The 5.8S pre‐rRNA maturation factor, M‐phase phosphoprotein 6, is a female fertility factor required for oocyte quality and meiosis

Abstract Objectives M‐phase phosphoprotein 6 (MPP6) is important for 5.8S pre‐rRNA maturation in somatic cells and was screened as a female fertility factor. However, whether MPP6 functions in oocyte meiosis and fertility is not yet known. We aimed to address this. Materials and Methods Mouse oocytes with surrounded nucleus (SN) or non‐surrounded nucleus (NSN) were used for all experiments. Peptide nanoparticle‐mediated antibody transfection was used to deplete MPP6. Immunofluorescence staining, immunohistochemistry and live tracker staining were used to examine MPP6 localization and characterize phenotypes after control or MPP6 depletion. High‐fidelity PCR and fluorescence in situ hybridization (FISH) were used to examine the localization and level of 5.8S rRNAs. Western blot was used to examine the protein level. MPP6‐EGFP mRNA microinjection was used to do the rescue. Results MPP6 was enriched within ovaries and oocytes. MPP6 depletion significantly impeded oocyte meiosis. MPP6 depletion increased 5.8S pre‐rRNA. The mRNA levels of MPP6 and 5.8S rRNA decreased within ageing oocytes, and MPP6 mRNA injection partially increased 5.8S rRNA maturation and improved oocyte quality. Conclusions MPP6 is required for 5.8S rRNA maturation, meiosis and quality control in mouse oocytes, and MPP6 level might be a marker for oocyte quality.


| INTRODUC TI ON
Based on recent knowledge, researchers agree that oocyte maturation should include cytoplasmic, nuclear and epigenetic maturation, [6][7][8] and further studies are required to completely resolve the mechanism. Recently, researchers identified many potential female fertility factors through transcriptome [8][9][10][11] and proteome-wide [12][13][14] analyses. For loss-of-function studies, besides small interfering RNA knockdown, the protein-depletion method using specific antibodies and trim21-mediated protein degradation provided a powerful tool, 15 which we have applied successfully to IVM oocytes. 16,17 Ribosomal RNAs (rRNAs), including 5S, 5.8S, 18S and 28S, are the main components of ribosomes in eukaryotes. rRNAs maintain the structure of ribosomes together with constitutive ribosomal proteins and also function as peptidyl transferases to catalyse the formation of peptide bonds between amino acids during protein translation. Mature rRNAs come from the multi-step splicing of pre-rRNAs. Specifically, from 5′ to 3′, the 45S pre-rRNA contains 18S, 5.8S and 28S rRNA, and intermediate sequences between them. 45S pre-rRNA is first spliced to separate out 18S rRNA, and then, the remaining part is spliced into 5.8S and 28S rRNAs. Many ribosome-associated proteins participate in this processes. 18 For example, in mitotic human somatic cells, poly(A)-specific ribonuclease participates in 18S rRNA maturation. Knockdown of poly(A)-specific ribonuclease or exogenous expression of a dead mutant (D28A) induces 18S pre-rRNA accumulation in both the cytoplasm and nucleus. 19 5′-3′ exonuclease Rrp17p binds to late pre-60S ribosomes and is required for maturation of the 5′ ends of 5.8S and 25S rRNA. 20 Ribosome synthesis factor Rrp5 participates in multiple steps of pre-rRNA splicing; the C-terminal domain is required for 18S rRNA synthesis, whereas the N-terminal domain is required for 5.8S and 25S rRNA maturation. 21 Studies of the involvement of specific proteins in rRNA maturation in meiotic oocytes are very scarce.
Recently, it was shown that DDB1 and cullin-4-associated factor 13 (DCAF13) were rich in the nucleolus of non-surrounded nucleolus (NSN) oocytes, but become undetectable in surrounded nucleolus (SN) oocytes. DCAF13 deletion inhibited nucleolus maturation through inhibiting protein synthesis without affecting mRNA transcription. The mechanism involved the participation of DCAF13 in 18S rRNA maturation. 22 M-phase phosphoprotein 6 (MPP6) was initially identified together with other MPPs by MPM2, a monoclonal antibody that recognizes a group of related M-phase phosphorylation sites, including F-phosphor-P-L-Q. 23,24 These MPPs mostly had characteristic and distinct localization patterns during mitosis compared with the patterns during interphase. However, these MPPs do not share high sequence similarity or conserved domains, indicating their diversified functions. 23,24 Notably, MPP6 is the only MPP to be screened as a "female fertility factor" in foxo3 -/mouse ovaries. 25 Several studies in somatic cells have shown that MPP6 plays an important role in the maturation of 5.8S rRNA, and the degradation of various other RNAs, by recruiting the nuclear exosome complex. [26][27][28] However, there are no MPP6 studies in oocyte meiosis. In the present study, we found that MPP6 was enriched within ovaries and oocytes, and important for 5.8S rRNA maturation within oocytes. Furthermore, MPP6 was important for normal meiosis, fertilization and oocyte quality. Finally, decreased MPP6 and its mis-localization appeared to be related to oocyte ageing.

