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Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells
Theriogenology 84 (2015) 811–817
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Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells Jin-You Zhang a, b, c, Yi Wu b, Shuan Zhao b, Zhen-Xing Liu b, Shen-Ming Zeng b, Gui-Xue Zhang a, * a
Laboratory of Animal Reproductive Science, College of Animal Science and Technology, Northeast Agricultural University, Harbin, China b National Key Laboratory of Animal Nutrition, Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, China Agricultural University, Beijing, China c Animal Husbandry and Veterinary Institute, Heilongjiang Academy of Land Reclamation Sciences, Harbin, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 November 2014 Received in revised form 14 May 2015 Accepted 20 May 2015
Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the ratelimiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol sidechain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Lysosome Low-density lipoprotein StAR Granulosa cell Bovine
1. Introduction Progesterone is one of the fundamental steroid hormones for the regulation of the bovine estrous cycle. The machinery of progesterone biosynthesis involves multiple critical components, such as cholesterol side-chain cleavage * Corresponding author. Tel.: þ86 451 55191421; fax: þ86 451 55103336. E-mail address: [email protected] (G.-X. Zhang). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.05.016
cytochrome P-450 (CYP11A1), which catalyzes the conversion of cholesterol to pregnenolone, and steroidogenic acute regulatory protein (StAR), which transports cholesterol from the outer to the inner mitochondrial membrane [1–4]. Before ovulation, estradiol is the primary steroid secreted by the follicles [5], while concentration of progesterone in the follicular fluid is low [6–8]. This may be due to the low to undetectable expression of StAR messenger RNA (mRNA) in follicular granulosa cells, although StAR and CYP11A1 are expressed in theca cells [5]. After ovulation, the
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preovulatory LH surge results in the luteinization of the residual granulosa and theca cells [9–11]. The ovarian follicle switches from mainly secreting estradiol to producing large amount of progesterone. The markedly enhanced progesterone biosynthesis is attributed to the increased expression of StAR and CYP11A1 [1–4]. The expression of StAR is regulated at different levels by many hormones and growth factors. Insulin, LH, FSH, insulinlike growth factor 1, transforming growth factor b, and forskolin have been shown to stimulate StAR mRNA expression in diverse species [12–17]. Numerous transcription factors, such as steroidogenic factor 1, GATA4/6, sterol regulatory element-binding proteins 1A, CCAAT/ enhancer-binding proteins, and cAMP response elementbinding protein, participate in StAR promoter activation [18–23]. Epigenetic mechanisms, including histone modifications, DNA methylation, and chromatin remodeling, are also involved in StAR gene expression in granulosa cells of bovine [24,25] and other species [26–28]. In addition, a recent study reported that StAR gene expression was downregulated under the adverse metabolic environment caused by lactation or nutritional restriction in cattle [29]. Moreover, accumulated evidence has shown that lipoprotein, as a nutritive material or metabolic substrate, is also involved in the regulation of StAR expression [30,31]. Progesterone biosynthesis requires cholesterol as a primary substrate. There are two major carriers of blood cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) [32]. The LDL is primarily used as a steroidogenic substrate for progesterone production by bovine granulosa cells [33]. Furthermore, LDL has been shown to have stimulatory effects on progesterone biosynthesis in cultured bovine luteal cells [34,35] and swine granulosa cells [36,37]. This stimulation of progesterone biosynthesis may be due to the transcriptional regulation effects of LDL on the expression of CYP11A1 protein as shown in bovine granulosa cells [38]. In addition, StAR expression in mouse nonovarian cells was upregulated by LDL [30,31]. However, it is currently unknown whether LDL has a similar role for increasing StAR expression in bovine granulosa cells. The uptake of LDL occurs through receptor-mediated endocytosis. Once LDL is internalized, the endosomes fuse with lysosomes, followed by lysosomal degradation of cholesteryl ester to release free cholesterol [39]. Inhibiting lysosomal function suppresses this process [40] and consequently blocks LDL-induced progesterone synthesis [41]. Thus, it seems reasonable to suggest that the lysosomal degradation pathway of LDL is indispensable for progesterone biosynthesis in granulosa cells after ovulation. However, the role of lysosomes in LDL regulation of StAR expression is largely unknown. Therefore, in this study, we test the hypothesis that LDL may promote StAR expression and lysosomes may participate in regulating this process. 2. Materials and methods
Low-density lipoprotein (95% purity, 78%–81% lipid; 19%– 22% protein, dissolved in 150-mM NaCl, 0.01% EDTA, pH 7.4) was purchased from Calbiochem (San Diego, CA, USA). Radioimmunoassay kits were obtained from the Beijing North Institute of Biological Technology. All other reagents were from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA), unless otherwise indicated. All plasticware was from Nalge Nunc International (Roskilde, Denmark). Chloroquine (CQ) was dissolved in water at a concentration of 50 mmol/ L as a 1000X stock solution. 2.2. Primary culture and treatment of granulosa cells Ovaries were collected from cows on Days 16 to 21 of the estrous cycle as described previously [42]. They were collected at a local abattoir within 20 minutes after slaughter, placed immediately in 0.9% NaCl at 37 C, and transported to the laboratory in 2 hours. Follicular fluid that contained granulosa cells was collected from a heterogonous population of follicles (diameter, 2–8 mm) by aspiration using a 20-ga needle. The fluid was transferred into 15-mL centrifuge tubes and left standing for at least 5 minutes to precipitate the cumulus–oocyte complexes. The supernatant was centrifuged at 200 g for 5 minutes to collect granulosa cells, which were then washed three times in serum-free medium. Petri dishes (35 mm) and coverslips were pretreated with DMEM/F12 containing 10% fetal bovine serum for 18 hours at 37 C in a humidified incubator, followed by three washes, to get coated by seral proteins. The granulosa cells (1 106 viable cells/dish) were cultured in humidified air with 5% CO2 at 37 C in DMEM/F12 for 12 hours either on the Petri dishes or on coverslips placed in the Petri dishes. Then, cells were washed twice with DMEM/F12 to remove nonadherent cells and further cultured with fresh control culture medium (DMEM/F12) or medium containing specific components for the indicated time. On the basis of previous reports examining plasma LDL concentrations and physiological effects of LDL on cells [30,34,39,43], 20 mg of protein/mL of LDL was used in this study. The concentration of lysosomal inhibitor CQ (50 mmol/L) used here was with low cytotoxicity, as reported in previous studies [40,44–46]. The following treatment groups were used: (1) control, DMEM/F12; (2) LDL, control culture medium plus LDL (20 mg of protein/mL); (3) LDL plus CQ, control culture medium plus LDL (20 mg of protein/mL) and CQ (50 mmol/L). Three independent replicates of culture were performed for each treatment. 