Surfactant protein secreted by the maturing mouse fetal lung acts as a hormone that signals the initiation of parturition

Abstract

Parturition is timed to begin only after the developing embryo is sufficiently mature to survive outside the womb. It has been postulated that the signal for the initiation of parturition arises from the fetus although the nature and source of this signal remain obscure. Herein, we provide evidence that this signal originates from the maturing fetal lung. In the mouse, secretion of the major lung surfactant protein, surfactant protein A (SP-A), was first detected in amniotic fluid (AF) at 17 days postcoitum, rising progressively to term (19 days postcoitum). Expression of IL-1beta in AF macrophages and activation of NF-kappaB in the maternal uterus increased with the gestational increase in SP-A. SP-A stimulated IL-1beta and NF-kappaB expression in cultured AF macrophages. Studies using Rosa 26 Lac-Z (B6;129S-Gt(rosa)26Sor) (Lac-Z) mice revealed that fetal AF macrophages migrate to the uterus with the gestational increase in AF SP-A. Intraamniotic (i.a.) injection of SP-A caused preterm delivery of fetuses within 6-24 h. By contrast, injection of an SP-A antibody or NF-kappaB inhibitor into AF delayed labor by >24 h. We propose that augmented production of SP-A by the fetal lung near term causes activation and migration of fetal AF macrophages to the maternal uterus, where increased production of IL-1beta activates NF-kappaB, leading to labor. We have revealed a response pathway that ties augmented surfactant production by the maturing fetal lung to the initiation of labor. We suggest that SP-A secreted by the fetal lung serves as a hormone of parturition.

Fig. 2.

Purified SP-A and SP-A-depleted preparation. SP-A isolated from the alveolar proteinosis material and the mannose-D-depleted SP-A preparation were analyzed for levels of SP-A by immunoblotting. The latter contained markedly reduced levels of SP-A.

Fig. 1.

IL-1β expression in AF and uterine macrophages increases in association with increased SP-A secretion into AF. (A) SP-A is first detected in mouse AF at 17 dpc and increases markedly toward term. AF isolated from ICR mice at 16-19 dpc was analyzed for SP-A by immunoblotting. Shown is a representative immunoblot of an experiment repeated in three gestational series of mice. (B) IL-1β protein increases to detectable levels in mouse AF macrophages toward term. AF macrophages were subjected to immunohistochemical analysis for IL-1β. Immunohistochemical analysis shown is representative of findings obtained using macrophages from three different gestational series of mice. (C) Macrophage infiltration of the pregnant uterus increases toward term. Uteri of 15, 17, and 19 dpc mice were analyzed for CD-68 levels by immunoblotting. The immunoblot shown is representative of findings obtained using uteri from three different gestational series of mice. (D) IL-1β levels increase in the pregnant uterus toward term. IL-1β protein levels in uteri of 15, 17, and 19 dpc mice were analyzed by immunoblotting. The immunoblot shown is representative of findings obtained using uteri from three different gestational series of mice.

Fig. 3.

IL-1β and NF-κB mRNA levels in mouse AF macrophages increase as term approaches and are stimulated by SP-A treatment. AF macrophages obtained from mice at 15, 17, and 19 dpc were incubated with or without 10 μg/ml SP-A for 30 min. IL-1β and p65 mRNA levels were assessed by RT-PCR. This experiment was repeated three times with similar results.

Fig. 4.

AF macrophages of fetal origin invade the maternal uterus near term. (A) AF macrophages are fetal in origin. β-gal staining was detected in cytoplasmic vacuoles of macrophages isolated from AF surrounding Lac-Z embryos at 17 dpc. (B) Cells of fetal origin infiltrate the maternal uterus near term. β-gal staining was detected in uteri of wild-type mice carrying Lac-Z embryos at 17 dpc (Left). Uteri from wild-type mice carrying wild-type pups were negative (Right). (C) Macrophages of fetal origin infiltrate the maternal uterus. Immunohistochemical analysis of serial sections of uteri from 17 dpc mice carrying Lac-Z embryos revealed that β-gal-expressing cells are immunopositive for F4/80. (D) β-gal and F4/80 colocalize in uterine cells. Dual immunofluorescence of uteri from 17 dpc mice carrying Lac-Z embryos revealed colocalization of β-gal and F4/80. (E) Cells of fetal origin infiltrate the maternal uterus in increasing numbers toward term. β-gal-expressing cells were first detectable in uteri of the wild-type ICR females carrying Lac-Z embryos at 17 dpc and increased to term. (F) SP-A injection into all amniotic sacs of one uterine horn on 15 dpc mice increases infiltration of fetal cells into the maternal uterus. Fifteen dpc mice carrying Lac-Z embryos were i.a. injected with SP-A or vehicle (Control). Postsurgery (4.5 h), the injected (inj) and noninjected (non inj) uterine horns were assayed for β-gal-positive cells.

Fig. 5.

Nuclear localization of NF-κB proteins increase in mouse uterus as term approaches and is further increased by SP-A injection into amniotic fluid. (A) NF-κB activation increases in the pregnant mouse uterus as term approaches. Nuclear and cytoplasmic fractions obtained from uteri of mice at 16, 17, 18, and 19 dpc were subjected to immunoblotting by using antibodies against p50 and p65. This immunoblot is representative of findings obtained using pregnant mice from three independent gestational series. (B) Injection of SP-A into all amniotic sacs of one uterine horn on 15 dpc increases activation of NF-κB in the pregnant uterus. Fifteen dpc mice were i.a. injected either with SP-A or vehicle (Control). The injected mice were killed 4.5 h after surgery. Uterine cytoplasmic and nuclear fractions prepared from the injected (inj) and contralateral noninjected (non inj) horns were analyzed for p65 by immunoblotting.