Deletion of TRPV3 and CaV3.2 T-type channels in mice undermines fertility and Ca2+ homeostasis in oocytes and eggs

Calcium (Ca2+) influx during oocyte maturation and after sperm entry is necessary to fill the internal Ca2+ stores and for complete egg activation. We knocked out the transient receptor potential vanilloid member 3 (TRPV3) and the T-type channel, CaV3.2 to determine their necessity for maintaining these functions in mammalian oocytes/eggs. Double knockout (dKO) females were subfertile, their oocytes and eggs showed reduced internal Ca2+ stores, and following sperm entry or PLCz cRNA injection, fewer dKO eggs displayed Ca2+ responses compared to wildtype (WT) eggs, which were also of lower frequency. These parameters were rescued and/or enhanced by removing extracellular Mg2+, suggesting the residual Ca2+ influx could be mediated by the TRPM7 channel, consistent with the termination of divalent-cation oscillations in dKO eggs by a TRPM7 inhibitor. In total, we demonstrated that TRPV3 and CaV3.2 mediate the complete filling of the Ca2+ stores in mouse oocytes and eggs. We also show they are required for initiating and maintaining regularly spaced-out oscillations, suggesting that Ca2+ influx through PM ion channels dictates the periodicity and persistence of Ca2+ oscillations during mammalian fertilization.

Cytoplasmic streaming drifts the polarity cue and enables posteriorization of the Caenorhabditis elegans zygote at the side opposite of sperm entry

The polarity cue moves before polarity establishment through a stochastic cytoplasmic streaming in C. elegans zygotes.

Inhibiting actin polymerization does not prevent the fast block to polyspermy in the African clawed frog, Xenopus laevis

Fertilization of an egg by more than one sperm presents one of the earliest and most prevalent obstacles to successful reproduction. As such, eggs employ multiple mechanisms to prevent sperm entry into the nascent zygote. The fast block to polyspermy is a depolarization of the egg membrane initiated by sperm entry and is employed by diverse external fertilizers including frogs and sea urchins. For some external fertilizers, sperm entry is associated with actin polymerization during the initiation of the fast block. We therefore sought to determine whether the fast block to polyspermy in the African clawed frog, Xenopus laevis, requires actin polymerization. Although actin polymerization is required for sperm entry into eggs from diverse external fertilizers, including sea urchins and zebrafish, here we demonstrate that actin polymerization is not required for the fast block to polyspermy in X. laevis.

Cytoplasmic streaming drifts the polarity cue and specifies the cell polarity in Caenorhabditis elegans zygotes

Cell polarisation is required to define body axes during development. The position of spatial cues for polarisation is critical to direct the body axes. In Caenorhabditis elegans zygotes, the sperm-derived pronucleus/centrosome complex (SPCC) serves as the spatial cue to specify the anterior–posterior axis. Approximately 30 minutes after fertilisation, the contractility of the cell cortex is relaxed near the SPCC, which is the earliest sign of polarisation and called symmetry breaking (SB). It is unclear how the position of SPCC at SB is determined after fertilisation. Here, we show that SPCC drifts dynamically through the cell-wide flow of the cytoplasm, called meiotic cytoplasmic streaming. This flow occasionally brings SPCC to the opposite side of the sperm entry site before SB. Our results demonstrate that cytoplasmic flow determines stochastically the position of the spatial cue of the body axis, even in an organism like C. elegans for which development is stereotyped.

Phosphatidylserine on viable sperm and phagocytic machinery in oocytes regulate mammalian fertilization

Fertilization is essential for species survival. Although Izumo1 and Juno are critical for initial interaction between gametes, additional molecules necessary for sperm:egg fusion on both the sperm and the oocyte remain to be defined. Here, we show that phosphatidylserine (PtdSer) is exposed on the head region of viable and motile sperm, with PtdSer exposure progressively increasing during sperm transit through the epididymis. Functionally, masking phosphatidylserine on sperm via three different approaches inhibits fertilization. On the oocyte, phosphatidylserine recognition receptors BAI1, CD36, Tim-4, and Mer-TK contribute to fertilization. Further, oocytes lacking the cytoplasmic ELMO1, or functional disruption of RAC1 (both of which signal downstream of BAI1/BAI3), also affect sperm entry into oocytes. Intriguingly, mammalian sperm could fuse with skeletal myoblasts, requiring PtdSer on sperm and BAI1/3, ELMO2, RAC1 in myoblasts. Collectively, these data identify phosphatidylserine on viable sperm and PtdSer recognition receptors on oocytes as key players in sperm:egg fusion.

