Cryopreservation and transplantation of spermatogonia stem cells in piracanjuba Brycon orbignyanus (Characiformes: Characidae), an endangered fish species

doi:10.21203/rs.3.rs-4266695/v1

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Cryopreservation and transplantation of spermatogonia stem cells in piracanjuba Brycon orbignyanus (Characiformes: Characidae), an endangered fish species

https://doi.org/10.21203/rs.3.rs-4266695/v1

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Piracanjuba (Brycon orbignyanus) is an endangered fish species from the Neotropical region. The establishment of a cryobank using spermatogonial stem cells (SSCs) and subsequent production of a germline chimera is thus a promising strategy for such species. In the present work, procedures for the isolation and cryopreservation of piracanjuba SSCs and subsequent transplantation into sterile recipients were established. The piracanjuba SSCs were obtained by Percoll density gradient centrifugation and differential plating. SSC fractions were evaluated by relative ddx4 expression, alkaline phosphatase activity, and light microscopy. SSC cryopreservation was performed using five cryoprotectants at three different concentrations. The mix of the cells from the 20% and 30% Percoll density gradients showed 58.35 ± 0.03% purity of SSCs. The purity of SSCs increased to 66.00 ± 0.01% after differential plating. The relative ddx4 expression was 3.5 times higher in cells from the Percoll density gradient centrifugation than in the gonad and cells after differential plating. Propanediol (1M) was the most effective cryoprotector evaluated (P = 1.000), showing 90.75 ± 1.85% cell viability. Freshly isolated and cryopreserved cells from the Percoll density gradient centrifugation were transplanted into a sterile male adult triploid hybrid with germ cell-less gonads. SSCs were observed in the germinal epithelium of the testes of recipients 20 days after transplantation. The results are promising for obtaining functional germline chimeras in Neotropical fish. Consequently, the procedures established here can be applied in future actions for the conservation and reconstitution of the piracanjuba in case of extinction.

conservation

spermatogonia

transplantation

gene bank

germline chimera

Brycon orbignyanus (Characidae, Bryconinae), popularly known as piracanjuba, is a migratory fish species distributed in the basin formed by the Uruguay and Paraná rivers (Zaniboni Filho et al. 2006). Although this species has a consumer market (BORBA et al. 2006) and relevance for fisheries and aquaculture, the wild populations have declined, mainly affected by riparian deforestation and the fragmentation of rivers caused by hydroelectric plants (Agostinho, Â. A., Thomaz, S. M., & Gomes 2005). Such events contributed to its current status on the IUCN Red List classified as EN A2c (EN – endangered; A2c - ≥50% reduction in observed, estimated, inferred, or suspected population over the last ten years or three generations, where there was a decline in area of occupancy, extent of observation, and habitat quality or both) (ICMBio 2018). Strategies have been implemented to maintain the natural populations of piracanjuba, such as captive breeding and restocking programs (Rodriguez-Rodriguez et al. 2010). However, restocking requires one to maintain the genetic variability and prevent a deviation in the sex ratio. Captive-bred Piracanjuba produces mostly males, hindering in situ maintenance and reducing restocking effectiveness (Lopera-barrero 2007; Rodriguez-Rodriguez et al. 2010; Zardo et al. 2021).

Long-term gene banking using cryopreserved samples is promising in the piracanjuba, especially using SSCs because a high percentage of males are available. In vitro cell preservation allows for subsequent transplantation use in the reconstitution of species by generating germline chimeras (xenogenesis) (Franěk et al. 2019; Marinović et al. 2019). This approach could be considered a solid strategy to conserve and reconstitute piracanjuba, preserving important genetic components (Cinalli et al. 2008).

Despite using sperm in cryopreservation for its ease of management and cryopreservation (Viveiros et al. 2012); reconstitution via androgenesis is difficult to achieve and presents problems regarding survival, genetic variability, and sex ratio (Yasui et al. 2010). Cryopreservation protocols for fish embryos and oocytes have been unviable due to their large size, high yolk content, and high sensitivity to cooling (Robles et al. 2009; Labbé et al. 2013).

Primordial germ cells, SSCs, and oogonial stem cells were successfully cryopreserved, transplanted, and used to produce a germline chimera for reconstitution. New individuals can be generated through cell transplantation using a sterile recipient to conserve endangered species (Hartung et al. 2014). Germ cell genetic banks were established in some fish species, such as Oncorhynchus mykiss (Okutsu et al. 2006), Danio rerio (Marinović et al. 2019), Tinca tinca (Linhartová et al. 2014; Marinović et al. 2017), and Oryzias latipes (Seki et al. 2017). Reconstitution through germ cell transplantation can be performed for blastula-stage embryos (Takeuchi et al. 2001), newly hatched larvae (Takeuchi et al. 2003), and adult animals (Lacerda et al. 2010). Regardless of the stage at which the germ cell transplant will be performed, recipients must be sterile and free of endogenous germ cells, which allows the proliferation and colonization of exogenous cells in the recipient’s gonad (Brinster et al. 2003; Lacerda, S.R. Batlouni, S.B.G. Silva, C.S.P. Homem 2006; Piva et al. 2018; Xu et al. 2019). Sterile recipients can be produced using techniques such as chromosome manipulation to generate triploids (Adamov et al. 2017; Do Nascimento et al. 2021), species hybridization (Piva et al. 2018; Xu et al. 2019), combination of both these methods (Piva et al. 2018; Xu et al. 2020), injection of morpholinos into embryos (dead end gene) (Franěk et al. 2022; Fujihara et al. 2022), as well as through high temperatures (Pandit et al. 2015; Nozu and Nakamura 2020) or busulfan chemical treatment associated with high temperatures (Lacerda et al. 2008; Majhi et al. 2017). Based on previous studies, cryopreservation and transplantation of germline cells are essential tools for the conservation of endangered species.

