Anidación de la tortuga marina Lepidochelys olivacea (Testudinata: Cheloniidae) en Campamento Tortuguero Cedeño, Honduras (2011-2021) / Nesting of olive ridley sea turtle Lepidochelys olivacea (Testudinata: Cheloniidae) in Campamento Tortuguero Cedeño, Honduras (2011-2021)

Introducción: El Campamento Tortuguero de Cedeño ha sido el sitio menos investigado del Golfo de Fonseca, donde se protege a la tortuga golfina en Honduras desde 1975. Objetivo: Evaluar la anidación de la tortuga Golfina (Lepidochelys olivacea) durante la temporada de veda entre el 2011 y 2021 en Campamento Tortuguero Cedeño, Choluteca, Honduras. Métodos: Entre 2011 a 2021, se llevó a cabo el monitoreo diario de las actividades de anidación durante la veda del 1 al 25 de septiembre. Los patrullajes se realizaron entre las 6:00-18:00 h, y las 18:00-5:00 h. Se registró el número total de tortugas que anidaban y se recogieron sus huevos, que se transportaron al criadero, donde se tabularon los resultados de las puestas y las crías. Resultados: Se registró un total 1 065 tortugas de L. olivacea, 95 051 huevos recolectados, 1 065 nidos marcados en tres playas que fueron reubicados en viveros artificiales y una eclosión exitosa de 62 747 neonatos. La playa Las Doradas fue el sitio con el mayor número de tortugas anidadoras, seguido de Los Delgaditos y por último Cedeño. El promedio de la frecuencia de anidación fue de 96 nidos. Del 2011 al 2021 el esfuerzo de recolección de los nidos aumentó en un 91.6 %, pasando de 84 a 161 nidos. El número de personas patrullando se asoció con la cantidad de nidos detectados en las playas. Conclusión: Los esfuerzos de monitoreo y conservación para la especie han indicado que ha habido un incremento en la anidación de L. olivacea en las tres playas, con un mayor incremento en Playa Las Doradas. Este escenario comprueba la funcionalidad de la veda en esta zona.

Introduction: The Cedeño Turtle Camp has been the least researched site in the Fonseca Gulf, where Olive Ridley Turtles in Honduras have been protected since 1975. Objective: To evaluate the nesting of Olive Ridley turtles (Lepidochelys olivacea) during the closed season from 2011 to 2021 in Campamento Tortuguero Cedeño, Choluteca, Honduras. Methods: From 2011 to 2021, daily monitoring of nesting activities was conducted during the closed season from the 1st to 25th of September. Patrols were conducted between 6:00-18:00 h, and 18:00-5:00 h. The total number of nesting turtles was recorded, and their eggs were collected and transported to the hatchery, where clutch and hatchling performance were tabulated. Results: A total of 1 065 L. olivacea turtles were recorded, 95 051 eggs collected, 1 065 nests marked on three beaches that were relocated in artificial hatcheries and a successful hatching of 62 747 hatchlings. Las Doradas beach was the site with the highest number of nesting turtles, followed by Los Delgaditos and lastly Cedeño. The average nesting frequency was 96 nests. From 2011 to 2021 the nest collection effort increased by 91.6 %, from 84 to 161 nests. The number of people patrolling was associated with the number of nests detected on the beaches. Conclusion: Monitoring and conservation efforts for L. olivacea in the Campamento Tortuguero Cedeño show a positive trend in nesting with a greater increase in Playa Las Doradas. This scenario proves the functionality of the closed season in this area.

Expression and characterization of the cyp19a gene and its responses to estradiol/letrozole exposure in Chinese soft-shelled turtle (Pelodiscus sinensis).

