MOLECULAR REPRODUCTION AND DEVELOPMENT 25:384-392 (1990)

Production and Characterization of Monoclonal Antibodies to the Mammalian Sperm Cytoskeleton THOMAS H. MAcRAE,~BOD0 M.H. LANGE? AND KEITH GULL3 'Department of Biology, Dalhousie Uniuersity, Halifax, Nova Scotia, Canada; 'Biological Laboratory, University of Kent, Canterbury, Kent, England; "Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Manchester, Manchester, England The cytoskeleton exerts a diABSTRACT rect effect on the function of sperm by influencing the distribution of subcellular organelles and plasma membrane molecules. We have prepared six monoclonal antibodies to Triton X - l 00-insoluble components of the bull sperm cytoskeleton. One of the antibodies reacts with a detachable portion of the bull sperm acrosome. The remainder include an antibody that recognizes the principal and end piece of the tail and another that is specific to the middle piece. Two of the antibodies yield dissimilar staining patterns of the neck region and the tail, and the final monoclonal antibody stains the subacrosomal region and a detachable acrosomal domain of bull sperm. The cross reactivities of the antibodies with hamster sperm and PtK, cells are described, as is the recognition of bull sperm polypeptides on western blots. The results suggest that these antibodies will provide interesting insights concerning the role of the cytoskeleton in sperm development and function.

Key Words: Immunofluorescence, Immunoblotting, Spermatozoa

INTRODUCTION Mammalian spermatozoa, although not all morphologically identical, are highly polarized cells divided into a small number of distinct structural domains (Fawcett, 1975; Friend, 1982; Longo et al., 1987; Alson et al., 1987). The head, consisting of the acrosomal, postacrosomal, and equatorial regions, is connected to the tail by the neck. The tail is divided into the middle piece adjacent to the head, the principal piece, and the end piece, as the most distal portion. Different subcellular organelles characterize each region of the sperm. The head is composed of the acrosome and the nucleus, both of which are enveloped by a complex system of membranes (reviewed in Fawcett, 1975; Longo e t al., 1987). Contained within the head are cytoskeletal elements apparently unique to the sperm, including the subacrosomal layer, the postacrosomal calyx, and a filamentous structure in the anterior region of the head,

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which is attached to the cytoplasmic surface of the outer acrosomal membrane (Longo et al., 1987; Olson e t al., 1987; Paranko et al., 1988). The neck contains centriole(s) and the connecting piece and is the location of the posterior ring, where the plasma membrane and the nuclear envelope fuse (Draber et al., 1986; Galfre et al., 1977). The posterior ring demarcates the hindmost end of the postacrosomal region. Nuclear pores are present in a so-called redundant portion of the nuclear envelope located behind the posterior ring, and the remainder of the membrane surrounding the condensed chromatin is devoid of pores (Fawcett, 1975; Suziki and Yanagimachi, 1986). The sperm tail has, as its innermost component, the axoneme, a microtubule-based organelle responsible for movement of the sperm. Adjacent to the axoneme and running almost the entire length of the sperm tail are the outer dense fibres (Fawcett, 1975; Olson and Winfrey, 1986; Olson et al., 1987). The middle piece of the tail is characterized by the presence of mitochondria and of the submitochondrial matrix (Olson and Winfrey, 1986; Olson et al., 1987), a cytoskeletal network lying next to the outer dense fibres and attached to the mitochondria. The principal piece of the tail possesses a fibrous sheath external to the outer dense fibres a s a t least one unique distinguishing cytoskeletal structure (Fawcett, 1975). Not only is the distribution of the major organelles within the sperm highly polarized, but the molecular constituents of the plasma membrane undergo the same type of regulated arrangement consistent with the functional regionalization of the sperm surface (Fawcett, 1975; Friend, 1982; Olson et al., 1987; Virtanen et al., 1984; Wolf, 1987). Molecules of the cytoplasmic membrane obtain specific locations during sperm development (Cowan et al., 1987; Lopez and Shur, 1987; Scully et al., 1987), and, when the sperm is mature, these must be maintained in the appropriate location. Examples include proteins that reside in specific domains of the sperm head and are involved in the acrosome reaction Received September 25, 1989; accepted November 16, 1989. Address reprint requests to Dr. Thomas H. MacRae, Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada.

