Effects of IL‐18 on the proliferation and steroidogenesis of bovine theca cells: Possible roles in the pathogenesis of polycystic ovary syndrome

Abstract Interleukin 18 (IL‐18) is a pleiotropic pro‐inflammatory cytokine and is associated with arrested follicle development and anovulation which are the typical pathological changes of PCOS. Theca cells (TCs) have a key role in follicular growth and atresia. But whether IL‐18 can directly affect ovarian TCs function is unknown. Therefore, the objective of this study was to determine the effect of IL‐18 on proliferation and steroidogenesis of bovine TCs and to explore the biological effect of IL‐18 on folliculogenesis. This work revealed that at 300‐1000 pg/mL, IL‐18 led to a time‐ and dose‐dependently increase in cell proliferation (P < .05). IL‐18 increased 17‐hydroxyprogesterone (17OHP4) and androstenedione (A2) secretion with up‐regulation of key steroidogenesis‐related genes CYP11A1 and CYP17A1 (P < .05). Furthermore, our data demonstrated that the IL‐18R protein is predominantly expressed in small‐follicle (3‐6 mm) TCs than large follicles (8‐22 mm) by immunohistochemistry. We also found that the stimulation effects of IL‐18 on TCs can be reversed with the addition of IL‐18BP as early as at 4 hours of culture and reached the peak at 16 hours. We conclude that IL‐18 appears to target TCs in bovine, and suggest an important role for this cytokine in ovarian function. Present findings further validate potential effects of IL‐18 in the conditions associated with follicular dysplasia and excessive growth of ovarian TCs (such as PCOS). But additional research is needed to further understand the mechanism of action of IL‐18 in theca cells as well as its precise role in folliculogenesis.

Interleukin 18 , formerly known as interferon-γ inducing factor, 7 is a pleiotropic, pro-inflammatory cytokine, which plays a key role in the host defence against several infectious agents. IL-18 modulates many genes involved in inflammation, infection and malignancy. Previous studies have shown that the follicular fluid (FF) of PCOS patients contains IL-18 or its mRNA, and our recent study also showed that IL-18 levels in FF of PCOS patients are higher than those in control women. 8 It is likely that locally produced IL-18 plays a physiological role in the pathogenesis of PCOS.
Typical pathological changes of PCOS include arrested follicle development and anovulation. IL-18 has been suggested to affect ovarian folliculogenesis. 9 Gutman et al 10  Abnormal function of T-I cells has been shown to be associated with pathological conditions, such as PCOS. Hyperplasia of the ovarian theca cells compartment is a key feature of the polycystic ovary phenotype. Ovarian theca cells are recognized as one of the primary sources of excess androgen biosynthesis in women with PCOS, 13 expressing a variety of genes encoding components of the steroidogenic pathway that are necessary for androgen and progestin biosynthesis. 14 Ovarian hyperandrogenism is associated with the increased number of androgen-producing cells contributing to increased androgen production, 15 and then leading to anovulation. 16 The biological functions of IL-18 are mediated through its receptor. IL-18 receptor (IL-18R) is a member of the IL-1 receptor family. It consists of a ligand-binding domain (α-chain), which binds IL-18 with low affinity, and a signal-transducing domain (β-chain). 17 Together, a formed high-affinity IL-18R complex transduces its signal to stimulate the MAPK pathway. 18 Cytokines are increasingly recognized as potentially important local regulators of ovarian function. 19 The expression of IL-18R has been determined in theca cells, but the effect of IL-18 on the proliferation and hormone secretion of TCs is not clear, and there is no related research report.
The aims of the present study were to: (a) examine the expression of IL-18 signalling receptors in TCs across different stages of bovine antral follicle development; (b) use non-luteinized bovine TC culture models to investigate whether IL-18 affects steroid production; (c) explore the potential biological effects of IL-18 on TCs proliferation in follicles sampled; (d) detect the effect of IL-18 on the key enzymes in steroid hormone synthesis of TCs; (e) determine whether the effect of IL-18 can be attenuated by IL-18BP; and (f) explore the possible roles of IL-18 in the pathogenesis of PCOS.

| Ethics statement
All procedures were reviewed and approved by the ethics committee of Tianjin Central Hospital of Gynecology Obstetrics, China (TJCHGO-2018-112). The local slaughterhouse in Tianjin provided ovaries for cattle aged 1-2 years. The ovaries were dissociated according to the laboratory standards of Nankai University.

