Gas6 Downregulation Impaired Cytoplasmic Maturation and Pronuclear Formation Independent to the MPF Activity

Previously, we found that the growth arrest-specific gene 6 (Gas6) is more highly expressed in germinal vesicle (GV) oocytes than in metaphase II (MII) oocytes using annealing control primer (ACP)-PCR technology. The current study was undertaken to investigate the role of Gas6 in oocyte maturation and fertilization using RNA interference (RNAi). Interestingly, despite the specific and marked decrease in Gas6 mRNA and protein expression in GVs after Gas6 RNAi, nuclear maturation including spindle structures and chromosome segregation was not affected. The only discernible effect induced by Gas6 RNAi was a change in maturation promoting factor (MPF) activity. After parthenogenetic activation, Gas6 RNAi-treated oocytes at the MII stage had not developed further and arrested at MII (90.0%). After stimulation with Sr2+, Gas6-silenced MII oocytes had markedly reduced Ca2+ oscillation and exhibited no exocytosis of cortical granules. In these oocytes, sperm penetration occurred during fertilization but not pronucleus (PN) formation. By roscovitine and colcemid treatment, we found that the Gas6 knockdown affected cytoplasmic maturation directly, independent to the changed MPF activity. These results strongly suggest that 1) the Gas6 signaling itself is important to the cytoplasmic maturation, but not nuclear maturation, and 2) the decreased Gas6 expression and decreased MPF activity separately or mutually influence sperm head decondensation and PN formation.


Introduction
Mammalian oocytes in ovarian follicles have arrested growth and a large nucleus known as a germinal vesicle (GV). The meiotic cell cycle is arrested at the diplotene stage of the first prophase, and some selective oocytes initiate growth following gonadotropin simulation [1,2]. Oocyte growth and maturation are long and requisite processes for fertilization and subsequent embryo development until embryonic genome activation starts. Oocyte maturation involves nuclear and cytoplasmic maturation. Although strictly linked, these are complex and different events [3,4,5].
The process of nuclear maturation, meiotic cell cycle, involves GV breakdown (GVBD), chromosome condensation and segregation, organization of microtubules, and release of the first polar body, after which oocytes are arrested again at metaphase II (MII) until fertilization [3]. This process is mainly controlled by a phosphorylation and/or dephosphorylation regulatory cascade of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) [6,7,8].
The process of cytoplasmic maturation involves organelle reorganization, cytoskeleton dynamics and molecular maturation during oocyte growth and meiosis [9]. Organelles such as mitochondria, ribosomes, endoplasmic reticulum, cortical gran-ules, and the Golgi complex redistribute to the cytoplasm during oocyte maturation. Cytoskeletal microfilaments and microtubules function in spindle formation and chromosome segregation.
Oocytes accumulate maternal mRNA, protein, and regulatory molecules that function in the completion of meiosis, fertilization, and early embryogenesis [10]. This process is mainly controlled by post-transcriptional regulatory mechanisms, such as RNA polyadenylation, localization, sorting, and masking, as well as protein phosphorylation [11]. Therefore, functional analysis of certain gene(s) in the oocyte should provide important information on the molecular regulatory mechanism of oocyte nuclear and cytoplasmic maturation, fertilization, and early embryogenesis [12,13,14].
Oocytes underwent nuclear and cytoplasmic maturation, resulting in arrest at meiotic MII. Sperm penetration breaks this arrest and requires recognition of the zona pellucida (ZP), dependent upon three ZP proteins (ZP1-3) [15]. Sperm undergo the acrosome reaction and penetrate the ZP [16]. Sperm bind the ooplasma through interactions with microvilli and associated membrane proteins and ultimately form a fusion pore [17]. The oscillatory Ca 2+ signal is necessary and sufficient for the resumption of meiosis and cortical granule release, resulting in the blockade of polyspermy and extrusion of the second polar body [18,19]. To complete the fertilization process, pronuclei (PNs) are formed through the remodeling of paternal and maternal chromatin. Subsequently, maternal and fraternal PNs migrate for the preparation of syngamy [20,21,22,23].
In a previous study, it was found that growth arrest-specific gene 6 (Gas6) is highly expressed in GV oocytes by using annealing control primer (ACP)-polymerase chain reaction (PCR) [24]. Gas6 is a member of the vitamin K-dependent protein family and a ligand for Axl, Tyro3, and Mertk receptor tyrosine kinases [25,26]. It has been reported that Gas6-mediated signaling is implicated in cell survival, growth arrest, proliferation, differentiation, and other cell type-specific functions [26,27,28,29]. Clinically, Gas6 plays an important role in hematosis and thrombosis [26]. At present, the function of Gas6 and receptor signaling has been studied in thrombosis and spermatogenesis, but not in oocytes and embryos [26,30]. Thus, the objective of the present study was to evaluate the roles of Gas6 in oocytes, completion of the meiotic cell cycle, and fertilization and embryo development.