MONOCLONAL ANTIBODIES TO THE SPERM CYTOSKELETON and fusion events during fertilization (Lopez and Shur, 1987; Peterson e t al., 1987; Primakoff et al., 1987).That the sperm should behave in this manner is especially important, since it is a cell of very limited biosynthetic potential, lacking the means to synthesize new molecules for subsequent intracellular routing and insertion into the appropriate membrane domain. Potential mechanisms for regulation of cell surface molecular architecture are the use of physical barriers to restrict lateral movement of molecules in the membrane or the attachment of membrane molecules to cytoskeletal elements (Cowan et al., 1987; Wolf, 1987). Are cytoskeletal elements commonly found in somatic cells also present in sperm? Microtubules (tubulin) are present but are apparently restricted to the axoneme and centrioles and to a role in sperm locomotion (Draber et al., 1986; Fawcett, 1975; Virtanen, 1984). Within the axoneme, tektins (Chang and Piperno, 1987), which share antigenic similarity with intermediate filament proteins and the nuclear membrane-associated lamins (Snow et al., 19871, are undoubtedly present. Actin has been localized, most often in monomer form (Camatini et al., 1986, 1987; Olson et al., 1987; Virtanen et al., 1984), as have myosin and spectrin (Virtanen e t al., 19841, but their roles in sperm organization are uncertain. It has, however, been indicated by Scully e t al. (1987) that actin microfilaments may influence the distribution of galactosyltransferase, involved in sperm-egg recognition, during sperm development. The intermediate filament proteins, cytokeratin and vimentin, have been found by some workers (Ochs et al., 1986; Virtanen et al., 19841, but others have noted their absence (Longo et al., 1987; Olson et al., 1987). Clearly, the story regarding the types of cytoskeletal protein to be expected in sperm is somewhat confusing, but there is evidence for common and unique cytoskeletal elements of restricted distribution within sperm. In this paper, we describe the preparation and characterization of monoclonal antibodies to the bull sperm cytoskeleton, defined as that portion of the cell remaining after extraction with the detergent Triton X-100. All the antibodies recognize discrete regions of the bull sperm cytoskeleton and some of them interact with hamster sperm and with subcellular components of PtK, cells. The availability of the antibodies, in conjunction with well-characterized antibodies to other cytoskeletal elements of mammalian cells, expands the opportunity for a more detailed analysis of cytoskeletal influences on sperm organization and function.

MATERIALS AND METHODS Preparation of Bull Sperm Cytoskeletons To prepare detergent-insoluble cytoskeletons, 1ml of bull sperm stored in milk a t 8 x lo7 cells/ml was removed from liquid nitrogen, thawed, layered on a 10 ml cushion of 30% sucrose prepared in 30 mM Tris HC1, pH 8.0, and centrifuged a t 1,500g for 10 rnin at room

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temperature. The sperm were resuspended for 1-2 min a t room temperature in 1 ml of Triton buffer, which consisted of 1% Triton X-100, 10 mM Tris HC1, 0.1 M KC1, 5 mM MgSO,, 0.5 mM ethylenediaminetetraacetic acid disodium salt (EDTA), and 1mM dithiothreitol (DTT), pH 7.0, layered on a 10 ml cushion of 30% sucrose and centrifuged as described above. The sperm pellet was resuspended in 1 ml of phosphate-buffered saline (PBS) and used to immunize mice, for immunofluorescence or for sodium dodecyl sulphate (SDS) gel electrophoresis and western blotting. In preparation for immunization of mice, bull sperm were sometimes disrupted in a Kinematica type PTA 10-35 polytron a t a control setting of 6 after resuspension in Triton buffer. In these cases, the sperm were subjected to two 30-40 sec treatments with cooling in a n ice bath between each treatment. Bull sperm were obtained from Bullpower, Milk Marketing Board, Cattle Breeding Centre, Whiligh Wallcrouch Wadhurst, Sussex, England (a gift of Mr. P.J. Townley, V.O.).

