Mouse Sperm Rosette: Assembling During Epididymal Transit, in vitro Disassemble, and Oligosaccharide Participation in the Linkage Material

Authors

  • María De Los Ángeles Monclus,

    Corresponding author
    1. Instituto de Histología y Embriología de Mendoza (IHEM), Histology and Embryology Area, Department of Morphology and Physiology, School of Medicine, National University of Cuyo—CONICET, Mendoza, Argentina
    • Instituto de Histología y Embriología, Área de Histología y Embriología, Departamento de Morfología y Fisiología, Facultad de Ciencias Médicas, Centro Universitario, Parque Gral, San Martín, Universidad Nacional de Cuyo, Casilla de Correo 56, Mendoza 5500, Argentina
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    • Fax: 54-261-4494117

  • Andreína Cesari,

    1. Instituto de Investigaciones Biológicas (IIB), School of Sciences, Mar del Plata National University—CONICET, Mar del Plata, Argentina
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  • María Eugenia Cabrillana,

    1. Instituto de Histología y Embriología de Mendoza (IHEM), Histology and Embryology Area, Department of Morphology and Physiology, School of Medicine, National University of Cuyo—CONICET, Mendoza, Argentina
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  • Paola Vanina Borelli,

    1. Instituto de Histología y Embriología de Mendoza (IHEM), Histology and Embryology Area, Department of Morphology and Physiology, School of Medicine, National University of Cuyo—CONICET, Mendoza, Argentina
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  • Amanda Edith Vincenti,

    1. Instituto de Histología y Embriología de Mendoza (IHEM), Histology and Embryology Area, Department of Morphology and Physiology, School of Medicine, National University of Cuyo—CONICET, Mendoza, Argentina
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  • Mario Héctor Burgos,

    1. Instituto de Histología y Embriología de Mendoza (IHEM), Histology and Embryology Area, Department of Morphology and Physiology, School of Medicine, National University of Cuyo—CONICET, Mendoza, Argentina
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  • Miguel Walter Fornés

    1. Instituto de Histología y Embriología de Mendoza (IHEM), Histology and Embryology Area, Department of Morphology and Physiology, School of Medicine, National University of Cuyo—CONICET, Mendoza, Argentina
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Abstract

In many mammals, sperm associations had been observed, but not in the mouse. In this work, mouse sperm rosettes are morphologically described inside the epididymis and during its dissolution in a culture medium. Also characterized are the saccharides present in the linking material. Sperm association and other epididymal actions are supported by sperm during epididymal transit and are verified at the caudal region, suggesting a relation between epididymal transit and sperm maturation. In drops of epididymal content obtained from distal (cauda), but not from proximal (caput and corpus) regions; dissolved in culture medium, rosettes appear to be 10 to 15 motile sperm joined by their heads. After 3 min, sperm progressively detach, disassembling the rosette. These structures are studied by several techniques, including optic, electronic (scanning electron microscopy and transmission electron microscopy), and video microscopy. At the ultrastructural level, a dense network of electron-dense material was observed between sperm heads, joining them. Based on previous works in rat, several lectins were used to characterize the type of saccharides present in this linking material. To avoid the contact between sperm and epididymal fluid from distal region—that probably exerts an influence on sperm association—a ligature was placed between caput and corpus. This epididymal content isolated from caput did not display any rosettes after 28 days. Anat Rec, 2007. © 2007 Wiley-Liss, Inc.

Mammalian spermatozoa support several changes during epididymal transit, collectively named sperm maturation (Yanagimachi, 1994). Morphological, biochemical, and functional changes had been described in individual sperm cells during the epididymal journey (Bedford, 1979). However, sperm association also takes place during sperm maturation in some species. This association consists in living sperm cells agglutinated by their heads described in several species (Guinea pig: Simeone and Young, 1931; Fawcett and Hollenberg, 1963; Martan and Shepherd, 1973; Yanagimachi and Mahi, 1976; squirrel: Martan and Hruban, 1970; naked tail armadillo: Heath et al., 1987; boars: Briz et al., 1995).

In guinea pig sperm rouleaux (GPSR), the sperm heads are stacked one upon the other, the curved apical segments fitting closely together. Interesting, between sperm head membranes a “filamentous material” was described. Moreover, the outer surface of the plasma membrane present junctional zones—like a “bridge”—between cells. On the contrary, tails can beat freely promoting the displacement of the sperm association (Fawcett and Hollenberg, 1963).

