Putrescine delays postovulatory aging of mouse oocytes by upregulating PDK4 expression and improving mitochondrial activity

Putrescine decreased the apoptotic index during the postovulatory aging of oocytes and alleviated morphological changes

After 24 h of in vitro culture (as the model of postovulatory aging), the aged oocytes in the control group showed various morphological defects, including degeneration, fragmentation and parthenogenetic activation when compared with the freshly ovulated oocytes. To explore the potential involvement of putrescine in the aging-induced morphological changes, we treated the MII oocytes cultured in vitro with 0 or 0.5 mM putrescine (Figure 1). When the MII oocytes were exposed to 0.5 mM putrescine in the medium, the number of morphological defects was significantly lowered when compared with the aged oocytes (Figure 1A). TUNEL analysis confirmed the increased level of apoptosis during the postovulatory aging of oocytes. Since the postovulatory aging process culminates in apoptosis [21], it is possible that putrescine acts as a protective agent to reduce the apoptotic level of aging oocytes. The apoptotic index was significantly decreased following putrescine treatment (Figure 1B). Moreover, we observed a significant rise in the level of caspase 3 activation in the aging oocytes. However, the level of caspase 3 activation was significantly decreased following putrescine treatment after 24 h of in vitro culture (Figure 1C). The MII oocytes were exposed to putrescine (or not) for 24 h and then immunolabeled for the analysis of spindle organization. As shown in Figure 1D, the fresh oocytes displayed the morphology of typical barrel-shaped spindles. However, the spindles in the aging oocytes became abnormal, and the microtubules were gradually lost from the spindle, while the aberrant chromosome alignment was increased. The analysis of the MII oocytes after the 24 h aging period showed that the proportion of abnormal spindles in the putrescine-treated group was significantly lower than that in the control group (Figure 1D).

Figure 1. Putrescine decreased the apoptosis index and alleviated morphological changes during the postovulatory aging of oocytes. (A) The morphological defects of oocytes during the postovulatory aging process. The proportion of abnormal oocytes was increased in the oocytes undergoing postovulatory aging. When the MII oocytes were exposed to 0.5 mM of putrescine, the rate of morphological defects was significantly lowered when compared with the aging oocytes. (B) The level of apoptosis in the oocytes during postovulatory aging. TUNEL analysis confirmed the increased apoptosis during the postovulatory aging of oocytes. The apoptosis was significantly inhibited by the 0.5 mM of putrescine that was added to the in vitro culture medium. (C) The activated caspase 3 showed the increased level of apoptosis in the oocytes during postovulatory aging. The level of cleaved caspase 3 was significantly increased in the aging oocytes. Putrescine significantly inhibited the activation of caspase 3 in the aging oocytes. (D) The morphological observation of spindles. In the aging oocytes, the spindles became elongated, the microtubules were gradually lost from the spindle, and the aberrant chromosomal alignment was increased. The proportion of abnormal spindles was significantly decreased in the putrescine-treated group. Put, putrescine. Compared with the fresh MII oocytes, *p<0.05; compared with the aging oocytes, # p<0.05.

ROS was accumulated in the aging oocytes, and putrescine decreased ROS by Sirt1-FOXO3a-SOD2 axis

Oxidative stress has been considered as a key mechanism of cellular aging [22,23]. Therefore, we measured the intracellular ROS level after 24 h of in vitro culture of the postovulatory oocytes. We found that ROS was accumulated in the oocytes during the postovulatory aging process. Putrescine treatment significantly decreased the ROS accumulation (Figure 2A). The expressions of SOD2 at both mRNA and protein levels were significantly decreased in the aging oocytes, while those expressions were partially rescued by putrescine treatment (Figure 2B). The Sirt1-FOXO3a-SOD2 pathway was reported to inhibit oxidative stress in embryonic cells [24,25]. To determine whether putrescine prevented the ROS accumulation in aging oocytes by this pathway, the postovulatory oocytes were treated with or without putrescine for 24 h of in vitro culture, and the expressions of Sirt1 and FOXO3a were then assessed. The protein expressions of Sirt1 and FOXO3a were significantly decreased in the postovulatory aging oocytes. Putrescine treatment partially rescued the expressions of Sirt1 and FOXO3a (Figure 2C).

