MOLECULAR REPRODUCTION AND DEVELOPMENT 28:380-393 (1991)

Formation of the Perinuclear Theca in Spermatozoa of Diverse Mammalian Species: Relationship of the Manchette and Multiple Band Polypeptides FRANK J. LONG0 AND SUSAN COOK Department of Anatomy, University of Iowa, Iowa City, Iowa

The perinuclear theca is a novel ABSTRACT cytoskeletal consisting of a densely layered lamina that surrounds the nucleus of mammalian sperm. Using antibodies specific for the multiple band polypeptides present in the perinuclear theca of bull sperm, we show that a heterogeneous group of immunological related proteins are present in the sperm heads of other mammals with greatly different morphologies, including guinea pig, hamster, rat, and mouse. In none of the species were identical groups of immunoreactive polypeptides found, although immunoreactive proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all species examined. lmmunoreactive proteins less than M, 55,000 were prominent in rat sperm heads and mouse sperm; guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately M, 50,000.Different immunoreactive proteins were present in isolated sperm tails.The perinuclear theca first appeared in the subacrosomal space of round to elongating spermatids. Later, with the caudal movement of the manchette, the postacrosomal segment of the perinuclear theca was deposited in a cephalad to caudal direction along the sperm nucleus. Concomitantly, the cytoplasmic space between the nuclear envelope and the plasma membrane narrowed such that only the theca occupied this portion of the sperm head. lmmunoreactivity accompanied the ultrastructural appearance of the subacrosomal layer and the postacrosomal segment. The periods of spermiogenesis, in which sub- and post-acrosomal components of the perinuclear theca are formed and the morphogenesis of sperm organelles with which these elements are associated, suggest that components of this cytoskeletal structure function to join the acrosome and the postacrosomal plasma membrane to the nucleus. Key Words: Cytoskeleton, Subacrosomal layer, Postacrosomal segment, Spermiogenesis, Multiple band polypeptides ~

INTRODUCTION The perinuclear theca comprises a varible number of dense layers that fill the thin cytoplasmic region surrounding the condensed sperm nucleus of mammalian sperm (Courtens et al., 1976, 1980; Bellve and O’Brien, 1983).On the basis of electron microscopic and cytochemical observations this extranuclear structure has been subdivided into an apical portion composed of

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dense amorphous material that lies within the thin cytoplasmic region between the acrosome and the nucleus, and is referred to as the subacrosomal layer (Courtens et al., 1976,1980; Bellve and O’Brien, 1983). The posterior or postacrosmal segment of the perinuclear theca consists of a funnel-shaped structure, the calyx, which can be further subdivided into a layer that is closely applied to the nuclear envelope and continuous with the subacrosomal layer, and a lamina, the postacrosomal sheath, which is connected to the plasma membrane by a paracrystalline sheet of ridges or filaments, 10 to 14 nm in diameter (Wooding and O’Donnell, 1971; Pedersen, 1972; Olson and Winfrey, 1985, 1988; Olson et al., 1983, 1987). These layers are believed to be involved in the dramatic shape changes spermatids undergo during spermiogenesis and in the development of the intimate association shared by the nucleus, acrosome, and plasma membrane. Consistent with this suggestion are reports that the perinuclear theca first appears at a time when dramatic architectural changes occur in the spermatid nucleus and cytoplasm (Courtens et al., 1976). Other cytoskeletal components have been postulated to be involved in the morphogenesis of the sperm head, such as actin (Vogl et al., 1983; Welch and O’Rand, 19851, intermediate filaments (Virtanen et al., 1984; Ochs et al., 19861,and microtubules (Fawcett et al., 1971; Cole et al., 1988); however, the mechanisms by which the spermatid nucleus and cytoplasm are modified during the course of spermiogenesis have not been determined. In studies of isolated perinuclear theca from bull sperm, a cytoskeletal element has been described that is resistant to extraction with detergents and high salt buffers and consists of two groups of basic proteins: a polypeptide of -Mr 60,000, termed calicin, and a set of different proteins, immunologically related to each other, but exhibiting significant peptide map differences and that are collectively referred to as multiple band polypeptides (Longo et al., 1987). These proteins appear to be unrelated to all cyto- and karyo-skeletal proteins described thus far. Based on similar electronmicroscopic appearances of the perinuclear theca in a

Received October 1, 1990; accepted November 27, 1990. Address reprint requests to Frank J. Longo, Department of Anatomy, University of Iowa, Iowa City, IA 52242.

