Chorionic Epithelial Cells Research Articles

Evidence for metabolism of creatine by the conceptus, placenta, and uterus for production of adenosine triphosphate during conceptus development in pigs†.

In pigs, the majority of embryonic mortality occurs when free-floating conceptuses (embryos/fetuses and associated placental membranes) elongate, and the uterine-placental interface undergoes folding and develops areolae. Both periods involve proliferation, migration, and changes in morphology of cells that require adenosine triphosphate (ATP). We hypothesize that insufficient ATP in conceptus and uterine tissues contributes to conceptus loss in pigs. Creatine is stored in cells as phosphocreatine for ATP regeneration through the creatine-creatine kinase- phosphocreatine pathway. However, the expression of components of this pathway in pigs has not been examined throughout gestation. Results of qPCR analyses indicated increases in AGAT, GAMT, CKM, CKB, and SLC6A8 mRNAs in elongating porcine conceptuses, and immunofluorescence microscopy localized guanidinoacetate N-methyltransferase, creatine kinase M, and creatine kinase B proteins to the trophectoderm of elongating conceptuses, to the columnar chorionic epithelial cells at the bottom of chorioallantoic troughs, and to endometrial luminal epithelium at the tops of the endometrial ridges of uterine-placental folds on Days 40, 60, and 90 of gestation. Guanidinoacetate N-methyltransferase protein is expressed in endometrial luminal epithelium at the uterine-placental interface, but immunostaining is more intense in luminal epithelium at the bottoms of the endometrial ridges. Results of this study indicate that key elements of the pathway for creatine metabolism are expressed in cells of the conceptus, placenta, and uterus for potential production of ATP during two timepoints in pregnancy with a high demand for energy; elongation of the conceptus for implantation and development of uterine-placental folding during placentation.

Functional complexity in the chorioallantoic membrane of an oviparous snake: Specializations for calcium uptake from the eggshell.

The chorioallantoic membrane of oviparous reptiles forms a vascular interface with the eggshell. The eggshell contains calcium, primarily as calcium carbonate. Extraction and mobilization of this calcium by the chorioallantoic membrane contributes importantly to embryonic nutrition. Development of the chorioallantoic membrane is primarily known from studies of squamates and birds. Although there are pronounced differences in eggshell structure, squamate and bird embryos each mobilize calcium from eggshells. Specialized cells in the chicken chorionic epithelium transport calcium from the eggshell aided by a second population of cells that secrete protons generated by the enzyme carbonic anhydrase. Calcium transporting cells also are present in the chorioallantoic membrane of corn snakes, although these cells function differently than those of chickens. We used histology and immunohistology to characterize the morphology and functional attributes of the chorioallantoic membrane of corn snakes. We identified two populations of cells in the outer layer of the chorionic epithelium. Calbindin-D28K , a cellular marker for calcium transport expressed in squamate chorioallantoic membranes, is localized in large, flattened cells that predominate in the chorionic epithelium. Smaller cells, interspersed among the large cells, express carbonic anhydrase 2, an enzyme not previously localized in the chorionic epithelium of an oviparous squamate. These findings indicate that differentiation of chorionic epithelial cells contributes to extraction and transport of calcium from the eggshell. The presence of specializations of chorioallantoic membranes for calcium uptake from eggshells in chickens and corn snakes suggests that eggshell calcium was a source of embryonic nutrition early in the evolution of Sauropsida.

Interleukin-10 and its receptors at the maternal-conceptus interface: expression, regulation, and implication for T helper 2 cytokine predominance and maternal immune tolerance in the pig, a true epitheliochorial placentation species†.