Figure 2. ROS accumulated in the aging oocytes and putrescine decreased ROS through the Sirt1-FOXO3a-SOD2 axis. (A) The accumulation of ROS in the oocytes during postovulatory aging. The level of ROS was significantly decreased in the putrescine-treated group. (B) The mRNA and protein expressions of SOD2. Both the mRNA and protein expressions of SOD2 were significantly decreased in the aging oocytes, while the expression of SOD2 was partially rescued by putrescine treatment. (C) The Sirt1-FOXO3a-SOD2 axis in the aging oocytes. The expressions of Sirt1, FOXO3a and SOD2 protein were significantly decreased in the postovulatory aging oocytes. Putrescine partially rescued the effects on the expressions of Sirt1, FOXO3a and SOD2 protein. Put, putrescine. Compared with the fresh MII oocytes, *p<0.05; compared with the aging oocytes, # p<0.05.

Functional degeneration of mitochondria in the aging oocytes

The decreased expression of Sirt1 has recently been found to be associated with the deterioration of oocyte quality by regulating the mitochondrial function during the postovulatory aging. The even distribution of mitochondria generally suggested a high mitochondrial activity, whereas the aggregated distribution of mitochondria always showed a functional degeneration [25]. In the fresh MII oocytes, most of the mitochondria were evenly distributed in the cytoplasm. The proportion of aggregated mitochondria was significantly increased in those aging oocytes. However, this proportion was significantly decreased by putrescine treatment (Figure 3A). The mitochondria membrane potential (MMP) was also tested by the JC-1 assay to evaluate the mitochondrial activity in our study. The ratio of red/green fluorescence intensity was used as the MMP index (Figure 3B). The MMP index was significantly reduced in the aging oocytes when compared with the fresh MII oocytes. Putrescine significantly increased the MMP index of those aging oocytes. Since Sirt1 induces autophagy by decreasing the activity of mTOR [26], it was of interesting to examine whether putrescine preserves autophagy during the postovulatory aging (Figure 3C). The autophagy level was evaluated using the total LC3 level as an indicator. We found that the autophagy was significantly decreased after 24 h of in vitro culture. However, the level of autophagy was restored when the aging oocytes were treated with putrescine (Figure 3C). In addition, the LC3-II/LC3-I ratio was significantly decreased, and the expression level of p-mTOR was increased in the aging oocytes, while putrescine treatment partially prevented those effects during the postovulatory aging of oocytes (Figure 3D).

Figure 3. Functional degeneration of mitochondria in the aging oocytes. (A) The distribution of mitochondria in the cytoplasm. The even distribution of the mitochondria generally suggested a high function, while the aggregated mitochondria always showed functional degeneration. In the fresh MII oocytes, most of the mitochondria were evenly distributed in the cytoplasm. The proportion of aggregated mitochondria was significantly increased in those aging oocytes. However, this number was significantly decreased by putrescine treatment. (B) The mitochondrial membrane potential (MMP) was tested by the JC-1 assay to evaluate the mitochondrial activity. The ratio of red/green fluorescence intensity was used as the MMP index. The MMP index was significantly reduced in the aging oocytes when compared with the fresh MII oocytes. Putrescine treatment significantly enhanced the MMP of the aging oocytes. (C) The expression level of total LC3 in oocytes during postovulatory aging. The autophagy level was evaluated with total LC3 as an indicator. The autophagy in the fresh MII oocytes was at a reasonable level. The autophagy was significantly decreased in oocytes during postovulatory aging. The autophagy level was partially rescued by putrescine in the aging oocytes. (D) mTOR and LC3 transform as autophagy-related factors. The expression of mTOR was increased and the LC3-II/LC3-I ratio decreased in the aging oocytes. These effects were partially rescued by putrescine treatment in the aging oocytes. Mito, mitochondria. Put, putrescine. Compared with the fresh MII oocytes, *p<0.05, ** p<0.01; compared with the aging oocytes, # p<0.05, ## p<0.01.

