Genetics: Early Online, published on January 18, 2017 as 10.1534/genetics.116.189092 Meiotic Consequences of Genetic Divergence across the Murine Pseudoautosomal Region Beth L. Dumont Department of Biological Sciences, Initiative in Biological Complexity, North Carolina State University, Raleigh, NC, 27695 Previous Address: North Carolina State University Initiative in Biological Complexity 112 Derieux Place 3510 Thomas Hall Campus Box 7614 Raleigh, NC 27695-7614 e-mail: [email protected] fax: 919-515-3355 Current Address: The Jackson Laboratory 600 Main Street Bar Harbor, ME 04609 email: [email protected] phone: 207-288-6647 Running Header: Sex chromosome meiosis in hybrid mice Key words: pseudoautosomal region, aneuploidy, recombination, meiosis, Mus musculus 1 Copyright 2017. 1 ABSTRACT 2 The production of haploid gametes during meiosis is dependent on the 3 homology-driven processes of pairing, synapsis, and recombination. On the mammalian 4 heterogametic sex chromosomes, these key meiotic activities are confined to the 5 pseudoautosomal region (PAR), a short region of near-perfect sequence homology 6 between the X and Y chromosomes. Despite its established importance for meiosis, the 7 PAR is rapidly evolving, raising the question of how proper X/Y segregation is buffered 8 against the accumulation of homology-disrupting mutations. Here, I investigate the 9 interplay of PAR evolution and function in two interfertile house mouse subspecies 10 characterized by structurally divergent PARs, Mus musculus domesticus and M. m. 11 castaneus. Using cytogenetic methods to visualize the sex chromosomes at meiosis, I 12 show that intersubspecific F1 hybrids harbor an increased frequency of pachytene 13 spermatocytes with unsynapsed sex chromosomes. This high rate of asynapsis is due, 14 in part, to the premature release of synaptic associations prior to completion of 15 prophase I. Further, I show that when sex chromosomes do synapse in intersubspecific 16 hybrids, recombination is reduced across the paired region. Together, these meiotic 17 defects afflict ~50% of spermatocytes from F1 hybrids and lead to increased apoptosis 18 in meiotically dividing cells. Despite flagrant disruption of the meiotic program, a subset 19 of spermatocytes complete meiosis and intersubspecific F1 males remain fertile. These 20 findings cast light on the meiotic constraints that shape sex chromosome evolution and 21 offer initial clues to resolve the paradox raised by the rapid evolution of this functionally 22 significant locus. 2 23 INTRODUCTION 24 The processes of meiotic pairing, synapsis, and recombination are essential for 25 progression through the first meiotic division and the formation of haploid gametes. 26 Unpaired, asynapsed chromatin is transcriptionally silenced (SHIU et al. 2001; BAARENDS 27 et al. 2005), and the aberrant repression of key genes regulating meiotic progression 28 can cause premature arrest of the meiotic cell cycle (TURNER et al. 2005; BURGOYNE et 29 al. 2009). A minimum of one crossover per bivalent is needed to ensure its stable 30 orientation at the metaphase plate (MATHER 1938; NICKLAS 1974), and homologous 31 chromosome pairs that lack an obligate crossover are susceptible to nondisjunction at 32 the first meiotic division (HAWLEY et al. 1994; LAMB et al. 1996; ROSS et al. 1996). As a 33 consequence, meiotic pairing, synapsis, and recombination defects are directly linked to 34 infertility and chromosome aneuploidy (HASSOLD and HUNT 2001; COHEN et al. 2006; 35 BURGOYNE et al. 2009; HANDEL and SCHIMENTI 2010), outcomes with severe 36 consequences for organismal fitness. 37 The mammalian sex chromosomes present a notable deviation to these general 38 rules of meiosis. Although the X and Y are derived from an ancestral pair of 39 homologous autosomes, the long-term suppression of recombination between them has 40 eroded sequence homology across most of their length (GRAVES et al. 1995; 41 CHARLESWORTH 1996; LAHN and PAGE 1999). In most mammals, the only vestige of this 42 ancestral identity is a short region of terminal homology known as the pseudoautosomal 43 region (PAR). The key homology-driven steps of meiotic pairing and recombination are 44 confined to this narrow zone of residual sequence identity (BURGOYNE 1982; MANGS and 45 MORRIS 2007), rendering the PAR one of the most functionally important regions of the 3 46 genome. Failure to initiate or maintain pairing across the PAR is associated with meiotic 47 arrest (MATSUDA et al. 1982, 1992) and reduced recombination across this region has 48 been directly linked to sex chromosome nondisjunction in humans (HASSOLD et al. 1991; 49 SHI et al. 2001). Expectedly, mutations that disrupt sequence homology across the PAR 50 or interfere with sex chromosome pairing are often associated with male infertility and 51 elevated rates of sex chromosome aneuploidy (GABRIEL-ROBEZ et al. 1990; MATSUDA et 52 al. 1992; MOHANDAS et al. 1992; KOROBOVA et al. 1998; BURGOYNE and EVANS 2000; 53 JORGEZ et al. 2011). 