# Sexual behavior in male mice is impaired by an obesogenic-diabetogenic diet and worsened by exposure to a relevant environmental mixture of phthalates

Mahiddine Lina, Parmentier Caroline, Touarssi Kaoutar, Grange-Messent Valérie, Mhaouty-Kodja Sakina, Hardin-Pouzet Hélène

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Mahiddine Lina, Parmentier Caroline, Touarssi Kaoutar, Grange-Messent Valérie, Mhaouty-Kodja Sakina, et al.. Sexual behavior in male mice is impaired by an obesogenic-diabetogenic diet and worsened by exposure to a relevant environmental mixture of phthalates. Environment International, 2025, 204, pp.109858. 10.1016/j.envint.2025.109858. hal-05377370

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# Sexual behavior in male mice is impaired by an obesogenic-diabetogenic diet and worsened by exposure to a relevant environmental mixture of phthalates

Mahiddine Lina¹, Parmentier Caroline¹, Touarssi Kaoutar¹, Grange-Messent Valérie¹, Mhaouty-Kodja Sakina¹, Hardin-Pouzet Hélène¹

Sorbonne Université, CNRS, Inserm, Développement Adaptation et Vieillissement, Dev2A, F-75005 Paris, France

## Acknowledgements

Special thanks to the animal facility of the Institute de Biologie Paris Seine, IBPS, for taking care of animals.

## Declaration of Interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Corresponding author address

Hélène Hardin-Pouzet, Sorbonne Université, CNRS, Inserm, Développement Adaptation et Vieillissement, Dev2A, F-75005 Paris, France

## Corresponding author email address

helene.pouzet@sorbonne-universite.fr

## Keywords

endocrine disruptors, phthalates, obesity, exposome, reproduction, male sexual behavior, hypothalamus

## Funding sources

This work was supported by Anses (ReprObesED – ANSES-22-EST-024), CNRS, INSERM and Sorbonne University.

## Highlights

* Phthalates exposure combined with HFHF diet strongly alters male sexual behavior.

* Phthalates exposure combined with HFHF diet alters the hypothalamic neural network.

* Public health must consider the specific vulnerability of male sexual behavior.
**Abstract**

The decrease in male fertility observed since the beginning of the 20th century in Western countries could be linked to changes in diets and exposure to molecules from industry, such as endocrine disruptors and, among them, phthalates. The combined effects of a high fat/high fructose diet (HFHF) and exposure to an environmental mixture of phthalates (mix) were evaluated on male murine sexual behavior. Four groups of adult male C57Bl/6J mice were exposed to a control diet (CD), a control diet coupled with the mix (CDmix), an HFHF diet, and an HFHF diet coupled with the mix (HFHFmix). Both HFHF diets induced metabolic alterations but only the HFHFmix diet altered the efficiency of sexual behavior, with a dramatic decrease in the number of animals reaching ejaculation and a subsequent increase in mating latency. This was associated with a decrease in testicular, seminal glands and Tyson glands weights and a loss in discrimination toward female counterparts in an olfactory preference test. No effects were recorded on locomotion activity. In the HFHFmix group, the protein amounts of synaptophysin, dopamine transporter (DAT), and glutamate receptor AMPA GluR1, known as modulatory pathways of sexual behavior, were modified in the hypothalamic medial preoptic area, the main integrating center in the control of this behavior. The additional effect of an HFHF diet and phthalates, at doses relevant for human exposure, on male sexual behavior, could be thus explained by an alteration in this neural network, revealing a specific vulnerability that requires special attention in term of public health recommendations.
<figure><img src="image_2.png" /><figcaption>→ worsened impairment of sexual behavior by the combination HFHFmix</figcaption></figure>
# 1. Introduction

The agricultural and industrial revolutions of the 20th century have changed Western eating habits (ANSES, 2017), as well as the environment of populations, especially exposure to chemical pollutants. Alongside these changes, an increase in the body weight (bw) of individuals, the incidence of metabolic diseases, and an alteration of reproductive health have been observed (“World Health Organization (WHO),” n.d.).

Among chemical pollutants, endocrine disruptors (EDs) require special attention because they can affect both the reproductive function and metabolic status of individuals (Encarnação et al., 2019). Phthalates constitute one ED subclass to which humans are most exposed, through plastic food packaging, paints, plastic toys, dust, and drinking water (Hahladakis et al., 2018). Indeed, di-(2-ethylexyl) phthalate (DEHP), dibutyl-phthalate (DBP), benzyl-butyl-phthalate (BBP), di-isobutyl Phthalate (DiBP), and di-ethyl phthalate (DEP) are abundantly detected in the environment. DEHP, DBP, and DiBP were classified as EDs by the European Chemical Agency (ECHA), and DEP is on the ED assessment list. A tolerable daily intake (TDI) dose of 50µg/kg of bw/day was established by the European Agency for Food Safety for DEHP, DBP, and BBP based on their ability to decrease fetal testosterone production (EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) et al., 2019). Several studies have confirmed the effect of phthalates on testosterone levels (Liu et al., 2024) and extended their effect on fertility via sperm alterations (Mondal et al., 2019). Our previous work has also identified that chronic adult exposure of male mice to DEHP alone at the TDI dose or at an environmental dose (5µg/kg/d) or in a phthalate mixture mimicking environmental exposure induced alterations to sexual behavior in male mice (Dombret et al., 2017; Ducroq et al., 2023). In addition to their reproductive effects, exposure to phthalates has also been shown to have metabolic effects: they contribute, both in humans and in animal models, to weight gain, insulin resistance, and diabetes (Heindel and Blumberg, 2019; Kahn et al., 2020; Patrick, 2020; Sharma et al., 2020) by interfering with adipocyte biology and glucose metabolism.

The Western lifestyle is characterized, in part, by the prevalence of obesity, which has tripled since 1975 due to a diet enriched in lipids and carbohydrates, often processed industrially (“World Health Organization (WHO)”). Obesity is responsible for an increase in inflammation and oxidative stress and is known to affect several physiological systems, including those involved in fertility (Cardoso et al., 2017; White et al., 2009). Several studies have reported the pathogenicity of obesity on the reproductive system. A decrease in testosterone circulating levels (Giagulli et al., 1994) and an increase in sperm disorders (Du Plessis et al., 2010; Mascarenhas et al., 2024; Swanson et al., 2019) were shown for obese men, and an impaired hypothalamic-pituitary-ovarian axis (Jungheim and Moley, 2010) has been documented for obese women. Studies on mice fed a high-fat diet have also demonstrated an alteration to male sexual behavior, with a decrease in the number of mounts and intromissions and an increase in the latency to first mount (Fernandez et al., 2011; Kacar et al., 2023; Takase et al., 2016; Yardimci et al., 2022).

Changes in lifestyle habits in Western countries are characterized, in particular, by calorie-enriched meals and exposure to industrial products and processes that are sources of exposure to ED (Heudorf et al., 2007; Lyche et al., 2009). Metabolic dysregulations and endocrine disruption share a community of cellular and molecular pathways that could explain the reproductive alterations described since the mid-20th century (Chen et al., 2009; Haverinen et al., 2021; Lubrano et al., 2013). This point has been
mainly investigated in terms of prenatal exposure (Barakat et al., 2024; de Jesus et al., 2015; Fan et al., 2022; Kannan et al., 2021; Kougias et al., 2018; Mirihagalle et al., 2019; Zhai et al., 2020) but the combined effect of adult exposure to phthalates and obesity on reproductive function still needs to be explored.

In this work, we assumed that the combination of a diet high in fat and sugar and environmental exposure to phthalates produces a greater effect than each treatment taken separately. To test this hypothesis, adult male mice were fed an obesogenic-diabetogenic diet (a high-fat, high-fructose diet [HFHF]) (Le Gléau et al., 2021; Panchal et al., 2011) combined with a phthalate mixture (mix) representative of environmental exposure and pertinent to replicating human exposure to phthalates, as previously described (Adam et al., 2021; Dewalque et al., 2014; Ducroq et al., 2023; Martine et al., 2013). Four groups of mice were formed: males fed with (1) a control diet (CD), (2) a control diet combined with the phthalate mixture (CDmix), (3) a high-fat, high-fructose diet (HFHF), or (4) a high-fat, high-fructose diet combined to the phthalate mixture (HFHFmix). Body weight, oral glucose tolerance, and insulin sensitivity were monitored throughout the study. From 12 weeks of treatment, the time required for metabolic deregulation to set in (Le Gléau et al., 2021; Panchal et al., 2011), sexual behavior, an essential component of reproductive function and thus of the survival of the species, was analyzed as well as olfactory preference for receptive females, and locomotor activity. In mice, the activation of sexual behavior is a complex process involving sex steroid hormones and various neurobiological and genetic mechanisms, where the medial preoptic area of the hypothalamus (mPOA) acts as an integrative center, connecting the structures involved in the perception of a receptive female (accessory and main olfactory circuit), the reward circuit, via the amygdala, and the executive areas of motor behavior (spinal cord) (for review see Jean et al., 2021). The amounts of different proteins, which are known to be involved in its plasticity, were evaluated in the mPOA.

