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. 2024 May 7;102:skae125. doi: 10.1093/jas/skae125

Follicular fluid meiosis-activating sterol prevents porcine ovarian granulosa cells from hypoxia-induced apoptosis via inhibiting STAT4 expression

Zhaojun Liu 1,#, Chengyu Li 2,#, Qianqian Chen 3, Chenyu Bai 4, Gang Wu 5, Chen Fu 6, Tong He 7, Ming Shen 8, Chungang Feng 9, Honglin Liu 10,✉
PMCID: PMC11245709  PMID: 38713167

Abstract

Follicular fluid meiosis-activating sterol (FF-MAS) is a small molecule compound found in FF, named for its ability to induce oocyte resumption of meiosis. Granulosa cells (GCs) within the follicle are typically located in a hypoxic environment under physiologic conditions due to limited vascular distribution. Previous research suggests that hypoxia-induced cell cycle arrest and apoptosis in GCs may be crucial triggering factors in porcine follicular atresia. However, the impact of FF-MAS on GCs within follicles has not been explored so far. In this study, we uncovered a novel role of FF-MAS in facilitating GC survival under hypoxic conditions by inhibiting STAT4 expression. We found that STAT4 expression was upregulated in porcine GCs exposed to 1% O2. Both gain and loss of function assays confirmed that STAT4 was required for cell apoptosis under hypoxia conditions, and that the GC apoptosis caused by hypoxia was markedly attenuated following FF-MAS treatment through inhibition of STAT4 expression. Correlation analysis in vivo revealed that GC apoptosis was associated with increased STAT4 expression, while the FF-MAS content in follicular fluid was negatively correlated with STAT4 mRNA levels and cell apoptosis. These findings elucidate a novel role of FF-MAS-mediated protection of GCs by inhibiting STAT4 expression under hypoxia, which might contribute to the mechanistic understanding of follicular development.

Keywords: apoptosis, FF-MAS, hypoxia, porcine follicle granulosa cells, STAT4

The development and survival of ovarian follicles are intricately regulated by various follicular fluid factors. By analyzing the molecular mechanisms of follicular development and atresia, we can increase ovulation rates and ultimately improve the reproductive performance of female livestock through molecular-based interventions.

Introduction

Ovary is the only organ in adult animals that retains the ability to remodel its vascular network (Robinson et al., 2009). Primordial and primary follicles lack an independent blood supply, but larger follicles are served by small arterioles that terminate in an anastomotic network just outside the basement membrane, preventing vessels from penetrating into the interior of the follicle (Redmer and Reynolds, 1996). In addition, the massive proliferation of GCs and the formation of fluid-filled follicular cavities keep the GCs away from the vasculature and lack an O2 supply. Many studies have demonstrated that the follicular interior of the ovary is a hypoxic environment (Bianco et al., 2005; Li et al., 2021a). Nishimura et al. reported that the oxygen concentrations in follicular fluid (FF) of large follicles were lower than those of small follicles in women, sows, and dairy cows (Nishimura and Okuda, 2015). The hypoxia environment within ovarian follicles is critical for determining the developmental fate of follicles. For instance, hypoxia is involved in the regulation of ovarian cell viability (apoptosis, proliferation, and autophagy), follicular angiogenesis, glucose metabolism, hormone synthesis, ovulation, et al. (Thompson et al., 2015; Zhou et al., 2018; Li et al., 2020a, 2020c; Wu et al., 2022).

Various environmental stresses, including oxidative stress and heat stress, have been known to negatively affect follicle development. Hypoxia, as a stress, also has a negative effect on cells. The adverse effects of hypoxia on ovarian function come from several sources, for example, exposure to hypoxia leads to follicular depletion (Temple-Smith et al., 2013), and hypoxia is a major inhibitory factor in follicles preventing cell cycle transition in ovarian GCs. Under hypoxic conditions, the JNK pathway is activated, inducing nuclear translocation of FOXO1. After FOXO1 enters the nucleus, on the one hand, it induces GC apoptosis by activating the mitochondrial apoptosis pathway, and on the other hand, it inhibits the activity of cyclin E/CDK2 complex resulting in G1-S arrest. (Liu et al., 2020; Li et al., 2021a). Li et al. found that FSH promotes mitophagy through the activation of the HIF-1α-PINK1-Parkin pathway, clearing damaged mitochondria and inhibiting cell apoptosis (Li et al., 2020c). Tao et al. reported that melatonin alleviates hypoxia-induced apoptosis of GCs by reducing ROS and activating MTNR1B-PKA pathway (Tao et al., 2021). In general, apoptosis can be initiated in response to stress or cellular injury via mitochondrial or death receptor pathways. The process of apoptosis is actually a cascade of amplified reactions of irreversible limited hydrolysis of substrates by caspases. Caspase-3 is considered to be the key enzyme leading to apoptosis after stimulation by a variety of inducing agents, and its activation heralds the beginning of the execution phase of apoptosis (Lossi et al., 2018; Martinvalet, 2019).

