Fam70A binds Wnt5a to regulate meiosis and quality of mouse oocytes

Abstract Objectives Little is known about the roles of integral membrane proteins beyond channels, carriers or receptors in meiotic oocytes. The transmembrane protein Fam70A was previously identified as a likely “female fertility factor” in Fox3a‐knockout mouse ovaries where almost all follicles underwent synchronous activation and the mice became infertile very early. However, whether Fam70A functions in oocyte meiosis remains unknown. Therefore, the present study aimed to address this question. Materials and Methods Co‐immunoprecipitation, immunogold labelling‐electron microscopy, co‐localization and yeast two‐hybrid assays were used to verify the interaction. Antibody or small interfering RNA transfection was used to deplete the proteins. Immunofluorescence, immunohistochemistry and live tracker staining were used to examine the localization or characterize phenotypes. Western blot was used to examine the protein level. Results Fam70A was enriched in oocyte membranes important for normal meiosis. Fam70A depletion remarkably disrupted spindle assembly, chromosome congression and first polar body extrusion, which subsequently increased aneuploidy and abnormal fertilization. Moreover, Fam70A directly bound Wnt5a, the most abundant Wnt member within oocytes. Depletion of either Fam70A or Wnt5a remarkably increased adenomatous polyposis coli (APC), which stabilizes active β‐catenin and microtubules. Consequently, depletion of either Fam70A or Wnt5a remarkably increased p‐β‐catenin (inactive form) and acetylated tubulin, while APC knockdown remarkably decreased these two. Furthermore, Fam70A depletion remarkably reduced Akt phosphorylation. Conclusions Fam70A regulates meiosis and quality of mouse oocytes through both canonical and non‐canonical Wnt5a signalling pathways.


| INTRODUC TI ON
Compared with somatic cells, meiotic oocytes are unique in several aspects. In addition to differences in cell division (meiosis vs mitosis), various maternal proteins exist in other tissues at low level whereas are uniquely or predominantly expressed and function within oocytes. For example, NLR family pyrin domain containing 5 (Nlrp5), also known as Mater protein homologue, belongs to the Nlrp family and is enriched within ovaries and oocytes. Nlrp5 is a subunit of the subcortical maternal complex that regulates cytoplasmic lattice formation, organelle positioning and distribution. 1 Nlrp5 depletion results in precocious sister chromatid separation and chromosome misalignment due to a reduction in centromere cohesion. 2 Notably, the Nlrp5 level is remarkably decreased in oocytes of ageing mice. 2 Furthermore, Nlrp5 is crucial for normal mitochondrial and endoplasmic reticulum distribution. 3 and calcium homoeostasis. 4 GDF9 and BMP15 are two well-known oocyte-predominant maternal proteins that are important for oocyte maturation. 5,6 However, a limited number of studies have investigated the functions of oocyte-predominant maternal proteins during meiosis.
Moreover, although integral membrane proteins account for about 30% of the total proteome, little is known about whether or how these proteins function during meiosis, except for those functioning as channels, carriers or receptors. Notably, a transcriptome-wide screening in Fox3a-knockout mouse ovaries identified 348 likely "female fertility factors," among which 13 are integral membrane proteins. 7 Fox3a is a well-known transcription factor downstream of the mTOR pathway, and it represses the expression of genes required for follicle activation and development. In Fox3a knockout mouse ovaries, almost all follicles undergo synchronous activation and the mice became infertile very early (4-month-old). 7 Therefore, genes with increased expression following Fox3a knockout may be important for follicle activation and development.  Figure S1A-D). We verified that Fam70A was more abundant in mouse ovaries than Fam70B ( Figure S1E). Furthermore, Fam70A but not Fam70B was screened as a "female fertility factor," suggesting that it serves a unique role in ovaries and oocytes. 7 The present study revealed that Fam70A directly bound an oocyte-predominant Wnt family member, Wnt5a, to regulate oocyte meiosis through the canonical Wnt-β-catenin pathway, which is a highly conserved signalling pathway important in a number of physiological processes, including early embryonic development, organ formation and tissue regeneration. [8][9][10] Furthermore, Fam70 was shown to regulate the activity of Akt, a well-known meiosis regulator. [11][12][13] 2 | MATERIAL S AND ME THODS

| General chemicals, reagents and animals
Chemicals and reagents were obtained from Millipore Sigma unless otherwise stated. Institute of Cancer Research (ICR) mice (female, age 3-4 weeks) used in this study were supplied by Vital River Experimental Animal Technical Co. All animal experiments were approved by the Animal Care and Use Committee of Nanjing Medical University and were performed in accordance with institutional guidelines.