Gas6 mRNA expression in oocytes and embryos
During oocyte maturation. During oocyte maturation, the level of polyadenylated mRNA is reduced by more than 50% through deadenylation [31,32]. Previously, it was found that the mRNA expression of certain genes required for oocyte maturation or embryogenesis, namely Sebox and Obox4, was The mRNA equivalent to a single oocyte taken after culture for 0, 2, 8, and 16 hours, corresponding to GV, GVBD, MI, and MII stages, respectively, was used for each lane. H1foo used as an internal control. GFP was used as an external control to measure equal recovery. (B) Expression of Gas6 during early embryogenesis. Relative gene expression of Gas6 in a single oocyte and single embryo throughout the developmental stages was measured by quantitative real-time PCR. Relative expression levels of Gas6 were calculated from C T values and normalized to added GFP synthetic RNA, and the expression ratio was calculated against Gas6 expression in the GV oocyte. Experiments were repeated at least three times, and data were expressed as mean 6 SEM. doi:10.1371/journal.pone.0023304.g001 markedly decreased during oocyte maturation [12,33]. However, the expression of Gas6 was constitutive throughout oocyte maturation (Fig. 1A). Therefore, the abundant Gas6 mRNA in the GV stage is not deadenylated during meiotic maturation and is maintained as polyadenylated mRNA in the MII stage.
During embryogenesis. The expression of Gas6 during preimplantational embryo development was evaluated by quantitative real-time PCR. In each reaction, the absence of nonspecific amplification was confirmed by checking the melting curve of each gene with a single peak. The Gas6 transcript was highly expressed in GV and MII oocytes, gradually decreased to an undetectable level from PN to 2-cell (2C) and 4-cell (4C) stage embryos, and slightly increased again from 8-cell (8C) to blastocyst stage embryos (Fig. 1B).
Gas6 RNAi had no effect on oocyte nuclear maturation but effect on oocyte cytoplasmic maturation RNAi by microinjection of double-stranded RNA (dsRNA) is a suitable tool for studying gene function in the oocytes and embryos of mammalian species [34,35,36]. In the present study, we demonstrated that microinjection of Gas6 dsRNA into the cytoplasm of GV oocytes led to selective interference of maternal mRNA and subsequent protein expression and that Gas6 RNAi inhibited the cytoplasmic maturation of oocytes and fertilization.
Oocyte maturation after Gas6 RNAi. After treatment with Gas6 RNAi, oocytes matured with similar MII rates (Gas6 RNAi; 81.9%) as those of the control (86.9%) and buffer-injected (78.4%) groups ( Table 1). The MII oocytes that developed from GV oocytes injected with Gas6 dsRNA were morphologically normal compared to the control oocytes ( Fig. 2A) despite the specific and marked depletion of Gas6 mRNA (Fig. 2B). To verify the sequence-specific knockdown of the target RNA, we measured the expression levels of targeted and untargeted genes and found that the expression of untargeted genes, such as Plat, Mos, and Gapdh, remained unchanged in Gas6-targeted MII oocytes despite the decrease in Gas6 mRNA expression. These results confirmed that Gas6 RNAi caused sequence-specific Gas6 knockdown (Fig. 2B). To confirm the complete knockdown of GAS6 protein expression, we performed Western blot analysis for GAS6 protein after treatment with Gas6 RNAi followed by 8 or 24 hours of in vitro culture of oocytes in 3-isobutyl-1-methyl-xanthine (IBMX) medium and in vitro maturation for 16 hours. High levels of endogenous GAS6 expression were detected in control MII oocytes that had matured in vitro for 16 hours (Fig. 2C, Lane 1). We found that GAS6 expression decreased after treatment with  (1,4,7) show the oocytes under bright field, whereas microphotographs in the middle panel (2,5,8) show the noninvasive analysis of spindle structure in same oocytes using Polscope. Microphotographs in the right panel (3,6,9) show the results of aceto-orcein staining of chromosomes in MII oocytes. (B) Immunofluorescence staining for spindle and chromosome morphology in control and Gas6 dsRNA-injected oocytes matured in vitro. MII oocytes were fixed in 4% paraformaldehyde, stained with a-tubulin antibody (Green), and counterstained with propidium iodide (Red) for DNA staining. Scale bars indicate 25 mm. doi:10.1371/journal.pone.0023304.g003 Gas6 RNAi (Fig. 2C, Lanes 2-4) and almost completely disappeared in MII oocytes following 8 or 24 hours of preincubation in IBMX (Fig. 2C, Lanes 3,4). These data suggest that the complete knockdown of GAS6 protein occurred after 8 hours of preincubation in IBMX-supplemented medium.
Spindle and chromosomal configuration. There was no change in the shape and localization of meiotic spindles and chromosomes in MII oocytes after Gas6 knockdown (Fig. 3A, 1-9).
The normal shape and size of the meiotic spindle and chromosomal alignments were additionally confirmed by immunofluorescence staining (Fig. 3B). Taken together, these results suggest that Gas6 is not involved in the resumption, progression, and completion of the normal meiotic cell cycle.
Decrease of MPF activity after Gas6 RNAi. Mouse oocyte maturation is regulated by changes in MPF and MAPK activities [37,38]. Therefore, we conducted a dual kinase activity assay to determine the effects of Gas6 RNAi on the activities of MPF and MAPK. The activity of MPF in MII oocytes was dramatically decreased by Gas6 RNAi treatment compared to the activity in the control group, whereas the activity of MAPK in MII oocytes was maintained at a level similar to that of the controls (Fig. 4A).
After confirming that Gas6 RNAi had inhibitory effects on MPF activity, we determined the time course of inhibition by assaying activity every 2 hours after Gas6 RNAi injection (Fig. 4B). In contol oocytes, MPF activity was increased after 2 hours, markedly decreased after 8-10 hours around the first polar body extrusion, and increased again and maintained through the MII stage. The level of MPF activity, therefore, closely corresponds to the nuclear events. In contrast, MPF activity in Gas6-depleted MII oocytes decreased 6-10 hours after in vitro maturation and did not increase again (Fig. 4C). These results suggest that Gas6 is important in the reactivation of MPF after the first polar body emission but is not essential for the progression of nuclear maturation in mouse oocytes.
Cyclin B1 and p34 cdc2 are essential components of active MPF. MPF activity is regulated by a translation-dependent mechanism that determines the level of cyclin B1 [39,40]. To determine whether the inactivation of MPF in Gas6-depleted MII oocytes is cyclin B1-dependent, we measured cyclinB1-p34 cdc2 expression by Western blotting. Interestingly, as shown in Fig. 4D, the expression of cyclin B1 was markedly decreased in Gas6 dsRNA-injected oocytes relative to its expression in control oocytes. These results depict that Gas6 RNAi caused MPF inactivation through cyclin B1 degradation. Moreover, although p34 cdc2 expression was unchanged, p34 cdc2 phospho-Tyr15 was upregulated in Gas6depleted MII oocytes (Fig. 4D). These findings suggest that Gas6 RNAi increased the phosphorylation of Tyr15 in p34 cdc2 , which resulted in MPF inactivation.
Parthenogenetic development after Gas6 RNAi. When cytoplasmic maturation is not completed, oocytes fail to undergo fertilization and early embryo development [41,42]. To confirm that normal cytoplasmic maturation was completed, parthenogenetic activation was performed using Sr 2+ to stimulate the MII oocytes after Gas6 RNAi treatment. Parthenogenetic activation in three control groups resulted in development to PN and 2C stages ( Table 2, Fig. 5A,B). Following parthenogenetic activation, control oocytes with cumulus ( Fig. 5Aa; 18.3% and 60.3%), control oocytes without cumulus ( Fig. 5Ab; 28.7% and 39.5%), and buffer-injected sham control oocytes ( Fig. 5Ac; 25.7% and 30.7%) developed to PN and 2C stages. However, 90% of the MII oocytes treated with Gas6 RNAi (closed black bar in Fig. 5B) were not activated and were arrested at the MII stage (Fig. 5Atd), suggesting that Gas6 plays a critical role in the initiation of cell cycle progression for early embryo development.

