CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
MlTOCHONDRI.AI. DIFFERENTIATION DURING SPERMATOGENESIS
1\
IN THE BROWN ALGA, FUCUS DISTTCHUS L.
A
thesis submitted in partial satisfaction
of the requirements for the degree of
Master of
Sci~nce
in Biology
by
Ronald Zachary Cassell
Ju.r1e 19 7 8
The Thesis of Ronald Zachary Cassell is approved:
California State University, Northridge
ACKNOWLEDGEMENTS
I happily acknowledge the patient assistance and
council rendered by Dr.· Edward G. Pollock, my major
professor.
His continued friendship and understanding
created the space for me to interact in the scientific
comllmnity as
11
colleague" as well as his student.
I
also
wish to express my sincere gratitude to Dr. Marvin H.
Cant.oT
J
\·lhose excel I ent comments on this vmrk as 1vell as
many hours of helpful discussions have contributed much
to my personal well being, thereby enabling me to avoid
many pitfalls and to reach some considerable heights!
Also, many thanks are due Dr. Steven B. Oppenheimer, whose
continued faith and moral support, not to mention excellent scientific comments, have always proven to be a
source of unending energy and strength.
Appreciation,
also, to Mr. .Jeff Brenneman for technical help, particu-
larly in the beginning, and for his friendship, both
invaluable commodities.
Lastly, I wish to thank my family, whose continued
faith and understanding brought this work to its final
happy conclusion.
iii
TABLE OF CONTENTS
List of Plates
Abstract .
.
.................
.. ................
INTRODUCTION
~~TERIALS
RESULTS . .
. .. . . .. .. .
-•
..
AND METHODS.
v
vii
1
5
...................
6
DISCUSSION
27
REFERENCES . . . • . . . . . .
35
iv
LIST OF PLATES
PLATE I
Figure 1.
Figure 2.
Figure 3.
Portion of a one-celled antheridium
with relatively pleomorphic mitochondria . . . . . . . • • . • • • • .
14
Young mitochondria from a fourcelled antheridium . . . . .
14
Mitochrondrion from an older antheridium than Figure 2 • . • . . . • . •
14
PLATE II
Figures 4-12.
Sections at various planes through
spheroidal mitochondria. . . . . •
16
Mitochondria of 64-celled antheridium . . . . . . . . . .
18
PLATE III
Figures 13-15.
Figure 16.
First appearance of intracristal
components. . . . . . . . . . • •
18
PLATE IV
Figure 17.
Mitochondria of near-mature sperm • . • 20
Figure 18.
Mitochondria of sperm within a
mature antheridium • • . • . . . • •• 20
PLATE V
Figure 19.
A pleomorphic mitochondrion from a
young antheridium. . • • • • • . • • . 22
LIST OF PLATES - continued
Figure 20.
Mitochondria-like body within
a young chloroplast of a 16-celled
antheridium • . . . . . . . . . • .
Figure 22a, b.
Two correlative sections through
the same mature sperm cell. • . . •
22
22
PLATE VI
Figure 23.
Figure 24.
Mitochondria of near-mature Fucus
--sperm
............
...
24
Mitochondria in fully mature,
swimming sperm.
. . . . . .. .... .
24
Thin section showing the association
of cristae with the outer compartment
at several places . . • • . • • . . • .
26
Cristae of near-mature mitochondria
with components • • • . • • . • . .
26
Branching of tubular cristae.
26
PLATE VII
Figure 25.
Figure 26.
Figure 27.
Figure 28a, b.
Two sections of the same
mitochondrion •
• . •
vi
26
ABSTRACT
MITOCHONDRIAL DIFFERENTIATION DURING SPERlviATOGENESIS
IN THE BROWN ALGA, FUCUS DISTICHUS L.
by
Ronald Zachary Cassell
Maiter of Science in Biology
Mitochondrial differentiation and development
within Fucus sperm is characterized by a series of internal
rearrangements and mass acquisitions relating to overall
form and organelle disposition within the mature sperm
cell.
Moreover, it seems clear that these changes are
e.ssential to the functional mode of these free swimming
cells in a hazardous environment.
structural changes are:
Among the more notable
increase in the number of cristae,
density changes of the matrix and cristae spaces, enhanced membrane staining, appearance and disappearance of
inclusions, cristae alignment and reorientation and, of
special interest, the formation of intracristal components
within all cristae of mature sperm.
The intracristal com-
uonent appears to signal functional capacity for these
cells, and its ubiquitous nature marks it f0r taxonomic
importance for this group of algae as well.
Collectively,
these changes represent multiple transformations of the
mitochondria in
_Fucu~
sperm cell development.
vii
INTRODUCTION
With some relatively rare exceptions, all groups of
animal and plant sperm contain mitochondria (49).
While
the details of sperm mitochondrial development differ
among species, most animal or plant sperm fall into two
general categories:
a) those which retain the normal
"somatic" or slightly altered mitochondrial morphology,
and b) those forming a "mitochondrial derivative" whereby
the typical morphology is lost (12),
This altered mor-
phology may be manifest in terms of loss of cristae, a
reorientation of cristae, or the appearance of paracrystalline depositions within the mitochondrial matrix.
Hence,
the mitochondria 1n their mature state may or may not resemble the original somatic configuration.
