Skip to main content
NCBI home page
Cell Cycle logoLink to Cell Cycle
. 2019 Sep 18;18(21):2986–2997. doi: 10.1080/15384101.2019.1666596

Involvement of CaMKII in regulating the release of diplotene-arrested mouse oocytes by pAkt1 (Ser473)

Lingling Liu a,b,c, Hanwen Li d, Ben Labbe e, Yang Wang b, Shitao Mao f, Yu Cao b, Mingjing Zhao f, Shuo Liu f, Hang Yu g, Xin Deng a,b,f,✉
PMCID: PMC6791694  PMID: 31530151

ABSTRACT

Calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII) had been reported to play a role in the process of fertilization. However, the role of CaMKII in the release of diplotene-arrested oocytes is poorly understood. In this study, we explored the potential effect of CaMKII on Akt1 and the relationship among CaMKII, Akt1 and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) during the meiotic resumption of mouse oocytes. We found that inhibition of CaMKII aggravated diplotene arrest. We detected the expression and distribution of pCaMKII (Thr286), pAkt1 (Ser473), Cdc25B and pCdc2 (Tyr15) when oocytes were treated with KN-93, SH-6, LY294002 or PIP3, respectively. Our data showed that down-regulated CaMKII by KN-93 decreased the levels of pAkt1 (Ser473) and rearranged the distribution of pAkt1 (Ser473). Meanwhile, down-regulated pAkt1 (Ser473) by SH-6 also decreased the levels of pCaMKII (Thr286), Cdc25B and pCdc2 (Tyr15) significantly and rearranged the distributions of pCaMKII (Thr286). Furthermore, our data showed that exogenous PIP3 up-regulated GVBD rates significantly and increased the levels of pCaMKII (Thr286) and pAkt1 (Ser473). On the contrary, down-regulation of PIP3 by LY294002 decreased GVBD rates and the levels of pCaMKII (Thr286) and pAkt1 (Ser473), respectively. Our results showed that Akt1 and CaMKII regulated each other, and PIP3 may be involved in these regulations during the release of mouse oocytes from diplotene arrest.

KEYWORDS: Calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII), protein kinase B alpha (PKBα/Akt1), mouse oocyte, diplotene arrest

1. Introduction

Up to the mid-two-cell stage (~27 h postfertilization), the embryo appears to rely largely upon protein and RNA synthesized during the course of oogenesis. As such, the development of oocytes is crucial for the first cleavage of fertilized eggs. The development of oocytes consists of successively meiotic events, which mainly occur in oocyte nucleus. Progression from germinal vesicle (GV) to germinal vesicle break down (GVBD) reinitiates the meiotic event, which ensures meiotic completion and embryonic development. Therefore, GVBD is the key node for the development of oocytes.

Previous reports indicated that Akt may cause the activation of Mitosis Promotion Factor (MPF) and strongly promotes the cleavage of mouse fertilized eggs by inducing Akt-dependent phosphorylation of Cdc25B [1–3]. On entry into M phase, Cdc25, a dual-specific phosphatase family, may dephosphorylate Cdc2 at Tyr15 to cause the activation of MPF [2,3]. As the catalytic subunit of MPF, dephosphorylation of Cdc2 at Tyr15 plays a key role in mitotic course [4,5]. Our previous reports also showed that inhibition of pAkt1 (Ser473) by SH-6 decreased GVBD rates [6], suggesting that pAkt1 (Ser473) promoted GVBD rates. However, whether pCdc2 (Tyr15) is one of the downstream factors of pAkt1 (Ser473) signaling pathway remains unclear during the course of diplotene arrest of mouse oocytes.

In addition, Akt1 activation relies on its pleckstrin homology domain (PHD) binding to phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Activated Akt then initiates a downstream cascade of phosphorylation and activation of the effector proteins [7–10]. PIP3 is produced by PIP2 which is phosphorylated by the class I phosphoinositide 3-kinase (PI3K), serving not only as components of biological membranes, but also as coordinators of diverse cellular events including proliferation, cell migration and ion transport [11–13]. Transition between PIP2 and PIP3 on cytomembrane is dependent on the phosphate group, which carries with negative charge, which is similar to ion exchanges inside and outside the cell. And these ion exchanges are similar to Ca2+ signaling events and resting potential [14]. As a result, the shape of GVBD oocytes is quite different from that of GV oocytes. During the change of shape, PIP3, one of the components in cytomembrane on the surface of oocytes, may be actively involved in structural change of cytomembrane. We suggest that the change of shape from GV to GVBD is closely related to PIP3 and Ca2+ signaling events.

