Sex-specific differences in mouse DMRT1 expression are both cell type- and stage-dependent during gonad development

Abstract

Immunohistochemistry was used to examine GCNA1, a germ cell-specific protein, together with DMRT1 (Doublesex and Mab-3-related transcription factor-1), a transcription factor implicated in Sertoli cell and germ cell function, in order to resolve DMRT1's cellular profile during pre- and postnatal gonad development in the mouse. In the indifferent gonad (10.5-11.5 days postcoitus [dpc]), DMRT1 localized to somatic cells and GCNA1(+) germ cells and was indistinguishable in males and females. By 12.5 dpc, a clear sexual preference for DMRT1 in male somatic cells was observed, with male DMRT1 localized to testicular cords and more abundant in Sertoli cells than in germ cells and female DMRT1 diffusely labeled and markedly lower in somatic cells than in germ cells. A male somatic preference continued throughout development, with DMRT1 evident in Sertoli cells at all ages examined and absent in ovarian somatic cells from 13.5 dpc onward. In contrast, expression in primordial germ cells was not sexually distinct, and both sexes showed DMRT1 increasing through 13.5 dpc and absent by 15.5 dpc. Notably, sexual differences in germ cell DMRT1 were detected after birth, when it was detected only in spermatogonia of the testis. Colocalization of DMRT1 with proliferation markers KI67 and proliferating cell nuclear antigen (PCNA) and stem cell markers OCT4 (also known as POU5F1) and NGN3 indicated that, in postnatal testes, DMRT1 was present in both stem and proliferating spermatogonia. Together, the findings implicate opposite functions for DMRT1 in somatic and germ cells of the testis. In Sertoli cells, DMRT1 expression correlated with differentiation, whereas in germ cells, it suggested a role in expansion and maintenance of undifferentiated spermatogonia.

Figure 1. DMRT1 antibody specifically

Immunohistochemical analysis of sequential sections from 15.5 dpc mouse testis using either A) DMRT1 immune serum or B) DMRT1 immune serum pre-adsorbed with its peptide antigen. C) Western blot analysis of lysates from MSC-1 cells transfected with a pcDNA3-based expression vector for rat DMRT1 (DMRT1) or empty pcDNA3 (vector), using the DMRT1 antibody (left) or pre-immune serum (right). D) Western blot analysis of tissue lysates from 17 day-old mice. Positions of the molecular weight markers (kDa) are indicated on the left. The membrane was stained with Ponceau S (left) prior to detection with the DMRT1 antibody (right). Both 10 and 20 micrograms of protein lysate (marked above the lane) were used for each indicated tissue. A and B; 20X magnification.

Figure 2. DMRT1 and GCNA1 expression in mouse embryonic gonads prior to sexual differentiation

Immunohistochemistry was used to examine DMRT1 and GCNA1 protein expression during gonad development. A) The DMRT1-specific antibody was used to reveal DMRT1 expression in 10.5 dpc XY embryo. Expression was detected by enzymatic staining for horse radish peroxidase. 4X magnification. B & C) Higher magnification of boxed region in A or B, respectively, showing expression in the genital ridge. Immunofluorescent detection of DMRT1 (green) and GCNA1 (red) in 11.5 dpc XY (D–F) and XX (G–I) gonads and 12.5 dpc XY (J–L) and XX (M–O) gonads. Insets show higher magnification with arrowheads indicating DMRT1⁺ germ cells, arrows DMRT1⁺ somatic cells, and diamonds DMRT1⁻ germ cells. Merged images (F, I, L, & O) show localization of DMRT1 in germ cells (orange/yellow). D–O were cropped from original 20X magnification.

Figure 3. DMRT1 expression in 13.5 dpc developing mouse gonads

Enzymatic (left) and immunofluorescent detection of DMRT1 (green) and GCNA1 (red) in gonads from 13.5 dpc XY (top) and XX (bottom) embryos. Insets show higher magnification with arrowheads indicating DMRT1⁺ germ cells and arrows DMRT1⁺ somatic cells. Merged images (right) show localization of DMRT1 in germ cells (orange/yellow). 20X magnification.

