1 Centro de Estudios en Ginecología y Reproducción, CEGyR, 1055-Buenos Aires, Argentina, 2 Departments of Obstetrics and Gynecology and Animal Sciences, University of Missouri-Columbia, S141 ASRC, Columbia, MO 652115300, 3 Oregon Health and Science University, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006, USA and 4 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: asthenozoospermia/dysplasia of the fibrous sheath/male infertility/sperm mitochondria/ubiquitin
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Introduction |
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Ubiquitin is a small, 8.5 kDa proteolytic polypeptide (Goldstein et al., 1975; Ciechanover et al., 1984
), involved in the regulation of proteolysis in a diverse array of cellular events. These include recycling of the outlived cytoplasmic proteins, endocytosis of membrane receptors and cell cycle control through the destruction of the cyclin component of maturation promoting factor (MPF) at the metaphase/anaphase transition (Hershko, 1998
; for review), but also the formation of Alzheimer's plaques (Perry et al., 1987
) and HIV viral infection (Strack et al., 2000
). Ubiquitination of multiple substrates occurs during the final stages of spermatogenesis (Bebington et al., 2001
; Sutovsky, 2002
) and mitochondria appear to be one of the primary targets of ubiquitination in normal sperm. During epididymal passage, the ubiquitinated substrates in the sperm mitochondria are sterically hidden from antibody detection by the formation of disulphide bonds in the mitochondrial membranes. The ubiquitination of sperm mitochondrial membrane proteins may be necessary for the recognition and proteolysis of sperm mitochondria inside the fertilized oocyte, thus promoting the maternal mode of mitochondrial DNA inheritance in mammals (Sutovsky et al., 1999
, 2000
). As it was reported (Sutovsky et al., 2001a
,b
), some epididymal and ejaculated sperm, particularly those with structural abnormalities, can display partial to entire cell ubiquitination. These sperm are found predominantly in the supernatants from Percoll separation gradients, which contain immotile, dead and defective sperm (Sutovsky et al., 2001a
). Failed disulphide bond stabilization and/or apoptosis-related alterations of the sperm plasma membrane could be a possible cause of the binding of epididymis-secreted ubiquitin-cross-reactive proteins to the surface of defective sperm (Sutovsky et al., 2001a
). Consequently, ubiquitin-cross-reactive substrates can be detected on the cell surface and in the mitochondria of the aldehyde fixed, or even unfixed, defective sperm without permeabilization, which is a necessary step for the detection of ubiquitinated mitochondrial substrates in normal, motile sperm. Based on these findings, a novel, ubiquitin-based, flow cytometric assay of semen quality has been developedsperm-ubiquitin-tag immunoassay (SUTI) (Sutovsky et al., 2001b
), with possible applications in the diagnostics of male infertility. The surprising phenomenon of extracellular sperm ubiquitination may be more widespread than originally thought, as the new evidence validating such hypothesis comes from the studies of ascidian fertilization (Sawada et al., 2002
).
In the present work, we report the presence and incidence of sperm-surface ubiquitination in sperm from patients with DFS. While the increased sperm ubiquitination was previously associated with male infertility (Sutovsky et al., 2001a), this is the first report showing this phenomenon in a group of patients suffering from a well defined, presumably heritable sperm anomaly.
