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Biology of Reproduction 63, 629-634 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Protein Composition of the Ventral Processes on the Sperm Head of Australian Hydromyine Rodents1

William G. Breed2,a, Dina Idrissb, and Richard J. Okob

a Department of Anatomical Sciences, The University of Adelaide, Adelaide SA 5005, Australia b Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6

ABSTRACT

The sperm head of the plains rat, an Australian hydromyine rodent, is highly complex in structure and contains, in addition to an apical hook, two large ventral processes (VPs) that extend from its upper concave surface and that are largely composed of a huge extension of the sperm head cytoskeleton surrounded by postacrosomal dense lamina. In this study we have attempted to determine their protein composition. For this, the VPs were isolated, the proteins within them separated by SDS-PAGE, and the resultant polypeptide bands Western blotted and probed with antibodies against laboratory rat perforatorial and bull perinuclear theca sperm proteins. Antibodies were also used to determine the perforatorial and perinuclear theca proteins by immunogold labeling of transmission electron microscopic sections. The results indicate that the material within the VPs is largely composed of perforatorial cross-reacting proteins together with F-actin with the dominant protein being PERF 15. The perinuclear theca proteins are, by contrast, restricted to a narrow region adjacent to the acrosomal and nuclear membranes. In conclusion, this study has shown that the VPs of the spermatozoa of Australian rodents are perforatorial-like appendages that contain similar proteins to the perforatorium of the apical hook together with F-actin; their functional significance remains unknown.

fertilization, sperm, sperm maturation

INTRODUCTION

Most species of eutherian mammals have a spatulate, or pear-shaped, sperm head that is mainly composed of a nucleus with highly condensed chromatin. Over the anterior two thirds of the nucleus an acrosomal cap occurs that contains a gamut of hydrolytic enzymes most of which are released at the time of the acrosome reaction. Between the inner acrosomal membrane and outer nuclear envelope a modest space is present, the subacrosomal space, that contains several proteins whose function, at least in part, may be to attach the inner acrosomal membrane to the outer nuclear envelope. This space extends posterior to the acrosomal cap where it gives rise to a postacrosomal sheath from which electron-dense material extends to, and stabilizes, the overlying plasmalemma.

Common laboratory rodents, unlike other groups of mammals, have a falciform or sickle-shaped sperm head. Although the same structural components of the sperm head are present as those in other eutherian mammals, they differ significantly in their organization. In these spermatozoa the nucleus extends into the apical hook where it is surrounded by a large extension of the cytoskeleton that is generally referred to as the perforatorium [1–6]. Early studies on the protein composition of the perforatorium suggested that it was primarily composed of a single 15-kDa protein [7], but subsequently it has been shown to contain several proteins whose composition differs somewhat from the postacrosomal sheath with which it is continuous [8–10]. More recently the 15-kDa protein present within the perforatorium has been cloned and characterized [11–13].

Within most Australian hydromyine rodents the sperm head is considerably more complex than that of the laboratory rat in that, in addition to the apical hook, there are two further processes that extend from the upper concave surface [14]. These processes develop late in spermiogenesis and are largely composed of electron-dense material that is continuous with the perforatorium in the apical hook [15–20]. The chemical composition of these processes is largely unknown although filamentous actin has been shown to be present [17, 18, 20–22] that is not present in the perforatorium of the mature sperm head of the laboratory rat [9, 23, 24]. The objective of the present study was to isolate the ventral processes (VPs) and determine their protein composition by SDS-PAGE and Western blotting, and by TEM after immunogold labeling with the use of antisera raised against proteins of the perforatorium in the laboratory rat and of the perinuclear theca in the bull.

MATERIALS AND METHODS

The species of hydromyine rodent used in this study was the plains rat, Pseudomys australis. This species has a sperm head morphology typical for this subfamily of murid rodents that contains both an apical hook and the two VPs that extend from its upper concave surface [14, 17, 18–22]. The animals occur within the arid region of South Australia and individuals used in the present investigation came from the breeding colony maintained within the Division of Animal Services at The University of Adelaide.

