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Embryo |
Departments of Animal Science3
Obstetrics and Gynecology,4 University of Missouri-Columbia, Columbia, Missouri 65211-5300
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, mitochondria, mitochondrial inheritance, proteasome, sperm, ubiquitin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The omnipresent ubiquitin system has been implicated in various steps of gametogenesis and fertilization (reviewed by Bebington et al. [4] and Sutovsky [5]), but reproductive ubiquitin research still lacks the attention it deserves. Our previous studies tied the ubiquitin system to the selective degradation of the sperm mitochondria inside the fertilized egg [6–8], a hypothesis providing feasible explanation for the maternal inheritance of mammalian mtDNA [9]. Initial evidence from those studies indicated the participation of the lysosomal pathway in the degradation of the ubiquitinated sperm mitochondria within the oocyte cytoplasm. Possible involvement of the proteasomal pathway of ubiquitin-mediated proteolysis was not examined in detail. The presence of proteasomal subunits in the sperm tail midpiece [10, 11], a site from which the sperm centriole is released during fertilization [6, 12], justified the suggestion that the ubiquitin-proteasome system facilitates the unmasking of the sperm centriole and sperm aster formation after mammalian fertilization [11]. Proteasomal subunits, component 2 and the zeta chain, were recognized by the antisperm antibodies in patients suffering from autoimmune fertility disorders [13]. Multiple proteasomal subunits were detected in the human [11, 14], ascidian [15, 16], and sea urchin [17] sperm acrosome. Fertilization was inhibited by anti-ubiquitin and anti-proteasome antibodies in ascidians [15] and pig (unpublished data). Finally, small peptides designed to target proteasomal activity, but not other protease activities, prevent fertilization in sea urchins [17], ascidians [16], mice [18], and pigs (this study).
Porcine in vitro fertilization (IVF; [19]) is an ideal system to study ubiquitin-dependent fertilization events. Unlike rodents, the porcine model provides an abundance of available gametes and a much larger time frame of oocyte maturation (42 h), fertilization (up to 6–8 h post-insemination), and time from fertilization to first embryonic cleavage (24–30 h). Moreover, the degradation of the sperm mitochondria inside the fertilized porcine egg occurs much faster than in the similarly convenient bovine and primate models. In the latter models, sperm mitochondria are still observed in 2–4 cell embryos [6–8], whereas the present study shows that the sperm mitochondrial degradation is largely accomplished prior to the first embryonic division in the pig. Taking full advantage of the porcine IVF system, the present study provides the first evidence for the role of the proteasome branch of the ubiquitin system in mammalian fertilization and mitochondrial inheritance.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries from prepubertal gilts were collected at a local abattoir and transported at 25°C to the laboratory in 0.9% saline containing 75 µg/ml penicillin-G, 50 µg/ml streptomycin sulfate, and 25 µg/ml gentamicin sulfate. Antral follicles between 3 and 6 mm in diameter were aspirated with an 18-gauge needle and 10-ml syringe. Cumulus cell oocyte complexes (COCs) were examined under a dissecting microscope, and those with multiple layers of intact cumulus cells were selected and washed three times in HEPES-buffered Tyrode lactate (TL-HEPES) medium containing 0.1% (w/v) polyvinyl alcohol.
In Vitro Maturation
Groups of 50 COCs were placed in 500 µl of serum-free modified tissue culture medium 199 (Gibco, Grand Island, NY) supplemented with 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 ng/ml epidermal growth factor, 0.5 µg/ml FSH, 0.5 µg/ml LH, 0.1% (w/v) polyvinyl alcohol, 75 µg/ml penicillin-G, and 50 µg/ml streptomycin sulfate [20] and matured for 22 h at 39°C and 5% CO2. COCs were washed in modified tissue culture medium 199 without FSH or LH and matured for an additional 20 h. Medium was pre-equilibrated under paraffin oil overnight at 39°C and 5% CO2. Following maturation, cumulus cells were removed by vortexing COCs in TL-HEPES medium containing 0.1% (w/v) hyaluronidase.
