Shinya Shikina

Production of Trout Offspring from Triploid Salmon Parents

Many salmonids have become at risk of extinction. For teleosts whose eggs cannot be cryopreserved, developing techniques other than egg cryopreservation to save genetic resources is imperative. In this study, spermatogonia from rainbow trout were intraperitoneally transplanted into newly hatched sterile triploid masu salmon. Transplanted trout spermatogonia underwent spermatogenesis and oogenesis in male and female recipients, respectively. At 2 years after transplantation, triploid salmon recipients only produced trout sperm and eggs. With use of these salmon as parents, we successfully produced only donor-derived trout offspring. Thus, by transplanting cryopreserved spermatogonia into sterile xenogeneic recipients, we can generate individuals of a threatened species.

Sexual plasticity of ovarian germ cells in rainbow trout

The sexual plasticity of fish gonads declines after the sex differentiation period; however, information about the plasticity of the germ cells themselves after sex differentiation is limited. Using rainbow trout (Oncorhynchus mykiss), we recently established a novel germ cell transplantation system that provides a unique platform with which to dissect the developmental and cellular mechanisms underlying gametogenesis. Using this technique, we show here that transplanted ovarian germ cells isolated from 6- to 9-month-old donors can colonize sexually undifferentiated embryonic gonads and resume gametogenesis. Ovarian germ cells containing oogonia and early oocytes isolated from female rainbow trout were transplanted into the peritoneal cavities of hatching-stage fry of both sexes and the behavior of the donor cells was observed. The transplanted ovarian germ cells migrated towards the recipient gonads, interacted with embryonic gonadal somatic cells, proliferated rapidly, and eventually differentiated into eggs in female recipients and sperm in male recipients. Furthermore, the donor-derived eggs and sperm obtained from the recipient fish were functional and were able to produce normal offspring. These findings indicate that mitotic germ cells, the oogonia, possess a high level of sexual plasticity.

Generation of functional eggs and sperm from cryopreserved whole testes

The conservation of endangered fish is of critical importance. Cryobanking could provide an effective backup measure for use in conjunction with the conservation of natural populations; however, methodology for cryopreservation of fish eggs and embryos has not yet been developed. The present study established a methodology capable of deriving functional eggs and sperm from frozen type A spermatogonia (ASGs). Whole testes taken from rainbow trout were slowly frozen in a cryomedium, and the viability of ASGs within these testes did not decrease over a 728-d freezing period. Frozen-thawed ASGs that were intraperitoneally transplanted into sterile triploid hatchlings migrated toward, and were incorporated into, recipient genital ridges. Transplantability of ASGs did not decrease after as much as 939 d of cryopreservation. Nearly half of triploid recipients produced functional eggs or sperm derived from the frozen ASGs and displayed high fecundity. Fertilization of resultant gametes resulted in the successful production of normal, frozen ASG-derived offspring. Feasibility and simplicity of this methodology will call for an immediate application for real conservation of endangered wild salmonids.

Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources

Recent progress in genome-based breeding has created various fish strains carrying desirable genetic traits; however, methods for the long-term preservation of their genetic resources have not yet been developed, mainly due to the lack of cryopreservation techniques for fish eggs and embryos. Recently, we established an alternative cryopreservation technique for fish spermatogonia using a slow-freezing method. Furthermore, we developed a transplantation system to produce functional eggs and sperm derived from spermatogonia. Spermatogonia isolated from the testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of triploid masu salmon (Oncorhynchus masou) hatchlings of both genders. The transplanted trout spermatogonia migrated towards the gonadal anlagen of the recipient salmon, into which they were subsequently incorporated. We confirmed that the donor-derived spermatogonia resumed gametogenesis, and produced sperm and eggs in male and female recipient salmon, respectively. Fertilization of the resultant eggs and sperm produced only rainbow trout in the first filial (F₁) generation, suggesting that the sterile triploid recipient salmon produced functional eggs and sperm derived from the trout donors. A combination of spermatogonial transplantation and cryopreservation could be a powerful tool for preserving valuable fish strains with desirable genetic traits and endangered species.

