Reproductive Isolation in Hybrid Mice Due to Spermatogenesis Defects at Three Meiotic Stages

Early in the process of speciation, reproductive failures occur in hybrid animals between genetically diverged populations. The sterile hybrid animals are often males in mammals and they exhibit spermatogenic disruptions, resulting in decreased number and/or malformation of mature sperms. Despite the generality of this phenomenon, comparative study of phenotypes in hybrid males from various crosses has not been done, and therefore the comprehensive genetic basis of the disruption is still elusive. In this study, we characterized the spermatogenic phenotype especially during meiosis in four different cases of reproductive isolation: B6-ChrX^MSM, PGN-ChrX^MSM, (B6 × Mus musculus musculus-NJL/Ms) F₁, and (B6 × Mus spretus) F₁. The first two are consomic strains, both bearing the X chromosome of M. m. molossinus; in B6-ChrX^MSM, the genetic background is the laboratory strain C57BL/6J (predominantly M. m. domesticus), while in PGN-ChrX^MSM the background is the PGN2/Ms strain purely derived from wild M. m. domesticus. The last two cases are F₁ hybrids between mouse subspecies or species. Each of the hybrid males exhibited cell-cycle arrest and/or apoptosis at either one or two of three distinct meiotic stages: premeiotic stage, zygotene-to-pachytene stage of prophase I, and metaphase I. This study shows that the sterility in hybrid males is caused by spermatogenic disruptions at multiple stages, suggesting that the responsible genes function in different cellular processes. Furthermore, the stages with disruptions are not correlated with the genetic distance between the respective parental strains.WHEN animals from genetically diverged populations hybridize, complete or partial sterility is often observed in the F₁ hybrids or in their descendants. This phenomenon belonging to postzygotic reproductive isolation accelerates irreversible genetic divergence by preventing free gene flow across the two diverging populations, and thereby plays a pivotal role in speciation. Sexual dimorphism is a general feature of reproductive isolation (Wu and Davis 1993; Laurie 1997; Orr 1997; Kulathinal and Singh 2008). In mammals, impairment is much more severe in males than in females, and in general the heterogametic sex is more sensitive to interspecific and intersubspecific genetic incompatibility. This phenomenon is well known as Haldane''s rule (Haldane 1922; Laurie 1997; Orr 1997).In many animals, the reproductive isolation is caused by spermatogenic disruptions characterized by reduced number of germ cells and small testis size. These animals include Drosophila (Joly et al. 1997), stickleback fish Pungitius (Takahashi et al. 2005), caviomorph rodent Thrichomys (Borodin et al. 2006), house musk shrew Suncus (Borodin et al. 1998), wallaby Petrogale (Close et al. 1996), and genus Mus (Forejt and Iványi 1974; Matsuda et al. 1992; Hale et al. 1993; Yoshiki et al. 1993; Kaku et al. 1995; Gregorová and Forejt 2000; Elliott et al. 2001, 2004; Good et al. 2008). Although reproductive isolation by spermatogenic impairment is a well-known phenomenon, its underlying genetic mechanism and molecular basis have remained elusive. The Dobzhansky–Muller model, which infers that hybrid sterility or inviability is caused by deleterious epistatic interactions between nuclear genes derived from their respective parent species or subspecies (Dobzhansky 1936; Muller 1942), is widely accepted in animals and plants and is also applicable to the sterility of hybrid animals in F₂ or backcross generations, so-called hybrid breakdown, in which the genes causing postzygotic reproductive isolation are partially recessive (Orr 2005).The genetic incompatibility between house mouse subspecies is an ideal animal model for studying the early stage of speciation. Two subspecies of mouse, Mus musculus domesticus and M. m. musculus, diverged from their common ancestor 0.3–1.0 MYA (Yonekawa et al. 1980; Moriwaki 1994; Bonhomme and Guénet 1996; Boursot et al. 1996; Din et al. 1996). M. m. domesticus ranges across western Europe and the Middle East, whereas M. m. musculus ranges throughout eastern Europe and northern Asia (Bonhomme and Guénet 1996). The two subspecies meet in a narrow hybrid zone, which is most likely maintained by a balance between dispersal and selection against hybrids (Hunt and Selander 1973; Bonhomme and Guénet 1996; Payseur et al. 2004). M. m. domesticus also displays reproductive isolation from the Japanese wild mouse, M. m. molossinus, which originated from hybridization of M. m. castaneus and M. m. musculus and its nuclear genome is predominantly derived from M. m. musculus (Yonekawa et al. 1980, 1988; Moriwaki 1994; Sakai et al. 2005). To investigate the reproductive isolation between M. m. domesticus and M. m. molossinus, we previously constructed a consomic strain B6-ChrX^MSM (Oka et al. 2004). This strain has the X chromosome from the MSM/Ms strain, which is derived from M. m. molossinus, in the genetic background of the laboratory strain C57BL/6J (B6), which is predominantly derived from M. m. domesticus (Moriwaki 1994). F₁ hybrid animals between B6 and MSM/Ms strains are fully fertile. On the contrary, B6-ChrX^MSM shows male-specific sterility characterized by a reduced sperm number and dysfunction of the sperm, including abnormal morphology and low motility, indicating that B6-ChrX^MSM is a model of hybrid breakdown in animals (Oka et al. 2004, 2007). Our previous study indicated that the abnormal morphology of the sperm head results from the genetic incompatibility between MSM/Ms-derived X-linked genes and B6 genes on autosomes including chromosomes 1 and 11 (Oka et al. 2007).In this study, to understand the genetic mechanism of reproductive isolation in mice, we first undertook in-depth characterization of phenotype for each B6-ChrX^MSM male especially during meiosis. Meiosis is a special cell division that produces four haploid cells after one round of chromosome replication and two rounds of chromosome segregation. During meiosis, homologous chromosomes pair, synapse, undergo crossing over, and achieve bipolar attachment to the spindle to segregate one set of chromosomes to each daughter cell. Homologous recombination is initiated during the leptotene stage of meiotic prophase I with the formation of DNA double-strand breaks (DSBs), which are repaired immediately during the zygotene stage or after crossing over of homologous chromosomes during the pachytene stage (Roeder 1997; Tarsounas and Moens 2001).During the first wave of spermatogenesis, most mitotic spermatogonia in the B6-ChrX^MSM testes fail to initiate meiotic DNA replication. Some proportion of those spermatogonia that enter into meiosis are again arrested and eliminated by apoptosis at the pachytene stage, resulting in the production of a small number of sperms. We extended the same analysis to three other cases of reproductive isolation. Another consomic strain PGN-ChrX^MSM has an MSM/Ms-derived X chromosome in the genetic background of the PGN2/Ms strain derived from wild mice (M. m. domesticus). PGN-ChrX^MSM males produce a small number of dysfunctional sperms as was the case with B6-ChrX^MSM males, but the former males show apoptosis mainly at metaphase of meiosis I. Furthermore, we examined F₁ hybrid males from intersubspecific cross of (B6 × M. m. musculus-NJL/Ms) and interspecific cross of (B6 × M. spretus). These F₁ hybrid males exhibited apoptosis at metaphase I and at the zygotene-to-pachytene stage of prophase I. As a whole, the postzygotic reproductive isolation in mice is caused by disruptions at a minimum of three different spermatogenic stages.