Spermatozoa HSP90b expression correlates with ROS generation and altered motility in response to methyl parathion treatment in vitro

INTRODUCTION

Agrochemicals like organophosphorus pesticides (OPs) are used worldwide as pesticides and are hazardous to human health. However, they are mainly known for their neurotoxic effects on non-target species through inhibition of acetylcholinesterase (AChE) activity together with the potential to generate reactive oxygen species (ROS) inducing oxidative stress during their metabolism.^[1] Based on this, majority of studies are focused on neurotoxicity of OPs in both non-target and model organisms. The previous studies on methyl parathion (MePa) revealed that it has adverse effects on semen quality in humans and animals.^[2,3] Reproductive potential principally in terms of semen quality and hormonal level are reported to be adversely impacted in response to OPs.^[4,5] Furthermore, epidemiological studies reveal multifarious reproductive disorders such as infertility and adverse pregnancy outcomes including perinatal mortality and congenital defect as a consequence of OP exposure.^[6] The OP pesticides alter reproductive functions primarily through reduction in the activity of brain AChE and, in turn, influence the gonads. Several studies on human exposure to OP pesticide have reported reduction in sperm count and motility, hormonal imbalances such as low testosterone and high luteinizing hormone levels, and increased sex chromosome aneuploidy in sperm.^[7] Decline in sperm chromatin integrity is observed in agricultural workers in response to OP exposure particularly MePa, methamidophos, and DZN.^[5]

MePa, a toxic organophosphate insecticide has great demand for its broad-spectrum activity. It plays a significant role in impairing sperm function. Changes in the histoarchitecture of testis of adult white throated munias (Lonchura malabarica) are observed on exposure to MePa (5–20 g/100 g body weight/day), where the higher doses reduced the testicular weight after 10 days. On the other hand, a marked decline in seminiferous tubules was evidenced after single administration of lower dose, thus implying impairment of the cholinergic functions of the testes by MePa.^[8] A recent study on HepG2 cells exposed to malathion toxicity reported DNA damage and oxidative stress by increase in the level of malondialdehyde (MDA) level, a by-product of lipid peroxidation.^[9] Although, Salazar-Arredondo et al. (2008) have reported MePa to decline the motility of human spermatozoa at a concentration of 750 μM, its mechanism of induction is largely unknown.^[10] Since oxidative stress and sperm motility are implicated in MePa toxicity, it will not be out of context to hypothesize the heat shock proteins (HSPs) involvement in this process. In fact, the previous proteomic studies by our group have reported the down regulation of different HSPs in human spermatozoa in various male infertility conditions.^[11,12] SagarePatil et al. (2017), have also reported a decline in HSP90 in oligoasthenozoospermic men and found a positive correlation with the number of motile spermatozoa. The authors also reported differential expression of HSPs. HSP90a was typically localized in residual nuclear envelope of capacitated human spermatozoa, whereas HSP90b isoform was predominantly flagellar. They also reported that HSP90 inhibition by geldanamycin or 17-AAG was able to suppress progesterone-mediated forward progressive motility, hyperactivation, and acrosome reaction.^[13] Under this scenario, the present study is designed to find out the effect of MePa on ROS generation, DNA damage vis-à-vis HSP90b expression, and its correlation with sperm motility in vitro.

MATERIAL AND METHODS

RESULTS

Sperm motility, ROS generation, sperm chromatin integrity, and expression of stress responsive molecular chaperone HSP90b were studied after exposure of motile human spermatozoa to different concentrations of MePa.

MePa exposure impaired sperm motility

Lower concentrations of MePa (i.e., 50 and 250 μM) for 60 min at 37°C did not show any effect on motility. However, a significant decrease in motility was observed at 500 and 750 μM MePa [Figure 1]. Since significant decrease in sperm motility was observed at 500 μM, time kinetics of effect of MePa on spermatozoa were studied at this concentration of MePa. When spermatozoa were exposed to 500 μM MePa for different time, their motility significantly decreased at 60 min [Figure 2].

Close

Figure 1:

Effect of different concentrations of methyl parathion (i.e., 50, 250, 500, and 750 μM) at 37^oC for 60 min on motility of human spermatozoa. *P < 0.05 with respect to control.

Export to PPT

Close

Figure 2:

Effect of exposure time (0, 15, 30, 45 and 60 min) of 500 μM methyl parathion at 37^oC for 60 min on motility (%) of human spermatozoa. *P < 0.05 with respect to control.

Export to PPT

Effect of MePa on ROS generation by spermatozoa

MePa induces increased generation of ROS in spermatozoa in a dose-dependent manner up to 750 μM [Figure 3] when incubated at 37°C for 60 min. A significant increase in the level of ROS was observed in higher treatment group (i.e., 500 and 750 μM) as compared to control, though no statistical significance was observed between the groups.