A recent report by Agamsu et al. proposed that upon epidermal growth factor (EGF) stimulation calmodulin (CaM) interacts with the PHD of Akt and mediates Akt translocation from cytoplasma to the cytomembrane for its activation [7]. CaM is then released upon binding of Akt to PIP3 on the plasma membrane. These results are consistent with our previous studies, suggesting that CaM regulated Akt activity by modulating its subcellular location [3,6,7]. Moreover, Ca2+/CaM-dependent protein kinase II (CaMKII) is necessary for Ca2+ homeostasis. Ca2+ signaling regulates a number of diverse events, such as cell migration and differentiation by diffusing a gradient across plasma membrane to bind to regulatory proteins, such as CaM or CaMKII [15–21]. Although CaMKII is closely related to Ca2+ and CaM, it remains unclear about the relationship among CaMKII, Akt1 and PIP3 during the release of mouse oocytes from diplotene arrest.

In this study, we investigated the relationship between CaMKII and Akt1 during the release of diplotene-arrested mouse oocytes using each specific inhibitors. We also measured the effect of PIP3 on GVBD, CaMKII and Akt1 during the release from diplotene arrest with exogenous PIP3 or LY294002, respectively. We observed that KN93 and SH-6 affected both the levels and distributions of Akt1 and CaMKII, respectively. And the changes of PIP3 were closely related to the levels of Akt1 and CaMKII. Our data provide new evidences on the relationship of Akt1 and CaMKII during the release of diplotene-arrested mouse oocytes.

2. Materials and methods

2.1. Animals

Kunming strain mice were obtained from the Department of Laboratory Animals, China Medical University (CMU). All experiments were performed at CMU in accordance with NIH Guidelines of the USA for Care and Use of Laboratory Animals. Protocols for animal handing and treatment procedures were reviewed and approved by the CMU Animal Care and Use Committee.

2.2. Reagents

Goat polyclonal antibody, Cdc25B, phospho-Cdc2 (Tyr15) monoclonal antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit/Tetramethylrhodamine (TRITC)-conjugated rabbit anti-goat IgG antibody, anti-GAPDH antibody and anti-CaMKII antibody were purchased from Proteintech Group Inc. Anti-CaMKII (Thr286) antibody was obtained from abcam. Anti-Akt1 (Ser473), total Akt1/2/3 and pPKCδ (Thr505) antibodies were from Cell Signaling. KN-92, KN-93, LY294002 and SH-6 were purchased from APExBio Technology, MedChemExpress, Selleckchem and abcam, respectively. Calmidazolium chloride was obtained from Amquar. D-myo-Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) were purchased from Echelon Biosciences Inc. Other reagents, unless otherwise specified, were purchased from Sigma-Aldrich, Shanghai, China.

2.3. Collection and culture of mouse oocytes

Immature GV-intact oocytes were collected from 3-week-old female Kunming mice. The ovaries were placed in M2 medium. Follicles were punctured with a fine needle to release cumulus-enclosed oocytes or naturally denuded GV-intact oocytes. GV-intact oocytes were released from attached follicular cells by repeated pipetting with a mouth-operated micropipette. Hanging drop culture of the oocytes was performed at 37°C, in a humidified atmosphere with 5% CO2. Oocytes culture was performed in M2 medium. Oocytes were collected and then stored at −80°C until use.

2.4. Observation of GVBD rates

CaMKII, pAkt1 (Ser473) and PIP3 special inhibitors, KN-93, SH-6 and LY294002 were treated into GV oocytes. Oocytes were placed in a drop of M2 medium under paraffin oil in the lid of a 9-cm Falcon culture dish. Oocytes in control groups were untreated. The GVBD rates of oocytes were counted under a dissecting microscope (40 × magnification) at 4 h at 37°C, in a humidified atmosphere with 5% CO2, after treatment of the special inhibitor and the results were analyzed statistically from three independent experiments.

2.5. Immunocytochemical analysis

GV-intact oocytes in the treated or untreated groups were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) with 0.5% bovine serum albumin (BSA) for 1 h at room temperature (RT) and permeabilized for 10 min in 0.1% TritonX-100 in PBS with 0.5% BSA at 37°C. These oocytes were stained overnight with pAkt Ser473 and pCaMKII (Thr286) antibodies diluted 1:100 at 4°C. After washing three times in PBS containing 0.5 mg/ml BSA, eggs were incubated for 2 h at RT in FITC (green fluorescence) or TRITC (red fluorescence)-conjugated goat anti-rabbit secondary antibody, followed by staining with Hochest33258 for 1 min at RT for chromatin visualization. Immunofluorescence images were obtained using a confocal laser scanning biological microscope (FLUOVIEW FV1000, OLYMPUS®) with FV10-ASW software.

