Expression and subcellular localization of ADD1 during mouse oocyte meiotic maturation
To detect the protein expression levels and dynamic subcellular localization of ADD1 in mouse oocytes at various stages of meiotic maturation, samples were collected after 0, 4, 8, 9.5, and 16 h of oocyte maturation culture, corresponding to germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase/telophase I (ATI), and metaphase II (MII) stages, respectively. Endogenous levels of ADD1 protein were detected by immunoblot analysis using an antibody against synthesized non-phosphopeptide around the phosphorylation site of serine 726 of ADD1 (T-P-S-P-F-L). The data demonstrated that ADD1 protein was expressed at all detected stages during mouse oocyte meiosis. Its expression level decreased to the lowest level in the MI stage, and then gradually increased with the progress of meiosis until the MII stage (Fig. 1A). Indeed, as shown in Fig. 1B, the protein level of ADD1 in MI oocytes was significantly lower than that in GV oocytes (n = 4, p < 0.05), but not significantly different from GVBD, ATI, and MII stage oocytes (n = 4, p > 0.05). To confirm the possible relationship between ADD1 and spindle apparatus, we double-stained with ADD1 and microtubule subunit α-tubulin antibodies. Immunofluorescent results demonstrated that ADD1 was uniformly and diffusely distributed throughout the cell except in the nucleoli in GV oocytes. Once the oocyte resumed meiosis and underwent GVBD, ADD1 migrated from the entire oocyte toward the perichromosomal region and accumulated around condensed chromosomes, where spindle microtubules were generated. When the cell cycle progressed to the MI stage, the chromosomes were aligned to the equatorial plate of the oocyte, and ADD1 was distributed on spindle poles with 1–3 spots clustered on each side. During the transition from the anaphase I (AI) to the telophase I (TI) stage (ATI), the homologous chromosomes were separated and segregated successively, and the aggregated ADD1 staining at the minus ends of the midzone microtubule bundle structure between segregating chromosomes was weakened to several faintly visible punctate staining. At the MII stage following the emission of the first polar body, the ADD1 signal concentrated at the spindle poles with a distribution similar to that in the MI stage (Fig. 1C). To further validate whether ADD1 functions as a microtubule minus end-binding protein or a component of the acentriolar microtubule-organizing centers (aMTOCs), we simultaneously labeled ADD1, α-tubulin, and γ-tubulin, a well-studied MTOC-specific protein that plays a critical role in microtubule nucleation and spindle formation in mammalian meiotic and mitotic cells [33], in oocytes. As shown in Fig. 1D, ADD1 colocalized with γ-tubulin at the spindle poles in MI and MII oocytes, suggesting that ADD1 may be one of the components of the oocyte microtubule organizing center to regulate spindle assembly. To illustrate the correlation between ADD1 and spindle microtubule dynamics, the effects of spindle-perturbing treatment with taxol and nocodazole on the localization of ADD1 in oocytes at MI and MII stages were investigated. After treatment of taxol, which can promote microtubule assembly and stabilize polymerized microtubules, the microtubule became excessively polymerized, and notably enlarged spindles and multiple asters were noticed in the cytoplasm. ADD1 signals were detected at the spindle poles and the minus ends of the astral microtubule fibers in the taxol-treated oocytes at both MI and MII stages (Fig. 1E). When oocytes were treated with nocodazole, a microtubule-depolymerizing agent, microtubule fibers were entirely disassembled, and no intact spindles were observed in these oocytes. The localization of ADD1 changed from being aggregated at the poles of the bipolar spindle to being dispersed around chromosomes in the nocodazole-treated oocytes at both MI and MII stages (Fig. 1F). These findings indicate that ADD1 is a component of the oocyte microtubule organizing center.
