| 1 |
Overview I |
Michael J. Soares,Joan S. Hunt |
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Abstract
The placenta is a specialized pregnancy-specific structure that develops concurrently with development of the embryo and fetus. From an evolutionary perspective, the placenta was the essential factor in permitting viviparity, a reproductive strategy in which fetal development proceeds within the female reproductive tract. Viviparous species are able to provide greater protection from environmental risks and can more precisely control the development of their progeny while they reside .. The placenta is comprised of numerous cell types. Among the cell types are specialized epithelioid cells, called trophoblast, that possess several important functions enabling viviparous development (.,.). Trophoblast cells play key roles in protecting the embryo/fetus from noxious substances, programming maternal support, and preventing maternal immune rejection while at the same time ensuring appropriate bidirectional nutrient/waste flow required for growth and maturation of the embryo. Although placenta functions are highly conserved, species-specific elements of placenta organization and activity are evident. Consequently, placental research has benefited and will continue to benefit from a comparative approach. Each species presents experimentally valuable attributes that can be exploited to better understand the biology of the placenta and viviparity.
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| 2 |
Methodologies to Study Implantation in Mice |
Kaushik Deb,Jeff Reese,Bibhash C. Paria |
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Abstract
Pregnancy begins with fertilization of the ovulated oocyte by the sperm. After fertilization, the egg undergoes time-dependent mitotic division while trying to reach the blastocyst stage and the uterus for implantation. Uterine preparation for implantation is regulated by coordinated secretions and functions of ovarian sex steroids. The first sign of contact between the blastocyst and the uterus can be detected experimentally by an intravenous blue dye injection as early as the end of day 4 or the beginning of day 5 of pregnancy. This blastocyst-uterine attachment reaction leads to stromal decidual reaction only at sites of implantation. The process of implantation can be postponed and reinstated experimentally by manipulating ovarian estrogen secretion. Stromal decidualization can also be induced experimentally in the hormonally prepared uterus in response to stimuli other than the embryo. Fundamental biological questions surrounding these essential features of early pregnancy can be addressed through the application of various techniques and manipulation of this period of early pregnancy. This chapter describes the routine laboratory methodologies to study the events of early pregnancy, with special emphasis on the implantation process in mice.
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| 3 |
Blastocyst Culture |
D. Randall Armant |
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Abstract
Experimental models of blastocyst development based on in vitro culture have played a prominent role in advancing our understanding of peri-implantation development, a process that is relatively inaccessible in vivo. Blastocyst culture provides a robust approach for examining embryonic interactions with the microenvironment under highly controlled conditions. Major events that occur . can be followed in vitro, including blastocyst expansion, hatching, and adhesion to extracellular matrices. This chapter will describe a method for obtaining and culturing mouse blastocysts. Morphological changes that occur during blastocyst culture will be discussed and related to the corresponding development .. Finally, quantitative assays will be detailed for monitoring peri-implanatation development of the trophoblast in vitro.
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| 4 |
Isolation of Hormone Responsive Uterine Stromal Cells |
Virginia Rider |
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Abstract
The female sex hormones estrogen and progesterone stimulate proliferation and differentiation of human and rodent uterine cells. The purpose of this chapter is to provide a method for isolating hormone-responsive rat uterine stromal cell lines that can be used to study steroid control of the cell cycle. Uteri from ovariectomized rats are differentially digested with trypsin to separate epithelial and stromal cells. The stromal cells are cultured in a standard growth medium containing 10% fetal bovine serum. After several passages, the purity of the stromal cell lines is determined using immunocytochemistry. Cell proliferation is studied by culturing the stromal cells in serum-free medium containing sex steroids and other mitogens. Cell cycle progression is assessed by flow cytometry, .H-thymidine and BrdU incorporation, whereas proliferation is monitored using the MTT assay. Cell cycle regulators are visualized by Northern and Western blotting whereas cyclin-cyclin-dependent kinase activity is monitored using immune complex kinase assays. Uterine stromal cell lines isolated using the methods reported in this chapter provide a suitable model system to investigate the signal transduction events that stimulate hormone-dependent control of the cell cycle.
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| 5 |
Rat Decidual Cell Cultures |
Yan Gu,Geula Gibori |
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Abstract
Pregnancy requires profound reorganization of the different tissues forming the uterus. Growth and differentiation of the uterine endometrial cells give rise to the decidual tissue, a transitory organ, which plays a key role in fetal survival. In this chapter, we describe a technique for the dispersion and the separation of the two different decidual cell sub-populations with high yield and viability. We also detail a cell culture method, which allows the maintenance of the function and life span of these highly purified decidual cells when cultured either separately or in a co-culture system.
