Estrogen dependent activation function of ERβ is essential for the sexual behavior of mouse females

Abstract

We previously generated and characterized a genuine estrogen receptor (ER) β-null mouse line (named ERβ_ST^L−/L−) and showed that ERβ_ST^L−/L− mice were sterile, due to an ovulation impairment in females and to an unknown reason in males, as their reproductive organs and spermatozoid motility appeared normal. We report here an assessment of the sexual behavior of ERβ_ST^L−/L− null mice. We found that ERβ_ST^L−/L− males display mildly impaired sexual behavior and that ERβ_ST^L−/L− females are significantly less receptive and less attractive than wild-type (WT) females. Decreased attractivity is also exhibited by ERβAF2⁰ but not by ERβAF1⁰ mutant females (females devoid of either AF2 or AF1 activation function of ERβ). Interestingly, by using an odor preference test, we have determined that the low attractiveness of ERβ_ST^L−/L− and ERβAF2⁰ females is related to a deficiency of a volatile chemosignal.

The role of estradiol (E2) and estrogen receptor α (ERα) on the sexual behavior of the mouse is well known (1). ERα-null females do not display sexual receptivity, although they are as sexually attractive as wild-type (WT) controls. ERα-null males exhibit severely reduced numbers of mounts, thrusts and intromissions, and very few ejaculate (2). In contrast, ERβ has not yet been reported to play an important role in mouse sexual behavior (3). Several mouse lines bearing partial null mutations of ERβ have been generated and named according to the laboratory of origin (4, 5): ERβKO_CH originated from a Chapel Hill laboratory (6), ERβKO_ST from a Strasbourg laboratory (7), and ERβKO_WY from Wyeth (8). The ERβKO_CH line has been duplicated and a second colony, named ERβKO_KI, has been established at the Karolinska Institut (9). A study performed on ERβKO_CH males and females showed that they performed normally in sexual behavior tests. Nevertheless, a delay in the age of the first ejaculation was reported in these mice, without further consequences in adults (10), as adults ERβKO_CH males were fertile and sired litters with the same efficiency as their WT counterparts (3). Contrasting with an apparent minor role in sexual behavior, ERβ appeared to be a major determinant of antianxiety behaviors in female mice (11, 12), and, in a transient manner, of aggressive behaviors in male mice (13). ERβ was also reported to be involved in male mouse brain defeminization (14).

We recently reported the generation and characterization of a complete ERβ-null mouse, the ERβ_ST^L−/L− mouse, of which the morphological phenotype contrasts in many aspects with that of the described ERβKO_KI mutant (5). ERβ_ST^L−/L− females were sterile, due to ovulation impairment, and males exhibited infertility from an unknown reason because their internal and external reproductive organs appeared morphologically normal. Because ERβ_ST^L−/L− mice of both sexes displayed reproductive anomalies, we performed sex behavioral tests to evaluate the extent of ERβ involvement in mouse reproductive functions. We found that ERβ is required for a normal sexual behavior in males that display delayed ejaculation, and in females that exhibit decreased receptivity and attractivity related to an alteration of a volatile chemical signal, most likely a pheromone.

Ligand-induced transcriptional activation by ERs involves two distinct activation functions, AF-1 and AF-2, acting synergistically (15). We investigated which one of these two ERβ functions could be responsible for or involved in the observed phenotype in ERβ_ST^L−/L− females. To this end, mice lacking either AF-1 or AF-2 transactivation function (ERβAF1⁰ and ERβAF2⁰ mutant mice, respectively) were generated. Our data show that AF-2 is involved in the phenotype of decreased attractivity of ERβ_ST^L−/L− females.

Results

Alteration of the Sexual Behavior of ERβ_ST^L−/L− Mutant Males.