Effects of Gas6 RNAi on fertilization and PN formation
Based on these results, we hypothesized that the reduction of Gas6 expression may have resulted in fertilization failure. Therefore, we conducted in vitro fertilization and evaluated the changes in Ca 2+ oscillation in MII oocytes and the rates of sperm penetration and PN formation. For measuring the exocytosis of cortical granules, fluorescein isothiocyanate (FITC)-Lens culinaris agglutinin (LCA) staining was performed following Sr 2+ -induced activation.

Sperm penetration but no PN formation after Gas6
RNAi. Concurrent with activation, sperm nuclear contents and paternal chromatin undergo biochemical remodeling via resources within the cytoplasm of oocytes [20]. Due to insufficient cytoplasmic maturation, oocytes failed to undergo fertilization. After several failures of in vitro fertilization with ZP-intact oocytes, we performed in vitro fertilization with ZP-free MII oocytes and monitored sperm penetration and PN formation. Following in vitro fertilization, uninjected and buffer-injected control oocytes exhibited high rates (.70%) of PN formation after sperm penetration (Fig. 6A, B). Because we used ZP-free oocytes, we could observe two or three PNs. In contrast, the majority of the eggs treated with Gas6 RNAi failed to undergo the remodeling of paternal and maternal chromatin and PN formation despite sperm penetration into the cytoplasm (Fig. 6A, B). We confirmed that sperm penetration had occurred in Gas6 RNAi-treated MII oocytes by finding partially decondensed paternal chromosomes in the cytoplasm (Fig. 6A, arrows). These results indicate that Gas6-silenced MII oocytes had insufficient cytoplasmic maturation that resulted in the failure of PN formation.

