Role of CYP51 in the Regulation of T₃ and FSH-Induced Steroidogenesis in Female Mice

Abstract

Cytochrome P450 lanosterol 14α-demethylase (CYP51) is a key enzyme in sterol and steroid biosynthesis that is involved in folliculogenesis and oocyte maturation, which is regulated by follicle-stimulating hormone (FSH), as a key reproductive hormone during follicular development. Thyroid hormone (TH) is also important for normal reproductive function. Although 3,5,3′-triiodothyronine (T₃) enhances FSH-induced preantral follicle growth, whether and how TH combines with FSH to regulate CYP51 expression during the preantral to early antral transition stage is unclear. The objective of this study was to determine the cellular and molecular mechanisms by which T₃ and FSH regulate CYP51 expression and steroid biosynthesis during preantral follicle growth. Our results indicated that CYP51 expression was upregulated in granulosa cells by FSH, and this response was enhanced by T₃. Moreover, knockdown CYP51 decreased cell viability. Meanwhile, gene knockdown also blocked T₃ and FSH-induced estradiol (E₂) and progesterone (P₄) synthesis. These changes were accompanied by upregulation of phospho-GATA-4 content. Results of small interfering RNA analysis showed that knockdown of GATA-4 significantly diminished CYP51 gene expression as well as E₂/P₄ levels. Furthermore, thyroid hormone receptor β was necessary to the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), which was required for the regulation of CYP51 expression; activated GATA-4 was also involved these processes. Our data demonstrate that T₃ and FSH cotreatment potentiates cellular development and steroid biosynthesis via CYP51 upregulation, which is mediated through the activation of the PI3K/Akt pathway. Meanwhile, activated GATA-4 is also involved in this regulatory system. These findings suggest that CYP51 is a mediator of T₃ and FSH-induced follicular development.

Thyroid hormone (TH) is important for normal reproduction function, and dysregulation of TH support is associated with reproductive disorders, including impaired follicular development. Hypothyroidism and hyperthyroidism are associated with dysregulation of the hypothalamic-pituitary axis and suppressed ovarian follicular growth and function (1). Follicle-stimulating hormone (FSH) increases antral follicle development and suppresses atresia by increasing granulosa cell mitotic activity and inhibiting apoptosis (2, 3). Moreover, FSH induces steroidogenesis in granulosa cells in vitro (4, 5). Although follicle development is markedly hampered in hypothyroidism, this ovarian condition can be markedly improved by TH, especially in the presence of gonadotropin (1, 6). In the presence of FSH, TH decreases atresia and significantly increases the numbers of healthy large antral follicles and ovulated oocytes (1, 7). In addition, our preliminary studies indicate that FSH increases preantral follicle growth in vitro, a response markedly enhanced by TH (8). Moreover, the gonadotropic regulation of granulosa cell survival and follicular growth involves the activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway (9, 10). And the interaction of FSH and 3,5,3′-triiodothyronine (T₃) on preantral follicular growth is mediated through activation of the PI3K/Akt pathway.

Cytochrome P450 lanosterol 14α-demethylase (CYP51) is a key enzyme in sterols and steroids biosynthesis, which has the characteristics of a housekeeping gene (11, 12). In the sterol biosynthetic pathway, lanosterol is metabolized by CYP51 to follicular fluid meiosis activating sterol, which is then converted to testis meiosis activating sterol by sterol 14-reductase (13, 14). CYP51 encodes lanosterol 14α-demethylase involved in the conversion of lanosterol into cholesterol. Ovarian CYP51 is expressed in oocytes and granulosa cells of primordial follicle and growing follicle (15). And CYP51 expression in prepubertal rats is low, which is induced by gonadotropin treatment (16). It has been reported that CYP51 is involved in the regulation of primordial folliculogenesis, and CYP51 is important in FSH-induced oocyte meiotic resumption (17). CYP51 small interfering RNA (siRNA) significantly reduces FSH-induced oocyte maturation (18). However, whether and how CYP51 is involved in granulosa cell development and steroidogenesis during preantral to early antral transition stage is not completely understood.

Transcription factors GATA-4 belongs to a six-member family of zinc-finger transcription factors recognizing a consensus GATA motif (A/T-GATAA/G) in the target gene promoters and enhancers. GATA-4 expression is activated in primary follicles along with activation of granulosa cell proliferation. GATA-4 acts also as an effector in the FSH signaling cascade, which is involved in follicle growth and steroid synthesis by regulating the related genes, including FSHR, LHβ-subunit, and CYP19 (19–21). Although GATA-4 plays important roles in the modulation of ovarian function, whether and how GATA-4 is involved in the regulation of CYP51 function remains to be determined.

In this study, we investigated the cellular and molecular mechanisms by which TH and FSH regulate CYP51 expression and steroid biosynthesis during preantral follicle growth. We demonstrated that T₃ and FSH cotreatment increases CYP51 expression, which is associated with increased steroidogenesis by granulosa cells. These responses are mediated by the PI3K/Akt pathway, and activation of GATA-4 is also involved these processes.

