Phenotypic Variation Through Ontogeny: Thyroid Axis Disruption During Larval Development in the Frog Pleurodema borellii

Studies of the effects of thyroid hormones on larval development in the frog Xenopus spp. have provided baseline information to identify developmental constraints and elucidate genetic and hormonal mechanisms driving development, growth, and life history transitions. However, this knowledge requires data based on other anurans to complete a comprehensive approach to the understanding of larval developmental diversity and phenotypic variation through ontogeny. Mesocosm experiments provide realistic data about environmental conditions and timing; this information is useful to describe anuran larval development and/or analyze endocrine disruption. In this study, mesocosm experiments of the larval development of the frog Pleurodema borellii were conducted to explore the consequences of thyroid axis disruption; the sensitivity of tadpoles to the methimazole (2.66 mg/l) and thyroxine (T4) (1.66 μg/l) was compared. These concentrations were selected based on previous studies in Pleurodema borellii. We test the effects of methimazole and thyroxine on development in early exposure (from beginning of larval development) and late exposure, 18 days after hatching, with doses administered every 48 h. Tadpoles were evaluated 31 days after hatching. Methimazole caused moderate hypertrophy of the thyroid gland, alteration in the growth rates, differentiation without inhibition of development, and an increase of developmental variability. Thyroxine produced slight atrophy of the thyroid gland, accelerated growth rates and differentiation, and minor developmental variability. In tadpoles at stages previous to metamorphose, skull development (differentiation of olfactory capsules, appearance of dermal bones, and cartilage remodeling) seemed to be unaltered by the disruptors. Moreover, similar abnormal morphogenesis converged in specimens under methimazole and thyroxine exposures. Abnormalities occurred in pelvic and pectoral girdles, and vent tube, and could have been originated at the time of differentiation of musculoskeletal tissues of girdles. Our results indicate that premetamorphic stages (Gosner Stages 25–35) are sensitive to minimal thyroid axis disruption, which produces changes in developmental rates; these stages would also be critical for appendicular musculoskeletal morphogenesis to achieve the optimal condition to start metamorphosis.

Comparative Milestones in Rodent and Human Postnatal Central Nervous System Development

Within the substantially different time scales characterizing human and rodent brain development, key developmental processes are remarkably preserved. Shared processes include neurogenesis, myelination, synaptogenesis, and neuronal and synaptic pruning. In general, altricial rodents experience greater central nervous system (CNS) immaturity at birth and accelerated postnatal development compared to humans, in which protracted development of certain processes such as neocortical myelination and synaptic maturation extend into adulthood. Within this generalization, differences in developmental rates of various structures must be understood to accurately model human neurodevelopmental toxicity in rodents. Examples include greater postnatal neurogenesis in rodents, particularly within the dentate gyrus of rats, ongoing generation of neurons in the rodent olfactory bulb, differing time lines of neurotransmitter maturation, and differing time lines of cerebellar development. Comparisons are made to the precocial guinea pig and the long-lived naked mole rat, which, like primates, experiences more advanced CNS development at birth, with more protracted postnatal development. Methods to study various developmental processes are summarized using examples of comparative postnatal injury in humans and rodents.

Modulation of lipid metabolism through multiple pathways during oocyte maturation and embryo culture in bovine

Lipid accumulation occurs in cultured embryos and is associated with reduced cryotolerance. Here we report the use of a multiple pathway lipid modulator cocktail (l-carnitine, linoleic acid and forskolin) to improve cryosurvival. First, we stained oocytes and embryos with Oil Red to examine the time course of lipid accumulation during in vitro fertilization (IVF) and embryo culture. Then we evaluated the effects of the lipid modulators cocktail on lipid content, developmental rates and survival after vitrification. In our conditions, lipid accumulation was detected (P < 0.05) at the end of in vitro maturation (IVM) and after 4 days of embryo culture (D4-D5). In experiment 1, we used lipid modulator cocktail during IVM. Reduced (P < 0.05) lipid accumulation was detected in oocytes (Control: 49.9 ± 1.6, Lip. Mod. IVM: 45.0 ± 1.8) but no changes were present at blastocyst stage (Control: 62.4 ± 2.6, Lip. Mod. IVM: 66.8 ± 2.7). Treated oocytes presented decreased (P < 0.05) blastocyst rates and lower (P < 0.05) re-expansion after vitrification. In experiment 2, lipid modulators cocktail was used during embryo culture (from D4–D7 or D6–D7). Treatment had an effect on lipid metabolism, as lipid content was increased (P < 0.05) in D7 blastocysts in treated groups (Control: 52.7 ± 3.1a, D4: 65.9 ± 2.6b, D6: 78.1 ± 2.7b). However, no effect was present for cleavage, blastocyst and cryosurvival rates. No difference was detected in mean cell number comparing the three groups (Control: 78.9 ± 9.6, D4: 82.6 ± 16.5, D6: 68.3 ± 7.8), but apoptosis rate was increased (P < 0.05) in vitrified-warmed blastocysts from treated groups (Control: 14.77*, D4: 22.28, D6: 22.22). We concluded that the combined use of lipid modulators was efficient to promote changes in lipid content of oocytes and embryos in bovine, but those changes did not reflect positively on embryo development or cryosurvival.

