Sturgeons are the most endangered species group and their wild populations continue to decrease. In this study, we apply intracytoplasmic sperm injection (ICSI), an assisted reproductive technology, for the first time in endangered and critically endangered sturgeons. Using various egg-sperm species combinations we performed different ICSI experiments with immobilized pre- or non-activated spermatozoa, single or many, fresh or cryopreserved. Then we evaluated the fertilization success as well as the paternity of the resultant embryos and larvae. Surprisingly, all experimental groups exhibited embryonic development. Normal-shaped feeding larvae produced in all egg-sperm species-combination groups after ICSI using single fresh-stripped non-activated spermatozoa, in one group after ICSI using single fresh-stripped pre-activated spermatozoa, and in one group after ICSI using multiple fresh-stripped spermatozoa. ICSI with single cryopreserved non-activated spermatozoa produced neurula stage embryos. Molecular analysis showed genome integration of both egg- and sperm-donor species in most of the ICSI transplants. Overall, ICSI technology could be used as an assisted reproduction technique for producing sturgeons to rescue valuable paternal genomes.

• Keywords
• assisted reproduction, embryo, intracytoplasmic sperm injection, larva, sturgeon,
• Publication type
• Journal Article MeSH

Several steps of sturgeon somatic cell nuclear transfer (SCNT) have been recently established, but improvements are needed to make it a feasible tool to preserve the natural populations of this group of endangered species. The donor cell position inside the recipient egg seems to be crucial for its reprogramming; therefore by injecting multiple donor somatic cells instead of a single cell with a single manipulation, we increased the potential for embryo development. Using the Russian sturgeon Acipenser gueldenstaedtii as a multiple cell donor and sterlet Acipenser ruthenus as the non-enucleated egg recipient, we obtained higher proportion of eggs developing into embryos than previously reported with single-SCNT. Molecular data showed the production of a specimen (0.8%) contained only the donor genome with no contribution from the recipient, while two specimens (1.6%) showed both recipient and donor genome. These findings are the first report of donor DNA integration into a sturgeon embryo after interspecific cloning. In all, we provide evidence that cloning with the multiple donor somatic cells can be feasible in the future. Despite the fact that the sturgeon cloning faces limitations, to date it is the most promising technique for their preservation.

• MeSH
• Embryonic Development * MeSH
• Genome * MeSH
• Cloning, Molecular methods   MeSH
• Endangered Species statistics & numerical data   MeSH
• Fishes embryology   genetics   MeSH
• Nuclear Transfer Techniques * MeSH
• Conservation of Natural Resources methods   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

To expand germ cell populations and provide a consistent supply for transplantation, we established basal culture conditions for sturgeon germ cells and subsequently increased their mitotic activity by eliminating gonad somatic cells, supplementing with growth factor, and replacing fetal bovine serum (FBS). The initial basal culture conditions were Leibovitz's L-15 medium (pH 8.0) supplemented with 5% FBS (p < 0.001) at 21 °C. Proliferation of germ cells was significantly enhanced and maintained for longer periods by elimination of gonad somatic cells and culture under feeder-cell free conditions, with addition of leukemia inhibitory factor and glial-cell-derived neurotrophic factor (p < 0.001). A serum-free culture medium improved germ cell proliferation compared to the L-15 with FBS (p < 0.05). Morphology remained similar to that of fresh germ cells for at least 40 d culture. Germline-specific gene expression analysis revealed no significant changes to germ cells before and after culture. Sterlet Acipenser ruthenus germ cells cultured more than 40 days showed development after transplant into Russian sturgeon Acipenser gueldenstaedtii. Polymerase chain reaction showed 33.3% of recipient gonads to contain sterlet cells after four months. This study developed optimal culture condition for sturgeon germ cells. Germ cells after 40 d culture developed in recipient gonads. This study provided useful information for culture of sturgeon germ cells.

• Keywords
• feeder cells, germ cell culture, glial-cell-derived neurotrophic factor, sturgeon, transplantation,
• Publication type
• Journal Article MeSH

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.

• MeSH
• Reproductive Techniques, Assisted veterinary   MeSH
• Breeding MeSH
• Diploidy MeSH
• Embryonic Development genetics   MeSH
• Fertilization genetics   MeSH
• Haploidy MeSH
• Models, Genetic MeSH
• Mosaicism * MeSH
• Endangered Species MeSH
• Fishes embryology   genetics   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• MeSH
• Species Specificity MeSH
• Genotype MeSH
• Haploidy MeSH
• Homozygote MeSH
• Cloning, Organism methods   MeSH
• Microsatellite Repeats MeSH
• Endangered Species MeSH
• Fishes embryology   genetics   growth & development   MeSH
• Nuclear Transfer Techniques * MeSH
• Tetraploidy MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Russia MeSH