Placental ontogeny in the Yucca Night Lizard, Xantusia vigilis.

Squamate placentas support physiological exchange between mothers and embryos. Uterine and embryonic epithelial cells provide sites for transporting mechanisms and extraembryonic membranes provide the scaffolding for embryonic epithelial cells and vascular systems. Diversity in placental structure involves variation in extraembryonic membrane development as well as epithelial cell specializations. Variation in placental ontogeny is known to occur and, although lineage specific patterns have been described, phylogenetic distribution of specific patterns is poorly understood. Xantusia vigilis is a viviparous lizard in a monophyletic clade, Xantusiidae, of predominantly viviparous species. Xantusiidae is one of two viviparous lineages within the clade Scincoidea that provides an important outgroup comparison for Scincidae, which includes the largest number of independent origins of viviparity among Squamata. Previous reports contain brief descriptions of placental structure of X vigilis but the developmental pattern is unknown including relevant details for comparison with skinks. We studied placental ontogeny in X. vigilis to address two hypotheses: (1) the pattern of development of placental architecture is similar to species of Scincidae and, (2) placental epithelial cell specializations are similar to species of Scincidae. The terminal placental stage of X. vigilis is similar to skinks in that it includes a chorioallantoic placenta and an omphaloplacenta. The chorioallantoic placenta is richly vascularized with thin, squamous epithelial cells separating the two vascular systems. This morphology differs from the elaborate epithelial cell specializations as occur in some skink species, but is similar to many species. Epithelial cells of the omphaloplacenta are enlarged, as they are in scincids, yet development of the omphaloplacenta includes a vascular pattern known to occur only in gerrhonotine lizards. Histochemical staining properties of the epithelium of the omphalopleure of the omphaloplacenta indicate the potential for protein transport, a function not previously reported for lizards.

Developmental energetics in the oviparous corn snake, Pantherophis guttatus, confirms a conservative evolutionary pattern in snakes.

Eggs of oviparous reptiles are ideal models for studying evolutionary patterns of embryonic metabolism since they allow tracking of energy allocation during development. Analyzing oxygen consumption of whole eggs throughout development indicates three patterns among reptiles. Embryos initially grow and consume oxygen exponentially, but oxygen consumption slows, or drops before hatching in some species. Turtles, crocodilians, and most lizards follow curves with initial exponential increases followed by declines, whereas embryonic snakes that have been studied exhibit a consistently exponential pattern. This study measured oxygen consumption of corn snake, Pantherophis guttatus, embryos to determine if this species also exhibits an exponential increase in oxygen consumption. Individual eggs, sampled weekly from oviposition to hatching, were placed in respirometry chambers for 24-h during which oxygen consumption was recorded. Embryos were staged and carcasses and yolk were weighed separately. Results indicate steady inclines in oxygen consumption during early stages of development, with a rapid increase prior to hatching. The findings support the hypothesis that embryonic oxygen consumption of snakes differs from most other non-avian reptiles. Total energy required for development was determined based on calorimetry of initial yolk compared to hatchlings and residual yolk and by integration of the area under the curve plotting oxygen consumption versus age of embryos. The cost of development estimates based on these two methods were 6.4 and 10.0 kJ, respectively. Our results emphasize the unique physiological aspects of snake embryogenesis and illustrate how the study of physiological characteristics can contribute to the broader understanding of reptilian evolution.

Functional complexity in the chorioallantoic membrane of an oviparous snake: Specializations for calcium uptake from the eggshell.

The chorioallantoic membrane of oviparous reptiles forms a vascular interface with the eggshell. The eggshell contains calcium, primarily as calcium carbonate. Extraction and mobilization of this calcium by the chorioallantoic membrane contributes importantly to embryonic nutrition. Development of the chorioallantoic membrane is primarily known from studies of squamates and birds. Although there are pronounced differences in eggshell structure, squamate and bird embryos each mobilize calcium from eggshells. Specialized cells in the chicken chorionic epithelium transport calcium from the eggshell aided by a second population of cells that secrete protons generated by the enzyme carbonic anhydrase. Calcium transporting cells also are present in the chorioallantoic membrane of corn snakes, although these cells function differently than those of chickens. We used histology and immunohistology to characterize the morphology and functional attributes of the chorioallantoic membrane of corn snakes. We identified two populations of cells in the outer layer of the chorionic epithelium. Calbindin-D28K , a cellular marker for calcium transport expressed in squamate chorioallantoic membranes, is localized in large, flattened cells that predominate in the chorionic epithelium. Smaller cells, interspersed among the large cells, express carbonic anhydrase 2, an enzyme not previously localized in the chorionic epithelium of an oviparous squamate. These findings indicate that differentiation of chorionic epithelial cells contributes to extraction and transport of calcium from the eggshell. The presence of specializations of chorioallantoic membranes for calcium uptake from eggshells in chickens and corn snakes suggests that eggshell calcium was a source of embryonic nutrition early in the evolution of Sauropsida.

