2.1 The screening of spindle-related proteins

Based on the importance of the spindle in oocyte meiotic division, we systematically compared the expression levels of spindle-related proteins during the MI and GV oocytes stages. The method was briefly described as follows. The proteomic data of oocytes at different stages was retrieved from the publicly available database by Li et al.,¹¹ and re-ranked the fold change ratios of protein expressions during the MI/GV periods from high to low. Then, based on published literature in the PubMed database and GeneCards (https://www.genecards.org/), we screened proteins that are potentially associated with spindle and have positional relationships. Furthermore, the top 10 proteins with the highest fold change ratios were selected and NUSAP1 had a much higher protein expression ratio (MI/GV) than other genes. The list of protein profile can be found in Table S1.

2.2 Animal

Approval for the animal experiments conducted in this study was obtained from the Institutional Animal Care and Use Committee of Nanjing Drum Tower Hospital (SYXK 2019-0059). Three-week-old female ICR mice were obtained from the Nanjing Medical University Animal Center and housed in the Nanjing Drum Tower Hospital Animal Center. The mice were kept on a 12/12 h light/dark cycle.

2.3 Oocyte collection and culture

Fully grown oocytes arrested at the GV stage were obtained from the ovaries of 3-week-old female mice. Superovulation was induced by intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin (PMSG) from Prospec, 46–48 h before oocyte collection. Cumulus-oocyte complexes were collected, and the surrounding cumulus cells were removed through repeated mouth pipetting.

The oocytes were then cultured in MEM-alpha supplemented with 3 mg/mL bovine serum albumin (BSA) from Sigma, and 5 mmol/L Milrinone from Calbiochem to prevent meiotic resumption. For in vitro maturation, the oocytes were incubated in MEM from Gibco, supplemented with 75 mg/mL penicillin G, 50 mg/mL streptomycin sulfate, 25 mg/mL pyruvate, 38 mg/mL ethylenediaminetetraacetic acid, and 3 mg/mL BSA. The incubation was carried out under mineral oil at 37°C in a 5% O₂, 5% CO₂, and 90% N₂ incubator.

2.4 Antibodies

The following antibodies were employed in this study: a rabbit polyclonal antibody against NUSAP1 (Cat#: 12024-1-AP; dilution 1:200; Proteintech), DDX6 (Cat#: 14632-1-AP; dilution 1:1000; Proteintech), EDC4 (Cat#: 17737-1-AP; dilution 1:1000, Proteintech), γ-tubulin (Cat#: 15176-1-AP; dilution 1:1000; Proteintech), a mouse monoclonal antibody conjugated with FITC against α-tubulin (Cat#: F2168; dilution 1:500; Sigma), a mouse monoclonal antibody against β-actin (Cat#: AF0003; dilution 1:1000; Beyotime), rhodamine-phalloidin (Cat#: YP0063-50T; dilution 1:1000; US EVERBRIGHT®INC), and Alexa Fluor Plus 555 donkey anti-rabbit immunoglobulin G (Cat#: A31572; dilution 1:1000; Thermo Fisher Scientific).

2.5 Microinjection

Mouse oocytes were subjected to microinjection with approximately 10 pl of small interfering RNA (siRNA) (25 μM) targeting NUSAP1 using a FemtoJet microinjector (Eppendorf). A pool of three distinct siRNAs was specifically employed to target Nusap1, as well as control siRNAs (GenePharma), were obtained and diluted in RNase-free water. The 5′-3′ sequences of the Nusap1 siRNAs were as follows: GGACTTCTATTGCAGTTAT, GCCAGTATTTGATCTCAAA, and GGAGAAGTAAATGACTTAT. Following injection, the oocytes were cultured in a medium supplemented with 5 μM milrinone to maintain GV arrest for 20 h. Subsequently, the oocytes were transferred to fresh medium and cultured under mineral oil in a 37°C incubator with an atmosphere of 5% O₂, 5% CO₂, and 90% N₂.