More recently, a similar sperm association was described in the rat (Fornés and Burgos, 1990). A great number of sperm associated in a close proximity, but not arranged in an orderly manner as in a rouleaux. The lateral faces of the sperm head are the regions used to associate cells instead the inner curved face of the apical acrosomal region in the GPSR. For this reason, it was called “rosettes” (rat sperm rosette = RSR). However, an electron-dense material (EDM) joining the sperm heads was also present. Schiff reaction (PAS) -positive material—in light and electron microscopy analysis—was detected in RSR (Fornés and Burgos, 1990).

Sperm association as well as other epididymal actions are supported by sperm during the epididymal transit and verified at the caudal region (Gatti et al., 2004). If epididymal transit is stopped by ligature, the maturational changes are not expressed (Cameo et al., 1971; Blaquier et al., 1972).

Dissociation of GPSR with trypsin or pronase indicated that proteins are involved in rouleaux or rosettes (Flaherty and Olson, 1991). Posterior studies showed that antibodies against specific membrane proteins, like WH-30 or autoantibody, can dissociate GPSR (Flaherty et al., 1993; Tung et al., 1980). Curiously, these autoantibodies raised to testis homogenates also dispersed the GPSR, but rouleaux are not arranged until the sperm cells reach the distal region (cauda epididymis), indicating that “sperm testis proteins” need a further processing during the epididymal trip (Tung et al., 1980). In RSR, antibodies against specific rat epididymal protein, DE glycoprotein, also recognize the linking material between sperm heads (Fornés and Burgos, 1994). Secreted epididymal glycoprotein binds to spermatozoa during epididymal maturation. In this sense, glycoprotein precursors radioactively labeled are trapped within sperm heads associations in mouse and guinea pig rosette or rouleaux (Kopecny et al., 1984). Sperm epididymal maturation is promoted by several factors secreted by the epididymal epithelium (Burgos et al., 1992; Cooper, 1998; Jones, 1998; Tulsiani, 2003). Some of these factors are glycosidases that might modified sperm membrane oligosaccharides involved in the interactions between sperms and others cells, e.g., sperm–egg interaction (Tulsiani et al., 1998; Talbot et al., 2003). All evidence indicates that glycoprotein or glycoproteins modified by glycosidase secreted by the epididymal epithelium are involved in sperm association during the epididymal trip. Surface sperm glycosides are important for sperm function (Skudlarek et al., 2000). One of them could also been involved in the assembling of sperm rosettes.

As it was stated in many mammals, during epididymal transit, sperm show cohesive properties producing sperm associations (Bedford, 1979; Phillips and Bedford, 1988). But the biological function of this sperm rouleaux or rosettes remains uncertain. Several speculations were made: a) membranes associated by specific junctional specialization in rouleaux could provide direction to membrane fusion events of the acrosomal reaction (Flaherty and Olson, 1991), b) it was also postulated that rouleaux preserve viability and prevent guinea pig sperm from undergoing a premature acrosomal reaction (Tung et al., 1980), c) others authors speculate that the associations have more implications in male's reproductive strategy (Cooper et al., 2000). Interesting examples could be found in the wood mouse (Apodemus sylvaticus). Sperm cells conform a “train” of sperm, which moves faster than one lone spermatozoon (Moore et al., 2002). This idea proposes that this behavior is altruistic, because each one of them helps the others to fertilize the egg by compromising their own fertilizing ability. A recent paper shows that the sperm association of some “promiscuous” species gives more chances of fertilization than the lone sperm if two males pair with the same female (Cooper et al., 2000).

The present work reports the detection of mouse sperm rosette (MSR) during epididymal transit. First, MSRs were studied in suspension by placing drops of epididymal fluid in a culture medium. Then, MSRs were analyzed inside the epididymal tubule, in situ. MSRs in suspension were performed in fixed cells (optic and electron microscope analysis and saccharides detection) and alive cells (recording of MSR disassembling in a culture medium). MSR in situ assembling was detected in ultrastructural sections of the main epididymal regions. Finally, the influence of “associating factors” secreted in corpus or cauda epididymis was studied by classical ligature experiment. If ligature is placed between caput and corpus, the sperm was retained in caput and no maturation take place or sperm association.

MATERIALS AND METHODS

Reagents

Reagents were from Sigma Chemical Co. (St. Louis, MO), except for electron microscopy reagents, which were from Pelco (Redding, CA). Some reagents from other sources were specified in the text.

Detection of MSR

Adult mice (Balb c) were obtained from our colony maintained under the guidelines of Bioethics Commission of the School of Medicine (Res. 32, 1995) that follow the Guidelines for the Care and Use of Laboratory Animals of the NIH (Bethesda, MD). They were killed by cervical dislocation, and the epididymis immediately was removed. Segments two, four, and six of the epididymis as described by Reid and Cleland (1957) were used. These segments correspond to the classically named caput, corpus, and cauda epididymis.

