All scientific investigations begin with distinct objectives: first is the hypothesis upon which studies are undertaken to disprove, and second is the overall aim of obtaining further information, from which future and more precise hypotheses may be drawn Studies focusing on the generation and use of gene-targeted animal models also apply these goals and may be loosely categorized into sequential phases that become apparent as the use of the model progresses Initial studies of knockout models often focus on the plausibility of the model based on prior knowledge and whether the generation of an animal lacking the particular gene will prove lethal or not Upon the successful generation of a knockout, confirmatory studies are undertaken to corroborate previously established hypotheses of the function of the disrupted gene product As these studies continue, observations of unpredicted phenotypes or, more likely, the lack of a phenotype that was expected based on models put forth from past investigations are noted Often the surprising phenotype is due to the loss of a gene product that is downstream from the functions of the disrupted gene, whereas the lack of an expected phenotype may be due to compensatory roles filled by alternate mechanisms As the descriptive studies of the knockout continue, use of the model is often shifted to the role as a unique research reagent, to be used in studies that 1) were not previously possible in a wild-type model; 2) aimed at finding related proteins or pathways whose existence or functions were previously masked; or 3) the subsequent effects of the gene disruption on related physiological and biochemical systems The alpha ERKO mice continue to satisfy the confirmatory role of a knockout quite well As summarized in Table 4, the phenotypes observed in the alpha ERKO due to estrogen insensitivity have definitively illustrated several roles that were previously believed to be dependent on functional ER alpha, including 1) the proliferative and differentiative actions critical to the function of the adult female reproductive tract and mammary gland; 2) as an obligatory component in growth factor signaling in the uterus and mammary gland; 3) as the principal steroid involved in negative regulation of gonadotropin gene transcription and LH levels in the hypothalamic-pituitary axis; 4) as a positive regulator of PR expression in several tissues; 5) in the positive regulation of PRL synthesis and secretion from the pituitary; 6) as a promotional factor in oncogene-induced mammary neoplasia; and 7) as a crucial component in the differentiation and activation of several behaviors in both the female and male The list of unpredictable phenotypes in the alpha ERKO must begin with the observation that generation of an animal lacking a functional ER alpha gene was successful and produced animals of both sexes that exhibit a life span comparable to wild-type The successful generation of beta ERKO mice suggests that this receptor is also not essential to survival and was most likely not a compensatory factor in the survival of the alpha ERKO In support of this is our recent successful generation of double knockout, or alpha beta ERKO mice of both sexes The precise defects in certain components of male reproduction, including the production of abnormal sperm and the loss of intromission and ejaculatory responses that were observed in the alpha ERKO, were quite surprising In turn, certain estrogen pathways in the alpha ERKO female appear intact or unaffected, such as the ability of the uterus to successfully exhibit a progesterone-induced decidualization response, and the possible maintenance of an LH surge system in the hypothalamus [ABSTRACT TRUNCATED]

Estrogen receptor null mice: what have we learned and where will they lead us?

Implantation initiation in eutherian mammals requires, at least in invasive types of implantation, that 1) the trophoblast or subpopulations of it have reached a state of "invasiveness" and, synchronously, 2) the endometrium has reached a state of "receptivity" ("implantation window"). The cell biological basis of "receptivity" as well as of "invasiveness" has long remained unknown, but recently it appears that the application of modern concepts of cell and developmental biology is opening new views of it, concentrating on cell adhesion and cell polarity phenomena. Implantation initiation requires that the trophoblast attaches via its apical plasma membrane to the apical plasma membrane of the uterine epithelium. Since apical plasma membranes of epithelia are normally non-adhesive, this represents a cell biological paradox. In development, cells can express two major phenotypes and switch between these: 1) the mesenchymal/fibroblastoid phenotype that is compatible with cells moving individually; 2) the epithelioid phenotype which is characterized by cells expressing apico-basal polarity and a strong association with neighbouring cells via various junctions, so that they can migrate as sheets but not as individual cells. Application of this concept to embryo implantation would seem to lead to the hypothesis that the trophoblast of blastocysts has to give up part of its typical epithelial organization when becoming invasive; it must express cell-cell adhesion molecules or matrix receptors non-typically at its apical plasma membrane, change the spectrum of expressed adhesion molecules, and alter its motility apparatus. This applies in a somewhat similar way to the uterine epithelium at "receptivity". Interestingly, recent data show that a substantial number of differentiation parameters of these cells change in addition. It appears that part of the epithelial differentiation program is down-regulated at this phase. This also suggests interesting new aspects of the basis of steroid hormone action at the endometrium, as well as of trophoblast invasiveness, possibly involving switches in the activity of regulatory "master" genes as are also involved in decision making during development.

Implantation: a cell biological paradox

Implantation involves the attachment of the blastocyst to the wall of the uterus with, in some cases, the embedding of the entire ovum in the uterine wall. Associated with it are very profound and characteristic changes in the endometrium both during preparation for implantation and following attachment of the blastocyst. In this chapter, the uterine changes that take place during preparation will be considered first and then the changes during the process of implantation. The former occur regularly and frequently throughout the life of most female mammals, whereas the latter are rarer events, especially in women. This division of the subject is also convenient for the consideration of the control mechanism involved. Whereas the preparative changes are largely directed by the varying levels of the hormones secreted by the ovary, once the implantation process has been initiated hormones play a largely permissive role.

The Implantation Reaction

Classical and contemporary studies have shown that endocrine regulation exerted by ovarian hormones priming the endometrium is essential for embryo implantation. Increasing evidence indicates that steroid-induced molecules acting as paracrine modulators are necessary for embryo-uterine interactions. That is the case for calcitonin, heparin-binding epidermal growth factor (EGF)-like growth factor, leukaemia inhibitory factor and other molecules. Furthermore, when the blastocyst enters the uterine cavity, it starts the complex signals that will drive embryo adhesion. The paracrinology of this process is based on the local interplay of molecules, such as the secretion of cytokines that may facilitate the acquisition of endometrial receptivity by controlling the expression of adhesion and anti-adhesion proteins. Finally, during the embryonic invasive phase, uterine stromal-trophoblast dialogue may modulate this self-controlled proteolytic and immunological process to avoid damage to both the embryo and maternal environment.

Paracrine regulators of implantation

Assessment of endometrial receptivity

Four-day-old mouse embryos were transferred to the uterine lumen of virgin cyclic and ovariectomized mice; the eggs 'implanted' and developed only in mice whose endometrium was previously traumatized with a glass scraper. The histology of the mechanically induced implantation sites is described and similarities to normal implantation sites are discussed. Implanted embryos developed only to stages equivalent to 5 to 9 days of normal pregnancy, but the trophoblast continued to proliferate and invaded the endometrium, eroding maternal blood vessels and distending the uterus. In five of eight ovariectomized mice, plaques of decidual-like cells were found near the trophoblast 7 to 12 days after transfer.

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Thomas P. Cowell

Papers

Implantation and development of mouse eggs transferred to the uteri of non-progestational mice.