On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTrackerTM and high resolution differential interference contrast microscopy

Peter Sutovsky2,5, João Ramalho-Santos2,4, Ricardo D. Moreno2, Richard Oko3, Laura Hewitson2 and Gerald Schatten1,2

1 Departments of Obstetrics and Gynecology, and Cell and Developmental Biology, Oregon Health Sciences University, and the 2 Oregon Regional Primate Research Center, Beaverton, OR 97006, USA and 3 Department of Anatomy and Cell Biology, Queens University, Kingston, Ontario K7L 3N6, Canada


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The selection of individual round spermatids for round spermatid injection (ROSI), a prerequisite for the successful application of this infertility treatment, has been hampered by the ambiguous definition of a round spermatid and the lack of specific vital and non-vital markers. Using cells from rhesus monkey and bull, we describe a non-invasive method for the on-stage selection of individual round spermatids for ROSI, based on the polarized patterns of mitochondria, visualized in live round spermatid cells by epifluorescence microscopy after incubation with MitoTrackerTM, a vital, mitochondrion-specific fluorescent probe. The correct identification of live round spermatid was confirmed by the presence of the acrosomal granule or acrosomal cap in parallel observations by Nomarski differential interference contrast microscopy. The existence of mitochondrial polarization was first established by the labelling of MitoTracker-tagged round spermatids with spermatid-specific antibodies against proteins of nascent sperm accessory structures combined with antibodies against a nuclear pore complex component, known to disappear at the round spermatid stage. Using an inverted microscope equipped with epifluorescence, the round spermatids can be individually selected from a heterogeneous population of testicular cells labelled with MitoTracker dyes. A major advantage of this approach is that the dyes are incorporated into the paternal mitochondria, destined for rapid elimination after fertilization. In addition, the relatively high excitation and emission wavelengths of MitoTracker dyes are less harmful to DNA after their photon excitation. Before the appropriate clinical testing is conducted, the MitoTracker-based round spermatid selection may be instrumental in the training of clinical staff.

Key words: fertilization/mitochondria/ROSI/round spermatid/spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Male primordial germ cells proceed through a series of mitotic divisions followed by two rounds of meiosis to reach the haploid stage at which they start to develop the accessory structures of a future spermatozoon (reviewed by Oko and Clermont, 1998Go). Such spermatogenic cells, termed round spermatids, acquire the acrosome and begin to form the sperm tail. The acrosome initially appears as a membrane-limited acrosomal granule that eventually becomes attached to the cytoskeletal structure covering the apical surface of the round spermatid nucleus, called the perinuclear theca (Courtens et al., 1976Go; Oko and Maravei, 1994Go). The sperm tail, or axoneme, possesses an elaborate structure composed of the mitochondrial sheath, fibrous sheath, outer dense fibres, microtubule doublets and, in most non-rodent mammals, a sperm centriole (Fawcett, 1975Go). Spermatogenic arrest occurring at the round spermatid stage of spermatogenesis presents a substantial health problem in men, which can be experimentally overcome by the isolation and injection of round spermatid or elongating spermatids into meiotically mature recipient oocytes. These treatments, termed ROSI (round spermatid injection) or ROSNI (round spermatid nucleus injection), are reported to have produced a limited number of pregnancies in humans (Fishel et al., 1995Go; Tesarik et al., 1995Go; Barak et al., 1998Go; reviewed by Vanderzwalmen and Schoysman, 1998Go), and viable offspring have been obtained after round spermatid fusion with mouse oocytes (Ogura et al., 1994Go) and after ROSI in the rabbit (Sofikitis et al., 1996Go). Embryonic development was also achieved after ROSI in the hamster (Ogura and Yanagimachi, 1993Go) and the sperm asters and seemingly normal mitotic spindles were reportedly found in ROSI-fertilized pig oocytes (Lee et al., 1998Go).

Although the isolation of testicular cells by testicular biopsy and the introduction of a single round spermatid into the cytoplasm of a recipient oocyte is technically feasible, a major obstacle hindering the success of such treatments is the unambiguous identification and selection of an appropriate cell type (Fishel et al., 1996Go; Tesarik, 1997Go; Silber et al., 1998Go). Apart from round spermatids, testicular biopsies also contain numerous spermatogonia, spermatocytes, blood cells and somatic testicular cells. Whereas some recent studies (e.g. Angelopoulos et al., 1997Go; Reyes et al., 1997Go) proposed new methods for the selection of round spermatids, the cell size and the presence of the acrosomal granule remain the principal criteria for round spermatid selection (Tesarik and Mendoza, 1996Go; Angelopoulos et al., 1997Go; Sofikitis et al., 1997Go; Yamanaka et al., 1997Go; Verheyen et al., 1998Go). Here, we present evidence that the polarization of spermatid mitochondria begins at the round spermatid stage of spermiogenesis, and can be used as a criterion for the non-invasive selection of round spermatids when visualized by the vital mitochondrion-specific fluorescent dye MitoTrackerTM. We used this probe in model animals, the rhesus monkey and bull, to demonstrate that individual round spermatids can be selected from a heterogeneous testicular cell population based on their exclusive patterns of mitochondrial polarization. In contrast to some other on-stage selection methods, our approach to round spermatid selection does not involve any DNA stains and targets the spermatid mitochondria, which, similar to those of mature spermatozoa, are not thought to contribute to the extranuclear genome of an embryo (Kaneda et al., 1995Go; Sutovsky et al., 1996bGo; Cummins et al., 1998Go). Though an extensive testing should precede its clinical use, our method may prove useful for the diagnosis of spermatogenic arrest and for the training of personnel involved in the still experimental ROSI/ROSNI procedures.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolation of spermatogenic cells and MitoTracker labelling
Adult bull testes, obtained from a slaughter house, were dissected and a small piece transferred to a Petri dish filled with TALP-HEPES [Bavister et al., 1983; Boatman, 1987; 10 mmol/l HEPES, 114 mmol/l NaCl, 3.16 mmol/l KCl, 2 mmol/l CaCl2, 0.5 mmol/l MgCl26H2O, 10 mmol/l Na lactate, 0.35 mmol/l NaH2PO4H2O, 5 mmol/l glucose, 2 mmol/l NaHCO3, 0.1 mmol/l pyruvate, 3 mg/ml bovine serum albumin (BSA)] medium and minced using two fine forceps. Rhesus monkey testes were obtained from males undergoing necropsy, and testicular cells were prepared as described above. The minced tissue was filtered through a fine mesh to remove the tissue debris and the cell suspension was centrifuged for 5 min at 700 g. The pellet was resuspended in 12 ml of warm TALP-HEPES and centrifuged one more time for 5 min at 700 g. Twenty-five microlitres of the loose final pellet were then resuspended in 975 µl of TALP-HEPES for MitoTracker labelling. A primary stock of 1 mmol/l MitoTracker CMTM Ros (Molecular Probes, Inc., Eugene, OR, USA) was prepared in dimethylsulphoxide (Sigma, St Louis, MO, USA). A secondary stock of 100 µmol/l MitoTracker was made from the primary stock in KMT medium (100 mmol/l KCl, 2 mmol/l MgCl2, 10 mmol/l Tris–HCl; pH 7.0) warmed to 37°C. This stock was added to 1 ml suspension of testicular cells to obtain a final concentration of 200 nmol/l and incubated for 20 min at 37°C. In some experiments, the round spermatids were incubated for 2 h with a vital, lysosome-specific probe LysoTracker Green DND-26 (Molecular Probes) at the concentration of 10 µmol/l, combined with 200 nmol/l MitoTracker CMTM Ros and 5 µg/ml Hoechst 33342 at 37°C. After incubation, the cells were collected by a 5 min centrifugation at 700 g, and washed by resuspension and centrifugation in 12 ml of TALP-HEPES medium. Peripheral blood was drawn from healthy rhesus males and stained as described above in order to demonstrate the difference between MitoTracker staining patterns in testicular and blood cells.

