL pregnant mare serum gonadotropin, 10 IU/mL human chorionic gonadotropin, 75 mg/mL penicillin G, and 50 mg/mL streptomycin sulfate under mineral oil for 44 h at 39°C in 5% CO2. For parthenogenetic activation, cumulus-free MII-arrested oocytes with the first polar body were selected and exposed to ionomycin prepared in North Carolina State University (NCSU)-23 medium for 5 min (activation = day 0). Following 3 h of culture in NCSU-23 medium supplemented with 7.5 μg/mL cytochalasin B (Sigma), porcine embryos were washed three times in NCSU-23 medium containing 0.4% BSA (w/v) and cultured in the same medium at 39°C in 5% CO2.

Experimental design and drug treatment

The ATM inhibitor 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-55933) (Selleckchem, Houston, TX, USA) was dissolved in dimethyl sulfoxide. The concentration of this solvent was maintained at 0.1% (v/v) in all experiments. First, we assessed whether KU-55933 affected cumulus expansion in a dose-dependent manner. The cumulus was classified as compact, partially expanded, or completely expanded. Partial expansion was defined as the dissociation of cells from the periphery of the cumulus, whereas complete expansion was defined as the dissociation of all cells, except for those of the corona. Second, to evaluate whether the effects of KU-55933 were reversible, oocytes were cultured in KU-55933 (100 µM)-containing or control IVM medium for 20 h, and then transferred to control or KU-55933-containing IVM medium, respectively. The developmental stage of the oocytes was determined by staining with 1 µg/mL 4′-6-diamidino-2-phenylindole (DAPI) for 10 min. In additon, oocytes were cultured in KU-55933 (0, 10, 50, 100 µM)-containing IVM medium for 20 h and 44 h to detect maternal mRNA and AKT pathway expression. Third, cumulus-free MII-arrested oocytes were selected after normal maturation for 44 h, parthenogenetically activated, and incubated in NCSU-23 medium containing KU-55933 (50 or 100 µM) and 0.4% BSA (w/v). The effects on embryo development and apoptosis in blastocysts were determined.

Quantitative PCR (qPCR) analysis

To analyze mRNA levels following KU-55933 treatment, total RNA was extracted from about 100 oocytes and from different tissues, followed by sequential purification with the Dynabeads mRNA DIRECT kit (Dynal Asa, Oslo, Norway). Oocytes RNA was reversetranscribed into cDNA using oligo(dT)12-18 and SuperScript II reverse transcriptase (Invitrogen). qPCR was performed using the DyNAmo HS SYBR Green qPCR kit (Finnzymes, Helsinki, Finland) and the DNA Engine OPTICON 2 continuous fluorescence detector (MJ Research, Minnesota, USA). For normalization, β-actin RNA was used as an internal control. Fold inductions are shown relative to the level of control RNA. Primer sequences used for real-time RT-PCR are listed in Table 1.

Immunofluorescence

Oocytes were washed with phosphate-buffered saline (PBS), fixed with 3.7% paraformaldehyde (w/v) prepared in PBS containing 0.1% PVA, and permeabilized with 1% Triton X-100 (v/v) for 30 min at 37°C. Samples were blocked with 1% BSA (w/v) for 1 h, incubated with an antibody at 4°C overnight in blocking solution, and washed with 1% BSA. The antibodies used were rabbit polyclonal anti-ATM (Genetex, San Antonio, TX, USA) and Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA) diluted 1:100 in PBS containing 1% BSA. Oocytes were counterstained with DAPI prior to mounting. For each group, the primary antibody was replaced with rabbit serum as a negative control. The samples were mounted onto glass slides and evaluated with a confocal laser-scanning microscope (Zeiss LSM 710 META).

