Comparative development of ruminant placentomes

Histological examination of placentomes from cows, sheep, deer, and several antelope species revealed a common pattern of development of the utero-placental junction. Chorionic membrane in contact with the uterine caruncles developed "milky patches" composed of a thick trophoblastic epithelium and multiple allantoic blood vessels, while caruncles formed simultaneously a network of crypts. The milky patches formed chorioallantoic villi that penetrated into the caruncular crypts usually simultaneously with both the villi and crypt formation but partial delay between the villi/crypt formation and penetration had no apparent detrimental effect on the fetus. The villi penetrated into caruncles in a row until they reached the dense basal layer separating caruncular mass from adjacent glandular endometrium. Further placentome growth continued by increasing the length, diameter, branching, and surface corrugation of the villi. Placentomes in different stages of development coexisted at different locations within the uterus throughout the pregnancy. During placental release after parturition, entire villi or only the villi mainstems can pull out of the maternal crypts, or the entire placentome mass can separate from the uterine wall. The remaining maternal portions of the placentomes are destroyed and sloughed down to the basal layer, leaving only a narrow band of the caruncular tissue for the regeneration of caruncles. The bare, wrinkled caruncular surface is then covered with a new epithelium and ultimately becomes smooth.     

Expression of major histocompatibility complex antigens on the bovine placenta

Immunohistochemistry was utilized to determine expression of the major histocompatibility complex (MHC) antigens on Day 8-9 hatched blastocysts and fetal membranes of mid- to late gestation cows and to examine the pattern of leucocytic infiltration into the gravid uterus. Hatched blastocysts were weakly positive for MHC class I antigens. In the mature placenta, chorioallantoic membranes in the interplacentomal area showed positive immunostaining for class I antigens on the chorionic epithelium but had no staining for class II antigens. There was an accumulation of lymphoid cells expressing class II antigens directly beneath the luminal epithelium of the endometrium. In addition, cells staining for leucocyte common antigen were present both within and beneath the luminal epithelium. Some cells positive for class II and leucocyte common antigen (CD45) were also associated with uterine glands. In the placentomes, class I antigens were expressed only on maternal caruncular septa. Fetal cotyledonary villi had no detectable immunostaining for class I and II antigens. No distinct pattern of leucocyte infiltration in the maternal caruncular tissue was observed; the caruncular septa contained some cells that were labelled for CD45 and a few class II-positive cells around blood vessels. The results indicate that the fetal placenta of the cow expresses MHC class I antigens in a regionally defined manner and there is a differential accumulation of lymphoid cells in the uterus.     

Ultrasonic evaluation of uterine involution in Bulgarian Murrah buffalo after administration of oxytocin

The aim of this study was to determine the time taken for complete uterine involution in Bulgarian Murrah buffaloes following normal parturition and oxytocin stimulated milking; and to establish the time course of the change in size of the uterine horns, the cervix and caruncles between parturition and involution by means of ultrasonography. There were 17 animals in the study aged 3-6 years and average parity of 2.17 ± 0.18. They were administered 20 IU oxytocin 15 min before each milking. Rectal palpation and transrectal ultrasonography were performed at 3 d intervals from Days 1 to 34 post partum. The involution of the non-gravid and gravid uterine horns, and the cervix was complete by Days 22 and 25 post partum when their diameters were 2.7 ± 0.4 cm, 2.8 ± 0.3 cm and 3.12 ± 0.4 cm, respectively. Caruncles underwent rapid regression until Day 10 post partum. It was not possible to determine the dimensions of the caruncles after that time. The cumulative percentage of animals whose uterus was located in the pelvic cavity increased from 24% at Day 10 post partum to 100% at Day 34 post partum. The combination of rectal palpation and transrectal ultrasonography provided a reliable method of evaluating changes in the uterus over time and determining the time of uterine involution. The present study showed that complete uterine involution, with the uterus located in the pelvic cavity, was achieved by Day 34 after parturition in all 17 Bulgarian Murrah buffaloes treated with oxytocin before milking.     

