Meiotic Recombination in the Mouse

In addition to our studies of yeast meiosis, we are also exploring the role of Spo11 in mammalian meiosis. We have cloned the mouse homolog of the yeast SPO11 gene and have shown that it is expressed primarily in reproductive tissues, as expected for a meiotic gene. We have also generated Spo11 knockout and transgenic mice to better understand this gene's role in meiotic recombination.

This project is an ongoing collaboration with other investigators at MSKCC:

• Dr. Maria Jasin
• Dr. Francesca Cole (Jasin lab)

[Enlarge Image] Figure 1 Lack of Recombination Complexes in Spo11 mutants Spread spermatocyte nuclei were stained with anti-Dmc1 antibodies (green) and with antibodies that recognize a component of the mouse chromosome axis, Scp3 (red). (A, B) A wild type zygotene nucleus shows numerous Dmc1 foci along the chromosome axes. Matched exposures of a Spo11 mutant nucleus (C, D) show an abnormal zygotene configuration with a complete lack of detectable Dmc1 foci.

We cloned cDNA and genomic DNA for a mouse gene encoding a protein with significant sequence similarity to conserved domains found in proteins of the Spo11 family. This mouse Spo11 gene is expressed primarily in meiotic cells in the testis and ovary. To test the function of the gene, we generated mice that are defective for Spo11 expression.

Disruption of SPO11 leads to severe gonadal abnormalities from deffective meiosis. Spermatocytes suffer apoptotic death during early prophase, at late zygotene, or early pachytene. Oocytes reach the diplotene/dictyate stage in nearly normal numbers but most die soon after birth. In normal animals, complexes of the DNA strand exchange proteins, Dmc1 and Rad51 form on meiotic chromosomes and are thought to represent the sites where recombination is occurring. These complexes are not observed in SPO11-defective mice (see Figure 1), similar to the situation in SPO11 mutants of yeast. These observations further support the view that Spo11 catalyzes double-strand break formation during mouse meiosis, just as it does in yeast.

We also find that Spo11-defective meiotic cells in the mouse display defects in homologous chromosome synapsis, similar to fungi but distinct from flies and nematodes. The available data, including the mouse knockout work described here, strongly supports the idea that recombination initiates prior to synaptonemal complex formation in mammals.

[Enlarge Image] Figure 3 DNA Damage-dependent and Independent Oocyte Loss Stages of oocyte development in wild type are diagrammed at the top, starting at early meiotic prophase in the fetal ovary. Meiotic arrest plus communication with somatic cells give rise to primordial follicles. Subsequent growth of follicles and oocytes is followed by resumption of meiosis, completion of the first division, and ovulation (not shown). Increased oocyte loss (gray arrows) in the Spo11 mutant is apparent during prenatal development and just after birth, during primordial follicle formation. More severe DNA damage-dependent oocyte loss occurs at/or prior to follicle formation in Dmc1-, Atm-, and Msh5-deficient animals.

Defects in meiotic recombination in many organisms result in arrest due to activation of a meiotic checkpoint(s). The proximal defect that triggers this checkpoint in mammalian germ cells is not understood but has been suggested to involve either the presence of DNA damage in the form of unrepaired recombination intermediates or defects in homologous chromosome pairing and synapsis independent of DNA damage per se.

To distinguish between these possibilities in the female germ line, we compared mouse oocyte development in a mutant that fails to form the double-strand breaks (DSBs) that initiate meiotic recombination (Spo11-/-) with mutants with defects in processing DSBs once they are formed (Dmc1-/- and Msh5-/-); and we examined the epistasis relationships between these mutations. Absence of DSB formation caused a partial defect in follicle formation, whereas defects in DSB repair caused earlier and more severe meiotic arrest, which could be suppressed by eliminating DSB formation. Our analysis reveals, therefore, that there are both DNA damage-dependent and DNA damage-independent responses to recombination errors in mammalian oocytes. Using these findings as a paradigm, we also examined oocyte loss in mutants lacking the DNA damage checkpoint kinase ATM. The absence of ATM caused defects in folliculogenesis that were similar to those in Dmc1 mutants and which could be suppressed by Spo11 mutation, implying that oocyte death in Atm-deficient animals is a response to defective DSB repair.