Determining the differentially expressed short peptides between human mature and immature follicular fluid by combined ultrafiltration and an LC–MS/MS based peptidomic strategy

A mature follicle and an immature follicle were collected from the same women. Five pairs of follicles obtained from five females (10 follicles in total) were subjected to peptide sequence analysis by using LC–MS/MS (Fig. 1A). The lengths of the peptides were in a normal distribution (Fig. 1B). The results from LC–MS/MS (Additional file 1) showed that a total of 362 short peptides were identified, in which 221 peptides were derived from immature FF, and 295 peptides were released by mature follicles. A Venn diagram shows the comparative difference analysis between the two groups, in which the overlapping circle indicates the common short peptides (154), while circles that do not overlap represent characteristic short peptides, with 67 in immature FF and 141 in mature FF (Fig. 1C). By comparing these 154 differentially expressed short peptides with immature FF, we found that 14 short peptides (about 9%) were significantly upregulated in mature FF, corresponding to 14 proteins and three variant sequences, while 7 short peptides (4.5%) were significantly downregulated in mature FF, corresponding to 7 proteins, and 133 short peptides (86%) were unchanged (Fig. 1D). A heat map shows the levels of the 21 differentially expressed short peptides between the five immature FF and five mature FF samples (Fig. 1E). The characteristics of the 21 differentially expressed short peptides, including the grand average of hydropathicity (GRAVY), isoelectric point (PI), instability index, and aliphatic index, are shown in Fig. 1F.

Fig. 1

Determining the differentially expressed short peptides between human mature and immature follicular fluid. A Workflow of the peptidomic analysis of human follicular fluid (FF). The mature (n = 5) and immature (n = 5) FF samples were collected according to a GnRH-ant protocol. The collected FF samples were subjected to ultrafiltration to separate the low-molecular-weight fraction (< 10 kDa) containing endogenous peptides (fragments of proteins). The peptides were prepared and analyzed by mass spectrometry. B Length distribution of fragments from FF. The x-axis represents the number of amino acids in each peptide, and the y-axis represents the amount of each peptide. C Venn plot of the FF peptides identified in this study. The left circle shows peptides from immature FF identified in the experiments (221 in total). The right circle shows peptides from mature FF identified in the experiments (295 in total). A total of 154 differentially expressed peptides between immature and maturated follicles were determined. D Volcano map of differentially expressed short peptides. Red represents the upregulated short peptides, and green represents the downregulated short peptides. E Heat map comparing hits of 21 candidate short peptides between immature FF 1–5 and mature FF 1–5. The color scale bar is located on the left, and red and blue indicate increased and decreased levels of the identified peptide, respectively. F Distributions of differentially expressed short peptides according to their isoelectric point (PI), instability index, aliphatic index, and grand average of hydropathicity (GRAVY)

After excluding precursor proteins with unclear functions, four short peptides upregulated in mature FF, namely, apolipoprotein A-I isoform 1 (APPOA1), complement C3 chain A (C3a), immunoglobulin heavy constant gamma 2 (IGHG2), and alpha-2-HS-glycoprotein (FETUA) (aa 20–46), indicated as red characters in Table 1, were selected for subsequent experiments.

Table 1 21 differential short peptides between immature and mature hFFThe C3a-peptide significantly increases the MII percentage of mouse oocytes without inducing chromosome aneuploidy

To evaluate the effects of these upregulated peptides from mature FF on oocyte development, they were synthesized and added to oocyte medium according to their maximum solubility. Subsequently, the growth and development of mouse oocytes were monitored by setting up four experimental groups, untreated, 1.5 nM, 1.5 μM and 1.5 mM treated group. The percentages of oocytes in the GVBD stage and MII stage were calculated (the numbers of oocytes were 128, 127, 128, 124 in four groups respectively). Of the four relatively upregulated peptides, only the C3a-peptide was found to produce an obvious effect on the growth and development of oocytes based on morphological evaluation (Fig. 2A, Additional file 2, Fig. 1S). The optimum concentration of the C3a-peptide was 1.5 μM (Fig. 2A). We found that 1.5 μM of C3a-peptide increased the percentage of oocytes in the MII stage from 80 to 90% (Fig. 2A and C). As for the GVBD stage, albeit not reaching significance, there was an increasing trend with an increasing C3a concentration. Once the concentration of C3a-peptide reached 1.5 mM, the percentage of oocytes at the MII stage was markedly reduced and the cytoplasm became dark, indicating toxic effects (Fig. 2B and C).