The apoptosis-related pathways in the aging oocytes and the effect of putrescine

An immunoblot analysis revealed that the levels of p-AKT, p-ERK1/2 and BCL-2 were significantly decreased and the level of BAX was increased during postovulatory aging. However, putrescine treatment significantly reduced those downregulations of p-AKT, p-ERK1/2 and BCL-2 and the upregulation of BAX (Figure 4A). Moreover, a significant rise in the level of caspase 9 activation was found in the aging oocytes; the level of caspase 9 activation was significantly reduced after 24 h of putrescine treatment in the in vitro culture (Figure 4B).

Figure 4. The apoptosis-related pathways in the aging oocytes and the effect of putrescine. (A) The phosphorylation levels of the AKT and ERK1/2 proteins and the expressions of BCL2 and BAX in the aging oocytes. The levels of p-AKT and p-ERK1/2 were significantly decreased in the oocytes during postovulatory aging, although the total level of AKT or ERK1/2 was not changed. The level of BCL2 was significantly decreased, and the level of BAX increased, as two subsequent factors of the AKT and ERK pathways. Putrescine rescued those effects in the aging oocytes.(B) The expression of caspase 9 as a key factor of the mitochondria-related apoptosis pathways. The level of cleaved caspase 9 was significantly increased in the aging oocytes. Putrescine significantly inhibited this incremental rise. Compared with the fresh MII oocytes, *p<0.05, ** p<0.01; compared with the aging oocytes, # p<0.05, ## p<0.01.

Effects of putrescine on PDK4 expression and oxidative stress

We found that the expression of PDK4 was significantly downregulated in the postovulatory aging oocytes in the in vitro culture. The putrescine treatment partially rescued this effect of PDK4 expression (Figure 5A). To confirm the function of putrescine on the postovulatory aging of oocytes by upregulating PDK4, the expression of PDK4 was downregulated by injecting siRNA-PDK4 into fresh oocytes with or without putrescine in the medium for 24 h of in vitro culture. Then, the level of oxidative stress, the mitochondrial distribution and the MMP index and the apoptotic index were analyzed. We found that the ROS level and the caspase 3 activition were significantly increased in those oocytes with siRNA-PDK4 (Figure 5B). The defects in mitochondrial distribution were also increased (Figure 5B). Following the in vitro aging process, the MMP index was significantly reduced in the PDK4-downregulated oocytes that were treated synchronously with putrescine, when compared to the putrescine-treated oocytes (Figure 5C). Meanwhile, putrescine significantly inhibited the caspase 3 activation in the postovulatory aging oocytes, and this effect of putrescine was partially blocked by the downregulation of PDK4 (Figure 5D).

Figure 5. Effects of putrescine on PDK4 expression, oxidative stress, and apoptosis in the oocytes during postovulatory aging. (A) The effect of putrescine on PDK4 expression was tested by immunofluorescence, qPCR and Western blot. Putrescine rescued the downregulation of PDK4 at both the mRNA and protein levels in the aging oocytes. (B) The effects of putrescine on the ROS level and mitochondrial distribution in the aging oocytes. Putrescine significantly reduced the ROS level and the number of aggregated mitochondria in the aging oocytes. If PDK4 expression was downregulated by siRNA, the above effects of putrescine were significantly weakened. (C) The effect of putrescine on the MMP was related to mitochondrial activity. Putrescine significantly raised the MMP in the aging oocytes. However, this effect was blocked by siRNA-PDK4. (D) The effect of putrescine on the cleaved caspase 3 was related to apoptosis in the aging oocytes. Putrescine significantly inhibited the increased level of cleaved caspase 3 in the oocytes during postovulatory aging. This effect of putrescine was partially blocked by the downregulation of PDK4. *p<0.05, ** p<0.01.

