Reprod Dom Anim 50, 256–262 (2015); doi: 10.1111/rda.12480 ISSN 0936–6768
Induction of Gynogenetic and Androgenetic Haploid and Doubled Haploid Development in the Brown Trout (Salmo trutta Linnaeus 1758) O Michalik1, S Dobosz2, T Zalewski2, M Sapota3 and K Ocalewicz3,4 1 Department of Molecular Evolution, University of Gdansk, Gdansk, Poland; 2Department of Salmonid Research, Inland Fisheries Institute in Olsztyn, Rutki, Zukowo, Poland; 3Department of Marine Biology and Ecology, Institute of Oceanography, University of Gdansk, Gdynia, Poland; 4 Department of Ichthyology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
Contents Gynogenetic and androgenetic brown trout (Salmo trutta Linnaeus 1758) haploids (Hs) and doubled haploids (DHs) were produced in the present research. Haploid development was induced by radiation-induced genetic inactivation of spermatozoa (gynogenesis) or eggs (androgenesis) before insemination. To provide DHs, gynogenetic and androgenetic haploid zygotes were subjected to the high pressure shock to suppress the first mitotic cleavage. Among haploids, gynogenetic embryos were showing lower mortality when compared to the androgenetic embryos; however, most of them die before the first feeding stage. Gynogenetic doubled haploids provided in the course of the brown trout eggs activation performed by homologous and heterologous sperm (rainbow trout) were developing equally showing hatching rates of 14.76 2.4% and 16.14 2.90% and the survival rates at the first feeding stage of 10.48 3.48% and 12.78 2.18%, respectively. Significantly, lower survival rate was observed among androgenetic progenies from the diploid groups with only few specimens that survived to the first feeding stage. Cytogenetic survey showed that among embryos from the diploid variants of the research, only gynogenetic individuals possessed doubled sets of chromosomes. Thus, it is reasonable to assume that radiation employed for the genetic inactivation of the brown trout eggs misaligned mechanism responsible for the cell divisions and might have delayed or even arrested the first mitotic cleavage in the androgenetic brown trout zygotes. Moreover, protocol for the radiationinduced inactivation of the paternal and maternal genome should be adjusted as some of the cytogenetically surveyed gynogenetic and androgenetic embryos exhibited fragments of the irradiated chromosomes.
Introduction In the latest decades, chromosome set manipulation techniques namely uniparental chromosome inheritance (gynogenesis and androgenesis) and multiplication of the chromosome sets have been developed in many fish species including those of the high economic importance as well as species recognized as biomedical models (Komen and Thorgaard 2007). Haploid development in the fish may be induced by radiation-induced damage of the nuclear DNA in either spermatozoa (gynogenesis) or eggs (androgenesis) before insemination. In turn, doubled haploids (DHs) are provided in the course of the three-step process including irradiation of the spermatozoa or the eggs (i), insemination (ii) and duplication of the maternal or the paternal haploid set of chromo-
somes (iii) accomplished by exposition of the haploid zygotes to the temperature or pressure shock suppressing the first mitotic cleavage (Pandian and Koteeswaran 1998). Although most of the haploid fish die during embryogenesis or soon after hatching, such embryos are excellent source of the haploid embryonic stem cells (Yi et al. 2009). Both haploids and doubled haploids are important in studies concerning recessive alleles. Viable doubled haploids have been used for the production of the fish homozygous clonal lines (Komen and Thorgaard 2007). Due to the lack of heterozygotes, haploids and doubled haploids significantly facilitate studies concerning genetic mapping and genome sequencing. This feature of the doubled haploids and clones simplifies detailed linkage analyses enabling identification of the chromosome regions connected with the quantitative traits loci (QTL) (Komen and Thorgaard 2007). Moreover, androgenetic and gynogenetic fish have been used in studies concerning genetic basis of the sex determination process and differentiation of the sex chromosomes (Ocalewicz et al. 2007; Chen et al. 2009). Androgenesis has been also applied to recover nuclear genomic information of the endangered fish lines or species from the normal and cryopreserved spermatozoa (Babiak et al. 2002a) and to investigate the effects of the cytoplasm and mitochondria on the embryo development (Brown and Thorgaard 2002). Unfortunately, despite the fact that doubled haploids survive better than haploids, high mortality observed among gynogenetic and androgenetic fish is still a limiting factor in their wide application. The low survival of the doubled haploids during embryogenesis and after hatching seems to be caused by expression of the lethal alleles, and the side effects of the manipulations performed on the eggs and the zygotes. Inactivation of the gametes with UV- or X-radiation and application of the heat and pressure shock to the zygotes probably affect also many other cellular organelles and developmental mechanisms what results in the various disturbances during the ontogeny and consequently increases mortality of the doubled haploid individuals. Early development stages in the fishes are solely directed by the maternal factors until the mid-blastula transition (Kane and Kimmel 1993; Pelegri 2003). Thus, radiationinduced misregulation of such mechanism may result in the lower survival of the androgenetic DHs when © 2015 Blackwell Verlag GmbH
Androgenetic and Gynogenetic Brown Trout
257
compared to the gynogenetic DHs that are developing in the non-irradiated eggs. To verify this hypothesis, we induced gynogenetic and androgenetic development of the haploid (H) and doubled haploid (DH) brown trout (Salmo trutta Linnaeus 1758). Survival of the androgenetic and gynogenetic specimens was monitored during their ontogeny until the swim-up stage. Moreover, to confirm ploidy level and to identify any residues of the irradiated paternal or maternal chromosomes, embryos from the haploid and doubled haploid variants of the experiment were cytogenetically studied. Homozygosity of the doubled haploids was assessed by the microsatellite DNA analysis. Consequences of the eggs irradiation and exposition of zygotes to the high pressure shock for the fish development are also discussed in the present study.
(approximately 600, 500 and 750 eggs in each, respectively). Inactivation of the parental nuclear DNA To inactivate maternal nuclear genome, eggs for the androgenetic variants of the research were transported on ice to the Clinic of Oncology and Radiotherapy, University Clinical Center, Medical University of Gdansk, and irradiated with 420 Gy of X-rays using linear accelerator Clinac 600 (Varian Medical Systems, Palo Alto, CA, USA) (Michalik et al. 2014). After the treatment, irradiated eggs carefully protected from light were transported back to the hatchery. Inactivation of the paternal nuclear DNA in the sperm was performed with UV light in the dose of 2075 lW/cm2 (Goryczko et al. 1991).
Material and Methods Gamete collection and experimental design Brown trout and rainbow trout (Oncorhynchus mykiss Walbaum 1792) gamete donors derived from the broodstocks kept at the Department of Salmonid Research, Inland Fisheries Institute in Olsztyn, Rutki, Poland. Diploid number of the chromosomes (2n) and number of the chromosome arms (FN) in the examined brown trout individuals equalled 80 and 100–102, respectively (Woznicki et al. 2000). Moreover, milt for the interspecies gynogenetic variant of the research was collected from the rainbow trout from Rutki strain characterized by the chromosome number varied from 2n = 59–62 and stable chromosome arm number FN = 104 (Ocalewicz 2002). Eggs of the highest quality provided from 15 brown trout females were pooled. Milt from four brown trout (B1, B2, B3, B4) and one rainbow trout (R) males was collected as individual lots. All gametes were kept at 2°C for the further use. In one of the gynogenetic variant, eggs were inseminated with the semen from the rainbow trout, and in the other gynogenetic variant, sperm derived from brown trout (B1) was used (Table 1). To inseminate eggs in the androgenetic variants, semen from three brown trout males (B2, B3 and B4) was used (Table 1). Every variant of the experiment contained control (C), haploid (h) and doubled haploid (d) groups Table 1. Induction of gynogenetic and androgenetic development of haploid and doubled haploid brown trout (Salmo trutta): summary of the experimental design Experimental variants
Gynogenesis Ia
Gynogenesis IIb
Males Experimental groups Controls Haploids Doubled haploids
R
B1
B2
B3
B4
Cg1 Gh1 Gd1
Cg2 Gh2 Gd2
Ca1 Ah1 Ad1
Ca2 Ah2 Ad2
Ca3 Ah3 Ad3
R, rainbow trout; B, brown trout. a gynogenesis with the heterologous sperm. b gynogenesis with the homologous sperm.
