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The dramatic increase in uterine growth during late pregnancy and the generation of labor contractions require dynamic remodeling of myometrial smooth muscle-ECM interactions. In many tissues, such interactions are provided by focal adhesions; however, there are no data as to the expression of focal adhesion proteins or of focal adhesion signaling in the myometrium. In this study, we show that tyrosine phosphorylation of myometrial FAK (FAK-P-Tyr) and of its downstream substrate, paxillin, exhibited a >10-fold increase during late pregnancy (days 15-22 of pregnancy) with each exhibiting a dramatic fall in P-Tyr on day 23 in association with the onset of labor. These changes in FAK-P-Tyr were paralleled by changes in FAK enzyme activity. Activated ERK1 and ERK2 expression remained relatively unchanged from day 15 to day 23, but decreased markedly 1 day post partum. Treatment of late pregnant rats with progesterone prevented the fall in FAK-P-Tyr/enzyme activity on day 23, and also blocked the onset of labor. These data suggest that progesterone (which decreases at term) modulates myometrial FAK activity/focal adhesion signaling and that these changes may underlie the tremendous remodeling that must occur in order for this muscle to develop optimal contractile activity during labor.

The underlying mechanisms controlling uterine contractions during labor are still poorly understood. Integrins are heterodimeric, transmembrane receptors composed of alpha and beta subunits that can be found in focal adhesions. Because these structures play an important role in the regulation of smooth muscle contractility and cell adhesion, we hypothesized that alpha5 integrin mRNA (Itga5) and protein (ITGA5) expression would be induced in the rat myometrium during late pregnancy and labor. Itga5 mRNA expression was significantly increased (P < 0.05) from Day 17 to labor, noticeably decreasing 1 day postpartum (PP). Immunoblot analysis illustrated a continual increase in ITGA5 levels during pregnancy, labor, and PP, with levels reaching significance at labor (P < 0.05). Analysis of ITGA5 expression by immunocytochemistry demonstrated that it is primarily localized to myometrial cell membranes in the longitudinal muscle layer of the myometrium from before pregnancy to Day 6, and in both the longitudinal and circular muscle layers from Day 15 to PP. Treatment of late-pregnant rats with progesterone blocked labor and resulted in sustained expression of Itga5 mRNA expression to Day 24. In addition, immunocytochemistry experiments showed ITGA5 was detectable at higher levels in cell membranes of both myometrial layers in progesterone-treated animals on Days 23 and 24, compared with vehicle controls. We propose that ITGA5, with its sole known partner, ITGB1, may be important in promoting cellular cohesion during late pregnancy. This process may aid the development of a mechanical syncytium for efficient force transduction during the sustained, coordinated, and powerful contractions of labor.

SUMMARY:Trophoblast differentiation during the first trimester of pregnancy involves cell proliferation and invasion and extracellular matrix (ECM) remodeling. Reports have indicated that, in a variety of cell types, processes such as proliferation, invasion, and ECM remodeling require the turnover of focal adhesions mediated by a cytoplasmic tyrosine kinase named focal adhesion kinase (FAK). Therefore, in the present study we examined the expression and spatial localization of FAK during early human placental development. Immunocytochemical and immunoblot analysis showed that FAK and a focal adhesionassociated protein named paxillin were highly expressed between the 5th and 8th weeks of gestation, specifically in villous cytotrophoblast and extravillous trophoblast (EVT) cells. Activated FAK, phosphorylated on Tyr-397, colocalized with ␣5 integrin and matrix metalloproteinase-2 (MMP2) expression in EVT cells within a previously characterized intermediate, invasiverestrained region. FAK and paxillin expression dramatically decreased after 10 to 12 weeks of gestation coincident with increasing pO 2 levels. Exposure of human villous explants of 5 to 8 weeks to a 3% O 2 environment resulted in increased trophoblast outgrowth, cell proliferation, and detection of ␣5 integrin and MMP2, as well as increased activation of FAK in EVT cells compared with explants grown in a 20% O 2 environment. To determine whether FAK was a key requisite for trophoblast differentiation, villous explants of 5 weeks gestation were grown in Matrigel in a 3% O 2 environment and incubated with 20-mer antisense FAK oligonucleotides. A dramatic reduction of trophoblast outgrowth was observed in antisense-treated explants compared with missense and control cultures, and, in addition, cell proliferation and MMP2 activity in antisense-treated explants were dramatically reduced. These data suggest that FAK is a key kinase involved in early trophoblast cell differentiation and plays a role in regulating cell proliferation and motility during early placental development. (Lab Invest 2001, 81:1469 -1483.

The fluid movements that arise during blastocyst formation (cavitation) are, at least in part, driven by the Na/K‐ATPase. In this study, the reverse transcriptase‐polymerase chain reaction (RT‐PCR) was used to survey bovine pre‐attachment embryos for transcripts encoding known isoforms of the Na/K‐ATPase α‐ and β‐ subunits, including isoforms not previously detected during the first week of mammalian development. Transcripts encoding the Na‐K‐ATPase α1, α2, α3 and β2 isoforms were detected throughout bovine preattachment development. This is the first indication that α2, α3 and β2 mRNAs are expressed during this early developmental interval. As in the mouse, β1‐subunit transcripts were not detected until the morula stage and were also present in blastocysts. Thus, in two mammalian species an increase in abundance of β1 isoform transcripts in the morula stage is coincident with the onset of cavitation. Transcripts encoding the recently characterized α4 isoform were not detected. The sensitivity of bovine blastocysts to ouabain (a potent inhibitor of Na/K‐ATPase) was determined by assessing the ability of bovine blastocysts to recover in ouabain supplemental culture medium following cytochalasin‐induced blastocyst collapse. Re‐expansion of bovine blastocysts was inhibited in all ouabain concentrations down to 10 9 M. Mouse blastocysts, in contrast, were sensitive to ouabain at or above 10 3M. These results have established that transcripts encoding multiple isoforms of both the α and β subunits of the Na/K‐ATPase are expressed throughout early bovine development and that bovine blastocysts display a greater sensitivity to ouabain than murine blastocysts. Future analysis will determine the possible individual and collective roles of these isoforms during blastocyst formation. Mol Reprod Dev 46:114–126, 1997. © 1997 Wiley‐Liss, Inc.

Na(+),K(+)-ATPase plays an essential role in mammalian blastocoel formation (cavitation) by driving trans-epithelial sodium transport. Previously, the alpha1 and beta1 subunit isoforms of this enzyme were identified in preimplantation mouse embryos and were assumed to be responsible for this function. Here we show that mRNAs encoding an additional alpha subunit isoform (alpha3) and the remaining two beta subunit isoforms are also present in preimplantation embryos. Whereas alpha3 mRNA accumulates between the four-cell and the blastocyst stages and thus results from embryonic transcription, the same could not be demonstrated for beta2 and beta3 mRNAs. Immunoblot analyses confirmed that these subunits are present in cavitating embryos. Using confocal immunofluorescence microscopy we found that alpha1 and beta1 subunits are concentrated in the basolateral membranes of the trophectoderm while being equally distributed in plasma membranes of the inner cell mass. In contrast, alpha3, beta2, and beta3 subunits were not detected in plasma membranes. Our current assessment, therefore, is that as many as six isozymes of Na(+),K(+)-ATPase could be involved in preimplantation development although it is primarily the alpha1beta1 isozyme that is responsible for blastocoel formation. Our findings imply that the regulation of sodium transport within the preimplantation mouse embryo is more complex than had been appreciated.