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.     

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.     

8-iso-prostaglandin F2alpha as a marker of tissue oxidative damage in bovine retained placenta

Retention of foetal membranes (RFM) in cows is supposed to be associated with the imbalance between production and neutralisation of reactive oxygen species (ROS). The consequence of uncontrolled ROS increase is oxidative damage to tissues, cells, and macromolecules. 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha) is considered as a marker of oxidative tissue damage. The aim of the study was to investigate whether the concentrations of 8-iso-PGF2alpha, in caruncles and cotyledons from the bovine placenta differ between retained and properly released foetal membranes. Placentomes were collected immediately after either spontaneous delivery at term via the vagina or caesarean section before as well as at term through the incision and divided into six groups consisting of eight cows each as follows: A-preterm caesarean section without RFM, B-preterm caesarean section with RFM, C-term caesarean section without RFM, D-term caesarean section with RFM, E-term spontaneous delivery without RFM, F-term spontaneous delivery with RFM. The concentrations of free and total 8-iso-PGF2alpha, were determined in caruncles as well as cotyledons by enzyme immunoassay and expressed in picogram per gram of wet weight of tissue. The concentrations of free and total 8-iso-PGF2alpha were lower (P < 0.05) in cotyledons than in caruncles in all groups examined, as well as they were higher (P < 0.05) in retained than in released placenta. The concentrations of both parameters were lower (P < 0.05) in term spontaneous delivery groups than in term caesarean section groups. The results indicate that oxidative tissue damage, which may be the result of ROS imbalance, appears during RFM. However, the dynamics of this damage requires further elucidation.     

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.     

Chemotactic activity of cotyledons for mononuclear leukocytes related to occurrence of retained placenta in dexamethasone induced parturition in cattle

Induction of parturition with glucocorticosteroids in cattle is used for research purposes, in diseased or injured pregnant cows, and as a management tool to time parturition. A negative side effect of induction of parturition with glucocorticosteroids is the high incidence of retained placenta that occurs after these calvings. Reaction of the maternal immune system against the ‘foreign’ foetal membranes contributes to the breakdown of the foetal-maternal attachment. Several studies indicate that failure of this immune assisted detachment increases the occurrence of retained placenta. We hypothesized that retained placenta occurring after induction of parturition with glucocorticosteroids is caused by failure of immune assisted detachment of the foetal membranes. The chemotactic activity of cotyledons for mononuclear leukocytes was used as a parameter to see whether immune assisted detachment of the foetal membranes had occurred. Cotyledons were collected from spontaneously calving non-retained placenta cows and from dexamethasone induced non-retained placenta and retained placenta cows. The study showed that the chemotactic activity of cotyledons for mononuclear leukocytes was lower (P < 0.001) in cotyledons obtained from retained placenta cows in which parturition was induced with dexamethasone compared to the chemotactic activity of cotyledons obtained from spontaneously calving non-retained placenta cows, whereas the chemotactic activity of cotyledons obtained from induced non-retained placenta cows was not lower (P = 0.10) than the chemotactic activity of cotyledons obtained from spontaneously calving non-retained placenta cows. We concluded that induction of parturition with dexamethasone causes a failure of immune assisted detachment of the foetal membranes and the accompanying release of chemotactic factors. As a result, the chemotactic activity of cotyledons for mononuclear leukocytes is lower in induced retained placenta cows than in cotyledons from non-retained placenta cows in which successful immune assisted detachment of the foetal membranes occurs.     

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.     

Monoclonal antibody (SBU-1 and SBU-3) identification of cells dissociated from the sheep placentomal trophoblast.

We studied the localization of alpha-keratin in the sheep placenta using an alpha-keratin-specific monoclonal antibody (MAb) SBU-1, and examined the feasibility of using this MAb as a marker for determining the purity of isolated uninucleate cells from the placentomal trophoblast. At about 30-50 days of gestation the placentomal and interplacentomal uninucleate cells and some binucleate cells were stained by SBU-1, whereas only the apical region of the syncytial cytoplasm was stained with this MAb. Other cells stained included the uterine and endometrial glandular epithelial cells and fibroblast-like cells in the endometrium and chorionic villi. At about 100-130 days of gestation only the trophoblast uninucleate cells were stained by SBU-1. Approximately 60% of cells isolated from placentomes at 100-130 days of gestation were stained by SBU-1, and they had similar morphological features to the trophoblast uninucleate cells. The number of binucleate cells present was confirmed by their affinity for MAb SBU-3. These results show that MAb SBU-1 is an excellent marker for trophoblast uninucleate cells from placenta of sheep at the later stages of pregnancy.     

