Maternal exposure to bisphenol a during late pregnancy resulted in an increase of Calbindin-D9k mRNA and protein in maternal and postnatal rat uteri

It has been reported that Calbindin-D9k (CaBP-9k) is rapidly and strongly induced by environmental estrogenic compounds, possibly through estrogen receptors (ERalpha) in the uterus of mammals. CaBP-9k can be evaluated as an early gene marker for assaying estrogenic effects of putative environmental chemicals in the rat uterus. This study was undertaken to investigate CaBP-9k mRNA and protein expression in the postnatal rat uterus following maternal exposure to 17beta-estradiol (E2) and bisphenol A (BPA) during the neonatal period. Treatment with a high dose of BPA (600 mg/kg body weight (BW) per day) resulted in a 3-fold increase in CaBP-9k mRNA expression for 3 days, while a single dose of E2 (40 microg/kg BW per day) induced 2-fold increase of this gene in the maternal uterus. In an agreement with maternal CaBP-9k mRNA, postnatal CaBP-9k mRNA in the uterus increased 4-fold when treated with BPA (600 mg/kg BW per day). In addition, treatment with increasing concentrations of BPA resulted in significant increases in CaBP-9k protein in the maternal rat uterus. It is of interest that increasing doses of BPA induced a significant ERalpha mRNA increase in the postnatal uterus. Furthermore, immunohistochemistry revealed that treatment with BPA induced CaBP-9k protein in the maternal uterus. We demonstrated that maternal exposure to BPA during late pregnancy induced CaBP-9k mRNA and protein in maternal and postnatal rat uteri. These results suggest that rapid absorption and distribution of environmental estrogenic compounds occurs in maternal and neonatal rat uteri and these chemicals can easily pass though the placenta during pregnancy to affect postnatal reproductive functions.     

Nutritional Programming of Intrauterine Development: A Concept Applicable to the Horse?

Human epidemiological observations and studies of experimental animals have shown that low birth weight is associated with adult phenotypes characterized by abnormalities in cardiovascular, metabolic, and endocrine function. In human populations, these changes can lead to overt degenerative diseases such as hypertension and type 2 diabetes. Because fetal growth depends primarily on the nutrient supply, the associations between birth weight and adult phenotype have been linked to poor nutrition in utero. The fetal supply of nutrients and oxygen depends on both the availability of these substances in the mother and the functional capacity of the placenta to supply them to the fetus. This review takes a comparative approach to examining the nutritional programming of adult physiological phenotype in mammals with emphasis on the horse where possible. It considers the role of maternal nutritional state and placental function in these processes and discusses the cellular and molecular mechanisms operating in utero that are responsible for the epigenetic regulation of phenotypical diversity.     

Effects of bisphenol A (BPA) on placentation and survival of the neonates in mice

The effects of bisphenol A (BPA) on placentation have not been fully determined. The aim of this study was to clarify the structural changes of the placenta, abortion rate, and survival of neonates after BPA administration in mice. BPA (10 mg/kg/day) was administered to pregnant mice (BPA mice) subcutaneously from the first day of pregnancy (Day 0) to Day 7 (8 days total). The number of embryos and weights of whole uteri were measured on Days 10 and 12. Morphological changes in the placentae were examined by light microscopy on the corresponding days of pregnancy. The number of neonates was also counted. Survival rates were periodically calculated for neonates from the first day after parturition (P-Day 0) to P-Day 56. The number of embryos and weight of the uterus on Days 10 and 12 were significantly decreased by BPA injection. No notable differences were recognized between the left and right uteri. The proportion of the labyrinthine zone per whole placenta in the BPA mice became lower than that in the controls, and that of the metrial gland was higher in the BPA mice. The intervillous spaces of the placenta were narrower in the BPA mice. Degenerative changes were found in the trophoblastic giant cells and spongiotrophoblast layers of the BPA mice. The number of BPA mouse neonates was drastically decreased within 3 days after birth, and no mice survived after P-Day 56. The results suggest that BPA not only disrupts placental functions and leads to abortion through chronic stimulation of gene expression by binding to DNA but that it also affects the mortality of neonates through indirect exposure of embryos.     