Antibodies and chemicals

All chemicals used in this study were purchased from Sigma-Aldrich unless otherwise stated. The rabbit polyoclonal anti-α-tubulin FITC-conjugated antibodies, the anti-p-mTOR, anti-mTOR, ani-Sirt1, anti-FOXO3a, anti-LC I/II, anti-p-AKT, anti-AKT, anti-p-ERK1/2 and anti-BCL2 antibodies were from Cell Signaling Technology (Beverly MA, USA). The rabbit polyclonal anti-PDK4 antibody was from Novus (Dallas, TA, USA). The mouse monoclonal anti-ACTIN antibody was from Sigma (St. Louis, MO, USA).

Animal care and welfare

The animals used for gamete collections herein were treated in accordance with the guidelines for the care and use of laboratory animals that were approved by the Animal Care Committee of Nanjing Medical University.

Oocyte collection and culture conditions

CD-1 female mice that were 8 weeks old were injected with 5 IU of pregnant mare serum gonadotropin (PMSG; Sigma, St Louis, MO). MII oocytes were obtained from the oviducts 14-16 h after the injection of 5 IU hCG, which was administered 48 h after the PMSG injection. The oocytes were released into HEPES-buffered M2 followed by treatment with 0.1% bovine testes hyaluronidase for 1 min to remove cumulus cells. The MII oocytes were thoroughly washed and transferred into 20 μl drops of M2 under paraffin oil at 37°C and in a humidified atmosphere containing 5% CO₂. The in vitro aging was accomplished by extending the culture time of oocytes after their collection in M2 medium with or without 0.5 mM putrescine to 24 h. The dosage of putrescine was referenced by Liu et al. (2017) .

Microinjection

The siRNAs were prepared as described previously [42]. Approximately 5-10 pl of siRNA were microinjected into the cytoplasm of oocytes using a Femto Jet microinjector (Eppendorf, Hamburg, Germany) with a Leica inverted microscope (DMIRB) that was equipped with a micromanipulator (Narishige, Tokyo, Japan). After injection, the oocytes were cultured in M2 medium at 37°C for 24 h in an atmosphere of 5% CO2 in air.

TUNEL assay

DNA fragmentation was detected by the TUNEL method with the use of an in situ cell death kit (Roche Diagnostic Gmbh, Mannheim, Germany). The oocytes were first fixed in 4% paraformaldehyde dissolved in PBS (pH 7.4) for 30 min and subsequently permeabilized with 0.5% Triton X-100 for 30 min. The experimental groups and control groups were incubated at 37°C for 1 h in 25 μl of the TUNEL reaction mixture containing dUTP-fluorescein isothiocyanate (FITC), terminal deoxynucleotydil transferase (TdT) enzyme, and reaction buffer. After they were washed three times, the samples were observed using a fluorescence microscope.

Immunofluorescence

The oocytes were fixed and permeabilized as described above. The oocytes were transferred into 5% normal goat serum for 1 h followed by incubation at 37°C. Then, the oocytes were incubated overnight at 4°C with the primary antibodies (1:50-1:100 dilution), followed by incubation with Alexa Fluor 594-conjugated secondary antibodies (1:1000, Thermo Fisher) for 1 h at room temperature. Hoechst 33342 (10 mg/ml in PBS) was used for the DNA counterstaining. To measure the immunofluorescence intensity, the signals from both control and experimental oocytes were acquired by performing the same immunostaining procedure and setting up the same parameters of confocal microscope. The data were analyzed by Image J software (National Institutes of Health, Bethesda, Maryland, USA).

