PDS 5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis
2020; Springer Nature; Volume: 21; Issue: 6 Linguagem: Inglês
10.15252/embr.201949273
ISSN1469-3178
AutoresAlberto Viera, Inés Berenguer, Miguel Ruiz‐Torres, Rocío Gómez, Andrea Guajardo-Grence, José Luís Barbero, Ana Losada, José Á. Suja,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle14 April 2020free access Transparent process PDS5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis Alberto Viera orcid.org/0000-0002-3602-4130 Unidad de Biología Celular, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Inés Berenguer Unidad de Biología Celular, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Miguel Ruiz-Torres Chromosome Dynamics Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain Search for more papers by this author Rocío Gómez Unidad de Biología Celular, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Andrea Guajardo Unidad de Biología Celular, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author José Luis Barbero Departamento de Biología Celular y Molecular, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain Search for more papers by this author Ana Losada Corresponding Author [email protected] orcid.org/0000-0001-5251-3383 Chromosome Dynamics Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain Search for more papers by this author José A Suja Corresponding Author [email protected] orcid.org/0000-0002-4266-795X Unidad de Biología Celular, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Alberto Viera orcid.org/0000-0002-3602-4130 Unidad de Biología Celular, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Inés Berenguer Unidad de Biología Celular, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Miguel Ruiz-Torres Chromosome Dynamics Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain Search for more papers by this author Rocío Gómez Unidad de Biología Celular, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Andrea Guajardo Unidad de Biología Celular, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author José Luis Barbero Departamento de Biología Celular y Molecular, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain Search for more papers by this author Ana Losada Corresponding Author [email protected] orcid.org/0000-0001-5251-3383 Chromosome Dynamics Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain Search for more papers by this author José A Suja Corresponding Author [email protected] orcid.org/0000-0002-4266-795X Unidad de Biología Celular, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Author Information Alberto Viera1, Inés Berenguer1,†, Miguel Ruiz-Torres2,†, Rocío Gómez1, Andrea Guajardo1, José Luis Barbero3, Ana Losada *,2 and José A Suja *,1 1Unidad de Biología Celular, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain 2Chromosome Dynamics Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain 3Departamento de Biología Celular y Molecular, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain †Present address: Department of Molecular Neuropathology, Centro de Biología Molecular Severo Ochoa, CBMSO, Madrid, Spain †Present address: Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA *Corresponding author. Tel: +34 91 7328000; E-mail: [email protected] *Corresponding author. Tel: +34 91 4978240; E-mail: [email protected] EMBO Rep (2020)21:e49273https://doi.org/10.15252/embr.201949273 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cohesin cofactors regulate the loading, maintenance, and release of cohesin complexes from chromosomes during mitosis but little is known on their role during vertebrate meiosis. One such cofactor is PDS5, which exists as two paralogs in somatic and germline cells, PDS5A and PDS5B, with unclear functions. Here, we have analyzed their distribution and functions in mouse spermatocytes. We show that simultaneous excision of Pds5A and Pds5B results in severe defects during early prophase I while their individual depletion does not, suggesting their functional redundancy. Shortened axial/lateral elements and a reduction of early recombination nodules are observed after the strong depletion of PDS5A/B proteins. Moreover, telomere integrity and their association to the nuclear envelope are severely compromised. As these defects occur without detectable reduction in chromosome-bound cohesin, we propose that the dynamic behavior of the complex, mediated by PDS5 proteins, is key for successful completion of meiotic prophase I. Synopsis Analysis of spermatogenesis in mice carrying conditional knock out alleles for cohesin cofactors PDS5A and PDS5B reveals the importance of cohesin dynamics for proper assembly of the synaptonemal complex, homolog recombination and telomere integrity. A single