1 Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80302 and 2 Colorado Reproductive Endocrinology, Rose Medical Center, Denver, CO, 80220, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: cleavage stage embryos/metabolism/mitochondria/microtubules/pronuclear embryos
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Here, it is investigated whether spatial differences in the organization of mitochondria at the pronuclear stage may be another developmentally significant characteristic of early human embryos that may determine competence. Preliminary studies of mitochondrial segregation between blastomeres in 2-4-cell human embryos (conventional IVF) demonstrated disproportionate patterns of mitochondrial inheritance (Van Blerkom, 2000). In cases where most mitochondria were partitioned into one blastomere, the deficient blastomere remained undivided and lysed during subsequent culture. In the present study, the objectives were to determine (i) whether differences in mitochondrial distribution occurred among individual blastomeres in cohorts of morphologically normal (unfragmented) cleavage stage embryos derived from `high-competence' pronuclear oocytes, (ii) whether differences in mitochondrial inheritance were related to blastomere ATP content, and (iii) whether a cytoarchitectural basis for specific patterns of mitochondrial organization existed at the pronuclear stage.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Microscopic analysis
Here, the analysis was confined to pronuclear oocytes (and resulting embryos) that, at 16-18 h after insemination, showed an equatorial nucleolar alignment and an unambiguous cytoplasmic flare or pronuclear halo, morphological characteristics that have been suggested to indicate high developmental competence. Analysis of mitochondrial fluorescence in living specimens used scanning laser confocal microscopy (SLCM) with temperature and atmosphere maintained during observation in DT culture dishes (Bioptechs, Inc., Butler, PA, USA) as previously described (Van Blerkom et al., 1995a). Research protocols that included patient consents permitted two types of analyses to be undertaken for normally fertilized oocytes: (i) analysis during the pronuclear stages up to syngamy and (ii) maintenance of embryos for up to 5 days in vitro. In the first case, appropriate pronuclear oocytes were stained with either R123 or NAO and examined in detail by SLCM through syngamy. After SLCM examination, specimens were fixed in formaldehyde, prepared for anti-b-tubulin immunofluorescence, and re-examined by SLCM. In the second case, which also included dispermic oocytes, stained specimens were observed at 16-18 h intervals from the pronuclear to the 16-cell stage. Unpenetrated, monopronuclear oocytes were examined at 16-18 h after insemination and subsequently fixed for immunostaining. Scans of pronuclear oocytes involved serial optical sections taken at intervals of 5 µm, which required 3.5 min to complete. Subsequent analyses of R123 or NAO fluorescence in intact cleavage stage embryos involved sections taken at intervals of 10 µm and <1 min to complete. After disaggregation, individual blastomeres used for ATP analysis were re-scanned to confirm relative intensities of fluorescence observed in the intact embryo. Representative intact embryos were fixed for anti-b-tubulin immunofluorescence. Analysis of mitochondrial and microtubular fluorescence used both serial sections and fully complied images, with blastomere nuclear and cytoplasmic volumes determined from digital images using ImageSpace software (v.3.10, Molecular Dynamics, Sunnyvale, CA, USA). Pseudo-colour images produced by SLCM were generated from specimens examined at the same photomultiplier detector gain and are presented as observed. The colour bar included in the figures represents the spectrum of fluorescence detected (lowest = blue, highest = white) with numerical values (relative fluorescent units) from 0 to 255 indicated.
ATP content
Individual blastomeres were rapidly frozen to 80°C in 200 ml of ultrapure water. ATP content was determined quantitatively by measurement of the luminescence (Berthold LB 9501 luminometer) generated in an ATP-dependent luciferin-luciferase bioluminescence assay as previously described (Van Blerkom et al., 1995b). A standard curve containing 14 ATP concentrations from 5 fmol/l to 5 pmol/l was generated for each series of analyses. To reduce potential variability between samples, blastomeres from different embryos were maintained at 80°C and analysed simultaneously. The ATP content in aliquots of selected blastomeres was determined both within and between bioluminescence assays.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
All unpenetrated monopronuclear oocytes in which a cytoplasmic flare developed (n = 6, asterisk, Figure 1F) showed a perinuclear accumulation of mitochondria (M, Figure 1F
) that was asymmetrical when observed by SLCM (Figure 1G and H
). In comparison to normally fertilized or dispermic oocytes, monopronuclear oocytes exhibited a perinuclear mitochondrial accumulation of mitochondria in consecutive sections and fully compiled images (e.g. compare Figure 1B and G with 1C and H
) that was less pronounced and largely localized to one portion of the single pronucleus. For all unpenetrated monopronuclear oocytes (n = 5) in which no cortical clearing was detected, mitochondria were uniformly distributed with no detectable perinuclear accumulation (comparable to image in Figure 1L
). The cleavage potential of unpenetrated monopronuclear oocytes was not determined, as all such oocytes were fixed for tubulin immunofluorescence analysis.
For nine 2-cell embryos that developed from pronuclear oocytes in which the perinuclear accumulation of mitochondria was relatively symmetrical (e.g. Figure 1B and 1C), both blastomeres showed similar intensities and distributions of mitochondrial fluorescence throughout the 2-cell stage (Figure 1O
). For eleven 2-cell embryos that developed from pronuclear oocytes with pronounced mitochondrial asymmetry and relatively large regions of cytoplasm devoid of mitochondrial fluorescence, differences in the intensity of R123 and NAO fluorescence between blastomeres were evident in all cases. For example, the representative embryo presented in Figure 1N
was examined during the first cleavage division and a very significant reduction in mitochondrial fluorescence is evident in one developing blastomere (asterisk, 10 µm SLCM section). Figure 1D
is representative of the type of asymmetrical mitochondrial distribution observed at the pronuclear stage which has been observed to be associated with profound distortions in the pattern of mitochondrial segregation and disproportionate inheritance between blastomeres at the first cell division. The white line in Figure 1D
indicates the plane of the first cleavage division observed in time-lapse recordings. Other 2-cell embryos in which the amount of disproportionate mitochondrial segregation was less severe than in the embryos shown in Figure 1D and N
, were examined during subsequent cleavage divisions (e.g. Figure 1P
).