Cytochrome P450 aromatase (CYP19) catalyzes the conversion of androgens to estrogens and is critical in sex differentiation. CYP19 exists as the ovarian type and brain type. Herein, we cloned the full-length ovarian cyp19a gene from the Chinese soft-shelled turtle, Pelodiscus sinensis (pscyp19a). We determined the distribution of pscyp19a in adult tissue and evaluated its expression during embryonic development, following treatment with 17ß-estradiol (E2) or letrozole (LE). The pscyp19a complementary DNA is 2,285 bp in length and comprises a 1,512 bp open reading frame that encodes a protein of 503 AA. The nucleotide sequence and amino acid of pscyp19a shared significant identity with other vertebrate sequences. Expression of pscyp19a was high in the ovary (p < 0.01), and exhibited modest expression in the female brain and intestine. Expression of pscyp19a displayed significant differences between sexes during early embryo development stages; expression increased gradually during embryonic development in females, but the opposite trend was observed in males. Female embryos treated with different concentrations of E2 and LE displayed altered pscyp19a expression compared with untreated individuals, and E2 clearly induced pscyp19a expression. These results indicate that pscyp19a gene plays important roles in early developmental stages in Chinese soft-shelled turtle, and may assist future studies on sex differentiation and sex control in this and similar species.

17ß-Estradiol modulates cell proliferation of medullary cords during ovarian differentiation of the Lepidochelys olivacea sea turtle.

In turtles undergoing temperature sex determination (TSD), bipotential gonads express Sox9 in medullary cords at both female- (FPT) and male-producing temperatures (MPT). Subsequently, when the sex fate of medullary cords becomes dimorphic, at FPT, Sox9 is downregulated, whereas at MPT, its expression is maintained. Medullary cords in the ovary turn into ovarian lacuna, whereas in the testis they differentiate as seminiferous cords. When embryos of Lepidochelys olivacea sea turtle are incubated at MPT and treated with estradiol, Sox9 expression persists in the medullary cords in the form of tiny ovotestis-like formations. The perturbed development of the treated gonads is due to a significant decrease in the number of proliferating cells. This suggests that the disturbed effect caused by exogenous estradiol may be due to a conflict between the gene networks regulated by temperature and the increased level of endogenous estrogens, induced by the treatment. Here, we decided to use fadrozole and fulvestrant, an aromatase inhibitor and an estrogen-receptor antagonist, respectively, to provide insights into the role played by endogenous estrogens in regulating the cell proliferation of the two main gonadal compartments: the medullary cords and the cortex. Comparing cell proliferation patterns, our current results suggest that the endogenous estrogens are involved in determining the sex fate of medullary cords, by repressing proliferation. Interestingly, our results showed that endogenous estradiol levels are unnecessary for the thickening of the ovarian cortex.

The evolution of basal progenitors in the developing non-mammalian brain.

The amplification of distinct neural stem/progenitor cell subtypes during embryogenesis is essential for the intricate brain structures present in various vertebrate species. For example, in both mammals and birds, proliferative neuronal progenitors transiently appear on the basal side of the ventricular zone of the telencephalon (basal progenitors), where they contribute to the enlargement of the neocortex and its homologous structures. In placental mammals, this proliferative cell population can be subdivided into several groups that include Tbr2(+) intermediate progenitors and basal radial glial cells (bRGs). Here, we report that basal progenitors in the developing avian pallium show unique morphological and molecular characteristics that resemble the characteristics of bRGs, a progenitor population that is abundant in gyrencephalic mammalian neocortex. Manipulation of LGN (Leu-Gly-Asn repeat-enriched protein) and Cdk4/cyclin D1, both essential regulators of neural progenitor dynamics, revealed that basal progenitors and Tbr2(+) cells are distinct cell lineages in the developing avian telencephalon. Furthermore, we identified a small population of subapical mitotic cells in the developing brains of a wide variety of amniotes and amphibians. Our results suggest that unique progenitor subtypes are amplified in mammalian and avian lineages by modifying common mechanisms of neural stem/progenitor regulation during amniote brain evolution.

Embryonic sex steroid hormones accumulate in the eggshell of loggerhead sea turtle (Caretta caretta).

Steroids hormones such as estradiol-17ß (E2) and testosterone (T) are involved in gonadal differentiation of oviparous animals with temperature-dependent sex determination (TSD), and are greatly distributed. This hypothesizes that these embryonic steroid hormones probably accumulate in the eggshell throughout blood or/and chorioallantoic fluid in sea turtle species with TSD, producing females at higher temperature. To demonstrate this hypothesis, concentrations of E2 and T in the blood plasma from the hatchling loggerhead sea turtle (Caretta caretta) and in their eggshells were measured by radioimmunoassay. In the present study we propose that both concentrations of E2 and T in the blood plasma are correlated with amounts of these sex steroids in the eggshell. Moreover, contents of E2 in the eggshell showed a significant positive correlation with mean incubation temperatures during a thermosensitive period in the experimental nests, whereas T contents in the eggshell did not. Taken together, these findings indicated that embryonic E2 and T that accumulated in the eggshell can be extracted and measured. Furthermore, the present study suggested that contents of E2 in the eggshell may differ between male and female, and monitoring of these steroids is a useful method to identify the sex of loggerhead sea turtle hatchling.

