International audience | Many toxicants are present in water as a mixture. Male infertility is one of the environmental impacts in developed countries. Using our rat seminiferous tubule culture model, we evaluated the effects of waters of different origins, on several parameters of the seminiferous epithelium. Concentrated culture medium was diluted with the waters to be tested (final concentrations of the tested waters were between 8 and 80%). The integrity of the blood-testis barrier was assessed by the trans-epithelial electric resistance (TEER). The levels of mRNAs specific of Sertoli cells, of cellular junctions, of each population of germ cells, of androgen receptor, of estrogen receptor α and of aromatase were also studied. We report, here, the results obtained with ten waters, some of them possessing a negative effect on spermatogenesis. The results showed that according to the tested waters, their effects on the parameters studied might be quite different indicating many different mechanisms of toxicity, including some endocrine disrupting effects. It has been reported that men with impaired semen parameters have an increased mortality rate suggesting semen quality may provide a fundamental biomarker of overall male health. Hence, we have developed a relevant in vitro bioassay allowing to evaluate the potential toxicity of different types of waters on male fertility and to assess some aspects of their mechanism of action. In addition to the TEER measure, the number and/or the identity of the studied mRNAs can be largely increased and/or modified, thus enhancing the possibility of using this model as a "warning system".

South Africa is the largest agrochemical user in sub-Saharan Africa, with over 3000 registered pesticide products. Although they reduce crop losses, these chemicals reach non-target aquatic environments via leaching, spray drift or run-off. In this review, attention is paid to legacy and current-use pesticides reported in literature for the freshwater environment of South Africa and to the extent these are linked to endocrine disruption. Although banned, residues of many legacy organochlorine pesticides (endosulfan and dichlorodiphenyltrichloroethane (DDT)) are still detected in South African watercourses and wildlife. Several current-use pesticides (triazine herbicides, glyphosate-based herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D) and chlorpyrifos) have also been reported. Agrochemicals can interfere with normal hormone function of non-target organism leading to various endocrine disrupting (ED) effects: intersex, reduced spermatogenesis, asymmetric urogenital papillae, testicular lesions and infertile eggs. Although studies investigating the occurrence of agrochemicals and/or ED effects in freshwater aquatic environments in South Africa have increased, few studies determined both the levels of agricultural pesticides present and associated ED effects. The majority of studies conducted are either laboratory-based employing in vitro or in vivo bioassays to determine ED effects of agrochemicals or studies that investigate environmental concentrations of pesticides. However, a combined approach of bioassays and chemical screening will provide a more comprehensive overview of agrochemical pollution of water systems in South Africa and the risks associated with long-term chronic exposure.

Environmental estrogens are capable of interfering with the spermatogenesis and fertility of fish. However in natural waters, these chemicals are more likely to occur as a combination rather than a single stressor. Whether and how the mixture of xenoestrogens with environmental relevant concentrations may affect fish spermatogenesis remains largely unknown. In this study, male zebrafish adults were administered to 17alpha-ethinylestradiol (EE2) and a mixture of xenoestrogens (Mix (E2, EE2, DES, 4-t-OP, 4-NP and BPA)), with the estrogenic potency equivalent to EE2. After a 60-day exposures, elevated mRNA levels of vitellogenin 1 (vtg1) and estrogen receptor 1 (esr1) in the liver of fish in both treated groups were observed. Moreover, the plasma level of E2 declined significantly in the Mix group and the ratio of 11-KT/E2 was significantly elevated in both treated groups. Consistently, the mRNA level of P450 side-chain cleavage (scc) in the EE2 group and ovarian type aromatase (cyp19a1a) in the Mix group was significantly suppressed. In addition, decreased gonadosomatic index and sperm count in the fish of Mix group were present. Furthermore, increased number of the proliferating germ cells (such as spermatogonia and spermatocytes) was observed in the fish of both groups, suggesting a stimulated germ cell proliferation and meiosis. Accordingly, both exposures significantly up-regulated the mRNA levels of genes in mitosis (cyclinb1) and meiosis (cyp26a1 in EE2 group, aldh1a2, cyp26a1, sycp3 and spo11 in Mix). In addition, decreased number of spermatozoa and increased number of TUNEL-positive signals were present in the testis of fish in the Mix group, indicating an enhanced apoptosis. Further analyses demonstrated the significant elevated expressions of tnfrsf1a and the ratio of tnfrsf1a/tnfrsf1b in the Mix group, suggesting an elevated apoptosis in the testis of fish in the Mix group via extrinsic pathway. The present study greatly extends our understanding of the underlying mechanisms of the reproductive toxicity of xenoestrogens on fish.

