Em lagartos, componentes de história de vida apresentam grande plasticidade fenotípica e evoluem de maneira correlacionada. O número de ovos, por exemplo, tende a ser correlacionado positivamente com o tamanho corpóreo das fêmeas grávidas, tanto em linhagens evolutivas quanto entre indivíduos, embora alguns tipos de hábito pareçam restringir ninhadas maiores. Há indícios de que outros fatores, como componentes climáticos, ou latitude, também sejam determinantes das características de história de vida do grupo, tanto por questões de fisiologia termal quanto ecológicas. Componentes climáticos influenciam ainda diferentes aspectos da reprodução em lagartos, como por exemplo a estrutura e morfologia da casca dos ovos de Squamata, conforme indicam diferenças nestas características entre linhagens de lagartos ovíparos. Neste contexto, o presente trabalho investigou aspectos evolutivos da história de vida de lagartos da subfamília Tropidurinae, e sua relação com componentes climáticos e latitude, através de métodos filogenéticos comparativos. Observou-se que o número de ovos da ninhada é positivamente correlacionado com o tamanho corpóreo ao longo da linhagem dos tropiduríneos. Por outro lado, esse padrão não é observado no caso de hábito escansorial e utilização de frestas de rochas, em razão de possíveis compromissos envolvendo peso e forma corpórea com efeitos na sobrevivência das fêmeas. O tamanho corpóreo em tropiduríneros é maior em regiões geográficas com maiores componentes de precipitação, possivelmente relacionado a disponibilidade de alimento. Há diminuição do tamanho corpóreo com incrementos na distância da linha do Equador, até aproximadamente 1500 km. A partir desse limite, o tamanho corpóreo aumenta. Adicionalmente foi estudada a morfologia e estrutura da casca de Tropidurus torquatus, uma das espécies com distribuição mais ampla dentro da subfamília. A morfologia da casca de Tropidurus torquatus segue o padrão geral para Squamata com algumas particularidades, mas estudos comparativos futuros é que elucidarão padrões evolutivos da morfologia da casca. / In lizards, life history components have great phenotypic plasticity and evolve in a correlated way. Clutch size, for example, tends to be positively correlated to body size of gravid females, when comparing different evolutive lineages or different individuals, though some kinds of habit seems to restrict larger clutches. There are indications that other factors, such as climate components or latitude, also determine characteristics of lizard life history, as a matter of thermal physiology or ecology. Climate factors also affect different aspects of lizards reproduction, for example the structure and morphology of Squamata eggshells, as differences observed in these factors between oviparous lizards lineages indicate. In this context, the present work investigated evolutive aspects of Tropidurinae lizards life history and its relation to climate components and latitude, using comparative phylogenetic methods. It has been observed that clutch size is positively correlated to body size in the Tropidurinae lineage. On the other hand, this pattern is not observed in lizards with scansorial habits or those that inhabit rock crests, maybe due to compromises involving the weight and shape of female bodies that can affect female survival. The body size in Tropidurinae is larger in geographical regions presenting higher precipitation, possibly due to increased food availability. Female body size decreases with increased distance from the Equator until approximately 1500 km. Beyond this limit, female body size increases. Additionally, morphology and structure of eggshells were studied on Tropidurus torquatus, one of the species within the Tropidurinae group with higher latitudinal range of distribution. Tropidurus torquatus eggshell morphology follows the general pattern described for Squamata with some particularities, and future comparative studies are needed to elucidate evolutive patterns of eggshell morphology in the group.