Close

Figure 3:

Effect of different concentrations of methyl parathion (i.e., 50, 250, 500, and 750 μM) at 37^oC for 60 min on reactive oxygen species generation (H2DCF-DA fluorescence). *P < 0.05 with respect to control.

Export to PPT

Alteration in the sperm chromatin integrity by MePa

As significant reduction in motility and augmented generation of ROS was observed in 500 μM treated group, so the effect of MePa on the sperm chromatin was evaluated by DFI at this concentration. It has been observed that MePa-treated spermatozoa have three-fold increase in DFI as compared to control [Figure 4].

Close

Figure 4:

Comparison of DNA fragmentation index between control and 500 μM methyl parathion treated group (Acridine Orange Test). *P < 0.05 with respect to control.

Export to PPT

Expression of HSP90β in MePa-treated spermatozoa

HSP90b is found to be primarily expressed in the head and neck region of the spermatozoa and its expression increased significantly when exposed to higher concentration of MePa, that is, 500 μM as evidenced by the difference in intensity of fluorescence measured by fluorescence microscope (Leica, Microscope) [Figure 5].

Close

Figure 5:

Expression profile of heat shock protein HSP90? In control and methyl parathion treated spermatozoa (500 μM) (a); corresponding box-whisker plot of fluorescence intensity of Immunocytochemistry (in arbitrary units) (b). *P < 0.05 with respect to control.

Export to PPT

DISCUSSION

OPs, including MePa, were found to be associated with reproductive failures with trans-generational effects. Their genotoxic effects on sperm are opined to be through oxidative damage.^[13,16] Pina-Guzman et al. studied on the exposure of MePa on male mice affecting spermatozoa functions decreasing its fertilization ability.^[17] Verma et al. (2009), reported that chronic oral toxicity of MePa causes lipid peroxidation in different parts of rat brain (i.e., fore-, mid-, and hind-brain) as evidenced by formation of MDA and 4-hydroxynonanal.^[18] In another study, very low doses of MePa (i.e., 0.5–1.5 mg/kg) on rats reported decreased sperm count, along with the formation of abnormal sperms.^[4] Similarly, in vitro exposure to MePa is reported to affect DNA integrity in human ejaculated spermatozoa without substantially affecting the motility until 750 μM concentration.^[10] Our results corroborated the findings of Salazar-Arredondo et al., with regard to sperm DNA integrity and induction of oxidative stress as revealed by augmented ROS generation. Furthermore, there is a significant decline in sperm motility at 500 and 750 μM MePa exposures with respect to control; although, no marked change was observed between the doses. The generation of ROS is a function of the source of production, for example, number of mitochondria present or number of moles of sperm NADH oxidase. Therefore, maximum rate of generation might have been achieved at 500 μM which did not increase further by augmenting the exposure level to 750 μM. It has been reported that in response to augmented ROS production, transcription and ATP production in mitochondria of boar spermatozoa are severely affected with a damage inflicted to electron transport chain proteins.^[19] Therefore, the observed decline in motility might be due to an energy deprived state along with a change in membrane fluidity due to ROS-mediated oxidative damage to the membrane lipids. Interestingly, we observed a sharp increase in HSP90b expression in response to 500 μM MePa exposure. HSPs are constitutively produced molecular chaperones which act in response to several environmental stressors.^[20] HSP90b is principally localized in the head (equatorial region). In a recent report, Profumo et al.^[21] have shown that induction of oxidative stress by H₂O₂ upregulated HSP90 on the surface of primary human endothelial cells. Therefore, it is natural to expect an upregulated expression of the chaperone in MePa-induced oxidative stress condition. Furthermore, oxidative damage to HSP90 is shown to cleave the protein into Hsp90cl resulting in degradation of its client proteins.^[22] On cleavage, Hsp90cl gains a new function that leads to the accumulation of actin aggregates.^[23] Actin polymerizationdepolymerization is suggested to regulate sperm motility including acrosome reaction and capacitation.^[24] Therefore, this may also contribute to the decline in sperm motility.

Overall, our study demonstrated that MePa is capable of generating ROS in vitro inflicting damage to the spermatozoa DNA and upregulates HSP90b expression resulting in impaired sperm motility.

CONCLUSION

The organophosphorus pesticides such as methylparathion alter the reproductive functions of animals and humans by inducing oxidative stress through the production of ROS. This ROS mediates the modulation of HSP90β expression which may affect the structural integrity of client proteins and causes oxidative injury to membrane lipids along with DNA integrity resulting in declined sperm motility in response to MePa.