2.6 Immunostaining

Following fixation in 4% paraformaldehyde (PFA) for 30 min, mouse oocytes were blocked with a solution containing 5% BSA and 0.5% Triton X-100 in phosphate buffered saline (PBS) at room temperature for 1 h. Subsequent overnight incubation with primary antibodies at 4°C was performed. After three washes, secondary antibodies were applied to the oocytes for 1 h at room temperature. Chromosome staining using DAPI was carried out for 10 min. Finally, the oocytes were mounted on glass slides and subjected to examination using a Carl Zeiss LSM 780 laser scanning confocal microscope. The criteria utilized to distinguish between normal and abnormal spindle and chromosome morphology in mouse oocytes are as follows: A normal spindle typically exhibits an elliptical or ovoid shape with well-defined poles and spindle fibers. Abnormal spindles may manifest as irregular shapes, distortions, excessive enlargement, or reduction in size. Normal chromosomes should be uniformly aligned in the central region of the spindle fibers, forming a well-organized metaphase plate. Abnormal chromosome alignment may involve scattered distribution, clustering, or misalignment.

2.7 Western blotting

Eighty mouse oocytes were lysed in sample buffer and boiled at 100°C for 5 min for Western blot analysis. The denatured proteins were then separated by 7.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. After blocking in PBST (PBS with 5% low-fat dry milk and 0.1% Tween-20) for 1 h at room temperature, the membranes were incubated overnight at 4°C with rabbit anti-NUSAP1 antibody (1:1000; Proteintech) and mouse anti-β-actin antibody (1:1000; Beyotime). Following three PBST washes, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies in blocking buffer for 1 h. Subsequently, the protein bands were visualized using the ECL Plus Western blotting Detection System (GE Healthcare) after three additional PBST washes.

2.8 RT-PCR

The complementary DNA (cDNA) from the oocyte/embryos lysate was amplified using the Single Cell Sequence Specific Amplification kit (P621; Vazyme) following the manufacturer's instructions. A brief description is as follows: The amplification primers for different target genes were mixed to create an Assay Pool (with a final concentration of each primer at 0.1 μM). For the assembly of the assay pool, 1 μL of oocyte lysate and primers specific to the target genes were used. Reverse transcription was carried out with 11 cycles as recommended in the manual. This step generated cDNA for subsequent analysis of differential gene expression. To reduce errors between experimental groups, we used at least 10 oocytes/embryos for each sample group. The resulting cDNA was subsequently diluted 100 times, and 2 μL of the dilution was utilized for quantitative PCR on a LightCycler 480 II (Roche). The expression levels of the genes were determined using the 2^−∆∆Ct method. The primer sequences employed are provided in Table S2.

2.9 Chromosome spreading

Chromosome spreading was conducted following a previously published protocol²² (29353819). To remove the zona pellucida of the oocytes, they were briefly exposed to Tyrode's buffer (pH 2.5) (T1788; Sigma) at 37°C for 30 s. After a 10-min recovery in fresh medium, the oocytes were fixed on a glass slide using a solution containing 1% PFA and 0.15% Triton X-100. Subsequently, the slides were air-dried, and the chromosomes were stained with Hoechst 33342. Finally, the slides were examined under a Carl Zeiss LSM 780 laser scanning confocal microscope.

2.10 RNA-Seq and gene enrichment analyses

After injection, GV-stage oocytes were cultured for 20 h in medium containing 5 μM milrinone to maintain GV arrest. Three sets of samples, each containing 10 control-siRNA and 10 NUSAP1-KD MI-stage oocytes, were collected for RNA-Seq analysis. Full-length cDNA amplification products were obtained using the Single Cell Full Length mRNA Amplification Kit (N712; Vazyme). Subsequently, the amplified cDNA was prepared into sequencing libraries specifically designed for the Illumina high-throughput sequencing platform using the TruePrep® DNA Library Prep Kit V2 for Illumina (TD503; Vazyme) following the manufacturer's instructions. The Illumina HiSeq X Ten platform was utilized to sequence the libraries, generating paired-end reads of 150 bp. Trim_galore (v0.6.4) was applied to remove adapter sequences and low-quality bases from the obtained reads. Trimmed read pairs were aligned to mouse genome(mm10) with STAR(v2.7.1a), and featureCounts (v2.0.0) was used to obtain read counts. The R package DESeq. 2 (v1.28.1) was used to analyze and normalize the differentially expressed genes. Gene enrichment analysis of the differentially expressed transcripts was performed using the DAVID (https://david.ncifcrf.gov/)²³ and Metascape (https://metascape.org/) platforms.²⁴