FORMATION AND CHARACTERIZATION OF PERINUCLEAR THECA broad range of mammalian species, calicin and multiple band polypeptides might be expected to have a widespread distribution among mammalian sperm. Conversely, sperm of mammalian species that differ markedly in size, shape, and intracellular arrays might be expected to possess dissimilar polypeptides, possibly reflecting their particular structural organization. Using antibodies specific for calicin the same, or an immunologically related polypeptide, has been detected in sperm heads of a variety of mammalian species with greatly different morphologies (Paranko et al., 1988). In the present study we present evidence that a heterogeneous group of proteins, differing in molecular weights but immunologically related to the multiple band polypeptides of bovine sperm are present in the perinuclear theca of a variety of mammalian sperm. These polypeptides appear during spermiogenesis in conjunction with the morphogenesis of the acrosome and manchette.

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obtained from dissociated testicular cells according to Meistrich (19771, was performed as described (Longo et al., 1987). For immunolocalization experiments at the light-microscopic level of observation, whole-mount preparations, suspensions of PBS washed sperm, and isolated testicular cells were placed on poly-l-lysinecoated coverslips and were rinsed briefly in PBS or were permeabilized by a brief incubation in 0.5%Triton X-100 in PBS. Specimens were either incubated with affinity purified antibody for 15 min or were first immersed in -2O’C acetone for 10 min. After washing in PBS, specimens were incubated for 15 min in FITClabeled secondary antibody. Controls consisted of either primary or secondary antibodies alone. For immunoelectron microscopy, the “pre-embedding method’ was employed (Longo et al., 19871, i.e., sperm adherent to poly-L-lysine coated coverslips were processed with affinity purified antibody followed by secondary antibody coupled to 5 nm colloidal gold particles (Jansen, Olen, Belgium) and then fixed and processed for elecMATERIAL AND METHODS tron microscopy (see below). Collection and Fractionation of Spermatozoa To determine the morphogenesis of the perinuclear Sperm were recovered from vasa and epididymides of theca during sperm development, testicular samples bull, guinea pig, hamster, and rat and fractioned into were fixed with 2.5% glutaraldehyde in 100 mM soheads and tails as previously described (Longo et al., dium cacodylate buffer (pH 7.2) for 60 min. Samples 1987).Sperm heads were extracted in 1%Triton X-100 were washed overnight in 100 mM cacodylate buffer, in phosphate-buffered saline (PBS). Mouse sperm, re- postfixed in 1%Os04 for 30 min, dehydrated in ascendcovered from isolated vasa and epididymides, were ing concentrations of ethanol, and embedded in Spurr’s washed in PBS and left intact. All solutions to isolate embedding medium. Thin sections, stained with uranyl sperm and to prepare sperm fractions contained 1 mM acetate and lead citrate, were examined in a Phillips phenylmethylsulfonyl fluoride and 1 pg/ml of each of 300 EM electron microscope. To demonstrate the apthe protease inhibitors: leupeptin, aprotinin, antipain, pearance of the perinuclear theca in differentiating spermatids, testes samples were silver stained after and pepstatin A. fixation according to Elder and Hsu (1981) and then Gel Electrophoresis and Immunoblotting processed for electron microscopy. Entire sperm, sperm head, or tail fractions were RESULTS boiled for 5 min in sample buffer [5% sodium dodecyl Morphogenesis of the Perinuclear Theca sulfate (SDS), 10% 2-P-mercaptoethanol, 10 mM sodium phosphate buffer; pH 7.21. SDS-polyacrylamide In all species examined a similar morphogenesis was gel electrophoresis (SDS-PAGE) was carried out ac- observed with respect to development of the perinucording to Laemmli (1970) using variable loads (up to clear theca; consequently, the following is a general 50 Fgilane). Gels were either stained with Commassie account applicable to each species. Specific features are blue or processed for immunoblotting (Towbin et al., considered in Figures 1to 11. Prior to the formation of 1979).Proteins transferred to nitrocellulose paper were the acrosomal vesicle and its close apposition to the visualized with Ponceau S. nuclear surface of round spermatids, there was little evidence of cytoplasmic specializations in the perinuAntibodies clear region. During the cap phase of round spermatids, Guinea pig antibodies were obtained after immuni- i.e., following the close apposition of the acrosome and zation with protein eluted from SDS-PAGE bands nucleus, two distinct perinuclear regions were discontaining the multiple band polypeptide obtained cerned: an “anterior region,” encompassing the subacfrom the calyx fraction of bull sperm heads (Longo rosomal space, which was delimited by the acrosome et al., 1987). Antibodies were affinity-purified as de- membrane and nuclear envelope and a “posterior rescribed (Krohne et al., 1982). Monoclonal antibody C23 gion” associated with the remainder of the spermatid (Lin et al., 1988) was used to detect nuclear lamins A nucleus (Figs. 1,2). The subacrosomal space had a and C in testis sections. constant width (-30 nm) and at its apex contained material that had the same texture and density of the Light and Electron Microscopic dense granular substance present within the acrosome Immunolocalization (Fig. 2). The posterior region lacked such material and Immunofluorescence microscopy employing cryostat was associated with cisternae of endoplasmic reticulum sections of testicular tissues and isolated spermatids, (Fig. 1).Nuclei of round spermatids at the cap phase

