Ho
Printed in Great Britain
J. Reprod. Fert. (1985) 74, 127-134 © 1985 Journals ofReproduction &Fertility Ltd
Photoelectron imaging of guinea-pig, hamster and human
spermatozoa
R. Mrsny and O. H. Griffith
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene,
Oregon 97403, U.S.A.
Summary. Photoelectron images of mammalian spermatozoa were obtained by
subjecting the specimens to u.v.-irradiation and focussing the emitted electrons by
electron optics (photoelectron microscopy). Guinea-pig, hamster and human sperm
atozoa were fixed in glutaraldehyde, deposited on conductive glass discs, and
dehydrated. Sufficient quantities of photoelectrons were released from the surface of
spermatozoa to produce images without staining, coating or metal shadowing. The
large planar heads of guinea-pig spermatozoa were easily resolved with good
delineation of acrosomal and postacrosomal regions. Residual vesicles could be
visualized on the surfaceof the inner acrosomal membrane of spermatozoa that had
undergone the acrosome reaction. Also detectable in these photoelectron images were
finer membrane surfacedetails, periodicities in the midpiece regionof the tail which
coincided with the distribution of mitochondria, and periodicities in the principal
piece which appeared to be related to fibrous sheath components. Hamster
spermatozoa were similarly well resolved but human spermatozoa were more difficult
to image because of their increased surface curvature. The mechanism responsible for
detection of these surface details is primarily topographical contrast rather than
material contrast, since spermatozoa coated with a thin layer of gold or platinum
exhibited similar features, althoughat reduced resolution, as the uncoated specimens.
Introduction
Photoelectron imaging can be thought of as the electron optical analogue of fluorescence
microscopy (Griffith et ai, 1972). Both techniques use an ultraviolet (u.v.) light source for
excitation. In fluorescence microscopy, photonsgiven up by molecules capable of fluorescence are
imaged with light optics to provide a patternof fluorescent molecules. Most biological molecules
are capable of photoemitting electrons after u.v. excitation. Photoelectron imaging uses electron
optics to accelerate, focus and image these emitted electrons to produce a representation of the
specimen. Photoelectron imaging is also called photoelectron microscopy (PEM) in the United
States and photoemission electron microscopy in Europe (PEM or PEEM). Its origins can be
traced, along with those of both transmission andscanning electron microscopy, to theearly 1930s
(Mollenstedt &Lenz, 1963; Schwarzer, 1981), butphotoelectron microscopy has only recently been
adapted for the imaging of biologicalspecimens (Griffith et ai, 1972) and it is best described as an
emerging technique, rather thananestablished method. Ofparticular interest isthe imaging ofcell
surfaces by this technique. Photoelectron images of whole cells obtained to date include those of
several lines of cultured fibroblasts, epithelial cells, and red cell ghosts (for reviews see Griffith,
Rempfer & Nadakavukaren, 1982; Griffith, Nadakavukaren &Jost, 1984). Spermatozoa havenot
been examined in detail. One early report contains a photoelectron micrograph of rabbit
spermatozoa which showed that the general shape of this cell type could be visualized by
photoelectron microscopy (Grund, Eichberg &Engel, 1979). Inprinciple, the photoelectron image
128 R. J. Mrsny and O. H. Griffith
ofspermatozoa contains useful information regarding fine topographical detail and organization of
the cell surface.
The purpose of the present report is to obtain higher resolution photoelectron images of the
spermatozoa of several species of mammal.