Phylogeny and evolutionary history of the amniote egg.

We review morphological features of the amniote egg and embryos in a comparative phylogenetic framework, including all major clades of extant vertebrates. We discuss 40 characters that are relevant for an analysis of the evolutionary history of the vertebrate egg. Special attention is given to the morphology of the cellular yolk sac, the eggshell, and extraembryonic membranes. Many features that are typically assigned to amniotes, such as a large yolk sac, delayed egg deposition, and terrestrial reproduction have evolved independently and convergently in numerous clades of vertebrates. We use phylogenetic character mapping and ancestral character state reconstruction as tools to recognize sequence, order, and patterns of morphological evolution and deduce a hypothesis of the evolutionary history of the amniote egg. Besides amnion and chorioallantois, amniotes ancestrally possess copulatory organs (secondarily reduced in most birds), internal fertilization, and delayed deposition of eggs that contain an embryo in the primitive streak or early somite stage. Except for the amnion, chorioallantois, and amniote type of eggshell, these features evolved convergently in almost all major clades of aquatic vertebrates possibly in response to selective factors such as egg predation, hostile environmental conditions for egg development, or to adjust hatching of young to favorable season. A functionally important feature of the amnion membrane is its myogenic contractility that moves the (early) embryo and prevents adhering of the growing embryo to extraembryonic materials. This function of the amnion membrane and the liquid-filled amnion cavity may have evolved under the requirements of delayed deposition of eggs that contain developing embryos. The chorioallantois is a temporary embryonic exchange organ that supports embryonic development. A possible evolutionary scenario is that the amniote egg presents an exaptation that paved the evolutionary pathway for reproduction on land. As shown by numerous examples from anamniotes, reproduction on land has occurred multiple times among vertebrates-the amniote egg presenting one "solution" that enabled the conquest of land for reproduction.

Morphological research on amniote eggs and embryos: An introduction and historical retrospective.

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.

Developmental morphology and evolution of extraembryonic membranes of lizards and snakes (Reptilia, Squamata).

Amniote embryos are supported and nourished by a suite of tissues, the extraembryonic membranes, that provide vascular connections to the egg contents. Oviparous reptiles share a basic pattern of development inherited from a common ancestor; a vascular chorioallantoic membrane, functioning as a respiratory organ, contacts the eggshell and a vascular yolk sac membrane conveys nutrients to the embryo. Squamates (lizards, snakes) have evolved a novel variation in morphogenesis of the yolk sac that results in a unique structure, the yolk cleft/isolated yolk mass complex. This structure is a source of phylogenetic variation in architecture of the extraembryonic membranes among oviparous squamates. The yolk cleft/isolated yolk mass complex is retained in viviparous species and influences placental architecture. The aim of this paper is to review extraembryonic membrane development and morphology in oviparous and related viviparous squamates to explore patterns of variation. The survey includes all oviparous species for which data are available (11 species; 4 families). Comparisons with viviparous species encompass six independent origins of viviparity. The comparisons reveal that both phylogeny and reproductive mode influence variation in extraembryonic membrane development and that phylogenetic variation influences placental evolution. Models of the evolution of squamate placentation have relied primarily on comparisons between independently derived viviparous species. The inclusion of oviparous species in comparative analyses largely supports these models, yet exposes convergent patterns of evolution that become apparent when phylogenetic variation is recognized.

Classics revisited, history of reptile placentology, part IV: Hanni Hrabowski's 1926 monograph on fetal membranes of lizards.