2.3. Radioimmunoassay Culture media obtained at the indicated time were analyzed for progesterone production using RIA kits. The intra-assay and interassay coefficients of variability were less than 10% and 6%, respectively. Assay sensitivity was 0.2 ng/mL. To check whether added LDL interferes the result of progesterone RIA, the unspent control and LDL-added media were also analyzed for progesterone using RIA kits.
2.1. Materials 2.4. Quantitative PCR Dulbecco’s modified Eagle’s medium containing Ham’s F12 (1:1 [v:v] DMEM/F12) and fetal bovine serum were obtained from Gibco Life Technologies (Carlsbad, CA, USA).
Quantitative polymerase chain reaction (PCR) was performed using real-time PCR Master Mix containing SYBR
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Green (Toyobo) and gene-specific primers on a Stratagene Mx3000P real-time quantitative PCR machine (Stratagene). Total RNA was isolated using Trizol reagent (Takara). The RNA samples were reverse transcribed into complementary DNA with a RevertAid First Strand cDNA Synthesis Kit (Fermentas). GAPDH was used as a housekeeping gene [15,26,47]. The specific primer sequences for StAR were forward: 50 CATGGTGCTCCGCCCCTTGG-30 and reverse: 50 -CGCTTGCGC AGGTGATTGGC-3’ (National Center for Biotechnology Information reference sequence: NM_174189.2). The specific primer sequences for CYP11A1 were forward: 50 -GCTCCAGAGGCAATAAAGAAC-30 and reverse: 50 -GACTCAAAGGCAAA GTGAAACA-30 (National Center for Biotechnology Information reference sequence: NM_176644.2). The specificity of the PCR products was verified using a melting curve analysis. Fold differences in mRNA levels over the control values were calculated using the 2DDCt method. The PCR reactions were run in triplicate for each sample, and at least three independent experiments were conducted.
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three washes with PBS, cells were incubated for 1 hour with a goat antirabbit Cy3-conjugated antibody (1:500, 111-165046; Jackson ImmunoResearch). The cell nuclei were counterstained with Hoechst 33342. The resulting fluorescence was visualized with a Nikon (TE2000) inverted C1 confocal microscope using Nikon EZ-C1 software and a Zeiss SM-510 laser scanning confocal system (Carl Zeiss). Fluorescence pixel intensities were measured as described by Peter Bankhead (available on http://blogs.qub.ac.uk/ccbg/ files/2013/06/Analyzing_fluorescence_microscopy_images. pdf). 2.8. Statistical analysis Data are presented as the mean standard error of the mean. Independent samples t tests and one-way ANOVA
2.5. Western blot analysis Western blot analyses were performed as described previously [48]. After blocking, the membranes were incubated with primary antibodies (4 C, overnight) against StAR (rabbit antihuman StAR antibody, 1:1000 dilutions, #8449; Cell Signaling Technology, Danvers, MA, USA) or b-actin (1:2000 dilutions, goat antirabbit; Zhongshan Biotechnology, Beijing, China). After 1 hour of incubation at room temperature with secondary antibodies (1:5000 dilutions, antigoat or antirabbit immunoglobulin G horseradish peroxidase linked, Zhongshan Biotechnology), the protein bands were visualized using enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China) and X-OMAT BT film (Eastman Kodak Co.). The films were digitized, and densitometry analysis was performed with ImageJ 1.44p software (National Institutes of Health). The relative intensities of the bands were quantified and normalized to the respective loading controls. 2.6. Acridine orange vital staining The cells grown on coverslips were vitally stained at the indicated time with acridine orange (AO) solution (10 mmol/L in complete medium) for 15 minutes at 37 C and then washed with PBS. The coverslips were mounted on a glass slide. Images were obtained using a Nikon (TE2000) inverted C1 confocal microscope with Nikon EZC1 software and a Zeiss SM-510 laser scanning confocal system (Carl Zeiss). 2.7. Immunofluorescence staining Cells were fixed with 4% paraformaldehyde in PBS for 10 minutes and washed twice with PBS. Cells were then permeabilized with 0.3% Tween-20 for 20 minutes and washed with PBS three times. Blocking was performed with 0.3 mol/L of glycine in 0.05% Tween-20 in PBS for 30 minutes. Cells were incubated with an anti-LAMP1 antibody diluted (1:1000; ab24170; Abcam, Cambridge, MA, USA) in 1% BSA in 0.05% Tween-20 in PBS overnight at 4 C. After
Fig. 1. Effects of low-density lipoprotein (LDL) on progesterone biosynthesis and expression levels of steroidogenic acute regulatory (StAR) messenger RNA (mRNA) and protein in bovine granulosa cells. Cells were cultured in DMEM/F12 for 12 hours and then cultured in control medium without (Con) or with LDL (20-mg protein/mL; LDL) for 24 hours. (A) Progesterone concentrations normalized to 1 105 cells for each sample are represented as the mean standard error of the mean (SEM) of five independent experiments. (B) The mRNA expression was analyzed using quantitative polymerase chain reaction (qPCR). Data are normalized to the internal control GAPDH mRNA and expressed as the fold change from control SEM for four independent experiments. (C) The immunoblots of StAR and b-actin protein expression levels; (D) StAR expression was normalized to b-actin expression after being quantified using ImageJ; statistical data from four independent experiments are shown as fold changes from control SEM. (E) The CYP11A1 mRNA expression was analyzed using qPCR. Data are normalized to the internal control GAPDH mRNA and expressed as the fold change from control SEM for three independent experiments. The asterisk (*) indicates a significant difference from the control group (P < 0.05).
