In externally fertilizing animals, such as sea urchins and frogs, prolonged depolarization of the egg immediately after fertilization inhibits the entry of additional sperm-a phenomenon known as the fast block to polyspermy. In the African clawed frog , this depolarization is driven by Ca-activated Cl efflux. Although the prominent Ca-activated Cl currents generated in immature oocytes are mediated by transmembrane protein 16a (xTMEM16A) channels, little is known about the channels that contribute to the fast block in mature eggs. Moreover, the gamete undergoes a gross transformation as it develops from an immature oocyte into a fertilization-competent egg. Here, we report the results of our approach to identify the Ca-activated Cl channel that triggers the fast block. By querying published proteomic and RNA-sequencing data, we identify two Ca-activated Cl channels expressed in fertilization-competent eggs: xTMEM16A and bestrophin 2A (xBEST2A). By exogenously expressing xTMEM16A and xBEST2A in axolotl cells lacking endogenous Ca-activated currents, we characterize the effect of inhibitors on currents mediated by these channels. None of the inhibitors tested block xBEST2A currents specifically. However, 2-(4-chloro-2-methylphenoxy)--[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9) and -((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA) each reduce xTMEM16A currents by more than 70% while only nominally inhibiting those generated by xBEST2A. Using whole-cell recordings during fertilization, we find that Ani9 and MONNA effectively diminish fertilization-evoked depolarizations. Additionally, these inhibitors lead to increased polyspermy in embryos. These results indicate that fertilization activates TMEM16A channels in eggs and induces the earliest known event triggered by fertilization: the fast block to polyspermy.

The prevention of polyspermy is essential for the successful progression of normal embryonic development in most sexually reproducing species. In external fertilizers, the process of fertilization induces a depolarization of the egg's membrane within seconds, which inhibits supernumerary sperm from entering an already-fertilized egg. This fast block requires an increase of intracellular Ca in the African clawed frog, , which in turn activates an efflux of Cl that depolarizes the cell. Here we seek to identify the source of this intracellular Ca Using electrophysiology, pharmacology, bioinformatics, and developmental biology, we explore the requirement for both Ca entry into the egg from the extracellular milieu and Ca release from an internal store, to mediate fertilization-induced depolarization. We report that although eggs express Ca-permeant ion channels, blockade of these channels does not alter the fast block. In contrast, insemination of eggs in the presence of Xestospongin C-a potent inhibitor of inositol 1,4,5-trisphosphate (IP)-induced Ca release from the endoplasmic reticulum (ER)-completely inhibits fertilization-evoked depolarization and increases the incidence of polyspermy. Inhibition of the IP-generating enzyme phospholipase C (PLC) with U73122 similarly prevents fertilization-induced depolarization and increases polyspermy. Together, these results demonstrate that fast polyspermy block after fertilization in eggs is mediated by activation of PLC, which increases IP and evokes Ca release from the ER. This ER-derived Ca then activates a Cl channel to induce the fast polyspermy block. The PLC-induced cascade of events represents one of the earliest known signaling pathways initiated by fertilization.

RNA sequencing (RNA-seq) is extensively used to quantify gene expression transcriptome-wide. Although often paired with polyadenylate (poly(A)) selection to enrich for messenger RNA (mRNA), many applications require alternate approaches to counteract the high proportion of ribosomal RNA (rRNA) in total RNA. Recently, digestion using RNaseH and antisense DNA oligomers tiling target rRNAs has emerged as an alternative to commercial rRNA depletion kits. Here, we present a streamlined, more economical RNaseH-mediated rRNA depletion with substantially lower up-front costs, using shorter antisense oligos only sparsely tiled along the target RNA in a 5-min digestion reaction. We introduce a novel Web tool, Oligo-ASST, that simplifies oligo design to target regions with optimal thermodynamic properties, and additionally can generate compact, common oligo pools that simultaneously target divergent RNAs, e.g. across different species. We demonstrate the efficacy of these strategies by generating rRNA-depletion oligos for Xenopus laevis and for zebrafish, which expresses two distinct versions of rRNAs during embryogenesis. The resulting RNA-seq libraries reduce rRNA to <5% of aligned reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available at https://mtleelab.pitt.edu/oligo to design antisense oligos for any taxon or to target any abundant RNA for depletion.

• Protein for the channels xBEST2A and xTMEM16A is present in X. laevis eggs. 40• The inhibitors MONNA and Ani9 effectively block xTMEM16A compared to xBEST2A. 41• Xenopus laevis fertilization opens TMEM16A to trigger egg depolarization.

After fertilization, maternally contributed factors to the egg initiate the transition to pluripotency to give rise to embryonic stem cells, in large part by activating de novo transcription from the embryonic genome. Diverse mechanisms coordinate this transition across animals, suggesting that pervasive regulatory remodeling has shaped the earliest stages of development. Here, we show that maternal homologs of mammalian pluripotency reprogramming factors OCT4 and SOX2 divergently activate the two subgenomes of Xenopus laevis, an allotetraploid that arose from hybridization of two diploid species ~18 million years ago. Although most genes have been retained as two homeologous copies, we find that a majority of them undergo asymmetric activation in the early embryo. Chromatin accessibility profiling and CUT&RUN for modified histones and transcription factor binding reveal extensive differences in enhancer architecture between the subgenomes, which likely arose through genomic disruptions as a consequence of allotetraploidy. However, comparison with diploid X. tropicalis and zebrafish shows broad conservation of embryonic gene expression levels when divergent homeolog contributions are combined, implying strong selection to maintain dosage in the core vertebrate pluripotency transcriptional program, amid genomic instability following hybridization.