Fig. 2

Differentially expressed short peptide, C3a-peptide, promotes oocyte maturation without chromosome aneuploidy change. A Morphological change of cultured mouse oocytes in response to treatment with different concentrations of C3a-peptide. B Calculation and statistical analysis of the percentage of cultured mouse oocytes at the GVBD stage in response to treatment with different concentrations of C3a-peptide (four independent biological replicates). C Calculation and statistical analysis of the percentage of cultured mouse oocytes at the MII stage in response to treatment with different concentrations of C3a-peptide (four independent biological replicates). D Mature mouse oocyte. E Immature mouse oocyte. F Morphological changes of cultured immature mouse oocytes in response to C3a-peptide treatment. G C3a-peptide treatment does not induce chromosome aneuploidy of mouse oocytes. Representative images of MII oocyte immunofluorescence stained with DAPI (blue) and anti-CREST (magenta) antibodies showing euploidy and aneuploidy in the oocytes, respectively (left). H Calculation and comparison of the chromosome aneuploidy rates between the control and C3a-peptide treatment groups (three independent biological replicates). I Calculation and statistical analysis of the percentage of cultured immature mouse oocytes at the GVBD stage in response to C3a-peptide treatment (thirteen independent biological replicates). J Calculation and statistical analysis of the percentage of cultured immature mouse oocytes at the MII stage in response to C3a-peptide treatment (thirteen independent biological replicates). K Morphological changes of an immature human oocytes after culture for 24 h. L Calculation and comparison of human oocyte maturation in response to C3a-peptide treatment (103 patients). Data are presented as the means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001 as compared to control cells

However, good quality GV oocytes are not an ideal model for studying the effect of C3a-peptide on the maturation of human oocytes. Our aim was to find a peptide that improved oocytes with poor quality and helped them to mature. Thus, as shown in Fig. 2D and E, the oocytes were divided into two main types: the high quality GV oocytes (clear, moderately granular cytoplasm; small perivitelline space; clear, smooth, colorless zona pellucida) and another oocyte group with a smaller size (rough and surrounded by cumulus cells; an irregular shape typically indicating poor developmental potential). Next, we focused on the effects of C3a-peptide on the oocytes with a smaller size. Although C3a-peptide did not significantly affect the percentage of GVBD oocytes compared with the untreated group (43.4% and 54.6%, n = 138) (Fig. 2F and I), the percentage of MII oocytes in the smaller sized group was significantly increased with C3a-peptide treatment, from 9.3% to 27.1% (n = 138) (Fig. 2F and J). This suggested that the C3a-peptide significantly increased the percentage of the oocytes at the MII stage as compared with those in the control group. Moreover, to ascertain whether C3a-peptide led to aneuploidy in mouse MII oocytes, chromosome aneuploidy rates were assessed. Specifically, kinetochores were quantified in MII oocytes after immunostaining with mouse anti-centromere CREST antibody (Fig. 2G). This analysis showed that overall, 18% of MII oocytes (4 of 22) were aneuploid in the untreated control group, whereas 19% of MII oocytes (4 of 21) were aneuploid in the C3a-peptide treatment group (Fig. 2H). Altogether, these results indicate that a suitable C3a-peptide concentration could increase the percentage of mouse oocytes at the MII stages, but not at the GVBD stage, and that, more importantly, the C3a-peptide did not elicit statistically significant changes in the chromosome aneuploidy rate during mouse oocyte maturation.

C3a-peptide supplementation enhances the developmental potential of human immature oocytes without increasing the rate of chromosome aneuploidy