In Vitro Fertilization
Cumulus-free oocytes were washed three times in 500 µl of modified tris-buffered medium consisting of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2, 20 mM tris, 11 mM glucose, 5 mM sodium pyruvate, 2 mM caffeine, and 0.2% (w/v) BSA [20], and up to 30 oocytes were placed in 50-µl drops of modified tris-buffered medium for IVF. Cryopreserved semen was thawed in 10 ml of Dulbecco PBS (Gibco) supplemented with 0.1% (w/v) BSA, and spermatozoa were washed two times by centrifugation (1000 x g for 4 min). To determine the fertilization rate and examine the degradation of the sperm mitochondria inside the oocytes, spermatozoa were prelabeled with 200 nM of a vital, mitochondrion-specific fluorochrome (MitoTracker Green FM or CMTM Ros; Molecular Probes, Eugene, OR) for 10 min at 37°C. Labeled spermatozoa were washed and resuspended in modified tris-buffered medium, sperm concentration was determined, and spermatozoa were added to the fertilization drops to yield a final concentration of 5 x 105 spermatozoa per milliliter. Gametes were coincubated for 6 h. Following IVF, the presumptive zygotes were washed three times in NCSU23 containing 0.4% (w/v) BSA, and groups of embryos were cultured in 500 µl of NCSU23 at 39°C and 5% CO2 for up to 6 days.
Inhibitor Treatments
To examine the role of the proteasome-ubiquitin system in mitochondrial degradation and embryo development, the effect of specific inhibitors of the proteasome's proteolytic activity, MG132 (reversible inhibitor; Biomol Research Labs., Plymouth Meeting, PA) and lactacystin (irreversible inhibitor; Biomol Research Labs.), was evaluated. IVF was performed as described above with the addition of MG132 (10 µM) or lactacystin (10 or 100 µM). Controls without an inhibitor included an addition of the appropriate solvent (MG132: 100% ethanol; lactacystin: H2O) at equivalent dilutions to the in vitro culture during IVF. Presumptive zygotes were cultured up to an additional 20–24 h, at which time they were fixed and immunofluorescence analysis was performed as described below to determine the incidence of fertilization and to evaluate sperm mitochondrial degradation. Four successive stages of mitochondrial degradation (see Fig. 1) were established, and the relative rates of the oocytes expressing such respective stages were accounted for. Treatment results were compared by chi-square test and by general linear model procedures of SAS 8.02 (SAS Institute Inc., Cary, NC).
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Transient Proteasome Inhibition
To examine if the degradation of sperm mitochondria after fertilization was dependent on the stage of embryonic development or embryo age (i.e., if there was a window of opportunity for the degradation of sperm mitochondria) and if inhibition of mitochondrial degradation was reversible, oocytes were fertilized in control medium, cultured in the presence of 10 µM MG132 for 18 h, recovered, and placed into inhibitor-free medium for an additional 96 h. In addition to transient exposure, some embryos were exposed to MG132 during the entire culture period and others were cultured without inhibitor. At the end of culture, embryos were fixed and mitochondrial degradation was analyzed as described below. Controls without inhibitor included the addition of the appropriate solvents to the inhibitors during embryo culture, at equivalent dilutions. Treatment results were compared by chi-square test and by general linear model procedures of SAS 8.02 (SAS Institute).