Baggs ewes adapt to maternal undernutrition and maintain conceptus growth by maintaining fetal plasma concentrations of amino acids

Adequate delivery of AA is essential for normal fetal growth and development. Recently, we reported that when ewes from the University of Wyoming flock (farm flock with adequate nutrition) were fed 50% (nutrient-restricted) or 100% (control-fed) of the NRC-recommended nutrient requirements between d 28 and 78 of gestation, fetal weights as well as concentrations of most AA in maternal and fetal blood were substantially reduced in nutrient-restricted vs. control-fed pregnancies. The current study utilized Baggs ewes, which were selected under a markedly different production system (range flock with limited nutrition), to test the hypothesis that adaptation of ewes to nutritional and environmental changes may alter placental efficiency and conceptus nutrient availability in the face of maternal nutrient restriction. Baggs ewes received 50 or 100% of the NRC nutrient requirements between d 28 and 78 of pregnancy. On d 78, maternal uterine arterial and fetal umbilical venous blood samples were obtained, and the ewes were euthanized. Amino acids and their metabolites (ammonia, urea, and polyamines) in plasma were analyzed using enzymatic and HPLC methods. The results showed that maternal plasma concentrations of 9 AA (Asp, Ile, Leu, Lys, Orn, Phe, Thr, Trp, and Val) as well as maternal and fetal plasma concentrations of ammonia and urea were reduced (P < 0.05) in nutrient-restricted compared with control-fed Baggs ewes. However, fetal plasma concentrations of all AA and polyamines did not differ (P = 0.842) between the 2 groups of ewes. Collectively, these findings suggest that Baggs ewes, by adapting to the harsh conditions and limited nutrition under which they were selected, were able to maintain fetal concentrations of AA in the face of a maternal nutrient restriction through augmenting placental efficiency.     

The uterus and early pregnancy failure in the mare

The environment the equine conceptus finds itself in when it arrives in the uterus some 6 days after ovulation will determine if it will thrive or die. The uterus will be its home for approximately the next 11 months and as such it needs to both coordinate the growth of the placenta and ensure there is enough nourishment passing to this organ for the fetus to develop normally. The trophoblast, endometrium, maternal ovaries and, later in pregnancy, the fetal gonads, all play roles in the hormonal changes that orchestrate these events. Although failures of these processes later in pregnancy can have catastrophic effects for the fetus, it is in early gestation that the foundations for a successful pregnancy are laid. This paper therefore concentrates on some of the noninfectious influences the uterus may have on survival of the young conceptus.     

The immunology of early pregnancy in farm animals

The mammalian conceptus undergoes development in the face of a functional immune system. This characteristic of viviparity creates opportunities and perils for the conceptus. In the period up to hatching from the zona pellucida, the conceptus appears immunologically inert with low expression of major histocompatibility complex (MHC) genes and little evidence for alterations in the function of maternal immune cells. The conceptus may benefit from cytokines produced by leucocytes resident in the reproductive tract or by other cells of the reproductive tract. One cytokine in particular, colony-stimulating factor 2, can promote preimplantation development and cause changes in conceptus function that increase the likelihood that the conceptus develops to term. It is not clear whether activation of specific types of immune responses in early pregnancy can enhance the likelihood of pregnancy success. Semen deposition causes inflammation in the reproductive tract, but there is little evidence that this process is beneficial to pregnancy unless the uterus was exposed to a prior inflammatory event. Around the time of placentation, the period of immunological inertness of the conceptus is replaced by a period of immune activation caused by the expression of interferon genes in ruminants and pigs and MHC class I genes in invasive trophoblast of the horse. The large-scale changes in the endometrium that occur as a result are likely to play an important role to ensure continued development of the conceptus. In contrast, other immune responses generated as a result of disease in the reproductive tract or other tissues (mastitis) can lead to death of the conceptus. Because of this hazard, as well as the possibility that cell-mediated immune responses against the conceptus could occur, the immune system is regulated by both the mother and conceptus to reduce immune responsiveness during pregnancy.     