Angiogenesis and morphometry of bovine placentas in late gestation from embryos produced in vivo or in vitro

The objective of this study was to determine the effects of in vitro embryo production on angiogenesis and morphometry of the bovine placenta during late gestation. Blastocysts produced in vivo were recovered from superovulated Holstein cows. Blastocysts produced in vitro were obtained after culture of in vitro-matured and -fertilized Holstein oocytes. Single blastocysts from each production system were transferred into heifers. Fetuses and placentas were recovered on Day 222 of gestation (in vivo, n=12; in vitro, n=12). Cotyledonary and caruncular tissues were obtained for quantification of vascular endothelial growth factor (VEGF) and peroxisome proliferator-activated receptor-gamma (PPARgamma) mRNA and protein. Tissue sections of placentomes were prepared for morphometric analysis. Fetuses and placentas were heavier from embryos produced in vitro than from embryos produced in vivo. More placentas from embryos produced in vitro had an excessive volume of placental fluid. There was no effect of treatment on the expression of mRNA for VEGF and PPARgamma in either cotyledonary or caruncular tissues. The expression of VEGF protein in cotyledons and caruncles as well as the expression of PPARgamma protein in cotyledons were not different between the in vitro and in vivo groups. However, caruncles from the in vitro group had increased expression of PPARgamma protein. The total surface area of endometrium was greater for the in vitro group compared with controls. In contrast, the percentage placentome surface area was decreased in the in vitro group. Fetal villi and binucleate cell volume densities were decreased in placentomes from embryos produced in vitro. The proportional tissue volume of blood vessels in the maternal caruncles was increased in the in vitro group. Furthermore, the ratios of blood vessel volume density-to-placentome surface area were increased in the in vitro group. In conclusion, these findings are consistent with the concept that compensatory mechanisms exist in the vascular beds of placentas from bovine embryos produced in vitro.     

Morphometric analysis of collagen in gestational and retained bovine placentomes

Bovine placentome collagen was quantified (P<0.01) at four gestational stages (90, 150, 210 and 270 d, n = 8 d ), at 2 h post partum without (n = 4) and at 2 and 12 h post partum with (n = 8) experimentally-induced placental retention. Placentome sections were fixed and stained for collagen. Fetal cotyledonary (FC) collagen volume fraction (V(V)) increased over days of gestation studied (V(V)=0.03+/-0.01, 0.06+/-0.01, 0.13+/-0.01 and 0.19+/-0.01). Fetal cotyledonary hydroxyproline (3.15+/-0.41, 4.55+/-0.41 and 7.04+/-0.41 mg/g) and FC protein (432.0+/-17.1, 479.9+/-17.1, 585.4+/-17.1 mg/g) increased over Days 90, 150 and 210 and were similar on Days 210 and 270. Fetal cotyledonary collagen V(V) and hydroxyproline did not differ between Day 270, retained and nonretained cotyledons. Protein concentration was higher in 2 h (578.1+/-18.5 mg/g) and 12 h (526.0+/-18.5 mg/g) retained versus nonretained (400.4+/-36.2 mg/g) cotyledons. Maternal caruncular (MC) collagen V(V) and protein concentration were higher on Days 90 and 150 than on Days 210 and 270. Maternal caruncular hydroxyproline was similar from Day 90 to 210 and increased from Day 210 to 270. Maternal caruncular collagen V(V), hydroxyproline and protein concentrations were similar on Day 270 and in 2 h and 12 h retained membrane caruncles. Gestational increases in placentome collagen occurred from FC sources. No difference in FC or MC collagen V(V) existed between Day 270, retained and nonretained placentomes.     

