Administration of follicle-stimulating hormone induces autophagy via upregulation of HIF-1α in mouse granulosa cells

Recent studies reported the important role of autophagy in follicular development. However, the underlying molecular mechanisms remain elusive. In this study, we investigated the effect of follicle-stimulating hormone (FSH) on mouse granulosa cells (MGCs). Results indicated that autophagy was induced by FSH, which is known to be the dominant hormone regulating follicular development and granulosa cell (GC) proliferation. The activation of mammalian target of rapamycin (mTOR), a master regulator of autophagy, was inhibited during the process of MGC autophagy. Moreover, MHY1485 (an agonist of mTOR) significantly suppressed autophagy signaling by activating mTOR. The expression of hypoxia-inducible factor 1-alpha (HIF-1α) was increased after FSH treatment. Blocking hypoxia-inducible factor 1-alpha attenuated autophagy signaling. In vitro, CoCl2-induced hypoxia enhanced cell autophagy and affected the expression of beclin1 and BCL2/adenovirus E1B interacting protein 3 (Bnip3) in the presence of FSH. Knockdown of beclin1 and Bnip3 suppressed autophagy signaling in MGCs. Furthermore, our in vivo study demonstrated that the FSH-induced increase in weight was significantly reduced after effectively inhibiting autophagy with chloroquine, which was correlated with incomplete mitophagy process through the PINK1-Parkin pathway, delayed cell cycle, and reduced cell proliferation rate. In addition, chloroquine treatment decreased inhibin alpha subunit, but enhanced the expression of 3 beta-hydroxysteroid dehydrogenase. Blocking autophagy resulted in a significantly lower percentage of antral and preovulatory follicles after FSH stimulation. In conclusion, our results indicate that FSH induces autophagy signaling in MGCs via HIF-1α. In addition, our results provide evidence that autophagy induced by FSH is related to follicle development and atresia.

Macroautophagy (hereafter referred to as autophagy) is a constitutive, dynamic, evolutionarily conserved catabolic process that involves the degradation and recycling of cellular constituents. 1,2 It maintains cell survival under various forms of stress conditions such as starvation, 3 hypoxia, 4 and interruption of growth signaling. 5 Autophagy is involved in many physiological and developmental processes such as cell survival, cell death, metabolism, and innate immunity. [6][7][8] Abnormalities in autophagy are implicated in numerous human diseases, including developmental disorders, neurodegenerative diseases, and cancer. 9 A granulosa cell or follicular cell is a somatic cell of the sex cord that is closely associated with the developing female gamete (called an oocyte or egg) in the mammalian ovary. Ovarian follicle atresia is a periodic process by which immature ovarian follicles degenerate and are reabsorbed during the follicular phase of the ovarian cycle. This complicated, dynamic process is triggered by GC apoptosis. 10,11 Recently, increasing evidence shows that autophagy plays a vital role in survival and proliferation of GCs. 12,13 In rat GCs, accumulation of autophagosomes induced by serum starvation activates cell apoptosis through decreased Bcl-2 expression and, subsequently, leads to caspase activation, 14 which suggests that a certain degree of autophagy may promote GC apoptosis. In a mouse model, inhibition of autophagy in IL-33 (-/-) mice leads to massive accumulation of tissue waste containing aging-related catabolic waste during folliculogenesis, indicating a role for autophagy in removing unnecessary cells and maintaining the metabolic balance in antral follicles. 15 Interestingly, specific knockout of autophagyrelated genes results in a significant decrease in primordial follicle pools, 16,17 which suggests an important role for autophagy in the regulation of follicular development and maintenance of the ovarian primordial follicle reserve.
Factors related to follicular development and GC proliferation have been extensively investigated, including death ligands and receptors, caspases, Bcl-2 family members, and gonadotropins. 18 Follicle-stimulating hormone (FSH) is synthesized and secreted by the gonadotropic cells of the anterior pituitary gland and plays an important role in follicular development, GC proliferation, and reproductive processes. Exogenous FSH is widely used to stimulate the development of mature follicles because of its effectiveness in preventing GC apoptosis and follicle atresia. Research indicated that almost all gonadotropins can significantly inhibit GC apoptosis and follicle atresia, with FSH exhibiting the highest efficiency. 19,20 Exposing immature rats to eCG can significantly decreased autophagy signaling at days 1 and 2, but increased it at day 3 and maintained it until day 5, 21 suggesting a complex regulation of autophagy during follicular development. However, the relationship between autophagy and inhibition of cell apoptosis and follicle atresia by FSH remains unclear. In this study, we investigated the molecular regulation of autophagy in FSH-treated MGCs to determine the role of FSH in GC autophagy and apoptosis.

