Mechanism of action of phthalate endocrine disruptors in male reproductive development.

Phthalates are plasticizers, commonly found in cosmetics, food-wrapping, personal care products and medical devices, and have recently been associated with reduced anogenital distance in male infants, reduced testosterone and altered behavior in boys. In rodents, in utero phthalate exposure disrupts the development of the internal and external male reproductive phenotype in the progeny. Testicular Leydig cells may be the primary target of phthalate action, since they often exhibit abnormal aggregation and reduced insulin-like factor 3 (INSL3). Moreover, microarray studies suggest that they are amongst the earliest testicular cell types affected by phthalate treatment, though acute effects on mature cells appear to be minor. The overall aim of this project was to assess the effect of phthalate, with the synthetic non-steroidal estrogen, diethylstilbestrol (DES) as control, on Leydig cell differentiation. First a time-resolved fluorescent immunoassay (TRFIA) for rodent INSL3, a specific marker of Leydig cell differentiation, was developed, which was highly specific for rat and mouse INSL3 in body fluids or in cell culture medium. Besides showing that INSL3 is a reliable marker for aging Leydig cells, it accurately reflected the differentiation of Leydig cells during puberty. The second aim of this project assessed whether the kinetics of new Leydig cell differentiation following their ablation by ethane dimethane sulfonate (EDS) was affected by dibutyl phthalate (DBP) treatment at an early stage of Leydig cell regeneration. mRNA expression of Leydig cell markers such as LH receptor (LHR), cytochrome P450 17-alphahydroxylase/17,20 lyase (Cyp17a1) and 11-beta-hydroxysteroid dehydrogenase type 1 (Hsd11b1) in the DBP-treated animals were increased, likely due to an increase in cell proliferation since Leydig cells in treated animals exhibited more clustering and higher numerical density compared to controls. The long-term effect of DBP on the adult Leydig cell population following in utero and lactational exposure was also investigated since most studies have concentrated on acute early effects of phthalates. Maternal DBP treatment appeared to accelerate Leydig cell development, especially when Leydig cells are actively proliferating or differentiating, largely due to an increased proliferation of Leydig cells. The consequences of such treatment do not persist to adulthood, since circulating INSL3 as well as mRNA expression of various Leydig cell markers were comparable in all treatment groups at postnatal day 90 (PND90). Finally, a long term in vitro model of Leydig cell differentiation was established, using undifferentiated adult-type Leydig cells isolated from day 10 rats. Preliminary experiments with DBP and its main metabolite, monobutyl phthalate (MBP), showed that both compounds were probably inhibitory to Leydig cell differentiation in culture in the presence of human chorionic gonadotropin (hCG). The precise mechanism by which these compounds slowed Leydig cell differentiation will be determined in future studies. Collectively, the findings of this thesis strongly imply that differentiating/developing adult-type Leydig cells are indeed direct targets of endocrine disruption. This thesis has also demonstrated that the EDS model and an in vitro Leydig cell differentiation model will be very useful in delineating the underlying mechanism of phthalate action.Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 201