Transplacental Transfer of Iron in the Water Buffalo (Bubalus bubalis): Uteroferrin and Erythrophagocytosis

The objectives of this investigation were to understand transplacental transport of iron by secreted uteroferrin (UF) and haemophagous areas of water buffalo placenta and clarify the role(s) of blood extravasation at the placental‐maternal interface. Placentomes and interplacentomal region of 51 placentae at various stages of gestation were fixed, processed for light and transmission electron microscopy, histochemistry and immunohistochemistry. Haemophagous areas were present in placentomes collected between 4 and 10 months of pregnancy. Perl’s reaction for ferric iron was negative in placentomes, but positive in endometrial glands. Positive staining for UF indicated areas in which it was being taken up by phagocytosis and/or fluid phase pinocytosis in areolae of the interplacentomal mesenchyme, with little staining in endometrial stroma. Imunohistochemistry detected UF in trophectoderm of haemophagous regions of placentomes and in other parts of the foetal villous tree, but the strongest immunostaining was in the epithelial cells and lumen of uterine glands. Ultrastructural analyses indicated that erythrophagocytosis was occurring and that erythrocytes were present inside cells of the chorion that also contained endocytic vesicles and caveolae. Results of this study indicate that both the haemophagous areas of placentomes and the areolae at the interface between chorion and endometrial glands are important sites for iron transfer from mother to foetal‐placental tissues in buffalo throughout pregnancy.     

Light and scanning electron microscopic study on the blood vascular system of the donkey placenta

The donkey placenta is diffuse and epitheliochorial with numerous microplacentomes consisting of a fetal microcotyledonary and a maternal microcaruncular part. The microplacentomal vasculature during the last third of pregnancy has been investigated by light microscopy in comparison to scanning electron microscopy of the materno-fetal contact surface and corrosion casts of blood vessels after plastic instillation from either the microcotyledonary or the microcaruncular side, and, for the first time in a perissodactyle, from both sides. Morphological data were semiquantitatively evaluated. The supplying parts of both, the microcotyledonary and the microcaruncular vascular system are strictly proximo-distally oriented, thus reaching the capillary systems or working parts in the shortest way possible. The straight course of the vasculature, particularly on the fetal side, suggests the occurrence of venulo-arteriolar back diffusion. The fetal capillary system consists of convolutes confronting the maternal septal capillary complexes in a countercurrent way. This materno-fetal blood flow interrelationship is highly efficient in terms of placental exchange, which is further supported (1) by dilations and increasing coiling of the fetal venular capillary limbs in particular and (2) by a decrease in the interhaemal distance from 12.5 to 7.2 microm between the two capillary systems. Besides the countercurrent blood flow interrelationship, some maternal branch arterioles reach the septal capillary system from the maternally oriented pole of the microplacentome or microcaruncle, respectively, resulting in the less efficient crosscurrent blood flow. Hence, in the donkey placenta fetal and maternal blood vessels meet in a mix of countercurrent and crosscurrent flow patterns.     

Administration of Thyroxine Affects the Morphometric Parameters and VEGF Expression in the Uterus and Placenta and the Uterine Vascularization but does Not Affect Reproductive Parameters in Gilts During Early Gestation

The aim of this study was to evaluate the effects of thyroxine administration on morphometric parameters, expression of vascular endothelial growth factor (VEGF) and vascularization in the uterus and placenta and reproductive parameters in gilts at 70 days of gestation. At 150 days of age, i.e., before first heat, 20 gilts were randomly divided into two experimental groups: treated (n = 10) and control (n = 10). The treated group received a daily dose of 400 μg of l ‐thyroxine (T₄) in their diet until slaughter and the control group received only typical meals. Before artificial insemination, blood was collected to determine plasma total T₄. The gilts were inseminated in the second oestrus and slaughtered at 70 days of gestation. The foetal thyroid follicular epithelium height, number, size and weight of foetuses; foetal myogenesis, corpora lutea number, embryonic mortality rate, uterine weight, placental weight and placental fluid volume were measured. Histomorphometric and immunohistochemical analysis of uterus and placenta were determined. The averages of all variables were compared by the Student’s t‐test. The gilts treated with thyroxine showed significant increase of plasma total T₄. At 70 days of gestation, the heights of the trophoblastic epithelium, endometrial epithelium and endometrial gland epithelium were significantly higher in the group treated with T₄. The expression of cytoplasmatic and nuclear VEGF in trophoblastic cells and the number of blood vessels per field in endometrial stroma were significantly higher in the gilts treated with T₄. No other significant differences between groups were obtained with respect to other parameters (p > 0.05). We conclude that oral administration of T₄ up to 70 days of pregnancy in gilts affects the morphometric parameters, the expression of placental VEGF and the uterine vascularization but does not affect reproductive parameters in gilts during early gestation.     

