| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Embryo; |
School of Biological Sciences,3 University of Southampton, Southampton SO16 7PX, United Kingdom
Institute of Developmental Sciences,4 Developmental Origins of Health and Disease Division, School of Medicine, and MRC Epidemiology Resource Centre,5 University of Southampton, Southampton General Hospital, Southampton SO16 0YD, United Kingdom
ABSTRACT
Poor maternal nutrition during pregnancy can alter postnatal phenotype and increase susceptibility to adult cardiovascular and metabolic diseases. However, underlying mechanisms are largely unknown. Here, we show that maternal low protein diet (LPD), fed exclusively during mouse preimplantation development, leads to offspring with increased weight from birth, sustained hypertension, and abnormal anxiety-related behavior, especially in females. These adverse outcomes were interrelated with increased perinatal weight being predictive of later adult overweight and hypertension. Embryo transfer experiments revealed that the increase in perinatal weight was induced within blastocysts responding to preimplantation LPD, independent of subsequent maternal environment during later pregnancy. We further identified the embryo-derived visceral yolk sac endoderm (VYSE) as one mediator of this response. VYSE contributes to fetal growth through endocytosis of maternal proteins, mainly via the multiligand megalin (LRP2) receptor and supply of liberated amino acids. Thus, LPD maintained throughout gestation stimulated VYSE nutrient transport capacity and megalin expression in late pregnancy, with enhanced megalin expression evident even when LPD was limited to the preimplantation period. Our results demonstrate that in a nutrient-restricted environment, the preimplantation embryo activates physiological mechanisms of developmental plasticity to stablize conceptus growth and enhance postnatal fitness. However, activation of such responses may also lead to adult excess growth and cardiovascular and behavioral diseases.
behavior, blastocyst, blood pressure, conceptus, developmental biology, embryo, environment, growth, low protein diet, megalin, preimplantation embryo, yolk sac
A critical objective in developmental biology and its relationship with adult health is to identify stages and causes of plasticity where environmental cues shape the inherent morphogenetic program to improve the competitive fitness of offspring. The developmental origins of health and disease hypothesis proposes that such evolutionary-conserved adaptive changes, induced in response to maternal diet and other environmental factors and involving a combination of epigenetic, cellular, and physiological mechanisms, ensure that the growth, metabolism, and phenotype of offspring match the prevailing nutrient availability. However, such "predictive" homeostatic processes may turn out to be unsuitable and lead to adult disease, including diabetes, obesity, and cardiovascular dysfunction, if gestational and postnatal nutrient availability are inconsistent [1–4].
Although there is good evidence to support the concept of developmental plasticity and its association with adult health and disease, the specific periods during development in which environmental factors may induce sustained changes in phenotype and the mechanisms by which such changes may propagate into postnatal life are poorly defined. The preimplantation embryo, in which founder cell lineages for fetal and extraembryonic tissues are established in the blastocyst, is known for its environmental sensitivity both in vivo and in vitro [5–7], in particular with respect to assisted reproductive treatments designed to overcome infertility or improve fecundity [8–12]. Previously, we have shown rat embryos to be sensitive to maternal protein undernutrition, leading to abnormal fetal development and postnatal metabolic disease [13–15]. Similarly, periconceptual maternal undernutrition in domestic animals has been found to induce fetal and postnatal changes that may be associated with later disease [16–21]. However, a mechanistic basis linking preimplantation environment in vivo with later ill health is yet to be realized.
In the current report, we investigate the sensitivity of preimplantation embryos to maternal protein undernutrition using a new mouse model with large sample size and with respect to diverse aspects of postnatal phenotype and health. We also evaluate the biological response made by embryos to dietary challenge and how this response may affect later development and the risk of adult disease. Our data reveal the capacity of preimplantation embryos to sense and respond to the quality of maternal diet and highlight a novel physiological mechanism mediated through the extraembryonic lineage by which environment during early development contributes to the etiology of adult disease.
