Formation and structure of egg envelopes in Russian sturgeon Acipenser gueldenstaedtii (Acipenseriformes: Acipenseridae)

The covering of the eggs in Russian sturgeon Acipenser gueldenstaedtii consists of three envelopes (the vitelline envelope, chorion and extrachorion) and is equipped with multiple micropyles. The most proximal to the oocyte is the vitelline envelope that consists of four layers of filamentous and trabecular material. The structural components of this envelope are synthesized by the oocyte (primary envelope). The chorion encloses the vitelline envelope. The extrachorion covers the external surface of the egg. Examination of the arrangement of layers that comprise the egg envelopes together with the ultrastructure of follicular cells revealed that the chorion and extrachorion are secondary envelopes. They are secreted by follicular cells and are built of homogeneous material. During formation of egg envelopes, the follicular cells gradually diversify into three morphologically different populations: 1) cells covering the animal oocyte region (cuboid), (2) main body cells (cylindrical) and (3) micropylar cells. The apical surfaces of follicular cells from the first two populations form processes that remain connected with the oocyte plasma membrane by means of gap junctions. Micropylar cells are located at the animal region of the oocyte. Their apical parts bear projections that form a barrier to the deposition of materials for egg envelopes, resulting in the formation of the micropylar canal.     

Aspects of early development in the Adriatic sturgeon Acipenser naccarii

This study investigates the development of the sensory-cutaneous apparatus and digestive tract of Adriatic sturgeon (Acipenser naccarii). Light and electron microscopy observations were carried out on various developmental stages, from hatching to 180 days old.
At hatching, sturgeon pre-larvae exhibit differentiation of olfactory sensory cells. By day 4 taste buds are differentiated on lips and barbels. At 12 days after hatching, the larvae are equipped with an extensive ventral cephalic sensory apparatus. Electrosensory organs are arranged in regular lines in the rostral ventral region, and taste receptors are organised in parallel rows on and inside the lips as well as on the external side of the barbel. Ventral free neuromasts are positioned in rostral grooves. The retina is completely differentiated in each stratum. At this stage, larvae show canine-like teeth on lips and pharynx, and the specific mucosae of the different digestive regions are differentiated. By day 36 the canine-like teeth are located exclusively on the tongue and roof of the buccal cavity, and the mouth is protrusible. At 180 days differentiation is still not completed, and although teeth have disappeared from the palate, they still persist along a central line on the tongue.     

Myotomal myogenesis of axial muscle in the sturgeon Acipenser baeri (Chondrostei, Acipenseriformes)

Compared to teleost fishes, a unique character of the myogenesis of the plesiomorphic A. baeri is the fusion of myoblasts derived from the somite, leading to the formation of multinucleate muscle lamellae. Then, the lamellae are converted into cylindrical muscle fibres. The mechanism of transformation of lamellae into fibres is still debatable. Early embryonic muscle growth is mainly due to the hypertrophy of somite-cell derived stock. After hatching, hypertrophic growth occurs parallel to hyperplastic growth. Proliferatively active mesenchymal cells, which migrate from the intermyotomal space into the myotomes, participate in both processes.     

Ovarian nests in cultured females of the Siberian sturgeon Acipenser baerii (Chondrostei,Acipenseriformes)

