BIOLOGY OF REPRODUCTION 48, 154-164 (1993)
Evidence that 68-Kilodalton and 54-51-Kilodalton Polypeptides Are Components
of the Human Sperm Fibrous Sheath'
KELLY L. BEECHER, 3 MONA HOMYK, 3 CHI-YU GREGORY LEE,4 and JOHN C. HERR2 '3
Department of Anatomy and Cell Biology and the Centerfor Recombinant Gamete Contraceptive
Vaccinogens,3 University of Virginia, Charlottesville, Virginia 22908
Andrology Laboratory,4 Department of Obstetrics and Gynecology, University of British Columbia
Vancouver, British Columbia, Canada
ABSTRACT
This study characterizes a common antigen recognized by two monoclonal antibodies (mAbs) that immunoreact with the
principal piece of the human sperm flagellum. By means of immunofluorescence microscopy, mAbs S69 (IgM) and S70 (IgG 1)
(WHO Taskforce nomenclature) were observed to bind to the principal piece of methanol- or detergent-permeablized human
spermatozoa, but did not react with live swimming spermatozoa as assessed by immunofluorescence microscopy. Faint immunofluorescence was also seen on the connecting piece in approximately 40% of the spermatozoa. Immunoreactivity in both
regions was resistant to sequential extraction with Triton X-100, sodium thiocynanate, and urea. Pre-embedding electron microscopic immunogold labeling of ejaculated spermatozoa with mAb S69 showed gold particles located on the fibrous sheath. Immunoreactive peptides of 68, 53, and 45 kDa were recognized by both S69 and S70 mAbs on immunoblots of nonreduced human
sperm extracts, while a 68-kDa band and a strongly immunoreactive triplet from 54 to 51 kDa were recognized in reduced sperm
extracts. Human fibrous sheaths were isolated by differential solubilization and centrifugation and characterized by transmission
electron microscopy. The 68-kDa and 54-51-kDa bands were enriched and found to be major polypeptides in the isolated fibrous
sheath fraction. These results suggest that the S69/S70 antigen, which we term SP (sperm protein) (68 kDa, 54-51 kDa), is a
component of the human fibrous sheath.
INTRODUCTION
thesis or assembly of the FS result in immotile sperm and
infertility [6, 7].
In rat sperm the FS consists of up to 17 polypeptides
ranging from 75 to 14.4 kDa [8], and also includes a major
protein of 80 kDa [9, 10]. Several antibodies that have been
generated to the FS have identified constituent proteins.
Monoclonal antibody K32 of Sakai and colleagues [11] recognizes sperm of mice, musk shrew, boar, and human, but
its cognate antigen has not been identified biochemically.
Monoclonal antibody ATC (IgM) of Fenderson et al. [12]
recognizes a 67-kDa antigen in rat and mouse [32], but not
human, sperm FS. Two antibodies synthesized by Jassim et
al. [13-15] recognize antigens in the human FS. One of these
antigens, a protein of 97 kDa, is the only human FS protein
whose apparent mass is known. Amino acid sequences for
FS components are currently not reported for any species.
Thus, possible similarities between FS proteins and other
cytoskeletal elements remain unknown.
The WHO Taskforce on Sperm Antigens recently assembled several new monoclonal antibodies (mAbs) to human
sperm [16], of which two, S69 and S70, demonstrated immunofluorescence of the tail. The present studies were undertaken, in view of the paucity of primary amino acid sequence information on proteins of the FS, to characterize
probes of possible use in cDNA cloning FS components.
We provide morphological and biochemical evidence that
the antigen recognized by S69 and S70, designated SP(68
kDa, 54-51 kDa), is a constituent of the human FS.
The flagellum of the mammalian spermatozoon contains
several cytoskeletal elements, including the fibrous sheath
(FS), the outer dense fibers (ODFs), and the 9 + 2 microtubules of the axoneme [1]. In the midpiece, the mitochondrial sheath surrounds nine ODFs, which in turn encircle the axoneme. The FS encases the ODFs and
microtubules in the principal piece and extends distally beyond the termination of the ODFs to end at the principal
piece/end piece junction. The axoneme, devoid of ODFs
and the FS, continues into the endpiece, completing the
flagellum.