The appropriate balance between pro-inflammatory and anti-inflammatory cytokines is important for the maternal immune tolerance during pregnancy in mammals. Among the various cytokines, interleukin (IL)-10 (IL10) plays an essential role in anti-inflammatory responses, while IL12 is involved in pro-inflammatory responses during pregnancy. However, the roles of IL10 and IL12 in the endometrium during pregnancy have not been studied in pigs. Thus, we investigated the expression of IL10, IL12 (IL12A and IL12B), and their receptors (IL10RA, IL10RB, IL12RB1, and IL12RB2) at the maternal-conceptus interface. IL10, IL12, and their receptors were expressed in the endometrium during the estrous cycle and pregnancy in a pregnancy stage-specific manner. During pregnancy, IL10 expression increased on Day 15, whereas the expression of IL12A and IL12B decreased after the implantation period. IL10 protein was localized to luminal epithelial (LE), stromal cells, and macrophages; IL10RA protein to LE, endothelial, stromal, and T cells; and IL10RB mRNA to LE cells in the endometrium. IL10 and IL10RA proteins and IL10RB mRNA were also localized to chorionic epithelial (CE) cells. In endometrial explants, the expression of IL10RA and IL10RB was induced by estradiol-17β, IL-1β, and/or interferon-γ. Heme oxygenase 1, an IL10-inducible factor, was expressed in the endometrium with the highest levels on Day 30 of pregnancy and was localized to LE and CE cells. These results in pigs suggest that conceptus-derived signals change the endometrial immune environment by regulating the expression of IL10 and IL10 receptors at the maternal-conceptus interface and that IL10 may provide anti-inflammatory conditions for the maternal immune tolerance.

Calcium-binding proteins S100A8, S100A9, and S100A12: expression and regulation at the maternal-conceptus interface in pigs†.

Among the many calcium-binding proteins, S100A8, S100A9, and S100A12 play important roles in inflammation, innate immunity, and antimicrobial function, but their expression, regulation, and function at the maternal-conceptus interface in pigs are not fully understood. Therefore, we determined the expression and regulation of S100A8, S100A9, S100A12, and their receptor AGER at the maternal-conceptus interface in pigs. We found that S100A8, S100A9, and S100A12 mRNAs were expressed in the endometrium during the estrous cycle and pregnancy, with the greatest levels on Day (D) 12 of pregnancy, and AGER appeared at greater levels on D15 and D30 of pregnancy than on other days. The expression of S100A8, S100A9, and S100A12 was predominantly localized to epithelial cells in the endometrium, and they were detected in early-stage conceptus and later chorioallantoic tissues during pregnancy. AGER expression was localized to endometrial epithelial and stromal cells and chorionic epithelial cells. In endometrial explant tissues, the expression of S100A8, S100A9, and S100A12 was induced by estrogen, S100A8 by interleukin-1β, and AGER by interferon-γ. We further found that on D12 of pregnancy, the expression of S100A8, S100A9, and S100A12 decreased significantly in the endometria of gilts carrying conceptuses derived from somatic cell nuclear transfer. These results indicate that the expression of S100A8, S100A9, and S100A12 is dynamically regulated in response to conceptus-derived signals at the maternal-conceptus interface, suggesting that S100A8, S100A9, and S100A12 could play a critical role in regulating endometrial epithelial cell function and conceptus implantation to support the establishment and maintenance of pregnancy in pigs.

Mycobacterium fortuitum abortion in a sow

Two aborted Chester White pig fetuses were presented to a veterinary diagnostic laboratory in Illinois. Postmortem examination identified no gross abnormalities. Histologic evaluation revealed multifocal necrosis of chorionic epithelial cells, coalescing areas of mineralization in the placenta, and focal accumulations of viable and degenerate neutrophils in the lung. Intra- and extracellular acid-fast bacilli were identified in the lesions in both the placenta and lungs. Bacterial culture of stomach contents yielded heavy growth of Mycobacterium fortuitum, a rapidly growing nontuberculous mycobacterium (NTM), which was further confirmed through whole-genome sequencing. NTM are opportunistic pathogens commonly found in the soil and in contaminated water supplies. In animals, M. fortuitum is typically introduced through cutaneous wounds leading to infections limited to the skin, with systemic infection being uncommon. To our knowledge, abortion caused by M. fortuitum has not been reported previously.

