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".

We investigated the in vitro effects of pyriproxyfen on ionic balance in the testis of the zebrafish by measuring ⁴⁵Ca²⁺ influx. In vivo pyriproxyfen treatment was carried out to study oxidative stress, and conduct morphological analysis of the testis and liver. Whole testes were incubated in vitro with/without pyriproxyfen (10⁻¹², 10⁻⁹ or 10⁻⁶ M; 30 min) and ⁴⁵Ca²⁺ influx determined. To study pyriproxyfen’s mechanism of action, inhibitors/activators of ionic channels or pumps/exchangers, protein kinase inhibitors or a calcium chelator were added 15 min before the addition of ⁴⁵Ca²⁺ and pyriproxyfen. We evaluated the in vivo effects of 7 day exposure to waterborne pyriproxyfen (10⁻⁹ M) on reactive oxygen species (ROS) formation, lipid peroxidation, and reduced glutathione content (GSH), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) and γ-glutamyltransferase (GGT) activity. Morphological analyses of the testis and liver were carried out after in vivo exposure of D. rerio to pyriproxyfen. Pyriproxyfen increased ⁴⁵Ca²⁺ influx by opening the voltage-dependent T-type channels (T-type VDCC), inhibiting sarco/endoplasmic reticulum ⁴⁵Ca²⁺-ATPase (SERCA) and the NCX exchanger (forward mode) and by mobilizing calcium from stores. The involvement of potassium channels and protein kinase C (PKC) was also demonstrated in pyriproxyfen-induced intracellular calcium elevation. In vivo pyriproxyfen treatment of D. rerio increased lipid peroxidation, decreased GSH content and increased GST activity in testes, in addition to increasing the number and size of spermatogonia cysts and inducing hepatocyte basophilia and dilation of blood vessels in the liver. The toxicity of pyriproxyfen is mediated by calcium overload, increased lipid peroxidation, and a diminished antioxidant capacity in the testis, due to GSH depletion, and altered spermatogenesis. The development of high basophilia in the liver suggests that pyriproxyfen may have estrogenic activity, possibly acting as an endocrine-disruptor. These findings indicate that these alterations may contribute to pyriproxyfen toxicity and spermatogenesis disruption.

One of the causes of the global decline of amphibians is agricultural activity, responsible for causing habitat fragmentation and bringing a range of agrochemicals and fertilizers in the environment, compounds with a potential disrupting effect on non-target organisms, such as frogs. Exposure to these compounds has numerous harmful effects on the testes of these animals, which can compromise reproduction and, consequently, the maintenance of their communities. In this context, we compared the morphology and morphometry of the testes of three species of neotropical anurans (Physalaemus cuvieri, Dendropsophus minutus, and Boana albopunctata) from an agricultural area and a conservation unit. Histologically, the testicular morphology of the species was similar for both environments; however, morphometrically, there was a difference in the measured testicular parameters (locular area and area of spermatogenic cysts). Physalaemus cuvieri presented higher averages of locular and spermatogonial area in the agricultural environment, whereas the area occupied by the spermatozoa was smaller. Additionally, the testicular pigmentation, which is only present in this species, was greater in animal from the agricultural area. In D. minutus, the locular, spermatogonial, and sperm areas showed lower values in the agricultural area, whereas in B. albopunctata, the opposite pattern was found, with the area of the locule, spermatocytes, and spermatozoids being higher. Agricultural activities influence the testicular metric parameters in different species, and our results suggest that D. minutus is most sensitive to anthropic pressures. The least sensitive species is B. albopunctata. We highlight the importance of evaluating different species, since each species responds differently to agricultural activities.

