BIOLOGY
OF
7,
REPRODUCTION
Histochemical
and
W.
H.
Department
145-159
(1972)
and
Ultracytochemical
Sperm
of Ascaris
CLARK,
of Biology,
R.
JR.,
U,,iversity
University
Received
L.
of
of
Iumbricoides
MORETTI,
Houston,
of california,
March
Studies
the
var.
W.
AND
Spermatids
suum1
W.
THOMSON
Houston,
Texas
and Department
Riverside,
California
30, 1971; accepted
August
of Life
Sciences,
24, 1971
Considerable
confusion
has existed concerning
the nature and function
of the refringent
cone and a series of vesicles found between the cone and the plasma membrane
in the sperm
of Ascaris
sp. (Nematoda).
In the present study histochemical
and ultracytochemical
techniques, at both the light and electron microscopic
levels, have been used in order to characterize these cellular components
in the spermatids
and spermatozoa
of Ascaris
lumbricoides
var.
suum
(A.
suum).
It has been demonstrated
that the vesicles, believed to be of Golgi origin, fuse with the
plasmalemma
of the mature sperm and undergo dehiscence
releasing their contents
into the
lumen of the female genital tract. These vesicles exhibit a positive PAS reaction at both the
light and electron microscope
levels. They further exhibit a positive reaction
for acid phosphatase. The refringent
cones do not exhibit a positive
reaction
for acid phosphatase
and
only show small loci of reaction product for the PAS test. The refringent
cones do exhibit an
orange fluorescence,
which is indicative
of RNA,
when the cells are stained with acridine
orange. However,
this fluorescence
is found in cells treated with ribonuclease;
but, is not
present in cells treated with trypsin.
While the refringent
cone resisted enzymatic
digestion
with ribonuclease,
electron dense spheres found associated
with the nucleus were partially
extracted.
The results of this study suggest that the vesicles (proacrosomal
vesicles) may be
acrosomal
in nature but lend no support to the thesis that the refringent
cone is the acrosome. This work further suggests that the sperm of A. simm contributes
RNA to an egg at
fertilization.
During
spermatogenesis
Ascaridoidea,
formed
in the
to coalesce
form
refringent
cytoplasm.
in the
the
large
mature
the
in members
large
These
spermatid
refringent
cone
spermatozoon.
greater
part
bodies
and
begin
cone
conical
nized
in
of the
1934;
Collier,
1956;
Nath,
Gupta
reasons
for
this
1937;
and
Nath
are
the
work
1
Science
morphology
of
was supported
Grant,
Foundation
The
two-fold.
in part
cence
© 1972 by
of
reproduction
The
Society
in any
acrosomes
Franklin,
by a National
origin
GB 4513.
for
form
the Study
reserved.
of
Reproduction
cones
as
and
the
nucleus.
bodies
first
Julin
in 1884,
recog-
were
1925;
and
1958) as are the prothe typical
flagellate
exhibited
demonstrated
in spermatozoa
(Colwin
and
145
Copyright
All rights
Beneden
the
to be of Golgi
origin
(Bowen,
1934;
Collier,
1937;
Nath
refringent
refringent
cone
superficially
the
refringent
the
although
to
sperm.
Other
investigators
have
not considered
the refringent
cone
analogous
to the acrosome
of flagellate
sperm.
For example,
the
Singh,
1961).
assumption
First,
the morphology
of the
of the mature
spermatozoon
resembles
and
Kochiar,
posterior
Singh,
1956; Hovasse,
acrosomal
vesicles
of
mature
spermatozoon
and
has
been
considered
analogous
to the acrosome
of flagellate spermatozoa
by many
workers
(Sturdivant,
spermatozoa,
found
by van
thought
Sturdivant,
the
occupies
portion
in flagellate
cone
is
Secondly,
are
eventually
found
This
of the
found
of the
bodies
signs
of dehis-
acrosomes
found
belonging
to other
Colwin,
1967;
Austin,
1970).
of the
no
by
In
refringent
addition,
bodies
the
was
phyla
1968;
actual
in ques-
CLARK,
146
tion until
croscopic
reported
complex
Favard
study
that
but
(1961),
in an electron
of Ascaris
megalocephala,
they arise not from
from
aggregations
endoplasmic
reticulum.
of the flagellate
sperm
as
exhibiting
MORETTI
a
Golgi
rough
reaction
for
the
test
(Leuchtenberger
Clermont
and Leblond,
refringent
bodies
of the
ascarid
spermatids
and the refringent
cone of
mature
ascarid
spermatozoa
are not positive
for this test. Instead
it has been shown
that
the
refringent
bodies
are
composed
THOMSON
in spermatids
from animals
held in saline
for prolonged
h) prior
to dissection
and
tion.
Thus,
be of
lemma
Also,
the acrosome
has been characterized
positive
periodic
acid-Schiff
and Schrader,
1950;
1955),
whereas
the
the
of
mi-
AND
of a pro-
undergo
acrosomal
(Nath
ei a!., 1961).
(1961)
showed
the
not of Golgi
origin,
particular
vesicles
similar
them
lieves
Their
view
vesicles
“membrane
in A. lumbricoides;
specializations”
he calls
and
be-
them
to be of mitochondrial
origin.
mitochondrial
origin
seems
unlikely
in
of Favard’s
(1961)
work
and a recent
study
by Beams
and Sekhon
(1970)
in which
similar
vesicles
were most convincingly
demonstrated
to be of Golgi
origin
in the male
gametes
of Rhabdilis
pci/jo.