© 2015 Blackwell Verlag GmbH
Androgenesis
Insemination and diploidization Batches of eggs were inseminated in the proportion of 0.1 ml semen for 200 eggs in the presence of Billard fertilization diluent (154 mM NaCl and 1 mM Ca2+, buffered to pH 9.0 with 20 mM Tris + 30 mM glycine) (Billard 1992). After 3 min, eggs were rinsed with the water. Controls for the gynogenetic (Cg1 and Cg2) and androgenetic (Ca1, Ca2, Ca3) variants and all haploid batches (Gh1, Gh2, Ah1, Ah2, Ah3) (Table 1) were then placed in the incubation apparatus. Batches intended for the diploid groups were further incubated at 10°C. To double parental set of chromosomes in the inseminated eggs from the gynogenetic and androgenetic diploid groups (Gd1, Gd2, Ad1, Ad2, Ad3) (Table 1) were treated with the high hydrostatic pressure (10 000 psi) applied 450 min after insemination and lasted 5 min (Babiak et al. 2002b; Preston et al. 2013). All eggs were incubated in three separate replicates at 6–8°C. Survival of androgenetic and gynogenetic embryos and hatchlings Fertilization rate was estimated 24 h after insemination. Afterwards, dead embryos and fry were removed from the batches every day until the end of experiment. First measurement of survival among embryos was performed 36 days post-fertilization (dpf) at the eyed stage. Living larvae were counted just after hatching (57 dpf) and finally at the swim-up stage (80 dpf). Cytogenetic analysis of embryos Metaphase plates were prepared from the haploid and the diploid eyed embryos from all experimental variants according in vivo (technique described by Michalik et al. (2014)). Chromosomes were stained with 40 , 6-diamidino-2-phenylindole DAPI (Vector, Burlingame, CA, USA) and observed under a Zeiss Axio Imager A1microscope equipped with a Zeiss EC Plan-Neofluar 100x/1.3 oil objective, a fluorescent lamp and a digital
258
O Michalik, S Dobosz, T Zalewski, M Sapota and K Ocalewicz
camera (Applied Spectral Imaging, Galilee, Israel). Capturing images and the electronic processing of the images were performed with the usage of BAND VIEW/FISH VIEW software (Applied Spectral Imaging, Galilee, Israel).
stage. On the contrary, hatching rates of the brown trout from the other control groups were very high varying from 82.52 3.82% (Ca3) to 91.60 2.60% (Cg2), and their survival did not decrease significantly during the yolk sac absorption (Fig. 1).
Microsatellite analysis Nuclear DNA for the molecular analysis was isolated from the parental fin tissues and body tissues of the gynogenetic (n = 22), androgenetic (n = 3) and control (n = 50) hatchlings. Moreover, eight 8 androgenetic embryos from diploid variants of the experiments were used for the nuclear DNA extraction. Extraction and purification of the DNA was performed using Genomic Mini AX Tissue Spin (A&A Biotechnology s.c., Gdynia, Poland). Parental and offspring genotypes were examined in two microsatellite loci, Strutta12 and Strutta58 (Poteaux et al. 1999). Reaction mixture contained 2.5 ll of 5X Green GoTaqâ Flexi Buffer (Promega, Madison, WI, USA), MgCl2 (2 mM), each dNTPs (0, 2 mM) (Promega), primers (F and R) (1 lM), 0, 5 U of Taq Polimerase (Thermo Fisher Scientific, Waltham, MA, USA), 3 ll of the template DNA and water added up to 25 ll. Amplification was performed in Biometraâ T1 Thermocycler with the initial denaturation step at 97°C for 4 min, 35 cycles of 94°C for 45 s, 56 °C for 30 s, 72°C for 45 s and the final elongation performed at 72°C and lasted 10 min. Products of amplification were separated in 3% agarose gel (Sigma-Aldrich, St. Louis, MO, USA) stained with ethidium bromide (0.05 mg/ml) and captured under UV transilluminator BioDoc-ItTM (Ultra-Violet Products Ltd, Cambridge, UK).
Survival rates of gynogenetic and androgenetic embryos at the eyed stage Embryo survival of the gynogenetic haploids to the eyed stage in the variants where rainbow trout (Gh1) and brown trout (Gh2) irradiated sperm was used to activate eggs equalled 87.83 2.15% and 84. 99 5.23%, respectively. Significantly reduced survivability was observed among androgenetic haploid eyed embryos from Ah1, Ah2 and Ah3 groups (58.49 3.06%, 62.71 7.00% and 63.18 5.53%, respectively) (p < 0.05) (Fig. 2a). Development of the doubled haploid embryos was impaired when compared to the haploid embryos (p < 0.05) (Fig. 2). Even though, DH eyed embryos from both gynogenetic groups exhibited quite high survival to the eyed stage (37.35 6.85% and 37.73 5.32%) (Fig. 2b). In turn, diploid androgenetic embryos from Ad1, Ad2 and Ad3 group showed much higher mortality and only 18.86 10.89%, 13.82 11.95% and 8.72 3.40%, respectively, survived to this stage of the embryonic development (Fig. 2b).
