Department of Biology, East Carolina University, Greenville 27858, NC, USA.
Department of Biology, East Carolina University, Greenville 27858, NC, USA.
Gen Comp Endocrinol. 2019 Jun 1;277:130-140. doi: 10.1016/j.ygcen.2019.04.003. Epub 2019 Apr 2.
Expression of adamts9 (A disintegrin and metalloprotease with thrombospondin type-1 motif, member 9) increases dramatically in the somatic cells surrounding oocytes during ovulation in vertebrates from zebrafish to human. However, the function of Adamts9 during ovulation has not been determined due to the embryonic lethality of knockouts in mice and Drosophila. To identify the role of Adamts9 during ovulation we generated knockout (adamts9) zebrafish using CRISPR/Cas9 and characterized the effects of the mutation. From 1047 fish generated by crossing adamts9 pairs, we found significantly fewer adult adamts9 fish (4%) than predicted by Mendelian ratios (25%). Of the mutants found, there was a significant male bias (82%). Only 3 female mutants were identified (7%), and they had small ovaries with few stage III and IV oocytes compared to wildtype (wt) counterparts of comparable size and age. Astoundingly, the remaining mutants (11%) did not appear to have normal testis or ovaries. Instead there was a pair of transparent, ovarian-like membranous shells that filled the abdominal cavity. Histological examination confirmed that shells were largely empty with no internal structure. Surprisingly, seminiferous tubules and various spermatocytes including mature spermatozoa were observed on the periphery of these transparent shells. No female or female like knockouts were observed to release eggs, and no ovulated oocytes were observed in histological sections. To our knowledge, this is the first report of an adamts9 global knockout model in any adult vertebrates and the first description of how gonadal sex and structure are affected- highlighting the importance of Adamts9 during gonadal development and the value of zebrafish as a model organism.
在从斑马鱼到人类的脊椎动物中，ADAMTS9（解整合素和金属蛋白酶与血小板反应蛋白 1 型基元 9）在卵母细胞周围的体细胞中的表达在排卵期间显著增加。然而，由于在小鼠和果蝇中敲除的胚胎致死性，ADAMTS9 在排卵中的功能尚未确定。为了确定 ADAMTS9 在排卵中的作用，我们使用 CRISPR/Cas9 生成了 ADAMTS9 敲除（adamts9）斑马鱼，并对突变的影响进行了特征分析。在通过交配 adamts9 对产生的 1047 条鱼中，我们发现成年 adamts9 鱼（4%）的数量明显少于孟德尔比率（25%）预测的数量。在发现的突变体中，存在显著的雄性偏倚（82%）。仅鉴定出 3 个雌性突变体（7%），与可比大小和年龄的野生型（wt）相比，它们的卵巢较小，且 III 期和 IV 期卵母细胞较少。令人惊讶的是，其余的突变体（11%）似乎没有正常的睾丸或卵巢。相反，有一对透明的、卵巢样的膜状壳充满了腹腔。组织学检查证实，壳基本上是空的，没有内部结构。令人惊讶的是，在这些透明壳的外围观察到了精小管和各种精母细胞，包括成熟的精子。没有观察到雌性或雌性样的敲除体释放卵子，也没有在组织学切片中观察到排卵的卵母细胞。据我们所知，这是首次在任何成年脊椎动物中报告 ADAMTS9 全局敲除模型，也是首次描述性腺性别和结构如何受到影响的报告——强调了 ADAMTS9 在性腺发育过程中的重要性以及斑马鱼作为模型生物的价值。
