Effect of Spag11a gene knockout on the epididymis in mice: A histopathological and molecular analyses.

The sperm-associated antigen 11a (Spag11a) gene is exclusively expressed in the caput epididymis. Our previous studies demonstrated that small interfering RNA (siRNA)-mediated ablation of this gene resulted in increased proliferation of epididymal epithelial cells. Further, active immunization-mediated ablation of SPAG11A protein increased the susceptibility of male reproductive tract tissues to diethylnitrosamine (DEN)-induced tumorigenesis. In this study, we report that the caput epididymis of Spag11a knockout mice displayed hyperplasia and inflammation, while the caput epididymis of wild-type mice exhibited normal anatomical structure. Global transcriptome analyses in the caput epididymis of knockout mice indicated differential expression of genes involved in a variety of cellular processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses suggested that the absence of Spag11a may activate microRNAs associated with cancer, chemical carcinogenesis-receptor activation, and chemical carcinogenesis-DNA adducts pathways; which may contribute to the promotion of tumorigenesis in the epididymis. The susceptibility of caput epididymis to chemically induced carcinogenesis in Spag11a knockout mice was analyzed. Histological analyses indicated that while the epididymis of wild-type mice did not show any signs of tumorigenesis, knockout mice displayed hyperplasia, anaplasia, dysplasia, neoplasia, and inflammation in the caput epididymis. Our results provide concrete evidence that deletion of Spag11a induces histopathological and molecular changes that contribute to tumorigenesis. It is possible that the expression of Spag11a gene could be one of the reasons for the rarity of epididymal cancers. The involvement of an epididymal gene in tumorigenesis is being demonstrated for the first time.

Ablation of the sperm-associated antigen 11A (SPAG11A) protein by active immunization promotes epididymal oncogenesis in the rat.

Incidence of cancer in the epididymis is very rare. It is proposed that proteins specific to this organ may contribute to this unique property. We previously demonstrated that siRNA-mediated knockdown of SPAG11A mRNA resulted in increased proliferation of epididymal epithelial cells, whereas overexpression of this gene caused reduced proliferation in immortalized cell lines. In this study, we evaluated the oncogenesis-related anatomical and transcriptome changes in the epididymis of SPAG11A-immunized rats challenged with a low dose of diethyl nitrosamine (DEN). DEN treatment or SPAG11A immunization alone did not cause any histopathological changes in the epididymis. Interestingly, indications of oncogenesis were observed in SPAG11A-immunized + DEN-treated rats. Using high throughput sequencing, we observed that 3549 transcripts that were differentially expressed in the caput epididymis of DEN only-treated rats displayed similar differential expression in the caput epididymis of SPAG11A-immunized rats, indicating that the microenvironment that contributes to oncogenesis sets in when SPAG11A protein is ablated. Differential expression of genes that are involved in 10 major cancer related pathways was also analyzed. Majority of the genes related to these pathways that were differentially expressed in the caput epididymis of DEN only-treated rats also showed similar pattern in the caput epididymis of SPAG11A-immunized rats. For the first time, results of our study demonstrate that ablation of SPAG11A by active immunization renders the epididymis susceptible to oncogenesis and that this protein may be one of the factors that contributes to the rarity of epididymal cancer.

Effect of a mixture of pyrethroids at doses similar to human exposure through food in the Indian context.

Residual amounts of pyrethroids were detected in rice and vegetables of the Indian market. Thus, consumers are exposed to a mixture of pyrethroids on a daily basis through food. Though a large number of studies reported the toxic effects of pyrethroids, there are no reports that used doses equivalent to human consumption. In this study, male Wistar rats were exposed daily to a mixture of pyrethroids for 1-15 months which is equivalent to the amount present in rice and vegetables consumed by an average Indian each day. The oxidant-antioxidant status (lipid peroxidation, nitric oxide; activities of catalase, glutathione peroxidase, glutathione S transferase, and superoxide dismutase) and anatomical changes in the general organs (liver, lung, and kidney) and male reproductive tract tissues (caput, cauda, testis, and prostate) were evaluated. Further, liver and kidney function tests, lipid profile, and complete blood picture were analyzed. Increased oxidative stress, perturbations in the antioxidant enzyme activities, and damage to the anatomical architecture were observed. Disturbances in the liver function and lipid profile were significant. Results of our study demonstrate that exposure to a mixture of pyrethroids at a dose that is equivalent to human consumption can cause systemic and reproductive toxicity, which may ultimately result in the development of lifestyle diseases. This first line of evidence will fuel further studies to determine the impact of food-based pyrethroid exposure on the long-term health of humans and to envisage policies to reduce pesticide content in food products.

