Extraembryonic Membranes and Placentation in the Mexican Snake Conopsis lineata.

Extraembryonic membranes provide protection, oxygen, water, and nutrients to developing embryos, and their study generates information on the origin of the terrestrial egg and the evolution of viviparity. In this research, the morphology of the extraembryonic membranes and the types of placentation in the viviparous snake Conopsis lineata are described through optical microscopy during early and late gestation. When embryos develop inside the uterus, they become surrounded by a thin eggshell membrane. In early gestation, during stages 16 and 18, the embryo is already surrounded by the amnion and the chorion, and in a small region by the chorioallantois, which is product of the contact between the chorion and the growing allantois. A trilaminar omphalopleure covers the yolk sac from the embryonic hemisphere to the level of the equator where the sinus terminalis is located, and from there a bilaminar omphalopleure extends into the abembryonic hemisphere. Thus, according to the relationship of these membranes with the uterine wall, the chorioplacenta, the choriovitelline placenta, and the chorioallantoic placenta are structured at the embryonic pole, while the omphaloplacenta is formed at the abembryonic pole. During late gestation (stages 35, 36, and 37), the uterus and allantois are highly vascularized. The allantois occupies most of the extraembryonic coelom and at the abembryonic pole, it contacts the omphaloplacenta and form the omphalallantoic placenta. This is the first description of all known placenta types in Squamata for a snake species member of the subfamily Colubrinae; where an eggshell membrane with 2.9 µm in width present throughout development is also evident. The structure of extraembryonic membranes in C. lineata is similar to that of other oviparous and viviparous squamate species. The above indicates not only homology, but also that the functional characteristics have been maintained throughout the evolution of the reproductive type.

Remodeling of Uterine Tissues During Gestation of Potamotrygon wallacei (Elasmobranchii), a Neotropical Freshwater Stingray Endemic to the Negro River, Central Amazonia.

Neotropical freshwater stingrays of the subfamily Potamotrygoninae exhibit aplacental viviparity with uterine trophonemata. In this reproductive mode, females nourish and provide oxygenation to the embryo via the mucosa of the uterine wall. The aim of this study was to describe and histologically quantify the tissue components of the gravid uterus in an Amazonian freshwater stingray. Adult females of Potamotrygon wallacei were studied in different reproductive periods: resting stage, pregnant, and postpartum. During reproductive rest, the left ovary has numerous follicles compared to the right side. Therefore, uterine fertility is usually higher on the left side. The presence of an embryo in the right uterus suggests that the right ovary is also functional, although this only occurs in larger females. In females at reproductive rest, the wall of the uterus is formed by a mucosal layer (without the trophonemata) that contributes 16.7% to the thickness, while the myometrium accounts for 83.3% of the thickness. The mass-specific volume of the mucosal layer, inner circular, and outer longitudinal smooth muscle sheets tend to increase in the gravid uterus, indicating hypertrophy and hyperplasia of these components. During pregnancy, the trophonemata undergo marked tissue remodeling. Epithelial cells are organized into glandular acini and have apical secretory vesicles; furthermore, peripheral blood vessels proliferate and become dilated. These characteristics demonstrate that the gravid uterus of P. wallacei presents intense uterolactation activity and provides oxygenation to the fetus. Tissue remodeling occurs only in the uterus with the presence of an embryo. During postpartum, females have low body condition factor indicating a high reproductive cost. This study contributes to the knowledge of the reproductive biology of this species and will help us understand the impacts of climate change on the breeding areas of potamotrygonids.

Microscopic Analysis of Nuclear Speckles in a Viviparous Reptile.

Nuclear speckles are compartments enriched in splicing factors present in the nucleoplasm of eucaryote cells. Speckles have been studied in mammalian culture and tissue cells, as well as in some non-mammalian vertebrate cells and invertebrate oocytes. In mammals, their morphology is linked to the transcriptional and splicing activities of the cell through a recruitment mechanism. In rats, speckle morphology depends on the hormonal cycle. In the present work, we explore whether a similar situation is also present in non-mammalian cells during the reproductive cycle. We studied the speckled pattern in several tissues of a viviparous reptile, the lizard Sceloporus torquatus, during two different stages of reproduction. We used immunofluorescence staining against splicing factors in hepatocytes and oviduct epithelium cells and fluorescence and confocal microscopy, as well as ultrastructural immunolocalization and EDTA contrast in Transmission Electron Microscopy. The distribution of splicing factors in the nucleoplasm of oviductal cells and hepatocytes coincides with the nuclear-speckled pattern described in mammals. Ultrastructurally, those cell types display Interchromatin Granule Clusters and Perichromatin Fibers. In addition, the morphology of speckles varies in oviduct cells at the two stages of the reproductive cycle analyzed, paralleling the phenomenon observed in the rat. The results show that the morphology of speckles in reptile cells depends upon the reproductive stage as it occurs in mammals.

