In the present study, we aimed to evaluate the effects of hatching enzymes on the egg envelope digestion during the hatching period in the male brooding seahorse. The complementary DNAs encoding two hatching-enzyme genes, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE), were cloned and functionally characterized from the lined seahorse (Hippocampus erectus). The genomic-synteny analysis confirmed that teleosts shared LCE gene synteny. In contrast, the genomic location of HCE was found to be conserved with pipefish, but not other teleosts, suggesting that translocation into a novel genomic location occurred. Whole-mount in situ hybridization showed that HCE and LCE mRNAs were expressed in hatching gland cells. To determine the digestion mechanisms of HCE and LCE in hatching, recombinant HCE and LCE were generated and their enzyme activities were examined using fertilized egg envelopes and synthetic peptides. Seahorse HCE and LCE independently digested and softened the egg envelopes of the lined seahorse. Although the egg envelope was digested more following HCE and LCE co-treatment, envelope solubilization was not observed. Indeed, both HCE and LCE showed similar substrate specificities toward four different synthetic peptides designed from the cleavage sites of egg envelope proteins. HCE and LCE proteins from other euteleostean fishes showed different specificities, and the egg envelope was solubilized by the cooperative action of HCE and LCE. These results suggest that the function of LCE was degenerated in the lined seahorse. Our results imply a digestion mechanism for evolutionary adaptation in ovoviviparous fish with male pregnancy.

中文翻译：

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孵化酶对雄性育雏海马卵壳消化的影响

在本研究中，我们旨在评估孵化酶对雄性育雏海马孵化期间卵壳消化的影响。编码两种孵化酶基因的互补 DNA，即高绒毛膜分解酶 (HCE) 和低绒毛膜分解酶 (LCE)，被克隆并从内衬海马 ( Hippocampus erectus ) 中进行功能表征。）。基因组同线性分析证实硬骨鱼共享 LCE 基因同线性。相比之下，发现 HCE 的基因组位置在尖嘴鱼中是保守的，而不是其他硬骨鱼，这表明发生了易位到新的基因组位置。全贴装原位杂交显示 HCE 和 LCE mRNAs 在孵化腺细胞中表达。为了确定 HCE 和 LCE 在孵化过程中的消化机制，产生了重组 HCE 和 LCE，并使用受精卵包膜和合成肽检查了它们的酶活性。Seahorse HCE 和 LCE 独立消化和软化内衬海马的蛋壳。尽管在 HCE 和 LCE 共同处理后卵壳被更多地消化，但没有观察到壳溶解。的确，HCE 和 LCE 对四种不同的合成肽都表现出相似的底物特异性，这些肽是由卵包膜蛋白的切割位点设计的。其他真真骨鱼类的 HCE 和 LCE 蛋白表现出不同的特异性，HCE 和 LCE 的协同作用使卵壳溶解。这些结果表明LCE的功能在衬里海马中退化了。我们的结果暗示了雄性妊娠卵胎生鱼类进化适应的消化机制。