Phylogenetic tree of stra8 and potential involvement of retinoic acid (RA) in male Atlantic salmon at puberty. (A) Phylogenetic tree produced from a multiple alignment of STRA8. Blocks of local alignment are shown as gray boxes along the multiple alignment. Support values (0–1) generated from 1000 bootstrap resamples are shown next to the branches in red. UniProtKB accession numbers are displayed in the tree next to the species name. (B)stra8 expression levels in the different adult tissues used for making the reference annotation of the Atlantic salmon genome (GenBank GBRB00000000.1) (Lien et al., 2016). Results are expressed as RNAseq normalized read counts (N = 1). Dashed line indicates 10 RNAseq reads. (C) Testicular stra8 expression in immature salmon males before and after smoltification. Results are shown as mean RNAseq normalized read counts ± SEM (N = 3–8) retrieved from GenBank PRJNA380580 data set (Kjærner-Semb et al., 2018) (16 m males) and own unpublished data (24 m males). (D) Testicular stra8 expression in wild-type (WT) and germ cell-free (GCF) dead end (dnd) knockout salmon males, GenBank PRJNA550414 (Kleppe et al., 2020). Data are shown as mean RNAseq normalized read counts ± SEM (N = 3–4; ***p < 0.001). Numbers in brackets indicate average read counts for each group. Dashed line indicates 10 RNAseq reads. (E) Representative testis morphology of postsmolt males before (gonado-somatic index [GSI] < 0.1%) and after starting pubertal development (GSI > 0.1%). Insets show testis tissue magnified from the marked area (black dashed line), and representative type A (SPG A) and type B spermatogonia (SPG B) are labeled. Scale bar, 25 μm. (F)In vivo testicular expression of genes involved in spermatogonial differentiation and RA testicular function in postsmolt males before and after starting pubertal development. Numbers in brackets indicate Cq values obtained by qPCR analysis. Data are shown as mean fold change ± SEM (N = 8–9; **p < 0.01), and expressed relative to the control condition, which is set at 1. (G) Modulation of sall4, dusp4, stra8 and rec8 mRNA levels upon RA synthesis inhibition. Testicular fragments from immature males were cultured for 4 days at 14°C in the absence or presence of DEAB (10 μM). Data are shown as mean fold change ± SEM (N = 8–9; *p < 0.05), and expressed relative to the control condition, which is set at 1.

Efficient disruption of salmon stra8 by CRISPR/Cas9 and stra8 germ cell-specific expression. (A) Design of guide RNA target sites on exon 3 and exon 6 targeting the stra8 gene. (B) CRISPR/Cas9-mediated decrease of WT stra8 transcript levels in both maturing and mature Atlantic salmon crispant males. Data are shown as mean relative expression ± SEM (N = 7–10; **p < 0.01; ***p < 0.001). WT, wild-type.

Evaluation of gonadal tissue and plasma androgen levels of WT and stra8 crispant salmon males. (A,C) Gonado-somatic indices (GSI) and plasma androgen (11-ketotestosterone; 11-KT) levels in WT and stra8 crispant males at samplings from May (maturing) and September 2017 (mature). (B,D) Histological images of maturing (B) and mature (D) Atlantic salmon testes obtained from WT and stra8 crispant fish. Insets show testis tissue magnified from the marked area (black dashed line). White arrowheads indicate apoptotic germ cells. Representative germ cell stages are shown in the upper right panels: type A spermatogonia (SPG A), type B spermatogonia (SPG B), spermatocytes (SPC), round spermatids (rSPT), elongated spermatids (eSPT) and spermatozoa (SPZ). WT, wild-type; Crispant, stra8 crispant. Scale bar, 50 μm.

Germ cell apoptosis and proliferation in WT and stra8 crispant salmon testis tissue. (A,B) Detection (A) and quantification (B) of germ cell apoptosis/DNA damage by TUNEL analysis. TUNEL + cells/cysts are shown in green and DAPI counterstain in blue. Representative germ cell stages are labeled: type A spermatogonia (SPG A), type B spermatogonia (SPG B), spermatocytes (SPC), spermatids (SPT) and spermatozoa (SPZ). Scale bar, 30 μm. Quantification results are shown as mean ± SEM (N = 3–5; *p < 0.05). (C,D) Detection (C) and quantification (D) of cell proliferation by pH3 analysis. White and black arrowheads indicate pH3 + type A undifferentiated and Sertoli cells, respectively (examples of proliferating cells are shown at higher magnification in Supplementary Figure 4C). Scale bar, 50 μm. WT, wild-type; Crispant, stra8 crispant.

Testicular gene expression in maturing WT and stra8 crispant fish. Transcript levels of selected markers for germ cells at different stages of development are shown as mean ± SEM (N = 7–10; *p < 0.05, **p < 0.01), and expressed relative to ef1a expression. WT, wild-type; Crispant, stra8 crispant; SPG, spermatogonia; SPC, spermatocytes; SPT, spermatids.