France

First identified as sexual hormones, estrogens were shown to have a key role in the development of gonads but also to be involved in the skeleton development and homeostasis. In mammals, cytosolic estrogen receptors (ERa and ERb) are expressed in osteoblasts, osteoclasts, osteocytes as well as chondrocytes and chondroblasts, regulating their activity to build up and maintain cartilage and bone. 
To expand our knowledge of estrogen signaling in skeletal development outside of mammals, we used the European sea bass Dicentrarchus labrax, to test the impact of two concentrations of the natural estrogen 17b estradiol (E2, 0.4 and 40 ng/L) on 6-to-23-day old post-hatching larvae. 
We evaluated the effects of E2 on the morphological characteristics of the larvae as well as on the mineralization of the cranium, vertebrae and fins using alizarin red staining. In addition, qPCR measurements were performed to analyze the expression of various genes known to be involved in the formation of cartilage and bone in vertebrates. We showed that the effect of estrogens varied depending on the developmental stage of the larvae and the state of mineralization of their skeleton at the time of treatment, and that these effects were dose-dependent.











SEB Annual Conference 2022

Estradiol effects on early skeletogenesis in the European sea bass Dicentrarchus labrax

Discover the art of creating extraordinary science at SEB’s Annual Conference at Le Corum Conference Center     in Montpellier, France from 5 to  8 July 2022.

SEB’s Annual Conference is celebrated for creating the perfect composition of research, new discoveries and collaborative connections. Be creative and innovative by exchanging ideas and knowledge with masters of biology from all over the world.

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2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years - Montpellier, France

technical paper

Anthropogenic changes in the environment are ubiquitos and affect organisms in many ways. Flexibility of the endocrine system may help organisms to response to those changes. However, disruption in the female endocrine function may lead to altered transfer of hormones in eggs or embryos, ie. transgenerational endocrine disruption, which can have long-lasting effects on offspring. Maternally derived thyroid hormones (THs) are essential for development, but poorly studied, especially in wild birds. Here, we provide three case examples of studies on transgenerational TH disruption. First, we studied how parental exposure to metal pollution, using repeated sampling of polluted and control areas across Europe, influenced egg TH levels in a passerine bird, the great tit. Second, we studied how long-term experimental parental herbicide exposure influenced egg THs and offspring traits in Japanese quails. Third, we studied experimetally how limitation of iodine, a key element of the TH molecule, influenced parental and egg THs in rock pigeons. We found no overall effect of parental metal exposure on egg THs, but the effect was dependent on calcium availability. Parental herbicide exposure did not influence egg THs, but affected hatchability and embryo traits. Parental iodine availability did not influence parental or egg TH levels, but decreased egg laying rate. Results of these case examples suggest that transgenerational endocrine disruption is a complex phenomena and should be further studied, as such legacy effects can delay adapting to environmental changes.

Transgenerational endocrine disruption - a potential mechanism for legacy effects of anthropogenic disturbance

Nocturnin (NOC) is a phosphatase involved in the transformation of NADP(H) to NAD(H), performing a key role in the regulation of metabolism. In mammals, it has been purposed that nocturnin expression is induced by E-box element activation. However, nothing is known about the possible elements that modulate its expression in fish. Our aim was to undertake the study of the possible endocrine regulation and the intracellular pathways involved in the gene expression of three noc paralogs (noc-a1, noc-a2 and noc-b1) expressed in goldfish (Carassius auratus). For this purpose, we studied the effect of glucagon, adrenaline and different activators and inhibitors of protein kinase A (PKA) and protein kinase C (PKC) pathways on noc paralogs expression in hepatopancreas organotypic cultures. We found a concentration-dependent induction of noc-a1 expression by both glucagon and adrenaline at 2 and 6-h incubation time. Forskolin, an adenylyl cyclase activator, induced a 5-fold increase of the relative abundance of this paralog, meanwhile a 2-fold increase was obtained in the presence of phorbol 12-myristate 13-acetate, a PKC activator. The effects of glucagon and forskolin were fully blocked by a PKA inhibitor (H89), and partially by a PKC inhibitor (chelerythrine) and by a PLC inhibitor (U73122), suggesting a possible interaction between PKA and PKC pathways in the intracellular signaling of glucagon on noc-a1 expression. Regarding noc-a2 mRNA expression, our results show an induction by adrenaline at 2 and 6-h incubation time but not by glucagon, forskolin or phorbol 12-myristate 13-acetate. Then, noc-b1 seems to be insensitive to both hormones, and to PKA and PKC activators. Altogether, these results suggest a possible functional specialization of noc paralogs in hepatopancreas and aim to investigate the possible role of other metabolic regulators in the control of noc expression in goldfish.
Supported by the Spanish MICINN (PID2019-103969RB-C32). D.M and A.A-G are predoctoral fellows, UCM (CT42/18-CT43/18) and FPI (BES-2017-081398), respectively.

