Zebrafish ( Danio rerio ) Gynogenetic Production by Heat Shock: Comparison Between Mitotic and Meiotic Treatment

Abstract

Inbred species are useful resources for a variety of biomedical research applications. To create isogenic zebrafish, it is feasible to stop meiosis II (repeatedly) or mitosis (two times) in a haploid embryo by applying pressure or by delivering a heat shock, respectively. In this study, to improve the repeatability, we suggest a less complicated approach based on sperm ultraviolet-C (UV-C) exposure for a shorter period followed by heat shock at various temperatures, eliminating the use of pressure in meiotic therapy since heat shock is more accessible to laboratories. In this study, the survivability rates of meiotic (Mei) and mitotic (Mit) gynogenesis offspring produced by various combinations of irradiation (28.5, 105, and 210 mJ/cm²) and temperature (Mei: 40.40°C, 40.60°C, or 40.90°C; Mt: 41.40°C, 41.90°C, or 42.45°C) were compared with diploid (C) and haploid (H) controls. Our findings demonstrated that 40.60°C and 41.90°C were the most suitable temperatures to produce meiotic and mitotic gynogenesis, respectively, whereas 28.5 mJ/cm² was more successful in ensuring haploid embryos. As a result, we deduced that meiotic gynogenesis produces more viable offspring than the mitotic approach and requires a lower temperature to maintain the second polar body.

Introduction

Genetically specified laboratory animals improve the repeatability of tests and also allow the use of a smaller number of animals and decrease false negative results.¹ But a careful strain identification is crucial in determining the generality of research findings.² The Zebrafish (Danio rerio), one of the most outstanding animal models, has gained popularity as an animal model for biological studies in areas, including neurobiology, genetics, developmental toxicity, and evolutionary biology due to its significant genetic similarity to humans.^3–8 Because laboratory animals are utilized as biological reagents, gynogenesis is one approach that provides isogeny faster than inbred normal diploid lineages, which is necessary to ensure the reproducibility of the tests.^2,9

Isogeny describes populations having a single allelic sample that are genetically homogenous.¹⁰ The Zebrafish exhibit inbreeding with 14 generations of brother reproduction.¹¹ However, isogenic animals may be created in two or three generations by using a variety of chromosomal alteration techniques, such as gynogenesis.¹² That technique has already been applied to several commercially important freshwater and marine fish species, including Carassius auratus, Oncorhynchus rhodurus, Oryzias latipes, Misgurnus anguillicaudatus, Oreochromis niloticus, Cyprinus carpio, Dicentrarchus labrax L., Petromyzon marinus, and Pseudosciaena croce.^12–22

When it comes to meiotic gynogenesis, after the end of meiosis II, which spontaneously happens after sperm fertilization, the second polar body is kept in place by pressure, as is frequently described in articles using zebrafish, or by heat shock, as described for other fish species.^23–29 In any event, centrosomes and microtubules may become unstable and disorganized as a result of these processes.¹¹ The genetic material from this corpuscle is preserved and united with the primary DNA to ensure the creation of a diploid zygote.³⁰

This approach ensures homozygous embryos at loci that did not undergo recombination during meiosis I but are susceptible to future crossing over.^31,32 In contrast, in mitotic gynogenesis, heat shocks halt the cell cycle and prevent the development of spindle fibers.³³ The turning zygote becomes diploid and hence homozygous for all loci, with the downside of having a poor survival rate as a result of the division stopping and daughter cells fusing.^12,33 This technique is used to create clonal lineages with a second generation of gynogenesis, and any crossing-over results in the same chromosome since they are identical.^34,35

Zebrafish inbreeding can be used for a variety of purposes, including cancer research, genetic mapping, heritability, sexual differentiation, and genetic improvement.^35–38 Therefore, the goal of this study is to evaluate the propensity of producing gynogenetic organisms in a comparative investigation of both meiotic and mitotic heat gynogenesis, taking into account the relevance of zebrafish as an animal model in current research and the distinct lack of genetically specified animals. To achieve this goal, three ultraviolet-C (UV-C) dosages were tested to examine sperm behavior and then temperatures were applied to freshly fertilized haploid embryos to ensure chromosomal diploidization by meiosis or mitosis interruption. Both methodologies were compared with each other.