These
vesicles
become
aligned
around
the
plasmalemma
of the
spermatid
during
its
growth
and are eventually
found
in the cytoplasm
between
the plasmalemma
and the Fefringent
cone
of the mature
spermatozoon.
It has been
shown
with
the plasmalemma
matozoon
suum),
of A.
undergo
contents
into
tract
(Clark,
Foor
vesicles
vesicle.
that
these
of the
lumbricoides
dehiscence,
vesicles
mature
var.
and
fuse
sper-
suum
emit
the lumen
of the female
Moretti
and Thomson,
(A.
their
genital
1967).
(1970)
has reporteJ
seeing
dehisced
in spermatids
from
the
seminal
We have only noted
this phenomenon
are
believed
they
exhibit
the
acrosomes
(Colwin
and
to
plasmaand
several
of flagellate
Colwin,
1967;
1970)
and
may
be
The purpose
of this investigation
was to
characterize
further,
using
morphological,
histochemical,
and
cytochemical
methods,
protein-lipid
complex
Although
Favard
fringent
bodies
were
function
to
has described
of
1968;
Franklin,
in nature.
Austin,
the
did not ascribe
any
particular
them.
Foor
(1968
and
1970)
vesicles
dehiscence,
properties
spermatozoa
(Faure-Fremiet,
1913)
believe
is a ribonucleo-
did
describe
small
vesicles
which
were
of
Golgi
origin.
He called
these vesicles
“Golgi
hats”
or proacrosomal
vesicles;
however,
he
the
Golgi
origin,
fuse
with
the
of the mature
spermatozoon,
tein called
ascaridine
which
some
workers
rehe
since
which
have been
periods
(24-48
subsequent
fixa-
inclusions
spermatids
their
and
and
organelles
spermatozoa
found
of A.
in
suum
the
with
emphasis
on the
proacrosomal
in order to more extensively
elucidate
role
in spermiogenesis
MATERIALS
AND
and
fertilization.
METHODS
Live adult A. suum
were collected
from the intestines of freshly killed pigs and returned
to the laboratory in normal saline @37-4OC.
Light
Microscopy
Material
was dissected within
1-3 h from the time
of collection
and fixed in neutral
buffered
formaldehyde, embedded in Paraplast,
sectioned at 2-6 1m and
stained by the periodic
acid-Schiff
(PAS) technique as
described
by Lillie (1965).
Acridine
orange, a fluorochroming
agent, was used
vitally and with fixed material.
For supravital
staining,
cells were placed in solution
of 0.01% acridine orange
(Basic
Orange
14, Matheson,
Coleman
and Bell) in
normal saline. The duration
of staining ranged from 5
to 15 mm. The cells were then washed with normal
saline and examined
with a fluorescent
dark field
microscope.
Other cells were smeared on glass slides,
air dried and ether-alcohol
fixed. After fixation
the
cells were hydrated
and stained in a 0.01% solution
of
acridine
orange in 0.05 si phosphate
buffer at pH 6.
Ribonuclease
and trypsin
incubations
(Pearse, 1960)
were performed
on some samples prior to fixation and
on others after fixation.
Electron
Microscopy
Male and female reproductive
systems were removed and immediately
placed in 2.5%
glutaraldehyde buffered with either 0.1 M phosphate
at pH 7.1,
0.1 M cacodylate
at pH 7.1, or 0.1 M cacodylate
at pH
MALE
7.1
with
7%
sucrose
added.
of reproductive
tract
placed
fixative
in
fresh
The
were
appropriate
for
I h. The
fixative
and
was
then
tissue
with
buffer
(phosphate
or cacodylate)
and
postflxed
with 1% phosphate
buffered osmium tetroxide for 30 miii. Tissue samples were dehydrated
in acetone series and embedded
in Maraglas.
Thin sections
were cut on a Porter-Blum
MT-2
or Porter-Blum
MT-I
ultramicrotome
and picked up on 400 mesh uncoated grids or 200 mesh Formvar
coated grids. Material was stained for I h with aqueous uranyl acetate
followed
by 30 mm with lead citrate or for 20 mm in
alcoholic
uranyl
acetate followed
by 5 mm with lead
All
electron
performed
with
electron
microscopic
an
Hitachi
HI.)
11 or
Acid
Tissue
samples
tissue
tissue
a Philips
with
30-50
microtome.
in cold 2.5(
0.1 M cacodylate
fixation
the tissue
buffer
slices,
7(
m,
These
for
20,
slices
for
40,
were
osmium
sucrose
were
tissue
for
and
and
for
either
were
series
and
unstained
above.
incubated
in media,
having
stained
by the
Periodic
in Maraglas.
procedures
Acid-Silver
Tissue
described
incubated
at
samples
in
l’
phosphate
rinsed
in
acetone
were
fixed
osmium
were
above.
of 7.1 for
buffer,
tions
were
picked
steel
grids.
These
1%
aqueous
I h in 2.5
tetroxide
at a pH
and
for
rn phosphate
butler
the
phosphate
series
sections
up on
grids
periodic
h. The
‘2
in
tissue
200
treated
was
at room
0.1
si
again
an
sec-
temperature,
with
then
washed
several
times
in distilled
water
and incubated
in a silver
methenamine
solution
(Rambourg
and
Leblond,
1967)
were
sodium
again
rinsed
counterstaining,
at
rinsed
60C
in
thiosulfate
for
I h. After
distilled
solution
in distilled
water
on a Philips
water
for
incubation
and
floated
5 mm.
