Testosterone recuperates deteriorated male fertility in cypermethrin intoxicated rats

Abstract

The present study investigates the protective effects of testosterone against reproductive toxicity induced by cypermethrin (50 mg/kg body weight) in rats. Significant reduction in the testicular and accessory sex organ weights were observed in cypermethrin-treated rats over controls. Cypermethrin intoxication significantly reduced testicular daily sperm count, epididymal sperm count, sperm motility, sperm viability and HOS-tail coiled sperm accompanied by significant reduction in the activity levels of testicular steroidogenic enzymes such as 3β- and 17β- hydroxysteroid dehydrogenases in rats as compared to controls. Further, qPCR studies indicated that the mRNA expression levels of steroidogenic acute regulatory protein (StAR) significantly decreased in cypermethrin-treated rats over controls. Molecular docking analysis indicated that the binding affinity of cypermethrin (− 11.2 kcal/mol) towards StAR protein was greater as compared to its natural ligand, cholesterol (− 8.2 kcal/mol) suggesting improper cholesterol channeling across the testis. Significant reduction in the circulatory levels of testosterone was also recorded in cypermethrin-exposed rats. An increase in pre- and post-implantation loss was observed in rats cohabited with cypermethrin-treated rats. On the other hand, testosterone (4.16 mg/kg body weight) treatment ameliorated cypermethrin-induced reprotoxic effects in rats. To conclude, cypermethrin-induced deterioration of suppressed reproductive performance in male rats could be linked to its antiandrogenic effects and on the other hand, testosterone-mediated protection of male reproductive health in cypermethrin-treated rats at least in part occurs via restoration of testosterone biosynthesis, spermatogenesis and sperm maturation events.

Introduction

There is a major concern towards the increased incidences of male reproductive abnormalities such as reduced semen quality and quantity and testicular cancer [1, 2]. Several studies indicated that a range of environmental toxicants including pesticides at least in part attributes to the deteriorated male reproductive health in humans and wild life [3, 4, 5]. Among a range of pesticides, synthetic pyrethroids are the diverse group of broad spectrum insecticides similar in structure to the natural pyrethrums [6]. Cypermethrin is one among the extensively used type II synthetic pyrethroid pesticide [7]. It consists of an alpha-cyano group attached to the benzylic carbon which enhances the insecticidal properties [8, 9]. The wide usage of cypermethrin to protect agricultural crops can lead to their accumulation and contamination of the environment and eventually interfered with the food chain. Exposure to cypermethrin causes several adverse reproductive abnormalities in humans and animal models [10]. Earlier, androgen receptor reported gene assays has also indicated that cypermethrin can able to induce anti-androgenic effects and estrogen transactivity [11, 12]. Cypermethrin exposure significantly decreased the weights of testes, epididymis, seminal vesicles and prostate in experimental animal models [13]. Studies also indicated that cypermethrin treatment causes reduced spermatogenesis, altered sperm maturation events and inhibition of testosterone levels [10, 14–18]. Many studies also claimed that cypermethrin treatment affect the ciruculatory levels of pituitary gonadotropins such as leutinizing hormone and follicle stimulating hormone in rodent models [10, 17, 19, 20]. Thus, it is apparent that cypermethrin treatment at least in part mediates inhibition of testosterone biosynthesis. On the other hand, testosterone plays a key role to sustain structural and functional integrity of reproductive organs in mammals [21].

Therefore, the central objective of this study was to investigate whether the testosterone administration restores cypermethrin-induced testicular toxicity and suppressed male fertility in rats and if so, what are the mechanisms that underpin protective effects of testosterone.

Materials and methods

Chemicals

Cypermethrin PESTANAL^® (CAS-No: 67375-30-8, with 99% purity by HPLC) was purchased from Sigma-Aldrich (St.Louis, Missouri, USA). All other reagents used in the experiments were analytical grade and purchased from local standard commercial Firms.

