Mol. Hum. Reprod. Advance Access originally published online on September 23, 2007
Molecular Human Reproduction 2007 13(11):759-770; doi:10.1093/molehr/gam068
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Mitochondrial signaling and fertilization
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA; Colorado Reproductive Endocrinology, Rose Medical Center, Denver, CO 80220, USA
1Correspondence address. E-mail: jonathan.vanblerkom{at}colorado.edu
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Abstract |
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The magnitude of the potential difference (polarity) across the inner mitochondrial membrane (
m) determines levels of several mitochondrial activities, including ATP generation, focal regulate calcium homeostasis and organelle volume homeostasis. We investigated whether a domain of mitochondria in the mouse oocyte, characterized by high m and a unique location in the subplasmalemmal cytoplasm, is involved in the earliest events of fertilization: sperm attachment, penetration and cortical granule exocytosis. Experimental manipulations of the magnitude of m and the distribution of mitochondria in zona-free MII oocytes, followed by insemination and culture, indicate that high-polarized mitochondria (HPM) are required for penetration and cortical granule exocytosis, but not for persistent attachment to the oolemma. The capacity of subplasmalemmal mitochondria to undergo transient reductions (dissipations) of m appears necessary for penetration and cortical granule exocytosis. We suggest that the HPM normally establish a continuous circumferential circuit of ‘reactive’ organelles capable of responding to and propagating, triggering or activating signals across the subplasmalemmal cytoplasm, such as those initiated by the fertilizing sperm at the site of penetration. The HPM in the oocyte and early embryo may have functions similar to those of their somatic cell counterparts and promote the focal regulation of developmental activities that are themselves spatially localized. The establishment of high m in the subplasmalemmal cytoplasm may be among the first steps in the preovulatory maturation of the oocyte and defects in this domain may result in fertilization failure or abnormality. Key words: oocyte/mitochondrial polarity/fertilization/cortical granule discharge/subplasmalemmal cytoplasm
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Introduction |
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The role of mitochondria during oocyte and early embryonic development in a mammal is usually viewed in terms of a singular purpose, the generation of ATP (Van Blerkom et al., 1995; Reynier et al., 2001; Shahinaz et al., 2006; May-Panloup et al., 2007; Shoubridge and Wai, 2007; Zeng et al., 2007). However, recent evidence demonstrates that in addition to their bioenergetic activity, somatic cell mitochondria have regulatory functions that include participation in calcium homeostasis, signal transduction and oxygen sensing (Pozzan et al., 2000; Bell et al., 2005; Zimijewski et al., 2005; Gutierrez et al., 2006; Quintero et al., 2006). Here, our intent is to review certain mitochondrial functions that may have similar roles during early mammalian development, and in particular, to present evidence that the state of mitochondrial polarity is an important factor during the initial stages of fertilization.
Although mitochondria in normal mouse and human oocytes and early embryos are largely morphologically equivalent, they can be classified into two spatially distinct populations by virtue of their state of polarization, i.e. the magnitude of the electrical potential across the inner mitochondrial membrane, commonly termed m (Van Blerkom et al., 2002, 2003, 2006). Differences in m can be clearly detected between mitochondria in living cells with potentiometric fluorescent reporters such as JC-1, which is one of the most specific stains currently used for this purpose (Reers et al., 1995; Salvioli et al., 1997). In the mature (MII) oocyte and early embryo, high-polarized mitochondria (HPM) normally occupy a circumferential domain immediately subjacent to the plasma membrane (Van Blerkom et al., 2002) and in total, represent 
5% organelles estimated to populate the mature mouse and human female gamete, respectively (Van Blerkom and Davis, 2006). These authors described the following unique characteristics of this domain: (i) it is spatially stable in the oocyte and after fertilization, segregated between daughter cells during early cleavage and (ii) adverse developmental consequences are associated with irreversible loss from one or more blastomeres as a result of minor fragmentation or by disproportionate inheritance at the first and second cell divisions. Van Blerkom and Davis (2006) used the term ‘vanguard mitochondria’ to distinguish this subset of high-polarized organelles from their more abundant but lower polarized counterparts that occupy virtually all of the cytoplasm, and to indicate the possibility that they may have specialized functions in early development.
We have previously suggested that certain developmental activities in the oocyte and preimplantation stage embryo may be spatially regulated by the magnitude of m (Van Blerkom et al., 2003, 2006) and could be adversely affected if vanguard mitochondrial polarity is subnormal, perturbed or aberrant in distribution (Van Blerkom and Davis, 2006). Here, we investigated whether some of the earliest processes in fertilization, namely, sperm attachment, penetration and cortical granule exocytosis, involve or require the presence of this domain. The results show that persistent sperm attachment to the oolemma does not require the presence of mitochondria in the subplasmalemmal cytoplasm, but a domain of HPM capable of undergoing transient dissipations of m is required for sperm penetration and cortical granule exocytosis. The findings are discussed with respect to the notion that this domain forms a continuous circumferential circuit of ‘reactive’ mitochondria that can respond to ionic (e.g. calcium) and electrical (changes in plasma membrane potential) signals and in this capacity, may participate in signal transduction within the subplasmalemmal cytoplasm, and if perturbed could be associated with fertilization and early developmental failure.
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Materials and Methods |
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Oocyte collection and in vitro fertilization
Normal-appearing MII stage oocytes were collected from the ampullary region of mice at 12–14 h after ovulation induction with 5 IU of pregnant mare’s serum gondotropin, which followed the administration of 5 IU of hCG by 48 h. After denudation of cumulus cells by exposure to hyaluronidase and mechanical removal by passage through a glass micropipet, oocytes with a distinct polar body and cytoplasm of uniform texture were placed in acidic Tyrode’s solution and constantly agitated by passage through a micropipet until the zona pellucida was completely dissolved. Zona-free oocytes were pooled and cultured in bovine serum albumin supplemented KSOM or HTF medium for 10–20 min prior to JC-1 staining, insemination, inhibitor exposure, centrifugation or quantitative analysis of net cytoplasmic ATP content, as described below.
