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Algae 2020, 35(4): 389-404
https://doi.org/10.4490/algae.2020.35.12.7
Open Access
Research Article
Copyright © 2020 The Korean Society of Phycology 389 http://e-algae.org pISSN: 1226-2617 eISSN: 2093-0860
Dynamics of spermatial nuclei in trichogyne of the red alga
Bostrychia moritziana (Florideophyceae)
Eunyoung Shim1, Hana Park1, Soo Hyun Im1, Giuseppe C. Zuccarello2 and Gwang Hoon
Kim1,*
1Department of Biological Sciences, Kongju National University, Gongju 32588, Korea
2School of Biological Sciences, Victoria University of Wellington, P.O. Box 600, Wellington 6140, New Zealand
Red algal fertilization is unusual and offers a different model to the mechanism of intracellular transport of nuclei
and polyspermy blocking. A female carpogonium (egg) undergoes plasmogamy with many spermatia (sperm) simul-
taneously at the receptive structure, trichogyne, which often contains numerous male nuclei. The pattern of selective
transport of a male nucleus to the female nucleus, located in the cell body of the carpogonium, remain largely unknown.
We tracked the movement of spermatial nuclei and cell organelles in the trichogyne after plasmogamy using time-lapse
videography and fluorescent probes. The fertilization process of Bostrychia moritziana is composed of five distinctive
stages: 1) gamete-gamete binding; 2) mitosis in the attached spermatia; 3) formation of a fertilization channel; 4) migra-
tion of spermatial nuclei into the trichogyne; and 5) cutting off of the trichogyne cytoplasm from the rest of the cell after
karyogamy. Our results showed that actin microfilaments were involved in the above steps of fertilization, microtubules
are involved only in spermatial mitosis. Time-lapse videography showed that the first (“primary”) nucleus which en-
tered to trichogyne moved quickly to the base of carpogonium and fused with the female nucleus. The transport of the
primary male nucleus to the egg nucleus was complete before its second nucleus migrated into the trichogyne. Male
nuclei from other spermatia stopped directional movement soon after the first one entered the carpogonial base and
oscillated near where they entered trichogyne. The cytoplasm of the trichogyne was cut off at a narrow neck connecting
the trichogyne and carpogonial base after gamete nuclear fusion but gamete binding and plasmogamy continued on
the trichogyne. Spermatial organelles, including mitochondria, entered the trichogyne together with the nuclei but did
not show any directional movement and remained close to where they entered. These results suggest that polyspermy
blocking in B. moritziana is achieved by the selective and rapid transport of the first nucleus entered trichogyne and the
rupture of the trichogyne after gamete karyogamy.
Key Words: Bostrychia moritziana; cytoskeleton; fertilization; polyspermy blocking; time-lapse videography
INTRODUCTION
Fertilization is one of the most important events of an
organism’s life cycle and consists of several well regu-
lated steps to guarantee successful zygote formation.
Polyspermy blocking, the inhibition of multiple sperm
nuclei fusing with the egg nucleus, is essential to make
viable zygotes. Fertilization of an egg nucleus by multiple
sperm nuclei is usually lethal (Brawley 1987, Mio et al.
2012). Polyspermy blocking is achieved through various
Received September 30, 2020, Accepted December 7, 2020
*Corresponding Author
E-mail: ghkim@kongju.ac.kr
Tel: +82-41-850-8504, Fax: +82-41-850-8479
This is an Open Access article distributed under the
terms of the Creative Commons Attribution Non-Com-
mercial License (http://creativecommons.org/licenses/by-nc/3.0/) which
permits unrestricted non-commercial use, distribution, and reproduction
in any medium, provided the original work is properly cited.
Algae 2020, 35(4): 389-404
https://doi.org/10.4490/algae.2020.35.12.7 390
tion and fusion has been reported as an intermediate
means of polyspermy block (e.g., Bianchi and Wright
2014). FUS1 and HAP2 proteins are two gamete‐specific
components essential for gamete fusion in Chlamydo-
monas (Ferris et al. 1996, Misamore et al. 2003, Liu et
al. 2008). These proteins become eliminated from the
plasma membrane after gamete fusion, which results in
the reduction in the fusion capacity of the gametes and
prevents polygamy (Liu et al. 2010).
Fertilization in red algae is unusual, partially due to
their lack of any flagellated stages (Picket-Heaps and
West 1998). The receptive area of the egg (carpogonium),
the trichogyne, is an elongated extension of the carpogo-
nium and relatively long-lived, and it is covered by cell
wall material, which is possibly an adaptation to passive
receiving of sperm (Kim and Fritz 1993a, 1993b). The
sperm (spermatia), also surround by wall material and
appendages, is passively transported to the trichogyne.