Immunofluorescence
Oocytes/embryos were fixed at indicated time points between 0 and 120 h after insemination with MitoTracker-labeled (MT CMTM Ros, unless indicated otherwise, Molecular Probes, Inc.) spermatozoa. Zona pellucida (ZP) was removed by a short incubation in TL-HEPES with 0.5% pronase (Protease; Sigma, St. Louis, MO). Oocytes were transferred into the first well of a nine-well Pyrex brand glass plate (Fischer Scientific, Brightwaters, NY) filled with 400 µl of 37°C warm PBS, and 100 µl of 10% formaldehyde was slowly added to bring the final concentration of formaldehyde to 2%. After 40 min fixation at room temperature, oocytes were washed in two wells of PBS, fixative was removed, and plates were wrapped in Handi-wrap plastic wrap (Fischer Scientific) and stored for 1–7 days in the dark at 4°C. Prior to immunofluorescence processing, oocytes were permeabilized in PBS with 0.1% Triton-X-100 for 40 min at RT and blocked for 25 min in 0.1 M PBS containing 5% normal goat serum and 0.1% Triton-X-100. Primary antibody incubation was performed for 40 min with rabbit anti-ubiquitin Ab 1690 (dilution 1/100; Chemicon Inc., Temecula, CA), known to cross-react with nuclear/nucleolar ubiquitin tail proteins [21], or mouse IgG mAb 414 against nuclear pore complex (dilution 1/200; BabCo/Covance, Berkeley, CA). After a wash, samples were incubated for 40 min with appropriate secondary antibodies, including goat anti-mouse IgG-FITC and goat anti-rabbit FITC (all 1/80; both from Zymed, Inc., San Francisco, CA). DNA stain DAPI was added at 2.5 µg/ml to second antibody solution. Negative controls (data not shown) were performed by first antibody omission (mAb 414) or by the incubation with preimmune rabbit serum in place of the first antibody (Ab 1690). Samples were mounted on microscopy slides in VectaShield mounting medium (Vector Labs, Burlingame, CA) and sealed with clear nail polish. Image acquisition was performed by a Nikon Eclipse 800 microscope (Nikon Instruments, Inc., Melville, NY) with CoolSnap camera (Roper Scientific, Tucson, AZ) and MetapMorh software (Universal Imaging Corp., Downington, PA). Data were archived on CD-R compact disks, edited using Adobe Photoshop 5.5 (Adobe Systems, Mountain View, CA), and printed on an Epson Stylus 1280 photo printer (Epson America, Inc., Long Beach, CA).
Electron Microscopy
Oocytes were fixed at 20 h post-insemination in 2% paraformaldehyde and 0.6% glutaraldehyde in cacodylate buffer, postfixed in 1% osmium tetroxide, dehydrated by an ascending ethanol series (30%–100%), and embedded in PolyBed 812 resin. Ultrathin sections were prepared on a Leica (Heidelberg, Germany) Ultracut UCT ultramicrotome, placed on 100 MESH copper grids, and stained in two steps with uranyl acetate and lead citrate. Serial sections of 10 oocytes were examined and photographed in a Jeol 1200 EX electron microscope. Negatives were scanned by a Umax Magic Scan flatbed scanner, recorded on a CD-R disk, and printed on an Epson Stylus 1280 photo printer using Adobe Photoshop 5.5 software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To study sperm mitochondrial degradation after fertilization, boar spermatozoa were prelabeled with a vital, fixable, mitochondrion-specific probe MitoTracker CMTM Ros (Fig. 1A) and used for IVF. Resulting zygotes were processed for immunofluorescence with the blue fluorescent DNA stain DAPI and antibodies against nuclear ubiquitin-tail proteins (Ab1690 [21]) and nuclear pore complexes (mAb 414 [22]) included for evaluating pronuclear development. Signs of sperm mitochondrial degradation were seen in some zygotes as early as 8 h after insemination, at which stage the male and female pronuclei appeared and the nuclear envelopes were reconstituted on them (Fig. 1B). Pronuclear apposition and progressive early stages of sperm mitochondrial degradation were observed at 12 h post-insemination (Fig. 1C). Complete removal of the sperm mitochondria, leaving behind the "naked" sperm axoneme with redundant outer dense fibers (ODF), was seen in some zygotes as early as 20 h post-insemination (Fig. 1D). The typical, "snake tongue" pattern resulting from the uptake of MitoTracker by sperm ODF was also seen in some of the two-cell embryos, emerging at 24–30 h post-insemination (Fig. 1E). Although it was possible to locate the remnants of ODF and the sperm-derived centriole/zygotic centrosome inside the zygotes at 20 h post-insemination (Fig. 2, A and C), there were no signs of the typical, oval sperm mitochondria that are morphologically distinct from the round-shaped oocyte mitochondria (Fig. 2, A and D). Sperm mitochondrial degradation was also observed in the pseudo-cleaving, polyspermic eggs (Fig. 1F). Interestingly, when the spermatozoa, differentially labeled with green and red fluorescent MitoTracker probes (Fig. 1G), were used for IVF at the 0 and 3 h insemination time points, the delay in the degradation of "red" sperm mitochondria seemed to mirror the time difference in insemination.