PDS5 protein is sufficient to complete prophase I successfully. Shortened axial/lateral elements and a reduction of early recombination nodules are observed after simultaneous depletion of the two PDS5 proteins without detectable reduction in cohesin levels. PDS5 proteins ensure the integrity of telomeres and their attachment to the nuclear envelope. Introduction Meiosis is a highly specialized cell division process that produces haploid gametes by performing two consecutive rounds of chromosome segregation after a single round of DNA replication. The accurate segregation of chromosomes during meiosis relies on the proper achievement of the preceding processes of homologous chromosome pairing, synapsis, and recombination 1. Meiotic recombination initiates in mammals during the leptotene stage of prophase I by the formation of programmed DNA double-strand breaks (DSBs) by the endonuclease SPO11 2, which leads to the phosphorylation of the histone variant H2AX at serine 139 (γ-H2AX) in the surrounding chromatin 3. After a 5′–3′ resection of DNA at DSBs, the recombinase RAD51, among other proteins, associates to the 3′ single-stranded DNA and promotes the invasion of the double-stranded DNA of the homolog, a process that facilitates the recognition and pairing of the homologs 4. Some of these events are finally processed as reciprocal crossovers via the participation of the DNA mismatch repair protein MLH1 5. Although meiotic recombination initiates early in meiosis, its resolution takes place at the pachytene stage after the homologs have achieved synapsis. Synapsis is mediated by the synaptonemal complex (SC), a tripartite proteinaceous structure specific of meiosis that assembles during prophase I 6. The SC assembly is initiated in leptotene, when an axial element (AE) is developed along each homolog over the trajectories of previously loaded cohesin complexes. During this stage, the telomeres attach to the nuclear envelope (NE), and by zygotene, they adopt a polarized "bouquet" configuration that promotes chromosome movements essential for accurate homolog pairing 7. The tethering of telomeres to the NE is mediated by several adaptor proteins whose deficiencies provoke alterations not only in telomere attachment but in pairing, synapsis and recombination leading in many cases to infertility 8. Subsequently, the so-called central element (CE) is formed between the AEs of the SC. At this time, the AEs are referred as lateral elements (LEs) and the CE, composed by transverse filaments, physically connects the LEs of the two homologs. By pachytene, the homologs have achieved synapsis along their entire length and thus each bivalent presents a fully developed SC. During the first and second meiotic divisions, recombined homologs and single chromatids migrate to opposite spindle poles, respectively, due to a sequential loss of arm and centromere sister-chromatid cohesion 9, 10. Cohesion is mediated in somatic vertebrate cells by ring-shaped cohesin complexes composed of two structural maintenance of chromosomes (SMC) proteins, SMC1α and SMC3, the kleisin subunit RAD21 and a STAG/SA subunit that can be SA1 or SA2 11. The dynamic loading, maintenance, and release of cohesin complexes from chromatin are mediated by cofactor proteins and some posttranslational modifications 12, 13. The functions of these cofactors have been characterized during the mitotic cycle, but much less is known of their behavior in meiosis. Once cohesin is loaded on chromatin in early G1 by the NIPBL-MAU2 heterodimer, PDS5 and WAPL bind to the complex and promote their dissociation 14, 15. During S-phase, as sister chromatids arise from the replication fork, cohesion is established by a fraction of cohesin complexes that are acetylated in their SMC3 subunit and bound by Sororin, which counteracts the cohesin-releasing activity of WAPL until mitotic prophase 16, 17. Two paralogs of the PDS5 protein exist in vertebrate cells, PDS5A and PDS5B, which are required both for cohesin dissociation together with WAPL, and for cohesin stabilization together with Sororin 14, 18-20. While the two PDS5 proteins present these activities, centromeric cohesion defects are more apparent in Pds5B-deficient cells in mitosis 19. In addition to the canonical cohesin subunits mentioned above, several meiosis-specific subunits have been described during mammalian meiosis. SMC1β and STAG3 are the paralogs of SMC1α and SA1/2, respectively, while REC8 and RAD21L are the meiotic counterparts of the kleisin subunit RAD21 10. The distribution and functions of the different cohesin subunits during mammalian meiosis have been extensively analyzed. All of them, both mitotic and meiosis-specific ones, are located at AEs/LEs during mouse prophase