Effect of Sildenafil on Pre‐Eclampsia‐Like Mouse Model Induced By L‐Name

N(omega)‐nitro‐L‐arginine methyl ester (L‐NAME) decreases the vasodilator effect of nitric oxide (NO) and induces pre‐eclampsia in mouse. Sildenafil inhibits the degradation of nitric oxide and increases vasodilation. This study aimed to determine the effects of sildenafil citrate on angiogenesis and oxidative stress at the maternal foetal interface on pre‐eclampsia‐like mouse model induced by L‐NAME. Twenty pregnant mice were divided into four groups: (i) vehicle control; (ii) L‐NAME; (iii) sildenafil; (4) L‐NAME+sildenafil. L‐NAME was administered from day 7 of pregnancy and sildenafil from day 8 until day 16; animals were euthanized on day 17. Placental and foetal sizes and weights were measured; lipid peroxide levels and catalase activity in placental homogenates were determined, and placental vascular endothelia were identified by lectin‐histochemistry using BSA‐I lectin. Western blot analysis was used to determine VEGF expression in placental homogenates. No changes were seen in placental and foetal development in mice with normal pregnancies treated with sildenafil. Treatments with L‐NAME reduced significantly the placental weight and average height and decreased the percentage of the endothelial surface. These alterations may be mediated by the reduction of NO levels in trophoblastic cells, due to the inhibitory effect of L‐NAME on nitric oxide synthase (NOS) synthesis. This effect was offset by the treatment with sildenafil, with an increase in the percentage of the endothelial surface. In conclusion, our results indicate that treatment with sildenafil on pre‐eclampsia mouse model can be used without adverse effects on the concept and its use in the treatment of pre‐eclampsia is promising.     

Immunohistochemical Localization of Vascular Endothelial Growth Factor (VEGF) and its Two Receptors (Flt-I and KDR) in the Endometrium and Placenta of the Mare During the Oestrous Cycle and Pregnancy

Polyclonal antisera for vascular endothelial growth factor (VEGF) and its two main receptor molecules, VEGF-I (Flt) and VEGF-II (KDR), were used in a conventional immunocytochemical staining method to localize these angiogenic ligand molecules in the endometrium and placenta of the mare during the oestrous cycle and pregnancy. The anti-VEGF and anti-Flt sera both labelled the lumenal and glandular epithelia of the endometrium throughout the oestrous cycle and both the invasive trophoblast cells of the endometrial cups and the non-invasive trophoblast of the allantochorion in pregnancy. The anti-KDR serum likewise stained the maternal and foetal epithelial layers during the oestrous cycle and pregnancy and it also labelled fibroblast-like cells in the endometrial and allantoic stromas and the endothelium of foetal and maternal capillaries. The results demonstrated that constant supplies of the principal vasculogenic and angiogenic factor, VEGF, and its two major receptors, Flt and KDR, are available on both the maternal and foetal sides of the placental barrier throughout gestation in the mare. They are presumed to facilitate the continuing development of the extensive foetal and maternal capillary networks that are such prominent features within the microplacentomes of the diffuse, epitheliochorial equine placenta.     

