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.     

Placental growth, angiogenic gene expression, and vascular development in undernourished adolescent sheep

Limiting maternal nutrient intake during ovine adolescent pregnancy progressively depleted maternal body reserves, impaired fetal nutrient supply, and slowed fetal soft tissue growth. The present study examined placental growth, angiogenic gene expression, and vascular development in this undernourished adolescent model at Days 90 and 130 of gestation. Singleton pregnancies were established, and ewes were offered an optimal control (C; n = 14) or low (L [0.7 x C]; n = 21) dietary intake. Seven ewes receiving L intakes were switched to C intakes on Day 90 of gestation (L-C). Fetal body weight (P < 0.01) and glucose concentrations (P < 0.03) were reduced in L versus C pregnancies by Day 130, whereas L-C group values were intermediate. Placental cellular proliferation, gross morphology, and mass were independent of maternal nutrition at both Day 90 and 130. In contrast, capillary area density in the maternal caruncular portion of the placentome was reduced by 20% (P < 0.001) at both stages of gestation in L compared with C groups. Caruncular capillary area density was equivalent in the L and L-C groups at Day 130. Placental mRNA expression of five key angiogenic ligands or receptors increased (P < 0.001) between Days 90 and 130 of gestation. VEGFA mRNA expression was higher (P < 0.04) in L compared with C and L-C pregnancies at Day 130, but otherwise gene expression of the remaining angiogenic factors and receptors analyzed was unaffected by maternal intake. Undernourishing the pregnant adolescent dam restricts fetal growth independently of changes in placental mass. Alterations in maternal placental vascular development may, however, play a role in mediating the previously reported reduction in maternal and hence fetal nutrient supply.     

Placental growth throughout the last two thirds of pregnancy in sheep: vascular development and angiogenic factor expression

Morphometric methodologies were developed and applied to investigate the patterns of vascular development in maternal (caruncular; CAR) and fetal (cotyledonary; COT) sheep placentas throughout the last two thirds of gestation. We also examined the expression levels of the major angiogenic factors and their receptors in CAR and COT sheep placentas. Although the vascularity of the CAR tissues increased continuously from Day 50 through Day 140 of pregnancy, those of the COT tissues increased at about twice the instantaneous rate (i.e., the proportionate increase/day) of the CAR. For CAR, vascularity increased 2-fold from Day 50 through Day 140 via relatively small increases in capillary number and 2- to 3-fold increases in capillary diameter. For COT, the increased vascularity resulted from a 12-fold increase in capillary number associated with a concomitant 2-fold decrease in capillary diameter. This large increase in fetal placental capillary number, which was due to increased branching, resulted in 6-fold increases in total capillary cross-sectional area and total capillary surface, per unit of COT tissue. Different patterns of expression of the mRNAs for angiogenic factors and their receptors were observed for CAR and COT. The dilation-like angiogenesis of CAR was correlated with the expression of vascular endothelial growth factor receptor-1 (FLT1), angiopoietin-2 (ANGPT2), and soluble guanylate cyclase (GUCY1B3) mRNAs. The branching-like angiogenesis of COT was correlated with the expression of vascular endothelial growth factor (VEGF), FLT1, angiopoietin-1 (ANGPT1), ANGPT2, and FGF2 mRNAs. Monitoring the expression of angiogenic factors and correlating the levels with quantitative measures of vascularity enable one to model angiogenesis in a spatiotemporal fashion.     

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.     

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.     

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.     

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.     

Cloned cattle fetuses with the same nuclear genetics are more variable than contemporary half-siblings resulting from artificial insemination and exhibit fetal and placental growth deregulation even in the first trimester