Nonflagellate
sperm and middle-piece formation in animals represent the
first type wherein the somatic or slightly altered mitochondrial configuration persists (12).
Neberikern
deriva~
tives of insects (36, 43) and crystalline formations of
snail sperm are indicative of the second type (39).
For the most part, variations in the structure of
plant sperm mitochondria closely parallel their animal
counterparts.
Relatively "undifferentiated" mitochondria
1
2
are present in the sperm of
~amia,
a Cycad (29).
Mito-
chondrial forms of intermediate differentiation are L,,;:nd.
in Pteridium, a fern (22), and Nitella, a Charophyte (45),
Also, a slightly altered mitochondrial morphology is found
in Marse:lia, a fern (27), and Equisetum, a pteridophyte
(10), and in the Charophyte, Chara (33), wherein either
several smaller mitochondria fuse to form a single mitochondrion or one small mitochondrion enlarges considerably.
Highly modified mitochondria are found in the
Liverwort, Sphaerocarpos (9) and "Nebenkern-like" d•>.rnrati ves are characteristic of the mosses, Bryum,
Funa!:'}~,
(42) and Polytrichum (31).
The mitochondria of Fucus sperm are among the most
highly differentiated of all the plant groups, while exhibiting characteristics somewhat intermediate between the
two extremes described for animals.
Moreover, Fucus mito-
chondriogenesis represents one of the rare instances among
all mitochondria types of the appearance of a "norm;; l"
intracristal inclusion.
Reports of "paracrystalline arrays" or inclusLms
within the mitochondrial matrix are numerous, while the
visualization of morphological entities within the intracristal space are rare (26).
Commonly, the intracristal
space is electron transparent and when, uncommonly, a
structural entity has been identified here, its presence
3
has been assigned an anomalous role.
Some of these struc-
tures arise through environmental stress put on the cells,
as in isolated mitochondria (7, 15, 19, 25, 40, 46), and
some are associated with certain pathologies (8, 17, 31,
41, 50).
Several papers have reported intracristal struc-
tures as normal constituents of healthy cells (3, 4, 19,
28, 38, 47).
The literature suggests a degree of uncer-
tainty in the interpretation of the origin of diverse intracristal morphologies, and this leaves quite unclear the
question of whether these structures are intrinsic components of the mitochondrion or extrinsically derived depositions seemingly not critical in respiratory functions.
Where an intracristal morphology appears as a persistent
structural entity, truly characteristic of the mitochondrion of a normal cell, it seems useful to suggest that
this entity may in some way reflect the total functional
capacity of the mitochondrion 1 rather than to consider it
as being anomalous or artifactual.
This thesis reports on fine structural changes in
sperm mitochondria of
;f_~:Icu~. ~ist~chus
L. during develop-·
ment, and on the nature of a persistent intracristal component of these mitochondria, which appears during terminal maturation.
norma~-
This structure is interpreted to be a
tubulo- filamentous entity and, as such, is one re-
flection of the functional state of the mitochondria of
4
these ~ells.
Changes in the general morphology of the
mitochondria, the extent and disposition of the cristae,
as well as special inclusions of the matrix, are also
described.
MATERIALS AND METHODS
Specimens of the monoecious brown alga Fucus
distichus L. were collected from Carmel Bay, California.
The collection and handling of plants for gametic discharge
was according to methods previously described (34).
Seg-
ments of fertile thallus tissue which included mature
conceptacles and freshly-released, free-swimming sperms,
were prepared for electron microscopy as follows.
After an initial washing of the materials with
sterile, filtered sea water, they were fixed in 4 percent
glutaraldehyde made up in cacodylate buffer at pH 7.0,
and with sucrose added to a final total concentration of
1.2 M.
Fixation was for 6-24 hours at 15° C.
The
materials were washed several times in cacodylate buffer
with decreasing concentrations of sucrose for about 2
hours.
The final wash consisted of 0.05 M buffer with
0.025 percent
~f
2 percent OsC
temperature.
0.1 N HCl added.
Post-fixation was with
in cacodylate buffer at pH 7.0 at room
:erial dehydration was with acetone and em-
bedding was in Epon-Araldite according to standard procedures (24, 34).
Sections were cut on an LKB ultramicro-
tome III and stained routinely .. The preparations were
examined with a Zeiss EM-9S2 electron microscope at 60 Kev.
5
".
RESULTS
Fucus sperm are produced within conceptacles of fertile thalli.
Antheridia arise £rom the germinal layer of
the conceptacle wall, and it is within this layer that
sperm arise.
Each sperm is one of 64 final products de-
rived from a primary nucleus.
The initial cell undergoes
meiosis, and then four subsequent mitoses (48).
While the
antheridia of a given conceptacle are not synchronous in
development, all of the sperm nuclei of a given antheridium
are.
As the sperm mature, the mitochondria, as well as
other sperm organelles pass through a distinct morphological sequence.
Discreet stages of organelle morpho-
genesis are related to discreet steps in overall spermatogenesis.
Consequently, following the phase of rapid
proliferation, i.e., once 64 spermatids have been formed,
it is convenient to refer to early, middle,
~l~t.!:!.ri~
lat~,
and
stages of sperm differentiation.
Few mitochondria are present in the initial antheredial cell prior to meiosis.
