Non-homologous chromosome synapsis during mouse meiosis : consequences for male fertility and survival of progeny

In the mouse, heterozygosity for several reciprocal and Robertsonian translocations is associated with impairment of chromosome synapsis and suppression of crossover formation in segments near the points of exchange during prophase of meiosis. This thesis describes the analysis of the consequences of the occurrence of non-homologous synapsis and/or suppression of meiotic crossover formation over many successive generations for male fertility and viability of the progeny.For studying chromosome synapsis, we modified a drying down technique which results in high yields of nuclei of all first meiotic prophase stages in both male and female from only small amounts of tissue (chapter 2). Preparations are suitable for synaptonemal complex (SC) analysis by normal light and electron microscopy (chapters 2, 3 and 7), for fluorescence immunocytochemistry and in situ hybridization (chapters 2, 8).In the study presented in chapter 3, we analysed the variation in male fertility of mice double heterozygous for two near identical reciprocal translocations T(1;13)70H and T(1;13)1Wa in relation to the synaptic behaviour of two differently sized heteromorphic bivalents during meiotic prophase. Male fertility rises when non-homologous synapsis in the small 1 13heteromorphic bivalent, leading to a "symmetrical" SC, is more frequent at the initial prophase stages. Based on the data presented, we favour the "unsaturated pairing site" model as the primary cause for male sterility.In T70H/T1Wa females not all heterologous synapsis within the small heteromorphic bivalent is effectuated during the early stages of meiosis; some is achieved lateron by the mechanism of "synaptic adjustment" (chapter 3). Each heteromorphic bivalent contains a copy of the chromosome 1 region between the T70H and T1Wa breakpoints which is about 10 cM in size (Δ1 segment). Although axial elements representing these Δ1 segments are seen to approach each other during early meiotic prophase stages, they never successfully constitute a synaptonemal complex in either sex (chapter 3). This agrees with the fact that in earlier cytogenetic studies quadrivalents were never seen at both male and female diakinesismetaphase 1.In chapter 7, we demonstrate that male fertility of the T70H/T1Wa mice is not only determined by the chromosomal constitution of the carrier but is additionally influenced by the pairing or synaptic history in previous meioses of especially the T70H and T1Wa short translocation chromosomes. Fertility of T70H/T1Wa males is more impaired after one or more successive transmissions of the T1Wa translocation chromosomes through a heteromorphic bivalent configuration, irrespective of the sex of the transmitting parent.Furthermore, we show that the introduction of the Robertsonian translocation Rb(l1.13)4Bnr into the T70H/T1Wa karyotype restores fertility of double heterozygous males by stimulating non-homologous synapsis of the small heteromorphic bivalent. We speculate that this Rb4Bnr effect is mediated by a prolongation of the early stages of meiotic prophase I.Successive female transmissions of the T1Wa translocation chromosomes in the presence of Rb4Bnr inititially resulted in an increase of the capacity for early meiotic nonhomologous synapsis within the small heteromorphic bivalent, leading to a restoration of fertility for the majority of carriers. Subsequently, a decrease of the capacity of the small heteromorphic bivalent to fully synapse was noticed, although a higher than original (F1) background level of male fertility remained.These variations in male fertility are most likely based on epigenetic variance, reflected as the capacity to engage into non-homologous synapsis early in male meiosis leading to a "symmetrical" SC, despite the different amounts of chromatin to accommodate.In chapter 4, the localization of several microsatellite markers and single copy genes relative to the T70H and T1Wa breakpoints, using quantitative PCR, quantitative Southern blotting and in situ hybridization, is described.In chapter 5, we investigated the level of suppression of meiotic recombination and impairment of chromosome synapsis in T70H heterozygotes in relation to the viability of the progeny. For T70H/+ females, the introgression of the D1Mit4, D1Mit20 and D1Mit122 microsatellite marker alleles positioned distal of the T70H breakpoint on the normal chromosome 1 into the 13 1T70H long translocation chromosome was suppressed in a distance dependent manner. This effect was more