ZSCAN4 functions as a safeguard to maintain centromere integrity during oocyte meiosis

Abstract

Background

Centromeres play a vital role in ensuring accurate chromosome segregation during meiosis by serving as the foundation for kinetochore assembly and microtubule attachment. In oocytes, maintaining centromere integrity is particularly critical due to the extended arrest period prior to meiotic resumption. However, the molecular safeguards that preserve centromere structure and function throughout oocyte maturation remain poorly understood.

Results

Here, we identify ZSCAN4 as an essential regulator of centromere integrity during mouse oocyte meiosis. ZSCAN4 depletion leads to a marked reduction in key centromeric and kinetochore proteins, including CENP-A, accompanied by aberrant centromere stretching under spindle tension. Mechanistically, ZSCAN4 promotes pericentromeric H3K9me3 enrichment, facilitating proper chromatin compaction and chromosome alignment. Moreover, ZSCAN4 contributes to genomic stability by mediating the chromosomal recruitment of the CIP2A complex in response to DNA damage during meiotic progression.

Conclusions

These findings establish ZSCAN4 as a critical factor in preserving centromere structure and function during oocyte meiosis, with potential implications for female reproductive health and developmental competence.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13059-025-03687-3.

Background

Centromeres are specialized chromosomal regions that play a critical role in accurate chromosome segregation during cell division [1, 2]. Centromeric DNA is organized into a high-order chromatin structure that forms what is called primary centromeric constriction. This region is epigenetically marked by the incorporation of specialized centromere-specific histone-variant CENP-A, which replaces the canonical histone H3 at centromeric nucleosomes. CENP-A localization to centromeres creates a platform for assembling kinetochores, large protein complexes that serve as attachment points for spindle microtubules. The interaction between kinetochores and spindle microtubules facilitates the proper alignment and segregation of chromosomes during mitosis and meiosis [3]. However, due to its repetitive sequences, centromeric DNA is intrinsically unstable and susceptible to the formation of secondary structures that could potentially induce replication fork stalling, topological problems, and high levels of recombination [4]. Therefore, maintaining centromere integrity is of paramount importance to ensure accurate transmission of genetic information during cell division and prevent errors in chromosome segregation.

During mitotic and meiotic entry, centromeres undergo compaction as they become enriched with cohesin and condensin, two ring-shaped complexes of structural maintenance of chromosomes (SMC) proteins [5–8]. While centromeric cohesin tethers sister chromatids during cell division, condensin provides stiffness to the centromeres [5, 9–11]. In addition to these complexes, pericentromeric heterochromatin, characterized by histone H3 lysine 9 trimethylation (H3K9me3), plays pivotal role in chromatin compaction and kinetochore integrity. This compaction enables accurate biorientation of chromosomes, a prerequisite for faithful chromosome segregation. The centromeric and surrounding pericentromeric regions form large domains of constitutive heterochromatin enriched with H3K9me3 [3, 8, 12, 13]. Although the importance of SMC complexes and H3K9me3 in maintaining centromere integrity is well established, the precise mechanisms by which they regulate centromeric chromatin remain a fundamental question in chromosome biology.

Zinc finger and SCAN domain-containing 4 (ZSCAN4) is a highly conserved transcription factor found in various organisms, including mice and humans [14–16]. Because of its specific expression during the first cycle of de novo transcription during zygotic genome activation (ZGA) and at the subpopulation of embryonic stem cells [16–18], ZSCAN4 has been implicated in the control of early mammalian embryogenesis, regulation of pluripotency, and maintenance of genome integrity in embryonic stem cells [16, 18]. Moreover, ZSCAN4 has been shown to regulate telomere length in embryonic stem cells [16, 17, 19]. Recently, ZSCAN4 has emerged as a key player in genome integrity by maintaining a nucleosome-rich state, safeguarding repetitive DNA [20]. Furthermore, ZSCAN4 has been found to interact with factors involved in the DNA damage response (DDR), promoting DNA repair and preventing the accumulation of DNA lesions [20–22]. Despite its pleiotropic roles in multiple cellular processes, the precise function of ZSCAN4 and the mechanism of its cellular effects remain largely unknown.

Mammalian oocytes arrest for a prolonged period at the first meiotic prophase, which is known as the germinal vesicle (GV) stage. At puberty, a hormonal surge triggers the resumption of meiosis, leading to meiotic maturation and progression to the metaphase of the second meiotic division (MII) [23]. Unlike cycling somatic cells, where CENP-A is deposited on newly replicated centromeric DNA during the S phase and replenished in the G1 phase [24–26], mammalian oocytes establish CENP-A nucleosomes at birth and maintain them throughout their lifespan without further assembly [27, 28]. This exceptional stability is critical for maintaining oocytes quality and enduring successful meiotic division, yet the underlying molecular mechanisms remain poorly understood. In this study, we investigated the role of ZSCAN4 in maintaining CENP-A centromeres and its contribution to centromere integrity during oocyte meiosis. We found that ZSCAN4 is specifically localized at centromeres and acts to protect centromere integrity during oocyte meiosis. Moreover, ZSCAN4 levels at centromeres increased in response to DNA damage, and ZSCAN4 depletion impaired DNA repair by disturbing chromosomal recruitment of the CIP2A complex. Our data are the first evidence that ZSCAN4 plays an essential role as a safeguard to maintain centromere integrity during meiotic maturation in mouse oocytes.

