Protective Effect of SCUBE1 on Oxidative Stress-induced Apoptosis in Human Ovarian Granulosa Cells

Background: Apoptosis of ovarian granulosa cells (GCs) is a sign of follicular atresia. This study aimed to explore the role and mechanism of signal peptide, CUB domain, epidermal growth factor-like protein1 (SCUBE1) in protecting GCs from apoptosis induced by hydrogen peroxide (H 2 O 2 ). Results: SCUBE1 was expressed in women of all ages and had the highest expression level in the ovaries in multiple organs and tissues of KM mouse. In vitro cell experiments show that SCUBE1 pretreatment reduced H 2 O 2 -induced apoptosis and improved cell viability. SCUBE1 also blocked the production of ROS in cells and improved mitochondrial membrane potential. After SCUBE1 pretreatment, anti-apoptotic protein Bcl-2 expression was upregulated, whereas the expression of the pro-apoptotic proteins Bax, Bax/Bcl-2, Caspase-3, and p53 were downregulated. Analysis of the impact of SCUBE1 (c.1169C >G, p.P390R) mutation from the aspect of mutation pathogenicity; protein stability; and gene haplotype insuciency, indicated that the p.P390R mutation is signicantly pathogenic. Conclusions: This is the rst time that the potential role of SCUBE1 in protecting GCs from H 2 O 2 -induced damage by blocking the production of ROS, increasing the mitochondrial membrane potential and regulating apoptosis-related proteins, attributing to POI, is studied. SCUBE1 (c.1169C >G, p.P390R) mutation has signicant pathogenicity but the specic harm needs to be conrmed by further studies.

Introduction encodes cell surface proteins and secreted proteins [8], and participates in in ammatory responses [9], cell proliferation repair [10], ischemia, hypoxia, and other pathophysiological processes, while OS plays an important role in the pathophysiological processes of in ammation, ischemia, and hypoxia. Recent studies have con rmed the involvement of the SCUBE1 gene in regulating the differentiation of porcine ovarian GCs, with subsequent veri cation by qRT-PCR showing SCUBE1 mRNA expression in porcine ovarian GCs [11]. SCUBE1 mRNA is widely expressed in human tissues and ranks second in ovarian tissues (The Human Protein Atlas: https://www.proteinatlas.org/ENSG00000159307-SCUBE1). However, it is unclear whether SCUBE1 can protect human GCs from the damage caused by OS.
The use of hydrogen peroxide (H 2 O 2 ) to induce oxidative damage in ovarian GCs in vitro and thus induce cell apoptosis, is a common, reasonable, and effective method for exploring the factors and mechanisms that affect follicular development and ovarian function [12].This study aimed to investigate the expression of SCUBE1 in the ovaries and the effects of SCUBE1 on ROS, mitochondrial membrane potential (ΔΨm), and related apoptosis factors in GCs treated with H 2 O 2 , to analyze the role of SCUBE1 in human GC apoptosis and provide new ideas for the prevention and treatment of POI.

Methods
All manufacturer guidelines were followed when using kits in this investigation unless otherwise speci ed.

Tissue samples Patients
The study protocol was approved by the ethics committee of Zhujiang Hospital of Southern Medical University, and all subjects signed an informed consent form before surgery. Participants were divided into a young group (18 ≤ group 1 ≤ 36 years old), middle-aged group (36 < group 2 ≤ 54 years old), and elderly group (group 3 > 54 years old), with ve cases in each group. Normal ovarian tissues were surgically removed and collected from the Department of Obstetrics and Gynecology, Zhujiang Hospital of Southern Medical University. The criteria for selected ovarian tissues included the exclusion of ovarian diseases, including ovarian cysts, ovarian benign tumors, and ovarian cancer. Postoperative pathology was con rmed by two professional pathologists. The ovarian tissue was placed in a liquid nitrogen tank within 30 min of being isolated until further processing.

