Influence of Glyphaea brevis twig extract on nucleus, tight junctions and expression of inhibin-β, stem cell factor, and androgen binding protein in TM4 Sertoli cells

Abstract

Objective: To examine the influence of Glyphaea (G.) brevis twig extract on the mitochondrial dehydrogenase activity, integrity of the tight junctions between adjacent cells, mitochondria, apoptosis, nucleus and expression of inhibin-ß, stem cell factor, and androgen binding protein in TM4 Sertoli cells. Methods: TM4 cell line was used in this study as it exhibited properties similar to the Sertoli cells. TM4 Sertoli cells were exposed to G. brevis twig extract (0.1, 1.0, 10.0, 100.0, or 1 000.0μg/mL) for 24, 48 and 72 h. Parameters studied included cell viability [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay], mitochondrial membrane potential (tetra methyl rhodamine ethyl ester dye), transepithelial electrical resistance, apoptosis (Annexin V Alexa Fluor®488/propidium iodide assay) and mRNA expression (quantitative reverse transcription polymerase chain reaction). Results: G. brevis twig extract had no cytotoxic impact on cell viability, thus, considerably increasing the activity of mitochondrial dehydrogenase enzyme after 24 and 72 h exposure. Transepithelial electrical resistance values revealed substantial (P<0.05) rise in treated groups, especially after 72 h of treatment. Moreover, there was a significant decrease in mitochondrial depolarization of TM4 Sertoli cells exposed to G. brevis twig extract when compared to controls. In addition, G. brevis twig extract significantly reduced necrosis and apoptosis of TM4 Sertoli cells when compared to control. Nevertheless, fluorescence microscopy revealed that the nuclei were egg-shaped and marked uniformly with consistent cell shape at the middle of the TM4 Sertoli cells. Significant stimulatory effects were observed on mRNA levels of inhibin-β , androgen binding protein and stem cell factor. Conclusions: G. brevis twig extract may increase the secretory roles of TM4 Sertoli cells, cells proliferation, as well as cell-cell tight junction integrity. Thus, G. brevis twig may enhance spermatogenesis.

Keywords

• glyphaea brevis twig
• tm4 sertoli cell line
• inhibin-β{androgen binding protein