Expression and subcellular localization of S726 phosphorylated ADD1 during mouse oocyte meiotic maturation
Previous studies have shown that ADD1 is distributed on the mitotic spindle [30], whereas S726-phosphorylated ADD1 (p-ADD1) is specifically localized on the centrosome in somatic cells [31]. To examine whether ADD1 is phosphorylated at S726 during oocyte meiotic maturation, the protein expression levels and subcellular localization of p-ADD1 were detected by immunoblotting and immunofluorescent staining with an antibody specific for S726 phosphorylated ADD1 in mouse oocytes, respectively. ADD1 phosphorylation at S726 occurred throughout the meiotic maturation of mouse oocytes. The expression level of p-ADD1 was significantly higher in MII oocytes than in MI and ATI oocytes (n = 3, p < 0.05), while it was not significantly different from GV and GVBD oocytes (n = 3, p > 0.05). In addition, there was no significant difference in the expression of p-ADD1 between MI and ATI oocytes (n = 3, p > 0.05) (Fig. 2A and B). Dynamic changes in relative protein expression levels of p-ADD1 and ADD1 during oocyte meiotic maturation were not identical (Figs. 1A, B, 2A, and B), implying that the post-translational phosphorylation of ADD1 is critical for regulating its function. Immunofluorescence data indicated that the subcellular localization of p-ADD1 was the same as that of ADD1 in the GV, GVBD, MI, and MII oocytes. Strikingly, during the transition from metaphase to telophase, p-ADD1 migrated from the spindle poles to the spindle plus ends and predominantly localized in the spindle midbody, while the signal of p-ADD1 at the minus ends of the spindle was attenuated to several faintly punctate stains, in AT1 oocytes. In contrast, ADD1 demonstrated a weaker or no signal on the spindle midbody in AT1 oocytes (Figs. 1D and 2C). To further investigate the association between p-ADD1 and microtubule assembly, spindle-perturbing drugs were employed. As with the effect of taxol or nocodazole on the subcellular localization of ADD1, taxol treatment of MI and MII oocytes caused p-ADD1 to accumulate at the poles of the enlarged bipolar spindle and the minus end of astral microtubule fibers (Figs. 1F and 2D), whereas nocodazole exposure of MI and MII oocytes resulted in a dispersed distribution of p-ADD1 around the chromosomes (Figs. 1G and 2E). These results imply that p-ADD1 is involved in the microtubule nucleation in mouse oocytes.
Disruption of ADD1 function impairs meiotic cell cycle progression in mouse oocytes
To clarify the function of ADD1 during mouse oocyte meiosis, a morpholino-based gene silencing approach, which provides much better specificity than RNAi, siRNA, and phosphorothioate-based oligos and greatly decreases the chance of catastrophic off-target antisense effects [34], was employed to perturb the function of ADD1. ADD1 protein expression was significantly reduced to approximately 40% in ADD1-morpholino-injected oocytes (MO-ADD1) compared to controls (MO-Control), revealing that ADD1-MO microinjection efficiently downregulated the expression of ADD1 protein in mouse oocytes (Fig. 3A and B). To investigate whether ADD1 disruption disturbs the meiotic cell cycle progression in mouse oocytes, we cultured ADD1-depleted oocytes for maturation in vitro. As shown in Fig. 3C and D, compared with the control group, ADD1 knockdown significantly reduced the proportion of oocytes with the first polar body (64.02 ± 4.81%, n = 122 vs 43.50 ± 3.28%, n = 121), increased the proportion of oocytes in AI or TI stage (3.19 ± 0.41%, n = 122 vs 11.88 ± 3.92%, n = 121).
ADD1 deficiency leads to aberrant spindle assembly and chromosome misalignment
To investigate the effect of ADD1 on spindle assembly and chromosome arrangement, the control- and ADD1-MO-injected oocytes were collected after maturation in vitro for double staining of α-tubulin and chromosomes. As shown in Fig. 3E, a typical barrel-shaped spindle apparatus with well-aligned chromosomes on the equatorial plate was observed in most of the control oocytes. By contrast, ADD1-MO-injected oocytes exhibited a variety of abnormal spindle morphologies, including elongated spindles, spindles with widened poles, multipolar spindles, and chromosomal alignment defects manifested as randomly scattered chromosomes and chromosome bridges. The rates of distorted spindles in the MO-ADD1 group (65.58% ± 10.54%, n = 108) were significantly higher than that in the MO-Control group (35.35% ± 7.68%, n = 103) (p < 0.01, N = 3) (Fig. 3F). Concomitantly, an obvious increase in chromosome misalignment incidence was observed in the ADD1-MO-injected oocytes (60.57% ± 1.74%, n = 165) compared to the control-MO-injected oocytes (34.28% ± 4.79%, n = 176), (p < 0.01, N = 3) (Fig. 3G). Therefore, these results suggest that ADD1 regulates the progression and quality of mouse oocyte meiotic maturation by mediating bipolar spindle assembly and chromosomal alignment.
