Toll-Like Receptor 4 Inhibits Estradiol Secretion via NF-κB Signaling in Human Granulosa Cells

Toll-like receptor 4 (TLR4) may play a critical role in regulating follicular development. Data are scarce on the role of TLR4 in the follicle. This study investigated the effects of TLR4 on steroidogenesis in human granulosa cells. Immunohistochemical analysis revealed stage-specific expression of TLR4 in the mouse ovarian cycle, and immunofluorescence showed TLR4 expression in the human granulosa-like tumor cell line (KGN). TLR4 agonist lipopolysaccharides (LPS) significantly inhibited follicular development and synthesis of estradiol (E2) in mice. In KGN cells, TLR4 activation significantly inhibited CYP19A1, FSHR and StAR, and TLR4 inhibition reversed these effects. TLR4 activation also inhibited forskolin-induced secretion of E2 by inhibiting CYP19A1, with no effect on progesterone. Further studies showed activation of p38, JNK and NF-κB signaling after TLR4 activation. Subsequent analyses showed that an NF-κB antagonist reversed the inhibitory effects on CYP19A1 expression and E2 secretion. Together, our results suggest that TLR4 activation may suppress CYP19A1 expression and E2 secretion via NF-κB signaling in human granulosa cells, with important implications for the regulation of ovarian pathophysiology.


INTRODUCTION
In mammals, ovaries undergo cyclic variation both in terms of morphology and function, characterized by folliculogenesis, ovulation and luteinization. Granulosa cells (GCs) play a crucial role in these processes and are critical for supporting ovarian function and determining follicular fate (1). Importantly, GCs regulate their own proliferation, responsiveness to gonadotropin, apoptosis and steroidogenesis (2).
Steroidogenesis under the control of follicle stimulating hormone (FSH), luteinizing hormone (LH) and local regulators is an important physiological process, which influences the maturation and ovulation of follicles. In addition, steroidogenesis was reported to be associated with embryo quality. Higher levels of follicular E2 correlated well with successful fertilization following artificial reproduction treatment (3). E2 is the main steroid produced by GCs and is controlled by several factors, among which FSH plays a central role (4). FSH protects GCs from oxidative injury, also rescues GCs from apoptosis and dominant follicle atresia (5). FSH induces the production of E2 via FSHR-cAMP-dependent signaling to induce the transcription of the CYP19A1 gene (6). This gene encodes the cytochrome P450 enzyme aromatase, which convert androgens to estrogens (7). Progesterone is an essential reproductive hormone that is well-known to be produced by GCs immediately prior to ovulation (8). In addition, progesterone synthesis was found to be promoted by FSH from human GCs without luteinization (9); however, premature increase of progesterone before ovulation trigger may reduce pregnancy rates in stimulated in vitro fertilization cycles (10). Therefore, steroidogenesis plays a critical role in follicular development, ovulation and luteinization, and abnormal steroidogenesis may lead to decreased follicle survival, ovulation rates and fertility.
Toll-like receptors (TLRs) are highly conserved proteins of the innate immune system that detect various pathogenassociated molecular patterns and play a role in the subsequent immune response (11). Previous studies have evaluated the expression of TLRs in GCs from human and bovine ovaries (12,13). Moreover, TLR signaling pathways were found to be induced in cumulus-oocyte complex samples collected from preovulatory follicles (13). Therefore, TLRs may play an important role in regulating follicular development and ovulation. TLR4 is a member of TLRs and is widely expressed on a variety of cell types in addition to immune cells. Ligand binding to TLR4 leads to the activation of the myeloid differentiation primary response 88 and TIR-domaincontaining adapter-inducing interferon-b pathways, which causes the activation of several transcription factors such as nuclear factor kB (NF-kB) and interferon response factor 3, thereby promoting the release of proinflammatory cytokines (14). Previous studies analyzed the role of TLR4 in immunosurveillance, with scarce data evaluating the influence on local cell populations. Whilst the expression of TLR4 in human GCs has been revealed, the influence on GC function remains unknown. Therefore, we aimed to explore the role of TLR4 in steroidogenesis in human ovarian GCs.

