Functional Study Of PP2A In The Reproductive System Of Male Mice

Protein phosphatase 2A(PP2A)family is one of the most abundant serine/threonine phosphatases of mammals.PP2A is a heterotrimer consisting of an A structural subunit,a C catalytic subunit and a B regulatory subunit that gives the complex substrate specificity.Four families of B subunits exist,each containing multiple members,while in higher eukaryotes,there are two isoforms of A and C subunits,a and ?.The combination of subunits gives rise to a number of PP2A variants,differing in substrate specificity,subcellular localization and tissue specificity.PP2A regulates multiple cell events that include DNA replication,oncogenic transformation,tumor suppression,nutrient sensing,cell cycle progression,RNA transcription,apoptosis and RNA splicing.Mouse gene ppp2ca encodes the a form of PP2A catalytic subunit.Conventional knockout of ppp2ca allele leads to embryonic lethality in mice.To investigate the function of PP2A in male mice reproductive system,we set up ppp2ca conditional knockout mice models by crossing four types of Cre tool mice with ppp2caflox/flox mice.We found that conditional knockout mice models established by Prm-Cre,Pgk2-Cre or TNAP-Cre showed grossly normal phenotype or only a small portion of defect,which may be result from the low efficiency of Cre expression that can not meet the needs for gene function study.In contrast,Ddx4-Cre showed a high expression efficiency,which made it possible to establish a successful germ cell specific knockout mice model to study the function of ppp2ca.We found that the germ cell specific ppp2ca null male mice was infertile,and showed defects in testicular and germ cell development.We observed that cells localized in thetestis tubules of null mice were of abnormal morphology and no sperm of normal morphology was produced,which indicate an arrest in the process of spermatogenesis.In addition,we observed that the apoptosis rate was higher in 17 days postnatal null mice.By analyzing how PP2A functions during spermatogenesis,we found that the expression level of cohesin subunit Rec8 was decreased in null mice,the expression level of BRCA1 which is associated with DNA repair was also decreased dramatically,while yH2AX showed an up-regulated expression level,and even an abnormal localization pattern on autosomal SCs(synaptonemal complex)during pachytene stage,all of which suggest the mechanisms of PP2A function may evolve a protection role on centromere cohesin of sister chromatids during meiosis I,and a role in maintaining the normal recombination repair process of homologue chormosomes that undergo a DSB(double-strand-break)during the prophase of meiosis I.In summary,our study proved the key function of PP2A in male mice reproductive system.By phenotyping the germ cell specific ppp2ca null mice,we determined that PP2A is evolved in the process of spermatogenesis,especially of vital function during meiosis I.