2.6. Western blot analysis

Cell lysates of oocytes were prepared by adding approximately 200 eggs in a minimal volume of collection medium to 20 µl of RIPA lysis buffer containing a protease inhibitor cocktail and 10 µg/ml PMSF. They were then separated on a 10% SDS-PAGE gel and, transferred to PVDF membranes. The membranes were blocked with 3% BSA or 5% Nonfat-Dried Milk in Tris-buffered saline containing 0.05% Tween 20, and probed with primary antibodies against pAkt1 (Ser473), Akt1/2/3, pCaMKII (Thr286), CaMKII, Cdc25B, pCdc2 (Tyr15), pPKCδ (Thr505), or GAPDH (1:400 dilution). Membranes were then incubated in HRP-conjugated secondary antibody at 1:5000 (Earthox, San Francisco, CA, USA). Proteins were detected using Tanon ECL detection system.

2.7. Statistical analysis

Data are presented as mean ± SEM of separate experiments (n > 3) and compared by one-way analysis of variance (ANOVA) with SPSS 13.0 software (SPSS, Chicago, IL, USA). P-values <0.05 were considered significant.

3. Results

3.1. Levels of CaMKII increased distinctly in GVBD stage and inhibition of CaMKII by KN-93 decreased GVBD rates

Previous reports showed that CaMKII involved in regulating Akt activation in Pancreatic Cancer Cells [7,21]. And our report also showed that Akt regulated the release of diplotene-arrested mouse oocytes [6]. However, it remains unclear whether CaMKII is involved in regulating Akt phosphorylation during release from diplotene arrest of mouse oocytes.

To explore the role of CaMKII in GVBD rates, firstly we detected the levels of CaMKII expressed in GV and GVBD with Western Blot Analysis (Figure 1(a)). Our results showed that the expression of CaMKII was weak in GV oocytes but increased distinctly in GVBD oocytes. And then, we cultured oocytes for 4 h to observe GVBD rates with KN-93, a special inhibitor of CaMKII. When oocytes were treated with KN-93 of 10, 25, 50, 125, 250 and 500 μM, respectively, we observed that GVBD rates decreased gradually with increases of KN-93 concentrations, especially when concentrations of KN-93 were more than 125 μM (Figure 1(b), p < 0.001).

Figure 1.

Figure 1.

Open in a new tab

CaMKII expressions in GV and GVBD oocytes and its role in GVBD rates. (a) Levels of pCaMKII (Thr286) in GV and GVBD oocytes were detected by Western Blot Analysis. (b) GV oocytes were treated with KN-93, an inhibitor of CaMKII for 4 h. GVBD rates were counted under microscope (40 × magnification).

3.2. Inhibition of CaMKII by KN-93 decreased levels of pAkt1 (Ser473) and pCaMKII (Thr286), and rearranged distributions of pAkt1 (Ser473)

To study the role of CaMKII in Akt activation, KN-93 of 50 and 250 μM was used to treat oocytes, respectively. We observed both the levels of pCaMKII (Thr286) and pAkt1 (Ser473) decreased significantly with the increases of KN-93 concentrations (p < 0.001, Figure 2(a,b)).

Figure 2.

Figure 2.

Open in a new tab

Inhibition of CaMKII by KN-93 changed the levels and distributions of pAkt1 (Ser473). (a-a) pAkt1 (Ser473) protein band of approximately 60 kDa was detectable by Western Blot Analyses. Anti-Akt1/2/3 and GAPDH antibodies recognized at 60 and 36 kDa as (control; lower panel). (a-b) Densitometric analyses of pAkt1 (Ser473) levels were reported as pAkt1 (Ser473)/Akt1/2/3. Error bar represents mean ± SEM of three independent experiments. (b-a) pCaMKII (Thr286) protein band of approximately 50 kDa from 200 mouse oocytes was detectable by Western Blot Analyses with an pCaMKII (Thr286) and CaMKII antibody. Anti-CaMKII and GAPDH antibodies recognized a lower molecular weight band of approximately 50 kDa and 36 kDa (control; lower panel). KN-92 was also used as control. (b-b) Densitometric analyses of pCaMKII (Thr286) levels were reported as pCaMKII (Thr286)/CaMKII. (c) Expressions of pAkt1 at Ser473 were fixed and detected with its antibodies and then treated with fluorescein isothiocyanate (FITC)-labeled antibody (green) or tetramethylrhodamine (TRITC)-labeled antibody (red). Nuclei were stained with Hochest33258 (blue). Scale bar = 20 μm. All images were magnified 60 times.

Furthermore, the distributions of pAkt1 (Ser473) in GV oocytes were in cytoplasma while around the nucleus in GVBD oocytes. However, the distributions of pAkt1 (Ser473) in GVBD oocytes were changed to cytoplasma under the treatment with KN-93 250 μM for 4 h, and the shape and diameter of oocytes in GV stage did change distinctly when oocytes were treated with KN-93 for 4 h, but DNA in nuclei of oocytes distributed toward to peripheral and cytoplasma showed no distinct polarity (Figure 2(c)). Our data suggested that CaMKII involved in regulating GVBD rates of oocytes by affecting the level and the distribution of pAkt1 (Ser473).