2.11 Immunoprecipitation (IP) coupled with mass spectrometry identification

Ovaries were collected from 3-week-old ICR female mice after injection of PMSG for 44–46 h. Ten ovaries were pooled as one group, and after sample lysis, IP of NUSAP1 was performed. The steps of IP were briefly described as follows: NUSAP1 antibody (Cat#: 12024-1-AP; Proteintech) was incubated with the ovarian lysate, followed by the addition of affinity beads to capture the antibody-antigen complex. The co-precipitated proteins were then analyzed by mass spectrometry (Thermo Scientific™ Q Exactive™ HF-X). Mass spectrometry data were searched using Proteome Discoverer 2.4, and the identified interacting proteins were listed in Table S3.

2.12 Statistical analysis

The experimental data are presented as the mean ± SEM, unless specified otherwise. Statistical analysis was conducted using the Student t test for comparing two groups and one-way analysis of variance followed by Tukey's HSD test for multiple group comparisons. Prism 5.0 software was utilized for the statistical analysis. The significance level was set at p ≤ 0.05.

3.1 Expression and localization of NUSAP1 during oocyte meiotic maturation

The spindle apparatus plays a vital role in the process of oocyte meiotic division, as its assembly and anchoring determine the proper segregation of chromosomes. By mining mass spectrometry data, we systematically compared the expression levels of spindle-related proteins during the GV, MI, and MII stages. Among spindle-related proteins, we observed that the protein expression of NUSAP1 during the MI stage was approximately fivefold higher than that during the GV stage, surpassing that of other spindle components¹¹ (Figure 1A). This unique phenomenon surrounding NUSAP1 piqued our interest. We discovered that Nusap1 exhibited dominant expression in oocytes, with both its mRNA and protein expression levels significantly higher than those in granulosa cells (Figure 1B,C). Consistent with the proteomic data, NUSAP1 demonstrated a threefold increase in protein levels in MI-stage oocytes compared to GV-stage oocytes (Figure 1D). During the fully grown stage of oocytes (FGO), DNA transcription ceases, mRNA no longer increases, and degradation becomes prevalent.²⁵ Correspondingly, as protein expression increased, the Nusap1 mRNA in oocytes rapidly decreased during the MI stage (Figure 1E). Furthermore, Nusap1 mRNA exhibits rapid degradation during the early stages of embryonic development following fertilization (Figure 1F).

The abundant expression of Nusap1 prompted us to investigate its localization during oocyte meiotic maturation. Immunofluorescence staining of oocytes at various stages (GV, Pro-MI, MI, AI/TI, MII) revealed that NUSAP1 is distributed within the nuclear membrane of immature oocytes with a nonsurrounded nucleolus configuration (GV-NSN). Conversely, in mature oocytes with a surrounded nucleolus configuration (GV-SN), it does not accumulate within the nuclear membrane. This localization may be associated with the SAP-like domain of NUSAP1, allowing interaction with chromosomes.¹³ Interestingly, we observed that NUSAP1 is localized near the spindle pole during the Pro-MI, MI, and AI/TI stages, with protein condensation particles present on both sides of the spindle apparatus. However, during the MII stage of oocytes, NUSAP1 became significantly enriched within the polar body (Figure 2). These findings suggest that NUSAP1 plays a crucial role in the process of oocyte meiotic maturation.