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Based on the above observations of rapid sperm mitochondrial degradation in porcine zygotes, the patterns of mitochondrial degradation seen inside the 1–2 cell embryos were divided into four stages/categories (Fig. 1, H–K). Type 1 represented a straight, rod-shaped mitochondrial sheath with little or no dents suggesting the removal of few sperm mitochondria. Type 2 was characterized by the recognizable mitochondrial sheath, with numerous missing mitochondria and a distorted, sometimes corkscrew-like shape. Type 3 sperm mitochondria were clumped or scattered around the remnants of axonemal ODF, which take up the MitoTracker dyes in a nonspecific manner. Type 4 represented the absence of detectable sperm mitochondria and often a typical pattern of split proximal ends of outer dense fibers (snake tongue; see also Fig. 1E).
Quantitative analysis revealed that at 27–30 h postinsemination, a vast majority (96%) of zygotes contained the partially or completely degraded types 3 and 4 sperm mitochondria (Table 1).
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Proteasomal Inhibitors Prevent the Degradation of the Sperm Mitochondria Inside the Fertilized Egg in a Reversible Fashion
To determine whether the degradation of the sperm mitochondria depends on the activity of the ubiquitin-proteasome pathway, oocytes were treated with 10 µM MG132, 10 µM lactacystin, or 100 µM lactacystin at insemination. Both inhibitors are small molecules that bind to the catalytic subunit of the proteasome and block its proteolytic activity specifically without affecting other, non-proteasomal protease activities [23]. Surprisingly, both treatments inhibited fertilization, and in the case of MG132, to a 100% effect (Table 1). This block was not due to changes in sperm motility, sperm-zona binding, or acrosome reaction (see Fig. 2E), as the inhibitors had no effect on the fertilization rates in zona free oocytes (see Discussion). This effect of proteasomal inhibitors on zona penetration required a change of treatment protocol in which the oocytes were treated with the above inhibitors 6 h post-insemination, a time sufficient for sperm penetration in approximately 71% of oocytes (comparable to an 80% fertilization rate in control oocytes). MG132 was used in most of these studies, as it is a more potent inhibitor of proteasomal activity than lactacystin, and in contrast to lactacystin is fully reversible (reviewed by Goldberg et al. [24]). Such treatment effectively blocked the degradation of the sperm mitochondria, wherein 86% of the treated oocytes contained type 1 or type 2 sperm mitochondria (Fig. 3, A–C). In a recovery experiment, oocytes were treated with 10 µM MG132 6 h postinsemination, the inhibitor was washed out 24 h after insemination (i.e., 18 h after the addition of MG132), and oocytes were allowed to develop for another 96 h. In parallel, one group of oocytes were left in the medium with MG132 for the duration of the trial (120 h) and the control, untreated oocytes were inseminated and cultured for the same amount of time. All oocytes cultured in the presence of MG132 for the entire culture period (120 h) remained in the one-cell stage (Table 2; Fig. 3, D–F) because of a known effect of the inhibition of cell cycle progression via the inhibition of ubiquitin- and proteasome-dependent cyclin degradation (reviewed in [1]). Invariably, the treated zygotes contained more or less intact type 1 or type 2 sperm mitochondria (Fig. 3F). In contrast, those oocytes that were washed from MG132 after 24 h showed a 53% cleavage rate comparable with 63% cleavage rates in the nonexposed control (Table 2). In none of the cleaved embryos were the sperm mitochondria detected (Fig. 3D), suggesting that the removal of MG132 not only allowed the progression of the embryonic cell cycle, but also the completion of the sperm mitochondrial degradation. Consistent with the 24-h shortened time for which the MG132 oocytes were allowed to develop, morulae were observed in this group at the time when some of the control, untreated embryos reached the blastocyst stage.