The number of mounts (F_(1,20) = 4.54, P < 0.05; Fig. 1A) as well as that of intromission episodes (F_(1,20) = 5.70, P < 0.05; Fig. 1B) were significantly increased in ERβ_ST^L−/L− mutant males compared with WT males. The latencies to the first mount and to the first intromission were similar for both genotypes [U ≤ 57, not significant (NS); Fig. 1C]. In addition, the duration of the first five intromission episodes (F_(1,15) = 0.48, NS; Fig. 1D) and of the last five intromission episodes (F_(1,11) = 0.75, NS; Fig. 1D) were similar. The percentage of ejaculating males was also similar for all trials after 2 h of testing (Chi2 ≤ 1.52; Fig. 1E). Nevertheless, 50% of WT males ejaculated within 30 min, whereas 60 min were required before 50% of ERβ_ST^L−/L− males ejaculated (Fig. 1E), indicating that ejaculation was delayed in mutant mice. The latency to ejaculation was significantly longer in KO males in Trial 1 (t₁₁ = 2.54, P < 0.05; Fig. 1F), but decreased with repetition, to become similar to that of WT males in Trial 2 and Trial 3 (t₁₁ > 1.50, NS; Fig. 1F). In general, we observed a significant decrease for some of the scored parameters between the first and the last test trial in both WT and ERβ_ST^L−/L− males, suggesting that the male sexual behavior is influenced by repetition [e.g., number of mounts F_Trial(2,40) = 4.86, P < 0.05 (Fig. 1A), and latency to ejaculate after the first intromission F_Trial(2,22) = 5.07, P < 0.05].

Fig. 1.

Sexual behavior of ERβ_ST^L−/L− mutant males. Number of mounts (A) and intromissions (B), mean latencies to the first mount and intromission over three trials (C), duration of first five and last five intromissions (D), time-course of ejaculation (E), and mean latency to ejaculation over three trials in WT and ERβ_ST^L−/L− males (F). Nine WT and 13 ERβ_ST^L−/L− males were used for A–D, and six WT and seven ERβ_ST^L−/L− males (which achieved ejaculation) for E and F. Results are expressed as mean ± SEM, except for E. *P ≤ 0.05 vs. WT (ANOVA results).

Alteration of the Sexual Behavior of ERβ_ST^L−/L− Mutant Females.

Naive ERβ_ST^L−/L− and WT-hormone–primed females were repeteadly tested in the presence of WT males. Video recordings showed that during the first and the second trial, 50% and 38%, respectively, of the ERβ_ST^L−/L− females were not mounted by males (Chi2 > 4.10, P < 0.05; Fig. 2A). Moreover, over the three trials, 25% of the ERβ_ST^L−/L− females never elicited male sexual behavior. The percentage of mount attempts increased in Trial 3, nearing that of WT females, suggesting that the sexual behavior is influenced by repetition (Chi2 < 2.55, NS; Fig. 2A). No active avoidance or aggressive behavior of the female toward the male and no aggressive behavior of the male toward the female were noticed. Males displayed normal social interactions with the females, as indicated by the repeated grooming and licking of the female, but no signs of sexual interest. These observations lead us to conclude that the reluctance of males to initiating sexual behavior could be related to impaired female attractivity.

Fig. 2.

Sexual behavior of ERβ_ST^L−/L− mutant females. Percentage of mount attempt (A), lordosis quotient (B), latency to the first mount attempt (C), and percentage of ejaculating males (D) in each trial. Eight WT and eight ERβ_ST^L−/L− females were used for (A and D) and eight WT and four, five, and six ERβ_ST^L−/L− for trials 1, 2, and 3, respectively, in (B and C; only females responding by a sexual behavior were considered: 50% in trial 1, 62% in trial 2, and 75% in trial 3). Results are expressed as percentage (A, B, and D) and as mean ± SEM (C). **P ≤ 0.01 vs. WT (Mann–Whitney results in C). *P ≤ 0.05; **, for P ≤ 0.01; ***P ≤ 0.001 vs. WT (χ² tests for A and D). *P ≤ 0.05 ; ***P ≤ 0.001 vs. WT (Student’s paired t test results).

In addition, when considering only the ERβ_ST^L−/L− females that engaged in sexual behavior, the lordosis quotient during the first 60 min of trials 1 and 2 for ERβ_ST^L−/L− females was significantly lower than in WT females (Trial 1: t₁₀ = 2.41, P < 0.05; Trial 2: t₁₁ = 5.43, P < 0.001; Fig. 2B) suggesting reduced receptivity in ERβ_ST^L−/L− females. This diminished receptivity may explain the increased latency to the first mount during the first trial (t₁₀ = 3.70, P < 0.01; Fig. 2C) as well as the significantly decreased percentage of males ejaculating successfully in the presence of ERβ_ST^L−/L− females (Chi2 > 10.58, P < 0.01; Fig. 2D).

ERβ_ST^L−/L− Females Are Less Attractive.

