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Gamete Biology |
Departments of Obstetrics and Gynecology and Cooperative Reproductive Science Research Center, Morehouse School of Medicine,3 Atlanta, Georgia 30310
Department of Zoology, Center for Neuroscience and Cell Biology, University of Coimbra,4 Largo Marques de Pombal, 3004-517 Coimbra, Portugal
Departments of Animal Sciences and Obstetrics and Gynecology, University of Missouri-Columbia,5 Columbia, Missouri 65211-5300
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, fertilization, gamete biology, sperm maturation, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ubiquitination is a versatile and universal mechanism for protein recycling, by which misfolded or outlived proteins are tagged for degradation by the covalent attachment of one or more molecules of ubiquitin [7]. Each molecule of ubiquitin added to a molecule of a substrate increases the substrate's mass by 8.5 kDa. Proteins marked with ubiquitin chains of at least four molecules (i.e., tetraubiquitin) are preferentially docked to 26-S proteasome and degraded. Monoubiquitinated proteins (i.e., those with only one ubiquitin moiety linked to their Lys residue) are typically internalized and degraded by the lysosome. During spermatogenesis, ubiquitination may also serve additional purposes, including cell signaling and regulation of gene transcription [8]. Spermatid histones H2B and H3 are ubiquitinated during spermatogenesis [9, 10], and several ubiquitin ligases are present in spermatogenic cells [11, 12]. Ubiquitin and ubiquitin-C-terminal hydrolase, PGP9.5, are also expressed in the epididymis [11], where ubiquitin seems to be secreted in the epididymal lumen [13] and preferentially binds to the defective spermatozoa [14]. Accordingly, ubiquitin is present in the human seminal plasma [15] and on the surface of defective human spermatozoa [16, 17].
A majority of published reports have demonstrated close associations between prohibitin protein and the mitochondria, with some suggesting a possible role of prohibitin as a chaperone to other mitochondrial membrane proteins during their controlled proteolysis (for review, see [18]). Prohibitin was first identified and described in the testis using subtractive hybridization, Northern and Western blot analysis, and immunocytochemical techniques [19]. It was reported that prohibitin mRNA and protein were expressed in Sertoli and Leydig cells. Moreover, they showed that prohibitin transcripts and protein were expressed differentially during germ cell development. Subsequently, we suggested that prohibitin, a conserved 30-kDa component of the inner mitochondrial membrane, is also expressed as an unusual, high-molecular-mass isoform in mammalian spermatozoa [2]. Little is known regarding the functional significance of prohibitin and ubiquitin in the male germ cell. The prohibitin gene was first identified in quiescent mammalian cells and has been shown to have antiproliferative activity in dividing eukaryotic cells [20, 21]. This gene product has been functionally associated with processes such as development [22], cell-cycle regulation [23], cell senescence [22], differentiation [24–26], and cellular immortalization [27, 28]. To our knowledge, the function of prohibitin within the mammalian system, despite its apparent ubiquitous expression, has not been elucidated.
In the present study, we investigated the possibility that the mitochondrial membrane protein prohibitin is one of the ubiquitinated substrates, the presence of which renders the paternal sperm mitochondria prone to rapid degradation after fertilization. Possible consequences of the ubiquitination of prohibitin and other sperm proteins in epididymal sperm quality control and protein recycling are discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Prohibitin was detected in bull and rhesus spermatozoa using antibodies Ab-1 (mouse immunoglobulin [Ig] G) and Ab-2 (rabbit polyclonal) from Neomarkers (Union City, CA). Mouse monoclonal antibody MK-12-3 (MBL, Nagoya, Japan) was raised against the purified bovine erythrocyte ubiquitin. Secondary antibodies were purchased from Zymed Labs (South San Francisco, CA). MitoTracker CMTM Ros and 4'6'-diamidino-2-phenylindole (DAPI) stains were purchased from Molecular Probes (Eugene, OR). Straws of frozen bull semen were purchased from ABS Global (De Forest, WI) and, where stated, were separated on a two-layer, 45%/90% Percoll gradient as described previously [14]. Some experiments were performed using fresh bull spermatozoa collected from the caput epididymis by dissection and release into culture medium.
Sperm heads and tails were separated by sonication and subsequent centrifugation on a sucrose-gradient as described previously [2]. Briefly, sperm heads and tails were separated by ultrasonication using a Branson Digital Sonifier (Danbury, CT). Samples were checked after three or four short pulses to ensure that no intact spermatozoa remained. Heads and tails were then purified by ultracentrifugation at 100 000 x g for 2 h (4°C) through a discontinuous sucrose density gradient using a Beckman TL-100 ultracentrifuge (Beckman Coulter, Inc., Fullerton, CA). Sperm heads were collected from the pellet and tails from the 15%–30% gradient interface. All samples were checked for purity by light microscopy and washed twice by ultracentrifugation to remove the contaminating sucrose (100 000 x g, 2 h, 4°C). Fresh rhesus semen was obtained by penile electroejaculation.