Effects of Gas6 RNAi on insufficient cytoplamic maturation
Calcium oscillations in Gas6-silenced MII oocytes. During fertilization, sperm break the arrest of the second meiotic cycle of the oocytes by inducing a series of repetitive Ca 2+ oscillations that persist for several hours and terminate upon PN formation [43,44]. This event has been confirmed in many rodents and in human oocytes [45]. To determine if a reduction in Gas6 expression affects the Ca 2+ oscillations in MII oocytes, MII oocytes were stimulated with SrCl 2 . When the Ca 2+ oscillation patterns in each group were analyzed ( Table 3), most of the uninjected and buffer-injected oocytes exhibited normal patterns of oscillatory Ca 2+ activity (Fig. 7A, B). In Gas6-scilenced oocytes, the frequency of the abnormal pattern of Ca 2+ oscillation increased (65.4%, Fig. 7C). Cortical granule exocytosis in Gas6-silenced MII oocytes. The normal Ca 2+ oscillation in eggs is essential for cortical granule release. In the present study, Gas6 RNAi caused low cytoplasmic Ca 2+ excitability. We further examined cortical granule exocytosis with SrCl 2 stimulation. Without SrCl 2 treatment, MII oocytes in all three groups exhibited the existence of intact cortical granules (green fluorescence) around the cortices of oocytes ( Fig. 8A-C). Upon treatment, SrCl 2 -treated in vivo and in vitro MII oocytes exhibited decreased green fluorescence around their cortices, indicating that cortical granule exocytosis had occurred (Fig. 8D, E). However, most of the Gas6-silenced MII oocytes exhibited intact green fluorescence, indicating unsuccessful cortical granule exocytosis in these oocytes (Fig. 8F). These results imply that decreased cytoplasmic Ca 2+ excitability after Gas6 RNAi prevented cortical granule release and caused fertilization failure.

Gas6
decrease directly affected failure of fertilization. To address whether the depletion of MPF activity due to reduction of Gas6 expression is indeed responsible for failure of fertilization, Gas6-expressed control and Gas6silenced oocytes were cultured with roscovitine (inhibit MPF activity) or colcemid (maintain MPF activity) after first polar body extrusion, as explained in the diagram Fig. 9A. We confirmed that the activity of MPF in Gas6-expressed MII oocytes with roscovitine treatment was markedly reduced, while the activity of MPF in colcemid-treated MII oocytes after Gas6 RNAi was rescued at a similar level to that of the control oocytes (Fig. 9B). When the oocytes were fertilized at this time point, 71.0% of control and 70.1% of Gas6-expressed, high MPF activity oocytes with colcemid treatment had PN formation. Also, 73.5% of Gas6-expressed, low MPF activity oocytes with roscovitine treatment had PN formation, but none of Gas6-sileced, high MPF activity oocytes with colcemid treatment had PN formation (Fig. 10A, B). Therefore, these results undoubtedly indicate that neither low MPF activity nor colcemid effect, but Gas6 knockdown resulted in the insufficient cytoplasm maturation and consequently the failure of PN formation.

