1 Center for Studies in Gynaecology and Reproduction, CEGyR, 1055-Buenos Aires and 2 Laboratory of Testicular Physiology and Pathology, Endocrinology Division, Children's Hospital, Buenos Aires, Argentina
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Abstract |
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Key words: dysplasia of the fibrous sheath/microtubules/mitochondrial sheath/spermatozoa
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Introduction |
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The main features of spermatozoa in DFS as evidenced by transmission electron microscopy studies are a marked hypertrophy and hyperplasia of random fibrous sheath constituents that form thick rings or broad meshes without the orderly disposition in longitudinal columns and transversal ribs that characterize the normal fibrous sheath. In some patients, the 9+2 axonemal structure is completely distorted while in some others it is preserved in the centre of dense rings of hyperplastic fibrous sheath. According to the prevalence of tail abnormalities, two groups of patients have been described. In some of them all spermatozoa are affected, while in some others the number of abnormal tails is about 7080% with 2030% in the normal configuration (Chemes et al., 1998). These two groups have distinct characteristics and correspond to the complete and incomplete form of DFS.
In the present report, we have analysed the distribution and incidence of tail structural disruptions associated with DFS. To this end we have used antibodies raised against different cytoskeletal components of the sperm tail or specific markers for mitochondria and studied them by phase contrast and fluorescent microscopy. This methodology, applied on sperm smears, provides a topographical view of different sperm components, not previously afforded by transmission electron microscopy.
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Materials and methods |
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Semen samples
A fresh semen sample was processed in each patient within 30 min of ejaculation. An aliquot of the semen sample was first washed with phosphate buffer (0.1 mol/l, pH 7.4), pelleted by centrifugation and resuspended in a volume of phosphate buffer. Viability was determined using the hypo-osmotic swelling test (HOST) (Jeyendran et al., 1994). Briefly, semen samples were incubated during 60 min at 37°C in a 1:10 dilution of hyposmotic solution and then the viability was assessed.
In all cases, a small aliquot of fresh semen was studied under phase contrast microscopy, and motility, viability and light microscopy morphology were investigated according to standard methods (World Health Organization, 1987).
Transmission and scanning electron microscopy
A fresh semen sample was processed in each of the six patients for transmission electron microscopy (TEM) within 30 min of ejaculation, according to methods previously described (Chemes et al., 1987). In brief, the spermatozoa were washed with phosphate buffer (0.1 mol/l, pH 7.4), pelleted by centrifugation and fixed in 3% glutaraldehyde followed by 1.3% osmium tetroxide. The pellets were embedded in Epon Araldite (Polysciences Inc., Warrington, PA, USA) and thin sections were examined and photographed in a Zeiss 109 electron microscope (Zeiss, Oberkochen, Germany) after double staining with uranyl acetate and lead citrate. In one patient, a second semen sample was processed and studied by scanning electron microscopy (SEM). The spermatozoa were fixed in suspension using the same fixatives with buffer washes between and after each step. Sperm cells were subsequently sedimented on poly-L-lysine-coated slide fragments, to assure sperm adherence to the glass, dehydrated in a graded series of ethanol followed by absolute acetone, dried in a Balzers CDP 030 critical point drying apparatus (Balzers Union Ltd, Balzers, Lichtenstein), using CO2 as transition fluid, coated with gold-palladium in a Balzers Union SCD 040, and observed in a Philips 515 scanning electron microscope (Philips Nederland BV, Eindhoven, The Netherlands).
For quantification of axonemal anomalies with the transmission electron microscope, at least 100 flagella were counted.
Mitochondrial sheath staining
Aliquots of semen samples processed using HOST were used to visualize the mitochondrial sheath. To this end, MitoTracker Green FMTM (Molecular Probes Inc., Eugene, OR, USA) was diluted to a concentration of 1 mmol/l in dimethylsulphoxide (Sigma Chemical, St Louis, MO, USA) and added to sperm medium to a final concentration of 400 nmol/l. The sperm suspension was incubated for 10 min at 37°C and subsequently mounted between a slide and a coverslip to be studied under UV excitation.
