Chorionic Girdle Research Articles

A Structural and Immunological Study of Chorionic Gonadotrophin Production by Equine Trophoblast Girdle and Cup Cells

Equine chorionic gonadotropin (eCG) production by the fetally derived endometrial cups appears to be necessary for the establishment and maintenance of normal equine pregnancy.Starting at about the 27th day of pregnancy, an equatorial band of trophectodermal cells on the surface of the spherical conceptus forms the chorionic girdle. This girdle consists initially of flat trophectodermal epithelium which corrugates into folds as the cells proliferate. The folds are then pressed against the uterine epithelium by expansion of the conceptus. The cells on the apices of the folds become binucleate before they start to invade the endometrium at days 35–37. Ultrastructural immunogold labelling shows that they begin to synthesize eCG as early as day 32, before they migrate into and through the maternal epithelium. Clusters of the girdle binucleate cells penetrate deep into the endometrium. The mature cup cell has a cytoplasm full of mitochondria, rough endoplasmic reticulum cisternae, a large Golgi apparatus and a strong immunoreactivity for the glucose transporter 1 isoform on its plasmalemma. Immunocytochemistry also demonstrates that eCG is localized in the Golgi cisternae, and in small dense granules similar to those found in the migrating girdle cell and present both in the Golgi region and at the peripheral plasmalemma. Release of eCG would therefore seem to be by the usual exocytotic mechanism as found for other protein hormones. The small size and absence of any significant accumulation of eCG-containing granules are in marked contrast to the numerous large luteinizing hormone (eLH) containing granules in the equine pituitary gonadotroph, although both hormones, eLH and eCG, show complete identity at the amino acid sequence level. These morphological indicators suggest that the cup cell secretes eCG constitutively (that is, continuously), with no requirement for secretagogues, whereas in the pituitary cell the regulated pathway is utilized capable of massive secretion under appropriate stimulation.

Ectopic transplantation of equine invasive trophoblast.

A system for transplanting invasive equine trophoblast (i.e., chorionic girdle) to ectopic sites has been developed as a means to study the differentiation of this tissue and to assess maternal immune responses to the conceptus tissue in a site outside the uterus. Chorionic girdle was isolated from Day 33 to 34 conceptuses and surgically placed into the vulvar mucosa or subdermal skin of recipient mares. Biopsy specimens of the graft sites for immunohistochemical staining were taken at weekly or biweekly intervals after grafting. Serum samples were collected from each recipient and tested for antibody to donor major histocompatibility complex (MHC) class I antigens using the lymphocyte microcytotoxicity assay. Transplanted trophoblast cells expressed differentiation markers associated with invading chorionic girdle and endometrial cup cells. The transplanted trophoblast cells were also labeled by an antibody to eCG. Strong cellular and humoral immune responses to the transplanted tissue were mounted by the recipients, similar to those occurring during normal equine pregnancy. Despite these responses, the invasive trophoblast transplants survived for at least 28 days after grafting and downregulated MHC class I antigens, as do the mature endometrial cup cells in equine pregnancy. These findings suggest that invasive equine trophoblast has the capacity to differentiate fully in equine nonuterine tissues, and that it can evade maternal immune responses independent of the physiological state of pregnancy and in sites other than the uterus.

Equine placentation.