The expression of Fas/FasL and apoptosis in yak placentomes

To clarify the status and distribution of Fas and Fas-Ligand (FasL) in yak's placentomes, immunohistochemistry (IHC) was carried out to analyze the expression and location of Fas and FasL in paraffin embedded sections. The area of positive stained sites was selected and measured using image analyses software (Image Pro-Plus 6.0). So the positive index (PI) was calculated to estimate the intensity of protein expression according to the percentage of positive area in corresponding compartment of the placentomes. In cotyledonary villi, Fas mainly presented on the villous trophoblast cells in early pregnancy. The positive index reached a maximum of 20.7±8.8 at the third month of pregnancy. Then Fas was declined rapidly along with the progress of gestation and the value was 2.8±1.3 after the 7th month of pregnancy. However, in caruncular crypts, Fas was mainly localized to isolated cells or clustered cells of the uterine stroma underlying the caruncular epithelium. The intensity was lower and the positive index was changed between 4.7±0.9 and 8.5±1.6 throughout gestation. For FasL, it gave a distinct immunostained distribution. In cotyledonary villi, FasL was localized dominantly and strongly in the cytoplasm of binuclear, mononuclear and trinuclear trophoblast giant cells (TGC). The positive index of FasL maintained a moderate level all through the gestation. In caruncular crypts, the expression of FasL was weak and the positive index was declined. Only in the first two months, maternal uterine epithelial cells intensely expressed FasL and the index reached to the maximum of 19.8±5.2. The result of subcellular localization of Fas ligand using immunoelectron microscopy technology indicated that FasL was subcellular located in some intracellular vesicles of TGC. This means the vesicles of trophoblast giant cells itself can express FasL. By the TUNEL method, apoptosis was detected in yak placentomes. The amount of apoptotic cells was rare. The fetal chorionic trophoblast cells and caruncular crypt epithelium cells demonstrated higher percentage of apoptosis in middle pregnancy, which suggested that apoptosis plays an important role in placental cellular regeneration. In addition, the apoptosis of maternal caruncular stromal cells provides a local mechanism for maternal immunotolerance to the fetus and this mechanism was mediated by Fas-FasL pathway.     

Expression of prostaglandin transporter in the bovine uterus and fetal membranes during pregnancy

Uteroplacental prostaglandins (PGs) play pivotal roles in the maintenance and termination of pregnancy in mammals. In the present study, we have characterized the expression of prostaglandin transporter (PGT) in placentome caruncles, intercaruncular tissues, fetal membranes, and utero-ovarian plexus during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. In caruncles and intercaruncular tissues, PGT mRNA (also known as SLC02A1) and PGT protein were highly expressed at the late stage of pregnancy compared to the early and mid stages, whereas the level of expression is constant and low in fetal membranes throughout pregnancy. PGT mRNA and PGT protein were expressed at a constant level in the utero-ovarian plexus both ipsilateral and contralateral to corpus luteum throughout the course of pregnancy. Overall, the relative expression of PGT mRNA and PGT protein were higher in caruncles than in intercaruncular tissue and fetal membranes, whereas no differences were detected between intercaruncular tissues and fetal membranes at any stage of gestation. Immunohistochemistry indicated that PGT was preferentially expressed in caruncular epithelial cells of placentomes and endometrial luminal epithelial and myometrial smooth muscle cells of the intercaruncular regions. The level of PGT expression was comparatively higher in maternal components than in fetal components. In conclusion, differential spatiotemporal tissue-specific expression of PGT in uterine and intrauterine tissues suggests a role for this transporter in the exchange of PGs between the maternal and the fetal compartments, as well as for intrauterine metabolism of PGs during pregnancy.     

Immunolocalization of progesterone receptors in bovine placentomes throughout mid and late gestation and at parturition.

The corpus luteum is the main source of progesterone (P(4)) responsible for maintenance of gestation in cattle. So far it has not been possible to assign any biological role to placental P(4), which contributes only marginally and temporarily to peripheral maternal blood levels. In order to identify possible P(4) target cells within the placenta, placentomes from 150-, 220-, 240-, and 270-day-pregnant cows and from parturient cows (3 animals per group) were screened immunohistochemically for expression of the progesterone receptor (PR). During gestation, PR-positive staining was found exclusively in the nuclei of caruncular stromal cells (CSC; maternal part of the placentome) and of caruncular vascular pericytes. In placentomes from parturient cows, occasional positive nuclear staining was also observed in the walls of small caruncular arteries. The percentage of PR-positive CSC increased slightly from 51.8 +/- 2.6% on Day 150 to 56.2 +/- 5.6% at Day 270 (p < 0.05) and was 58.9 +/- 1.8% at parturition. These results suggest that in pregnant cattle, CSC are under the control of P(4) of placental rather than luteal origin. Thus, whereas luteal P(4) may regulate "coarse" systemic progestational functions in the maternal compartment in the classical hormonal manner, placental P(4) may act as a paracrine factor involved in the local regulation of caruncular growth, differentiation, and functions.     

