Interactions between trophoblast and uterine epithelium: monitoring of adhesive forces

At embryo implantation, it is postulated that the initial contact between blastocyst and maternal tissues is by adhesion of the trophoblast to the uterine epithelium. This cell-to-cell interaction is thought to be critical for implantation, although the actual adhesive forces have never been determined. In the present study, the atomic force microscope (AFM) was used to study the adhesion between human uterine epithelial cell lines (HEC-1-A; RL95-2) and human trophoblast-type cells (JAR). Specific interaction forces of these epithelia via their apical cell poles were determined on the basis of approach-and-separation cycles. For this purpose, the AFM tip was functionalized with JAR cells, then brought to the surface of uterine epithelial monolayers and was kept in contact for different periods of time (ms, 1, 10, 20, 40 min). The approach force curves displayed repulsive interactions for both HEC-1-A and RL95-2 cells. However, RL95-2 cells (with a smooth surface structure and a thin glycocalyx) showed lower values of the repulsive regime than HEC-1-A cells (with a rough surface structure and a thick glycocalyx). After having overcome repulsive interactions, the initial contact was followed by adhesive interactions. For contact times of 20 and 40 min, RL95-2 cells, but not HEC-1-A cells, showed specific JAR binding, i.e. the separation force curves displayed repeated rupture events in the range of 1-3 nN with a distance between 7-15 microm and, thereafter, a final rupture event at a distance of up to 45 microm. These features point to the formation of strong cell-to-cell bonds. Collectively, these studies provide the first definition of interaction forces between the trophoblast and the uterine epithelium, and are consistent with the hypothesis that an RL95-2-like architecture of uterine epithelial cells, i.e. an non-polarized phenotype, is essential for apical adhesiveness for the human trophoblast.     

The late component of L-type calcium current during guinea-pig cardiac action potentials and its contribution to contraction

The fetus must transport considerable and increasing amounts of calcium across the placental trophoblast epithelium to support growth and development and bone formation. Active calcium transport across epithelia has been shown to correlate with calbindin D9k or 28k content. This study examined the distribution of calbindin D9k (9CBP) protein and mRNA during pregnancy in the bovine placenta to determine its possible role in calcium transport in this system. The immunocytochemical results show 9CBP in an increasing percentage of interplacentomal uninucleate trophoblast cells until, at term, all show a level at least eight times that of any other placental cell. There is a similar, although smaller, rise in their 9CBP mRNA content. The mature interplacentomal binucleate cell ( approximately 5% of the total) contains no 9CBP at any stage of pregnancy. In interplacentomal uterine epithelium, 9CBP protein and mRNA decrease to zero in late pregnancy but the glands maintain constant low levels throughout. In the placentome trophoblast, uninucleate cells show insignificant amounts but binucleate cells (15-20% of the total trophoblast cells) contain considerable levels of both 9CBP protein and mRNA, as do all the uninucleate uterine epithelial cells. The placentomal binucleate cells show peak values at mid-pregnancy; the placentomal uterine epithelium shows only small changes in levels in the second half of pregnancy. Increase in fetal calcium demand in the second half of pregnancy therefore correlates with a major increase in 9CBP only in the interplacentomal trophoblast, as we have also shown in the sheep and goat, indicating an important role for this region in active calcium transport by the ruminant placenta. The 9CBP is distributed uniformly in the cytosol and nucleoplasm, supporting a role in facilitated diffusion of calcium through the cell rather than a vesicular shuttle system.     