Next, we proceeded to validate the effect of C3a-peptides on human immature oocyte development in a clinical assisted reproductive technology (ART) practice. A total of 320 GV oocytes were collected and were randomly divided into two groups, the control (n = 152) and C3a-peptide-treated groups (n = 168), for subsequent IVM experiments. After 24 h of culture, we found that the maturation rate of oocytes in the C3a-peptide-treated group was significantly higher than that in the control group based on the observed first polar body (Fig. 2K and L) (58.3% [98/168] vs 41.4% [63/152], P = 0.0026). Considering the C3a-peptide as a potential therapeutic drug, a total of 30 human in vitro matured oocytes were subjected to chromosome aneuploidy analysis, including 15 in the control group and 15 in the C3a-peptide-treated group. The results from array comparative genomic hybridization revealed 12 oocytes with a 46, XX karyotype, 1 oocyte with a 47, XX karyotype, and 2 oocytes that could not be successfully karyotyped in the C3a-peptide-treated group, whereas there were 11 oocytes with a 46, XX karyotype, 2 oocytes with a 45, XX karyotype, 1 oocyte with a 43, XX karyotype, and 1 oocyte with a 47, XX karyotype in the control group. Therefore, we did not find any statistical difference in chromosome aneuploidy between the two groups (Table 2). In summary, our data demonstrated that the C3a-peptide indeed increased the percentages of human MII oocytes without increasing the chromosome aneuploidy rate.

Table 2 Array CGH of 30 human oocytes in control and C3a-peptide treated groupsC3a-peptide plays pivotal roles during maturation of oocytes through C3aR

Each peptide results from the enzymatic processing of large intact protein precursors. We found that the C3a-peptide has high homology between mice and humans, and the sequence of C3a-peptide matched that of the human complement C3a, as determined through a BLAST comparison of the short peptide sequence with the GenBank database (Additional file 3, Fig. S2, A-C). Given that C3a-peptide corresponded to C3a, we wondered whether C3a-peptide exerted the various effects of the native C3a by binding to its receptor, C3aR. A series of experiments were conducted. As shown in Additional file 4, Fig. S3, A, we first observed that after FITC-labeled C3a-peptide was added to the oocyte medium at a concentration of 1.5 μM, positive signals were evenly distributed in the cytoplasm of oocytes, indicating that C3a-peptide penetrated the membrane and successfully entered the cytoplasm of the oocytes. Next, to determine whether the C3a-peptide could bind with C3aR, we constructed the expression plasmids, pcDNA3.1-C3a-peptide-Flag and pcDNA3.1-C3a-Flag. We performed transfection and co-immunoprecipitation (Co-IP) on 293 T cells and used anti-Flag magnetic beads to pull down the expression proteins. As shown in Fig. 3A, C3aR and Flag-tagged proteins were expressed well in the input sample. Moreover, C3aR was present in the immunoprecipitated proteins of 293 T cells transfected with pcDNA3.1-C3a-peptide-Flag and pcDNA3.1-C3a-Flag, indicating that C3a-peptide directly interacted with C3aR. To investigate the expression level and location of C3aR in oocytes, qRT-PCR and immunofluorescence staining were performed. A total of 30 retrieved oocytes were used to extract total RNA in every stage. Compared with the expression level at the GV stage, C3AR1 (encoding C3aR) mRNA levels reached a peak at the GVBD stage. Then, the transcription of C3AR1 gradually decreased with maturation and reached the lowest level at the MII stage, suggesting that C3aR was required for mouse oocyte meiotic maturation (Fig. 3B). To clarity the C3aR location, double immunofluorescence staining was conducted. Specially, the specificity of primary antibody C3aR was confirmed through peptide blocking assays (Additional file 4, Fig. S3, D, bottom row). The results from double immunofluorescence staining showed that C3aR positive signals were evenly distributed in the cytoplasm of oocytes at the GV and GVBD stages. Interestingly, after entering the MI and MII stages, C3aR and β-tubulin positive signals were entirely co-localized on the spindles (Fig. 3C). This result was independently verified by using antibodies originating from different companies (Additional file 4, Fig. S3, B). The co-localization phenomenon of C3aR and β-tubulin was also observed in 293 T cells (somatic cell line) (Additional file 4, Fig. S3, C).

Fig. 3

C3a-peptide increases the percentage of oocytes at the MII stage by directly interacting with C3aR. A Confirming the direct interaction between C3a-peptide and C3aR by Co-IP. B RT-PCR results show the C3AR1 mRNA expression levels of mouse oocytes at different stages (three independent biological replicates). C Double immunofluorescences staining reveals that C3aR and β-tubulin co-localized in the cytoplasm at the GV and GVBD stages and co-localized to spindles at the MI and MII stages (three independent biological replicates). D Morphological changes of mouse oocytes during maturation in response to C3a-peptides combined with C3aR antagonist treatment at different concentrations. E Calculation and statistical analysis of the percentage of cultured mouse oocytes entering the MII stage in response to C3a-peptide combined with C3aR antagonist treatment at different concentrations (five independent biological replicates). F Calculation and statistical analysis of the percentage of cultured mouse oocytes entering the GVBD stage in response to C3a-peptide combined with C3aR antagonist treatment at different concentrations (five independent biological replicates). Data are presented as the means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001 as compared to control cells

