GAS6 ameliorates advanced age-associated meiotic defects in mouse oocytes by modulating mitochondrial function

Gas6 expression in mouse oocytes decreased with maternal aging

In mouse oocytes, mRNA and protein expression levels are altered with aging. We examined Gas6 expression in young (3 weeks) and aged (12 months) mice using qRT-PCR and Western blot analyses to determine whether age affects Gas6 expression in the mouse oocyte. Briefly, abundant Gas6 mRNA levels in young oocytes did not change during oocyte meiotic maturation (Figure 1A); thus, the GAS6 protein level was maintained in the young MII oocytes (Figure 1B). However, abundant Gas6 transcript level observed in the germinal vesicle (GV) stage in aging mice was markedly reduced during meiosis, resulting in a decrease in the Gas6 mRNA level and translational repression in aged MII oocytes (Figure 1B). Thus, the expression of Gas6 mRNA and protein was lower in the MII oocytes of aging mice than in those of young mice (Figure 1A, 1B). Previous studies showed that the disruption of oocyte Gas6 alone leads to fertilization failure caused by insufficient oocyte cytoplasmic maturation [24, 26]. These results revealed that inadequate Gas6 expression in oocytes due to maternal aging led to impaired oocyte cytoplasmic maturation.

Figure 1. Reduced expression of Gas6 and increased meiotic errors in oocytes with maternal aging. (A, B) Typical expression patterns of the Gas6 transcripts (A) and protein (B) in GV and MII oocytes from young and aged female mice, respectively. Gas6 expression was significantly decreased in aged oocytes. The levels of the GAS6 protein are presented in a bar graph. The data are presented as the means ± SEM. Different letters indicate significant differences at p<0.05. Young oocytes were obtained from 3-week-old female mice; aged oocytes were obtained from 12-month-old female mice. (C) Maternal aging causes spindle organization defects and chromosome misalignment during oocyte maturation. Green, spindle; blue, chromosome; red, mitochondria; red box, oocyte with abnormal spindle and chromosome alignment. The scale bars represent 25 μm.

An increasing maternal age may result in a gradual decrease in oocyte quality and developmental potential. Oocyte competence is also determined by spindle morphology and chromosome alignment [27]. Spindles and chromosomes were visualized using immunofluorescence staining to observe changes in the spindle shape and chromosome configuration in oocytes with aging. Young and aged oocytes developed to the MII stage. Young MII oocytes exhibited barrel-shaped spindles with slightly pointed and well-aligned regular chromosomes along spindle equatorial plane (Figure 1C). However, some of the aged MII oocytes presented spindles with an irregular microtubule distribution and reduced length and chromosomes that did not align on the spindle equatorial plane (Figure 1C, red box). Aged MII oocytes might be unable to maintain the spindle morphology and the connection between the spindle and chromosomes.

GAS6 alleviates advanced age-associated meiotic defects

Female fecundity is known to decline with age. In Figure 1, we noted reduced Gas6 expression with maternal aging. We conducted rescue experiments to observe whether increasing GAS6 in aged oocytes would improve their deficient phenotypes. The GAS6 protein was injected into aged GV oocytes and meiotic progression was analyzed to address this question. As shown in Figure 2A, almost all of the GAS6-rescued aged oocytes developed to the MII stage. The maturation rate of the GAS6-rescued aged oocyte group (93.7%) was not significantly different from that of control aged oocyte group (97.6%; Figure 2B). Indeed, expression of the GAS6 protein in these MII oocytes was increased compared with that in the control aged MII oocytes (Figure 2C, 2D). Most importantly, GAS6 restoration in oocytes significantly reduced maternal age-associated meiotic defects compared with those observed in the control aged oocytes (8.6% vs. 40.5%; Figure 2E, 2F). Notably, the restoration of GAS6 in aged MII oocytes resulted in the formation of a bipolar spindle and chromosome assembly at the equatorial plane (Figure 2E). In contrast, we observed that control aged MII oocytes showed a high frequency of meiotic defects with disorganization of the spindle and chromosomal architecture (Figure 2E, red box and Figure 2F, black bar). These observations suggest that GAS6 in aged oocytes is crucial for proper organization of the spindle and normal chromosome architecture.