pronounced in T70H/+ females, additionally homozygous for Rb4Bnr. The delay in introgression was paralleled by a reduction of the frequency and extent of non-homologous synapsis in segments near the T70H breakpoints of the pachytene translocation multivalents in T70H/+ and Rb4BnT70H/Rb4Bnr+ males. The extend of non-homologous synapsis around the centre of the synaptic cross configuration in these males correlated with fluctuations in prenatal viability of segregating translocation homozygotes in crosses between (Rb4Bnr)T70H homozygous males and heterozygous females when meiotic drive at the female second meiotic division is excluded. The reduction in viability is explained by the gain of mutations resulting from incorrect processing of recombination intermediates which is due to non-homologous synapsis around the translocation breakpoints.In chapter 6, we analysed the consequences of the absence of crossing over for regions between the T70H and T1Wa breakpoints (Δ1 and Δ13 segments) of the Rb4BnrT1Wa translocation chromosomes, which have been transmitted for over 20 generations via heteromorphic bivalents in Rb4BnrT70H/Rb4BnrT1Wa females. Survival of heterozygous and homozygous carriers for these segments was taken as the phenotypic endpoint. The viability of progeny of crosses between Rb4BnrT70H homozygous males and Rb4BnrT70H/Rb4BnrT1Wa females, of which the latter principally produce 4 types of gametes, was estimated using a haplotype analysis of microsatellites in the Δ1 segment for genotyping (see chapter 4). We observed no differences in the pre- and postnatal survival rates of the double heterozygous and 13 1H, 13 1H, 1 13Wa 1 13H "duplication" progeny in which the Δ1 and Δ13 segments of the T1Wa translocation chromosomes had either no, an onegeneration or a multi-generation history of non-homologous synapsis in heteromorphic bivalents during previous female meioses. In addition, intercrossing of Rb4BnrT70H/Rb4BnrT1Wa double heterozygotes after genetic isolation of these Δ1 and Δ13 segments for 20 to 22 generations, showed that the viability of the Rb4BnrT1Wa homozygotes was not different from the Rb4BnrT70H homozygous and Rb4BnrT70H/Rb4BnrT1Wa karyotypes generated by this cross. Thus, exclusion of the Δ1 and Δ13 segments from meiotic crossing over within non-homologous synapsed heteromorphic bivalents during 20 to 25 successive generations does not result in an accumulation of recessive lethal mutations or an increased susceptibility for gaining dominant lethal mutations.For the D1Mit122 microsatellite used in offspring haplotyping a higher mutation frequency was observed after transmission through a double heterozygous than after transmission through a T70H homozygous karyotype (chapter 6). On the basis of the identity of the mutations, the ectopic pairing of the St2 gene copies (containing D1Mit122) during meiosis of T70H/T1Wa males (chapter 8) and the observation of ectopic homologous contacts of the Δ1 segments during the zygotene stage without SC formation (chapter 3), we speculate that these mutations are the result of ectopic homologous gene conversion events most likely occurring in the absence of a synaptonemal complex.The crossover suppressive influence of the Rb translocation on the Δ1 segment (chapter 5) enabled us to analyze the effects of introgression of genetic material from the Swiss +/+ stock into the translocation karyotypes. Introgression of "new" genetic material correlated with an increase in littersize of Rb4BnrT70H homozygotes (chapter 5), an improvement of the life expectancy of Δ1 duplication offspring from double heterozygous mothers (chapter 6) and a clear improvement of male fertility in double heterozygous and T70H homozygous males also carrying Rb4Bnr (chapter 7). These pleiotrophic findings are discussed in chapter 8 in terms of genetic versus epigenetic mechanisms of inheritance.Finally, when T1Wa was backcrossed for many generations to the Rb4BnrT70H/Rb4BnrT70H karyotype, essentially precluding genetic recombination in the Δ1 and Δ13 segments, or when T1Wa was combined with Rb4Bnr after many successive transmissions via alternating T1Wa heterozygotes and homozygotes, stable Rb4BnrT1Wa homozygous lines could not be bred (chapter 8). Especially female reproductive performance decreases after repeated male and female homologous meiosis. As non-homologous synapsis in the centre of the synaptic cross configuration in T1Wa/+ males is common too (unpublished results), more work into the genetic stability of chromosome segments, that have a history of hindered homologous interaction, is indicated (chapter 8).