Identification of Stem Leydig Cells Derived from Pig Testicular Interstitium

Stem Leydig cells (SLCs), located in the testicular interstitial compartment in the mammalian testes, are capable of differentiating to testosterone-synthesizing Leydig cells (LCs), thus providing a new strategy for treating testosterone deficiency. However, no previous reports have identified and cultured SLCs derived from the pig. The aim of the current study was to isolate, identify, and culture SLCs from pigs. Haematoxylin and eosin staining and immunochemical analysis showed that SLCs were present and that PDGFRα was mainly expressed in the pig testicular interstitium, indicating that PDGFRα was a marker for SLCs in the neonatal pig. In addition, reverse transcription-PCR results showed that SLC markers were expressed in primary isolated LCs, indicating that they were putative SLCs. The putative SLCs were subsequently cultured with a testicular fluid of piglets (pTF) medium. Clones formed after 7 days and the cells expressed PDGFRα. However, no clones grew in the absence of pTF, but the cells expressed CYP17A1, indicating that pTF could sustain the features of porcine SLCs. To summarize, we isolated porcine SLCs and identified their basic characteristics. Taken together, these results may help lay the foundation for research in the clinical application of porcine SLCs

A screening for optimal selenium enrichment additives for selenium-enriched fish production: Application of a HPLC-ICP-MS method

The production of selenium-enriched fish contributes to alleviating selenium deficiency for humans. In this study, selenium nanoparticles (SeNPs) comparable in bioavailability to selenomethionine (SeMet), increased SeMet content in O. macrolepis (Onychostoma macrolepis) muscle. Additionally, dietary SeNPs significantly enhanced selenocysteine (SeCys2) and methylselenocysteine (MeSeCys) levels in O. macrolepis muscle. The effect of SeNPs on selenium speciation in grass carp muscle was consistent with O. macrolepis results. SeCys2 and MeSeCys showed antioxidant capacity in HEK293T cells, indicating enhanced health benefits of Se-enriched fish produced using SeNPs. Furthermore, the addition of 0.3 mg/kg SeNPs significantly improved the flesh quality of O. macrolepis by reducing the content of crude fat and heavy metals, as well as increasing the levels of crude protein, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and the ratio of n-3/n-6 polyunsaturated fatty acids (PUFAs). Therefore, selenium-enriched fish produced from SeNPs is a good source for improving human dietary selenium intake

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

<p>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×).</p

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.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092483#pone.0092483-Companyo1" target="_blank">[32]</a>. 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).</p

Sequence of primers used for RT-PCR.

<p>F, Forward primer; R, reverse primer. GAPDH as an internal standard <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092483#pone.0092483-Tabuchi1" target="_blank">[28]</a>.</p

Effects of trehalose on transfer of pEGFP-C1 into the mouse epididymis <i>in vivo</i>.

<p>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.</p

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

<p>Cells were cultured in IMDM supplemented with 120(approximately at 6^th day of subculture), and then harvested for RNA isolation. mRNA levels of <i>rE-RABP, AR and ER-beta</i> were determined by RT-PCR and mRNA of <i>GAPDH</i> 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.</p

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

<p>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. * (<i>p</i><0.05) and ** (<i>p</i><0.01) indicated significant difference between 0 mM and other concentrations of trehalose. n = 4.</p