| Chemicals and antibodies
All chemicals and reagents were obtained from Sigma unless otherwise stated.

| Oocyte collection and culture
GV oocytes were obtained from the ovaries of 3-week-old ICR mice supplied by the Animal Core Facility of Nanjing Medical University.
The mice were euthanized by CO 2 inhalation and cervical dislocation, and ovaries were isolated and placed in operation medium

| In vitro Fertilization (IVF)
Spermatozoa were obtained from the epididymis of 10-weekold B6 x DBA2 F1 male mice and were then capacitated in 1ml MEM + for 1 hour. Control or MPP6-DE oocytes were washed rapidly for 3 times with MEM + medium to remove FBS right before fertilization. Next, 10 µl of a sperm suspension containing 5-10 × 10 6 /mL spermatozoa was added to 490 µl MEM + medium, and FBS-free oocytes were added. 9 hours later, the oocytes were processed for immunofluorescence and examined to determine the frequency of successful fertilization, by the identification of the formation of pronuclei.

| Detection of ROS generation
The ROS Assay Kit (Cat#: S0033, Beyotime) was used to detect ROS generation in oocytes. Briefly, oocytes were incubated with dichlorofluorescein diacetate (DCFH-DA) probe for 20 min at 37°C in the dark, washed and mounted on slides for confocal imaging. After washing, oocytes were mounted onto glass slides, and images were obtained as above.

| Immunofluorescence staining
Oocytes were briefly washed in PBS with 0. were positioned between the slide and cover glass. Images were obtained as above.

| Fluorescence in situ hybridization (FISH)
FISH was performed with the antisense oligonucleotide probes listed in Table S2. The probes were synthesized by Genewiz

| mRNA production and microinjection
For mRNA production, MPP6 and EGFP full-length coding sequences were amplified and cloned into pBluescript II SK (+) at BamHI/EcoRV and EcoRV/XhoI restriction sites for a fusion expression of MPP6-EGFP. The constructed plasmid was digested and linearized by PsiI, purified as a DNA template for MPP6-EGFP mRNA transcription. T3 mMessage mMachine Ultra Kit (Ambion) was used to transcribe the initial MPP6-EGFP mRNA, and then, Poly(A) Tailing Kit (Ambion) was used to elongate the 3' UTR and stabilize the mRNA.
For microinjection, a thin-wall glass tube with a built-in guide wire (1.0 mm outer diameter, 0.8 mm inner diameter; World Precision Instruments) was pulled into two needles with Micropipette Puller P-97 (Sutter Instrument). Then, the needle tip was bent on a Micro Forge (Narishige) at 30 degrees. MPP6-EGFP mRNA (500-1000 ng/µl) was loaded into the front part of the tip by guide wire-assisted siphonage. Then, the needle was loaded onto the 3-D electromechanical arm of a micromanipulator (Narishige) installed on a Ti-S inverted fluorescence microscope (Nikon) and connected with a nitrogen-driven programmable injector (Narishige). The injection time was about 10-20 ms, and the injection volume was about 10-20 pl.

| Data analysis and statistics
All experiments were repeated at least three times. Measurements of immunohistochemistry images, immunofluorescence images, FISH images, Western blot and PCR bands were all performed with ImageJ (NIH). Net intensity was obtained by subtracting mean object intensity by mean background intensity around the object. Data were presented as mean ± SEM. Statistical comparisons between two groups were made with Student's t test. Statistical significance was set at P < .05.