A tough, elastic glycoprotein capsule envelops the equine blastocyst between Days 6 and 23 after ovulation. It maintains the spherical configuration of, and provides physical support for, the embryo as it traverses the entire uterine lumen during Days 6-17, propelled by myometrial contractions that are stimulated by pulsatile release of prostaglandin F2alpha and prostaglandin E2. The capsule also accumulates constituents of the exocrine secretions of the endometrial glands ('uterine milk') as nutrients for the mobile embryo as it releases its antiluteolytic maternal recognition-of-pregnancy signal to the whole of the surface of the endometrium. Mobility ceases abruptly on Day 17 with a sudden increase in uterine tonicity that 'fixes' the conceptus at the base of one of the uterine horns. At Day 35, the trophoblast of the spherical conceptus has separated into its invasive and non-invasive components. The former, distinguished as the thickened, annulate chorionic girdle, invades the maternal endometrium to form the unique endometrial cups. These secrete a chorionic gonadotrophin that synergizes with pituitary follicle-stimulating hormone to induce secondary luteal development in the maternal ovaries. The cup cells express foreign fetal antigens that stimulate strong maternal humoral and cell-mediated immune responses, which curtail their lifespan. The non-invasive trophoblast of the allantochorion establishes a stable microvillous contact with the endometrial epithelium around Day 40 and, over the next 100 days, develops a complex multibranched interdigitation with the endometrium to form the microcotyledonary haemotrophic exchange units that cover the entire surface of the diffuse epitheliochorial placenta. Reduction in the effective total area of fetomaternal contact at this placental interface, by competition between twin conceptuses for the limited area of available endometrium, by attachment of the allantochorion to an imperfect endometrium in a mare with endometrosis, or following cross-breeding or embryo transfer between a sire and dam of dissimilar size, will all induce intrauterine growth retardation of the fetus and runting of the foal, which persists into adult life. Over 40 years ago, Professor Roger Short and his colleagues determined that the high concentrations of conventional and unique ring B unsaturated oestrogens in the blood and urine of mares during the second half of pregnancy stem from placental aromatization of large quantities of C-19 precursor molecules secreted by the temporarily hypertrophic fetal gonads. Placental production of progesterone and 5alpha-reduced progestagens, on the other hand, depends on both maternal and fetal adrenal sources of pregnenelone.

Cell proliferation patterns during development of the equine placenta.

Placentation involves considerable growth and reorganization of both maternal and fetal tissues. In this investigation, immunohistochemical localization of the proliferation marker Ki-67 antigen was used to monitor cell division during placentation in mares. Endometrial biopsies were obtained from eight mares between day 14 and day 26 of pregnancy and from eight anoestrous mares that had been treated with various combinations of progesterone and oestrogen. Samples of endometrium and fetal membranes were obtained from 19 mares carrying normal horse conceptuses between day 30 and day 250 of gestation and from three failing extraspecific donkey-in-horse pregnancies. Proliferation in the superficial strata of the endometrium was increased by day 18 of gestation and this effect could be mimicked by supplementing with oestradiol benzoate during the last 6 days of a prolonged period (18-36 days) of progesterone administration. Fetal chorionic girdle cells were proliferating vigorously at days 30-32 of gestation, but stopped dividing after they invaded the endometrium, while the trophoblast cells of the allantochorion showed an increase in mitotic activity after day 38. The luminal epithelium of the endometrium started to proliferate only after the primary villi of the true epitheliochorial placenta had been formed, and during days 58-70 this effect was seen only in the pregnant horn in which placentation was further advanced. During the second half of gestation, most of the mitotic activity was confined to the periphery of the microcotyledons which were still growing. In the donkey-in-horse pregnancies, proliferation rates of the maternal and fetal epithelial at day 70 of gestation were markedly reduced in areas of heavy endometrial lymphocyte infiltration and poor placentation. These results provide a basis for further studies on factors that influence invasive and non-invasive placentation.

Equine Placental Cup Cells show Glycan Expression Distinct from that of Both Chorionic Girdle Progenitor Cells and Early Allantochorionic Trophoblast of the Placenta

Using lectin histochemistry on plastic-embedded material, the glycosylation patterns of equine girdle and cup cells, and associated endometrial glands, have been investigated from 37 to 67 days gestation. Results were compared with the glycosylation of the 50-day allantochorionic trophoblast of the established equine placenta that will later form the microcotyledons. The differentiated cup cells, which secrete equine chorionic gonadotropin (eCG), showed a pattern of glycosylation that was distinct both from the progenitor girdle cells and the allantochorionic trophoblast, with granules that bound lectins indicating high levels of α2,6 and α2,3-linked sialic acid, N-acetyllactosamine and bi/tri antennary non-bisected and bisected complex N-glycan. This is consistent with the known carbohydrate content of eCG. In contrast, the allantochorionic trophoblast at 50 days lacked detectable amounts of sialic acid and showed high levels of tri/tetra-antennary non-bisected complex N-glycan and N-acetyl galactosamine which was absent in the cup cells. During the process of girdle cell migration into maternal tissues, the uterine glands became greatly enlarged and dilated basally, with increased amounts of glycosylated secretory products revealed by lectins, which often seeped out into the extracellular space via ruptures in the apical regions of the gland wall.

Interspecific and extraspecific pregnancies in equids: anything goes.