Nest site selection by individual leatherback turtles (Dermochelys coriacea, Testudines: Dermochelyidae) in Tortuguero, Caribbean coast of Costa Rica / Selección del sitio de anidación de individuos de tortuga baula (Dermochelys coriacea, Testudines: Dermochelyidae) en Tortuguero, costa Caribe de Costa Rica

Nest site selection for individual leatherback sea turtles, Dermochelys coriacea, is a matter of dispute. Some authors suggest that a female will tend to randomly scatter her nests to optimize clutch survival at a highly dynamic beach, while others suggest that some site fidelity exists. It is also possible that both strategies exist, depending on the characteristics of each nesting beach, with stable beaches leading to repeating nest site selections and unstable beaches leading to nest scattering. To determine the strategy of the Tortuguero population of D. coriacea, female site preference and repetition were determined by studying whether females repeat their nest zone choices between successive attempts and whether this leads to a correlation in hatching and emergence success of subsequent nests. Nesting data from 1997 to 2008 were used. Perpendicular to the coastline, open sand was preferred in general, regardless of initial choice. This shows a tendency to scatter nests and is consistent with the fact that all vertical zones had a high variability in hatching and emergence success. It is also consistent with nest success not being easily predictable, as shown by the lack of correlation in success of subsequent nesting attempts. Along the coastline, turtles showed a preference for the middle part of the studied section of beach, both at a population level and as a tendency to repeat their initial choice. Interestingly, this zone has the most artificial lights, which leads to slightly lower nest success (though not significantly so) and hatchling disorientation. This finding merits further study for a possibly maladaptive trait and shows the need for increased control of artificial nesting on this beach.

La selección de sitio de anidación por parte de individuos de la tortuga baula, Dermochelys coriacea, es tema de discusión. Algunos autores sugieren que una hembra dispersa sus nidos al azar para maximizar su éxito en las inestables playas que elige para anidar, mientras que otros sugieren que existe cierta fidelidad al sitio de anidación. También es posible que ambas estrategias existan, dependiendo de las características de la playa donde anidan, así playas estables llevarían a las hembras a repetir la selección de sitio mientras que las playas inestables las llevarían a dispersar sus nidos. Para determinar la estrategia que usan las hembras de D. coriacea de Tortuguero, se estudió la preferencia de sitio de las hembras así como su repetición en este sitio, desde 1997 hasta 2008. Para la selección de sitio vertical (perpendicular a la costa), las hembras en general prefirieron anidar en arena abierta, independientemente de su escogencia inicial. Esto muestra una tendencia a dispersar los nidos y es consistente con la alta variación en los éxitos de eclosión en todas las zonas, así como la falta de relación entre los éxitos de eclosión de nidos subsecuentes, lo cual muestra que el éxito de los nidos no es predecible. Para la selección horizontal (a lo largo de la costa) se prefirió la parte media de la sección estudiada de la playa, tanto a nivel poblacional como individual (repetición de selección de sitio). Interesantemente, esta zona tiene la mayor cantidad de luces artificiales lo cual lleva a éxitos de eclosión ligeramente más bajos (aunque no significativamente) y a la desorientación de neonatos. Este resultado amerita estudios futuros para determinar si se trata de una tendencia maladaptativa en la población y muestra la importancia de controlar la cantidad de luz artificial en esta playa como medida de conservación.