Antimicrobial peptides cathelicidin, PMAP23, and PMAP37: Expression in the endometrium throughout the estrous cycle and at the maternal-conceptus interface during pregnancy and regulation by steroid hormones and calcitriol in pigs

The endometrium, which regulates the reproductive cyclicity and the establishment and maintenance of pregnancy, is a type of mucosal tissue and is involved in the regulation of immunity. Antimicrobial peptides, called cathelicidins, play critical roles in innate immunity by killing various microbes and modulating host immune properties. The expression and regulation of cathelicidin in female reproductive tissues have been studied in some species, but not determined in pigs, which form a true epitheliochorial placenta. Thus, we determined the expression of Cathelicidin (the conserved common cathelin domain for all porcine cathelicidin family members), PMAP23, and PMAP37 in the endometrium throughout the estrous cycle and pregnancy. The expression of Cathelicidin, PMAP23, and PMAP37 in the endometrium changed during the estrous cycle and pregnancy in a stage-specific manner. The mRNA expression of Cathelicidin, PMAP23, and PMAP37 was also detected in conceptus and chorioallantoic tissues during pregnancy. Cathelicidin protein was mainly localized to luminal epithelial cells and some immune cells in blood vessels and to chorionic epithelial cells of chorioallantoic membranes during mid-to late pregnancy. The expression of Cathelicidin was induced by calcitriol but increasing doses of estradiol-17β and progesterone did not affect the expression of Cathelicidin, PMAP23, or PMAP37 in endometrial tissues. These results indicate that members of the cathelicidin family were dynamically expressed at the maternal-conceptus interface in pigs, suggesting that the cathelicidin family may play important roles in the regulation of innate immunity by mediating antimicrobial activity to protect the maternal and/or conceptus tissues to conserve pregnancy capacity in pigs.

Changes in calcium levels in the endometrium throughout pregnancy and the role of calcium on endometrial gene expression at the time of conceptus implantation in pigs.

Calcium plays an essential role in regulating many cellular functions, including proliferation, differentiation, and apoptosis. In spite of its importance in the establishment and maintenance of pregnancy, changes in calcium levels at the maternal-conceptus interface during pregnancy and its action on endometrial gene expression are not well understood. Thus, we examined changes in calcium levels in the endometrium during pregnancy, calcium deposition at the maternal-conceptus interface during pregnancy, and the role of calcium on the expression of endometrial genes related to conceptus implantation during early pregnancy in pigs. The amounts of endometrial calcium increased during mid- to late pregnancy, and calcium deposition was mainly localized to endometrial and chorionic epithelial cells at the maternal-conceptus interface during pregnancy and conceptus tissues during early pregnancy. The amounts of total recoverable calcium in uterine flushings were greater on Day 12 of pregnancy than Day 12 of the estrous cycle, and estrogen increased absorption of calcium ions by endometrial tissues. Increasing endometrial calcium levels by treatment with A23187, a calcium ionophore, decreased the expression of the estrogen-responsive endometrial genes AKR1B1, ESR1, FGF7, IL1RAP, LPAR3, S100G, SPP1, and STC1 and increased the expression of genes related to prostaglandin synthesis and transport, namely PTGES, PTGS2, and SLCO5A1. These data suggest that calcium ions at the maternal-conceptus interface play a critical role in the establishment and maintenance of pregnancy in pigs by regulating the expression of endometrial genes involved in conceptus implantation, as well as the attachment of endometrial epithelial and conceptus trophectoderm/chorionic epithelial cells during pregnancy.

Expression and regulation of prostaglandin transporters, ATP-binding cassette, subfamily C, member 1 and 9, and solute carrier organic anion transporter family, member 2A1 and 5A1 in the uterine endometrium during the estrous cycle and pregnancy in pigs.