Materials and Methods

Reagents and antibodies

Cell culture media (M199) were purchased from Gibco Bethesda Research Laboratories (Grand Island, NY). The fetal bovine serum, penicillin and streptomycin, l-glutamine, sodium pyruvate, and trypsin were purchased from Invitrogen (Burlington, Canada). The enhanced chemiluminescence detection kit was obtained from Amersham Life Science (Oakville, ON, Canada). Acrylamide (electrophoresis grade), N,N′-methylene-bis-acrylamide, ammonium persulfate, glycine, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis prestained molecular weight standards were products of Bio-Rad (Richmond, CA). PI3K inhibitor LY294002 (LY) and Akt inhibitor API-2 were purchased from Selleck (Selleck Chemicals, Houston, TX). The Cell Counting Kit-8 (CCK-8) was used to analyze cell viability, which was purchased from Dojindo (Dojindo, Kumamoto, Japan). The Cell-Light™ EdU Apollo^® 488 In Vitro Imaging Kit was purchased from RiboBio (Guangzhou, China). Lipofectamine 3000 was purchased from Invitrogen (Carlsbad, CA). Rabbit polyclonal anti-GATA-4 antibody (ab84593), rabbit polyclonal antithyroid hormone receptor (TR) β antibody (ab5622), mouse monoclonal anti-PI3 kinase p85 antibody (ab189403), and rabbit polyclonal anti-GAPDH (ab9485) were purchased from Abcam (Cambridge, MA). Rabbit polyclonal antiphospho-Akt antibody (#9271) and rabbit polyclonal anti-Akt antibody (#9272) were from Cell Signaling Technology (Danvers, MA). Goat polyclonal anti-CYP51 (sc-160263), mouse monoclonal antiphospho-GATA-4 antibody (sc-377543), horse radish peroxidase-conjugated antirabbit, and antimouse immunoglobulin G were from Santa Cruz Biotechnology, Inc. (Beijing, China). The above antibodies were used to detect protein level by Western blot or immunofluorescence analysis. RevertAid First Strand cDNA Synthesis Kit and TRIzol Reagent were obtained from Thermo Fisher Scientific (Waltham, MA), Inc. SYBR Green polymerase chain reaction (PCR) kit was purchased from Bio-Rad. PCR primers for CYP51 and 18S ribosomal RNA (rRNA) were from Beijing Sunbiotech Co., Ltd. (Beijing, China). The other chemicals and culture media components used in the current study were purchased from Sigma-Aldrich Corp. (Santa Clara, CA) unless otherwise specified.

Animal preparation

All animal experiments were approved by the Institutional Animal Care and Use Committee of Capital Normal University and were conducted in accordance with the Principles of the Care and Use of Laboratory Animals and China Council on Animal Care. Immature 12- to 14-day-old Kunming White female mice (outbreed strain) were purchased from the Beijing Vital Laboratory Animal Technology Co. (Beijing, China). Mice were maintained under constant conditions of temperature (24–26°C) and humidity (60% ± 2%) with a 12/12-hour light/dark cycle and received pathogen-free water and food for maintenance except special requirement. Mice were injected subcutaneously with diethylstilbestrol (1 mg/d; 3 days), and ovaries were collected by cervical dislocation at 72 hours after euthanization.

Primary culture of mouse granulosa cells

Granulosa cells were released by follicular puncture with a 26.5-gauge needle, washed, and centrifuged (900g, 5 minutes). The oocytes and cell clumps were removed by filtering the cell suspensions through a nylon cell strainer (40 μm; Becton, Dickinson, and Co., no.352340). Cell number and viability were estimated by Trypan blue dye-exclusion test. Granulosa cells (9 × 10⁵ per well in a six-well plate) were plated with 2 mL of M199 medium [supplemented with HEPES (10 mM), streptomycin (100 μg/mL), penicillin (100 U/mL), and fungizone (0.625 μg/mL)] containing fetal bovine serum (10%, w/v) under a humidified atmosphere of 95% air and 5% CO₂. The media were then replaced with serum free M199 supplemented as above for 12 hours thereafter, and cells were treated with FSH (100 ng/mL) with or without T₃ (1.0 nM). In some experiments, cells were pretreated with TR antagonists MLS000389544 (10⁻⁵ mol/L), PI3K inhibitor LY (10 μM), or Akt inhibitor API-2 (10 μM) 30 minutes or 1 hour before FSH with or without T₃ treatment, respectively.

RNA interference

The siRNA was transfected into granulosa cells according to the instruction of the manufacturer’s protocol. Granulosa cells were transfected (48 hours) with GATA-4, CYP51, or TRβ siRNA (GenePharma) and scrambled sequence control (GenePharma), using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.