First, we present studies done in suspension (MSR in suspension) with material coming from puncture of epididymis, including saccharide detection and characterization by specific inhibitors, and then in situ (MSR in situ) observations of epididymal sections. Epididymal fluid containing sperm was obtained by puncture the epididymal duct at the principal regions—caput, corpus, and cauda—in 10 adult mice as described previously (Fornés and Burgos, 1990, 1994). A new needle (27G 1/2″) was used in each set of experiments. Immediately after puncture, a dense drop of epididymal fluid appears. Epididymal drops were obtained and immediately placed in 40 μl of warm (36°C) HM culture medium (modified Krebs-Ringer bicarbonate medium, Hepes buffered) on biologically cleaned slides (Lutz and Inoué, 1986). HM was made thawing 5 ml of 3× HM stock solution and adding sodium pyruvate (1.65 mg), 17 mM CaCl2 (1.5 ml), and 8.5 ml of double-distilled water. Finally, the pH was adjusted to 7.4 with 10 M NaOH (HM 1 × consists of 25 mM Hepes, 109 mM NaCl, 4.77 mM KCl, 1.19 mM MgSO4, .7 H2O, 1 mg ml−1 glucose, 3.7 μl of Na-lactate (60% syrup), 1.19 mM KH2PO4; Visconti et al, 1995).

As soon as the epididymal drop was placed in the HM, the epididymal fluid dispersion began. For this reason, the procedure was performed under stereomicroscope (200 ×, Zeiss stemi 1000 stereomicroscope, Germany) or microscope (250 ×, bench top Bausch & Lomb, USA)—without cover glass—to verify the pattern of sperm dispersion, rosette appearance, and sperm motility. Once the MSRs were recognized—in drops from cauda epididymis—all of them were fixed except for video microscopy or light microscopy of live MSRs. In these cases, epididymal drops were placed in chambers previously prepared. Samples were fixed adding to the suspension equal volume of fixative solution consisting of 4% paraformaldehyde (w/v), 4% glutaraldehyde (w/v), and 20 % saturated picric acid (v/v) as it was described by Mollenhauer et al. (1976) in phosphate buffer saline (PBS). PBS was prepared from Sigma tablets (pH 7.4; 0.001 M phosphate; 0.0027 M ClK and 0.137 M ClNa).

Ten minutes after fixation, MSRs (cauda samples) or groups of sperm—see below—(caput and corpus) were harvested with a Pasteur pipette and transferred into 2-ml polypropylene centrifuge tubes. These “groups of sperms” was also included because they sometimes have a rosette-like appearance (called rosette-like). Samples were centrifuged 10 min at 500 × g (IEC, centrifuge, Newark, NY) to concentrate the MSR or rosette-like to obtain a pellet suitable for electron microscopy. Samples from the three epididymal regions were then washed twice by centrifugation: resuspension in PBS for equal time, and centrifugal force. The pellets were resuspended in 100 μl of PBS and prepared for scanning electron microscopy (SEM) or transmission electron microscopy (TEM).

SEM of Fixed MSRs in Suspension

Twenty microliters of fixed-washed MSRs or rosette-like were collected with an automatic pipette from the centrifuge tube and placed on mica grids (ø: 4 mm, Pelco) coated with gelatin. Jelly coated were prepared by immersing clean and new mica grids in 1% gelatin in double-distilled water (w/v). Grids were then placed onto cover slip and left to dry at room temperature overnight protected from dust. After 10 min, when the sperm adhered to the grid, they were dehydrated by immersing the cover slip containing grids in grades of ethanol–acetone (up to absolute acetone, Merck®) and dried by the critical point method with liquid CO2 (critical point dried equipment, Sorvall, Newtown, CN). After drying, cells were coated with gold (approximately 20 nm, Balzers Union sputter device) and preserved under vacuum equipment. Studies were performed in an Austoscan ETEC scanning electron microscope.

TEM of Fixed MSRs in Suspension

MSRs or rosette-like in PBS suspension (see above) were centrifuged for 10 min at 500 × g (IEC, centrifuge), and pellets were re-fixed adding 30 μl of 1% OsO4 (w/v) overnight at 4°C. Osmified samples were dehydrated in ethanol–acetone (up to absolute acetone, Merck®) and embedded in Epon 812 (Pelco). Ultrathin sections were obtained in an Ultracut, (Leica ultramicrotome) and observed in Zeiss 900 electron microscope.

