N-acetyl-L-cysteine (NAC) delays post-ovulatory oocyte aging in mouse

Treatment with NAC delays formation of spindle anomalies during post-ovulatory oocyte aging in vitro

Increasing concentrations (0.3mM, 0.6mM, 1.0mM) of NAC were supplied to culture medium (M2) for in vitro oocyte culture to test whether NAC treatment could delay aging-induced changes in spindle anomalies. Oocytes aged in vitro were collected after 24h of culture for immunofluorescence analysis. The results showed that fresh oocytes displayed bipolar spindles with focused poles. Various formations of severely abnormal spindles were found in aged oocytes, including elongated spindles and spindles with abnormal poles, with the major spindle defect being spindles with no apparent poles. We found significant differences in oocytes treated with 0.3mM, 0.6mM, and 1.0 mM NAC displaying a lower rate of abnormal spindles compared to oocytes without NAC treatment (Figure 1), suggesting that NAC delayed post-ovulatory oocyte aging in vitro. Due to fewer numbers of spindle anomalies caused by 0.6mM NAC, we used this concentration for all subsequent experiments.

We then investigated the morphological alterations of spindles during oocyte aging for different times with or without NAC treatment. Confocal microscopy showed that various morphological spindle structures were formed during post-ovulatory oocyte aging (Figure 2A). The percentages of abnormal spindles were analyzed at 6h, 12h, 18h, and 24h of postovulatory oocyte aging in vitro. Oocytes cultured for 12h, 18h and 24h showed increased percentages of abnormal spindles compared to fresher oocytes which were cultured for 6h, in both NAC-treated and control group. When MII oocytes were exposed to 0.6mM NAC, the percentage of abnormal spindles observed at 18h or 24h was significantly lower compared to that in control untreated oocytes, indicating that NAC treatment could delay the changes in spindle anomalies at 18h and 24h of post-ovulatory oocyte aging in vitro (Figure 2B). There was no significant difference between the NAC treatment group and control group at 12h during post-ovulatory oocyte aging in vitro.

Next, live cell imaging was performed to analyze the dynamic changes of spindles after treatment with or without NAC. In control aging oocytes, spindles gradually lose poles. At around 14h and 40 minutes of live cell imaging, spindles without one pole were observed. At 20h and 40 minutes, both poles disappeared. In contrast, normal morphological spindles were observed at 14h and 40 minutes when oocytes were treated with NAC. At 20h and 40 minutes of live cell imaging, the two poles could still be seen in the spindles (Figure 2C). The results showed that NAC treatment could delay the changes in spindle anomalies during post-ovulatory oocyte aging in vitro.

NAC administration maintains the normal distribution of CGs

It has previously been shown that post-ovulatory oocyte aging causes morphological changes in the spindle, as well as abnormal distribution in CGs. We investigated the distribution of CGs during post-ovulatory oocyte aging in vitro with or without NAC treatment. Confocal microscopy showed that different distribution patterns of CGs appeared during post-ovulatory oocyte aging in vitro. We determined that oocytes displayed a layer of CGs beneath the oolemma and a distinct CG-free domain near the area of the spindle apparatus that we called normal distribution in fresh oocytes. On the other hand, during post-ovulatory oocyte aging, CG congregation occurred. We found two major types of abnormal distribution: type I, all CGs moved to the cortex of oocytes without leaving CG-free domains; Type II, CGs moved to CG-free domains near the chromosomes (Figure 3A), and the percentage of abnormal distribution of CGs clearly increased obviously at 18h and 24h of in vitro culture in contrast to fresh oocytes. However, compared with control oocytes, the oocytes treated with 0.6mM NAC showed a lower percentage of abnormal CG distribution at 18h and 24h (Figure 3B), indicating its protective effect on post-ovulatory oocyte aging in vitro. However, no significant difference was observed between the NAC supplementation group and the control group at 12h of culture in vitro.

Supplementation of NAC decreases the intra-cellular ROS level

Cellular increase in reactive oxygen species (ROS) level is further considered to be an important change in post-ovulatory oocyte aging. So we further investigated whether NAC treatment could protect oocyte quality by decreasing the intra-cellular ROS level. Because we found protective effects of NAC supplementation on spindle integrity and CG distribution at 18h and 24h during post-ovulatory oocyte aging in vitro, the ROS level was analyzed at 18h and 24h of culture. Oocytes collected and analyzed at 24h of post-ovulatory aging showed increased ROS levels when compared to oocytes aged for 18h. When MII oocytes were exposed to 0.6mM NAC, the ROS level observed at 18h or 24h was significantly lower compared to oocytes in the control group (Figure 4), indicating reduced ROS by NAC supplementation.

NAC treatment protects the function of mitochondria during post-ovulatory oocyte aging in vitro

We next examined whether the function of mitochondria could be maintained effectively with NAC supplementation. Two experiments were designed to test the function of mitochondria. The first experiment was to show the distribution of mitochondria by fluorescence staining, and the second was to analyze the cellular ATP level. Confocal microscopy showed different distribution patterns of mitochondria in oocytes aging in vitro (Figure 5A). Mitochondria of fresh oocytes displayed even distribution, referred to as normal distribution while aged oocytes showed asymmetric clustered distribution. At 18h or 24h of post-ovulatory oocyte aging, the proportion of the abnormal distribution pattern was significantly decreased in the NAC-treated group when compared to the control group (Figure 5B). Oocytes were collected at 12h, 18h and 24h of aging for ATP content analysis. No significant difference was found between control groups and treatment groups at 12h. However, the ATP level at 18h and 24h was clearly higher in NAC-treated oocytes than in oocytes without NAC treatment (Figure 5C). These results suggest that treatment with NAC during post-ovulatory oocyte aging can help to maintain normal mitochondrial functions.