Implantation and decidualization in rodents

This article reviews the main events of embryo-implantation and decidualization in rodents. In common laboratory rodents the embryo attaches to the uterine epithelial lining, usually on days 4 to 6 of pregnancy. A progressive degree of proximity between trophoblast and epithelium occurs until the epithelial cells undergo apoptosis and detach from the basement membrane. During the attachment stage, the spindle-shaped connective tissue cells that underlie the epithelium next to the embryos transform into polyhedral and closely packed decidual cells. Following the epithelial detachment and the breaching of the basement membrane the embryo is thus in direct contact with decidual cells. These cells accumulate organelles associated with synthesis of macro-molecules, intermediate filaments, and eventually lipid droplets and glycogen. Another remarkable feature of decidual cells is the establishment of gap and adherens intercellular junctions. Differentiation of fibroblasts into decidual cells advances antimesometrially and mesometrially, creating in the endometrium several regions of cells with different morphology. The whole phenomenon of decidualization which is normally triggered by the embryo can be artificially induced in pseudo-pregnant or hormonally-prepared animals with the use of diverse stimuli. The uterine epithelium is probably responsible for the transduction of the initial stimulus. Prostaglandins have been shown to be important in the induction of decidualization. More recently other substances such as leukotrienes, platelet-activating factor (PAF), and transforming growth factor (TGF) have been thought to play a role in induction. Much evidence points to prostaglandin production by the decidual cells. New proteins such as a luteotropic factor, desmin, and other molecules were shown to be produced after rat stromal cells undergo decidual transformation. The extracellular matrix of the mouse decidua contains very thick collagen fibrils. Mouse decidual cells are also very active in phagocytosing the thick fibrils, contributing to the remodeling and involution of the decidua that accompanies embryonic growth. Radioautographic data indicates that mouse decidual cells produce and secrete collagen and sulfated proteoglycans.     

Modulation of murine Lyme borreliosis by interruption of the B7/CD28 T-cell costimulatory pathway

Mammalian uteri are unreceptive to blastocyst implantation except during a relatively brief period. The transmembrane, cell surface mucin, Muc1, is present on epithelial cells of nonreceptive uteri in various species and has been demonstrated to have antiadhesive properties. These activities of Muc1 may prevent interaction of the embryonic trophoblast cells with the uterine epithelium. A previous study indicated that Muc1 expression in the rabbit, as in primates, is up-regulated by progesterone. This response would be expected to create a nonadhesive uterine surface during the progesterone-dominated receptive phase. In the current study, Northern blot analysis was used to evaluate Muc1 messenger RNA expression in the endometrium of estrous and progesterone-treated estrous rabbits and in endometrium from different stages of pregnancy or pseudopregnancy. Steady state levels of Muc1 messenger RNA were increased 10-fold when estrous animals were treated with progesterone for 5 days. Muc1 message was elevated 2- to 6-fold over estrous levels in endometrium of pseudopregnant females and 30-fold in preimplantation stage (6.75 days postcoitum) uteri. During implantation (7.25 day postcoitum), the high level of Muc1 expression continued in nonimplantation regions, but was dramatically reduced in endometrium from implantation sites. Using immunofluorescence localization, Muc1 protein was present on the apical surface of epithelial cells of estrous, pseudopregnant (4 and 6.75 days), preimplantation (6.75 days), and implantation (7.25 day) stage uteri. At the latter stage, luminal epithelium apposed to blastocysts had a marked reduction or absence of Muc1 immunostaining. Muc1-immunoreactive cells included luminal and cryptal epithelium in pregnant/pseudopregnant uteri, whereas the glandular cells stained weakly. Short term coculture of uterine epithelial cells with trophoblastic vesicles derived from 6.75-day blastocysts also resulted in a local reduction in apical epithelial Muc1 staining. These findings demonstrate that Muc1 expression is up-regulated by progesterone in the rabbit uterine epithelium and increases incrementally during pre- and periimplantation stages. Removal of Muc1 from the epithelial surface at implantation sites is accomplished locally via signals apparently produced by the blastocyst.     

Biochemical evidence for autocrine/paracrine regulation of apoptosis in cultured uterine epithelial cells during mouse embryo implantation in vitro

During embryo implantation, apoptosis is observed morphologically at the implantation site of endometrium. The objectives of this study were to demonstrate biochemical evidence of apoptosis and quantitative assessment of DNA fragmentation in uterine epithelial cells using a mouse implantation model, and to investigate the autocrine/paracrine regulation of apoptosis in uterine epithelial cells during blastocyst outgrowth. Blastocysts from day 4 pregnant mice were cultured on uterine epithelial cells for 96 h. Uterine epithelial cells dislodged by trophoblasts in endometrium-trophoblast unit demonstrated morphological features of apoptosis by Acridine Orange staining. Electrophoresis demonstrated DNA ladder and DNA fragmentation by enzyme-linked immunosorbent assay markedly increased after 48 h period of incubation. Apoptosis increased in an exponential way in accordance with trophoblast outgrowth. In addition, DNA fragmentation was shown in the epithelial cells by adding embryo-conditioned medium (CM) and the effect of embryo CM on apoptosis was significantly inhibited by anti-transforming growth factor (TGF)-beta antibody. Delayed outgrowth was observed after 48 h of incubation in the blastocysts cultured with anti-TGF-beta antibody. These results suggest there is autocrine/paracrine regulation of apoptosis in uterine epithelial cells at mouse embryo implantation and that TGF-beta might play an important role in the occurrence of apoptosis in the endometrium-trophoblast unit.     

