Resumption of donor-origin spermatogenesis in senescent goldfish Carassius auratus (Linnaeus, 1758) following spermatogonial cell therapy

Ethics statement

This study was approved by the Animal Ethics Committee of National Bureau of Fish Genetic Resources (#G/CPCSEA/IAEC/2015/2).

Animals, age, and rearing protocols

Male goldfish (C. auratus; n = 60; mean body weight ± standard deviation [SD] of 230 ± 20.5 g) were procured from Maharashtra, India, which were reported to be approximately 10 years old by the providers. The age of the fish was evaluated and confirmed by counting the growth rings located at scale (the growth rings from the fish raised in the farm with known age was used as reference; Fig. S1). The fish were stocked in 500 L tanks after procuring them from the farm at the density of 5.0 kg of fish per m³ and reared in flow-through fresh water system (temperature: 25 °C ± 2 °C; dissolved oxygen: 5.3–6.1 ppm; pH: 7.5–8.0; hardness: 40–45 ppm) under a constant light cycle (12 h light and 12 h dark). The fish were reared for an additional two years before confirmation of their senility through histological analysis and germ-cell-specific vasa gene expression studies. The prepubertal donor C. auratus males (three months old) were produced at the rearing facilities of the National Bureau of Fish Genetic Resources, Lucknow. Both groups of animals were fed a commercially available pelleted diet twice a day to satiation.

Gonad histology

After two years of rearing, five fish were randomly sampled and sacrificed using an overdose of anaesthesia (2-phenoxyethanol; HiMedia, India), and their body weights were recorded. The gonads of the fish were excised and weighed, macroscopically examined, and photographed using a digital camera. The middle portion of the right and left gonads from each fish were then immersed in Bouin’s fixative for 24 h and preserved in 70% ethanol. The gonads were processed for microscopic examination following routine histological procedures up to the stage of preparing 5-µm thick cross-sections and staining them with hematoxylin–eosin. Histological sections from each fish were examined under a microscope at magnifications between 10× and 60×.

Gene expression analysis

Samples for real-time reverse transcription polymerase chain reaction (RT-PCR) of vasa gene expression were obtained from the anterior region of the testes after the 2-year rearing period (n = 3) and at six months after spermatogonial cell therapy (n = 3). The samples were stored in RNAlater (Sigma–Aldrich, St.Louis, MO, USA) at −80 °C until further processing. RNA was extracted using TRIzol (Invitrogen Life Technology, Carlsbad, CA, USA) according to the manufacturer’s protocol. cDNA was synthesised using a first-strand cDNA synthesis kit (Thermoscientific, USA). The primers for real-time RT-PCR were (5′-AACCCTCATGTTCAGCGCCAC-3′ and 5′-TGGTTTCAACAAAGACCATCGTGC-3′). The real-time PCRs were run in an ABI PRISM 7300 (Applied Biosystems, USA) system using Power SYBR® Green PCR Master Mix in a total volume of 15 µL, which included 7.5 µL of 2 × Maxima™ SYBR Green qPCR Master Mix (Thermoscientific, USA), 20 ng of first-strand cDNA and 5 pmol L⁻¹ of each primer. β-actin (5′-GAC TTC GAG CAG GAG ATG G-3′ and 5′-CAA GAA GGA TGG CTG GAA CA-3′) was used as an endogenous control. The comparative Ct method was used for vasa mRNA quantification. The fold change in the expression level was calculated using the 2−ΔΔCt method (Whelan, Russell & Whelan, 2003).