Production of Monoclonal Antibodies BALB/c mice were immunized with bull sperm cytoskeletons prepared a s described above. In the first injection, sperm cytoskeletons emulsified in Freund's complete adjuvant were administered intraperitonally. This was followed, a t 2 week intervals, with three or four injections of sperm cytoskeletons in Freund's incomplete adjuvant followed by a final intravenous injection of cytoskeletons in PBS. After 4-6 days, spleens were removed from the immunized mice and the splenocytes fused, using PEG 1,500 (Galfre et al., 1977), with Sp2-OIAg myeloma cells at about one-third the number of the splenocytes. Cells from each fusion were plated in 4-24 well tissue culture plates, with spleens from nonimmunized mice used for preparation of feeder cells. To test for antibody production, medium was removed from the wells and screened by immunofluorescence on bull sperm cytoskeletons. For cloning, cells from positive wells were plated by limiting dilution a minimum of three times. Antibody subclass was determined by double immunodiffusion with specific antisera from ICN Immunobiologicals. Immunofluorescence Bull sperm cytoskeletons, prepared a s previously described, were resuspended in 1 ml of PBS. Fifteen microliters of the cytoskeleton suspension were spread on each poly-L-lysine (Sigma)-coated slide and allowed to settle for 1 h r at 25°C in a moist chamber. The slides were rinsed briefly in PBS, fixed in methanol at -20°C for 2 min, and the cells were then rehydrated in PBS a t room temperature for 5 min. Culture medium or other antibody solutions were applied, the slides were incubated at 25°C for 1h r in a moist environment and then given three washes of 10 min each in PBS at room temperature. The fluorescent isothiocyanate ( F I T 0 conjugated secondary antibody (rabbit antimouse im-

