Phosphoserine-86-HSPB1 (pS86-HSPB1) is cytoplasmic and highly induced in rat myometrium at labour.

Uterine myocytes during pregnancy proceed through a series of adaptations and collectively transform into a powerfully contractile tissue by term. Previous work has indicated that members of the heat shock protein (HSP) B family of stress proteins are associated with the process of adaptation and transformation. Utilizing immunoblot analyses, widefield epifluorescence and total internal reflection (TIRF) microscopy, this study investigated the temporal and spatial detection of HSPB1 phosphorylated on serine-86 (pS86-HSPB1) in rat myometrium during pregnancy, the role of uterine distension in regulation of pS86-HSPB1, and the comparative localization with pS15-HSPB1 in rat myometrial tissue as well as in an immortalized human myometrial cell line. Immunoblot detection of pS86-HSPB1 was significantly elevated during late pregnancy and labour. In particular, pS86-HSPB1 was significantly increased at day (d)22 and d23 (labour) compared with all other timepoints assessed. Localization of pS86-HSPB1 in myometrium became prominent at d22 and d23 with cytoplasmic detection around myometrial cell nuclei. Furthermore, pS86-HSPB1 detection was found to be significantly elevated in the gravid rat uterine myometrium compared with the non-gravid tissue at d19 and d23. Both widefield epifluorescence and TIRF microscopy examination of human myometrial cells demonstrated that pS15-HSPB1 was prominently localized to focal adhesions, while pS82-HSPB1 (homologous to rodent pS86-HSPB1) was primarily located in the cell cytoplasm. Our data demonstrate that levels of phosphorylated HSPB1 increase just prior to and during labour, and that uterine distension is a stress-inducing signal for HSPB1 phosphorylation. The exact roles of these phosphorylated forms in myometrial cells remain to be determined.

Examination of FERMT1 expression in placental chorionic villi and its role in HTR8-SVneo cell invasion.

Transmembrane integrin receptors mediate cell-extracellular matrix as well as cell-cell adhesion. As placental trophoblast cells undergo differentiation they display changes in integrin expression or switching, but the mechanism(s) of integrin activation that supports this differentiation is still unknown. The Fermitin family of adapter proteins (FERMT 1-3) are integrin activators that mediate integrin-mediated signaling. In this study, we examined the spatiotemporal pattern of expression of FERMT1 in human chorionic villi throughout gestation and its role in HTR8-SVneo substrate adhesion and invasion. Placental villous tissue was obtained from patients undergoing elective terminations at weeks 8-14, as well as from term deliveries at weeks 37-40 and analyzed by immunofluorescence. Additionally, HTR8-SVneo trophoblast cells were transfected with FERMT1-specific siRNA or non-targeting siRNA (control) and used in cell-substrate adhesion as well as invasion assays. FERMT1 was primarily localized to membrane-associated regions at the base or around the periphery of the villous cytotrophoblast and proximal as well as distal cell column trophoblast. FERMT1 was also localized to endothelial cells of blood vessels in chorionic villi. siRNA-mediated depletion of FERMT1 in HTR8-SVneo cells did not markedly alter HTR8-SVneo cell-substrate adhesion but did significantly decrease invasion (P < 0.05) compared to control cells. These novel findings identify the presence of the integrin activator FERMT1 in trophoblast cells and that FERMT1 can regulate HTR8-SVneo cell invasion. FERMT1 may directly influence integrin activation and the subsequent integrin-mediated signaling and differentiation that underlies the acquisition of the invasive trophoblast phenotype in vivo.

Inducible heat shock protein A1A (HSPA1A) is markedly expressed in rat myometrium by labour and secreted via myometrial cell-derived extracellular vesicles.

