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| Influence of Interfering Ion Channels of Antler Stem Cells on Morphogenesis of Deer Antlers (Cervus nippon) |
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Abstract The theory of bioelectric code is a remarkable achievement for life sciences. It is unknown whether this theory could apply to mammals. In the present study, sika deer (Cervus nippon) antlers were used as a model to investigate if morphogenesis of mammalian organs was also related to bioelectricity. Deer antlers are complex mammalian organs. Morphogenetic primordia of deer antlers is antlerogenic periosteum (AP). Deer antler morphogenesis can be altered artificially by manipulating AP conveniently. Ten pieces of APs were collected through surgery from 3 male and 3 female sika deer calves before antler generation. These APs were cultured in sterile Dulbecco's modified eagle medium (DMEM) medium (containing 10% fetal calf serum (FBS) and 2% penicillin-streptomycin) in vitro and treated with four kinds of drugs for 2.5 d respectively. These drugs were ivermectin, MS-222, concanamycin A and SCH-28080 which were known to be able to alter bioelectricity state of tissues/cells through interrupting ion channels. Before the culture, APs were punctuated with a beam of needles to facilitate the drugs to diffuse into the tissue. After the culture, APs were implanted back to their original places. Subsequent development of antlers was observed weekly and photographed when necessary. Two months later, following results were obtained. Irrespective of the depolarized or hyperpolarized status, antler development was affected. The results from male deer were consistent with those from female deer (mechanical stimulation could induce female deer to grow antlers). The role of mechanical stimulation was impaired by drug treatment in female deer in this particular case. For the purpose of testing the effect of drug, AP cells were isolated from AP tissue and cultured in sterile DMEM medium (containing 10% FBS and 2% penicillin-streptomycin) in vitro. MS-222 was selected as a representative drug and added to DMED medium. Three groups of AP cells were designed and treated with MS-222 for 12, 24 and 48 h, respectively. The same treatments were carried out but without MS-222 as controls. The intracellular sodium (Na) ion of AP cells were strained with CoroNa green indicator dye. The difference of intracellular Na ion concentration was analyzed with fluorescent density. Results showed that intracellular Na+ concentration was decreased by MS-222 treatment, indicating that MS-222 played its proper effect on AP cells. Cell growth rate was measured with (3-(4,5)-dimethylthiahiazo(-z-y1)-2,5-di- phenytetrazoliumromide) MTT assay. Results showed that growth rate of AP cells was not influenced by MS-222 treatment, indicating that inhibition of antler development was not induced by cytotoxicity of drug but may be interfering polarization state of AP. All results indicated antler development was inhibited by treatment of interfering ion channels of AP cells. Therefore, a specific bioelectricity state of AP may be critical to antler development, as ion channel is the main factor to maintain the bioelectricity state of cells. This study provides theoretical basis for further verification of bioelectricity encoding mammalian organ morphogenesis.
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Received: 21 March 2017
Published: 16 June 2017
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