VARIATION IN OFFSPRING SIZE WITH BIRTH ORDER IN PLACENTAL FISH: A ROLE FOR ASYMMETRIC SIBLING COMPETITION?

Asymmetric sibling competition arises when siblings with different competitive abilities share a limited resource. Such competition occurs in species with postnatal parental care and may also occur when mothers provision embryos between fertilization and birth (matrotrophy). We hypothesized that the combination of matrotrophy and the simultaneous provisioning of embryos in different stages of development (superfetation) leads to asymmetric competition between sibling embryos. Moreover, we expect the intensity of this competition to increase with the level of superfetation as high levels of superfetation result in greater temporal overlap between broods. This hypothesis predicts that offspring from early broods, which predominantly compete with less‐developed siblings, will be larger at birth than offspring from later broods, which experience competition from more and less‐developed siblings. Data on offspring size at birth from two populations of the highly matrotrophic fish, Heterandria formosa, and similar studies of poeciliid fish spanning a range of life histories are consistent with our hypothesis. Together these results suggest that sibling competition is a direct consequence of the evolution of matrotrophy and superfetation in poeciliid fish.     

DO EMBRYOS INFLUENCE MATERNAL INVESTMENT? EVALUATING MATERNAL‐FETAL COADAPTATION AND THE POTENTIAL FOR PARENT‐OFFSPRING CONFLICT IN A PLACENTAL FISH

The evolution of matrotrophy introduces the potential for genomic conflicts between mothers and embryos. These conflicts are hypothesized to accelerate the evolution of reproductive isolation and to influence the evolution of life-history traits, reproductive structures, and genomic imprinting. These hypotheses assume offspring can influence the amount of maternal investment they receive and that there is a trade-off between maternal investment into individual offspring and maternal survival or fecundity. We used field data and laboratory crosses to test whether these assumptions are met in the matrotrophic poeciliid fish Heterandria formosa . Comparisons of life histories between two natural populations demonstrated a trade-off between the level of maternal investment into individual embryos and maternal fecundity. Laboratory crosses between individuals from these populations revealed that offspring genotype exerts an influence on the level of maternal investment and affects maternal fecundity through higher rates of embryo abortion and lower numbers of full-term offspring. Our results show that the prerequisites for parent–offspring conflict to be a potent evolutionary force in poeciliid fish are present in H. formosa. However, determining whether this conflict has shaped maternal investment in nature will require disentangling any effects of conflict from those of several ecological factors that are themselves correlated with the expected intensity of conflict.     

Interpopulation variation in life-history traits of Poeciliopsis prolifica: implications for the study of placental evolution

Placental reproduction is widespread across vertebrate taxa, but little is known about its life-history correlates and putative adaptive value. We studied variation in life-history traits in two populations of the placental poeciliid fish Poeciliopsis prolifica to determine whether differences in post-fertilization maternal provisioning to embryos have a genetic basis and how food availability affects reproduction. Life histories were characterized for wild-caught females and for second-generation lab-born females raised under two levels of food availability. We found that the two populations did not differ significantly in the wild for any life-history traits except for the lipid dry weight in females and in embryos at an advanced stage of development. When environmental effects were experimentally controlled, however, populations exhibited significant differences in several traits, including the degree of maternal provisioning to embryos. Food availability significantly affected female size at first parturition, brood size and offspring dry weight at birth. Altogether, these results demonstrate that population differences in maternal provisioning and other life-history traits have a genetic basis and show a plastic response to food availability. We infer that phenotypic plasticity may mask population differences in the field. In addition, when comparing life-history patterns in these two populations with known patterns in placental and non-placental poeciliids, our results support the hypotheses that placentation is an adaptive reproductive strategy under high-resource conditions but that it may represent a cost under low-food conditions. Finally, our results highlight that age at maturity and reproductive allotment may be key life-history traits accompanying placental evolution.     

Parent–offspring conflict and selection on egg size in turtles

The trade‐off between offspring size and number can present a conflict between parents and their offspring. Because egg size is constrained by clutch size, the optimal egg size for offspring fitness may not always be equivalent to that which maximizes parental fitness. We evaluated selection on egg size in three turtle species (Apalone mutica, Chelydra serpentina and Chrysemys picta) to determine if optimal egg sizes differ between offspring and their mothers. Although hatching success was generally greater for larger eggs, the strength and form of selection varied. In most cases, the egg size that maximized offspring fitness was greater than that which maximized maternal fitness. Consistent with optimality theory, mean egg sizes in the populations were more similar to the egg sizes that maximized maternal fitness, rather than offspring fitness. These results provide evidence that selection has maximized maternal fitness to achieve an optimal balance between egg size and number.     