Microtubules and fibrous sheath identification
Spermatozoa were attached to slides, fixed in 2% formaldehyde in 0.1 mol/l phosphate-buffered saline (PBS) and permeabilized in 0.1% Triton X-100 in 0.1 mol/l PBS. To detect microtubules, spermatozoa were incubated for 40 min at room temperature with a 1:100 dilution of a mouse monoclonal antibody (IgG) raised against -acetylated tubulin (diluted in PBS containing 0.1% BSA and 0.02% sodium azide). The slides were washed in PBS, and further incubated in 1:500 fluorescein-conjugated goat anti-mouse IgG. Fibrous sheaths were visualized by an incubation of sperm for 40 min at room temperature with a 1:10 dilution of a mouse monoclonal antibody (IgM) against FSC1 protein. Then they were washed in PBS, and further incubated in 1:40 TRITC-conjugated goat anti-mouse IgM, for 40 min at room temperature. Both anti-
-acetylated tubulin and anti-FSC1-treated spermatozoa were washed in PBS, in some cases counterstained with Hoescht 33258 (1 µg/ml) for 5 min at room temperature, washed in PBS and mounted between a slide and a coverslip. Samples were examined using an Olympus epifluorescent microscope and photographed with Ektachrome film (1600 ASA). Images were processed using Adobe Photoshop 5.0 software (Adobe System Inc.). For control staining, non-immune serum + PBS + BSA replaced the specific antibody solution. Anti-
-acetylated tubulin antibodies and reagents used were obtained from Sigma. The anti-fibrous sheath antibody (anti-FSC1 protein, 5A8) was kindly supplied by Dr Mich Eddy.
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Results |
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Results of semen analysis done the day of the fluorescence study are summarised in Table I. Under light microscopy, spermatozoa had short, rigid and thick flagella, frequently displaying irregular contours and/or coiled tails.
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The typical configuration of the tails was evident in studies with SEM. Thick, short and irregular tails were noted. In some spermatozoa tails were longer and the fibrous sheath was thickened in some areas and missing in others (Figures 1 and 3).
Fluorescence analysis
Mitochondrial sheath patterns
Human normal sperm prelabelled with a final concentration of 400 nmol/l MitoTracker displayed intense and uniform labelling of the mitochondrial sheath covering both the connecting piece and mid piece of the sperm axoneme. Strong green fluorescence was observed in the form of a smooth cylindroconical rod, which thinned toward the tail (Figure 2). Conversely, mitochondrial sheaths were mainly disrupted or absent in spermatozoa with DFS. Five different configurations of MitoTracker staining were discerned. In the complete form of DFS the prevalent pattern (5060%) consisted in a few, sometimes a single mitochondrion, at the neck region (Figure 2
). The next pattern in frequency in the complete form (2027%) was that of a `necklace' formed by a few mitochondria surrounding the connecting piece (Figure 2
). Less frequent configurations (215%) were those characterized by either absent mitochondria or mixed patterns. Finally, between 2.5 and 17% spermatozoa displayed well-preserved mitochondrial sheaths, similar to those observed in normal spermatozoa. In patients with the incomplete form, the predominant pattern was that of normal mitochondrial sheaths (5575%) with the abnormal types comprising the remaining spermatozoa. The incidence of each pattern in the patients studied is shown in Table II
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Microtubules
Three main patterns of microtubule immunostaining were observed (Figure 3). In the complete form of DFS many sperm tails (~50%) showed the label only over the final portion of the tail (end piece). The second distribution (2030%) consisted of a discontinuous signal over the length of the tail. Finally, a continuous staining, reminiscent of the normal pattern was detected in 1530% of sperm tails (Figure 3
and Table II
). This last pattern predominated in cases of incomplete DFS (5065%). However, even in cases when the microtubules could be observed along the whole length of the tail, the label was weaker than in normal spermatozoa, probably indicating a reduction in the microtubular material of the sperm axoneme (Figure 3
).
