Untersuchungen zum Vorkommen von Wachstumsfaktoren (aFGF, bFGF, TGF-[alpha]) und deren Rezeptoren (FGF-R, EGF-R) in der Plazenta des Rindes

Abstract

This study examines the expression and the distribution of the growth factors aFGF, bFGF and TGF-[alpha] and their receptors FGF-R and EGF-R during the second half of pregnancy and throughout parturition. Placentomes were collected from cows at days 150, 220, 240 and 270 of pregnancy and from normal-term placentas, these latter extracted by caesarean section. Each group consisted of three animals. After removal placentomes were fixed in formalin and embedded in paraffin.

As long as the right reagents and pre-treatments of the probes were chosen, the immunohistochemical technique using biotinylated secondary antibodies and the ABC-complex was the appropriate one for detecting aFGF, bFGF, FGF-R and EGF-R, although controls were essential in order to distinguish between specific and non-specific immunolabelling (isotypic control of antibodies, known positive/negative tissues). To reduce extraneous influences on the immunolabelling, all tests were prepared by myself under the same conditions. In this investigation specific detection of TGF-[alpha] was not possible.

Immunolabelling of aFGF did not depend on the stage of pregnancy, but resulted in nearly the same staining pattern in each probe and detected the growth factor in each cell type. It seems that in bovine placentomes aFGF does not act as a mitogen, but activates non-mitogenic cell functions (cellular differentiation, modulation of cellular metabolism, and cellular motility and migration).

The growth factor bFGF was detected in bovine placentomes by two different antibodies, which gave different staining patterns. While the monoclonal antibody showed cytoplasmic immunostaining of epithelial cells, the polyclonal antibody located weak to intense immunostaining of the nuclei, concurrent with weak cytoplasmic reaction in these cells. Additionally, both antibodies were immunoreactive in nuclei of stromal cells and showed a reduction of immunoreactivity at day 220. Stromal reactions of bovine placentomes indicate that bFGF stimulates cell proliferation and angiogenesis, while epithelial reactions are connected with non-mitogenic cell functions (changes in differentiated cell function, modulation of specific cellular protein synthesis). The different staining patterns could be due to the recognition of different isoforms of bFGF.

The present study detected the expression of FGF-R in bovine placentomes during the second half of pregnancy to parturition and proved that the staining pattern varied according to the different stages of pregnancy. Outer membranes and cytoplasm of the maternal stromal cells stained with a weak to intensive signal, while on days 220, 240 and at parturition the number of positive cells was higher than on days 150 and 270. At each investigated stage of pregnancy foetal stromal cells had nuclear and perinuclear immunostaining, which started with extra-weak to weak reaction on day 150, became stronger as pregnancy progressed and ended with weak to strong reactions from day 240 to parturition. Cytoplasmic signals of the caruncular epithelium cells were weak and overlapped with weak reaction of the nuclei; on day 240 many cells reacted positive while on day 270 only a few cells stained. Remarkably, some parts of the probes were absolutely negative. The two trophoblastic cell types (BNC and columnar trophoblastic cells) reacted in the same way to positive cell membranes or cytoplasm; on days 150, 220 and 270 many cells stained, while on day 240 and at parturition almost all cells were positive. Additionally, there was a weak nuclear signal of single cells on day 220. This latter signal was most commonly found beside greater stromal areas. There is no possibility of differentiation between FGF-R 1 and FGF-R 2.

EGF-R expression was also detected, whereas the staining reaction was restricted to the epithelial parts of placentomes. Intensity and distribution of the signals did not vary only according to the stage of pregnancy but also between different animals at the same stage of pregnancy. This probably reflects the different concentrations of EGF-R/ligand in the individual specimen. The loss of signals in at-term placentomes can be explained by the progressive degeneration of the caruncular epithelium, while loss of signals on day 220 corresponds to the observations at bFGF.

In conclusion, during the second half of gestation and under parturition the growth factors aFGF and bFGF, as well as their receptors FGF-R and EGF-R, are expressed in bovine placentomes. Therefore, these proteins are important for the development and function of bovine placentomes.