This study aimed to establish procedures for isolating and cryopreserving SSCs from piracanjuba (Brycon orbignyanus). Additionally, the effectiveness of transplanting SSCs into the sterile testes of adult triploid hybrid recipients (Astyanax altiparanae female X Astyanax fasciatus male) was evaluated.

2.1 Ethical considerations for the use of animals

Animal collection and experiments were carried out at the National Center for Research and Conservation of Continental Aquatic Biodiversity of the Chico Mendes Institute for Biodiversity Conservation (CEPTA/ICMBio) in Pirassununga – São Paulo, Brazil. The experiments were conducted with the approval of the Animal Ethics Committee from CEPTA. (CEUA/CEPTA #02031.000088/2021-61).

2.2 Histological analysis of the testes of piracanjuba (Brycon orbignyanus)

To determine the appropriate time of SSC obtention from piracanjuba, testes were collected from sexually mature juveniles at 15 months (n = 2) and adults at 8 years old. Testes of juveniles were collected in March 2020 (resting period). For adults, they were collected (n = 2) at the height of the breeding season in December and in June (n = 2) in the winter season. Before test collection, the animal was euthanized using a solution of 200 mg/L eugenol (Biodynamic #10298550063, Ibipora, Brazil). The collected gonads were fixed in Bouin fixative for 24 hours at room temperature and then transferred into 70% ethanol. Subsequently, the material was processed with conventional histology procedures, using a series of dehydration steps in increasing concentrations of ethanol (70%, 80%, 90%, and 100%), bleaching in xylene, and embedding in paraplast® (Sigma # SLBS8607, St. Louis, USA). 5 µm thick histological sections were obtained using a microtome equipped with a steel blade (Leica RM2235, Lincolnshire, USA). Slides were stained with hematoxylin and eosin, analyzed by microscopy (Nikon-Eclipse Ni, Tokyo, Japan), and photographed (Nikon DSRi2, Nikon, Tokyo, Japan). The gonadal development spermatogenesis analyses were evaluated based on previous studies Brown-Peterson et al (2011).

2.3 Isolation of spermatogonial stem cells

One male of piracanjuba (Brycon orbignyanus) collected in June weighing 1.345 kg and a standard length of 44 cm, was used for isolatation of the SSCs. The male was euthanized as mentioned in the previous section. The testis was collected, weighing 0.9858 g, and two small parts were separated: one was fixed in Bouin (Exodus Scientific #FB08835SO, Sumaré, Brazil) for 24 hours for further histological analysis; and the other part was stored at -80°C for gene expression analysis. Most of the testis was used to isolate SSCs through enzymatic dissociation and Percoll density gradient centrifugation (see below).

The testis was transferred into a 90 × 15 mm sterile Petri dish containing Leibovitz cell culture medium (L-15, Gibco #21083027, Grand Island, USA) supplemented with 0.5% fetal bovine serum, 2 mM glutamine (Sigma #G7513, St. Louis, USA), 1 mM pyruvate (Sigma #S8636, St. Louis, USA), 1× MEM vitamin solution (Sigma #M6895 St. Louis, USA), 1× nonessential amino acid MEM solution (Sigma #M7147, St. Louis, USA), and 1× antibiotic and antimycotic solution (Sigma #A5955, St. Louis, USA). In a laminar flow cabinet (FUH12, VECO, Campinas - SP, Brazil), the testis was minced using a scalpel and washed three times with a supplemented Leibovitz (L-15) medium to remove excess blood cells. The minced testis was transferred to 15 mL centrifuge tubes, adding 10 mL of supplemented Leibovitz (L-15) medium for each 1 g of tissue and 2 mg/mL collagenase type I from Clostridium histolyticum (Sigma #C0130, St. Louis, USA). The centrifuge tube was placed in an orbital shaker at 60 r/min (PHOENIX, São Paulo, Brazil) and incubated for 2 hours at room temperature (~ 27°C). Then, 20 µg/mL DNase I (Sigma‒Aldrich #SLBT5559, St. Louis, USA) was added and incubated for an additional hour on a shaker. The cell suspension was filtered through a 50-µm nylon mesh to eliminate debris and transferred to a 15-mL tube. The final volume was adjusted to 10 mL with supplemented Leibovitz (L-15) medium and centrifuged at 300 x g for 5 minutes (Eppendorf 5702, Hamburg, Germany) at 25°C. The cell pellet was resuspended in 10 mL of supplemented Leibovitz (L-15) medium and centrifuged at 300 × g for 5 minutes to remove the excess collagenase and DNase. This step was performed twice.

SSCs were fractionated using the Percoll density gradient centrifugation in concentrations of 10, 20, 30, and 40% Percoll® Plus (Sigma‒Aldrich # SLBH8181 V, St. Louis, USA). The Percoll density gradient centrifugation was performed in 15-mL tubes. The cell suspension was carefully pipetted on the surface of the 10% density gradient Percoll solution and centrifuged at 800 × g for 30 minutes at 25°C. The collected fractions were resuspended in 10 mL of supplemented Leibovitz (L-15) medium and centrifuged at 300 × g for 8 minutes at 25°C to remove the excess Percoll. The cells from the resultant cell pellet were suspended in 1 mL of supplemented Leibovitz (L-15) medium. Cell suspensions were analyzed by microscopy to assess the fractions containing the highest percentage of SSCs.

