IMMUNOCYTOCHEMICAL LOCALIZATION OF CYTOCHROMES-P450 17-ALPHA-HYDROXYLASE AND AROMATASE IN EMBRYONIC-CELL LAYERS OF ELONGATING PORCINE BLASTOCYSTS (VOL 135, PG 2248, 1994)

Localized expression of cytochromes P450 17 alpha-hydroxylase (P450,17 ) 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 parafor maldehyde, embedded in paraffin, sectioned, and stained using immunogo ld- and peroxidase-based techniques. Staining for both P450(c17) and P 450(arom) was intense in spherical 7- to 10-mm blastocysts, but was ab sent in earlier stage 2- to 4-mm blastocysts and less intense or absen t in later stage 20-mm and filamentous embryos. Cytochrome P450(c17) w as limited to the trophoblast of all blastocysts expressing the enzyme , and in spherical 7- to 10-mm blastocysts, essentially all cells of t he trophoblast layer stained positively for P450(c17). However, as elo ngation became apparent in 10-mm blastocysts, the cells of the trophob last became flattened, and the expression of P450(c17) declined partic ularly in those trophoblast cells adjacent to the embryonic disc where mesoderm outgrowth was occurring. In fact, two distinct populations o f trophoblast cells became obvious: one that maintained P450(c17) expr ession, and one that did not. Moreover, those trophoblast cells expres sing P450(c17) were less flattened than neighboring cells in which P45 0(c17) expression was absent. These two morphologically and functional ly 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 embry onic disc or the mesoderm of the expanding blastocyst. These functiona l and structural changes in the embryonic cell layers of the elongatin g conceptus may be associated with the transient synthesis and secreti on of estrogen that occur at the time of maternal recognition of pregn ancy in the-pig.