PLK1 regulates centrosome migration and spindle dynamics in male mouse meiosis
2021; Springer Nature; Volume: 22; Issue: 4 Linguagem: Inglês
10.15252/embr.202051030
ISSN1469-3178
AutoresEnrique Alfaro, Pablo López‐Jiménez, José González‐Martínez, Marcos Malumbres, José Á. Suja, Rocío Gómez,
Tópico(s)Plant Molecular Biology Research
ResumoArticle21 February 2021Open Access Source DataTransparent process PLK1 regulates centrosome migration and spindle dynamics in male mouse meiosis Enrique Alfaro Enrique Alfaro orcid.org/0000-0002-4681-147X Departamento de Biología, Facultad de Ciencias, Unidad de Biología Celular, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Pablo López-Jiménez Pablo López-Jiménez orcid.org/0000-0002-6673-5996 Departamento de Biología, Facultad de Ciencias, Unidad de Biología Celular, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author José González-Martínez José González-Martínez orcid.org/0000-0002-3850-6803 Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Marcos Malumbres Marcos Malumbres orcid.org/0000-0002-0829-6315 Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author José A Suja José A Suja orcid.org/0000-0002-4266-795X Departamento de Biología, Facultad de Ciencias, Unidad de Biología Celular, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Rocío Gómez Corresponding Author Rocío Gómez [email protected] orcid.org/0000-0003-4408-9812 Departamento de Biología, Facultad de Ciencias, Unidad de Biología Celular, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Enrique Alfaro Enrique Alfaro orcid.org/0000-0002-4681-147X Departamento de Biología, Facultad de Ciencias, Unidad de Biología Celular, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Pablo López-Jiménez Pablo López-Jiménez orcid.org/0000-0002-6673-5996 Departamento de Biología, Facultad de Ciencias, Unidad de Biología Celular, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author José González-Martínez José González-Martínez orcid.org/0000-0002-3850-6803 Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Marcos Malumbres Marcos Malumbres orcid.org/0000-0002-0829-6315 Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author José A Suja José A Suja orcid.org/0000-0002-4266-795X Departamento de Biología, Facultad de Ciencias, Unidad de Biología Celular, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Rocío Gómez Corresponding Author Rocío Gómez [email protected] orcid.org/0000-0003-4408-9812 Departamento de Biología, Facultad de Ciencias, Unidad de Biología Celular, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Author Information Enrique Alfaro1, Pablo López-Jiménez1, José González-Martínez2, Marcos Malumbres2, José A Suja1 and Rocío Gómez *,1 1Departamento de Biología, Facultad de Ciencias, Unidad de Biología Celular, Universidad Autónoma de Madrid, Madrid, Spain 2Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain *Corresponding author. Tel: +34 91 4978242; Fax: +34 91 4978344; E-mail: [email protected] EMBO Reports (2021)22:e51030https://doi.org/10.15252/embr.202051030 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 Cell division requires the regulation of karyokinesis and cytokinesis, which includes an essential role of the achromatic spindle. Although the functions of centrosomes are well characterised in somatic cells, their role during vertebrate spermatogenesis remains elusive. We have studied the dynamics of the meiotic centrosomes in male mouse during both meiotic divisions. Results show that meiotic centrosomes duplicate twice: first duplication occurs in the leptotene/zygotene transition, while the second occurs in interkinesis. The maturation of duplicated centrosomes during the early stages of prophase I and II are followed by their separation and migration to opposite poles to form bipolar spindles I and II. The study of the genetic mouse model Plk1(Δ/Δ) indicates a central role of Polo-like kinase 1 in pericentriolar matrix assembly, in centrosome maturation and migration, and in the formation of the bipolar spindles during spermatogenesis. In addition, in vitro inhibition of Polo-like kinase 1 and Aurora A in organotypic cultures of