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Growth differential factor-9 inhibits testosterone production in mouse theca interstitial cells
Growth differential factor-9 inhibits testosterone production in mouse theca interstitial cells Ming-hui Chen, M.D., Tao Li, Ph.D., Chen-hui Ding, Ph.D., Yan-wen Xu, Ph.D., Lu-yan Guo, M.Sc., and Can-quan Zhou, M.D. Reproductive Medicine Center, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, People's Republic of China
Objective: To explore the effect of growth differential factor-9 (GDF-9) alone on cell proliferation, cell viability, steroidogenesis, and hormone-stimulated gene expression in cultured mouse theca interstitial cells. Design: Basic research. Setting: University hospital. Animal(s): Immature 3- to 4-week-old SPF KM mice obtained from the Laboratory Animal Center of Sun Yat-Sen University. Intervention(s): Addition of GDF-9 at different dosages to primary culture of mouse theca interstitial cells. Main Outcome Measure(s): Cell number, cell viability, progesterone and testosterone levels, and hormone-stimulated gene mRNA abundance. Result(s): Growth differential factor-9 mildly increased the number of mouse theca interstitial cells and cell viability in a dosedependent manner and mildly inhibited the production of progesterone in mouse theca interstitial cells. Administration of GDF-9 at the dosages of 200 ng/mL and 400 ng/mL resulted in a significant decrease in the testosterone level compared with the control group by 60.42% and 68.76%, respectively. Growth differential factor-9 significantly suppressed Lhcgr mRNA by 47.36%, Cyp11a1 mRNA by 62.30%, and Cyp17a1 mRNA by 55.39%, but had only a mild effect on Star gene expression. Conclusion(s): Growth differential factor-9 can inhibit the production of testosterone in mouse Use your smartphone theca interstitial cells and suppress the corresponding gene expression. (Fertil SterilÒ 2013;100: to scan this QR code 1444–50. Ó2013 by American Society for Reproductive Medicine.) and connect to the Key Words: Gene expression, growth differential factor-9, hormone, theca Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/chenm-growth-differential-factor-9-theca/
P
olycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders, but its etiology and pathogenesis are still unclear. Previous studies have demonstrated that the growth and development of PCOS follicles stop at the diameter range of 4 to 7 mm, resulting in the accumulation of a large number of small antral follicles and thereby
the generation of the polycystic ovary phenotype (1, 2). Recent studies on the role of oocytes in folliculogenesis have revealed that the oocytes can secrete growth factors and directly induce the development of follicles (3–5). One of these oocyte-derived factors, namely, growth differentiation factor-9 (GDF-9), has a critical impact on folliculogenesis (6, 7).
Received April 21, 2013; revised June 23, 2013; accepted July 10, 2013; published online August 15, 2013. M.-h.C. has nothing to disclose. T.L. has nothing to disclose. C.-h.D. has nothing to disclose. Y.-w.X. has nothing to disclose. L.-y.G. has nothing to disclose. C.-q.Z. has nothing to disclose. Supported by grants from the Key Laboratory of Reproductive Medicine of Guangdong Province and the National Basic Research Program of China (973 Program, 2012CB947600), People's Republic of China. Reprint requests: Can-quan Zhou, M.D., Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-Sen University, 58 2nd Zhongshan Road, Guangzhou, GD510080, People's Republic of China (E-mail: [email protected]). Fertility and Sterility® Vol. 100, No. 5, November 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2013.07.200 1444
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Accumulating evidence has shown that an adequate amount of GDF-9 is required for the growth of follicles; female GDF-9 knockout mice are infertile with a complete block in folliculogenesis at the primary stage (8). The dysregulation of oocyte GDF-9 expression may contribute to aberrant folliculogenesis in PCOS because GDF-9 expression is decreased in PCOS ovaries when compared with normal controls in oocytes at all developmental stages from the transition to Graafian follicles (9). Another line of evidence indicating that GDF-9 is potentially associated with PCOS comes from a genomic study that identified five novel missense mutations of GDF-9 in PCOS patients (10). Excessive production of ovarian androgen by theca interstitial cells is a VOL. 100 NO. 5 / NOVEMBER 2013
Fertility and Sterility® principal feature of PCOS. There is evidence to indicate that GDF-9 plays an important regulatory role in theca recruitment, differentiation, and proliferation during early folliculogenesis: follicles from GDF-9 null mice and sheep lack supporting theca cells (8, 11–13). In vitro studies have shown that GDF-9 can inhibit the production of progesterone and stimulate the proliferation of bovine and human theca cells (14, 15). However, the effect of GDF-9 on androgen production in theca cells is controversial. Growth differential factor-9 has been found to enhance both basal and forskolinstimulated androstenedione accumulation in primary and immortalized rat theca interstitial cells (16), but that study did not exclude the interference of theca cell proliferation on androgen accumulation in the culture medium. Excluding the interference factor of theca cell proliferation, Spicer et al. (15) found that recombinant rat GDF-9 could inhibit the androgen production induced by insulin-like growth factor I (IGF-I) and luteinizing hormone (LH) and decrease the expression of LH receptor and CYP11A1 in bovine theca cells. Growth differential factor-9 was first discovered from mouse genomic DNA via polymerase chain reaction (PCR) by use of degenerate oligonucleotides corresponding to conserved regions of known transforming growth factor b (TGF-b) family members (17). In addition, although mice are common animal models for PCOS research, the effect of GDF-9 on mouse theca interstitial cells has not been studied. We explored the effect of GDF-9 alone on cell proliferation, cell viability, steroidogenesis, and hormone-stimulated gene expression in cultured mouse theca interstitial cells.