The family of signal transducers and activators of transcription has now been identified in seven isoforms: STAT1-4, STAT5a, STAT5b, and STAT6. signal transducer and activator of transcription as transcription factors are located in the cytoplasm and can be phosphorylated by a variety of cytokines to form homodimers that are translocated into the nucleus and can bind to the promoters of target genes to regulate their expression (Villarino et al., 2015). Among them, STAT4 can be activated by a variety of inflammatory mediators such as IL-12 (interleukin 12), IL-2, and IFN-α/β (interferon α/β), which regulate T cells and NK cells (Iida et al., 2011). STAT4 plays an important biological function in cytokine-induced cell proliferation, apoptosis, invasion, and metastasis, and participates in a variety of pathophysiological processes, such as inflammation, immunity, and tumor (Miyagi et al., 2007; Glosson-Byers et al., 2014). STAT4 is abnormally highly expressed in most tumor cells and tissues (Zhao et al., 2017; Li et al., 2021b) and tumors are internally ischemic and hypoxic microenvironments. It suggests that the abnormal expression of STAT4 may be correlated with hypoxia. Jiang et al. reported that interfering with the expression of STAT4 significantly stimulates the synthesis of E2 and the anti-apoptotic effect of KISS1 in porcine ovarian GCs (Jiang et al., 2020).

FF-MAS (Follicular fluid meiosis-activating sterol) is a sterol that is secreted by cumulus cells and is an intermediate in the cholesterol biosynthesis pathway. FF-MAS is a product of the demethylation of lanosterol by 14α-demethylase (CYP51) and was first found in human FF and bovine testis, termed FF-MAS and T-MAS, respectively (Byskov et al., 1997; Gatticchi et al., 2017). In most tissues and body fluids, sterol intermediates are rapidly converted to cholesterol, resulting in very low concentrations of sterol intermediates; however, in the ovary, elevated levels of FSH and LH stimulate the synthesis of FF-MAS, and in particular, FF-MAS accumulates in large quantities (human: 0.3 to 5.3 μM) in the follicle prior to ovulation in the presence of LH, which induces oocyte resumption of meiosis (Grøndahl et al., 2003). Therefore, some researchers speculated that FF-MAS may be a signaling molecule in the process of gonadotropin-induced oocyte maturation. Cavilla et al. used chemically synthesized FF-MAS to treat human oocytes matured in vitro and found that FF-MAS significantly increased the in vitro survival rate of oocytes derived from patients with ovarian cysts (PCO) but did not significantly affect their maturation ratios (Cavilla et al., 2001). Although the research related to FF-MAS has made great progress in recent years, little research has been done on the signaling mechanism of its action, with even fewer studies on its regulation of ovarian GC. Therefore, it is interesting to study the molecular mechanisms of FF-MAS in regulating GC.

In this study, we determined the effects of FF-MAS treatment on GC apoptosis and molecular component of apoptotic signaling during hypoxia stress via RNA-seq. The results demonstrated that STAT4 plays a pivotal role in FF-MAS-mediated suppression of apoptosis in hypoxic porcine GCs.

Materials and Methods

Ethics statement

All porcine ovaries used in this study followed humane slaughter practices. All experiments and treatments followed the recommendations of the Regulations for the Administration of Affairs Concerning Experimental Animals of China and were approved by the Animal Ethics Committee at Nanjing Agricultural University (SYXK2022-0031).

Reagents and antibodies

FF-MAS (700077P) was bought from Avanti Polar Lipids, inc. Antibodies against Caspase-3 (9,662) and α-Tubulin (2,125) were purchased from Cell Signaling Technology. Antibodies against FLAG (20,543-1-AP) were purchased from Proteintech. Antibodies against STAT4 (A6991) were obtained from ABclonal Technology. TUNEL cell apoptosis Detection Kit (KGA702) were purchased from KeyGEN BioTECH. Annexin V-FITC/PI Apoptosis Detection Kit (A211-02) was purchased from Vazyme. E2, P4, and FF-MAS ELISA kits were purchased from Ai Fang.

