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Formation of the outer dense fibers during spermiogenesis in the rat.

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T H E ANATOMICAL RECORD 202463-471 (1982)
Formation of the Outer Dense Fibers
During Spermiogenesis in the Rat
MARGARET J. IRONS A N D W E S CLERMONT
Department of Anatomy, McGill Uniuersity, Montreal, Quebec, Canada H3A
2B2
ABSTRACT
The morphogenesis of the outer dense fibers (ODF)in rat spermatids has been studied by electron microscopy, and the synthesis and incorporation of proteins into the ODF during this process have been followed by radioautography using 3H-prolineand 3H-cystineas precursors for ODF proteins. In the
first phase (steps 8-14), nine very fine fibers termed anlagen of the ODF develop in
association with the microtubule doublets. These first appear along the most proximal portion of the axoneme in step 8 of spermiogenesis; during steps 9-14 they
gradually increase in length in a proximal-to-distal direction, being first observable along the forming midpiece and later along the principal piece as well. In the
second phase (steps 15-16), the rudimentary fibers suddenly increase in diameter,
with the most dramatic growth occurring in step 16, and assume a close resemblance to the mature ODF. This striking transformation, which appears to result
from simultaneous deposition of electron-dense material along the length of
anlagen of the ODF, coincides with a period of rapid incorporation of 'H-prolineand 3H-cystine-containingproteins, which become permanent structural components of the ODF. These proteins, which comprise the bulk of the ODF, are synthesized in the cytoplasm of spermatids during the acrosome and early maturation
phases. In the final phase (steps 17-19) the fibers continue to enlarge very slowly,
assuming their definitive form in step 19of spermiogenesis. Thus formation of the
ODF in the rat is a lengthy multistep procedure, requiring from step 8-19 of spermiogenesis and utilizing proteins synthesized throughout most of this period.
The outer dense fibers are a set of nine proteinaceous columns that surround the axoneme along much of the length of the flagellum
of mammalian spermatozoa. Each longitudinally oriented fiber is largest at the proximal
end of the midpiece where it is continuous with
one of the nine striated columns of the connecting piece and tapers progressively along the
tail, ending at a point along the principal piece
where it is attached to one of the doublets of
the axoneme. Each outer dense fiber has a distinctive size and cross-sectional profile, and
those designated 1, 5, and 6 are generally
larger than the others. (see review by Fawcett,
'75). The use of selective staining procedures
for electron microscopy has revealed that the
outer dense fibers are comprised of two layers-a narrow, electron-dense cortex, and a
central medulla (Telkka et al., '61; Bawa, '63;
Gordon and Bensch, '68; Fawcett, '70; Olson
and Sammons, '80). In several mammalian species, the cortex of the outer dense fibers displays, in surface replicas, a regular pattern of
0003-276X/82/2024-0463$03.00 0 1982 ALAN R. LISS. INC.
oblique striations (Woolley,'71; Pedersen, '72;
Baccetti et al., '73; Pihlaja and Roth, '73;
Phillips and Olson, '74; Espevik and Elgsaeter,
'78). In the rat, the cortex is reportedly composed of 6-8 nm globular subunits forming a
single continuous layer over the abaxial surface of the fibers, but is replaced by longitudinally oriented satellite fibrils facing the axoneme (Fawcett, '75; Olson and Sammons, '80).
Biochemical analyses of isolated outer dense fibers reveal four major polypeptide bands in
spermatozoa of rats and bulls (Price, '73; Baccetti et al., '73; '76 a,b; Calvin et al., '75; Olson
and Sammons, '80),and at least six bands in
mice (Bradley et al., '81). In each case, one high
molecular weight component, and several low
Keceived J u l y 30. I Y f i I . accepted A u p s t 24. I Y X I
Margaret J. Irons's present address i s Department of Anatomy,
Division of Histology. University of Toronto. Toronto, Ontario M5S
1A8.
Address reprint requests to Dr. Yves Clermont. Department of
Anatomy, McGill University. 3640 University Street. Montreal.
Quebec, H3A 2B2.
