Introduction
When a somatic diploid nucleus is transplanted into an enucleated oocyte, the successful development is conditioned by the nucleus reprogramming degree, in most cases showing an incomplete reprogramming (Latham, Reference Latham2005; Ng and Gurdon, Reference Ng and Gurdon2005, Reference Ng and Gurdon2008). However, it has recently been observed that developmental rates of reconstructed embryos by nuclear transplant (NT) can be improved, in medaka fish, using non-enucleated diploidized oocytes as recipients as the more stable ploidy condition reduces the formation of haploid cells in reconstructed embryos (Bubenshchikova et al., Reference Bubenshchikova, Kaftanovskaya, Motosugi, Fujimoto, Arai, Kinoshita, Hashimoto, Ozato and Wakamatsu2007). However, cell mosaicism is still observed (Kaftanovskaya et al., 2005; Bubenshchikova et al., Reference Kaftanovskaya, Motosugi, Kinoshita, Ozato and Wakamatsu2007).
Zebrafish haploid larvae (both gynogenotes and androgenotes) can survive for up to 5 days (morphogenesis already completed; Nüsslein-Volhard & Dahm, Reference Nüsslein-Volhard and Dahm2002). This fact allowed us to derive cell cultures in vitro from these larvae (unpublished results). If these somatic cultured cells were transplanted into non-enucleated oocytes, the diploid condition would be reconstructed and, possibly, avoid any possible mosaicism in reconstructed embryos.
So, the aim of the present work was to test the developmental ability of reconstructed embryos using cells derived from both androgenetic and gynogenetic haploid larval cultures as donor nuclei.
Materials and methods
Gold zebrafish strain was used in all experiments.
Recipient eggs
Eggs were obtained by gentle extrusion of the ovary (Westerfield, Reference Westerfield2007) and kept inactivated in egg medium (Hanks’ saline medium supplemented with 1.5% (v/v) of BSA and 0.1 g of NaCl/100 ml of Hanks’ saline; pH: 7.4; osmolarity: 310–320 mOsm) at 8 °C (Cardona-Costa et al., Reference Cardona-Costa, Pérez-Camps, García-Ximénez and Espinós2009).
Haploid somatic larval cultures
Gynogenetic
Sperm was recovered from the genital male pore in individual glass microcapillaries (Westerfield, Reference Westerfield2007) and kept in egg medium at 8 °C (Cardona-Costa et al., Reference Cardona-Costa, Pérez-Camps, García-Ximénez and Espinós2009). A sample of 100 μl of the sperm diluted in Hanks’ balanced solution (Cardona-Costa et al., Reference Cardona-Costa, Pérez-Camps, García-Ximénez and Espinós2009) was irradiated using a UV germicide lamp (General Electric, 30 W). Irradiation was carried out at 62 cm of focus-object distance, applying a radiation dose of 0.529 mW/cm2, measured with a USB 4000 (Miniature Fiber Optic Spectrometer; Ocean Optics Inc. First In Photonics,). A vortex (MS1; IKA-Works Inc.) at 200 rpm was also used with the aim of homogenising the irradiation area during UV exposure (Francisco-Simão et al., Reference Francisco-Simão, Cardona-Costa, Perez Camps and García-Ximénez2010). The sperm irradiation time was 1 min.
Androgenetic
Eggs were collected as described in ‘Recipient eggs’. Then, they were UV irradiated by the same procedure as used with sperm, but without shaking, the irradiation time being 12 min.
In vitro fertilization of non-irradiated oocytes with irradiated sperm and vice-versa was carried out as Westerfield (Reference Westerfield2007) described. Briefly, non-activated eggs and sperm were mixed in a 35 mm Petri-dish and both gametes activated by the addition of a 1 ml of system water. After 2–3 min, the dish was filled with system water to achieve well developing embryos. Further embryo culture was done at 28.5 °C. Larvae at 24 h stage that showed the ‘haploid syndrome’ (Westerfield, Reference Westerfield2007) were selected for in vitro culture.
Haploid larvae were mechanically dechorionated, disinfected with 0.2% v/v bleach/system water and washed in Hanks’ buffered salt solution. Both zebrafish androgenetic and gynogenetic larvae were cultured separately in Leibovitz medium supplemented with 20% of FBS and 0.036 g/100 ml of glutamine (L15-FBS) at 28.5°C (Westerfield, Reference Westerfield2007).
Somatic cell nuclear transplant
Somatic cells used as nuclear donors came from primary cultures derived from both gynogenetic and androgenetic larvae.
The two somatic cell nuclear transplant techniques, as well as the equipment employed were described by Pérez-Camps et al. (Reference Pérez-Camps, Cardona-Costa, Francisco-Simao and García-Ximénez2010a).
NT before egg activation
The somatic cell nucleus was inserted in the central region of the egg. To prevent egg activation, the transplant was performed in a handling drop composed of egg medium and the micromanipulation area was cooled down to 8 °C. Non-activated eggs were activated by fresh water after their NT.
NT during egg activation
The donor nucleus was injected in the incipient animal pole, just at the position where zygote nucleus was being constituted. The handling drop was composed of fresh water so that the eggs became the activation and the micromanipulation area was not cooled (room temperature).
Experimental design
Experiment 1
Primary cell cultures derived from haploid androgenetic larvae were used as nuclear donors. Two differenced groups were established depending on the nuclear transplant methodology: NT before egg activation and NT during egg activation.
Experiment 2
Due to the extremely ‘bad’ results obtained when cell donor nuclei were from androgenetic larvae (Experiment 1) and as zebrafish gynogenetic diploid specimens reach adult stages and become fertile (Nüsslein-Volhard & Dahm, Reference Nüsslein-Volhard and Dahm2002), in Experiment 2, cells derived from primary haploid gynogenetic larvae cultures were used as nuclear donors. Two differentiated groups were also established depending on the nuclear transplant methodology: ‘NT before egg activation’ and ‘NT during egg activation’.