seminiferous tubules points out to a prominent role of both kinases in the regulation of the formation of meiotic bipolar spindles. Synopsis Polo-like Kinase 1 (PLK1) has a key role in the regulation of centrosome dynamics in male mouse meiosis. PLK1 participates in pericentriolar matrix (PCM) assembly, centrosome maturation and migration, and in the formation of the meiotic bipolar spindle. Centrosomes duplicate twice during male mouse meiosis: in the leptotene/zygotene transition and during interkinesis. Meiotic centrosomes separate during diplotene and during interkinesis/prophase II to facilitate the assembly of bipolar meiotic spindles. Genetic ablation or chemical inhibition or PLK1 result in monopolar spindles in both meiotic divisions in mouse spermatocytes. In vitro inhibition of PLK1 and AURKA suggest an important role of both kinases in formation of meiotic bipolar spindles. Inhibition of PLK1 and AURKA impairs KIF11 localization to monopolar spindles. Introduction Cell division requires equal segregation of the genetic material, karyokinesis, and the cytoplasmic material, cytokinesis, to both somatic daughter cells. During karyokinesis, chromosomes are driven by microtubules (MTs) to the metaphase plate and, once aligned, they are pulled towards the cell poles (Kline-Smith & Walczak, 2004). MTs are also involved in the subsequent process of cytokinesis, forming the mid-body that allows the separation of cytoplasmic material between daughter cells (Khodjakov & Rieder, 2001; Piel et al, 2001). MTs together with MT-organising centres (MTOCs) comprise a complex structure essential for cell division in mammals: the achromatic spindle. Centrosomes behave as the major MTOCs in most animal cells, whereby polymerisation of MTs is implicated in cell division as well as functions such as the formation of the basal body of cilia and flagella, and the maintenance of cell shape and polarity (Azimzadeh & Marshall, 2010). Typically, centrosomes are formed by a pair of centrioles, each one composed of nine MT triplets, surrounded by an electrodense pericentriolar material (PCM). The PCM harbours not only proteins important for MT nucleation, but also regulators of the cell cycle and its checkpoints, in line with important roles for centrosomes in intracellular signalling (Woodruff et al, 2014). Centrioles have important roles in most microtubule-related processes, including cell motility, division and signalling. However, centrioles are not essential for MTOCs in mammalian oocytes (Dumont & Desai, 2012), nor in some insect spermatocytes (Steffen et al, 1986), and are absent in the first division of rodent zygotes (Coelho et al, 2013). Almost all land plant MTOCs also lack centrioles (Sluder, 2014). On the contrary, centrosome matrix has an evolutionarily conserved PCM protein composition (Sluder, 2014; Woodruff et al, 2014; Conduit et al, 2015). The centrosome function is tightly coordinated with cell-cycle progression (Nigg & Holland, 2018). The centriole pair, called diplosome, undergoes a duplication during S phase, thus enabling the formation of two centrosomes that will occupy opposite poles of the dividing cell (Arbi et al, 2018). Duplicated centrosomes are connected by a proteinaceous link between the parental centrioles which includes centrosome-associated protein CEP250 (CEP250/CNAP1) and rootletin (Mayor et al, 2000). The pair of centrosomes must mature and migrate before entering mitosis in order to carry out nucleation of the spindle MTs. This maturation consists of the recruitment and accumulation of PCM proteins, such as pericentrin (PCNT; Bornens, 2012), CEP192 and CDKRAP2/CEP215, which are required for the formation of the γ-Tubulin ring complex (γTuRC; Haren et al, 2006). PCM reaches its maximum volume and maturation in the G2-M transition (Fujita et al, 2016), when γTuRC is organised around the centrosome and allows nucleation of MTs (Zheng et al, 1995). At the onset of mitotic cell division, duplicated centrosomes migrate to cell poles separated by the microtubule-dependent motor kinesin-like protein KIF11 (kinesin-related motor protein EG5) to promote the formation of a