engleski

Cep63 deficient mice reveal an essential role for the centrosome in male meiosis

Significant overlap in the pathology of human diseases caused by defects in the DNA damage response (DDR) or centrosome homeostasis suggests that they may have similar underlying mechanisms. Seckel syndrome is caused by deficiency in the DDR proteins ATR or CTIP or centrosomal proteins, such as PCNT or CenpJ. Seckel patients present with severe dwarfism, microcephaly, mental retardation and metabolic abnormalities. In addition, a number of Seckel mouse models have been shown to have defects in fertility. We have been investigating the functions of Cep63, a centrosomal protein that was identified as an ATR substrate and has recently been implicated in Seckel syndrome, as it provides a potential link between the DDR and the centrosome. To determine the consequences of Cep63 deficiency, we have generated animals lacking expression of all known isoforms of Cep63. We find that the DNA damage response is largely intact, however Cep63 deficient cells display abnormal centrosome configurations and chromosomal instability. Animals lacking Cep63 recapitulate the mild Seckel syndrome reported in human patients as they are runted and develop microcephaly that is p53 dependent. In addition, we have found that Cep63 deficient male mice are infertile and have severe defects in testicular development. In contrast, Cep63 females are fertile with normal litter sizes and Mendelian ratios. The drastic decrease in Cep63 deficient testes size is accompanied by an increase in apoptotic tubules that is independent of p53. Moreover, Cep63 mice do not produce any mature sperm, although some elongated spermatids are detected in testes sections. Further analysis revealed that Cep63 deficiency provokes defects in the progression through meiotic prophase I, with enrichment of cells in leptonema and zygonema. Cep63 deficient spermatocytes also progress poorly through pachynema with only few cells reaching diplotene stage. To address the effects of Cep63 deficiency on meiotic recombination, we have analyzed the induction of double strand breaks (DSB’s) by scoring RAD51 and DMC1 foci, cytological markers of DSB’s. The timing of breaks in Cep63 deficient mice is normal, peaking at early zygonema and declining by pachynema, except that they have increased numbers of both markers per nuclei from late leptonema to late zygonema, compared to wild- type mice. The increased number of DSBs results in more crossovers per autosome, as quantified by MLH1 foci along the synaptonemal complex (SC). Moreover, Cep63 deficient pachynema cells with bright MLH1 had longer SCs. However, the total portion of Cep63 pachytene cells with bright MLH1 foci was significantly diminished, consistent with problems progressing normally through pachynema. As the major functions of the Cep63 protein appear to be involved in centriole duplication, with deficient cells showing severe defects in centriole and centrosome numbers, we reason that aberrant male meiosis in Cep63 may be a consequence of defective microtubule dynamics during prophase I. Microtubule dynamics and centrosome positioning are important for the chromosome homology search and bouquet formation during meiosis. We propose that in addition to providing a model system to understand the etiology of microcephaly, Cep63 animals provide a model system for understanding the functions of the centrosome in male fertility. Current results from the ongoing characterization of these animals will be presented.

centrosome; microcephaly; meiosis; mitosis

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