Synaptonemal complexes , transverse filaments and interference in mouse meiotic recombination; an immunocytological study = Synaptonemale complexen, transversale filamenten en interferentie bij de meiotsche recombinatie bij de muis; een immuuncytologische studie

During the prophase of the first meiotic division, homologous chromosomes (homologs} recognize each other and form stable pairs (bivalents). Subsequently non-sister chromatids of homologous chromosomes exchange corresponding parts (crossing over). These events are accompanied by the formation of a ladder like protein structure, the synaptonemal complex (SC), between the paired homologs. First, the two sister chromatids of each chromosome form a common axis, the axial element (AE)1 and subsequently the AEs of homologous chromosomes are connected by numerous transverse filaments (TFs), a process called "synapsis". AEs and TFs together form the SC.In addition, protein complexes are formed during meiotic prophase that are involved both in homologous chromosome pairing and in crossover formation. These protein complexes can be visualized at the light microscope level as foci by immunofluorescence labeling. Previous research has shown that the composition of these foci changes during the course of meiotic prophase, which reflects the order of proteins that are involved in successive steps in meiotic recombination. Initially, one finds only RAD51/DMC1 foci; subsequently, the recombination proteins RPA and MSH4 become components of the foci, while RAD51 and DMCl disappear; finally MLHl appears, whereas RPA and MSH4 are lost. In the mouse, foci are initially formed in much greater numbers per nucleus than the number of crossovers (COs) that is eventually formed. Accordingly, most foci in early meiotic prophase are not involved in CO formation, but in recombinational interactions that promote homologous chromosome pairing. However, at the transition from RPA/MSH4 to MLHl foci, the number of foci per nucleus decreases drastically, so that eventually the number of MLHl nucleus fits closely with the number of COs per nucleus. Also, the spatial distribution of MLH1 foci along the bivalents closely corresponds to the spatial distribution of COs along the bivalents.Early in meiosis, the RAD51/DMC1 foci associate already with the AEs and the SCs that are being formed, and this association persists in subsequent stages. This elicited various questions regarding the relation between foci and the SC: do the recombination protein complexes that are marked by the foci have a role in homologous chromosome pairing and the assembly of the SC, and/or is the SC involved in meiotic recombination? In this thesis I focused mainly on the second question: Is the SC involved in meiotic recombination, and if yes, how? Following a primarily immunocytological approach, I analyzed this question in the mouse, because foci can be analyzed well in this species, whereas antibodies against most known components of foci and SCs were already available.In chapter 1 I summarize the course of meiosis, and provide an overview of the roles of various recombination proteins and SC components, as far as these were known when I started my thesis investigation. Although most information has been obtained by research in yeast, it seems likely that many recombination proteins fulfill similar roles in yeast and mouse meiosis. However, there are also some differences. For instance, mouse meiocytes form far more MSH4 foci than COs, while this is not so in yeast. Furthermore, yeast can form two types of meiotic COs, class I and class U COs, whereas the mouse forms (almost) exclusively class 1 COs. Class I COs display interference, i.e., they are more evenly spaced along the bivalents was to be expected if they would be placed randomly along the bivalents. One important question is, whether SCs have a role in the formation of COs and the positioning of COs along the bivalents.In chapter 2 we focus on the role of the TFs. The gene encoding TF protein SYCPl is disrupted, and the effects of the mutation on meiotic recombination and chromosome behavior are analyzed. Mice that are homozygous for the SYCPI disruption (Sycpl" mice) were infertile. Sycpl'" spermatocytes formed morphologically normal axial elements (AEs), and homologous AEs were properly aligned, but, as expected, no SC was formed. Most SycpI' ~ spermatocytes were blocked in the pachytene stage of meiotic prophase, and subsequently entered apoptosis. A small proportion, however, reached a later stage of meiotic prophase, diplotene, or, exceptionally, the metaphase of the first meiotic division (metaphase I). However, we found almost no chiasmata in these Sycpl~'~ metaphase I cells. Chiasmata are the cytological manifestations of crossing over. In wild type metaphase I cells one finds the same number of chiasmata as the number of COs that is formed. The