54 Despite its central role in mammalian meiosis, the PAR is rapidly evolving, both 55 at the level of DNA sequence and structure (KIPLING et al. 1996; PERRY and ASHWORTH 56 1999; SCHIEBEL et al. 2000; FILATOV and GERRARD 2003; BUSSELL et al. 2006; WHITE et 57 al. 2012a). A comparative sequence analysis of human-orangutan orthologs found that 58 non-coding divergence in PAR-linked genes is more than twice the rate of evolution at 59 autosomal genes (FILATOV and GERRARD 2003). Similarly, the rate of nucleotide 60 substitution across the mouse PAR is several-fold higher than that for adjacent X-linked 61 regions (PERRY and ASHWORTH 1999; WHITE et al. 2012a). PAR length polymorphisms 62 have been characterized in both human and mouse (KIPLING et al. 1996; WHITE et al. 63 2012a; MENSAH et al. 2014), and there is rapid structural rearrangement of the PAR in 64 closely related vole species (ACOSTA et al. 2011; BORODIN et al. 2012). Owing to 65 challenges associated with genotyping and sequencing this region of the genome, the 66 full extent of natural genetic variation in the mammalian PAR is still in active discovery 67 phase (MENSAH et al. 2014). 68 Together, the PAR’s essential meiotic roles and dramatic evolutionary trends 4 69 present an intriguing biological conundrum: How does such a functionally significant 70 region of the genome – and, notably, one whose function is directly dependent upon the 71 preservation of homology between two chromosomes – evolve so rapidly? Answers to 72 this question could provide important new insights into the general requirements for 73 sequence homology and pairing at meiosis, as well as clues into the selective forces 74 that constrain the evolution of sex chromosomes. 75 Toward this goal, one potentially informative experimental approach is to directly 76 assess the meiotic consequences of PAR divergence in hybrids characterized by 77 distinct X- and Y-linked PAR sequences. House mice of the genus Mus represent an 78 especially powerful model system for studying the interplay of PAR function and 79 evolution from this perspective. The pseudoautosomal boundary (PAB) has been 80 subject to considerable repositioning across the Mus phylogeny, including significant 81 PAR divergence between inter-fertile taxa. Within the M. musculus species complex, 82 two closely related subspecies, M. m. domesticus and M. m. castaneus, are 83 characterized by a ~430kb shift in the pseudoautosomal boundary (PAB) (WHITE et al. 84 2012a). The M. m. domesticus PAB, which appears to coincide with that of the standard 85 laboratory mouse strain C57BL6/J, is located ~700kb from the distal end of the X 86 chromosome in intron 3 of Mid1 (PALMER et al. 1997). In contrast, the M. m. castaneus 87 PAR is ~1.1Mb long and encompasses the entire Mid1 locus (WHITE et al. 2012a). 88 Numerous indel mutations within introns of Mid1 further distinguish the M. m. 89 domesticus and M. m. castaneus PARs (WHITE et al. 2012a). 90 The marked shift in the PAB between M. m. castaneus and M. m. domesticus is 91 associated with a reduced frequency of X/Y synapsis in spermatocytes from F1 hybrids 5 92 (WHITE et al. 2012b). Whereas 95% of heterogametic sex chromosomes are synapsed 93 along their PARs at late pachytene in both M. m. castaneus and M. m. domesticus, the 94 X and Y are synapsed in only ~70% of F1 spermatocytes at this early meiotic sub-stage. 95 Surprisingly, however, reduced X/Y synapsis does not appear to elicit downstream 96 consequences for F1 hybrid fertility. F1 animals have testis weights, sperm densities, 97 and measures of sperm morphology that are comparable to or exceed fertility 98 parameters in the inbred parental strains (WHITE et al. 2012b). These findings are in line 99 with observations from other intersubspecies house mouse hybrids, in which the X and 100 Y experience premature dissociation at metaphase I, without obvious repercussions for 101 male fertility (IMAI et al. 1981; MATSUDA et al. 1982, 1983). However, given the 102 established significance of synapsis and recombination for meiotic progression, it 103 remains puzzling that organisms with impaired meiotic sex chromosome associations 104 suffer no apparent fitness consequences. One possible explanation is that animals with 105 reduced XY pairing exhibit subtle spermatogenic defects, but that the associated 106 reduction in fertility is matched or exceeded by fitness gains associated with 107 outbreeding. A second alternative is that the resulting gametes are of reduced genetic 108 quality, harboring elevated rates of sex chromosome aneuploidy. 109 Here, I apply cytogenetic methods and immunofluorescence imaging to conduct 110 an in-depth study of sex chromosome synapsis, recombination, meiotic progression, 111 and aneuploidy in M. m. domesticus, M. m. castaneus, and their reciprocal inter- 112 subspecific F1 hybrids. My findings reveal the phenotypic consequences of PAR 113 divergence on X/Y dynamics at meiosis, with important implications for our 114 understanding the functional constraints governing the evolution of the heterogametic 6 115 mammalian sex chromosomes.