The fraction from the 20% and 30% Percoll density gradients containing the highest concentrations of SSCs were combined, and 5 mL of D-MEM media culture was added (Dulbecco's Modified Eagle Medium, Gibco #31053028, Grand Island, USA). The cells were centrifuged twice at 300 × g for 5 minutes to remove the fetal bovine serum from the previous solution, and this permitted the labeling of stem cells with alkaline phosphatase, a marker of pluripotent stem cells and SSC (Hong et al. 1996, 2004). The cells were resuspended in 500 µL of D-MEM containing 1 µL of alkaline phosphatase substrate (Life Technologies #A14353, Frederick, USA). Fluorescent cells were verified by fluorescence microscopy (Nikon-Eclipse Ni, Tokyo, Japan), and digital images were obtained (Nikon DSRi2, Nikon, Tokyo, Japan). After confirming the alkaline phosphatase activity of the SSCs, 10 µL of cell suspension was used to quantify the cell concentration using a hemocytometer. The viability of cells was assessed in triplicate using 30 µL of cell suspension and 5 µL of 0.4% trypan blue (Gibco #15250061, Grand Island, USA). Cell counting was performed using the method suggested by Louis and Siegel (2011).

The cell suspension from the Percoll density gradient centrifugation was used for the differential plating. The culture media was composed of Dulbecco's modified Eagle’s medium (high glucose DMEM) (Gibco # 11965, Grand Island, USA) supplemented with 10% fetal bovine serum, 2 mM glutamine (Sigma #G7513, St. Louis, USA), 1 mM pyruvate (Sigma #S8636, St. Louis, USA), 1× MEM vitamin solution (Sigma #M6895 St. Louis, USA), 1× MEM nonessential amino acid solution (Sigma #M7147, St. Louis, USA), and 1× antibiotic and antimycotic solution (Sigma #A5955, St. Louis, USA). The cells were cultured at a concentration of 1.5×10⁷ SSCs/mL in a 90 x 15 mm sterile Petri dish with agar 1% for 14 h in an atmosphere of 5% CO₂ and 100% humidity at 30°C (Sanyo Electric, MCO-20AIC, Sakata, Japan). Then, the cells in suspension were recovered, centrifuged at 300 × g for 8 minutes at 25°C, and resuspended in supplemented Leibovitz (L-15) medium solution. The percentages of SSCs, viability, and alkaline phosphate activity were determined as described for the cells from the Percoll density gradient centrifugation step. These steps are illustrated in Fig. 1.

2.4 Characterization of isolated spermatogonial stem cells

The testis and cells from the purification steps, a mix of the bands from the 20% and 30% Percoll density gradient centrifugation and differential plating, were sampled. All samples were stored at -80°C until determination of the relative ddx4 expression, using β-actin as an endogenous gene. Ddx4 is considered a marker gene of SSCs.

2.5 Primer design for the gene of interest and endogenous gene

Primers were designed from the alignment of the β-actin and ddx4 coding region sequences from different fish species deposited at the National Center for Biotechnology Information (NCBI). Astyanax mexicanus β-actin and ddx4 sequences were input into the Nucleotide BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and sequences with more significant identities were selected primarily from the Characiforme order and after from others, such as Siluriformes and Cypriniformes. Coding regions were obtained by the Open Reading Frame Finder program (https://www.ncbi.nlm.nih.gov/orffinder/). The coding regions were aligned using the Multalign program (http://multalin.toulouse.inra.fr/multalin/). The conserved regions were preferentially selected for the design of specific primers by the Primer-BLAST program (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), using A. mexicanus sequences as a reference.

2.6 cDNA synthesis, primer specificity analysis, and sequencing

Total RNA extraction from the samples was performed with TRIzol® Reagent (Ambion #15596026, Carlsbad, USA) following the manufacturer's instructions. RNA was quantified using a QIAxpert spectrophotometer (Qiagen, Hilden, Germany). Before cDNA synthesis, 1 µg of RNA from each sample was treated with DNAse I (Sigma‒Aldrich #SLBR4100 V, St. Louis, USA) and used for first-strand cDNA synthesis with a SuperScript III First-Strand Synthesis System kit (Invitrogen #18080051, Vilnius, Lithuania), according to the manufacturer instructions.

The cDNA was used as a template for amplifying the regions specified by the β-actin and ddx4 primers by conventional PCR. PCR was performed by recombinant Taq DNA polymerase (Invitrogen #11615-010, Itapevi, Brazil) according to the manufacturer's instructions using 1 µL of cDNA from the samples and the corresponding designed primers. The cycling parameters used were 95°C for 5 minutes, followed by 35 cycles of amplification (95°C for 45 seconds, 55°C for 30 seconds, and 72°C for 30 seconds) with a final step at 72°C for 10 minutes. The reaction was performed in a Multigene thermocycler (Labnet International, Inc.). After amplification, the products were assessed for primer specificities by visualizing them on a 2% agarose gel. The recovered products were separated from the gel using the EZNA Gel Extraction Kit (OMEGA), cloned into the pGEM-T easy vector (Promega), and then sent for sequencing to verify the gene sequence.

2.7 Relative ddx4 expression analysis

The relative ddx4 expression analysis was performed using β-actin as an endogenous gene. Standard curves for the genes of interest and endogenous genes were established using serial dilution (1:3) of a mixture of cDNA from all samples. Samples were diluted 1:5 for the gene expression analysis. A negative reaction control (NRC) was used for each gene. All samples were evaluated in triplicate; for each reaction, 10 µL of Quanti Nova SYBR Green PCR Master Mix (Qiagen #208054, Hilden, Germany), 8 µL of nuclease-free water, 1 µL of primer mix for target genes, and 1 µL of cDNA were used. The curves were made with each point in duplicate and showed efficiency between 0.9 and 1.1 and R ≅ of 0.99. The primers used in the qPCR are shown in Table 1.

Table 1

Sequences of primers used in qPCR for gene expression of piracanjuba SSCs (*Brycon orbygnianus*). Since *b-actin* is an endogenous gene and *ddx4* is specific for spermatogonia.
Genes	Sequence (5´-3´)	Amplicon size (bp)
ß-actin	F: 5´- CGTGCTGTCTTCCCATCCA-3´	86
ß-actin	R: 5´- TCACCAACGTAGCTGTCCTTCTG-3´	86
ddx4	F: 5´- AAGACCACAGGAACTGAGCG-3´	118
ddx4	R: 5´- CCCGGTCTCCATGAATGCTT-3´	118

The amplification conditions were 95°C for 2 minutes, followed by 40 cycles of amplification (95°C for 5 seconds and 60°C for 15 seconds). The melting curve was performed from 60 to 95°C. The reaction was carried out in a Rotor-Gene Q thermocycler (Qiagen, Hilden, Germany). Relative gene expression analysis was evaluated by the 2^−ΔΔCT method Larionov et al. (2005).