Maintenance of animals

Adult albino male rats (90 days old, weighing 150 ± 10 g) of Wistar strain were purchased from an authorized vendor (M/S Sri Venkateswara Enterprises, Bengaluru, India). Rats were maintained in sterilized polypropylene cages (18″ × 10″ × 8″) provided with paddy husk as bedding material. The animals were acclimatized for 10 days under temperature 25 ± 2 °C; light 12 h light/12 h dark cycle and the relative humidity 50 ± 5%. The rats were fed with standard rodent chow and water was ad libitum throughout the experimental period. All the provided experiments performed in this study were carried out in accordance with the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals, Government of India [22] and approved by the Institutional Animal Ethical Committee resolution at Sri Padmavathi Mahila Visvavidyalayam, Tirupati (No:1677/PO/Re/S/2012/CPCSEA/ IAEC-39 dated 06 May 2016).

Treatment schedule

Healthy adult male rats were randomly assigned into four groups of eight animals in each group. Group I animals administered with saline and treated as control group and remaining II, III and IV groups treated as experimental groups. Group II injected with testosterone at a dose of 4.16 mg/kg body weight on 1st, 7th and 14th day through intraperitoneally. Group III was administered with 50 mg/kg body weight with cypermethrin for 60 days and group IV was maintained as regimen with groups III and II.

Fertility studies

After completion of experimental period, control and experimental male rats were allowed to mate with normal cycling female rats to assess reproductive performance. The conception time, the interval between the first day of cohabitation and the day of vaginal plug/or sperm in vaginal smear was recorded for each female. Pregnant rats were moved into separate cages and housed individually. The number of pregnant rats for each experimental and control group was recorded to determine the mating and fertility index. On the 8th day of gestation, six animals from each group were sacrificed to determine the number of corpora lutea/rat, and number of pre implantations/rat and the pregnant rats sacrificed on 18th day of gestation from each group were used to determine the number of live fetuses/rat and post-implantation loss.

Necropsy and evaluation of organ indices

After fertility assays the male rats were fasted overnight, and the weights were recorded. Rats from control and experimental groups were humanely sacrificed by cervical dislocation. Rats were dissected and isolated testis, epididymis and other reproductive organs immediately, removed the adhered materials and blotted with blot paper. After blotting, the isolated organs were weighed to the nearest milligram using Shimadzu electronic balance (Model no: BL-220 H, Shimadzu, Japan). Tissue somatic indices were determined using the formula:

$$\text{TSI}=\frac{\text{Weight of the tissue}}{\text{Weight of the rat}}\times 100$$

Testicular daily sperm production

Testicular daily sperm production was determined according to the method of Blazak et al. [23]. The testes were decapsulated and homogenized in 50 mL ice-cold 0.9% sodium chloride solution containing 0.01% Triton X-100 and allowed to settle for 1 min. After thorough mixing of each sample, the number of sperm heads was counted in four chambers of an improved hemocytometer. The number of sperm produced per gram of testicular tissue per day was calculated and the units of DSP were expressed as millions/gm tissue [24].

$$\text{DSP}=\left(\text{No}.\text{of spermatids}\times \text{dilution factor}\right)/\left(\text{Wt}.\text{of testis}\times \text{time divisor}\right)$$

Epididymal sperm analysis

The cauda part of epididymis was isolated immediately, thoroughly minced in 2 mL of physiological saline (0.9% NaCl) at 37 °C to obtain sperm suspension and used for the determination of sperm count and sperm motility according to Belsey et al. [25]. Total sperm number was enumerated using a Neubauer chamber and the sperm count was expressed as millions/ml. The ratio of live and dead spermatozoa (Viability of sperm) was determined using 1% trypan blue solution by the method of Talbot and Chacon [26]. The sperm membrane integrity was determined by exposing the sperms to hypo-osmotic solution (HOS) and observed for coiled tails under the microscope and the percent of tail coiled sperm was determined by the method of Jeyendran et al. [27]. The motility, viability and HOS-coiled sperms were expressed as a percentage of total sperm counted. All the sperm observations were examined under the OPTICA microscope (Model no: B-350, Italy) at 40X.

