Trehalose maintains vitality of mouse epididymal epithelial cells and mediates gene transfer

Abstract

In the present study, trehalose was utilized to improve primary culture of mouse epididymal epithelial cells in vitro, and to enhance naked DNA delivery in epididymis in vivo. During the six-day culture, the proliferation activity of the cells in the medium with addition of trehalose was higher than that of those cells cultured in absence of trehalose (p<0.01). To determine the optimal concentration for cell proliferation, a series of trehalose concentrations (0, 60, 120, 180 mM) were tested, and the result indicated that the cell in the medium with 120 mM trehalose showed the highest proliferation potential. The epididymis epithelial cells were cultured in the medium containing 120 mM trehalose upon 16th passage, and they continued expressing markers of epididymal epithelial cell, such as rE-RABP, AR and ER-beta. Our study also indicated that trehalose concentrations of 120-240 mM, especially 180 mM, could effectively enhance DNA delivery into the mouse epididymis epithelial cell in vitro. Moreover, trehalose could induce in vivo expression of exogenous DNA in epididymal epithelial cells and help to internalize plasmid into sperm,which did not influence motility of sperm when the mixture of trehalose (180 mM) and DNA was injected into epididymal lumen through efferent tubule. This study suggested that trehalose, as an effective and safer reagent, could be employed potentially to maintain vitality of mouse epididymal epithelial cells during long-term culture in vitro and to mediate in vitro and in vivo gene transfer.

Figure 1. Effects of trehalose at different concentrations on proliferation of mouse epididymal epithelial cells in vitro.

A, B) Isolation and CK-18 expression of epithelial cells from forty day mouse epididymis. A: First passage cells cultured in the medium with 120 mM trehalose. B: CK-18 expression of the cells. C) Growth curve of epididymal epithelial cells in the medium with different trehalose concentration. Bars represent means±S.D. * (p<0.05) and ** (p<0.01) indicated significant difference between 0 mM and other concentrations of trehalose. n = 4.

Figure 2. Morphology and cell cycle of the cells at 6^th day after subculture in the medium with 120 mM or without trehalose.

Cell morphology (A) and cell cycle (C) of the cells in IMDM with 120 mM trehalose; Morphology (B) and cell cycle (D) of the cells in IMDM without trehalose. Bar: 20 μm. Inserts in A, B showed morphology of cellular nuclear using PI staining under fluorescence microscopy (400×).

Figure 3. RT-PCR analysis of rE-RABP, AR, and ER-beta mRNA levels in isolated epididymal cells (P0), and cells at first, 4^th, 16^th passage culture in presence of trehalose.

Cells were cultured in IMDM supplemented with 120(approximately at 6^th day of subculture), and then harvested for RNA isolation. mRNA levels of rE-RABP, AR and ER-beta were determined by RT-PCR and mRNA of GAPDH was used as an inner control. P0, P1, P4, P16 represented the isolated epidiymal cells, the cells at the first passage, the 4^th passage, and the 16^th passage culture, respectively.

Figure 4. Effects of trehalose on transfer of pEGFP-C1 into the epididymal epithelial cells in vitro.

A) EGFP positive cells were determined at 72 h after transfection. 1) The green fluorescent cells transfected pEGFP-C1 with lipofectamine 2000; 2) The morphology of cells transfected pEGFP-C1 without lipofectamine 2000 or trehalose; 3) The morphology of cells transfected pEGFP-C1 with 60 mM trehalose; 4) The morphology of cells transfected pEGFP-C1 with 180 mM trehalose; 5) The percent of GFP positive cells was determined by flow cytometry. Lipo represents complex of lipofectamine 2000 and DNA; 0, 60, 120, 180, and 240 represent the complex of different concentration trehalose and DNA, respectively. Different letters indicated significant difference (p<0.05), means±S.D, n = 5. B) Effect of 180 mM trehalose-DNA on proliferation of the cells by MTT analysis as described in Materials and Methods. The cells were cultured in the medium with 120 mM trehalose as a control. Lipo-DNA represented the transfection cells with the complex of lipofectamine-2000 and DNA; DNA-Tre indicated that the transfection cells with the complex of 180 mM trehalose and DNA; 120 mM Tre represented control; Different letters (a, b, c) indicated significant differences (p<0.05); and A, B indicated highly significant differences (p<0.01). Bars represent means±S.D, n = 5.

Figure 5. Effects of trehalose on transfer of pEGFP-C1 into the mouse epididymis in vivo.

A, B, and C) fluorescence appeared in different segments of mouse epididymis at 3^rd day after the complex of trehalose-DNA was injected into mouse efferent duct. A) The morphology of mouse epididymis after injecting the complex. B) The mouse epididymis under fluorescence microscopy; C) Localization of GFP protein in epithelial cells and lumen of epididymal caput by immunohistochemistry. D, E, and F) fluorescence only appeared in mouse epididymal caput at 3^rd day after Tre-DNA was injected into mouse epididymal caput interstitial tissue. D) The morphology of mouse epididymis after injecting the complex in light view. E) The mouse epididymis under fluorescence microscopy; F) GFP protein expressed in epithelial cells and intercellular cells of epididymal caput by immunohistochemistry. G, H, and I) Little fluorescence appeared in different segments of mouse epididymis at 3^rd day after the pEGFP-C1 plasmid was injected into mouse efferent duct. G) The morphology of mouse epididymis after injecting the plasmid in light view. H) The mouse epididymis under fluorescence microscopy; I) No GFP positive signal appeared in the epididymal caput by immunohistochemistry. Bar: 40 μm. J) GFP mRNA expression was detected in mouse epididymal caput, corpus and cauda at 3^rd day after treatment by RT-PCR.

Figure 6. Internalization of plasmid DNA into sperm and analysis of sperm membrane fluidity at 3^rd day after the complexes injected into mouse epididymal efferent tubule.

A) Detection of internalization of plasmid DNA into sperm by PCR. M: molecular mark; 1^st lane: plasmid DNA; 2^nd lane: the sperm samples from wildtype mouse; 3^rd, 5^th and 7^th lane: the sperm samples from mouse injected Lipo-DNA; 4^th, 6^th, and 8^th lane: the sperm samples from mouse injected Tre-DNA. B) Membrane futility of sperm was measured by fluorescence polarization of DPH according to Companyo, M et al . The plasma membrane fluidity increasing, while the DPH fluorescence polarization decreasing. Control: sperm from common mouse; Tre-DNA: sperm from mouse injected complex of trehalose and plasmid DNA; Lipo-DNA: sperm from mouse injected complex of lipofectamine 2000 and plasmid DNA. * indicated significant differences (p<0.05,n = 8).