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Inhibition of insulin signaling impacts the competence of porcine granulosa cells
in steroidogenesis
Jing Cong, Xue-min Qiu, Yang Liu, Xiaoke Wu
Department of Obstetrics and Gynecology, First Affiliated Hospital, HeilongjiangUniversity
of Chinese Medicine, Harbin, China;
Abstract
Objective: To investigate inhibition of insulin signaling pathway directly contribute to alteration of granulosa cells in sex steroid production.
Design: In vitro cell model
Setting: University-affiliated laboratory
Intervention: Granulosa cells from porcine follicles were isolated and cultured in vitro, and inhibition of insulin signaling was induced in cultured granulosa cells with a specific inhibitor of phosphatidylinositol-3-kinase (PI-3K), wortmannin or a specific inhibitor of mitogen-activated protein kinase (MAPK) kinase, PD98059.
Main outcome measures: All hormones were determined using RIA, Cytochrome P450 17a-hydroxylase (CYP17) and cytochrome P450 aromatase (CYP19) mRNA were assayed by reverse transcriptase polymerase chain reaction (RT-PCR).
Results: Basal and forskolin-stimulated testosterone accumulation associated with CYP17 mRNA expression were increased with treatment by wortmannin or PD98059 in cultured granulosa cells. Wortmannin alone increased CYP19 gene expression and estrodiol production, while decreased with forskolin plus wortmannin treatment. Incubation of granulosa cells with PD98059 indicated to cause steroid production enhanced together. However, the basal and forskolin-stimulated CYP19 mRNA had a differential response to PD98059 treatment.
Conclusions: Inhibition of insulin signaling pathways induced by wortmannin or PD98059 could directly exaggerate androgenesis potentially in granulosa cells, suggesting alteration of steroidogenesis occurred in cultured porcine granulosa cells in a state of impairment of insulin signaling.
Key Words: Insulin signaling, PI-3K, MAPK, granulosa cell, androgenesis.
Introduction
Sex steroid synthesis in the ovary occurs in both the granulosa and theca cells. Normally, androstenedione come from the theca cell and oestrogen from the granulosa cell according to the two-cell two-gonadotropin theory. Furthermore, the various steroidogenic enzymes expression is thought to determine unique steroidgenesis in granulosa and theca cells. Ovarian hyperandrogenism is associated with a variety of insulin-resistant states[1]. We focus the impact of altered insulin signaling on ovary steroidogenesis, thus exploring the mechanism of hyperandrogenism in PCOS. In insulin resistant states ovaries remain sensitive to insulin because insulin can activate alternative signaling pathways, such as phosphatidylinositol-3-kinase (PI-3K) and mitogen-activated protein kinase (MAPK) pathways. The former is necessary for insulin-stimulated glucose uptake and
transport-called metabolic pathway (2). The latter is involved in regulating mitogenesis-
called mitogenic pathway (3). Both the PI-3K and MAPK pathways are thought to be involved in insulin-mediated steroidogenesis . We have previously confirmed that the insulin resistant model can be established in cultured porcine ovarian granulosa cells by treatment with wortmannin, as demonstrated by defects in glucose uptake. Our hypothesis is that in state of impairment of insulin signaling, granulosa will convert to produce androgens like the theca cell. In this study, we induce a defective insulin signaling in cultured granulosa cells by treatment with wortmannin (WM), a special inhibitor of PI3K signaling cascade (4), and MAPK signaling inhibition with pharmacological inhibitor of MAPK, PD98059 (PD) , and then we assess in vitro effects of WM or PD on steroidogenesis and cytochrome P450 17a-hydroxylase (CYP17), cytochrome P450 aromatase (CYP19) gene expression.
Materials and methods
Granulosa Cell Culture
Granulosa cells were prepared from porcine ovaries according to previous studies[5, 6]. Porcine ovaries were obtained from a slaughterhouse at the follicular stage, maintained in a warm 9 g/L sodium chloride solution, and taken back to the laboratory within 2 hours. Granulosa cells from antral follicles (diameter 4-6 mm) were harvested by puncturing the follicles allowing pulsion of the cells. The cell suspension was collected, pelleted, and washed three times in Ham's F-12/DMEM supplemented with 10% fetal calf serum (FCS; HyClone, Logan, UT). Granulosa cells were collected by centrifugation for 5 minutes at 1,000 rpm and cultured in a culture flask (Orange Scientific, Braine-l'Alleud, Belgium) in Ham's F-12/DMEM at a density of 1 x 106 cells/mL in a humidified 95% air, 5% CO2 incubator at 37°C for 3 days.