Isolation of the VPs from Plains Rat Spermatozoa

Spermatozoa were obtained from the caudae epididymides of nine adult males for isolation and extraction of the proteins from the VPs. Because there are between 500 x 106 and 800 x 106 sperm stored in the caudae epididymides of an adult plains rat [25–27] this resulted in about 5000 x 106 for extraction. The sperm were sonicated on ice in 20 mM Tris-HCl 0.9% NaCl, pH 7.4 (TBS), to break the heads from tails. The sonicated sperm suspension was then washed several times by low-speed centrifugation and the final pellet resuspended in 80% sucrose, TBS, and centrifuged at 280 000 x g for 1 h in a 60 Ti angle rotor (Beckman, Mississauga, ON, Canada). The oblong sperm head pellet on the centrifugal side of the tube containing mostly heads was resuspended in TBS and checked for sperm head purity by phase contrast microscopy. If necessary, the ultracentrifugation step was repeated until the fraction contained >99% sperm heads.

Isolated sperm heads were exposed to two successive extraction steps consisting of incubations in 0.2% Triton-X-100 and 1 M NaCl for 1 h, each step being followed by a TBS wash. The final pellet was then resuspended in 1 M NaOH for 10 min followed by passing the suspension through a 20-gauge needle several times. The resultant shearing force detached the VPs that were subsequently separated from the sperm head by centrifugation at 100 000 x g through a 20/80% sucrose gradient. The VPs, collected from the sucrose interface, were then diluted in TBS, pelleted by centrifugation, and analyzed by electron microscopy, SDS-PAGE, and Western blotting.

SDS-PAGE and Western Blotting

Isolated VPs were solubilized in 2% SDS/5% ß-mercaptoethanol and run on a linear gradient (8–18%) polyacrylamide gel according to Laemmli [28]. Proteins were electrophoretically transferred from the gels to immobilin-P (0.45 µm pore size; Millipore, Bedford, MA) in a solution of 25 mM Tris-HCl, 192 mM glycine, and 10% methanol (pH 8.3) using a Hoefer transfer apparatus.

Immunoblotting

The reactivity of the antilaboratory rat perforatorial and antibull perinuclear theca antibodies was investigated on the Western blots of the plains rat VPs, using a secondary antibody (alkaline phosphatase-conjugated goat antirabbit IgG; Sigma, St. Louis, MO) to detect the signal according to the method of McGadey [29].

Immunogold Labeling of Thin Sections and Electron Microscopy

For obtaining mature spermatids and cauda spermatozoa for transmission electron microscopy and immunogold labeling, small pieces of tissue or sperm pellets were immersed in 3% paraformaldehyde/0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. After fixation for several hours, the tissue was osmicated, dehydrated, and embedded in Lowicryl. Thick, 0.5- to 1-µm, sections were cut with an ultramicrotome, and when appropriate regions were obtained, ultrathin sections were cut and stained with uranyl acetate and lead citrate. These were then incubated with one of the several polyclonal antibodies raised against laboratory rat perforatorial proteins [8, 9] as well as bull perinuclear theca proteins [30]. An antiactin monoclonal antibody (anti-C4) kindly donated by Dr. James Lessard was also used [31].

RESULTS

Isolation of the VPs

Two VPs, that are a characteristic feature of the sperm head of most Australian hydromyine rodents including the plains rat (Fig. 1), form during late spermiogenesis. They develop as extensions from the upper concave surface of the sperm head, are joined basally, and differ a little in size with the more apical process being slightly larger [14, 19] (Fig. 2, A to D). The nucleus protrudes into the base of the processes (Fig. 2A) distal to which there are two fingerlike acrosomal extensions (Fig. 2B). These are surrounded by large amounts of cytoskeletal material and they become progressively more bilaterally flattened toward their tips (Fig. 2, C and D). They contain an extension of the postacrosomal dense lamina beneath which filamentous actin occurs [21, 22] (Fig. 3).



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  		FIG. 1. Nomarski differential interference micrograph (A) and scanning electron micrograph (B) of sperm head of the plains rat showing that, in addition to an apical hook (AH), two VPs extend from the upper concave surface; VS = ventral spur. Transmission electron micrograph of section through a-a is shown in Fig. 2C and b-b in Fig. 2D. Bars: A = 3.5 µm; B = 1.0 µm



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  		FIG. 2. Transmission electron microscopy of sperm head of plains rat. A) Two large VPs extend from the upper concave surface of sperm head and the nucleus protrudes into the base of these processes. B) Distal to the nucleus a fingerlike acrosomal projection occurs (arrow). This process is, however, very largely composed of electron-dense material that is an extension of the cytoskeleton, and transverse sections show that both processes become progressively bilaterally flattened distally (C and D). AH, Apical hook; Ac, acrosome; VS, ventral spur. Bars: A = 850 nm, B = 450 nm, C = 100 nm, and D = 140 nm



