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First published online 15 September 2004
doi: 10.1242/dev.01368

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Potential role of protein tyrosine phosphatase nonreceptor type 13 in the control of oocyte meiotic maturation

Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305, USA

View larger version (33K): [in a new window]   Fig. 1. Screening for potential PKA substrates from a mouse oocyte library. (A) Radiolabeled protein pools were generated and analyzed by a SDS-gel mobility shift assay. Asterisks indicate the proteins where mobility was shifted by incubation with PKA. (B) Radiolabeled proteins derived from isolated single clones (1-4B, 1-5B, 3-7F and 14-12D) were incubated with or without the PKA catalytic subunit, followed by a SDS-gel mobility shift assay. (C) mRNA from each independent clone were synthesized in vitro. Twenty nanograms of each mRNA (in 20 nl of H2O) or 20 nl of H2O (vehicle) were injected into 30 Xenopus oocytes 12 hours before 500 nM progesterone stimulation. Percentage of GVBD was measured by counting white spots on the animal pole of Xenopus oocytes.

View larger version (60K): [in a new window]   Fig. 2. Structure of PTPN13. (A) Full-length mouse PTPN13 and C-terminus Xenopus PTPN13. The full-length PTPN13 contains N-terminal FERM domain (also known as band 4.1 homology membrane-binding domain), five PDZ domains and a protein tyrosine phosphatase (PTPase) domain. (B) Alignment of predicted PTPN13 catalytic domain amino acid sequences of Xenopus (xPTPN13), and the sequences of mouse (mPTPN13) and human (hPTPN13). C terminus Xenopus PTPN13 is based on RT-PCR analysis, and the sequences are from databases (GenBank Accession Numbers BJ052929, BJ063819 and BJ070269). A solid line indicates the protein tyrosine phosphatase domain. An asterisk indicates the catalytically important residue in this domain that we used for mutagenesis in this report.

View larger version (59K): [in a new window]   Fig. 3. PTPN13 expression in the oocytes. (A) The expression of PTPN13 mRNA in Xenopus oocytes was determined by RT-PCR. Minus RT was used as a negative control. (B) Immunohistochemistry of mouse ovary was performed using a polyclonal antibody against mouse PTPN13. Arrows indicate the cytoplasm of the oocytes.

View larger version (31K): [in a new window]   Fig. 4. Phosphorylation of PTPN13 by PKA in vitro and in vivo. (A) HEK293 cell lysate was subjected to immunoprecipitation using control IgG (lane 1-4) or anti-human PTPN13 antibodies (lane 5-12). Immunoprecipitates were then incubated with or without PKA catalytic subunits for the indicated time followed by 6% SDS-PAGE analysis. Radiolabeled PTPN13 was detected by autoradiography. (B) [32P]-metabolic-labeled HEK293 cells were stimulated with 100 µM forskolin for the indicated time. Harvested cell lysates were then subjected to immunoprecipitation using an anti-human PTPN13 antibody or control IgG, followed by 6% SDS-PAGE analysis. Radiolabeled and total PTPN13 were detected by autoradiography (upper panel) or western blotting analysis (lower panel). Densitometric analysis was performed on four independent experiments and the average and SEM are reported in the bar graph. (C) Human PTPN13 was immunoprecipitated by anti-PTPN13 antibody or normal rabbit IgG from HEK293 cell lysates. Immunoprecipitated PTPN13 was then subjected to in vitro phosphorylation with or without PKA catalytic subunit (PKA), PKA + PKI, or heat-inactivated PKA (HI-PKA). After washing three times with lysis buffered solution, tyrosine phosphatase activity in the immunoprecipitates was measured.

View larger version (15K): [in a new window]   Fig. 5. Effects of C-PTPN13 injection on progesterone-inducing Xenopus oocyte maturation. (A-C) Twenty nanograms of HA-tagged C-PTPN13 mRNA (in 20 nl H2O) or 20 nl of H2O were injected into 30 Xenopus oocytes and 12 hours later oocytes were stimulated with the indicated concentration of progesterone. Resumption of meiosis was scored at the indicated time by the appearance of a white spot on the animal pole of the oocyte. (D) Different amounts (as indicated) of HA-tagged C-PTPN13 mRNA were injected into Xenopus oocytes and 12 h later oocytes were stimulated with 500 nM progesterone. Results shown are representative of the two independent experiments performed.

View larger version (22K): [in a new window]   Fig. 6. Effect of C-PTPN13 injection on progesterone-dependent changes in signaling molecules in Xenopus oocytes. Twenty nanograms of HA-tagged C-PTPN13 mRNA (in 20 nl H2O) or 20 nl of H2O were injected into Xenopus oocytes and 12 hours later oocytes were stimulated with 100 nM progesterone for 8 hours. The oocyte lysates were subjected to 8% SDS-PAGE and the expression and phosphorylation of each protein were determined. Similar results were observed in three independent experiments.

View larger version (14K): [in a new window]   Fig. 7. Effect of phosphatase-dead C-PTPN13 injection on Xenopus oocyte maturation. (A) Schematic diagram of full-length mouse PTPN13 (mPTPN13), HA-tagged C-terminus mPTPN13 (C-PTPN13 or C-PTPN13 WT) and HA-tagged C-terminus C2374S mutant mPTPN13 (C-PTPN13 PD), a construct where the conserved cysteine 2374 was mutated to serine. (B) mRNA coding for a HA-tagged C-PTPN13 (C-PTPN13; C-PTPN13 PD) or vehicle (H2O) was injected into Xenopus oocytes. Twelve hours later, oocyte extracts were prepared and the expression of the two constructs was detected using HA antibodies. (C) Twelve hours after injection of these mRNAs, oocytes were treated with 500 nM progesterone and resumption of meiosis was scored at different times of incubation. Results shown are representative of three independent experiments.

View larger version (35K): [in a new window]   Fig. 8. Effects of PTPN13 siRNA injection on Xenopus oocyte maturation. Ten nanograms of specific PTPN13 siRNAs (A) or scrambled siRNAs (B), or indicated amounts of PTPN13 siRNA-1 (C) or scrambled siRNA-1 (D) were injected into Xenopus oocytes. After 12 hours, oocytes were collected and total RNA was isolated. Two micrograms of total RNA was used for semiquantitative RT-PCR. After 12 hours, oocytes were stimulated by 500 nM progesterone for 10 hours (A,B) or the indicated numbers of hours (C,D) and resumption of meiosis was scored. These experiments were repeated at least five times and representative results are shown.

View larger version (38K): [in a new window]   Fig. 9. Effects of PTPN13 siRNA injection on progesterone-dependent changes in signaling molecules in Xenopus oocytes. Ten nanograms of PTPN13 siRNA-1 or scrambled siRNA-1 were injected into Xenopus oocytes. After 12 hours, oocytes were stimulated with 500 nM progesterone for 8 hours. The oocyte lysates were subjected to 8% SDS-PAGE, and the expression and phosphorylation of each protein were detected by western blotting analysis.

View larger version (14K): [in a new window]   Fig. 10. Cdc25B overexpression, but not PDE3A overexpression, overcomes the meiotic block induced by PTPN13 siRNA. Ten nanograms of siRNA-1, scrambled siRNA-1 or vehicle were injected into Xenopus oocytes. After 12 hours, oocytes were stimulated with (A) 500 nM progesterone, or injected with (B) 1 ng of Cdc25B mRNA or (C) 20 ng of PDE3A mRNA to induce oocyte maturation, and resumption of meiosis was scored. Results shown are representative of the two experiments performed.

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