UPREGULATION OF NEUROGLOBIN PROMOTES TM 3 LEYDIG CELL VIABILITY

Neuroglobin (NGB) is a globin family member protein expressed in the nerve system and prevents oxidative stress and apoptosis induced death in neurons. The anti-oxidant role of NGB against oxidative stress and reactive oxygen species (ROS) brings up its promising protective role in other tissues. The overexpression of NGB in testosterone producing Leydig cells might be a solution to hypoxia related male infertility. In the current study, NGB was overexpressed in Leydig cells by using viral transduction methods and the cell proliferation, gene expression and anti-oxidant enzyme levels were analyzed. Upregulation of NGB increased anti-apoptotic Bcl-2, cell proliferation and anti-oxidant enzyme levels and decreased the expression of apoptotic genes BAX, p53 and caspase 3. In addition, NGB transduced cells proliferated and expressed less apoptotic genes after H2O2 exposure. In conclusion, NGB might be a target for androgen deficiency related male infertility and could be used in clinics in the future.


Introduction
Neuroglobin (NGB) was identified as a vertebrate nerve globin in neural tissues by Burmester et al. (2000).The NGB protein consists of 150 amino acids and is similar to myoglobin structurally (Burmester et al. 2000, Dewilde et al. 2001).Expression of NGB is observed in central and peripheral nervous system and some endocrine tissues (Reuss et al. 2002, Burmester et al. 2000).
The neuroprotective activity of NGB through prevention of apoptosis and oxidative stress is well documented (Amri et al. 2017).NGB is able bind to O2, CO or NO and prevents oxidative stress (Dewilde et al. 2001).The potential pathways and regulatory roles of NGB are providing O2 supply, preventing reactive oxygen species (ROS) damage and hypoxia (Burmester & Hankeln 2009).NGB acts as a respiratory protein with an O2 binding affinity which resembles to myoglobin oxygen-binding capacity (Hundahl et al. 2006).Localization of NGB in specific tissues and cellular regions that are metabolically active supports its O2 binding ability (Schmidt et al. 2003).Interaction of NGB with O2 enables the regulation of NGB expression in hypoxic conditions (Schmidt-Kastner et al. 2006).NGB acts as NO-dioxygenase when O2 levels are low and react with NO2 to form NO (Petersen et al. 2008).NGB expression in human cell cultures is not only triggered by hypoxia (Haines et al. 2012 The harmful effects of oxidative stress are not only observed in neural tissues but also disrupt functions in many tissues of the systems of the body one of which is the reproductive system.Oxidative stress induced DNA damage in sperm function is one of the most important reasons for male fertility (Bisht et al. 2017).In addition to spermatogenesis, Leydig cell steroidogenesis is also sensitive to oxidative stress.A number of internal and external factors, e.g.age, infection, diabetes, temperature, testis diseases, toxin exposure and hormonal changes cause ROS production in the testes leading to male fertility problems (Asadi et al. 2017).The antioxidant defense system in Leydig cells protects testicular tissue and provides appropriate testosterone production and sperm generation (Aitken & Roman 2008).Leydig cells are the primary sources for testosterone production which is required for male reproduction.Therefore, identification of new protective mechanisms that control oxidative stress mechanisms in Leydig cells might be valuable for future therapeutic applications.Although large amounts of NGB were detected in the nervous system and brain, endocrine organs including testis and pituitary and adrenal glands also express NGB (Burmester et al. 2000, Reuss et al. 2002, Zhang et al. 2002).Because NGB is expressed in testis tissue, we hypothesized that overexpression of NGB might protect testis cells against stress.Protective activity of NGB against oxidative stress in tissues such as that of the nervous system where high amounts of localization are observed gave rise to the idea of potential protective effect of NGB in other tissues.In the current study, the protective role of NGB in TM3 mouse Leydig cells was evaluated by using a genetic manipulation approach.

Determination of H2O2 toxic dose
TM3 Leydig cells were seeded onto 96-well plates at a cell density of 5000 cells/well.One day later, cells were treated with various concentrations (100µM, 200µM, 300µM, 400µM) of H2O2 to determine the toxic dose for further experiments.Cell viability was measured by MTS assay and absorbance was measured at 490nm using an ELISA plate reader.

TUNEL assay
TdT-mediated dUTP nick-end labeling (TUNEL) assay was performed in TM3-NGB and TM3-GFP cells by using a TUNEL Assay kit (Roche Molecular Biochemicals, Indianapolis, IN) to detect the apoptotic cell number.Briefly, transduced cells were collected and suspended in 50µl of TUNEL reaction mixture (Labeling solution + Enzyme solution, supplied with the kit), incubated at 37°C and suspended in PBS for flow cytometry analysis using Becton Dickinson FACS Calibur (Becton Dickinson, San Jose, CA) flow cytometry system.

Gene expression analyses
Quantitative RT-PCR experiments were conducted according to the previously described protocol (Apdik et al. 2015).Primer sequences for NGB, p53, caspase3, Akt, BAX, Bcl-2 and β-actin were designed by IDT primer Quest software.β-Actin was used as housekeeping gene for normalization of the data.Total RNA was isolated form transduced cells and H2O2 administered cells by using a RNA-easy plus mini kit (Qiagen, Hilden, Germany) and cDNA was synthesized using High Fidelity cDNA synthesis kit (Roche, USA).qPCR experiments were conducted by SYBR Green using the CFX96 RT-PCR system (Bio-Rad, Hercules, CA).
SOD and GPx activity SOD (19160, Sigma, MO, USA) and GPx (CGP1, Sigma, MO, USA) enzyme activities were determined in TM3-NGB and TM3-GFP cells according to the manufacturer's instructions.Protein samples were isolated from TM3-NGB and TM3-GFP cells by RIPA buffer and used for enzyme activity analysis.Absorbances of SOD and GPx assays were measured at 450nm and 340nm, respectively by using an ELISA plate reader.