Sialyl-Lewis x and Sialyl-Lewis a are associated with MUC1 in human endometrium

Endometrial epithelial cells express MUC1 with increased abundance in the secretory phase of the menstrual cycle, when embryo implantation occurs. MUC1 is associated with the apical surface of epithelial cells and is also secreted, being detectable in uterine fluid at elevated levels in the implantation phase. However, its physiological role is uncertain; it may either inhibit intercellular adhesion by steric hindrance or carry carbohydrate recognition structures capable of mediating cell-cell interaction. Here we show that endometrial epithelium expresses both Sialyl-Lewis x (SLex) and Sialyl-Lewis a (SLea), with a distribution and pattern of menstrual cycle regulation similar to that of MUC1. Using Western blotting and double determinant ELISA of uterine flushings, we demonstrate that SLex is associated with MUC1 core protein. The endometrial carcinoma cell lines HEC1A and HEC1B are shown to express MUC1 in a mosaic pattern, while three other cell lines express much lower amounts. HEC1A expresses both SLex and SLea while HEC1B expresses SLea only. Immunoprecipitation has been used to demonstrate that SLea is associated with MUC1 in HEC1B cells, and both SLex and SLea are associated with MUC1 in HEC1A cells.     

Effects of feeder cells (human cancer cell lines) on the development of mouse embryos by co-culture

The participation of apical membranes of uterine epithelial cells in the process of blastocyst adhesion makes them an interesting object in the study of changes occurring during early pregnancy. In the study of these changes alkaline phosphatase (AIP), a typical brush border enzyme, was chosen for demonstration with the scanning electron microscope (SEM) by means of a backscatter detector. Thus the temporal and spatial pattern of enzyme activity on the uterine luminal surface was made visible with lead salt procedures. AIP activity was shown to be located on apical membranes and microvilli of endometrial epithelial cells with high activity on day 2 of pregnancy decreasing to virtually no activity on day 5. This decrease in overall AIP activity was shown to be asymmetrical with respect to the uterine cavity. It begins on the antimesometrial half of the uterine lining on day 2. A distribution pattern demarcating a presumptive implantation site along the uterine horn was not found. However, on day 5 of pregnancy, a characteristic pattern of surface folds was found, dividing the uterine horn into 'implantation segments'. In addition, SEM investigation revealed a marked variation of AIP activity from one individual cell to the next on day 2 of pregnancy resulting in a mosaic-like pattern. This pattern is lost with the decrease of AIP activity on day 5. Thus heterogeneity of uterine epithelial cells in AIP activity is apparently a feature of nonreceptive epithelium in contrast to the homogeneous epithelium on day 5. It is proposed that epithelial cell homogeneity could be a marker for uterine receptivity.     

Differentiation of oxyntic cells and cell-matrix interactions during avian gastric gland morphogenesis

The relation between the expression of the oxyntic cell phenotype and the modifications of the extracellular matrix during development of the gastric glands, was studied in 10 to 21 day-old chick embryos. Cytodifferentiation of the oxyntic cells was established by ultrastructural methods, while the expression of pepsinogen, mitochondrial enzyme markers and apical secretory membranes was determined by histochemical and biochemical procedures. Results show that the morphogenesis of the glandular lobules occurs between days 8 and 15 of gestation. Later on, the lobules enlarge but maintain their basic morphology. Until day 13, the developing glands consist of primary tubes lined by a stratified columnar epithelium. The apical poles of the cells that contact the lumen show cytoplasmic processes, and Mg-ATPase activity and F-actin are concentrated at the apical cell borders. From day 13 on, the cells of the simple epithelium that lines secondary tubules budding from the primary tube, show all the features that define differentiated oxyntic cells. The synthesis of glycosaminoglycans during glandular morphogenesis was studied measuring the incorporation of radioactive sulfate into developing chick embryo proventriculi. An important increase in isotope incorporation was found between days 13 and 18 of development. Histochemical localization of these macromolecules shows that glycosaminoglycans are closely associated with the developing glandular lobules. Variations in the structure of epithelial cells undergoing morphogenesis and in the composition of the extracellular matrix are synchronous, suggesting that interactions between them may be significant in terms of the establishment and maintenance of the adult gastric gland phenotype.     

