Influence of proline-rich inositol polyphosphate 5-phosphatase, on early development of fertilized mouse eggs, via inhibition of phosphorylation of Akt

Abstract

Objectives: Proline-rich inositol polyphosphate 5-phosphatase (PIPP) is one of the signal-modifying enzymes that play pivotal regulatory roles in PI3K signalling pathway. The aim of this study was to determine the role of PIPP in early development of fertilized mouse eggs, via inhibition of Akt activity and subsequent downstream signalling events.

Materials and methods: The mRNA transcript levels of endogenous PIPP and Akt1, Akt2, Akt3 were detected in G(1) , S, G(2) and M phases of fertilized mouse eggs by RT-PCR. Levels of exogenous PIPP, phosphorylated Akt at Ser473, dephosphorylated cdc2 at Tyr15 and levels of CCNB1, were detected respectively by immunoblotting. Changes in Akt localization were observed by fluoroimmunoassay; meanwhile, changes in activity of Akt and its downstream MPF were detected. Percentages of cells undergoing division were determined by counting, using a dissecting microscope.

Results: PIPP and Akt1 transcripts were detectable in G(1), S, G(2) and M phases of fertilized mouse eggs, but Akt2 and Akt3 were not. We also observed that overexpression of PIPP in fertilized eggs decreased expression of phosphorylated Akt at Ser473 and altered membrane localization of phosphorylated Akt at Ser473 specifically. Furthermore, overexpression of PIPP resulted in decreases in mitosis-phase promoting factor activity, level of dephosphorylated cdc2 at Tyr15 and cleavage rate of fertilized mouse eggs.

Conclusions: Our data suggest, for the first time, that PIPP may affect development of fertilized mouse eggs by inhibition of level of phosphorylated Akt at Ser473 and subsequent inhibition of downstream signal cascades.

Figure 1

Development model of fertilized mouse eggs. G1 phase: 0–9 h post fertilization (11–20 h after hCG injection); S phase: 9–15 h post fertilization (20–26 h after hCG injection); G2 phase: 15–18 h post fertilization (26–29 h after hCG injection); M phase: 18–21 h post fertilization (29–30 h after hCG injection).

Figure 2

RT‐PCR analyses of Akt1, Akt2 and PIPP mRNA expression in fertilized mouse eggs. Line 1: GV‐intact oocytes; Line 2: fertilized eggs at G₁ phase; Line 3: fertilized eggs at S phase; Line 4: fertilized eggs at G₂ phase; Line 5: fertilized eggs at M phase. GV‐intact oocytes and β‐actin were served as controls.

Figure 3

Effect of PIPP overexpression on phosphorylation of Akt at Ser473 in fertilized mouse eggs. (A) Expression of phosphorylated Akt at Ser473. (A‐a) Immunoblot analysis of phosphorylation of Akt at Ser473. β‐actin and GV‐intact oocytes were used as control. (A‐b) Densitometric analyses of 60 kDa phospho‐Akt at Ser473 (pAkt Ser473) expression were reported as phosphorylated Akt at Ser473/β‐actin ratio. Asterisk represents level of significance between experimental groups versus control groups (***P < 0.001). Error bar represents mean ± SEM of three independent experiments. (B) Expression of exogenous PIPP in fertilized mouse eggs and inhibition of overexpression of PIPP on phosphorylation Akt at Ser473. Fertilized mouse eggs were either not microinjected or microinjected with plasmids DNA (FLAG‐vector or FLAG‐PIPP). Eggs were immunoblotted with antibodies specific for FLAG tag or phospho‐Akt at Ser473. β‐actin served as loading control.

Figure 4

Effects of PIPP on distribution of phosphorylated Akt at Ser473 and Akt activity in fertilized mouse eggs. (A) Expression of phospho‐Akt at Ser473 was fixed and detected with pAkt Ser473 antibody and then treated with goat anti‐rabbit fluorescein isothiocyanate (FITC)‐labelled antibody (green, left). Nuclei were stained with propidium iodide (PI, red, middle). Panels (right) showed mergers of FITC (green) and PI (red). Scale bar = 75 μm. (A‐a) Localization of phospho‐Akt Ser473 during G₁, S, G₂ or M phases in fertilized mouse eggs. (A‐b) Localization of phospho‐Akt Ser473 in GV‐intact oocytes or in fertilized eggs microinjected with FLAG vector, FLAG‐PIPP or wortmannin. (B) Variation in Akt activities in GV‐intact oocytes, untreated fertilized eggs, or fertilized eggs treated with FLAG vector, FLAG‐PIPP or wortmannin.

Figure 5

Effects of PIPP on mitotic division of fertilized mouse eggs. (A) Effect of PIPP on MPF activity during G₂/M transition of fertilized mouse eggs. MPF activity was determined with histone H1 as substrate. Eggs were lysed and examined by scintillation counting and autoradiography. Each value was expressed as mean ± SEM from three independent experiments. Significant difference between experimental groups versus control groups is indicated by asterisk (***P < 0.001). (B) Effect of PIPP on phospho‐cdc2 at Tyr15 during G₂/M transition of fertilized mouse eggs. (B‐a) Immunoblot analysis of dephosphorylation status of phospho‐cdc2 at Tyr15 with the anti‐phospho‐cdc2 Tyr15 antibody (upper panel). Unphosphorylated cdc2 was detected using anti‐cdc‐2 antibody (lower panel). (B‐b) Densitometric analyses of phospho‐cdc2 at Tyr15 expression were reported as phospho‐cdc2 at Tyr15/Total Cdc2 ratio). Significant difference between experimental groups versus control groups is indicated by asterisk (***P < 0.001). Error bars represent mean ± SEM of three independent experiments. (C) Expression of CCNB1 in fertilized mouse eggs. Eggs were untreated, treated with TE buffer, FLAG vector, FLAG‐PIPP or wortmannin. (D) Division rate in cultured fertilized mouse eggs after FLAG‐PIPP microinjection. Percentage of division was calculated after counting under a dissecting microscope 30 h after injection of hCG in female mice. Data were pooled from three experiments and are expressed as mean ± SEM. Significant difference between experimental groups versus control groups is indicated by asterisk (***P < 0.001).

Figure 6

Domain organization of the mannalian proline‐rich inositol polyphosphate 5‐phosphatases (PIPP) and protein kinase B (Akt). The proline‐rich domain of PIPP may hydrolyze 5‐position phosphates of PtsIna(3,4,5)P₃ and/or PtsIna(4,5)P₂ forming PtsIna(3,4)P₂ and/or PtsIna(4)P. The PH domain of Akt may bind PtdIns(3,4,5)P₃.