Intrinsic and extrinsic apoptotic pathways are involved in rat testis by cold water immersion-induced acute and chronic stress

Abstract

Testicular apoptosis is activated by stress, but it is not clear which signaling pathway is activated in response to stress. The aim of this study was to investigate whether intrinsic, extrinsic, or both apoptotic signaling pathways are activated by acute and chronic stress. Adult male rats were subjected to cold water immersion-induced stress for 1, 20, 40, and 50 consecutive days. The seminiferous tubules:apoptotic cell ratio was assayed on acute (1 day) and chronic (20, 40, 50 days) stress. Apoptotic markers, including cleaved-caspase 3 and 8, the pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins were also determined after acute and chronic stress induction. Additionally, epididymal sperm quality was evaluated, as well as corticosterone and testosterone levels. An increase in tubule apoptotic cell count percentage after an hour of acute stress and during chronic stress induction was observed. The apoptotic cells rate per tubule increment was only detected one hour after acute stress, but not with chronic stress. Accordingly, there was an increase in Bax, cleaved caspase-8 and caspase-3 proapoptotic proteins with a decrease of anti-apoptotic Bcl-2 in both acutely and chronically stressed male testes. In addition, sperm count, viability, as well as total and progressive motility were low in chronically stressed males. Finally, the levels of corticosterone increased whereas testosterone levels decreased in chronically stressed males. Activation of the extrinsic apoptotic pathway was shown by cleaved caspase-8 increase whereas the intrinsic apoptotic pathway activation was determined by the increase of Bax, along with Bcl-2 decrease, making evident a cross-talk between these two pathways with the activation of caspase-3. These results suggest that both acute and chronic stress can potentially activate the intrinsic/extrinsic apoptosis pathways in testes. Chronic stress also reduces the quality of epididymal spermatozoa, possibly due to a decrease in testosterone.

Stress and Cell Death in Testicular Cells

“…Changes in the integrity of the outer mitochondrial membrane promote the release of molecules with proapoptotic activity, such as cytochrome C, from the intermembranal space to the cytoplasm, leading to a marked activation of apoptosis in the testicular cells of males exposed to stress by immersion in cold water [49]. It is likely that cold water immersion stress may be related to the activation of apoptosis in testicular cells through increased corticosterone concentration as well as increased ROS and oxidative stress in the testes.…”

“…Data obtained in this work, propose that both the intrinsic and extrinsic pathways could be involved in this process, due to the increase in expression and activity of caspases 8, 9 and 3 [64]. These alterations are frequently associated with male infertility and the increased apoptosis frequency in the germinal cells of the seminiferous epithelium [65] as well as with the decrease in the concentration of mature sperm stored in the tail of the epididymis [12,49,66]. In the epididymis, sperm undergo changes during their maturation process; they acquire motility, final condensation of the nucleus and the ability to fertilize.…”

“…In E) representative gels of five proteins at each time interval. Each point represents the mean+standard error (X+E.E) (n=5) * p<0.05 compared to the control [49].…”

“…Caspase 3 can be activated by increasing the concentration of active caspase 8 (extrinsic label), either by increasing the Bax content as well as by decreasing Bcl-2 concentration (Figure 4) [49]. Cell death is likely to start when FasL, produced by Sertoli cells, binds to the Fas receptor [30,50], located on the cytoplasmic membrane of germ cells.…”

“…These processes are all androgendependent, mainly testosterone and dihydrotestosterone (DHT). Chronic exposure to stress causes decreased testosterone secretion as well as increased corticosterone [49,66,67]. The reduction in the sperm concentration of the epididymis may be related to the decrease in testosterone, since the decrease of this hormone affects the conversion of round to elongated spermatids during spermiogenesis in stages VllVlll and causes its premature detachment, inhibiting its elongation process [13], this may lead to an increase in the incidence of apoptosis in germ cells, leading to a large loss of germ cells [23].…”