Saccharide Detection in Fixed MSRs in Suspension

Drops of caudal epididymal content were obtained as described above and placed in 30 μl of HM media onto clean slides maintained on a hot plate (36°C). After 3 min, 30 μl of 4% freshly prepared paraformaldehyde (w/v) in PBS was added. Ten minutes later, MSR were harvested under stereomicroscope observation with automatic pipette. Then 20 to 30 μl of the fixed MSR suspension were finally placed onto cleaned slides avoiding dryness by keeping glasses in a moist chamber. The slide had a delineated zone traced with a diamond pencil to keep the suspension inside the area. The next day, slides were rinsed by immersion in a beaker containing 40 ml of PBS. Under stereomicroscope, the MSRs were check and the slides were dried outside the sample area with tissue papers (Kimwipes papers, Kimberley Clark, Neenah, WI). Immediately, 20 μl of different lectin solutions were placed onto the samples. Lectin coupled with fluorescein isothiocyanate (FITC): Pisum sativum agglutinin (PSA); Arachis hypogaea (PNA), and Ricinus communis (RCA120) or colloidal gold coupled with Canavalia ensiformis (Concanavalin A, Con A) were used. Fluorescent lectins were applied in a PBS solution, 1 mg ml−1 (w/v) and Con A coupled with 20-nm colloidal gold was prepared in the lab following Handley (1989) and Benhamou (1989). In the fluorescence experiments, before 30 min at room temperature in the dark, slides were immersed in PBS for 1–3 min. Then, slides were dried with tissue paper, except the sample area, and cover slips was added with glycerol, and samples were observed in a fluorescence-inverted microscope (Nikon Eclipse TE 300). Digital images were obtained and stored in a PC with Metamorph 4.5® software. In the case of colloidal gold, before 60 min in the dark and in a moist chamber, slides were immersed in double-distilled water and developed by silver stain according to previous papers (Hacker, 1989; Pietrobon et al., 2003). Several controls must be performed in these series of experiments: (1) Slides were also incubated with colloidal gold alone to test for any nonspecific deposits onto sperm cells, and then processed; (2) Slides were also exposed only to the Silver Enhancer kit for gold, also to check unspecific staining by silver.

Presence of Saccharide in Rosette Analyzed by Specific Glycosidase Inhibitors

To confirm the role of α-D-glucose and/or α-D-mannose detected by lectin staining in the linking material (see above), we performed a MSR dissociation assay in the presence of different glycosidases inhibitors in HM. We evaluated the effect of Swainsonine (SW) a mannosidase II inhibitor (Skudlarek et al., 1991); 1,4-dideoxy-1,4-imino-D-mannitol (DIM), mannosidae I and II inhibitor (Winchester et al., 1993), and 1-deoxymannojirimycin (DMJ), mannosidase I and glucosidase inhibitor (Mulakala et al., 2006) at different concentrations. Several epididymal drops containing MSR were obtained by puncture of cauda epididymal region. Drops were immediately placed in 50 μl of warm (36°C) HM. The media were supplemented in separate experiments with increasing concentrations off each inhibitor (Table 1). After 3 min at 36°C, the epididymal fluid dispersion was microscopically examined (×400) and the MSRs remaining in the media were tabulated. As a control, epididymal drops were run in parallel without inhibitors. Each microscopic field contains an equal amount of motile sperm (120 cells ± 10/field).

Table I. Glycosidase inhibitors vs. number of MSRs in suspension after 3 min
Glycosidase inhibitorFinal concentrationNo. of MSR/microscopic field
  1. MSR, mouse sperm rosette.

Swainsonine (SW)0 μM (control)4,25 (±1)
10 μM9,5 (±1)
50 μM11,89 (±2)
100 μM7,41 (±1)
1,4-Dideoxy-1,4-imino-D-mannitol (DIM)0 μM (control)4 (±1)
1μM4 (±1)
5 μM2,66 (±1)
10 μM3,94 (±1)
1-Deoxymannojirimycin (DMJ)0 mM (control)4 (±1)
0.125 mM5 (±1)
0.250 mM4 (±1)
0.500 mM5 (±1)

Video Microscopy of Living MSRs in Suspension

For these experiments, chambers on slides were prepared the previous day and maintained in a clean box. Chambers were made as described in the previous reports (Fornés and Burgos, 1990) following the instructions in “Techniques for observing living gametes and embryos” (Lutz and Inoué, 1986). Briefly, one biologically cleaned cover slip was mounted over two sheets of plastic paper (thickness ∼ 20 μm), located at the center of the slides and kept in the position with small balance weight. Bee wax and solid Vaseline mixed (1:1) was used to seal the space between cover and slides using electric soldering. Then, both papers were taken away and two small “U” were made in the place that was occupied by the sheets. At the time of the experiments, slide chambers were filled—vacuumed three times with 100 μl of HM and maintained on a hot plate (36°C). Immediately after the epididymis was separated, drops of epididymal content were obtained, as described above, and placed in the entrance of the chamber. In different experiments, drops from caput, corpus, and cauda epididymis were obtained and placed in the entrance of the slide chamber and immediately observed. Sperm and MSRs in suspension were recorded for 20 min by equipment made up of a SIT 66 camera (MTI) attached to a Leitz microscope with differential interference contrast microscopy and connected to a Sony monitor assisted by a video cassette recorder (Panasonic VCA). The microscope was kept warm under a tent ventilated by an automatically controlled heater to maintain an environment of 36°C. The images were digitalized by a facility off campus (MVS Company, Mendoza) and further analyzed and processed (QuickTime®).

