Initiative on Avian Primordial Germ Cell Cryobanking in Thailand.

Background: Biobanking the reproductive tissues or cells of animals preserves the genetic and reproductive ability of the species in long-term storage and promotes sharing of reproductive materials. In avian species, the primordial germ cell (PGC) is one of the most promising reproductive cells to be preserved in biobanks, due to self-renewal properties and direct access to the germ line mediated by PGC transfer. Methods: To conserve the genetic resource of local chicken breeds that are of conservation importance, we systematically isolated two types of pregonadal PGCs from chicken embryos-circulating and tissue PGCs. PGCs of individual embryos were separately isolated, cultured, and cryopreserved. Characteristics of cultured PGCs are described and evaluated. Results: The efficiency of PGC isolation from individual embryos was 98.9% (660/667). In most cases, both matching circulating and tissue PGC lines were isolated from the same embryo (68.2%, 450/660), whereas the remaining lines were from a single source, being either tissue (30.6%, 202/660) or circulating (1.2%, 8/660). Efficient PGC isolation and proliferation can be expected in cultures of circulating PGCs (68.7% and 64.3%, respectively) and tissue PGCs (97.8% and 80.7%, respectively). Following cryopreservation, recovered cells sustained PGC identities including expression of chicken vasa homolog and deleted in azoospermia-like proteins and migration ability to recipient embryonic gonads. Culture conditions equally supported proliferation of circulating and tissue PGCs from both sexes. Combining tissue PGC culture in the regimen prevented 30.3% loss of PGC cultures in the case where circulating PGC culture was ineffective. Cultured circulating and tissue PGCs were similar in morphology, but optimal culture characteristics were different. Conclusion: We applied the approach of PGC isolation from blood and tissue origins on a wide scale and demonstrated its efficiency for biobanking chicken PGCs. The workflow can be operated effectively almost year-round in a tropical climate. It was also described in ample and practical details, which are suitable for adoption or optimization in other conditions.

Primordial germ cells isolated from individual embryos of red junglefowl and indigenous pheasants of Thailand.

Interspecific germline chimerism mediated by transplantation of primordial germ cells (PGCs) of wild species to domestic hosts promises the conservation of wild birds. Cryopreservation of avian eggs and embryos is impracticable, and currently only frozen PGCs enable conservation of both the male and female descendants. Purebred offspring have been obtained from germline chimeras of wild avian species, proving the feasibility of such technology. In vitro propagation has been optimized for avian PGCs of domestic species; however, evidence is rather limited for successful isolation as well as long-term culture from a single embryo of wild species. With accelerating biodiversity loss, we have committed to preserving current genetic resources by freezing PGCs isolated from individual embryos in addition to their genetic material. We have devised a reliable protocol for the isolation and proliferation of PGCs from wild fowls in the family Phasianidae that are conserved in captive breeding (red junglefowl, bar-tailed pheasant, kalij pheasant, Siamese fireback pheasant, and silver pheasant). We obtained individual isolates of cultured circulating PGCs (49.7%, 79/155) as well as tissue PGCs (92.9%, 144/155). The specific co-culture conditions of autologous embryonic cells, without additional growth factors, facilitated the proliferation of so-called tissue PGCs (the remaining PGCs in embryonic tissue following blood aspiration). Only circulating PGCs left in blood vessels and of PGCs migrating to developing gonads have been previously reported. However, the present study is the first to report on the harvest of ectopic PGCs. The defined conditions sustained continuous proliferation of tissue PGCs for at least six months and maintained PGC identity following cryopreservation. Cultured tissue PGCs of these wild species were extensively characterized for their expression of the germ cell-specific proteins, chicken vasa homolog (CVH) and deleted in azoospermia-like (DAZL), as well as the ability to colonize chicken embryonic gonads. The novel protocol is practical for generating enough PGCs for cryopreservation, transplantation, and additionally, it enables isolation of PGCs from both blood circulation and embryonic tissue simultaneously. For conservation purposes, this approach is potentially applicable more widely to other non-domestic birds than those in the family Phasianidae that were investigated in the present study.

Comparative analysis of single-cell transcriptomics in human and Zebrafish oocytes.

