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 pro-apoptotic 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.

• Apoptosis
• extrinsic pathway
• germ cells
• intrinsic pathway
• stress
• testis

Acknowledgments

The authors want to express their gratitude to Dr. Miguel Betancourt for his support in the use of the fluorescence microscope.

Declaration of interest

This study was partially supported by CONACyTPIFOP-CONACYT-SEP 309-0 (C/PFPN-2002-35-32). PROMEP, grant 1035/09/1247. The authors report no conflict of interest.

Author contributions

Study design: Conceived, designed, and coordinated the study, evaluated steroid hormones, analysis and interpretation of data, and participated in the critical review of the manuscript: MSIRM; Carried out TUNEL, western blot, sperm analysis, statistical data analysis, and drafted the manuscript: ALJR; Participated in the critical review of the manuscript: MGL, RMVV, AAM, LEGQ. All authors read and approved the final version of the manuscript.