MSRs In Situ: TEM of Epididymis

Segments two, four, and six of the epididymis, from five adult males, as described by Reid and Cleland (1957) were excised with a new razor blade in pieces of 5 × 5 mm and immersed in the fixative solution (see above) in a relation 1 to 10 (v/v) by 2 hr. Fixed epididymal segments were rinsed twice in 20 ml of PBS by 1 hr and then were re-fixed in 1% OsO4 (w/v) overnight and dehydrated through ethanol–acetone. Finally, samples were embedded in Epon 812 and thin sectioned in an ultramicrotome, and sections stained with uranyl acetate and lead citrate. Observations and micrographs were performed and observed by means of a Siemens Elmiskop I or Zeiss 900 electron microscope.

Epididymal Ligature

Five adult mice were anesthetized under ether atmosphere, and both epididymides were exposed. A ligature was placed between caput and corpus in one of them using sterile nylon (000) to avoid the transit between caput to corpus. The other epididymis was used as a control. Twenty eighth days later, mice were killed and the caput epididymides of both sides were used to prepare ultrathin sections of caput epididymis as it was described in the MSRs In Situ: TEM of Epididymis section.

RESULTS

Detection of MSRs

Observation under stereomicroscopy or microscopy permits the following of the epididymal drop dispersion. When drops from the principal epididymal regions were placed in HM medium, the dense clump of sperm cells and epididymal fluid began to disperse. The dispersion of the fluid varied depending on the origin of the drops (compare Figs. 1A, 1B, and 1C). Drops coming from caput and corpus epididymis were dispersed completely in the culture medium in few seconds (Fig. 1A,B). Instead, drops obtained from cauda epididymis were dispersed in successive steps delimiting an interface zone between culture medium and the border of the initial drops. The sperm from the border detach progressively as rosettes (MSR), and in a few seconds (20 sec), they became free and highly motile sperm (Fig. 1C, delimited area). The remainder of the drop was progressively dispersing, and the interface was less clear. Remarkably, the interface zone could not be recognized in caput or corpus drops (compare Figs. 1A and 1B). Typical MSRs were not detected in caput or corpus epididymal drops even at more magnification (Fig. 1D,E). The interface zone contains numerous free sperm and MSR (Fig. 1F) in cauda samples. Figure 1G shows an MSR during its dissociation in a caudal epididymal sample.

Figure 1.

A–G: Light microscopy of epididymal fluid drop dissolution. A and D correspond to caput, B and E to corpus, and C, F, and G to cauda. Sperm cells are distributed at random in A, D, B, and E. Some sperm cells were very closely arranged. This sperm association was called rosette-like. They are shown at the center of D and E. Sperm from caudal epididymal drop disperse progressively into the culture medium. An interface zone was always observed between culture medium and the border of the initial drop. This area was delimited with a box in C and magnified in F. The arrangement of sperm cell in D and E differs with the typical mouse sperm rosette (MSR). In F (arrows) and G, typical MSR were shown. Note that the initial caudal epididymal drop is on the left side of the picture (dense sperm clump) and the culture medium containing free sperm is on the right. In G, a MSR in suspension (after 90 sec) located at the outer limit of the interface zone is shown with high magnification. The MSR was composed of normal sperm stacked by the lateral faces of the sperm heads. Tails were not in contact. Some individual sperm cell detaching from the MSR can be observed. Original magnification: ×100 in A–C, ×250 in D–F, ×630 in G.

Light Microscopy of an Alive MSR in Suspension

The MSR was only detected when the drops came from cauda epididymis (Fig. 2). The morphology of the MSR changes during the dissolution. The number of associated sperm decreased while they were moving away from the initial drop (compare the sequence shown in Fig. 2). The MSR was composed of several sperm stacked by the lateral faces of the sperm head. Tails were beating vigorously and moved the MSR away from the initial drop. While the MSR is moving, some individual sperm cells or discrete groups of sperm detach from the initial MSR (compare Fig. 2A, B and C). During the MSR dissociation, the initial arrangement is lost. As a consequence, head and tail were oriented at random.

Figure 2.