These are
dist~ibuted
random-
ly in the cytoplasm, are small, highly pleomorphic, and
have relatively few cristae (Figures 1, 19).
6
At this
7
stage the matrix is generally more electron dense than
the intracristal spaces, although some regions of the
matrix are almost electron transparent.
Some cristae
membranes are continuous with the inner mitochondrial
membrane so that the intracristal spaces are continuous
with the outer compartment.
Prior to
rap~d
proliferation, but when meiosis is
complete, the mitochondria have elongated to some extent
and the cristae are more numerous.
The matrix stains more
intensely than in earlier stages, and appears somewhat
granular in nature.
Osmiophilic bodies also appear for
the first time in the matrix (Figure 2).
During the first
few mitotic divisions, the mitochondria also proliferate,
possibly by way of division or possibly by an alternative
mechanism which may involve the chloroplast.
Double mem-
brane, "mitochondrial-like bodies", having cristae, have
been obseived within the stroma of young chloroplasts
(Figure 20).
These forms are fairly common during the
first three mitotic divisions, but are not seen 1n later
stages of development.
By the end of the third mitosis
presumptive sperm cells may be recognized.
And while
karyokinesis is normal, cytokinesis is not complete, so
that cytoplasmic bridges are retained between the developing sperm until the late spermatid stage.
The extracellu-
lar matrix becomes more apparent and may be seen to
8
contain small membrane bound vesicles of varied size and
of possible Golgi origin (Figure 12).
During these later
stages of active mitosis, the mitochondria are spheroidal
and appear "donut-shaped" in thin sections.
However,
sections through several mitochondria strongly suggest
they are really hollow sphere.s formed by way of an invagination of the outer mitochondrial wall (Figures 3, 4, 11).
This results in the confinement of cytoplasm within the
mitochondrial involution, except for one rather small
opening.
The confined cytoplasm is strikingly different
from the cell cytoplasm being less electron dense; due
possibly to smaller numbers of what appear to be polyribosomes (Figures 11, 12).
With the conclusion of pro-
liferation (i.e., the beginning of the early stage), the
mitochondria, as well as the other sperm organelles, come
to lie adjacent to individual nuclei.
As the sperm cell
begins to elongate, the "donut-shaped" feature of the
mitochondria is no longer apparent; although for a short
time they still appear to be spheroidal (Figures 13, 15).
As cell elongation proceeds, figures are observed within
the matrix.
They are 120
Xin
diameter and are thus com-
parable to those observed in the cells of the snake,
plaphe (47).
However, they are of a transient nature and
of uncertain function (Figure 21).
The sperm cytoplasm
is reduced in volume and a portion of the smooth
9
endoplasmic reticulum appears closely appressed to the
mitochondria just inside the outer cell membrane
(Figure 16).
Mi9. . -s_!:.age spermatids are characterized by 64 nuclei
connected by thinner cytoplasmic bridges.
Mitochondria of
these spermatids clearly show an inner membrane which is
more electron dense than the other mitochondrial membranes
(Figures 13, 15).
This difference in membrane staining
actually is first noticeable in the very young mitochondria
of single nucleate antherida.
now enhanced.
However, this feature is
The cristae have become longer 7 show an ex-
tensive degree of twisting, and, when cut in cross-section 7
are clearly less electron dense than the matrix.
The
matrix contains numerous, large, osmiophilic bodies as
well as smaller, medium, electron-dense granules which
later appear within the intracristal spaces (Figure 14).
This material seems to coalesce subsequently and may possibly contribute to the first appearance of the intracrista! component (Figure 16).
Cristae which appear to be
continuous with the inner mitochondrial membrane are much
rarer than in the phase of proliferation.
Those cristae
which can be shown to associate with the inner mitochondrial membrane do so by way of pedicels (Figure 15).
The late and maturing stages of spermatid development
are marked by elongate mitochondria which come to lie
10
parallel to the long axis of, and within a groove beneath,
the nucleus.
The mature sperm usually have three or four
mitochondria visible in a given median--longitudinal section, although up to eight smaller ones have been observed.
These have normal fine structural architecture, i.e.,
an outer membrane (SO ~) separated from an inner membrane
(60
i)
by an outer compartment (40
XJ.
The inner membrane
evaginates into numerous tubular cristae of relatively
even diameter.
In addition, the cristae of mature and
swimming sperm come to be arranged in a manner which is,
more or less, parallel to the long axis of the mitochondrion (Figures 17, 18, 24).
The majority of the cristae
lumina do not show continuity with the outer compartment
in thin sections.
They appear to be associated with the
inner membrane by way of pedicules so that many cristae
appear to end abruptly at the inner membrane (Figure 25).
Occasionally cristae branch, but the lumina of the branches
appear to be cut off from the lumen of the main crista
(Figure 27).
While the mitchondrial matrix is quite
electron dense, the cristae are of lesser density and the
outer compartment is electron transparent.
The cristae of well-differentiated sperms are further
characterized by a tubule-filamentous component which runs
the full length of the cristae and measure 110
meter.
R in
dia-
Since the inside diameter of the cristae is about
ll
535
X,
the component appears to be free to orient randomly
within the intracristal lumen.