Assay of testicular steroidogenic marker enzymes activity levels

The activity levels of testicular steroidogenic marker enzymes, 3β hydroxysteroid dehydrogenase (3β HSD) and 17β hydroxysteroid dehydrogenase (17β HSD) was determined as per the protocol described by Bergmeyer [28]. Briefly, testis was homogenized in ice cold 20 mM Tris–HCl buffer (pH 6.8, 5% w/v) and centrifuged to separate microsomal fraction which was used as an enzyme source. The reaction mixture (2 ml) contains 0.08 μmol of steroid substrate (dehydroepiandrosterone for 3β HSD and androstenedione for 17β HSD), 100 μmol of cofactors (NADPH for 17β HSD and NAD for 3β HSD) and 0.1 M pyrophosphate buffer (pH 7.4) and 20 mg of enzyme source. The incubation mixture deprived of substrate(s) was used as reagent blank. The absorbance was recorded at 340 nm on a spectrophotometer. The enzyme assays were performed under the conditions following zero order kinetics after preliminary standardization regarding linearity with respect to the time of incubation and enzyme concentration. The units for 3β- and 17β-HSDs were expressed as nmol of NAD converted to NADH/mg protein/min and nmol of NADPH converted to NADP/mg protein/min, respectively.

StAR gene expression

Total RNA was isolated from rat testis by GeneJET RNA Purification kit (K0731, Thermo Scientific) according to the manufacturer’s instructions. In brief, the testis tissue (30 mg) grinded thoroughly with mortar and pestle in the presence of liquid nitrogen and transferred immediately into a 1.5 ml micro centrifuge tube containing 300 µl of lysis buffer supplemented with β-mercaptoethanol. This was followed by vortexing mixture for 10 s and 600 μl of Proteinase K was added and placed the tube on vortex to mix thoroughly and incubated at 15–25 °C for 10 min. The contents were centrifuged at 12000g for 10 min followed by the transfer of supernatant into a new RNase-free microcentrifuge tube. To this, 450 μL of ethanol (96–100%) was added and mixed well. RNA was extracted as per the instructions given in the manufacturer’s protocol (GeneJET RNA Purification kit). The purified RNA stored used for the synthesis of cDNA.

cDNA synthesis (RT-PCR)

RT reaction was performed using Revert Aid First Strand cDNA synthesis kit (K1621, Thermo Scientific). The reaction mixture was prepared according to manufacture protocol, mixture was mixed gently and incubated for 60 min at 42 °C. The reaction was terminated by heating at 70 °C for 5 min. The reverse transcription reaction product directly used in Real time PCR reaction.

qPCR studies

Real-time fluorescence-monitored PCRs were performed using Step One Real-time PCR System (Agilent Technologies, Stratagene Mx3005P). The detection of StAR gene expression was performed using gene specific primers and SYBR green Universal master mix (Thermo Fisher, SKU No: 4344463). The expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. The reactions were set up in optical 48-well reaction plates using 25 μl of a mixture containing 12.5 μl of SYBR green Universal PCR Master Mix, 1.0 µl of Forward primer 5′-TTGGGCATACTCAACAACCA-3′ and 1.0 µl of Reverse primer 5′-ATGACACCGCTTTGCTCAG-3′ of both GAPDH, StAR genes, (according to the manufacturer’s instructions), and 5 μl of nuclease-free water. 5 µl of the cDNA samples were subsequently added to each well.

