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.

Profile of protein kinase C isozymes and their possible role in mammalian egg activation

“…Eggs arrested at metaphase II (MII) were isolated from immature superovulated rats as previously described [12]. Samples of 200 MII eggs were collected in a minimal volume of medium (5–10 μl) and a lysis buffer was added as described earlier [20]. The extracts were thawed and refrozen twice and stored at −70°C.…”

“…The extracts were thawed and refrozen twice and stored at −70°C. SDS‐PAGE was performed on 10% polyacrylamide minigel and transferred to nitrocellulose membranes [20]. Approximate molecular masses were determined by comparison with the migration of prestained protein standards (Sigma, St. Louis, MO).…”

In vitro fertilization by intracytoplasmic sperm injection

“…2B), a less invasive method of micromanipulation-assisted fertilization in which the interaction between gamete surfaces is not suppressed. Most investigators adhere to one of two major hypotheses on this subject and attribute the main role either to an oocyte surface receptor coupled to a G-protein- (19) or tyrosine-kinase-mediated (20) signalling pathway on the one hand, or to a direct action of a soluble sperm factor in the oocyte cytoplasm on the other. (16) In contrast, no Ca 2ϩ oscillations were observed after sham injection of oocytes with sperm-free culture medium, (15) demonstrating the decisive role of sperm factor(s) in this process.…”

Gamete membrane microdomains and their associated molecules in fertilization signaling

“…Sperm‐induced tyrosine phosphorylation and its physiological importance were also first shown in this animal (Kinsey, 1984, 1996; Ribot et al, 1984; Kamel et al, 1986; Ciapa and Epel, 1991; Abassi and Foltz, 1994; Shearer et al, 1999; Abassi et al, 2000; Kinsey and Shen, 2000). The following studies using other model organisms such as ascidian (Ueki and Yokosawa, 1997; Runft and Jaffe, 2000), fish (Wu and Kinsey, 2000; Kinsey et al, 2003; Sharma and Kinsey, 2006), frog (Ferrell et al, 1991; Yim et al, 1994; Sato et al, 1998; Glahn et al, 1999; Runft et al, 1999), mammals (Ben‐Yosef et al, 1998; Mehlmann et al, 2001; Sette et al, 2002; Kurokawa et al, 2004; Talmor‐Cohen et al, 2004; Mehlmann and Jaffe, 2005; McGinnis et al, 2007, 2009; Levi and Shalgi, 2010), and starfish (Carroll et al, 1997; Giusti et al, 1999a,b, 2000) have also demonstrated that these animals employ protein–tyrosine phosphorylation for successful oocyte maturation, fertilization, and/or activation of development (Kinsey, 1997; Sato et al, 2000a). By the late 1990s, the involvement of the sperm MDs in fertilization events (e.g., capacitation, as described above) had already been documented.…”