For 14 embryos observed by SLCM during the first cleavage division, mitochondria segregated in a polarized manner such that the highest intensity of fluorescence was initially detected in the apical cytoplasm of daughter blastomeres (asterisk, Figure 1M). After completion of cytokinesis, a pronounced perinuclear accumulation of mitochondria was evident in all blastomeres (e.g. Figure 1O
). Blastomeres with reduced inheritance exhibited pronuclear mitochondrial aggregation, but of lower intensity (e.g. upper blastomere, Figure 1P
).
Four- to 16-cell stage
At the 4-cell stage (2PN, derived from: n = 8; 3PN, n = 12), blastomere-specific differences in the relative intensity of mitochondrial fluorescence were clearly evident for 14 embryos that showed similar differences at the 2-cell stage, and which developed from pronuclear oocytes in which mitochondria were asymmetrically distributed. For some embryos (n = 6/20), a reduced amount of mitochondrial fluorescence was observed in one cell (asterisk, Figure 1Q). In order to preclude the possibility that differential mitochondrial fluorescence observed in intact embryos was related to blastomere position during SLCM analysis, these embryos were disaggregated either partially or completely into component blastomeres and rescanned. The representative embryo presented in Figure 1Q-S
shows blastomere-specific mitochondrial fluorescence in a fully compiled image of an intact 4-cell embryo from a monospermic fertilization (Figure 1Q
) followed by analysis where three blastomeres were left intact (Figure 1R
) and the fourth examined in isolation (Figure 1S
). The blastomere with reduced mitochondrial fluorescence detected in the intact embryo and after removal of a single blastomere is indicated by an asterisk in Figure 1Q and R
, respectively. Sequential fluorescence analysis performed on intact embryos and disaggregated blastomeres at the 4-cell stage (n = 4) showed one or two blastomeres with significantly reduced intensities of NAO fluorescence. All of these embryos had developed from pronuclear oocytes in which a pronounced asymmetry in peripronuclear R123 or NAO fluorescence was evident. Other intact embryos with mitochondrially deficient blastomeres were maintained in culture and examined by SLCM at 24 h intervals (see below).
Shortly after completion of the second cleavage division, the intracellular distribution of mitochondrial fluorescence in each blastomere of the 4-cell embryo was polarized. The highest intensity of R123 or NAO (Figure 1Q and R) was usually detected in the apical cytoplasm and reduced intensity of fluorescence common to those regions of the cytoplasm where blastomeres were in opposition (M, Figure 1R
). For some blastomeres, however, the highest concentration of mitochondrial fluorescence was not positioned apically, such as shown in Figure 1R
. In these instances, differences in the relative position of the mitochondrial aggregate may be related to rotations blastomeres can undergo during early cleavage. In contrast, R123 or NAO fluorescence was uniformly distributed in blastomeres that exhibited low levels of mitochondrial fluorescence (similar to light blue cells in Figure 1Y1
). For 4-cell embryos that developed from pronuclear oocytes in which mitochondrial distribution was relatively symmetrical (e.g. Figure 1B
), similar intensities of mitochondrial fluorescence were detected between blastomeres (images shown at 8-cell stage, Figure 2A1-8
). Monospermic (n = 6) and dispermic (n = 5) embryos with similar intensities of R123 fluorescence between blastomeres were retained in culture and examined at the 8-16-cell stage for blastomere-specific mitochondrial fluorescence and ATP content.
|
The development of 39 normal-appearing monospermic 46-cell embryos (day 3) where half of the blastomeres showed low intensity R123 fluorescence (e.g, Figure 1Y1) was determined during an additional 2 days of culture. Fifty-six per cent (22/39) arrested cleavage at the 810-cell stage. Forty-four per cent (17/39) contained between 16 and 21 cells on day 5 and were classified as stage-inappropriate. During additional culture, some blastomeres in which very low R123 or NAO intensities were detected at the 68-cell stage arrested division and subsequently lysed. Blastomere lysis was indicated by the elaboration of plasma membrane blebs followed by blastomere swelling and subsequent lysis several hours later. For example, Figure 1V and W
are time-lapse images of a 6-cell embryo which appeared normal at 50 h post-insemination (day 2.5), but where the relative intensity of R123 fluorescence in one blastomere was clearly reduced at the 4-cell stage when compared to other cells in the embryo (similar to Figure 2B1
). At 58 h, small plasma membrane blebs were observed on cell surface of this blastomere, some of which eventually detached and were detectable as discrete entities in the perivitelline space (arrows, Figure 1V
). At 65 h, this blastomere appeared to swell (asterisk, Figure 1V
) and its increased size was accompanied by a reduction in cytoplasmic density. Fourteen hours after blebs were first detected (72 h), and 6 h after swelling was initially observed, this blastomere abruptly burst, leaving behind a cytoplasmic matrix (asterisk, Figure 1W
) enclosed by remnants of the plasma membrane and small membraneous ghosts in the perivitelline space. Cell division continued in this representative embryo and 18 mononucleated blastomeres were counted on day 5. The embryo shown in Figure 2B15
is also representative of an apparently normal appearing monospermic 8-cell embryo that developed from a `high competence' pronuclear embryo with asymmetrically localized mitochondria. For embryos of this type, where several blastomeres showed low R123 (or NAO) intensity, development beyond the 810-cell stage was not observed after 2.5 additional days, at which time culture was terminated. To date, the developmental capacity of 16 monospermic embryos with comparable levels of blastomere R123 fluorescence (similar to Figure 2A18
) at the 8-cell stage has been examined during 6 days of culture, as described previously (Van Blerkom, 1993
). Eleven embryos (68%) developed into blastocysts, two (13%) showed signs of cavitation, and three were compacted morulae (19%). If confirmed, this preliminary finding suggests that mitochondrial inheritance among blastomeres may be one factor that influences the ability of the human embryo to develop progressively.