A first experiment was carried out to evaluate the efficiency of the odor preference test. We used a pair of C57BL6/J females in which one female was ovariectomized (ovx female, the putative nonattractive individual) and one ovariectomized and pharmacologically treated (hormone-primed female, the putative attractive individual). Naïve age-matched C57BL/6J males exhibited more numerous and longer odor exploration episodes with hormone-primed than with ovx females (t₉ ≥ 5.84, P < 0.001; Fig. 3 A and B), whereas the number of rears and groomings were not significantly affected (t₉ ≤ 1.76, NS; Fig. 3 A and B). WT naïve age-matched males were exposed to the odor of ERβ_ST^L−/L− and WT naïve hormone-primed females. Odor exploration episodes were significantly increased in number and duration with odor from WT females compared with ERβ_ST^L−/L− females (t₉ ≥ 3.00, P < 0.05 and P < 0.001 respectively; Fig. 3 C and D). The number of rears and groomings and the time spent grooming were not affected (t₉ ≤ 1.11, NS; Fig. 3 C and D). Taken together, these results show that the odor of hormone-primed ERβ_ST^L–/L– mutant females is a significantly less attractive stimulus for WT males than that of hormone-primed WT females. We next wondered whether multiple progesterone injections eliciting sexual receptivity, performed 3 d apart before the odor preference test (Materials and Methods), may restore the attractiveness of ERβ_ST^L−/L− females. No effect on the attractiveness of ERβ_ST^L−/L− female odor was noted, because WT males continued to prefer the WT female odor, as demonstrated by the significant difference between the time spent in exploring the two different odors (t₉ ≥ 4.83, P < 0.001; Fig. 3E). Moreover, repeated progesterone administration had no effect on WT female attractiveness, because the mean exploration time of odor from WT females receiving one progesterone injection was similar to that of WT females receiving four progesterone injections (Fig. 3E).

Fig. 3.

Female sexual attractiveness. Number of odor exploration, rears, and grooming episodes (A) and duration of odor exploration and grooming (B) of WT males exploring odors of two C57Bl6/J (ovx and hormone-primed) females. This test was repeated twice by using independent groups of males and females. Number of odor exploration, rears, and grooming episodes (C) and duration of odor exploration and grooming (D) of WT males exploring odors of ERβ_ST^L−/L− and WT females. We performed these experiment with five independent pairs of ERβ_ST^L−/L− and WT hormone-primed females and five independent groups of males. Duration of odor exploration of males exploring odors of ERβ_ST^L−/L− and WT females after repeated progesterone administration (E). Results are expressed as mean ± SEM *P ≤ 0.05 ; ***P ≤ 0.001 vs. WT (Student’s paired t test results).

Sexual Attractiveness of ERβAF1⁰ and ERβAF2⁰ Females.

Odor preference tests were performed to assess the attractiveness of mutant females. WT naïve age-matched males were exposed to the odor of ERβAF1⁰, ERβAF2⁰, or WT hormone-primed females. WT males had significantly fewer exploration episodes, both in number and duration, when exposed to odor of ERβAF2⁰ female than to that of WT females (t₉ ≥ 4.93, P < 0.001; Fig. 4 A and B). In contrast, WT males showed no odor preference when pairs of WT and ERβAF1⁰ female mice were tested (t₉ ≤ 0.42, NS; Fig. 4 A and B). Thus, the estrogen-dependent transactivating function 2 (AF2) of ERβ appears to be indispensible for female mouse attractiveness.

Fig. 4.

Sexual attractiveness of ERβAF1⁰ and ERβAF2⁰ females. Number (A) and duration (B) of odor explorations of ERβAF1⁰, ERβAF2⁰, and WT females by naïve age-matched WT males. These experiments were repeated twice with two independent pairs of ERβAF1⁰ or ERβAF2⁰ and WT females and two independent groups of naive males. Results are expressed as mean ± SEM ***P ≤ 0.001 vs. control (Student’s paired t test results).

Discussion

We reported that ERβ_ST^L−/L− of both sexes are sterile, males for an unknown reason and females because of ovulation impairment (5). This result prompted us to explore whether the lack of and/or abnormal sexual behavior could be the cause of this male infertility. To this end, we performed sexual behavior tests by mating ERβ_ST^L−/L− males with hormone-primed WT females and recording their behavior. We found that ERβ_ST^L−/L− mutant males exhibited increased number of mounts and intromissions and a delayed ejaculation compared with WT males.