proper bipolar spindle for karyokinesis (Blangy et al, 1995; Kapitein et al, 2005). Finally, the generation of astral MTs, which emanate from the centrosome and are connected to the cell membrane, favours the maintenance of the bipolar spindle (Kline-Smith & Walczak, 2004). Once established, the kinetochore MTs exert the driving force that allows chromosome congression and segregation. Afterwards, interzonal MTs will be responsible for executing cytokinesis (Nigg & Holland, 2018). In 1988, Sunkel and Glover described a Drosophila melanogaster mutant presenting defects on cell pole establishment during cell division (Sunkel & Glover, 1988). This mutant, called Polo, was defective in the Plk1 fly ortholog and allowed the description of a family of protein kinases well conserved in the evolution of eukaryotes (Vaid et al, 2016). Polo-like kinases are serine/threonine kinases (de Carcer et al, 2011). In mammals, this protein family is formed by up to five different proteins that appeared along the eukaryote evolution and present structural differences and diverse functions (Archambault & Glover, 2009). These proteins are implicated in DNA replication and repair, centrosome duplication and maturation; and cell division regulation and cytokinesis (Schmucker & Sumara, 2014). In mitosis, kinases CDK1 and Aurora A (AURKA) are essential for Polo-like kinase 1 (PLK1) regulation (Lukasiewicz & Lingle, 2009; Nikonova et al, 2013). The location, activation and function of PLK1 are highly regulated, and defects on these processes are involved in the appearance of aneuploidies and carcinogenesis (Liu et al, 2017). The PLK1 protein has three domains, a catalytic N-terminal domain, a C-terminal Polo-box type domain and a binding domain of these two previous ones (de Carcer et al, 2011). The N-terminal domain corresponds to its active site, involved in its kinase function, and in the regulation of its activation loop; the Polo-box domain in turn participates in ligand recognition (Schmucker & Sumara, 2014). Residues serine 137 (S137) and threonine 210 (T210) are located in the activation loop, and their phosphorylation is essential for the regulation of PLK1 (Tsvetkov & Stern, 2005). During mitosis, PLK1 participates in centrosome maturation and the establishment of the spindle (Zitouni et al, 2014), as well as cohesin complex removal (Hauf et al, 2005) and cytokinesis (Fabbro et al, 2005). Mitotic PLK1 is localised at centrosomes, kinetochores and mid-body (Barr et al, 2004). The dissolution of the link between duplicated centrioles at the onset of mitosis, a process termed centrosome separation, is dependent on PLK1 and AURKA kinases (Fujita et al, 2016). Once the duplicated centrosomes have separated, their migration towards the cell poles can occur mediated by kinesin KIF11, which is also regulated by PLK1 and CDK1 (Agircan et al, 2014). Although there is great knowledge about the functional role of PLK1 during mitosis, much less is known about its importance during meiosis, the special cell division process that allows the formation of haploid gametes. Meiosis implies two rounds of chromosome segregation after a single round of DNA replication. During the first meiotic division, chromosomes undergo a process of DNA exchange called recombination, which provides genetic diversity to the resulting gametes (Miller et al, 2013). In prophase I, homologous chromosomes pair, synapse, recombine and desynapse in a coordinated process that requires the formation of a tripartite protein structure called the synaptonemal complex (SC; Page & Hawley, 2004; Hunter, 2015), followed by the reductional segregation of recombined homologous chromosomes during anaphase I. During the second meiotic division, sister chromatids segregate in anaphase II in an equational manner to allow the formation of haploid gametes. Several important roles for PLK1 have been described in mammalian oogenesis, where progression is arrested during prophase I at the stage of dictyotene. PLK1 is needed for the resumption of cell division before ovulation (Hunt & Hassold, 2002), and for the establishment of the female acentriolar meiotic