464
M.J. IRONS AND Y . CLERMONT
MW polypeptides have been identified. The
low MW proteins are rich in cysteine and proline (Price, '73; Baccetti et al., '76a; Olson and
Sammons, '80),and are thought to be present
in the medulla of the fibers (Olson and Sammons, '80).
Despite the considerable effort that has been
directed toward ultrastructural and biochemical characterization of the outer dense fibers,
investigation of their mode of formation is an
area that previously has been largely neglected. Changes in the appearance of the fibers
have been noted anecdotally in several ultrastructural studies of spermiogenesis (Challice,
'52; Yasuzumi, '56; de Kretser, '69; Fawcett and
Phillips, '69, '70; Sapsford et al., '70; Fawcett et
al., '71; Dooher and Bennett, '73). but there has
not been a systematic investigation of their
formation in any mammalian species. From
the past studies it has been demonstrated that
the outer dense fibers arise during the acrosome phase as very slender fibers in intimate
relation to the axonemal doublets. These fine
fibers subsequently thicken late in spermiogenesis to form the definitive fibers characteristic of the mature spermatozoon. The
details of these events, however, such as the
precise points during spermiogenesis at which
the fibers first appear and are subsequently
transformed, the duration of the entire process
and the mode of assembly of the fibers at different levels along the flagellum, are lacking.
Furthermore, very little is known of the origin
of the proteins which comprise the outer dense
fibers, either in terms of 1)when they are synthesized during spermiogenesis, 2) where they
are synthesized within the cell, or 3) how they
are transported and subsequently assembled
into the definitive fibers. The present investigation was undertaken in an attempt to answer these questions. The sequence of events
in the formation of the outer dense fibers during spermiogenesis in the rat was determined
by electron microscopy. In addition, the synthesis and incorporation of proteins into the
outer dense fibers was followed by light and
electron microscope radioautography after injection of tritiated amino acids.
MATERIALS AND METHODS
Ultrastruct ural studies
Testes of adult Sherman rats (350 g) were
perfused through the abdominal aorta with 5%
glutaraldehyde buffered with 0.1 M sodium
cacodylate, pH 7.4, and small blocks of the fixed tissue were washed in buffer, postfixed in
1%OsO,, dehydrated, and embedded in Epon.
Semithin (half-micrometer thick) and thin 'sections were cut on an ultramicrotome. The
semithin sections stained with toluidine blue
were used for identification of the 14 stages of
the cycle of the seminiferous epithelium
(Leblond and Clermont, '52) and the steps of
spermiogenesis.' Thin sections of seminiferous
tubules in each stage of the cycle were routinely stained with uranyl acetate and lead citrate
and examined in a Siemens Elmiskop 1A at 80
kV .
Preparation of LM and E M radioautographs
The radioautographic experiments were designed to follow the synthesis and incorporation of proteins into the outer dense fibers.
Because the low MW proteins of these fibers
are known to contain unusually high proportions of proline and cysteine (Price, '73; Baccetti et al., '76a; Olson and Sammons, '80) these
amino acids were selected as suitable precursors for the outer dense fiber proteins. Because
'H-cysteine was not available, 'H-cystine was
used in its place. It was assumed that this
would be converted to cysteine within the cells.
Radiolabeled amino acids L-[2,3 - 'H(N)]proline (New England Nuclear) and L-[3,3' - 'HI
cystine (Amersham Corp.) were evaporated to
dryness, resuspended in lactated Ringer's solution, and used for intratesticular injections into adult Sherman rats. Each injection, having
a volume of 0.1 ml, contained 1.0 mCi of radioactivity. Sixteen rats were divided into four
groups, given an intratesticular injection of
'H-proline, and subsequently perfused with
glutaraldehyde as described above, at intervals of 1 hour, and 2,4, and 13 days postinjection. Prior to fixation, animals in the 1-hour interval were first perfused briefly with a large
excess of cold L-proline to prevent nonspecific
binding of radioactive proline to the tissues.
One rat was injected with 1.0mCi of 'H-cystine
and perfused 1 hour later. Samples of fixed tissues were taken from the injected testes of all
experimental animals and from the epididymides of the 13-day group and processed for
light and electron microscope radioautography. Semithin sections from each experimental animal were stained with iron hematoxylin,
'Rat spermiogenesis. as seen with the light microscope in toluldine
blue stained semithin Epon sections. is illustrated in Figure 1 of Clermont and liamhourg. 197X. This drawing shows the main changes taking place in the spermatids during the 19 stcps of spermiogenesis.
