Chronic Exposure of Recipient Mice to Dibutyl Phthalate Affects Maturation of Testis Xenografts from Infant Rhesus Monkeys.

Abstract

Phthalic acid esters, such as dibutyl phthalate (DBP) are widely used as plasticizers in many daily-used products from which they leach into the environment. In rodents, gestational exposure implicated phthalates in the testicular dysgenesis syndrome. However, little is known regarding the effects of phthalate exposure on postnatal testis develoment, particularly in primates. Testis tissue xenografting allows recapitulating testis development of larger animals in mice, and is therefore an attractive strategy to study testis development and spermatogenesis of large species. The aim of this study was to evaluate the effect of exposure to DBP on postnatal testis development of primates. Fragments of testes from 6-mo old rhesus macaques (n=12) were transplanted into castrated, immunodeficient mice (8 pieces/mouse, 12 mice/donor), which were assigned to three treatments (4 mice/treatment): i) Low Dose (LD): 10 mg DBP/kg, ii) High Dose (HD): 500 mg DBP/kg, and iii) Control (CTL): no DBP; administered in corn oil by daily gavage for 14 (mice from 7 donors) and 28 weeks (mice from 5 donors), starting one week after surgery. From week 4, mice received 10 IU of hCG twice/week to stimulate xenograft development. At 14 weeks, while body weight of LD mice was not different to those of HD and CTL mice, weight of HD mice was lower than that of CTL mice (P<0.05). At 28 weeks, there was no effect of treatment on mouse body weight (P>0.05). At both collection times, there was no effect on liver weight (P>0.05). However, seminal vesicles in HD mice were smaller than those of LD and CTL mice (P<0.05) indicating lower levels of circulating androgens. At 14 weeks, there was no effect on xenograft weight (P>0.05), but at 28 weeks xenograft weight in HD mice was lower, with no difference between xenografts of LD and CTL mice (P<0.05). At 14 weeks, xenografts did not show germ cell differentiation beyond the spermatogonial stage. Number of tubules with UCH-L1 positive-cells (undifferentiated spermatogonia) was lower in xenografts than in donor tissue, and this number was lower in xenografts of HD mice than in those of LD and CTL mice (P<0.05). While the number of these cells per tubule cross-section was lower in HD xenografts than in CTL (P<0.05), in LD xenografts this number was not different to HD and CTL xenografts (P>0.05). At 28 weeks, in mice from 3/5 donors, xenografts developed to full spermatogenesis in LD and CTL mice, while those in HD mice did not developed beyond meiosis. Number of tubules with spermatocytes and UCH-L1 positive-cells was lower in HD xenografts than in LD and CTL xenografts (P<0.05). Number of UCH-L1 positive-cells per tubule cross-section was also lower in xenografts from HD mice than in those of LD and CTL mice (P<0.05). There was no difference in the number of tubules with round and elongated spermatids between xenografts from LD and CTL mice (P>0.05). These results indicate that chronic exposure of the immature primate testis to DBP in the xenografting model resulted in impaired testis development. The underlying mechanisms are currently under investigation. Supported by 1 R21 ES014856-01A2 and CIHR.