Increased superfetation precedes the evolution of advanced degrees of placentotrophy in viviparous fishes of the family Poeciliidae.

The causes and consequences of the evolution of placentotrophy (post-fertilization nutrition of developing embryos of viviparous organisms by means of a maternal placenta) in non-mammalian vertebrates are still not fully understood. In particular, in the fish family Poeciliidae there is an evolutionary link between placentotrophy and superfetation (ability of females to simultaneously bear embryos at distinct developmental stages), with no conclusive evidence for which of these two traits facilitates the evolution of more advanced degrees of the other. Using a robust phylogenetic comparative method based on Ornstein-Uhlenbeck models of adaptive evolution and data from 36 poeciliid species, we detected a clear causality pattern. The evolution of extensive placentotrophy has been facilitated by the preceding evolution of more simultaneous broods. Therefore, placentas became increasingly complex as an adaptive response to evolutionary increases in the degree of superfetation. This finding represents a substantial contribution to our knowledge of the factors that have shaped placental evolution in poeciliid fishes.

Isotopic (δ15 N) relationship of pregnant females and their embryos: Comparing placental and yolk-sac viviparous elasmobranchs.

Nitrogen stable isotopes ratios (δ15 N) were determined for selected tissues (muscle, liver, blood and yolk) of pregnant females and their embryos of a placental viviparous species, the Pacific sharpnose shark (Rhizoprionodon longurio), and a yolk-sac viviparous species, the speckled guitarfish (Pseudobatos glaucostigmus). The R. longurio embryo tissues were 15 N enriched compared to the same tissues in the pregnant female, using the difference in δ15 N (Δδ15 N) between embryo and adult. Mean Δδ15 N was 2.17‰ in muscle, 4.39‰ in liver and 0.80‰ in blood. For P. glaucostigmus, embryo liver tissue was significantly 15 N enriched in comparison with liver of the pregnant female (Δδ15 N mean = 1.22‰), whereas embryo muscle was 15 N depleted relative to the muscle of the pregnant female (Δδ15 N mean = -1.22‰). Both species presented a significant positive linear relationship between Δδ15 N and embryo total length (LT ). The results indicated that embryos have different Δδ15 N depending on their reproductive strategy, tissue type analysed and embryo LT .

Resource availability and its effects on mother to embryo nutrient transfer in two viviparous fish species.

Different hypotheses have been proposed to explain the adaptive value of matrotrophy, which is the postfertilization maternal provisioning to developing embryos. The Trexler-DeAngelis model proposes that matrotrophy provides fitness advantages when food abundance is high and availability is constant. If food availability is low or unpredictable, prefertilization maternal provisioning (lecithotrophy) should be favored over matrotrophy. In this study, we tested this model in two fish species from the family Poeciliidae, Poeciliopsis gracilis and P. infans, using field and laboratory data. In the field study, we explored the effects of population, season, and food abundance on the degree of matrotrophy. In P. infans, we found evidence that supports this model: In the population where food abundance decreased during the dry season, females reduced the amount of postfertilization provisioning and thus exhibited a more lecithotrophic strategy. In P. gracilis, we observed patterns that were partially consistent with this model: Food abundance decreased during the wet season in three populations of this species, but only in one of these populations, females exhibited less postfertilization nutrient transfer during this season. In the laboratory study, we tested the effects of constant, fluctuating, and low food availability on the relative amounts of pre- and postfertilization provisioning of P. infans. Our laboratory results also support the Trexler-DeAngelis model because both low and fluctuating food regimes promoted a more lecithotrophic strategy. Together, our findings indicate that the benefits of matrotrophy are more likely to occur when females have constant access to food sources.

Change of lecithotrophic to matrotrophic nutrition during gestation in the viviparous teleost Xenotoca eiseni (Goodeidae).

