Journal of Artificial Organs

, Volume 16, Issue 2, pp 229–233

Research on the steroidogenesis of proliferated Leydig cells in vitro

Authors

  • Liang Zhong
    • Department of Urology, Shanghai First People’s HospitalShanghai Jiaotong University School of Medicine
  • Jie Sun
    • Department of Urology, Shanghai Children’s Medical CenterShanghai Jiaotong University School of Medicine
  • Guo-hua Liu
    • Department of Urology, Shanghai First People’s HospitalShanghai Jiaotong University School of Medicine
    • Department of Urology, Shanghai First People’s HospitalShanghai Jiaotong University School of Medicine
    • Department of Urology, Shanghai First People’s HospitalShanghai Jiaotong University School of Medicine
Original Article

DOI: 10.1007/s10047-012-0684-y

Cite this article as:
Zhong, L., Sun, J., Liu, G. et al. J Artif Organs (2013) 16: 229. doi:10.1007/s10047-012-0684-y

Abstract

Several treatments for patients with primary hypogonadism are available, but these are associated with major complications. In this study, we explored the possibility of testosterone secretion by proliferated Leydig cells embedded in Matrigel with the aim of developing a source of endogenous testosterone supplement for recipients while reducing the need for donor material. Leydig cells were isolated and proliferated in vitro. The expression of 3β-hydroxysteroid dehydrogenase, cholesterol side-chain cleaving enzyme (CYP11A1), and 17α-hydroxylase/17,20-lyase (CYP17A1) was analyzed to confirm the purity and steroidogenesis capability of Leydig cells. The proliferated cells were then embedded in three-dimensional Matrigel, and following culture the supernatant medium was collected for measurement of testosterone concentration by radioimmunoassay. The biological behavior of the Leydig cells in the Matrigel was carefully observed under the microscope. Approximately 6.0 × 105 Leydig cells were obtained from one testis after primary culture in vitro. Aliquots of 1.0 × 105 Leydig cells were mixed with Matrigel, with the amount of cells in one pellet being equal to that in an adult testis. Leydig cells gradually formed aggregates when maintained in Matrigel. A rapid and constant linear increase in testosterone levels was detected in the supernatant medium. Our results demonstrate that Matrigel is a perfect support matrix for Leydig cells. Proliferated Leydig cells embedded in Matrigel have a great steroidogenesis reserve. In our study, they contributed to continuous steroidogenesis, which implies that the pellet may provide the physiological demand for endogenous androgen once engrafted in vivo. This system may ultimately provide a novel alternative treatment for people who are in need of androgen replacement.

Keywords

Leydig cellsTestosteroneIn vitroAndrogen replacement

Introduction

Primary hypogonadism, a clinical syndrome that results from the failure of the testes to produce physiological levels of testosterone, is commonly encountered in clinical practice [1]. Androgen replacement has been the most widely used clinical treatment to maintain secondary sexual characteristics. However, there are multiple testosterone delivery systems, and it is difficult to coincide the treatment with the physiological requirement and the patient’s compliance. Such treatments are also associated with potential health risks, which also restrict the application of androgen replacement. Research in this field has focused on developing a more suitable physiological method that will consistently maintain the hormonal level of the patient at the physiological level. Testis transplantation may provide endogenous testosterone [2, 3], but it comes with assorted ethics problems. As Leydig cells are the terminal site of the hypothalamic–pituitary–gonadal axis, our group has attempted to mimic natural testosterone fluctuations by Leydig cells transplantation, as reported in a previous study [4]. While this approach avoids the ethics issue, insufficient donor sources remain an unsolved problem in the clinical situation. In 2011, we succeeded in isolating and proliferating mice leydig cells of a higher purity and vitality [5]. The biological behavior of Leydig cells in vitro indicated that Matrigel was ideal support matrix for these Leydig cells [6].

In this study, we explored the capability of the proliferated Leydig cells embedded in Matrigel to secrete testosterone. Our expectation was that this would provide a novel method of testosterone supplement to recipients who require androgen replacement and concomitantly solve the problem of donor deficiency.

Materials and methods

Animals and reagents

C57BL/6 mice were purchased from the Shanghai Slack Experimental Animal Center (Shanghai, China). The experiment was approved by the Research Ethics Committee of Shanghai Jiaotong University Affiliated First People’s Hospital. NB4 collagenase was purchased from Serva (Heidelberg, Germany); dehydroepiandrosterone (DHEA), nitro blue tetrazolium, and β-nicotinamide adenine dinucleotide were purchased from Sangon Biotech (Shanghai, China); antihuman cytochrome P450 (CYP) 11A1 (sc-18043), antihuman 3-β-hydroxysteroid dehydrogenase (3β-HSD; sc-30820), and antimouse CYP17A1 (sc-46081) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor® 488 dye-conjugated secondary antibody (Invitrogen, Carlsbad, CA) were used for detection. Leydig cells were maintained in low glucose Dulbecco's Modified Eagle Medium (DMEM; Gibco, Invitrogen) with 10 % fetal calf serum (SAFC; Sigma, St. Louis, MO). Matrigel (354262) was purchased from Becton Dickinson (Franklin Lakes, NJ). The gamma radioimmunoassay counter was from USTC Chuangxin Co., Ltd. Zonkia Branch (Hefei, China), and the Iodine-125I-Testosterone Radioimmunoassay kit was from Union Medical & Pharmaceutical Technlogy Tianjin Ltd.(Tianjin, China).