Western blot analysis

Western blot analysis was performed as described previously (Lin et al., 2013b). Briefly, 200 porcine oocytes were thawed at room temperature (RT) and added to 20 µl of 1× SDS sample buffer [62.5 mM Tris-HCl (pH 6.8) at 25°C containing 2% SDS (w/v), 10% glycerol (v/v), 50 mM DTT, and 0.01% bromophenol blue or phenol red (w/v)]. Samples were then heated at 95°C for 5 min. SDS-PAGE was performed using a Criterion precast gel (Bio-Rad, Richmond, CA, USA) for 2 h at 100 V, followed by electrophoretic transfer to a PVDF membrane using the iBlot system (Invitrogen, Grand Island, NY, USA) for 2.5 h at 200 mA and 4°C. The membrane was blocked with 5% low-fat milk (w/v) prepared in TBST [20 mM Tris (pH 7.4) containing 137 mM NaCl and 0.1% Tween-20 (v/v)] for 2 h at RT and then incubated with an anti-ATM (Genetex, San Antonio, TX, USA) or an anti-p-AKT (Ser473, Thr308) (Cell Signaling Technology, Danvers, MA, USA) antibody diluted 1:1000 in blocking buffer overnight at 4°C. After three washes with TBST (10 min each), the membrane was incubated with horseradish peroxidase-linked anti-rabbit IgG (Cell Signaling Technology, Beverly, MA) diluted 1:2000 in TBST for 1 h. The membrane was washed three times with TBST, and proteins were visualized using an enhanced chemiluminescence detection system (Invitrogen). An anti-β-actin antibody [(I-19) (Santa Cruz Biotechnology, Santa Cruz, CA)] was used as the loading control. The experiment was repeated at least three times using different samples.
Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay Blastocysts were washed three times with PBS (pH 7.4) containing 1 mg/ml polyvinyl pyrrolidone (PBS/PVP). After fixation with 3.7% paraformaldehyde prepared in PBS for 1 h at RT, the embryos were washed with PBS/PVP and permeabilized by incubation in 0.5% Triton X-100 for 1 h at RT. The embryos were then washed twice with PBS/PVP and incubated with fluorescein-conjugated dUTP and the terminal deoxynucleotidyl transferase enzyme (In Situ Cell Death Detection Kit, Roche, Mannheim, Germany) in the dark for 1 h at 37°C. Blastocysts were counterstained with Hoechst 33342 (bisBenzimide H33342 trihydrochloride, Sigma Life Science) to label all nuclei, washed with PBS/PVP, mounted with slight coverslip compression, and examined under an Olympus fluorescence microscope.

Statistical analysis

All percentage data were subjected to arcsine transformation prior to statistical analysis. Data were analyzed by analysis of variance using SPSS. p < 0.05 was considered significant.

Results

Expression and subcellular localization of ATM

We examined the expression and subcellular localization of ATM in porcine tissues and oocytes. Real-time qPCR revealed that the mRNA level of ATM was significantly higher in oocytes than in tissues (Fig. 1A). ATM was expressed throughout IVM of porcine oocytes, gradually increasing from the GV stage to the GVBD stage, and then decreasing during the MI and MII stages (Fig. 1B). The pattern of ATM gene expression in oocytes was in accordance with its protein expression (Fig. 1C). ATM protein predominantly localized in nuclei during GV arrest, began to accumulate near condensed chromosomes after GVBD, and was enriched in the spindle during the MI and MII stages (Fig. 1D). Morphological changes of COCs during IVM and the effect of KU-55933 on cumulus expansion
Treatment with KU-55933, even at a concentration of 100 µM, had no observable effect on cumulus expansion (Fig. 2A); however, meiotic progression was inhibited. The percentages of oocytes that underwent GVBD and polar body extrusion (PBE) were lower among KU-55933-treated oocytes than among control oocytes, and these percentages decreased as the KU-55933 concentration increased (Fig. 2B, C). The durations of GVBD and PBE were higher in KU-55933-treated oocytes than in control oocytes. However, the percentage of oocytes that underwent PBE was not significantly affected by treatment with a low concentration of KU-55933 (10 µM) (Fig. 2C). After incubation in KU-55933 (100 µM)-containing or control IVM medium for 20 h, oocytes were transferred to control or KU-55933-containing IVM medium, respectively. COCs were recovered and meiosis resumed in both groups (percentage of oocytes that underwent PBE was 78.3% in the former group and 79.1% in the latter group) to a level comparable to that in the untreated group (Fig. 2D). These results suggest that the effects of KU-55933 treatment are reversible and that this inhibitor primarily affects oocyte maturation prior to GVBD stage.

