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Biology of Reproduction 63, 582-590 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Ubiquitinated Sperm Mitochondria, Selective Proteolysis, and the Regulation of Mitochondrial Inheritance in Mammalian Embryos1

Peter Sutovskya, Ricardo D. Morenoa, João Ramalho-Santosb, Tanja Dominkoa, Calvin Simerlya, and Gerald Schatten2,,b

a Oregon Regional Primate Research Center, Departments of Cell-Developmental Biology and Obstetrics-Gynecology, Oregon Health Sciences University, Beaverton, Oregon 97006 b Center for Neuroscience and Cell Biology of Coimbra, Department of Zoology, University of Coimbra, Portugal

ABSTRACT

The strictly maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) in mammals is a developmental paradox promoted by an unknown mechanism responsible for the destruction of the sperm mitochondria shortly after fertilization. We have recently reported that the sperm mitochondria are ubiquitinated inside the oocyte cytoplasm and later subjected to proteolysis during preimplantation development (P. Sutovsky et al., Nature 1999; 402:371–372). Here, we provide further evidence for this process by showing that the proteolytic destruction of bull sperm mitochondria inside cow egg cytoplasm depends upon the activity of the universal proteolytic marker, ubiquitin, and the lysosomal apparatus of the egg. Binding of ubiquitin to sperm mitochondria was visualized by monospecific antibodies throughout pronuclear development and during the first embryonic divisions. The recognition and disposal of the ubiquitinated sperm mitochondria was prevented by the microinjection of anti-ubiquitin antibodies and by the treatment of the fertilized zygotes with lysosomotropic agent ammonium chloride. The postfecundal ubiquitination of sperm mitochondria and their destruction was not seen in the hybrid embryos created using cow eggs and sperm of wild cattle, gaur, thus supporting the hypothesis that sperm mitochondrion destruction is species specific. The initial ligation of ubiquitin molecules to sperm mitochondrial membrane proteins, one of which could be prohibitin, occurs during spermatogenesis. Even though the ubiquitin cross-reactivity was transiently lost from the sperm mitochondria during epididymal passage, likely as a result of disulfide bond cross-linking, it was restored and amplified after fertilization. Ubiquitination therefore may represent a mechanism for the elimination of paternal mitochondria during fertilization. Our data have important implications for anthropology, treatment of mitochondrial disorders, and for the new methods of assisted procreation, such as cloning, oocyte cytoplasm donation, and intracytoplasmic sperm injection.

conceptus, fertilization, gametogenesis, sperm, spermatogenesis

INTRODUCTION

The strictly maternal inheritance of mitochondrial DNA (mtDNA) in mammals [1, 2] is a developmental paradox because the fertilizing spermatozoon introduces up to 100 functional mitochondria into the oocyte cytoplasm at fertilization. However, the mandatory destruction of sperm mitochondria appears to be an evolutionary and developmental advantage [3, 4], because the paternal mitochondria and their DNA (mtDNA) may be compromised by the action of reactive oxygen species encountered by the sperm during spermatogenesis and fertilization [5]. Although a number of studies supported the notion that sperm mitochondria are actively destroyed by the egg [6–9], the actual mechanism of this process is not known. Earlier claims that the sperm mitochondria disperse evenly throughout embryonic cytoplasm [10] and the misconception about sperm mitochondria not entering the egg (as scrutinized by Ankel-Simon and Cummins [3]), were overturned by new research. The dilution of paternal mtDNA in the maternal cytoplasmic genome [11] and the oxidative damage of sperm mitochondria during fertilization [12] were also implicated in this process but were not adequately supported by experimental data. The studies of intraspecific and interspecific mouse crosses suggest that a nuclear-encoded, proteinaceous component of the sperm mitochondrial membrane, rather than the sperm mtDNA itself, is recognized by the destruction mechanism residing in the egg cytoplasm [13]. In our previous studies, we observed that the elimination of the sperm mitochondrial sheath is somehow tied to the progression of the embryonic cell cycle, and we speculated that both processes are guided by ubiquitination [8], a process that is a universal substrate-tagging step in proteolysis. Although the role of ubiquitin in cell cycle regulation via the destruction of cyclin B at metaphase-anaphase transition is well known [14, 15], we reported very recently that the ubiquitination of sperm mitochondrial sheath can be visualized in fertilized cow and rhesus monkey eggs [16].