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Figs. 1and 2. Bovine round spermatid at the cap stage in which the acrosomal vesicle (A) has become closely appressed to the anterior portion of the nucleus. A thin layer of condensed chromatin lines the nuclear envelope, subjacent to the subacrosomal region (arrows). The posterior of the spermatid nucleus is associated with cisternae of endoplasmic reticulum. The nuclear envelope in this region is lined with aggregations of condensed chromatin that form a discontinuous layer. Figure 2 is a t higher magnification depicting the structural relationship of the acrosomal membrane (AM), subacrosomal space (3,and nuclear surface. The subacrosomal space contains some

electron-dense material (arrow)directly subjacent to the aggregation of dense material present within the acrosome. Figure 1, x 18,000; Figure 2, x 48,500. Fig. 3. Elongating rat spermatid. At the posterior aspect of the acrosome is located the nuclear ring (NR) from which microtubules (M) of the manchette eminate. The subacrosomal space (arrow) contains dense material, a portion of the developing perinuclear theca. The nuclear envelope associated with the subacrosomal region is lined by a thin layer of condensed chromatin. x 14,800.

demonstrated a distinctive internal polarity with respect to the anterior and posterior perinuclear regions. Immunofluorescence observations indicated an absence of nuclear lamins A and C along the inner margin of the entire nuclear envelope (see also Moss et al., 1987). A structure characteristic of the fibrous or nuclear lamina observed in most cells (Gerace, 1986) was also absent; however, a thin, fairly continuous layer of condensed chromatin lined the anterior region

of the nucleus, while the posterior region possessed a discontinuous layer of chromatin aggregates (Fig. 1). During spermatid elongation modifications became apparent along the anterior and posterior perinuclear regions. There was an accumulation of dense material to form a distinctive layer within the subacrosomal space, particularly along the posterior margin of the acrosome where patches of electron dense material become organized along the inner acrosomal membrane

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Figs. 4 and 5. Posterior aspect of acrosomal vesicles (A), the nuclear rings (NR), and microtubules (M)comprising the manchettes of hamster (Fig. 4) and mouse (Fig. 5) elongating spermatids. In both cases electron-dense material in the subacrosomal space has aggregated into discrete deposits (arrows) which line the acrosomal mem-

brane. In Figure 5 short filaments connect manchette microtubules to the surface of the nuclear envelope. Figures 4 and 5, x 54,000. Fig. 6. Posterior aspect of a guinea pig elongating spermatid nucleus depicting manchette microtubules (M) associated with the nuclear envelope (NE) by numerous cross-bridges (arrows). x 66,000.

(Figs. 3-5; Sandoz, 1970). Patch size and organization varied depending on the species and are not detailed here. Just subjacent to the caudal portion of the acrosome cap was located the nuclear ring, an aggregation of the amorphousigranular material that girdled the nucleus and from which microtubules of the manchette originated (Fig. 3; Rattner and Olson, 1973). The plasma membrane was closely applied to the cephalic portion of the nuclear ring, as well as the acrosome, so that there was little subjacent cytoplasm in the two areas (Figs. 3-5). The remainder of the differentiating sperm head had an abundant cytoplasm in which was located the manchette (Fig. 3). Manchette microtubules were organized into sheets that paralleled the surface of and projected beyond the caudal pole of the elongating nucleus. The space between the manchette and the nucleus was filled with short filaments or cross bridges approximately 75 nm in length that formed a striated layer connecting the microtubules t o the nuclear envelope (Figs. 5,6).The caudal pole of elongating spermatid nuclei was associated with cisternae of endoplasmic reticulum and elements of the developing sperm tail (Fig. 3). Early elongating spermatid nuclei possessed a homogeneous textured chromatin and lacked the large, dense chromatin aggregations present in round spermatids (Figs. 1,3),as well as nuclear lamins A and C. A thin layer of condensed chromatin lined the inner