Results
FSH promotes MGC autophagy in vivo. To determine whether the function of FSH is correlated with autophagy in MGCs, we measured autophagy signaling during 48 h after FSH treatment in vivo. Immunohistochemical analysis indicated that FSH injection increased endogenous LC3 expression when compared with the control group (0 h; Figure 1a). In particular, LC3-positive staining was concentrated in the MGCs of antral and preovulatory follicles, both FSH-sensitive follicles. In addition, we labeled and tracked acidic organelles using lysotracker red staining. Results demonstrated that the fluorescence intensity was higher in follicles of FSH-treated mice ( Figure 1b). Western blot results showed that the lipid conjugation of free LC3-I to the autophagic membraneassociated LC3-II was enhanced in MGCs following FSH treatment and that degradation of the autophagy receptor SQSTM1 (p62) was enhanced (Figures 1c and d). Thus, after intraperitoneal injection of FSH, autophagy signaling in MGCs from antral and preovulatory follicles was significantly increased and remained at a relatively high level.
FSH promotes MGC autophagy through the AKT-mTOR pathway. To further investigate the molecular mechanism by which FSH induces autophagy in MGCs, we focused on molecular regulation within the 12 h following administration. As shown in Figures 2a and b, western blot results showed that LC3-II protein expression was significantly increased after FSH treatment and peaked at 6 h. Interestingly, the expression of LC3-I was significantly increased at 1.5 h (data not shown). We hypothesize that the activation of LC3-I is a preparation for cell autophagy because LC3-II can be distinguished from LC3-I by its increased mobility. The expression of p62 was increased within 3 h after administration and decreased over the next 9 h. These results demonstrated that autophagy is rapidly activated and maintained at a high level up to 12 h after administration. FSH activates multiple downstream signaling pathways in GCs, including PKA, PI3K, AKT-mTOR, p38-MAPK, and ERK1. 22 Linking them, mTOR acts as a central sensor of growth factors, nutritional condition, and energy status, and plays a dominant role in autophagy. 23 Our results indicated that AKT, mTOR, and S6K1 (downstream effector of mTOR) expression levels were enhanced by FSH stimulation when compared with the control group. The expression of p-AKT was induced at 1.5 h after FSH stimulation, but returned to the basal level at 9 h. p-mTOR and p-S6K1 expression levels were also induced at 1.5 h after FSH stimulation and then decreased significantly when compared to the control group (Figure 2a, bottom, Figure 2c). In addition, the effect of the mTOR activator, MHY1485, (10 mg/kg, 2 days) before FSH treatment was investigated. The results suggested that MHY1485 blocked the autophagy signaling induced by FSH. p-mTOR and p-S6K1 expression levels were maintained at a high level in the presence of MHY1485 (Figure 2d, bottom, Figure 2f), whereas LC3 expression showed no marked  Figure 2e). These findings demonstrated that FSH induces MGCs autophagy through the AKT-mTOR signaling pathway and initiates a dynamic process occurring within 12 h posttreatment.
