Effect of Vanadate on the Chromatin Configuration in Pig GV-oocytes

Abstract

Abstract.Vanadate, an inhibitor of tyrosine phosphatases, has been reported to prevent germinal vesicle breakdown in mammalian oocytes. We examined the effect of vanadate on the chromatin configuration of fully grown pig oocytes. In the presence of human menopausal gonadotropin (hMG), vanadate (0.5-5 mM) resulted in a dose-dependent change in oocyte chromatin in germinal vesicles from the condensed state to a decondensed filamentous or stringy configuration. The effect of vanadate and hMG on chromatin configuration could be replicated with 2 mM dibutyryl cyclic AMP (dbcAMP) in place of hMG. Western blot analysis showed that vanadate caused a massive accumulation in the oocytes of tyrosine-phosphorylated proteins with a range of molecular weights that was enhanced by both hMG and dbcAMP in a similar manner. These results suggest that inhibition of tyrosine phosphatase(s) in the presence of an effective level of cAMP induces a change in chromatin configuration of pig oocytes. Key words: Cyclic AMP (cAMP), Oocyte Maturation, Pig, Vanadate (J. Reprod. Dev. 55: [367][368][369][370][371][372] 2009) ully grown mammalian oocytes arrested at prophase I resume meiosis spontaneously in vitro when they are released from the inhibitory influences of their surrounding follicles [1]. Because meiotic resumption is inhibited by membrane-permeable cAMP analogs, such as dibutyryl cAMP (dbcAMP), and inhibitors of cyclic nucleotide phosphodiesterase, such as 3-isobutyl-1-methylxanthine (IBMX), it is thought that a sustained high level of cAMP inhibits this process in oocytes within the follicles [2]. In this environment, meiotic resumption is triggered by the preovulatory gonadotropic surge from the pituitary [3]. Gonadotropins promote meiotic resumption of oocytes via an indirect mechanism mediated by surrounding cumulus granulosa cells rather than by a direct action on the oocytes [4].A burst of phosphorylation of a range of proteins occurs in oocytes during meiotic resumption [5,6]. The key kinase that phosphorylates many cellular substrates is a maturation-promoting factor (MPF), a serine/threonine protein kinase consisting of a catalytic subunit, Cdc2, and a regulatory subunit, cyclin B [7,8]. Protein tyrosine phosphatases (PTPases) have also been suggested to be involved in meiotic resumption of oocytes. Vanadate, a potent inhibitor of tyrosine phosphatases, has been used to study the role of PTPases in oocytes and somatic cells [9]. In mouse and rat oocytes, vanadate prevents germinal vesicle breakdown by inhibiting tyrosine dephosphorylation of Cdc2 [10,11]. In pig oocytes, Aquino et al. have reported that vanadate inhibits germinal vesicle breakdown almost completely at concentrations of 250 μM and 500 μM [12].We repeated this experiment using pig oocytes and found that vanadte induced changes in chromatin configuration in arrested germinal vesicles. In the present study, we cultured fully grown pig oocytes with vanadate at different concentrations and examined the precise change in chromatin configuration. The results s...

Isolation of female germline stem cells from porcine ovarian tissue and differentiation into oocyte-like cells

“…3A, B). Filamentous configuration is defined as the stage when filamentous chromatin is decondensed and distributed throughout the germinal vesicle (GV) [23]. In the third group of follicles, 1 mm in size, the oocytes also contained a nucleolar-like body.…”