Primary culture of mouse Leydig cells

Leydig cells were isolated from testes of 8-week-old mice as described previously [5]. In brief, mice were sacrificed by cervical dislocation and the testes extracted. The testes were then incubated in a centrifuge tube containing 0.03 % collagenase NB4 and digested for 15 min at 37 °C with shaking at 150 rpm. The supernatant was then discarded, and a fresh collagenase NB4 solution was added for a second 15-min’s digestion, during which time the rotation speed was lowered to 130 rpm. Cells in the supernatant were then centrifuged, resuspended in low-glucose DMEM supplemented with 10 % fetal bovine serum, and cultured and proliferated in vitro for 7 days before being mixed with Matrigel. Histochemical staining for 3β-HSD was conducted to determine cell purity according to the method described by Klinefelter et al. [7]. The expression of CYP11A1, CYP17A1, and 3β-HSD was detected using the immunofluorescence method described in our previous study [5].

Leydig cells embedded in Matrigel

Leydig cells were collected after the 7-day culture using 0.25 % trypsin and examined by trypan blue exclusion for viability. The cells were then resuspended in ice-cold low-glucose DMEM supplemented with 10 % fetal bovine serum (FBS; 200 μl medium) at a density of 1 × 105 cells, and this cell suspension (200 μl) was mixed with 200 μl of Matrigel by gentle agitation on ice. The mixtures were then poured into individual wells of an 6-well chamber and heated to 37 °C in an incubator for 10 min to form gels. Low-glucose DMEM supplemented with 10 % FBS was added to the chamber. To ensure conformity of medium volume, moderately fresh medium was added into the chamber every 2 days. As the negative control, 200 μl Matrigel was cultured in medium alone. Images were captured using phase contrast microscope.

Radioimmunoassay of testosterone

The medium from each well was collected and stored at −20 °C until the analysis of testosterone. Testosterone content was determined using an I125-radioimmunoassay kit in accordance with the procedures supplied by the manufacturer. The radioactivity of the medium was measured on a automatic gamma counter.

Results

Primary culture of Leydig cells

Leydig cells grew into typical epithelioid-like cells by the third day of culture. Abundant lipid drops were present in the cytoplasm, mainly surrounding the nucleus (Fig. 1a). Prior to being embedded in Matrigel, both the purity and survival rates of the Leydig cell preparations were >90 %, as determined by 3β-HSD histochemical staining and trypan blue dye exclusion, respectively (Fig. 1b). Immunofluorescence staining for CYP11A1, CYP17A1, and 3β-HSD of the Leydig cells was positive (Fig. 2).
https://static-content.springer.com/image/art%3A10.1007%2Fs10047-012-0684-y/MediaObjects/10047_2012_684_Fig1_HTML.jpg
Fig. 1

The primary culture of Leydig cells. a Abundant lipid drops, which are the source material for testosterone synthesis, are present in the cytoplasm, mostly surrounding the nuclei. b Histochemical staining for 3-β-hydroxysteroid dehydrogenase (3β-HSD) shows bluish violet particles in the cytoplasm of the Leydig cells. The purity of the Leydig cell preparations was about 90 % before they were embedded in Matrigel

https://static-content.springer.com/image/art%3A10.1007%2Fs10047-012-0684-y/MediaObjects/10047_2012_684_Fig2_HTML.jpg
Fig. 2

Enzyme for steroidogenesis is expressed in Leydig cells. a Positive immunofluorescence for cytochrome P450 (CYP) 11A1: blue nuclei, green CYP11A1 in cytoplasm. b Positive immunofluorescence for 3β-HSD: blue nuclei, green 3β-HSD in cytoplasm. c Positive immunofluorescence for CYP17A1: blue nuclei, green CYP17A1 in cytoplasm (color figure online)

Leydig cells in three-dimensional culture

To create a three-dimensional (3D) culture, we embedded the Leydig cells in a thick layer (200 μl) of Matrigel. The mixtures were approximately 1 cm in diameter and 1.5 mm in thickness and remained in gel form in studied conducted at 37 °C (Fig. 3a). The Leydig cells were predominantly oval-shaped within the Matrigel (Fig. 3b). Microscopic observations showed no obvious signs of apoptosis and cell debris. The cells gradually formed aggregates that became suspended in different bedding planes of the Matrigel (Fig. 3c). This distribution is similar to that of Leydig cells in testes, with clustering in the interstitium (data not shown).
https://static-content.springer.com/image/art%3A10.1007%2Fs10047-012-0684-y/MediaObjects/10047_2012_684_Fig3_HTML.jpg
Fig. 3

Leydig cells embedded in Matrigel. a Gross examination of pellet which is configured by Leydig cells and Matrigel. b Leydig cells are predominantly oval-shaped and scattered within the Matrigel. c Leydig cells gradually formed aggregates over time when suspended in Matrigel

