Steroid metabolism in gonads of turtle embryos as a function of the incubation temperature of eggs

In embryos of many reptiles, the sexual differentiation of gonads is temperature-dependent. In the turtle Emys orbicularis, all individuals become phenotypic males at 25 degrees C, whereas 100% phenotypic females are obtained at 30 degrees C. Steroid metabolism in embryonic gonads was studied at both temperatures, during and after the thermosensitive period for sexual differentiation. Pools of gonads were incubated for various times, with 3 beta-hydroxy-5-pregnen-20-one (pregnenolone), progesterone, dehydroepiandrosterone or 4-androstene-3,17- dione as substrates. The analysis of metabolites combined two successive chromatographies (HPLC and TLC) and autoradiography. Conversion of pregnenolone to progesterone and of dehydroepiandrosterone to 4-androstene-3,17-dione was more important in testes at 25 degrees C than in ovaries at 30 degrees C. In ovaries, a large amount of 5-pregnene- 3 beta,20 beta-diol was formed from pregnenolone, and 5-androstene-3 beta,17 beta-diol was produced from dehydroepiandrosterone. In both testes and ovaries, 5 alpha-pregnane and 5 alpha-androstane derivatives were the main metabolites obtained from progesterone and 4-androstene-3,17-dione, respectively. Progesterone was also converted to 20 beta-hydroxy-4-pregnen-3-one. Dehydroepiandrosterone and 4-androstene-3,17-dione were also metabolized into 11 beta-hydroxy-4-androstene-3,17-dione (only in testes), testosterone, 11 beta,17 beta-dihydroxy-4-androstene-3-one, 17 beta-hydroxy-4-androstene-3,11-dione (low amounts in testes, traces in ovaries), 17 alpha-hydroxy-4-androstene-3-one, estrone and estradiol-17 beta (traces).     

Aromatase activity in fetal gonads of mammals

This is a review of work demonstrating aromatase activity in both ovaries and testes of mammalian fetuses. Studies on the regulation of this enzyme activity by gonadotrophins and other factors have also been reviewed. Finally, the significance of aromatase activity in conjunction with the role of estrogen in ovarian and testicular development has been considered.     

Determination of temperature sensitive stages for sexual differentiation of the gonads in embryos of the turtle,Emys orbicularis

In order to determine the temperature sensitive stages for sexual differentiation of the gonads in Emys orbicularis, eggs of this turtle were shifted at different stages of embryonic development from the male-producing temperature of 25°C to the female-producing temperature of 30°C and reciprocally. Based on the series of developmental stages described by Yntema (′68) for Chelydra serpentina, temperature begins to influence sexual differentiation of Emys orbicularis at stage 16, a stage in which the gonads are still histologically undifferentiated. Its action lasts over the first steps of histological differentiation of the gonads. The minimal exposure at 25°C required for male differentiation of all individuals extends from stage 16 to somewhat before stage 21. For 100% female differentiation, incubation at 30°C must be longer, from stage 16 to somewhat before stage 22. Shorter exposures at 25°C or 30°C during these periods result in different percentages of males, females, and intersexes. Our results show that there is a critical stage (stage 16) which is the same for both male and female differentiation of the gonads. The thermosensensitive periods are rather long, corresponding to 11–12 days at 25°C and 30°C.     

Optimal sex ratio as a function of EGG incubation temperature in the crocodilians

Recently, there has been a growing consensus as to the adaptive significance of temperature-dependent sex determination in the Crocodilia. The observationally and experimentally motivated hypotheses are that male fitness depends more strongly on quality of incubation environment than female fitness, and that there is a strong correlation between a female's egg incubation temperature choice and her own egg incubation temperature. A population genetics model based on these hypotheses is derived. A method for finding the optimal sex ratio as a function of temperature, which is an evolutionary stable strategy (ESS), is stated and applied under various assumptions. This extends ESS theory to thefunctional case. Cases where there is no ESS and the population sex ratio oscillates in evolutionary time are discovered. Numerical computation is needed to solve the full problem and the resulting optimal sex ratio is compared to laboratory sex ratio data. The general pattern of TSD in crocodilians (female-male-female with female biased overall sex ratio) agrees well with the theory, but details of the pattern are problematic.     