Glucagon and adrenaline: two modulators of nocturnin expression in goldfish?

How limited resources are physiologically directed towards somatic growth is a simple question, but poorly understood. The hormone - insulin-like growth factor 1 (IGF-1) - is an evolutionarily conserved hepatic peptide that is one of the more important hormones of growth regulation. For example, administration of IGF-1 to the nestlings of the pied flycatcher (Ficedula hypoleuca) induced growth to bigger pre-fledging body size. Moreover, administrating an IGF-1 receptor inhibitor (OSI-906) resulted in reduced growth. The growth rate is age-dependent and we investigated how IGF-1 levels changed with age and growth conditions in nestlings of two passerine species – great tits (Parus major) and jackdaws (Corvus monedula) – with different life histories. We showed experimentally in both species that levels of IGF-1 change positively with the improving food availability, both when observed in the short- (within 1h) and long-term (during the nestling phase). However, the effects of nestling age on IGF-1 levels as well as IGF-1 growth associations contrasted strongly between these species. Thus, IGF-1 appears to respond to environmental cues uniformly across species, but the timing of IGF-1 mediated investments into growth is dependent on the particular growth strategy. These findings together show that IGF-1 variability provides a promising avenue for studies of animals’ growth, of which the exploration has only just begun.

Physiology of somatic growth: insulin-like growth factor 1 and its variability in passerine species with different life-history

Shark skin is a composite of mineralized dermal denticles embedded in an internal collagen fiber network. Shark skin is sexually dimorphic; females have skin that is thicker, denticle density and degree of overlap is greater, and denticle morphology differs from males. Shark skin behaves with mechanical anisotropy, showing greater extensibility when tested along the longitudinal axis and increased stiffness along the hoop axis. As a result, shark skin has been hypothesized to function as an exotendon. The goal of this study was to quantify mechanical properties (tensile strain at maximum load (%), strength (MPa), stiffness (MPa), and toughness (MPa)) of shark skin between sexes and testing directions (longitudinal and hoop). We hypothesized that female sharks would have greater skin mechanical properties due to observed skin morphological and dermal density differences between sexes. We also hypothesized that skin tested in the hoop direction would be stiffer than skin tested longitudinally. We tested skin from two juvenile male and two juvenile female sharks from three species (bonnethead, Sphyrna tiburo; bull, Carcharhinus leucas; silky, Carcharhinus falciformis) in tension with 250N load cell on an Instron E1000 at a 2 mm/s strain rate. Refuting our hypothesis, we found that male shark skin was stiffer and stronger than female skin. Supporting our hypothesis, we found skin was stiffer when tested in the hoop direction compared to the longitudinal direction (across species and sexes). Our study provides evidence of sexual variation in shark skin mechanical behavior and further supports the exotendon hypothesis of shark skin.