The cloning of cattle by somatic cell nuclear transfer (NT) is associated with a high incidence of abnormal placentation, excessive fluid accumulation in the fetal sacs (hydrops syndrome), and fetal overgrowth. Fetal and placental development was investigated at Day 50, during placentome formation; at Day 100, when placentation was completed; and at Day 150, when the hydrops syndrome frequently develops. The NT fetuses were compared with contemporary half-siblings generated from in vitro-produced embryos or by artificial insemination (AI). Fetal cotyledon formation and vascularization of the chorioallantoic membranes was initiated normally in NT conceptuses, but fewer cotyledons successfully formed placentomes. By Day 100, the mean number of placentomes was significantly lower in surviving NT fetuses. Only those with normal placentome numbers were represented in surviving NT pregnancies at Day 150. The mean total caruncle tissue weight of the placentomes was significantly higher in the surviving NT groups at Days 100 and 150, irrespective of the placentome numbers, indicating that increased NT placental weight was caused by excessive uterine tissue growth. By Day 100, NT fetuses exhibited growth deregulation, and those that survived to Day 150 were 17% heavier than contemporary AI controls. Placentome, liver, and kidney overgrowth accompanied the hydrops syndrome at Day 150. The NT fetal overgrowth was not a consequence of in vitro embryo culture and showed no correlation with placental overgrowth. However, in vitro culture and incomplete reprogramming of the donor genome are epigenetic effects that may override genetic traits and contribute to the greater variability in placental and fetal development in the NT group compared with AI half-siblings.     

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.     

Determination of gestational age in sheep and goats using transrectal ultrasonographic measurement of placentomes

Three experiments were conducted to determine gestational age in the ewe and doe by measuring placentomes with a B-mode ultrasonograph and a 5 MHz transducer. Transrectal measurements were obtained by placing the female over a bale of hay. In Experiment 1, ewes (n = 12) and does (n = 15) were examined by transrectal ultrasonography every week from breeding to parturition to determine the growth pattern of placentomes during pregnancy. In Experiment 2, placentomes from 132 ewes and 169 does were measured between 30 and 90 d of gestation. A linear regression relationship between fetal age in days and placentome size in mm was calculated and adjusted for does (gestational age = 28.74 + 1.80PL + e, r(2) = 70.34) and for ewes (age = 47.98 + 0.62PL + e, r(2) = 15.59). In Experiment 3, the placentomes of 63 does were measured to validate this relationship by using linear regression. Gestational age was determined correctly in 66% of the does, with a range of +/- 7 d and in 96% with a margin of +/- 14 d. In conclusion, transrectal ultrasonography allowed for the measurement of placentome size, which increased rapidly during the first 70 to 90 d of gestation in ewes and does. In ewes, however, there was a poor correlation of placentome size with gestational age, while in goats, measurement of placentomes could be used along with pregnancy diagnosis by transrectal ultrasonography as an indication of gestation age.     

Pregnancy rates and gravid uterine parameters in single, twin and triplet pregnancies in naturally bred ewes and ewes after transfer of in vitro produced embryos

The objectives of this study were to: (1) evaluate the pregnancy rates after transfer of embryos produced in the presence or absence of epidermal growth factor (EGF) during in vitro maturation, and (2) compare several variables of the gravid uterus on day 140 after fertilization in single, twin and triplet pregnancies in ewes (n = 12) bred naturally and in ewes (n = 18) after transfer of embryos produced in vitro. Oocytes collected from FSH-treated ewes (n = 18) were collected from all visible follicles and cultured in maturation medium with or without EGF. Oocytes were then fertilized in vitro by frozen-thawed semen. On day 5 after fertilization, embryos with > or = 16 cells were transferred to recipient ewes (n = 39). In addition 12 ewes were bred naturally. Pregnancy was verified by real-time ultrasonography on day 45 or later after embryo transfer (ET) or breeding. On day 140 of pregnancy, the reproductive tract was collected from all ewes and the following parameters were determined: the number, sex, weight and crown to rump length (CRL) of fetuses, weights of gravid uterus and fetal membranes, and weight and number of placentomes. Presence of EGF in maturation medium increased (P < 0.04) cleavage rates (78% versus 59%) and percentage of > or = 16 cell embryos on day 5 after fertilization (62% versus 40%). Pregnancy rates tended to be greater (P < 0.1) after transfer of embryos matured in the presence of EGF (52%) than in the absence of EGF (39%). EGF presence in maturation medium did not affect any variables of gravid uterus or fetal weight. For single pregnancies in naturally bred ewes and ewes after ET all uterine variables were similar. For twin pregnancies, weight of gravid uterus, weight of uterus plus fetal membranes, total weight of placentomes/ewe, mean weight of individual placentome, mean weight of fetus, total fetal weight/ewe and CRL were greater (P < 0.0001-0.04) for ewes after ET than for ewes bred naturally. The weights of gravid uterus, fluid, uterus plus fetal membranes, fetal membranes, total placentomes/ewe, mean weight of individual placentome and total fetal weight/ewe were greater (P < 0.0001-0.08) for triplet pregnancies in ewes after ET than single and twin pregnancies in ewes naturally bred or after ET. The number of placentomes/fetus was greatest (P < 0.0001-0.06) in single pregnancies in ewes bred naturally and after ET fewer in twin pregnancies in ewes bred naturally and after ET and fewest in triplet pregnancies in ewes after ET. The total number of placentomes/ewe was greatest (P < 0.0001-0.06) for twin pregnancies in ewes naturally bred, fewer in single pregnancies in ewes naturally bred and twin and triplet pregnancies after ET, and fewest in single pregnancies in ewes after ET. The mean weight of fetus was greater (P < 0.0001-0.07) in single pregnancies in ewes naturally bred or after ET than in twin or triplet pregnancies in ewes naturally bred or after ET. The CRL was the lowest (P < 0.01) in twin pregnancies in ewes bred naturally. For pregnancies after natural breeding and after ET, the number of fetuses/ewe was negatively correlated (P < 0.03-0.0001) with the weight of placentomes/fetus, the number of placentomes/fetus, the mean weight of the fetus and CRL, and was positively correlated (P < 0.0001-0.05) with weight of gravid uterus, the total number of placentomes/ewe and total fetal weight/ewe. These data demonstrate that the presence of EGF in maturation medium increases the rates of cleavage and early embryonic development, and has a tendency to enhance rates of pregnancy but does not affect variables of the gravid uteri in ewes after transfer of in vitro produced embryos. Transfer of embryos produced in vitro affected some uterine variables in twin but not single pregnancies to compare with pregnancies after natural breeding. In addition, culture conditions in the present experiment did not create large offspring syndrome. The low number of placentomes/fetus seen in triple pregnancies appears to be compensated for by the increase in the weight of each individual placentome.     