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Fig. 2. Effect of low-density lipoprotein (LDL) on LAMP1 distribution in bovine granulosa cells. Cells grown on coverslips were cultured in control medium (A, Con) or control medium containing 20-mg protein/mL of LDL (A, LDL) for 24 hours. The cells were fixed, permeabilized, and stained for LAMP1. (B) Bar graph showing the average intensity (mean standard error of the mean) of LAMP1 fluorescence (red) of 15 different cells for each culture from four independent experiments. Scale bar: 10 mm. The asterisk (*) indicates a significant difference (P < 0.05) from the control group. (For interpretation of the references to color in this figure, the reader is referred to the Web version of this article.)
were performed using the SPSS 16.0 software package (Chicago, IL, USA) for statistical data analysis. A value of P < 0.05 was considered statistically significant. 3. Results 3.1. LDL enhances StAR expression and progesterone production in cultured bovine granulosa cells To characterize the role of LDL in progesterone synthesis, we first examined its effects on granulosa progesterone production and expression of StAR, the critical gene positively promoting this process. The treatment with LDL (20 mg of protein/mL) for 24 hours stimulated more progesterone production (155.61 35 ng/105 cells) in granulosa cells than in untreated control cells (30.31 6.2 ng/ 105 cells; P < 0.05; Fig. 1A). Compared with the control, LDL treatment also induced approximately 2.29- and 2.71-fold increases in StAR mRNA and protein levels, respectively (P < 0.05; Fig. 1B, C, D). In addition, the expression of
CYP11A1 mRNA was also estimated. The result showed that CYP11A1 mRNA was increased by LDL treatment up to approximately 2.33-fold compared with the control (P < 0.05; Fig. 1E). In general, LDL exhibits stimulatory effects on biosynthesis of progesterone, which may be undertaken through upregulating StAR and CYP11A1 gene expression. 3.2. LDL promotes the increasing of lysosomes in cultured bovine granulosa cells Because the lysosomal degradation pathway of LDL is indispensable for progesterone biosynthesis, we next studied the changes of lysosomes. We visualized lysosomes using two different approaches: AO staining and immunostaining with anti-LAMP1 antibodies. As shown in Figure 2, LDL induced a wider distribution of LAMP1 fluorescence (Fig. 2A) in granulosa cells than in control cells, and the fluorescence intensity (Fig. 2B) was significantly increased in the LDL-treated cells (0.93 0.02) compared
Fig. 3. Effect of low-density lipoprotein (LDL) on acridine orange (AO)–stained lysosomes in bovine granulosa cells. Cells grown on coverslips were cultured in control medium (A, Con) or control medium containing 20-mg protein/mL of LDL (B, LDL) for 24 hours. Cells were subsequently loaded with AO. (C) Bar graph showing the number (mean standard error of the mean) of red granules (AO-stained lysosomes) per cell in 15 different cells for each culture from four independent experiments. Scale bar: 10 mm. The asterisk (*) indicates a significant difference (P < 0.05) from the control group.