For normally developing, stage-appropriate embryos, comparable intensities of R123 fluorescence that were previously observed between blastomeres at the 4- and 8-cell stages were no longer apparent at 1216-cell stage. While only four monospermic 1216-cell embryos of this type were examined, R123 fluorescence was clearly detectable in all mono- (e.g. Figure 2C2, 4 and 11) and binucleated (Figure 2C3
) blastomeres and differed in intensity between blastomeres, as shown for 12 cells of one such embryo in Figure 2C112
. Blastomeres from these embryos were used for ATP determination (see below). However, the position of individual cells in the intact embryos (inside or outside) was not correlated with specific blastomeres after disaggregation. The intensity of R123 fluorescence was examined in 13 binucleated blastomeres (e.g. Figure 2C3
) obtained from 13 normal appearing 812-cell monospermic embryos. The relative intensities of mitochondrial fluorescence in these cells were largely comparable to those observed in mononucleated blastomeres in the same embryo (e.g. compare Figure 2C3 and 4
).
Quantitative determinations of blastomere-specific ATP content in normal and slow developing 810-cell embryos
Whether the net ATP content of an individual blastomere and apparent intensities of mitochondrial R123 fluorescence were related was determined by measuring blastomere-specific net ATP content after SLCM analysis. The values presented in Table I are representative results from two classes of cleavage stage embryos that were available for analysis: (i) intact, stage-appropriate embryos (810 cells on day 3, n = 9) with comparable intensities of mitochondrial fluorescence between blastomeres determined by sequential analysis from the 2-cell stage (e.g. Figure 2A1-8
), and (ii) intact (unfragmented) monospermic embryos that were classified as `slow developing' and stage inappropriate (e.g. 8-cell on day 3.5, Figure 2B15
, n = 9; 1012-cell on day 4.5, n = 11). Slow-developing embryos were derived from pronuclear oocytes with asymmetric mitochondrial distribution and different intensities of R123 fluorescence were detected between blastomeres at the 2-cell stage.
|
Microtubule organization in pronuclear and early cleavage stage embryos
All 10 unpenetrated monopronuclear oocytes examined in this study that exhibited an asymmetrical perinuclear aggregation of mitochondria (e.g. Figure 1G, H) and a cytoplasmic flare (asterisk, Figure 1F
) showed a corresponding asymmetry in the distribution of microtubular arrays that extended into the cytoplasm from the vicinity of the nuclear membrane (Figure 3A
). Cytoplasmic regions with reduced intensities of mitochondrial fluorescence were largely devoid of microtubules (asterisk, Figure 3A
). For these activated oocytes, SLCM analysis showed several intense foci of tubulin fluorescence associated with the nuclear membrane with arrays of microtubules emanating from these structures. Serial SLCM sections through the nuclear region indicated that these foci were not uniformly distributed but rather clustered to one region of the nuclear membrane (arrows, Figure 3B
). For all six unpenetrated monopronuclear oocytes in which no cytoplasmic flare was evident, a uniform cytoplasmic distribution of mitochondrial fluorescence was observed. The cytoplasm was devoid of detectable microtubules, and no perinuclear foci of tubulin immunofluorescence was detected (Figure 3C
).
|
For all 24 high competence pronuclear oocytes examined, increased intensities of mitochondrial R123 and NAO fluorescence were associated with regions of the cytoplasm containing the highest apparent density of microtubules. For example, the region indicated by an asterisk in Figure 3G was virtually devoid of mitochondrial fluorescence similar to the image shown in Figure 1D
. The relative symmetry of peripronuclear mitochondrial fluorescence was associated with the pattern and density of microtubular arrays extending into the cytoplasm from the nuclear region, including those originating from distinct perinuclear foci (e.g. arrows, Figure 3D and E
).
During formation of the 2-cell embryo (n = 6), the highest density of microtubules was located in the apical cytoplasm with virtually no detectable microtubules in basal regions where segregation of the cytoplasm into daughter blastomeres was taking place (asterisks, Figure 3H). This arrangement of microtubules was correlated with the distribution of mitochondrial fluorescence at this stage (e.g. Figure 1M
). The varying patterns and intensities of perinuclear mitochondrial fluorescence observed during the 2-cell stage (Figure 1O and P
) correspond to differences in the organization and density of perinuclear microtubules that appear to be cell cycle-related. For example, perinuclear mitochondrial fluorescence that was relatively intense and symmetrically distributed (e.g. Figure 1O
) occurred in blastomeres where arrays of nuclear membrane-associated microtubules radiated into the adjacent cytoplasm in a largely radial fashion during the early 2-cell stage (e.g. Figure 3I
).