MF1 mice, under U.K. Home Office license and local ethics approval, were bred in-house (University of Southampton Biomedical Research Facility) on a 0700–1900 light cycle with standard chow. Virgin females (7–8.5 wk) were mated naturally overnight with MF1 studs. Plug-positive females were housed individually the following morning and assigned without preference to either (a) normal protein diet (NPD, 18% casein) throughout gestation, (b) low protein diet (LPD, 9% casein) throughout gestation, or (c) LPD for 3.5 days before being switched to NPD for the remainder of gestation (Emb-LPD). Diet composition [13, 22] is shown in Supplemental Table 1 (available at www.biolreprod.org) with constituents supplied by Special Diets Service, Sigma, and ICN Biomedicals. All pregnancies developed to term. Mothers and all offspring, after weaning at 3 wk, were fed standard chow. Litter size was adjusted to six (three per gender) after birth weight determination by selecting pups closest to the mean litter male or female birth weight, the sexes caged separately from weaning. The design included 19 litters per treatment and animal coding such that all assays were conducted "blind" to ensure objectivity.
|
Offspring were weighed at birth and weekly up to 28 wk. Systolic blood pressure was determined at 9, 15, and 21 wk by tail-cuff plethysmography between 1000 and 1400 h as previously described [12, 23]. Behavioral tests were conducted at 4, 5, 6, 8, 11, 14, 17, and 20 wk (between 1000 and 1200 h, or overnight where stated), with Weeks 4–6 being acclimatizing and Weeks 8–20 as assay tests. These tests included burrowing, nesting, and glucose solution consumption for affective behavior, as described previously [24, 25], and an illuminated, open field (white PVC arena, 30 cm2) test for anxiety-related behavior [24, 25], composed of 3-min observations with automated recording of total distance traveled, number of hind rears and jumps, time spent resting, and average velocity. At 28 wk, mice were weighed and culled, and their organs (liver, left and right kidneys, heart, lungs, brain, cerebellum) were weighed and freezer stored. Some offspring were used at different times up to 28 wk in alternative assays other than those reported here, which, together with rare mortalities, required their removal from the study. Litter and actual offspring numbers used in postnatal assays are provided in relevant figure and supplemental table legends (available online at www.biolreprod.org).
Mice at 3.5 days after mating and diet treatment were culled, and their blastocysts were collected by uterine flushing and transiently held in T6 medium (
1 h) before insertion into the uterine lumen of synchronized pseudopregnant MF1 recipients using standard techniques [12]. Six blastocysts derived from NPD-treated or Emb-LPD-treated mothers were transferred to apposing uteri of foster mothers, and conceptus growth was assessed at Day 17 postcoitus in multiparous horns by dissection and weighing of entire conceptus and component parts (fetus and placenta, separate from yolk sac).
Yolk sacs, immediately after dissection from diet-treated mothers, were cultured for 5 h in Medium 199 (Life Sciences) containing 10% fetal calf serum (pH 7.4, 5% CO2, 37°C) and [14C]-sucrose (Amersham Radiochemicals), a marker for fluid-phase endocytosis in rodent yolk sacs [26]. Yolk sacs were subsequently washed x5 in PBS, digested overnight in 1 ml 0.25 M NaOH, and neutralized in 1 ml 0.25 M HNO3, before total protein determination (Biorad) and scintillation counting of samples and medium. The endocytic index (µl fluid captured hr/mg protein) was determined [27].
Yolk sacs, upon isolation from mothers, were processed for electron microscopy by fixation in 3% glutaraldehyde, 4% formaldehyde in 0.1 M PIPES, postfixation in 1% osmium tetroxide in PIPES, ethanol dehydration, and TAAB epoxy resin embedding. Ultrasections were stained and examined on a Hitachi H7000 transmission electron microscope. Micrographs at standardized magnification were analyzed using Interactive Biological Analysis System software (Imaging Associates Ltd.) to determine visceral yolk sac endoderm (VYSE) cell surface area and vesicle dimensions.
Yolk sacs were individually homogenized in 1% SDS buffer, boiled, centrifuged (13 000 rpm, 5 min), and stored at –80°C before thawing, determining total protein (Biorad), solubilizing in NuPAGE 4x LDS sample buffer, conducting SDS-PAGE using NuPAGE 3%–8% Tris acetate gels (Invitrogen) with equal total protein loading for all samples, immunoblotting onto PVDF membranes (Millipore), detecting megalin using a mouse monoclonal antibody [28], and determining integrated density values in ratio to E-cadherin using Odyssey infrared imaging (Licor). E-cadherin is highly expressed in yolk sac and was used as a control because we found its expression level did not change with respect to maternal diet.
