Phosphorylation of the anaphase promoting complex activator FZR1/CDH1 is required for meiosis II entry in mouse male germ cell (本文)

APC/C activity during mitotic cell cycle.However, the physiological significance of FZR1 phosphorylation is yet to be examined in vertebrate mitotic tissues in vivo.The meiotic cell cycle consists of one round of DNA replication followed by two rounds of chromosome segregation, producing haploid gametes from diploid cells.Although study in budding yeast elucidated that the role of APC/C in meiosis relies on the meiosis-specific co-activator AMA1 18,19 , its homolog or counterpart does not exist in mammals.Instead, CDC20 and FZR1 contribute to the regulation of APC/C activity in mammalian meiosis.In mice, it was demonstrated that loss of FZR1 led to abnormal spermatogonial proliferation and defects in progression of meiotic prophase I in both male and female germ cells 20 .In female meiosis I, FZR1 plays crucial roles in sustaining dictyate arrest of germinal vesicle (GV) oocytes [21][22][23] and in regulating chromosome segregation [24][25][26][27][28] .These studies indicate that FZR1 is required for mammalian meiosis, but it remains elusive how APC/C activity regulated by phosphorylation of FZR1 is involved in meiotic cell cycle.Here, we examined whether phosphorylation of FZR1 is required for the regulation of APC/C activity during mitotic and meiotic cell cycle in vivo, using knockin mice that carries non-phosphorylatable mutations of FZR1.Our study demonstrates that phosphorylation of FZR1 is required for maintenance of spermatogonia for a long period of time.Furthermore, we show that FZR1 phosphorylation has a crucial role in the regulation of APC/C activity during meiosis I-meiosis II transition in juvenile male but not in female.Sexual dimorphism in the requirement of FZR1 phosphorylation raised an insight into different modes of meiosis I-to-meiosis II progression in spermatocytes and oocytes. Results Generation of Fzr1 9A/9A knockin mice.It has been shown that human FZR1 is phosphorylated during mitotic cell cycle.Ectopic expression of non-phosphorylatable FZR1 mutant in HeLa cells resulted in formation of constitutively active APC/C FZR1 and a reduction of G2/M phase 17 .To analyze the physiological role of FZR1phosphorylation, we generated a mouse model, Fzr1 9A/9A knockin (Fzr1 9A/9A KI) mice, in which the nine putative CDK-phosphorylation sites of Ser/Thr residues in Fzr1 were substituted with alanine amino acids (Fig. 1A-C).Fzr1 9A/9A KI allele was generated by Cre-mediated site-specific recombination of full length cDNA encoding mutant Fzr1 9A into the endogenous Fzr1 locus using an exchangeable gene-trap (GT) line 29,30 .For the control mouse line, full length cDNA cassette encoding wild type (WT) FZR1 was inserted into the targeted locus in the same manner, generating Fzr1 Gt wt/Gt wt KI mice (Fig. 1A).To distinguish Fzr1 Gt wt KI allele from natural WT Fzr1 allele, hereafter we refer to natural WT Fzr1 allele as Fzr1 + .The expression levels of FZR1-Gt wt and FZR1-9A proteins in the corresponding KI testes were comparable to the FZR1 expression level in natural WT Fzr1 +/+ testes at postnatal day18 (P18) (Fig. 1D).Further, CDC20, APC/C subunits (CDC27/APC3) and the canonical substrates of APC/C (Cyclin B1, PLK1) overall showed similar expression levels in natural WT Fzr1 +/+ , Fzr1 9A/9A KI and Fzr1 Gt wt/Gt wt KI testes (Fig. 1D).We also confirmed Fzr1 Gt wt/Gt wt KI mice showed normal fertility as natural WT Fzr1 +/+ , indicating Fzr1 Gt wt KI allele functions in a manner indistinguishable from natural WT Fzr1 + allele.Immunoprecipitation of CDC27/APC3, a core subunit of APC/C, demonstrated that the non-phoshorylatable FZR1-9A and control FZR1-Gt wt proteins expressed from the corresponding KI alleles were indeed incorporated in APC/C of Fzr1 9A/9A and Fzr1 Gt wt/Gt wt KI testes, respectively (Fig. 1E).We noticed that the level of CDC20 incorporated in APC/C in Fzr1 9A/9A testis was less than that in natural WT Fzr1 +/+ and Fzr1 Gt wt/Gt wt KI testes.This implies that persistent inclusion of FZR1-9A in APC/C impedes the association of CDC20 to APC/C.

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