We firstly examined the localization of FKBP25 at different meiotic stages. As shown in Figure 1A, FKBP25 is predominantly distributed in the nucleus at GV stage. However, with the meiotic resumption, FKBP25 resides in the cytoplasm and significantly accumulated on the spindle region from pre-metaphase I (Pre-MI) to metaphase II stage (arrowheads). Furthermore, we confirmed that FKBP25 was colocalized with the spindle in oocytes by performing the double staining (Figure 1B). The dynamic distribution pattern implies that FKBP25 may play an important role in regulating oocyte meiotic maturation.

To investigate the function of FKBP25 in meiosis, GV stage oocytes were microinjected with FKBP25 siRNAs. After injection, the GV oocytes were cultured in M16 medium supplemented with milrinone for 20 h to facilitate endogenous FKBP25 mRNA degradation. The amount of FKBP25 protein level was dramatically decreased following knockdown (KD; Figure 2A). The results showed that FKBP25 knockdown had no effect on GVBD, shown by similar GVBD rate (Figure 2B). In contrast, FKBP25-KD oocytes exhibited a lower Pb1 extraction rate compared with controls after 14 h of mature culture (Figures 2C–E, asterisks). Oocytes with abnormal division, particularly large polar body extrusion, were frequently observed when FKBP25 was knocked down (Figures 2C,D, arrowheads). In addition, FKBP25-KD oocytes were significantly arrested at MI stage as compared with controls identified by immunofluorescence staining (Figure 2F). These observations strongly suggest that FKBP25 is required for meiotic divisions.

The specific localization of FKBP25 in meiotic process prompted us to investigate whether FKBP25 knockdown affects the meiotic apparatus in oocytes. To gain insight into this issue, we stained oocytes with anti-tubulin antibody and propidium iodide (PI) to visualize spindle and chromosomes, respectively. Using confocal microscope, we found a high defect rate in spindle assembly and chromosome organization in FKBP25-KD oocytes, showing the elongated/multipolar spindles (yellow arrows) and scattered chromosomes (white arrowheads; Figures 3A,B). By contrast, MII oocytes in control groups generally showed a typical barrel-shape spindle and well-aligned chromosomes. Moreover, the karyotype of MII oocytes was analyzed by chromosome spreading and kinetochore labeling. As shown in Figures 3C,D, we found that FKBP25 depletion resulted in about 3-fold increase in incidence of aneuploidy compared to controls. These results suggest that FKBP25 depletion disrupts spindle/chromosome organization in meiotic process, elevating the incidence of aneuploidy.

Kinetochore, an important target connecting spindles and chromosomes, within a right location ensures that chromosomes are aligned at the equator and segregation precisely. Stable kinetochore-microtubule (K-MT) attachment is qualified to the spindle assembly checkpoint (SAC; Cheeseman, 2014; Tauchman et al., 2015). Given the disorganization of chromosome/spindle in FKBP25-KD oocytes, we hypothesized that FKBP25 knockdown may affect K-MT attachments. For this purpose, the kinetochores, MTs and chromosomes of MI oocytes were visualized by staining with anti-CREST antibody, anti-tubulin antibody and Hoechst 33342, respectively (Figures 4A–C). In most control oocytes, kinetochores were properly attached to MTs (amphitelic attachment). However, a prominently increased incidence of two forms of mismatch (lost attachment and monotelic attachment) was detected in FKBP25-KD oocytes relative to control oocytes (Figure 4C).

Spindle assembly checkpoint, a ubiquitous trigger that ensures the precise separation of chromosomes, monitors the right connections between kinetochores and MTs. The SAC signal is strongly expressed when the kinetochores is not yet integrated with the MT. When the kinetochores fused with the tubulin, the signal is quenched (Sun and Kim, 2012; Polanski, 2013; Sacristan and Kops, 2015). BubR1 (budding uninhibited by benzimidazole-related 1), an important integral element of the SAC, is often used to evaluate the status of SAC (Sudakin et al., 2001; Lara-Gonzalez et al., 2012; Overlack et al., 2015; Touati and Wassmann, 2016). In normal oocytes, BubR1 was detected during pre-MI, and then disappeared at MI stage. However, in FKBP25-KD oocytes, BubR1 signals were still present at MI stage (Figures 4D,E). Together, the defective K-MT attachments may be the main reason why chromosomes cannot align properly in FKBP25-KD oocytes.

