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Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development.

Smith CA, Shoemaker CM, Roeszler KN, Queen J, Crews D, Sinclair AH - BMC Dev. Biol. (2008)

Bottom Line: Inhibition of estrogen synthesis with a specific aromatase inhibitor results in a decline in chicken RSPO1 expression, indicating that RSPO1 is influenced by estrogen.These results indicate that RSPO1 is up-regulated in the embryonic gonads of female vertebrates with different sex-determining mechanisms.These findings suggest that R-SPONDIN1 is an ancient, conserved part of the vertebrate ovary-determining pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: The University of Melbourne Department of Paediatrics and Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, 3052, Australia. craig.smith@mcri.edu.au

ABSTRACT

Background: R-Spondin1 (Rspo1) is a novel regulator of the Wnt/beta-catenin signalling pathway. Loss-of-function mutations in human RSPO1 cause testicular differentiation in 46, XX females, pointing to a role in ovarian development. Here we report the cloning and comparative expression analysis of R-SPONDIN1 orthologues in the mouse, chicken and red-eared slider turtle, three species with different sex-determining mechanisms. Evidence is presented that this gene is an ancient component of the vertebrate ovary-determining pathway.

Results: Gonadal RSPO1 gene expression is female up-regulated in the embryonic gonads in each species at the onset of sexual differentiation. In the mouse gonad, Rspo1 mRNA is expressed in the somatic cell lineage at the time of ovarian differentiation (E12.5-E15.5), with little expression in germ cells. However, the protein is localised in the cytoplasm and at the cell surface of both somatic (pre-follicular) and germ cells. In the chicken embryo, RSPO1 expression becomes elevated in females at the time of ovarian differentiation, coinciding with female-specific activation of the FOXL2 gene and estrogen synthesis. RSPO1 protein in chicken is localised in the outer cortical zone of the developing ovary, the site of primordial follicle formation and germ cell differentiation. Inhibition of estrogen synthesis with a specific aromatase inhibitor results in a decline in chicken RSPO1 expression, indicating that RSPO1 is influenced by estrogen. In the red-eared slider turtle, which exhibits temperature-dependent sex determination, up-regulation of RSPO1 occurs during the temperature-sensitive period, when gonadal development is responsive to temperature. Accordingly, RSPO1 expression is temperature-responsive, and is down-regulated in embryos shifted from female- to male-producing incubation temperatures.

Conclusion: These results indicate that RSPO1 is up-regulated in the embryonic gonads of female vertebrates with different sex-determining mechanisms. In all instances, RSPO1 is expressed in the incipient ovary. These findings suggest that R-SPONDIN1 is an ancient, conserved part of the vertebrate ovary-determining pathway.

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Gonadal expression of chicken WNT4, assessed by in situ hybridization. A) cWNT4 is expressed in the gonads of both sexes at E4.5 (prior to sexual differentiation) (arrows). Expression is similar between the sexes at E6.5 (near the beginning of sexual differentiation), but is down-regulated in males (ZZ) by E8.5. B) Transverse sections of over-stained E8.5 whole mounts showing cWNT4 expression in the outer region of the female gonad. Weak staining in the male gonad represents background.
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Figure 4: Gonadal expression of chicken WNT4, assessed by in situ hybridization. A) cWNT4 is expressed in the gonads of both sexes at E4.5 (prior to sexual differentiation) (arrows). Expression is similar between the sexes at E6.5 (near the beginning of sexual differentiation), but is down-regulated in males (ZZ) by E8.5. B) Transverse sections of over-stained E8.5 whole mounts showing cWNT4 expression in the outer region of the female gonad. Weak staining in the male gonad represents background.

Mentions: The RSPO1 expression profile in embryonic chicken gonads was compared with that of WNT4, which also signals through β-catenin and is known to be important for proper ovary development in mammals. As assessed by whole mount in situ hybridisation, cWNT4 was expressed in the gonads of both sexes from E4.5, prior to gonadal sex differentiation. Expression became sexually dimorphic during gonadal sex differentiation (E6.5 – 8.5), being up-regulated in females and down-regulated in males. Little expression was observed in the adjoining mesonephros (Fig. 4A). In sections of over-stained tissues, WNT4 mRNA was localised towards the outer (cortical) region of the developing ovary (Fig. 4B).


Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development.