Protein extraction and Western blotting

Western blot analysis was performed as described previously (22). Whole-cell lysates were prepared by incubating cell pellets in lysis buffer [30 mM NaCl, 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.4) containing a cocktail of protease and phosphatase inhibitor (Sigma-Aldrich, Corp.)] for 30 minutes at 0°C. After insoluble fractions were removed by centrifugation (15,000g, 4°C, 30 minutes), supernatant was collected and protein concentration was determined with the BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China), following the manufacturer’s instructions. Thirty micrograms (depending on individual experiments) of cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 4.5% stacking and 10% separating gels. Proteins were electrophoretically transferred to nitrocellulose membrane. The membranes were then blocked in tris buffered saline with Tween (20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20; pH 7.5) buffer containing 5% dehydrated nonfat milk at room temperature for 1 hour and subsequently incubated (4°C, overnight) with diluted primary antibody [polyclonal anti-CYP51 (1:1000), polyclonal anti-GATA-4 (1:1000), polyclonal antiphospho-GATA-4 (1:500), polyclonal anti-Akt (1:1000), polyclonal antiphospho-Akt (Ser473; 1:1000), or GAPDH (1:10,000)], followed by horse radish peroxidase-conjugated secondary antibody (1:1000–1:10,000; 1.5 hours, room temperature). Peroxidase activity was visualized with the enhanced chemiluminescence kit according to the manufacturer’s instructions. Protein content was determined by densitometrically scanning the exposed x-ray film. Immunoreactions signals were analyzed using Gel-pro Analyzer 4.0.

Real-time quantitative PCR analysis

The 0.2 μg total RNA was used to synthesize the complementary DNA, which was extracted with TRIzol Reagent (Invitrogen). The messenger RNA (mRNA) abundance of target genes was analyzed by real-time PCR and normalized to 18S rRNA. Specific primer pairs used in the experiments are listed in Table 1. Data were analyzed by the 2^−ΔΔCT method (23).

Table 1.

Primer Sequence Used for Real-Time Quantitative PCR

Target Gene	GenBank Accession No.	Primer Sequence	Product Size, bp	Annealing Temperature, °C
CYP51	NM_020010.2	Forward: 5′-TGGAGCGAAAAGTCCACCAC-3′	172	60
		Reverse: 5-TGCATCACTCCCCAGAAGGTA-3′
GATA-4	NM_008092.4	Forward: 5′-CCCTACCCAGCCTACATGG-3′	139	60
		Reverse: 5′-ACATATCGAGATTGGGGTGTCT-3′
18S rRNA	NM_008084.3	Forward: 5′-TGGCCTTCCGTGTTCCTAC-3′	178	60
		Reverse: 5′-GAGTTGCTGTTGAAGTCGCA-3′

Hormone level analysis

Spent medium of cultured granulosa cells were collected and centrifuged (900g, 5 minutes) for progesterone (P₄) and estradiol (E₂) analysis by using commercial radioimmunoassay kits (Research Institute of North Beijing, China). The sensitivity of P₄ and E₂ were <0. 2 ng/mL and <5 pg/mL, respectively. The intra-assay coefficients of variation were <10% for hormones (24).

Analysis of cell viability

The CCK-8 (Dojindo, Kumamoto, Japan) was use to measure cell viability (25). Briefly, CCK-8 solution (10 µL) was added to each well after hormone incubation, and the cultures were incubated for an additional 2 hours at 37°C. The optical density values were recorded using a microplate reader at 450 nm. The mean optical density values for each treatment were used as the index of cell viability.

5-Ethynyl-2’-deoxyuridine incorporation assay

5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay was conducted according to the manufacturer’s protocol (RiboBio) (26). Briefly, granulosa cells were cultured as before, and then were incubated with 50 μM EdU for 2 hours. After the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes and permeated in 0.5% Triton X-100 for 10 minutes, they underwent EdU staining. Hoechst 33342 was counterstained for 30 minutes to detect cell nuclei. EdU-positive nuclei were determined under a Laser Scanning Microscope LSM 780 (ZEISS, Jena, Germany). The cell proliferation rate was calculated as the proportion of nucleated cells incorporating EdU in five high-power fields per well.

Immunofluorescence cell staining

Granulosa cells were cultured in poly-d-lysine (0.05% w/v; Sigma-Aldrich Corp.) coated eight-well glass culture slides (Becton, Dickinson, and Co.) with or without FSH and T₃ for 30 minutes. After the stimulation, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 minutes, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes, and then blocked with 5% bovine serum albumin in PBS for 30 minutes. And then, cells were incubated with monoclonal anti-P85 (1:100 dilution in blocking solution) or polyclonal anti-TRβ (1:100 dilution in blocking solution), respectively, at 4°C for overnight. Cells were washed with PBS three times and incubated with Alexa Fluor 488-conjugated secondary antibody (1:400 dilution in blocking solution; Jackson ImmunoResearch) for 1.5 hours at room temperature. Cells were washed with PBS three times and incubated with 4′,6-diamidino-2-phenylindole for 10 minutes at room temperature. After washing with PBS, the coverslips were mounted on object slides using fluorescent mounting medium. Immunofluorescence was visualized using an immunofluorescence microscope (Olympus BX51), and images were recorded by using Laser Scanning Microscope LSM 780 (ZEISS). To minimize observe bias, the observers did not know the treatment of the cells they were scoring.

