CISD2 regulates oxidative stress and mitophagy to maintain the balance of the follicular microenvironment in PCOS

ABSTRACT

Objectives

To observe the CISD2 expression among PCOS patients and to explore its profound impact on the follicular microenvironment. Moreover, we want to elucidate the intricate mechanistic contribution of CISD2 to the onset and progression of PCOS.

Methods

Oxidase NOX2, mitophagy-related proteins, and CISD2 were detected by WB. The changes in mitochondrial structure and quantity were observed by transmission electron microscopy. Mitochondrial and lysosome colocalization was used to detect the changes of mitophagy. MDA kit, GSH and GSSG Assay kit and ROS probe were used to detect oxidative stress damage.

Results

We found that CISD2, mitophagy and oxidase in the GCs of PCOS patients were significantly increased. Testosterone stimulation leads to the increase of oxidase, mitophagy, and CISD2 in KGN cells. CISD2 inhibition promoted the increase of mitophagy, and the activation of mitochondria-lysosome binding, while alleviating the oxidative stress.

Conclusions

Inhibition of CISD2 can improve the occurrence of oxidative stress by increasing the level of mitophagy, thus affecting the occurrence and development of PCOS diseases.

1. Introduction

Polycystic ovary syndrome (PCOS) is the most prevalent endocrine metabolic disorder among women of reproductive age [1]. Ovarian dysfunction, hyperandrogenemia (HA), infertility resulting from oligoovulation or anovulation, and menstrual cycle disturbances are recognized as the primary characteristics of PCOS [2–4]. Patients with PCOS exhibit increased cystic follicles, disorganized arrangement of granulosa cells (GCs), thickened thecal cell layer, and impaired conversion of androgens to estrogens, which may contribute to HA. Numerous studies have speculated that HA could be the principal cause of PCOS [5]. Excessive androgens impair ovarian function and disrupt the follicular microenvironment, ultimately leading to abnormal oxidative stress and formation of intracellular reactive oxygen species (ROS). Studies have revealed a significant increase in oxidative stress levels in individuals with PCOS [6], further suggesting its association with the pathogenesis of this condition.

Mitochondria play a crucial role in maintaining cellular homeostasis and are involved in regulating various cellular functions [7] such as including aerobic phosphorylation, electron transport, calcium homeostasis [8], and signal transduction. These functions are essential for the body’s physiological activities. Mitochondria serve as the primary sites for cellular energy transformation and biological oxidation, generating most of the cell’s energy to sustain life, while also producing ROS [9–11]. Under normal physiological conditions, intracellular ROS participate in cell signal transduction and regulate bodily functions. However, excessive ROS accumulation because of mitochondrial dysfunction [12] can lead to oxidative stress. Increased ROS levels result in the accumulation of mitochondrial DNA mutations [13], reduction of mitochondrial membrane potential, and damage to mitochondrial structure and function. Mitophagy is primarily responsible for selectively removing damaged or dysfunctional mitochondria to maintain mitochondrial homeostasis [14]. Damaged or excess mitochondria are enclosed within autophagosomes and subsequently fused with lysosomes for degradation through regulation of the Pink1-Parkin pathway. When cellular oxidative stress causes mitochondrial damage, mitophagy is activated to remove abnormal mitochondria and prevent them from harming cells. Studies have shown that the level of mitophagy in PCOS patients is higher than that in the normal population [15], and appropriately increased mitophagy may alleviate cell damage caused by oxidative stress [16] and ameliorate the occurrence and development of PCOS.

CDGSH iron sulfur domain 2 (CISD2), a member of the NEET protein family, encodes nutrient deprivation autophagy factor-1 (NAF-1), which localizes to the mitochondrial outer membrane (MOM), endoplasmic reticulum (ER), and mitochondria-associated membrane (MAM) [17,18]. It plays a crucial role in regulating aging, cancer, autophagy, neurodegeneration, and apoptosis [19–24]. Recent studies have revealed that CISD2 plays a pivotal role in various cellular processes including the regulation of cellular iron levels, maintenance of ROS homeostasis, and control of mitophagy [25–28]. CISD2 is a redox-active [2Fe–2S] cluster protein involved in these processes. The biochemical properties of the [2Fe–2S] cluster are modulated by its redox state [29]. This unique structure demonstrates its pivotal role in redox regulation and iron regulation. In recent years, numerous studies have showed that CISD2 is a highly conserved gene widely expressed in various tissues and cells throughout the human body and plays a critical role in diverse pathological and physiological processes, such as maintaining mitochondrial structural integrity, regulating mitochondrial function, controlling mitophagy, as well as modulating Ca²⁺ homeostasis and redox homeostasis [30,31]. However, whether it is involved in the pathogenesis of PCOS is still unclear and necessitates further investigation.