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| 6 |
The Immortalization of Human Endometrial Cells |
Graciela Krikun,Gil Mor,Charles Lockwood |
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Abstract
The loss of replicative potential with each cell division has been attributed to the progressive shortening of telomeres. This “mitotic clock” occurs because most normal human cells are telomerase-negative. Telomerase is a multicomponent enzyme that prevents loss of telomeric DNA associated with normal cell division. Transfection of cells with vectors expressing the catalytic subunit of human telomerase (hTERT) is often sufficient for immortalization. In this article, we will address this approach in the establishment of immortalized endometrial cells and its value in facilitating in vitro studies.
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| 7 |
Sheep Uterine Gland Knockout (UGKO) Model |
Thomas E. Spencer,C. Allison Gray |
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Abstract
Endometrial gland development is a postnatal event in the ovine uterus that can be inhibited epigenetically by chronic exposure of ewe lambs to a synthetic progestin after birth. The uterus of neonatally progestinized ewes lack endometrial glands and display a uterine gland knockout (UGKO) phenotype. Progestin ablation of endometrial gland development is specific, because it does not affect development of extra-uterine reproductive tract structures or the hypothalamic—pituitary—ovarian axis. The UGKO ewe is a useful model for study of uterine development and the role of endometrial glands in uterine function during the estrous cycle and pregnancy. UGKO ewes exhibit altered estrous cycles due to the inability of the uterus to produce luteolytic pulses of prostaglandin F2α. UGKO ewes are infertile, and blastocysts hatch normally but fail to survive or elongate during early pregnancy. This pregnancy defect is primarily due to the absence of endometrial glands and their secretions rather than alterations in expression of either anti-adhesive or adhesive molecules on the endometrial epithelium. Genomics and proteomics are being used to identify specific components of histotroph that are absent or diminished in the UGKO ewe and will serve as markers of endometrial function and uterine receptivity.
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| 8 |
A Baboon Model for Inducing Endometriosis |
Asgerally T. Fazleabas |
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Abstract
Endometriosis is a disease that is associated with severe pelvic pain and is a major cause of infertility in women. It is an enigmatic disease whose etiology and pathophysiology has been studied to a limited extent. The events associated with the establishment of the disease and mechanisms associated with infertility are difficult to assess in a systemic manner in women. In order to understand the early and progressive events associated with the establishment of the disease, we have developed a baboon model in which the disease can be induced. This induction manifests itself in a manner that recapitulates the spontaneous disease. The advantage of the induced model is that the progressive changes in both the ectopic and eutopic endometrium can be studied in a nonhuman primate model at specific times during the menstrual cycle and as the disease process continues.
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| 9 |
A Baboon Model for Simulating Pregnancy |
Asgerally T. Fazleabas |
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Abstract
Estrogen and progesterone secreted by the corpus luteum regulate the function of the uterine endometrium in preparation for pregnancy. Embryonic signals superimposed on this steroid hormone-primed uterus further modulate the uterine environment to make it conducive to pregnancy. Understanding the signaling mechanisms that initiate the embryonic-maternal dialog in humans is not feasible. In an effort to elucidate the role of chorionic gonadotropin as a mediator of endometrial function in addition to its luteotrophic role, we have developed a simulated pregnant model in the baboon. Infusion of chorionic gonadotropin in a manner that mimics blastocyst transit induces major changes in the morphology and secretory activity of the endometrium. This model provides a method by which the function of various embryonic factors on endometrial can be tested in an in vivo model.
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| 10 |
The Common Marmoset Monkey as a Model for Implantation and Early Pregnancy Research |
Almuth Einspanier,Kai Lieder,Ralf Einspanier,Bettina Husen |
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Abstract
This chapter describes methods used to investigate implantation in the common marmoset monkey, .. A reverse-transcriptase polymerase chain reaction-strategy with which to detect transcripts for steroid receptors and enzymes involved in estradiol biosynthesis is described, and an immunohistochemistry approach for detecting proteins within the implantation site is presented.
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| 11 |
Mouse Trophoblast Stem Cells |
Jennifer Quinn,Tilo Kunath,Janet Rossant |
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Abstract
The trophectoderm is one of the earliest cell types to differentiate in the forming mammalian embryo. It is responsible for the initial implantation and the formation of the trophoblast components of the placenta, an organ essential for nutrient and waste exchange between the fetus and its mother. The trophoblast can be modeled in vitro using trophoblast stem cells. Trophoblast stem cells require fibroblast growth factor (FGF)4, heparin, and contact with embryonic fibroblasts, or fibroblast-conditioned medium. They grow as tight epithelial colonies, which express markers of the early trophectoderm and have been shown to contribute to all of the components of the placenta through chimera studies. These cells can be passaged indefinitely and can be differentiated by removal of FGF4 and fibroblasts and will express genetic markers of later placental cell types. This chapter will discuss the initial derivation of trophoblast stem cells from the blastocyst stage, maintenance, differentiation, flow cytometry and transfection techniques that can be used with these cells.