It has been well elucidated that female fertility decreases with maternal age on account of the maturation defects in oocytes (Holubcova et al., 2015; Duncan et al., 2017; Miao et al., 2020). Given that the FKBP25-KD oocytes exhibited the similar phenotypes as those in old oocytes, we decided to detect FKBP25 expression in oocytes from young and old mice. As shown in Figure 5A, remarkable reduction of FKBP25 protein was detected in oocytes from old mice compared with young oocytes at GV stage. We also assessed the FKBP25 distribution and accumulation in old oocytes via immunostaining. There was no significant alteration in the distribution pattern of FKBP25 between young and old oocytes (Figure 5B). However, the average florescence intensity of FKBP25 protein was dramatically decreased in both GV and MII oocytes from old mice (Figure 5C). In order to evaluate the relationship between FKBP25 protein level and the phenotypes of old oocytes, we performed the rescue experiments through microinjection of cRNA encoding FKBP25 into fully grown old oocytes (Figure 6A). Exogenous FKBP25 protein was efficiently expressed in oocytes (Figures 6B,C). It is worth noting that ectopic expression of FKBP25 ameliorated the maturation defects in old oocytes (Figures 6D,E). Furthermore, as shown in Figures 6F,G, overexpression of FKBP25 in old oocytes significantly reduced the incidence of aneuploidy. These results indicate that depletion of FKBP25 is one of key factors inducing meiotic defects in oocytes from aged mice.

During oocytes maturation, there is a dramatically change in protein phosphorylation and dephosphorylation. A wealth of these phosphorylation and dephosphorylation events are key to regulate meiotic process, chromosome dynamics, and spindle assembly (McGinnis and Albertini, 2010; Schindler, 2011). To test whether FKBP25 phosphorylation affects meiotic process in oocytes, we constructed the site-specific mutants targeting Serine 163 (S163) of FKBP25 (Dilworth et al., 2018). Ser163 was mutated to an alanine residue (S163A), to preclude phosphorylation, or to an aspartate residue (S163D), to mimic permanent phosphorylation (Hou et al., 2015). We injected the mRNA encoding FKBP25 mutants into GV oocytes for analysis. As shown in Figure 7A, immunoblotting verified that FKBP25-S163 mutant was efficiently expressed in oocytes. Compared with control groups, S163A mutant led to an almost 3-fold increase in meiotic abnormalities, such as spindle defects and chromosome misalignment (Figure 7B). Subsequently, we checked whether S163D mutant could rescue some phenotypic abnormalities of aged mouse oocytes. We injected the S163D mutant into old oocytes, and then they were in vitro matured for analysis (Figure 7C). It is worth noting that those meiotic phenotypes of old oocytes could be partially prevented by overexpression of phospho-mimetic S163D (Figures 7D,E). The results suggest that FKBP25 phosphorylation is essential for mediating the effects of aging on oocyte quality.

Three-4-week female mice were used as a control in this study. 42–45-week-old mice which near the end of their reproductive lifespan were used as a reproductive aging model. All animal work and experiments were carried out according to relevant ethical guidelines and regulations, and approved by the Animal Care and Use Committee of Nanjing Medical University.

Rabbit polyclonal anti-FKBP25 antibody were purchased from Abcam (Cat#: ab16654; 1:150); Mouse monoclonal FITC conjugated anti α-tubulin antibody were purchased from Sigma (Cat#: F2168; 1:500); human anti-centromere CREST antibody was purchased from Antibodies Incorporated (Cat#: 15234; 1:500); Cy5-conjugated donkey anti-human IgG and FITC-conjugated donkey anti-goat IgG were purchased from Jackson Immuno-Research Laboratory (Cat#: 709605149 and 705095147; 1:500); Goat polyclonal anti-BubR1 antibody and mouse monoclonal anti-Myc tag antibody were purchased from Abcam (Cat#: ab28193 and ab18185; 1:250); and FITC conjugated goat anti-rabbit IgG purchased from Thermo Fisher Scientific (1:300).

