This web only provides the extract of this article. If you want to read the figures and tables, please reference the PDF full text on Blackwell Synergy. Thank you.

- Original Article -

Cox7a2 mediates steroidogenesis in TM3 mouse Leydig cells

Liang Chen¹, Zhong-Cheng Xin¹, Xin Li², Long Tian¹, Yi-Ming Yuan¹, Gang Liu¹, Xue-Jun Jiang³, Ying-Lu Guo¹

¹Andrology Center of Peking University First Hospital, Beijing 100009, China

²Department of Surgery, People's Hospital of Zhangqiu, Zhangqiu 250200, China

³Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China

Abstract

Aim: To investigate the regulatory function of Cox7a2 on steroidogenesis and the mechanism involved in TM3 mouse Leydig cells.  Methods: The cDNA of Cox7a2 was cloned from TM3 mouse Leydig cells. It was subcloned to pDsRed-Express-N1 and transfected back into TM3 mouse Leydig cells for Cox7a2 overexpression by transient gene transfection. Steroidogenesis affected by overexpressed Cox7a2 was studied by ELISA. To elicit the mechanism of this effect, expression of steroidogenic acute regulatory (StAR) protein and reactive oxygen species (ROS) were examined by Western blot and fluorometer, respectively.  Results: The cDNA of Cox7a2 (249 bp) was cloned from Leydig cells and confirmed by DNA sequencing. After constructed pDsRed-Express-N1-Cox7a2 was transfected back into TM3 mouse Leydig cells, Cox7a2 inhibited not only luteinizing hormone (LH)-induced secretion of testosterone but also the expression of StAR protein. At the same time, Cox7a2 increased the activity of ROS in TM3 mouse Leydig cells.  Conclusion: Cox7a2 inhibited LH-induced StAR protein expression, and consequent testosterone production, at least in part, by increasing ROS activity in TM3 mouse Leydig cells. (Asian J Androl 2006 Sep; 8: 589_594)

Keywords: Cox7a2; fusion protein; steroidogenesis; steroidogenic acute regulatory protein; reactive oxygen species; Leydig cell

Correspondence to: Dr Zhong-Cheng Xin, Andrology Center of Peking University First Hospital, Beijing 100009, China.

Tel/Fax: +86-10-6618-2822
E-mail: xinzc@bjmu.edu.cn
Received 2005-10-31 Accepted 2006-03-18
DOI: 10.1111/j.1745-7262.2006.00178.x

1 Introduction

Progressive decrease of testosterone production is a common phenomenon in aging men, and is accompanied by a series of clinical manifestations, such as decreased libido, erectile dysfunction (ED), osteoporosis, fatigue, depression and alterations in mood and cognition [1, 2]. The syndrome of these clinical symptoms is referred to as partial androgen deficiency in aging male (PADAM) [2, 3]. Although it has been suggested that the decline of serum testosterone plays a major role in PADAM, other factors might also be involved [3_6]. Leydig cells are the main producer of testosterone in mammalian testes; however, the mechanism underlying the age-related functional deficiency of Leydig cells in steroidogenesis remains unclear [2_4].

It is well established that mitochondria contain proteins and enzymes essential for testosterone biosynthesis. For example, steroidogenic acute regulatory (StAR) protein, which mediates the rate-limiting step in steroidogenesis, is located in the inner membrane of mitochondria [7_9].

During mitochondrial electron transport reactions, the by-products, reactive oxygen species (ROS), can be produced and cumulated continuously [4_6]. Prolonged exposure to ROS can cause cumulative oxidative damage, which is thought to be one of the major causes of cellular aging and reductive ability to produce testosterone in Leydig cells [5, 6].

Cox7a2 is the seventh subunit of the nuclear-coded polypeptide chains of cytochrome c oxidase in the mitochondrial respiratory chain [10, 11]. Published studies demonstrate that Cox7a2 gene is significantly upregulated in testes of aged men [10], but the connection between the increase of Cox7a2 and the decrease of testosterone production in testes of aged men is unknown.