ObjectiveProstaglandins (PGs) function in various reproductive processes, including luteolysis, maternal pregnancy recognition, conceptus development, and parturition. Our earlier study has shown that PG transporters ATP-binding cassette, subfamily C, member 4 (ABCC4) and solute carrier organic anion transporter family, member 2A1 (SLCO2A1) are expressed in the uterine endometrium in pigs. Since several other PG transporters such as ABCC1, ABCC9, SLCO4C1, and SLCO5A1 are known to be present in the uterine endometrium, this study investigated the expression of these PG transporters in the porcine uterine endometrium and placenta.MethodsUterine endometrial tissues were obtained from gilts on day (D) 12 and D15 of the estrous cycle and days 12, 15, 30, 60, 90, and 114 of pregnancy.ResultsABCC1, ABCC9, SLCO4C1, and SLCO5A1 mRNAs were expressed in the uterine endometrium, and levels of expression changed during the estrous cycle and pregnancy. Expression of ABCC1 and ABCC9 mRNAs was localized mainly to luminal and glandular epithelial cells in the uterine endometrium, and chorionic epithelial cells during pregnancy. Conceptuses during early pregnancy and chorioallantoic tissues from mid to late pregnancy also expressed these PG transporters. Estradiol-17β increased the expression of ABCC1 and SLCO5A1, but not ABCC9 and SLCO4C1 mRNAs and increasing doses of interleukin-1β induced the expression of ABCC9, SLCO4C1, and SLCO5A1 mRNAs in endometrial explant tissues.ConclusionThese data showed that several PG transporters such as ABCC1, ABCC9, SLCO4C1, and SLCO5A1 were expressed at the maternal-conceptus interface, suggesting that these PG transporters may play an important role in the establishment and maintenance of pregnancy by regulating PG transport in the uterine endometrium and placenta in pigs.

Chemokine (C-C motif) Ligand 28 and Its Receptor CCR10: Expression and Function at the Maternal-Conceptus Interface in Pigs.

Many chemokines are present at the maternal-fetal interface and play important roles in the establishment and maintenance of pregnancy. Our study demonstrates that a chemokine, chemokine (C-C motif) ligand 28 (CCL28), is expressed in the uterine endometrium during early pregnancy in pigs. Thus, we investigated expression of CCL28 and its receptors, chemokine (C-C motif) receptor type 3 (CCR3) and 10 (CCR10), in the uterine endometrium during the estrous cycle and pregnancy and the function of CCL28 at the maternal-fetal interface during early pregnancy. Levels of CCL28 mRNAs were highest on Day 10 of pregnancy and decreased thereafter during pregnancy and CCL28 was localized mainly to endometrial glandular epithelial cells. The presence of the CCL28 protein in uterine flushings was confirmed on Day 12 of the estrous cycle and pregnancy. Endometrial tissues expressed CCR3 and CCR10 during pregnancy. The CCR10 protein was localized to endometrial luminal and glandular epithelial cells, chorionic epithelial cells, and the allantoic membrane during pregnancy. Conceptuses during early pregnancy expressed CCL28 and CCR10, but not CCR3, and chorioallantoic tissues expressed CCR10 at increasing levels towards term. Treatment with recombinant CCL28 increased the proliferation and migration of a porcine trophectoderm cell line. These results indicated that the CCL28 chemokine and its receptors, CCR3 and CCR10, are expressed at the maternal-conceptus interface, and CCL28 induces the proliferation and migration of trophectoderm cells through CCR10, suggesting that CCL28 may play a critical role in the establishment and maintenance of pregnancy in pigs.