In this study, we observed significantly elevated levels of oxidative stress and mitophagy in ovarian granulosa cells (GCs) obtained from patients diagnosed with PCOS, as compared to those from individuals without PCOS. Moreover, increased expression of CISD2 was detected among PCOS patients. By inhibiting the expression of CISD2 in the testosterone-stimulated human ovarian granulosa cell line (KGN), we effectively alleviated oxidative stress by promoting enhanced levels of mitophagy. These findings suggest that CISD2 may play a crucial role in maintaining the delicate balance within the follicular microenvironment during the development of PCOS through its regulation of both oxidative stress and mitophagy.

2. Materials and methods

2.1. Clinical specimens

The study received approval from Taizhou People’s Hospital Institutional Review Committee (KY2022-162–01). Participants for this research included control subjects and individuals diagnosed with PCOS and HA between ages 20–35 years old, recruited from Taizhou People’s Hospital Reproductive Center. Prior to starting the study, informed consent was obtained from all participants. The recruitment process for PCOS patients followed Rotterdam inclusion/exclusion criteria established in 2004 which required confirmation of HA through either oligo-ovulation/anovulation or ultrasound examination showing ovarian polycystic changes. The control group comprised male/tubal infertility patients undergoing IVF treatment with normal ovarian function and endocrine indexes. Individuals with infectious diseases or previous/current antibiotic usage during IVF treatment were excluded.

2.2. Collection and isolation of follicular fluid (FF) and GCs

All subjects received the same GnRH antagonist ovulation induction protocol. Ovulation was induced by intramuscular injection of 10,000 IU human chorionic gonadotropin (HCG) when subjects developed dominant follicles with a diameter measuring ≥18 mm. Oocytes retrieval was performed vaginally 36 h later, and both the accumulated cells and FF were aspirated. The collected FF was transferred to sterile centrifuge tubes. After centrifugation, the sediment was resuspended in phosphate-buffered saline (PBS) and transferred to sterile centrifuge tubes containing 50% Percoll (v/v) (50 mL Percoll + 50 mL ddH₂O). After centrifugation, cells in the middle layer of the isolated solution were collected, suspended in PBS, and centrifuged at 2000 rpm for 5 min at room temperature. The pellet was resuspended in red blood cell lysate, lysed at 4°C for 5 min, and centrifuged at 1500 rpm for 5 min. After washing twice with PBS, the obtained cell precipitates were stored at −80°C for subsequent protein and nucleic acid extraction.

2.3. Cell culture

The KGN cells (ZQ0916) were supplied by Zhongqiao Xinzhou Biotechnology (Shanghai, China). KGN cell lines were cultured in a 5% CO2 incubator at 37°C in DMEM/F12 medium (Gibco) containing 10% fetal bovine serum (FBS, Sigma) and 1% penicillin/streptomycin. To simulate the hyperandrogen state in PCOS patients, KGN cells were treated with 10 μM testosterone (APExBIO, C6163) for 12 h or 24 h. Testosterone concentrations were based on previous studies. To investigate the relationship between mitophagy and oxidative stress, we used the mitophagy inhibitor mdivi-1 to inhibit the occurrence of mitophagy. We treated KGN cells with 50 μM mdivi-1 (MCE, HY-15886, China) for 24 h. Mdivi-1 concentrations are based on previous studies. For experiments with chemical inhibitors, cells were pretreated with NAC (MCE, HY-B0215, 10 mM, 30 min) or Mito-TEMPO (MCE, HY-112879, 10 μM, 2 h).

2.4. Transfection of CISD2 interference (CISD2i)

The shRNA-CISD2 (10⁷ TU/mL, Genechem, Shanghai, China) loaded in lentivirus (LV) supplemented with 1× HitransG A reagent (Genechem) was transfected in KGN cells for 12 h when the confluence reached 30%. Then, complete medium was cultured for 60 h.

2.5. Transmission electron microscope

Transmission electron microscopy of KGN cells was performed according to the manufacturer’s instructions. The sections were further stained with 0.3% lead citrate(v/v) (3 µL lead citrate +997 µL ddH₂O) and imaged using an electron microscope (HITACHI, Tokyo, Japan).