Teleosts possess unique features of the female reproductive system compared with the rest of vertebrates, features that define the characteristics of their viviparity. Viviparity involves new maternal-embryonic relationships detailing the most diverse structures during gestation that include embryonic nutrition. In order to analyze the morphological features of the complex nutrition in viviparous teleosts during intraovarian gestation, this study utilizes the goodeid Xenotoca eiseni as a model. Ovarian gestation in X. eiseni, as in all goodeids, is intraluminal; the early embryo moves from the follicle to the ovarian lumen where gestation continues. The scarce yolk in the oocytes implies that the initial lecithotrophy is replaced by matrotrophy, with nutrients provided via maternal tissues. The nutrients are absorbed by the embryo mainly by trophotaenia, extensions of the embryonic intestine into the ovarian lumen. This histological study analyses the structures involved in these two types of nutrition and when they occur during gestation in X. eiseni. The morphology displayed in this study demonstrated the extended simultaneity of lecithotrophy and matrotrophy during gestation with the progressive reduction of lecithotrophy and increase of matrotrophy. Similarly, it describes the development of complex embryonic structures for metabolic exchange with the maternal tissues associated with matrotrophy; specifically the branchial placenta and mainly the trophotaenia.

Proteomic Profile of Mabuya sp. (Squamata: Scincidae) Ovary and Placenta During Gestation.

Reptiles are one of the most diverse groups of vertebrates, providing an integrated system for comparative studies on metabolic, animal physiology, and developmental biology. However, the molecular data available are limited and only recently have started to call attention in the "omics" sciences. Mabuya sp. is a viviparous placentrotrophic skink with particular reproductive features, including microlecithal eggs, early luteolysis, prolonged gestation, and development of a highly specialized placenta. This placenta is responsible for respiratory exchange and the transference of all nutrients necessary for embryonic development. Our aim was to identify differentially expressed proteins in the ovary and placenta of Mabuya sp. during early, mid, and late gestation; their possible metabolic pathways; and biological processes. We carried out a comparative proteomic analysis during gestation in both tissues by sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis, and matrix-assisted laser desorption/ionization. Differential protein expression in both tissues (Student's t-test P < 0.05) was related to several processes such as cell structure, cell movement, and energy. Proteins found in ovary are mainly associated with follicular development and its regulation. In the placenta, particularly during mid and late gestation, protein expression is involved in nutrient metabolism, transport, protein synthesis, and embryonic development. This work provides new insights about the proteins expressed and their physiological mechanisms in Mabuya sp. placenta and ovary during gestation.

How does a viviparous semifossorial lizard reproduce? Ophiodes intermedius (Squamata: Anguidae) from subtropical climate in the Wet Chaco region of Argentina.

The best predictors of reproductive patterns are commonly associated with climate factors, but evolutionary history also plays an important role. The semifossorial and viviparous lizard Ophiodes intermedius from the Wet Chaco region of Argentina showed an annual cycle with asynchrony between males and females and an unusual pattern for subtropical climates, with vitellogenesis beginning in autumn, ovulation and copulation in spring, and births occurring in summer. Males exhibited annual variation of testicular size associated with spermatogenic activity, reaching their maximum gonadal activity in late summer (March), but sperm storage in the epididymis and/or deferent duct occurred throughout the year. Females showed an extended reproductive cycle beginning in mid-autumn (May) with vitellogenesis and finishing with births from late spring to mid-summer (December to February). Litter size varied from 4 to 9 offspring. Females reached sexual maturity at a larger snout-vent length and, overall, showed greater body size than males, while males exhibited larger heads than females. Fat body cycles indicated that females use lipid reserves to support vitellogenesis and embryo development, while males allocate lipid resources to the search for females, courtship and copulation rather than to gametogenesis. Ophiodes intermedius differed from other species of the genus in litter size, gestation period, timing of birth and the minimum size at sexual maturity, probably as a result of the influence of ecological, historical and phylogenetic factors.

Spatial and temporal variation in superfoetation and related life history traits of two viviparous fishes: Poeciliopsis gracilis and P. infans.

Superfoetation is the ability of females to simultaneously bear multiple broods of embryos at different developmental stages. Most studies on the phylogenetic distribution of superfoetation and on the factors that potentially promote superfoetation ignore variation within species. Here, we studied 11 populations of two species of viviparous fishes of the family Poeciliidae (Poeciliopsis gracilis and Poeciliopsis infans) and document wide variation in superfoetation and in three related life history traits: brood size, individual embryo mass and total reproductive allotment. We found significant differences in the average number of simultaneous broods among populations of P. gracilis but not among populations of P. infans. In addition, we found even greater variation between months within populations for both species, although no specific pattern of temporal variation was evident. Instead of the expected consistency of seasonal differences in superfoetation across populations, we found that large variation among months within seasons and the amount and direction of this monthly variation differed widely between populations. Our results emphasize the importance of including intraspecific variation in superfoetation and other life history traits in studies that aimed at finding general explanations of life history trait evolution.

[Fishery and biology of Rhinobatospercellens (Rajiformes: Rhinobatidae) caught by the artisanal fishery at La Pared beach, Venezuela]. / Pesquería y biologia de Rhinobatos percellens (Rajiformes: Rhinobatidae) capturados por la pesquería artesanal de playa La Pared, Venezuela.