2.8 Cryopreservation of spermatogonial stem cells

For the SSCs isolation and cryopreservation experiments, four adult males approximately four years old, weighing 739.29 ± 2.16 g, were used. Animals were euthanized using a solution of 200 mg/L eugenol. The testes collected had a mean weight of 5.13 ± 0.018 g. The purification of SSCs was carried out as described in the previous section “Isolation of spermatogonial stem cells”. The concentration of SSCs was determined microscopically using a hemocytometer. Before the cryopreservation trials, the cell viability of cultured cells was evaluated using Trypan Blue dye (Gibco #15250061, Grand Island, USA) (see above).

Before freezing, the cell suspension was diluted 1:1 in cryo-solution using 50 µL of cell suspension at a concentration of 1.4×10⁷ SSCs/mL and 50 µL of cryoprotectant solution. The cryoprotectant solutions were composed of 100 mM glucose and 0.5% bovine albumin serum (Inlab confidence # 1870, SP, Brazil), and the cryoprotectants ethylene glycol (Sigma # SHBB4592 V, St. Louis, USA), glycerol (Sigma # 72996TMV, St. Louis, USA), dimethylsulfoxide (Sigma # SHBG9653 V, St. Louis, USA), dimethylacetamide (Sigma # STBC4643 V, St. Louis, USA), and propanediol (Sigma # MKBF9063 V, St. Louis, USA). The cryoprotectant concentrations were 2 M, 3 M, and 4 M. The cell suspension and cryoprotectant solutions were diluted 1:1. The mixture of cryo-solutions and cells was loaded into 250-µL cryopreservation straws (IMV Technologies, France) with the total volume adjusted to 70 µL by cutting. The straws were sealed with polyvinyl alcohol (Sigma # 089K0037, St. Louis, USA), placed in cryotubes (Corning Incorporated # 430659, DF, Mexico), and transferred to a freezing container (CoolCell™ LX Freezing Container, BioCision). The freezing container containing the straws was transferred to an ultrafreezer at -80°C and kept for two hours (estimated period to reach − 80°C). The freezing rate was measured using a thermal recorder (OM-EL-USB-TC, Omega Instruments, Norwalk, USA) with a K-type thermocouple (OM-EL-USB-TC, Omega Instruments®, Norwalk, USA), which was inserted inside the straw. After freezing at -80°C, the cryotubes were immersed in liquid nitrogen at -196°C for storage, approximately for one week.

Each sample was thawed in a water bath at 30°C for one minute. The straw was cut at the ends, and the cell suspension was transferred to a 1.5 mL centrifuge microtube and centrifuged at 350 x g for 5 minutes at 25°C (Hermle Labnet # Z326K, Wehingen, Germany). The supernatant was discarded, the pellet was resuspended in 30 µL of Hank’s solution, and 5 µL of Trypan Blue dye was added for subsequent viability analysis, which was performed in triplicates (see above).

2.9 Transplantation of spermatogonial stem cells

Four eight-year-old male piracanjuba were used as cell donors for the transplantation of SSCs into adult triploid hybrid recipients. The animals were euthanized with 200 mg/L eugenol (Biodynamic #10298550063, Ibipora, Brazil). The testes were collected and weighed, presenting an average of 0.43 ± 0.01 g. After isolation (see procedures above), SSCs from two donors were used for in vitro cryopreservation using the best cryoprotectant solution (Propanediol 1M).

The SSCs were stained using the fluorescent membrane marker PKH26 (Sigma‒Aldrich #MKCK8658, St. Louis, USA). The cell pellet was suspended in 1 mL of supplemented L-15 medium, added to 8 µL of PKH26 dye, and incubated for 10 minutes. After this period, 1 mL of fetal bovine serum was added and incubated for one minute to inactivate the fluorescent marker. The volume of the cell suspension was adjusted to 10 mL with supplemented L-15 medium and centrifuged at 300 × g for 5 minutes. Three washes were performed with the same medium to eliminate excess dye and fetal bovine serum.

Sterile male adult triploid hybrid recipients (Astyanax altiparanae female X Astyanax fasciatus male) and germ cell-free gonads at 11 months of age were used for the transplantation of SSCs (Piva et al. 2018). A total of 24 animals were divided into two groups (n = 12). In the first group, transplantation was performed using freshly isolated SSCs from the Percoll density gradient centrifugation at a concentration of 2.3×10⁶ SSCs/mL, resulting in 92% cell viability. In the second group, cryopreserved SSCs at 2.9×10⁶ SSCs/mL with 88% cell viability from the Percoll density gradient were used in transplantation. An insulin syringe coupled with a tip was used to perform the transplant via the urogenital papilla. Approximately 70 µL of the cell suspension contained approximately 1.9×10⁵ freshly isolated SSCs, and 2.4×10⁵ cryopreserved SSCs were injected into each recipient. For transplantation, the recipients were anesthetized in a solution containing 100 mg/L eugenol (Biodynamic #10298550063, Ibipora, Brazil).