Involvement of oestrogens in sexual differentiation of gonads as a function of temperature in turtles

Abstract. To investigate the possible involvement of oestrogens in the phenomenon of temperature sex-reversal in the turtle Emys orbicularis , the effects of oestrone, oestradiol and an antioestrogen. tamoxifen, on sexual differentiation of gonads were examined at a male-producing temperature of 25° C and at a female-producing temperature of 30° C. When oestrone or oestradiol were injected into eggs incubated at 25° C just before or at the beginning of the thermosensitive period, the gonads differentiated into ovaries instead of testes. Conversely, when tamoxifen was injected, at the same stages, into eggs incubated at 30° C, epithelial cords or tubes, similar to potential seminiferous cords, differentiated in the interior part of the gonads. However, an ovarian-like cortex persisted at their surface. At 25° C, treatment with tamoxifen or with both tamoxifen and oestradiol also resulted in differentiation of ovotestes. These experiments show that tamoxifen binding to oestrogen receptors prevented the inhibitory action of oestrogens on testicular cord development. Maintenance (at 30° C) or development (at 25° C) of ovarian cortex in the presence of tamoxifen can be expected from an agonistic action of this drug, as already described. Preliminary data on steroid content in the gonads indicate that, during the early stages of the thermosensitive period, the level of estrogens is higher at 30° C than at 25° C. It is proposed that in species displaying temperature sensitivity for the sexual differentiation of gonads, temperature acts on the processes regulating the synthesis or the activity of cyto-chrome-P450 aromatase.     

Molecular sensors for temperature detection during behavioral thermoregulation in turtle embryos

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Prolonged incubation in seawater induces a DNA-dependent protein phosphorylation activity in Arbacia punctulata eggs

Various protein kinases are activated in eggs in response to fertilization. We have previously shown that the induction of DNA-dependent protein phosphorylation activity in the sea urchin eggs is triggered by fertilization. The present study demonstrates that the activation of a DNA-dependent serine/threonine kinase in unfertilized eggs of Arbacia punctulata can be achieved without fertilization. Prolonged incubation in seawater resulted in the activation of the eggs with concomitant induction of DNA-dependent protein phosphorylation activity. The activated eggs when fertilized show a slight increase in the phosphorylation activity 10-min post-insemination. The activity gradually declines as the first and second cleavages proceed. The cytoplasmic extracts of the blastulae, gastrulae, and plutei lack the enzyme activity. These findings reveal that not only fertilization but also egg activation serves as a signal for the induction of a DNA-dependent protein phosphorylation activity in sea urchin eggs suggesting that sperm-entry is not required for the induction of the enzyme activity.     

Oxidative activity of poikilotherm mitochondria as a function of temperature

In Newell & Northcroft (1967) evidence was presented which showed that the metabolism of certain intertidal invertebrates at rest does not vary greatly with temperature change. The following work was undertaken to determine whether temperature-independent metabolism could be demonstrated in suspensions of mitochondria extracted from poikilotherm tissue and whether it could be inferred that there was any difference in the ability of poikilotherms from different habitats to adjust toshort-term temperature change.
It was found that the rate of reaction/temperature curve of mitochondria isolated from the skate Raia had a flattened region below 10°C but above this the rate of oxidation of succinate and pyruvate increased regularly with temperature. The curve for intertidal forms had a gradual slope extending as high as 20°C in the mussel Mytilus edulis while in the terrestrial snail Helix aspersa the gradual slope reached 27.5°C after which the rate of oxidation of substrate increased sharply before declining towards higher temperatures. Finally, the curve for mitochondria isolated from the desert locust Schistocerca gregaria showed a gradual slope up to 35°C after which a sharp increase in the rate of oxidation of substrate occurred. Thus the extent of the gradual slope corresponds with the normal environmental temperature range to which the tissues of the poikilotherms are subjected and the general form of the R-T curve is such that rapid fluctuations within the normal environmental temperature range have relatively little effect on the rate of mitochondrial activity.     

