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Cadmium Toxicity Towards Testicular Cell Lines
Reproduction, sexual development, environmental compound exposure
Date : 15/12/2016
Author Information
Uploaded by : Biola
Uploaded on : 15/12/2016
Subject : Medicine
Cadmium exposure is a risk factor for male
infertility. I examined the effects of cadmium
chloride on the viability, proliferation, morphology and migration of mouse reproductive cell lines. Both
cell lines exhibited time and/or concentration dependent reductions in cell
viability in response to cadmium as determined by series of assays
including MTT reduction, lactate dehydrogenase release, trypan blue dye
exclusion and live/dead assays. Analysis of haematoxylin and eosin (H & E) and Coomassie Brilliant Blue
stained cell monolayers indicated that exposure to 1 M
and 12 M concentrations of cadmium for 4 h induced morphological
changes consistent with reduced formation of the leading edge and an increased
population of rounded cells in both cell lines. Analysis of fixed cell
monolayers by indirect immunofluorescence using antibodies against cytoskeletal
proteins revealed disruption of the microfilament and microtubule networks in
cells treated with both concentrations of CdCl2 cells, but no
obvious effect on intermediate filament organisation. Cell migration was
inhibited in both cell lines by 1 and 12 M CdCl2 treatments as
determined by live cell imaging in a 24 h scratch assay. Western blotting analysis suggested that CdCl2
induced a gradual reduction in the levels of actin, although 1 M CdCl2 had no significant
effect in Sertoli cells. However, a transient rise and fall in the levels of
cofilin and p-cofilin (suggesting increased actin binding activity),
respectively, were observed after 4 h exposure to this concentration of Cd2+,
followed by a decrease in both (i.e. decreased activity) after 24 h in Sertoli
cells. Leydig cells showed a significant decrease in the level of actin only
with 12 M Cd2+at the two time points. However, there was a
transient fall and rise in the levels of cofilin and p-cofilin, respectively
after 24 h. Further analysis of Western blots of 1 M CdCl2 treated
Sertoli cells suggested slight decreases in the levels of tubulin and stathmin
1, the latter showing increased levels of phosphorylation (suggesting overall
decreased tubulin binding activity) over 24 h. In contrast, tubulin levels were
significantly increased by both Cd2+ concentrations after 4 h and by
1 M at 24 h in Leydig cells. Data indicate that stathmin 1 levels were
significantly decreased and increased by 12 M Cd2+ at 4 h and 24 h
respectively, with a significant increase and decrease in phosphorylation state
under the same conditions respectively in the same cell line. This indicates
that Cd2+ interferes with the ability of stathmin to destabilise
MTs. Further analysis of Western blots from CdCl2 treated cell
lysates indicated that the two concentrations of CdCl2 caused
significant increases in HSP70 from 4 h to 24 h and in HSP90 at 24 h in Sertoli
cells. HSP60, however, showed no significant changes under all conditions
tested in this cell line. In contrast, in Leydig cells, HSP60, HSP70, and HSP90
were significantly increased by exposure to 12 M Cd2+ at both time
points. Whilst there was no change in the HSP90 with 1 M Cd2+,
HSP60 and HSP70 were significantly increased by exposure to the same M Cd2+
concentration at 4 h and 24 h, respectively.
Although
there were changes in the levels of both total and phosphorylated forms of
MAPKs examined, evaluation of phosphorylation ratios showed increased phosphorylation
and activation of p38 under all conditions tested in both cell lines except
with 12 M Cd2+ at 24 h, which showed no
significant effect on p38 phosphorylation in Leydig cells. While MEK and ERK increased activation was observed with 12 M
Cd2+ at 4 h and 24 h in Sertoli cells,
their phosphorylation states were differentially affected under the same
conditions in Leydig cells Interestingly, JNK phosphorylation was significantly
reduced with 12 M CdCl2 at all time points and with 1 M
Cd2+ at 24 h in Sertoli cells.
Similarly, it was significantly reduced by both CdCl2 concentrations
at 4 h and 24 h in Leydig cells. This indicates that Cd2+
exposure disrupts key signalling pathways involved in the function and survival of testicular cell lines Proteomic analysis of 1 M CdCl2 treated Sertoli cell
lysates showed that at least 14 proteins were differentially
expressed after either 4 or 24 h exposure, four of which were tentatively
identified, one of which is important in spermatogenesis. In conclusion, the two testicular cell lines
exhibit similar levels of sensitivity to CdCl2 but differ in their
precise molecular and morphological responses to the cytotoxic effects of this
heavy metal.
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