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Materials and methods |
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Clinical evaluation of sperm ubiquitination by immunofluorescence with anti-ubiquitin antibodies
Sperm were attached to microscopy coverslips coated with 1% Poly-L-lysine and fixed in 2% formaldehyde in 0.1 mol/l phosphate buffered saline (PBS) (Sigma St. Louis, MO, USA) (Sutovsky et al., 2001a,b
). To detect the ubiquitin signal in sperm mitochondria, sperm were permeabilized for 40 min in PBS with 0.1% Triton X-100 (TX-100), and incubated overnight at 4°C with mouse monoclonal immunoglobulin M (IgM) antibody KM 691 (Kamiya Biomedical Comp., Seattle, WA, USA), raised against human recombinant ubiquitin. KM 691 was diluted 1:100 in PBS containing 0.1% bovine serum albumin (BSA), 0.02% sodium azide and 0.1% Triton X-100. Samples were washed in PBS, and further incubated in TRITC-conjugated goat anti-mouse IgM (diluted 1:40; Zymed Inc., San Francisco, CA, USA) for 40 min at room temperature. For negative control staining, PBS + BSA alone replaced the first antibody solution. A parallel positive control with sperm from three fertile donors (nos. 68) was performed for comparison. Coverslips with sperm were washed in PBS and counterstained with Hoechst 33258 (1 mg/ml) for 5 min at room temperature, washed again in PBS, mounted on slides, and sealed with a clear nail polish. Samples for clinical evaluation (patient nos. 15 and fertile donor nos. 68) were examined using an Olympus BX-40 epifluorescence microscope and photographed on Kodak Ektachrome 1600 film. Images were scanned and processed using Adobe Photoshop 5.0 software (Adobe System Inc., Mountain View, CA, USA). To ascertain that this labelling was indeed confined to the sperm mitochondria, unfixed sperm were labelled with a vital mitochondrial probe, MitoTracker Green FM (Molecular Probes Inc., Eugene, OR, USA) as described previously (Sutovsky et al., 2000
).
Flow cytometric SUTI assay
SUTI was performed as described previously (Sutovsky et al., 2001b). Sperm samples (250 µl) were pelleted by a 5 min centrifugation at 500 g in TALP-HEPES medium and fixed for 40 min in 2% formaldehyde in PBS. No permeabilization was performed. Samples were blocked for 25 min in 5% normal goat serum (NGS) in PBS and processed with anti-ubiquitin antibody KM691 (1/100) and FITC-conjugated goat anti-mouse IgM (1/80). All washings were performed by resuspension/centrifugation in PBS with 1% of NGS. At the end, the samples were resuspended in 500 µl of pure PBS without serum. Blank, control samples were prepared by omitting the primary, anti-ubiquitin antibody.
Ubiquitin median values (channel number at which half the cells are dimmer and half the cells are brighter), the histograms of relative fluorescence and the diagrams of the visible light scatter, were generated by FACS Calibur Analyzer (Becton Dickinson, San Diego, CA, USA) at 488 nm wavelength. Relative levels of fluorescence in 5000 individual cells per sample were recorded in each of the three repeats. A blank sample from the corresponding donor/patient, labelled with secondary antibody alone, was measured prior to each anti-ubiquitin-labelled sample. Samples from five DFS patients (nos. 15) and five fertile donors (nos. 913) were measured and compared.
Sperm samples already processed for flow cytometry were also screened by high resolution epifluorescence microscopy. Representative images are shown in Figure 3. Briefly, 23 µl of the processed sperm suspension in PBS were pipetted onto a microscopy slide in a 10 µl of VectaShield (Vector Labs, Burlingame, CA, USA) mounting medium containing 5 µg/ml of DNA stain, Hoechst 33258. Drops of sperm were covered with a coverslip and sealed with a clear nail polish. Samples were examined and photographed with a Nikon Eclipse 800 biological research microscope equipped with infinity-corrected, planar, apochromatic lenses (CFI60 Plan Apo Series, Nikon), and a CoolSnap HQ CCD camera (Roper Scientific, Tucson, AZ, USA), operated by MetaMorph 4.6.5 software (Universal Imaging Corp., Downington, PA, USA). Images were edited using Adobe Photoshop 6.0 software. Most reagents were purchased from Sigma and secondary antibodies were obtained from Zymed.