To unequivocally confirm that the C3a-peptide played a pivotal role through C3aR, oocytes were treated with both C3a-peptide and the C3aR antagonist SB290157 (0, 1 nM, 100 nM, and 10 μM, 225 oocytes were used in every group). The results showed that C3a-peptide alone increased the percentage of oocytes in the MII stages, and the percentage of MII stages was significantly reduced once combined use of SB290157 with C3a-peptide at any of the concentrations tested. In addition, only 10 μM SB290157 was needed to suppress the otherwise increased GVBD percentage of oocytes induced by C3a-peptide (Fig. 3D–F). Our data suggested that the C3a-peptide played pivotal roles during maturation of oocytes through its receptor C3aR, which might be involved in spindle formation during either meiosis of oocytes or mitosis of somatic cells.

C3aR morpholino inhibition results in disrupted F-actin aggregation and spindle migration

To further determine the effect of C3aR on mouse oocyte maturation, a C3aR morpholino was introduced. The strategy is shown in the schema graph (Fig. 4A). First, mouse GV oocytes were injected with a C3aR morpholino. The knockdown efficiency was verified by Western blotting (a total of 500 oocytes were retrieved for preparing the protein samples in every group) (Fig. 4B). Then, the growth and development of the oocytes were evaluated, and the percentages at the GVBD and MII stages in untreated (n = 320), negative control (n = 257) and C3aR- morpholino groups (n = 235) were calculated (Fig. 4C). The results showed that although the C3aR morpholino slightly decreased the percentage of oocytes at the GVBD stage compared with the untreated group, there was no statistical difference between the experimental and negative control groups (Fig. 4D). The percentage of oocytes at the MII stage was significantly reduced compared with either the untreated or negative control groups once C3aR was inhibited, while there was no statistical difference between the untreated or negative control groups (Fig. 4E), indicating that C3aR indeed affected mouse oocyte maturation.

Fig. 4

C3aR promotes oocyte maturation by enabling F-actin aggregation and spindle migration. A Diagram depicting C3aR morpholino delivery into oocytes. B Western blotting shows the validation of the inhibitory efficiency of the C3aR morpholino (two independent biological replicates). C Morphological changes of mouse oocytes during maturation in response to C3aR morpholino injection. D C3aR morpholinos significantly reduce the percentage of oocytes entering the GVBD stage (five independent biological replicates). E C3aR morpholinos significantly reduce the percentage of oocytes entering the MII stage (five independent biological replicates). F Triple immunofluorescence staining shows that C3aR morpholino injection suppressed C3aR expression (red) and inhibited F-actin aggregation in the sub-cortical and spindle regions (pink) (three independent biological replicates). G Diagram of six quantitative indexes: (a) ratio of the spindle width to oocyte diameter (W/D); (b) ratio of the spindle inter-polar distance to oocyte diameter (L/D); (c) length from the spindle to the cortex (S-C); (d) relative intensity of F-actin in the subcortical region (S1); (e) relative intensity of F-actin around the spindle (S2); (f) cortical thickness. H W/D was analyzed and compared between the negative control and C3aR morpholino-injected oocytes. I L/D was analyzed and compared between the negative control and C3aR morpholino-injected oocytes. J S-C was analyzed and compared between the negative control and C3aR morpholino-injected oocytes. K S1 was analyzed and compared between the negative control and C3aR morpholino-injected oocytes. L S2 was analyzed and compared between the negative control and C3aR morpholino-injected oocytes. M Cortical thickness was analyzed and compared between the negative control and C3aR morpholino-injected oocytes. Data are presented as the means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001 as compared to control cells