Sperm Entry into the Egg Cell Induces the Progression of Karyogamy in Rice Zygotes

Karyogamy is a prerequisite event for plant embryogenesis, in which dynamic changes in nuclear architecture and the establishment of appropriate gene expression patterns must occur. However, the precise role of the male and female gametes in the progression of karyogamy still remains elusive. Here, we show that the sperm cell possesses the unique property to drive steady and swift nuclear fusion. When we fertilized egg cells with sperm cells in vitro, the immediate fusion of the male and female nuclei in the zygote progressed. This rapid nuclear fusion did not occur when two egg cells were artificially fused. However, the nuclear fusion of two egg nuclei could be accelerated by additional sperm entry or the exogenous application of calcium, suggesting that possible increase of cytosolic Ca2+ level via sperm entry into the egg cell efficiently can facilitate karyogamy. In contrast to zygotes, the egg–egg fusion cells failed to proliferate beyond an early developmental stage. Our transcriptional analyses also revealed the rapid activation of zygotic genes in zygotes, whereas there was no expression in fused cells without the male contribution. Thus, the male sperm cell has the ability to cause immediate karyogamy and to establish appropriate gene expression patterns in the zygote.

Analytic solutions for calcium ion fertilisation waves on the surface of eggs

The evolution of calcium fertilisation waves on the cortex of amphibian eggs can be described by a nonlinear reaction-diffusion process on the surface of a sphere. Here, we use the nonclassical symmetry technique to find an exact analytic solution that describes the evolution of the calcium concentration. The solutions presented compare well with published experimental results. The analytic solution can be used to give insight into the processes governing the fertilisation wave, such as the flow of calcium ions from the sperm entry point. By finding a spiral solution to an approximate equation linearised near saturation, we also demonstrate how solutions with other properties may be constructed using this technique.

The Ly6/uPAR protein Bouncer is necessary and sufficient for species-specific fertilization

Fertilization is fundamental for sexual reproduction, yet its molecular mechanisms are poorly understood. We found that an oocyte-expressed Ly6/uPAR protein, which we call Bouncer, is a crucial fertilization factor in zebrafish. Membrane-bound Bouncer mediates sperm-egg binding and is thus essential for sperm entry into the egg. Remarkably, Bouncer not only is required for sperm-egg interaction but is also sufficient to allow cross-species fertilization between zebrafish and medaka, two fish species that diverged more than 200 million years ago. Our study thus identifies Bouncer as a key determinant of species-specific fertilization in fish. Bouncer’s closest homolog in tetrapods, SPACA4, is restricted to the male germline in internally fertilizing vertebrates, which suggests that our findings in fish have relevance to human biology.

The Ly6/uPAR protein Bouncer is necessary and sufficient for species-specific fertilization

Fertilization is fundamental for sexual reproduction, yet its molecular mechanisms are poorly understood. Here, we identify an oocyte-expressed Ly6/uPAR protein, which we call Bouncer, as a crucial fertilization factor in zebrafish. We show that membrane-bound Bouncer mediates sperm-egg binding and is thus essential for sperm entry into the egg. Remarkably, Bouncer is not only required for sperm-egg interaction, but also sufficient to allow cross-species fertilization between zebrafish and medaka, two fish species that diverged over 150 million years ago. Our study thus identifies Bouncer as a key determinant of species-specific fertilization in fish. Bouncer’s closest homolog in tetrapods, SPACA4, is restricted to the male gonad in internally fertilizing vertebrates, suggesting that our findings in fish have relevance to human biology.