2.10 Evaluation of chimerism by histochemistry

The testes (n = 2) of transplanted recipients (freshly isolated SSCs and cryopreserved SSCs) at 10 and 20 days after transplant were collected and fixed in Bouin’s fixative (Exodus Científica #FB08835SO, Sumaré, Brazil) for 24 hours. The samples were dehydrated using increasing concentrations of ethanol (from 70–100%) and embedded in paraplast (Sigma # SLBS8607, St. Louis, USA). The paraffin blocks were cut at 5 µm using a microtome equipped with a steel blade (Leica RM2235, Lincolnshire, USA). The cut material was examined by fluorescence microscopy (Nikon-Eclipse Ni, Tokyo, Japan) and photographed (NikonDSRi2, Nikon, Tokyo, Japan). The slides were visualized by the fluorescent marker PKH26 and counterstained with hematoxylin and eosin to identify cell types.

2.11 Statistical analysis

Data were evaluated in triplicate for each cryoprotectant at different molar concentrations. Data were reported as the mean and standard error and analyzed for homogeneity using the Levene test (Brown and Forsythe 1974), and for normality, the Cramer‒von Mises test was used (Tamura 2008). Then, they were evaluated by ANOVA and Tukey’s test (α = 0.05). Statistical analyses were performed using STATISTICA 7.0 software.

3.1 Histological characterization of the testis of piracanjuba

The evaluated testes of the juvenile piracanjuba were small and translucent, presenting a gonadosomatic index (GSI) of 0.01 ± 0.00. The testes were identified in the development phase. Spermatogonia, a few cysts of spermatocysts, spermatids, and some spermatozoa isolated in the testis were observed in the germinal epithelium (Fig. 2A-B). The testes of adult males collected in the winter were translucent and easily visualized in the abdominal cavity, presenting a GSI of 0.07 ± 0.00. A spermatogenesis in the regeneration phase was observed, showing a large number of spermatogonia and some cysts of spermatocysts, spermatids, and isolated spermatozoa in the testis (Fig. 2C-D). The testes of adult males collected in the breeding season were large, firm, and had a blanched color, presenting a GSI of 0.86 ± 0.13. The spermatogenesis was active with the spermatozoa in the testicular lumen, where cysts of spermatocytes, spermatids, and a few spermatogonia were observed (Fig. 2E-F). In addition, the animals released semen when slight manual pressure was applied to the coelomic cavity in the craniocaudal direction.

3.2 Isolation of spermatogonial stem cells

The cell mix from the 20% and 30% bands of the Percoll density gradient centrifugation showed 58.35 ± 0.03% SSCs, 30.54 ± 0.02% spermatocytes, and 11.11 ± 0.05% sperm (Fig. 3A). After differential plating, 66.00 ± 0.01% SSCs, 33.23 ± 0.01% spermatocytes, and 0.78 ± 0.02% sperm were obtained (Fig. 3B). The cell viability of SSCs from the Percoll density gradient centrifugation was 92.94 ± 0.06%, increasing to 94.59 ± 0.13% after plating (Fig. 4). SSCs from the Percoll density gradient centrifugation and differential plating showed higher alkaline phosphatase activity than spermatocytes when visually compared to the intensity of fluorescence from both cells. This result indicates that the purified cells were SSCs (Fig. 5).

3.3 Analysis of piracanjuba gene sequences

The sequencing of the amplified regions of the Brycon orbignyanus β-actin and ddx4 with 86 and 118 bp showed 99% and 92.37% identity, respectively, for Astyanax mexicanus sequences (NCBI Reference Sequence: XM_007247821.3; XM_022681259.1). Figures 6A-B, show the alignments of the ddx4 and B-actin sequences from Astyanax mexicanus and B. orbignyanus. The products of the amplifications were visualized on a 2% agarose gel, showing the specificity of the primers (Fig. 6C).

3.4 Relative ddx4 expression analysis

The standard curves for the β-actin and ddx4 genes showed amplification efficiencies for the gene of interest and the endogenous control was as expected (0.9 to 1.1). The sampling points were located within the limits of the curves. The melting curve showed only one peak at a specific temperature for each amplification product, where no dimer formation was observed for the evaluated genes. The results of the relative ddx4 expression indicated that the expression of ddx4 was approximately 3.5 times higher in cells from the Percoll density gradient centrifugation (cell mix from the 20% and 30% bands) than in the testis and cells after differential plating (Fig. 7). This result suggests a decrease in the relative ddx4 expression in cultured cells from differential plating, even with higher SSC purity in differential plating.

3.5 Viability of spermatogonial stem cells after cryopreservation and freezing rate

The freezing rate of each treatment is shown in Table 2. The cooling rate was similar between the different cryoprotectant solutions, between a minimum of 6.33 ± 0.27 minutes (Etileglycol 1.5M) and a maximum of 9.67 ± 0.72 minutes (DMA 2M) (Table 3). While the exothermic peak was observed the solution without cryoprotectant had a lower exothermic peak with − 3.06 ± 0.62 (°C) and a time of 4.52 ± 0.30 (min) Table 2. As expected, control group cells did not show cell viability after thawing without any cryoprotectant (P = 0.0000) (Table 3). For 1M propanediol, the cell viability was 94.74 ± 1.85% (P = 1.0000) after thawing (Table 3), this solution was considered the best cryoprotective solution. Followed by Propanediol 1.5M, 1M DMA and 2M DMSO presented a viability of 67.57 ± 8.15% (P = 0.2842), 67.41 ± 12.85% (P = 0.0001) and 62.15 ± 6.45% (P = 1.0000) respectively after thawing (Table 3). Ethylene glycol had viability below 60% in the three molarities with values of 36.06 ± 4.18% (P = 0.9999) (1M), 35.35 ± 13.76% (P = 1.0000) (1.5M), and 45.66 ± 8.43% (P = 0.9999) (2M) (Table 3).

