Tyrosyl-phosphorylated proteins are involved in regulation of meiosis in the rat egg

Abstract

Fertilization in invertebrates results in tyrosine (Tyr) phosphorylation of several egg proteins. However, the involvement of Tyr phosphorylation in mediating mammalian egg activation has not yet been investigated. Using an antibody specific for phosphotyrosine (P‐Tyr), immunoblotting, and densitometric analysis, we found that maturation of the oocyte is accompanied by a generalized increase in the P‐Tyr content of almost all egg proteins detected. After sperm penetration, 5 of the 17 protein bands detected demonstrated a small increase in their P‐Tyr content, while at the pronuclear (PN) stage the signal was markedly reduced. Ionomycin emulated the changes observed at fertilization in most protein bands detected, demonstrating a small increase in their P‐Tyr content within 15 min of exposure. Analysis of the involvement of the tyrosyl‐phosphorylated, mitogen‐activated protein (MAP) kinase during meiosis revealed comigration of the phosphotyrosyl bands with the protein and a good correlation with its enzyme activity. Maturation was accompanied by an increase in MAP kinase activity. The activity dropped partially after sperm penetration and furthermore later at the PN stage. A larger quantity accompanied by a more significant change in the P‐Tyr content implies for extracellular regulated kinase (ERK) 2 being the dominant isoform present in the rat egg. Our results indicate that fertilization in mammals involves changes in activity of protein tyrosine kinases (PTKs) or in the balance between PTKs and protein tyrosine phosphatases. The single, ionomycin‐induced Ca2+ rise is sufficient to imitate fertilization‐induced changes in MAP kinase activity, as well as in tyrosine phosphorylation of other proteins within the egg. Mol. Reprod. Dev. 49:176–185, 1998. © 1998 Wiley‐Liss, Inc.

Dynamics of protein phosphorylation during meiotic maturation

“…1, 2, 3 and 4). This disagrees with previous studies showing no change in overall levels of pTyr proteins between GV and MII [16]. Interestingly, a recent study examining changes in protein phosphorylation and the kinome of porcine oocytes during maturation found that the number of tyrosine phosphorylated proteins increased was equal to the number that decreased during transitions from GV-MI and MI-MII [22].…”

“…To reinitiate the meiotic cell cycle PKA migrates from the cytoplasm to the mitochondrial membrane where it is sequestered by one of the many forms of A-Kinase anchoring proteins (AKAP1) [15]. And as mentioned above, tyrosine kinase activity has recently been shown to increase significantly following fertilization [16] but the origins of these changes during meiotic maturation have not been rigorously studied. Thus, the importance of kinase regionalization and the lack of information on the relative contributions of tyr or ser/thr kinases has prompted this investigation.…”

De novo synthesis of protein phosphatase 1A, magnesium dependent, alpha isoform (PPM1A) during oocyte maturation

“…Germinal vesicle (GV) oocytes were isolated from ovaries of untreated mice into M2 medium [36] supplemented with 1 μM milrinone (Sigma) to prevent resumption of meiosis and maintain the oocytes at the GV stage [37]. Oocytes arrested at metaphase of the second meiotic division (MII): Female mice were injected with 7 IU of hCG, 48 h after administration of 5 IU PMSG.…”

Myristoylated alanine-rich C kinase substrate, but not Ca2+/calmodulin-dependent protein kinase II, is the mediator in cortical granules exocytosis

“…Lysis buffer was added to samples of 300 MII eggs collected in 5-10 ml of TH medium (Ben-Yosef et al 1998). After two cycles of freezing/thawing, the extracts were stored at K70 8C.…”