Results

ZSCAN4 is a novel centromere protein indispensable for meiotic maturation in oocytes

ZSCAN4, a protein widely recognized as a marker for the ZGA in early embryos, is expressed in GV oocytes [18, 29], implying a role in oocyte meiosis. Therefore, to investigate the function of ZSCAN4 in oocytes, we examined its expression and localization during meiotic maturation. We found that ZSCAN4 is constantly expressed in oocytes throughout meiotic maturation, with specific localization to the centromeres (Fig. 1A, Additional file 1: Fig. S1A–B). The centromeric localization of ZSCAN4 prompted us to further investigate its function during meiotic maturation. To that end, we injected GV-arrested oocytes with either a scrambled control morpholino (Control MO) or a ZSCAN4-targeting morpholino (ZSCAN4 MO). Immunoblot analysis revealed that ZSCAN4 MO reduced endogenous ZSCAN4 levels by approximately 75% compared with the control MO (Fig. 1B–C). Consistently, immunostaining of chromosome spreads showed an ~ 70% reduction in ZSCAN4 intensity at the centromeres following ZSCAN4 MO injection (Additional file 1: Fig. S1C). We next examined the effects of ZSCAN4 depletion on meiotic maturation. ZSCAN4-depleted oocytes exhibited a significant delay in GV breakdown (GVBD) and a markedly reduced the rate of polar body extrusion (PBE) at 16 h after meiotic resumption (Fig. 1D–E). In addition, these oocytes at the metaphase I (MI) stage exhibited severe chromosome displacement from the metaphase plate (Fig. 1F).

Fig. 1.

ZSCAN4 is a centromeric protein related to the proper maturation of mouse oocytes. A Subcellular localization of ZSCAN4 during mouse oocyte meiotic maturation. Oocytes were collected after 8 and 16 h of culture, corresponding to Metaphase I (MI) and Metaphase II (MII), respectively. Immunofluorescence staining was performed using ZSCAN4 and ACA antibodies. Scale bar, 10 μm. B, C Immunoblot analysis of ZSCAN4 expression in control and ZSCAN4-depleted oocytes. Oocytes were collected at the MI stage and subjected to immunoblot analysis with ZSCAN4 antibody. Each lane includes 50 oocytes, and α-tubulin was used as a loading control. Quantification of ZSCAN4 levels is shown in (C). Data are presented as the mean ± SEM of three independent experiments. ***p < 0.0001. D Quantification of the GVBD rate in ZSCAN4-depleted conditions. The GVBD rate was measured every 1 h from GV oocytes. Data are presented as the mean ± SEM of four independent experiments. E Quantification of the PBE rate in ZSCAN4-depleted conditions. The PBE rate was measured as the proportion of oocytes that had been incubated for 16 h and had a visible polar body identified on a DIC image. Data are presented as the mean ± SEM of four independent experiments. ****p < 0.00001. F Representative images and quantification of DNA area at the MI stage. DNA area was measured as the area of a circle that includes all the aligned chromosomes. Data are presented as the mean ± SEM of three independent experiments. **p < 0.001. G Quantification of the PBE rate with ZSCAN4 depletion and treatment with the MPS1 inhibitor AZ3146. Data are presented as the mean ± SEM of two independent experiments. ns, not significant. **p < 0.001. H Representative images of cold shock–treated control and ZSCAN4-depleted oocytes stained with acetylated alpha-tubulin and ACA antibodies at the MI stage. Scale bar, 10 μm. I Quantification of the rate of kMT attachment. Each centromere was scanned to determine whether it was attached to tubulin. Data are presented as the mean ± SEM of three independent experiments

To validate this phenotype using an independent approach, we employed the Trim-Away technique to acutely deplete ZSCAN4 protein in GV-stage oocytes [30]. We found that Trim-Away efficiently reduced ZSCAN4 levels by approximately 60% within 2 h (Additional file 1: Fig. S2A–C). Similar to the morpholino-mediated knockdown, Trim-Away–mediated depletion of ZSCAN4 also resulted in severe chromosome displacement, confirming that the observed phenotype is specifically due to ZSCAN4 depletion (Additional file 1: Fig. S2D–E). To assess the consequence of this chromosome misalignment, we evaluated aneuploidy in MII oocytes and observed an increase in aneuploidy incidence upon ZSCAN4 depletion, indicating defects in chromosome segregation (Additional file 1: Fig. S2F–G).

Chromosome misalignment during meiosis is often associated with unattached kinetochores, which activate the spindle assembly checkpoint (SAC) and lead to MI arrest [31]. To test whether SAC activation contributed to the increased MI arrest observed in ZSCAN4-depleted oocytes, we inhibited SAC using the MPS1 inhibitor AZ3146. However, SAC inactivation did not rescue the MI arrest (Fig. 1G). Further analysis revealed that kinetochore-microtubule (kMT) attachments were unaffected by ZSCAN4 depletion (Fig. 1H–I). Moreover, we found that spindle morphology and pole integrity remained intact, despite a modest decrease in overall spindle intensity (Additional file 1: Fig. S3). These findings indicated that ZSCAN4 is a novel centromere protein essential for regulating chromosome dynamics during meiotic maturation in oocytes.

ZSCAN4 depletion disrupts CENP-A centromere integrity and causes centromere stretching

Given the centromeric localization of ZSCAN4, we performed a detailed analysis of centromere morphology in oocytes following ZSCAN4 depletion. We found that centromeres exhibited an unusually elongated and stretched appearance with sharp ends following ZSCAN4 depletion (Fig. 2A). To quantify these structural changes, we measured centromere circularity, which ranges from 0 (highly elongated or irregular) to 1 (perfectly circular) (Fig. 2B). ZSCAN4 depletion significantly decreased centromere circularity, indicating stretching (Fig. 2C). Additionally, the intensity of ACA, a marker of centromere, was markedly reduced (Fig. 2D). Similar results were obtained when ZSCAN4 was depleted by Trim-Away (Additional file 1: Fig. S2H–J), reinforcing the specificity of the phenotype and validating the knockdown effect, thereby strengthening our findings.