A mixture of pyrethroids induces reduced fecundity and increased testicular genotoxicity in rats.

To achieve crop protection and higher agricultural yield, pesticides are used; and among them pyrethroid based ones are the most preferred choice because of their specificity on the pests. Uncontrolled use of pesticides resulted in contamination of food products. Presence of residual amounts of pyrethroids was reported in agricultural products sold in Indian market, indicating that humans are constantly exposed to these chemicals through food on a routine basis. Studies that determine the toxic effects of pyrethroids at doses equivalent to human exposure are rare. We orally administered a mixture of pyrethroids (detected in the rice and vegetables of Indian market) to male rats for 15 months to mimic the long-term exposure in humans. We observed reduced fecundity, sperm count and 13ß-hydroxysteroid dehydrogenase enzyme activity. The serum concentrations of hormones involved in male reproductive function were altered. Further, testicular genotoxicity as reflected by perturbations in the expression pattern of genes involved in the molecular processes of gametogenesis was evident. Such toxic effects may also be occurring in humans who consume agricultural products that contain residual amounts of pyrethroids on a regular basis throughout their lifetime.

Pyrethroid based pesticides - chemical and biological aspects.

Human and animal welfare primarily depends on the availability of food and surrounding environment. Over a century and half, the quest to identify agents that can enhance food production and protection from vector borne diseases resulted in the identification and use of a variety of pesticides, of which the pyrethroid based ones emerged as the best choice. Pesticides while improved the quality of life, on the other hand caused enormous health risks. Because of their percolation into drinking water and food chain and usage in domestic settings, humans unintentionally get exposed to the pesticides on a daily basis. The health hazards of almost all known pesticides at a variety of doses and exposure times are reported. This review provides a comprehensive summation on the historical, epidemiological, chemical and biological (physiological, biochemical and molecular) aspects of pyrethroid based insecticides. An overview of the available knowledge suggests that the synthetic pyrethroids vary in their chemical and toxic nature and pose health hazards that range from simple nausea to cancers. Despite large number of reports, studies that focused on identifying the health hazards using doses that are equivalent or relevant to human exposure are lacking. It is high time such studies are conducted to provide concrete evidence on the hazards of consuming pesticide contaminated food. Policy decisions to decrease the residual levels of pesticides in agricultural products and also to encourage organic farming is suggested.

Effect of long-term treatment with a mixture of pyrethroids on the expression of genes that govern male germ cell production in rats.

Humans are exposed to pyrethroid-based pesticides through agricultural produce. In this study, male Wistar rats were orally treated for 9 to 12 months with a mixture of pyrethroids that is equivalent to one-fifth (high dose; HD) or one-twenty fifth (low dose; LD) of the amount of pyrethroids present in the cereals and rice consumed by an average Indian. In rats treated for 9 months, the spermatogenesis-associated genes Abp, Ar, Cd9, Dax1, Dazap1, Ddx3y, Gdnf, Gfra1, Grth, Inhb, Ovol1, P1, Plzf, Pygo2, Scf, Tgfb1, Tp1, Tp2, and Vim1 were downregulated in both LD and HD groups. In rats treated for 12 months Gdnf, Hsf2, Inhb, Tgfb1, Thy1, and Ybx2 expression was downregulated in both LD and HD groups. Steroidogenesis-associated genes 17-ß-Hsd, Gata4, Hmgcr, Hmgcs1, Pde4b, and Tspo gene expression were reduced in both LD- and HD-treated groups treated for 9 months. In 12-month-treated rats, Creb1 expression decreased in both LD and HD groups. The epigenetic reprogramming-associated genes, Dnmt1, Dnmt3a, Dnmt3b, Hdac10, Hp1bp3, Kat3a Kat3b, Mch2ta, Ncoa7, and Sirt1 were downregulated in both HD and LD groups of 9-months-treated rats. In rats treated for 12 months, Hdac10, Mch2ta, Ncoa7, and Sirt1 messenger RNA levels decreased in both the HD and LD groups. Thus, we demonstrate that long-term exposure to a mixture of pyrethroids caused aberrations in the transcriptome of factors involved in sperm production and development.

