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.     

Expression of matrix metalloproteinase (MMP)-2, MMP-14 and tissue inhibitor of matrix metalloproteinase (TIMP)-2 during bovine placentation and at term with or without placental retention

Matrix metalloproteinases (MMPs) and counteracting tissue inhibitors of metalloproteinases (TIMPs) are balancing extracellular matrix (ECM) formation and degradation. The latter is believed to be an important aspect for the detachment of fetal membranes postpartum when loosening the feto-maternal connection which is a prerequisite to avoid placental retention a common disease in cows leading to considerable economic loss. Membrane-type (MT) MMPs have been suggested as potential activators controlling ECM remodelling. In particular, MT1-MMP (MMP-14) is able to degrade ECM substrates and activate MMP-2 through binding TIMP-2 at the cell surface. Since the connection between the trophoblast and the maternal caruncular epithelium is supported by integrin receptors bound to ECM, we hypothesize that impaired modulation of the ECM by TIMPs/MMPs participates in the aetiology of bovine retained fetal membranes. To analyse this involvement, placentomes were collected from cows after term parturition and timely release of fetal membranes (n = 4) and cows with retained fetal membranes after various treatments for the induction of parturition using progesterone antagonist (aglepristone), PGF_2α analogue, glucocorticoid, and after elective caesarean sections (each group n = 3). The expression of MMP-14, MMP-2 and of TIMP-2 was examined by real-time-PCR, immunohistochemistry, Western blot and zymography. The relative mRNA expression levels of MMP-14 remained unchanged, while the expression levels of TIMP-2 and MMP-2 partly increased in animals with induced parturition and retention of fetal membranes compared to animals without placental retention. MMP-14 protein was expressed in cells of the uninucleated trophoblast, the fetal mesenchyme and maternal stroma. TIMP-2 was present exclusively in trophoblast giant cells, while MMP-2 could be detected in uninucleated trophoblast cells and the fetal mesenchyme. The presence of the activated enzyme was confirmed by zymography. In conclusion, MMP-14, MMP-2 and TIMP-2 are co-localized in the fetal compartment and therefore could influence the timely release of fetal membranes in cattle.     

Implantation and placental development in somatic cell clone recipient cows

Successful somatic cloned animal production has been reported in various domesticated species, including cattle; however, it is associated with a high rate of pregnancy failure. The low cloning yield could possibly arise from either an abnormal and/or poorly developed placenta. In comparison to control cows, fewer placentomes were found in somatic cell nuclear recipient (NT) cows at day 60 of gestation, suggesting a retardation of fetal/placental growth in these animals. NT cows not only had fewer numbers of chorionic villi but also had poorly developed caruncles. Macroscopic examination revealed atypical development of the placentome in terms of shape and size. Histological disruption of chorionic villi and caruncular septum was found in NT cows. Of particular interest was that the expression of genes, as well as proteins in the placentome, was disparate between NT and artificially inseminated cows, especially placental lactogen (PL) and pregnancy-associated glycoprotein (PAG). In contrast, prolactin-related protein-1 (PRP-1) signals were comparable across cows, including NT cows carrying immotile fetuses. The expression of extracellular matrix degrading molecule, heparanase (HPA), in NT cows was divergent from that of control cows. Microarray data suggest that gene expression was disorientated in early stages of implantation in NT cows, but this was eliminated with progression of gestation. These findings strongly support a delay in trophoblast development during early stages of placentation in NT cows, and suggest that placental specific proteins, including PLs, PAGs, and HPA, are key indicators for the aberration of gestation and placental function in cows.     

Involution and regeneration of the endometrium following parturition in the ewe

Involution and regeneration of the endometrium after parturition in the ewe, were studied by light- and electron microscopy. The luminal epithelium in intercaruncular regions of the endometrium remained intact at all stages, but degeneration and death of many glandular epithelial cells were observed on the day after parturition. Glandular regeneration had commenced by 8 d post partum, and the glands were substantially regenerated by 15 d. Caruncular epithelial cells on the maternal side of the placentomes, between the bases of the maternal septa, persisted during the period of degeneration of the foetal and maternal tissues of the placentomes. Epithelial cells from this source contributed to the regeneration of the caruncular epithelium following shedding of plaques of degenerate placental tissue from the caruncles, which commenced after 8 d and was completed before 31 d. Thus, ingrowth of epithelium from the edges of the caruncles, as previously proposed, was not the sole source of new caruncular epithelium. The additional source of regenerating epithelium identified here may account for the rapidity with which epithelium appears in the centres of some caruncles, several millimetres in diameter, during endometrial regeneration. However, in some caruncles, regeneration of the epithelium was not completed until after 31 d post partum.     