| The female fertility factor MPP6 is important for 5.8S rRNA maturation in FGOs
Nuclear exosome complex is required for the maturation of 5.8S rRNA in human somatic cells, while MPP6 is a nucleolus-specific exosome co-factor that binds preferentially to poly(C) and (U)rich ITS2 region to recruit the exosome. [26][27][28] For SN FGOs that are transcriptionally quiescent, some researches suggested that rDNA-containing nucleolus-like bodies did not contain transcribed rRNA genes, pre-rRNAs or pre-ribosomes. [29][30][31] Thus, we first wanted to verify whether pre-rRNA existed within SN FGOs.
Due to the low homology of the ITS2 region between human and mouse ( Figure S1), we could not deduce which subsection of the ITS2 region MPP6 might bind in the mouse. Thus, we designed two forward primers (F0 and F1) inside the mature 5.8S rRNA, and eight reverse primers within mature 5.8S rRNA (R0) or different poly(C) or (U)-rich ITS2 regions of pre-rRNA (R1-R7, Figure 1A, Table S2). We also designed a pair of primers for the 5'ETS and ITS1 regions of pre-rRNA.
We first strictly subtyped FGOs into NSN and SN FGOs  Table S2). (B) FGOs (fully grown oocytes) from large antral follicles were stained with live-cell dye hoechst 33 258 and then strictly subtyped into NSN (non-surrounded nucleus) FGOs and SN (surrounded nucleus) FGOs for subsequent experiments. (C and D) Sanger sequencing showed that PCR product with 5'ETS (C) or F0 + R3 (D) primer in the SN FGOs was identical to the 5'ETS pre-rRNA or 5.8S pre-rRNA. (E) RT-PCR showed that strong bands can be detected by any of primer pairs above (A) in both NSN and SN, but only products with 5'ETS primer and F0 + R2, not with ITS1, significantly decreased in SN FGOs. GAPDH was the loading control. (F) Quantification of E. (G) Fluorescence in situ hybridization (FISH) of pre-5.8S rRNA with FAM-R2 probe showed that in NSN oocytes, 5.8S pre-rRNA was enriched within nucleus; while in SN oocytes, 5.8S pre-rRNA significantly decreased within nucleus but tended to increased within the cytoplasm around nucleus. (H) Quantification of FAM-R2 intensity of oocytes (Oo), nucleus (Nu) and cytoplasm (Cyto). (I) Representative intensity curve of cytoplasmic FAM-R2 showed that FAM-R2 intensity was higher around the nucleus in SN oocytes than in NSN oocytes. (J) FISH of pre-rRNA with TRMRA-5'ETS probe showed that in NSN oocytes, 5'ETS pre-rRNA was enriched within nucleus; while in SN oocytes, 5'ETS pre-rRNA significantly decreased within nucleus but tended to increased within the cytoplasm around nucleus. (K) Quantification of TRMRA-5'ETS intensity of oocytes (Oo), nucleus (Nu) and cytoplasm (Cyto). (L) Representative intensity curve of cytoplasmic TRMRA-5'ETS showed that TRMRA-5'ETS intensity was higher around the nucleus in SN oocytes than in NSN oocytes. Scale bar, 20 µm. *P < .05 that nuclear rRNA intensity was significantly higher in the NSN oocytes than in the SN oocytes for both probes (FAM-R2 nuclear intensity, Figure 1G

| MPP6 is rich in oocytes and important for their in vitro maturation
MPP6 was screened as a female fertility factor and found to be abundant in ovaries and oocytes in a transcriptome study. 25 However, there are no studies to verify this finding. We found that MPP6 was abundant within ovaries at both the mRNA and protein levels (Figure 2A,B). Immunohistochemistry also showed that MPP6 was enriched in oocytes ( Figure 2C), and the abundance remained constant as the follicles developed ( Figure 2C,D). Furthermore, MPP6 was more abundant within oocytes than in granulosa cells ( Figure 2E,F). These findings suggested that MPP6 might be important for oocyte maturation.
Immunofluorescence showed that MPP6 was enriched within nuclei at the GV stage, and within spindles at the MI and MII stages.
The abundance remained constant during meiosis, indicating that it might be involved in this process ( Figure 3A,B).