Fig. 2.

ZSCAN4 depletion causes centromere stretching due to microtubule pulling forces. A Representative images of cold shock–treated control and ZSCAN4-depleted oocytes stained with acetylated alpha-tubulin and ACA antibodies at the MI stage. Scale bar, 10 μm. B Schematic diagram depicting centromere circularity. C–D Quantification of ACA circularity and ACA intensity. Data are presented as the mean ± SEM of three independent experiments. ***p < 0.0001. ****p < 0.00001. E Representative live images of control and ZSCAN4-depleted oocytes targeting major satellite repeat sequences and H2B histone clusters. Scale bar, 10 μm. F-G Quantification of fluorescence intensity and area in major satellite repeat. The total major satellite signal was measured as the centromere area. Data are presented as the mean ± SEM of three independent experiments. H Quantification of the average time to undergo GVBD and establish centromere foci. Data are presented as the mean ± SEM of three independent experiments. I Representative images of control and ZSCAN4-depleted oocytes treated with DMSO or nocodazole (NOC) at the MI stage. After treatment, nocodazole was washed out with nocodazole-free medium for 2 h for recovery. Oocytes were stained with acetylated alpha-tubulin and ACA antibodies. Scale bar, 10 μm. J Quantification of ACA circularity. Data are presented as the mean ± SEM of three independent experiments. ns, not significant. ****p < 0.00001. K Schematic diagram depicting how ZSCAN4 protects the centromere from microtubule-derived pulling forces

To further investigate this phenomenon, we tracked centromeres in live oocytes during meiotic maturation using fluorescent transcription activator–like effector (TALE) constructs targeting major satellite repeat sequences that are enriched in pericentromeric regions [32]. In control oocytes, centromeres became condensed and compacted in each individualized bivalent after GVBD. However, in ZSCAN4-depleted oocytes, the fluorescence intensity of centromeres at the GV stage decreased, the formation of individualized bivalents was delayed after GVBD, and the centromeres became stretched, with an increase in overall centromere area during meiotic maturation (Fig. 2E–H, Additional file 2: Video S1–S2).

The observation that centromere stretching was directed toward the spindle poles led us to hypothesize that pulling forces exerted by microtubules attached to kinetochores are responsible for this phenomenon. Indeed, we observed that centromere stretching was partially rescued after destabilizing microtubules with nocodazole, and when nocodazole was washed out, centromere stretching reappeared concomitant with microtubule polymerization (Fig. 2I–J). It is also notable that the stretched centromeres frequently observed in ZSCAN4-depleted oocytes were not detectable in chromosome spreads, supporting the notion that microtubule pulling forces are responsible for centromere and kinetochore stretching in ZSCAN4-depleted oocytes (Additional file 1: Fig. S4). Therefore, our results suggest the critical role of ZSCAN4 in maintaining centromere integrity by counteracting the stretching induced by microtubule-generated forces (Fig. 2K).

CENP-A is essential for maintaining centromere identity and function, prompting us to investigate whether ZSCAN4 contributes to stabilizing CENP-A at centromeres during oocyte meiosis. We found that ZSCAN4 depletion caused a significant decrease in CENP-A intensity and circularity at the centromeres (Fig. 3A–C). Given that the centromere serves as a platform for kinetochore assembly, we investigated whether ZSCAN4 depletion influences kinetochore assembly. To that purpose, we examined levels of the kinetochore proteins HEC1 and BubR1 and observed a significant decrease in their intensity and circularity after ZSCAN4 depletion (Fig. 3D–I). These results suggest that ZSCAN4 is essential for the assembly and maintenance of centromeres and kinetochores in oocytes. To further clarify our findings, we performed chromosome spreading and validated the dissociation of CENP-A, HEC1, and BubR1 at centromeres after ZSCAN4 depletion (Additional file 1: Fig. S4A–I). Overall, our findings reveal that ZSCAN4 depletion leads to the dissociation of CENP-A from centromeres, disrupting the recruitment of key kinetochore proteins HEC1 and BubR1. These results suggest the critical role of ZSCAN4 in preserving centromere integrity and facilitating proper kinetochore assembly.

Fig. 3.

ZSCAN4 depletion impairs the recruitment of CENP-A, HEC1, and BubR1 at centromeres and kinetochores. A, D, G Representative images of control and ZSCAN4-depleted oocytes stained with CENP-A (or HEC1 or BubR1) at the MI stage. Scale bar, 10 μm. B, E, H Quantification of CENP-A (or HEC1 or BubR1) circularity. Data are presented as the mean ± SEM of two independent experiments. ****p < 0.00001. C, F, I Quantification of CENP-A (or HEC1 or BubR1) intensity. Data are presented as the mean ± SEM of two independent experiments. ****p < 0.00001

ZSCAN4 depletion impairs pericentric heterochromatin and disrupts chromosome architecture

The proper assembly of pericentric heterochromatin, marked by H3K9me3, is crucial for establishing and maintaining centromere integrity [13, 33, 34]. Therefore, we investigated whether the centromere stretching observed in ZSCAN4-depleted oocytes is associated with impaired pericentric heterochromatin assembly. Although ZSCAN4 depletion did not abolish H3K9me3 distribution along entire chromosomes, its intensity at the pericentromere was significantly reduced in ZSCAN4-depleted oocytes. Notably, pericentric H3K9me3 signals were elongated toward the chromosome ends, similar to centromere stretching, after ZSCAN4 depletion, indicating compromised pericentric heterochromatin compaction (Fig. 4A–E). The reduction in pericentric H3K9me3 intensity was further validated in chromosome spreads (Additional file 1: Fig. S4J–L). It is also notable that pericentromere stretching was absent in chromosomes spreads, corroborating the notion that microtubule pulling forces are responsible for centromere stretching in ZSCAN4-depleted oocytes (Additional file 1: Fig. S4J–L). Therefore, our results suggest that ZSCAN4 depletion decreases H3K9me3 levels at the pericentric region, leading to a reduction in centromere compaction and integrity.