Table 2

Freezing rates of cryoprotectant solutions used in freezing spermatogonia stem cells of piracanjuba (*Brycon orbignyanus*) over two hours at -80°C. The cryoprotectant solutions were loaded in straws and frozen through slow cooling (approximately − 1°C/min). Data were obtained from three replicates and are expressed as the mean ± standard error.
	Cooling rate (-1°C/min) (20°C to 0°C)			Freezing rate (-1°C/min) (0°C to -50°C)			Freezing temperature			Exoterm delay (min)			Exoterm peak
CONTROL	7.67	±	0.72	35.67	±	1.09	-0.50	±	0.00	4.52	±	0.30	-3.06	±	0.62
PROPANEDIOL 1M	7.33	±	0.27	28.00	±	0.47	-3.83	±	0.14	3.18	±	0.16	-11.12	±	1.02
PROPANEDIOL 1.5M	7.67	±	0.54	31.33	±	0.72	-5.83	±	0.59	3.23	±	0.09	-13.29	±	1.86
PROPANEDIOL 2M	8.00	±	0.47	29.00	±	1.70	-8.33	±	0.27	3.04	±	0.11	-15.53	±	1.28
DMA 1M	8.00	±	0.82	29.67	±	1.36	-3.50	±	0.24	2.98	±	0.06	-10.63	±	1.14
DMA 1.5M	7.67	±	0.54	31.00	±	0.94	-6.83	±	0.49	2.79	±	0.10	-15.13	±	1.15
DMA 2M	9.67	±	0.72	32.67	±	1.09	-9.67	±	0.27	3.15	±	0.10	-18.73	±	1.09
DMSO 1M	7.33	±	0.27	31.00	±	0.94	-4.33	±	0.27	3.18	±	0.07	-11.40	±	1.22
DMSO 1.5M	8.00	±	0.94	31.33	±	0.27	-5.50	±	0.24	3.39	±	0.20	-10.45	±	1.45
DMSO 2M	8.67	±	1.19	31.67	±	1.19	-8.67	±	0.54	3.04	±	0.13	-15.57	±	0.35
GLYCEROL 1M	7.33	±	0.27	34.33	±	1.19	-4.00	±	0.24	2.84	±	0.04	-10.92	±	0.78
GLYCEROL 1.5M	7.00	±	0.47	33.00	±	0.47	-6.83	±	1.11	3.11	±	0.19	-15.34	±	1.53
GLYCEROL 2M	6.67	±	0.27	31.00	±	0.82	-6.83	±	0.36	3.31	±	0.12	-15.76	±	0.87
ETILEGLYCOL 1M	7.00	±	0.00	30.00	±	1.41	-3.67	±	0.14	3.52	±	0.18	-9.96	±	0.50
ETILEGLYCOL 1.5M	6.33	±	0.27	28.33	±	0.72	-5.00	±	0.24	3.12	±	0.09	-11.37	±	0.62
ETILEGLYCOL 2M	8.00	±	0.82	31.00	±	1.25	-8.17	±	0.36	3.19	±	0.08	-17.27	±	1.16

Table 3

Viability of SSCs before freezing and post-thawing of piracanjuba (*Brycon orbignyanus*). Cell suspensions were diluted 1:1 in each cryo-solution, loaded in straws, and frozen through slow cooling (approximately − 1°C/min). Data were obtained from four replicates and are expressed as the mean ± standard error. Values with different letters in the same column indicate a significant difference.
Treatment		Viability (%)
PRE-FREEZING CONTROL		87.68	±	5.22^a
PROPANEDIOL	1 M	90.74	±	1.85^a
	1.5 M	67.57	±	8.15^ab
	2 M	55.25	±	12.41^abc
DMA	1 M	67.41	±	12.85^ab
	1.5 M	40.99	±	6.47^bcd
	2 M	29.89	±	4.04^bcd
DMSO	1 M	34.85	±	5.96^bcd
	1.5 M	51.01	±	10.94^abc
	2 M	62.15	±	6.45^abc
GLYCEROL	1 M	52.49	±	22.07^abc
	1.5 M	21.73	±	5.23^bcd
	2 M	18.04	±	6.5^cd
ETILENOGLICOL	1 M	36.03	±	4.18^bcd
	1.5 M	35.35	±	13.76^bcd
	2 M	45.66	±	8.43^abc
POST-DEFROST CONTROL		0.00	±	0.00^d
P - value		0.0161

3.6 Evaluation of SSCs transplantation into the testis of adult triploid hybrid recipients

In the testes of recipients transplanted (n = 2) with freshly isolated SSCs, PKH26-positive cells were visualized ten days after transplantation (Figs. 8A and 8B). Through counterstaining with hematoxylin and eosin, the initial development of the germinal epithelium was verified in the testes of the transplanted recipients, given that the triploid hybrids were germ cell-free testes (Figs. 8C and 8D). At 20 days after transplantation (n = 1) with freshly isolated and cryopreserved SSCs (n = 1), the initial spermatogenesis process was verified in the transplanted testes, observing the presence of SSCs and some spermatocytes (Fig. 9A-D). Without transplantation, the individuals in the control group had testes without germ cells (Fig. 9A). Some of the recipients' animals died in the days following the transplant (n = 3) for freshly isolated SSCs and cryopreserved SSCs. After a prolonged period of hormonal inductions, some of the transplanted animals were able to produce sperm, but the molecular analysis was inconclusive due to DNA degradation in the samples. The semen was used for in vitro fertilization of piracanjuba oocytes, as well as intracytoplasmic microinjection, but embryo development was not observed. In microscopy analysis, a few spermatozoids were found, but they were not activated. Other hormonal inductions were attempted, but they did not result in successful sperm production. The procedures were not included in the material and methods once the results were negative.

The evaluation of spermatogenesis stages in juveniles and adult piracanjuba collected in the winter and summer allowed for the identification of the adequate season and spermatogenesis stage to perform the isolation of SSCs. Adults collected in the winter were allowed to obtain a higher cellular concentration of purified SSCs for later use in cryopreservation or cell transplantation. The testes of juvenile animals were small, presenting a GSI of 0.01 ± 0.00, and even with SSCs, many animals are necessary to obtain an adequate quantity of cells to perform cryopreservation or transplantation in adult animals, as described in other studies (Majhi et al. 2014; Xu et al. 2019). Piracanjuba is an endangered species; therefore, the use of juveniles is unsuitable for the isolation of SSCs for cryopreservation or transplantation into adult recipients. In the absence of adults, however, testes from juveniles could be used in tissue cryopreservation and SSC isolation for transplantation in larvae (Marinović et al. 2019; Zhou et al. 2021).

