SPP1 (Osteopontin) and SPARC (Osteonectin) May Interact Developmentally During Mouse Pregnancy.

Secreted phosphoprotein 1 (SPP1, osteopontin) is an extracellular matrix (ECM) protein that binds integrins to affect cell-cell and cell-matrix interactions including adhesion, migration, proliferation and survival. Secreted-protein-acidic-and-rich-in-cysteine (SPARC, osteonectin) is a calcium-binding matricellular glycoprotein known to regulate ECM interaction during tissue remodeling and repair. We developed a Spp1-/-/Sparc-/- double null mouse in which 50% fewer pups are present at birth than are found in utero on days 18-20 of gestation due to postpartum maternal cannibalization of the pups, suggesting that these pups are stillborn or exhibit severe birth defects. Indeed, hemorrhages were observed prominently in lower right limbs and distal region of the tail in many embryos; in a few embryos, hemorrhages were present the intestine and right foreheads. In order to gain insight into the developmental abnormalities and decreased litter sizes seen in double null litters, we evaluated the expression of both Spp1 and Sparc in the mouse uterus throughout pregnancy. Ten-week old outbred CD-1 female mice were mated to an intact male of the same strain (copulatory plug = Day 1 of gestation). Pregnant females were sacrificed and uteri collected on Day 4, 4.5, 5, 9, 10, 11, 12, and 16 of pregnancy. Cell-specific expression of Spp1 and Sparc mRNA in serial sections of mouse uteri was determined by radioactive in situ hybridization analysis. On Day 4 of pregnancy, Spp1 was expressed in central cells of the inner cell mass (ICM) of the embryo, in scattered immune cells in uterine stroma, and in luminal epithelium (LE) directly contacting the embryo within the implantation chamber. LE expression within the implantation chamber was absent by Days 4.5-5 of pregnancy, but remained in LE adjacent to the implantation chamber and in interimplantation sites, suggesting that Spp1 may be involved in adhesion during the initial phase of embryo attachment to the LE, but subsequently must be down-regulated at sites of attachment to allow embryo invasion. Sparc was expressed diffusely in the uterine stroma, in peripheral cells of the ICM, and was not expressed in LE through Days 4.5-5. When we utilized a model of delayed implantation to determine the effect of nidatory estrogen, localization of Spp1 and Sparc was identical to that of pregnancy. On Day 9 of gestation, Spp1 was expressed by uterine natural killer (NK) cells and LE, whereas Sparc was expressed by decidual cells and LE. On Day 10, NK cells and LE continued to express Spp1, while Sparc was localized to the stroma directly beneath the LE. On Day 12, Spp1 was also expressed by trophoblast giant cells, and Sparc was expressed in the Reichert's Membrane adjacent to the trophoblast giant cells. By Day 16 of pregnancy, both Spp1 and Sparc were expressed in adjacent but non-overlapping regions of developing fetal bone and cartilage. Because Spp1 and Sparc often have antagonistic roles during tissue remodeling and are localized to adjacent but different cell types during mouse pregnancy, we hypothesize that simultaneous deletion of both Spp1 and Sparc leads to disruption of cell-cell, cell-matrix and inter-tissue communication essential to normal conceptus development that is not observed when the genes are deleted individually. This study was supported by NIH grants R01 CA90920 and RO1 CA137091. (platform)