Apoptosis durante el desarrollo embrionario de la retina de tortuga (Trachemys scripta elegans) / Apoptosis in turtle embryonic retina (Trachemys scripta elegans)

La apoptosis o muerte celular programada es un proceso que ocurre durante el desarrollo del sistema nervioso. El objetivo de este estudio fue observar los patrones de apoptosis que se producen durante el desarrollo embrionario de la retina, desde el estadio S16 al momento del nacimiento, mediante miscoscopía óptica y electrónica. Se utilizaron retinas de embriones de tortuga. Nuestros datos muestran que los primeros signos de apoptosis comienzan en el estadio S16, en la capa nuclear interna y alcanzan su máxima densidad tanto en la capa nuclear interna como en la capa de células ganglionares en S20, para extinguirse, prácticamente, en el momento del nacimiento. Por otra parte, la apoptosis sigue un gradiente centro-periferia.

Apoptosis or programmed cell death is a process that occurs during development of the nervous system. The aim of this study was to observe the patterns of apoptosis that occur during embryonic development of the retina from the stage S16 at birth, by light and electron miscoscopia. Turtle embryonic retinas were used for the study. Our data show that the first signs of apoptosis begins at stage S16 in the inner nuclear layer and reaches maximum density both in the inner nuclear layer and the ganglion cell layer in S20 until they practically disappear at the time of birth. Furthermore, apoptosis follows a gradient center-periphery.

Chronology, magnitude and duration of expression of putative sex-determining/differentiation genes in a turtle with temperature-dependent sex determination.

The red-eared slider turtle (Trachemys scripta) possesses temperature-dependent sex determination (TSD) in which the incubation temperature determines gonadal sex. Although a number of mammalian gene homologues have been identified in reptiles with TSD, the exact sex-determining trigger(s) is not known. To date, the current study represents the most comprehensive simultaneous evaluation of the chronology of mRNA expression profiles of putative sex-determining/differentiation genes (Dmrt1, Sox9, Amh, Lhx9, and Foxl2) from gonads incubated at male- and female-producing temperatures in T. scripta. Additionally, sex-reversing treatments with 17ß-estradiol and letrozole were examined. At a male-producing temperature, Dmrt1 expression was sexually dimorphic by stage 17, Sox9 by 19 and Amh by 21. In contrast, Foxl2 did not significantly increase until after the thermosensitive period at a female-producing temperature. Treatment with 17ß-estradiol resulted in reduced gonad size and/or inhibited gonadal development and differentiation. Gene expression was subsequently low in this group. Sex reversal utilizing letrozole failed to produce testes at a female-producing temperature and as such, gene expression was comparable to ovary. These results indicate that Dmrt1 and Sox9 are potential triggers for testis differentiation and Amh, Lhx9 and Foxl2 represent a conserved core set of genes in the sex-determining/differentiation pathway of TSD species.

Adjustments in cholinergic, adrenergic and purinergic control of cardiovascular function in snapping turtle embryos (Chelydra serpentina) incubated in chronic hypoxia.

Adenosine is an endogenous nucleoside that acts via G-protein coupled receptors. In vertebrates, arterial or venous adenosine injection causes a rapid and large bradycardia through atrioventricular node block, a response mediated by adenosine receptors that inhibit adenylate cyclase and decrease cyclic AMP concentration. Chronic developmental hypoxia has been shown to alter cardioregulatory mechanisms in reptile embryos, but adenosine's role in mediating these responses is not known. We incubated snapping turtle embryos under chronic normoxic (N21; 21 % O2) or chronic hypoxic conditions (H10; 10 % O2) beginning at 20 % of embryonic incubation. H10 embryos at 90 % of incubation were hypotensive relative to N21 embryos in both normoxic and hypoxic conditions. Hypoxia caused a hypotensive bradycardia in both N21 and H10 embryos during the initial 30 min of exposure; however, f H and P m both trended towards increasing during the subsequent 30 min, and H10 embryos were tachycardic relative to N21 embryos in hypoxia. Following serial ≥1 h exposure to normoxic and hypoxic conditions, a single injection of adenosine (1 mg kg(-1)) was given. N21 and H10 embryos responded to adenosine injection with a rapid and large hypotensive bradycardia in both normoxia and hypoxia. Gene expression for adenosine receptors were quantified in cardiac tissue, and Adora1 mRNA was the predominant receptor subtype with transcript levels 30-82-fold higher than Adora2A or Adora2B. At 70 % of incubation, H10 embryos had lower Adora1 and Adora2B expression compared to N21 embryos. Expression of Adora1 and Adora2B decreased in N21 embryos during development and did not differ from H10 embryos at 90 % of incubation. Similar to previous results in normoxia, H10 embryos in hypoxia were chronically tachycardic compared to N21 embryos before and after complete cholinergic and adrenergic blockade. Chronic hypoxia altered the development of normal cholinergic and adrenergic tone, as well as adenosine receptor mRNA levels. This study demonstrates that adenosine may be a major regulator of heart rate in developing snapping turtle embryos, and that chronic hypoxic incubation alters the response to hypoxic exposure.