ADD1 is necessary for the assembly of interpolar microtubules
To further investigate the role of ADD1 in maintaining proper morphology and function of the meiotic spindle, we examined the impact of ADD1 knockdown on the stability of K-fibers and interpolar microtubules in mouse oocytes. To assess K-fibers' stability, we treated MII oocytes, which had been injected with either control- or ADD-MO, ice-cold for 15 min to depolymerize dynamic microtubules within the meiotic spindle with preserving the stable kinetochore-bound microtubules [35, 36]. K-fibers were comparable between the control- and ADD1-MO-injected eggs (p > 0.05, data not shown), which implies that the connections of the k-fibers to the kinetochores and spindle poles were still intact after ADD1 deficiency by approximately 60% in mouse oocytes. To evaluate the stability of interpolar microtubules, which are the most dynamic and abundant subclass of spindle microtubules and comprise the main body of the mature spindle [37], oocytes were exposed to calcium to depolymerize fragmentary or free microtubules within the acentriolar spindle while preserving both K-fibers and stable interpolar microtubules [35, 36]. As shown in Fig. 4A and B, the fluorescence intensity of the stable interpolar microtubules in the ADD1-MO-injected oocytes (1950.16 ± 179.64 A.U., n = 46) was significantly lower than that in control-MO-injected oocytes (2660.85 ± 161.59 A.U., n = 53) (p < 0.01, N = 4). Overall, ADD1 is essential for interpolar microtubule stability in mouse oocyte spindles. In addition, the length and width of the spindles were marginally shorter and wider in the calcium-treated ADD1-depleted oocytes compared to their control oocytes (p > 0.05, N = 3; data not shown).
ADD1 is required for the maintenance of euploidy in MII oocytes
Aberrant spindle assembly and chromosomal misalignment usually lead to aneuploidy, which causes spontaneous abortion, embryonic lethality, and ovarian teratoma [38, 39]. To elucidate whether ADD1 knockdown disrupts chromosome separation and segregation leading to aneuploidy during meiosis in mouse oocytes, we, therefore, analyzed the karyotype of MII oocytes injected with either control- or ADD1-MO by chromosome spreading. As expected, ADD1 disruption significantly increased the incidence of aneuploid with more or less 20 chromosomes, from 25.00% ± 4.81% (n = 26) in oocytes injected with control-MO to 62.39% ± 4.27% (n = 38) in the oocytes injected with ADD1-MO (p < 0.01, N = 3; Fig. 5A, B), suggesting that ADD1 is essential for accurate chromosome segregation during mouse oocyte meiosis.
Expression and subcellular localization of TPX2 during mouse oocyte meiotic maturation
Given that ADD1 phosphorylation at S726 is important for its interaction with TPX2, which is crucial for spindle pole integrity in somatic cells [31], this prompted us to detect the regulatory mechanisms by which ADD1 and TPX2 regulate the acentrosomal spindle assembly in mouse oocytes. To achieve this, we examined the expression and subcellular localization of TPX2 during oocyte meiotic maturation. As shown in Fig. 6A and B, the results of western blotting revealed that the overall trend of TPX2 protein level was a gradual increase with the cell cycle progression of oocyte meiosis. Quantitative analysis of western blot band density manifested that the level of TPX2 protein in MII oocytes was significantly higher than that in GV, GVBD, and ATI oocytes (N = 3, p < 0.05), while there was no significant difference in the level of TPX2 protein among the GV, GVBD and ATI oocytes (N = 3, p > 0.05). The TPX2 protein level in MI oocytes was significantly higher than that in GV and GVBD oocytes (N = 3, p < 0.05), whereas no significant difference was found between MI and ATI oocytes or MI and MII oocytes (N = 3, p > 0.05) (Fig. 6B). To further determine the relationship between the spatial distribution of TPX2 and the meiotic spindle, we double-stained oocytes at different stages with antibodies against the microtubule subunit α-tubulin and TPX2. The data of immunofluorescence demonstrated no obvious TPX2-positive signal in GV oocytes. Around GVBD, TPX2 was concentrated on newly generated spindle microtubules in perichromosomal regions. At the pro-MI or MI stage, TPX2 was localized along the spindle microtubules and was more abundant in spindle poles or minus end of spindle microtubules and less in regions close to aligned chromosomes. TPX2 staining appeared at the minus ends of the midzone microtubule bundle structure around two separate chromosomal clumps at the AI stage. It then moved to a region close to the chromosomes between two separate chromosomal clusters at the TI stage. TPX2 signaling flanked chromosomes in MII oocytes with a distribution similar to that in MI oocytes (Fig. 6C). To elucidate the correlation between TPX2 and meiotic spindle microtubule dynamics, the effects of the microtubule polymerization stabilizer taxol and the microtubule depolymerizing agent nocodazole on the subcellular localization of TPX2 in the MI and MII oocytes were investigated. TPX2 signal was detected at the minus end of both the enlarged spindle and astral microtubules in oocytes exposed to taxol (Fig. 6D). Whereas when oocytes were treated with nocodazole, the localization of TPX2 changed from being aggregated at the poles of the bipolar spindle to being randomly distributed on one or both sides of the chromosomes (Fig. 6E). These findings confirm that TPX2 is associated with microtubule nucleation and bipolar spindle assembly in mouse oocytes.