Adult testes collected in June were found in the regeneration stage of spermatogenesis, presenting a GSI of 0.07 ± 0.00. In this stage, SSCs were abundant in the gonads. Therefore, it is the most recommended site to isolate SSCs to reach high quantities of isolated SSCs. In the testes of adults collected in December, spermatozoa were observed in abundance in the lumen, ready to be released, and there was a low quantity of SSCs. Therefore, this stage of spermatogenesis is unfavorable for the isolation of SSCs; once the number of spermatozoa is more significant than SSCs, it makes cellular purification difficult. Studies involving the histological characterization and isolation of SSCs are essential to identify the moment the testes produce higher quantities of SSCs. Identifying this spermatogenesis stage is essential to obtain a higher concentration and better purification of SSCs, which can be used later in cell transplantation, cryopreservation, or in vitro cultivation (Poursaeid et al. 2020; Zupa et al. 2020).

The regeneration spermatogenesis stage of adult piracanjuba was used to isolate SSCs. SSCs were isolated by enzymatic dissociation, Percoll density gradient centrifugation, and differential plating. This presented a degree of purity after differential plating of approximately 8% greater than the Percoll density gradient centrifugation, also obtaining superior viability. It was possible to eliminate mainly spermatocytes and sperm through differential plating, which was reduced from approximately 11% in the Percoll density gradient centrifugation to 0.78% after differential plating. Differential plating was applied as a purification step for the isolation of SSCs in other species, such as O. mykiss (Shikina et al. 2008), Oreochromis niloticus (Lacerda et al. 2014), Rhamdia quelen (Silva et al. 2016), and Prochilodus lineatus (Dias et al. 2020). Differential plating is considered an important step to increase the purity of germ stem cells during the purification step. The purification of SSCs is essential for the generation of high-quality in vitro genetic banks, which preserve the genetic material of endangered species, such as piracanjuba (B. orbignyanus).

The relative ddx4 expression analysis of the testis and cells from the purification steps indicated that the Percoll density gradient centrifugation increased relative gene expression when compared with testis and differential plating. This result may be attributed to the increased concentration of SSCs obtained in the Percoll density gradient centrifugation (20% and 30% bands) compared to the whole testis. A higher percentage of SSCs was obtained in the differential plating step. However, the relative ddx4 expression was lower than that of cells from the Percoll density gradient centrifugation. This is because ddx4 is expressed in germline cells throughout all stages (Hartung et al. 2014). After differential plating, the expression was lower due to the loss of type B spermatogonia and spermatocytes. Ddx4 is mainly expressed in SSCs of O. niloticus, with slight expression observed in primary spermatocytes (Kobayashi et al. 2000). In Neotropical species like R. quelen and Colossoma macropomum, the ddx4 gene is primarily expressed in type A and B spermatogonia, with slight expression in spermatids and no expression in spermatozoa. In C. macropomum, ddx4 expression was reported in type A oogonia and oocytes, probably due to the accumulation of germplasm RNA (Ricci et al. 2018; Vasconcelos et al. 2019; Borella et al. 2020). In R. quelen, other genes showed high expression levels in cell fractions enriched by type A spermatogonia, such as plzf and pou5f3 (Lacerda et al. 2019).

To identify SSCs in Percoll density gradient centrifugation and differential plating, we used alkaline phosphatase observing cells positive for this marker. Previously, alkaline phosphatase has been used to identify type A spermatogonia in vitro cultures of mice (Van Der Wee et al. 2001), as well as in fish such as O. latipes (Hong et al. 2004), Epinephelus coioides (Zhong et al. 2022) and Opsariichthys bidens (Chen et al. 2022).

Cell cryopreservation is an essential strategy to preserve material with high genetic value. However, freezing is potentially fatal for most living cells, mainly due to the formation of intra- and extracellular ice crystals that lead to cell injury (Chowdhury and Ghosh 2021; Mahmud 2021). Furthermore, cells from different tissues or cells within the same tissue may have different rates of water efflux across the cell membrane; thus, the optimal cooling rate needs to be determined for each type of cell to be cryopreserved (Hoon et al. 2017). The cooling curve is an essential parameter for cell survival, and there is little information about the ideal cooling rate for SSCs (Redden et al. 2009). In this work, different cryoprotectants at different molarities were evaluated, as well as their respective freezing rates for cryopreservation of SSCs isolated from piracanjuba. The best cryoprotectant solution found was 1 M propanediol, presenting a viability of 90.74 ± 0.19 after thawing, a freezing temperature of -3.83 ± 0.14°C, a freezing rate of 28.00 ± 0.4 minutes, and an exothermic peak of -11 ± 1.02°C at 3.18 ± 0.16 minutes.

Isolation and cryopreservation of SSCs have been successfully performed in various fish species. For instance, in R. quelen, 67% of cell viability was achieved after thawing by using MeOH 1.3 M (Rosa et al. 2023). Similarly, in T. tinca, the viabilities obtained were 57% using 1.5 M glycerol (Linhartová et al. 2014) and 50% at 3 M DMSO (Marinović et al. 2019). In a study by Patra et al. (2016), Labeo rohita had a 70% viability rate after being thawed using 1.4 M DMSO. Meanwhile, Zupa et al. (2020) found that Argyrosomus regius had a viability rate of 28.5% after being thawed using 10% de DMSO. The cryoprotectant and the molar concentration are species-specific; therefore, cryopreservation protocols are necessary for each species (Pšenička et al. 2016). The results obtained in this study were highly efficient for the cryopreservation of piracanjuba SSCs, reaching a high percentage of viability compared with the results of other studies. In addition, there are few cryopreservation protocols developed for SSCs of Neotropical fish species; this protocol shows promise for establishing future in vitro genetic banks.

