Exposure to Endosulfan can result in male infertility due to testicular atrophy and reduced sperm count

Abstract

Endosulfan (ES) is a widely used organochlorine pesticide and is speculated to be detrimental to human health. However, very little is known about mechanism of its genotoxicity. Using mouse model system, we show that exposure to ES affected physiology and cellular architecture of organs and tissues. Among all organs, damage to testes was extensive and it resulted in death of different testicular-cell populations. We find that the damage in testes resulted in qualitative and quantitative defects during spermatogenesis in a time-dependent manner, increasing epididymal reactive oxygen species levels, affecting sperm chromatin integrity. This further culminated in reduced number of epididymal sperms and actively motile sperms. Finally, we show that ES exposure affected fertility in male but not in female mice. Therefore, we demonstrate that ES exerts pathophysiological changes in mice, induces testicular atrophy, affects spermatogenesis, reduces quantity and vigour of epididymal sperm and leads to infertility in males.

Figure 1

Evaluation of physiological effects of Endosulfan in mice. (a) Body weight distribution of the ES-treated animals (n=5 per group) following exposure to different doses of Endosulfan (0, 0.33, 1, 3 and 9 mg/kg body weight). Mean body weight of the group is plotted against days after the offset of treatment. (b) Graph derived from the area of the peak in HPLC profile, showing the bioavailability of ES in mice after oral ingestion of ES (3 mg/kg) against time. (c) Bar graphs indicating enzymatic activities reflective of liver and kidney function at 1 and 21 days after ES treatment (3 mg/kg, n=5). Alkaline phosphatase and Alanine aminotransferase are markers of liver function; urea and creatinine are of kidney function. IU corresponds to international unit. n=5 in all groups. (d) Bar graphs showing RBC, WBC and platelet counts at different time points (1, 11, 21, 31 days) after ES treatment (3 mg/kg) completion. NS, not significant.

Figure 2

Histopathological examination of organs from Endosulfan exposed mice and rat. (a) Histopathology of liver, testes and lungs of mice following ES treatment. Control in all panels indicates respective tissues from mice with no treatment and ES represents tissues from ES-treated mice (20×) (3 mg/kg, 1st day). (b) Histopathology of rat testes after ES treatment (10×) (3 mg/kg, 1st day). (c) Bar graph showing the relative difference in the diameter of seminiferous tubules of testes from control group and Endosulfan-treated rats (3 mg/kg). n=20 tubules in each group.

Figure 3

Endosulfan-induced cell death is specific to testes. (a) TUNEL assay showing DNA fragmentation in testicular cells following ES treatment (3 mg/kg) in male mice. Green colour indicates methyl green counter-stained nuclei and the brown spots are broken DNA stained with diaminobenzidine (DAB). Control sections are devoid of TUNEL-positive cells. (b) TUNEL assay performed on sections obtained at 11th day post-ES treatment showing TUNEL-positive cells. indicating persisting cell death. (c) TUNEL assay showing no stained cells in control and ES-treated mice lungs (1st day). (d) TUNEL assay in brain sections showing absence of positively stained cells (×20).

Figure 4

Evaluation of changes during spermatogenesis following ES exposure. (a) Schematic showing different stages of spermatogenesis in mice. (b) Representative testicular FACS profile of an untreated mouse with cell cycle phases marked. 1n indicates round, elongating and elongated spermatids, which constitutes the majority of cells in testis. G1 peak consists of somatic cells in testis, secondary spermatocytes and spermatogonial mother cells at their G1 phase. S and G2/M phases are constituted solely by the mother cells at their different divisional phases, since somatic cells in testis do not divide. (c) Representative FACS analysis of PI-stained testicular cells from ES-treated mice (3 mg/kg) at different time points after treatment completion showing G₀, 1n, G1, S and G2/M populations. (d) Graphs indicate the time-dependent changes in testicular-cell populations on ES treatment obtained using FACS analysis (3 mg/kg, n=5 in each group). Separate controls were acquired along with samples belonging to different time points. 1n and G1 represent spermatids and diploid cells, respectively. S and G2/M phases indicate spermatogonial mother cells undergoing division. (e) ES-induced, time-dependent testicular-cell death, relative to the control obtained from testicular FACS analysis (n=5 per group).

Figure 5

Evaluation of effect of ES on epididymal sperm integrity. (a) Microscopic analysis of sperm morphology on ES exposure (3 mg/kg) at different time points (0, 10, 20 and 30 days) post-treatment completion. Samples are stained with Eosin Y. (b) Representative FACS analysis of sperm chromatin structure assay. P2 represents red-high sperm indicating loss of chromatin integrity in epididymal sperm cells. Bar graphs show cumulative red-high sperm obtained from ES-treated mice (3 mg/kg) at various time points (10, 20 and 30 days; n=5 per group). (c) Bar graphs indicating the epididymal sperm ROS levels following exposure to ES (3 mg/kg) measured using DCFDA staining followed by FACS (n=9 and 11 for control and ES, respectively). (d) Propidium Iodide-stained sperm heads after sperm dispersion assay (halosperm assay) performed on the epididymal sperm sample that exhibited highest ROS (detected in FACS) following treatment with ES (3 mg/kg). Bar graphs indicate the quantitative representation of the sperm halo area in control and treated groups (n=210 and 179 in control and treated, respectively).

Figure 6

Quantitative analyses of epididymal sperms on Endosulfan exposure. (a–d) Analyses of epididymal sperm collected from ES-treated mice (3 mg/kg) at various time points (11, 26 and 36 days) after treatment completion (n=9 and 10 for control and treated, respectively) showing total sperm count (a), actively motile sperms (b), sluggish motile sperms (c) and non-motile sperms (d). NS, not significant.

Figure 7

Evaluation of effect of Endosulfan on fertility in mice. (a) Bar graphs show average litter size and fertility in males and females at different mating intervals (5–10, 20–25 and 30–35 days) after ES treatment completion (3 mg/kg). (b) Comparison of difference in fertility levels when ES was given only to males (n=10) or to both males and females (n=10 each) (3 mg/kg). Mating window is 5–10 days after treatment completion. NS, not significant.