R. percellens is incidentally caught in Venezuela by the artisanal fishery using bottom gillnets. This species was classified by the IUCN as being "near threatened", and there is poor knowledge about its biology and fishery in Venezuela. For this reason, we analyzed the specimens caught by the artisanal fleet in playa La Pared, once a week, from January to December 2007. We determined total length, sex and maturity for each captured specimen. A total of 210 specimens were analyzed, 159 females and 51 males. The 81% of all specimens caught were adults, but within this group 27% were pregnant. The average size of sexual maturity was found between 51 and 52cm for females and males, respectively. Adult females were found all year round, and pregnant females in seven of the months sampled; highest numbers were found between February-March and September. The analyzed females had a total of 96 embryos with a size range between 2 and 19cm, with a maximum fertility of four embryos per litter. February and June-July showed the maximum breeding time, and considering the broad range of embryos sizes and the frequency of pregnant females along the different months, allow us to believe that they reproduce throughout the year. Fishing regulation including minimum catch size in relation to maturity length and release of pregnant females in water is recommended.

Pesquería y biología de Rhinobatos percellens (Rajiformes: Rhinobatidae) capturados por la pesquería artesanal de playa La Pared, Venezuela / Fishery and biology of Rhinobatos percellens (Rajiformes: Rhinobatidae) caught by the artisanal fishery at La Pared beach, Venezuela

R. percellens is incidentally caught in Venezuela by the artisanal fishery using bottom gillnets. This species was classified by the IUCN as being near threatened, and there is poor knowledge about its biology and fishery in Venezuela. For this reason, we analyzed the specimens caught by the artisanal fleet in playa La Pared, once a week, from January to December 2007. We determined total length, sex and maturity for each captured specimen. A total of 210 specimens were analyzed, 159 females and 51 males. The 81% of all specimens caught were adults, but within this group 27% were pregnant. The average size of sexual maturity was found between 51 and 52cm for females and males, respectively. Adult females were found all year round, and pregnant females in seven of the months sampled; highest numbers were found between February-March and September. The analyzed females had a total of 96 embryos with a size range between 2 and 19cm, with a maximum fertility of four embryos per litter. February and June-July showed the maximum breeding time, and considering the broad range of embryos sizes and the frequency of pregnant females along the different months, allow us to believe that they reproduce throughout the year. Fishing regulation including minimum catch size in relation to maturity length and release of pregnant females in water is recommended.

Rhinobatos percellens es capturada en Venezuela por la flota artesanal que opera con redes de fondo. Está clasificada por la IUCN como casi amenazada, y no existen estudios sobre su biología o pesquería en el país. Por ello, se analizaron desembarques de la pesca artesanal de playa la Pared (isla de Margarita) durante enero-diciembre 2007. A cada ejemplar capturado se le determinó la longitud total, sexo y madurez. Fueron analizados 210 ejemplares, 159 hembras y 51 machos. El 81% de los ejemplares se encontraban maduros y un 27% de este grupo correspondió a hembras grávidas. La talla media de madurez sexual quedó ubicada en 51 y 52cm para hembras y machos, respectivamente. Se detectaron hembras maduras todo el año, y hembras grávidas en siete meses. Fueron analizados 96 embriones con tallas entre 2-19cm, siendo la fecundidad máxima cuatro embriones. La variación de las longitudes de los embriones entre los meses del año indican dos picos máximos de alumbramiento al año pero no existe una sincronía en la cópula ya que la especie se reproduce prácticamente todo el año. Se recomienda elaborar regulaciones pesqueras que incluyan una talla mínima de captura en función de la talla de madurez señalada y liberar las hembras grávidas al agua.

Uterine and chorioallantoic angiogenesis and changes in the uterine epithelium during gestation in the viviparous lizard, niveoscincus conventryi (Squamata: Scincidae).