From blastocyst to placenta: the morphology of implantation in the baboon

Implantation and placentation in the baboon share many morphological features with other primates, as well as having some specific distinctions. The ability to use deturgescence of the sex skin as a method of timing ovulation and the ease with which the uterine lumen can be flushed have been used to examine morphological aspects of blastocyst differentiation and implantation in this species. Preimplantation blastocysts were obtained by non-surgical flushing of the uterus 6-8 days after ovulation, and implantation sites were excised from uteri removed on days 10-16 of gestation. All tissues were prepared for electron microscopy by aldehyde fixation and plastic embedding. Maturation of trophoblast from the compacted morula stage to the expanded blastocyst stage includes increase in numbers of polyribosomes, changes in conformation of mitochondria, and development of an effective endocytic apparatus. An endodermal layer forms beneath the inner cell mass prior to loss of the zona pellucida, and parietal endodermal cells extend beyond the inner cell mass. Azonal blastocysts have regions of syncytial trophoblast adjacent to the inner cell mass, and they may represent adhesion stages of early implantation. In early postimplantation stages, trophoblast replaces the uterine epithelium and processes of syncytial trophoblast invade dilated superficial maternal vessels. In subsequent lacunar stages there is rapid elevation of the developing conceptus above the uterine surface as the lacunae enlarge. Cytotrophoblast rapidly enters maternal vessels, and arterioles are partially or completely occluded by migrating cytotrophoblast. The early access to controlled maternal blood flow apparently allows trophoblastic lacunae to expand superficially as opposed to more extensive endometrial invasion.     

Two-parameter flow fluorescence analysis of human chromosomes. Quantitative processing]

Adrenomedullin (AM) is a newly discovered hypotensive peptide which is believed to play an important role for blood pressure control in the adult. Although it has been well established that a major production site of AM is vascular endothelial cells, we now show that AM is most highly expressed in trophoblast giant cells, which are derived from the conceptus and are directly in contact with maternal tissues at the implantation site. Northern blot and in situ hybridization analyses show that the AM mRNA begins to be detected just after implantation and its level peaks at 9.5 days postconception (d.p.c.) in those cells. Expression then falls dramatically after 10.5 d.p.c., coincident with the completion of the mature chorioallantoic placenta. Immunohistochemical analyses show that the AM peptide is secreted from the trophoblast giant cells into the surrounding tissues, i.e., embryo, decidua, and maternal circulation. In contrast, the expression of an AM receptor was not detected by Northern blot analyses in either embryo or trophoblast giant cells at 7 d.p.c., when the AM gene is most highly expressed in the trophoblast giant cells. This suggests that the AM produced and secreted from the embryo's trophoblast giant cells acts on the maternal tissues rather than on the embryonic tissues. Based on these results, we propose that the high production of AM may be the mechanism by which the embryos survive at the early postimplantation period by pooling maternal blood in the implantation site in order to secure nutrition and oxygen before the establishment of efficient embryo-maternal circulation through the mature placenta.     

Distribution of type-I interferon-receptors in human first trimester and term placental tissues and on isolated trophoblast cells

PROBLEM: Type-I interferon (IFN) is the protein recognizing pregnancy in ruminants. Although IFN is secreted in early pregnancy, its role is not still clear in other species. Like other cytokines, IFN exerts its biological functions through specific membrane receptors. We have investigated the potential action of IFN in human pregnancy by studying the distribution of the receptors in the human placenta. METHOD: Reactivity to monoclonal antibodies (mAbs) to the type-I IFN-receptor (R) was analyzed by immunohistochemistry in human placental tissues and in cytospins of first trimester trophoblast cells. RESULTS: Type-I IFN-R immunoreactivity was observed mostly in first trimester villous cytotrophoblasts and in the cytotrophoblast cell columns. Trophoblast in the decidua, the epithelium of the uterine glands, and most of the isolated trophoblast cells were also immunoreactive. CONCLUSION: The expression of type-I IFN-R in the highly proliferating and migrating trophoblast suggests that this cytokine has a role in trophoblast growth and invasion.     