Epididymal sperm were collected in 1 ml of HTF supplemented with 0.4% BSA, cultured for 10–30 min, and then added to the same medium at a concentration of approximately 5000 ml–1 (Quinn et al., 1985). Zona-free oocytes were co-incubated with sperm for varying lengths of time (see below), and depending upon the study, the dynamics of oocyte–sperm interaction recorded for up to 24 h by time-lapse microscopy on optical memory discs, with images taken at 10 min intervals, or by continuous recordings on DVDs (Van Blerkom, 2007). A standard interval of gamete co-incubation was derived for untreated, inhibitor treated and centrifuged oocytes that was based on the timing of maximal sperm binding to the oolemma, as determined by the formation of stable attachments observed by both recording methods and quantified as described below. Insemination and culture used T dishes maintained at a constant 37°C by means of a T controller, which regulated the electrical current passed through a thermo-optically treated glass coverslip integrated into the bottom of a plastic culture dish (Bioptics, USA), as previously described (Van Blerkom et al., 1998). After insemination, oocytes were cultured in KSOM (Lawitts and Biggers, 1991).
Staining with fluorescence probes for high m mitochondria, DNA and cortical granules
All oocytes were stained for 30 min with JC-1 (5,5'6,6'-tetrachloro-1,1,3,3'- tetraethylbenzimidazolycarbocyanine iodide, Molecular Probes, USA) at a concentration of 1 µmol/l (Van Blerkom et al., 2002). After staining, oocyes were washed through sequential changes of medium, examined by light microscopy and photographed under epifluorescence illumination, first in the fluorescein isothiocynate channel (FITC), and then in the rhodamine isothiocynate channel as described previously (RITC; Van Blerkom et al., 2006). After insemination or culture (see below), oocytes, pronuclear eggs and cleavage stage embryos were fixed in PBS containing 3.7% formaldehyde, washed in PBS and stained for DNA with DAPI (4,6-diamidino-2 phenylindole diacetate, 10 µg/ml for 15 min), or for cortical granules with biotinylated Lens culinaris agglutinin (LCA) and Texas Red-streptavidin (Van Blerkom et al., 2003). For microscopic examination, fixed and stained oocytes were mounted between glass coverslips without compression.
To determine the number of sperm firmly bound to the oolemma, prior to fixation, oocytes were washed through several changes of medium with vigorous and repeated passages through a glass micropipet (Van Blerkom, 2007). After fixation and DAPI staining, the number of sperm attached to the oolemma was determined by optical sectioning and fluorescent microscopy. The fluorescent images of mitochondria are presented without manipulation and as shown, closely represent the intensity and color of J-aggregate fluorescence observed.
Experimental treatments
Zona-free oocytes were cultured, inseminated and stained with JC-1 at normal (37°C, NT) and reduced temperatures (25°C, RT), after exposure to FCCP (carbonyl cyanide 4-trifluoro-methoxyphenylhydrazone, 100 µmol/l, Sigma Chem, USA), or during treatment with Bongkrekic acid (BA, 50 µmol/l, Calbiochem, USA), as previously described (Van Blerkom et al., 2003). Culture and insemination at subnormal temperatures used a modular incubator chamber (Billups-Rothenberg, USA) equilibrated with a premixed atmosphere (5% O2, 5% CO2, 90% N2) maintained in an incubator at 25°C. After inhibitor treatment, oocytes were returned to normal medium and temperature and examined by epifluorescence microscopy at the timed intervals described below.
Each experimental treatment included a microdroplet of oocytes cultured under normal conditions, and for insemination studies, a microdroplet with identically treated oocytes was cultured in the absence of sperm. Mitochondrial compartmentalization involved centrifugation of zona-free oocytes at 19 600 g through 1.0 ml of Percoll (Sigma Chem, USA) containing equal parts of 40% and 80% solution (Van Blerkom et al., 1998). As noted in this study, centrifugation for 20 min elongated oocytes while separation into mitochondria enriched cytoplasts and mitochondria-deficient karyoplasts required 60–90 min.
Cytoplasmic ATP content
The net average ATP content of zona-free MII oocytes was determined quantitatively (Van Blerkom et al., 1995, 2003, 2006). Briefly, groups of 5–6 control or treated oocytes were rapidly frozen to –80°C in 200 µl of ultrapure water and ATP levels quantified by measuring the luminescence (Berthold LB9501 luminometer, Berthold Technologies, USA) generated in an ATP-dependent, luciferin–luciferase bioluminescence assay (Bioluminescence Somatic Cell Assay System; Sigma Chem, USA). A standard curve containing 14 ATP concentrations from 1 fmol to 5 pmol was generated for each series of analyses. ATP levels (fmol/oocyte) were analysed statistically by the 
2-test and unless indicated, were considered significant at P < 0.01.
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Results |
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Zona-free oocytes were used in this study in order to preclude oocyte-specific differences that may influence sperm passage through the zona, and by exposing the entire oolemma, enabled the surface area available for attachment to be maximized. This was the most effective method we found to determine whether differences in attachment, penetration and cortical granule exocytosis existed within regions of the oolemma, and the subplasmalemmal cytoplasm where mitochondria were high or low polarized, or absent after experimental treatment. In preliminary studies, sperm attachment was determined after 5, 15, 30, 60 and 90 min co-incubation. Continuous video-microscopic recordings of living oocytes, and timed fluorescent microscopic analysis of fixed oocytes, showed that attachment in untreated oocytes was relatively immediate after co-incubation and under the conditions used, reached a maximal number of firmly adherent sperm within 15 min. As a result, quantitation of bound sperm was standardized at 15 min for control MII stage oocytes after they were repeatedly and vigorously passed through a glass micropipet, fixed in formaldehyde, and stained for DNA with the fluorescent probe DAPI. The same protocol was used for oocytes exposed to agents or conditions that influenced
m. The high density of bound sperm often obscured direct visualization of the subjacent subplasmalemmal cytoplasm. In order to determine the timing, intensity and distribution of J-aggregate fluorescence, especially during the recovery of high m, a separate dish containing identically treated JC-1 stained oocytes, cultured in the absence of sperm, was included in each experiment.
High-polarized subplasmalemmal mitochondria and sperm attachment to the oolemma of zona-free MII mouse oocytes
JC-1 and J-aggregate fluorescence report the presence of mitochondria in living cells that are relatively low- (green-fluorescent) or high-polarized (red-fluorescent, hyperpolarized), respectively. When viewed in the FITC channel, high-polarized, J-aggregate positive mitochondria fluoresce orange against a green background (Van Blerkom et al., 2006). Fig. 1B–D are three representative images of circumferential subplasmalemmal J-aggregate fluorescence (HPM) detected in the FITC channel that were typical of MII stage mouse oocytes (Fig. 1A, n = 148) at 30 min, 3 and 12 h after zona removal. The distribution and intensity of J-aggregate fluorescence were similar between oocytes and equivalent to images obtained for intact oocytes (Van Blerkom et al., 2002; Van Blerkom and Davis, 2006).