Percoll dissolved in DPBS was pipetted into a sterile plastic centrifuge tube (BD Falcon; BD Biosciences), and 1 mL of 40% Percoll dissolved in DPBS was layered on top of the 50% Percoll solution; the dispersed cells were carefully layered on top of the 40% Percoll solution. The tubes were centrifuged at 400 g at 4 C for 20 minutes, and the purified theca interstitial cells were collected by aspiration inside the 40% Percoll layer. The cells were washed three times in DPBS. The purified theca interstitial cells were counted with a hemacytometer, and the cell viability was determined by trypan blue exclusion (GIBCO). The cell viability was in the range of 90% to 95%.
Cell Culture Theca interstitial cells were plated on 12-well plates (2.0 105/mL, 1 mL/well) for RNA extraction and on 96-well plates (5 104/mL, 0.1 mL/well) (Corning Incorporated) for cell counting and Cell Counting Kit-8 (CCK-8) test in DMEM-F12 medium supplemented with penicillin (100 IU/mL), streptomycin (100 mg/mL), and 10% fetal bovine serum (FBS) (GIBCO), and cultured in a 37 C humidified incubator with 5% CO2. The treatments were applied after the first 48 hours of culture in medium that was changed at every 24 hours. The dosages of GDF-9 that we used were determined in a previous experiment. The medium was collected for a steroid chemiluminescence immunoassay, and the cells were collected for enumeration or RNA extraction.
Immunofluorescence
MATERIALS AND METHODS Animals Immature 3- to 4-week-old SPF KM mice obtained from the laboratory animal center of Sun Yat-Sen University were killed by cervical dislocation with a procedure approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University. Ethics approval for this study was obtained from medical ethics committee of the First Affiliated Hospital of Sun Yat-Sen University.
Isolation and Purification of Theca-Interstitial Cells The ovaries were collected in Dulbecco's phosphatebuffered saline (DPBS) and freed from their connective tissues under stereomicroscope. The granulosa cells and oocytes were released by puncturing the follicles with a sterile hypodermic needle. The remaining ovary tissues were cut into 1-mm3 fragments using scissors and were pipetted to facilitate cell dispersion in Dulbecco's modified essential medium-F12 medium (DMEM-F12; GIBCO) containing collagenase type I (5 mg/mL) (GIBCO). The suspended ovarian fragments were incubated at 37 C for 90 minutes and pipetted every 10 minutes. Undigested ovarian fragments were then removed with a 40-mm cell strainer (BD Falcon; BD Biosciences). The dispersed cells were washed three times in DPBS, subsequently reconstituted in 1 mL of DPBS, and purified using a discontinuous Percoll gradient (Sigma-Aldrich). In brief, 1 mL of 50% VOL. 100 NO. 5 / NOVEMBER 2013
Cells were cultured on a 13 13 mm sterile cover glass placed on the 12-well plate for 48 hours as described earlier. Glass/ cells were fixed in 20 C methanol for 10 minutes, washed three times with phosphate-buffered saline (PBS), and blocked for 30 minutes in PBS containing 5% donkey serum (Santa Cruz Biotechnology). Glass/cells were then incubated in PBS (negative control) or in the presence of goat antimouse Cyp17a1 polyclonal antibody and rabbit anti-mouse follicle-stimulating hormone receptor (Fshr) polyclonal antibody (Santa Cruz Biotechnology) at the dilution ratio of 1:1:100 (diluted in PBS containing 5% donkey serum) overnight at 4 C. After rinsing in PBS, the glass/cells were incubated with donkey anti-goat IgG-fluorescein isothiocyanate conjugate (FITC) and donkey anti-rabbit IgG-Texas Red (TR) (Santa Cruz Biotechnology) at a dilution ratio of 1:1:100 (diluted in PBS containing 5% donkey serum) for 60 minutes at 37 C, washed three times in PBS, stained with 40 ,6diamidino-2-phenylindole (DAPI; Aquarius/Cytocell Ltd.), and mounted. The cells were visualized using a fluorescence microscope (Axio Observer Z1; Carl Zeiss) and confocal laser scanning microscope (LSM710; Carl Zeiss). A positive reaction was evaluated by a green fluorescence for Cyp17a1 and red fluorescence for Fshr.
Determination of Cell Number and Steroid Concentration The medium was collected from individual wells and frozen at 20 C for subsequent determination of the concentrations of 1445
ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE testosterone, progesterone, and estradiol by chemiluminescent immunoassay kit (USCN Life Science Inc.) according to the manufacturer's protocol for the microplate reader (Infinite F500; TAKAN) with same inter-assay and intra-assay variation coefficients of less than 12% and 10%. The detection limit of testosterone, progesterone, and estradiol were 52.8, 317.5, and 4.85 pg/mL, respectively. Cells were then washed with DPBS. Trypsin-EDTA (0.25%) (GIBCO) solution was added to cover the cells at room temperature for 5 minutes. After the trypsin-EDTA was removed, the medium containing 10% FBS (0.2 mL/well) was added to inhibit trypsin activity. Each well was pipetted 10 times to disperse the cells. Cells were counted with a hemacytometer.
RNA Extraction and Quantification We extracted RNA with the High Pure RNA Isolation Kit (Roche Diagnostics) according to the manufacturer's instructions, and quantitated RNA by 260/280 ultraviolet spectrophotometry (NanoDrop ND-1000). Approximately 500 ng to 1 mg RNA was subjected to reverse transcription (RT) with Oligo-dT primer using Roche Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics). The differential expression of the target gene mRNA in theca interstitial cells was quantified using TaqMan Gene Expression Assays (Applied Biosystems, Life Technologies) to analyze Lhcgr (Mm00442931_m1), Star (Mm00441558_m1), cyp11a1 (Mm00490735_m1), and cyp17a1 (Mm00484040-m1). Quantification was accomplished with an Applied Biosystems 7500 real-time RT-PCR machine using TaqMan Fast Advanced Master Mix (Applied Biosystems/Life Technologies). The relative mRNA level was calculated by comparative Ct method using Hprt1 (Mm00446968) as a reference gene.
hours. Cell viability was subsequently measured by the Cell Counting Kit-8 (CCK-8) (Beyotime Institute of Biotechnology). The samples were stained with 10 mL of CCK-8 solution and were incubated at 37 C for 2 hours. The absorbance at 450 nm was measured with a microplate reader (Bio-Rad Laboratories). Experiment 4 was designed to elucidate the effect of GDF-9 on hormone-stimulated gene expression of the LH receptor (Lhcgr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage cytochrome P450 (Cyp11a1), and Cytochrome P450 17A1 (Cyp17a1) in theca interstitial cells. After the first 48 hours of culture, the cells in the 12-well plate were washed twice with serum-free medium and then were cultured for 24 hours without or with 400 ng/mL GDF-9 in serum-free medium. Cellular RNA was collected and extracted for RT-PCR.