Oocytes were retrieved from female mice at the age of 3–4 week (young mice) and 42–45 week (reproductively old mice). To collect fully grown GV oocytes, female mice were superovulated with 5 IU pregnant mare serum gonadotropin (PMSG) by intraperitoneal injection. After 48 h, cumulus-oocyte complexes were acquired by manually rupturing of antral ovarian follicles. Cumulus cells were removed by repeatedly mouth pipetting. For in vitro maturation, GV oocytes were cultured in M16 medium under mineral oil at 37°C in a 5% CO2 incubator.

Total RNA was extracted from 100 mouse oocytes using Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, CA, United States), and the cDNA was generated with QIA quick PCR Purification Kit (Qiagen, Germany). Plasmid construction and cRNA synthesis were conducted as we reported previously (Zeng et al., 2018). For the synthesis of cRNA, plasmids were linearized by NotI, and cRNAs were made using in vitro transcription with SP6 mMessage mMachine (Ambion, CA, United States) according to the manufacturer’s instruction and purified by RNeasy Micro Kit (Qiagen, Germany). Synthesized RNA was aliquoted and stored at −80°C. The related primer sequences can be found in Supporting Information Supplementary Table 1.

Microinjection experiments were conducted using a Narishige microinjector. Microinjection of siRNA or cRNA was used to knock down or overexpress FKBP25 in mouse oocytes, respectively. 2.5 pl FKBP25 siRNA (1 mM) was injected into oocytes for knockdown analysis, or an equivalent amount of negative siRNA. 10 pl cRNA (10 ng/μl) was microinjected into GV oocytes for overexpression experiments. Following multiple washes, oocytes were arrested at GV stage in M16 medium containing 2.5 μM milrinone for 20 h to promote mRNA degradation or translation, and then cultured in M16 medium without milrinone for further experiments. The related primer sequences can be found in Supporting Information Supplementary Table 1.

A total of 100 GV oocytes were lysed in 2× Laemmli sample buffer containing protease inhibitor, and denatured at 95°C for 5 min, then frozen at −20°C until use. Total oocyte proteins were separated by 10% SDS-PAGE and electrophoretically transferred to PVDF membrane. Membranes were blocked with 10% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and then probed with primary antibodies (Myc antibody, 1:1,000; FKBP25 antibody, 1:1,000) at 4°C overnight. After washes in TBST for three times, the membranes were incubated with HRP-conjugated secondary antibodies. Then, the protein bands were visualized using an ECL Plus Western Blotting Detection System (GE Healthcare, Little Chalfont, United Kingdom). Tubulin was used as a loading control.

Oocytes were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 20 min at room temperature, permeabilized in 0.5% Triton-X 100 for 15 min at RT. Then, oocytes were blocked with 1% BSA-supplemented PBS for 1 h at RT and were subjected to indirect immunofluorescence staining by incubating with primary antibodies overnight at 4°C as follows: anti-FKBP25 antibody. To visualize spindle, oocytes were probed with FITC-conjugated tubulin antibody. Oocytes were co-labeled with CREST (1:500) to detect kinetochores. Following three washes, oocytes were labeled with secondary FITC- or Cy5-conjugated anti-body for 1 h at room temperature. Chromosomes were evaluated by staining with PI or Hoechst 33342 for 10 min. After washed in PBS, oocytes were mounted on antifade medium (Vectashield, Burlingame, CA, United States) and examined under a laser scanning confocal microscope (LSM 700, Zeiss, Germany).

Chromosome spread was conducted as described previously (Gao et al., 2018). To remove zona pellucida, MII oocytes were placed in Tyrode’s buffer (pH 2.5) for about 30 s at 37°C. After recovery in M16 medium for 10 min, oocytes were fixed in a drop of 1% paraformaldehyde with 0.15% Triton X-100 on a glass slide. After air drying, oocytes were incubated with CREST overnight at 4°C and then Cy5-conjugated secondary antibody for 1 h for kinetochore labeling. Chromosomes were stained with Hoechst 33342. Samples were examined under a laser scanning confocal microscope.

All experiments were replicated at least three times, and the data obtained were subjected to statistical analysis. Data are presented as mean ± SEM, unless otherwise indicated. Differences between two groups were analyzed by Student’s t test. Multiple comparisons between more than two groups were analyzed by one-way ANOVA test using GraphPad Prism 8. P < 0.05 was considered to be significant.