Board-invited review: intrauterine growth retardation: implications for the animal sciences

Intrauterine growth retardation (IUGR), defined as impaired growth and development of the mammalian embryo/fetus or its organs during pregnancy, is a major concern in domestic animal production. Fetal growth restriction reduces neonatal survival, has a permanent stunting effect on postnatal growth and the efficiency of feed/forage utilization in offspring, negatively affects whole body composition and meat quality, and impairs long-term health and athletic performance. Knowledge of the underlying mechanisms has important implications for the prevention of IUGR and is crucial for enhancing the efficiency of livestock production and animal health. Fetal growth within the uterus is a complex biological event influenced by genetic, epigenetic, and environmental factors, as well as maternal maturity. These factors impact on the size and functional capacity of the placenta, uteroplacental blood flows, transfer of nutrients and oxygen from mother to fetus, conceptus nutrient availability, the endocrine milieu, and metabolic pathways. Alterations in fetal nutrition and endocrine status may result in developmental adaptations that permanently change the structure, physiology, metabolism, and postnatal growth of the offspring. Impaired placental syntheses of nitric oxide (a major vasodilator and angiogenic factor) and polyamines (key regulators of DNA and protein synthesis) may provide a unified explanation for the etiology of IUGR in response to maternal undernutrition and overnutrition. There is growing evidence that maternal nutritional status can alter the epigenetic state (stable alterations of gene expression through DNA methylation and histone modifications) of the fetal genome. This may provide a molecular mechanism for the role of maternal nutrition on fetal programming and genomic imprinting. Innovative interdisciplinary research in the areas of nutrition, reproductive physiology, and vascular biology will play an important role in designing the next generation of nutrient-balanced gestation diets and developing new tools for livestock management that will enhance the efficiency of animal production and improve animal well being.     

Endometrial gland surface density and hyperaemia of the endometrium during early pregnancy in the mare

REASON FOR PERFORMING STUDY: The morphology and surface density of endometrial glands responsible for the nutrition of the placenta requires further study employing computer assisted morphometric analysis. OBJECTIVES: To analyse changes in the morphology and surface density of endometrial glands situated adjacent to, and remote from, the conceptus during the first 30 days of gestation. METHODS: Endometrial biopsies were recovered, under videoendoscopic control, from both nonpregnant and pregnant mares 12 days after ovulation, as well as from pregnant mares on Days 16, 20, 25 and 30 of gestation. One biopsy was taken at the base of the nongravid uterine horn and the other from beneath the conceptus at the base of the gravid horn. The samples were evaluated by computer assisted morphometric analysis to determine the surface area per unit volume (Sv) of endometrial glands in the stratum spongiosum. RESULTS: No differences in morphology or density of the glands were discernable between pregnant and nonpregnant mares on Day 12 day after ovulation. On Day 16, however, the glands beneath the conceptus appeared to be at a significantly lower density than the clearly more tortuous glands at the base of the nongravid horn. Between Days 20 and 30 of gestation the endometrium in direct contact with the conceptus was visibly more reddened and hyperaemic than elsewhere in the uterus. However, the Sv of glands beneath the conceptus had now increased again so they appeared similar to those in the nongravid horn, due mainly to a reduction in the level of stromal oedema in the former. CONCLUSIONS AND POTENTIAL RELEVANCE: The embryo-maternal interactions that are vital during early pregnancy to suppress the cyclical luteolytic cascade and support nidation of the conceptus, were characterised here with local endometrial hyperaemia, histotroph production and a decrease in endometrial gland Sv at the commencement of the stationary phase. Both the components secreted by the endometrial glands and the factors liberated by the conceptus during early pregnancy need further investigation.     

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.     