Statistical Analysis Data are presented as the least squares mean ( standard error of the mean) of measurements from three replicate experiments. Steroid production was expressed as pg/104 cells, and the cell number at the termination of the experiment was used for this calculation. Statistical analysis was conducted using SPSS 13.0 for Windows (SPSS, Inc.). We applied the Kolmogorov-Smirnov test to analyze the distribution of data in the two-group comparison. Differences within two groups were accessed by Student's t test if the data were in normal distribution or by Mann-Whitney U test if the data were not in normal distribution. Differences within more than two groups were accessed by analysis of variance (ANOVA) followed by Tukey's multiple-comparison post hoc tests if equal variances were assumed or by Dunnett's T3 post hoc tests if equal variances were not assumed to identify individual differences between the means. P< .05 was considered statistically significant.
Experimental Design Experiment 1 was designed to evaluate the purity of theca interstitial cells by immunofluorescent staining of Cyp17a1 and Fshr and by measuring the expression levels of testosterone and estradiol in culture medium by chemiluminescent immunoassay. Experiment 2 was designed to evaluate the effect of GDF-9 on proliferation and steroidogenesis of theca interstitial cells. After the first 48 hours' culture in a 96-well plate with a cell population of 5 103/well, the cells were washed twice with serum-free medium and then were cultured for 24 hours without or with GDF-9 (50, 200, and 400 ng/mL) in serum-free medium. Then the cells were counted, and the medium was collected from individual wells and frozen at 20 C for subsequent concentration determination of testosterone and progesterone. Experiment 3 was designed to investigate the effect of GDF-9 on cell viability. The cells (5 103 cells/well) were plated onto 96-well plates at 37 C in medium with 10% FBS. After the first 48 hours' culture, the cells were washed twice with serum-free medium and then were cultured in the presence or absence of GDF-9 at gradient concentrations (10, 100, 200, and 400 ng/mL) in serum-free medium for 24 1446
RESULTS Characterization of Theca Interstitial Cells The theca interstitial cells were 95.0% 0.6% for positive staining of Cyp17a1 and 0 for positive staining of Fshr. The level of testosterone reached 219.95 28.28 pg/104 cells in the culture medium, but no estradiol was detected. The confocal laser scanning microscope results revealed that adhered mouse theca interstitial cells present as spindle shapes with green fluorescence-labeled Cyp17a1 granules in cytoplasm (Fig. 1A and B).
Effect of GDF-9 on Proliferation of Theca Interstitial Cells The treatment with GDF-9 resulted in an increase in the theca interstitial cell number in a dose-dependent manner when the dosage of GDF-9 was less than 200 ng/mL. At the doses of 200 ng/mL and 400 ng/mL, GDF-9 increased the cell number by 2.5-fold when compared with the control. However, GDF-9 at various concentrations had no statistically significant impact on the proliferative effect of theca interstitial cells (Fig. 2A). VOL. 100 NO. 5 / NOVEMBER 2013
Fertility and Sterility®
FIGURE 1
The expression of Cyp17a1 in theca interstitial cells. Adhered mouse theca interstitial cells reveal a spindle shape and granular aggregation of Cyp17a1 labeled with green fluorescence scattered in cytoplasm. (A) Observation under fluorescence microscope (400 magnification). (B) Observation under confocal laser scanning microscope (1,000 magnification). Chen. GDF-9 inhibits testosterone production. Fertil Steril 2013.
Effect of GDF-9 on Viability of Theca Interstitial Cells Treatment with GDF-9 caused an increase of cell viability in a dose-dependent manner without being statistically significance. The treatment of GDF-9 at the dose of 400 ng/mL revealed the most obvious effect on cell viability; the cells exposed to this treatment displayed a viability that was 33.33% greater than that of the control group (Fig. 2B).
of theca interstitial cells by 60.42% (P¼ .038) and 68.76% (P¼ .012), respectively, when compared with the control group (Fig. 3A). Treatment with GDF-9 at the dose %200 ng/mL caused a dose-dependent decrease of progesterone production in the theca interstitial cells. The progesterone level was at its lowest level (993.21 617.76 pg/104 cells) in the 200 ng/mL GDF-9 treatment group. The inhibition effect of GDF-9 on progesterone production in theca interstitial cells was not statistically significant (Fig. 3B).
Effect of GDF-9 on Steroidogenesis of Theca Interstitial Cells Treatment with GDF-9 resulted in the inhibition of testosterone production in a dose-dependent manner in theca interstitial cells. At the doses of 200 and 400 ng/mL, GDF-9 statistically significantly inhibited testosterone production
Effect of GDF-9 on Hormone-Stimulated Gene Expression in Theca Interstitial Cells Treatment with GDF-9 led to a decrease in Lhcgr mRNA abundance by 47.36% (P¼ .00), Cyp11a1 mRNA by 62.30%
FIGURE 2
Theca interstitial cells were treated with GDF-9 at various doses for 24 hours, then the number of theca interstitial cells was counted or the cell viability was determined by CCK8 kit. Each data point represents the mean standard error of the mean of replicate experiments. (A) The stimulatory effect of GDF-9 on the proliferation of theca cells. No statistically significant difference in cell number between the groups with different doses was observed. The P value between the 0 ng/mL and 200 ng/mL groups was 0.059. (B) Effect of GDF-9 on theca interstitial cell viability. No statistically significant difference in cell viability between groups with various doses was observed. Chen. GDF-9 inhibits testosterone production. Fertil Steril 2013.