3.3. Inhibition of pAkt1 (Ser473) by SH-6 regulated the expression of pCaMKII, Cdc25b and pCdc2 (Tyr15), and the distribution of pCaMKII (Thr286)

The above results indicate that inhibition of CaMKII decreased GVBD rates and levels of pAkt1 (Ser473). Although our previous report showed that Akt1 involved in promoting the release from diplotene arrest [6], it is unclear whether Akt1 regulates the levels of pCaMKII (Thr286), Cdc25B or pCdc2 Tyr15.

To study the relationship between CaMKII and Akt, we employed SH-6, a special inhibitor of pAkt1 (Ser473) to treat oocytes for 4 h for observing the effect of inhibition of pAkt1 (Ser473) on CaMKII. We thought that if inhibition of pAkt1 (Ser473) by SH-6 did not change the levels of pCaMKII (Thr286), CaMKII may be an upstream regulator of Akt1. However, if inhibition of pAkt1 (Ser473) by SH-6 decreased the levels of pCaMKII (Thr286), pAkt1 (Ser473) may regulate CaMKII.

With the treatment of SH-6, our data showed that the levels of pCaMKII (Thr286) and pAkt1 (Ser473) were decreased significantly in a dose-dependent manner (Figure 3(a,b)) during release from diplotene arrest of mouse oocytes, suggesting that the levels of pCaMKII (Thr286) were regulated significantly by positive feedback of pAkt1 (Ser473). For the reason that the results in Figure 2(a) showed that KN-93 decreased the levels of pAkt1 (Ser473), we combined the two results of Figures 2(a) and 3(a) to deduced that Akt1 and CaMKII may positively regulate each other during the release from diplotene arrest of mouse oocytes.

Figure 3.

Figure 3.

Open in a new tab

Inhibition of pAkt1 (Ser473) by SH-6 changed the levels and distribution of pCaMKII (Thr286). (a-a) Oocyte extracts were incubated with anti-pCaMKII (Thr286) antibodies. The presence of pCaMKII (Thr286) was analyzed by Western Blot. (a-b) Densitometric analyses of pCaMKII (Thr286) levels were reported as pCaMKII (Thr286)/CaMKII. Error bar represents mean ± SEM of three independent experiments. (b-a) Oocyte extracts were incubated with anti-pAkt1 (Ser473) antibodies. The presence of pAkt1 (Ser473) was analyzed by Western Blot. (b-b) Densitometric analyses of pAkt1 (Ser473) levels were reported as pAkt1 (Ser473)/Akt1/2/3. Error bar represents mean ± SEM of three independent experiments. (c) Oocytes were fixed and detected with pCaMKII antibodies and then treated with fluorescein isothiocyanate (FITC)-labeled antibody (green) or tetramethylrhodamine (TRITC)-labeled antibody (red), respectively. Nuclei were stained with Hochest33258 (blue). Scale bar = 20 μm.

Furthermore, with the treatment of SH-6, we also observed that the distribution of pCaMKII (Thr286) was also changed clearly. Our data showed that pCaMKII (Thr286) distributed normally in cell nucleus and cortical area of GV oocytes. And its distribution was rearranged around cell nucleus of GVBD oocytes. When oocytes were treated with SH-6 for 4 h, the distributions were rearranged to cytoplasma. Meanwhile, the volume and diameter of oocytes were shrunk distinctly by SH-6, compared with normal oocytes and oocytes treated by KN-93 (Figure 3(c), lowest panels), suggest that pAkt1 (Ser473) may be involved in regulating these proteins related with cell skeleton but not CaMKII.

In addition, previous report showed that Cdc25B, a dual-specific phosphatase, dephosphorylate Cdc2 on Tyr15, causing the activation of MPF on entry into M phase [2]. And Cdc25B also act as a potential target of Akt [2]. However, it still needs to be clarified about if Akt regulates Cdc25B during release from diplotene arrest. Here, we used SH-6 to inhibit pAkt1 (Ser473) specifically and then observed the changes of Cdc25B or pCdc2 (Tyr15) during the release from diplotene arrest of mouse oocytes (Figure 4(a,b)). Our results showed that the inhibitions of pAkt1 (Ser473) decreased the levels of Cdc25B and the dephosphorylations of Cdc2 at Tyr15 significantly (Figure 4(a-c)), but did not affect the levels of pPKCδ (Thr505) (Figure 4(d)), suggesting that pAkt1 (Ser473) promote release from diplotene arrest in mouse oocytes by regulating the levels of Cdc25B and pCdc2 (Tyr15) but not PKCδ (Figure 4).

Figure 4.

Figure 4.

