Effects Of Triazole Fungicides On Reproductive Functions And Mechanism Of Male Caenorhabditis Elegans

ObjectiveIn the study, Caenorhabditis elegans were used as a model to investigate the toxicity effects induced by triazole fungicides and to explore the adverse effects on spermatogenesis, spermiogenesis, mating behaviors and the regulatory mechanisms in those process.Methods1 The brood sizes, generation time and the number of outcross progeny were measured to assess the fecundity of male nematodes after triazole fungicide exposure.2 The number of germ cells and spermatids, the mitotic germ cell proliferation and entry into meiosis assays in the male nematodes were measured to assess the effects of spermatogenesis of male nematodes after triazole fungicide exposure. Further, the gene relative expressions of germ cell proliferation and differentiation signal pathways TGF-P (daf-7, daf-1, daf-4, daf-3, and daf-5) and IGF-1 (daf-2, age-1, akt-1, akt-2, and daf-16) were analyzed after triazole fungicide exposure.3 The spermatids diameter and cross-sectional area and the percentage of spermatids normal morphology, spermatids activated and sperm normal transfer of male nematode exposure to triazole fungicide were tested to explore the adverse effects on spermiogenesis. Further, the gene relative expressions of male sperm activation pathway SWM-1 (swm-1, try-5, spe-6, spe-4,fer-1) were analyzed after triazole fungicide exposure.4 The number of hermaphrodities contacted and turns, vulva location efficiency, and mating efficiency were used to explore the adverse effects of male mating behavior after triazole fungicide exposure. Further, the gene expression levels of male mating behavior were analyzed after exposure to triazole fungicide.Results1 Effects of triazole fungicides on the fecundity of male nematodesThe brood sizes at concentrations more than 10.0 ?g/L and the number of outcross progeny at concentrations more than 1.0?g/L were decreased, however, the generation time were not changed compared with the control group after exposure to tebuconazole, epoxiconazole and prothioconazole(0.1,1.0,10.0 ?g/L).2 Effects of triazole fungicides on spermatogenesis of male nematodesThe data demonstrated that the number of total germ cells, mitotic cells, meiotic cells and spermatids were less than control group's both in tebuconazole, epoxiconazole and prothioconazole exposure at concentrations more than 10.0 ?g/L, sperm counts was decreased at concentrations more than 1.0 ?g/L after exposure to epoxiconazole. The decrease of mitotic cells also indicated that germ cell proliferation was inhibited by tebuconazole, epoxiconazole and prothioconazole. The process of entry into meiotic was inhibited after exposure to tebuconazole and epoxiconazole at concentration more than 10.0 ?g/L. It was suggested that tebuconazole and epoxiconazole induced the mitotic germ cell proliferation arrest and premature entry into meiosis. Besides, the results showed that tebuconazole, epoxiconazole and prothioconazole did activate the TGF-? and IGF-1 signal pathways and increase the genes expression levels.3 Effects of triazole fungicides on spermiogenesis of male nematodesThe results showed that compared with the control group, spermatids diameter and cross-sectional area were both decreased after exposure to tebuconazole, epoxiconazole and prothioconazole at concentrations 0.1?1.0 and 10.0?g/L, while the spermatids morphology was not observed abnormal changes; the percentage of spermatids normal morphology were decreased exposure to tebuconazole, and epoxiconazole at concentrations more than 10.0 ?g/L, while exposure to prothioconazole did not noticeably decrease; sperm transfer were not affected exposure to tebuconazole, epoxiconazole and prothioconazole. Besides, the results showed that tebuconazole and epoxiconazole did activate the SWM-1 signal pathways and increase the genes expression levels.4 Effects of triazole fungicides on the mating behavior of male nematodesThe results showed that the number of hermaphrodities contacted were not changed, the number of turns was increased at the concentration of 10.0 ?g/L after tebuconazole exposure and at the concentrations of 10.0?g/L after epoxiconazole and prothioconazole exposure; vulva location efficiency were reduced exposure to tebuconazole and prothioconazole at concentrations more than 10.0 ?g/L and epoxiconazole at concentrations more than 1.0?g/L; mating efficiency were reduced exposure to epoxiconazole and prothioconazole at concentrations more than 10.0 ?g/L, while exposure to tebuconazole did not observe noticeably changes. The results showed that osm-5, lov-1, pkd-2, unc-29, unc-103 genes expression levels were increased after exposure to tebuconazole, epoxiconazole and prothioconazole, respectively.ConclusionGerm cells development can be affected by tebuconazole, epoxiconazole and prothioconazole though activating the TGF-? and IGF-1 signal pathways, reducing the numbers of germ cells and spermatids and defecting spermatogenesis; tebuconazole, epoxiconazole and prothioconazole induce smaller sperm, the SWM-1 signal pathway also be activated after exposure to tebuconazole and epoxiconazole with the decreased activated spermatids number and the spermiogenesis was disturbed as a result; the genes osm-5?lov-1?pkd-2?unc-29?unc-103 associated with mating behavior were induced up-regulation, disturbing the turns, vulva location efficiency, mating efficiency of male nematodes and decreasing the number of offspring after exposure to tebuconazole, epoxiconazole and prothioconazole.