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.     

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.     

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.     

TRPV6 and Calbindin-D9k-expression and localization in the bovine uterus and placenta during pregnancy

ABSTRACT: BACKGROUND: Transient receptor potential channel type 6 (TRPV6) and Calbindin-D9k (CaBP-9 k) are involved in the active calcium (Ca2+) transport mechanism in many tissues including placenta and uterus, suggesting a role in the establishment and maintenance of pregnancy. Moreover, TRPV6 and CaBP-9 k seem to support the materno-fetal Ca2+ transport that is crucial for fetal Ca2+ homeostasis, bone growth and development. However, it is unknown if these proteins are also involved in the aetiology of pathologies associated with parturition in cows, such as retained fetal membranes (RFM). The aim of the current study was to create an expression profile of uterine and placentomal TRPV6 and CaBP-9 k mRNAs and proteins during pregnancy and postpartum in cows with and without fetal membrane release. METHODS: Uteri and placentomes of 27 cows in different stages of pregnancy and placentomes of cows with and without RFM were collected. Protein and mRNA expression of TRPV6 and CaBP-9 k was investigated by real-time PCR, immunohistochemistry and Western blot. RESULTS: In the uterine endometrium, highest TRPV6 and CaBP-9 k expression was found in the last trimester of pregnancy, with a particular increase of protein in the glandular epithelium. In the placentomes, a gradual increase in TRPV6 mRNA was detectable towards parturition, while protein expression did not change significantly. Placentomal CaBP-9 k expression did not change significantly throughout pregnancy but immunohistochemistry revealed an increase in staining intensity in the maternal crypt epithelium. Immunohistochemical, stronger placental CaBP-9 k signals were seen in animals with RFM compared to animals with an undisturbed fetal membrane release, while protein levels, measured by Western blot analyses did not change significantly. CONCLUSIONS: The results of the present study demonstrate a dynamic expression of TRPV6 and CaBP-9 k during pregnancy in the bovine uterine endometrium and placentomes, suggesting a functional role for these proteins in Ca2+ metabolism during pregnancy. The temporal and spatial expression patterns indicate that TRPV6 and CaBP-9 k may be involved in materno-fetal Ca2+ transport, mainly through an interplacentomal transport, and that both proteins may participate in physiological processes that are crucial for fetal and placental development. However, neither TRPV6 nor CaBP-9 k seem to be causative in the retention of fetal membranes.     

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.     

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.     

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.     

Effect of morphology on placentome size, vascularity, and vasoreactivity in late pregnant sheep

Ovine placentomes vary in shape, with type A placentomes being concave, type D convex, and types B and C intermediate in morphology. It has been speculated that as placentomes advance in type they differ in vascularity and nutrient transport capacity. Our objective was to determine cellularity and vascularity measurements, angiogenic factor expression, and arterial vasoactivity within different morphologic types of placentomes. On Day 130 of gestation, placentomes were collected from multiparous ewes (n = 38) and were evaluated for size, cellularity estimates, angiogenic factor mRNA expression, capillary vascularity (capillary size, capillary surface density [CSD], capillary number density [CND], and capillary area density [CAD]), and vasoreactivity to potassium chloride and angiotensin II. The average weight and size of type A and B placentomes were less (P < 0.01) than those of type C and D placentomes. Placentome morphology did not affect (P > or = 0.24) cotyledonary or caruncular cellularity estimates or percentage of cellular proliferation. Placentome morphology affected (P > or = 0.41) neither caruncular CAD, CND, CSD, or capillary size nor cotyledonary CND, CSD, or capillary size. Cotyledonary CAD was increased (P < 0.01) in type B and D placentomes compared with type A placentomes. Furthermore, placentome type did not affect (P > or = 0.06) angiogenic factor gene expression in the cotyledon or the caruncle. Size, but not morphologic type of placentome, was associated with greater caruncular artery contractility to potassium chloride and angiotensin II (P < 0.01 for both). Placentome size, but not morphologic type, may be important for vascularity and nutrient transfer in the placenta of the pregnant ewe.     