absence of chiasmata from Sycpl~'~ spermatocytes might indicate that intact SCs are required for CO formation; alternatively, the SYCPl protein is involved in CO formation besides its role in the assembly of TFs and SCs.To analyze the CO defect in Sycpl~'' metaphase E spermatocytes in more detail, we performed an imunocytochemical analysis of a series of proteins that are involved in successive steps in meiotic recombination. Foci of proteins that are involved in early and intermediate steps in meiotic recombination, such as RAD51 and MSH4 foci, were formed in normal numbers, and were positioned normally relative to the AEs. However, they persisted much longer than in wild type spermatocytes, and MLHl foci were not formed. Apparently, the first steps in meiotic recombination occur normally in Sycpl' spermatocytes, but subsequently the SYCPl protein is required for the resolution of the recombination intermediates, either by formation of COs, or in other ways that do not yield COs.One of the first markers for the presence of a meiotic recombination intermediate is ??2??, a phosphorylated form of histone H2A variant H2AX. Ear!y in meiotic prophase of SycpI" spermatocytes, ??2?? appeared throughout the nucleus, as in wild type. However, subsequently yH2AX disappeared much more slowly from the nuclei of SycpI" spermatocytes than from wild type spermatocyte nuclei, whereas in later stages of meiotic prophase, a limited number of distinct yH2AX domains persisted on each bivalent, presumably at sites of unrepaired, blocked recombination intermediates. The ATR protein, which phosphorylates H2AX to generate vH2AX, displayed a similar aberrant localization pattern as ??2??. Notably, the XY bivalent also displayed a limited number of distinct yH2AX domains, whereas in wild type, yH2AX was present throughout the XY bivalent from pachytene on. In other respects the XY bivalent also behaved aberrantly in Sycpl~'~ spermatocytes: whereas the XY bivalent in wild type formed a compact, condensed chromatin domain (the sex vesicle), this did not happen in Sycpl' spermatocytes. We suppose that in wild type, synapsis is accompanied by repair of recombination intermediates, and that ATR disappears from the synapsed portions of AEs as soon as repair has been completed. The ATR that has been released from the synapsed portions of AEs might subsequently relocate to the remaining asynaptic portions of AEs, including those of the XY bivalent, and it might phosphorylate H2AX at these sites. On the XY bivalent, this might result in the formation of a compact chromatin structure, the sex vesicle. In Sycpl' ~ spermatocytes, there is no synapsis, and most recombination intermediates are presumably not repaired. Possibly, ATR is sequestered at the sites of unrepaired DNA, so that insufficient ATR can relocate to the XY bivalent, and no sex vesicle is formed.In chapter 3 we focus on the spatial distribution of recombination complexes along the bivalents in wild type and Sycpl' spermatocytes and oocytes. Whi!e characterizing the Sycpl' mutant, we noticed that the distinct yH2AX domains, which persisted in Sycpl~ ~ spermatocytes, were more evenly spaced along the bivalents than was to be expected if they were placed randomly, in other words, they displayed (positive) interference. This was remarkable for two reasons: firstly, interference had only been described for COs, and secondly, particularly in older models for the interference mechanism, an important role was ascribed to the SCs and/or synapsis. Therefore, we decided to analyze systematically in wild type and Sycpl' spermatocytes when interference is first detectable, and whether interference manifests itself at a constant level during the course of meiotic prophase. In pachytene of wild type, we found strong interference among MLHl foci, which mark virtually all meiotic COs in the mouse. However, in an earlier stage of meiotic prophase, late zygotene, interference was already detectable among MSH4 foci, which mark intermediate steps in the recombination process. RPA foci, which represent slightly earlier steps in recombination than MSH4 foci, also displayed already interference in late zygotene. Thus, the phenomenon of interference is not limited to COs. The level of interference among RPA or MSH4 foci was lower than the level of interference among MLHl foci. In SycpI''' mutants, interference among MSH4 or RPA foci was as strong as interference among these foci in wild type, which shows that interference can occur without synapsis and without SC. Because the Sycpl^^ mutant does not make MLHl foci, we could not analyze whether the SYCPl protein is required for the strong interference among MLHl foci.In chapter 4 we consider the question whether the weak interference among MSH4 foci is imposed by the same mechanism that causes the strong interference among MLHlfoci. In particular, we wondered