Fig. 4.

ZSCAN4 depletion disrupts pericentric heterochromatin assembly and chromosome architecture. A Representative images of control and ZSCAN4-depleted oocytes stained with H3k9me3 antibody at the MI stage. Scale bar, 10 μm. B Plot graph depicting H3K9me3 intensity from one chromosome end (−20) to the opposite end (20) (yellow arrow). Ten representative chromosomes from control and ZSCAN4-depleted oocytes were selected for the plot. C Quantification of ratio intensity of H3k9me3 at centromeres over each chromosome (centromere/chromosome). Data are presented as the mean ± SEM of two independent experiments. ****p < 0.00001. D Quantification of centromeric H3K9me3 length (white arrow). Data are presented as the mean ± SEM of two independent experiments. ****p < 0.00001. E A scatter plot of control and ZSCAN4-depleted oocytes depicting centromeric H3K9me3 intensity relative to centromeric H3K9me3 length. Each symbol represents an individual centromere. F, J Representative chromosome spread images of control and ZSCAN4-depleted oocytes stained with SMC3 (or SMC4) and ACA antibodies at the MI stage. Scale bar, 10 μm. (G, K) Plot graph depicting SMC3 (or SMC4) and ACA intensities from one chromosome end (−20) to the opposite end (20) (yellow arrow). H, L Quantification of the SMC3 (or SMC4) intensity of each chromosome. Data are presented as the mean ± SEM of three independent experiments. **p < 0.001. I, M Quantification of SMC3 (or SMC4) intensity on centromeres. Data are presented as the mean ± SEM of three independent experiments. ***p < 0.0001. ****p < 0.0001

Because cohesin and condensin are fundamental factors in establishing the robust structure of chromosomes and are enriched at pericentromeres during both mitosis and meiosis [5–8], we examined whether the loss of centromere integrity caused by ZSCAN4 depletion affects the recruitment of these complexes to centromeric heterochromatin regions. We found that cohesin and condensin levels were significantly decreased in ZSCAN4-depleted oocytes (Additional file 1: Fig. S5). Moreover, analysis of chromosome spreads revealed a significant decrease in the abundance of cohesin and condensin not only at the chromosome arms, but also at the centromere region after ZSCAN4 depletion (Fig. 4F–M). Therefore, our data suggest that ZSCAN4 plays a critical role in H3K9me3 levels at pericentric heterochromatin, facilitating the recruitment of cohesin and condensin complexes and ultimately ensuring proper chromosome architecture and segregation during oocyte meiosis.

Given the observed reduction in the intensity of centromeres in ZSCAN4-depleted oocytes at the GV stage (Fig. 2E), we wondered whether the loose condensation of centromeres in mouse oocytes originates during prophase I. To investigate that, we examined centromeres at the GV stage of oocytes and found reduced centromere intensity and fewer foci in ZSCAN4-depleted oocytes at this stage (Fig. 5A–C). A 3D analysis further showed that ZSCAN4 depletion led to a significant reduction in the volume of centromeres in decondensed chromosomes at the GV stage (Fig. 5D–E, Additional file 2: Video S3–S4). These findings indicate that ZSCAN4 is critical for maintaining centromere integrity during the prolonged GV arrest. Consequently, ZSCAN4 depletion likely disrupts chromosome reorganization during meiotic maturation by impairing pericentric heterochromatin assembly, ultimately affecting chromosome condensation and cohesion.

Fig. 5.

ZSCAN4 is essential for maintaining centromere integrity during prophase I arrest. A Representative images of control and ZSCAN4-depleted oocytes stained with ACA and anti-CENP-A antibodies at the GV stage. Scale bar, 10 μm. B Quantification of the number of CENP-A foci. Data are presented as the mean ± SEM of two independent experiments. ****p < 0.00001. C Quantification of CENP-A foci intensity. Data are presented as the mean ± SEM of two independent experiments. ****p < 0.00001. D Representative 3D reconstruction of CENP-A spots originally shown in Fig. 5A. Scale bar, 10 μm. E Quantification of CENP-A foci volume. Data are presented as the mean ± SEM of two independent experiments. ***p < 0.0001

ZSCAN4 depletion impairs DNA repair by disrupting the chromosomal relocation of CIP2A complexes

Recently, centromeres have emerged as crucial structural hubs for chromosomal recruitment of CIP2A-MDC1-TOPBP1 complexes from spindle poles in response to DNA damage during oocyte meiosis [35]. Given the essential role of ZSCAN4 in maintaining centromere integrity, we sought to determine whether ZSCAN4 is involved in the DDR in oocytes. To investigate that, we introduced DNA double-strand breaks (DSBs) in MI stage oocytes using etoposide (ETP) and examined ZSCAN4 levels. We found that ZSCAN4 signals increase significantly on chromosomes, particularly at the centromeres, following DSB induction (Additional file 1: Fig. S6). Interestingly, this ETP-induced increase in ZSCAN4 levels was blocked by PLK1 inhibition but not ATM inhibition, suggesting that PLK1 activity regulates ZSCAN4 recruitment on chromosomes in response to DNA damage in oocytes (Fig. 6A–B).