Statistical analysis

Results are presented as mean ± standard error of the mean (SEM) of at least three independent experiments, as detailed in the figure legends. All data were analyzed using GraphPad Prism 5.0 statistical software (GraphPad Software, Inc., San Diego, CA). The statistical differences between treatments were subjected to two- or three-way (repeated-measure) analysis of variance. When significant differences were found, means were compared by the Bonferroni posttest. P values < 0.05 were considered to be statistically significant.

Results

T₃ increased FSH-induced steroidogenesis in granulosa cells and cellular development

Our previous report showed that T₃ enhances FSH-induced preantral follicle growth and granulosa cell development in vitro (25, 27, 28). To confirm whether T₃ regulates FSH-induced steroidogenesis, granulosa cells were cultured with or without T₃. As observed in Fig. 1, FSH marked stimulated E₂ secretion in granulosa cells, a response significantly increased by exogenous T₃ [18.94 ± 1.48 (FSH + T₃) vs 11.04 ± 1.09 (FSH), P < 0.01; Fig. 1A]. Similarly, T₃ also increased FSH-induced P₄ secretion (10.69 ± 0.85 (FSH + T₃) vs 5.44 ± 0.69 (FSH), P < 0.01; Fig. 1B]. To examine whether hormones affected granulosa cell growth, we investigated cellular development after hormone treatment. As shown in Fig. 1C and 1D, T₃ enhanced FSH-induced cellular development [cell viability: 1.99 ± 0.06 (FSH + T₃) vs 1.45 ± 0.05 (FSH), P < 0.01; EdU: 28.62 ± 1.35 (FSH + T₃) vs 14.14 ± 1.29 (FSH), P < 0.01; Fig. 1C and 1D].

Figure 1.

Effect of T₃ and/or FSH on steroidogenesis in granulosa cells in vitro. Immature mice were treated with diethylstilbestrol (1 mg/d, 3 consecutive days) prior to granulosa cell isolation from preantral and early antral follicles. The cells were cultured for up to 24 hours in the absence or presence of FSH (100 ng/mL) and/or T₃ (1.0 nM). Testosterone (0.5 μM) was added to serve as substrate of aromatase during culture. (A) E₂ and (B) P₄ secreted into the medium were measured by enzyme-linked immunosorbent assay. (C and D) The cellular growth was analyzed by CCK-8 assay and EdU measurement, respectively. Data are presented as mean ± SEM of four independent experiments. *P < 0.05; **P < 0.01 compared with control (CTL); ⁺⁺P < 0.01 compared with FSH alone. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Effect of T₃ and FSH on CYP51 expression

It has been reported that CYP51 is expressed in ovarian cells and plays important roles in ovarian cell growth (15). To determine whether its expression is dependent on the duration time of FSH, granulosa cells were cultured with FSH, and protein content of CYP51 was analyzed by Western blot. As shown in Fig. 2A, FSH induced increase in CYP51 protein expression at 6 hours, and expression peaked at 24 hours [0.84 ± 0.04 (FSH) vs 0.41 ± 0.01 (control), P < 0.05]. To evaluate the effects of T₃ on FSH-induced CYP51 expression, T₃ was cocultured with FSH for 24 hours. Although T₃ alone was ineffective, a further and significant increase in CYP51 content was detected in the presence of both FSH and T₃ compared with the FSH alone group [1.20 ± 0.14 (FSH + T₃) vs 0.66 ± 0.05 (FSH), P < 0.01; Fig. 2B]. Two-way analysis of variance indicates a significant interaction between T₃ and FSH (P < 0.05). Moreover, CYP51 mRNA abundance is also significantly higher in FSH and T₃ cotreatment than that in the FSH alone group [3.79 ± 0.18 (FSH + T₃) vs 2.52 ± 0.32 (FSH), P < 0.01; Fig. 2C]. CYP51 protein content increased along with the upregulation of CYP51 mRNA abundance, suggesting that increased gene transcription may at least partially account for the increased protein content induced by hormonal cotreatment.

Figure 2.

Effect of T₃ and/or FSH on CYP51 expression in granulosa cell. (A) Granulosa cells were cultured with FSH for different duration, and CYP51 expression was assessed by Western blot analysis. (B) Granulosa cells were cultured with FSH and/or T₃ for 24 hours, and CYP51 protein was assessed by Western blot analysis. (C) Cells were harvested at 24 hours for mRNA analysis by real-time PCR. Data are presented as mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01 compared with control; ⁺⁺P < 0.01 compared with FSH alone.