Fresh epididymal and testicular spermatozoa and testicular cells were obtained by mincing of the appropriate bovine tissues purchased from a local slaughterhouse. Tissues were minced with two pairs of fine forceps, a procedure releasing mainly spermatids and fully differentiated spermatozoa. Such cell suspensions were collected by 5-min centrifugation at 350 x g, loaded onto 45%–90% Percoll gradient, and centrifuged for 10 min at 500 x g. Spermatozoa were collected from pellet and spermatids from the 45%–90% interface. Differential interference contrast microscopy, allowing recognition of the developing acrosomal cap and acrosomal granule in the round spermatids, was used to check the purity of fractions. Cell suspensions were washed in SpermTL medium (modified Tyrode-lactate-Hepes medium).
Immunofluorescence
Spermatozoa were attached to poly-L-lysine-coated microscopy coverslips and fixed for 40 min in 2% formaldehyde in PBS. In some experiments (Fig. 1, a–c), spermatozoa on coverslips were treated for 15 min with 1 mM or 10 mM dithiothreitol (DTT; Sigma, St. Louis, MO) before fixation. Samples were blocked and incubated with antibody Ab-1 (1:100 dilution), washed, and incubated for 40 min with fluorescein isothiocyanate-conjugated goat anti-mouse IgM (1:80 dilution) and a blue-fluorescent DNA-binding dye, DAPI, as described previously [14]. MitoTracker CMTM Ros labeling and subsequent immunofluorescence processing was performed as described previously [2]. Images were captured using a Nikon Eclipse 800 microscope and CoolSnap HQ RTE/CCD 1217 digital camera operated by MetaMorph 4.6 software (Universal Imaging Corp., Downington, PA), edited by Adobe Photoshop 5.5, and printed by Epson Stylus 1200 photo printer. Negative controls were performed by omitting first antibody (Ab-1; MK-12-3), and by replacing first antibody with a preimmune rabbit serum (Ab-2). Image acquisition times were comparable to those of labeled samples. Positive control was performed using a rabbit serum raised against the synthetic peptide corresponding to a sequence of the C-terminal domain of bull sperm arylsulfatase A (GTGKSPRRTL), an enzyme present in the sperm tail principal piece and in the sperm acrosome. Two repeats were performed.
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Electron Microscopy
Spermatozoa in solution were processed with antibody Ab-1 with permeabilization in Triton X-100, as described for immunofluorescence, except that the fluorescently conjugated secondary antibodies were replaced by a 10-nm gold-conjugated goat anti-mouse IgG (Jackson Immunochemicals, West Grove, PA). The labeled cells were collected by centrifugation for 5 min at 350 xg, fixed in glutaraldehyde/paraformaldehyde fixative, postfixed in 1% osmium tetroxide, dehydrated, and embedded in Epon 812. Ultrathin sections were cut on a Sorvall MT-5000 ultramicrotome (Ivan Sorvall, Inc., Norwalk, CT), stained by uranyl acetate and lead citrate, and photographed in Phillips EM 300 electron microscope. Negatives were scanned by Umax Powerlook 3000 flat-bed scanner (Umax Technologies, Inc., Fremont, CA) and edited by Adobe Photoshop 5.5.
Western Blot Analysis
Twenty-five micrograms of total protein extracts from Percoll-separated sperm samples were subjected to one-dimensional gel electrophoresis. The procedures were described in detail previously [26]. Proteins separated by 12% SDS-PAGE were subsequently transferred to 0.2-µm nitrocellulose membranes (Sigma) using the Royal Genie electrophoretic blotter (Idea Scientific, Minneapolis, MN) at 350 mA for 5 h. Blots were preincubated in Tris-buffered-saline containing 0.05% Tween-20 and 5% nonfat dried milk, after which membranes were incubated overnight at 4°C with the antiprohibitin Ab-1 or Ab-2 (both 1:2000) or antiubiquitin MK-12-3 (1:2000). Membranes were incubated with the appropriate secondary antibody (goat anti-rabbit IgG-horseradish peroxidase [HRP] or goat anti-mouse IgG-HRP) for 2 h at room temperature, and antibody binding was detected by chemiluminescence (Amersham, Arlington Heights, IL). Negative controls were performed by the omission of first antibody and preabsorption of antibody MK-12-3 with purified bovine erythrocyte ubiquitin (Sigma).