Sexual dimorphisms in the mechanical properties of shark skin

The vocal repertoire of birds is incredibly diverse. They vocalize using a specific organ, the syrinx, producing a primary vibration at the origin of a primary sound. This sound propagates through the suprasyringeal system, composed of the trachea, the larynx, and the oral cavity. The large volume of this suprasyringeal system, compared to mammals of similar size, suggests that it plays a role in the modulation of the sound initially emitted. To investigate this, we filmed 21 species of dead birds and manipulated them under biplanar X-ray. Markers were implanted in tissues (e.g., on the trachea contour, glottis, beak) to calculate the passive deformations and volume changes of the vocal tract during a range of movements of the neck/head system. Our results show that neck extension and beak closing are associated with an elongation of the trachea of up to 20% of its initial length. We tested the effect of such elongation on sound modulation using computational simulations. Our work shows that the complexity of bird vocalizations is not only related to the syrinx, but also to the entire upper vocal system. This work provides new elements to better understand complex communication, one of the amazing characteristics we share with birds.

Influence of the avian upper vocal tract on sound modulation

Muscle shortening underpins most skeletal motion, and ultimately animal performance. Shortening is usually assumed to be homogenous within a muscle. However, in swimming fish, the whole body deforms like a bending beam: as the vertebral column flexes laterally, muscle shortening increases along a medio-lateral gradient. Recently it has been shown that fish also bend their bodies dorsally during feeding, resulting in dorsoventral shortening gradients in some regions. This suggests these dorsal motions also follow beam-like bending, where the curvature of the vertebral column predicts the linear, dorsoventral shortening gradient in the muscle. We tested this hypothesis by measuring vertebral curvature and dorsal body (epaxial) muscle shortening during feeding motions in rainbow trout (Oncorhynchus mykiss). We used XROMM to measure dorsal and lateral curvature across the anterior vertebral column and used fluoromicrometry to measure shortening between markers throughout the epaxial muscles. Vertebral curvature and muscle marker position were used to predict muscle strain following beam theory. Predicted strains were compared to the measured strains from fluoromicrometry. Trout flexed the anterior vertebrae dorsally and laterally during feeding strikes. When curvature and marker position in both planes were included, the model’s predicted strain matched the measured strain well across most vertebral regions. Our model confirms dorsal flexion during feeding in trout can be modelled with beam theory, even when accompanied by lateral flexion. We find evidence of linear dorsoventral and mediolateral shortening gradients in the epaxial muscles, although it remains to be determined if these reflect shortening of the fibres within these muscles.

Testing the relationship between muscle deformation and vertebral curvature during feeding in rainbow trout (Oncorhynchus mykiss).

Nuclear Pore Complexes (NPCs) are large macromolecular assemblies embedded into the nuclear envelope (NE). They are involved in the selective nucleocytoplasmic transport of macromolecules, chromatin organization, and DNA repair. NPCs are organized into sub-complexes composed of repetitions of about 30 highly conserved nucleoporins (NUPs). The NUP107–160 complex (also known as Y complex) is located in the outer ring and is conserved between distantly related eukaryotes such as yeast and mammals. Here, we are going to focus on two Arabidopsis mutants deficient for this sub-complex: Atnup160 and Atnup96, also known as suppressor of auxin resistance1 (sar1) and sar3, respectively. NUP160 and NUP96 are critical for the nucleocytoplasmic transport of mRNAs and play important functions in auxin signalling, plant growth and flowering time regulation, as well as in immune defence and cold stress tolerance. In the context of our global study about the possible role of NPCs during meiosis (the specialised cell division that leads to gamete formation), we decided to explore the potential function of NUP160 and NUP96 during this cell division because the corresponding mutants show reduced fertility. The results of our study have demonstrated that meiosis is altered in a percentage of the meiocytes from these mutants, which would explain the reduction in the number of seeds. Our observations have revealed that the alteration of the Y complex causes problems in meiotic recombination, the formation of meiotic structures (such as the synaptonemal complex), and viability of the meiocytes. The possible origins of these problems will be discussed.