We used immunofluorescent confocal microscopy and scanning electron microscopy to quantify uterine vascularity and to describe uterine surface morphology during gestation in pregnant females of the lecithotrophic lizard Niveoscincus coventryi. As uterine angiogenesis and epithelial cell morphology are thought to be under progesterone control, we studied the effect of a progesterone receptor antagonist (mifepristone) on uterine and chorioallantoic microvasculature and features of the uterine epithelial surfaces. Although intussuceptive angiogenesis was observed in both, uterine and chorioallantoic, vascular beds during gestation, the only significant increases were in the diameters of the uterine vessels. An ellipsoid vessel-dense area grows in the mesometrial hemisphere of the developing conceptus, which parallels the expansion of the allantois to form the chorioallantoic placenta. Uterine surface topography changed during gestation. In particular, uterine blood vessels bulge over the luminal surface to form marked ridges on the uterine embryonic hemisphere, especially during the last stage of pregnancy, and ciliated cells are maintained in the embryonic and abembryonic hemispheres but disappear in both the mesometrial and antimesometrial poles. This distinct regionalization of uterine ridges and ciliated cells in the uterine surface and in the shape of the epithelial component of the chorion might be related to the function of both chorioallantoic and yolk sac placentae during gestation. There was no significant difference between females treated with or without mifepristone, which may be related to the partial function of mifepristone as a progestin antagonist and/or with the function and time of action of progesterone in the uterus during gestation in N. coventryi. Differences in the pattern of angiogenesis and uterine surface morphology during gestation among squamates may be related to the functional diversity of the uterine component of the different placentae and probably reflect its diverse evolutionary history.

Transplacental nutrient transfer during gestation in the Andean lizard Mabuya sp. (Squamata, Scincidae).

Mabuya skinks have the most specialized allantoplacenta and the greatest degree of placentotrophy known among the Reptilia. Their recently ovulated eggs are microlecithal (1-2 mm) and lack fatty yolk platelets; thus, virtually all of the nutrients for embryonic development must pass across the placenta. We quantified the net uptake of nutrients during gestation in an Andean population of Mabuya and compared these results with other matrotrophic skinks and eutherian mammals. Total dry and wet masses, ash and organic content, ions (calcium, potassium, sodium, magnesium, iron), lipids (cholesterol, vitamin E and fatty acids in the major lipid classes-triacylglycerol, phospholipids, cholesteryl ester, free fatty acids), nitrogen (an index of protein) were measured during the different developmental stages throughout gestation, and in neonates. A significant net uptake of inorganic and organic matter was found. This uptake begins slowly during early gestation but accelerates during the last third of gestation when the growth of the embryo is maximal and the allantoplacenta has developed its greatest complexity. The drastic reduction of egg size in this clade is related to the great reduction in the contribution of lecithotrophic nutrients to the embryo, an obligatory placentotrophy from early developmental stages, and the highest placental complexity known in the Reptilia. All of these features converge with features found in eutherian mammals.

Toxic hydrogen sulfide and dark caves: life-history adaptations in a livebearing fish (Poecilia mexicana, Poeciliidae).

Life-history traits are very sensitive to extreme environmental conditions, because resources that need to be invested in somatic maintenance cannot be invested in reproduction. Here we examined female life-history traits in the Mexican livebearing fish Poecilia mexicana from a variety of benign surface habitats, a creek with naturally occurring toxic hydrogen sulfide (H2S), a sulfidic cave, and a non-sulfidic cave. Previous studies revealed pronounced genetic and morphological divergence over very small geographic scales in this system despite the absence of physical barriers, suggesting that local adaptation to different combinations of two selection factors, toxicity (H2S) and darkness, is accompanied by very low rates of gene flow. Hence, we investigated life-history divergence between these populations in response to the selective pressures of darkness and/or toxicity. Our main results show that toxicity and darkness both select for (or impose constraints on) the same female trait dynamics: reduced fecundity and increased offspring size. Since reduced fecundity in the sulfur cave population was previously shown to be heritable, we discuss how divergent life-history evolution may promote further ecological divergence: for example, reduced fecundity and increased offspring autonomy are clearly beneficial in extreme environments, but fish with these traits are outcompeted in benign habitats.

Outbred embryos rescue inbred half-siblings in mixed-paternity broods of live-bearing females.

Females commonly mate with more than one male, and polyandry has been shown to increase reproductive success in many species. Insemination by multiple males shifts the arena for sexual selection from the external environment to the female reproductive tract, where sperm competition or female choice of sperm could bias fertilization against sperm from genetically inferior or genetically incompatible males. Evidence that polyandry can be a strategy for avoiding incompatibility comes from studies showing that inbreeding cost is reduced in some egg-laying species by postcopulatory mechanisms that favour fertilization by sperm from unrelated males. In viviparous (live-bearing) species, inbreeding not only reduces offspring genetic quality but might also disrupt feto-maternal interactions that are crucial for normal embryonic development. Here we show that polyandry in viviparous pseudoscorpions reduces inbreeding cost not through paternity-biasing mechanisms favouring outbred offspring, but rather because outbred embryos exert a rescuing effect on inbred half-siblings in mixed-paternity broods. The benefits of polyandry may thus be more complex for live-bearing females than for females that lay eggs.