FSH upregulates HIF-1α and AMPK in MGCs. FSH is a powerful growth factor that promotes GC proliferation, 24,25 as confirmed by our CCK-8 results during the 12 h period following FSH treatment (Supplementary Figure S1). Cell autophagy and apoptosis are tightly linked to cell metabolism. Excessive cell proliferation causes metabolic stress, including hypoxia and nutrition stress, promoting cell autophagy and death. 26 Therefore, we investigated the expression of HIF-1a and AMPK by using qPCR and western blot. The results demonstrated that HIF-1α mRNA and protein expression was significantly upregulated (Figures 3a and c). Activation of AMPK was significantly enhanced between 3 and 12 h after FSH injection (Figures 3d and e), while total AMPK expression did not change (Figures 3b and d). In addition, the expression of a downstream factor, Beclin1, was also increased after FSH administration ( Figure 3f). Recent reports indicated that reactive oxygen species (ROS) may lead to damage of cellular components and subsequently induce cell autophagy. 27,28 Therefore, we measured the intracellular ROS level in MGCs after FSH injection within 12 h. The level of intracellular ROS did not change significantly ( Figure 3g). However, the mRNA levels of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX), increased ( Figure 3h). These results demonstrated that FSH leads to hypoxia and reduces nutritional status in MGCs. Moreover, FSH plays a role in protecting GCs against the effect of ROS by activating the antioxidant enzyme system. HIF-1α is the critical factor in MGC autophagy. To determine the effect of FSH-mediated HIF-1α and AMPK activation on cell autophagy, MGCs, with or without FSH, were treated with HIF-1α (Px-478) and AMPK inhibitors (Compound C), and cell autophagy signaling was detected by western blot. The experimental protocol is described in Supplementary Figure S2. After pretreating mice with Px-478, the expression of HIF-1α was significantly decreased at days 2 and 3 (Figures 4a and b). The LC3-II/LC3-I ratio was also significantly decreased at 12 h compared with that at 3 h after pretreatment with Px-478 (Figure 4c, top). In contrast, the expression of p62 was maintained at a high level after pretreatment with Px-478 (Figure 4c, bottom). Subsequently, we measured autophagy signaling in MGCs after AMPK inhibition. The results showed that the expression level of p-AMPK was inhibited by Compound C injection (Figures 4d and e) and total AMPK expression was inhibited. However, the LC3-II/LC3-I ratio and the degradation of p62 did not change compared with those in the groups only treated with FSH ( Figure 4f), suggesting that the AMPK signaling pathway is not crucial for the promotion of cell autophagy although p-AMPK is highly expressed following FSH injection. These results demonstrated that HIF-1α is primarily involved in FSH-regulated MGC autophagy.
FSH enhances MGC autophagy through HIF-1α in vitro. To further confirm the effects of HIF-1α on MGC autophagy, we monitored this process in MGC primary cultures in vitro. Since HIF-1α is unstable under conditions of normoxia, we used a chemical inducer of HIF-1α, which acts by stabilizing HIF-1α transcription factor, inhibiting its degeneration under normoxia. As shown in Figures 5a and b, FSH in combination with CoCl 2 significantly increased HIF-1α expression, suggesting that FSH functions as a positive regulator of HIF-1α expression. The ratio of LC3-II/LC3-I and p62 degradation increased in FSH-treated MGCs compared with that in the CoCl 2 -only group ( Figure 5c). Consistent with the results presented in Figure 3f, in the presence of CoCl 2 , FSH significantly increased Beclin1 expression (Figure 5d, left). We also detected the expression of Bnip3, a member of the BH3-only protein family that contains a hypoxia response element in the promoter 29 and is associated with autophagy in the hypoxia model. 30 The results demonstrated that Bnip3 expression was significantly enhanced after treatment with FSH and CoCl 2 compared to that in MGCs treated with CoCl 2 alone (Figure 5d, right). Immunofluorescence studies indicated that FSH-induced HIF-1α significantly increased the formation of GFP-LC3 puncta, suggesting increased autophagy signaling under these conditions (Figures 5e and g). Moreover, we used the autophagy-flux inhibitor, Bafilomycin A1, which prevents lysosome degradation, thus increasing punctate GFP-LC3 exclusively when autophagy is active. Bafilomycin A1 treatment indicated that FSH significantly increased the autophagy flux, as monitored by GFP-LC3 puncta (Figures 5f and g). In addition, Bafilomycin A1 significantly increased the GFP-LC3 puncta in Cocl 2 treated cells regardless of FSH. These results demonstrated that FSH promotes MGC hypoxia, further enhancing autophagy in vitro.
