MutS[gamma] FUNCTION DURING MOUSE MEIOSIS
Milano, Carolyn Rose
MutSγ is a highly conserved heterodimer comprised of MSH4 (MutS homolog 4) and MSH5 (MutS homolog 5). MutSγ complex is widely conserved across many lineages of eukaryotes with few exceptions. Unlike other MutS complexes, MSH4-MSH5 function is specific to, and essential for meiosis. Prophase I of meiosis harbors two events critical for proper segregation of homologs at the end of meiosis I: pairing/synapsis and homologous recombination. Loss of MutSγ function results in severe defects in one or both of these events in budding yeast (S. cerevisiae), in the nematode (C. elegans), among some plants including A. thaliana, and in mammals such as M. musculus. Specifically, in M. musculus loss of either MSH4 or MSH5 results in loss of meiocytes prior to pachynema. This suggests a function for MutSγ preceding pachynema has a critical role for cell vitality in mammals. The loss of meiocytes prior to pachynema in Msh5-/- or Msh4-/- makes it impossible to understand the function of MutSγ in crossover regulation. To further establish a function for mammalian MSH5, this work looks at two mouse lines, each with a different mutation in the Msh5 coding sequence. The first mouse (Msh5GA/GA) harbors a point mutation in the ATP binding domain of MSH5, predicted to disrupt binding. The second line (Msh5ΔC/ΔC) contains a deletion of the MSH5 c-terminus. Both of these mouse lines show a less severe phenotype in spermatogenesis than does Msh5-/-, allowing for phenotypic analysis of homologous recombination events throughout prophase I. A proportion of Msh5GA/GA spermatocytes can progress to diakinesis of MI, thus alleviating the early loss of spermatocytes in Msh5-/- animals. Chromosome spread analysis of Msh5GA/GA spermatocytes reveals abnormal pairing/synapsis between non-homologous chromosomes. Across eukaryotes MutSγ is associated with class I CO repair events. Surprisingly in Msh5GA/GA spermatocytes, all chiasmata are lost or fail to form in these mutant spermatocytes. These findings identify MutSγ as essential to all CO regulation in mouse. The Msh5ΔC/ΔC mice confer infertility phenotypes, a result which identifies this domain as essential for proper MutSγ function. A proportion of Msh5ΔC/ΔC spermatocytes achieve full autosomal synapsis during pachynema, suggesting the c-terminal domain is less important for homolog synapsis. In the Msh5ΔC/ΔC pachynema spermatocytes there is a reduction of class I CO establishment in, identifying this domain as necessary for normal CO establishment. These findings provide the first direct evidence that MutSγ is necessary for the normal recruitment of class I CO machinery during prophase I. Taken together the results suggest that MutSγ function is integral to not only homolog pairing and synapsis, but also to the establishment of all crossovers in mouse.
Genetics; crossover; Molecular biology; meiosis; fertility
Alani, Eric E.; Wolfner, Mariana Federica; Chappie, Joshua S.
Genetics, Genomics and Development
Ph.D., Genetics, Genomics and Development
Doctor of Philosophy
dissertation or thesis