Materials and Methods
Media. The minimal capacitation medium with lactate and pyruvate (MCM-PL) prepared was a
slight modification ofa Ca2+-free medium used byHyne &Garbers (1982). MCM-PL consisted of
126 mM-NaCl, 1 mM-MgCl2, 25 mM-NaHC03, 0-25 mM-sodium pyruvate and 20 mM-L( + )lactic
acid(neutralized withNaOH)at 295 mosmol/kg and pH 7-4. For capacitation, CaCl2 wasaddedto
Medium MCM-PL to a final concentration of 1-7 mM. Phosphate-buffered saline with sucrose
(PBS-S) and modified Tyrode's bicarbonate buffer (TALP) used for capacitation were prepared
essentially as described by Mrsny & Meizel (1981). Medium PBS-S consisted of 72mM-NaCl, 4-25
mM-KCl, 3-2 mM-Na2HP04,0-8 mM-KH2P04,0-46 mM-CaCl2,0-2 mM-MgCl2 and 132mM-sucrose
at 290 mosmol/kg and pH 7-3. Medium TALPcontained 121 mM-NaCl, 0-4 mM-NaH2P04, 5 mM-
KCl, 25 mM-NaHCOj, 2-4 mM-CaCl2, 0-4 mM-MgCl2, 5 mM-glucose, 12-5 mM-L( + )lactic acid
(neutralized withNaOH), 0-25 mM-sodium pyruvate, 12 mgbovine serum albumin/ml (purified by
trichloroacetic acid precipitation, ethanol resolubilization and extensive dialysis againstH20 and
PBS) and 0-5 mM-taurine. Hepes-buffered saline (HBS) consisted of 50mM-Hepes, 3-4 mM-CaCl2,
6-7 mM-KCl, 0-6 mM-MgS04 and 137 mM-NaCl at 310 mosmol/kg and at pH 7-2. Allsolutions were
passed through 0-22 urn Millex-GS filter units (obtained from Millipore) before use.
Preparation of spermatozoa. Sexually mature guinea-pigs (Topeka strain) were anaesthetized
with ether and killed by cervical dislocation. Cauda epididymal spermatozoa, obtained from
punctured tubules, were layered on 10 ml ofMedium MCM-PL orHBS. Spermatozoa were washed
twice bycentrifugation (500# for 15 min) and resuspended in the buffer used initially, MCM-PL or
HBS. In some instances, after the second centrifugation in Medium MCM-PL, spermatozoa were
resuspended in Medium MCM-PL containing Ca2+ at 5 x 106/ml. To initiatecapacitation, 0-1 ml
aliquants of this sperm suspension were incubated at 37°C.
Sexually mature golden hamsters were anaesthetized with ether and killed by cervical
dislocation. Spermatozoa obtainedfrom punctured tubules of the caudaepididymidis werelayered
on 10mlof Medium PBS-S or HBS.Spermatozoawerewashed twice bycentrifugation (800g for 10
min) and resuspended in the buffer used initially, PBS-S or HBS. In some instances, after the
second centrifugation, spermatozoa, washed in Medium PBS-S were resuspended in Medium
TALP at 5 x 106/ml. To initiate capacitation, spermatozoa were incubated in 0-1 ml volumes at
37°C.
Semen from a healthy manof known fertility wasobtainedafter48h of abstention. The semen
was allowed to liquefy at room temperature for1h, andaliquants (0-5 ml) were thenplaced under5
ml warm Medium HBS and incubated for 2 h at 37°C. The spermatozoa that had swum into the
Medium HBSwere washed twice in Medium HBS (500 g for 15min) and resuspended in Medium
HBS.
Assay ofsperm motility andacrosome reaction. Samples (~ 50 ul) were removed from suspensions
ofwashed guinea-pig and hamster spermatozoa orfrom suspensions ofincubated spermatozoa and
examined by phase-contrast and dark-field microscopy. Estimates ofmotility and hyperactivated
motility (whiplash-like flagellar movement characteristic of capacitation in some spermatozoa)
were assessed according to previously described criteria (Yanagimachi, 1969, 1970, 1972). An
objective count was performed ofat least 100 motile cells todetermine the percentage ofcells that
hadundergone theacrosome reaction. Noattempt was made tocapacitate thehuman spermatozoa.