Animals
The study protocol was approved by the Ethics Committee of the Zhujiang Hospital of Southern Medical University. Kunbai strain healthy female mouse (KM mouse) aged 3-5 weeks and 10-11 weeks old were purchased from the Experimental Animal Center of Southern Medical University. They were reared in a single cage in a conventional manner under a 14/10 h light/dark environment at a room temperature of 22°C. For the experiment, 6-week-old (n = 5) and 12-week-old (n = 5) mice were selected. After the mouse were sacri ced, both ovaries were immediately removed. One ovary was xed with 4% paraformaldehyde for 24-48 h. The other ovary and the removed brain tissue, heart, lung, liver, spleen, stomach, small intestine, and muscle were placed in a liquid nitrogen tank.

Hematoxylin and eosin (HE) staining and immunohistochemistry
Fixed KM mouse ovaries were para n-embedded. Serial sections of the wax block (4 µm thick) were mounted on a glass slide, and four sets of sections were prepared for HE staining (tissue location control), SCUBE1 immunohistochemical analysis (observation of SCUBE1 distribution), PBS replaces the primary antibody (blank control), and one sheet for standby application. The sections were depara nized in xylene, hydrated in gradient alcohol, and then immersed in citric acid-EDTA antigen retrieval solution (Beyotime, Shanghai, China); the mixture was placed in a microwave oven on high heat for 5 minutes, left to rest for 3 minutes, returned to a medium heat for 5 minutes, and then naturally cooled to room temperature. After blocking with endogenous peroxidase, the sections were incubated with an anti-SCUBE1 antibody (1:400, bs-9903R, Bioss, Beijing, China), followed by incubated the secondary antibody conjugated with biotin and the horseradish enzyme-labeled streptavidin working solution (ZSGB-BIO, Beijing, China) for 1 h at room temperature. The immunoreactivity of SCUBE1 in ovarian cells was then analyzed and scored independently by three observers. The immune response to SCUBE1 was evaluated according to the intensity of the brown color. The immunoreactivity corresponding to the degree of brown color from light to dark was weak, medium, and strong.

Quantitative real time PCR (qRT-PCR)
A TRIzol reagent kit (Takara, Japan) was used to extract total RNA from human ovarian tissue and different organs and tissues of KM mouse, according to the manufacturer's instruction. The absorbance at 260/280 nm was measured using an ultra-micro high-precision spectrophotometer Nanodrop ND-2000 (Thermo, USA) ratio to determine the concentration and purity of RNA. After reverse transcription of total RNA (1 µg), FastFire qPCR PreMix (SYBR Green) (Tiangen Biotech, Beijing, China) was used for real-time PCR analysis. The primer sequences for SCUBE1 and GAPDH are listed in Table 1. Table 1 Primers for SCUBE1 and GAPDH used for qRT-PCR containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (HyClone, USA) at 37°C in a 5% CO 2 atmosphere, and cultivated in an incubator with 95% humidity.

Cell treatment
Cells were seeded in a 6-well plate or Petri dish and divided into three groups (control, model, and experimental groups). Twenty-four hours after cell inoculation, the experimental group was treated with 5 ng/mL SCUBE1 human recombinant protein (rhSCUBE1, Abnova, USA), and the remaining two groups were replaced with the same volume of serum-containing DMEM/F12 medium. After another 24 h, the model and experimental groups replaced the medium with 0.3 mmol/L (mM) H 2 O 2 , and the control group replaced the same volume of serum-containing DMEM/F12 medium. After incubation at 37°C in an incubator with a 5% CO 2 atmosphere for 24 h, one step of processing was performed.

Analysis of cell viability
Cells were inoculated in 96-well plates (5000 cells/100 µL/well), incubated at 37°C in an incubator with a 5% CO 2 atmosphere for 24 h, the culture medium was discarded, H 2 O 2 (0.01, 0.1, 0.3, 0.5, 0.8, 1.0, and 1.5 mM) and an equal volume of serum-containing DMEM/F12 medium was added to the control group. Cells were incubated for 24 h, the medium was aspirated, and 100 µL of medium containing 10% CCK-8 solution (Apexbio, USA) was added to each well, with each plate incubated at 37°C in an incubator with a 5% CO 2 atmosphere for 3 h. A microplate reader was used to measure the optical density (OD) of each well at a 450 nm wavelength. All experiments were repeated three times, analyzed, and graphed using GraphPad Prism 8 software. The concentration of H 2 O 2 was determined based on less than or close to the half-maximal inhibitory concentration (IC50). The inhibition rate of cell proliferation (%) = (mean OD value of control group -mean OD value of experimental group) / (mean OD value of control group -mean OD value of blank group) × 100. in the rest of the groups it was changed using the same volume of serum-containing DMEM medium.
These groups were all incubated at 37°C in an incubator with a 5% CO 2 atmosphere for 24 h, with the respective culture media discarded, and the cells tested using the CCK-8 assay.