The surface of the trichogyne, in many red algae, can ac-
commodate a large number of attached spermatia (Pick-
et-Heaps and West 1998).
There is no apparent change in cell membrane poten-
tial after gamete binding in red algae. Numerous sperma-
tia simultaneously bind to trichogyne and develop fertil-
ization channels, which leads to multiple plasmogamy,
therefore there is also no apparent changes in extracel-
lular matrix to block additional binding (Kim and Fritz
1993a, Mine and Tatewaki 1994, Picket-Heaps and West
1998). Each spermatium that attaches to the trichogyne
undergo mitosis and discharges two nuclei together with
other cell organelles into trichogyne. It has been sug-
gested that an actin-myosin machinery is involved in the
migration of spermatial nuclei into and along the tricho-
gyne (Kim and Kim 1999a, Wilson et al. 2002). Previous
study using time-lapse videography in the red alga Bos-
trychia moritziana showed that two spermatial nuclei
move in different directions within trichogyne, one go-
ing to the base of carpogonium, potentially fusing with
the egg nucleus, and the other migrates to the opposite
direction, towards a tip of the trichogyne. It has been
suggested that the two nuclei in each spermatium are
differentiated so that only one is capable of fertilization,
differentiation being visibly expressed in their directional
movement (Picket-Heaps and West 1998). This scenario
would indicate that when multiple plasmogamy occur
several spermatial nuclei could move to the carpogoni-
um cell body, and potentially fuse with the egg nucleus,
producing polyspermic fertilization.
In this study we examined the fertilization process of
B. moritziana to answer the following questions: What
mechanisms. Two types of mechanisms for polyspermy
blocking have been reported in mammals: the “oocyte
membrane block” to sperm penetration and the “zona
reaction.” The former response involves a depolarization
of the egg membrane caused by the influx of Na+, which
changes the potential of the egg membrane from negative
to positive inhibiting sperm fusion. The latter response
occurs in the ‘zona pellucida’ in animals, once the first
sperm has attached to egg membrane, changing its prop-
erties and blocking any further sperm penetration (Coy
and Avilés 2009). Polyspermy blocking in sea urchins and
sea worms depends on a change in the electrical charge
across the surface of the egg, which is caused by the fu-
sion of the first sperm with the egg (Jaffe 1976, Gould-
Somero et al. 1979). Unfertilized sea urchin eggs have a
negative charge inside, but the charge becomes positive
upon fertilization. When the first sperm contacts the egg
and causes the electrical change, subsequent sperms are
prevented from fusing. The mechanisms of nuclear fu-
sion and maternal inheritance of cell organelles are not
fully answered yet.
Plants also have evolved a battery of mechanisms that
potentially act as polyspermy barriers (e.g., Tekleyohans
et al. 2017, Tekleyohans and Groß‐Hardt 2019). In most
sexually reproducing plants, pollen grows a pollen tube
which delivers one pair of sperm nuclei that ultimately
fertilize two female nuclei, the egg and the two nuclei
of the central cell (Russell 1993, Johnson et al. 2019).
Sperm cell release and gamete fusion trigger sequential
disintegration of both synergids such that typically only
a single pollen tube is attracted to one ovule (Völz et al.
2013, Maruyama et al. 2015). Apart from the regulation
of pollen tube growth, the plant egg is subjected to fur-
ther modifications after fertilization. In rice and maize,
a shielding material accumulates on the cell wall of egg
and prevents supernumerary sperm cell fusion in 10-20
min after the first sperm fuses with egg (Kranz et al. 1995,
Toda et al. 2016).
In fucoid brown algae, polyspermy is lethal with zy-
gote development ceasing at the four‐cell stage (Brawley
1987). To circumvent the fatal consequences of super-
numerary gamete fusion, fucoid algae have evolved a
sodium‐dependent polyspermy block similar to the “oo-
cyte membrane block” in animals (Brawley 1987, 1991).
Secretion of cell wall material upon exposure of calcium
ionophores has been reported in these algae suggesting
that calcium mediates vesicle release into the egg extra-
cellular matrix thereby establishing a permanent block to
polyspermy (Brawley and Bell 1987, Brawley 1990, 1991).
In green algae, the proteins involved in gamete recogni-
Shim et al. Dynamics of Spermatial Nuclei in Bostrychia moritziana
391 http://e-algae.org
Microscopy, uorescent probes and image
analysis
Samples prepared as above were examined on an
Olympus BX51 research microscope (Tokyo, Japan)
equipped with differential interference contrast (DIC)
optics and Samsung iPolis camera (Samsung, Suwon,
Korea), with an oil-immersion condenser. Single frame
photographs were taken every 3 s for 120 min and all the
frames were amalgamated into video clips using a time-
lapse program (VideoVelocity, CandyLabs, Vancouver,
Canada).