The aim of the present study is to investigate the effect of Cox7a2 on steroidogenesis and the involved mechanism. TM3 mouse Leydig cells (TM3 cells) are used for the study of steroidogenesis because these cells express luteinizing hormone (LH) receptors and StAR protein and are responsive to LH [12, 13]. Cox7a2 overexpressed in TM3 cells by transient transfection of recombinant Cox7a2 cDNA plasmid. LH-induced testosterone production is observed in these cells overexpressing Cox7a2. To elicit the influence of Cox7a2 on steroidogenesis, ROS level and the expression of StAR protein are also measured.

2 Materials and methods

2.1 Materials and apparatus

TM3 mouse Leydig cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Oligo-dT and Pfu DNA polymerase were obtained from Promega (Madison, WI, USA). Restriction endonucleases were from New England Biolabs (Beverly, MA, USA). Recombinant plasmid DNA purification kit and effectene transfection reagent were from Qiagen (Valencia, CA, USA). CM-H2DCFDA was from Molecular Probes (Invitrogen, Carlsbad, CA, USA). Fluorescence imaging microscopy system (ECLIPSE TE2000) was the product of Nikon (Tokyo, Japan). Rabbit anti-StAR antibody was from Santa Cruz (Santa Cruz, CA, USA). Testosterone ELISA kit was from DRG (Marburg, Germany). LH was bought from Sigma (St. Louis, MO, USA).

2.2 Methods

2.2.1 Plasmid construction

TM3 cells were cultured in F12-DME medium supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, 5% horse serum and 2.5% fetal calf serum. 5.0 × 10⁶ cultured TM3 cells were washed with PBS solution and were collected by centrifugation (1 000 × g for 3 min). Total RNA of TM3 cells was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA). Three micrograms of total RNA were reversely transcribed to first strand cDNA using oligo (dT) as the primer and M-MLV reverse transcriptase (Promega, Madison, WI, USA) [14]. The coding region of mouse Cox7a2 cDNA was obtained by polymerase chain reaction (PCR) using the first strand cDNA as the template. The forward primer was 5’-gaagatctatgttgc-ggaatctgctggcc-3’ and the reverse primer was 5’-cggaatt-cgcgttctgcttcttgggga-3’. After being digested with EcoRI and BglII, the PCR product was cloned into the plasmid pDsRed-Express-N1 (BD Biosciences Clontech, Franklin Lakes, NJ, USA). The recombinant plasmid was further confirmed by dideoxynucleotide sequencing.

2.2.2 Cell transfection

TM3 cells were cultured in the above medium and split into 60 mm dishes. When the cells reached 65% confluence, the transfection was performed using effectene transfection reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions, with vector as a control. Cox7a2-pDsRed-Express-N1 (1.5 µg) and pDsRed-Express-N1 (1.5 µg) were diluted separately in Buffer EC (the enhancer and the DNA-condensation buffer) to a total vo-lume of 150 µL and 8 µL enhancer was added and mixed by vortexing for 1 s. After incubating at 20ºC for 5 min, the 25 µL effectene transfection reagent was added to the DNA-enhancer mixture and the mixture was vortexed for 10 s. During the waiting time, the growth medium of the plates was gently aspirated and 3 mL of fresh growth medium was added to the cells. While being incubated at 20ºC for 10 min, 1 mL growth medium was added to the transfection complexes, which were then added to the cells in the 60 mm dishes. Finally, the cells with the transfection complexes were incubated under an atmosphere containing 95% air and 5% CO₂ at 37ºC. After transfected for 12 h, the cells were treated with LH 12 h (100 ng/mL) for another 12 h. Then the red fluorescence was acquired through Cy3 channel (excitation: 557 nm; emission: 579 nm). The transfection efficiency was estimated by cell counting under fluorescence microscope, and Cox7a2 expression was determined by reverse transcriptase (RT)-PCR (forward primer: 5’-cgtcagattgggcagag-3’; reverse primer: 5’-ggaaatga agccacagc-3’). The same procedure as described above were performed for total RNA extraction and RT-PCR. The cell transfection was repeated for three times.