Inhibition of Insulin Signaling Pathway and Treatment with Forskolin
To investigate the direct effect of insulin signaling pathway on steroidogenesis, we used PD98059 (Sigma Chemical) -stimulated and Wortmannin (Sigma Chemical) -stimulated porcine granulosa cells as in vitro models. After an initial 96-hour culture to allow for anchorage,
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the attached granulosa cells were exposed to Ham's F-12/DMEM containing 1% BSA (Biosource International, Camarillo, CA) with or without 50 цМ PD98059 and 250 nM Wortmannin as previously reported. In brief, the porcine granulosa cells with PD98059 or Wortmannin treatment and control cells (Con) were incubated at 37°C in F-12/DMEM (0.86 ^mol/L) with 1% bovine serum albumin (BSA) per well. With PD98059 or Wortmannin induction of 48 hours, the media were further treated for an additional 48 hours with 20цМ Forskolin (Sigma Chemical) in the PD, Wor and Control groups, respectively. At the end of treatment, the cultured media were collected and stored at -20° for steroid assays. The cells were also collected for study of their molecular biology.
Steroid Biosynthesis
The medium estradiol, progesterone and testosterone levels were assayed with standard RIA kits (Heilongjiang Provincial Hospital, Harbin, China).
Reverse Transcriptase Polymerase Chain Reaction
Total RNA was extracted from the cells prepared with TRIzol reagents (Invitrogen, Carlsbad, CA). Ribonucleic acid was assayed with the ThermoScript RT-PCR (reverse transcriptase polymerase chain reaction) System (Invitrogen). The PCR re-action was performed by a thermal cycler (Eppendorf, Ger-many). The sequences of the sense/antisense primers to amplify a 210-base pair (bp) fragment for P450arom were 5'- GGC TCG CAT ACA CTA CTT CC -3' /5' - AAC TCC CTT CCC TGC TCA AA -3' . The sequences of the sense/antisense primers to amplify a 131 bp of CYP17 fragment were 5' - TGC TTA TTA AGA AGG GCA AGG ACT T -3' /5' - TGT TGG ACG CGA TGT CTA GAG T -3' . The sequences of the sense/antisense primers to amplify a 110 bp fragment for p-actin were 5' - ATG GTG GGT ATG GGT CAG AA -3' /5' - ATG TCG TCC CAG TTG GTG AT -3'. The amplification was followed by 35 cycles of denaturation at 94° for 30 seconds, annealing for 30 seconds, extension at 72° for 30 seconds, and telo-extension at 72° for 10 minutes. All reactions were followed during 28 cycles. The PCR products were then electrophoresed on 2% agarose gel by 100 Vand visualized by staining with ethidium bromide.The intensity of the ethidium bromide luminescence was measured by a laser densitometer (Bio-Rad, Hercules, CA).The computational method is as follows: intensity of objective gene*area/(P-actin intensity^area).
Results and discussion
In vitro effects of PI3K inhibitor on steroidogenesis and CYP17 and CYP19 mRNA level in porcine granulosa cells.
To determine whether the inhibition of PI-3K signaling pathway directly influence steroid biosynthesis, we examined the effect of the PI-3K specific inhibitor, wortmannin on steroidogenesis. Dose response studies revealed that 250 nM wortmannin was maximally effective in stimulating granulose cell steroidogenesis after 48 h when combined with 20 цМ forskolin (data not shown). Thus, 250nM wortmannin and 20 цМ forskolin were used to examine possible stimulatory effects in the present study. We observed an approximately 4-fold increase in testosterone production in response to 48 h of wortmannin alone treatment, as compared with the basal granulosa cells ( without any treatment). There were more than 4-fold increase in testosterone production in wortmannin plus forskolin group compared to both the basal and forskolin-stimulated groups. Wortmannin alone increased estradiol production, while media level of estradiol was much lower in the wortmannin plus forskolin treated group than the basal group. However, there is no significant difference in progesterone accumulation in the media among four trial groups (P>0.05).
