Single-cell insights into development of the bovine placenta†.

A central determinant of pregnancy success is proper development of the conceptus (embryo/fetus and associated extraembryonic membranes including the placenta). Although the gross morphology and histology of the bovine placenta have been well studied, the cellular and molecular mechanisms regulating placenta development and trophoblast differentiation and function remain essentially undefined. Here, single-cell transcriptome (scRNA-seq) analysis was performed on the day 17 bovine conceptus and chorion of day 24, 30, and 50 conceptuses (n = 3-4 samples per day) using the 10X Genomics platform. Bioinformatic analyses identified cell types and their ontogeny including trophoblast, mesenchyme, and immune cells. Loss of interferon tau-expressing trophoblast uninucleate cells occurred between days 17 and 30, whereas binucleate cells, identified based on expression of placental lactogen (CSH2) and specific pregnancy-associated glycoprotein genes (PAGs), first appeared on day 24. Several different types of uninucleate cells were present in day 24, 30, and 50 samples, but only one (day 24) or two types of binucleate cells (days 30 and 50). Cell trajectory analyses provided a conceptual framework for uninucleate cell development and binucleate cell differentiation, and bioinformatic analyses identified candidate transcription factors governing differentiation and function of the trophoblasts. The digital atlas of cell types in the developing bovine conceptus reported here serves as a resource to discover key genes and biological pathways regulating its development during the critical periods of implantation and placentation.

The ruminant placental trophoblast binucleate cell: an evolutionary breakthrough.

Viviparity and the development of a placenta are two of the major reasons for the success of the mammals in colonizing all habitats, both terrestrial and aquatic. The placenta is an apposition of fetal to maternal tissue which serves two main, but competing functions: to maximize oxygen transfer and the acquisition of nutrients from the mother, but to minimize immunological rejection by the maternal immune system. This has resulted in the evolution of four main types differing in the degree of loss of the maternal uterine epithelial (UE) barrier: epitheliochorial, synepitheliochorial, endotheliochorial, and hemochorial, all providing a successful safe balance between the needs of mother and fetus. Epitheliochorial is the least invasive, a simple apposition and microvillar interdigitation of the apices of uterine epithelium and trophoblast. It is suggested to have evolved as a response to the increase in the size of the animal to provide a sufficiently long gestation to produce a single altricial (run/swim-soon-as-born) neonate as in the Cetartiodactyla. The mother needs to have good control of the fetal demands so the UE barrier is maintained. However, in the synepitheliochorial placenta, characteristic of all ruminants, the fetus has evolved a means of increasing, or at least maintaining, demand without the need for invasion. This has been achieved by the development of the trophoblast binucleate cell which, uniquely, can fuse with a UE cell to form fetomaternal hybrid tissue. This can maintain some maternal barrier function but also deliver fetally synthesized immunomodulatory and metabolic messages to the maternal circulation. This review provides the evidence for this remarkable evolutionary step and also considers an alternative explanation for the formation of the structure of the ruminant placenta.

Incidence of micronuclei in human peripheral blood lymphocytes exposed to modulated and unmodulated 2450 MHz radiofrequency fields.

Peripheral blood samples from four healthy volunteers were collected and aliquots were exposed in vitro for 2 h to either (i) modulated (wideband code division multiple access, WCDMA) or unmodulated continuous wave (CW) 2450 MHz radiofrequency (RF) fields at an average specific absorption rate of 10.9 W/kg or (ii) sham-exposed. Aliquots of the same samples that were exposed in vitro to an acute dose of 1.5 Gy ionizing gamma-radiation (GR) were used as positive controls. Half of the aliquots were treated with melatonin (Mel) to investigate if such treatment offers protection to the cells from the genetic damage, if any, induced by RF and GR. The cells in all samples were cultured for 72 h and the lymphocytes were examined to determine the extent of genetic damage assessed from the incidence of micronuclei (MN). The results indicated the following: (i) the incidence of MN was similar in incubator controls, and those exposed to RF/sham and Mel alone; (ii) there were no significant differences between WCDMA and CW RF exposures; (iii) positive control cells exposed to GR alone exhibited significantly increased MN; and (iv) Mel treatment had no effect on cells exposed to RF and sham, while such treatment significantly reduced the frequency of MN in GR-exposed cells.

