Localization and binding of transforming growth factor-beta isoforms in mouse preimplantation embryos and in delayed and activated blastocysts.

The stage and cell-specific accumulation of mammalian isoforms of transforming growth factor-beta (TGF-beta 1, TGF-beta 2, and TGF-beta 3) and TGF-beta binding were examined in the preimplantation embryo and in progesterone (P4)-treated delayed or P4 plus estradiol-17 beta (E2)-treated activated blastocysts in the mouse. Immunocytochemical studies revealed that while all three immunoreactive TGF-beta isoforms were present in one-cell embryos, very little or no immunostaining was observed in two-cell embryos. However, distinct immunostaining of these isoforms was again observed in four-cell embryos and persisted through the blastocyst stage. Among the isoforms studied, TGF-beta 2 immunostaining showed a unique pattern in late morulae. In many of these morulae, the staining was primarily observed in outside cells. However, in blastocysts, immunostaining for all three isoforms was present both in the inner cell mass (ICM) and trophectoderm (Tr). Immunostaining in sectioned blastocysts and immunosurgically isolated ICMs confirmed immunostaining in Tr and ICM cells. To ascertain whether preimplantation embryos can produce TGF-beta isoforms, immunostaining was performed in embryos grown in vitro from two-cell stage in simple balanced salt solution. Immunoreactive TGF-beta s 1-3 were present in embryos at all stages of development examined (four-cell embryos through blastocysts). The virtual absence of immunoactive TGF-beta s in two-cell embryos but their accumulation in embryos at later stages of development in vitro provides evidence that these growth factors were produced by embryos. In order to assess at what stages of development preimplantation embryos could be responsive to TGF-beta s, specific binding of [125I]TGF-beta 1 and [125I]TGF-beta 2 was performed in embryos and examined by autoradiography. Low levels of binding were first detected in eight-cell embryos. The binding increased in morulae followed by a further increase in blastocysts. Analysis of binding of [125I]TGF-beta 2 in immunosurgically isolated ICMs indicated that binding was primarily evident in Tr cells. Affinity labeling of TGF-beta 1 or TGF-beta 2 in Day 4 blastocysts revealed three classes of binding proteins with approximate molecular sizes of 65 kDa (type I), 90 kDa (type II), and greater than 250 kDa (type III), in addition to a doublet of 130 and 140 kDa proteins. This observation is similar to those reported for other cell types. The data suggest that embryos are likely to be responsive to TGF-beta s after the third cleavage.(ABSTRACT TRUNCATED AT 400 WORDS)