Researches have shown that silica nanoparticles (SiNPs) could reduce both the quantity and quality of sperm. However, the mechanism of toxicity induced by SiNPs in the male reproductive system is still unclear. In this study, male mice were randomly divided into a control group, and SiNPs treated group (20 mg/kg dose; n = 30 per group). Half of the mice per group were sacrificed on 35 days and the remaining on 50 days of the SiNPs exposure. SiNPs were found to decrease sperm count and mobility, increase the sperm abnormality rate, and damage the testes' structure. Furthermore, SiNPs decreased the protein levels of Protamine 1(PRM1) and elevated the histones' levels and suppressed the chromatin condensation of sperm. There was a significant reduction of the ubiquitinated H2A (ubH2A)/H2B (ubH2B) and RING finger protein 8 (RNF8) levels in the spermatid nucleus, while the RNF8 level in the spermatid cytoplasm increased evidently. The protein expression levels of PIWI-like protein 1(MIWI) in the late spermatids significantly increased on day 35 of SiNPs exposure. After 15 days of the withdrawal, the sperm parameters and protamine levels, and histones in the epididymal sperm were unrecovered; however, the changes in testis induced by SiNPs were recovered. Our results suggested that SiNPs could decrease the RNF8 level in the nucleus of spermatid either by upregulating of the expression of MIWI or by inhibiting its degradation. This resulted in the detention of RNF8 in the cytoplasm that maybe inhibited the RNF8-mediated ubiquitination of ubH2A and ubH2B. These events culminated in creating obstacles during the H2A and H2B removal and chromatin condensation, thereby suppressing the differentiation of round spermatids and chromatin remodeling, which compromised the sperm quality and quantity.

It has been found that polystyrene microplastics (PS-MPs) exposure leads to decreased sperm quality and quantity, and we aim to explore the underlying mechanisms. Therefore, we gave 20 mg/kg body weight (bw) and 40 mg/kg bw 4 μm and 10 μm PS-MPs to male Balb/c mice by gavage. RNA sequencing of testes was performed. After PS-MPs exposure, blood-testis barrier (BTB) integrity was impaired. Since cytoskeleton was closely related to BTB integrity maintenance, and cytoskeleton disorganization could be induced by PS-MPs exposure in the testis, which resulted in the truncation of actin filaments and disruption of BTB integrity. Such processes were attributed to the differential expression of Arp3 and Eps8 (two of the most important actin-binding proteins). According to the transcriptome sequencing results, we examined the oxidative stress level in the testes and Sertoli cells. We found that PS-MPs exposure induced increased reactive oxygen species (ROS) level, which destroyed the balance between mTORC1 and mTORC2 (the mTORC1 activity was increased, while the mTORC2 activity was decreased). In conclusion, PS-MPs induced the imbalance of mTORC1 and mTORC2 via the ROS burst, and altered the expression profile of actin-binding proteins, resulting in F-actin disorganization and reduced expression of junctional proteins in the BTB. Eventually PS-MPs led to BTB integrity disruption and spermatogenesis dysfunction.

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.

Phenanthrene (Phe) is a tricyclic polycyclic aromatic hydrocarbon with high bioavailability under natural exposure. However, there are few studies on the reproductive toxicity of Phe in mammals. In this study, male Kunming mice were gavaged once every two days with Phe (5, 50, and 500 ng/kg) for 28 weeks. The accumulation levels of Phe in the testis were dose-dependently increased. Histopathological staining showed that Phe exposure reduced the number of spermatogonia, sperm and Sertoli cells. The percentage of testicular apoptotic cells was significantly increased, which was further verified by the upregulated BAX protein. The expression of the GDNF/PI3K/AKT signaling pathway was downregulated, which might suppress the self-renewal and differentiation of spermatogonial stem cells. Meanwhile, Phe exposure inhibited the expression of Sertoli cell markers (Fshr, WT1, Sox9) and the Leydig cell marker Cyp11a1, indicating damage to the function of Sertoli cells and Leydig cells. Serum estrogen and testicular estrogen receptor alpha were significantly upregulated, while androgen receptor expression was downregulated. These alterations might be responsible for impaired spermatogenesis. This study provides new insights for evaluating the reproductive toxicity and potential mechanisms of Phe in mammals.

