Sex differences in the cerebellum and frontal cortex: roles of estrogen receptor alpha and sex chromosome genes

Abstract

Most neurobehavioral diseases are sexually dimorphic in their incidence, and sex differences in the brain may be key for understanding and treating these diseases. Calbindin (Calb) D28K is used as a biomarker for the well-studied sexually dimorphic nucleus, a hypothalamic structure that is larger in males than in females. In the current study weanling C56BL/6J mice were used to examine sex differences in the Calb protein and message focusing on regions outside of the hypothalamus. A robust sex difference was found in Calb in the frontal cortex (FC) and cerebellum (CB; specifically in Purkinje cells); mRNA and protein were higher in females than in males. Using 2 mouse lines, i.e. one with a complete deletion of estrogen receptor alpha (ERα) and the other with uncoupled gonads and sex chromosomes, we probed the mechanisms that underlie sexual dimorphisms. In the FC, deletion of ERα reduced Calb1 mRNA in females compared to males. In addition, females with XY sex chromosomes had levels of Calb1 equal to those of males. Thus, both ERα and the sex chromosome complement regulate Calb1 in the FC. In the CB, ERα knockout mice of both sexes had reduced Calb1 mRNA, yet sex differences were retained. However, the sex chromosome complement, regardless of gonadal sex, dictated Calb1 mRNA levels. Mice with XX chromosomes had significantly greater Calb1 than did XY mice. This is the first study demonstrating that sex chromosome genes are a driving force producing sex differences in the CB and FC, which are neuoranatomical regions involved in many normal functions and in neurobehavioral diseases.

Fig. 1

ISH of calbindin mRNA shows sex differences in the CB (a–d), the FC (e, f), and the SDN of the hypothalamus (g, h). Bright-field photomicrographs of coronal sections show Calb1 in juvenile mouse CB in low (a, b) and high (c, d) magnifications. Photographs of males (a, c, e, g) and females (b, d, f, h). Single arrows in a and b show regions pictured in c and d. Double arrows indicate neurons in the dorsal peduncular area of the FC (bregma 1.42 mm). Scale bars = 100 μm.

Fig. 2

Protein quantification of calbindin reveals sex differences in the cortex and CB. Calbindin protein quantification in CB and FC by Western blots. Each lane represents an individual animal. The ratio of calbindin to β-actin for each sample was measured (n = 5; animals were analyzed for each sex and region). Histograms represent the mean + SEM of the calbindin/β-actin ratio designating the protein level. * Females expressed significantly more calbindin protein (p < 0.05) than did males in both the CB and the FC.

Fig. 3

In the FC, ERα and the sex chromosome complement influence Calb1 mRNA in females. Mean + SEM Calb1 in the FC of WT and ERαKO (KO) mice (a) and in FCG mice (b). n = 5–6 mice per group. * Significantly different from all other groups (p < 0.05).

Fig. 4

In the CB, ERα modulates Calb1 mRNA in both sexes, but sex chromosome genes modulate the sexual dimorphism. Mean + SEM Calb1 in the CB of WT and ERαKO (KO) mice (a) and FCG mice (b). n = 5–6 mice per group. * Females had significantly greater concentrations of Calb1 mRNA than did males (p < 0.015). ** WT littermates had significantly more Calb1 mRNA than did ERαKO mice (p < 0.007). *** XX mice had significantly more Calb1 than did XY mice (p < 0.0001).