全 文 ：水蕨卵膜的形成及其超微结构的观察 ?
曹建国 , 杨耐英 , 王全喜
(上海师范大学 生命与环境科学学院 , 上海 200234)
摘要 : 蕨类植物成熟卵的周围有一层卵膜 , 但其细微结构和形成过程仍不清楚 , 本研究应用透射电镜技术
对水蕨 ( Ceratopteris thalictroides) 卵细胞发育过程中卵膜的形成及超微结构进行了观察。结果表明水蕨卵细
胞在发育中期开始形成卵膜 , 卵上方的卵膜十分显著 , 是由多层嗜锇性内质网片层附着于质膜内表面形成
的 , 成熟时卵上方的卵膜中心部分厚 , 向边缘逐渐变薄 , 在嗜锇性片层之间填充有嗜锇性物质。比较而
言 , 卵下方及侧面的卵膜薄 , 由两层紧密连接的嗜锇性膜构成。首次阐明了蕨类植物卵膜形成的超微结
构 , 并对卵膜的一些功能进行了探讨。
关键词 : 蕨 ; 水蕨 ; 卵膜 ; 超微结构
中图分类号 : Q 944 文献标识码 : A 文章编号 : 0253 - 2700 (2008) 05 - 543 - 06
Observations on the Formation and Ultrastructure of
the Egg Membrane in the fern Ceratopteris
thalictroides (Parkeriaceae)
CAO Jian-Guo, YANG Nai-Ying, WANG Quan-Xi
( Collegeof Lifeand Environment Science, Shanghai Normal University, Shanghai 200234 , China)
Abstract : Thematureeggof ferns possesses a layer of eggmembrane in the periphery . However, thefinetrastructureand
the formation of theeggmembrane in the egg of ferns are still unclear . The present paper described theformation and the
ultrastructure of the eggmembrane in the egg of the fern Ceratopteris thalictroides using transmission electron microscopy
( TEM) . The resultsshowthat theeggmembrane beginsto format the stageof thematuringegg . The eggmembrane in the
upper sideof theegg is prominent . It isformed by attachingof osmiophilic sheetsof endoplasmic reticulumto the inner sur-
face of the plasmalemma . When mature the eggmembrane in the upper side of the egg is much thick in thecentral region
and it becomes thinner gradually towards themargin . Some osmiophilic materials are filled in the spaces betweenthesheets
of endoplasmic reticulum . On the contrary, the eggmembrane in the lower side of the egg is thin . It is composed of two
osmiophilic membranes which are associated with each other closely . Theformation andthe ultrastructure of the eggmem-
brane are described for the first time . Some functions of the egg membrane are also discussed .
Key words: Fern; Ceratopteris thalictroides; Eggmembrane; Ultrastructure
Bell and Mühlethaler (1962 ) found an egg mem-
brane in the mature egg of Pteridiumaquilinum ( L .)
Kuhn . Similar membrane has been detected in eggs of
many ferns including Dryopteris filix-mass (Menke and
Fricke, 1964 ) , Histiopteris incise ( Bell , 1980 ) ,
Todea ( Bell , 1986 ) , Osmunda cinnamomea var. as-
iatica (Bao et al. , 2003) and Dryopteris crassirhizoma
(Bao et al. , 2005) . The egg membrane was consid-
ered to be derived from the degenerated organelles or
thevesicles in thecytoplasmof the egg and formedout-
云 南 植 物 研 究 2008 , 30 (5) : 543～548
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2008.07323
? ?Foundation item: The National Natural Science Foundation of China ( 30670128)
Received date: 2008 - 01 - 08 , Accepted date: 2008 - 03 - 20
作者简介 : 曹建国 (1968 - ) 男 , 副教授 , 主要从事蕨类植物发育与生殖生物学研究。E-mail : cao101 @ shnu. edu. cn
side the plasmalemma ( Bell and Mühlethaler, 1962;
Bell , 1986; Bao et al. , 2003; Bao et al. , 2005 ) . It
has been proved that the eggmembrane around the egg
of P . aquilinum is lipid in nature ( Cave and Bell ,
1974) . However, the formation and the ultrastructure
of the egg membrane in the egg of ferns are still un-
clear . In the present paper, the ultrastructure and the
formation process of the eggmembranein the eggof the
fern Ceratopteris thalictroideswasobserved by transmis-
sion electron microscope . Some new phenomena about
the formation and the structure of egg membrane, dif-
ferent fromthe previous studies, are described .
