Anti-Müllerian hormone may regulate the number of calbindin-positive neurons in the sexually dimorphic nucleus of the preoptic area of male mice

Abstract

Background: The male brain is putatively organised early in development by testosterone, with the sexually dimorphic nucleus of the medial preoptic area (SDN) a main exemplifier of this. However, pubescent neurogenesis occurs in the rat SDN, and the immature testes secrete anti-Müllerian hormone (AMH) as well as testosterone. We have therefore re-examined the development of the murine SDN to determine whether it is influenced by AMH and/or whether the number of calbindin-positive (calbindin+ve) neurons in it changes after pre-pubescent development.

Methods: In mice, the SDN nucleus is defined by calbindin+ve neurons (CALB-SDN). The number and size of the neurons in the CALB-SDN of male and female AMH null mutant (Amh-/-) mice and their wild-type littermates (Amh+/+) were studied using stereological techniques. Groups of mice were examined immediately before the onset of puberty (20 days postnatal) and at adulthood (129-147 days old).

Results: The wild-type pre-pubertal male mice had 47% more calbindin+ve neurons in the CALB-SDN than their female wild-type littermates. This sex difference was entirely absent in Amh-/- mice. In adults, the extent of sexual dimorphism almost doubled due to a net reduction in the number and size of calbindin+ve neurons in females and a net increase in neuron number in males. These changes occurred to a similar extent in the Amh-/- and Amh+/+ mice. Consequently, the number of calbindin+ve neurons in Amh-/- adult male mice was intermediate between Amh+/+ males and Amh+/+ females. The sex difference in the size of the neurons was predominantly generated by a female-specific atrophy after 20 days, independent of AMH.

Conclusions: The establishment of dimorphic cell number in the CALB-SDN of mice is biphasic, with each phase being subject to different regulation. The second phase of dimorphism is not dependent on the first phase having occurred as it was present in the Amh-/- male mice that have female-like numbers of calbindin+ve neurons at 20 days. These observations extend emerging evidence that the organisation of highly dimorphic neuronal networks changes during puberty or afterwards. They also raise the possibility that cellular events attributed to the imprinting effects of testosterone are mediated by AMH.

Figure 1

The sexually dimorphic nucleus of the medial preoptic area. A cryosection of an adult Amh^+/+ male mouse was stained with an antibody to calbindin. The CALB-SDN (dashed line) is an ellipsoid-shaped cell cluster close to the third ventricle (3V) extending in a dorsolateral direction to the anterior commissure (ac). The scale bar represents 500 μm.

Figure 2

The dimorphism in the CALB-SDN varies with age and Amh genotype. The images are photomicrographs of the CALB-SDN illustrating the appearance of the nucleus in pre-pubescent (20 days old) and adult mice. All sections were stained with anti-calbindin antibodies. The location of the CALB-SDN relative to the anterior commissure and third ventricle is illustrated in Figure 1, with the quantitative estimates of the number and size of the calbindin^+ve neurons illustrated in Figures 3 and 4, respectively. ♂, male; ♀, female.

Figure 3

The CALB-SDN has pre-pubescent and adult forms that are differentially regulated. The bars are the mean number of calbindin^+ve neurons ± the standard error of the mean of six mice. The Amh genotype (^+/+ or ^-/-) is shown beneath each bar. (A) Twenty-day-old mice. *1: There was a significant effect of sex (p = 0.001), genotype (p = 0.008) and sex × genotype interaction (p = 0.022, two-way ANOVA). *2: The Amh^+/+ males were significantly different to Amh^-/- males (p = 0.004) and both female groups (p < 0.002 Amh^+/+, p < 0.001 Amh^-/-, Student’s t test). (B) Adult mice. *3: There was a significant effect of sex (p < 0.001), genotype (p = 0.002) and sex × genotype interaction (p = 0.003, two-way ANOVA). *4: The Amh^+/+ males were significantly different to all other adult groups (p = 0.002 to the Amh^-/- males and p < 0.001 to both female groups, Student’s t test). *5: The adult Amh^-/- males were also significantly different to both of the adult female groups (p < 0.001 Amh^+/+, p < 0.001 Amh^-/-, Student’s t test). (C) The bars illustrate the mean change in cell number after 20 days. In two-way ANOVAs of age and genotype, there was a significant effect of age (p = 0.007) and genotype (p < 0.001) for the male mice and a significant effect of age (p < 0.001) for the female mice. The adult mice were significantly different to the corresponding 20-day-old mice (p = 0.037 for the Amh^+/+ males, p = 0.035 for the Amh^+/+ females and p = 0.017 for the Amh^-/- females, Student’s t test). ♂, male; ♀, female.

Figure 4

The size of neuronal soma in the CALB-SDN is dimorphic after puberty. The bars are the mean size of the cell body of the calbindin^+ve neurons ± the standard error of the mean of six mice. The Amh genotype (^+/+ or ^-/-) is shown beneath each bar. (A) Twenty-day-old mice. There is no effect of sex or genotype at this age. (B) Adult mice. *1: There was a significant effect of sex (p < 0.001, two-way ANOVA) but not of genotype. *2: The Amh^+/+ males were significantly different to both of the female groups (p = 0.005 Amh^+/+ and p = 0.004 Amh^-/-, Student’s t test). *3: The adult Amh^-/- males were also significantly different to both of the adult female groups (p = 0.011 Amh^+/+, p = 0.008 Amh^-/-, Student’s t test). (C) The bars illustrate the mean change in cell size after 20 days. In the female mice, the change in size was marginally significant (p = 0.048, two-way ANOVA of age and genotype). ♂, male; ♀, female.