They
the
on
were
and examined,
without
300 electron
microscope
operated
at an accelerating
voltage
of 40 kv. Controls
were treated
in the same manner
as experimental
tissue
that
the
periodic
acid
by
either
M sodium
were
the
samples
lacking
bath was deleted.
of the
the
nucleus
proce-
consisted
of
substrate
or
fluoride
spheres
and
layer
of varying
5).
The
spherical
a
spermatid
added.
(Fig.
extremely
is not
dense
apparent;
is bordered
electron-dense
nucleus
have
(Fig.
found
1). Large
in the cell
often
and
contains
density
(Figs.
is surrounded
l-2
matrices
mitochondria,
which
granular
I) is a
nucleus.
how-
by an irregular,
which
size
by
.im
I
large
in diameter,
and
few cristae
regions
of cytoplasm
are
which
are devoid
of any in-
clusions
or organelles.
These
regions
represent the pseudopodia
of the late spermatid.
The cytoplasm
of these
pseudopodial
areas
4).
of varying
length
5 nm in diameter
Microtubules
the
pseudopod-like
relatively
present
areas
electron
in the
cytoplasm
The vesicles
brane
are
which
material
the
large,
inclusions
seen
1) correspond
with the light
vesicles
are
containing
present
in the
(Fig.
bodies
2).
in
Several
translucent
of the spermatid
(Figs.
I and 4).
are bounded
by a tripartite
memis asymmetric
bordering
finger-like
membrane
protrude
and
4).
are
1, 3
distributed
except
(Fig.
cytoplasm
dense
layer
Numerous
vesicle
which
(Figs.
found
cytoplasm,
to the refringent
microscope
(Fig.
Membrane-bound
whorls
of fibrillar
stainless-
20 mm
center
throughout
Thin
mesh
for
stained
of Spermatids
small
(1-1.5
Mm),
A nuclear
membrane
and
through
Maraglas.
uncoated
were
acid
with
dehydrated
embedded
glutaral-
at pH of 7.1.
tissue was post-
buffered
sections
OBSERVATIONS
contain
fibrils
approximately
Met/zenan,ine
dehyde
buffered
with
0.1
After
rinsing
in phosphate
fixed
Thin
in acetone
Thin
Control
0.01
tissue
buffered
dehydrated
or
described
ever,
TC-2
of
embedded
incubation,
phosphate
30 mm,
dures
or tissue
After
in Maraglas.
tissue
lead,
cut
and
l
with
embedded
examined
and
60 mm.
postfixed
In the
24 h at 4C.
cut on a Sorvall
slices
washed
ribonuclease,
tetroxide
series
glutaral-
at pH 7.1 and
was washed
in
ribonuclease
(Cal Biochem),
0.5 mg
water or 0.1 si cacodylate buffer
at pH 7.1. Before
use, the incubation
medium
was heated to 60C, to inactivate
any traces of
deoxyribonuclease.
Incubations
were carried
out for
periods of , 1, 2, and 3 h. After incubation,
tissue
slices were postflxed
with
phosphate
buffered
osmium
tetroxide
for 30 mm, dehydrated
in acetone
except
out
prefixed,
300
pancreatic
ml, in distilled
in solutions
3%
procedure
Ulirasiruciure
were dissected
0.1 M cacodylate
grids
as described
in
incubated
in a
modified
Gomori
medium
according
to the method
of
Harka and Anderson
(1962). Incubations
were carried
was
the
were
E.viraction
dehyde
buffered
in
fixed
for I h. After
37C
Phiospizatase
microscope.
Ribomicleic
Thin
examinations
147
suwn
Tissue
washed
citrate.
Ascaris
Acid
sections
in the
dissected
OF
GAMETES
microtubules
with
the
the cytoplasm
invaginations
into
the
often
matrix
appear
thicker,
(Fig.
of
4).
the
of
the
to be
S
1
4
1
#{149} ‘.
A
4
I
L
I
Fto.
chondria
podial
FIG.
acridine
fluoresce
I.
Electron
(M),
micrograph
refringent
extension
(Ps).
green
3.
(arrow).
Electron
(R),
a spermatid
and
containing
numerous
a centrally
proacrosomal
vesicles
located
(P).
nucleus
The
cell
(N),
numerous
mito-
contains
a large
pseudo-
x8,700.
2.
Micrograph,
from
orange.
The large light
FIG.
of
bodies
Xl
a darkfleld
fluorescent
microscope,
showing
five spermatids
vitally
stained
areas represent
the orange
fluorescence
of the refringent
bodies
while
the
,400.
micrograph
showing
the
fibrillar
148
material
found
in the
pseudopodia.
X90,500.
with
nuclei
-
.-...
,
...
:‘
:.-
‘-#{149}‘
#{149}f
.e
4.
.
-
:-
“
____
_____
Fio.
points
4.
to cross
acrosomal
FIG.
Closely
FIG.
defined
An
body
5.
An
electron
micrograph
sections
(P).
Pseudopodia
with
6.