OUTER DENSE FIBER FORMATION
dipped in Kodak NTB-2 emulsion, stored for
various durations at 4°C in dry light-proof containers, and developed in Amidol. Exposure
times were 29 days for the 1-hour interval, 10
days for the 2-, 4- and 13-day intervals after
3H-prolineinjection, and 15 days following administration of 3H-cystine.For preparation of
electron microscope radioautographs, thin sections were cut from selected portions of radioactive seminiferous tubules in all 14 stages of
the cycle from each experimental group and
placed on celloidin-coated glass slides that
were subsequently coated with a thin ( 5 nm)
layer of carbon. These slides were then dipped
in Ilford L4 emulsion, exposed for 60 days at
4"C, and developed in Kodak D19b developer
according to the method of Kopriwa ('73).
Following development, the celloidin coat was
removed by brief immersion in amyl acetate or
glacial acetic acid, and the sections were
routinely stained for electron microscopy and
examined in the EM.
RESULTS
Electron microscopic appearance of the outer
dense fibers during spermiogenesis
During steps 1-7 of spermiogenesis in the
rat, the spermatid flagellum is composed simply of an axoneme ensheathed in a sleeve of
cytoplasm delimited by the plasma membrane.
During step 8, anlagen of the outer dense fibers
begin to develop in intimate association with
the axoneme. In cross section, these slender fibers appear as electron-dense masses immediately adjacent to the outer aspect of each microtubule doublet (Fig. l). From the beginning
the anlagen of fibers 1,5, and 6 are larger than
the others and have an ovoid cross-sectional
profile; the anlagen of the remaining six fibers
are represented by minute studlike processes
that project from between the paired microtubules (Fig. 1).At the time of their first appearance in step 8, the rudimentary fibers are
present exclusively along the most proximal
portion of the future midpiece of the flagellum-that is, that part closest to the nucleus.
By the beginning of step 12 they are observable along the entire length of the midpiece,
and by the end of step 14 they are also found
along the proximal segment of the principal
piece, which has an ovoid cross-sectional
outline (Fig. 2). At the level of the principal
piece, the anlagen of fibers 3 and 8 join the axonemal doublets to the longitudinal columns of
the developing fibrous sheath and do not
change further (Figs. 2,3). As seen in the midpiece, the anlagen of fibers 1,5, and 6 are largcr
465
than the others in the proximal segment of the
principal piece; the remaining fibers associated
with doublets 2, 4, 7, and 9 are very small and
poorly resolved at this level (Fig. 2). In the
distal segment of the principal piece, which is
circular in cross section, none of the rudimentary fibers is clearly discernible (Fig. 3).
During step 15 of spermiogenesis, the anlagen of the outer dense fibers begin to increase in diameter and change shape. Fibers 1,
5 , and 6 remain larger than the others and assume a kidney-shaped cross-sectional appearance. The rest of the fibers, formerly seen as
studlike projections, at this stage become
clearly visible as hemicylindrical electrondense masses joined to the doublets (Figs. 4,5).
From the proximal to distal end of the principal piece, the fibers taper progressively and
terminate at various levels, the largest ones (1,
5, and 6) extending the greatest distance, but
ending proximal to the round segment of the
principal piece (Fig. 6).Thus from their first appearance in step 8 until the end of step 15, the
outer dense fibers exist as miniscule fibers intimately related to the axoneme and as such
bear little resemblance to the outer dense fibers of the mature spermatozoon (Fig. 9).
In step 16 of spermiogenesis, immediately
following the formation of the midpiece, the
anlagen of the outer dense fibers enlarge very
rapidly, suddenly taking on an appearance
closely similar to that of the fully differentiated outer dense fibers (Fig. 7). This striking
transformation appears to result from rapid
deposition of a large amount of electron-dense
material onto the abaxial surfaces of the fiber
anlagen. As in the mature spermatozoon, the
transformed fibers in a step 16 spermatid are
most massive at the level of the midpiece and
taper progressively along the. principal piece
(Figs. 7, 8).During the remaining steps 17-19
of spermiogenesis the fibers continue to grow
very slowly, and with the development of the
satellite fibrils in step 19, assume the definitive characteristics of the outer dense fibers of
the rat spermatozoon (Fig. 9).