Expression of aromatase in the embryonic brain of the olive ridley sea turtle (Lepidochelys olivacea), and the effect of bisphenol-A in sexually differentiated embryos.

Brain aromatase participates in several biological processes, such as regulation of the reproductive-endocrine axis, memory, stress, sexual differentiation of the nervous system, male sexual behavior, and brain repair. Here we report the isolation and expression of brain aromatase in olive ridley sea turtle (Lepidochelys olivacea) embryos incubated at male- and female-promoting temperatures (MPT and FPT, respectively), at the thermosensitive period (TSP) and the sex-differentiated period. Also, aromatase expression was assessed in differentiated embryos exposed to bisphenol-A (BPA) during the TSP. BPA is a monomer of polycarbonate plastics and is considered an endocrine-disrupting compound. Normal aromatase expression was measured in both forebrain and hindbrain, showing higher expression levels in the forebrain of differentiated embryos at both incubation temperatures. Although no significant differences were detected in the hindbrain, expression was slightly higher at MPT. BPA did not affect aromatase expression neither in forebrains or hindbrains from embryos incubated at MPT, whereas at FPT an inverted U-shape curve was observed in forebrains with significant differences at lower concentrations, whereas in hindbrains a non-significant increment was observed at higher concentrations. Our data indicate that both incubation temperature and developmental stage are critical factors affecting aromatase expression in the forebrain. Because of the timing and location of aromatase expression in the brain, we suggest that brain aromatase may participate in the imprinting of sexual trends related to reproduction and sexual behavior at the onset of sex differentiation, and BPA exposure may impair aromatase function in the female forebrain.

Sulfonation of maternal steroids is a conserved metabolic pathway in vertebrates.

All vertebrate embryos develop in the presence of maternally derived steroids, and maternal steroids have been hypothesized to link phenotype of the offspring to maternal physiology. In placental vertebrates, it is known that maternally derived steroids are metabolized during development via the sulfonation pathway. We used eggs from the red-eared slider turtle (Trachemys scripta) to determine whether the same metabolic pathway is used to metabolize maternally derived steroids in an oviparous vertebrate. To examine the relationship between estradiol and estrogen sulfates during development, levels of maternally derived estradiol were compared with levels of estradiol sulfate, estrone sulfate, and estriol sulfate at oviposition and after 20 days of embryonic development. Estrone sulfate was the only detectable estrogen sulfate. At oviposition, levels of both estradiol and estrone sulfate varied seasonally with clutches from later in the nesting season having significantly higher concentrations of both steroids. Levels of estrone sulfate increased during development, demonstrating that the sulfonation of maternally derived steroids occurs in oviparous vertebrates as well as in placental vertebrates. We also found that exogenous estrone sulfate increases the production of female hatchlings, thereby demonstrating the ability of this metabolite to influence embryonic development. To examine the role of sulfonation in the metabolism of maternal progesterone and testosterone, we characterized the metabolic fate of both steroids by applying tritiated forms of each steroid at oviposition and characterizing metabolites after 20 days of incubation. Similar to what was demonstrated for estradiol, both progesterone and testosterone are converted to sulfonated metabolites during embryonic development. These data suggest that steroid sulfates, both those that are maternally derived and those resulting from the metabolism of maternal steroids, are a key component of the mechanism underlying steroid-mediated maternal effects.

A little winning streak: the reptilian-eye view of gastrulation in birds.