The FS consists of two longitudinal columns connected
by circumferential ribs. The longitudinal columns connect
to the axoneme, replacing ODFs 3 and 8 in the principal
piece [1]. The FS has been reported to be a relatively insoluble structure with extensive disulfide crosslinks [2, 3].
Proteins that make up the FS in rat sperm are assembled
in a distal-proximal direction and are synthesized throughout stages 8-17 of spermiogenesis [4, 5]. Defects in the synAccepted August 22, 1992.
Received April 8, 1992.
'Supported by NIH HD 16767; HD 23789; HD 29099;
Ortho Pharmaceuticals, Contraceptive Research and Development Program
(CONRAD-009); E. VA Med School, under a cooperative agreement with the USAID
(dEP-3044-A-00-6063-00); MRC 5-99802; and the Network of Center of Excellence
(Canada Genetic Diseases Network, No 5-90490). The views expressed by the authors do not necessarily reflect the views of AID or CONRAD.
ZCorrespondence: John C. Herr, Ph.D., Box 439 Medical Center, University of
Virginia, Charlottesville, VA 22908. FAX: (804) 982-3912.
154
HUMAN SPERM FIBROUS SHEATH
MATERIALS AND METHODS
Production of mAbs
S69 and S70 are two anti-human sperm mAbs secreted
by hybridomas generated to acrosome-reacted sperm according to reported procedures [17-19]. Antibody-secreting hybrid clones were screened initially by an indirect immunofluorescence assay using methanol-permeablized
human sperm. Among these antibodies, S69 and S70 were
shown to belong to IgM and IgG 1 immunoglobulin subclasses, respectively, and to immunoreact with the human
sperm flagellum. These antibodies are part of the second
WHO Sperm Antigen Workshop (Dr. D. Anderson, Harvard
Medical School) [16].
Immunofluorescence Microscopy
Motile spermatozoa. Ejaculated spermatozoa were obtained from healthy donors, washed twice in Ham's F-10
medium, and incubated for 30 min in Ham's F-10 at 370C
with 5% CO 2 to allow the live spermatozoa to swim to the
top. Live spermatozoa were collected and the percentage
of motile spermatozoa was determined. A suspension of 1
x 106 sperm (>95% motile) was incubated for 1 h at 4°C
with S69 or S70 mAbs or control IgM (anti-parotid secretory membrane protein; a gift from Dr. Susan Laurie, Department of Anatomy and Cell Biology, University of Virginia, Charlottesville, VA) or IgG antibody (null ascites; a
gift from Debra Koons, Hybridoma Facility, University of
Virginia) at 1:40 in Ham's F-10. After washing, sperm were
incubated for 1 h with a 1:40 dilution of goat anti-mouse
IgM/IgG-fluorescein isothiocyanate (FITC, Jackson Immuno Research Laboratories, West Grove, PA) at 4C in the
dark. After being washed twice, samples were observed as
wet mounts. Percentage of labeled, motile spermatozoa was
determined after the spermatozoa had warmed to room
temperature on the microscope stage.
Fixation,permeablization, and extraction of spermatozoa. Spermatozoa were washed as above and fixed for 5
min in 2% paraformaldehyde. Excess paraformaldehyde was
neutralized using 0.2 M glycine, and spermatozoa were pelleted at 500 x g and resuspended in PBS. Spermatozoa were
cytocentrifuged onto microscope slides and air-dried. Rat,
mouse, musk shrew, boar, and frog spermatozoa from epididymides were collected according to an established protocol [20] and were prepared as above. Some slides were
treated with one or all of the following for 30 min at room
temperature: 1% Triton X-100, 0.6 M potassium thiocyanate
(KSCN), 4 M urea [12]. Other slides were treated with 100%
methanol or left untreated. After washing, slides were blocked
for 30 min in 10% normal goat serum (NGS). Samples were
incubated with a 1:40 dilution of S69, S70, or control IgM
or IgG in 1% NGS for 30 min, washed, and incubated with
the secondary antibody, IgG/IgM-FITC, at 1:40 for 30 min.
After washing, coverslips were mounted using 90% glycerol
and 10% 0.1 M Tris (pH 7.5) with n-propylgallate added to
155
prevent fading. Slides were viewed and photographed immediately on a Zeiss microscope with Nomarski and epifluorescent optics using a 100x objective.