Figure 2. GAS6 ameliorates age-associated meiotic defects in oocytes. (A) Micrographs of aged MII oocytes treated without or with the GAS6 protein. Control, nontreated MII oocyte; GAS6-rescued, MII oocyte treated with the GAS6 protein. The scale bar represents 100 μm. (B) In vitro maturation rates of aged mouse oocytes after the microinjection of the GAS6 protein into GV-stage oocytes. After GAS6 restoration, these oocytes developed into morphologically normal MII oocytes, similar to the control groups. The data are presented as the means ± SEM. (C, D) Microinjection of the GAS6 protein led to a partial recovery of the decreased GAS6 levels induced by maternal aging. α-TUBULIN was used as a loading control. Relative expression levels of the GAS6 protein are presented in a bar graph and compared to the control young oocyte group. The data are presented as the means ± SEM. Different letters indicate significant differences at p<0.05. Control, nontreated oocyte; GAS6-rescued, oocyte treated with the GAS6 protein; Young, oocytes obtained from 3-week-old female mice; Aged, oocytes obtained from 12-month-old female mice. (E) Immunofluorescence staining for spindles and chromosomes in aged MII oocytes after injection of the GV oocytes without (control) or with the GAS6 protein (GAS6-rescued), which were then allowed to mature in vitro. Green, spindle; blue, chromosome; red, mitochondria; red box, oocyte with abnormal spindle and chromosome alignment. The scale bars represent 25 μm. (F) Proportions of control or GAS6 protein-injected MII oocytes with defects in spindle assembly and chromosome configuration.

GAS6 reverses maternal aging-induced mitochondrial deterioration and metabolic dysfunction in oocytes

In mice and humans, mtDNA copy numbers and mitochondrial function are closely related to oocyte quality [15]. As previously reported, mtDNA copy numbers are reduced in oocytes from aged mice [28]. Because a reduction in Gas6 expression resulted in a decreased mtDNA content in young MII oocytes, we examined whether the restoration of GAS6 would rescue the mtDNA copy number in oocytes with maternal aging. We therefore examined the mtDNA content in aged oocytes with or without GAS6 treatment using qRT-PCR analysis. Young control MII oocytes contained an average of 117,579 ± 13,922 mtDNA copies per oocyte (Figure 3A). However, control aged MII oocytes contained an average of 48,650 ± 4,840 mtDNA molecules (Figure 3A). The mtDNA copy number decreased by approximately 50% with oocyte aging. Importantly, GAS6-rescued aged oocytes contained an average of 74,040 ± 7,750 mtDNA molecules (Figure 3A). As shown in Figure 3B, the transcript levels of the mtDNA-encoded genes Mtnd1 and Mtapt6 were reduced in aged MII oocytes. After GAS6 protein microinjection in aged MII oocytes, however, the Mtnd1 and Mtapt6 transcripts were significantly increased to levels similar to those observed in young control group (Figure 3B). Mitochondrial function is an important prerequisite for oocyte cytoplasmic maturation and subsequent fertilization and embryo development [10, 15, 16]. Accordingly, we used a mitochondrial tracker to examine active mitochondria in aged oocytes after restoring GAS6. Red and green signals indicated mitochondria with a high ΔΨm and low ΔΨm, respectively (Figure 3C). As shown in Figure 3C, 3D, the red/green intensity ratio increased with advanced maternal age. In contrast, ΔΨm was apparently reduced in GAS6-rescued aged oocytes, similar to the young control group (Figure 3C, 3D). Aged oocytes display numerous abnormalities, including a reduction in the ATP content. The decrease in ATP levels exerts a deleterious effect on fertilization and early embryogenesis. We also evaluated mitochondrial ATP production in aged oocytes treated with GAS6. Compared with young control MII oocytes, aging of oocytes resulted in significantly decreased intracellular ATP levels (Figure 3E). However, restoration of GAS6 in aged oocytes resulted in a marked increase in intracellular ATP (Figure 3E). Mitochondrial dysfunction is one of the major factors that induces an increase in ROS levels; therefore, we assessed changes in ROS levels associated with maternal aging and GAS6 treatment in oocytes. As shown in Figure 3F, compared to young control MII oocytes, ROS levels were markedly increased in control aged MII oocytes, while intracellular ROS levels were considerably reduced after GAS6 restoration in aged MII oocytes. Intracellular glutathione (GSH) contributes to oxidative stress resistance and protects oocytes from oxidative damage. Indeed, GSH levels reflect oocyte competence and quality. However, intracellular GSH levels in oocytes gradually decrease with increasing maternal age [29]. We analyzed intracellular GSH levels in MII oocytes treated with or without the GAS6 protein to examine the effect of GAS6 on GSH synthesis during the aging process. As expected, GSH levels and the GSH/GSSG ratio were decreased in the aged oocytes (Figure 3G, 3H). However, both GSH contents and GSH/GSSG ratio in aged MII oocytes were markedly increased after GAS6 restoration, similar to the young control group (Figure 3G, 3H). Altogether, these observations indicated that GAS6 prevented maternal aging-induced mitochondrial dysfunction in oocytes and increased the quality of aged oocytes by modulating metabolism.