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FIGURE 3
Effects of GDF-9 on steroidogenesis of theca interstitial cells. Theca interstitial cells were treated with various doses of GDF-9 for 24 hours then the (A) testosterone or (B) progesterone level in the medium was measured by chemiluminescent immunoassay. Each data point represents the mean standard error of the mean of replicate experiments. Asterisks indicate that the mean differs from the control group (P<.05). Chen. GDF-9 inhibits testosterone production. Fertil Steril 2013.
(P¼ .01), Cyp17a1 mRNA by 55.39% (P¼ .01), and Star mRNA by 45.66% (P¼ .11) (Fig. 4).
DISCUSSION Our study has revealed that [1] GDF-9 mildly increases mouse theca interstitial cell number and cell viability, [2] GDF-9 statistically significantly inhibited the production of testosterone in theca interstitial cells and mildly inhibited
progesterone production when compared with the control, and [3] GDF-9 statistically significantly suppressed hormone-stimulated expression of Lhcgr, Cyp11a1, and Cyp17a1 genes but had no significant effect on the expression of Star. As far as we know, ours is the first confocal laser scanning microscope photograph of mice theca cells stained for Cyp17a1 with immunofluorescence. Our study identified theca interstitial cells by immunofluorescent staining for Cyp17a1, quantitative real-time-PCR
FIGURE 4
Effects of GDF-9 on hormone-stimulated gene expression in theca interstitial cells. Theca interstitial cells were treated for 24 hours with serum-free medium alone (control) or with GDF-9 (400 ng/ml), then the mRNA was quantified by qRT-PCR. Each data point represents the mean standard error of the mean of replicate experiments. Asterisks indicate that the mean differs from the control group (P<.05). (A) Lhcgr. (B) Star. (C) Cyp11a1. (D) Cyp17a1. Chen. GDF-9 inhibits testosterone production. Fertil Steril 2013.
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Fertility and Sterility® (qRT-PCR) for Cyp17a1 gene expression, and chemiluminescent immunoassay for testosterone. No Fshr in isolated cells and no estradiol in culture medium were detected, indicating little possibility of granulosa contamination. The histochemical evaluation of theca interstitial cells subjected to the purification with similar methods showed that 90% to 95% of the cells were positive for the mesenchymal marker vimentin, 5% to 10% of cells were positive for the epithelial marker cytokeratin, and less than 5% cells were positive for the endothelial marker factor VIII (18, 19). Thus, approximately 5% of the cells with negative staining for Cyp17a1 may be epithelial or endothelial cells. It is known that the endoplasmic reticulum of theca cell contains Cyp17a1. Accordingly, our observations via confocal laser scanning microscope showed that Cyp17a1 marked with green fluorescence aggregated to granules and scattered in the theca interstitial cell's cytoplasm. Our study shows that GDF-9 at the highest dose increases the number of theca interstitial cells by 2.5-fold, which is consistent with previous results that GDF-9 at the highest dose can enhance the number of theca cells from small bovine follicles by 2.5- to 3.5-fold. Spicer et al. (15) suggested that theca cells from large preovulatory follicles had minor response to GDF-9. Dose-dependent increasing tendency in cell viability is good agreement with the change in cell number of theca interstitial cells in the present study. The elevation degree of cell viability was smaller than that of cell number of theca interstitial cells. It may be related to effect of GDF-9, a member of TGF-b family which can change mitochondrial function. CCK8 assay used to detect cell viability in present study measures cellular metabolic activity via NAD(P)H-dependent cellular oxidoreductase enzymes in mitochondria. A recent study showed that TGF-b had an obvious effect on mitochondrial biogenesis (20). Oocytes may secrete cell factors to inhibit luteinization, because the removal of an oocyte from a preovulatory follicle can initiate luteinization (21, 22). In our study, although GDF-9 decreased the production of progesterone in theca interstitial cells, the lowest progesterone level still reached up to 993.21 617.76 pg/104 cells after GDF-9 treatment, indicating that GDF-9 alone is not sufficient to inhibit luteinization completely. In addition, we found that GDF-9 suppressed the expression of Cyp11a1 and Cyp17a1 genes, and inhibited the production of testosterone in mouse theca interstitial cells. These results indicate that GDF-9 inhibits androgen production in theca interstitial cells by changing gene expression. Our results are in accord with those of Spicer et al. (15) except for the effect of GDF-9 on the gene expression of Cyp17a1; one possible explanation is their application of recombination rat GDF-9 in bovine theca cells. The effect of GDF-9 on androgen production in theca cells is similar in different species such as mice, bovines, and humans. The decreased GDF-9 expression has been found in PCOS ovaries when compared with normal oocytes at all developmental stages from the transition to Graafian follicles (9). According to our results, decreased secretion of GDF-9 will result in an increased expression of Lhcgr, Cyp11a1, and Cyp17a1 genes, subsequently increasing the sensitivity VOL. 100 NO. 5 / NOVEMBER 2013
to LH and androgen production in theca interstitial cells and finally forming hyperandrogenism phenotype in PCOS. In conclusion, GDF-9 can mildly enhance the proliferation and viability of mouse theca interstitial cells; it can significantly inhibit the production of testosterone and mildly inhibit the generation of progesterone in theca interstitial cells. Moreover, it can significantly suppress hormonestimulated gene expression of Lhcgr, Cyp11a1, and Cyp17a1, and has no significant effect on the expression of Star. Dysregulation of GDF-9 causes abnormal androgen production in theca interstitial cells. Acknowledgments: The authors thank Yan-hong Zeng, Ping-ping Hong, Xin-peng Qiu, Wen-bo Zhu for generous technical assistance; Medical Science Experimental Center of Zhonshan School of Medicine of Sun Yat-sen University for assistance with the confocal laser scanning microscopy and capture of fluorescent images; and Professor Guanglun Zhuang for his insightful suggestions on the manuscript.