Ubiquitin is a universal marker of proteolysis and protein recycling, unique because of its versatility and high degree of evolutionary conservativism. The wide array of physiological stimuli and structure motifs in substrate proteins that regulate ubiquitination includes phosphorylation, the short destruction motifs in the protein molecule (e.g., cyclin's destruction box), endocytotic motifs on membrane receptors, short hydrophobic protein domains with lysine residues, and the so-called N-end rule that determines the half-life of proteins inside the cell (reviewed in [17–19]). In addition, the misfolding or denaturation of a protein molecule is enough to trigger its ubiquitination [17]. Ubiquitin is present in the cell cytoplasm as an inactive pool that has to be activated by ubiquitin-activating enzyme E1 and attached to the lysine residue of the substrate protein by a complex of ubiquitin carrier E2 and ubiquitin ligase E3 (reviewed in [20, 21]). A new ubiquitin ligase, E4, was recently described in yeast [22]. After the attachment of a single ubiquitin molecule to the Lys residue of the substrate, referred to as monoubiquitination, one or more molecules can be attached to this monoubiquitin and form a long tail of ubiquitin residues (hence the term polyubiquitination). When separated and labeled by SDS-PAGE and Western blotting using monospecific antibodies, the ubiquitinated substrates display a typical "ladder" of labeled bands separated from each other by the molecular weight of one ubiquitin molecule of 8.5 kDa, in addition to the expected single band of nonubiquitinated substrate protein. Although polyubiquitination is the most common way of targeting obsolete or damaged proteins toward the proteolytic apparatus, mono- and di-ubiquitination are often sufficient for this targeting step to occur [17]. The end point for ubiquitinated substrates is either in the lysosome or in a proteasome (reviewed in [23]).

In this study, we show that the sperm mitochondria acquire the ubiquitin tag during spermatogenesis, most likely at the secondary spermatocyte/round spermatid stages. The ubiquitinated epitopes are then masked by disulfide-bond cross-linking during epididymal passage, just to be exposed again by the egg-induced disulfide-bond reduction after fertilization. Subsequently, the increase in the binding of ubiquitin to the sperm mitochondrial sheath is observed inside the egg cytoplasm, suggesting further ubiquitination of the sperm mitochondrial substrates. To our knowledge, this is the first study providing experimental support for active proteolysis of the paternal mitochondria after animal fertilization, though both ubiquitin [24] and the process of proteolytic destruction [25] were recently implicated in the regulation of mitochondrial inheritance in yeast and protozoa, respectively.

MATERIALS AND METHODS

Gamete and Embryo Procedures

Motile bull sperm (ABS, DeForest, WI) were separated on a two-layer Percoll gradient and labeled with 400 nM MitoTracker Green FM (Molecular Probes Inc., Eugene, OR), a vital mitochondrial dye [8]. Oocytes were aspirated from ovaries obtained from a local abattoir, matured in vitro, fertilized with MitoTracker-tagged sperm at a final concentration of 1 x 106 sperm/ml, and incubated at 39°C in a humid atmosphere of 5% CO2 as described elsewhere [26]. Zygotic lysosomes were visualized by the 1-h incubation of the zona-free zygotes with the red-fluorescent, vital lysosomal dye LysoTracker DND 99 (1 µM concentration; Molecular Probes) and the blue DNA stain Hoechst 33342 (5 µg/ml; Boehringer Mannheim Corp., Indianapolis, IN). Gaur sperm, kindly donated by Dr. Naida Loskutoff (Henry Doorly Zoo, Omaha, NE), were tagged with MitoTracker Green FM and used at the concentration of 3 x 106 to fertilize domestic cow oocytes as described above. Antibody injections (antibody MK-12-3; see below) were performed essentially as described previously for lectin injections into bovine eggs [27]. Testicular spermatogenic cells and epididymal spermatozoa were isolated by mincing the fresh testicular and epididymal tissues, respectively. In some experiments, the resultant testicular cell suspensions were incubated for 15 min with 400 nM MitoTracker CMTM Ros (Molecular Probes) and fixed for immunofluorescence. Treatment of bull sperm with DTT dissolved in alkaline KMT medium was performed as described elsewhere [28]. Reducing treatment was not necessary to detect ubiquitin in human, rhesus, and mouse sperm, known for their lesser degree of disulfide-bond cross-linking. Sperm heads and tails were separated by sonication, followed by centrifugation on a sucrose gradient as described by others [29, 30]. Parthenogenetic activation with ionomycin and 6-DMAP was performed as described previously [28]. Rhesus in vitro fertilization (IVF) was performed as described previously [31, 32].

Immunofluorescence

Spermatozoa were attached to the poly-L-lysine-coated coverslips, fixed in 2% formaldehyde in 0.1 M PBS, and permeabilized in 0.1% Triton-X 100 in 0.1 M PBS. Ubiquitin in the ejaculated and epididymal bull sperm was detected by a sequential, three-step incubation with the bovine erythrocyte ubiquitin-specific antibody MK-12-3 (clone 2C5; MBL Co. Ltd., Nagoya, Japan; diluted 1:50), biotinylated anti-mouse IgG (Jackson Immunochemicals; diluted 1:80), and FITC-conjugated streptavidin (Jackson Immunochemicals; diluted 1:100). The DNA was counterstained with DAPI (Molecular Probes). Ubiquitin in bovine and mouse zygotes was detected using the antibody MK-12-3 as described above, except that the primary antibody was followed directly by an FITC-conjugated anti-mouse IgG (Zymed Labs, Burlingame, CA). Other anti-ubiquitin antibodies (AB 1690 from Chemicon International, Inc., Temecula, CA; and UCBA798/R5H from Accurate Chemical & Scientific Corp., Westbury, NY) were used in bovines with similar results (data not shown). Ubiquitin labeling in rhesus zygotes and primate sperm was performed with monoclonal antibody KM691 against human recombinant ubiquitin (Kamiya Biomedical Co., Seattle, WA) by a two-step labeling process with appropriate fluorescent conjugates. A sampler of 17 proteasome-specific antibodies (Affinity Research Products, Mamhead, UK) was used to examine the association of 26-S-proteasome with the incorporated sperm mitochondria, but no positive results were obtained (not shown). The samples were examined on a Zeiss Axiophot microscope with an RTE/CCD 1217 camera (Princeton Instruments, Inc., Trenton, NJ) operated by MetaMorph software (Universal Imaging Corp., West Chester, PA). Images were archived on recordable CDs and printed on Sony UP-D-8800 video printer by using Adobe Photoshop 4.0 software (Adobe Systems Inc., Mountain View, CA).