spindle, localising to the MTOCs (Xiong et al, 2008). In addition, PLK1 carries out specific functions such as nuclear envelope breakdown (NEBD), and activation of the anaphase promoting complex and chromosome condensation maintenance (Solc et al, 2015). Nevertheless, knowledge about the extended roles of PLK1 in vertebrate spermatogenesis is considerably limited. This work is focused on addressing the role of PLK1 and AURKA in centrosome regulation and in the establishment of male mouse meiotic spindles. Mammalian male meiotic centrosomes present centrioles and act as MTOCs. These centrioles are needed for the formation of spermatozoa flagellum and, in many mammals including human, are inherited by the zygote to constitute the first centrioles of an organism (Coelho et al, 2013). Therefore, correct MTOC and spindle formation and regulation in meiosis are essential for male fertility. Results Centrosome migration starts at the diplotene and prophase II stages In order to learn about male mouse meiotic centrosomes, we have studied its composition and dynamics on squashed spermatocytes since this technique does not disturb the tridimensionality of cell morphology, neither chromosome condensation and protein distribution in prophase I and dividing spermatocytes (Page et al, 1998). We first determined the distribution of PCM components. For this, we performed a double immunofluorescence staining of PCNT, the major structural protein of PCM (Delaval & Doxsey, 2010), together with SYCP3, a component of the SC commonly used as marker of meiosis progression (Parra et al, 2004). In early prophase I (leptotene and zygotene), a small and round PCNT signal appears externally but highly proximal to the nucleus. In these stages, SYCP3, a component of the SC, marked the newly formed axial elements (AEs) and lateral elements (LEs) in those places where synapses have already been established between the homologues (Fig 1A). In pachytene, a large and diffuse PCNT signal is observed, while SYCP3 marks the fully synapsed autosomal chromosome LEs and the AEs and the pseudoautosomal region of the XY sex pair (Fig 1B). In diplotene, SYCP3 marks the desynapsed LEs, and two compact signals of PCNT are clearly distinguished. These signals are comparatively smaller and more compact than the pachytene signal and can be observed very close to each other. This result suggests that both signals correspond to the two centrosomes, which are already identified separately (Fig 1C), indicating the onset of the separation of the centrosomes. At diakinesis, PCNT foci begin to separate, although they remain very close to the nuclear periphery. At this stage, SYCP3 marks synapsed and desynapsed LEs and small cytoplasmic agglomerations (Fig 1D), in accordance with previous results (Parra et al, 2004). In prometaphase I, two intense PCNT signals are oriented towards the poles of the cell, although bivalents are not yet fully aligned (Fig 1E). In metaphase I, two PCNT signals are located at the opposite poles of the cell (Fig 1F). SYCP3 labels the inner centromeric domain and the interchromatid domain (Fig 1E and F). In telophase I, two groups of PCNT signals are observed, one at each pole, in which several small PCNT foci can be observed. SYCP3 is observed as small bars on segregated homologous chromosomes (Fig 1G). Figure 1. Distribution of PCNT and SYCP3 during the first meiotic division A–G. Double immunolabelling of pericentrin -PCNT- (green) and SYCP3 (red) on squashed WT mouse spermatocytes on (A) zygotene, (B) pachytene, (C) diplotene, (D) diakinesis, (E) prometaphase I, (F) metaphase I and (G) telophase I. Chromatin has been stained with DAPI (blue). Scale bar in G represents 10 μm. Download figure Download PowerPoint In interkinesis, SYCP3 is presented as bars and PCNT conforms two small foci very close to each other (Fig 2A). During the second meiotic division, at early prophase II, two small signals of PCNT are still observed (Fig 2B). Following this stage, SYCP3 is no longer detected, as previously described (Parra et al, 2004). It is in late prophase II that the two signals of PCNT begin to distance themselves (Fig 2C). In