Effect of KU-55933 on maternal mRNA expression during porcine oocyte IVM

Expression of BMP15, GDF9, CDC2, cyclinB1, and the checkpoint kinases CHK1, AKT, and PKC was analyzed after 20 and 44 h of porcine oocyte maturation using real-time qPCR. KU-55933 treatment significantly decreased the mRNA levels of CDC2, cyclinB1, AKT, and PKC, and increased the level of CHK1 at 20 h (Fig. 3A, B). At 44 h, the mRNA levels of CHK1 and PKC were consistently higher in KU-55933-treated oocytes than in control oocytes, whereas the mRNA levels of BMP15, GDF9, CDC2, and cyclinB1 were significantly lower in the KU-55933-treated oocytes than in control oocytes.
We next investigated whether ATM signaling in porcine oocytes regulates maternal genes related to the DNA damage response. ATM inhibition attenuated expression of the maternal DNA damage response genes BRCA1, BUB1, and P53 (Fig. 3D), whereas MDC1 and BRCC36 expression was consistently higher in KU-55933-treated oocytes than in control oocytes. This suggests that ATM acts upstream of these maternal genes and that its signaling regulates the DNA damage response pathway in porcine oocytes during IVM.

KU-55933 potentially regulates the AKT pathway to influence porcine oocyte IVM

ATM reportedly regulates the phosphorylation of AKT at Ser473 in response to insulin and ionizing radiation (Viniegra et al., 2005). We examined the effect of KU-55933 treatment on AKT phosphorylation. After 20 h of culture in medium containing KU-55933 (100 µM), AKT phosphorylation at Thr308 was markedly decreased (Fig. 4A) and the level of AKT Ser473 phosphorylation was reduced by 30% (Fig. 4A-2). A similar response was observed at 44 h (Fig. 4B); AKT phosphorylation at Ser473 and Thr308 decreased in response to KU-55933 treatment in a concentration-dependent manner. These data suggest that the ATM inhibitor KU-55933 modulates phosphorylation of AKT at Ser473 and Thr308, and that this phosphorylation is regulated by ATM in porcine oocytes.

Effect of KU-55933 on early embryo development and apoptosis in blastocysts

The effects of KU-55933 on porcine early embryos were investigated after parthenogenetic activation. The percentage of embryos that reached the blastocyst stage was significantly lower among embryos treated with a high concentration of KU-55933 (100 µM) than among control embryos (0 µM) and those treated with a low concentration of KU-55933 (50 µM) (Fig. 5). There were more TUNEL-labeled nuclei, indicative of apoptotic cells, in KU-55933-treated blastocysts than in control blastocysts (Fig. 6).