Experimental Neospora Caninum Infection in Pregnant Dairy Heifers Raises Concentrations of Pregnancy‐Associated Glycoproteins 1 and 2 in Foetal Fluids

Plasma concentrations of PAG‐1 are used for pregnancy diagnosis and as a marker of placental/foetal well‐being, while those of PAG‐2 may be an indicator of abortion risk in Neospora caninum‐infected cows. Studies have shown that N. caninum infection modifies PAG‐1 and PAG‐2 patterns in maternal blood plasma. However, no prior work has examined the effects of N. caninum infection on concentrations of PAGs in foetal fluids. In this study, PAG‐1, PAG‐2 and pH levels were determined in the amniotic and allantoic fluids of foetuses collected at 152 days of gestation from control uninfected dams and from dams experimentally infected with N. caninum on Day 110 of gestation. Foetal fluids from infected foetuses had significantly higher PAG‐2 concentrations (p = 0.026) and pH values (p = 0.02) than fluids from non‐infected foetuses. In infected foetuses, significantly higher concentrations of PAG‐1 (p < 0.001) and PAG‐2 (p < 0.001) were detected in fluid samples showing antibodies against N. caninum than those without antibodies. Moreover, pH values were significantly higher (p = 0.011) in foetal fluid samples with antibodies than in samples from non‐infected foetuses. In conclusion, this is the first report on the effect of N. caninum infection on PAG levels in foetal fluids. Our results indicate that following the experimental infection of dams with N. caninum on Day 110 of gestation, foetal fluids collected from the infected foetuses of these dams featured higher PAG‐1 and PAG‐2 levels and pH values than fluids from non‐infected controls, provided that the samples tested showed the presence of antibodies. The clinical implications of these findings are that following infection with N. caninum, most cows will experience some level of placental damage and that this injury correlates with foetal fluid PAG levels and pH.     

Cellular Remodelling by Apoptosis During Porcine Placentation

The mechanisms that regulate the apoptosis are essential to the normal development and maintenance of homoeostasis and play an important role in placental development in mammals. During porcine pregnancy, there must be a proper cellular remodelling to achieve a normal gestational development. Knowledge of pig physiology during pregnancy will explore options to increase the productivity of this species of high economical value. The purpose of this work was to study the cell morphology and apoptosis of porcine placentas from early, mid and late pregnancy. For that purpose, high‐resolution light microscopy and transmission electron microscopy were performed to the study of cell morphology. TUNEL, the apoptosis index (IAp) and the expression of c‐FLIP through immunohistochemistry technique were used to the study of apoptosis. High‐resolution light microscopy and transmission electron microscopy confirmed the presence of placental cells with ultrastructural apoptotic features. Apoptotic nuclei were detected by TUNEL in different placental structures and phagocytes containing apoptotic bodies. The IAp in villi was 9.34% at early, 0.82% at mid and 23.85% at late pregnancy. Statistically significant differences were found between periods (p < 0.05). In previous studies, we determined a differential induction of the apoptotic routes in the placental villi in agreement with the gestational period. A co‐expression of receptors and mitochondrial proteins in placental connective tissue was detected, but the immunolocalization of c‐FLIP would indicate an endogenous inhibition of the extrinsic pathway. In conclusion, in swine there exists differential activation of inducing apoptotic pathways in different placental structures according to the gestational period.     

Uterine serpin (SERPINA 14) correlates negatively with cytokine production at the foetal–maternal interface but not in the corpus luteum in pregnant dairy heifers experimentally infected with Neospora caninum

This study examines gene expression patterns in dairy heifers experimentally infected with N. caninum during on Day 110 of pregnancy with live foetuses at euthanasia, 42 days later. The study population was constituted of four non‐infected controls and three infected dams. Gene expression was determined on gamma interferon (IFNγ), (Th1 pro‐inflammatory cytokine), interleukin‐4 (IL4) (Th2 pro‐gestation cytokine) or interleukin‐10 (IL10) (T regulatory cytokine) and the serine peptidase inhibitor SERPINA14 in intercaruncular, placental, uterine lymph node (UTLN) and luteal tissue samples. Intercaruncular SERPINA14 expression was negatively correlated with IFNγ expression in cotyledon samples and with IL4 expression in UTLN. No relationships were detected between cytokine gene expression at the foetal–maternal interface and SERPINA14 expression in the luteal samples. Our findings suggest that gene expression of the uterine serpin SERPINA14 correlates negatively with the expression of Th1 and Th2 cytokines at the foetal–maternal interface but not in the corpus luteum.     