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Objective: To explore the effect of growth differential factor-9 (GDF-9) alone on cell proliferation, cell viability, steroidogenesis, and hormone-stimulated gene expression in cultured mouse theca interstitial cells. Design: Basic research. Setting: University hospital. Animal(s): Immature 3- to 4-week-old SPF KM mice obtained from the Laboratory Animal Center of Sun Yat-Sen University. Intervention(s): Addition of GDF-9 at different dosages to primary culture of mouse theca interstitial cells. Main Outcome Measure(s): Cell number, cell viability, progesterone and testosterone levels, and hormone-stimulated gene mRNA abundance. Result(s): Growth differential factor-9 mildly increased the number of mouse theca interstitial cells and cell viability in a dosedependent manner and mildly inhibited the production of progesterone in mouse theca interstitial cells. Administration of GDF-9 at the dosages of 200 ng/mL and 400 ng/mL resulted in a significant decrease in the testosterone level compared with the control group by 60.42% and 68.76%, respectively. Growth differential factor-9 significantly suppressed Lhcgr mRNA by 47.36%, Cyp11a1 mRNA by 62.30%, and Cyp17a1 mRNA by 55.39%, but had only a mild effect on Star gene expression. Conclusion(s): Growth differential factor-9 can inhibit the production of testosterone in mouse Use your smartphone theca interstitial cells and suppress the corresponding gene expression. (Fertil SterilÒ 2013;100: to scan this QR code 1444–50. Ó2013 by American Society for Reproductive Medicine.) and connect to the Key Words: Gene expression, growth differential factor-9, hormone, theca Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/chenm-growth-differential-factor-9-theca/
P
olycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders, but its etiology and pathogenesis are still unclear. Previous studies have demonstrated that the growth and development of PCOS follicles stop at the diameter range of 4 to 7 mm, resulting in the accumulation of a large number of small antral follicles and thereby
the generation of the polycystic ovary phenotype (1, 2). Recent studies on the role of oocytes in folliculogenesis have revealed that the oocytes can secrete growth factors and directly induce the development of follicles (3–5). One of these oocyte-derived factors, namely, growth differentiation factor-9 (GDF-9), has a critical impact on folliculogenesis (6, 7).
Received April 21, 2013; revised June 23, 2013; accepted July 10, 2013; published online August 15, 2013. M.-h.C. has nothing to disclose. T.L. has nothing to disclose. C.-h.D. has nothing to disclose. Y.-w.X. has nothing to disclose. L.-y.G. has nothing to disclose. C.-q.Z. has nothing to disclose. Supported by grants from the Key Laboratory of Reproductive Medicine of Guangdong Province and the National Basic Research Program of China (973 Program, 2012CB947600), People's Republic of China. Reprint requests: Can-quan Zhou, M.D., Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-Sen University, 58 2nd Zhongshan Road, Guangzhou, GD510080, People's Republic of China (E-mail: [email protected]). Fertility and Sterility® Vol. 100, No. 5, November 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2013.07.200 1444
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Accumulating evidence has shown that an adequate amount of GDF-9 is required for the growth of follicles; female GDF-9 knockout mice are infertile with a complete block in folliculogenesis at the primary stage (8). The dysregulation of oocyte GDF-9 expression may contribute to aberrant folliculogenesis in PCOS because GDF-9 expression is decreased in PCOS ovaries when compared with normal controls in oocytes at all developmental stages from the transition to Graafian follicles (9). Another line of evidence indicating that GDF-9 is potentially associated with PCOS comes from a genomic study that identified five novel missense mutations of GDF-9 in PCOS patients (10). Excessive production of ovarian androgen by theca interstitial cells is a VOL. 100 NO. 5 / NOVEMBER 2013
Fertility and Sterility® principal feature of PCOS. There is evidence to indicate that GDF-9 plays an important regulatory role in theca recruitment, differentiation, and proliferation during early folliculogenesis: follicles from GDF-9 null mice and sheep lack supporting theca cells (8, 11–13). In vitro studies have shown that GDF-9 can inhibit the production of progesterone and stimulate the proliferation of bovine and human theca cells (14, 15). However, the effect of GDF-9 on androgen production in theca cells is controversial. Growth differential factor-9 has been found to enhance both basal and forskolinstimulated androstenedione accumulation in primary and immortalized rat theca interstitial cells (16), but that study did not exclude the interference of theca cell proliferation on androgen accumulation in the culture medium. Excluding the interference factor of theca cell proliferation, Spicer et al. (15) found that recombinant rat GDF-9 could inhibit the androgen production induced by insulin-like growth factor I (IGF-I) and luteinizing hormone (LH) and decrease the expression of LH receptor and CYP11A1 in bovine theca cells. Growth differential factor-9 was first discovered from mouse genomic DNA via polymerase chain reaction (PCR) by use of degenerate oligonucleotides corresponding to conserved regions of known transforming growth factor b (TGF-b) family members (17). In addition, although mice are common animal models for PCOS research, the effect of GDF-9 on mouse theca interstitial cells has not been studied. We explored the effect of GDF-9 alone on cell proliferation, cell viability, steroidogenesis, and hormone-stimulated gene expression in cultured mouse theca interstitial cells.
Percoll dissolved in DPBS was pipetted into a sterile plastic centrifuge tube (BD Falcon; BD Biosciences), and 1 mL of 40% Percoll dissolved in DPBS was layered on top of the 50% Percoll solution; the dispersed cells were carefully layered on top of the 40% Percoll solution. The tubes were centrifuged at 400 g at 4 C for 20 minutes, and the purified theca interstitial cells were collected by aspiration inside the 40% Percoll layer. The cells were washed three times in DPBS. The purified theca interstitial cells were counted with a hemacytometer, and the cell viability was determined by trypan blue exclusion (GIBCO). The cell viability was in the range of 90% to 95%.