# 2. Material and Methods

## 2.1 Animals and treatment

The experiments were conducted in compliance with French and European legal requirements (Decree 2010/63/UE) and received approval from the local Ethical Committee and the French Ministry of Research (APAFIS#27976-2020102606318963_v4). The experimental procedures were described following the Animal Research: Reporting in Vivo Experiments (ARRIVE) guidelines. Eight-week-old C57BL/6J mice (Janvier Labs) were used in this study and housed in nest-enriched polysulfone cages with polysulfone bottles. Mice were kept at 22 °C under an inverted light schedule: i.e., the dark period began at 1:00 P.M. with a 12:12 h light–dark cycle.

Four groups of mice were fed ad libitum with one of four diets: (1) a control chow diet (the CD group), (2) a chow diet combined with a phthalate mixture (the CDmix group), (3) a high-fat, high-fructose diet (the HFHF group), and (4) a high-fat, high-fructose diet combined with a phthalate mixture (HFHFmix) throughout the 17 weeks of the study. The chow diet was an A03-10 Safe Diet containing 5% kcal fat. The HFHF diet was a D12492 Research Diet with 60% kcal fat associated with 30% fructose (Sigma Aldrich) in drinking water (Le Gléau et al., 2021; Panchal et al., 2011).

Exposure to the phthalate mixture was performed as previously described (Adam et al., 2021; Ducroq et al., 2023). The composition of the phthalate mixture (DEHP (CAS 117-81-7) at 5 µg/kg/d, DBP (CAS
84-74-2) at 0.5 µg/kg/d, BBP (CAS 85-68-7) at 0.5 µg/kg/d, DiBP (CAS 84-69-5) at 0.5 µg/kg/d, and DEP (CAS 84-66-2) at 0.25 µg/kg/d; Sigma Aldrich) was based on French and European studies that demonstrated external co-exposure to these compounds (Martine et al., 2013) and detected their metabolites in urinary samples (Anses, 2015; Dewalque et al., 2014). The ratio of DEHP to the other phthalates was determined based on the estimated daily intake in France and Europe (Dewalque et al., 2014; Martine et al., 2013). Phthalates were dissolved in ethanol and water (1% and 40% of prepared food, respectively), mixed by the experimenter into powdered food, and subsequently reformed into pellets. For the CD and HFHF groups, similar volumes of vehicles (water and ethanol) were included in the pellets. Mice were weighed weekly, and phthalate doses were adjusted to their body weight and calculated for a daily food intake of 6 g per animal.

Experiments were conducted on two cohorts of male mice (n=95), with each cohort including the four treated groups. Both cohorts were subjected to metabolic tests at week 0 (W0), W7, W12, and W16. Behavioral analyses were conducted after 12 weeks of exposure on 23 males from the CD group, 25 males from the CDmix group, 25 males from the HFHF group, and 22 males from the HFHFmix group. At the end of the behavioral analyses (W17), the males were sacrificed by decapitation, their blood was collected, and the weights of their reproductive organs were measured. Brains were processed for western blot analyses (see timeline in Figure S1).

## 2.2 Body and organs weight measurements

The body weights of the male mice in the two cohorts were monitored weekly. Testis, seminal vesicles, and Tyson glands were collected at the end of the study. Weights were expressed as absolute body weights and absolute and relative organ weights.

## 2.3 Circulating hormone levels

Blood was collected and stored at RT for 2 h. Samples were centrifugated 15 min at 3,000 RPM, and serum was stored at -80 °C. Circulating levels of leptin were measured using Elisa Multiplex (Tebubio). Data were normalized upon positive control and expressed as relative fluorescence intensity.

## 2.4 Metabolic experiments

2.4.1 **Oral Glucose Tolerance Test (OGTT).** Glucose tolerance was tested after a 12 h fasting period. Mice were orally given (via gavage) 2 g/kg bw of glucose. Glycemia (mg/dL) was measured from a drop of blood at the tail using a glucometer (AccuCheck Performa, Roche) at 0 (basal glucose level), 10, 30, 60, 90, and 120 min after the glucose administration. OGTT was performed before the beginning of the treatments (W0) and at 7, 12, and 16 weeks of treatment.

2.4.2 **Insulin Tolerance Test (ITT).** Insulin sensitivity was tested after a 3 h fasting period. Mice were administered with insulin (0.5 UI/kg bw) via intraperitoneal injection. Glycemia (mg/dL) was measured from a drop of blood taken at the tail using a glucometer (AccuCheck Performa, Roche) at 0 (basal glucose level), 10, 15, 30, 45, 60, 90, and 120 min after the insulin injection. ITT was performed at W7, W12, and W16.
## 2.5 Behavioral tests

Tests were conducted under red light 2 h after lights-off (Jean et al., 2017a) and recorded for later analysis using LabWatcher (ViewPoint), as previously described (Adam et al., 2021; Capela et al., 2018; Ducroq et al., 2023; Jean et al., 2017a). The mazes and equipment used for mating, the olfactory preference test, and locomotor activity were cleaned with 10% ethanol after use by each animal.

**2.5.1 Preparation of sexually receptive females.** C57BL/6J female mice were ovariectomized under general anesthesia (Isoflurane 4%, Isovet, Osalia) and implanted with SILASTIC implants (Dow Corning) containing 50 µg of estradiol-benzoate (Sigma Aldrich) in 30 µL of sesame oil. Two weeks post-surgery, and 5 h prior to their first sexual experience and before each behavioral test where females were used as stimuli, females received a subcutaneous injection of 1 mg progesterone (Sigma Aldrich) in 100 µL of sesame oil. Female receptivity was confirmed using sexually experienced males before the experiments.

**2.5.2 Mating behavior.** Two weeks prior to the first mating (Test 1), at week 11, males were housed individually. At W13, they were then paired with sexually receptive females for 10 h and allowed to reach ejaculation. This initial mating aimed to enhance their olfactory preference toward receptive females, USV emission, and mating behaviors (Dombret et al., 2017; Jean et al., 2017a; Picot et al., 2014). Two weeks later (i.e., W15 of the treatment), males were subjected to a second mating experience with an unknown receptive female (Test 2). The percentage of males doing at least one mount, doing at least one intromission, and reaching ejaculation, along with mating duration and the latency to the first mount, first intromission, and ejaculation, were scored. In the absence of sexual behavior, latencies were set at 600 min, corresponding to the total duration of the recording.

**2.5.3 Olfactory preference.** At W14, olfactory preference was evaluated using an enclosed plexiglass Y-maze, as previously described (Adam et al., 2021; Capela et al., 2018; Dombret et al., 2017; Picot et al., 2014). Male mice were acclimated to the maze, which contained two empty perforated goal boxes at each end, for 10 min over two consecutive days. On the day of the test, males were given the choice between a sexually receptive female and a gonadally intact male, each of which was placed in one of the goal boxes. Stimuli were anesthetized (ketamine 100 mg/kg/xylasine 10 mg/kg IP) to prevent social interaction. The exposed males could not directly access the stimuli, but the perforated walls of the goal boxes allowed air to flow into the maze. The total time spent in chemo-investigation and the number of entries into each arm of the maze were scored during the 10 min test.

**2.5.4 Locomotor activity.** The circular corridor used to measure activity consisted of two concentric cylinders intersected by four diametrically opposite infrared beams (Dombret et al., 2017; Jean et al., 2017b). Locomotor activity was recorded when animals interrupted two successive beams, indicating that they had traveled a quarter of the circular corridor. Activity was monitored for 120 min and expressed as the cumulative activity over the entire 120 min test (as the number of crossed beams/120 min).
**2.6 Protein analysis by Western Blot**

Brains were collected at W17 and sliced into coronal sections of 500 µm thickness using a Vibroslice (World Precision Instrument) in cold 0.1 M phosphate buffer. The mPOA of the hypothalamus was punched out with 1 mm diameter canula using anterior commissure as a landmark. The punched samples were homogenized in 50 mM Tris (pH 7.2), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 10 mM EDTA, 10 mM EGTA, 1% Triton-X100, and 1% protease inhibitor (Roche). Proteins were quantified (Bradford Assay; Thermo Fisher), denatured in Laemmli buffer, separated (5 µg) on a 4–12% Bis-Tris NuPAGE (Thermo Fisher), and transferred onto a PVDF membrane (Millipore).

Blots were probed overnight at RT with primary antibodies against ERK, AR, ERα, Synaptophysin, AMPA GluR1, DAT, GFAP, Iba1, spinophilin, and PSD 95 (Table S2). Then, incubation with peroxidase-conjugated secondary antibodies diluted at 1:2500 (Table S2) was performed at RT for 2 h. Signals were detected with the Super Signal West Pico or Femto detection kits (Thermo Fisher), visualized using Amersham Imager 600 (GE Healthcare Bio-Sciences AB), quantified with Fiji ImageJ software (National Institutes of Health), and normalized with ERK42 (Jean et al., 2017b).

