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Spermatogonial Stem Cell Sensitivity to Capsaicin: An in Vitro Study

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Spermatogonial Stem Cell Sensitivity to Capsaicin: An in Vitro Study

Sefika C Mizrak et al. Reprod Biol Endocrinol.

Erratum in

  • Reprod Biol Endocrinol. 2011;9:17

Abstract

Background: Conflicting reports have been published on the sensitivity of spermatogenesis to capsaicin (CAP), the pungent ingredient of hot chili peppers. Here, the effect of CAP on germ cell survival was investigated by using two testis germ cell lines as a model. As CAP is a potent agonist of the transient receptor potential vanilloid receptor 1 (TRPV1) and no information was available of its expression in germ cells, we also studied the presence of TRPV1 in the cultured cells and in germ cells in situ.

Methods: The rat spermatogonial stem cell lines Gc-5spg and Gc-6spg were used to study the effects of different concentrations of CAP during 24 and 48 h. The response to CAP was first monitored by phase-contrast microscopy. As germ cells appear to undergo apoptosis in the presence of CAP, the activation of caspase 3 was studied using an anti activated caspase 3 antibody or by quantifying the amount of cells with DNA fragmentation using flow cytometry. Immunolocalization was done with an anti-TRPV1 antibody either with the use of confocal microscopy to follow live cell labeling (germ cells) or on Bouin fixed paraffin embedded testicular tissues. The expression of TRPV1 by the cell lines and germ cells was confirmed by Western blots.

Results: Initial morphological observations indicated that CAP at concentrations ranging from 150 uM to 250 uM and after 24 and 48 h of exposure, had deleterious apoptotic-like effects on both cell lines: A large population of the CAP treated cell cultures showed signs of DNA fragmentation and caspase 3 activation. Quantification of the effect demonstrated a significant effect of CAP with doses of 150 uM in the Gc-5spg cell line and 200 uM in the Gc-6spg cell line, after 24 h of exposure. The effect was dose and time dependent in both cell lines. TRPV1, the receptor for CAP, was found to be expressed by the spermatogonial stem cells in vitro and also by premeiotic germ cells in situ.

Conclusion: CAP adversely affects spermatogonial survival in vitro by inducing apoptosis to those cells and TRPV-1, a CAP receptor, may be involved in this effect as this receptor is expressed by mitotic germ cells.

Figures

Figure 1
Figure 1
Photomicrograph of Gc-5spg stained with an anti-activated caspase 3 antibody and counterstained with Haemaluin as described in Materials and Methods. A, Control culture treated with DMSO at the same concentration as that used in the 250 uM CAP solution; B, Culture treated with 250 uM CAP during 48 h. Arrow shows apoptotic cells. Bars represent 15 um
Figure 2
Figure 2
Flow cytometric determination of apoptotic germ cells. Representative dot blot histograms of Gc-5spg cells cultured in control medium (left panel) and in medium ontaining 200 μM CAP during 48 h (right panel). On the x-axis the FL1 fluorescence (autofluorescence) and on the y-axis the FL3 fluorescence (for PI) are indicated. The population of apoptotic cells (with hypodiploid DNA, arrow) becomes clear after incubation with CAP.
Figure 3
Figure 3
CAP triggers in a dose-dependent manner apoptosis of the Gc-5spg and Gc-6spg cell lines. Quantification of CAP induced apoptosis per time point and doses was based on data obtained from the flow cytometry analysis as illustrated in Fig. 2. A, Gc-5spg B, Gc-6spg. a represents statistically significant difference between control and the doses applied and b represents statistically significant difference between 24 h. of treatment and 48 h. of treatment.
Figure 4
Figure 4
Images of Gc-5spg (A) and Gc-6spg (B) cells obtained after immunofluorescent labelling of the cells with an anti-TRPV1 antibody and CLS microscopy as described in Materials and Methods. Labelling was observed on the plasma membrane of the germ cells Bars: 20 um.
Figure 5
Figure 5
Detection of TRPV1 on Gc-5spg and Gc-6spg cell lines using Western Blotting. Lane 1, A10-85 Glioma cell line (positive control); lane 2, Gc-6spg; lane 3, Gc-5spg.
Figure 6
Figure 6
Photomicrograph of a section through an adult rat testis showing TRPV1 labelling of premeiotic germ cells, at stage II of the seminiferous epithelium. Arrow, undifferentiated spermatogonia; arrow head, early pachytene spermatocytes; asterisk, Sertoli cells. Bar represents 10 um.

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