| Antibodies
The following primary Abs were used: Rabbit anti-Fam70a Ab (Cat#:

| Oocytes collection and culture
Fully grown immature oocytes arrested in GV stage were obtained from the ovaries of 3-4-week-old ICR female mice. Oocytes were re- were cultured at 37.0°C, 5% O 2 , 5% CO 2 in a humidified atmosphere.

| Antibody transfection
Chariot™ Protein Delivery Reagent (Active Motif) was used for antibody transfection. Abs for transfection have been thoroughly buffer exchanged (>10 4 dilutions of original buffer) into PBS/50% glycerol with a size-exclusion spin column (cutoff, 100 kD a ; spin speed, 5000 rpm; Millipore, Sigma) to remove antiseptics (usually N a N 3 ) in the original package. In brief, two tubes, one of which contained 1 µL chariot (1 mg/mL in 50% DMSO) in 5 µL sterile water, the other 1 µg Ab in PBS (final volume, 6 mL), were first set up. Solutions from the two tubes were mixed gently and incubated at room temperature for 30 minutes to allow for the formation of the chariot-IgG complex.
The complex solution was added into a 100 mL MEM + drop that contained 50 oocytes. After 12-14 hours of treatment, oocytes were washed to remove the complex-containing MEM+. Oocytes were then prepared for WB or transferred into milrinone-free MEM+ and cultured for 8-16 hours for phenotype analysis-related experiments below. During the entire treatment, 2.5 mmol/L milrinone was included to prevent the resumption of meiosis.

| Small interfering RNA knockdown
Sequences of all DNA templates for small interfering RNA (siRNA) were designed by using the online siRNA design service, BLOCK-iT RNAi Designer (http://rnaid esign er.invit rogen.com/rnaie xpres s/) with some modification. Sequence specificity was confirmed by a blast homology search. The control template is a mock sequence that was not complementary to any mouse mRNA. Detailed sequence information is in Tables S1 and S2.
Small interfering RNAs were produced by using the T7 RiboMAX Express RNAi System (Promega) according to the manufacturer's instructions. In brief, for each double-stranded siRNA against one of the four target gene regions, two pairs of synthesized complementary single-stranded DNA oligonucleotides were first annealed to form 2 double-stranded DNA templates. Subsequently, two complementary single-stranded siRNAs were separately synthesized in accordance with these two templates and then annealed to constitute a final double-stranded siRNA. siRNA was then purified by conventional phenol/chloroform/isopropanol precipitation, which was aliquoted and stored at −80°C after a quality check on an agarose gel. A ready-to-use siRNA mixture was set by mixing siRNAs against four distinct target regions at an equal molar ratio to a final concentration of 5 µmol/L. During the entire siRNA treatment, typically 36-44 hours long, 2.5 mmol/L milrinone was included to prevent the resumption of meiosis. Oocytes were then collected for experiments described in data analysis and statistics.

| In vitro fertilization
Spermatozoa were obtained from the epididymis of 10-18-weekold B6-DBA2 F1 male mice and were then capacitated in 1 mL MEM + for 1 hour. Subsequently, 10 µL of the suspension containing 5-10 x 10 6 /mL spermatozoa was added to 490 µL MEM + medium, and oocytes washed off FBS were added. Five hours later, the spermatozoa remaining on the surface of oocytes were washed off by pipetting. After another 4 hours, the oocytes were processed for immunoassaying to determine the frequency of successful fertilization, by the identification of the formation of pronuclei.

