C-phycocyanin protects against low fertility by inhibiting reactive oxygen species in aging mice
Part 2 of 2
Yan-Jiao Li1, Zhe Han1, Lei Ge1, Cheng-Jie Zhou1, Yue-Fang Zhao1, Dong-Hui Wang1, Jing Ren1, Xin-Xin Niu1 and Cheng-Guang Liang1
1The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, The Research Center for Laboratory Animal Science, College of Life Science, Inner Mongolia University, Hohhot, Inner Mongolia, People’s Republic of China
Correspondence to: Cheng-Guang Liang, email: firstname.lastname@example.org
Keywords: ovarian aging, oocyte, oxidative stress, C-phycocyanin, D-galactose, Gerotarget
Women over 35 have higher rates of infertility, largely due to deterioration of oocyte quality characterized by fragmentation, abnormal meiotic spindle-chromosome complexes, and oxidative stress. C-phycocyanin (PC) is a biliprotein enriched in Spirulina platensis that is known to possess antioxidant, anti-inflammatory, and radical-scavenging properties. D-galactose-induced aging acceleration in mice has been extensively used to study aging mechanisms and for pharmaceutical screening. In this study, adult female B6D2F/1 mice injected with D-galactose were used as a model to test the age-reversing effects of C-phycocyanin (PC) on degenerated reproductive ability. Our results show that PC can prevent oocyte fragmentation and aneuploidy by maintaining cytoskeletal integrity. Moreover, PC can reverse the expression of antioxidant genes, increase superoxide dismutase (SOD) activity and decrease methane dicarboxylic aldehyde (MDA) content, and normalize mitochondria distribution. PC exerts its benefit by inhibiting reactive oxygen species (ROS) production, which decreases apoptosis. Finally, we observe a significant increase in litter size after C-phycocyanin (PC) administration to D-galactose-induced aging mice. Our study demonstrates for the first time that D-galactose-induced impaired female reproductive capability can be partially rescued by the antioxidant effects of C-phycocyanin (PC).
In this study, we investigated the ability of C-phycocyanin (PC) to reverse D-gal-induced reproduction impairments in mice. Our results indicate that consecutive subcutaneous D-gal injections had toxic effects on the female reproductive system. Notably, C-phycocyanin (PC) protects against the D-gal-induced reduction of fertility in mice.
C-gal-induced aging is associated with oxidative stress and AGE generation . ROS play key roles in this process as their accumulation leads to the induction and maintenance of cellular senescence . In humans, aging is associated with increased ROS levels and decreased antioxidant levels in oocytes, cumulus cells, and follicular fluid [45-47], which impair multiple physiological processes from oocyte maturation to fertilization, embryo development, and pregnancy .
We observed that D-gal treatment reduced ovary and oocyte quality. These data concur with previous reports that D-gal can damage ovarian function in mice  and that oocyte quality decreases with age . Failure of polar body extrusion is often correlated with abnormal spindle formation and chromosome congression. We observed disrupted oocyte SCC integrity and an increased percentage of aneuploidy after D-gal treatment. This is probably a consequence of increased ROS levels. In natural ovulation, aging decrease oocyte number by altering hormone levels . Unexpectedly, D-gal or PC administration did not influence oocyte number after gonadotropin stimulation. We propose that in gonadotropin-treated mice, age-related immature follicles will respond to this stimulation, inducing more oocyte to ovulate . Interestingly, ROS levels were decreased after C-phycocyanin treatment, and this was accompanied by improved ovarian function and oocyte quality. Therefore, C-phycocyanin appears to protect against D-gal-induced ovarian aging by reducing ROS levels.
Figure 10: Experimental design. B6D2F/1 mice at 8 weeks of age were used.
(1) The control group was subcutaneously injected with 0.9% saline (green) and intragastrically administered ultrapure water daily (blue).
(2) The D-gal group was subcutaneously injected with 250 mg/kg/day D-gal solution (orange) and intragastrically administered ultrapure water daily. (3) The D-gal+PC group was subcutaneously injected with 250 mg/kg/day D-gal solution and intragastrically administered 500mg/kg/day of C-phycocyanin (PC) daily (yellow).