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The extent of sperm attachment to the oolemma after a 15 min co-incubation was assessed in living oocytes by light microscopy (e.g. Fig. 1E) and after fixation, the number of DAPI stained sperm heads that were clearly attached to the oolemma after vigorous washings was determined by epifluorescence microscopy (e.g. Fig. 1F). The average number of attached sperm was 80 ± 15 (n = 118). The average net ATP content of untreated, zona-free MII oocytes (n = 161) was
740 ± 80 fmol (Fig. 4).
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Effects of FCCP exposure on
m and sperm attachmentIn agreement with earlier findings (Van Blerkom et al., 2002, 2003), after a 15 min incubation in the presence of the
m-dissipating proton ionophore FCCP followed by JC-1 staining (Fig. 1G), only green (JC-1) monomeric fluorescence was observed throughout the cytoplasm (Fig. 1H). No sperm attached to the oolemma when inseminations were performed at 10 min intervals for up to 40 min in the presence of FCCP, despite the persistence of motility. Culture of zona-free oocytes in the presence of FCCP significantly reduced the net average cytoplasmic ATP content after 15 and 30 min by 35% (479 ± 36 fmol; n = 130; Fig. 4) and 80% (155 ± 38 fmol; n = 125) of normal, respectively, as previously reported (Van Blerkom et al., 2003). The reduction in cytoplasmic ATP content was not associated with a detectable disruption of the MII spindle, which remained intact and showed no evidence of chromosomal displacement or detachment during a 1-h exposure (n = 46; e.g. Fig. 1I). Approximately 10 min after transfer to the normal medium, focal regions of subplasmalemmal J-aggregate fluorescence were detectable (arrows, Fig. 1J) and sperm attachment was initially observed at these regions by light (SP, Fig. 1K) and fluorescence microscopy (similar to Fig. 1F). Between 15 and 20 min, the pattern of circumferential J-aggregate fluorescence in most oocytes (n = 27/31; similar to Fig. 1D) returned to normal, although some (n = 4/31) showed subplasmalemmal zones of discontinuous or scant J-aggregate fluorescence that remained in this state for several hours when restained with JC-1 (e.g. 3 h, arrows, Fig. 1L). By 20 min, the intensity of circumferential subplasmalemmal J-aggregate fluorescence was normal in the uninseminated group of oocytes (n = 55; images similar to Fig. 1B and D) and the relative number of sperm attached to the oolemma in the corresponding group of inseminated oocytes (n = 63; Fig. 1M) was similar to untreated, zona-free oocytes (e.g. Fig. 1E). At the time of sperm attachment at normal levels, measurements of ATP contents in an identically treated cohort of uninseminated oocytes (n = 150) that had been exposed to FCCP for 30 min, showed levels that were approximately 50% of the normal (381 ± 27 fmol, –FCCP, Fig. 4). Measurements of ATP content were not made in the inseminated group because of the high density of firmly bound sperm.
Effects of reduced culture temperature on m and sperm attachment
The above findings suggested that the capacity of the oolemma to bind sperm could be related to the presence of HPM in corresponding subplasmalemmal cytoplasm, or require a threshold net cytoplasmic ATP content, or both. To distinguish between these possibilities, intact (n = 80; Fig. 1N) and zona-free oocytes (n = 95) were preincubated in JC-1 at 25°C for 30–45 min. At this temperature, oocyte mitochondria fluoresce green (Fig. 1O) indicating the incorporation of the JC-1 monomer, but high-polarity, as reported by the multimerization of JC-1 into red-fluorescent J-aggregates, was undetectable in the subplasmalemmal domain (RITC channel, Fig. 1P), as previously described (Van Blerkom et al., 2003). The specificity of mitochondrial JC-1 staining at 25°C was confirmed by differences in fluorescent patterns detected between oocytes within the same cohort, where the cytoplasmic distribution of mitochondria normally occurs as two distinct phenotypes: (i) a relatively uniform dispersion and (ii) with small clusters dispersed throughout the cytoplasm (Van Blerkom and Runner, 1984; Van Blerkom et al., 2002). Fig. 1O shows a representative example of each, and for the clustered phenotype in the upper portion of this figure, distinct foci of green JC-1 fluorescence (asterisks, Fig. 1Q) correspond to mitochondrial aggregates that where internally located and normally low-polarized at 37°C. Green-fluorescent mitochondrial aggregates in the subplasmalemmal cytoplasm are indicated by arrows in lower oocyte in Fig. 1O. These mitochondria are normally high-polarized at 37°C and fluoresce orange–red in the FITC and RITC channels, respectively (e.g. arrows, Fig. 1D).
Zona-free oocytes were exposed to sperm at 25°C and the levels of binding determined after 15 min were similar to those measured at 37°C. Although previously high-polarized subplasmalemmal mitochondria were J-aggregate-negative (asterisk, Fig. 1O), mitochondria in the midpiece of sperm bound to the oolemma (MP, Fig. 1Q) fluoresced orange, indicating that exposure at this subnormal temperature did not reduce their polarity below the threshold potential for J-aggregate formation. Sperm remained attached and motile at 25°C for at least 16 h, (e.g. 10 h, Fig. 2A) but in the absence of subplasmalemmal J-aggregate fluorescence, no evidence of penetration was observed (n = 87; Fig. 2B). After 2- or 3-h incubation at this temperature, the net cytoplasmic ATP content had declined from normal levels by 8% (675 ± 31 fmol, n = 115; RT, Fig. 4) and 13% (641 ± 28 fmol, n = 127), respectively, but these values were not significant.
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Within 10–15 min after culture at 37°C, regions of the subplasmalemmal cytoplasm showed a J-aggregate positive signal (HPM, Fig. 2C, RITC channel) and by 30 min, the normal intensity and circumferential distribution of HPM were completely restored (arrows Fig. 2D, RITC channel). The net cytoplasmic ATP content returned to normal levels (750 ± 25 fmol, n = 120) within 15 min of culture at 37°C (Fig. 4). Penetration by motile sperm that had previously attached at the reduced temperature occurred within 30 min of the restoration of high
m in the subplasmalemmal cytoplasm, as discussed below.