Cell Culture Theca interstitial cells were plated on 12-well plates (2.0 105/mL, 1 mL/well) for RNA extraction and on 96-well plates (5 104/mL, 0.1 mL/well) (Corning Incorporated) for cell counting and Cell Counting Kit-8 (CCK-8) test in DMEM-F12 medium supplemented with penicillin (100 IU/mL), streptomycin (100 mg/mL), and 10% fetal bovine serum (FBS) (GIBCO), and cultured in a 37 C humidified incubator with 5% CO2. The treatments were applied after the first 48 hours of culture in medium that was changed at every 24 hours. The dosages of GDF-9 that we used were determined in a previous experiment. The medium was collected for a steroid chemiluminescence immunoassay, and the cells were collected for enumeration or RNA extraction.
Immunofluorescence
MATERIALS AND METHODS Animals Immature 3- to 4-week-old SPF KM mice obtained from the laboratory animal center of Sun Yat-Sen University were killed by cervical dislocation with a procedure approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University. Ethics approval for this study was obtained from medical ethics committee of the First Affiliated Hospital of Sun Yat-Sen University.
Isolation and Purification of Theca-Interstitial Cells The ovaries were collected in Dulbecco's phosphatebuffered saline (DPBS) and freed from their connective tissues under stereomicroscope. The granulosa cells and oocytes were released by puncturing the follicles with a sterile hypodermic needle. The remaining ovary tissues were cut into 1-mm3 fragments using scissors and were pipetted to facilitate cell dispersion in Dulbecco's modified essential medium-F12 medium (DMEM-F12; GIBCO) containing collagenase type I (5 mg/mL) (GIBCO). The suspended ovarian fragments were incubated at 37 C for 90 minutes and pipetted every 10 minutes. Undigested ovarian fragments were then removed with a 40-mm cell strainer (BD Falcon; BD Biosciences). The dispersed cells were washed three times in DPBS, subsequently reconstituted in 1 mL of DPBS, and purified using a discontinuous Percoll gradient (Sigma-Aldrich). In brief, 1 mL of 50% VOL. 100 NO. 5 / NOVEMBER 2013
Cells were cultured on a 13 13 mm sterile cover glass placed on the 12-well plate for 48 hours as described earlier. Glass/ cells were fixed in 20 C methanol for 10 minutes, washed three times with phosphate-buffered saline (PBS), and blocked for 30 minutes in PBS containing 5% donkey serum (Santa Cruz Biotechnology). Glass/cells were then incubated in PBS (negative control) or in the presence of goat antimouse Cyp17a1 polyclonal antibody and rabbit anti-mouse follicle-stimulating hormone receptor (Fshr) polyclonal antibody (Santa Cruz Biotechnology) at the dilution ratio of 1:1:100 (diluted in PBS containing 5% donkey serum) overnight at 4 C. After rinsing in PBS, the glass/cells were incubated with donkey anti-goat IgG-fluorescein isothiocyanate conjugate (FITC) and donkey anti-rabbit IgG-Texas Red (TR) (Santa Cruz Biotechnology) at a dilution ratio of 1:1:100 (diluted in PBS containing 5% donkey serum) for 60 minutes at 37 C, washed three times in PBS, stained with 40 ,6diamidino-2-phenylindole (DAPI; Aquarius/Cytocell Ltd.), and mounted. The cells were visualized using a fluorescence microscope (Axio Observer Z1; Carl Zeiss) and confocal laser scanning microscope (LSM710; Carl Zeiss). A positive reaction was evaluated by a green fluorescence for Cyp17a1 and red fluorescence for Fshr.
Determination of Cell Number and Steroid Concentration The medium was collected from individual wells and frozen at 20 C for subsequent determination of the concentrations of 1445
ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE testosterone, progesterone, and estradiol by chemiluminescent immunoassay kit (USCN Life Science Inc.) according to the manufacturer's protocol for the microplate reader (Infinite F500; TAKAN) with same inter-assay and intra-assay variation coefficients of less than 12% and 10%. The detection limit of testosterone, progesterone, and estradiol were 52.8, 317.5, and 4.85 pg/mL, respectively. Cells were then washed with DPBS. Trypsin-EDTA (0.25%) (GIBCO) solution was added to cover the cells at room temperature for 5 minutes. After the trypsin-EDTA was removed, the medium containing 10% FBS (0.2 mL/well) was added to inhibit trypsin activity. Each well was pipetted 10 times to disperse the cells. Cells were counted with a hemacytometer.
RNA Extraction and Quantification We extracted RNA with the High Pure RNA Isolation Kit (Roche Diagnostics) according to the manufacturer's instructions, and quantitated RNA by 260/280 ultraviolet spectrophotometry (NanoDrop ND-1000). Approximately 500 ng to 1 mg RNA was subjected to reverse transcription (RT) with Oligo-dT primer using Roche Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics). The differential expression of the target gene mRNA in theca interstitial cells was quantified using TaqMan Gene Expression Assays (Applied Biosystems, Life Technologies) to analyze Lhcgr (Mm00442931_m1), Star (Mm00441558_m1), cyp11a1 (Mm00490735_m1), and cyp17a1 (Mm00484040-m1). Quantification was accomplished with an Applied Biosystems 7500 real-time RT-PCR machine using TaqMan Fast Advanced Master Mix (Applied Biosystems/Life Technologies). The relative mRNA level was calculated by comparative Ct method using Hprt1 (Mm00446968) as a reference gene.
hours. Cell viability was subsequently measured by the Cell Counting Kit-8 (CCK-8) (Beyotime Institute of Biotechnology). The samples were stained with 10 mL of CCK-8 solution and were incubated at 37 C for 2 hours. The absorbance at 450 nm was measured with a microplate reader (Bio-Rad Laboratories). Experiment 4 was designed to elucidate the effect of GDF-9 on hormone-stimulated gene expression of the LH receptor (Lhcgr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage cytochrome P450 (Cyp11a1), and Cytochrome P450 17A1 (Cyp17a1) in theca interstitial cells. After the first 48 hours of culture, the cells in the 12-well plate were washed twice with serum-free medium and then were cultured for 24 hours without or with 400 ng/mL GDF-9 in serum-free medium. Cellular RNA was collected and extracted for RT-PCR.