Megalin (LRP2) localization was determined in sections of 4%-formaldehyde-fixed, Histoclear-embedded yolk sacs (Raymond A. Lamb), using specific antibody [28] after blocking endogenous IgG and standard immunoperoxidase.
Gestation length, mean litter size at birth, and gender ratio were analyzed using one-way ANOVA (SigmaStat, version 3.5). All other postnatal data were analyzed using multilevel random effects regression (SPSS, version 14) to account for their hierarchical structure. Maternal and offspring effects were estimated simultaneously, incorporating between-mother and within-mother variation and different parameters measured from individual animals [12, 23, 29]. Thus, differences identified between treatment groups are independent of maternal origin of litter, litter size, and, for nongrowth data, body weight. Growth data were additionally converted to Z-scores prior to statistical analysis, using SPSS, to standardize the entire data set to the same scale (mean 0, SD 1) to compare entire growth curves across treatment groups. Correlations were analyzed using Pearson correlation (SPSS, version 14). Significant differences between correlations were analyzed using random effects regression. For all tests, sample numbers and P values are included in text, figure legends, or supplemental tables (available online at www.biolreprod.org).
Maternal Protein Diet During Preimplantation Period Affects Postnatal Health
To investigate the importance of maternal diet during preimplantation development on adult disease risk, we mated virgin female mice, raised on standard laboratory chow, with males overnight. The following morning we assigned pregnant females to either (a) control, 18% casein, NPD throughout gestation; (b) 9% casein, LPD, made isocaloric to NPD by increased starch and sucrose, throughout gestation; or (c) LPD for just 3.5 days corresponding to the preimplantation period, followed by NPD for the remainder of gestation (Emb-LPD) (Fig. 1A). LPD used here is a mild diet restriction, sufficient for a nonpregnant rodent, and all diets contain equivalent vitamin and micronutrient levels [22] (see Supplemental Table 1 for composition). At term, litters (19 per treatment) were standardized to six, and all offspring were fed standard chow up to culling at 28 wk. No significant between-treatment differences were evident for gestation length, litter size, or gender ratio (Table 1).
|
Birth weight and subsequent weekly weight increases were similar in NPD and LPD offspring. However, Emb-LPD offspring were heavier at birth than NPD (males and females combined P = 0.036, females P = 0.026, males P > 0.05), and, after litter size adjustment, Emb-LPD females remained heavier throughout postnatal life (Fig. 1B). This difference, together with other outcomes reported later, was independent of potential hierarchical factors including individual mother origin and gestational litter size. Offspring systolic blood pressure (SBP) was measured at 9, 15, and 21 wk, and the mean of these for each mouse was termed "life SBP". Both male and female offspring from LPD and Emb-LPD treatments exhibited raised SBP (P < 0.05) compared with NPD (Fig. 1C). Offspring behavior was also analyzed, using tests of species-typical behaviors that inform on affective-related and anxiety-related activity [24, 25], repeated at Weeks 8, 11, 14, 17, and 20. Although burrowing, nesting, and glucose solution consumption assays did not indicate differences in affective behavior between groups (Supplemental Fig. 1, available online at www.biolreprod.org), female Emb-LPD animals showed differences in open field activity, rearing and jumping, with respect to NPD controls (P < 0.04, Fig. 2). Terminal analysis of organ allometry at 28 wk showed organ size to be proportionate to body size for males and females from all treatments, except for the heart, which was undersized in female LPD and Emb-LPD offspring (Fig. 1D, Supplemental Table 2, available online at www.biolreprod.org). Moreover, heart:body weight ratio in Emb-LPD females was negatively correlated with their life SBPs (r2 = –0.294, P = 0.039).
|
These data show that poor maternal diet during preimplantation development is associated with broad changes in postnatal phenotype, affecting growth, cardiovascular, and behavioral criteria. To evaluate the mechanisms, we reasoned that maternal protein undernutrition (LPD, Emb-LPD) may induce compensatory responses in the early embryo to promote subsequent prenatal growth in conditions of low nutrient availability. When protein restriction is consistent throughout pregnancy (LPD group), normal perinatal weight results. However, when early responses become inappropriate for conditions in later gestation (Emb-LPD), excess perinatal growth will ensue.