**2.7 Statistics**

Data were expressed as means ± SEM, except for those reporting the percentage of males exhibiting at least one mount, one intromission, or ejaculation, and analyzed by GraphPad Prism 10 (GraphPad Software). Normality test (Shapiro–Wilks test) was checked and Bartlett's test showed homoscedasticity. For body weight, OGTT and ITT curves, since the data presented no missing values, a repeated measure two-way ANOVA, sphericity was tested and the Geisser-Greenhouse epsilon was applied if necessary. Sources of variation were investigated: time x column factor, time alone, column factor alone, subject. Finally, the different group means were tested using a Tukey test. For AUC of OGTT and ITT, either a one-way ANOVA, followed by Tukey's post-hoc test (parametric data) or a Kruskal-Wallis followed by a Dunn's test (non-parametric data) was used to determine group differences. The number of entries in the stimulus compartment and the time spent in investigation for olfactory preference test, and the number of events in the locomotor test were analyzed with a two-way ANOVA and a Tukey's test. Chi-square was used to analyze the percentage of individuals reaching ejaculation and doing at least one mount and one intromission. Kruskal-Wallis test followed by Dunn's test was performed to analyze circulating leptin levels, absolute and relative organ weights, mating duration, latencies of first mount, first intromission, and ejaculation, total time in investigation, cumulative locomotor activity and quantitative protein amounts in western blots. P-values of less than 0.05 were considered significant.

**3. Results**

**3.1 Effects of the combined HFHF diet and phthalate mixture exposure on the metabolic profile of male mice**

Mice were weighed weekly, and all groups had similar body weights (bw) at the start of the treatment (W0; Figure 1A). The HFHF and HFHFmix diets increased bw in a significant matter starting at W2 compared to the CD group (CD vs. HFHF: P=0.0006; CD vs. HFHFmix: P=0.002) and the CDmix group
(CDmix vs. HFHF: P=0.001; CDmix vs. HFHFmix: P=0.006). This increase in bw in the HFHF and HFHFmix groups persisted until the end of the treatment (45.05 ± 0.58 g and 43.87 ± 0.58 g for HFHF and HFHFmix, respectively, at W16 vs. 29.93 ± 0.54 g and 29.58 ± 0.58 g for CD and CDmix, respectively, at W16). The effect of time x treatment was found significant ($F_{(48, 1456)} = 60.19, P < 0.0001$). The addition of the mix of phthalates to either diet had no effect on bw throughout the 16 weeks of exposure.

Baseline glycemia was comparable across groups at W0 (P=0.5; Figure 1B). At W16, the HFHF and HFHFmix groups showed a fasting hyperglycemia, a significant decrease in glucose tolerance (Figure 1C, D), and a decrease in insulin sensitivity compared to the CD and CDmix groups (Figures 1E, 1F). The difference was already present at W7 and was accentuated throughout the treatment (W12, Figure S3).

Serum was collected after the sacrifice (W17), and leptin dosage was performed. The HFHF and HFHFmix diets induced a significant sevenfold increase in the circulating leptin levels compared to the CD and CDmix groups (P<0.0001; Figure 1G).

To summarize, both the HFHF and HFHFmix treatments induced an altered metabolic status from W7 characterized by overweight, fasting hyperglycemia, reduced glucose tolerance, and insulin sensitivity, which are reminiscent of an obesogenic-diabetogenic profile. Co-exposure with phthalates did not show an aggravation of this metabolic status.

## 3.2 Effects of the combined HFHF diet and phthalate mixture exposure on male sexual behavior

Male mice with prior sexual experience were evaluated for their mating behavior with receptive females. The whole behavioral sequence was analyzed during the 10 h test.

Figure 2A indicates that most CD (96%; 22/23) and CDmix (96%; 22/23) males exhibited at least one mount. This rate tended to be reduced for the HFHF group (80%; 20/25) and was significantly decreased in the HFHFmix group (73%; 16/22) compared to the CD (P=0.03) and CDmix (P=0.03) groups. The CD (91%; 21/23) and CDmix (87%; 20/23) groups had also similar percentages of individuals performing at least one intromission (Figure 2B). By contrast, the percentage of males exhibiting one intromission was decreased for the HFHF (60%; 15/25) and HFHFmix groups (41%; 9/22) compared to the CD (CD vs. HFHF: P=0.01; CD vs. HFHFmix: P=0.0003) and CDmix (CDmix vs. HFHF: P=0.03; CDmix vs. HFHFmix: P=0.001; Figure 2B) groups. Concerning the number of males reaching ejaculation, no significant difference was found between the CD (83%; 19/23) and CDmix (57%; 13/23) groups (P=0.054; Figure 2C). The number of HFHF males reaching ejaculation was significantly decreased (28%; 7/25) compared to the CD (P=0.0001) and CDmix (P=0.04) groups. This proportion was dramatically reduced for the HFHFmix group, where almost no males reached ejaculation (5%; 1/22) compared to the CD (P<0.0001), CDmix (P=0.0002), and HFHF (P=0.03) groups.

To clarify the characteristics of the behavioral alteration, the different mating phases corresponding to motivation and copulation were analyzed. The mating duration was prolonged in the CDmix (+257%; P=0.03), HFHF (+411%; P<0.0001), and HFHFmix (+585%; P<0.0001) groups compared to the CD group (Figure 2D). Although it was not statistically significant, a tendency to increase mating duration was observed between the HFHF and HFHFmix groups (P=0.06). The latency to first mounting the female was significantly increased in the HFHF (+297%; P=0.02) and HFHFmix (+332%; P=0.02) groups
compared to the CD group (P=0.02 vs. CD for both groups; Figure 2E). The latency to first intromission was also significantly increased in the HFHFmix group compared to the CD (+422%; P=0.0002) and CDmix (+258%; P=0.005) groups (Figure 2F). For ejaculation latency, a tendency to increase, although not statistically significant, was observed between the CD and CDmix groups (P=0.056; Figure 2G). By contrast, ejaculation latency was increased in the HFHF group compared to the CD group (+284%; P=0.0009) and in the HFHFmix group compared to the CD (+370%; P<0.0001) and CDmix groups (+189%; P=0.0007), consistent with the drastic reduction in males having a complete sexual behavior.

In summary, these results suggest an effect of the HFHF diet on both the motivational and copulatory phases, and these effects were aggravated with the co-exposure, leading to the almost complete impairment of sexual behavior.

### 3.3 Effects of combined HFHF diet and phthalate mixture exposure on olfactory preference for receptive females

During the motivational phase of sexual behavior, males and females emit pheromones that stimulate the sexual partner, which exhibits an olfactory preference towards the opposite sex. In the olfactory preference test, treated males were given choice between an anesthetized receptive female and an anesthetized gonadally intact male in a Y-maze paradigm (Figure 3A). The four groups exhibited similar total time spent in investigation (P=0.552; Table 1) and a similar number of entries into the stimulus compartments (P=0.761; Table 1). Males from the CD (P=0.015), CDmix (P<0.0001) and HFHF (P=0.001) groups all displayed a preference for females over males (Figure 3B). By contrast, males exposed to the combination HFHFmix exhibited no significative preference toward females (P=0.158).

### 3.4 Effects of combined HFHF diet and phthalate mixture exposure on locomotor activity

A possible explanation for impaired sexual behavior could be related to male difficulties pursuing and mounting the female due to their overweight. Locomotor activity was thus evaluated in a circular corridor. No effect of the treatments was observed on the activity measured during the 120 min test (P=0.1; Figure S4) or on the cumulative activity (P=0.4; Figure S4), thereby excluding this hypothesis.

### 3.5 Effects of combined HFHF diet and phthalate mixture exposure on reproductive organs

Androgen-dependent tissues were weighed at the end of the experiment (W17). The absolute and relative weights of the testis, Tyson glands, and seminal vesicles were compared among the four groups. The absolute weight of testis and Tyson glands were unchanged (Table 2). Absolute seminal vesicle weights, however, were significantly increased in the HFHFmix group compared to the CD (p=0.001) and CDmix (p=0.03) groups. The seminal vesicle weights of the HFHF group were also increased compared to the CD group (p=0.003).

By contrast, when organ weights were calculated as a percentage of corresponding bw, reduced relative weights of testis, Tyson glands and seminal vesicles were observed for the HFHF and HFHFmix groups compared to the CD and CDmix groups, reflecting the effect of the HFHF and HFHFmix diets on bw.
## 3.6 Effects of combined HFHF diet and phthalate mixture exposure on protein markers in the medial preoptic area of the hypothalamus (mPOA)

The preoptic area of the hypothalamus, the mPOA, is the main integrating center of the neural circuit of male sexual behavior. To decipher the underlying mechanisms of the behavioral impairment, the amounts of several proteic markers, already known to be involved in its plasticity (Jean et al., 2021), were analyzed.

The levels of AR and ERα, the receptors for sex hormones, were analyzed, considering their importance in the expression of male sexual behavior (Raskin et al., 2009; Trouillet et al., 2022). Western blot quantification showed a significant decrease in the AR protein amount (p=0.02) for the CDmix (-48.8%), HFHF (-48.7%), and HFHFmix (-48.1%) groups compared to the CD control group (Figure 4A). The ERα level remained similar for all groups (P=0.7; Figure 4B).

Proteins involved in the cellular, structural, and functional arrangement of the mPOA were then analyzed since a plasticity of this neural network has been demonstrated in response to individual living conditions. The protein levels of the glial markers GFAP and IBA1 remained unchanged (Figure S5 A, B), suggesting that the treatments rather affect neurons. The neuronal pre-synaptic marker synaptophysin protein level was significantly increased in the CDmix (+189%; p=0.02) and HFHFmix (+213%; p=0.03) groups but not in the HFHF group (P=0.07) compared to the CD group (Figure 5A). No effect of the treatments was observed in the level of the post-synaptic proteins PSD95 or spinophilin (Figure S5 C, D). By contrast, the level of the dopamine transporter (DAT), involved in dopaminergic signaling, which facilitates the appetitive aspects of male sexual behavior, was significantly decreased in the mPOA of male HFHFmix mice compared to the control CD group (-66%; p=0.03; Figure 5B). In addition, the protein level of the AMPA GluR1 receptor was significantly decreased in the mPOA of the HFHFmix groups compared to the CD group (-60%; P=0.008; Figure 5C).