Figure 4. DMRT1 expression in 15.5 dpc developing mouse gonads

Enzymatic (left) and immunofluorescent detection of DMRT1 (green) and GCNA1 (red) in gonads from 15.5 dpc XY (top) and XX (bottom) embryos. Insets show higher magnification with arrowheads indicating DMRT1⁺ germ cells and arrows DMRT1⁺ somatic cells. Merged images (right) show localization of DMRT1 in germ cells (orange/yellow). Top, 20X magnification. Bottom, 32X magnification.

Figure 5. DMRT1 expression during postnatal development of the mouse ovary

Enzymatic (left) and immunofluorescent detection of DMRT1 (green, 2^nd column) and GCNA1 (red, 3^rd column) individually, or as merged images (far right column) in ovaries isolated from newborn (P 0.5), 17 days postpartum (17dpp), and adult mice. No specific staining for Dmrt1 was detected in images for Dmrt1, either alone (2^nd column from left) or merged with GCNA (right). 0.5 dpp, 32X magnification. 17 dpp, 20X magnification. Adult enzymatic 10X magnification. Adult immunofluorescence, 6.4X magnification.

Figure 6. DMRT1 expression during postnatal development of the mouse testis

Dual immunofluorescent staining for DMRT1 (green) and GCNA1 (red) in testes isolated from 0.5 days postpartum (dpp), 2 dpp, 7 dpp, 8 dpp, 10 dpp, 20 dpp, 35 dpp, and adult mice. Merged images show DMRT1 expressed in Sertoli cells (green) and co-expressed with the germ cell marker GCNA (orange/yellow cells) at all stages of postnatal testis development. At pre-pubertal stages from 8 to 20 dpp, individual tubules were designated according to the number of positive germ cells and their DMRT1 levels. The noted patterns were designated H (abundant, high-expressing and few negative cells), M (abundant, moderately-expressing cells) and L (predominantly DMRT1⁻germ cells). 0.5 dpp, 40X magnification. All others 20X magnification.

Figure 7. DMRT1 expression in mitotically active germ cells of the postnatal developing mouse testis

Dual immunofluorescent staining was performed for DMRT1 (green) and KI67 (red) in testes isolated from 0.5 dpp and adult mice. Likewise, triple immunofluorescent staining was performed for DMRT1 (green), GCNA (red), and PCNA (blue) in testes isolated from 2 dpp, 7 dpp, 8 dpp, and 10 dpp. Shown are merged images, which indicate DMRT1 in proliferating Sertoli cells at 0.5 dpp (yellow/orange) through 8 dpp (light blue), and in proliferating germ cells from 2 dpp through adult. In merged images of 2 dpp, 7 dpp, 8 dpp, and 10 dpp testes, DMRT1⁺ and DMRT1⁻ proliferating germ cells project as white/light pink and dark pink cells, respectively. In the adult, DMRT1⁺ proliferating germ cells are depicted as yellow/orange cells in the merged images. 0.5 dpp, 40X magnification. Others, 20X magnification.

Figure 8. DMRT1 and OCT4 expression during postnatal development of the mouse testis

DMRT1 (green) and OCT4 (blue) were visualized by dual immunofluorescence and DNA by TOTO-3 counter-staining (white/grey) in testes isolated from 0.5 dpp, 1 dpp, 2 dpp, 7 dpp, 10 dpp, and adult mice. Merged images show DMRT1⁺/OCT4⁺ germ cells (aqua) at all ages examined. DMRT1 (red) and NGN3 (green) were also visualized by dual immunofluorescence in testes from 7 dpp mice, revealing DMRT1⁺/NGN3⁺ germ cells. DMRT1⁻/OCT4⁺ (dark blue) or DMRT1⁻/NGN3⁺ (green) cells were infrequent or not observed at the time points tested. 0.5 dpp, 2dpp, & 7dpp are 40X; 10 dpp, & adult 60X; Ngn3/7 dpp 20X.