Genomic and proteomic analyses reveal non-neofunctionalized vitellogenins in a basal clupeocephalan, the Atlantic herring, and point to the origin of maturational yolk proteolysis in marine teleosts.

Oocyte hydration is a unique event in oviparous marine teleosts that provides the single-celled egg with an essential pool of water for survival during early development in the saline oceanic environment. A conserved mechanism of maturational yolk proteolysis of a neofunctionalized vitellogenin (VtgAa) has been shown to underlie the hydration event in all teleosts that spawn pelagic eggs (pelagophils), and is argued to be a key adaptation for teleost radiation in the oceanic environment 55 Ma. We have recently shown that a small pool of free amino acids (FAAs) significantly contributes to the osmolarity of the ovulated egg in an ancestral marine teleost, the Atlantic herring that spawns benthic eggs (benthophil). To determine whether multiple forms of vtg exist and whether neofunctionalization of the gene products are related to the egg FAA pool in this species, genomic sequences conserved between the exons of Atlantic herring and zebrafish were amplified. This approach identified a small polymorphic intron between exons 9 and 10 in Atlantic herring and demonstrated that two closely related major vtg transcripts (chvtgAc1 and chvtgAc2) are expressed during oogenesis. A separate polymerase chain reaction-based approach identified a more ancestral phosvitinless transcript (chvtgC). Proteomic analyses of the translated products of the major vtg forms demonstrated that the yolk proteins are similarly processed during deposition, and oocyte maturation and reveal that vtgs have duplicated but not neofunctionalized in this species. Phylogenetic analyses consistently clustered the transcripts and proteins as the basal sister group to the Ostariophysi in full congruence with the Clupeocephalan rank, and suggest that expansion of ostariophysan vtgAo1 and vtgAo2 genes occurred in a lineage-specific manner after separation from the Clupeiformes. Three-dimensional modeling of the ChvtgAc1 sequence against the resolved lamprey lipovitellin module revealed that the tertiary structure is highly conserved, with most substitutions occurring on the outside of the molecule. The data indicate that the phosvitin domain, the smallest yet reported for teleosts, and an N-terminal fragment of the lipovitellin light chain contribute to the FAA pool. The present findings thus show that yolk proteolysis and the generation of an organic osmolyte pool of FAAs was an adaptive response to spawning in seawater also for the Clupeiformes, but that this process was not evolutionarily successful in terms of biodiversity until vtg gene neofunctionalization occurred in the Acanthomorpha.

Vertebrate vitellogenin gene duplication in relation to the "3R hypothesis": correlation to the pelagic egg and the oceanic radiation of teleosts.

The spiny ray-finned teleost fishes (Acanthomorpha) are the most successful group of vertebrates in terms of species diversity. Their meteoric radiation and speciation in the oceans during the late Cretaceous and Eocene epoch is unprecedented in vertebrate history, occurring in one third of the time for similar diversity to appear in the birds and mammals. The success of marine teleosts is even more remarkable considering their long freshwater ancestry, since it implies solving major physiological challenges when freely broadcasting their eggs in the hyper-osmotic conditions of seawater. Most extant marine teleosts spawn highly hydrated pelagic eggs, due to differential proteolysis of vitellogenin (Vtg)-derived yolk proteins. The maturational degradation of Vtg involves depolymerization of mainly the lipovitellin heavy chain (LvH) of one form of Vtg to generate a large pool of free amino acids (FAA 150-200 mM). This organic osmolyte pool drives hydration of the ooctye while still protected within the maternal ovary. In the present contribution, we have used Bayesian analysis to examine the evolution of vertebrate Vtg genes in relation to the "3R hypothesis" of whole genome duplication (WGD) and the functional end points of LvH degradation during oocyte maturation. We find that teleost Vtgs have experienced a post-R3 lineage-specific gene duplication to form paralogous clusters that correlate to the pelagic and benthic character of the eggs. Neo-functionalization allowed one paralogue to be proteolyzed to FAA driving hydration of the maturing oocytes, which pre-adapts them to the marine environment and causes them to float. The timing of these events matches the appearance of the Acanthomorpha in the fossil record. We discuss the significance of these adaptations in relation to ancestral physiological features, and propose that the neo-functionalization of duplicated Vtg genes was a key event in the evolution and success of the teleosts in the oceanic environment.