Electron Microscopic Immunocytochemistry:
Pre-EmbeddingAntibody Staining
Human spermatozoa were washed as above and incubated in PBS (pH 8.4) containing 1% NGS and S69, S70, or
a negative control ascites fluid at a 1:100 dilution for 30
min at 20 0C (RT). Goat anti-mouse IgM or IgG conjugated
to 10-nm gold particles (diluted 1:50 with PBS, pH 8.4, containing 0.5% BSA) was incubated with the spermatozoa for
30 min at RT. After two washes, the sperm pellet was taken
up in half-strength Karnovsky's fixative for 30 min at RT.
This was followed by two washes in 0.1 M cacodylate buffer
(pH 7.4) and incubation with 1% osmium tetroxide for 1
h at RT. The spermatozoa were serially dehydrated and
embedded in Araldite (Electron Microscopy Sciences, Fort
Washington, PA). Grids were counterstained with 7% uranyl acetate in methanol followed by 1% lead citrate and
were viewed on a JOEL 100CX transmission electron microscope (Joel Ltd., Tokyo, Japan).
Western Blots
Proteins from washed sperm pellets were extracted using 4% SDS with or without -mercaptoethanol. Insoluble
material was removed by centrifugation; supernatants were
diluted 1:1 with Laemmli buffer [21] and analyzed by SDSPAGE. Twenty micrograms of purified tubulin (a gift from
Dr. Gary Gorbsky, Dept. of Anatomy and Cell Biology, University of Virginia) in Laemmli buffer was also electrophoresed. Electrotransfer of polypeptides to nitrocellulose was
conducted according to Towbin et al. [22]. Total protein
and molecular mass standards were stained with 0.1% amido
black. The immunoblotting method has been described
previously [23]; in our protocol the primary antibody incubation time was 18 h. Primary antibodies used included
S69 and S70, control IgM or IgG, monoclonal mouse antitubulin IgG (a gift from Dr. Tony Frankfurter, Dept of Biology, University of Virginia), and polyclonal rabbit anti-actin (Boehringer-Mannheim, Indianapolis, IN).
Periodate Oxidation Test for CarbohydrateEpitopes
To test whether the epitopes recognized by the S69 and
S70 mAbs were carbohydrates, the method of Woodward
et al. [24] was followed. Briefly, proteins electroblotted onto
nitrocellulose were incubated with periodic acid followed
by sodium borohydride. The nitrocellulose was then blocked
and incubated with S69, S70, or control mAb followed by
peroxidase-conjugated goat anti-mouse IgM or IgG. As a
positive control, a Chlamydomonas flagellar extract was also
periodate-treated; polypeptides in this extract were recognized by a mouse anti-carbohydrate IgG mAb (FlAb#8). Both
flagellar extract and antibody were gifts from Dr. Robert A.
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BEECHER ET AL.
Bloodgood (Dept. of Anatomy and Cell Biology, University
of Virginia).
Spermatozoa Fractionationand Isolation of Human FS
FS isolation. The method of Olson et al. [9] for fractionation of rat spermatozoa and isolation of the FS was
modified to isolate the human sperm FSs. Washed sperm
pellets were extracted in 2% Triton X-100, 2 mM dithiothreitol (DTT) in 50 mM sodium borate (pH 9.0) for 1 h, followed by extraction in 0.3 M sodium thiocyanate, 2 mM
DTT in 50 mM Tris-HCl (pH 8.0) for 2 h. The final extraction was in 4 M urea, 25 mM DTT in 50 mM Tris-HCl (pH
8.0) for 4-5 h. All incubations were done at 4C with agitation. FSs were first separated from heads by centrifugation at 3000 x g for 10 min and then were pelleted from
the supernatant by centrifugation at 10 000 x g for 30 min.
