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.

Evolutionary conservation of maternal RNA localization in fishes and amphibians revealed by TOMO-Seq.

Asymmetrical localization of biomolecules inside the egg, results in uneven cell division and establishment of many biological processes, cell types and the body plan. However, our knowledge about evolutionary conservation of localized transcripts is still limited to a few models. Our goal was to compare localization profiles along the animal-vegetal axis of mature eggs from four vertebrate models, two amphibians (Xenopus laevis, Ambystoma mexicanum) and two fishes (Acipenser ruthenus, Danio rerio) using the spatial expression method called TOMO-Seq. We revealed that RNAs of many known important transcripts such as germ layer determinants, germ plasm factors and members of key signalling pathways, are localized in completely different profiles among the models. It was also observed that there was a poor correlation between the vegetally localized transcripts but a relatively good correlation between the animally localized transcripts. These findings indicate that the regulation of embryonic development within the animal kingdom is highly diverse and cannot be deduced based on a single model.

Author Correction: Application of interspecific Somatic Cell Nuclear Transfer (iSCNT) in sturgeons and an unexpectedly produced gynogenetic sterlet with homozygous quadruple haploid.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

First evidence of viable progeny from three interspecific parents in sturgeon.

Polyspermy is the most commonly observed cause of embryonic abnormalities in fertilization, often resulting in death. In sterlet (Acipenser ruthenus), however, polyspermic embryos have high survival (similar to a control group) and morphological development is similar to monospermic larvae. Ploidy of these individuals is n/2n mosaic (whereas the normal state for A. ruthenus is a functional diploid). This study was undertaken to test whether sturgeon eggs can be fertilized by several spermatozoa from different species to produce viable offspring from three interspecific parents: A. ruthenus (2n), A. gueldenstaedtii (4n), and A. baerii (4n). Four trials were performed: (1) and (2) A. baerii eggs were fertilized with a mixture of A. ruthenus and A. gueldenstaedtii sperm; (3) A. gueldenstaedtii eggs were fertilized with a mixture of A. baerii and A. ruthenus sperm; and (4) A. gueldenstaedtii eggs were fertilized with a mixture of A. gueldenstaedtii and A. ruthenus sperm. Fertilized embryos with abnormal cleavage (3, 5, 6, 7, 9, and 10 cells) were collected and kept separately until 14 days post-fertilization. Ploidy level of 25 larvae (hatched from abnormal cleaved embryos) was evaluated by flow cytometry. Forty-four percent of observed hybrids had a mosaic 2n/3n ploidy. Five larva were processed further with microsatellite analysis and demonstrated that three specimens were heterospecific polyspermic larvae, containing alleles from three parents, and two specimens were conspesific polyspermic larvae from two parents. This astonishing phenomenon was emphasized by the fact that it was generated without any significant intervention.

Application of interspecific Somatic Cell Nuclear Transfer (iSCNT) in sturgeons and an unexpectedly produced gynogenetic sterlet with homozygous quadruple haploid.

Somatic cell nuclear transfer (SCNT) is a very promising cloning technique for reconstruction of endangered animals. The aim of the present research is to implement the interspecific SCNT (iSCNT) technique to sturgeon; one fish family bearing some of the most critically endangered species. We transplanted single cells enzymatically isolated from a dissociated fin-fragment of the Russian sturgeon (Acipenser gueldenstaedtii) into non-enucleated eggs of the sterlet (Acipenser ruthenus), two species bearing different ploidy (4n and 2n, respectively). Up to 6.7% of the transplanted eggs underwent early development, and one feeding larva (0.5%) was successfully produced. Interestingly, although this transplant displayed tetraploidism (4n) as the donor species, the microsatellite and species-specific analysis showed recipient-exclusive homozygosis without any donor markers. Namely, with regards to this viable larva, host genome duplication occurred twice to form tetraploidism during its early development, probably due to iSCNT manipulation. The importance of this first attempt is to apply iSCNT in sturgeon species, establishing the crucial first steps by adjusting the cloning-methodology in sturgeon's biology. Future improvements in sturgeon's cloning are necessary for providing with great hope in sturgeon's reproduction.

Polyspermy produces viable haploid/diploid mosaics in sturgeon.

Most of sturgeon species (Acipenseridae) are currently critically endangered. Attempts to revive these populations include artificial breeding in hatcheries. However, under artificial reproduction, sturgeon embryos occasionally develop atypically, showing 3, 5, 6, 7, 9, or 10 cells at the 2- to 4-cell stage. This study was undertaken with the objective of understanding the mechanism of the atypical division (AD) in embryos during artificial breeding. Using several sturgeon species, we tested two hypotheses: (1) polyspermy and (2) retention of the second polar body. We found that (1) AD embryos survive similar to controls, (2) the ratio of AD embryos is positively correlated with the amount of sperm used for fertilization, (3) the number of micropyles and the area covered by them in AD embryos is significantly greater when compared to controls, (4) numerous spermatozoa nuclei are in the cytoplasm after fertilization, (5) all AD embryos are mosaics, and (6) AD fishes with n/2n ploidy contain diploid cells from maternal and paternal genetic markers, while the haploid cells contained only paternal ones. These results clearly indicate that AD embryos arise from plasmogamy where the accessory spermatozoon/spermatozoa entry the egg and develop jointly with zygotic cells. This suggests that a well-controlled fertilization procedure is needed to prevent the production of sturgeon with irregular ploidy, which can have detrimental genetic effects on sturgeon populations. On the other hand, if AD fish can produce haploid-derived clonal gametes, induction of multiple-sperm mosaicism might be a useful tool for the rapid production of isogenic strains of sturgeons.

Elimination of primordial germ cells in sturgeon embryos by ultraviolet irradiation.

A technique for rescuing and propagating endangered species involves implanting germ line stem cells into surrogates of a host species whose primordial germ cells (PGCs) have been destroyed. We induced sterilization in sterlet (Acipenser ruthenus) embryos by means of ultraviolet (UV) irradiation at the vegetal pole, the source of early-stage PGCs of sturgeon eggs. The optimal cell stage and length of UV irradiation for the effective repression of the developing PGCs were determined by exposing embryos at the one- to four-cell stage to different doses of irradiation at a wavelength of 254 nm (the optimal absorbance spectrum for germplasm destruction). The vegetal pole region of the embryos was labeled immediately upon irradiation with GFP bucky ball mRNA to monitor the amount of germ plasm and FITC-dextran (M.W. 500,000) to obtain the number of PGCs in the embryos. The size of the germ plasm and number of surrounding mitochondria in the irradiated embryos and controls were observed using transmission electron microscopy, which revealed a drastic reduction in both on the surface of the vegetal pole in the treated embryos. Furthermore, the reduction in the number of PGCs was proportional to the dose of UV irradiation. Under the conditions tested, optimum irradiation for PGCs removal was seen at 360 mJ/cm2 at the one-cell stage. Although some PGCs were observed after the UV irradiation, they significantly reduced in number as the embryos grew. We conclude that UV irradiation is a useful and efficient technique to induce sterility in surrogate sturgeons.