Western Blotting

Sperm were lysed in 0.5 ml of a sample buffer (1 M NaCl; 20 mM imidazole; 1 mM EDTA; 5 mM benzamidine HCl; 5 mg/ml leupeptin; 1 mg/ml pepstatin A; and 1% Triton-X 100; pH 6.0), run on a 10% SDS-PAGE under reducing and denaturant conditions (except for the controls in nonreducing conditions), transferred to Hybond sheets (Amersham, Buckinghamshire, UK) using a dry system at 0.8 mA per cm2, blocked with 2% PBS-BSA for 1 h, incubated overnight at 4°C with the mouse monoclonal antibody MK-12-3 against bovine erythrocyte ubiquitin (MBL; diluted 1:200), washed, and incubated with goat anti-mouse IgG/horseradish peroxidase (Sigma, St. Louis, MO; diluted 1:2000). The bands were developed using the ECLplus system (Amersham) following the manufacturer's directions. Protein concentration was determined by Pierce bicinchoninic acid method (Pierce, Rockford, IL) according to the manufacturer's specifications. Negative controls included omission of the first antibody, preimmune serum-antibody replacement, and preabsorption of antibody MK-12-3 with purified bovine erythrocyte ubiquitin (Sigma). Western blotting and immunofluorescence of prohibitin was performed with antibodies RB-292/Ab-2 and MS-261/Ab-1 (both from NeoMarkers, Inc., Union City, CA) with similar results (only RB-292 data are shown). In some experiments, the immunostained gels were stripped with glycine (pH 2.7, 2 h at room temperature) and reprobed with anti-ubiquitin antibody MK-12-3 (Fig. 5A, lane 2).



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  		FIG. 5. Detection of prohibitin, a candidate-ubiquitin substrate, in bull sperm mitochondria. A) Western blotting of sperm extracts with anti-prohibitin antibody RB-292 (lane 1). Blot was stripped and reprobed with anti-ubiquitin antibody MK-12-3 (lane 2). Note that the double, high-molecular mass band in lane 1, but not the common, 30-kDa prohibitin band (asterisk), overlaps (double-headed arrows) with ubiquitinated bands in lane 2. B) Immunofluorescent detection of prohibitin in bull sperm pretreated with 10 mM DTT. Scale bar = 5 µM

Electron Microscopy

Bovine zygotes were fixed for 90 min in 2.5% glutaraldehyde and 0.6% paraformaldehyde in 0.25 M cacodylate buffer (pH 7.2), washed in 0.1 M cacodylate buffer containing 0.2 M sucrose, and postfixed for 1 h in 1% osmium tetroxide. Following the dehydration by an ascending ethanol series (30–100%), the samples were infiltrated in a mixture of propylene oxide and Polybed 812 (Polyscience, Warrington, PA) and were embedded in PolyBed 812. Ultrathin sections were cut on a Sorval MT2B ultramicrotome, placed on 100 MESH copper grids, stained with uranyl acetate and lead citrate, and photographed on a Phillips 300 electron microscope. Negatives were scanned by the Umax Sprint Scan flatbed scanner, recorded onto a Jaz disk, and printed by using Adobe Photoshop 4.0 software.

RESULTS

Sperm Mitochondrial Ubiquitination after Fertilization

To monitor the ubiquitination of paternal mitochondria after fertilization, we used bull sperm prelabeled with the vital fluorescent probe MitoTracker Green FM [8] for IVF of bovine oocytes. The resultant zygotes were labeled with red-fluorescent conjugates of anti-ubiquitin antibodies and blue-fluorescent DNA stain DAPI. Although recently incorporated sperm with swollen nuclei did not display any labeling (Fig. 1A), ubiquitin was detected on the incorporated sperm mitochondria at the late stages of pronuclear development (Fig. 1, B and C). The intensity of ubiquitin labeling of the mitochondrial sheath increased with the progress of pronuclear apposition (Fig. 1C; 16 h post-insemination [p.i.]) and culminated during first mitotic division (Fig. 1, D and E). At the metaphase of first mitosis, the ubiquitinated mitochondrial sheath was found at one spindle pole (Fig. 1D; 24 h p.i.), and distinct sequestration of ubiquitin, similar to that seen in the meiotic spindles of rhesus (see Fig. 1M) and cow (not shown) oocytes, was seen. Deformed mitochondrial sheath was found in only one of the two blastomeres after the completion of first embryonic mitosis (Fig. 1, F and G; 40 h p.i.). The sperm mitochondria typically disappeared at the four- to eight-cell stage of preimplantation development (Fig. 1H; 48 h p.i.). Oocyte mitochondria did not contain ubiquitin even after parthenogenetic activation (Fig. 1, I and I'). The antibodies (see Materials and Methods) against bovine erythrocyte ubiquitin also cross-reacted with the sperm tail midpiece inside mouse zygotes (Fig. 1J), and the antibodies against recombinant human ubiquitin detected incorporated sperm mitochondria in rhesus monkey zygotes (Fig. 1, K–N). Similar to the case of bovine zygotes, ubiquitin was not detected on rhesus sperm mitchondria at the early stages after incorporation (Fig. 1L), whereas it was present throughout subsequent pronuclear development (Fig. 1, K–N). The ubiquitination of the second meiotic spindle and its midbody was regularly found in early rhesus pronuclear zygotes (Fig. 1M) and is consistent with the known role of ubiquitin in cell cycle regulation [15]. Red background labeling in the egg cytoplasm was most likely due to the ubiquitinated cytoplasmic substrates and/or to the presence of unconjugated ubiquitin in the cytosol.