2.6. Western blot and antibodies

The protein extraction was performed on ice. The cells were washed three times with pre-cooled PBS, and the petri dishes were added with RIPA buffer (APPLYGEN, Beijing, China) and protease inhibitor benzoyl fluoride (PMSF) (Beyotime, Shanghai, China) and cracked on ice for 30 min. The cleavage products were lightly scraped with a cell scraper and collected in a microfuge tube and centrifuged at 12000 rpm for 15 min. The supernatant was transferred into a new microfuge tube, and the protein concentration of the supernatant was determined using the bicinchoninic acid (BCA) kit (Beyotime, China). SDS-PAGE Protein loading buffer (5×) (Beyotime, China) was mixed and heated in a metal bath at 100°C for 10 min. Gel electrophoresis was performed with 10% gel(v/v) (26.6 mL ddH₂O + 33.3 mL 30%Acr-Bis(29:1) + 38 mL 1M Tris + 0.1 g SDS + 0.1 g APS + 40 µL TEMED), and the proteins were then transferred to PVDF membranes and sealed with 5% Skim milk(v/v) (5 g Skim milk + 95 mL TBST) at room temperature for 2 h and washed thrice in TBST. The film was then incubated with the primary antibody at 4℃ overnight. The next day, the film was washed thrice in TBST, 5 min each time. The membrane was then combined with HRP-conjugated Goat Anti-Rabbit IgG (AS014, dilution: 1: 5000, ABclonal) or HRP-conjugated Goat Anti-Mouse IgG (AS003, dilution: 1:5000, ABclonal) were incubated at room temperature for 2 h. After three rinses in TBST, the film was incubated with the exposure solution for 1 min, and the strips were exposed with the LAS-4000 chemiluminescence capture machine. ImageJ software was used to quantify the strength of the western blot bands. The antibody for Pink1 (ab23707, dilution: 1:1000) was purchased from Abcam (Cambridge, UK). Parkin (Abcam, ab77924, dilution: 1:2000); LC3B (CST, 83506, dilution: 1:1000); P62 (Abcam, ab109012, dilution: 1:10000); CISD2 (CST, 60758, dilution: 1:1000); SOD2 (CST, 13141, dilution: 1:1000); NOX2 (SAB, 32357, dilution: 1:1000); and NrF2 (Abcam, ab62352, dilution: 1:1000) antibodies were purchased from Abcam (Cambridge, UK).

2.7. Immunofluorescence staining

The cells were inoculated in 12-well plates at a density of 1 × 10⁵ cells/well. When the cell adhesion reached about 80%, the medium was discarded and cleaned with PBS thrice. After fixing in 4% paraformaldehyde(v/v) (4 mL paraformaldehyde +96 mL ddH₂O) for 15 min, the cells were washed thrice with PBS, and then permeated with 0.5% Triton X-100 (v/v) (5 µL Triton X-100 + 995 µL ddH₂O) (Beyotime, Shanghai, China) on ice for 15 min, and again washed thrice with PBS. After being blocked by the blocking solution for 1 h, the cell slides were incubated with the primary antibody at 4°C overnight. After three washes with PBS on the second day, the cell slides and the second antibody were incubated in a wet box at room temperature and away from light for 1 h. After that, it was washed thrice with PBS and incubated with DAPI for 5 min in dark light. Finally, the cells were cleaned with PBS thrice, sealed with fluorescence quencher (Beyotime, P0126, Shanghai, China), and observed under fluorescence microscope.

2.8. RNA isolation and real-time quantitative PCR (qPCR) analysis

KGN cells were seeded in 6-well plates and mixed with Trizol for 5 min for cell lysis. Then, 0.2 mL chloroform was added to every 1 mL Trizol, shaken for 15 s, incubated on ice for 10 min, and centrifuged at 12000 rpm at 4°C for 15 min. The upper aqueous phase was drained into a new EP tube, and isopropanol in equal volume was added, mixed well, place on ice for 10 min, and centrifuged at 12000 rpm at 4°C for 10 min. After discarding the supernatant, 1 mL 75% ethanol(v/v) (75 mL ethanol +25 mL ddH₂O) was added into the precipitation, mixed well, and centrifuged at 7500 rpm at 4°C for 5 min. These steps were repeated; the supernatant was discarded, and the residue was air dried at room temperature for 5–15 min. Finally, 20 µL diethyl pyrocarbonate water (DEPC) was added to re-suspend the RNA. FastKing gDNA Dispelling RT SuperMix (Tiangen Biotech, Beijing) was used to convert RNA to cDNA. Real-time quantitative PCR (qPCR) was performed with SuperReal PreMix Plus (SYBR Green) (Tiangeng) on a Roche LightCycler Real-time qPCR System (Roche, Basel, Switzerland). The threshold period value (Ct) was used to determine the levels of CISD2. Then, using β-actin as the unified parameter, the equation 2^-△△Ct was used for calculation. The relative gene expression level was compared with that of the control group, and all real-time qPCRs were performed in triplicate. The primers sequences used in this study are as follows: CISD2 (NM_001008388.5) (forward: 5′-GTGGCCCGTATCGTGAAGG-3′, reverse: 5′-CTAGCGAACCCGGTAATGCTT-3′); β-actin (NM_001101.5) (forward: 5′-CATGTACGTTGCTATCCAGGC-3′, reverse: 5′-CTCCTTAATGTCACGCACGAT -3′).

2.9. Mitochondrial membrane potential (MMP) detection

Cultured KGN cells were seeded at a density of 1.5 × 10⁵ cells/well in 12-well plates. Next day, cells were treated with vehicle or testosterone (10 µM) for 24 h. Then, the culture medium was discarded, and the cells were washed in PBS once. Then, 1 mL cell culture medium and 1 mL JC-1 dyeing solution (Beyotime, C2003, Shanghai, China) were added to the cells and mixed thoroughly. The cells were incubated at 37°C for 20 min. After incubation at 37°C, the supernatant was removed, and the cells were washed with JC-1 staining buffer twice. The buffer was removed, 2 mL of cell culture medium was added, and then observed under a fluorescence microscope.

