Study On The Role And Mechanisms Of Interleukin-18 And Interleukin-18 Binding Protein In Regulating Ovarian Theca Cell Function

Polycystic ovary syndrome (PCOS) is the most common cause of oligomenorrhea, amenorrhea, and anovulatory infertility, and appears to be one of the most common endocrinopathy affecting women in their reproductive years. These patients are at higher risk of developing infertility, dysfunctional uterine bleeding, and a number of metabolic disorders. The prevalence of PCOS in reproductive-aged women is estimated to be about 4-8%, and account about 50-70% of the anovulatory infertility, which have greatly influenced the quality of life and mental status of young women. Although a series of study have carried out on the etiopathogenisis of PCOS, such as genetic factors, functional disorders of HPO axis, disfunction of adrenal and ovarian enzymics, insulin resistance and obesity, the precise aetiology is still not known.There is accumulating evidence indicating chronic low-grade inflammation in PCOS, including increased levels of CRP, increased leukocyte count, and increased pro-inflammatory cytokines (i.e. IL-6 and IL-18). These data suggest that the pathogenesy of PCOS may be associated with chronic low-grade inflammation and activation of the innate immune system. IL-18 is a new proinflammatory cytokine, discovered recently that induces the production of TNF-a, which in turn promotes the synthesis of IL-6, and IL-6 regulates thesynthesis of CRP in liver. Interleukin-18 (IL-18), initially known as interferon-y inducing factor, is a member of the IL-1 cytokine family and has been found to play an important role in innate and acquired immunity and inflammation response. Recently, elevated IL-18 levels were found in serum from some PCOS women, and the IL-18 levels were positively correlated with androgen levels, but were inversily correlated with insulin sensitivity. It indicates that IL-18 may be a contributing factor linking the onset of PCOS.PCOS ovaries are characterized by the accumulation of small follicles 4-7mm in diameter, with hyperplasia of the theca interna layers and excessive production of androgens by these cells. Thecal cells control follicle growth and atresia, regulate ovarian seroidogenesis, and are recognized as one of the primary sources of excess androgen biosynthesis in women with PCOS. Dysregulation of the thecal cells compartment will encountered in pathological conditions such as PCOS and hyperthecosis which are characterized by thecal and stromal hyperplasia and steroidogenic hyperactivity. The IL-18 receptor has been confirmed to be exist in thecal cells, and may be participant in follicle development process, although the exactly influence is still unknown.Extracelllular IL-18 exerts its biological effects by binding the heterodimeric IL-18 receptor complex. IL-18-binding protein(IL-18BP) is a naturally occurring protein that binds and neutralizes IL-18. It can binds IL-18 with specificity and high affinity and blocks its biological activities. Therefore, in this study, we examined whether or not IL-18 and its receptor were implicated in the proliferation of thecal cells in culture. In addition, we also analyzed the possibly mechanism of the modulating effect, and the possible protect effect of IL-18BP to thecal cells. Our work will provide not only new experimental data for furthering the pathogenesis of inflammation in PCOS in depth but also theoretical basis for setting up new therapies.Our research is divided into four parts. In the first part, we used fresh bovine ovaries to cultivate original bovine thecal cells, so that there were enough thecal cells to investigate. In the second part, we investigate the alteration of proliferation and steroidogenesis of thecal cells with different concentration of IL-18 with different time, and analyse the function of IL-18 in the morbidity of PCOS. In the third part, we investigate the possible mechanism of IL-18 effecting thecal cells. In the last part, we studied the effects of IL-18BP-Fc on the proliferation and steroidogenesis induced by IL-18 on thecal cells. To study the molecular mechanism of inflammation in PCOS and offer helpful experiments data for the treatment of PCOS by IL-18BP. Objective:In order to investigate the effect of IL-18 on cell proliferation and steroidogenisis of thecal cells, we did original cultivation of bovine thecal cells.Methods:We obtained fresh bovine ovaries to cultivate original bovine thecal cells through enzyme digestion and Percoll density gradient centrifugation. Then we used immune cytochemistry and immunofluorescence to identify thecal cells.Results:The cells we cultured was in good condition, monolayer, and short fusiform shaped. About 90% of the cells we cultivated were alive. The results of immune cytochemistry showed vimentin positively stained while keratinose andⅧfactor negatively stained. Under fluorescence microscope, the thecal cells were positively stained with CYP17A1. The cells we cultivited was thecal cells.Conclusions:We have successfully cultivated original bovine thecal cells. Objective:In the present study, we investigated the cell proliferation and steroidogenisis on original cultured bovine thecal cells induced by IL-18.Methods:Original thecal cells were cultured in free serum DMEM/F12 medium, and treated with