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  		FIG. 1. Ubiquitination of the sperm mitochondria inside the oocyte cytoplasm in bovine (A–J), mouse (K), and rhesus monkey (L). Ubiquitin (red) was detected with monospecific antibodies in the oocytes fertilized by the sperm preincubated with a vital fluorescent probe MitoTracker Green FM (green; A–G; I, K–N) in bovine zygotes shortly after sperm incorporation (A; no ubiquitin is found on the mitochondrial sheath at this stage); during pronuclear apposition (B, C); at the metaphase (D; note the sequestration of ubiquitin in the metaphase spindle) and anaphase (E) of the first mitosis; in the interphase, two cell embryos (F, G); and in an eight-cell embryo in which the sperm mitochondria were not detected (H). The MitoTracker-labeled mitochondria of parthenogenetically activated eggs (I) did not contain ubiquitin (I'), whereas it was detected on the axonemal midpiece in pronuclear mouse (J; arrows) and rhesus monkey (K–N) embryos. Note the labeling of second meiotic spindle in M. F, M) Arrows point to sperm mitochondrial sheath. The presence of unconjugated ubiquitin and/or ubiquitinated substrates in the cytosol most likely accounts for the red background labeling in the egg cytoplasm. Scale bars: F and H = 15 µm; I and I' = 2 µm; J = 5 µm; N and M = 10 µm. Magnification for A, x2000; B, x1500; C–E, x1000; G, x1000; J, x500; K, x750; L, x250.

The ultimate mechanism for sperm mitochondrial destruction after ubiquitination might involve proteasomes, lysosomes, or both. Although the ubiquitination pathway is typically linked to proteasome-mediated proteolysis [20], none of 17 proteasome-specific antibodies (Affinity) detected proteasomes in association with the incorporated bovine sperm mitochondria (not shown). In contrast, electron-microscopic analysis revealed abundant lysosomal vacuoles and multivesiculated bodies surrounding the incorporated sperm mitochondrial sheaths (Fig. 2, A–E). This finding was further supported by the labeling of fertilized eggs with the vital lysosomal dye LysoTracker DND 99 (Fig. 2F). Ten millimoles of NH4Cl, a lysosomotropic agent, prevented the destruction of the sperm mitochondria in all eight-cell embryos screened in two experiments (Fig. 2G) when the oocytes were treated with the compound 20 h after insemination (Fig. 2H; a total of 214 embryos were examined). Clumped remnants of the fluorescent mitochondrial membranes were found in 25% of control eight-cell embryos, as compared with the sperm tails found in 100% of embryos treated with NH4Cl (Fig. 2H).



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  		FIG. 2. Destruction of the sperm tail mitochondria in homologous bovine embryos (A–K) and in interspecies crosses between domestic cows and gaur bulls (L–N). Electron micrographs (A–E) show an intact sperm tail (A) at the early stage of zygotic development (approximately 8 h after fertilization) and the disintegrating sperm tails (B–E) surrounded by lysosomes (arrows) and multivesiculated bodies (arrowheads) 20 h after fertilization. Lysosomes labeled with a vital probe LysoTracker (arrowheads) are associated with sperm mitochondria inside the zygotic cytoplasm (F). Persistence of an intact sperm mitochondrial sheath (arrow) in an eight-cell embryo that was developing in the presence of 10 mM lysosome antagonist NH4Cl (G) and the diagram illustrating the persistence of sperm tails in bovine embryos cultured in presence of this agent (H, {Sigma}n = 214). The rate of one-cell embryos containing the sperm tail is less than 100% because this group includes the oocytes that remained unfertilized 48 h after insemination. Sperm mitochondrial sheaths (arrows) were present in metaphase II-arrested zygotes (I, J) fertilized 1 h after microinjection of anti-ubiquitin antibody MK-12-3 and fixed 50 h after fertilization (a stage that corresponds with 8- to 16-cell embryos under control conditions), whereas the control, sham-injected embryos (K) were free of sperm mitochondria. Sperm mitochondria (arrows) persisted in the cytoplasm of the embryos from interspecies crosses between domestic cattle and gaur (L–N). L, M, and N) Two-, four-, and eight-cell embryos, respectively. Scale bars: A = 1 µm; B = 500 nm; C–E = 200 nm; G = 15 µm; N, I, and K–M = 20 µm. F) Magnification x1200. J) Magnification x1000