Ultrastructure of the fetal membranes of the oviparous kingsnake, Lampropeltis getula (Colubridae) as revealed by scanning electron microscopy

ABSTRACTIn reptilian sauropsids, fetal (extraembryonic) membranes that line the eggshell sustain developing embryos by providing for gas exchange and uptake of water and eggshell calcium. However, a scarcity of morphological studies hinders an understanding of functional specializations and their evolution. In kingsnakes (Lampropeltis getula), scanning electron microscopy reveals two major fetal membranes: the chorioallantois and yolk sac omphalopleure. In early development, the chorioallantois contains tall chorionic epithelial cells, avascular connective tissue, and enlarged allantoic epithelial cells. During its maturation, the chorionic and allantoic epithelia thin dramatically and become underlain by a rich network of allantoic capillaries, yielding a membrane ideally suited for respiratory gas exchange. Yolk sac development initially is like that of typical lizards and snakes, forming an avascular omphalopleure, isolated yolk mass (IYM), and yolk cleft. However, unlike the situation in most squamates studied, the omphalopleure becomes transformed into a “secondary chorioallantois” via three asynchronous events: flattening of the epithelium, regression of the IYM, and vascularization by the allantois. Progressive expansion of chorioallantois parallels growing embryonic needs for gas exchange. In early through mid‐development, external surfaces of both the chorionic and omphalopleure epithelium show an abundance of irregular surface protrusions that possibly increase surface area for water absorption. We postulate that the hypertrophied allantoic epithelial cells produce allantoic fluid, a viscous substance that facilitates water uptake and storage. Our findings are consistent with a previous study on the corn snake Pantherophis guttatus, but include new observations and novel functional hypotheses relevant to a reconstruction of basal squamate patterns. J. Morphol. 276:1467–1481, 2015. © 2015 Wiley Periodicals, Inc.

Ultrastructure of the fetal membranes of the oviparous kingsnake, Lampropeltis getula (Colubridae) as revealed by scanning electron microscopy

In reptilian sauropsids, fetal (extraembryonic) membranes that line the eggshell sustain developing embryos by providing for gas exchange and uptake of water and eggshell calcium. However, a scarcity of morphological studies hinders an understanding of functional specializations and their evolution. In kingsnakes (Lampropeltis getula), scanning electron microscopy reveals two major fetal membranes: the chorioallantois and yolk sac omphalopleure. In early development, the chorioallantois contains tall chorionic epithelial cells, avascular connective tissue, and enlarged allantoic epithelial cells. During its maturation, the chorionic and allantoic epithelia thin dramatically and become underlain by a rich network of allantoic capillaries, yielding a membrane ideally suited for respiratory gas exchange. Yolk sac development initially is like that of typical lizards and snakes, forming an avascular omphalopleure, isolated yolk mass (IYM), and yolk cleft. However, unlike the situation in most squamates studied, the omphalopleure becomes transformed into a "secondary chorioallantois" via three asynchronous events: flattening of the epithelium, regression of the IYM, and vascularization by the allantois. Progressive expansion of chorioallantois parallels growing embryonic needs for gas exchange. In early through mid-development, external surfaces of both the chorionic and omphalopleure epithelium show an abundance of irregular surface protrusions that possibly increase surface area for water absorption. We postulate that the hypertrophied allantoic epithelial cells produce allantoic fluid, a viscous substance that facilitates water uptake and storage. Our findings are consistent with a previous study on the corn snake Pantherophis guttatus, but include new observations and novel functional hypotheses relevant to a reconstruction of basal squamate patterns.