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  		FIG. 3. Fluorescent light micrograph of cauda epididymal sperm head of plains rat stained with propidium iodide (a nuclear stain) and Bodipy-phallacidin (a stain for filamentous actin). Note the protrusion of the nucleus into the base of the VPs distal to which abundant F-actin occurs, as shown by intensely fluorescent staining with Bodipy-phallacidin

Incubation of sonicated cauda epididymal spermatozoa in 1 M NaOH followed by the passing of the sperm suspension through a 20-gauge needle confirmed that this treatment resulted in the detaching of the VPs from the rest of the sperm head [19] (Fig. 4). These VPs were subsequently isolated and collected at the 20/80% sucrose interface. Although some protein may be lost during the isolation procedure, the VPs retained their shape and most of their electron density suggesting much of the proteinaceous material was retained.



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  		FIG. 4. Separation of VPs from isolated sperm heads of plains rats. Intact isolated sperm head showing apical process (AP) and two VPs (a); x1250. Sperm head after exposure to IM NaOH and shearing through a 20-gauge needle showing that the VPs detach from the head (b); x1000. Phase contrast survey micrograph of detached VP (c); x500. Electron micrograph of detached VPs isolated on a discontinuous sucrose gradient (d). Note that the VPs retain their density and shape and show their nuclear attachment site (arrow); x20 000

Identification of Proteins in the VPs

The isolated sample of VPs was then denatured under reducing conditions and run on SDS-PAGE. When stained with either Coomassie or silver a major component was found to have a molecular weight of 15 kDa (Fig. 5). A second minor band of 34 kDa was also sometimes seen when the gels were stained with silver; whether this was visible was dependent on the sample load. Because these molecular weights are identical to the most prominent proteins present in the laboratory rat perforatorium, the proteins were transferred to immobilin-P and probed with antibodies raised against laboratory rat PERF 15 and PERF 34. Western blotting showed clear immunocross-reactivity to laboratory rat anti-PERF 15 and anti-PERF 34 (Fig. 6) and no reactivity to immune serum (anti-1449) raised against the bull perinuclear theca.



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  		FIG. 5. Silver-stained SDS-PAGE of denatured proteins derived from isolated VPs. Lane S, molecular mass standards; x1000. Lane 1, polypeptide profile of VPs showing prominent band of 15 kDa and a minor band at 34 kDa



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  		FIG. 6. Western blot of VP polypeptides immunostained with antiperforatorial antibodies. Lane S, molecular mass standards; x1000. Lane 1, Coomassie blue-stained VPs polypeptides. Lane 2, VP polypeptides immunostained with preimmune serum from rabbit used to obtain antiperforatorial serum. Lane 3, VP polypeptides immunostained with antiperforatorial serum affinity purified on isolated 15-kDa perforatorial protein of laboratory rat (anti-PERF 15). Note cross-reactivity with the major 15-kDa protein of VPs. Lane 4, VP polypeptides immunostained with affinity purified anti-34 kDa perforatorial serum. The serum cross-reacts with both 34- and 15-kDa proteins of VPs. Lane 5, VP polypeptides immunostained with antiserum raised against bull perinuclear theca extract (anti-1449) that normally labels several prominent bull perinuclear theca proteins. No reactivity is evident. The parallel marks in lanes 3 and 5 at the 30-kDa level are artifacts of preparation and do not immunostain

To determine the distribution of the 15- and 34-kDa proteins within the VPs immunogold labeling with anti-PERF 15 and anti-PERF 34 was carried out together with another antibody raised against a less prominent perforatorial protein, anti-PERF 57. It was found that labeling of both VPs took place as well as the perforatorium of the apical hook after incubation with antibodies to the 15-, 34-, and 57-kDa proteins (Fig. 7, A to D and Fig. 8, A to C). When the sections were incubated with antiactin antibody (C4) labeling also occurred throughout much of the VPs but this did not take place over the perforatorium (Fig. 8, D to F). However when the sections were incubated with anti-1449 serum, raised against the bull perinuclear theca, labeling occurred close to the nucleus and/or acrosome (Fig. 9).