Statistical Analysis Data are presented as the least squares mean ( standard error of the mean) of measurements from three replicate experiments. Steroid production was expressed as pg/104 cells, and the cell number at the termination of the experiment was used for this calculation. Statistical analysis was conducted using SPSS 13.0 for Windows (SPSS, Inc.). We applied the Kolmogorov-Smirnov test to analyze the distribution of data in the two-group comparison. Differences within two groups were accessed by Student's t test if the data were in normal distribution or by Mann-Whitney U test if the data were not in normal distribution. Differences within more than two groups were accessed by analysis of variance (ANOVA) followed by Tukey's multiple-comparison post hoc tests if equal variances were assumed or by Dunnett's T3 post hoc tests if equal variances were not assumed to identify individual differences between the means. P< .05 was considered statistically significant.
Experimental Design Experiment 1 was designed to evaluate the purity of theca interstitial cells by immunofluorescent staining of Cyp17a1 and Fshr and by measuring the expression levels of testosterone and estradiol in culture medium by chemiluminescent immunoassay. Experiment 2 was designed to evaluate the effect of GDF-9 on proliferation and steroidogenesis of theca interstitial cells. After the first 48 hours' culture in a 96-well plate with a cell population of 5 103/well, the cells were washed twice with serum-free medium and then were cultured for 24 hours without or with GDF-9 (50, 200, and 400 ng/mL) in serum-free medium. Then the cells were counted, and the medium was collected from individual wells and frozen at 20 C for subsequent concentration determination of testosterone and progesterone. Experiment 3 was designed to investigate the effect of GDF-9 on cell viability. The cells (5 103 cells/well) were plated onto 96-well plates at 37 C in medium with 10% FBS. After the first 48 hours' culture, the cells were washed twice with serum-free medium and then were cultured in the presence or absence of GDF-9 at gradient concentrations (10, 100, 200, and 400 ng/mL) in serum-free medium for 24 1446
RESULTS Characterization of Theca Interstitial Cells The theca interstitial cells were 95.0% 0.6% for positive staining of Cyp17a1 and 0 for positive staining of Fshr. The level of testosterone reached 219.95 28.28 pg/104 cells in the culture medium, but no estradiol was detected. The confocal laser scanning microscope results revealed that adhered mouse theca interstitial cells present as spindle shapes with green fluorescence-labeled Cyp17a1 granules in cytoplasm (Fig. 1A and B).
Effect of GDF-9 on Proliferation of Theca Interstitial Cells The treatment with GDF-9 resulted in an increase in the theca interstitial cell number in a dose-dependent manner when the dosage of GDF-9 was less than 200 ng/mL. At the doses of 200 ng/mL and 400 ng/mL, GDF-9 increased the cell number by 2.5-fold when compared with the control. However, GDF-9 at various concentrations had no statistically significant impact on the proliferative effect of theca interstitial cells (Fig. 2A). VOL. 100 NO. 5 / NOVEMBER 2013
Fertility and Sterility®
FIGURE 1
The expression of Cyp17a1 in theca interstitial cells. Adhered mouse theca interstitial cells reveal a spindle shape and granular aggregation of Cyp17a1 labeled with green fluorescence scattered in cytoplasm. (A) Observation under fluorescence microscope (400 magnification). (B) Observation under confocal laser scanning microscope (1,000 magnification). Chen. GDF-9 inhibits testosterone production. Fertil Steril 2013.
Effect of GDF-9 on Viability of Theca Interstitial Cells Treatment with GDF-9 caused an increase of cell viability in a dose-dependent manner without being statistically significance. The treatment of GDF-9 at the dose of 400 ng/mL revealed the most obvious effect on cell viability; the cells exposed to this treatment displayed a viability that was 33.33% greater than that of the control group (Fig. 2B).
of theca interstitial cells by 60.42% (P¼ .038) and 68.76% (P¼ .012), respectively, when compared with the control group (Fig. 3A). Treatment with GDF-9 at the dose %200 ng/mL caused a dose-dependent decrease of progesterone production in the theca interstitial cells. The progesterone level was at its lowest level (993.21 617.76 pg/104 cells) in the 200 ng/mL GDF-9 treatment group. The inhibition effect of GDF-9 on progesterone production in theca interstitial cells was not statistically significant (Fig. 3B).
Effect of GDF-9 on Steroidogenesis of Theca Interstitial Cells Treatment with GDF-9 resulted in the inhibition of testosterone production in a dose-dependent manner in theca interstitial cells. At the doses of 200 and 400 ng/mL, GDF-9 statistically significantly inhibited testosterone production
Effect of GDF-9 on Hormone-Stimulated Gene Expression in Theca Interstitial Cells Treatment with GDF-9 led to a decrease in Lhcgr mRNA abundance by 47.36% (P¼ .00), Cyp11a1 mRNA by 62.30%
FIGURE 2
Theca interstitial cells were treated with GDF-9 at various doses for 24 hours, then the number of theca interstitial cells was counted or the cell viability was determined by CCK8 kit. Each data point represents the mean standard error of the mean of replicate experiments. (A) The stimulatory effect of GDF-9 on the proliferation of theca cells. No statistically significant difference in cell number between the groups with different doses was observed. The P value between the 0 ng/mL and 200 ng/mL groups was 0.059. (B) Effect of GDF-9 on theca interstitial cell viability. No statistically significant difference in cell viability between groups with various doses was observed. Chen. GDF-9 inhibits testosterone production. Fertil Steril 2013.