Effects of Bongkrekic acid on m and sperm attachment
The finding that high-polarity in the subplasmalemmal mitochondrial domain was not required for persistent sperm attachment was unexpected in view of the results obtained with FCCP. To further investigate the association between sperm binding, m and cytoplasmic ATP content, oocytes were cultured and inseminated in the presence of Bongkrekic acid (BA), an inhibitor of the mitochondrial megachannel (permeability transition pore) that prevents the collapse of m and for the mouse oocyte has been shown to maintain a constitutive high-polarity under conditions that would normally dissipate m and depress oxidative phosphorylation (Van Blerkom et al., 2003). Fig. 2E shows the typical pattern of J-aggregate fluorescence detected in the FITC channel in zona-free oocytes cultured in the presence of BA for 20–30 min at 37°C. Although quantitative analyses were not undertaken, the intensity of J-aggregate fluorescence appeared higher (HPM, Fig. 2E) than was typical for untreated oocytes (e.g. Fig. 1B–D), and in some oocytes, a low level of punctate orange fluorescence extended into the interior of the cytoplasm. Internal J-aggregate fluorescence of this type was rarely observed in previous studies (Van Blerkom et al., 2002, 2003) or in the control oocytes examined here (n = 114), suggesting that the influence of BA may not be limited to subplasmalemmal mitochondria. In agreement with earlier findings (Van Blerkom et al., 2003), the net average cytoplasmic ATP content was approximately 75% (520 ± 23 fmol, n = 92, Fig. 4) and 65% (477 ± 19 fmol, n = 75) of normal after a 15 and 30 min exposure to this inhibitor, respectively.
BA-treated oocytes inseminated at 15 and 30 min showed levels of sperm binding that were similar to untreated, zona-free oocytes (n = 60; 30 min, Fig. 2F). Oocytes exposed to BA at 25°C showed normal levels of cytoplasmic JC-1 monomeric fluorescence, which included green-fluorescent mitochondrial clusters throughout the cytoplasm (asterisk, Fig. 2G) and within the subplasmalemmal domain (arrows, Fig. 2G), but no detectable J-aggregate signal. After a 30 min pre-exposure to BA at 25°C followed by insemination in the presence of the inhibitor, sperm attachment was immediate and after 15 min the density was similar to normal levels seen in untreated oocytes (Fig. 2H), despite reductions in net cytoplasmic ATP content (n = 95; 440 ± 21 fmol) and the absence of a domain of HPM.
Are subplasmalemmal mitochondria required for sperm attachment?
Taken together, the above results indicate that persistent sperm attachment to the mouse oolemma is independent of the both a corresponding subplasmalemmal domain of HPM and a normal cytoplasmic ATP content. To investigate whether the presence of mitochondria in the subplasmalemmal cytoplasm was required for attachment, zona-free oocytes (n = 116) were centrifuged under conditions that either (i) elongated oocytes and segregated mitochondria into enriched (+M) and deficient (–M) compartments (Fig. 2I) or (ii) resulted in the formation of separated mitochondria-deficient karyoplasts and mitochondria-enriched cytoplasts (+M, Fig. 2I), as previously described (Van Blerkom et al., 1998). The polarized accumulation of a ‘dense material’ and a translucent cytoplasm were characteristic features of mitochondrial deficient regions in intact oocytes, and readily distinguished karyoplasts from cytoplasts at the light microscopic level (Fig. 2I and L). This dense material contains pelleted ‘fibrillar lattices’ (FL, Fig. 2I and L) that are ubiquitous in the mouse ooplasm (Van Blerkom et al., 1998). In intact oocytes, J-aggregate fluorescence was detected in the portion of the oocyte enriched for mitochondria and was especially evident as a focal band of intense fluorescence between the enriched and deficient compartments (arrow, Fig. 2J, FITC channel; Fig. 2K, RITC channel). J-aggregate fluorescene was detected only in separated cytoplasts (+M, Fig. 2K).
Within 15 min of co-incubation with sperm, elongated oocytes and separated karyoplasts and cytoplasts showed sperm attachment to the oolemma at high density. A representative example of persistent sperm binding to a compartmentalized oocyte after a 10 min exposure to sperm and imaged by light and fluorescent microscopy (DAPI stained) is shown in Fig. 2L and M, respectively. For intact oocytes and cytoplasts, the relatively high background of DAPI fluorescence in the compartment devoid of the filamentous sediment (e.g. +M, Fig. 2M) represents mitochondrial DNA staining. These results indicate that the presence of mitochondria in the subplasmalemmal cytoplasm, regardless of whether high or low polarized, is not a prerequisite for persistent sperm attachment to the oolemma. In addition, persistent sperm attachment occurred with karyoplasts whose ATP content was <15% of normal (82 ± 21 fmol, n = 54; Fig. 4).
High-polarized subplasmalemmal mitochondria are required for sperm penetration and cortical granule exocytosis
Under normal conditions, penetration of untreated zona-free oocytes occurred within 3 h after co-incubation with spermatozoa. Evidence of post-penetration activation of oocytes was indicated by the formation of a second polar body (Fig. 2N), sperm DNA decondensation (Fig. 2O) and the absence of cortical granules (Fig. 3B). Fig. 3A is a representative example of cortical granule fluorescence after staining uninseminated zona-free MII oocytes with Texas-red tagged LCA. Although cortical granule fluorescence was largely undetectable after penetration, for approximately 20% (26/120) of penetrated zona-free oocytes, focal areas of LCA fluorescence remained (arrow, Fig. 3B), indicating that cortical granule exocytosis was incomplete. The frequency of penetration for zona-free oocytes was virtually 100% (n = 123/124), and >90% of penetrations were di- or tri-spermic (e.g. Fig. 2O, 110/123).
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After penetration, most attached sperm were displaced from the oolemma, presumably in response to cortical granule exocytosis and changes in membrane organization (Fig. 2N and O). All penetrated oocytes (n = 93) stained with JC-1 showed a normal domain of HPM (images comparable to Fig. 1B). Approximately 53% (33/62) of fertilized eggs divided to the 2-cell stage (Fig. 2P) and virtually all contained multinucleated blastomeres (Fig. 2Q). Multinucleated blastomeres were also observed at the 4-cell stage (image not shown). The relatively few oocytes that evolved a single male pronucleus and were presumed penetrated by a single spermatozoon produced mononucleated blastomeres at the 2- and 4-cell stage (Fig. 2R). However, each embryo contained one or more blastomeres with a condensed sperm nucleus (arrow, Fig. 2R) indicating that in these cases, penetration had been polyspermic.