2.10. Mitochondrial tracker staining

Cultured KGN cells were seeded at a density of 1.5 × 10⁵ cells/well on 12-well plates. Next day, cells were treated with vehicle or testosterone (10 µM) for 24 h. The cell culture medium was discarded, and a Mito-Tracker dye working medium (Beyotime, C1048, C1049B, Shanghai, China) at a final concentration of 20 nM was added for pre-warming at 37°C and incubated with the cells for 30 min at 37°C. The Mito-Tracker dye was then discarded and fresh cell culture solution pre-warmed at 37°C was added. The cells were then observed under a fluorescence microscope.

2.11. Lysosome tracker staining

Cultured KGN cells were seeded at a density of 1.5 × 10⁵ cells/well on 12-well plates. The next day, cells were treated with vehicle or testosterone (10 µM) for 24 h. When cells have reached the desired confluence, remove the medium from the dish and add the prewarmed (37°C) probe-containing medium (Invitrogen, L7528). The cells were incubated for 30 min under growth conditions appropriate for the particular cell type. Then, the loading solution was replaced with fresh medium, and the cells were observed using a fluorescence microscope fitted with the correct filter set.

2.12. Immunoprecipitation (COIP)

KGN cells were lysed with protein lysis buffer on ice for 30 min and centrifugated at 12,000 ×g at 4°C for 10 min. Then, 50 μL of the supernatant containing protein was collected as the ‘input’ group, while the remaining supernatant was probed with Protein G Sepharose® 4 Fast Flow beads (GE Healthcare Sverige AB, Stockholm, Sweden) coated with an antibody against CISD2 at 60 rpm and 4°C overnight, on a rotary table. The beads were boiled twice in 2× loading buffer at 100°C for 5 min and then washed at 120 rpm and 4°C.

2.13. Determination of MDA levels

Estimate MDA levels using commercial kits (Beyotime, S0131, Shanghai, China). Different groups of cells were collected, washed twice with PBS, and lysis buffer was added to lysis cells respectively. The lysed cells were centrifuged (10,000×g, 10 min) to remove debris and retain the supernatant. The contents of MDA in supernatant were determined by microplate reader. In addition, MDA levels were normalized according to protein concentration.

2.14. Determination of GSH/GSSG levels

GSH is the main source of sulfhydryl groups in most living cells, plays an important role in maintaining the proper REDOX state of sulfhydryl groups in proteins, and is a key antioxidant in animal cells. The specific experimental steps were carried out according to the GSH and GSSG Assay Kit instructions (Beyotime, S0053, Shanghai, China). The absorbance was measured at 412 nm and calculated based on the standard curve of GSH and GSSG.

2.15. ROS measurement

Cultured KGN cells were seeded at a density of 1.5 × 10⁵ cells/well on 12-well plates. The next day, cells were treated with vehicle or testosterone (10 µM) for 24 h. Then, the cell culture medium was discarded and 500 μL of DCFH-DA diluent (Beyotime, S0033, Shanghai, China) at a final concentration of 10 µM was added. The cells were incubated at 37°C for 20 min. A fluorescence microscope was used to observe the fluorescence intensity.

2.16. Mito-SOX measurement

Cultured KGN cells were seeded at a density of 1.5 × 10⁵ cells/well on 12-well plates. The next day, cells were treated with testosterone (10 µM) or mdivi-1 (50 µM) for 24 h. Then, the cell culture medium was discarded and 500 μL of Mito-SOX Red (MCE, HY-D1055, China) at a final concentration of 2 µM was added. The cells were incubated at 37°C for 20 min. A fluorescence microscope was used to observe the fluorescence intensity.

2.17. Statistical analysis

All data were derived from at least three replicates. Experimental data are presented as means ± standard deviation (SD) with analysis by Prism software (GraphPad, San Diego, CA, USA). Normal distribution of all data was confirmed by using the Shapiro – Wilk test. When the data were normally distributed, differences between the two groups were analyzed by Student’s t-test. One-way or two-way analysis of variance (ANOVAs) and Student–Newman–Keuls multiple comparison tests were applied to analyze differences among three or more groups. P value < 0.05 was considered statistically significant.

3. Results

3.1. Excessive oxidative damage is exhibited in the GCs of individuals with PCOS

Numerous studies have demonstrated that excessive oxidative stress may significantly contribute to follicular dysfunction. However, the presence of evident lipid peroxide damage in the follicles of patients with PCOS remains a subject of controversy. Therefore, we conducted a comparative analysis between women diagnosed with PCOS and healthy controls undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment to assess the levels of malondialdehyde (MDA), an indicator of the degree of lipid peroxidation, and NOX2, a key oxidase enzyme, in GCs. Our findings showed a significant elevation in the MDA content within GCs from PCOS patients (Figure 1(A)) accompanied by an increase in NOX2 expression (Figure 1(B and C)). These results suggest an increased oxidase activity and lipid peroxide damage in the follicular microenvironment of PCOS patients, which may play a crucial role in PCOS-related follicular dysplasia.