Integrin activation in bovine placentomes and in caruncular epithelial cells isolated from pregnant cows

In the bovine synepitheliochorial placenta, restricted trophoblast invasion requires complex interactions of integrin receptors with proteins of the extracellular matrix (ECM) and integrin receptors of neighboring cells. Activated integrins assemble to focal adhesions and are linked to the actin cytoskeleton via signaling molecules including alpha-actinin (ACTN), focal adhesion kinase (PTK2 or FAK), phosphotyrosine, and talin (TLN1). Aims of this study were to assess integrin activation and focal adhesion assembly within epithelial cells of bovine placentomes and low-passage (not transformed) placentomal caruncular epithelial cells cultured on dishes coated with ECM proteins. Immunofluorescence analysis was performed to colocalize the signaling molecules ACTN, PTK2, phosphotyrosine, and TLN1 with each other and with beta(1)-integrin (ITGB1) in placentomal cryosections throughout pregnancy and in caruncular epithelial cells in vitro. Antibody specificity was confirmed by Western blot. Cells were cultured on uncoated dishes, and the dishes were coated with fibronectin (FN), laminin (LAMA), and collagen type IV (COL4), thereby statistically assessing cell number and qualitatively assessing the expression pattern of ITGB1, phosphotyrosine, and TLN1. Results demonstrated integrin activation and focal adhesion assembly in the placentome and that low-passage caruncular epithelial cells maintain integrin-associated properties observed in vivo. Expression and/or colocalization of signaling molecules with ITGB1 confirmed, for the first time, integrin activation and participation in "outside-in" and "inside-out" signaling pathways. The prominent role of ECM, and FN in particular, in integrin signaling is supported by the in vitro enhancement of proliferation and focal adhesion expression. Thus, this in vitro model provides excellent potential for further mechanistic studies designed to elucidate feto-maternal interactions in the bovine placentome.     

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.     

