Impaired spermatogenesis and associated endocrine effects of azole fungicides in peripubertal Xenopus tropicalis.

Early life exposure to endocrine disrupting chemicals (EDCs) has been suggested to adversely affect reproductive health in humans and wildlife. Here, we characterize endocrine and adverse effects on the reproductive system after juvenile exposure to propiconazole (PROP) or imazalil (IMZ), two common azole fungicides with complex endocrine modes of action. Using the frog Xenopus tropicalis, two short-term (2-weeks) studies were conducted. I: Juveniles (2 weeks post metamorphosis (PM)) were exposed to 0, 17 or 178 µg PROP/L. II: Juveniles (6 weeks PM) were exposed to 0, 1, 12 or 154 µg IMZ/L. Histological analysis of the gonads revealed an increase in the number of dark spermatogonial stem cells (SSCs)/testis area, and in the ratio secondary spermatogonia: dark SSCs were increased in all IMZ groups compared to control. Key genes in gametogenesis, retinoic acid and sex steroid pathways were also analysed in the gonads. Testicular levels of 3ß-hsd, ddx4 were increased and cyp19 and id4 levels were decreased in the IMZ groups. In PROP exposed males, increased testicular aldh1a2 levels were detected, but no histological effects observed. Although no effects on ovarian histology were detected, ovarian levels of esr1, rsbn1 were increased in PROP groups, and esr1 levels were decreased in IMZ groups. In conclusion, juvenile azole exposure disrupted testicular expression of key genes in retinoic acid (PROP) and sex steroid pathways and in gametogenesis (IMZ). Our results further show that exposure to environmental concentrations of IMZ disrupted spermatogenesis in the juvenile testis, which is a cause for concern as it may lead to impaired fertility. Testicular levels of id4, ddx4 and the id4:ddx4 ratio were associated with the number of dark SSCs and secondary spermatogonia suggesting that they may serve as a molecular markers for disrupted spermatogenesis.

Pubertal sexual development and endpoints for disrupted spermatogenesis in the model Xenopus tropicalis.

Peripubertal models to determine effects of anti-androgenic endocrine disrupting chemicals are needed. Using the toxicological model species Xenopus tropicalis, the aims of the study were to 1) provide data on sexual maturation and 2) characterise effects of short-term exposure to an anti-androgenic model substance. Juvenile (2.5 weeks post metamorphosis old) X. tropicalis were exposed to 0, 250, 500 or 1000 µg flutamide/L (nominal) for 2.5 weeks. Upon exposure termination, histology of gonads and Müllerian ducts was characterised in detail. New sperm stages were identified: pale and dark spermatogonial stem cells (SSCs). The testes of control males contained spermatozoa, indicating pubertal onset. The ovaries were immature, and composed of non-follicular and pre-vitellogenic follicular oocytes. The Müllerian ducts were more mature in females than males indicating development/regression in the females and males, respectively. In the 500 µg/L group, the number of dark SSCs per testis area was decreased and the number of secondary spermatogonia was increased. No treatment effects on ovaries or Müllerian ducts were detected. To conclude, our present data provide new knowledge on spermatogenesis, and pubertal onset in X. tropicalis. New endpoints for evaluating spermatogenesis are suggested to be added to existing assays used in endocrine and reproductive toxicology.

Pilot survey reveals ophidiomycosis in dice snakes Natrix tessellata from Lake Garda, Italy.

Ophidiomycosis is an emerging infectious disease caused by the fungus Ophidiomyces ophidiicola (Oo). To date, Oo presence or associated disease condition has been recorded in wild and/or captive snakes from North America, Europe, Asia and Australia, but the data is still scarce outside the Nearctic. Although Italy is a country with a high snake biodiversity in the European panorama, and animals with clinical signs compatible with Oo infection have been documented, to date no investigations have reported the disease in the wild. Therefore, a pilot survey for the Italian territory was performed in conjunction with setting up a complete diagnostic workflow including SYBR Green-based real-time PCR assay for the detection of Oo genomic and mitochondrial DNA combined with histopathology of scale clips. Oo presence was investigated in 17 wild snake specimens from four different species. Four snakes were sampled in a targeted location where the mycosis was suspected via citizen science communications (i.e. North of the Lake Garda), whereas other ophidians were collected following opportunistic sampling. Oo genomic and mitochondrial DNA were detected and sequenced from all four Lake Garda Natrix tessellata, including three juveniles with macroscopic signs such as discolouration and skin crusts. From histopathological examination of scale clips, the three young positive individuals exhibited ulceration, inflammation and intralesional hyphae consistent with Oo infection, and two of them also showed the presence of arthroconidial tufts and solitary cylindrical arthrospores, allowing "Ophidiomycosis and Oo shedder" categorisation. For the remaining snake samples, the real-time PCR tested negative for Oo. This pilot survey permitted to localise for the first time Oo infection in free-ranging ophidians from Italy. Ophidiomycosis from Lake Garda highlights the need to increase sampling efforts in this area as well as in other northern Italian lakes to assess the occurrence of the pathogen, possible risk factors of the infection, its impact on host population fitness and the disease ecology of Oo in European snakes.