Open in a new tab

Inhibition of pAkt1 (Ser473) by SH-6 changed the levels of Cdc25B and pCdc2 (Tyr15) but not pPKCδ (Thr505). (a) and (b) GV oocytes were treated with SH-6 for 4 h and then oocyte extracts were incubated with anti-pCdc2 (Tyr15) or Cdc25B antibodies. The presence of pCdc2 (Tyr15) or Cdc25B was determined by Western Blot Analysis. (c) Densitometric analyses of 60-kDa Cdc25B or 34-kDa pCdc2 (Tyr15) expressions were reported as pCdc2 (Tyr15) or Cdc25B/GAPDH. (d) GV oocytes were treated with SH-6 of 10 μM and 100 μM for 4 h and then oocyte extracts were incubated with anti-pPKCδ (Thr505) antibodies. The presence of pPKCδ (Thr505) was determined by Western Blot Analysis.

3.4. Inhibition of PIP3 by LY294002 decreased GVBD rates, and the levels of pAkt1 (Ser473) and pCaMKII (Thr286) during release from diplotene arrest in mouse oocytes

Nothing is reported on the role of PIP3 during release from diplotene arrest of mouse oocytes. To evaluate the effects of down-regulating PIP3 on GVBD rates, we used LY294002, a PI3K inhibitor, to indirectly inhibit PIP3 production. Our data showed that GVBD rates decreased gradually with the increases of LY294002 concentrations (p < 0.001, Figure 5(a)). When the concentration of LY294002 was 0.5 mM, GVBD rates and levels of pCaMKII (Thr286) were less than half of the original quantity. When its concentration was 1 mM, GVBD rates and pCaMKII (Thr286) levels were much less than a quarter of original quantity (Figure 5(a,c)).

Figure 5.

Figure 5.

Open in a new tab

Inhibition of PIP3 by LY294002 decreased GVBD rates and the levels of pAkt1(Ser473) or pCaMKII (Thr286). (a) GV oocytes were treated with LY294002 for 4 h. GVBD rates were counted using a microscope (80 ×). (b-a) and (c-a) Oocyte extracts were incubated with anti-pAkt1 (Ser473) or pCaMKII (Thr286) antibodies. The presences of pAkt1 (Ser473) or pCaMKII (Thr286) were analyzed by Western Blotting Analysis. (b-b) and (c-b) Densitometric analyses of pAkt1 (Ser473) levels were reported as pAkt1 (Ser473) or pCaMKII (Thr286)/GAPDH. Data are presented as mean ± SEM of three independent experiments.

Our data also showed that the levels of pCaMKII (Thr286) and pAkt1 (Ser473) were decreased gradually when oocytes were treated with 0.5 mM and 1 mM LY294002 (Figure 5(b,c)). And when the concentration of LY294002 was 1 mM, the levels of pCaMKII (Thr286) and pAkt1 (Ser473) were decreased distinctly, especially the levels of pCaMKII (Thr286) were scarce. Our results suggested that levels of PIP3 are closely relative with Akt1 and CaMKII, especially for CaMKII.

3.5. Exogenous PIP3 increased GVBD rates, and the levels of pAkt1 (Ser473) and pCaMKII (Thr286) during release from diplotene arrest in mouse oocytes

To further explore the direct role of PIP3 during release from diplotene arrest of mouse oocytes, we employed exogenous PIP3 to treat oocytes. We observed that GVBD rates enhance distinctly when oocytes were treated with exogenous PIP3 of 1 μM for 1, 2 and 3 h. Although GVBD rate was highest at the 3 h than these at the 1 and 2 h. However, the changes between control group and experimental group were in peak at the 2 h, not at the 3 h (Figure 6(a)). Our results suggested that PIP3 involves in regulating release from diplotene arrest of mouse oocytes in a time-dependent manner.

Figure 6.

Figure 6.

Open in a new tab

Up-regulated PIP3 increased GVBD rates and the levels of pAkt1(Ser473) or pCaMKII (Thr286). GV oocytes were treated with exogenous PIP3 for 4 h. A. GVBD rates were counted using a microscope (80 ×). (b-a) and (c-a) Oocyte extracts were incubated with anti-pAkt1 (Ser473) or pCaMKII (Thr286) antibodies. The presences of pAkt1 (Ser473) or pCaMKII (Thr286) were analyzed by Western Blotting Analysis. (b-b) and (c-b) Densitometric analyses of pAkt1 (Ser473) levels were reported as pAkt1 (Ser473) or pCaMKII (Thr286)/GAPDH. Data are presented as mean ± SEM of three independent experiments.

Furthermore, when oocytes were treated with PIP3 of 1 μM for 2 h, the levels of pAkt1 (Ser473) and pCaMKII (Thr286) were increased significantly (Figure 6(b,c)). Moreover, our data also showed that the levels of pAkt1 (Ser473) were increased significantly at 2 h treatment by 0.5 μM or 1 μM PIP3 but not 0.1 μM and 2 μM (Figure 6(b)). These data suggested that PIP3 may also regulate the levels of pAkt1 (Ser473) and pCaMKII (Thr286) in a dose-dependent manner.