A–C: Disassembling of a mouse sperm rosette (MSR) in suspension. The morphology of the MSR is changing with time and distance from the initial drop. The number of associated sperm decreased as the distance from the initial drop increased (compare the sequence A, B, and C). While the MSR is moving, some individual sperm cells or discrete groups of sperm were detaching from the initial MSR (compare A, B, and C). Original magnification, ×400.

SEM of Fixed MSRs in Suspension

Drops coming from caput or corpus epididymis show some rosette-like structure. Sperm heads and tails have random distributions (Fig. 3A). However, the MSRs are clearly observed in drops from cauda epididymis (Fig. 3B–D). When the samples were harvested from the interface zones (delimited area in Fig. 1C) and viewed by SEM, the sperm heads are associated in discrete groups (approximately 10 cells) and the tails are orientated centripetally from the MSR (Fig. 3B). If the MSRs were picked up far from the initial drop, the number of associated sperm cell was reduced, progressively (Fig. 3C). At higher magnification, the heads appear tightly associated by their head-tips and lateral faces (Fig. 3D). Note the presence of “sticking material” between the sperm heads.

Figure 3.

Scanning electron microscopy of fixed mouse sperm rosette (MSR) in suspension. A: Epididymal drop content obtained from the caput epididymis. B–D: Epididymal drop content obtained from the cauda epididymis. Sperm cells are randomly distributed in A. However, typical MSRs were clearly visible with this magnification in B (arrows), C, and D. In B, sperm cells are tightly associated by their head-tips and lateral faces. But sperm tails are orientated centripetally from the MSR. These MSRs were harvested at the interface zone, the box in Figure 1C. The number of associated sperm reduces progressively with the distance from the MSR and the initial drop (C). Note the presence of “sticking material” between sperm heads in the MSR (arrows in D). Original magnification: ×4,000 in A, ×5,000 in B, ×10,000 in C, ×12,000 in D.

A–D: Transmission electron microscopy of a fixed mouse sperm rosette (MSR) in suspension. Sperm heads proximally distributed are observed sometimes in samples from the caput epididymis (A, rosette-like, ×7,500). Scarce electron-dense material (EDM) between cells can be found, but it does not form a dense mesh around heads or tails (compare A,B with C,D). Some EDM between cells was detected in samples from the corpus epididymis (B, ×12,000). Typical MSRs (cauda sample) fixed in suspension show a discrete number of sperm heads closely distributed and abundant EDM between sperms cells (C,D, ×8,000). The lateral head faces stack sperm cells, and the sperm heads inside the MSR are oriented in the same way. EDM forms cores (filled arrows, C,D) and networks between heads and tails (empty arrows C).

TEM of Fixed MSRs in Suspension

The rosette-like or MSR ultrastructural appearance, fixed in suspension, corresponded to normal and well-preserved sperm. Sperm heads proximally distributed are sometimes observed (Fig. 4A) in samples isolated from caput. But the EDM between them did not conform to a dense mesh around heads or tails (Compare Fig. 4A–D). If a rosette-like comes from the corpus epididymis, it is possible to observe some EDM like a mesh accumulated between two heads or a tail and head (Fig. 4B). MSRs fixed in suspension show a discrete number of sperm heads closely distributed and abundant EDM between sperm cells (arrows, Fig. 4C,D). EDM forms cores and networks between heads and tails. Sperm heads are stacked by the lateral faces, and the sperm heads inside the MSRs are oriented in the same way.

Saccharide Detection in Fixed MSRs in Suspension

The linking material presents an intense fluorescence with PSA–FITC distributed on rosettes, over sperm heads, and less intense over the sperm cells (Fig. 5A). The other lectins—PNA and RCA120—also showed fluorescence over the whole spermatozoa, located both inside and out of rosette, but not over the linking material (Fig. 5B and C, respectively). Dark silver deposits indicating the location of Con A–colloidal gold complexes cover the rosettes. The amount of silver deposition was less abundant in the rosettes located far from the epididymal drop, probably because the linking material was dissociated while the sperms detached from the rosette (compare Fig. 6A and B). Control slides were not labeled (Fig. 6C).

Figure 5.

Saccharide detection in fixed mouse sperm rosette (MSR) in suspension, fluorescent lectins. A The linking material presents an intense fluorescence with Pisum sativum agglutinin–fluorescein isothiocyanate (PSA-FITC) distributed on rosettes—over sperm heads—and less intense over the sperm cells. B,C The other lectins—PNA and RCA120—also showed fluorescence throughout the whole spermatozoa, located both inside and out of the rosette, but not over the linking material. Original magnification, ×450.

Saccharide detection in fixed mouse sperm rosettes (MSRs) in suspension, gold-coupled lectin. A: Dark silver deposits indicate the location of Concanavalin A–colloidal gold complexes over the rosettes. B The amount of silver deposition was less abundant in the rosettes located far from the epididymal drop, probably because the linking material was dissociated while the sperm cells detached from the rosette (compare A and B). C Control slides were not labeled. Original magnification, ×400.

