Estrogen modulates neuronal movements within the developing preoptic area-anterior hypothalamus

Abstract

The preoptic area-anterior hypothalamus (POA-AH) is characterized by sexually dimorphic features in a number of vertebrates and is a key region of the forebrain for regulating physiological responses and sexual behaviours. Using live-cell fluorescence video microscopy with organotypic brain slices, the current study examined sex differences in the movement characteristics of neurons expressing yellow fluorescent protein (YFP) driven by the Thy-1 promoter. Cells in slices from embryonic day 14 (E14), but not E13, mice displayed significant sex differences in their basal neuronal movement characteristics. Exposure to 10 nm estradiol-17beta (E2), but not 100 nm dihydrotestosterone, significantly altered cell movement characteristics within minutes of exposure, in a location-specific manner. E2 treatment decreased the rate of motion of cells located in the dorsal POA-AH but increased the frequency of movement in cells located more ventrally. These effects were consistent across age and sex. To further determine whether early-developing sex differences in the POA-AH depend upon gonadal steroids, we examined cell positions in mice with a disruption of the steroidogenic factor-1 gene, in which gonads do not form. An early-born cohort of cells were labelled with the mitotic indicator bromodeoxyuridine (BrdU) on E11. More cells were found in the POA-AH of females than males on the day of birth (P0) regardless of gonadal status. These results support the hypothesis that estrogen partially contributes to brain sexual dimorphism through its influence on cell movements during development. Estrogen's influence may be superimposed upon a pre-existing genetic bias.

Figure 1. Fluorescence microscopy reveals two distinct groups of cells expressing YFP in the developing POA/AH

Panels A and B show low magnification fluorescent photomicrographs of the POA/AH at E13 and E14, respectively. At both ages two distinct groups of YFP+ cells are evident, designated dorsal (dashed box) and ventral (solid box). Panels C and D show higher magnification photomicrographs of the field of view used for video acquisition. AC, anterior commissure; V, third ventricle. The scale bar in A represents 250μm for panels A and B and the scale bar in C represents 40μm for panels C and D.

Figure 2. Neuronal movement characteristics differ between cells in slices taken from males and those in slices taken from females at E14 in the ventral cell group

Representative cell tracking schematics show that YFP+ cells in slices taken from females (panels A and B) move faster (average distance between any two points) and more frequently (average number of points) than YFP+ cells in slices taken from males (panels C and D) in both baseline and 10nM E2 conditions. Each track represents the path taken by a given cell in 1.5h, with each point showing a tracked position of the cell and the open point showing the starting position. Although E2 increased the average speed and probability of movement in both sexes, a sex difference exists in both conditions. Scale bars show dimension in x and y orientations.

Figure 3. Administration of 10nM Estradiol-17β, but not 100nM DHT, caused an increase in the average speed of YFP+ cells in the ventral cell group independent of age or sex

E2 administration significantly increased the average speed of YFP+ cells in slices taken from either males or females at E13 or E14 (black arrows in A; p<0.05). A sex difference in the basal average speed and frequency of movement emerges at E14 with YFP+ cells in slices taken form females moving significantly faster and more often (* over bracket in panel B; p<0.05) than YFP+ cells in slices taken from males. Panel C shows that YFP+ cells in slices taken from females at E14 respond to E2 but not DHT with increased speed and frequency of movement (** over bracket; p<0.01). Values are mean ± SEM for female YFP+ cells (n=13) and male YFP+ cells (n=11).

Figure 4. Administration of 10nM Estradiol-17β decreases the average speed and movement speed of YFP+ cells in the dorsal cell group independent of age or sex

E2 administration (A) significantly decreases the average speed (black arrows; p<0.05) of YFP+ cells and (B) also decreases the average speed of movement (black arrows; p<0.01) of YFP+ cells in slices taken from either males or females at E13 or E14. Values are mean±SEM for female YFP+ cells (n=10) and male YFP+ cells n=22.

Figure 5. The effect of estradiol-17β on YFP+ cells was relatively rapid in the dorsal group

Schematic, cumulative distance graphs of two representative cells in the dorsal group, in which E2 decreases the average speed, from a slice taken from a female at E14 demonstrate the speed of action of E2. The same two cells are shown before and after E2 administration. The slope of each line represents the average speed of that particular cell; level portions of the graph show that the cell did not move between those frames. Each line represents 1.5h of video.

Figure 6. BrdU birthdate labeling in the POA shows a significant effect of sex with no effect of genotype

Males (A,B) had fewer cells labeled for BrdU than females (C,D) when BrdU was administered at embryonic day 11 and examined at postnatal day 0. Wildtype males (A) had similar numbers of BrdU labeled cells to SF-1 knockout (KO) males (B), and wildtype females (C) had similar numbers of BrdU labeled cells to SF-1 knockout females (D). AC, anterior commisure; V, third ventricle. * p < 0.01.

Figure 7. Immunocytochemistry reveals the phenotype of the YFP+ cells

(A) ERα immunoreactive (red) and YFP-immunoreactive (green) cells were both easily detected in the dorsal region, but not in the same cells, as early as E13. (B) nNOS-immunoreactive (red) and YFP-immunoreactive (green) cells were detectable in the caudal dorsal region in an overlapping, though not co-localizing pattern as early as E13. (C) In caudal sections, some but not all YFP+ (green) cells also contained immunoreactive calbindin (red). (D) Graphic representation of the typical distribution of specific expression patterns in the POA/AH. AC=anterior commisure; V=third ventricle.