Induced Autoimmunity against Gonadal Proteins Affects Gonadal Development in Juvenile Zebrafish

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 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. 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.

水产养殖鱼类的繁殖抑制乃至完全消除方法存在迫切需求。现有相关手段存在一定应用局限性。迄今为止，尚未开发出可影响幼龄动物性腺发育的免疫干预策略。我们提出假说：多种疾病中自发出现的自身免疫机制，可通过靶向免疫诱导产生。本研究旨在探究，对幼龄斑马鱼性腺内特定靶点进行免疫，是否会引发自身免疫应答，进而干扰性腺发育。

性腺体细胞衍生因子（Gonadal soma-derived factor, Gsdf）、生长分化因子（growth differentiation factor, Gdf9）以及淋巴细胞抗原75（lymphocyte antigen 75, Cd205/Ly75）均对早期性腺发育至关重要，本研究通过5组免疫实验对其进行靶向干预。实验共使用329尾斑马鱼，于受精后6周进行首次免疫注射，15天后施加加强免疫，并于注射后30天完成取样。

研究人员通过原位杂交（in situ hybridization）定位靶向蛋白的转录本，利用实时荧光定量PCR（quantitative real-time PCR）检测免疫相关、凋亡相关及性腺相关基因的表达量，并针对促凋亡蛋白Bcl2相互作用杀伤蛋白（Bcl2-interacting-killer, Bik）开展性腺组织学观察与全标本免疫组化（whole-mount immunohistochemistry）实验。结果显示，干预措施成功诱导自身免疫反应，引发性腺发育迟缓、大量细胞凋亡、细胞闭锁以及转录产物生成紊乱。抗Gsdf免疫干预组的睾丸发育显著滞后；抗Gdf9免疫干预可诱导睾丸细胞凋亡，并导致卵巢发育异常；抗Cd205免疫干预在雌雄个体中均引发强烈免疫应答，导致卵母细胞闭锁以及支持体细胞的大量凋亡。该免疫干预的效应不依赖于促卵泡激素（FSH）。此外，针对生殖细胞蛋白的免疫干预会干扰支持体细胞的发育。

本研究首次证实，靶向自身免疫可干扰幼龄鱼类的性腺发育，为开发基于自身免疫的技术以抑制鱼类性成熟前的繁殖提供了直接可行的思路。但不同干预组与个体间的实验结果存在显著差异，提示需对该技术进行大量优化，方能充分发挥其应用潜力。