Figure 1.

Oxidative damage, mitophagy, and CISD2 are increased in the GCs of PCOS. (A) Representative MDA levels in GCs from controls and PCOS patients. (B-C) The protein level of NOX2 detected by western blot was increased in GCs of the PCOS group compared with those in the control group. (D-F) GCs were analyzed by transmission electron microscopy. Red arrowheads represent mitochondrial disruption and mitophagy-like structures. (G-I) The protein levels of Pink1 and Parkin detected by western blot were increased in GCs of the PCOS group compared with those in the control group. (J-K) The protein level of CISD2 detected by western blot was increased in the GCs of the PCOS group compared with those in the control group. The error bars represent the SD (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

3.2. Mitophagy and CISD2 levels are elevated in the GCs of individuals with PCOS

Mitochondria play a crucial role as primary sites for oxidative stress, while mitophagy has been shown to regulate ROS generation and contribute to follicle function regulation. Therefore, we conducted a comparative analysis using electron microscopy to investigate the subcellular structure of GCs in individuals with and without PCOS. Our findings revealed significant deviations in abnormal mitochondrial morphology along with an increased occurrence of mitophagy within the GCs obtained from PCOS patients. Specifically, we observed contraction of the mitochondrial ridge resulting in reduced mitochondrial size alongside an elevated presence of autophagosomes compared to controls (Figure 1(D–F)). Furthermore, western blot analysis showed a substantial upregulation in protein levels of Pink1 and Parkin (two pivotal regulators involved in mediating mitophagy) in the GCs from the PCOS group (Figure 1(G–I)), indicating a significant increase in mitophagy in PCOS patients.

Being closely associated with mitochondria, multiple studies have consistently demonstrated the involvement of CISD2 in modulating cellular oxidative stress levels, maintaining mitochondrial quality control, and regulating autophagy processes. Henceforth, it is postulated that CISD2 likely exerts its influence on the pathogenesis of PCOS through its impact on cellular oxidative stress levels and mitophagy regulation. To substantiate this hypothesis, we conducted an investigation into the expression profile of CISD2 in GCs derived from PCOS patients undergoing IVF/ICSI treatment. The results obtained from western blotting analysis showed a significant upregulation in protein levels for CISD2 within the PCOS group (Figure 1(J and K)). Collectively, these findings provide compelling evidence for an augmented expression level of CISD2 in PCOS pathology, while potentially playing a pivotal role in governing mitophagy processes.

3.3. HA induces oxidative stress and impairs mitochondrial function in KGN cells

HA is a crucial clinical characteristic of PCOS, and previous research has demonstrated that HA plays a central role in the development of follicular dysplasia in PCOS. To further investigate the relationship between HA, oxidative stress, and mitochondrial damage in the GCs of PCOS, we subjected KGN cells to an in vitro treatment with a high concentration of testosterone (10 μM), aiming to replicate the hyperandrogenic environment observed in PCOS patients. As anticipated, exposure to testosterone significantly increased the levels of oxidative damage marker MDA in stimulated KGN cells (Figure 2(A)), and western blot analysis also revealed an upregulation of NOX2 protein expression following 24 h of testosterone stimulation (Figure 2(B and C)). At the same time, the cell ROS assay also showed that ROS levels in KGN cells were significantly increased after exposure to testosterone stimulation, and ROS levels were sharp decreased after adding ROS scavenger NAC (Figure 2(D and E)).

Figure 2.

Testosterone induces oxidative stress and impairs mitochondrial function in KGN cells. (A) Representative MDA levels in testosterone-stimulated KGN cells after 12 and 24 h. (B-C) The protein level of NOX2 detected by western blot was increased in testosterone-stimulated KGN cells after 24 h compared with those in the control group. (D-E) Visualized by fluorescent microscopy, the green fluorescence intensity of ROS in the Ctrl group was weaker than that in the T group. The blue fluorescence signal indicates cell nucleus stained by DAPI. Scale bars: 100 μm. (F-H) Mitochondrial membrane potential (MMP) detection in testosterone-stimulated KGN cells after 24 h. Scale bar = 50 μm. The error bars represent the SD (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Previous studies have demonstrated that an increase in oxidative stress can impact the structure and function of mitochondria. Therefore, we assessed changes in mitochondrial membrane potential in KGN cells using a JC-1 fluorescent probe. Our results show that JC-1 aggregates were transformed into monomers in testosterone-stimulated KGN cells (Figure 2(F–H)), indicating significant reduction and damage to mitochondrial membrane potential compared to the normal group. These findings suggest that oxidative stress damage is significantly increased in testosterone-stimulated KGN cells, which also affects the structure and function of mitochondria.