Ovaries of Acipenser baerii are of an alimentary type and probably are meroistic. They contain ovarian nests, individual follicles, inner germinal ovarian epithelium, and fat tissue. Nests comprise cystoblasts, germline cysts, numerous early previtellogenic oocytes, and somatic cells. Cysts are composed of cystocytes, which are connected by intercellular bridges and are in the pachytene stage of the first meiotic prophase. They contain bivalents, finely granular, medium electron dense material, and nucleoli in the nucleoplasm. Many cystocytes degenerate. Oocytes differ in size and structure. Most oocytes are in the pachytene and early diplotene stages and are referred to as the PACH oocytes. Oocytes in more advanced diplotene stage are referred to as the DIP oocytes. Nuclei in the PACH oocytes contain bivalents and irregularly shaped accumulation of DNA (DNA‐body), most probably corresponding to the rDNA‐body. The DNA‐body is composed of loose, fine granular material, and comprises multiple nucleoli. At peripheries, it is fragmented into blocks that remain in contact with the inner nuclear membrane. In the ooplasm, there is the rough endoplasmic reticulum, Golgi complexes, free ribosomes, complexes of mitochondria with cement, fine fibrillar material containing granules, and lipid droplets. The organelles and material of nuclear origin form a distinct accumulation (a granular ooplasm) in the vicinity of the nucleus. Some of the PACH oocytes are surrounded by flat somatic cells. There are lampbrush chromosomes and multiple nucleoli present (early diplotene stage) in the nucleoplasm. These PACH oocytes and neighboring somatic cells have initiated the formation of ovarian follicles. The remaining PACH oocytes transform to the DIP oocytes. The DIP oocytes contain lampbrush chromosomes and a DNA‐body is absent in nuclei. Multiple nucleoli are numerous in the nucleoplasm and granular ooplasm is present at the vegetal region of the oocyte.     

Gonadal development and sex differentiation in the cichlid fish Cichlasoma dimerus (Teleostei, Perciformes): a light- and electron-microscopic study

Although the overall pattern and timing of gonadal sex differentiation have been established in a considerable number of teleosts, the ultrastructure of early stages of gonadal development is not well documented. In this study, gonads from larval and juvenile stages of laboratory-reared Cichlasoma dimerus were examined at the light-microscopic and ultrastructural levels. This freshwater species adapts easily to captivity and spawns with high frequency during 8 months of the year, providing an appropriate model for developmental studies. Larvae and juveniles were kept at a water temperature of 26.5 +/- 1 degrees C and a 12:12 hour photoperiod. Gonadal development was documented from 14-100 days postfertilization, covering the period of histologically discernible sex differentiation. Gonadal tissue was processed according to standard techniques for light and electron microscopy. C. dimerus, a perciform teleost, is classified as a differentiated gonochorist, in which an indifferent gonad develops directly into a testis or ovary. On day 14, the gonadal primordium consists of a few germ cells surrounded by enveloping somatic cells. Ovarian differentiation precedes testicular differentiation, as usual in teleost fishes. The earliest signs of differentiation, detected from day 42 onward, include the onset of meiotic activity in newly formed oocytes, which is soon accompanied by increased oogonial mitotic proliferation and the somatic reorganization of the presumptive ovary. The ovarian cavity is completely formed by day 65. Numerous follicles containing perinucleolar oocytes are observed by day 100. In contrast, signs of morphological differentiation in the presumptive testis are not observed until day 72. By day 100, the unrestricted lobular organization of the testis is evident. The latest stage of spermatogenesis observed by this time of testicular development is spermatocyte II.     

Isolation and characterization of microsatellites in the Adriatic sturgeon (Acipenser naccarii)

We isolated and characterized seven polymorphic microsatellite loci from a partial genomic library of the Adriatic sturgeon (Acipenser naccarii) enriched for GATA repeats. Variability was tested in 20 specimens of this endangered species. Our data support the tetraploid condition for A. naccarii and will be useful for the investigation of the remnant genetic variability of this species as well as for genetic tagging studies.     

Previtellogenic and vitellogenic oocytes in ovarian follicles of cultured siberian sturgeon Acipenser baerii (Chondrostei,Acipenseriformes)