To visualize actin microfilaments, female with tricho-
gynes and attached spermatia were fixed for 30 min in
3.7% (w/v) formaldehyde diluted in microfilament-
stabilizing buffer (MFSB) consisting of 10 mM EGTA, 5
mM MgSO4, and 100 mM PIPES-KOH (pH 6.9) (Traas et
al. 1987). The trichogynes were rinsed three times with
MFSB, and then placed for 30 min in 0.5% (v/v) Triton
X-100 diluted in MFSB. The algae were washed three
times with MFSB before being placed in a solution of
BODIPY FITC-phallacidin (Invitrogen, Carlsbad, CA,
USA) (Wieland et al. 1983) for 3-6 h at 4°C in the dark.
FITC-phallacidin was prepared as a stock solution of 300
units mL-1 in methanol and was stored at -20°C in the
dark. The stock was diluted with MFSB to a final con-
centration of 1.5 units mL-1. For dual staining of nucleus,
Hoeschst33342 stain was diluted in IMR medium as 1 μL
mL-1 and cells were stained in 1.5 mL tube for 15 min in
the dark before observation under microscope.
For microtubule labelling, female trichogynes with
attached spermatia were fixed in 3.7% formaldehyde
diluted with phosphate-buffered saline (PBS; 8 mM
Na2HPO4, 2 mM NaH2PO4, 140 mM NaCl; pH 7.4) for 30
min and then rinsed three times in the same buffer. The
trichogyne were cut off from the female plant with a ra-
zor blade to allow antibody entry into the cytoplasm, and
incubated overnight in the dark with the monoclonal
anti-alpha-tubulin antibody conjugated to fluorescein
isothiocyanate (Sigma-Aldrich, St. Louis, MO, USA), di-
luted at a 1 : 40 ratio in PBS. For dual staining of nucleus,
Hoeschst33342 was added to the solution (1 μL mL-1) for
15 min before observation.
For the general stain for vacuolar inclusions in sper-
matia and trichogynes DRAQ5 (Thermo Fisher, Seoul,
Korea) solution diluted as 1 μL 1 mL-1 in IMR medium
and applied to the sample after gamete mixing. DRAQ5
is originally developed as a cell permeable far-red fluo-
rescent DNA dye that can be used in fixed or live cells. We
found this drug stains all vacuolar structures of B. morit-
mechanism is involved in spermatial nuclear movement
and what is the fate of nuclei that do not fuse with the
carpogonial nucleus? We used time-lapse videography
combined with image analysis system and fluorescent
probes to trace the dynamics of spermatial organelles in-
side the carpogonium.
MATERIALS AND METHODS
Algal cultures
Gametophytes (male and female) of B. moritziana
were obtained from the John A. West culture collection
(culture number #2746) and samples deposited in Na-
tional Marine Biodiversity Institute of Korea (MABIK)
(KNU culture No. KNU000027, KNU000028) were used.
The thalli were maintained in unialgal cultures in IMR
medium (Kim et al. 2005) at 20°C in a 16 : 8 h light : dark
cycle with illumination of >20 μmol photons m-2 s-1 pro-
vided by cool-white fluorescent lighting. The plants were
transferred every 1-2 weeks to new IMR medium. For fer-
tilization experiments, subcultures of 5 to 10 shoot tips
approximately 1 cm long were transferred to fresh me-
dium in 100 mL dishes a week before use. These plants
were checked for growth and reproduction daily. Male
and female gametophytes actively developing sper-
matangial stichidia and carpogonial branches with vis-
ible trichogynes, respectively, were used.
Preparations for microscopy
To induce spermatial release individual male shoot
tips with well-developed spermatangial stichidia were
exposed to osmotic shock by placing into distilled wa-
ter for 30 s (Picket-Heaps and West 1998). The male was
then removed from the suspension of spermatia and
several female shoot tips with visible trichogynes were
added and the dish was gently agitated to promote mix-
ing and contact of spermatia with the trichogynes. The
female branches were then quickly placed in seawater on
a well-cleaned glass slide and a coverslip added. Excess
water was blotted from the edge and the whole mount
was sealed with Valap (1 : 1 : 1 : 1 mixture of paraffin wax,
lanolin and Vaseline, melted at about 40°C (Picket-Heaps
and West 1998). Within 2-5 min of spermatial contact the
slide was placed on the video microscopy system.
Algae 2020, 35(4): 389-404
https://doi.org/10.4490/algae.2020.35.12.7 392
over time. When the nucleus moved more quickly, the
spots marked on nuclei traveled a greater distance in a
given time. The final distance of each nucleus from the
fertilization channel to the base of carpogonium was
measured and presented as a bar graph.
