1. Summary

Fish germline stem cells (FGSCs), cultured and expanded in vitro, have been shown to subsequently be able to develop into mature eggs upon transplant into the ovaries of recipient animals. Whereas this technology has value for the preservation of endangered species, it also has potential as a platform for a bioengineering approach to the scalable production of animal-free, mature oocytes of Beluga sturgeon as a luxury foodstuff. Novel 3D bioprinting and stem cell-based tissue engineering technologies, proprietary to Geneus Biotech (patent filings P34641PC00 and GB. 2103868.2), will be brought to bear on the challenge of identifying growth conditions that adequately recapitulate the ovarian microenvironment that surrounds FGSCs in order to stimulate mature oocyte formation ex vivo. 


2. Scientific and technical state of the art

Fish are able to produce large numbers of eggs throughout their reproductive life and maintain a high level of fecundity [1]. Genetic studies of zebrafish [2], Danio rerio, a species proven to be biocompatible with Russian sturgeon and beluga, Huso huso, with respect to in vitro co-culture, have provided evidence for the existence of a population of female germline stem cells (FGSCs) that is maintained in the adult fish ovary and is responsible for the continuous production of oocytes. The existence of fish FGSCs in the adult ovary has been further confirmed by cell transplantation studies with trout [4] and zebrafish [5] showing that donor ovarian germ cells can colonize the gonads of sterile recipient fish and produce functional gametes in the adult chimeras.

Stable cultivation of homogenous, genetically modified FGSCs has been established [6]. Such FGSCs have also been cultivated in vitro using a feeder layer derived from Sertoli cells and a culture medium containing trout plasma [7] to successfully maintain stem cell activity and potency to produce functional eggs. In alternative approaches, a genetically modified feeder cell line was co-cultured with the FGSCs, harbouring transgenes to direct secretion of zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and glial-cell-line derived neurotrophic factor (Gdnf). The presence of these feeder cells was shown to significantly enhance FGSC growth and survival in culture, and competency to differentiate into oocytes.


3.FGSCs as an unlimited source of mature oocytes.

Fish germline stem cells (FGSCs) have the potential for self-renewal and differentiation. Work with FGSCs started in the 1990s using zebrafish and medaka fish species. Since then it has been demonstrated that cultivated Medaka FGSCs can be transplanted to the ovary of a recipient animal and where they will develop into viable eggs that can be fertilised and development into viable offspring. Attempts at germline transmission of FGSC cell cultures and gene targeting modifications have also been reported in zebrafish. Recent years have witnessed rapid progress in the identification of biomarkers and procedures for FGSC cell characterization.


4.Technical implementation – 3D printed skein mimic

The sturgeon ovary is composed of two compartments; the stromal compartment, also known as the skein, in which folliculogenesis and oocyte maturation proceed and the ovarian cavity, into which the mature eggs are ovulated and then subsequently flow through specialized tubes, the paramesonephric or müllerian ducts, for release outside of the body cavity (spawning). Mature eggs of the Beluga sturgeon are not preferred for the luxury foodstuff, caviar, which in fact consists of still-maturing oocytes that are manually separated from a layer of intact follicular tissues that surround oocytes and anchor them to ovarian membranes. We will obtain donor immature oocytes from ovarian tissue of a range of model fish species, including Beluga sturgeon, and use proprietary Geneus Biotech techniques to co-culture them with FGSCs within 3D bioprinted scaffolds optimised to provide a functional analogue of the ovarian microenvironment. In the synthetic ovary analogue microenvironment, immature oocytes will be co-cultured with genetically modified feeder cells, which harbour synthetic gene networks that direct oscillatory pulses of secretion of gonadotropin-releasing hormone (GnRH), gonadotropins (GtH), estradiol (E2), 17 hydroxy-progesterone (17-OHP), maturation-inducing steroid (MIS), vitellogenin (Vg) and prostaglandin (PG) to stimulate oocyte maturation. A design of experiments (DOE) approach [8] will also be applied to identify optimal conditions and cultivation durations needed to maximise mature oocyte yield.


5.Concluding remarks of technological and scientific feasibility study 

Maturation of viable fish eggs ex vivo remains a challenging, but realistic objective, for the conservation and research community. Arguably a less technically ambitious aim in biological terms is to construct an ex vivo microenvironment that favours development of FGSCs into mature oocyte that can match the taste and mouthfeel of caviar. A proprietary, 3D-printed, ex vivo fish ovary analogue also represents a valuable platform technology in the fields of aquaculture and conservation of biodiversity. 

The Acipenseridae is an ancient species that faces internal and external threats including the loss of species genetic integrity through frequent interspecific hybridization, habitat degradation, and overfishing for their roe processed into caviar. Dramatic decrease in sturgeon populations attracted attention of the International Union for Conservation of Nature (IUCN) that categorized them as the most critically endangered, more than any other group of species. All 27 sturgeon species are on the IUCN Red List of threaten species with 17 categorized as critically endangered and four considered to be extinct.
Fig.1 Young Sturgeon whilst our mucosal DNA sampling January 2021


We obtained (cruelty free)  cells from 100 Sturgeon females, partially with a mucosa swab,  removed  partially debris, cultured harvested cells and will co-culture with FGSCs to generate immortalized ovarian cell lines. A design of experiments (DOE) approach will then be applied to identify conditions and cultivations durations that favour differentiation of cultured cells into mature eggs.


 
Image 2. Histology of a H. huso (Beluga Sturgeon) early female gonad. Kuhl et al. 2020

References

1. McMillan DB (2007) Ovarian Follicles. Fish Histology: Female Reproductive systems. Dordrecht, The Netherlands: Springer. 67–208.

2. Draper BW, McCallum CM, Moens CB (2007) nanos1 is required to maintain oocyte production in adult zebrafish. Dev Biol 305: 589–598.

3. Nakamura S, Kobayashi K, Nishimura T, Higashijima S, Tanaka M (2010) Identification of germline stem cells in the ovary of the teleost medaka. Science 328: 1561–1563.

4. Yoshizaki G, Ichikawa M, Hayashi M, Iwasaki Y, Miwa M, et al. (2010) Sexual plasticity of ovarian germ cells in rainbow trout. Development 137: 1227–1230.

5. Wong TT, Saito T, Crodian J, Collodi P (2011) Zebrafish germline chimeras produced by transplantation of ovarian germ cells into sterile host larvae. Biol Reprod 84: 1190–1197.

6. Wong T-T, Tesfamichael A, Collodi P (2013) Production of Zebrafish Offspring from Cultured Female Germline Stem Cells. PLoS ONE 8(5): e62660. doi:10.1371/journal.pone.0062660

7. Iwasaki-Takahashi, Y., Shikina, S., Watanabe, M. et al. Production of functional eggs and sperm from in vitro-expanded type A spermatogonia in rainbow trout. Commun Biol 3, 308 (2020). https://doi.org/10.1038/s42003-020-1025-y

8. Konstantinidis, S., Kong, S. and Titchener‐Hooker, N. (2013), Identifying analytics for high throughput bioprocess development studies. Biotechnol. Bioeng., 110: 1924-1935. doi:10.1002/bit.24850