From 1 to 7 filaments can
be seen within the lumina of cross-sectioned cristae
(Figures 27, 28).
In single longitudinal sections, fre-
quently only 1 or 2 axial filamentous components are seen
(Figures 23, 24).
(Figure 28).
Rarely, however, up to 4 may be seen
In some sections, the component appears to
dichotomize within individual cristae.
This is not true
branching, but rather the crossing over of filaments in
and out of the plane of section.
Crossed-over filaments
are always of equal diameter throughout their entire
length (Figure 23).
The intracristal component, which usually runs along
the central axis of the crista lumen, ends abruptly at the
inner membrane in cristae lumina closed to the outer compartment.
Some cristae lumina, however, appear to be
somewhat open to the outer compartment, and here also the
the component appears to end abruptly at the proximal
stalk (Figure 25).
While it does not enter the outer com-
partment, at high magnification there is a suggestion that
it may associate rather closely with one or several points
of the inner face of the crista! membrane within the
proximal region of the crista (Figures 25, insert, and 26).
No comparable close association of the component with the
inner crista! membranes has been seen in other regions of
the cristae.
There is the suggestion of a cross-band
appearance or periodicity along the proximal axis of the
component with a center to center spacing of about 80 ~Each period appears to be the locus of a cross structure
of medium electron density, running at right angles to
the long axis of the component, but these are rather
vague (Figure 25, insert).
Preliminary high resolution
studies using a Zeiss EM-10 at 80 Kev. and tilting techniques thus far have failed to add substantially to these
observations.
This work is continuing.
In branching
cristae, there does not appear to be any continuity between
intracristal components (Figure 27).
An interesting feature which may relate to the mainterrance of shape and position of the mitochondria in
mature sperm has come to light in a rather fortuitous
manner.
Two sections from a correlative series demonstrate
"tight" connections between the nuclear and mitochondrial
membranes.
These were enhanced by slight cell shrinkage
due to fixation of the mature sperm in some early samples
(Figures 22a, 22b).
13
PLATE I
Figure 1.
Portion of a one-celled antheridium with rela-
tively pleomorphic mitochondria (M) with few digital
cristae.
nucleus;
Figure 2.
C. young chloroplast; G, golgi complex; N,
(+) smooth endoplasmic reticulum.
X26,000.
Young mitochondria from a four-celled antheri-
dium showing medium dense matrix and rather transparent
intracristal spaces.
Most of the intracristal spaces
are continuous with the outer compartment in young mitochondria (arrows).
Figure 3.
XSO,OOO.
Mitochondrion from an older antheridium than
Figure 2, with an invagination of the surrounding membranes
(arrow).
Note the enhanced density of the matrix and in-
creased extent of the twisted cristae.
X82,000.
c;
.:. ~~ '~·_?.
15
PLATE II
Figures 4-12.
Sections at various planes through
spheroidal mitochrondria characteristic of 16- and 32celled antheridia.
The mitochondria have deep, well-
expanded involutions with one narrow opening to the ·cell
cytoplasm at the surface (large arrow).
Section plane
tangential to, but just outside, the involution (small
arrow)
(Figure 4).
Section plane barely passes through
the perimeter of the involution (small arrow)
(Figure 5).
Section plane passes progYessively deeper into the involution (small arrow)
(Fiiures 6-10).
Note the difference
between the cytoplasm within the involution (EC) and that
without in Figures 9-12.
EM, extracellular matrix.
Figures 4-10, X29,000; Figure 11, X45,000; Figure 12,
XS4,000.
17
PLATE III
Figures 13-15.
Mitochondria of 64-celled antheridium mid-
way to terminal differentiation.
Note the denser staining
of the inner mitochondrial membrane (large arrow) as compared with the outer membrane and cristae membranes.
Dense, osmiophilic bodies appear within the matrix (small
arrows) (Figure 13).
Smaller osmiophilic granules within
the cristae (arrows) (Figure 14).
A pedicle-type attach-
ment (small arrow) of cristae to the inner membrane and
coalescence of intracristal material (large arrow) (Figure
15).
X83,000.
Figure 16.
First appearance of intracristal components
(arrows) within the mitochondria of a 64-celled antheridium, but later in development than Figures 13-15.
Some
smooth endoplasmic reticulum is closely appressed to the
mitochondrial periphery (large arrow).
X55,000.
C, chloroplast.
19
PLATE IV
Figure 17.
Mitochondria of near-mature sperm.
Digital
cristae arranged both at right angles and more or less
parallel to the long axes of the mitochondria.
At this
stage all cristae contain tubulo-filamentous components
(IC).
C, chloroplast; F, flagellum;
IC, intracristal
component.
X80,000.
Figure 18.
Mitochondria of sperm within a mature antheri-
dium, but not released as yet from the thallus.
Most of
the cristae with intracristal components are aligned
parallel to the long axes of the mitochondria.
nucleus.
XSO,OOO.
N,
21
PLATE V
Figure 19.
A pleomorphic mitochondrion from a young
antheridium (enlarged from Figure 1).
(arrows) along a
narro~
Osmiophilic bodies
constriction of the mitochondrion.
XS4,000.
Figure 20.
Mitochondria-like body (arrow) within a young
chloroplast of a 16-celled antheridium.
A mitochondrion
within the cytoplasm provides a basis for comparison and
reveals many similarities with this inclusion.