RESULTS
The cytoskeleton during the fertilization process
Early in gamete binding, microfilaments in spermatia
were distributed evenly on the periphery of the sperma-
tium, surrounding the central nucleus, and microfila-
ments in the trichogyne are also fairly evenly distribut-
ed in the cytoplasm (Fig. 1A). When spermatia became
closely appressed to the trichogyne, they enter mitosis
within 20 min. At this time, microfilaments accumulated
at the center plate separating the two daughter nuclei
(Fig. 1B). A fertilization channel, between spermatia
and trichogyne, began to develop around 30-40 min af-
ziana cells in blue under a light microscope. We used it as
a counter stain to observe nuclear movement in tricho-
gyne using DIC optics.
Chromeo a live cell mitochondrial staining kit (Santa
Cruz Biotechnology, Santa Cruz, CA, USA) was stored in
a 100 mM stock solution in solubilizing buffer at -20°C.
To observe mitochondria, the stock solution was diluted
to 1 μL 1 mL-1 in IMR medium and cells were stained for
30 min prior to observation on a fluorescent microscope.
An image analysis program (Image-pro plus 7.0; Media
Cybernetics, Warrendale, PA, USA) was used to measure
the direction and velocity of male nuclei and organelles
movement in the trichogyne. To measure distance of
male organelle movement, the video clips taken from
each experimental set up were analyzed using a motion
tracking system as follows: A spot was marked on the
nuclei or male organelles appearing in the frame of the
time-lapse clip, and distance traveled by each spot in 1
min was shown as a peak in the graph. Each line in the
graph represents the distance that each spot traveled.
Each peak that one spot made was traced as a line graph
Fig. 1. Microfilaments during fertilization in Bostrychia moritziana. (A) Microfilaments distribute evenly on the periphery of spermatium at
time of attachment. (B) Spermatium enter mitosis soon after attachment. Microlaments separate two daughter nuclei. (C) Fertilization channel
developed and microlaments of spermatium and trichogyne connected through it. (D) Spermatial nucleus migrates into trichogyne. (E) Sper-
matial nucleus enclosed by microlaments moving towards the base of the carpogonium. (F) Spermatial nucleus migrated into the base of the
carpogonium. Microlaments connecting trichogyne and carpogonial base became thinner and disconnected. Spermatial nuclei stained blue
with Hoeschst33342 and microlaments stained green with FITC-phallacidin. N, spermatial nucleus; T, trichogyne; CB, carpogonial base. Scale bar
represents: 10 μm.
AC
D
B
E F
Shim et al. Dynamics of Spermatial Nuclei in Bostrychia moritziana
393 http://e-algae.org
trichogyne (Fig. 1E). After a spermatial nucleus migrated
into the base of carpogonium, the microfilaments in the
region connecting the trichogyne and carpogonial base
became thin and fragmentary (Fig. 1F).
Microtubules were observed only in spermatia (Fig.
2). Extensive amount of tubulin (microtubules) was ob-
ter gamete binding and microfilament bridged between
the two cells through this fertilization channel (Fig. 1C).
When spermatial nuclei migrate into the trichogyne
some spermatial microfilaments remain in the sperma-
tia body (Fig. 1D). Spermatial nuclei became elongated
and surrounded with microfilaments while moving in the
Fig. 2. Microtubules during the early stages of fertilization. (A) Even distribution of microtubules around periphery of the spermatium during
the initial stage of gamete binding. (B) Microtubules now found in center of the cell close to metaphase plate. (C) At metaphase, microtubules
located at each polar region of dividing nucleus forming the spindle. (D) At telophase, microtubule accumulated at the center plate between the
two daughter nuclei. (E) During the migration of spermatial nuclei into the trichogyne microtubules in spermatium degenerated. Some microtu-
bules remained in the peripheral region of the spermatium. Spermatial nuclei stained blue with Hoeschst33342 and microtubules stained green
with FITC-conjugated alpha-tubulin antibody. Micrographs of Hoeschst33342 staining (DNA), tubulin and merged image. The surface of tricho-
gyne was marked with dashed line. N, spermatial nucleus; T, trichogyne. Scale bar represents: 5 μm.
A
C
D
B
E
DAPI Anti-tubulin Merge
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Fig. 3. Time-lapse videograph of multiple plasmogamies and the migration of spermatial nuclei in a trichogyne. (A) Mitosis occurred in at-
tached spermatia and multiple plasmogamy occurred nearly simultaneously along the surface of trichogyne. (B) The rst nucleus (n1) to enter
trichogyne followed by the rst nucleus from the other spermatium (n2) also entered. (C) The rst nucleus (n1) moved towards the base of carpo-
gonium, but the second nucleus (n2) did not move in that direction but oscillated near the fusion area. (D) The rst nucleus (n1) reached to the
base of carpogonium and the second nucleus (n1`) from the same spermatium entered the trichogyne. The nucleus from the third spermatium
(n3) entered the trichogyne. (E) The rst nucleus (n1) entered the carpogonial base and all other spermatial nuclei remained around where they
entered. (F) The other spermatial nuclei (n1`, n2, n2`, n3, n3`) showed slow oscillation in the trichogyne. Scale bar represents: 10 μm.