Placentation and hormonal maintenance of pregnancy in the impala (Aepyceros melampus).

INTRODUCTION: The impala is a widely distributed African ungulate. Detailed studies of the placenta and ovaries in impala undertaken in the 1970s did not address the endocrine functions of the placenta. METHODS: The uteri of 25 pregnant impala estimated to be between 49 and 113 days of the 190 day gestation were examined grossly, histologically and immunohistochemically. RESULTS: A single corpus luteum was present in either maternal ovary but the conceptus was always situated in the right uterine horn. The fetal membranes extended to the tips of both uterine horns. The amnion was in intimate contact with, but not fused to, the allantochorion. Placentation was typically ruminant with fetal macrocotyledons attached to the rows of maternal caruncles. The fetal villi were highly branched, especially in the centre of each placentome where the attenuated maternal epithelium lining the placental crypts was absent in some places. Both the corpus luteum and the uninucleate trophoblast cells of the interplacentomal allantochorion stained strongly for 3-ß hydroxysteroid dehydrogenase, and progestagen concentrations in allantoic and amniotic fluids increased significantly as gestation progressed, with a tendency to do likewise in maternal serum. Binucleate trophoblast cells stained positively for bovine placental lactogen, but neither the placenta nor the maternal corpus luteum showed evidence of oestrogen synthesis. DISCUSSION: Despite exhibiting the same basic type of placentation, both the gross and histological structure of the impala placenta, along with its immunohistochemical properties, demonstrates that great variation exists across ruminant placentas.

Glycosylation and immunocytochemistry of binucleate cells in pronghorn (Antilocapra americana, Antilocapridae) show features of both Giraffidae and Bovidae.

Although the pronghorn (Antilocapra americana) resembles an antelope, its nearest relatives are the giraffe and okapi. In this study we have examined the placentae of 6 pronghorns using lectin- and immunocytochemistry to identify giraffid and bovid features. Binucleate cells (BNC) of the placenta exhibited features intermediate between those of the giraffe and bovine; Dolichos biflorus agglutinin binding - strong in the bovine BNC and absent in the giraffe - was evident in only a subpopulation of BNC while binding to blood vessels, as in the giraffe. Binding of Phytolacca americana agglutinin resembled that of the giraffe and okapi whereas many other glycans were found in all four clades. PAG antigens were similar to bovine and okapi but not giraffe. In summary, although the pronghorn outwardly resembles an antelope, placental BNC show giraffid features. Although each clade has its own individual characteristics, there are far more similarities than differences between them, emphasizing the common ancestry of all four clades.

Experimental Neospora caninum infection modifies trophoblast cell populations and plasma pregnancy-associated glycoprotein 1 and 2 dynamics in pregnant dairy heifers.

Neospora caninum is an obligate intracellular protozoan that causes abortion in cattle worldwide. Plasma concentrations of pregnancy-associated glycoprotein 1 (PAG-1), produced by binucleate trophoblast cells, are used for pregnancy diagnosis and as a marker of foetal-placental well-being, while PAG-2, produced by both mono- and binucleate trophoblast cells, may serve as an indicator of abortion risk. In prior work, natural N. caninum infection was found to modify plasma PAG-1 and PAG-2 patterns. The present study examines PAG-1 and PAG-2 dynamics and trophoblast cell populations following experimental infection with N. caninum. The study population was comprised of 17 N. caninum seronegative Holstein-Friesian heifers. On day 110 of gestation, 6 heifers were inoculated intravenously with 10(7) taquyzoites of N. canimum. Plasma samples for PAG-1 and PAG-2 determinations were collected on days 0, 7, 14, 21 and 42 post infection. During the study course, pregnancy was normally expressed in all controls while three infected heifers lost their foetuses. All heifers were euthanised on day 42 post infection and placentome samples from the 14 non-aborting heifers were collected to examine trophoblast cell populations. Plasma PAG-1 and PAG-2 concentrations in non-infected heifers increased until the day of euthanasia while non-aborting infected heifers showed a temporary fall in PAG-1 (P<0.004) and PAG-2 (P<0.002) concentrations from 7 to 14 days post infection (dpi). The two dams aborting at 14 and 21 dpi and a third dam with a mummified foetus upon euthanasia showed dramatic PAG-1 and PAG-2 reductions from 14 dpi to undetectable levels upon euthanasia. A stereological study of placentomes revealed significantly higher relative proportions of mono- (P=0.035) and binucleate (P=0.029) trophoblast cells at 42 dpi in non-infected heifers than infected non-aborting heifers. According to our findings, following experimental N. caninum infection on day 110 of gestation, non-aborting heifers showed a brief reversible drop in plasma PAG-1 and 2 concentrations two weeks later and reduced proportions of bi- and mono-nucleate trophoblast cells 42 days after infection. In aborting dams, dramatically reduced PAG levels were related to severe placental damage and a non-viable pregnancy.

