Regulation of human placental development by oxygen tension

Science

Volumn 277, Issue 5332, 1997, Pages 1669-1672

  Genbacev, Olga ^a   Zhou, Yan ^a   Ludlow, John W ^b   Fisher, Susan J ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b UNIVERSITY OF ROCHESTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; CELL DIFFERENTIATION; CELL HYPOXIA; CELL PROLIFERATION; CYTOTROPHOBLAST; HUMAN; HUMAN TISSUE; OXYGEN TENSION; PLACENTA CIRCULATION; PLACENTA DEVELOPMENT; PREGNANCY; PRIORITY JOURNAL; UTERINE ARTERY;

ANTIGENS, CD; CELL DIFFERENTIATION; CELL DIVISION; CELL HYPOXIA; CHORIONIC VILLI; CYCLIN-DEPENDENT KINASE INHIBITOR P21; CYCLINS; FEMALE; HUMANS; INTEGRIN ALPHA1; MITOSIS; ORGAN CULTURE TECHNIQUES; OXYGEN; PLACENTA; PLACENTAL LACTOGEN; PREGNANCY; S PHASE; TROPHOBLASTS;

EID: 0030879909     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5332.1669     Document Type: Article

References (25)

1. S. J. Fisher and C. H. Damsky, Semin. Cell Biol. 4, 183 (1993).
2. R. Schwarting, Lab. Invest. 68, 597 (1993).
3. C. H. Damsky, M. L. Fitzgerald, S. J. Fisher, J. Clin. Invest. 89, 210 (1992).
4. C. L. Librach et al., J. Cell Biol. 113, 437 (1991).
5. M. T. McMaster et al., J. Immunol. 154, 3771 (1995).
6. R. J. Kurman et al., Int. J. Gynecol. Pathol. 3, 101 (1984).
7. F. Rodesch, P. Simon, C. Donner, E. Jauniaux, Obstet. Gynecol. 80, 283 (1992).
8. O. Genbacev, R. Joslin, C. H. Damsky, B. M. Polliotti, S. J. Fisher, J. Clin. Invest. 97, 540 (1996).
9. 3H]thymidine (44 Ci/mM; Amersham) was added for 24 hours. The villi were then washed with phosphate-buffered saline (PBS) containing unlabeled thymidine (5 μg/ml), treated with Dispase (Collaborative Biomedical Research, Bedford, MA), and lysed; radioactivity was measured after trichloroacetic acid precipitation.
10. R. W. King, P. K. Jackson, M. W. Kirschner, Cell 79, 563 (1994).
11. 4, 1 mM NaF, 1 mM phenyl-methylsulfonyl fluoride, 25 μg/ml aprotinin, 25 μg/ml leupeptin, and 10% (w/v) glycerol. Samples containing 100 μg of protein, determined as described by M. M. Bradford [Anal. Biochem. 72, 248 (1976)], were separated by SDS-10% polyacrylamide gel electrophoresis [U. K. Laemmli, Nature 227, 680 (1970)]. The separated proteins were transferred to nitrocellulose paper [H. Towbin, T. Staehelin, J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979)]. Blots were blocked in TBST [20 mM tris-HCl (pH 8.0), 120 mM NaCl, 0.1% Tween-20] containing 4% nonfat dry milk. The replicas were incubated overnight with primary antibodies in TBST containing 2% nonfat dry milk. Anti-cyclin B (Pharmingen, San Diego, CA) was used at 2.5 μg/ml and anti-p21 (Oncogene Research Products, Cambridge, MA) at 2 μg/ml. Antibody binding was assessed with the use of horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection (Amersham). Densitometric analysis was performed using the NIH Image program.
12. 4, 1 mM NaF, 1 mM phenyl-methylsulfonyl fluoride, 25 μg/ml aprotinin, 25 μg/ml leupeptin, and 10% (w/v) glycerol. Samples containing 100 μg of protein, determined as described by M. M. Bradford [Anal. Biochem. 72, 248 (1976)], were separated by SDS-10% polyacrylamide gel electrophoresis [U. K. Laemmli, Nature 227, 680 (1970)]. The separated proteins were transferred to nitrocellulose paper [H. Towbin, T. Staehelin, J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979)]. Blots were blocked in TBST [20 mM tris-HCl (pH 8.0), 120 mM NaCl, 0.1% Tween-20] containing 4% nonfat dry milk. The replicas were incubated overnight with primary antibodies in TBST containing 2% nonfat dry milk. Anti-cyclin B (Pharmingen, San Diego, CA) was used at 2.5 μg/ml and anti-p21 (Oncogene Research Products, Cambridge, MA) at 2 μg/ml. Antibody binding was assessed with the use of horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection (Amersham). Densitometric analysis was performed using the NIH Image program.