Nucleus
and
less dense
zone
of spermatid
than
numerous
aligned
(Ps)
micrograph
this
I
_____________
illustrating
of microtubules
electron
associated
.
t1-
and
around
dense
treated
in untreated
the cytoplasmic
mitochondria
illustrating
are
microtubules
(M)
a spermatid
spheres.
are also
nucleus
in the cytoplasm
side
of the
present.
_______
of a spermatid.
limiting
membrane
Arrow
of a pro-
X30.200.
surrounded
by
an
electron
dense
zone.
X33,500.
with
ribonuclease.
tissue.
x33500.
149
Arrows
indicate
spheres
which
now
appear
poorly
CLARK,
150
aligned
around
vesicles
the
correspond
bodies
or
hats”
his
The
of the
centrally
green
stained
study
located
in fresh
with
2).
within
this
zone
appear
(Fig.
6)
than
in
electron-translucent
less
size and
refringent
indica-
treated
the
dense
zone
dense
spheres
electron
refringent
more
peripheral
zones
of silver
periodic
acid-silver
and in some cases
in their
exhibit
matrix
deposition
(Figs.
7 and
8). They
reaction
product
when
tested
phatase
activity.
When
stained
orange
the
bright
refringent
orange
the
first
with
with
orange
by
results
in an
cence.
absence
These
earlier
results
with
periphery
of the
The
of the
orange
fluores-
vesicles
the
periodic
for
8)
exhibit
and
phosphatase
of the
show
method
(Clark,
positive
these
around
(Figs.
10 and
the
(Fig.
granules
correvesicles.
silver
depositions
reaction
11).
(Figs.
7 and
for
acid
the
still
matrix
is
in the
The
with
mito-
the
nu-
and
occasionally
apical
end of the
refringent
cone
and
the
12). Also
found
in this
region
are the proacro-
cytoplasmic
Many
of
these
plasmalemma
vesicles
and
have
undergone
and
20).
to have
dehisced
(Figs.
contain
a fibrillar
and
13).
Although
the
the
above
structure,
contain
areas
matrix
spermatozoa
erable
variability.
gent cone
is no
Those
spermatozoa
generally
they
may
show
In many
cases
longer
apparent
not
exhibiting
of
poorly
12
have
consid-
the refrin(Fig.
16).
a cone
defined,
electron-
dense
material
which
probably
represents
remnants
of the cone
(Fig.
16). In some
instances
the cytoplasm
of the conical
portion
of spermatozoa
without
refringent
cones
exhibit
strands
material
orange
acridine
is not
of electron-dense
(Figs.
Cytochemistry
As
matid
methenamine
microscopy
a positive
are
cytoplasm
acid-silver
electron
Moretti,
proacrosomal
heavy
of our
granules
microscopy
of
to that
These
some
spermatid’s
position
sponds
with
light
trypsin
orange
studies
prominent
with
acridine
to
noted
spermatids.
region
of the
the
the
was
associated
(Fig.
with
same
dehiscence
(Figs.
12, 14, 16, 18
Those
vesicles
which
do not appear
then
their
the
found
vesicles.
fused
acridine
and
contradicted
preliminary
phos-
with
Thomson,
1968).
A series of small PAS
narrow
of
Incubation
staining
somal
spermatids
the
their
than
between
show
no
however,
of
are
plasmalemma
retain
cristae;
cytoplasmic
the periphery
sperm
is suggestive
orange
fluorescence.
followed
the
a
ribonuclease
acridine
clear
along
fluoresce
However,
few
method
are seen
acid
with
2) which
of RNA.
incubated
stained
for
bodies
(Fig.
presence
with
of about
spermaInstead
are found
(Fig. 12). The
have
coalesced
to form
a
electron-dense
chondria
narrow
methenamine
small depositions
areas
mitochondria
spermatids
bodies
in this zone in the
no longer
present.
distribution
bodies
contain
opaque
untreated
reorganization
mature
spermatozoon
the
spermatid.
The
located
and bordered
zone;
but
the dense
large
cone
with
its base
proximal
nucleus
(Fig.
12). The
mitochondria
elec-
5).
The
9).
fluosper-
with
tron
microscope
the peripheral
around
the nucleus
and
the
and
sper-
fixed
orange,
viewed
cytoplasmic
is demonstrated
in the
when
compared
with
nucleus
is still centrally
by an electron-dense
nuclei
In spermatids
and
of spermatozoa
spheres
observed
tids (Fig.
5) are
or
acridine
(Fig.
ribonuclease
(Fig.
of
THOMSON
Considerable
Favard
Spermatids
small,
of DNA
with
by
inegalocephala.
A.
a bright
matids
described
AND
Ultrastructure
(Fig.
4). These
the
proacrosomal
ultrastructural
in
Cyiochemistry
resce
to
“Golgi
(1961)
in
matogenesis
tive
vesicles
MORETTI
with
the
16 and
of mature
particulate
17).
spermatozoa
the refringent
bodies
of the sperrefringent
cone
exhibits
a bright
fluorescence
when
orange
(Fig.
15).
affected
by treatment
treated
This
with
fluorescence
ribonuclease
with
FIG.
7.
An electron
micrograph
showing
a spermatid
stained
by the periodic
acid-silver
methenamine
method.
The proacrosomal
vesicles (arrows)
show extremely
heavy silver depositions.
The refringent
bodies (R)
exhibit
narrow zones of reaction
product
around their periphery;
small loci of reaction
product are sometimes
present in their matrix.