Materials and Methods

The groups in this study are called meiotic gynogenesis (Mei), mitotic gynogenesis (Mit), haploid control (H), and diploid control (C). The reproductions happened in triplicate. This research was approved by the Federal University of São Carlos' Animal Use Ethics Committee (authorization 6211040219 and 8222200722). All animal care and health measures were followed under literature-standard procedures.³⁹

At first, the sensitivity of the spermatozoa of the males was verified through the analysis of sperm motility using three different dosages of ultraviolet light: 28.5 mJ/cm² (20 s of exposition), 105 mJ/cm² (78 s of exposition) and 210 mJ/cm² (148 s of exposition). With the use of this assessment, it was possible to determine the dosage that would damage the DNA without shortening the motility time by >10% compared with unirradiated sperm, besides producing haploid embryos.

For gynogenesis, four wild phenotype males were anesthetized with tricaine (400 mg/L), and the sperm were placed in a modified Ringer's solution (128.3 mM NaCl, 2.6 mM KCl, 1.8 mM CaCl₂, 2.1 mM MgCl₂, and 2.4 mM NaHCO₂) on ice. Altogether, they were tested different temperatures for meiotic and mitotic gynogenesis 41.4°C, 40.4°C, 40.6°C, and 40.9°C, and 41.4°C, 41.9°C, and 42.45°C, respectively.

Golden-type Zebrafish females were used as a visual control of the success of the procedure.⁴⁰ Each reproduction originated from all groups. Gamete activation was carried out by applying E3 embryonic medium at 28°C. At 4 min after fertilization, haploid embryos destined for Mei were placed in a water bath at 40.6°C for 2 min, then returned to the embryonic medium at 28°C. At 18 mpf haploid embryos Mit were put in a 41.9°C water bath for 2 min, then returned to 28°C. To determine the efficacy of denaturing the paternal genetic material, a fraction of the haploid embryos was not duplicated.

The effectiveness of the Mit therapy was evaluated by comparing it with other treatments during the period of four-cell duplication. The viability analyzed included just survival and the occurrence of abnormalities in groups included developmental alterations; therefore, the correct development of somites, notochord, eyes, brain, heart, ears, spine, anal opening, swim bladder inflation, and haploid syndrome, as shown in Ref.⁴¹ So, larvae that achieved 120 hpf without the aforementioned abnormalities were regarded as healthy.

Analysis of variance two-way and the Tukey tests were used to examine all the parameters (viability, malformations, and healthy), with p < 0.05 being regarded as statistically significant. In addition, the average and confidence interval of 95% of the proportion of animals that were alive in each gynogenesis between 24 and 120 h after fertilization were examined. Only viability was taken into account at this time because defects are just clearly visible once the embryos hatch. Tests of Between-Subjects Effects were applied. All calculations and percentages were made using the total number of fertilized eggs as a base.

The confirmation of the successful inactivation of the sperm's genetic material was evaluated by the presence of haploid syndrome from 48 hpf, chromosome counts at 26 hpf and phenotypical analysis at 48 hpf in the H group.^31,37,38 The haploid syndrome was characterized by a shortened body, reduced size of the eyes, pericardial edema, and failure in the circulation of the trunk.^38,42 The methodology described in Ref.,³⁸ with adjustments was used to perform metaphase chromosomal counts in part H.

To do this, 10 randomly chosen embryos were used and prefixation was employed using 500 μL of fixative Carnoy 2 (3:1 methanol to acetic acid) while the embryos were still in sodium citrate for 10 min before the fixation suggested by the authors. In addition, cell waves were generated using the resuspension method to burst the cells. The absence of spots across the body, as well as black eyes, was used to phenotypically assess the only maternal component in the progeny. Once animals with the golden phenotype homozygous for the gold mutation and have genotypes that are recessive to the wild-type genotype, this happens.

Chromosome count at 26 hpf and phenotypic analysis at 48 hpf in the Mit and Mei groups, in the identical manner as indicated for the haploid control, were used to determine whether or not gynogenesis had been effective.