Previtellogenic and vitellogenic oocytes in ovarian follicles from cultured Siberian sturgeon Acipenser baerii were examined. In previtellogenic oocytes, granular and homogeneous zones in the cytoplasm (the ooplasm) are distinguished. Material of nuclear origin, rough endoplasmic reticulum, Golgi complexes, complexes of mitochondria with cement and round bodies are numerous in the granular ooplasm. In vitellogenic oocytes, the ooplasm comprises three zones: perinuclear area, endoplasm and periplasm. The endoplasm contains yolk platelets, lipid droplets, and aggregations of mitochondria and granules immersed in amorphous material. In the nucleoplasm, lampbrush chromosomes, nucleoli, and two types of nuclear bodies are present. The first type of nuclear bodies is initially composed of fibrillar threads only. Their ultrastructure subsequently changes and they contain threads and medium electron dense material. The second type of nuclear bodies is only composed of electron dense particles. All nuclear bodies impregnate with silver, stain with propidium iodide, and are DAPI‐negative. Their possible role is discussed. All oocytes are surrounded by follicular cells and a basal lamina which is covered by thecal cells. Egg envelopes are not present in previtellogenic oocytes. In vitellogenic oocytes, the plasma membrane (the oolemma) is covered by three envelopes: vitelline envelope, chorion, and extrachorion. Vitelline envelope comprises four sublayers: filamentous layer, trabecular layer 2 (t2), homogeneous layer, and trabecular layer 1 (t1). In the chorion, porous layer 1 and porous layer 2 are distinguished in most voluminous examined oocytes. Three micropylar cells that are necessary for the formation of micropyles are present between follicular cells at the animal hemisphere. J. Morphol. 278:50–61, 2017. ©© 2016 Wiley Periodicals,Inc.     

Cytochrome P-450 levels and EROD activity in the Adriatic sturgeon (Acipenser naccarii, Chondrostei) and its induction by β-Naphthoflavone

The cytochrome P-450 enzyme system was studied in juvenile Acipenser naccarii (317.6±77 g initial live weight) reared at 23°C and at a standard diet over a period of two month before using for experiments (final weight 546.3±148.8 g). While determining normal levels of cytochrome P-450 in liver tissue its induction was also studied under exposure to P-naphthoflavone (BNF; single dose at 80 mg/Kg body weight 52 hrs prior to sampling). Control fish showed total cytochrome P-450 levels in liver microsomes around 0.40±0.12 nmoles/mg proteins (n=12) while in BNF exposed fish the values increased significantly (0.55±0.11nmoles/mg proteins; n=12). Ethoxyresorufin-O-deethylase (EROD) in control fish was 0.015±0.02 nmoles/mg proteins/min (n=12); it was found to be NADPH dependent and inducible by BNF. Western Immunoblotting and enzyme-linked-immunosorbent (ELISA) analyses of liver microsomes from untreated and BNF-exposed fish, using an antibody to rat cytochrome P-450 1A1 isoform (P-450 1A1) (Nelson et al.,1996) suggested the presence of a constitutive and BNF-inducible cytochrome P-450 homologous to rat P-450 protein.
Measurement of cytochrome P-450-dependent EROD activity and P-450 1A1 expression levels in A. naccarii may be a useful parameter in monitoring environmental pollution.     

Sex differentiation and aspects of gametogenesis in shortnose sturgeon Acipenser brevirostrum Lesueur

Shortnose sturgeon Acipenser brevirostrum gonad samples were collected from industry-reared fish and wild broodstock at various developmental stages to elucidate patterns of gonadal differentiation and maturation. Genital ridges, containing germ cells, were present in 26 day-old fish and distinct gonads were present by day 54. Sturgeon gonads are known to consist of two tissue types (adipose and gametogenic) and both were present at 72 day. Anatomical differentiation of gonads occurred by 6 months and was advanced by 15 months. Ovaries had distinct lamellae while testes remained non-lamellate. Gonial proliferation had occurred by 15 months, but the cells were not identifiable as spermatogonia or oogonia. Small white 'pinhead' oocytes were macroscopically visible in ovaries as early as 36 months. At 43 months ovaries were clearly organized, with some areas containing only immature oocytes and other containing oocytes apparently developing as cohorts. Individual fish showed considerable variation: the level of development remained unchanged at 84 months in some females, while others showed clear progression towards sexual maturation at 48 months. Sperm cells were present in males as early as 52 months. Advanced development of ovarian follicles was observed only in biopsies of re-conditioned broodstock of wild origin. In the year before spawning, the most advanced oocytes became pigmented, the chorion thickened, the nucleus (germinal vesicle) migrated towards the micropyle complex at the animal pole, and ovulation occurred in May under appropriate environmental conditions.     

