The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.
Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.
Novel trends in science and biotechnology have led to the creation of thousands of new mutant lines of mice, Drosophila, zebrafish and other species used as model organisms in biomedical and other sciences1. Furthermore, as new technologies are developed and become available, the numbers of mutant lines steadily increase2. This leads to the necessity for a safe storage of genetic resources beyond the common practices of keeping breeding colonies. As a method which enables safe storage of genetic resources for an indefinite period of time, cryopreservation offers many advantages such as extension of reproductive season, circumvents the need for continuous maintenance of broodstock, and it is more cost- and labor-efficient2.
Protocols for sperm cryopreservation developed during the past several years2,3,4 offer the opportunity for successful storage of zebrafish male genetic material. However, cryopreservation of eggs or embryos in fish is not yet possible due to their complex structure and large amounts of yolk material. Recently, the practice of transplantation of primordial germ cells (PGCs) or spermatogonial stem cells (SSCs) offers a bypass to this barrier by developing into functional sperm and eggs after transplantation5. Therefore, cryopreservation of SSCs offers a new frontier in conservation of rare and valuable genetic resources.
Even though cryopreservation offers many advantages, the slow-rate freezing process generates several conditions that may lead to cell damage2. These include intracellular and extracellular ice formation, dehydration, cryoprotectant toxicity and others. Intracellular ice damages the cells, extracellular ice may lead to mechanical crushing of cells, while water diffusion from the cells during slow-rate freezing may lead to dehydration6. Recently, vitrification as a technique which prevents the negative effects of ice formation has been applied in the cryopreservation of fish gametes7,8,9. It presents an ultra-rapid cooling technique through which the internal and external media turn into an amorphous/glassy state without crystalizing into ice7,10. Successful vitrification of testicular and ovarian tissue has been evidenced in avian and mammalian species10,11,12, thus opening possibilities for its application in fish, as well.
In this study, we present the needle immersed vitrification (NIV) procedure for the cryopreservation of whole zebrafish testes. We demonstrate a reliable method for the isolation of zebrafish early-stage germ cells without contamination and a cryopreservation process that yields relatively high amounts of early-stage germ cells with a low presence of other cells, especially spermatozoa. To the best of our knowledge, this is the first study to demonstrate a detailed visualized protocol for ultra-rapid cooling of fish gonadal tissue and zebrafish germline cells. Additionally, repeatability of the method is demonstrated in five different zebrafish strains: AB wild type, casper (roy-/-; nacre-/-), leopard (leot1/t1), vasa [Tg(vas::eGFP)] and Wilms tumor [Tg(wt1b::eGFP 1)] transgenic line.
All methods described here have been approved by the Hungarian Animal Welfare Law.
1. Reagent Preparation
2. Testes Collection
3. Ultra-Rapid Cooling of the Testicular Tissue
4. Warming Procedure
5. Tissue Digestion
6. Viability Evaluation and Cell Counting
On average, the number of early-stage germ cells isolated from a single fresh zebrafish testis varied between 40,000 and 200,000 cells depending on the size of the fish. When digesting fresh zebrafish testes in all 5 strains, early-germ cells were not the only cells present in the cell suspensions (Figure 2). Beside the early-stage germ cells, numerous spermatozoa were found as well. On the other hand, there were far less spermatozoa after digestion of cryopreserved testes. This indicated that spermatozoa do not survive this ultra-rapid cooling protocol, and that they are most likely eliminated during the digestion process, which yields a much cleaner suspension of early-stage germ cells.
There were no significant differences in the number of early-stage germ cells between the left and right testes (1 ± 0.5 × 105 vs 1.1 ± 0.7 × 105) of a single individual demonstrated by digesting fresh testes of three AB zebrafish males (one-way ANOVA; F(1,4) = 0.04, p = 0.85). In all zebrafish lines used in this study, the current ultra-rapid cooling protocol yielded average viability rates higher than 50% (Table 1).
AB wild type | Casper | Leopard | Vasa transgenic | Wilms tumor transgenic | ||
Number of live cells (×104) | Fresh testis | 14.5 ± 3.9 | 9.5 ± 2.3 | 12.7 ± 2.4 | 14.5 ± 5.6 | 4.8 ± 1.8 |
Vitrified testis | 8.7 ± 2.8 | 7.2 ± 3.0 | 8.8 ± 1.7 | 7.6 ± 3.7 | 2.4 ± 0.7 | |
Viability (%) | 58 ± 9 | 72 ± 13 | 69 ± 1 | 50 ± 6 | 53 ± 13 |
Table 1. Number of live cells and viability percentages (mean ± SD) obtained from fresh or vitrified/warmed zebrafish testis. Results are presented for the five tested zebrafish lines: AB wild type, casper (roy-/-; nacre-/-), leopard (leot1/t1), vasa [Tg(vas::eGFP)] and Wilms tumor [Tg(wt1b::eGFP 1)] transgenic line.
Figure 1. Dissected adult zebrafish demonstrating the position of various anatomical structures. Please click here to view a larger version of this figure.
Figure 2. Cells suspensions prepared from fresh and vitrified/warmed zebrafish testicular tissue from five tested zebrafish lines (AB wild type, casper (roy-/-; nacre-/-), leopard (leot1/t1), vasa [Tg(vas::eGFP)] and Wilms tumor [Tg(wt1b::eGFP 1)] transgenic line) imaged with phase-contrast microscopy. Scale bar: 40 µm. Please click here to view a larger version of this figure.