Relative quantification, fold changes were calculated by following the 2^−∆∆Ct method. Cycle threshold (Ct) is the cycle number at which fluorescence signal to cross the Threshold, generated by the cleavage of the dye, crosses the threshold. For each sample, the Ct value of target gene mRNA was normalized to the GAPDH endogenous control as ∆Ct (∆Ct = Ct target gene—CtGAPDH). The fold change of the target gene mRNA in the experimental sample relative to control sample was determined by

$$2^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}=\mathrm{\Delta }{\text{C}}_{\text{t}}\text{Experimental}-\mathrm{\Delta }{\text{C}}_{\text{t}}\text{Control}$$

Molecular docking

StAR protein (Protein Data Bank [PDB] ID: 3P0L) was subjected to energy minimization by steepest descent, using GROMOS96 43b1 force field. The structures of Cholesterol (CID: 5997) and cypermethrin (Pub chem ID: 2912) were downloaded from PubChem in SDF format and then converted into PDB format using openbabel. For rat StAR protein homology modeling, 3P0L was selected as the template. To investigate the conserved domains and the gaps between the rat StAR sequence and the 3P0L, multi align tool was used. The modelled protein sequence of the rat StAR protein was used to dock with the selected ligands obtained the 3D ligand site prediction tool. For docking analysis between the modeled rat StAR protein and the selected ligands were performed by using PyRx (0.8 version). The docked structures were visualized using the tools PyMol, Chimera and Discovery studios.

Statistical analysis

The data were analyzed using one-way analysis of variance (ANNOVA) followed by Dunnett’s multiple comparison test. The data were presented as the mean ± SD and p < 0.05 was considered statistically significant. The statistical analysis was performed by using SPSS (version 16.0; Chicago, IL, USA).

Results

General toxicity observations

The rats exposed to cypermethrin for 60 days did not show any clinical signs of toxicity. During dosage period all the animals were absolutely normal and there were no mortalities observed in both controls and experimental in the entire dosage period and none of the animals were exempted from the treatment schedule. Both control and experimental male rats expressed high signs of sexual stimulus as soon as the female was introduced.

Fertility efficiency

The reproductive end point was studied, all treated and control male rats were able to impregnate the female, had copulatory plugs and appearance of sperm in vaginal smears of females mated with treated males suggests that sexual behavior is not effected due to cypermethrin toxicity. But significant decrease in the fertility index, prolonged conception time, reduced no. of implantations, marked decrease in live pups associated with increased pre and post implantation loss and significant decrease in fetal birth weight observed. Whereas testosterone injected to cypermethrin exposed rats took less conception time to impregnate females when compared to cypermethrin alone exposed males (Table 1). An increase in number of implantations and decrease in pre- and post-implantation loss in females mated with rats co-administered with cypermethrin and testosterone (Fig. 1). No significant changes were observed in fertility parameters in normal rats injected with testosterone when compared with control rats (Table 1).

Table 1.

Effect of testosterone (T) on fertility output of adult male rats exposed to cypermethrin (CYP)

Parameters	Control	Control + T	CYP	CYP + T
Conception time (Days)@	1.30a ± 0.36	1.08a ± 0.05 (− 16.92)	4.96b ± 0.24 (281.53)	1.75c ± 0.21 (− 64.71)
Mating index (%)	100 (12/12)	100 (12/12)	100 (12/12)	100 (12/12)
Fertility index (%)	100 (12/12)	100 (12/12)	58.33 (7/12)	91.66(11/12)
No. of corporalutea/rat*	12.92a ± 0.27	12.97a ± 0.12 (0.38)	12.22b ± 0.10 (− 5.41)	12.94a ± 0.02 (5.89)
No. of implantations/rat*	11.65a ± 0.38	13.32b ± 0.22 (14.33)	3.09c ± 0.07 (− 73.47)	8.55d ± 0.40 (176.69)
Pre-implantation loss (%)	4.66	2.04	69.28	33.92
No. of live fetuses/rat*	11.11a ± 0.08	13.29b ± 0.20 (19.62)	3.09c ± 0.08 (− 72.18)	8.01d ± 0.07 (159.22)
Post-implantation loss (%)	4.63	1.89	84.84	19.64
Fetal weight (g)#	5.54a ± 0.35	5.98a ± 0.17 (7.94)	2.98b ± 0.39 (− 46.20)	4.92c ± 0.19 (65.10)

Values are mean ± SD; ^@n = 12; *^{, #}n = 6. For calculation of percentage change for CYP + T treated group respective CYP treated group considered as control. For the remaining groups untreated rats (control) considered as control. Values in the parentheses are percentage change from that of control

^a−dDifferent superscripts in the same row differ significantly from each other at (p < 0.001)

Fig. 1.

