Zglp-1 zinc finger domains are well conserved in vertebrates. A Sequence alignment of the ZGLP-1 proteins. Amino acids that are identical are denoted by black letters, whereas those with a higher degree of conservation are represented by green background. Additionally, the sequences corresponding to the zinc finger domain are underlined for emphasis. B Comparison of ZGLP-1 proteins. The secondary structural details of ZGLP-1 proteins determined by employing the SMART program. Utilizing PHYRE2 analysis, the three-dimensional configurations of ZGLP-1 proteins from humans, mice, and zebrafish were predicted. C A syntenic map was crafted to depict the specific genomic region where zglp-1 is located within the chromosomes of zebrafish and other vertebrates. Rectangles denote genes, and the orientation of each rectangle signifies the transcriptional direction of the gene. Sequence source: Homo sapiens ZGLP-1: NP_001096637.1; Mus musculus Zglp-1: NP_001096638; Danio rerio Zglp-1: NP_001038914.1

The lack of females observed in zglp-1^−/− mutants is attributed to sex reversal. A The CRISPR/Cas9 target location and zebrafish zglp-1 mutant varieties. a. The intended knockout site for the zglp-1 gene was located within exon 1, denoted by the boxed exons. The translated regions are marked by yellow squares, whereas the untranslated regions are indicated by white squares. The folded line represents introns. b and c. In comparison to the wild type, mutant 1 exhibited a deletion of 31 bases, resulting in premature translation termination at the 233rd amino acid. d and e. Mutant 2 differed from the wild type by the insertion of two bases and the substitution of one base, causing translation to halt prematurely at the 244th amino acid. B The expression of zglp-1 in wild-type and zglp-1 mutant zebrafish by quantitative real-time PCR. actb1 served as the loading control in our study. The data presented here represent the mean values with standard deviations (mean ± SD). Statistical analysis was performed using one-way ANOVA. (**P < 0.01, ***P < 0.001). C Details of the counts (n) and proportions (%) of female and male offspring resulting from a cross between zglp-1 heterozygous mutant zglp-1^{± +2} males and zglp-1^{± +2} females, categorized accordingly. D Primary and secondary sex characteristics of zglp-1 male mutants. The body (a–e), caudal fin (f–j), genital papilla (k–o), anal fin (p–t), and gonad at the cloaca (u–y) were observed (n = 10). E Primary and secondary sex characteristics of zglp-1 female mutants. The body (a–c), caudal fin (d–f), genital papilla (g–i), anal fin (j–l), and gonad at the cloaca (m–o) were observed (n = 10)

zglp-1^−/− mutants show a male expression profile. A Sex determination and gonad development in zebrafish. The development of gonads first passes through an ovary-like stage (Ye et al. 2020). B KEGG analysis of differentially expressed genes between wild-type female zebrafish and zglp-1^{−/− +2} zebrafish at 35 dpf. C GO analysis histogram of differentially expressed genes between wild-type male zebrafish and zglp-1^{−/− +2} zebrafish at 35 dpf

zglp-1^−/− mutants show a male expression profile. A A cluster analysis was conducted to identify differentially expressed genes among the three distinct groups: zglp-1^−/− zebrafish (KO), wild-type male zebrafish (WM), and wild-type female zebrafish (WF). B A comparative analysis was performed to assess the expression levels of amh, dmrt1, cyp19a1a, and foxl2a genes in WM, WF, and zglp-1^−/− zebrafish at 35 dpf. actb1 was used as a control. Data are presented as mean ± SD. The data were subjected to statistical analysis using one-way ANOVA (**P < 0.01, ***P < 0.001)

Zglp-1 inhibits the transcription of amh activated by Sf-1 in its zinc finger domain region. Aamh promoter activity test. The amh promoter demonstrated transcriptional activity in contrast to the control. B The regulatory relationships among Zglp-1, Sf-1, and the amh promoter determined by a dual-luciferase reporter gene assay. The activation of the amh promoter was suppressed when Zglp-1 and Sf-1 were co-transfected into HEK293T cells. C The functional domain of Zglp-1 that blocks the ability of Sf-1 to activate the amh promoter. a. The domain structure of zebrafish Zglp-1, as predicted by the SMART program, revealed two distinct sections within its amino acid sequence. In particular, amino acids 227–374 constituted the Zglp-1-ZnF region, which encompassed the zinc finger domain. Amino acids 1–226 formed the Zglp-1-N segment. b. The regulatory relationships among Zglp-1-N, Sf-1, and the amh promoter. c. The regulatory relationships among Zglp-1-ZnF, Sf-1, and the amh promoter. Zglp-1-ZnF, rather than Zglp-1-N, suppressed the ability of Sf-1 to activate the amh promoter. Data are presented as the mean ± SD. ***P < 0.001; ns, no statistical difference