which metric applies to the regulation of the distances between foci, and whether the same metric applies to the regulation of the distances between MSH4 foci (the inter-MSH4 distances) as to the regulation of the inter~MLHl distances: are the interfocus distances regulated in terms of micrometers SC length (?m SC), percentage of the entire SC or chromosome (% SC), or does another metric apply? We assumed that the average length of the interfocus distances and the strength of interference among foci (i.e., the evenness of spacing of foci along the SCs) are regulated by one single mechanism, which we will denote as "the interference mechanism". The question was therefore whether the interference mechanisms for MSH4 and MLHl foci were the same, or at least employed the same interference metric. From comparisons of inter-MSH4 distances on long chromosomes with those on short chromosomes we inferred that within spermatocytes, the proportion of "??? SC length" to the interference metric for MSH4 foci was constant, whereas this did not hold true for the proportion of"% SC length" to the interference metric MSH4 foci; within oocytes we found the same. "%SC" was therefore not the interference metric for MSH4 foci. Furthermore, we concluded, based on comparisons of inter-MSH4 distances in spermatocytes with those in oocytes, that the proportion ??"??? SC length" and the interference metric for MSH4 foci in spermatocytes differs from the corresponding proportion in oocytes. Therefore, "?m SC" also dropped out as possible interference metric for MSH4 foci. We argue that the number of chromatin loops between adjacent MSH4 foci could be the interference metric for MSH4 foci.We followed the same approach to learn more about the interference metric for MLHl foci. We found that within oocytes and within spermatocytes, the proportion of"% SC" to the interference metric for MLHl foci was fixed, whereas this did not hold true for the proportion of "?m SC" to the interference metric for MLHl foci. Apparently, "?m SC" is not the interference metric for MLHl foci, and in addition, the interference metric for MLHl differs from that for MSH4 foci. That would imply that the interference mechanism for MLHl foci differs from that for MSH4 foci.In chapter 5 we analyze whether intact SCs and/or intact AEs are required for the strong interference among MLHl foci. For this purpose we used the mouse Sycp3" mutant, which lacks a protein component of the axial element (AE), SYCP3. We performed the analysis in Sycp3';' oocytes, because spermatocytes of this mutant enter apoptosis, presumably before the stage of meiolic prophase when MLHl foci are assembled. During meiotic prophase of Sycp3' mutants, the two sister chromatids of each chromosome form a common axial structure, which lacks besides SYCP3 another AE protein, SYCP2. Like the AEs in wildtype, the axial structures in Sycp3~'~ mutants contain the proteins that keep the sister chromatids together: the cohesins. Therefore we denote the axial structures in Sycp3~" meiocytes as "cohesin axes". In spread preparations of Sycp3~" spermatocytes or oocytes, the cohesin axes, as visualized by immunofluorescence labeling of cohesins. had a stretched and fragmented appearance. The cohesin axes in Sycp3" mutants displayed synapsis, albeit incomplete and discontinuous.Sycp3~'~ oocytes formed MLHl foci in slightly smaller numbers than wildtype oocytes; the MLHl foci usually occurred al sites where the two cohesin axes were connected by synapsis, but in some instances also at sites where two cohesin axes converged, but were not connected by synapsis. Interference among MLHl foci was as strong in Sycp3'' oocytes as it was in wild type, so intact AEs are not required for wildtype levels of interference among MLHl foci. Furthermore, interference among MLHl foci that were separated by stretches of asynapsis or discontinuous synapsis of the cohesin axes was as strong as interference among MLHl foci that were separated by stretches of continuous synapsis of the cohesin axes, so intact SCs and continuous synapsis are not required either for wildtype levels of interference among MLHl foci.In chapter 6 we provide an overview of the possible functions of transverse filaments (TFs) of SCs. Evidence is accumulating that TFs and SCs have no role in CO interference, and the work described in this thesis indicates the same. However, TFs appear to enhance the formation of class I COs, and appear to be important for homologous pairing of chromosomes along their entire length. Perhaps, TFs were originally important for the formation of stable chromosome pairs during meiosis I. Later in evolution, homologous recombination might have become more important for the recognition of homologous chromosomes and the formation of stable bivalents; the function of TFs might then have been reduced to a supporting role in these processes.