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with that in control cells (0.57 0.14, P < 0.05). The AO staining (Fig. 3) showed that more lysosomes (red granules) (58.2 5.5/cell) appeared in the granulosa cells treated with LDL than in control cells (25.4 3.0/cell, P < 0.05). These results indicate that the added LDL promotes more lysosomes appearing in cytoplasm. 3.3. Lysosomal activity is essential for StAR protein expression and progesterone production in cultured bovine granulosa cells To determine whether the synthesis of LDL-induced StAR and progesterone was mediated by lysosome activation, we used a specific inhibitor CQ to inhibit lysosomal activity. As a diprotic weak base, CQ is a lysosomotropic agent. The unprotonated form of CQ preferentially accumulates in lysosomes, increasing pH values in lysosomes and inhibiting lysosomal enzyme activities. As shown in Figure 4A, CQ blocked the LDL-induced progesterone synthesis. Treatment with CQ strikingly abolished the stimulation of progesterone production by LDL (19.2 7.5 ng/105 vs. 178.5 9.4 ng/105 cells, P < 0.05), close to the baseline level observed in the control cells (27 4.7 ng/105 cells, P > 0.05). In addition, as shown in Figure 4B, C, CQ inhibited the LDL-induced StAR protein expression. These results indicate that lysosomal activity is essential for the induction of progesterone synthesis and StAR expression by LDL. 4. Discussion Our present study provides new insights into the effects of LDL to promote progesterone synthesis in bovine granulosa cells. The major changes that occur during the process of luteinization are marked increases in progesterone synthesis and the levels of both StAR and cytochrome P-450 [49,50]. Although LH, FSH, and cAMP induce the synthesis of progesterone and the expression of StAR and cytochrome P450 in cultured granulosa cells, these inductive effects in the follicle mostly occur before ovulation under physiological
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condition [6,49,51]. In the follicular phase, granulosa cells do not have access to LDL because the content of LDL in follicular fluid is very low compared with that in plasma [43]. However, after ovulation, with the rupture of the follicular wall and angiogenesis, granulosa cells are immersed in a high LDL microenvironment. Similar to previous reports in bovine [34,35] and swine [36,37], our results showed that LDL promoted progesterone production in cultured bovine granulosa cells, whereas the unspent LDL in medium did not interfere in progesterone RIA (data not shown). One report showed that synthesis of CYP11A1 protein was induced by LDL in cultured bovine granulosa cells [38], and our study also showed that expression of CYP11A1 mRNA was increased by LDL. These results suggest that LDL is involved in the regulation of progesterone synthesis process. So this outcome of LDL promoting progesterone production is attributed to more than just an increase in available cholesterol derived from LDL for progesterone production. In addition, our study has revealed for the first time that expression of the StAR mRNA and protein was upregulated by LDL in cultured bovine granulosa cells. Others have reported similar results in nonovarian cells [30,31]. A portion of the cholesterol derived from LDL can be oxidized by specific hydroxylases to generate oxysterols in cells. Evidence indicates that StAR protein levels are positively regulated by oxysterols [31,52,53]. However, the regulation of StAR expression by LDL is not mediated by an oxysterol intermediate. A possible explanation for the result is that LDL increases StAR expression through a mechanism that is dependent on the upregulation of the StAR promoter and transcription factor steroidogenic factor 1 activity [30]. According to a study examining the rat StAR promoter, the increased cholesterol may regulate StAR transcription through a pathway involving the binding of sterol regulatory element-binding proteins to sterol regulatory elements located in the promoter regions of StAR genes [54]. Cells derive cholesterol from LDL through lysosomal degradation. Lysosomes have long been regarded as dynamic and terminal organelles that receive and degrade
Fig. 4. Effects of chloroquine (CQ) on low-density lipoprotein (LDL)–induced synthesis of progesterone and expression of steroidogenic acute regulatory (StAR) protein. Granulosa cells were cultured with control medium (Con), or control medium containing 20 mg of protein/mL of LDL (LDL) or 20 mg of protein/mL of LDL plus 50 mmol/L of CQ (LDL þ CQ) for 24 hours. (A) Progesterone concentrations were normalized to 1 105 cells for each sample and are represented as the mean standard error of the mean (SEM) for five independent experiments. (B) The immunoblots of StAR and b-actin protein expression levels. (C) The data were normalized to b-actin expression after being quantified by ImageJ; statistical data from four independent experiments are shown as fold changes from control SEM. An asterisk (*) indicates a significant difference (P < 0.05) from the other two groups.
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materials from the secretory, endocytic, autophagic, and phagocytic membrane-trafficking pathways in animal cells [55]. A previous study showed that treatment of porcine granulosa cells with CQ completely blocked LDL-induced stimulation of progesterone synthesis [37]. Our study revealed that once the function of lysosomes was inhibited, the stimulatory effect of LDL on StAR protein expression was suppressed, and progesterone production by granulosa cells also decreased. Intriguingly, our studies using AO staining and immunostaining with anti-LAMP1 antibodies showed that the number of lysosomes was increased by LDL treatment in cultured bovine granulosa cells. A similar result was shown in another study, which found that LDL enlarged endolysosomes and increased LAMP1 expression in cultured embryonic rat neurons [56]. Lysosome biogenesis requires complex integration of the endocytic and biosynthetic pathways of the cell. The best understood pathway is the mannose-6-phosphate receptor (M6PR)– mediated transport of lysosomal hydrolases [56,57]. In addition, the M6PR-independent pathways, lysosomal integral membrane protein 2 and sortilin were recently reported [58]. By contrast, very little is known about the structural and molecular machinery for the transport of lysosomal membrane proteins to lysosomes. Although the exact mechanism for the increase in the number of lysosomes by LDL is still unknown, it can be concluded that lysosomes mediate the effect of LDL on promoting progesterone synthesis and StAR expression in bovine granulosa cells and that cholesterol may be an important factor in this process. In conclusion, lysosomes mediate the effects of LDL to promote progesterone synthesis and StAR expression in bovine granulosa cells. The results of this study extend our knowledge about the luteotrophic effects of LDL on granulosa cells after ovulation in vivo. After ovulation, luteinizing granulosa cells take up plasma LDL, which consequentially induces more lysosomes, resulting in marked increases of StAR and CYP11A1 expression and progesterone synthesis. Acknowledgments The authors thank Haq Ihtesham and Dr Fei Gao (Cincinnati Children’s Hospital Medical Center, USA) for critical reading of the article. This work was supported by a grant from the National Natural Science Foundation of China (Grant 31172209).