Structure and functions of the placenta in common minke (<i>Balaenoptera acutorostrata</i>), Bryde’s (<i>B. brydei</i>) and sei (<i>B. borealis</i>) whales

The structure and functions of placentas were examined in 3 species of rorqual whales, common minke (Balaenoptera acutorostrata), Bryde’s (B. brydei) and sei (B. borealis) whales, with the aim of confirming the structural characteristics of the chorion, including the presence of the areolar part, and clarifying steroidogenic activities and fetomaternal interactions in the placentas of these whales. Placentas were collected from the second phase of the Japanese Whale Research Program under Special Permit in the North Pacific (JARPN II). Histological and ultrastructural examinations revealed that these whale placentas were epitheliochorial placentas with the interdigitation of chorionic villi lined by monolayer uninucleate cells (trophoblast cells) and endometrial crypts as well as folded placentation by fold-like chorionic villi. Moreover, well-developed pouch-like areolae were observed in the placentas, and active absorption was suggested in the chorionic epithelial cells of the areolar part (areolar trophoblast cells). Berlin blue staining showed the presence of ferric ions (Fe3+) in the uterine glandular epithelial cells and within the stroma of chorionic villi in the areolar part. An immunohistochemical examination revealed tartrate-resistant acid phosphatase (TRAP; known as uteroferrin in uteri) in the cytoplasm of glandular cells and areolar trophoblast cells. This result suggested that, in cetaceans, uteroferrin is used to supply iron to the fetus. Furthermore, immunoreactivity for P450scc and P450arom was detected in trophoblast cells, but not in areolar trophoblast cells, suggesting that trophoblast cells synthesize estrogen in whale placentas. Therefore, we herein immunohistochemically revealed the localization of aromatase and uteroferrin in cetacean placentas during pregnancy for the first time.

Chorioallantoic Placentation in Viperid Snakes

Chorioallantoic Placentation in Viperid Snakes

Placental development and expression of calcium transporting proteins in the extraembryonic membranes of a placentotrophic lizard

Pseudemoia pagenstecheri is a viviparous Australian scincid lizard in which the maternal-embryonic placental interface is differentiated into structurally distinct regions. The chorioallantoic placenta contains an elliptical-shaped region, the placentome, characterized by hypertrophied uterine and embryonic epithelial cells supported by dense vascular networks. The remainder of the chorioallantoic placenta, the paraplacentome, is also highly vascularized but uterine and chorionic epithelia are thin. An omphaloplacenta with hypertrophied epithelia is located in the abembryonic hemisphere of the egg. There is extensive placental transport of organic and inorganic nutrients, e.g., 85-90% of neonatal calcium is received via placental transfer. Calcium uptake by extraembryonic membranes of squamates correlates with expression of the intracellular calcium binding protein, calbindin-D(28K) , and plasma membrane calcium ATPase (PMCA) is a marker for active calcium transport. We estimated expression of calbindin-D(28K) and PMCA in the chorioallantoic membrane in a developmental series of embryos using immunoblotting and used immunohistochemistry to define the cellular localization of calbindin-D(28K) to test the hypotheses that 1) expression of calcium transporting proteins is coincident with placental transport of calcium and 2) the placenta is functionally specialized for calcium transport in regions of structural differentiation. Calbindin-D(28K) and PMCA were detected at low levels in early stages of development and increased significantly prior to birth, when embryonic calcium uptake peaks. These data support the hypothesis that placental calcium secretion occurs over an extended interval of gestation, with increasing activity as embryonic demand escalates in late development. In addition, calbindin-D(28K) expression is localized in chorionic epithelial cells of the placentome and in the epithelium of the omphalopleure of the omphaloplacenta, which supports the hypothesis that regional structural differentiation in the placenta reflects functional specializations for calcium transport.

A case of equine abortion caused by Encephalitozoon sp.

A Lippizan mare aborted a male fetus a few days before the expected foaling date without showing any clinical sings. Focal lympho-histiocytic hepatitis in the foal and multiplex focal lympho-histiocytic villitis accompanied by villus necroses and marked hypertrophy of chorionic epithelial cells in the arcades were observed. Elongated nucleated organisms were seen in groups in vacuoles or solitarily located in the cytoplasm of the chorionic epithelial cells. The organisms were in large numbers and often extracellularly in areas of villitis and villus necroses. They were Gram-positive, stained with haematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Giemsa, weakly with Warthin-Starry silver stain but not with Gömöri's methenamine-silver stain. By ultrastructural and immunohistochemical examinations, the organisms were identified as microsporidia belonging to the genus Encephalitozoon. No Encephalitozoon organisms were detected in the fetal organs. This is the first reported case of equine abortion induced by Encephalitozoon sp. in Europe. Although abortion induced by Encephalitozoon is rare, microsporidia should be considered a differential diagnosis for intracellular organisms observed in the chorionic epithelial cells of horses.