In mammals, male sexual behavior is characterized by several phases, namely precopulatory, which includes the presence of sexual interest (libido) and sexual arousal, mounting, intromission, ejaculation, and postejaculatory behavior (16). The regulation of these phases is mediated by various central nervous systems structures. The medial preoptic area of the hypothalamus is involved in the control of libido and ejaculation, the locus coeruleus in the mediation of sexual arousal, and the nucleus paragigantocellularis and the paraventricular nucleus of hypothalamus in ejaculation control (17–20). Interestingly, the ERβ protein is detectable in the preoptic area, the paraventricular nucleus of the hypothalamus, the dorsal raphe, and locus coeruleus (21, 22). Moreover, ERβ protein is present in the preoptic area and the bed nucleus of stria terminalis that are important for processing of pheromone-induced signals.

The sexual interest quantified by the latency to the first mount appears to be normal in ERβ_ST^L−/L− males. ERβ_ST^L−/L− males perform intromission episodes of comparable length to those performed by WT males but require longer latencies to ejaculation than WT males. These observations suggest that ERβ_ST^L−/L− males have difficulties in relieving the inhibition of the ejaculatory reflex, leading to delayed ejaculation. The serotoninergic system has been involved in the control in the sexual behavior in mice and humans (17, 23). Serotonin acts as an inhibitor of the ejaculation (17, 23, 24). Because a decrease in tryptophan hydroxylase (the enzyme catalyzing the rate-limiting step in the synthesis of serotonin) mRNA in the dorsal raphe has been reported in ERβKO_KI mice (25), our present behavioral results are not in keeping with this molecular data. However, the medial preoptic area and the paraventricular nucleus of hypothalamus appear to have excitatory influences on ejaculation. The neurotransmitters in these pathways are dopamine and oxytocin, respectively. Several pieces of evidence suggest an involvement of ERβ in the regulation of dopamine D2 receptor, dopamine transporters (DAT), and oxytocin (26–28). In the absence of estradiol action via ERβ in ovariectomized female rats, D2 and DAT expression decrease and this decrease is prevented by treatment with either estradiol or the selective ERβ agonist DPN (27). In the absence of ERβ in male mice, β-estradiol 3-benzoate, an ER agonist, fails to increase oxytocin mRNA levels in the paraventricular nucleus of hypothalamus, but not in the medial preoptic area (28). It is therefore possible that there is a decrease of D2, DAT, and oxytocin expression in the brain of ERβ_ST^L−/L− males leading to a reduced excitatory influence of medial preoptic area and paraventricular nucleus on the ejaculatory reflex and, as a consequence, to a delayed ejaculation.

In summary, absence of ERβ in males alters their sexual behavior. However, these new data do not explain the previously reported male infertility (5) because the behavior of ERβ_ST^L−/L− male is not drastically affected. One possibility to explain why we concluded to the infertility of ERβ_ST^L−/L− males could be that these males were not left for a long enough period with WT females. For fertility assay of ERβ_ST^L−/L− males, the mice were crossed with two C57BL6/J females. The presence of vaginal plugs was checked during 15 consecutive days followed by a mating period up to 16 wk. At the end of the 16-wk period, because no litters were found, females were removed and crossed with males of known fertility to exclude female sterility. Two new females were placed with the mutant male and the same follow-up procedure was performed. Five ERβ_ST^L−/L− males were tested and no vaginal plug and no litter were ever found.

Female ERβ_ST^L−/L− also display abnormal sexual behavior. Fifty percent and 38% of the ERβ_ST^L−/L− females did not elicit male sexual behavior in trials 1 and 2, respectively, suggesting impaired attractivity of these females. Note that the effect of ERβ ablation on female attractiveness appears to be specific to this ER isotype, because ERα-null mutants display normal attractiveness, but impaired receptivity to stud males (29). Moreover, the 50% and 62% of the ERβ_ST^L−/L− females that elicited male sexual behavior in trials 1 and 2, respectively, displayed reduced lordosis quotients, suggesting a diminished receptivity of these females. This difference becomes less pronounced in the third trial, suggesting an effect of experience, with ERβ_ST^L−/L− females being slower learners compared with WT females. Altogether, our data lead us to conclude that ERβ_ST^L−/L− females displayed impaired attractivity and receptivity, defects that are partially corrected by experience.

