Gonadal expression of Foxo1, but not Foxo3, is conserved in diverse Mammalian species

Abstract

The Foxos are key effectors of the PI3K/Akt signaling pathway and regulate diverse physiologic processes. Two of these factors, Foxo1 and Foxo3, serve specific roles in reproduction in the mouse. Foxo3 is required for suppression of primordial follicle activation in females, while Foxo1 regulates spermatogonial stem cell maintenance in males. In the mouse ovary, Foxo1 is highly expressed in somatic cells (but not in oocytes), suggesting an important functional role for Foxo1 in these cells. Given that invertebrate model species such as Caenorhabditis elegans and Drosophila melanogaster harbor a single ancestral Foxo homolog, these observations suggest that gene duplication conferred a selective advantage by permitting the Foxos to adopt distinct roles in oogenesis and spermatogenesis. Our objective was to determine if the remarkably specific expression patterns of Foxo1 and Foxo3 in mouse gonads (and, by inference, Foxo function) are conserved in diverse mammalian species. Western blotting was used to validate isoform-specific antibodies in rodents, companion animals, farm animals, nonhuman primates, and humans. Following validation of each antibody, immunohistochemistry was performed to ascertain Foxo1 and Foxo3 gonadal expression patterns. While Foxo1 expression in spermatogonia and granulosa cells was conserved in each species evaluated, Foxo3 expression in oocytes was not. Our findings suggest that Foxo3 is not uniquely required for primordial follicle maintenance in nonrodent species and that other Foxos, particularly Foxo1, may contribute to oocyte maintenance in a functionally redundant manner.

FIG. 1

Comparative analysis of Foxo1 and Foxo3 proteins in diverse vertebrate species. A) Scientific and common names of the species included in this study. Pairwise alignment scores (HomoloGene, NCBI database) were used to calculate the identity (%) shared with the human FOXO1 and FOXO3 homologs. Sequence data were not available for deer mouse, cat, pig, and baboon. B) Phylogrammatic representation of the data (ClustalW2) reveals that each Foxo protein is more similar to its cross-species orthologs (e.g., mouse vs. human Foxo1) than to the other Foxos within the same species (e.g., mouse Foxo1 vs. mouse Foxo3).

FIG. 2

Validation of Foxo1 antibody in diverse mammalian species. Tissue homogenates and cell lysates were subjected to SDS-PAGE and Western blot analysis. A) The Foxo1 (rabbit monoclonal) antibody employed in this study detected 72- to 75-kDa proteins consistent with Foxo1. Note that the image is uncropped to show all detected molecular species; the minor secondary bands appear to be related to Foxo1. The membrane was then stripped and reprobed with actin and GAPDH control antibodies. Although actin and GAPDH are highly conserved phylogenetically, the respective antibodies did not detect the proteins in all species. B) The same Foxo1 antibody failed to detect a protein in Foxo1 null cells. The principal band remained detectable in untreated cells as well as control homogenate (liver).

FIG. 3

Validation of Foxo3 antibody in diverse mammalian species. Tissue homogenates and cell lysates were subjected to SDS-PAGE and Western blot analysis. A) The Foxo3 (rabbit polyclonal) antibody employed in this study for IHC detected 80- to 86-kDa proteins consistent with Foxo3. In one species (cat), the Foxo3 protein was of lower apparent molecular weight (65 kDa, asterisk). The secondary bands in this uncropped image appear to be degradation products or variants of Foxo3. The membrane was stripped and reprobed with the control antibodies actin and GAPDH. B) The same Foxo3 antibody failed to detect a protein in Foxo3 null cells. C) Western blotting with an unrelated Foxo3 antibody (ab2, rabbit monoclonal) confirmed that the cat 65-kDa protein detected in A is indeed Foxo3 (asterisk). The principal bands detected for mouse, dog, and human were of the expected molecular weight range.

FIG. 4

Foxo1 expression in spermatogonial stem cells is conserved across all mammalian species studied. A) Immunohistochemical detection and localization of Foxo1 protein in tissue sections from paraffin-embedded testes. Foxo1 is clearly localized to a rare population of cells that rest on the seminiferous tubule basement membrane, consistent with undifferentiated spermatogonia. Bar = 25 μm. B) Foxo1 and Plzf immunofluorescence in tissue sections from paraffin-embedded testes. The two markers were coexpressed in all species studied. Representative fields from four species are shown. All images are at ×63 magnification except mouse (×40).

FIG. 5

Foxo1 expression in ovarian granulosa cells is conserved across all mammalian species studied. Foxo1 has been previously shown to be the principal Foxo in ovarian granulosa cells in mice. At low magnification (upper panels), immunohistochemistry against Foxo1 showed expression in developing follicles in all species. Granulosa cell expression was induced at the primary to secondary follicle stage and remained prominent in antral/preovulatory follicles in all species. Higher magnification (lower panels) contrasts the absence of expression in pregranulosa cells in primordial follicles (insets) with prominent granulosa cell expression in adjacent, more advanced follicles. Bars = 200 μm and 50 μm for upper and lower panels, respectively.

FIG. 6

Foxo3 expression in primordial oocytes and Foxo3 nuclear-to-cytoplasmic translocation following primordial follicle activation is conserved only in rodents and not other mammalian species. Foxo3 is the principal Foxo in the mouse oocyte, and its nuclear-to-cytoplasmic translocation is believed to be an important regulator of primordial follicle activation. A) Immunohistochemistry against Foxo3 shows exclusive nuclear expression in primordial follicle oocytes in mouse, rat, and deer mouse. Nuclear-to-cytoplasmic translocation was evident in larger primary follicles in all three species, followed by degradation during the secondary follicle stage, as previously demonstrated in mouse (not shown). In companion and farm animals (B) and primates, including human (C), no specific Foxo3 localization was evident. Only a faint and likely nonspecific signal was apparent in some nonrodent species. D) Staining in vascular smooth muscle in all species served as an internal positive control. Examples for dog and cow are shown. Bars = 10 μm.

FIG. 7

Foxo1 is expressed in bovine primordial oocytes. Immunohistochemistry against Foxo1 shows nuclear expression within primordial follicle oocytes. Expression persisted in some primary follicles, whereas it was absent in others. Evidence of nuclear-to-cytoplasmic translocation was detected in some primary follicles. Nuclear Foxo1 expression was variable in the oocytes of secondary follicles. Bars = 25 μm.