Comparison of RNA localization during oogenesis within Acipenser ruthenus and Xenopus laevis

The oocyte is a unique cell, from which develops a complex organism comprising of germ layers, tissues and organs. In some vertebrate species it is known that the asymmetrical localization of biomolecules within the oocyte is what drives the spatial differentiation of the daughter cells required for embryogenesis. This asymmetry is first established to produce an animal-vegetal (A-V) axis which reflects the future specification of the ectoderm, mesoderm, and endoderm layers. Several pathways for localization of vegetal maternal transcripts have already been described using a few animal models. However, there is limited information about transcripts that are localized to the animal pole, even though there is accumulating evidence indicating its active establishment. Here, we performed comparative TOMO-Seq analysis on two holoblastic cleavage models: Xenopus laevis and Acipenser ruthenus oocytes during oogenesis. We found that there were many transcripts that have a temporal preference for the establishment of localization. In both models, we observed vegetal transcript gradients that were established during either the early or late oogenesis stages and transcripts that started their localization during the early stages but became more pronounced during the later stages. We found that some animal gradients were already established during the early stages, however the majority were formed during the later stages of oogenesis. Some of these temporally localized transcripts were conserved between the models, while others were species specific. Additionally, temporal de novo transcription and also degradation of transcripts within the oocyte were observed, pointing to an active remodeling of the maternal RNA pool

Migratory patterns and evolutionary plasticity of cranial neural crest cells in ray-finned fishes

The cranial neural crest (CNC) arises within the developing central nervous system, but then migrates away from the neural tube in three consecutive streams termed mandibular, hyoid and branchial, respectively, according to the order along the anteroposterior axis. While the process of neural crest emigration generally follows a conserved anterior to posterior sequence across vertebrates, we find that ray-finned fishes (bichir, sterlet, gar, and pike) exhibit several heterochronies in the timing and order of CNC emergence that influences their subsequent migratory patterns. First, emigration of the cranial neural crest in these fishes occurs prematurely compared to other vertebrates, already initiating during early neurulation and well before neural tube closure. Second, delamination of the hyoid stream occurs prior to the more anterior mandibular stream; this is associated with early morphogenesis of key hyoid structures like external gills (bichir), a large opercular flap (gar) or first forming cartilage (pike). In sterlet, the hyoid and branchial CNC cells form a single hyobranchial sheet, which later segregates in concert with second pharyngeal pouch morphogenesis. Taken together, the results show that despite generally conserved migratory patterns, heterochronic alterations in the timing of emigration and pattern of migration of CNC cells accompanies morphological diversity of ray-finned fishes

Standardization of sperm motility analysis by using CASA-Mot for Atlantic salmon (Salmo salar), European eel (Anguilla anguilla) and Siberian sturgeon (Acipenser baerii)

[EN] It is essential to define an optimized standard method to assess the fish sperm quality to minimize the differences between the results obtained by different laboratories. Only this optimization and standardization can make them useful from academia to industry. This study presents the validation of sperm motility assessment using a CASA-Mot system for three endangered diadromous fish species: European eel (Anguilla anguilla), Atlantic salmon (Salmo salar) and Siberian sturgeon (Acipenser baerii). To attain this goal, different technical and data processing methods were tested: 1) magnification lens (×10 and ×20), 2) Spermtrack® reusable chambers (10 and 20¿¿m depth) and 3) different frame rates (50¿¿¿FR¿¿¿250). The results suggested that the sperm motility assessment for eel, salmon and sturgeon should be performed at 200, 250 and 225¿frames¿s¿1, respectively. Moreover, to obtain a high number of analysed spermatozoa in less time and a natural movement of the sperm cells, it is recommended to use ×10 objective and 20¿¿m depth. In conclusion, different technical settings influence sperm kinetic parameters and should be validated for each fish species to allow the comparison of results between laboratories.This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No [642893]. AV is granted by the CONICIT and MICITT, Costa Rica. The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic projects "CENAKVA" (No. CZ.1.05/2.1.00/01.0024), "CENAKVA II" (No. LO1205 under the NPU I program), project Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370), by the Czech Science Foundation (project No. 17-19714Y).Caldeira, C.; Hernández-Ibáñez, S.; Valverde, A.; Martin, P.; Herranz-Jusdado, JG.; Gallego Albiach, V.; Asturiano Nemesio, JF.... (2019). Standardization of sperm motility analysis by using CASA-Mot for Atlantic salmon (Salmo salar), European eel (Anguilla anguilla) and Siberian sturgeon (Acipenser baerii). Aquaculture. 502:223-231. https://doi.org/10.1016/j.aquaculture.2018.12.001S22323150

Non-motile tetraploid spermatozoa of Misgurnus loach hybrids

We have compared various properties of spermatozoa from the wild diploid male pond loach Misgurnus anguillicaudatus to those from the interspecific male hybrid of the cross between a female M. anguillicaudatus and a male mud loach M. mizolepis. Our results show that spermatozoa from this interspecific hybrid had poor motility, low viability, abnormal morphology, a larger volume of mitochondrial mass per cell and higher ATP content of spermatozoa with tetraploid DNA content, and they were present at a low concentration. The interspecific hybrid males produced spermatozoa with a larger head, with either no flagellum (36.4 %), one flagellum (46.7 %) or two flagella (16.9 %). These flagella were shorter than those of the normal wild-type male M. anguillicaudatus and often presented with abnormalities in microtubule structure. An abnormally shorter flagellum has difficulty in propelling tetraploid spermatozoa with an increased head size in normal progressive motility, although they had higher energy, as shown by their larger volume of mitochondrial mass and higher ATP content. These tetraploid spermatozoa are likely produced by the arrest of the regular meiotic division after chromosomal replication, followed by abnormal spermiogenesis