Immunofluorescence and cell imaging
Testicular cells were stained with MitoTracker CMTMRos as described above and attached to poly-L-lysine-coated coverslips in a drop of warm KMT medium. Coverslips were fixed for 40 min in 2% formaldehyde in 0.1 mol/l phosphate-buffered saline (PBS) and permeabilized overnight in 0.1% Triton X-100 in 0.1 mol/l PBS. Non-specific antibody binding was blocked by a 1 h incubation in 0.1 mol/l PBS containing 10% normal goat serum (NGS). Coverslips with rhesus cells were then incubated with a mixture of anti-outer dense fibre antibody pAb 4443 (Oko, 1988Go; diluted 1/200) and a nucleoporin-specific antibody mAb 414 (Meier et al., 1995Go; BabCo, Berkeley, CA, USA; diluted 1/200) in 0.1 mol/l PBS containing 0.05% NaN3, 2 mmol/l EGTA, 1 % NGS and 0.1 % Triton-X-100 (further referred to as labelling solution). After a short wash in labelling solution, the coverslips were incubated for 40 min with a mixture of fluorescein isothiocyanate (FITC)-conjugated goat-anti rabbit IgG and Cy5-conjugated goat anti-mouse IgG (Zymed Labs Inc., South San Francisco, CA, USA; diluted 1/40). Five micrograms per ml of 4',6-diamidino-2-phenylidone (DAPI; Molecular Probes Inc., Eugene, OR, USA) were added to the labelling solution 10 min before the end of incubation. At the end of incubation, the coverslips were washed in PBS and mounted on microscope slides in a VectaShield mounting medium (Vector Labs, Burlingame, CA, USA). Bull testicular cells were processed as described above except that the perinuclear theca-specific antibody pAb 427 (Oko and Maravei, 1994Go; diluted 1/200) was used instead of pAb 4443. The antibody pAb 427 also cross-reacts with the perinuclear theca of rhesus monkey spermatids (data not shown). In some experiments, the rhesus monkey testicular cells were double-labelled with a mixture of anti-syntaxin rabbit polyclonal antibody (Conner et al., 1997Go; diluted 1/200; kindly donated by Dr Garry Wessel) and mAb 414, or with a mixture of anti-syntaxin and mouse monoclonal anti-acrosin antibodies (De Ioannes et al., 1990Go; diluted 1/50; kindly donated by Dr Claudio Barros), followed by appropriate secondary antibodies. Both anti-syntaxin and anti-acrosin antibodies cross-reacted with the acrosome of rhesus monkey round spermatids.

Slides were examined on a Zeiss Axiophot microscope and images were recorded by a cooled CCD camera (Princeton Instruments, Inc., Trenton, NJ, USA) operated by Metamorph software (Universal Imaging Corp., West Chester, PA, USA). Original data were archived on recordable compact disks. Images were pseudocoloured and contrast-enhanced using Adobe Photoshop 4.0 software (Adobe Systems Inc., Mountain View, CA, USA) and printed on a Sony UP-D-8800 colour video printer. Figure 1Go is a composite image of the individually photographed round spermatids, printed using Adobe Photoshop.



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Figure 1. Identification of the round spermatids in the cellular populations isolated from bull (upper panel) and rhesus monkey (lower panel) testes. Cells were pre-labelled with a fixable mitochondrion-specific vital probe, MitoTracker CMTM Ros (red), then fixed and labelled with a nuclear pore complex-specific antibody mAb414 (white), a DNA-specific stain DAPI (blue) and a perinuclear theca-specific antibody pAb 427 (green; upper panel), or an outer dense fibre-specific antibody pAb 4443 (green; lower panel). The presumed round spermatids (right) display a reduced number of nuclear pores (white), strongly polarized mitochondria (red) and the presence of either the nascent outer dense fibres (green in rhesus monkey), or the nascent subacrosomal perinuclear theca (green in bull). Each cell in this image was photographed individually at a constant magnification of x1000 and pasted onto this plate using Adobe Photoshop software. Scale bar = 5 µm

 
Transmission electron microscopy
Testicular cells were pelleted by a 10 min centrifugation in TALP-HEPES at 700 g, and fixed for 1 h in 2.5% glutaraldehyde and 0.6% paraformaldehyde in 0.25 mol/l cacodylate buffer (pH 7.2). After washing in 0.1 mol/l cacodylate buffer containing 0.2 mol/l sucrose, the cells were pelleted again, the washing buffer was carefully removed and cells were embedded in hot 1% agar. After cooling, the agar pellets were post-fixed for 1 h in 1% osmium tetroxide. Following dehydratation by an ascending ethanol series (30–100%), the pellets were infiltrated in a mixture of propylene oxide and PolyBed 812 (Polyscience, Warrington, PA, USA) and embedded in PolyBed 812. Ultrathin sections were cut on a Sorval MT2B ultramicrotome, placed on 100-mesh copper grids and stained in two steps with uranyl acetate and lead citrate. Serial sections were examined and photographed in a Phillips EX 120 STEM electron microscope. Negatives were scanned by an Umax Magic Scan flat bed scanner, saved on Jazz disks and printed as described for immunofluorescence.