Exploring the function of components of the Nuclear Pore Complex during meiosis: SAR1 (NUP160) and SAR3 (NUP96)

Mitochondria play a central role in ATP provisioning, redox and Ca2+ homeostasis and stress signaling of aerobic organisms. Animal mitochondria are extremely sensitive to fluctuating oxygen (O2) levels such as occur during tissue ischemia and/or environmental hypoxia. In hypoxia-sensitive organisms, hypoxia and especially post-hypoxic reoxygenation cause mitochondrial injury due to the elevated production of reactive oxygen species, Ca2+ overload and damage to the metabolic machinery; yet many hypoxia-tolerant species (including intertidal invertebrates) endure frequent hypoxia-reoxygenation cycles without apparent ill effects. The mechanisms of such exceptional mitochondrial robustness are not yet fully understood. I will discuss the mitochondrial responses to intermittent hypoxia in marine intertidal mollusks emphasizing the potentially adaptive functional and proteomic changes of the mitochondria and integration of the mitochondrial responses with cellular protection and stress response pathways (including antioxidant defense, autophagy and apoptosis) that might contribute to high tolerance to fluctuating O2 levels and support recovery in these organisms. I will also discuss the current knowledge gaps with an outlook to future studies needed to shed light on mitochondrial adaptations and evolution of metabolism in the environments with highly variable oxygen levels such as the intertidal zone.

Mitochondrial mechanisms of tolerance to fluctuating oxygen conditions: What can we learn from intertidal animals?

Understanding molecular mechanisms underlying physiology benefits from studies of isolated and/or cultured cells. However, virtually all experiments in cell culture are done in the absence of O2 regulation, resulting in incubator headspace O2 levels of 18-19%. In vivo, O2 levels in heart, skeletal muscle, brain, liver, and indeed most of the mammalian body, are typically 2-6%. The supra-physiological O2 levels in cell culture drive increased cellular reactive oxygen species (ROS) production, affecting many aspects of cell physiology. We have shown that supra-physiological O2 targets mitochondria, with important effects on bioenergetic function and network dynamics. Cellular responses to hormones like 17β-estradiol are significantly different in 18%, versus 5%, O2. Our RNAseq investigations show widespread effects of 18%, versus 5% O2 on diverse molecular processes ranging from DNA repair and cell cycle to energy metabolism to the viral response. Since supra-physiological O2 affects both transcriptional and translational processes, there are additional effects on relative protein abundance that are evident in proteomic analyses. The effects of supra-physiological O2 we have characterized are generally cell line-specific, making them difficult to predict. Given these myriad cellular effects of O2, it is important to reference hypoxia experiments in cell culture to physiological normoxia (e.g. 2-6%), but the vast majority of CO2 incubators used by cell culturists are not capable of regulating O2. We have developed a low-cost (&lt;1000 CAD) cell culture incubator capable of regulating O2, CO2, and temperature in their appropriate physiological ranges. Improved control over O2 levels will improve data quality in hypoxia experiments.

Importance of maintaining physioxia in cell culture

The crucian carp has the rare ability to survive days to months of anoxia, depending on temperature. Transcriptome analyses show that this is accompanied by widespread changes in gene expression. However, the importance of changes on the mRNA level can of course be questioned because it is the proteins that ultimately matter. We have therefore, in addition to global analyses of transcription and translation (ribosomal footprint profiling), measured the levels of proteins and phosphopeptides in tissues of crucian carp exposed to one week of anoxia and subsequent reoxygenation at 8°C. A general conclusion is that the transcriptome is a good indicator for the direction of change but that rather few proteins show a detectable change in their concentration during one week of anoxia and subsequent reoxygenation (e.g. 66 out of 3304 proteins in brain), which probably reflects that protein synthesis is slow and occur on top of a background of existing protein pools. Moreover, most proteins may have structural or housekeeping functions that are not altered during anoxia, and making major changes in protein concentrations would be energetically expensive, and energy use needs to be suppressed in anoxia. Phosphorylation of proteins, on the other hand, is an instant response, but also here relatively few changes were seen. In brain 109 out of 4316 phosphopeptides were regulated, while in liver 395 out of 1293 were regulated. The changes found in the proteome and phosphoproteome are likely to point at important adaptive mechanisms, which include mitochondrial adjustments, AMPK signalling and glucose metabolism.

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Estradiol effects on early skeletogenesis in the European sea bass Dicentrarchus labrax

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