First identification of caldesmon transcripts in bovine oviduct epithelial cells in vitro by means of an RNA differential display technique examining culture-induced expression changes.

Innovative molecular biology techniques enable the quick evaluation of distinct gene expression pattern of cells or tissues. Hitherto, a cell-type-specific behaviour has been difficult to evaluate. In addition to standard morphological and immunological criteria in this study the expression of in vitro-cultured bovine oviduct epithelial cells (BOECs) was compared with fresh cells by RNA arbitrarily primed polymerase chain reaction (RAP-PCR). The cultured cells showed mitotic activity during the whole culture period (6 days) until they had reached about 80% confluency. Remarkable results of a random transcript screening using fresh versus cultured BOECs were reported, in which a single PCR product appeared during culture, but was absent in fresh cells. Sequence analysis of the culture-induced 522 bp fragment revealed a high homology (87%) to caldesmon (CaD) of various species and a 92% homology to a short cDNA fragment of a bovine non-muscle CaD. Specific, cross-species PCR primers were used to elongate this partial sequence (1,036 bp). This resulting cDNA showed an open reading frame and was identified as a bovine non-muscle CaD isoform. When compared with human non-muscle CaDs (89% homology) a deletion of 2 codons was observed. According to sequential culture experiments, CaD expression was not found in fresh BOECs but specific transcripts appeared within 48-113 h under specific culture conditions. It is likely that augmented CaD expression in cultured BOECs may reflect the cell effort adapting to specific culture conditions. The hypothesis that increased CaD levels could be important to facilitate adherence and spreading by formation of new stress fibres can be favoured. This first identification of caldesmon expression being specifically induced during in vitro culture demonstrates the potential of RAP-PCR for the analysis and validation of cell culture techniques.

Growth factors and extracellular matrix proteins in interactions of cumulus-oocyte complex, spermatozoa and oviduct.

The expression and localization of selected growth factor systems and extracellular matrix (ECM) components that may influence oocyte maturation and fertilization within the mammalian oviduct are reported. Fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) systems could be detected by use of RT-PCR, RNase protection assay (RPA) and immunohistochemistry in bovine follicles, bovine cumulus-oocyte complexes (COC) and bovine and marmoset oviducts. Two different subtypes of the FGF receptor (FGFR-1 and -2) were identified in distinct cell types, indicating a functional difference. A complete epidermal growth factor (EGF) system was found in the porcine, but not in the bovine, oviduct. There were additional differences between bovine and primate oviducts: FGF-1/2 and FGFR were increased in the marmoset around ovulation, in contrast to an increase in FGF-1 in the cow. Immunohistochemistry revealed accumulation and storage of FGF and VEGF on the surface of the epithelium, possibly due to their binding property on heparanglycoproteins. Other ECM components, matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase 1 (TIMP-1), were found to be modulated in the ovarian follicle, COC and oviduct during the cycle. An oviduct-mediated depletion of sperm surface proteins (BSP1-3) was discovered as well as a sperm-induced novel oviductal mRNA related to an anti-oxidant protein family. Associated systems of growth factors and ECM components can be suggested as paracrine or autocrine mediators during fertilization in a species-, cycle- and tissue-dependent manner.

Growth factors and components for extracellular proteolysis are differentially expressed during in vitro maturation of bovine cumulus-oocyte complexes.

In the present study, the time-dependent collagenolytic potential and mRNA transcription of extracellular matrix (ECM)-degrading components, transforming growth factor beta1 (TGFbeta1), and both basic fibroblast growth factor (bFGF) and FGF receptors (FGFR) in bovine cumulus-oocyte complexes (COCs) were investigated during 24 h of in vitro maturation (IVM). COCs were collected from 2- to 6-mm follicles, cultured in maturation medium, and sequentially removed at 3-h intervals for analysis. From 285 oocytes matured under these conditions, 114 (40.0%) developed to blastocysts on Day 9 after fertilization. Gelatin zymograms revealed protease activity at about 55 kDa for COCs matured for at least 3 h and two additional proteolytic zones at about 70 kDa after at least 9 h of IVM. The mRNAs of ECM-degrading components urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI1), matrix metalloproteinase 1 (MMP1), and tissue inhibitor of metalloproteinases 1 (TIMP1), as well as TGFbeta1, bFGF, and FGFR, were detected during IVM in a factor-specific pattern: all transcript levels found at COC 0 generally increased after 3 h of maturation and either remained high or decreased thereafter. On the basis of the chosen reverse transcription-polymerase chain reaction technique, one could suggest relative higher mRNA concentrations for TIMP1, PAI1, and both growth factors compared to uPA, MMP1, and FGFR. Our results suggest a finely tuned extracellular proteolysis during IVM of bovine COCs, for which the concerted action of modulating growth factors like bFGF and TGFbeta1 may be essential.