Retention of functional characteristics by bovine oviduct and uterine epithelia in vitro

The composition of fluids within the bovine oviduct and uterine lumen, important in fertilisation and early embryonic development, is ultimately determined by the transport properties of the epithelial cells which line the lumen. A preparation has therefore been devised to study the role of these cells in oviduct and uterine fluid formation. Pure preparations of epithelial cells, as judged immunocytochemically, were isolated by enzyme digestion, and grown on collagen filters in primary culture. The cells re-establish intercellular junctions to form a confluent epithelial layer. Serial samples from the apical and basal media were analysed for K+, Na+, Ca2+, glucose and lactate. Bovine oviduct epithelial cells maintained gradients of K+ and Ca2+ (apical > basal) for up to 14 days after confluence, while bovine uterine epithelial cells maintained apical > basal gradients of K+. Both types of epithelium exhibited a small transepithelial electrical potential difference and a higher uptake of glucose and production of lactate in the basal, as opposed to apical medium. There were no consistent differences in any of these parameters with the stage of the oestrous cycle at which the cells were removed. The data indicate that bovine oviduct and uterine epithelia may be isolated and grown as polarised layers in primary culture. The preparations will now enable the mechanisms underlying the secretion of ions and non-electrolytes to be determined.     

Studies on the mechanism of embryo implantation

Implantation is a complex process accomplished by synchronization and interactions between embryo and endometrium by local exchange of signals including a number of cytokines and growth factors and direct cell-cell and cell-matrix contact. However, the research in early events of human implantation is still in its infancy. This presentation comprises the results of our attempts to investigate the mechanisms of human implantation process in its early stage by cell-biological method, including establishment of experimental implantation model in vitro. 1. Human trophoblast of early stage of gestation showed active cell locomotion, active endocytosis, and invasion of endometrial cell monolayer in mixed cultures. Trophoblast invasion was later arrested by transformed endometrial cells similar to decidual cells in vivo. These results appeared to indicate the interactions between trophoblast and endometrial cells in implantation. 2. Coculture system of rabbit preimplantation blastocyst and endometrial epithelium reformed from isolated endometrial epithelial cells on basement membrane matrix (Matrigel) simulated the in vivo rabbit implantation processes. This coculture system may provide a useful experimental implantation model. 3. A human trophoblast cell line was established from chorionic tissues of normal early pregnancy. These cells were cytotrophoblast-like morphology and endocrine functions. They formed the villous structures similar to those in vivo in culture on Matrigel and invasion of Matrigel was observed. These indicated the extracellular matrix may affect the morphology and function of invading trophoblast in implantation site. 4. Human endometrial epithelial single cells were cultured on Matrigel. Reconstruction of gland followed by epithelium formation quite similar to in vivo structures by migration and proliferation of isolated cells was demonstrated. Height of gland was promoted by estrogen and initiation of epithelization was upregulated by platelet-derived growth factors. This system revealed the extracellular matrix regulated morphogenesis of endometrial epithelium in vivo and is an essential substrate in experimental implantation model of endometrial epithelium. 5. Parallel cultures of endometrial epithelial cells on Matrigel were carried out with the IVF. ET patients to evaluate the endometrial morphology at time of ET. Endometrial cultures were initiated in previous cycles on Matrigel and the sera of patients were added to her own cultures from 1st day of IVF treatment cycle. Evaluation of reformed epithelium revealed the apparently unsuitable morphology for implantation in group of patients who eventually failed in pregnancy. This system may provide a useful measures in evaluation of endometrial receptivity and modality of treatment for endometrial aberrations. 6. Cyclic changes of extracellular matrix components in endometrium were investigated. Collagen I, III, IV, V were immunohistochemically estimated. Relative levels of all types of collagen except for collagen V declined at early secretory phase. In rodents, not only collagen but also laminin and fibronectin levels declined at early secretory phase. These changes may facilitate trophoblast invasion of endometrium. Collagen V distributed in myometrial surface was found to consist of subunit (alpha 1)2 alpha 2 and trophoblast growth was inhibited on substrate of alpha 1 subunit. Collagen V in myometrial surface may have a role in blocking trophoblast invasion. 7. HGF (hepatocyte growth factor) mRNA was demonstrated to be expressed during menstruation and secretory phase in endometrium distinctly and its receptor in endometrial epithelial cells and decidual cells. Positive correlation between plasma HGF levels and ultrasonographic thickness of endometrium was observed at late secretory phase. Recombinant HGF promoted proliferation of endometrial epithelial cells and decidual cells and upregulated initiation of endometrial epithelization of Matrigel.     