margin of the nuclear envelope in association with the subacrosomal space. This layer was discernible until shaping of the sperm head was nearly completed. Deposition of the perinuclear theca within the postacrosomal region of elongate spermatid nuclei did not occur until the manchette moved caudally (Figs. 7-9). With this movement there was a cephalad to caudal accumulation of electron-dense material along the surface of the nuclear envelope just superior to the nuclear ring; concomitantly, the plasma membrane was brought into close proximity to the nuclear surface. Subsequent to its deposition, the postacrosomal segment of the perinuclear theca differentiated into discrete layers: an amorphous lamina closely associated with the nuclear envelope and continuous with material filling the subacrosomal space, and a prominent stratum of dense material, closely associated with the plasma membrane (Fig. 10a-e). In most cases the latter consisted of linear repeating elements spaced 10 to 14 nm from one another (Fig. 10d). Silver stain, which has been shown to react with components within the perinuclear theca (Elder and Hsu, 1981; Krimer and Esponda, 1988), revealed the deposition of a thin layer within the sub- and postacrosomal regions of elongating spermatids and spermatozoa (Fig. 11).Prior to the caudal displacement of the manchette, dense silver staining was confined primarily to the nuclear ring (Fig. l l a ) . As the

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Fig. 7. Elongated bull spermatid in which the nuclear ring (NR) and manchette (MN) have initiated their caudal movement. A portion of the posterior segment of the perinuclear theca (arrow) has been deposited between the acrosome (A) and the nuclear ring. In the subacrosomal space the perinuclear theca (S)is present as a dense lamina. The chromatin has condensed into thickened fibers. x 36,000: Figs. 8 and 9. Stages in the caudal movement of the manchette of differentiating hamster (Fig. 8) and bull (Fig. 9) spermatids. The narrow postacrosomal space between the posterior aspect of the acrosome (A) and the nuclear ring (NR) is filled with electron-dense material comprising the perinuclear theca (postacrosomal segment, arrows).The plasma membrane (PM)has become closely applied to the

nuclear surface in the region where thecal material has been deposited. Figure 8, x 36,000; Figure 9, x 44,000. Fig. 10. Structure of the postacrosomal segment in bovine la), guinea pig (b),hamster (c), rat (d),and mouse (e) mature sperm. The nuclear envelope is lined by amorphous material continuous with that in the subarachnoid space. Superficial to this amorphous material is an electron-translucent space, which may be an artifact of tissue processing. A relatively thick linear array of filamentous material extends from the posterior margin of the acrosome (A) and is closely associated with the plasma membrane. a, x 54,000; b, x 52,000; c, x 36,000; d, x 66,000; e, x 49,000.

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Fig. l l a and b. Silver stained elongating guinea pig spermatid (a) and hamster spermatozoon (b).Silver deposits are associated with the nuclear ring (NR)and in the postacrosomal region (arrows) vacated by the manchette of the differentiating spermatid. The mature sperm

possess silver deposits in the postacrosomal region (arrows) and along the surface of the acrosome (A). Figure l l a , x 52,000; Figure l l b , x 33,000.

manchette moved caudally, agyrophilic material filled the cytoplasmic space between the acrosome and nuclear ring. In mature sperm, dense silver deposits were present within the postacrosomal segment, while lesser accumulations of stain were observed along the periphery of the entire acrosome (Fig. l l b ) .

affinity purified antibody showed a strong reactivity to a band at M, 66,000 and lesser reactivity to bands a t 41, 43, 44, 51, and 75 kDa from isolated sperm heads. In hamsters, affinity purified antibody reacted with polypeptides from isolated sperm heads having M, of 50,000,74,000,76,000,and 97,000; in the rat, bands a t 30 and 49 kDa were highly reactive with the affinity purified antibody with less intense bands a t 66,74, and 76 kDa. Affinity purified antibody reacted with proteins of M, 36,000, 37,000, 50,000, and 74,000 from whole mouse sperm. Polypeptides isolated from sperm tails also reacted to affinity purified antibody (Fig. 13). Isolated preparations virtually consisted entirely of tails; occasionally, however, a n intact sperm or sperm head was encountered upon microscopic examination of isolates. Contamination with whole sperm or sperm heads was estimated to be less than 0.196, i.e., one head or whole spermilOO0 tails. Protein bands of M, 64,000, 69,000, 76,000,81,000, and 84,000 from isolate bull sperm tails showed strong reactivity to purified antibody with lesser reactivity to a band a t 29,000. Immunoblots of proteins from guinea pig sperm tails showed a slightly reactive band at M, 27,000, which corresponded to an intense Coomassie-blue-stained band in SDS-polyacrylamide gels. A band at M, 36,000 from hamster and rat tails demonstrated an intense reactivity to affinity purified antibody, while less reactivity was associated with a band migrating with an apparent molecular