Flagellar labeling patterns during
spermiogenesis
The incorporation of protein into the forming
outer dense fibers was investigated by a light
microscopic analysis of flagellar labeling patters in radioautographed testis sections after
administration of 3H-prolineor 3H-cystine.One
hour after intratesticular injection of either radiolabeled amino acid, the flagella of step 8-15
spermatids appeared weakly labeled but, in
466
M.J. IRONS AND Y. CLERMONT
Figs. 1-9. Cross sections through the future midpiece
(MP) and proximal (PPP) and distal (DPP)segments of the
principal piece of rat spermatids showing the appearance of
the forming outer dense fibers a t the indicated steps of sper-
miogenesis. A) anlagen of the outer dense fibers: LCI
longitudinal column of the fibrous sheath: SF) satellite
fibrils. x 49,800.
contrast, those of step 16 spermatids were intensely labeled (Fig. 10). As in step 8-15 spermatids, the tails of more advanced spermatids
(steps 17-19) were also weaklylabeled after the
1-hourinterval. The flagella of these older spermatids became heavily labeled, however, with
increasing time intervals after injection. For
example, 2 or 4 days after injection the flagella
were heavily labeled in spermatids in step 1618, or 16-19, respectively (Fig. 11).Similarly,
at 13 days postinjection, spermatozoa with
heavily labeled tails were abundant in radioautographed sections of the caput epididymis.
In order to determine the site of the radioactivity in the labeled flagella, thin sections of
seminiferous tubules known to contain spermatids with heavily labeled tails were analyzed by E M radioautography. The flagella of
these cells appeared highly labeled in the electron microscope. In longitudinal sections revealing all three components of the midpiecei.e., the outer dense fibers, mitochondrial
OUTER DENSE FIBER FORMATION
sheath, and axoneme-the majority of the
silver grains was centered over the outer dense
fibers; however, a number of grains also a p
peared over the latter two structures (Fig. 12).
In grazing longitudinal sections that passed
through only the mitochondrial sheath, how
ever, the midpiece was weakly labeled relative
t o that of more deeply sectioned flagella, indicating that the mitochondrial sheath was not
the main source of radioactivity in these highly
labeled tails (Fig. 13).
DISCUSSION
Time course and mode of formation of the
outer dense fibers
The morphological findings presented here
indicate that formation of the outer dense
467
fibers in rat spermatids is a lengthy process
that begins in step 8 and ends in step 19 of
spermiogenesis hence having a duration of
about 13 days (Clermont et al., '59). I t may be
subdivided into three distinct phases: the
period of formation of the anlagen of the outer
dense fibers (steps 8-14), the period of growth
in diameter of the anlagen (steps 15- 16),and
the period of formation of the definitive outer
dense fibers (steps 17-19). As in other rodent
species studied (de Kretser, '69;Sapsford et al.,
'70; Fawcett and Phillips, '70; Dooher and Bennett, '73), the anlagen of the dense fibers in the
rat form in association with the doublets of the
axoneme. It was not previously appreciated,
however, that their growth along the axoneme
is unidirectional. Fawcett and Phillips ('70)
suggested that the initiation of the develop-
Fig. 10. Light microscope radioautograph of a
Fig. 11. LM radioautograph from the 4day interval
seminiferous tubule in stage I1 of the cycle showing the after injection of 'H-proline. Note the heavily labeled
labeling patterns of the various classes of cells within the flagella of the nearly mature step 19 spermatids projecting
seminiferousepithelium 1 hour after injection of 3H-proline. into the lumen of the seminiferous tubule. x 680.
A step 16 spermatid with a highly labeled flagellum is seen
in longitudinal section in the center of the field (arrows).x
680.
468
M.J. IRONS AND Y. CLERMONT
Fig. 12. EM radioautograph prepared from the same
tubule depictad in Figure 11. The majority of the silver
grains seen over the heavily labeled flagellum of this step 19
spermatid appears to overlie the outer dense fibers (ODF);
however, several also reside over the axoneme (AX)and
mitochondria1 sheath (MS). x 26,145.