| Immunofluorescence
After washed in PBS with 0.05% polyvinylpyrrolidone (PVP) briefly, oocytes were permeabilized in 0.5% Triton X-100/PHEM (60 mmol/L PIPES, 25 mmol/L HEPES pH 6.9, 10 mmol/L EGTA, 8 mmol/L MgSO 4 ) for 5 minutes and washed three times rapidly in PBS/ PVP. Next, the oocytes were fixed in 3.7% paraformaldehyde (PFA)/PHEM for 20 minutes and blocked in 1% BSA/PHEM with 100 mmol/L glycine at room temperature for 1 hour. Then, the oocytes were incubated at 4°C overnight with primary antibody diluted in blocking buffer. After being washed three times (10 minutes each) in PBS with 0.05% tween-20, the oocytes were incubated at room temperature for 45 minutes with secondary antibody diluted in blocking buffer (1:750 in all cases). Finally, chromosomes were stained by 10 µg/mL Hoechst 33342 (Sigma) for 10 minutes and the oocytes were mounted onto a slide with mounting medium (0.5% proposal gallate, 0.1 mol/L Tris-Hcl, pH 7.4, 88% Glycerol) and covered with a cover glass (0.13-0.17 µm thick). To maintain the dimension of the oocytes, two strips of double-stick tap (90 µm thick) were stuck between the slide and cover glass. The oocytes were examined with an Andor Revolution spinning disk confocal workstation (Oxford instruments).

| Chromosome spread
Oocytes were exposed to Tyrode's buffer (pH 2.5) for about 40 seconds to remove zona pellucida and then fixed in a drop of 1% PFA with 0.15% Triton X-100 (pH = 9.2) on a glass slide. Kinetochore and chromosome were stained as that of immunofluorescence. The Andor Revolution spinning disk confocal workstation was used to examine chromosome and kinetochore numbers in oocytes.

Fam70A-interacting proteins
For silver staining, immunocomplex beads from the control IgG or

| Yeast two-hybrid assays
The coding sequence of Fam70A was cloned into pGADT7, and the coding sequences of Wnt5a were cloned into pGBKT7. Two plasmids were together transformed into yeast strain AH109. Empty vectors were used as negative controls. The transformed yeast was grown on synthetically defined SD/-Trp-Leu dropout medium plates for 2-4 days. The yeast cells with co-transformation of the two fusion plasmids were further dropped on SD/-Trp-Leu-His-Ade dropout medium plates for further verification of the interactions.

| Immunogold-electron microscopy of oocytes
Metaphase I oocytes were blocked with 1% BSA, incubated with Fam70A Ab (4°C, overnight) and next labelled with Donkey anti-rabbit IgG/Gold (35 nm, room temperature for 2 hours). Then, oocytes were re-blocked with 1% BSA (to eliminate the cross-reaction between two antibodies), incubated with Wnt5a Ab (4°C, overnight) and then labelled with Donkey anti-rabbit IgG/Gold (15 nm, room temperature for 2 hours).
After being labelled, oocytes were fixed in 2.5% glutaraldehyde for 2 hours at 4°C, washed three times with PBS and stained with eosin for 2 minutes to facilitate the oocyte positioning by eye. Oocytes were then placed in 2% agarose and spun for 5 minutes at 13 000 rpm and held overnight at 4°C. The next day, the agarose piece with eosin-stained oocytes (red in the piece) was trimmed and sent to the Testing and Analysis Center (Nanjing Medical University) for sample preparation for transmission electron microscopy. Electron microscopy pictures were obtained with a transmission electron microscope (FEI Tecnai G2 Spirit Bio Twin; Thermo Fisher Scientific).

| Data analysis and statistics
All experiments were repeated at least three times. Measurement of Western blot and confocal images was conducted with Image J.
(National Institutes of Health). Data are presented as mean ± SEM.
Statistical comparisons between 2 groups were made with the Student's t test of the Excel program (Microsoft). Multiple comparisons were made by using the Kruskal-Wallis one-way nonparametric ANOVA (Prism; GraphPad Software). Values of P < .05 were considered statistically significant.

