Neural Mechanisms Underlying the Disruption of Male Courtship Behavior by Adult Exposure to Di(2-ethylhexyl) Phthalate in Mice

Abstract

Background: Courtship behavior plays a critical role in attracting females and reproduction success. However, the effects of exposure to a ubiquitous contaminant di(2-ethylhexyl) phthalate (DEHP) on these behaviors and, in particular, on courtship vocalizations have not been examined.

Objective: The effects of adult exposure to DEHP on courtship and mating behaviors and gonadotropic axis and neural mechanisms involved in DEHP-induced effects were analyzed in male mice.

Methods: Adult C57BL/6J males were orally exposed to DEHP (0, 0.5, 5, and 50μg/kg/d) for 4 wk. Olfactory preference, ultrasonic vocalizations (USVs), partner preference and mating, as well as locomotor activity and motor coordination, were measured. The kisspeptin system and testosterone levels were analyzed. Proteomic and molecular studies were conducted on the hypothalamic preoptic nucleus, the key region involved in sexual motivation to vocalize and mate.

Results: DEHP at 50μg/kg/d reduced the emission of USVs, whereas lower doses changed the ratio of syllable categories. This was associated with diminished sexual interest of female partners toward males exposed to 5 or 50μg/kg/d and increased latency to mate, despite normal olfactory preference. The kisspeptin system and circulating testosterone levels were unaffected. In DEHP-exposed males, proteomic analysis of the preoptic nucleus identified differentially expressed proteins connected to the androgen receptor (AR). Indeed, exposure to 5 or 50μg/kg/d of DEHP induced selective AR downregulation in this nucleus and upstream chemosensory regions. The involvement of AR changes in the observed alterations was further supported by the reduced emission of courtship vocalizations in males with disrupted neural AR expression.

Conclusions: These data demonstrate the critical role of neural AR in courtship vocalizations and raises the possibility that the vulnerability of this signaling pathway to exposure to endocrine disrupters may be detrimental for courtship communication and mating in several species. https://doi.org/10.1289/EHP1443.

Figure 1.

Effects of phthalate di(2-ethylhexyl) phthalate (DEHP) on olfactory preference and courtship vocalizations in male mice. (A) Total time spent in the chemoinvestigation of male (♂) and sexually receptive female (♀) stimuli by males exposed to the vehicle (Veh) or DEHP (0.5, 5, or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$). (B) Number of entries into the male or female arm of the Y-maze. (C) Percentage of time spent in investigating males (♂) vs. sexually receptive females (♀). Data are expressed as the means $\pm \mathrm{SEM}$ of 11–12 males per treatment group, *$p<0.05$ vs. the male arm. (D–E) Representative sound waveform for males exposed to vehicle (D) or DEHP at $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$ (E) in the presence of a sexually receptive female. (F–G) Total number (F) and duration (G) of ultrasonic vocalizations (USVs) produced during the 4-min test by males exposed to the vehicle or DEHP at 0.5, 5, or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$. Data are expressed as the means $\pm \mathrm{SEM}$ of 11–12 males per treatment group; *$p<0.05$, **$p<0.01$.

Figure 2.

Quantitative and qualitative analyses of syllables emitted by males exposed to di(2-ethylhexyl) phthalate (DEHP). (A) Total number of each syllable type produced in the presence of a sexually receptive female during the 4-min recording for males exposed to the vehicle (Veh) or DEHP at 0.5, 5, or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$. One-way analysis of variance (ANOVA) showed an effect of DEHP treatment (#) on short ($p<0.0001$), flat ($p=0.0008$), downward ($p=0.007$), modulated ($p=0.03$), complex ($p=0.01$), mixed ($p=0.05$), one-jump ($p=0.013$), and frequency jump (0.019) syllables. Post hoc comparisons are indicated: *$p<0.05$, **$p<0.01$, ***$p<0.001$. Data are expressed as the means $\pm \mathrm{SEM}$ of 11–12 males per treatment group. (B) Percentage of courtship vocalizations per category (simple, complex, and with frequency jumps). (C) Total duration of each syllable type. One-way ANOVA showed an effect of DEHP treatment (#) on short ($p=0.0003$), modulated ($p=0.047$), complex ($p=0.035$), one-jump ($p=0.035$), and frequency jump (0.05) syllables. Post hoc comparisons are indicated: *$p<0.05$, **$p<0.01$, ***$p<0.001$.

Figure 3.

