The chorioallantoic membrane resides adjacent to either the inner surface of the egg shell or uterine epithelium in oviparous and viviparous reptiles, respectively. Chorionic cells face the shell or uterine epithelium and transport calcium to underlying embryonic capillaries. Calcium transport activity of the chorioallantois increases in the final stages of development coincident with rapid embryonic growth and skeletal ossification. We excised embryos from viviparous Zootoca vivipara females at a stage prior to significant calcium accumulation and incubated them ex utero with and without calcium to test the hypothesis that chorioallantois calcium transport activity depends on developmental stage and not calcium availability. We measured calcium uptake by monitoring incubation media calcium content and chorioallantois expression of calbindin-D28K, a marker for transcellular calcium transport. The pattern of calcium flux to the media differed by incubation condition. Eggs in 0 mM calcium exhibited little variation in calcium gain or loss. For eggs in 2 mM calcium, calcium flux to the media was highly variable and was directed inward during the last 3 days of the experiment such that embryos gained calcium. Calbindin-D28K expression increased under both incubation conditions but was significantly higher in embryos incubated with 2 mM calcium. We conclude that embryos respond to calcium availability, yet significant calcium accumulation is developmental stage dependent. These observations suggest the chorioallantois exhibits a degree of functional plasticity that facilitates response to metabolic or environmental fluctuations.

O objetivo do presente estudo foi investigar a estratégia nutricional neonatal das raias Rhinoptera bonasus e Rhinoptera brasiliensis, analisando suas interações tróficas e dependência do recurso materno durante as fases iniciais da vida. Lipídios, ácidos graxos (FA) e isótopos estáveis (SI) foram analisados em diferentes tecidos, como ferramentas para avaliar as alterações durante o crescimento em raias jovens-de-ano (YOY), divididas em dois grupos, YOY I (<=50 cm) e YOY II (50-70 cm). O registro de YOY em três anos consecutivos (2015, 2016 e 2017) e a permanência dos filhotes durante o ano, confirmam que a região de Bertioga é uma área de berçário para as espécies R. bonasus e possivelmente para R. brasiliensis. Em R. bonasus, as concentrações plasmáticas de triacilglicerol e &#946;ß-hidroxibutirato não diferiram entre YOY I e II. No entanto, a concentração plasmática de colesterol (CHOL) foi maior em YOY I quando comparado com YOY II. O perfil de FA foi similar no fígado, músculo e plasma de R. bonasus, com predomínio de FA polinsaturados (PUFA), seguidos por FA saturados (SFA) e monoinsaturados (MUFA). No músculo de R. bonasus ocorreu uma diminuição de DHA (ácido docosahexaenoico) e ARA (ácido araquidônico) e um aumento de EPA (ácido eicosapentaenoico) com o aumento do tamanho corporal, reduzindo a relação DHA/EPA (indicador de posição trófica). Diferenças na composição de FA do fígado e músculo entre R. bonasus e R. brasiliensis foram encontradas na somatória de SFA, MUFA e PUFA, sugerindo que as espécies apresentam diferentes dietas ou diferentes períodos de nascimento. Em R. bonasus os valores de &#948;15N diminuíram com o aumento do tamanho corpóreo, e os valores mais altos foram encontrados na nadadeira dorsal e hemácias (RBC) de YOY I comparados a YOY II. Em YOY I e II, o &#948;13C da nadadeira foi maior que nas RBC. Comparando-se as duas espécies, R. bonasus apresenta maior valor de &#948;15N e &#948;13C nas RBC que R. brasiliensis, indicando que particionam recursos alimentares, com diferentes níveis tróficos e/ou forrageiam em diferentes locais, apesar das duas espécies serem simpátricas na região de estudo. Esses dados combinados demonstram que estas raias investem significativamente nos jovens e, consequentemente, em melhores condições nutricionais no nascimento, pois os dados encontrados sugerem que filhotes não mostraram deficiência de FA essenciais (EFA), como observado em tubarões placentários. Valores elevados de CHOL, ARA e DHA em YOY I, e posterior redução com o aumento do tamanho corpóreo, sugere o uso destes substratos em processos metabólicos. Os dados sugerem ainda que animais maiores aumentam as habilidades de forrageamento, demonstradas pelo aumento plasmático de EFA, tais como EPA e DHA e uma diminuição de SFA em YOY II. Tais aspectos metabólicos ligados à estratégia nutricional neonatal são fundamentais para compreender os padrões de investimento materno e recursos energéticos necessários nas fases iniciais de vida de elasmobrânquios vivíparos / The aim of the present study was to investigate the neonatal nutritional strategy adopted by species R. bonasus and R. brasiliensis, related to its trophic interactions and maternal resource dependency during the early stages of life. Lipids, fatty acids (FA) and stable isotope (SI) were assessed in different tissues, as tools to evaluate biomarkers changes during growth in young-of-the-year (YOY) rays, divided into two stages, YOY I (<=50 cm) and YOY II (50-70 cm). The registration of YOY in three consecutive years (2015, 2016 and 2017) and the permanence of the YOY during the year, confirms that the region of Bertioga is a nursery area for R. bonasus and possibly to R. brasiliensis. In R. bonasus the plasma triglycerides and &#948;-hydroxybutyrate did not differ between YOY I and II. Plasma cholesterol (CHOL) was higher in YOY I than in YOY II. FA profiles were similar in liver, muscle and plasma, with a predominance of polyunsaturated FA (PUFA), followed by saturated FA (SFA) and monounsaturated FA (MUFA). In R. bonasus there was a reduction of C22:6n3 (DHA - docosahexaenoic acid) and C20:4n6 (ARA - arachidonic acid), and an increase in C20:5n3 (EPA - eicosapentaenoic acid) in muscle, with increasing body size, resulting in a decrease in DHA/EPA ratio (used as an indicator of trophic position). In terms of SI, &#948;15N values decreased with increasing body size with higher &#948;15N values found in dorsal fin and red blood cells (RBC) of YOY I compared to YOY II animals. In YOY I and II, the &#948;13C of fin tissue was higher than RBC. Comparing both species, R. bonasus presents higher &#948;15N and &#948;13C values in the RBC than R. brasiliensis, suggesting the partition of food resources, at different trophic levels and/or foraging in different locations, despite the two species being sympatric in the region. These combined data demonstrate that cownose rays significantly invest in their young and consequently better nutritional conditions at birth, since the data suggest that the pups did not show essential FA (EFA) deficiency, as observed in placental sharks. High values of DHA, ARA and CHOL in YOY I, and subsequent decrease with increasing body size, confirms the use of these substrates in metabolic processes. In addition, data indicate that larger animals have improved foraging skills, demonstrated by increased plasma levels of EFA, such as DHA, EPA and a decrease of &#931;SFA in YOY II. Such metabolic aspects linked to neonatal nutritional strategy are fundamental factors for understanding the patterns of maternal investment and energy resources required in the early life stages of viviparous elasmobranchs