| The "female fertility factor" Fam70A is important for the meiotic progression of IVM mouse oocytes
The integral membrane protein Fam70A was previously identified as a "female fertility factor" and is the only Fam70 family member with a predominant expression in the ovaries. 7 Therefore, we hypothesized that this protein may be important for oocyte meiosis.
We first examined the localization and expression of Fam70A within mouse oocytes. Results showed that Fam70A was more highly expressed within oocytes than granulosa cells ( Figure 1A,B).
The expression in the ovaries increased sharply as follicles were initially recruited (PND 21) ( Figure 1C). During meiosis, Fam70A exhibited a constant expression level ( Figure 1D) and was exclusively concentrated on oocyte membranes ( Figure 1E,F).
Next, we examined whether Fam70A knockdown affected oocyte meiosis. Fam70A protein levels were remarkably reduced with peptide-mediated antibody transfection (Figure 2A,B), as previously described. 14,15 We found that at 7.5 hours of in vitro maturation F I G U R E 1 The "female fertility factor" Fam70A is enriched on the oocyte membrane. A, Immunohistochemistry showed that Fam70A was enriched on the oocyte membrane of growing oocytes. Fam70A was developed in brown; DNA was stained with haematoxylin. Two secondary follicles were shown. B, Western blot showed that Fam70A was more abundant in oocytes (Oos) than in granular cells (GCs). C, Western blot showed that the Fam70A protein level remarkably elevated as follicles were initially recruited (PND 21). D, Western blot showed that the Fam70A protein level kept constant during meiosis. E, Z-slices of Fam70A immunofluorescent image showed that Fam70A was enriched on the oocyte membrane. F, Immunofluorescence showed that Fam70A remained on the oocyte membrane during meiosis. Scale bar, 20 µm. GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II; PND, post-natal day (IVM), spindle organization was severely disrupted and chromosome congression was impeded. Furthermore, the percentage of These results indicated that Fam70A, an integral membrane protein that was screened as a "female fertility factor," was important for the meiotic progression of IVM oocytes.

F I G U R E 2
Fam70A is important for normal meiosis and fertilization of in vitro maturation (IVM) mouse oocytes. A, Western blot showed that Fam70A could be remarkably reduced by chariot-mediated antibody transfection. B, Immunofluorescence showed that Fam70A staining was remarkably reduced by chariot-mediated antibody transfection. C, Immunofluorescence showed that Fam70A depletion (70A-DE) remarkably disrupted chromosome congression at 7.5 h of IVM. D, Quantification of (C). E, Immunofluorescence showed that Fam70A depletion retarded 1 pb (first polar body) extrusion at 12 h of IVM. F, Representative bright-field images of oocytes at 14.5 h of IVM. Numbers in the images indicate the number of MII oocytes/number of total oocytes. G, Quantification of (F). H, Immunofluorescence showed that Fam70A depletion remarkably increased oocytes with aneuploidy after Fam70A depletion. I, Quantification of (H). J, Immunofluorescence of in vitro fertilization oocytes showed that Fam70A depletion remarkably reduced fertility rate and 2-PN (pro-nucleus) rate. K, Quantification of (J). Except for (B) (Fam70A in green), tubulin in green, kinetochores in red, DNA in blue. Scale bar in (C) and (H), 5 µm; Scale bar in other panels, 20 µm. *P < .05. MII, metaphase II

| Fam70A is important for oocyte quality
The discrepancy between MII, fertilization and 2-PN rates suggested that certain Fam70A-depleted MII oocytes were not fully competent. Mitochondria are responsible for ATP production, and mitochondrial dysfunction may decrease oocyte viability. 16 Autophagy is an important process wherein LC3B-labelled cellular waste is encapsulated in autosomes and subsequently transported to the lysosomes for degradation. Decreased autophagy prevents the removal of waste, thereby reducing oocyte viability. 17 Therefore, mitochondrial dysfunction and autophagy were further investigated. First, mitochondria distribution analysis showed that mitochondria formed several large aggregates in Fam70A-depleted oocytes ( Figure 3A Over-aggregation of mitochondria or lysosomes suggested that mitochondrial function and normal autophagy were severely disrupted in Fam70A-depleted oocytes, as also demonstrated by decreased ATP and increased LC3B levels, and may adversely affect oocyte quality.