Effects of reduced temperature on m, penetration and cortical granule exocytosis
For at least 16 h of culture at 25°C, sperm remained firmly adhered to the oolemma and retained motility (8 h, n = 42, Fig. 2S; 12 h, n = 29; Fig. 2U), but no evidence of penetration was observed either by second polar body formation or the presence of sperm nuclei within the ooplasm after fixation and DAPI staining (Fig. 2T and V). However, after sequential washes to remove non-adherent sperm, followed by culture at 37°C, penetration by two or more sperm was detected within 90 min in oocytes previously cultured at 25°C for up to 5 h, and in these instances, penetration involved sperm that had previously attached to the oolemma at the lower temperature.
The time of penetration following culture at 37°C could be estimated in living oocytes by the displacement of most accessory sperm from the oolemma (Fig. 3C) and confirmed after fixation by (i) the absence of cortical granule LCA fluorescence (similar to Fig. 3B) and (ii) the presence of decondensing sperm nuclei in the ooplasm (arrow, Fig. 3C). In all cases (n = 89), penetration coincided with the reappearance of subplasmalemmal J-aggregate fluorescence and initially, the site of penetration seemed to correspond to regions where high-polarity first returned. However, this was difficult to confirm in each instance by coincident analysis of JC-1 and DAPI fluorescence because of the rapidity with which normal intensities of J-aggregate fluorescence returned at 37°C and the high density of sperm binding. Penetration-induced oocyte activation was indicated by formation of a second polar body, followed by the development of multiple pronuclei (e.g. Fig. 3D), and continued culture was associated with the occurrence of multinucleated blastomeres at the first cleavage division (e.g. Fig. 3E).
Oocytes previously cultured at reduced temperatures for >6 h (n = 56) mostly remained unpenetrated when returned to 37°C (52/56), despite the presence of motile sperm that had attached to oolemma during the first 15 min of culture at 25°C (images similar to Fig. 1E and F). Unpenetrated oocytes showed scant or discontinuous subplasmalemmal J-aggregate fluorescence, and in each instance, the retention of cortical granules was confirmed by LCA fluorescence after fixation (similar to Fig. 3A). In contrast, all penetrated oocytes that were either untreated (after zona removal) or penetrated at 37°C after culture at 25°C showed a normal intensity and circumferential distribution of subplasmalemmal J-aggregate fluorescence. At the time penetration was confirmed, the net ATP content of the uninseminated cohort of oocytes previously cultured at 25°C hours (e.g. 5 h, n = 111) was similar to levels measured in untreated MII oocytes (725 ± 38 fmol). Prolongation of culture at reduced temperatures appears to be inconsistent with penetration at 37°C despite the restoration of high polarity in the subplasmalemmal domain and normal levels of cytoplasmic ATP.
Effects of FCCP exposure on m, penetration and cortical granule exocytosis
As described above, oocytes cultured in the presence of FCCP showed no J-aggregate fluorescence or sperm attachment to the oolemma. However, within 10 min of insemination under normal conditions, persistent attachment was observed at different sites along the oolemma in all oocytes (n = 60; arrows, Fig. 3F); by 90 min, 70% (43/60) of oocytes were penetrated, and by 3–4 h, multiple male pronuclei were evident in each case (Fig. 3G). In all instances (n = 43), sperm penetration coincided with the detection of J-aggregate fluorescence in the subplasmalemmal cytoplasm.
Unlike penetrations that occurred in untreated oocytes, and oocytes cultured at reduced temperature, the disassociation of accessory sperm from the oolemma was not complete in some FCCP-exposed oocytes, with portions of the oolemma showing persistent sperm binding during the pronuclear (arrows, Fig. 3H) and early cleavage stages (image not shown). However, in locations where sperm remained attached, the status of J-aggregate fluorescence and cortical granules could not be determined definitively for every oocyte because the co-localization of these elements in the subplasmalemmal cytoplasm resulted in overlap of similar red-fluorescent emission signals (e.g. Fig. 3I and J). This association could be made by examining penetrated FCCP-exposed oocytes for either cortical granule (n = 52; Fig. 3I) or J-aggregate fluorescence (n = 51; HPM, Fig. 3J, RITC channel), especially in regions where sperm remained attached to the overlying oolemma. For newly penetrated oocytes previously exposed to FCCP (n = 43/60), cortical granules persisted in the subplasmalemmal cytoplasm (CG, Fig. 3K) where the overlying oolemma contained bound sperm (SP, Fig. 3L) and in sibling oocytes, the corresponding domain of mitochondria was found to be low polarized (e.g. LPM, Fig. 3J). In these instances, condensed sperm nuclei and the initial stages of male pronuclear formation occurred in proximity to a domain of high-polarized, J-aggregate positive mitochondria.
The possibility that sperm penetration and cortical granule exocytosis may require a corresponding domain of HPM was further suggested by the retention of cortical granules, and scant or undetectable subplasmalemmal J-aggregate fluorescence, in 80% (49/60) of FCCP-exposed oocytes that were unpenetrated after placement in normal medium, despite sperm attachment throughout the oolemma. For uninseminated oocytes exposed to FCCP for 30 min (n = 137), the average net cytoplasmic ATP content measured in the absence of the inhibitor at the same time penetration was observed in the corresponding group of inseminated siblings, 90 min after return to normal conditions, was 65% (510 ± 30 fmol) of normal.
Effects of BA m, penetration and cortical granule exocytosis
The above findings indicated that the presence of a domain of HPM might be more critical for penetration and cortical granule exocytosis than a normal net cytoplasmic ATP content. This notion was investigated further by culture of MII oocytes in the presence of BA. It was anticipated that by maintaining a high m in the subplasmalemmal domain, penetration rates would be similar to controls, and to FCCP exposed oocytes after high polarity was restored. For the latter, penetration occurred at a subnormal ATP level that was similar to the one measured in BA treated oocytes at the time of sperm attachment (Fig. 4). However, while extensive sperm attachment was evident in the presence of BA (Figs 2F and 3M), penetration and cortical granule exocytosis occurred only after the inhibitor was removed (n = 55/60; images similar to Fig. 3B). At penetration, the average ATP content of BA exposed oocytes was approximately 60–65% of normal. For example, after a 30 min exposure to BA (n = 120) followed by insemination in the absence of the inhibitor, penetration occurred with a net average cytoplasmic ATP content of 450 ± 28 fmol (Fig. 4). However, the timing and extent of polyspermic penetration (Fig. 3N), detachment of accessory spermatozoa from the oolemma and cleavages producing multinucleated blastomeres at the 2- and 4-cell stage were similar to untreated oocytes and to oocytes that fertilized after recovery from FCCP or reduced temperature.