Sperm head isolation. To obtain an enriched head
fraction, further modifications of this procedure were necessary. Heads were dissociated from flagella [25] as follows:
washed human sperm pellets were treated for 15 min with
a 1:30 dilution of n-butylamine in 0.1 M Tris (pH 7.2) containing 0.9% NaCl and 5 mM EDTA. Samples were sonicated
gently for 3 sec at 20% output to dissociate heads from
flagella. Head and flagellar fractions were isolated by centrifugation at 136 000 x g (Rm) for 1 h through a sucrose
step gradient of 1.06, 2.05, and 2.2 M sucrose, as described
by Calvin [26]. The head pellet was washed, then sonicated
three times for 20 sec to break nuclei; deoxyribonuclease
(DNase; in 20 mM Tris-HCI with 10 mM MgCI2) was added
to digest nucleic acids. Proteins were extracted from head
pellets using 4-strength Laemmli buffer [21].
Fraction analysis. The head and FS pellets were analyzed for subcellular organelles by transmission electron
microscopy, and the integrity of the FS was analyzed by
scanning electron microscopy. Supernatants from each extraction step were concentrated by dialysis and lyophilized.
Proteins from the supernatants and pellets were analyzed
by SDS-PAGE and blotted as previously described.
Transmission electron microscopy and morphometric
analysis. Pellets were fixed in half-strength Karnovsky's
fixative for 1 h and embedded in Araldite as previously described. Sections were counterstained using 5% aqueous
uranyl acetate followed by 1% lead citrate.
Fraction percent contamination was determined by
counting the number of FSs, ODFs, axonemes, and heads
in 25 representative sections, then dividing by the total
number of organelles counted per fraction. Each cross section, longitudinal section, or transverse section of a structure was counted as one organelle. Mitochondrial membrane contamination in the FS fraction was determined by
morphometric analysis using a Compaq Diskpro 286 computer and the Bioquant System IV program (R & M Biometrics, Nashville, TN). The areas enclosed by either mitochondrial membranes or FS were traced via transmission
electron micrographs of 10 random fields of the FS fraction
sections. The total area occupied by the mitochondrial
membranes was divided by the total area occupied by both
mitochondrial membranes and FS, resulting in a percent
area of mitochondrial contamination.
RESULTS
Monoclonal antibodies S69 and S70 did not react with
live swimming or intact spermatozoa (negative data not
shown), but did label human spermatozoa made permeable by air-drying, methanol, or Triton X-100 treatment. As
shown in Figure 1, Triton X-100-extracted, formaldehydefixed human spermatozoa were reacted with either S69, S70,
or a control IgG or IgM antibody, followed by a fluorescein-conjugated murine IgG + M secondary antibody. The
immunofluorescence pattern of both S69 and S70 was limited to the principal piece of the sperm flagellum. The head,
midpiece, and endpiece remained unstained. Ninety-six
percent of the spermatozoa had principal piece staining. Of
this 96%, 41% of the spermatozoa also had faint staining at
the connecting piece. The latter staining could be enhanced
by prolonged extraction in Triton and was more pronounced when the S69 antibody was used. Other minor
patterns of immunofluorescence included principal piece
and diffuse head staining (3%) as well as principal piece
and equatorial band staining (2%) (data not shown). Immunofluorescence of the principal piece was retained when
air-dried spermatozoa were sequentially extracted with Triton X-100, sodium thiocyanate, and urea, indicating that the
epitopes of S69 and S70 resisted solubilization with these
agents. S69 and S70 did not react with spermatozoa from
several other species, including rat, mouse, boar, musk shrew,
and frog; preparation of these was identical to that of human spermatozoa (negative data not shown).
At the ultrastructural level, pre-embedding immunogold
labeling indicated that the S69 antigen is associated with
the FS. This association was specific to the surface of the
FS and occurred only when the overlying plasma membrane had been removed. It was not possible to determine
whether this association extended integrally within the FS.
Due to the limitations presented by pre-embedding staining, antigenic sites on the interior of the sperm are inaccessible to the gold probe and thus remain unlabeled despite antibody presence. In Figure 2, both a cross section
and a longitudinal section of the principal piece are shown.
The gold particles can be seen along both the longitudinal
columns and ribs in the cross-sectional micrograph (Fig.
2A) and along the ribs in the longitudinal section (Fig. 2C),
as indicated by the arrows.
