Introduction

Faced with the need to develop biotechnologies that act in the conservation and reconstitution of endangered fish species, as well as assisting production practices, several methodologies have been developed. These include the generation of germline chimeras that compromise the transplantation of different cell types, such as PGCs embryonic (Saito et al., 2008; Yasui et al., 2011), spermatogonial (Franěk et al., 2019) and oogonial stem cells (Pšenička et al., 2015) from adults. The heterologous propagation of gametes via germline chimeras in fish can be used as a tool for the propagation of species in which reproduction is more critical, such as endangered species with high commercial value or with a long period of gonadal maturation (Robles et al., 2017; Siqueira-Silva et al., 2018). Studies in domestic species often point to the need for more advanced studies in more critical species, such as endangered fish.

The use of primordial germ cells (PGCs) in genebanks and species reconstitution has become a very promising approach (Goto and Saito, 2019; Siqueira-Silva et al., 2018). This is because they are the only precursor cells of the entire germ line and subsequent gamete formation, and they preserve maternal constituents such as germplasm and mitochondrial DNA (Yamaha et al., 2007; Yoshizaki et al., 2003). Although PGCs have favorable characteristics for their use, transplantation involving this cell type covers more complex paths, since it requires previous knowledge of donor and receptor reproduction and also embryo development (Arashiro et al., 2018; Silva et al., 2017) for the successful application of germline chimera production technology (Kawakami et al., 2010; Saito et al., 2011; Yasui et al., 2011). This includes the characterization of the origin, specification, and migration routes of PGCs in different species (Coelho et al., 2021, 2019; Linhartova et al., 2014; Saito et al., 2006).

PGCs can be transplanted into a sterile host organism, such as triploids generated by chromosome manipulation (Adamov et al., 2016; Arai and Fujimoto, 2013; Nascimento et al., 2017). The germline chimera will then produce gametes from the donors species (xenogenesis) (Saito et al., 2008; Yamaha et al., 2003; Yasui et al., 2011).

The yellowtail tetra, Astyanax altiparanae, is known as “lambari” and “tambiú”. The species is distributed in rivers and streams of the upper Paraná River basin of Brazil; it can reach 5-20 cm in length and weigh up to 60 g as an adult (Lobón-Cerviá and Bennemann, 2000; Porto-Foresti et al., 2005). As an omnivorous species, it easily accepts commercial pellets for consumption and adapts easily in captivity (Hayashi et al., 2004). Gonadal maturation is precocious; within four months, this species reaches the adult stage, starting reproduction. It is a species with high fecundity and easy larviculture (Porto-Foresti et al., 2005; Yasui et al., 2020).

Astyanax altiparanae presents simpler in vitro fertilization procedures, which makes it a good model species for development of biotechnologies. Recently, research efforts have been carried out on basic techniques for artificial reproduction of A. altiparanae (Yasui et al., 2015), in addition to studies of fertilization events, sperm morphology and motility, and embryonic development (Pereira-Santos et al., 2016). With this set of information, it was possible to establish a chromosomal manipulation protocol (Adamov et al., 2016; Piva et al., 2018), in order to ensure the sterility of recipients, and this has been used as an important factor for the success of PGC transplantation. Based on these previous steps, the yellowtail tetra can be considered an important experimental model for the development of biotechniques involving PGC transplantation to produce germinative chimeras. Other Characiformes species may be produced from the procedures established in the yellowtail tetra.

Results

For transplantation, regardless of the medium in which the PGCs were cultivated, post-transplant survival showed significant differences during embryonic development and in the percentages of normal and abnormal larvae, compared with the control groups. Only the condition where transplants had triploid recipient associated with cells cultivated in saline solution was there no significant differences between groups (Tables 1, 2, 3, and 4).