Females attractiveness involves pheromones in addition to behavioral display and vocalizations (30). Pheromones are eliminated through urine and belong to the family of major urinary proteins (MUPs), major histocompatibility complex (MHC) peptides, sulfated steroids, and a variety of volatile compounds [e.g., (methylthio)methanethiol, 2,3-dehydro-exo-brevicomin] (31, 32). The sulfated steroids are only present in female urine and appear to account for the stimulation of the majority of vomeronasal sensory neurons (31). The use of steroids as pheromones has also been found in lampreys and goldfish (33, 34), but little is known about the factors influencing their production. It has been reported that the production and elimination of sulfated steroids in goldfish coincides with the ovulatory period (35). We investigated whether the impaired attractivity of ERβ_ST^L−/L− females could be due to a perturbation of urinary pheromones. To this purpose, we performed odor preference tests. These tests clearly point out that WT males prefer WT females odor to ERβ_ST^L−/L− females odor. Furthermore, we showed that the diminished attractivity of the odor of ERβ_ST^L−/L− females is attributable to the lack of the ligand-dependent AF2 transactivation function, because ERβAF2⁰, but not ERβAF1⁰ females odor, is significantly less preferred by an WT male than that of WT females in odor preference tests. Assuming that the normal ovarian hormone production during ovulation is required for the production of sulfated steroids pheromones, then both ERβ_ST^L−/L− or ERβAF2⁰ females are clearly deficient for this production because their ovulation and corpora lutea formation are impaired (5). Interestingly, repeated progesterone administration to E2-primed ERβ_ST^L−/L− females could not rescue the diminished attractivity phenotype of ERβ_ST^L−/L−, suggesting that the constitutive absence of ERβ has irreversible effects on the pathways involved in pheromone production in female mice.

Female receptivity was reported to be a result of hormone priming and experience (36) (37). In our experimental conditions, repeated testing and progesterone priming were not sufficient to improve ERβ_ST^L−/L− females receptivity to WT levels. However, lack of ERβ is responsible for increased anxiety in female mice (11, 12) and has been reported to interfere with spatial learning (38). In this context, we can hypothesize that the deficits in acquiring sexual receptivity displayed by ERβ_ST^L−/L− females can be explained, at least partly, by their anxiety and by an already existing learning deficit. Nevetheless, this assumption needs further experimental support. In summary, it appears that in female mice, absence of ERβ impairs acquisition of the sexual behavior and their ability to produce WT-quality pheromones. In addition, the deletion of ERβ AF2 function recapitulates the effects of ERβ absence in odor preference tests.

In conclusion, our present work demonstrates that ERβ is involved in a subtle manner in the sexual behavior of males and females, in addition to its well-demonstrated effects on ovarian homeostasis (5, 6). It is noteworthy that this work was performed by using mutant mice bearing a germ-line inactivation of ERβ in which the receptor is absent from the embryonic period. The effects observed can be related to altered developmental mechanisms involving CNS and extra-CNS systems.

Materials and Methods

Mice.

ERβ_ST^L−/L− mutant mice (C57BL6/J background) were generated as described (5). ERβAF1⁰ and ERβAF2⁰ mutant mice were generated and phenotypically characterized for reproductive function. For behavioral tests, 3- to 4-mo-old mutant and WT littermates males and females were housed in individual cages (Techniplast) for 3 wk before experiments, maintained on a reverse light cycle (light on at 1900/light off at 0700) to allow testing during their active period, with food and water available ad libitum. All animal care and used procedures were in accordance with the European legislation. Animal facility is licenced by the French Ministry of Agriculture (Agreement B67-218-5). Experiments were supervised by H.M. (Agreement 67-172).

Surgery and Pharmacological Treatment.

Eight-week-old female mice were ovariectomized to ensure all females have adequate levels of steroid hormones. A pellet of 17β-estradiol (35 mg per pellet 60-d release, SE-121; Innovative Research of America) was intrascapular s.c. implanted. Ovariectomy and pellet implantation were performed during the same surgical session under isofluran anesthesia. Surgery was followed by a recovery period of 5 d. Four hours before testing, females (named hormone-primed) were s.c. injected with 400 μg of progesterone (Sigma-Aldrich) in 50 μL of sunflower oil to obtain sexual receptivity, and bedding of their cages was changed.

Behavioral Procedures.

Sexual behavior test.

