Isolation and characterization of vascular smooth muscle cell growth promoting factor from bovine ovarian follicular fluid and its cDNA cloning from bovine and human ovary.

A protein possessing vascular smooth muscle cell (SMC) growth-promoting activity (VSGP) was purified from bovine ovarian follicular fluid. The purified protein showed a broad band on SDS-PAGE with an apparent molecular mass of 90-100 kDa. The purified protein was characterized by amino acid sequence analysis of its N-terminal and internal peptides. Based on the information of the peptide sequences, bovine ovarian cDNA library was screened and cDNA clones encoding the protein were isolated. Human homolog of the protein was also cloned from human ovarian cDNA library. Nucleotide sequence analysis revealed that bovine VSGP transcript has a 2421-bp open reading frame, which encodes a protein of 807 amino acid residues. A homology search indicated that bovine and human VSGP are counterparts of rat F-spondin, which has been previously identified as a promoter molecule of neurite extension in rat fetal floor plate. RNA blot analysis showed wide distribution of VSGP/F-spondin transcripts in fetal and adult human tissues. Especially the expression was highest in the adult human ovary. The purified bovine VSGP/F-spondin showed vascular SMC growth promoting activity with an ED(50) value of 10(-8) M. Together with these findings, we demonstrated here that VSGP/F-spondin is a major factor for vascular SMC proliferation in the ovary. In conclusion, our present study provides a distinct and important function of VSGP/F-spondin as a strong VSMC proliferation promoting factor, in addition to the previously proposed function in neuronal system, and also provides insight into mechanisms underlying vascular SMC proliferation during ovarian folliculogenesis.

[1]  K. Miyamoto,et al.  Regulation and localization of midkine in rat ovary , 1995, FEBS letters.

[2]  J. Findlay.  Angiogenesis in reproductive tissues. , 1986, The Journal of endocrinology.

[3]  A. Frumkin,et al.  F-Spondin Is Required for Accurate Pathfinding of Commissural Axons at the Floor Plate , 1999, Neuron.

[4]  Takashi Suzuki,et al.  Cyclic changes of vasculature and vascular phenotypes in normal human ovaries. , 1998, Human reproduction.

[5]  L. Nelson,et al.  Human follicle fluid vascular endothelial growth factor concentrations are correlated with luteinization in spontaneously developing follicles. , 1998, Human reproduction.

[6]  H. Phillips,et al.  Vascular endothelial growth factor is expressed in rat corpus luteum. , 1990, Endocrinology.

[7]  D. Sherer,et al.  Angiogenesis of the ovary. , 2000, American journal of obstetrics and gynecology.

[8]  H. Leffers,et al.  The tetramethylammonium chloride method for screening of cDNA libraries using highly degenerate oligonucleotides obtained by backtranslation of amino-acid sequences. , 1993, Journal of biochemical and biophysical methods.

[9]  H. Phillips,et al.  Vascular endothelial growth factor messenger ribonucleic acid expression in the primate ovary. , 1992, Endocrinology.

[10]  M. Mueller,et al.  Vascular endothelial growth factor in reproductive biology. , 1999, Current opinion in obstetrics & gynecology.

[11]  B. Dastugue,et al.  SCO-spondin: a new member of the thrombospondin family secreted by the subcommissural organ is a candidate in the modulation of neuronal aggregation. , 1996, Journal of cell science.

[12]  H. Dvorak,et al.  Secretion of vascular endothelial growth factor/vascular permeability factor from human luteinized granulosa cells is human chorionic gonadotrophin dependent. , 1998, Molecular human reproduction.

[13]  K. Miyamoto,et al.  Regulation of midkine messenger ribonucleic acid levels in cultured rat granulosa cells. , 1996, Biochemical and biophysical research communications.

[14]  Josephine C. Adams,et al.  The thrombospondin type 1 repeat (TSR) superfamily: Diverse proteins with related roles in neuronal development , 2000, Developmental dynamics : an official publication of the American Association of Anatomists.

[15]  D. Redmer,et al.  Angiogenesis in the ovary. , 1996, Reviews of reproduction.

[16]  S. Fujii,et al.  Co-expression of integrin-associated protein (IAP/CD47) and its ligand thrombospondin-1 on human granulosa and large luteal cells. , 1999, Molecular human reproduction.

[17]  E. Sage,et al.  Secreted Protein, Acidic and Rich in Cysteine (SPARC) and Thrombospondin in the Developing Follicle and Corpus Luteum of the Rat , 1998, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[18]  M. Kozak.  An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. , 1987, Nucleic acids research.

[19]  S. Scherer,et al.  Accumulation of F-Spondin in Injured Peripheral Nerve Promotes the Outgrowth of Sensory Axons , 1998, The Journal of Neuroscience.

[20]  T. Burstyn-Cohen,et al.  F-Spondin, Expressed in Somite Regions Avoided by Neural Crest Cells, Mediates Inhibition of Distinct Somite Domains to Neural Crest Migration , 1999, Neuron.

[21]  K. Miyamoto,et al.  Isolation and identification of midkine and pleiotrophin in bovine follicular fluid , 1994, Molecular and Cellular Endocrinology.

[22]  J. Lahav,et al.  Differentiation-controlled synthesis and binding of thrombospondin to granulosa cells. , 1992, Endocrinology.

[23]  T. Jessell,et al.  F-spondin: A gene expressed at high levels in the floor plate encodes a secreted protein that promotes neural cell adhesion and neurite extension , 1992, Cell.

[24]  R. Nakano,et al.  The vascular endothelial growth factor/fms-like tyrosine kinase system in human ovary during the menstrual cycle and early pregnancy. , 1999, The Journal of clinical endocrinology and metabolism.

[25]  M. Breckwoldt,et al.  Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) production by luteinized human granulosa cells in vitro; a paracrine signal in corpus luteum formation. , 1998, Gynecological endocrinology : the official journal of the International Society of Gynecological Endocrinology.

[26]  G. Blobel,et al.  SIGNAL SEQUENCES FOR EARLY EVENTS IN PROTEIN SECRETION AND MEMBRANE ASSEMBLY , 1980, Annals of the New York Academy of Sciences.

[27]  R. Einspanier,et al.  Expression and Tissue Concentration of Vascular Endothelial Growth Factor, Its Receptors, and Localization in the Bovine Corpus Luteum During Estrous Cycle and Pregnancy1 , 2000, Biology of reproduction.

[28]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[29]  P. Bornstein,et al.  Control of smooth muscle cell growth by components of the extracellular matrix: autocrine role for thrombospondin. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[30]  V. Dixit,et al.  Cell surface thrombospondin is functionally essential for vascular smooth muscle cell proliferation , 1988, The Journal of cell biology.