The eggshell of lizards is a complex structure composed of organic and inorganic molecules secreted by the oviduct, which protects the embryo by providing a barrier to the external environment and also allows the exchange of respiratory gases and water for life support. Calcium deposited on the surface of the eggshell provides an important nutrient source for the embryo. Variation in physical conditions encountered by eggs results in a tradeoff among these functions and influences eggshell structure. Evolution of prolonged uterine egg retention results in a significant change in the incubation environment, notably reduction in efficiency of gas exchange, and selection should favor a concomitant reduction in eggshell thickness. This model is supported by studies that demonstrate an inverse correlation between eggshell thickness and length of uterine egg retention. One mechanism leading to thinning of the eggshell is reduction in size of uterine shell glands. Saiphos equalis is an Australian scincid lizard with an unusual pattern of geographic variation in reproductive mode. All populations retain eggs in the uterus beyond the embryonic stage at oviposition typical for lizards, and some are viviparous. We compared structure and histochemistry of the uterus and eggshell of two populations of S. equalis, prolonged egg retention, and viviparous to test the hypotheses: 1) eggshell thickness is inversely correlated with length of egg retention and 2) eggshell thickness is positively correlated with size of shell glands. We found support for the first hypothesis but also found that eggshells of both populations are surprisingly thick compared with other lizards. Our histochemical data support prior conclusions that uterine shell glands are the source of protein fiber matrix of the eggshell, but we did not find a correlation between size of shell glands and eggshell thickness. Eggshell thickness is likely determined by density of uterine shell glands in this species.

The evolutionary process leading to the emergence of viviparity in Squamata consists of lengthening the period of egg retention in utero coupled with marked reduction in the thickness of the eggshell. We used light microscopy and scanning electron microscopy to study uterine structure during the reproductive cycle of oviparous and viviparous females of the reproductively bimodal Lacerta vivipara. We compared the structure of the uterine shell glands, which secrete components of the eggshell, during preovulatory and early gestation phases of the reproductive cycle and also compared histochemistry of the eggshells. The uterine glands of both reproductive forms undergo considerable growth within a period of a few weeks during folliculogenesis and vitellogenesis preceding ovulation. The majority of the proteinaceous fibers of the shell membrane are secreted early in embryonic development and the uterine glands regress shortly thereafter. This supports previous observations indicating that, in Squamata, secretion of the shell membrane occurs very rapidly after ovulation. The most striking differences between reproductive modes were larger uterine glands at late vitellogenesis in oviparous females, 101 μm compared to 60 μm in viviparous females, and greater thickness of the shell membrane during early gestation in oviparous females (52-73 μm) compared to viviparous females (4-8 μm). Our intraspecific comparison supports the conclusions of previous studies that, prior to ovulation, the uterine glandular layer is less developed in viviparous than in oviparous species, and that this is the main factor accounting for differences in the thickness of the shell membrane of the two reproductive forms of squamates.

Evolution of the terrestrial egg of amniotes (reptiles, birds, and mammals) is often considered to be one of the most significant events in vertebrate history. Presence of an eggshell, fetal membranes, and a sizeable yolk allowed this egg to develop on land and hatch out well-developed, terrestrial offspring. For centuries, morphologically-based studies have provided valuable information about the eggs of amniotes and the embryos that develop from them. This review explores the history of such investigations, as a contribution to this special issue of Journal of Morphology, titled Developmental Morphology and Evolution of Amniote Eggs and Embryos. Anatomically-based investigations are surveyed from the ancient Greeks through the Scientific Revolution, followed by the 19th and early 20th centuries, with a focus on major findings of historical figures who have contributed significantly to our knowledge. Recent research on various aspects of amniote eggs is summarized, including gastrulation, egg shape and eggshell morphology, eggs of Mesozoic dinosaurs, sauropsid yolk sacs, squamate placentation, embryogenesis, and the phylotypic phase of embryonic development. As documented in this review, studies on amniote eggs and embryos have relied heavily on morphological approaches in order to answer functional and evolutionary questions.

The mineralized eggshell of Reptilia was a major innovation in the evolution of the amniotic egg. Inorganic components strengthen the eggshell and are a potential source of nutrients to developing embryos. Embryos of oviparous reptiles do extract calcium from eggshells but vary interspecifically in exploitation of this resource. The pattern of embryonic calcium nutrition of the corn snake, Pantherophis guttatus, is similar to a diversity of squamate species: embryos obtain most calcium from yolk, yet also mobilize calcium from the eggshell. We tested the hypothesis that embryonic development is not dependent on eggshell calcium by manipulating calcium availability. We peeled away the outer calcareous layer of the eggshell of recently oviposited eggs; control eggs were left intact. Eggs were sampled periodically and calcium content of egg compartments (embryo, yolk, eggshell) was measured. We also analyzed skeletal development and size of hatchlings. There was no difference in survivorship or length of incubation between treatments. However, hatchlings from intact eggs contained more calcium and were larger in mass and length than siblings from peeled eggs. There were no observable differences in ossification but hatchlings from intact eggs had larger skeletal elements (skull, vertebrae). Our results indicate that mobilization of eggshell calcium is not a requirement for embryonic development of P. guttatus and that embryos augment yolk calcium by extracting calcium from the eggshell. This pattern of embryonic calcium nutrition would favor embryos with a greater capacity to mobilize calcium from the eggshell by promoting growth and thereby potentially enhancing hatchling fitness.