All blastomeres of 4-cell (n = 6) and 8-cell embryos (n = 5) derived from pronuclear oocytes with symmetrically distributed mitochondria exhibited similar patterns of microtubular distribution. Typically, the density of the microtubular network in these embryos was reduced in one portion of each blastomere (MT, Figure 3J and K). Each nucleus was associated with at least one focus of intense tubulin fluorescence (arrows, Figure 3J and K
). For each blastomere at the 4-cell stage, the region of cytoplasm associated with the highest intensities of mitochondrial fluorescence (e.g. Figure 1R and S
) occurred in regions containing the highest density of microtubules as observed in serial SLCM sections and fully complied images. At the 8-cell stage for both monospermic (n = 3) and dispermic embryos (n = 8), microtubules were found throughout the cytoplasm (Figure 3L
). However, microtubules were often deficient in basal regions of the cytoplasm where blastomeres were opposed (asterisks, Figure 3L
). For these blastomeres, mitochondrial fluorescence was also diminished or absent in these regions (e.g. Figure 2A4 and 5, and B2 and 4
). For 4- (n = 4) and 8-cell embryos (n = 5) that developed from pronuclear oocytes with highly asymmetric mitochondrial distributions (e.g. Figure 3L
), the perinuclear and cytoplasmic distribution of microtubules in blastomeres with relatively normal levels of R123 fluorescence was comparable to organizations detected in cleavage stage embryos that developed from pronuclear oocytes with symmetrically distributed mitochondria. In contrast, for blastomeres with very low intensity R123 or NAO fluorescence (e.g. asterisk Figures 1N and 2B1
), the cytoplasm was devoid of microtubules detectable by SLCM (similar in appearance to Figure 3C
).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Here, evidence is provided that mitochondrial distribution at the pronuclear stage may be another epigenetic factor related to the organization of the 1-cell embryo that is developmentally relevant with respect to embryo competence. The recognition that mitochondria have a fundamental role in the normality and success of early human development has emerged recently as one possible explanation for early developmental failure in general and the progressively higher frequencies of failure observed in women of advanced reproductive age in particular (Jansen and de Boer, 1998). One current view of mitochondrial function is that reduced meiotic competence and fertilizability for the oocyte (Barritt et al., 1999
; Perez et al., 2000
) and developmental failure in the preimplantation embryo could result from pre-existing oocyte mitochondrial DNA (mtDNA) defects, or from an age-related accumulation of mtDNA mutations (Keefe et al., 1995
). Presumably, mtDNA defects that reduce oxidative phosphorylation capacity could have adverse developmental consequences if accompanied by diminished ATP. An inability to regulate or mobilize intracellular calcium could be a functional defect in genetically altered mitochondria which may affect cell cycle regulation in the early embryo (Sousa et al., 1997
). However, the extent (if any) to which specific human oocyte mtDNA defects are proximate causes of early reproductive failure or are related to specific aetiologies of infertility has yet to be determined unambiguously (see below, and Brenner et al., 1998).
The relative contribution of mitochondrial oxidative phosphorylation to ATP production during the preimplantation stages has been examined in several mammals, including mice, rats, cows, and humans (Magnusson et al., 1986; Hardy et al., 1989
; Gott et al., 1990
, Brison and Leese, 1994
; Houghton et al., 1996
; Thompson et al., 1996
, 2000
), and the findings indicate that ATP generation during pre-compaction stages is largely dependent upon oxidative phosphorylation, with a shift to glycolysis occurring during cavitation and the blastocyst stages (see review by Van Blerkom et al., 1998). In support of this notion is the birth of mice that developed from embryos exposed to inhibitors of mitochondrial protein (31.2 µg/ml, chloramphenicol) and RNA synthesis (0.1 µg/ml, ethidium bromide) during the preimplantation stages (Piko and Chase, 1973
). Because these biosynthetic activities are normally activated between 8- and 16-cell stages in the mouse (Piko and Chase, 1973
), pre-existing (oocyte) mitochondrial proteins and transcripts are likely utilized prior to cavitation to generate ATP. At higher concentrations, Piko and Chase (1973) reported developmental delay or arrest, suggesting that mitochondrial sensitivity to these inhibitors may exist in early cleavage stage embryos. Van Blerkom et al. (1995b) showed that mouse oocytes exposed to uncouplers of mitochondrial oxidative phosphorylation matured in vitro with net ATP contents significantly below values present in untreated controls. While treated oocytes were fertilizable, development was severely compromised with most arresting during cleavage. Other species, such as the bovine, appear to be less dependent upon mitochondrially generated ATP during cleavage, at least in vitro, as indicated by the ability of early embryos to up-regulate glycolysis in the presence of inhibitors of oxidative phosphorylation (Brison and Leese, 1994
; Thompson et al., 1996
, 2000
).
For the human, mitochondrial oxidative phosphorylation may be the primary mechanism of ATP generation in the oocyte, newly fertilized oocyte and pre-compaction cleavage stage embryo. In this respect, it has been suggested that mitochondrial `transfusion' into MII oocytes by cytoplasmic donation (Cohen et al., 1998) or by direct injection of these organelles (Van Blerkom et al., 1998
) may be one factor associated with an apparent improvement in the normality of development during early cleavage and outcome after embryo transfer in some women who previously failed to conceive (with their untreated gametes). Decreased mitochondrial inheritance in early blastomeres owing to disproportionate segregation may account for the reduced ATP content detected in cells with diminished or poor R123 or NAO fluorescence. The extent to which mitochondrially deficient blastomeres may be able to compensate for reduced oxidative phosphorylation capacity by up-regulating glycolytic activity, as recently reported for cultured bovine oocytes (Krisher and Bavister, 1999
) and embryos (Thompson et al., 2000
), is unclear. However, the arrest of cell division and eventual cell death for affected blastomeres suggests that diminished ATP generating capacity can have adverse developmental, if not lethal consequences.