High prevalence of chlamydial (Chlamydophila psittaci) infection in fetal membranes of aborted equine fetuses

Seventy-seven cases of equine abortion from 49 Hungarian farms that occurred between 1998 and 2000 were investigated for the presence of chlamydiae by immunohistochemistry, PCR and/or MZN staining. Evidence of the presence of these bacteria was obtained in 64 cases (83.1%) from 41 (83.7%) different farms. Partial ompA gene sequencing of PCR products revealed that the agent was Chlamydophila psittaci. Based on the findings of microbial diagnosis, pathology and case history, chlamydial infection was considered to be the most likely cause of abortion in at least 11 (14.3%) cases. In the remaining 53 Chlamydophila-positive cases, either other bacterial and viral agents (n = 22 or 28.6%) as well as non-infectious factors (n = 14 or 18.2%) were identified as more probable primary causes of disease, or the role of chlamydiae remained unclear because lesions in fetuses and fetal membranes were absent (n = 17 or 22.1%). When chlamydial antigen was detected in aborted equine placental tissue using immunohistochemistry it was seen only in the chorionic epithelial cells, but not in other parts of the fetal membranes nor in any of the fetal tissues. In conclusion, chlamydial infection of the genital tract should be considered a possible factor in equine reproductive disorders.

Proliferation and apoptosis in bovine placentomes during pregnancy and around induced and spontaneous parturition as well as in cows retaining the fetal membranes.

Placental growth can be achieved by either cellular proliferation or hypertrophy. Tissue regeneration and the nutrition of the fetus via embryotrophe require high rates of cellular turnover and the so-called pre-term 'maturation' of the placenta is correlated with a reduction of maternal crypt epithelial cells. Placentomes of 45 pregnant cows were collected from an abattoir to assess the role of proliferation and apoptosis in placental physiology and pathology. Placentomes were also taken from five cows undergoing premature Caesarean section and from ten naturally calving cows immediately after the expulsion of the fetus. Five of these animals had not released the fetal membranes after 12 h. Tissue sections of placentome were assessed for the Ki-67 protein; the TUNEL procedure was performed and verified by transmission electron microscopy. The maternal crypt epithelium and the fetal chorionic epithelium had a higher percentage of Ki-67-positive cells than the stroma. The percentage of Ki-67-positive cells increased significantly during pregnancy in fetal chorionic epithelium and was significantly decreased in fetal chorionic epithelium and maternal crypt epithelium after the expulsion of the fetus in comparison with tissue from month 9 of pregnancy. The number of apoptotic cells increased significantly during pregnancy in maternal crypt epithelium, maternal stroma and fetal chorionic epithelium as detected in slaughtered animals. Significantly more apoptotic fetal chorionic epithelial cells were found in animals retaining their fetal membranes in comparison with prepartum cattle during month 9 of pregnancy, at premature section and in animals releasing the fetal membranes completely. The results strongly indicate that bovine placentomes have cell type-specific rates of cellular turnover reflecting tissue growth, embryotrophe and placental maturation. Retention of fetal membranes is characterized by a large number of fetal chorionic epithelial cells undergoing apoptosis immediately after the expulsion of the fetus. This finding indicates that incomplete maturation of placentomes plays an important role in fetal membrane retention and that massive apoptosis after the expulsion of the fetus should be the consequence of diminished blood supply to the uterus, as verified in a recent study.

Placentation in garter snakes. II. Transmission EM of the chorioallantoic placenta of Thamnophis radix and T. sirtalis.