Changes of sperm morphology, volume, density and motility and seminal plasma composition in

Eighteen spermiating males were randomly selected from a hatchery-reared stock and electronically tagged to record changes in their sperm quality parameters (spermatozoa morphology, ultrastructure and motility, ionic composition and osmolality of the seminal plasma, and sperm volume and density) during the spawning season. Stripping was performed at the beginning of March, April and May. The Barbus barbus spermatozoon has a head without acrosome, a midpiece with 4–6 mitochondria and proximal and distal centrioles, and a flagellum with the typical 9+2 pairs of microtubules. Apart from posterior width of the midpiece, morphological and ultrastructural parameters changed significantly during the reproductive season; generally by decreasing toward the end of reproductive season. Sperm volume also decreased from 0.42 in March to 0.15 ml in May, and density from 18.81 in March to 12.45 × 109 spz ml−1 in May. Osmolality (mOsmol kg−1) was 268 ± 4, 276 ± 2 and 268 ± 2 in March, April and May respectively. Chloride, sodium, calcium and potassium ion concentrations (mM) did not show significant differences between March and April (Cl−: 125.3 vs. 120.5, Na+: 75.7 vs. 69.7, Ca2+: 0.4 vs. 0.3 and K+: 84.7 vs. 84.0). The percentage of motile spermatozoa at 15 s post activation did not show a significant difference between dates, but the highest spermatozoa velocity at 15 s post activation was observed in April (91.4 ± 3.2 µm s−1) and then decreased significantly towards the end of the reproductive season (80.6 µm s−1 in May). However, lowest spermatozoa velocity was measured in March (70.4 ± 1.9 µm s−1). This study supports the hypothesis that longer spermatozoa swim faster. Within one stripping, velocity and percentage motility decreased significantly with time post activation. In conclusion, changes observed in B. barbus sperm parameters during the reproductive season, suggest there is association between such changes and spermatozoa aging processes

Volume changes during the motility period of fish spermatozoa: Interspecies differences

The aim of this study was to describe spermatozoa volume changes during the motility period of fish species with either osmotic (common carp Cyprinus carpio) or with ionic (sterlet Acipenseri ruthenus and brook trout Salvelinus fontinalis) modes of motility activation. Nephelometry, light microscopy, and spermatocrit methods were used for quantitative assessment of cell volume changes in media of different osmolalities. Significant correlation (R-2 = 0.7341; P < 0.001) between parameter of volume changes measured using nephelometry and light microscopy methods confirmed nephelometry as a sufficiently sensitive method to detect changes of spermatozoa volume. The spermatocrit alteration method resulted in a large proportion of damaged and potentially immotile spermatozoa in media of osmolality less than 150 mOsm/kg in carp and osmolalities from 10 to 300 mOsm/kg in sterlet and brook trout. Therefore, this method is not reliable for assessing spermatozoa swelling in hypotonic solutions, because the integrity of the cells is not fully preserved. Increase in carp spermatozoa (osmotic activation mode) volume occurred during the motility period in hypotonic conditions, but no indications of volume changes were found in sterlet and brook trout spermatozoa (ionic activation mode) associated with environmental osmolality alteration. Accordingly, we conclude that sperm volume changes are differentially involved in the motility activation process. Species-specific differences in spermatozoa volume changes as a response to a hypotonic environment during the motility period are discussed in relation to their potent physiological role. (c) 2013 Elsevier Inc. All rights reserved

Image1_Comparison of RNA localization during oogenesis within Acipenser ruthenus and Xenopus laevis.pdf

The oocyte is a unique cell, from which develops a complex organism comprising of germ layers, tissues and organs. In some vertebrate species it is known that the asymmetrical localization of biomolecules within the oocyte is what drives the spatial differentiation of the daughter cells required for embryogenesis. This asymmetry is first established to produce an animal-vegetal (A-V) axis which reflects the future specification of the ectoderm, mesoderm, and endoderm layers. Several pathways for localization of vegetal maternal transcripts have already been described using a few animal models. However, there is limited information about transcripts that are localized to the animal pole, even though there is accumulating evidence indicating its active establishment. Here, we performed comparative TOMO-Seq analysis on two holoblastic cleavage models: Xenopus laevis and Acipenser ruthenus oocytes during oogenesis. We found that there were many transcripts that have a temporal preference for the establishment of localization. In both models, we observed vegetal transcript gradients that were established during either the early or late oogenesis stages and transcripts that started their localization during the early stages but became more pronounced during the later stages. We found that some animal gradients were already established during the early stages, however the majority were formed during the later stages of oogenesis. Some of these temporally localized transcripts were conserved between the models, while others were species specific. Additionally, temporal de novo transcription and also degradation of transcripts within the oocyte were observed, pointing to an active remodeling of the maternal RNA pool.</p