Determination of endocrine disruptors and endogenic androgens and estrogens in rat serum by high-performance liquid chromatography-tandem mass spectrometry.

To simultaneously measure some targeted endocrine disruptors and several forms of sex hormones in rat serum, an accurate analytical procedure was developed. First, a comparison between a polymeric-based solid-phase extraction (SPE) and a micro-extraction by packed sorbent was performed to choose the optimal method to extract and concentrate the analytes: bisphenol A, atrazine, vinclozolin metabolite, testosterone, androstenedione, estrone, estradiol, estrone-sulfate and glucuronide and estradiol-sulfate and glucuronide. The analyses were then performed by high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with electrospray ionisation in positive and negative modes. The protocol based on SPE was validated using the ICH/2005 guidelines. The validation demonstrated good performance in terms of linearity (R(2)>0.99), recovery (71-90%) and repeatability (relative standard deviation: 1-18%). The method was sensitive with LOQ comprised between 0.1 and 0.4 ng/ml for androgens and between 0.098 and 10.2 ng/ml for estrogens. The results obtained on the serum of rats exposed to the targeted endocrine disruptors showed the suitability of this analytical strategy.

An engineered 3D blood-testis barrier model for the assessment of reproductive toxicity potential.

We have developed an in vitro model that replicates the composition, organization, and barrier and spermatogenesis functions of the in vivo rat blood-testis barrier. This engineered blood-testis barrier (eBTB) is based on a three-dimensional (3-D) culture in a bicameral chamber of testicular cells isolated from 18-day-old rats. Peritubular cells were cultured on the bottom of the insert. On the top of the insert, a mixture of Sertoli and germ cells were coated within an artificial extracellular matrix, thereby mimicking the basement membrane. The matrix composition was defined to obtain a cord-like organization. This structure was revealed depending on morphogenetic gradients, and was made of polarized Sertoli cells and germ cells in the center of the structure. The in vivo functionality of the BTB was characterized by tight junctions between Sertoli cells. Claudin-11 protein immunodetection suggests that these junctions were also implicated in vitro in the cord-like structure, suggesting the presence of a physical compartment with apical and basal spaces. Measurement of the trans-epithelial electrical resistance characterized the relationship between the Sertoli cells, peritubular cells, and matrix/cells that influenced the tightness of their junctions during the course of the culture. In vitro germ cell differentiation was confirmed with the detection of haploid cells. The development of the eBTB under optimum conditions addresses the involvement of new models, testing the barrier and spermatogenesis functions that are sensitive to chemical compounds from the environment. In this way, the eBTB could be used as an alternative method to animal reprotoxicity studies, and would be of high interest in the scope of regulatory requests for chemical risk assessment.

Aromatase gene expression in the stallion.

Adult stallion secretes very high estrogen levels in its testicular vein and semen, and the responsible enzyme cytochrome P450 aromatase (P450 arom) is known to be present mainly in Leydig cells. We studied in further details the distribution of equine aromatase in various adult tissues including the brain (hypothalamic area), liver, kidney, small intestine, muscle, bulbourethral gland and testes. The aromatase mRNA was essentially detected by RT-PCR in testis (169+/-14 amol of aromatase mRNA per microg of total RNA) and was barely detectable in brain, or below 0.1 amol/microg RNA in other tissues. This range of expression was confirmed by ELISA (50+/-7 pg/microg total protein) in the testis, and by immunoblot, evidencing a 53 kDA specific protein band in testis and brain only. The corresponding aromatase activity was well detected, by 3H(2)O release from 1beta, 2beta(3)H-androstenedione, in testis and brain (200+/-23 and 25+/-6 pmol/min per mg, respectively) and below 3 pmol product formed/min per mg in other tissues. This study indicates that the testis, among the tissues analyzed, is the major source of aromatase in the adult stallion, and that the aromatase gene expression is specifically enhanced at this level, and is responsible for the high estrogen synthesis observed. Moreover, the study of aromatase in one colt testis has shown lower levels of transcripts, protein and enzyme activity, evidencing that aromatase is regulated during the development and may serve as a useful marker of testicular function. As the second organ where aromatase mRNA and activity are both well detected is brain, this study also underlines the possible role of neurosteroids in stallion on behaviour, brain function or central endocrine control.