Animals
Immature (3-week-old) female C57BL/6 mice were obtained from the SLAC Company (Shanghai, China). This study was approved by the experimental animal ethics committee of Fudan University and all experiments were performed under the guidelines of the animal care regulations of Fudan University. All mice were housed under controlled conditions on a 12h light/ 12h dark cycle and had free access to food and water. All agents were injected intraperitoneally. Mice were randomly divided into six groups as follows:

Immunohistochemical Analysis
Immunohistochemical analysis was performed to analyze the localization of TLR4 in the ovaries. Ovaries were immersed in 4% paraformaldehyde for fixation, and then embedded in paraffin and cut into 4 mm sections for histological analysis. The slides were incubated in citrate buffer (pH 6.0) and heated at 98°C for 30 mins for antigen retrieval. After recovering to room temperature, the slides were incubated with 3% H 2 O 2 for 10 mins to inhibit peroxidase activity followed by blocking with 10% goat serum for 1 h. Slides were then incubated with primary antibody against TLR4 (GB11519, 1:500) overnight at 4°C. For the negative control, IgG from rabbit serum was used instead of primary antibodies. The slides were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (GB23303, 1:500) for 1h at room temperature. Immunoreactive signals were observed using 3,3-diaminobenzidine. And the slides were counterstained with hematoxylin. Images of the stained slides were obtained using an Olympus BX53 microscope (Olympus, Tokyo, Japan).
Immunofluorescence KGN cells were seeded on cover slips in 6-well plates and cultured for 24 h. Cells were then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 on ice, followed by blocking with goat serum. Then, the cells were incubated with TLR4 antibody (GB11519, 1:1000) overnight at 4°C. The negative control was incubated with rabbit IgG isotype control (A7016, 1:1000). The next day, cells were incubated with FITC-conjugated goat anti-rabbit secondary antibodies (1:500, A0562) for 1 h at room temperature. Then nuclei were stained with DAPI (1.5 mM, Beyotime, Shanghai, China) for 10 mins. Images were obtained using Nikon fluorescence microscopy (Nikon, Tokyo, Japan).

Histology
Ovaries were fixed with 4% paraformaldehyde and embedded in paraffin, serially sectioned in the longitudinal plane with a thickness of 4mm. Every 15 th section was stained with hematoxylin and eosin (HE). The numbers of primordial, primary, secondary and antral follicles were counted. Follicles with a single layer of flattened GCs were classified as primordial follicles. Follicles with a single layer of cuboidal GCs were counted as primary follicles. Follicles with multiple layers of cuboidal GCs were classified as secondary follicles. Follicles with two or more layers of GCs and A fluid-filled antral space were counted as antral follicles (18).

Quantitative Real-Time PCR
Total RNA was isolated with TRIZOL regent (Invitrogen, Carlsbad, USA), and cDNA was synthesized with PrimeScript ™ RT Reagent Kit (Takara, Otsu, Japan). The respective primers are shown in Table 1. Quantitative realtime PCR (qRT-PCR) was carried out using the TB Green ™ Premix Ex Taq ™ II Kit (Takara, Otsu, Japan) in the ABI 7900 real-time PCR system (Applied Biosystems Inc., Foster City, USA). The 2 −DDCt method was used to evaluate the fold change at the transcriptional level.

Enzyme-Linked Immunosorbent Assay
KGN cells were seeded in 24-well plates at a density of 4×10 4 cells per well in 1 ml of DMEM/F12 without red phenol and with charcoaled-treated 10% fetal bovine serum. Cells were cultured with rFSH (1IU, Merck-Serono, Geneva, Switzerland) or FSK

Statistical Analyses
Data were obtained from at least three independent experiments and expressed as mean ± S.D. Student t-test, one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test or Dunnett's multiple comparisons and Kruskal-Wallis test followed by Dun's multiple comparisons were used to analyze the differences between groups. The threshold for statistical significance was P<0.05.

TLR4 Expression and Localization in Mouse Ovaries and Human Granulosa Cells
Immunohistochemistry confirmed that within the ovary TLR4 is predominantly located in GCs and luteal cells. As shown in Figures 1A-H, TLR4 was detectable in GCs at different stages of follicles. It was relatively weakly expressed in GCs from mature follicles ( Figures 1C-D), whereas it significantly increased both in GCs and luteal cells in response to hCG treatment ( Figures  1E-F). In contrast, TLR4 was significantly decreased in the corpus luteum in the hCG 12 h group ( Figures 1G-H).
These findings indicate that TLR4 shows stage-specific expression in GCs and the corpus luteum, which may suggest a role for TLR4 in regulating follicular development and luteinization. Previous studies have shown the expression of TLR4 in human GCs (20). We further examined TLR4 localization in KGN cells, As shown in Figure 1J, green immunofluorescence confirmed the expression of TLR4 in KGN cells. In addition, we further examined the effects of TLR4 agonist LPS, FSH and FSK on TLR4 expression in KGN cells.  As evidenced in Figure 1K, the protein levels of TLR4 decreased after treatment of LPS for 24 h (P=0.0317). No obvious changes were observed in TLR4 expression after the stimulation of FSH and FSK for 24 h (Figures 1L-M).