On-stage identification and selection of individual round spermatids
One microlitre of testicular cell suspension was diluted in a 150 µl drop of TALP-HEPES medium in a large Petri dish or a glass-bottom Petri dish (see below), transferred onto a preheated stage of a Nikon Diaphot inverted microscope equipped with micromanipulators and appropriate filter sets, and the patterns of mitochondrial distribution were screened and the round spermatids were searched for using a 20x/0.75 NA (numerical aperture) Fluor oil lens and a 40x/1.30 NA Fluor oil lens (both from Nikon). Patterns of MitoTracker labelling were the only guidance in these studies. The selected cells were used for ROSI and nuclear transfer experiments to be reported separately. For image recording for the purpose of this publication, 35 mm glass-bottom Petri dishes (MatTek Corp., Ashland, MA, USA) were used on a Nikon Eclipse 300 inverted microscope equipped with an environmental chamber (5% CO2, 37°C; Nikon), a Hamamatsu C 4742–95 digital camera, Nomarski DIC and appropriate filter sets, and the images were recorded using a 40x/1.30 NA Fluor oil lens and a 60x/1.4 NA infinity-corrected Plan Apo lens (both from Nikon). A 20x/0.75 NA Fluor lens was used to search for the round spermatids to be photographed. At that primary magnification, the polarized patterns of the mitochondrial distribution, and the nuclear uptake of MitoTracker dye were the only criteria used for the identification of individual round spermatids. The correct identification of round spermatids was confirmed by the presence of an acrosomal granule/acrosomal cap seen in differential interference contrast (DIC) with x40 or x60 lenses. Images were recorded on a PC using MetaFluor 3.5 imaging software (Universal Imaging Corp.) and edited by Adobe Photoshop 4.0, as described for immunofluorescence data. Cell suspensions used for all studies described in this paper were not pre-sorted and contained all cell types that can be found in the testis, including spermatogonia, spermatocytes, spermatids, Sertoli cells, stromal cells and leukocytes.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cytological characterization of round spermatids in bull and rhesus monkey and the round spermatid-specific patterns of mitochondrial polarization
MitoTracker probes revealed the polarization of round spermatid mitochondria in a distinct subpopulation of rhesus monkey and bovine testicular cells with an approximate diameter of 8–10 µm. To establish that these cells were round spermatids, we selected a set of round spermatid markers to be used in combination with MitoTracker labelling. An antibody mAb 414 was used to visualize a subset of nuclear pore complex components (NPC), including a major NPC protein, nucleoporin 153 (Nup 153; Meier et al., 1995Go). Perinuclear theca-specific antibody pAb 427 (Oko and Maravei, 1994Go) and an antibody pAb 4443 against the outer dense fibres of the sperm axoneme (Oko, 1988Go) were used to label the nascent sperm accessory structures in the round spermatids. The above antibodies were chosen based on the evidence that mammalian spermatozoa lose a majority of their NPC during spermatogenesis (Fawcett, 1975Go), and acquire sperm accessory structures such as the acrosome, perinuclear theca and axoneme (Oko and Clermont, 1988Go, 1998Go; Oko and Maravei, 1995Go). Among bull testicular cells (Figure 1Go, upper panel), only the round spermatids displayed the loss of NPC, visible as the mAb 414-positive patches adjacent to the base of their nuclei. Other cell types present in the testicular cell suspensions contained a continuous ring of NPCs around their nuclei (Figure 1Go, upper panel). The loss of NPCs in bull round spermatids was accompanied by the formation of the subacrosomal layer of perinuclear theca, visualized by the pAb 427 as a distinct cap on the apical pole of the round spermatid nucleus. In the rhesus monkey (Figure 1Go, lower panel), the cells losing the NPCs invariably contained the outer dense fibres of the developing sperm axoneme, visualized by pAb 4443. This was visible as a single dot and/or as a distinct short sperm tail in the basal pole of the cells at a more advanced stage of spermiation. The dot-like pattern seen in some cells (Figure 1Go, lower panel) probably corresponds with the labelling of cytoplasmic granular bodies, that are known to contain outer dense fibres proteins (Clermont et al., 1990Go). In both rhesus monkey and bull, only the cells displaying both the loss of NPC and the formation of the sperm accessory structures (i.e. perinuclear theca or outer dense fibres) displayed strong polarization of mitochondria visualized by MitoTracker labelling. This round spermatid-specific redistribution of mitochondria probably corresponds with the recruitment of mitochondria into a compact mitochondrial sheath at this stage of spermatogenesis (Figure 2A–CGo).



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Figure 2. Morphological characteristics of rhesus monkey round spermatids demonstrating the development of the sperm accessory structures used as spermatid-specific markers in this study. (A–C) show progressive condensation of the round spermatid nucleus accompanied by the formation of the acrosomal cap and redistribution of the mitochondria (arrows). (D) The acrosome is initially composed of a granule (asterisk) adjacent to the subacrosomal layer of perinuclear theca (arrows), that attracts the cytoplasmic membrane vesicles (arrowheads). These vesicles eventually fuse into a continuous acrosomal cap (E). The sperm axoneme is formed by a single centriole (arrow) found in the cytoplasm at an early stage of spermiogenesis (F). (G) This centriole (arrow) later becomes attached to the nucleus and serves as a mould for the formation of the axonemal microtubule doublets (arrowheads). (H) A cross-section of the nascent sperm axoneme with the microtubule doublets parallelled by the outer dense fibres (arrow). Scale bars = 1 µm in A–C, 500 nm in D, E and 200 nm in F–H. (IK) These figures complement the ultrastructural studies of acrosomal development in rhesus monkey by showing the immunofluorescent localization of a membrane fusion protein syntaxin in the developing acrosomal cap (I, J), as opposed to the localization of nuclear pore complexes (NPC) at the base of the nucleus (I') and the labelling of acrosin in the acrosomal granule (J'). Note that the distribution of acrosin and syntaxin in round spermatids in part overlaps with that of subacrosomal perinuclear theca (PT), as shown in Figure 1Go. (K and K') The double labelling of live round spermatids with a lysosomal probe LysoTracker Green DND-26 (K) and Mitotracker CMTM Ros (K'). DNA was labelled with DAPI in I'', J'' and with Hoechst 33342 in K''. Scale bar = 5 µm.

