Three-Dimensional Scaffold, Light Emitting Diode (LED) And Melatonin Effects on Cell Proliferation and Testosterone Secretion in TM3 Leydig Cells

Different effects of brin scaffold, green LED and melatonin on cell proliferation and differentiation have received extensive attentions. Testosterone, the most important sex hormone is secreted by Leydig cells to regulate spermatogenesis and many other phenomena in the body organs. The aim of the present study was to evaluate the proliferation of Leydig cells and their testosterone secretion under the inuence of three-dimensional scaffold culture, green light irradiation and melatonin. The experimental groups, were TM3 cells incorporated in 3D scaffold culture, exposed to green light and also exposed to melatonin individually, in pairs and three factors together. Cell proliferation and testosterone secretion were measured after 72 hr. Cell cycle genes including PCNA, CYCLIND1, CDC2 and CDKN1B and, testosterone related genes; GATA4 and RORα were also examined. In general, three-dimensional scaffold increased Leydig cells proliferation, testosterone secretion and the expression of the related genes. On the other hand, melatonin decreased cell proliferation and testosterone synthesis. Green light did not signicantly change the results but slightly decreased cell proliferation and testosterone synthesis. Combination of green light with melatonin signicantly reduced the outcome while, light with scaffold increased the results. Application of light + scaffold + melatonin covered the decreasing effect of melatonin.


Introduction
Testes, the organs of male sex hormones secretion, play an important role in the maintenance and maturation of germ cells. Leydig and Sertoli cells of testes, work together to orchestrate and regulate spermatogenesis 1 . Leydig cells promote the proliferation and development of germ cells in the seminiferous tubules and maintain the high level of androgen concentrations including testosterone 2,3 ; the most abundant sex hormone in the body, which regulates spermatogenesis. In the absence of testosterone or androgen receptors, spermatogenesis will cease at the meiotic stage 4 .
Melatonin, a lipophilic and hydrophilic hormone which is mainly secreted by the pineal gland is involved in the regulation of circadian rhythms. It could pass through the blood-testis barrier, enter the testicular cells and act through speci c receptors including membrane melatonin receptors 1 (MT1) and 2 (MT2). MT1 and MT2 play some role in testosterone synthesis by regulating cAMP signal transduction cascades thorough coupling with G-protein. Melatonin can bind to retinoic acid receptor-related orphan receptor A (RORα), which has been identi ed in many mammalian cell types. RORα regulates the levels of reproductive hormones and animal reproduction at the transcriptional level through a nuclear receptor depending on physiological conditions and species of animals 5 .
In vitro three-dimensional (3D) culture mimics the physiologic milieu of the in vivo conditions as it could ll the gap between two-dimensional (2D) cell culture and animal tissues, and has been suggested as a solution for 2D culture insu ciencies 6 . To achieve a 3D system, the cells are seeded onto a porous matrix, known as scaffolds, to attach and colonize 7 . The most common materials utilized as scaffold are metals such as titanium; synthetic organic materials such as polymers; synthetic inorganic materials such as hydroxyapatite, and natural organic materials such as collagen and brin 8 . Fibrinogen is a blood plasma glycoprotein that is essential for wound healing and other biological phenomena. Fibrinogenbased 3D scaffold is a biologic network assembled by brinogen polymerization 9 in which stem and somatic cells proliferate and differentiate more successfully 10 .
Some physical factors including light emitting diodes (LEDs) irradiations have currently been introduced to potentially affect proliferation and differentiation of various cell types, mostly the mesenchymal stem cells 11,12 . Although the mechanism is not fully revealed, it has been postulated to be due to an increase in the oxidative function of mitochondria [13][14][15][16][17] . Considering the signi cance of testosterone in sexual activities and the probable effect of melatonin on the function and level of testosterone, and also the effects of 3D culture and green light emitted from a LED, on the proliferation and differentiation of different cells, the aim of the present study was to examine the effects of melatonin, 3D culture condition and green light irradiation on the cell proliferation, testosterone secretion and gene expression of a cellular model of testosterone production; TM3 cells.