The polypeptides recognized by these two mAbs were
analyzed by means of SDS-PAGE and Western blotting. Due
to the relative insolubility of the protein in 1% SDS, 4%
SDS extracts of human sperm samples were required in
these studies. Figure 3 shows the immunoreactive banding
patterns resulting from staining both nonreduced and re-
HUMAN SPERM FIBROUS SHEATH
157
FIG. 1. Immunofluorescence micrographs of mAbs S69 (A) and S70 (C), and corresponding Nomarski images (B and D), indicate the localization of
the antigen recognized by S69 and S70 on permeablized human spermatozoa. Staining by S69 and S70 is limited to the principal piece of the flagellum.
E and F are negative controls. White arrows (fluorescence) and black arrows (Nomarski) indicate the principal piece/end piece junction. x1024.
duced extracts with S69 and S70. Both mAbs recognized the
same polypeptide bands. In the nonreduced extracts, major
polypeptides at 68, 53, and 45 kDa were immunoreactive,
while reduced bands at 68 and a triplet at 54-51 kDa were
observed. The difference in intensity between nonreduced
and reduced polypeptides, especially noticeable for the 45kDa band, may indicate the presence of intrachain disulfide
bonds. The overall intensity of the S70 reactive bands was
slightly lower than that of the S69 reactive bands.
Because of the localization of the S69/S70 antigen in the
principal piece, as well as resistance of the epitopes to solubilization by non-ionic detergents and strong chaotropes,
we considered that the S69/S70 antigen was most likely a
cytoskeletal element. Therefore, immunological cross-reac-
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BEECHER ET AL.
FIG. 2. Electron microscopic immunogold localization of S69 antigen on human spermatozoa. Cross sections (A
and B) and longitudinal sections (C and D of the principal piece of human sperm labeled with either S69 (A and C)
or a negative control IgM antibody, followed by a gold-conjugated secondary antibody. Immunoreactivity is seen only
along the FS at regions where the plasma membrane has been removed (arrows). No staining is seen on the negative
controls. A) x94 118; B) x133 330; C) x79 266; D) x48 898.
tivity studies with other cytoskeletal proteins were performed. In human spermatozoa, two cytoskeletal proteins
have been reported to have immunofluorescence patterns
and molecular masses similar to those of the protein recognized by S69 and S70. These are tubulin [27] and actin
[28], which have molecular masses of 55 kDa and 42 kDa,
respectively. Neither S69 nor S70 recognized purified tubulin on immunoblots, as shown in Figure 4A. When Western blots of identical sperm extracts were incubated with
anti-tubulin, anti-actin, or S69, the results indicated that neither tubulin nor actin were being recognized by S69 (Fig.
4B). The apparent masses of tubulin or actin in the sperm
extract were different from the mass of the S69 antigen.
The diffuse tubulin band, at 55 kDa, was of larger apparent
mass than the major 51-54-kDa triplet; and the 42-kDa actin
band electrophoresed lower than the 45-kDa band. Both
cytoskeletal antibodies (anti-tubulin and anti-actin) were
specific for epitopes common to all forms of tubulin or
actin.
In order to investigate whether the epitopes recognized
by S69 and S70 were carbohydrate or protein, a periodate
oxidation method was used [24]. An antibody, FIAB#8, which
recognizes a carbohydrate epitope of a glycoprotein found
in Chlamydomonas flagellar membranes, was used as a
positive control (Fig. 5, lane 1). Following periodate treatment, the FlAb#8 antibody no longer recognized its anti-
HUMAN SPERM FIBROUS SHEATH
R
NR
R
NR
R
159
NR
R
NR
kDa
11775.548-
28-
19-
AMIDO
S69
S70
NULL
FIG. 3. Western blot analysis of reduced (R) and nonreduced (NR) SDS extracts of human spermatozoa. Total
protein was revealed by amido black stain. Monoclonal antibodies S69 and S70 labeled polypeptides of 68, 54, 53,
51, and 45 kDa in the nonreduced extracts, while the 68-kDa polypeptide and the 51-54-kDa triplet predominated in
the reduced extracts. Control IgM (null) showed no reaction.
gen, due to the modification of the carbohydrate residues
necessary for integrity of its epitope (Fig. 5, lane 2). In contrast, periodate treatment did not affect the ability of the
S69 or S70 mAbs to recognize their cognate epitopes (Fig.