Sample collection, cell culture, and treatment

Ovaries from mature sows collected at a local slaughterhouse were transported to the laboratory while maintained in 0.9% sodium chloride solution at room temperature. 45 follicles of the same size were isolated from ovarian antral follicles with small forceps and scalpels. Individual follicle was punctured with needles to collect FF and then the mural GCs were obtained by scraping the follicular wall. GCs were immediately processed for the determination of cell apoptosis, or qRT-PCR. For cell culture, primary GCs were obtained by aspirating 3 to 5 mm antral follicles of porcine ovaries. Cells were then cultured in DMEM/F-12 medium containing 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco). Cells were maintained at 37°C in a 5% CO2, 20% O2 humidified atmosphere for control group and cells were maintained in 1% O2, 5% CO2, and 94% N2, 37 °C incubator for hypoxic group. To induce cell apoptosis, cells at 60% to 70% confluency were treated with 1% O2 for 48 h.

Plasmids, siRNA, and transfection

Sus scrofa STAT4 cDNA was subcloned into the pcDNA3.1 vector containing the FLAG tag to create the pcDNA3.1-STAT4-FLAG plasmid. For overexpression, GCs were transfected with the pcDNA3.1-STAT4-FLAG plasmid or the blank pcDNA3.1 vector for 48 h, and then exposed to 21% O2 or 1% O2 for the indicated time. For RNA interference, GCs were transfected with STAT4 siRNA or scrambled control siRNA for 48 h, and then exposed to 21% O2 or 1% O2 for the indicated time. siRNA sequences were shown in Supplementary Table S1. All GCs were transiently transfected using Lipofectamine 3,000 (ThermoFisher Scientific) following the manufacturer’s instructions.

Transcriptome sequencing

Primary GCs were harvested with Trizol reagent (Invitrogen) after the desired treatments. Our cDNA library construction is based on the TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer’s protocol. After filtering out low-quality reads and removing the adaptor sequences, we aligned RNA-seq reads sequenced through an Illumina HiSeq X platform (Illumina) to pig genome (Sscrofa11.1, NCBI) with Hisat2 software and calculated the fragments per kilobase of transcript per million (FPKM) of each gene using Cufflinks. Differentially expressed genes (DEGs) and corresponding adjusted P-values were determined using DEseq2. DEGs were assessed using edgeR at a P-value of < 0.05, and FC of > 2 or < 0.5.

Western blot

Total proteins from cultured primary GCs were extracted using pre-chilled RIPA lysis buffer (Beyotime, P0013B) containing protease inhibitor cocktail. After 12,000 g centrifugation for 15 min, supernatant concentration was determined using BCA kit (Beyotime, P0012), and the rest of the supernatants were proceeded to denaturation, electrophoresis on a PAGE gel (Genscript) and transferred to PVDF membranes (Millipore) by electroblotting. PVDF membranes were blocked in 5% BSA for 1 h at room temperature and then incubated in primary antibodies (1:1000) overnight at 4 °C. After rinsing three times with TBST, protein bands were incubated for 2 h at ambient temperature with secondary antibody (Abcam,1:2,000). The immunoblotting signals were visualized using ECL HRP substrate kit (Advansta). The relative band intensity was quantified using the Image-J software. TUBA1A was used as an endogenous control for cell total protein.

Quantitative PCR

GCs treated with 1% O2 or 10 μM FF-MAS were collected at the indicated timepoints. Total RNA was extracted with Trizol (Invitrogen) and 1μg RNA was reverse transcribed using cDNA Synthesis SuperMix for qPCR (YEASEN). Real-time PCR were performed according to the protocol of SYBR Green Master Mix (YEASEN). For primer sequences see Supplementary Table S2.

Flow cytometry

Apoptosis in GCs was assessed using the Annexin V-FITC/propidium iodide Apoptosis Detection Kit (Vazyme). Briefly, GCs were digested by 0.25% trypsin without EDTA. After washing twice with ice-cold PBS, cells were suspended in 100 μL binding buffer and then stained with 5 μL Annexin V-FITC and 5 μL propidium iodide for 10 min in the dark at room temperature. 400 μL binding buffer were added and samples were detected by flow cytometry in 1 h. GCs apoptosis rates were analyzed using FlowJo.