Steroidogenic enzymes is largely a reflection of mRNA levels. We examined the effect of wortmannin on the key steroidogenic genes, including CYP17 and CYP19. As expected, basal CYP17 mRNA was increased by forskolin treatment and further increased by concomitant treatment with wortmannin. Notably, the levels of CYP17 and CYP19 mRNA were both increased in response to wortmannin alone at 48h, especially CYP19. In contrast with the result of CYP17
gene expression in cultured granulosa cells with forskolin plus wortmannin treatment, CYP19 mRNA levels were decreased at the same condition.
In vitro effect of PD98059 on steroidogenesis and CYP17 and CYP19 mRNA in porcine granulosa cells
To examine whether the MAPK pathway is also involved in regulation of steroidogenesis in granulosa cells, we incubated the porcine granulosa cells with the MAPK inhibitor,PD98059 to investigate the effect of PD98059 on steroidogenesis. The effects of PD98059 and forskolin on steroidogenesis were concentration dependent (data not shown). The optimal concentration of PD98059 and forskolin for the present study was determined to be 50^M and 20^M, respectively. PD98059 alone stimulated basal progesterone, testosterone, and estradiol accumulation in culture media at the 48-h time point. As anticipated, forskolin alone increased the accumulation of steroid in the media. Co-treatment with PD98059 and forskolin result in much higher accumulation of steroid compared to treatment with forskolin alone. Previous studies demonstrated that human granulosa cells with treatment of MAPK kinase inhibitor PD98059 in vitro resulted in a significant increment in CYP17 mRNA levels and androstenedione production [7]. In human theca cells, activation of MAPK signaling directly inhibits CYP17 mRNA and androgen biosynthesis [8]. Data from our study demonstrated that PD98059 increased basal testosterone production associated with an increase in CYP17 mRNA expression, in agreement with other studies in human theca cells cultured with PD98059 [8].
As differential expression of the steroidogenic genes studied in granulosa cells was greatly pronounced at 48 hours in vitro culture, and differential regulation of steroidogenesis by a MAPK-dependent pathway also was observed at this time point, the question arises whether or not the MAPK-dependent pathway also simultaneously involved in the differential regulation of steroidogenic gene expression. To evaluate this possibility, we first investigated the stimulatory effect of PD98059 on the expression of CYP17 mRNA in granulosa cells at 48 hours in vitro culture. PD98059 dramatically and significantly promoted forskolin-stimulated elevation of CYP17 mRNA above forskolin alone and basal groups. In contrast to the effects of PD98059 alone on CYP17 mRNA, treatment of PD98059 alone decreased the expression of CYP19 mRNA. However, addition of PD98059 to forskolin had a positive effect on CYP19 mRNA expression.
Cytochrome P450 aromatase (Cyp19) is a key enzyme in ovarian steroidogenesis, which is expressed mainly in granulosa cells and convert theca-derived androgens to oestrogens. Follicle-stimulating hormone (FSH) is the primary stimulator of granulosa CYP19 expression[9], which is mediated by the cAMP/protein kinase A (PKA) pathway, however other pathways such mitogen and extracellular signal-regulated kinase (MEK/ERK), p38 mitogen-activated protein kinases (p38-MAPK) and phosphatidylinositol-3-kinase (PI3K) are all involved in positively and negatively regulating gonadotrophin-stimulated steroidogenesis [10, 11, 12]. Data from several researches have indicated that FSH stimulates Akt/protein kinase B (PKB) phosphorylation and activation of PI 3-kinase in granulose cells.[13]. Our study found that the expression of CYP19 mRNA was significantly increased in wortmannin-stimulated granulosa cells, as well as the level of estradiol in media was higher than the basal group, namely, the excessive estradiol was synthesized in wortmannin-treated granulosa cells. This demonstrates that PI-3K pathway plays a negative role in regulating CYP19 mRNA and estrodiol production in porcine granulosa cells. However, CYP19 mRNA and estrodiol production were decreased by concomitant treatment with wortmannin and forskolin, indicating some interaction between the signaling pathways in porcine granulosa cells. It is interesting to note that the effect of MEK pathway inhibition on CYP19 mRNA and estrodiol is just the opposite. These findings suggest that PI3K and MAPK signaling pathways exert different effect on steroidogenesis in granulosa cells, although the delicate interaction between the two pathways cannot be determined. Forskolin, like FSH, is capable of inducing the differentiation of cultured rat granulosa cells by itself, and potentiate FSH hormonal action [14]. Wortmannin alone promoted estradiol synthesis in basal granulosa cells but decreased forskolin-stimulated estradiol synthesis and CYP19 mRNA, implying that the similar FSH effect of forskolin was inhibited.