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An alternative hypothesis, based on the meeting reports of paternal mtDNA presence in the human placenta, suggests that the sperm mtDNA could escape degradation after fertilization and ultimately drift toward extra-embryonic tissues. Although such an intriguing hypothesis warrants detailed examination [34], it must be supported by a detailed, peer-reviewed report to win acceptance. In addition, the seeming mtDNA recombination in the placenta and other tissues may be due to the ongoing intra-organelle mtDNA repair [35], rather than recombination of maternal and paternal mtDNA. The first credible case of human paternal mitochondrial inheritance was reported in a recent case study [36], yet no evidence of paternal-maternal mtDNA recombination was found. Sadly for the subject, although intriguing for a scholar studying mitochondrial inheritance, this paternal transmission was observed in a case of severe mitochondrial myopathy accompanied by a de novo 2-base pair (bp) deletion of the mitochondrial ND2 gene, which was carried by the patient but not by his father.
Although further studies on possible recombination between maternal and paternal mtDNA are warranted, more attention should be paid to cell biology of the sperm mitochondrial degradation after fertilization. Due to abundant ultrastructural [6, 37, 38] and genetic [39, 40] evidence of sperm mitochondrion degradation inside the zygotic cytoplasm, it is less likely that mtDNA would be liberated from the degraded organelles. If a recombination event was to occur, it would most likely be mediated by the fusion of intact maternal and paternal mitochondria. At present, we cannot rule out that at least some sperm mitochondria could escape proteolytic degradation. However, this is a fairly unlikely scenario considering the presence of the proteolytic signal, ubiquitin, in the sperm mitochondrial membrane [7, 8] and the evidence implicating both lysosomes [8] and proteasomes (this study) in the degradation of sperm mitochondria inside the zygotic cytoplasm. In our previous studies using the bovine model, we were able to delay sperm mitochondrial degradation by a general lysosomal inhibitor, ammonium chloride [8]. This was also attempted in the course of our present studies (data not shown). Although we noticed some delay in the degradation of the fertilizing spermatozoon's mitochondria, the treatments seemed to block embryonic development irreversibly and eventually caused embryonic death (data not shown). On the other hand, treatment with MG132, a specific proteasomal inhibitor, prevented degradation of the sperm mitochondrial reversibly and without affecting the rates of embryonic development. Figure 3D shows a four-cell embryo on Day 5 after a 24-h exposure to MG132, a treatment applied on Day 1 of the IVF cycle. Although this embryo did not develop into a morula or a blastocyst as other MG132-treated embryos did after reversal of this treatment, it serves as a good example of the full reversibility of MG132 treatment and shows that the MG132-treated embryos resumed the degradation of the paternal mitochondria immediately after the removal of blocking peptide. Such a result favors a role of the proteasome pathway over lysosomal degradation in the proteolysis of sperm mitochondria after fertilization. However, it is possible that lysosomal inhibitors more specific than ammonium chloride would have yielded better results, i.e., a more efficient block of sperm mitochondrion degradation in our porcine studies. Interspecies differences should also be considered, implying that different routes of sperm mitochondrial degradation could be employed by the porcine (this study) and bovine [8] zygotes. The degradation of the paternal mitochondria is species-specific and because of unknown incompatibilities does not occur in the mouse interspecies crosses [7, 39, 40]. A possible contribution from a 15-lipoxygenase pathway for the degradation of the lipid components of the mitochondrial membrane, known to intersect with ubiquitin-dependent organelle/mitochondrial degradation during reticulocyte differentiation [41, 42], is under examination.