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munoglobulin; Dakopatts) was applied and the slides antibodies that gave strong immunofluorescence stainwere incubated for 1 h r at 25°C in a moist chamber ing of discrete regions of the sperm, and these were followed by washing in PBS a s described for prelimi- accordingly cloned by limiting dilution. The result is a nary antibody. The slides were mounted in mowial con- panel of six monoclonal antibodies that recognize diftaining p-phenylene diamine to prevent fading of the ferent epitopes of sperm cytoskeletal elements. Of fluorescence. Hamster sperm used for immunofluores- these antibodies, BS1 is a n IgG,, BS5 is a n IgM, and cence staining were a gift from Dr. H. Moore (Regents the remainder are IgG2b. The reactivities of the antiPark, London) and were prepared as described for bull bodies with hamster sperm, with a somatic cell line (PtK,), and with bull sperm polypeptides transferred to sperm. PtK, cells were grown on coverslips in Dulbecco’s nitrocellulose membranes have been determined. modified Eagle’s medium (DMEM), with 10% foetal boImmunofluorescence Staining of Sperm vine serum and 1%nonessential amino acids. For imBS1 stained the principal and end piece of the bull munof luorescence, the coverslips were washed in PBS, fixed in methanol a t -2O”C, exposed to antibodies, and sperm tail very brightly and did not stain any other mounted as for sperm cytoskeletons. If required, the part of the sperm (Fig. la,a’).The antibody BS2 stained DNA intercalating dye 4,6-diamidino-2-phenylindole the middle piece of bull sperm (Fig. lb,b’). The charac(DAPI), a t 1 p.g/ml, was applied to the slide after the teristic staining pattern was a pair of parallel lines immunofluorescence staining, followed by brief wash- running the length of the middle piece with a region of ing in PBS. Detergent extraction involved incubation lighter stain in between indicating a cytoskeletal eleof the coverslips in buffer containing 5 mM piperazine- ment that extends, as a tube, along the middle piece. N-Nf-bis(2-ethanesulphonic acid) (PIPES), 2 mM eth- BS3 stained the acrosomal cap region of bull sperm ylene glycol-bis-(P-amino ethyl ether)N,N,N’,-tetra- (Fig. lc,c’). Some bull sperm were not stained by BS3 acetic acid disodium salt (EDTA), and 1%Triton X-100, presumably due, since acrosomal caps not attached to pH 6.7, for 1 min at room temperature, followed by sperm were visible on the slides, to the loss of their caps washing in PBS after the cells were fixed in methanol in preparation for staining (Fig. lc,c’,d,d’).The monoclonal antibody, BS5, recognized the neck region of bull at -20°C. sperm in addition to yielding a speckled pattern along SDS-Polyacrylamide Gel Electrophoresis the principal piece of the tail (Fig. le,e’). The pattern of Electrophoresis of sperm prepared as previously de- speckles along the principal piece became more disscribed was on 10% SDS-polyacrylamide gels, essen- persed toward the end piece of the tail and the end tially as described by Laemmli (1970) and using SDS piece was not stained. A weaker, more irregular stainfrom Sigma (Clayton et al., 1980). Polypeptides were ing of the middle piece of bull sperm by BS5 was also stained with Coomassie blue R250 or were transferred apparent. The acrosomal cap and the postacrosomal reto nitrocellulose membranes. The markers used were gion of the bull sperm head were stained with BS6 (Fig. myosin, 200,000; p-galactosidase, 116,000; phosphory- lf,f‘,g,g‘).The cap staining, unlike that for antibody lase B, 97,000; serum albumin, 66,000; ovalbumin, BS3, was speckled and was most intense a t the anterior end of the sperm head. Sperm were visible that lacked 43,000; and carbonic anhydride, 31,000. a fluorescently stained cap (Fig. lg,g’) and caps were Immunoblotting present in the absence of sperm (Fig. lf,f‘, arrow). The After one-dimensional electrophoresis on 10% SDS- staining of the bull sperm postacrosomal region was polyacrylamide gels, polypeptides were transferred to most obvious as a discrete band in the posterior region nitrocellulose (Schleicher and Schuell) and the blots of the head, with a lighter but still intense staining were probed with primary and secondary antibodies extending forward to the equatorial plate. BS7 gave a (Dakopatts) essentially as described by Birkett et al. strong staining of the neck region and the principal (1985). To ensure that transfer of polypeptides had oc- piece of the bull sperm with a very light staining of the curred, the membrane was stained with Ponceau S and postacrosomal region, middle and end piece (Fig. the gel with Coomassie blue subsequent to blotting. lh,h’). In comparison to BS5, the staining of the neck was more extensive, and it progressed further into the RESULTS head region to yield a bar-like structure a t the anterior Complex cytoskeletal immunogens, consisting of Tri- limit of the stained region. The staining of the tail was ton X-100-extracted bull sperm, were used to immunize not as punctate as with BS5 and appeared more solid. BALB/c mice. The splenocytes of the mice were fused Thus i t seemed that BS7 and BS5 were recognizing with Sp2-O/Ag myeloma cells followed by selection in different antigenic determinants. HAT medium. The resulting hybridomas, which seHamster sperm were recognized by BS2, BS5, BS6, creted antibody to sperm cytoskeletal elements, were and BS7 but not by the remaining two antibodies identified by a n immunofluorescence screening proce- raised to bull sperm. The staining patterns obtained for dure employing detergent-extracted sperm as the tar- BS2 and BS5 with hamster sperm were the same a s for get. Of particular interest were hybridomas producing bull. However, with BS6 we were able to demonstrate

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Fig. 1. Immunofluorescence and phase micrographs of detergentextracted bull sperm reacted with the monoclonal antibodies raised to sperm cytoskeletons. a-h show bull sperm immunofluorescently stained as described in the Materials and Methods, whereas a'-h' are corresponding phase micrographs. The antibodies were a, BS1; b,

BS2; c, d, BS3; e , BS5; f, g, BS6; h, BS7. The arrows in c and f indicate detached acrosomal regions recognized by the antibodies BS3 and BS6, respectively. Bar = 10 IJ-m(all figures are a t the same magnification).

staining of only the hamster sperm acrosomal cap region and not the back of the head. BS7, on the other hand, stained a region in the anterior, concave portion of the falciform hamster sperm head as well a s areas corresponsing to those stained in bull sperm (not shown). The very different morphologies of bull and hamster sperm heads makes a direct comparison of their staining regions difficult.

recognized a doublet a t 80,000, another a t 36,000, and a diffuse band a t 32,000. Other minor bands were apparent on the blot probed with BS7 (Fig. 2). The observation that BS7 interacted with a polypeptide on a western blot, but BS5 did not, confirmed our earlier proposal that these two antibodies, although labeling the same general areas of bull sperm, were in fact recognizing different epitopes.