Purification and characterization of the hemoglobin components of Adriatic sturgeon (Acipenser naccarii) blood

The oxygen binding properties of Acipenser naccarii hemoglobins have been investigated, and were found not to differ significantly from those shown by blood, intact erythrocytes and hemolysate in the presence of the physiological cofactors GTP and chloride ions. In particular, the oxygen equilibrium reveal a very low sensitivity of the hemolysate to the chloride ions and temperature while in the presence of organic phosphates their oxygen affinity decreased strongly. The electrophoretic analysis of the hemolysate of the sturgeon showed the presence of two hemoglobin components, each with a considerable globin multiplicity. The partial amino acid sequence of the a and P chain of the single hemoglobins was also established.     

Histochemical and immunohistochemical investigation of muscle fibres in the sturgeon (Chondrostei; Acipenser)

The arrangement and innervation of lateral muscle and adductor mandibulae (AM) complex were examined histochemically and immunohistochemically in the sturgeon Acipenser transmontanus. Lateral muscle was arranged in myotomes in which the superficial slow-red muscle layer and the deep fast-white muscle layer were separated by an intermediate layer of pink muscle. The AM complex was composed of a small slow-red muscle layer, but the bulk of the muscle was composed of fast-white fibres. Pink fibres appeared both in an intermediate layer and scattered throughout the fast-white muscle. The innervation appeared to resemble that of elasmobranchs and some teleosts. Slow-red fibres of lateral muscle were multiply innervated, whereas fast-white muscle fibres were focally innervated at the myoseptal end. Pink fibres of lateral muscle and fast-white fibres of AM were focally innervated in the mid-region of the fibres. There were no significant differences between 5 month old and adult sturgeons except for a much better developed intermediate (pink) layer surrounding the lateral line in the adult.     

Influence of sampling conditions on blood chemistry values of Adriatic sturgeon Acipenser naccarii (Bonaparte, 1836)

Data on the blood chemistry of a chondrostean fish, the Adriatic sturgeon ( Acipenser naccarii ), are reported as measured with different sampling procedures, and as related to rearing conditions and age. Serum Cortisol, glucose, osmolality, Na⁺, Cl^-, Ca²⁺ and total protein concentrations were measured. Reference values for the blood chemistry of farmed sturgeon were measured on samples from resting undisturbed animals collected via a chronic indwelling catheter in the dorsal aorta that was implanted under anaesthesia. Following 24h recovery from catheterization, serum Cortisol, glucose and osmolality levels were 9.4 ng/ml, 58.8 mg/dl and 261.4 mOsm/kg, respectively. Furthermore, blood samples collected with the chronic indwelling catheters indicated that the surgical procedure of cannulation caused a stress response, with physiological changes that followed a pattern like that described in teleosts. Cortisol, glucose and osmolality were more sensitive to stress than the other variables measured. Sampling by cardiac puncture tended to be associated with elevated serum Cortisol levels in older, larger sturgeon, but not in young fish. Greater capture, confinement and handling stress in older, larger, sturgeon may have been responsible for this and other age-related differences in blood chemistry values measured following cardiac puncture. Within the same age class, both rearing conditions and temperature affected Cortisol, sodium and total protein concentrations significantly. Anaesthesia did not appear to reduce the degree of stress associated with cardiac puncture but altered serum ion concentrations.     