13. 4, 1 mM NaF, 1 mM phenyl-methylsulfonyl fluoride, 25 μg/ml aprotinin, 25 μg/ml leupeptin, and 10% (w/v) glycerol. Samples containing 100 μg of protein, determined as described by M. M. Bradford [Anal. Biochem. 72, 248 (1976)], were separated by SDS-10% polyacrylamide gel electrophoresis [U. K. Laemmli, Nature 227, 680 (1970)]. The separated proteins were transferred to nitrocellulose paper [H. Towbin, T. Staehelin, J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979)]. Blots were blocked in TBST [20 mM tris-HCl (pH 8.0), 120 mM NaCl, 0.1% Tween-20] containing 4% nonfat dry milk. The replicas were incubated overnight with primary antibodies in TBST containing 2% nonfat dry milk. Anti-cyclin B (Pharmingen, San Diego, CA) was used at 2.5 μg/ml and anti-p21 (Oncogene Research Products, Cambridge, MA) at 2 μg/ml. Antibody binding was assessed with the use of horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection (Amersham). Densitometric analysis was performed using the NIH Image program.
14. A. L. Gartel, M. S. Serfas, A. L. Tyner, Proc. Soc. Exp. Biol. Med. 213, 138 (1996).
15. C. H. Damsky et al., Development 120, 3657 (1994).
16. A. T. Hertig, Anat. Rec. 82, 420 (1942).
17. T. G. Graeber et al., Nature 379, 88 (1996).
18. J. D. Boyd and W. J. Hamilton, J. Obstet. Gynaecol. Br. Commonw. 74, 161 (1967).
19. Y. Zhou et al., J. Clin. Invest. 99, 2139 (1997).
20. J. Stone et al., J. Neurosci. 15, 4738 (1995).
21. Y. Zhou, C. H. Damsky, K. Chiu, J. M. Roberts, S. J. Fisher, J. Clin. Invest. 91, 950 (1993).
22. Placental-bed biopsy specimens were obtained as previously described (5, 17). Sections of these specimens were incubated with anti-Ki67 (Dako, Carpinteria, CA) and anti-human cytokeratin (7D3) [S. J. Fisher et al., J. Cell Biol. 109, 891 (1989)]. Antibody binding was detected with fluorescein-conjugated rabbit anti-mouse immunoglobulin G (IgG) and rhodamine-conjugated goat anti-rat IgG (Jackson Immunoresearch). Tissue blocks of cultured anchoring villi were prepared like the biopsy specimens. Sections cut from these blocks were double stained with 7D3 and an anti-integrin α1 mouse IgG (T Cell Diagnostics, Woburn, MA). Different sections were stained with an anti-HLA-G mouse monoclonal antibody produced in our laboratory (5), anti-integrin α5 (3), anti-αv/β3 (LM609, D. Cherish, Scripps Research Foundation), anti-human placental lactogen (Harlan Bioproducts, Indianapolis, IN), anti-cyclin B, and anti-p21 (both from Santa Cruz Biotechnology, Santa Cruz, CA). Antibody binding was detected as described above.
23. 2 at the cell-medium interface, measured using a micro-oxygen electrode (MI-730; Microelectrodes, Inc., Londonderry, NH), was 20% (98 mm Hg) under standard tissue culture conditions, and either 6% (40 mm Hg) or 2% (14 mm Hg) in hypoxic conditions.
24. After 48 hours, the culture medium was aspirated, and medium containing 1 μM BrdU (Sigma) was added. After 24 hours, villi on filter substrates were washed with PBS for 20 min, fixed for 1 hour (4°C) in 4% paraformaldehyde, embedded in optimal-cutting-temperature medium, and frozen in liquid nitrogen (5, 17). Sections cut from these blocks were incubated with a fluorescence-labeled antibody to BrdU (Boehringer Mannheim, Indianapolis, IN). Adjacent sections were stained with 7D3.
25. Supported by NIH grants HD30367 (S.J.F.) and CA 56940 (J.W.L.) and grant 3RT-0324 from The University of California Tobacco-Related Disease Program (S.J.F.). We thank R. Joslin for excellent technical assistance and E. Leash for excellent editorial assistance.