Immunoblot of Bull Sperm The antibodies BS1-BS3 and BS5 failed to detect any polypeptides on western blots of bull sperm from SDSpolyacrylamide gels. On the other hand, BS6 reacted with several polypeptides of molecular weights greater than 80,000 and another a t about 40,000, whereas BS7

Immunofluorescence Staining of PtK, Cells To determine if structures within cells other than sperm were recognized by these monoclonal antibodies and to gain a n initial appreciation of the biochemical nature of the labeled sperm cytoskeletal elements, we exposed PtK, cells to the various antibodies, followed

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Fig. 2. Western blot showing the polypeptides recognized by the antibodies BS6 and BS7. Detergent-extracted bull sperm were boiled in SDS, electrophoresed on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and reacted with antibody. Lane 1 is from a Coomassie blue-stained gel. Lanes are blots stained, respectively, with BS6, BS7, and secondary antibody in the absence of primary are a t left. antibody. The molecular weight markers ( x

by poststaining with FITC-conjugated antibodies. BS1BS3 did not recognize any subcellular components within the PtK, cells, whereas the remaining antibodies gave very definite staining patterns. Labeling of PtK, cells with BS5 revealed a filamentous network similar in overall character to that obtained by exposing this cell type to the antibody, LE41, which recognizes the intermediate filament protein, cytokeratin (Fig. 3a,b). LE41 was the kind gift of Dr. Birgitte Lane (I.C.R.F., London). BS6 labeled the nucleus and the cytoplasm of PtK, cells, but upon detergent extraction only the nucleus of the cells remained stained in a punctate pattern similar to what might be expected if the nuclear pores were recognized by the antibody (Fig. 4a, b). That the labeled structure is the nucleus was verified by the positional correspondence, in double-stained cells, of DAPI and BS6-derived fluorescence (Fig. 4c,d). Focusing of the microscope through the cell revealed that the punctate pattern was visible only at locations presumably corresponding to the nuclear envelope and was not found throughout the interior of the nucleus. Both the cytoplasm and the

Fig. 3. Micrographs of immunofluorescence staining patterns of PtK, cells probed with BS5 (a) and LE41 (b). Bar = 15 km (both figures are a t the same magnification).

nucleus of PtK, cells contain components recognized by BS7, but cytoplasmic staining disappeared upon detergent extraction of the cells (Fig. 5a,b). The nuclear staining pattern was definitely different from that obtained with BS6, tending to be much more irregular in appearance. Of particular interest, the areas of most intense fluorescence in BS7-stained cells were those that tended not to be labeled by DAPI (Fig. 5c,d). In the absence of primary antibody, there was no immunofluorescent staining of PtK, cells.

DISCUSSION The role of the cytoskeleton in organization of the internal organelles and the cytoplasmic membrane molecules of the mammalian sperm has attracted a n increasing amount of attention. Some work, a t times with conflicting results, has centered on the analysis of cytoskeletal elements such a s the microtubules, microfilaments, and intermediate filaments, which are found

MONOCLONAL ANTIBODIES TO THE SPERM CYTOSKELETON

Fig. 4. Micrographs of PtK, cells immunofluorescently stained with BS6 ( a 4 and with DAPI, a DNA intercalating dye (d).The cell in a was not detergent extracted, whereas the cell in b was, and it clearly reveals a punctate nuclear staining pattern. c and d are pic-

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tures of the same cells, extracted with detergent and double labeled with BS6 and DAPI to demonstrate that the epitope recognized by BS6 in detergent-extracted cells is in the nucleus. Bar = 10 p m (a,b) and 25 pm (c, d).