Statistical analysis Survival rates of the gynogenetic and androgenetic doubled haploids and haploids were calculated as number of the embryos and the larvae in relation to number of the fertilized eggs. Calculations were made at three stages of the development: eyed stage, hatching and swim-up stage. Differences between survivability were compared using Shapiro–Wilk test. All calculations were made using Statistica software version 10.1 (StatSoft, Tulsa, OK, USA). A value of p < 0.05 was considered statistically significant. All values in the text were expressed as averages standard deviations (SD)
Hatching rates and survival of gynogenetic and androgenetic larvae at the swim-up stage Hatching rates of the gynogenetic and the androgenetic haploids did not differ significantly. Gynogenetic haploid brown trout progenies hatched from the eggs activated by the rainbow trout and the brown trout sperm with the rates equalled 13.74 7.34% and 16.21 4.64%, respectively (Fig. 2a), which was com-
Results Survival of the embryos and larvae from the control groups Embryo survival to the eyed stage in the control groups equalled 82.88 14.63% (Cg1), 94.95 1.18% (Cg2), 92.63 1.74% (Ca1), 88.11 2.50% (Ca2) and 84.16 5.11% (Ca3). Only few (0.22 0.38%) of the brown trout 9 rainbow trout hybrids (Cg1) hatched, but none of the hybrid larvae survived to the swim-up
Fig. 1. Survival of the cross between brown trout (Salmo trutta) female and the rainbow trout (Oncorhynchus mykiss) male (Cg1) and the brown trout from the other control groups of the experiment: Cg2, Ca3, Ca4 and Ca5
© 2015 Blackwell Verlag GmbH
Androgenetic and Gynogenetic Brown Trout
parable with the survival rates of the gynogenetic doubled haploids (16.14 2.90% and 14.76 2.4%) (Fig. 2b). Among androgenetic haploid progenies, the lowest survival at hatching was observed among fish from Ah1 group (7.19 4.54%). Survival rates of the hatched androgenetic haploid progenies of two other brown trout males were 16.08 8.56% (Ah2) and 20.61 9.83% (Ah3) (Fig. 2a). On the contrary, significantly reduced survival at hatching was observed among androgenetic doubled haploids: 1.64 1.24% (Ad1), 1.84 0.60% (Ad2) and 1.58 0.68% (Ad3) (Fig. 2b). Survival of the gynogenetic and androgenetic haploid hatchlings decreased radically to the swim-up stage, and only n = 17 (2.93 0.80%) and n = 10 (1.83 1.76%) of the gynogenetic larvae with fully absorbed yolk sacs were counted in the Gh1 and Gh2 groups, respectively. The only androgenetic swimming larvae that survived to this stage were one larva from the Ah2 group (0.29 0.50%), one larva from Ad1 group (0.14 0.24%) and four larvae from Ad2 group (0.58 0.29%). On the
(a)
259
contrary, survival of the gynogenetic doubled haploids did not decrease substantially since hatching, and n = 89 (12.78 2.18%) and n = 72 (10.48 3.48%) of the swimming larvae were observed in the Gd1 and Gd2 variants of the experiment, respectively (Fig. 2b). Cytogenetic analysis of the haploid and diploid embryos Cytogenetic survey of the androgenetic embryos from the haploid groups enabled identification of eighteen embryos with 40 chromosomes and one embryo with both haploid (40 chromosomes) and diploid cells (80 chromosomes). All fourteen cytogenetically inspected gynogenetic embryos from the haploid groups exhibited 40 chromosomes. Among embryos from the diploid groups, all karyologically tested androgenotes were found to be haploids (n = 8), and all studied gynogenotes showed diploid number of chromosomes (Fig. 3). Apart from the intact chromosomes, small chromosome fragments were identified in approximately 21% and 25% of the cytogenetically examined androgenetic haploid and diploid embryos, respectively. The number of chromosome fragments varied intra-individually from 1 to 9 and from 1 to 3 within the androgenetic embryos from the haploid and diploid groups. Among surveyed gynogenetic embryos, two individuals from the haploid group and one individual from the diploid group showed cells with 1 or 4 chromosome fragments. Observed chromosome fragments were smaller than the intact chromosomes. Most of the chromosome fragments were too small for the precise description of their morphology (Fig. 3). Molecular verification of the androgenetic and gynogenetic embryos and larvae As expected, all studied androgenetic and gynogenetic progenies were homozygous in the tested loci showing only one of the paternally or maternally (respectively) inherited alleles (Fig. 4).
(b)
Discussion
Fig. 2. Survival of the gynogenetic (G) and androgenetic (A) haploid (Gh1Gh2, Ah1Ah2, Ah3) (a) and doubled haploid (Gd1Gd2, Ad1Ad2, Ad3) (b) brown trout (Salmo trutta)
© 2015 Blackwell Verlag GmbH
Development of the doubled haploids has been induced in several fish species; however, expression of the recessive alleles and side effects of the treatments applied to the gametes and the zygotes result in the high mortality among mitotic gynogenetic and androgenetic embryos and larvae. Yields of the hatched salmonid gynogenetic and androgenetic DHs rarely exceed 10% (Chourrout 1984; Babiak et al. 2002b). Moreover, high mortality among the doubled haploids continues during the further steps of the ontogeny, and generally, only few such fish survive to maturity (Babiak et al. 2002a). However, in several fish species, gynogenetic or/and androgenetic doubled haploids reached reproduction age and have been utilized to produce clonal lines (Komen and Thorgaard 2007).
260
O Michalik, S Dobosz, T Zalewski, M Sapota and K Ocalewicz
(a)
(b)
(c)
(d)
(e)
(f) Fig. 3. Metaphase plates from embryos of the brown trout (Salmo trutta) gynogenetic haploids (a, b), gynogenetic diploid (c), androgenetic haploid (d) and androgenetic haploid/diploid mosaic (e, f). Red arrows indicated UV-radiation-induced chromosome fragments of the paternal origin while white arrows showed X-radiation-induced chromosome fragments of the maternal origin. Bars represent 5 lm
In the common carp (Cyprinus carpio Linnaeus 1758), survival of the gynogenetic and the androgenetic doubled haploids has not differed substantially (Komen et al. 1991; Tanck et al. 2001); however, results of our research performed on the brown trout are in the opposition to such observation. At the eyed stage, brown trout gynogenetic haploid embryos survived much better than androgenetic haploids. Among brown trout doubled haploids, differences in the survival rates of the gynogenetic and androgenetic specimens also appeared during embryogenesis and deepen along their further development. Radiation used in order to inactivate nuclear DNA in the eggs can be harmful to mRNAs deposited in the fish egg cytoplasm during oogenesis. In fish, such maternal transcripts govern embryonic development before mid-blastula transition
during which zygotic transcript is activated (Pelegri 2003). Thus, radiation-induced modification of the maternal mRNA in the egg cytoplasm may impair early development and decrease survival of the androgenetic embryos. Appearance of the haploids among embryos from the androgenetic diploid variants in our experiment indicates radiation-induced egg enucleation misaligned cellular mechanism controlling brown trout cell division and delay the first cleavage. The radiationinduced mitotic delay caused that high pressure shock implemented to restore diploidization state was more efficient in the case of the brown trout zygotes, which were developing in the eggs not subjected to any radiation. Similar was observed in the sea urchin where radiation-induced gynogenetic haploids divide exactly at the same time as normal diploid eggs while androgenetic © 2015 Blackwell Verlag GmbH
Androgenetic and Gynogenetic Brown Trout
261
(a)
(b) Fig. 4. Heterozygous genotypes of the brown trout females (lanes 3, 4) and trout male (lane 5) and their homozygous gynogenetic (lanes 6–9) and androgenetic (lanes 10 and 11) offspring from the diploid variants of the experiments for the microsatellite DNA loci Strutta12 (a) and Strutta58 (b). Lane 1: O’GeneRulerTM 100 bp DNA Ladder (Fermentas, Vilnius, Lithuania), lane 2: pUC/MspI DNA Ladder (A&A Biotechnology s.c., Poland)
haploids exhibit a characteristic delay of the first division (Rustad 1970). It has been also observed that ionizing radiation induces overduplication of the centrosomes in the human tumour cells exposed to the radiation dose much lower that these applied to the fish eggs (Sato et al. 2000). Thus, it is reasonable to assume that part of the vast mortality observed among androgenetic fish embryos may also result from the radiationinduced defects in the centrosome functions. Higher mortality among doubled haploids when compared to the survival rates of the haploid embryos at the eyed stage might be caused by the pressure shock applied to recover a diploid state in the zygotes. Hydrostatic pressure disturbs chromosome segregation by destabilizing of the spindle microtubules. However, in the fish eggs microtubules play also some role in the transportation of the cytoplasmic particles and factors committed to the early cellular differentiation of the blastomeres (Webb et al. 1995). Changes in the microtubule structure caused by the high pressure shock may thus impair early development of the gynogenetic and androgenetic specimens. In the goldfish and crucian carp (Carassius auratus Linnaeus 1758), high pressure shock applied to the fertilized eggs results in the formation of the thin blastodiscs with poorly developed cytoplasm, delay of epiboly and suppression of the dorso ventral differentiation (Yamaha et al. 2002). Furthermore, high pressure that has been proved to damage DNA-protein structure and affect gene expression (Fernandes et al. 2004; Sironen et al. 2002) may be also harmful for the maternal transcripts in the egg cytoplasm. Yields of the hatched brown trout gynogenetic DHs were comparable with the number of DHs provided in the course of the mitotic gynogenesis in other fish species (Komen and Thorgaard 2007). Although, hybrids provided in the course of crossing brown trout
References Babiak I, Dobosz S, Goryczko K, Kuzminski H, Brzuzan P, Ciesielski S, 2002a: Androgenesis in rainbow trout using
© 2015 Blackwell Verlag GmbH
female and rainbow trout male are unviable and die before hatching (Fig. 1), UV-inactivated rainbow trout sperm was successfully employed for the brown trout egg activation. Moreover, brown trout gynogenetic doubled haploids were developing equally regardless of whether the sperm used for the insemination was either homologous or heterologous. Low doses of UV- and X-radiation applied to damage nuclear DNA in the spermatozoa and eggs resulted in the incomplete genetic inactivation of the brown trout gametes. Residues of the irradiated chromosomes may interfere with the cell cleavages and provoke consequent chromosome rearrangements what in turn may increase mortality among the doubled haploids (Ocalewicz et al. 2012). Furthermore, genes from the radiation-induced chromosome fragments may contain mutated genetic information that is harmful for the haploid and doubled haploid individuals. Thus, it is reasonable to improve protocols for the radiation-induced genetic inactivation of the brown trout gametes that may be used in the genome manipulation experiments. Acknowledgements We thank Rafa Ro_zy nski from the Department of Salmonid Research, Inland Fisheries Institute in Olsztyn, Rutki, for his technical assistance during the experiments. This research was supported by the National Science Centre (Poland), grant No. N N311 525240.