The myometrium goes through physiological, cellular and molecular alterations during gestation that necessitate effective cellular proteostasis. Inducible heat shock protein A1A (HSPA1A) is a member of the 70-kDa heat shock protein A (HSPA) family, which acts as a chaperone to regulate proteostasis; however, HSPA1A also participates as a cytokine in inflammatory regulation, leading to its designation as a chaperokine. This study examined the spatiotemporal expression of HSPA1A protein in the rat myometrium throughout gestation and assessed whether it is secreted as cargo of myometrial cell-derived extracellular vesicles (EVs). Immunoblot analysis demonstrated that HSPA1A expression was markedly elevated during late pregnancy and labour and increased by uterine distension. Myometrial HSPA1A expression insitu increased in myocytes of longitudinal and circular muscle layers from Day 19 through to postpartum, specifically in the cytoplasm and nuclei of myocytes from both muscle layers, but frequently detectable just outside myocyte membranes. Scanning electron microscopy examination of samples isolated from hTERT-HM cell-conditioned culture medium, using EV isolation spin columns, confirmed the presence of EVs. EV lysates contained HSPA8, HSPA1A and the EV markers apoptosis-linked gene 2-interacting protein X (Alix), the tetraspanin cluster of differentiation 63 (CD63), tumour susceptibility gene 101 (TSG101) and HSP90, but not the endoplasmic reticulum protein calnexin. These results indicate that HSPA1A may act as a chaperokine in the myometrium during pregnancy.

The porcine trophoblast cell line PTr2 is susceptible to porcine reproductive and respiratory syndrome virus-2 infection.

INTRODUCTION: Porcine reproductive and respiratory syndrome virus-2 (PRRSV-2) breaches the maternal-fetal interface (MFI) to infect porcine fetuses, yet the exact mechanism(s) of transmission is not understood. The objective of this study was to determine the susceptibility of porcine trophoblast cell line (PTr2) to PRRSV-2 infection to understand the potential role of the trophoblast in viral transmission to fetuses in vivo. METHODS: PTr2 cells were exposed in vitro to PRRSV-2 and then subjected to immunofluorescence analysis (IF), flow cytometry (FCM), real-time quantitative PCR (RT-qPCR), transmission electron microscopy (TEM) and immunogold electron microscopy (IEM) to assess viral infection. The effects of PRRSV-2 on PTr2 cell cycle progression and apoptosis, as well as the ability of PTr2 cells to produce infectious viral particles were also examined. RESULTS: PRRSV-2 was readily detected in PTr2 cells by IF, FCM, RT-qPCR, TEM and IEM techniques. RT-qPCR and FCM results of a time course of infection of PTr2 cells indicated PRRSV-2 load decreased over time after initial infection up to 72 h. PRRSV-2 infection altered PTr2 cell cycle with a selective increase of cells within the G2/M phase and also induced apoptosis. TEM and IEM demonstrated PRRSV-2 within and on the surface of PTr2 cells and PRRSV-2 virions released from PTr2 cells infected naïve MARC-145 cells inducing cytopathic effects. DISCUSSION: Trophoblast cells are susceptible to PRRSV-2 infection and release live virions capable of inducing cytopathic effects in naïve cells. This suggests a possible mechanism by which PRRSV-2 can breach the MFI resulting in fetal infection and death.

Spatiotemporal immunofluorescent evaluation of porcine reproductive and respiratory syndrome virus transmission across the maternal-fetal interface.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes severe reproductive failure characterized by high fetal morbidity and mortality leading to substantial economic losses to the swine industry. Evaluation of spatiotemporal transmission of PRRSV at the maternal-fetal interface (MFI) is critical for understanding fetal infection. Localization of PRRSV-2 strain NVSL 97-7895 at different regions of the MFI in 20 pregnant gilts at 2, 5, 8, 12 and 14 days post-inoculation (dpi) were analyzed by immunofluorescence (IF). Samples of MFI were collected from 15 inoculated and 5 control gilts and transplacental PRRSV transmission assessed in randomly selected fetuses from each litter. Localization of NVSL 97-7895 antigen immunoreactivity in the MFI was focused in three major areas: endometrial connective tissues (ENDO), the feto-maternal junction (FMJ) and fetal placenta (PLC). NVSL 97-7895 was detected at the FMJ by 2 dpi. At 2, 5 and 8 dpi, NVSL 97-7895 was localized within the ENDO and FMJ, whereas at 12 and 14 dpi, it was mainly localized in the PLC. Using a novel IF strategy for counting and size sorting NVSL 97-7895 viral antigen in situ, results of this study indicate that non-cell-associated mechanisms are involved in PRRSV transmission across the MFI.