Results
Cell proliferation assay by MTT Proliferation of TM3 cells in the different groups was evaluated using MTT assay. Each of the interventions including melatonin administration and LED irradiation reduced the cell proliferation rate compared with the control group. The least proliferation rate was detected in LED+MEL group (47%, P<0.001), followed by LED (75%, P<0.001) and MEL (97%) groups. The use of scaffold signi cantly increased cell proliferation rate in the related groups. The highest proliferation rate was observed in SC+ MEL group (147%, P<0.001), followed by SC and LED+MEL+SC (137%, P<0.001), and LED+SC (125%, P<0.01) ( Figure 1).

Trypan blue cell viability assessment
Cell viability was measured using trypan blue staining method in which the highest cell viability rate (93%) was in CTRl group, while the lowest viability rate was detected in MEL group (76%, P<0.001), followed by LED+MEL (77%, P<0.001) and LED (81%, P<0.01) None of the treatments resulted in a signi cant difference among the treated groups ( Figure 2). It should be noted that the viability assay was not performed in the scaffold-containing groups due to the limitations of trypan blue method.
Testosterone level in the different groups The amount of testosterone in the supernatant of the cultured TM3 cells in all groups was measured 24 hours after a hCG challenge, using an ELISA kit. The highest amount of testosterone was detected in LED + SC group (3634 ±285 ng/l), followed by 3614±220 ng/l for LED + MEL + SC, 3563±91 ng/l for LED + MEL, 3389±367 ng/l for CTRL, 3338±132 ng/l for LED, 3328±204 ng/l for SC and 3134±71 ng/l for SC +MEL groups. However, the values in the various groups were not signi cantly different compared with the control and other treatment group ( Figure 3).

Gene expression analysis
Expression of different genes associated with the cell cycle and testosterone synthesis was evaluated by qRT-PCR. The expression pattern of the genes that are involved in the cell cycle mechanism including PCNA, CYCLIN D1 and CDC2 was evaluated and the results showed that exposure of TM3 cells to a 3D condition generally increased the expression level of these genes compared with the scaffold-free environments (2D) (Figure 4: a, b and c).
The expression level of PCNA gene, which is involved in the stage S of the cell cycle, increased in the scaffold-containing groups compared with the control group and the scaffold-free groups. It was signi cantly (P<0.05) higher in the SC + Mel group compared with the MEL group and also it was signi cantly (P<0.05) higher in the LED+ SC+ MEL group compared with the control group (P <0.05) (Figure 4, a).
The expression level of CDC2 gene which encodes cdk1 protein and is highly involved in cell cycle progression, increased in the scaffold-containing groups compared with the scaffold-free groups. It was signi cantly (P < 0.05) different in the SC group compared with the control group. It also decreased signi cantly and nonsigni cantly in the LED + MEL (P<0.05), LED (P<0.05) and MEL groups compared with the control group ( Figure 4, b).
The expression level of CYCLIN D1 gene, which is involved in the promotion of G1 to S stage transition, non-signi cantly increased in the scaffold-containing groups compared with the scaffold-free and the control groups ( Figure 4, c).
The expression level of CDKN1B gene, that controls the cell cycle progression at G1 through its inhibitory effect on Cyclin D1, were contrary to the results of the Cyclin D1 gene expression as in the almost all scaffold-containing groups it signi cantly (P<0.05) reduced compared with the scaffold-free and the control groups ( Figure 4, d).
The expression of the genes involved in steroidogenesis procedures including GATA4 and RORα increased signi cantly in the scaffold-containing groups, compared with the control and scaffold-free groups. RORα expression was signi cantly (P<0.05) higher in the LED + SC + MEL group compared with the LED + MEL and the control group. The Expression of GATA4 also followed the pattern of gene expression of RORα. Similarly, scaffold-containing groups had higher expression of GATA4 in comparison with the scaffold-free groups. The highest expression was detected in the LED+SC+MEL group followed by SC, SC+MEL and LED+SC groups ( Figure 5: a, b).