Physiological development of the equine fetus during late gestation

In many species, the pattern of growth and physiological development in utero has an important role in determining not only neonatal viability but also adult phenotype and disease susceptibility. Changes in fetal development induced by a range of environmental factors including maternal nutrition, disease, placental insufficiency and social stresses have all been shown to induce adult cardiovascular and metabolic dysfunction that often lead to ill health in later life. Compared to other precocious animals, much less is known about the physiological development of the fetal horse or the longer-term impacts on its phenotype of altered development in early life because of its inaccessibility in utero, large size and long lifespan. This review summaries the available data on the normal metabolic, cardiovascular and endocrine development of the fetal horse during the second half of gestation. It also examines the responsiveness of these physiological systems to stresses such as hypoglycaemia and hypotension during late gestation. Particular emphasis is placed on the role of the equine placenta and fetal endocrine glands in mediating the changes in fetal development seen towards term and in response to nutritional and other environmental cues. The final part of the review presents the evidence that the early life environment of the horse can alter its subsequent metabolic, cardiovascular and endocrine phenotype as well as its postnatal growth and bone development. It also highlights the immediate neonatal environment as a key window of susceptibility for programming of equine phenotype. Although further studies are needed to identify the cellular and molecular mechanisms involved, developmental programming of physiological phenotype is likely to have important implications for the health and potential athletic performance of horses, particularly if born with abnormal bodyweight, premature or dysmature characteristics or produced by assisted reproductive technologies, indicative of an altered early life environment.     

Early maternal changes contributing to the formation of the chorioallantoic and yolk sac placentas in rat: a morphological study

In order to determine the maternal changes contributing to the formation of the chorioallantoic and yolk-sac placentas, rat gestation sites were examined by light and electron microscopy on days 7 through 10 of pregnancy. On day 7, the implantation chamber showed different compartments and contained the blastocyst in the antimesometrial chamber. The epithelial lining of the implantation chamber disappeared at the antimesometrial chamber, transformed into disintegrated cells in the mesometrial chamber, and showed signs of the programmed cell death in the decidual crypt. On day 8, the mesometrial chamber lumen contained red blood cells and it was continuous with subepithelial sinusoids. The endothelial cells lining the mesometrial sinusoids also showed some characteristics of the sprouting type angiogenesis such as hypertrophy and cell proliferation. While the yolk-sac placental circulation was more obvious with participation of the giant trophoblasts at the antimesometrial pole of the conceptus on day 9, the antimesometrial cells showed autophagic degeneration after the formation of the chorioallantoic placenta on day 10. The contribution of the regional cell death and angiogenesis to form both of the two placentas are discussed.     

Differential effects of the endocrine-disrupting compounds bisphenol-A and octylphenol on gonadotropin secretion, in prepubertal ewe lambs

This study examined the effects of long-term exposure to the endocrine-disrupting compounds (EDCs) Bisphenol-A (BPA) and Octylphenol (OP) on gonadotrophin secretion in pre-pubertal female sheep. Four-week-old, female lambs were randomly allocated to four groups (n=6), and twice each week treated with i.m. injections of either corn oil (vehicle controls), diethylstilbestrol (DES; 0.175mg/kg), BPA (3.5mg/kg) or OP (3.5mg/kg). After 5 weeks of treatment, animals were ovariectomized (ovx) and ovary weights recorded. Two weeks later, blood samples were collected from lambs every 15min for 6h, for LH pulse analysis. Animals were then euthanased and adrenal and kidney weight recorded. An age-related increase in tonic LH secretion was noted in Control, BPA- and OP-treated lambs, but was absent in DES-treated lambs. Following ovx, LH secretion increased in all except DES-treated lambs; FSH concentrations increased in all groups. BPA and DES significantly suppressed LH pulse frequency (C: 6.7+/-0.3pulses/6h, DES: 1.5+/-0.8pulses/6h, BPA: 2.3+/-0.8pulses/6h) and amplitude (C: 7.1+/-1.0ng/ml, DES: 1.9+/-0.6ng/ml, BPA: 1.6+/-0.4ng/ml). OP had no effect on LH secretion (Frequency: 5.8+/-0.5pulses/6h, amplitude: 8.0+/-2.0ng/ml). Ovary weight was similar among all groups. Results show that chronic in vivo exposure of prepubertal female lambs to BPA, at levels lower than those reported previously, can have significant effects on LH secretion that are comparable to those seen following exposure to the known xenoestrogen, DES. Exposure to an equal dose of the EDC OP, over the equivalent period of time was without effect on gonadotropin secretion in the prepubertal ewe lamb. These results indicate that exposure of prepubertal female lambs to the EDC BPA can induce significant effects on gonadotropin secretion, the potential long-term effects of exposure and the effects of these changes on reproductive performance and efficacy, therefore, merit further study.     