Induction of expression and phosphorylation of heat shock protein B5 (CRYAB) in rat myometrium during pregnancy and labour.

During pregnancy the myometrium undergoes a programme of differentiation induced by endocrine, cellular, and biophysical inputs. Small heat shock proteins (HSPs) are a family of ten (B1-B10) small-molecular-weight proteins that not only act as chaperones, but also assist in processes such as cytoskeleton rearrangements and immune system activation. Thus, it was hypothesized that HSPB5 (CRYAB) would be highly expressed in the rat myometrium during the contractile and labour phases of myometrial differentiation when such processes are prominent. Immunoblot analysis revealed that myometrial CRYAB protein expression significantly increased from day (D) 15 to D23 (labour; P<0.05). In correlation with these findings, serine 59-phosphorylated (pSer59) CRYAB protein expression significantly increased from D15 to D23, and was also elevated 1-day post-partum (P<0.05). pSer59-CRYAB was detected in the cytoplasm of myocytes within both uterine muscle layers mid- to late-pregnancy. In unilaterally pregnant rats, pSer59-CRYAB protein expression was significantly elevated in the gravid uterine horns at both D19 and D23 of gestation compared with non-gravid horns. Co-immunolocalization experiments using the hTERT-human myometrial cell line and confocal microscopy demonstrated that pSer59-CRYAB co-localized with the focal adhesion protein FERMT2 at the ends of actin filaments as well as with the exosomal marker CD63. Overall, pSer59-CRYAB is highly expressed in myometrium during late pregnancy and labour and its expression appears to be regulated by uterine distension. CRYAB may be involved in the regulation of actin filament dynamics at focal adhesions and could be secreted by exosomes as a prelude to involvement in immune activation in the myometrium.

Distension of the uterus induces HspB1 expression in rat uterine smooth muscle.

The uterine musculature, or myometrium, demonstrates tremendous plasticity during pregnancy under the influences of the endocrine environment and mechanical stresses. Expression of the small stress protein heat shock protein B1 (HspB1) has been reported to increase dramatically during late pregnancy, a period marked by myometrial hypertrophy caused by fetal growth-induced uterine distension. Thus, using unilaterally pregnant rat models and ovariectomized nonpregnant rats with uteri containing laminaria tents to induce uterine distension, we examined the effect of uterine distension on myometrial HspB1 expression. In unilaterally pregnant rats, HspB1 mRNA and Ser(15)-phosphorylated HspB1 (pSer(15) HspB1) protein expression were significantly elevated in distended gravid uterine horns at days 19 and 23 (labor) of gestation compared with nongravid horns. Similarly, pSer(15) HspB1 protein in situ was only readily detectable in the distended horns compared with the nongravid horns at days 19 and 23; however, pSer(15) HspB1 was primarily detectable in situ at day 19 in membrane-associated regions, while it had primarily a cytoplasmic localization in myometrial cells at day 23. HspB1 mRNA and pSer(15) HspB1 protein expression were also markedly increased in ovariectomized nonpregnant rat myometrium distended for 24 h with laminaria tents compared with empty horns. Therefore, uterine distension plays a major role in the stimulation of myometrial HspB1 expression, and increased expression of this small stress protein could be a mechanoadaptive response to the increasing uterine distension that occurs during pregnancy.

Spatiotemporal expression of alpha(1), alpha(3) and beta(1) integrin subunits is altered in rat myometrium during pregnancy and labour.

