Inhibitor of differentiation (Id) genes are expressed in the steroidogenic cells of the ovine ovary and are differentially regulated by members of the transforming growth factor-beta family

Abstract

Inhibitor of differentiation (Id) proteins act during embryogenesis and development to repress gene transcription required for lineage commitment, while promoting cell growth. Growth factors belonging to the TGFbeta superfamily of signaling molecules, notably the bone morphogenetic proteins (BMPs) and activin, can regulate Id expression in these tissues. Id expression and function in adult physiology is less well determined, and we hypothesized a role for Id proteins in the adult mammalian ovary. Immunohistochemistry for Id1, Id2, Id3, and Id4 in the sheep ovary revealed consistent expression in granulosa and thecal cells of ovarian follicles throughout development. In atretic follicles, Id proteins were selectively down-regulated in thecal cells (P < 0.0001). Additionally, Id1 was universally up-regulated in the cumulus cells adjacent to the oocyte. Immunohistochemistry for phospho (p)-smad 1/5/8 signaling components (stimulated by BMPs) showed a punctate pattern of expression whereas p-smad 2/3 (stimulated by activin) was ubiquitously expressed in follicles. Neither pathway, however, displayed differential staining in line with Id1 cumulus-specific expression, suggesting a more complex relationship between Id1 expression and TGFbeta signaling in these cells. Nevertheless, in vitro, stimulation of ovine granulosa cells with BMP6 or activin A led to a respective increase and decrease in Id1 (P < 0.0001), Id2 (P < 0.0001), Id3 (P < 0.0001), and Id4 (P < 0.05) transcripts, and Id1 gene expression was further manipulated by the oocyte-secreted factors BMP15 and growth differentiation factor 9 (P < 0.001). These data confirm that TGFbeta signaling can regulate Id gene expression in the sheep ovarian follicle and suggest a functional role for the Id family in the mammalian ovary.

Figure 1

Immunohistochemistry for Id1 (a-c), Id2 (d-f), Id3 (g-i) and Id4 (j-l) protein (brown) in the sheep ovary, illustrates from left to right, staining in primordial or primary, secondary or early antral, and tertiary or late antral follicles. Arrow in b) indicates intense peri-oocytic Id1 expression. Insets in c), f), i) and l), are negative controls using the respective Id antibody binding protein to confirm antigen specific binding. Scale bars = 50μm.

Figure 2

Id2 expression in adjacent healthy and atretic follicles at low (a) and high (b) magnification and activated caspase-3 protein expression (c) in the corresponding ovine follicles. Arrows in a) show thecal layer and in c) depict positive caspase immunoreactivity indicating apoptosis in those cells. Scale bars = 50μm. Absent, partial or intense Id1 (d), Id2 (e), Id3 (f) and Id4 (g) expression was classified in thecal cells of healthy and atretic, pre-antral and antral follicles and is illustrated for each Id. Chi-squared statistical analysis showed a significant difference was between the healthy and atretic categories for Id1 (P<0.0001), Id2 (P<0.0001), Id3 (P<0.0001) and Id4 (P<0.0001).

Figure 3

Immunohistochemistry (brown) for p-smad 1/5/8 (a and b), p-smad 2/3 (d and e), smad 6 (g and h) and smad 7 (j and k) in the sheep ovary. Arrow in g) identifies smad 6 positive immunostaining in granulosa cells and in h) indicates staining in a blood vessel. Respective negative controls in c), f), i) and l) represent omission of the primary antibody. Scale bars = 50μm.

Figure 4

Co-immunofluorescence of Id1 (red) with p-smad 1/5/8 (green) (a) and smad 6 (green) (b) in follicles of the sheep ovary. Merged pictures include DAPI nuclear counterstain (blue). Scale bars = 100μm.

Figure 5

Effect of activin A and/or BMP6 (a), and GDF-9 and/or BMP15 (b) on Id1 gene expression, and activin A and/or BMP6 on Id2 (c), Id3 (d) and Id4 (e) gene expression, in ovine granulosa cells cultured for 24 h. Values are mean ± sem of relative mRNA levels standardised to control values (n=3; three experiments using cells from a total of ten sheep). An ANOVA analysis revealed overall significant differences for each gene; Id1 (a; P<0.0001 and b; P<0.001), Id2 (P<0.0001), Id3 (P<0.0001) and Id4 (P<0.05) and a pair-wise comparison to the control column using the Bonferroni post test are illustrated. *P<0.05, **P<0.01, ***P<0.001.