 
The immunofluorescence data were supported by the ultrastructural studies conducted in the rhesus monkey. Initially, most mitochondria were seen in the apical cytoplasm, next to the developing acrosome (Figure 2AGo). Progressive condensation of the spermatid nucleus was paralleled by the relocation of mitochondria to the basal pole of the round spermatid cytoplasm (Figure 2B,CGo). Similar to other mammalian species, the acrosome of rhesus monkey round spermatids appears to arise from a Golgi-derived acrosomal granule (Figure 2DGo), emerging at an early stage of spermiogenesis (Moreno and Schatten, 1998Go). This acrosomal granule becomes attached to the nascent subacrosomal perinuclear theca, which appears to attract the cytoplasmic membrane vesicles (Figure 2DGo), eventually fusing into a continuous acrosomal cap (Figure 2EGo). This event is paralleled by the attachment of one of the two spermatid centrioles (Figure 2FGo) to the developing basal plate–implantation fossa complex at the base of the round spermatid nucleus. The second centriole (Figure 2GGo) serves as a mould for the formation of microtubule doublets paralleled by the outer dense fibres (Figure 2HGo). Additional markers of the round spermatids that were used for immunofluorescence localization of the acrosome in rhesus monkey round spermatids included a polyclonal anti-syntaxin antibody (Figure 2I,JGo), a monoclonal anti-acrosin antibody (Figure 2JGo') and a vital, lysosome-specific fluorescent probe Lyso-Tracker Green DND-26 (Figure 2KGo). Syntaxin (Figure 2I,JGo), a membrane fusion protein (Ramalho-Santos et al., 1998Go), displayed distribution patterns identical to those of perinuclear theca proteins, suggesting that it may be involved in the fusion of membrane vesicles into a continuous acrosomal cap, as shown by in Figure 2DGo. In contrast, the acrosomal matrix component, acrosin (Figure 2JGo'), appears to be restricted to the acrosomal granule of the developing round spermatids (Moreno et al., 1998). LysoTracker staining (Figure 2KGo) seems to be concentrated in the developing acrosome of rhesus monkey round spermatids and will be evaluated as an alternative to MitoTracker probes for the purpose of round spermatid selection.

On-stage visualization of mitochondria in live testicular cells of the rhesus monkey
Based on the presence of the nascent sperm accessory structures and the absence of NPCs, the above observations established that round spermatids contain highly polarized mitochondria. Due to its phylogenetic and reproductive similarities with humans, the rhesus monkey was selected as a model for further studies. The MitoTracker CMTM Ros-labelled testicular cells were examined on the stage of two different epifluorescence-equipped inverted microscopes (see Materials and methods), the second one equipped with Nomarski DIC and a digital camera for the purpose of confirming the correctness of round spermatid selection and for image recording, respectively. Acrosomal granule-stage round spermatids (Figure 3A,AGo') were revealed by the fluorescently tagged cluster of mitochondria seen next to the acrosomal granule and subacrosomal perinuclear theca visualized on the apical surface of the round spermatids nucleus by DIC. The polarization of round spermatid cytoplasm and relocation of round spermatid mitochondria were concomitant with the attachment of the acrosomal granule to the nucleus and with the formation of a distinct acrosomal cap (Figure 3B–EGo'). At the same time, the progressive condensation of the sperm nucleus caused an increased nuclear retention of MitoTracker dye (Figure 3B–EGo'), that therefore appears to be a helpful additional criterion for the identification of such cells. The elongation of the sperm nucleus was accompanied by further polarization of the cytoplasm and by the clustering of MitoTracker-tagged mitochondria in it (Figure 3F–HGo'). Some unusually large round spermatids, containing one or two nuclei with distinct acrosomal caps, were occasionally found (Figure 3I,IGo'). In general, the x20 lens did not provide sufficient resolution to detect the acrosomal cap in round spermatids by Nomarski DIC, though it allowed us to recognize the polarized mitochondrial distribution therein by epifluorescence microscopy. The round spermatids were readily identifiable by both DIC and epifluorescence using the 40x lenses (Figure 4A–CGo'), and best results were obtained with the infinity-corrected x60 lens (Figure 3Go).



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Figure 3. On-stage identification of live rhesus monkey round spermatids on the basis of vital staining with MitoTracker CMTM Ros combined with epifluorescence (A–I) and differential interference contrast (DIC) (A'–I') microscopy. (A, A') Acrosomal granule (A, arrowhead) and the nascent subacrosomal perinuclear theca in an early stage round spermatid; MitoTracker staining (A') reveals the onset of mitochondrial polarization, at this stage seen near the apex of the nucleus. (B, B') Acrosomal cap (arrowhead in B) stage round spermatid with polarized cytoplasm and mitochondria (B', arrowheads) clustered near the base of its nucleus. Note the beginning of MitoTracker sequestration in the nucleus. C, C') Onset of spermatid elongation, accompanied by the condensation and flattening of the nucleus (C, arrowhead points to the acrosomal cap), and an increased nuclear retention of MitoTracker dye (C'; arrowheads point to the polarized cytoplasm with mitochondria). (D, D') In addition to a round spermatid with typical acrosomal cap (D; arrowhead), a second cell of similar shape and size is shown (arrow), that does not contain an acrosomal cap or polarized mitochondria (arrows in D' point to those in a round spermatid). (E, E') Late round/early elongating spermatid with protruding nucleus covered by a typical acrosomal cap (E, arrowhead), and a developing sperm tail mitochondrial sheath, seen as a clump of fluorescent mitochondria in E'. (F, F') An elongating spermatid with the acrosomal cap (F, arrowhead) and the strong nuclear and mitochondrial (F'; arrowheads) uptake of MitoTracker. (G, G') An elongating spermatid with the acrosomal cap (G, arrowhead) and clumped mitochondria (G', arrowhead). (H, H') Late elongating spermatid with a flat acrosomal cap (H, arrowhead), polarized cytoplasm and a condensed nucleus (H'). (I, I') Three testicular cells including a round cell (asterisk) with no distinct mitochondrial staining (I', asterisk), seen next to a typical round spermatid featuring the acrosomal cap (I, arrow) and polarized cytoplasm and mitochondria (I', arrowheads on the right). Note an unusually large round spermatid (I, arrow) with strongly polarized mitochondria (I', arrowheads on the left) and cytoplasm. Scale bar = 10 µm.

