Fractionation of a Sex-inducing Substance from Flatworms Using Open-column Chromatography and Reverse-phase High-performance Liquid Chromatography

Overview

Journal STAR Protoc

Publisher Cell Press

Specialties Biology
Biomedical Engineering
Science

Date 2024 Nov 4

PMID 39491554

Authors

Kiyono Sekii

Taro Watanabe

Riku Ito

Akitoshi Yoshikawa

Madoka Ichikawa-Seki

Kimitoshi Sakamoto

Kazuya Kobayashi

Affiliations

Soon will be listed here.

Abstract

A substance that sexualizes planarians, an ancestral group of parasitic flatworms, is widely present in planarians and parasitic flatworms. Here, we present a protocol for extracting and purifying the active fraction with sex-inducing activity. We describe steps for homogenization of flatworms, sample concentration, open-column chromatography, and reverse-phase high-performance liquid chromatography. We then detail a feeding bioassay to confirm sex-inducing activity. The obtained active fraction may positively affect parasitic flatworm sexual maturation and can be tested by adding it into the culture media. For complete details on the use and execution of this protocol, please refer to Sekii et al. (2023)..

References

Kobayashi K, Hoshi M . Switching from asexual to sexual reproduction in the planarian Dugesia ryukyuensis: change of the fissiparous capacity along with the sexualizing process. Zoolog Sci. 2002; 19(6):661-6. DOI: 10.2108/zsj.19.661. View

Hase S, Kobayashi K, Koyanagi R, Hoshi M, Matsumoto M . Transcriptional pattern of a novel gene, expressed specifically after the point-of-no-return during sexualization, in planaria. Dev Genes Evol. 2003; 212(12):585-92. DOI: 10.1007/s00427-002-0288-2. View

Kobayashi K, Hoshi M . Sex-inducing effect of a hydrophilic fraction on reproductive switching in the planarian Dugesia ryukyuensis (Seriata, Tricladida). Front Zool. 2011; 8:23. PMC: 3215924. DOI: 10.1186/1742-9994-8-23. View

Sekii K, Miyashita S, Yamaguchi K, Saito I, Saito Y, Manta S . Sex-inducing effects toward planarians widely present among parasitic flatworms. iScience. 2023; 26(1):105776. PMC: 9804148. DOI: 10.1016/j.isci.2022.105776. View

Egger B, Lapraz F, Tomiczek B, Muller S, Dessimoz C, Girstmair J . A transcriptomic-phylogenomic analysis of the evolutionary relationships of flatworms. Curr Biol. 2015; 25(10):1347-53. PMC: 4446793. DOI: 10.1016/j.cub.2015.03.034. View

Wang B, Collins 3rd J, Newmark P . Functional genomic characterization of neoblast-like stem cells in larval Schistosoma mansoni. Elife. 2013; 2:e00768. PMC: 3728622. DOI: 10.7554/eLife.00768. View

van Wolfswinkel J, Wagner D, Reddien P . Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment. Cell Stem Cell. 2014; 15(3):326-339. PMC: 4171737. DOI: 10.1016/j.stem.2014.06.007. View

Hori M, Shozugawa K, Sugimori K, Watanabe Y . A survey of monitoring tap water hardness in Japan and its distribution patterns. Sci Rep. 2021; 11(1):13546. PMC: 8242065. DOI: 10.1038/s41598-021-92949-8. View

Ramm S . Exploring the sexual diversity of flatworms: Ecology, evolution, and the molecular biology of reproduction. Mol Reprod Dev. 2016; 84(2):120-131. DOI: 10.1002/mrd.22669. View

10.

Collins 3rd J, Wang B, Lambrus B, Tharp M, Iyer H, Newmark P . Adult somatic stem cells in the human parasite Schistosoma mansoni. Nature. 2013; 494(7438):476-9. PMC: 3586782. DOI: 10.1038/nature11924. View

11.

Iyer H, Collins 3rd J, Newmark P . NF-YB Regulates Spermatogonial Stem Cell Self-Renewal and Proliferation in the Planarian Schmidtea mediterranea. PLoS Genet. 2016; 12(6):e1006109. PMC: 4909293. DOI: 10.1371/journal.pgen.1006109. View

12.

Wang B, Lee J, Li P, Saberi A, Yang H, Liu C . Stem cell heterogeneity drives the parasitic life cycle of . Elife. 2018; 7. PMC: 6039179. DOI: 10.7554/eLife.35449. View

13.

Grasso M, BENAZZI M . Genetic and physiologic control of fissioning and sexuality in planarians. J Embryol Exp Morphol. 1973; 30(2):317-28. View

14.

Li P, Nanes Sarfati D, Xue Y, Yu X, Tarashansky A, Quake S . Single-cell analysis of Schistosoma mansoni identifies a conserved genetic program controlling germline stem cell fate. Nat Commun. 2021; 12(1):485. PMC: 7817839. DOI: 10.1038/s41467-020-20794-w. View

15.

Nakagawa H, Sekii K, Maezawa T, Kitamura M, Miyashita S, Abukawa M . A comprehensive comparison of sex-inducing activity in asexual worms of the planarian : the crucial sex-inducing substance appears to be present in yolk glands in Tricladida. Zoological Lett. 2018; 4:14. PMC: 5996458. DOI: 10.1186/s40851-018-0096-9. View

16.

Newmark P, Sanchez Alvarado A . Not your father's planarian: a classic model enters the era of functional genomics. Nat Rev Genet. 2002; 3(3):210-9. DOI: 10.1038/nrg759. View