Discussion
In this study, we investigated the expression of Gas6 in mouse oocytes and embryos, and demonstrated that Gas6 RNAi caused the disturbance of oocyte cytoplasmic maturation with a concurrent failure of fertilization. These results strongly suggest that Gas6 is a new candidate mammalian maternal effect gene.
Translation in oocytes is controlled by cytoplasmic polyadenylation and deadenylation of maternal transcripts, which are associated with the addition or loss of adenosine residues on the 39-untranslated region of mRNA [46]. We observed that polyadenylated Gas6 mRNA is highly expressed in MII oocytes, and this constitutively abundant Gas6 mRNA throughout oocyte maturation may play an essential role in oocyte cytoplasmic maturation and is directly related to early embryo development until zygotic gene activation starts. Previously, we observed that the mRNA expression of Bcl2l10 is also highly expressed in MII oocytes compared with its expression in GV oocytes, but Bcl2l10 RNAi treatment resulted in metaphase I (MI) arrest [14]. At this time, however, we cannot explain why Bcl2l10 RNAi resulted in MI arrest whereas Gas6 RNAi resulted in MII arrest.
Studies in mammalian oocytes revealed that the dynamic events of polymerization of microtubules and microfilaments are crucial in spindle formation, chromosome segregation, and extrusion of the polar body during oocyte meiotic maturation [39,47]. In the present study, despite the significant reduction of the transcript and protein content of Gas6, oocytes developed to a morphologically normal MII stage with the first polar body. Additionally, we observed completion of meiosis with normal meiotic spindle organization and chromosome arrangement. Consequently, we concluded that Gas6 is not involved in oocyte meiotic maturation, especially in the dynamics of microtubules and microfilaments.
MPF, a key regulator of both mitotic and meiotic cell cycles, consists of a catalytic subunit (p34 cdc2 ) and a regulatory subunit (cyclin B1) [7]. Cyclin B1 plays an important role in regulating MPF action. Degradation of cyclin B1 and dissociation of the p34 cdc2 -cyclin B1 complex without cyclin B1 proteolysis leads to MPF inactivation. It also has been reported that phosphorylation of Thr14 and Tyr15 in p34 cdc 2 inhibits MPF activity [48]. In the present study, Gas6 knockdown in MII oocytes led to cyclin B1 degradation and subsequent MPF inactivation. Moreover, we observed that Gas6-silenced MII oocytes had increased phosphorylation of Tyr15 in p34 cdc2 . Therefore, we concluded that the decrease in cyclin B1 levels and increased phosphorylation of Tyr15 in p34 cdc2 reduced MPF activity in Gas6-silenced MII oocytes.
When MPF activation was inhibited in oocytes, meiotic resumption was prevented, and oocytes arrested at the GV stage [49]. Blocking MPF activity in oocytes before or immediately after the first polar body extrusion also prevented MII entry and led the oocytes into interphase, as manifested by the presence of a welldefined nucleus and decondensed chromosome. These findings suggest that MPF reactivation is absolutely necessary for preventing oocyte entry into interkinesis and the MII stage [50]. Surprisingly, however, Gas6-silenced oocytes with decreased MPF activity progressed into the MII stage with morphological demonstration of the first polar body emission. That is because the activity of MPF of the Gas6-silenced MII was reduced after extrusion of the first polar body; thus, it did not affect the nuclear maturation of oocytes.
After polar body emission, cyclin B degradation stops, and the anaphase promoting complex/cyclosome is reactivated upon entry into the second meiotic metaphase by high levels of MPF. The equilibrium between the slow degradation and the continuous synthesis of cyclin B depends on cytostatic factor (CSF), and MPF activity is maintained at high levels by CSF in MII oocytes [39,51]. The cell cycle progression of unfertilized oocytes is arrested at the MII stage until fertilization or activation occurs [52]. The maintenance of MII arrest requires CSF activity through a Mos/MEK/MAPK/p90 rsk -dependent pathway. In Gas6-silenced MII oocytes, spontaneous activation did not occur, and oocytes maintained long-term MII stage arrest (data not shown), suggesting that Gas6 RNAi may not affect CSF activity.
Moreover, we observed that meiosis was completed with normal meiotic spindle organization and chromosome segregation in Gas6-silenced MII oocytes. The results indicate that Gas6 is crucial for cytoplasmic maturation excluding cytoskeletal systems. In this study, Gas6-silenced mouse oocytes could complete nuclear maturation with decreased MPF activity near the time of the first polar body extrusion, and MPF activity remained low thereafter. We confirmed that these Gas6-silenced oocytes had abnormal oscillation patterns of intracellular Ca 2+ , no cortical granule exocytosis, and failure of fertilization due to the lack of PN formation. This indicated that nuclear and cytoplasmic maturation are uncoupled in Gas6-silenced oocytes, but both nuclear and cytoplasmic maturation are necessary to achieve complete developmental competence for fertilization. Cytoplasmic maturational events are required to initiate sperm head decondensation, DNA synthesis, and PN formation [53,54].
After fertilization, oocytes undergo an increase in intracellular Ca 2+ excitability, cortical granule exocytosis, and meiotic resumption with concurrent MPF inactivation and PN formation. Mammalian oocytes were arrested at metaphase of the second meiotic division. After sperm penetration, entailing sperm release sperm-specific phospholipase C to activate the phosphatidylinositol bisphosphate cascade, resulting in the production of inositol triphosphate (IP3) and diacylglycerol (DAG) [55]. IP3 stimulates the endoplasmic reticulum to release stored Ca 2+ and facilitate oscillation [56]. Rescovitine, a specific inhibitor of p34cdc2/ Cyclin B kinase inhibitor, was used to inactivate MPF in mouse oocytes, and suppressed fertilization-induced Ca 2+ oscillations in normal MII eggs after first 1,2 Ca 2+ spikes. Therefore, inhibition of MPF activity in MII oocytes inhibits sperm-induced Ca 2+ oscillations, suggesting that MPF plays an important role in regulation of the cytoplasmic Ca 2+ excitability in mouse oocytes [57]. In this study, the low levels of MPF activity by Gas6-silencing and may be the low level of Gas6 expression itself may alter the pattern of Sr 2+ -induced Ca 2+ oscillations due to insufficient cytoplasmic maturation. The pattern of repetitive Ca 2+ oscillations in mice egg is essential for both early events of egg activation, such as the resumption of meiosis, cortical granules release, recruitment of maternal mRNA, and the subsequent normal developmental program [58,59,60]. After egg activation, concentrations of DAG and Ca 2+ can activate protein kinase C (PKC). In conjunction with PKC, Ca 2+ induces cortical granule exocytosis and the blockage of polyspermy [61]. When the calcium chelator BAPTA-AM was applied, no cortical granule exocytosis and no completion of meiosis occurred [62]. However, when a Ca 2+ ionophore was applied, MII oocytes underwent cortical granule exocytosis, second polar body emission, and PN formation [63]. In this study, we observed that Gas6-silenced oocytes exhibited apparently low cytoplasmic Ca 2+ excitability, followed by reduced cortical granule exocytosis and resulting in the failure of fertilization.
Initial sperm processing entails disassembly of the sperm nuclear envelop, which releases sperm components into the cytoplasm of oocytes, and this process is dependent upon oocyte-derived PKC [20]. Oocyte-derived glutathione and MPF activity are required for sperm decondensation through the reduction of protamine disulfide bonds [22,64]. Subsequently, maternally supplied histones from oocyte cytoplasm are needed to recondense paternal chromatin via the assistance of various protein kinases and phosphatases [21,23]. Previous research indicated that paternal chromatin remodeling is dependent upon the maturity of oocyte cytoplasm [65]. Indeed, Ca 2+ oscillations promote histone assembly onto paternal chromatin [23]. In the present study, Gas6-silenced oocytes exhibited sperm penetration but no PN formation in the cytoplasm (Fig. 6). Due to the insufficient cytoplasmic maturation of oocytes after Gas6 RNAi treatment, Gas6-silenced MII oocytes exhibited decreased MPF activity and concurrent cytoplasmic changes that require further study to reveal in the effects on paternal and maternal chromatin remodeling. In addition, Gas6-silenced, but MPF activity-rescued MII oocytes failed to undergo PN formation despite sperm penetration into the cytoplasm (Fig. 10A, B). These results suggest that the reduction of Gas6 expression itself sufficient to affect cytoplasm maturation to induce failure of PN formation.
In conclusion, this is the first report of the expression and function of Gas6 in the nuclear and cytoplasmic maturation of oocytes and further process of fertilization. We propose Gas6 as a new candidate mammalian maternal effect gene that is pivotal for oocyte cytoplasmic maturation and normal fertilization, especially for the low cytoplasmic Ca 2+ excitability, and a lack of cortical granule exocytosis, paternal chromosome decondensation, and PN formation (Fig. 11).