Telomere dysfunction may contribute to reproductive aging-associated meiotic defects, miscarriage, and infertility [53-55]. Unexpectedly, we did not observe any D-gal- or PC-induced changes in telomerase activity or the T/S ratio in ovaries. We propose that telomere dysfunction is mainly correlated with natural aging and might be observed over a longer term. There may not have been enough time for telomeres to shorten after only 40 days of D-gal treatment in our protocol.
Mammalian ovaries possess antioxidant defenses including the antioxidant tripeptide glutathione (GSH), and ROS-scavenging enzymes such as SOD, GSH-Px, CAT, glutathione S-transferase (GST), and peroxiredoxin (PRDX) [4, 56, 57]. We hypothesized that ovarian aging is associated with decreased expression of ovarian antioxidant genes and lower antioxidant enzyme levels, resulting in oxidative damage to ovarian function. This was confirmed by the observed variation of SOD activity and oxidative stress marker MDA content after D-gal treatment. Consistently, D-gal increased the expressions of Gclc and Gpx3 and decreased that of Cat. C-phycocyanin was able to reverse these changes, indicating that its likely rescue mechanism is related to the inhibition of ROS production.
To date, there is no evidence that C-phycocyanin directly affects mitochondrial distribution. We speculate that C-phycocyanin improved mitochondrial distribution mainly via its inhibition of ROS. A previous study reported that mitochondrial distribution is a cytoskeleton-dependent intracellular traffic behavior that relies on microtubule organization . The disruption of mitochondrial distribution is therefore a consequence of malfunctioned
ROS deteriorate microtubule dynamics and lead to their instability , which further deranges mitochondrial distribution and function. Galactitol is a secondary metabolite of D-gal formed by the reduction of D-gal after cellular metabolism. Gradual intracellular accumulation of galactitol can increase osmotic stress and elevate ROS levels . Binding between C-phycocyanin and ROS can form non-radical products, thus preventing ROSmediated damage of organelles including microtubules . Therefore, C-phycocyanin treatment can prevent ROS from decreasing microtubule stability and help maintain proper mitochondrial distribution. ROS serve as both key signaling molecules in physiological processes such as meiotic resumption and as indicators of cell apoptosis . Apoptotic pathways are also activated by ROS production . Our results indicate that D-gal treatment in mice triggers early stage apoptosis in MII oocytes, possibly due to ROS accumulation. Any treatment that inhibits ROS production, including C-phycocyanin, may decrease apoptosis.
Although no significant differences were found in birth weight, the results of our oocyte and ovary analyses suggest that PC can rescue D-gal-induced impairment of reproduction, especially small litter size. In conclusion, our results show that C-phycocyanin can help maintain reproductive performance in a D-gal-induced aging model. It is important to note that data obtained from a mouse model may not extrapolate directly to human reproduction and natural aging, and more extensive research is needed in a natural aging model before any clinic trials are to be attempted.
MATERIALS AND METHODS
All experiments adhered with the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Inner Mongolia University.
Experimental design, mouse feeding, mating, and offspring assessment
Female B6D2F1 mice were purchased from the Research Center for Laboratory Animal Science of Inner Mongolia University and housed under 12:12-h light:dark cycles in a specific pathogen-free animal facility at the Research Center for Laboratory Animal Science of Inner Mongolia University. During the whole experiment, mice had free access to food and water. At the age of 8 weeks, adult female mice were randomly divided into three groups and treated as follows. In the control group, mice were subcutaneously injected daily with 0.1 mL 0.9% saline for up to 40 days. During the last 30 days, 0.4 mL ultrapure water was intragastrically administered each day. For the D-gal group, mice were subcutaneously injected with 250 mg/kg/day D-gal (Sigma Aldrich, St. Louis, MO, USA) solution for up to 40 days. During the last 30 days, 0.4 mL ultrapure water was administered intragastrically each day. For the D-gal+PC group, mice were subcutaneously injected with 250 mg/kg/day D-gal solution for up to 40 days. During the last 30 days, 500 mg/kg/day C-phycocyanin (Zhejiang Binmei Biotechnology Co.,Ltd, Linhai, China) was intragastrically administered each day. A schematic of the experimental design is shown in Figure 10. Unless otherwise stated, all chemicals and media were purchased from Sigma Aldrich.