3.4. Mitophagy and CISD2 expression are increased in testosterone-stimulated KGN cells

To further investigate the occurrence of mitophagy in testosterone-stimulated KGN cells, western blot analysis was carried out to examine the expression levels of mitophagy-related proteins, including Pink1 and Parkin. The results revealed a significant increase in both Pink1 and Parkin levels in the testosterone-stimulated group (Figure 3(A–C)). Moreover, immunofluorescence analysis demonstrated a substantial enhancement in fluorescence intensity for Pink1 and Parkin following testosterone treatment (Figure 3(D–F)). Subsequently, co-labeling of cells with a mitochondrial tracker and lysosome tracker facilitated direct observation of changes in mitophagy among different treatment groups. The findings exhibited a notable increase in autophagic lysosomes after testosterone treatment (Figure 3(G and H)). These findings suggest that stimulation by testosterone promotes an upregulation of mitophagy in KGN cells.

Figure 3.

Mitophagy is abnormally increased in testosterone-stimulated KGN cells. (A-C) The protein levels of Pink1 and Parkin detected by western blot were increased in testosterone-stimulated KGN cells after 24 h compared with those in the control group. (D-F) Immunohistochemical staining in testosterone-stimulated KGN cells after 24 h. Scale bar = 50 μm. (G-H) Testosterone-induced colocalization of mitochondria and lysosome was assayed 24 h later by confocal fluorescence microscopy using the fluorescent probes LysoTracker (red fluorescence) and MitoTracker (green fluorescence). Scale bar = 10 μm. The error bars represent the SD (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Similarly, western blot analysis was used to detect the protein levels of CISD2 in KGN cell models following testosterone stimulation, revealing a significant increase in CISD2 expression after 24 h of testosterone treatment (Figure 4(A and B)). Moreover, immunostaining confirmed the upregulation of CISD2 protein induced by testosterone (Figure 4(C and E)). Additionally, our findings demonstrated the mitochondrial localization of CISD2, which was consistent with previous literature reports and may be implicated in the regulation of mitochondrial function.

Figure 4.

Testosterone induces the expression of CISD2. (A-B) The protein level of CISD2 detected by western blot was increased in testosterone-stimulated KGN cells after 24 h compared with those in the control group. (C-E) Immunohistochemical staining in testosterone-stimulated KGN cells after 24 h. Scale bar = 100 μm. (F) The mRNA level of CISD2 in KGN cells treated with different CISD2-shRNA. (G-H) The protein level of CISD2 was decreased in KGN cells treated with different CISD2-shRNA compared with those in the Ctrl-shRNA. The error bars represent the SD (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

3.5. Downregulation of CISD2 promotes mitophagy via the Pink1-Parkin pathway

Subsequently, to investigate the regulatory role of CISD2 in testosterone-stimulated KGN cells, we established a cellular model with knockdown of CISD2 and assessed the levels of mitophagy-related proteins. As shown in the Figure 4(F–H), the protein expression efficiency of CISD2 has been knocked down to about 32.55%. Knockdown of CISD2 resulted in increased protein levels of Pink1, Parkin and LC3B and decreased expression of autophagy-consuming substrate P62 in testosterone-stimulated KGN cells (Figure 5(A–E)). Additionally, our results showed that mitochondrial autophagosomes exhibited increased co-labeling with a mitochondrial probe and lysosomal probe in the CISD2 knockdown group (Figure 5(G and H)). These findings suggest that downregulating CISD2 can potentiate mitophagy through the Pink1-Parkin pathway in KGN cells.

Figure 5.

Inhibition of CISD2 promotes mitophagy by Pink1-Parkin pathway. (A) The protein levels of Pink1, Parkin, P62, and LC3B were detected by western blot. (B–E) The protein level of P62 was decreased, whereas those of Pink1, Parkin, and LC3B were increased in the CISD2-shRNA + T group compared with those in the Ctrl-shRNA + T group. (F) Representative protein bands of CISD2, Pink1, Parkin, and LC3B in KGN cells after IP assay using an antibody against CISD2. (G-H) Different group colocalization of mitochondria and lysosome was assayed by confocal fluorescence microscopy using the fluorescent probes LysoTracker (red fluorescence) and MitoTracker (green fluorescence). Scale bar = 10 μm. The error bars represent the SD (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Furthermore, the COIP experiment was conducted to investigate the regulatory role of CISD2 in mitophagy regulation via the Pink-Parkin pathway. Our analysis revealed a specific interaction between CISD2 and Pink1, while no direct interactions were observed with Parkin and LC3B (Figure 5(F)). These findings suggest that CISD2 may exert a regulatory function in mitophagy through its binding with Pink1.