A total of 86 transplants were performed by using PGCs cultivated in saline solution into triploid recipients, where 36 transplanted embryos subsequently hatched (47.5 ± 4.5%) and of which 20 (52.4 ± 11.0%) were normal (Table 1 and Fig. 1). For the 125 transplants that were performed with triploid hybrid recipient, 76 (58.5 ± 4.5%) hatched, and 56 (73.6 ± 6.3%) were normal. (Table 2 and Fig. 2).

Fig. 1. Triploid embryos transplanted with GFP positive primordial germ cells (PGCs) from A. altiparanae, cultivated in saline.

(A,B) Embryos with 20 somites. (C,D) Embryo in the hatching phase. (E) Detail of the highlighted region in (D). (PGCs, arrowhead). (B,D) Images captured under fluorescence of (A,C), respectively.

Figure

Fig. 1. Triploid embryos transplanted with GFP positive primordial germ cells (PGCs) from A. altiparanae, cultivated in saline.

(A,B) Embryos with 20 somites. (C,D) Embryo in the hatching phase. (E) Detail of the highlighted region in (D). (PGCs, arrowhead). (B,D) Images captured under fluorescence of (A,C), respectively.

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Fig. 2. Triploid hybrid embryos transplanted with A. altiparanae GFP positive primordial germ cells (PGCs), cultivated in saline.

(A,B) Embryos in gastrula stage with 30% epiboly. (C,D) Embryos in gastrula stage with 90% epiboly. (E,F) Embryos with 16 somites. (G,H) Larvae with 1-day post-hatch. (PGCs, arrowhead). (B,D,F and H) Images captured under fluorescence of (A,C,E, and G), respectively.

Figure

Fig. 2. Triploid hybrid embryos transplanted with A. altiparanae GFP positive primordial germ cells (PGCs), cultivated in saline.

(A,B) Embryos in gastrula stage with 30% epiboly. (C,D) Embryos in gastrula stage with 90% epiboly. (E,F) Embryos with 16 somites. (G,H) Larvae with 1-day post-hatch. (PGCs, arrowhead). (B,D,F and H) Images captured under fluorescence of (A,C,E, and G), respectively.

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In the experiment using PGCs cultivated in supplemented medium and triploid receptors, 94 transplants were performed, where 22 embryos hatched (34.4 ± 10.1%), and 6 presented normal morphology (20.8 ± 12.3%) (Table 3 and Fig. 3). At the same time, 62 transplants were performed for PGCs cultivated in supplemented culture medium and transplanted into triploid hybrid recipients, where 31 embryos hatched (53.8 ± 10.6%) with 12 being normal (41.8 ± 13.8%) (Table 4 and Fig. 4).

Fig. 3. Triploid embryos transplanted with PGCs from A. altiparanae, grown in supplemented cell culture medium.

(A,B) Embryos in the final stage of blastula. (C,D) Embryos in gastrula stage with 90% epiboly. (E,F) Embryos with 12 somites. (G,H) Embryos at hatch. (I) Detail of the highlighted region in (H). (PGCs, arrowhead). (B,D,F, and H) are images captured under fluorescence of (A,C,E and G), respectively.

Figure

Fig. 3. Triploid embryos transplanted with PGCs from A. altiparanae, grown in supplemented cell culture medium.

(A,B) Embryos in the final stage of blastula. (C,D) Embryos in gastrula stage with 90% epiboly. (E,F) Embryos with 12 somites. (G,H) Embryos at hatch. (I) Detail of the highlighted region in (H). (PGCs, arrowhead). (B,D,F, and H) are images captured under fluorescence of (A,C,E and G), respectively.

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Fig. 4. Triploid hybrid embryos transplanted with PGCs from A. altiparanae, grown in supplemented cell culture medium.

(A,B) Embryos in early gastrula stage. (C,D) Embryos with 8 somites. (E,F) Embryos at hatch. (G) Detail of the highlighted region in (F). (H,I) – Larva with 1-day post-hatch. (J) Detail of the highlighted region in (I). (PGCs, arrowhead). (B,D,F, and I) are images captured under fluorescence of (A,C,E, and I), respectively.