1 Materials and methods
The spores of C . thalictroides (L .) Brongn . were collected
from plant grown in the Botanical Garden of Shanghai Normal
University . The culture of gametophyteswereobtainedas follow:
Themature spore, soaked in sterilizedwater for 24 h in advance,
was sterilizedwith 5% sodium hypochlorite solution for 5 minutes
andthen washed with sterilized water 3 times . The spores were
sown in theculturemedium of modified Knop′s solution, solidi-
fied with 1 .5 % agar . The materialswere cultured at the condi-
tion of about 25℃ , fluorescent light ( ca . 35μmol·m- 2·s- 1 )
with 18 h illumination and 6 h night . After about 3 to 4 weeks
later, the gametophyte containing archegonia was picked up and
plunged into3 % glutaraldehyde in 0 .1 mol?L phosphate buffer at
roomtemperature for 6 - 12 h . The materials were subsequently
washedwith the same buffer for three times and fixed in osmic
acid (2% aqueous solution) for overnight and then washed with
thesame buffer 3 times . After dehydration in a graded acetone
series, the material was infiltrated with mixture of acetone and
spurr′s resin and then embedded in the pure resin . Specimens
werethick sectioned firstly for the presence of the archegonium
and then thin-sectioned with a diamond knife on an Ultracut-E
ultramicrotome and mounted on grid . The thin sections were
stained with uranyl acetate and lead citrate . All specimens were
observed with H-600 electronmicroscope .
2 Results of observation
2 .1 The development of egg and the formation of
the egg membrane
The development of the egg of ferns is generally
divided into 3 stages, i . e . the young egg, the matur-
ing egg and themature egg ( Bell and Duckett, 1976) .
In the young egg of C. thalictroides, there are abun-
dant organelles, includingplastids, mitochondria, ves-
icles and endoplasmic reticula in the cytoplasm . These
organelles are basically distributed through out the cy-
toplasm of the egg except the endoplasmic reticula,
which are principally located at the periphery (fig. 1) .
The plasmalemma of the egg can be identified clearly
(fig. 1 , arrowhead) . The cell wall between theeggand
the adjacent cells is well defined and devoid of plas-
modesmata . At the latestageof theyoung egg, a sepa-
rate cavity (fig. 1 , SC) is formed between the plasma-
lemma of the egg and the wall of the archegonium as
the eliminatingof vesicles fromthe cytoplasmof the egg
cell . No additional membrane was formed around the
egg in this stage .
In thestageof thematuring egg, astriking feature
of the egg of C. thalictroides is that a layer of osmio-
philic membrane is formed around the egg . Theosmio-
philic membrane is not existed around the ventral and
the neck canal cells . So themembrane is named as an
eggmembrane (fig.2 , EM) .The eggmembrane in the
upper side of the egg is prominent ( figs. 3 - 5 ) . Most
likely the endoplasmic reticula are involved in the for-
mation of the egg membrane in this stage . Firstly,
many sheets of endoplasmic reticulum in the egg cyto-
plasm lie closely and parallel to the plasmalemma
( fig. 3 ) . When a sheet of endoplasmic reticulum
(fig. 3 , arrow) is attached to the inner surface of the
plasmalemma, both the sheet of endoplasmic reticulum
and the plasmalemma, become osmiophilic . The two
osmiophilic membranes formthe eggmembrane (fig. 3 ,
EM) . At the side and the lower part of the egg, al-
though some endoplasmic reticula lie closely to the in-
ner surface of the egg, no additional sheet of endoplas-
mic reticulumwas seen to be attached to the plasmale-
mma (figs. 4 , 5) . The osmiophilic membrane, cover-
ing the lower surfaceof the egg, seems to be a double-
layered membrane, occasionally two membranes can be
detected in places (fig. 4 , arrow) .
With the developing of the egg cell , more sheets
of endoplasmic reticulum are attached to the inner sur-
face of the upper egg membrane . As soon as the new
sheets of endoplasmic reticulum are attached to old
ones, they also becomeosmiophilic (fig. 6 , arrow) . In
addition, the osmiophilic sheets, constituting the egg
445 云 南 植 物 研 究 30 卷
Figs . 1 - 7 Theformation of the egg membrane in the egg of Ceratopteris thalictroides
1 . A part of young egg contains plentyof organelles including mitochondria (M ) , plastid ( P ) , vesicles ( V ) and endoplasmic reticula (ER) , which are
principally located in theperiphery of the egg . A separate cavity ( SC) formed between the plasmalemma (arrowhead) and thewall ( W) of the archego-
nium; 2 . A maturing egg ( E) is surrounded by a layer of osmiophilic eggmembrane ( EM) . Theventral canal cell (VCC) is degenerating; 3 . Magnifi-
cation of figure 2 showing the eggmembrane (EM) in theupper sideof the egg . Theeggmembraneformed by attaching of asheet of endoplasmic reticu-
lum (arrow) to theplasmalemma ( Pl) ; 4 . Thesame egg cell with figure2 showing the egg membrane (EM ) and endoplasmic reticula in sideof the egg;
5 . The same egg cell with figure 2 showing the egg membrane (EM) and endoplasmic reticula in lower sideof the egg; 6 . The egg membrane ( EM )
develops further by attaching of more sheets of endoplasmic reticulum (arrow) to the inner surface of the egg membrane; 7 . The lower part of the
same egg cell with figure 6 showing the egg membrane (EM) in the lower of the egg unchanged . Abbreviation : N , nucleus; G, Golgi body
5455 期 CAO Jian-Guo et al . : Observations on the Formation and Ultrastructure of the Egg Membrane in the . . .