Considering that the C3a-peptide might promote mouse oocyte maturation mediated by C3aR by influencing spindle formation or migration, triple immunofluorescence staining with antibodies raised in the same species was used to study the complex distributions of F-actin (pink color), C3aR (red color), and β-tubulin (green color). The results showed that the cloud of F-actin around the spindle disappeared, and the positive signals of a cortex localization of F-actin sharply weakened in C3aR morpholino-injected oocytes compared with the negative control oocytes. Both C3aR and β-tubulin were completely co-located on the spindle. Notably, with C3aR knocked down, the intensity and distribution of β-tubulin did not change (Fig. 4F). To further quantify the influence of C3aR on spindle assembly, migration, and microfilament polymerization, oocytes were divided into two groups (morpholino group, n = 44; negative control group, n = 46) and six indexes were measured and evaluated, such as the ratio of the short diameter of the spindle (W) to the diameter of the oocyte (W/D) (Fig. 4G-a), the ratio of the long diameter of the spindle (the distance between the two poles, L) to the diameter of the oocyte (L/D) (Fig. 4G-b), the distance from the spindle center to the cortex (S-C) (Fig. 4G-c), the relative F-actin fluorescence intensity in the subcortical region (S1) (Fig. 4G-d), the relative F-actin fluorescence intensity around the spindle (S2) (Fig. 4G-e), and the cortical thickness (red line showed in Fig. 4G-f).

We noticed that there were no statistical differences in the W/D ratio (0.24 ± 0.025 vs 0.24 ± 0.04) (Fig. 4H) and in the mean S-C (27.4 ± 4.96 μm vs 26.09 ± 5.27 μm) (Fig. 4J) between the negative control group and the morpholino group (P > 0.05). The mean L/D was slightly shorter (0.35 ± 0.055) in the morpholino group than in the negative control group (0.39 ± 0.067, P < 0.01) (Fig. 4I). Moreover, S1 and S2 were significantly weakened with C3aR morpholino treatment. Specifically, S1 in the morpholino group was 31.47 ± 9.322, while the mean S1 in the negative control group was 39.67 ± 9.62 (P < 0.001) (Fig. 4K); the mean S2 in the morpholino group was 27.69 ± 7.40, whereas the mean S2 in the negative control group was 34.98 ± 13.14 (P < 0.05) (Fig. 4L), suggesting that C3aR inhibition might suppress F-actin polymerization. In addition, the mean cortical thickness in the morpholino group (1.73 ± 0.59 μm) was significantly lower than that in the control group (2.03 ± 0.58 μm) (P < 0.05) (Fig. 4M). Altogether, these data indicate that C3aR is partially responsible for polymerization of F-actin and the migration of spindles and that spindle assembly is not affected by C3aR.

C3a-peptide promotes F-actin aggregation and spindle migration, and this effect of C3a-peptide was attenuated by C3aR morpholino inhibition

To reveal the changes of F-actin polymerization and distribution during the development of mouse oocytes cultured with C3a-peptide, we first constructed F-actin probes in living cells, pcDNA3.1-3xmscarleti_Utrch, which could specifically bind to F-actin without changing its kinetic characteristics. The results showed that at the GVBD stage, F-actin polymerization around the spindle was more obvious in C3a-peptide-treated oocytes than in untreated oocytes and that at the MII phase, the fluorescence intensities of microfilaments in the cortical region and cytoplasm, especially surrounding the spindle, were stronger in the C3a-peptide-treated oocytes than in the untreated group (Fig. 5B). These results suggest that C3a-peptide promoted F-actin polymerization and regulated the distribution of F-actin. With delivery of C3a-peptide, triple immunofluorescence staining showed enhanced F-actin positive signals in the sub-cortical region and around the spindle compared with the untreated group (Fig. 5A).

Fig. 5

C3a-peptide combined with C3aR promotes F-actin aggregation and spindle dynamics by directly binding to MYO10. A Triple immunofluorescence staining shows that C3a-peptide treatment increased C3aR expression (green) and enhanced F-actin aggregation in the cytoplasm, sub-cortical regions, and around the spindle (pink). C3aR and tubulin co-localized to the spindle (three independent biological replicates). B F-actin probes in living cells revealed that C3a-peptide treatment enhanced F-actin aggregation in the sub-cortical regions and around the spindle. C W/D, L/D, S-C, S1, S2, and cortical thickness were calculated and compared between the untreated and C3a-peptide-treated groups. D Triple immunofluorescence staining showed that the enhanced F-actin positive signals in the cytoplasm, sub-cortical regions, and around the spindle (pink) caused by C3a-peptide treatment were restored with a C3aR morpholino injection (three independent biological replicates). E Direct interaction between C3aR and MYO10 in mouse oocytes was determined with a proximity ligation assay (PLA) using rabbit anti-C3aR and anti-MYO10 antibodies. After staining, the oocytes were imaged by confocal and differential interference contrast (DIC) microscopy. Scale bar, 20 μm. F W/D, L/D, S-C, S1, S2, and cortical thickness were calculated and compared between the C3a-peptide-treated group and the combined C3a-peptide-treated and C3aR morpholino-injected group. G Confirming the direct interaction between C3aR and MYO10 by Co-IP (three independent biological replicates). Data are presented as the means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001 as compared to control cells