Integrins are transmembrane extracellular matrix (ECM) receptors composed of alpha- and beta-subunits. Integrins can cluster to form focal adhesions and, because there is significant ECM remodelling and focal adhesion turnover in the rat myometrium during late pregnancy, we hypothesised that the expression of alpha(1), alpha(3) and beta(1) integrin subunits in the rat myometrium would be altered at this time to accommodate these processes. Expression of alpha(1) and beta(1) integrin subunit mRNA was significantly increased on Days 6-23 of pregnancy compared with non-pregnant (NP) and postpartum (PP) time points (P < 0.05). In contrast, alpha(3) integrin subunit mRNA expression was significantly increased on Days 14, 21 and 22 compared with NP, Day 10, 1 day PP and 4 days PP (P < 0.05). A relative gene expression study revealed that, of the integrins studied, the expression of beta(1) integrin mRNA was highest in pregnant rat myometrium. The alpha(1), alpha(3) and beta(1) integrin subunit proteins became immunolocalised to myocyte membranes in situ by late pregnancy and labour in both myometrial muscle layers. Increased alpha(1), alpha(3) and beta(1) integrin gene expression during gestation and the specific detection of these subunits in myocyte membranes during late pregnancy and labour may contribute to the cell-ECM interactions required for the development of a mechanical syncytium.

Expression of small heat shock-related protein 20 (HSP20) in rat myometrium is markedly decreased during late pregnancy and labour.

The underlying mechanisms regulating uterine contractions during labour are still poorly understood. Heat shock protein 20 (HSP20) is known to be present at high levels in smooth muscle and implicated in muscle relaxation, but HSP20 expression in the myometrium is completely undetermined. Since HSP20 has been implicated in smooth muscle relaxation, we hypothesized that HSP20 would be highly expressed in the rat myometrium during early and mid-pregnancy when the myometrium is relatively quiescent. Northern blot analysis particularly demonstrated that HSP20 mRNA detection was significantly decreased from day (d) 22 of pregnancy to 1-day post-partum (PP) compared with d6 (P < 0.05). HSP20 mRNA detection was also significantly decreased from d22 to d23 of gestation compared with non-pregnant (NP) samples. Immunoblot analysis showed that detection of HSP20 was significantly decreased at d23 compared with d12 and d15 (P < 0.05). HSP20 detection also significantly decreased at PP compared with d15 (P < 0.05). Immunofluorescence analysis demonstrated that after d15, plasma membrane-associated localization of HSP20 decreased markedly in both circular and longitudinal muscle layers. In addition, HSP20 was detectable near cell membranes at much higher levels in the longitudinal muscle layer of progesterone-treated pregnant rats (delayed labour) at all gestational time points examined, compared with controls. Our results demonstrate that HSP20 mRNA and protein are highly expressed during early and mid-pregnancy and then the expression markedly decreases during late pregnancy and labour. The observed patterns of HSP20 expression are consistent with a potential role for HSP20 in facilitating myometrium quiescence during early and mid-pregnancy.

Integrin-linked kinase (ILK) is highly expressed in first trimester human chorionic villi and regulates migration of a human cytotrophoblast-derived cell line.