Discussion

In this study, we investigated the role of ATM in the meiotic maturation of porcine oocytes. First, we showed that KU-55933 treatment remarkably decreased the percentages of oocytes that underwent GVBD and PBE, suggesting that ATM contributes to the regulation of porcine oocyte maturation. However, cumulus expansion was not significantly affected by KU-55933 treatment, suggesting that KU-55933 affects oocytes directly, rather than cumulus cells. Second, we showed that KU-55933 inhibited the development of GVBD and MII stage oocytes in a dose-dependent manner at concentrations lower than 100 µM, and such treatment also induced apoptosis in blastocysts. KU-55933 regulates cell survival and cell cycle checkpoints that modulate apoptosis (Bozulic et al., 2008). Therefore, we speculate that KU-55933 can induce cell cycle arrest and apoptosis during IVM of porcine oocytes and porcine early embryo development. Defective DNA replication can result in genomic instability, cancer, and developmental defects, and the DNA damage response maintains genomic stability (Warmerdam et al., 2009). Analysis of cell division and DNA damage markers (BRCA1, BRCC36, and BUB1) (Wells et al., 2005) showed that checkpoint proteins are activated and the cell cycle resumes following DNA DSB repair. MDC1 (Stewart et al., 2003) and DDIT3 (David et al., 2005) are regulators of the mammalian DNA damage checkpoint, and p53 is thought to be phosphorylated by ATM in response to DNA damage in melanoma cells (Banin et al., 1998; Lin et al., 2014b). In the current study, BRCA1, BUB1, and TP53 were down-regulated by KU-55933 treatment, which indicates that the activity of ATM was inhibited. This would perturb DNA stabilization and slightly decrease the activities of genes associated with the DNA damage response.
In oocytes with DSBs, ATM is activated and phosphorylates CHK1, causing arrest at the GV stage (Sorensen et al., 2005). Similarly, overexpression of CHK1 causes the arrest of oocytes at the GV stage (Chen and Sanchez, 2004). Treatment with KU-55933 for 20 or 44 h led to the arrest of oocytes at GVBD and increased the expression of CHK1. Oocyte maturation is primarily controlled by the activity of maturation-promoting factor (Lin et al., 2013a), which is a heterodimer of cyclinB1 and CDC2 and an essential regulator of meiotic resumption. Dynamic changes in the cyclinB1 transcript level are reportedly controlled by cytoplasmic polyadenylation during mouse (Tay et al., 2000) and bovine (Tremblay et al., 2005) oocyte maturation; however, it remains to be determined whether this also occurs in porcine oocytes. GDF9 and BMP15 belong to the transforming growth factor-β superfamily, are secreted by oocytes, and are involved in oocyte maturation and the cooperative regulation of granulosa cells (McNatty et al., 2005; Lin et al., 2013a). The levels of cAMP and cAMP-dependent protein kinases, such as PKC, also influence oocyte maturation (Peyton and Thomas, 2011). Degradation of cAMP reportedly leads to a decrease in PKA activity and initiates meiotic resumption in rodent oocytes (Kovo et al., 2006). In the current study, IVM of porcine oocytes was affected by KU-55933 treatment, as indicated by the reduced percentages of oocytes that underwent GVBD and
PBE. This effect was correlated with the downregulation of CDC2, cyclinB1, AKT, and PKC expression, which we speculate blocks the activity of ATM. Primary oocytes typically arrest at prophase of meiosis I. The serine/threonine protein kinase AKT can initiate meiotic G2/M transition (Okumura et al., 2002). The two principal phosphorylation sites of AKT, Ser473 and Thr308, are phosphorylated at different stages of meiosis (Kalous et al., 2009). In the current study, the ATM-specific inhibitor KU-55933 blocked phosphorylation of these residues, and AKT activity was markedly diminished at MII stage and was inversely correlated with the KU-55933 concentration.
KU-55933 inhibits the migration and invasion of glioma cells mediated by phosphorylation of AKT at Ser473 and Thr308 (Golding et al., 2009). AKT induces the meiotic G2/M transition and activated AKT is necessary for successful meiotic maturation (Okumura et al., 2002). The current study shows that the activity of AKT is not essential for the induction of GVBD in porcine oocytes, but plays a substantial role during the progression to the MI/MII stage. Therefore, we hypothesize that ATM can regulate the activity of AKT and influence the IVM of porcine oocytes at the GVBD and MI/MII stages. Taken together, our data suggest that ATM regulates the meiotic and cytoplasmic maturation of porcine oocytes, probably by decreasing their sensitivity to DNA DSBs, stimulating the AKT pathway, and/or altering the expression of other maternal genes.

