Correlations (Pearson's <i>r</i>) between the relative expressions of the investigated genes.

<p>The pattern of correlations is similar in controls (n = 32) and in the treated groups (n = 77). Significant <i>r</i>-values (<i>p</i><0.05) are marked with bold font. Significant correlations among male-enriched and female-enriched transcripts are denoted with M or F letters, respectively.</p><p>Correlations (Pearson's <i>r</i>) between the relative expressions of the investigated genes.</p

Retardation in testis development.

<p>A) Representation of normally developing testis in a control male (78.0 mg, 21.5 mm) at 15 days post treatment (dpt). B) Retarded development in an anti-Gsdf-a treated male at 30 dpt (132.0 mg, 25.5 mm): initial phase of differentiation with only undifferentiated gonocytes visible. C) Testis of an anti-Gsdf-b treated fish at 30 dpt (108.5 mg, 24.0 mm): the testis consisted predominantly of spermatogonia with the start of the spermatocyte phase visible. D) Testis of an anti-Gdf9-a treated fish at 30 dpt (89.4 mg, 22.4 mm): spermatocytes reached the zygotene stage of meiotic prophase. SgA – spermatogonia type A, SgB – spermatogonia type B, Scl – spermatocytes, leptotene of meiotic prophase, Scz – spermatocytes, zygotene of meiotic prophase, Scp – spermatocytes at pachytene stage, Sd – spermatids, Gc – undifferentiated gonocytes. All scalebars represent 50 µm.</p

Induced Autoimmunity against Gonadal Proteins Affects Gonadal Development in Juvenile Zebrafish (Dataset)

<p>A method to mitigate or possible eliminate reproduction in farmed fish is highly demanded. The existing approaches have certain applicative limitations. In mammals, immunocontraception has been applied to control wildlife populations with some success. However, such a strategy is not relevant in fish, where reducing gonadal growth, rather than preventing fertilization is required. So far, no immunization strategies affecting gonadal development in juvenile animals have been developed. We hypothesize that autoimmune mechanisms, occurring spontaneously in a number of diseases, could be induced by targeted immunization. We have asked whether the immunization against specific targets in a juvenile zebrafish gonad will produce an autoimmune response, and consequently, disturbance in gonadal development. Gonadal soma-derived factor (Gsdf), growth differentiation factor (Gdf9), and lymphocyte antigen 75 (Cd205/Ly75), all essential for early gonad development, were targeted with 5 self-vaccine tests. Zebrafish (n=329) were injected at 6 weeks post fertilization, a booster injection was applied 15 days later, and fish were sampled at 30 days. We localized targeted protein transcripts by in situ hybridization, quantified expression of immune-, apoptosis-, and gonad-related genes with quantitative real-time PCR, and performed gonadal histology and whole-mount immunohistochemistry for Bcl2-interacting-killer (Bik) pro-apoptotic protein. The treatments resulted in an autoimmune reaction, gonad developmental retardation, intensive apoptosis, cell atresia, and disturbed transcript production. Testes were remarkably underdeveloped after anti-Gsdf treatments. Anti-Gdf9 treatments promoted apoptosis in testes and abnormal development of ovaries. Anti-Cd205 treatment stimulated a strong immune response in both sexes, resulting in oocyte atresia and strong apoptosis in supporting somatic cells. The effect of immunization was FSH-independent. Furthernmore, immunization against germ cell proteins disturbed somatic supporting cell development. This is the first report to demonstrate that targeted autoimmunity can disturb gonadal development in a juvenile fish. It shows a straightforward potential to develop auto-immunization-based technologies to mitigate fish reproduction before they reach maturation. However, the highly variable results between treatments and individuals suggest significant optimization should be performed to achieve the full potential of this technology.</p

Expression of marker genes at 15 and 30 days post treatment (dpt).

<p>The relative gene expression is shown as fold change compared to control fish. The values in bold represent a significant difference in expression (ANOVA and LSD test at p<0.05). Different antigens for the same target protein are indicated using –a or –b following the target protein name. All data were normalized using the reference genes <i>rpl13α</i> and <i>ef1α</i>.</p><p>Expression of marker genes at 15 and 30 days post treatment (dpt).</p

Sections of testis from 7 month-old zebrafish.

<p>A) Respresentative haematoxylin-eosin (HE) staining of the mature zebrafish testis; spermatogonia (Sg), spermatocytes (Sc), spermatids (Sd), and spermatozoa (Sp) could all be positively identified. B) <i>in situ</i> hybridization (ISH) of <i>gsdf</i> transcripts in the testis; staining shows strong signal throughout the testis, although it is unclear from which cell type it originates; no staining is visible in spermatocytes or spermatids. Spermatogonia and spermatozoa could not be identified. C) ISH of <i>gdf9</i> transcripts in testis; faint signal was found throughout the testis. It is unclear which cell types the signal is localized to. D) ISH of <i>cd205</i> transcripts in the testis; the signal is strongest in spermatogonia, while it is not visible in spermatocytes or spermatids. Spermatozoa could not be identified. All scalebars represent 100 µm.</p

Immunohistochemistry of Bcl2-interacting-killer (Bik) protein in testes of control and treated zebrafish of the <i>tg</i>(<i>vas::egfp</i>) line.