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  		FIG. 7. Transmission electron micrographs of transverse sections through apical hook (AH) and VPs of sperm heads from cauda epididymides after staining with antibody to 15-kDa (A and B) and 34-kDa perforatorial proteins (C and D). Note abundance of gold particles over both perforatorium of apical hook and material in both of the VPs (VP1 and VP2). Bars: A = 170 nm, B = 170 nm, C = 130 nm, and D = 110 nm



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  		FIG. 8. Transmission electron micrographs of transverse sections through apical hook (AH) and VPs (VP1 and VP2) of sperm heads from cauda epididymides after staining with antibody to the 57-kDa polypeptide indicating heavy labeling over the material in the VPs as well as the perforatorium in the apical hook but not over the nucleus (A–C), and antiactin antibody C4 with labeling over the VPs but not over the perforatorium in the apical hook (D–F). Bars: A = 170 nm, B = 130 nm, C = 170 nm, D = 120 nm, E = 120 nm, and F = 90 nm



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  		FIG. 9. Triton-X100- and NaCl-extracted plains rat sperm head immunogold labeled with anti-1449 immune serum raised against bull perinuclear theca proteins. Labeling is only evident along the periphery of acrosome (arrows) and nucleus (arrowheads) but absent in central core of VPs and apical process (AP). N, Nucleus; VS, ventral spur. Bar = 300 nm

DISCUSSION

The non-Rattus native rodents of Australia are a distinct group of murid rodents that constitute an assemblage of about 60 species ranging from small mouselike mammals to others that have body mass of about 1 kg [32]. Many other members of this group are also present in New Guinea [33], but no close relatives have been found in southeast Asia. Due to their distinctiveness and separate evolutionary history from that of other murids, these rodents have been placed within a separate subfamily, the Hydromyinae [34–36]. Studies on spermatozoa of these species have shown that most members of this group have a very distinctive morphology. Like those of most murid rodents, as exemplified by the laboratory rat and mouse [1–6], the sperm head is falciform in shape in which there is an apical hook containing a perforatorium [3–6]. However, in addition to this structure, two further processes extend from the upper concave surface that have been termed VPs [14, 15, 17–19]. Early transmission electron microscopic observations suggested that these structures were an extension of the subacrosomal space [15, 16, 19], although later work on their morphogenesis indicated that there were two regions of electron density with the peripheral material being shown to be part of the postacrosomal dense lamina [20, 21]. These VPs stain positively with NBD and Bodipy phallacidin, thus demonstrating that F-actin occurs within them [17, 21, 22] (see Fig. 3). So far, however, no other proteins have been identified. That there is other material present is evident from ultrastructural observations of changes that take place during spermiogenesis where, concomitant with their formation, microfilaments of actin can be seen laterally with amorphous material centrally. Furthermore, as maturation proceeds, the processes change in shape coincident with more electron-dense material accumulating within these structures so that, shortly before spermiation, the actin filaments are no longer visible [17, 20, 21].

The present study has been carried out to determine what other proteins are present. In the laboratory rat the perforatorium has been shown to contain several proteins that differ somewhat from those in the postacrosomal sheath [8, 9] and immunolabeling with antibodies raised against several high molecular weight proteins (34, 43, 57, and 63 kDa) demonstrated these proteins to be present throughout the perforatorium, as well as the inner layer of the ventral spur, but no labeling was present over the postacrosomal sheath. Three further low molecular weight proteins (13, 13.4, and 15 kDa) were found to be restricted to the thicker apical parts of the perforatorium as well as within the inner layer of the ventral spur [9].

In the present study antibodies to the 15-kDa (PERF 15) and 34-kDa proteins of the laboratory rat perforatorium cross-reacted strongly with the only two proteins of the same molecular weight recovered from the NaOH isolated VPs. Combined with the ultrastructural observation that the core density of the isolated processes is preserved, immuno-Western blot data indicate a similarity in composition between the respective structures with PERF 15 being the dominant shared protein. Subsequent immunostaining of the thin sections of cauda epididymal spermatozoa with antibodies to both the 15-kDa and 34-kDa proteins confirmed the presence of these proteins in the VPs as well as the 57-kDa protein. These results show that, even though the VPs are surrounded by an extension of the postacrosomal dense lamina, they internally contain material that is composed of at least some of the proteins that are present in the perforatorium, analogous to the ventral spur of the laboratory rat sperm [9]. They are not, however, an extension of this structure as scanning and transmission electron microscopy of spermatozoa of the plains rat clearly demonstrate its presence separate from the VPs [15–17, 19] (see Figs. 1 and 2). Furthermore our results indicated that the VPs are composed of perforatorial proteins (i.e., PERF 15 and 34) that are not found in spatulate spermatozoa of the bull, strongly suggesting that these proteins are specific to members of the murid family of rodents. Figure 10 summarizes our data on the distribution of perforatorial proteins in the plains rat sperm head.