Quantitative analysis of messenger RNA expression of matrix metalloproteinases (MMP-2 and MMP-9), tissue inhibitor-2 of matrix metalloproteinases (TIMP-2), and steroidogenic enzymes in bovine placentomes during gestation and postpartum

The relationship between the mRNA expression of proteolytic and steroidogenic enzymes in bovine placentomes was examined. Caruncle and cotyledon tissues were collected every 6 hr after spontaneous parturition until the fetal membranes were released. Based on the time of fetal membrane release after parturition, the specimens were classified as follows: (1) the early group, in which the fetal membranes were released within 6 hr after parturition; and (2) the late group, in which the fetal membranes were released 6-12 hr after parturition. The placentomes from a slaughterhouse were additionally collected as samples for the examination of enzymes during the gestation period. The mRNA expression of steroidogenic enzymes in the cotyledon was observed to be higher than that in caruncle tissues; however, the mRNA expression patterns of P450scc and StAR tended to be similar in both placental tissues. On the other hand, although the expression levels of TIMP-2 mRNA in both caruncle and cotyledon tissues were similar, during gestation and postpartum the expression levels of MMP-2 and MMP-9 mRNA were approximately 10 times higher in caruncle than in cotyledon tissue. Marked contrasting changes in mRNA expression patterns between pre- and postpartum periods were observed for MMP-2 and MMP-9 in caruncle tissues and for MMP-9 and TIMP-2 in cotyledon tissues. The present study provides the first evidence that MMP-2, MMP-9, and TIMP-2 mRNAs are expressed in bovine placentomes during the gestational and postpartum periods and suggests that these enzymes, in conjunction with steroidogenic enzymes, mediate fetal membrane detachment after parturition.     

Placental synthesis of estrogens at parturition and during placental retention in the cow

Nine cows were submitted to lutectomy at 250 or 270 d of pregnancy and catheters were implanted in the jugular, carotid, uterine artery and uterine vein to determine endocrine changes following lutectomy and throughout parturition. Blood samples were collected at 8-h intervals and assayed for estrogens. Fetal and maternal placental tissues were also collected at parturition and 3 d postpartum for incubation studies on estrogen synthesis. Based on plasma concentrations, the uterus is able to secrete considerable quantities of unconjugated and conjugated estrone (E1) and estradiol 17 μ (E2α) at both 250 and 270 d of gestation. In vitro conversion of androstendione to total estrogens averaged 32.4% and 16.8% for fetal and maternal tissues at parturition, respectively. Incubation of placental tissues collected from animals with placental retention on Day 3 postpartum resulted in conversion of 3.2 and 4.6% of androstendione by fetal and maternal tissues, respectively. One cow which retained the placenta was sampled until 3 d postpartum and assay of the plasma estrogen content indicated that there was always a higher concentration of estrogen in the uterine vein than in the uterine artery, supporting the in vitro incubation data.     

Effects of embryo culture on angiogenesis and morphometry of bovine placentas during early gestation

The objective of this study was to determine the effects of undefined and semidefined culture systems for in vitro embryo production on angiogenesis and morphometry of bovine placentas during early gestation. Blastocysts produced in vivo were recovered from superovulated Holstein cows and served as controls. Blastocysts produced in vitro were exposed to either serum-supplemented medium with cumulus cell coculture (in vitro-produced with serum; IVPS) or modified synthetic oviductal fluid medium without serum or coculture (mSOF). Single blastocysts from each production system were transferred into heifers. Fetuses and placentas were recovered on Day 70 of gestation. Cotyledonary tissues were obtained for quantification of vascular endothelial growth factor (VEGF) and peroxisome proliferator-activated receptor-gamma (PPARG) mRNA and protein. Samples of placentomes were prepared for immunocytochemistry and histological analysis. Placentas from the mSOF group were heavier and had the fewest placentomes, least placental fluid, and lowest placental efficiency (fetal weight/placental weight) compared with the in vivo and IVPS groups. There was no effect of embryo culture system on volume densities of fetal villi or maternal endometrium within placentomes. The volume density of fetal pyknotic cells was increased in placentomes in the mSOF group compared with the in vivo and IVPS groups. Placentomes in the mSOF group had decreased densities of blood vessels and decreased levels of VEGF mRNA in cotyledonary tissue. In conclusion, compared with placentas from embryos produced in vivo or in vitro using an undefined culture system, placentas from embryos produced in vitro using a semidefined culture system exhibited a greater degree of aberrant development of the placenta during early gestation.     