In 1926, the German biologist Johanna (Hanni) Hrabowski published a study of the morphology and development of the fetal placenta in lizards that has proven to be of historical importance. Her anatomical descriptions and interpretations identified developmental patterns that differ from other amniotes -- features now recognized as unique attributes of squamate (lizards and snakes) development. Her 1926 monograph presented the first histological comparison of fetal membranes in closely-related oviparous and viviparous reptiles, thereby establishing a comparative framework for understanding placental specializations for viviparity. Hrabowski reported that yolk sac development did not differ between oviparous and viviparous species. The novel, shared components of yolk sac development she identified are now recognized as the foundation for the unique yolk sac placenta of reptiles, the omphaloplacenta. In addition, Hrabowski's extensive ontogenetic sampling and the detail and accuracy of her anatomical descriptions set high standards for subsequent studies of comparative evolutionary embryology.

A developmental synapomorphy of squamate reptiles.

The reptilian clade Squamata is defined primarily by osteological synapomorphies, few of which are entirely unambiguous. Studies of developing squamate eggs have revealed a uniquely specialized feature not known to occur in any other amniotes. This feature-the yolk cleft/isolated yolk mass complex-lines the ventral hemisphere of the egg. During its formation, extraembryonic mesoderm penetrates the yolk and an exocoelom (the yolk cleft [YC]) forms in association with it, cutting off a thin segment of yolk (the "isolated yolk mass" [IYM]) from the main body of the yolk. The YC-IYM complex has been observed and described in more than 65 squamate species in 12 families. In viviparous species, it contributes to the "omphaloplacenta," a type of yolk sac placenta unique to squamates. The only squamates known to lack the IYM are a few highly placentotrophic skinks with minuscule eggs, viviparous species in which it clearly has been lost. Given its absence in mammals, chelonians, crocodylians, and birds, the YC-IYM complex warrants recognition as a developmental synapomorphy of the squamate clade. As in extant viviparous lizards and snakes, the YC-IYM complex presumably contributed to the placenta of extinct viviparous squamates.

Facultative mobilization of eggshell calcium promotes embryonic growth in an oviparous snake.

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.

Novel placental structure in the Mexican gerrhonotine lizard, Mesaspis viridiflava (Lacertilia; Anguidae).

The evolution of viviparity alters the physical relationship between mothers and offspring and the prevalence of viviparity among squamate reptiles presents an opportunity to uncover patterns in the evolution of placental structure. Understanding the breadth of this diversity is limited because studies of placental structure and function have emphasized a limited number of lineages. We studied placental ontogeny using light microscopy for an embryological series of the Mexican gerrhonotine lizard, Mesaspis viridiflava. This species develops an elaborate yolk sac placenta, an omphaloplacenta, which receives vascular support arising in a structure known only from other gerrhonotine lizards. A prominent feature of the omphaloplacenta is a zone of uterine and embryonic epithelial cell hyperplasia located at the upper shoulder of the yolk mass, often extending above the yolk mass. The omphaloplacenta covers more than one-half of the surface area of maternal-embryonic contact. The chorioallantoic placenta has a more restricted distribution because the allantois remains in the embryonic hemisphere of the egg throughout development and lies internal to the vascular support for the omphaloplacenta in areas where they overlap. The structural profile of the chorioallantoic placenta indicates a potential for respiratory exchange and/or hemotrophic nutritive transport, while that of the omphaloplacenta suggests that nutritive transfer is primarily via histotrophy. An eggshell is present in the earliest embryonic stages examined but regresses relatively early in development. Placental specializations of this species are consistent with a pattern of matrotrophic embryonic nutrition and have evolved in a unique lineage specific developmental pattern.

Yolk sac development in lizards (Lacertilia: Scincidae): New perspectives on the egg of amniotes.

Embryos of oviparous reptiles develop on the surface of a large mass of yolk, which they metabolize to become relatively large hatchlings. Access to the yolk is provided by tissues growing outward from the embryo to cover the surface of the yolk. A key feature of yolk sac development is a dedicated blood vascular system to communicate with the embryo. The best known model for yolk sac development and function of oviparous amniotes is based on numerous studies of birds, primarily domestic chickens. In this model, the vascular yolk sac forms the perimeter of the large yolk mass and is lined by a specialized epithelium, which takes up, processes and transports yolk nutrients to the yolk sac blood vessels. Studies of lizard yolk sac development, dating to more than 100 years ago, report characteristics inconsistent with this model. We compared development of the yolk sac from oviposition to near hatching in embryonic series of three species of oviparous scincid lizards to consider congruence with the pattern described for birds. Our findings reinforce results of prior studies indicating that squamate reptiles mobilize and metabolize the large yolk reserves in their eggs through a process unknown in other amniotes. Development of the yolk sac of lizards differs from birds in four primary characteristics, migration of mesoderm, proliferation of endoderm, vascular development and cellular diversity within the yolk sac cavity. Notably, all of the yolk is incorporated into cells relatively early in development and endodermal cells within the yolk sac cavity align along blood vessels which course throughout the yolk sac cavity. The pattern of uptake of yolk by endodermal cells indicates that the mechanism of yolk metabolism differs between lizards and birds and that the evolution of a fundamental characteristic of embryonic nutrition diverged in these two lineages. Attributes of the yolk sac of squamates reveal the existence of phylogenetic diversity among amniote lineages and raise new questions concerning the evolution of the amniotic egg. J. Morphol. 278:574-591, 2017. © 2017 Wiley Periodicals, Inc.