Effect of oral administration of a mixture of pyrethroids at doses relevant to human exposure on the general and male reproductive physiology in the rat.

Studies on the effects of unintentional intake of pyrethroid pesticides that are akin to actual human exposure settings are very rare. Such an exposure is primarily by consuming the food products as routine diet that contain residual levels of pyrethroids. In this study, rats were orally administered for 15 months with a mixture of pyrethroids at a dose that is one-fifth (high dose; HD) or one-twenty fifth (low dose; LD) of the residual levels commonly present in the average amount of rice and vegetables consumed by Indian population. Lipid profile, kidney and liver function were assessed. Lipid peroxidation, nitric oxide, antioxidant enzyme activities and histopathological changes were analyzed in the liver, lung, kidney, pancreas, testes, caput, cauda and prostate. The effect on the male reproductive system as a function of sperm count, enzyme activity of 3ß-HSD and 17ß-HSD and the expression profile of genes involved in spermatogenesis, steroidogenesis, genetic reprogramming and apoptosis of male gametes were evaluated. Significant increase in the relative organ weight, perturbations in the activities of antioxidant enzymes, lipid profile and liver function were observed in both LD and HD groups. Damage to the anatomical architecture was evident in all the tissues due to pyrethroid toxicity. Exposure to LD and HD of pyrethroid mixture resulted in decreased sperm count, activities of 3ß-HSD and 17ß-HSD, impaired capacitation and acrosome reaction and perturbations in the expression of genes that govern male gamete production. Results of our study indicate that exposure to pyrethroids for longer durations even at doses that are far below the residual levels present in the food consumed will result in severe damage to general physiological processes as well as reproductive function.

siRNA-mediated knockdown of sperm-associated antigen 11a (Spag11a) mRNA in epididymal primary epithelial cells affects proliferation: a transcriptome analyses.

Differential expression of a variety of proteins in the four major regions of the epididymis contributes to maturation of spermatozoa and region-specific cellular functions as well. Proliferation of epithelial cells of the epididymis is highly controlled and thus is one of the major reasons for the nonoccurrence of cancers in this organ system. The molecular mechanisms and the contribution of region-specific genes in epithelial cell proliferation are not yet fully understood. In this study, for the first time, we analyzed the role of sperm-associated antigen 11a (Spag11a), a caput-specific beta-defensin-like antimicrobial gene in governing epididymal cell proliferation and global gene expression. siRNA-mediated knockdown of Spag11a mRNA in epididymal primary epithelial cells resulted in increased cell proliferation. Out of the 68,842 genes analyzed, 4182 genes were differentially expressed (2154 upregulated and 2028 downregulated). A variety of genes that participate in different cellular processes and pathways were differentially regulated. Genes that are important for epithelial cell proliferation were found to be differentially regulated and these changes were confirmed by real-time PCR. Overexpression of Spag11a in immortalized rat caput epididymal cells resulted in decreased proliferation capacity. Results of this study indicate that Spag11a plays a crucial role in governing epididymal epithelial cell proliferation.

Uroplakin expression in the male reproductive tract of rat.