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Results |
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The analysis of DFS semen samples by SUTI posed a unique challenge due to the presence of an unusually large number of cells other than normal sperm. Initially, the median values were obtained for the whole samples (R0: Table IIA, Figure 4A
) and then the cells within the screened pool were divided into two groups, based on the arbitrary subdivision of scatter diagrams of the cell size measured in the visible light spectrum (Figure 4B
). (i) Cells of prevailing size, considered to be normal size sperm (R2), which formed a tight focus in the centre of the scatter diagrams (Figure 4B
); and (ii) cells of either small or large size (R1; Figure 4B
), scattered outside the R2 region. High median values for all cells measured (R0) were detected in all five patients (19.837.9; Table IIA
). The fertile samples displayed significantly lower overall median values (9.316.6; mean 13.4), as well as lower fluorescence of normal size-cells (R2; 8.715.4; as opposed to 16.627.4 in DFS samples) and somewhat lower fluorescence of the large cells (R1, 16.652.3 in the fertile samples; 22.967.3 in DFS samples). Overlapping of the flow cytometric histograms of overall fluorescence (R0) of all five patients with that of the fertile donor with highest ubiquitin median (donor no. 7) revealed a shift of the curve towards the highly fluorescent cells in all patients (examples shown in Figure 4A
).
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Discussion |
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With regard to the ubiquitination of the sperm tail structures in these DFS and other patients, it is important to distinguish between two different steps of ubiquitination that occur during spermatogenesis in the testis and during sperm maturation/storage in the epididymis respectively. At the round spermatid stage of spermiogenesis, the mitochondria in both normal and defective sperm cells acquire ubiquitin cross-reactivity. This ubiquitin tag is probably masked by disulphide bond formation in the caput epididymis (Sutovsky et al., 2000), and later may serve as a signal for the recognition and elimination of the sperm mitochondria at fertilization (Sutovsky et al., 1999
, 2000
). By immunofluorescence, the mitochondrial sheath is the only sperm structure displaying a detectable ubiquitin cross-reactivity in human and animal testicular sperm prior to epididymal passage (Sutovsky et al., 2000
, 2001a
). In the defective sperm, such a ubiquitination pattern may diverge. The failure of disulphide bond cross-linking and/or the irreversible changes of the sperm plasma membrane may render the defective sperm prone to further ubiquitination by the ubiquitin cross-reactive proteins inside the epididymal lumen (Sutovsky et al., 2001a
). Thus the cross-reactivity of the ubiquitinated substrates in the mitochondria of DFS sperm may be the results of failed cross-linking of the mitochondrial membranes, and/or de-novo ubiquitination of the mitochondrial membranes during epididymal passage. The ubiquitin cross-reactive proteins, detected in DFS samples on the surface of the sperm tail principal-piece and on the sperm head, most likely originate from the epididymal secretion (Fraile et al., 1996
; Sutovsky et al., 2001a
). The microscopic assessment of ubiquitin-cross-reactivity in sperm tail mitochondria could therefore be a good marker of infertility wherever flow cytometry is not available. More sensitive tests could be performed using flow cytometric SUTI assay, as described here and previously (Sutovsky et al., 2001b
), or by other immunological methods (Western blotting, ELISA, proteomics).
Sperm evaluation by SUTI poses a unique challenge in DFS patients because of the high number of cells other than mature sperm present in their semen samples. On the one hand, these cells may affect the overall reading of ubiquitin medians as their median fluorescence may be lower than that of the defective sperm (see Figure 3). On the other hand, due to their large size and dense cytoplasm, these cells may cause a higher autofluorescence in blank, negative control samples than the normal and defective sperm. It was therefore useful to subdivide the scatter diagrams generated by flow cytometer into areas representing normal/prevailing size sperm cells (R2 in Figure 4A
) and small/large cells (R1 in Figure 4A
). The standard, fertile sperm samples showed the lowest overall ubiquitin medians, as well as the lowest ubiquitin medians of prevailing-size cells. While the median values for the small and large, R1 cells were lower in the fertile samples than in the DFS semen, the fluorescence levels were comparable. It has been shown previously that the leukocytes, residual bodies and cellular debris in human semen contain both intracellular and cell surface-bound ubiquitin-cross-reactive proteins (Sutovsky et al., 2001b
).