In addition to supporting the role of the ubiquitin-proteasome pathway in the sperm mitochondrial degradation, the purpose of MG132 recovery experiments was to examine whether there is a window of opportunity for the targeting and removal of paternal mitochondria during the early stages of zygotic development. Should that be the case, concern could arise that when the plasma membrane-intact human or animal spermatozoa are injected directly in the egg cytoplasm by intracytoplasmic sperm injection (ICSI), the presence of the plasma membrane, normally removed during sperm-oolemma fusion, could delay the degradation of the sperm mitochondria and result in heteroplasmy caused by the carryover of paternal mtDNA from the injected sperm. Present results in the pig do not justify such a concern and neither do the few existing studies of mtDNA inheritance in ICSI babies (reviewed in [5, 33, 43]). Further studies in other mammalian species and a wide-scale screening of ICSI children may still be warranted to completely rule out such a possibility.
The finding of this study that the proteasomal inhibitors prevent the penetration of zona pellucida by the fertilizing boar spermatozoa deserves further attention. Although this may sound sensational at first, there are at least three lines of evidence supporting the role of sperm proteasomes in animal fertilization, acrosome reaction, and zona penetration. First, proteasomal subunits can be detected in the acrosome of human [14] and boar (unpublished data) spermatozoa. Second, sequential ubiquitination and acrosomal-proteasomal degradation of the putative sperm receptor protein HrVC70, the ascidian homologue of mouse ZP3-sperm receptor, has been shown on the ascidian vitelline envelope, an analogue of mammalian ZP [16]. Similarly, ubiquitin immunoreactive proteins can be detected on the outer face of the pig ZP (unpublished data). Third, zona penetration can be blocked by proteasomal inhibitors in mouse [18], pig (this study), and invertebrate [15, 17] oocytes, and by anti-proteasome antibodies in pig (unpublished data). It should also be considered that the knockouts of several acrosomal proteins with predicted enzymatic, zona-digesting activity did not yield the targeted animals infertile, and it is still not known which acrosomal component digests the ZP after acrosomal exocytosis [44, 45]. It is very important to emphasize that the proteasomal inhibitors used in this study did not directly affect the viability of either spermatozoa or oocytes during and after IFV. Zona-free oocytes fertilized in the presence of lactacystin or MG132 invariably showed high levels of polyspermic fertilization (data not shown). In zona-intact oocytes, the reversibility of MG132 treatment (lactacystin is known to be irreversible) and embryo viability have been demonstrated (Table 2).
Detailed discussion of the proteasomal involvement in acrosomal exocytosis and zona penetration is beyond the focus of the present study. It appears that the sperm-ZP interactions involve sperm proteasomes present in the acrosomal matrix, and an ubiquitin-immunoreactive substrate presents exclusively on the outer face of the ZP. Further effort is being focused on the characterization of individual proteasomal subunits present in the porcine acrosome and on the isolation and sequencing of the ubiquitin immunoreactive substrates in the outer layer of pig ZP.
Taken together, the present studies provide the first evidence for the role of proteasomal degradation of ubiquitinated proteins in the control of mammalian mitochondrial inheritance. At the same time a new role for the ubiquitin-proteasome pathway is invoked in the process of zona penetration by the fertilizing spermatozoon.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Peter Sutovsky, University of Missouri-Columbia, S141 ASRC, 920 East Campus Drive, Columbia, MO 65211-5300. FAX: 573 884 5540; sutovskyp{at}missouri.edu
Received: 29 October 2002.
First decision: 14 November 2002.
Accepted: 5 December 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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