Mitochondria
(M) do not exhibit reaction product.
X9,800.
FIG.
8. An electron micrograph
showing
proacrosomal
bodies (P) and a mitochondrion
(M) from a spermatid
FIG.
stained
by the periodic
9.
Light
micrograph
spermatids.
acid-silver
methenamine
demonstrating
PAS
method.
X22,500.
positive
granules
situated
X2,100.
151
around
the
peripheries
of the
FIG.
vesicles
10.
(I’
chondria
FIG.
(P)
from
An
electron
contain
(M)
II.
exhibit
A high
spermatids
micrograph
lead deposition
no
lead
deposition.
magnification
treated
showing
indicating
for
treated
acid
phosphatase.
The
The cytoplasm,
for
refringent
bodies
proacrosomal
(R) and mito-
xlO,800.
electron
acid
a spermatid
acid phosphatase.
micrograph
phosphatase.
demonstrating
X40,200.
152
lead
deposition
in proacrosomal
vesicles
:1
:‘-
FiG.
12.
portion
An
electron
of the oviduct.
cytoplasmic
region
micrograph
A large
between
vesicles
(P). These
vesicles
the vesicles
have undergone
surrounded
FIG.
still
by numerous
13-
contains
FIG.
14.
spermatazoon.
FIG.
cones
15.
fluoresce
A
high
the
of a mature
spermatozoon,
cone
is present
refringent
refringent
often
contain
dehiscence
cone
a fibrillar
matrix.
A high
magnification
and
the
stained
for
in the conical
plasnialemma
are
acid
(M.
phosphatase,
portion
from
of the sperm.
mitochondria
deposits
of lead. indicatir
the presence
as indicated
by arrows.
In the middle
of
mitochondria
magnification
(Rc)
(M)
of acid
the cell
and
the
dista
In the narrow
proacrosomal
phosphatase.
Some of
is a dense nucleus
(N)
x21.750.
micrograph
showing
a proacrosomal
vesicle,
from
a mature
sperm,
which
X53,000.
micrograph
illustrating
dehiscence
ci
a proacrosomal
vesicle
acridine
The
in
a mature
X 52,000.
A light
a bright
micrograph
orange.
showing
The
nuclei
spermatozoa
fluoresce
vitally
green
stained
(arrow).
with
>< 1,600.
orange.
large
refringent
FIG.
16.
An
with
correspond
electron
the
micrograph
refringent
showing
cone.
A
a mature
centrally
flbrillar
sperm.
cytoplasm.
Proacrosomal
vesicles
(P) are
In some instances
the bounding
membranes
sperm.
The
microvilli
pseudopodial
(Mv)
of the
FIG.
17.
portion
uterine
Insert
of the cell
epithelial
from
Fig.
spermatozoon.
located
nucleus
located
around
of these vesicles
is composed
cells.
X31,700.
16 illustrating
of a granular
the
I4
granularity
and
Arrows
indicate
numerous
material
mitochondria
believed
are
found
the periphery
of the conical
portion
are fused with the plasma
membrane
matrix.
of the
The
cell
cytoplasm.
is closely
X95,I00.
apposed
ta
in a
of the
of the
to
the
I.--
-
periodic
the conical portion
of a mature spermatozoon.
A large refrinof the refringent
cone and the plasmalemma
are several mitovesicles. Some of these vesicles have undergone
dehiscence.
X2l 500.
19.
An electron
micrograph
showing
proacrosomal
vesicles from a spermatozoon
stained
by the
acid-silver
methenamine
techniuue.
Some of the vesicles exhibit very dense silver deposition
(arrows).
X49,600.
FIG.
20.
FIG.
chondria
FIG.
tion
do
An electron
micrograph
is present
(Rc).
Between
and numerous
proacrosomal
18.
gent cone
not
border
A micrograph
showing
the conical portion
of two spermatids
treated for acid phosphatase.
Reaccan be seen localized in the proacrosomal
vesicles (P). Those vesicles (arrows)
which have dehisced
product
refringent
showing
the
appear
cones
to
contain
(Rc)
as much
exhibit
reaction
no reaction
product
product.
as the
Unstained
155
vesicles
tissue.
which
have
X47,000.
not
undergone
dehiscence.
The
156
but
CLARK,
is lost
after
acrosomal
action
mine
trypsin
vesicles
for the
test (Fig.
positive
exhibit
The
pro-
a positive
re-
periodic
acid-silver
methena19). They
also demonstrate
reaction
ever,
vesicles
less
reaction
which
have
digestion.
still
for
acid
AND
MORETTI
phosphatase;
a
how-
undergoing
dehiscence
product
than
those
not undergone
dehiscence
exhibit
vesicles
(Fig.
20).
Fertilization
Figure
21 shows
spermatozoon
membrane.
The
matozoon
numbers
ciated
a fertilized
lying
inside
the
fertilization
of
(Figs.
21 and
lacking,
particles
21 and
22).
contents
into
since
nants
gamete
of
spermatozoon’s
contents
(Figs.
the cytoplasmic
zoon was carried
the
the
unaffected
are
labile
but
are
large
asso-
Although
clease
was
and
of
the
rem-
the male
ooplasm
22).
refringent
Beneden
‘achromatic”
(Scheben,
gested
as
1905
it was
1905).