5, lanes 4 and 6).
Both the immunofluorescence and ultrastructural data
suggested that the protein epitopes recognized by mAbs
S69 and S70 were components of the FS. To further investigate this hypothesis, we modified an FS isolation procedure of Olson et al [9] and two procedures for dissociation
of sperm heads and flagella [25, 26] in order to fractionate
human spermatozoa and isolate a fraction enriched in the
human FS (see Materialsand Methods). The procedure resulted in the following fractions: a Triton X-100 soluble
fraction, a sodium thiocyanate-soluble fraction, a urea-soluble fraction, a head pellet, and a urea-insoluble flagellar
pellet.
The subcellular content of the various fractions and the
purity of the separation of heads from flagella were monitored by electron microscopy. The head and flagellar pel-
lets were fixed and embedded for transmission electron
microscopy. In Figure 6, A and B, the transmission electron
micrographs of the FS and head pellet are shown. It was
determined that there was 0.5% contamination of heads and/
or other flagellar components in the FS fraction. Mitochondrial membranes accounted for the majority of the contamination (30.8%) of the FS fraction. The head fraction contained 7% contamination of flagellar components.
Figure 7 presents a Western blot of those fractions obtained in the sperm fractionation procedure that were immunoreacted with mAb S69. Each fraction was either stained
for total protein with amido black (lanes 1, 3, 5, 7, 9) or
immunoreacted with S69 (lanes 2, 4, 6, 8, 10). Fractions
containing sperm components solubilized by Triton (lane
2) and urea (lane 6) showed no immunoreactivity. The sodium thiocyanate-soluble fraction contained two immunoreactive peptides at 53 kDa and 34 kDa (lane 4). The head
fraction contained immunoreactive peptides of 128 kDa and
95-84.5 kDa (lane 8). These bands were not visible in nonDNase-treated head fractions. The urea-insoluble flagellar
160
BEECHER ET AL.
munofluorescence microscopy indicated that the antigen was
not present in either endpiece or midpiece, and was therefore not a component of the axoneme, outer dense fibers,
or mitochondria. Ultrastructural localization of gold particles further indicated that the antigen was internal and flagellar and also revealed this antigen as a component of both
longitudinal columns and circumferential ribs of the FS. Additional evidence that an FS protein was recognized by antibodies S69 and S70 was derived from immunochemical
analysis of the fractions obtained by sequential extraction.
Strong immunoreactivity was noted in the fraction that contained predominantly FSs. We have designated these im-
FIG. 4. Comparison of the S69 and S70 antigen with tubulin and actin.
A) Western blots of purified bovine brain tubulin reacted with amido black
protein stain (lane 1), S69 (lane 2), or S70 (lane 3). Neither antibody reacted
with the purified tubulin. B) Western blots of reduced sperm extracts reacted with amido black stain (lane 1), monoclonal anti-tubulin (lane 2), S69
(lane 3), or polyclonal anti-actin (lane 4). Tubulin runs as a diffuse band
centering around 55 kDa, which is slightly higher than the central triplet of
the S69-labeled bands. The 42-kDa actin band is lower than the lowest band
(45 kDa) of the S69 pattern.
fraction, which contained mainly FS (see Fig. 6A), showed
the 68-kDa and 54-51-kDa pattern of polypeptides (lane
10).
The major polypeptides detected by amido black staining in the insoluble flagellar fraction were wide bands centered at 108 kDa, 88 kDa, 75 kDa, and 53 kDa (lane 9).
These bands, resulting from extraction of purified human
FSs in 4% SDS and -mercaptoethanol (lane 9), showed
indistinct boundaries and minor polypeptides of varying
apparent mass, most likely reflecting the relative insolubility of the FS under aqueous conditions and its partial resistance to detergent solubilization and reduction. The immunoreactive 68-kDa and 54-51-kDa polypeptides (lane 10)
correspond to several of the major amido-stained polypeptides (lane 9). There is no doubt that the 53-kDa immunoreactive complex in lane 10 corresponds to the 53-kDa
band in lane 9. However, the 68-kDa immunoreactive band
in lane 10 runs at the base of a wide, 68-74-kDa band with
indistinct boundaries in lane 9.
