Embryonic modulation of maternal steroids in European starlings (Sturnus vulgaris)

In birds, maternally derived yolk steroids are a proposed mechanism by which females can adjust individual offspring phenotype to prevailing conditions. However, when interests of mother and offspring differ, parent–offspring conflict will arise and embryonic interests, not those of the mother, should drive offspring response to maternal steroids in eggs. Because of this potential conflict, we investigated the ability of developing bird embryos to process maternally derived yolk steroids. We examined how progesterone, testosterone and oestradiol levels changed in both the yolk/albumen (YA) and the embryo of European starling eggs during the first 10 days of development. Next, we injected tritiated testosterone into eggs at oviposition to characterize potential metabolic pathways during development. Ether extractions separated organic and aqueous metabolites in both the embryo and YA homogenate, after which major steroid metabolites were identified. Results indicate that the concentrations of all three steroids declined during development in the YA homogenate. Exogenous testosterone was primarily metabolized to an aqueous form of etiocholanolone that remained in the YA. These results clearly demonstrate that embryos can modulate their local steroid environment, setting up the potential for parent–offspring conflict. Embryonic regulation must be considered when addressing the evolutionary consequences of maternal steroids in eggs.     

Glucocorticoid metabolism in the in ovo environment modulates exposure to maternal corticosterone in Japanese quail embryos (Coturnix japonica)

Maternal effects have gained attention as a method by which mothers may alter the physiological condition and phenotype of their offspring based upon current environmental conditions. The physiological and phenotypic outcomes of glucocorticoid-mediated maternal effects have been extensively studied in a variety of vertebrates; however, the underlying mechanism is currently unclear. Here, we injected tritiated corticosterone into the yolks of freshly laid Japanese quail eggs (Coturnix japonica) and traced its movement and metabolism through the in ovo development period. We found that corticosterone was extensively conjugated throughout the egg by the end of development, and while minimal corticosterone was detected within the embryo during development, accumulation of a conjugated metabolite in the embryo started to occur on day 6 of development. Because no movement and metabolism of corticosterone occurred in infertile eggs, our findings suggest that embryos are not passive recipients of maternal steroids, but instead appear to possess extensive metabolic capabilities, which may modulate their exposure to maternal steroids.     

The nature and origin of the glycosaminoglycans of the embryonic chick vitreous body

Glycosaminoglycans of the embryonic chicken vitreous were characterized and then were used as markers to establish which tissues synthesize the vitreous humor during development. The glycosaminoglycans are predominantly chondroitin sulfates by several criteria. They are resistant to streptomyces hyaluronidase, an enzyme which degrades only hyaluronate, and are digested by testicular hyaluronidase and chondroitinase AC, enzymes which degrade hyaluronate plus chondroitin 4- and 6-sulfates. On electrophoresis on cellulose acetate in 0.15 M phosphate buffer, pH 6.7, the vitreous glycosaminoglycans migrate slightly slower than authentic chondroitin sulfate, but, in 0.1 N HCl, they migrate very close to chondroitin sulfate standards. Finally, the disaccharides produced by digestion of these radioactively labeled glycosaminoglycans with chondroitinases AC and ABC were identified as Δdi-4S and Δdi-6S, as expected for chondroitin 4- and 6-sulfate. By using incorporation of radioactive precursors into chondroitin sulfates in vitro, we than determined which tissues synthesize the vitreous humor in the developing eye. Late in development, on Day 12–13, the isolated vitreous is very active in chondroitin sulfate synthesis, while the neural retina, the lens, and the pecten are less active and produce a high proportion of enzyme-resistant GAG. The eye tissues isolated from embryos labeled in ovo retain similar amounts and types of glycosaminoglycans, indicating that cells within the vitreous synthesize the vitreous humor glycosaminoglycans at this time. Earlier in development, from Days 6 to 8, the isolated vitreous incorporates very low levels of radioactivity into GAG, but the neural retina incorporates high levels of radioactivity into chondroitin sulfate. When the embryos are labeled in ovo and the same tissues are isolated following incorporation, the vitreous retains more radioactive chondroitin sulfate than does the neural retina. Thus, the vitreous humour glycosaminoglycan is initially synthesized by the neural retina and is secreted into the vitreous space.     

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes,Followed by Intracytoplasmic Sperm Injection,to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.     