Insulin-like growth factor (IGF) family genes are aberrantly expressed in bovine conceptuses produced in vitro or by nuclear transfer

Embryos produced through somatic cell nuclear transfer (NT) or in vitro production (IVP) are often associated with increased abortion and abnormalities thought to arise from disruptions in normal gene expression. The insulin-like growth factor (IGF) family has a major influence on embryonic, fetal and placental development; differences in IGF expression in NT- and IVP-derived embryos may account for embryonic losses during placental attachment. In the present study, expression of IGF-I, IGF-II, IGF-I receptor (IGF-IR), and IGF-IIR mRNAs was quantitated in Day 7 and 25 bovine embryos produced in vivo, by NT, IVP, or parthenogenesis, to further understand divergent changes occurring during development. Expression of the IGF-I gene was not detected in Day 7 blastocysts for any treatment. However, there were no differences (P>0.10) among Day 7 treatments in the amounts of IGF-IR, IGF-II, and IGF-IIR mRNA. For Day 25 conceptuses, there was higher expression of IGF-I mRNA for NT and IVP embryonic tissues than for in vivo embryonic tissues (P<0.05). Furthermore, embryonic tissues from NT-derived embryos had higher expression of IGF-II mRNA than IVP embryonic tissues (P<0.05). Placental expression of IGF-IIR mRNA was greater for NT-derived than in vivo-derived embryos (P<0.05). There were no differences in IGF-IR mRNA across all treatments and tissues (P>0.10). In conclusion, these differences in growth factor gene expression during early placental attachment and rapid embryonic growth may directly or indirectly contribute to increased losses and abnormalities in IVP- and NT-derived embryos.     

Placental polar lipid composition is associated with placental gene expression and neonatal body composition

The polar-lipid composition of the placenta reflects its cellular heterogeneity and metabolism. This study explored relationships between placental polar-lipid composition, gene expression and neonatal body composition.Placental tissue and maternal and offspring data were collected in the Southampton Women's Survey. Lipid and RNA were extracted from placental tissue and polar lipids measured by mass spectrometry, while gene expression was assessed using the nCounter analysis platform. Principal component analysis was used to identify patterns within placental lipid composition and these were correlated with neonatal body composition and placental gene expression.In the analysis of placental lipids, the first three principal components explained 19.1%, 12.7% and 8.0% of variation in placental lipid composition, respectively. Principal component 2 was characterised by high principal component scores for acyl-alkyl-glycerophosphatidylcholines and lipid species containing DHA. Principal component 2 was associated with placental weight and neonatal lean mass; this component was associated with gene expression of APOE, PLIN2, FATP2, FABP4, LEP, G0S2, PNPLA2 and SRB1. Principal components 1 and 3 were not related to birth outcomes but they were associated with the gene expression of lipid related genes. Principal component 1 was associated with expression of LEP, APOE, FATP2 and ACAT2. Principal component 3 was associated with expression of PLIN2, PLIN3 and PNPLA2.This study demonstrates that placentas of different sizes have specific differences in polar-lipid composition and related gene expression. These differences in lipid composition were associated with birth weight and neonatal lean mass, suggesting that placental lipid composition may influence prenatal lean mass accretion.     

Do density‐driven mating system differences explain reproductive incompatibilities between populations of a placental fish?