Secretion of testosterone by proliferated Leydig cells

Testosterone levels are positively correlated with the functioning of Leydig cells embedded in Matrigel. As expected, the concentration of testosterone in the supernatant medium significantly increased in a linear manner over time being about 59.86 ± 16.82 ng/ml on the first day and 363.87 ± 112.34 ng/ml on the fifth day. There was no change in the concentration of testosterone in the medium cultured with only the Matrigel (Fig. 4).
https://static-content.springer.com/image/art%3A10.1007%2Fs10047-012-0684-y/MediaObjects/10047_2012_684_Fig4_HTML.gif
Fig. 4

The concentration of testosterone in supernatant medium significantly increased in a linear manner over time in the experimental group (medium + Matrigel + Leydig cells) compared to the control group (medium + Matrigel only)

Discussion

Androgen replacement is currently the mainstay of treatment for patients with primary hypogonadism. However, exogenous testosterone replacement is associated with several potential health risks, including acne and male pattern baldness, gynecomastia, erythrocytosis, suppression of spermatogenesis, liver toxicity, and cardiovascular event [814].

Tissue-engineered testicular prostheses may be a novel approach [15]. Although the technology may benefit patients who require testicular prostheses, it provides testosterone by passive release. Testis transplantation can provide endogenous testosterone, but it is associated with ethics issues. As Leydig cells are the terminal site of the hypothalamic–pituitary–gonadal axis, a procedure involving the transplantation of Leydig cells may be a promising approach. In a previous study, we succeeded in mimicking natural testosterone fluctuations by transplanting Leydig cells of the mature rat into castrated prepubertal recipients [4]. However, this approach is completely dependent on the availability of donor testis. In the present study, we explored the possibility of using testosterone secretion tissue obtained in a system of proliferated Leydig cells and Matrigel.

In this study, we constructed the pellet through the entrapment of 1.0 × 105 Leydig cells into 200 μl Matrigel; as such, the amount of Leydig cells in one pellet is equal to that in an adult testes. This means that Leydig cells isolated and proliferated from a single testes can be used to construct several allotments of testosterone secretion tissue. This is a fundamental and essential criterion for further application of this approach.

Matrigel, which has a high biocompatibility, is liquid at 4 °C but solidifies rapidly at 37 °C [1618]. Leydig cells maintained in Matrigel gradually formed aggregates over time, which may possibly be due to cell migration through the matrix [6]. This migration is a interesting phenomenon because in vivo Leydig cells cluster together in the central interstitium of testes [19]. We assumed that cell aggregates may greatly contribute to the endocrine function of the Leydig cells. A pilot study found that Leydig cells are functional and positively express key enzymes in Matrigel as in the primary culture [20]. This capacity may guarantee that the cells have sufficient capability for testosterone synthesis. Moreover, the use of Matrigel as support matrices is attractive because it prevents the Leydig cells from being displaced when engrafted in vivo.

In terms of the conformity of supernatant medium volume, a rapid increase in testosterone level in consistently linear fashion can mostly be ascribable to constant secretion from Leydig cells over the entire study period rather than medium evaporation. We added 2 ml of medium, which is equivalent to a circulation volume of a mature C57BL/6 mouse, at regular intervals to maintain the testosterone concentration at the prescribed level. The testosterone level in the medium quickly increased to 363.87 ± 112.34 ng/ml without stimulation from luteinizing hormone and negative feedback by adenohypophysis or being metabolized, as in vivo. The final concentration of testosterone on the fifth day was hundreds of fold greater than the serum testosterone concentration in a mature mouse (approx. 1 ng/ml) [21]. These results confirmed the great steroidogenesis reserve of Leydig cells embedded in Matrigel in vitro. In addition, the Leydig cells may possibly fulfill the physiological demand for androgen in recipients in vivo if the experimental conditions are carefully controlled. This may benefit more men in need of androgen replacement and diminish the donor requirement.

Conclusion

Testosterone secretion tissue can be developed by using Matrigel and proliferated Leydig cells in vitro. These cells can provide endogenous androgen and may reconstruct the hypothalamic–pituitary–gonadal axis in the recipient. Moreover, this technology can maximally reduce the need for donor testes. Thus, upcoming experiments in vivo and clinical applications are promising because both patients and donor will benefit from this novel therapy. Future research by our group will involve the grafting of these mixtures constructed by Leydig cells and Matrigel into castrated mice to monitor the change in serum testosterone in vivo.

Acknowledgments

This study was supported by grants from the Research Program of Science and Technology Commission of Shanghai Municipality (10411967200), the Shanghai Song-Jiang Health Bureau (2011PD06), and the National Natural Science Foundation of China (81170642). The authors also received a Shanghai Shen Kang Platform Grant (SHDC12007206). We thank Dr. Yilin Cao, Dr. Wei Liu, Dr. Guangdong Zhou (Department of Plastic and Reconstructive Surgery, Shanghai 9th People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai) and the National Tissue Engineering Center of China (Shanghai) for technical assistance.

Conflict of interest

None.

Copyright information

© The Japanese Society for Artificial Organs 2013