membrane, are interconnected with each other . Final-
ly, a multilayeredosmiophilic egg membrane is formed
inside the plasmalemma ( fig. 6 ) . In the cytoplasm,
some redundant endoplasmic reticula are also existed
(fig. 6 ) . By contrast, the egg membrane in the side
and lower part of the egg remain almost unchanged
(fig.7 ) .
In the latestageof thematuring egg, another con-
spicuous featureof the egg is the occurrence of numer-
ous small vesicles containing the strongly osmiophilic
materials . Thesevesicles, with a diameter about 0 .3 -
0 .5μm, areprincipally located in the cytoplasmabove
the cup-liked nucleus ( Figs. 8 , 9 , Vo) . The vesicles
are undoubtedly involved in the formation of the egg
membrane in the upper side of the egg . The vesicles
move firstly to the inner surface of theupper eggmem-
brane and soon these vesicles are attached to the egg
membrane ( fig. 9 , Vo) . Occasionally, the fusion of
vesicles with the egg membrane is observed clearly . At
the same time, the osmiophilic materials within the
vesicles begin to decondense intosmall granules, which
are undoubtedly incorporated into the egg membrane
( fig. 10) . Thus, theeggmembrane completes its forma-
tion . The maturing egg also contains plenty of or-
ganelles, including the degenerated plastids, the mito-
chondria and Golgi bodies (figs. 3 - 5) .
2 . 2 The structure of the egg membrane in the
mature egg
In thematurestage, the egg completes the forma-
tion of the egg membrane . No sheets of endoplasmic
reticulum are attached to the egg membrane . A con-
spicuous featureof the egg is that the endoplasmic re-
ticula, previously lying closely to the periphery of the
egg, now formstacks in the cytoplasm ( figs. 11 - 14 ) .
The structure of the egg membrane in the upper sideof
the egg can be identified clearly . The outmost layer of
the egg membrane is undoubtedly the plasmalemma
which ismuchsmoother than theosmiophilic sheets be-
neath it (fig. 11 ) . The osmiophilic sheets of endoplas-
mic reticulum beneath the plasmalemma, with an un-
dulated and rough shape, are interconnected with each
other . Thespaces between thesheets arefilled withos-
miophilic materials, which are condensed again after
being incorporated into the eggmembrane (fig. 11 , ar-
row) . Another feature of the mature egg is that the
thickness of the egg membrane in theupper sideof the
egg is not equal . In the central portion, the thickness
of the eggmembrane reaches or exceeds 0 .5μmand it
becomes thinner gradually towards the margin ( figs. 8 ,
12) .
The egg membrane in the side and lower part of
the egghas a relatively simple structure . It is a layerof
osmiophilic membrane at first sight . But at highmagni-
fication, two layers of osmiophilic membrane can be
detected clearly ( fig. 12 ) . The thickness of the egg
membrane increases toabout 70 - 80 nm . Occasionally,
small osmiophilic granules are seen being attached to
the egg membrane ( fig. 13 , arrow) . In the mature
egg, some large lumps of osmiophilic materials are of-
ten seen in the cavity between the egg membrane and
thewall of the archegonium (fig. 14 , arrow) .
3 Discussion
3 .1 The formation and development of the egg
membrane
The mature egg of C . thalictroides possesses a
layer of egg membrane, which has been proved to be
existed in egg of other homosporous ferns including
Pteridiumaquilinum ( Duckett and Bell , 1972; Cave
and Bell , 1974 ) , Histiopteris incise ( Bell , 1980 ) ,
Osmunda cinnamomea var . asiatica ( Bao et al. ,
2003) , Dryopteris crassirhizoma ( Bao et al. , 2005 ) .