2.2.3 Evaluation of StAR protein and testosterone production

Samples of culture medium in process 2.2.2 were assayed for testosterone release using a testosterone ELISA kit (DRG, Marburg, Germany), according to the manufacturer’s instructions. Testosterone level was normalized to protein concentration for each sample and was expressed as nanogram/mL media. Each sample was quadruplicated for testosterone measurement and the experiment was repeated for three times. Part of the cells was lysed in cold TGH lysis buffer (1% Triton X-100, 10% glycerol, 20 mmol/L Hepes [pH 7.4], 100 mmol/L NaCl, 1 mmol/L phenylmethy-lsulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mmol/L Na₃VO₄ and 50 mmol/L NaF). The lysate was separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was incubated in a blocking buffer (Tris Buffered Saline [TBS] containing 5% nonfat dry milk) for 1 h, followed by wash with TBS containing 0.1% Triton X-100 (TBST) (3 × 10 min). Subsequently, the membranes were probed with rabbit anti-StAR antibody (incubation overnight at 4ºC, 1:500 TBST, 1% milk), rabbit anti-b-actin antibody (incubation overnight at 4ºC, 1:5 000 TBST, 1% milk). The secondary antibody was the horseradish peroxidase conjugated goat anti-rabbit IgG antibody. Blotted antibodies were then visualized by enhanced chemoluminescence (Amersham, Piscataway, NJ, USA). The experiment was repeated for three times. To determine the linear range of the chemiluminescence signals, several X-ray radiographs were analyzed, and densitometry was performed for quantification by using AlphaImager 2200 and Alpha Ease image software (Alpha Technology, San Francisco, CA, USA). StAR protein expression level was normalized to that of b-actin and was expressed as the optical density ratio of the StAR band to the b-actin band for further analysis.

2.2.4 Measurement of reactive oxygen species (ROS) production

CM-H2DCFDA (Invitrogen, Carlsbad, CA, USA), an acceptable cell-permeant indicator of ROS, was used to assess the ROS level within the TM3 cells [5]. Cells in process 2.2.2 were harvested by centrifugation (1 000 × g for 3 min) and washed in serum free medium (pH 7.2, buffered in Hepes) three times. The cells were re-suspended in serum free culture medium to 10⁵/mL. The cells were added 2 µL of 0.5 mmol/L CM-H2DCFDA (final concentration) and incubated at 37ºC for exact 10 min. Finally, the cells were plated into the 96-well plate. The cells incubated with 1 mmol/L H₂O₂ for 30 min at 37ºC were used as positive controls [15, 16]. The relative level of ROS within the cells was analyzed by microplate fluorometer (excitation: 488 nm, emission: 535 nm) (BMG labtech-nology, Offenburg, Germany), and the data were expressed as the relative numerical values by detecting the increase of fluorescence. Each sample was quadruplicate for ROS measurement and the experiment was replicated three times.

2.2.5 Statistical analysis

Data were presented as means ± SME of testosterone production and ROS relative level in three independent experiments carried out in quadruplicate under each sample or of the ratio of StAR to b-actin integrated density of three separate experiments. To compare the significance of the two means obtained from LH-treated and LH-untreated samples, statistical analysis was performed using the unpaired t-test. The significant differences between various means (more than two means) were determined by analysis of variance using the statistical package SPSS 11.0 for Windows (SPSS, Chicago, IL, USA). P < 0.05 was considered as statistically significant.

3 Results

3.1 Expression of fusion fluorescence protein in TM3 cells

An image of red fluorescence Cox7a2 was observed 24 h post transfection, as shown in Figure 1. Among 200 cells counted, 85% of them were stained with red fluorescence. The transfection efficiency of the three separate experiments was similar to each other. The expression of Cox7a2 was also determined by RT-PCR. An approximate four-fold increase of expressing Cox7a2 was demonstrated (data not shown).

3.2 Cox7a2 inhibited LH-induced increase of testosterone synthesis

Upon overexpressed Cox7a2, we examined the testosterone synthesis in TM3 cells in presence or absence of LH. Testosterone synthesis induced by LH stimulation was inhibited in TM3 cells overexpressing Cox7a2 (P < 0.01, compared with non-transfected and vector-transfected TM3 cells with LH stimulation). Testosterone concentration in the media did increase dramatically in non-transfected or vector-transfected TM3 cells with LH stimulation (P < 0.01, compared with their respective counterparts without LH stimulation) (Figure 2). Each experiment was performed independently for three times and the results were similar.