CYP51 knockdown attenuate hormone-induced steroidogenesis and cell development

To investigate the function of CYP51 in steroidogenesis and granulosa cell development, CYP51 gene was knockdowned by siRNA. As shown in Fig. 3A, knockdown of CYP51 significantly decreased FSH-induced E₂ levels [9.77 ± 1.12 (FSH) vs 3.92 ± 0.20 (FSH + siRNA), P < 0.05], which was further downregulated in the presence of T₃ [18.94 ± 1.48 (FSH + T₃) vs 5.87 ± 0.90 (FSH + T₃ + siRNA), P < 0.001]. Moreover, gene knockdown also reduced P₄ concentration [6.06 ± 0.94 (FSH) vs 2.38 ± 0.48 (FSH + siRNA), P < 0.05; 10.69 ± 0.85 (FSH + T₃) vs 2.85 ± 0.26 (FSH + T₃ + siRNA), P < 0.01; Fig. 3B]. Meanwhile, we also assessed the regulatory role of CYP51 in cell development. As shown in Fig. 3C and 3D, CYP51 downregulation by siRNA significantly decreased cell viability [1.33 ± 0.03 (FSH) vs 0.86 ± 0.14 (FSH + siRNA), P < 0.05; 1.75 ± 0.08 (FSH + T₃) vs 0.97 ± 0.09 (FSH + T₃ + siRNA), P < 0.01; Fig. 3C] and proliferation [14.80 ± 2.53 (FSH) vs 3.91 ± 0.93 (FSH + siRNA), P < 0.01; 28.28 ± 1.68 (FSH + T₃) vs 5.38 ± 1.92 (FSH + T₃ + siRNA), P < 0.001; Fig. 3D]. However, these responses induced by CYP51 knockdown were reversed by the supplementation with E₂ (P > 0.05; Fig. 4A) or P₄ (P > 0.05; Fig. 4B), respectively. Moreover, we further investigated whether CYP51 regulated CYP19A1 expression; the latter converts T to E₂. The results showed that siRNA of CYP51 had no significant effect on CYP19A1 expression (Supplemental Fig. 1).

Figure 3.

Role of CYP51 in hormones-induced steroidogenesis and cellular development. Granulosa cells were transfected with CYP51 siRNA [scrambled sequence as control (SC)] for 12 hours using Lipofectamine 3000, and then treated with T₃ in the presence of FSH for another 24 hours. (A and B) E₂ and P₄ concentrations in culture medium were measured by enzyme-linked immunosorbent assay. (C and D) Granulosa cells were treated as previously described, and cell viability and proliferation were analyzed by CCK-8 assay and EdU measurement, respectively. Data are presented as mean ± SEM of four independent experiments. *P < 0.05; **P < 0.01 compared with FSH + SC; ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 compared with FSH + T₃ + SC.

Figure 4.

E₂/P₄ reversed CYP51 knockdown-inhibited cellular development. After CYP51 siRNA transfection, cells were treated with FSH/T₃ with or without E₂/P₄ for another 24 hours. (A and B) The cellular growth was analyzed by CCK-8 assay and EdU measurement, respectively. *P < 0.05; **P < 0.01 compared with FSH + scrambled sequence as control (SC); ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 compared with FSH + T₃ + SC.

Phospho-GATA-4 mediated hormone-induced genes expression, steroidogenesis, and cell development

To determine whether T₃/FSH activate granulosa cells GATA-4 in vitro, granulosa cells were incubated with FSH and/or T₃ for 12 hours and phospho-GATA-4 content was determined. We found that phospho-GATA-4 content was significantly increased in granulosa cells incubated with FSH, a response significantly enhanced by T₃ [1.39 ± 0.22 (FSH + T₃) vs 0.62 ± 0.07 (FSH), P < 0.01; Fig. 5B], although T₃ alone was ineffective (P > 0.05; Fig. 5B). To test the hypothesis that phospho-GATA-4 is involved the regulation of cell CYP51 expression, and consequently promoting cell survival, granulosa cells were transfected with GATA-4 siRNA, and then treated with FSH and/or T₃ for 24 hours (Fig. 5C and 5D). Although CYP51 protein content was upregulated by the presence of FSH and T₃, this response was significantly attenuated by GATA-4 knockdown [1.68 ± 0.22 (FSH) vs 1.01 ± 0.09 (FSH + siRNA), P < 0.05; 2.40 ± 0.21 (FSH + T₃) vs 1.09 ± 0.09 (FSH + T₃ + siRNA), P < 0.01; Fig. 5C]. Moreover, FSH and T₃-induced steroidogenesis were also abolished by gene knockdown (P < 0.05; P < 0.01; P < 0.001; Fig. 6A and 6B). GATA-4 siRNA also abrogated the positive effect of FSH+T₃ (P < 0.05; P < 0.01; P < 0.001; Fig. 6C and 6D).

Figure 5.

Role of GATA-4 in hormones-induced CYP51 expression. (A) Granulosa cells were treated with FSH at different time points. The phospho-GATA-4 and GATA-4 content were detected by Western blot analysis. (B) Granulosa cells were cultured in the presence or absence of FSH and/or T₃ for 12 hours. The protein levels of p-GATA-4 and GATA-4 were quantified. (C and D) Granulosa cells were transfected with GATA-4 siRNA, and then treated with FSH and/or T₃ for 24 hours. CYP51 protein and mRNA content were detected by Western blot and real-time PCR analysis, respectively. (A and B) *P < 0.05 compared with control (CTL); ⁺⁺P < 0.01 compared with FSH alone; (C and D) *P < 0.05 compared with FSH + scrambled sequence as control (SC); ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 compared with FSH + T₃ + SC. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 6.