| Isolation and culture of TCs
Bovine TCs were isolated from the ovaries of randomly cycling cattle obtained from the slaughterhouse. Freshly collected ovaries were placed on ice in saline with antibiotics (0.9% saline solution with 1% penicillin-streptomycin) and transferred to the laboratory within 2 hours after collection. The follicles were dissected from the ovaries using surgical tweezers and striped until the surface was smooth.
Follicles were then cut into halves under a stereomicroscope (Nikon) and the granulosa cells (GCs) were dislodged using a plastic inoculation loop. The follicle halves were shaken vigorously in a DMEM/F12 medium (GIBCO) to remove any remaining GCs. The theca internal layer was peeled away from the basement membrane and then was torn into small pieces, incubated for 30 minutes at 37°C with DMEM/ F12 medium containing 1.0 mg/mL collagenase (170.0 U/mg, type 4; GIBCO), 0.2% glucose pH7.4 (Sigma-Aldrich Inc), and 0.4% bovine serum albumin (GIBCO). 20 The nondigested tissue was filtered out through sterile syringe filter holders with metal screens of 149 μm mesh. The isolated cells were pelleted twice in phosphate-buffered saline (PBS) solution by centrifugation (800g for 10 minutes) to lyse any red blood cells. 21 Cell viability was assessed by trypan blue exclusion and averaged above 90%. Then the TCs were re-suspended in serum-free medium containing collagenase (1.25 mg/mL) and DNase (0.5 mg/mL) to prevent cell clumping. 22,23 The resultant TCs preparations obtained using this method were judged to have <5% contamination with GCs. 24 On average, 2.0 × 10 5 viable cells were plated on 24-well Falcon multiwell plates in 1 mL of medium and cultured in an environment of 38.5°C with 5% CO2 and 95% air in 10% FCS for the first 48 hours until cells reached 80% confluency. Cells were washed twice with 0.5 mL of serum-free medium, treatments were applied in serum-free medium for an additional 24 hours or 48 hours, and medium was either aspirated or collected from each well depending on the particular experiment. The concentration of LH was selected based on previous studies. 25 This culture system was developed to yield hormonally responsive nonluteinized TC. 26,27 First, progesterone production does not increase with time using this culture paradigm. 27 Secondly, the morphology of the TC retains a fibroblastic appearance. 28 Third, the TC remain responsive to LH in terms of CYP17A1 mRNA and androstenedione production. 29

| Immunofluorohistochemistry detection of IL-18R in TCs
The expression of IL-18R on the TC was detected by immunofluorescence histochemistry (IF). The cells on the slide were fixed in 10% formalin for 10 minutes, permeabilized using 0.01% Triton X-100 (Sigma

| Cell viability and proliferation assay
Theca cells were seeded into 96-well plates and cultured overnight with McCoy's medium containing 0.1% BSA. After attachment, cells were pretreated with IL-18 (300 pg/mL) for 1 hour followed by LH (150 pg/mL) for 24 hours. After the treatment periods, cell viability was determined by MTT assay. 31

| Steroid immunoassays
Steroid concentrations in cell culture media were determined by ELISA. The 17-hydroxyprogesterone (17OHP4) assay had a detection limit of 20 pg/mL and intra-and inter-assay CVs were 8% and 10% respectively. The androstenedione (A2) had a detection limit of 30 pg/mL and intra-and inter-assay CVs were 7% and 10% respectively. The results, expressed in ng 17OHP4/pg A2 per μg protein, were then normalized to the control condition of each experiment and presented as mean ± SEM of four experiments with three replicates per condition.

| RT-PCR and quantitative (Q)-PCR
Total RNA from TCs was extracted using the TRIzol reagent (Roche) according to the manufacturer's instructions. cDNA was synthesized from 1 μg of RNA using the AB High-Capacity cDNA synthesis kit (Thermo Fisher Scientific) with random hexamers. Real-time PCR was performed using SYBR green master mix on LightCycler ® 96 System (Roche Diagnostics Ltd). The sequences of the primers used were given in Table 1. qPCR was carried out using QuantiTect SYBR Green master mix (Qiagen) and an AB StepOne plus thermal cycler (Applied Biosystems). The PCR parameters used were a 10 seconds denaturation cycle at 95°C, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. PCR efficiencies were detected using a relative standard curve derived from diluted cDNA reaction mixture (a 2-fold dilution series with five measuring points). The R2 values for all standard curves generated ranged between 0.997 and 0.999 and PCR efficiencies were between 90% and 110%. The β-actin as an inner control was applied to normalize the mRNA expression results using 2 − ΔΔC t method.