LPS Inhibited Follicular Development in Mice
To examine the effects of TLR4 activation on follicular development, TLR4 agonist LPS was co-administered with PMSG. In this way, the mice were exposed to LPS during the growth period of follicles. Results showed that numerous large antral follicles are observed in the control group after 48 h treatment of PMSG (Figure 2A). Correspondingly, few antral follicles were observed in the LPS group ( Figure 2B). As shown in Figures 2C-D, the LPS group had significantly fewer antral follicles (P=0.0028) and E2 levels (P=0.0113) than the control group, but the two groups did not differ significantly in numbers of pre-antral follicles. For 15 h after hCG treatment, the ovulated oocytes were collected and counted. And we observed that the number of ovulated oocytes is significantly decreased in the LPS group (P=0.0034). These results suggest that TLR4 activation inhibited follicular development in mice.

TLR4 Signaling Regulated Steroidogenic Genes in KGN Cells
Steroidogenesis is essential for follicular development and selection, so we examined whether the activation of TLR4 signaling is involved in the regulation of steroidogenic genes, levels of CYP19A1, StAR, CYP11A1 and FSHR, which are the key genes regulating E2 and progesterone in GCs, were investigated by qRT-PCR. As shown in Figure 3A,  Figures 3F-H). These results indicate that TLR4 signaling is involved in the regulation of steroidogenic genes.

Effects of TLR4 Activation on rFSH-Stimulated Estradiol and Progesterone Production in KGN Cells
We further evaluated the effect of TLR4 activation on rFSHstimulated steroid hormone production. Results showed that 48 h incubation with rFSH induced a 3-fold increase in E2 expression (P<0.0001) and no significant increase in progesterone levels ( Figure 4A). However, TLR4 activation significantly inhibited the rFSH-induced production of E2 (P=0.0305). There was a rising trend for progesterone production among incubation of rFSH with LPS compared to rFSH alone, but this trend was not significant ( Figure 4A).

TLR4 Activation Suppressed Estradiol Secretion Through Inhibition of CYP19A1
To explore the potential causes underlying the decreased E2 secretion, we examined the expression changes of the steroidogenic enzymes, StAR and CYP19A1, which are key regulators of E2 and progesterone production by GCs. Results showed that incubation with rFSH for 24 h led to significant increases in StAR and CYP19A1 mRNA expression (P=0.0455 and P=0.0027, respectively). However, the activation of TLR4 signaling significantly decreased rFSH-induced CYP19A1 mRNA expression (P=0.0028). In addition, StAR mRNA expression induced by rFSH was not affected ( Figure 4B). Considering the inhibition of TLR4 activation on FSHR, we used the adenylate cyclase activator FSK to confirm the inhibitory effect of TLR4 activation on CYP19A1. As shown in Figure 4C, FSK led to 11fold increase in E2 expression (P=0.0001) and no significant increase in progesterone, and TLR4 activation significantly inhibited the FSK-induced production of E2 (P=0.0214). Similar to rFSH, FSK facilitated the transcription of CYP19A1 and StAR (P=0.0002 and P=0.0007, respectively). TLR4 activation also decreased the transcription (P=0.0098, Figure 4D) and protein level (P=0.0298, Figure 4E) of CYP19A1 induced by FSK, with no inhibitory effect on StAR. These results indicate that TLR4 activation suppresses E2 secretion through inhibition of CYP19A1.