Equids possess the unusual ability to interbreed freely among the phenotypically and karyotypically diverse member species of the genus to produce viable, but usually infertile, offspring. The mule (female horse x male donkey) was humanity's first successful attempt at genetic engineering and its clear expression of both parental phenotypes has contributed much to our understanding of genetic inheritance over the centuries. Even more surprising, mares and donkeys have been shown to be capable of carrying to term a range of true, xenogeneic extraspecies pregnancies created by embryo transfer, including Przewalski's horse (Equus prezwalskii; 2n = 66)-in-horse, (E. caballus; 2n = 64), and Grant's zebra (E. burchelli; 2n = 44)-in-horse pregnancies. Fetal genotypes has a marked influence on placental development in equids, especially on the width and general development of the annulate chorionic girdle, progenitor tissue of the gonadotrophin (eCG)-secreting endometrial cups. However, transfer of intact and bisected demi-mule embryos (E. mulus; 2n = 63) to Jenny donkeys (E. asinus; 2n = 62) showed convincingly that maternal uterine environment, probably mediated by intrauterine growth factor production, can exert an overriding influence on chorionic girdle development and its invasion of the maternal endometrium. Transfer of donkey embryos (2n = 62) to horse mares (2n = 64) results in the development of an exceptionally small chorionic girdle that completely fails to invade the endometrium to form endometrial cups. Around 70% of these donkey-in-horse pregnancies are aborted between days 80 and 85 of gestation in conjunction with delayed and abnormal placental attachment combined with a vigorous maternal cell-mediated reaction against the xenogeneic donkey trophoblast. This model of pregnancy loss shows strong evidence of immune memory and the rate of fetal death is reduced by immunization of the surrogate mare against donkey lymphocytes. The findings suggest an important role for the invasive trophoblast cells of the equine placenta in initiating and driving attachment and interdigitation of the non-invasive placenta for fetal sustenance, and in modulating materno-fetal immunological interaction to enable survival of the antigenetically foreign fetus in the uterus.

Placental localization of relaxin in the pregnant mare

In situ hybridization employing a cRNA probe derived from a 428-bp fragment of equine relaxin was used to localize relaxin mRNA, and immunocytochemistry was used to localize relaxin itself, in tissues of the placenta-endometrium interface recovered between 33 and 153 days of gestation from mares carrying intraspecific horse, interspecific mule and extraspecific donkey conceptuses. Immunocytochemical staining was also used to localize trophoblast-specific and class I major histocompatibility complex (MHC) antigens on some specimens, Relaxin mRNA and relaxin were both present in the single-cell non-invasive trophoblast layer of the allantochorion between 45 and 153 days of gestation in all three types of equine pregnancy examined, Both, however, were absent from the invasive trophoblast cells of the progenitor chorionic girdle and the differentiated trophoblast cells of the endometrial cups throughout the latters' 60–80-day period of development and regression. Discrete and irregularly spaced clusters of elongated pseudostratified trophoblast cells on the allantochorion remained negative for relaxin mRNA and ligand, but stained strongly for equine trophoblast-specific antigens. These areolae-like structures of the mature horse placenta overlie the mouths of endometrial glands between adjacent microcotyledons and they are clearly involved with the uptake of uterine milk for fetal sustenance. It is speculated that their loose attachment to the endometrium and weak expression of class I MHC antigens may serve to tolerize the mother to the paternally-inherited histocompatibility antigens of the fetus.

Susceptibility of equine chorionic girdle cells to lymphokine-activated killer cell activity.

Equine chorionic girdle cells are a subpopulation of highly invasive trophoblast cells that attach and invade the uterine epithelium on Day 35 (Day 0 = day of ovulation). These invading chorionic girdle cells form endometrial cups that are associated with a marked local maternal leukocytic response that may result in the demise of the cups at Day 120 of pregnancy. Once endometrial cups become established in the uterine wall they do not express MHC antigens, and therefore may only be susceptible to non-MHC restricted cytotoxic cells. The susceptibility of cultured chorionic girdle cells to LAK cell cytotoxicity was tested in order to evaluate the role of this type of cytotoxicity in the life-cycle of endometrial cup tissue. Chorionic girdle cells from ten Day 34 conceptuses were collected, cultured, and used as a target cell in a lymphokine activated killer assay to determine if these cells were susceptible to lymphokine activated killing. Cultured chorionic girdle cells demonstrated similar in vitro morphological features and patterns of antigen expression to those seen in vivo in endometrial cups. Cultured chorionic girdle cells were susceptible to lymphokine activated killing. Lymphokine activated killer cells may play a role in the degeneration of endometrial cup tissue. The potential mechanisms of regulation of uterine LAK cell activity in the horse are discussed.