TUNEL assay

The TUNEL cell apoptosis detection kit is used to detect the fragmentation of cellular nuclear DNA during the apoptosis process. The principle is that biotin-labeled dUTP can be incorporated into the 3ʹ-OH ends of fragmented DNA in apoptotic cells under the action of terminal deoxynucleotidyl transferase (TdT Enzyme). It can specifically bind with streptavidin-horseradish peroxidase (Streptavidin-HRP) linked to biotin, and in the presence of the horseradish peroxidase substrate diaminobenzidine (DAB), a strong color reaction (dark brown) is generated, accurately and specifically locating the cells undergoing apoptosis. Briefly, GCs were cultured on sterilized glass coverslips, fixed with 4% paraformaldehyde for 30 min at ambient temperature and washed with PBS three times. The cells were then permeabilized using ice-cold 1% Triton-100 for 5 min. After blocking with 3% H2O2, 50 mL of TdT enzyme reaction liquid was added to each cell coverslip. The cells were incubated at 37 °C in the dark for 1 h. The cell samples were then incubated with Streptavidin-HRP working solution at 37 °C away from light for another 1 h. After washing with PBS, the samples were incubated with 50 mL DAB solution at ambient temperature for 3 min and the cell nuclei were stained with hematoxylin for 3 min. The immunostaining signals of Cells were observed under a light microscope (Olympus Corporation, Tokyo, Japan).

ELISA assay of E2 and P4 content

The levels of E2 and P4 were determined using a double antibody sandwich ELISA kit (Nanjing Aifang Biological Technology Co, Ltd) following the manufacturer’s instructions. Briefly, the samples of FF were centrifuged at 2,000 g for 20 min. The diluted supernatants (1:5) were added to the microplates embedded with E2 or P4 specific antibodies, followed by incubation at 37 °C for 30 min with a horseradish peroxidase–conjugated antibody against E2 or P4. After washing five times, the substrate solution TMB was added to trigger the chromogenic reaction. The absorbance was measured at 450 nm by using a TECAN microplate reader. The concentration of E2 or P4 was calculated from the standard curve.

ELISA assay of FF-MAS

The level of FF-MAS was determined using a competitive ELISA kit (Nanjing Aifang Biological Technology Co, Ltd) following the manufacturer’s instructions. Briefly, the supernatants were added to the microplates embedded with FF-MAS-specific antibodies, followed by incubation at 37 °C for 30 min with a horseradish peroxidase–conjugated antibody against FF-MAS. After washing five times, the substrate solution TMB was added to trigger the chromogenic reaction. After washing five times, the substrate solution TMB was added to trigger the chromogenic reaction. The absorbance was measured at 450 nm by using a TECAN microplate reader. The concentration of FF-MAS was calculated from the standard curve.

Statistical analysis

GraphPad Prism (version 7.0.0) was used for the visualization of graphs and statistical analyses. All experiments were performed in triplicate and repeated three times. The data were presented as the mean ± standard error of the mean (mean ± SEM). Differences between the two groups were assessed using the Student’ t test. One-way analysis was used to compare multiple data groups. Correlation analyses were performed by Pearson’s correlation analysis. A value of P < 0.05 was considered to be statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001

Results

FF-MAS inhibits porcine GC apoptosis during hypoxic stress

Granulosa cell apoptosis is believed as the hallmark of follicular atresia and our many previous researches demonstrated that hypoxia initiates apoptosis of porcine GCs (Li et al., 2020c; Liu et al., 2020; Tao et al., 2021). Therefore, we sought to explore whether FF-MAS has an impact on apoptosis of GCs. We first performed terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to detect GC apoptosis. As shown in Figure 1A and B, 1% O2 incubation significantly increases the GC apoptosis rate. In contrast, the proportion of apoptotic GCs was markedly decreased in hypoxic GCs following FF-MAS supplementation, which was correlated with attenuated apoptosis signals as evidenced by the results obtained from flow cytometry analysis (Figure 1C and D). We also detected the apoptotic marker protein Caspase-3 using immunoblotting. Western blot assay showed hypoxia stimulation significantly increased the levels of cleaved Caspase-3 protein. However, FF-MAS treatment obviously inhibited the elevated expression of Caspase-3 cleavage in hypoxic GCs (Figure 1E and F). Collectively, these findings suggested that FF-MAS might have the potential to alleviate apoptosis in hypoxic GCs.

Figure 1.

Figure 1.