Uroplakins (UPKs) play an important role in the normal and pathophysiology of the urothelium. They protect the urothelium and play a crucial role during urothelial infections by Uropathogenic E. coli. However, their functions beyond this organ system remain unexplored. A wide variety of proteins secreted in the male reproductive tract tissues contribute to spermatogenesis, sperm maturation, fertilization and innate immunity. However, the presence of UPKs and their possible contribution to the male reproductive tract physiology is not yet reported. Hence, in this study, we characterized UPKs in the male reproductive tract of rats. To the best of our knowledge, for the first time, we report the expression of UPKs in the male reproductive system. Upk1a, Upk1b, Upk2 and Upk3b mRNA and their corresponding proteins were abundantly expressed in the caput, cauda, testis, seminal vesicles and the prostate. Their expression was not developmentally regulated. UPK protein expression was also localized on the spermatozoa, suggesting a role for these proteins in sperm function. To study the role of UPKs in innate immunity, Upk mRNA expression in response to endotoxin challenge was evaluated in vitro and in vivo. In the rat testicular and epididymal cell lines, Upk mRNA levels increased in response to lipopolysaccharide challenge. However, in the caput, cauda, testes, seminal vesicle and prostate obtained from LPS treated rats, Upk mRNA expression was significantly reduced. Results of this study indicate a role for UPKs in male reproductive physiology and innate immune responses.

Tlr1-13, Nod1/2 and antimicrobial gene expression in the epididymis and testis of rats with alloxan-induced diabetes.

Pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) and nuclear oligomerization domain (NOD) receptors along with antimicrobial proteins and peptides (AMPs) are crucial for innate immunity. The pathology of insulin-dependent diabetes mellitus is associated with the disrupted expression of TLRs, NODs and AMPs in the kidney, lungs and other organs. However, such a relation in the male reproductive tract is not yet investigated. In this study, we analysed the expression pattern of Tlr1-13, Nod1/2 receptors and AMPs (ß-defensins and defensin-like proteins of the Sperm-Associated Antigen 11 (Spag11) family) in the male reproductive tissues (caput, cauda and testis) obtained from diabetic or insulin-treated diabetic or untreated control rats. Alterations in the expression pattern of Tlr1-13, Nod 1/ 2, Defb1, 2, 21, 24, 27, 30 and Spag11a/ c/ t were observed under diabetic conditions. Administration of insulin to diabetic rats could modulate the expression pattern of only some these genes. Results of our study indicate perturbed gene expression profile of Tlrs, Nod1/2, Defbs and Spag11 isoforms in the epididymis and testis of diabetic rats, and this could be one of the important reasons for the increased risk of infections in the male genital tract.

Exposure to allethrin-based mosquito coil smoke during gestation and postnatal development affects reproductive function in male offspring of rat.

The threat of zika virus looms throughout the world and the use of allethrin-based mosquito coils to prevent mosquito bites during and postpregnancy is on the rise. The aim of this study was to analyze the toxic effects of exposure to allethrin-based mosquito coil smoke in rats under conditions that reflect human settings. Pregnant female rats were exposed to mosquito coil smoke and same was continued to the male pups up to 111 days postparturition (21-day weaning plus up to 90 days postweaning). Increased oxidative stress, distorted antioxidant enzyme status, downregulation of genes involved in spermatogenesis, sperm maturation and steroidogenesis was observed. Daily sperm production, total sperm count and acrosome reaction was compromised. Results of our study indicate the toxic effects of exposure to allethrin-based mosquito coil smoke in male offspring and calls for preventing mosquito coil use during pregnancy and postnatal development. Community-based programs that will encourage general population to use classical methods such as use of mosquito nets, keeping the surroundings clean and use of natural mosquito repellents should be conducted.

Allethrin toxicity causes reproductive dysfunction in male rats.