Genetically targeting adenosine deaminase to trophoblasts of the murine placenta rescues adenosine deaminase deficient fetuses from perinatal lethality

s: R.T.C. and T.G.W.M.S. Canada 1996 Genetically Targeting Aakrwsine Deaminase to Trophoblasts of the Murine Placenta Rescues Adenosine Deaminase Deficient Fetuses from Perinatal Lethality. mlackburn, R.E. Kellems, Department of Biochemistry, Baylor College of Medicine, Houston, TX, USA. Adenosine deaminase (ADA) is an essential enzyme of purine metabolism that is expressed at very high levels in trophoblasts of the muring placenta where it accounts for over 95% of the ADA present at the fetal gestation site. ADA deficient fetuses, which also lack ADA in their adjoining placentas, die during late fetal development in association with profound purine metabolic disturbances and hepatocellular impairment. To investigate the potential importance of placental ADA we genetically restored the enzyme to placentas of ADA deficient fetuses. This was accomplished by using regulatory elements from the murine Ada gene to drive the murine ADA cDNA specitically to the placenta prenatally in transgenic mice. These mice were then bred to mice heterozveous for the null Ada allele, generating progeny heterozygous ?or both the placental snecific ADA miniaene and an Ada null allele. Intercrosses between these mice s 072” *The Babraham Institute, Cambrrdge, CB2 : . . A unique feature of equine placentation is the annulate chorionic girdle of specialised trophoblast cells that invade the maternal endometrium around day 36 of pregnancy (term=320-340 days). This chorionic girdle develops through hyperplasia of trophoblast cells on the region of chorion adjacent to its point of fusion with the aliantois, and we have proposed a model to explain its formation that involves local stimuli from the allantoic mesenchyme. The most important of these appear to be the growth factors, insulin-like growth factor II (IGF II) and hepatocyte growth factor-scatter factor (HGF-SF), the latter having morphogenic, motogenic and mitogenic actions. We have now examined the effects of these growth factors on equine uophoblast cells in vitro by means of a colourimetric mitogenic assay using pure populations of growth-arrested equine trophoblast cells in serum-free medium. The cells demonstrated significant dose-related mitogenic responses to both growth factors with the maximum response to HGF-SF being 63% of that obtained with fetal calf serum compared to only 20% for IGF II. Furthermore, HGFSF stimulated an increased secretion of equine chorionic gonadotrophin, a marker of trophoblast differentiation. These results support our hypothesis that the allantoic mesenchyme-derived growth factors, IGF II and HGFSF, are important for the growth and differentiation of equine trophoblast.

Analysis of MHC class I expression in equine trophoblast cells using in situ hybridization

Down-regulation of major histocompatibility complex (MHC) genes by trophoblast cells is considered to be a primary mechanism preventing maternal immune rejection of the fetal-placental unit in mammalian pregnancy by rendering these cells, which form the primary barrier between mother and fetus, relatively non-antigenic. In situ hybridization with probes encoding human and horse MHC class I genes was used to characterize the pattern of MHC class I mRNA expression in the various forms of horse trophoblast. Strong hybridization signals were observed in the invasive trophoblast cells of chorionic girdle tissue. In contrast, no hybridization signal specific for MHC class I mRNA transcripts was observed in the descendent endometrial cup trophoblast cells. In the non-invasive trophoblast cells of the allantochorion, no hybridization signals specific for horse MHC class I mRNA transcripts were consistently detected. In parallel to the in vivo results, strong hybridization signals were observed in the small, mononuclear cells present in chorionic girdle cell explant cultures, but not in the population of large binucleate cells corresponding to endometrial cup cells. The results obtained using in situ hybridization are consistent with the hypothesis that expression of MHC class I genes may be controlled at the transcriptional level in horse invasive and non-invasive trophoblast cells, and suggest that down-regulation of MHC class I antigen expression in endometrial cup cells may be accomplished by the same mechanisms in vivo and in vitro.