Fig. 6.

ZSCAN4 depletion impairs DNA repair by disrupting chromosomal relocation of the CIP2A-MDC1-TOPBP1 complex. A Representative images of chromosome spread stained with ZSCAN4 antibody in control and ZSCAN4-depleted oocytes after treating ATM (ATM i) or PLK1 (PLK1 i) inhibitors. Oocytes were exposed to etoposide (ETP) to induce DSBs. Control oocytes were treated with DMSO. Scale bar, 10 μm. B Quantification of ZSCAN4 intensity. Data are presented as the mean ± SEM of two independent experiments. ns, not significant. **p < 0.001. ****p < 0.00001. C Representative images of control and ZSCAN4-depleted oocytes stained with phospho-MDC1 and CIP2A antibodies after ETP treatment and recovery (ETP + R). For recovery, ETP was washed out using ETP-free medium for 2 h. Scale bar, 10 μm. D–E The ratio of p-MDC1 and CIP2A intensities at chromosomes over the spindle poles. Data are presented as the mean ± SEM of three independent experiments. ns, not significant. *p < 0.01. ***p < 0.0001. F Representative images of chromosome spread showing TUNEL signals in control and ZSCAN4-depleted oocytes after ETP treatment and recovery. Scale bar, 10 μm. G Quantification of TUNEL intensity. Data are presented as the mean ± SEM of three independent experiments. ns, not significant. ***p < 0.0001

To further investigate the role of ZSCAN4 in DSB repair in oocytes, we examined the chromosomal recruitment of CIP2A and MDC1 complexes. In control oocytes, CIP2A and MDC1 signals appeared on chromosomes after ETP treatment, and they decreased after recovery for 2 h in ETP-free medium (Fig. 6C–E). In striking contrast, ZSCAN4-depleted oocytes exhibited impaired chromosomal relocation of CIP2A and MDC1 complexes after ETP treatment. Notably, however, CIP2A and MDC1 signals did appear on chromosomes after recovery from ETP (Fig. 6C–E). Consistent with this delayed recruitment of CIP2A and MDC1 complexes, TUNEL signals remained high on chromosomes after ZSCAN4 depletion and did not decrease even after recovery (Fig. 6F–G). These results suggest that impaired centromere integrity in ZSCAN4-depleted oocytes perturbs recruitment of the DNA repair complex to chromosomes, leading to defects in DNA repair. Taken together, our results suggest that ZSCAN4 is essential for preserving centromere integrity and safeguarding against DNA damage, maintaining genomic integrity during oocyte meiosis.

Discussion

In this study, we identified ZSCAN4 as a novel centromeric protein that plays a critical role in maintaining centromere integrity during meiotic maturation in mouse oocytes. Our findings show that ZSCAN4 depletion leads to a coordinated reduction in key centromeric and kinetochore-associated proteins, including CENP-A, HEC1, and BubR1, along with defects in chromosome condensation, cohesion, and pericentromeric heterochromatin formation (Fig. 7). These results establish ZSCAN4 as an essential factor in sustaining the structural integrity of the centromere and ensuring faithful chromosome segregation during oocyte meiosis.

Fig. 7.

ZSCAN4 functions as a safeguard to maintain centromere integrity during oocyte meiosis. A schematic model illustrating the role of ZSCAN4 in maintaining centromeric integrity during meiosis. In mouse oocytes, ZSCAN4 facilitates proper CENP-A loading and recruitment of kinetochore proteins. It also maintains pericentric heterochromatin by recruiting H3K9me3, cohesin, and condensin, ensuring centromere compaction. Thus, ZSCAN4 is vital for centromere and kinetochore formation during oocyte meiosis. Collectively, ZSCAN4 is required to maintain centromere integrity by preventing the centromere from elongating and stretching under microtubule forces

Centromeres are dynamic chromatin regions that undergo compaction at the onset of meiosis and mitosis, forming a higher-order structure necessary for chromosome cohesion and condensation [1]. This process involves the recruitment of cohesin and condensin complexes, which stabilize chromosomal architecture, particularly under tension from spindle microtubules [36]. In somatic cells, centromeric proteins are continuously replaced throughout the cell cycle to maintain centromere function. In contrast, mammalian oocytes establish centromere integrity at birth and maintain it throughout their lifespan without further protein assembly [27, 28]. This exceptional stability is crucial for ensuring successful meiotic division and maintaining oocyte quality over time. While our experiments involved short-term perturbations, they served as effective models to identify key factors necessary for the long-term maintenance of centromere integrity. Oocytes, unlike cycling somatic cells, do not undergo continuous centromere protein turnover. By examining the immediate effects of ZSCAN4 depletion, we inferred its essential role in sustaining centromere structure over extended periods, thereby providing insights into how centromere stability is preserved throughout the reproductive lifespan.

