Intracellular Pathways of Holothuroid Oocyte Maturation Induced by the Thioredoxin Trx-REES

In holothuroids, oocyte maturation is stopped in ovaries at the prophase I stage of meiosis. In natural conditions, the blockage is removed during the spawning by an unknown mechanism. When oocytes are isolated by dissection, the meiotic release can be successfully induced by a natural inducer, the REES (i.e., Rough Extract of Echinoid Spawn) that is used in aquaculture to obtain viable larvae in mass. A thioredoxin has recently been identified in the REES as the molecule responsible for holothuroid oocyte maturation. As a redox-active protein, thioredoxin is thought to reduce target proteins within the oocyte membrane and initiate an intracellular reaction cascade that leads to the unblocking of the oocyte meiosis. Our results allow us to understand additional steps in the intracellular reaction cascade induced by the action of thioredoxin on oocytes. Pharmacological agents known to have activating or inhibiting actions on oocyte maturation have been used (Forskolin, Isobutylmethylxanthine, Hypoxanthine, 6-dimethyaminopurine, Lavendustin, Genistein, Roscovitine, Cycloheximide). The effects of these agents were analysed on oocytes of the holothuroid Holothuria tubulosa incubated with or without REES and were compared to those obtained with another reducing agent, the dithiothreitol. Our results demonstrated that, at the opposite of dithiothreitol-induced oocyte maturation, thioredoxin-induced oocyte maturation is cAMP independent, but dependent of the presence of calcium in the seawater. Both pathways of induction require the activation of protein serine/threonine kinases.

Metaphase spreads of human oocytes

This protocol outlines the generation of metaphase spreads of a single oocyte at either the immature metaphase I (MI) or mature metaphase II (MII) stage. This protocol utilizes formaldehyde fixation and therefore allows for further immunofluorescence analysis. These spreads can also be used to identify aneuploidy and other large-scale structural chromosomal abnormalities. However, this assay is prone to false positives for whole chromosome aneuploidy losses, due to either fixation failure or excessive spreading leading to an inability to find all the chromosomes. Additionally, success rates can be low using human oocytes compared to similar protocols for mouse oocytes due to difficulty bursting the oocyte membrane and providing sufficient dispersion of the chromosomes for individual analysis. The protocol duration is two days, however, immunofluorescence staining will add to the total time before final analysis.

Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm−oocyte fusion in mice

Sperm−oocyte membrane fusion is one of the most important events for fertilization. So far, IZUMO1 and Fertilization Influencing Membrane Protein (FIMP) on the sperm membrane and CD9 and JUNO (IZUMO1R/FOLR4) on the oocyte membrane have been identified as fusion-required proteins. However, the molecular mechanisms for sperm−oocyte fusion are still unclear. Here, we show that testis-enriched genes, sperm−oocyte fusion required 1 (Sof1/Llcfc1/1700034O15Rik), transmembrane protein 95 (Tmem95), and sperm acrosome associated 6 (Spaca6), encode sperm proteins required for sperm−oocyte fusion in mice. These knockout (KO) spermatozoa carry IZUMO1 but cannot fuse with the oocyte plasma membrane, leading to male sterility. Transgenic mice which expressed mouseSof1,Tmem95,andSpaca6rescued the sterility ofSof1,Tmem95, andSpaca6KO males, respectively. SOF1 and SPACA6 remain in acrosome-reacted spermatozoa, and SPACA6 translocates to the equatorial segment of these spermatozoa. The coexpression of SOF1, TMEM95, and SPACA6 in IZUMO1-expressing cultured cells did not enhance their ability to adhere to the oocyte membrane or allow them to fuse with oocytes. SOF1, TMEM95, and SPACA6 may function cooperatively with IZUMO1 and/or unknown fusogens in sperm−oocyte fusion.

Spermatozoa lacking Fertilization Influencing Membrane Protein (FIMP) fail to fuse with oocytes in mice

Sperm–oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm–oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm–oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testis-specific gene 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm–oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm–oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm–oocyte fusion process.

The N terminus of α-ENaC mediates ENaC cleavage and activation by furin

Epithelial Na+ channels comprise three homologous subunits (α, β, and γ) that are regulated by alternative splicing and proteolytic cleavage. Here, we determine the basis of the reduced Na+ current (INa) that results from expression of a previously identified, naturally occurring splice variant of the α subunit (α-ENaC), in which residues 34–82 are deleted (αΔ34–82). αΔ34–82-ENaC expression with WT β and γ subunits in Xenopus oocytes produces reduced basal INa, which can largely be recovered by exogenous trypsin. With this αΔ34–82-containing ENaC, both α and γ subunits display decreased cleavage fragments, consistent with reduced processing by furin or furin-like convertases. Data using MTSET modification of a cysteine, introduced into the degenerin locus in β-ENaC, suggest that the reduced INa of αΔ34–82-ENaC arises from an increased population of uncleaved, near-silent ENaC, rather than from a reduced open probability spread uniformly across all channels. After treatment with brefeldin A to disrupt anterograde trafficking of channel subunits, INa in oocytes expressing αΔ34–82-ENaC is reestablished more slowly than that in oocytes expressing WT ENaC. Overnight or acute incubation of oocytes expressing WT ENaC in the pore blocker amiloride increases basal ENaC proteolytic stimulation, consistent with relief of Na+ feedback inhibition. These responses are reduced in oocytes expressing αΔ34–82-ENaC. We conclude that the α-ENaC N terminus mediates interactions that govern the delivery of cleaved and uncleaved ENaC populations to the oocyte membrane.