Microinjection of anti-ubiquitin antibody MK-12-3 into cow oocytes prior to insemination was attempted in order to block sperm mitochondrion destruction. As expected, these microinjections blocked the exit of fertilized oocytes from metaphase II arrest, presumably by blocking cyclin destruction by ubiquitin [15]. In most cases, the antibody injection also prevented the destruction of sperm mitochondria, resulting in triploid metaphase plates flanked by a sperm mitochondrial sheath seen as late as 40 h p.i. (Fig. 2, I and J), whereas the sham-injected control oocytes reached the four- to eight-cell stage and lost the sperm mitochondria (Fig. 2K). It is not clear, however, whether this block of mitochondrial destruction was caused directly by the binding of the injected antibodies to the ubiquitinated epitopes in the sperm mitochondria or indirectly by the block of cell cycle progression that could control the activation of ubiquitin and lysosomal machinery during fertilization. Remarkably, paternal mitochondria and mtDNA persist in the interspecies crosses, a phenomenon documented previously in murine hybrids [13, 33]. When oocytes from the domestic cow (Bos taurus) were inseminated with the sperm of the Asian cattle, gaur (Bos gaurus), ubiquitin was not detected on the gaur sperm mitochondria incorporated within the resulting hybrid embryos, despite the fact that the antibody MK-12-3 does cross-react with gaur sperm ubiquitin (not shown). Furthermore, the nearly intact sperm tails were found in two-cell (Fig. 2N), four-cell (Fig. 2M), and 8- to 16-cell hybrid embryos (Fig. 2N). Our preliminary data confirmed these observations by detecting gaur mtDNA in hybrid eight-cell embryos using a highly sensitive, paternal mtDNA-specific nested PCR (unpublished data).

Sperm Mitochondrial Ubiquitination During Spermatogenesis

The ubiquitin signal appears to be present in bull sperm mitochondria prior to IVF, though its intensity is so low that it can only be detected by fluorescence amplification with avidin-biotin conjugates (see Materials and Methods) or by Western blotting. Numerous ubiquitinated bands were revealed by Western blot analysis of motile sperm after disulfide bond reduction (Fig. 3A), whereas only a few bands were present under nonreducing conditions (see Fig. 3C). Sperm tails and heads separated by sonication displayed some common and some distinct ubiquitinated bands (Fig. 3B), suggesting that some of the ubiquitinated substrates in the sperm head, perhaps histones H2A [34, 35] and histone H3 [36], differ from those in the sperm tail. Preabsorption of the ubiquitin antibody with purified bovine erythrocyte ubiquitin eliminated all cross-reactivity in Western blots of bull sperm extracts (Fig. 3C). By immunofluorescence, sperm mitochondrial ubiquitination was detected by biotin-streptavidin amplification in the sperm from caput epididymis (Fig. 3E) but not in those from corpus and cauda epididymis (not shown). Positive results (Fig. 3D) were also obtained after the decondensation of ejaculated sperm in vitro with 10 mM dithiothreitol (DTT; [28, 37]), suggesting that sperm mitochondrial ubiquitination begins in the male reproductive tract and that the ubiquitinated epitopes are masked by disulfide bond cross-linking that occurs during epididymal passage [38]. In addition to bull sperm mitochondria, ubiquitin was detected by immunofluorescence in the mitochondrial sheath of mouse (Fig. 3F), rhesus monkey (Fig. 3G), and human sperm (Fig. 3H). Reducing treatment was not necessary to detect ubiquitin in sperm of those species. In rhesus sperm, the most prominent labeling was seen in the connecting piece, where the sperm centriole is found. This finding is consistent with the known association of ubiquitin with the somatic cell centrosome [39], as well as with centrosome reduction during spermatogenesis (reviewed in [40]).



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  		FIG. 3. Detection of the ubiquitinated substrates in bull (A–E), mouse (F), rhesus (G), and human (H) sperm. Western blot of the motile bull sperm fraction with anti-ubiquitin antibody MK-12-3 (A) shows several ubiquitinated substrates. Western blot of sperm heads (B, lane 1) and tails (B, lane 2) isolated by sonication of bull sperm demonstrates the prominent ubiquitinated bands of ~80 kDa and ~50 kDa that are predominantly found in the head and tail fractions, respectively. Some of the ubiquitinated substrates may have been lost or reduced during separation and fractionation, whereas cross-contamination cannot be ruled out completely. Control blot for antibody specificity (C), using whole sperm samples run under nonreducing (lanes 2, 4) and reducing (lanes 3, 5) conditions and antibody MK-12-3 preabsorbed with purified bovine erythrocyte ubiquitin (C; lanes 2, 3) or untreated MK-12-3 (C; lanes 4, 5). Lane 1 shows molecular weight markers. Detection of ubiquitin (green) with antibody MK-12-3 amplified by streptavidin-biotin reaction in the ejaculated bull sperm treated with 10 mM DTT (D) and in the untreated sperm from caput epididymis (E) are shown. Detection of ubiquitin by a simple, two-step procedure without reducing treatment, is shown in mouse (F), rhesus (G), and human (H) sperm. Nuclear DNA in all sperm samples was counterstained with DAPI. Bars = 5 µm

