SCP2, a major protein component of the axial elements of synaptonemal complexes

Synaptonemal complexes (SCs) are ladderlike protein structures, which are formed between homologous chromosomes during the prophase of the first meiotic division. SCs consist of two axial elements, one along each chromosome, and transverse filaments (TFs), which connect the axial elements. On the TFs, between the axial elements, there is a third longitudinal structure, the central element (CE). Possible functions of the axial elements include organization of the chromatin in loops, providing sister chromatid cohesion, stabilization of chiasmata and regulation of recombination. These possible functions are explained in chapter 1 . In axial elements of SCs of rat, two major protein components have been identified, named SCP2 and SCP3, with relative electrophoretic mobilities ( Mr ) of 190,000 and 30,000-33,000. This study mainly focused on the analysis of the function of SCP2.In chapter 2 we describe how we isolated and sequenced the cDNA encoding SCP2 of the rat (rnSCP2). The protein predicted from the nucleotide sequence is basic and has a mass of 173 kDa. At the C-terminus, SCP2 contains a region, which is predicted to be capable of forming a coiled-coil structure. Furthermore, SCP2 contains two clusters of S/T-P motifs, which are common in DNA-binding proteins, and several potential phosphorylation sites. The SCP2 gene is expressed predominantly in meiotic prophase cells.In order to identify conserved domains within SCP2, we isolated and analyzed the human SCP2 cDNA (hsSCP2). This is described in chapter 3 . Although there is only 63% identity at the amino acid level between hsSCP2 and rnSCP2, several structural features and amino acid sequence motifs are conserved; hsSCP2 contains S/T-P motifs, which are commonly found in DNA-binding proteins, in the same domains as rnSCP2 and has a predicted coiled-coil region at its C-terminus. The structural organization of hsSCP2 and rnSCP2 resembles the structural organization of Red1, a protein component of the axial elements in S. cerevisiae . We speculate that Red1 and SCP2 are functional homologues. The hsSCP2 gene was assigned to chromosome 20q13.33 by fluorescence in situ hybridization.The ultrastructural organization of SCP2 and SCP3 within SCs was analyzed by immunogold labeling of surface-spread spermatocytes and sections of testicular tissue as described in chapter 4 . The immunogold-labeling patterns of spreads and sections differed significantly and we attributed this difference to artifacts, caused by the surface-spreading technique. Therefore we relied on sections for the precise localization of SCP2 and SCP3. The immunogold-label distribution of anti-SCP2 and anti-SCP3 antibodies coincided with the axial elements and therefore we conclude that both SCP2 and SCP3 are structural components of the axial elements. The distribution of anti-SCP2 label had a shoulder at the innerside of the axial element, which was not observed with the anti-SCP3 label. Because we observed fuzzy connections between the axial elements that were labeled by anti-SCP2 but not anti-SCP3 antibodies, we attributed the shoulder in the gold label distribution of anti-SCP2 antibodies to the labeling of these connections.To study the localization of SCP2 and SCP3 in successive stages of meiosis, we performed immunofluorescence labeling of dry-down spread preparations of spermatocytes with anti-SCP2 and anti-SCP3 antibodies ( chapter 5 ). SCP2 and SCP3 colocalize along the axial elements from leptotene up till and including diplotene. After diplotene, SCP2 and SCP3 concentrate at the centromeres and small amounts of SCP2 and SCP3 are retained along the chromosome arms until metaphase I. From these results we conclude that both SCP2 and SCP3 are involved in sister chromatid cohesion and chiasma stabilization. SCP2 and SCP3 are furthermore present on connections between desynapsed axial elements in late diplotene. SMC1 and SMC3, which are involved in sister chromatid cohesion during mitosis, and are present in dots along the axial elements during meiosis, are also present along these connections. We think that these connections represent the sites of crossing-over and that they stabilize recombination intermediates between homologous chromosomes, and ensure the continuity of the cores of recombining chromatids.In chapter 6 we consider possible functions of SCP2 and of axial elements.