Egg envelope formation of medaka Oryzias latipes requires ZP proteins originating from both the liver and ovary.

Teleost oocytes are surrounded by a structure called chorion or egg envelopes, which is composed of zona pellucida (ZP) proteins. As a result of the gene duplication in teleost, the expression site of the zp genes, coding the major component protein of egg envelopes, changed from the ovary to the maternal liver. In Euteleostei, there are three liver-expressed zp genes, named choriogenin (chg) h, chg hm, and chg l, and the composition of the egg envelope is mostly made up of these Chgs. In addition, ovary-expressed zp genes are also conserved in the medaka genomes, and their proteins have also been found to be minor components of the egg envelopes. However, the specific role of liver-expressed versus ovary-expressed zp genes was unclear. In the present study, we showed that ovary-synthesized ZP proteins first form the base layer of the egg envelope and then Chgs polymerize inwardly to thicken the egg envelope. To analyze the effects of dysfunction of the chg gene, we generated some chg knockout medaka. All knockout females failed to produce normally fertilized eggs by the natural spawning. The egg envelopes lacking Chgs were significantly thinner, but layers formed by ZP proteins synthesized in the ovary were found in the thin egg envelope of knockout as well as wildtype eggs. These results suggest that the ovary-expressed zp gene is well conserved in all teleosts, including those species in which liver-derived ZP proteins are the major component, because it is essential for the initiation of egg envelope formation.

Male parental assistance in embryo hatching of barred-chin blenny Rhabdoblennius nitidus.

Most teleostean embryos develop and hatch without parental assistance, though some receive parental care. We focused on a paternal brood-care species, the barred-chin blenny (Rhabdoblennius nitidus [Günther, 1861]). As hatching approached, fanning behavior by the male parent drastically increased and then embryos hatch. In the absence of the male parent, most embryos failed to hatch. However, the hatching rate was greatly assisted by introducing an artificial water current, suggesting that paternal assistance other than for aeration is required for successful embryo hatching. Next, we analyzed genes for the hatching enzyme and egg-envelope protein, which were successfully cloned from barred-chin blenny, and found the expression patterns differed from those of other euteleosts. Generally, high choriolytic enzyme swells the intact egg envelope, and then low choriolytic enzyme solubilizes the swollen envelope. The expression levels of both the enzymes, but especially the latter, were much lower in barred-chin blenny that is known in most other oviparous species. In addition, the main component of the egg envelope was changed into ChgHm and choriogenin L (ChgL) in barred-chin blenny, whereas ChgH and ChgL for other euteleosts. These in barred-chin blenny would result in ineffective egg-envelope digestion because the posthatching egg envelopes were observed to be swollen but not solubilized. Male parental assistance by fanning until hatching may compensate for this insufficiency. Our study illustrates an example of the evolution of parent-embryo interaction built on a novel relationship: Degradation of the hatching enzyme/egg-envelope digestion system, accompanied by male parental hatching assistance.