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. 2022 Aug 19;12:14162. doi: 10.1038/s41598-022-18422-2

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Isolation of testicular haploid germ cells and ejaculated spermatozoa from the seabream and RNA extraction. (a, b) Representative flow cytometry plots of the seabream cell populations in the whole testis before and after centrifugation generated with the MoFlo XDP cell sorter software (https://www.beckman.es/). In a, the populations of testicular haploid and diploid cells from the testis crude extract before centrifugation are encircled. In b, after centrifugation two subpopulations of haploid germ cells (HGCs) are recovered, one corresponding to a mix of type II spermatocytes (SPC II) and spermatids (SPD), and the other to intratesticular spermatozoa (SPZ_I). (c) Representative immunostaining of Lys⁹ acetylated histone 3 (H3K9ac), meiotic recombination protein Spo11 and α-tubulin (Tuba) in sorted HGC shown in b and ejaculated spermatozoa (SPZ_EJ). For each cell type the brightfield (left panels) and epifluorescence (right panels) images are shown. SPD_R, round spermatids; SPD_E, elongating spermatids. Scale bars, 2 and 5 µm. (d, e) Electrophoretic size distribution of RNAs in representative samples of SPCII + SPD (d) and SPZ_EJ (e) populations analyzed and plotted by the Agilent Bioanalyzer 2100 Expert Software (https://www.agilent.com/). For each sample, fluorescence is plotted against mRNA length (in nucleotides, nt). The RNA integrity number (RIN) is indicated for each sample.