The failure of penetration to occur in the presence of BA, despite the persistence of high m, suggested that penetration and cortical granule exocytosis might require the ability of HPM to undergo a transient reduction/dissipation of polarity, which as discussed below, is a normal characteristic of active mitochondria that BA inhibits. This notion was tested by culturing zona-free oocytes (n = 55) in the presence of BA for 30 min, followed by the addition of the oocyte-activating calcium ionophore, A23187.
[GenBank]
Exposure of oocytes to A23187
[GenBank]
in the presence of BA failed to induce detectable changes in cortical granule density (Fig. 3O), despite the presence of high m (e.g. Fig. 2E). However, within 15 min after the removal of BA and culture under normal conditions, the addition of the ionophore initiated cortical granule discharge (Fig. 3P). Similar to the situation observed other normal and experimentally treated oocytes, focal regions of retained cortical granules were evident (e.g. CG, Fig. 3P).
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Reduced ATP generation by mitochondrial oxidative phosphorylation is a common etiology of certain maternally inherited metabolic disorders (termed OXPHOS-diseases) that originate from oocyte mitochondria carrying specific pathogenetic defects that adversely effect respiratory capacity (McFarland et al., 2007). Because all mitochondria are maternally inherited, the proportion of genetically compromised mitochondria capable of replication (the mutant load) largely determines the extent to which post-implantation development may be compromised, and the probability that certain cytopathologies will develop later in life (Christodoulou, 2000; Jacobs et al., 2006; McFarland et al., 2007). Similarly, metabolic deficits associated with a subnormal mitochondrial complement (Santos et al., 2006; Shahinaz et al., 2006; Shoubridge and Wai, 2007; Zeng et al., 2007), or the occurrence of structural defects that compromise respiration (Muller-Hocker et al., 1996), have been suggested etiologies of failures during preovulatory oocyte maturation, fertilization and preimplantation embryogenesis (for review, Van Blerkom, 2004).
Although some proportion of early developmental failure and demise may be related to a high mutant load for certain mtDNA defects, especially in women of advanced reproductive age (Chan et al., 2005), such pathogenic defects are uncommon and unlikely to be the primary cause of developmental incompetence. In most instances, a mitochondrial contribution to developmental abnormality or embryo demise is more likely to be a metabolic deficit (Shoubridge and Wai, 2007). For example, disproportionate mitochondrial segregation during cleavage in the human (Van Blerkom et al., 2000) and pig embryo (Shahinaz et al., 2006) is associated with arrested cytokinesis and lysis in the blastomere(s) that inherited a significantly reduced organelle complement. In these instances, developmental incompetence that can be related to mitochondria is considered in the context of the total bioenergetic capacity of the oocyte or the individual cells that comprise the early embryo.
A relatively small subset of mitochondria, characterized by a comparatively high m and a stable (persistent) localization in the subplasmalemmal cytoplasm, has been proposed to have specialized functions in the oocyte and early embryo that in addition to a metabolic contribution, may involve the local regulation of important developmental activities (Van Blerkom et al., 2003; Jones et al., 2004; Van Blerkom and Davis, 2006; Van Blerkom, 2004, 2007). At the blastocyst stage, mitochondria in mural trophectodermal cells are virtually all high-polarized, whereas few in the polar trophectoderm and virtually none in the in inner cell mass exhibit a high m (Van Blerkom et al., 2002, 2006), suggesting that their activity or function may be location and cell-type specific during the peri-implantation stage. The importance of the magnitude of m as a determinant of mitochondrial activity and function has been demonstrated in somatic cells by its association with the following:
- levels of ATP production and maintenance of oxidative phosphorylation activity (Gottlieb, 2001),
- rates of cytoplasmic protein uptake by and transport within mitochondria (Huang et al., 2002; Pfanner and Truscott, 2005),
- regulation of mitochondrial volume homeostasis (Safiulina et al., 2006),
- focal control of ambient free calcium levels (Harris, 1979; Loew et al., 1994),
- responsiveness to electrical and ionic fluxes (e.g. calcium-induced, calcium release; Ichas et al., 1997; Diaz et al., 1999),
- participation in signal transduction pathways (Gutierrez et al., 2006; Quintero et al., 2006).
m and sperm penetration
Here, we report that for the mature oocyte, high m in the so-called ‘vanguard mitochondrial’ domain (Van Blerkom and Davis, 2006) is not required for persistent sperm attachment to the oolemma, but appears to be required for penetration and cortical granule exocytosis. Indeed, the findings demonstrate that attachment can occur in the absence of mitochondria in this domain. In treated oocytes where m was significantly reduced, sperm penetration occurred after the restoration of high-polarity and was followed shortly thereafter by cortical granule exocytosis and pronuclear evolution. In treated oocytes where the recovery of high-polarity in the subplasmalemmal cytoplasm was discontinuous, penetration was observed where mitochondria with high m were localized. However, this spatial correspondence was difficult to confirm in all instances in the same oocyte(s) because the high density of attached sperm often obscured fluorescent microscopic characterizations of m in the subjacent mitochondria, and in other instances, high-polarity returned relatively rapidly throughout the subplasmalemmal domain. On the basis of the present findings, we suggest that sperm attachment is independent of the polarity of the corresponding subplasmalemmal mitochondria, but high-polarity seems to be a prerequisite for penetration.
m and cortical granule exocytosis
Studies of zona-free oocytes showed that accessory sperm detached from the oolemma shortly after penetration, indicating that their displacement could be a consequence of the cortical granule reaction (exocytosis). This interpretation was supported by (i) the absence of LCA fluorescence were sperm were displaced and (ii) the persistence of attachment of cortical granules remained after penetration in untreated oocytes, oocytes cultured at reduced temperature but penetrated at 37°C, and those exposed to FCCP prior to penetration. In each instance where sperm remained bound to the overlying oolemma and the corresponding subplasmalemmal cytoplasm could be clearly visualized, cortical granules were retained in the same region where mitochondria were characterized as low polarized owing to the absence of J-aggregate fluorescence. Findings from intact (zona-enclosed) oocytes after FCCP exposure showed that penetration did not occur until subplasmalemmal J-aggregate fluorescence was restored (Van Blerkom, unpublished), indicating that the zona-free oocyte is a relevant model for the processes described in the present study.