3.6. CISD2 knockdown mitigates testosterone-induced oxidative stress

Previous studies have shown the crucial role of mitophagy in regulating mitochondrial function and ROS production across various tissues. However, it remains unclear whether CISD2 inhibition exerts a similar influence on the follicular microenvironment of PCOS. Therefore, we further investigated whether CISD2 knockdown modulates the level of oxidative stress induced by HA in KGN cells. Subsequent analysis revealed a significant reduction in ROS levels following CISD2 knockdown in KGN cells (Figure 6(A and B)). Moreover, quantification of GSH/GSSG and MDA revealed an increase in GSH/GSSG content and a decrease in MDA content after silencing CISD2 (Figure 6(C and D)). Finally, additional examination was conducted to evaluate the expression of NOX2 oxidase as well as antioxidant enzymes SOD2 and NrF2 in KGN cells. The results exhibited a notable decrease in NOX2 oxidase expression, while there was a substantial upregulation of antioxidant enzymes SOD2 and NrF2 upon CISD2 knockdown compared to the control group (Figure 6(E–H)).

Figure 6.

Inhibition of CISD2 suppresses testosterone-induced oxidative stress. (A-B) Visualized by fluorescent microscopy, the green fluorescence intensity of ROS in the CISD2-shRNA + T group was weaker than that in the Ctrl-shRNA + T group. The blue fluorescence signal indicates cell nucleus stained by DAPI. Scale bars: 50 μm. (C-D) Representative GSH/GSSG and MDA levels in KGN cells. (E) The protein levels of NOX2, SOD2, and NrF2 were detected by western blot. (F-H) The protein level of NOX2 was decreased, whereas those of SOD2 and NrF2 were increased in the CISD2-shRNA + T group compared with those in the Ctrl-shRNA + T group. The error bars represent the SD (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

3.7. Inhibition of mitophagy aggravates the occurrence of oxidative stress

In order to investigate the relationship between the increase of mitophagy promoted by knockdown CISD2 and the decrease of oxidative stress in the testosterone induced PCOS model, we added testosterone (T, 10 μM) and mdivi-1 (MD, 50 μM) to KGN cells with knockdown CISD2 for 24 h, and then observed the changes of mitophagy and oxidative stress related indexes. Our experimental study found that WB results showed that the expression of mitophagy associated protein (Pink1, LC3B) was significantly reduced after mdivi-1 treatment (Figure 7(A–C)), while the expression of oxidase NOX2 was increased and the expression of antioxidant oxidase SOD2 was decreased (Figure 7(D–F)). Next, Mito-SOX probe staining was performed on the cells of different treatment groups, and the results showed that Mito-SOX increased significantly after mdivi-1 treatment (Figure 7(G and H)). These findings suggest that promoting mitophagy through CISD2 knockdown effectively mitigates the intracellular oxidative stress induced by HA in KGN cells by suppressing ROS production and enhancing cellular antioxidant capacity.

Figure 7.

Inhibition of mitophagy increases oxidative stress. (A-C) The protein levels of Pink1 and LC3B detected by western blot were decreased in mdivi-1-stimulated KGN cells after 24 h. (D-F) The protein level of NOX2 was increased, whereas SOD2 was decreased in mdivi-1-stimulated KGN cells after 24 h. (G-H) Visualized by fluorescent microscopy, the red fluorescence intensity of Mito-SOX in the CISD2-shRNA + T + MD group was strengthener than that in the CISD2-shRNA + T group. The blue fluorescence signal indicates cell nucleus stained by DAPI. Scale bars: 100 μm. The error bars represent the SD (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

4. Discussion

PCOS is one of the most common endocrine disorders that affects women worldwide, seriously affecting the physical and mental health of women of childbearing age. A growing body of research evidence shows that HA and oxidative stress play a major role in the occurrence and development of PCOS. Several studies have shown that there is a high level of ROS production in PCOS patients [32], and the over-androgen-stimulated ROS production leads to mitochondrial dysfunction [33], which is the most important site for ROS production. An increase in the number of damaged mitochondria exacerbates oxidative stress, creating a vicious cycle [34]. At the same time, other reports have found that the level of mitophagy in PCOS patients is also higher than that in normal people [35], and improved mitophagy can help cells remove excess or dysfunctional mitochondria [36], thus improving the intracellular microenvironment. Therefore, research into treatments targeting mitochondrial dysfunction has the potential to break this vicious cycle and prevent or mitigate oxidative stress and the progression of PCOS. CISD2 is a mitochondrial protein that plays a crucial role in mitochondrial functional regulation, energy metabolism regulation, Ca²⁺ homeostasis regulation, and ROS production. In this study, we found that the expression of CISD2 is elevated in PCOS patients, and inhibition of mitochondria-associated protein CISD2 has protective effects on oxidative stress and mitophagy under excessive androgen stimulation.