Increased Perinatal Weight is Predictive of Adult Disease
The relationship between increased perinatal growth seen in Emb-LPD offspring and other later phenotypic outcomes was evaluated to assess whether the growth increase during pregnancy might be a key factor in the onset of adult disease. Positive correlations, usually significant (P < 0.05), existed in males and females from all treatment groups between body weight, either at lactation (3 wk, when individual animals were tail-marked) or immediately thereafter (4–6 wk) and adult weight, either when SBP was determined (9, 15, 20 wk) or at time of culling (28 wk) (Fig 3A, Supplemental Table 3 [available online at www.biolreprod.org]). For Emb-LPD females, the increased weight compared with NPD and LPD groups observed during postnatal life (Fig. 1B) derives from continuous, usually week-by-week, relative weight gain over other groups, rather than a growth spurt over any specific time period (Fig. 3B). Body weight at 3 wk was also strongly correlated (P < 0.035) with subsequent life SBP for males and females from all treatments. Although in NPD controls this was a negative correlation, reflecting the expected association between low weight in early life and elevated SBP in later life [1–4], both LPD and Emb-LPD offspring exhibited positive correlations, different (P < 0.05) from controls (Fig. 3C), indicating an association between excess perinatal weight and later hypertension. Correlations between behavioral outcomes and either early body weight or later SBP were less evident (Supplemental Table 4, available online at www.biolreprod.org). However, adult body weight in Emb-LPD females correlated positively with open-field activities (rears, jumps; both P < 0.05) and negatively at trend level with open-field time spent resting (P < 0.069). Lastly, birth weight and 3-wk weight both correlated with gestational litter size (Fig. 3D), independently of the differences in weight induced by diet discussed earlier. Thus, the expected strong negative correlation between birth weight and litter size was seen in males and females in NPD and LPD groups; however, this correlation was lost in Emb-LPD offspring, reflecting the capacity for increased perinatal growth to occur especially in litters of larger size.
|
These results demonstrate that the increase in perinatal weight induced in response to Emb-LPD and LPD treatments is indeed predictive of increased weight and/or hypertension in adult life and, in Emb-LPD females, may indirectly lead to altered behavior through increased adult weight. Subsequent studies, therefore, focused on the cause of the increased perinatal weight.
Maternal Protein Diet Induces Adaptive Response Within the Blastocyst
We next investigated whether the apparent induction of adaptive responses to maternal protein undernutrition resided in the embryo and its derivative tissues or depended upon continued interaction with maternal signals and physiological status. We collected blastocysts from mothers at 3.5 days following NPD and Emb-LPD treatments and immediately transferred them into apposed uterine horns of synchronized pseudopregnant NPD recipient females. These pregnancies were allowed to proceed to Day 17 before conceptuses were collected and weighed. We found no difference in horn litter size between treatments, but conceptus weight at Day 17 was increased (P < 0.05) when derived from Emb-LPD blastocysts compared with NPD blastocysts (Fig. 4A). Moreover, although not significant, mean fetal and placental weights were also higher in Emb-LPD conceptuses, indicating a proportionate increase in growth of components such that the fetal:placental weight ratio was unchanged between treatments. Thus, the growth adaptations induced in response to maternal diet resided specifically within the early embryo at the blastocyst stage, independent of continued maternal environment.
|
Maternal Protein Diet Alters Nutrient Retrieval Capacity of the Visceral Yolk Sac
The preimplantation embryo from the blastocyst stage onward differentiates and segregates embryonic (fetal) and extraembryonic (placental, yolk sac) lineages. Because the increases in perinatal growth in response to Emb-LPD and LPD treatments are predictive of later disease (Fig. 3), we next investigated the physiological basis for this increase. The extraembryonic lineages segregated at the blastocyst stage are likely contributors to preimplantation responses to maternal diet because they control the maternal-fetal nutrient transport capacity. Here, we focus on the yolk sac, which derives from the blastocyst inner cell mass and gives rise to the VYSE that, in rodents, surrounds the conceptus from early postimplantation development onward throughout gestation. The VYSE functions, in part, in histotrophic nutrition through endocytic uptake and lysosomal digestion of maternal proteins and delivery of released amino acids for fetal growth [30]. The contribution of rodent VYSE cells to fetal nutrition extends from before development of the chorio-allantoic placenta until term, and in late gestation a significant proportion of fetal amino acids is thought to derive from VYSE histotrophic activity [30, 31].