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Follicular fluid (FF)-MAS inhibits hypoxia-induced porcine GC apoptosis. (A) Apoptotic GCs after exposure to hypoxia (1% O2) with (or) 10 μM FF-MAS (TUNEL assay). Images scale bars, 100 μm. (B) Percentage of TUNEL-positive porcine GCs. (C, D) Cell apoptosis rates were detected by FACS. (E) Cleaved Caspase-3 protein expression in GCs treated with FF-MAS under hypoxia (1% O2) for 24 h were determined by western blot. (F) Quantification of total and cleaved Caspase-3 expression. TUBA1A served as the control for loading. Data above are represented as mean ± S.E.M.; *P < 0.05, **P < 0.01, ***P < 0.001

STAT4 is involved in FF-MAS signaling in hypoxic porcine GCs

In order to dissect the underlying mechanism of how FF-MAS functioned in preventing apoptosis in hypoxic GCs, we performed RNA sequencing of GCs in control, hypoxia, and FF-MAS plus hypoxia groups. The transcription levels of several randomly selected genes in each group were verified using quantitative real-time PCR, indicating the reliability of the RNA-seq data (Supplementary Figure S1A). PCA (principal component analysis) showed there is better repeatability within groups, with very similar sample data, while there is better differentiation between groups (Supplementary Figure S1B). It suggests that the sample treatments are effective. Heatmap and volcano plot data reflected that significant difference between each of the two groups. (Supplementary Figure S1C and D) 449 transcripts were downregulated and 758 transcripts were upregulated in hypoxic GCs. In addition, FF-MAS supplementation upon hypoxia displayed 17 downregulated DEGs and 93 upregulated DEGs in comparison with hypoxic GCs, as well as 695 upregulated DEGs and 410 downregulated DEGs in comparison with control group. To characterize potential targets involved in FF-MAS-regulated survival of hypoxic GCs, we identified 7 genes by overlapping the three groups of DEGs (Supplementary Figure S1E). These 7 genes show differential expression in comparisons of hypoxia vs. control and hypoxia vs. hypoxia plus FF-MAS, while they do not exhibit differential expression in comparisons of control vs. hypoxia plus FF-MAS. Among the seven identified genes, STAT4 has been suggested as an important factor controlling cell apoptosis.

Next, to examine the expression pattern of STAT4 under hypoxia, we subjected GCs to a time gradient of hypoxic incubation. We found the levels of STAT4 mRNA and protein in hypoxia-treated GCs were significantly increased in a time-dependent manner (Figure 2A to C). We next investigated the regulatory effect of FF-MAS on STAT4 under hypoxia. Quantitative RT-PCR (qRT-PCR) and immunoblotting results showed that FF-MAS strongly decreased the accumulation of STAT4 mRNA and protein in GCs exposed to hypoxia (Figure 2D to F). These results further suggested that STAT4 is regulated in FF-MAS signaling in hypoxic porcine GCs.

Figure 2.

Figure 2.

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STAT4 is involved in follicular fluid (FF)-MAS signaling in hypoxic porcine GCs. (A) mRNA of STAT4 in GCs subjected to hypoxia (1% O2) at different times. (B, C) Protein levels and quantification of STAT4 in GCs. (D-F) mRNA and protein levels of STAT4 in GCs treated with hypoxia and FF-MAS. Data above are represented as mean ± S.E.M; *P < 0.05, **P < 0.01, ***P < 0.001

Disturbing STAT4 expression alleviates hypoxia-induced GCs apoptosis

We asked whether hypoxia-induced GCs apoptosis is correlated with elevated STAT4 expression. We first performed a knockdown method to suppress STAT4 expression. We selected a highly potent siRNA to effectively silence STAT4 (Figure 3A). As shown in Figure 3B to D, silencing of STAT4 in hypoxic GCs led to decreased levels of Caspase-3 cleavage. Using flow cytometry, we further confirmed that knocking down STAT4 mitigated hypoxia-induced apoptosis proportion (Figure 3E and F). These data suggested that high levels of STAT4 promote GC apoptosis upon oxygen deprivation.

Figure 3.

Figure 3.

