CHARACTERIZATION AND EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH-FACTOR(VEGF) IN THE OVINE CORPUS-LUTEUM

The corpus luteum undergoes tremendous growth, development and regress ion each oestrous or menstrual cycle. These changes are reflected by e qually impressive growth and regression of the luteal vasculature. We have previously shown that angiogenic factors from corpora lutea are p rimarily heparin binding and that one of these factors is similar to v ascular endothelial growth factor (VEGF). In an effort to identify thi s factor, and to define its role in luteal vascular development, the c DNA for the coding region of ovine VEGF was sequenced and a sensitive RNase protection assay was developed to quantitate mRNA encoding VEGF in luteal tissues from ewes in the early (days 2-4), mid- (day 8) and late (days 14-15) stages of the oestrous cycle. In addition, an N-term inal peptide was synthesized from the translated ovine cDNA sequence f or VEGF and an antiserum was raised against this peptide for use in we stern immunoblotting procedures. Nested reverse transcriptase (RT)-PCR of RNA from ovine corpora lutea resulted in three products that corre spond in size to the alternatively spliced variants of VEGF (VEGF(120) , VEGF(164), and VEGF(188)) predicted from other species. The RNase pr otection assay revealed that the proportion of mRNA encoding VEGF was 2- to 3-fold greater on days 2-4 than on day 8 or days 14-15. Densitom etric analysis of gels from the RNase protection assay showed that VEG F(120) represented approximately one third of the total mRNA encoding VEGF in the corpus luteum and that this proportion did not vary with s tage of the oestrous cycle. SDS-PAGE and western immunoblot analysis o f a homogenate from corpora lutea showed a single 18 kDa protein. Thes e data demonstrate that VEGF is expressed in luteal tissue throughout the ovine oestrous cycle and that expression of mRNA encoding VEGF is upregulated during the period of rapid luteal development, when luteal vascular growth is at its maximum.