During spermatogenesis, mitochondrial ubiquitination was detected using biotin-streptavidin amplification as early as at the secondary spermatocyte stage (Fig. 4A), then in round spermatids (Fig. 4B), elongated spermatids (Fig. 4C), and in fully differentiated testicular spermatozoa of bull (Fig. 4D). Ubiquitinated mitochondria were also found in the sperm residual bodies (Fig. 4E). At the concomitant stages of spermatogenesis, we were able to detect the ubiquitin-conjugating enzyme E2, which first appears as a combination of dot-like and diffuse cytoplasmic stains around the mitochondria in secondary spermatocytes (Fig. 4F) and later migrates into the acrosomal cap in round and elongating spermatids (Fig. 4, G and H). Enzyme E2 is no longer detectable in mature testicular spermatozoa (Fig. 4I), although it can still be detected in the residual bodies (Fig. 4J).



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  		FIG. 4. Ubiquitination of the mitochondria in bovine spermatogenic cell lineage. A–E) Colocalization of ubiquitin (green) with the mitochondria (red) in secondary spermatocytes (A), round spermatids (B), elongating spermatids (C), fully differentiated testicular spermatozoa (D), and rejected residual bodies (E). F–J) Localization of the ubiquitin-conjugating enzyme E2 in the corresponding stages of bull spermatogenesis. E2-Labeling (green) complements, rather than overlaps with, the mitochondrial signal in the cytoplasm of a secondary spermatocyte (F), and in the residual cytoplasmic droplet (J). Note the sequestration of E2 in the developing acrosome of the round (G) and elongating (H) spermatids, and the complete loss of E2 cross-reactivity from the fully differentiated testicular spermatozoa (I). Spermatogenic cells were isolated, labeled with MitoTracker CMTM Ros (red), and processed for immunofluorescence with the anti-ubiquitin antibody MK-12-3 (green in A–E) amplified by avidin-biotin reaction or with the anti-E2 antibody (green in F–J) by a simple two-step procedure. DNA (blue) was stained with DAPI; bar = 5 µm. Ubiquitination of the mitochondria is shown in bull spermatogenic cells. Colocalization of ubiquitin (green) with the mitochondria (red) in secondary spermatocytes (A), round spermatids (B), elongating spermatids (C), fully differentiated testicular spermatozoa (D), and rejected cytoplasmic droplets with discarded mitochondria (E). Bars = 5 µm

We have identified one of the potential ubiquitinated sperm mitochondrial substrates as prohibitin, an evolutionarily conserved, 30-kDa integral protein of inner mitochondrial membrane [41]. In Western blots of SDS-reduced bull sperm extracts, two different anti-prohibitin antibodies cross-reacted with the expected band of ~30 kDa, as well as with two higher molecular weight bands (Fig. 5A, lane 1). These two bands appear to be ubiquitinated, as demonstrated by stripping and relabeling a prohibitin gel with anti-ubiquitin antibodies (Fig. 5A, lane 2). Similar to the case in ubiquitin, we were only able to detect prohibitin in the mitochondria of mature bull sperm after disulfide bond reduction with 10 mM DTT (Fig. 5B).

DISCUSSION

In this study, we demonstrate that ubiquitination of sperm mitochondria is involved in the control of mitochondrial inheritance in mammals. Sperm mitochondria in rat [7], bovine [8], and mouse [13, 42] embryos are destroyed prior to or during the third embryonic cleavage, and the ubiquitination of the sperm mitochondria during spermatogenesis in bovine, mouse, rhesus and humans (this study) probably marks these mitochondria for selective recognition and destruction by the proteolytic machinery of the egg cytoplasm. The turnover of other egg substrates, including the components of pronuclear nuclear envelope [27], could be affected by ubiquitination, thus accounting for the patches of cytoplasmic ubiquitin-labeling seen in most fertilized eggs. Our data offer a feasible explanation for maternal mtDNA inheritance in mammals. The alternative theory of mtDNA dilution (discussed by Ankel-Simons and Cummins [3]), based on the high theoretic ratio of paternal versus maternal mitochondria (~1:1000; see [11]), does not explain the transmission of paternal mtDNA occurring in interspecies crosses in mice [13, 33, 43] and the persistence of sperm mitochondria in hybrid cattle embryos (Fig. 2, L–N). The oxidative-damage theory [12] suggests that the sperm mitochondria are recognized by oocyte cytoplasm because of the oxidative damage they suffer during passage through the female genital tract. Even though mtDNA is clearly susceptible to mutagenesis by reactive oxygen species [5, 44], such oxidative damage is minimal during IVF, and thus, the elimination of paternal mitochondria after IVF is unexpected with regard to this hypothesis. Species specificity of sperm mitochondrion destruction may reside in the amino acid sequence of ubiquitin, as shown in yeast, wherein the substitution of single-lysine residue (Lys-63) for arginine effectively derailed both ubiquitination and proper mitochondrial transmission to buds [24]. Similarly, the ubiquitin-activating and -conjugating enzymes (E1-E4 or Ubcs) in the egg cytoplasm may not be compatible across mammalian species and thus may fail to recognize and recycle foreign sperm mitochondria in the mouse and bovine interspecies hybrids. Similar to sperm mitochondrial elimination, ubiquitination is involved in the removal of mitochondria from differentiating red blood cells [45], and mitochondrial inheritance in yeast relies on ubiquitin and ubiquitin ligase Rsp5p for normal mitochondrial transmission to buds [24]. A recent study in yeast further supports this theory by showing the necessity of a proteolytic event for normal mitochondrial inheritance [25].

