Cryopreservation of testicular tissue from Murray River Rainbowfish, Melanotaenia fluviatilis
Animal husbandry and sample collection
All animal handling and experimental procedures were approved by the Animal Ethics Committee B at Monash Medical Centre (MMCB/2017/39) and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Melanotaenia fluviatilis (Aquarium Industries, Victoria, Australia) were held at 25 °C ± 1 °C on a 12:12 light–dark cycle. At the time of experimentation, fish 5.76 cm ± 1.00 cm in length and weighing 3.25 g ± 1.38 g, were humanely killed by anesthetic overdose using aquatic anaesthetic AQUI-S (Primo Aquaculture, Queensland, Australia) and death was confirmed by destruction of the brain. The gonads were removed and placed into handling medium composed of Eagles minimum essential media (EMEM, SigmaAldrich) supplemented with 5% FBS (ThermoFisher Scientific, Victoria Australia), and 25 mM HEPES (ThermoFisher Scientific; pH 7.8) and kept on ice.
Histology and immunohistochemistry
Whole testes were fixed in 10% neutral buffered formalin (Merck, Victoria, Australia) for 48 h and processed by the Monash Histology Platform which included standard hematoxylin and eosin staining. Unstained sections were stained for Vasa using a zebrafish-specific anti-Vasa antibody (Sapphire Bioscience Pty. Ltd, New South Wales, Australia) and counter-stained with Hoechst (ThermoFisher Scientific). De-paraffinised sections were rehydrated through changes of xylene and a standard series of decreasing ethanol dilutions before antigen retrieval in 10 mM citrate buffer (pH 6), microwaved to boiling point for 10 min. Sections were rested in citrate buffer for 30 min prior to blocking with CAS Block (Invitrogen) for one hour followed by incubation with anti-Vasa antibody (1:200) in 5% BSA in PBS at 4 °C overnight. Sections were washed in PBS and incubated with secondary antibody, Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500; Invitrogen), and Hoechst nuclear counterstain (1:1000) in 5% BSA and PBS for one hour at room temperature.
Images were captured using the EVOS FL Auto 2 Imaging system (ThermoFisher Scientific) and an Olympus BX43 Upright Microscope with an X-Cite Series 120 Q laser (Lumen Dynamics). Approximate cell sizes were measured using cellSens Standard imaging software (Software version: 1.16, build 15,404, Olympus) and images were analysed in FIJI23 (Software version: 2.0.0-rc-69/1.52p, Image J).
Validation of size-based cell sorting by flow cytometry
Using cell measurements taken from histological analysis as a guide, a size-based cell sorting method was developed to isolate our target spermatogonial cells. A set of five size-specific beads (16.5 μm, 10.2 μm, 7.56 μm, 5.11 μm, 3.3 μm, Spherotech, Lake Forest, IL, USA) were analysed on a FACS Aria Fusion flow cytometer (BD Biosciences, New South Wales, Australia). These sizes cover the range of cell sizes seen in the testis, with sperm heads being approximately 2–3 μm and spermatogonia being over 10 μm in M.fluviatilis. Due to differences in the light scattering properties of plastic beads in comparison to live cells, these bead sizes can only be interpreted as a guide of scale and not as an exact size indication for cells in suspension. Using the scatter profile produced by these beads, two gates were set: the “A” gate surrounded events in the high forward scatter region on the scatter plot, approximately 9 μm and larger to capture larger cells such as spermatogonia; the “B” gate surrounded events in a low forward scatter region, between 2—5 μm, to capture smaller germ cells such as spermatids and spermatocytes. An unstained cell suspension was then sorted through these gates and sorted cells were pelleted by centrifugation (500 g for 15mins). Images were taken of live cells in suspension using the EVOS FL Auto 2 Imaging system (ThermoFisher Scientific) and cell sizes were measured in FIJI. Samples were then fixed in 2% PFA (Thermo Fisher Scientific) for 10 min and suspended in PBS.