A
C
D
B
E
F
Shim et al. Dynamics of Spermatial Nuclei in Bostrychia moritziana
395 http://e-algae.org
numerous spermatia, attached to a trichogyne, started
mitosis simultaneously and all developed fertilization
channels (Fig. 3A). The first nucleus to enter the tricho-
gyne from the ‘primary’ spermatium began to move to-
wards the base of carpogonium soon after it entered the
trichogyne (Fig. 3B). Often the first nucleus from the
other spermatium (n2) entered the trichogyne before
the second nucleus (n1`) of the primary spermatium en-
tered the trichogyne (Fig. 3B). The second nucleus (n1`)
eventually followed the first nucleus towards the base of
carpogonium but did not pass the narrow neck that con-
nects the trichogyne with the carpogonial base (Supple-
mentary Video S1). Although several nuclei entered the
trichogyne over a short interval only the first one (pri-
mary nucleus) to enter migrated all the way to the base of
carpogonium (Fig. 3C-E). The other nuclei in the tricho-
gyne did not show any particular directional movement
but showed slow oscillations, after the primary nucleus
had entered the carpogonial base (Fig. 3F). Often the cy-
toplasm at the base of the trichogyne cut off at the nar-
served on the surface of spermatia at the initial stage of
gamete binding (Fig. 2A). During prometaphase micro-
tubules disappeared from the surface and moved to the
center close to the metaphase plate (Fig. 2B). At meta-
phase microtubules moved to polar region of dividing nu-
clei (Fig. 2C). Microtubule began to appear at the center
plate between the two daughter nuclei during telophase
(Fig. 2D). There was no cytokinesis, so the spermatium
remained binucleate. When spermatial nuclei migrated
into the trichogyne, microtubules began to disappear
and remnant microtubules remained in the spermatium
(Fig. 2E). Microtubules were not observed in the tricho-
gyne surface nor on the fertilization channel during the
fertilization processes (Fig. 2E).
Movement of spermatial nuclei and organelles
within the trichogyne
Multiple plasmogamy occurred almost simultane-
ously between a trichogyne and spermatia (Fig. 3). Often
Fig. 4. Image analysis of time-lapse footages on spermatial movement in trichogyne. (A) Moving distance by time, measured as number of
frames, of each male nucleus inside trichogyne. Note the rst male nucleus move much faster and further than the other nuclei. (B) Comparison
of moving distance of spermatial nuclei in order of the entrance to trichogyne. The rst male nucleus moves much longer distance than the oth-
ers. (C) Velocity of the spermatial nuclei in order of the entrance to trichogyne.
A
C
B
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Fig. 5. Movement of male nucleus in trichogyne with DRAQ5 staining. Membranous organelles in spermatium and trichogyne were stained
blue. (A) The male nucleus (*) was in contact with blue-stained organelles in trichogyne. (B) Some stained membranous organelles in the tricho-
gyne were pushed towards the carpogonial base together with the male nucleus. (C) Primary spermatial nucleus proceed around the organelles.
(D) Primary spermatial nucleus (*) entered the base of carpogonium and female organelles moved back close to original position. Scale bar repre-
sents: 10 μm.
A
C
D
B
Shim et al. Dynamics of Spermatial Nuclei in Bostrychia moritziana
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clei (Fig. 6). Often, some male organelles were discharged
before the first nucleus migrated into the trichogyne (Fig.
6A & B), but as this first nucleus proceed towards the car-
pogonial base, it left these organelles behind (Fig. 6C &
D, Supplementary Video S3). The shape of mitochondria
in the spermatia was distinctive and each mitochondria
was discrete at the initial stage of fertilization (Fig. 7A),
but they became smaller and often aggregated into ir-
regular shaped masses at later stages, e.g., mitosis in
spermatia (Fig. 7B & C). Some spermatial mitochondria
followed the migrating male nucleus into the trichogyne
after plasmogamy, but remained close to the fertilization
channel (Fig. 7D).