in most eukaryotic cells (Camatini et al., 1986, 1987; main uncertain. By immunization of mice with Triton Draber et al., 1986; Ochs e t al., 1986; Olson et al., 1987; X-100-extracted bull sperm and subsequent fusion of Scully et al., 1987; Virtanen et al., 1984). The proce- their splenocytes with Sp2-0IAg myeloma cells we have dure has generally been to use antibodies prepared prepared six monoclonal antibodies to the bull sperm against cytoskeletal proteins from cells other than cytoskeleton. The antibodies have been characterized sperm or to use fluorescently labeled chemicals that in relation to the region of bull and hamster sperm they react specifically with known components of the cy- recognize, their cross reactivity with PtK, cells and the toskeleton, a n example being 7-nitrobenz-1,3-diazole- polypeptides they detect on western blots. The antibodphallacidin (NBD-phallacidin) for filamentous actin ies BS1 and BS2 labeled regions of the sperm tail. The (Olson et al., 1987; Virtanen et al., 1984). Two prob- only known unique cytoskeletal element in the princilems exist with this approach, these being the cross pal piece, which stained strongly with BS1, is the f i reactivity of dissimilar proteins due to a single shared brous sheath, but it does not extend into the end piece epitope and the inability to identify proteins unique to (Fawcett, 1975; Olson et al., 1987). The middle piece the sperm. A second method has been to extract sperm contains the mitochondria and, a t least in hamster with varying degrees of severity and to characterize sperm, the submitochondrial reticulum (Olson and the structural and molecular components that remain, Winfrey, 1986; Olson e t al., 1987). The submitochonor are extracted, by ultrastructural, immunological, drial reticulum is resistant to mild Triton X-100 exand biochemical techniques (Longo et al., 1987; Olson traction (0.1% for 1 min), a s are the mitochondria and Winfrey, 1986; Olson et al., 1987; Paranko et al., (Olson and Winfrey, 1986), and the reticulum forms a 1988). Novel cytoskeletal elements, as defined by their relatively thin cylindrical cytoskeletal structure. Visuability to resist extraction from sperm by detergent alization of a pair of parallel lines with a lighter stainand/or salt, have been found, but their functions re- ing in between, a s shown for BS2, is indicative of the

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T.H. MACRAE ET AL. type of subcellular arrangement exhibited by the submitochondria1 reticulum or of another structure arranged in the same way. The absence of cytoplasmic staining in PtK, cells suggests that the mitochondria of the sperm that remain after the relatively mild extraction procedure we employed are not being stained by BS2. BS3 recognizes a detachable portion of the sperm acrosomal region. There are no previous reports, to our knowledge, of a loosely attached cytoskeletal component located a t the anterior region of the acrosomal cap other than in the falciform hamster sperm. Hamster sperm are not, however, recognized by BS3. There are novel cytoskeletal components in the subacrosomal layer of the perinuclear theca of mammalian sperm heads (Longo et al., 1987; Paranko et al., 1988), but they would not be expected to detach from the sperm by the procedure we used to prepare sperm for immunofluorescence. The monoclonal antibodies BS5 and BS7 stain the collar and principal piece of bull sperm most prominently, but the patterns they generate are different. The staining of the collar region by BS5 is less extensive than for BS7, and BS5 yields a punctate pattern of interaction along the principal piece whereas BS7 labeling is more solid in appearance. In addition, BS7, but not BS5, stains a discrete region of the anterior portion of the hamster sperm head (not shown), with no obvious equivalent immunoreactive area in bull sperm. Olson et al. (1987) have shown a filamentous cytoskeleta1 structure in the same region of the hamster sperm head as labeled by BS7, but i t is not known if a morphologically similar structure is present in bull sperm. When PtK, cells are exposed to BS5, a pattern similar to that obtained for cytokeratin staining is observed. In contrast, BS7 stains the entire cell in a fuzzy pattern before extraction with detergent and only the nucleus after extraction. The areas of most intense fluorescence in the nucleus upon interaction with BS7 are those not staining with DAPI, a DNA intercalating dye. Different antibodies to flagella tektin C stain intermediate filament proteins on the one hand and nuclear envelopes, through binding to lamins, on the other (Chang and Piperno, 1987). Thus i t is not unprecedented for antibodies such as BS5 and BS7, which recognize flagella components, to stain other cytoplasmic organelles. This does not necessarily indicate, however, that constituent proteins of those organelles are in the flagellum. Indeed, Longo et al. (1987) have stated quite