The apparent reliance on oxidative phosphorylation in maturing oocytes and early embryos may explain why, in some species, mitochondria undergo redistribution at these stages. During meiotic maturation of the mouse oocyte, mitochondria are translocated to the nuclear region during germinal vesicle break down and MI spindle formation (Van Blerkom and Runner, 1984; Tokura et al., 1993
) along arrays of microtubules emanating from perinuclear microtubule organizing centres (Van Blerkom, 1991
). In pronuclear hamster oocytes and early cleavage stage embryos, stage-specific mitochondrial redistributions include a pronounced translocation to the nuclear region, which is apparently mediated by cytoarchitectural elements (Barnett et al., 1996
). Perturbations from normal patterns of mitochondrial translocation and distribution during oocyte maturation in the mouse (Van Blerkom, 1991
) and early cleavage in the hamster (Barnett et al., 1997
) have been shown to have lethal consequences. It seems that the maturing oocytes and newly fertilized oocytes of several species have developed a common strategy to adjust mitochondrial density to different intracellular regions where locally high concentrations of ATP (Van Blerkom and Runner, 1984
) or mobilized calcium (Sousa et al., 1997
) may be required at different stages of development. The possibility that a similar pattern of mitochondrial redistribution occurs during early human development and may be of developmental significance is discussed below.
Disproportionate mitochondrial inheritance occurs during cleavage
Here, fluorescent probe analysis by SLCM demonstrates that development in newly fertilized oocytes exhibiting morphodynamic characteristics suggestive of competence is associated with a pronounced ellipsoidal accumulation of mitochondria around the opposed pronuclei. Peripronuclear aggregation is accompanied by the depletion of mitochondria from the cortical cytoplasm where a cytoplasmic flare is detectable or circumferential clearing of the cytocortex produces a pronuclear halo. However, the results also demonstrate that significant differences in peripronuclear mitochondrial organization occur between oocytes in the same and different cohorts, which range from relatively symmetrical to grossly asymmetrical. When pronuclear oocytes with a pronounced asymmetrical distribution of mitochondria were examined during syngamy, mitochondrial distribution remained asymmetrical and when re-examined after the first cell division mitochondrial inheritance, as indicated by the intensity of R123 or NAO, fluorescence differed between daughter blastomeres. If cleavage progressed to the 46-cell stage, one or two blastomeres showed intensities of mitochondrial fluorescence that were reduced considerably when compared to other blastomeres in the same embryo. Analysis of ATP content demonstrated comparatively low amounts in blastomeres with reduced mitochondrial fluorescence. As reported elsewhere (Van Blerkom, 2000), blastomeres that are profoundly deficient in mitochondrial fluorescence at the first or second cleavage division remain undivided and often die during subsequent culture.
The current protocol does not permit transfer of embryos exposed to fluorescent probes, and, consequently, whether outcome is related to specific patterns of mitochondrial inheritance observed during early cleavage is unknown. However, the occurrence of a single blastomere with poor mitochondrial fluorescence and reduced ATP content at 46-cell stage does not necessarily indicate a premorbid or developmentally adverse condition because cell division continued in other blastomeres. In contrast, competence was impaired significantly in embryos derived from pronuclear oocytes where pronounced mitochondrial asymmetry was associated with half of the blastomeres in 6- and 8-cell stage embryos exhibiting poor R123 or NAO fluorescence. In these instances, cell division arrested for the affected blastomeres, and, for the embryo, development either arrested or progressed slowly, with cell numbers clearly inappropriate for days 35 of culture. It is suggested that the number of blastomeres affected by disproportionate mitochondrial inheritance may be a proximal determinant of developmental competence.
Blastomere-specific differences in R123 intensity were detected in all 1216-cell embryos that were stage appropriate with respect to cell number on days 3 and 4 of culture. Because none of these embryos showed pronounced asymmetry in mitochondrial distribution at the pronuclear stage, it is unlikely that these differences are related to aberrant segregation patterns at the first cell division. However, neither the ATP content nor the relative intensity of R123 fluorescence detected in these blastomeres occurred at the reduced amounts observed in arrested blastomeres. The variability in R123 intensity and ATP content detected in presumably normal 1216 cell embryos may indicate stage-specific differences in amounts of ATP generation between blastomeres, perhaps reflecting differential activation of glycolysis, or possibly cell position, which may influence relative substrate availability, especially for those cells in the interior of the embryo.
The human mitochondrial respiratory chain involves 83 polypeptides, 70 of which are encoded by nuclear genes and 13 by mtDNA (Leonard and Schapira, 2000). Normal mitochondrial function and replication are dependent upon the interaction of nuclear and mitochondrial genes, and abnormalities of either genome can result in mitochondrial disease (Larsson and Clayton, 1995
; Chinnery and Turnbull, 1999
). An essential role for mitochondrially generated ATP in early human development would seem to be negated by the birth of infants with mitochondrial diseases (OXPHOS diseases) inherited from the oocyte and which effect the efficiency of oxidative phosphorylation. While some of these diseases are lethal during gestation or shortly after birth, others result in debilitating or fatal pathophysiologies associated with progressive tissue and organ degeneration later in life (DiMauro, 1998
). Although it is unknown which, if any, mitochondrial diseases are lethal prior to implantation, the occurrence of individuals with OXPHOS disorders would tend to suggest that early cleavage may not have an absolute requirement for this mode of ATP generation. However, it is possible that if embryo mitochondrial respiration occurs at a reduced level, a threshold may exist that is consistent with early embryogenesis as long as mitochondrial segregation between blastomeres was largely equivalent. It is unknown whether rates of cell division, blastocyst expansion and hatching in affected embryos may be slower than normal or delayed, as has been shown for mouse embryos cultured in the presence of inhibitors of mitochondrial transcription and translation (Piko and Chase, 1973
). In this respect, the pathogenic effects of OXPHOS defects, whether involving proteins encoded by nuclear or mtDNA, may not compromise preimplantation development, but will have downstream consequences associated with cell-type or organ-specific dysfunction that are related to tissue-specific requirements for mitochondrially derived ATP (Schon and Grossman, 1998
; Leonard and Schapira, 2000
). For normal cleavage stage embryos, it is proposed that blastomere arrest or demise occurs when disproportionate mitochondrial inheritance is accompanied by a diminished capacity to generate ATP at threshold values. In these instances, the response of the affected blastomere(s) may be similar to that experienced by specific tissues in individuals with OXPHOS diseases that perturb normal function or are ultimately fatal in certain cell types (e.g. nerve, muscle; Hammans, 1994). This notion is further supported by previous findings that correlated implantation potential and embryo ATP content (Van Blerkom et al., 1995b
).