Pyrethroids are widely used for domestic and agricultural purposes and their use is increasing, especially in developing countries. Uncontrolled use of these insecticides resulted in their entry into the food chain thereby causing toxicity to different organ systems. Allethrin is one of the widely used pyrethroids, but its toxicological effects are underreported when compared to other pyrethroids. Further, its effects on the male reproductive tract remain uncharacterized. In this study, its toxicity on the male reproductive tract was evaluated by administering 25-150 mg/kg body weight allethrin to adult rats for 60 days. The mRNA expression of factors that are important in spermatogenesis (Scf, c-Kit, Hsf2, Ovol1, Brdt, Kdm3A, Ybx-2, and Grth) and steroidogenesis (StAR, 3ß-HSD, 17ß-HSD) was significantly downregulated. Decreased levels of testosterone, reduced sperm count and daily sperm production was also observed due to allethrin toxicity. However, sperm quality parameters assessed by computer-assisted sperm analyzer were not affected. Spermatozoa obtained from allethrin-treated rats failed to undergo acrosome reaction. Results of this study indicate that allethrin affects spermatogenesis and sperm function, thus lending further support to the growing evidence of its toxicity.

Transcriptional regulation of the rat sperm-associated antigen 11e (Spag 11e) gene during endotoxin challenge.

The lipopolysaccharide (LPS) inducible expression of antimicrobial proteins of the Sperm-Associated Antigen 11 (Spag11) family is dependent on nuclear factor-κB (NF-κB) activation and epigenetic factors. However, the regulatory mechanisms that govern their gene expression during endotoxin challenge are unknown. In this study, we demonstrate that the Spag11e gene upstream sequence contains binding sites for androgen receptor (AR), NF-κB, nuclear factor-1, E-twenty-six and activator protein 2. The role of these transcription factors in inducing Spag11e gene during LPS challenge was analysed by measuring luciferase activity in HEK cells transiently transfected with deletion constructs that lacked one or more of the binding sites. Deletion of AR-binding site resulted in loss of luciferase activity and no further decrease was observed when progressive deletions of the other transcription factor binding sites were made. Mutations in AR or NF-κB binding site resulted in loss of luciferase activity. Electrophoretic gel-mobility shift assays indicated that AR and NF-κB proteins bind to the synthesised radio-labelled oligomers used as probes and the mobility shifted when respective antibodies were added. Results of this study indicate the direct involvement of AR and NF-κB in LPS-induced Spag11e expression, thereby expanding our understanding of antimicrobial gene expression during endotoxin challenge.

Allethrin induced toxicity in the male reproductive tract of rats contributes to disruption in the transcription of genes involved in germ cell production.

Pyrethroids are known to be neurotoxic. However, their toxic effects including that of allethrin on the male reproductive tract are not elucidated. Adult male rats were treated orally with 25, 50, 100, and 150 mg/kg body weight allethrin every day for 60 days. Lipid peroxidation was increased (p < 0.001) in the caput, cauda, and testes. Nitric oxide production was increased (p < 0.001) in the caput, but unaltered in the cauda and testes. The activities of catalase, glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase were decreased in the caput and cauda where as a decrease was observed in the testis obtained from allethrin treated rats. In the epididymides and testes, damage to tubular architecture, congestion, degeneration of epithelial cell lining, intestinal edema, and presence of dead or degenerating spermatids were observed in a dose dependent manner. The expression profile of genes involved in spermatogenesis (Tgf-beta1), sperm maturation (Spag11e), and sperm function (Defb22) were reduced (p < 0.001) in allethrin rats. The expression of p53 gene was decreased and increased phosphorylation of MAPK (p42/p44) expression was observed the male reproductive tract tissues of allethrin treated rats. Although earlier studies have reported the effects of allethrin inhalation because of the use of mosquito coils and vaporizers, our results for the first time prove that oral exposure to allethrin could affect fertility and may contribute to deregulation of cell cycle in the male reproductive tract.

Lipopolysaccharide induces epididymal and testicular antimicrobial gene expression in vitro: insights into the epigenetic regulation of sperm-associated antigen 11e gene.