Interaction of Zglp-1 and Sf-1 occurs between their zinc finger domains. A Co-localization of Zglp-1 and Sf-1 was observed in the nucleus of HEK293T cells. Confocal microscopy images taken 24 h after co-transfection revealed the expression of Zglp-1 (depicted in green) and Sf-1 (shown in red) within the cells. Nuclei were counterstained with DAPI (blue). B An interaction analysis between Zglp-1 and Sf-1 was performed through co-immunoprecipitation and immunoblot assays in HEK293T cells co-transfected with Zglp-1-HA (2 μg) and Sf-1-Myc (2 μg). C Domain structure of zebrafish Zglp-1 and Sf-1 predicted by the SMART program. The amino acid sequence of Zglp-1 was divided into two sections. Amino acids 227–374 were designated Zglp-1-ZnF. Amino acids 1–226 were designated Zglp-1-N. The Sf-1 amino acid sequence was segmented into two distinct regions: amino acids 1–163, designated as Sf-1-ZnF, and amino acids 164–502, constituting the Sf-1-C segment. D Interaction studies were conducted in HEK293T cells co-transfected with combinations of Zglp-1-ZnF-HA (2 μg) and Sf-1-Myc (2 μg), or Zglp-1-N-HA (2 μg) and Sf-1-Myc (2 μg). These interactions were analyzed using co-immunoprecipitation and immunoblot techniques. E Similar interaction analyses were performed in HEK293T cells co-transfected with Sf-1-ZnF-Myc (2 μg) and Zglp-1-ZnF-HA (2 μg), or Sf-1-C-Myc (2 μg) and Zglp-1-ZnF-HA (2 μg). Co-immunoprecipitation and immunoblotting were employed to assess the interactions between these proteins

sf-1 homozygous mutant zebrafish lost their gonads. A Generation of sf-1^−/− zebrafish via CRISPR/Cas9 technology. a. The target gene for knockout was located within exon 3. Exons are denoted by blue boxes, while introns are represented by folded lines. The white squares indicate untranslated regions. b and c. In comparison to the wild type, the mutant exhibits an insertion of 13 bases, resulting in premature termination of translation at the 59th amino acid. B Histological identification of the first and second sexual characteristics in wild-type and sf-1 mutant zebrafish. The body (a–e), pelvic fin (f–j), caudal fin (k–o), anal fin (p–t), genital papilla (u–y), and gonad at the cloaca (I–IV) were observed (n = 10). C HE staining of the gonads in wild-type and sf-1 heterozygous mutant zebrafish. sg, spermatogonia; sc, spermatocyte; sp, sperm cell. I–IV refers to oocytes of various stages

Zglp-1 regulates expression of genes involved in the proliferation and apoptosis of gonadal cells. A, B A comparative analysis was conducted to assess the expression levels of cdc25 and cdca9 genes in wild-type zebrafish versus sf-1^−/− zebrafish at 19 dpf and 35 dpf, respectively. C, D Similarly, the expression of the vasa gene was compared between wild-type and sf-1^−/− zebrafish at both 19 dpf and 35 dpf. E, F Furthermore, the expression of the tp53 gene was evaluated in wild-type and sf-1^−/− zebrafish at 19 dpf and 35 dpf. actb1 was used as a control. Data are presented as mean ± SD. The data were subjected to one-way ANOVA analysis; “ns” indicates the absence of any statistically significant difference, *P < 0.05, **P < 0.01

A model for the regulation of Zglp-1 in sex differentiation in zebrafish. The zglp-1 mutant zebrafish exhibited a male-specific expression pattern, resulting in a phenotypic transition from female to male, indicating a sex reversal phenotype. sf-1 homozygous mutant zebrafish lost their gonads