Quantitative Changes in Gene Expression in Fetal Rat Testes following Exposure to Di(n-butyl) Phthalate

Abstract

Di(n-butyl) phthalate (DBP) alters male reproductive development by decreasing testicular testosterone (T) production when fetuses are exposed on gestation days (GD) 12-21. Previous studies have shown altered gene expression for enzymes in the T biosynthetic pathway following exposure to DBP. The objectives of this study were to develop a more detailed understanding of the effect of DBP on steroidogenesis, using a robust study design with increased numbers of dams and fetuses, compared with previous studies, and to explore messenger RNA (mRNA) expression for other critical genes involved in androgen biosynthesis and signaling. Additionally, immunohistochemical localization of protein expression for several key genes was performed to further confirm mRNA changes. Fetal Leydig cell lipid levels were also examined histochemically, using oil red O. Six to seven pregnant Crl:CD(SD)BR rats per group were gavaged with corn oil or DBP at 500 mg/kg/day on GD 12-19. Testicular RNA isolated from three randomly selected GD 19 fetuses per litter was used for real-time RT-PCR for the following genes: scavenger receptor class B-1 (SRB1), steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450c17, 17beta-hydroxysteroid dehydrogenase (17beta-HSD), androgen receptor (AR), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), stem cell factor tyrosine kinase receptor (c-kit), stem cell factor (SCF), proliferating cell nuclear antigen (PCNA), and testosterone-repressed prostate message-2 (TRPM-2). mRNA expression was downregulated for SRB1, StAR, P450scc, 3beta-HSD, P450c17, and c-kit following DBP exposure, and TRPM-2 was upregulated. 17beta-HSD, AR, LHR, FSHR, and PCNA were not significantly changed. Immunohistochemical staining for c-kit was seen in fetal Leydig cells, which has not been previously reported. Downregulation of most of the genes in the T biosynthetic pathway confirms and extends previous findings. Diminished Leydig cell lipid content and alteration of cholesterol transport genes also support altered cholesterol metabolism and transport as a potential mechanism for decreased T synthesis following exposure to DBP.

Impact of dibutyl phthalate and benzyl butyl phthalate on motility parameters of sperm from the European pikeperch Sander lucioperca (L.)

“…It was demonstrated that when these phthalates are delivered per os to pikeperch during the sex differentiation stage, there is a delay in testis development and the feminization of the male gonads is induced (Jarmo³owicz et al, unpublished data). Additionally, there is a link between the presence of some phthalates (including DBP) in mammals and oxidative stress caused by excess reactive oxygen species (ROS) (Barlow et al 2003, Lehmann et al 2004, Liu et al 2005. In humans, ROS can cause lipid peroxidation in sperm cell membranes, changes in the structure of membrane receptors, and in enzyme and transport proteins, and it can also increase anomalies in chromatin structure and breaks in sperm DNA strands (Hughes et al 1996).…”

The effects on steroidogenesis and histopathology of adult male Japanese quails (Coturnix coturnix japonica) testis following pre-pubertal exposure to di(n-butyl) phthalate (DBP)

“…A number of endocrine disrupting chemicals (EDCs), including DBP, are known to be anti-androgenic ). The androgenic effects of DBP include decrease in testosterone synthesis possibly due to reductions in the expression of genes involved in cholesterol transport and downstream synthesis (Barlow et al, 2003) or include the inhibition of the expression of a number of key enzymes involved in cholesterol uptake or transport and steroidogenesis (Stocco, 2001;Stocco et al, 2005) with downstream reproductive consequences.…”

“…Consequently, intermediate stages of crowing development could be associated with reduced testosterone production stemming from social stimulation (Groothuis). Several reports (Gray et al, 1982;Barlow et al, 2003) have indicated that testosterone effects on germinal tissue are in fact, responsible for the observed decrease in seminiferous tubular height and the corresponding decrease in testis weight. In this present study, there was no difference between the mean body weight and testicular weight.…”

“…Since it has been shown that phthalates do not directly bind to the androgen receptor (Parks et al, 2000;Foster et al, 2001), the anti-androgenic effects of DBP are suggested to be due to a decreased testosterone synthesis that arises from the reduction in the expression of genes involved in cholesterol transport and testosterone synthesis (Shultz et al, 2001;Barlow et al, 2003). Laboratory studies have shown that the male Japanese quail is sensitive to the effects of exogenous hormones during sexual differentiation (Ottinger et al, 2002).…”

A study of the consequences of early gestational ethanol and/or nicotine exposure in the mouse

“…Following from this, in the field of DOHaD it is common to sample only a few offspring per litter, with the assumption that the findings from these individuals are representative of the characteristics of their littermates (Haseman and Hogan, 1975). However, there is an increasing body of evidence to suggest that there is greater intra-litter variability than inter-litter variability in fetal outcomes following many prenatal exposures (van der Schoot, 1992; Howdeshell and Saal, 2000; Barlow et al, 2003;Godin et al, 2010;Kietzman et al, 2014;Han et al, 2016).…”