Infections of the male reproductive tract lead to infertility, and the molecular mechanisms that operate under these conditions are not well studied. Using epididymal and testicular tissues cultured in vitro, we demonstrate that lipopolysaccharide (LPS) induces the mRNA expression of beta-defensins, Spag11s, and pro-inflammatory cytokines in the rat caput, cauda, and testes. LPS-induced antimicrobial gene expression involved NF-kB activation and decreased levels of histone deacetylase 1 (HDAC1) and DNA methyltransferase (DNMT), all of which possibly allow antimicrobial gene transcription. Inhibition of endogenous HDAC1 and DNMT1 resulted in higher antimicrobial gene expression when compared to the LPS alone treated conditions. Increased trimethylation of histone 3, its binding to the upstream region of Spag11e gene, and demethylation of this region were observed during endotoxin challenge. We demonstrate that antimicrobial gene expression in the male reproductive tract tissues during endotoxin challenge involves NF-kB activation and epigenetic changes.

Characterization of SPINK2, SPACA7 and PDCL2: Effect of immunization on fecundity, sperm function and testicular transcriptome.

Testicular factors play a vital role in spermatogenesis. We characterized the functional role of rat Spink2, Spaca7 and Pdcl2 genes. Their primary, secondary and tertiary structure were deduced in silico. The genes of rat Spink2, Spaca7 and Pdcl2 mRNA were predominantly expressed in the testis. SPINK2, SPACA7 and PDCL2 protein expression was evident in all the cell types of testis and on spermatozoa. Ablation of each of these proteins by active immunization resulted in reduced fecundity and sperm count. Damage to the anatomical architecture of testis and epididymis was evident. In SPINK2 immunized rats, 283 genes were differentially regulated while it was 434 and 872 genes for SPACA7 and PDCL2 respectively. Genes that were differentially regulated in the testis of SPINK2 immunized rats primarily belonged to extracellular exosome formation, extracellular space and response to drugs. SPACA7 ablation affected genes related to extracellular space, oxidation-reduction processes, endoplasmic reticulum membrane and response to drugs. Differential gene expression was observed for nuclear function, protein binding and positive regulation of transcription from RNA polymerase II promoter in testis of PDCL2 immunized rats. Results of our study demonstrate the role of SPINK2, SPACA7 and PDCL2 in spermatogenesis and in important molecular processes that may dictate testicular function and other physiological responses as well.

Uroplakin 1a Interacts with Regucalcin and Proteasome Subunit Beta 1.

Uroplakins (UPKs) are specialized proteins that plan an important role in protecting the epithelium of the bladder from toxic waste. We recently demonstrated the expression pattern of UPKs in the male reproductive tract and their importance in sperm function in murine models. However, the exact mechanisms through which UPKs affect spermatogenesis are not reported. In this study, using yeast two-hybrid screening was conducted to determine the interaction partners of Uroplakin 1a (UPK1A). Y2H Gold yeast strain overexpressing UPK1A was mated with Y187 yeast strain overexpressing human testis cDNA library and the mutants were plated on SD agar plates containing selection media. Colonies that grew on SD/-Trp, SD/-Leu, SD/-His, and SD/-Ade plates were isolated and evaluated to identify the interacting partners of UPK1A. Regucalcin (RGN) and proteasome subunit beta 1 (PSMB1) were identified as potential interaction partners. Using HEK cells that overexpress UPK1A and RGN or PMSB1, the co-localization and interaction were estimated with high-resolution microscopy and Pearson's coefficient. In light of the fact that UPK1A knockout caused subfertility and that the role of RGN and PSMB1 in spermatogenesis is documented, an interaction between UPK1A and RGN or PSMB1 could be required for spermatogenesis.

Uroplakin 1a Knockout Mice Display Marginal Reduction in Fecundity, Decreased Bacterial Clearance Capacity, and Drastic Changes in the Testicular Transcriptome.

