G. Morgan

Functional specialization in the ruminant placenta : Evidence for two populations of fetal binucleate cells of different selective synthetic capacity

Trophoblast binucleate cells (BNC) in the ruminant placenta demonstrate a characteristic development, mature structure and migratory capacity whether situated in cotyledonary or intercotyledonary regions of the placenta. However, previous immunocytochemical studies demonstrated clear differences in gene expression in granule contents of BNC according to their anatomical location with some proteins being expressed in all BNC (e.g. ovine placental lactogen) whereas others were unique to a particular origin (e.g. SBU3 antigen in cotyledonary BNC only). We have used enriched preparations of binucleate cells and showed differences in steroid metabolic capacity in vitro which is more related to their species origin (sheep or goat) than to their anatomical location. The predominant product from [3H]pregnenolone is progesterone (sheep) and 5 beta-pregnane-3 alpha, 20 alpha-diol (goat) and the amount formed (corrected for the number of BNC) is similar irrespective of whether BNC were derived from the cotyledonary or intercotyledonary regions. These studies indicate specific forms of regional functional specialization of BNC and emphasize their multifunctional role in the ruminant placenta.

Calcium transport and the localisation of calbindin-D9k in the ruminant placenta during the second half of pregnancy

Abstract.In late pregnancy the sheep fetus requires 3 g of calcium per day, all of which must be transported across the trophoblast epithelium of the placenta. Such high levels of calcium transport across other epithelia are normally associated with the presence of calbindin-D9 or -28k. Our immunocytochemical results show that ovine, bovine and caprine interplacentomal trophoblast have high levels of calbindin-D9k, about eight to ten times more than in the placentomal region. The protein is detectable only in the uninucleate trophoblast cells in sheep and goat, the frequent binucleate cells show none. The calbindin-D9k is also present in the maternal glandular epithelium but not the surface epithelium of the uterus. The cellular distribution of the calbindin-D9k immunoreactivity suggests a soluble protein homogenously distributed through cytosol and nucleoplasm but absent from all organelles and intercellular spaces. In contrast, the uterine milk protein(s) are localised in Golgi cisternae and secretory vesicles in gland cells and in apical small endocytic vesicles and lysosomes in the uninucleate trophectodermal cells. The distribution of calbindin-D9k supports the concept that it mediates the high calcium flux by facilitated diffusion and not via any vesicular, membrane-bounded system.

Light and electron microscopic studies of cellular localization of oPL with monoclonal and polyclonal antibodies

Accurate knowledge of placental lactogen localization is fundamental to any hypothesis of its synthesis and secretion. We used locally generated monoclonal and polyclonal antibodies from three separate sources to localize ovine placental lactogen immunoreactivity on light and electron microscope Lowicryl K4M sections of ovine placentomes of 97-145 days of gestation, using immunogold techniques. All antibodies demonstrated that immunoreactivity was exclusively localized in the trophoectoderm binucleate cell Golgi body and granules and in granules in the syncytium derived from binucleate cell migration. No evidence was found to support a recent claim that monoclonal antibodies to oPL that were produced in Canada indicated a predominant localization of ovine placental lactogen to uninucleate trophectodermal cells.

Membrane dynamics during migration of placental cells through trophectodermal tight junctions in sheep and goats

Binucleate cells in ruminant trophectodermal epithelium are unique in that they form part of the tight junction as they migrate across it, maintaining the ionic barrier seal to the internal milieu of the fetus. Such participation imposes considerable constraints on the cell migration because membrane cannot flow through a tight junction. We report quantitative ultrastructural immunocytochemical evidence for vesicle membrane insertion into the binucleate cell plasmalemma which allows the cells to form a pseudopodium past the tight junction. This pseudopodium increases continuously in area by vesicle insertion and develops a close apposition to the plasmalemma of the fetomaternal syncytium which constitutes the fetomaternal boundary in the placenta of the sheep and goat. Enventually the apposed membranes of the binucleate cell pseudopodium and the syncytium fuse by vesiculation and the cytoplasm and nuclei of the binucleate cell merge into the fetomaternal syncytium. The binucleate cell plasmalemma remaining on the trophectodermal side of the tight junction is blebbed off into, and phagocytosed by, the uninucleate trophectodermal cells between which the binucleate cell passed. This process permits the delivery of the binucleate cell granules to the maternal side of the placenta but none of the fetal molecules expressed on the plasma membrane of the binucleate cells are exposed to potential maternal immunological rejection.