Presence of Saccharide in Rosette Analyzed by Specific Glycosidase Inhibitors

The number of remaining MSRs—as a consequence of glycosidases inhibitors in the culture medium—were tabulated after 3 min (Table 1). Controls (without inhibitors) were also checked at the same periods. Data were expressed as MSRs counted in ×400 microscope field and 120 (± 10 cells) live sperm. The counts of MSRs vary from 11.89 (± 2) in the presence of 50 μM SW (final concentration) to 4 or 5 (± 1) with DIM and DIJ. Control experiments did not show statistical difference with DIM and DIJ.

Video Microscopy of Live MSRs in Suspension

Observing the dissolution of the dense clump of sperm coming from cauda epididymis in HM, MSRs can be easily found. They were clearly detected at the interface zones (delimited area in Fig. 1C). Highly motile MSRs could be observed and video recorded. Most of the MSRs disappear in less than 3 min. The MSR trajectory in the medium is erratic and slower than the motile sperm alone. A sequence of pictures of rosettes disassembling is shown in Figure 7A–D.

Figure 7.

Rosettes dissociation under video microscopy observation. One rosette was followed up during its dissolution (1 min and 30 sec). The white square marks the position of the initial rosette during their “whole life.” Note that, in the same frame (time), other rosettes are composed of fewer numbers of sperm. Original magnification, ×450).

MSRs In Situ: TEM of Epididymis

Rosettes appear immediately as the epididymal content was suspended in the culture medium. For this reason, it is possible that they were already assembled inside the epididymal tubules. To confirm this idea, samples of intact epididymis were examined. Sperm cells inside the epididymal lumen of the caput region are distributed at random. Sometimes, sperm heads were found close together but the EDM between the cells was scarce (Fig. 8A). Epididymal lumen from corpus shows sperm also distributed at random, but EDM increases conforming a network (Fig. 8B). Instead, close appositions of sperm heads joined by a compact network of EDM were observed in all caudal epididymal tubules (Fig. 8C,D). Some of these associations were in contact with epithelium stereocilia.

Figure 8.

Micrograph of mouse sperm rosettes (MSRs) inside the epididymal lumen. A,B Thin sections of caput (A, ×12,000) and corpus epididymis (B, ×4,400), respectively. Note than sperm heads are close to each other, but there is no presence of electron-dense material (EDM) between them. C,D In C (×17,000) and D (×18,000), corresponding to caudal thin sections, MSRs are associated by EDM between sperm heads (arrows). Note the presence of stereocilia of the principal epididymal cells.

Epididymal Ligature

It is possible to avoid the maturation of gametes by surgically barring the sperm journey along the epididymis. Sperm were imprisoned in the caput milieu. Under this condition, drops of caput epididymis did not show any MSRs (picture not shown). Epididymal lumen diameters were increased and several empty spaces were present in the caput regions at the TEM level (Fig. 9). Sperm cells and EDM form big patches. Inside these patches, some sperms are close to each other, but a typical MSR was not observed. This ultrastructural observation substantiates the absence of rosettes within the caput of ligated epididymis.

Figure 9.

Picture of a representative caput epididymal lumen (thin section) obtained from a ligated epididymis. The lumen diameter of epididymal tubules was increased and presented empty spaces (asterisk). Sperm cells formed small patches, but in any patch, a typical rosette was observed. Original magnification, ×4,000.

DISCUSSION

In this study, we have shown for the first time the appearance of associated living spermatozoa, the mouse sperm rosette (MSR), when the caudal epididymal content was suspended in culture medium. This new sperm association was also observed inside the epididymal tubules from the same region, but not in the proximal regions, caput or corpus. Moreover, the ligature of the epididymal tubule between caput and corpus avoid the appearance of MSRs between retained sperm. This association consists of several motile sperm cells stacked together by the lateral face of their heads. The assemblage of the MSR depends on the epididymal transit, but this special cell-to-cell association is transient because their suspension in the culture medium promotes dispersion as single motile cells in less than 3 min. Simultaneous with MSR detection during epididymal transit, an EDM between sperm form a network that trapped sperm cells. The network material contains α-D-glucose and/or α-D-mannose analyzed by two different methods: glycoside detection by specific staining with lectins and indirect demonstration avoiding MSR disassembly by glycosidase-specific inhibition.