Male mice at 12 weeks of age with proven fertility were used for mating. Late in the afternoon, a single female mouse was placed in the cage of one male for mating. On the following morning, successfully mated female mice with a plug were returned to their cages for pup delivery. Female without mating plugs were not used for breeding experiments. The number and weight of offspring were recorded immediately after delivery. Body weight was measured and recorded once a week through the eighth week.
Ovary, liver, spleen, and kidney weight and oocyte collection
GV-stage oocytes were collected by puncturing the follicles of ovaries 48 hours after injection of pregnant mare serum gonadotropin (PMSG, SanSheng, Ningbo, China). Cumulus cells were removed by gentle pipetting. Oocytes were washed thoroughly and cultured in ChatotZiomet-Bavister (CZB) under liquid paraffin oil at 37°C in an atmosphere of 5% CO2 in air for 14 hours until they reached the MII stage. For in vivo MII-stage oocyte collection, mice were superovulated with 10 IU PMSG followed 48 h later by 10 IU human chorionic gonadotropin (hCG, SanSheng). MII oocytes with cumulus mass were released from the oviduct ampullae 14 h after hCG injection. Cumulus cells were dispersed by 0.3 mg/mL hyaluronidase in HEPES-M2 medium. Oocytes were cultured in CZB medium for 30 min of recovery. All oocytes from in vivo or in vitro maturation were examined for PB1 extrusion and fragmentation. The criteria for fragmentation evaluation were based on previous reports [61, 62]. Body weight and organ (ovary, liver, and spleen) wet weight were measured and recorded at the end of treatment, and ovaries were kept frozen at -80°C for further experiments.
Immunofluorescence microscopy and chromosome spreading
Oocytes were exposed to acidic tyrode solution (pH 2.5) for a few seconds to remove the zona pellucida followed by three washes in M2 medium. Oocytes were then fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in phosphate-buffered saline (PBS) at room temperature for 30 min, followed by permeabilization in PBS containing 0.5% Triton X-100 for 2 h at room temperature. Sample blocking was conducted with 1% bovine serum albumin (BSA, Amresco, Solon, OH, USA) in PBS containing 1/1000 Tween-20 (Amresco) and 1/10,000 Triton X-100. After blocking, samples were incubated with primary antibodies overnight at 4°C. For primary antibodies, we used mouse anti-alpha tubulin (1:2000, Abcam, Cambridge, UK) and anti-gamma tubulin (1:500, Abcam). For secondary antibodies, we used DyLight 549-conjugated donkey anti-mouse (1:100, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). DNA was stained with 4’,6-diamidino-2phenylindole (DAPI, 5 µg/mL, Roche, Mannheim, Germany) for 10 min. After staining and washing, samples were mounted on glass slides using Vectashield mounting medium (Vector Labs, Burlingame, CA, USA) and examined with a confocal laser-scanning microscope (Nikon, A1R, Tokyo, Japan). Images were analyzed with NIS-Element AR 3.0 software. Chromosome spreading analysis was performed as we described previously .
Telomere length measurement and telomerase activity assay
The average telomere length was measured from total genomic DNA of ovaries using a real-time PCR assay. The reactions were performed with telomeric primers for a reference control gene (the mouse 36B4 single-copy gene) using PCR settings as previously described . For each PCR reaction, a standard curve was made by serial dilutions of known amounts of DNA from the same tissues. The telomere signal was normalized to the signal from the single-copy gene to generate a T/S ratio indicative of relative telomere length. The telomerase activity of ovaries was measured with a telomerase enzyme-linked immunosorbent assay (ELISA) kit (Cusabio, Wuhan, China). The experiments were performed according to the manufacturer’s instructions. In brief, a telomerase-specific antibody was pre-coated onto a microplate. Standards and samples were pipetted into the wells, and any telomerase present was bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for telomerase was added to the wells. After washing, avidin-conjugated horseradish peroxidase was added to the wells. Following a wash to remove any unbound avidinenzyme reagent, a substrate solution was added, and color developed in proportion to the amount of telomerase bound in the initial step. After color development was terminated, color intensity was measured with a highsensitivity luminometer (Thermo Scientific, Waltham, MA, USA).