In
structure
suggested
that
the
1911
was
Romieu
composed
sugof
“nucleo-albumens.”
Pasteels
(1948)
histochemically
demonstrated
the
presence
of
ribonucleoproteins
cells of Parascaris
unable
later
in the
equorum;
to demonstrate
stages
of the
their
spermatid
spermatozoon.
Nath
quent
histochemical
et
a!.
study
young
male germ
however,
he was
presence
and
the
in the
mature
(1961)
in a subsereported
the
presence
of RNA
in the refringent
bodies
the spermatid
and
refringent
cone
of
of
the
spermatozoon
of Porrocaecu,n
angusticolle.
However,
the presence
of RNA
in the mature
gametes
could
only
be demonstrated
after
treatment
with
thus,
suggested
cone,
zone
trichloroacetic
that
the
RNA
acid.
was
Nath,
being
by ribonuclease
to proteolytic
ribonuof an
rich in
no
seen
ing that
composed
of RNA
treatment
detectable
effect
in the refringent
these
bodies
of ribonucleic
in nematode
and
the
exhibit
stained
proper-
digestion.
of ribonubodies
or
it did affect
the spheres
and
associated
with the nucleus,
peripheral
suggesting
are,
at least
acid. The
spermatozoa
in part,
presence
is ex-
tremely
interesting
since
the evidence
obtained
from studies
of other
phyla
has characterized
the
spermatozoon
as lacking
in
RNA.
Recent
bodies,
first
described
by van
and
Julin
(1884),
consisted
of
material
of nuclear
origin
the
fringent
bodies
of the
spermatid
refringent
cone
of the spermatozoon
a positive
reaction
for RNA
when
with acridine
orange,
these
staining
are
at
Panijel
male ga-
acid,
linked
to purine
groups.”
The
results
seem
to be in accord
with
(1950)
findings.
Although
the re-
but
DISCUSSION
As early
aspartic
present
Panijel’s
ties
of
complex.
of the
metes
of A. megalocephala
found
no
cleoprotein
but reported
the “presence
acid protein
of high nitrogen
content,
sper-
cytoplasmic
vesicles
within
“masked”
by a lipoprotein
(1950)
in a chemical
study
the
plasma
The majority
of the
spermatoegg’s cytoplasm
mitochondria
proacrosomal
are
apparent
with
egg’s
plasmalemma
is completely
of ribosomal
with
egg
the
ThOMSON
increase
lowing
work
has
in RNA
fertilization.
ported
herein
ribosome-like
indicated
that
there
is an
in the nematode
ovum
The
observations
demonstrate
particles
the
associated
folre-
presence
with
of
the
spermatozoon
within
the ovum.
The appearance
of these
ribosomal-like
particles
has
also
been
reported
by Foor
(1968,
1970).
Panijel
and Pasteels
(1951)
demonstrated
the
presence
Kaulenas
chemical
increase
ization
sented
nome
results
mature
of this
histochemically
evidence
indicating
is responsible
for
indicate
that
sperniatazoon
The relationship
sudden
increase
ovum
following
However,
RNA
while
and
Fairbairn
(1968),
using
biotechniques,
demonstrated
a sudden
in ribosomal
RNA
following
fertilin the ova of A. suum.
They
pre-
it seems
that the male gethis synthesis.
Our
RNA
is present
in the
prior
to fertilization.
between
this
in the ribosomal
fertilization
possible
that
RNA
and the
RNA
in the
is not
clear.
this
synthesis
FIG.
tions
FIG.
present
21.
An electron
of ribosomal-like
22.
are
A high
the
micrograph
particles
magnification
remnants
of
of a fertilized
are associated
micrograph
proacrosomal
egg with
with
the
illustrating
vesicles
(P),
the spermatozoon
spermatozoon.
the
sperm
X42,500.
157
ribosomal-like
mitochondria
in the ooplasm
Dense
accumula-
x7,800.
particles
(sM)
and
shown
egg
in
Fig.
mitochondria
21.
Also
(eM).
158
CLARK,
may be initiated
as suggested
found
in the
matozoa.
ticulate
by
to sperm
particulate
cytoplasm
of some
mature
cone.
From
to hypothesize
precursors
ribosomes.
Although
nature
further
a
complete
Hirschler
small
in the
ameboid
situated
of
the
must
await
that the cone
(1913)
and
granules,
apical
portion
spermatozoon.
in the narrow
These
region
ma-
granules
of cyto-
and their existence
was placed
in doubt
until
Favard
(1961),
in an ultrastructural
study
of
portion
of the
firmed
the
and Collier
similar
brasiliensis.
vesicles
appeared
membrane
However,
in the sperm
In some
to
fuse
ticular
function.
In
were found
to dehisce
in
in
in the
have
female
an ultrastructural
A. lumbricoides
dehisced
termed
The
thus
con-
of Nippostroninstances
these
with
of the mature
Jamuar
did not
cence and considered
the
mitochondria”
to which
were
and
observations
of Hirschler
(1913)
(1937).
Jamuar
(1966)
has noted
vesicles
gylus
described
a series
of vesiin the apical
cytoplasmic
spermatozoon
the
plasma
shown
activity
tract.
study
observed
of
the
Foor
and
Thus
to be positive
for
the
undergo
dehiscence
when
with
the
spermatozoon
dehiscence
occurs
when the spermatozoa
are
in the female
genital
tract.