Conflict of interest None of the authors have any conflict of interest to declare.
Author contributions All authors were involved in designing and performing the experiments. KO, OM and MS were also involved in data analysis, writing and editing the manuscript.
cryopreserved spermatozoa: the effect of processing and biological factors. Theriogenology 57, 1229–1249. Babiak I, Dobosz S, Kuzminski H, Goryczko K, Ciesielski S, Brzuzan P, Urba-
nyi B, Horvath A, Lahnsteiner F, Piironen J, 2002b: Failure of interspecies androgenesis in salmonids. J Fish Biol 61, 432–447.
262 Billard R, 1992: Reproduction in rainbow trout: sex differentiation, dynamics of gametogenesis, biology and preservation of gametes. Aquaculture 100, 263–298. Brown KH, Thorgaard GH, 2002: Mitochondrial and nuclear inheritance in an androgenetic line of rainbow trout, Oncorhynchus mykiss. Aquaculture 204, 323–335. Chen S-L, Tian Y-S, Yang T-J, Shao C-W, Ji X-S, Zhai J-M, Liao X-L, Zhuang Z-M, Su P-Z, Xu J-Y, Sha Z-X, Wu P-F, Wang N, 2009: Artificial gynogenesis and sex determination in half-smooth tongue sole (Cynoglossus semilaevis). Mar Biotechnol 11, 243–251. Chourrout D, 1984: Pressure induced retention of 2nd polar body and suppression of 1st cleavage in rainbow trout production of all triploids all tetraploids and heterozygous and homozygous diploid gynogenetics. Aquaculture 36, 111–126. Fernandes P, Domitrovic T, Kao CM, Kurtenbach E, 2004: Genomic expression pattern in Saccharomyces cerevisiae cells in response to high hydrostatic pressure. FEBS Lett 556, 153–160. Goryczko K, Dobosz S, M€akinen T, Tomasik L, 1991: UV-irradiation of rainbow trout sperm as a practical method for induced gynogenesis. J Appl Ichthyol 7, 136–146. Kane DA, Kimmel CB, 1993: The zebrafish midblastula transition. Development 119, 447–456. Komen H, Thorgaard G, 2007: Androgenesis, gynogenesis and the production of clones in fishes: a review. Aquaculture 269, 150–173. Komen J, Bongers ABJ, Richter CJJ, Van Muiswinkel WB, Huisman EA, 1991: Gynogenesis in common carp Cyprinus carpio L. II: the production of homozygous gynogenetic clones and F1 hybrids. Aquaculture 92, 127–142. Michalik O, Dobosz S, W ojcik I, Zalewski T, Ocalewicz K, 2014: Use of eggs derived
O Michalik, S Dobosz, T Zalewski, M Sapota and K Ocalewicz from the interspecific charr hybrids to induce androgenetic development of the brook charr (Salvelinus fontinalis Mitchill 1814). Reprod Domest Anim 49, 191–196. Ocalewicz K, 2002: Cytogenetic markers for X chromosome in karyotype of rainbow trout from Rutki strain. Folia Biol (Krakow) 50, 10–14. Ocalewicz K, Babiak I, Kasprzycka B, Dobosz S, Kuzminski H, Goryczko K, 2007: Occurrence of two forms of Y chromosome in rainbow trout (Oncorhynchus mykiss) males from Rutki strain. Aquaculture 270, 546–551. Ocalewicz K, Dobosz S, Kuzminski H, 2012: Distribution of telomeric DNA sequences on the X-radiation-induced chromosome fragments in the genome of androgenetic brook trout (Salvelinus fontinalis, Mitchill 1814). Cytogenet Genome Res 137, 1–6. Pandian TJ, Koteeswaran R, 1998: Ploidy induction and sex control in fish. Hydrobiologia 384, 167–243. Pelegri F, 2003: Maternal factors in zebrafish development. Dev Dyn 228, 535–554. Poteaux C, Bonhomme F, Berrebi P, 1999: Microsatellite polymorphism and genetic impact of restocking in Mediterranean brown trout (Salmo trutta L.). Heredity 82, 645–653. Preston AC, Taylor JF, Craig B, Bozzolla P, Penman DJ, Migaud H, 2013: Optimisation of triploidy induction in brown trout (Salmo trutta L.). Aquaculture 414–415, 160–166. Rustad RC, 1970: Variations in the sensitivity to x-ray-induced mitotic delay during the cell division cycle of the sea urchin egg. Radiat Res 42, 498–512. Sato N, Micumoto K, Nakamura M, Tanaka M, 2000: Radiation-induced centrosome overduplication and multiple mitotic spindles in human tumor cells. Exp Cell Res 255, 321–326.
Sironen R, Karjalainen H, T} orr} onen K, Elo M, Kaarniranta K, Takigawa M, Helminen H, Lammi M, 2002: High pressure effects on cellular mRNA stability. A cDNA array analysis. Biorheology 39, 111–117. Tanck MWT, Palstra AP, Van de Weerd M, Leffering CP, Van der Poel JJ, Bovenhuis H, Komen J, 2001: Segregation of microsatellite alleles and residual heterozygosity at single loci in homozygous androgenetic common carp (Cyprinus carpio L.). Genome 44(5), 743–751. Webb TA, Kowalski WJ, Fluck RA, 1995: Microtubule-based movements during ooplasmic segregation in the medaka fish egg (Oryzias latipes). Biol Bull 188, 146–156. Woznicki P, Sanchez L, Martinez P, Pardo BG, Jankun M, 2000: A population analysis of the structure and variability of NOR in Salmo trutta by Ag, CMA3 and ISH. Genetica 108, 113–118. Yamaha E, Otani S, Minami A, Arai K, 2002: Dorso-ventral axis perturbation in goldfish embryos caused by heat- and pressure-shock treatments for chromosomes set manipulation. Fish Sci 68, 313–319. Yi M, Hong N, Hong Y, 2009: Generation of medaka fish haploid embryonic stem cells. Science 326, 430–433.