4. Discussion

The process of oocyte maturation is companied with a number of signaling processes, such as a reduction of cGMP in response to luteinizing hormone or the activation of the Akt1 through PI3K pathway. In this pathway, Akt1 phosphorylates and activates Cdc25B [1,22–31]. Cdc25B has been reported to potentially enhance the rate of oocyte maturation by dephosphorylating Cdc2 at Tyr15 [32–40]. Recent reports also indicated that CaM may promote Akt(PHD) binding to PIP3, and then Akt localizing to the cytomembrane [4].

CaM, a multifunctional calcium-binding messenger protein in all eukaryotic cells. Ca2+/calmodulin-dependent kinase II (CaMKII), a serine/threonine kinase, is necessary for the maintenance of Ca2+ homeostasis. Its autoinhibition is governed by a threonine 286 residue. CaMKII is activated by the binding of Ca2+/CaM [41–46]. When CaMKII was phosphorylated at Thr286, it activates permanently. So, as a down-stream protein of CaM [21], we deduced that CaMKII may be closely relative with Akt.

To explore the relationship between pCaMKII (Thr286) and pAkt1 (Ser473) during the release of mouse oocytes from diplotene arrest, we have developed a method for the comprehensive functional mapping of the interaction between protein and protein and have applied the method to the connections between Akt1 and CaMKII during the release from diplotene arrest. We thought that there are three relationships between two proteins in all biological cells. They are interactive (including direct or indirect), up (or down) stream and no relationship. We deduced if two proteins interact, both of them should be decreased by their special inhibitor, respectively. If one protein is the upstream of the other protein, only one inhibitor could decrease the levels of the other protein. If two proteins are no relationship, their special inhibitors did not affect the other protein. Certainly, this method may confirm only indirect interaction of two proteins but cannot confirm if they were direct interaction.

In the present study, we used the special inhibitors of pCaMKII (Thr286) or pAkt1 (Ser473), KN-93 or SH-6, to treat oocytes, respectively. We observed that downregulations of pCaMKII (Thr286) or pAkt1 (Ser473) decreased the levels of pAkt (Ser473) or pCaMKII (Thr286) (Figures 2(a) and 3(a)) and rearranged their distributions, respectively. Furthermore, our data showed that down-regulation of pAkt (Ser473) also decreased the levels of Cdc25B and dephosphorylation of Cdc2 (Tyr15) during release from diplotene arrest of mouse oocytes. So we concluded that CaMKII involved in regulating the release from diplotene arrest of mouse oocytes via pAkt1 (Ser473).

Moreover, our data also showed that down-regulation of PIP3 not only decreased GVBD rates and the level of pAkt (Ser473), but also decreased the level of pCaMKII (Thr286) significantly. Conversely, up-regulation of PIP3 increased those of them. For the reason that phosphorylation of Akt1 at Ser473 produced by its PH domain binding with PIP3 is closely related to GVBD rates. And CaM mediates the phosphorylation. So we deduced that GVBD rates may be closely related to the interaction among Akt1, PIP3, CaM and CaMKII.

In conclusion, we reported for the first time that pCaMKII (Thr286) involves in regulating the release of mouse oocytes from diplotene arrest by interacting with pAkt1 (Ser473). And PIP3 is also involved in regulating the levels of pCaMKII (Thr286) and pAkt (Ser473) during the transition from GV to GVBD.

Funding Statement

This work was supported by the National Nature Science Foundation of China under Grant number [81370712 and 81700063].

Acknowledgements

We are grateful to ProfessorZhenbo Wang (Institute of Zoology, Chinese Academy of Sciences) for critical comments on the manuscript and DirectorQin Li (The Fourth Affiliated Hospital of China Medical University) for his warm assistance in experimentation.

Disclosure statement

No potential conflict of interest was reported by the authors.