Photoelectron imaging of mammalian spermatozoa 129
Sperm preparation for photoelectron microscopy. Round glass coverslips (5 mm) were used as
sample mounts after being first made conductive with a layer of Sn02 (Griffith et ai, 1982).
Adhesion ofcells to these discs was facilitated bydipping thediscs in 1mg/ml aqueous solutions of
alcian blue, cytochrome c, or poly-L-lysine (6000 average mol. wt, brought to pH 13 with NaOH)
and air-drying. Washedspermatozoa wereaffixed tocoatedglassdiscsbyexposure for 15min, with
excess spermatozoa being drawn off. Adhering spermatozoa were fixed for 1 h in 2-5%
glutaraldehyde at room temperature and thediscs were washed withH20. Spermatozoa incubated
for capacitation were collected, washed once in Medium MCM (guinea-pig) or Medium PBS
(hamster) and fixed as above. Nodifferences were observed in preparations when the sperm cells
were fixed before placement on the glass discs. Post-fixation with 1% Os04 for 1 h at room
temperature provided no improvement of the photoelectron images.
For photoelectron microscopy, the fixed cells were dehydrated through a graded series of
ethanol (acetone produced equivalent results) followed byair-drying. Replacement ofethanol with
freshly distilled Freon 113 followed by air-drying frequently caused loss of tail and some head
membranes of spermatozoa. After dehydration through ethanol, some specimens were critical-
point dried from C02 but judged to be inferior due to shrinkage artefacts.
After ethanol dehydration and air-drying, some specimens were then over-coated with a 15nm
layer of gold or platinum in a Varian FC-12 ultrahigh vacuum chamber (metal-sealed, oil-free).
Current was passed through a loosely coiled tungsten filament wrapped with a 26-gauge wire
composed of gold or platinum (wires were rinsed and stored in distilled acetone) which was
positioned directly above the specimens at a distance of 10 cm to minimize shadowing. The
rotoevaporated metal film thickness was monitored using an Inficon XTM quartz crystal oscillator
which had been previously calibrated with an interference microscope (Varian A-scope).
The photoelectronmicroscope used in this study is an ultrahigh vacuum instrument built at the
University of Oregon (Griffith, Rempfer & Lesch, 1981). The u.v.-illumination sources were two
Osram HBO 100 short-arc mercury lamps. Accelerating voltage was 30 kV. Photoelectron
micrographs were takenon Kodak 4489 electron microscopy film withexposure times of 10-30 sec
for uncoated specimens and 1-5 sec for gold- or platinum-coated samples. All micrographs were
taken using a 50 urn objective aperture.
Results
Guinea-pig
Photoelectron images of guinea-pig spermatozoa are shown in Plates 1 and 2. The acrosomal
cap of the sperm head in PI. 1, Fig. 2 was representative of many relatively flat spermatozoa
PLATES 14
Abbreviations: AC, acrosomal cap; C,capitulum; CD, cytoplasmic droplet; EP, end piece of
tail; ES, equatorial segment; H,head;IF, implantation fossa; IM,inneracrosomal membrane;
MP, midpiece of tail; N, neck; P, perforatorium; PA, postacrosomal region; PP, principal
piece of tail; SC, segmented column.
PLATE 1
Fig. 1. Photoelectron micrograph of an entire guinea-pig spermatozoon. The morphological
regions of this cell are identified. Bar, 5 urn. x 1200.
Fig. 2. Higher magnification ofa guinea-pig spermatozoon showing greater detail of head and
neck regions. Note the topographical relief of the anterior acrosomal region (acrosomal cap)
caused by thenon-symmetric nature ofitscontents. Also note theridge caused by a perinuclear
thickening at the anterior margin (arrow heads) ofthe equatorial segment. The topographical
contrast at theposterior margin (arrows) oftheequatorial segment iscaused by thethickening
of the cell where the acrosomal membrane folds back onto itself. Bar, 2 um. x 8500.