Bongso et al.(1989) demonstrated that human oocytes can be fertilized successfully with immotile spermatozoa by micro-injection. The use of intracytoplasmic sperm injection (ICSI) has resulted in pregnancies in 60% of patients with DFS (Stalf et al., 1995
; Terriou et al., 1995; Chemes et al., 1998
; Brugo Olmedo et al., 1997; 2000). Although having an elevated rate of epifluoresecence-assessed sperm mitochondrial ubiquitination (~10x higher than standard sperm) and higher sperm surface ubiquitination (~2x higher than standard samples), treatment by ICSI in patient no. 1 led to a triplet pregnancy, and birth of one boy and two girls (unpublished results). This implies that DFS sperm could be used for ICSI, though all necessary precautions should be taken with regard to possible embryonic abnormalities. It is yet to be determined what happens with the ubiquitinated epitopes on the sperm surface once they are injected into the oocyte cytoplasm. Since the sperm plasma membrane, mitochondria, axoneme and perinuclear skeleton are disposed of after natural fertilization (Sutovsky and Schatten, 2000
; review), it is possible that their ubiquitination prior to sperm injection into the oocyte may not interfere with normal zygotic and embryonic development. However, there could be some cases in which the sperm centriole, an organelle required for successful pronuclear apposition in humans, could also be compromised (V.Rawe et al., unpublished observations).
In summary, the present study demonstrates that the anti-ubiquitin antibodies recognize defective sperm in a presumably heritable, male infertility syndrome, DFS. Ubiquitin thus may be an efficient marker of defective sperm in humans regardless of the cause of infertility. Ubiquitin-based screening may not be necessary for the diagnosis of DFS, as the gross morphological defects in this particular type of infertility are obvious from routine microscopic sperm evaluation. However, this is the first study to show increased sperm ubiquitination in any type of presumably inherited fertility disorder, and the SUTI assay could be useful for revealing other heritable infertility cases, should these be associated with less conspicuous sperm abnormalities.
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Acknowledgements |
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Notes |
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References |
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Baccetti, B., Burrini, A.G., Capitani, G. Collodel, G., Moretti, E., Piomboni, P. and Renieri, T. (1993) Notulae seminologicae. 2 The `short tail' and `stump' defect in human sperm. Andrologia, 25, 331335.[ISI][Medline]
Barth, A.D. and Oko, R. J. (1989) Defects of the sperm tail. In Abnormal Morphology of bovine Spermatozoa. Iowa State University Press, Ames, p. 214.
Bebington, C., Doherty, F.J. and Fleming, S.D. (2001) The possible biological and reproductive functions of ubiquitin. Hum. Reprod. Update, 7, 102111.
Bisson, J.P., Leonard, C. and David, G. (1979) Caractere familial de certaines perturbations morphologiques des spermatozoides. Arch. Anat. Cytol. Path., 27, 230233.
Blom, E. (1976) A sterilizing tail stump sperm defect in a Holstein-Friesian bull. Nord. Vet. Med., 28, 295298.[ISI][Medline]
Bongso, T., Sathananthan, A., Wong, P. C., Ratnam, S.S, Ng, S.C., Anandakumar, C. and Ganatra, S. (1989) Human fertilization by microinjection of immotile spermatozoa. Hum. Reprod., 4, 175179.[Abstract]
Brugo Olmedo, S., Nodar, F., Chillik, C. and Chemes, H. E. (1997) Successful intracytoplasmic sperm injection with spermatozoa from a patient with dysplasia of the fibrous sheath and chronic respiratory disease. Hum. Reprod., 7, 14971499.