The presence of a non-dividing binucleated blastomere(s) appears to be a common feature of midlate cleavage stage human embryos that does not seem to adversely effect competence (Tesarik, 1994). In this study, the possibility that multinucleation occurs in blastomeres where a reduced ATP environment resulted from mitochondrial malsegregation during previous divisions was examined. The rationale for this analysis was the assumption that a threshold amount of ATP related to the mitochondrial content of a particular blastomere may be permissive for DNA replication and nuclear membrane assembly but not for cytokinesis. Here, relative levels of R123 fluorescence and ATP content were determined for 13 binucleated blastomeres in thirteen 1216-cell embryos. The findings demonstrated that ATP contents and fluorescent intensities in binucleated blastomeres were largely comparable to amounts detected in mononucleated cells from the same and from different embryos. It appears that diminished ATP production is an unlikely cause of binucleation. However, whether other blastomere-specific chromosomal defects such as aneuploidy and chaotic mosaicisms may be related to reduced ATP generation associated with disproportionate mitochondrial segregation remains to be determined.
Cytoarchitecture and mitochondrial distribution
Owing to the reported relationship between cytoplasmic microtubular organization and stage-specific mitochondrial distributions in rodent oocytes and cleavage stage embryos, studies were performed to examine whether a similar dynamic process may be involved in the generation of disproportionate mitochondrial inheritance during early cleavage in the human. Unpenetrated, monopronuclear oocytes were particularly informative because development of microtubular configurations reflects inherent oocyte capacities, i.e. those which arise in the absence of a sperm centrosome contribution. Under the light microscope, a cytoplasmic flare or cortical clearing was evident in some monopronuclear examined during the first 24 h after insemination, while others showed no change in cytoplasmic organization. The distribution of mitochondria was associated with the presence or absence of cortical clearing and was asymmetric with respect to the maternal pronucleus in oocytes with focal cortical clearing, or uniformly distributed throughout the cytoplasm in oocytes in which cortical cytoplasmic organization remained unchanged. In oocytes that showed cortical clearing, asymmetrical arrays of microtubules extended into the cytoplasm from the nuclear membrane. In these oocytes, serial SLCM sections demonstrated intense foci of tubulin fluorescence asymmetrically localized on the nuclear membrane from which arrays of microtubules emanated. In contrast, unpenetrated monopronuclear oocytes that showed uniform mitochondrial distributions were virtually devoid of detectable microtubules and, in this respect, resembled the cytoplasm of the metaphase II human oocyte (Van Blerkom et al., 1995a). These findings suggest that stage-specific changes in cytoplasmic organization that affect the formation of microtubular arrays and the distribution of mitochondria represent an inherent developmental ability expressed in human oocytes that activate spontaneously or as a consequence of sperm penetration. The absence of cytoplasmic arrays of microtubules and no detectable perinuclear mitochondrial translocation in monopronuclear oocytes without cortical clearing could indicate that the ability to promote microtubule polymerization, but not pronuclear formation, may have failed to develop during meiotic maturation.
Pronuclear oocytes with high competence characteristics showed differences in the distribution of mitochondria that correlated with the organization of microtubules extending from the vicinity of the pronuclear membranes. An asymmetrical distribution of mitochondria was associated with a dense microtubular network localized to one portion of the oocyte with relatively large regions of cytoplasm devoid of either fluorescent signal. The extent to which arrays of microtubules that develop from the sperm centrosome contribute to perinuclear mitochondrial aggregation is unknown. However, previous studies suggest that it may be significant (Ash et al., 1995; Van Blerkom and Davis, 1995
; Van Blerkom et al., 1995a
) and in this study could account for the more pronounced aggregation of mitochondria observed in fertilized oocytes, as opposed to unpenetrated monopronuclear oocytes. Regions of the cleared cortical cytoplasm involved in the cytoplasmic flare or perinuclear halo were devoid of detectable mitochondrial fluorescence and tubulin immunofluorescence. In the developing cleavage stage embryo, regions deficient in mitochondrial fluorescence were also deficient in microtubules, and a transient perinuclear accumulation of mitochondria detected in early cleavage-stage blastomeres was associated with the presence of radial arrays of nuclear membrane-associated microtubules. These results indicate that dynamic and stage-specific changes in mitochondrial distributions during early human development are directed or influenced by intrinsic patterns of microtubular distribution and orientation.