Tolerance of whitefish embryos to Pseudomonas fluorescens linked to genetic and maternal effects,and reduced by previous exposure

Juvenile or adult fish can alter their behaviour and rely on an innate and adaptive immune system to avoid/counteract pathogens, while fish embryos have to depend on egg characteristics and may be partly protected by their developing immune system that is building up from a certain age on. We developed an infection protocol that allows testing the reaction of individual whitefish embryos (Coregonus palaea) to repeated exposures to Pseudomonas fluorescens, an opportunistic bacterial fish pathogen. We used a full-factorial in vitro breeding design to separately test the effects of paternal and maternal contributions to the embryos' susceptibility to different kinds of pathogen exposure. We found that a first non-lethal exposure had immunosuppressive effects: pre-exposed embryos were more susceptible to future challenges with the same pathogen. At intermediate and high levels of pathogen intensity, maternal effects turned out to be crucial for the embryos' tolerance to infection. Paternal (i.e. genetic) effects played a significant role at the strongest level of infection, i.e. the embryos' own genetics already explained some of the variation in embryo susceptibility. Our findings suggest that whitefish embryos are largely protected by maternally transmitted substances, but build up some own innate immunocompetence several days before hatching.     

Arrested embryonic development: a review of strategies to delay hatching in egg-laying reptiles

Arrested embryonic development involves the downregulation or cessation of active cell division and metabolic activity, and the capability of an animal to arrest embryonic development results in temporal plasticity of the duration of embryonic period. Arrested embryonic development is an important reproductive strategy for egg-laying animals that provide no parental care after oviposition. In this review, we discuss each type of embryonic developmental arrest used by oviparous reptiles. Environmental pressures that might have directed the evolution of arrest are addressed and we present previously undiscussed environmentally dependent physiological processes that may occur in the egg to bring about arrest. Areas for future research are proposed to clarify how ecology affects the phenotype of developing embryos. We hypothesize that oviparous reptilian mothers are capable of providing their embryos with a level of phenotypic adaptation to local environmental conditions by incorporating maternal factors into the internal environment of the egg that result in different levels of developmental sensitivity to environmental conditions after they are laid.     

Studies on the metabolism of oestrone sulphate. Comparative perfusions of oestrone and oestrone sulphate through isolated rat livers

The metabolism of [4-(14)C]oestrone and of [6,7-(3)H(2)]oestrone sulphate was studied during cyclic perfusion and once-through perfusion of the isolated rat liver. The following results were obtained. 1. As shown by once-through perfusion, the two steroids are metabolized differently during the first passage through the organ. [4-(14)C]Oestrone was taken up by the liver and partly delivered as oestradiol-17beta and oestriol into the medium. After uptake of [6,7-(3)H(2)]oestrone sulphate, only oestrone, liberated by hydrolysis, was delivered into the medium; no oestradiol-17beta or oestriol could be detected in the medium after one passage through the organ. This indicates that intracellular oestrone, which was taken up as such, and oestrone, which derived from intracellular hydrolysis, may be metabolized in different compartments of the liver cell. 2. The results of the cyclic perfusion showed that intracellular oestrone is preferentially conjugated with glucuronic acid, and subsequently excreted into the bile. Intracellular oestrone sulphate is preferably reduced to oestradiol sulphate, thus indicating that oestrone sulphate is a better substrate for the 17beta-hydroxy steroid oxidoreductase than is oestrone. 3. Albumin-bound oestrone sulphate acts as a large reservoir, and in contrast with free oestrone is protected from enzyme attack by its strong binding to albumin. 4. Oestrone sulphate is partly converted into the hormonally active oestrone by liver tissue. This suggests that liver not only inactivates oestrogens, but also provides the organism with oestrone, which is subsequently readily taken up by other organs.     

Ionic and osmotic environment of developing elasmobranch embryos

Synopsis The elasmobranchs display a variety of ionic and osmotic environments for developing embryos. Oviparous species protect their eggs with a tough, fibrous capsule which is highly permeable to ions and urea even at oviposition. Thus the embryonic tissues are bathed by a solution ionically similar to sea water. In the more advanced reproductive style ofSqualus acanthias (a lecithotrophic live bearer) early embryos in egg capsules are retained in utero and bathed in a solution osmotically similar to maternal plasma. Several months into the 22 month gestation period the embryos can iono- and osmoregulate in a uterine solution resembling sea water. Embryos of more advanced viviparous species develop in a solution that is ionically and osmotically similar to maternal plasma. Iono- and osmoregulation by these embryos would appear to be unnecessary. Clearly, in the oviparous elasmobranchs, the ability of the embryo to regulate salts and urea is present at the earliest stage of development. The need for elasmobranch embryos to regulate osmolytes was reduced or delayed as viviparity evolved.     