m and focal levels of ATP and free calcium
In comparison to their lower polarized cytoplasmic counterparts, HPM in the subplasmalemmal cytoplasm of the mouse and human oocyte may be more energetic with respect to ATP generation, or have a greater capacity to be involved in the focal regulation of calcium homeostasis, or both (Van Blerkom et al., 2002, 2003). The physical association between clusters of HPM and cisternae of the smooth endoplasmic reticulum in the pericortical cytoplasm may be of particular relevance in this regard (Van Blerkom et al., 2002; Van Blerkom, 2004; Makabe and Van Blerkom, 2006). Dumollard et al. (2003, 2007) reported that mitochondrial ATP production in mouse oocytes was likely under local control and that up- or down-regulation of metabolic activity involved local changes in free calcium that in turn, were mediated by proximal elements of the ER. They proposed that calcium-mediated crosstalk between these organelles may tightly couple ATP production to changing focal demands without involving or requiring an upregulation of activity in mitochondria from more distant sites. In this context, although determinations of total cytoplasmic ATP content may provide information on the overall metabolic state of individual oocytes or blastomeres (Van Blerkom et al., 1995, 2000), the levels measured offer little insight into processes that may be spatially localized or under local control (Aw, 2000).
Here, we propose that a domain of HPM comprising a small fraction of the total mitochondrial complement and positioned in the subplasmalemmal cytoplasm may, by virtue of these properties, have a specialized role in the local regulation of developmental processes such as penetration and cortical granule exocytosis. The present findings also suggest that their participation in these processes may require the capacity to transiently dissipate m.
m and mitochondrial activity during the earliest stages of fertilization
Fully developed mitochondria in differentiated cells have been shown to be ‘excitable’ organelles that change levels of activity and polarity in response to local ionic and electrical fluxes (Ichas et al., 1997; Diaz et al., 1999). Although transient fluctuations in m are a dynamic and normal aspect of mitochondrial activity, they are not spontaneous and can be triggered by very small changes in levels of ambient free calcium that originate from the ER, or from other mitochondria through the (mitochondrial) calcium-induced calcium-release pathway (Harris, 1979; Loew et al., 1994). Transient changes in m can be accompanied by the efflux (lower polarity) or influx (higher polarity) of calcium through the megachannel (permeability transition pore complex, PTP), and their ability to regulate calcium may protect cells and these organelles from calcium overload (Harris, 1979). However, in order to maintain m at a level sufficient to drive ATP synthesis by oxidative phosphorylation, mitochondria normally function with the PTP closed (Gottlieb, 2001; Ly et al., 2003). Where mitochondria occur in highly elongated forms that can be visualized individually and in detail at the light and fluorescent microscopic levels, transient fluctuations in m have been detected between mitochondria in the same cell, and within the same mitochondrion, where regions of high and low polarity likely reflect differential activities and PTP state transitions (Smiley et al., 1991).
Although numerous, the mitochondria of mouse and human oocytes are spherical and structurally underdeveloped organelles that are <1 µm in diameter and occur in the subplasmalemmal cytoplasm in small clusters (Van Blerkom et al., 2002). These properties are problematic for investigating the extent to which fluctuations in m may occur, and whether putative changes in polarity are spatially localized and responsive to triggering signals, such as changes in free calcium (Day et al., 2000) or electrical currents across the oolemma or within the ooplasm (Gianaroli et al., 1994). However, the following results suggest that HPM in the mature oocyte have responses and characteristics that are similar to their fully developed, somatic cell counterparts.
FCCP is a proton ionophore that uncouples oxidation from phosphorylation by dissipating the chemiosmotic gradient that creates m (Mitchell and Moyle, 1967), but leaves the electron transport system functional. BA binds to the adenine nucleotide transporter (translocase, ANT) located on the inner mitochondrial membrane and inhibits phosphorylation of exogenous ADP (adenosine diphosphate) and dephosphorylation of ATP (Stoner and Sirak, 1973). ANT is a component of the mitochondrial megachannel and its inhibition by BA prevents its opening and the dissipation of m. By inhibiting mitochondrial depolarization, and in some cells maintaining m at a constitutive high state, BA has been shown to have antiapoptotic properties (Mastrangelo et al., 1999; Muranyi and Li, 2005) and in healthy cells, has been reported to slow the active uptake and efflux of calcium by mitochondria (Harris, 1979).
Although oocyte mitochondria are currently not amenable to the same types of polarity studies performed with somatic cells, when considered as a group, subplasmalemmal HPM share the following behaviors typical of their somatic cell counterparts (Van Blerkom et al., 2003): (i) they dissipate m in the presence of FCCP, (ii) maintain high-polarity in the presence of BA and (iii) at subnormal temperatures, J-aggregate formation that reports a hyperpolarized state is precluded. The reversible inhibition of penetration and cortical granule exocytosis seen with BA and FCCP would seem paradoxical because of the opposite effects these inhibitors have on the magnitude of m. However, a common response for the oocyte could be explained if, as the present findings indicate, these processes require the presence of HPM in the subplasmalemmal cytoplasm that can be triggered by exogenous factors to undergo changes in state. The latter notion is supported by preliminary findings in which the calcium ionophore A23187
[GenBank]
was ineffective in promoting cortical granule discharge in J-aggregate positive oocytes if megachannel opening in subplasmalemmal HPM was inhibited by BA.
Caveats related to manipulations of m and ATP content
The focus of this study was limited to the attachment and penetration phases of fertilization and the extent to which high m in subplasmalemmal mitochondria may be involved or required. However, deriving definitive conclusions concerning the potential role of mitochondrial polarity in these events can be complicated by the differential effects each experimental treatment may have on cytoplasmic activities, including bioenergetic status as indicated by net cytoplasmic ATP contents. As previously reported (Van Blerkom et al., 2003) and confirmed here, relatively short-term culture in the presence of FCCP or BA significantly reduced the average net ATP content of MII mouse oocytes, but only in the case of the former was attachment precluded. The absence of J-aggregate fluorescence during FCCP exposure is unlikely to be related to attachment failure as firm binding occurred in other experimental treatments where high m was similarly affected. Transient reductions in ATP content also seem unrelated to sperm attachment, as shown by findings with BA, culture under reduced temperatures and oocyte segmentation into cytoplasts and karyoplasts. FCCP may have pleiotropic effects on cytoplasmic activities that coincidentally affect the oolemma and its ability to bind sperm; these putative effects are reversible, as shown by sperm attachment at normal levels shortly after the inhibitor was removed.
