Subzonal fertilization of mouse round spermatids

Mouse round spermatids were electrofused with homologous mature oocytes to examine the behaviour of their nuclei within the ooplasm and the abilities of development. A single spermatid was injected in the perivitelline space of a mature oocyte and an electron fusion pulse was given. The best round spermatid-oocyte pairs (RS-O) fusion took place at 20–30 s AC (1 MHz, 50V/cm) followed by a single fusion DC pulse (3 700-3 800 V/cm, 25 μs) and another 30 s AC current. The total survival rate and fusion rate of RS-O were 89.0% (575/646) and 61.9 % (356/575), respectively. 49.2 % (175/356) of fused oocytes developed to 2PN stage. The concentration of Ca2+ in the fusion medium produced no significant effect on the above targets. The 2PN development rate of the fused RS-O from the oocytes collected 14–16 h after hCG injection was higher than others. 32.6% (57/175) of the 2PN oocytes had fully developed spermatid (male) and oocyte (female) pronuclei. The rest spermatid-derived pronuclei remained small in size throughout the pronuclear stage. 192 fertilized eggs were transferred surgically into the oviducts of the pseudopregnant female mouse. 12 offspring were produced. Project supported by the State Key Laboratory of Reproductive Biology.