<p>Merged images combine the eGFP filter (green; germ cell lineage), and dsRed filter (red; Bik protein) under epifluorescent light. A) Control fish 30 days post treatment (dpt) (D; 59.3 mg, 21.3 mm): only a few small points of Bik signal were detected. The cell lineage from which the signal originates could not be determined. The testes were developing normally with clusters of spermatogonia (sg) and spermatocytes (sc) visible. B) Anti-Gsdf-a treated fish 15 dpt (27.3 mg, 16.0 mm): The testis was extremely early in development with only a few spermatogonia expressing weak eGFP signal. No Bik signal was detected. C) Anti-Gsdf-b treated fish 30 dpt (86.4 mg, 23.4 mm): Only a very weak Bik signal is detected in the testes. Only presumptive spermatogonia (sg) and undifferentiated gonocytes (gc) were identified. D and E) Anti-Gdf9-a treated fish at 15 and 30 dpt, respectively (D: 46.2 mg, 20.3 mm, E: 138.7 mg, 26.5 mm): Bik signal was localized to clusters of spermatogonia. In contrast, the clusters of spermatocytes (sc) and spermatids (st) did not show Bik signal. F) Anti-CD205 treated fish at 30 dpt (128.4 mg, 25.1 mm): a strong Bik signal was detected throughout the testis. However, resolution was insufficient to determine from which cell types it originated. All scalebars represent 100 µm. White boxes identify area being shown with dsRed filter only.</p

Overview of number (n) and lines of fish used in the experiment.

<p>Fish were sampled at 15 and 30 days post-treatment (dpt). Zebrafish lines used were: TAB (T), nacre -/- (N), and tg(vasa::vasa-EGFP) (V).</p><p>Overview of number (n) and lines of fish used in the experiment.</p

Histological comparison of ovary development in control and experimental fish.

<p>A) A representative ovary from a control fish (36.8 mg, 17.2 mm) at 15 days post treatment (dpt); primary oocytes at stage 2 (Poc2) are located predominantly in the center, whereas primary oocytes at stage 1 (Poc1) and oogonia (Oo) are located in the periphery of the ovary. B) A representative ovary from a control fish (216.0 mg, 29.9 mm) at 30 dpt; stage 4 vitellogenic primary oocytes (Poc4) have a thick <i>zona radiata</i> (arrowhead) surrounded by an even layer of follicular cells. Stage 3 previtellogenic primary oocytes lack the thick <i>zona radiata</i>. C) Ovary from an anti-Gsdf-a treated fish at 30 dpt (326.3 mg, 31.9 mm): stage 4 and 5 (Poc5) primary oocytes weredeveloped. An atretic oocyte is identified by the thick arrow. Granulomatous inflammatory reaction as a consequence of invasion of peritonial cells is identified by the black star. D) Ovary from an anti-Cd205 treated fish at 30 dpt (241.0 mg, 30.5 mm): <i>zona radiata</i> is apparently thinner than in controls, and some invaginations are evident (arrowhead), indicating the onset of the atresia process. An irregular follicular layer also indicates atresia (thick arrow). Scalebars represent 100 µm.</p

Induced Autoimmunity against Gonadal Proteins Affects Gonadal Development in Juvenile Zebrafish

<div><p>A method to mitigate or possibly eliminate reproduction in farmed fish is highly demanded. The existing approaches have certain applicative limitations. So far, no immunization strategies affecting gonadal development in juvenile animals have been developed. We hypothesized that autoimmune mechanisms, occurring spontaneously in a number of diseases, could be induced by targeted immunization. We have asked whether the immunization against specific targets in a juvenile zebrafish gonad will produce an autoimmune response, and, consequently, disturbance in gonadal development. Gonadal soma-derived factor (Gsdf), growth differentiation factor (Gdf9), and lymphocyte antigen 75 (Cd205/Ly75), all essential for early gonad development, were targeted with 5 immunization tests. Zebrafish (n = 329) were injected at 6 weeks post fertilization, a booster injection was applied 15 days later, and fish were sampled at 30 days. We localized transcripts encoding targeted proteins by <i>in situ</i> hybridization, quantified expression of immune-, apoptosis-, and gonad-related genes with quantitative real-time PCR, and performed gonadal histology and whole-mount immunohistochemistry for Bcl2-interacting-killer (Bik) pro-apoptotic protein. The treatments resulted in an autoimmune reaction, gonad developmental retardation, intensive apoptosis, cell atresia, and disturbed transcript production. Testes were remarkably underdeveloped after anti-Gsdf treatments. Anti-Gdf9 treatments promoted apoptosis in testes and abnormal development of ovaries. Anti-Cd205 treatment stimulated a strong immune response in both sexes, resulting in oocyte atresia and strong apoptosis in supporting somatic cells. The effect of immunization was FSH-independent. Furthermore, immunization against germ cell proteins disturbed somatic supporting cell development. This is the first report to demonstrate that targeted autoimmunity can disturb gonadal development in a juvenile fish. It shows a straightforward potential to develop auto-immunization-based technologies to mitigate fish reproduction before they reach maturation. However, the highly variable results between treatments and individuals suggest significant optimization should be performed to achieve the full potential of this technology.</p></div

Total length (A) and weight (B) of fish over the course of the experiment.

<p>Weight at the start of treatment (day 0) was not measured. Stars represent a statistically significant difference from the control group (ANOVA, LSD test at <i>p</i><0.05) at the given time point. Error bars represent standard deviation.</p