Lymphocyte Antigen 75 (Ly75/CD205) Is a Surface Marker on Mitotic Germ Cells in Rainbow Trout

In mammals, several cell surface molecular markers have been characterized in order to identify the mitotic germ cells. However, little is known in fish about their cell surface antigen. In this study, we identified lymphocyte antigen 75 (Ly75/CD205) as a germ cell-specific cell surface marker by combination expressed sequence tag analysis of purified type A spermatogonia (A-SG) from immature testis, in silico prediction of membrane proteins, and expression studies. The ly75 transcripts were abundant in the testis and gills, and weak signals were detected in the head kidney and brain. In addition, ly75 mRNA was predominantly localized in the primordial germ cells of newly hatched embryos, A-SG in testis, oogonia, and chromatin nucleolus-stage oocytes in the ovary. In contrast, ly75 mRNA was not detected in spermatocytes, spermatids, spermatozoa, vitellogenic oocytes, or gonadal somatic cells from either males or females. The expression profile of Ly75 protein was similar to that of the mRNA. Furthermore, identification of various fish homologs of ly75 confirmed that their amino acid sequences are well conserved. Therefore, Ly75 may be appropriate for use as a versatile cell surface marker for mitotic germ cells in fish.

Improved In Vitro Culture Conditions to Enhance the Survival, Mitotic Activity, and Transplantability of Rainbow Trout Type A Spermatogonia

Spermatogenesis originates from a small population of spermatogonial stem cells, which have the ability to both self-renew and produce differentiated germ cells. We previously established a surrogate broodstock technique using xenotransplantation of spermatogonia in salmonids. This technique promised to be an efficient tool for producing target seeds that are valuable to markets or endangered species. We have been attempting to establish a technique to produce seeds by transplanting spermatogonia proliferated in culture dishes. However, our previous methods for culturing spermatogonia had several defects. First, residual testicular somatic cells infiltrated excessively proliferating cultures and eventually outcompeted spermatogonia. Second, the total number of spermatogonia gradually decreased during culture periods even though mitosis was confirmed. Third, the cultured spermatogonia were less able to be incorporated into the recipient gonads following transplantation as compared to the ability of intact spermatogonia. To overcome these defects, in the present study we improved upon spermatogonia culture conditions. The overgrowth of testicular somatic cells could be suppressed by adjusting fetal bovine serum concentration in the medium to 1%. The addition of soluble factors, such as bovine serum albumin, adenosine, and salmonid serum, to the medium would enhance spermatogonial survival, mitotic activity, and transplantability. Under newly developed conditions, we extended the culture periods. Furthermore, a transplantation assay showed that spermatogonia cultured in the modified medium for 42 days still possessed their transplantability. The present study represents valuable steps toward establishing a culture method enabling spermatogonia to expand in vitro for use in seed production with surrogate broodstock technology.

Yolk formation in a stony coral Euphyllia ancora (Cnidaria, Anthozoa): insight into the evolution of vitellogenesis in nonbilaterian animals.

Vitellogenin (Vg) is a major yolk protein precursor in numerous oviparous animals. Numerous studies in bilateral oviparous animals have shown that Vg sequences are conserved across taxa and that Vgs are synthesized by somatic-cell lineages, transported to and accumulated in oocytes, and eventually used for supporting embryogenesis. In nonbilateral animals (Polifera, Cnidaria, and Ctenophora), which are regarded as evolutionarily primitive, although Vg cDNA has been identified in 2 coral species from Cnidaria, relatively little is known about the characteristics of yolk formation in their bodies. To address this issue, we identified and characterized 2 cDNA encoding yolk proteins, Vg and egg protein (Ep), in the stony coral Euphyllia ancora. RT-PCR analysis revealed that expression levels of both Vg and Ep increased in the female colonies as coral approached the spawning season. In addition, high levels of both Vg and Ep transcripts were detected in the putative ovarian tissue, as determined by tissue distribution analysis. Further analyses using mRNA in situ hybridization and immunohistochemistry determined that, within the putative ovarian tissue, these yolk proteins are synthesized in the mesenterial somatic cells but not in oocytes themselves. Furthermore, Vg proteins that accumulated in eggs were most likely consumed during the coral embryonic development, as assessed by immunoblotting. The characteristics of Vg that we identified in corals were somewhat similar to those of Vg in bilaterian oviparous animals, raising the hypothesis that such characteristics were likely present in the oogenesis of some common ancestor prior to divergence of the cnidarian and bilaterian lineages.

Short-term in vitro culturing improves transplantability of type A spermatogonia in rainbow trout (Oncorhynchus mykiss).

