Molecular cloning and partial characterization of a low‐density lipoprotein receptor‐related protein 13 (Lrp13) involved in vitellogenin uptake in the cutthroat trout (Oncorhynchus clarki)

Abstract

Multiple ovarian membrane proteins that bind vitellogenin (Vtg) have been detected in teleosts. One of these Vtg receptors was recently identified as low-density lipoprotein receptor-related protein 13 (lrp13/Lrp13) in perciform species, but little is known about this Vtg receptor in salmonid fish. In this study, a cDNA encoding a putative Vtg receptor with 13+1 ligand binding repeats (lr13+1) was cloned from the ovary, and identified as an lrp13 ortholog for cutthroat trout (Oncorhynchus clarki). This lrp13 was predominantly expressed in the pre-vitellogenic stage ovary, and its expression decreased during vitellogenesis. Ovarian localization of Lrp13 was observed by immunohistochemistry using specific antiserum against recombinant Lrp13. Lrp13 immunoreactivity was observed at the oolemma, throughout the zona radiata, and within the perivitelline space between the zona radiata and granulosa cells in ovarian follicles at both the lipid-droplet and vitellogenic stages of growth-an expression pattern that mimics that of a lr8/LR8-type Vtg receptor in this species and of lrp13/Lrp13 in Morone species. Six discrete Vtg-binding proteins were detected in cutthroat trout ovarian membrane proteins when probing with a digoxygenin-labeled salmonid A-type Vtg (VtgAs) followed by chemiluminescent ligand detection. Western blotting using the anti-Lrp13 serum revealed a broad signal consisting of two proteins with masses ranging from ∼190 to ∼210 kDa, which corresponded with some of the VtgA-binding proteins. These findings suggest that, in addition to lr8/LR8, lrp13/Lrp13 acts as a VtgA receptor in trout.

Resolving the complexity of vitellogenins and their receptors in the tetraploid Atlantic salmon (Salmo salar): Ancient origin of the phosvitin‐less VtgC in chondrichthyean fishes

“…The relative expression levels of the vtg and vtgr genes identified in Atlantic salmon were determined by quantitative real‐time PCR, using Elongation factor 1α ( ef1α) as the reference gene (Mushirobira et al, ). The ef1α gene was determined to be the most‐stable gene among six different genes tested for eight distinct tissues in the Atlantic salmon (Olsvik, Lie, Jordal, Nilsen, & Hordvik, ); ef1α transcript abundance did not differ across various ovarian stages when mRNA prepared from the ovaries of rainbow trout was used as template (Luckenbach, Iliev, Goetz, & Swanson, ).…”

“…Whereas a single oocyte receptor seems to mediate uptake of Vtgs in tetrapods, multiple Vtg receptor (VtgRs) were reported for teleosts (Stifano, Barber, Nimpf, & Schneider, ; Stifano, Nimpf, & Schneider, ; Hiramatsu et al, ). Six discrete Vtg‐binding ovarian proteins were detected by ligand labeling in cutthroat trout ( Oncorhynchus clarkii ), while white perch ( Morone americana ), and rainbow trout ( O. mykiss ) were found to possess four VtgRs (Mushirobira et al, ; Reading, Hiramatsu, & Sullivan, ; Reading et al, ; Tyler & Lubberink, ). The Very‐low‐density lipoprotein receptor (Vldlr) is characterized by eight ligand‐binding repeats (LR8), and is encoded within a single vldlr , or vtgr , gene in oviparous vertebrates; two distinct vtgr genes were reported in parallel studies of rainbow trout (Davail et al, ; Prat et al, ).…”

“…Two splice variants of VtgR are widely distributed in teleosts and tetrapods, and the ovarian form lacking the O ‐linked sugar domain is probably responsible for Vtg uptake (Bujo et al, ; Mizuta et al, ; Okabayashi et al, ; Prat et al, ). In addition to the classical LR8 type, the LDLR‐related protein 13 (named Lrp13) was shown to bind the cutthroat trout VtgAs and the VtgAa in perch and tilapia, and was implicated as an important mediator of yolk deposition in other oviparous vertebrates (Hiramatsu et al, ; Mushirobira et al, ; Reading et al, ).…”

Oogenesis and Egg Quality in Finfish: Yolk Formation and Other Factors Influencing Female Fertility

“…Lack of Lr8 gene expression during vitellogenesis suggests that the receptors are recycled to the oocyte membrane after binding vitellogenins and are reused throughout vitellogenic growth. A second form of vitellogenin receptor called Lrp13 also has been reported in fishes and in some species the Lr8 and Lrp13 bind different vitellogenin ligands [13][14][15]20]. Generally, the Lrp13 gene and protein expression mirrors that of the Lr8 receptor in Acanthomorph [14] and salmonid [20] species.…”

“…Vitellogenin receptors cluster into clathrin coated vesicles that are endocytosed and vitellogenins are processed into yolk proteins for storage in yolk granules located throughout the ooplasm (Yolk Deposition). The figure summarizes data from: [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]25,[28][29][30][31]134].…”