This
dehiscence
occurs
in many
spermatozoa
even
though
each may not be proximal
to an ovum.
Perhaps,
in the case
of internal
fertilization,
such
as
of these
in A. suum,
the
vesicles
serves
an
can
combined
reaction
to establish
and
environment
in which
fertiliza-
occur.
REFERENCES
A. C.,
enzymes
AND
in
W.
urchin
activity
of
siruct.
Res.
AUSTIN,
tion.”
BARKA,
(1968).
III.
T.,
Acid
spermatozoa
25,
AND
their
Fert. 21,
and
Lysopossible
501-5
Cytochemistry
and
15.
of
alkaline
phosphatase
fertilization.
J.
“Ultrastructure
and Winston,
ANDERSON,
for
and
acid
of
New
P. J. (1962).
using
phosphatase
sea
Ultra-
1-14.
C. R. (1968).
Holt,
Reinhart
cal methods
acrosome
J. .Reprod.
A.
gametes.
E. F. (1970).
HARTREE,
the
role in fertilization.
PAS
sperma-
spermatozoa
of A. suum.
in their
Golgi
origin,
fusion
ANDERSON,
of
demonstrated
of the
contacts
an ovum
or is in the presence
of
substances
evolved
from
the surface
coats
of the ovum
(Colwin
and
Colwin,
1967;
Austin,
1968;
Franklin,
1970).
In A. suum
somal
he
been
vesicles
the plasmalemma,
dehiscence
prior
to fertilization,
and their
histochemical
and cytochemical
properties,
these
vesicles
are similar to the acrosome
of flagellate
spermatozoa. The acrosomes
of flagellate
spermatozoa
in
which
fertilization
occurs
externally
ALLISON,
(1968)
fertilization
presence
has
proacrosomal
vesicles
“specialized
he ascribed
no par-
genital
Schrader,
found
to be present
in the acrosomes
from
sperm
of a newt
(Buongiorno-Nardelli
and
Bertolini,
1967)
sea
urchin
(Anderson,
1968),
and
several
mammals
(Allison
and
Hartree,
1970).
In the
present
study
acid
tion
A. suum
these
vesicles
when the spermatozoa
and
and Leblond,
1955).
Jamuar
a similar
PAS
positive
revesicles
he described
in N.
The
results
described
herein
show
these
proacrosomal
vesicles
to be
positive.
Acid
phosphatase
has been
also
PAS
maintain
spermatozoon.
note
any dehis-
proacrosomal
vesicles
which
“surface
membrane”
specializations.
acrosomes
of flagellate
spermatozoa
been
Clermont
described
for the
brasiliensis.
tids
plasm
between
the plasma
membrane
and
the
border
of the refringent
cone.
Subsequent
investigators
(Nath,
Gupta
and Kochhar,
1961; Nath
and Singh,
1956) were
unable to confirm
the presence
of these granules
A. megalocephala,
des of Golgi
origin
(Leuchtenberger
1950;
(1966)
action
in the
Collier
“Golgi
of the
THOMSON
phosphatase
in nature.
The proacrosomal
do have
properties
which
are acrosomal.
(1937)
described
remnants,”
it is
may
of
definition
of the
refringent
cone
investigations,
it is clear
is not acrosomal
vesicles,
however,
sper-
such parlack
a
these
results
that
the cone
for the synthesis
AND
reaction
penetration
material
Spermatozoa
containing
material
characteristically
refringent
tempting
contribute
ture
were
prior
the
MORETTI
FertilizaYork.
Histochemi-
hexazonium
MALE
GAMETES
OF
pararosanilin
as coupler.
J. Histoc/zem.
Cytoc/zem.
10, 741-753.
BEAMS,
H. W., AND SEKHON,
S. S. (1970).
Spermiogenesis in the nematode,
Rlzabditis
pellio. 101/I Ann.
Meet. Amer. Soc. Ce/I Biol. 47, 16a.
BOWEN,
R. H. (1925). Further
notes on the acrosome
of the animal sperm. The homologies
of non-flagellated sperms. Anal.
Rec. 31, 201-23
1.
M.,
BUONGIORNO-NARDELLI,
(1967).
Subcellular
drolases
in
8,
chemie
CLARK,
the
of some
spermatozoa.
localization
Triluros
cristarus
B.
BERTOLINI,
acid
by-
Hislo-
H.
MORErrI,
JR.,
(1967).
presence
Electron
of
R. L.,
acrosomal
an
AND
microscopic
reaction
in
for
Ascaris
var. suum. Exp. Cell Res. 47, 643-647.
W. H. JR., THOMSON,
W. W., AND Mogzrn,
R. L. (1968). Electron
microscope
and histochemical characterization
of the proacrosomal
vesicles
found in the gametes of Ascaris lumbricoides
var.
suum. J. Cell Biol. 39, 25a.
CLERMONT,
Y., AND LEBLOND,
C. P. (1955). Spermiogenesis
of man,
monkey,
ram and other mammals
as shown by the “periodic
acid-Schiff”
technique.
lumbricoides
J. Anal.
96,
229-253.
Sci.
78,
L. H.,
AND
Microsc.
397-418.
A. H. (1967). Membrane
association.
In
“Fertilization.”
(Metz, C. B. and Monroy,
A., eds.),
Chap. 7. Academic
Press, New York.