One key observation from our study is that ZSCAN4 depletion disrupts centromere stability, as evidenced by impaired chromosome condensation and cohesion at the onset of GVBD. Consequently, centromeres became decondensed and structurally weakened, making them prone to abnormal stretching during spindle formation [28, 37]. Our live imaging analysis and nocodazole treatment further supported this hypothesis. Disrupting microtubules partially rescued condensation defects and reduced centromere stretching, indicating that compromised centromeric architecture, rather than primary microtubule attachment defects, is responsible for the stretching phenotype. Despite these structural abnormalities, our analysis showed that kMT attachments were largely preserved in ZSCAN4-depleted oocytes. Although we did not observe a statistically significant reduction in attachment frequency, we acknowledge that our current analysis may not fully capture subtle or unstable interactions. Moreover, spindle intensity was modestly reduced upon ZSCAN4 knockdown, and HEC1 levels at kinetochores were significantly decreased, suggesting potential compromises in the quality or stability of kMT attachments. It is possible that residual HEC1 protein is sufficient to support a minimal threshold of attachment, or that compensatory mechanisms maintain basic attachment function despite weakened kinetochore structure. However, this attachment may not be sufficiently robust to ensure faithful chromosome segregation, as indicated by the increased incidence of aneuploidy. These interpretations highlight the need for future studies using higher-resolution and dynamic assays to assess the mechanical robustness of kMT attachments under compromised centromere conditions.

Our findings also revealed a significant reduction in BubR1 localization at kinetochores in ZSCAN4-depleted oocytes. While BubR1 is known to decrease upon stable kMT attachments [38], previous studies have shown that loss of centromere or kinetochore integrity can also reduce BubR1 recruitment independently of attachment status [39, 40]. Thus, we interpret the reduction in BubR1 as a consequence of impaired kinetochore assembly secondary to centromere disruption. Notably, despite this reduction, SAC function appeared to remain at least partially intact. This is consistent with previous reports demonstrating that partial BubR1 depletion does not abolish SAC activity [41, 42]. It is also noteworthy that cytoplasmic BubR1 not targeting kinetochores could rescue impaired kMT attachment in BubR1 knockout oocytes [43], raising the possibility that kMT attachments in ZSCAN4-depleted oocytes may be maintained, at least in part, by a cytoplasmic pool of BubR1. Nevertheless, we acknowledge that our current dataset may not fully resolve the functional consequences of reduced BubR1 levels, and future studies employing more sensitive SAC activity assays will be required to further clarify this relationship.

Furthermore, we observed defective pericentric heterochromatin establishment, marked by reduced H3K9me3 levels in ZSCAN4-depleted oocytes. Although ZSCAN4 localizes at the centromere, its depletion impacts broader pericentric domains. One possible explanation is that stable centromeric nucleosomes, including those containing CENP-A, provide structural support for recruiting chromatin-modifying enzymes such as Suv39h, necessary for H3K9me3 deposition [13, 44–46]. However, further research is needed to elucidate whether ZSCAN4 directly interacts with these enzymes or influences pericentric heterochromatin maintenance through structural stabilization alone.

Once considered to be passive regions of the genome, centromeres have emerged as active players in the DDR. The accumulation of DNA damage at centromeres triggers a cascade of signaling events that induce cell cycle arrest, DNA repair, and chromatin remodeling [47, 48]. Recent studies have demonstrated that unique chromatin features and protein compositions at centromeres contribute to the recruitment and activation of DDR factors [4, 48], and dysfunction of DDR signaling at centromeres has been implicated in genome instability [35, 49, 50]. In line with that notion, we observed an increased level of ZSCAN4 at centromeres following DNA damage, which suggests that ZSCAN4 plays a critical role in centromere stability and integrity in response to DNA damage. Considering that ZSCAN4 protects repetitive DNA by maintaining a nucleosome-rich state [20], it is reasonable to postulate that ZSCAN4 prevents broken centromeres from separating by structurally compacting chromosomes. In addition to its role in centromere compaction in response to DNA damage, ZSCAN4 depletion impairs the chromosomal recruitment of CIP2A complexes, suggesting that ZSCAN4 actively participates in DNA damage repair. Consistent with our observations, ZSCAN4 expression has been shown to be upregulated in response to DNA damage induced by genotoxic chemotherapeutic drugs in human stromal cells [51]. Therefore, ZSCAN4 is likely to act as a safeguard to maintain centromere integrity against DNA damage. It is also notable that ZSCAN4 has been shown to be transiently expressed in several cancer cell lines [52, 53]. Given that damaged centromeres are frequently observed in cancer cells [4], it would be interesting to determine a potential link between ZSCAN4 expression and centromere fragility in cancer cells. In addition to cancers, centromere fragmentation has been observed in aged oocytes [54], and reduced cohesin levels have been demonstrated at centromeres and chromosome arms with oocyte aging [55]. This decline in cohesin levels and centromere integrity in aged oocytes is considered to weaken sister chromatid cohesion, thereby increasing the risk of chromosome missegregation. Therefore, it is plausible to speculate that ZSCAN4 expression is associated with oocyte aging. Further studies are required to clarify the precise roles of ZSCAN4 in cancers and aged oocytes.

Conclusions

Extensive genome-wide studies have greatly contributed to our understanding of the critical role of ZSCAN4 in the pluripotency of stem cells. However, most of those studies have focused on its function as a transcription factor, and its involvement in cellular processes associated with chromosome dynamics during cell division has remained largely unexplored. In this study, we demonstrated for the first time that ZSCAN4 is specifically localized at centromeres and functions as a safeguard to maintain centromere integrity during meiotic maturation in oocytes. These findings underline the pivotal role of ZSCAN4 in preserving the structural and functional integrity of centromeres, ensuring the fidelity of chromosome segregation during oocyte meiosis.

