Identification of Drosophila-based endpoints for the assessment and understanding of xenobiotic-mediated male reproductive adversities

Abstract

Men are at risk of becoming completely infertile due to innumerable environmental chemicals and pollutants. These xenobiotics, hence, should be tested for their potential adverse effects on male fertility. However, the testing load, a monumental challenge for employing conventional animal models, compels the pursuit of alternative models. Towards this direction, we show here that Drosophila melanogaster, an invertebrate, with its well characterized/conserved male reproductive processes/proteome, recapitulates male reproductive toxicity phenotypes observed in mammals when exposed to a known reproductive toxicant, dibutyl phthalate (DBP). Analogous to mammals, exposure to DBP reduced fertility, sperm counts, seminal proteins, increased oxidative modification/damage in reproductive tract proteins and altered the activity of a hormone receptor (estrogen related receptor) in Drosophila males. In addition, we show here that DBP is metabolized to monobutyl phthalate (MBP) in exposed Drosophila males and that MBP is more toxic than DBP, as observed in higher organisms. These findings suggest Drosophila as a potential alternative to traditional animal models for the prescreening of chemicals for their reproductive adversities and also to gain mechanistic insights into chemical-mediated endocrine disruption and male infertility.

Keywords: Drosophila; endocrine disruption; estrogen related receptor; male infertility; phthalate.

FIG. 1.

Metabolism of DBP in Drosophila and the effect of DBP and MBP on Drosophila male fertility. To determine the chemical load in exposed organisms, the level of DBP within control and exposed males was estimated through GC-MS/MS. Panel (A) includes the total ion current chromatograms from GC-MS depicting MBP and DBP peaks (as labeled) in control males (control), flies exposed to 100μM or 1mM DBP and also with reference standard (Standard). Subsequently, fertility (the number of progeny produced over a period of 10 days) of females mated to males exposed to different concentrations of (B) DBP or (C) MBP throughout their development was analyzed. Developmental exposure of males to 100μM of DBP reduced (***p < 0.001) the fertility of their mates while MBP hampered fertility (**p < 0.01 when compared with vehicular control, DMSO) even at a concentration 10 times lower than that of DBP. All experiments were repeated three times (N = 15–20 males/mates per replicate/group).

FIG. 2.

Reduced reproductive performance of males exposed to DBP during development. The effect of DBP on the reproductive performance of exposed males was analyzed by mating control or exposed males to wild-type virgin females. We measured (A) the overall fecundity (number of eggs laid/ female over a period of 10 days) of females mated to control or exposed males (10 or 100μM DBP) and (B) daywise fecundity with transfer of mated females to fresh food every 24 h for 10 days (D1–D10). Subsequently, we scored these vials for the (C) overall fertility (number of progeny/female/10 days), and (D) daywise fertility of females mated to males exposed to 10 or 100μM DBP or their controls. We determined (E) overall percentage hatchability based on the proportion of eggs that reach adult stage and (F) at 24 h intervals for 10 days. The significant differences in fecundity, fertility and percentage hatchability between controls and exposed groups are denoted by *p < 0.05, **p < 0.01 or ***p < 0.001. All experiments were repeated three to four times (N = 15–20 males/mates per replicate/group).

FIG. 3.

Fewer mature sperm and late spermatogenesis stages in males developmentally exposed to DBP. To determine the effect of DBP on spermatogenesis, we counted the number of sperm in seminal vesicles and sperm bundles and canoe stages in testes of control and developmentally exposed w; P{Protamine B-EGFP} males after recurrent matings over 3 days followed by 48 h relaxation. The number of sperms in the seminal vesicle (A) and the number of sperm bundles in testes (B) of Protamine B-EGFP males exposed developmentally to 100μM DBP were significantly reduced (***p < 0.001; *p < 0.05) whereas the number of canoe stages (C) were similar to controls. All these experiments were carried out twice and sperm were counted in tissues twice with a repeatability index of 92% (N = 10–15/replicate/group).

FIG. 4.

Alteration of a few genes, expressed in the reproductive tract or encoding seminal proteins in males exposed to 100μM DBP during development. To validate the semiquantitative PCR data, we analyzed the transcript levels of candidate genes (Ovulin, CG11864, CG10586, CG4605, CG11598, CG17673, GLD, ERR, and CG17843) through real-time PCR. Ovulin, CG11864, CG10586, CG11598, GLD, and ERR were significantly down-regulated, and transcript levels of CG17673 were significantly higher when compared with control (*p < 0.05; **p < 0.01). The Δct values were determined through normalization against RPL32, which was used as an internal control for the quality of the template.

FIG. 5.