Submitted: 1 Oct 2014; Accepted: 4 Dec 2014 Author’s address (for correspondence): K Ocalewicz, Department of Marine Biology and Ecology, Institute of Oceanography, University of Gdansk, 81-378 Gdynia, Poland. Emails: [email protected]; konrad. [email protected]
© 2015 Blackwell Verlag GmbH
Induction of Gynogenetic and Androgenetic Haploid and Doubled Haploid Development in the Brown Trout (Salmo trutta Linnaeus 1758) O Michalik1, S Dobosz2, T Zalewski2, M Sapota3 and K Ocalewicz3,4 1 Department of Molecular Evolution, University of Gdansk, Gdansk, Poland; 2Department of Salmonid Research, Inland Fisheries Institute in Olsztyn, Rutki, Zukowo, Poland; 3Department of Marine Biology and Ecology, Institute of Oceanography, University of Gdansk, Gdynia, Poland; 4 Department of Ichthyology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
Contents Gynogenetic and androgenetic brown trout (Salmo trutta Linnaeus 1758) haploids (Hs) and doubled haploids (DHs) were produced in the present research. Haploid development was induced by radiation-induced genetic inactivation of spermatozoa (gynogenesis) or eggs (androgenesis) before insemination. To provide DHs, gynogenetic and androgenetic haploid zygotes were subjected to the high pressure shock to suppress the first mitotic cleavage. Among haploids, gynogenetic embryos were showing lower mortality when compared to the androgenetic embryos; however, most of them die before the first feeding stage. Gynogenetic doubled haploids provided in the course of the brown trout eggs activation performed by homologous and heterologous sperm (rainbow trout) were developing equally showing hatching rates of 14.76 2.4% and 16.14 2.90% and the survival rates at the first feeding stage of 10.48 3.48% and 12.78 2.18%, respectively. Significantly, lower survival rate was observed among androgenetic progenies from the diploid groups with only few specimens that survived to the first feeding stage. Cytogenetic survey showed that among embryos from the diploid variants of the research, only gynogenetic individuals possessed doubled sets of chromosomes. Thus, it is reasonable to assume that radiation employed for the genetic inactivation of the brown trout eggs misaligned mechanism responsible for the cell divisions and might have delayed or even arrested the first mitotic cleavage in the androgenetic brown trout zygotes. Moreover, protocol for the radiationinduced inactivation of the paternal and maternal genome should be adjusted as some of the cytogenetically surveyed gynogenetic and androgenetic embryos exhibited fragments of the irradiated chromosomes.
Introduction In the latest decades, chromosome set manipulation techniques namely uniparental chromosome inheritance (gynogenesis and androgenesis) and multiplication of the chromosome sets have been developed in many fish species including those of the high economic importance as well as species recognized as biomedical models (Komen and Thorgaard 2007). Haploid development in the fish may be induced by radiation-induced damage of the nuclear DNA in either spermatozoa (gynogenesis) or eggs (androgenesis) before insemination. In turn, doubled haploids (DHs) are provided in the course of the three-step process including irradiation of the spermatozoa or the eggs (i), insemination (ii) and duplication of the maternal or the paternal haploid set of chromo-
somes (iii) accomplished by exposition of the haploid zygotes to the temperature or pressure shock suppressing the first mitotic cleavage (Pandian and Koteeswaran 1998). Although most of the haploid fish die during embryogenesis or soon after hatching, such embryos are excellent source of the haploid embryonic stem cells (Yi et al. 2009). Both haploids and doubled haploids are important in studies concerning recessive alleles. Viable doubled haploids have been used for the production of the fish homozygous clonal lines (Komen and Thorgaard 2007). Due to the lack of heterozygotes, haploids and doubled haploids significantly facilitate studies concerning genetic mapping and genome sequencing. This feature of the doubled haploids and clones simplifies detailed linkage analyses enabling identification of the chromosome regions connected with the quantitative traits loci (QTL) (Komen and Thorgaard 2007). Moreover, androgenetic and gynogenetic fish have been used in studies concerning genetic basis of the sex determination process and differentiation of the sex chromosomes (Ocalewicz et al. 2007; Chen et al. 2009). Androgenesis has been also applied to recover nuclear genomic information of the endangered fish lines or species from the normal and cryopreserved spermatozoa (Babiak et al. 2002a) and to investigate the effects of the cytoplasm and mitochondria on the embryo development (Brown and Thorgaard 2002). Unfortunately, despite the fact that doubled haploids survive better than haploids, high mortality observed among gynogenetic and androgenetic fish is still a limiting factor in their wide application. The low survival of the doubled haploids during embryogenesis and after hatching seems to be caused by expression of the lethal alleles, and the side effects of the manipulations performed on the eggs and the zygotes. Inactivation of the gametes with UV- or X-radiation and application of the heat and pressure shock to the zygotes probably affect also many other cellular organelles and developmental mechanisms what results in the various disturbances during the ontogeny and consequently increases mortality of the doubled haploid individuals. Early development stages in the fishes are solely directed by the maternal factors until the mid-blastula transition (Kane and Kimmel 1993; Pelegri 2003). Thus, radiationinduced misregulation of such mechanism may result in the lower survival of the androgenetic DHs when © 2015 Blackwell Verlag GmbH
Androgenetic and Gynogenetic Brown Trout
257
compared to the gynogenetic DHs that are developing in the non-irradiated eggs. To verify this hypothesis, we induced gynogenetic and androgenetic development of the haploid (H) and doubled haploid (DH) brown trout (Salmo trutta Linnaeus 1758). Survival of the androgenetic and gynogenetic specimens was monitored during their ontogeny until the swim-up stage. Moreover, to confirm ploidy level and to identify any residues of the irradiated paternal or maternal chromosomes, embryos from the haploid and doubled haploid variants of the experiment were cytogenetically studied. Homozygosity of the doubled haploids was assessed by the microsatellite DNA analysis. Consequences of the eggs irradiation and exposition of zygotes to the high pressure shock for the fish development are also discussed in the present study.
(approximately 600, 500 and 750 eggs in each, respectively). Inactivation of the parental nuclear DNA To inactivate maternal nuclear genome, eggs for the androgenetic variants of the research were transported on ice to the Clinic of Oncology and Radiotherapy, University Clinical Center, Medical University of Gdansk, and irradiated with 420 Gy of X-rays using linear accelerator Clinac 600 (Varian Medical Systems, Palo Alto, CA, USA) (Michalik et al. 2014). After the treatment, irradiated eggs carefully protected from light were transported back to the hatchery. Inactivation of the paternal nuclear DNA in the sperm was performed with UV light in the dose of 2075 lW/cm2 (Goryczko et al. 1991).
Material and Methods Gamete collection and experimental design Brown trout and rainbow trout (Oncorhynchus mykiss Walbaum 1792) gamete donors derived from the broodstocks kept at the Department of Salmonid Research, Inland Fisheries Institute in Olsztyn, Rutki, Poland. Diploid number of the chromosomes (2n) and number of the chromosome arms (FN) in the examined brown trout individuals equalled 80 and 100–102, respectively (Woznicki et al. 2000). Moreover, milt for the interspecies gynogenetic variant of the research was collected from the rainbow trout from Rutki strain characterized by the chromosome number varied from 2n = 59–62 and stable chromosome arm number FN = 104 (Ocalewicz 2002). Eggs of the highest quality provided from 15 brown trout females were pooled. Milt from four brown trout (B1, B2, B3, B4) and one rainbow trout (R) males was collected as individual lots. All gametes were kept at 2°C for the further use. In one of the gynogenetic variant, eggs were inseminated with the semen from the rainbow trout, and in the other gynogenetic variant, sperm derived from brown trout (B1) was used (Table 1). To inseminate eggs in the androgenetic variants, semen from three brown trout males (B2, B3 and B4) was used (Table 1). Every variant of the experiment contained control (C), haploid (h) and doubled haploid (d) groups Table 1. Induction of gynogenetic and androgenetic development of haploid and doubled haploid brown trout (Salmo trutta): summary of the experimental design Experimental variants
Gynogenesis Ia
Gynogenesis IIb
Males Experimental groups Controls Haploids Doubled haploids
R
B1
B2
B3
B4
Cg1 Gh1 Gd1
Cg2 Gh2 Gd2
Ca1 Ah1 Ad1
Ca2 Ah2 Ad2
Ca3 Ah3 Ad3
R, rainbow trout; B, brown trout. a gynogenesis with the heterologous sperm. b gynogenesis with the homologous sperm.