References

Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings BA (1996) Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15: 6541–51.
Banin S, Moyal L, Shieh SY, Taya Y, Anderson CW, Chessa L, Smorodinsky NI, Prives C, Reiss Y, Shiloh Y, Ziv Y (1998) Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281: 1674–77.
Bozulic L, Surucu B, Hynx D, Hemmings BA (2008) PKB alpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. Mol Cell
Cortez D, Wang Y, Qin J, Elledge SJ (1999) Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks. Science 286: 1162–66.
David AW, Erin LC, Kristy AW, Fadel E, Sadik AK, James CW (2005) ABCC5, ERCC2, XPA and XRCC1 transcript abundance levels correlate with cisplatin chemoresistance in non-small cell lung cancer cell lines. Mol Cancer 4: 18.
Feng J, Park J, Cron P, Hess D, Hemmings BA (2004) Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J Biol Chem 279: 41189–96.
Golding SE, Rosenberg E, Valerie N, Hussaini I, Frigerio M, Cockcroft XF, Chong WY, Hummersone M, Rigotrau L, Menear KA, O’Connor MJ, Povirk LF, Meter T, Valerie K (2009) Improved ATM kinase inhibitor KU-60019 radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvival signaling, and inhibits migration and invasion. Mol Cancer Ther 8: 2894–902.
Hashiba Y, Asada Y, Heikinheimo O, Lanzendorf SE, Mizutani S (2001). Microinjection of antisense c-mos oligonucleotides prevents the progression of meiosis in human and hamster oocytes. Fertil Steril 76: 143–7.
Kalous J, Kubelka M, Šolc P, Šušor A, Motlík J (2009) AKT (protein kinase B) is implicated in meiotic maturation of porcine oocytes. Reprod 138: 645–654.
Katayama K, Fujita N, Tsuruo T (2005) Akt/protein kinase B-dependent phosphorylation and inactivation of WEE1Hu promote cell cycle progression at G2/M transition. Mol Cell Biol 25: 5725–37.
Lin ZL a, Li YH, Xu YN, Wang QL, Namgoong S, Cui XS, Kim NH (2014) Effects of growth differentiation factor 9 and bone morphogenetic protein 15 on the in vitro maturation of porcine oocytes. Reprod Domest Anim 49(2): 219–27.
Lin ZL b, Xu YN, Namgoong S, Kim NH (2014) JMY functions as actin nucleation- promoting factor and mediator for p53-mediated DNA damage in porcine oocytes. Plos one 9: e109385.
Lukas J, Lukas C, Bartek J (2011) More than just a focus: the chromatin response to DNA damage and its role in genome integrity maintenance. Nat Cell Biol 13: 1161–69.
Marangos P, Carroll J (2012) Oocytes progress beyond prophase in the presence of DNA damage. Curr Biol 22: 989–94.
McNatty KP, Juengel JL, Reader KL, Lun S, Myllymaa S, Lawrence SB, Western A, Meerasahib MF, Mottershead DG, Groome NP, Ritvos O, Laitinen MP (2005) Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulose cell function in ruminants. Reprod 129: 481–87.
McLendon RE, Turner K, Perkinson K, Rich J (2007) Second messenger systems in human gliomas. Arch Pathol Lab Med 131: 1585–90.
Ohashi S, Naito K, Sugiura K, Iwamori N, Goto S, Naruoka H, Tojo H (2003) Analyses of mitogen-activated protein kinase function in the maturation of porcine oocytes. Biol Reprod 68: 604–9.
Peyton C, Thomas P (2011) Involvement of epidermal growth factor receptor signaling in estrogen inhibition of oocyte maturation mediated through the G protein-coupled estrogen receptor in zebrafish. Biol Reprod 85: 42–50.
Shiloh Y (2003) ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 3: 155–68.
Shtivelman E, Sussman J, Stokoe D (2002) A role for PI 3-kinase and PKB activity in the G2/M phase of the cell cycle. Curr Biol 12: 919–24.
Sorensen CS, Hansen LT, Dziegielewski J, Syljuasen RG, Lundin C, Bartek J (2005) The cell cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair. Nat Cell Biol 7: 195–201.
Sowd GA, Li NY, Fanning E (2013) ATM and ATR activities maintain replication fork integrity during SV40 chromatin replication. PLoS Pathog 9 (4): e1003283.
Surucu B, Bozulic L, Hynx D, Parcellier A, Hemmings BA (2008) In vivo analysis of PKB/Akt regulation in DNA-PKcs-null mice reveals a role for PKB/Akt in DNA damage response and tumorigenesis. J Biol Chem 283: 30025–33.
Stewart GS, Wang B, Bignell CR, A, Taylor MR, Elledge SJ (2003) MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature 421: 961–66.
Tay J, Hodgman R, Richter JD (2000) The control of cyclinB1 mRNA translation during mouse oocyte maturation. Dev Biol 221: 1–9.
Tremblay K, Vigneault C, McGraw S, Sirard MA (2005) Expression of cyclin B1 messenger RNA isoforms and initiation of cytoplasmic polyadenylation in the bovine oocyte. Biol Reprod 72: 1037–44.
Valerie K, Yacoub A, Hagan MP, Curie D, Fisher PB, Grant S, Dent P (2007) Radiationinduced cell signaling: inside-out and outside-in. Mol Cancer Ther 6: 789–801.
Viniegra JG, Martínez N, Modirassari P, Hernández LJ, Parada CC, Sánchez-Arévalo LVJ, Aceves LCI, Alvarez-Vallina L, Ramón CS, Rojas JM, Sánchez-Prieto R (2005) Full activation of PKB/Akt in response to insulin or ionizing radiation is mediated through ATM. J Biol Chem 280: 4029–36.
Warmerdam DO, Kanaar R (2009) Dealing with DNA damage: relationships between checkpoint and repair pathways. Mutat Res 704: 2–11.
Wells D, Bermudez MG, Steuerwald N, Thornhill AR, Walker DL, Malter H, Delhanty JDA, Cohen J (2005) Expression of genes regulating chromosome segregation, the cell cycle and apoptosis during human preimplantation development. Hum Reprod 20: 1339–48.
Yang F, Nickols NG, Lia BC, Marinovc GK, Saidd JW, Dervana PB (2013) Antitumor activity of a pyrrole-imidazole polyamide. Proc Natl Acad Sci 110: 1863–1868.