3.3 Cox7a2 inhibited LH-induced increase of StAR protein

StAR protein mediates the transfer of cholesterol from outer mitochondrial membrane to inner mitochondrial membrane where the cholesterol is cleaved to pregnenolone, a rate-limiting step in testosterone synthesis [7_9]. Therefore, we investigated the amount of StAR protein in TM3 cells overexpressing Cox7a2 in presence or absence of LH. Expression of StAR protein did increase considerably in non-transfected or vector-transfeced TM3 cells with LH stimulation (P < 0.01, compared with their respective counterparts without LH stimulation). The induced increase of StAR protein by LH stimulation did not occur any more in TM3 cells overexpressing Cox7a2 (P < 0.01, compared with non-transfected and vector-transfected TM3 cells with LH stimulation) (Figure 3). Each experiment was performed independently for three times and the results were similar.

3.4 Cox7a2 enhanced ROS production

ROS, mainly produced in the respiratory chain of mitochondria, are involved in negatively regulating the expression of StAR protein [5, 6, 15, 16]. We examined the possible connection between ROS level and Cox7a2 overexpression. As shown in Figure 4, stimulated by LH, the level of ROS increased significantly in TM3 cells overexpressing Cox7a2 (P < 0.05, compared with TM3 cells that overexpressed Cox7a2 fusion protein without LH stimulation; P < 0.01, compared with non-transfected and vector-transfected cells with LH stimulation). In non-transfected and vector-transfected cells, the production of ROS changed little with or without LH stimulation. Each experiment was performed independently for three times and the results were similar.

4 Discussion

Testosterone production in Leydig cells depends on LH stimulation. Upon binding to its receptor, LH initiates the translocation of cholesterol into the mitochondria and triggers the process of androgen biosynthesis with the involvement of upregulated StAR protein [7_9, 17, 18]. Our findings (Figures 2, 3) on testosterone production in TM3 cells in response to LH treatment supports the previous observations. Most importantly, we find that Cox7a2 plays a role in mediating testosterone synthesis. Cox7a2 is able to inhibit androgen production induced by LH (Figure 2).

At the same time, our findings also demonstrate that Cox7a2 inhibits the expression of StAR protein induced by LH in TM3 cells (Figure 3). Considerable evidences indicate that the expression of StAR protein is the key regulatory event delivering cholesterol to the inner mitochondrial membrane, which constitutes the rate-limiting step in testosterone synthesis [6_9]. Therefore, our results suggest that the inhibiting effect of Cox7a2 on testosterone production might be, at least, a result of the disruption of the expression of StAR protein (Figure 3), that is, the disruption of the cholesterol-transferring process. StAR protein is primarily translated as a larger molecule (37 kDa) containing mitochondria targeting precursor in the amino terminus [8, 9]. During the cholesterol transferring process, the larger StAR molecule is transferred into mitochondria and transformed into the mature form (30 kDa) [8, 9]. In the present study, we used rabbit anti-StAR antibody, which recognized the 30 kDa StAR protein. Therefore, the translocation of StAR protein onto mitochondria might also be influenced by Cox7a2, which needsto be further validated.

The data suggest that the inhibiting effect of Cox7a2 on StAR protein expression might be partially involved in the enhancement of ROS level. Former studies indicate that ROS mediates mitochondrial perturbation of Leydig cell, decreases the expression of StAR protein and results in the inhibition of testosterone production [15, 16]. Our findings on TM3 cells overexpressing Cox7a2 are consistent with these observations. By analysis of ROS level, we reveal that the decrease of testosterone production and expression of StAR protein upon overexpression of Cox7a2 are possibly because of the increased production of ROS in TM3 cells (Figure 4).

However, the mechanism of steroidogenesis in Leydig cells is complicated and remains obscure so far [6, 7]. Some recent reports indicate that the peripheral-type benzodiazepine receptor is also an indispensable element mediating the delivery of cholesterol to the inner mitochondrial membrane [19, 20]. Further research should be performed to fully understand the steroidogenic process.

In summary, data presented here reveal an unknown role of Cox7a2 in the regulation of the expression of StAR protein, and in its consequent mediating androgen biosynthesis. In TM3 cells, the negative regulatory effect of Cox7a2 on steroidogenesis is, at least, a result of the increase of ROS production.

Acknowledgment

This work was supported by the "211" Project of the "Tenth-Five" Program for Peking University Health Science Center, China (No 219). We would like to thank Professor Ding-Fang Bu (China) for his critical reading of this paper.