Localization of TGF-ß and TGF-ß receptor in bovine term placentome and expression differences between spontaneous and induced parturition.

INTRODUCTION: Mechanisms of detachment of fetal membrane after parturition in cattle are poorly understood. Glucocorticoids trigger the initiation of parturition and may facilitate the placental maturation. We compared the disappearance of trophoblast binucleate cells (BNCs) and expression of transforming growth factor-ß (TGFB) in term placentomes between spontaneous and induced parturition to investigate the influences of glucocorticoids on the placental maturity. METHODS: Cows were delivered spontaneously (SP group) or after the administration of prostaglandin (PG) F(2)α (PG group); dexamethasone, PGF(2)α, and estriol (DEX group); and triamcinolone acetonide, PGF(2)α, and betamethasone (BET group) and placentomes were collected immediately after parturition. The number of BNCs in hematoxylin and eosin stained section was examined. Protein localization and mRNA levels of TGFB and its receptor (TGFBR) were analyzed using immunohistochemistry and qRT-PCR, respectively. RESULTS: TGFB1 is characteristically localized in the maternal septum in caruncle in contrast to TGFB2 and TGFB3, which are mainly found in cotyledonary villi and maternal epithelial cells. TGFBR1 and TGFBR2 colocalized in BNCs. The number of BNCs was lower in the SP group than in PG and DEX groups. mRNA levels of TGFB1, TGFBR1 and TGFBR2 in the SP group differed from PG and DEX groups. There was no difference between SP and BET groups in all analyses. DISCUSSION: These results indicate that parturition inductions using PGF(2)α or dexamethasone were not able to induce disappearance of BNCs and change of TGFB signaling. Results in the BET group suggest that investigation into types, dose, and dosage schedule of glucocorticoids may facilitate placental maturation.

Ochratoxin-induced toxicity, oxidative stress and apoptosis ameliorated by quercetin--modulation by Nrf2.

Ochratoxin (OTA) is one of the most abundant food contaminating mycotoxins and is commonly present in the food chain. Many of the effects associated with OTA, appear to be mediated through oxidative stress. Although the toxicity of OTA is fairly well characterized, antidotes for alleviating the toxicity are sparsely reported. Dietary antioxidants have gained much importance in the recent years for their antioxidative and therapeutic properties. In the present study the therapeutic strategy was directed towards use of quercetin, a dietary antioxidant to combat OTA-induced toxicity in Vero cell line. Our results demonstrate that quercetin pre-treatment suppressed OTA-induced cytotoxicity and oxidative stress. It modulated OTA-induced alteration on the antioxidant defence through activation of Nrf2 pathway. Morphological studies by scanning electron microscopy (SEM) and cell cycle analysis indicated that quercetin prevented OTA-induced apoptosis. It also inhibited the activation of caspase cascade that leads to DNA fragmentation. Quercetin also exhibited antigenotoxic potential by attenuating OTA-induced DNA damage and micronucleus (MN) formation. The results of the study demonstrate for the first time that quercetin pre-treatment prevents OTA-induced oxidative stress and apoptosis in Vero cell line.