Effects of bisphenol A and estradiol in adult rat testis after prepubertal and pubertal exposure.

Over the past few decades, male fertility has been decreasing worldwide. Many studies attribute this outcome to endocrine disruptors exposure such as bisphenol A (BPA), which is a chemical compound used in plastics synthesis and exhibiting estrogenic activity. In order to assess how the window of exposure modulates the effects of BPA on the testis, prepubertal (15 dpp to 30 dpp) and pubertal (60 dpp to 75 dpp) male Sprague-Dawley rats were exposed to BPA (50 µg/kg bw/day), 17-ß-estradiol (E2) (20 µg/kg bw/day) as a positive control, or to a combination of these compounds. For both periods of exposure, the rats were sacrificed and their testes were collected at 75 dpp. The histological analysis and the quantification of the gene expression of testis cell markers by RT-qPCR confirmed the complete spermatogenesis in all groups for both periods of exposure. However, our results suggest a deleterious effect of BPA on the blood-testis barrier in adults after pubertal exposure as BPA and BPA+E2 treatments induced a decrease in caveolin-1 and connexin-43 gene expression; which are proteins of the junctional complexes. As none of these effects were found after a prepubertal exposure, these results suggested the reversibility of BPA's effects. Caution must be taken when transposing this finding to humans and further studies are needed in this regard. However, from a regulatory perspective, this study emphasizes the importance of taking into account different periods of exposure, as they present different sensitivities to BPA exposure.

Interactions between oestrogen and 1α,25(OH)2-vitamin D3 signalling and their roles in spermatogenesis and spermatozoa functions.

Oestrogens and 1α,25(OH)2-vitamin D3 (1,25-D3) are steroids that can provide effects by binding to their receptors localised in the cytoplasm and in the nucleus or the plasma membrane respectively inducing genomic and non-genomic effects. As confirmed notably by invalidation of the genes, coding for their receptors as tested with mice with in vivo and in vitro treatments, oestrogens and 1,25-D3 are regulators of spermatogenesis. Moreover, some functions of ejaculated spermatozoa as viability, DNA integrity, motility, capacitation, acrosome reaction and fertilizing ability are targets for these hormones. The studies conducted on their mechanisms of action, even though not completely elicited, have allowed the demonstration of putative interactions between their signalling pathways that are worth examining more closely. The present review focuses on the elements regulated by oestrogens and 1,25-D3 in the testis and spermatozoa as well as the interactions between the signalling pathways of both hormones.

L'œstradiol et la 1α,25(OH)2-vitamin D3 (1,25-D3 ou calcitriol) sont respectivement la forme la plus active des œstrogènes et la forme hormonalement active de la vitamine D. Ces stéroïdes peuvent exercer leurs effets biologiques après fixation à des récepteurs localisés dans le cytoplasme et le noyau (récepteurs dit nucléaires) ou par fixation à des récepteurs localisés à la membrane plasmique (récepteurs membranaires) à l'origine d'effets appelés génomiques et non génomiques respectivement. Bien que les œstrogènes aient longtemps été considérés comme uniquement des hormones féminines, de nombreux travaux ont permis de montrer leur importance dans le bon déroulement de la spermatogenèse et la qualité des gamètes. De même, la 1,25-D3 est capable de réguler les fonctions testiculaires suggérant son importance dans la fertilité. Les études réalisées sur leurs mécanismes d'action, bien qu'ils ne soient pas complètement élucidés, ont permis de mettre en évidence des interactions entre les voies de signalisation de ces deux hormones. Cette revue est centrée sur les évènements régulés par les œstrogènes et la 1,25-D3 dans les testicules et les spermatozoïdes et les interactions entre leurs voies de signalisation.

Differential effects of bisphenol A and estradiol on rat spermatogenesis' establishment.

Several studies have highlighted the negative effects of bisphenol A (BPA), a chemical compound with estrogenic activity, on reproductive health. To elucidate the impact of BPA on spermatogenesis' establishment and mechanisms of action of BPA and 17ß-estradiol (E2), as both can be found in the environment, we exposed rats to BPA (50µg/kg bw/day of BPA), E2 (20µg/kg bw/day of E2) and BPA+E2 from 15 to 30days post-partum. Histological and gene expression studies revealed that BPA and BPA+E2 exposures promoted spermatogenesis establishment whereas E2 alone delayed it. Then, a decrease in gene expression of blood-testis-barrier (BTB) proteins was observed in all treated groups. Therefore, our study has demonstrated a differential effect of BPA and E2 exposures on spermatogenesis establishment in prepubertal rats and a deleterious effect of these chemicals on BTB establishment. Thus, the effects of BPA seem to be mediated by receptors other than estrogen receptors.