BACKGROUND: Zebrafish is a popular model organism, which is widely used in developmental biology research. Despite its general use, the direct comparison of the zebrafish and human oocyte transcriptomes has not been well studied. It is significant to see if the similarity observed between the two organisms at the gene sequence level is also observed at the expression level in key cell types such as the oocyte. RESULTS: We performed single-cell RNA-seq of the zebrafish oocyte and compared it with two studies that have performed single-cell RNA-seq of the human oocyte. We carried out a comparative analysis of genes expressed in the oocyte and genes highly expressed in the oocyte across the three studies. Overall, we found high consistency between the human studies and high concordance in expression for the orthologous genes in the two organisms. According to the Ensembl database, about 60% of the human protein coding genes are orthologous to the zebrafish genes. Our results showed that a higher percentage of the genes that are highly expressed in both organisms show orthology compared to the lower expressed genes. Systems biology analysis of the genes highly expressed in the three studies showed significant overlap of the enriched pathways and GO terms. Moreover, orthologous genes that are commonly overexpressed in both organisms were involved in biological mechanisms that are functionally essential to the oocyte. CONCLUSIONS: Orthologous genes are concurrently highly expressed in the oocytes of the two organisms and these genes belong to similar functional categories. Our results provide evidence that zebrafish could serve as a valid model organism to study the oocyte with direct implications in human.

A 13-plex of tetra- and penta-STRs to identify zebrafish.

The zebrafish species Danio rerio has become one of the major vertebrate model organisms used in biomedical research. However, there are aspects of the model that need to be improved. One of these is the ability to identify individual fish and fish lines by DNA profiling. Although many dinucleotide short tandem repeat (diSTR) markers are available for this and similar purposes, they have certain disadvantages such as an excessive polymerase slippage ("stutter") that causes difficulties in automated genotyping and cross-laboratory comparisons. Here we report on the development of a 13-plex of tetranucleotide and pentanucleotide STRs (tetraSTRs and pentaSTRs, respectively) that have low stutter. The system uses an inexpensive universal primer labelling system, which can easily be converted to a direct labeling system if desired. This 13-plex was examined in three zebrafish lines (NHGRI-1, kca33Tg, and kca66Tg, originally obtained from ZIRC). The average observed heterozygosity (Ho) and expected heterozygosity (He) in these highly inbred lines were 0.291 and 0.359, respectively, which is very similar to what has been found with diSTRs. The probability of identity (PI) for all fish tested was 2.1 × 10-5 and the PI for siblings (PIsib) was 6.4 × 10-3, as calculated by the Genalex package. Ninety percent of the fish tested were correctly identified with their respective strains. It is also demonstrated that this panel can be used to confirm doubled-haploid cell lines. This multiplex should find multiple uses for improving the accuracy and reproducibility of studies using the zebrafish model.

Use of soluble sperm extract to improve cloning efficiency in zebrafish.

During somatic cell nuclear transfer (SCNT), egg activation is required to initiate embryonic development. In zebrafish cloning, the reconstructed egg is activated by exposing it to hypotonic water. Egg activation using water-only is not capable of activating the same intracellular calcium release as fertilization which is required for proper embryonic development. Here we test whether the use of soluble sperm extract (SSE) can properly modulate the activation of reconstructed eggs during SCNT. We microinjected SSE from genomic-inactivated zebrafish sperm into unfertilized eggs and reconstructed eggs right after somatic cell nuclear transfer. We also evaluated the most effective approach for SSE microinjection. Microinjection of SSE (with 0.68 mg/ml of protein concentration) into non-activated eggs through the micropyle induced parthenogenetic development beyond the blastula stage, whereas all water-only activated eggs failed to enter the cleavage period. Microinjection of SSE at 1 mg/ml of protein concentration into non-activated reconstructed egg improved the developmental rate of cloned embryos in comparison to non-injected control clones. The cumulative survival time of cloned embryos injected with SSE was significantly longer than reconstructed eggs activated following sham injection (P<0.01). No significant difference was found among controls (P=0.32). SSE benefits both parthenogenesis and the survival cloned embryos which have never been reported in zebrafish. Further work is necessary to define the functional component(s) of SSE as well as the physiological pathway, to understand its principle of action and advance the utilization of SSE in cloning.

Optimized Protocol of Zebrafish Somatic Cell Nuclear Transfer (SCNT).

Zebrafish (Danio rerio) is an established animal model to study developmental biology as well as a wide array of human diseases. Here we describe a protocol for somatic cell nuclear transfer (SCNT). This protocol can be used to introduce genetic modifications in zebrafish and for the study of cell plasticity.