Methods

Oocyte collection and culture

Three- to four-week-old female CD1 mice sourced from a local company (Koatech, Korea) were used in all experiments. All animal care and use procedures complied with institutional guidelines and were approved by the Institutional Animal Care and Use Committee of Sungkyunkwan University (approval ID: SKKUIACUC2023-09–31-1). Female mice were injected intraperitoneally with 5 IU of pregnant mare serum gonadotropin 46–48 h before oocyte isolation. Fully grown GV oocytes were retrieved from ovarian follicles, the cell cycle being arrested in M2 medium (M7167, Sigma) supplemented with 200 μM 3-isobutyl-1-methylxanthine (IBMX; I5897, Sigma). The selected oocytes were washed three times in IBMX-free M2 medium under mineral oil (M5310, Sigma) and incubated at 37 °C in a 5% CO2 atmosphere for 0, 8, or 16 h to obtain GV, MI, and MII stage oocytes, respectively.

Chemical and cold shock treatment

MI oocytes were incubated in M2 medium containing 20 μg/ml nocodazole (M1404, Sigma) for 15 min to depolymerize spindle microtubules. DNA damage was induced by treating MI oocytes to 50 μg/ml ETP (E1383, Sigma) for 30 min. After treatment with nocodazole or ETP, oocytes were washed and cultured in fresh M2 medium for 2 h to allow recovery, if necessary. To inhibit ATM and PLK1, oocytes were treated with 10 μM ATM inhibitor (KU55933, Selleckchem) and 200 nM PLK1 inhibitor (BI2536, Selleckchem) for 2 h. Control oocytes were treated with an equivalent volume of DMSO.

For analysis of kMT attachment, MI oocytes were incubated in an ice-cold M2 medium for 8 min immediately before fixation.

Plasmid construct, RNA preparation, and morpholino

Full-length cDNA encoding TRIM21 was purchased from the Korea Human Gene Bank and subcloned into the pRN3-mCherry vector. pTALYM3B15 clones for TALE-mClover targeting the mouse major satellite sequence were acquired from Addgene (#47,878). The pRN3-H2B-mCherry plasmid had been previously generated in an earlier study [56]. According to the manufacturer's instructions, all mRNAs were transcribed in vitro using a T3 mMESSAGE mMACHINE kit (Ambion). Purified mRNAs were diluted to a final concentration of approximately 500 ng/μl and stored at −80 °C.

For ZSCAN4 knockdown at the GV stage, ZSCAN4 morpholino (MO) (5′-TGCCTGCTGTGAAGCCATTGT-3′; Gene Tools) was used along with a control MO (5′-CCTCTTACCTCATTACAATTTATA-3′).

Microinjection

For microinjection, 5–10 pl of a solution containing ~ 500 ng/µl mRNA or 1 mM MO was introduced into the cytoplasm of fully grown GV oocytes using a FemtoJet microinjector (Eppendorf, Germany). The injection procedure was performed using a Leica inverted microscope (DMIRB) paired with a Narishige micromanipulator (Japan). Following microinjection, the GV oocytes were cultured for 24 h in M2 medium supplemented with 200 μM IBMX for knockdown or overexpression.

Trim-Away-mediated ZSCAN4 depletion

For ZSCAN4 depletion via Trim-Away experiment, GV-arrested oocytes were microinjected with mRNA encoding TRIM21-mCherry along with ZSCAN4 antibody (NBP1-77,120, NOVUS). Control oocytes were injected with an equivalent concentration of normal IgG antibody (sc-2024, Santa Cruz Biotechnology). Following microinjection, oocytes were allowed to recover for 2 h in M2 medium containing 200 μM IBMX, and then cultured in IBMX-free medium for up to 16 h to allow meiotic progression to the MII stage. Depletion of ZSCAN4 was confirmed by immunostaining using a different ZSCAN4 antibody (1:250, ab4340, Sigma). After culture, oocytes at the MI or MII stages were subjected to immunostaining or chromosome spreading.

Immunostaining

Oocytes were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.1% Triton X-100 and 0.01% Tween-20 for 20 min in PBS at room temperature. Following permeabilization, the oocytes were blocked with 3% BSA in PBS for 1 h. Primary antibody incubation was carried out overnight at 4 °C using the following antibodies: anti-ZSCAN4 (1:250, ab4340, Sigma), anti-centromere (1:100, 15–234, Antibodies Incorporated), anti-acetylated α-tubulin (1:1000, T7451, Sigma), anti-HEC1 (1:250, ab3613, Abcam), anti-BubR1 (1:250, ab28193, Abcam), anti-CENP-A (1:200, 2048S, Cell Signaling), anti-histone 3 lysine 9 trimethylation (1:200, ab8898, Abcam), anti-SMC3 (1:100, ab128919, Abcam), anti-SMC4 (1:100, NBP1-86,635, Novus Biologicals), anti-CIP2A (1:500, sc-80662, Santa Cruz), and anti-p-MDC1 (1:250, ab35967, Abcam). After being washed three times, the oocytes were incubated with secondary antibodies for 2 h at room temperature. The secondary antibodies used were Alexa Fluor 488‐conjugated anti-mouse IgG (1:500, 115–545-146, Jackson ImmunoResearch), Alexa Fluor 594‐conjugated anti-mouse IgG (1:500, 115–585-044, Jackson ImmunoResearch), Alexa Fluor 488‐conjugated anti-rabbit IgG (1:500, 111–545-003, Jackson ImmunoResearch), Alexa Fluor 594‐conjugated anti-rabbit IgG (1:500, 111–585-144, Jackson ImmunoResearch) and rhodamine (TRITC)-conjugated anti-human IgG (1:100, 109–025-088, Jackson ImmunoResearch). Following three additional washes, DNA was counterstained with PBS containing 1.6 μM Hoechst 33,342 (H33342) (14,533, Sigma) and observed under a Zeiss LSM 900 laser scanning confocal microscope using a C‐Apochromat 40 ×/1.2 water immersion objective.