Determination of intracellular ROS level
Cells were washed three times with PBS and incubated with DCFH-DA (10 µmol/L) (Beyotime, Shanghai, China) in a 37°C incubator for 20 min. Cell culture medium was washed 3 times to remove excess DCFH-DA, followed by an analysis of cells uorescence intensity with a ow cytometer (BD Biosciences, USA).

Measurement of mitochondrial membrane potential
The changes in mitochondrial membrane potential (ΔΨm) were measured using the uorescent dye rhodamine123 (Rh123, Beyotime, Shanghai, China). Cells were digested and collected by trypsin (Gibco, USA), washed twice with PBS, with Rh123 added at a nal concentration of 10 mM. Cells were, incubated at 37°C for 30 min, followed by washing with PBS three times. Immediately after, ow cytometry was performed to measure the average uorescence intensity of the cells.

Analysis of apoptosis by ow cytometry
Treated cells were collected, washed, centrifuged, and resuspended in Binding Buffer, and then 5 µL Annexin V-FITC and 10 µL PI (CWBIO, Jiangsu, China) were added, mixed, and incubated at room temperature for 15 min in the dark for analysis with ow cytometry.

Western blotting
Total protein extract was obtained from human ovarian tissues and cultured KGN cells. The BCA protein quanti cation kit (CWBIO, Jiangsu, China) was used to detect the protein concentration and adjust it to the same level. The protein sample (20 µg) was loaded onto an SDS-polyacrylamide gel for electrophoresis and then transferred to a PVDF membrane. After blocking non-speci c protein binding sites with 5% skimmed milk for 1.5 h, they were combined with the corresponding primary antibodies [β- According to the Exome Aggregation Consortium (ExAC) PLI (loss-intolerance) [15] the score used to evaluate the possibility of SCUBE1 loss of function (LOF) mutation ranges from 0 to 1. The higher the score, the lower the tolerance.
Align-GVGD [16] is a free online tool for the physical and chemical characterization of mutations. It combines the GVs and GDs for the prediction. The predicted categories range from C0 to C65, and the higher the level, the greater the probability that the mutation will interfere with protein function.
iStable [17] is an integrated prediction tool that predicts the results of MUPRO and I-Mutant2.0. Using the sequence of the protein, we predicted the changes in stability due to speci c substitutions.
PyMol software was used to analyze the local spatial con guration changes in the wild-type and mutanttype SCUBE1 models.

Statistical analysis
The experiments were repeated at least three times. Data are expressed as mean ± SD, and SPSS software (Version 23.0, SPSS, Inc., Chicago, USA) was used for analysis. One-way analysis of variance was used to determine the statistical differences. Non-normally distributed data were analyzed with a non-parametric Kruskal-Wallis test. A P value < 0.05 was considered statistically signi cant (* P < 0.05, **P < 0.01).

Expression of SCUBE1 in women ovaries
Firstly, the total RNA and protein of the human ovaries in the young, middle-aged, and old groups were extracted, and the expression levels of SCUBE1 mRNA and protein in each group were detected by qRT-PCR and western blotting, respectively. Results showed that SCUBE1 was expressed in women of all ages, and the expression level was higher in the elderly group (P > 0.05) (Fig. 1B, C), consistent with the qRT-PCR results (Fig. 1A).

Localization of SCUBE1 in KM mouse ovaries
Next, due to the limited availability of human ovarian tissue and other tissues, we conducted related research on KM mouse. The expression and localization of SCUBE1 in the ovaries of the KM mice was determined. HE staining ( Fig. 2A, G) indicated that hematoxylin resulted in a purple-blue nucleus while eosin gave the cytoplasm and extracellular matrix components a red color.
Results showed that SCUBE1 had similar expression patterns in the ovaries of KM mice at 6 and 12 weeks old (corresponding to puberty and sexual maturity, respectively) (Fig. 1B, H). SCUBE1 was strongly expressed in stromal cells and the surrounding area rich in blood vessels, moderately expressed in the corpus luteum, and there was no obvious expression in the follicles (Fig. 2C-F, I-L). SCUBE1 mRNA expression levels in the different organs and tissues of the mice were as follows: ovary > lung > brain > stomach > spleen > small intestine > liver > heart > thigh muscles (Fig. 3).