Matrotrophy, the provisioning of embryos between fertilization and birth, creates the potential for conflict between mothers and embryos over the level of maternal investment. This conflict is predicted to drive the evolution of reproductive isolation between populations with different mating systems. In this study, we examine whether density‐driven mating system differences explain the patterns of asymmetric reproductive isolation observed in previous studies involving four populations of the matrotrophic least killifish, Heterandria formosa. Minimum sire number reconstructions suggested that two populations characterized by low densities had lower levels of concurrent multiple paternity than two populations characterized by high densities. However, low levels of genetic variation in the low‐density populations greatly reduced our probability of detecting multiple mating in them. Once we took the lower level of genetic variation into account in our estimations, high levels of multiple paternity appeared the rule in all four populations. In the population where we had the greatest power of detecting multiple mating, we found that multiple paternity in H. formosa typically involves multiple sires contributing to offspring within the same brood instead of different fathers contributing to distinct, simultaneously provisioned broods. Paternity was often skewed towards one sire. Our results suggest that differences between H. formosa populations in the levels of multiple paternity are not sufficient to explain the reproductive isolation seen in previous studies. We suggest that other influences on maternal–foetal conflict may contribute to the pattern of reproductive isolation observed previously. Alternatively, the asymmetric reproductive isolation seen in previous studies might reflect the disruption of maternal–foetal coadaptation.     

Cross-generational environmental effects and the evolution of offspring size in the Trinidadian guppy Poecilia reticulata

Abstract The existence of adaptive phenotypic plasticity demands that we study the evolution of reaction norms, rather than just the evolution of fixed traits. This approach requires the examination of functional relationships among traits not only in a single environment but across environments and between traits and plasticity itself. In this study, I examined the interplay of plasticity and local adaptation of offspring size in the Trinidadian guppy, Poecilia reticulata. Guppies respond to food restriction by growing and reproducing less but also by producing larger offspring. This plastic difference in offspring size is of the same order of magnitude as evolved genetic differences among populations. Larger offspring sizes are thought to have evolved as an adaptation to the competitive environment faced by newborn guppies in some environments. If plastic responses to maternal food limitation can achieve the same fitness benefit, then why has guppy offspring size evolved at all? To explore this question, I examined the plastic response to food level of females from two natural populations that experience different selective environments. My goals were to examine whether the plastic responses to food level varied between populations, test the consequences of maternal manipulation of offspring size for offspring fitness, and assess whether costs of plasticity exist that could account for the evolution of mean offspring size across populations. In each population, full‐sib sisters were exposed to either a low‐ or high‐food treatment. Females from both populations produced larger, leaner offspring in response to food limitation. However, the population that was thought to have a history of selection for larger offspring was less plastic in its investment per offspring in response to maternal mass, maternal food level, and fecundity than the population under selection for small offspring size. To test the consequences of maternal manipulation of offspring size for offspring fitness, I raised the offspring of low‐ and high‐food mothers in either low‐ or high‐food environments. No maternal effects were detected at high food levels, supporting the prediction that mothers should increase fecundity rather than offspring size in noncompetitive environments. For offspring raised under low food levels, maternal effects on juvenile size and male size at maturity varied significantly between populations, reflecting their initial differences in maternal manipulation of offspring size; nevertheless, in both populations, increased investment per offspring increased offspring fitness. Several correlates of plasticity in investment per offspring that could affect the evolution of offspring size in guppies were identified. Under low‐food conditions, mothers from more plastic families invested more in future reproduction and less in their own soma. Similarly, offspring from more plastic families were smaller as juveniles and female offspring reproduced earlier. These correlations suggest that a fixed, high level of investment per offspring might be favored over a plastic response in a chronically low‐resource environment or in an environment that selects for lower reproductive effort     

Effects of feed restriction during pregnancy on maternal reproductive outcome,foetal hepatic IGF gene expression and offspring performance in the rabbit

Primiparous female rabbits have high nutritional requirements and, while it is recommended that they are subjected to an extensive reproductive rhythm, this could lead to overweight, affecting reproductive outcomes. We hypothesised that restricting food intake during the less energetic period of gestation could improve reproductive outcome without impairing offspring viability. This study compares two groups of primiparous rabbit does in an extensive reproductive programme, one in which feed was restricted from Day 0 to Day 21 of gestation (R021), and another in which does were fed ad libitum (control) throughout pregnancy. The mother and offspring variables compared were (1) mother reproductive outcomes at the time points pre-implantation (Day 3 postartificial insemination [AI]), preterm (Day 28 post-AI) and birth; and (2) the prenatal offspring characteristic IGF system gene expression in foetal liver, liver fibrosis and foetus sex ratio, and postnatal factor viability and growth at birth, and survival and growth until weaning. Feed restriction did not affect the conception rate, embryo survival, or the number of morulae and blastocysts recovered at Day 3 post-AI. Preterm placenta size and efficiency were similar in the two groups. However, both implantation rate (P < 0.001) and the number of foetuses (P = 0.05) were higher in the R021 mothers than controls, while there was no difference in foetal viability. Foetal size and weight, the weights of most organs, organ weight/BW ratios and sex ratio were unaffected by feed restriction; these variables were only affected by uterine position (P < 0.05). Conversely, in the R021 does, foetal liver IGBP1 and IGF2 gene expression were dysregulated despite no liver fibrosis and a normal liver structure. No effects of restricted feed intake were produced on maternal fertility, prolificacy, or offspring birth weight, but control females weaned more kits. Litter weight and mortality rate during the lactation period were also unaffected. In conclusion, pre-implantation events and foetal development were unaffected by feed restriction. While some genes of the foetal hepatic IGF system were dysregulated during pregnancy, liver morphology appeared normal, and the growth of foetuses and kits until weaning was unmodified. This strategy of feed restriction in extensive reproductive rhythms seems to have no significant adverse effects on dam reproductive outcome or offspring growth and viability until weaning.     