Chorionic girdle cell invasion and serum factors in mares

To assess whether the expression of connexins (Cx) by keratinocytes is altered under conditions of abnormal epidermal differentiation, we have compared Cx26, Cx32, Cx37, Cx40, and Cx43 in the epidermis of 11 psoriatic patients who had not been treated for at least 1 mo and of seven healthy individuals. In all samples of fully mature psoriatic plaques, we have observed a massive expression of Cx26, as judged at both the transcript level (northern blot) and the protein level (immunofluorescence). This protein became consistently detected between keratinocytes of the basal and granular layers at the periphery of psoriatic plaques and in all layers of fully developed psoriatic epidermis, except in regions of parakeratosis. None or a minimal amount of Cx26 was observed in both control and nonlesional regions of psoriatic epidermis. Psoriatic plaques also contained Cx43, the prominent gap junction protein in the interfollicular epidermis of normal human skin. The levels of this protein appeared to be slightly higher in psoriatic than in control skin, as judged at both the transcript level (northern blot) and the protein level (immunofluorescence). Three other connexins (Cx32, Cx37, and Cx40), which are not observed in control interfollicular epidermis, were not induced in either nonlesional or lesional regions of psoriatic skin. The data indicate that selective changes in the normal expression of connexins by keratinocytes are associated with the changes in the proliferation and differentiation program that these cells undergo in psoriasis.

Expression of major histocompatibility complex antigen and timing of invasion by equine chorionic girdle cells cultured on Matrigel.

Chorionic girdle cells are a highly invasive subpopulation of trophoblastic cells of the horse conceptus that adhere to the uterine epithelium and begin to invade the endometrium on Days 34-36 (Day 0 = day of ovulation). Just prior to and during invasion (Days 32-36), chorionic girdle cells express high levels of major histocompatibility complex (MHC) I, but expression of this antigen decreases by Days 40-45 and is lost by Day 55. The mechanisms involved in the control of chorionic girdle cell invasion and altered MHCI expression over time are not known. The objective of this study, therefore, was to determine the timing of invasion and the characteristics of MHC expression by girdle cells cultured on Matrigel to determine whether chorionic girdle cell behavior in this environment is similar to the behavior in vivo. Chorionic girdles from four conceptuses were collected on each of Days 30, 31, and 32 and placed in Matrigel invasion chambers for 48 h and in additional duplicate chambers for a time period equivalent to the number of days from the day of collection to Day 36 (6, 5, and 4 days, respectively). After these culture times, the area of the filter covered by invasive cells was determined through use of the software program NIH image. At 48 h, Day 31 and Day 32 girdle cell preparations had invaded Matrigel; but only one Day 30 girdle cell preparation showed invasion into Matrigel, and the extent of invasion was limited. Girdle cells collected on all 3 days had invaded Matrigel by the time equivalent to Day 36. Additionally, chorionic girdles from six conceptuses were collected on Day 34 and placed in Matrigel invasion chambers. Three of these were examined for surface antigen expression of MHCI, MHCII, and a trophoblast-specific antigen (102.1) by immunocytochemistry (ICC) every 48 h to a time point equivalent to Day 44. The remaining three were examined for these same antigens at times equivalent to Days 36, 40, 50, and 60. Invasive Day 34 girdle cells expressed MHCI and trophoblast-specific antigen, but not MHCII, at each time examined (Days 36-60). Neither the timing of invasion nor MHCI expression by chorionic girdle cells on Matrigel mimicked what occurs in vivo. Therefore, it is likely that the in utero environment plays a role in regulating these two characteristics of equine chorionic girdle cells.

Anti-paternal cytotoxic lymphocyte (CTL) activity during allogeneic or xenogeneic pregnancy in equids

In situ hybridization employing a cRNA probe derived from a 428-bp fragment of equine relaxin was used to localize relaxin mRNA, and immunocytochemistry was used to localize relaxin itself, in tissues of the placenta-endometrium interface recovered between 33 and 153 days of gestation from mares carrying intraspecific horse, interspecific mule and extraspecific donkey conceptuses. Immunocytochemical staining was also used to localize trophoblast-specific and class I major histocompatibility complex (MHC) antigens on some specimens, Relaxin mRNA and relaxin were both present in the single-cell non-invasive trophoblast layer of the allantochorion between 45 and 153 days of gestation in all three types of equine pregnancy examined, Both, however, were absent from the invasive trophoblast cells of the progenitor chorionic girdle and the differentiated trophoblast cells of the endometrial cups throughout the latters' 60–80-day period of development and regression. Discrete and irregularly spaced clusters of elongated pseudostratified trophoblast cells on the allantochorion remained negative for relaxin mRNA and ligand, but stained strongly for equine trophoblast-specific antigens. These areolae-like structures of the mature horse placenta overlie the mouths of endometrial glands between adjacent microcotyledons and they are clearly involved with the uptake of uterine milk for fetal sustenance. It is speculated that their loose attachment to the endometrium and weak expression of class I MHC antigens may serve to tolerize the mother to the paternally-inherited histocompatibility antigens of the fetus.