Effect of rhSCUBE1 treatment on KGN cell viability and apoptosis
Considering that the apoptosis of GCs is a sign of follicular atresia, KGN cells were used as the research object to explore the effect and mechanism of SCUBE1 on the apoptosis of human GCs cultured in vitro.
When KGN cells were exposed to different concentrations of H 2 O 2 for 24 h, cell viability decreased in a concentration-dependent manner (Fig. 4A) (Fig. 4B). Therefore, in subsequent experiments, cells were pretreated with 5 ng/mL rhSCUBE1.
Annexin V binds to FITC and speci cally binds to phosphatidylserine residues in apoptotic cells. Results showed that the apoptosis of KGN cells increased after exposure to 0.3 mM H 2 O 2 (P = 0.001), while 5 ng/mL rhSCUBE1 pretreatment reduced H 2 O 2 -induced cell apoptosis (P = 0.018) (Fig. 4C and D).
rhSCUBE1 reduced the generation of intracellular ROS and increased the mitochondrial membrane potential Classic DCFH-DA staining was used to determine the changes in ROS levels in KGN cells. Results showed that 0.3 mM H 2 O 2 could induce intracellular ROS to increase to 1.25 times that of the control (P = 0.000), and the rhSCUBE1 pretreatment could signi cantly reduce intracellular ROS levels (P = 0.000) (Fig. 5A and  B).
Early stages of apoptosis caused a drop in mitochondrial membrane potential. It was found by Rh123 staining that 0.3 mM H 2 O 2 could cause the mitochondrial membrane potential to drop to 0.63 times that of the control (P = 0.000), and the rhSCUBE1 pretreatment reversed this loss (P = 0.022) (Fig. 5C and D). rhSCUBE1 exerts cytoprotective effects via the mitochondrial pathway To study the protective mechanism of rhSCUBE1, the expression of pathway-related proteins in KGN cells were detected by western blotting (Fig. 6A and B). We found that SCUBE1 protein was expressed on KGN cells. It was clear that H

SCUBE1(c.1169C > G, p.P390R) pathogenicity and stability analysis
Based on software analysis of the bioinformatic changes before and after gene point mutations, the various effects of the p.P390R mutation on SCUBE1 could be more clearly understood. Sanger sequencing was performed on 20 POI samples [5], of which three patients were consequently found to have heterozygous mutations in SCUBE1 (c.1169C > G, p.P390R) (Fig. 7A). The p.P390R mutation resulted in a proline mutation (Fig. 7B, a) to arginine (Fig. 7B, b) at position 390. Based on PyMol software, analysis of the SCUBE1 homologous protein model (Template PDB Code 4xbmB), there were changes from proline to arginine in the local space of SCUBE1 (Fig. 7B, c, and d).
Data from SIFT, LRT, SNAP, and PANTHER software also suggested that this mutation seriously affects protein function (Table 2). According to ExAC, the PLI score of SCUBE1 was 0.96. SCUBE1 cannot tolerate LOF mutations, that is, SCUBE1 has a single dose under-dose, which further supports the pathogenicity of the p.P390R mutation. Both iStable and i-Mutant2.0 in the iStable tool predicted that p.P390R is a destabilizing activity ( Table 3). The Align-GVGD server was further used to predict the destabilizing effect of the p.P390R mutation on SCUBE1 and the results showed that the mutation was classi ed as Class C65, which signi cantly hindered protein function (Table 4).

