Until now, complete ex vivo spermatogenesis has been reported only in the mouse. In this species, the duration of spermatogenesis is 35 days, whereas it is 54 days in the rat and 74 days in humans. We performed long-term (until 60 days) cultures of fresh or frozen rat or human seminiferous tubule segments in a bioreactor made of a hollow cylinder of chitosan hydrogel. Testicular tissues were obtained from 8- or 20-day-old male rats or from adult human subjects who had undergone hormone treatments leading to a nearly complete regression of their spermatogenesis before bilateral orchiectomy for gender reassignment. The progression of spermatogenesis was assessed by cytological analyses of the cultures; it was related to a dramatic increase in the levels of the mRNAs specifically expressed by round spermatids, Transition protein 1, Transition protein 2, and Protamine 3 in rat cultures. From 2% to 3.8% of cells were found to be haploid cells by fluorescence in situ hybridization analysis of human cultures. In this bioreactor, long-term cultures of seminiferous tubule segments from prepubertal rats or from adult men allowed completion of the spermatogenic process leading to morphologically mature spermatozoa. Further studies will need to address the way of optimizing the yield of every step of spermatogenesis by adjusting the composition of the culture medium, the geometry, and the material properties of the chitosan hydrogel bioreactors. Another essential requirement is to assess the quality of the gametes produced ex vivo by showing their ability to produce normal offspring (rat) or their biochemical normality (human).
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".
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