3.1.1 Nusap1 depletion reduced the GVBD rate and polar body extrusion (PBE) rate of mouse oocytes

To investigate the role of NUSAP1 in the process of oocyte meiotic maturation, we utilized siRNA interference during the GV stage of oocytes to reduce Nusap1 expression (Figure 3A). Following microinjection, GV-stage oocytes were cultured in medium containing milrinone for 20 h to facilitate the degradation of Nusap1 mRNA. In NUSAP1-KD oocytes, the expression level of Nusap1 mRNA decreased by more than 90% (Figure 3B). Additionally, the expression level of NUSAP1 protein was reduced by over 50% in NUSAP1-KD oocytes (Figure 3C,D). Following NUSAP1 KD, the rate of GVBD was significantly delayed compared to that of the control group. After 120 min of maturation culture, less than 40% of NUSAP1-KD oocytes resumed meiosis, while the GVBD rate approached 90% in the control group (Figure 3E). The rate of PBE in NUSAP1-KD oocytes was also significantly lower than that in the control group (Figure 3F). The NUSAP1-KD oocytes exhibited various phenotypes, including arrest at the GV stage, failed extrusion of the first polar body, and higher percentage of enlarged polar bodies [NUSAP1-KD (15.0 ± 1.0%) vs. Control (6.7 ± 1.0%)] (Figure 3G,H). These findings strongly indicate that NUSAP1 serves as a critical regulatory molecule in oocyte meiotic maturation.

3.1.2 Nusap1 depletion leads to aberrant meiosis and increases aneuploidy in MII-stage oocytes

To further investigate the impact of NUSAP1 KD on oocyte maturation, we conducted immunofluorescence staining on oocytes cultured for 14 h in vitro. Consistent with the reduced rate of Pb1 observed in NUSAP1-KD oocytes, we found that almost no oocytes in the NUSAP1-KD group exhibited a normal MII morphology (Figure 4A). Upon Nusap1 depletion, approximately 25% of oocytes were arrested at the AI/TI stage, and approximately 25% exhibited disorganized chromosome alignment in the MI stage (Figure 4B,C). Even among the approximately 40% of oocytes that extruded Pb1, these oocytes displayed typical DNA lagging (Figure 4D). Due to the presence of elevated levels of chromosome misalignment in NSUAP1-KD oocytes, we performed karyotype analysis on MII-stage oocytes through chromosome spreading. Consistent with our expectations, there was a substantial increase in the proportion of aneuploidy in NUSAP1-KD oocytes (Figure 4E,F). These results suggest that NUSAP1 KD significantly affects chromosome arrangement.

3.1.3 Nusap1 depletion disrupts spindle assembly during mouse oocyte meiosis

The unique localization of NUSAP1 near the spindle poles during oocyte maturation prompted us to investigate how Nusap1 depletion affects spindle assembly. After 8 h of in vitro maturation of oocytes, we labeled the spindles using tubulin antibodies. As expected, we observed a significant reduction in spindle pole width in NUSAP1-KD oocytes. Quantitative analysis revealed that the width of spindle poles in NUSAP1-KD oocytes was almost half that in the control group, where the abnormal spindle pole width was approximately 4 μm (Figure 5A,B).

Due to the enrichment of NUSAP1 at the spindle poles (Figure 2) and the prominent phenotype observed in NUSAP1-KD oocytes, which primarily manifests as spindle pole defects, we further investigated the relationship between NUSAP1 and MTOC markers (such as γ-tubulin) in terms of spindle pole localization (Figure 5C). We found that although both NUSAP1 and γ-tubulin were enriched near the spindle poles, they did not exhibit co-localization. In NUSAP1-KD oocytes, although the protein expression levels of MTOC markers (such as γ-tubulin) remained unchanged, the distribution of γ-tubulin was affected, no longer displaying the characteristic crescent-shaped clustering (Figure 5D,E). These results indicate that the loss of NUSAP1 significantly affects spindle assembly during oocyte maturation.