Autophagy signaling was inhibited, even under conditions of hypoxia as indicated by decreased LC3-II/LC3-I ratio, inhibited p62 degradation, and decreased formation of GFP-LC3 puncta (Figures 6e and f). In addition, the differences between FSH treated and untreated HIF1α, Beclin1, and FSH has a protective effect on follicular development by increasing the activity of mitochondrial clearance. We next investigated the relationship between FSH-induced autophagy and follicular development. Chloroquine, an  autophagy inhibitor, induced the accumulation of LC3-II and p62 in FSH-treated GCs by blocking the later stage of autophagy ( Figure 7a). Interestingly, FSH treatment signifi-cantly increased the size and weight of the ovary, which were reduced by chloroquine (Figures 7b and c). However, MGC apoptosis was not significantly affected by autophagy FSH induces granulosa cell autophagy via HIF-1α J Zhou et al inhibition, assessed by monitoring caspase-3 activity, and the expression of apoptosis-related genes (Figure 7d and Supplementary Figure S4). Previous reports indicated a mutual regulation between apoptosis and autophagy in the death signaling process mediated by mitochondria. 31,32 Thus, we investigated whether FSH-mediated autophagy affected mitochondrial membrane potential (ΔΨ (m)). JC-1 staining experiments indicated that the increased mitochondrial membrane potential by FSH was abolished by autophagy inhibition (Figures 7e and f). In addition, we assessed the expression of the PINK1-Parkin system that belongs to the mitophagy pathway. Western blot indicated that PINK1 expression increased in MGC treated with FSH, and PINK1 level further increased after chloroquine treatment ( Figures  7g and h). However, no marked change of Parkin expression was detected (Figure 7g), as Parkin could promote mitophagy by changing the mitochondrial location. 33 Moreover, the loss of mitochondria resulting from mitophagy was decreased, which was indicated by mitochondrial membrane protein, Tom20 protein expression (Figures 7g and h). Taken together, these data demonstrated that mitophagy induction via the PINK1-Parkin pathway is an important mechanism of FSHmediated follicular growth and development.
FSH-induced autophagy in MGCs is associated with cell proliferation. Autophagy is required for the cyclic phase of GC proliferation and differentiation. 34 Thus, we investigated the potent function of autophagy in FSH-mediated cell proliferation. qPCR results revealed that cell proliferation was inhibited, mainly reflected in cyclinA2 and cyclinD2 expression (Figure 8a). To better assess cell proliferation deregulation, cell cycle, and cell proliferation were investigated. Flow cytometry showed that FSH and chloroquine co-treatment resulted in a small population of cells in the G2/ M phase when compared with the FSH-treated group (Figure 8b), suggesting a cell cycle delay in S phase caused by autophagy inhibition. As shown in Figure 8c, chloroquine significantly attenuated the increased number of EdU-labeled cells following FSH treatment (Figures 8c and d), as well as the number of living/viable cells detected by CCK-8 assay (Supplementary Figure S5). Therefore, FSH-induced autophagy promotes MGC proliferation and follicular development.
In addition, the expression of genes involved in estrogen biosynthesis and steroidogenic regulation associated with follicle development was detected by qPCR. Interestingly, the expression of 3β-HSD was enhanced. In contrast, INHα expression was decreased compared with FSH only treatment (Figure 8e). To further demonstrate that autophagy is necessary for the development of follicles, we performed a hematoxylin and eosin (H&E) staining assay. The increased number of antral follicles and preovulatory follicles after FSH treatment was reduced by chloroquine (Figures 8f and g). Overall, these results provided evidence that FSH-mediated autophagy is associated with GC proliferation and follicular development.