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Discussion |
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It has been shown that the assembly of the fibrous sheath during spermiogenesis is preceded by the appearance of a granular material in the sperm flagellum which labels positively with anti-fibrous sheath antibodies (Irons and Clermont, 1982; Sakai et al., 1986
). In humans, a flagellar tubulofilamentous complex of undefined function (the spindle shaped body) is transiently observed during fibrous sheath assembly (Wartenberg and Holstein, 1975
). The presence of simultaneous axonemal and peri-axonemal anomalies in mice and humans suggest that a proper association with the axoneme controls fibrous sheath spatial organization (Escalier and Serres, 1985
; Phillips et al., 1993
). These concepts are challenged by observations of distorted fibrous sheath surrounding well-structured axonemes in some spermatozoa with DFS, and by the existence of non-specific axonemal anomalies associated with normal fibrous sheaths (Chemes et al., 1998
).
One of the most severe abnormalities of sperm flagellar structure is the DFS. Although electron microscopy studies of semen samples have characterized in detail serious distortions of the fibrous sheath constituents in DFS (Chemes et al., 1987, 1990
, 1998
) and even pregnancies have been established (Brugo Olmedo et al., 1997
), to date, no reports are available on the incidence and spatial configuration of sperm tail structures in humans with DFS. The few reported immunocytochemical studies of the fibrous sheath have been directed mainly to normal rodents (Fenderson et al., 1988
; Oko and Clermont, 1989
; Eddy et al., 1991
; Carrera et al., 1994
). The use of epifluorescence microscopy applied to pathological human spermatozoa has allowed us to study DFS from a different point of view. As expected, the incidence of flagellar distortions correlated with the ultrastructural findings and, once again, differences between the complete and incomplete forms were evident. A range of 90.3100% affected spermatozoa was observed in the complete form and 66.568.6% in the incomplete form, suggesting that fibrous sheath biogenesis is quite differently affected.
As previously described, DFS affects various cytoskeletal components such as microtubules and outer dense fibres (Chemes et al., 1998). Electron microscopy studies showed that in some cases the 9+2 axonemal structure is completely distorted whereas in some others it is preserved in the centre of dense rings of hyperplastic fibrous sheath; central pairs were missing in about half of the cases. Using immunofluorescence we were able to determine microtubule patterns with different prevalence depending on the form studied. The abnormal patterns of microtubular labelling seem to depend on the extension of the fibrous sheath hyperplasia since this configuration can have a `masking effect' on microtubules which were evident only in places where the fibrous sheath was not very thick, probably allowing a good accessibility of the antibody. Alternatively, since the microtubular labelling was frequently weak in DFS spermatozoa it may also reflect the absence or scarcity of microtubules among abnormal fibrous sheath constituents, as has been evidenced by previous electron microscopy studies.
Another structure frequently distorted in DFS spermatozoa is the mitochondrial sheath. Electron microscopy studies revealed severe alteration or absence as a consequence of a failure in the migration of the annulus, which remains in a cranial position. The use of mitochondria-specific probes affords a three-dimensional view of this intracellular structure. A complete range of altered mitochondrial sheaths was visualized in DFS spermatozoa. Fluorescence studies of the mitochondrial pattern in whole spermatozoa proved to be especially useful, since some of these configurations (e.g. the `necklace like' or the mixed patterns) are difficult or even impossible to detect in thin sections studied by electron microscopy.
We found the immunofluorescence approach an excellent technique to study specific organelles in DFS. While ultrastructural studies of thin sections allow an in-depth knowledge of the internal organization of the sperm tail, fluorescence labelling of selected sperm components affords a unique view of the whole flagellum including topographical relationships of various organelles. The combination of these different approaches is essential for a comprehensive understanding of this particular pathology.
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Acknowledgements |
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Notes |
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References |
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Submitted on June 8, 2000; accepted on January 31, 2001.