We first investigated the endocytic capacity of the VYSE in vitro at 12 and 17 days postcoitus following isolation and immediate culture in standard medium containing [14C]-sucrose, a marker of fluid-phase endocytosis for this tissue [26]. Comparing LPD and NPD treatments, the rate of fluid-phase endocytosis (µl captured hr/mg protein) was increased (P < 0.05) at Day 17 in the LPD yolk sacs (Fig. 4B). Moreover, morphometric analysis of the ultrastructure of the polarized VYSE epithelial cells revealed more numerous (P < 0.05) apical endocytic vesicles in Day 17 LPD samples than in NPD cells (Fig. 4, C and D). The VYSE employs apical membrane receptors, in particular megalin (LRP2), a large (600 Mr x 10–3) transmembrane glycoprotein belonging to the low density lipoprotein receptor gene family, for endocytosis of many plasma proteins to supply fetal growth [32, 33]. Megalin protein expression and localization were examined in conceptuses following maternal diet treatments. Megalin protein expression was upregulated (P < 0.05) in LPD and Emb-LPD yolk sacs at Day 17 compared with NPD yolk sacs (Fig. 4E), however, its distribution was not affected, it being preferentially localized to the apical membrane and associated cytoplasm in all treatments (Fig. 4F).
Our data firstly illustrate the importance of maternal nutrition during early embryo development in the establishment and control of the growth trajectory of the conceptus and in the onset of disease in adult life. The range of adverse outcomes identified following maternal LPD during the preimplantation period was broad, involving different body systems, and it affected growth, cardiovascular physiology, behavior, and organ allometry. Thus, the mouse embryo shows sensitivity to early nutritional restriction similar to that of the rat embryo with respect to excess postnatal growth and hypertension, as we have shown previously [13]. However, the changes observed here in offspring open-field activity in response to Emb-LPD treatment are the first to associate adult anxiety-related behavior with nutrition during early development. Learning and behavior changes in mice have previously been demonstrated in response to embryo culture protocols [8, 9], indicating further consistency in long-term outcomes to different forms of preimplantation experience [5–7, 12]. We found that overweight and behavioral outcomes affected female offspring preferentially. Although the basis for this gender distinction is currently unknown, possible causes may include gonadal hormone influences on appetite regulation and anxiety-mediated behavior [34].
A second critical conclusion from our large data set, made possible by individual animal coding from the time of weaning to relate different outcomes, is that the dietary-induced change in perinatal growth detected at 3 wk in the Emb-LPD group is a strong predictor of those animals that will exhibit overgrowth and hypertension in later life (Fig. 3). This is consistent with the central concept of the developmental origins of health and disease hypothesis derived from epidemiological research that susceptibility to adult disease, although having adult lifestyle risk factors, correlates independently and more strongly with early life factors such as weight and body form at birth [1–4]. Using embryo transfer techniques, we show that the growth enhancement detected in late pregnancy following Emb-LPD treatment appears to be already induced or "programmed" in the blastocyst, independent of a continued maternal LPD environment during subsequent gestation. This novel finding suggests that the recently segregated embryonic and extraembryonic cell lineages of the blastocyst may be sensitive to nutritional cues for their subsequent maturation as well as to classical, intrinsic mechanisms. Thus, our data not only support the developmental origins of health and disease hypothesis concept that mismatches between gestational and postnatal diets are contributory to disease outcomes [1–4] but also that there is a critical requirement for consistency in maternal diet between preimplantation and postimplantation periods. Thus, when consistency is present, adaptive responses induced in the blastocyst extraembryonic lineages (discussed later) by maternal protein undernutrition (LPD group) lead to normal fetal and postnatal growth, likely to protect offspring from loss in reproductive fitness. However, in the absence of such consistency (Emb-LPD group), adaptive responses by the blastocyst become inappropriate, leading to overgrowth. Critically, because perinatal growth (3 wk postnatal) is a positive predictor of later hypertension in both Emb-LPD and LPD groups (Fig. 3C), it is the activation of the blastocyst response to stimulate conceptus growth rather than its appropriateness with respect to later nutrient availability that predisposes to adult disease. However, the disease phenotype appears exacerbated following Emb-LPD treatment in females because hypertension is accompanied by overweight and abnormal behavior.