Discussion
We studied the expression of SCUBE1 in ovarian tissue and found that SCUBE1 is expressed in human and mouse ovaries. Although SCUBE1 is mainly expressed in several highly vascularized tissues such as liver, kidney, lung, spleen and brain [18], its expression levels in other organs were lower than those of the ovaries in KM mouse. It is speculated that the continuous expression of SCUBE1 in human ovaries is likely to be an important guarantee for maintaining normal ovarian function. As a secreted protein, it may regulate the growth and development of follicles through paracrine and autocrine functions.
At present, one of the focuses of the etiology of POI is the molecular biological mechanism of granulosa cell apoptosis during follicular atresia. Studies have shown that ROS levels in POI patients with normal cytogenetics are signi cantly higher than those in normal controls [19], and ROS plays an important role in antral follicular apoptosis [20]. According to reports, SCUBE1 may be a potential serological marker of in ammatory diseases (acute appendicitis [21]), ischemic hypoxic diseases (acute mesenteric ischemia [22], acute coronary syndrome and ischemic stroke [23], ovarian torsion [24]). In rats with ischemiareperfusion kidney injury, SCUBE1 can promote the proliferation of renal tubular epithelial cells and antagonize apoptosis [25][26]. However, whether SCUBE1 can protect human GCs from damage by H 2 O 2 has not yet been reported.
The key to H 2 O 2 inducing apoptosis of GCs lies in the abnormal increase of ROS, which causes mitochondrial dysfunction, and the decrease in mitochondrial membrane potential is a landmark event in the early stage of apoptosis [27]. Nishi et al. [28]  used for 24 h for inducing apoptosis of KGN cells; as the cell morphology changed accordingly, the intracellular ROS level increased, and the mitochondrial membrane potential was reduced. rhSCUBE1 antagonized these changes after pretreatment. This suggests that SCUBE1 can protect KGN cells from OS damage, and this protection is closely related to the normal operation of mitochondrial functions.
The mitochondrial apoptotic pathway is one of the key pathways by which OS promotes the apoptosis of GCs. It mediates the expression of Bcl-2 and Bax, leading to a decrease in mitochondrial membrane potential and the opening of mitochondrial permeability transition pores, releasing cytochrome C (Cyt-c), and activating the caspase family cascade [29]. These results showed that after H 2 O 2 cell treatment, the apoptosis rate was increased by ow cytometry, while the apoptosis rate decreased after rhSCUBE1 pretreatment. At the same time, apoptosis-related proteins Bax, Bax/Bcl-2 ratio, and cleaved Caspase-3 showed similar trends. This suggested that H 2 O 2 activates the ROS-mitochondrial-Caspase-3 pathway to induce KGN cell apoptosis, and rhSCUBE1 can protect KGN cells from OS damage through the mitochondrial pathways. Additionally, OS activates p53 protein, which activates the Bcl-2 family and plays a role in promoting apoptosis and anti-proliferation [30]. In this study, the upregulation of p53 expression after H 2 O 2 treatment was not signi cant, but pretreatment with rhSCUBE1 signi cantly downregulated the p53 protein. This showed that the p53 protein is also a regulatory target of rhSCUBE1 used to protect KGN cells.
Finally, analysis of the impact of SCUBE1 (c.1169C > G, p.P390R) mutation from the aspect of mutation pathogenicity; protein stability; and gene haplotype insu ciency, indicated that the p.P390R mutation is signi cantly pathogenic. In this study, the direct effect of the p.P390R mutation on GCs or the effect on phenotypic changes in animals and mice was not determined, however, this study remains signi cant as there is limited research on SCUBE1 and GCs, and the mechanisms regulating the proliferation and apoptosis of GCs have not previously been reported. These ndings will lay a theoretical and practical foundation for further in-depth research, and at the same time provide new ideas for POI research.

Conclusions
In summary, we speculate that the continuous expression of SCUBE1 is an important guarantee for maintaining normal ovarian function, as SCUBE1 can protect ovarian (KGN) cells from OS damage. The role of p.P390R mutation of SCUBE1 in causing local spatial conformation changes, weakening protein stability, hindering protein function, and causing high pathogenicity, is indicative of the signi cant role that SCUBE1 can play as a biomarker for genetic manipulation in POI research studies. However, further rigorous and scienti c experiments are required for veri cation.   SCUBE1 mRNA has the highest expression in the ovarian tissue of KM mouse. The mRNA expression of SCUBE1 was normalized to RNA loading for each sample using GAPDH mRNA as an internal standard.