| Fam70A directly interacts with oocytepredominant Wnt5a
Next, we performed IP-MALDI to identify proteins that interact with Fam70A ( Figure 4A; Figure S2A). A PubMed search revealed that four proteins, which were specifically enriched within the Fam70A IP complex but not in the control, might be correlated with the Wnt-βcatenin pathway ( Figure S2B; Table S1). This suggested that Fam70A may interact with Wnt signalling. By systematic comparison of the abundance of all Wnt family members, we found that Wnt5a is the most abundant member within mouse oocytes ( Figure 4B,C). Costaining showed that Fam70A and Wnt5a co-localized ( Figure 4D).
Co-IP in oocytes and transfected NIH3T3 cells showed that the two proteins interacted with each other ( Figure 4E,F), while a yeast two-hybrid assay confirmed their direct interaction ( Figure 4G).

Furthermore, immunogold-electron microscopy showed that
Fam70A and Wnt5a were located to each other (<20 nm) on oocyte membranes and microvilli ( Figure 4H). These results indicated that Fam70A may directly bind Wnt5a during meiosis.

| Fam70A and Wnt5a regulate the APC level and microtubule stability
The

| Fam70A and Wnt5a regulate β-catenin activity
The transcription factor β-catenin is a major downstream effector of the canonical Wnt signalling pathway. Therefore, we examined whether Fam70A and Wnt5a regulate its activity. The results showed that either Fam70A or Wnt5a could co-immunoprecipitate with β-catenin ( Figure 6A,B). Furthermore, depletion of either Fam70A ( Figure 6C-E) or Wnt5a ( Figure 6F,G) remarkably increased p-β-catenin levels. Conversely, APC knockdown F I G U R E 3 Fam70A is important for oocyte quality. A, Fluorescent live tractor staining showed that Fam70A depletion caused mitochondria over-aggregation. Mitochondria in red, DNA in blue. B, Quantification of (A). C, Fam70A depletion remarkably reduced ATP concentration within oocytes. D, Fluorescent live tractor staining showed that Fam70A depletion remarkably increased lysosome aggregation. Lysosome in red, DNA in blue. E, Quantification of (D). F, Western blot showed that LC3B remarkably increased in Fam70A-depleted oocytes. G, Quantification of (F). Scale bar, 20 µm. *P < .05 remarkably reduced p-β-catenin levels ( Figure 6H,I). These results suggested that Fam70A and Wnt5a function in a canonical Wnt-βcatenin signalling pathway.

| Fam70A and Wnt5a regulate the activity of Akt involved in normal meiosis
In mitotic somatic cells, Wnt regulates several kinases independently of the APC-β-catenin pathway, which is termed the "non-canonical Wnt pathway." 21,22 The present study examined whether Fam70A functions through a similar non-canonical Wnt pathway. Akt is the upstream regulator of several kinases, including mTOR, which regulates follicle activation and oocyte quality. 23,24 Furthermore, Akt is known to regulate oocyte quality and meiosis. [11][12][13] Therefore, Akt was investigated in the present study.
Western blot and immunofluorescence staining showed that depletion of Fam70A dramatically reduced the phosphorylation (activation) of Akt at both S473 ( Figure 7A-C) and T308 ( Figure 7D-F).
These results suggested that Fam70A may also function through a non-canonical Wnt pathway.

