Functional studies of the egg cortical alveolus proteases on fertilization of medaka

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Functional studies of the egg cortical alveolus
proteases on fertilization of medaka
(メ ダ カ の 受 精 に お け る 卵 表 層 胞 局 在 プ ロ テ ア ー ゼ の 機
能解明)
FU Bo (傳 博 )

論 文 内 容 の 要 旨
In most organisms, fertilization is a starting point of embryonic development, in which
dormant eggs are activated by sperm to trigger rapid changes for early development and
ontogenesis. In fish eggs, the egg plasma membrane is surrounded by chorion, a th ick
extracellular proteinaceous layer. Under the egg plasma membrane, there exist secretory
vesicles called cortical alveoli (CA) in the cortical cytoplasm. Immediately after
fertilization, the CA fuse with the plasma membrane to undergo exocytosis and dis charge
of their contents into the perivitelline space (PVS), a space between the plasma membrane
and the chorion. Subsequent enlargement of the PVS and the concomitant elevation of the
chorion are accompanied by a chorion hardening in which the soft chorio n changes to the
hard chorion. A series of these events are called the cortical reaction. One of the prominent
events is a proteolytic processing of the CA component, hyosophorin. Hyosophorin is a
major sialic acid-rich glycoprotein, and is present as a hi gh-molecular-weight form (Hhyosophorin), consisting of tandem -repeat structure of the glycononapeptide. During the
cortical reaction, H-hyosophorin undergoes proteolytic depolymerization to the least
repeating unit, L-hyosophorin. An enzyme responsible fo r the depolymerization of H- to
L-hyosophorin was named as hyosophorinase, which has remained unidentified yet.
Another drastic event during the cortical reaction is chorion hardening. It has been shown
that a CA-localized protease, alveolin, is involved i n chorion hardening in medaka.
Alveolin is known to cleave the ZPB, a major chorion component; however, how alveolin
facilitates the chorion hardening in vivo has remained unknown. It has also remained
unknown if alveolin is also involved in the depolymerization of hyosophorin. Thus, the
objective of this study is to understand how CA-localized proteases are involved in
hyosophorin depolymerization and chorion hardening during cortical reaction, and to

attain this, focusing on alveolin and its related prote ases in medaka eggs, the following
experiments were carried out: (1) Search for CA -localized proteases and generation of
their deficient medaka; (2) Functional analysis of alveolin in vivo using alveolin-deficient
medaka; (3) Identification of the hyosophorinase; (4) Analysis of multiple genes for the
major CA-localized glycoprotein hyosophorin.
(1) Search for CA-localized proteases and generation of their deficient medaka (Chapter
2): Although alveolin was already identified to be involved in chorion hardening in
medaka, it shared the common properties with presumptive hyosophorinase: (a) CA - and
PVS-localization before and after fertilization, respectively; (b) increased activity at
fertilization; (c) specific cleavage of the peptide bond before Asp residue. Accordingly,
gene databases were searched for alveolin -like proteases. As a result, a zinc
metalloproteinase Nas-4 showing 45% identity in the amino acid sequence was found.
Then, the alveolin-deficient medaka (Alv-KO) and the Nas-4-deficient medaka (Nas-4KO) were generated using the CRISPR/Cas 9 gene editing technology to evaluate effects
on the depolymerization of hyosophorin at the organism level. In both Alv-KO and Nas4-KO medaka, the sperm could fertilize the egg, and the embryo normally developed,
grown up, and produced offspring.
(2) Functional analysis of alveolin in vivo using alveolin-deficient medaka (Chapter 3):
Since the most prominent feature of Alv-KO embryos was that chorion was mechanically
fragile, properties of the chorion from Alv-KO and wild-type medaka (WT) were analyzed
by the measurement of mechanical toughness, light and transmission electron microscope
(TEM) observations, the measurement of permeability, and biochemical analysis of
chorionic components. First, the chorion diameter of Alv-KO was larger than that of WT,
due to the expansion of PVS. Second, the thickness of chorion was thinner in Alv-KO than
WT, although the number of multiple layers remained unchanged. Interestingly, the TEM
observation showed that the chorion structure of Alv-KO was collapsed at the outer part
of the inner layers. In addition, the soft chorion was permeable to at le ast 10 kDa FITCdextran, in contrast with the WT chorion. Moreover, although the crosslink between ZPB
and ZPC happened in the Alv-KO like WT, crosslinking process was extremely delayed in
Alv-KO. However, the delayed hardening process was accelerated in the balanced salt
solution (BSS), compared with the case in water, although its acceleration was far slow
than the WT case. Notably, alveolin processed not only ZPB, but also the chorionlocalized transglutaminase at fertilization, which was a new finding.
(3) Identification of the hyosophorinase ( Chapter 4): To analyze if alveolin and Nas-4
are involved in the depolymerization of H -hyosophorin, the H- and L-hyosophorin
fractions in the fertilized embryos were quantified for Alv-KO and Nas-4-KO. In Nas-4KO, H-hyosophorin was not depolymerized, while in Alv-KO, it was still depolymerized,
although its amount was decreased. Interestingly, the decreased H -hyosophorin in Alv-KO

embryos was found in the culture medium, because the Alv -KO chorion was leaky to
hyosophorin. These results indicate that Nas -4 is the hyosophorinase that depolymerizes
H-hyosophorin during cortical reaction.
(4) Analysis of structural features of hyosophorin family in fish (Chapter 5): The
recently updated databases were surveyed to find the hyosophorin gene not only in medaka
and rainbow trout, but also in carp, northern pike, and tilapia. It has been shown that
hyosophorin has multiple genes, each of which consists of three regions, including Nregion, a major R-region consisting of tandem-repeats of completely the same sequences,
and C-region. In rainbow trout, 54 hyosophorin genes found in this study contained largely
the same gene structure as those reported previously. On the other hand, 18 genes were
detected in medaka and half of them contained significant mutations in the R -region,
which does not match the common concept of the hyosophorin gene. These results suggest
that at least two different features in terms of conservation of the R-domain structure in
evolution, which might be interesting to understand the significance of the CA contents
and conditions of fertilization in various fish.
In conclusion, this study first demonstrates that Nas-4 is the hyosophorinase responsible
for the proteolytic depolymerization of hyosophorin during cortical reaction in medaka.
It also demonstrates that alveolin facilitates rapid chorion hardening via proteolytic
cleavage of the ZPB and a processing transglutaminase, followed by the ZPB -ZPC
crosslink. Thus, this study for the first time clarified the significance of cortical alveolus
proteases at fish fertilization at the organism level. The data obtained in this study have
not only advanced the knowledge of biology of fertilization, but also have enhanced the
usefulness of medaka as a model animal for biological and pharmacological applications.