Among vertebrates, oviparity, a condition in which young at least partially develop outside the mother's body and are supported by yolk, is the ancestral state to viviparity, the reproductive mode in which embryos are fully developed at birth. Viviparity in reptiles is found only in the squamates. Among the more than 100 origins of viviparity in squamates, many have occurred fairly recently and as a result some species contain populations of both reproductive modes, i.e., are reproductively bimodal. The evolution of viviparity is associated with many changes including an increase in oviductal egg retention times, a decrease in thickness of the eggshell and a decrease in secretion of the uterine shell glands that are responsible for eggshell secretion. The uterine morphology of the oviparous Saproscincus mustelinus and the reproductively bimodal Saiphos equalis were compared to study the uterine shell gland cycle.

Pseudemoia pagenstecheri is a viviparous Australian scincid lizard in which the maternal-embryonic placental interface is differentiated into structurally distinct regions. The chorioallantoic placenta contains an elliptical-shaped region, the placentome, characterized by hypertrophied uterine and embryonic epithelial cells supported by dense vascular networks. The remainder of the chorioallantoic placenta, the paraplacentome, is also highly vascularized but uterine and chorionic epithelia are thin. An omphaloplacenta with hypertrophied epithelia is located in the abembryonic hemisphere of the egg. There is extensive placental transport of organic and inorganic nutrients, e.g., 85-90% of neonatal calcium is received via placental transfer. Calcium uptake by extraembryonic membranes of squamates correlates with expression of the intracellular calcium binding protein, calbindin-D 28K, and plasma membrane calcium ATPase (PMCA) is a marker for active calcium transport. We estimated expression of calbindin-D 28K and PMCA in the chorioallantoic membrane in a developmental series of embryos using immunoblotting and used immunohistochemistry to define the cellular localization of calbindin-D 28K to test the hypotheses that 1) expression of calcium transporting proteins is coincident with placental transport of calcium and 2) the placenta is functionally specialized for calcium transport in regions of structural differentiation. Calbindin-D 28K and PMCA were detected at low levels in early stages of development and increased significantly prior to birth, when embryonic calcium uptake peaks. These data support the hypothesis that placental calcium secretion occurs over an extended interval of gestation, with increasing activity as embryonic demand escalates in late development. In addition, calbindin-D 28K expression is localized in chorionic epithelial cells of the placentome and in the epithelium of the omphalopleure of the omphaloplacenta, which supports the hypothesis that regional structural differentiation in the placenta reflects functional specializations for calcium transport.