Figure

Fig. 4. Triploid hybrid embryos transplanted with PGCs from A. altiparanae, grown in supplemented cell culture medium.

(A,B) Embryos in early gastrula stage. (C,D) Embryos with 8 somites. (E,F) Embryos at hatch. (G) Detail of the highlighted region in (F). (H,I) – Larva with 1-day post-hatch. (J) Detail of the highlighted region in (I). (PGCs, arrowhead). (B,D,F, and I) are images captured under fluorescence of (A,C,E, and I), respectively.

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In experiments carried out using the transplantation of PGCs cultivated in saline medium, it was observed that the PGCs transplanted in all embryos presented ectopic migration, regardless of the type of receptor, and GFP-positive cells were visualized in the cephalic region, yolk, and musculature (Figs. 2 and 3). Only for triploid hybrid recipients, the migration of PGCs from the donor was verified, being very close to the region of formation of the gonadal ridge (Fig. 3). Transplants were performed using 1 to 3 donor PGCs, and cell division was observed in 2 (5.5%) embryos of triploid recipient and in 1 (1.3%) hybrid triploid embryos. For these embryos in the hatching phase, between 4 to 6 GFP positive cells were observed. The presumptive chimeric embryo did not observe cell division.

In transplantation experiments of PGCs cultivated in supplemented medium, directed migration to the region of gonadal ridge formation was observed in 1 (4.5%) triploid transplanted embryo, and this embryo presented abnormal hatching (Table 3 and Fig. 3). However, when transplantation was performed in triploid hybrid receptors, GFP-positive cells with migration directed to the region of the gonadal ridge were observed in 6 embryos (19.3%), where 2 of them presented normal hatching and 4 abnormal hatching (Table 4 and Fig. 4).

Cellular proliferation of transplanted cells was observed in 3 (13.6%) transplanted triploid embryos and in 5 (16.1%) triploid hybrids, regardless of the final migration site of the PGCs. At hatching, 4 to 6 GFP positive cells were observed, except for two transplanted triploid hybrids. PGCs in the region of the gonadal ridge that hatched with 9 and 12 cells presented embryos with a drastic drop in GFP expression 1-day post-hatching (Fig. 4).

Gene expression

The relative gene expression results demonstrate differences in the expression levels of the genes evaluated between different samples (Fig. 5). An increase in the expression level of genes cyclin B1, dnd1, dazl, and ddx4 was observed in PGCs cultivated in supplemented medium, while the expression levels of cxcr4b and nanos genes showed a decrease (Fig. 5). The dazl and ddx4 genes did not show significant differences.

Fig. 5. Relative expression of cxcr4b, cyclin B1, dazl, ddx4, dnd1, and nanos genes in samples of primordial germ cells (PGCs) of Astyanax altiparanae under different culture conditions.

PGCs from saline medium were used as expression calibrators.

Figure

Fig. 5. Relative expression of cxcr4b, cyclin B1, dazl, ddx4, dnd1, and nanos genes in samples of primordial germ cells (PGCs) of Astyanax altiparanae under different culture conditions.

PGCs from saline medium were used as expression calibrators.

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The greatest increase in gene expression level was observed for the dnd1 gene, where there was about 68% higher in PGC in supplemented medium than in saline. Cyclin B1 had an increase of about 33% greater in PGCs for the supplemented medium compared to PGCs from the saline medium. Although no significant difference was found, the dazl gene showed, on average, about 24% increase in expression in the supplemented medium, while ddx4 showed about 12% increase in expression in relation to the saline medium. These results indicate a trend in the increase of gene expression in the PGCs of the genes evaluated in the supplemented medium in relation to the saline medium (Fig. 5).

Differing from the tendency to increase the level of gene expression in the genes evaluated for the supplemented medium, the cxcr4b and nanos genes showed a decrease in the expression level in PGCs from the supplemented medium compared with PCGs from the saline medium. For cxcr4b, a decrease of around 23% was observed in the level of gene expression in the supplemented medium in relation to the saline medium; and for nanos, the fall was drastic, approximately 92%. (Fig. 5).