TA B L E 1 Primer sequences
Reverse

| Statistical analysis
All data were analysed using SPSS Statistics 22 software (IBM

| Purification of TCs
To evaluate the purity of isolated TCs and rule out fibroblast contamination, TCs were incubated with rabbit anti-vimentin

| Expression of IL-18R in bovine TCs
To further reveal the manner of IL-18 functions in follicles, we localized IL-18R protein in TCs by IF. By means of Immunofluorescence, we were able to prove that bovine follicular TCs express IL-18R. Figure 2A demonstrates the immunoreactive signal of IL-18R was expressed in both the membrane and cytoplasm of theca cells, and the expression was mainly in the cell membrane. Bovine TCs from small and large follicles both expressed the IL-18R. In small follicle ( Figure 2C), the expression of IL-18R was significantly higher than that in large follicle ( Figure 2D). The fluorescence intensity was analysed by Image J and the results also showed that the expression of IL-18R decreased gradually during follicular development ( Figure 2E).
The present data suggested that IL-18 from TCs or serum could mediate the biological effects in the cells. Furthermore, IL-18 may play an essential role in regulation of the growth and development of the follicles via an intrafollicular paracrine/autocrine manner.

| Effect of IL-18 on the proliferation of TCs
The initial experiments examined toxicity of IL-18 treatment in cultured TCs using cell viability assay. To test this, cultured TCs were preincubated with or without IL-18 (300 pg/mL) for 1 hour followed by treatment with LH for 24 hours. Cell viability was analysed by MTT assay. The results presented in Figure 3A show that IL-18 To identify the exact effect of IL-18 on the TC proliferation, BrdU assay was utilized to assess the cell proliferation. Cultured TCs isolated from bovine ovaries were treated with different concentration of IL-18 (0, 10, 30, 100, 300, 500 and 1000 pg/mL) for 24, 48, and 72 hours in the presence of BrdU. As shown in Figure 3B, the results showed that treatment with IL-18 in TCs caused a biphasic dose-response, with 10-100 pg/mL were without effect, whereas 300-1000 pg/mL in dose and time-dependent promotes the incorporation of BrdU into newly synthesized DNA in TCs (P < .05), suggesting that IL-18 stimulates TCs proliferation. The curve of cell proliferation percentage showed that at the highest dose tested, IL-18 increased cell numbers by 1.5-to 2-fold ( Figure 3C).

| Activation of JNK by IL-18-induced cell proliferation in bovine theca cells
The JNK signal pathway appears to play a significant role in IL-18 signalling. Therefore, this study examined whether or not IL-18 actually activates this kinase in bovine theca cells. The theca cells were treated with high-dose IL-18 during a 3-d culture results in strikingly enhanced activation of JNK, which correlated with increased expression of the anti-apoptotic factor Bcl-xL, and cell cycle-related factors Cdk6 and cyclin D3 ( Figure 4B,C). Furthermore, pretreatment of IL-18-stimulated cultures with the JNK inhibitor SP600125 revealed that IL-18-dependent increases in cell proliferation is particularly sensitive to JNK inhibition ( Figure 4A), suggesting that enhanced JNK activation may be the key molecular mechanism of IL-18 stimulation on proliferation of theca cell.
Direct evidence for JNK activation was provided by immunoblotting the whole cell lysated with anti-phospho-JNK antibody after the IL-18 treatment. As shown in Figure 4B, the IL-18 treatment increased the level of JNK phosphorylation. This suggests that IL-18 enhances theca cell proliferation by activating JNK.

| Effect of IL-18 on basal and LH-induced steroid secretion by TCs
To examine the effects of IL-18 on steroid biosynthesis, cultured TCs

| Effects of IL-18 on TC expression of steroidogenesis-related transcripts
To test the cellular mechanisms underlying IL-18 stimulation of A2 and 17OHP4 biosynthesis, the ability of IL-18 to alter abundance of LHR, CYP11A1, and CYP17A1 mRNA and protein were analysed in cultured TCs.  As shown in Figure 6A, the results showed that treatment with IL-18BP caused a biphasic dose-response, with 1 ng/mL was

F I G U R E 4 Involvement of JNK pathway for IL-18-induced cell proliferation in bovine theca cells. A, Theca cells were pretreated with
or without SP600125 (10 μmol/L) for 1 h before being treated with IL-18, and then incubated with 300 pg/mL IL-18 for 24 h in serum-free media. DMSO was used as the control for SP600125. After incubation, cells were harvested and the percentage of cell proliferation was detected by BrdU incorporation. Three independent experiments were carried out in triplicate; bars, mean ± SEM. *P < .05. B and C, Theca cells were treated with IL-18 (300 pg/mL) for 0, 12, 24, 48 and 72 h. The cells were lysed, and the level of Bcl-xL, Cdk6, cyclin D3 and JNK 1 phosphorylation was examined by Western blot. The total JNK 1 was detected to confirm the equal volume of the cell lysates. Three independent experiments were carried out in triplicate; bars, mean ± SEM. *P < .05 compared between groups

CO N FLI C T S O F I NTE R E S T
The authors have no conflicts of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets are available under reasonable request.