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Free access

Sook-Young Yoon and Rafael A Fissore

During fertilization of mammalian eggs a factor from the sperm, the sperm factor (SF), is released into the ooplasm and induces persistent [Ca2+]i oscillations that are required for egg activation and embryo development. A sperm-specific phospholipase C (PLC), PLCz, is thought to be the SF. Here, we investigated whether the SF activity and PLCζare simultaneously and completely released into the ooplasm soon after sperm entry. To accomplish this, we enucleated sperm heads within 90 min of intracytoplasmic sperm injection (ICSI) and monitored the persistence of the [Ca2+]i oscillations in eggs in which the sperm had been withdrawn. We also stained the enucleatedsperm heads to ascertain the presence/absence of PLCζ. Our results show that by 90 min all the SF activity had been released from the sperm, as fertilized enucleated eggs oscillated as fertilized controls, even in cases in which oscillations were prolonged by arresting eggs at metaphase. In addition, we found that the released SF activity became associated with the pronucleus (PN), as induction of PN envelope breakdown evoked comparable [Ca2+]i responses in enucleated and non-manipulated zygotes. Lastly, we found that PLCzlocalized to the equatorial area of bull sperm and to the post-acrosomal region of mouse sperm and that by 90 min after ICSI all the sperm’s PLCζimmunoreactivity was lost in both species. Altogether, our findings show that during fertilization the SF activity and PLCζimmunoreactivity are simultaneously released from the sperm, suggesting that PLCζmay be the only [Ca2+]i oscillation-inducing factor of mammalian sperm.

Free access

Neha Gupta, Hiroki Akizawa, Hoi Chang Lee, and Rafael A Fissore

The discovery of PLCZ1 nearly 20 years ago as the primary Ca2+ oscillation-inducing factor in the sperm of mammals represented a significant breakthrough in our quest to elucidate the molecules and pathways that promote egg activation during fertilization. The advent of the intracytoplasmic sperm injection (ICSI) technique, which made fertilization possible without sperm capacitation, acrosome reaction, and gamete fusion, strengthened the research that led to the discovery of PLCZ1 and became an essential clinical tool for humans. The use of ICSI combined with the detection of PLCZ1 expression and mutations in infertile patients established the fundamental role of PLCZ1 in human fertility while leading to the discovery of novel components of the perinuclear theca, the site of the residence of PLCZ1 in sperm before fertilization. Remarkably, the more extensive use of ICSI in species other than humans and mice revealed poor success and exposed gaps in our understanding of PLCZ1 release and/or activation. Similarly, fertilization using sperm from mouse models lacking Plcz1 has produced striking results whose true implications are yet to be determined. Nevertheless, answers to these unresolved questions will produce a complete picture of the adaptations and molecular players that mammalian species employ to ensure the success of the triggering event of embryo development that has linked generations since the beginning of times.

Free access

Manabu Kurokawa, Ken-ichi Sato, Jeremy Smyth, Hua Wu, Kiyoko Fukami, Tadaomi Takenawa, and Rafael A Fissore

Recent evidence in marine invertebrate, frog, and zebrafish eggs suggests the involvement of a Src family kinase (SFK) in fertilization-induced Ca2+ release. In the present study, we have investigated whether activation of an SFK is required for initiation of intracellular Ca2+ ([Ca2+]i) oscillations in mouse fertilization. We detected a Hck-like protein and tyrosine-phosphorylated proteins in soluble and insoluble sperm fractions, respectively. However, the presence of these proteins did not correspond to the active fractions of porcine sperm extracts (pSE). Moreover, [Ca2+]i oscillations induced by pSE in mouse eggs were unaltered by pre-incubation of pSE with specific SFK inhibitors such as 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]-pyrimidine (PP2) or lavendustin A, despite the fact that the inhibitors were shown to be active both in vivo and in vitro. Another SFK inhibitor, peptide A, blocked oscillations when incubated with pSE prior to injection into eggs, but this inhibition required more than ten times the concentration reportedly required to inhibit SFK activity. In addition, pre-injection or pre-incubation of eggs with these inhibitors did not affect the ability of pSE to trigger [Ca2+]i oscillations in mouse eggs. Microinjection of a recombinant c-Src protein or mRNAs encoding constitutively active Src proteins did not induce [Ca2+]i release. Finally, when sperm and eggs, both of which were pre-treated with PP2, were fertilized, [Ca2+]i oscillations occurred normally. We can therefore conclude that activation of an SFK is neither necessary nor sufficient for triggering fertilization-induced [Ca2+]i oscillations.