Platelet-activating factor receptor (PAF-R) and acetylhydrolase (PAF-AH) are co-expressed in immature bovine trophoblast giant cells throughout gestation, but not at parturition

Platelet-activating factor (PAF) was associated with successful implantation in the cow, trophoblast invasiveness and angiogenesis. Bovine placentation is characterized by the limited invasion of trophoblast giant cells (TGC) into the maternal caruncular epithelium. TGC exhibit both endocrine activity and properties of tumor cells and may thus be targets of and mediators for the action of PAF. We examined PAF-receptor (PAF-R) and PAF-acetylhydrolase (PAF-AH) gene expression and localized mRNA and corresponding proteins in bovine placentomes throughout gestation and at parturition. PAF-R and PAF-AH protein and mRNA were highly expressed and colocalized in immature TGC from early gestation until near term, while mature TGC were negative. After the onset of parturition both PAF-R and PAF-AH were expressed in the maternal stroma, predominantly endothelial cells. The expression of PAF-R and PAF-AH in immature but not mature TGC during gestation implicates a role for PAF in the differentiation, maturation and function of bovine placentomal TGC. Placentomal angiogenesis could be mediated by binding of PAF to PAF-R present in endothelial cells. The parturition-related "switch" of PAF-R and PAF-AH from TGC to the maternal stroma suggests that PAF may participate in the regulation of parturition and in prepartum tissue programming.     

Stromal-to-Epithelial Transition during Postpartum Endometrial Regeneration

Endometrium is the inner lining of the uterus which is composed of epithelial and stromal tissue compartments enclosed by the two smooth muscle layers of the myometrium. In women, much of the endometrium is shed and regenerated each month during the menstrual cycle. Endometrial regeneration also occurs after parturition. The cellular mechanisms that regulate endometrial regeneration are still poorly understood. Using genetic fate-mapping in the mouse, we found that the epithelial compartment of the endometrium maintains its epithelial identity during the estrous cycle and postpartum regeneration. However, whereas the stromal compartment maintains its identity during homeostatic cycling, after parturition a subset of stromal cells differentiates into epithelium that is subsequently maintained. These findings identify potential progenitor cells within the endometrial stromal compartment that produce long-term epithelial tissue during postpartum endometrial regeneration.     

Light and electron microscopic immunolocalization of bovine pregnancy-associated glycoprotein in the bovine placentome.

A bovine pregnancy-associated glycoprotein (bPAG) of 67 kDa has previously been isolated from bovine fetal cotyledons. The objective of this study was to determine the cytological localization of that protein in the placentomes and possibly the cells responsible for its production. Highly specific antisera raised against pure bPAG were used to demonstrate the cellular localization of the protein in bovine placentomes by light and electron microscopic techniques. Strong immunostaining was observed exclusively in the cytoplasm of large binucleate cells present predominantly in fetal cotyledonary tissue (villi). Some smaller weakly immunostained cells were also present in caruncular epithelium. By ultrastructural immunogold procedures, the protein was detected only within amorphous cytoplasmic granules. Granules of identical size, but weakly labeled, were found on the maternal side. All cells containing labeled granules were binucleate. These results suggest that bPAG is probably synthesized by trophoblast binucleate cells and stored in granules prior to delivery into the maternal circulation after cell migration.     

Validation of primary epitheloid cell cultures isolated from bovine placental caruncles and cotyledons

In order to study feto-maternal interactions in the bovine synepitheliochorial placenta primary cell cultures of both placentomal components throughout pregnancy, namely caruncular epithelial cells and trophoblast cells were developed. The aim of this study was to validate and improve a method to culture caruncular epithelial cells and fetal trophoblast from manually separated placentomes. Prior to seeding the presence of fetal cells in caruncular samples and vice-versa could be demonstrated by the detection of the Y-chromosome via fluorescence in situ hybridization (FISH) provided the fetus was male. Epitheloid shaped cells present in both cultures (cotyledon and caruncle) were characterized on a morphological basis as well as by immunofluorescence and Western blot thereby detecting cytokeratin, zonula occludens-1 and vimentin but not alpha-smooth muscle actin and desmin. The absence of the Y-chromosome demonstrated the caruncular origin of epitheloid cells. In addition, a population of polygonally shaped cells derived from the cotyledon was propagated and displayed the same cytoskeletal characteristics as described above. The presence of the Y-chromosome confirmed the fetal origin of these cells and the lacking uptake of fluorescence conjugated low density lipoprotein, specific for endothelial cells, identified polygonally shaped cells as fetal trophoblast cells. In conclusion, the cross-contamination of maternal and fetal cells in manually separated placentomes should be considered in future experiments as it may lead to false positive results dependent on the sensitivity of the method applied. This study highlights the importance of an appropriate cell characterization and identification, especially when isolating primary cells.     