Mitochondrial distribution and competence
It has previously been reported that the extensive network of cytoplasmic microtubules detected during the pronuclear stages disassembles during syngamy with microtubular arrays confined to the nascent mitotic spindle (Van Blerkom et al., 1995a). This change in cytoplasmic microtubule organization together with cytoplasmic turbulence (Edwards and Beard, 1997
) may account for the progressive redistribution of perinuclear mitochondria detected between syngamy and the first cleavage division. There may be no adverse developmental consequences if the meridional plane of the first cell division partitions a mitochondrial aggregate where peripronuclear asymmetry was not pronounced. Where, mitochondrial aggregation was largely asymmetrical and this asymmetry persisted during syngamy, an epigenetic basis for the observed non-equivalence of mitochondrial distribution at the first cell division may occur if the meridional plane of cleavage bisects the syngamic oocyte in the normal fashion (Edwards and Beard, 1997
). For the affected blastomere and its progeny, if any, a significantly reduced capacity to generate ATP by oxidative phosphorylation may be one developmental consequence of disproportionate mitochondrial segregation at the first cell division, and, with continued culture, arrested cell division and death may be the outcome. The absence of cytoplasmic arrays of microtubules in blastomeres with poor mitochondrial inheritance is consistent with compromised cell function. For the embryo, the results of the current study suggest that arrested development occurs when disproportionate mitochondrial inheritance during early cleavage results in about half of the blastomeres exhibiting reduced mitochondrial fluorescence. The extent to which differences in competence are related to specific patterns of mitochondrial inheritance and attendant metabolic capacities will need to be determined clinically with mitochondrial distributions assessed non-invasively. One method that may be applicable to the human is confocal microscopy at the 1047 nm wavelength, which has been recently used to study stage-specific mitochondrial distributions during the development of the preimplantation hamster embryo (Squirrell et al., 1999
).
In the present study, only those intact embryos that developed from pronuclear oocytes with an equatorial nucleolar alignment and a cytoplasmic flare or perinuclear halo were examined. However, even when these apparent characteristics of competence were present, during cleavage, differences in mitochondrial inheritance were observed between blastomeres; it is suggested that depending upon degree, they are significant with respect to the ability of the affected embryo to develop progressively. Currently, studies are being performed to investigate whether differences in the extent/pattern of cortical clearing (focal or circumferential) are related to cytoplasmic and peripronuclear mitochondrial distributions at the 1-cell stage that could influence mitochondrial segregation during the first cleavage division. Whether the absence of a cytoplasmic flare or perinuclear halo, which has been correlated with poor implantation potential (Scott and Smith, 1998), is associated with patterns of mitochondrial distribution and microtubular configurations that are different from those observed in `high competence' pronuclear oocytes remains to be determined. However, an association between mitochondrial organization and competence, especially one that can be determined non-invasively during the earliest stages of development, would go a long way in establishing an epigenetic basis for unanticipated developmental arrest in vitro and differences in outcome observed with human embryos that appear normal at transfer.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barnett, D., J., Kimura, J. and Bavister, B. (1996) Translocation of active mitochondria during hamster preimplantation embryo development studied by confocal scanning microscopy. Dev. Dyn., 205, 6472.[ISI][Medline]
Barnett, D., Clayton, M., Kimura, J. and Bavister, B. (1997) Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including the distribution of active mitochondria. Mol. Reprod. Dev., 48, 227237.[ISI][Medline]
Barritt, J., Brenner, C., Cohen, J. and Matt, D. (1999) Mitochondrial DNA rearrangements in human oocytes and embryos. Mol. Hum. Reprod., 5, 927933.
Brenner, C., Wolney, Y., Barritt, J. et al. (1998) Mitochondrial DNA deletion in human oocytes and embryos. Mol. Hum. Reprod., 4, 887892.[Abstract]
Brison, D. and Leese, H. (1994) Blastocoel cavity formation by preimplantation rat embryos in the presence of cyanide and other inhibitors of oxidative phosphorylation. J. Reprod. Fertil., 101, 305309.[Abstract]
Chinnery, P. and Turnbull, D. (1999) Mitochondrial DNA and disease. Lancet, 354, si17-21.
Cohen, J., Scott, R., Alikani, M. et al. (1998) Ooplasmic transfer in mature human oocytes. Mol. Hum. Reprod., 4, 269280.[Abstract]
Coulam, C., Goodman, C. and Rinehart, J. (1999) Color Doppler indices of follicular blood flow as predictors of pregnancy after in-vitro fertilization and embryo transfer. Hum. Reprod., 14, 19791982.
DiMauro, S. (1998) Mitochondrial diseases: clinical considerations. BioFactors, 7, 277285.[Medline]
Edwards, R.G., and Beard, H.K. (1997) Oocyte polarity and cell determination in early mammalian embryos. Mol. Hum. Reprod., 3, 863905.[Abstract]
Garello, C., Baker, H., Rai, J., Montgomery, S. et al. (1999) Pronuclear orientation, polar body placement, and embryo quality after intracytoplasmic sperm injection and in-vitro fertilization: further evidence for polarity in human oocytes? Hum. Reprod., 14, 25882595.
Gott, A., Hardy, K., Winston, R., and Leese, H. (1990) Non-invasive measurement of pyruvate and glucose uptake and lactate production by single human preimplantation embryos. Hum. Reprod., 5, 104108.[Abstract]
Gregory, L., Walker, S. and Shaw, R. (1999) The use of transvaginal power Doppler ultrasonography to evaluate the relationship between perifollicular vascularity and outcome in in-vitro fertilization treatment cycles. Hum. Reprod., 14, 939945.
Hammans, S. (1994) Mitochondrial DNA and disease. Essays Biochem., 28, 99112.[Medline]
Hardy, K., Hooper, M., Handyside, A. et al. (1989) Non-invasive measurement of glucose and pyruvate uptake by individual human oocytes and preimplantation embryos. Hum. Reprod., 4, 188191.[Abstract]
Houghton, F., Thompson, J., Kennedy, C. and Leese, H. (1996) Oxygen consumption and energy metabolism of the early mouse embryo. Mol. Reprod. Dev., 44, 476485.[ISI][Medline]
Huey, S., Abuhamad, A., Barros, G. et al. (1999) Perifollicular blood flow Doppler indices, but not follicular pO2, pCO2, or pH, predict oocyte developmental competence in in vitro fertilization. Fertil. Steril., 72, 707712.[ISI][Medline]
Jansen, R. and de Boer, K. (1998) The bottleneck: mitochondrial imperatives in oogenesis and ovarian follicular fate. Mol. Cell Endocrinol., 145, 8188.[ISI][Medline]
Keefe, D., Niven-Fairchild, T., Powell, S. and Buradagunta, S. (1995) Mitochondrial deoxyribonucleic acid deletions in oocytes of reproductively aging women. Fertil. Steril., 64, 577583.[ISI][Medline]
Krisher, R. and Bavister, B. (1999) Enhanced glycolysis after maturation of bovine oocytes in vitro is associate d with increased developmental competence. Mol. Reprod. Dev., 53, 1926.[ISI][Medline]
Larsson, N. and Clayton, D. (1995) Molecular genetic aspects of human mitochondrial disorders. Ann. Arev. Genet., 29, 151178.