Fig. 13. EM radioautograph from the 1-hour interval
after injection of 'H-proline showing the midpiece region of
two adjacent late step 15 spermatids in longitudinal section.
The flagellum on the left has been sectioned through the
newly formed mitochondrial sheath, whereas the plane of
section passed deeper in the tail on the right, revealing the
axoneme and enlarging outer dense fibers a s well as the
mitochondria. Note that the relative intensity of labeling is
much greater in the flagellum on the right. x 14,805.
OUTER DENSE FIBER FORMATION
ment of these fibers took place at about the
same time along their entire length. In contrast, the present observation that the anlagen
of the outer dense fibers appear at increasingly
more distal points along the flagellum in successively older spermatids strongly indicates
that growth of the fiber anlagen is in a proximal-to-distal direction during steps 8-14. I t is
intriguing to note that the anlagen of the longitudinal columns of the developing fibrous
sheath, which also form in association with two
of the axonemal doublets (3 and 8),grow along
the tail in a distal-to-proximal direction during
steps 2-17 (Irons and Clermont, '82).
The striking growth of the outer dense fibers
reported to occur late in spermiogenesis in the
human and the guinea pig (de Kretser, '69;
Fawcett and Phillips, '69) takes place shortly
after formation of the midpiece, i.e., in step 16
of spermiogenesis in the rat. Indeed, this was
noted in two early electron microscopic studies
of rat spermiogenesis in which the enlarged
outer dense fibers became visible only at this
time (Challice,'52; Yasuzumi, '56). Similarly in
the bandicoot rat, the outer dense fibers
assume the shape of the mature fibers immediately after midpiece formation (Sapsford
et al., '70). The remarkable transformation of
the fibers in step 16 spermatids appears to
result from the relatively rapid deposition of
electron-dense material onto the surface of the
anlagen of the outer dense fibers; indeed, the
duration of step 16 (29 hours) is short compared to the 20-21-day duration of spermiogenesis in the rat (Clermontet al., '59).This
deposition process appears to take place simultaneously along the length of the fibers, as suggested by Fawcett and Phillips ('69, '70). In
contrast to what these investigators observed
in several other species, in the rat the dense
fibers appear to remain closely associated with
the axonemal doublets following the period of
enlargement.
Source of radioactivity in labeled flagella
One hour after injection of labeled amino
acids, the flagella of spermatids seen in radioautographed semithin sections appeared virtually unlabeled in steps 8-15 and remarkably
heavily labeled in step 16. This striking initiation of flagellar labeling coincided precisely
with the time of rapid growth of the outer
dense fibers observed by electron microscopy.
It was therefore tempting to think that the
labeling of the flagellum in step 16 was due to
incorporation of proline- and cysteine-containing proteins into the outer dense fibers. In-
469
deed, in E M radioautographs the majority of
the silver grains seen over the midpiece was
located over the outer dense fibers; however,
owing to the limited resolution of this technique, these grains could also have been due to
a radioactive source in either of the other two
flagellar structures present at this level -i.e.,
the mitochondrial sheath or the axoneme. The
latter possibilities are improbable, however,
since the mitochondrial sheath alone, as seen in
grazing sections through the midpiece, was
very weakly labeled in E M radioautographs
and the axoneme, which is fully formed very
early in spermiogenesis, was essentially unlabeled during steps 8-15 and hence would be
unlikely to become labeled in step 16. Thus it
seems reasonable to conclude that the observed flagellar labeling was mainly due to
radioactive proteins in the outer dense fibers.
That these proteins did indeed become permanent structural elements of the flagellum was
indicated by the flagellar labeling patterns at
longer intervals after injection. The observation that the most advanced cells with labeled
flagella at 2,4, and 13 days postinjection were
respectively step 18 spermatids, step 19 spermatids and caput epididymal spermatozoa
corresponded precisely to the expected progression of these cells through spermiogenesis,
calculated on the basis of the known kinetics of
spermiogenesis in the rat (Clermont et al., '59).
