Immunocytochemical localization of cytochromes P450 17α-hydroxylase and aromatase in embryonic cell layers of elongating porcine blastocysts

Localized expression of cytochromes P450 17α-hydroxylase (P450(c17)) and aromatase (P450(arom)) was investigated in embryonic cell layers of elongating porcine blastocysts by immunocytochemistry. Blastocysts were flushed from the uterus on day 12 of pregnancy, fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained using immunogold- and peroxidase-based techniques. Staining for both P450(c17) and P450(arom) was intense in spherical 7- to 10-mm blastocysts, but was absent in earlier stage 2- to 4-mm blastocysts and less intense or absent in later stage 20-mm and filamentous embryos. Cytochrome P450(c17) was limited to the trophoblast of all blastocysts expressing the enzyme, and in spherical 7- to 10-mm blastocysts, essentially all cells of the trophoblast layer stained positively for P450(c17). However, as elongation became apparent in 10-mm blastocysts, the cells of the trophoblast became flattened, and the expression of P450(c17) declined particularly in those trophoblast cells adjacent to the embryonic disc where mesoderm outgrowth was occurring. In fact, two distinct populations of trophoblast cells became obvious: one that maintained P450(c17) expression, and one that did not. Moreover, those trophoblast cells expressing P450(c17) were less flattened than neighboring cells in which P450(c17) expression was absent. These two morphologically and functionally distinct trophoblastic cell populations were most obvious in areas furthest from the embryonic disc. Cytochrome P450(arom) was expressed in the trophoblast as well as the hypoblast under the embryonic disc. Neither P450(c17) nor P450(arom) appeared to be expressed in the embryonic disc or the mesoderm of the expanding blastocyst. These functional and structural changes in the embryonic cell layers of the elongating conceptus may be associated with the transient synthesis and secretion of estrogen that occur at the time of maternal recognition of pregnancy in the pig.