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Introduction: Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood., Objective: While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens., Methods: A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections., Results: The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion., Discussion/conclusion: Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst's regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The expression pattern of caveolin-1 and the distribution of caveolae in the murine placental labyrinth and visceral yolk sac have been determined. Immunoblot analysis demonstrates that both placenta and yolk sac express the protein caveolin-1. Immunofluorescence microscopy was used to determine which cell types in the placental labyrinth and yolk sac express caveolin-1. In yolk sac, detectable caveolin-1 was restricted to endothelial cells and smooth muscle cells of the vitelline vasculature and to mesothelial cells. Endoderm, the major cell type in the yolk sac, does not express caveolin-1 as assessed by this assay. In the labyrinth region of the placenta, endothelial cells express caveolin-1 but this protein was not detectable in any of the three trophoblast layers. These tissues were also examined by electron microscopy to determine which cell types contain the specialized plasma membrane microdomains known as caveolae. Morphologically detectable caveolae were present in endothelial and smooth muscle cells, as well as mesothelial cells of the yolk sac and in endothelial cells of the placental labyrinth. Neither endodermal cells of the yolk sac nor trophoblastic cells in the placental labyrinth contained caveolae-like structures. We conclude that caveolin-1 and caveolae have restricted distribution in the murine placenta and yolk sac and that this parallels the situation in human placenta., (Copyright 2009. Published by Elsevier Ltd.)

Dysferlin (DYSF) and myoferlin (MYOF), members of the ferlin family of membrane proteins, are co-expressed in human placental syncytiotrophoblast (STB). Although the role of these ferlin proteins in the placenta has yet to be established, it has been suggested that DYSF and MYOF may contribute to the stability of the apical STB plasma membrane. The release of STB-derived cellular debris increases in the setting of preeclampsia (PE), suggesting relative destabilization of the hemochorial interface. To test whether PE was associated with alterations in placental expression of DYSF and/or MYOF, a cross-sectional study was performed using specimens of villous placenta collected form women with severe PE (n=10) and normotensive controls (n=10). DYSF and MYOF expression were examined using quantitative real-time RT-PCR, immunoblotting, and immunofluorescence labeling of tissue specimens. Placental DYSF expression was 57% lower at the mRNA level (p=0.03) and 38% lower at the protein level (p=0.026) in severe PE as compared to normotensive subjects. There were no differences in placental MYOF protein or mRNA expression between these groups. No appreciable changes in the distribution of DYSF or MYOF within placental villi was observed in PE relative to control specimens. We conclude that DYSF expression is reduced in severe PE relative to gestational age-matched controls. As DYSF has a role in membrane repair, these data suggest a role for DYSF in the stability of the apical STB plasma membrane and may account, at least in part, for the increased shedding of microparticles from this membrane in PE.

Proteomics analysis of biological samples has the potential to identify novel protein expression patterns and/or changes in protein expression patterns in different developmental or disease states. An important component of successful proteomics research, at least in its present form, is to reduce the complexity of the sample if it is derived from cells or tissues. One method to simplify complex tissues is to focus on a specific, highly purified sub-proteome. Using this approach we have developed methods to prepare highly enriched fractions of the apical plasma membrane of the syncytiotrophoblast. Through proteomics analysis of this fraction we have identified over five hundred proteins several of which were previously not known to reside in the syncytiotrophoblast. Herein, we focus on two of these, dysferlin and myoferlin. These proteins, largely known from studies of skeletal muscle, may not have been found in the human placenta were it not for discovery-based proteomics analysis. This new knowledge, acquired through a discovery-driven approach, can now be applied for the generation of hypothesis-based experimentation. Thus discovery-based and hypothesis-based research are complimentary approaches that when coupled together can hasten scientific discoveries.

Proteomics is an area of study that sets as its ultimate goal the global analysis of all of the proteins expressed in a biological system of interest. However, technical limitations currently hamper proteome-wide analyses of complex systems. In a more practical sense, a desired outcome of proteomics research is the translation of large protein data sets into formats that provide meaningful information regarding clinical conditions (e.g., biomarkers to serve as diagnostic and/or prognostic indicators of disease). Herein, we discuss placental proteomics by describing existing studies, pointing out their strengths and weaknesses. In so doing, we strive to inform investigators interested in this area of research about the current gap between hyperbolic promises and realities. Additionally, we discuss the utility of proteomics in discovery-based research, particularly as regards the capacity to unearth novel insights into placental biology. Importantly, when considering under studied systems such as the human placenta and diseases associated with abnormalities in placental function, proteomics can serve as a robust 'shortcut' to obtaining information unlikely to be garnered using traditional approaches.

In the third trimester, human placental endothelial cells express Fc gamma receptor IIb (FcgammaRIIb). This expression is unique because FcgammaRIIb is generally expressed on immune cells and is typically undetectable in adult endothelial cells. Recently, we found a novel FcgammaRIIb-defined, IgG-containing organelle in placental endothelial cells; this organelle may be a key structure for the transcytosis of IgG across the endothelial layer. In this study, we verify the expression of FcgammaRIIb in endothelial placenta cells and use reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing analyses to define the expressed FCGR2B mRNA transcript variant. We also investigated the distribution of FCGR2B mRNA and protein within the vascular tree of the full-term human placenta by RT-PCR and quantitative microscopy. The mRNA sequence of FCGR2B expressed specifically in placental endothelial cells is that of transcript variant 2. FcgammaRIIb expression and synthesis occur throughout the placental vascular tree but do not extend into the umbilical cord. This study provides additional information on FcgammaRIIb expression in the human placenta.

In this report, we describe procedures for correlative fluorescence and electron microscopy in immunocytochemical studies on the human placenta. Ultrathin cryosections of placenta were used for detection of the distribution of antigens by immunofluorescence and subsequently by immunoelectron microscopy of the same ultrathin cryosection. This methodology has certain advantages over conventional immunohistochemistry and immunoelectron microscopy. The advantages are, most notably, that the same exact structures are examined by both imaging modalities. In addition, since the tissue is physically sectioned (50-100 nm thickness), greater resolution for fluorescence can be obtained in the z-dimension than can be obtained by optical sectioning in confocal microscopy. This last point is of particular importance for discriminating between structures closely stacked in the z-dimension. In this report, we have determined the distribution of caveolin-1 in ultrathin cryosections of terminal villi of the human term placenta. We demonstrate that the use of ultrathin cryosections is a powerful approach for immunofluorescence and correlative microscopy for the in situ localization of antigens.

Correlative microscopy is a collection of procedures that rely upon two or more imaging modalities to examine the same specimen. The imaging modalities employed should each provide unique information and the combined correlative data should be more information rich than that obtained by any of the imaging methods alone. Currently the most common form of correlative microscopy combines fluorescence and electron microscopy. While much of the correlative microscopy in the literature is derived from studies of model cell culture systems we have focused, primarily, on correlative microscopy in tissue samples. The use of tissue, particularly human tissue, may add constraints not encountered in cell culture systems. Ultrathin cryosections, typically used for immunoelectron microscopy, have served as the substrate for correlative fluorescence and electron microscopic immunolocalization in our studies. In this work, we have employed the bifunctional reporter FluoroNanogold. This labeling reagent contains both a fluorochrome and a gold-cluster compound and can be imaged by sequential fluorescence and electron microscopy. This approach permits the examination of exactly the same sub-cellular structures in both fluorescence and electron microscopy with a high level of spatial resolution. [ABSTRACT FROM AUTHOR]