© 2015 Blackwell Verlag GmbH
Androgenesis
Insemination and diploidization Batches of eggs were inseminated in the proportion of 0.1 ml semen for 200 eggs in the presence of Billard fertilization diluent (154 mM NaCl and 1 mM Ca2+, buffered to pH 9.0 with 20 mM Tris + 30 mM glycine) (Billard 1992). After 3 min, eggs were rinsed with the water. Controls for the gynogenetic (Cg1 and Cg2) and androgenetic (Ca1, Ca2, Ca3) variants and all haploid batches (Gh1, Gh2, Ah1, Ah2, Ah3) (Table 1) were then placed in the incubation apparatus. Batches intended for the diploid groups were further incubated at 10°C. To double parental set of chromosomes in the inseminated eggs from the gynogenetic and androgenetic diploid groups (Gd1, Gd2, Ad1, Ad2, Ad3) (Table 1) were treated with the high hydrostatic pressure (10 000 psi) applied 450 min after insemination and lasted 5 min (Babiak et al. 2002b; Preston et al. 2013). All eggs were incubated in three separate replicates at 6–8°C. Survival of androgenetic and gynogenetic embryos and hatchlings Fertilization rate was estimated 24 h after insemination. Afterwards, dead embryos and fry were removed from the batches every day until the end of experiment. First measurement of survival among embryos was performed 36 days post-fertilization (dpf) at the eyed stage. Living larvae were counted just after hatching (57 dpf) and finally at the swim-up stage (80 dpf). Cytogenetic analysis of embryos Metaphase plates were prepared from the haploid and the diploid eyed embryos from all experimental variants according in vivo (technique described by Michalik et al. (2014)). Chromosomes were stained with 40 , 6-diamidino-2-phenylindole DAPI (Vector, Burlingame, CA, USA) and observed under a Zeiss Axio Imager A1microscope equipped with a Zeiss EC Plan-Neofluar 100x/1.3 oil objective, a fluorescent lamp and a digital
258
O Michalik, S Dobosz, T Zalewski, M Sapota and K Ocalewicz
camera (Applied Spectral Imaging, Galilee, Israel). Capturing images and the electronic processing of the images were performed with the usage of BAND VIEW/FISH VIEW software (Applied Spectral Imaging, Galilee, Israel).
stage. On the contrary, hatching rates of the brown trout from the other control groups were very high varying from 82.52 3.82% (Ca3) to 91.60 2.60% (Cg2), and their survival did not decrease significantly during the yolk sac absorption (Fig. 1).
Microsatellite analysis Nuclear DNA for the molecular analysis was isolated from the parental fin tissues and body tissues of the gynogenetic (n = 22), androgenetic (n = 3) and control (n = 50) hatchlings. Moreover, eight 8 androgenetic embryos from diploid variants of the experiments were used for the nuclear DNA extraction. Extraction and purification of the DNA was performed using Genomic Mini AX Tissue Spin (A&A Biotechnology s.c., Gdynia, Poland). Parental and offspring genotypes were examined in two microsatellite loci, Strutta12 and Strutta58 (Poteaux et al. 1999). Reaction mixture contained 2.5 ll of 5X Green GoTaqâ Flexi Buffer (Promega, Madison, WI, USA), MgCl2 (2 mM), each dNTPs (0, 2 mM) (Promega), primers (F and R) (1 lM), 0, 5 U of Taq Polimerase (Thermo Fisher Scientific, Waltham, MA, USA), 3 ll of the template DNA and water added up to 25 ll. Amplification was performed in Biometraâ T1 Thermocycler with the initial denaturation step at 97°C for 4 min, 35 cycles of 94°C for 45 s, 56 °C for 30 s, 72°C for 45 s and the final elongation performed at 72°C and lasted 10 min. Products of amplification were separated in 3% agarose gel (Sigma-Aldrich, St. Louis, MO, USA) stained with ethidium bromide (0.05 mg/ml) and captured under UV transilluminator BioDoc-ItTM (Ultra-Violet Products Ltd, Cambridge, UK).
Survival rates of gynogenetic and androgenetic embryos at the eyed stage Embryo survival of the gynogenetic haploids to the eyed stage in the variants where rainbow trout (Gh1) and brown trout (Gh2) irradiated sperm was used to activate eggs equalled 87.83 2.15% and 84. 99 5.23%, respectively. Significantly reduced survivability was observed among androgenetic haploid eyed embryos from Ah1, Ah2 and Ah3 groups (58.49 3.06%, 62.71 7.00% and 63.18 5.53%, respectively) (p < 0.05) (Fig. 2a). Development of the doubled haploid embryos was impaired when compared to the haploid embryos (p < 0.05) (Fig. 2). Even though, DH eyed embryos from both gynogenetic groups exhibited quite high survival to the eyed stage (37.35 6.85% and 37.73 5.32%) (Fig. 2b). In turn, diploid androgenetic embryos from Ad1, Ad2 and Ad3 group showed much higher mortality and only 18.86 10.89%, 13.82 11.95% and 8.72 3.40%, respectively, survived to this stage of the embryonic development (Fig. 2b).