M, mitochondrion.
F.iguTe 21.
G, golgi;
X40,000.
0
Myelin-like figures 120 A in diameter (arrow)
within mitochondria of a 64-celled antheridium.
Figure 22a,b.
X42,000.
Two correlative sections through the same
mature sperm cell showing a tight junction-like association between the mitochondrial membranes and the nuclear
membranes (arrows).
The firmness of this association
appears to be enhanced due to shrinkage of cell components
during fixation.
X30,000.
23
PLATE VI
Figure 23.
Mitochondria of near-mature Fucus sperm.
The
intracristal component is characterized as 1 to 5 cylindrical strands in cross-sectioned tubular cristae (arrows).
In longitudinal sections of cristae the component usually
appears as 1 or 2 strands (D).
ponents is common (Y).
nucleus.
Figure 24.
Crossing over of 2 com-
Ma, mitochondrial matrix; N,
X99,000.
Mitochondria in fully mature, swimming sperm.
The cristae are oriented more or less parallel to the long
axis of the mitochondrion and reveal 1 or 2 strands of
the intracristal component (IC) per crista.
X84,000.
N, nucleus.
.
'
-
Q.1U
PLATE VII
Figure 25.
Thin section showing the association of
cristae with the outer compartment (OC) at several places.
The association of the intracristal component (IC) by
faint cross strands (F} to the inner faces of the cristae
membranes appears to be tenuous at best, even though the
cristae lumina may be continuous with the outer compartment (insert).
Other cristae lumina appear to end
abruptly at the inner membrane (large arrows).
Mt,
microtubules; N, nucleus, X99,000; insert, Xl42,000.
Figure 26.
Cristae of near-mature mitochondria with com-
ponents and definitely open to the outer compartment (OC).
Xll3,000.
Figure 27.
Branching (B) of tubular cristae; but in this
case no apparent branching continuity of the intracristal
component.
X99,000.
Figure 28a,b.
Two sections of the same mitochondrion.
(a) Seven tubule-filamentous components in cross section
(arrow).
(b) About four components in longi-section
(arrow).
X79,000.
DISCUSSION
There are four mitotic divisions following meiosis
to form 64 sperm cells.
Consequently, the total number
of mitochondria in maturing antheridia must increase well
above that present in the original cell.
Apparently, this
is accomplished by way of mitochondrial division, and the
evidence is good that this manner of mitochondrial production occurs in several other systems (20, 23).
Although
the mitochondrial population increases overall throughout
the phase of rapid proliferation;, the number of mitochondria per cell remains relatively constant.
Throughout this
phase and the ensuing phases of differentiation and maturation, the mitochondria undergo a series of morphological
changes as well as associations with other cellular components.
In fact, the unique association of _!lucus sperm
mitochondria with the developing chloroplast suggests an
alternative mechanism for mitochondriogenesis.
We have
observed organelle associations which may be interpreted
as a kind of budding phenomenon of the chloroplast which
gives rise to mitochondria (21).
Fine structural details
of this relationship are reported elsewhere (6).
2 ?i
28
Although definitive histochemical work is required to
verify this interpretation, intrinsic mitochondrial changes
include the increase in the number of cristae, density
changes of the matrix and cristae
spaces~
alterations in
membrane staining, changes in the relationship of the
intracristal space with the outer compartment, appearance
of inclusions, and cristae alignment or reorientation
within the mitochondria.
Collectively, these changes rep-
resent multiple transformations of the mitochondria in
· Fuc~_:~. sperm cell development.
Pleomorphic mitochondria were first reported for
sperm cells by Andre (1, 2).
Such
mitochon~ria
to be a rather common finding in animal
sp~
have been reported for other cell types as
now appear
cells and
~ell
(26).
However, no data are available which relate mitochondrial
pleomorphisms to a known function.
On the other hand, in
contrast to the more typical, cigar-shaped mitochondria,
pleomorphic mitochondria have an increased surface area.
Also, the enclosed cytoplasm is in a more confined relationship with the surface mitochondrial membrane.
This
condition may facilitate the exchange of metabolites between the enclosed cytoplasm and the mitochondrion.
!:_ucu~,
In
the enclosed cytoplasm contains polyribosomes and
is quite different than the outer cell cytoplasm in overall staining.
Since pleomorphic mitochondria are
29
characteristic of the phase of cellular proliferation,
where there is a concurrent increase in the overall mitechondrial population, any condition which enhances mitechondrial protein synthesis would be desirable.
Maturation and refinement of structure appears to be
the main feature of the mitochondria during the postmitotic phase of cellular differentiation.
Increase in
matrix density and the remarkably higher staining intensity of the inner mitochondrial membrane versus the outer
membrane relates well to the enhanced protein content of
these regions (30).
Appearance of the intracristal component seems to be
related to a number of smaller granules which are first
~pparent
during the mid-phase of differentiation and then
appear to coalese; overlapping with the first appearances
of the intracristal component.
Since this component be-
comes a permanent structure of from 1 to 7 tubular filarnents in all of the cristae, one can be suspicious that
its presence relates to the functional requirements of
these cell-s.
An intracristal component which comprises
the normal architecture of cell mitochondria is rather
uncommon.