Perinuclear Theca Polypeptides Reacting With Antibodies to Multiple Band Polypeptides of Bovine Sperm Dot-blot assays of proteins excreted from bovine sperm heads revealed the presence of specific antibodies in immune serum with sufficient reactivity that the sperm could be diluted up to 10,000-fold (Fig. 12a). No reactivity was detected in preimmune serum. Specificity of the affinity purified immune serum was determined in immunoblots of bovine sperm head proteins run on two-dimensional gels (Fig. 12b). In addition to an intense reaction with multiple band proteins of M, 64,000 69,000,76,000,81,000,and 84,000 from isolated bull sperm heads, affinity purified antibody reacted with lower intensity to a group of polypeptides of higher molecular weights (97,000, 120,000, 140,000, and 150,000; Fig. 12). When affinity purified antibody was reacted with polypeptides from isolated sperm heads and sperm from guinea pig, hamster, rat, and mouse, groups of reactive polypeptides from each species were identified that were heterogeneous with respect to number and molecular weight (Fig. 12).In guinea pigs,

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PI

r

NEPHGE

Fig. 12a. Dot blot assay of bovine sperm head proteins reacted with decreasing concentrations of immune serum. PI, preimmune serum diluted to 10 z. Fig. 12b. Immunoblot of a two-dimensional polyacrylamide gel

electrophoresis of proteins from bovine sperm heads reacted with affinity purified immune serum to multiple band proteins. First dimension, NEPHGE; second dimension, SDS-PAGE.

weight of 25,000. When immunoblots of hamster and rat tails were compared to corresponding Coomassieblue-stained gels a band migrating with an apparent molecular weight of 36,000 was barely detectable, whereas a dense Coomassie-staining band was present at 25,000. Affinity purified antibody was used for staining of whole sperm and testicular cells in fluorescent and electron-microscopic preparations and similar results were found in each of the species examined. Fluorescent staining was first observed in round and elongating bull spermatids as a cup-shaped area that corresponded to the subacrosomal region (Fig. 14c). A similar staining pattern was also present in spermatids of guinea

pig, rat, hamster, and mouse (Figs. 17-20). In sections of bovine testes stained with antibodies to tubulin and multiple band polypeptides, elongating spermatids were observed that demonstrated the sequential deposition of perinuclear thecal components in the sub- and post-acrosomal regions. That is, antitubulin staining was present in the manchette, just inferior to the subacrosomal space that was reactive to antibodies to multiple band proteins (Fig. 15). A similar staining pattern was also observed in isolated, elongating mouse spermatids where regions of the developing sperm head, anterior to the manchette were positive for multiple band proteins (Fig. 16). Staining for multiple band polypeptides was present

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Fig. 12c and d. ( c ) SDS-PAGE of proteins from sperm heads of bull (lane 11, guinea pig (lane 2), hamster (lane 3) and rat (lane 41, and entire sperm of mouse (lane 5) stained with Coomassie blue and corresponding immunoblot (d; autoradiographic detection of lZ5Ilabeled secondary antibodies to affinity purified antibodies to multiple band proteins of bovine sperm, lanes 1’-5’j. Reference proteins (lane R, specific proteins are marked by a dot) from top to bottom; 0-galactosidase, M, 116,000; phosphorylase b, M, 97,000; bovine serum albumin, M, 66,000; egg albumin, M, 45,000; carbonic anhydrase, M, 29,000. Reactive bands are present in all species tested.

Fig. 13a and b. (a) SDS-PAGE of proteins from sperm tails of bull (lane l),guinea pig (lane2j, hamster (lane3),and rat (lane4) stained with Coomassie blue and corresponding immunoblot (b; lanes 1’-4’) probed with affinity purified antibodies to multiple band proteins. With the exception of proteins M, 64,000 to 84,000 from bull, reactive bands of the tail fractions differ from those present in the head. The positions of the same reference proteins used in Figure 12c are indicated by the dashes.