Inclusions in the trichogyne cytoplasm changed after
the first spermatil nucleus passed the narrow neck of
carpogonium (Fig. 8, Supplementary Video S4). The cy-
toplasm initially was filled with many small vesicle-like
structures which oscillated all the time (Fig. 8A-C). These
row neck connecting the trichogyne to the carpogonial
cell body after the first spermatial nucleus passed that
point. Image analysis result showed that the first sper-
matial nucleus moved faster and further than the other
nuclei inside the trichogyne (Fig. 4A & B).
The cytoplasm of trichogyne was filled with numerous
membranous organelle-like inclusions which oscillated
all the time. These organelles were not distinctive in the
light microscope unless stained with DRAQ5 and became
blue. When the first spermatial nucleus proceed to these
obstacles the organelles were pushed towards the base of
carpogonium (Fig. 5A & B). The spermatial nucleus left
them behind when it passed the narrow neck connect-
ing the trichogyne to the carpogonial base (Fig. 5C & D,
Supplementary Video S2). In spermatia, these blue stain-
ing bodies mostly degenerated during the development
of the fertilization channel, but some survived until plas-
mogamy and entered to trichogyne together with the nu-
Fig. 6. Movement of spermatial membranous organelles during plasmogamy. (A) When male nucleus entered the trichogyne some organelles
entered also. (B) Spermatial organelles trapped between two spermatial nuclei. (C) Spermatial organelles remain where they were discharged
when the rst spermatial nucleus entered the carpogonial base. (D) At late stage of fertilization, spermatial organelles still staying where they
were discharged. *, the spermatial nuclei; red arrow, the spermatial organelles. Scale bar represents: 5 μm.
A
CD
B
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of the trichogyne was cut off at the narrow neck and there
was a total collapse of the trichogyne cytoplasm after the
first nucleus entered (Supplementary Video S5). Sper-
matial binding, as well as nuclear division, still occurred
when newly released spermatia were added to the tricho-
gyne even after the cytoplasm had collapsed, but all sper-
matia aborted without the development of a fertilization
channel (Supplementary Video S6).
structures disappeared at later stages of fertilization and
many large vacuoles appeared in the cytoplasm (Fig. 8D
& E). The other male nuclei were trapped between these
vacuoles (Fig. 8F, Supplementary Video S4). When plas-
mogamy occurred simultaneously, the first nucleus from
the secondary spermatium followed the first primary
nucleus to the base of the carpogonium but did not enter
the cell body (Supplementary Video S5) as the cytoplasm
Fig. 7. Spermatial mitochondria stained with uorescent probe Chromeo. (A) At the initial stages of gamete binding mitochondria in sperma-
tium are distinctive and separate from each other. (B) When spermatium enters mitosis, the number of mitochondria decreases and some of them
agglutinate to form irregular masses. (C) Most mitochondria degenerated or fused during mitosis. (D) Some mitochondria entered the trichogyne
through the fertilization channel. Spermatial nuclei stained blue with Hoeschst33342 and mitochondria stained green with Chromeo, light image
also shown. Scale bar represents: 5 μm.
A
C
D
B
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399 http://e-algae.org
Fig. 8. The changes in the cytoplasm of trichogyne during fertilization. (A) Trichogyne cytoplasm was lled with small vesicle-like structures
showing oscillating movements. (B) Primary spermatial nucleus moved into the trichogyne (n1) not blocked by these vesicles. (C) Movement of
male nucleus (n1) continues toward the base of trichogyne. (D) Many vesicles disappeared after the primary male nucleus enters the carpogonial
base. (E) Most vesicles disappeared in the trichogyne cytoplasm and the space began to be lled with large vacuoles. (F) Before the collapse of
the trichogyne, the whole trichogyne cytoplasm was lled with swollen vacuoles. n1, the rst male nucleus entered trichogyne; n1`, the second
nucleus of the primary spermatium; n2, the rst nucleus of the secondary spermatium; n3, the rst nucleus of the tertiary spermatium. Scale bar
represents: 10 μm.
A
C
D
B
E
F
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plasmogamy has been reported in some animals (Hem-
mings and Birkhead 2015). Bird eggs are penetrated by
multiple sperms without any polyspermy blocking, and
polyspermy is somehow crucial to the development of
the bird embryo. Although multiple plasmogamy occurs
in the cell membrane egg nucleus fuses with only one
sperm nucleus to form a viable zygote. When the egg of
chicken was inseminated with low dose of sperm and few
sperm penetrated the egg, the bird embryo was unlikely
to survive, but the embryo survived better when the egg
was penetrated with multiple sperms (Hemmings and
Birkhead 2015). It has been suggested that extra sperm
may somehow support cell cycles necessary for early
embryo development. It is an interesting topic for future
study to determine if the number of spermatia attached
to one trichogyne has something to do with the success
of fertilization or development of zygotes.