The effect of fluoride as an ongoing topic has attracted much attentions due to the decline in overall human fertility worldwide. However, whether fluorine causes a temporary stimulus or permanent damage to the male reproductive system, as well as the mechanism of fluoride influencing spermatogenesis remained unclear. 48 adult male rats were randomly divided into four groups (twelve each). Control group received the distilled water, while the other three groups were treated with 25, 50, 100 mg/L NaF via drinking water for 8 weeks. Six rats from each group were selected randomly to detect the levels of various indices related to spermatogenesis. The remaining rats were given only distilled water and left for recovery of a period of 2 weeks. Results showed that the levels of serum CK, ALP, CHE, BUN, UA, and Cr, testis morphology and the ultrastructure of sperm acrosome and chromatoid body (CB) were significantly changed by fluoride. Interestingly, the elongated spermatid counts, spermatids elongation ratio, and mRNA expressions of Prm1/2 and MIWI, TDRD1, TDRD 6, TDRD7, PABP, and Hsp72 related to CB decreased markedly in fluoride treatment groups compared to the control. Furthermore, the expression levels of DDX25 and associated regulatory proteins like CRM1, HMG2, H4, TP2, and PGK2 were down-regulated by fluoride. After 2-weeks withdrawal period, out of the 19 altered spermatogenesis indicators, 15 indicators in 100 mg/L group and 3 indicators in 50 mg/L group still exhibited a significant change, while none showed change in 25 mg/L group. These results proved that the reversibility of fluoride toxicity is dose-dependent on the male reproductive system. Meanwhile, fluoride caused unrestored arrest during the haploid period of spermatogenesis, where reduced DDX25 and associated regulatory proteins play a crucial role in this process, which could provide the underlying insights to the toxic mechanism of fluoride induced male reproductive toxicity.

Bisphenol A (BPA) is an endocrine disruptor whose ubiquitous presence in the environment has been related with impairment of male reproduction. BPA can cause both transcriptomic and epigenetic changes during spermatogenesis. To evaluate the potential effects of male exposure to BPA, adult zebrafish males were exposed during spermatogenesis to doses of 100 and 2000 μg/L, which were reported in contaminated water bodies and higher than those allowed for human consumption. Fertilization capacity and survival at hatching were analysed after mating with untreated females. Spermatogenic progress was analysed through a morphometrical study of testes and apoptosis was evaluated by TUNEL assay. Testicular gene expression was evaluated by RT-qPCR and epigenetics by using ELISA and immunocytochemistry. In vitro studies were performed to investigate the role of Gper. Chromatin fragmentation and the presence of transcripts were also evaluated in ejaculated sperm. Results on testes from males treated with the highest dose showed a significant decrease in spermatocytes, an increase in apoptosis, a downregulation of ccnb1 and sycp3, all of which point to an alteration of spermatogenesis and to meiotic arrest and an upregulation of gper1 and esrrga receptors. Additionally, BPA at 2000 μg/L caused missregulation of epigenetic remodelling enzymes transcripts in testes and promoted DNA hypermethylation and H3K27me3 demethylation. BPA also triggered an increase in histone acetyltransferase activity, which led to hyperacetylation of histones (H3K9ac, H3K14ac, H4K12ac). In vitro reversion of histone acetylation changes using a specific GPER antagonist, G-36, suggested this receptor as mediator of histone hyperacetylation. Males treated with the lower dose only showed an increase in some histone acetylation marks (H3K14ac, H4K12ac) but their progeny displayed very limited survival at hatching, revealing the deleterious effects of unbalanced paternal epigenetic information. Furthermore, the highest dose of BPA led to chromatin fragmentation, promoting direct reproductive effects, which are incompatible with embryo development.

T-2 toxin is an unavoidable contaminant in human food, animal feeds, and agricultural products. T-2 toxin has been found to impair male reproductive function. But, few data is available that reveals the reproductive toxicity mechanism. In the study, male Kunming mice were orally administrated with T-2 toxin at the doses of 0, 0.5, 1 or 2 mg/kg body weight for 28 days. The body and reproductive organs weight, the concentration, malformation rate and ultrastructure of sperm in cauda epididymis were detected. Oxidative stress biomarkers and apoptosis were also measured in testes. Histological change of testes was performed by H&E and TUNEL staining. T-2 toxin down-regulated body and reproductive organs (testis, epididymis and seminal vesicle) weight, sperm concentration, increased sperm malformation rate and damaged the ultrastructure of sperm and structure of testes. T-2 toxin treatment increased the reactive oxygen species (ROS) and malondialdehyde content, while, decreased the total anti-oxidation capacity (T-AOC) and the superoxide dismutase activity in testes. T-2 toxin exposure increased the TUNEL-positive germ cells, the activities and mRNA expressions of caspase-3, caspase-8 and caspase-9, the mRNA expression of Bax, and inhibited the Bcl-2 mRNA expression. Furthermore, the expressions of caspase-3, caspase-8 caspase-9 and Bax were positively correlated with ROS level, but negatively correlated with T-AOC in testis. In summary, T-2 toxin caused spermatogenesis disorder associated with the germ cell apoptosis medicated by oxidative stress, impairing the male reproductive function.