The primitive streak is where the mesoderm and definitive endoderm precursor cells ingress from the epiblast during gastrulation. It is often described as an embryological feature common to all amniotes. But such a feature has not been associated with gastrulation in any reptilian species. A parsimonious model would be that the primitive streak evolved independently in the avian and mammalian lineages. Looking beyond the primitive streak, can one find shared features of mesoderm and endoderm formation during amniote gastrulation? Here, we survey the literature on reptilian gastrulation and provide new data on Brachyury RNA and laminin protein expression in gastrula-stage turtle (Pelodiscus sinensis) embryos. We propose a model to reconcile the primitive streak-associated gastrulation in birds and the blastopore-associated gastrulation in extant reptiles.

Characterizing the metabolism and movement of yolk estradiol during embryonic development in the red-eared slider (Trachemys scripta).

Eggs of oviparous amniotes can contain substantial quantities of several steroids at the time of oviposition. These maternally derived steroids appear to affect the phenotype of developing offspring, but not all steroid sensitive traits are affected by maternal steroids, and little is known about how these effects may arise. In this study, we applied tritiated estradiol to the eggs of red-eared sliders (Trachemys scripta) at the time of oviposition and characterized the subsequent metabolism and movement throughout embryonic development. Results indicate that very early in development, estradiol is converted to a variety of water-soluble estrogen sulfates that reside in the yolk and extraembryonic fluids until late in development. Within the final stages of development, we observe a significant decline in the total amount of metabolites present in the yolk and extraembryonic fluids and a significant increase in the amount of metabolites present in the embryo. While estradiol metabolism occurs during the early stages of development, the later stages appear to be the most dynamic with regards to the movement of estradiol metabolites. Our findings have important implications for studies investigating the effect of maternally derived steroids on offspring development.

Changes in cortical interneuron migration contribute to the evolution of the neocortex.

The establishment of the mammalian neocortex is often explained phylogenetically by an evolutionary change in the pallial neuronal progenitors of excitatory projection neurons. It remains unclear, however, whether and how the evolutionary change in inhibitory interneurons, which originate outside the neocortex, has been involved in the establishment of the neocortex. In this study, we transplanted chicken, turtle, mouse, and marmoset medial ganglionic eminence (MGE) cells into the embryonic mouse MGE in utero and compared their migratory behaviors. We found that the MGE cells from all of the species were able to migrate through the mouse neocortical subventricular zone and that both the mouse and marmoset cells subsequently invaded the neocortical cortical plate (CP). However, regardless of their birthdates and interneuron subtypes, most of the chicken and turtle cells ignored the neocortical CP and passed beneath it, although they were able to invade the archicortex and paleocortex, suggesting that the proper responsiveness of MGE cells to guidance cues to enter the neocortical CP is unique to mammals. When chicken MGE cells were transplanted directly into the neocortical CP, they were able to survive and mature, suggesting that the neocortical CP itself is essentially permissive for postmigratory development of chicken MGE cells. These results suggest that an evolutionary change in the migratory ability of inhibitory interneurons, which originate outside the neocortex, was involved in the establishment of the neocortex by supplying inhibitory components to the network.

Expression of Dazl and Vasa in turtle embryos and ovaries: evidence for inductive specification of germ cells.

In bilaterian animals, germ cells are specified by the inductive/regulative mode or the predetermined (germ plasm) mode. Among tetrapods, mammals and urodeles use the inductive mode, whereas birds and anurans use the predetermined mode. From histological data it has been predicted that some reptiles including turtles use the inductive mode. Examining turtle oocytes, we find that Dazl RNA, Vasa RNA, and Vasa protein are not localized, suggesting that germ plasm is not present. In turtle embryos at somite stages, primordial germ cells (PGCs) expressing Dazl lie on a path from the lateral posterior extraembryonic endoderm through the gut to the gonad as previously described. In gastrulating embryos, cells expressing Dazl are found in the blastoporal plate and subsequently below the blastoporal plate, indicating that PGCs are generated at the equivalent of the early posterior primitive streak of mammals. Vasa RNA is expressed in somatic cells of gastrula to early somite stages, and Vasa RNA and protein are expressed in PGCs of later embryos. Taken together the evidence strongly suggests that turtles, and other reptiles (lacertoid lizards) with the same location of PGCs in embryos, use the inductive mode of germ cell specification. Phylogenetic analysis of the available evidence supports the following hypotheses: (1) the inductive mode is basal among reptiles, indicating that this mode was maintained as basal tetrapods evolved to amniotes, (2) the predetermined mode arose twice within reptiles, and (3) the induced mode may be used in several lepidosaurs whose PGCs are located in an unusual pattern distributed around the embryo.