Role of GATA-4 in hormones-induced steroidogenesis and cellular development. (A and B) The cells were transfected with GATA-4 siRNA, and E₂ and P₄ concentrations in culture medium were measured by enzyme-linked immunosorbent assay. (C and D) GATA-4 was knockdown, and cell development were analyzed by CCK-8 assay and EdU measurement, respectively. Data represent the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01 compared with FSH + scrambled sequence (SC) as control; ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 compared with FSH + T₃ + SC.

TRβ involved the activation of PI3K/Akt pathway

Akt activation is essential for granulosa cell development. Our previous report showed that PI3K/Akt pathway mediated FSH and T₃-induced granulosa cell development (28). Consistent with the previous results, FSH significantly increased phospho-Akt content, which was dramatically enhanced by T₃ [1.29 ± 0.04 (FSH + T₃) vs 0.90 ± 0.02 (FSH), P < 0.01; Fig. 7A] in the current study. In addition, hormone-induced phosphorylation of Akt was blocked by MLS000389544 (Fig. 7B, P < 0.01), the antagonists of TRs and gene knockdown (Supplemental Figs. 2 and 3). To further confirm whether TRβ shuttled between the nucleus and the cytoplasm, immunofluorescence was used to detect the location of TRβ (Supplemental Figs. 4 and 5). The results showed that there was a dramatic shift of receptor distribution to the cytoplasm after the cells were treated with FSH and T₃, which was colocalized with p85 (Fig. 7C).

Figure 7.

TRβ involved FSH/T₃-induced p-Akt. (A) Granulosa cells were treated with FSH for 30 minutes, and then protein content of p-Akt was assessed by Western blot analysis. (B) Cells were pretreated for 30 minutes with MLS000389544 (10⁻⁵ mol/L), and the p-Akt level was detected after hormone treatment. (C) The combination of FSH and T₃ induced the shift of receptor distribution to the cytoplasm, which was colocalized with p85. *P < 0.05 compared with FSH alone; ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 compared with FSH + T₃. Bar, 10 µm. CTL, control; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

PI3K/Akt pathway positively regulates genes expression, steroidogenesis, and cell development

To determine whether PI3K/Akt signaling is necessary for GATA-4-mediated CYP51 expression, we applied LY or API-2 1 hour before FSH and T₃ treatment. Western blot analysis carried out after 12 hours revealed that the level of p-GATA-4 was significantly lower than in the group untreated with inhibitors [0.44 ± 0.07 (FSH) vs 0.23 ± 0.03 (FSH + LY), 0.22 ± 0.02 (FSH + API-2), P < 0.01; 1.28 ± 0.15 (FSH + T₃) vs 0.25 ± 0.01 (FSH + T₃ + LY), 0.24 ± 0.02 (FSH + T₃ + API-2), P < 0.001; Fig. 8A]. We also detected the expression of CYP51 after hormone treatment of 24 hours. We found the same effects were also showed on CYP51 expression [0.43 ± 0.05 (FSH) vs 0.23 ± 0.01 (FSH + LY), 0.23 ± 0.01 (FSH + API-2), P < 0.01; 0.89 ± 0.07 (FSH + T₃) vs 0.26 ± 0.04 (FSH + T₃ + LY), 0.28 ± 0.06 (FSH + T₃ + API-2), P < 0.001; Fig. 8B]. Furthermore, we found that inhibitors significantly decreased steroidogenesis levels [E₂: 10.71 ± 1.69 (FSH) vs 4.48 ± 0.75 (FSH + LY), 3.17 ± 0.49 (FSH + API-2), P < 0.01; 18.29 ± 2.65 (FSH + T₃) vs 6.96 ± 1.28 (FSH + T₃ + LY), 4.97 ± 0.75 (FSH + T₃ + API-2), P < 0.01, P < 0.001; Fig. 9A; P₄: 6.35 ± 0.71 (FSH) vs 3.64 ± 0.35 (FSH + LY), 1.85 ± 0.25 (FSH + API-2), P < 0.05, P < 0.01; 11.02 ± 1.09 (FSH + T₃) vs 6.09 ± 1.46 (FSH + T₃ + LY), 4.18 ± 1.05 (FSH + T₃ + API-2), P < 0.05, P < 0.01; Fig. 9B] and cellular development [cell viability: 1.43 ± 0.04 (FSH) vs 0.96 ± 0.09 (FSH + LY), 0.93 ± 0.13 (FSH + API-2), P < 0.05; 1.82 ± 0.08 (FSH + T₃) vs 1.05 ± 0.04 (FSH + T₃ + LY), 1.05 ± 0.11 (FSH + T₃ + API-2), P < 0.01; Fig. 9C; EdU: 16.14 ± 2.07 (FSH) vs 2.71 ± 0.71 (FSH + LY), 2.29 ± 0.42 (FSH + API-2), P < 0.001; 29.95 ± 2.79 (FSH + T₃) vs 2.42 ± 0.43 (FSH + T₃ + LY), 3.20 ± 0.51 (FSH + T₃ + API-2), P < 0.001; Fig. 9D].

Figure 8.