In the testes of adult triploid hybrid animals that received transplantation of freshly isolated and cryopreserved SSCs, the cells were visualized up to 20 days after transplantation by histological analysis. The combination of the two sterilization methods, hybridization between species and chromosomal manipulation, was efficient in producing recipient testes without germ cells, making them ideal recipients. Initial development of the germinal epithelium in the testes of the recipients indicated that the transplanted cells were proliferating, mainly due to the presence of spermatogonia and spermatocytes. The fact that cryopreserved germ cells colonize the recipient testes makes it possible to establish procedures that can be used in the reconstitution of piracanjuba in the event of extinction, as was done for other species (Yoshizaki and Lee 2018; Franěk et al. 2019).

Some animals that received transplants were evaluated for semen production by hormonal induction. Despite obtaining sperm, in vitro fertilization and intracytoplasmic microinjection tests were unsuccessful. Additionally, it was not feasible to determine through molecular analysis whether the semen belonged to piracanjuba. This outcome created uncertainty about whether the sperm cells generated could have originated from the transplanted cells or if numerous hormonal stimulations enabled the receiving gonad to produce endogenous semen. Although it did not successfully obtain functional gamete production from the donor species, this work presents promising results for establishing procedures for the reconstitution of piracanjuba. The procedures performed are the first established for Neotropical species using adult triploid hybrid receptors.

Transplantation of piracanjuba SSCs via urogenital papilla was previously performed using A. altiparanae male diploid recipients sterilized with busulfan and temperature techniques, producing endogenous and exogenous sperm (de Siqueira-Silva et al. 2019). In other studies, spermatogenesis was depleted using busulfan treatment, and SSC transplantation was performed, resulting in endogenous and exogenous spermatozoa production (Majhi et al. 2009; Lacerda et al. 2010). However, using 100% sterile animals, as in the case of triploid hybrids in which the testes are constituted only by support cells, is advantageous to these works cited since the competition for space and nutrients between endogenous and exogenous cells is eliminated (Dobrinski 2006). Therefore, some published works tested the production of recipients completely devoid of germ cells (Piva et al. 2018; Yoshikawa et al. 2018; Ponjarat et al. 2019). This strategy in some studies was successful in germ stem cell transplantation, where gametes were produced only from the donor species (Takeuchi et al. 2018; Xu et al. 2019).

In this study, effective procedures for gene banking were established for the first time in an endangered neotropical fish species, the piracanjuba (B. orbignyanus). These procedures include isolation, cryopreservation, and transplantation. The procedure was optimized by identifying the period of highest abundance of SSCs in the testis of adult animals. The purification methods which involved Percoll density gradient and differential plating have resulted in high purity and viability of SSCs. It has been observed that the SSCs fractioned have shown high alkaline phosphatase activity in comparison to spermatids and spermatocytes. The relative ddx4 expression analysis has indicated a decrease in cells from differential plating as compared to those from the Percoll density gradient. The best cryoprotectant solution for piracanjuba SSCs was a 1 M propanediol solution. Transplantation of freshly isolated and cryopreserved cells resulted in functional SSCs that developed the germinal epithelium in the testes of adult triploid hybrid recipients. Despite the initial evaluations suggesting low gamete production and no observed fertilization, further investigations are required to achieve functional gamete production. The gene banking procedures developed are innovative and have the potential for application in aquaculture and the conservation of endangered species like piracanjuba.

Acknowledgments

Authors are grateful to FAPESP (Young Investigator Award #2010/17429-1) which permitted the initial works in the yellowtail tetra and China Three Gorges Brasil (CTG Brasil, Project ANEEL #PD-00387-0418/2019) for the full funding of studies on the species. We also acknowledge CEPTA / ICMBio for kindly providing the facilities and experimental fish.

Competing interests

The authors declare that they have no competing interests.

Funding

The project was supported by the China Three Gorges Brasil (CTG Brasil) (Research & Development, Project ANEEL #PD-00387-0418/2019).

Data Availability

All data generated or analyzed during the current study are included in this published article.

Ethics approval

The experiments were conducted following the Animal Ethics Committee from the National Center for Research and Conservation of Continental Aquatic Biodiversity (CEUA/CEPTA # 02031.000088/2021-61). The authenticity of the document can be verified on the website https://sei.icmbio.gov.br/autenticidade informing the verification code: 9643544 and the code CRC: EC53598A.

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Cryopreservation and transplantation of spermatogonia stem cells in piracanjuba Brycon orbignyanus (Characiformes: Characidae), an endangered fish species

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Ethical considerations for the use of animals

2.2 Histological analysis of the testes of piracanjuba (Brycon orbignyanus)

2.3 Isolation of spermatogonial stem cells

2.4 Characterization of isolated spermatogonial stem cells

2.5 Primer design for the gene of interest and endogenous gene

2.6 cDNA synthesis, primer specificity analysis, and sequencing

2.7 Relative ddx4 expression analysis

2.8 Cryopreservation of spermatogonial stem cells

2.9 Transplantation of spermatogonial stem cells

2.10 Evaluation of chimerism by histochemistry

2.11 Statistical analysis

3. Results

3.1 Histological characterization of the testis of piracanjuba

3.2 Isolation of spermatogonial stem cells

3.3 Analysis of piracanjuba gene sequences

3.4 Relative ddx4 expression analysis

3.5 Viability of spermatogonial stem cells after cryopreservation and freezing rate

3.6 Evaluation of SSCs transplantation into the testis of adult triploid hybrid recipients

4. Discussion

Conclusion

Declarations

References

Additional Declarations

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