Insights into the mechanisms of aquaporin-3 inhibition by gold(III) complexes: the importance of non-coordinative adduct formation

A series of six new Au(III) coordination compounds with phenanthroline ligands have been synthesized and studied for the inhibition of the water and glycerol channel aquaporin-3 (AQP3). From a combination of different experimental and computational approaches, further insights into the mechanisms of AQP3 inhibition by gold compounds at a molecular level have been gained. The results evidence the importance of noncoordinative adduct formation, prior to “covalent” protein binding, to achieve selective AQP3 inhibition

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

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

Contribution of small and large Leydig cell aggregates to the total Leydig cell aggregate area per testis in e21.5 rat testes after <i>in utero</i> exposure to vehicle (control) or dibutyl phthalate (DBP-500 or 750 mg/kg), dexamethasone (Dex 100 µg/kg) or DBP-500+Dex from e13.5–e20.5 (full treatment window), e15.5–e18.5 (MPW window) or e19.5–e20.5 (late window).

<p>Values are Means ± SEM for 8–15 animals from 3–5 litters per treatment group. ***p<0.001, in comparison with controls; ^ap<0.001 in comparison with Dex group (except p<0.05 when Dex is compared with DBP-500 late window treatment); ^bp<0.05 in comparison with DBP-500 late window group; ^cp<0.001 in comparison with DBP-750 late window group; ^dp<0.01 in comparison with DBP-750 MPW window group; ^ep<0.05 in comparison with DBP-750 full treatment window group.</p

Relationship between Leydig cell (LC) aggregation ( = focal dysgenesis) and anogenital distance (AGD) (A, B) or intratesticular testosterone (ITT) at e21.5 (C, D) or between AGD and ITT at e21.5 (E, F) in animals exposed <i>in utero</i> to vehicle (control), dibutyl phthalate (DBP-500 or 750 mg/kg), dexamethasone (Dex 100 µg/kg) or DBP-500+Dex during all treatment windows (A, C, E), or during the full treatment window (e13.5–e20.5) only (B, D, F).

<p>Dysgenesis is negatively correlated with AGD irrespective of treatment period (A, B) whereas all other correlations were affected by the treatment period (full details in text).</p

Relationship between Leydig cell (LC) aggregation ( = focal dysgenesis) and anogenital distance (AGD) at postnatal day (pnd) 8 after <i>in utero</i> exposure to vehicle (control), dibutyl phthalate (DBP-500 mg/kg), dexamethasone (Dex 100 µg/kg) or DBP-500+Dex from e13.5–e21.5.

<p>(A) Average size of the 3 largest Leydig cell aggregates is shown as Means ± SEM for 8–17 animals from 3–6 litters. ***p<0.001, in comparison with controls; other comparisons are indicated by capped lines. (B) Correlation between dysgenesis and anogenital distance (AGD) in pnd8 animals.</p

Anogenital distance (AGD) and intratesticular testosterone (ITT) in rats at e21.5 after <i>in utero</i> exposure to vehicle (control), dibutyl phthalate (DBP-500 or 750 mg/kg), dexamethasone (Dex 100 µg/kg) or DBP-500+Dex from e13.5–e20.5 (full treatment window), e15.5–e18.5 (MPW window) or e19.5–e20.5 (late window).

<p>Only treatments which included the masculinization programming window (MPW) resulted in a significant reduction in AGD in animals (A), whereas ITT was maximally reduced when treatment included the late (e19.5–e20.5) window (B). Values are Means ± SEM for 18–39 animals from 3–7 litters per group. ***p<0.001, in comparison with controls; ^ap<0.001 in comparison with Dex group; ^bp<0.001 in comparison with DBP-500 late window group; ^cp<0.001 in comparison with DBP-750 late window group; ^dp<0.01 in comparison with DBP-750 MPW window group; ^ep<0.05 in comparison with DBP-500 full treatment window group.</p

Immunohistological analysis of focal dysgenetic areas in rat testes exposed to vehicle (control) or dibutyl phthalate (DBP).

<p>(A–B) Double immunofluorescence for 3β-HSD (blue) and Sox-9 (red) on e21.5 testis sections from (A) vehicle (control) and (B) DBP-exposed (750 mg/kg/) animals, illustrating focal dysgenesis in which Leydig cell aggregates contain ectopically localized Sertoli cells. Green depicts DAPI nuclear counterstain. SC = seminiferous cords. Scale bar = 20 µm. (C1–C6) Example of sections stained for 3β-HSD (brown) used for Leydig cell aggregate analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030111#pone-0030111-g003" target="_blank">Figure 3</a>). Arrows indicate large Leydig cell aggregates, asterisks indicate seminiferous cords. Scale bar = 200 µm. (D–E) Double immunohistochemistry for 3β-HSD (blue) and SMA (brown) on postnatal day (pnd) 8 testis sections from (D) vehicle (control) and (E) DBP-exposed (500 mg/kg/) animals, illustrating focal dysgenesis after DBP-exposure, with large Leydig cell aggregates and malformed seminiferous cords and intratubular Leydig cells (arrows). Scale bar = 50 µm.</p