different concentration of IL-18(10,30,50,100,300,500,1000 ng/ml). Culture solution were collected after treated for 24h,48h and 72h, respectively. Then, cell proliferation was tested by MTT assay, androstenedione and 17-hydroxyprogesterone were detected with radio-immunity.Results:There were no statistically difference between the control group and the groups with IL-18 in concentration of 10ng/ml-100ng/ml (P>0.05). Compared with the control group, the OD value and androstenedione and 17-hydroxyprogesterone concentrations were significantly different with cells treated with IL-18 between 300ng/ml and 1000ng/ml(P<0.01); The OD value of IL-18 in concentration of 300ng/ml,500ng/ml and 1000ng/ml decreased significantly (P<0.01), and the androstenedione and 17-hydroxyprogesterone concentrations of them incerased significantly (P＜0.01), as the concentration of IL-18 upgraded.The OD value and androstenedione and 17-hydroxyprogesterone concentrations were compared between the control group and experimental groups after treated with IL-18(<100ng/ml) for 24,48 and 72h, respectively, there were no significantly difference (P>0.05). While OD value was significantly decreased and androstenedione and 17-hydroxyprogesterone concentration were significantly increased in the experimental groups with the concentration of IL-18 was>300ng/ml, as the time prolonged.Conclusions:There was no difference in the cell proliferation and androstenedione and 17-hydroxyprogesterone concentrations between control group and groups treated with IL-18 with concentration lower than 100ng/ml. With concentrations between 300ng/ml and 1000ng/ml,IL-18 could dose and time dependently induce cell preliferation and steroidgenesis of the thecal cell. Objective:The present study was to investigate the effect of IL-18 on the protein and mRNA expression of cholesterol side-chain cleavage enzyme(P450scc), cytochrome P450(P450c) 17-hydroxylase, and LH-R. We also investigate the differences of the expression of protein and mRNA of IL-18 R between big and small ovary follicles. The aim of this study was to demonstrate the mechanism of the IL-18 resulting in PCOS, and the possible pathyway to anovulation.Methods:Cattle thecal cells were treated with IL-18 with the concentration of 1000 pg/ml. The cultured media were collected after 72h for detection. P450scc, P450c17-hydroxylase and LH-R protein were detected by Western bolt. CYP11A1, CYP17A1 and LH-R mRNA were detected by RT-PCR.Based on surface diameter, the thecal cells were collected from small (<8mm) and large (8-20mm) follicles respectively. Then we used Western blot and RT-PCR to investigate the differences of protein and mRNA of IL-18R between the two groups.Results:Significantly increased of P450scc(P<0.01), P450c17-hydroxylase (P<0.01) and LH-R(P＜0.01) protein expression were observed in cattle thecal cells that was treated with IL-18. Also, IL-18 has caused an increased effect of mRNA expression of CYP17A1(P<0.01), CYP11A1(P<0.01) and LH-R(P<0.01). The expression of IL-18R protein(P<0.05) and mRNA(P<0.05) was significantly different between the big and small ovarian follicles.Conclusions:IL-18 can directly effect the thecal cells, include the increased testosterone secretion by inducing the expression of CYP17A1, CYP11A1 and LH-R in thecal cells. The results of the increased recruitment of small follicles and anovulation maybe related with the overexpression of IL-18 R on small follicles. All of these changes may related to the disruption of steroid hormone production and hyperandrogenism likely happend in PCOS. Objective:A naturally occurring protein, interleukin 18 binding protein (IL-18BP), which is normally present in serum can bind IL-18 and block its bioavailability and subsequent function. Therefore, to determine the effects of IL-18BP-Fc on proliferation and steroidogenesis of thecal cells induced by IL-18, experiments were conducted in the last part of our experiment.Methods:Original cultured thecal cells was induced by IL-18 (1000ng/ml) alone or in combination with IL-18BP-Fc in different concentrations (1ng/ml, 10ng/ml, 100ng/ml and 1000ng/ml). Then to collect the supernate for detection. MTT was used to elucidate the effects of IL-18BP-Fc on proliferation of thecal cells, and radioimmunity was used to detect the content of androstenedione and 17-hydroxyprogesterone in the supernate.Results:There was no difference between IL-18 group and group treated with IL-18BP-Fc with concentration of lng/ml in the cell proliferation and the concentrations of androstenedione and 17-hydroxyprogesterone. With concentrations between 10ng/ml and 1000ng/ml, IL-18BP-Fc could time dependently inhibit cell preliferation and steroidgenesis induced by IL-18, and to a top role at 16 hours. Otherwise, there was an evident dose-effect ralationship of IL-18BP-Fc on the thecal cell proliferation and androstenedione and 17-hydroxyprogesterone concentrations induced by IL-18. After treated with IL-18BP-Fc with concentration of 1000ng/ml for 16 hours, the depressive effect could achieve to about 90 percent, and no statistically significant with control group.Conclusions:The stimulating effect to thecal cells by IL-18 could be inhibit by IL-18BP-Fc and the depressive effect was time and dose dependently. The IL-18BP-Fc was avirulent to thecal cells and with a long half life.