Reduced ovulin levels in the accessory glands of ovulin-GFP transgenic males developmentally exposed to DBP. To determine if changes in transcript levels are reflected at the protein level, we visualized the ovulin protein based on the GFP fluorescence in the accessory glands of (A) control, (B) males exposed to 100μM DBP, and (C) males exposed to 1mM DBP through confocal microscopy. Panel (D) indicates the significant reduction in the fluorescence intensity of ovulin-GFP, in 100μM DBP exposed males and 1mM DBP exposed males (***p < 0.001). Anti-GFP antibody detected ovulin-GFP (63 kDa) in protein samples from the male accessory glands of control and exposed males on Western blots (panel E, ovulin-GFP). Upon normalization of ovulin-GFP intensities with corresponding α-tubulin signals (panel E, α-tubulin), the levels of ovulin-GFP were found to be significantly reduced in males exposed to 100μM DBP (*p < 0.05) or 1mM DBP (**p < 0.01), when compared with those in control (panel F). The data depicted is the average of five replicates from as many independent blots.

FIG. 6.

Oxyblot of reproductive tract proteins from males exposed to DBP and their controls. The “+” lane represents protein samples wherein protein carbonyl functional groups were derivatized with DNPH (“+” lanes) whereas samples without DNPH served as derivatization controls (“–” lanes) for the corresponding batches. These samples were probed with anti-DNP antibody (panel A, DNP). Western blot of α-tubulin (panel A, α-tubulin) served as the loading control. Analysis of signal intensities of 98 and 64 kDa through densitometry, after normalization with loading control, revealed a dose-dependent increase in the extent of oxidative modification of 98 (panel B; *p < 0.05) and 64 kDa (panel C; *p < 0.05) in males exposed to 100μM or 1mM DBP when compared with controls. The data depicted is the average of three replicates from a minimum of three independent blots.

FIG. 7.

Exposure to DBP modulates the activity of estrogen related receptor (ERR) in Drosophila. Spatial patterns of ERR activity were visualized through X-gal stainingof male reproductive tract tissues from (A) control males, (B) males exposed to 100μM DBP, and (C) males exposed to 1mM DBP. Panel (D) represents the number of X-gal positive spots observed in reproductive tissues of males exposed to 100μM (**p < 0.01), and 1mM (***p < 0.001) in comparison to controls. The cumulative intensities of X-gal spots observed in the reproductive tracts of males exposed to 100μM or 1mM in comparison to control (***p < 0.001) are represented in panel (E). All experiments were repeated three times with 10–15 replicates per group.

FIG. 8.

Exposure to DBP influences the fate of sperm in Drosophila. To determine the effect of DBP on the fate of sperm in the female milieu, each of the control or exposed Protamine B-EGFP male was presented with broods of three virgin females every 24 h for 3 days. The first mated female of brood1 (considered as mate I) of exposed male, had significantly fewer sperm in SR (seminal receptacle; A) at 2 h ASM whereas the sperm counts in SP (spermathecae; B) did not differ from those in mate I of control males. However, mate IV (from brood 2) and mate VII (from brood 3) of male exposed to 100μM DBP contained fewer sperms in both seminal receptacle (C and E) and spermathecae (D and F) when compared with those mates in control broods (*p < 0.05; **p < 0.01; N = 10–15/replicate). In addition, sperm in seminal receptacles (SR) from X mates of control (G) or exposed (H) Protamine B-EGFP males were counted and we observed significant reduction in the number of sperms in seminal receptacle of females mated to Protamine B-EGFP males, developmentally exposed to 100μM in comparison to control (*p < 0.05; N = 10–15; panel I). Panels (J) and (K) depict sperm in the spermathecae of X mates of control or exposed Protamine B-EGFP males, respectively. Panel (L) presents the number of sperm stored in spermathecae of females mated to exposed males, when compared with their controls (*p < 0.05; N = 10–15/replicate).

FIG. 9.

Ovulin in the reproductive tracts of females mated to DBP exposed males or their controls. Panel (A) represents the Western blot showing ovulin-GFP (tagged with GFP) at 1 h ASM in the reproductive tracts of females, which received seminal proteins and sperm from ovulin-GFP control males (lane control), or from males developmentally exposed to either 100μM (lane 100μM) or 1mM (lane 1mM). The blots were probed with α-tubulin (α-tubulin) as control for protein loading. Semi-quantitative analysis through densitometry revealed lower levels of ovulin in the female tracts of those mated to males exposed developmentally either to 100μM or 1mM DBP (**p < 0.01), when compared with control (panel B). The data depicted is the average of three replicates from a minimum of three independent blots.