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
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Abstract |
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Key words: fertilization/mitochondria/ROSI/round spermatid/spermatozoa
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Introduction |
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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., 1996; Tesarik, 1997
; Silber et al., 1998
). 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., 1997
; Reyes et al., 1997
) 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, 1996
; Angelopoulos et al., 1997
; Sofikitis et al., 1997
; Yamanaka et al., 1997
; Verheyen et al., 1998
). 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., 1995
; Sutovsky et al., 1996b
; Cummins et al., 1998
). 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.
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Materials and methods |
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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, 1988; diluted 1/200) and a nucleoporin-specific antibody mAb 414 (Meier et al., 1995
; 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, 1994
; 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., 1997
; 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., 1990
; 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 1 is a composite image of the individually photographed round spermatids, printed using Adobe Photoshop.
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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 474295 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.
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Results |
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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,A') 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 3BE
'). At the same time, the progressive condensation of the sperm nucleus caused an increased nuclear retention of MitoTracker dye (Figure 3BE
'), 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 3FH
'). Some unusually large round spermatids, containing one or two nuclei with distinct acrosomal caps, were occasionally found (Figure 3I,I
'). 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 4AC
'), and best results were obtained with the infinity-corrected x60 lens (Figure 3
).
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Discussion |
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The present study suggests that the round spermatid-specific polarization of the mitochondria (see Figure 6), 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., 1970
; Grabske et al., 1975
; Romrell et al., 1976
), Percoll separation (Bucci et al., 1986
) and cell sorting (Gledhill et al., 1990
; Aslam et al., 1998
). 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, 1996
; Sofikitis et al., 1997
; Vanderzwalmen et al., 1997
; Yamanaka et al., 1997
). 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 3D
). 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, 1999
) 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, 1998
; 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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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., 1997). 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, 1993
) 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., 1988
). 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., 1998
). 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, 1996
). 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., 1996b
; Cummins et al., 1997
).
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., 1994; Kaneda et al., 1995
; Sutovsky et al., 1996a
,b
; Cummins et al., 1997
). Similarly, the mitochondria of round spermatids appear to be destroyed in the oocyte cytoplasm after ROSI, at least in mice (Cummins et al., 1998
). Ubiquitination of the mitochondrial proteins was proposed to play a role in the elimination of sperm mitochondria after fertilization (Sutovsky et al., 1996b
), and our new data strongly support this hypothesis (Sutovsky et al., 1998
). 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,A
'), 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., 1986
). Pre-labelling of bull (Sutovsky et al., 1996b
) and mouse (Kaneda et al., 1995
) 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 (x1040) 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.
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Acknowledgments |
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Notes |
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5 To whom correspondence should be addressed
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References |
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Submitted on March 5, 1999; accepted on May 28, 1999.