Differences in offspring size predict the direction of isolation asymmetry between populations of a placental fish

Crosses between populations or species often display an asymmetry in the fitness of reciprocal F₁ hybrids. This pattern, referred to as isolation asymmetry or Darwin''s Corollary to Haldane''s Rule, has been observed in taxa from plants to vertebrates, yet we still know little about which factors determine its magnitude and direction. Here, we show that differences in offspring size predict the direction of isolation asymmetry observed in crosses between populations of a placental fish, Heterandria formosa. In crosses between populations with differences in offspring size, high rates of hybrid inviability occur only when the mother is from a population characterized by small offspring. Crosses between populations that display similarly sized offspring, whether large or small, do not result in high levels of hybrid inviability in either direction. We suggest this asymmetric pattern of reproductive isolation is due to a disruption of parent–offspring coadaptation that emerges from selection for differently sized offspring in different populations.     

Knockdown of CDKN1C (p57kip2) and PHLDA2 Results in Developmental Changes in Bovine Pre-implantation Embryos

Imprinted genes have been implicated in early embryonic, placental, and neonatal development and alterations in expression levels of these genes can lead to growth abnormalities and embryonic lethality. However, little is known about the functions of bovine imprinted genes during the pre-implantation period. Therefore, the objective of this study was to assess the influence of altered expression of imprinted genes on developmental progress of embryos using small interfering RNA (siRNA). Expression levels of 18 imprinted genes (MAGEL2, UBE3A, IGF2R, NAP1L5, TSSC4, PEG3, NDN, CDKN1C, PHLDA2, MKRN3, USP29, NNAT, PEG10, RTL1, IGF2, H19, MIM1, and XIST) were compared between embryos reaching the blastocyst stage and growth-arrested embryos (degenerates) using quantitative real-time PCR (qRT-PCR). Ten genes were found to be differentially expressed between blastocysts and degenerates. The CDKN1C gene showed the highest upregulation in blastocysts whereas PHLDA2 was highly expressed in degenerates. To assess whether the observed differential gene expression was causative or resultant of embryo degeneration, these genes were selected for functional analysis using siRNA. Injection of siRNA specific to PHLDA2 into one-cell zygotes resulted in a substantial increase in blastocyst development, whereas injection of CDKN1C-specific siRNA resulted in a 45% reduction (P = 0.0006) in blastocyst development. RNA-Seq analysis of CDKN1C-siRNA-injected vs. non-injected embryos revealed 51 differentially expressed genes with functions in apoptosis, lipid metabolism, differentiation, and cell cycle regulation. Gene ontology analysis revealed nine pathways related to cell signaling, metabolism, and nucleic acid processing. Overall, our results show that proper expression levels of the imprinted genes CDKN1C and PHLDA2 are critical for embryo development, which suggests that these genes can be used as markers for normal blastocyst formation.     

Prefeeding larval development time is not correlated with egg size in regular echinoids (Strongylocentrotus species)

Summary

We compared prefeeding development times, from fertilized egg to prism larva, for Strongylocentrotus embryos from four clutches of eggs (each from a different species) differing in size. Development times did not vary consistently with egg diameter, and trends among eggs of different sizes varied with stage of development. In some cases, development times for eggs of intermediate diameter (S. franciscanus) were longer than those for larger or smaller eggs. Although mean egg diameters in clutches ranged from 84 μm (S. purpuratus) to 162 μm (S. pallidus), differences in development time to the last embryonic stage (prism) were very small. We conclude that the inverse relationship between parental investment in offspring and premetamorphic development time in echinoids depends only on the functional consequences of reduced size of feeding larval stages: effects of egg size on prefeeding development time are not evident.     