Oxidative activity of poikilotherm mitochondria as a function of temperature

In Newell & Northcroft (1967) evidence was presented which showed that the metabolism of certain intertidal invertebrates at rest does not vary greatly with temperature change. The following work was undertaken to determine whether temperature-independent metabolism could be demonstrated in suspensions of mitochondria extracted from poikilotherm tissue and whether it could be inferred that there was any difference in the ability of poikilotherms from different habitats to adjust to short-term temperature change.
It was found that the rate of reaction/temperature curve of mitochondria isolated from the skate Raia had a flattened region below 10C but above this the rate of oxidation of succinate and pyruvate increased regularly with temperature. The curve for intertidal forms had a gradual slope extending as high as 20C in the mussel Mytilus edulis while in the terrestrial snail Helix aspersa the gradual slope reached 27.5C after which the rate of oxidation of substrate increased sharply before declining towards higher temperatures. Finally, the curve for mitochondria isolated from the desert locust Schistocerca gregaria showed a gradual slope up to 35C after which a sharp increase in the rate of oxidation of substrate occurred. Thus the extent of the gradual slope corresponds with the normal environmental temperature range to which the tissues of the poikilotherms are subjected and the general form of the R-T curve is such that rapid fluctuations within the normal environmental temperature range have relatively little effect on the rate of mitochondrial activity.     

Incubation temperature and energy expenditure during development in loggerhead sea turtle embryos

The choice of a suitable nest habitat by oviparous reptiles that deposit eggs into a nest and provide no subsequent parental care is likely to play a major role in the survival of the offspring. In particular variations in nest temperature may influence the rate at which embryos utilise their yolk energy.The effects of nest temperature on total energy use are however complex. High temperatures may advance development and shorten the time to hatching, thereby reducing energy use, but they also stimulate metabolic rate increasing energy use. The net effect of temperature on total energy demands is therefore uncertain.Oxygen consumption (VO₂) and carbon dioxide production (VCO₂) were measured by open-flow respirometry during the incubation of loggerhead sea turtle eggs at three temperatures (27.6, 30.0 and 31.8 °C).At each temperature, VO₂ and VCO₂ showed a peak followed by a decline to hatching. Incubation temperature was negatively related to incubation duration and positively related to the maximum metabolic rate of the embryos. Peak VO₂ was 74.8 ml/egg/day at 27.6 °C, 91.9 ml/egg/day at 30.0 °C, and 97.9 ml/egg/day at 31.8 °C. Peak VO₂ occurred closer to hatching in eggs incubated at higher temperatures.Total energy expenditure was greatest at the lowest incubation temperature and lowest at the highest temperature. Total VO₂ and VCO₂ were 1777 ml/egg and 1226 ml/egg, respectively, at 27.6 °C, 1680 ml/egg and 1235 ml/egg at 30.0 °C, and 1613 ml/egg and 1191 ml/egg at 31.8 °C. Using the actual RQ values, this corresponds to a cost of development of 34,963 J/egg at 27.6 °C, 33,403 J/egg at 30.0 °C, and 32,107 J/egg at 31.8 °C.At all temperatures, the calculated respiratory quotient values did not suggest that yolk substrates were oxidised proportionately, but more likely indicated their sequential use.Nest temperatures may play a key role in energy use, with cooler temperature nests increasing the overall energy demands placed on developing embryos.     

Forecasting the viability of sea turtle eggs in a warming world

Animals living in tropical regions may be at increased risk from climate change because current temperatures at these locations already approach critical physiological thresholds. Relatively small temperature increases could cause animals to exceed these thresholds more often, resulting in substantial fitness costs or even death. Oviparous species could be especially vulnerable because the maximum thermal tolerances of incubating embryos is often lower than adult counterparts, and in many species mothers abandon the eggs after oviposition, rendering them immobile and thus unable to avoid extreme temperatures. As a consequence, the effects of climate change might become evident earlier and be more devastating for hatchling production in the tropics. Loggerhead sea turtles (Caretta caretta) have the widest nesting range of any living reptile, spanning temperate to tropical latitudes in both hemispheres. Currently, loggerhead sea turtle populations in the tropics produce nearly 30% fewer hatchlings per nest than temperate populations. Strong correlations between empirical hatching success and habitat quality allowed global predictions of the spatiotemporal impacts of climate change on this fitness trait. Under climate change, many sea turtle populations nesting in tropical environments are predicted to experience severe reductions in hatchling production, whereas hatching success in many temperate populations could remain unchanged or even increase with rising temperatures. Some populations could show very complex responses to climate change, with higher relative hatchling production as temperatures begin to increase, followed by declines as critical physiological thresholds are exceeded more frequently. Predicting when, where, and how climate change could impact the reproductive output of local populations is crucial for anticipating how a warming world will influence population size, growth, and stability.     