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Disturbing STAT4 expression rescues hypoxia-induced GCs apoptosis. (A) The gene silencing efficiency of siRNAs against STAT4. Primary GCs were transfected with STAT4 siRNAs or scrambled control siRNA for 48 h, and then collected for western blot detection of the protein levels. (B) STAT4, total and cleaved caspase-3 protein expression in GCs. GCs transfected with STAT4 siRNA or scramble control siRNA for 48 h were cultured with or without follicular fluid-MAS for an additional 24 h under normoxia or hypoxia (1% O2) conditions. (C, D) Quantitative analysis of protein levels in B. (E, F) Cell apoptosis rate were detected by FACS. Data above are represented as mean ± S.E.M; *P < 0.05, **P < 0.01, ***P < 0.001

Overexpressing STAT4 attenuates the protective effect of FF-MAS

Next, to further investigate the effects of STAT4 on GCs apoptosis during hypoxia, we adopted the overexpression strategy by transfecting FLAG-tagged STAT4 overexpression plasmids into GCs. As shown in Figure 4A to C, GCs with enforced expression of STAT4 exhibited further increased levels of cleaved Caspase-3 during hypoxia exposure regardless FF-MAS treatment. In accordance with this, data from flow cytometry assay further indicated that STAT4 overexpression aggravated hypoxia-triggered apoptosis and dampened the anti-apoptotic effects of FF-MAS. (Figure 4D and E). These findings indicated that STAT4 plays a central role in FF-MAS-mediated GCs protection against oxygen deprivation.

Figure 4.

Figure 4.

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Overexpressing STAT4 attenuates the protective effect of FF-MAS (A) STAT4, FLAG, total, and cleaved Caspase-3 protein expression in GCs. GCs transfected with pcDNA3.1-STAT4-FLAG plasmid or blank pcDNA3.1 vector for 48 h were cultured with or without FF-MAS for an additional 24 h under normoxia or hypoxia (1% O2) conditions. (B, C) Quantitative analysis of protein levels in A. (D, E) Cell apoptosis rates were detected by FACS. Data above are represented as mean ± S.E.M; *P < 0.05, **P < 0.01, ***P < 0.001

In vivo, results suggested that the levels of STAT4 and FF-MAS are correlated with GC apoptosis

To investigate the potential contribution of STAT4 to GCs apoptosis within follicles, GCs were isolated from medium antral follicles of similar diameters in porcine ovaries (approximately 4 mm). Using qRT-PCR, we assessed the mRNA levels of apoptosis-related genes including Caspase-3, TRAIL, FASL, and STAT4 in GCs. Additionally, flow cytometry was employed to determine the proportion of apoptotic cells. As depicted in Figure 5A, there was a significant positive correlation between the mRNA levels of STAT4 in GCs and the mRNA levels of the apoptotic genes Caspase-3, TRAIL, and FASL. Moreover, the apoptosis rate of GCs within the follicles showed a positive correlation with the mRNA levels of STAT4 (Figure 5B). Subsequently, we analyzed the concentrations of progesterone (P4) and estrogen (E2) in FF. As illustrated in Figure 5C, the ratio of P4 to E2 exhibited a positive correlation with the GC apoptosis rate, suggesting that this ratio could serve as an indicator of follicular health status to some extent. Finally, we investigated the levels of FF-MAS in the FF. Correlation analysis revealed a negative correlation between FF-MAS levels and GC apoptosis, as well as between FF-MAS concentration and STAT4 mRNA levels (Figure 5D). Taken together, these results suggest that the STAT4 gene may be predictive of GC apoptosis in follicles, and FF-MAS is an intracellular pro-survival factor that acts by repressing STAT4.

Figure 5.

Figure 5.

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In vivo correlation analysis of STAT4 and FF-MAS on apoptosis in ovarian follicular GCs. (A) The relationship between STAT4 mRNA and apoptosis-related gene Caspase-3, TRAIL, and FasL were analyzed by linear regression in follicles GCs. (B) The relationship between STAT4 mRNA and GC apoptosis rate was analyzed by linear regression in follicles. (C) The relationship between P4/E2 and apoptosis rate were analyzed by linear regression in follicles GCs. (D) The relationship between apoptosis rate, STAT4 mRNA and FF-MAS concentration were analyzed by linear regression in follicles GCs. Data above are represented as mean ± S.E.M; *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