Materials and Methods
Animals ICR mice were obtained from Koatech (Pyeoungtack, Korea) and mated to male mice of the same strain to produce embryos in the breeding facility at the CHA Stem Cell Institute of Pochon CHA University. All described procedures were reviewed and approved by the University of Science Institutional Animal Care and Use Committee and performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.

Isolation of oocytes and embryos
To isolate GV oocytes from preovulatory follicles, 4-week-old female ICR mice were injected with 5 IU of eCG and sacrificed after 46 hours later. Cumulus-enclosed oocyte complexes (COCs) were recovered from ovaries by puncturing the preovulatory follicles with 27-gauge needles. M2 medium containing 0.2 mM IBMX was used to inhibit GVBD. Isolated oocytes were snapfrozen and stored at 270uC prior to RNA isolation.
To obtain MII oocytes, we injected female mice with 5 IU eCG, followed by 5 IU hCG after 46 hours. Superovulated MII oocytes were obtained from the oviduct 16 hours after hCG injection. Cumulus cells surrounding MII oocytes were removed by treating COCs with hyaluronidase (300 U/ml). Female mice were superovulated and mated, and embryos were obtained at specific time points after hCG injection as follows: PN 1-cell embryo at 18-20 hours, 2C embryos at 44-46 hours, 4C embryos at 56- Roscovitine, a specific inhibitor of p34 cdc2 -cyclin B1 kinase, was used to inactivate MPF. Control oocytes were cultured in M16 medium for 10 hours, and then transferred in M16 medium containing roscovitine, when first polar body was emitted. Colcemid, a drug for preventing the degradation of Cyclin B, was used to maintain MPF activity. Control and Gas6-dsRNA injected oocytes were cultured in M16 medium for 10 hours until the first polar body extrusion followed by culture in M16 medium containing colcemid for 6 hours. (B) Dual kinase activity analysis to determine the activation levels of the MPF and MAPK was performed. Roscovitine-treated oocytes suppressed activity of MPF and colcemid-treated oocytes maintained activity of MPF. doi:10.1371/journal.pone.0023304.g009 58 hours, 8C embryos at 68-70 hours, morula stage at 80-85 hours, and blastocyst stage at 96-98 hours.

