Exposure to Methylmercury Does Not Change the Sexual Preference of C57BL Male Mice

Mercury is a powerful neurotoxin, teratogen, and endocrine disruptor in animals. Methylmercury (MeHg) is the most biologically available and toxic form of mercury 1, 2, 3. It has been reported that exposure to MeHg can change the sexual preference of white ibises 4, 5. It is unclear whether MeHg can change the sexual preference in higher mammals. Here, we used C57BL male mice to examine the pairing behavior outcomes of exposure to different concentrations of MeHg.

C57BL mice (male, 1 month old; female, 3 month old) were purchased from Sino‐British SIPPR/BK Laboratory Animals (Shanghai, China). All animal procedures were approved by the ethics committee of the Second Military Medical University and performed in compliance with the institutional guide for care and use of laboratory animals.

First, sesame oil preference and fox urine avoidance experiments were performed in all male mice to test the olfactory function by using a T maze (n = 50) 6. One horizontal arm of the T maze was empty, and the other was set 1.5 mL sesame oil or 1.5 mL fox urine. The mouse was put in the vertical arm. After it entered the horizontal arms, the door between the vertical and horizontal arms was closed. We recorded the time that the mouse spent in each horizontal arm for 5 min. The mice with normal olfactory function preferred the sesame oil versus air and preferred air versus fox urine (170 ± 6.5 seconds to sesame oil vs. 130 ± 6.5 seconds to air, 224 ± 8.2 seconds to air vs. 76 ± 8.2 seconds to fox urine, P < 0.01, Figure 1A). The mice with olfactory dysfunction were discarded in the following experiments (n = 2).

Figure 1.

Olfactory function test and body weight record. (A) Sesame oil preference and fox urine avoidance experiments. (B) Body weight record at 1‐, 2‐ and 3‐month time points. Data are expressed as the mean ± SD. Data are analyzed with Student's t‐test and one‐way analysis of variance (ANOVA). **P < 0.01.

The other 48 male C57BL mice were divided into three groups (control and two doses of MeHg exposure groups). The control group was feed with normal food fixed with vehicle (corn oil) (n = 16). In MeHg groups, MeHg was fixed into the animal food with two different doses (1 and 10 parts per million (ppm) of food), and administrated consecutively for 3 months. The doses we set were environmentally relevant concentrations that mammals may be exposed in MeHg‐polluted regions 4. Mice were started the MeHg treatment from 1 month old rather than from nursing period because they were more tolerant at that time. We measured the body weight every month. There is no difference in the body weight between the control group and two MeHg groups (P > 0.05, Figure 1B).

After 3 months feed, the mating choice, bedding preference, and genital odor preference experiments were performed accordingly to examine the effect of MeHg exposure on the sexual preference 6. In the mating choice experiment, a sexually naive male C57BL mouse and an oestrous female C57BL mouse were placed into the home cage of the test male for 5 min. Mouse–mouse mounting activities were recorded and analyzed using a Mouse and Rat Spontaneous Activity Video Analysis System (JLBehv‐LAG‐4, Shanghai Jiliang Software Technology Co., Ltd., Shanghai, China). In each group, 100% of males preferred to mounting female targets. There is no difference between the control group and MeHg groups (P > 0.05, Figure 2A).

Figure 2.

Methylmercury (MeHg) exposure does not change the sexual preference of C57 male mice. (A) Mating choice percentage on female target mouse. (B) Bedding preference experiment: the upper panel shows the device used in this assay, and the lower panel shows time spending on female and male bedding. (C) Time spending on sniffing female and male genitals. Data are expressed as the mean ± SD. Data are analyzed with Student's t‐test and one‐way analysis of variance (ANOVA). **P < 0.01, n.s. not significant.

In the bedding preference experiment, a cage was divided into two sides and was covered with female or male bedding on each side. Bedding was not changed for 3 days from group‐housed adult C57BL mice. Male and female bedding were prevented from mixing by two plastic bars with a hole which the mice can freely cross. A wire netting separated the test mice from the bedding (the upper panel of Figure 2B). The test mouse was placed in the center, and we accounted the time that the mouse spent on the two side for 5 min. After each experiment, the cage was washed with alcohol and water to remove odor. All the three groups of males prefer female bedding (control group, 95.5 ± 19.1 seconds to male vs. 205 ± 19.1 seconds to female, P < 0.01; 1 ppm group, 97.1 ± 19.8 seconds to male vs. 203 ± 19.8 seconds to female, P < 0.01; 10 ppm group, 99.8 ± 20.6 seconds to male vs. 200 ± 20.6 seconds to female, P < 0.01). Compared with the control group, MeHg exposure (1 ppm, 10 ppm) did not change the time on male bedding (P > 0.05, the lower panel of Figure 2B).

In the genital odor preference experiment, a slide with one side spreading with female genitals and the other side with male genitals was introduced to a test male. We accounted the sniffing time of both sides for 30 seconds. All the three groups of male mice prefer sniffing female genitals (control group, 5.3 ± 1.7 seconds to male genitals vs. 24.8 ± 1.7 seconds to female, P < 0.01; 1 ppm group, 5.5 ± 2.1 seconds to male vs. 24.5 ± 2.1 seconds to female, P < 0.01; 10 ppm group, 5.1 ± 1.9 seconds to male vs. 24.9 ± 1.9 seconds to female, P < 0.01). MeHg exposure (1 ppm, 10 ppm) does not change the time sniffing male genitals with the control group (P > 0.05, Figure 2C).

Our results show that exposure to MeHg does not change the sexual preference of C57BL male mice. Studies of animals with different sexual preferences are essential for understanding the seemingly simple pairing behavior. The mechanisms under mammalian sexual preference should be further studied.

Conflict of Interest

The authors declare no conflict of interest.