Ultraviolet light (UV) and hydrogen peroxide (H2O2) decrease eggshell bacteria. However, when combined, the optimum amount of each and effects on hatchability are unknown. In Experiment 1, when compared to other concentrations of H2O2 and lengths of UV, the combination of 1.5% H2O2 and 8 minutes of UV yielded optimum results with a 3 log10 CFU/egg reduction in bacteria on the eggshell. In Experiment 2, exposing eggs to this optimum combination yielded a 1000 fold reduction in eggshell bacteria but only a numerical increase in hatch of set and hatch of fertile. In Experiment 3, eggs exposed to repetitive treatments of H2O2 and UV yielded a 4 log reduction in eggshell bacteria but no differences in hatchability or chick characteristics. In conclusion, the combination of H2O2 and UV proved to be effective for eggshell sanitization, especially when used repetitively, and did not alter hatchability.

It is generally accepted that populations which experience severe bottlenecks have a reduction in fitness. One of the most frequently reported fitness costs is increased hatching failure in bottlenecked populations of birds. The mechanism responsible for increased hatching failure is unknown. Research on other animals suggest that reduced population numbers cause unavoidable inbreeding that in turn leads to abnormalities in the gametes. In this thesis I examine some of the possible causes for increased hatching failure in severely bottlenecked populations of introduced birds in New Zealand. I look at three traits identified as a cause for infertility or hatching failure previously and determine whether there is a link with the size of a population s bottleneck. It is possible that reduced numbers of sperm reaching the site of fertilisation is a primary cause of hatching failure. I examined the perivitelline membrane of various species of introduced birds and counted the total number of sperm present to compare to how many would be expected in non-bottlenecked species. Although there was no relationship between the size of the bottleneck and the number of sperm present, all species had lower than expected sperm counts. In many species of mammals, a reduction in the quality of sperm is attributed to inbreeding depression bought about by genetic bottlenecks. I next compared the level of sperm abnormalities, variation in midpiece size sperm, and sperm motility with the size of the bottleneck each species passed through when introduced to New Zealand. There was no significant correlation between either the variation in midpiece size or sperm motility with bottleneck size. However, there was a trend for species that passed through more severe bottlenecks to have a slightly higher level of midpiece size and lower motility. Finally, I examined whether there was a link between abnormalities in the eggshell and the size of the respective bottleneck. There was no significant change in eggshell thickness or any change in the number of pores associated bottleneck size. However, there was a decreased number of round pores in severely bottlenecked species, although the consequences of this are unknown. My findings do not directly link a single cause for increased hatching failure in bottlenecked species of birds, but they do highlight the need for monitoring of reproductive traits in endangered species that have experienced a population bottleneck.

This dissertation is an investigation into the effect of different zinc sources and levels on eggshell quality and microstructure, as well as keel bone damage. Eggshell function is two-fold; eggshells function to protect the developing embryo, as well as act as a barrier against bacterial penetration, optimizing food safety of the egg for human consumption (Mabe et. Al., 2003). Two small trials were conducted in order to determine differences in eggshell microstructure of eggs produced from hens at peak lay (26 weeks of age) and at the end of lay (88 weeks of age). Two groups of hens were fed a calcium sufficient or a calcium deficient diet. From this it was determined that eggs with higher breaking strengths had some differences in eggshell microstructure. Eggs with greater breaking strengths had a greater density of ‘normal’ structures, compared to ‘abnormal’ structures. Additionally, eggs requiring a greater breaking force, had a thicker micro-structure, compared to shells requiring less breaking force. With this knowledge on microstructure, a larger, 36-week study was conducted using different zinc sources. Every four weeks, eggs were collected and standard egg quality measurements were taken and keel bones were scored. At the end of the study, keel bones were collected from randomly selected hens representing each treatment. Picture of these keel bones were taken and measurements were taken to determine type and degree of deformation, in comparison to scores taken on the live bird. Pens selected for keel bone analysis, were the same pens that eggs were taken for imaging by the scanning electron microscope, to determine eggshell microstructure. From this data, it was determined that egg quality differences were detected, as well as differences in eggshell microstructure. Additionally, keel bone scores progressively worsened throughout the 36-week long study, with type and degree of deformation differing depending on zinc source.