Testicular damage has been described as a common side effect of cisplatin (CDDP), which limits its clinical uses. Since oxidative injury and inflammatory response are the most pathological impact, estimation of natural antioxidant and anti-inflammatory agents like trans-ferulic acid (TFA) could protect against CDDP-induced testicular damage. In the current investigation, rats were assigned into four groups: normal, TFA (50 mg/kg/day, P.O), CDDP (10 mg/kg) as single intraperitoneal (I.P) injection at the end of the 5th day, and TFA+CDDP where TFA was administered 5 days before CDDP injection and 5 days after. Interestingly, TFA significantly restored testosterone levels and abrogated oxidative stress injury. Additionally, TFA effectively suppressed inflammatory cytokines. It also counteracted the inflammation via downregulation of TLR4 and IRF3, P38-MAPK, NF-κB-p65, JAK1, STAT3, ERK1, and ERK2. Besides, TFA can modulate AKT and p-AKT protein expressions. In parallel, TFA mitigated the histopathological aberration of the testis and prevented spermatogenesis disruption. On the other hand, TFA augmented the in vitro CDDP cytotoxicity on Caco-2 and MCF-7 cells. Interestingly, TFA enhanced the cytotoxic effect of CDDP via apoptosis induction in both the early and late stages of apoptosis. Collectively, TFA exhibited a potential protective effect against CDDP-induced testicular injury by inhibiting oxidative stress as well as TLR4/IRF3/INF-γ, P38-MAPK/NF-κB-p65/TNF-α, and JAK1/STAT-3/ERK1/2 inflammatory signaling pathways with enhancing its in vitro cytotoxic activity.

Amphibians find a suitable habitat in rice agroecosystems, but their viability is affected by the sublethal effects of agrochemicals. Animal condition, external morphology, and liver and male gonad tissue histology were examined on Lysapsus limellum and Rhinella bergi, common species that occurs in rice fields (RF) and reference sites (RS) from Santa Fe and Chaco provinces, Argentina, respectively. Animal condition factor showed no significant differences between environments in either species. Males of both species from RF showed asymmetrical testis size. A higher proportion of cysts with early spermatogenesis cell stages were observed in RF specimens than in RS individuals. The proportion of tubules with free spermatozoa differed between environments for L. limellum. Bidder organ presented vacuoles, atretic follicles, and pigmentary cells in R. bergi from Chaco RF. Liver tissues of both species from RF exhibited sinusoid enlargement, hypervascularization, hepatocyte vacuolization, and dilated blood vessels. R. bergi had a higher number of hepatic melanomacrophages in RF than in RS. The histological biomarker was more sensitive than the morphological one to evaluate chronic pesticide exposure using anuran non-target organs (liver, gonads) and Bidder organ. The potential effect of those alterations on the reproductive performance of anurans that commonly occur in agroecosystems needs further research.

Zearalenone (ZEA) and Deoxynivalenol (DON) are two mycotoxins highly detected in agricultural products and feed. Both mycotoxins produce reproductive toxicity and pose a serious threat to human and animal health, among which pigs are the most sensitive animals. Sertoli cells (SCs) play an important role in spermatogenesis; however, the combined toxicity of ZEA and DON and the screening of effective protective agents remains to be determined. By studying the effects of N-acetylcysteine (NAC) on the cells exposed to 20 μM of ZEA and 0.6 μM of DON, we explored the protective mechanism of NAC (4 mM) on the cytotoxic injury of piglets SCs induced by both mycotoxins. The results showed that the combination of ZEA and DON destroy organelles and SCs structures, NAC significantly alleviates the damage caused by ZEA and DON. NAC also significantly increased the expression and distribution of zonula occludens 1 (ZO-1), decreased the relative mRNA and protein expression levels of Bax, Bid, caspase-3, and caspase-9, and increased Bcl-2 expression level and inhibited the decrease of mitochondrial membrane potential. Further, NAC also eases the cell cycle arrest and oxidative stress caused by ZEA and DON. In summary, our results show that NAC could alleviate SCs injury via reducing the oxidative damage and apoptosis caused by ZEA and DON.