Statistical analysis Survival rates of the gynogenetic and androgenetic doubled haploids and haploids were calculated as number of the embryos and the larvae in relation to number of the fertilized eggs. Calculations were made at three stages of the development: eyed stage, hatching and swim-up stage. Differences between survivability were compared using Shapiro–Wilk test. All calculations were made using Statistica software version 10.1 (StatSoft, Tulsa, OK, USA). A value of p < 0.05 was considered statistically significant. All values in the text were expressed as averages standard deviations (SD)
Hatching rates and survival of gynogenetic and androgenetic larvae at the swim-up stage Hatching rates of the gynogenetic and the androgenetic haploids did not differ significantly. Gynogenetic haploid brown trout progenies hatched from the eggs activated by the rainbow trout and the brown trout sperm with the rates equalled 13.74 7.34% and 16.21 4.64%, respectively (Fig. 2a), which was com-
Results Survival of the embryos and larvae from the control groups Embryo survival to the eyed stage in the control groups equalled 82.88 14.63% (Cg1), 94.95 1.18% (Cg2), 92.63 1.74% (Ca1), 88.11 2.50% (Ca2) and 84.16 5.11% (Ca3). Only few (0.22 0.38%) of the brown trout 9 rainbow trout hybrids (Cg1) hatched, but none of the hybrid larvae survived to the swim-up
Fig. 1. Survival of the cross between brown trout (Salmo trutta) female and the rainbow trout (Oncorhynchus mykiss) male (Cg1) and the brown trout from the other control groups of the experiment: Cg2, Ca3, Ca4 and Ca5
© 2015 Blackwell Verlag GmbH
Androgenetic and Gynogenetic Brown Trout
parable with the survival rates of the gynogenetic doubled haploids (16.14 2.90% and 14.76 2.4%) (Fig. 2b). Among androgenetic haploid progenies, the lowest survival at hatching was observed among fish from Ah1 group (7.19 4.54%). Survival rates of the hatched androgenetic haploid progenies of two other brown trout males were 16.08 8.56% (Ah2) and 20.61 9.83% (Ah3) (Fig. 2a). On the contrary, significantly reduced survival at hatching was observed among androgenetic doubled haploids: 1.64 1.24% (Ad1), 1.84 0.60% (Ad2) and 1.58 0.68% (Ad3) (Fig. 2b). Survival of the gynogenetic and androgenetic haploid hatchlings decreased radically to the swim-up stage, and only n = 17 (2.93 0.80%) and n = 10 (1.83 1.76%) of the gynogenetic larvae with fully absorbed yolk sacs were counted in the Gh1 and Gh2 groups, respectively. The only androgenetic swimming larvae that survived to this stage were one larva from the Ah2 group (0.29 0.50%), one larva from Ad1 group (0.14 0.24%) and four larvae from Ad2 group (0.58 0.29%). On the
(a)
259
contrary, survival of the gynogenetic doubled haploids did not decrease substantially since hatching, and n = 89 (12.78 2.18%) and n = 72 (10.48 3.48%) of the swimming larvae were observed in the Gd1 and Gd2 variants of the experiment, respectively (Fig. 2b). Cytogenetic analysis of the haploid and diploid embryos Cytogenetic survey of the androgenetic embryos from the haploid groups enabled identification of eighteen embryos with 40 chromosomes and one embryo with both haploid (40 chromosomes) and diploid cells (80 chromosomes). All fourteen cytogenetically inspected gynogenetic embryos from the haploid groups exhibited 40 chromosomes. Among embryos from the diploid groups, all karyologically tested androgenotes were found to be haploids (n = 8), and all studied gynogenotes showed diploid number of chromosomes (Fig. 3). Apart from the intact chromosomes, small chromosome fragments were identified in approximately 21% and 25% of the cytogenetically examined androgenetic haploid and diploid embryos, respectively. The number of chromosome fragments varied intra-individually from 1 to 9 and from 1 to 3 within the androgenetic embryos from the haploid and diploid groups. Among surveyed gynogenetic embryos, two individuals from the haploid group and one individual from the diploid group showed cells with 1 or 4 chromosome fragments. Observed chromosome fragments were smaller than the intact chromosomes. Most of the chromosome fragments were too small for the precise description of their morphology (Fig. 3). Molecular verification of the androgenetic and gynogenetic embryos and larvae As expected, all studied androgenetic and gynogenetic progenies were homozygous in the tested loci showing only one of the paternally or maternally (respectively) inherited alleles (Fig. 4).
(b)
Discussion
Fig. 2. Survival of the gynogenetic (G) and androgenetic (A) haploid (Gh1Gh2, Ah1Ah2, Ah3) (a) and doubled haploid (Gd1Gd2, Ad1Ad2, Ad3) (b) brown trout (Salmo trutta)
© 2015 Blackwell Verlag GmbH
Development of the doubled haploids has been induced in several fish species; however, expression of the recessive alleles and side effects of the treatments applied to the gametes and the zygotes result in the high mortality among mitotic gynogenetic and androgenetic embryos and larvae. Yields of the hatched salmonid gynogenetic and androgenetic DHs rarely exceed 10% (Chourrout 1984; Babiak et al. 2002b). Moreover, high mortality among the doubled haploids continues during the further steps of the ontogeny, and generally, only few such fish survive to maturity (Babiak et al. 2002a). However, in several fish species, gynogenetic or/and androgenetic doubled haploids reached reproduction age and have been utilized to produce clonal lines (Komen and Thorgaard 2007).
260
O Michalik, S Dobosz, T Zalewski, M Sapota and K Ocalewicz
(a)
(b)
(c)
(d)
(e)
(f) Fig. 3. Metaphase plates from embryos of the brown trout (Salmo trutta) gynogenetic haploids (a, b), gynogenetic diploid (c), androgenetic haploid (d) and androgenetic haploid/diploid mosaic (e, f). Red arrows indicated UV-radiation-induced chromosome fragments of the paternal origin while white arrows showed X-radiation-induced chromosome fragments of the maternal origin. Bars represent 5 lm
In the common carp (Cyprinus carpio Linnaeus 1758), survival of the gynogenetic and the androgenetic doubled haploids has not differed substantially (Komen et al. 1991; Tanck et al. 2001); however, results of our research performed on the brown trout are in the opposition to such observation. At the eyed stage, brown trout gynogenetic haploid embryos survived much better than androgenetic haploids. Among brown trout doubled haploids, differences in the survival rates of the gynogenetic and androgenetic specimens also appeared during embryogenesis and deepen along their further development. Radiation used in order to inactivate nuclear DNA in the eggs can be harmful to mRNAs deposited in the fish egg cytoplasm during oogenesis. In fish, such maternal transcripts govern embryonic development before mid-blastula transition
during which zygotic transcript is activated (Pelegri 2003). Thus, radiation-induced modification of the maternal mRNA in the egg cytoplasm may impair early development and decrease survival of the androgenetic embryos. Appearance of the haploids among embryos from the androgenetic diploid variants in our experiment indicates radiation-induced egg enucleation misaligned cellular mechanism controlling brown trout cell division and delay the first cleavage. The radiationinduced mitotic delay caused that high pressure shock implemented to restore diploidization state was more efficient in the case of the brown trout zygotes, which were developing in the eggs not subjected to any radiation. Similar was observed in the sea urchin where radiation-induced gynogenetic haploids divide exactly at the same time as normal diploid eggs while androgenetic © 2015 Blackwell Verlag GmbH
Androgenetic and Gynogenetic Brown Trout
261
(a)
(b) Fig. 4. Heterozygous genotypes of the brown trout females (lanes 3, 4) and trout male (lane 5) and their homozygous gynogenetic (lanes 6–9) and androgenetic (lanes 10 and 11) offspring from the diploid variants of the experiments for the microsatellite DNA loci Strutta12 (a) and Strutta58 (b). Lane 1: O’GeneRulerTM 100 bp DNA Ladder (Fermentas, Vilnius, Lithuania), lane 2: pUC/MspI DNA Ladder (A&A Biotechnology s.c., Poland)
haploids exhibit a characteristic delay of the first division (Rustad 1970). It has been also observed that ionizing radiation induces overduplication of the centrosomes in the human tumour cells exposed to the radiation dose much lower that these applied to the fish eggs (Sato et al. 2000). Thus, it is reasonable to assume that part of the vast mortality observed among androgenetic fish embryos may also result from the radiationinduced defects in the centrosome functions. Higher mortality among doubled haploids when compared to the survival rates of the haploid embryos at the eyed stage might be caused by the pressure shock applied to recover a diploid state in the zygotes. Hydrostatic pressure disturbs chromosome segregation by destabilizing of the spindle microtubules. However, in the fish eggs microtubules play also some role in the transportation of the cytoplasmic particles and factors committed to the early cellular differentiation of the blastomeres (Webb et al. 1995). Changes in the microtubule structure caused by the high pressure shock may thus impair early development of the gynogenetic and androgenetic specimens. In the goldfish and crucian carp (Carassius auratus Linnaeus 1758), high pressure shock applied to the fertilized eggs results in the formation of the thin blastodiscs with poorly developed cytoplasm, delay of epiboly and suppression of the dorso ventral differentiation (Yamaha et al. 2002). Furthermore, high pressure that has been proved to damage DNA-protein structure and affect gene expression (Fernandes et al. 2004; Sironen et al. 2002) may be also harmful for the maternal transcripts in the egg cytoplasm. Yields of the hatched brown trout gynogenetic DHs were comparable with the number of DHs provided in the course of the mitotic gynogenesis in other fish species (Komen and Thorgaard 2007). Although, hybrids provided in the course of crossing brown trout
References Babiak I, Dobosz S, Goryczko K, Kuzminski H, Brzuzan P, Ciesielski S, 2002a: Androgenesis in rainbow trout using
© 2015 Blackwell Verlag GmbH
female and rainbow trout male are unviable and die before hatching (Fig. 1), UV-inactivated rainbow trout sperm was successfully employed for the brown trout egg activation. Moreover, brown trout gynogenetic doubled haploids were developing equally regardless of whether the sperm used for the insemination was either homologous or heterologous. Low doses of UV- and X-radiation applied to damage nuclear DNA in the spermatozoa and eggs resulted in the incomplete genetic inactivation of the brown trout gametes. Residues of the irradiated chromosomes may interfere with the cell cleavages and provoke consequent chromosome rearrangements what in turn may increase mortality among the doubled haploids (Ocalewicz et al. 2012). Furthermore, genes from the radiation-induced chromosome fragments may contain mutated genetic information that is harmful for the haploid and doubled haploid individuals. Thus, it is reasonable to improve protocols for the radiation-induced genetic inactivation of the brown trout gametes that may be used in the genome manipulation experiments. Acknowledgements We thank Rafa Ro_zy nski from the Department of Salmonid Research, Inland Fisheries Institute in Olsztyn, Rutki, for his technical assistance during the experiments. This research was supported by the National Science Centre (Poland), grant No. N N311 525240.