Bisphenol A (BPA) is an endocrine disruptor used in manufacturing of plastic devices, resulting in an ubiquitous presence in the environment linked to human infertility, obesity or cardiovascular diseases. Both transcriptome and epigenome modifications lie behind these disorders that might be inherited transgenerationally when affecting germline. To assess potential effects of paternal exposure on offspring development, adult zebrafish males were exposed to BPA during spermatogenesis and mated with non-treated females. Results showed an increase in the rate of heart failures of progeny up to the F2, as well as downregulation of 5 genes involved in cardiac development in F1 embryos. Moreover, BPA causes a decrease in F0 and F1 sperm remnant mRNAs related to early development. Results reveal a paternal inheritance of changes in the insulin signaling pathway due to downregulation of insulin receptor β mRNAs, suggesting a link between BPA male exposure and disruption of cardiogenesis in forthcoming generations.

The interaction of xenobiotics is common in aquatic ecosystems; therefore, we wanted to evaluate if trenbolone (TB) modulates the effects of 17α-ethinylestradiol (EE2). Male eelpout (Zoarces viviparus) were exposed to 5 ng L−1 EE2 continuously for 19 d (EE2-C) or discontinuously (11 d, EE2-D) alone or in combination with low (50 ng L−1, TBL) or high (500 ng L−1, TBH) concentrations of TB (19 d). Exposure to EE2 caused reduced gonadosomatic index, increased plasma vitellogenin concentrations, up-regulated vtg and era mRNA expression and severe alterations in gonadal histology. TBL and TBH did not affect plasma vitellogenin, era or vtg mRNA expression. TBL and TBH did not counteract the EE2-induced increase in plasma vitellogenin and reduction in 11-ketotestosterone whereas TBH counteracted the EE2 induced increase in vtg and era mRNA expression. Exposure to TBH and EE2-C + TBH lead to severe gonadal histology alterations. TBL and EE2-D + TBH exposed fish showed less histopathological alterations.

Testicular junctions are pivotal to male fertility and regulated by constituent proteins. Increasing evidence suggests that environmental chemicals, including bisphenol A (BPA), may impact these proteins, but whether the impacts persist for generations is not yet known. Here, we investigate the effect of BPA (a ubiquitous endocrine-disrupting chemical) on testis and sperm functions and whether the effects are transferred to subsequent generations. Male mice (F0) were exposed to corn oil (Control) or 5 or 50 mg BPA/kg body weight/day from 6 to 12 weeks of age. The F0 were mated with wild-type females to produce the first filial (F1) generation. F2 and F3 were produced using similar procedures. Our results showed that BPA doses decreased the levels of some junctional proteins partly via binding with estrogen receptors (ERα and Erβ), upregulation of p-ERK1/2, P85, p-JNK and activation of p38 mitogen-activated protein kinase signaling. Consequently, testicular histological abnormalities, disrupted spermatogenesis, decreased sperm count, and inability to fertilize eggs were observed in mice exposed to BPA. These effects were transferred to successive generations (F2), partly through DNA methylation, but mostly alleviated in F3 males. Our findings suggest that paternal exposure to chemicals promoting alteration of testicular junctional proteins and its transgenerational inheritance is a key component of the origin of male reproductive health problems.

Environmental cadmium (Cd) or high-fat diet (HFD) exposure alone are risk factors of male infertility. However, the effect and mechanism of co-exposure to HFD and Cd on sperm quality remain unclear. This study was aimed to explore the combined effects of HFD and Cd on spermatogenesis as well as its m6A-dependent mechanism in vivo and in vitro. As a result, co-exposure of HFD and Cd resulted in a significant decrease in the number of mature testicular seminiferous tubules and epididymis sperm quantity in mice, compared with Cd or HFD exposure alone. Correspondingly, the mRNAs expression of Smc3(spermatocytes marker), Acrv1(round spermatids marker) and Lzumo3(elongated spermatids marker) were downregulated in HFD and Cd group. Furthermore, combined exposure downregulated the expression of meiosis-related proteins (STRA8 and SYCP3), increased the m6A level of Stra8, and upregulated the expression of m6A-related proteins (METTL3 and YTHDF2) in mouse spermatocytes. Mechanistically, the above-mentioned impacts caused by co-exposure were markedly restored by Mettl3 siR and Ythdf2 siR. In addition, RNA stability assay showed that Ythdf2 siR obviously reversed co-exposure-increased Stra8 mRNA degradation rate in actinomycin-D-treated mouse spermatocytes. Meanwhile, excess ROS was observed in combined-exposure group, and a free radical scavenger N-tert-Butyl-α-phenylnitrone (PBN) attenuated co-exposure-upregulated expression of METTL3 and YTHDF2 in mouse spermatocytes. These results suggested that combination of HFD and Cd impaired spermatogenesis by degrading Stra8 in an m6A-YTHDF2-dependent manner via ROS activation.