## 4. Discussion

The aim of this study was to analyze the effect of a combined obesogenic-diabetogenic diet and endocrine-disruptor exposure, as a model of environmental contamination and a Western lifestyle, on reproductive behavior. To our knowledge, this study is the first to demonstrate that adult male mice fed with an HFHF diet and exposed to a mixture of phthalates at environmentally relevant doses experienced a drastic impairment of their sexual behavior, with a clear worsening effect when both factors were combined. These behavioral modifications were associated with structural and molecular changes in the medial preoptic area of the hypothalamus (mPOA), the main integrative area for the central control of sexual behavior in male rodents.

The HFHF and HFHFmix diets were composed of 60% kcal fat associated with 30% fructose, while the CD diet contained only 5% kcal fat. High fructose was preferred to high sucrose because it induced a more rapid obesogenic-diabetogenic effect (Dornas et al., 2015; Pereira et al., 2017; Toop and Gentili, 2016). From the second week of exposure, the HFHF and HFHFmix diets induced rapid weight gain (+108% for HFHF and +107% for HFHFmix by the end of the experiment). From the seventh week, the animals presented with fasting hyperglycemia, reduced glucose tolerance, higher resistance during an
insulin test, and, at W17, an elevated level of circulating leptin. All these elements were reminiscent of an obesogenic-diabetogenic profile, as previously described by Le Gléau et al. (2021) and Panchal et al. (2011). The addition of phthalate mixture to the diet, whether CD or HFHF, did not lead to metabolic aggravations. This finding confirms our previous results reporting no differences in the body weights of mice exposed to the same mixture of phthalates compared to the control group (Ducroq et al., 2023). In this study, we extended this analysis with the metabolic profile of mice subjected to the phthalate mixture and showed that phthalates did not alter metabolism. Finally, the comparison of the results obtained among the different groups suggests that the observed metabolic alterations are mainly due to the HFHF diet.

The metabolic results contrast with those obtained by examining the sexual behavior of male mice exposed to the combination of the HFHF diet and phthalate-mixture exposure. Indeed, even if either the HFHF diet or exposure to phthalates did separately alter the sexual performance of males (Ducroq et al., 2023; Yardimci et al., 2022), the combination of the two induced a dramatic effect, with only one male out of 22 having a complete set of sexual behavior resulting in ejaculation.

The sexual impairment of the HFHFmix males was characterized not only by an alteration of the copulatory phase, since they did not reach ejaculation, but also by a disturbance of the motivational phase, with a decreased latency in the first mount, suggesting that HFHFmix mice tried less to mount the females during the 10 h mating period. This hypothesis was confirmed with the results of the olfactory preference test, showing that HFHFmix mice had lost their preference for receptive female mice without an alteration of their interest in social interaction, suggesting a reduction in their ability to discriminate between the sensory signals emitted by males and females.

Locomotor activity was not affected by either the exposure to the phthalate mixture (as previously shown by Ducroq et al. (2023) or the HFHF diet, or to the combination of both, as demonstrated by the results obtained with the circular corridor test. In addition, mice in the HFHFmix group had weights similar to those of the HFHF mice but a much greater alteration of their sexual behavior. It is therefore unlikely that a reduction in the male ability to mount females explains their observed altered sexual behavior.

In mice, the activation of sexual behavior is tightly regulated by sex steroid hormones. We thus analyzed androgen-dependent tissues as an index of steroid hormone production and sensitivity (Chai, 1956; Chapin and Creasy, 2012). Exposure to the environmental mix of phthalates had no effect on the relative weight of testis or androgen-sensitive tissue (i.e., Tyson gland, seminal vesicles), as described by Dombret et al. (2017). The HFHF and HFHFmix groups demonstrated reduced relative weights in their Tyson glands and seminal vesicles compared to those in mice in the CD and CDmix groups, which could contribute to their decreased sexual performance. As the magnitude of this reduction was comparable between the HFHF and HFHFmix groups, however, this is not sufficient to explain the drastic effect of the combination observed in the alteration of HFHFmix sexual behavior.

Therefore, the data from locomotion and peripheral organs lead us to seek the origin of the profound alteration of male sexual behavior in the central component of the control of reproduction. The expression of reproductive behavior in male mice results in the activation of a set of central structures interacting with each other and whose integrative center is constituted by the hypothalamic medial preoptic area (mPOA). The functionality of the mPOA neural circuit relies on its structural organization and the interactions among several signaling pathways, under the control of sex steroid hormones,
dopamine, and glutamate (for review, see Jean et al., 2021). To further understand the underlying modifications resulting from HFHFmix exposure, we analyzed different proteic markers as an index of the cellular and molecular neural network in the mPOA.

Androgen receptor (AR) and estrogen receptor α (ERα) are the two main sex steroid hormone receptors present in the male mPOA (Mhaouty-Kodja, 2018; Torres et al., 2024). ERα protein levels were not affected by the different treatments, confirming that it is not the main signaling pathway triggering male sexual behavior (Trouillet et al., 2022). By contrast, AR levels were reduced in the CDmix group, confirming the anti-androgenic effect of phthalates and consistent with our previous results obtained on male mice exposed to DEHP alone or in a phthalate mixture (Capela et al., 2018; Dombret et al., 2017; Ducroq et al., 2023). AR levels were also reduced in the HFHF and HFHFmix groups, with a similar amplitude as in the CDmix group. These results indicate for the first time that an HFHF diet affects the AR signaling pathway in the mPOA. These will be confirmed in further studies by measuring mRNA expression levels or by electrophysiological recordings of mPOA neurons. There is, however, no cumulative effect of the combined HFHF and phthalate-mixture exposure. Thus, the reduction in AR protein levels alone is not sufficient to explain the dramatic effect on sexual performance of the HFHFmix group.

The levels of several proteins organizing the structure of the mPOA neuronal network were then analyzed. Plasticity in the mPOA has been shown to involve both the neurons and the glial cells (Ahmadpour et al., 2021; Jean et al., 2017a). In our case, glial cells seemed not to be targeted by the treatments because the levels of GFAP, an astrocytic marker, and Iba1, specific to microglia, were not modified. We then looked at pre- and post-synaptic structural proteins, and functional neuronal markers related to neurochemical pathways. Neither the levels of spinophilin nor those of PSD95, which are involved in the plasticity of the mPOA network (Jean et al., 2017a), were affected. By contrast, the level of synaptophysin, a membrane protein of presynaptic vesicles, was increased in the CDmix and HFHFmix groups but not in the HFHF group. These data on PDS95, synaptophysin, and spinophilin are consistent with previous results obtained on the mPOA of male mice exposed to DEHP alone or in a mixture (Ducroq et al., 2023). Synaptophysin is a synaptic vesicle protein crucial for neurotransmitter release. Decrease of its expression has been reported in different models of neuronal plasticity impairment, such as neuroinflammation (Chugh et al., 2013; Liu et al., 2018), ageing (Head et al., 2009; Liu et al., 2010), or Terahertz waves stimulation (Song et al., 2025). Alternatively, synaptophysin knockout mutant mice demonstrate increased efficiency in synaptic transmission (Raja et al., 2019). Thus, the increase observed in the phthalate-exposed groups may indicate lower efficiency in the synaptic transmission of the mPOA neuronal network. Again, however, this cannot alone explain the alteration in sexual behavior exhibited by the HFHFmix group only.

From a functional point of view, the mPOA neuronal network is specifically affected by co-exposure, as demonstrated by the decrease in the levels of the dopamine transporter protein (DAT) and the AMPA receptor GluR1 only in the HFHFmix group but not in the CD, CDmix, or HFHF groups. These changes could explain, at least in part, the major impairment of sexual behavior. Indeed, the main neurotransmitters involved in mPOA activity are dopamine and glutamate. Dopamine facilitates copulation and genital responses in rats (Hull et al., 1995), regulating the motivational and copulatory components of sexual behavior (Simerly et al., 1986). In DAT-KO mutants, sexual performance was decreased (Sanna et al., 2020), thus suggesting that the reduced DAT protein levels may contribute to the impairment of the HFHFmix males' sexual performance. Glutamate also stimulates sexual behavior
(Drago and Busă, 1997), notably via AMPA receptors since the administration of an AMPA GluR1 receptor antagonist alters sexual behavior (Powell et al., 2003). The decrease in AMPA GluR1 levels in the HFHFmix could therefore be another component explaining their reduced sexual behavior.

