Comparative Analysis of Anti-Mouse SRY Antibodies

Abstract

The Y chromosome gene SRY is the initiator of male sexual differentiation in mammals, but the molecular and cellular mechanisms operating downstream of SRY remain undefined. A deeper understanding of these issues relies on the ability to visualize SRY protein endogenously under a number of experimental conditions. Here we compare the specificity and effectiveness of several available antibodies to mouse SRY. Two antibodies cross-reacted with other SOX proteins in immunofluorescence analyses of transfected cells, and one of these two was unable to detect SRY on Western blots. A third antibody was both avid and specific, and was able to detect endogenous SRY in developing Sertoli cells in mouse genital ridges. Our findings underline the need to distinguish between useful and spurious reagents for biochemical and immunolocalization studies involving mouse SRY protein.

Loss of Mitogen-Activated Protein Kinase Kinase Kinase 4 (MAP3K4) Reveals a Requirement for MAPK Signalling in Mouse Sex Determination

“…Fgf9 transcript levels appeared to be reduced in XY byg/byg gonads, although this difference was not statistically significant. We then studied the expression of SRY protein in mutant and control gonads at the same stage using a specific antibody to SRY [39],[68]. Expression of SRY was observed in somatic cells of the developing gonad at 11.5 dpc in control XY gonads (Figure 5D and 5F).…”

Loss of Mitogen-Activated Protein Kinase Kinase Kinase 4 (MAP3K4) Reveals a Requirement for MAPK Signalling in Mouse Sex Determination

“…Fgf9 transcript levels appeared to be reduced in XY byg/byg gonads, although this difference was not statistically significant. We then studied the expression of SRY protein in mutant and control gonads at the same stage using a specific antibody to SRY [39],[68]. Expression of SRY was observed in somatic cells of the developing gonad at 11.5 dpc in control XY gonads (Figure 5D and 5F).…”

NRG1 signalling regulates the establishment of Sertoli cell stock in the mouse testis

“…An extended time of expression was evident in immunofluorescence analysis and SRY-positive cells were readily detectable in XY Wt1-Cre Tg/ + ; Nrg1 fl/fl gonads in comparison with controls at 12.5 dpc (Fig. 3C) (Bradford et al, 2007). Following Sry expression, the Sertoli differentiate and synthetizes the growth factor FGF9, that in turn stimulates (caption on next page) the proliferation of the progenitor cells.…”

Testicular Differentiation Occurs in Absence of R-spondin1 and Sox9 in Mouse Sex Reversals

“…For paraffin-embedded and Cryomount-embedded samples, sections of 5 and 8 µm thickness were processed, respectively. The following dilutions of primary antibodies were used: AMH/MIS (C-20, sc-6886, Santa Cruz), 1∶200; AR (sc-816, SantaCruz), 1∶100; DMRT1 (kindly provided by David Zarkower), 1∶500; FOXL2 (ab5096, Abcam), 1∶250; γH2AX (U5-636, Upstate), 1∶500; GATA1 (sc-265, SantaCruz), 1∶50; SDMG1 (a kind gift from Ian Adams) 1∶2000; SF1 (kindly provided by Ken Morohashi) 1∶1500; SOX9 (HPA-001758, Sigma) 1∶250 and SRY [59], [66] 1∶100, STRA8 (ab49602, Abcam), 1∶100. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) was used to detect nuclei (in blue).…”

Loss of Mitogen-Activated Protein Kinase Kinase Kinase 4 (MAP3K4) Reveals a Requirement for MAPK Signalling in Mouse Sex Determination

“…Fgf9 transcript levels appeared to be reduced in XY byg/byg gonads, although this difference was not statistically significant. We then studied the expression of SRY protein in mutant and control gonads at the same stage using a specific antibody to SRY [39],[68]. Expression of SRY was observed in somatic cells of the developing gonad at 11.5 dpc in control XY gonads (Figure 5D and 5F).…”

NRG1 signalling regulates the establishment of Sertoli cell stock in the mouse testis

“…An extended time of expression was evident in immunofluorescence analysis and SRY-positive cells were readily detectable in XY Wt1-Cre Tg/ + ; Nrg1 fl/fl gonads in comparison with controls at 12.5 dpc (Fig. 3C) (Bradford et al, 2007). Following Sry expression, the Sertoli differentiate and synthetizes the growth factor FGF9, that in turn stimulates (caption on next page) the proliferation of the progenitor cells.…”

Testicular Differentiation Occurs in Absence of R-spondin1 and Sox9 in Mouse Sex Reversals

“…For paraffin-embedded and Cryomount-embedded samples, sections of 5 and 8 µm thickness were processed, respectively. The following dilutions of primary antibodies were used: AMH/MIS (C-20, sc-6886, Santa Cruz), 1∶200; AR (sc-816, SantaCruz), 1∶100; DMRT1 (kindly provided by David Zarkower), 1∶500; FOXL2 (ab5096, Abcam), 1∶250; γH2AX (U5-636, Upstate), 1∶500; GATA1 (sc-265, SantaCruz), 1∶50; SDMG1 (a kind gift from Ian Adams) 1∶2000; SF1 (kindly provided by Ken Morohashi) 1∶1500; SOX9 (HPA-001758, Sigma) 1∶250 and SRY [59], [66] 1∶100, STRA8 (ab49602, Abcam), 1∶100. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) was used to detect nuclei (in blue).…”

Loss of Mitogen-Activated Protein Kinase Kinase Kinase 4 (MAP3K4) Reveals a Requirement for MAPK Signalling in Mouse Sex Determination

“…Fgf9 transcript levels appeared to be reduced in XY byg/byg gonads, although this difference was not statistically significant. We then studied the expression of SRY protein in mutant and control gonads at the same stage using a specific antibody to SRY [39],[68]. Expression of SRY was observed in somatic cells of the developing gonad at 11.5 dpc in control XY gonads (Figure 5D and 5F).…”

NRG1 signalling regulates the establishment of Sertoli cell stock in the mouse testis

“…An extended time of expression was evident in immunofluorescence analysis and SRY-positive cells were readily detectable in XY Wt1-Cre Tg/ + ; Nrg1 fl/fl gonads in comparison with controls at 12.5 dpc (Fig. 3C) (Bradford et al, 2007). Following Sry expression, the Sertoli differentiate and synthetizes the growth factor FGF9, that in turn stimulates (caption on next page) the proliferation of the progenitor cells.…”

Testicular Differentiation Occurs in Absence of R-spondin1 and Sox9 in Mouse Sex Reversals

“…For paraffin-embedded and Cryomount-embedded samples, sections of 5 and 8 µm thickness were processed, respectively. The following dilutions of primary antibodies were used: AMH/MIS (C-20, sc-6886, Santa Cruz), 1∶200; AR (sc-816, SantaCruz), 1∶100; DMRT1 (kindly provided by David Zarkower), 1∶500; FOXL2 (ab5096, Abcam), 1∶250; γH2AX (U5-636, Upstate), 1∶500; GATA1 (sc-265, SantaCruz), 1∶50; SDMG1 (a kind gift from Ian Adams) 1∶2000; SF1 (kindly provided by Ken Morohashi) 1∶1500; SOX9 (HPA-001758, Sigma) 1∶250 and SRY [59], [66] 1∶100, STRA8 (ab49602, Abcam), 1∶100. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) was used to detect nuclei (in blue).…”