Assessments
Survival rates from experimental groups were evaluated at mid blastula transition stage (MBT), 100% epiboly, 24 h and 5 days. To assess egg quality of recipient eggs, fertilizability control groups were immediately carried out after extracting eggs, whatever the time and method of transplant carried out in each experiment. Nuclear transplant sessions were considered only when the initial fertilization rates were higher than 70%.
At least three replicates were done in all experimental groups. Results were analysed using the chi-squared test. When a single degree of freedom was involved, Yates’ correction for continuity was performed.
Results
Experiment 1
From the six NT sessions (both before and during egg activation) using cell cultures derived from androgenetic haploid larvae, no reconstructed embryos reached upper developmental stages to MBT; in fact, only two out of 15 embryos reached this stage.
In the light of results obtained from these experimental sessions, the heteroparental reconstruction was aborted.
Experiment 2
Regarding the developmental rates of reconstructed embryos, when donor cells were derived from gynogenetic haploid larval cultures, significant differences were observed at the MBT stage (p = 0.074; <0.1) in favour of the NT performed ‘during egg activation’ (Table 1). These differences became more evident (p<0.05) at the 100% epiboly stage. In this group, 20% of reconstructed embryos developed and reached the 100% epiboly stage, although embryos transplanted before activation stopped their development at MBT stage (Table 1). Five of the surviving embryos at 100% epiboly continued developing to 24 h (26 somites stage) but only two survived for up to 5 days. In this case, these larvae showed morphologic anomalies, although they presented a heartbeat.
Survival rates of reconstructed embryos derived from the transplant of somatic cell nuclei derived from haploid gynogenetic larvae of 24 h stage into metaphase II eggs or early zygotes in zebrafish
a, b, x, yData in columns with different superscripts are statistically different (p < 0.05).
*Data statistically differ at 10% (p < 0.1).
Discussion
Survival and developmental degree rates obtained from the transplant of nuclei derived from haploid gynogenetic larval cultures were, unexpectedly, much higher than those transplants from androgenetic cultures, also being higher than those results obtained by the transplantation of diploid somatic nuclei into non-enucleated oocytes (Pérez-Camps et al., Reference Pérez-Camps, Cardona-Costa and García-Ximénez2010b). These results could be explained in different ways: (i) They could be due to a possible gametic imprinting. However, on the one hand, in fish, diploids of both gynogenetic and androgenetic condition are viable (Nüsslein-Volhard & Dahm, Reference Nüsslein-Volhard and Dahm2002), and on the other, although this homoparental condition is possible, the expected results pointed to reaching better efficiencies in reconstructing the heteroparental condition. (ii) The culture conditions may also explain the present results. Haploid androgenetic larval cultures showed lower growth rates than haploid gynogenetic (non-published results), which could be an indication of the need for differential culture requirements, possibly due to the existence of a genomic imprinting (Tsalavouta et al., Reference Tsalavouta, Astudillo, Byrnes and Nolan2009; Jiang et al., Reference Jiang, Yu, Yang, Agar, Frado and Johnson2010). (iii) Finally, this difference could be due to the process of obtaining androgenetic larvae. The oocyte UV irradiation method may cause cellular damage which could result in a cell penalization from the outset in haploid androgenetic cultures (Nüsslein-Volhard & Dahm, Reference Nüsslein-Volhard and Dahm2002), which may imply lower in vitro growth rates and lower NT efficiencies.
The NT of nuclei from cells derived from haploid gynogenotes larvae during egg activation by water overcome the MBT developmental barrier, in contrast to that observed when transplants were done before oocyte activation by water. Given that the MBT is the stage at which the zygote transcription begins in zebrafish (Kane & Kimmel, Reference Kane and Kimmel1993; Korz, Reference Korzh2009), the higher developmental stages reached by NT reconstructed embryos (100% epiboly, 24 h and even until 5 days) suggest a complete oocyte activation. However, the reprogramming or integrating degree of transplanted nuclei was not enough to achieve an adult development.
On the other hand, the characterization of recessive mutations with haploid cells has been known for many years in yeast (Botstein & Fink, Reference Botstein and Fink1988). Application of this cell ploidy condition is of early use in vertebrate cells, mainly because of the effectiveness in obtaining and culturing these cells has been highly difficult (Yi et al., Reference Yi, Hong and Hong2009). To our knowledge, one human near-haploid cell line has been derived from carcinogenetic haematopoietic origin to investigate the host/pathogen interaction (Carette et al., Reference Carette, Guimaraes, Varadarajan, Park, Wuethrich, Godarova, Kotecki, Cochran, Spooner, Ploegh and Brummelkamp2009) and one haploid ESCs line was established in medaka fish (Yi et al., Reference Yi, Hong and Hong2009). In this way, zebrafish cell cultures from haploid larvae used in this work could open the possibility to carry out genetic screening of somatic cells of non-tumoural origin in the widely used zebrafish model system. These haploid cell cultures could enable the direct genetic analyses of recessive phenotypes (Yi et al., Reference Yi, Hong and Hong2009), and mutagenesis-based genetic approaches, which should also allow the generation of null mutants for most non-essential genes in vertebrate cells (Carette et al., Reference Carette, Guimaraes, Varadarajan, Park, Wuethrich, Godarova, Kotecki, Cochran, Spooner, Ploegh and Brummelkamp2009).
Author disclosure statement
The authors declare that no conflicting financial interests exist.
Acknowledgements
We would like to thank Mr Javier Rubio Rubio for technical support. The authors also thank Mr Neil Macowan for revising the English of this manuscript.