A third novel finding in our study is the elucidation of a physiological mechanism by which the blastocyst may respond to nutrient signals by altering the development and function of the extraembryonic yolk sac. We investigated the yolk sac because it has been shown to contribute to early fetal growth and derives from blastocyst morphogenesis [30]. Our data, showing enhanced endocytosis, increased presence of endocytic vesicles, and stimulated megalin receptor expression in yolk sacs at 17 days postcoitus, a time coincident with maximal fetal growth, in response to LPD or Emb-LPD treatments, identify one mechanistic pathway apparently linking blastocyst nutritional environment with later capacity for fetal growth. Although this manifestation of the response to maternal diet by the yolk sac is appropriately timed to influence fetal growth, the proximate components of this proposed mechanism and its gender relatedness still need to be identified. In late gestation, rodent yolk sac processing of maternal proteins and delivery after digestion of liberated amino acids to the fetal compartment is considered an important nutrient source for fetal growth alongside the chorioallantoic placenta [30, 31]. Our data are the first to show that yolk sac histotrophic function is subject to developmental plasticity mediated by diet and that it provides a means by which embryos may respond to poor nutrition to protect fetal growth and competitive fitness. Although the importance of the yolk sac in rodent fetal nutrition has been established, we consider our findings may have wider relevance. The human yolk sac, although organized differently from that of the rodent, has been ascribed an important role in maternal-fetal nutrient exchange during organogenesis and the first trimester [35]. We are currently examining the association between blastocyst environment and yolk sac function at molecular and epigenetic levels to determine the potential causes of this physiological mechanism.
In summary, we find that maternal diet during preimplantation development acts as a signaling input to the early embryo to regulate its future growth, mediated at least in part through the extraembryonic yolk sac lineage and its efficiency in maternal-fetal nutrient transport. Although this physiological pathway is likely to be protective in conditions of poor nutrient availability, the resulting enhancement in growth of the conceptus predisposes to adult disease and may have health implications across species.
ACKNOWLEDGMENTS
We thank M.K. Pratten, A. Page, E. Bishop, V. Pepper, C. Cartledge, and P. Ariyaratnam for assistance or advice with experimental work and the staff of the Biomedical Facility, University of Southampton for their technical support.
FOOTNOTES
1Supported by NIH as part of the NICHD National Cooperative Program on Female Health and Egg Quality under cooperative agreement U01 HD044635 and with partial support from the Medical Research Council, UK (G9800781). R.P. is in receipt of a Medical Research Council, UK, postgraduate studentship; M.A.H. is supported by the British Heart Foundation. 
Correspondence: 2Tom P. Fleming, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, SO16 7PX, United Kingdom. FAX: 44 2380 594459; e-mail: tpf{at}soton.ac.uk
Received: 12 July 2007.
First decision: 21 August 2007.
Accepted: 28 October 2007.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  J.J. Eppig, M.J. O'Brien, K. Wigglesworth, A. Nicholson, W. Zhang, and B.A. King Effect of in vitro maturation of mouse oocytes on the health and lifespan of adult offspring Hum. Reprod., April 1, 2009; 24(4): 922 - 928. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Mitchell, S. L. Schulz, D. T. Armstrong, and M. Lane Metabolic and Mitochondrial Dysfunction in Early Mouse Embryos Following Maternal Dietary Protein Intervention Biol Reprod, April 1, 2009; 80(4): 622 - 630. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Fernandez-Gonzalez, J. de Dios Hourcade, I. Lopez-Vidriero, A. Benguria, F. R. De Fonseca, and A. Gutierrez-Adan Analysis of gene transcription alterations at the blastocyst stage related to the long-term consequences of in vitro culture in mice Reproduction, February 1, 2009; 137(2): 271 - 283. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. D. Morgan, X. L. Jin, A. Li, E. Whitelaw, and C. O'Neill The Culture of Zygotes to the Blastocyst Stage Changes the Postnatal Expression of an Epigentically Labile Allele, Agouti Viable Yellow, in Mice Biol Reprod, October 1, 2008; 79(4): 618 - 623. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Watkins, A. Wilkins, C. Cunningham, V. H. Perry, M. J. Seet, C. Osmond, J. J. Eckert, C. Torrens, F. R. A. Cagampang, J. Cleal, et al. Low protein diet fed exclusively during mouse oocyte maturation leads to behavioural and cardiovascular abnormalities in offspring J. Physiol., April 15, 2008; 586(8): 2231 - 2244. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
trend at P < 0.1 compared with NPD.