References

  • [1].Kang H, Hwang SC, Park YS, et al. Cdc25B phosphatase participates in maintaining metaphase II arrest in mouse oocytes. Mol Cells. 2013;35(6):514–518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Chen F, Aiming Y, Ying L, et al. Involvement of protein kinase B/AKT in early development of mouse fertilized eggs. Biol Reprod. 2007;77(3):560–568. [DOI] [PubMed] [Google Scholar]
  • [3].Deng X, Feng C, Wang EH, et al. Influence of proline-rich inositol polyphosphate 5-phosphatase, on early development of fertilized mouse eggs, via inhibition of phosphorylation of Akt. Cell Prolif. 2011;44(2):156–165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Fei H, Zhou Y, Li R, et al. HBXIP, a binding protein of HBx, regulates maintenance of the G2/M phase checkpoint induced by DNA damage and enhances sensitivity to doxorubicin-induced cytotoxicity. Cell Cycle. 2017;16(5):468–476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Wood E, Nurse P.. Pom1 and cell size homeostasis in fission yeast. Cell Cycle. 2013;12(9):3228–3236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Liu L, Li S, Li H, et al. Protein kinase Cδ (PKCδ) involved in the regulation of pAkt1 (Ser473) on the release of mouse oocytes from diplotene arrest. Cell Biochem & Funct. 2018;36(4):221–227. [DOI] [PubMed] [Google Scholar]
  • [7].Agamasu C, Ghanam RH, Xu F, et al. The interplay between calmodulin and membrane interactions with the pleckstrin homology domain of Akt. J Biol Chem. 2017;292(1):251–263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Constance A, Ghanam RH, Saad JS. Structural and biophysical characterization of the interactions between calmodulin and the pleckstrin homology domain of Akt. J Biol Chem. 2015;290(45):27403–27413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Wang X, Boyken SE, Hu J, et al. Calmodulin and PI(3,4,5)P3 cooperatively bind to the Itk pleckstrin homology domain to promote efficient calcium signaling and IL-17A production. Sci Signal. 2014. August 5;7(337):ra74. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Lemmon MA. Pleckstrin homology (PH) domains and phosphoinositides. Biochem Soc Symp. 2007;74(74):81–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Manna P, Jain SK. PIP3 but not PIP2 increases GLUT4 surface expression and glucose;metabolism mediated by AKT/PKC zeta/lambda phosphorylation in 3T3L1;adipocytes. Mol Cell Biochem. 2013;381(1–2):291–299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Kurokawa T, Takasuga S, Sakata S, et al. 3ʹ Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP). PNAS. 2012;109(25):10089–10094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Lianzhi D, Luhua Z, Bingchang Z, et al. DAG/PKCδ and IP3/Ca(2+)/CaMK IIβ operate in parallel to each other in PLCγ1-driven cell proliferation and migration of human gastric adenocarcinoma cells, through Akt/mTOR/S6 pathway. Int J Mol Sci. 2015;16(2):28510–28522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Hashad AM, Mazumdar N, Romero M, Nygren A, Bigdely-Shamloo K, Harraz OF, Puglisi JL, Vigmond EJ, Wilson SM, Welsh DG Interplay among distinct Ca2+ conductances drives Ca2+ sparks/spontaneous transient outward currents in rat cerebral arteries. J Physiol. 2017;595(4):111–1126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Backs J, Stein P, Backs T, et al. The gamma isoform of CaM kinase II controls mouse egg activation by regulating cell cycle resumption. PNAS. 2010;107(1):81–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Chang JY, Parra-Bueno P, Laviy T, et al. CaMKII autophosphorylation is necessary for optimal integration of Ca2+, signals during LTP induction, but not maintenance. Neuron. 2017;94(4):800–808.e4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Dong B, Valencia CA, Liu R. Ca2+/calmodulin directly interacts with the pleckstrin homology domain of AKT1. J Biol Chem. 2007;282(34):25131–25140. [DOI] [PubMed] [Google Scholar]
  • [18].Lim GB. Gene therapy to inhibit CaMKII in CPVT. Nat Rev Cardiol. 2019;16(8):456. [DOI] [PubMed] [Google Scholar]
  • [19].Obashi K, Matsuda A, Inoue Y, et al. Precise temporal regulation of molecular diffusion within dendritic spines by actin polymers during structural plasticity. Cell Rep. 2019;27(5):1503–1515. [DOI] [PubMed] [Google Scholar]
  • [20].Koval OM, Nguyen EK, Santhana V, et al. Loss of MCU prevents mitochondrial fusion in G1-S phase and blocks cell cycle progression and proliferation. Sci Signal. 2019;12(579):Pii: eaav1439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Ghimire K, Zaric J, Alday-Parejo B, et al. MAGL1 medicates eNOS activation and NO production in endothelial cells in response to fluid shear stress. Cells. 2019;8(5):pii: E388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Aoki M, Fujishita T. Oncogenic roles of the PI3K/AKT/mTOR axis. CurrTop Microbiol Immunol. 2017;407:153–189. [DOI] [PubMed] [Google Scholar]
  • [23].Toker A, Rameh L. PIPPing on AKT1: how many phosphatases does it take to turn off PI3K? Cancer Cell. 2015;28(2):143–145. [DOI] [PubMed] [Google Scholar]