Secretion of angiogenic activity by placental tissues of cows at several stages of gestation

Samples of maternal and fetal placental tissues were obtained from cows on Days 100 (N = 4), 150 (N = 5), 200 (N = 6) and 250 (N = 6) of gestation and incubated for 24 h. Conditioned media from caruncular explants were mitogenic for bovine aortic endothelial cells (BAEC) on all days of gestation. Media from intercaruncular endometrium were stimulatory for proliferation of BAEC on Day 100 but inhibitory on Days 150, 200 and 250. Media from cotyledonary and intercotyledonary tissues inhibited proliferation of BAEC on all days. Caruncular-conditioned media stimulated migration of BAEC on Days 150, 200 and 250. Cotyledonary-conditioned media inhibited migration of BAEC on all days. Effects of media from intercaruncular and intercotyledonary tissues on migration of BAEC varied with stage of gestation. Angiogenic activity of media from caruncular (all stages) and intercaruncular (Day 100) tissues appeared to have an Mr greater than 100,000. In cows, therefore, the maternal placentome (caruncle) appears to be the primary source of placental angiogenic activity throughout gestation. The fetal placentome (cotyledon) secretes activity which inhibits two major components of angiogenesis (proliferation and migration of endothelial cells) throughout gestation. Intercaruncular and intercotyledonary tissues may modulate placental angiogenesis throughout gestation. Placental vascular development in the cow is therefore probably controlled by an interaction between stimulatory and inhibitory factors produced by the placenta itself.     

Progesterone receptors, oestrogen receptor alpha and glucocorticoid receptors in the bovine intercaruncular uterine wall around parturition

The bovine intercaruncular uterine wall expresses steroid hormone receptors throughout pregnancy. Concentrations of specific hormones undergo massive changes during the peripartal period and modulate the synthesis of their own receptors. This is well documented for the placentome, but respective data concerning the intercaruncular uterine wall are completely lacking. Thus, intercaruncular uterine wall segments from cows (I) being 8 and 9 months pregnant (slaughtered cows) and (II) cows undergoing a premature caesarean section 269-282 days after artificial insemination (AI) with (IIa, b) or without (IIc) induction of birth with PGF(2alpha) agonist or (III) receiving a caesarean section during severe dystocia (n=6, 5, 5, 5, 6 and 4 animals, respectively) were studied. In four naturally calving cows (IV) endometrial biopsies were obtained within 30 min after the expulsion of the calf. All tissue probes were fixed for 24h in 4% formaldehyde, routinely embedded in paraffin, and cut at 4 microm. Progesterone receptors (PR), estrogen receptor alpha (ERalpha) and glucocorticoid receptors (GR) were assessed using specific antibodies and staining intensities were documented employing an immunoreactive score (IRS). PR, ERalpha and GR exhibited cell type- and location-specific distribution patterns. IRS for PR and ERalpha did not differ between groups. GR-IRS of endometrial stromal cells, however, were higher in animals undergoing premature caesarean section after induction of birth compared to animals slaughtered during month 8 or 9 of pregnancy or animals receiving caesarean section following dystocia. Results of the present study indicate that steroid hormone receptor amounts within the intercaruncular uterine wall do not (PR, ERalpha) - or in a tissue-specific manner (GR) only - change during the peripartal period, although respective hormones undergo massive changes during this period. This is in strict contrast to the placentome. Comparatively lower local tissue estrogen concentrations around term may be one cause for this difference.     

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.