Both attachment and penetration occurred in experimental treatments that transiently reduced m and where net cytoplasmic ATP levels were well below normal. Although cytoplasmic ATP levels were near normal at a reduced culture temperature, penetration failure could be associated with temperature-sensitive alterations in oolemmal structure or with perturbed molecular activities necessary to promote penetration and cortical granule exocytosis. However, in all instances, the ability of an oocyte to be penetrated and to discharge cortical granules was present only after high-polarity was restored in subplasmalemmal mitochondria. In this regard, extensive sperm binding but no penetration was observed in J-aggregate-positive, BA-treated oocytes. The ATP content of these oocytes was similar to the subnormal level measured at penetration in oocytes previously exposed to FCCP. Penetration and cortical granule exocytosis occurred in BA treated oocytes shortly after the removal of the inhibitor and while the ATP content was subnormal. This finding confirms that a reduced ATP content may not be a proximate cause of penetration failure. Rather, fertilization failure in the presence of BA may be associated with the inability of HPM to dissipate m in response to triggering signals that may originate with the attached sperm.
Taken together, the present results suggest the following: (i) persistent sperm attachment to the oolemma is independent of the state of polarity of the subjacent mitochondria and as demonstrated by attachment to mitochondria-deficient karyoplasts, may be unrelated to, or require a very low threshold ATP content (Shahinaz et al., 2006) and (ii) high m in the subplasmalemmal domain may be a prerequisite for sperm penetration and cortical granule exocytosis.
Developmental implications of a subplasmalemmal domain of HPM
Typically, after entrance into the cytosol, ATP is utilized almost immediately and within a few microns of the site of generation (Aw, 2000). In the context of the findings reported here and elsewhere (Van Blerkom et al., 2003, 2006; Van Blerkom, 2004), it seems reasonable to speculate that local mitochondrial activity may be a critical factor in the regulation of developmental events that are themselves spatially localized to the oolemma or subplasmalemmal cytoplasm. This notion would seem to be particularly applicable in the case of fully grown oocyte, which at 80–100 µm in diameter in the mouse and human, respectively, is the largest cell in the body. We suggest that the domain of vanguard mitochondria normally forms a largely continuous ‘circuit’ of reactive elements that may locally respond to and propagate triggering signals within the subplasmalemmal cytoplasm. During the earliest stages of development, this response may involve electrical and ionic fluctuations associated with the attachment, penetration or incorporation of the fertilizing spermatozoon (Gianaroli et al., 1994, Sousa et al., 1996, 1997). Evidence suggestive of such a role was the detection of discontinuities in this ‘circuit’ represented by J-aggregate negative regions where (i) the corresponding oolemma was refractory to penetration despite sperm binding and (ii) if the oocyte was penetrated, cortical granules persisted where subplasmalemmal mitochondria were low polarized. In this regard, HPM may be among the first elements in the oocyte to respond to the fertilizing sperm, perhaps by undergoing a transient reduction in m with a coincident calcium discharge, as previously suggested (Van Blerkom et al., 2003).
Implications for clinical IVF
The above results suggest that a relatively small number of high-polarized ‘vanguard mitochondria’ may have specialized developmental functions in the oocyte and early embryo that are location based. Whether the inability of the subplasmalemmal cytoplasm to respond to the presence of a bound or penetrating spermatozoon is related to, or influenced by, the polarity of the corresponding mitochondria, or their capacity to transition between states of m, as suggested here, warrants further investigation. If confirmed, the presence of a non-responsive population of mitochondria in the subplasmalemmal cytoplasm could provide new insights into the origin of certain fertilization failures, such as those observed in conventional clinical IVF with fresh (Van Blerkom et al., 2002) and thawed oocytes (Jones et al., 2004) where defects in this domain have been reported.
The present results suggest that while the contribution of HPM to the net cytoplasmic ATP content of the oocyte may be negligible, as might be expected from the proportion of the organelle complement involved, they may have specialized functions or activities in the oocyte that are location-based. For example, locally elevated levels of ATP produced by high m subplasmalemmal mitochondria may drive stage-specific phosphorylation(s) of developmentally significant proteins, such as transmembrane receptors, or activate molecules involved in signal transduction (Quintero et al., 2006). Another speculative possibility is that they are involved in the modification of lipids (Zimijewski et al., 2005; Gutierrez et al., 2006), which if precursor components of the oolemma could alter its organization, making it permissive for penetration or cortical granule exocytosis.
The importance of m in early development is also suggested by the study of Muller-Hocker et al. (1996), who correlated fertilization failures in the oocytes of women of advanced maternal age with the occurrence of swollen mitochondria. Mitochondrial swelling results from an inability to maintain m at levels sufficient to sustain normal mitochondrial volume homeostasis and is associated with metabolic and ionic regulatory dysfunctions (Safiulina et al., 2006). We are investigating whether mitochondria in oocytes with an absent or deficient subplasmalemmal domain are swollen or show fine structural defects similar to those reported by Muller-Hocker et al. (1996). Confirmation of such a defect would support our current interpretation that the inability to establish or maintain a domain of HPM can be a proximate cause of penetration and fertilization failure.
m and cytoplasmic maturation of the preovulatory oocyte
The interruption of direct intercellular communication between the oocyte and somatic cell compartment is largely considered the first step in the initiation of preovulatory maturation (Albertini, 2004). It has been suggested that subplasmalemmal mitochondria shift from low- to high-polarity after the spontaneous withdrawal of transzonal coronal cell processes (TZPs) from the surface of the germinal vesicle stage oolemma (Van Blerkom et al., 2002). These authors reported that where TZPs persisted on the oolemma, the underlying mitochondria were J-aggregate negative, but became positive after their mechanical disruption. If subplasmalemmal mitochondria have specialized roles in fertilization-related processes, as the present results indicate, an increase in m within this domain might also represent an essential first step in cytoplasmic maturation. Although the notion of a domain of HPM that becomes functional during preovulatory maturation to form a signaling circuit in the subplasmalemmal cytoplasm is a speculative one, confirmation of such a capacity may offer new insights into cellular processes that lead to the acquisition of fertilization capacity and normal developmental competence.
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Submitted on August 17, 2007; accepted on September 13, 2007.
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