Nuclear division in the spermatium following its at-
tachment to the trichogyne is regarded as a common
feature of sexual reproduction in Ceramiales red algae
(e.g., Kim and Kim 1999a, Wilson et al. 2002). Why do
red algal spermatia enter mitosis before plasmogamy
while only one nucleus is needed to fuse with the female
nucleus? Picket-Heaps and West (1998) suggested that
two spermatial nuclei may be differentiated after the
mitosis because both nuclei from each spermatium en-
ter the trichogyne but only one was transported to the
base of carpogonium and fertilizes the carpogonial nu-
cleus. They reported that differentiation of the two nuclei
was usually immediately apparent from the direction in
which they move, which in all cases was in opposite di-
rections along the cytoplasm of the trichogyne. Whatever
transport systems operate within the trichogyne, sibling
nuclei appeared to engage them independently so that
their destination and fates are different (Picket-Heaps
and West 1998). Our results showed that it is not probable
and not even necessary that two spermatial nuclei from
a spermatium are differentiated. If each spermatium dis-
charges one nucleus which can be engaged with female
transport system, we should observe several male nuclei
move together to the carpogonial base when multiple
plasmogamy occurred in short intervals. We observed
that only the first nucleus that entered the trichogyne
showed rapid directional movement to the base of the
carpogonium. It was the only nucleus to proceed around
the vacuole-like organelles which fill the space in tricho-
gyne cytoplasm. The second nucleus as well as some nu-
clei from the other spermatia also initially showed direc-
tional movements toward the carpogonial base but were
trapped between these obstacles and stopped moving
DISCUSSION
The fertilization process was followed in close detail
in B. moritziana and showed features that have not been
reported before. Our results showed that both spermatial
nuclei could move towards the same direction but only
the first (primary) nucleus to enter the trichogyne was
transported rapidly and directionally to the base of car-
pogonium while the other nuclei including nuclei from
other spermatia stopped directional movement when the
first one entered the carpogonial base. This is contrary to
previous reports which suggest that two nuclei in each
spermatium are differentiated to move to opposite direc-
tions so that only one is capable of fertilization (Pickett-
Heaps and West 1998).
Our observation of multiple plasmogamy in tricho-
gynes, some even occurring after putative karyogamy,
suggests that polyspermy blocking works by another
mechanism in red algae different from other organisms.
There was no apparent change in the cell membrane
potential of a carpogonium after the gamete binding
and plasmogamy in B. moritziana. In many organisms,
including brown algae, polyspermy blocking is achieved
by an electric depolarization of the oocyte membrane
within seconds after gamete fusion (e.g., Brawley 1991).
It could be suggested that the non-motility of gametes of
red algae may have led to the loss of this electrical po-
larity block to polyspermy. Although red algal spermatia
have long appendages to increase effective reach and en-
hance gamete binding (Kugrens 1980, Fetter and Neushul
1981, Magruder 1984, Pueschel 1990, Broadwater et al.
1991, Kim and Fritz 1993a, 1993b), it may not be efficient
enough compared to flagella-based directional move-
ments which specifically bring both gametes together. As
fertilization channels between spermatia and trichogyne
are easy to collapse during the development the presence
of some back-up spermatia undergoing plasmogamy
may be helpful to ensure successful fertilization. Picket-
Heaps and West (1998) suggested that a collapse of the
trichogyne after fertilization may be an active means of
preventing polyspermy in B. moritziana. Our results par-
tially support this, we observed that there was a block,
possibly involving microfilament disruption, at the neck
between the trichogyne and carpogonial cell body, after
the primary nucleus entered the base. It is also possible
that the increase in vacuoles, and blocking of movement
within the trichogyne cytoplasm, after fertilization are a
secondary means of reducing the chance of polyspermy.
Multiple binding of spermatia may be needed for the
success of fertilization. Fertilization involving multiple
Shim et al. Dynamics of Spermatial Nuclei in Bostrychia moritziana
401 http://e-algae.org
advantage of existing water movement as they have non-
motile gametes. Spermatia only bind to female tricho-
gynes through a specific lectin-carbohydrate comple-
mentary system (Kim and Fritz 1993b, Kim et al. 1995,
Kim and Kim, 1999b, Han et al. 2012, Shim et al. 2012). To
compensate the inefficiency of fertilization resulted from
non-motility of gametes, red algae have a complicated
life cycle involving parasitic carposporophyte on female
gametophyte (Searles 1980). Multiple plasmogamy oc-
curring in the early stage of fertilization may be neces-
sary to prepare more back-up spermatia in case collapse
of fertilization channel before the carpogonial nucleus
get fertilized. If there is no highly selective transport sys-
tem, it will cause serious problem in the formation of vi-
able zygote.