This test evaluates the behavior of a mouse in the presence of a congener of opposite sex and known normal sexual behavior. Mutant males and females were paired with WT females and males, respectively. Testing was performed in a cage [M.I.C.E. (Micro-environmental comfort, Isolation, Containment, Enrichment), Charles River; internal dimensions: width × length × height: 324 × 163 × 148 to 190 mm] containing clean bedding and separated in two equivalent compartments by a vertical metallic grid (3). Before testing, the male was placed in one compartment and the hormone-primed female in the other one for a habituation period of 10 min. The grid was then removed, allowing direct contact between the two animals, and their behavior was recorded for 2 h by using a digital camera on infrared under low light conditions (<10 Lux). The following parameters were defined and scored for male sexual behavior: (i) latency to the first mount, intromission episode, and ejaculation; (ii) total number of mounts, intromission episodes, and ejaculations; and (iii) duration of each intromission episode. For mounts and intromissions, latencies were considerated as the time interval between the beginning of the test trial and the first occurrence of the respective behavior. For ejaculation, latency was considered as the time interval between the first intromission and the ejaculation. For female sexual behavior, the receptive stands allowing male intromission and the number of mount attempts were scored. Individual sexual receptivity was measured by the lordosis quotient, LQ [LQ = (stands/mounts) × 100]. At the end of the recording period, mice were separated with the grid, and replaced in their respective home cages. Each mouse was subjected to three test trials performed at least 3 d apart. Test cages were cleaned after each trial. Data from 13 ERβ_ST^L−/L− and 9 WT males, and 8 ERβ_ST^L−/L− and 8 WT females, were analyzed.

Female sexual attractiveness (odor preference test).

This test evaluates a possible difference in attractiveness of two females of two different genotypes (WT and mutant), as perceived by a sexually naïve age-matched WT male. The procedure was designed to allow free exploration by the male mouse of two different odors coming from unreachable compartments placed at the extremities of a corridor in which female mice and their respective beddings had been placed. The test was performed in a transparent Plexiglas corridor (87 × 9 cm) (Fig. 5) with an odor compartment (10 × 9 cm) (Fig. 5, white area) at each extremity of the runway closed by holed transparent separators allowing odor sniffing (hole width 0.5 cm, hole diameter 0.8 cm, distance between centers of two holes 2 cm) (Fig. 5, dotted lines). Goal boxes neighboring the odor compartments were defined (12 × 9 cm in front of the separator) (Fig. 5, dark gray area) in the video tracking software. A start box (10 × 9 cm) (Fig. 5, black area) was set at the center of the corridor, closed by removable metallic grids. The experiment was performed as follows: (i) day 1, habituation of males. To reduce neophobia and to evaluate any potential preference between the two arms of the corridor, each male was placed in the start box during 15 s and then was allowed to freely explore the corridor during 15 min. The corridor was cleaned with tap water and alcohol after each male run. (ii) day 2, male testing. Before male testing, a pair of hormone-primed females, one mutant and one WT, and their respective cage bedding, were randomly placed in the odor compartments 10 min before the test. Females mice were removed and replaced in their respective home cages. For testing, each male mouse was placed in the start box during 15 s and then was allowed to explore the corridor for 10 min. Occurrence and duration of sniffing episodes through the separators of odor compartments, as well as grooming and rears were visually scored in the goal boxes. The experiment was reproduced five times by using five different pairs of females, each pair being tested with 10 sexually naïve age-matched WT males.

Fig. 5.

Schematic representation of the corridor used for odor preference tests. Illustration of different zones of interest in behavioral evaluation. The wall thickness is 0.5 cm. The size of the different compartments (width x length x height) is given in centimeters. At the extremities, the white areas (9 × 10 × 16 cm) represent the compartments, closed by lids, in which WT or ERβ_ST^L−/L− female and their respective bedding were placed for odor impregnation (odor compartments). The different parameters were scored in the dark gray area (9 × 12 × 16, goal boxes). White and dark gray zones are separated by holed separators (dotted black line). Males are placed in the central black compartment (9 × 10 × 16, start box) before starting the odor exploration test.

Data Analysis.

Data are represented as mean ± SEM or percentage, except for latencies that are represented as box plot of the mean values over three trials. The box plot represents the 25th to 75th percentile range, the median is displayed as an horizontal line in the box, and bottom and top whiskers indicate the 10th and 90th percentile, respectively. ANOVA for repeated measurements was used to study the effects of genotype, repetition and their interaction on the occurencies, and durations of observed behaviors. Student’s unpaired t test or χ² test were used to study the genotype effect for each trial and for mean value. For latency analysis, Mann–Whitney test was used to study genotype effect. The level of significance was set at P < 0.05. In the sexual attractiveness assay, Student’s paired t tests were performed to study the preference for one corridor arm in each of the parameters scored.