Rapid decline in the concentrations of three yolk steroids during development: is it embryonic regulation?

Maternally derived yolk steroids have been found to elicit both short-term and long-term effects on offspring phenotype. Paradoxically, their effects can be strikingly specific given the often substantial concentrations present at oviposition, and they do not appear to uniformly affect all steroid-sensitive processes. To better understand the dynamics of yolk steroids across embryonic development, we quantified levels of progesterone, testosterone, and estradiol at 5-day intervals throughout development in eggs of the red-eared slider turtle (Trachemys scripta) incubated at both male- and female-producing temperatures. We also assessed the effect of season on yolk steroid levels. For all steroids assayed, the concentrations in yolk declined significantly by day 15 of embryonic development despite large differences in initial concentrations among steroids. We found that estradiol was the only steroid whose initial concentration varied significantly with season, while only the decline in testosterone was affected by incubation temperature. These findings illustrate the complex nature of yolk steroid dynamics and suggest that maternal steroids may be rapidly degraded or subject to embryonic processing, emphasizing the need for studies aimed at understanding the mechanisms through which yolk steroids may elicit their effects.

Estrogen inhibits caudal progression but stimulates proliferation of developing müllerian ducts in a turtle with temperature-dependent sex determination.

Müllerian duct development appears to be similar in many vertebrate groups, and previous studies have shown that this development is estrogen sensitive. For example, embryonic exposure to diethylstilbestrol (DES) in humans and mice, and estrogen exposure in chickens, can have multiple, usually adverse, effects on müllerian duct differentiation and growth. The current study investigates 17beta-estradiol's effects on müllerian duct development in a reptile, the turtle Trachemys scripta. In T. scripta, normal müllerian duct development proceeds cranially to caudally over developmental stages 15 to 21. To ascertain 17beta-estradiol's effect on this process, four groups of eggs were incubated at a female-producing temperature. Each group was treated with 50 mug of 17beta-estradiol or a vehicle control at one of four stages (15, 17, 19, 21). The degree of müllerian duct development was assessed by examining gross morphology and histology. Results showed that estradiol-17beta blocked development of the müllerian duct if applied before differentiation began. If applied afterwards, 17beta-estradiol caused hypertrophy in the differentiated portion, but prevented further differentiation of the müllerian duct in more caudal regions. Therefore, reptilian müllerian ducts in T. scripta are estrogen sensitive and estrogen's effects may be similar to those reported for birds.

Effect of cadmium on gonadal development in freshwater turtle (Trachemys scripta, Chrysemys picta) embryos.

Prior studies on painted turtle (Chrysemys picta) sub-populations near the Massachusetts Military Reservation (MMR), a Superfund site on Cape Cod, Massachusetts, USA, suggest several reproductive deficits which may be related to xenobiotics. Several heavy metals, including cadmium, have been detected in Cape Cod surface water and sediments. The present study was carried out to investigate the effect of an environmentally relevant dose of cadmium on gonadal development during the end of the germ cell migration phase and post-natal gonadal maturation in freshwater turtles. Comparison of cadmium concentration in eggs of C. picta from Cape Cod showed that eggs from the impacted site animals had significantly higher cadmium in yolk than eggs from the reference site animals (7.23 +/- 1.95 ng/g vs. 1.31 +/- 0.50 ng/g). Gonadal structure and the number of proliferating germ cells of neonates derived from eggs of adult females from these sites showed no marked difference between sites. However, apoptosis of oocytes was significantly increased in neonate C. picta from the impacted pond compared to the reference pond. The effect of an administered environmentally relevant dose of cadmium on germ cell number and oocyte apoptosis was subsequently assessed in lab-reared Trachemys scripta, a closely related freshwater turtle species. Assessment of isotopic cadmium transmission showed that 6.29% of cadmium applied to the eggshell was transmitted through the eggshell to the yolk. The results showed that the total number of germ cells in cadmium-treated (1 microg/g) embryos was less than half that found in control embryos. The reduced germ cell number in Cd-treated embryos suggests that cadmium may reduce proliferation and/or delay migration of germ cells to the genital ridge. The effects of cadmium on turtle gonadal development were found to extend into 3 months post hatch. Proliferation of oocytes was not influenced by exposure to cadmium in ovo. In contrast, apoptosis of oocytes was significantly increased in cadmium exposed T. scripta. Since a lesion at the gonial stages will lead eventually to the depletion of more mature germ cells, the results for neonate turtles suggest that an environmental impact due to a xenobiotic mixture may enhance the rate of apoptosis, thus resulting in the reduction in follicle number seen in adult turtles from the impacted site. Overall, the data provide evidence that environmentally relevant doses of cadmium may affect gonadal developmental processes of freshwater turtles during embryonic and post-natal stages that may result in disruption of reproductive processes later in life.