An overview of molecular and cellular mechanisms associated with porcine pregnancy success or failure

Prenatal mortality remains one of the major constraints for the commercial pig industry in North America. Twenty to thirty per cent of the conceptuses are lost early in gestation and an additional 10-15% is lost by mid-to-late gestation. Research over the last two decades has provided critical insights into how uterine capacity, placental efficiency, genetics, environment, nutrition and immune mechanisms impact successful conceptus growth; however, the exact cause and effect relationship in the context of foetal loss has yet to be determined. Similar to other mammalian species such as the human, mouse, rat, and primates, immune cell enrichment occurs at the porcine maternal-foetal interface during the window of conceptus attachment. However, unlike other species, immune cells are solely recruited by conceptus-derived signals. As pigs have epitheliochorial placentae where maternal and foetal tissue layers are separate, it provides an ideal model to study immune cell interactions with foetal trophoblasts. Our research is focused on the immune-angiogenesis axis during porcine pregnancy. It is well established that immune cells are recruited to the maternal-foetal interface, but their pregnancy specific functions and how the local milieu affects angiogenesis and inflammation at the site of foetal arrest remain unknown. Through a better understanding of how immune cells modulate crosstalk between the conceptus and the mother, it might be possible to therapeutically target immune cells and/or their products to reduce foetal loss. In this review, we provide evidence from the literature and from our own work into the immunological factors associated with porcine foetal loss.     

Differences in placental structure during gestation associated with large and small pig fetuses

The efficiency of nutrient transport from the pregnant female pig to the developing fetus depends on the size and function of the placenta. It has been reported that maternal and fetal blood vessels are arranged in a cross-countercurrent arrangement within placental microscopic folds. Thus, the blood supplies are in close apposition to each other within these microscopic folds, and maternal and fetal blood flows in approximately opposite directions perpendicular to the plane of the placenta. This arrangement indicates that the width of the microscopic folds influences placental efficiency. The objective of this study was to determine whether differences in pig placental microscopic fold development are associated with differences in fetal size or are influenced by selection for ovulation rate or uterine capacity. Gilts from a randomly selected control line, a line selected for ovulation rate, and a line selected for uterine capacity were slaughtered, and uterine wall samples were collected within the placentas associated with the largest and smallest fetuses in each litter on d 45, 65, 85, and 105 of gestation. The uterine wall samples were processed for histology and analyzed using computer-assisted morphometry. Average width of the placental folds and average width of the placental stroma above the folds were measured. To measure fold complexity, the length of the epithelial bilayer for a given length of placenta was also measured. The width of the folded bilayer increased significantly from d 65 to 105 and was greater in placentas associated with small fetuses compared with large fetuses on d 105 of gestation. In contrast, the width of the placental stroma above the folded bilayer decreased with gestation and decreased more rapidly in placenta associated with the smallest compared with the largest fetus. These results indicate that the width of the microscopic folds of the placental trophoblast/endometrial epithelial bilayer is increased in placenta associated with small fetuses, which we hypothesize will increase the surface area for interaction between maternal and fetal blood supplies, thus improving placental efficiency in response to reduced placental size.     

Development of the areola in the early placenta of the one-humped camel (Camelus dromedarius): a light, scanning and transmission electron microscopical study

This study aimed to elucidate development of the areola in the early dromedary placenta in comparison with that of the pig and mare. Placental tissues from 25 pregnant camels were obtained from Cairo abattoir and prepared for light, scanning and transmission electron microscopy by routine methods. Vascular casts were made by injection of 4 : 1 liquid plastic mixture of mercox and methylmethacrylate. Areolar formation was first observed at 4.5 cm curved-crown-rump CVR length, while by 5-9 cm CVR length, the endometrial surface was uneven and studded with numerous uterine gland openings, where corresponding foetal areolae were barely detectable and the foetal areolar cells were of variable appearance and covered with long microvilli. At 10-13 cm CVR length the uterine gland openings developed irregular folds and the maternal areolar cells showed numerous apical blebs. At 14-29 cm CVR length the foetal areolae showed a great increase in height at the expense of their width. At 30-34 cm (CVR) length the maternal areolae appeared discoid and sharply demarcated from the surrounding inter-areolar tissues and the foetal areolae were rounded to irregular in shape with well-developed areolar rims. The vascular casts showed a widely meshed capillary network on the maternal areola, connecting with the pre- and post-capillary vessels, whereas the foetal side showed a relatively dense capillary meshwork. These studies indicate that the areola in the placenta of the one-humped camel is of the regular type like in the pig, and is poorly vascularized.     