The eggmembrane is believed to be formed by thedep-
osition of materials which are derived fromthedegener-
ated organelles or vesicles in thecytoplasmof theyoung
egg on the outside of the plasmalemma . Although the
young egg of C . thalictroides possesses plenty of vesi-
cles, no osmiophilic materials within the vesicles are
observed in theyoung egg . There are also no evidence
that thevesicles arederived fromthe degeneratedmito-
chondria or plastids . So we considered that the egg
membrane is not formed by the osmiophilic materials
within the vesicles in the young egg . In this study, we
described the detailed formation processes of the egg
membrane in theupper sideof the egg . It isproved that
the eggmembrane, especially the upper side, is formed
645 云 南 植 物 研 究 30 卷
directly by sheets of the endoplasmic reticulum . As ear-
ly as in theyoung egg, thesheet of endoplasmic reticu-
lum is observed to lie closely to the periphery of the
egg . As soon as the sheet of endoplasmic reticulumis
Figs . 8 - 14 The formation of the egg membrane in the egg of Ceratopteris thalictroides
8 . Numerous vesicles containing strongly osmiophilic materials ( Vo) occurring in the cytoplasm at the late stage of thematuring egg; 9 . Magnification
of the same egg cell with figure 8 , showing thevesiclescontaining osmiophilic materials ( Vo) move to the eggmembrane ( EM ) ; 10 . Thevesiclescon-
taining osmiophilic materials (Vo) are fused with the egg membrane; 11 . Themature egg membrane in the upper side of the egg, consisting of mem-
branous sheets and osmiophilic materials ( arrow) ; 12 . The eggmembrane (EM) in the side of themature egg, two layers of membrane can bedetected
clearly; 13 . Thesmall osmiophilic material granules are attached to the egg membrane (arrow) . Stacksof endoplasmic reticula ( ER) areoften seen in the
cytoplasm of the mature egg; 14 . Some lumps of osmiophilic materials (arrow) areoften seen in the cavity between the egg membrane and thewall of the
archegonium in the mature egg . Abbreviation: E, egg; M , mitochondrion; N , nucleus; P, plastid; Pl , plasmalemma; VCC , ventral canal cell
7455 期 CAO Jian-Guo et al . : Observations on the Formation and Ultrastructure of the Egg Membrane in the . . .
attached to the inner surface of plasmalemma, both of
them become osmiophilic and form the eggmembrane .
In succession, more sheets of endoplasmic reticulum
will be attached to the inner surface of the egg mem-
brane . Thus themembranous eggmembrane is formed .
After that, it is also demonstrated that the new formed
vesicles containing the strongly osmiophilic materials in
the cytoplasmof the eggare involved in theformationof
the egg membrane . The distinct fusion of the vesicles
with the egg membrane suggested theosmiophilic mate-
rials within the vesicles are incorporated into the egg
membrane ( fig. 10 ) . However, the origin of the new
formed vesicle is still uncertain . We propose that the
occurrence of the vesicles may be relative to the abun-
dant endoplasmic reticulum in the cytoplasm of the
egg .
3 . 2 ?The structure and function of the egg mem-
brane
The detail structure of the egg membrane is still
uncertain in the ferns so far reported ( Bell and Duck-
ett, 1976; Bell , 1980; Bao et al. , 2003; Bao et al. ,
2005) . In present investigation, the fine structure of
the upper egg membrane in the fern C . thalictroides is
described for the first time . The detailed observations
about the formation of the eggmembranesuggested that
theoutmost layer of the upper egg membrane is undo-
ubtedly theplasmalemma and beneath it are theosmio-
philic sheetsof endoplasmic reticulum . Here, it is sug-
gested that the sheets beneath the plasmalemma are the
fundamental skeleton of the egg membrane . After the
membranous skeleton was formed, the osmiophilic ma-
terials within the vesicles are incorporated into the egg
membrane . By contrast, the egg membrane in lower
part of the egg ismuch simple, only inhigh magnifica-
tion, the two osmiophilic membranes can be detected .
What role the special egg membrane plays is much in-
teresting . It is known that the egg membrane around
the egg of P . aquilinum is made up of lipid material ,
which may be used as a barrier to protect the egg from
the ingress of metabolites or“ informational”molecules
from elsewhere in the gametophyte ( Cave and Bell ,
1974; Bell and Duckett, 1976) . The eggmembraneof
the egg of C . thalictroides is much prominent . Its role
may be the same with that in P . aquilinum . In addit-
ion, the distinct differences in morphology and struc-
tureof the egg membrane between the upper part and
the lower partof theeggmay mean thegreat differences
in function of them . The thick upper egg membrane,
exposed to the sperms, suggests that it may be used to
prevent thesperms fromenteringthe egg . The entrance
of the spermmay be a pore, which is found recently in
the egg membrane ( unpublished) . A further research
will be conducted about the function of the egg mem-
brane . Moreover, theosmiophilic lumps in the separate
cavity are also encountered frequently in the mature
egg, which may also be used to prevent the sperms
from entering the egg in the lateral side .
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