Isolation and labelling of donor cells

The donor C. auratus males (n = 3) were sacrificed using an anaesthetic overdose, and their testes were excised and rinsed in phosphate buffered saline (PBS; pH = 8.2). The testicular tissue was finely minced and incubated in a dissociating solution containing 0.5% trypsin (pH 8.2; Worthington Biochemical Corp., Lakewood, NJ), 5% foetal bovine serum (JRH Biosciences, Lenexa, KS), and 1mmol L⁻¹ Ca²⁺ in PBS (pH 8.2) for 2 h at 22 °C. The dispersed testicular cells were sieved through a nylon screen (mesh size 50 µm) to eliminate the non-dissociated cell clumps, suspended in discontinuous percoll (Sigma–Aldrich, St. Louis, MO, USA) gradients of 50%, 25%, and 12% and centrifuged at 200 × g for 20 min at 20 °C (Majhi et al., 2014). The bottom phase containing predominantly spermatogonial cells (determined during preliminary trials which involved cell size measurement) was harvested, and the cells were subjected to rinsing as well as a cell viability test through trypan blue (0.4% w/v) exclusion assay. The cells were then exposed to the PKH-26 Cell Linker (Sigma–Aldrich, St.Louis, MO, USA) at the concentration of 8 µmol/mL (room temperature, 10 min) to label the cells for tracking their behaviour inside the senescent (recipient) C. auratus gonads. The staining procedure was stopped by the addition of an equal volume of heat-inactivated foetal bovine serum. The labelled cells were rinsed three times to remove the unincorporated dye, suspended in Dulbecco Modified Eagle Medium (Life Technologies, Rockville, MD) with 10% foetal bovine serum, and placed on ice until transplantation.

Cell therapy procedure

Forty fishes were first anaesthetised using 200 ppm phenoxyethanol and placed on a cell transplantation platform, where they received a constant flow of oxygenated water containing 100 ppm of the anaesthetic drug. To prevent desiccation, the surface of the fish was kept moisturised during the entire procedure of cell transplantation, which lasted for approximately 5–7 min per fish on average. A micro syringe was used to inject the cell suspension into the testicular lobe through genital papilla (Fig. S2). Each fish was injected with 100 µL of a cell suspension containing approximately 5 × 10⁴ cells, at the flow rate of approximately 20 µL/min. Trypan blue was added to the injection medium prior to transplantation to facilitate visualisation of the cell suspension inside the needle and inside the gonads after injection. The genital papilla region was topically treated with 10% isodine solution and the fish were returned to clean water.

Analysis of donor cells post therapy

The fate of the donor cells after transplantation was analysed through fluorescent microscopy at six and 12 weeks after injection. Then, the testes from the five animals chosen randomly during each sampling were removed, washed in PBS (pH 8.2), macroscopically examined for the degree of dispersion of the cell suspension (Fig. 1), and immediately frozen in liquid nitrogen. Cryostat (Leica CM 1500, Germany) section 8 µm thick were cut from the representative portions of these testes and observed under a fluorescent microscope (Nikon Eclipse E600, Tokyo, Japan) for detecting the presence of PKH-26-labelled donor cells.

The fate of the donor cells was then examined by measuring the gonado-somatic index (GSI; n = 5) and sperm density at six months after therapy. On each occasion, 20–30 µL of sperm was collected from each of the 25 therapy-treated males and five control males. Sperm was manually extracted by applying gentle abdominal pressure after careful removal of urine and wiping the genital papilla. The sperm samples (10 µL) were then diluted 1,000 times with PBS and the density of spermatozoa was counted using a haemocytometer (Marienfeld, Germany) under a microscope. Some of the spreads of sperm used for counting were also observed under the fluorescent microscope for the detection of PKH-26-labelled cells.

Sperm and blood samples were collected at 18 months from the spermatogonia cell transplanted senescent male (n = 25). Total DNA was extracted using PureLink Genomic DNA kit (Invitrogen Life Technology, Carlsbad, CA, USA) according to the manufacturer’s protocol and DNA typing was done using C. auratus SSR locus J60 * (forward 5′-CTGGCTGTCTGATCCTGCTGAT-3′ and reverse 5′-TGGCCAGAGTTTAAAAACCAGTCC-3′) (Yue & Orban, 2002). PCR reaction was performed in 25 µL consisted of 1x Taq buffer, 1.5 mM MgCl₂, 0.5 µm each primer, 200 µM of dNTP, 0.75 U Taq DNA polymerase and 80 ng of the DNA template. The amplification was done in a thermal Cycler (ThermoFisher Scientific, USA) and consisted of an initial denaturation at 94 °C for 5 min, 25 cycles of 94 °C for 30 sec, 62 °C for 30 sec and 72 °C for 30 sec, followed by elongation at 72 °C for 10 min. Amplified products was visualized by 10% polyacrylamide gel electrophoresis (PAGE) and silver staining. Amplicon sizing was done on gel imaging and analysis system (UVP) using Msp I digested pBR322 as ladder.