These data therefore supported the concept
that the radioactive constituent that was incorporated into the flagellum in step 16 r e
mained associated with it during subsequent
steps of spermiogenesis and after release from
the seminiferous epithelium. Thus the evidence strongly favors the conclusion that the
source of radioactivity in the highly labeled
flagella was proline- and cysteine-containing
proteins which became permanent constituents of the outer dense fibers. The observation
that the sudden incorporation of proteins by
the outer dense fibers in step 16 coincides with
a period of rapid addition of electron-dense
material onto the surface of the fiber anlagen
suggests that at the time of incorporation,
these proteins may become polymerized into a
form visible by electron microscopy.
Origin of flagellar proteins
The radioautographic data provided new information on the timing of flagellar protein
synthesis during spermatogenesis. One hour
after injection of 3H-prolineor 3H-cystine, the
outer dense fibers of step 16 spermatids were
heavily labeled, indicating that large amounts
470
M.J. IRONS AND Y. CLERMONT
of proteins were newly synthesized, transported, and incorporated into the outer dense
fibers within the step 16 spermatids themselves. Radioautographic data from the longer
intervals showed, however, that not all of the
proteins incorporated into the outer dense
fibers in step 16 were synthesized during that
step of spermiogenesis. Two or four days after
injection of 3H-proline, as after 1 hour, the
spermatid tails were weakly labeled in step
8-15, but heavily labeled beginning in step 16.
However, in this case the labeled proteins in
the step 16 flagella had been synthesized at the
time of the injection 2 or 4 days previously, i.e.,
when the cells were respectively in steps 12
and 15 of spermiogenesis. Thus at least some
of the proteins destined for incorporation into
the outer dense fibers in step 16 were synthesized during the acrosome phase as well as early in the maturation phase. Whereas the
proteins incorporated during step 16 clearly
represented the bulk of the outer dense fiber
proteins, no doubt other of the proteins synthesized during steps 8-15 were utilized in earlier
stages, during the formation of the anlagen of
the outer dense fibers, as well as other flagellar
components that are assembled during this
time. Indeed, proteins that become incorporated into the forming connecting piece and
fibrous sheath during steps 8-17 have been
shown by radioautography to originate in the
cytoplasm of step 8-17 spermatids.’ Using a
different approach, OBrien and Bellve (’SO)
came to a similar conclusion-i.e., that the
SDS-insoluble proteins of the sperm tail (components of the basal plate, connecting piece,
outer dense fibers, fibrous sheath, and outer
mitathondrial membranes) are synthesized by
mouse spermatids, with a peak of activity during midspermiogenesis, at a time that would
appear to correspond to the acrosome and early maturation phases.
The answers to the questions of where within
the spermatids the flagellar proteins are synthesized and how they are transported to the
flagellum remained elusive. From the previous
radioautographic analysis of protein synthesis
it was clear that these proteins were synthesized in the general cytoplasm of the spermatid
rather than within the periaxonemal cytoplasm, which is in fact devoid of ribosomes.
Within the cytoplasmic lobule of step 8-17
’Irons. Margaret .J. 1980 Formation of the flagellum in the rat spermatid. Ph.D. thesis, McGill University. A more complete analysis of
protein synthesis during spermiogenesis in the rat, as visualized hy
radioautography. will be presented separately.
spermatids, however, no particular structure
or localized region of the cell could be implicated in flagellar protein synthesis or assembly.
No obvious precursor of any flagellar structure
was recognizable by electron microscopy, and
the distribution of labeled proteins, as visualized by LM and EM radioautography after injection of 3H-proline or 3H-cystine, was
remarkably uniform throughout the cytoplasmic lobules of these cells. The radioautographic observations could be interpreted as
evidence for synthesis or storage of flagellar
proteins throughout the spermatid cytoplasm;
however, owing to the nonspecific nature of the
precursors, it is not possible by this technique
to distinguish flagellar proteins from any other
proteins within the cytoplasm. Clearly the development of more specific markers for detection of flagellar proteins will be required for the
further pursuit of knowledge in this area.
ACKNOWLEDGMENTS
The assistance of Dr. M. Lalli during the
course of this work is acknowledged.
This work was supported by a grant from the
Medical Research Council of Canada.
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