Fig. 5

Fig. 5. Micrographs of PtK, cells immunofluorescently stained with BS7 ( a 4 and DAPI (d). The cell in a was not detergent extracted, whereas those in b were, revealing the retention of the antibody in the nucleus of the detergent-extracted cells. c and d show the same cell double stained with BS7 and DAPI, respectively. The areas of most intense fluorescence in c are not stained or stained very lightly with DAPI in d. Bar = 10 km (all figures are at the same magnification).

MONOCLONAL ANTIBODIES TO THE SPERM CYTOSKELETON emphatically that cytokeratins and lamin-like proteins of somatic cells cannot be detected in rat and bull sperm with well-characterized antibodies. BS6 stains the postacrosomal region of bull sperm in patterns similar to those reported by other workers studying the subacrosomal layer and calyx of the perinuclear theca (Longo e t al., 1987; Paranko et al., 1988). However, staining in a detachable portion of the anterior acrosomal region of the head was not reported by other workers. Moreover, the molecular weights of the proteins we detect on blots of sperm with BS6 do not correspond to those for calicin or the multiple-band polypeptides found in the perinuclear theca of sperm. When applied to PtK, cells after detergent extraction, BS6 stains nuclei in a pattern reminiscent of nuclear pores, and on western blots it reacts with a family of relatively high molecular weight proteins as previously reported for other monoclonal antibodies that recognize nuclear pore complex glycoproteins (Snow e t al., 1987). However, nuclear pores are reported to reside in the redundant nuclear membrane of the sperm in a location behind the posterior ring (Fawcett, 1975; Suziki and Yanagimachi, 1986) and not in the postacrosomal or acrosomal regions recognized by BS6. The exact nature of the subcellular component recognized by BS6 is uncertain but it is extremely interesting that it should stain regions in sperm and PtK, cells apparently occupied by very different cytoskeletal structures.

CONCLUSIONS Several antibodies recognizing components of the sperm cytoskeleton are described. None of the immunofluorescence staining patterns or immunoblotting results are exactly the same as those obtained when antibodies to common somatic cell cytoskeletal elements are used to study sperm. Some of the antibodies stain regions that correspond to the locations of novel sperm cytoskeletal elements, whereas the immunofluorescence patterns generated by other antibodies suggest the presence of new, previously undescribed components of the cytoskeleton in sperm. It is with caution, of course, that results of immunofluorescence staining from one laboratory and those from another or even from one type of sperm to another in the same laboratory are compared. Differences in detergent extraction and cell fixation procedures for immunofluorescence may alter the staining patterns seen (Ochs et al., 1986; Virtanen, 1984). Variations in sperm morphology make it difficult to compare the locations of the same proteins within these cells, and it is known that immunologically related polypeptides occur in different domains of sperm (Paranko, 1988). However, in spite of the difficulties, the availability of the monoclonal antibodies we have described will, with the application of high-resolution immunogold staining and molecular technologies, permit a detailed analysis of various aspects of the sperm cytoskeleton. The information ob-

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tained should then lead to a better understanding of cytoskeletal influences on the polarization of sperm components and ultimately on sperm function.

ACKNOWLEDGMENTS We thank Ms. M.R. Wilcox for excellent technical assistance, Mr. P.J. Towley for the bull sperm, Dr. H. Moore for the hamster sperm, and Dr. Birgitte Lane for the antibody (LE41) to cytokeratin. The financial support of the Science and Engineering Research Council, the Natural Sciences and Engineering Research Council, and the Royal Society is gratefully acknowledged.