Aliquots of each sample (A gate, B gate and an unsorted control) were smeared onto Superfrost Plus slides (ThermoFisher Scientific), baked overnight at 37 °C and stained with anti-Vasa antibody to determine the number of Vasa-positive cells in each sample. Briefly, the slides were washed with MilliQ water to remove any salt that was present and irrigated with wash buffer (0.1% BSA in PBS) before blocking with 10% goat serum, 0.1% Triton X in PBS for 45 min. Sections were stained with anti-Vasa antibody (1:200) in PBS containing 5% BSA for 1 h at room temperature, washed with wash buffer, incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500), and counterstained with Hoechst (1:1000). Sections were imaged on the EVOS FL Auto 2 and analysed using FIJI.
Cryopreservation protocol
This cryopreservation method was adapted from research described by Lee et al.14,15. Whole gonads weighing 0.0124 g ± 0.0095 g were transferred into 1.2-ml CryoTubes with 500 μl of cryomedia containing a permeating cryoprotectant, dimethyl sulfoxide (DMSO), ethylene glycol (EG), methanol or glycerol (all purchased from Merck), at concentrations ranging between 1.0 M and 2.0 M, with 0.1 M trehalose (Merck), and 1.5% BSA (Bovogen Biologicals Pty. Ltd, Victoria, Australia) in a mixed salt solution (~ 296 mOsm, pH 7.8) previously described by Lee et al.14. Control samples contained all components except the permeating cryoprotectant. Samples were equilibrated on ice for one hour and then cooled at a rate of -1 °C/minute in a CoolCell (Merck) in a -80 °C freezer for at least 3 h before being plunged into liquid nitrogen. Samples were held in liquid nitrogen for at least 24 h before thawing.
Thawing and cell suspension preparation
Samples were thawed in a 30 °C water bath for 1 min. The gonad was removed and gently blotted on a Kim-wipe to remove excess cryoprotectant residue and then rehydrated in three changes of handling medium (as described under “Animal husbandry and sample collection”) for 20 min per change (60 min total). After rehydration, the testis was placed in a tissue grinder with 500 μl of PBS and crushed. The tissue grinder was washed with another 500 μl of PBS resulting in a final volume of 1 ml. The cell suspension was passed through a 40 μm nylon filter to remove any large particulates prior to flow cytometry.
Viability assessment by flow cytometry
Cell suspensions were stained with the LIVE/DEAD Sperm Viability Kit (ThermoFisher Scientific) which included a membrane-permeating SYBR14 nucleic acid dye for detecting live cells and membrane-impermeable Propidium Iodide (PI) nucleic acid dye to detect membrane-compromised, presumably dead cells. SYBR14 was added and incubated for 5 min in the dark, followed by PI for a further 5-min incubation.
Prior to the assessment of experimental samples, the sized beads (Spherotech) were analysed on the FACS Aria Fusion flow cytometer. Using these beads as a guide, a gate was set for the approximate size of the spermatogonial cells based on our own histological analysis of this species and previous publications on fish in general24. An unstained control and two single stain controls (PI only or SYBR14 only) were included with the experimental samples in the analysis. The sample used for the PI-only control was flash frozen in liquid nitrogen three times to ensure a high percentage of dead cell to provide an adequate count for PI staining. Flow cytometry output was analysed in FlowJoTM25. Events captured by the gate were analysed for SYBR14 and PI spectra and divided into quartiles based on the absorbance of single stain controls (Fig. 1).
Figure 1
Flow cytometry scatter plots and gating method. (a) Analysis of size-specific beads shows five distinct clusters. (b) A gate is set to capture events from the 9 μm measurement and above. (c) Events detected in this region are replotted to determine SYB14 and PI absorbance. Events in the Q3 region are SYB14 positive and PI negative and therefore viable. In samples treated with a negative control (d), the majority of events falls in the Q1 region, with only propidium iodide detected (e).
Full size image
Statistical analysis
Statistical analysis was performed using GraphPad Prism version 8.1.2 for MacOS, GraphPad Software, La Jolla California USA, www.graphpad.com. Data is presented as mean ± standard deviation, with a p-value less than 0.05 considered statistically significant.
For cell gating data, the proportion of cell sizes in live cell suspensions in each treatment group was analysed using a chi-square. The percentage of Vasa-positive cells in the unsorted sample and the “A” gate was analysed using an un-paired t-test; data for the “B” gate was excluded as no Vasa-positive cells were detected.
For percentage viability data assumptions for normality and variance were met using the Shapiro–Wilk test and the Brown-Forsythe test, respectively. Following this, treatment groups were compared by one-way ANOVA and Tukey’s post hoc test. More