DISCUSSION
The antigen recognized by S69 and S70, SP(68 kDa, 5451 kDa), was localized to the FS of the principal piece. Im-
FIG. 5. Carbohydrate analysis of S69 and S70 epitopes by periodate
oxidation. Experimental lanes (E) were incubated with periodate followed
by sodium borohydride (see Materials and Methods); in control lanes (C),
the periodate step was omitted. Lanes 1 and 2 are Chlamydomonas flagellar extracts used as a positive control for a periodate-sensitive epitope
of the mAb, FIAb#8. This antibody recognized a series of carbohydratecontaining polypeptides from 200 kDa to 60 kDa in the control lane (1),
which were no longer immunoreactive following the modification of carbohydrates by periodate (lane 2). Lanes 3-6 are reduced human sperm extracts reacted with S70 (3, 4), or S69 (5, 6). While the FIAb#8 antibody
failed to recognize the flagellar epitope after periodate oxidation (lane 2),
the S69/S70 polypeptides continued to be immunoreactive following periodate treatment (lanes 4 and 6).
HUMAN SPERM FIBROUS SHEATH
FIG. 6. A) Transmission electron micrograph of enriched FS fraction. All internal flagellar structures have been
solubilized, leaving only an intact FS. Mitochondrial membranes are also visible in this fraction (arrows). x21 000. B)
Transmission electron micrograph of enriched head fraction. x10 500.
161
162
BEECHER ET AL.
FIG. 7. A) Western blot of fractionation of human spermatozoa by differential solubilization. Lanes 1 and 2, Triton
X-100-soluble fractions; lanes 3 and 4, sodium thiocyanate-soluble fractions; lanes 5 and 6, urea-soluble fractions;
lanes 7 and 8, head fractions; lanes 9 and 10, urea-insoluble flagellar fractions. Lanes 1, 3, 5, 7 and 9 show total
protein by amido black staining; lanes 2, 4, 6, 8, and 10 show S69 immunoreactivity. The Triton and urea fractions
did not react with S69; the sodium thiocyanate, head, and FS fractions did react. The sodium thiocyanate lane has
bands at 53 kDa and 34 kDa (lane 4), and the head fraction has bands at 128.5 kDa and a cluster from 95 to 84.5 kDa
(lane 8). The FS fraction has the characteristic pattern of bands at 68 kDa and 54-51 kDa seen in previous blots.
Amido black staining of the FS fraction revealed wide major bands centered at 108, 88, 75, and 53 kDa (lane 9), two
of which correspond to immunoreactive polypeptides of similar mass in the immunoblot of the FS fraction (lane 10).
munoreactive bands SP(68 kDa, 54-51 kDa), since these
are the dominant polypeptide bands seen in most preparations.
SP(68 kDa, 54-51 kDa) consists of immunoreactive polypeptides at 68, 54-51, and 45 kDa when extracted by 4%
SDS from whole sperm or urea-insoluble flagellar pellets.
Nonreduced conditions reveal an immunoreactive pattern
of 68 kDa, 53 kDa, and 45 kDa. SP(68 kDa, 54-51 kDa) is
highly insoluble, as indicated by the need for a high concentration of SDS for solubilization of quantities sufficient
for immunoblotting as well as by its relative insolubility in
Triton X-100, sodium thiocyanate, and urea. Insolubility is
a characteristic of a number of cytoskeletal proteins, such
as keratins, and also of previously reported FS-associated
proteins in other species [9, 12]. Presence of disulfide bonds
has also been reported for a number of FS and other sperm
cytoskeletal components [2, 9]. SP(68 kDa, 54-51 kDa) con-
tains disulfide bonds, as indicated by the marked decrease
in intensity of the 45-kDa band in reduced preparations as
well as by the higher concentrations of immunoreactive
polypeptides extracted when reducing agents were used.