Unindicated preoperative testing: ASA physical status and financial implications

Adenoid basal carcinoma of the uterine cervix is rare and its cell origin is still obscure. We report a case of adenoid basal carcinoma of the uterine cervix discovered incidentally in a 69-year-old woman who had been hysterectomized due to endometrial adenocarcinoma of the uterine corpus. Histologically, small round-to-oval cancer cell nests with peripheral cell palisading were seen budding from the basal cell layer of the uterine cervix showing carcinoma in situ. Immunohistochemically, the basaloid cells of the adenoid basal carcinoma were positive for keratins 14, 17 and 19 and resembled reserve cells of the cervical epithelium. The results of this study clearly demonstrated that adenoid basal carcinoma shows a phenotype similar to reserve cells of the uterine cervix. A review of the literature indicated that this tumor has a favorable prognosis and should be clearly separated from adenoid cystic carcinoma, which has a much poorer outcome.     

Nicotine administration decreases the number of binding sites and mRNA of M1 and M2 muscarinic receptors in specific brain regions of rat neonates

Mucins, best known as the major constituent of mucus, are a family of highly glycosylated, high molecular weight (> or = 200 kDa) glycoproteins present on the surface of human epithelial cells. MUC1 has the features of an integral membrane protein. It has an extracellular tandem repeat domain that forms the major part of the core protein, and results in a highly repetitive structure, which is extremely immunogenic. In the protein there is also a proteolytic cleavage site reported in the proximal extracellular domain. The secreted form of MUC1 lacks the cytoplasmic tail, but it is not clear whether this results from alternative splicing or proteolysis and release of the free extracellular domain. The locus of the MUC1 gene is on band 21 of the long arm of chromosome 1 (1q21). Anti-adhesion properties of this mucin are probably the result of the unique structure of the molecule. In mouse uterine epithelium, the homologue MUC1 is regulated with reduced expression in the implantation period, but in humans, expression is high during the peri-implantation period. MUC1 may inhibit the interaction between trophoblast and apical epithelium adhesion molecules at the time of implantation, giving the possibility of forming a uterine barrier for implantation.     

Quantitative assay system for specific enzyme activity using antibody: the case of protease, subtilisin BPN''

Syndecan-1 is a cell surface heparan sulphate proteoglycan, which binds to the extracellular matrix (ECM), growth factors and antithrombin III. The early expression of syndecan-1 during mouse embryonic development suggests a potential role in the communication between the embryo and the ECM of decidua. Using immunohistochemical methods, the present study showed that the expression of syndecan-1 in the trophoblast cells changes along trophoblast differentiation. The syncytiotrophoblasts in the chorionic villi exhibited an apical expression of syndecan-1. This suggests that the expression is restricted to non-migrating, non-proliferating trophoblasts. The mode of syndecan-1 expression by human placental trophoblasts is independent of gestational age. The expression is not changed in miscarriages. In pre-eclampsia, the staining for syndecan-1 on the villous syncytiotrophoblast is weaker compared to normal pregnancy, but in placental bed the expression is similar. The unique apical localization of syndecan-1 in chorionic villi, not detected in any other tissues, suggests a potential role in fetomaternal communication probably via growth factor binding and in anticoagulation of intervillous circulation.     

Age of onset of drug use and its association with DSM-IV drug abuse and dependence: results from the National Longitudinal Alcohol Epidemiologic Survey