Our results suggest a pathway in which the paternal mitochondria are already tagged with ubiquitin in haploid spermatocytes, with the bulk of the mitochondria lost to the residual bodies that eventually become reabsorbed by Sertoli cells. The remaining sperm mitochondria form the sperm's mitochondrial sheath and retain their nascent, perhaps mono-ubiquitin or di-ubiquitin tag, on one or more mitochondrial membrane proteins. This tag becomes undetectable as disulfide bond cross-linking of mitochondrial membranes occurs during the transit through the male reproductive tract. The ubiquitination of the sperm mitochondria is supported by our direct observations of ubiquitin colocalization with mitochondria in spermatogenic cell lineage. In addition, expression of ubiquitin at the round spermatid stage was previously shown in roosters [46], humans and mice [47], and the ubiquitin-activating enzyme E1 [48], and ubiquitin carriers E2 (this study; [49]) and UBC 4 [50, 51] are active during mammalian spermatogenesis. After the incorporation into oocyte cytoplasm during fertilization, perhaps concordant with the glutathione-induced reduction of the sperm disulfide bonds [37, 52], the sperm mitochondrial ubiquitination is again detectable and appears to increase in intensity. The paternal mitochondria may therefore be distinguished from the maternal mitochondria by ubiquitination, and both the ubiquitin and the MitoTracker signals are lost by the eight-cell stage in most embryos. Although the ubiquitin tag appears enhanced after sperm incorporation, the approaches employed cannot determine whether this is the result of polyubiquitination of the substrates previously mono- or di-ubiquitinated during spermatogenesis or whether additional sperm epitopes are ubiquitinated. Alternatively, the conformational changes of the ubiquitinated sperm epitopes inside the egg cytoplasm (e.g., disulfide bond reduction) alone could account for the increased cross-reactivity of anti-ubiquitin antibodies with the incorporated mitochondrial sheath.

Studies of murine interspecies crosses established that the proteinaceous component(s) of the sperm mitochondrial membrane, rather than sperm mtDNA itself, are recognized by egg cytoplasm during the elimination of paternal mtDNA [13]. By back-crossing the hybrids of domestic mouse and wild mice Mus spretus, these authors showed that males carrying sperm mitochondria with mtDNA of M. spretus and membrane proteins of domestic mouse were not able to propagate their mtDNA when back-crossed with domestic mice. Our studies suggest that prohibitin, an evolutionarily conserved, 30-kDa integral protein of inner mitochondrial membrane [53], is among the possible substrates for ubiquitination in sperm mitochondria during spermatogenesis and after fertilization. The 30-kDa isoform of prohibitin disappears at the round spermatid stage in the rat [41], perhaps because of the loss of cross-reactivity caused by posttranslational modification such as ubiquitination. Though the anti-prohibitin antibodies used in our studies recognized the 30-kDa prohibitin in mature bull sperm, they were also able to detect two additional bands in the approximate 47- to 50-kDa range. These bands also cross-reacted with ubiquitin, and if our calculations are correct, the lower band of ~47 kDa could be the di-ubiquitinated (ligation of two ubiquitin molecules, 8.5 kDa each, per molecule of prohibitin) isoform of 30-kDa prohibitin. The upper band of ~49–50 kDa could be a result of phosphorylation, which is known to play an important role in the process of ubiquitin-substrate targeting (reviewed in [17]). If this is indeed true, the partial amino acid sequence of two upper bands should yield two additional amino-termini and sequence motifs homologous to the known ubiquitin sequence. Similar to ubiquitin, prohibitin in ejaculated bull sperm is only detectable by immunofluorescence after DTT treatment and by Western blotting in reducing conditions. In vivo, the reduction of disulfide bonds, known to destabilize the sperm mitochondrial sheath [28] and to cause the rearrangement of the inner and outer mitochondrial membrane domains in vitro [54], could cause the rearrangement of the sperm mitochondrial membranes and the exposure of ubiquitin-tagged prohibitin on it to the egg cytoplasm. Depletion of the oocyte's intrinsic reducing tripeptide glutathione retarded the remodeling of sperm mitochondrial sheath occurring at an early stage of fertilization in bovines [37]. An additional reason for the ubiquitination and destruction of prohibitin-carrying sperm mitochondria is the ability of prohibitin to block ("prohibit"; hence the name of this protein) S-phase entry [55]. The release of prohibitin into egg cytoplasm could collide with subsequent pronuclear development and embryonic cleavage. It is noteworthy that prohibitin, similar to ubiquitin, has been implicated in a pathway that controls mitochondrial inheritance in yeast [56]. Other possible substrates for ubiquitination include the disulfide-bond cross-linked proteins in sperm mitochondrial membranes [57], the selenium-rich protein of sperm mitochondrial capsule (SMCP; [58, 59]), and the new kinesin light-chain protein (KLCt) that seems to provide the link between sperm mitochondria and axonemal outer dense fibers [60].