FAvARD,
P. (1961).
Evolution
Des Ultrastructures
Cellulaires
au Cours
de La Spermatogenese
De
l’Ascaris.
Ann. Sci. Nat. Zoo!.
Ser. 12, 53-1 52.
FAURE-FREMtET,
E. (1913).
Le cycle
germinatif
chez
fusion
!‘Ascaris
Morp/zol.
in
COLWIN,
relation
to
sperm-egg
mega!ocepha!a.
Exp.
Arc/i.
Anal.
Microsc.
15, 435-758.
W. E. (1968).
Zygote
formation
in Ascaris
lumbricoides
(Nematoda).
J. Cell Biol. 39, 119-134.
Fo0R, W. E. (1970). Spermatozoan
morphology
and
zygote
formation
in nematodes.
Bio!.
Reprod.
Suppl. 2, 177-202.
FRANKLIN,
1. E. (1970). Fertilization
and the role of
the acrosomal
region
in non-mammals.
Biol. .Reprod.
Suppl. 2, 159-176.
HIRSCHLER,
J. (1913).
Ueber
die Plasmastrukturen
(Mitochondrien,
Golgischer
Apparat)
in
den
Geschlechtse-zellen
der Ascariden.
Arc/i.
Zellforsc/i.
9, 351-398.
HovAssE,
R. (1958).
Etude
au microscope
electronique
des phenomenes
cytoplasmiques
de Ia spermatogenese
chez l’Ascaris
(Parascari
equorum).
XV:h
mt. Congr. Zool. Sec. IX Paper 22. 708-709.
FOOR,
Studies
of spermiogenesis
in a
J. Cell Biol.
Nippostrongylus
brasiliensis.
381-396.
KAULENAS,
M.
A.
metabolism
AND
of
during
FAIRBAIRN,
fertilized
uterine
D.
Ascaris
(1968).
RNA
eggs
lumbricoides
development.
Exp.
Cell
52,
Res.
233-251.
C.,
LEUCHTENBERGER,
chemical
nature
Proc.
cells.
R.
LILLIE,
NA-rn
AND
of
Nat.
Acad.
E. (1965).
acrosome
Sci.
U. S.
Univ.
the
36,
3rd
Co.,
New
Res.
germ
Technique
and
677-683.
ed.
The
pp.
198-199.
nematode
sperm.
121-1 35.
B. L., AND
V. 91,
Bull.
The
male
York.
S. (1956).
SINGU,
Punjab
in
“Histopathologic
Book
AND
F. (1950).
SCHRADER,
the
Histochemistry.”
AND
SINGH,
S., GUPTA,
KOCHHAR,
D. M. (1961).
The histochemistry
of the male germ
cells of the nematode
Porracaecum
angustico/le,
a
parasite
in the vulture.
Quart.
J. Microsc.
Sci. 102,
NATH
39-50.
J. (1950).
PANUEL,
signification
Researches
de
d’Ascaris
la
proteine
sur
gram
megalocephala.
Biochim.
Ia
nature
et
du
gamete
male
Biophys.
Ia
Acta
6,
79-93.
J.,
PANIJEL,
V. (1937). Studies on the cytoplasmic
components
in fertilization
I. Ascaris suilla. Quart.
J.
COLLIER,
OLWIN,
nematode,
McGraw-Hill
THOMSON,
evidence
CLARK,
Amer.
M. P. (1966).
JAMuAR,
31,
159
suwn
Practical
34-44.
W.
W. W.
AND
Ascaris
AND
J.
PASTEELS,
(1951).
Analyse
cyto-
chimique
de certains
phenomenes
de recharge
en
ribonucleoproteines.
Le Cas del’oeuf de Parascaris
equorum
lors
de la fecondation.
Arch.
Biol. 62,
353-370.
PASTEEL.S,
J. (1948).
nucleiques
Biol.
Etude
dans
development
chez
59,
Applied.”
and
Co.,
le cycle
germinal
des
acides
las spermatogenese
Parascaris
et le
Goeze.
equorum
Arch.
405-447.
A. G. E. (1961).
and
sur
cytochimique
l’oogneese,
L (Leige)
PEARSE,
2nd
“Histochemistry,
ed.,
pp.
Theoretical
916-917.
Little,
Brown
Boston.
A., AND LEBLOND,
observations
RAMBOURG,
microscope
cell
Recherches
l’Ascaris
chez
coat
present
at
the
C. P. (1967).
on
the
surface
Electron
carbohydrate-rich
of
cells
in
the
rat.
J. Cell Biol. 32, 27-53.
ROMIEU,
M. (1911). La sperrninogenese
chez l’Ascaris
megalocephala.
Arch.
Zellforsch.
6, 254-325.
L.
SCHEBEN,
tozoons
A. 79,
Ab:.
(1905).
von
zur
kenntnis
megalocephala.
der
Z.
Wiss.
spermaZoo!.
397.
H.
STURDIVANT,
Beitrage
Ascaris
P.
(1934).
Studies
on
the
spermato-
special
reference to the central
bodies,
Golgi
complex
and
mitochondria.
J. Morphol.
55, 534-565.
VAN BENDEN,
E., AND JULIN,
C. (1884).
La spermatocyte
genese
Roy.
divisions
chez
BeIg.
in Ascaris
l’ascaride
7, 3 12-342.
megalocephala
with
megalocephale.
Bull.
Acad.