However, it now appears that this structure is
a uniquely consistent feature of the reproductive cells
of brown algae (5, 11).
0
'
30
The intracristal component reported here is determined to be tubulofilamentous in form.
This conclusion
is based primarily upon its appearance in thin sections
of tubular cristae where two or more such filaments coexist within a single crista.
Moreover, the filaments
cross over randomly showing that they are somewhat independent of each other.
There are few reports on the intracristal components
for normal mitochondria of healthy cells, and perhaps one
or two at best are comparable in some way to the component
reported here.
A component of the mitochondria of sperma-
tozoa of the ostracod,
one in Fucus.
Cyprid~psis,
looks similar to the
There is only 6ne filament per tubular
crista and it occupies the axial plane of the crista!
lumen.
It is a dense filament about one-half the width
(40--60 ~) of the Fucus
- - component and the authors did not
elaborate on its nature or significance (38).
A reticu-
lum of narrow tubules about 120 ~ in diameter has been
reported within the intracristal space of the mitochondria
of the snake, Elaphe, but these are not comparable in
appearance or arrangement (47).
Several other filaments
and tubes have been reported as mitochondri\].1 inclusions
of normal cells, but these are larger or smaller than the
one reported here, have dissimilar arrangements, and most
are found within the mitochondria of harshly treated cells
(19, 26).
Recently, from 1 to 3 intracristal components
were reported for the heterothallic, Fucus yesiculosus,
although their structure and disposition were not characterized (5).
However, they are presumed to be comparable
to those described here for the homothallic, Fucus
distichus.
Attempts made to describe the origin of these
inclusions or to discern their function are frustrated by
the absence of chemical data.
The two more promising interpretations concerning the
site of origin of intracristal componetits to date are (a)
that they represent aberrations of the outer leaflet of the
cristae membranes, such as the cqming together of two membrane leaflets within the crista lumen, or (b) that they
are real, non-membranous depositions (perhaps enzymes)
within the intracristal space; e.g, paracrystalline arrays,
lamellar arrays, etc.
(19).
These two views are not mutu-
ally exclusive and both may invoke only intrinsic structures of the mitochondria.
In all probability, the intracristal component reported here is partially formed from intrinsic mitochond~ial
structures plus other depositions which are either
intrinsic to the mitochondrion or of extramitochondrial
origin, or both.
Other types of intracristal morphologies
have been defined by invoking only intrinsic mitochondrial
structures, but in these systems the intracristal membranes
32
are in close basepiece-to-basepiece apposition (19, 26).
The basepieces of the intracristal membranes of Fucus
0
sperm mitochondria are at least 500 A apart throughout
the cristae.
Hence, it seems reasonable to assume that
other depositions contribute to the final nature of the
intracristal component in Fucus.
Many investigators have postulated the chemical nature of mitochondrial inclusions, e.g., interference with
basic enzyme activity or changes in phospholipid content.
All such explanations account for these inclusions as
aberrancies in mitochondrial function (44).
Although it
is not possible to gain insight about their function without histochemical data, the observations reported here
leads to the conclusion that the intracristal component of
Fucus is at least a normal, rather than a fortuitous or
anomalous structure.
Tubular cristae of Fucus sperm mitochondria are very
much akin to those of protozoans rather than to the mitochondria of plants.
Perhaps this feature relates well to
the independent mode of activity of these cells.
Evidence
has accumulated which suggests a correlation between the
environment wherein fertilization occurs, the general
morphological features of the mature sperm, and the final
morphology of these mitochondria (12, 13, 14).
The cristae are long, somewhat twisted, and wellexpanded in mature, swimming sperms of Fuc:us (18, 26, 32).
Moreover, the intracristal component appears to be a normal feature of the cristae, being present in all cristae
of the mitochondria of mature cells observed in thin section.
Since this
~omponent
is not present in younger,
less-differentiated sperms, its presence in older cells
correlates well with the functional requirements of freeswimming cells.
Its apparent absence in mitochondria of
thallus cells, the germinal layer, oogonia, and the
sterile paraphases further supports this contention.
More-
over, it now appears that intracristal components of sperm
mitochondria are possibly a consistent structural feature
of the brown algae.
The significance of other types of
inclusions which appear more sporadically in these cells
is not clear.
Cristae orientation within the mature Fucus sperm is
consistently parallel to the long axis of the mitochondria.
This structural shift from random orientation of less
mature cells to order in mature cells is unique for Fucus
sperm, but has also been reported for the "starved" mitochondria of the proximal tubule cells of the nephron of
the frog, Rana pipiens, for many neurons and motor end
plates, for parafollicular cells of rat thyroid, and for
cells of the ovatestis of Helix aspersa (26).
It has been
34
suggested that this type of cristae orientation correlates
with loss of cytochrome oxidase activity in some systems
(16).
However, its significance in Fucus sperm is not
clear.
With the tight packing and specific positioning of
the organelles in mature sperm, it becomes clear that the
membranes of the various organelles are very cl?sely
associated.
The "tight-junction-like" contact between
the outer mitochondrial membrane and the nuclear envelope
is the best example of this.
Consequently, organelle
dispositions and organelle-organelle communications are
likely maintained in this manner, and may be necessary for
sperm function.