Pre-implantation sites of female rats mated with a control, b control + testosterone, c cypermethrin, d cypermethrin + testosterone on eighth day of pregnancy. Post-implantation sites of female rats mated with e control, f control + testosterone, g cypermethrin, h cypermethrin + testosterone on eighteenth day of pregnancy

Body weight and tissue indices

No significant changes were observed in the body weights of experimental rats when compared with controls. However, a significant (p < 0.01) reduction in the testicular and accessory sex organ indices was observed in cypermethrin exposed rats when compared with control rats (Table 2). Whereas injection of testosterone to cypermethrin exposed rats showed an increase in the weight of testes and other accessory sex organ indices (Table 2). Similarly, a significant (p < 0.05) changes were observed in the indices of reproductive organs in testosterone alone injected rats as compared to control rats (Table 2).

Table 2.

Effect of testosterone (T) on body weight and tissue somatic indices (g %) in rats exposed to cypermethrin (CYP)

Parameter/organ	Control	Control + T	CYP	CYP + T
Body weight	295.62a ± 10.56	295.98a ± 8.25 (0.12)	290a ± 9.48 (− 1.90)	294.25a ± 9.00 (1.46)
Testis	1.15a ± 0.02	1.24a ± 0.06 (7.82)	0.56b ± 0.12 (− 51.30)	0.84c ± 0.15 (50)
Epididymis	0.23a ± 0.04	0.25a ± 0.04 (8.69)	0.07b ± 0.02 (− 69.56)	0.11c ± 0.03 (57.14)
Vas deferens	0.14a ± 0.03	0.15a ± 0.02 (7.14)	0.06b ± 0.02 (− 57.14)	0.10c ± 0.01 (66.66)
Seminal vesicles	0.44a ± 0.02	0.49a ± 0.06 (11.36)	0.30b ± 0.01 (− 31.81)	0.45c ± 0.07 (50)
Prostate	0.18a ± 0.01	0.19a ± 0.04 (5.55)	0.05b ± 0.01 (− 72.22)	0.08c ± 0.01 (60)

Values are mean ± SD of 8 rats. For calculation of percentage change for CYP + T treated group respective CYP treated group considered as control. For the remaining groups untreated rats (control) considered as control

Values in the parentheses are percent change from that of control

Mean values with same superscripts in a row do not differ significantly from each other at p < 0.05

Sperm parameters

A significant (p < 0.01) decrease in daily sperm production, sperm count, sperm motility, sperm viability, and sperm coiling percentage was observed in rats exposed to cypermethrin when compared to controls (Table 3). Whereas injection of testosterone to cypermethrin exposed rats showed an increase in the sperm count, sperm motility, sperm viability, and sperm HOS coiling when compared to cypermethrin alone treated rats (Table 3).

Table 3.

Effect of testosterone (T) on sperm production and quantity in cauda epididymis of adult male rats exposed to cypermethrin (CYP)

	Control	Control + T	CYP	CYP + T
DSP (millions/gm tissue)	25.4a ± 1.9	31.12b ± 3.33 (22.51)	12.03c ± 1.10 (− 52.65)	20.39d ± 1.56 (69.49)
Sperm count (millions/ml)	66.04a ± 2.25	76.44b ± 2.34 (15.74)	25.70c ± 1.93 (− 61.08)	42.86d ± 6.06 (66.77)
Sperm motility (%)	72.24a ± 2.33	80.13b ± 3.85 (10.92)	28.03c ± 3.18 (− 61.19)	43.21d ± 5.39 (54.15)
Sperm viability (%)	77.10a ± 1.70	87.45b ± 1.96 (13.42)	37.59c ± 4.36 (− 51.24)	60.03d ± 4.93 (59.69)
HOS tail coiled sperm (%)	70.96a ± 1.60	81.03b ± 3.06 (14.19)	28.88c ± 4.64 (− 59.30)	43.04d ± 4.34 (49.03)