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| 12 |
Connexins and Trophoblast Cell Lineage Development |
Mark Kibschull,Elke Winterhager |
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Abstract
The mouse is a valuable model for studying basic mechanisms of gene regulation in trophoblast cell lineage differentiation. Elements of placental development are conserved across species, including trophoblast proliferation, differentiation, migration, and vessel invasion. Among the regulatory processes, direct intercellular communication between trophoblast cells via gap junction channels seems to play a crucial role in placental development and physiology. Here we describe in detail the generation of trophoblast stem (TS) cell lines from connexin-deficient mice. The design of differentiation and proliferation assays are specified including marker gene sets which are important for analyzing and comparing the differentiation capacity of the connexin-deficient TS cell lines. Furthermore, we show that TS cells are capable of forming tumors after subcutaneous injection into nude mice, providing the opportunity to investigate trophoblast invasion into host vessels in vivo.
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| 13 |
Rcho-1 Trophoblast Stem Cells |
Namita Sahgal,Lindsey N. Canham,Brent Canham,Michael J. Soares |
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Abstract
The biology of trophoblast cell development can be investigated using in vitro model systems. The Rcho-1 trophoblast stem cell line was derived from a rat choriocarcinoma and is an effective tool for elucidating regulatory mechanisms controlling trophoblast cell differentiation. In this chapter, we describe methods used in the maintenance and manipulation of the Rcho-1 trophoblast cell line.
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| 14 |
Bovine Trophoblast Cell Culture Systems |
Kazuyoshi Hashizume,Arata Shimada,Haruo Nakano,Toru Takahashi |
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Abstract
Bovine trophoblastic cells are the first cells to differentiate during embryogenesis and play pivotal role in morphological and physiological development of the placenta. We have developed culture systems for bovine trophoblast stem cells isolated from in vitro fertilized blastocysts in the absence of feeder cells. These cells continuously proliferate in Dulbecco’s modified Eagle’s/F12 culture medium supplemented with bovine endometrial fibroblast-conditioned medium. The cells possess epithelial morphology, express cytokeratin, and form dome-like structures (vesicles). Methods for the maintenance, subculture, storage, and measurement of bovine trophoblast stem cell growth are described. The cells exhibit characteristics of bovine trophoblastic stem cells and possess the ability to differentiate into binucleate cells and express placental lactogen, prolactin-related protein-1, pregnancy-associated glycoprotein-1, and interferon τ.
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| 15 |
In Vitro Induction of Trophoblast from Human Embryonic Stem Cells |
Ren-He Xu |
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Abstract
Human embryonic stem (ES) cells can proliferate without a known limit and can form advanced derivatives of all three embryonic germ layers. What is less widely appreciated is that human ES cells can also form the extra-embryonic tissues that differentiate from the embryo before gastrulation. The use of human ES cells to derive early human trophoblast is particularly valuable, because it is difficult to obtain from other sources and is significantly different from mouse trophoblast. Here we describe a method by using bone morphogenetic protein (BMP)4 , a member of the transforming growth factor (TGF)-β superfamily, to induce the differentiation of human ES cells to trophoblast. Immunoassays (as well as DNA microarray and reverse-transcription polymerase chain reaction analyses—data not shown) demonstrate that the differentiated cells express a range of trophoblast markers and secrete placental hormones. When plated at low density, the BMP4-treated cells form syncytia that express chorionic gonadotrophin (CG). This technique underscores fundamental differences between human and mouse ES cells, which differentiate poorly, if at all, to trophoblast. Human ES cells thus provide a tool for studying the differentiation and function of early human trophoblast and could provide a new understanding of some of the earliest differentiation events of human postimplantation development.
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| 16 |
Isolation and Culture of Term Human Trophoblast Cells |
Margaret G. Petroff,Teresa A. Phillips,Hakhyun Ka,Judith L. Pace,Joan S. Hunt |
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Abstract
Experimentation with most human cell types is restricted to the use of cell lines, and this limits our ability to extrapolate interpretations to the in vivo condition. However, in studying human trophoblast cells, we have a unique opportunity to obtain large quantities of readily available human tissue. In this chapter, we outline the methodology for purification of human trophoblast cells from term placentas. The procedures are based on enzymatic dissociation of villous placental tissue, followed by gradient centrifugation and immunomagnetic bead purification. Purity may be assessed by immunocytochemistry or flow cytometry using a number of markers to identify both cytotrophoblast cells and cellular contaminants. The resulting cytotrophoblast cell populations have excellent viability and purity, and may be subjected to long-term culture.
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发表于 2025-3-21 16:23:46