3.1.4 NUSAP1 KD alters transcriptome dynamics in mouse oocytes

To explore the underlying mechanisms causing abnormal development in NUSAP1-KD oocytes, we conducted transcriptomic analysis on MI-stage NUSAP1-KD and control oocytes. Upon NUSAP1 KD, the volcano plot of the transcriptome analysis revealed drastic changes in mRNA within the oocytes (Figure 6A). A total of 860 transcripts showed significant alterations, with the number of upregulated transcripts being twice as high as that of downregulated transcripts. Among them, 598 transcripts were significantly upregulated, while 262 transcripts were significantly downregulated (Figure 6B). Consistent with the previously observed abnormal spindle assembly (Figure 5), further gene set enrichment analysis (GSEA) indicated significant inhibition of the “establishment of spindle orientation” signaling pathway. Additionally, the blockade of the “attachment of spindle microtubules to kinetochore” pathway and the activation of the “spindle assembly checkpoint” pathway suggested abnormal connections between the spindle and chromosomes (Figure 6C–E). Furthermore, Gene Ontology (GO) enrichment analysis bubble plots revealed significant differential changes in nearly 30 crucial transcripts associated with “spindle assembly” processes (Figure 6F). Based on the impact of NUSAP1 KD on spindle assembly, we categorized some important spindle-related genes into four groups: spindle pole-, MTOC-, kinetochore-, and MT-related genes. We further validated these findings through RT-PCR, which confirmed that the majority of spindle-related genes changed significantly (Figure 7). Among them, 13 spindle-related genes with differential changes were consistent with the dynamic trends (up- or downregulation) revealed by RNAseq (Figure 7). These findings suggest that NUSAP1 KD alters transcriptome dynamics in mouse oocytes.

3.1.5 NUSAP1 plays a critical role in actin assembly during oocyte meiosis

The localization of the spindle in mouse oocytes relies on dynamic changes in actin filaments.⁸ In mouse oocytes, the typical MT spindle is enveloped by actin filaments, known as spindle-actin. The interaction between spindle-actin, spindle MTs, and chromosomes is essential for meiotic division.⁷ When NUSAP1 was knocked down in mouse oocytes, the “actin filament assembly” signaling pathway was significantly inhibited (Figure 8A). The fluorescence signals of actin were greatly reduced in NUSAP1-KD oocytes. The distribution of actin in the cortical and cytoplasmic regions of oocytes is further magnified through line graphs. Compared to the control group, the fluorescence intensity of actin was significantly decreased in NUSAP1-KD oocytes (1 vs. 0.25 ± 0.12, p < 0.05) (Figure 8B–D). These findings suggest that NUSAP1 KD affects actin assembly during oocyte meiotic maturation.

3.2 The depletion of RNA-binding protein (RBP) NUSAP1 affected the dynamics of processing bodies—the key components involved in mRNA homeostatic regulation

Given the impact of NUSAP1 functional loss on RNA stability, we further investigated the relationship between NUSAP1 and RNA stability. By examining publicly available human and mouse datasets of RBPs extracted from the EMBL RBPbase (https://rbpbase.shiny.embl.de/), we identified the RNA-binding capability of NUSAP1^26-29 (Figure 9A, Table S3). Moreover, through a comparative analysis of 137 NUSAP1-interacting proteins in ovary identified here and the available human and mouse RBP datasets, we identified 31 proteins with overlapping interactions.^26-29 Within the NUSAP1 interactomes, RBPs accounted for 41% (56/137). GO analysis of the 56 common NUSAP1 interactomes revealed enrichment for RNA binding (Figure 9B). Processing bodies (P-bodies) represent a prominent category of cytoplasmic ribonucleoprotein (RNP) granules, where messenger RNAs (mRNAs) are intricately associated with proteins involved in various cellular processes, including translational repression, deadenylation, mRNA decapping, and 5′-3′ decay.³⁰ Core proteins such as EDC4 and DDX6 are among these components.^{31, 32} Notably, significant differential changes were observed in the main constituents of processing (P)-bodies, such as DDX6 and EDC4, in NUSAP1-KD oocytes (Figure 9C,D). These findings suggest that the RBP NUSAP1 may directly or indirectly regulate mRNA homeostasis by affecting the dynamics of processing (P)-body components.