Values are mean ± SD of 8 rats. For calculation of percentage change for CYP + T treated group respective CYP treated group considered as control. For the remaining groups untreated rats (control) considered as control

Values in the parentheses are percentage change from that of control

Mean values are significantly differ from control at p < 0.001

Activity levels of testicular steroidogenic marker enzymes

The activity levels of testicular 3β-HSD and 17β-HSD were significantly decreased (p < 0.01) in rats exposed to cypermethrin when compared to control rats. Injection of testosterone to cypermethrin exposed rats showed an increase in the activity levels of testicular 3β-HSD and 17β-HSD (Fig. 2a). A significant (p < 0.05) increase was observed in the activity levels of testicular 3β-HSD and 17β-HSD in testosterone injected animals as compared to control rats (Fig. 2a).

Fig. 2.

a Effect of Testosterone (T) on testicular 3β and 17β-hydroxysteroid dehydrogenase activity levels in the male rats exposed to cypermethrin (CYP). Bars are mean ± S.D. of 8 rats. Bars with different superscripts differ from respective controls at p < 0.001. For calculation of significance for Control+T group and CYP groups, control rats considered as controls; for CYP + T treated groups, CYP rats served as controls. b The mRNA levels of StAR gene expression in Testes of rats obtained after normalizing with GAPDH mRNA levels. Bars are mean ± SD of six rats. Bars with different superscripts differ significantly from respective controls at p < 0.05. For calculation of significance for Control+T group and CYP groups, control rats considered as controls; for CYP + T treated groups, CYP rats served as controls

StAR expression

The expression levels of StAR mRNA in the testes were significantly (p < 0.05) decreased in rats exposed to cypermethrin when compared with the levels of StAR mRNA in controls, whereas co-administration of testosterone and cypermethrin resulted in significant (p < 0.05) increase in the expression levels of StAR mRNA when compared with their respective cypermethrin treated rats (Fig. 2b).

Molecular docking studies

The correctness in the built protein (StAR) was validated using the chimera software pipeline GA341 (value: 1.0) and DOPE scores (− 1.19 kcal/mol) and galaxyrefine tool. PROCHECK analysis indicated that the structural quality of the modelled protein was stable as indicated by the Ramachandran plot analysis (approximately 96% of the amino acids occur in the allowed regions and none of the amino acids in the disallowed regions. Moreover structural analysis from ProSA (Z-score − 5.95) and the quality of protein using ProQ (LG score = 4.256) showed that the selected modelled protein was good in stability and quality. Docking was performed between the modelled rat StAR protein and selected ligands (cholesterol and cypermethrin) using the Autodock Vina available in the PyRX 0.8 version. The binding energy between the modelled rat StAR protein and cholesterol was − 8.2 kcal/ mol, whereas, the binding energy between the modelled rat StAR protein and cypermethrin was − 11.2 kcal/ mol, suggesting a greater affinity between the modelled rat StAR protein and the pyrethroid. This was not surprising because, as the interactions between the rat StAR protein and cholesterol were found to be hydrophobic (Fig. 3a), while the interactions between the rat StAR protein and cypermethrin were found to contain hydrogen bonds and hydrophobic interactions (Fig. 3b). The hydrophobic interactions between the modelled rat StAR protein and cholesterol comprises of the following amino acids Leu136, Val137, Met140, Met143, Val150, Glu168, Phe183, Arg187, Thr189, Leu198, Ala198, His219, Met224, Trp240, Leu242, Ile244, Thr262, Phe266. On the other hand, the hydrophobic interactions between the modelled rat StAR protein and cypermethrin comprises of the following amino acids: Met143, Asn149, Val150, Glu168, Ala170, Phe183, Arg187, Leu198, Ala199, Gly200, His219, Thr222, Met224, Ile244, Leu259, Thr262, Phe266 and the amino acids involved in the hydrogen bonding interactions with the cypermethrin were Asn147 and Trp240 (Table 4). The length of the both hydrogen bond was 2.83A in interactions between the rat StAR protein and cypermethrin (Fig. 3a, b).