Continuous production of sperm within the testes is supported by spermatogonial stem cells capable of both self‐renewal and the production of numerous differentiated germ cells. We previously demonstrated that a subpopulation of trout type A spermatogonia transplanted into the body cavity of a recipient embryo incorporated into the genital ridge, where they produced functional gametes within the gonads. Various cell‐surface proteins could have played a role in the incorporation of spermatogonia into recipient genital ridges. During the preparation of cell suspensions for transplantation in our experimental protocol, however, dissociation of testis by strong proteases was unavoidable. This was problematic as cell‐surface proteins may have been at least partially digested by protease activity. In the present study, recovery of spermatogonial surface proteins using short‐term culture prior to transplantation was attempted. It was found that spermatogonia cultured in vitro could be harvested by ethylenediaminetetraacetic acid (EDTA) instead of protease treatment. Furthermore, when cultured spermatogonia collected by EDTA treatment were maintained for 24 hr in vitro, they exhibited high adhesiveness. These cultured spermatogonia also possessed higher survival of transplantation compared to spermatogonia newly dispersed by trypsin treatment. These results indicated that spermatogonia possess a reduced ability to migrate toward, adhere to, and/or be incorporated into the recipient genital ridge immediately after protease treatment. Short‐term in vitro culturing, however, could allow spermatogonia to recover the surface proteins required for successful incorporation into the recipient genital ridge. Mol. Reprod. Dev. 80: 763–773, 2013.

Germ cell development in the scleractinian coral Euphyllia ancora (Cnidaria, Anthozoa).

Sexual reproduction of scleractinian coral is among the most important means of establishing coral populations. However, thus far, little is known about the mechanisms underlying coral gametogenesis. To better understand coral germ cell development, we performed a histological analysis of gametogenesis in Euphyllia ancora and characterized the coral homolog of the Drosophila germline marker gene vasa. The histological analysis revealed that E. ancora gametogenesis occurs in the mesenterial mesoglea between the mesenterial filaments and the retractor muscle bands. The development of germ cells takes approximately one year in females and half a year in males. Staining of tissue sections with an antibody against E. ancora Vasa (Eavas) revealed anti-Eavas immunoreactivity in the oogonia, early oocyte, and developing oocyte, but only faint or undetectable reactivity in developing oocytes that were >150 µm in diameters. In males, Eavas could be detected in the spermatogonia and primary spermatocytes but was only faintly detectable in the secondary spermatocytes, spermatids, and sperms. Furthermore, a reverse transcription-polymerase chain reaction analysis and Western blotting analysis of unfertilized mature eggs proved the presence of Eavas transcripts and proteins, suggesting that Eavas may be a maternal factor. Vasa may represent a germ cell marker for corals, and would allow us to distinguish germ cells from somatic cells in coral bodies that have no distinct organs.

Production of germ cell-deficient salmonids by dead end gene knockdown, and their use as recipients for germ cell transplantation

We previously established a spermatogonial transplantation model in fish using triploid recipients. Although triploid salmonids are sterile, they carry a limited number of immature triploid germ cells that potentially compete with the donor‐derived germ cells for their niche. We therefore assessed the biological characteristics of germ cell‐deficient gonads in rainbow trout for their suitability as recipients for germ cell transplantation in this study. Antisense morpholino oligonucleotides against the dead end gene were microinjected into the fertilized eggs of rainbow trout to eliminate endogenous germ cells, leaving only their supporting cells. Unlike similar approaches performed in zebrafish and medaka, these germ cell‐deficient rainbow trout did not show a male‐biased sex ratio. Approximately 30,000 spermatogonia were then transplanted into the body cavities of both germ cell‐deficient and control recipients. The donor‐derived germ cells showed significantly higher proliferation in the gonads of germ cell‐deficient recipients than those in the gonads of the control recipients. Finally, the applicability of the germ cell‐deficient recipients for xenogeneic transplantation was evaluated by transplanting rainbow trout spermatogonia into germ cell‐deficient masu salmon recipients. The resulting recipient salmon matured normally and produced trout gametes, and early survival of the resulting trout offspring was as high as that of the control offspring. Thus, dead end‐knockdown salmonids appear to be ideal recipients for the intraperitoneal transplantation of spermatogonia. Mol. Reprod. Dev. 83: 298–311, 2016.