Zebrafish embryos obtained by UV-irradiated spermatozoa are gynogenic haploids
UV-irradiated zebrafish spermatozoa were obtained by exposing spermatozoa to varying durations of UV rays as follows: 30 s (6 millijoules (mJ)/cm2), 60 s (12 mJ/cm2), 90 s (18 mJ/cm2), 120 s (24 mJ/cm2) and 150 s (30 mJ/cm2). Zebrafish embryos were then generated by in vitro fertilisation (IVF) of mature eggs using either non-UV-treated or UV-treated spermatozoa from different exposures. We refer to the embryos obtained using non-irradiated spermatozoa as control or diploid embryos and those obtained using irradiated spermatozoa as UV-treated or haploid embryos hereafter.
We first assayed the DNA content of control embryos and UV-treated embryos by fluorescence-assisted cell cycle profiling at ~24 h post fertilisation (hpf) using Propidium Iodide (PI) staining. As seen in the PI fluorescence intensity plots, control embryos show peak PI fluorescence intensity at ~25 arbitrary units (a.u), while in UV-treated embryos the peak fluorescence decreased by half to ~12 a.u (Fig. 1A and Suppl. Fig. 1A). PI is a double-stranded nucleic acid binding dye and its fluorescence intensity can be correlated with the DNA content of the tissue being analysed. In our experiments, the PI intensity in control embryos is twice that of the PI intensity in UV-treated embryos from each exposure, indicating that the DNA content in control embryos is twice that of UV-treated embryos. Since zebrafish embryos are naturally diploid, our cell cycle profiling data leads us to conclude that control embryos are diploid and the UV-treated embryos are haploids. We performed metaphase chromosome spreads, which allow direct counting of chromosomes to verify the cell cycle profiling data. Control embryos had a chromosome count of 50 while all UV-treated embryos had a chromosome count of 25 (Suppl. Fig. 1B), validating that the UV-treated embryos were haploid.
Mouse and frog haploids harbour smaller sub-nuclei in addition to the zygotic nuclei, which are postulated to be the inert paternal chromatin. Live haploid zebrafish embryos are morphologically indistinguishable from control diploid embryos for the first 10–12 h of development (data not shown). Immunolabelling of control diploid embryos for α-tubulin (microtubules), γ-tubulin (centrosomes) and DAPI (DNA) at 45 min post fertilisation (mpf) revealed cytokinetic microtubular arrays and the dividing zygotic nuclei (Suppl. Fig. 1C). UV-treated haploid embryos from all durations of UV exposures had the cytokinetic microtubular array as was expected. However, UV-treated haploid embryos from the 30 to 90 s exposure had an inactive chromatin streak stretched across the cytokinetic microtubular array. This streak of inactive chromatin was visible as condensed inactive chromatin in UV-treated haploid embryos from the 120 to 150 s exposure. We postulate that the extra chromatin seen in the haploids is the UV irradiated paternal chromatin and we categorize it as cell biologically inactive due to its inability to interact with microtubules and pericentriolar material. A cytokinesis time-course study revealed the presence of the inactive paternal chromatin in all haploids at 60 mpf (data not shown) and we hypothesize that it is eventually extruded from the developing embryo, similar to the polar body chromatin.
Haploid zebrafish embryos manifest a pleiotropic developmental syndrome known as the haploid syndrome
Live haploids are morphologically indistinguishable from control diploid embryos during the first 20 h of early development (data not shown). By 24 hpf, zebrafish embryos are larvae with a functional circulatory system and well-developed axis with a long tail (Fig. 1B). At ~24–30 hpf, haploid embryos from all UV exposures were morphologically different from control embryos. All haploids could be sorted into two phenotypic categories based on the severity of the haploid syndrome (Fig. 1C, D). The striking feature of embryos with mild haploid syndrome was a shorter body axis and smaller heads. These embryos had mild pericardial edema but were not cyclopic and had no curvature of the tail (Fig. 1C). In comparison, haploid embryos manifesting severe haploid syndrome had cyclopia, severe pericardial edema and a severe bending of the tail in addition to the shortened body axis (Fig. 1D). While control embryos become free-swimming larvae and inflated swim bladders by 4–5 days post fertilisation (dpf), haploid embryos with severe haploid syndrome die by 3 dpf, while those manifesting mild haploid syndrome survive for up to 5–6 dpf, but do not inflate swim bladders.
Haploid zebrafish embryos generated from shorter UV exposures have higher survival indices and lower incidence of severe haploid syndrome
Since we could reliably categorize haploid embryos based on the severity of the haploid syndrome, we quantified the occurrence of each phenotype category across all UV exposures (Fig. 1E). Control embryos did not manifest any phenotypic abnormalities or lethality. In UV-treated embryos, we observed a trend of decrease in incidence of mild haploid syndrome with increasing duration of UV exposures from 60 to 150 s. Similarly, there was a decrease in the incidence of severe haploid syndrome with increasing duration of UV exposures. However, we found that lethality increased with increasing duration of UV exposures. Since the objective was to identify a UV exposure threshold that would generate healthier haploids, we binned the lethality and severe haploid syndrome as a combinatorial output of the increasing UV exposure. Our data reveal that ~80% of UV-treated embryos from 60 and 90 s exposures and ~90% of UV-treated embryos from the 120 and 150 s exposures either die or manifest severe haploid syndrome. The remaining ~20% embryos from 60 and 90 s exposures and ~10% embryos from the 120 and 150 s exposures manifest the mild haploid syndrome. Strikingly, >95% of embryos from the shortest UV exposure of 30 s either die or manifest severe haploid syndrome. Thus our data show that the clutch of embryos obtained from 60–90 s UV exposures are healthier with significantly lower clutch lethality when compared to the ones generated from 120–150 s exposures. Furthermore, though haploid zebrafish embryos generated from 60 and 90 s UV exposures both yield ~20% embryos with mild haploid syndrome, we conclude that the 60 s exposure is better due to the lower lethality in the clutch in comparison with the 90 s exposure.
The progressive increase in embryonic lethality in gynogenic haploids generated from spermatozoa exposed to UV rays for longer than 60 s suggests that the sperm potentially contributes transcripts or other molecular information for embryonic development in zebrafish. It is unlikely that the degraded paternal DNA is the cause of embryonic lethality as it is not inherited by all cells of the early zygote. Additionally, if the lethality is due to gene dosage errors it would be uniform across all durations of UV exposures as gynogenotes obtained at all UV exposures are haploids. Thus the sperm must contribute information in addition to DNA and centrioles that are potentially progressively degraded with increasing UV exposures, which manifests as UV dose-dependent increase in haploid lethality. It would be interesting to identify such paternal transcripts that the zygote potentially inherits, which are required for normal vertebrate development and survival.
It would be ideal to eliminate the occurrence of the haploid syndrome completely in the gynogenic haploid embryos. A transient heat shock paradigm has been used to induce instant duplication of the genome in zebrafish to generate tetraploids from diploids. It is possible to diploidize haploids by a transient heat shock during the first zygotic mitosis (data not shown) and reduce or eliminate the occurrence of the developmental syndrome completely in such gynogenic diploids, particularly if the haploids are generated using spermatozoa UV-irradiated for 60–90 s.