prometaphase II, two signals of PCNT are already oriented towards the opposite poles of the cell and close to the chromosomes (Fig 2D). In metaphase II, chromosomes are aligned, and PCNT signals are observed in each pole (Fig 2E). In telophase II, we observe a PCNT signal in each pole of the cell along with the two daughter nuclei (Fig 2F). Figure 2. Distribution of PCNT and SYCP3 during interkinesis and the second meiotic division A–F. Double immunolabelling of pericentrin -PCNT- (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) interkinesis, (B) early prophase II, (C) late prophase II, (D) prometaphase II, (E) metaphase II and (F) telophase II. Chromatin has been stained with DAPI (blue). Scale bar in F represents 10 μm. Download figure Download PowerPoint Centrosomes duplicate twice during male mouse meiosis We then focused on analysing the timing of centrosome duplication during meiosis. To do this, we immunolabelled a structural component of centrioles with anti-Centrin-3 antibody together with SYCP3. In early prophase I, Centrin-3 (CETN3) is detected as two small and very proximal dots near the nucleus in leptotene (Fig 3A and A´), while it clearly shows four small signals, two pairs, in zygotene (Fig 3B and B´) and pachytene (Fig 3C and C´). This suggests that centrosome duplication occurs in the transition from leptotene to zygotene. Centrioles are always seen near the nucleus; however, as the result of z-projection, some centrioles appear immersed in DAPI signals in the images. CETN3 is clearly seen as two dots per opposite poles in metaphase I (Fig 3D and D'). When bivalents are segregated in telophase I, two CETN3 dots are visible in each pole (Fig 3E and E´). In early interkinesis, two small signals of CETN3 are detected (Fig 3F and F') and four dots are clearly seen in late interkinesis (Fig 3G and G'). This suggests a second centrosome duplication between the first and the second meiotic divisions. The newly duplicated centrosomes start separating again in prophase II (Fig 3H and H'). Two clear signals of CETN3 at the poles are also visible in metaphase II (Fig 3I and I´) and in telophase II (Fig 3J and J´). Spermatids show a pair of centrioles in early (Fig 3K and K´) and late (Fig 3L and L´) maturation stages. These results indicate that centrosomes duplicate twice during male mouse meiosis: in leptotene-zygotene transition and during interkinesis. Figure 3. Distribution of CETN3 and SYCP3 during spermatogenesis A–L. Double immunolabelling of CETN3 (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) leptotene, (B) zygotene, (C) pachytene, (D) metaphase I, (E) telophase I, (F) early interkinesis (F), late interkinesis (G), prophase II (H), metaphase II (I), telophase II (J), round spermatid (K) and elongated spermatid (L). Amplifications at 300% magnification for the centrosomic CETN3 signals are presented for each image. Chromatin has been stained with DAPI (blue). Scale bar in L represents 10 μm. Download figure Download PowerPoint To further confirm these results, a double immunolabelling of γ-Tubulin, a centrosome marker, together with SYCP3 was analysed in both meiotic divisions (Fig EV1). γ-Tubulin was appreciated as a single round mark in pachytene (Fig EV1A), while it appeared as two signals when centrosomes migrated in diplotene and diakinesis (Fig EV1B and C). γ-Tubulin is detected at the bipolar spindles at the first (Fig EV1D) and second (Fig EV1F) meiotic divisions. Two signals of γ-Tubulin are also detected in late interkinesis (Fig EV1A). All data of γ-Tubulin are in accordance with PCNT and CETN3 patterns of localisation (Figs 1-3). Click here to expand this figure. Figure EV1. Distribution of γ-Tubulin and SYCP3 during male mouse meiosis A–F. Double immunolabelling of γ-Tubulin (green) and SYCP3 (red) on squashed control mouse spermatocytes at (A) pachytene, (B) diplotene, (C) diakinesis, (D) prometaphase I, (E) interkinesis and (F) metaphase II. Chromatin has been stained using DAPI (blue). Insets correspond to centrosomes at 200% magnification. White arrows indicate the location of centrosomes. Scale bar in F represents 10 μm. Download figure