Localization and Function of Cyclin B1 and Cyclin B2 during Porcine Oocyte Maturation

Abstract

Oocyte maturation is regulated by maturation/Mphase promoting factor (MPF), a crucial M-phase regulating enzyme composed of a catalytic subunit, p34 cdc2 , and a regulatory subunit, cyclin B. The amount of p34 cdc2 is almost constant during oocyte maturation, and the amount of cyclin B is the principal factor determining MPF activity [1]. The presence of two types of cyclin B, cyclin B1 and cyclin B2, has been shown in vertebrates. In human cells, cyclin B1 can cause chromosome condensation, reorganization of the microtubules, and disassembly of the nuclear lamina and of the Golgi apparatus, whereas the role of cyclin B2 is restricted only to disassembly of the Golgi apparatus [2,3]. In maturing oocytes, differences between cyclin B1 and cyclin B2 functions have been reported in the first meiotic spindle formation and the second metaphase arrest in frog and mouse oocytes, respectively [4][5][6]. In our laboratory, we have studied cyclin B functions during porcine oocyte maturation for the past several years. The present review describes our recent observations with regard to protein levels, intracellular localizations and roles of cyclin B. We focus here on the differences between cyclin B1 and cyclin B2. Protein Levels of Cyclin B1 and Cyclin B2 during Meiotic Maturation of Porcine OocytesWe used immunoblotting to examine the protein levels of cyclin B1 and cyclin B2, looking at the density of the bands at 62 kDa and 45 kDa, respectively. In immature porcine oocytes, the presence of a small amount of pre-MPF, a hyperphosphorylated inactive-MPF, has been suggested [7][8][9], but the relative amount and the kind of cyclin B comprising the pre-MPF have never been clarified. Our resent experiment with 200 porcine oocytes revealed that cyclin B1 was absent from the immature oocytes, whereas the amount of cyclin B2 in immature oocytes was between 1/20 and 1/ 40 of that in the first metaphase oocytes (the intensity of the cyclin B2 band of 200 non-cultured oocytes was between 5 and 10 oocytes in oocytes cultured for 24-h: Fig. 1A).Cyclin B1 and cyclin B2 were detected just before germinal vesicle breakdown (GVBD), which was induced in most of the oocytes at 20-24 h in the present culture conditions [10][11][12]. Thereafter, cyclin B1 gradually increased, with a transient decrease at the first polar body extrusion, and reached its peak level at the second meiotic metaphase (48 h of culturing). In contrast, cyclin B2 reached its peak level at the first metaphase, then decreased abruptly in correlation with the first polar body extrusion and maintained a low level during the second meiosis (Fig. 1B). In Xenopus oocytes, the degradation of cyclin B2 at the first meiosis/ second meiosis transition and the requirement of cyclin B1 synthesis for the second meiosis induction have been reported [13]. Except for the cyclin B levels in immature oocytes, the cyclin B fluctuation patterns in porcine oocytes agreed well with those reported in Xenopus and mouse oocytes [14][15][16]. These results suggest that cyclin B2 works during ...

Activities of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are not required for the global histone deacetylation observed after germinal vesicle breakdown (GVBD) in porcine oocytes

“…Scale bar: 30 mm. and MAPK phosphorylation, as reported previously (Inoue et al 1995, Kuroda & Naito 2003. This result suggests that the mixing of GV contents and oocyte cytoplasm by the destruction of the GV membrane did not activate MPF and MAPK after the 6-h culture.…”

Meiotic Resumption of Porcine Immature Oocytes is Prevented by Ooplasmic Gs.ALPHA. Functions

“…As shown in Fig. 4A, both cyclins B1 and B2 were undetectable in non-cultured oocytes, but were dramatically increased after culture without IBMX for 24 and 30 h; the CDC2 level remained unchanged, as reported previously [30,32]. Cyclin B synthesis was delayed by the IBMX treatment, and only faint bands were observed in the cyclin B1 and B2 of normal rabbit IgG-injected oocytes until 30 and 24 h, respectively, which is consistent with the inhibitory effect of IBMX on meiotic resumption.…”

Critical effect of pigWee1B on the regulation of meiotic resumption in porcine immature oocytes

“…We previously reported that cyclin B synthesis was started at 18 h of culture in porcine oocytes, 21,33 and in the present study cyclin B synthesis was similarly initiated at 24 h of culture. The addition of 0.5 mM cAMP delayed the onset of cyclin B synthesis until 30 h of culture, and this period correlated well with the timing of MPF activation.…”

“…18,19 Porcine immature oocytes, in which cyclin B1 is absent and the cyclin B2 level is very low, also require protein synthesis for GVBD, and their cyclins B1 and B2 syntheses start around GVBD. [20][21][22] Because protein synthesis is prerequisite for the meiotic resumption in mammalian species other than rodents, it remains possible that the inhibitory phosphorylation of Cdc2 is not required for the meiotic arrest of follicular oocytes. However, the GVBD induction has been reported in porcine oocytes following okadaic acid-induced activation of cdc25, which dephosphorylates the inhibitory cdc2 phosphorylation sites, 22 indicating the involvement of cdc2 phosphorylation in the meiotic arrest of porcine immature oocytes.…”