Embryos of oviparous squamate reptiles obtain all organic and most inorganic nutrients from yolk; yolk provides 19-86% of hatchling calcium content. The remaining calcium is extracted from the eggshell. Yolk calcium provision to viviparous embryos also is variable and includes three patterns. The contribution of yolk to embryonic development for most viviparous squamates is similar to oviparous species, but the attenuated eggshell of viviparous species is a poor source of calcium because it lacks an outer layer of calcium carbonate, and embryos supplement yolk calcium via placental transfer. In a second pattern, yolk provides all organic nutrients and calcium. The final pattern occurs in viviparous species that are substantially placentotrophic and placental transfer accounts for most organic and inorganic nutrients, including calcium. The many independent evolutionary transitions to viviparity among squamates have inspired interest in a possible link to patterns of embryonic calcium nutrition. A prominent model predicts that the pattern of maternal provision and embryonic uptake of calcium unique to squamates facilitates the evolution of viviparity. A primary assumption of the model is that the evolution of viviparity precedes the evolution of calcium placentotrophy. An alternative model predicts that viviparity and placentotrophy evolve concurrently because mechanisms for nutrient provision and mobilization are not dependent on reproductive mode. These hypotheses have not been tested directly but review of the literature indicates that neither fully explains the diversity of squamate embryonic calcium nutrition. Viviparous species differ from oviparous species primarily in the timing of uterine calcium secretion and structure of eggshell calcium. Future studies should focus on the mechanisms that promote these differences.

A prominent scenario for the evolution of viviparity and placentation in reptiles predicts a step-wise pattern with an initial phase of prolonged oviductal egg retention accompanied by progressive reduction in eggshell thickness culminating in viviparity; calcium placentotrophy evolves secondarily to viviparity. Saiphos equalis is an Australian scincid lizard with a reproductive mode that is uncommon for squamates because eggs are retained in the oviduct until late developmental stages, and the embryonic stage at oviposition varies geographically. We studied calcium mobilization by embryos in two populations with different oviductal egg retention patterns to test the hypothesis that the pattern of nutritional provision of calcium is independent of the embryonic stage at oviposition. Females from one population are viviparous and oviposit eggs containing fully formed embryos, whereas embryos in oviposited eggs of the second population are morphologically less mature, and these eggs hatch several days later. The reproductive mode of this population is denoted as prolonged oviductal egg retention. Yolk provided the highest proportion of calcium to hatchlings in both populations. Eggs of both populations were enclosed in calcified eggshells, but shells of the population with prolonged egg retention had twice the calcium content of the viviparous population and embryos recovered calcium from these eggshells. Placental transfer accounted for a substantial amount of calcium in hatchlings in both populations. Hatchling calcium concentration was higher in the population with prolonged egg retention because these embryos mobilized calcium from yolk, the eggshell and the placenta. This pattern of embryonic calcium provision in which both a calcified eggshell and placentotrophy contribute to embryonic nutrition is novel. The reproductive pattern of S. equalis illustrates that calcified eggshells are compatible with prolonged oviductal egg retention and that viviparity is not requisite to calcium placentotrophy.