along the entire nucleus of testicular and epididymal bull sperm and was particularly heavy in the region of the calyx (Figs. 14,211. Less reactivity was present within the proximal portion of the tail (Fig. 14a), specifically in association with the dense fibers (Fig. 22). Antibody staining of guinea pig sperm was most prominent in the region of the equatorial segment, while lesser reactivity was present in the subacrosomal space and the proximal portion of the tail (Figs. 17,231. In the rat spermatozoon heavy antibody staining was localized throughout the perinuclear theca (Fig. 18); the greatest reactivity was present in the postacrosomal dense lamina (Fig. 24; Oko and Clermont, 1988). The tail was also reactive along its entire length (Fig. 18).Antibody reactivity was present along the subacrosomal space of hamster sperm; the greatest reactivity was concentrated along a band at the base of the nucleus (Figs. 25,261. Staining was also

associated with the hamster sperm tail (Fig. 19). In the mouse antibody reactivity was confined to the subacrosomal space and the proximal portion of the tail (Figs. 20,271. In all of the species examined precise localization of colloidal gold to a specific region of the perinuclear theca was uncertain, since the method of labeling resulted in disruption of the plasma membrane. Sperm and testis sections that were prepared for electron microscopy with aldehyde fixatives and then processed for antibody staining failed to react with affinity purified antibody.

DISCUSSION The present observations demonstrate the morphogenesis of the perinuclear theca in the subacrosomal and postacrosomal regions of diverse mammalian species and indicate that this cytoplasmic component forms in stages coincident with changes in the ac-

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Figures 14-17. (Figure 17 legend on page 390.)

FORMATION AND CHARACTERIZATION OF PERINUCLEAR THECA rosome, nucleus, and manchette. Western blots of proteins from guinea pig, hamster, rat, and mouse sperm, probed with affinity purified antibodies generated to perinuclear theca protein from bull sperm, revealed the presence of a heterogenous group of polypeptides that differed widely in molecular weights but were immunologically related to the multiple band polypeptides of bull sperm. Using affinity purified antibodies, the appearance and localization of proteins immunologically related to the multiple band polypeptides of bovine sperm have been demonstrated. During the period in which the subacrosomal layer formed, staining with affinity purified antibodies was localized to a thin cytoplasmic compartment between the nuclei and acrosomes of round and elongating spermatids. Later with the caudal movement of the manchette, the postacrosomal segment formed and staining for multiple band polypeptides appeared in the postacrosomal region. It is noteworthy that the extended family of related proteins comprising the multiple band polypeptides of bovine sperm was not present in Coomassie-bluestained gel and in immunoblots of the other mammals examined. In none of the species examined were identical groups of polypeptides found; however, proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all of the species examined. In addition, the sperm heads of all species except bull possessed molecular weight proteins lower than 50 kDa. Immunoreactive proteins less than M, 55,000 were prominent in rat sperm heads and mouse sperm; guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately M, 50,000. These

