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Follicular fluid C3a-peptide promotes oocyte maturation through F-actin aggregation - PubMed

️Sun Jan 01 2023

Follicular fluid C3a-peptide promotes oocyte maturation through F-actin aggregation

Ye Yang et al. BMC Biol. 2023.

Abstract

Background: Immature cumulus-oocyte complexes are retrieved to obtain mature oocytes by in vitro maturation (IVM), a laboratory tool in reproductive medicine to obtain mature oocytes. Unfortunately, the efficiency of IVM is not satisfactory. To circumvent this problem, we therefore intended to commence with the composition of ovarian follicular fluid (FF), an important microenvironment influencing oocyte growth. It is well known that FF has a critical role in oocyte development and maturation. However, the components in human FF remain largely unknown, particularly with regard to small molecular peptides.

Results: In current study, the follicular fluid derived from human mature and immature follicles were harvested. The peptide profiles of FF were further investigated by using combined ultrafiltration and LC-MS/MS. The differential peptides were preliminary determined by performing differentially expressed analysis. Human and mouse oocyte culture were used to verify the influence of differential peptides on oocyte development. Constructing plasmids, cell transfecting, Co-IP, PLA etc. were used to reveal the detail molecular mechanism. The results from differentially expressed peptide as well as cultured human and mouse oocytes analyses showed that highly conserved C3a-peptide, a cleavage product of complement C3a, definitely affected oocytes development. Intriguingly, C3a-peptide possessed a novel function that promoted F-actin aggregation and spindle migration, raised the percentage of oocytes at the MII stage, without increasing the chromosome aneuploidy ratio, especially in poor-quality oocytes. These effects of C3a-peptide were attenuated by C3aR morpholino inhibition, suggesting that C3a-peptide affected oocytes development by collaborating with its classical receptor, C3aR. Specially, we found that C3aR co-localized to the spindle with β-tubulin to recruit F-actin toward the spindle and subcortical region of the oocytes through specific binding to MYO10, a key regulator for actin organization, spindle morphogenesis and positioning in oocytes.

Conclusions: Our results provide a new perspective for improving IVM culture systems by applying FF components and also provide molecular insights into the physiological function of C3a-peptide, its interaction with C3aR, and their roles in enabling meiotic division of oocytes.

Keywords: C3a-peptide; C3aR; Human follicular fluid; Liquid chromatography-tandem mass spectrometry; MYO10; Oocyte in vitro maturation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

**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)

**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

**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

**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

**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

**Fig. 6**
Sketches illustrating the molecular mechanisms of C3a-peptide and C3aR action during meiotic division of oocytes. C3a-peptide accumulates in the follicular fluid during the follicular maturation process and then enters the cytoplasm of the oocytes, where it exerts its effects on maturation by binding to C3aR. C3aR and β-tubulin co-localize on the spindles and directly interact with an intermediate molecule, MYO10. MYO10 contains two domains, the head motor and MyTH4-FERM domain, which bind to F-actin and microtubules, respectively. Our result revealed that C3aR can bind to MYO10. Thus, these molecules collaborate to participate in F-actin aggregation and spindle dynamics in meiotic oocytes

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Follicular fluid C3a-peptide promotes oocyte maturation through F-actin aggregation - PubMed