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  		FIG. 10. Summary of distribution of perforatorial proteins (orange) in a diagrammatic sagittal section through the sperm head of the plains rat accompanied by two representative cross-sectional views. Perforatorial proteins are found throughout the dense core of VPs and apical process (AP). A relatively thin cortex (in yellow border dotted) surrounds the dense core of the VPs and is an extension of the postacrosomal dense lamina (PDL). Labels: A = acrosome, stippled regions; N = nucleus; NP = neck piece. The white dashed line denotes where the caudal-lateral extent of the acrosome would be present on the exterior of the sperm head. Compare with illustrative diagram of laboratory rat spermatozoon (cf. plate 1 [10])

In our previous fluorescent light microscopic investigations of the structural organization of the sperm head of the plains rat using NBD phallacidin we found that the VPs fluoresced brightly, demonstrating the presence of filamentous actin [17, 18, 21, 22] (see also Fig. 3). In spermatozoa of most mammals, including those of the laboratory rat, it has been found that the filamentous actin in the subacrosomal space of late spermatids depolymerizes prior to the release of sperm from the testis [23, 24, 37–39]. Thus the retention of filamentous actin in the mature sperm of the plains rat is unusual. This presence of F-actin in epididymal sperm was subsequently confirmed by immunogold labeling with a mouse monoclonal antiactin antibody [21], and it has now been demonstrated with another mouse antiactin antibody, C4, whereas no staining occurs over the perforatorium as previously found [9]. Actin's absence in PAGE analysis of the VPs is explainable by its tendency to solubilize in NaOH (R.J.O., personal observation), the solution used in this study to detach the VPs from the sperm head.

What is the function of these VPs and the material within them? Previously, it has been suggested that the material in the perforatorium of the laboratory rat spermatozoon may either bind the inner acrosomal membrane to the underlying nuclear envelope and/or maintain stability of the sperm head membranes during penetration of the egg coats [9, 10]. The cytoskeletal material in the sperm head VPs of the plains rat could not perform the former function as these processes do not lie beneath the acrosome, which, in fact, only occurs as two small protrusions surrounded by the cytoskeletal material near the base of the VPs. Furthermore, these VPs could hardly have evolved to maintain the bulk of the sperm head shape as they extend up to several micrometers distally from the main body of the sperm head. In the recent past several functions have, in fact, been suggested for the VPs, but there is minimal evidence for any of these functions. It would appear that the processes make contact with the matrix of the zona pellucida during zona binding and penetration and studies on oocytes of recently mated animals have clearly indicated that these processes become incorporated in the egg cytoplasm at the time of fertilization without undergoing any change in form or apparent structural organization [17, 21]. Thus, as a working hypothesis, it is proposed that the VPs of these Australian hydromyine rodents may form stabilized structures to maintain a region of the cell membrane in which ligands for sperm/zona pellucida binding occur. In vitro and immunogold labeling studies with antibodies to sperm surface molecules involved in sperm/egg interactions are now required to investigate this possibility.

Conclusion

The sperm head of most of the Australian hydromyine rodents is one of the most morphologically complex sperm types to have evolved in eutherian mammals due to the presence of two massive extensions of the cytoskeleton that form processes that extend from its upper concave surface. In this study, immunolabeling with polyclonal antibodies to the laboratory rat perforatorial proteins 15, 34, and 57 kDa, as well as with an antibody to mouse actin C4, showed that all four proteins occur throughout the length of these processes that are surrounded by an extension of the postacrosomal dense lamina. The function of these processes has yet to be determined, but molecules on the plasmalemma surrounding them may participate in sperm/zona binding.

ACKNOWLEDGMENTS

We thank Chris Leigh for technical assistance and Esther Breed for typing the manuscript.

FOOTNOTES

First decision: 20 January 2000.

1 This work was supported by grants from ARC of Australia to W.G.B., and NSERC and MRC of Canada to R.J.O. Back

2 Correspondence: FAX: 61 8 8303 4398; william.breed{at}adelaide.edu.au Back

Accepted: March 5, 2000.

Received: December 20, 1999.

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