Cadaveric sperm induces intergeneric androgenesis in the fish, Hemigrammus caudovittatus

Abstract

Intergeneric androgenetic golden Buenos Aires tetra (BT), Hemigrammus caudovittatus was generated using sperm drawn from post-mortem males preserved at -20°C for 10, 20, 30 and 40 days or fresh sperm to activate the UV-irradiated oocytes of black widow tetra (WT), Gymnocorymbus ternetzi. UV-irradiation (4.2W/m²) of the oocytes for 3 min inactivated their nuclear genome. Fry hatched out from these activated oocytes were haploids; suffering haploid syndrome, they died before or within 48h after hatching. Fresh BT sperm activated 95% oocytes; however, the sperm drawn from post-mortem males preserved at -20°C for 60 (within glycerol packing) and 30 days (without glycerol packing) activated only 24 and 19% oocytes, respectively. Following activation, diploidy was restored by shocking the 25-min-old embryos at 41°C for 2min. Nuclear genomic inactivation of the oocytes was confirmed by (i) production of 100% haploids, (ii) karyotype and erythrocyte measurements, (iii) phenotypic markers, (iv) progeny testing and (v) species-specific marker. At hatching, survival of androgenotes decreased from 11% for those induced with fresh sperm to 4% for those generated using sperm from 30-day-old post-mortem males. Reproductive performance of the 'fresh' and 'cadaveric' F₀ and F₁ androgenetic males (Y²Y²) was superior to the control (X¹Y²). Crosses involving homozygous (Y²Y²) 'fresh' F₀ androgenetic males with heterozygous females (X¹X²) and F₀ homozygous males (Y²Y²) with females (X²X²) produced 2-4% unexpected female progenies. Paternal autosomes, inherited by the homozygous androgenetic female (X²X²), induced the production of female progenies in significantly less number of crosses than the crosses with heterozygous females (X¹X²), which carried equal number of paternal and maternal autosomes. PCR analyses of the genomic DNA of normal male and unexpected F₁ and F₂ female progenies amplified by DMRT 1 specific primer produced bands of 237 and 300bp length, and thereby confirmed that these unexpected females were genetic males. RAPD analyses of the androgenetic progenies showed that their genome was not contaminated with maternal genome.