130 R. J- Mrsny and O. H. Griffith
Fig. 3. Early stage of the guinea-pig sperm acrosome reaction induced by incubation in
Medium MCM-PL with Ca2+for 2-5h. Fenestrations of the plasma membrane at the anterior
edge of the acrosomal cap and the area immediately anteriorto the crescent-shaped marginof
the nucleus are identified by arrows. Bar, 2 um. x 5000.
Fig. 4. Late stage oftheguinea-pig sperm acrosome reaction induced byincubation for2-5 h in
Medium MCM-PL with Ca2+. Note the vesicles (arrows) present on the surface of the inner
acrosomal membrane. A lip of topographical contrast at the rostral edge of the equatorial
segment (arrow heads) marks the line of fusion between the remaining outer acrosomal
membrane and plasma membrane. The perforatorium is observable at this stage of the
acrosome reaction. Bar, 2 um. x 5000.
Fig. 5. Photoelectron micrograph of an acrosome-reacted guinea-pig spermatozoon following
lossof acrosome reaction vesicles. This spermatozoon was prepared as in PI. 1, Figs 1-4 except
that critical-point drying was performed rather than air drying. Note the few remaining
vesicles (arrows) and complete exposure of the inner acrosomal membrane anterior to the
equatorial segment. Theridge offusion between theremaining outer acrosomal membrane and
plasma membrane (arrow heads) is still evident. Bar, 2 um. x 5000.
PLATE 2
Fig. 6. Photoelectron micrograph of the posterior head region, neck and proximal midpiece
region ofa guinea-pig spermatozoon. Note theprominent ridge produced bytheimplantation
fossa (IF) at the head-neckjunction.The segmented columns (SC) of the connecting pieceare
also observable. The midpiece shows an irregular pattern of periodicity representing
mitochondria. Bar, 1 um. x7000.
Fig. 7. Head, neck and midpiece regions of guinea-pig spermatozoon showing neck surface
topography due to capitulum and prominent cytoplasmic droplet (CD). The cytoplasmic
droplet was observed at various positions along the midpiece and was absent in most
spermatozoa after capacitation. Bar, 1 um. x 7500.
Fig. 8. Principal piece region ofaguinea-pig spermatozoon. Theprincipal piece showed ridges
and a partly resolved transverse periodicity representing the longitudinal columns (arrow) and
ribs(arrow heads) of the fibrous sheath,respectively, which liebeneaththe plasma membrane.
Bar, 0-5 um. x 17 000.
Fig. 9. Principal piece ofguinea-pig spermatozoon prepared asin Fig. 8except forreplacement
of ethanol with Freon 113 before air drying. Note the increased topographical resolution
(compared with Fig. 8)ofthelongitudinal column (arrow) andribs (arrow heads) ofthefibrous
sheathdueto removal of the plasma membrane. In the lower portion of the micrograph the tail
has twisted through 180°. Bar, 1 um. x 12 000.
Fig. 10. Distal portion ofguinea-pig sperm tail showing transition from principal piece toend
piece. Bar, 0-5 um. x 11 000.
Fig. 11. Distal portion ofguinea-pig sperm tail prepared as inFig. 10 except for replacement of
ethanol with Freon 113 before air drying. Note the detailed resolution of the microtubules
(arrows) in a frayed pattern following disruption of the axoneme due to loss of the plasma
membrane. Bar, 1 um. x 12 500.
Fig. 12. Photoelectron micrograph ofguinea-pig spermatozoon with anintact acrosome after
coating with a thinlayer ofplatinum. Note thedecreased surface detail incomparison with PI.
1, Fig. 2. Bar, 2 um. x5000.
Fig. 13. Platinum-coated guinea-pig spermatozoon which has undergone the acrosome
reaction. The line of fusion between remaining outer acrosomal and plasma membranes is
identified by arrow heads. Bar, 2 um. x 5000.