Histological Changes of the Feline Cervix, Endometrium and Placenta after Mid-gestational Termination of Pregnancy with Aglepristone

This study was conducted to investigate endometrial and placental structural changes that occurred in response to mid‐gestational termination of pregnancy in queens using aglepristone, a progesterone receptor antagonist. Thirteen European Shorthair pregnant queens were either treated with aglepristone (10 mg/kg body weight; subcutaneously) twice on days 25 and 26 after first mating (am; group I; n = 9), or remained untreated and served as control (group II; n = 4). Queens of group I were ovariohysterectomized between days 30 and 41 am, either at the onset (n = 3) or during (n = 1) abortion and 12 h (n = 1), 24 h (n = 3) or 10 days after abortion (n = 1). Queens of group II were ovariohysterectomized on day 30 am. Tissue was collected from the cervix, and the interplacental zone as well as the paraplacenta/placental girdle of the uterus, subjected to standard histological procedures and evaluated using light microscopy. During abortion, gaps appeared within the paraplacenta and the placental girdle which were filled with blood, leading to an embryo‐maternal disconnection. Blood was also observed within the uterine lumen as well as the interstitial mucosal stroma of the cervix and the placental girdle zone and probably originated from damaged venules, whilst arterioles remained intact. As the interval between abortion and surgery increased, the interstitial and luminal haemorrhages became less pronounced and completely disappeared except interstitial remnants 10 days after abortion. The endometrial regeneration was not fully completed on day 10 after abortion and a few cystically dilated glands were evident. In conclusion, abortion of queens through aglepristone given during mid‐gestation is assumed to be the result of damage of uterine venules. This leads to an interstitial haemorrhages and bleeding into the uterine lumen, subsequently resulting in utero‐placental detachment.     

Flutamide Influences Placental Aldo–Keto Reductase Family 1 Member C1 (AKR1C1) Expression in Pigs

Aldo–keto reductase family 1 member C1 (AKR1C1) catalyses the conversion of progesterone into inactive 20α‐dihydroxyprogesterone. It is suggested that AKR1C1 expression in the placenta prevents from the cytotoxic effect of progesterone on foetuses during late pregnancy. The aim of the study was to determine whether the anti‐androgen flutamide administered during late pregnancy (83–89 days of gestation) or before parturition (101–107 days of gestation) influences AKR1C1 expression in the porcine placenta. AKR1C1 mRNA and protein levels were measured using real‐time PCR and western blotting, respectively. Immunolocalization of AKR1C1 within placentas was also performed. Flutamide significantly increased AKR1C1 mRNA (p = 0.008) and protein (p = 0.019) expression only during the pre‐parturient period in pigs. AKR1C1 protein was immunolocalized in the epithelial and stromal cells of foetal and maternal part of placenta at both stages of gestation. Following flutamide treatment, the intensity of staining was higher (p = 0.045) on day 108 of gestation. In conclusion, porcine placental AKR1C1 expression seems to be regulated by an androgen signalling pathway and may be involved in foetal survival by preventing the detrimental effect of progesterone.     

Extracellular matrix in epitheliochorial,endotheliochorial and haemochorial placentation and its potential application for regenerative medicine

A placenta is defined as structural approximation of maternal and foetal tissues to perform physiological exchange. Associated processes of differentiation and the establishment of its cells take place within the extracellular matrix (ECM) that provides a rich environment of collagens, fibronectins, cytokines and other components. Placental ECM is promising for tissue regeneration purposes, because it has immune tolerance capacities that may cause only minimal rejections of transplants with immunological differences between donor and recipient. However, specific characteristics of ECM during evolution of the structurally very diverse mammalian placenta are not yet revealed. We here address the major aspects of placental types, that is non‐invasive (epitheliochorial), medium (endotheliochorial)‐to‐high (haemochorial) invasive nature of the interhemal barrier between the foetal and maternal blood system as well as their main components of ECM with special reference to species that are commonly used as animal models for human placentation and in the potential applications for regenerative medicine.     

The maternal to fetal transfer of immunoglobulins associated with placental lesions in sheep.