Discussion
During follicular development, a proportion of ovarian follicles are removed by atresia prior to maturation in order to promote energy investment and ovulation of viable follicles. Autophagy captures and degrades intracellular components such as abnormal proteins and damaged organelles to sustain metabolism and homeostasis. In particular, autophagy is closely related with the remodeling of follicle cells during the process of follicular development. 35,36 Signs of autophagy depend on gonadotropin dose, age, and body weight in freshly harvested human GCs, 37 suggesting that autophagy is common in mammalian ovaries where most follicles and cells are in a highly regulated state, balancing hormonal and environmental stimulus. However, the underlying molecular mechanism is still unknown. FSH is an important survival factor leading to selection and survival of growing follicles during development. 38 The physiological functions of FSH are achieved by activating several signaling cascades in GCs, including PKA, PKB, p38-MAPK, and ERK1/2, which in turn modulate 4100 different target genes. 39 These downstream factors are directly or indirectly involved in autophagy regulation. [40][41][42][43] In the mouse ovary, TGFβ signaling was activated after exposure to FSH and LH. 44 Inhibition of TGFβ signaling by blocking TGFβR2 significantly decreased FSH-mediated autophagy signaling. 45 In porcine GCs, FSH increased the level of inhibitor of NF-κB (IκB) protein and subsequently increased autophagy via JNK signaling. 46 In this study, we demonstrated that FSH administration enhanced autophagy in MGCs. Injection of FSH increased endogenous LC3 staining, the LC3-II/LC3-I ratio, p62 degradation, and enhanced lysotracker staining in MGC. These are the most widely used tests for autophagy determination. 47 mTOR is a downstream regulator of PKB, which senses nutrient, energy and oxygen availability, and growth factor signaling, and plays an important role in autophagy. 48 In this study, we introduced mTOR as the main factor affecting autophagy induced by FSH in MGCs. FSH activates the expression of p-mTOR and promotes the accumulation of LC3-I within 1.5 h. After 3 h, FSH significantly enhanced signs of autophagy by inhibiting the expression of p-mTOR. In addition, the mTOR activator, MHY1485, suppressed the autophagy level following FSH treatment. These results suggest that FSH regulates autophagy through mTOR inactivation. Hypoxia is a pathological condition in which the body or a region of the body is deprived of an adequate oxygen supply. Evidence suggests that HIF-1α plays an essential role in angiogenesis, 49 cell proliferation/survival, 50 and glucose/iron metabolism. 51,52 Consistent with our results (Figure 2a,  Figures 3a and c), a previous study indicated HIF-1α is FSH induces granulosa cell autophagy via HIF-1α J Zhou et al activated in FSH-stimulated ovarian cancer cells, SKOV-3, and that the PI3K/AKT signaling is upregulated. 53 In the ovary, excessive cell proliferation induced by gonadotropins promotes the accumulation of HIF-1α and leads to hypoxia. 54 Here, we identified HIF-1α as an inducible factor after FSH treatment. Injection of FSH significantly increased HIF-1α expression in vivo and vitro. Our results are consistent with a recent report demonstrating that HIF-1α is a downstream factor of FSH in rat GCs. 55 Remarkably, HIF-1α mRNA decreased at 6 h without reflecting a modulation of HIF1-α protein level until 12 h (Figures 3a and c), indicating that HIF-1α transcriptional and post-transcriptional regulation is also involved in this process. Recent studies indicated that autophagy has a complex and close link with hypoxia. 56,57 Evidence from various mammalian cell types indicates that HIF-1α induces autophagy by activating Bnip3. 58,59 The expression of this conserved member of the BH3 only subfamily of the pro-apoptotic Bcl-2 family proteins correlates with the induction of autophagy in different cell models. [60][61][62][63] Moreover, Bnip3 can dissociate Beclin1 from the Bcl-2-Beclin1 complex, 64 consistent with our results showing that Bnip3 knockdown affects Beclin1 expression (Figure 6d). Beclin1 is an important component of the class III PtdIns3K complex, required for the induction of autophagy. Hypoxia preconditioning in hepatocellular carcinoma significant activated autophagy, but this process can be attenuated by Beclin1 knockdown. 65 In this study, Bnip3 and Beclin1 expression was significantly enhanced in MGCs subjected to hypoxia. Furthermore, Bnip3 or Beclin1 knockdown decreased autophagy signaling in MGCs, suggesting that Bnip3 and Beclin1 regulation is involved in hypoxiainduced autophagy.
During follicular development, the constituent cells, including theca cells, GCs, and oocytes, exhibit a series of functional gene activation. A cohort of follicles that have undergone initial development are stimulated to develop further by rising concentrations of gonadotropins. The GCs become more responsive to FSH and show a higher rate of proliferation once a follicle is selected as a dominant follicle. Therefore, autophagy appears to be an important, evolutionarily conserved mechanism for maintaining homeostasis and providing energy. 66 A previous report showed that autophagy was upregulated during cyclic phases of cell proliferation and differentiation. 34 In addition, Atg3-, Atg5-, or Pik3c3/Vps34deficient T cells cannot efficiently proliferate because of loss of the cell cycle inhibitor, CDKN1B/p27Kip1, which is selectively degraded by autophagy. 67 In this study, inhibition of autophagy reduced the proliferation rate of MGCs, further decreasing the number of antral follicles and preovulatory follicles.
