br Materials and methods br Results br Discussion BTB

Materials and methods
Results
Discussion BTB, as a necessary structure for the spermatogenic epithelium, provides an important internal environmental for spermatogenesis and its disruption leads to male infertility (Qiu et al., 2016). GJs and TJs which are mainly components of BTB play essential roles in many biological processes on cell development, proliferation and differentiation (Steuer et al., 2016). In decades, the mechanism of MC-LR-induced BTB toxicity mainly focused on TJs (Chen et al., 2017; Zhou et al., 2018), however, the relationship between MC-LR and GJs has not been uncovered yet. Therefore, it was intriguing to investigate whether and how GJs could contribute to the reproductive function of BTB upon exposure to MC-LR. In our present work, it first found the evidence of GJ-mediated MC-LR-induced BTB toxicity in vivo and in vitro. Several observations supported this conclusion: (1) MC-LR disrupted GJIC between Sertoli UNC669 sale by alerting the expression and distribution of Cx43, (2) the Akt and Erk were involved in MC-LR-impaired the function of GJs, and (3) the pharmacological inhibition of Akt could attenuate MC-LR-induced GJs injury. First, to establish in vivo model of MC-LR-induced male reproductive injury, mice were orally administrated MC-LR at the doses ranging from 1 to 100 μg/L for 90 days. Dose selection was based on the recommended level of MCs (1 μg/L) by The World Health Organization (WHO) (Chen et al., 2011). In considering the difference of body surface area between human and mouse, the highest dosage (100 μg/L) would be 100-fold higher than that WHO recommended level. One literature reference suggests mouse mean daily water intakes 7 mL/30 g body weight (Bachmanov et al., 2002). In our experiments the mouse body weight ranged from 28 to 32 g. The average concentrations of MC-LR was ingested by mice at 0.23, 2.3 and 23 μg/kg body weight per day. There evidence demonstrates that MC-LR is detected in rat testes after injected intraperitoneally at dose of 300 μg/kg per day for 6 days (Wang et al., 2013). However, MC-LR was not detected in testes of exposed animals due to detection limits and lower exposure doses in present study. Ito et al. found that MC-LR entering via an oral route caused histological damage to the small intestine and then entered the blood via capillaries (Ito et al., 2001). Thus, it can therefore be assumed that MCs would enter the testis with blood and accumulate in it due to routinely intake of contaminated food and water. Our data showed that MC-LR significantly induced the loss in BTB integrity, suggesting that the mouse-based model of MC-LR-induced male reproductive injury was successfully established. In addition, the formula weight of MC-LR is 995.2 g/mol according to manufacture instructions. Thus, the in vivo exposure concentrations were approximately equivalent to 0.001, 0.01 and 0.1 μM. In considering the accumulation of MC-LR in vivo study and the sensitive features of cell line (Li et al., 2019), the highest dosage (10 μM) in in vitro would be 100-fold higher than that in in vivo study. Concentrations of MC-LR applied in the study, results of which are presented here, were not only selected for other MC toxicity studies but also related to the environmental accumulation of MC in cyanobacteria blooms where the concentration of MC may range from as low as less than 20 nM to more than 100 μM as reported (Roegner et al., 2014). As a predominate component of GJs in the testis, Cx43 is pivotal in spermatogenesis and testis development through controlling the integrity of BTB, which is also a sensitive target of environmental toxicants (Pointis et al., 2011). To verify the involvement of Cx43 in alteration of BTB permeability induced by MC-LR, the levels of Cx43 were investigated in testis and TM4 cells. MC-LR dramatically enlarged the expression of Cx43. Results of heavy metals toxicological studies, which were consistent with ours, have shown that elevating of Cx43 might be a possible protection in response to cell damage (Ramos-Trevino et al., 2017). There evidence suggests that the function of GJIC is proportional to the content of Cx43 (Li et al., 2016a). GJIC plays an essential role in spermatogenesis by establishing directly communication between Sertoli cell (Nielsen et al., 2012). Steuer et al. have proven that benzo pyrene (BAP) compromise spermatogenesis through impairing GJIC function of Sertoli contribute to the decline of Cx43 (Sobarzo et al., 2009; Steuer et al., 2016). The results demonstrated that GJIC between Sertoli cells was blocked by MC-LR using SLDT method. These results implied that Cx43 can regulate cell physiology independent of GJIC.