Search

Background: Phthalate exposure induces germ cell effects in the fetal rat testis. Although experimental models have shown that the human fetal testis is insensitive to the steroidogenic effects of phthalates, the effects on germ cells have been less explored., Objectives: We sought to identify the effects of phthalate exposure on human fetal germ cells in a dynamic model and to establish whether the rat is an appropriate model for investigating such effects., Methods: We used immunohistochemistry, immunofluorescence, and quantitative real-time polymerase chain reaction to examine Sertoli and germ cell markers on rat testes and human fetal testis xenografts after exposure to vehicle or di(n-butyl) phthalate (DBP). Our study included analysis of germ cell differentiation markers, proliferation markers, and cell adhesion proteins., Results: In both rat and human fetal testes, DBP exposure induced similar germ cell effects, namely, germ cell loss (predominantly undifferentiated), induction of multinucleated gonocytes (MNGs), and aggregation of differentiated germ cells, although the latter occurred rarely in the human testes. The mechanism for germ cell aggregation and MNG induction appears to be loss of Sertoli cell-germ cell membrane adhesion, probably due to Sertoli cell microfilament redistribution., Conclusions: Our findings provide the first comparison of DBP effects on germ cell number, differentiation, and aggregation in human testis xenografts and in vivo in rats. We observed comparable effects on germ cells in both species, but the effects in the human were muted compared with those in the rat. Nevertheless, phthalate effects on germ cells have potential implications for the next generation, which merits further study. Our results indicate that the rat is a human-relevant model in which to explore the mechanisms for germ cell effects.

Environmental factors are implicated in increased incidence of human testicular germ-cell cancer (TGCC). TGCC has foetal origins and may be one component of a testicular dysgenesis syndrome (TDS). Certain phthalates induce TDS in rats, including effects on foetal germ cells (GC). As humans are widely exposed to phthalates, study of the effects of phthalates on foetal rat GC could provide an insight into the vulnerability of foetal GC to disruption by environmental factors, and thus to origins of TGCC. This study has therefore characterized foetal GC development in rats after in utero exposure to di(n-butyl) phthalate (DBP) with emphasis on GC numbers/proliferation, differentiation and time course for inducing effects. Pregnant rats were treated orally from embryonic day 13.5 (e13.5) with 500 mg/kg/day DBP for varying periods. GC number, proliferation, apoptosis, differentiation (loss of OCT4, DMRT1 expression, DMRT1 re-expression, GC migration) and aggregation were evaluated at various foetal and postnatal ages. DBP exposure reduced foetal GC number by ∼60% by e15.5 and prolonged GC proliferation, OCT4 and DMRT1 immunoexpression; these effects were induced in the period immediately after testis differentiation (e13.5-e15.5). In contrast, DBP-induced GC aggregation stemmed from late gestation effects (beyond e19.5). Foetal DBP exposure delayed postnatal resumption of GC proliferation, leading to bigger deficits in numbers, and delayed re-expression of DMRT1 and radial GC migration. Therefore, DBP differentially affects foetal GC in rats according to stage of gestation, effects that may be relevant to the human because of their nature (OCT4, DMRT1 effects) or because similar effects are demonstrable in vitro on human foetal testes (GC number). Identification of the mechanisms underlying these effects could give a new insight into environment-sensitive mechanisms in early foetal GC development that could potentially be relevant to TGCC origins., (© 2011 The Authors. International Journal of Andrology © 2011 European Academy of Andrology.)

We have shown previously that deficient androgen action within a masculinization programming window (MPW; e15.5-e18.5 in rats) is important in the origin of male reproductive disorders and in programming male reproductive organ size, but that androgen action postnatally may be important to achieve this size. To further investigate importance of the MPW, we used two rat models, in which foetal androgen production or action was impaired during the MPW by exposing in utero to either di(n-butyl) phthalate (DBP) or to flutamide. Reduced anogenital distance (AGD) was used as a monitor of androgen production/action during the MPW. Offspring were evaluated in early puberty (Pnd25) to establish if reproductive organ size was altered. The testes, penis, ventral prostate (VP) and seminal vesicles (SV) were weighed and penis length measured. Both DBP and flutamide exposure in the MPW significantly reduced penis, VP and SV size along with AGD at Pnd25; AGD and organ size were highly correlated. In DBP-, but not flutamide-, exposed animals, testis weight was also reduced and correlated with AGD. Intratesticular testosterone was also measured in control and DBP-exposed males during (e17.5) or after (e21.5) the MPW and related to AGD at e21.5. To evaluate the importance of postnatal androgen action in reproductive organ growth, the effect of combinations of prenatal and postnatal maternal treatments on AGD and penis size at Pnd25 was evaluated. In prenatally DBP-exposed animals, further postnatal exposure to either DBP or flutamide significantly reduced AGD and penis size in comparison with prenatal DBP exposure alone. In comparison, rats exposed postnatally to testosterone propionate after prenatal vehicle-exposure showed considerable increase in these parameters vs. controls. In conclusion, we show that the size of all male reproductive organs is programmed by androgen exposure in the MPW, but that growth towards this size is dependent on androgen action postnatally.

Common male reproductive abnormalities including cryptorchidism, hypospadias, and low sperm counts may comprise a testicular dysgenesis syndrome (TDS), resulting from fetal testis dysfunction during a critical developmental period involving reduced androgen production/action. The recent increase in TDS prevalence suggests environmental/lifestyle factors may be etiologically important. The developing fetus is exposed to multimodal challenges, and we hypothesized that exposure to a combination of factors rather than single agents may be important in the pathogenesis of TDS. We experimentally induced fetal testis dysfunction in rats via treatment of pregnant females daily from embryonic day (e) 13.5 to e21.5 with vehicle, 100 or 500 mg/kg . d dibutyl phthalate (DBP), 0.1 mg/kg . d dexamethasone (Dex), or a combination of DBP + Dex. In adulthood, penile length/normality, testis weight/descent, prostate weight, and plasma testosterone levels were measured plus anogenital distance (AGD) as a measure of androgen action within the masculinization programming window. Intratesticular testosterone and steroidogenic enzyme gene expression were measured in fetal testes at e17.5. High-dose DBP reduced fetal intratesticular testosterone and steroidogenic gene expression; induced mild hypospadias (31%) and cryptorchidism (53%); and reduced penile length, AGD, and testis and prostate weight in adulthood. Dex alone had no effect except to reduce birth weight but amplified the adverse effects of 500 mg/kg . d DBP and exacerbated the effects of 100 mg/kg . d DBP. All adverse effects were highly correlated to AGD, emphasizing the etiological importance of the masculinization programming window. These findings suggest that exposure to common environmental chemicals in combination with, for example, maternal stress, may increase the risk of common male reproductive abnormalities, with implications for human populations.