Expression of target genes in tissues

In adult A. altiparanae, cxcr4b, cyclin B1, and nanos (Fig. 6 C,D,H) were expressed in a wide range of tissues, including brain, kidney, liver, and gonads. cxcr4b and cyclin B1 expression was observed in muscle only (Fig. 6 C,D). Dazl and ddx4 are specific for male and female gonads (Fig. 6 E,F). Dnd1 showed weak expression in both male and female gonads (Fig. 6G).

Fig. 6. 2% agarose gel in semi-quantitative PCR analysis of gene expression using cDNA from tissues of Astyanax altiparanae.

(A) b-actin; (B) cxcr4b; (C) cyclin B1; (D) dazl; (E) ddx4; (F) dnd1; (G) nanos, (1) muscle; (2) brain; (3) kidney; (4) liver; (5) testicle; (6) ovary.

Figure

Fig. 6. 2% agarose gel in semi-quantitative PCR analysis of gene expression using cDNA from tissues of Astyanax altiparanae.

(A) b-actin; (B) cxcr4b; (C) cyclin B1; (D) dazl; (E) ddx4; (F) dnd1; (G) nanos, (1) muscle; (2) brain; (3) kidney; (4) liver; (5) testicle; (6) ovary.

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Discussion

The transplanted PGCs presenting effective migration showed similar behavior to the PGCs of the species A. altiparanae (unpublished data). In the screening studies of PGCs in A. altiparanae embryos at the 6 to 19 somites stage, the stage used for transplantation, 3 to 12 PGCs were visualized placed in groups or spread over the yolk extension. While in the hatching phase, between 3 to 13 PGCs were found positioned mostly in the yolk's extension, where some cases were in the upper part of the yolk. These data are similar to those found in transplant receptors.

Successful transplantation is well known to be related with the correct migration of donor cells to the region of gonadal formation in the recipients. In addition, there is also an adequate time range for transplantation, due to their ability to migrate, which can decrease over time (Takeuchi et al., 2003; Yamaha et al., 2007).

The efficiency of germ cell transplantation is also related to previous sterilization of the recipient. The infertility of the recipient species contributes to the decrease in competition between endogenous and transplanted germ cells. Previous sterilization work was carried out with the studied species, using hybridization and chromosomal manipulation techniques. Nascimento et al., 2017 shows that triploid females are sterile, on the other hand, triploid males were not sterile. In later studies, using triploid hybrids of crossing A. altiparanae x A. fasciatus generated sterile offspring, with progenies that did not present a germ lineage in the gonads (Piva et al., 2018). This is in agreement with the results observed in our studies using triploid receivers and triploid hybrids.

In species frequently used in micromanipulation (experimental), such as Danio rerio, Carassius auratus, Misgurnus anguillicaudatus, Danio albolineatu, and trout, the embryos and isolated cells develop in a saline media, such as Ringer's solution. This solution is used for chorion removal, embryonic culture, and PGC culture; it is usually added with penicillin and streptomycin (Higaki et al., 2010; Kawakami et al., 2010; Saito et al., 2008) and fetal bovine serum or albumin (Fujimoto et al., 2006; Naya et al., 2020; Saito et al., 2010; Yoshizaki et al., 2005). However, it has been verified that in neotropical species, the best conditions for embryonic culture vary highly from one species to another, which often makes it difficult to choose the best solution when carrying out the PGCs cultivation (Coelho et al., 2019, 2021; unpublished data). In this study, it was verified that the PGCs presented directed migration when using adequate cell culture medium, enriched with amino acids, vitamins, glutamine, pyruvate, and fetal bovine serum, in addition to being cultivated in a CO₂ incubator. This report is important, as it indicates that the success in transplanting and achievement germline chimeras can be influenced by the culture medium.