Results

The average motility of intact spermatozoa was 125.2 s, whereas 25 mJ/cm² averaged 114 s, 105 mJ/cm² averaged 93.8 s, and 210 mJ/cm² was lethal. With these results, only sperm subjected to 25 mJ/cm² were used for fertilization, since this dosage guaranteed sufficient gametic viability time for fertilization rate >85% and to create haploid embryos with severe haploid syndrome.

In mitotic gynogenesis, 41.4°C was lethal for the embryos even at 24 h, as well as 40.9°C; 40.4°C was insufficient for duplication and 40.6°C showed better results. For mitotic gynogenesis, 41.4°C was insufficient to interrupt mitosis, whereas 42.45°C was lethal; 41.9°C showed better results.

While C, H, and Mei had four cells, 95% of Mit only had two at 1.3 h (Fig. 1). At 24 h all H had malformation and no tail detachment (Fig. 1—24 h). While all of H had 25 chromosomes, ∼93% and 91% of Mit and Mei metaphases, respectively, had 50 chromosomes, as C. This demonstrates that the meiosis and mitosis duplication procedures were effective and that the sperm DNA was damaged. Although chromosomal doubling failed and Mit and Mei metaphases with 25 chromosomes were observed, there was no aneuploidy in the results (Fig. 2).

FIG. 1.

Microscopy photos of all stages (lines) and treatments (columns). Line 1.3 h is the embryo's development throughout 1 h and 20 min, displaying the number of cells in each group: C, H, and Mei each have four cells, whereas Mit has just two cells. The second line is 24 hpf embryo's development. C, Mei, and Mit had normal development with eye formation and tail detachment. H had a short tail and malformation of eyes. The third line is the 48 hpf embryo's development showing the C, Mei, and Mit groups with normal development and H group with malformation (Haploid Syndrome). C had pigmentation characterized by black eyes and black points in all the body, whereas H, Mei, and Mit had no black points in the body and the eyes are not black. The fourth line is the 72 hpf larval development showing: C groups with normal development; H groups with haploid syndrome characterized by a shortened body, reduced eye size, edema of the pericardium, and trunk circulation failure; two samples of larvae from Mei and Mit groups with normal development (Mei A and Mit A), and lordosis, pericardial edema, hatch delay, respectively (Mei B and Mit B). The fifth line is the 120 hpf larval development showing: C groups with normal development; two samples of larvae from Mit and Mei (Mei A and Mit A) groups with normal and without inflated anatomy bladder, respectively (Mei B and Mit B).

FIG. 2.

Microscopic pictures (objective of 100 × ) of embryos 26 hpf with metaphase chromosomes, with the C, Mit, and Mei groups each containing 50 chromosomes and the H group having 25. Were used 10 embryos each clutch. While all of H had 25 chromosomes, ∼93% and 91% of Mit and Mei metaphases, respectively, had 50 chromosomes, as C. 10 μm scale.

While H, Mit, and Mei did exhibit body and eye hypopigmentation 48 h after fertilization, C did wild pigmentation (Fig. 1—48 h). All C had normal development at 72 hpf and all H displayed severe haploid syndrome, as demonstrated in Ref.³⁸ (Fig. 1—72 h).

At 120 hpf, all C had inflated swim bladder, but not all Mit and Mei presented such condition (Fig. 1—120 h). Each therapy shown had different effects on deformities, survival, and health. The Mit group has the lowest average survival, followed by H and Mei, according to graph (A) (Fig. 3). The rate of survival for the control group was 100%. The H group has the highest average malformation, followed by Mei and Mit, as shown in graph (B) (Fig. 3). The malformation-free group was the control. The Mei group, followed by Mit and H, has the largest percentage of healthy larvae, according to graph (C) (Fig. 3). All the animals in the control group were in good health.

FIG. 3.

