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Involvement of phospholipase Cgamma1 in mouse egg activation induced by a truncated form of the C-kit tyrosine kinase present in spermatozoa.

Sette C, Bevilacqua A, Geremia R, Rossi P - J. Cell Biol. (1998)

Bottom Line: A GST fusion protein containing the SH3 domain of PLCgamma1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective.A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit-induced egg activation, showing that the effect of the SH3 domain of PLCgamma1 is specific.These data indicate that tr-kit activates PLCgamma1, and that the SH3 domain of PLCgamma1 is essential for tr-kit-induced egg activation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Sanitá Pubblica e Biologia Cellulare, Sezione di Anatomia, Universitá di Roma Tor Vergata, Rome, Italy.

ABSTRACT
Microinjection of a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa (tr-kit) activates mouse eggs parthenogenetically, and tr-kit- induced egg activation is inhibited by preincubation with an inhibitor of phospholipase C (PLC) (Sette, C., A. Bevilacqua, A. Bianchini, F. Mangia, R. Geremia, and P. Rossi. 1997. Development [Camb.]. 124:2267-2274). Co-injection of glutathione-S-transferase (GST) fusion proteins containing the src-homology (SH) domains of the gamma1 isoform of PLC (PLCgamma1) competitively inhibits tr-kit- induced egg activation. A GST fusion protein containing the SH3 domain of PLCgamma1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective. A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit-induced egg activation, showing that the effect of the SH3 domain of PLCgamma1 is specific. Tr-kit-induced egg activation is also suppressed by co-injection of antibodies raised against the PLCgamma1 SH domains, but not against the PLCgamma1 COOH-terminal region. In transfected COS cells, coexpression of PLCgamma1 and tr-kit increases diacylglycerol and inositol phosphate production, and the phosphotyrosine content of PLCgamma1 with respect to cells expressing PLCgamma1 alone. These data indicate that tr-kit activates PLCgamma1, and that the SH3 domain of PLCgamma1 is essential for tr-kit-induced egg activation.

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Quantitative analysis of  recombinant tr-kit microinjected  into MII-arrested mouse oocytes.  Western blot analysis with an anti– c-kit antibody was performed on 25  μl (50 μg of total proteins) of extracts from COS cells expressing recombinant tr-kit or on extracts from  3 × 106 sperms. The expected major  immunoreactive band of ∼28 kD  was present in both samples in a 3:1  ratio (see Materials and Methods).  Since in our microinjection experiments we inject into mouse eggs  ∼1–2 pg of proteins from extracts of tr-kit–expressing COS cells,  we can calculate that we inject between 0.2 and 0.4 sperm equivalents of recombinant tr-kit. This experiment was performed twice  with similar results.
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Figure 1: Quantitative analysis of recombinant tr-kit microinjected into MII-arrested mouse oocytes. Western blot analysis with an anti– c-kit antibody was performed on 25 μl (50 μg of total proteins) of extracts from COS cells expressing recombinant tr-kit or on extracts from 3 × 106 sperms. The expected major immunoreactive band of ∼28 kD was present in both samples in a 3:1 ratio (see Materials and Methods). Since in our microinjection experiments we inject into mouse eggs ∼1–2 pg of proteins from extracts of tr-kit–expressing COS cells, we can calculate that we inject between 0.2 and 0.4 sperm equivalents of recombinant tr-kit. This experiment was performed twice with similar results.

Mentions: Cell lysate from 3 × 106 spermatozoa and 50 μg of proteins from mock- and tr-kit–transfected COS cell extracts were separated on a 10% SDS-PAGE gel under denaturing conditions, blotted onto a nitrocellulose membrane, and then analyzed by Western blot using an anti–c-kit antibody as described below. Intensity of the bands corresponding to tr-kit were quantified by optical densitometry using the Molecular Analyst program and a GS-700 Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA). 50 μg of tr-kit–transfected COS cell extracts contained an amount of tr-kit threefold higher than that present in 3 × 106 spermatozoa (see Fig. 1). In microinjection experiments we injected 5 pl of a 0.2–0.4 μg/μl solution of tr-kit cell extracts (1–2 pg of proteins), an amount corresponding to 0.2–0.4 sperm equivalents of tr-kit.