Detection of intracellular ROS level

To identify the oxidative stress/ROS levels in aged oocytes, 50-carboxy-20,70- difluorodihydrofluorescein diacetate (carboxy-DFFDA; Molecular Probes), a fluorescent probe capable of detecting powerful oxidants such as H2O2 and peroxynitrite, was utilized. The oocytes were incubated in a 10 μM solution of carboxy-DFFDA in the M2 medium for 30 min at 37°C under 5% CO2 gas. Oocytes were then washed three times in the M2 medium before they were mounted on a glass slide for microscopy.

Detection of mitochondrial distribution

For the mitochondrial staining, oocytes were incubated for 30 min at 37°C in an M2 medium that was supplemented with 200 nM MitoTracker Red (Invitrogen, Carlsbad, CA, USA). Next, the oocytes were mounted on glass slides and examined under a laser scanning confocal microscope (Nikon C2 Plus, Tokyo, Japan). A pixel intensity value was calculated for each oocyte using ImageJ software (National Institutes of Health).

JC-1 assay for mitochondrial activity

The membrane potential sensitive dye JC-1 (Molecular Probes, Eugene, Oregon, USA) was used to estimate the functional activity of mitochondria. A 2 mM stock solution was prepared in DMSO and kept at −20°C. Before use, the stock solution was diluted to 2 μM in M2. The oocytes were incubated with the working solution for 30 min at 37°C before the confocal observation. All of the oocytes were captured under the same exposure intensity. To calculate the confocal ratio of red to green images, the intensities of green and red fluorescence were quantified separately with Image J software.

Western blotting

Cell lysates from 100 cumulus-free oocytes were prepared by adding 10 μl of 2X sample buffer (SB) (Laemmli 1970) as described previously [43]. The samples were boiled for 5 min and loaded onto 4–20% Tris-Acetate gels (Laemmli 1970), and the proteins were separated using electrophoresis and transferred onto PVDF membranes (Micron Separations, Westboro, MA). The membranes were blocked and then probed with specific anti-Sirt1 (1:500), anti-FOXO3a (1:500), anti-SOD2 (1:500), anti-LC I/II (1:500), anti-p-mTOR (1:500), anti-mTOR (1:1000), anti-p-AKT (1:500), anti-AKT (1:1000), anti-p-ERK1/2 (1:1000), anti-ERK1/2 (1:1000), anti-BCL2 and anti-ACTIN (1:1000) antibodies at 4°C overnight. After the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h, an enhanced chemiluminescence (ECL) kit was used to detect the signals. The bands were semiquantified with the analysis software that was provided by the imaging system.

Real-time RT-PCR analysis

Total RNA was extracted from 100 oocytes using a RNeasy Plus Micro Kit (Qiagen, Hilden, Germany). First-strand cDNA was generated using a cDNA synthesis kit (Qiagen, Hilden, Germany). GAPDH was selected as the reference gene. The primer sequences are as follows: PDK4 forward: 5’-TGATTGGCTACTGTAAAAGTCCCGC-3’; reverse: 5’-ATCCCAGGTCCCTAGGACTTCAGG-3’; SOD2 forward: 5’-GGGGGCCGGCCGAGAGCAGCGGT

CGTGTA-3’; reverse: 5’-GGGGGCGCGCCATGTGGCCGTGAGTGAG-3’. The SYBR® FAST qPCR Kits (Takara, Wilmington, MA, USA) were used in combination with a Real-Time PCR Detection System (A, Hercules, CA, USA). The relative gene expression was calculated by the 2^ΔΔCT method.

Statistical analyses

Statistical analysis was performed using SPSS 17 Software. All of the experiments were repeated at least three times, and each experimental group included at least 20 oocytes unless otherwise specified. The significance of the differences between groups was analyzed by ANOVA. Data are expressed as the means ± SEM; differences with p values <0.05 were considered statistically significant.