Calbindin-D9k expression in the pregnant cow uterus and placenta

Calbindin-D_9k (CaBP_9k) is a vitamin D-dependent, calcium binding protein first identified in the cytoplasm of the intestinal epithelial cell. Using biotin-streptavidin immunohistochemistry, CaBP_9k was localized to the maternal caruncular epithelium, fetal chorionic epithelium, and trophoblastic binucleated cells of the bovine placenta. Within the maternal epithelium the intensity of staining increases from second trimester pregnancies to term pregnancies, indicating a higher intracellular concentration of CaBP_9k in the epithelium at term. Luminal and glandular epithelium of the non-caruncular endometrium also stained positively for CaBP_9k in all stages of pregnancy observed. No CaBP_9k was identified within the stroma or myometrium of the pregnant cow uterus. The increased level of CaBP_9k in the caruncular epithelium during the last trimester is hypothesized to be in response to the rising demand for calcium to aid in the mineralization of the fetal skeleton. CaBP_9k may play a role in enhancing calcium transport across the placenta in cattle.     

The role of endogenous estrogens in the maturation process of the bovine placenta

The plasma estrogen and progesterone concentrations of 19 pregnant cows (average duration of pregnancy 266.0 +/- 2.3 d at the start of the study) were determined daily from Day 6 pre partum to Day 1 post partum. Parturition was induced in all cows by administration of 10 mg i.m. flumethasone. Values were centered around the delivery date (Day 0) following either induced normal calving (n = 3) or surgical delivery (n = 16). In animals showing spontaneous expulsion of the fetal membranes (Group 1, n = 6) the average total estrogen concentration increased significantly from Day 6 until Day 1 before parturition (1329.2 +/- 317.9 to 3719.8 +/- 951.2 pg/ml in total estrogens). A marked decrease was observed on Day 1 post partum (459.4 +/- 344.2 pg/ml). In comparison with Group 1, animals showing either a delayed or partial expulsion of the fetal membranes, or in which the placenta could be withdrawn 16 h after calving (Group 2, n = 5), had consistently lower total estrogen concentrations between Day 6 (595.4 +/- 174.8 pg/ml) and Day 1 (1884.3 +/- 565.1 pg/ml) before parturition. The estrogen values of the cows with retained placenta (Group 3, n = 8) from Days 6 to 0 pre partum were significantly lower than those of Group 2. Total estrogen concentrations of the three groups 1 d post partum did not differ significantly. It is generally recognized that estrogens play an important role in the maturation process of the placentomes. Our investigation demonstrates that not only is the magnitude of the prepartum rise in estrogens of great influence of the maturation process but the duration of this rise is likewise important. These two factors are vital for a normal expulsion of the fetal membranes.     

Histopathology of retained bovine fetal membranes

Placentomes were obtained from 20 cows with retained placenta (9 following normal birth, 5 after abortion and 6 with dystocia), and this material was examined by light microscopy. Histologic changes that were consistently seen in placentomes of cows with retained placenta after normal birth included vascular changes (edema, thrombosis and vasculitis) and the presence of numerous clumps of bacterialcolonies in the connective tissue of the caruncles and cotyledons. Only a few binucleate cells were seen in these cases. In placentomes obtained from cows with retained placenta after abortion, there were instances of focal necrosis of the fetal villi and the presence of variable numbers of binucleate cells. Vascular changes and numerous clumps of bacterial colonies in the caruncles and cotyledons were also noted. The changes in placentomes obtained from cows with retained placenta and dystocia included the presence of numerous binucleate cells, infiltration of polymorphonuclear cells in the connective tissue of both the fetal and maternal villi, vascular changes, and the presence of extensive necrosis and numerous clumps of bacterial colonies. Significant differences (P < 0.05) were encountered in the number of binucleate cells in the various groups. Binucleate cells appear to be involved in the process of placental separation in cows with retained placenta.     