Fig. 14. Photoelectron micrograph of a guinea-pig spermatozoon which has undergone the
acrosome reaction. The sample has been coated with a thin layerof gold. The line of fusion
between remaining outeracrosomal membrane and plasma membrane is identified by arrow
heads. Bar, 2 um. x 5000.
i
PLATE 1
(Facing p. 130)
PLATE 2
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PLATE 3

IPhotoelectron imaging of mammalian spermatozoa 131
observed. There was a gradation of surface topography with some spermatozoa exhibiting an
undulated acrosomal region. PI. 1, Figs 3-5 illustrate sperm heads in various stages ofacrosomal
loss induced by incubation for several hours in a capacitating medium. The fenestrations of the
surface membrane at the tip of the acrosomal cap and adjacent to the crescent-shaped anterior
margin ofthe nuclear region (PI. 1, Fig. 3)have been observed previously as the initial fusion and
vesiculation sites between the outer acrosomal membrane and its overlying plasma membrane
PLATE 3
Fig. 15. Photoelectron micrograph ofgolden hamster spermatozoa, showing thehead and tail
Bar, 5 um. x 1600.
Fig. 16. Higher magnification ofgolden hamster spermatozoon showing greater detail ofhead
regions. Note the prominent curved shape (broad anterior margin) of the acrosome. The
topographical relief ofthe anterior margin (arrow heads) ofthe equatorial segment isdue toa
thickening ofthe perinuclear theca. The posterior margin (arrows) ofthe equatorial segment is
observed as the topographical contrast caused by the acrosomal membrane folding back onto
itself beneath the plasma membrane. Bar, 2 um. x 9000.
Fig. 17. Early stage ofthe golden hamster sperm acrosome reaction. The specimen had been
incubated in acapacitating medium for 4h. Note the ruffled appearance ofthe acrosomal cap
and the fenestrations ofthe plasma membrane (arrows) atthe anterior margin ofthe equatorial
segment. The sperm head inthis field is lying atop (arrow head) theprincipal piece ofanother
spermatozoon, affecting the topography (bright area)of its postacrosomal region. Bar, 2 um
x5000. p
Fig. 18. Late stage ofhamster sperm acrosome reaction. The acrosomal ghost (arrows) has a
foamy appearance as it is shed during the acrosome reaction. The newly exposed inner
acrosomal membrane (IM) is now evident. Bar, 2 urn. x 5000.
Fig. 19. Photoelectron micrograph ofanacrosome-reacted hamster spermatozoon after loss of
the acrosomal ghost. Note the long perforatorium (formed by an extension ofthe perinuclear
theca) and the fusion point ofthe remaining outer acrosomal membrane and plasma membrane
(arrow heads). Bar, 2 um. x 5000.
PLATE 4
Fig. 20. Photoelectron micrograph ofthe distal head, neck and proximal midpiece ofa golden
hamster spermatozoon. The neck region shows topographical relief suggesting the sub-
plasmalemmal structure of the implantation fossa (IF). This midpiece shows an irregular
pattern of periodicity representing mitochondrial deposition. Bar, 0-5 urn. x 16000.
Fig. 21. A golden hamster spermatozoon showing the midpiece periodicity of a helical
arrangement of mitochondria. The capitulum (C) and segmental columns (SC) of the
connecting piece are also observable. Bar, 0-5 um. x 12 000.
Fig. 22. A golden hamster spermatozoon showing a prominent cytoplasmic droplet
approximately half-way down the midpiece region of the tail. Cytoplasmic droplets were
observed atvarious positions along the midpiece but were less frequently observed after several
hours in a capacitating medium. Bar, 1 um. x 7000.
Fig. 23. Principal piece region of a hamster spermatozoon. The topographical contrast
represents the longitudinal column (arrow) and ribs (arrow heads) of the fibrous sheath Bar
0-25 um. x 24 000.
Fig. 24. Distal portion of a hamster sperm tail showing the transition (arrow) from the
principal piece of the distal end piece. Bar, 1 um. x 10000.