The proliferation inhibitory proteins p27(Kip1) and retinoblastoma are involved in the control of equine lymphocyte proliferation

Observations in early equine pregnancy clearly reveal maternal immune recognition of and response to the presence of the conceptus. Nevertheless, both maternal cellular and humoral responses appear ineffective in destroying the developing placenta and fetus in early pregnancy. Our previous studies had shown that the pre-conditioned medium generated from the culture of equine invasive trophoblast inhibited mitogen-induced lymphocyte proliferation and the expression of cytokine messenger RNA in vitro. Those findings also suggested that lymphocytes might have been halted in the G0/G1 phase of the cell cycle. To characterize the cell cycle and the intracellular mechanisms involved in the inhibition of lymphocyte proliferation, equine peripheral blood lymphocytes were cultured in the presence or absence of pokeweed mitogen (PWM) in fresh medium, or in medium pre-conditioned through cell culture of invasive trophoblast cells or fetal fibroblasts. Two-color flow cytometric analysis for bromodeoxyuridine (BrdU) incorporation by stimulated lymphocytes, and concomitant DNA staining with 7-amino-actinomycin D (7-AAD), indicated that a greater proportion of lymphocytes were found in the G0/G1 phase of the cell cycle when cultured in the invasive trophoblast cell pre-conditioned medium compared to controls. Analysis using carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescence intensity demonstrated that lymphocytes cultured in the presence of invasive trophoblast cell pre-conditioned medium had fewer cells going through division, but that those fewer cells sustained similar numbers of cell divisions as in control cultures. Hypophosphorylated retinoblastoma (Rb) protein expression was increased and p27Kip1 expression was maintained at higher levels in lymphocytes cultured in invasive trophoblast pre-conditioned medium compared to fresh medium. In agreement with these data, flow cytometric measurement of the Ki-67 protein expression in lymphocytes cultured in invasive trophoblast pre-conditioned medium was lower in comparison to controls. These findings suggest that the equine lymphocyte proliferation is at least partially regulated by the expression of proliferation inhibitory proteins such as p27Kip1 and hypophosphorylated Rb. These proteins seem to be important regulators of cell cycle transition between G1 and S phase in equine lymphocytes.     

The impacts of uterine environment and fetal genotype on conceptus size and placental vascularity during late gestation in pigs.

Meishan and Yorkshire gilts were bred exclusively to Yorkshire and Meishan boars, respectively, resulting in similar Meishan x Yorkshire fetuses gestating in the uteri of both maternal breeds. Gilts of both breeds were slaughtered on d 90 and 110 of gestation, and the weight and volume of each uterine horn was determined. Fetal weights and crown-rump lengths were recorded. A section of the chorioallantoic-endometrial attachment site was collected for each conceptus and evaluated for placental and endometrial vascular density. An intact placenta was recovered for each conceptus and weighed, and its surface area was determined. Fetal weight and crown-rump length increased (P < .001) markedly between d 90 and 110 of gestation in Meishan and in Yorkshire uteri, but they were markedly less (P < .001) in Meishan than in Yorkshire uteri. Placental weight and placental surface area were reduced by 40% in Meishan, compared with Yorkshire, uteri; however, placental size did not increase between d 90 and 110 in either uterine type. Placental vascular density and associated endometrial vascular density were similar (P > .20) for conceptuses in Meishan and Yorkshire uteri on d 90 and 110 of gestation. Additionally, even though the ratio of fetal weight: placental weight (placental efficiency) increased between d 90 and 110, placental efficiency was similar for conceptuses in Meishan and Yorkshire uteri. In a previous study using straightbred Meishan or Yorkshire conceptuses gestated in either a Meishan or Yorkshire uterus, we found Meishan conceptuses had a markedly greater placental efficiency than did Yorkshire conceptuses, regardless of the type of uterus in which they were gestated. These data indicate that uterine type determines conceptus size and conceptus genotype controls placental efficiency.