Effects of di(2-ethylhexyl) phthalate (DEHP) treatment on female interest in males and latency to intromission and ejaculation. (A–B) The partner preference test was performed in a three-chamber paradigm. (A) The number of entries of sexually receptive females into the chamber of the vehicle (Veh) vs. the chamber of the DEHP-exposed male. (B) The percentage of time spent investigating the vehicle (Veh) vs. the DEHP-exposed male. Data are expressed as the means $\pm \mathrm{SEM}$ of 11–12 males per treatment group, *$p<0.05$ vs. the vehicle male. (C–D) In the mating test, one-way analysis of variance (ANOVA) showed no effect of DEHP treatment on the latency to the first anogenital chemoinvestigation towards sexually receptive females (C), but an effect (#) on the latencies to first intromission and ejaculation (D). Data are expressed as the means $\pm \mathrm{SEM}$ of 11–12 males per treatment.

Figure 4.

Effects of di(2-ethylhexyl) phthalate (DEHP) on kisspeptin, green fluorescent protein (GFP)/androgen receptor (AR) and $\text{GFP}/\text{estrogen}\text{receptor}\left(\text{ER}\right)\mathrm{\alpha }$ immunoreactivity in wild type and Kiss1-creGFP male mice. Mice were treated with DEHP (0.5, 5, or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$) or the vehicle (Veh). Analyses were performed in the rostral periventricular area of the third ventricle (RP3V) and arcuate nucleus. (A–C) Representative immunolabelling (upper panels) and quantification (lower panels) of the number of kisspeptin-immunoreactive neurons in the RP3V (A) and fiber density in the RP3V (B) and arcuate nucleus (C) in wild type mice. Data are expressed as the means $\pm \mathrm{SEM}$ of six males per treatment group. (D) Upper panels: Representative immunolabelling of GFP- (left), AR-immunoreactivity (middle), and the merge (right) in the RP3V of Kiss1-creGFP males. Lower panels: quantification of the number of GFP-cells (left), GFP/AR- (middle), and $\text{GFP}/\text{ER}\mathrm{\alpha }$-coexpressing cells (right). Data are expressed as the means $\pm \mathrm{SEM}$ of five to six males per treatment group. (E) Upper panels: Representative immunolabelling of GFP- (left), AR-immunoreactivity (middle), and the merge (right) in the arcuate nucleus of Kiss1-creGFP males. Lower panels: quantification of the number of GFP-cells (left), GFP/AR-(middle), and $\text{GFP}/\text{ER}\mathrm{\alpha }$-coexpressing cells (right). Data are expressed as the means $\pm \mathrm{SEM}$ of five to six males per treatment group.

Figure 5.

Proteome analysis of the medial preoptic nucleus (MPN) in di(2-ethylhexyl) phthalate (DEHP)–exposed males. (A) Classification of differentially expressed proteins between the exposed groups into six major categories based on cellular localization and function. The percentages of proteins in each category are indicated. (B) Androgen-related protein interaction network generated by Ingenuity Pathway Analysis.

Figure 6.

Validation of proteomic data and characterization of androgen receptor (AR) and $\text{estrogen}\text{receptor}\left(\text{ER}\right)\mathrm{\alpha }$ expression in the medial preoptic nucleus (MPN). (A) 3-D view of the spot ID:0656, corresponding to GFAP, shown for three animals exposed to the vehicle (Veh) or DEHP at 5 or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$. (B–C) Upper panels: Representative Western blots of GFAP (B), $\mathrm{\beta }-\text{actin}$ (C), and GAPDH used as a protein reference, in the MPN of the Veh, DEHP-5, and DEHP-50 groups. Lower panels: quantification of the protein levels normalized to GAPDH. Data are expressed as the means $\pm \mathrm{SEM}$ of four males per treatment group, *$p<0.05$ compared to the vehicle group. (D–F) Representative AR-immunolabeling of the medial preoptic nucleus of males exposed to the vehicle (Veh) and DEHP at 5 or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$ (D) and quantitative data for AR- (E) and $\text{ER}\mathrm{\alpha }-\text{immunoreactive}$ cells (F). 3V: Third ventricle. Data are expressed as the means $\pm \mathrm{SEM}$ of four to six males per treatment group, *$p<0.05$ vs. the vehicle group. (G) AR gene expression in males exposed to the vehicle and DEHP at 5 or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$. Data are expressed as the means $\pm \mathrm{SEM}$ of six to eight males per treatment group, *$p<0.05$ compared to the vehicle group.

Figure 7.

Effect of neural androgen receptor (AR) invalidation on ultrasonic vocalizations (USVs). (A) Representative sound waveform for a control (${\text{AR}}^{\text{lox}}/\mathrm{Y}$, left) and mutant male (${\text{AR}}^{\text{NesCre}}$, right) in the presence of a sexually receptive female. (B–C) Number (B) and duration (C) of syllable types by category produced during the 4-min recording in the presence of a sexually receptive female. Data are expressed as the means $\pm \mathrm{SEM}$ of nine males per treatment group, *$p<0.05$, **$p<0.01$ compared to control littermates.