## 5. Conclusion

In conclusion, the HFHF diet combined with an environmentally relevant mixture of phthalates induces an enhanced alteration in male sexual behavior, almost eliminating it entirely. This combination aggravates the effects of each of the treatments done separately. The HFHF diet induced a decrease in testicular, seminal glands and Tyson glands relative weights suggesting a probable decrease in testosterone sensitivity, and a decrease in AR levels in the mPOA. Phthalate exposure also affected mPOA AR and synaptophysin levels. Finally, co-exposure induced a decrease in AMPA GluR1, and DAT levels, which were not affected when each treatment was done separately. Since these specific protein changes occur only in combined exposure, this suggests molecular interactions between their signaling pathways that could explain the results observed in HFHFmix males. The accumulation of changes in AR, synaptophysin, DAT, and AMPA GluR1 levels could lead to a drastic decrease in the functionality of the mPOA neuronal network, which could explain the behavioral changes specifically observed in the HFHFmix co-exposed group. Further functional studies restoring neural network integrity may clarify the underlying mechanisms.

The observed strong alterations in sexual behavior by co-exposure must be confronted with the similar metabolic effects in the HFHF and HFHFmix groups; this highlights a specific vulnerability of the central circuit controlling sexual behavior and therefore reproduction. Since the neuroendocrine control of male reproductive processes is conserved across mammals, this point should attract particular attention for studies of the effects of the exposome on the reproductive health of Western societies. It could suggest recommendations for nutritional prevention not only in terms of food safety and packaging toxicity but also in terms of changes in consumer behavior.

This work also draws attention to the harm caused by adult exposure to endocrine disruptors, here phthalates, at doses comparable to environmental contamination but well below the reference doses ("Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) related to Bis(2-ethylhexyl)phthalate (DEHP) for use in food contact materials | EFSA," 2005; US EPA). Consideration may, therefore, be needed of the re-evaluation of these reference doses in terms of population protection.
References

Adam, N., Brusamonti, L., Mhaouty-Kodja, S., 2021. Exposure of Adult Female Mice to Low Doses of di(2-ethylhexyl) Phthalate Alone or in an Environmental Phthalate Mixture: Evaluation of Reproductive Behavior and Underlying Neural Mechanisms. Environ. Health Perspect. 129, 17008. https://doi.org/10.1289/EHP7662

Ahmadpour, D., Mhaouty-Kodja, S., Grange-Messent, V., 2021. Effects and underlying cellular pathway involved in the impairment of the neurovascular unit following exposure of adult male mice to low doses of di(2-ethylhexyl) phthalate alone or in an environmental phthalate mixture. Environ. Res. 112235. https://doi.org/10.1016/j.envres.2021.112235

Anses, 2015. Connaissances relatives à la réglementation, à l'identification, aux propriétés chimiques, à la production et aux usages des composés de la famille des Phtalates (Tome 3).

ANSES, A.N. de S.S. de l'Alimentation, de l'Environnement et du Travail, 2017. Etude individuelle nationale des consommations alimentaires (INCA3), Rapport d'expertise collective.

Barakat, R., Lin, P.-C.P., Bunnell, M., Oh, J.-E., Rattan, S., Arnieri, C., Flaws, J.A., Ko, C.J., 2024. Prenatal exposure to Di(2-ethylhexyl) phthalate and high-fat diet synergistically disrupts gonadal function in male mice†. Biol. Reprod. 110, 1025-1037. https://doi.org/10.1093/biolre/ioae029

Capela, D., Dombret, C., Poissenot, K., Poignant, M., Malbert-Colas, A., Franceschini, I., Keller, M., Mhaouty-Kodja, S., 2018. Adult male mice exposure to nonylphenol alters courtship vocalizations and mating. Sci. Rep. 8, 2988. https://doi.org/10.1038/s41598-018-21245-9

Cardoso, A.M., Alves, M.G., Mathur, P.P., Oliveira, P.F., Cavaco, J.E., Rato, L., 2017. Obesogens and male fertility. Obes. Rev. Off. J. Int. Assoc. Study Obes. 18, 109-125. https://doi.org/10.1111/obr.12469

Chai, C.K., 1956. Seminal vesicle growth as a function of androgen stimulation in mice. Am. J. Physiol. 186, 463-467. https://doi.org/10.1152/ajplegacy.1956.186.3.463

Chapin, R.E., Creasy, D.M., 2012. Assessment of circulating hormones in regulatory toxicity studies II. Male reproductive hormones. Toxicol. Pathol. 40, 1063-1078. https://doi.org/10.1177/0192623312443321

Chen, J.-Q., Brown, T.R., Russo, J., 2009. Regulation of energy metabolism pathways by estrogens and estrogenic chemicals and potential implications in obesity associated with increased exposure to endocrine disruptors. Biochim. Biophys. Acta 1793, 1128-1143. https://doi.org/10.1016/j.bbamcr.2009.03.009

Chugh, D., Nilsson, P., Afjei, S.-A., Bakochi, A., Ekdahl, C.T., 2013. Brain inflammation induces post-synaptic changes during early synapse formation in adult-born hippocampal neurons. Exp. Neurol. 250, 176-188. https://doi.org/10.1016/j.expneurol.2013.09.005

de Jesus, M.M., Negrin, A.C., Taboga, S.R., Pinto-Fochi, M.E., Góes, R.M., 2015. Histopathological alterations in the prostates of Mongolian gerbils exposed to a high-fat diet and di-n-butyl
phthalate individually or in combination. Reprod. Toxicol. Elmsford N 52, 26-39.
https://doi.org/10.1016/j.reprotox.2015.02.005

Dewalque, L., Charlier, C., Pirard, C., 2014. Estimated daily intake and cumulative risk assessment of phthalate diesters in a Belgian general population. Toxicol. Lett. 231, 161-168.
https://doi.org/10.1016/j.toxlet.2014.06.028

Dombret, C., Capela, D., Poissenot, K., Parmentier, C., Bergsten, E., Pionneau, C., Chardonnet, S., Hardin-Pouzet, H., Grange-Messent, V., Keller, M., Franceschini, I., Mhaouty-Kodja, S., 2017. Neural Mechanisms Underlying the Disruption of Male Courtship Behavior by Adult Exposure to Di(2-ethylhexyl) Phthalate in Mice. Environ. Health Perspect. 125, 097001.
https://doi.org/10.1289/ehp1443

Dornas, W.C., de Lima, W.G., Pedrosa, M.L., Silva, M.E., 2015. Health implications of high-fructose intake and current research. Adv. Nutr. Bethesda Md 6, 729-737.
https://doi.org/10.3945/an.114.008144

Drago, F., Busă, L., 1997. Dose-dependent effects of acute kainic acid treatment on sexual behavior of male rats. Brain Res. 778, 135-139.

Du Plessis, S.S., Cabler, S., McAlister, D.A., Sabanegh, E., Agarwal, A., 2010. The effect of obesity on sperm disorders and male infertility. Nat. Rev. Urol. 7, 153-161.
https://doi.org/10.1038/nrurol.2010.6

Ducroq, S., Duplus, E., Grange-Messent, V., Trivelloni, F., Penalva-Mousset, L., Petropoulos, I., Mhaouty-Kodja, S., 2023. Cognitive and hippocampal effects of adult male mice exposure to environmentally relevant doses of phthalates. Environ. Pollut. 323, 121341.
https://doi.org/10.1016/j.envpol.2023.121341

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP), Silano, V., Barat Baviera, J.M., Bolognesi, C., Chesson, A., Cocconcelli, P.S., Crebelli, R., Gott, D.M., Grob, K., Lampi, E., Mortensen, A., Rivière, G., Steffensen, I.-L., Tlustos, C., Van Loveren, H., Vernis, L., Zorn, H., Cravedi, J.-P., Fortes, C., Tavares Poças, M. de F., Waalkens-Berendsen, I., Wölfle, D., Arcella, D., Cascio, C., Castoldi, A.F., Volk, K., Castle, L., 2019. Update of the risk assessment of di-butylphthalate (DBP), butyl-benzyl-phthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP), di-isononylphthalate (DINP) and di-isodecylphthalate (DIDP) for use in food contact materials. EFSA J. 17, e05838. https://doi.org/10.2903/j.efsa.2019.5838

Fan, Y., Xu, Qiaoqiao, Qian, H., Tao, C., Wan, T., Li, Z., Yan, W., Niu, R., Huang, Y., Chen, M., Xu, Qiujin, Martin, E.M., Wang, X., Qin, Y., Lu, C., 2022. High-fat diet aggravates prenatal low-dose DEHP exposure induced spermatogenesis disorder: Characterization of testicular metabolic patterns in mouse offspring. Chemosphere 298, 134296.
https://doi.org/10.1016/j.chemosphere.2022.134296

Fernandez, C.D., Bellentani, F.F., Fernandes, G.S., Perobelli, J.E., Favareto, A.P.A., Nascimento, A.F., Cicogna, A.C., Kempinas, W.D., 2011. Diet-induced obesity in rats leads to a decrease in sperm motility. Reprod. Biol. Endocrinol. RBE 9, 32. https://doi.org/10.1186/1477-7827-9-32
Giagulli, V.A., Kaufman, J.M., Vermeulen, A., 1994. Pathogenesis of the decreased androgen levels in obese men. J. Clin. Endocrinol. Metab. 79, 997–1000.
https://doi.org/10.1210/jcem.79.4.7962311

Hahladakis, J.N., Velis, C.A., Weber, R., Iacovidou, E., Purnell, P., 2018. An overview of chemical additives present in plastics: Migration, release, fate and environmental impact during their use, disposal and recycling. J. Hazard. Mater. 344, 179–199.
https://doi.org/10.1016/j.jhazmat.2017.10.014