Metalloproteinase activity has a role in equine chorionic girdle cell invasion.

Chorionic girdle cells are a highly invasive subpopulation of trophoblast cells of the equine conceptus. By Day 35 (Day 0 = day of ovulation), cells of the chorionic girdle adhere to the uterine epithelium and begin to invade the endometrial wall. Invasive cells must attach to extracellular matrix proteins, secrete proteinases capable of degrading matrix, and migrate through the degraded matrix; invasion is largely dependent on the proteinase activity of the cells. The objective, therefore, was to develop an in vitro system to examine the mechanisms of equine chorionic girdle cell invasion through extracellular matrix. Day 34 chorionic girdle cell preparations were cultured on Matrigel Invasion Chambers. The cultured invasive cells were binucleate with prominent nucleoli and were often highly vacuolated, consistent with in vivo cup cell morphology. In addition, the cultured cells produced eCG. Additional Day 34 chorionic girdle cell preparations were cultured on Matrigel Invasion Chambers with or without proteinase inhibitors (aprotinin, bestatin, 1,10-phenanthroline) to determine the proteinase activity associated with girdle cell invasion. Only the metalloproteinase inhibitor, 1,10-phenanthroline, inhibited chorionic girdle cell invasion through Matrigel. Chorionic girdle cell supernatants were characterized by zymography, and the proteinases produced by these cells were confirmed to be metalloproteinases at approximate molecular masses of 72 and 95 kDa. The results indicated that equine chorionic girdle cells have matrix-degrading capabilities through metalloproteinase activity. Similar metalloproteinase activity has been reported to be necessary for mouse and human trophoblast invasion, suggesting a similar mechanism of implantation.

Horse Trophoblasts Produce Tumor Necrosis Factor α but not Interleukin 2, Interleukin 4, or Interferon γ1

The distribution of four cytokines was analyzed in the endometrium and trophoblast of the horse between Days 30 and 55 of gestation. Endometrial tissues, invasive trophoblast (chorionic girdle), and noninvasive trophoblast (chorion and allantochorion) were examined separately. Cytokine expression was determined by amplification of specific mRNA via the reverse transcriptase polymerase chain reaction (RT-PCR). Messenger RNA for interleukin 2 (IL-2), interleukin 4 (IL-4), and interferon gamma (IFN gamma) was detected in endometrial tissues, unstimulated peripheral blood lymphocytes, and control kidney tissue, but not in trophoblasts. leukocytes resident in the endometrium or traversing the uterus via blood vessels might be the source of these cytokines. Endometrial tissues and invasive and noninvasive trophoblasts expressed mRNA for tumor necrosis factor alpha TNF alpha). Immuonoreactive TNF alpha protein was detected in different cell types of the endometrium and in the invasive and noninvasive trophoblast. The ubiquitous expression of TNF alpha by the endometrium and trophoblasts suggests that this cytokine might have an important role in regulating placental growth and differentiation or maternal leukocyte responses to trophoblasts. IL-2, IL-4, and IFN gamma might have important immunoregulatory roles within the endometrium.

The equine endometrial cup reaction: a review.

The function of eCG in equine pregnancy is far from clear but it has become evident that eCG has little or no FSH activity in the horse and is therefore probably not responsible for the secondary ovulations. eCG does have luteotrophic activity and it could play a role in the resurgence of the primary corpus luteum (1,7,44). Some evidence exists that the receptor population on the equine gonads is heterogenous in a way that makes it possible to distinguish eCG from eLH, resulting in different post-receptor effects (7). There is also evidence that eCG itself is heterogenous, both in glycosylation and in primary structure, not only between different individual animals but also within one animal during different stages of gestation. The differences could simply reflect the difference between stored and secreted hormone, but on the other hand the release of different eCG forms could be under endocrine control, allowing the mare to produce forms appropriate to specific biological needs (74). Thus some forms of eCG could play a role in immunological events taking place at the foeto-maternal interface. The role of cytotoxic antibodies in the equine pregnancy is not understood. The fact that they are not harmful to the pregnancy can be explained by the fact that their target, the paternal MHC molecules, are withdrawn from the endometrial cup tissue by the time the antibodies start appearing in the circulation. This unique way of regulation of MHC expression is also poorly understood.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor II gene expression in the fetus and placenta of the horse during the first half of gestation.