Statistical analysis
Results are expressed as mean ± standard deviation.Standard errors and t-test values were calculated using the GraphPad Prism 5 (GraphPad, La Jolla, CA) software.Differences were considered to be statistically significant at P values of less than 0.05 (P < 0.05).

TUNEL assay
The apoptotic status of NGB overexpressing cells were detected by TUNEL assay.Although 27% of the TM3-GFP cells were apoptotic after transduction, only 12% of the TM3-NGB-GFP cells were positively stained (Fig. 2D).

SOD and GPx enzyme activity
Potential role of NGB overexpression on antioxidant enzyme activities was detected by SOD and GPx activity measurements.NGB overexpression increased the antioxidant enzyme activities significantly compared to TM3-GFP cells.TM3-NGB-GFP cells exerted approximately 2 fold higher antioxidant enzyme activity.SOD and GPx enzyme activities of TM3-GFP cells were determined as 60%±3.1 and 57%±3.2,respectively while activities of both were 100% in TM3-NGB-GFP cells (Fig. 5).

Discussion
Free radicals generated through cellular metabolism cause oxidative stress in tissues such as testis and induce cell death.Oxidative stress induced ROS disrupts reproductive system, prevents androgen (testosterone) secretion and spermatogenesis (Aitken et al. 2008).Leydig cell dysfunction due to membrane lipid peroxidation upon Lipopolysaccharide (LPS) application has been observed previously (Husain & Somani 1998).As Leydig cells produce androgens such as testosterone which binds to the androgen receptor and activates sperm generation (Dohle et al. 2003), they are potential targets for oxidative stress related male fertility treatments.Understanding the protective mechanisms against oxidative stress and identifications of new potential pathways are among the recent aims of studies addressing development of new therapeutic targets.
In the present study, we evaluated the role of NGB in Leydig cells by using an overexpressing gene editing approach.Because NGB protects many tissues against oxidative stress and are expressed in testis tissue, overexpression of NGB in Leydig cells could be promising to prevent harmful effects of oxidative stress.Proliferation of NGB overexpressing mouse TM3 Leydig cells were higher compared to control cells indicating the potential protective activity.The activity of NGB on cell proliferation and protection has previously been shown in neurons and cancer cells (Fiocchetti et (Liu et al. 2015).Anti-apoptotic activity was confirmed by gene expression analysis of pro-and antiapoptotic genes including BAX, p53, caspase-3 and Bcl-2.Enhanced Bcl-2 and low levels of BAX and caspase-3 have been reported after spinal cord injury in NGB overexpressing animals (Lan et al. 2014).Because Leydig cells are responsible for testosterone production and highly crucial for spermatogenesis, anti-apoptotic function of NGB to block apoptosis is highly important for potential therapeutic options.Same observations for cell proliferation and gene expression were reported after H2O2 exposure indicating the protective effect against ROS and oxidative stress.Induction of SOD and GPx in testis tissue (Kaur et al. 2006) and Leydig cells (Baek et al. 2007) is a defense mechanism after oxidative stress.Therefore, we evaluated the effect of NGB overexpression on anti-oxidant enzyme activities.NGB overexpression upregulated the SOD and GPx levels as reported in the literature.Although there is not an evidence in the literature for NGB overexpression in Leydig cells, transgenic mice overexpressing NGB showed high SOD and GPx levels in hippocampal tissues (Li et al. 2010).
In conclusion, we demonstrated, for the first time, the promising protective role of NGB in Leydig cells against oxidative stress.NGB gene could be a potential target for oxidative stress induced male infertility and might be used for therapy in future.Further experiments explaining the molecular mechanism of NGB in Leydig cells should be conducted both in vitro and in vivo.
) but also induced by H2O2 (De Marinis et al. 2013) indicating the regulatory role in O2 metabolism.17β-estradiol (E2) induced NGB upregulation in neurons has been shown to be neuroprotective (De Marinis et al. 2013).Moreover, NGB was shown to translocate into mitochondria upon hormone and H2O2 stimulation and prevent cytochrome C release to help overcoming stress induced programmed cell death (De Marinis et al. 2013, Brittain et al. 2010).NGB overexpression in transgenic animals prevented heart and brain injury after ischemia or stroke indicating the tissue protective role of NGB after oxidative stress (Sun et al. 2003, Khan et al. 2006, Jin et al. 2010).Endogenous NGB in neural tissues exerted 116 A. Doğan neuroprotective roles against oxidative stress indicating the possible protective role of NGB in neurodegenerative disorders (Ye et al. 2009, Fiocchetti et al. 2013).
al. 2017, Greenberg et al. 2008, Zhang et al. 2018) which supports our results.NBG overexpressing transgenic mice can rescue under hypoxic stress and NGB induces neural protection in vitro against hypoxic injury (Greenberg et al. 2008).Overexpression of NGB increased cell viability in MCF-7 breast cancer cells against nutrient deprivation through an anti-apoptotic Bcl-2 expression (Fiocchetti et al. 2017).Similarly, both overexpression and knockdown of NGB in glioblastoma cells regulated the cell proliferation by activating the PI3K/AKT pathway (Zhang et al. 2018).Promoting activity of NGB in cell proliferation and viability might be useful to increase Leydig cell number and to treat oxidative stress related male fertility in the future clinical applications.Additionally, low levels of TUNEL positive cells in NGB transduced cells demonstrated the anti-apoptotic role of NGB in Leydig cells.Knockdown of NGB in neurons increased TUNEL positive cells in arsenite induce toxicity indicating the protective role of NGB against apoptosis