Successful Production of Offspring Derived from Phospholipase C Zeta-Deficient Sperm by Additional Artificial Activation

During mammalian fertilization, repetitive rises of intracellular calcium called calcium oscillations are required for full activation of oocytes. Therefore, oocytes such as round spermatid injected or somatic cell nuclear transferred require additional artificial activation which mimics the calcium oscillations. It is well recognized that sperm specific phospholipase C (PLCζ) is a strong candidate as the sperm factor which can induce calcium oscillations and, at least in mammals, the genetic mutation of PLCζ in human causes male infertility due to the lack of calcium oscillations in the oocytes. Recent studies showed that the sperm lacking PLCζ (Plcz1^-/-) still could induce rise(s) of intracellular calcium in the oocytes after IVF but not intracytoplasmic sperm injection (ICSI). In the ICSI oocytes, no pronuclear formation or development to the two-cell stage was observed. However, it is still unclear whether additional activation treatment can rescue the low developmental ability of Plcz1^-/--sperm-derived oocytes after ICSI. In this study, we examined whether oocytes injected with a Plcz1^-/- sperm can develop to term by additional artificial activation. In oocytes injected a Plcz1^-/- sperm and Plcz1^-/- and eCS (another candidate of the sperm factor) double knockout sperm (Plcz1^-/-eCS^-/-), the rates of pronuclear formation were very low (2.0 ± 2.3% and 6.1 ± 3.7%, respectively) compared to control (92.1 ± 2.6%). However, these rates were dramatically improved by additional procedures of PLCζ-mRNA injection or SrCl₂ treatment (Plcz1^-/- sperm + PLCζ mRNA, Plcz1^-/- sperm + SrCl₂ and Plcz1^-/-eCS^-/- sperm + PLCζ mRNA; 64.2 ± 10.8%, 89.2 ± 2.4% and 72.6 ± 5.4%, respectively). Most of the oocytes were developed to the two-cell stage. After embryo transfer, healthy pups were obtained in all these groups (Plcz1^-/- sperm + PLCζ mRNA:10.0 ± 2.8%, Plcz1^-/- sperm + SrCl₂:4.0 ± 4.3% and Plcz1^-/-eCS^-/- sperm + PLCζ mRNA: 10.0 ± 5.7%). The rate in Plcz1^-/- sperm + SrCl₂ group was significantly lower than that in control (26.0 ± 2.4%). Taken together, our present results show that additional activation treatment such as SrCl₂ and PLCζ mRNA can fully support to develop to term even in oocyte injected Plcz1^-/- sperm. In addition, PLCζ-induced oocyte activation is more suitable for successful development to term compared to that such as phenomenon induced by SrCl₂. These findings will contribute to improvement for male-dependent human infertility and reproductive technologies in other mammalian species.