Effect of PI3K/Akt pathway on FSH/T₃-induced p-GATA-4 and CYP51 expression. Granulosa cells were cultured as previously described and pretreated for 1 hour with inhibitors LY294002 (LY; 10 µM) or API-2 (10 µM). (A) Cells were then treated with T₃ and FSH for 12 hours prior to analysis of GATA-4 by Western blot analysis. (B and C) Cells were pretreated with inhibitors and then cultured with FSH and T₃ for 24 hours. CYP51 protein levels and mRNA content were detected, respectively. **P < 0.01 compared with FSH alone; ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 compared with FSH + T₃. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 9.

Effect of PI3K/Akt pathway on hormone-induced steroidogenesis and cell development. Granulosa cells were cocultured with inhibitors LY294002 (LY; 10 µM) or API-2 (10 µM) for 1 hour before hormone treatment. (A) E₂ and (B) P₄ secreted into the medium were measured by enzyme-linked immunosorbent assay. (C) Cell viability and (D) proliferation were also detected. *P < 0.05; **P < 0.01, ***P < 0.01 compared with FSH alone; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 compared with FSH + T₃.

Discussion

In the current study, we have demonstrated that CYP51 is a positive regulator of FSH and T₃-induced granulosa cell growth. Our studies demonstrate that T₃ combined with FSH increased CYP51 expression at both transcriptional and translational levels, which induced steroidogenesis in mice granulosa cells. These responses are mediated by phospho-GATA-4, which are activated by the PI3K/Akt signaling pathway, and TRβ was also involved these regulations. This study proves that CYP51 has important roles in T₃/FSH-induced steroidogenesis and development in granulosa cells at the preantral to early antral transition stage.

It is well known that CYP51 is a critical enzyme, which converts lanosterol to cholesterol. The latter is the substrate of steroid biosynthesis. The ovarian cells could synthesize three major sex steroids, including E₂, testosterone (T), and P₄. E₂ is one of the most important ovarian hormones and mainly produced in granulosa cells, which is converted to E₂ from T by aromatase (29). And granulosa cells are considered as the main resource for P₄, whereas T is synthesized by theca cells. The steroidogenic activity of all types of cells differs at different stages of follicle development. Previous research characterized that CYP51 is expressed in oocytes and granulosa cells of primordial follicle and growing follicle (15). Ovarian CYP51 expression in prepubertal rats is low and expressed dominantly in follicles, which is induced by gonadotropin treatment (16, 30). Several lines of evidence have shown that CYP51 is important to FSH-induced oocyte maturation (17, 18, 31). These facts suggest that ovarian CYP51 is regulated by pituitary gonadotropins and that CYP51 may have a follicular stage-specific function. However, the function of CYP51 during preantral and early antral follicular development is unclear. In the current study, T₃ enhanced FSH-induced CYP51 expression in granulosa cells. Our results also showed that T₃ and FSH significantly stimulated E₂ production, and an increasing tendency was observed in P₄ secretion in association with the increase of CYP51 gene expression. The elevated content of CYP51 could promote steroid hormone production in granulosa cells because cholesterol is thought to be the precursor for steroid biosynthesis. Furthermore, the observations of the presence of CYP51 siRNA had shown that gene knockdown attenuated hormone-induced steroid biosynthesis. Meanwhile, CYP51 knockdown also increased granulosa cell apoptosis and decreased cell growth. The supplementation with E₂ or P₄ rescued cell viability and proliferation after CYP51 knockdown. However, CYP51 knockdown failed to change the expression of aromatase enzyme (CYP19A1), and the latter converts T to E₂, which indicate that CYP51 mainly affects the synthesis of cholesterol. These results suggest that CYP51 might play an important role in the follicular development of early stage.

It has been reported that GATA-4 is a member of the GATA family of zinc finger transcription factors. Accumulating evidence suggests that GATA-4 is linked to reproductive development or function in mammals (32, 33). Promoter regions capable of GATA-4 binding are required for activation of StAR transcription in FSH-treated granulosa cells (34, 35). Moreover, GATA-4 is also involved in the regulation of genes related to follicle growth and steroid synthesis, including FSHR, LHβ-subunit, and CYP19 (19–21). Our results are consistent with the earlier studies that FSH increased phosphorylated GATA-4 protein (20). We now present data that the ability of T₃ to augment FSH-stimulated activation of GATA-4 was accompanied by T₃-enhancement of FSH-stimulated CYP51 upregulation. Moreover, GATA-4 knockdown by siRNA could inhibit CYP51 expression. The results provided evidence that GATA-4 directly affected CYP51 expression. It is not surprising that E₂ and P₄ concentrations in culture medium were also reduced by siRNA. Follicular atresia via apoptosis is associated with decreased expression of GATA-4 (36), which is consistent with the current data that gene knockdown induced cell apoptosis.