Discussion
Androgenic hormones including testosterone have a pivotal role on the development and progression of male primary and secondary sexual characteristics and also sexual activities in life. Since many factors are involved in the steroidogenesis procedures which could affect testosterone production and function, in the present study, we investigated the proliferation and testosterone production of TM3 Leydig cells in the three-dimensional culture, green light irradiation, and melatonin administration.
Proliferation of Leydig cells in 2D and 3D culture conditions, was evaluated by MTT assay and q-RT-PCR analysis of the genes involved in the cell cycle including PCNA, CDC2, CYCLIND1, and its inhibitor: CDKN1B. Comparison of the results obtained from 2D culture with 3D culture indicated an enhancement in the proliferation of TM3 cells in the 3D culture. This increase was also observed after combination of showed that suppression of melatonin receptors, MT1 and MT2, stopped the genes in the testosterone synthesis pathway 30 . These con icting results require closer attention especially considering that in our study when melatonin was concurrently administered with brin scaffold and LED green light, the genes involved in proliferation and also the genes involved in steroidogenesis were highly expressed; concluding that a 3D system would have lessened the inhibitory effects of melatonin on cell proliferation and testosterone production.

Conclusion
We may conclude that the use of three-dimensional brin scaffold has a signi cant role in cell proliferation, testosterone secretion and the expression of the genes related to the testosterone synthesis pathway; GATA4 and RORα, in TM3 cells. In contrast, melatonin has a reducing role in Leydig cell proliferation and testosterone secretion. Although visible light radiation did not cause noticeable changes on cell proliferation and testosterone secretion, but in some cases caused a signi cant decrease. Light irradiation accompanied with a brin scaffold follows the additive effects of scaffold on the two mentioned processes and with melatonin follows the decreasing effects of melatonin. On the other hand, the simultaneous use of three variables could signi cantly mitigate the decreasing effect of melatonin and the overall result was an increase in the cell proliferation and testosterone secretion. Before the ndings of the present study could be used for the treatment of patients suffering from low androgen levels, further studies on the different scaffolds and LED irradiation to promote testosterone secretion is needed. Cell culture and experimental groups TM3 cells were cultured in DMEM-F12 supplemented with 10% FBS, 1% penicillin & streptomycin and 1% amphotericin (complete culture medium) at 37ºC with 5% CO 2 in the atmosphere. After reaching 80% con uence, the cells were harvested by trypsinization and different treatments were enrolled according to the following groups (table 1).

2D culture
The harvested cells were cultured at 1×10 4 in 96 well culture plates for 24 h in DMEM-F12 supplemented with 10% FBS and 1% antibiotics at 37ºC with 5% CO 2 in the humidi ed atmosphere as 2D control group.
Some wells received 10 -7 M melatonin as MEL group.
Fibrin 3D culture Ten μl of brinogen extracted from human plasma, 10 μl cell suspension (1×10 4 ) in PBS and 10 μl thrombin were transferred into a 1.5 ml microtube and mixed thoroughly. The samples were then incubated in a 37ºC incubator with 5% CO 2 in the air for 10 min. After brin clot formation, it was pushed out of microtube and transferred into 96 well culture plate (3D, SC group). 10 -7 M melatonin was added to some wells 24 h later as SC+MEL group.

