Cold-shock eliminates female nucleus in fertilized eggs to induce androgenesis in the loach (Misgurnus anguillicaudatus), a teleost fish

Abstract

Background: Androgenesis (all-male inheritance) is generally induced by means of irradiating the eggs to inactivate the maternal genome, followed by fertilization with normal sperm. In fish, the conventional technique for induced androgenesis has been applied for rapid fixation to traits, recovery of cryopreserved genotypes, sex-control, etc. A new method of androgenesis that eliminates the need to irradiate the egg was proposed using the loach, Misgurnus anguillicaudatus (a teleost fish).

Results: When the eggs of wild-type females were fertilized with sperm of albino or orange phenotype males and cold-shocked at 0 to 3°C for 60 min duration just after fertilization, generally more than 30% (with a peak of 100%) of the hatched progeny were androgenotes. While a few of them were the normal diploid, most of them turned out to be abnormal haploid. All-male inheritance was verified by the expression of the recessive color trait (albino or orange) and microsatellite genotypes comprising only paternally derived alleles. Nuclear behavior after the cold-shock treatment was traced by microscopic observation of DAPI (4'6-diamidino-2-phenylindole)-stained samples and hematoxylin-eosin stained histological sections, and the extrusion of egg (maternal) nucleus was observed in eggs treated in the optimum timing.

Conclusion: In this paper, we demonstrate that cold-shock treatment (at 0 and 3°C) of loach eggs for 60 min just after fertilization successfully induces androgenetic haploid development. The most likely mechanism of cold-shock induced androgenesis is an elimination of the egg nucleus together along with the second polar body and subsequent development of a decondensed sperm nucleus or male pronucleus.

Figure 1

Fertilization (A) and hatching rates (B) relative to total eggs used in control and 0, 3, 6 and 9°C cold-shocked groups from five different crosses. Histograms and bars denote means and SD, respectively. Different letters mean significant differences (P < 0.05).

Figure 2

External appearance of larvae developing from the control and cold-shocked eggs in the loach. A: Wild-type fry in the control group with normal external appearance; B: Albino fry from the 0°C cold-shocked group with abnormal external appearance; C: Wild-type fry from the 0°C cold-shocked group with normal external appearance; D: Wild-type (upper) and albino (lower) fry from the 3°C cold-shocked group with normal external appearance. Scales denote 1 mm.

Figure 3

Frequencies of four kinds of phenotypes, normal wild-type, abnormal wild-type, normal albino and abnormal albino in hatched fry from control and 0, 3, 6 and 9°C cold-shocked groups from five different crosses. Histograms and bars denote means and SD, respectively. Different letters mean significant differences (P < 0.05).

Figure 4

Relative DNA contents of fry developing from the control and cold-shocked groups. A: Wild-type diploid of the control group; B: Wild-type triploid from the 0°C cold-shocked group; C: Albino haploid from the 0°C cold-shocked group; D: Hyper diploid showing wild-type phenotype from the 0°C cold-shocked group; E: Euploid mosaic (diploid and triploid) with wild-type phenotype from the 6°C cold-shocked group; F: Aneuploid mosaic (1.3n-2.9n) with wild-type phenotype from the 9°C cold-shocked group.

Figure 5

Fertilization (A) and hatching rates (B) relative to total eggs used in control and 3°C cold-shocked groups from six different crosses. Histograms and bars denote means and SD, respectively. Different letters mean significant differences (P < 0.05).

Figure 6

Frequencies of four kinds of phenotypes, normal wild-type, abnormal wild-type, normal albino and abnormal albino in hatched fry from control and 3°C cold-shocked groups from six different crosses. Histograms and bars denote means and SD, respectively. Different letters mean significant differences (P < 0.05).

Figure 7

DAPI-stained nuclear behavior in eggs after fertilization in the control. A (10 min after fertilization; af): three condensed nuclei, sperm nucleus (sn), egg nucleus (en) and second polar body nucleus (2 pb); B (20 min af) two decondensed pronuclei, female pronucleus (fpn) and male pronucleus (mpn); C (30 min af) decondensed and fused one pronucleus; D (40 min af) anaphase of the first cleavage. Scale bars denotes 10 μm.

Figure 8

DAPI-stained nuclear behavior in cold-shocked eggs after fertilization. Arrows indicate condensed nuclei. Arrowheads indicate decondensed pronuclei. A (60 min after fertilization (af); just after the cold-shock treatment): two condensed nuclei, egg nucleus and sperm nucleus; B (70 min af): two condensed nuclei, sperm nucleus (sn) and egg nucleus (en) or second polar body nucleus (2 pb); C (80 min af): three condensed nuclei, sperm nucleus, egg nucleus and second polar body nucleus; D (70 min af): one decondensed male pronucleus, section from the same egg shown in E; E (70 min af): two condensed nuclei, egg nucleus and second polar body nucleus; F (90 min af): one condensed nucleus and one decondensed pronucleus; G (90 min af): two decondensed pronuclei; H (90 min af): one decondensed pronucleus. Scale bars denote 10 μm.

Figure 9

Histological sections of control eggs after fertilization. A (10 min after fertilization: af): second polar body released and egg nucleus present just underneath the polar body; B (10 min af): condensed sperm nucleus; C (20 min af): decondensed female and male pronuclei; D (30 min af): metaphase of first cleavage; E (40 min af): anaphase of first cleavage; F (50 min af): prophase of second cleavage. Scale bars denote 10 μm.

Figure 10

Histological sections of cold-shocked eggs after fertilization. A (70 min after fertilization: af): second polar body released and egg nucleus present just underneath the polar body; B (70 min af) second polar body present, but no egg nucleus; C (70 min af): abnormal extrusion of second polar body (note abnormal metaphase equator or nuclear substances ranging from egg surface to polar body); D (70 min af): extrusion of second polar body suppressed; E (70 min af): condensed sperm nucleus present; F (80 min af): decondensed male pronucleus appears; G (90 min af): two asters seen; H (100 min af): anaphase of first cleavage. Scale bars denote 10 μm.

Figure 11

Histological sections showing abnormalities in cold-shocked eggs after fertilization (af). A (80 min af) anuclear cell with one aster (arrow); B (90 min af) anuclear cell with two asters (arrows); C (100 min af) anuclear cell with two asters (arrows); D (120 min af) cell with a clumped nucleus (arrowhead) and a tripolar spindle with three asters (arrows); E (120 min af) cell with a clumped nucleus (arrowhead) and four asters (arrows). Scales denote 10 μm.

Figure 12

Presumptive cytological mechanisms for androgenesis induced by cold-shock treatment for just-fertilized eggs of the loach.