Disorders of human male reproductive health include cryptorchidism, hypospadias, infertility/subfertility, testicular germ cell cancer and primary hypogonadism. The ‘testis dysgenesis syndrome’ hypothesis proposes that ALL of these problems have a shared origin during fetal life: if testis development is perturbed during a critical window of time whilst in the womb, reproductive health and function is affected. These disorders are escalating at such high rates that it is presumed that environmental causes are to blame - the key suspect our increasing exposure to ‘endocrine disrupting chemicals’, particularly during fetal life.
We have previously shown that the presence of signalling molecule retinoic acid (RA) is detrimental to development of three major cell types of the mouse fetal testis – germ cells, Sertoli cells and fetal Leydig cells. Normally, P450 enzyme CYP26B1 degrades RA, but if CYP26B1 does not function, ectopic RA causes germ cells to aberrantly enter meiosis, the testis is partially feminized, and secondary sexual structures are perturbed.
Combining developmental biology, mouse transgenic expertise, and reproductive toxicology, we have developed a novel ex-vivo testis culture system to read-out RA-Cyp26b1 signalling perturbation. We have used this system to evaluate the molecular effects of a common agricultural azole, Flusilazole, and related compounds. Flusilazole is a fungicide that works by inhibiting the fungal P450 enzyme CYP51, though it is likely that it can also inhibit mammalian CYP enzymes.
Transgenic fetal testes at E12.5 were cultured in hanging drops for 48hrs in the presence/absence of a panel of azole chemicals before harvesting for qRT-PCR analysis, histological examination, staining or imaging. We found that ectopic RA signalling could be detected in response to azole exposure at a range of concentrations, indicating possible consequences for reproductive development and function. Our ongoing work will have future translational importance, in particular for the refinement of current chemical screening methodologies.