Molecular Mechanism of Oocyte Activation in Mammals: Past, Present, and Future Directions.

During mammalian fertilization, repetitive intracellular Ca2+ increases known as Ca2+ oscillations occur. These oscillations are considered crucial for successful fertilization and subsequent embryonic development. Numerous researchers have endeavored to elucidate the factors responsible for inducing Ca2+ oscillations across various mammalian species. Notably, sperm-specific phospholipase C zeta (PLCζ) emerged as a prominent candidate capable of initiating Ca2+ oscillations, particularly in mammals. Genetic mutation of PLCζ in humans results in the absence of Ca2+ oscillations in mouse oocytes. Recent studies further underscored PLCζ's significance, revealing that sperm from PLCζ-deficient (Plcz1-/-) mice fail to induce Ca2+ oscillations upon intracytoplasmic sperm injection (ICSI). Despite these findings, observations from in vitro fertilization (IVF) experiments using Plcz1-/- sperm revealed some residual intracellular Ca2+ increases and successful oocyte activation, hinting at potential alternative mechanisms. In this review, we introduced the current hypothesis surrounding oocyte activation in mammals, informed by contemporary literature, and probed into the enigmatic mechanisms underlying mammalian fertilization-induced oocyte activation.

Recent advances of oocyte/embryo vitrification in mammals from rodents and large animals.

Vitrification is a valuable technology that enables semipermanent preservation and long-distance or international transportation of genetically modified and native animals. In laboratory mice, vitrification maintains and transports embryos, and many institutions and companies sell vitrified embryos. In contrast, despite numerous papers reporting on vitrification in livestock over the past decade, practical implementation has yet to be achieved. However, with advances in genome editing technology, it is anticipated that the number of genetically modified domestic animals will increase, leading to a rise in demand for vitrification of oocytes and embryos. Here, we provide an objective overview of recent advancements in vitrification technology for livestock, drawing a comparison with the current developments in laboratory animals. Additionally, we explore the future prospects for vitrification in livestock, focusing on its potential benefits and drawbacks.

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.