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Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells Jin-You Zhang a, b, c, Yi Wu b, Shuan Zhao b, Zhen-Xing Liu b, Shen-Ming Zeng b, Gui-Xue Zhang a, * a
Laboratory of Animal Reproductive Science, College of Animal Science and Technology, Northeast Agricultural University, Harbin, China b National Key Laboratory of Animal Nutrition, Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, China Agricultural University, Beijing, China c Animal Husbandry and Veterinary Institute, Heilongjiang Academy of Land Reclamation Sciences, Harbin, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 November 2014 Received in revised form 14 May 2015 Accepted 20 May 2015
Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the ratelimiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol sidechain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Lysosome Low-density lipoprotein StAR Granulosa cell Bovine
1. Introduction Progesterone is one of the fundamental steroid hormones for the regulation of the bovine estrous cycle. The machinery of progesterone biosynthesis involves multiple critical components, such as cholesterol side-chain cleavage * Corresponding author. Tel.: þ86 451 55191421; fax: þ86 451 55103336. E-mail address: [email protected] (G.-X. Zhang). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.05.016
cytochrome P-450 (CYP11A1), which catalyzes the conversion of cholesterol to pregnenolone, and steroidogenic acute regulatory protein (StAR), which transports cholesterol from the outer to the inner mitochondrial membrane [1–4]. Before ovulation, estradiol is the primary steroid secreted by the follicles [5], while concentration of progesterone in the follicular fluid is low [6–8]. This may be due to the low to undetectable expression of StAR messenger RNA (mRNA) in follicular granulosa cells, although StAR and CYP11A1 are expressed in theca cells [5]. After ovulation, the
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preovulatory LH surge results in the luteinization of the residual granulosa and theca cells [9–11]. The ovarian follicle switches from mainly secreting estradiol to producing large amount of progesterone. The markedly enhanced progesterone biosynthesis is attributed to the increased expression of StAR and CYP11A1 [1–4]. The expression of StAR is regulated at different levels by many hormones and growth factors. Insulin, LH, FSH, insulinlike growth factor 1, transforming growth factor b, and forskolin have been shown to stimulate StAR mRNA expression in diverse species [12–17]. Numerous transcription factors, such as steroidogenic factor 1, GATA4/6, sterol regulatory element-binding proteins 1A, CCAAT/ enhancer-binding proteins, and cAMP response elementbinding protein, participate in StAR promoter activation [18–23]. Epigenetic mechanisms, including histone modifications, DNA methylation, and chromatin remodeling, are also involved in StAR gene expression in granulosa cells of bovine [24,25] and other species [26–28]. In addition, a recent study reported that StAR gene expression was downregulated under the adverse metabolic environment caused by lactation or nutritional restriction in cattle [29]. Moreover, accumulated evidence has shown that lipoprotein, as a nutritive material or metabolic substrate, is also involved in the regulation of StAR expression [30,31]. Progesterone biosynthesis requires cholesterol as a primary substrate. There are two major carriers of blood cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) [32]. The LDL is primarily used as a steroidogenic substrate for progesterone production by bovine granulosa cells [33]. Furthermore, LDL has been shown to have stimulatory effects on progesterone biosynthesis in cultured bovine luteal cells [34,35] and swine granulosa cells [36,37]. This stimulation of progesterone biosynthesis may be due to the transcriptional regulation effects of LDL on the expression of CYP11A1 protein as shown in bovine granulosa cells [38]. In addition, StAR expression in mouse nonovarian cells was upregulated by LDL [30,31]. However, it is currently unknown whether LDL has a similar role for increasing StAR expression in bovine granulosa cells. The uptake of LDL occurs through receptor-mediated endocytosis. Once LDL is internalized, the endosomes fuse with lysosomes, followed by lysosomal degradation of cholesteryl ester to release free cholesterol [39]. Inhibiting lysosomal function suppresses this process [40] and consequently blocks LDL-induced progesterone synthesis [41]. Thus, it seems reasonable to suggest that the lysosomal degradation pathway of LDL is indispensable for progesterone biosynthesis in granulosa cells after ovulation. However, the role of lysosomes in LDL regulation of StAR expression is largely unknown. Therefore, in this study, we test the hypothesis that LDL may promote StAR expression and lysosomes may participate in regulating this process. 2. Materials and methods
Low-density lipoprotein (95% purity, 78%–81% lipid; 19%– 22% protein, dissolved in 150-mM NaCl, 0.01% EDTA, pH 7.4) was purchased from Calbiochem (San Diego, CA, USA). Radioimmunoassay kits were obtained from the Beijing North Institute of Biological Technology. All other reagents were from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA), unless otherwise indicated. All plasticware was from Nalge Nunc International (Roskilde, Denmark). Chloroquine (CQ) was dissolved in water at a concentration of 50 mmol/ L as a 1000X stock solution. 2.2. Primary culture and treatment of granulosa cells Ovaries were collected from cows on Days 16 to 21 of the estrous cycle as described previously [42]. They were collected at a local abattoir within 20 minutes after slaughter, placed immediately in 0.9% NaCl at 37 C, and transported to the laboratory in 2 hours. Follicular fluid that contained granulosa cells was collected from a heterogonous population of follicles (diameter, 2–8 mm) by aspiration using a 20-ga needle. The fluid was transferred into 15-mL centrifuge tubes and left standing for at least 5 minutes to precipitate the cumulus–oocyte complexes. The supernatant was centrifuged at 200 g for 5 minutes to collect granulosa cells, which were then washed three times in serum-free medium. Petri dishes (35 mm) and coverslips were pretreated with DMEM/F12 containing 10% fetal bovine serum for 18 hours at 37 C in a humidified incubator, followed by three washes, to get coated by seral proteins. The granulosa cells (1 106 viable cells/dish) were cultured in humidified air with 5% CO2 at 37 C in DMEM/F12 for 12 hours either on the Petri dishes or on coverslips placed in the Petri dishes. Then, cells were washed twice with DMEM/F12 to remove nonadherent cells and further cultured with fresh control culture medium (DMEM/F12) or medium containing specific components for the indicated time. On the basis of previous reports examining plasma LDL concentrations and physiological effects of LDL on cells [30,34,39,43], 20 mg of protein/mL of LDL was used in this study. The concentration of lysosomal inhibitor CQ (50 mmol/L) used here was with low cytotoxicity, as reported in previous studies [40,44–46]. The following treatment groups were used: (1) control, DMEM/F12; (2) LDL, control culture medium plus LDL (20 mg of protein/mL); (3) LDL plus CQ, control culture medium plus LDL (20 mg of protein/mL) and CQ (50 mmol/L). Three independent replicates of culture were performed for each treatment. 