Brugo Olmedo, S., Rawe, V.Y., Nodar, F.N., Galaverna, G.D., Acosta, A.A. and Chemes, H.E. (2000) Pregnancies established through Intracytoplasmic Sperm Injection (ICSI) using spermatozoa with Dysplasia of the Fibrous Sheath. Asian J. Androl., 2, 125130.[ISI][Medline]
Chemes, H.E. (1991) The significance of flagellar pathology in the evaluation of asthenozoospermia. In Baccetti B. (ed.) Comparative Spermatology 20 years later. Serono Symposia Publications, Raven Press, New York, 815 pp.
Chemes, H.E., Brugo Olmedo, S., Carrere, C. Oses, R., Carizza, C., Leisner, M. and Blaquier, J. (1998) Ultrastructural pathology of the sperm flagelum: association between flagellar pathology and fertility prognosis in severely asthenozoospermic men. Hum. Reprod., 9, 25212526.
Chemes, H.E., Brugo, S., Zanchetti, F. Carrere, C. and Lavieri, J.C. (1987) Dysplasia of the fibrous sheath. An ultrastructural defect of human spermatozoa associated with sperm immotility and primary sterility. Fertil. Steril., 48, 664669.[ISI][Medline]
Chemes, H.E., Morero, J.L. and Lavieri, J.C. (1990) Extreme asthenozoospermia and chronic respiratory disease. A new variant of the immotile cilia syndrome. Int. J. Androl., 13, 216222.[ISI][Medline]
Ciechanover, A., Finley, D. and Varshavsky, A. (1984) Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutants. Cell, 37, 5766.[ISI][Medline]
Coubrrough, R.I. and Barker, C.A.V. (1964) Spermatozoa: an unusual middle piece abnormality associated with sterility in bulls. Proc. 5 Int. Congr. Animal Reprod. (Trento), 5, 219229.
Eliasson, R., Mossberg, B., Cammer, P., and Afzelius, B.A. (1977) The immotile cilia syndrome: a congenital ciliary abnormality as an etiology factor in chronic airway infections and male sterility. N. Engl. J. Med., 297, 16.[Abstract]
Fraile, B., Martin, R., De Miguel, M.P., Arenas, M.I., Bethencourt, F.R., Peinado, F., Paniagua, R. and Santamaria L. (1996) Light and electron microscopic immunohistochemical localization of protein gene product 9.5 and ubiquitin immunoreactivities in the human epididymis and vas deferens. Biol. Reprod., 55, 291297.[Abstract]
Goldstein, G., Scheid, M., Hammerling, U., Schlesinger, D.H., Niall, H.D. and Boyse, E.A. (1975) Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells. Proc. Natl Acad. Sci., USA, 72, 1115.[Abstract]
Hershko, A. (1998) The ubiquitin system: Past, present and future perspectives. In Peters, J.-M., Harris, J. R. and Finley, D. (eds) Ubiquitin and the biology of the cell. Plenum Press New York., pp. 117
Jeyendran, R., Van der Ven, H., Perez-Pelaez, M., Crabo, B.G. and Zaneveld, L.J.D. (1984) Development of an assay to assess the functional integrity of human sperm membranes and its relationship to other semen characteristics. J. Reprod. Fertil., 70, 219228.[Abstract]
Kastury, K., Taylor, W.E., Arver, S., Shen, R., Gutierrez, M., Fisher, C.E., Coucke, P.J., Van Hauwe, P., Van Camp, G., and Bhasin, S. (1997) cDNA cloning, chromosomal mapping and tissue distribution of the human axonemal dynein light chain gene for the immotile cilia syndrome. J. Androl., (Suppl. ), p. 56.
Maqsood, M. (1951) An abnormality of mammalian spermatozoa. Experientia, 7, 304.
Perry, G., Friedman, R., Shaw, G., and Chau, V. (1987) Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains. Proc. Natl Acad. Sci. USA, 84, 30333036.[Abstract]
Rawe, V.Y., Brugo Olmedo, S., Galaverna, G. D., Olmedo, S.B. and Chemes, H.E. (2001) Incidence of tail structure distortions associated with Dysplasia of the Fibrous Sheath in human spermatozoa. Hum. Reprod., 5, 879886.