Costs and benefits of polyandry in a placental poeciliid fish Heterandria formosa are in accordance with the parent-offspring conflict theory of placentation

In viviparous species, a conflict over maternal resource allocation may arise between mothers and embryos, between siblings, and between maternal and paternal genes within an embryo due to relatedness asymmetries. We performed two experiments to study the effects of polyandry and brood relatedness on offspring growth in a placental fish (Heterandria formosa). Polyandry was beneficial as it increased the probability of pregnancy, possibly to avoid genetic incompatibility. However, females mated to four males produced offspring that had a longer maturation time than those of monandrous females. When within-brood relatedness was manipulated, the size of the newborn offspring decreased with time in low-relatedness treatment, whereas in highly related broods, offspring size was constant. Low within-brood relatedness may lead to less cooperative offspring in terms of resource extraction from the mother, which may lead to impaired development during gestation. Offspring conflict may thus reduce the benefits of polyandry in viviparous species.     

A novel pattern of placental leucine transfer during mid to late gestation in a highly placentotrophic viviparous lizard

Placentotrophy is the nourishment of embryos by resources provided via the placenta during gestation. The magnitude and timing of placental nutrient support during pregnancy are important for embryonic growth, especially in highly placentotrophic animals such as mammals. However, no study has yet investigated how placental organic nutrient support may change during pregnancy in highly placentotrophic viviparous reptiles. Amino acids are essential nutrients for embryonic growth and leucine is a common amino acid. The magnitude and timing of placental leucine transfer may affect embryonic growth and mass and, therefore, offspring phenotype. In this study, female Pseudemoia entrecasteauxii, a highly placentotrophic viviparous skink, were collected throughout gestation. We injected (3)H-leucine into these gravid females and assessed the transfer of (3)H-leucine into maternal compartments (i.e., the blood and the liver), and into embryonic compartments (i.e., the embryo, the yolk, and the amniotic fluid). At either 60 or 120 min post-injection, the radioactivity in each sample was extracted and then counted, and the transfer ratio was calculated. Our results provide direct evidence that circulating maternal leucine passes through the placenta into the embryos in this species. The relative rate of placental leucine transfer did not alter during mid to late gestation. This suggests the steady somatic growth of the embryos during mid-late pregnancy is dependent upon the placental transfer of nutrients rather than yolk stores. This pattern of placental nutrient support may determine offspring body size at birth and, therefore, offspring fitness in P. entrecasteauxii.     

Depression in pregnancy,infant birth weight and DNA methylation of imprint regulatory elements

Depressed mood in pregnancy has been linked to low birth weight (LBW, < 2,500 g), a risk factor for adult-onset chronic diseases in offspring. We examined maternal depressed mood in relation to birth weight and evaluated the role of DNA methylation at regulatory sequences of imprinted genes in this association. We measured depressed mood among 922 pregnant women using the CES-D scale and obtained birth weight data from hospital records. Using bisulfite pyrosequencing of cord blood DNA from 508 infants, we measured methylation at differentially methylated regions (DMRs) regulating imprinted genes IGF2/H19, DLK1/MEG3, MEST, PEG3, PEG10/SGCE, NNAT and PLAGL1. Multiple regression models were used to examine the relationship between depressed mood, birth weight and DMR methylation levels. Depressed mood was associated with a more that 3-fold higher risk of LBW, after adjusting for delivery mode, parity, education, cigarette smoking, folic acid use and preterm birth. The association may be more pronounced in offspring of black women and female infants. Compared with infants of women without depressed mood, infants born to women with severe depressed mood had a 2.4% higher methylation at the MEG3 DMR. Whereas LBW infants had 1.6% lower methylation at the IGF2 DMR, high birth weight (> 4,500 g) infants had 5.9% higher methylation at the PLAGL1 DMR compared with normal birth weight infants. Our findings confirm that severe maternal depressed mood in pregnancy is associated with LBW, and that MEG3 and IGF2 plasticity may play important roles.