Suppression subtractive hybridization identified a marked increase in thrombospondin-1 associated with parturition in pregnant sheep myometrium.
Changes in several extracellular matrix proteins, such as fibronectin in fetal membrane and cervical collagens, occur at term and preterm delivery. However, no studies have evaluated changes in extracellular matrix proteins, in relation to myometrial activation recorded using invasive techniques during parturition in any species. We used suppression subtractive hybridization (SSH), to demonstrate the dramatic increases in one extracellular matrix protein, thrombospondin-1 (TSP1), in the pregnant ovine myometrium associated with parturition. Myometrial poly-A- RNA, extracted from term control ewes not in labor at 143-147 days of gestational age (dGA, n = 4), and from ewes in spontaneous term labor (STL) at 145-147 dGA (n 4), was subjected to SSH to construct a subtracted myometrial complementary DNA library. A complementary DNA clone from myometrial subtracted library, representing differentially expressed gene in the pregnant sheep myometrium during STL, was identified as TSP1 by sequence analysis and Blastn search. This cloned TSP1 was used to perform Northern blot analysis on total RNA isolated from five early controls, not in labor, at 130 dGA, five pregnant ewes in betamethasone-induced premature labor (BPL) at 130 dGA (betamethasone administered i.v. to the fetus at 0.48 mg over 48 h), six term-control-not-in-labor ewes at 143-147 dGA, and six pregnant ewes in STL. Northern blot analysis demonstrated that TSP1 increased significantly, associated with both BPL and STL. TSP1 protein level paralleled the increase of TSP1 messenger RNA during BPL and STL. To determine whether increase in this gene paralleled the increased strength of myometrial contractility, six ewes were treated with nimesulide, a selective PG synthase 2 inhibitor, at 147-148 dGA. Nimesulide infusion to the ewe i.v. to inhibit myometrial contraction (30 mg bolus, followed by 5 h infusion, 30 mg/h) commenced 9 h after onset of labor at 147-148 dGA. TSP1 in the myometrium decreased when myometrial contraction was inhibited by nimesulide. Both in situ hybridization and immunocytochemistry demonstrated that fibroblasts and the smooth muscle cells contained TSP1 messenger RNA and protein. TSP1 was also localized in the extracellular matrix. Our conclusions are: 1) our data provide the first evidence that changes in TSP1 are associated with myometrial activation in pregnant sheep during term and preterm labor; 2) myometrial fibroblasts and the smooth muscle cells are responsible for producing TSP1; and 3) SSH is a powerful technique that enables us to study differentially regulated genes during labor.
Wu
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WX
Department Physiology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401, USA.
Zhang
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Ma
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XH
Unno
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Nathanielsz
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PW
D000818:Animals; D001623:Betamethasone / Q000008:administration & dosage; D015152:Blotting, Northern; D015153:Blotting, Western; D018076:DNA, Complementary / Q000032:analysis; D005260:Female; D015723:Gene Library; D007150:Immunohistochemistry; D007751:Labor, Induced; D007743:Labor, Obstetric / Q000502:physiology*; D009215:Myometrium / Q000378:metabolism*; D009693:Nucleic Acid Hybridization / Q000379:methods; D007752:Obstetric Labor, Premature / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis; D012756:Sheep; D019699:Thrombospondins / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D012333:RNA, Messenger; D019699:Thrombospondins; D001623:Betamethasone