Smith CA, Shoemaker CM, Roeszler KN, Queen J, Crews D, Sinclair AH - BMC Dev. Biol. (2008)

Gonadal expression of chicken WNT4, assessed by in situ hybridization. A) cWNT4 is expressed in the gonads of both sexes at E4.5 (prior to sexual differentiation) (arrows). Expression is similar between the sexes at E6.5 (near the beginning of sexual differentiation), but is down-regulated in males (ZZ) by E8.5. B) Transverse sections of over-stained E8.5 whole mounts showing cWNT4 expression in the outer region of the female gonad. Weak staining in the male gonad represents background.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2519078&req=5

Figure 4: Gonadal expression of chicken WNT4, assessed by in situ hybridization. A) cWNT4 is expressed in the gonads of both sexes at E4.5 (prior to sexual differentiation) (arrows). Expression is similar between the sexes at E6.5 (near the beginning of sexual differentiation), but is down-regulated in males (ZZ) by E8.5. B) Transverse sections of over-stained E8.5 whole mounts showing cWNT4 expression in the outer region of the female gonad. Weak staining in the male gonad represents background.
Mentions: The RSPO1 expression profile in embryonic chicken gonads was compared with that of WNT4, which also signals through β-catenin and is known to be important for proper ovary development in mammals. As assessed by whole mount in situ hybridisation, cWNT4 was expressed in the gonads of both sexes from E4.5, prior to gonadal sex differentiation. Expression became sexually dimorphic during gonadal sex differentiation (E6.5 – 8.5), being up-regulated in females and down-regulated in males. Little expression was observed in the adjoining mesonephros (Fig. 4A). In sections of over-stained tissues, WNT4 mRNA was localised towards the outer (cortical) region of the developing ovary (Fig. 4B).

Bottom Line: Inhibition of estrogen synthesis with a specific aromatase inhibitor results in a decline in chicken RSPO1 expression, indicating that RSPO1 is influenced by estrogen.These results indicate that RSPO1 is up-regulated in the embryonic gonads of female vertebrates with different sex-determining mechanisms.These findings suggest that R-SPONDIN1 is an ancient, conserved part of the vertebrate ovary-determining pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: The University of Melbourne Department of Paediatrics and Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, 3052, Australia. craig.smith@mcri.edu.au

ABSTRACT

Background: R-Spondin1 (Rspo1) is a novel regulator of the Wnt/beta-catenin signalling pathway. Loss-of-function mutations in human RSPO1 cause testicular differentiation in 46, XX females, pointing to a role in ovarian development. Here we report the cloning and comparative expression analysis of R-SPONDIN1 orthologues in the mouse, chicken and red-eared slider turtle, three species with different sex-determining mechanisms. Evidence is presented that this gene is an ancient component of the vertebrate ovary-determining pathway.

Results: Gonadal RSPO1 gene expression is female up-regulated in the embryonic gonads in each species at the onset of sexual differentiation. In the mouse gonad, Rspo1 mRNA is expressed in the somatic cell lineage at the time of ovarian differentiation (E12.5-E15.5), with little expression in germ cells. However, the protein is localised in the cytoplasm and at the cell surface of both somatic (pre-follicular) and germ cells. In the chicken embryo, RSPO1 expression becomes elevated in females at the time of ovarian differentiation, coinciding with female-specific activation of the FOXL2 gene and estrogen synthesis. RSPO1 protein in chicken is localised in the outer cortical zone of the developing ovary, the site of primordial follicle formation and germ cell differentiation. Inhibition of estrogen synthesis with a specific aromatase inhibitor results in a decline in chicken RSPO1 expression, indicating that RSPO1 is influenced by estrogen. In the red-eared slider turtle, which exhibits temperature-dependent sex determination, up-regulation of RSPO1 occurs during the temperature-sensitive period, when gonadal development is responsive to temperature. Accordingly, RSPO1 expression is temperature-responsive, and is down-regulated in embryos shifted from female- to male-producing incubation temperatures.

Conclusion: These results indicate that RSPO1 is up-regulated in the embryonic gonads of female vertebrates with different sex-determining mechanisms. In all instances, RSPO1 is expressed in the incipient ovary. These findings suggest that R-SPONDIN1 is an ancient, conserved part of the vertebrate ovary-determining pathway.

Show MeSH