Gonadal morphogenesis and sex differentiation in intraovarian embryos of the viviparous fish Zoarces viviparus (Teleostei, Perciformes, Zoarcidae): a histological and ultrastructural study

It is essential to know the timing and process of normal gonadal differentiation and development in the specific species being investigated in order to evaluate the effect of exposure to endocrine-disrupting chemicals on these processes. In the present study gonadal sex differentiation and development were investigated in embryos of a viviparous species of marine fish, the eelpout, Zoarces viviparus, during their intraovarian development (early September to January) using light and electron microscopy. In both sexes of the embryos at the time of hatching (September 20) the initially undifferentiated paired bilobed gonad contains primordial germ cells. In the female embryos, ovarian differentiation, initiated 14 days posthatch (dph), is characterized by the initial formation of the endoovarian cavity of the single ovary as well as by the presence of some early meiotic oocytes in a chromatin-nucleolus stage. By 30 dph, the endoovarian cavity has formed. By 44 dph and onward, the ovary and the oocytes grow in size and at 134 dph, just prior to birth, the majority of the oocytes are at the perinucleolar stage of primary growth and definitive follicles have formed. In the presumptive bilobed testis of the male embryos, the germ cells (spermatogonia), in contrast to the germ cells of the ovary, remain quiescent and do not enter meiosis during intraovarian development. However, other structural (somatic) changes, such as the initial formation of the sperm duct (30 dph), the presence of blood vessels in the stromal areas of the testis (30 dph), and the appearance of developing testicular lobules (102 dph), indicate testicular differentiation. Ultrastructually, the features of the primordial germ cells, oogonia, and spermatogonia are similar, including nuage, mitochondria, endoplasmic reticulum, and Golgi complexes.     

Differentiation of the cardiac outflow tract components in alevins of the sturgeon Acipenser naccarii (Osteichthyes, Acipenseriformes): implications for heart evolution

Previous work showed that in the adult sturgeon an intrapericardial, nonmyocardial segment is interposed between the conus arteriosus of the heart and the ventral aorta. The present report illustrates the ontogeny of this intermediate segment in Acipenser naccarii. The sample studied consisted of 178 alevins between 1 and 24 days posthatching. They were examined using light and electron microscopy. Our observations indicate that the entire cardiac outflow tract displays a myocardial character during early development. Between the fourth and sixth days posthatching, the distal portion of the cardiac outflow tract undergoes a phenotypical transition, from a myocardial to a smooth muscle-like phenotype. The length of this region with regard to the whole outflow tract increases only moderately during subsequent developmental stages, becoming more and more cellularized. The cells soon organize into a pattern that resembles that of the arterial wall. Elastin appears at this site by the seventh day posthatching. Therefore, two distinct components, proximal and distal, can be recognized from the fourth day posthatching in the cardiac outflow tract of A. naccarii. The proximal component is the conus arteriosus, characterized by its myocardial nature and the presence of endocardial cushions. The distal component transforms into the intrapericardial, nonmyocardial segment mentioned above, which is unequivocally of cardiac origin. We propose to designate this segment the "bulbus arteriosus" because it is morphogenetically equivalent to the bulbus arteriosus of teleosts. The present findings, together with data from the literature, point to the possibility that cells from the cardiac neural crest are involved in the phenotypical transition that takes place at the distal portion of the cardiac outflow tract, resulting in the appearance of the bulbus arteriosus. Moreover, they suggest that the cardiac outflow tract came to be formed by a bulbus arteriosus and a conus arteriosus from an early period of the vertebrate evolutionary story. Finally, we hypothesize that the embryonic truncus of birds and mammals is homologous to the bulbus arteriosus of fish.     

Managing polyploidy in ex situ conservation genetics: the case of the critically endangered Adriatic sturgeon (Acipenser naccarii)