The placenta represents a critically important fetal-maternal interaction. Trophoblast migration and invasion into the uterine wall is a precisely controlled process and aberrations in these processes are implicated in diseases such as preeclampsia. Integrin-linked kinase (ILK) is a multifunctional, cytoplasmic, serine/threonine kinase that has been implicated in regulating processes such as cell proliferation, survival, migration, and invasion; yet the temporal and spatial pattern of expression of ILK in human chorionic villi and its role in early human placental development are completely unknown. We hypothesized that ILK would be expressed in trophoblast subtypes of human chorionic villi during early placental development and that it would regulate trophoblast migration. Immunoblot analysis revealed that ILK protein was highly detectable in placental tissue samples throughout gestation. In floating branches of chorionic villi, from 6 to 15 wk of gestation immunofluorescence analysis of ILK expression in placental tissue sections demonstrated that ILK was highly detectable in the cytoplasm and membranes of villous cytotrophoblast cells and in stromal mesenchyme, whereas it was barely detectable in the syncytiotrophoblast layer. In anchoring branches of villi, ILK was highly localized to plasma membranes of extravillous trophoblast cells. Transient expression of dominant negative E359K-ILK in the villous explant-derived trophoblast cell line HTR8-SVneo dramatically reduced migration into wounds compared to cells expressing wild-type ILK or empty vector. Therefore, our work has demonstrated that ILK is highly expressed in trophoblast subtypes of human chorionic villi during the first trimester of pregnancy and is a likely mediator of trophoblast migration during this period of development.

Expression of alpha5 integrin (Itga5) is elevated in the rat myometrium during late pregnancy and labor: implications for development of a mechanical syncytium.

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.

Small heat shock protein 27 (Hsp27) expression is highly induced in rat myometrium during late pregnancy and labour.

The underlying mechanisms that regulate uterine contractions during labour are still poorly understood. A candidate regulatory protein is heat shock protein 27 (Hsp27). It belongs to the small heat shock protein family and can regulate actin cytoskeleton dynamics, act as a chaperone, and may regulate contractile protein activation. As a result, we hypothesized that Hsp27 expression would be highly induced during late pregnancy and labour. Hsp27 mRNA expression was significantly elevated (P <0.05) on days 17 to 22 of gestation. In addition, immunoblot analysis demonstrated that detection of total Hsp27 increased (P <0.05) between day 21 and 1 day post-partum (PP) inclusive. Since phosphorylation of Hsp27 has been reported to be a prerequisite for smooth muscle contraction, we examined the temporal and spatial expression of Ser-15 phosphorylated Hsp27. Immunoblot analysis showed that the detection of Ser-15 phosphorylated Hsp27 significantly increased (P <0.05) between days 19 and 23 (active labour) inclusive, in parallel with detection of total Hsp27. Immunocytochemical analysis of Ser-15 phosphorylated Hsp27 expression in situ demonstrated that phosphorylated Hsp27 in circular muscle became detectable in peri-nuclear and membrane regions on days 19 to 22, but was primarily restricted to the cytoplasm on days 23 to PP. In contrast, phosphorylated Hsp27 in longitudinal muscle was primarily detected in myocyte membranes on days 15 to 22, and then also became detectable in the cytoplasm of myocytes on days 23 and PP. Our results demonstrate that Hsp27 expression is highly upregulated during late pregnancy and labour and suggest that Hsp27 is a potential candidate contraction-associated protein.

Focal adhesion kinase is a key mediator of human trophoblast development.

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 adhesion-associated 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 alpha5 integrin and matrix metalloproteinase-2 (MMP2) expression in EVT cells within a previously characterized intermediate, invasive-restrained 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 alpha5 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.

Expression and localization of the contractile prostaglandin F receptor in pregnant rat myometrium in late gestation, labor, and postpartum.

A polyclonal antibody was raised against amino acids 7-18 in the first extracellular loop of rat prostaglandin F (FP) receptor to monitor expression and localization in pregnant rat myometrium at Gestational Days 16, 18, 20, 21, 21.5, 22 (delivery), and 23 (1-day postpartum; n = 5 per group). The antibody recognized a protein of approximately 43 kDa on Western blot analysis in both membrane (soluble and nonsoluble) and cytosolic fractions of myometrium on each day of gestation. Expression of FP protein increased significantly (P < 0.05) during late gestation in both soluble membrane and cytosolic fractions, being significantly greater at Day 21.5 than at Day 20 of gestation in the soluble membrane fraction and in the cytosolic fraction of tissues collected during labor compared with those obtained before labor. The total concentration of FP receptor in the membrane (soluble plus nonsoluble) remained high throughout late gestation and fell significantly (P < 0.05) in the postpartum period. The FP receptor in the soluble membrane fraction (compared to the total membrane FP receptor) was significantly (P < 0.05) higher in late gestation than earlier, whereas the ratio of FP protein in cytosolic to that in the total membrane was significantly (P < 0.05) higher on Day 23 than earlier in gestation, suggesting a dynamic movement of FP with advancing gestational age. Immunoreactive FP receptor localized to circular and longitudinal smooth muscle at all gestational ages, but changes in intracellular localization were observed in late gestation with a staining pattern similar to alpha-actin, suggesting an association with myofibrils. Our study suggests an increase in FP-receptor protein in myometrium with advancing gestation and a marked elevation at term. This supports a role for uterine FP receptors in mediation of uterine contractility at term.

