The rat chorioallantoic placenta is comprised of two morphologically distinct regions: the junctional zone and the labyrinth zone. The purpose of this investigation was to determine the relative contributions of trophoblast cells in each of these regions to the expression of placental lactogen-II (PL-II) and PRL-like protein-A (PLP-A) during development, mRNA expression was estimated by Northern blot analysis, whereas, protein expression was estimated by electrophoresis, immunoblotting, and immunocytochemical analyses. The immunochemical analyses used antipeptide antisera directed to amino acids 56-70 of PL-II and amino acids 152-164 of PLP-A. Northern and immunoblotting analyses indicated that both PL-II mRNA and protein expression were maximal in the junctional zone on day 13 of gestation and declined as gestation proceeded. In contrast, PL-II mRNA and protein expression in the labyrinth zone were low on day 13 and increased as gestation advanced. PL-II was specifically localized to giant cells. At midgestation, PL-II-positive giant cells were identified bordering the uterine decidua in the junctional zone and choriovitelline placenta. As gestation advanced. PL-II-positive cells were also localized to the labyrinth zone. Immunoreactivity was restricted to the cytoplasm of PL-II-positive cells. PLP-A mRNA and protein were predominantly expressed in the junctional zone of the chorioallantoic placenta. Expression of PLP-A increased as gestation advanced. PLP-A was specifically localized to giant and spongiotrophoblast cells of the junctional zone. Immunoreactivity was found in both cytoplasmic and nuclear compartments of PLP-A-positive cells. In summary, PL-II expression shifts from the junctional to the labyrinth zone during pregnancy, whereas PLP-A is predominantly expressed in the junctional zone during the latter third of pregnancy. Both hormones are produced by giant cells of the junctional zone, but only PL-II is expressed by choriovitelline and labyrinthine trophoblast cells. PLP-A is also expressed by spongiotrophoblast cells of the junctional zone. These findings provide insights into the process of placental morphogenesis.
We have examined the effect of differentiation on the expression of different members of the transforming growth factor type-beta (TGF-beta) family using embryonal carcinoma (EC) cells and early mammalian embryos. We determined that TGF-beta activity increases approximately 25-100% when the mouse EC cell line, F9, is induced to differentiate with retinoic acid (RA). Interestingly, the increased TGF-beta activity reflects the induction of TGF-beta 2 secretion following differentiation of both F9 EC cells and the human EC cell line, NT2/D1. Using the technique of reverse transcription-polymerase chain reaction (RT-PCR), we have verified that differentiation induces the expression of TGF-beta 2 as well as a distant member of the TGF-beta family, Vgr-1. Transcripts for TGF-beta 2 and Vgr-1 were readily detected in the differentiated cells of F9 and PC-13 but not in their undifferentiated counterparts. Moreover, TGF-beta 2 mRNA was readily detected in NT2/D1 cells following differentiation. In addition, transcripts for TGF-beta 2 were detected by RT-PCR in mouse morulae, preimplantation blastocysts and cultured blastocysts. Based on the data presented, it appears that the expression of both TGF-beta 2 and Vgr-1 is closely associated with the induction of differentiation during early development.
We have previously shown that differentiation of embryonal carcinoma (EC) cells leads to both increased binding of FGF (fibroblast growth factor) and suppression of k-FGF expression. In the current study, we examined the expression of FGF receptors by EC cells, EC-derived differentiated cells and early mammalian embryos using the technique of reverse transcription-polymerase chain reaction (RT-PCR). We determined that both mouse, F9, and human, NT2/D1, EC cells as well as their differentiated counterparts express transcripts for two forms of FGF receptors, bek (bacterially expressed kinase) and flg (fms-like gene). In addition, we determined that mouse blastocysts express flg transcripts. The presence of FGF receptor transcripts in early embryos and the previous finding of FGF-related activity in medium conditioned by mouse blastocysts argue that the FGF family plays important roles during early mammalian development.
Utilizing the technique of reverse transcription-polymerase chain reaction (RT-PCR), we have examined the expression of transforming growth factor-beta 3 (TGF-beta 3) by embryonal carcinoma (EC) cells, EC-derived differentiated cells and early mammalian embryos. Using a TGF-beta bioassay, we determined that PYS-2 cells express considerable TGF-beta activity that cannot be completely neutralized by antibodies specific for TGF-beta 1 and TGF-beta 2. We also have determined that PYS-2 cells, as well as F9 EC cells and their differentiated cells, express transcripts for TGF-beta 3. In addition, we have determined that blastocysts, cultured for three days in serum-containing medium, express TGF-beta 3 transcripts. Thus, our data suggest that expression of TGF-beta 3 is initiated during early stages of mammalian development.
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