Figure 3. Improvements in mitochondrial function in oocytes during aging after GAS6 supplementation. (A) Measurements of the mtDNA copy numbers in aged oocytes expressing GAS6. The mtDNA copy number was higher in GAS6-rescued aged oocytes than in control aged oocytes. Different letters indicate significant differences at p<0.05. Control, nontreated oocyte; GAS6-rescued, oocyte treated with the GAS6 protein; Young, oocytes obtained from 3-week-old female mice; Aged, oocytes obtained from 12-month-old female mice. (B) Expression of Mtnd1 and Mtatp6 in aged MII oocytes from the control and GAS6-rescued groups. Different letters indicate significant differences at p<0.05. (C) Representative images of ΔΨm in aged MII oocytes after GAS6 supplementation. ΔΨm indicates the ratio of RITC (J-aggregate, high membrane potential) to FITC (J-monomer, low membrane potential) intensity in GAS6-rescued aged MII oocytes. Scale bars represent 50 μm. (D) Graphic representation of the results shown in C. The data are presented as the means ± SEM. Different letters indicate significant differences at p<0.001. (E) Effects of GAS6 restoration on ATP levels in aging oocytes. Increasing GAS6 expression resulted in elevated mitochondrial ATP production. Different letters indicate significant differences at p<0.05. (F) Treatment with GAS6 reduced ROS levels in young and aged MII oocytes. After treatment, MII oocytes were cultured in M16 medium supplemented with DCFH-DA to evaluate ROS levels. Scale bars represent 100 μm. (G) GAS6 treatment increased oocyte GSH levels with aging. The data are presented as the means ± SEM. Different letters indicate significant differences at p<0.05. (H) The ratio of GSH to GSSG in GAS6-rescued aged MII oocytes was calculated. The GSH/GSSG ratio is a biochemical marker of oxidative stress. The GSH/GSSG ratio was increased in aged MII oocytes after the restoration of GAS6. Thus, GAS6 reduces the oxidative stress caused by maternal aging. Different letters indicate significant differences at p<0.05.

GAS6 improves the maternal aging-induced decrease in fertility and promotes the fertilization ability of young oocytes

Gas6-silenced oocytes failed to undergo normal fertilization [24]. Because Gas6 expression decreases in oocytes with age, contributing to a decrease in the oocyte quality, we sought to determine whether the fertilization ability would be restored by the GAS6 treatment. We increased GAS6 levels by injecting the GAS6 protein to address these questions. Control aged oocytes exhibited a markedly lower fertilization rate than young control oocytes (23.1% vs. 53.3%; Figure 4A, 4B). As expected, in the GAS6-rescued aged oocyte group, the rate of PN formation was effectively increased compared with that in control aged oocytes (Figure 4B). After GAS6 rescue, the percentage of PN formation was approximately double that in the control aged oocytes (23.1% vs. 45.0%; Figure 4B). Remarkably, the percentage of PN formation in the GAS6-rescued aged oocytes was not substantially different than that in the control young oocytes (53.3% vs. 45.0%; Figure 4B). Collectively, these findings strongly suggest that GAS6 protects oocytes from the deterioration of oocyte competence during reproductive aging and improves fertilization ability (Figure 5).

Figure 4. GAS6 restores the Gas6 silencing- and/or maternal age-induced decline in fertility. (A) Microphotographs of PN embryos after in vitro fertilization. Aged oocytes were treated without (control) or with the GAS6 protein (GAS6-rescued) and then fertilized after in vitro culture. White triangles indicate PN formation. Scale bars represent 100 μm. (B) Percentage of oocytes showing PN formation after in vitro fertilization. Experiments were repeated at least three times, and data are presented as the means ± SEM. Different letters indicate significant differences at p<0.05.

Figure 5. GAS6 attenuates the decrease in the quality of aged oocytes. Aged oocytes showed increased mitochondrial deterioration, elevated oxidative stress and insufficient cytoplasmic maturation due to decreased Gas6 expression, resulting in meiotic defects and consequential PN formation failure. Moreover, GAS6-rescued aged oocytes collectively exhibited metabolic alterations, indicating better-quality mitochondrial functions. Therefore, the age-associated decreases in oocyte quality and fertility were prevented by restoring GAS6 levels.