Artificial fertilization and induced spawning

After 6 months, the senescent therapy-treated C. auratus males (n = 25) resumed production of spermatozoa. Artificial insemination and natural spawning were performed using eggs from wild C. auratus females. Approximately 20 µL of milt from each of the ten therapy-treated males was used to fertilise a batch of C. auratus eggs. The batches of eggs were then incubated under flowing water at 25 °C until hatching. In the natural spawning trials, ten senescent therapy-treated males and wild females were paired in a 100 L glass aquarium and reared in fresh water (Temperature: 25 °C ± 2 °C; dissolved oxygen: 5.3–6.1 ppm; pH: 7.5–8.0; hardness: 40–45 ppm) under a constant light cycle (12 h light and 12 h darkness). Every, morning between 10:00 and 11:00 h, the tanks were examined for spawning and embryos were collected. The fertilised eggs obtained from each cross were incubated at 25 °C and observed under a light microscope for the assessment of fertilisation, embryonic development, and hatching.

Statistical analysis

The statistical significance of the differences in vasa gene expression levels, GSI values and sperm densities among the groups is evaluated using one-way analysis of variance and Tukey’s multiple comparison test. Graphpad Prism ver. 4.00 (Graphpad Software, San Diego, Carlifornia, USA) is used for statistical analyses. Data are presented as mean ± SD and the differences among the groups are considered as statistically significant at p < 0.05.

Histological observation of germ cells

Microscopic examination of the testes revealed that all the five senescent C. auratus males aged 12 years exhibited shrunken gonads (Fig. 1A) and complete disappearance of all the stages of germ cells (Figs. 2A–2B) in all sections examined before stem cell transplantation. By contrast, the control males (age >1 year) exhibited active spermatogenesis with a large cyst of spermatogonial cells, all stages of germ cells (Figs. 2C–2D) and their efferent duct contained spermatozoa. These histological observations were also corroborated by the results of GSI evaluation and real-time RT-PCR analysis, which showed that both GSI (Fig. 3) values and vasa gene transcript levels (Fig. 4) were significantly lower in the senescent males compared to the controls (p < 0.05).

Fate of transplanted cells in senescent testes

The transplanted donor spermatogonial cells were found randomly distributed throughout the spermatogenic lobules in all the five senescent males examined 6 weeks after the therapy. At 12 weeks after the therapy, the donor spermatogonial stem cells had reached the blind end of the lobules (cortical region of the testis; Figs. 5A–5D) and had undergone proliferation to form a network along the testicular lobule; this stage was observed in all the sampled fish (n = 5; Fig. 5E). At six months after the therapy, the sperm could be collected by applying gentle pressure on the abdomen of all 25 senescent C. auratus males (Fig. 5F). The sperm density, which was not detectable before the therapy, had significantly increased after the therapy and was comparable with that of the reproductive control males (Fig. 6).

Production of viable gametes and progeny from senescent males

Six months after cell therapy, the senescent C. auratus males resumed spermatogenesis and produced spermatozoa from the transplanted cells; the origin of the spermatozoa (from the transplanted cells) was confirmed by the existence of two different genotypes and presence of the red fluorescent labels in the cells (Fig. 5F). Locus J60* differentiated amplicons from blood (recipient) and sperm (donor cell) DNA (Figs. 7 and 8). These therapy-treated males were then used for artificial fertilization and for trials of induced spawning of eggs obtained from young C. auratus females (Tables 1–2). These crosses resulted in normal embryonic development and hatching, which were similar to those in the control fish. Breeding between the spermatozoa from therapy-treated males and eggs from wild C. auratus females resulted in 88.6%–97.5% hatching whereas 93.8%-97.7% result was obtained in the control (Table 1). On the other hand, when the senescent males were coupled with wild C. auratus females for induced spawning, the breeding resulted in 95.5%–99.5% hatching with normal embryonic development and viable progeny; oppose to 97.7%–98.4% in control (Table 2). These observations suggest the viability of the proposed approach in revitalising the reproductive competence of the males from commercially valuable fish species that have aged and can no more impregnate the females.