Effects of Cobalt on Gene Expression of Zinc Transporters in TM4 Sertoli Cells

Rapid Communication

Austin J Pharmacol Ther. 2016; 4(1).1080.

Effects of Cobalt on Gene Expression of Zinc Transporters in TM4 Sertoli Cells

Williams M¹, Coleman TL² and Pedigo NG²*

¹Department of Biological Sciences, Claflin University, USA

²Department of Pharmaceutical Sciences, Presbyterian College School of Pharmacy, USA

*Corresponding author: :Pedigo NG, Department of Pharmaceutical and Administrative Sciences, Presbyterian College School of Pharmacy, 307 N. Broad St., Clinton, SC 29325, USA

Received: October 15, 2015; Accepted: May 24, 2016; Published: May 30, 2016


The in vivo administration of cobalt in male mice causes reproductive toxicity resulting in decreased testicular weight followed by reduced sperm concentration and fertility. However, the co-administration of zinc with cobalt in vivo mitigates reproductive toxicity and histological damage to the germinal epithelium. Zinc is a key signaling molecule which is tightly controlled intracellularly by two families of zinc transporters, ZNT 1-10 and ZIP 1-14. They regulate intracellular zinc levels and distribution of zinc into organelles. Since Sertoli cells regulate spermatogenesis and are one of the last surviving cells observed histologically in the cobalt-treated testis, we investigated the mechanism of cobalt reproductive toxicity using a TM4 Sertoli cell line. Cobalt treatment of TM4 cells altered over 5500 genes as measured by microarray analysis. Validation of specific changes by quantitative PCR showed significant changes in the gene expression of slc39a9 and a13 (ZIP 9 and13) and slc30a4, a5, a7 (ZNT 4, 5 and 7) zinc transporters. After 24 hours of treatment with Co 33 μM, maximum reduction of gene expression of slc30a4, a5 and a7 efflux transporters to 4.08, 2.86 and 2.67% of controls was observed, respectively. Expression of slc39a9 and a13 influx transporter genes was maximally decreased to 3.22 and 2.79% of controls, respectively. These data support the hypothesis that cobalt reproductive toxicity may be mediated, in part, by alteration of zinc transport and homeostasis in TM4 Sertoli cells.

Keywords: Metal toxicity; Zinc signaling; Zinc transporter; Cobalt


Co: Cobalt; Zn: Zinc; PCR: Polymerase Chain Reaction; MT-1 or -2: Metallothioneins 1 or 2; GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase


The reproductive toxicity of metals, including Cobalt (Co) [1,2] and Cadmium (Cd) [3,4] in male rodents is well established in vivo. The toxic effects observed with chronic administration of Co include decreased testicular weight, degeneration of the germinal epithelium, and reduced sperm production resulting in decreased fertility. These reproductive effects of Co are slowly reversible over 20 weeks after discontinuation of administration [2,5]. Histopathology of the testis after chronic Co treatment shows ubiquitous damage to the germinal epithelium with replacement of spermatocytes, spermatids and spermatozoa by Giant cells and fatty depositions. However, the Sertoli cells remain intact even with severe damage to the germinal epithelium [5]. Measurement of Co levels in the whole testis shows an elevation of Co in the testis with chronic treatment in the virtual absence of germinal epithelium, suggesting that Co is associated with the remaining somatic cells, e.g. Sertoli cells [6].

Interestingly, mice treated concomitantly with Zn and Co in vivo exhibit partial mitigation of cobalt’s toxic reproductive effects in the testis [7]. Zinc (Zn) is a cofactor for over 300 enzymes critical for biochemical processes, and is required for function of more than 2000 transcription factors [8], including the metal responsive element-binding transcription factor MTF-1 [9]. Zn binds to about 10% of proteins in humans. Therefore, Zn must be tightly regulated at the cellular level because disruption of Zn homeostasis can cause pathological conditions, including male infertility. Zn is necessary for normal spermatogenesis and reproductive function in the male [10]. Both Zn deficiency and excess cause impairment of spermatogenesis in mice [11] and humans [12].

There are two major families of Zn transporters in mammalian species that control cytoplasmic Zn levels and subcellular distribution of Zn within the cell, ZNT (slc30) and ZIP (slc39) transporters, which function to move Zn in opposite directions. ZNT family of transporters (1-10) moves Zn out of the cytoplasm across the cellular membrane into the extracellular space, or sequesters Zn into intracellular compartments [13]. ZIP transporter family (ZIP1-14) primarily moves Zn from the extracellular space or from intracellular stores into the cytoplasm [8].