It may be concluded that Fucus sperm mitochondria are
highly differentiated organelles which are more closely
akin to the mitochondria of flagellate protozoans than to
plants.
Moreover, they are among the most highly differ-
entiated mitochondria present in all plant sperm cells.
Lastly, these features, among others, suggest that
sperm cells are structurally
s~ited
Fti~tis
to the functional
demands of an external, oogamous fertilization scheme in
a marine environment.
REFERENCES
1.
Andre J.
1958. Decouverte chez le scorpion
Euscorpius flavicaudis d'une nouvelle ultrastructure mitochondriale.
C. r. hebd. Seanc.
Acad. Sci. Paris 247:1232-1235.
·
2.
Andre J.
1959. ftude au microscope electronique
de !'evolution du chondriom pendant la spermatogen~se du scorpion Euscorpius flavicaudis.
J.
Ultrastruc. Res. 2:288-308.
3.
Andre J.
1962. Contribution § la connaissance du
condriome. ftude de ses modifications ultrastructurales pendant la spermatogen~se. J.
Ultrastruct. Res., Suppl. 3:1-185.
4.
Braak, H.
1967. Elektronenmikroakopische untersuchungen an catecholarninkernen im hypothalamus vom
Goldfisch (Carassius auratus).
Z. Zellforsch.
Mikrosk. Anat. 83:398-415.
5.
Brawley, S. H., R. Wetherbee, and R. Quatrano.
1976.
Fine-structural studies of the gametes and embryo of Fucus vesiculosus L. J. Cell Science
20:233-254.
6.
Cassell, R. Z. and Pollock, E. G. (in preparation).
Chloroplast and mitochondrial relationships in
Fucus during spermatogenesis.
7.
Cheah, K. S., A.M. Cheah, and C. A. Voyle.
1973.
Paracrystalline arrays in mitochondria following
aging of mitochondria in situ. J. Bioenergetics
4:383-389.
- --
8.
Chou, S. M.
1969. "Megaconial" mitochondria observed
in a case of chronic polymyositis. Acta Neuropath. (Berlin) 12:68-89.
9.
Diers, L.
1967. Der feinbau des spermatozoids von
Sphaerocarpos donnellii Aust. (Hepaticae).
Planta. (BerTin) 72:119-145.
35
10.
Duckett, J. G. 1973. An ultrastructural study of
the differentiation of the spermatozriid of
Equisetum. J. Cell Science 12:95-129.
11.
Evans, L. V. 1966. Distribution of pyrenoids among
some brown algae. J. Cell Science 1:449-454.
12.
Favard, P. and J. Andr§. 1970. The mitochondria
of spermatozoa.
In Comparative Spermatology
(B. Bacetti, ed.), Academic Press, New York.
pp. 415-4 2 9.
13.
Fawcett, D. W. 1970.
ultrastructure.
127.
A comparative view of sperm
Reproduct. Suppl. 2:90-
Bio~.
14.,
Franzen, A. 1970. Phylogenetic aspects of the
morphology of spermatozoa and spermiogenesis.·
In Comparative Spermatology (B. Bacetti, ed.),
Academic Press, New York. pp. 29-46.
15.
Hall, J. D. and F. L. Crane. 1970. An intracristal
structure in beef heart mitochondria. Exptl.
Cell Res. 62:480-483.
16.
Karnovsky, M. J. 1962. Mitochondrial changes and
cytochrome oxidase in the frog nephron. Proc.
5th Int: Congr. Electron Microscopy 2, Q9.
17.
Ketelsen, U. P., H. Berger, and E. Freund-Molbert.
1968. Feinstrukturelle befunde bei der progressiuen okul~ren muskeldystrophie unter besonderer berilck sichtigung der mitochrondrien~
ver~nderungen.
Beitr. Path. Anat. 138:223-242.
18.
Korman, E. F., A. D. F. Addink, T. Wakabayashi, and
D. E. Green. 1970. A unified model of mito.chondrial morphology. J. Bioenergetics 1:9-32.
19.
Korman, E. F., R. A. Harris, C. H. Williams, T.
Wakabayashi, D. E. Green, and E. Valdivia.
1970. Paracrystalline arrays in mitochondria.
J. Bioenergetics 1:387-404.
20.
Luck, D. J. L. 1965. Formation of mitochondria in
:Neurospora crass a. J. Cell !3-~~_logy 24:461-4 70.
21.
1\faltzahn, K. von, and K. Muhlethaler. 1962. Observations on the division of mitochondria in
37
dedifferentiating cells of Splachnum ampullaceum.
Experientia 18:315-316.
22.
Manton. L.
1959. Observations on the microanatomy
of the spermatozoid of the braken fern
(Pteridium aquilinum). J. Biophysic and Biochem. Cytol. 6(3):413-418.
23.
Manton, I. 1961. Some problems of mitochrondrial
growth.
J. Exp. Bot. 12:421-429.
24.
Mollenhauer, H. H. 1964. Plastic embedding mixtures
for use in electron microscopy.
Staip Technology
39:111-114.
25.
Morton, D. J., R. W. D. Rowe, and J. J. Macfarlane.
1973. The formation of intracristal structures
induced in skeletal muscle mitochrondria by
high pressure. J. Bioene~getics 4:445-453.
26.