Involvement of phospholipase Cgamma1 in mouse egg activation induced by a truncated form of the C-kit tyrosine kinase present in spermatozoa.

Sette C, Bevilacqua A, Geremia R, Rossi P - J. Cell Biol. (1998)

Quantitative analysis of  recombinant tr-kit microinjected  into MII-arrested mouse oocytes.  Western blot analysis with an anti– c-kit antibody was performed on 25  μl (50 μg of total proteins) of extracts from COS cells expressing recombinant tr-kit or on extracts from  3 × 106 sperms. The expected major  immunoreactive band of ∼28 kD  was present in both samples in a 3:1  ratio (see Materials and Methods).  Since in our microinjection experiments we inject into mouse eggs  ∼1–2 pg of proteins from extracts of tr-kit–expressing COS cells,  we can calculate that we inject between 0.2 and 0.4 sperm equivalents of recombinant tr-kit. This experiment was performed twice  with similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132866&req=5

Figure 1: Quantitative analysis of recombinant tr-kit microinjected into MII-arrested mouse oocytes. Western blot analysis with an anti– c-kit antibody was performed on 25 μl (50 μg of total proteins) of extracts from COS cells expressing recombinant tr-kit or on extracts from 3 × 106 sperms. The expected major immunoreactive band of ∼28 kD was present in both samples in a 3:1 ratio (see Materials and Methods). Since in our microinjection experiments we inject into mouse eggs ∼1–2 pg of proteins from extracts of tr-kit–expressing COS cells, we can calculate that we inject between 0.2 and 0.4 sperm equivalents of recombinant tr-kit. This experiment was performed twice with similar results.
Mentions: Cell lysate from 3 × 106 spermatozoa and 50 μg of proteins from mock- and tr-kit–transfected COS cell extracts were separated on a 10% SDS-PAGE gel under denaturing conditions, blotted onto a nitrocellulose membrane, and then analyzed by Western blot using an anti–c-kit antibody as described below. Intensity of the bands corresponding to tr-kit were quantified by optical densitometry using the Molecular Analyst program and a GS-700 Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA). 50 μg of tr-kit–transfected COS cell extracts contained an amount of tr-kit threefold higher than that present in 3 × 106 spermatozoa (see Fig. 1). In microinjection experiments we injected 5 pl of a 0.2–0.4 μg/μl solution of tr-kit cell extracts (1–2 pg of proteins), an amount corresponding to 0.2–0.4 sperm equivalents of tr-kit.

Bottom Line: A GST fusion protein containing the SH3 domain of PLCgamma1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective.A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit-induced egg activation, showing that the effect of the SH3 domain of PLCgamma1 is specific.These data indicate that tr-kit activates PLCgamma1, and that the SH3 domain of PLCgamma1 is essential for tr-kit-induced egg activation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Sanitá Pubblica e Biologia Cellulare, Sezione di Anatomia, Universitá di Roma Tor Vergata, Rome, Italy.

ABSTRACT
Microinjection of a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa (tr-kit) activates mouse eggs parthenogenetically, and tr-kit- induced egg activation is inhibited by preincubation with an inhibitor of phospholipase C (PLC) (Sette, C., A. Bevilacqua, A. Bianchini, F. Mangia, R. Geremia, and P. Rossi. 1997. Development [Camb.]. 124:2267-2274). Co-injection of glutathione-S-transferase (GST) fusion proteins containing the src-homology (SH) domains of the gamma1 isoform of PLC (PLCgamma1) competitively inhibits tr-kit- induced egg activation. A GST fusion protein containing the SH3 domain of PLCgamma1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective. A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit-induced egg activation, showing that the effect of the SH3 domain of PLCgamma1 is specific. Tr-kit-induced egg activation is also suppressed by co-injection of antibodies raised against the PLCgamma1 SH domains, but not against the PLCgamma1 COOH-terminal region. In transfected COS cells, coexpression of PLCgamma1 and tr-kit increases diacylglycerol and inositol phosphate production, and the phosphotyrosine content of PLCgamma1 with respect to cells expressing PLCgamma1 alone. These data indicate that tr-kit activates PLCgamma1, and that the SH3 domain of PLCgamma1 is essential for tr-kit-induced egg activation.

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