REFERENCES Birkett CR, Foster KE, Johnson L, Gull K (1985): Use of monoclonal antibodies to analyze the expression of a multi-tubulin family. FEBS Lett 187:211-218. Camatini M, Anelli G, Casale A (1986):Identification of actin in boar spermatids and spermatozoa by immunoelectron microscopy. Eur J Cell Biol 42:311-318. Camatini M, Casale A, Cifarelli M (1987): Immunocytochemical identification of actin in rabbit spermiogenesis and spermatozoa. Eur J Cell Biol 45:274-281. Chang X-j, Piperno G (1987):Cross-reactivity of antibodies specific for flagellar tektin and intermediate filament subunits. J Cell Biol 104:1563-1568. Clayton L, Quinland RA, Roobol A, Pogson CI, Gull K (1980):A comparison of tubulins from mammalian brain and Physarum polycephalum using SDS-polacrylamide gel electrophoresis and peptide mapping. FEBS Lett 115:301-305. Cowan AE, Myles DG, Koppel DE (1987): Lateral diffusion of the PH-20 protein on guinea pig sperm: Evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm. J Cell Biol 104:917-923. Draber P, Draberova E, Zicconi D, Sellitto C, Viklicky V, Cappuccinelli P (1986): Heterogeneity of microtubules recognized by monoclonal antibodies to alpha-tubulin. Eur J Cell Biol 4132-88. Fawcett DW (1975):The mammalian spermatozoon. Dev Biol44:394436. Friend DS (1982):Plasma-membrane diversity in a highly polarized cell. J Cell Biol 93:243-249. Galfre G, Hove SC, Milstein C, Butcher GW, Howard J C (1977): Antibodies to a major histocompatibility antigen produced by hybrid cell lines. Nature 226:550-552. Laemmli UK (1970):Cleavage of structural proteins during assembly stage of bacteriophage T,. Nature 227:680-685. Longo FJ, Krohne G, Franke WW (1987):Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: A novel class of cytoskeletal elements. J Cell Biol 105:1105-1120. Lopez LC, Shur BD (1987): Redistribution of mouse sperm surface galactosyltransferase after the acrosome reaction. J Cell Biol 105: 1663-1670. Ochs D, Wolf DP, Ochs RL (1986): Intermediate filament proteins in human sperm heads. Exp Cell Res 167:495-504. Olson GE, Winfrey VP (1986):Identification of a cytoskeletal network adherent to the mitochondria of mammalian spermatozoa. J Ultrastruct Mol Struct Res 94:131-139. Olson GE, Winfrey VP, Flaherty SP (1987):Cytoskeletal assemblies of mammalian spermatozoa. Ann NY Acad Sci 513:222-246. Paranko J, Longo F, Potts J, Krohne G, Franke WW (1988): Widespread occurrence of calicin, a basic cytoskeletal protein of sperm cells, in diverse mammalian species. Differentiation 38:21-27. Peterson RN, Gillott M, Hunt W, Russell LD (1987):Organization of the boar spermatozoa plasma membrane: Evidence for separate domains (subdomains) of integral membrane proteins in the plasma membrane overlying the principal segment of the acrosome. J Cell Sci 88:343-349.

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Primakoff P, Hyatt H, Tredick-Kline J (1987): Identification and purification of a sperm surface protein with a potential role in spermegg membrane fusion. J Cell Biol 104:141-149. Scully NF, Shaper JH, Shur BD (1987): Spatial and temporal expression of cell surface galactosyltransferase during mouse spermatogenesis and epididymal maturation. Dev Biol 124:lll-124. Snow CM, Senior A, Gerace L (1987):Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J Cell Biol 104: 1143-1 156.

Suziki F, Yanagimachi R (1986): Membrane changes in Chinese hamster spermatozoa during epididymal maturation. J Ultrastruct Mol Struct Res 96:91-104. Virtanen I, Badley RA, Paasivuo R, Lehto V-P (1984): Distinct cytoskeletal domains revealed in sperm cells. J Cell Biol 99:10831091. Wolf DE (1987): Overcoming random diffusion in polarized cellsCorralling the drunken beggar. Bioessays 6:116-121.

Production and characterization of monoclonal antibodies to the mammalian sperm cytoskeleton.

The cytoskeleton exerts a direct effect on the function of sperm by influencing the distribution of subcellular organelles and plasma membrane molecul...
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