Graphs displaying the mean (with standard deviation) (A) values of survival rates, (B) malformations, and (C) the rate of healthy larvae at 120 hpf. Data were analyzed using the two-way Analysis of variance test, followed by Tukey's post hoc test. Different letters represent significant differences with a p-value <0.05. The viability analyzed was just survival and the occurrence of abnormalities in groups included developmental alterations, therefore, the correct development of somites, notochord, eyes, brain, heart, ears, spine, anal opening, swim bladder inflation, and haploid syndrome. So, larvae that achieved 120 hpf without the aforementioned abnormalities were regarded as healthy. All calculations and percentages were made using the total number of fertilized eggs as a base.

When analyzing each condition over the time of initial development (Fig. 4), we see that the biggest drop in survival in meiotic occurs until 48 h (84.57% ± 2.38% in 24 h, 69.85% ± 3.0% in 48 h and, after that, tends to remain constant). As for the mitotic, there is a marked mortality up to 72 h (48.17% ± 1.80% in 24 h, 35.70% ± 1.44% in 48 h, and 26.07% ± 1.32% in 72 h), for which the survival curve softens (Fig. 4). In addition, the statistical test showed a significant difference between groups and between embryonic periods (p < 0.01).

FIG. 4.

Graph displaying the mean (with 95% confidence interval) values of viability rates between 24 and 120 h postfertilization. Only viability was taken into account at this time because defects are just clearly seen once the embryos hatch. All calculations and percentages were made using the total number of fertilized eggs as a base.

Discussion

To create gynogenetic Zebrafish, this study proposes a unique systematization based on heat shock. Distinct methods using thermal and pressure shock to produce different fishes with mitotic and meiotic gynogenesis were described previously.^{28,32,43–46} The discussion on the optimum strategy for creating lineages with defined genetics and the comparison of the survivability and health of animals produced by meiotic and mitotic gynogenesis are unique contributions made by this study. The distinct UV-C dosages exposed in this study, using a dosage attained in a shorter amount of time than those published in Refs.^38,42 ensures that the spermatozoa are vulnerable for a shorter amount of time in a hostile environment. In addition, for the creation of meiotic gynogetics, all published protocols to date have exclusively utilized pressure shock in Zebrafish, the methodology used to create the first gynogenetic lineage by Ref.⁴⁷

We used a different time indicated for pressure as used in Refs.^39,47 and we proposed the same time described in Ref.⁴⁸ that used other species and techniques. We introduce the use of heat shock meiotic treatment, before used only in pisciculture to produce triploid.^49–52 Our study supports and extends approaches that are easily accessible in many laboratories that wish to use the basic concept of gynogenesis in zebrafish for a wide range of research purposes, such as maternal mutation and haploid screening, as done in Refs.^32,53,54 in addition to the production of a controlled genetic lineage. Finally, adding at previous studies such as Ref.⁵⁵ we found that there is a narrow temperature range that allows the duplication of maternal genetic material during the transient window of resilience of early development.

In this context, when performed three or more times, meiotic gynogenesis may be more advantageous than mitotic gynogenesis for the creation of a genetically known lineage.¹² If the meiotic gynogenesis is repeated in the progeny, the distal and proximal loci will be fixed as a continuous heterozygous and homozygous condition, resulting in a heterozygous clone.^12,56 This study showed that meiotic gynogenesis has this advantage since it generates more healthy embryos and has reduced offspring mortality (Fig. 3). In subsequent generations, the survival rate of meiotic gynogenetic individuals appears to increase, which may be related to the decline in recessive lethal genes.⁵⁷

The probability of death increases with temperature, as is the case, for instance, in the herein analyzed mitotic group. There is greater survival but a heightened incidence of deformities developing when the temperature is lower, as is required in the case of the meiotic group. In this context, our results show that the total mortality in mitotic gynogenesis is greater than mortality added to malformations in meiotic gynogenesis, resulting in fewer healthy animals at the end of 120 hpf, complementing other investigations.^11,12,23 Reduced gene expression was also seen in gynogenetic lineages produced at high temperatures.^58–60

When analyzing day by day both gynogenesis we see that there are differences in the pattern. The consequences of heat shock are more marked in mitotic gynogenesis-average survival <50% in the first 24 h-but it is less impactful in the first 24 h of meiosis, results that complement the study of Ref.⁵⁵ The temperature inherent to each process, added to the expression of deleterious genes when in homozygosis, explains this tendency to have a sudden drop in the survival of mitotic up to 72 h of life. However, the mild temperature and greater chances of crossing over in the miotics ensure that the decrease in survival takes place within 48 h. In the same context, keeping the polar body requires a lower temperature than stopping a cell cycle, implying that early embryos are fragile and cannot withstand the same temperatures as slightly older embryos. So, we demonstrated that a brief time is essential to determining the fragility of the embryos due to the rapid development of zebrafish.