Immunocytochemical and In situ hybridization studies of the distribution of calbindin D9k in the bovine placenta throughout pregnancy.

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 (∼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.

Calcium localization in lactating rabbit mammary secretory cells.

Several methods were investigated for calcium localization. Only glutaraldehyde fixation followed by osmium plus pyroantimonate produces a consistent localization of precipitate in lactating rabbit mammary secretory cells. The precipitate is found only in vesicles and cisternae of the Golgi apparatus and is shown by electron microscope microanalysis to consist of calcium and antimony. The origin of the calcium is briefly discussed.

“Sunbursts” and “christiesomes”: Cellular fragments in normal cow and goat milk

SummaryGoats milk includes numerous cell fragments (“christiesomes”) which originate from the mammary secretory cells, contain well preserved endoplasmic reticulum, mitochondria and lipid droplets, and are responsible for the considerable triglyceride synthesising capacity of fresh goat milk. Cows milk shows a few such particles only after repeated oxytocin-aided milkings. Cows milk does contain quite different particles which have a dense content with a few small vesicles and numerous microvillus-like protrusions on one side (“sunbursts”). These have not been found in goats milk. Cytoplasmic particles similar to sunbursts have been found on the surface of the mammary secretory epithelium. It is suggested that they are residues of dead cells.

Cell migration in the ruminant placenta: A freeze-fracture study

In the ruminant placenta, binucleate cells normally migrate out of the trophectodermal epithelium throughout pregnancy to fuse with the syncytium bounding the maternal connective tissue. This study shows that they form part of the trophectodermal tight junction as they migrate, thus maintaining the barrier function while penetrating the junction. This method of migration seems to be unique and the means by which it is achieved is briefly discussed.

Quantitative analysis throughout pregnancy of intraepithelial large granular and non-granular lymphocyte distributions in the synepitheliochorial placenta of the cow

Intraepithelial lymphocytes are a constant feature of ruminant uterine epithelium. Light microscope quantitation on semithin sections of resin embedded perfused material from pregnant and non-pregnant cows shows that although the proportion of large granular lymphocytes to non-granular lymphocytes in interplacentomal areas increases during pregnancy, the total number of lymphocytes in these areas remains at a similar level. However all types of lymphocytes are eliminated from the caruncular uterine epithelium of the cow by 28 days of pregnancy at the initiation of placentomal development. Despite the subsequent enormous growth in area of this region during pregnancy, no lymphocytes are found in the bovine placentomal cellular uterine epithelium. This pattern is similar to that in the ewe and goat although in these species the placentomal uterine epithelium is modified to maternofetal hybrid syncytial plaques. However, in the deer, a similar cellular placentomal epithelium has numerous intraepithelial lymphocytes and large granular lymphocytes are closely associated with fetomaternal hybrid trinucleate cells formed by binucleate cell migration throughout pregnancy. Cow interplacentomal large granular lymphocytes and trinucleate cells show similar apposition but the formation and fate of cow placentomal trinucleate cells does not involve lymphocytes. Since the caruncular (placentomal) area is 10-20 times that of the intercaruncular this suggests a very different function for intraepithelial lymphocytes in the deer compared with the other ruminant species.

Genetic regulation of placental function: a quantitative in situ hybridization study of calcium binding protein (Calbindin-D9k) and calcium ATPase mRNAs in sheep placenta

The calcium requirement of the ovine fetus increases progressively throughout pregnancy. The 9-kDa calcium binding protein (calbindin-D9k; 9CBP) is considered to be a reliable marker for epithelia mediating calcium transport. This quantitative in situ hybridization study shows that the levels of 9CBP mRNA show a pregnancy stage-related increase which correlates with fetal calcium demand only in maternal endometrial gland and fetal interplacentomal trophoblast epithelia. Levels of 9CBP mRNA in the placentome, which has by far the greater area of maternofetal contact, show no changes during pregnancy. mRNA for the CaATPase enzyme, a second requirement for calcium transport, is shown to be present in epithelia in interplacentomal and placentomal regions but shows no change in concentration as pregnancy progresses. Results with the 9CBP and CaATPase mRNAs confirm our recent immunocytochemical results with ruminant placenta and indicate the basis for a cellular calcium transport system analogous to that in the enterocyte. The interplacentomal trophoblast system appears to be eminently suitable for investigations of details of the cellular mechanism and control of epithelial calcium transport.