The conditions and time of culture of these cells may be related to the viability and expression of genes that are important for the maintenance of PGCs and, consequently, in the migration rates. Several genes are related to differentiation, maintenance, and migration of PGCs, and low levels of expression can lead to loss of identity, germ cell apoptosis, or ectopic migration (Barton et al., 2016; Doitsidou et al., 2002; Gross-Thebing et al., 2017; Knaut et al., 2003)

Gene expression in isolated PGCs showed lower level of expression in cxcr4b in PGCs grown in supplemented culture medium. The reduced level of cxcr4b can maintain the mobile behavior of cells; however, they can reach ectopic sites in the embryo due to undirected migration (Doitsidou et al., 2002; Raz, 2003; Raz and Reichman-Fried, 2006), which may also be related to the high number of ectopic migrations observed in the transplants performed in this work. However, even with the reduction of expression in PGCs cultivated in supplemented medium, the levels of cxcr4b in these cells still allowed migration to occur. This fact may indicate that other factors may be involved in migration and must have been optimized with the cultivation in the supplemented medium, such as the increased expression of dnd1, ddx4, and dazl, genes also responsible for the migration of PGCs. This hypothesis can be reinforced by the fact that even with higher levels of cxcr4b in cells cultivated in saline solution and lower levels of dnd1, ddx4, and dazl, the migrations observed were all ectopic.

It was reported that the deficiency of cxcr4b proteins and its ligand sdf-1 and/or dnd1 affect the migration of PGCs, which are located in ectopic positions or activate the apoptosis process (Barton et al., 2016; Gross-Thebing et al., 2017; Herpin et al., 2008; Sánchez-Sánchez et al., 2010; Weidinger et al., 2003). According Gross-Thebing et al., 2017, PGCs depleted of cxcr4b, but still expressing dnd1, can be located ectopically and maintain their morphology. Depletion of dnd1 can cause changes in the morphology of PGCs, leading to a gradual decrease in the expression of germ cell markers, such as vasa (ddx4) and nanos3, or causing apoptosis.

Studies involving mutations of the ddx4 gene in Danio rerio resulted in offspring consisting only of sterile males (Hartung et al., 2014). In Oryzias latipes, the ddx4 gene knockout did not affect PGC proliferation, survival, and motility; however, when used in transplantation, they did not show adequate migration. This indicates that ddx4 inhibition would result in the loss of PGC response to migration signaling towards the gonad (Li et al., 2009). However, the dazl gene knockout significantly reduced the number of PGCs and, in some cases, completely prevented the formation of PGCs (Li et al., 2016).

The nanos gene, also essential for the development of PGCs, at reduced levels can result in premature mitosis of PGCs, ectopic migration, and loss of their identity (Hashimoto et al., 2010; Köprunner et al., 2001). In this study, the expression level of nanos was drastically reduced in PGCs cultivated in supplemented medium when compared to PGCs from saline solution. It has been reported in Drosophila that Nanos and Pumilio proteins are involved in binding the 3'UTR region of cyclin B1 mRNA and repressing its translation. Cyclin B1 is an active protein in the mitotic phase, and its regulation is important so that PGCs do not proliferate prior to migration (Asaoka-taguchi et al., 1999; Hashimoto et al., 2010; Kadyrova et al., 2007). The gene expression results obtained from PGCs indicated a low level of nanos mRNA and a higher level of cyclin B1 in PGCs cultivated in supplemented medium compared with PGCs grown in saline solution. As described in the transplantation studies present in the literature (Saito et al., 2010, 2008), migration of the transplanted cells with little or no cell division was observed. PGCs from saline, which showed higher mRNA levels for nanos and lower for cyclin B1, show lower rates of cell division, ranging from 1.3 to 5.5%. While for the PGCs from the supplemented culture medium that showed a lower level of nanos expression and higher expression of cyclin B1, the cell division found were higher than those of the saline solution, ranging from 13.6 to 16.1%. These cell division and the mRNA levels of nanos and cyclin B1 appear to be related. Despite a decrease in the expression of nanos, an increase in cyclin B1, and an increase in the rate of division of PGCs, the PGCs from the supplemented culture medium were those capable of performing the correct migration to the gonadal ridge. This suggests that this decrease at the mRNA level of nanos still allows for the correct migration of PGCs.