LED irradiation
A handmade LED device with green light emission was used in the LED exposure groups (LED, MEL+LED, SC+LED and LED+SC+MEL). Twenty-four hr later the medium was refreshed and the samples were once irradiated for 10 minutes at radiation energy of 1.5 J/cm 2 , and 530 nm wavelength 31 . All the exposure procedures were carried out inside a 37ºC CO 2 incubator. In the MEL+LED group the cells/scaffolds were treated with 10 -7 M melatonin and were then irradiated as explained above.
TM3 proliferation assessment MTT assay was used to estimate the proliferation of TM3 cells. Brie y, cell density was adjusted to 7500 cells per well/ brin scaffold in 96-well plates. After different treatments, the cells/scaffolds were incubated for 72 hr at 37 ºC under 5% CO2 in the air. Twenty µl MTT solution (5 mg/ml) was added into each well and incubation was continued for 4 hr. The supernatant was discarded afterward and 150 µl DMSO was added into each well. The absorbance of each well was measured at 490 nm against a reference wavelength of 630 nm. All the experiments were done in triplicates 32 .
Cell viability assessment 3×10 4 TM3 cells were cultured in the 24 well plates with 300 µl complete culture medium. After 72 hr of the different treatments, the supernatants containing oating cells were transferred into 1.5 ml microtubes. The attached cells were washed with PBS, detached by trypsin EDTA solution, and added to the microtubes with gently pipetting. Ten µl of the cell suspension was mixed with 10 µl of trypan blue, loaded onto a Neubauer chamber and the number of viable and dead cells were counted in 4 large square under an inverted microscope. This assessment was not applicable for the scaffold-containing groups.
The cell viability rate was assessed using the following formula: cell viability rate = number of viable cells/total cell number Testosterone assessment The level of testosterone in the supernatant was assessed using an ELISA kit was purchased from ZellBio GmbH (Germany). In brief, 3×10 4 viable TM3 cells were cultured in 24 well plates with complete culture medium for 24 hr, and different treatments were applied according to the study groups. 48 hr later, 10 IU/ml hCG was added to each well, and 24 h later the supernatant was removed and stored at -20 ºC for testosterone measurement. The testosterone level was evaluated according to the protocol provided by the manufacturer 33 .

RNA extraction and cDNA synthesis
Total RNA was isolated using RNX plus (1 ml) and chloroform (200 μl) solutions at room temperature. After centrifugation at 12000 rpm at 4°C for 15 min, the aqueous phase was replaced into a new microtube, cold isopropanol solution was added and was then gently inverted 10 times. The tubes were transferred to a -20 °C freezer overnight. The next day, after centrifugation at 12000 rpm (4°C) for 10 min, one ml 75% ethanol was rinsed on the pellet, the tubes were centrifuged at 7500 rpm (4°C) for 5 min, the ethanol was removed and the pellet was dissolved in diethyl pyrocarbonate (DEPC) water and stored at -20 °C. The concentration and purity of the isolated RNA were assessed by absorbance readings on a UV spectrophotometer (Thermo Scienti c™ NanoDrop 2000) at the wavelengths of 260 and 280 nm to obtain a ratio of 1.81 ± 0.06. The RNA integrity was then determined by 1% agarose gel electrophoresis. The cDNA synthesis was performed using cDNA synthesis kit (Yektatajhiz azma, Tehran, Iran) according to the manufacturer's protocol: 70 °C for 5 min (RNA denaturing), 42°C for 60 min (cDNA synthesis) and then 70 °C for 10 min (enzyme inactivation).
Quantitative real time PCR Quantitative real time PCR was carried out to evaluate the mRNA expression level of PCNA, CYCLIND1, CDC2, CDKN1B, GATA4 and RORα genes in the TM3 cells by using magnetic induction cycler (mic) Realtime PCR system (Australia). For the ampli cation reactions, the synthetized cDNA solution (1 μl) was mixed with speci c primers (1 μl) and SYBR Green I Master (10 μl) at a total volume of 20 μl based on the following PCR program (table 2). Primer sequences are described in Table 3. GAPDH was used as a housekeeping gene to normalize the qRT-PCR reactions. The 2 −ΔΔCT method was used to measure gene expression levels.

Declarations
Con ict of interest: The authors do not have any con ict of interest to declare.
Contributions: B. Alavi, designed the study and carried out the laboratory activities; M. Shojaei, prepared the LED device, supervised LED irradiation part of the study and prepared the manuscript: T. haghpanah, designed the study and supervised the molecular and statistical tests; V. Mirzaei, interpreted the data and prepared the manuscript; M. Abedini esfahlani, designed the study and carried out some laboratory activities; M. Jalalkamali, carried out some irradiation procedure and helped in manuscript preparation; F. Seyedi, designed the study and supervised scaffold preparations; SN. Nematollahi-mahani, designed and supervised the study, provided funding and prepared the manuscript.