  • [24].Rodgers SJ, Ferguson DT, Mitchell CA, et al. Regulation of PI3K effector signalling in cancer by the phosphoinositide phosphatases. Biosci Rep. 2017;37(1):pii.BSR20160432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Di Paolo G, De Camilli P. Phosphoinositides in cell regulation and membrane dynamics. Nature. 2006;443(7112):651–657. [DOI] [PubMed] [Google Scholar]
  • [26].Balla T. Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol Rev. 2013;93(3):1019–1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Huang H, Li L, Shi W, et al. The multifunctional effects of nobiletin and its metabolites In Vivo and In Vitro. Evid Based Complement Alternat Med. 2016;2016:2918796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Li F, Malik KU. Angiotensin II-induced Akt activation is mediated by metabolites of arachidonic acid generated by CaMKII-stimulated Ca2(+)-dependent phospholipase A2. Am J Physiol Heart Circ Physiol. 2005;288(5):H2306–H2316. [DOI] [PubMed] [Google Scholar]
  • [29].Jun M, Cui C, Liu Y, et al. The role of 14-3-3ϵ interaction with phosphorylated CDC25B at its Ser321 in the release of the mouse oocyte from prophase I arrest. PloS One. 2013;8:e53633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Boutros R, Lobjois V, Ducommun B, et al. CDC25 phosphatases in cancer cells: key players? Good targets? Nature Rev Cancer. 2007;7(7):495–507. [DOI] [PubMed] [Google Scholar]
  • [31].Bischof J, Brand CA, Somogyi K, et al. A cdk1 gradient guides surface contraction waves in oocytes. Nat Commun. 2017;8(1):849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Adhikari D, Busayavalasa K, Zhang J, et al. Inhibitory phosphorylation of Cdk1 mediates prolonged prophase I arrest in female germ cells and is essential for female reproductive lifespan. Cell Res. 2016;26(11):1212–1225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Toyoshima-Morimoto F, Taniguchi E, Shinya N, et al. Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase. Nature. 2001;410(6825):215–220. [DOI] [PubMed] [Google Scholar]
  • [34].Akera T, Chmatal L, Trimm E, et al. Spindle asymmetry drives non-Mendelian chromosome segregation. Science. 2017;358(6363):668–672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Baldin V, Theis-Febvre N, Benne C, et al. PKB/Akt phosphorylates the CDC25B phosphatase and regulates its intracellular localisation. Biol Cell. 2003;95(8):547–554. [DOI] [PubMed] [Google Scholar]
  • [36].Kin J, Singh AK, Takata Y, et al. LSD1 is essential for oocyte meiotic progression by regulating CDC25B expression in mice. Nat Commun. 2015. December 2;6: 10116. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [37].Mcdougall A, Chenevert J, Dumollard R. Cell-cycle control in oocytes and during early embryonic cleavage cycles in Ascidians. Int Rev Cell Mol Biol. 2012;297:235–264. [DOI] [PubMed] [Google Scholar]
  • [38].Reikvam H, Tamburini J, Skrede S, et al. Antileukaemic effect of PI3K-mTOR inhibitors in acute myeloid leukaemia-gene expression profiles reveal CDC25B expression as determinate of pharmacological effect. Br J Haematol. 2014;164(2):200–211. [DOI] [PubMed] [Google Scholar]
  • [39].Xiao J, Liu C, Hou J, et al. Ser149 is another potential PKA phosphorylation target of CDC25B in G2/M transition of fertilized mouse eggs. J Biol Chem. 2011;286(12):10356–10366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Zhang Y, Zhang Z, Xu XY, et al. Protein kinase A modulates CDC25B activity during meiotic resumption of mouse oocytes. Dev Dyn. 2008;237(12):3777–3786. [DOI] [PubMed] [Google Scholar]
  • [41].Kishimoto T. MPF-based meiotic cell cycle control: half a century of lessons from starfish oocytes. Proc Jpn Acad Ser B Phys Biol Sci. 2018;94(4):180–203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [42].Chang JY, Nakahata Y, Hayano Y, et al. Mechanisms of Ca2+/calcium-dependent kinase II activation in single dendritic spines. Nat Commun. 2019. June 25;10(1):2784. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [43].Oh JS, Susor A, Conti M. Protein tyrosine kinase Wee 1B is essential for metaphase II exit in mouse oocytes. Science. 2011;332(6028):462–465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [44].Ito J, Kaneko R, Hirabayashi M. The regulation of calcium/calmodulin-dependent protein kinase II during oocyte activation in the rat. J Reprod Dev. 2006;52(3):439–447. [DOI] [PubMed] [Google Scholar]
  • [45].Heim A, Tischer T, Mayer TU. Calcineurin promotes APC/C activation at meiotic exit by acting on both XErp1 and Cdc20. EMBO Rep. 2018;19(12):pii. E46433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [46].Zhang Z, Ahmed-Braimah YH, Goldberg ML, et al. Calcineurin-dependent protein phosphorylation changes during egg activation in drosophila melanogaster. Mol Cell Proteomics. 2019. March 15;18(Suppl 1):S145–S158. . [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Cell Cycle are provided here courtesy of Taylor & Francis

RESOURCES