In this study we evaluated maternofetal transmission of immunoglobulins in ewes under conditions of altered placental morphology. Intravenous injection of human red blood cells was used to induce immunoglobulins in pregnant ewes. The hemagglutination test was used to detect antibody in maternal serum, fetal and placental fluids. Placental injury was induced by intravenous inoculation of Escherichia coli endotoxin or spores of Aspergillus fumigatus into pregnant ewes at days 99 or 100 of gestation respectively. Placental infarction, thrombosis of maternal placental vessels and variable neutrophil infiltrate characterized lesions produced by A. fumigatus. Endotoxin treated ewes developed marked placental edema, congestion, hemorrhage and focal loss of uterine epithelium. Human red blood cell agglutinating antibody was not detected in placental or fetal fluids obtained from ewes with either of the above placental lesions. Placentitis of undetermined etiology was observed in seven ewes. Two ewes had received A. fumigatus, two had received endotoxin and three were untreated ewes. Histological examination of their placentas revealed trophoblastic and endometrial epithelial necrosis and necrotizing vasculitis of the chorioallantois. Human red blood cell agglutinating antibody was detected only in the fetal and placental fluids of the seven ewes with these placental lesions. The nature of these lesions would have produced a functional confluence of the maternal and fetal circulations. Antibody transfer from dam to fetus was observed only in association with placental lesions which produced this confluence of circulations. The character of the placental lesions, rather than the mere presence of placental lesions apparently determined the transfer of immunoglobulins.(ABSTRACT TRUNCATED AT 250 WORDS)     

哺乳动物胎盘营养感应研究进展

妊娠期间母体营养的改变会影响胎儿的生长发育,而胎盘是母体-胎儿间进行气体、营养和代谢物交流的重要枢纽,哺乳动物胎盘营养感应系统响应母体和胎儿营养信号的变化,保证母体健康和胎儿生长发育。鉴于胎盘营养感应系统对哺乳动物繁育的重要性,本文从胎盘营养感应和胎盘养分分配方面综述了哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、磷酸腺苷活化蛋白激酶(AMP-activated protein kinase,AMPK)、氨基己糖(Hexosamine)信号通路、糖原合成酶3(glycogen synthase kinase,GSK-3)、胰岛素/胰岛素样生长因子(insulin/insulin-like growth factor signaling pathway,IIS)等信号通路及其对妊娠期养分响应和对胎儿发育的影响,以期为哺乳动物的繁育提供参考依据。     

The effect of divergent selection for uterine capacity on fetal and placental development at term in rabbits: maternal and embryonic genetic effects

The aim of this work was to study the effects of the genotype of the dam, the embryo, or their interactions on prenatal growth by performing double-reciprocal embryo transfers between two lines of rabbits divergently selected for uterine capacity. Females from high (n = 53) and low (n = 48) lines were slaughtered at 72 h of gestation, and recovered embryos were transferred to the oviducts of recipient does from the high (n = 23) and low (n = 19) lines. Each recipient doe received eight embryos from the high line into one oviduct and eight embryos from the low line into the other. Recipient does were slaughtered on d 28 of gestation. The percentages of live fetuses at 28 d of gestation were 89.2 and 74% for high and low recipient lines, respectively. Length and weight of the empty uterine horn and weight of the full uterine horn were not affected by either the recipient or by donor line. Fetal weight was affected by the recipient line but not by the donor line. Fetuses gestated in high recipient does were 7% heavier (P < 0.10) than those in the low recipient does. There was a donor and a donor x recipient interaction effect on fetal placental weight. Fetal placental weight was heavier (7%, P < 0.01) for embryos from the low line. Embryos from the high line gestated in low-line uteri showed a lower fetal placenta weight than did low-line embryos gestated in high-line uteri and low-line uteri (P < 0.05). Linear regression coefficients of fetal weight at term on fetal placental weights differed (P < 0.05) for the high and low donors (4.33 +/- 0.28 and 3.41 +/- 0.29 respectively). A significant effect of the donor genotype on individual placental length was observed (P < 0.05), which might have resulted from a smaller individual placental length of low-line embryos gestated high-line uteri (P < 0.10). Neither donor nor recipient lines affected maternal placental weight or available space for fetuses. Fetuses and their fetal placentae were heavier when receiving more than four blood vessels than when receiving less than three blood vessels (13 and 17% respectively, P < 0.05). Neither recipient nor donor genotype affected the number of blood vessels arriving at each live fetus. Thus, fetal weight depends on the maternal genotype, whereas fetal placental weight depends on the embryo genotype in these two lines of rabbits divergently selected for uterine capacity.     