I. Spermatocytes from mice deficient for the meiosis-specific cohesin subunits SMC1β 21-24, REC8 25, 26, RAD21L 27, and STAG3 28-31 arrest at different stages of prophase I and display severe defects in the assembly and pairing of AEs/LEs, recombination and, in some cases, show altered telomere structure. However, there are few studies describing the dynamics and function of cohesin cofactors during mammalian meiosis. WAPL decorates the AEs/LEs in mouse oocytes 32 and spermatocytes, where it is involved in the removal of arm cohesion by the end of prophase I 33. Unexpectedly, Sororin has been detected at the SC central region, unlike the cohesin subunits and WAPL 34, 35. The single orthologs of PDS5 in Sordaria macrospora (Spo76p) and budding yeast (Pds5) are located at AEs/LEs during prophase I stages 36-38. Analyses of meiosis in Pds5 mutants in Sordaria, budding and fission yeasts, and the worm Caenorhabditis elegans, indicate that this protein is involved in several processes including cohesion, formation of AEs/LEs, condensation, homolog pairing, and repair of DSBs 36-42. In contrast, simultaneous ablation of four out of the five Pds5 genes present in Arabidopsis thaliana does not have a relevant impact in meiotic chromosome structure, or the progression of meiosis 43. In mammalian meiosis, like in mitosis, both PDS5A and PDS5B are present. While nothing is known about the localization of PDS5A, PDS5B has been detected at AEs/LEs in mouse spermatocytes 44. In the present study, we report the distribution of PDS5A and PDS5B during male mouse meiosis. Moreover, taking advantage of conditional knock out (cKO) mouse models previously generated 19, we have analyzed the meiotic phenotype of spermatocytes after drastic depletion of one or both PDS5 proteins. Our results indicate that a single PDS5 protein, either PDS5A or PDS5B, is sufficient for prophase I progression. However, their simultaneous depletion results in severe meiotic defects including the formation of shortened AEs/LEs, reduced formation of early recombination nodules, and alterations in the structure of telomeres and their attachment to the NE during prophase I. Results Different localization and dynamics of PDS5A and PDS5B in mouse spermatocytes We analyzed the distribution of PDS5A and PDS5B proteins on spread mouse spermatocytes by immunofluorescence using specific antibodies for these proteins (Fig EV1A–C) and for SYCP3, a structural component of the AEs/LEs, to identify the different meiotic stages. PDS5A was undetectable during leptotene when SYCP3-labeled AEs started to organize (Fig 1A and B), but colocalized with SYCP3 along the synapsing AEs/LEs of both autosomes and sex chromosomes during zygotene (Fig 1C and D) and early pachytene (Fig 1E and F). By mid-pachytene, however, the PDS5A labeling was dispersed throughout the chromatin and was less intense on the sex body (Fig 1G and H). In diplotene, lack of staining was additionally observed around the ends of the desynapsing autosomal LEs which corresponded to DAPI-positive regions, namely the chromocenters (Fig 1I–L). This distribution of PDS5A persisted in diakinesis although the overall signal intensity was slightly reduced (Fig 1M and N). By metaphase I, PDS5A was found at centromeres (Fig 1O and P). In side-viewed centromeres, PDS5A was present as a T-shaped signal below the closely associated sister kinetochores stained with an ACA serum (Fig 1O; bottom inset), while viewed from the top PDS5A encircled sister kinetochores (Fig 1O; top inset). This localization is similar to that of SYCP3 at the inner centromere domain 45, 46, but different from that of the meiosis-specific cohesin subunit REC8 which localized in small patches along the interchromatid domain at the arms (Fig 1Q and R). Both PDS5A and REC8 were detected at the inner centromere domain, but in side-viewed centromeres, the PDS5A signals were larger than those of REC8 (Fig 1Q, inset). In metaphase II PDS5A was also present at the centromeres (Fig 1S and T). In side-viewed centromeres, two PDS5A signals were detected below kinetochores (Fig 1S; bottom inset), while in top-viewed centromeres, a single PDS5A ring encircled kinetochores (Fig 1S; top inset). Click here to expand this figure. Figure EV1. Characterization of mouse models A. Schematic representation of the conditional alleles (loxfrt or lox) and the null allele (−) obtained upon Cre-mediated recombination for Pds5A (top) and Pds5B (bottom). The position of the primers used for genotyping is indicated (blue arrows). B. Example of genotyping PCRs for Pds5A (top) and Pds5B (bottom) alleles using DNA obtained from testis of the indicated mice as well as DNA from mouse embryo fibroblasts as control. cKO mice carry the conditional