Although the fluorescence patterns of S69 and S70 were
similar to those reported previously for certain tubulin [27]
and actin [28] antibodies, the S69 and S70 reagents did not
recognize either tubulin or actin. Other cytoskeletal proteins previously reported in human spermatozoa include
myosin, a 210-kDa protein found mainly in the neck region;
vimentin, a 58-kDa protein found in the equatorial segment
of the head; and a p230 immunoanalogue of ct-spectrin found
in both head and principal piece [29]. Also, Ochs and colleagues [30] reported a 53-kDa protein that stained acrosomal cap in human spermatozoa and also stained epithelial cells in a keratin-like pattern, although normal anti-keratin
antibodies (usually at 68 and 57 kDa) did not recognize the
HUMAN SPERM FIBROUS SHEATH
sperm protein. Some investigators [2, 29] have speculated
that the FS, in view of its insolubility and disulfide binding,
contains keratin-like proteins; but apparently the FS proteins do not cross-react with keratin antibodies. Other intermediate filament antibodies do not react with human
spermatozoa [29], with the exception of the neurofilament
antibody used by Jassim et al. [14] to characterize the 97kDa FS protein.
Although SP(68 kDa, 54-51 kDa) was not analyzed for
carbohydrate content, neither the epitope recognized by S69
nor the epitope recognized by S70 was affected by periodate modification. It is likely that these antibodies recognize
a peptide epitope rather than a carbohydrate epitope, and
are therefore potentially useful as cloning probes to screen
cDNA testis libraries.
The 68-kDa immunoreactive polypeptide and a large
quantity of the 54-51-kDa immunoreactive polypeptides were
observed within the insoluble flagellar fraction, which was
found ultrastructurally to be predominantly FS. Another immunoreactive band of a smaller apparent mass (34 kDa)
was present in the sodium thiocyanate-soluble fraction along
with a 53-kDa band. This mass difference is probably due
to proteolysis of the 68-kDa polypeptide during extraction
or dialysis, resulting in two smaller 34-kDa polypeptides.
Another possibility is that this is a minor component that
either 1) needs the chaotrope for extraction, or 2) was
present at a concentration too low for detection in whole
sperm extracts, but sufficient for detection by the extraction
procedure.
A cluster of immunoreactive peptides, much larger in
molecular mass (128.5 kDa, 95-84.5 kDa), was released from
the highly enriched head fraction by DNAse treatment. This
group of bands was seen only on head preparations when
DNAse was used; it was absent in other extracts even when
the gel was overloaded with protein. Therefore, the polypeptides in this fraction appear to be nuclear in origin. It
is possible that these polypeptides, if nuclear, are part of
the nuclear cytoskeleton and that they contain contain an
epitope that cross-reacts with the S69 antibody. The head
fraction immunoreactivity may alternatively indicate a precursor protein or an aggregate of the 68-kDa and 54-51kDa polypeptides covalently bonded to another protein found
in the sperm head. On this latter point it is interesting to
note that the ATC mAb to mouse and rat FS [12, 32] also
recognizes a protein showing variable mass, in this case
correlating with the type of spermatogenic cells. ATC recognizes an Mr 67 000 protein in round spermatids, in mixtures of condensing spermatids and residual bodies, and in
isolated FSs; but it reacts with an Mr 78 000 protein in mixtures of leptotene and zygotene primary spermatocytes,
pachytene spermatocytes, and round spermatids [32].
Although SP(68 kDa, 54-51 kDa) has one band of 68
kDa, similar in molecular mass to the 67-kDa protein found
in rat sperm FS [12], our antibodies did not cross-react with
FSs of several other species including rat, nor did the
163
monoclonal reagent of Fenderson [12] cross-react with human spermatozoa. Any similarities between FS proteins
in
humans and those in other animals will be elucidated
only
when cDNAs of the respective proteins can be
obtained.
The deduced amino acid sequence, when compared
with
others, may give information concerning the
similarities
between FS proteins and other cytoskeletal elements,
including proteins of the ODFs, for which a cDNA
has been
reported [31]. Sequence data may indicate whether
spermatozoa utilize unique cytoskeletal proteins in the FS;
such
studies may also provide sequence information that can
be
used to investigate the genetic basis of those flagellar
abnormalities of the FS that contribute to infertility
[6, 7].
ACKNOWLEDGMENTS
The authors would like to thank Jan Redick, BonnieSheppard,
and Peter Lauren
for technical assistance; Barbara Kurth, Jim Foster, Ken
Klotz, and Richard Wright for
helpful advice; and Charles Flickinger and E. Mitch
Eddy for critical reading of this
manuscript.
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