Polyspermy is not the only issue. Our results also sug-
gest the mechanism of organelle inheritance in red al-
gae. In molecular studies investigating the inheritance
of organelles in B. moritziana, it appears that organelle
(plastids and mitochondria) inheritance is maternal
(Zuccarello et al. 1999a, 1999b). Our observations of the
movement of spermatial mitochondria suggests that they
do not move far from the fertilization channel, explain-
ing their maternal inheritance, as they never reach the
developing zygote. Following the fate of plastids in this
fertilization system would be worthwhile also pursuing.
We cannot tell whether the events we describe above
are common to sexual reproduction in other red algae.
Neither do we know what cytoskeletal motor elements
are involved in this rapid directional movement. Further
studies combining genome data with cytological obser-
vation may reveal the transportation machinery working
in red algal fertilization.
ACKNOWLEDGEMENTS
We thank Kongju National University internal grant for
the sabbatical leave of GHK. This research was supported
by Marine Biotechnology Program of the Korea Institute
of Marine Science and Technology Promotion (KIMST)
funded by the Ministry of Oceans and Fisheries (MOF)
(No. 20170431) and the National Research Foundation of
Korea (NRF) grant (No. 2019M3C1B7025093) to GHK.
SUPPLEMENTARY MATERIALS
Supplementary Video S1. Mitosis, plasmogamy and
movement of spermatial nuclei in trichogyne of Bostry-
when the first one entered the carpogonial base. This is
contrary to previous reports which suggest that two nu-
clei in each spermatium move to opposite directions; one
goes to carpogonial base and the other up to the tip of
trichogyne (Pickett-Heaps and West 1998).
A distinctive change in microfilaments was observed
in different stages of fertilization. Microfilaments bridge
between the cytoplasm of the two fused gametes, en-
closed male nucleus while it is transported in trichogyne
and degenerated at the neck of carpogonium after puta-
tive karyogamy. A transportation system based on actin-
myosin has been proposed for bringing a male nucleus
to a female nucleus (Kim and Kim 1999a, Wilson et al.
2002). To infer myosin activity in these studies, indirect
evidence using non-specific myosin inhibitors were
used. However, genome studies showed that there is no
myosin homologue in any reported red algal genomes,
which limits interpretation of cytoplasmic dynamics in
red algae as microfilament-mediated phenomena (e.g.,
Brawley et al. 2017). Accumulating genomic evidences
showed that red algae lack the complexity and diversity
of cytoskeletal elements present in other multicellular
lineages (Matsuzaki et al. 2004, Bhattacharya et al. 2013,
Collén et al. 2013, Nakamura et al. 2013, Schönknecht et
al. 2013, Brawley et al. 2017). It is probable that a novel
cytoskeletal protein fills the role of myosin during fertil-
ization because there is a transportation system work-
ing in bringing a male nucleus to carpogonial nucleus in
anyways.
Microtubules within spermatia in the early stage of red
algal fertilization were observed for the first time. Micro-
tubules were not observed anywhere else in gamete fu-
sion. There are few ultrastructural studies on visualizing
the cytoskeleton of red algae during fertilization as con-
ventional fixation methods cannot preserve structures
including trichogyne adequately (Picket-Heaps and West
1998). Broadwater and Scott (1982), using electron mi-
croscopy, described parallel microtubules along the long
axis of trichogyne, this is the only report of microtubules
in the trichogyne so far. Microtubule inhibition experi-
ments disrupted spermatial mitosis (Wilson et al. 2002)
but not nuclear transport inside trichogynes (Kim and
Kim 1999a, Wilson et al. 2002). Our results also showed an
extensive network of microtubules in spermatia during
mitosis. However, we did not observe any microtubules
in the trichogyne or the fertilization channels between
gametes. Further studies on the cytoskeletonin red algal
fertilization will reveal very noble genes and cytoskeletal
motor proteins involved in intracellular movement.
Red algae have evolved a fertilization system that takes
Algae 2020, 35(4): 389-404
https://doi.org/10.4490/algae.2020.35.12.7 402
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trichogyne; n1`, the second nucleus of the spermatium
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(https://e-algae.org).
Supplementary Video S2. Movement of male nucleus
in trichogyne. The first nucleus proceed around female
organelles (https://e-algae.org).
Supplementary Video S3. Movement of male organ-
elles in trichogyne (https://e-algae.org).
Supplementary Video S4. The changes occurring to
inclusions in the trichogyne cytoplasm after the first
male nucleus passed the narrow neck of carpogonium
(https://e-algae.org).
Supplementary Video S5. Trichogyne cytoplasm cut
off after the first male nucleus enter the carpogonial base
(https://e-algae.org).
Supplementary Video S6. Spermatial binding and mi-
tosis occurring on the trichogyne after the cytoplasm cut
off (https://e-algae.org).
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