These results suggest that CYP51 is involved in T₃ and FSH-induced steroid hormone synthesis and cell development, which is mediated via activated GATA-4. However, the activated-GATA-4 by T₃ and FSH were attenuated by LY and API-2, respectively. These indicate that activation of GATA-4 by hormones is mediated by PI3K/Akt pathway. The gonadotropic regulation of granulosa cell survival and follicular growth involves the activation of the PI3K/Akt pathway (9, 10). Although TH regulates gene expression by binding to its specific nuclear TRs, several rapid effects of TH are mediated by other nongenomic pathways. Our previous results have demonstrated that the action of FSH and T₃ on preantral follicular growth is mediated via activation of the PI3K/Akt pathway (27). In the current study, ovarian cell growth observed in the presence of both TH and FSH is a consequence of PI3K/Akt-mediated CYP51 expression. We also show the association between the p85 regulatory subunit of PI3K and TRβ, the latter shuttled between the nuclear and the cytoplasm after hormone treatment, which indicates a nongenomic action of T₃ on granulosa cell survival (37). Although T₃ combined with FSH to induce CYP51 expression, steroid hormone synthesis, and cellular development via the PI3K/Akt/GATA-4 signal pathway, the precise mechanisms of CYP51 on regulating granulosa cell growth still remain to be determined.

It is well known that gonadotropins are important for follicle development. However, TH has been highlighted as possible paracrine factors involved in ovarian function. Dysregulation of the hypothalamic-pituitary axis is associated with reproductive disorders, including impaired follicular development (1, 6, 24), and reproductive hormones play very important roles in the follicular development. It has been reported that TSH, FSH, and luteinizing hormone are glycoprotein pituitary hormones, which share the same glycoprotein α chain (CGA) as part of their protein structure (38, 39). It is possible that TH disorder impairs the synthesis and secretion of FSH and luteinizing hormone by regulating CGA expression (39). The in vitro findings in the current study may partially explain the in vivo observations showing that ovarian follicles of TH dysregulation in animals failed to mature.

In conclusion, our findings demonstrate that CYP51 is a positive regulator on T₃ and FSH-induced granulosa cell development. As shown in the hypothetical model (Fig. 10), T₃ enhanced FSH-induced CYP51 expression, which contributes to steroidogenesis and cell growth. TRβ-dependent activation of the PI3K/Akt signal pathway is regulated by T₃/FSH. Meanwhile, phospho-GATA-4 is also involved in these processes. This study substantially improves our understanding of the role of CYP51 in ovarian cells.

Figure 10.

Schematic diagram of the role of FSH and T₃ on CYP51 expression.

Appendix. Antibody Table

Peptide/Protein Target	Antigen Sequence	Name of Antibody	Manufacturer, Catalog No.	Species Raised in; Monoclonal or Polyclonal	Dilution Used	RRID
p-GATA4		Anti-p-GATA-4 antibody	Santa Cruz Biotechnology, sc-377543	Mouse; monoclonal	1:500	AB_2651181
GATA-4	Synthetic peptide conjugated to KLH derived from within residues 100–200 of human GATA4	Anti-GATA-4 antibody	Abcam, ab84593	Rabbit; polyclonal	1:1000	AB_10670538
CYP51A1		Anti-CYP51A1 antibody	Santa Cruz Biotechnology, sc-160263	Goat; polyclonal	1:1000	AB_2651182
CYP19A1		Anti-CYP19A1 antibody	Santa Cruz Biotechnology, sc-14245	Goat; polyclonal	1:1000	AB_2088684
GAPDH		Anti-GAPDH antibody	Abcam, ab9485	Rabbit; polyclonal	1:10,000	AB_2651183
Akt/phospho-Akt		Akt/phospho-Akt (Ser473) antibody	Cell Signaling, 9272/9271	Rabbit; polyclonal	1:1000	AB_329827/AB_329825
TRβ	Synthetic peptide corresponding to human TRβ aa 62–81. With an N-terminal added cysteine	Anti-TRβ antibody- chromatin immunoprecipitation grade	Abcam, ab5622	Rabbit; polyclonal	1:100	AB_304991
p85	Recombinant fragment corresponding to human PI3K p85 aa 159–388.expressed in Escherichia coli sequence	Anti-PI3K p85 antibody	Abcam, ab189403	Mouse; monoclonal	1:100	AB_2651184

Abbreviation: RRID, Research Resource Identifier.

Abbreviations:

 
• Akt

  protein kinase B

 
• CCK-8

  Cell Counting Kit-8

 
• CYP51

  cytochrome P450 lanosterol 14α-demethylase

 
• E₂

  estradiol

 
• EdU

  5-ethynyl-2’-deoxyuridine

 
• FSH

  follicle-stimulating hormone

 
• LY

  LY294002

 
• mRNA

  messenger RNA

 
• P₄

  progesterone

 
• PBS

  phosphate-buffered saline

 
• PCR

  polymerase chain reaction

 
• PI3K

  phosphoinositide 3-kinase

 
• rRNA

  ribosomal RNA

 
• SEM

  standard error of the mean

 
• siRNA

  small interfering RNA

 
• T

  testosterone

 
• T₃

  3,5,3′-triiodothyronine

 
• TH

  thyroid hormone

 
• TR

  thyroid hormone receptor.

Acknowledgments

Financial Support: This work was supported by National Natural Science Foundation of China Grants 31671555 and 31300958 and Beijing Natural Science Foundation Grant 5142003. This project was also supported by Scientific Research Program of Beijing Municipal Commission of Education Grant KM201610028011. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Disclosure Summary: The authors have nothing to disclose.