Cell division, chromosome count, phenotype, and haploid syndrome are all indicators of the success of gynogenesis-related parameters and point to the proper application of the methodology as performed in other studies^31,37,39 and expanding the methods of verifying the success of the procedure. After reaching the ideal temperature shock for complete maternal genome duplication, our Mit embryos exhibited two cells, whereas Mei, C, and H embryos revealed four. This most likely occurred as a result of the heat shock disrupting the Mit embryos' embryonic cell cycle and delaying cellular division. This does not occur in Mei because when the second polar body is retained for diploidization using this approach, cell duplication cycles are not disrupted, proving that the mitotic gynogenesis operation was successful as shown in Ref.³⁷

The percentage of two-cell Mit group embryos is similar to the percentage of 2N metaphases, which complements the works^37–39 and show the effectiveness of using these combined methodologies to achieve greater reliability of the results. About the golden phenotype, in addition to serving as a visible indicator of gynogenesis in this context, the condition is used in studies on nuclear transplants or even the analysis of cryopreserved gametes.^38,61 Thus, our study extends the definition of effective gynogenesis to also include chromosome counting, female recessive mutations, and haploid syndrome.

Tests carried out at 42.15°C in our study also produced positive results in the mitotic clutch, so these findings corroborate the work,⁵⁵ and we add to this result that there is a temperature range in which it is possible to have chromosomal duplication of haploid embryos in 18 min after the fertilization. In the same vein, our findings show that diploidization of haploid embryos at 41.4°C is not feasible between 18 and 20 min after fertilization.

When exposed to a higher temperature and subsequent shock, our results indicate that the offspring have a higher survival chance until maturity with good take care during the larviculture: ∼10%–14% as opposed to previous investigation that estimate <5%,³⁸ and corroborating with Ref.⁵⁵ In this context, other gynogenetic fish lineages showed changes in the expression of genes linked to genome stability and DNA repair.^58,62

When applied the identical settings as in this study for mitotic gynogenesis in lineage from another origin, 100% of animals died in <96 h of life. Thus, even if it is not the main topic of discussion in other studies in the scientific literature, the issue of the intrinsic sensitivity of each strain to radiation and thermal shock in each study is relevant and significant in studies such as this one. Everything from food to water temperature that is used to create and maintain these creatures has the potential to have an impact on physiological and behavioral features.^63–65 In this way, each researcher must examine and assess these factors and adjust the methodology, knowing the narrow temperature range that exists as explored in this study.

Finally, once the larvae reach 120 h age without any type of malformation, these animals have the potential to reach adulthood, and the success of growth depends on larviculture. This stage is an important component of the Zebrafish model because of, among other things, the fish's tiny size and restricted swimming range at the start of feeding and for the technique to be successful, researchers must take into account elements such as the behavior and dietary requirements.⁶⁶ Thus, this study compared the health, survival, and malformations of both gynogenesis during, exclusively, the period that least depends on external care and is closely linked to the gynogenesis process itself.

Conclusions

We demonstrate how to build this method utilizing only thermal shock as opposed to pressure shock, introducing new suitable methods to maximize the health of the progeny. We discovered that retaining the second polar body in producing meiotic gynogenetic requires lower temperatures than producing mitotic gynogenetic, which requires the interruption of a cell cycle. Offspring of meiotic gynogenesis also seem to be more viable.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

The authors would like to thank FAPESP (São Paulo Research Foundation), file 2018/18910-6, and CNPq (National Council for Scientific and Technological Development), file 425668/2018-5.

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