allele(s) in homozygosis and a Cre-ERT2 transgene and have been treated with TX to promote the translocations of the Cre recombinase to the nucleus. In most cKO mice, excision of the targeted exon is not complete and a weak band corresponding to the conditional allele can be still be detected in addition to the stronger band of the null allele. The sizes of the different PCR products are as follows: Pds5A wild-type (+) 872 bp, loxfrt 778 bp and null (−) 414 bp; for Pds5B wild-type (+) 706 bp, lox 859 bp and null (−) 415 bp. C. Immunoblot analysis of total extracts prepared from testes of mice of the indicated genotypes. Decreasing amounts of extract from testes obtained from wild-type mice (WT) were loaded for comparison. Overall, elimination of Pds5B is less efficient than elimination of Pds5A both in the single and in the double cKO mice. D–M. Double immunolabeling of PDS5A (green, D–H) or PDS5B (green, I–M) and SYCP3 (red) in Pds5A cKO spread spermatocytes at the indicated prophase I stages (D–G and I–L) and in metaphase I bivalents (H and M). N–W. As in (D–M), but in Pds5B cKO spread spermatocytes at the indicated prophase I stages (N–Q and S–V), and in metaphase I bivalents (R and W). Data information: In both cases, from mid-pachytene onwards PDS5 proteins are not depleted. Sex bivalents (XY) are indicated. Scale bar, 10 μm. Download figure Download PowerPoint Figure 1. PDS5A distribution in mouse spermatocytes A–N. Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in leptotene (A, B), zygotene (C, D), early pachytene (E, F), mid-pachytene (G, H), diplotene (I–L), and diakinesis (M, N). Arrows in (I–L) indicate the position of some chromocenters. O, P. Double immunolabeling of PDS5A (green) and kinetochores revealed with an ACA serum (red) in a metaphase I spermatocyte. Q, R. Double immunolabeling of PDS5A (green) and REC8 (pseudocolored in purple) in a metaphase I spermatocyte. S, T. Double immunolabeling of PDS5A (green) and kinetochores (ACA, red) in a metaphase II spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes. Insets included in (O, Q, and S) correspond to a 300% magnification. Scale bar, 10 μm. Download figure Download PowerPoint Unlike PDS5A, PDS5B colocalized with SYCP3 along the AEs/LEs of the autosomes and sex chromosomes from leptotene up to pachytene (Fig 2A–F). In addition, rounded PDS5B signals protruded from the AEs/LEs ends in pachytene (Fig 2E and F). REC8 colocalized with PDS5B along the AEs/LEs, but was not present at the two close PDS5B signals at SC ends (Fig 2G and H). Instead, PDS5B signals at AEs/LEs ends colocalized with TRF1, a telomere binding protein (Fig 2I and J). To validate PDS5B location at telomeres, we also analyzed its distribution in spermatocytes from mice deficient for the meiosis-specific cohesin subunits SMC1β 23 and REC8 47, which present variable degrees of telomere abnormalities. Smc1β−/− spermatocytes present, among other phenotypes, telomere signals that are split, extended, absent, or disconnected from SC ends 23. Our results showed that in Smc1β−/− pachytene-like spermatocytes, PDS5B localized along the shortened SCs and unsynapsed AEs/LEs (Appendix Fig S1A and B), and as altered signals at SC ends (Appendix Fig S1B and C), similar to those reported for telomeric DNA or telomere proteins in Smc1β−/− mouse spermatocytes 23. Telomere alterations are less severe in Rec8−/− spermatocytes and only 3–4 chromosomes per spermatocyte present decreased levels of telomeric DNA and telomere proteins 47. Accordingly, in Rec8−/− zygotene-like spermatocytes, we found PDS5B at shortened unsynapsed AEs and at most telomeres (Appendix Fig S1D and E), and only a low number of AEs ends presented a highly reduced PDS5B labeling (Fig EV2E and F). Altogether our results indicate that PDS5B accumulates at telomeres during prophase I in mouse spermatocytes. Figure 2. PDS5B distribution in prophase I spermatocytes A–F. Double immunolabeling of PDS5B (green) and SYCP3 (red) in spread spermatocytes at the indicated stages. G–L. Immunolabeling of PDS5B (green) and either REC8 (pseudocolored in blue, G, H) or SYCP3 (red) and TRF1 (pseudocolored in yellow, I, J) or ACA (pseudocolored in purple, K, L) onto a spread pachytene spermatocyte. M–P. Double immunolabeling of PDS5B (green) and SYCP3 (red) on diplotene (M, N) and diakinesis (O, P) spread nuclei. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. Insets included in (C–P) correspond to a 300% magnification. Scale bar, 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Reduction of PDS5 proteins in Pds5AB cKO spermatocytes A, B. Representative images of Pds5AB cKO spread spermatocytes at the indicated stages double immunolabeled for SYCP3 (red) and either PDS5A (green, A) or PDS5B (green, B). C, D. Box whiskers plot analyses of corrected total nuclear fluorescence of PDS5A (C) and PDS5B (D) in a given wild-type (WT, green) or Pds5AB cKO (white) spermatocyte at the indicated stages (n = 10 for each stage). Whiskers indicate the minimum to maximum values, first and third quartiles are depicted by the box, and the horizontal lines in the boxes indicate the median value. Statistical significance was assessed using an unpaired two-tailed t-test function (*****P < 0.00001; ns, not significant difference). E. Schematic representation of cell progression and timing throughout the indicated stages of spermatogenesis in TX-treated Pds5AB cKO testes. In mouse, the transition from a differentiating spermatogonia (A1) into a preleptotene cell lasts around 8.6, 8.6 days more to become a pachytene spermatocyte, and finally 17.2 days to progress to elongated spermatids. Note that in seminiferous tubules, this process is continuous and different cell populations coexist. Under our experimental conditions, if the targeted exons were excised in spermatogonial cells, depletion or extreme reduction of PDS5 protein levels would only be accomplished in spermatocytes up to the early pachytene stage (highlighted in red). F. Percentage of fluorescence intensity, relative to their respective maximum values, for PDS5A (yellow) and PDS5B (blue) in wild-type (top) and Pds5AB cKO (bottom) spermatocytes at the indicated stages (n = 10 for each stage). Download figure Download PowerPoint Consistent with the telocentric nature of mouse chromosomes, kinetochore signals were always internally located in relation to PDS5B telomeric ones at the centromeric end of the pachytene SCs (Fig 2K and L). By diplotene, PDS5B persisted along the desynapsing LEs and at telomeres (Fig 2M and N). In some images, we could detect two closely associated PDS5B signals at desynapsed LE ends corresponding to telomeres of sister chromatids (Fig 2M and N, insets). During diakinesis, PDS5B labeling began to disappear from the autosome LEs but not from telomeres (Fig 2O and P). In metaphase I, large PDS5B signals colocalized with SYCP3 at the inner centromere domains (Fig 3A–F). Interestingly, smaller PDS5B signals were located in pairs at the distal chromosome ends (Fig 3C–F), that are reminiscent of the location of distal telomeres in metaphase I bivalents 48. In metaphase II chromosomes, large PDS5B signals were found at each centromere between the kinetochores (Fig 3G–J). Altogether, our results indicate that PDS5A and PDS5B are present along the AEs/LEs in prophase I, similar to cohesin subunits, although the two paralogs display somehow different dynamics. Moreover, only PDS5B appears at telomeres. Figure 3. PDS5B distribution in condensed meiotic chromosomes A–F. Double immunolabeling of PDS5B (green) and SYCP3 (red) in a metaphase I spermatocyte (A, B) and selected bivalents (C–F).The sex bivalent (XY) is indicated. G–J. Double immunolabeling of PDS5B (green) and kinetochores (ACA, red) and staining of the chromatin (DAPI) in a metaphase II spermatocyte. Data information: Insets included in (I) correspond to a 300% magnification. Scale bar, 10 μm. Download figure Download PowerPoint Conditional knock out mouse models for PDS5 protein depletion The different localization patterns of PDS5A and PDS5B during prophase I stages suggest that they may have specific functions in meiosis. To address this possibility, we took advantage of Pds5-deficient mice generated previously 19. Since constitutive knock out alleles for each gene are lethal in homozygosis, we used male mice carrying cKO alleles for Pds5A, Pds5B or both (Pds5AB hereafter) in combination with a Cre recombinase allele that can be induced by tamoxifen (TX) supplemented in the diet (Fig EV1A). The health condition of Pds5AB cKO mice was critically deteriorated after 2 weeks of treatment. Thus, in order to ensure the animal's welfare and to standardize the experimental conditions, mice of all genotypes were fed TX diet for 15 days, and then, testes were extracted for analyses. Efficient, and almost complete, excision of the corresponding targeted exon(s) in testis from TX-treated Pds5A cKO, Pds5B cKO, and Pds5AB cKO mice was confirmed by PCR. However, immunoblot analyses of testis extracts showed only a partial decrease of protein levels (Fig EV1B and C). It must be noted that testis comprises a mixture of heterogeneous and asynchronous cell populations, both at the seminiferous tubules (i.e. Sertoli cells, spermatocytes, and spermatids) and at the testis interstitium (i.e. Leydig cells and fibroblasts), which could present different turnover rates of PDS5 protein levels. To assess the depletion of PDS5 proteins in individual spermatocytes, we