Download PowerPoint Genetic depletion of Plk1 indicates a role of PLK1 in PCM assembly and in the regulation of centrosome migration In order to verify the localisation of PLK1 at the centrosomes in male mouse meiosis, we first detected its phosphorylated modification PLK1S137P in squashed spermatocytes, since it was previously described in mouse oocytes (Du et al, 2015). In pachytene, PLK1S137P diffusely decorated the nucleoplasm, showing intense signals at the chromocentres, which represent clustered centromeres (Fig 4A). In addition, two signals are detected near to the nucleus. These signals colocalise with γ-Tubulin (Fig 4B), indicating the position of the centrosome. In late diplotene, PLK1S137P signals are slightly separated from each other (Fig 4C) at the onset of centrosome migration. In diakinesis, separated signals are clearly detected (Fig 4E). This same separation is observed with γ-Tubulin (Fig 4D and F). When bivalents are aligned in the metaphase I plate, intense PLK1S137P and γ-Tubulin signals at the centrosomes are clearly located at opposite poles. In addition, centromeric signals of PLK1S137P are detected (Fig 4G and H). The same pattern is observed during the second meiotic division, with a second centrosome migration during prophase II and the location of PLK1S137P and γ-Tubulin in opposite poles at metaphase II (Fig 4I and J). The PLK1S137P pattern of distribution was also confirmed in spread spermatocytes (Fig EV2E–J). Distribution of PLK1T210P, another modification of PLK1 (Du et al, 2015), was also studied in squashed (Fig EV2A and B) and spread spermatocytes (Fig EV2C and D); however, no centrosomic signals were detected with this modification. Figure 4. PLK1S137P distribution during male mouse meiosis A–J. Double immunolabelling of PLK1S137P (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) pachytene, (C) diplotene, (E) diakinesis, (G) metaphase I and (I) metaphase II. Chromatin has been stained with DAPI (blue). Fourth column shows the double immunolabelling of PLK1-S137P (green) and γ-Tubulin (magenta) on squashed WT mouse spermatocytes at (B) pachytene, (D) diplotene, (F) diakinesis, (H) metaphase I and (J) metaphase II. Insets correspond to centrosomes at 200% magnification. White arrows indicate the location of centrosomes. Scale bar in J represents 10 µm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Distribution of PLK1S137P and PLK1T210P A, B. Double immunolabelling of PLK1T210P (green) and SYCP3 (red) on squashed spermatocytes at (A) metaphase I and (B) metaphase II. Chromatin has been stained using DAPI (blue). C, D. Double immunolabelling of PLK1T210P (green) and SYCP3 (red) on spread spermatocytes at (C) metaphase I and (D) metaphase II. Chromatin has been stained using DAPI (blue). E–J. Double immunolabelling of PLK1S137P (green) and SYCP3 (red) on spread spermatocytes at (E) metaphase I, (F) anaphase II, (G) interkinesis, (H) prophase II, (I) metaphase II and (J) telophase II. Chromatin has been stained using DAPI (blue). Insets correspond to centrosomes at 200% magnification. Data information: White arrows indicate the location of centrosomes. Scale bar in D and J represents 10 μm. Download figure Download PowerPoint We then performed a genetic approach examining Plk1-deficient mice generated previously (Wachowicz et al, 2016; de Carcer et al, 2017), whose meiotic phenotype has not yet been described. Since constitutive knock-out alleles for each gene are lethal in homozygosis, we used male mice carrying conditional knock-out (cKO) alleles in combination with a Cre recombinase allele that can be induced by tamoxifen (TX) (Fig EV3). We analysed the centrosome dynamics in Plk1(lox/lox) mice after a 7 day of TX treatment, Plk1(Δ/Δ), since the health of the mice deteriorated critically leading to death after this period (Wachowicz et al, 2016; de Carcer et al, 2017). These Plk1(Δ/Δ) mice reduce 86% Plk1 levels in testis (Wachowicz et al, 2016). Gene strategy from Wachowicz et al. is displayed in Fig EV3 A. Efficient excision of the corresponding targeted exon in testis from TX-treated Plk1(Δ/Δ) mice was confirmed by PCR (Fig EV3B). Immunofluorescence