PMC

A schematic diagram of the hypothesized model for the leading role of germ cells on gonadal differentiation in the gynogenetic fish. The process of gonadal development is divided into three stages: before gonadal differentiation, during gonadal differentiation and mature. Before gonadal differentiation, there are PGCs and somatic precursor cells in WT gonads, whereas no PGCs in germ cell-deficient gonads. During gonadal differentiation, there are PGCs and somatic precursor cells, and early oocytes with granulosa cells and thecal cells on their surface, and estrogen-producing cells in WT gonads; however, in the germ cell-deficient gonads, there are somatic precursor cells, Sertoli cells, Leydig cells and androgen-producing cells. At mature stage, there are many mature occytes and several early occytes, granulosa cells and thecal cells surround around the occytes, and estrogen-producing cells are distributed among oocytes in WT gonads; whereas, there are many Sertoli cells arrayed into circles, Leydig cells and androgen-producing cells in the germ cell-deficient gonads

Testis differentiation-related genes are up-regulated in dnd-MO gonads during gonadal differentiation. a Transcriptomic analysis of gonadal development-related genes expressing in WT and dnd-MO gonads from 25 dph to 60 dph (left) and in mature testis relative to mature ovary (right). b-i RT-PCR detection of the indicated genes above. b Wt1a, c dmrt1, d sox9, e amh, f hsd3b, g hsd11b2, h cyp17a, i cyp19a1a, j foxl2, k gdf9, l h2af1o and m zp3. n Schematic diagram of gene expression pathways during gonadal differentiation from 25 dph to 60 dph in WT gynogenetic fish and the germ cell-depleted gynogenetic fish. The gonadal differentiation-related genes (red) are shown in the corresponding positions

Expression changes of sex-biased genes during gonad differentiation. a Pie charts representing the main GO terms (biological process) associated with the DEGs in germ cell-depleted gonads. Only GO terms with >800 DEG sequences are shown (Except for DEGs in germ cell-depleted gonads at 25 dph). b Venn diagram shows the commonly and differentially DEGs existed in the germ cell-depleted gonads at 35 dph, 45 dpg and 60 dph. c Sex-biased gene expression patterns in germ cell-depleted gonads in comparison to WT gonads from 35 dph to 60 dph. d Venn diagram shows the overlap association between common DEGs in germ cell-depleted gonads from 35 dph to 60 dph and sex-biased genes in normal testis and ovary. e Expression pattern of sex-biased genes in common DEGs in germ cell-depleted gonads from 35 dph to 60 dph

Gonadal and histological structures of WT and dnd-MO gynogenetic fish during gonadal differentiation. Gonadal and histological structures of WT and dnd-MO gonads at 25 dph (a-f), 35 dph (g-l), 45 dph (m-r) and 60 dph (s-x). The external morphology of WT gonads (a, g, m and s), and dnd-MO gonads (b, h, n and t). Haematoxylin–eosin and immunofluorescence staining of WT gonads (c, d, i, j, o, p, u and v) and dnd-MO gonads (e, f, k, l, q, r, w and x). White arrow indicates PGC, arrow head indicates differentiating oogonia, star indicates primary oocyte and black arrow indicates cavity. [Scale bars, 500 mm (a, b, g and h), 1 cm (m, n, s and t), 20 μm (c and d) and others 50 μm

Identification and distribution of vasa-positive PGCs by whole mount in situ hybridization in WT (a-e) and dnd-MO injected (2000 pg/per embryo) (f-j) embryos from 50 % epiboly to 36 hpf. a and f 50 % epiboly stage, b and g bud stage, c and h 3-somite stage, d and i 24 hpf, and e and j 36 hpf. (Scale bar, 250 μm). k Average number of vasa-positive PGCs/per embryo in embryos injected with con-MO (circle) or dnd-MO (triangle) from sphere to 24 hpf. N ≥ 30, and the statistical data are presented as mean ± standard deviation (SD), P < 0.001

Gonadal structures and gene expression patterns in adult germ cell-depleted gynogenetic gonads. a-d The external morphology of 1 year to 3 years old germ cell-depleted gonads. a 1-year-old germ cell-depleted gonads. b 2-year-old germ cell-depleted gonads. c and d 3-year-old germ cell-depleted gonads. e-h Cross section of germ cell-depleted gonads in above box 1 (e), 2 (f), 3 (g) and 4 (h). Green arrow indicated the cavity. L, Leydig cells; S, Sertoli cells. [Scale bars, 2 cm (a-d) and 50 μm (e-h). i RT-PCR analysis of somatic gene expression in control testis (Con-testis) from sexual reproduction, control ovary (Con-ovary) and the 3-year-old germ cell-depleted gonad (Morphant gonad). The gene expression level is related to β-actin and Rpl13a

Phenotypic masculinization and their gonadal morphology in the germ cell-depleted adults. a-e, f-j and k-o Representative images of control gynogenetic females, control males from sexual reproduction, and the germ cell-depleted gynogenetic males, respectively. a, f and k Body shape. b, g and l Gill cover. c, h and m Anus. d, i and n Images of mature ovary, testis, and 1-year-old germ cell-depleted gonad, respectively. e, j, o HE staining of control ovary, control testis, and 1-year-old germ cell-depleted gonad, respectively. Green circle indicates the cavity, inset shows the whole cross section of 1-year-old germ cell-depleted gonad. Pm, Peritoneal membrane; Sg, spermatogonia; Sc, spermatocytes; Sz, spermatozoa; L, Leydig cells; S, Sertoli cells; My, peritubular myoid cells. [Scale bars, D, I and N, 2 cm; E 100, μm, J and O 50 μm]