Given the great diversity in reproductive strategies, spawning pattern, gamete size, specific temperature range for embryonic development, and sensitivity for handling in micromanipulation stages, the methodologies developed for the production of germline chimera can vary and require adaptations according with the species employed. Thus, each species may present a different challenge during the application of the transplant technique, which in turn is already invasive.

For the transplantation of PGCs, it is essential to have previous mastery of in vitro reproduction to obtain the labeling and isolation of PGCs, which is difficult in most species at risk of extinction. In addition, PGC transplantation requires developmental standardization between donor and recipient species, in which each species has a specific temperature range for optimal embryo development (Arashiro et al., 2018; Coelho et al., 2019; Pereira-Santos et al., 2016; Silva et al., 2017).

The size and timing of embryonic development of the species may limit the applicability of some transplantation techniques. The present model species Astyanax altiparanae and other species such as Danio rerio, Oryzias latipes, and Misgurnus anguillicaudatus present small eggs and larvae; this makes it difficult to use some methods used in trout (Takeuchi et al., 2003). The embryonic development velocity of neotropical species can vary from 11 to 29 h (Arashiro et al., 2018; Coelho et al., 2019; Pereira-Santos et al., 2016; Silva et al., 2017), differing from the species most commonly used in transplant works; this can take from 72 h to days for hatching (Fujimoto et al., 2006; Iwamatsu, 2004; Kimmel et al., 1995). In salmonids, for example, where cell transplantation techniques are quite advanced (Kobayashi et al., 2007; Nagasawa et al., 2013; Takeuchi et al., 2004), there are intrinsic biological facilities that optimize transplantation, such as slow embryo development (32-83 days, which increases transplant period), larger embryo size (facilitating visualization and micromanipulation), acclimatization in a micromanipulation system (Petri dish with static water), in addition to scientific support, which, as a group established in the basic sciences and in aquaculture, has important information regarding genetics (gene sequences and their expressions), reproduction, physiology, among other important areas for PGC transplantation. In the case of native species, as is the case of the present work, although great progress has been made in recent works (Arashiro et al., 2018; Bertolini et al., 2017; Coelho et al., 2021, 2019; Nascimento et al., 2020, 2017; Pereira-Santos et al., 2016; Piva et al., 2018; Yasui et al., 2020, 2015), basic information to support these advanced chimerism techniques is still lacking.

This is the first study to report PGC transplantation in order to obtain a germline chimera in a neotropical species. Gene expression in isolated PGCs is a good indicator that the supplementation of the culture medium provided better conditions for PGCs, in addition to confirming the identity of isolated cells for transplantation. Knowledge about the behavior of model native species in micromanipulation systems helps in the use of the technique for the conservation of endangered species. However, there is still a need for improvements in the stages of transplantation and knowledge about cell behavior in culture systems, time for transplantation, recipient conditions, the intercellular interaction between germ cells (donated) and somatic cells (receiver), and the performance of related gene maintenance and characterization of primordial germ cells in native species.

Material and Methods

The experimental procedures and analyses were carried out at the Laboratory of Fish Biotechnology, National Center for Research and Conservation of Continental Fish, Chico Mendes Institute of Biodiversity Conservation (ICMBio/CEPTA) - Pirassununga-SP, in line with the Ethics Committee for the Use of Laboratory Animals from the Faculty of Veterinary Medicine and Animal Science – University of São Paulo (CEUA/FMVZ n° 4534110820).

Acknowledgements

We are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) and Sao Paulo Research Foundation (FAPESP) (2010/17429-1 and 2011/11664-1) for the financial support of this research. We also acknowledge CEPTA/ICMBio for generously providing the facilities and experimental fish and Michael Stablein for the review of this manuscript.

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