| D ISCUSS I ON
The present study revealed that the "female fertility factor" Fam70A may regulate meiosis through multiple pathways.
Furthermore, to the best of our knowledge, we were the first to show that Wnt5a may be the main Wnt family member that regulates the APC expression level and β-catenin activity during meiosis in oocytes. Moreover, we showed that Fam70A directly binds Wnt5a to regulate both canonical and non-canonical Wnt signalling pathways.
The roles of integral membrane proteins in meiosis in IVM oocytes have not been widely investigated. One reason may be that it is thought that IVM oocytes do not require membrane-initiating signals, since IVM cumulus-free oocytes are able to undergo meiosis in protein-free media. However, other studies suggested that membrane-initiating signals are important for in vitro female meiosis. For example, adenosine present in mouse follicular fluid has been shown to prevent oocyte maturation in vitro. However, this effect was not due to adenosine transport into oocytes or adenosine-induced ATP level increment. It was proposed that adenosine exerts a maturation-inhibitory effect in oocyte membranes. 25 Calreticulin, a protein F I G U R E 4 Fam70A interacts directly with oocyte-predominant Wnt5a. A, SDS-PAGE and silver staining of Fam70A antibody or control IgG IP complex showed many distinct bands; we cut and sent these distinct bands for IP-MALDI to identify the potential interacting proteins of Fam70A (see also Figure S2A). B, Among all Wnt members, we found that Wnt5a (red dot-line square) is the most abundant within mouse oocytes. C, Quantification of (B). D, Immunofluorescence showed that the Fam70A signal on the oocyte membrane highly overlapped with Wnt5a. Fam70A in green, Wnt5a in red, DNA in blue. E, Co-IP in oocyte lysate showed that endogenous Fam70A and Wnt5a interacted with each other. F, Co-IP in the lysate of NIH3T3 cells co-transfected with Fam70A and Wnt5A plasmids showed that exogenous Fam70A and Wnt5a also interacted with each other. G, Yeast 2-hybrid assays showed that Fam70A directly binds Wnt5a on both Trp/Leu-deficient and Trp/Leu/His/Ade-deficient Agar plate. H, Immuno-EM showed that Fam70A localized close (<20 nm) to Wnt5a on oocyte membrane or microvillus. Fam70A and Wnt5a were labelled with 35 and 15 nm gold particles, respectively. Scale bar in E, 20 µm. Scale bar in F, 400 nm. GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II best known as an endoplasmic reticulum chaperone, is present on the extracellular surface of the plasma membrane of mouse oocytes.
Calreticulin inhibition altered cortical actin distribution and meiosis resumption independently of intercellular calcium, suggesting that calreticulin mediates transmembrane signalling linked to meiosis resumption. 26 In the present study, we showed that The Wnt-β-catenin signalling pathway is highly conserved and serves important roles in early embryonic development, organ formation, tissue regeneration and other physiological processes. [8][9][10] When Wnt signalling is inactivated, APC complexes phosphorylate β-catenin, leading to the degradation of β-catenin, which prevents the nuclear deposition of β-catenin and related transcriptional regulation. [8][9][10] The Wnt family includes 16 members and has distinct expression within mouse tissues, 29 and not surprisingly, different tissues express distinct Wnt members. For example, Wnt9b is predominantly found in the kidney and is important for the development of mesonephric and metanephric tubules, and for caudal extension of the Müllerian duct through the non-canonical Rho/Jnk branch of the Wnt pathway. 30 Wnt5a is enriched in lungs, and its expression is significantly higher in non-small-cell lung cancer and was positively correlated with that of β-catenin, vascular endothelial growth factor A and Ki-67. 31 Reduction in both canonical and non-canonical Wnt5a F I G U R E 5 Fam70A and Wnt5a regulate adenomatous polyposis coli (APC) level and MT stability. A, Western blot showed that APC protein remarkably increased after Fam70A depletion. B, Quantification of (A). C, Immunofluorescence showed that APC was enriched within spindles and remarkably increased after Fam70A depletion. APC in green, tubulin in red, DNA in blue. D, Western blot showed that Wnt5a was efficiently reduced by siRNA (Table S2). E, Western blot showed that APC remarkably increased after Wnt5a knockdown. F, Quantification of (E). G, RT-PCR showed that APC was efficiently reduced by siRNA (Table  S3). H, Fam70A depletion and Wnt5a knockdown both remarkably increased acetylated tubulin level within spindles, while APC knockdown remarkably reduced acetylated tubulin level. Acetylated tubulin in green, DNA in blue. I, Quantification of (H). Scale bar, 5 µm. *P < .05 signalling is linked to impaired lung repair in chronic obstructive pulmonary disease. 32 However, to the best of our knowledge, the specific Wnt member present in oocytes had not been previously determined. In the present study, we found that Wnt5a was the most abundant Wnt protein in oocytes and that Wnt5a knockdown altered the APC level and β-catenin phosphorylation. Collectively, the results suggested that Wnt5a is the primary Wnt protein involved in oocyte meiosis.