Our study showed that the expression of oxidase NOX2 and mitophagy-related proteins Pink1 and Parkin were significantly increased in follicular fluid granule cells of PCOS patients, and the expression of CISD2 protein was also significantly increased. These data suggest that CISD2 may play a key role in the occurrence and development of oxidative stress and mitophagy in PCOS diseases. Interestingly, our results showed that when we inhibited CISD2 with shRNA in the testosterone-stimulated model, the expressions of mitophagy-related proteins Pink1, Parkin and LC3B were significantly increased, and mitochondrial and lysosome colocalization was also significantly activated. These results suggest that inhibition of CISD2 as a regulator of intermitochondrial connectivity can enhance mitophagy mediated by Pink1-Parkin. In our subsequent study, we found that the oxidative stress damage of cells was significantly improved when the expression of CISD2 was inhibited to activate mitophagy. Inhibiting the expression of CISD2 can reduce the production of ROS, increase the expression of antioxidant enzyme SOD2, and improve the intracellular environment, thus maintaining the stability of the follicular microenvironment. A large number of studies have shown that mitophagy is double-sided, and a certain degree of mitophagy can devour damaged mitochondria [37,38] and reduce the production of ROS [39] to maintain cell homeostasis. However, excessive mitophagy can cause cell apoptosis or pyroptosis [40]. Our study found that in the PCOS disease model, the level of mitophagy was increased in the presence of CISD2-shRNA, and the enhanced mitophagy would phagocytose the damaged mitochondria, improve overall mitochondrial function, reduce the production of ROS, enhance the expression of antioxidant enzymes, and improve cellular oxidative stress. Other studies observed that CISD2 overexpression protects the liver from oxidative stress and reduces the occurrence of mitochondrial DNA deletions [28]. The reason for these varied results may be that CISD2 has a special [2Fe-2S] cluster whose biological function involves the transfer of electrons and Fe-S clusters [41,42], so CISD2 acts differently depending on the redox conditions in the cell. CISD2 is unable to transfer [2Fe-2S] clusters when the intracellular environment is in a reductive state. However, only when cells are under oxidative stress does CISD2[2Fe–2S] in the mitochondria transfer [2Fe–2S] clusters to apoproteins, and electrons from NADH to oxygen or ubiquinone [43–45]. In an oxidizing environment, CISD2 contributes to oxidative stress and production of superoxide radicals (O²⁻) by transferring iron to the mitochondrial matrix and electrons to oxygen through the oxidation of NADH (the electron donor) [46]. These results suggest that inhibiting CISD2 expression can save ROS production and mitochondrial dysfunction in oxidative state, improve mitochondrial quality control, and play a protective role in cells. Therefore, our findings provide a new possible etiology for PCOS disease, and CISD2 may be a therapeutic molecular target for mitochondrial dysfunction during PCOS.

Our study has some limitations, including the lack of in vivo experiments and study of specific signaling pathways in which CISD2 likely plays a role. In future research, we plan to establish a CISD2 conditional knockout mouse model to study its main physiological functions and specific signaling pathways in mice with PCOS, for a more comprehensive evaluation. In addition, CISD2 is located in the ER-mitochondria contacts (MERC) and has important regulatory effects on the mitochondria and ER [47]. Our previous studies have also found that HA induces ER stress in the FF of GCs from PCOS patients by causing inflammation, thus inducing NLRP3 expression and pyroptosis [48]. Whether CISD2 regulates the occurrence of endoplasmic reticulum stress in PCOS still needs further study (Figure 8).

Figure 8.

The follicular microenvironment in PCOS is modulated by CISD2, which exerts its influence through the regulation of oxidative stress and mitophagy. Hyperandrogenemia induces an upregulation of CISD2 expression in ovarian GCs, subsequently leading to the inhibition of mitophagy activation. This disruption creates an imbalance between mitophagy and oxidative stress within these cells, ultimately impacting the equilibrium of the follicular microenvironment in PCOS patients.

5. Conclusions

Our findings suggest that CISD2 may play a pivotal role in affecting the intricate balance of the follicular microenvironment during PCOS development by regulating oxidative stress and mitophagy. Therefore, targeting CISD2 could potentially serve as an effective therapeutic strategy for managing PCOS.

Funding Statement

This work was supported by Innovative Research Group Project of the National Natural Science Foundation of China.

Author contributions

Study conception and design: HSG, QZ; experiment implementation: HHW, QZ, NL, TYX; data analysis: ZCH, JYC, LLW; manuscript draft: HSG, QZ, YX; study supervision: HSG. All authors approved the final version of the manuscript.

Glossary

PCOS

Polycystic ovary syndrome

HA

Hyperandrogenemia

IVF

In vitro fertilization

GCs

Granulosa cells

FF

Follicular fluid

HCG

Human chorionic gonadotropin

MAM

Mitochondria-associated membrane

MMP

Mitochondrial membrane potential

MOM

Mitochondrial outer membrane

ER

Endoplasmic reticulum

T

Testosterone

MD

Mdivi-1

MDA

Malondialdehyde

GSH

Reduced glutathione

GSSG

Oxidized glutathione

FBS

Fetal bovine serum

PBS

Phosphate-buffered saline

ROS

Reactive oxygen species

Mito-SOX

Mitochondrial Superoxide

SD

Standard deviation

WB

Western blot

Data availability

Data will be made available on request.

Disclosure statement

No potential conflict of interest was reported by the author(s).