Uroplakins (UPKs) form physical and chemical barriers in the bladder and other urinary tract tissues. We previously reported the identification and localization of UPKs in the male reproductive tract of rat. In this study, we characterized Upk1a knockout mice and report a marginal reduction in fecundity associated with significant decrease in sperm count. Upk1a mice had lower bacterial clearance capacity when challenged with uropathogenic Escherichia coli for 1 to 5 days. High-throughput analyses of testicular transcriptome indicated that 1128 genes that are expressed in testis of wild-type mice were completely absent in the knockout, while 2330 genes were found to be expressed only in the testis of knockout mice. Furthermore, differential regulation of 148 (67 upregulated and 81 downregulated) was observed. Gene ontology analyses indicated that processes related to integral components of membrane (plasma membrane), G-protein receptor activity and signaling, olfactory receptor activity and perception of smell, organization of extracellular space/region, immune and inflammatory responses to pathogens, spermatid development, meiotic cell cycle, and formation of synaptonemal complex were affected. Results of this study provide evidence on the possible multi-functional role of Upk1a in male reproductive tract and in other tissues as well.

Effect of continuous inhalation of allethrin-based mosquito coil smoke in the male reproductive tract of rats.

OBJECTIVES: Continuous inhalation of allethrin-based mosquito coil smoke may affect fertility, an aspect that has not received much attention. In this study, we attempt to understand the harmful effects on the male reproductive system caused by continuous exposure to allethrin-based mosquito coil smoke. METHODS: Adult Wistar rats were allowed to inhale mosquito coil smoke for 15-180 days, and male reproductive tract tissues (caput, cauda, and testes) were collected. Using standard biochemical techniques, changes in oxidative stress (lipid peroxidation) and antioxidant status was measured. Histopathological analyses were carried out to assess pathomorphological damage in the caput, cauda, and testis. Real-time polymerase chain reaction was carried out to determine the expression pattern of the stress-response gene, p53, and the spermatogenic factors c-Kit, Scf, and Tgf-ß1. RESULTS: In rats exposed to allethrin-based mosquito coil smoke for 15-180 days, compared to the unexposed controls, lipid peroxidation was increased in the cauda and testes. The activity of antioxidant enzymes remained largely unchanged in the all the tissues analyzed. Histopathological analyses revealed loss of tubule architecture, epithelial cell disruption, increase in lumen size, interstitial edema, and presence of dead spermatozoa. p53 gene expression was differentially altered in the epididymis and testes. The expression of spermatogenic factors, namely, stem cell factor and its ligand c-Kit was unaltered though decreased levels of Tgf-ß1 were observed. CONCLUSION: Results of this study demonstrate that prolonged exposure to allethrin-based mosquito coil smoke could lead to oxidative stress and compromise germ cell production.

Transgenerational effects on the fecundity and sperm proteome in rats exposed to a mixture of pyrethroids at doses similar to human consumption.

Pyrethroid based pesticide usage for crop protection resulted in percolation of these compounds into the food chain. Toxicological studies that reflect exposure to pyrethroids through food in the human settings are rare. We conducted animal experimentations using a mixture of pyrethroids that is equivalent to the amount consumed by average individual through rice and vegetables in the Indian context. Male rats treated with a mixture of pyrethroids for 1-12 months displayed decreased transgenerational fecundity, sperm count, activities of 3ß- and 17ß-HSD and perturbed hormonal profile. At the transcriptome level, the expression of genes involved in spermatogenesis, steroidogenesis, germ cell epigenetic modulators and germ cell apoptosis were altered in the testis. In the sperm lysates of control rats, 506 proteins identified by mass spectrometry. The differential expression of these proteins (treated/control ratio) in the pyrethroid exposed rats was analyzed. Among the 506 proteins, 153 had a ratio of 0; 41 had a ratio ranging from >0 to <0.5; and 10 had a ratio >2.0. Interestingly, the differential expression was transgenerational. 26 proteins that were differentially expressed in the sperm of F0 treated rats continued to remain the same in the F1, F2 and F3 generations, while the differential expression was maintained up to F2 and F1 generations for 46 and 2 proteins respectively. Some of the proteins that continued to be differentially expressed in the later generations are reported to have critical roles in male reproduction. These results indicate that the reduced fecundity observed in the later generations could be due to the continued differential expression that was initiated by pyrethroid treatment in the F0 rats. Results of our study, for the first time, provide evidence that long-term exposure to pyrethroids affects transgenerational fecundity manifested by changes in sperm proteome.