NF-kB Signaling Mediated LPS-Induced Inhibition on CYP19A1
The downstream signaling of the TLR4 pathway includes the MAPK and NF-kB pathways. To determine the downstream signaling cascade in KGN cells, we examined the activation of NF-kB, p38 and JNK after LPS treatment by Western blotting. The phosphorylation of NF-kB p65, p38 MAPK and JNK were enhanced after LPS treatment (P=0.019, P=0.016, and P=0.019, respectively; Figures 5A-B), indicating that LPS could activate NF-kB signaling pathway in KGN cells, as well as the p38 MAPK and JNK signaling pathways. We next investigated the potential mechanisms by which TLR4 signaling inhibits CYP19A1. We explored NF-kB, p38 MAPK and JNK signaling using specific inhibitors. The inhibitory effect on CYP19A1 mRNA induced by LPS stimulation was markedly reversed by pretreatment with the NF kB inhibitor JSH-23 (P=0.0004, Figure 5C). Similar results were observed in protein levels of CYP19A1 by pretreatment with JSH-23 (P=0.0095, Figure 5F). When incubated alone, p38 MAPK inhibitor SB 203580 substantially inhibited the transcription of CYP19A1 (P=0.0175, Figure 5D), while the JNK inhibitor SP600125 significantly promoted the transcription of CYP19A1 (P<0.0001, Figure 5E). Coincubation of SP600125 with LPS inhibited the facilitatory effect of SP600125 on CYP19A1 mRNA levels (P<0.0001). These results demonstrate that NF-kB signaling plays a key role in the inhibitory effects of TLR4 activation on CYP19A1 expression. Moreover, JSH-23 dramatically reversed the inhibitory effects of LPS on FSKinduced secretion of E2 (P=0.0486, Figure 5G), as well as transcription and protein levels of CYP19A1 (P=0.0429, P=0.0473, Figures 5H-I). Taken together, these results suggest that NF-kB signaling mediates the inhibition of TLR4 activation on CYP19A1 and E2 secretion.