 


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Figure 4. Suspensions of testicular cells visualized by differential interference contrast (DIC) (A–C) and epifluorescence with MitoTracker CMTM Ros (A'–C'), shown at three different secondary magnifications. Arrows and inserts in A, A' show two round spermatids with acrosomal caps and polarized mitochondria. Arrow in B, B' points to a round spermatid with a small clump of fluorescent mitochondria at the base of its nucleus. The arrowhead points to an elongating spermatid with distinct constriction between its cytoplasm and nucleus. Arrow in C, C' points to a round spermatid, which in contrast to the surrounding testicular cells shows strong polarization of its mitochondria and distinct uptake of MitoTracker dye into its nucleus. Scale bars = 5µm

 
Blood cells, suggested to be a possible source of bias during the on-stage identification of round spermatids (Vanderzwalmen et al., 1997Go), were incubated with MitoTracker CMTM Ros under conditions identical to testicular cell labelling. Most blood cells such as erythrocytes did not incorporate MitoTracker dye, and only a limited number of cell types seemed to be able to take up the dye into their mitochondria, or nuclei (Figure 5AGo). Three basic patterns of MitoTracker uptake by blood cells were observed: (i) cells with the segmented nuclei, most likely granulocytes or monocytes, displayed nuclear labelling that more or less completely masked the cytoplasmic labelling (Figure 5A,BGo'); (ii) cells with few mitochondria strongly stained with MitoTracker, most likely immature erythrocytes (Figure 5C,CGo'); and (iii) cells with a single nucleus stained by MitoTracker, most likely lymphocytes, with no distinct mitochondrial labelling (Figure 5D,DGo'). Therefore it appears that the patterns of MitoTracker uptake by blood cells do not resemble those seen in the spermatogenic cells, and particularly in the round and elongating spermatids. The condensed chromatin of white blood cells appears to retain MitoTracker CMTM Ros.



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Figure 5. Patterns of Mitotracker CMTM Ros (A'D') uptake by blood cells visualized by differential interference contrast (AD). (AB') MitoTracker-free erythrocytes mixed with MitoTracker-stained white blood cells, most likely granulocytes or monocytes with segmented nuclei. (C, C') Small clusters of strongly labelled mitochondria in two blood cells (arrow and arrowhead) classified as immature erythrocytes. (D, D') Small round cell, probably a small lymphocyte with very little cytoplasm, showing diffuse MitoTracker labelling. Scale bar = 7 µm.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An unambiguous on-stage method for the selection of individual round spermatids has been sought ever since the ROSI procedure emerged as a potential clinical treatment. Angelopoulos et al. (1997) used Testsimplets, a staining kit composed of N-methylene blue and cresyl violet acetate, to identify round spermatids and other types of spermatogenic cells from human testicular biopsies. Although not fluorescent, this dye strongly stains the nucleus and might be harmful if the stained cells are injected into oocyte cytoplasm and embryonic development is induced. Subsequently, the additional criteria such as cell size and shape and the presence of the acrosomal granule were taken into account in the above study, and the final selection was performed by means of phase contrast microscopy without cell staining. The main value of this method appears to be its use in the diagnosis of spermatogenic arrest rather than in the selection of round spermatids for ROSI. Similarly, the methods used for the staining of fixed testicular cell smears, including the Papanicolaou staining method, fluorescent-labelled Pisum sativum agglutinin, and anti-acrosin immunolabelling alone or combined with the autosomal DNA in-situ hybridization, were helpful in evaluating the occurrence of spermatids in the testis of men with non-obstructive azoospermia (Mendoza and Tesarik, 1996Go; Mendoza et al., 1996Go).

The present study suggests that the round spermatid-specific polarization of the mitochondria (see Figure 6Go), as revealed by the vital, mitochondrion-specific probe, MitoTracker, can be used as a reliable criterion for the selection of round spermatids in assisted reproduction. Stage-specific selection of the individual spermatogenic cell types was previously achieved by gravity sedimentation (Lam et al., 1970Go; Grabske et al., 1975Go; Romrell et al., 1976Go), Percoll separation (Bucci et al., 1986Go) and cell sorting (Gledhill et al., 1990Go; Aslam et al., 1998Go). Using large samples of testicular tissue, such methods yield cellular populations enriched in the desired spermatogenic cell type. However, such methods cannot be applied to diagnostics and clinical treatment of human subjects limited by a minute size of tissue samples obtained by testicular biopsy. Therefore, selection of single cells has to be performed, most frequently guided by the size and shape of individual testicular cell types and by the presence of the acrosomal granule revealed by phase contrast or DIC (Tesarik and Mendoza, 1996Go; Sofikitis et al., 1997Go; Vanderzwalmen et al., 1997Go; Yamanaka et al., 1997Go). Blood cells, renewing spermatogenic stem cells and somatic cells from testicular stroma are often similar to round spermatids in their size and shape (see Figure 3DGo). Furthermore, recognizing the acrosomal granule or the acrosomal cap at low magnification is complicated due to the low resolution of light microscopy. These and other factors (reviewed by Sutovsky and Schatten, 1999Go) may increase inaccuracies in the selection of round spermatids and consequently contribute to the ambiguity in interpreting the results of spermatid selection and ROSI. Spermatid elongation as an additional criterion for selection may not be available in patients suffering from spermatogenic arrest that prevents the elongation step of spermiogenesis. From this point of view, the combination of epifluorescence and DIC microscopy of testicular cells labelled with MitoTracker dyes appears to be advantageous, providing two new criteria for round spermatid identification, i.e. mitochondrial polarization and nuclear dye uptake, in addition to cell size and acrosomal cap visualization by DIC. The possibility of using the acrosome-sequestrated lysosomal dyes such as LysoTracker (Moreno and Schatten, 1998Go; this study) as an additional criterion for round spermatids selection is currently being explored by our laboratory, though there is an obvious disadvantage of slow incorporation and consequently long incubation times.



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Figure 6. Schematic diagram of round spermatid (RS) development applicable to both mammalian species described in this study. The subacrosomal layer of perinuclear theca (green) forms at an early stage of round spermatid development, marked by the presence of a large Golgi complex, formation of the acrosomal granule, and onset of mitochondrial and cytoplasmic polarization. Polarization of the spermatid mitochondria advances concomitantly with the formation of the acrosome and sperm axoneme, arising from the spermatid's distal centriole. At a final stage, i.e. spermatid elongation (far right), half of the mitochondria form the compact mitochondrial sheath, although the other half are rejected with the cytoplasmic droplet. At this stage, the perinuclear theca covers almost the whole surface of the sperm nucleus, the acrosome and the nucleus acquire their final shape and the distal sperm centriole undergoes degeneration.