Fig. 3.

a Interactions between the modelled rat StAR protein and the Cholesterol. b Interactions between the modelled rat StAR protein and the cypermethrin

Table 4.

Binding affinities and interacting aminoacids of StAR with cholesterol and cypermethrin

Ligand	Binding affinity	Interacting aminoacids
Cholesterol	− 8.2 kcal/mol	Leu136, Val137, Met140, Met143, Val 150, Glu168, Phe183, Arg187, Thr189, Leu198, Ala198, His219, Met224, Trp240, Leu242, Ile244, Thr262, Phe 266
Cypermethrin	− 11.2 kcal/mol	Met143, Asn149, Val150, Glu168, Ala170, Phe183, Arg187, Leu198, Ala199, Gly200, His219, Thr222, Met224, Ile244, Leu259, Thr262, Phe266

Discussion

The results of the present study indicated that testosterone treatment showed ameliorative effects on male reproductive health in rats intoxicated with cypermethrin as evidenced by (a) significant increase in the weights of reproductive organs, (b) restoration of spermatogenesis and sperm maturation events, (c) improvement in testicular steroidogenesis and (d) fertility efficacy.

In the present study, no significant differences were observed in the body weights of cypermethrin treated rats over controls, reflecting no overt toxicity in cypermethrin treated rats. However, we did notice a significant reduction in the weights of testis, epididymis, seminal vesicles, vas deferens and prostate gland suggesting inadequate supply of androgens. The results are in agreement with previous studies Joshi et al. [29]. The weight of the testes is completely dependent on the mass of differentiated spermatogenic cells and marked decrease in the weight of testes observed in this study may be due to reduced seminiferous tubule size, decreased number of germ cells, Leydig cells and elongated spermatids [10, 20, 30]. Adequate bioavailability of androgens is one of the prerequisites for proper maintenance of reproductive organ weights and their structural and functional integrity [21]. Accordingly, testosterone supplementation showed restoration of reproductive organ weights in cypermethrin treated rats. The results are in agreement with previous studies wherein, testosterone supplementation improves the weights of reproductive organ weights in rats intoxicated with di-n-butylpthalate [31], lead [32] and cadmium [33].