Transmission electron microscopy was used to examine the ultrastructure of the allantoplacenta of garter snakes during the last half of gestation. This placenta occupies the dorsal hemisphere of the egg and is formed through apposition of the chorioallantois to the inner lining of the uterus. The uterine epithelium consists of flattened cells with short, irregular microvilli and others that bear cilia. The lamina propria is vascularized and its capillaries lie at the base of the uterine epithelial cells. The chorionic epithelium consists of a bilayer of squamous cells that are particularly thin superficial to the allantoic capillaries. Neither the chorionic epithelium nor the uterine epithelium undergoes erosion during development. Although a thin remnant of the shell membrane intervenes between fetal and maternal tissue at mid-gestation, it undergoes fragmentation by the end of gestation. Thus, uterine and chorionic epithelial are directly apposed in some regions of the allantoplacenta, forming continuous cellular boundaries at the placental interface. During development, capillaries proliferate in both the uterine and chorioallantoic tissues. By late gestation, the interhemal diffusion distance has thinned in some areas to less than 2 microm through attenuation of the uterine and chorionic epithelia. Morphologically, the allantoplacenta is well adapted for its function in gas exchange. However, the presence of cytoplasmic vesicles, ribosomal ER, and mitochondria in the chorionic and uterine epithelial cells are consistent with the possibility of additional forms of placental exchange.

In situ hybridization and immunogold localization of vascular endothelial growth factor receptor-2 on the pericytes of the chick chorioallantoic membrane.

This paper describes the expression of VEGF and of VEGFR-2 in the vasculature of the chorioallantoic membrane (CAM) as revealed by in situ hybridization and immunoelectron microscopy. Results showed that VEGFR-2 is expressed in both the endothelial cells and the pericytes, while VEGF in the chorionic epithelial cells. VEGF may therefore be released to promote both angiogenesis, by initiating an angiogenic response by endothelial cells expressing VEGFR-2, and the recruitment of pericytes along the capillary wall, playing also a crucial role in maturation and stabilization of the CAM blood vessels.

Novel activator protein-2alpha splice-variants function as transactivators of the ovine placental lactogen gene.

Activator protein-2 (AP-2) has been implicated as a transactivator of the human and ovine placental lactogen (oPL) genes. Transcriptional enhancement through an AP-2 cis-acting element has been described for other genes expressed in the placenta, but the AP-2 isoform enhancing expression is species dependent. Transactivation of the oPL minimal promoter (-124 bp to +16 bp) by AP-2 was confirmed by mutational analysis in transiently transfected human choriocarcinoma cells (BeWo). AP-2alpha was localized in ovine chorionic epithelial cells by immunohistochemistry and a 3-kb transcript was identified by Northern hybridization. Four nearly full-length AP-2 cDNAs were isolated from an ovine placenta cDNA library. Nucleotide sequencing these cDNAs revealed that the AP-2 mRNA expressed in the ovine placenta shares identity with human AP-2alpha, but variations in the predicted N-terminus were observed, and three unique AP-2alpha splice-variants were identified. Expression of AP-2alpha variants in HepG2 cells, devoid of endogenous AP-2, indicates that enhancement through the AP-2 element in the oPL gene minimal promoter was variant dependent. RNA transcripts for all of the ovine AP-2alpha splice-variants were confirmed in ovine placenta by RT-PCR, and homologs for two variants were found in human placenta. However, only one AP-2alpha transcript, which shares identity to Xenopus AP-2alpha, was expressed in BeWo cells. Immunoblot analysis confirmed AP-2alpha variants in ovine chorionic binucleate cell nuclear extracts, one of which migrates similar to the AP-2alpha variant identified in BeWo cell nuclear extracts. These data indicate the presence of new mammalian AP-2alpha splice-variants that augment transactivation of the oPL gene in ovine chorionic binucleate cells.