Bainbridge RE et al. (2023), Xenopus laevis lack the critical sperm factor P...

XB-ART-59607

bioRxiv 2023 Feb 03; doi: 10.1101/2023.02.02.526858.

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Xenopus laevis lack the critical sperm factor PLCζ.

Bainbridge RE , Rosenbaum JC , Sau P , Carlson AE .

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Fertilization of eggs from the African clawed frog Xenopus laevis is characterized by an increase in cytosolic calcium, a phenomenon that is also observed in other vertebrates such as mammals and birds. During fertilization in mammals and birds, the transfer of the soluble PLCζ from sperm into the egg is thought to trigger the release of calcium from the endoplasmic reticulum (ER). Injecting sperm extracts into eggs reproduces this effect, reinforcing the hypothesis that a sperm factor is responsible for calcium release and egg activation. Remarkably, this occurs even when sperm extracts from X. laevis are injected into mouse eggs, suggesting that mammals and X. laevis share a sperm factor. However, X. laevis lacks an annotated PLCZ1 gene, which encodes the PLCζ enzyme. In this study, we attempted to determine whether sperm from X. laevis express an unannotated PLCZ1 ortholog. We identified PLCZ1 orthologs in 11 amphibian species, including 5 that had not been previously characterized, but did not find any in either X. laevis or the closely related Xenopus tropicalis . Additionally, we performed RNA sequencing on testes obtained from adult X. laevis males and did not identify potential PLCZ1 orthologs in our dataset or in previously collected ones. These findings suggest that PLCZ1 may have been lost in the Xenopus lineage and raise the question of how fertilization triggers calcium release and egg activation in these species.

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	Figure 2. Comparison of our RNA-seq data with two other published datasets. a Plot showing transcripts per million (TPM) for matched transcripts between this study (X-axis) and two datasets in a previous study (Session et al., 2016). b Array representing the Spearman rank correlation between each dataset in (a).
	Figure 3. Domain architecture of PLC isoforms PLCδ and PLCζ. Schematic representation of the domain structures: pleckstin homology (PH) domain, EF hands, X and Y catalytic domains, and the C-terminal C2 domain.
	Figure 4. Heatmap of PLC expression in anuran and mouse testis.a Heatmaps of the expression levels (shown as log2 transformed transcripts per million) of annotated PLC genes from the indicated animals. The X. laevis (outbred) data obtained here, X. laevis J strain from GSE73419 (Session et al., 2016), X. tropicalis from GSM5230669 (unpublished), and B. orientalis from GSE163874 (unpublished). b Expression levels of PLC isoforms in the testis of adult (6 month) mice (GSE181426) (Huang et al., 2021).