Fig. 25. Distal portion of the principal piece of a hamster sperm tail. In this instance,
membrane damage had occurred as assessed by the extrusion offine filaments (arrows) which
are probably components of the tail motility apparatus. Bar, 1 um. x 9500.
Fig. 26. Photoelectron micrograph ofhuman spermatozoa depicting regions ofthe head and
tail. Bar, 5 um. x 3000.
Fig. 27. A human spermatozoon coated with a thin layer (15 nm) ofgold. Bar, 5um. x3000.
Fig. 28. Platinum-coated (15 nm thickness) human spermatozoon. Bar, 5 um. x3000.
132 R. J. Mrsny and O. H. Griffith
during an early stage of the acrosome reaction (Friend, Orci, Perrelet & Yanagimachi, 1977).
Acrosomal vesicles, hybrid vesicles composed of both outer acrosomal and plasma membranes, are
formed by the multiple fusion and vesiculation events of the acrosome reaction (Meizel, 1984).
These vesicles were present on the surface of the newly exposed inner acrosomal membrane at late
stages of the acrosome reaction (PI. 1, Fig. 4). At such a stage of the acrosome reaction the
perforatorium was clearly visible and observation of the fusion between the remaining outer
acrosomal membrane and plasma membrane at the anterior edge of the equatorial segment was
possible. Following loss of these vesicles, a clear view of the inner acrosomal membrane was
attainable (PI. 1, Fig. 5).
The periodic bright and dark bands in the midpiece region, which began posterior to the
connecting piece and stopped at the end of the midpiece (the annulus), were irregular in their
disposition, but the distance between adjacent bright bands was about 0-3 um. This periodicity
coincides with the distribution of mitochondria in mammalian spermatozoa (Fawcett, 1975).
Coating of spermatozoa with a thin layer of metal increased the brightness of the photoelectron
images, but reduced the resolution of the fine surface detail (PI. 2, Figs 12-14).
Hamster
Photoelectron micrographs of golden hamster spermatozoa are shown in Plates 3 and 4. The
very different head morphology of these cells provides a separate test of photoelectron imaging.
The equatorial segment of the head was delineated by topographical relief at its margins. The
anterior margin is the ridgeof thickened perinuclearmaterial (part of the theca) and the posterior
margin being the siteof reflection of the acrosomal membrane (Franklin, Barros & Fussell, 1970).
Golden hamster spermatozoashowedno variation in topographyof the intact acrosomalcap region
(PI. 3, Figs 15, 16). During the acrosome reaction the acrosome wasfoamy in appearance and the
acrosomal ghost (Yanagimachi & Phillips, 1984) lifted away to expose the inner acrosomal
membrane (PI. 3, Figs 17-19).
The periodicity ofthegolden hamster sperm midpiece region showed an irregular (PI. 4,Fig. 20)
or a helical (Pi. 4, Fig. 21) pattern of banding.
Human
Human spermatozoa weremoredifficult to image by photoelectron microscopy (PI. 4, Fig. 26)
than were guinea-pig or hamster spermatozoa. Even under the best conditions, some image
distortion occurred over the human sperm head. Coating with a thin layer of gold or platinum did
notdramatically reducethe imagedistortionwhichmustthereforehave beendue to the topography
of the spermatozoa and not specimencharging. Thosespermatozoathat couldbe viewed, however,
did showthe morphological variation of the sperm head observed in this species (Bedford, Bent &
Calvin, 1973). As with guinea-pig and hamster spermatozoa, human sperm tail structures
(midpiece, principal piece and end piece) were well imaged (PI. 4, Figs 26-28).
Discussion
Photoelectron imaging is still under development as a technique for relating ultrastructure to cell
function. The present results demonstrate the application of photoelectron imaging at its present
state of development for the study of the surfaces of mammalian spermatozoa.
