In vitro production of functional gametes can revolutionize reproduction by reducing generation intervals and accelerating genetic breeding in aquaculture, especially in fish with relatively long generations. Nevertheless, functional sperm production from in vitro-cultured spermatogonia remains a challenge in most aquaculture fish. In this study, we isolated and characterized premeiotic spermatogonia from marine four-eyed sleepers ( Bostrychus sinensis), which are prone to ovotesticular or sterile testicular development, and induced the differentiation of the spermatogonia into flagellated sperm in a three-dimensional (3D) culture system. Artificial insemination indicated that the in vitro-derived sperm were capable of fertilizing mature oocytes to develop into normal larvae. Furthermore, melatonin significantly promoted spermatogonia proliferation and differentiation through the ERK1/2 signaling pathway, and thus increased the efficiency in functional sperm production. The 3D culture system and resulting functional sperm hold great promise for improving the genetic breeding of aquaculture fish.
体外培养生殖干细胞产生功能性配子快速获得种质资源，可缩短育种间隔。在性成熟周期较长的养殖鱼类中，它有望作为一种遗传育种的潜在策略。但在养殖鱼类中体外培养精原细胞产生功能性精子仍然是一种挑战。在该研究中，利用Percoll密度梯度离心从中华乌塘鳢精巢中分离了雄性生殖细胞。经细胞形态和分子标记鉴定表明，得到的细胞主要为减数分裂前精原细胞。随后，通过三维（3D）添加激素（Hor）的培养方法，对分离的高纯度中华乌塘鳢精原细胞进行培养，诱导产生游动精子，其比例为培养细胞的9.4%。人工授精实验结果表明，这些体外产生的精子能与中华乌塘鳢成熟卵子受精并产生正常的后代。该研究还发现，在培养系统中添加褪黑素，可显著提高精子产生效率，达到约16%。进一步研究表明，褪黑激素可能通过ERK1/2信号通路激活细胞周期、精子发生和减数分裂相关信号通路，促进精原细胞分化成功能性精子。该鱼类精原细胞3D培养系统有望作为一种新的干细胞育种策略，加速养殖鱼类遗传育种和种质改良。.
Keywords: 3D culture model; Four-eyed sleeper; Genetic breeding; In vitro spermatogenesis; Melatonin; Spermatogonia.