Free access

Pablo J Ross, Ramon M Rodriguez, Amy E Iager, Zeki Beyhan, Kai Wang, Neli P Ragina, Sook-Young Yoon, Rafael A Fissore, and Jose B Cibelli

The production of cloned animals by the transfer of a differentiated somatic cell into an enucleated oocyte circumvents fertilization. During fertilization, the sperm delivers a sperm-specific phospholipase C (PLCZ) that is responsible for triggering Ca2 + oscillations and oocyte activation. During bovine somatic cell nuclear transfer (SCNT), oocyte activation is artificially achieved by combined chemical treatments that induce a monotonic rise in intracellular Ca2 + and inhibit either phosphorylation or protein synthesis. In this study, we tested the hypothesis that activation of bovine nuclear transfer embryos by PLCZ improves nuclear reprogramming. Injection of PLCZ cRNA into bovine SCNT units induced Ca2 + oscillations similar to those observed after fertilization and supported high rates of blastocyst development similar to that seen in embryos produced by IVF. Furthermore, gene expression analysis at the eight-cell and blastocyst stages revealed a similar expression pattern for a number of genes in both groups of embryos. Lastly, levels of trimethylated lysine 27 at histone H3 in blastocysts were higher in bovine nuclear transfer embryos activated using cycloheximide and 6-dimethylaminopurine (DMAP) than in those activated using PLCZ or derived from IVF. These results demonstrate that exogenous PLCZ can be used to activate bovine SCNT-derived embryos and support the hypothesis that a fertilization-like activation response can enhance some aspects of nuclear reprogramming.

Open access

Dalileh Nabi, Davide Bosi, Neha Gupta, Nidhi Thaker, Rafael Fissore, and Lynae M Brayboy

In brief

Oocyte quality remains the most important and unsolved issue in reproduction. Our data show that multidrug resistance transporters and oocyte mitochondria are involved in determining oocyte quality in a mouse model.


Multidrug resistance transporter-1 (MDR-1) is a transmembrane ATP-dependent effluxer present in organs that transport a variety of xenobiotics and by-products. Previous findings by our group demonstrated that this transporter is also present in the oocyte mitochondrial membrane and that its mutation led to abnormal mitochondrial homeostasis. Considering the importance of these organelles in the female gamete, we assessed the impact of MDR-1 dysfunction on mouse oocyte quality, with a particular focus on the meiotic spindle organization, aneuploidies, Ca2+ homeostasis, ATP production and mtDNA mutations. Our results demonstrate that young Mdr1a mutant mice produce oocytes characterized by lower quality, with a significant delay in the germinal vesicle to germinal vesicle breakdown transition, an increased percentage of symmetric divisions, chromosome misalignments and a severely altered meiotic spindle shape compared to the wild types. Mutant oocytes exhibit 7000 more SNPs in the exomic DNA and twice the amount of mitochondrial DNA (mtDNA) SNPs compared to the wild-type ones. Ca2+ analysis revealed the inability of MDR-1 mutant oocytes to manage Ca2+ storage content and oscillations in response to several stimuli, and ATP quantification shows that mutant oocytes trend toward lower ATP levels compared to wild types. Finally, 1-year-old mutant ovaries express a lower amount of SIRT1, SIRT3, SIRT5, SIRT6 and SIRT7 compared to wild-type levels. These results together emphasize the importance of MDR-1 in mitochondrial physiology and highlight the influence of MDR-1 on oocyte quality and ovarian aging.

Free access

Luis Águila, Ricardo Felmer, María Elena Arias, Felipe Navarrete, David Martin-Hidalgo, Hoi Chang Lee, Pablo Visconti, and Rafael Fissore

The efficiency of intracytoplasmic sperm injection (ICSI) in the bovine is low compared to other species. It is unknown whether defective oocyte activation and/or sperm head decondensation limit the success of this technique in this species. To elucidate where the main obstacle lies, we used homologous and heterologous ICSI and parthenogenetic activation procedures. We also evaluated whether in vitro maturation negatively impacted the early stages of activation after ICSI. Here we showed that injected bovine sperm are resistant to nuclear decondensation by bovine oocytes and this is only partly overcome by exogenous activation. Remarkably, when we used heterologous ICSI, in vivo-matured mouse eggs were capable of mounting calcium oscillations and displaying normal PN formation following injection of bovine sperm, although in vitro-matured mouse oocytes were unable to do so. Together, our data demonstrate that bovine sperm are especially resistant to nuclear decondensation by in vitro-matured oocytes and this deficiency cannot be simply overcome by exogenous activation protocols, even by inducing physiological calcium oscillations. Therefore, the inability of a suboptimal ooplasmic environment to induce sperm head decondensation limits the success of ICSI in the bovine. Studies aimed to improve the cytoplasmic milieu of in vitro-matured oocytes and to replicate the molecular changes associated with in vivo capacitation and acrosome reaction will deepen our understanding of the mechanism of fertilization and improve the success of ICSI in this species.