Haverinen, E., Fernandez, M.F., Mustieles, V., Tolonen, H., 2021. Metabolic Syndrome and Endocrine Disrupting Chemicals: An Overview of Exposure and Health Effects. Int. J. Environ. Res. Public. Health 18, 13047. https://doi.org/10.3390/ijerph182413047

Head, E., Corrada, M.M., Kahle-Wrobleski, K., Kim, R.C., Sarsoza, F., Goodus, M., Kawas, C.H., 2009. Synaptic proteins, neuropathology and cognitive status in the oldest-old. Neurobiol. Aging 30, 1125–1134. https://doi.org/10.1016/j.neurobiolaging.2007.10.001

Heindel, J.J., Blumberg, B., 2019. Environmental Obesogens: Mechanisms and Controversies. Annu. Rev. Pharmacol. Toxicol. 59, 89–106. https://doi.org/10.1146/annurev-pharmtox-010818-021304

Heudorf, U., Mersch-Sundermann, V., Angerer, J., 2007. Phthalates: toxicology and exposure. Int. J. Hyg. Environ. Health 210, 623–634. https://doi.org/10.1016/j.ijheh.2007.07.011

Hull, E.M., Du, J., Lorrain, D.S., Matuszewich, L., 1995. Extracellular dopamine in the medial preoptic area: implications for sexual motivation and hormonal control of copulation. J. Neurosci. Off. J. Soc. Neurosci. 15, 7465–7471.

Jean, A., Bonnet, P., Liere, P., Mhaouty-Kodja, S., Hardin-Pouzet, H., 2017a. Revisiting medial preoptic area plasticity induced in male mice by sexual experience. Sci. Rep. 7.
https://doi.org/10.1038/s41598-017-18248-3

Jean, A., Mhaouty-Kodja, S., Hardin-Pouzet, H., 2021. Hypothalamic cellular and molecular plasticity linked to sexual experience in male rats and mice. Front. Neuroendocrinol. 63, 100949.
https://doi.org/10.1016/j.yfrne.2021.100949

Jean, A., Trouillet, A.-C., Andrianarivelo, N.A., Mhaouty-Kodja, S., Hardin-Pouzet, H., 2017b. Phospho-ERK and sex steroids in the mPOA: involvement in male mouse sexual behavior. J. Endocrinol.
https://doi.org/10.1530/JOE-17-0025

Kacar, E., Bulmus, O., Ercan, Z., Buran Kavuran, I., Zorlu, G., Tan, F., Serhatlioglu, I., Kelestimur, H., 2023. Treadmill exercise has healing effects on obesity-induced sexual behavior disorder through kisspeptin and kiss1R expression in male rats. Cell. Mol. Biol. Noisy--Gd. Fr. 69, 8–12.
https://doi.org/10.14715/cmb/2023.69.3.2

Kahn, L.G., Philippat, C., Nakayama, S.F., Slama, R., Trasande, L., 2020. Endocrine-disrupting chemicals: implications for human health. Lancet Diabetes Endocrinol. 8, 703–718.
https://doi.org/10.1016/S2213-8587(20)30129-7
Kannan, A., Davila, J., Gao, L., Rattan, S., Flaws, J.A., Bagchi, M.K., Bagchi, I.C., 2021. Maternal high-fat diet during pregnancy with concurrent phthalate exposure leads to abnormal placentation. Sci. Rep. 11, 16602. https://doi.org/10.1038/s41598-021-95898-4

Kougias, D.G., Cortes, L.R., Moody, L., Rhoads, S., Pan, Y.-X., Juraska, J.M., 2018. Effects of Perinatal Exposure to Phthalates and a High-Fat Diet on Maternal Behavior and Pup Development and Social Play. Endocrinology 159, 1088-1105. https://doi.org/10.1210/en.2017-03047

Le Gléau, L., Rouault, C., Osinski, C., Prifti, E., Soula, H.A., Debédat, J., Busieau, P., Amouyal, C., Clément, K., Andreelli, F., Ribeiro, A., Serradas, P., 2021. Intestinal alteration of α-gustducin and sweet taste signaling pathway in metabolic diseases is partly rescued after weight loss and diabetes remission. Am. J. Physiol. Endocrinol. Metab. 321, E417-E432.
https://doi.org/10.1152/ajpendo.00071.2021

Liu, J., Chang, L., Roselli, F., Almeida, O.F.X., Gao, X., Wang, X., Yew, D.T., Wu, Y., 2010. Amyloid-β induces caspase-dependent loss of PSD-95 and synaptophysin through NMDA receptors. J. Alzheimers Dis. JAD 22, 541-556. https://doi.org/10.3233/JAD-2010-100948

Liu, L.-L., Yue, J.-Z., Lu, Z.-Y., Deng, R.-Y., Li, C.-C., Yu, Y.-N., Zhou, W.-J., Lin, M., Gao, H.-T., Liu, J., Xia, L.-Z., 2024. Long-term exposure to the mixture of phthalates induced male reproductive toxicity in rats and the alleviative effects of quercetin. Toxicol. Appl. Pharmacol. 483, 116816.
https://doi.org/10.1016/j.taap.2024.116816

Liu, Y., Zhang, Y., Zheng, X., Fang, T., Yang, X., Luo, X., Guo, A., Newell, K.A., Huang, X.-F., Yu, Y., 2018. Galantamine improves cognition, hippocampal inflammation, and synaptic plasticity impairments induced by lipopolysaccharide in mice. J. Neuroinflammation 15, 112.
https://doi.org/10.1186/s12974-018-1141-5

Lubrano, C., Genovesi, G., Specchia, P., Costantini, D., Mariani, S., Petrangeli, E., Lenzi, A., Gnessi, L., 2013. Obesity and metabolic comorbidities: environmental diseases? Oxid. Med. Cell. Longev. 2013, 640673. https://doi.org/10.1155/2013/640673

Lyche, J.L., Gutleb, A.C., Bergman, A., Eriksen, G.S., Murk, A.J., Ropstad, E., Saunders, M., Skaare, J.U., 2009. Reproductive and developmental toxicity of phthalates. J. Toxicol. Environ. Health B Crit. Rev. 12, 225-249. https://doi.org/10.1080/10937400903094091

Martine, B., Marie-Jeanne, T., Cendrine, D., Fabrice, A., Marc, C., 2013. Assessment of adult human exposure to phthalate esters in the urban centre of Paris (France). Bull. Environ. Contam. Toxicol. 90, 91-96. https://doi.org/10.1007/s00128-012-0859-5

Mascarenhas, C., Sousa, A.C.A., Rato, L., 2024. Effects of Pharmaceutical Substances with Obesogenic Activity on Male Reproductive Health. Int. J. Mol. Sci. 25, 2324.
https://doi.org/10.3390/ijms25042324

Mhaouty-Kodja, S., 2018. Role of the androgen receptor in the central nervous system. Mol. Cell. Endocrinol. 465, 103-112. https://doi.org/10.1016/j.mce.2017.08.001

Mirihagalle, S., You, T., Suh, L., Patel, C., Gao, L., Rattan, S., Qiao, H., 2019. Prenatal exposure to di-(2-ethylhexyl) phthalate and high-fat diet synergistically disrupts mouse fetal oogenesis and affects folliculogenesis. Biol. Reprod. 100, 1561-1570. https://doi.org/10.1093/biolre/ioz051
Mondal, S., Ghosh, S., Bhattacharya, S., Mukherjee, S., 2019. Chronic dietary administration of lower levels of diethyl phthalate induces murine testicular germ cell inflammation and sperm pathologies: Involvement of oxidative stress. Chemosphere 229, 443-451.
https://doi.org/10.1016/j.chemosphere.2019.05.017

Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) related to Bis(2-ethylhexyl)phthalate (DEHP) for use in food contact materials | EFSA [WWW Document], 2005. URL https://www.efsa.europa.eu/en/efsajournal/pub/243 (accessed 1.26.25).

Panchal, S.K., Poudyal, H., Iyer, A., Nazer, R., Alam, A., Diwan, V., Kauter, K., Sernia, C., Campbell, F., Ward, L., Gobe, G., Fenning, A., Brown, L., 2011. High-carbohydrate high-fat diet-induced metabolic syndrome and cardiovascular remodeling in rats. J. Cardiovasc. Pharmacol. 57, 51-64. https://doi.org/10.1097/FJC.0b013e3181feb90a

Patrick, L., 2020. Diabetes and Toxicant Exposure. Integr. Med. Encinitas Calif 19, 16-23.

Pereira, R.M., Botezelli, J.D., da Cruz Rodrigues, K.C., Mekary, R.A., Cintra, D.E., Pauli, J.R., da Silva, A.S.R., Ropelle, E.R., de Moura, L.P., 2017. Fructose Consumption in the Development of Obesity and the Effects of Different Protocols of Physical Exercise on the Hepatic Metabolism. Nutrients 9, 405. https://doi.org/10.3390/nu9040405

Picot, M., Naulé, L., Marie-Luce, C., Martini, M., Raskin, K., Grange-Messent, V., Franceschini, I., Keller, M., Mhaouty-Kodja, S., 2014. Vulnerability of the neural circuitry underlying sexual behavior to chronic adult exposure to oral bisphenol a in male mice. Endocrinology 155, 502-512. https://doi.org/10.1210/en.2013-1639

Powell, W.S., Dominguez, J.M., Hull, E.M., 2003. An NMDA antagonist impairs copulation and the experience-induced enhancement of male sexual behavior in the rat. Behav. Neurosci. 117, 69-75.