2.3. Radioimmunoassay Culture media obtained at the indicated time were analyzed for progesterone production using RIA kits. The intra-assay and interassay coefficients of variability were less than 10% and 6%, respectively. Assay sensitivity was 0.2 ng/mL. To check whether added LDL interferes the result of progesterone RIA, the unspent control and LDL-added media were also analyzed for progesterone using RIA kits.
2.1. Materials 2.4. Quantitative PCR Dulbecco’s modified Eagle’s medium containing Ham’s F12 (1:1 [v:v] DMEM/F12) and fetal bovine serum were obtained from Gibco Life Technologies (Carlsbad, CA, USA).
Quantitative polymerase chain reaction (PCR) was performed using real-time PCR Master Mix containing SYBR
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Green (Toyobo) and gene-specific primers on a Stratagene Mx3000P real-time quantitative PCR machine (Stratagene). Total RNA was isolated using Trizol reagent (Takara). The RNA samples were reverse transcribed into complementary DNA with a RevertAid First Strand cDNA Synthesis Kit (Fermentas). GAPDH was used as a housekeeping gene [15,26,47]. The specific primer sequences for StAR were forward: 50 CATGGTGCTCCGCCCCTTGG-30 and reverse: 50 -CGCTTGCGC AGGTGATTGGC-3’ (National Center for Biotechnology Information reference sequence: NM_174189.2). The specific primer sequences for CYP11A1 were forward: 50 -GCTCCAGAGGCAATAAAGAAC-30 and reverse: 50 -GACTCAAAGGCAAA GTGAAACA-30 (National Center for Biotechnology Information reference sequence: NM_176644.2). The specificity of the PCR products was verified using a melting curve analysis. Fold differences in mRNA levels over the control values were calculated using the 2DDCt method. The PCR reactions were run in triplicate for each sample, and at least three independent experiments were conducted.
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three washes with PBS, cells were incubated for 1 hour with a goat antirabbit Cy3-conjugated antibody (1:500, 111-165046; Jackson ImmunoResearch). The cell nuclei were counterstained with Hoechst 33342. The resulting fluorescence was visualized with a Nikon (TE2000) inverted C1 confocal microscope using Nikon EZ-C1 software and a Zeiss SM-510 laser scanning confocal system (Carl Zeiss). Fluorescence pixel intensities were measured as described by Peter Bankhead (available on http://blogs.qub.ac.uk/ccbg/ files/2013/06/Analyzing_fluorescence_microscopy_images. pdf). 2.8. Statistical analysis Data are presented as the mean standard error of the mean. Independent samples t tests and one-way ANOVA
2.5. Western blot analysis Western blot analyses were performed as described previously [48]. After blocking, the membranes were incubated with primary antibodies (4 C, overnight) against StAR (rabbit antihuman StAR antibody, 1:1000 dilutions, #8449; Cell Signaling Technology, Danvers, MA, USA) or b-actin (1:2000 dilutions, goat antirabbit; Zhongshan Biotechnology, Beijing, China). After 1 hour of incubation at room temperature with secondary antibodies (1:5000 dilutions, antigoat or antirabbit immunoglobulin G horseradish peroxidase linked, Zhongshan Biotechnology), the protein bands were visualized using enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China) and X-OMAT BT film (Eastman Kodak Co.). The films were digitized, and densitometry analysis was performed with ImageJ 1.44p software (National Institutes of Health). The relative intensities of the bands were quantified and normalized to the respective loading controls. 2.6. Acridine orange vital staining The cells grown on coverslips were vitally stained at the indicated time with acridine orange (AO) solution (10 mmol/L in complete medium) for 15 minutes at 37 C and then washed with PBS. The coverslips were mounted on a glass slide. Images were obtained using a Nikon (TE2000) inverted C1 confocal microscope with Nikon EZC1 software and a Zeiss SM-510 laser scanning confocal system (Carl Zeiss). 2.7. Immunofluorescence staining Cells were fixed with 4% paraformaldehyde in PBS for 10 minutes and washed twice with PBS. Cells were then permeabilized with 0.3% Tween-20 for 20 minutes and washed with PBS three times. Blocking was performed with 0.3 mol/L of glycine in 0.05% Tween-20 in PBS for 30 minutes. Cells were incubated with an anti-LAMP1 antibody diluted (1:1000; ab24170; Abcam, Cambridge, MA, USA) in 1% BSA in 0.05% Tween-20 in PBS overnight at 4 C. After
Fig. 1. Effects of low-density lipoprotein (LDL) on progesterone biosynthesis and expression levels of steroidogenic acute regulatory (StAR) messenger RNA (mRNA) and protein in bovine granulosa cells. Cells were cultured in DMEM/F12 for 12 hours and then cultured in control medium without (Con) or with LDL (20-mg protein/mL; LDL) for 24 hours. (A) Progesterone concentrations normalized to 1 105 cells for each sample are represented as the mean standard error of the mean (SEM) of five independent experiments. (B) The mRNA expression was analyzed using quantitative polymerase chain reaction (qPCR). Data are normalized to the internal control GAPDH mRNA and expressed as the fold change from control SEM for four independent experiments. (C) The immunoblots of StAR and b-actin protein expression levels; (D) StAR expression was normalized to b-actin expression after being quantified using ImageJ; statistical data from four independent experiments are shown as fold changes from control SEM. (E) The CYP11A1 mRNA expression was analyzed using qPCR. Data are normalized to the internal control GAPDH mRNA and expressed as the fold change from control SEM for three independent experiments. The asterisk (*) indicates a significant difference from the control group (P < 0.05).