Zn transporters are ubiquitously expressed in mammalian tissues including the testis, and specifically Sertoli cells. Metal ions including Zn, Cd and Co have been shown to induce or repress gene and protein expression of both families of Zn transporters [8,11,13- 16], as well as metal binding proteins metallothioneins 1 and 2 (MT1 and MT 2) [17]. Mutations of the Zn transporters are associated with disease states including a subtype of Ehlers-Danlos syndrome in mice and humans (ZIP13, slc39a13) [18] and lethal milk disorder in mice (ZNT4, slc30a4) [19].

Since the Sertoli cell regulates the process of spermatogenesis [20] and remains intact during Co reproductive toxicity, the role of the Sertoli cell in Co toxicity was investigated in vitro using TM4 Sertoli cells, a murine derived epithelial cell line [21]. Because Co levels are elevated in vivo in the testis with chronic toxicity, we explored the effect of Co on gene expression in TM4 Sertoli cells. We also measured the cellular levels of Co and Zn after Co treatment in vitro. Since previous studies showed concurrent Zn treatment in vivo reduced Co reproductive toxicity in vivo, the impact of Co on Zn transporters is of particular interest. We conducted these studies to determine if Co toxicity is mediated through Sertoli cell mechanisms, and whether disruption of Zn homeostasis is involved.

Materials and Methods

TM4 Sertoli cell line obtained from ATCC was grown to confluence in Ham’s F-12/DMEM with 5% (v/v) FBS in a Forma Scientific 5% CO2 incubator in T75 cm2 flasks. Early passages cells (6.5 X 105) were plated into 6-well (SA-9.6 cm2) culture ware (Falcon B-D) for individual treatments. Non-confluent cultures were treated with control (fresh media), or Co media containing cobaltous chloride hexahydrate (Sigma Aldrich) at a concentration of 10, 33, or 100 μM for 24 hours. Cells were harvested by detaching cells with a sterile scraper from well surface, and collected in cell lysis solution. Cells were then disrupted by passing the lysis suspension with cells rapidly through a 23G needle 3 times.

RNA Isolation

Total RNA isolation was performed using RNeasy RNA kit (Qiagen). DNA was then digested using an RNase-free DNase (Invitrogen). Total RNA was stored at -20°C until reverse transcribed and assayed for expression of specific messages by microarray analysis or quantitative PCR.

Reverse transcription

Isolated total RNA from each sample (1.5 μg) was used as the template for reverse transcription to generate cDNA using random hexamer primers with 1 μL reverse transcriptase (iScript, BioRad) under standard synthesis conditions.

Microarray analysis

In collaboration with Dr. Jamie Barth of the Proteogenomics Core Facility at the Medical University of South Carolina (MUSC) the research resources at the core facilities were utilized to carry out this study. The facility is equipped with a complete Affymetrix system, comprising a GeneChip® Scanner 3000 7G with AutoLoader, hybridization oven and fluidics workstation. This is complemented by a BioRadiCycler real time PCR machine and an Agilent 2100 Bioanalyzer. Bioinformatic analysis of significant changes was evaluated in collaboration with the Microarray facility to identify important pathways involved in the Co effects on gene expression. Affymetrix Murine microarray 2.0 ST was used to evaluate gene expression changes in response to cobalt treatment in TM4 cells. Single channel microarrays with 30,000 transcripts were used for screening control and cobalt-treated TM4 Sertoli cells. Three samples from the treatment groups 0, 10 and 33 μM Co were analyzed on triplicate microarrays.

Real-time PCR

Quantitative PCR was performed using Eva Green SsofastSupermix (BioRad) under cycling conditions optimized for each specific primer target. Specific forward and reverse primers were designed for each target using Primer3 software and evaluated for specificity for target using PrimeBlast (NCBI) (Table 2). Primers synthesized by Integrated DNA Technologies (IDT) were used to amplify cDNA from genes affected by cobalt treatment as identified by microarray analysis. PCR assays were done with a BioRad C1000 Thermal cycler with CFX96 Real-time PCR system, with data management software CFX Manager used for analysis. PCR products were analyzed using a high resolution Melt Curve Analysis program (BioRad). Changes in gene expression were normalized to GAPDH and expressed as per cent control or folds change over control. Gene expression was statistically evaluated by ANOVA, using a significance level of p < 0.05.