Although adaptive explanations for menstruation go back at least twenty-five hundred years, in the last decade two new hypotheses have been advanced. The first suggests that menstruation evolved to cleanse the uterus of pathogens introduced by sperm, and the second argues that the function of endometrial regression (with the associated menstruation in humans) is to save energy by getting rid of tissue, rather than maintaining it in the absence of an available blastocyst. Both these suggestions may be questioned on the grounds that they do not take into account the physiology of the reproductive processes involved. Menstruation is not an independent physiological process and is unlikely to have been selected for independently of the evolutionary events that led to it. Furthermore, most primitive menstruating animals would have menstruated infrequently, and many may have reproduced or died without ever menstruating. In order to provide a context for understanding how menstruation may have come about, the evolution of the female vertebrate reproductive tract is briefly reviewed. In later stages, the coevolution of the embryo and uterus resulted in an intimate association between the trophoblast and the uterine blood vessels. As the embryo became more invasive, the uterus responded with increased cellular growth and differentiation of the endometrium to accommodate it. This reached its peak in mammals (such as rodents and humans), where the embryo passes through the epithelium into the endometrial stroma, which responds with differentiation of cells and blood vessels. Progesterone, secreted after ovulation, plays a crucial role in preparation for pregnancy. In addition to its well-known effects on the uterus, progesterone may be important in suppressing the inflammatory reaction that would be expected in response to the presence of a foreign body, such as an embryo. It is also suggested that vascular and cellular differentiation of the endometrial stroma has evolved by adaptation of the inflammatory (granulation tissue) reaction. When progesterone levels fall at the end of the cycle, there is tissue breakdown and bleeding. The uterus then reforms for the next ovulatory cycle. It is shown that the female reproductive tract has multiple functions that must occur in sequence. The coevolution of the embryo and maternal tract thus led to the close contact of two genetically different tissues, and problems such as the inflammatory reaction had to be overcome. Menstruation is a necessary consequence of these evolutionary changes, and needed no adaptive value in order to evolve.     

Receptivity is a polarity dependent special function of hormonally regulated uterine epithelial cells

Useful knowledge of the mechanisms which regulate ovoreceptivity and implantation remains elusive in spite of increasing efforts to apply the technology of biochemistry and to a lesser extent, cellular and molecular biology to the analysis of the problem. Existing models used to analyze interactions of the blastocyst and endometrial cells of the uterus have been unable to account for nongenotypic embryonic losses, particularly those following in vitro fertilization and embryo transfer. Separation of endometrial uterine epithelial (UE) and uterine stromal (US) cells was used to demonstrate that each cell type responds independently and interdependently to the same regulatory signals. Cultured by classical techniques UE cells proved unable to respond to steroid hormone signals. For this reason UE cell cultures could not be used to develop an experimental cell system that mimicked growth and development of UE cells in utero. The failure of classical UE cell cultures derived from their inability to maintain epithelial cell polarity. Polarity, the spatial asymmetry of plasma membrane domains, is intrinsic to the structure and function of an epithelial cell. Apical and basolateral surfaces have different lipid and protein compositions which are correlates of the special functions of that epithelial cell. As epithelial cells differentiate they must, in response to regulatory cues, direct the flux of membrane components moving into and out of each surface in order to establish the polarity characteristic of each stage specific expression. The acquisition of receptivity by the apical surface of the UE cell may be considered to be such a special function. To prove this hypothesis polarized cultures of primary UE cells had to be developed that were hormonally responsive. Such an experimental cell system could serve as a model for in vitro implantation. This essay describes such a culture system in which blastocysts cocultured with UE cells in the presence of estrogen, will as predicted, fail to attach. This polarized UE cell system provides a functional in vitro model to study ovoreceptivity. It is now feasible to initiate studies of hormonal regulation of the composition and function of UE cell plasma membranes as they reflect the nonreceptive, receptive, and refractory nature of its apical surface.     

Morphogenesis of the C. elegans hermaphrodite uterus

We have undertaken electron micrographic reconstruction of the Caenorhabditis elegans hermaphrodite uterus and determined the correspondence between cells defined by their lineage history and differentiated cell types. In this organ, many cells do not move during morphogenesis and the cell lineage may function to put cells where they are needed. Differentiated uterine cell types include the toroidal ut cells that make structural epithelium, and specialized utse and uv cells that make the connection between the uterus and the vulva. A cell fate decision in which the anchor cell (AC) induces adjacent ventral uterine intermediate precursor cells to adopt the pi fate, rather than the ground state rho, has profound consequences for terminal differentiation: all pi progeny are directly involved in making the uterine-vulval connection whereas all rho progeny contribute to ut toroids or the uterine-spermathecal valve. In addition to specifying certain uterine cell fates, the AC also induces the vulva. Its multiple inductions thereby function to coordinate the connection of an internal to an external epithelium. The AC induces the pi cells and ultimately fuses with a subset of their progeny. This is an example of reciprocal cell-cell interaction that can be studied at single cell resolution. The AC is thus a transitory cell type that plays a pivotal role in organizing the morphogenesis of the uterine-vulval connection.