Cypermethrin treatment showed reduction in the testicular daily sperm count and epididymal sperm parameters such as sperm motility, sperm viability and number of tail coiled sperm in rats, reflecting diminished spermatogenesis and sperm maturation events. Earlier, several studies reported that exposure of cypermethrin significantly reduced the sperm count and motility associated with reduced circulatory levels of testosterone in rats [18, 19, 34]. The structural integrity of testis with proper number of Sertoli cells is required for the spermatogenesis. Previously, cypermethrin treatment showed abnormal structural aspects in rats with lumen devoid of sperm or with few sperm and with few Sertoli cell number [20]. The Sertoli cell functions in turn depend on the adequate supply of androgens and published reports indicated that cypermethrin treatment negatively affects the testosterone biosynthesis thereby interferes with spermatogenesis [12]. The machinery of testicular steroidogenesis comprises of proteins and cascade of marker enzymes, 3β-HSD and 17β-HSD [35]^. Steroidogenic acute regulatory protein (StAR) is a key protein that controls a rate-limiting step, proper channeling of cholesterol from outer to inner mitochondrial membrane. Thus, interruption at this critical step may lead to reduced production of testosterone. In the present study, we found a significant reduction in the activity levels of testicular steroidogenic cascade enzymes: 3β-HSD and 17β-HSD associated with a significant reduction in the expression levels of StAR mRNA levels in cypermethrin-intoxicated rats over a period of 60 days. These events consequently leads to reduced testicular steroidogenesis in rats treated with cypermethrin. Previously, we demonstrated that cypermethrin-intoxication inhibits circulatory levels of testosterone in rats [20]. Further, it has been shown that exposure to cypermethrin inhibits the activity levels of 3β-HSD and 17β-HSD in rats [10, 36, 37]. In silico analysis also revealed that, the binding affinity between the cypermethrin and rat-StAR was greater than the cholesterol, a natural ligand and rat-StAR protein. Thus, it is clear from the above results that cypermethrin treatment could be toxic at the level of testicular Sertoli- and Leydig-cells. Surprisingly, injection of testosterone to cypermethrin treated rats showed enhancement of testicular steroidogenesis as indicated by increased activity levels of 3β-HSD and 17β-HSD accompanied elevated expression levels of StAR mRNA levels. These events could ameliorate the testosterone levels thereby sperm production and its maturation events in cypermethrin treated rats. The exact reason(s) for the amelioration of spermatogenesis in testosterone administered cypermethrin treated rats can be determined from this study. In this study, we found an increase in the activity levels of 3β-HSD and 17β-HSD accompanied elevated expression levels of StAR mRNA levels in rats subjected to testosterone plus cypermethrin. Similar results were reported by Rangel et al. [38] who have shown that the supplementation of testosterone promotes the progesterone production via stimulation of StAR, P450 cholesterol side chain cleavage and LH receptor mRNA expression in granulosa cells of Gallus gallus. Published reports also indicated that testosterone plays a key role in the feed-back regulation of testicular StAR mRNA expression [39]. Though, we did not determined these aspects, the results of this study have shown that supplementation of testosterone stimulated the expression levels of testicular StAR mRNA levels in rats exposed to cypermethrin. Piecing the results, it can be speculated that the supplementation of testosterone promotes StAR mRNA expression which in turn facilitated the transport of cholesterol and consequently testicular steroidogenesis. These events further stimulated the spermatogenesis in rats subjected to both testosterone plus cypermethrin [13].

All treated and control male rats were able to impregnate the female, had copulatory plugs and appearance of sperm in vaginal smears of females mated with treated males suggests that sexual behavior is not effected in rats treated with cypermethrin. However, a significant decrease in the fertility index, prolonged conception time, reduced no. of implantations, marked decrease in live pups associated with a significant decrease in fetal birth weight was observed in cypermethrin treated rats. Further, a significant increase in pre- and post-implantation loss in rats cohabited with cypermethrin treated rats associated with reduced sperm motility, sperm viability and number tail-coiled sperm might reflect compromised sperm fertility. It is known well that an adequate number of motile sperm, viable sperm and sperm with intact membrane integrity determine the male fertility efficacy in rats [40, 41]. This is because motile sperm can able to reach the zona pellucida thereby fertilization process occurs in mammals. Thus, reduced motile sperm associated with reduced fertility in cypermethrin treated rats reflects suppressed male reproductive performance. Previously, it has been shown that cypermethrin induced sperm chromatin damage, low sperm quality and quantity are the reasons for decreased fertility and spermatogenesis [29, 42, 43]. On the other hand, testosterone injection significantly enhanced fertility efficacy in male rats intoxicated with cypermethrin. This could be attributed to enhanced spermatogenesis and sperm maturation events or increased testicular steroidogenesis and/or both. It was also reported that exogenous supplementation of testosterone can partially maintain spermatogenesis in rats treated with chemotherapeutic agent, carboplatin [44]. The present results also support the studies of Reshma and Reddy [32]. Studies of Reshma and Reddy [32] demonstrated that the supplementation of testosterone restores fertility efficacy in lead-intoxicated rats.

Based on the findings, it is clear that the cypermethrin toxicity at the level of testis and epididymis could lead to spermatotoxicity, reduced biosynthesis of testosterone and altered sperm maturation events, respectively. In contrast, administration of testosterone sustains the physiological levels in cypermethrin treated rats thereby supports the structural and functional integrity of testis and epididymis.

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Conflict of interest

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.