 
Besides using MitoTracker labelling as a selection criterion, our method was substantially improved by using glass-bottom Petri dishes. These can be purchased (see Materials and methods for manufacturer information), or simply made by drilling a hole into a conventional Petri dish and gluing a conventional microscopy coverslip on the bottom of it. Also, using x40 and x60 lenses and the infinity-corrected x60 lenses appears to be an advantage over the low magnification x20 lenses. Low magnification lenses (x10–40), on the other hand, are most frequently used for micromanipulations such as ROSI. At these magnifications, the acrosomal granule/cap may not be recognizable in microscopes equipped with Nomarski DIC, phase or Hoffman modulation contrast. In contrast, MitoTracker labelling is apparent even when low magnification x20 lenses are used. Such an improved method thus eliminates the necessity for an expensive confocal setup (e.g. Sofikitis et al., 1997Go), with the risk of damage to cellular structure from the laser beam.

A method for the selection of round spermatids and other types of spermatogenic cells was reported based on estimation of their DNA content (Reyes et al., 1997Go). This study, however, employed the DNA-binding fluorescent dyes, such as Hoechst 33342 and ethidium bromide, the photon excitation of which may cause DNA breaks (Simerly and Schatten, 1993Go) and therefore renders such dyes unsuitable for assisted reproduction procedures. Furthermore, it should be emphasized that relatively long exposure times are necessary for the on-stage selection of spermatogenic cells with fluorescent dyes. Red dyes with longer emission wavelength seem to cause substantially less damage to the cellular structures than the short wavelength dyes such as blue-emitting DNA stains and green-emitting dyes such as FITC (Vigers et al., 1988Go). Phase contrast microscopy is less invasive than confocal or fluorescent DNA-stain microscopy and thus appears to be more appropriate for the selection of round spermatids in clinical practice (Verheyen et al., 1998Go). Though MitoTracker CMTM Ros (but not its green equivalent MitoTracker Green FM; P.Sutovsky, unpublished data) is sequestered in the nuclei of round spermatids beyond the acrosomal granule stage, it may not directly bind to DNA. The emission wavelength of Mitotracker CMTM Ros peaks at 576 nm, as opposed to the 461 nm emission of DNA dye Hoechst 33342 (Haugland, 1996Go). An added advantage of MitoTracker dyes for research is that they persist in the stained live cells, including the spermatozoa and oocytes, for several days and are fixable and permeabilization-resistant, if properly fixed (Sutovsky et al., 1996bGo; Cummins et al., 1997Go).

During natural fertilization, the sperm mitochondria become metabolically inactive shortly after their incorporation into oocyte cytoplasm and are subsequently targeted for destruction (Shalgi et al., 1994Go; Kaneda et al., 1995Go; Sutovsky et al., 1996aGo,bGo; Cummins et al., 1997Go). Similarly, the mitochondria of round spermatids appear to be destroyed in the oocyte cytoplasm after ROSI, at least in mice (Cummins et al., 1998Go). Ubiquitination of the mitochondrial proteins was proposed to play a role in the elimination of sperm mitochondria after fertilization (Sutovsky et al., 1996bGo), and our new data strongly support this hypothesis (Sutovsky et al., 1998Go). Therefore, it appears that no paternal mtDNA is inherited by an embryo during mammalian fertilization. If the major concern for the MitoTracker-assisted selection of round spermatids is that the excitation of the mitochondrion-bound fluorescent dye may cause structural damage to the mitochondrial proteins and mtDNA, the method may not pose a threat to an embryo relying exclusively on the maternal mitochondrial pool. Although more research and meticulous testing will be necessary to prove the safety of our method, the above data on mitochondrial inheritance suggest that, even if MitoTracker dye damages paternal mitochondria, such damage is not harmful to an embryo. Nuclear uptake of MitoTracker by late round spermatids may be a concern, yet even this aspect of Mitotracker-assisted selection may not necessarily rule out the use of this method if appropriate pre-clinical testing is conducted. Early stage round spermatids do not display the nuclear uptake of MitoTracker CMTM Ros (see Figure 3A,AGo'), and the shorter wavelength dye, MitoTracker Green FM, shows similar mitochondrial labelling in the absence of nuclear uptake (data not shown). Furthermore, this nuclear uptake does not appear to be the result of a specific MitoTracker binding to DNA or nucleosome, as it is completely eliminated by the fixation and permeabilization of the cells. In our preliminary experiments with ROSI, we did not observe the persistence of MitoTracker in the round spermatid nuclei injected into oocyte cytoplasm. Similarly, MitoTracker is rapidly excluded from the nuclei of mature spermatozoa injected into bovine and rhesus monkey oocytes. Pre-labelling of hamster and rabbit sperm with the fluorescent agent, monobromide, that binds to the whole sperm tail including mitochondria, as well as to the nucleus, preceded normal embryonic development and birth of healthy offspring in these species (Fleming et al., 1986Go). Pre-labelling of bull (Sutovsky et al., 1996bGo) and mouse (Kaneda et al., 1995Go) sperm with the mitochondrion-specific fluorescent dyes MitoTracker Green FM and Rhodamine123, respectively, did not interfere with fertilization and embryonic cleavage. It should be emphasized, however, that the fluorescently labelled, oocyte-incorporated spermatozoa were not exposed to fluorescent excitation in these studies.

In conclusion, we have developed a new method for the on-stage selection of mammalian round spermatids based on the round spermatid-specific patterns of mitochondrial polarization, as revealed by the fluorescent dye, MitoTracker CMTM Ros, and confirmed by high resolution DIC microscopy. This method is fast and reliable, and can be used for very small testicular samples, such as testicular biopsies. Furthermore, no DNA-binding dyes are used. From this preclinical investigation, we suggest that high numerical aperture oil immersion objectives (magnification x60 or higher; NA >1.3) appear to provide sufficient discrimination for the selection of round spermatids by DIC microscopy. However, the routine use of low magnification (x10–40) dry lenses with low NA (<1.2) may well benefit from round spermatid selection assisted by MitoTracker dyes. Since the paternal mitochondria appear to be discarded during preimplantation embryonic development, this method may present no harm to the round spermatids and resultant embryos and could be used in human assisted reproduction techniques, if the method is appropriately tested. In the meantime, the MitoTracker staining of testicular cells may become a useful tool for the training of clinical staff and for the diagnosis of human spermatogenic arrest. Experiments are underway to test the viability of the oocytes fertilized with the MitoTracker-labelled round spermatids, that were exposed to fluorescent excitation light during the on-stage selection. This method may also be useful for the selection of primordial germ cells from fetal and juvenile gonads in animal research and for cell sorting of testicular cells.