Quantitative real-time RT-PCR and enzyme assay
For quantitative real-time RT-PCR, total RNA was extracted from ovaries using the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. cDNA was synthesized using the PrimeScript RT reagent Kit (TaKaRa) following the manufacturer’s instructions. The primer sequences were obtained from a previous publication . RT-PCR was performed with the SYBR Green kit (TaKaRa). The comparative Ct method was used for data analysis, and Gapdh was used as an internal control.
For ovarian enzyme activity assays and MDA measurement, all procedures were performed according to the manufacturer’s instructions with kits purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China) unless noted otherwise.
ATP levels in oocytes and cumulus cells were measured using a kit (FL-ASC from Sigma Aldrich) as previously described with a minor change . Briefly, 30 denuded oocytes or cumulus cells collected from 30 cumulus-oocyte complexes in each mouse were snapfrozen in a microfuge tube containing 50 μL water and stored at -80°C. For ATP assays, 50 μL of each thawed sample solution was added to 100 μL ice-cold Cell ATP-Releasing Reagent and incubated on ice for 5 min, followed by the addition of 100 μL ice-cold ATP Assay Mix (1:25 diluted in assay mix buffer). The reaction mixture was then incubated for 10 min in the dark at room temperature for the initial chemiluminescence flash period. The bioluminescence of each sample was measured with a high-sensitivity luminometer (Thermo Scientific).
Evaluation of mitochondrial distribution
Denuded oocytes were fixed in 4% paraformaldehyde in PBS for 30 min in a humidified chamber, washed, and incubated in 25 nM Mitotracker Green-Fluorescence Mitochondria (Mitotracker Green FM; Molecular Probes, Eugene, OR, USA) in PBS-BSA for 30 min in the dark. After several washes, the oocytes were transferred into a drop of medium containing DAPI for 10 min. After staining, the samples were mounted on glass slides using Vectashield (Vector Labs) mounting medium and examined with a confocal laser-scanning microscope (Nikon). Images were analyzed with NISElement AR3.0 software.
Determination of ROS generation
To assess ROS production, cumulus-denuded oocytes were incubated with an oxidation-sensitive fluorescent probe (dichlorofluorescein, DCFH) for 30 min at 37°C in CZB containing 10 μM DCFH-DA (Nanjing Jiancheng Bioengineering Institute). Then the oocytes were washed three times with D-PBS with 0.1% BSA and mounted on glass slides. Florescence intensity in each oocyte was measured with a confocal system (Nikon) with the same scan settings for each sample.
Apoptosis detection was performed with an annexin-V staining kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Briefly, oocytes were washed twice in PBS and stained for 10 min in the dark with 100 mL binding buffer that contained 10 mL annexin-V-FITC. The samples were observed immediately after staining. Fluorescent signals were measured using a fluorescent microscope (Nikon) with 450-490 nm (excitation) and 520 nm (emission) filters.
At least six replicates were conducted for each treatment. Results are shown as means ± SEMs. Statistical comparisons were made using analysis of variance (ANOVA), and differences between treatment groups were assessed with Newman-Keul’s multiple comparison post hoc tests. All analyses were performed using GraphPad Prism 5.0 statistical software (GraphPad Software Inc., La Jolla, CA, USA). P < 0.05 was considered significant.
We thank Dr. Qing-Yuan Sun and Dr. Morigen for valuable suggestions and Angeleem Lu, Hong-Xia Zhou, and Xiang-Wei Kong for their kind assistance with oocyte collection.
CONFLICTS OF INTEREST
The authors have no competing interests to declare.
This study was supported by the NSF of China (31160243, 31371454); Program of Science and Technology Supporting for Homecoming People in Inner Mongolia; Natural Science Foundation of Inner Mongolia (2015JQ02).
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