The cohesin complex is essential for mitosis and meiosis. distributed throughout

The cohesin complex is essential for mitosis and meiosis. distributed throughout the chromatin and SYCP1 which normally marks synapsed axes is largely absent. Centromeric and telomeric sister chromatid cohesion are impaired. Centromere and telomere clustering happens in the absence of STAG3 and telomere structure is not seriously affected. Additional cohesin proteins are present localize throughout the STAG3-devoid chromatin and form complexes with cohesin SMC1β. No other deficiency in one meiosis-specific cohesin causes a phenotype as drastic as STAG3 deficiency. STAG3 emerges as the key STAG cohesin involved in major functions of meiotic cohesin. cohesin complexes (Losada (Michaelis gene that causes a frameshift was found in patients of a family affected by premature ovarian failure. If translated and stable this would lead to a small truncated protein of 194 amino acids of 1 1 225 amino acids (Caburet of the only meiosis-specific STAG protein STAG3. It remained unclear whether STAG3 is essential for meiosis and whether it functions in one or several of the meiotic processes mentioned above. Further it was unfamiliar whether STAG3-connected cohesin complexes represent a major functional portion of the cohesin complexes in mammalian meiocytes. Consequently we set out to investigate the role of STAG3 using a STAG3-deficient mouse strain and revealed an essential function of STAG3 in meiosis. STAG3-deficient spermatocytes and oocytes suffer from an absence of chromosome axes and impaired sister chromatid cohesion and are eliminated during meiosis. Thus STAG3 which is present in the most prominent types of cohesin complexes in mammalian meiocytes represents the key STAG protein acting in major functions of meiotic cohesin. Results Infertility Capsaicin of STAG3-deficient mice The embryonic stem cell mutant strain. This strain carries a knockout-first cassette designed to block gene expression after its insertion through providing a splice acceptor in the lacZ component of the place from which no further splicing occurs (Supplementary Fig?S1). Capsaicin We bred this strain to homozygosity (named to indicate its ‘knockout-first’ design). Male and female were infertile but normally healthy. The testes of mice were less than half the size and weight of those of wild-type (wt) mice (Fig?1A). The presence of mRNA was assessed by RT-PCR diagnostic for transcription and/or splicing across or flanking the knockout-first insertion (Supplementary Fig?S1). This confirmed the disruption of gene expression (Fig?1B). STAG3 protein was also absent in spermatocyte chromosome spreads further corroborated the absence of STAG3 in spermatocytes (Fig?1D). In addition cohesin immunoprecipitation (IP) affirmed the lack of STAG3 protein (observe below). Physique 1 Characterization of spermatogenesis in mice Meiotic arrest in Capsaicin STAG3-deficient spermatocytes To determine the stage of meiotic arrest testis sections were prepared from STAG3-deficient and STAG3-proficient mice and stained for the AE and SC component SYCP3. The sections were also stained for γH2AX which marks unsynapsed regions of chromosomes and with DAPI (Fig?2A; Supplementary Fig?S2 provides examples of individual tubules and their staging). The diameter of the tubules of mice was reduced by about half. testis tubules in stages I and IV of the seminiferous epithelium cycle harbored cells that showed Vegfc some patches of SYCP3 staining and of γH2AX. Generally the transmission intensity for γH2AX decreased with progression from stages I to IV and thus we consider cells with less widespread γH2AX signals more advanced. As visible in Fig?1C no or only very short SYCP3-made up of axial structures were observed in the cells of any stage. The presence of SYCP3 indicated cells in leptonema and possibly subsequent stages. The absence of AEs however renders precise staging of the cells based on chromosome structure difficult. Therefore we also analyzed the developmental stages of individual tubules based on their cell associations (Supplementary Capsaicin Fig?S2). Progression up to tubular stage IV was observed and was not grossly perturbed. Analysis of the first wave of meiosis in young males also showed spermatocytes at days 11 13 and 15?pp when they would.