Immunohistochemical localization of integrin alpha V beta 3 and osteopontin suggests that they do not interact during embryo implantation in ruminants

Abstract

Background: It has been suggested that trophoblast attachment requires co-expression of integrin alpha V beta 3 and its ligand osteopontin at the fetal-maternal interface. Until now the expression patterns of integrin alpha V beta 3 and osteopontin in the pregnant bovine uterus were unknown. The objectives of this study were to localize integrin alpha V beta 3 and osteopontin in bovine and sheep endometrium during the periimplantation period and to compare the distribution patterns using antibodies that had not yet been tested in sheep.

Methods: Cell compartments within endometrial tissue sections were scored for immunohistochemical staining intensity and data were analyzed to determine the effects of day of pregnancy or cycle.

Results: In pregnant bovine endometrium, integrin alpha V beta 3 was detected in luminal epithelium, stroma, myometrium and smooth muscle. A strong band of immunoreactivity was observed in the subepithelial stroma of intercaruncular regions, but there was reduced reactivity in the caruncles and glands. Bovine trophoblast did not express integrin alpha V beta 3 at any stage of pregnancy. In ovine endometrium a different pattern of staining for integrin alpha V beta 3 was observed. Reactivity was not present in the luminal epithelium or trophoblast. There was strong staining of the deep glands and no reactivity in the superficial glands. Osteopontin distribution was similar for sheep and cattle. For both species, apical staining was present on the luminal epithelium and glands and on embryonic tissues.

Conclusion: In ruminants, integrin alpha V beta 3 and osteopontin do not co-localize at the fetal-maternal interface indicating that these proteins could not interact to facilitate embryo attachment as has been proposed in other species.

Figure 1

Immunohistochemical localization of integrin α_Vβ₃ in cryostat cross sections of bovine endometrial tissue during the estrous cycle (A and C, day 18 cyclic, B, day 16 cyclic) and at different stages of pregnancy (D, day 16 pregnant, E, day 18 pregnant, F, day 21 pregnant, G, day 24 pregnant, H, day 30 pregnant). Positive antibody reactivity (shown as brown) was strongest in intercaruncular (ICAR) subepithelial stroma (SES) as indicated by the arrow, except at day 16 of the estrous cycle (B) as reported previously 14. Note the low reactivity in the caruncles (CAR), glands (G), superficial gland (SG) and trophoblast (T). A section treated with mouse IgG instead of primary antibody (negative control) is shown in panel A. Bar = 50 μm.

Figure 2

Effect of pregnancy stage on the median number of rows of subepithelial (SE) stromal cells showing strong reactivity to anti-α_Vβ₃ antibody. Acetone fixed cyrostat cross sections from three cows each at days 16, 18, 21, 24 and 30 of pregnancy were used. The number of rows of positively stained SE stromal cells (score ≥ 4) underlying the intercaruncular luminal epithelium were counted by two independent observers and analyzed to determine if there was an effect of stage of pregnancy. * indicates the median was significantly different from other medians (P < 0.05).

Figure 3

Immunohistochemical localization of integrin α_Vβ₃ in cryosections of ovine endometrial tissue during the estrous cycle (A, day 13 cyclic, B, day 17 cyclic) and at day 13 of pregnancy (C-F, day 13 pregnant). Positive reactivity (brown) was strongest in the glands (G) and blood vessels, while very weak or no reactivity was detected in the superficial glands (SG) or luminal epithelium (LE). Panels A, C and D illustrate the differences in subepithelial stroma (SES) reactivity to anti-integrin α_Vβ₃ in caruncular (CAR) and intercaruncular (ICAR) regions at different magnifications. Intercaruncular SES reactivity varied among the sheep, ranging from moderate (D) to relatively weak and diffuse as seen in panel E. Control sections (F) were treated with mouse IgG substituted for primary antibody. Stroma (S). Bar = 50 μm.

Figure 4

Immunohistochemical localization of osteopontin in cryostat cross-sections of bovine endometrial tissue during the estrous cycle (A, day 1 cyclic, B, day 14 cyclic, C, day 18 cyclic) and early pregnancy (D, day 18 pregnant, E, day 21 pregnant, F, day 24 pregnant, G, day 30 pregnant). Osteopontin (positive reactivity is red) was detected at the apical surface of glandular (G) and luminal epithelium (LE), associated with blood vessels (V) and on extraembryonic membranes (arrow) luminal to the trophoblast (T), as well as trophoblast itself. A representative control section, treated with rabbit IgG instead of primary antibody, is shown in H. mLE – LE modified by binucleate cell migration. Bar = 50 μm.

Figure 5

Estrous cycle (A) and pregnancy (B) effects on osteopontin levels inbovine endometrium. Tissue sections from stages 1 (metestrus), 2 (early diestrus), 3 (late diestrus), and 4 (proestrus/estrus) of the estrous cycle and from early pregnancy (days 16, 18, 21, 24 and 30) were scored by two independent observers for staining intensity of the luminal epithelium, superficial glands and deep glands Osteopontin reactivity in luminal epithelium decreased in late diestrus. As pregnancy progressed, apical cell reactivity to osteopontin antibody decreased in the glands. * means significantly different from other stages (P < 0.05).

Figure 6

Immunohistochemistry for detection of osteopontin in endometrial tissue collected from cyclic (A, day 13) and pregnant (B, day 13) sheep. Moderate to strong apical cell reactivity to osteopontin antibodies (shown in red) was detected on glands (G), luminal epithelium (LE) and extraembryonic membranes (arrow). No reactivity was observed on trophoblast (T). A representative control section treated with rabbit IgG substituted for primary antibody is shown in C. Bar = 50 μm.