Oocyte collection

6–8 week-old ICR mice were used in this study, and they were kept in accordance with policies promulgated by the Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences. To induce superovulation, female mice received intraperitoneal injection of 8 IU PMSG followed 48 h later by 8 IU hCG. The superovulated mice were killed 12-14h after hCG injection and the oviductal ampullae were broken to release the cumulus-oocyte complexes (COCs). The cumulus cells were removed by pipetting the M2 medium, containing 0.1% hyaluronidase; only denuded MII oocytes were used for the experiments. We set the time 0 hour at 12-14 h of hCG injection. Oocytes were placed in M2 medium under liquid paraffin oil at 37 °C in an atmosphere of 5% CO2 in air.

Oocyte treatment

Collected MII oocytes were washed at least three times and immediately cultured in M2 medium supplemented with or without different concentrations of NAC (Cat #S A7250 Sigma-Aldrich). The optimal concentration of NAC was obtained from previously published studies [24, 30–32]. The fresh oocytes, which were collected after 12–14 h of hCG injection without any culture or treatment, were used as control in our study.

Immunofluorescence microscopy

Immunofluorescent staining of spindles was performed as described previously [33]. Briefly, oocytes were fixed in 4% paraformaldehyde in PBS buffer for 30 minutes at room temperature. After being permeabilized with 0.5% Triton X-100 for 20 minutes, they were then blocked in 1% BSA-supplemented PBS for 1 hour at room temperature. For single staining of α-tubulin, oocytes were incubated overnight at 4°C with 1:800 anti-α-tubulin-FITC antibodies (Cat #2125, Beverly, MA). The method for immunofluorescent staining of CGs was performed according to a previous study [34]. Briefly, the zona pellucida was removed by treating the oocytes with Tyrode’s Solution for 1 minute. After being washed three times in a washing solution (M2 supplemented with 0.3% BSA and 0.01% TritonX-100), oocytes were fixed with 3.7% paraformaldehyde in M2 for 30 min at room temperature. The oocytes were then blocked three times (5 min each time) in a blocking solution (M2 containing 0.3% BSA and 100 mM glycine). After permeabilization for 5 min in M2 containing 0.1% Triton X-100, oocytes were washed two additional times (5 min each) in blocking solution and then cultured with 1:200 lens culinaris (LCA)-FITC for 2 hours at room temperature. The DNA was stained in the final incubation step for 15 min with Hoechst 33342. For mitochondrial staining, oocytes were incubated for 20 min at 37°C in M2 supplemented with 200 nM MitoTracker Red (Cat #M7512, Invitrogen, USA). After washing, oocytes were stained with Hoechst 33342 (10 mg/ml) for 10 min. Finally, oocytes were mounted on glass slides and viewed under a confocal laser scanning microscope (Zeiss LSM 780).

Time-lapse live imaging experiments

After microinjecting MAP4-eGFP mRNA and H2B-mCherry mRNA, oocytes were incubated for 1h in M2 medium. Microtubule and chromosome dynamics were filmed on a Perkin Elmer precisely Ultra VIEW VOX Confocal Imaging System. Oocytes were exposed once an hour for 10h, and then exposed every 20 min for an extra 12h. The acquisition of digital time-lapse images was controlled by IP Lab (Scanalytics) or AQM6 (Andor/Kinetic-imaging) software packages. Confocal images were acquired with a 20x oil objective on a spinning disk confocal microscope.

Detection of intra-cellular ROS level

Method for ROS level detection in oocytes was conducted as described in a previous study [34]. The procedure is briefly summarized as follows. Oocytes were collected at different aging times with or without NAC treatment, and then incubated for 30 min at 37°C in M2 supplemented with 10 mM carboxy-H2DCF diacetate (Cat #S0033, Beyotime). After washing in M2 medium for at least 3 times, oocytes were observed under a spinning disk confocal microscope (Perkin Elmer) with identical settings. Images were analyzed by a Perkin Elmer precisely Ultra VIEW VOX Confocal Imaging System. The fluorescence intensity analysis for each oocyte was conducted using ZEN (2012) software.

Measurement of cytoplasmic ATP content in oocytes

The measurement was performed using a Berthold Lumat LB 9501 luminometer (Berthold Technologies, Bad Wildbad, Germany) and a commercial assay kit based on the luciferin–luciferase reaction (Bioluminescent Somatic Cell Assay Kit, Sigma) following the method described previously [35] following the manufacturer’s recommendations. A standard curve containing 11 ATP concentrations from 10 fmol to 10 pmol was first generated before the detection of ATP content in oocytes. For each experiment, one hundred oocytes were loaded in 1.5 ml PCR tubes, and then stored at -80°C until measurement of ATP content. The ATP content was finally calculated using the formula derived from the linear regression of the standard curve.

Statistical analysis

For each experiment, at least 3 replicates were performed. Statistical analyses were conducted by analysis of variance. Differences between two groups were compared by using an unpaired Student’s t-test. In all analyses, data were expressed as means S.E.M, and a p < 0.05 was considered significant.