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Fig. 2. Effect of low-density lipoprotein (LDL) on LAMP1 distribution in bovine granulosa cells. Cells grown on coverslips were cultured in control medium (A, Con) or control medium containing 20-mg protein/mL of LDL (A, LDL) for 24 hours. The cells were fixed, permeabilized, and stained for LAMP1. (B) Bar graph showing the average intensity (mean standard error of the mean) of LAMP1 fluorescence (red) of 15 different cells for each culture from four independent experiments. Scale bar: 10 mm. The asterisk (*) indicates a significant difference (P < 0.05) from the control group. (For interpretation of the references to color in this figure, the reader is referred to the Web version of this article.)
were performed using the SPSS 16.0 software package (Chicago, IL, USA) for statistical data analysis. A value of P < 0.05 was considered statistically significant. 3. Results 3.1. LDL enhances StAR expression and progesterone production in cultured bovine granulosa cells To characterize the role of LDL in progesterone synthesis, we first examined its effects on granulosa progesterone production and expression of StAR, the critical gene positively promoting this process. The treatment with LDL (20 mg of protein/mL) for 24 hours stimulated more progesterone production (155.61 35 ng/105 cells) in granulosa cells than in untreated control cells (30.31 6.2 ng/ 105 cells; P < 0.05; Fig. 1A). Compared with the control, LDL treatment also induced approximately 2.29- and 2.71-fold increases in StAR mRNA and protein levels, respectively (P < 0.05; Fig. 1B, C, D). In addition, the expression of
CYP11A1 mRNA was also estimated. The result showed that CYP11A1 mRNA was increased by LDL treatment up to approximately 2.33-fold compared with the control (P < 0.05; Fig. 1E). In general, LDL exhibits stimulatory effects on biosynthesis of progesterone, which may be undertaken through upregulating StAR and CYP11A1 gene expression. 3.2. LDL promotes the increasing of lysosomes in cultured bovine granulosa cells Because the lysosomal degradation pathway of LDL is indispensable for progesterone biosynthesis, we next studied the changes of lysosomes. We visualized lysosomes using two different approaches: AO staining and immunostaining with anti-LAMP1 antibodies. As shown in Figure 2, LDL induced a wider distribution of LAMP1 fluorescence (Fig. 2A) in granulosa cells than in control cells, and the fluorescence intensity (Fig. 2B) was significantly increased in the LDL-treated cells (0.93 0.02) compared
Fig. 3. Effect of low-density lipoprotein (LDL) on acridine orange (AO)–stained lysosomes in bovine granulosa cells. Cells grown on coverslips were cultured in control medium (A, Con) or control medium containing 20-mg protein/mL of LDL (B, LDL) for 24 hours. Cells were subsequently loaded with AO. (C) Bar graph showing the number (mean standard error of the mean) of red granules (AO-stained lysosomes) per cell in 15 different cells for each culture from four independent experiments. Scale bar: 10 mm. The asterisk (*) indicates a significant difference (P < 0.05) from the control group.
