Expression of Penaeus monodon ortholog of Niemann-Pick type C-2 in the spermatic tract, and its role in sperm cholesterol removal.

Protein and lipid composition of sperm plasma membrane are modified as these gametes continue to mature during their transit along the spermatic tract. Our previous study revealed that during its journey through the spermatic duct of the black tiger prawn, Penaeus monodon, sperm cholesterol content decreases through the action of lipid-binding proteins within the luminal environment. In this study, the full cDNA sequence of epididymal secretory protein E1 (HE1), or Niemann-Pick C2 (NPC2), was cloned from P. monodon (termed Pmnpc2), and its conserved cholesterol/lipid-binding domain was characterized. The putative tertiary structure of PmNPC2 showed high similarity with the structure of Bos taurus NPC2. Pmnpc2 is expressed in many tissues, including the spermatic tract (i.e., testis, vas deferens, terminal ampoule) and the female thelycum. In situ hybridization revealed the presence of Pmnpc2 transcripts in the vas deferens, terminal ampoule, and thelycum epithelia, suggesting that PmNPC2 could be secreted into the lumen of the spermatic duct. A recombinant hexahistidine-tagged PmNPC2 (rPmNPC2-6His) was able to bind cholesterol and sperm lipid extracts, while co-incubation of sperm from the vas deferens with rPmNPC2-6His resulted in the depletion of cholesterol from these gametes. Together, these results suggest that PmNPC2 participates in sperm cholesterol efflux during the sperm maturation process in P. monodon. Mol. Reprod. Dev. 83: 259-270, 2016. © 2016 Wiley Periodicals, Inc.

Insights into eyestalk ablation mechanism to induce ovarian maturation in the black tiger shrimp.

Eyestalk ablation is commonly practiced in crustacean to induce ovarian maturation in captivity. The molecular mechanism of the ablation has not been well understood, preventing a search for alternative measures to induce ovarian maturation in aquaculture. This is the first study to employ cDNA microarray to examine effects of eyestalk ablation at the transcriptomic level and pathway mapping analysis to identify potentially affected biological pathways in the black tiger shrimp (Penaeus monodon). Microarray analysis comparing between gene expression levels of ovaries from eyestalk-intact and eyestalk-ablated brooders revealed 682 differentially expressed transcripts. Based on Hierarchical clustering of gene expression patterns, Gene Ontology annotation, and relevant functions of these differentially expressed genes, several gene groups were further examined by pathway mapping analysis. Reverse-transcriptase quantitative PCR analysis for some representative transcripts confirmed microarray data. Known reproductive genes involved in vitellogenesis were dramatically increased during the ablation. Besides these transcripts expected to be induced by the ablation, transcripts whose functions involved in electron transfer mechanism, immune responses and calcium signal transduction were significantly altered following the ablation. Pathway mapping analysis revealed that the activation of gonadotropin-releasing hormone signaling, calcium signaling, and progesterone-mediated oocyte maturation pathways were putatively crucial to ovarian maturation induced by the ablation. These findings shed light on several possible molecular mechanisms of the eyestalk ablation effect and allow more focused investigation for an ultimate goal of finding alternative methods to replace the undesirable practice of the eyestalk ablation in the future.