Sawada, H., Sakai, N., Abe, Y., Tanaka E., Takahashi Y., Fujino J., Kodama E., Takizawa S. and Yokosawa, H. (2002) Extracellular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor. Proc. Natl Acad. Sci. USA, 99, 12231228.
Stalf, T., Sanchez, R., Kohn, F.M. et al. (1995) Pregnancy and birth after intracytoplasmic sperm injection with spermatozoa from a patient with tail stump syndrome. Hum. Reprod., 10, 21122114.[Abstract]
Strack, B., Calistri, A., Accola, M.A., Palu, G. and Gottlinger, H.G. (2000) A role for ubiquitin ligase recruitment in retrovirus release. Proc. Natl Acad. Sci. USA, 97, 1306313068.
Sutovsky, P., Moreno, R., Ramalho-Santos, J., Dominko, T., Simerly, C. and Schatten, G. (1999) Ubiquitin tag for sperm mitochondria. Nature, 402, 371372.[ISI][Medline]
Sutovsky, P. and Schatten, G. (2000) Paternal contributions to the mammalian zygote: fertilization after sperm-egg fusion. Int. Rev. Cytol., 195, 165.[ISI][Medline]
Sutovsky, P., Moreno, R., Ramalho-Santos, J., Dominko, T., Simerly, C. and Schatten, G. (2000) Ubiquitinated sperm mitochondria, selective proteolysis and the regulation of mitochondrial inheritance in mammalian embryos. Biol. Reprod., 63, 582590.
Sutovsky, P. (2002) Ubiquitin-dependent proteolysis in mammalian spermatogenesis, fertilization, and sperm quality control: killing three birds with one stone. Micros. Res. Tech., In press.
Sutovsky, P., Moreno, R., Ramalho-Santos, J., Dominko, T., Thompson, W.E. and Schatten, G. (2001a) A putative, ubiquitin-dependent mechanism for the recognition and elimination of defective spermatozoa in the mammalian epididymis. J. Cell. Sci., 114, 16651675.
Sutovsky, P., Terada, Y. and Schatten, G. (2001b) Ubiquitin-based sperm assay for the diagnosis of male factor infertility. Hum. Reprod., 16, 250258.
Terriou, P., Giorgetti, C., Hans, C., Spach, J.L., Salzmann, J., Carlon, N., Navarro, A. and Roulier, R. (1993) Subzonal sperm insemination and total or extreme asthenozoospermia: an effective technique for an uncommon cause of male infertility. Fertil. Steril., 60, 10571061.[ISI][Medline]
Torikata, C., Kawai, T., Nogawa, S. Ikeda, K., Shimizu, K. and Kijimoto, C. (1991) Nine Japanese patients with immotile-dyskinetic cilia syndrome: an ultrastructural study using tannic acid-containing fixation. Hum. Pathol., 22, 830836.[ISI][Medline]
Turner, R.M., Musse, M.P., Mandal, A., Klotz, K., Jayes, F.C., Herr, J.C., Gerton, G.L., Moss, S.B. and Chemes, H.E. (2001) Molecular genetic analysis of two human sperm fibrous sheath proteins, AKAP4 and AKAP3, in men with dysplasia of the fibrous sheath. J. Androl., 22, 302315.
World Health Organization (1987) Laboratory Manual for the Examination of Human Semen and Semen Cervical Mucus Interaction. Cambridge University Press, Cambridge, UK.
Xu, X., Toselli, P.A., Russell, L.D. and Seldin, D.C. (1999) Globozoospermia in mice lacking the casein kinase II alpha catalytic subunit. Nature Genet., 23, 118121.[ISI][Medline]
Submitted on November 30, 2001; resubmitted on February 22, 2002; accepted on April 25, 2002.