Messenger RNA isolation
Messenger RNA was isolated from oocytes and embryos by using the Dynabeads mRNA DIRECT kit (Invitrogen Dynal AS, Oslo, Norway) according to the manufacturer's instructions. To evaluate recovery, 0.1 ng of Green Fluorescent Protein (GFP) synthetic RNA were added to each oocyte or embryo prior to mRNA extraction [12]. Briefly, oocytes were resuspended in 300 ml of lysis/binding buffer (100 mM Tris-HCl [pH 7.5], 500 mM LiCl, 10 mM EDTA, 1% LiDS, 5 mM DTT) for 5 minutes at room temperature. After vortexing, 20 ml of prewashed Dynabeads oligo dT 25 were mixed with lysate and annealed by rotating for 5 minutes at room temperature. The beads were separated with a Dynal MPC-S magnetic particle concentrator, and poly (A) + RNAs were eluted by incubation in 10 ml of Tris-HCl (10 mM Tris-HCl, pH 7.5) at 65uC for 2 minutes.  Control and Gas6-expressed colcemid or roscovitine treatment eggs had PN formation, while colcemid-treated Gas6-silenced eggs exhibited no PN formation. After in vitro fertilization, eggs were fixed in 4% paraformaldehyde and stained with propidium iodide (red). White dot circles indicate PN formation, and white arrows indicate not fully decondensed paternal chromatin. Control, uninjected oocytes; Gas6-expressed colcemid-treated, control oocytes cultured in M16 medium for 10 hours followed by 6 hours of culture in colcemid containing medium; Gas6-expressed rocovitine-treated, control oocytes cultured in M16 medium for 10 hours followed by 6 hours of culture in roscovitine containing medium; Gas6-silenced colcemid-treated, Gas6 dsRNA-injected oocytes cultured in M16 medium for 10 hours followed by 6 hours of culture in colcemid containing medium. doi:10.1371/journal.pone.0023304.g010 and primer sequences for the encoding genes are listed in Table 4. Set-A primers were used for dsRNA preparation, whereas Set-B was used for analyzing RNAi-mediated gene-specific knockdown by RT-PCR. Relative gene expression levels were normalized to the expression of Gapdh. All experiments were repeated in triplicate.

Preparation of Gas6 dsRNA
For RNAi experiments, we prepared dsRNA for Gas6 (561 bp). Two different primer sets, Set-A and Set-B, were used for dsRNA preparation and confirmation of knockdown of endogenous transcripts, respectively (Table 4). PCR for Gas6 was performed using Set-A primers and resulted in a 561-bp product. Each Gas6 cDNA was cloned into pGEMH-T Easy (Promega) and linearized with Spe I. Insert orientation was confirmed by PCR amplification by using the T7 primer with each Set-A primer. Single stranded RNA (ssRNA) for each orientation was synthesized using the MEGAscript RNAi Kit (Ambion, Austin, TX) and T7 RNA polymerase. Complementary RNAs were mixed and incubated for annealing at 75uC for 5 minutes, then cooled to room temperature. Formation of dsRNA was confirmed by 1% agarose gel electrophoresis, in which the mobility of dsRNA was compared to that of ssRNA. For microinjection, RNAs were diluted to a final concentration of 2 mg/ml.

Microinjection and in vitro culture
GV oocytes were microinjected with Gas6 dsRNA in M2 medium containing 0.2 mM IBMX. An injection pipette containing dsRNA solution was inserted into the cytoplasm of an oocyte, and 10 pl of dsRNA were microinjected using a constant flow system (Transjector; Eppendorf, Hamburg, Germany). To assess injection damage, oocytes were injected with elution buffer alone and used as sham controls. To determine the rate of in vitro maturation, oocytes were cultured in M16 medium containing 0.2 mM IBMX for 8 hours, followed by culture in M16 alone for 16 hours in 5% CO 2 at 37uC. After RNAi experiments, in vitro maturation rates and morphological changes were recorded as previously described [12]. Briefly, the maturation stage of oocytes was scored by the presence of a germinal vesicle (GV oocyte), a polar body (MII oocyte), or neither a germinal vesicle nor a polar body (MI oocyte).
To confirm the effect of Gas6, oocytes were cultured in M16 medium containing 0.2 mM IBMX for 8 hours, followed by culture in M16 alone for 10 hours. After emission of first polar body, control oocytes were cultured in M16 containing 100 mM roscovitine or 1 mg/ml colcemid, while Gas6-silenced MII stage oocytes were cultured in M16 containing 1 mg/ml colcemid for 6 hours in 5% CO 2 at 37uC. Roscovitine, a purine derivative, inhibit specifically the activity MPF [66], while colcemid, a well known microtubule-depolymerizing drug, maintain MPF activity by preventing the degradation of cyclin B in the mouse oocytes [67].