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variations in polypeptide content are consistent with the expectation that mammalian sperm heads would possess proteins reflective of their markedly different shapes and intracellular arrays (Longo et al., 1987). Recent investigations by Bellve et al. (1990) have characterized a family of proteins (M, 75,000 to 80,000) in mouse spermatids called thecins, which appear to be processed to 48 and 50 kDa proteins after sperm have moved into the epididymis. The possible relationship of these proteins to those described here, specifically the 50 and 74 kDa proteins, is intriguing and worthy of further study. Other proteins have been characterized that are unique to the perinuclear theca of mammalian sperm and include a 13 kDa polypeptide, located in mouse and rat perforatoriums (Oko and Clermont, 1988; Pruslin and Rodman, 1985) and a 60 kDa protein, calicin (Longoet al., 1987; Parenko et al., 1988; Olson and Winfrey, 1988). Reports of other polypeptides localized to the cytoplasmic region of the sperm head include actin, vimentin, keratin, myosin, and spectrin (Flaherty et al., 1988; deCurtis et al., 1986; Virtanen et al., 1984; Ochs et al., 1986). The presence and polarized distribution of these cytoskeletal components are to be involved in the regulation of cell surface events associated with the acrosome reaction and fertilization (Virtanen et al., 1984). Immunoblots of tail proteins demonstrated reactivity of affinity purified antibodies to shared polypeptides of M, 23,000 to 29,000 in bull, guinea pig, hamster, and rat, and 36,000 in hamster and rat. The reactive bands of 64 to 84 kDa in isolated bull sperm tails may reflect the actual presence of these proteins within the sperm flagellum or the result of contaminating heads and whole sperm. Fluorescent staining of sperm tails was variable in that different lengths and regions of the flagellum reacted with purified antibody. ElectronFig. 14. Immunolocalization (a-c epifluorescence optics; a'-c' dense material, which persists after detergent extracphase contrast optics) of multiple band proteins in whole mount tion, has been identified in mammalian sperm tails preparations of epididymal sperm (a,a') and in frozen sections of (Olson et al., 1987; Oko and Clermont, 1988). Further seminiferous tubules containing sperm (b,b') and spermatids (c,c') from bull. Intense reactivity to affinity purified antibody is present efforts are needed to characterize such tail proteins and within the subacrosomal region of differentiating spermatids (arrow) their possible relationship to components present in and has a cup-like organization. The entire perinuclear theca of sperm heads. testicular sperm possesses intense reactivity, while less reactivity is Fluorescence and immunogold staining localized propresent in the anterior portion of the tail. Epididymal sperm show a teins immunologically related to multiple band polysimilar labeling pattern to testicular sperm with intense reactivity in the postacrosomal region and less reactivity in the subacrosomal peptides in perinuclear thecal components of mature region and anterior portion ofthe tail. Figure 14a, x 1,300;Figure 14b and differentiating sperm of all of the species examand c, x 675. ined. The staining patterns obtained with affinity Fig. 15. Immunolocalization (a and b, epifluorescence; c, phase purified antibody were similar t o those shown previcontrast) of proteins reacting with affinity purified antibodies to multiple band proteins (a) and with antitubulin antibody (b) in a ously by other investigators using silver solutions to section of bull testis. The streaks at the arrows in b depict portions of stain the perinuclear theca (Courtens et al., 1976; the manchette that are subjacent to areas reacting with affinity Krimer and Esponda, 1978; Courtens and Loir, 1981; purified antibody to multiple band polypeptides (arrows in a). x 1,500. Elder and Hsu, 1981; Flaherty and Breed, 1987). The Fig. 16. Immunolocalization of multiple band proteins (a)and to microtubules (b) in a n isolated, elongating mouse spermatid. Speci- presence of immunoreactive material corresponded mens were reacted with affinity purified multiple band proteins the appearance of electron dense and agyrophilic substances within the sub- and post-acrosomal spaces of antisera followed by FITC-labeled, secondary antibody (a), tubulin antisera followed by rhodamine-labeled, secondary antibody (b),and differentiating sperm (Lalli and Clermont, 1981). DAPI for DNA (c).Components that react to multiple band protein These observations indicate that formation of the perantibodies are located primarily along the anterior and dorsal aspects of the nucleus (a), i.e., in areas other than those occupied by the inuclear theca is a two stage process correlated with manchette (b). The arrows point to microtubules of the manchette. spermatid morphogenesis. First is the deposition of the x 1,050. subacrosomal layer; this is followed by formation of the

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Figs. 17-20. Immunolocalization (a-c, epifluorescence, a"', phase contrast) of substances reacting with affinity purified antibodies to multiple band proteins in whole mount preparations of epididyma1 sperm (a,a') and in frozen sections of seminiferous tubules containing sperm (a,a') and spermatids (c,c'), from guinea pig (Fig. 17), rat (Fig. 181, hamster (Fig. 19), and mouse (Fig. 20).

Reactivity to affinity purified antibody is associated with the nuclei in differentiating spermatids and in the perinuclear theca of testicular and epididymal sperm. Staining of tails is variable: Anterior tail only, mouse; entire tail, guinea pig, hamster and rat. Figures 17a-Z0a, x 1,350; Figures 17b,c-ZOb,c, x 675.

FORMATION AND CHARACTERIZATION OF PERINUCLEAR THECA

Figs. 21 and 22. Immunoelectron microscopy of bull sperm head (Fig. 21) and proximal tail (Fig. 22) depicting the localization of substances reacting with affinity purified antibodies to multiple band proteins detected by secondary antibodies coupled to 5 mm gold particles. A positive reaction is associated with electron dense material comprising the perinuclear theca. Label within the tail is associated with the dense fibers. M, mitochondria. Figure 21, x 70,000; FiEure 22, x 52,000. Figs. 23-27. Immunolocalization of substances reacting with affinity purified antibodies to multiple band proteins in preparations of epididymal sperm. Fig. 23. Portion of a guinea pig sperm nucleus depicting colloidal gold labeling similar to that seen in fluorescent preparations, i.e., relatively heavy labeling along the anterior region and equatorial

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segment of the sperm nucleus and lesser staining along the posterior region. A relatively heavy deposit of colloidal gold label along that region corresponding to the equatorial segment and less reactivity along its base. x 54,000. Fig. 24. Subacrosomal (a)and postacrosomal dense lamina (b)of a rat sperm depicting different degrees of colloidal gold labeling associated with these areas. x 54,000. Figs. 25 and 26. Subacrosomal (Fig. 25) and postacrosomal (Fig. 26) regions of hamster sperm showing differences in the concentration of label associated with these areas. Figure 25, x 42,000; Figure 26, x 54,000. Fig. 27. Colloidal gold label associated with the perinuclear theca of a mouse sperm. x 54,000.