The main purpose of this study was to adapt the needle immersed ultra-rapid cooling procedure developed for avian and mammalian species10,11,12 to the cryopreservation of fish testis (zebrafish as a model organism). Most of the previous studies regarding cryopreservation of zebrafish genetic resources were primarily focused on cryopreservation of zebrafish sperm2,3,4. However, protocols for cryopreservation of mature oocytes and embryos have not been developed yet, even though some recent studies demonstrate that cryopreservation of early-stage oocytes is plausible14,15. In this study we demonstrated successful cryopreservation of zebrafish spermatogonia which offers new possibilities for the storage of valuable genetic resources, especially since after transplantation they can mature into both male and female gametes5.
To the best of our knowledge, this is the first study dealing with the ultra-rapid cooling of zebrafish tissue by the NIV method. Similar studies included vitrification of zebrafish testes in 0.25 mL plastic straws16 and vitrification of zebrafish ovaries in closed metal containers14. The main advantage of NIV compared to the two mentioned containers is the direct exposure of testes to liquid nitrogen with minimal volumes of cryoprotectants being attached to them, therefore maximizing the cooling rate10. The increase in cooling rate reduces the required concentration of cryoprotectants, thereby reducing their toxicity. Furthermore, all tissue pieces can be exposed to the cryoprotectants and liquid nitrogen synchronously. However, direct exposure to liquid nitrogen may have one disadvantage with regard to cross-contamination. It is possible that bacteria or viruses are present in the liquid nitrogen and that direct tissue exposure may lead to contamination. Therefore, we would suggest to refrain from reusing liquid nitrogen when conducting NIV and to discard the used liquid nitrogen after cooling. Proposed metal containers offer advantages in this regard14, however they were custom made and are not easily accessible to all laboratories.
When comparing ultra-rapid cooling to slow-rate freezing, one crucial advantage of ultra-rapid cooling is the absence of spermatozoa after digestion. Slow-rate freezing methods demonstrated in some cyprinid fish species that similar protocols yield comparable viability of both early-stage germ cells and spermatozoa17 resulting in high spermatozoa numbers after digestion of the cryopreserved tissue. Our results in zebrafish (present study), common carp and goldfish (unpublished results) indicate that there are very few spermatozoa after digestion of the warmed tissues and that they do not survive this procedure and get digested which simplifies downstream applications since no additional enrichment procedures are needed.
There are several critical steps within this protocol. The first is to prevent any contamination during the isolation process since it may lead to a decrease in the number of cells obtained and may hinder any downstream applications. One of the crucial steps is excision of testes where bacterial contamination might occur from damaged guts (therefore it is best not to injure or completely remove the intestines) or from the skin if using the same dissection tool for cutting the skin and removing the testes. Secondly, take care when pinning testes to the acupuncture needle since it is possible that the testes fall off or slide down. Currently, we are testing different methods to prevent the testes from sliding during the vitrification procedure (liquid nitrogen temperatures). Third, pay attention to the period of exposure to the cryoprotectants. Exposure of testes (and thus cells) to high cryoprotectant concentrations (especially in the vitrification solution) for too long may lead to cell death due to cryoprotectant toxicity. Also, take care when plunging the needles into liquid nitrogen; wipe off the excess of VS since it may affect the cooling process and plunge the needles quickly in order to avoid exposure to liquid nitrogen vapor. Lastly, transfer the testes from liquid nitrogen into the warming media quickly in order to avoid premature warming in the air during transfer.
In the present paper we present the procedure for cryopreservation of testes from five zebrafish strains by using MeOH, PG and Me2SO as permeating cryoprotectants. The method yields reliable results for all tested zebrafish strains with a cell viability of above 50%. It is possible to use this method with modifications for other species, as well. Firstly, each cryopreservation protocol is species specific, and different cryoprotectants yield different efficiencies in different species (i.e. ethylene glycol yielded the highest viability in acipenserids18 while Me2SO yielded the highest viability in salmonids19 and cyprinids17). Furthermore attention should be given to the concentrations used as well. The present study and the study conducted on brown trout Salmo trutta ovaries15 demonstrate that the best results are obtained by using the same concentration of two cryoprotectants, however this may vary in other species. Lastly, zebrafish testes are very small, and it is even possible to pin several testes on one needle. When working with species with larger testes, the size of testicular pieces which are used is a very important factor. We suggest to increase exposure times for larger tissue pieces taking into account cryoprotectant toxicity and efficiency.
The authors have nothing to disclose.
This study was supported by the National Research, Development and Innovation Office of Hungary (grant 116912 to ÁH), the COST office (Food and Agriculture COST Action FA1205: AQUAGAMETE), the Stipendium Hungaricum Scholarship Programme (grant to ZM) and the New Hungarian National Excellence Predoctoral Fellowship (grant to EK).
Leibovitz media (L-15) | Sigma-Aldrich | L1518 | Supplemented with L-glutamine |
Fetal bovine serum (FBS) | Sigma-Aldrich | F9665 | |
Tricaine methanesulfonate (MS-222) | Sigma-Aldrich | E10521 | |
HEPES | Sigma-Aldrich | H3375 | |
Sucrose | Acros Organics | 57-50-1 | |
Trehalose | Acros Organics | 99-20-7 | Dihydrate |
Methanol | Reanal | 20740-0-08-65 | |
Propylene glycol | Reanal | 08860-1-08-65 | |
Dimethyl sulfoxide | Reanal | 00190-1-01-65 | |
Collagenase | Gibco | 9001-12-1 | |
Trypsin | Sigma-Aldrich | T8003 | |
DNase I | Panreac AppliChem | A3778 | |
Trypan blue | Sigma-Aldrich | T6146 | |
Phospate buffered saline (PBS) | Sigma-Aldrich | P4417 | Tablets for preparation of 200 ml PBS solution |