Similar to our study in bovine granulosa cells, oestradiol secretion was reduced from FSH-stimulated cells after culture with PI3K inhibitors, despite maintained CYP19 mRNA levels [15]. The authors demonstrated that that PI3K pathways favor oestradiol secretion at a site distal to CYP19 gene expression [16]. Conclusions
In summary, our results support a possibility that alteration of steroidogenesis in granulosa cells in the state of inmpairment of PI3K and MAPK pathways could be an important mechanism for abnormal steroid production in ovarian cells, Although the phenomenon is distinct from the traditional two-cell two-gonadotropin model of follicular steroidogenesis, it is contributed to explain the mechanism of hyperandrogenia observed in PCOS with insulin-resistant state. References
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Acupuncture and Clomiphene Citrate for Anovulatory Infertility in Polycystic Ovary Syndrome: Study Design of a Randomized Controlled Trial
Hongying Kuang1,Yan Li1,Xiaoke Wu1*, Lihui Hou1, Taixiang Wu2, Jianping Liu3, Ernest Hung Yu Ng4,ElisabetStener-Victorin5, Richard S. Legro6, Heping Zhang7
1 Department of Obstetrics and Gynecology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin 150040, China
Chinese Clinical Registry, Chinese Cochrane Centre, Chinese Evidence-Based Medicine Centre, INCLEN CERTC, West China Hospital, Sichuan University, Chengdu 610041, China.
3 Centre for Evidence-Based Chinese Medicine, Beijing University of Chinese Medicine, Bei San Huan Dong Lu 11, Chaoyang District, Beijing 100029, China.
4 Department of Obstetrics and Gynecology, the University of Hong Kong, Queen Mary Hospital, Hong Kong Special Administrative Region, People's Republic of China
5 Institute of Neuroscience and Physiology, Department of Physiology, Sahlgrenska Academy, University of Gothenburg, 405 30 Gothenburg, Sweden
6 Department of Obstetrics and Gynecology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
7 Department of Biostatistics, Yale University School of Medicine, New Haven, Connecticut, USA
Abstract
Acupuncture is an alternative therapy to induce ovulation in women with polycystic ovary syndrome (PCOS)but there is no study reporting the live birth rate following ovulation induction by acupuncture or its potential as an adjuvant treatment to clomiphene citrate (CC). We assess the efficacy of acupuncture with or without CCin achieving live births among 1000 infertile PCOS women in China. This article reports the methodology of an ongoing multi-center randomized controlled trial. The randomization scheme is coordinated through the central mechanism and stratified by the participating site. Participantswill be randomized into one of the four treatment arms: A) true acupuncture and CC, B) control acupuncture and CC, C) true acupuncture and placebo CC, and D) control acupuncture and placebo CC. Acupuncture will be administered twice a week for up to 16 weeks, starting on day 3 after a spontaneous period or a withdrawal bleeding. Either CC or placebo CC 50 mg will be given dailyfrom day 3 to day 7 of the cycle and the dose will be increased in subsequent cycles for non-respondents up to 150 mg/day. To ensure the quality and integrity of the trial we have developed a unique multinational team of investigators and Data and Safety Monitoring Board. Up to the end of April 2013, 326 subjects were recruited. In conclusion, the success of this trial will allow us to evaluate the potential benefit of acupuncture beyond the first line medicine for infertility treatment in PCOS women in an unbiased manner.The trial has been registered at clinicaltrials.gov (NCT01573858) as well as the Chinese clinical trial registration site (http://www.chictr.org/cn/; ChiCTR-TRC-12002081), prior to the start of randomization.
1. Introduction