Loss of TPX2 results in oocyte meiotic cell cycle arrest and bipolar spindle assembly failure
To explore the function of TPX2 in mouse oocyte meiosis, we investigated the effects of TPX2 depletion via TPX2-targeting morpholino (MO-TPX2) on oocyte meiotic cell cycle progression, spindle assembly, and chromosomal alignment. Compared with MO-Control-injected oocytes, TPX2 protein expression was significantly reduced in MO-TPX2-injected oocytes, indicating that MO-TPX2 microinjection can effectively knockdown the expression level of TPX2 protein in mouse oocytes (Fig. 7A and B). Cell cycle assays (Fig. 7C and D) showed that 67.57 ± 5.12% (n = 99) oocytes in the MO-Control-injected group could develop to the MII stage, while in the MO-TPX2-injected group, only 28.11 ± 11.44% (n = 99) oocytes could develop to MII stage, and the remaining 62.62 ± 12.38% (n = 99) oocytes were arrested at or before MI stage. Statistical analysis demonstrated that TPX2 depletion resulted in mouse oocyte meiotic arrest in the MI stage (N = 3, p < 0.01). Spindle immunofluorescence assay revealed that in the MO-TPX2-injected group, 62.32 ± 14.22% (n = 86) oocytes had no or little α-tubulin signal, 29.30 ± 8.73% oocytes had monopolar aster-like spindle, and only 8.39 ± 7.20% oocytes had a normal-shaped spindle, which was significantly lower than the proportion of oocytes with a normal-shaped spindle in the MO-Control-injected group (77.07 ± 21.17%, n = 88) (N = 3, p < 0.01; Fig. 7E and F).
TPX2 regulates the spindle assembly in mouse oocytes by mediating the expression and subcellular localization of S726-phosphorylated ADD1
To further reveal the regulatory relationship between ADD1 and TPX2 during spindle assembly in mouse oocytes, we examined the effect of the knockdown of ADD1 or TPX2 on each other's subcellular localization and expression levels. Our data demonstrated that the deficiency of ADD1 protein by approximately 60% had no or litter effect on the subcellular localization and the levels of TPX2 protein (data not shown). Our criteria for judging the effect of TPX2 knockdown on the localization of ADD1 or p-ADD1 were as follows: If add1 or p-ADD1 fluorescence signals existed on both sides of the chromosomes, and the sum of the fluorescence intensity on one side was comparable to the sum of the fluorescence intensity on the other side, it was considered that the localization of add1 or p-ADD1 is normal. Otherwise, add1 or p-ADD1 localization was considered abnormal if any of the conditions above were not met, or there was no visible fluorescent signal in the whole cell. Interestingly, TPX2 depletion disrupted the subcellular localization of p-ADD1 (Fig. 7E and G), reduced the expression levels of p-ADD1 (Fig. 7H, I), increased the protein content of ADD1 (Fig. 7J, K), but had no apparent effect on the subcellular localization of ADD1 (Additional file 1: Fig. S1A and S1B). These results suggest that the biological function of ADD1 requires its phosphorylation by TPX2 at S726 during mouse oocyte meiotic maturation.