Immunoprecipitation
Sperm extracts were precleared with protein-A agarose beads for 30 min at room temperature on a rotating wheel. Agarose beads were pelleted at 200 x g, and supernatant was removed. Subsequently, 3 µl of polyclonal antiprohibitin (Ab-2) or antitubulin antibodies were allowed to mix for 1 h, then 50 µl of protein-A agarose beads were added and mixed at 4°C overnight on a rotating wheel. Immunoprecipitated complexes were washed four times and dissociated by boiling in SDS-PAGE sample buffer. Western blot analysis next examined the protein solutions. Immunoblots were probed with antiubiquitin antibody, and antibody binding was subsequently revealed with chemiluminescence.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Prohibitin was found mainly on the mitochondrial membrane of bull and rhesus monkey spermatozoa, with some labeling in the matrix (Fig. 2, a–d) using colloidal gold immunocytochemistry. Prohibitin immunoreactivity was not observed on other parts of spermatozoa (Fig. 2, a–d) or in negative preparations with omission of first antibody (Fig. 2e). Localization of prohibitin to the sperm mitochondria was further supported by Western blot analyses of isolated bull sperm head and tail fractions. Both the expected 30-kDa prohibitin isoform and a 64-kDa isoform suggestive of tetraubiquitination were largely expressed in the sperm tail fraction and were absent from the sperm head fraction (Fig. 3a).
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Rat and bovine granulosa cells, which are known to contain high levels of prohibitin [26], displayed a single 30-kDa antiprohibitin immunoreactive band when probed with antiprohibitin antibody Ab-1 (Fig. 3a). To confirm that prohibitin in the sperm mitochondria is modified by ubiquitin, we reprobed the antiprohibitin blot shown in Figure 3b with antiubiquitin antibody MK-12-3, which was shown previously to cross-react with bull sperm ubiquitin [14]. Similar to the antiprohibitin immunoreactivity described above, the high-molecular-mass bands of approximately 64–200 kDa were revealed in the bull sperm lysates by this antiubiquitin antibody (Fig. 3c). The 64-kDa band and the bands migrating above the 119-kDa marker are consistent with polyubiquitin ladders. Accordingly, the 30-kDa prohibitin band did not bind antiubiquitin antibodies (Fig. 3b).
The indication that prohibitin is ubiquitinated in the sperm mitochondria was further supported by the results of immunoprecipitation experiments. When prohibitin immunocomplexes obtained from the bull sperm lysates were probed with antiubiquitin, a predominant 64-kDa band, as well as two other less prominent bands of approximately 55 and 46 kDa, were detected (Fig. 4, lane 1). The same extracts were also immunoprecipitated with antibodies against tubulin. Although a number of immunoreactive bands were detected, most of the antitubulin immunocomplexed proteins did not overlap with those detected in the antiprohibitin immunocomplexes (Fig. 4, lane 2). Tubulin and microtubule-associated proteins are the likely ubiquitinated substrates in the defective spermatozoa, which are known to be present in bull sperm pellets and lysates [14, 29], as also shown below.
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Consistent with our previous [2] and present immunolocalization studies, ubiquitinated prohibitin molecules were identified in the lysates of spermatids and testicular spermatozoa (Fig. 5a), and increased in the immotile sperm fraction after Percoll-gradient separation, probed with a polyclonal antiprohibitin antibody Ab-2 (Fig. 5b). Based on our previous studies of such defective/immotile sperm fractions, we proposed that the defective spermatozoa become ubiquitinated during epididymal passage [14]. The accumulation of high-molecular-weight prohibitin species in the defective sperm fractions (Fig. 5c) coincided with an increased number of surface-ubiquitinated defective spermatozoa [14, 16]. Such a putative mechanism of ubiquitin-dependent sperm quality control presumes that ubiquitin in its monomeric form (monoubiquitin) is secreted by the principal cells of epididymal epithelium via small exocytotic vesicles, apical blebs, or epididymosomes [13]. Ubiquitin molecules within the epididymal lumen bind predominantly to the defective spermatozoa showing signs of apoptotic process, such as altered plasma membrane [14, 16] and DNA fragmentation [29].
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Posttranslational modifications and rapid proteolysis of the sperm-mitochondrial prohibitin is justified in the context of early embryonic development. Prohibitin has been named for its antiproliferative activity [21]. Should such a protein be released from the degradation-prone sperm mitochondria after fertilization, it would likely impede the progression of embryonic development beyond the 1-cell stage. Whereas prohibitin may be present in the oocyte mitochondria [25], these are not subject to the dramatic changes seen in the mitochondria of the fertilizing spermatozoa. Previous studies suggest that loss of prohibitin results in destabilization of the mitochondrial membrane, which could facilitate abortive apoptosis in the defective preimplantation embryos [26, 35, 36].