Changes in the proportion of type I and type III collagen in the developing and retained bovine placentome

The proportions of Type I and Type III collagen were evaluated from gestational, postpartum-retained, and released bovine placental membranes. Placentomes were excised at 90, 150, 210, and 270 days of gestation (n = 32) and from postpartum-retained (2 and 12 h, n = 8) and released (2 h, n = 4) membranes. Placentome components were processed for collagen, hydroxyproline, protein, and dry weight determination. Collagen extracts were separated by SDS-PAGE. Densitometry was used to establish the proportions of collagen alpha chains (Type I = 2 alpha 1 + 1 alpha 2; Type III = 3 alpha 1). No difference in the proportion of maternal caruncular Type I and Type III collagen was found. The proportion of Type I fetal cotyledonary collagen was lowest (p less than 0.05) at Day 90 of gestation but did not differ between Days 150, 210, 270, or between retained and released fetal membranes. The proportion of Type III fetal cotyledonary collagen was greatest (p less than 0.05) at Day 90. Retained fetal cotyledons had a greater (p less than 0.05) proportion of Type III collagen than did released fetal cotyledons. Therefore, although hydroxyproline content was not different between retained and released fetal membranes, the retained bovine fetal cotyledon was characterized by disproportionate amounts of Type III collagen as compared to the fetal cotyledon that was not retained.     

Cotyledonary responses to maternal selenium and dietary restriction may influence alterations in fetal weight and fetal liver glycogen in sheep

To examine the effects of maternal supranutritional selenium (Se) and nutrient restriction during mid and late gestation on placental characteristics and fetal liver glycogen, ewes received either adequate Se (ASe) or high Se (HSe) prior to breeding. On d 64 of gestation, ASe and HSe ewes remained at 100% of requirements (controls; CON) or were restricted (RES; 60% of requirements). On d 135 of gestation, fetal weight (P ≤ 0.08) was greatest in both HSe and CON ewes. Placentome number, mass, and caruncular and cotyledonary weight were not different (P ≥ 0.17) among treatments. Fetal mass:placental mass ratio was less (P = 0.06) in RES compared to CON ewes. Compared to ASe, HSe exhibited increased (P ≤ 0.08) cellular proliferation and DNA concentration and decreased (P = 0.07) cellular size in cotyledonary tissue. Nutritional restriction decreased (P ≤ 0.08) cotyledonary protein concentration and cellular size. VEGF receptor 1 (Flt) mRNA in cotyledonary tissue was greater in HSe compared with ASe ewes (P = 0.06) and in RES compared with CON ewes (P = 0.08). There was no effect of diet on caruncular growth variables (P ≥ 0.13) or on placental vascularity (P ≥ 0.11). Progesterone was greater (P ≤ 0.08) in ASe–RES ewes compared to all groups at d 90 and ASe–CON and HSe–CON at d 104. Although fetal glucose and cortisol concentrations were not affected by diet, fetal liver glycogen was greater (P = 0.04) in ASe–RES compared to ASe–CON and HSe–RES ewes with HSe–CON being intermediate. Both Se and nutritional plane may impact placental function and fetal growth, as fetal weight and liver glycogen are altered despite similar placental vascularity measurements.     

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.     

Clinical,hormonal, and hematologic characteristics of bovine calves derived from nuclei from somatic cells

Although healthy animals are born after nuclear transfer with somatic cells nuclei, the success of this procedure is generally poor (2%-10%) with high perinatal losses. Apparently normal surviving animals may have undiagnosed pathologies that could develop later in life. The gross pathology of 16 abnormal bovine fetuses produced by nuclear transfer (NT) and the clinical, endocrinologic (insulin-like growth factors I and II [IGF-I and IGF-II], IGF binding proteins, post-ACTH stimulation cortisol, leptin, glucose, and insulin levels), and biochemical characteristics of a group of 21 apparently normal cloned calves were compared with those of in vitro-produced (IVP) controls and controls resulting from artificial insemination. Oocytes used for NT or IVP were matured in vitro. NT to enucleated oocytes was performed using cultured adult or fetal skin cells. After culture, Day 7, grade 1-2 embryos were transferred (one per recipient). All placentas and fetuses from clones undergoing an abnormal pregnancy showed some degree of edema due to hydrops. Mean placentome number was lower and mean placentome weight was higher in clones than in controls (69.9 +/- 9.2 placentomes with a mean weight of 144.3 +/- 21.4 g in clones vs. 99 and 137 placentomes with a mean individual weight of 34.8 and 32.4 g in two IVP controls). Erythrocyte mean cell volume was higher at birth (P < 0.01), and body temperature and plasma leptin concentrations were higher and T4 levels were lower during the first 50 days and the first week (P < 0.05), respectively, in clones. Plasma IGF-II concentrations were higher at birth and lower at Day 15 in clones (P < 0.05). Therefore, apparently healthy cloned calves cannot be considered as physiologically normal animals until at least 50 days of age.