Proportional accumulation of yolk proteins derived from multiple vitellogenins is precisely regulated during vitellogenesis in striped bass (Morone saxatilis)

Abstract

We quantified three vitellogenins (VtgAa, VtgAb, VtgC) or their derived yolk proteins (YPs) in the liver, plasma, and ovary during pre-vitellogenic (PreVG), mid-vitellogenic (MVG), and late-vitellogenic (LVG) oocyte growth and during post-vitellogenesis (PostVG) in the striped bass (Morone saxatilis) using label-free quantitative mass spectrometry (MS). Western blotting of the samples using antisera raised against gray mullet (Mugil cephalus) lipovitellins derived from VtgAa, VtgAb, and VtgC confirmed the MS results. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed liver as the primary site of expression for all three Vtgs, with extra-hepatic transcription weakly detected in ovary, foregut, adipose tissue, and brain. Quantitative real-time RT-PCR confirmed vtgAb to be primarily expressed in liver and VtgAb proteins were predominant in liver and plasma from MVG to PostVG. However, the primary period of deposition into oocytes of VtgAb occurred up until MVG, whereas VtgAa was primarily deposited from MVG to LVG. The VtgC was gradually taken up by oocytes throughout vitellogenesis and was detected at trace levels in plasma. The ratio of yolk proteins derived from VtgAa, VtgAb, VtgC (YPAa/YPAb/YPC) in PostVG ovary is 1.4:1.4:1, which differs from ratios previously reported for other fish species in that YPC comprises a greater proportion of the egg yolk. Our results indicate that proportional accumulation of multiple Vtgs in the yolk may depend both on the precise rates of their hepatic secretion and specific uptake by oocytes. Furthermore, composition of the Vtg-derived yolk may vary among Acanthomorph fishes, perhaps reflecting their different early life histories and reproductive strategies.

Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes

“…Samples were measured in triplicate using a 7300 real-time PCR system (Applied Biosystems) and gene expression was reported as copy number calculated from serially diluted plasmid DNA standard curves. Picha and colleagues ( 38,39 ) show that normalization of target RNA to total RNA shows similar results to that for normalization to 18S RNA in hybrid striped bass, and we also show this trend when normalization of target RNA to total RNA is compared with normalization to ribosomal protein L9 in striped bass ( 37 ). Therefore, we used total RNA for normalization of lr8 Ϫ and lrp13 gene expression.…”

“…For the largest group of fi shes (Acanthomorpha), a system of dual ovarian Vtgrs (Lr8 Ϫ and Lrp13) could explain, in part, the disparate accumulation and hydrolysis of egg yolk derived from the functionally distinct VtgAa and VtgAb, which are critical processes governing acquisition of proper egg buoyancy and provision of different types of nutrients to embryos and larvae ( 37,57,58 ). During maturation, yolk proteins derived from VtgAa are disproportionally hydrolyzed to free amino acids, which osmotically drive oocyte hydration and are nutrients selectively utilized by early embryos.…”

A Diverse Family of Host-Defense Peptides (Piscidins) Exhibit Specialized Anti-Bacterial and Anti-Protozoal Activities in Fishes

“…All qPCR products were electrophoresed on agarose gels and sequenced to verify that each primer set specifically amplified a single target piscidin. Data was normalized to the starting total RNA concentration as we have previously described [42]. Pooled total RNA samples from hybrid striped bass not used in these experiments were included on each plate to normalize the data between plates.…”

Vitellogenesis and choriogenesis in fishes

“…This is particularly true for Salmonidae. In addition, in fish species where the complete form of Vtg has been further classified into VtgAa and VtgAb, the ratio of subtypes present in the blood depends on each species [117,118]. Because of the large molecular weight differences, the ratio of complete to incomplete forms of Vtg can be readily determined by electrophoresis and similar methods.…”

“…Our recent research on the physiological functions of each type of Vtg during oocyte maturation and embryogenesis following fertilization suggested that egg quality is directly dependent on the relative abundance (accumulation ratio) of each Vtg within the ovulated eggs [117,118]. Assuming that this hypothesis is correct, when EEDCs disrupt the ratios of different forms of Vtgs found in the blood or accumulated in the egg, this suggests a potential direct impact on fertilization or survival rates.…”