The ion, energy, lipid, nitrogen and fat-soluble vitamin contents of freshly ovulated eggs and neonates of the viviparous lizard Niveoscincus metallicus were measured to quantify uptake of nutrients across the placenta. This species is particularly interesting because it has a chorio-allantoic placenta that is intermediate in complexity compared to viviparous species that have been the focus of other studies. Newly ovulated eggs have a wet mass of 79.6±4.6 mg and a dry mass of 41.8±2.8 mg, compared to the neonates that have a wet mass of 224.2±8.2 mg and dry mass of 37.9±1.2 mg. Thus, there is no significant net uptake of dry matter across the placenta. Neonates have significantly less lipid (6.2±0.4 mg) than eggs (12.7±0.5 mg) but no significant difference in nitrogen (4.1±0.3 mg) compared to eggs (4.5±0.2 mg). Energy densities reflect the protein and lipid composition and the relative dry masses of the eggs and neonates. There is significantly more energy (1029.1±80.0 J) in the egg than in the neonate (858.2±36.6 J). The increase in the ash content of the neonates (2.9±0.2 mg) compared to fresh eggs (2.1±0.3 mg) was not significant, even though there was an approximately threefold increase in the amount of sodium (0.11±0.01 mg in neonates, 0.34±0.01 mg in eggs) and potassium (0.12±0.017 in neonates, 0.40±0.01 mg in eggs) in neonates compared to eggs. There was no significant uptake of calcium and magnesium during development. The egg lipids consisted of triacylglycerol (66.7±2.3%), phospholipid (18.9±0.7%), cholesteryl ester (4.9±1.6%) and free cholesterol (5.6±1.5%). The egg phospholipid contained comparatively high proportions of arachidonic and eicosapentanoic acids but low levels of docosahexaenoic acid (DHA), whereas the phospholipid of the neonate was greatly enriched in DHA. In the egg, the predominant vitamin E was α-tocopherol (62.6±3.4 mg g-1), although there was some γ-tocotrienol (3.5±0.3 mg g-1), and vitamin A was present (1.5±0.2 mg g-1). The ratio of neonate dry mass to egg dry mass of N. metallicus (0.91) lies between that of species with type I (0.78) and type III (1.70) chorio-allantoic placentae, confirming our conclusion that the placenta of N. metallicus is functionally intermediate, as well as intermediate in complexity, between these other two types.

Yolk supplies the majority of embryonic calcium in snakes. Oviparous and viviparous snakes also receive calcium late in development from the eggshell and placenta, respectively. The pattern and mechanism of calcium transport are partly understood for oviparous snakes. I studied a viviparous snake, Virginia striatula, to determine the pattern of embryonic calcium accumulation as well as the ontogenetic expression of calcium transporting proteins in extraembryonic tissues. The pattern of embryonic calcium uptake of V. striatula occurs late in development, during the phase of highest embryonic growth. Calbindin-D28k, Ca2+ ATPase, and carbonic anhydrase II are expressed in chorioallantoic membrane, while yolk sac only expresses calbindin-D28k, coincident with the timing of calcium transport in embryos of V. striatula. Thus, the pattern of embryonic calcium accumulation in V. striatula is similar to that of oviparous snakes. Although calbindin-D28k and Ca2+ ATPase are likely active in embryonic calcium transport, the role of carbonic anhydrase II remains less clear.

Within the Sceloporus scalaris species group, some species are oviparous while others are viviparous, suggesting the origin of viviparity is relatively recent. This interpopulation comparison focused on Sceloporus scalaris in Arizona because low elevation (1500 m) and high elevation (2500-2900 m) populations exhibit short and long periods of egg retention, respectively, and increased periods of egg retention are presumably an intermediate step in the evolution of viviparity. Low elevation populations had life histories typical of lowland Sceloporus: eggs are laid at embryonic stages 32-33, clutch sizes are relatively large, hatchlings are relatively small, and more than one clutch is produced per season. In contrast, montane populations retain eggs to embryonic stages 36-38, clutch sizes are relatively small, hatchlings are relatively large, and only one clutch is produced per season. In accord with their lengthy retention, eggshells of montane populations were thinner than those of lowland populations. The assumption that prolonged egg retention is mechanistically associated with relatively thin eggshells was tested by comparing the development of embryos in eggs retained in utero with that of control embryos in eggs incubated in Simulated nests where water and gases were presumably not limited. For the low elevation population, growth rates of embryos retained in utero as long as stage 39 were identical to those of control eggs at the same stages. However, water uptake by retained eggs was confined to the embryo whereas water uptake of control eggs was associated with both the embryo and extraembryonic tissues. These results suggest that the length of egg retention by S. scalaris is not constrained by eggshell thickness, and that the capacity to regulate the water balance of eggs may be an important component of the evolution of viviparity. / Master of Science

One hypothesis explaining the numerous independent evolutionary transitions from oviparity to viviparity among squamates (snakes and lizards) proposed that squamate embryonic development is independent of eggshell calcium. Recent research showed at least 25% of the calcium in hatchling oviparous squamates is extracted from the shell. Though not a direct test, these results are inconsistent with the hypothesis. To directly test the hypothesis, we removed eggshell calcium (through peeling) early in development of Pantherophis guttatus (corn snake) eggs. Survivorship to hatching did not differ between peeled and intact eggs. Yet hatchlings from peeled eggs were shorter (273.6 ± 3.4 vs. 261.0 ± 3.7 mm, p=0.0028, n=16), lighter (6.36 ±0.22 vs. 5.75 ± 0.23 g, p=0.0158, n=16), and had reduced calcium (40.8 ± 1.7 vs. 30.5 ± 1.8 mg, p