Placentation in equids involves two types of trophoblast: a minor invasive component, the chorionic girdle, that gives rise to transient endocrine structures known as endometrial cups, and a major non-invasive component, the allantochorion, that forms the diffuse, microcotyledonary placenta. Growth factors are likely to be important in controlling these complex events at implantation and this study describes the use of in situ hybridization and northern blotting techniques to monitor expression of insulin-like growth factor II (IGF-II) in the fetus and placenta of the horse (Equus caballus), using 12 conceptuses recovered between 14 and 150 days of gestation (term is about 340 days). The anti-sense, but not the sense, ovine IGF-II oligonucleotide probe (45 mer) hybridized to a total of seven IGF-II mRNA transcripts (6.2-1.3 kb) in RNA extracted from horse fetal liver, demonstrating the specificity and validity of the probe for equine IGF-II mRNA. In situ hybridization demonstrated that the IGF-II gene was expressed intensely in the fetus at all stages examined, predominantly in tissues of mesodermal origin, but also in the endoderm-derived liver and epithelia of the gut and lung bronchioles, and the ectoderm-derived facial mesenchyme and choroid plexus. High concentrations of IGF-II mRNA were also detected in the extraembryonic mesoderm, invasive chorionic girdle and mature endometrial cup tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors Affecting the Maturation and Invasiveness In Vitro of Chorionic Girdle Cells of Different Genotypes1

Factors Affecting the Maturation and Invasiveness In Vitro of Chorionic Girdle Cells of Different Genotypes1

Paternal and maternal major histocompatibility complex class I antigens are expressed co-dominantly by equine trophoblast

Invasive equine trophoblast cells of the chorionic girdle express high levels of paternally inherited Major Histocompatibility Complex (MHC) class I antigens prior to migration into the endometrium to form the so-called endometrial cups. Three groups of experiments were performed to determine if maternally inherited MHC class I antigens are expressed on chorionic girdle cells. Results indicated that maternally and paternally inherited MHC class I antigens are co-dominantly expressed by cells of the invasive equine trophoblast, and therefore, that the expression of polymorphic equine MHC class I genes does not appear to be affected by genomic imprinting in this tissue. The demonstration that cells of the chorionic girdle were immunogenic supports the hypothesis that invasion of the maternal endometrium by chorionic girdle cells stimulates the production of anti-paternal alloantibodies normally observed in early horse pregnancy. The co-dominant expression of MHC class I antigens by invasive chorionic girdle cells has important implications for the mechanism of recognition of allogeneic fetal MHC class I antigens by the maternal immune system.

Chorionic gonadotropin (α and β subunit RNA's are present in equine placental membranes by day 30 of pregnancy

Summary Northern analysis was used to investigate timing of transcrip tion of the genes for α and β subunits of chorionic gonadotropin (CG) by equine conceptuses. Two conceptuses were collected on each of days 26, 28, 30 and 32 after ovulation. Conceptus mem branes and embryos were separated and frozen immediately on dry ice. Total RNA was extracted, electrophoretically separated on agarose gels, and RNA was transferred to nitrocellulose membranes by osmotic blotting. Total RNA from equine liver, pituitary, testis and from a day 32 embryo sewed as control. The α and β subunits were both first detected in RNA extracted from day 30 conceptus membranes. The α subunit was detected in placental membrane RNA from both samples at day 30 and in both samples at day 32. The β subunit was detected in RNA in only one sample at day 30 and in both samples at day 32. Both α and β subunits were detected in equine pituitary RNA, but neither was detected in embryo, liver or testis RNA. These results indicate that transcription of the genes for α and β subunits of CG has begun by day 30 of gestation. This is well before the chorionic girdle cells invade the endometrium to form endometrial cups, and earlier than has been reported for presence of the hormone in serum or for in vitro production by girdle cells.