| MPP6 is important for oocyte quality in multiple ways
The meiotic spindle is not only the important apparatus for chromosome segregation, but also an important cytoskeletal structure for various cellular processes, including activation and inactivation of diverse microtubule-associated kinases. Thus, presumably, spindle disruption caused by MPP6 depletion could affect various important cellular processes and meiotic kinases, and consequently affect oocyte quality.
To assess these possibilities, we examined the levels of reactive oxygen species (ROS), annexin V, and ATP, and the distribution of mitochondria, indices that are closely correlated with overall oocyte quality. ROS are toxic by-products of aerobic metabolism, an increased level of ROS indicates abnormal aerobic metabolism. 33 Annexin V is a marker of early apoptosis, and an increased level of annexin V indicates an abnormally high apoptotic level. 34 Mitochondria are the factory that generates ATP, over-aggregated mitochondria indicate impeded mitochondrial autophagy, and the cellular ATP level might decrease consequently. 35 Our results showed that MPP6 depletion significantly elevated the ROS ( Figure 6A Next, we examined the activities of key meiotic kinases. MPF, composed of p-cdk1 and cyclin B1, is the master promoter of meiosis. Low MPF activity caused by increased p-cdk1 or decreased cyclin B1 could retard meiosis. 36,37 Akt is an important meiotic kinase that regulates the progression of meiosis in FGOs. 38,39 Retarded or disrupted meiosis could subsequently affect many aspects of oocyte quality. Western blot showed that MPP6 depletion significantly increased p-cdk1 by almost 100% (Figure 7A,B, p-Cdk1 intensity, Ctrl vs MPP6-DE, 1.00 vs 1.94) and decreased cyclin B1 by more than 50% ( Figure 7A,B, Cyclin B1 intensity, Ctrl vs MPP6-DE, 1.00 vs 0.44), indicating that MPF activity was significantly reduced. MPP6 depletion also significantly reduced p-Akt by 50% ( Figure 7C-E, p-Akt intensity, Ctrl vs MPP6-DE, 1.00 vs 0.49). Overall, these results indicate that MPP6 is important for oocyte quality in various ways.

| MPP6 is important for 5.8S rRNA maturation
From the above evidence, and previous reports in somatic cells, it appears that MPP6 is related to 5.8S rRNA processing in oocytes.
Next, we examined whether MPP6 depletion affected 5.8S rRNA maturation and which region it affected. RT-PCR showed that MPP6 depletion specifically and significantly increased 5.8S pre-rRNA amplified with the F + R2 primer ( Figure 8A

| The ageing-associated decrease in oocyte quality partially correlates with a reduced MPP6 level
Numerous clinical studies have shown that the fertility of women starts to decrease at the age of 35, and, concomitantly, the oocyte quality also starts to decline. [40][41][42][43] A transcriptome study showed that 40 of 78 downregulated genes in the ovaries of ageing mice were ribosome-related genes, 43 indicating that downregulation of ribosome function might have a significant impact on fertility. Thus, we next investigated whether ageing could alter 5.8S rRNA maturation and whether the level of MPP6, the 5.8S pre-rRNA maturation factor, could be affected by ageing.
Oocytes from 8-month-old retired mice, which corresponds to 35-year-old women, were used as ageing oocytes. Oocytes from Immunofluorescence also showed that the MPP6 protein signal within spindles was significantly decreased ( Figure 9E). To further support the correlation between 5.8S pre-rRNA level and MPP6 level, we injected in vitro-transcribed MPP6-EGFP mRNA into oocytes to successfully overexpress (OE) MPP6 protein ( Figure 9F).
RT-PCR showed that the 5.