Leonard, J. and Schapira, A. (2000) Mitochondrial respiratory chain disorders I. mitochondrial DNA defects. Lancet, 355, 299304.[ISI][Medline]
Magnusson, C. Hillensjo, T., Hamberger, L. and Nilsson, L. (1986) Oxygen consumption by human oocytes and blastocysts grown in vitro. Hum. Reprod., 1, 183184.[Abstract]
Payne, D., Flaherty, S., Barry, M. and Matthews, C. (1997) Preliminary observations of polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum. Reprod., 12, 532541.[ISI][Medline]
Perez, G., Trbovich, A., Godsen, R. and Tilly, J. (2000) Mitochondria and death of oocytes. Nature, 403, 500501.[ISI][Medline]
Piko, L. and Chase, D. (1973) Role of mitochondrial genome during early development in mice. J. Cell Biol., 58, 357378.
Schon, E. and Grossman, M. (1998) Mitochondrial diseases: genetics. BioFactor, 7, 191195.[Medline]
Scott, L. and Smith, S. (1998) The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum. Reprod., 13, 10031013.[Abstract]
Sousa, M., Barros, A., Silva, J. and Tesarik, J. (1997) Developmental changes in calcium content of ultrastructurally distinct subcellular compartments of pre-implantation human embryos. Mol. Hum. Reprod., 3, 8390.[Abstract]
Squirrell, J., Wokosin, D., White, J. and Bavister, B. (1999) Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability. Nat. Biotechnol., 17, 763-767.[ISI][Medline]
Tesarik, J. (1994) Developmental failure during the preimplantation period of human Embryogenesis. In Van Blerkom, J. (ed.), The Biological Basis of Early Human Reproductive Failure: Applications to Medically Assisted Conception. Oxford University Press, Oxford, pp. 327344.
Tesarik, J. and Greco, E. (1999) The probability of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology. Hum. Reprod., 14, 13181323.
Thompson, J., Parttridge, R., Houghton, F. et al. (1996) Oxygen uptake and carbohydrate metabolism by in vitro derived bovine embryos. J. Reprod. Fertil., 106, 299306.[Abstract]
Thompson, J., McHaughton, C., Gasparrini, B. et al. (2000) Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos cultured in vitro. J. Reprod. Fertil., 118, 4755.
Tokura, T., Noda, Y., Goto, Y. and Mori, T. (1993) Sequential observations of mitochondrial distribution in mouse oocytes and embryos. J. Assist. Reprod. Genet., 10, 417426.[Medline]
Van Blerkom, J. (1991) Microtubule mediation of cytoplasmic and nuclear maturation during the early stages of resumed meiosis in cultured mouse oocytes. Proc. Natl. Acad. Sci. USA, 88, 50315035.[Abstract]
Van Blerkom, J. (1993) Development of human embryos to the hatched blastocyst stage in the presence and absence of a monolayer of Vero cells. Hum. Reprod., 8, 15251539.[Abstract]
Van Blerkom, J. (2000) Intrafollicular influences on human oocyte developmental competence: Perifollicular vascularity, oocyte metabolism and mitochondrial function. Hum. Reprod., 15 (Suppl.2), 173188.
Van Blerkom, J. and Runner, M. (1984) Mitochondrial reorganization during resumption of arrested meiosis in the mouse oocyte. Am. J. Anat., 171, 335355.[ISI][Medline]
Van Blerkom, J. and Davis, P. (1995) Evolution of the sperm aster after microinjection of isolated human sperm centrosomes into meiotically mature human oocytes. Mol. Hum. Reprod., 1, see Hum. Reprod., 10, 21792182.[Abstract]
Van Blerkom, J., Davis, P. and Merriam, J. (1994) A retrospective analysis of unfertilized and presumed parthenogenetically activated human oocytes demonstrated a high frequency of sperm penetration. Hum. Reprod., 9, 23812388.[Abstract]
Van Blerkom, J., Davis, J.P., Merriam, J. and Sinclair, J. (1995a) Nuclear and cytoplasmic dynamics of sperm penetration, pronuclear formation, and microtubule organization during fertilization and early preimplantation development in the human. Hum. Reprod. Update, 1, 429461.[Abstract]
Van Blerkom, J., Davis, P. and Lee, J. (1995b) ATP content of human oocytes and developmental potential and outcome after in-vitro fertilization and embryo transfer. Hum. Reprod., 10, 415424.[Abstract]
Van Blerkom, J., Antczak, M. and Schrader, R. (1997) The developmental potential of the human oocyte is related to the dissolved oxygen content of follicular fluid: association with vascular endothelial growth factor levels and perifollicular blood flow characteristics. Hum. Reprod., 12, 10471055.[ISI][Medline]
Van Blerkom, J., Sinclair, J. and Davis, P. (1998) Mitochondrial transfer between oocytes: potential applications of mitochondrial donation and the issue of heteroplasmy. Hum. Reprod., 13, 28572868.
Submitted on June 19, 2000; accepted on September 21, 2000.