analyzed preparations from TX-treated Pds5A cKO, Pds5B cKO, and Pds5AB cKO mice by immunofluorescence with antibodies against PDS5A, PDS5B, and SYCP3. In leptotene, zygotene and early pachytene spermatocytes from Pds5A cKO (Fig EV1D–F and I–K), Pds5B cKO (Fig EV1N–P and S–U), and Pds5AB cKO mice (Figs 4A–C and F–H, and EV2A and B), the levels of the PDS5 protein(s) whose gene had been presumably excised were below the detection threshold. However, PDS5A and PDS5B labeling were indistinguishable between wild-type and Pds5 cKO spermatocytes in later meiotic stages (Figs EV1, EV2A, and B, and 4). Quantification of nuclear PDS5A and PDS5B fluorescence intensities demonstrated the strong depletion of both PDS5 proteins in individual leptotene-like, zygotene-like and early pachytene-like Pds5AB cKO spermatocytes compared to wild-type levels (Fig EV2C and D). We determined a 3.25-, 9.14- and 11-fold reduction of PDS5A fluorescence in leptotene-like, zygotene-like and early pachytene-like Pds5AB cKO spermatocytes (Fig EV2C and F). Similarly, PDS5B fluorescence was 8.03-, 11-, and 10.08-fold reduced in the same stages (Fig EV2D and F). These results indicate an almost complete absence of PDS5 proteins in these Pds5AB cKO early prophase I spermatocytes. On the other hand, the nuclear fluorescence levels of both PDS5 proteins in Pds5AB cKO mid-pachytene spermatocytes were similar to those found in wild-type ones (Fig EV2A–D and F). Figure 4. Reduced AEs/LEs length in Pds5AB cKO spermatocytes A–J. Double immunolabeling of either PDS5A (green, A–E) or PDS5B (green, F–J) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages. K–M. Preparations from the same mice were immunolabeled with the testis-specific histone variant H1t (green) and SYCP3 (red). N. Scatter dot-plot graph of total SC length measured in the autosomal pairs of 33 early pachytene cells from three different individuals of each genotype. Bars indicate mean and standard error. Statistical significance was assessed using an unpaired two-tailed t-test function (****P < 0.0001). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm. Download figure Download PowerPoint We reckon that PDS5 proteins are highly stable, as previously noticed in mouse embryo fibroblasts in which protein elimination required at least 5 days following Cre activation 19. Moreover, cohesin mediating cohesion in mammalian oocytes shows very little turnover over several months 49 and the same could be true for cohesin cofactors. Thus, in A1 spermatogonia entering meiosis during the TX treatment, the corresponding targeted exon(s) would be excised and they would develop into spermatocytes in which PDS5 proteins are absent, or strongly depleted under detection limits. These spermatocytes could only progress up to early pachytene during the TX treatment, given that an A1 spermatogonia takes approximately 17 days to progress into a pachytene spermatocyte 50 (Fig EV2E). In cells already coursing meiosis at the beginning of the TX treatment, the prior synthesis of highly stable PDS5 proteins would allow them to progress normally throughout spermatogenesis (Fig EV2E). This idea, consistent with the immunofluorescence analyses, can also explain the immunoblot detection of considerable amounts of PDS5 proteins in testis extracts after the effective excision of Pds5 genes. In conclusion, two different populations of spermatocytes, clearly recognizable by immunofluorescence, coexist in Pds5A cKO, Pds5B cKO, and Pds5AB cKO testes after the 15-day-long TX treatment. Under our experimental design, mouse models allowed us to analyze the effects of the strong depletion of PDS5 proteins from leptotene up to early pachytene stages. A single PDS5 paralog protein is sufficient for prophase I progression Spermatocytes from TX-treated Pds5A cKO and Pds5B cKO mice developed normal SYCP3-labeled AE/LEs despite extreme reduction of PDS5A and PDS5B, respectively, observed before mid-pachytene stages (Fig EV1D–F and N–P). In later stages of meiosis, PDS5A and PDS5B staining were indistinguishable between wild-type and Pds5-deficient cells (Fig EV1G, H, Q and R). Interestingly, the reduction of one PDS5 paralog did not affect the distribution of the other paralog (Fig EV1D–W). Furthermore, the analysis of seminiferous tubules from these mice treated for 15 days with TX demonstrated that spermatocytes completed all meiotic and spermiogenic stages without noticeable failures (Appendix Fig S2). Since PDS5B has been specifically involved in centromere cohesion in MEFs 19, we next studied centromere distribution in Pds5A or Pds5B deficient spermatocytes. Our results did not evidence appreciable alterations of centromere cohesion or ar
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