DNA content and the endometrial cellular mitotic activity in the phase of menstrual bleeding

Endometrium obtained during menses from 46 healthy women in reproductive age was investigated morphologically and cytospectrophotometrically in order to solve the problem on the source of the cells reepithelizing the uterine mucous membrane after desquamation. It was stated that desquamation takes place not in the whole functional layer of endometrium, some mucous fragments, covered with persisting luminal epithelium, are always preserved. During endometrial regeneration the cells of the luminal and glandular epithelia and those of endometrial stroma are predominantly diploid. The amount of premitotic cells in population is so small that they cannot secure any intensive cellular proliferation. Mitogenesis in endometrium is stimulated only after a complete restoration of the epithelial layer. It is suggested that persisting luminal epithelium is the source of cells for reepithelization; they migrate towards endometrial "wounds" and repair defects in the uterine mucosa during the regeneration phase.     

Immunohistochemical localization of prostaglandin H synthase in the sheep placenta from early pregnancy to term.

Prostaglandin H synthase (PGHS) activity within intrauterine tissues is considered to catalyze a critical step in prostaglandin (PG) biosynthesis at parturition. In sheep, the placenta is a major site of PG production throughout pregnancy, but little information is available concerning the cells that are responsible. Therefore we determined the distribution of immunoreactive (IR-) PGHS in ovine placental tissue obtained at different times of pregnancy using immunohistochemical techniques. In placentomes from early pregnancy (Days 30-54), IR-PGHS was present in maternal epithelial syncytium, but was not detectable in trophoblast cells. Between Day 54 and Day 100, the number of cells that stained positive for PGHS declined in the maternal epithelial layer in the body of the placenta, but IR-PGHS was present in maternal epithelial cells overlying the vascular cones of the placental hemophagous zone. It was also present in the chorionic fibroblasts, but remained undetectable from all classes of trophoblast cells. IR-PGHS was first detectable in the trophoblastic epithelium by Day 114. Between Day 119 and term the trophoblast mononuclear epithelial cells were intensely immunopositive for PGHS, although immunonegative binucleate cells were present. The maternal epithelium was immunonegative except during the last 7-10 days of pregnancy when PGHS immunostaining appeared in both basal and apical regions of the placenta. Thus, the cellular localization of IR-PGHS changes during ovine pregnancy, from predominantly maternal during the first half of gestation to undetectable and then to predominantly trophoblastic between Day 114 and term, suggesting a gestation-dependent change in sites of PG production during ovine pregnancy. Appearance of IR-PGHS in the trophoblast precedes activation of the fetal hypothalamic-pituitary-adrenal axis, generally considered to provide the trigger to the onset of parturition in sheep, and would therefore appear to be regulated through alternative pathways or mechanisms.     

Comparative histology of antelope placentomes

The histological structure of ruminant (family: Bovidae) placentomes in eight antelope species was compared to that of domestic cattle and sheep. The chorioallantoic villi differed in degree of branching, surface corrugation, and complexity of utero-placental junction. All species had the epitheliochorial type of placenta, with the epithelial lining of maternal caruncular crypts varying between cellular and syncytial types.     

Characterization of glycoconjugates in the bovine endometrium and chorion by lectin histochemistry

Lectin binding patterns were examined on normal bovine endometrium and bovine placentomes during four stages of pregnancy using 13 biotinylated lectins. Lectin binding intensity increased in early pregnancy for many lectins, whereas binding to fucosyl residues decreased. Persistence of strong lectin binding later in pregnancy usually was limited to the arcade and intercotyledonary trophoblastic cells. Binding of some lectins to cell surfaces was prominent, particularly in early pregnancy. A few lectins were excellent markers for binucleate trophoblastic cells. These distinctive surface and binucleate cell binding patterns on placentomes and endometrial epithelium are useful markers of trophoblastic cell-endometrial epithelial cell surface interactions and of binucleate cell differentiation.