Do female newts modify thermoregulatory behavior to manipulate egg size?

Reproductive females manipulate offspring phenotypes by modifying conditions during embryogenesis. In ectotherms, the environmental control over embryogenesis is often realized by changes in maternal thermoregulation during gravidity. To determine if reproduction influences thermoregulatory behavior in species where females lay eggs shortly after fertilization (strict oviparity), we compared preferred body temperatures (T_p) between reproductive (egg-laying) and non-reproductive female newts, Ichthyosaura alpestris. Next, we exposed reproductive females to temperatures mimicking T_p ranges of reproductive and non-reproductive individuals to find out whether the maternally modified thermal regime influences ovum and jelly coat volume, and early cleavage rates at the time of oviposition. In the thermal gradient, reproductive females maintained their body temperatures within a narrower range than non-reproductive individuals. The exposure of ovipositing females to temperatures preferred during their reproductive and non-reproductive period had a negligible influence on egg size and early cleavage rates. We conclude that the modification of maternal thermoregulatory behavior provides a limited opportunity to manipulate egg traits in newts.     

Maternally derived hormones,neurosteroids and the development of behaviour

In a wide range of taxa, there is evidence that mothers adaptively shape the development of offspring behaviour by exposing them to steroids. These maternal effects have major implications for fitness because, by shaping early development, they can permanently alter how offspring interact with their environment. However, theory on parent–offspring conflict and recent physiological studies showing that embryos rapidly metabolize maternal steroids have placed doubt on the adaptive significance of these hormone-mediated maternal effects. Reconciling these disparate perspectives requires a mechanistic understanding of the pathways by which maternal steroids can influence neural development. Here, we highlight recent advances in developmental neurobiology and psychiatric pharmacology to show that maternal steroid metabolites can have direct neuro-modulatory effects potentially shaping the development of neural circuitry underlying ecologically relevant behavioural traits. The recognition that maternal steroids can act through a neurosteroid pathway has critical implications for our understanding of the ecology and evolution of steroid-based maternal effects. Overall, compared to the classic view, a neurosteroid mechanism may reduce the evolutionary lability of hormone-mediated maternal effects owing to increased pleiotropic constraints and frequently influence long-term behavioural phenotypes in offspring.     

Evidence of divergent growth rates among populations of the lizard Anolis carolinensis based on experimental manipulations of egg size

Geographic variation in body size is of special interest because it affects nearly all aspects of an organism’s life. I examined whether differences in body size among four populations of the green anole lizard, Anolis carolinensis, were attributable to maternal investment in egg size and/or growth rates of embryos and juveniles. Larger body size and larger egg size relative to female size in the northern part of the range have been documented in this species, and suggested to be adaptive responses to more extreme winters. The current study confirmed the trends in adult size and egg size in the north, but rejected the trend of larger egg size relative to body size in the south. To control for differences in maternal investment in egg size among populations, I performed yolk removals on eggs from two northern populations to produce comparably sized eggs relative to one southern population. This manipulation was designed to minimize the confounding effect of maternal investment in yolk, the primary energy reserves for eggs, so that intrinsic differences in embryonic growth due to metabolism could be investigated. I found that differences in juvenile and, potentially, embryonic growth rates existed among populations of A. carolinensis, both due to and independent of differences in egg size. Juveniles from the northernmost population were bigger not only due to larger egg size, but also due to faster juvenile growth and possibly differences in developmental stage of oviposition or conversion of egg mass to hatchling mass. Larger body size may hold a number of advantages in northern populations of this species, including starvation resistance through winters and better competitive access to food resources and warmer microhabitats.     

Ion and Osmoregulation in Prenatal Elasmobranchs: Evolutionary Implications

The elasmobranchs represent a fascinating series of experimentsin the evolution of maternal support for developing embryos.In oviparous species, eggs are enclosed in a tough, fibrouscapsule. The capsule is very permeable and the embryonic tissuesare bathed in a solution ionically similar to sea water withinhours of oviposition. In the primitively viviparous speciesSqualus acanthias, early embryos in egg capsules are retainedin utero and are bathed in a solution similar to maternal plasma.Several months into the 22 month gestation period the embryosare capable of independent iono- and osmoregulation in a uterinesolution that resembles sea water. Embryos of more advancedviviparous species develop in a solution that is similar tomaternal plasma. Iono- and osmoregulation by these embryos wouldappear to beminimal. It is clear that in the oviparous elasmobranchs,the ability of the egg/embryo to maintain salts and urea atappropriate levels is present at the earliest stage of development.The ability of prenatal elasmobranch embryos to iono- and osmoregulatewould allow the evolution ofa diverse array of reproductivestrategies in the elasmobranchs.     