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FIGURE 3
Effects of GDF-9 on steroidogenesis of theca interstitial cells. Theca interstitial cells were treated with various doses of GDF-9 for 24 hours then the (A) testosterone or (B) progesterone level in the medium was measured by chemiluminescent immunoassay. Each data point represents the mean standard error of the mean of replicate experiments. Asterisks indicate that the mean differs from the control group (P<.05). Chen. GDF-9 inhibits testosterone production. Fertil Steril 2013.
(P¼ .01), Cyp17a1 mRNA by 55.39% (P¼ .01), and Star mRNA by 45.66% (P¼ .11) (Fig. 4).
DISCUSSION Our study has revealed that [1] GDF-9 mildly increases mouse theca interstitial cell number and cell viability, [2] GDF-9 statistically significantly inhibited the production of testosterone in theca interstitial cells and mildly inhibited
progesterone production when compared with the control, and [3] GDF-9 statistically significantly suppressed hormone-stimulated expression of Lhcgr, Cyp11a1, and Cyp17a1 genes but had no significant effect on the expression of Star. As far as we know, ours is the first confocal laser scanning microscope photograph of mice theca cells stained for Cyp17a1 with immunofluorescence. Our study identified theca interstitial cells by immunofluorescent staining for Cyp17a1, quantitative real-time-PCR
FIGURE 4
Effects of GDF-9 on hormone-stimulated gene expression in theca interstitial cells. Theca interstitial cells were treated for 24 hours with serum-free medium alone (control) or with GDF-9 (400 ng/ml), then the mRNA was quantified by qRT-PCR. Each data point represents the mean standard error of the mean of replicate experiments. Asterisks indicate that the mean differs from the control group (P<.05). (A) Lhcgr. (B) Star. (C) Cyp11a1. (D) Cyp17a1. Chen. GDF-9 inhibits testosterone production. Fertil Steril 2013.
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Fertility and Sterility® (qRT-PCR) for Cyp17a1 gene expression, and chemiluminescent immunoassay for testosterone. No Fshr in isolated cells and no estradiol in culture medium were detected, indicating little possibility of granulosa contamination. The histochemical evaluation of theca interstitial cells subjected to the purification with similar methods showed that 90% to 95% of the cells were positive for the mesenchymal marker vimentin, 5% to 10% of cells were positive for the epithelial marker cytokeratin, and less than 5% cells were positive for the endothelial marker factor VIII (18, 19). Thus, approximately 5% of the cells with negative staining for Cyp17a1 may be epithelial or endothelial cells. It is known that the endoplasmic reticulum of theca cell contains Cyp17a1. Accordingly, our observations via confocal laser scanning microscope showed that Cyp17a1 marked with green fluorescence aggregated to granules and scattered in the theca interstitial cell's cytoplasm. Our study shows that GDF-9 at the highest dose increases the number of theca interstitial cells by 2.5-fold, which is consistent with previous results that GDF-9 at the highest dose can enhance the number of theca cells from small bovine follicles by 2.5- to 3.5-fold. Spicer et al. (15) suggested that theca cells from large preovulatory follicles had minor response to GDF-9. Dose-dependent increasing tendency in cell viability is good agreement with the change in cell number of theca interstitial cells in the present study. The elevation degree of cell viability was smaller than that of cell number of theca interstitial cells. It may be related to effect of GDF-9, a member of TGF-b family which can change mitochondrial function. CCK8 assay used to detect cell viability in present study measures cellular metabolic activity via NAD(P)H-dependent cellular oxidoreductase enzymes in mitochondria. A recent study showed that TGF-b had an obvious effect on mitochondrial biogenesis (20). Oocytes may secrete cell factors to inhibit luteinization, because the removal of an oocyte from a preovulatory follicle can initiate luteinization (21, 22). In our study, although GDF-9 decreased the production of progesterone in theca interstitial cells, the lowest progesterone level still reached up to 993.21 617.76 pg/104 cells after GDF-9 treatment, indicating that GDF-9 alone is not sufficient to inhibit luteinization completely. In addition, we found that GDF-9 suppressed the expression of Cyp11a1 and Cyp17a1 genes, and inhibited the production of testosterone in mouse theca interstitial cells. These results indicate that GDF-9 inhibits androgen production in theca interstitial cells by changing gene expression. Our results are in accord with those of Spicer et al. (15) except for the effect of GDF-9 on the gene expression of Cyp17a1; one possible explanation is their application of recombination rat GDF-9 in bovine theca cells. The effect of GDF-9 on androgen production in theca cells is similar in different species such as mice, bovines, and humans. The decreased GDF-9 expression has been found in PCOS ovaries when compared with normal oocytes at all developmental stages from the transition to Graafian follicles (9). According to our results, decreased secretion of GDF-9 will result in an increased expression of Lhcgr, Cyp11a1, and Cyp17a1 genes, subsequently increasing the sensitivity VOL. 100 NO. 5 / NOVEMBER 2013
to LH and androgen production in theca interstitial cells and finally forming hyperandrogenism phenotype in PCOS. In conclusion, GDF-9 can mildly enhance the proliferation and viability of mouse theca interstitial cells; it can significantly inhibit the production of testosterone and mildly inhibit the generation of progesterone in theca interstitial cells. Moreover, it can significantly suppress hormonestimulated gene expression of Lhcgr, Cyp11a1, and Cyp17a1, and has no significant effect on the expression of Star. Dysregulation of GDF-9 causes abnormal androgen production in theca interstitial cells. Acknowledgments: The authors thank Yan-hong Zeng, Ping-ping Hong, Xin-peng Qiu, Wen-bo Zhu for generous technical assistance; Medical Science Experimental Center of Zhonshan School of Medicine of Sun Yat-sen University for assistance with the confocal laser scanning microscopy and capture of fluorescent images; and Professor Guanglun Zhuang for his insightful suggestions on the manuscript.
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