While the current expansion of conservation genetics enables to address more efficiently the management of threatened species, alternative methods for genetic relatedness data analysis in polyploid species are necessary. Within this framework, we present a standardized and simple protocol specifically designed for polyploid species that can facilitate management of genetic diversity, as exemplified by the ex situ conservation program for the tetraploid Adriatic sturgeon Acipenser naccarii. A critically endangered endemic species of the Adriatic Sea tributaries, its persistence is strictly linked to the ex situ conservation of a single captive broodstock currently decimated to about 25 individuals, which represents the last remaining population of Adriatic sturgeon of certain wild origin. The genetic variability of three F1 broodstocks available as future breeders was estimated based on mitochondrial and microsatellite information and compared with the variability of the parental generation. Genetic data showed that the F1 stocks have only retained part of the genetic variation present in the original stock due to the few parent pairs used as founders. This prompts for the urgent improvement of the current F1 stocks by incorporating new founders that better represent the genetic diversity available. Following parental allocation based on band sharing values, we set up a user-friendly tool for selection of candidate breeders according to relatedness between all possible parent-pairs that secures the use of non-related individuals. The approach developed here could also be applied to other endangered tetraploid sturgeon species overexploited for caviar production, particularly in regions lacking proper infrastructure and/or expertise.     

Morphology and fine structure of Acipenser persicus (Acipenseridae, Chondrostei) spermatozoon: Inter-species comparison in Acipenseriformes

This study describes morphology and fine structure of the Persian sturgeon (Acipenser persicus) (Acipenseridae, Chondrostei) spermatozoon. The results show that the spermatozoon of A. persicus is differentiated into an elongated head (length: mean±SD: 7.1±0.5μm) with an acrosome (length: 1.2±0.2μm), a cylindrical midpiece (length: 1.8±0.5μm), a flagellum (length: 50.3±5.9μm) and a total length of 59.2±6.2μm. Ten posterolateral projections (PLPs) arise from the posterior edge of the acrosome and there were 3 endonuclear canals that traversed the nucleus from the acrosomal end to the basal nuclear fossa region. Three to six mitochondria were in peripheral midpiece and the proximal and distal centrioles were located near to "implantation fossa" and basement of the flagellum. The axoneme has a typical eukaryotic structure composed of 9 peripheral microtubules and a central pair of single microtubule surrounded by the plasma membrane. Lateral fins were observed along the flagellum. The fins started and ended at 0.5-1μm from midpiece and at 4-6μm from the end of flagellum. There were significant differences in the size of almost all measured morphological parameters between males and flagellar, midpiece and nucleus characters were more isolated parameters that can be considered for detecting inter-individual variations. This study showed that sperm morphology and fine structure are similar among sturgeon species, but the dimensions of the parameters may differ.     

Development of the skull and pectoral girdle in Siberian sturgeon,Acipenser baerii,and Russian sturgeon,Acipenser gueldenstaedtii (Acipenseriformes: Acipenseridae)

The head is considered the major novelty of the vertebrates and directly linked to their evolutionary success. Its form and development as well as its function, for example in feeding, is of major interest for evolutionary biologists. In this study, we describe the skeletal development of the cranium and pectoral girdle in Siberian (Acipenser baerii ) and Russian sturgeon (A. gueldenstaedtii ), two species that are commonly farmed in aquaculture and increasingly important in developmental studies. This study comprises the development of the neuro‐, viscero‐ and dermatocranium and the dermal and chondral components of the pectoral girdle, from first condensation of chondrocytes in prehatchlings to the early juvenile stage and reveals a clear pattern in formation. The otic capsules, the parachordal cartilages, and the trabeculae cranii are the first centers of chondrification, at 8.4mm TL. These are followed by the mandibular, then the hyoid, and later the branchial arches. Teeth form early on the dentary, dermopalatine, and palatopterygoid, and then appear later in the buccal cavity as dorsal and ventral toothplates. With ongoing chondrification in the neurocranium a capsule around the brain and a strong rostrum are formed. Dermal ossifications start to form before closure of the dorsal neurocranial fenestrae. Perichondral ossification of cartilage bones occurs much later in ontogeny. Our results contribute data bearing on the homology of elements such as the lateral rostral canal bone that we regard homologous to the antorbital of other actinopterygians based on its sequence of formation, position and form. We further raise doubts on the homology of the posterior ceratobranchial among Actinopteri based on the formation of the hyoid arch elements. We also investigate the basibranchials and the closely associated unidentified gill‐arch elements and show that they are not homologous. J. Morphol. 278:418–442, 2017. © 2017 Wiley Periodicals, Inc.