Raja, M.K., Preobraschenski, J., Del Olmo-Cabrera, S., Martinez-Turrillas, R., Jahn, R., Perez-Otano, I., Wesseling, J.F., 2019. Elevated synaptic vesicle release probability in synaptophysin/gyrin family quadruple knockouts. eLife 8, e40744. https://doi.org/10.7554/eLife.40744

Sanna, F., Bratzu, J., Serra, M.P., Leo, D., Quartu, M., Boi, M., Espinoza, S., Gainetdinov, R.R., Melis, M.R., Argiolas, A., 2020. Altered Sexual Behavior in Dopamine Transporter (DAT) Knockout Male Rats: A Behavioral, Neurochemical and Intracerebral Microdialysis Study. Front. Behav. Neurosci. 14, 58. https://doi.org/10.3389/fnbeh.2020.00058

Sharma, A., Mollier, J., Brocklesby, R.W.K., Caves, C., Jayasena, C.N., Minhas, S., 2020. Endocrine-disrupting chemicals and male reproductive health. Reprod. Med. Biol. 19, 243-253.
https://doi.org/10.1002/rmb2.12326

Simerly, R.B., Gorski, R.A., Swanson, L.W., 1986. Neurotransmitter specificity of cells and fibers in the medial preoptic nucleus: an immunohistochemical study in the rat. J. Comp. Neurol. 246, 343-363. https://doi.org/10.1002/cne.902460305
Song, L., Dong, J., Cheng, W., Fei, Z., Wang, R., He, Z., Pan, J., Zhao, L., Wang, H., Peng, R., 2025. The Role of Glutamatergic Neurons in Changes of Synaptic Plasticity Induced by THz Waves. Biomolecules 15, 532. https://doi.org/10.3390/biom15040532

Swanson, G.M., Estill, M., Diamond, M.P., Legro, R.S., Coutifaris, C., Barnhart, K.T., Huang, H., Hansen, K.R., Trussell, J.C., Coward, R.M., Zhang, H., Goodrich, R., Krawetz, S.A., 2019. Human chromatin remodeler cofactor, RNA interactor, eraser and writer sperm RNAs responding to obesity. Epigenetics 15, 32–46. https://doi.org/10.1080/15592294.2019.1644880

Takase, K., Tsuneoka, Y., Oda, S., Kuroda, M., Funato, H., 2016. High-fat diet feeding alters olfactory-, social-, and reward-related behaviors of mice independent of obesity. Obes. Silver Spring Md 24, 886–894. https://doi.org/10.1002/oby.21441

Toop, C.R., Gentili, S., 2016. Fructose Beverage Consumption Induces a Metabolic Syndrome Phenotype in the Rat: A Systematic Review and Meta-Analysis. Nutrients 8, 577. https://doi.org/10.3390/nu8090577

Torres, T., Adam, N., Mhaouty-Kodja, S., Naulé, L., 2024. Reproductive function and behaviors: an update on the role of neural estrogen receptors alpha and beta. Front. Endocrinol. 15, 1408677. https://doi.org/10.3389/fendo.2024.1408677

Trouillet, A.-C., Ducroq, S., Naulé, L., Capela, D., Parmentier, C., Radovick, S., Hardin-Pouzet, H., Mhaouty-Kodja, S., 2022. Deletion of neural estrogen receptor alpha induces sex differential effects on reproductive behavior in mice. Commun. Biol. 5, 383. https://doi.org/10.1038/s42003-022-03324-w

US EPA, O., n.d. Di (2-ethylhexyl)phthalate (DEHP) CASRN 117-81-7 | DTXSID5020607 | IRIS | US EPA, ORD [WWW Document]. URL https://cfpub.epa.gov/ncea/iris2/chemicalLanding.cfm?substanceNmbr=14 (accessed 3.29.22).

White, C.L., Pistell, P.J., Purpera, M.N., Gupta, S., Fernandez-Kim, S.-O., Hise, T.L., Keller, J.N., Ingram, D.K., Morrison, C.D., Bruce-Keller, A.J., 2009. Effects of high fat diet on Morris maze performance, oxidative stress, and inflammation in rats: contributions of maternal diet. Neurobiol. Dis. 35, 3–13. https://doi.org/10.1016/j.nbd.2009.04.002

World Health Organization (WHO) [WWW Document], n.d. URL https://www.who.int (accessed 1.26.25).

Yardimci, A., Ulker, N., Bulmus, O., Sahin, E., Alver, A., Gungor, I.H., Turk, G., Artas, G., Kaya Tektemur, N., Ozcan, M., Kelestimur, H., Canpolat, S., 2022. Irisin Improves High-Fat Diet-Induced Sexual Dysfunction in Obese Male Rats. Neuroendocrinology 112, 1087–1103. https://doi.org/10.1159/000523689

Zhai, L.-L., Zhao, J., Bai, Y.-L., Wei, W., Sun, Q., Jia, L.-H., 2020. Combined effects of obesity and di-(2-ethylhexyl) phthalate on testosterone levels and kisspeptin/GPR54 expression in hypothalamus and testes of male mice. J. Chin. Med. Assoc. JCMA 83, 1020–1028. https://doi.org/10.1097/JCMA.0000000000000402
<table><caption>Table 1</caption><thead><tr><th></th><th>CD<br>(N = 12)</th><th>CDmix<br>(N = 16)</th><th>HFHF<br>(N = 15)</th><th>HFHFmix<br>(N = 14)</th></tr></thead><tbody><tr><td>Total time in investigation (s)</td><td>68.19 ± 6.33</td><td>67.61 ± 4.73</td><td>59.08 ± 6.35</td><td>64.70 ± 6.21</td></tr><tr><td>Number of entries in stimulus compartment</td><td>22.61 ± 0.96</td><td>20.31 ± 1.31</td><td>18.90 ± 1.10</td><td>20.03 ± 0.56</td></tr></tbody></table>
<table><caption>Table 2</caption><thead><tr><th>Weights</th><th>CD<br>(N = 23)</th><th>CDmix<br>(N = 25)</th><th>HFHF<br>(N = 25)</th><th>HFHFmix<br>(N = 22)</th></tr></thead><tbody><tr><td>Body weight (bw, g)<sup>a</sup></td><td>29.93 ± 0.54</td><td>29.58 ± 0.58</td><td>45.05 ± 0.58 ****</td><td>43.87 ± 0.58 ****</td></tr><tr><td>Testis (mg)<sup>a</sup></td><td>208.77 ± 3.74</td><td>211.13 ± 2.51</td><td>216.34 ± 3.14</td><td>207.35 ± 6.61</td></tr><tr><td>Testis (%bw)<sup>b</sup></td><td>6.8 ± 0.13</td><td>7.0 ± 0.11</td><td>4.8 ± 0.10****</td><td>4.7 ± 0.16****</td></tr><tr><td>Tyson glands (mg)<sup>a</sup></td><td>62.9 ± 4.4</td><td>64.5 ± 2.38</td><td>62.3 ± 3.29</td><td>63 ± 3.96</td></tr><tr><td>Tyson glands (%bw)<sup>b</sup></td><td>2.0 ± 0.14</td><td>2.1 ± 0.07</td><td>1.4 ± 0.08***</td><td>1.4 ± 0.09***</td></tr><tr><td>Seminal vesicles (SV, mg)<sup>a</sup></td><td>326.5 ± 8.14</td><td>353.4 ± 15.18</td><td>384.5 ± 15.09**</td><td>399 ± 16.92***</td></tr><tr><td>SV (%bw)<sup>b</sup></td><td>10.6 ± 0.22</td><td>11.6 ± 0.43</td><td>8.6 ± 0.3****</td><td>8.9 ± 0.32***</td></tr></tbody></table>
**Figure 1: Effects of an adult exposure to a combination of a HFHF and phthalate mixture on the metabolic state of male mice.**

(A) Body weight (bw, g) throughout the total duration of the exposure. Data are expressed as mean ± SEM. Post hoc comparisons are shown. Repeated measures from a two-way ANOVA of the weekly body-weight measure showed an effect of time ($F_{(4,296,390.9)}=611.2, p<0.0001$) treatment ($F_{(3,91)}=103.6, p<0.0001$) and interaction of time and treatment ($F_{(48,1456)}=60.19, P<0.0001$). (B) Blood glucose levels during OGTT in week 0. Repeated measures from a two-way ANOVA of the blood glucose levels in the OGTT at W0 showed an effect of time ($F_{(3,061,272.4)}=325.9, P<0.0001$) but no effect of treatment ($F_{(3,89)}=0.4185, P=0.7$), with fasting blood glucose levels comparable across groups at W0. (C) Blood glucose levels during OGTT in week 16. Repeated measures from a two-way ANOVA of the blood glucose levels in the OGTT at W16 showed an effect of time ($F_{2,196,144.9)}=120.7; P<0.0001$) and of treatment ($F_{(3,66)}=29.94, P<0.0001$). (D) Area under the curve (AUC) for OGTT W16. (E) Blood glucose levels during ITT in week 16. Repeated measures from a two-way ANOVA of the blood glucose levels in the ITT at W16 showed an effect of time ($F_{(2,970,228.8)}=200.6, P<0.0001$) and treatment ($F_{(3,75)}=17.06, P<0.0001$). (F) Glucose area under the curve (AUC) during ITT W16. (G) Circulating levels of Leptin at W17 (normalized fluorescence intensity, AU).