Sex-determining potencies vary among female incubation temperatures in a turtle

Reptiles whose sex is determined by incubation temperature typically exhibit all-male or all-female sex ratios over a wide range of incubation temperatures. The question arises as to whether the various all-female temperatures (or the various all-male temperatures) are equivalent in their "potency", or capacity to effect female determination. In map turtles, warm incubation temperatures produce all females and cool ones produce all males. We compared sex determining potencies of two all-female temperatures, 31 degrees C and 32.5 degrees C, by incubating eggs first at a male-producing temperature (26 degrees C) and then shifting them to the warm temperatures. The resulting sex ratio was significantly more male biased in the 26 degrees C----31 degrees C shift than in the 26 degrees C----32.5 degrees C shift, indicating that 32.5 degrees C has the greater female potency. These results point to the possibility that sex determination depends on a quantitative rather than qualitative level of gene expression.     

The influence of temperature on the water exchange in amphibian eggs and embryos

The water exchange has been measured in oocytes of siredon mexicanum and rana temporaria, and in unfertilized eggs and early gastrulae of the former species, by recording the D₂O? H₂O exchange with the cartesian diver balance. In oocytes, where no diffusion barrier to water is demonstrable, the temperature coefficient Q₁₀ for the exchange of water is about 1.3–1.4, corresponding to that of free diffusion. in unfertilized eggs, and in early gastrulae the exchange is considerably slowed down, indicating that a surface membrane to some extent limits the rate of exchange. at the same time the Q₁₀ value is increased, lying in the range 2.3–3.8. since it is most likely that the exchange even in this case occurs by diffusion, but through membrane pores, it is concluded that the area available for diffusion (pore size or number, or both) increases with temperature.     

Effect of incubation temperature on the hatching of rotifer resting eggs collected from sediments

Sediment samples from Loch Leven, Scotland, were incubated at 5°C, 10°C and 15°C to induce hatching of rotifer resting eggs. The emergent animals were identified and counted. The temperature which induced hatching varied among the nine species studied. These results are discussed in relation to seasonality and temperature preferences previously recorded for the most abundant species. Resting egg densities of 2.2–13.9 eggs cm^–3 were recorded in the upper 5 cm of sediment.     

The effect of incubation media on the water exchange of snapping turtle (Chelydra serpentina) eggs and hatchlings

Summary The effect of two different incubation media, sand and vermiculite, on the water exchange of eggs and the mass of hatchlings of snapping turtles (Chelydra serpentina) was assessed. The eggs were incubated fully buried in either sand or vermiculite at 30 °C and egg mass was measured periodically throughout incubation. The wet and dry masses of each hatchling and its residual yolk were measured at the end of incubation. The media had similar water potentials () but their thermal conductivities differed 2.8-fold. The eggs experienced a net water gain during incubation. The rates of water uptake between treatments were not statistically different throught the first 36 days of incubation but were statistically different thereafter, with eggs incubating in sand taking up water at about twice the rate of eggs incubating in vermiculite. Hatchling masses were similar to both media but hatchling water contents were significantly different. Hatchlings incubated in sand had lower water contents than hatchlings incubated in vermiculite even though the eggs in sand took up more water. Hatchling mass was correlated with egg water exchange for eggs incubated in vermiculite but not for eggs incubated in sand. The difference in egg water exchange in the two media appears to be attributable to differences in the thermal conductivity of the media. The presence of such a thermal effect supports the hypothesis that the eggs exchanged water with the media as water vapor. Egg water exchange was limited by the shell and shell membranes and not by the media. The shell and shell membranes appear to present an effective barrier to water uptake.Abbreviations M _{H 2 O} water flux (cm³·day^-1) - L _p hydraulic conductivity (cm·day^-1·kPa^-1) - A shell area (cm²) - A _p pore area (cm²) - l shell thickness (cm) - r pore radius (cm) - viscosity (kPa·day) - P _{EH 2 O} egg water potential (kPa) - P _{AH 2 O} medium water potential (kPa) - G _{H 2 O} water vapor conductance (cm³·day^-1·kPa^-1) - D _{H 2 O} diffusion coefficient (cm²·day^-1) - R gas constant (cm³·kPa·K^-1·cm^-3) - T temperature (K) - P _{EH 2 O} egg water vapor pressure (kPa) - P _{AH 2 O} medium water vapor pressure (kPa) - d egg diameter - K soil hydraulic conductivity (cm²·day^-1·kPa^-1) - DHM hatchling dry mass - WHM hatchiling wet mass - WU water uptake - IM initial egg mass