Epididymal sperm associations were reported in several species (Simeone and Young, 1931; Fawcett and Hollenberg, 1963; Biggers and DeLamater, 1965; Martan and Hurban, 1970; Martan and Shepherd, 1973; Bedford, 1979; Phillips and Bedford, 1988; Briz et al., 1995). Recently, these descriptions were extended to rat sperm (Fornés and Burgos, 1990, 1994). The arrangement of sperm heads in the sperm association varied between species. Probably, sperm use the bigger surface to obtain close interactions between cells. For those species in which sperm have a spoon-like sperm head, cell association is by the concavo-convex surface of the sperm head (Guinea pig, squirrel). But in rat or mouse, the bigger surface is the lateral face of the sperm head. At the beginning of the caudal drop dissolution or inside the epididymis, MSRs appeared stacked by the lateral face of the head. But during dissolution and sperm movement, the head position in relation with the neighbor sperm changed to a random sperm head distribution. This behavior gives the idea that MSRs are not arranged orderly. Due to this behavior, a pellet of isolated MSRs at the light and electron microscopic level show sperm cells joined but not regularly organized.

Sperm heads were joined by a network of EDM that was viewed at SEM as a filamentous material. In other species, it was also described as a periodic array of fine bridging elements that link the plasma membrane (Flaherty and Olson, 1991). But in this paper, it is clearly demonstrated that an extra cellular material fills the spaces between sperm heads. When the rosette is placed in the medium, the distance between the sperm increases and the network loosened. These changes are probably due to the initiation of sperm motility and dissolution of epididymal fluid. Conversely, when rosettes are inside the epididymal lumen (in situ), the spermatozoa and the network of EDM are tightly packed. Probably, when sperm initiate their motility, they release themselves from the EDM and move as free cells. It is also probable; that the EDM has less affinity with the sperm tail membrane and is the first to release from the rosette.

The question formulated by Tung et al. (1980): Why sperm do not form rosettes until they have reached the cauda epididymis? The answer could be: Because sperm cells should make contact with EDM, which contains α-D-glucose and/or α-D-mannose, which is not present in caput or initial corpus lumen.

It is difficult to know the biochemical composition of EDM on the basis of morphological data, but in rat, the material between heads is PAS-positive (Fornés and Burgos, 1990), indicating glycoside structure, probably glycoprotein. In this sense, another epididymal protein, DE, was also found in rat rosette (Fornés and Burgos, 1994). In the present work, we show that glycosides—α-D-glucose and / or α-D-mannose—recognized by PSA and Concanavalin A are present in EDM. We feel tempted to propose that glycoproteins secreted by epididymal epithelium are involved in rosette assembly, but glycosidases—also secreted into the epididymal fluid—might modify the carbohydrate surface domains, thus permitting the rosette assembly. On the basis of the ligature experiments, it is possible to conclude that, if the sperms cannot arrive to more distal epididymal fluid and contact with unknown factors, they cannot associate. It is interesting to speculate that EDM is/are the factor/s contributed by the epididymal epithelium. But more studies must be done.

The transient sperm association is an intriguing topic, and some suggestions for such types of cell–cell interactions were offered: (1) protection of the acrosome from a premature reaction (Phillips and Bedford, 1988) and (2) some advantages given by the fact that sperm association, in wood mouse (Apodemus sylvaticus), conforms a “train” of sperm, which moves faster than a lone spermatozoa (Moore et al., 2002). This idea proposes that this behavior is altruistic, because each one of them helps the others to fertilize the egg, but compromises their own fertilizing ability. One interesting difference is that wood mouse spermatozoa are released as a single cell suspension and then engaged to form a motile clump of cells. Consistent with this idea Taggart et al. (1993) and Moore and Taggart (1995) also propose than paired marsupial sperm showed significant motility advantages over single sperm. Rosettes in rat or mouse did not swim faster than sperm alone and are present since the beginning of epididymal drop dissolution. Free and motile sperm were observed far from the rosettes. For this reason, there could be a different phenomenon. Another head-to-head sperm association was carefully studied in boars and was named agglutination. Head to head agglutination takes place after extensive dilution and washing of epididymal sperm. That behavior was thought to be due to the removal of antiagglutinins from the sperm cells (Dacheaux et al., 1983, Harayama et al., 1996). However, in the mouse, rosettes are present inside the epididymis and they are recognized since the beginning of epididymal drop dissolution in culture medium. Probably this is a different sperm association. Notwithstanding the above-mentioned ideas, further work seems desirable for a clear explanation both of transient sperm head-to-head association and the biological significance of rosettes.

Rosettes are present in many different species; they are a transient association and appear at the end of epididymal transit, which may mean that they are a consequence of epididymal maturation probably due to epididymal glycoside-containing secretions.

Acknowledgements

The authors thank Dr. Fabio Sacerdote for his kind linguistic assistance and Mr. Alejandro Sabez for his technical assistance.

Ancillary