Animals

Female imprinting control region (ICR; young, 3 weeks; aged, 12 months) mice, which were exclusively provided by Koatech (Pyeoungtack, Korea) and Envigo (Horst, Netherlands). We used 200 female mice at 12 months of age for the GAS6 rescue experiments. All procedures were performed in accordance with institutional guidelines and had been approved by the Institutional Animal Care and Use Committee of CHA University.

Reagents and antibodies

Unless otherwise stated, chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Goat anti-GAS6 (sc-1936) and mouse anti-α-TUBULIN (sc-8035) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Isolation of oocytes by ovarian stimulation

Oocyte isolation was performed as previously reported [25]. The ICR mice strain was used for all animal experiments. Female mice were sacrificed 46 hours after the intraperitoneal injection of PMSG. To prevent meiotic progression during oocyte collection and microinjection, we used M2 medium containing 0.2 mM IBMX. The isolated ovaries were dissociated in M2 medium containing IBMX. After the cumulus-oocyte complexes had been collected, cumulus cells were mechanically detached from the oocytes, and fully grown denuded GV oocytes were collected for microinjection experiments. The cumulus-oocyte complexes were cultured to obtain MII oocytes using a previously described protocol [25]. After washing with PBS-PVA, 20 or 50 oocytes from each group were transferred into a microcentrifuge tube and immediately snap frozen. The oocytes were stored at -80° C until the experiments were performed.

Oocyte microinjection

Recombinant mouse GAS6 protein was purchased from R&D Systems (8310-GS; Minneapolis, MN, USA). The GAS6 protein was diluted to final concentration of 5 ng/μl. Aged GV oocytes were randomly grouped and microinjected with the GAS6 protein using a microinjector (Transjector; Eppendorf, Hamburg, Germany) for the GAS6 rescue assay. After protein microinjection, the oocytes were thoroughly washed and cultured in M16 medium supplemented with IBMX for 4 to 6 hours with 5% CO₂ at 37° C. The GAS6-rescued aged oocytes were washed and then cultured for 16 hours in M16 medium. To determine the oocyte stage, morphological changes were documented as previously described [24].

RNA isolation from oocytes and qRT-PCR

To detect Gas6 transcript expression during the aging process, mRNA was isolated from 20 GV-stage oocytes using the Dynabeads mRNA DIRECT kit (Ambion, Austin, TX, USA). The mRNA isolated from each sample (20 oocytes) was reverse transcribed for cDNA synthesis using M-MLV RT (Promega) with oligo (dT) primer. The synthesized cDNA was assessed by qRT-PCR. All assays used cDNA from a single oocyte for each PCR sample. The sequences of the gene-specific primers are listed in Supplementary Table 1. The expression of Gas6 and mitochondrially encoded transcripts was measured using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) with a CFX96 Touch^TM Real-Time PCR Detection System (Bio-Rad), and then results were assessed using CFX Maestro software (Bio-Rad). Melting curve analysis was used to determine the specificity of the amplified products. H1foo was used to normalize the expression of each gene. Relative changes in gene expression were quantified using the comparative C_T method.

Western blotting

In samples containing an equal number of oocytes, protein expression was evaluated by Western blotting using a standard protocol. The bands were viewed using the ChemiDoc XRD+ system (Bio-Rad). α-TUBULIN served as a control. The relative intensity of each band was evaluated using Image Lab software (Bio-Rad).

Mitochondrial staining with mitotracker and JC-1

Mitochondria were examined using MitoTracker Orange CMTMRos (Molecular Probes, Eugene, OR, USA). Briefly, oocytes were cultured in M16 medium containing 300 nM MitoTracker for 30 minutes with 5% CO₂ at 37° C. After washing with PBS-PVA, the oocytes were fixed in 3.7% paraformaldehyde for 1 hour and permeabilized in PBS-PVA-BSA containing Triton X-100 for 30 minutes. After blocking, the oocytes were stained overnight at 4° C with an anti-α-TUBULIN antibody and then incubated in PBS-PVA-BSA containing Alexa Fluor 488-conjugated secondary antibody. After washing, the DNA was stained with DAPI, and the oocytes were mounted on glass sides. The mitochondrial distribution was observed with a Leica confocal microscope (Wetzlar, Germany). At least 30 oocytes per group were assessed in three different replicates.