signals for PLK1 at centrosomes in Plk1(Δ/Δ) metaphases presented levels below the detection threshold, while CETN3 confirmed the position of centrosomes. Quantification analysis offers significance in comparison to control metaphases (Fig EV3C). Although mice were not able to survive longer periods of Plk1 ablation, having proliferative tissue seriously affected, the histological sections of Plk1(Δ/Δ) seminiferous tubules showed minimal apoptosis (Fig EV3D). The level of apoptosis detected by immunolabelling cleaved caspase-3 (caspase-3) showed no significant differences between Plk1(+/+) and Plk1(Δ/Δ) mice (Fig EV4F). Altogether, these data suggest that somatic division in proliferative tissues severely alters the animal's health (Wachowicz et al, 2016; de Carcer et al, 2017), yet Plk1 ablation can be studied in testis. Click here to expand this figure. Figure EV3. Gene strategy and PCR analysis for Plk1(Δ/Δ) mouse model Schematic representation of the conditional alleles (loxfrt or lox) and the null allele (–) obtained upon Cre-mediated recombination for Plk1, extracted from Wachowicz et al 2016. Example of genomic genotyping PCRs for Plk1 alleles (lox and Δ alleles) obtained from testis of the indicated mice. Immunolabelling of PLK1 (grey/green), CETN3 (red) and chromatin counterstained using DAPI (blue) in metaphases I from control -Plk1(+/+)- and Plk1(Δ/Δ) mice. Scale bar represents 10 μm. Experiments were conducted for two biological replicates. Number of cells analysed: control (n = 36), Plk1(Δ/Δ) (n = 35). Data are mean ± SD; ****P < 0.0001, Student's t-test. Histological section of seminiferous tubules stained with H/E staining in control (a) and Plk1(Δ/Δ) (c). ["L" indicates the lumen of the seminiferous tubule; black arrow indicates the position of an apoptotic cell]. Histological section of liver stained with H/E staining in control (e) and Plk1(Δ/Δ) (f). Cryosection of testis and counterstaining with DAPI in control (b) and Plk1(Δ/Δ) (d). Yellow arrows indicate the presence of aligned metaphases (a). Pink arrows indicate the presence of apparently monopolar metaphases (d). Scale bars represent 20 μm. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Optimisation of in vitro studies A, B. Organotypic cultures of seminiferous tubules. (A) X40 microscope field stained with DAPI (blue) and (B) caspase-3 (green) and SYCP3 (red) in control culture of seminiferous tubules in DMSO. Scale bars represent 10 μm. C. Graphic representation of the percentage of apoptotic cells in cultured seminiferous tubule. Caspase-3-positive labelling for the comparison between control (WT freshly squashed spermatocytes) and spermatocytes from different time organotypic control cultures of seminiferous tubules. Three biological replicates were conducted for each condition. Data are mean ± SD; **P < 0.01, ****P < 0.0001, Student's t-test. D. Graphic representation of the percentage of monopolar spindles at different timings. Data are presented for 2 h, 4 h and 8 h for control cultures spermatocytes, 100 µM of BI2536 and 10 µM of MLN8237. Three biological replicates were conducted for each condition, except MLN837 (2 h and 4 h) that were conducted in two replicates. Data are mean ± SD; ****P < 0.0001, Student's t-test. E. Graphic representation of the percentage of monopolar spindles in the different conditions in relation to concentration. Data are presented for 8 h for the different concentration trials for BI2536 and MLN8237 treatments used for the optimisation of the methodology. At least two biological replicates were conducted per each condition. Data are mean ± SD; ***P < 0.0001, ****P < 0.0001, one-way ANOVA with Turkey's multiple comparisons test. F. Graphic representation of the percentage of apoptotic spermatocytes in the different conditions of this study. Data are presented for 100 µM of BI2536 and 10 µM of MLN8237 for 8-h treatment. Three biological replicates were conducted per each condition. Data are mean ± SD. Source data are available online for this figure. Download figure Download PowerPoint Plk1(+/+) mice (onwards
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