Identification and characterization of lipocalin-type prostaglandin D2 synthase homologs in the urine of male rockfish.

Black rockfish (Sebastes schlegelii) and its relatives are viviparous marine fish. Males produce urinary proteins during the copulation season; however, the identity of these proteins was unknown. In this study, we focused on high-molecular-weight urinary proteins (HMWups) in male black rockfish. The HMWups were identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS) of urine. In silico analyses of RNA-seq data predicted the tissue distribution of candidate HMWup transcripts and their gene structures. Candidate cDNAs were cloned and a recombinant protein of a major candidate was prepared. Western blotting of urine using an antiserum against the recombinant protein was performed to reconfirm the LC-MS/MS results. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry were employed to validate the prediction by RNA-seq and identify the cells producing HMWups, respectively. LC-MS/MS, in conjunction with Western blotting and cDNA cloning, identified the HMWups as lipocalin-type prostaglandin D2 synthase (l-PGDS) homologs. RNA-seq analyses and qRT-PCR revealed that the l-PGDS homolog transcripts were dominantly expressed in the testis and male kidney; Sertoli cells and epithelial cells in the renal tubules were immunoreactive. These results indicated that major protein components in the urine of male black rockfish are l-PGDS homologs, potentially produced by the renal tubules in the kidney. Male rockfish (genus Sebastes) are thought to release unknown pheromone substances during mating behavior. The knowledge and tools obtained in this study empower research into the role(s) of HMWups in pheromone systems underlying rockfish reproduction. No protein-type teleost pheromone has heretofore been discovered.

Changes in the Hepatic Expression of Three Vitellogenin Subtype Genes During Ovarian Development in Female White-Edged Rockfish (Sebastes taczanowskii).

Viviparous fish, including white-edged rockfish (Sebastes taczanowskii), accumulate substantial yolk mass in the oocytes; however, the details of the molecular mechanisms underlying yolk formation are not yet fully understood, especially concerning multiplicity in the yolk precursor vitellogenin (Vtg). The present study aimed to reveal the hepatic transcriptional profiles of multiple vtg gene transcripts (vtgAa, vtgAb, vtgC) during the reproductive cycle in captive female white-edged rockfish reared in an aquarium under natural photo-thermal conditions. The serum estradiol-17ß concentration and the hepatic transcript levels of all vtg subtypes increased with the progress of vitellogenesis; both levels decreased at the beginning of oocyte maturation and remained low during the gestation period. Considering the similarity in the transcriptional profiles of vtg subtypes between Sebastes and Oncorhynchus, along with the differences between Sebastes and Morone, it is suggested that the transcription patterns of multiple vtg genes relate to neither their reproductive modes (viviparity versus oviparity) nor to teleost phylogeny.

Molecular characterization of fshb and lhb subunits and their expression profiles in captive white-edged rockfish, Sebastes taczanowskii.

Fundamental knowledge on the regulation of reproduction by gonadotropins (Gths) is quite limited in viviparous fishes. In the present study, we performed molecular cloning and characterization of cDNAs encoding two Gth subunits (fshb and lhb) from the pituitaries of viviparous white-edged rockfish, Sebastes taczanowskii; expression profiles of both gene transcripts were elucidated in the pituitaries of reproductive males and females which were kept in a captive environment. The cloned fshb and lhb fragments exhibited high sequence identities with corresponding ß-subunit sequences from black rockfish, S. schlegelii. Notably, the fshb of white-edged rockfish appeared to lack a putative N-glycosylation site, whereas lhb conserved it. Expression of fshb and lhb transcripts in the rockfish pituitaries largely changed in synchrony but for minor exceptions. In males, levels of both transcripts increased with progression of spermatogenesis, although the peak for fshb (October) appeared slightly earlier than that for lhb (November). In females, both gene transcripts exhibited synchronous bimodal changes. High expression of fshb and lhb transcripts in the female pituitary during the gestation period, followed by the drastic decrease at parturition, suggest their possible involvement in regulation of gestation of this species. The knowledge gained for Sebastes in this study superimposes fundamental information necessary for further physiological understanding of viviparity in teleost fish.

Knock out of a major vitellogenin receptor gene with eight ligand binding repeats in medaka (Oryzias latipes) using the CRISPR/Cas9 system.

Recent studies of vitellogenesis engendered a novel model of teleost yolk formation in which multiple yolk precursors, vitellogenins (Vtgs), and their receptors (Vtgrs) interact to ensure proper yolk composition for embryonic development and larval growth. As a step toward verification of this concept, we examined the role of one candidate Vtgr, termed low-density lipoprotein receptor relative with eight ligand-binding repeat (Lr8), in the medaka, a representative teleost and established laboratory model. A homozygous lr8 knock out (lr8-KO) medaka was produced to perform reverse-genetic functional analyses. In ovaries of wild type (WT) medaka, Western blotting detected a putative Lr8 protein band at ~130 kDa, while immunohistochemistry detected the putative Lr8 signal at the periphery of the oocyte underneath the zona radiata. These signals disappeared in ovaries of the lr8-KO group. Offspring of lr8-KO medaka exhibited decreased survival rate compared to WT fish, but KO of lr8 was not 100% lethal. There was no significant difference in total yolk protein content or size of eggs between WT and lr8-KO fish. However, LC-MS/MS analyses revealed a remarkable decrease in the relative abundance of yolk proteins derived from VtgAb in lr8-KO eggs, in conjunction with a compensatory increase in proteins derived from VtgAa1. These findings strongly support the conclusion that Lr8 is an important receptor for VtgAb in medaka. The disruption of proper yolk composition by lr8-KO is possibly one cause of the low offspring survival.