Limits...
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.

Show MeSH
Time-course of the cell cycle events  induced by microinjection of recombinant tr-kit  into MII-arrested mouse oocytes. This pattern  was constantly observed in the tr-kit–activated  oocytes. Eggs were fixed and stained with  Hoechst (10 μg/ml) either before microinjection  (A), or after increasing times from microinjection  of extracts from COS cells expressing tr-kit (200  μg/ml), 1 h (B), 4 h (C), and 6 h (D). Bar, 30 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132866&req=5

Figure 2: Time-course of the cell cycle events induced by microinjection of recombinant tr-kit into MII-arrested mouse oocytes. This pattern was constantly observed in the tr-kit–activated oocytes. Eggs were fixed and stained with Hoechst (10 μg/ml) either before microinjection (A), or after increasing times from microinjection of extracts from COS cells expressing tr-kit (200 μg/ml), 1 h (B), 4 h (C), and 6 h (D). Bar, 30 μm.

Mentions: We have previously shown that microinjection of either cell extracts expressing recombinant tr-kit or synthetic tr-kit mRNA is able to induce complete parthenogenetic activation of mouse eggs and cleavage to the two-cell stage (Sette et al., 1997). To compare the amount of recombinant tr-kit capable of activating mouse eggs with the amount carried by a single mouse sperm, we performed Western blot analysis (Fig. 1). Densitometric analysis of immunoreactive bands indicated that the amount of tr-kit protein obtained from extracts of 3 × 106 sperm was nearly threefold smaller than that from 50 μg of extracts of tr-kit– expressing COS cells (see also Materials and Methods). As shown in Fig. 2 and Table I, injection of ∼0.2–0.4 sperm equivalents of recombinant tr-kit is sufficient to exert activation of 60–70% mouse eggs. Injection of ∼0.1 sperm equivalents of tr-kit reduced the activation rate to 40–50%, and injection of ∼0.01 sperm equivalents did not result in significant activation of mouse eggs above the background reported in Table I (not shown). Although the timing of egg activation triggered by microinjection of recombinant tr-kit is not completely synchronous, the typical time course and pattern of the cell cycle events (Fig. 2) closely resemble those observed at fertilization of the mouse eggs (Mori et al., 1988). 1 h after injection, 60–70% of the eggs underwent metaphase–anaphase transition and initiated polar body extrusion (Fig. 2, A and B). At 4 h, the polar body was extruded in all activated eggs, chromosome decondensation had begun and an incipient pronucleus was evident (Fig. 2 C). The size of the pronucleus progressively increased from the time of appearance to reach its full size after 6–7 h from the injection in all the activated eggs (Fig. 2 D).


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)

Time-course of the cell cycle events  induced by microinjection of recombinant tr-kit  into MII-arrested mouse oocytes. This pattern  was constantly observed in the tr-kit–activated  oocytes. Eggs were fixed and stained with  Hoechst (10 μg/ml) either before microinjection  (A), or after increasing times from microinjection  of extracts from COS cells expressing tr-kit (200  μg/ml), 1 h (B), 4 h (C), and 6 h (D). Bar, 30 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Time-course of the cell cycle events induced by microinjection of recombinant tr-kit into MII-arrested mouse oocytes. This pattern was constantly observed in the tr-kit–activated oocytes. Eggs were fixed and stained with Hoechst (10 μg/ml) either before microinjection (A), or after increasing times from microinjection of extracts from COS cells expressing tr-kit (200 μg/ml), 1 h (B), 4 h (C), and 6 h (D). Bar, 30 μm.
Mentions: We have previously shown that microinjection of either cell extracts expressing recombinant tr-kit or synthetic tr-kit mRNA is able to induce complete parthenogenetic activation of mouse eggs and cleavage to the two-cell stage (Sette et al., 1997). To compare the amount of recombinant tr-kit capable of activating mouse eggs with the amount carried by a single mouse sperm, we performed Western blot analysis (Fig. 1). Densitometric analysis of immunoreactive bands indicated that the amount of tr-kit protein obtained from extracts of 3 × 106 sperm was nearly threefold smaller than that from 50 μg of extracts of tr-kit– expressing COS cells (see also Materials and Methods). As shown in Fig. 2 and Table I, injection of ∼0.2–0.4 sperm equivalents of recombinant tr-kit is sufficient to exert activation of 60–70% mouse eggs. Injection of ∼0.1 sperm equivalents of tr-kit reduced the activation rate to 40–50%, and injection of ∼0.01 sperm equivalents did not result in significant activation of mouse eggs above the background reported in Table I (not shown). Although the timing of egg activation triggered by microinjection of recombinant tr-kit is not completely synchronous, the typical time course and pattern of the cell cycle events (Fig. 2) closely resemble those observed at fertilization of the mouse eggs (Mori et al., 1988). 1 h after injection, 60–70% of the eggs underwent metaphase–anaphase transition and initiated polar body extrusion (Fig. 2, A and B). At 4 h, the polar body was extruded in all activated eggs, chromosome decondensation had begun and an incipient pronucleus was evident (Fig. 2 C). The size of the pronucleus progressively increased from the time of appearance to reach its full size after 6–7 h from the injection in all the activated eggs (Fig. 2 D).

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.

Show MeSH