Parthenogenetic activation and culture of activated oocytes
Mature oocytes were activated parthenogenetically by culturing for 2 hours in Ca 2+ -free KSOM medium supplemented with 10 mM SrCl 2 and 5 mg/ml cytochalasin B. Modified Chatot, Ziomek, and Bavister (CZB) medium was used for the culture of parthenogenetically activated oocytes. Activated oocytes were cultured in CZB medium at 37uC in an air atmosphere containing 5% CO 2 to monitor the development of activated oocytes to the 2C stage.

Noninvasive examination of spindle structure
Spindle structure observation of living cells was performed using the LC Polscope optics and controller system combined with a Figure 11. Gas6 RNAi affects MPF activity and PN formation. When Gas6 was silenced, oocytes developed to the MII stage with low MPF activity. These MII oocytes exhibited altered Ca 2+ excitability and markedly reduced cortical granule exocytosis. In addition to these two well-known cytoplasmic changes related to the decreased MPF activity (black lines), novel mechanisms caused by Gas6 knockdown (red line) may induce further aberrant changes of cytoplasmic maturation that permits sperm penetration but not PN formation. Gray circles with dotted lines designate failure of male and female pronuclei production. Green vesicles around the ooplasma membrane designate remained intact cortical granules. doi:10.1371/journal.pone.0023304.g011 computerized image analysis system (Oosight TM Meta Imaging System, CRI Inc., MA).

Aceto-orcein staining
Oocytes were fixed with aceto-methanol (acetic acid:methanol = 1:3) solution for 6 hours at 4uC. Fixed oocytes were transferred onto a slide and covered with a clean cover glass. Aceto-orcein solution was inserted between the slide and the cover glass, and the oocytes were photographed.

In vitro fertilization
Sperm were collected from the cauda epididymis of 8-week-old male ICR mice (Koatech). The tissues were incised, and the sperm were allowed to swim out into M16 medium. Sperm were incubated in M16 medium for 1 hour to allow capacitation. The ZP was removed from oocytes by dissolution in Tyrode's solution (pH 2.5). When ZP thinning was observed through the microscope, oocytes were transferred to M16 medium, and the cellular mass was washed out of the ZP by gentle pipetting. ZP-free MII eggs, which had been placed previously in a 200-ml M16 medium droplet under mineral oil, were inseminated with 2.5610 4 /ml sperm for 2 hours, and then oocytes were washed free of unbound sperm. Washing and transfer of the oocyte were necessary to avoid excessive polyspermy of ZP-free oocytes. The oocytes were transferred into M16 medium for culture in 5% CO 2 at 37uC for 5 hours to observe PN formation.

Measurement of intracellular Ca 2+ concentration
Changes in the intercellular calcium concentration, [Ca 2+ ] i , were measured by loading oocytes with 1 mM of the Ca 2+ -sensitive indicator dye Fluo-4AM (Molecular Probes, Eugene, OR) supplemented with 0.02% pluronic acid (Molecular Probes) in HEPES-buffered Tyrode's lactate solution. Changes in [Ca 2+ ] i were monitored using an Axiovert 200M microscope fitted with a 106 objective lens and CCD camera controlled by Axiovision software 4.8.1 (Carl Zeiss, Jena, Germany). Intracellular calcium concentrations were monitored by measuring fluorescence from individual oocytes loaded on a temperature-controlled chamber dish (SEC, Seoul, Korea) coated with CellTakH (BD Biosciences,

Observation of exocytosis of cortical granules
Oocytes were labeled with lectins according to the previously reported method [68]. Immediately after removal of the ZP, oocytes were fixed in 3% paraformaldehyde in PBS for 30 minutes at 20uC. After fixation, oocytes were rinsed with blocking solution, PBS containing 1% BSA, and 100 mM glycine, for 10 minutes to remove aldehyde. Oocytes were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes. After washing twice in blocking solution, oocytes were incubated with solution of 200 mg/ml FITC-conjugated LCA lectin for 30 minutes. Oocytes were rinsed thoroughly with PBS and transferred to PBS-PVA. Chromatin condensation was visualized by costaining with 10 mg/ml 4,6-diamidino-2-phenyl indole for 10 minutes. Oocytes were mounted between a slide and coverslip.

Statistical analysis
Statistical analyses of real-time PCR data were evaluated using one-way analysis of variance and a log linear model. Data were presented as mean 6 SEM, derived from at least 3-5 separate and independent experiments, and a value of p,0.05 was considered statistically significant.