Embryonic responses to variation in oviductal oxygen in the lizard Sceloporus undulatus from New Jersey and South Carolina, USA

Viviparity in reptiles is hypothesized to evolve in cold climates at high latitudes and high elevations through selection for progressively longer periods of egg retention. Oxygen consumption of embryos increases during development and therefore longer periods of egg retention should be associated with maternal or embryonic features that enhance embryonic oxygen availability. We tested the hypotheses that embryos of the oviparous lizard Sceloporus undulatus from a high-latitude population in New Jersey are oviposited at more advanced developmental stages and have a higher growth rate at low oxygen partial pressures ( p O₂) than embryos from a low-latitude population in South Carolina. These hypotheses were rejected; embryos from the two populations did not differ in embryonic stage at oviposition, survival, rate of differentiation or growth in mass when incubated under simulated in utero conditions at low oxygen concentrations. We also estimated the effective p O₂ experienced by lizard embryos in utero . At an effective p O₂ of 8.6 kPa (9% O₂), development of S. undulatus embryos is arrested at Dufaure and Hubert stage 30 and at a dry mass of 0.8 mg. Physiological and morphological features of gravid females, embryos, or both, that facilitate oxygen uptake for developing embryos appear to be a critical early step during the evolution of reptilian viviparity. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 83 , 289–299.     

Steroid pathway and oestrone sulphate production in canine inflammatory mammary carcinoma

Spontaneous canine mammary inflammatory carcinoma (IMC) shares epidemiologic, histopathologic and clinical characteristics with the inflammatory breast carcinoma (IBC) disease in humans. We have analysed the steroids levels in serum and in tissue homogenates of IMC, the expression of two of their receptors (androgen and β-estrogen) and of three enzymes included in the steroidogenesis pathway (aromatase (CYP19A1), steroid sulphatase (STS) and estrogen sulfotransferase (EST)) trying to explain the specific accumulation of steroids in IMC tissues generating deposits in the form of lipid droplets whose presence can be attributed to steroids secreted by IMC cells. According to our working hypothesis, oestrone sulphate would be the main component of these lipid droplets. The presence of these steroid deposits would contribute to the intense proliferation and invasive behaviour of IMC and IBC, although their involvement in angiogenesis is yet to be demonstrated.     

The use of nonradiolabelled steroid infusions to investigate the origin of oestrone sulphate in postmenopausal women

Infusion of nonradiolabelled dehydroepiandrosterone sulphate (DHA-S) has been used to investigate the possible formation of oestrone sulphate via a sulphated conjugate of androstenedione. The metabolic clearance rate (MCR) of DHA-S also was measured and the mean value (25 1/24h) was similar to values reported using isotopic techniques. Although conversion of DHA-S to 5-androstenediol, a steroid with oestrogenic properties, was detected during infusion of DHA-S, there were no significant increases in plasma levels of conjugated androstenedione or oestrone sulphate. The MCR's oestrone sulphate measured using infusion of nonradiolabelled steroid in two menopausal women were 99 1/24h and 121 1/24h. For one woman, the production rate of oestrone sulphate, calculated from the conversion of oestrone and oestradiol to oestrone sulphate (151 nmol/day) was similar to the measured production rate of oestrone sulphate (144 nmol/day). It is concluded that in menopausal women, oestrone sulphate is derived from conversion of oestrone and oestradiol with no formation occurring via conjugated androstenedione.     

The effect of glucocorticoids on the in vivo conversion of androstenedione to oestrone

In vitro studies have previously shown that the activity of the aromatase enzyme system, which is responsible for the conversion of androstenedione to oestrone, can be stimulated by natural and synthetic glucocorticoids and also by ACTH. In view of the potential physiological importance of such a regulatory mechanism we have examined the effect of administration of dexamethasone, and of ACTH on the conversion of androstenedione to oestrone in vivo. The transfer constants for the conversion of androstenedione to oestrone [( rho]AEBU) measured in two women before administration of dexamethasone were 1.0% and 1.1% and after were 0.9% and 1.2%. Similarly no increase in conversion of androstenedione to oestrone [( rho]AE1BB) was detected after ACTH stimulation (pre = 0.74%, post = 0.77%). It is concluded from this study that glucocorticoids and ACTH do not have a role in regulating aromatase activity in vivo.