DISCUSSION
Earlier studies showed that TLR4 is expressed in human cumulus cells. Recently, a study highlighted TLR4 expression in human primordial and primary follicles, with apparent staining in GCs (21). Consistent with these reports, we observed TLR4 expression in mouse GCs at different stages. In addition, the TLR4 agonist LPS can stimulate TLR4 target genes in ovaries in vitro, as well as cumulus cells from cumulus-oocyte complexes (22). These findings indicate that TLR4 signaling in human GCs is complete and functional. However, previous studies looked more closely at role of TLR4 in immunosurveillance, with scarce data evaluating the influence on functionality of local cell populations. Moreover, the influence of TLR4 activation on steroidogenesis in human GCs is less well understood. Incubation of rFSH with LPS reduced rFSH-induced expression of CYP19A1 mRNA, with no effect on StAR mRNA. (C) FSK-induced secretion of E2 was also reduced when incubated with LPS, and there was no difference in progesterone secretion. (D) Incubation of LPS with FSK reduced FSK-induced transcription of CYP19A1, with no effect on StAR mRNA. (E) Incubation of LPS with FSK reduced FSK-induced protein level of CYP19A1, while there was no difference in StAR. All data are means ± S.D. n=3. One-way ANOVA followed by Tukey's multiple comparisons test were performed to analyze the differences. *P<0.05, **P < 0.01, ***P < 0.001 and ****P<0.0001 vs control group. # P < 0.05 and ## P < 0.01 vs FSH or FSK groups.
We present evidence that TLR4 activation suppresses follicular development in mouse ovary. And in the human GCs, the basal expression of CYP19A1 is significantly inhibited. Indeed, some studies in mammals found that LPS decreases CYP19A1 expression in vitro (12,23,24). In addition, TLR4 was involved in the downregulation of CYP19A1 (23). Our data are in accordance with these findings, further confirming the inhibitory effect of TLR4 on CYP19A1. Moreover, we illustrated for the first time that TLR4 activation also inhibits rFSH-and FSK-induced expression of CYP19A1, as well as E2 The results are expressed as means ± S.D. Student t-test, one-way ANOVA followed by Tukey's multiple comparisons test and Kruskal-Wallis test followed by Dun's multiple comparisons were used to analyze the differences. *P<0.05, **P < 0.01, ***P < 0.001 and ****P<0.0001 vs control or FSK group. # P < 0.05, ## P<0.01 and ### P < 0.001 vs LPS or FSK+ LPS group. $$$$ P < 0.0001 vs SP600125 group. secretion in human GCs. In addition, TLR4 signaling may suppress FSH activity by inhibiting the expression of FSHR. Though FSH is a major survival factor for antral follicles and GCs, activation of TLR4 signaling compromised the ability of rFSH to stimulate CYP19A1 and consequent E2 production. Thus, the development of follicles can be restricted. Furthermore, TLR4 activation led to the activation of NF-kB, JNK and p38 MAPK signaling. According to our data, although JNK and p38 MAPK may be involved in the regulation of CYP19A1, NF-kB predominantly inhibited TLR4 activation of CYP19A1 and E2 secretion. Collectively, these data suggest that TLR4 signaling impacts E2 production and FSH response in GCs ( Figure 6).
Our study suggests a new role for TLR4 in ovulation. Ovulation is well known to be linked to the inflammatory response, with many genes associated with immune surveillance induced in cumulus cells, including TLR4 signaling (22). TLR4 signaling is thought to play critical role in surveillance during ovulation (25). We did observe the enhancement of TLR4 expression after ovulation triggering, further suggesting a role of TLR4 in ovulation. Importantly, after the ovulatory LH surge, the expression of CYP19A1 is rapidly suppressed, and GCs shift from estrogen to progesterone synthesis, which is crucial for ovulation and following luteinization (26). Though LPS is not part of normal mammalian physiology, TLR4 can also be activated by numerous endogenous ligands. Currently, the role of TLR4 in ovulation is not well understood. And the ligand which may regulate the TLR4 signaling in the process is uncertain. We speculate that TLR4 is likely to play a role in the regulation of steroidogenesis in ovulatory GCs, but further studies are needed to verify this speculation.
Our study also implies that TLR4 signaling may be involved in abnormal follicular development and selection in some inflammatory diseases. It is well known that the production of estrogen is essential for follicular development and selection. A previous study demonstrated that estrogen promotes survival and growth of follicles at the preantral to early antral stage (27), and disruption of these processes may drive follicular atresia. LPS is a major component of Gram-negative bacteria, and the bacteria is closely associated with pelvic inflammatory disease (PID), which is a common gynecological disease. And TLR4 can recognize Gramnegative bacteria through LPS. Apart from PID, LPS was also found elevated in obese women with PCOS (28). Abnormal elevated LPS in circulation or the microenvironment can activate the TLR4 signaling in GCs. Our data showed that TLR4 is detectable in GCs at different stages of follicles. We also present evidence that numbers of pre-antral follicles are unchanged, but follicular development can be disturbed after LPS stimulation. On the other hand, upregulated TLR4 and its downstream targets can also cause TLR4 hyperactivation, leading to detrimental effects (12). A previous report revealed that GCs from women with polycystic ovary syndrome (PCOS) express higher levels of TLR4, which is associated with lower embryo quality (20). TLR4 can be upregulated in several cases. For example, our data showed increased expression of TLR4 after stimulation of hCG. Moreover, lipid challenge was found to upregulate TLR4 expression in mononuclear cell in PCOS patients (28). Therefore, upregulated expression of TLR4 and its downstream targets, as well as abnormally elevated LPS may inhibit the production of E2 in GCs and influence the development of follicles. Our findings indicate that hyperactivation of TLR4 signaling may be implicated in the abnormal follicular development observed in PCOS. FIGURE 6 | Schematic presentation of TLR4 activation in steroidogenesis in human granulosa cells. The TLR4 agonist LPS inhibited the expression of CYP11A1, StAR, FSHR and CYP19A1. The TLR4 inhibitor TAK-242 reversed the inhibition of LPS on CYP19A1, FSHR and StAR. TLR4 activation also inhibited FSH-and FSKinduced expression of CYP19A1 and estradiol secretion, with no effect on progesterone secretion. Importantly, TLR4 activation led to the activation of NF-kB signaling. The NF-kB inhibitor, JSH-23, rescued the inhibition on both basal and FSK induced expression of CYP19A1, as well as FSK-induced estradiol secretion.

CONCLUSIONS
Our data demonstrate for the first time that TLR4 activation inhibits follicular development in mice, suppresses basal and FSK-induced CYP19A1 expression, as well as E2 secretion in human GCs. In addition, TLR4 activation also compromised FSH activity by inhibiting the expression of FSHR. Furthermore, we showed the involvement of NF-kB signaling in mediating the inhibition on CYP19A1 and E2 secretion. Thus, TLR4 activation may affect reproductive capacity, which suggests a novel role of TLR4 signaling in ovarian pathophysiology.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by the experimental animal ethics committee of Fudan University.