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with that in control cells (0.57 0.14, P < 0.05). The AO staining (Fig. 3) showed that more lysosomes (red granules) (58.2 5.5/cell) appeared in the granulosa cells treated with LDL than in control cells (25.4 3.0/cell, P < 0.05). These results indicate that the added LDL promotes more lysosomes appearing in cytoplasm. 3.3. Lysosomal activity is essential for StAR protein expression and progesterone production in cultured bovine granulosa cells To determine whether the synthesis of LDL-induced StAR and progesterone was mediated by lysosome activation, we used a specific inhibitor CQ to inhibit lysosomal activity. As a diprotic weak base, CQ is a lysosomotropic agent. The unprotonated form of CQ preferentially accumulates in lysosomes, increasing pH values in lysosomes and inhibiting lysosomal enzyme activities. As shown in Figure 4A, CQ blocked the LDL-induced progesterone synthesis. Treatment with CQ strikingly abolished the stimulation of progesterone production by LDL (19.2 7.5 ng/105 vs. 178.5 9.4 ng/105 cells, P < 0.05), close to the baseline level observed in the control cells (27 4.7 ng/105 cells, P > 0.05). In addition, as shown in Figure 4B, C, CQ inhibited the LDL-induced StAR protein expression. These results indicate that lysosomal activity is essential for the induction of progesterone synthesis and StAR expression by LDL. 4. Discussion Our present study provides new insights into the effects of LDL to promote progesterone synthesis in bovine granulosa cells. The major changes that occur during the process of luteinization are marked increases in progesterone synthesis and the levels of both StAR and cytochrome P-450 [49,50]. Although LH, FSH, and cAMP induce the synthesis of progesterone and the expression of StAR and cytochrome P450 in cultured granulosa cells, these inductive effects in the follicle mostly occur before ovulation under physiological
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condition [6,49,51]. In the follicular phase, granulosa cells do not have access to LDL because the content of LDL in follicular fluid is very low compared with that in plasma [43]. However, after ovulation, with the rupture of the follicular wall and angiogenesis, granulosa cells are immersed in a high LDL microenvironment. Similar to previous reports in bovine [34,35] and swine [36,37], our results showed that LDL promoted progesterone production in cultured bovine granulosa cells, whereas the unspent LDL in medium did not interfere in progesterone RIA (data not shown). One report showed that synthesis of CYP11A1 protein was induced by LDL in cultured bovine granulosa cells [38], and our study also showed that expression of CYP11A1 mRNA was increased by LDL. These results suggest that LDL is involved in the regulation of progesterone synthesis process. So this outcome of LDL promoting progesterone production is attributed to more than just an increase in available cholesterol derived from LDL for progesterone production. In addition, our study has revealed for the first time that expression of the StAR mRNA and protein was upregulated by LDL in cultured bovine granulosa cells. Others have reported similar results in nonovarian cells [30,31]. A portion of the cholesterol derived from LDL can be oxidized by specific hydroxylases to generate oxysterols in cells. Evidence indicates that StAR protein levels are positively regulated by oxysterols [31,52,53]. However, the regulation of StAR expression by LDL is not mediated by an oxysterol intermediate. A possible explanation for the result is that LDL increases StAR expression through a mechanism that is dependent on the upregulation of the StAR promoter and transcription factor steroidogenic factor 1 activity [30]. According to a study examining the rat StAR promoter, the increased cholesterol may regulate StAR transcription through a pathway involving the binding of sterol regulatory element-binding proteins to sterol regulatory elements located in the promoter regions of StAR genes [54]. Cells derive cholesterol from LDL through lysosomal degradation. Lysosomes have long been regarded as dynamic and terminal organelles that receive and degrade
Fig. 4. Effects of chloroquine (CQ) on low-density lipoprotein (LDL)–induced synthesis of progesterone and expression of steroidogenic acute regulatory (StAR) protein. Granulosa cells were cultured with control medium (Con), or control medium containing 20 mg of protein/mL of LDL (LDL) or 20 mg of protein/mL of LDL plus 50 mmol/L of CQ (LDL þ CQ) for 24 hours. (A) Progesterone concentrations were normalized to 1 105 cells for each sample and are represented as the mean standard error of the mean (SEM) for five independent experiments. (B) The immunoblots of StAR and b-actin protein expression levels. (C) The data were normalized to b-actin expression after being quantified by ImageJ; statistical data from four independent experiments are shown as fold changes from control SEM. An asterisk (*) indicates a significant difference (P < 0.05) from the other two groups.
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materials from the secretory, endocytic, autophagic, and phagocytic membrane-trafficking pathways in animal cells [55]. A previous study showed that treatment of porcine granulosa cells with CQ completely blocked LDL-induced stimulation of progesterone synthesis [37]. Our study revealed that once the function of lysosomes was inhibited, the stimulatory effect of LDL on StAR protein expression was suppressed, and progesterone production by granulosa cells also decreased. Intriguingly, our studies using AO staining and immunostaining with anti-LAMP1 antibodies showed that the number of lysosomes was increased by LDL treatment in cultured bovine granulosa cells. A similar result was shown in another study, which found that LDL enlarged endolysosomes and increased LAMP1 expression in cultured embryonic rat neurons [56]. Lysosome biogenesis requires complex integration of the endocytic and biosynthetic pathways of the cell. The best understood pathway is the mannose-6-phosphate receptor (M6PR)– mediated transport of lysosomal hydrolases [56,57]. In addition, the M6PR-independent pathways, lysosomal integral membrane protein 2 and sortilin were recently reported [58]. By contrast, very little is known about the structural and molecular machinery for the transport of lysosomal membrane proteins to lysosomes. Although the exact mechanism for the increase in the number of lysosomes by LDL is still unknown, it can be concluded that lysosomes mediate the effect of LDL on promoting progesterone synthesis and StAR expression in bovine granulosa cells and that cholesterol may be an important factor in this process. In conclusion, lysosomes mediate the effects of LDL to promote progesterone synthesis and StAR expression in bovine granulosa cells. The results of this study extend our knowledge about the luteotrophic effects of LDL on granulosa cells after ovulation in vivo. After ovulation, luteinizing granulosa cells take up plasma LDL, which consequentially induces more lysosomes, resulting in marked increases of StAR and CYP11A1 expression and progesterone synthesis. Acknowledgments The authors thank Haq Ihtesham and Dr Fei Gao (Cincinnati Children’s Hospital Medical Center, USA) for critical reading of the article. This work was supported by a grant from the National Natural Science Foundation of China (Grant 31172209).
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