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postacrosomal segment in conjunction with dynamic changes of the manchette. Although the composition of the cross-bridges that join the manchette microtubules to the nuclear envelope is unknown, their structure and location are consistent with the notion that they are microtubule associated proteins involved with movements of the manchette (Fawcett et al., 1971). The function of the manchette is controversial. It has been postulated to be involved in elongation and shaping of the sperm head (Fawcett et al., 1971; Meistrich et al., 1987; Cole et al., 1988) and there is evidence suggesting that constrictive forces exerted by this organelle modify mechanisms that contribute to normal head shaping (Wolosewick and Bryan, 1977; Cherry and Hus, 1984; Cole et al., 1988). Fawcett et al. (1971) contend, however, that other factors modulate the shape of the sperm head and that the manchette functions to transfer cytoplasm from the cephalic to the caudal pole of the cell during spermatid elongation. As demonstrated here the accumulation of components that comprise the postacrosomal segment, as well as the organization of the plasma membrane along the lateral aspect of the sperm head, are spatially related to the morphogenesis of the manchette. Together, the manchette and the deposition of perinuclear thecal components may contribute to the reduction of cytoplasm in the differentiating sperm head. The perinuclear theca represents the principal cytoi karyo-skeletal structure of the mammalian sperm head (Bellve et al., 1975; Bellve and O’Brien, 1983; Longo et al., 1987) and although its components share structural features with cytoskeletal elements found in somatic cells this may not be their only role (Lalli and Clermont, 1971; Longo et al., 1987; Olson et al., 1987; Olson and Winfrey, 1988). Components of the perinuclear theca are closely apposed to membranes of the sperm head and may act on neighboring components. The sperm plasma membrane has unique surface properties and antigens (Myles et al., 1981; Primakoff and Myles, 1983) and distinctive arrangements of intramembranous particles (Fawcett, 1975; Olson et al., 19831,which undergo considerable rearrangement during capacitation and the acrosome reaction (Nicolson et al., 1977; Friend, 1980).Such redistributions may be modulated by an anchoring framework of the underlying cytoplasm (Olson et al., 1987; Olson and Winfrey, 1988; Olson et al., 1983).In this context components of the perinuclear theca may be involved in establishing the topology and properties of sperm membranes and important in determining specific domains within the plasma membrane (Virtanen et al., 1984) and in the regulation of intracellular calcium during the acrosome reaction (Ruknudin, 1989). Although the definitive shape of the sperm nucleus is established later in spermiogenesis, spermatid elongation occurs prior to the formation of the postacrosomal sheath and, consequently, the extended structure and axis of the sperm nucleus are fixed before the perinuclear theca is fully formed. Furthermore, the perinu-

clear theca can be solubilized without significantly affecting the overall shape of the sperm nucleus (Longo et al., 1987). It has been suggested that the perinuclear theca may function as a dense web connecting the acrosome, nucleus, and the postacrosomal plasma membrane, thereby stabilizing the association of these organelles and maintaining the overall shape of the sperm head (Courtens et al., 1976; Lalli and Clermont, 1981; Bellve and O’Brien, 1983; Longo et al., 1987). The present observations, demonstrating the appearance of sub- and post-acrosomal components of the perinuclear theca concomitant with the close apposition of the acrosome and the plasma membrane to the nucleus, are consistent with this proposal. Previous studies have documented dramatic changes in the sperm plasma membrane a t the time of the acrosomal reaction involving the translocation of specific proteins from one domain to another (Friend, 1980; Myles et al., 1981). I t is noteworthy that with the dehiscence of the acrosome the entire membrane delimiting the anterior of the sperm head is lined by perinuclear theca. Perinuclear thecal polypeptides may be involved in establishing the topology of membranes and important in determining domains that come to delimit the spermatozoon at the time of fertilization (see also Virgil, 1989). Although the contribution of perinuclear thecal components to gamete fusion (Yanagimachi, 1988) and subsequent stages of sperm entry has not been determined. Courtot et al. (1987) have observed that abnormalities of the postacrosomal segment are negatively correlated with the percentage of oocytes in contact with sperm and that fertilize.

ACKNOWLEDGMENTS The authors thank Mark Brachtenbach and Becky Hurt for their assistance. This work was supported by funds from an NIH grant (HD15510).

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