    Acknowledgments
 
We are very thankful to Michelle Emme, Crista Martinovich and Diana Takahashi for their clerical and technical assistance and to Dr Grayson Scott and Michael Webb for their assistance with EM. Stimulating discussions with Drs Jim Cummins (Murdoch University, Western Australia), Tanja Dominko (ORPRC), Cal Simerly (ORPRC) and Justin St John (University of Birmingham, UK), as well as the assistance of Dr Marc Luetjens with DIC microscopy, are gratefully acknowledged. All experiments described in this study were performed in compliance with the principles detailed in the Guide for the Care and Use of Animals, the Animal Welfare Act. All protocols were approved by an Institutional Animal Care and Use Committee (IACUC). This work was supported in part by grants from NIH and USDA to G.S., grants from NCERC and MRC to R.O., by fellowships from the Fogarty International Center, NIH, to R.M. and P.S., and by a fellowship from FCT, Portugal to J.R.-S.


    Notes
 
4 Present address: Center for Neuroscience, Department of Zoology, University of Coimbra, Portugal Back

5 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Angelopoulos, T., Krey, L., McCullough, A. et al. (1997) A simple and objective approach to identifying human round spermatids. Hum. Reprod., 12, 2208–2216.[Abstract]

Aslam, I., Robins, A., Dowell, K. et al. (1998) Isolation, purification and assessment of viability of spermatogenic cells from testicular biopsies of azoospermic men. Hum. Reprod., 13, 639–645.[Abstract]

Barak, Y., Kogosowski, A., Goldman, S. et al. (1998) Pregnancy and birth after transfer of embryos that developed from single-nucleated zygotes obtained by injection of round spermatids into oocytes. Fertil. Steril., 70, 67–70.[ISI][Medline]

Bavister, B.D., Boatman, D.E., Leibfried, L. et al. (1983) Fertilization and cleavage of rhesus monkey oocytes in vitro. Biol. Reprod., 28, 983–999.[ISI][Medline]

Boatman, D.E. (1987) In vitro growth of non-human primate pre- and peri- implantation embryos. In Bavister, B.D. (ed.), The Mammalian Preimplantation Embryo. Plenum Press, New York, pp. 273–308.

Bucci, L.R., Brock, W.A., Johnson, T.S. et al. (1986) Isolation and biochemical studies of enriched populations of spermatogonia and early primary spermatocytes from rat testes. Biol. Reprod., 34, 195–206.[Abstract]

Clermont, Y., Oko, R. and Hermo, L. (1990) Immunocytochemical localization of proteins utilized in the formation of outer dense fibers and fibrous sheath in rat spermatids: an electron microscope study. Anat. Rec., 227, 447–457.[ISI][Medline]

Conner, S., Leaf, D. and Wessel, G. (1997) Members of the SNARE hypothesis are associated with cortical granule exocytosis in the sea urchin egg. Mol. Reprod. Dev., 48, 106–118[ISI][Medline]

Courtens, J.L., Courot, M. and Flechon, J.E. (1976) The perinuclear substance of boar, bull, ram and rabbit spermatozoa. J. Ultrastruct. Res., 57, 54–64.[ISI][Medline]

Cummins, J.M., Wakayama, T. and Yanagimachi, R. (1997) Fate of microinjected sperm components in the mouse oocyte and embryo. Zygote, 5, 301–308.[ISI][Medline]

Cummins, J.M., Wakayama, T. and Yanagimachi, R. (1998) Fate of microinjected spermatid mitochondria in the mouse oocyte and embryo. Zygote, 6, 213–222.[ISI][Medline]

De Ioannes, A.E., Becker, M. I., Perez, C. et al. (1990) Role of acrosin and antibodies to acrosin in gamete interaction. In Alexander, N. J. et al. (ed.), Gamete Interaction: Prospects of Immunocontraception. Wiley, New York, pp. 185–195.

Fawcett, D.W. (1975) The mammalian spermatozoon. Dev. Biol., 44, 394–436.[ISI][Medline]

Fishel, S., Green, S., Bishop, M. et al. (1995) Pregnancy after intracytoplasmic injection of spermatid. Lancet, 345, 1641–1642.

Fishel, S., Aslam, I. and Tesarik, J. (1996) Spermatid conception: a stage too early, or a time too soon? Hum. Reprod., 11, 1371–1375.[Free Full Text]

Fleming, A.D., Cummins, J.M., Kuehl, T.J. et al. (1986) Normal development of hamster and rabbit eggs fertilized by spermatozoa labeled with the fluorescent thiol alkylating agent, monobromobimane. J. Exp. Zool., 237, 383–390.[ISI][Medline]

Gledhill, B.L., Evenson, D.P. and Pinkel, D. (1990) Flow cytometry and sorting of sperm and male germ cells. In Melamed, M., Lindmo, T. and Mendelsohn, M. (eds.), Flow Cytometry and Sorting. Willey-Liss,New York, pp. 531–551.

Grabske, R.J., Lake, S., Gledhill, B.L. et al. (1975) Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity. J. Cell Physiol., 86, 177–189.[ISI][Medline]

Haugland, R. P. (1996) Handbook of Fluorescent Probes and Research Chemicals, 6th edn. Molecular Probes Inc., Eugene, OR, USA.

Kaneda, H., Hayashi. J., Takahama, S. et al. (1995) Elimination of paternal mitochondrial DNA in intraspecific crosses during early mouse embryogenesis. Proc. Natl. Acad. Sci. USA, 92, 4542–4546.[Abstract]

Lam, D.M.K., Furrer, R. and Bruce, W.R. (1970) The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse. Proc. Natl. Acad. Sci. USA, 65, 192–199.[Abstract]

Lee, J.W., Kim, N.H., Lee, H.T. et al. (1998) Microtubule and chromatin organization during the first cell-cycle following intracytoplasmic injection of round spermatid into porcine oocytes. Mol. Reprod. Dev., 50, 221–228.[ISI][Medline]

Mendoza, C. and Tesarik, J. (1996) The occurrence and identification of round spermatids in the ejaculate of men with nonobstructive azoospermia. Fertil. Steril., 66, 826–829.[ISI][Medline]

Mendoza, C., Benkhalifa, M., Cohen-Bacrie, P. et al. (1996) Combined use of proacrosin immunocytochemistry and autosomal DNA in situ hybridisation for evaluation of human ejaculated germ cells. Zygote, 4, 279–283.[ISI][Medline]

Meier, E., Miller, B.R. and Forbes, D.J. (1995) Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation. J. Cell Biol., 129, 1459–1472.[Abstract]

Moreno, R. and Schatten, G. (1998) Fate of acrosomal contents and matrix after fertilization and ICSI. Mol. Biol. Cell, 9 (Suppl.), 66a.