Photoelectron imaging of mammalian spermatozoa 133
The photoelectron image is formed by collecting all of the electrons released from the surface at
the same time rather than point by point as in a scanning electron microscope. Two sources of
contrast, material and topographical, can contribute to the photoelectron image of a cell surface
(Rempfer, Nadakavukaren & Griffith, 1980; Griffith etai, 1982). Material contrast is not a major
contributor to these photoelectron images of spermatozoa, as judged by comparing the uncoated
and metal coated specimens. The image is sensitive to very fine topographic detail because the low
energy electrons emitted from the surface are readily influenced by the accelerating field across the
cathode-anode gap. Electrons emitted from sloping surfaces of protrusions or depressions are
deflected, making it possible to detect very small cell surface features in the image. It is this
topographical contrast mechanism that gives the photoelectron micrographs their three
dimensional appearance. No image processing is required. The topographical and other image
information is recorded directly onto a photographic film as in transmission electron microscopy.
In these photoelectron micrographs the major known structural features of the mammalian
spermatozoon, such as the acrosomal cap, equatorial segment, postacrosomal region, neck,
midpiece, principal piece, and end piece are well resolved and fine surface detail is visible. In
theory, very small steps of surface topography can be detected by this approach.
The high sensitivity to topographic contrast places a limit on the range of topography that can
beaccommodated bythis technique. The headsof humanspermatozoa are at the limitof this range
because theyare more rounded than are the guinea-pig or hamstersperm heads. Allof the major
known topographic features of guinea-pig and hamster spermatozoa are readily identified in the
photoelectron images which provide a faithful representation of the surface and correlate well
with the morphology previously established by other microscopic techniques such as optical
microscopy, thin section and replica transmission electron microscopy and scanning electron
microscopy.
Each microscopic technique has a different physical basis for image formation and therefore
provides a different information content. For example, the energy source for photoelectron
production, the u.v. light, penetrates deeply into biological specimens. However, these lowenergy
electrons are easily scattered by organic material and the electrons actually escaping and
contributingto the photoelectron imageoriginate primarilyin the top surfacelayer(Houle, Engel,
Willig, Rempfer & Griffith, 1982). This is normally the plasma membrane unless it is disrupted or
removed as, for example, a consequence of the acrosome reaction. As a result of the short escape
depth of the photoelectrons the surface detail is sharply imaged. This has been described as a high
depth of resolution or a short depth of information (Houle et al., 1982). Objects beneath the surface
can alsocontribute to the image by inducing topography at the surface. For example, marking the
rostral edge of the equatorial segment of spermatozoa is a ridge of the perinuclear theca which is
beneath the plasma membrane, cytoplasm, outer acrosomal membrane, acrosomal matrix, and
inner acrosomal membrane (Fawcett, 1975). This ridge is observed in the photoelectron
micrographs (PI. 1,Fig. 2; PI. 3, Fig 15)not because the electrons forming the image originate from
this perinuclear materialbut because the plasmamembraneand underlying membranesare draped
over it, producing an elevation at the surface. Another example is provided by the photoelectron
images of the midpiece region. The observed periodicity isa resultof the plasmamembranedraped
overthemitochondria whichare knownto becircumferentially orientatedaround the corecomplex
in a helical or irregular pattern (Phillips, 1977).
As the techniques of photoelectron imaging and photoemissive marker technology are
developed further they may contribute to our understanding of the molecular events of fertilization
by allowing the locations of specific cell surfacecomponents of the spermatozoon to be mapped.
We thank Douglas Habliston for the photoelectron micrographs, Lori Evans for preparation of
the figures; and Walter Skoczylas and Dr Gertrude F. Rempfer for continued improvement to the
University of Oregon photoelectron microscope. This work was supported by PHS Grant CA
11695. R.J.M is the recipient of NIH postdoctoral fellowship GM 08712.
134 R. J. Mrsny and O. H. Griffith
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Received 6 August 1984