Gonadal expression of Sf1 and aromatase during sex determination in the red-eared slider turtle (Trachemys scripta), a reptile with temperature-dependent sex determination.

Many egg-laying reptiles have temperature-dependent sex determination (TSD), where the offspring sex is determined by incubation temperature during a temperature-sensitive period (TSP) in the middle third of development. The underlying mechanism transducing a temperature cue into an ovary or testis is unknown, but it is known that steroid hormones play an important role. During the TSP, exogenous application of estrogen can override a temperature cue and produce females, while blocking the activity of aromatase (Cyp19a1), the enzyme that converts testosterone to estradiol, produces males from a female-biased temperature. The production of estrogen is a key step in ovarian differentiation for many vertebrates, including TSD reptiles, and temperature-based differences in aromatase expression during the TSP may be a critical step in ovarian determination. Steroidogenic factor-1 (Sf1) is a key gene in vertebrate sex determination and regulates many steroidogenic enzymes, including aromatase. We find that Sf1 and aromatase are differentially expressed during sex determination in the red-eared slider turtle, Trachemys scripta elegans. Sf1 is expressed at higher levels during testis development while aromatase expression increases during ovary determination. We also assayed Sf1 and aromatase response to sex-reversing treatments via temperature or the modulation of estrogen availability. Sf1 expression was redirected to low-level female-specific patterns with feminizing temperature shift or exogenous estradiol application and redirected to more intense male-specific patterns with male-producing temperature shift or inhibition of aromatase activity. Conversely, aromatase expression was redirected to more intense female-specific patterns with female-producing treatment and redirected toward diffuse low-level male-specific patterns with masculinizing sex reversal. Our data do not lend support to a role for Sf1 in the regulation of aromatase expression during slider turtle sex determination, but do support a critical role for estrogen in ovarian development.

Steroid signaling system responds differently to temperature and hormone manipulation in the red-eared slider turtle (Trachemys scripta elegans), a reptile with temperature-dependent sex determination.

Many reptiles, including the red-eared slider turtle (Trachemys scripta elegans), exhibit temperature-dependent sex determination (TSD). Temperature determines gonadal sex during the middle of embryogenesis, or the temperature-sensitive period (TSP), when gonadal sex is labile to both temperature and hormones--particularly estrogen. The biological actions of steroid hormones are mediated by their receptors as defined here as the classic transcriptional regulation of target genes. To elucidate estrogen action during sex determination, we examined estrogen receptor alpha (Esr1, hereafter referred to as ERalpha), estrogen receptor beta (Esr2, hereafter referred to as ERbeta), and androgen receptor (Ar, hereafter referred to as AR) expression in slider turtle gonads before, during and after the TSP, as well as following sex reversal via temperature or steroid hormone manipulation. ERalpha and AR levels spike at the female-producing temperature while ovarian sex is determined, but none of the receptors exhibited sexually dimorphic localization within the gonad prior to morphological differentiation. All three receptors respond differentially to sex-reversing treatments. When shifted to female-producing temperatures, embryos maintain ERalpha and AR expression while ERbeta is reduced. When shifted to male-producing temperatures, medullary expression of all three receptors is reduced. Feminization via estradiol (E(2)) treatment at a male-producing temperature profoundly changed the expression patterns for all three receptors. ERalpha and ERbeta redirected to the cortex in E(2)-created ovaries, while AR medullary expression was transiently reduced. Although warmer incubation temperature and estrogen result in the same endpoint (ovarian development), our results indicate different steroid signaling patterns between temperature- and estrogen-induced feminization.