Although originally discovered as a GPI-anchored cell-surface protein involved in neuronal differentiation and cell-cell contact in the developing nervous system (13
), it is now evident that Dragon can also act as a co-receptor for BMP 2 and 4 (17
). In addition to previously reported sites of expression in neural tissues (13
), our studies demonstrate that Dragon is also expressed in many specific cell types throughout the reproductive system. In addition, we found that Dragon was expressed in numerous cell lines derived from reproductive tissues, and that Dragon expression enhanced responsiveness of Ishikawa and KGN cells to BMP2 and 4. Thus, our results suggest that Dragon may have important roles in mediating BMP signaling in reproduction.
To define specific sites where Dragon-mediated BMP signaling might be important in reproduction, we explored cell-specific expression in the male and female reproductive tracts. In males, BMP 8a and b are expressed in maturing spermatocytes, and BMP8b knockout males are infertile due to developmental arrest and degeneration of spermatocytes (11
), suggesting that these BMP’s are critical for normal spermatocyte development. BMP receptors ALK3 and BMPRII are localized in postnatal spermatogonia and BMP4 is produced by Sertoli cells very early in postnatal development, consistent with an ongoing requirement for BMP signaling in the testis (30
). In addition, BMP2 primarily stimulates spermatogonial proliferation while BMP7 acts mainly on Sertoli cells in the testis from 7-day-old mice (31
). Our results extend these findings to a novel BMP co-receptor which enhances BMP signaling, since in 3 day old male mice, Dragon was highly expressed in gonocytes before and after they migrated from the tubule lumen to their basal position, with this immunoreactivity being maintained as spermatogonia in Day 9 animals. By day 21, staining in spermatogonia was substantially diminished but gonocytes remaining within the tubule lumen were still positive. In adults, Dragon staining appeared in maturing spermatocytes but not in other testicular cell types. The shift in expression from gonocytes and spermatogonia in juvenile animals to spermatocytes in mature males suggests that the role of BMPs may change as the testes mature to produce active sperm. Taken together, these results suggest a critical role for BMPs in regulating testis development and spermatogenesis and suggest that Dragon may be an important mediator of these processes.
BMP 4, 7 and 8A are expressed in the epididymis, and knockout of each gene by itself resulted in degeneration of the epididymal epithelium (10
). These results indicate a role for BMPs in the control of epididymal function. Interestingly, Dragon was strongly expressed on the apical surface of polarized epididymal epithelium in immature and mature males consistent with a role for Dragon in enhancing this essential BMP signaling.
In females, both BMPs and their receptors have been identified in numerous ovarian cell types, including oocytes and granulosa cells (8
). In vitro
studies have demonstrated that BMP 2, 4, 6 7 and 15 regulate granulosa cell functions and BMP4 and 7 promote the primordial-to-primary follicle transition during follicle maturation (reviewed in (8
)). The significance of BMP signaling in ovarian function is also underscored by the altered ovulation rates in Inverdale sheep with a natural point mutation in the BMP 15 gene (32
) and in Booroola sheep with a point mutation in the BMPR-IB gene (33
). Our results demonstrate that in the ovary, Dragon is expressed exclusively in oocytes, and most prominently in oocytes within secondary follicles. This is a time of oocyte growth and cytoplasmic maturation (34
), suggesting that BMP signaling in general, and Dragon enhancement of this signaling in particular, may be important for growth and maturation of oocytes.
BMP signaling components are expressed in a variety of cells within the rat uterus (9
). BMP2 mRNA is restricted to periluminal stroma and BMP7 is expressed in periluminal stroma and glandular epithelial cells while BMP 4 and BMP 6 are expressed in blood vessels in the uterus. BMPIA, BMPRIB and BMPRII are expressed in a number of cell types in the uterus including luminal and glandular epithelial cells. We observed Dragon expression in luminal and glandular epithelial cells of the mouse endometrium, suggesting that Dragon may enhance BMP signals involved in regulating uterine maturation in preparation for implantation.
BMP 2, 4, 6, 7 and 15 are expressed in mouse pituitary and BMP 6, 7 and 15 have been shown to stimulate FSH synthesis and secretion (25
). BMP 6 and 7 can also stimulate FSH mRNA biosynthesis in LβT2 mouse pituitary cells in culture (27
). We observed Dragon expression in LβT2 cells, as well as in numerous cells within the mouse pituitary, some of which also stained for FSH. These results suggest that BMPs may act in an autocrine manner to modulate FSH biosynthesis and that Dragon may enhance this process.
In cell culture studies using cell lines from the reproductive tract, we found that Dragon expression enhanced the response to endogenous BMP ligand, as well as low doses of exogenous BMP2 and 4, results that agree with our earlier observations in non-reproductive cell lines (36
). While the precise mechanism for this signaling enhancement has not yet been fully elucidated, our immunocytochemical analysis indicates that Dragon is located on the plasma membrane in discrete patches, consistent with its belonging to the class of GPI-anchored proteins which are known to localize in lipid rafts (37
). Moreover, our earlier studies indicate that Dragon can interact directly with BMPRII, ActRII, and the Alk3 and Alk6 type I receptors (36
). Taken together, these results suggest a model for enhanced BMP signaling in which Dragon acts as a BMP co-receptor in collecting type II and type I receptors into lipid rafts where they are optimized to respond to low doses of BMP ligands. Since Dragon can bind BMP 2 and 4 directly, it is also possible that Dragon acts to stabilize the ligand-receptor complex in lipid rafts, thereby facilitating endocytosis and signaling. Of course, these two possibilities are not mutually exclusive. Based on the localized expression of Dragon in developing and maturing germ cells, as well as specific epithelial cells within the reproductive tract that are known to be BMP-responsive, our results support the concept that BMP’s play an important role in regulating reproduction in mammals and that this role may be regulated by Dragon.