Ogura, A. and Yanagimachi, R. (1993) Round spermatid nuclei injected into hamster oocytes form pronuclei and participate in syngamy. Biol. Reprod., 48, 219–225.[Abstract]

Ogura A., Matsuda, J. and Yanagimachi, R. (1994) Birth of normal young after electrofusion of mouse oocytes with round spermatids. Proc. Natl. Acad. Sci. USA, 91, 7460–7462.[Abstract]

Oko, R. (1988) Comparative analysis of proteins from the fibrous sheath and outer dense fibers of rat spermatozoa. Biol. Reprod., 39, 169–182.[Abstract]

Oko, R., and Clermont, Y. (1988) Isolation, structure and protein composition of the perforatorium of rat spermatozoa. Biol. Reprod., 39, 673–687.[Abstract]

Oko, R., and Clermont, Y. (1998) Spermiogenesis. In Knobil, E. and Neil, J. D. (eds), Encyclopedia of Reproduction. Academic Press, San Diego, Vol. IV, pp. 602–609.

Oko, R. and Maravei, D. (1994) Protein composition of the perinuclear theca of bull spermatozoa. Biol. Reprod., 50, 1000–1014.[Abstract]

Oko, R. and Maravei, D. (1995) Distribution and possible role of perinuclear theca proteins during bovine spermiogenesis. Microsc. Res. Tech., 32, 520–532.[ISI][Medline]

Ramalho-Santos, J., Moreno, R.D., Sutovsky, P. et al. (1998) Do SNARE proteins mediate membrane fusion events in mammalian spermatozoa? Mol. Biol. Cell, 9 (Suppl.), 332a (Abstract 1929).

Reyes, J.G., Diaz, A., Osses, N. et al. (1997) On stage single cell identification of rat spermatogenic cells. Biol. Cell, 89, 53–66.[ISI][Medline]

Romrell, L.J., Bellve, A.R. and Fawcett, D.W. (1976) Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization. Dev. Biol., 49, 119–131.[ISI][Medline]

Shalgi, R., Magnus, A., Jones, R. et al. (1994) Fate of sperm organelles during early embryogenesis in the rat. Mol. Reprod. Dev., 37, 264–271.[ISI][Medline]

Silber, S.J., Verheyen, G. and Van Steirteghem, A.C. (1998) Spermatid conception. Hum. Reprod., 13, 2976–2979.[Free Full Text]

Simerly, C. and Schatten, G. (1993) Techniques for localization of specific molecules in oocytes and embryos. Methods Enzymol., 225, 516–553.[ISI][Medline]

Sofikitis, N.V., Toda, T., Miyagawa, I. et al. (1996) Beneficial effects of electrical stimulation before round spermatid nuclei injections into rabbit oocytes on fertilization and subsequent embryonic development. Fertil. Steril., 65, 176–185.[ISI][Medline]

Sofikitis, N., Yamamoto, Y., Isoyama, T. et al. (1997) The early haploid male gamete develops a capacity for fertilization after the coalescence of the proacrosomal granules. Hum. Reprod., 12, 2713–2719.[Abstract]

Sutovsky, P. and Schatten, G. (1999) Paternal contributions to the mammalian zygote: fertilization after sperm–egg fusion. Int. Rev. Cytol., In press

Sutovsky, P., Hewitson, L., Simerly, C.R. et al. (1996a) Intracytoplasmic sperm injection (ICSI) for Rhesus monkey fertilization results in unusual chromatin, cytoskeletal, and membrane events, but eventually leads to pronuclear development and sperm aster assembly. Hum. Reprod., 11, 1703–1712.[Abstract]

Sutovsky, P., Navara, C.S. and Schatten, G. (1996b) Fate of the sperm mitochondria, and the incorporation, conversion, and disassembly of the sperm tail structures during bovine fertilization. Biol. Reprod., 55, 1195–1205.[Abstract]

Sutovsky, P., Moreno, R. and Schatten, G. (1998) Sperm mitochondrial ubiquitination and a model explaining the strictly maternal mtDNA inheritance in mammals. Mol. Biol. Cell, 9 (Suppl.), 309a.[ISI]

Tesarik, J. (1997) Sperm or spermatid conception? Fertil. Steril., 68, 214–216.[ISI][Medline]

Tesarik, J. and Mendoza, C. (1996) Spermatid injection into human oocytes. I. Laboratory techniques and special features of zygote development. Hum. Reprod., 11, 772–779.[Abstract]

Tesarik, J., Mendoza, C. and Testart, J. (1995) Viable embryos from injection of round spermatids into oocytes. N. Engl. J. Med., 333, 525.[Free Full Text]

Vanderzwalmen, N.M., and Schoysman, P. (1998) Selection of viable spermatozoa from immature germ cells and non-germ cells. In Semen Evaluation, Testing and Selection: A New Look in the 2000's. Pre-Congress Training Course, Genk, Belgium, pp. 61–67.

Vanderzwalmen, P., Zech, H., Birkenfeld, A. et al. (1997) Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation. Hum. Reprod., 12, 1203–1213.[ISI][Medline]

Verheyen, G., Crabbé, E., Joris, H. et al. (1998) Simple and reliable identification of the human round spermatid by inverted phase-contrast microscopy. Hum. Reprod., 13, 1570–1577.[Abstract]

Vigers, G.P.A., Coue, M. and McIntosh, J.R. (1988) Fluorescent microtubules break up under illumination. J. Cell Biol., 107, 1011–1024.[Abstract]

Yamanaka, K., Sofikitis, N.V. and Miyagawa, I. et al. (1997) Ooplasmic round spermatid nuclear injection procedures as an experimental treatment for nonobstructive azoospermia. J. Assist. Reprod. Genet., 14, 55–62.[ISI][Medline]

Submitted on March 5, 1999; accepted on May 28, 1999.