To evaluate the change in ΔΨm in the oocytes, oocytes were stained with MitoProbe JC-1 (Thermo Fisher Scientific, Waltham, MA, USA). Oocytes were cultured in M16 medium supplemented with JC-1 at 1 μg/mL for 30 minutes and then washed with PBS-PVA. The oocytes were mounted and immediately imaged in the red and green fluorescence channels under a confocal microscope. For quantitative analysis, LAS AF Lite software (Leica) was used to measure the signal intensities, and ΔΨm was calculated as the ratio of the red/green signal.

Intracellular ATP levels in oocytes

Based on the ATP-dependent luciferin-luciferase reaction, intracellular ATP levels (10-20 oocytes) were examined using a Bioluminescent Somatic Cell Assay Kit (FL-ASC), as we described in a previous report [37]. Serial dilutions of an ATP standard (range of 0-10 pmol) and negative controls were prepared for each assay. The relative signal intensity of each group was assessed using a luminometer (Centro XS3 LB 960; Berthold Technologies, Bad Wildbad, Germany). A standard curve was generated using linear regression, and the intracellular ATP content per oocyte was calculated from the standard curve.

Reduced GSH (GSH), oxidized GSH (GSSH) and ROS levels in oocytes

The levels of GSH and GSSH in the oocytes were measured using an EnzyChrom™ GSH and GSSG Assay Kit (EGTT-100; Bioassay Systems, Hayward, CA, USA). Oocytes were vortexed in cold buffer with or without a scavenger. After centrifugation, the oocyte lysates were deproteinated using metaphosphoric acid. A detection reagent containing assay buffer, NADPH, DTNB and glutathione reductase was added to deproteinate the oocyte samples. Upon the reaction of DTNB and GSH, the sample turned yellow. Finally, the optical density was measured at 412 nm using an Epoch microplate spectrophotometer. A standard curve was generated, and the GSH and GSSH levels per oocyte were calculated.

To determine ROS levels in the oocytes, intracellular ROS production was evaluated using an Oxiselect ROS assay kit (STA 342; Cell Biolabs, San Diego, CA, USA). Oocytes were cultured in M16 medium supplemented with the ROS indicator 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) for 1 hour at 37° C in the dark. After washing with M16 medium, the oocytes were transferred to a 96-well dish and immediately visualized using fluorescence microscopy.

mtDNA content in the oocytes

Mitochondrial DNA was extracted from 10 MII oocytes subjected to GAS6 rescue experiments using a mitochondrial DNA isolation kit (Abcam). The mitochondrial DNA content in the oocytes was evaluated by qRT-PCR as we reported previously [41]. qRT-PCR analysis used mtDNA at an amount equivalent to a single oocyte per PCR sample. The sequences of the primers specific to Mtnd1 are listed in Supplementary Table 1. A standard curve was generated from tenfold dilutions of purified plasmid DNA for each assay. The mtDNA copy number per single oocyte was determined from the standard curve. Each sample was analyzed in triplicate.

In vitro fertilization (IVF)

For IVF experiments, M16 medium (with or without BSA) and mineral oil were prewarmed and equilibrated prior to use. Male ICR mice at 10 weeks of age were sacrificed. Sperm were obtained from the caudal epididymis (Koatech) and underwent capacitation by incubation in a 100-μl droplet of M16 medium supplemented with BSA for 1 hour with 5% CO₂ at 37° C. To remove the zona pellucida, the oocytes were carefully and rapidly treated in acid Tyrode’s solution (pH 2.5). Zona pellucida-free MII oocytes (10-15 oocytes) were placed in a 200-μl droplet of M16 medium and incubated for 30 minutes with 5% CO₂ at 37° C. After capacitation, 10 μl of 2.5×10⁴ sperm/ml was added to a droplet containing zona pellucida-free MII oocytes and subsequently cultured for 4 hours. To remove the sperm, the oocytes were washed extensively with M16 medium and then transferred to prewarmed plain M16 medium. After 3-4 hours, PN formation was examined under a microscope.

Data analysis and statistics

Each experiment was repeated independently at least three times unless indicated otherwise. The results are reported as the mean ± standard error of the mean (SEM). Statistical analyses between the control aged oocyte group and GAS6-rescued aged oocyte group were performed using Student’s t-test. In addition, comparisons of multiple groups were carried out with one-way ANOVA. Differences for which p<0.05, as determined by statistical analysis, were deemed to be significant.