Accumulation of the ubiquitinated prohibitin species in the immotile sperm fractions suggests and supports the existence of the ubiquitin-dependent mechanism for the recognition of defective spermatozoa in the epididymis [14]. According to this hypothesis, the ubiquitination of prohibitin could occur at three distinct steps in the life of a spermatozoon: in the testis, in the epididymis, and in the oocyte cytoplasm. The ubiquitin tag of sperm mitochondria, which is acquired during spermatogenesis, may be masked by the disulfide bond cross-linking during epididymal passage and could be exposed by the disulfide bond-reduction activities in the egg cytoplasm after fertilization. The exposed ubiquitin-prohibitin complexes may thus target sperm mitochondria toward the 26-S proteasome, where they are destroyed, or they may facilitate further polyubiquitination of other mitochondrial substrates. At the same time, the rapid degradation of sperm prohibitin protects the embryo from its antiproliferative effect. In defective spermatozoa, the surface ubiquitination occurs in the epididymis, which is consistent with the increase in the number of ubiquitinated substrates in the defective sperm fraction. Thus, two different pathways exist for sperm mitochondrial ubiquitination, depending on whether the spermatozoa are normal or defective. In the normal spermatozoa, prohibitin is ubiquitinated constitutively in the testis and is, perhaps, concealed by disulfide bond cross-linking to subsequently serve as a proteolytic signal after fertilization. In the defective spermatozoa, however, prohibitin and other mitochondrial membrane proteins may be further ubiquitinated, assuring their degradation during epididymal passage.
The identification of prohibitin as the ubiquitinated sperm mitochondrial protein has significant implications for the understanding of mitochondrial inheritance, evolution, assisted reproduction, and infertility: Constitutive ubiquitination of sperm mitochondrial proteins, such as prohibitin, suggests a role for ubiquitin and prohibitin in the regulation of mitochondrial inheritance in mammals. Ubiquitin-prohibitin complexes may provide the signal on which the oocyte's proteolytic degradation system specifically targets the sperm mitochondria, without affecting the maternal mitochondria. Intriguingly, both ubiquitin and prohibitin are highly conserved, and both are involved in the control of mitochondrial inheritance in the budding yeast [37, 38]. The evolutionary conservation and widespread expression of ubiquitin and prohibitin does not contradict the existence of such a mechanism in higher eukaryotes. Because the ubiquitin system is highly substrate specific [7, 30], this substrate specificity could actually provide a feasible explanation for the species-specificity observed for sperm mitochondrion degradation by the zygote [1, 4, 5]. Intriguingly, prohibitin has been proposed to act as a chaperone of other mitochondrial membrane proteins during their controlled degradation by a proteolysis-dependent quality control mechanism [18].
It is plausible that a failure or delay of sperm mitochondrion degradation by the oocyte could provide a window of opportunity for the survival of paternal mitochondria, resulting in the heteroplasmic propagation of both maternal and paternal mtDNA in the embryo. This scenario is applicable after assisted reproductive techniques, such as during in vitro fertilization procedures, in which early steps of fertilization and sperm disassembly are bypassed. However, heteroplasmy, or the propagation of donor cell mtDNA, has not been observed after injection of mitochondria from spermatogenic cells that are already tagged with ubiquitin or, to our knowledge, after human infertility treatment by intracytoplasmic sperm injection (for review, see [39]). In contrast, heteroplasmy has been demonstrated in animals cloned from somatic cells (e.g., [40]). Based on the present results, we can speculate that prohibitin in the mitochondrial membranes of donor cells used for nuclear transfer could be genetically modified (e.g., rendered destructible by the insertion of additional ubiquitination sites) to promote the timely destruction of such donor mitochondria and the elimination of foreign mitochondrial genes.
This report provides evidence that prohibitin is one of the components of the sperm mitochondrial membrane that undergoes ubiquitination during spermatogenesis. Further ubiquitination of prohibitin and other substrates may occur in the defective spermatozoa during epididymal passage. We have already demonstrated the usefulness of ubiquitin-based assays in the diagnostics of human male infertility [16, 17] and in the semen analysis in farm animals [29]. The present study thus provides additional evidence for a link between ubiquitin, prohibitin, sperm mitochondria, and male infertility. The most recently published study [17] showed that an increased unmasking of the ubiquitin-immunoreactive substrates occurs in the sperm mitochondria of infertile, teratospermic men with defects of the sperm tail and sperm mitochondrial sheath. It is therefore conceivable that differential ubiquitination of prohibitin, which is observed between the semen samples of fertile and infertile men, can be used for semen analysis in human infertility diagnostics.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Peter Sutovsky, Assistant Professor, University of Missouri-Columbia, S141 ASRC, 920 East Campus Drive, Columbia, MO 65211-5300. FAX: 573 884 5540; sutovskyp{at}missouri.edu
Received: 4 September 2002.
First decision: 25 September 2002.
Accepted: 5 March 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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