Characterization of the ubiquitination pathways in the testis and in the zygote may have important implications for the prevention and treatment of hereditary mitochondrial diseases, as well as for the new methods of assisted procreation, such as cloning, oocyte cytoplasm donations, intracytoplasmic sperm injection (ICSI), and round spermatid injection (ROSI). By each of the above assisted reproductive technologies (ART), foreign mitochondria are introduced into the oocyte cytoplasm, where they may reproduce autonomously unless they are recognized and properly processed by the egg's cytoplasmic machinery. Although the first reports on mtDNA inheritance after ICSI suggest that such human embryos can properly recognize and process the mtDNA of the injected sperm [61, 62], other studies [63] show that even normal, let alone multipronuclear embryos generated by ART, may promote the persistence of paternal mtDNA in their cytoplasm. It is possible that the intact plasma membrane on the surface of the microinjected sperm can delay the processing of the sperm axoneme in a manner similar to the aberrant remodeling of the sperm nucleus after ICSI [31, 32]. In a limited extent, human sperm mitochondria can indeed survive and repopulate other cells [64]. Furthermore, the diminished reducing potential and the immaturity of lysosomal machinery may occur in the superovulated eggs harvested from infertility patients and may further jeopardize normal development and mtDNA inheritance after ART. In ROSI, the injected round spermatids should carry mitochondria that are already imprinted with ubiquitin, and the initial studies in mice indeed suggest that their spermatid mitochondria can be recognized and eliminated by egg cytoplasm [65]. Similarly, spermatocyte mitochondria and mtDNA became undetectable when spermatocyte cytoplasts were injected into mouse pronuclear embryos [66]. With regard to cloning, it was suggested that the mismatched mitochondrial gene products may contribute to the high mortality of cloned offspring [67]. High postnatal mortality was indeed reported in calves cloned from a somatic cell [68–70]. Although Dolly [71] and calves from some cloning trials [72] reportedly do not carry donor cell mtDNA, other teams have created animals with a variable and often very high degree of heteroplasmy due to the presence of donor cells' mtDNA [73–76]. Although it is possible that some cloning protocols inadvertently prevent the survival of mitochondria from donor cells, it should also be cautioned that there may be differences in the sensitivity and controls of the PCR-based approaches used to detect such foreign mtDNAs. Furthermore, such analyses were carried out using a limited number of tissue types, whereas the foreign mtDNAs are known to be distributed unevenly among individual tissues of heteroplasmic organisms [77]. Foreign mitochondria are also introduced into human embryos by cytoplasmic transfer therapy of aging oocytes, and the first babies born after cytoplasmic donation evidently carry foreign mtDNA from donor oocyte cytoplasm [78]. The consequences of such "homologous" (egg/egg) heteroplasmy are yet to be determined.

Finally, our data on the proteolysis of paternal mitochondria may influence the perception of mtDNA-based phylogenetic studies and the ongoing debate [79] about the possibility of recombination between human sperm and egg mtDNA [80, 81]. Although our proposed ubiquitin-based proteolytic mechanism for the elimination of sperm mitochondria (and mtDNA) after fertilization indirectly supports the conservative view that there is or was no recombination among human mtDNA lines, skeptics may argue that any specific cellular mechanism has inherent potential for a failure. Consequently, we cannot rule out that under certain conditions, some sperm mitochondria may escape proteolytic death and intermix with egg mitochondria.

ACKNOWLEDGMENTS

We thank Dr. Naida Loskutoff (Henry Doorly Zoo, Omaha, NE) for the gift of gaur sperm samples; Dr. Richard Oko (Queens University, Kingston, ON) for additional Western blotting controls; N. Duncan, M. Emme, C. Martinovich, B. McVay, D. Takahashi, and H. Wilson for technical and clerical support; and M. Webb for the preparation of samples for electron microscopy. Inspirational discussions with Drs. Jim Cummins, Richard Oko, and Justin St. John are gratefully acknowledged.

FOOTNOTES

First decision: 27 January 2000.

1 This work was supported by the New Investigator Award/Animal Reproductive Efficiency Grant from USDA-NRI to P.S., by grants from NIH and USDA to G.S., and by a Fogarty International Research Fellowship from NIH to R.M. J.R.-S. received financial support from FCT (Praxis XXI postdoctoral fellowship), Portugal. The ORPRC infrastructure is supported as an NIH/NCRR regional primate research center. Back

2 Correspondence: Gerald Schatten, Oregon Regional Primate Research Center, 505 NW 185th Ave., Beaverton, OR 97006. FAX: 503 614 3725; schatten{at}ohsu.edu Back

Accepted: March 23, 2000.

Received: December 7, 1999.

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