Expression of the Vascular Endothelial Growth Factor (VEGF‐A) and its Receptors in the Umbilical Cord in the Course of Pregnancy in the Pig

The umbilical cord (UC) and the placenta are important organs through which respiratory gases, nutrients, wastes and biologically active substances are exchanged between the maternal and the foetal system. A rapid placental vascularization observed in the second half of pig pregnancy is positively correlated with the mRNA expression of the vascular endothelial growth factor (VEGF). Based on these findings, we hypothesized that VEGF may have a stimulatory effect in the dynamically growing UC. To further understand the role of the VEGF–VEGFR system during UC development, mRNA and protein expression as well as the cellular localization of VEGF‐A, VEGFR‐1 and VEGFR‐2 in UC were examined on days 40, 60, 75 and 90 of pregnancy and after physiological delivery in the pig (day 114 of pregnancy). Real Time RT‐PCR analysis showed an increase in the mRNA levels of VEGF120 and VEGF164 from day 90 of pregnancy. VEGFR‐1 mRNA expression was significantly increased on day 75 of pregnancy. No significant changes in VEGFR‐2 mRNA expression were detected. In turn, western blot analysis revealed an increase in VEGF‐A protein expression on day 40, compared to the later days of pregnancy. A rapid increase in the VEGFR‐1 protein level was noted on day 75 and 90 of gestation. No significant changes in VEGFR‐2 protein expression were detected on any of the analysed days of pregnancy. Immunohistochemical staining enabled detection of VEGF–VEGFR system, in endothelial and tunica media cells of the umbilical vessels and in allantoic duct and amniotic epithelium on all analysed days of pregnancy. Positive reactions for VEGF‐A and VEGFR‐1, but not VEGFR‐2, were also observed in myofibroblasts. In conclusion, this data shows that members of the VEGF–VEGFR system are temporally and spatially well localized for playing key roles during umbilical cord formation and its intensive growth observed after day 75 of pregnancy.     

Effect of early gestational undernutrition on angiogenic factor expression and vascularity in the bovine placentome

The effect of early gestation maternal undernutrition followed by realimentation on placentomal vascular growth and angiogenic factor expression was determined in multiparous beef cows bred to the same bull. Cows gestating only female fetuses (n = 30) were fed in equal numbers to meet the NRC requirements (control) or were fed below the NRC requirements to lose BW (nutrient restricted; NR) from d 30 to 125 of gestation. After slaughter on d 125 of gestation, 10 control and 10 NR cows were necropsied. The remaining NR cows (n = 5) were then fed to achieve a BCS equal to their control group contemporaries (n = 5) by d 220 of gestation. All cows were fed the control diet from d 220 until 250 of gestation, when the remaining control and NR cows were slaughtered and necropsied. At necropsy, placentomes were fixed via perfusion of the caruncular and cotyledonary arteries to determine capillary vascular density. Cotyledonary (fetal placental) and caruncular (maternal placental) tissues also were snap-frozen in liquid nitrogen, and mRNA concentrations of vascular endothelial growth factor and its 2 specific receptors, fms-like tyrosine kinase and kinase insert domain containing receptor, as well as placental growth factor, were determined. There was no effect of diet or day of gestation on the percentage of proliferating caruncular cells. Although diet did not impact cotyledonary cellular proliferation, there was an increase (P < 0.05) in the percentage of proliferating cells on d 250 compared with d 125 of gestation. Nutrient restriction from d 30 to 125 increased (P < or = 0.10) placental mRNA concentrations of placental growth factor and fms-like tyrosine kinase; however, there was no alteration in vascularity. By d 250 of gestation, NR cows had increased (P < 0.05) caruncular capillary surface density and decreased (P < 0.05) cotyledonary capillary area density, capillary number density, and capillary surface density compared with control cows. Although nutrient restriction had little effect on placental vascularity by d 125, upon realimentation, alterations in vascularity became apparent by d 250 of gestation, suggesting a placental programming effect.