| D ISCUSS I ON
MPP6 was previously screened but never verified as a female fertility factor. The results of the present study demonstrated, for the first time, that MPP6 is important for oocyte meiosis, quality and F I G U R E 8 MPP6 is important for 5.8S rRNA maturation. (A) RT-PCR showed that MPP6 depletion specifically increased 5.8S pre-rRNA amplified with F0 + R2 primer pair. (B) Quantification of (A). (C) FISH of pre-rRNA with FAM-R2 probe in NSN oocytes showed that MPP6 depletion caused a significant increase of 5.8S pre-rRNA within oocytes, nucleus and cytoplasm. (D) Quantification of (C). (E) FISH of pre-rRNA with FAM-R2 probe in SN oocytes showed that MPP6 depletion caused a significant increase of 5.8S pre-rRNA within oocytes, nucleus and cytoplasm. (F) Quantification of (E). (G) FISH of pre-rRNA with TRMRA-5'ETS probe in NSN oocytes showed that MPP6 depletion did not cause a significant increase of 5'ETS pre-rRNA within oocytes, nucleus and cytoplasm.
(H) Quantification of (G). (I) FISH of pre-rRNA with TRMRA-5'ETS probe in SN oocytes showed that MPP6 depletion did not cause a significant increase of 5'ETS pre-rRNA within oocytes, nucleus and cytoplasm. (J) Quantification of I. Scale bar, 20 µm. *P < .05 fertilization. And we also identified the pre-rRNA region where MPP6 might bind and act. Moreover, we also found the correlations between oocyte ageing and the 5.8S rRNA level, oocyte ageing and the MPP6 level, MPP6 level and 5.8S rRNA maturation. Overall, these findings indicated that MPP6 might be an important female fertility factor.
FGOs store numerous maternal proteins to accomplish many cellular processes before fertilization. However, for many dynamic proteins, such as cyclin B1, active translation is still required for meiosis, 44 probably to meet the increased level of cyclin B1 needed before MI or to replenish used "old" proteins. 45 To ensure the active translation of maternal proteins, the ribosome must function normally; therefore, enzymatic rRNA should still exist in SN FGOs. Notably, several studies proposed that SN FGOs do not contain transcribed rRNA genes, pre-rRNAs or pre-ribosomes. [13][14][15] However, these researchers only analysed the nuclear signal. A previous study showed that protein synthesis during MI of oocytes increased 3-fold upon entry into meiosis 46 and, correspondingly, cytoplasm at MI stored more ribosomes. 47 These findings are consistent with our FISH finding that SN oocytes have increased 5.8S rRNA in the cytoplasm.
Evidence that rRNA and pre-rRNA exist in SN FGOs provided a basis for the function of MPP6 in SN FGOs because MPP6 is known to be a 5.8S rRNA processing factor. Our data showed that 5.8S pre-rRNA, amplified specifically by F + R2, was significantly increased after MPP6 depletion. Subsequently, the localization of a checkpoint protein F I G U R E 9 Ageing-associated quality decrease of oocytes is partially correlated with reduced MPP6 level. (A) RT-PCR showed that pre-rRNA with 5'ETS primer pair and mature 5.8S rRNA with F0 + R0 primer pair both significantly decreased within ageing oocytes, and 5.8S pre-RNA with F0 + R2 remained unchanged. (B) Quantification of (A). (C) RT-PCR showed that the MPP6 mRNA level significantly decreased in ageing oocytes. (D) Quantification of (C). (E) Immunofluorescence staining showed that the MPP6 level significantly decreased within ageing oocytes. DNA in blue, tubulin in green, MPP6 in red. (F) Western blot showed that good overexpression of exogenous MPP6-EGFP protein could be achieved by mRNA injection into oocytes. Red arrows marked endogenous MPP6 and exogenous MPP6-EGFP. (G) RT-PCR showed that MPP6-EGFP overexpression significantly reduced 5.8S pre-rRNA amplified with F0 + R2 primer pair, while did not change 5'ETS pre-rRNA. (H) Quantification of (G). (I) ROS level in ageing oocytes was significantly higher than in young oocytes, while injection of MPP6-EGFP mRNA into ageing oocytes significantly reduced the ROS level. (J) Quantification of (I). (K) Mitochondria aggregation in ageing oocytes was significantly higher than in young oocytes, while injection of MPP6-EGFP mRNA into ageing oocytes significantly reduced the mitochondria aggregation. (L) Quantification of (K). Scale bar, 20 µm. *P < .05 (BubR1), the activities of multiple meiotic kinases (MPF and Akt), and the levels of several indices of function (ROS, annexin V, mitochondria and ATP) all became abnormal. These changes may be due to damaged protein biogenesis of the ribosomes due to the reduction of 5.8S rRNA.
Finally, we found that the levels of both 5.8S pre-rRNA and MPP6 were significantly decreased in oocytes from 8-month-old mice. Furthermore, supplementation with exogenous MPP6 could partially rescue oocyte quality, indicating that the reduction of rRNA and MPP6 might be at least partially responsible for the decreased oocyte quality in aged mice. A transcriptome study showed that 40 of 78 downregulated genes in the ovaries of ageing mice were ribosome-related genes, such as Rps12, Rpl22l1, Rrs1 and Brix1. 43 This supports the concept that ribosomal dysfunction is a major cause of the low quality of ageing oocytes.
Overall, our study, for the first time, shows that pre-rRNA and rRNA exist in SN FGOs, and the pre-rRNA maturation factor, MPP6, is important for the level of mature 5.8S rRNA and subsequent oocyte quality and meiosis. Therefore, MPP6 might be a potent marker for oocyte quality.

ACK N OWLED G EM ENTS
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.