Furuno N et al. (2003), Expression of cell-cycle regulators during Xeno...

XB-ART-5391

Gene Expr Patterns 2003 May 01;32:165-8. doi: 10.1016/s1567-133x(03)00037-1.

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Expression of cell-cycle regulators during Xenopus oogenesis.

Furuno N , Kawasaki A , Sagata N .

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In full-grown Xenopus oocytes, cell-cycle regulators and an inactive form of maturation/M phase promoting factor (pre-MPF) are stored ready to bring about a specific cell cycle for oocyte maturation. We examined the expression pattern of these cell-cycle regulators as well as pre-MPF formation during oogenesis. Cdc2 and Cyclin B2 were already present in stage I oocytes and pre-MPF formation was also detected in stage I oocytes. Some negative regulators of MPF, Myt1 and Chk1, were synthesized early in oogenesis. In contrast, positive regulators of MPF, MEK, MAPK and Cdc25C, were mainly synthesized late in oogenesis. Northern blotting analysis suggested that the synthesis of these cell-cycle regulators was controlled by translation.

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Species referenced: Xenopus
Genes referenced: ccnb2 cdc25c cdk1 cdk2 cds1 chek1 mapk1 mos myt1 pold1 wee1
GO keywords: oocyte maturation [+]

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	Fig. 1. Expression of Cyclin B2 and Cdc2, and pre-MPF formation during oogenesis. (A) Western blotting analysis of Cyclin B2 and Cdc2. Oocyte extracts prepared from the smallest stage I oocytes to stage VI oocytes were subjected to Western blotting analysis with anti-PSTAIR (Furuno et al., 1994) or anti-Cyclin B2 antibody (Yoshitome et al., 1998), respectively. Routinely, oocyte extracts equivalent to one half of a stage VI oocyte were used in this assay. I, II, III, IV, V and VI indicate stages I, II, III, IV, V and VI, respectively. Since the size of each stage varied widely, we chose the oocytes according to the following criteria: oocytes of stages I, II, III, IV, V and VI were about 50 μm in diameter and transparent, about 300 μm and translucent, about 500 μm and light brown, of minimum size with differentiated animal and vegetal hemispheres, about 1000 μm without a white band and 1200 μm with a white band, respectively. Cyclin B2 was detected in two forms, as previously reported in stage VI oocytes. The band indicated by a filled triangle corresponds with the phosphorylated form of Cdc2 on Thr14 and Tyr15, whose residues become phosphorylated when Cdc2 associates with Cyclin B (Solomon et al., 1990). (B) Pre-MPF formation during oogenesis. Extracts of oocytes from each stage were subjected to Western blotting with Anti-phospho-Tyr (Cdc2) (purchased from Cell Signal Technology). The phosphorylated form of Cdc2 is indicated by a filled triangle. (C) Northern blotting analysis of Cyclin B2 and Cdc2. Total RNA was extracted from 100 oocytes of stages I and II, 20 oocytes of stage III and 10 oocytes of stages IV to VI. Total RNA equivalent to that of two and a half oocytes prepared from oocytes of each stage was subjected to Northern blotting using Cdc2 or Cyclin B2 cDNA as the probe. Usually, total RNA of the same number of oocytes of the same stages is used for Northern blotting analysis or RNase protection assays (Sagata et al., 1988, Kobayashi et al., 1991, Paris et al., 1991), because the amount of rRNA is greatly increased during oogenesis. Mos mRNA was used for the loading control. The amount of this mRNA is reported to remain fairly constant during oogenesis (Sagata et al., 1988). Pure Cyclin B2 or Cdc2 mRNA transcribed in vitro was used as a standard for estimating the mRNA concentration.
	Fig. 2. Expression of negative regulators for the initiation of oocyte maturation during oogenesis. (A) Western blot analysis of Wee1, Myt1, Chk1 and Cds1. Western blotting was performed with anti-XeWee1 (Nakajo et al., 2000), anti-XeMyt1 (Nakajo et al., 2000), anti-XeChk1 (Nakajo et al., 1999) or anti-XeCds1 antibody (Gotoh et al., 2001), respectively. (B) Northern blotting analysis of Wee1, Myt1, Chk1 and Cds1. Total RNA was extracted from oocytes of each stage and the mRNA of Wee1, Myt1, Chk1 and Cds1 was detected by using each of the cDNAs as the probe. Pure Wee1, Myt1, Chk1 or Cds1 mRNA made in vitro was used as a standard for estimating the mRNA concentration.
	Fig. 3. Expression of positive regulators of MPF during oogenesis. (A) Western blotting analysis of Cdc25C, MEK and MAPK. Western blotting was carried out with anti-XeCdc25C (Nakajo et al., 1999), MEK (purchased from Santa Cruz) or MAPK antibody (purchased from Santa Cruz), respectively. (B) Northern blotting analysis of Cdc25C, MEK and MAPK. The mRNA of Cdc25C, MEK and MAPK was detected by using Cdc25C, MEK or MAPK cDNA as the probe. Pure MEK, MAPK or Cdc25C mRNA transcribed in vitro was used as a standard for estimating the mRNA concentration.
	Fig. 4. Expression of Cdk2, Nek2B and Cyclin B1 during oogenesis. (A) Western blotting analysis of Cdk2, Nek2B and Cyclin B1. Western blotting was performed with anti-Cyclin B1 (Yoshitome et al., 1998), Nek2B (Uto et al., 1999) or Cdk2 antibody (Furuno et al., 1997), respectively. (B) Northern blotting analysis of Cdk2, Nek2B and Cyclin B1. Total RNA extraction and Northern blotting were performed as described in Fig. 1. Each mRNA was probed by using Cyclin B1, Nek2B or Cdk2 cDNA. Pure Cyclin B1, Nek2B or Cdk2 mRNA made in vitro was used as a standard for estimating the mRNA concentration.