Differential involvement of Na(+),K(+)-ATPase isozymes in preimplantation development of the mouse.

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.

Focal adhesion signaling in the rat myometrium is abruptly terminated with the onset of labor.

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.

Evidence for the presence of sodium- and potassium-dependent adenosine triphosphatase alpha1 and beta1 subunit isoforms and their probable role in blastocyst expansion in the preattachment horse conceptus.

The unusual hypotonicity of equine blastocyst fluid has prompted us to investigate the role of sodium- and potassium-dependent adenosine triphosphatase (Na+,K+-ATPase) in the process of fluid accumulation in the horse conceptus. Nine mares were used for the experiments. Reverse transcriptase polymerase chain reaction was conducted on two sets of five conceptuses recovered between 12 and 28 days (+/- 1 day) after ovulation. Messenger RNAs encoding the alpha1 and beta1 subunit isoforms of Na+,K+-ATPase were detected in all embryonic tissues examined. Western blot analysis showed that alpha1 and beta1 subunits are both present in Day 15 conceptuses. Trophoblast tissues from 19 conceptuses between 8 and 31 days after ovulation were stained immunohistochemically using primary antibodies against the alpha1 and beta1 subunit isoforms of the Na+,K+-ATPase. Both isoforms were detected in all sections. Trophoblastic vesicles, prepared from 6 conceptuses between 12 and 14 days after ovulation, were used to investigate the inhibition of blastocyst expansion with ouabain after collapse induced with cytochalasin D. In normal medium there was a mean 3-fold increase, and in ouabain (10(-6) M) a mean 3-fold decrease, in the volume of vesicles that had been partially collapsed with cytochalasin D. We therefore conclude that, despite the hypotonicity of the blastocyst fluid in the early horse conceptus, the Na+,K+-ATPase plays a role in its accumulation, as in other species.

Ouabain sensitivity and expression of Na/K-ATPase alpha- and beta-subunit isoform genes during bovine early development.

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 alpha- and beta-subunits, including isoforms not previously detected during the first week of mammalian development. Transcripts encoding the Na-K-ATPase alpha 1, alpha 2, alpha 3 and beta 2 isoforms were detected throughout bovine preattachment development. This is the first indication that alpha 2, alpha 3 and beta 2 mRNAs are expressed during this early developmental interval. As in the mouse, beta 1-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 beta 1 isoform transcripts in the morula stage is coincident with the onset of cavitation. Transcripts encoding the recently characterized alpha 4 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(-3)M. These results have established that transcripts encoding multiple isoforms of both the alpha and beta 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.

Evidence that disruption of connexon particle arrangements in gap junction plaques is associated with inhibition of gap junctional communication by a glycyrrhetinic acid derivative.

Glycyrrhetinic acid exhibits many pharmacological activities, including the ability to block gap junctional communication. However, the mechanism of glycyrrhetinic action is not clear. Others have shown that glycyrrhetinic acid apparently binds to a single proteinatious binding site in the plasma membrane. We present evidence that while exposure to glycyrrhetinic acid derivatives may not affect protein synthesis or location, it does seem to alter connexon particle packing in gap junction plaques.