Munn, E. A. 1974. The Structure of Mitochrondri&
Academic Press,-New York.
27.
Myles, D. G. and P. R. Bell. 1975. An ultrastructural study of the spermatozoid of the fern,
Marselia vestita.
J. Cell Science 17:633-645.
28.
Newcomb, E. H., M. W. Steer, P. K. Hepler, and W. P.
Wergin. 1968. An atypical crista resembling
a "tight junction" in bean root mitochrondria.
J. Cell Biology 39:35-42.
29.
Norstog, K.
1967. Fine structure of the spermatozoid
of Zamia with special reference to the flagellar
apparatus. Am. J. Bot. 54(7):831-840.
30.
Opik, H.
1974. Mitochondria.
In Dynamic Aspect~
of Plant Ultrastructure (A. W. Rubards, ec.t.),
McGraw Hill (U.K.), pp. 52-83.
31.
Paolillo, D. J., Jr., G. L. Kreitner, and J. A.
Reighard. 1968a.
Spermatogenesis in Polytrichum juniperinum.
I.
The origin of the apical
body and the elongation of the nucleus. Planta
(Berlin) 78:226-247.
38
32.
Penniston, J. T., R. A. Harris, J. Asai, and D. E.
Green. 1968. The conformational basis of
energy transformations in membrane systems. I.
Conformational changes in mitochondria. Proc.
Natl. Acad. Sci. USA 59:624-631.
33.
Pickett-Heaps, J. D. 1968. Ultrastructure and
differentiation in Chara (Fibrosa). IV.
Spermatogenesis. Aust. J. Bioi. Sci. 21:655690.
34.
Pollock, E. G. 1970. Fertilization in Fucus.
Planta (Berlin) 92:85-99.
35.
Pollock, E. G. and R. Z. Cassell. An Intracristal
Component of Fucus Sperm Mitochondria. J.
UltrastrucL R~-58 (2): 172-177.
36.
Pratt, S. A. 1970. Formation and differentiation
of the Nebenkern in spermatids of an Hempteran
insect, Murgantia histronica. In Comparative
Spermatology (B. Bacetti, ed.), Academic Press,
New York. pp. 301-310~
37.
Price, H. M. 1967. In Exploratory Concepts in
Muscular Dystrophy and Related Disorders--TA. T.
fVfifnarot, ed.), Excerpta Medica Foundation, New
York. pp. 341-350.
38.
Reger, J. F. and N. T. Florendo. 1969. Studies on
motile, non-tubule-containing filiform spermatozoa of the Ostracod, Cypridopsis sp. II.
Mature spermatozoa. J. Ultrastruct. Res. 28:
250-258.
39.
Ritter, C. and J. Andr~. 1975. Presence of a complete set of cytochromes despite the absence of
cristae in the mitochondrial derivative of snail
sperm. Experimental Cell Res. 92:95-101.
40.
Saito, A., M. Smigel, and S. Fleischer. 1974. Membrane junctions in the intermembrane space of
mitochondria from mammalian tissues. J. Cell
Bioi. 60:653-663.
41.
Shy, G. M., N. K. Gonatas, and M. Perez. 1966. Two
childhood myopathies with abnormal mitochondria.
I. Megaconial myopathy. II. Pleconial myopathy. Brain 89:133-158.
,..
3 :J
42.
39
Sun, C. N.
1964. Fine structure of the spermatozoid
of the mosses, with special reference to the
so-called "Nebenkern." Protoplasma 58:663-666.
43.
Tokuyasu, K. T. 1975. Dynamics of spermiogenesis
in Drosophila ~~lanogaste~. VI.
Significance
of "Onlon" Nebenkern formation.
.J. Ultrastruct.
Res. 53:93-112.
44.
Tuchweber, B., K. Kovacs, J. D. Khandekar, and B.
1972.
Intramitochondrial lamellar
D. Garg.
formations induced by Pregnenolone-16-aCarbonitrile in the hepatocytes of pregnant
rats. U. Ultrastruct. Res. 39:456-464.
45.
Turner, F. R. 1968. An ultrastructural study of
plant spermatogenesis.
Spermatogenesis in
Nitella. J. Cell Biol. 37:370-393.
46.
Wakabayashi, T., J. M. Smoly, 0. Hatase, and D. E.
Green.
1971. A lattice structure in beef
heart mitochondria induced by phosphotungstic
acid. J. Bioenergetics 2:167-182.
47.
Yamamoto, T., T. Ebe, and S. Kobayashi. 1969.
Intra.mitochondrial inclusions in various cells
of a snake (Elaphe quadrivirgata).
Z. Zellforsch. Mikrosk. Anat. 99:252-262.
48.
Yamanouchi, S. 1909.
47(3):173-197.
49.
Yasuzumi, G. 1974. Electron microscope studies on
spermogenesis in various animal species.
International Review of Cytology 37:53-119.
50.
Zintz, R.
Mitosis in Fucus. Bot. Gaz.
1966. Dystrophische ver~nderungen in
augenmuskeln und schuttermuskeln bei
der sog. progressiven graefeschen ophthalrnophegie.
In Progres s.i ve Muskeldystrophie
Myotonie-Myasthenie (E. Kuhn, ed.), SpringerVerlag~ Berlln.
pp. 109-114.
~ussern