Results are expressed as mean ± SEM. Post hoc comparisons are shown (*p<0.05, **<0.01, ***p<0.001, ****p<0.0001; a : p<0.05 for CD vs CDmix, b : p<0.05 for CD vs HFHF, c : p<0.05 for CD vs HFHFmix, d : p<0.05 for CDmix vs HFHF, e : p<0.05 for CDmix vs HFHFmix, f : p<0.05 for HFHF vs HFHFmix). bw: body weight; AU: arbitrary unit. Arrow shows glucose or insulin administration.

**Figure 2: Effects of adult exposure to a combination of HFHF and phthalate mixture on male sexual behavior.**

(A)Percentage of males performing at least one mount, (B) performing at least one intromission, and (C) reaching ejaculation during the 10 h mating event. Data were compared with Chi-square analysis. (D) Mating duration (ejaculation latency – first mount latency) in minutes. (E) Latency for the first mount (time of the first mount – start of the mating event), (F) latency to first intromission (time of the first intromission – start of the mating event), and (G) ejaculation latency (time of ejaculation – start of the mating event) in minutes. In the absence of sexual behavior, latencies were set at 600 min, corresponding to the total duration of the recording (10h).

Data expressed as means ± SEM. Post-hoc comparisons are shown (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

**Figure 3: Effects of adult exposure to a combination of HFHF and phthalate mixture on male olfactory preference.**

(A) Olfactory preferences of males toward an anesthetized male and female were measured in a Y-maze paradigm. (B) Time spent in investigation of male vs. female stimuli. Two-way ANOVA of the time
spent investigating the two stimuli showed no effect of treatment (F<sub>(3,53)</sub>=0.5112; P=0.6) but an effect
of sex of the stimulus (F<sub>(1,53)</sub>=47.73; P<0.0001) and of the subject (F<sub>(53,53)</sub>=2.934; P<0.0001).

Data expressed as means ± SEM. Post-hoc comparisons are shown (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

**Table 1:** Total time (seconds) spent in investigation (male + female stimuli), and number of entries in each stimulus compartment. Two-way ANOVA and Tukey's test of the number of entries in stimulus compartments showed no effect of treatments (F<sub>(3,53)</sub>=1.522; P=0.2) but did show an effect of sex of the stimulus (F<sub>(1,53)</sub>=15.40; P=0.0003) and of subject (F<sub>(53,53)</sub>=6.369; P<0.0001). The two-way ANOVA also showed an effect of the interaction (sex of the stimulus x treatment), although it was not statistically significant (F<sub>(3,53)</sub>=0.3892; P=0.7).

<caption>**Table 2:** Effects of adult exposure to a combination of HFHF and phthalate mixture on absolute (a) and relative (% bw, b) organ weights of adult male mice.</caption>

Results are expressed as mean ± SEM. Post hoc comparisons are shown (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

bw: body weight, SV : seminal vesicle

<caption>**Figure 4:** Effects of adult exposure to a combination of HFHF and phthalate mixture on the protein expression of steroid hormones receptors in the mPOA.</caption>

(A) Quantification of protein amounts for AR, and (B) ERα normalized to the subunit 42 of ERK in the mPOA. Data are expressed as the means ± SEM of six to eight males per treatment group. Post-hoc comparisons are shown (*p<0.05, **p<0.01). AU : arbitrary unit.

<caption>**Figure 5:** Effects of an adult exposure of a combination of a HFHF and phthalate mixture on the expression of protein markers in the mPOA.</caption>

(A) Quantification of protein amounts for synaptophysin, (B) the dopamine transporter DAT and (C) the glutamatergic receptor subunit AMPA GluR1 normalized to the subunit 42 of ERK in the mPOA. Data are expressed as the means ± SEM of six to eight males per treatment group. Post-hoc comparisons are shown (*p<0.05, **p<0.01).
## Supplementary data

**Figure S1:** Mice from both cohorts were exposed from eight weeks of age. The duration of exposure is indicated in green, with males exposed for eight weeks before the first analysis and the treatments maintained until sacrifice at W17. OGTT: oral glucose tolerance test, ITT: insulin tolerance test, OP: olfactory preference test.

<table><caption>**Table S2:** Antibodies used for western blot analysis</caption><thead><tr><td>Protein</td><td>Species</td><td>Dilution</td><td>Reference</td></tr></thead><tbody><tr><td>ERK</td><td>Rabbit</td><td>1/1000</td><td>Cell Signaling (9102S)</td></tr><tr><td>AR</td><td>Rabbit</td><td>1/500</td><td>Abcam (Ab133273)</td></tr><tr><td>ERα</td><td>Rabbit</td><td>1/200</td><td>Santa Cruz (SC-542)</td></tr><tr><td>Synaptophysin</td><td>Mouse</td><td>1/500</td><td>Abcam (Ab8049)</td></tr><tr><td>AMPA GluR1</td><td>Rabbit</td><td>1/5000</td><td>Millipore (04-855)</td></tr><tr><td>DAT</td><td>Mouse</td><td>1/500</td><td>Abcam (Ab128848)</td></tr><tr><td>GFAP</td><td>Mouse</td><td>1/500</td><td>Sigma (G3893)</td></tr><tr><td>IBA1</td><td>Rabbit</td><td>1/500</td><td>Abcam (108539)</td></tr><tr><td>Spinophilin</td><td>Mouse</td><td>1/1000</td><td>Millipore (06-852)</td></tr><tr><td>PSD95</td><td>Mouse</td><td>1/500</td><td>Santa Cruz (SC32290)</td></tr><tr><td>Peroxidase-conjugated secondary antibody</td><td>Rabbit</td><td>1/2500</td><td>Jackson Immuno research (111-035-144)</td></tr><tr><td>Peroxidase-conjugated secondary antibody</td><td>Mouse</td><td>1/2500</td><td>Jackson Immuno research (115-035-062)</td></tr></tbody></table>
**Figure S3: (A) Blood glucose levels during OGTT in week 7 and area under the curve (AUC) ; (B) Blood glucose levels during ITT in week 7 and area under the curve ; (C) Blood glucose levels during OGTT in week 12 and area under the curve (AUC) ; (D) Blood glucose levels during ITT in week 12 and area under the curve.**

Results are expressed as mean ± SEM. Post-hoc comparison are shown (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; a : p<0.05 for CD vs CDmix, b : p<0.05 for CD vs HFHF, c : p<0.05 for CD vs HFHFmix, d : p<0.05 for CDmix vs HFHF, e : p<0.05 for CDmix vs HFHFmix, f : p<0.05 for HFHF vs HFHFmix). AU: arbitrary unit. Arrow shows glucose or insulin administration.

**Figure S4: Effects of an adult exposure to a combination of a HFHF and phthalate mixture on locomotor activity.**

(A) Number of locomotor events per 20 min within 120 min. (B) Cumulative locomotor activity during the 120 min test. Data expressed as means ± SEM. Repeated measures from a two-way ANOVA of the number of movements during the locomotor test showed an effect of time ($F_{(3.136,257.1)}=80.01$; $P<0.0001$) but no effect of treatment ($F_{(3,82)}=1.755$; $P=0.1$).

**Figure S5: (A) Quantification of protein amounts for GFAP, (B) IBA1, (C) spinophilin, and (D) PSD95 are normalized to the subunit 42 of ERK in the mPOA.**

Data are expressed as the means ± SEM of six to eight males per treatment group.
<figure><img src="image_3.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>
A

<figure><img src="image_11.png" /></figure>

B

<figure><img src="image_12.png" /></figure>
<figure><img src="image_13.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_19.png" /></figure>
A

<figure><img src="image_20.png" /></figure>

B

<figure><img src="image_21.png" /></figure>
<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>
<figure><img src="image_25.png" /></figure>
<caption>A</caption>

Blood glucose levels (mg/dl)

<figure><img src="image_26.png" /><figcaption>B</figcaption></figure>

Blood glucose levels (mg/dl)

<figure><img src="image_27.png" /><figcaption>C</figcaption></figure>

Blood glucose levels (mg/dl)

<figure><img src="image_28.png" /><figcaption>D</figcaption></figure>

Blood glucose levels (mg/dl)

<figure><img src="image_29.png" /></figure>

Area under the curve (AU)

<figure><img src="image_30.png" /></figure>

Area under the curve (AU)

<figure><img src="image_31.png" /></figure>

Area under the curve (AU)

<figure><img src="image_32.png" /></figure>

Area under the curve (AU)

<figure><img src="image_33.png" /></figure>

p<0.05

a : CD vs CDmix

b : CD vs HFHF

c : CD vs HFHFmix

d : CDmix vs HFHF

e : CDmix vs HFHFmix

f : HFHF vs HFHFmix
A

<figure><img src="image_34.png" /></figure>

B

<figure><img src="image_35.png" /></figure>
A

<figure><img src="image_36.png" /></figure>

B

<figure><img src="image_37.png" /></figure>

C

<figure><img src="image_38.png" /></figure>

D

<figure><img src="image_39.png" /></figure>