Due to the physiology of the follicle, follicular development, growth, ovulation, and subsequent early luteal development are under localized hypoxic or anoxic conditions (Kowalewski et al., 2015). Hypoxia of the GCs occurs simultaneously with follicular atresia in vivo. Our lab has done many in vitro studies on hypoxia, which has shown that hypoxia leads to cell cycle arrest, decreased proliferation, and even apoptosis in pig GCs (Li et al., 2021a; Tao et al., 2021; Zhang et al., 2022). Apoptosis and prolonged stagnation of GC can inhibit the maturation ability of follicles, ultimately leading to the blockage of immature follicles (Shi et al., 2023). We found that some FF factors (FSH, IGF-I, and melatonin) can maintain normal follicular and GC function in response to stimulation by a low-oxygen environment (Li et al., 2020b, 2020c; Tao et al., 2021). However, what is the effect of FF-MAS, one of the FF components, on GCs in a low-oxygen condition and the underlying mechanisms are so far understudied. In this study, we showed for the first time that hypoxia-induced porcine GCs apoptosis by promoting STAT4 protein and that FF-MAS could reverse this phenomenon via inhibiting STAT4 expression, thereby protecting porcine GCs from hypoxia-induced apoptosis.

The vast majority of follicles (>99%) in mammalian ovaries undergo a degenerative process known as follicular atresia at various stages of development. Inappropriate follicular atresia is the cause of certain anovulatory disorders, and low follicular utilization, such as polycystic ovary syndrome (PCOS) and premature ovarian failure. It is well-known that GC apoptosis is a key triggering factor for inducing follicular atresia (Shen et al., 2017). Research has found that the proportion of GC apoptosis in atresia follicles is significantly higher than that in mild atresia follicles and healthy follicles. The levels of estradiol and the ratio of estradiol to progesterone in healthy follicles are significantly higher than those in atresia follicles (Yu et al., 2004; Nishimoto et al., 2009). Estrogen can stimulate GC proliferation and prevent apoptosis. Low concentrations of progesterone contribute to the formation of the luteinizing hormone peak, thereby inducing ovulation of mature follicles. Changes in hormone levels can reflect the degree of follicular atresia (Silva et al., 2011; Shi et al., 2023). The P4/E2 ratio is frequently employed in research on apoptosis-related factors. And it is also commonly used to indicate the health status of follicles, with an increase in the ratio indicating a transition of the follicles towards atresia(Lewis-Wambi and Jordan, 2009; Phillipps et al., 2011). Our result in Figure 5C showed the P4/E2 ratio was positively correlated with GC apoptosis in follicles.

Tissue or cell-type-specific accumulation of sterol intermediates correlates with the regulation of physiologically relevant cellular functions (Tacer et al., 2002; Mitsche et al., 2015; Ačimovič et al., 2016). FF-MAS, one of the FF components, has been reported to increase the survival rate of oocytes, improve fertilization success, and reduce the number of fertilization errors by inhibiting their degeneration (Cukurcam et al., 2007; Lundin et al., 2007). FF-MAS is extremely difficult to dissolve in water, resulting in numerous studies focusing on reducing or increasing the accumulation of FF-MAS by inhibiting its production and metabolism (Hao et al., 2013, 2015). Leonardo et al. used cholesterol biosynthesis inhibitors AY9944 and17OHP to induce MAS accumulation and FF-MAS accumulation inhibits proliferation and cell cycle progression in HepG2 cells. It is reported that FF-MAS is a ligand of Liver X Receptor α (LXRα), but LXRα is highly expressed in the liver, kidney, gut, spleen, and adipose tissue (Janowski et al., 1996). FF-MAS accumulates in ovaries and reaches a high concentration in the preovulatory human FF. However, it remains unclear whether FF-MAS is required for GC survival during follicle development. In the present study, we exogenously added FF-MAS into the culture medium and detected that the addition of FF-MAS significantly reduced the percentage of GC apoptosis under hypoxic conditions by immunoblotting, flow cytometry, and TUNEL assay. These findings suggest that FF-MAS has a role in protecting porcine GC from hypoxia-induced apoptosis. Notably, the in vivo results (Figure 5D) showed a negative correlation between the FF-MAS content within the FF and the level of GC apoptosis, and this further confirmed the protective effect of FF-MAS on hypoxic GCs.

