Effect of interleukin-3 on ovine trophoblast interferon during early conceptus development

Abstract

The effect of interleukin-3 (IL-3) on conceptus production of ovine interferon tau (olFNτ) was examined using two dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE), western blot, northern/slot blot,in situ hybridization and immunohistochemical methodologies. Ovine conceptuses (day 17,n=14) were culturedin vitro for 24 h in Eagle's Minimum Essential Medium (MEM) containing various doses of human recombinant interleukin-3 (hIL-3). At 75 and 150 colony forming units (units)/ml hIL-3, amounts of oIFNτ released into the culture media increased 3-8-fold over controls while at 300 units/ml (60 pM), hIL-3 did not enhance oIFNτ production. These variations in production of oIFNτ parallel amounts of oIFNτ mRNA detected by northern and slot blot analyses. Ovine IL-3 mRNA was primarily localized at luminal and glandular epithelia with minor staining at stromal regions of uterine endometria collected from day 17 pregnant ewes but its polypeptide was localized in the luminal epithelial region. However, such signals were not detected in day 17 conceptuses. Ovine IL-3 mRNA and its polypeptide were also localized at the same regions of the endometrium obtained from cyclic ewes on days 8 and 12. The effect of hIL-3 on the enhancement of oIFNτ productionin vitro could be mimicked by the addition of phorbol 12-myristate 13-acetate (PMA). The present observations demonstrate that the hemopoietic cytokine, IL-3, stimulates conceptus production of oIFNτin vitro and that ovine IL-3 and its mRNA are localized in the maternal endometrium. These results suggest that a cytokine, IL-3, produced by the maternal endometrium, is involved in conceptus production of oIFNτ which may result from the activation of a second messenger system, protein kinase C.

Regulation of Blastocyst Migration, Apposition, and Initial Adhesion by a Chemokine, Interferon γ-inducible Protein 10 kDa (IP-10), during Early Gestation

“…In Vitro Culture-Whole conceptuses (day 14, n ϭ 3) and minced conceptuses (days 17 and 20, ϳ200 mg/culture dish, n ϭ 3 each) were cultured under conditions essentially described previously (30).…”

Enhancement of ovine trophoblast interferon by granulocyte macrophage-colony stimulating factor: possible involvement of protein kinase C

“…To monitor any subsequent estrous activity following breeding, two vasectomized rams fitted with marking harnesses were penned with the ewes; eight ewes not showing behavioral estrus during the next 16-day period were subjected to the study. On day 16 of gestation, ewes were anesthetized as previously described (Imakawa et al 1995), and the reproductive tracts were collected and placed under ice. Conceptuses were flushed from the uteri with 20 ml sterile PBS (pH 7·2), rinsed twice in PBS and placed in Eagle's minimum essential medium (MEM; Sigma Chemical Co., St Louis, MO, USA).…”

“…After the 12-h preincubation, fresh media containing the following treatments were added: (1) MEM alone (controls, n=4); (2) 150 units/ml hGM-CSF (Genetics Institute, Cambridge, MA, USA, n=4); (3) 10 n PMA (n=4); (4) 150 units/ ml hGM-CSF+50 n calphostin C (a PKC inhibitor, n=4); (5) 150 units/ml hGM-CSF (in PKC-deficient tissues, n=4); or (6) 150 units/ml hGM-CSF+10 n PMA (n=4). Conceptus tissues were then cultured for an additional 24 h at 37 C under conditions previously described (Imakawa et al , 1995. At the end of the culture period, conceptus tissues were collected by centrifugation at 2500 r.p.m.…”

“…The enhanced production of oIFN by the conceptuses in vitro in response to hGM-CSF and hIL-3 was mimicked by a PKC activator, phorbol 12-myristate 13-acetate (PMA) (Imakawa et al 1994(Imakawa et al , 1995. Therefore, the present experiments seeked to define molecular mechanisms by which GM-CSF and/or PMA up-regulate oIFN .…”

“…Similarly, it is conceivable that oIFN production occurs in vitro without maternal factors, but a progesteronedominated uterine environment is required for sufficient production of oIFN to allow maternal recognition of pregnancy. In fact, oIFN production in vitro is enhanced by the addition of human granulocyte macrophage-colony stimulating factor (hGM-CSF) or interleukin-3 (hIL-3) (Imakawa et al 1994(Imakawa et al , 1995; GM-CSF and IL-3 are produced by the ovine endometrium and are readily available to the conceptus (Imakawa et al 1994(Imakawa et al , 1995.…”