The basic mechanism of the Wnt-β-catenin pathway is that β-catenin acts as a transcription factor that enters the nucleus and modulates transcription. However, in fully grown IVM oocytes, the transcription activity is quiescent, suggesting that β-catenin is not likely to affect meiosis, but rather serves important roles in subsequent early embryo development. [8][9][10] However, APC, the intermediate protein of the Wnt-β-catenin pathway, may affect in vitro female meiosis. For example, in PTK2 cells, APC was found to bind to microtubules and increased microtubule stability in vivo and in vitro.
Deletion of its microtubule binding site from the C-terminal domain did not eliminate its binding but decreased its capacity to stabilize microtubules. 18 In MCF-7 cells, APC is the direct binding partner of ends-binding 1 and regulates microtubule stability; APC knockdown reduced microtubule stability and cell migration. 19 The present study revealed that depletion of either Fam70A or Wnt5a remarkably increased APC expression and acetylated tubulin levels within the F I G U R E 6 Fam70A and Wnt5a regulate β-catenin activity. A, Co-IP showed that Fam70A interacted with β-catenin within oocytes. B, Co-IP showed that Wnt5a interacted with β-catenin within oocytes. C, Western blot showed that Fam70A depletion remarkably increased p-βcatenin level. D, Quantification of (C). E, Immunofluorescence showed that Fam70A depletion remarkably increased p-β-catenin at spindle poles. F, Western blot showed that Wnt5a knockdown remarkably increased p-β-catenin level. G, Quantification of (F). H, Western blot showed that APC knockdown remarkably decreased p-β-catenin level. I, Quantification of (H). Scale bar, 5 µm. *P < .05 F I G U R E 7 Fam70A regulates Akt activity. A, Western blot showed that Fam70A depletion dramatically decreased p-Akt S473 level. B, Quantification of (A). C, Immunofluorescence showed that Fam70A depletion dramatically decreased p-Akt S473 level within spindles. p-Akt S473 in green, tubulin in red, DNA in blue. D, Western blot showed that Fam70A depletion dramatically decreased p-Akt T308 level. E, Quantification of (D). F, Immunofluorescence showed that Fam70A depletion dramatically decreased p-Akt T308 level within spindles. p-Akt T308 in green, tubulin in red, DNA in blue. Scale bar, 5 µm. *P < .05 spindles. APC knockdown remarkably reduced acetylated tubulin levels. Several studies showed that overstable microtubules could impede mitotic cell division. 33,34 Taken together, the results indicated that APC, as a downstream effector of Fam70A and Wnt5a, may be one of the important modulators of their roles in female meiosis.
In mitotic somatic cells, Wnt functions through not only the canonical Wnt-β-catenin pathway, but also the non-canonical Wnt pathways. The extracellular domain of Ptk7 acts as an important regulator of both non-canonical Wnt/planar cell polarity (PCP) and canonical Wnt/β-catenin signalling in zebrafish embryo development. 21 In basal breast cancer, the archetypal Wnt/PCP protein VANGL PCP protein 2 (VANGL2) is upregulated and is associated with poor prognosis and tumour growth through the VANGL2-p62/ SQSTM1-JNK pathway. 22 In the present study, Fam70A depletion also reduced Akt activity. Akt activation is important for mouse oocyte meiosis. [11][12][13] Thus, Fam70A may also regulate female meiosis through the non-canonical Wnt pathway.
In all, the present study revealed that Fam70A is a "female fertility factor" that may bind Wnt5a to regulate the APC level and Akt activity through canonical and non-canonical signalling, respectively ( Figure 8). However, Fam70A may also work independently of Wnt5a to regulate important meiotic kinases, such as Akt. This needs further investigation.

ACK N OWLED G EM ENT
This work was financially supported by the National Key Research

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Dong Zhang
https://orcid.org/0000-0002-0257-4441 F I G U R E 8 Fam70A working model. At cell membrane, Fam70A directly binds Wnt5a, which results in the inhibition of APC and activation of Akt (p-Akt). APC promotes the phosphorylation (inactive form) of β-catenin and stabilizes microtubules as well. β-Catenin (active form) can get into the nucleus to promote gene transcription (but not in fully grown oocytes). Proper microtubule stability and p-Akt together promote meiosis and quality of mouse oocytes