Once the dominant follicle is mature, GCs begin to express aromatase, to secrete estradiol and increasing the amount of inhibin B. Thus, maintaining the homeostasis of the ovary to produce more mature follicles and prevent follicles from entering atresia is important. Previous results highlighted a critical role for autophagy in the function of GCs. Autophagy inhibition by conditional knockout of Beclin1 in the ovarian GC population causes a defect in progesterone production and a subsequent preterm labor phenotype. 68 In porcine GC, autophagy activated through NF-κB inhibition promotes steroidogenesis. 46 In this study, there was a change in the transcription of progesterone biosynthesis genes, 3 betahydroxysteroid dehydrogenase (3β-HSD) and inhibin alpha subunit (INHα), representing a crosslink with the molecular regulation of autophagy. Although no change was observed in the levels of CYP19A1, controlling estradiol production, autophagy may still be involved in estradiol signaling, as demonstrated in different cell lines. [69][70][71] Reports revealed that autophagy is associated with GC apoptosis and follicle atresia throughout the reproductive period. 13,18 In this study, although no significant change was observed in apoptosis-related gene expression after autophagy inhibition, the mitochondrial membrane potential was significantly changed through the PINK1-Parkin system, indicating that a selectively autophagy, mitophagy was involved in this process. Since mitophagy facilitates cell death programs, 72 we speculate that FSH-induced mitophagy has a protective role in damaged mitochondrial clearance. Interestingly, a recent report indicated that defect of mitochondrial fusion protein Mfn2 impaired autophagy-induced degradation, subsequently decreasing mitochondrial oxygen consumption rate and cell glycolysis, reducing ATP production, and suppressing cell proliferation. 73 Therefore, FSH-induced autophagy is a regulatory mechanism that ensure the order and timing of cell cycle transition by mitophagy activation through the PINK1-Parkin pathway (Figure 8h), which may help reducing follicle atresia and GC apoptosis. 74 In summary, FSH treatment promotes the activation of autophagy via upregulation of HIF-1α in MGCs. FSH-mediated autophagy has a protective role on GC proliferation and follicle development through the selective degradation of damaged mitochondria. Blocking FSH-induced autophagy influences steroid production and antral/preovulatory follicle numbers. Overall, our study highlights a mechanism by which FSH regulates MGC autophagy, which may be a novel strategy to reduce follicle atresia and degeneration.

Materials and Methods
Animal treatment. All animal experiments were approved by Nanjing Agricultural University, Animal Research Institution Committee. Three to 4-weekold female ICR mice (Nanjing Qinglongshan Experimental Animal Center) were housed, five per cage, in a temperature controlled (22 ± 2°C) room with a 12: 12 h light: dark cycle (lights on from 07 00 to 1900 hours) and free access to water and food. To induce MGC autophagy, mice were injected i.p. with FSH (Ningbo Second inhibitor, Px-478, and AMPK inhibitor, compound C, (Selleck Chemicals, Houston, TX, USA) were injected before FSH treatment and the experiment protocol is described in Supplementary Figure S2.
GFP-LC3 assay. MGCs were seeded into 24-well plates post-treatment, the coverslips were washed, mounted on slides, and inspected under a confocal laser scanning microscope (Carl Zeiss, Göttingen, Germany). Several bright green fluorescent puncta were observed in the cells. One punctum was considered equal to one autophagosome. The results are presented as the average number of puncta per cell.
JC-1 assay. The lipophilic cation JC-1 was used to assess the mitochondrial status of MGC. According to the manufacturer (Beyotime Institute of Biotechnology), JC-1 reversibly changes its fluorescence from green (monomeric status) to orange (multimeric status) when the mitochondrial membrane potential is high. After treatment, 1 × 10 6 cells of each group were collected and incubated with 10 μg/ml of 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylimidacarbocyanine iodide (JC-1) at 37°C, 5% CO2 for 30 min. The cells were analyzed by using a BD FACScan system (Becton Dickinson, Franklin, NJ, USA). FSH-mediated cell proliferation promotes HIF-1α expression, which further leads to autophagy in hypoxic condition. Autophagy transfers the hypoxic stress through the mitophagy pathway and participates in the regulation of the cell cycle, which has a positive feedback on cell proliferation