Unlike the C3aR morpholino injection group, the fluorescence intensities of subcortical F-actin and around the spindle were stronger in the C3a-peptide group than in the untreated group (P < 0.001). The fluorescence intensity S1 in the C3a-peptide group was 33.16 ± 9.45, and in the untreated group, it was 24.69 ± 10.00 (Fig. 5C-d). S2 in the C3a-peptide group was 23.92 ± 9.50, and in the untreated group, it was 17.04 ± 6.70 (Fig. 5C-e). Furthermore, compared with the untreated group (n = 45), a much shorter S-C was found in the C3a-peptide group (25.54 ± 6.20 μm vs 20.07 ± 4.4 μm, P < 0.001, respectively) (n = 35) (Fig. 5C-c). There were no significant changes in the W/D (0.23 ± 0.03 vs 0.22 ± 0.03), L/D (0.31 ± 0.04 vs 0.32 ± 0.05), or cortical thickness (1.87 ± 0.59 vs 2.10 ± 0.48) between the untreated group and C3a-peptide group respectively (Fig. 5C-a, b, and f).

Importantly, as expected, the enhanced relative fluorescence intensities of F-actin in the sub-cortical region and surrounding the spindle, as well as the upregulated expression of C3aR caused by C3a-peptide, significantly decreased with C3aR morpholino administration (Fig. 5D). Quantitatively, the altered S1, S2, and S-C intensities caused by the C3a-peptide were partially restored by injecting the oocyte with C3aR morpholino; the mean S1 declined from 40.18 ± 12.0 to 32.2 ± 13.87 (P < 0.01) (Fig. 5F-a); the mean S2 decreased from 33.5 ± 9.00 to 22.0 ± 9.65 (P < 0.001) (Fig. 5F-b); and the S-C was lengthened from 24.3 ± 6.52 μm to 27.8 ± 4.65 μm (P < 0.01) (Fig. 5F-c) between the negative control and morpholino-treated groups. There were still no obvious changes in the W/D (0.22 ± 0.040 vs 0.23 ± 0.035, respectively, P > 0.05) (Fig. 5F-d), L/D (0.38 ± 0.056 vs 0.39 ± 0.045, respectively, P > 0.05) (Fig. 5F-e), and the cortical thickness (1.975 ± 0.87 vs 2.178 ± 0.83, respectively, P > 0.05) (Fig. 5F-f) between the negative control group (n = 41) and the C3aR morpholino-treated group (n = 43). To summarize, these observations indicated that the C3a-peptide recruited F-actin to the subcortex and around the spindle, accelerating F-actin aggregation and spindle migration, but did not affect spindle formation and that C3a-peptide exerted its biologic role primarily through its receptor, C3aR.

C3a-peptide/C3aR promotes F-actin aggregation and spindle migration during oocyte maturation by interacting with Myo10 but does not impact spindle formation

Next, we explored how C3a-peptide/C3aR influenced spindle migration and F-actin aggregation. Published studies showed that MYO10 directly links F-actin to spindle microtubules in mouse oocytes and thus participates in spindle off-centering. Proximity ligation assays (PLAs) provide highly specific and sensitive in situ detection of protein interactions within a complex [9]. We therefore investigated whether there was a direct interaction between MYO10 and C3aR by conducting PLAs and Co-IP assays. The results showed that the intensities of positive signals in C3a-peptide-treated oocytes were stronger than those in normal oocytes (Fig. 5E), indicating that C3a-peptide indeed enhanced the interactions between MYO10 and C3aR. Co-IP was carried out with C3aR antibody to verify this interaction, and IgG was used as a negative control (Fig. 5G). The results indicated that more MYO10 was immunoprecipitated in C3a-peptide-treated cells compared with untreated cells. Combining all these data, we can conclude that C3a-peptide delivery prompted F-actin aggregation and spindle migration by enhancing the interaction between MYO10 and C3a-peptide/C3aR.