Immunohistochemical localization of prostaglandin G/H synthase 1 and 2 in sheep placenta after glucocorticoid-induced and spontaneous labour

Enhanced prostaglandin production and release by the placenta is an essential element in the normal transition to labour in many animal species. In sheep, expression of prostaglandin G/H synthase (PGHS) is the central enzyme regulating this process. In this study immunohistochemistry was used to examine the distribution of cells expressing PGHS-1 and PGHS-2 in ovine placenta in association with spontaneous parturition (n = 6) and glucocorticoid-induced labour (n = 5). Labour was induced in ewes after the intrafetal injection of betamethasone on day 131 of gestation. Animals administered an intrafetal injection of isotonic saline (n = 5) acted as non-labour controls. In placentomes collected from all groups, immunoreactive PGHS-1 was present in the mononuclear trophoblast cells of the fetal placenta. Cells in the maternal mesenchyme and epithelial syncytium were weakly immunopositive for this enzyme. PGHS-1 immunoreactivity was also demonstrated in the endothelial cells of the chorionic vessels. The PGHS-2 isozyme was localized exclusively to the trophoblast epithelial cells. Immunoreactive PGHS-2 was not detectable in the maternal epithelial syncytium or the stroma of the cotyledons. The binucleate cells of the fetal placenta were consistently immunonegative for both PGHS isozymes. These results indicate that the cellular localization of PGHS-1 and PGHS-2 in ovine placenta does not change during the last 15 days of pregnancy. Co-localization of these isozymes indicates that the source of arachidonic acid and the site of prostanoid formation are the same. Quantitation of the percentage area of positive staining for PGHS-1 and PGHS-2 using image analysis software demonstrated a significant increase in PGHS-2 in the fetal trophoblast after glucocorticoid-induced labour and spontaneous parturition. This finding indicates that increased formation of the PGHS-2 isozyme is responsible for the large increase in prostaglandin production by the ovine placenta at term labour.     

Bovine placental prostaglandin synthesis: principal cell synthesis as modulated by the binucleate cell

Bovine placentomes were collected during late gestation, prepartum, and immediately postpartum. Postpartum tissues were collected prior to fetal placental release. A procedure for separating fetal placental principal cells from fetal binucleate cells (BNC) was developed. Dispersed fetal placental cells (mixed types), principal cells, and BNC were each examined for their ability to produce prostaglandins (PGs) from arachidonic acid (AA) and to metabolize prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) in vitro. Dispersed fetal placental cells obtained prepartum produced predominantly PGs of the E series (PGEs) from AA (p less than 0.05). PGE synthesis predominated (p less than .05) in cells from postpartum tissue if the fetal placental membranes were subsequently retained, whereas synthesis of PGs of the F series (PGFs) predominated (p less than 0.05) if the fetal membranes were subsequently released. Principal cells were the primary source of fetal placental PG synthesis from AA (p less than 0.05). BNC exhibited a lesser ability to synthesize PGs from AA (p less than 0.05), but were able to convert PGF2 alpha to PGE2. Dispersed fetal placental cells exhibited greater ability to convert PGF2 alpha to PGE2 (p less than 0.05) than did the separated cells. These data suggest the function of a two-cell system within the fetal villi such that the BNC modulate the output of principal cell PG synthesis and/or metabolism.     

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.     

Incidence of Some Diseases in Connection with Parturition in Dairy Cows

The incidence of dystocia, uterine, prolapse, retained placenta, puerperal metritis and vaginal prolapse was investigated in 283 herds over a period of eight years (about 2,590 cows per year). The incidence of dystocia (0.012 cases per cow and year), of uterine prolapse (0.002) and puerperal metritis (0.002) varied with the monthly variation in parturition. Retained placenta (incidence 0.025) occurred more frequently in March-June relative to parturition. The cows then calving were older than average. Vaginal prolapse (incidence 0.002) was also commoner in the spring months. Herds with a high incidence of retained placenta also showed more mastitis, ketosis and parturient paresis.     

Relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in postpartum cows with dystocia or retained placenta

A study was conducted to investigate the relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in cows that experienced a dystocia or retained their placenta. Fifteen healthy cows, 31 cows with retained placenta (RP) and 13 cows that had dystocia were clinically examined 1 or 2 days after parturition when a uterine swab for bacteriological examination was taken. In addition, plasma and uterine lochia samples were collected to determine lipopolysaccharide (LPS) and the plasma IgG anti-LPS concentrations. Subsequently, 15 RP and 6 dystocia cows were initially left untreated and another uterine swab was collected at 2 and 4 wk postpartum. Immediately after calving, RP cows had significantly higher LPS levels in uterine lochia (average of 2.24 x 10(4) Endotoxin Units (EU)/mL) as compared to dystocia and healthy postpartum cows (average of 0.10 and 0.26 EU/mL, respectively). However, plasma LPS levels were below the detection limit (<0.036 EU/mL platelet-rich plasma) in all groups of cows. IgG anti-LPS levels in plasma were not significantly different between the 3 groups immediately postpartum (average of 26, 16 and 44 Median Units (MU)/mL) for healthy, dystocia and RP cows, respectively), but they were significantly lower when compared to plasma IgG anti-LPS levels of healthy cows at more than 2 months postpartum (mean 83 MU/mL). High LPS levels in lochia at 1 or 2 days postpartum were significantly related to abnormal cervical discharge, the presence of Escherichia coli, black pigmented gram-negative anaerobes and Clostridium spp. shortly after calving, and Arcanobacterium pyogenes and gram-negative anaerobes in the uterus at 14 days postpartum. These results suggest that the presence of E. coli and LPS (endotoxins) in lochia early postpartum favor the development of uterine infections by A. pyogenes and gram-negative anaerobes later postpartum. LPS were not observed in plasma, suggesting that either they are not absorbed into the blood, or they are efficiently detoxified by IgG anti-LPS or other detoxification mechanisms.     

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.     

Dexamethasone inhibition of cyclooxygenase expression in bovine term placenta.

Since both prostaglandin (PG) F2 alpha and corticosteroids are elevated in mammals before the onset of parturition, we studied the effect of the synthetic corticosteroid dexamethasone on PGF2 alpha accumulation and cyclooxygenase (prostaglandin synthase, PGS) expression in the bovine fetal placenta. Cultures were prepared from cotyledons at different stages of gestation. The effect of dexamethasone on PGF2 alpha accumulation and PGS expression was determined by radioimmunoassay and [35S]methionine metabolic labeling followed by immunoprecipitation with specific anti-cyclooxygenase antibodies, respectively. Data demonstrate that in fetal placental cells at term, both PGF2 alpha accumulation and cyclooxygenase expression are significantly inhibited after 18 hours of dexamethasone treatment (150 nM). In contrast, neither first nor second trimester cells were sensitive to dexamethasone treatment. Dexamethasone inhibition of PGF2 alpha synthesis in fetal cells at term was abolished in the presence of RNA or protein synthesis inhibitors (actinomycin D or puromycin, 10 micrograms/ml each). Neither progesterone nor 17 beta-estradiol accumulation were affected by dexamethasone treatment at any stage of gestation. Data suggest that corticosteroids play a role in parturition through PGF2 alpha synthesis regulation by fetal placental cells. Since abnormalities during parturition e.g. retained placenta, are common following dexamethasone induction of labor in cows, we postulate that the local inhibition of PGF2 alpha accumulation by cotyledon cells after corticosteroid administration, may be involved in placental retention.     

Peripartum changes in the bovine placenta related to fetal membrane retention

To understand fetal membrane retention in dairy cattle, we examined these tissues in the immediate peripartum period before tissue separation. Placentomes were collected at 270-280 d of gestation from pre-partum Holstein cows (n = 5) and at 1, 3, 6, and 12 h postpartum from a) cows releasing fetal membranes in less than 12 h (n = 7), b) cows retaining fetal membranes for more than 12 h (n = 5), and c) cows induced to calve with dexamethasone (releasing membranes in more than 12 h; n = 5). Subjective evaluations of necrosis, distribution, and condition of binucleate giant and of principal cells were made. Necrotic foci and binucleate giant cells were counted for each interval for which tissue was available. Necrosis, existing prepartum, was identical to that observed at 1 h postpartum in all treatment groups (P > 0.05). Necrosis gradually increased in all treatment groups with time (P < 0.05); thus necrosis is unrelated to initiation of calving and is indirectly related to fetal membrane retention. Many binucleate giant cells were observed prepartum. In cows releasing fetal membranes normally, a significant (P < 0.05) decline in these cell numbers had occurred by 1 h postpartum. When fetal membrane retention occurred, no such decrease in binucleate giant cells was observed before 12 h postpartum. Loss of binucleate giant cells appears necessary for, or must occur with, separation of fetal membranes.     

