PKC isotypes in post-activated and fertilized mouse eggs: association with the meiotic spindle

Abstract

Several isotypes of protein kinase C (PKC) have been reported to be expressed in mammalian eggs, but it is unknown whether these isotypes have a common function in the egg during or within the first few hours of fertilization. Here we show that the isotypes of PKC exhibit distinct patterns of enrichment immediately after mouse egg activation. PKCalpha and gamma accumulate in the egg cortex 25 min post-activation, while only PKCalpha accumulates at the contractile ring of the forming second polar body about 1.5 h post-activation. PKCzeta exhibits some unique features that resulted in it being the focus of more extensive analysis. PKCzeta is tightly associated with the meiotic spindle as determined by detergent extraction and is closely associated with alpha-tubulin as determined by FRET analysis in the metaphase II (MII) egg. In addition, after egg activation, PKCzeta remains associated with the spindle as it transits into anaphase II and later telophase II, becoming associated with the midzone microtubules. Antibodies to the active form of PKCzeta are enriched on the spindle poles and later in development on the midzone microtubules. Active PKCzeta also is enriched in both pronuclei in the 6-h post-fertilization and in the 14-h post-fertilization embryo as well as in the nuclei of the two-cell embryo. Inhibition of PKCzeta, but not inhibition of other isotypes of PKC, results in rapid disruption of the meiotic spindle. This study suggests that PKCzeta has a role in spindle stability, while other PKC isotypes have different roles in the conversion of the egg to the zygote.

Actin cytoskeleton in cell polarity and asymmetric division during mouse oocyte maturation

“…The Par proteins have been demonstrated to be crucial for polarity establishment in many cell types [Cowan and Hyman, 2004;Goldstein and Macara, 2007]. In mouse oocytes, PAR1, PAR4, and aPKC are localized to meiotic spindles [Baluch et al, 2004;Vinot et al, 2004;Duncan et al, 2005;Moore and Zernicka-Goetz, 2005;Szczepanska and Maleszewski, 2005;Na and Zernicka-Goetz, 2006]. aPKC accumulates at spindle poles and may play a role in regulating spindle MTs, as disruption of this distribution by Cdc42 perturbation results in abnormal spindle morphology [Na and Zernicka-Goetz, 2006].…”

Identification of a Spindlin homolog in gibel carp (Carassius auratus gibelio)

“…In this study, we further demonstrate from the GST pull-down assay that CagSpin directly or indirectly associates with h-tubulin, and therefore provide the first biochemical evidence for the specific interaction of Spindlin protein and h-tubulin, the microtubule component. Interestingly, some important proteins that play crucial functions during oocyte maturation and egg fertilization, such as p39 mos (Zhou et al, 1992;Haccard et al, 1993;Verlhac et al, 1996), p34 cdc2 of metaphase promoting factor (MPF) (Langan et al, 1989;Peter et al, 1990;Verde et al, 1990;Zhou et al, 1991Zhou et al, , 1992, cyclin B, the regulatory component of MPF (Pines and Hunter, 1991;Ookata et al, 1993), MAP kinase (Verlhac et al, 1993) and PKC (Page Baluch et al, 2004), have been also confirmed to associate with microtubules. Interestingly, three putative PKC phosphorylation sites have been revealed in the CagSpin sequence (Fig.…”

PKC Regulation of Gametogenesis and Early Development

“…Total PKC remained behind in the cytoskeleton after the detergent extraction process; the closeness of this binding was confirmed by FRET analysis between -tubulin of the meiotic spindle and the PKC isotypes. FRET analysis demonstrated a close molecular proximity not only between -tubulin and PKC , but also between -tubulin and PKC (Baluch et al, 2004, Baluch & Capco, 2008. Other investigations have shown that PKC exists in the cytoplasm of the fertilization-competent egg (Raz et al, 1998).…”

“…Total antibody, that is, antibody that detects both the phosphorylated and unphosphorylated form of the kinase, directed against PKC , -, -and -were found not only in the cytoplasm, but also enriched on the spindle apparatus of the fertilization-competent egg (Baluch et al, 2004). The tightness of this PKC binding was assessed by detergent extraction, a process which removes the soluble components of the cell and retains the egg's cytoskeleton and associated proteins.…”

“…Upon fertilization PKC , -, and -localize to the plasma membrane (Raz et al, 1998;Baluch et al, 2004, Halet et al, 2004. Here these conventional PKCs are poised to become active, as they are in a prime location to interact with their co-factors (i.e.…”