Inter-Relationship between Testicular Dysgenesis and Leydig Cell Function in the Masculinization Programming Window in the Rat

Abstract

The testicular dysgenesis syndrome (TDS) hypothesis proposes that maldevelopment of the testis, irrespective of cause, leads to malfunction of the somatic (Leydig, Sertoli) cells and consequent downstream TDS disorders. Studies in rats exposed in utero to di(n-butyl) phthalate (DBP) have strongly supported the TDS concept, but so far no direct evidence has been produced that links dysgenesis per se to somatic cell dysfunction, in particular to androgen production/action during the ‘masculinization programming window’ (MPW; e15.5–e18.5). Normal reproductive tract development and anogenital distance (AGD) are programmed within the MPW, and TDS disorders arise because of deficiencies in this programming. However, DBP-induced focal testicular dysgenesis (Leydig cell aggregation, ectopic Sertoli cells, malformed seminiferous cords) is not evident until after the MPW. Therefore, we used AGD as a read-out of androgen exposure in the MPW, and investigated if this measure was related to objectively quantified dysgenesis (Leydig cell aggregation) at e21.5 in male fetuses exposed to vehicle, DBP (500 or 750 mg/kg/day) or the synthetic glucocorticoid dexamethasone (Dex; alone or plus DBP-500) from e15.5–e18.5 (MPW), e13.5–e20.5 or e19.5–e20.5 (late window). Dysgenesis was found only in animals exposed to DBP during the MPW, and was negatively correlated (R2 = −0.5) with AGD at e21.5 and at postnatal day 8, irrespective of treatment period. Dysgenesis was also negatively correlated (R2 = –0.5) with intratesticular testosterone (ITT) at e21.5, but only when treatments in short windows (MPW, late window) were excluded; the same was true for correlation between AGD and ITT. We conclude that AGD, reflecting Leydig cell function solely within the MPW, is strongly related to focal dysgenesis. Our results point to this occurring because of a common early mechanism, targeted by DBP that determines both dysgenesis and early (during the MPW) fetal Leydig cell dysfunction. The findings provide strong validation of the TDS hypothesis.

Experimentally induced testicular dysgenesis syndrome originates in the masculinization programming window

“…This is supported by the fact that focal dysgenesis is inversely correlated with AGD in fetal life (this study and ref. 40) and adulthood (this study).…”

“…Time-mated females were allocated randomly to receive vehicle control treatment or 750 mg/kg DBP (Sigma-Aldrich; 99% pure according to the supplier) in 1 ml/kg corn oil daily by oral gavage. The dose of DBP was chosen as it induces a high incidence of focal dysgenesis as well as profoundly suppressing testosterone production by fetal Leydig cells (40). Three DBP treatment groups were used in this study: DBP-FW (E13.5-E21.5), DBP-MPW (E15.5-E18.5), and DBP-LW (E19.5-E21.5).…”

“…Identification of the MPW in rodents was based on use of the androgen receptor antagonist flutamide (20), but in humans TDS is considered to result from maldevelopment and consequent malfunction of the fetal testis (3,6), so that a model with such changes was needed. Our earlier studies suggested that exposure of pregnant rats to the environmental chemical dibutyl phthalate (DBP) might be a potential model for TDS in humans, as the testis differentiates normally after DBP exposure (40), but this treatment induces focal dysgenesis (40)(41)(42) and reduces fetal testis testosterone levels via effects on steroidogenic gene expression (43)(44)(45), which then causes TDS disorders (41,42,46). However, such studies were undertaken before the importance of the MPW had been identified.…”

Abolition of prenatal lipopolysaccharide-induced reproductive disorders in rat male offspring by fulvestrant

“…AGD was increased after postnatal fulvestrant treatment as compared to LPS‐treated group. Animal model studies have identified that AGD may provide the means for retrospectively determining foetal androgen exposure during masculinisation and male reproductive tract development (van den Driesche, Kolovos, Platts, Drake, & Sharpe, ; Welsh, ). AGD decreased in male mice at PND26 after maternal LPS treatment from E13 to E17 (Wang et al, ).…”

Anti‐Androgenic Chemicals

“…Certain similarities in humans of these malformations in rodents, described as testicular dysgenesis syndrome (TDS), are speculated to be caused by exposure to anti-androgenic chemicals, which could result from reduced androgen action during fetal development (Skakkebaek et al, 2001;Wohlfahrt-Veje et al, 2009). For example, it has been shown that exposure of rats in utero to DBP can induce a TDS-like syndrome in the male offspring (Mylchreest et al, 2000;Fisher et al, 2003;Foster, 2006;Dean et al, 2012;van den Driesche et al, 2012), which is manifested as dose-dependent induction of cryptorchidism, hypospadias, and impaired spermatogenesis/infertility ( Fig. For example, it has been shown that exposure of rats in utero to DBP can induce a TDS-like syndrome in the male offspring (Mylchreest et al, 2000;Fisher et al, 2003;Foster, 2006;Dean et al, 2012;van den Driesche et al, 2012), which is manifested as dose-dependent induction of cryptorchidism, hypospadias, and impaired spermatogenesis/infertility ( Fig.…”