Factors related to the etiology of retained placenta in dairy cattle

Birth records of 369 288 calvings of 160 188 Meuse-Rhine-Yssel cows were analysed to assess the influence of factors associated with retained placenta. Special emphasis was placed on the analysis of a subset containing data on births involving a single live calf and an easy or normal calving process. The overall rate of incidence of retained placenta was 6.6%. The rate increased during the years studied. Abortion, stillbirth and multiple birth caused a marked increase in rate, as did difficult calving, caesarean section and fetotomy. After adjusting for these factors, analysis of the corrected subset showed that the rate of incidence increased with age of the dam. Gestation length prior to retention and birth weight were also associated with higher rates. The combination of short gestation length (<270 days) and low birth weight (⩽37 kg) was associated with the highest risk of retained placenta. High birth weights mainly caused higher rates when related to dystocia. The incidence rate in cows delivering a male calf was only slightly higher than in cows delivering a female calf. Cows having retained placenta for a first or second time were three and six times, respectively, as likely to do so again at a subsequent parturition when compared with cows which had not had retained placenta previously.     

Insulin-like growth factor-I and binding proteins 1, 2, and 3 in bovine nuclear transfer pregnancies

In cloned pregnancies, placental deficiencies, including increased placentome size, reduced placentome number, and increased accumulation of allantoic fluid, have been associated with low cloning efficiency. To assess differences in paracrine and endocrine growth regulation in cloned versus normal bovine placentomes and pregnancies, we have examined the expression of insulin-like growth factor (IGF)-I and -II and their binding proteins (IGFBP)-1 through -3 in placentomes of artificially inseminated (AI), in vitro-produced (IVP), and nuclear transfer (NT) pregnancies at Days 50, 100, and 150 of gestation. Fetal, maternal, and binucleate cell counts in representative placentomes were performed on Days 50-150 of gestation in all three groups. Increased numbers of fetal, maternal, and binucleate cells were present in NT placentomes at all stages of gestation examined. Immunolocalization studies showed that spatial and temporal patterns of expression of IGFBP-2 and -3 were markedly altered in the placentomes of NT pregnancies compared to AI/IVP controls. Concentrations of IGF-I in fetal plasma, as determined by RIA, were significantly higher (P = 0.001) in NT pregnancies (mean +/- SEM, 30.3 +/- 2.3 ng/ml) compared with AI (19.1 +/- 5.5 ng/ml) or IVP (24.2 +/- 2.5 ng/ml) pregnancies on Day 150 of gestation. Allantoic fluid levels of IGFBP-1 were also increased in NT pregnancies. These findings suggest that endocrine and paracrine perturbations of the IGF axis may modulate placental dysfunction in NT pregnancies. Furthermore, increased cell numbers in NT placentomes likely have significant implications for fetomaternal communication and may contribute to the placental overgrowth observed in the NT placentomes.     

The levels of lipid peroxidation products in bovine retained and not retained placenta

Unsaturated fatty acids undergo peroxidation processes. The presence of elevated levels of metabolites of lipid peroxidation processes may cause alterations in metabolic pathways which may lead to disturbances and clinical symptoms of illnesses. One such illness may be retention of fetal membranes (RFM) in cows. The aim of the study was the determination of thiobarbituric acid (TBA) reactive substances, conjugated dienes and hydroperoxides in bovine retained and not retained placenta in order to describe the oxidative status of cows affected with RFM. Placental samples were collected immediately after spontaneous delivery or caesarian section before term and at term and divided into 6 groups, with 16 animals in each group, as follows: A) caesarian section before term without RFM: B) caesarian section before term with RFM: C) caesarian section at term without RFM: D) caesarian section at term with RFM: E) spontaneous delivery at term without RFM; F) spontaneous delivery with RFM. The levels of TBA reactive substances, conjugated dienes and hydroperoxides were measured spectrophotometrically. The levels of all parameters were statistically significantly higher in the maternal than in the fetal part of placenta and the lowest in preterm groups. Statistically significantly higher concentrations in retained placenta samples than in control animals were observed. In conclusion, the metabolites of lipid peroxidation processes are elevated in cases of retained placenta and their presence might influence directly and/or indirectly the improper release of fetal membranes in cows.