Conflict of interest None of the authors have any conflict of interest to declare.
Author contributions All authors were involved in designing and performing the experiments. KO, OM and MS were also involved in data analysis, writing and editing the manuscript.
cryopreserved spermatozoa: the effect of processing and biological factors. Theriogenology 57, 1229–1249. Babiak I, Dobosz S, Kuzminski H, Goryczko K, Ciesielski S, Brzuzan P, Urba-
nyi B, Horvath A, Lahnsteiner F, Piironen J, 2002b: Failure of interspecies androgenesis in salmonids. J Fish Biol 61, 432–447.
262 Billard R, 1992: Reproduction in rainbow trout: sex differentiation, dynamics of gametogenesis, biology and preservation of gametes. Aquaculture 100, 263–298. Brown KH, Thorgaard GH, 2002: Mitochondrial and nuclear inheritance in an androgenetic line of rainbow trout, Oncorhynchus mykiss. Aquaculture 204, 323–335. Chen S-L, Tian Y-S, Yang T-J, Shao C-W, Ji X-S, Zhai J-M, Liao X-L, Zhuang Z-M, Su P-Z, Xu J-Y, Sha Z-X, Wu P-F, Wang N, 2009: Artificial gynogenesis and sex determination in half-smooth tongue sole (Cynoglossus semilaevis). Mar Biotechnol 11, 243–251. Chourrout D, 1984: Pressure induced retention of 2nd polar body and suppression of 1st cleavage in rainbow trout production of all triploids all tetraploids and heterozygous and homozygous diploid gynogenetics. Aquaculture 36, 111–126. Fernandes P, Domitrovic T, Kao CM, Kurtenbach E, 2004: Genomic expression pattern in Saccharomyces cerevisiae cells in response to high hydrostatic pressure. FEBS Lett 556, 153–160. Goryczko K, Dobosz S, M€akinen T, Tomasik L, 1991: UV-irradiation of rainbow trout sperm as a practical method for induced gynogenesis. J Appl Ichthyol 7, 136–146. Kane DA, Kimmel CB, 1993: The zebrafish midblastula transition. Development 119, 447–456. Komen H, Thorgaard G, 2007: Androgenesis, gynogenesis and the production of clones in fishes: a review. Aquaculture 269, 150–173. Komen J, Bongers ABJ, Richter CJJ, Van Muiswinkel WB, Huisman EA, 1991: Gynogenesis in common carp Cyprinus carpio L. II: the production of homozygous gynogenetic clones and F1 hybrids. Aquaculture 92, 127–142. Michalik O, Dobosz S, W ojcik I, Zalewski T, Ocalewicz K, 2014: Use of eggs derived
O Michalik, S Dobosz, T Zalewski, M Sapota and K Ocalewicz from the interspecific charr hybrids to induce androgenetic development of the brook charr (Salvelinus fontinalis Mitchill 1814). Reprod Domest Anim 49, 191–196. Ocalewicz K, 2002: Cytogenetic markers for X chromosome in karyotype of rainbow trout from Rutki strain. Folia Biol (Krakow) 50, 10–14. Ocalewicz K, Babiak I, Kasprzycka B, Dobosz S, Kuzminski H, Goryczko K, 2007: Occurrence of two forms of Y chromosome in rainbow trout (Oncorhynchus mykiss) males from Rutki strain. Aquaculture 270, 546–551. Ocalewicz K, Dobosz S, Kuzminski H, 2012: Distribution of telomeric DNA sequences on the X-radiation-induced chromosome fragments in the genome of androgenetic brook trout (Salvelinus fontinalis, Mitchill 1814). Cytogenet Genome Res 137, 1–6. Pandian TJ, Koteeswaran R, 1998: Ploidy induction and sex control in fish. Hydrobiologia 384, 167–243. Pelegri F, 2003: Maternal factors in zebrafish development. Dev Dyn 228, 535–554. Poteaux C, Bonhomme F, Berrebi P, 1999: Microsatellite polymorphism and genetic impact of restocking in Mediterranean brown trout (Salmo trutta L.). Heredity 82, 645–653. Preston AC, Taylor JF, Craig B, Bozzolla P, Penman DJ, Migaud H, 2013: Optimisation of triploidy induction in brown trout (Salmo trutta L.). Aquaculture 414–415, 160–166. Rustad RC, 1970: Variations in the sensitivity to x-ray-induced mitotic delay during the cell division cycle of the sea urchin egg. Radiat Res 42, 498–512. Sato N, Micumoto K, Nakamura M, Tanaka M, 2000: Radiation-induced centrosome overduplication and multiple mitotic spindles in human tumor cells. Exp Cell Res 255, 321–326.
Sironen R, Karjalainen H, T} orr} onen K, Elo M, Kaarniranta K, Takigawa M, Helminen H, Lammi M, 2002: High pressure effects on cellular mRNA stability. A cDNA array analysis. Biorheology 39, 111–117. Tanck MWT, Palstra AP, Van de Weerd M, Leffering CP, Van der Poel JJ, Bovenhuis H, Komen J, 2001: Segregation of microsatellite alleles and residual heterozygosity at single loci in homozygous androgenetic common carp (Cyprinus carpio L.). Genome 44(5), 743–751. Webb TA, Kowalski WJ, Fluck RA, 1995: Microtubule-based movements during ooplasmic segregation in the medaka fish egg (Oryzias latipes). Biol Bull 188, 146–156. Woznicki P, Sanchez L, Martinez P, Pardo BG, Jankun M, 2000: A population analysis of the structure and variability of NOR in Salmo trutta by Ag, CMA3 and ISH. Genetica 108, 113–118. Yamaha E, Otani S, Minami A, Arai K, 2002: Dorso-ventral axis perturbation in goldfish embryos caused by heat- and pressure-shock treatments for chromosomes set manipulation. Fish Sci 68, 313–319. Yi M, Hong N, Hong Y, 2009: Generation of medaka fish haploid embryonic stem cells. Science 326, 430–433.
Submitted: 1 Oct 2014; Accepted: 4 Dec 2014 Author’s address (for correspondence): K Ocalewicz, Department of Marine Biology and Ecology, Institute of Oceanography, University of Gdansk, 81-378 Gdynia, Poland. Emails: [email protected]; konrad. [email protected]
© 2015 Blackwell Verlag GmbH