A series of studies have recently recommended that the STAT family might get involved in apoptosis in mammals (Dalgıç et al., 2015; Groner and von Manstein, 2017). Cryptotanshinone inhibits cell progression of lung tumors by increasing CD4+ T cell toxicity through activation of the JAK2/STAT4 pathway (Man et al., 2016). Silencing of STAT4 stimulates the production of NO in HUVECs and inhibits apoptosis (Jin et al., 2021). However, whether STAT4 is involved in FF-MAS-mediated regulation of GC protection during hypoxic stress is currently unknown. In this study, we used RNA-seq to identify STAT4 signaling as a key mediator of GC maintenance by FF-MAS during hypoxia exposure. Through qRT-PCR and immunoblotting assay, we found that STAT4 was highly expressed in hypoxia-treated GCs. The analysis of Annexin V-FITC flow cytometry showed that the pcDNA3.1-STAT4 promoted the apoptosis of GCs in the hypoxia group and STAT4 siRNA decreased hypoxia-induced apoptosis. FasL and TRAIL are important proapoptotic genes in porcine GCs, which as death receptor ligand promotes apoptosis via a death receptor signaling pathway (Shen et al., 2012). In vivo, correlation tests by analyzing STAT4 gene and apoptosis-related genes Caspase-3, TRAIL, and FasL showed that STAT4 is associated with apoptosis in GCs (Figure 5A). Correlation analysis of flow cytometry and STAT4 mRNA yielded similar results (Figure 5B). These results suggested that STAT4 may be involved in GC apoptosis and possibly serve as a marker for follicular atresia. In agreement with these findings, Jiang et al. Jiang et al. reported that the overexpression of exogenous STAT4 led to a significant increase in apoptosis of porcine granulosa cells (GCs), whereas knocking down STAT4 inhibited GC apoptosis (Jiang et al., 2020). In addition, they observed a significant decrease in STAT4 mRNA levels during the development from immature follicles to mature follicles. Interfering with STAT4 expression was found to stimulate the synthesis of estrogen (E2). However, our results did not reveal a significant correlation between STAT4 and E2 levels. Next, we further confirmed that STAT4 is a downstream target of FF-MAS-regulated apoptotic signaling in hypoxic GCs by overexpression of STAT4. Furthermore, data from the correlation test showed there is a negative correlation between FF-MAS content in FF and STAT4 mRNA.

The findings of this study further enrich the molecular mechanisms of hypoxia regulation in follicle GC apoptosis. However, there are some limitations in this study, such as how hypoxia signaling and FF-MAS regulate the STAT4 gene, and how STAT4 plays a role in apoptotic signaling pathways.

Conclusion

To explore the effect of FF-MAS on GCs of follicles, we performed gene expression profiling in the domestic pig. We discovered STAT4 is required for GC apoptosis under hypoxia conditions both in vitro and in vivo and FF-MAS prevents porcine ovarian GCs from hypoxia-induced apoptosis via inhibiting STAT4 expression.

Supplementary Material

skae125_suppl_Supplementary_Data
skae125_suppl_supplementary_data.zip (669.7KB, zip)

Glossary

Abbreviations

DEGs

differentially expressed genes

E2

estrogen

FasL

factor-related apoptosis ligand

FPKM

fragments per kilobase of transcript per million

FF-MAS

follicular fluid meiosis-activating sterol

GCs

granulosa cells

IFN-α/β

interferon α/β

IL-12

interleukin 12

P4

progesterone

PCA

principal component analysis

qRT-PCR

quantitative real-time PCR

STAT4

signal transducer and activator of transcription 4

TUNEL

TdT-mediated dUTP Nick-End Labeling

TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand

Contributor Information

Zhaojun Liu, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.

Chengyu Li, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.

Qianqian Chen, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.

Chenyu Bai, Beijing 101 High School, Beijing, 100084, China.

Gang Wu, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.

Chen Fu, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.

Tong He, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.

Ming Shen, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.

Chungang Feng, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.

Honglin Liu, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.

Author contributions

Zhaojun Liu: Conceptualization, methodology, data curation and analysis, and writing—original draft. Chengyu Li: Funding acquisition and writing—review & editing. Qianqian Chen: Methodology and data curation. Chenyu Bai: Data curation. Gang Wu: Data curation and software. Chen Fu: Methodology and data curation. Tong He: Methodology. Ming Shen: Funding acquisition and methodology. Chungang Feng: Methodology and supervision. Honglin Liu: Funding acquisition and supervision.

Conflict of interest statement

The authors declare no conflict of interest.

Funding

This work was supported by China Postdoctoral Science Foundation (2022M721650), Jiangsu Funding Program for Excellent Postdoctoral Talent (2023ZB300), the Fundamental Research Funds for the Central Universities (KYT2023002), the National Natural Science Foundation of China (31972571, 31972564), and the “JBGS” Project of Seed Industry Revitalization in Jiangsu Province (JBGS (2021) 026).

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