Immunoblotting

Oocytes were lysed in SDS sample buffer and subjected to SDS-PAGE. After transfer, the membranes were blocked in 3% BSA-supplemented TBS-T for 1–2 h at room temperature. Primary antibody incubation was carried out overnight at 4 °C using the following antibodies: anti-ZSCAN4 (1:1000, ab4340, Sigma) and anti-α-tubulin (1:5000, ab7291, Abcam). After being washed three times, the membranes were incubated with secondary antibodies for 1 h at room temperature. The secondary antibodies used were anti-mouse IgG (1:5000, 715–005-150 or Jackson ImmunoResearch) or anti-rabbit IgG (1:5000, 711–005-152, Jackson ImmunoResearch). Following three additional washes, the blots were developed with Pierce™ ECL western blotting substrate (32,109, Thermo Scientific).

Chromosome spreads and aneuploidy analysis

MI or MII oocytes were treated with acidic Tyrode's solution for 2 min to remove the zona pellucida. After a brief recovery in fresh M2 medium for 10 min, the oocytes were fixed on slides following lysis using a fixation solution containing 1% paraformaldehyde, 0.15% Triton X-100, and 3 mM dithiothreitol in distilled water (pH 9.2). The slides were dried slowly in a humid chamber for several hours. Spread oocytes were blocked with 3% BSA in PBS for 1 h, incubated with primary antibodies overnight at 4 °C, and then treated with secondary antibodies for 2 h at room temperature. Antibody concentrations matched those used for immunofluorescence. DNA was counterstained with DAPI (H-1200–10, Vectashield), and the slides were observed under an LSM 900 laser scanning confocal microscope (Zeiss) with a C‐Apochromat 63 ×/1.4 oil immersion objective.

To assess aneuploidy, dispersed chromosomes were stained with anti-centromere antibody (1:100, 15–234, Antibodies Incorporated) to clarify individual chromosomes. By identifying distinct centromere signals, the total number of chromosomes per oocyte was determined. Oocytes exhibiting 20 pairs of centromeres (n = 40) were classified as euploid, while any deviation from this number was considered as aneuploidy. All samples, including controls and ZSCAN4-depleted oocytes, were processed in parallel under identical conditions and analyzed in a blinded fashion.

TUNEL assay

Double-strand breaks (DSBs) were detected using the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, performed with an in situ cell death detection kit (Roche) following the manufacturer’s protocol. This assay specifically labels the 3’-OH ends of DNA with fluorescently modified nucleotides. The oocytes were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.1% Triton X-100 and 0.01% Tween-20 for 1 h on ice. The oocytes were washed and incubated with fluorescently tagged terminal deoxynucleotide transferase dUTP for 2 h at 37 °C. After being washed three times, the oocytes were mounted on glass slides and counterstained with DAPI. The fluorescence signal was observed under an LSM 900 laser scanning confocal microscope (Zeiss) with a C‐Apochromat 63 ×/1.4 oil immersion objective.

Time-lapse imaging

For time-lapse imaging to monitor chromosomes and pericentromeres, oocytes were microinjected with mRNA solutions containing approximately 200 ng/μl H2B-mCherry and 500 ng/μl TALE-mClover, targeting the mouse major satellite sequence. Following microinjection, the oocytes were incubated in M2 medium supplemented with 200 μM IBMX for 1 h to allow recovery, then transferred to IBMX-free M2 medium for release. Time-lapse imaging was performed for 8 h using a Nikon Eclipse Ti inverted microscope equipped with a DS-Qi1Mc CCD-cooled camera (Nikon). A temperature controller (CU-301, Live Cell Instrument) was connected to the microscope setup to maintain a stable temperature of 37 °C during imaging.

Quantification of fluorescence intensity

All images were acquired at a resolution of 1024 × 1024 pixels and presented as maximum intensity Z-projections using a Zeiss LSM 900 laser scanning confocal microscope. Images were captured with consistent laser power settings for immunofluorescence intensity measurements, and mean fluorescence intensity was quantified in arbitrary units. All fluorescence signals were normalized to DAPI or Hoechst 33,342 intensity to control noises across samples, with the average intensity of the control group set to 1.0 to facilitate comparison. Images were processed as needed to reduce background noise before quantification. Data analysis was performed using ZEN 3.4 Blue (Zeiss) and ImageJ software (National Institutes of Health), with consistent processing parameters applied throughout.

Statistical analysis

All statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software). Data represent at least three independent experiments unless stated otherwise, with each experimental group consisting of a minimum of 10 oocytes. Differences between two groups were evaluated using Student's t-test, while comparisons among multiple groups were analyzed with one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A p-value of less than 0.05 was considered statistically significant.

Authors' contributions

J.S.O. conceived the study and designed the experiment; D.Y.C. and J.L. performed the experiments and generated the data with assistance from C.L.; J.S.O drafted the manuscript with input from D.Y.C. and J.L.; J.S.O. organized the data and finalized the manuscript. All authors have read and approved the manuscript.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2017R1A6A1A03015642 and RS-2025–00513287).

Data availability

Supplementary figures and videos are available in the additional files. All study data are included in the article and supporting information. No custom code was used in the analysis. The immunofluorescence images used in our study, including full image sets contributing to quantitative analyses, have been deposited in Figshare [57] with a reserved DOI: 10.6084/m9.figshare.29400161.v2.

Declarations

Ethics approval and consent to participate

Animal experiments were performed in strict compliance with institutional guidelines and were approved by the Institutional Animal Care and Use Committee of Sungkyunkwan University (approval ID: SKKUIACUC2023-09–31-1).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.