Ex utero culture of viviparous embryos of the lizard, Zootoca vivipara, provides insights into calcium homeostasis during development.

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 0mM calcium exhibited little variation in calcium gain or loss. For eggs in 2mM calcium, calcium flux to the media was highly variable and was directed inward during the last 3days of the experiment such that embryos gained calcium. Calbindin-D28K expression increased under both incubation conditions but was significantly higher in embryos incubated with 2mM 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.

Thirty Years of Cystocele/Rectocele Repair in the United States.

OBJECTIVE: A growing body of evidence suggests an increased role for apical support in the treatment of pelvic organ prolapse regardless of phenotype. The objective of this study was to determine whether changes in cystocele/rectocele diagnosis and surgical management for the last 30 years reflect this changing paradigm. METHODS: Data from the National Hospital Discharge Survey were mined from 1979 to 2009 for diagnosis and procedure codes. Records were categorized according to predefined combinations of diagnosis and procedure codes and weighted according to the National Hospital Discharge Survey data set. Pearson χ test was used to evaluate the changes in population proportions during the study interval. RESULTS: The proportion of isolated cystocele/rectocele diagnoses decreased from 1979 to 2009 (56.5%, n = 88,548, to 34.8%, n = 31,577). The proportion of isolated apical defect diagnoses increased from 1979 to 2009 (38.4%, n = 60,223, to 60.8%, n = 55,153). There was a decrease in the frequency of isolated cystocele/rectocele repair procedures performed from 1979 to 2009 (96.3%, n = 150,980, to 67.7%, n = 61,444), whereas there was an increase in isolated apical defect repair procedures (2.5%, n = 3929, to 22.5%, n = 20,450). The proportion of cystocele/rectocele plus apical defect procedures also increased (1.2%, n = 1879, to 9.7%, n = 8806). Furthermore, 87.0% of all studied diagnostic groups were managed by cystocele/rectocele repair alone. CONCLUSIONS: Surgeons have responded to the increased contribution of apical support defects to cystocele/rectocele by modifying their diagnostic coding practices. Unfortunately, their surgical choices remain largely rooted in an older paradigm.

Placental specializations in lecithotrophic viviparous squamate reptiles.

Squamate reptiles have been thought to be predisposed to evolution of viviparity because embryos of most oviparous species undergo considerable development in the uterus prior to oviposition. A related hypothesis proposes that prolonged intrauterine gestation, an intermediate condition leading to viviparity, requires little or no physiological adjustment, other than reduction in thickness of the eggshell. This logical framework is often accompanied by an assumption that mode of parity (oviparity, viviparity) and pattern of embryonic nutrition (lecithotrophy, placentotrophy) are independent traits that evolve in sequence. Thus, specializations for viviparity should be absent in some lecithotrophic viviparous species. Studies of species of lizards with geographic variation in mode of parity challenge this scenario by demonstrating that placental specializations are correlated with viviparity. Uterine specializations for placental transport of calcium to viviparous embryos alter uterine physiology compared to oviparous females. In addition, comparative studies of oviparous and viviparous species, i.e., in which gene flow is disrupted, reveal that both uterine and embryonic structural modifications are commonly associated with viviparity, suggesting relatively rapid evolution of placental specializations. Studies of squamate reproductive biology support two hypotheses: 1) evolution of viviparity requires physiological adjustments of the uterine environment, and 2) evolution of viviparity promotes relatively rapid adaptations for placentation. Models for the evolution of viviparity from oviparity, or for reversals from viviparity to oviparity, should reflect current understanding of squamate reproductive biology and future studies should be designed to challenge these models.