A Phenological Model for Olive (Olea europaea L. var europaea) Growing in Italy

The calibration of a reliable phenological model for olive grown in areas characterized by great environmental heterogeneity, like Italy, where many varieties exist, is challenging and often suffers from a lack of observations, especially on budbreak. In this study, we used a database encompassing many phenological events from different olive varieties, years, and sites scattered all over Italy to identify the phases in which site-enlarged developmental rates can be well regressed against air temperature (Developmental Rate function, DR) by testing both linear and nonlinear functions. A K-fold cross-validation (KfCV) was carried out to evaluate the ability of DR functions to predict phenological development. The cross-validation showed that the phases ranging from budbreak (BBCH 01 and 07) to flowering (BBCH 61 and 65) and from the beginning of flowering (BBCH 51) to flowering can be simulated with high accuracy (r2 = 0.93-0.96; RMSE = 3.9–6.6 days) with no appreciable difference among linear and nonlinear functions. Thus, the resulting DRs represent a simple yet reliable tool for regional phenological simulations for these phases in Italy, paving the way for a reverse modeling approach aimed at reconstructing the budbreak dates. By contrast, and despite a large number of phases explored, no appreciable results were obtained on other phases, suggesting possible interplays of different drivers that need to be further investigated.

N-acetyl-L-cysteine Improves the Developmental Competence of Bovine Oocytes and Embryos Cultured In Vitro by Attenuating Oxidative Damage and Apoptosis

Oxidative stress has been suggested to negatively affect oocyte and embryo quality and developmental competence, resulting in failure to reach full term. In this study, we investigated the effect of N-acetyl-L-cysteine (NAC), a cell-permeating antioxidant, on developmental competence and the quality of oocytes and embryos upon supplementation (0.1–10 mM) in maturation and culture medium in vitro using slaughterhouse-derived oocytes and embryos. The results show that treating oocytes with 1.0 mM NAC for 8 h during in vitro maturation attenuated the intracellular reactive oxygen species (ROS) (p < 0.05) and upregulated intracellular glutathione levels (p < 0.01) in oocytes. Interestingly, we found that NAC affects early embryonic development, not only in a dose-dependent, but also in a stage-specific, manner. Significantly (p < 0.05) decreased cleavage rates (90.25% vs. 81.46%) were observed during the early stage (days 0–2), while significantly (p < 0.05) increased developmental rates (38.20% vs. 44.46%) were observed during the later stage (from day 3) of embryonic development. In particular, NAC supplementation decreased the proportion of apoptotic blastomeres significantly (p < 0.05), resulting in enhanced hatching capability and developmental rates during the in vitro culture of embryos. Taken together, our results suggest that NAC supplementation has beneficial effects on bovine oocytes and embryos through the prevention of apoptosis and the elimination of oxygen free radicals during maturation and culture in vitro.

Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres

Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the species to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. For this purpose, we compared in vitro development of synchronic (Sync) or asynchronic (Async) and asynchronic with a tetraploid (Async4n) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA fragmentation. Additionally, the developmental rates of Async4n aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. Async aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by Async4n aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by Async4n aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of two-cell-fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos.

Dietary-based developmental plasticity affects juvenile survival in an aquatic detritivore

Developmental plasticity is ubiquitous in natural populations, but the underlying causes and fitness consequences are poorly understood. For consumers, nutritional variation of juvenile diets is probably associated with plasticity in developmental rates, but little is known about how diet quality can affect phenotypic trajectories in ways that might influence survival to maturity and lifetime reproductive output. Here, we tested how the diet quality of a freshwater detritivorous isopod ( Asellus aquaticus ), in terms of elemental ratios of diet (i.e. carbon : nitrogen : phosphorus; C : N : P), can affect (i) developmental rates of body size and pigmentation and (ii) variation in juvenile survival. We reared 1047 individuals, in a full-sib split-family design (29 families), on either a high- (low C : P, C : N) or low-quality (high C : P, C : N) diet, and quantified developmental trajectories of body size and pigmentation for every individual over 12 weeks. Our diet contrast caused strong divergence in the developmental rates of pigmentation but not growth, culminating in a distribution of adult pigmentation spanning the broad range of phenotypes observed both within and among natural populations. Under low-quality diet, we found highest survival at intermediate growth and pigmentation rates. By contrast, survival under high-quality diet survival increased continuously with pigmentation rate, with longest lifespans at intermediate growth rates and high pigmentation rates. Building on previous work which suggests that visual predation mediates the evolution of cryptic pigmentation in A. aquaticus , our study shows how diet quality and composition can generate substantial phenotypic variation by affecting rates of growth and pigmentation during development in the absence of predation.