References

1 Lunenfeld B. Aging men-challenges ahead. Asian J Androl 2001; 3: 161_8.

2 Morales A, Heaton JP, Carson CC 3rd. Andropause: a misnomer for a true clinical entity. J Urol 2000; 163: 705_12.

3 Wang C, Leung A, Sinha-Hikim AP. Reproductive aging in the male Brown Norway rat: a model for the human. Endocrino-logy 1993; 133: 2773_81.

4 Walker DW, Benzer S. Mitochondrial "swirls" induced by oxygen stress and in the Drosophila mutant hyperswirl. Proc Natl Acad Sci USA 2004; 101: 10290_5.

5 Hoffmann S, Spitkovsky D, Radicella JP, Epe B, Wiesner RJ. Reactive oxygen species derived from the mitochondrial respiratory chain are not responsible for the basal levels of oxidative base modifications observed in nuclear DNA of Mammalian cells. Free Radic Biol Med 2004; 36: 765_73.

6 Zirkin BR, Chen H. Regulation of Leydig cell steroidogenic function during aging. Bio Reprod 2000; 63: 977_81.

7 Christenson LK, Strauss JF 3rd. Steroidogenic acute regulatory protein (StAR) and the intramitochondrial translocation of cholesterol. Biochim Biophys Acta 2000; 1529: 175_87.

8 Stocco DM. Recent advances in the role of StAR. Rev Reprod 1998; 3: 82_5.

9 Stocco DM. StARTing to understand cholesterol transfer. Nat Struct Biol 2000; 7: 445_7.

10 Xin Z, Liu W, Tian L, Yuan Y, Xin H, Fu J, et al. Studies on the testis regression related gene profile in aged male. Beijing Da Xue Xue Bao 2003; 35: 364_8.

11 Chrzanowska-Lightowlers ZM, Turnbull DM, Bindoff LA, Lightowlers RN. An antisense oligodeoxynucleotide approach to investigate the function of the nuclear-encoded subunits of human cytochrome c oxidase. Biochem Biophys Res Commun 1993; 196: 328_35.

12 Musa FR, Takenaka I, Konishi R, Tokuda M. Effects of luteinizing hormone, follicle-stimulating hormone, and epidermal growth factor on expression and kinase activity of cyclin-dependent kinase 5 in Leydig TM3 and Sertoli TM4 cell lines. J Androl 2000; 21: 392_402.

13 Mather JP. Establishment and characterization of two distinct mouse testicular epithelial cell lines. Biol Reprod 1980; 23: 243_52.

14 Zheng Y, Zhou ZM, Min X, Li JM, Sha JH. Identification and characterization of the BGR-like gene with a potential role in human testicular development/spermatogenesis. Asian J Androl 2005; 7: 21_32.

15 Diemer T, Allen JA, Hales KH, Hales DB. Reactive oxygen disrupts mitochondria in MA-10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis. Endocrinology 2003; 144: 2882_91.

16 Margolin Y, Aten RF, Behrman HR. Antigonadotropic and antisteroidogenic actions of peroxide in rat granulosa cells. Endocrinology 1990; 127: 245_50.

17 Sultana T, Svechnikov KV, Gustafsson K, Wahlgren A, Tham E, Weber G, et al. Molecular identity, expression and functional analysis of interleukin-1alpha and its isoforms in rat testis. Asian J Androl 2004; 6: 149_53.

18 Moundipa PF, Beboy NS, Zelefack F, Ngouela S, Tsamo E, Schill WB, et al. Effects of Basella alba and Hibiscus macranthus extracts on testosterone production of adult rat and bull Leydig cells. Asian J Androl 2005; 7: 411_7.

19 Hauet T, Yao ZX, Bose HS, Wall CT, Han Z, Li W, et al. Peripheral-type benzodiazepine receptor-mediated action of steroidogenic acute regulatory protein on cholesterol entry into leydig cell mitochondria. Mol Endocrinol 2005; 19: 540_54.

20 Hauet T, Liu J, Li H, Gazouli M, Culty M, Papadopoulos V. PBR, StAR, and PKA: partners in cholesterol transport in steroidogenic cells. Endocr Res 2002; 28: 395_401.