Natural bone is a composite of collagen based hydrogel and inorganic dahilite crystals. The unusual combination of a hard inorganic material and an underlying elastic hydrogel network endows native bone with unique mechanical properties, such as low stiffness, resistance to tensile and compressive forces and high fracture toughness. Throughout the cavities of the bone, there are bone cells and myriads of soluble and extracellular matrix components that are constantly involved in the bone formation and remodeling process. Among the extra cellular component the acidic matrix proteins that are attached to the collagen scaffold play important templating and inhibitory roles during the mineralization process. It would be interesting to generate such functional scaffolds that mimic a template driven mineralization and which can assist cell adhesion, proliferation, migration and differentiation. Towards this direction, we have chosen one synthetic (Nylon 66 membrane) and one natural (eggshell membrane) scaffold and carried out a template driven mineralization of CaCO₃ as model systems. The surface modifications were carried out by the pre-adsorption of acidic polymers before the deposition of the CaCO₃. The deposition of the crystalline calcium carbonate on these modified templates were archived from a supersaturated solution of Ca(HCO₃)₂. / Singapore-MIT Alliance (SMA)

Lipids carry out important structural as well as signaling functions in the cell. In recent years, enzymes that metabolize lipids have been emerging as key regulators of basic cellular functions and developmental processes. In order to study metabolism of lipids, we have focused our research on a class of proteins: the cytochrome P450s (CYPs), which are involved in lipid production in many organisms. We have used C. elegans, a classical genetic model system, to investigate lipid metabolism because this nematode offers several technical advantages that render it suitable for our investigations. The aim of our project was to identify and characterize essential lipids for the development of worms. We have performed RNAi (RNA interference) against C. elegans CYP31A, and found that silencing of this enzyme leads to the arrest of embryonic development. Further characterization of this embryonic lethal phenotype revealed that it is caused by problems in establishment of polarity and failure in the extrusion of a polar body. Moreover, we found that embryos depleted of CYP31A are osmotic sensitive and their eggs are permeable to dyes (hoechst, FM 4-64 etc.). The defects described above are common to a class of mutants that received the denomination of POD (for Polarity and Osmotic Defects). Analysis by electron microscopy demonstrated that cyp31A(RNAi) embryos exhibit an improperly constructed eggshell. Further functional studies have demonstrated that the defects observed in cyp31A(RNAi) embryos can be ascribed to the malfunctioning of one of the three layers of the eggshell: the lipid-rich layer, but additional problems in the assembling of the other two layers are also present. In order to identify the product of CYP31A, we set up a bioassay in which we tested the capability of lipidic extract from wild type embryos to rescue the embryonic lethality. The bioassay provided a method to track the activity and allowed us to enrich the metabolic product of CYP31A by the fractionation of the total lipid extract. Another POD gene, emb-8, codes for an NADPH CYP reductase. This 4 protein supplies electrons to the CYPs for their metabolic reactions. A mutant of emb-8 (emb-8(hc69)), gives a similar phenotype as the knockdown CYP31A. With the aim to test if EMB-8 and CYP31A act in the same pathway we extracted lipids from emb-8TS mutants. We tested in the bioassay if extracts from emb-8(hc69) mutants, containing the metabolic product of CYP31A, can rescue cyp31A(RNAi) phenotype. The results obtained suggest that EMB-8 and CYP31A work in the same metabolic pathway. Conclusively, CYP31A and EMB-8 cooperate to produce a class of lipids that are required for the construction of a functional eggshell. A defective eggshell causes failure in polarity establishment, extrusion of the polar bodies, osmotic sensitivity and permeability and eventually it leads to the arrest of the development of C. elegans embryos.