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Interactions between embryos and the culture milieu
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INTERACTIONS BETWEEN EMBRYOS AND THE CULTURE MILIEU B.D. Bavister Department of Animal Health & Biomedical Sciences, University of Wisconsin-Madison, Madison, WI USA ABSTRACT Although in vitro production of embryos up to the blastocyst stage is now possible in numerous species, the quality and quantity of embryos are still not satisfactory. Clearly, culture conditions do not yet replace all of the benefits of development within the female reproductive tract. Analysis of the interactions between embryos and the components of culture media provides insights into regulatory mechanisms and how they are perturbed in vitro, and also offers some clues about the nature of the support provided to early embryos by the female tract. Further elucidation of these events and their underlying regulation will be helpful for improving culture media formulations to support normal embryo development in vitro. C 1m by S-Ier Sc:lence Inc.
Key words: preimplantation embryos, mitochondria, intracellular pH, calcium, oviduct INTRODUCTION During the early stages of embryo development, fundamental changes take place that are crucial for subsequent normal development. These changes include combination of the parental genomes, activation of the embryonic genome, alterations of energy-generating pathways, and at least in some species, activation of intracellular homeostatic mechanisms and reorganization of the cytoplasm and organelles. The significance of the cytoplasmic reorganizations, which have been described predominantly in the hamster (3,4,13), is unknown but these events occur in vivo as well as in vitro in this species, and correlate strongly with developmental ability of these embryos. To date, migration of active mitochondria to a central (peri-/pro-nuclear) position is the best documented structural change. Preliminary data indicate that similar though not identical changes occur in other species. Alteration of the normal pattern of mitochondrial distribution correlates strongly with developmental competence, although whether this disturbance is a cause or a symptom of perturbed development is not certain. When regulation of ion homeostasis is disturbed in cultured embryos, development is severely compromised or blocked completely. Most recently, it was found that alteration of intracellular pH (PHi) using weak acids or bases disrupted mitochondrial distribution and also severely inhibited embryo development (9t These Acknowledgments Work from my laboratory described here was supported by the NIH (grant no. HD22023) as part of the National Program on Non-Human In Vitro Fertilization and Embryo Development, and by the USDA (grant no. CSREES 9602156). a unpublished data. Thertogenology 63:619-626. 2000
o 1999 by ElHYIer SCIence Inc.
0093-691X100l$-lee front matter PII 50093-691 X(99)OO262-9
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results suggest that (i) maintenance of pHj is critically important for development, (ii) pHi is easily disturbed in culture, so mechanisms that regulate the H+ gradient can be ovetWhelmed or attenuated, (iii) pHj and mitochondrial distribution are linked, so perhaps loss of pHj regulation alters mitochondrial function, i.e., oxidative metabolism, (iv) in designing culture media for preimplantation embryos, we should endeavor to assist embryos through the critical early development period in ways that mimic the help normally provided by the oviduct. ORGANIZATIONAL CHANGES IN EARLY EMBRYOS Changes in Distribution of Active Mitochondria During Early Development. Metabolic studies on embryos of several species indicate that mitochondria are less important in the earliest stages of embryo development, since oxidative metabolism becomes much more pronounced at the morula and blastocyst stages (2). Nevertheless, the mitochondria in the oocyte, fertilized ovum and/or cleavage stage embryos may be already preparing for later role(s) in development. For example, in the golden hamster, soon after egg activation the mitochondria change from a homogeneous distribution in the cytoplasm and cluster around the pronuclei. Although the functional significance of this change is unknown, in hamsters it occurs during normal development in vivo, and it correlates strongly with the developmental competence of the egg or early embryo. Mitochondrial re-distribution has also been described in other species, including cattle and primates, although the timing and distribution patterns differ from that described for the hamster. Distribution of Active Mitochondria in Fertilized Hamster Ova Our laboratory has used confocal microscopy to examine ova and embryos stained with Rhodamine 123 or Mitotracker, fluorescent dyes that stain active mitochondria. The measured response is the pixel intensity calculated by the image processing program. Either ova undergoing fertilization or cleaved embryos were flushed from the reproductive tracts of mated female hamsters, and stained within 15 min in gas-equilibrated culture medium. The mitochondria are uniformly distributed in the cytoplasm in unfertilized ova, but by about 6 h post-egg activation (PEA) by sperm, they migrate towards the center of the ovum (4). By the time the pronuclei have become apposed just before syngamy, that is about 12 h PEA, the mitochondria have become arranged in a dense cluster encircling the pronuclei. This clustering of mitochondria persists around the nuclei of the cleaving embryo at least up to the 4-cell stage. In hamsters, this relocation of is remarkably consistent: all the embryos from all females examined exhibited this event during the same collection time period. However, this is not true with embryos from other species that are generated from in vitro matured and/or fertilized ova, as discussed later. Other cytoplasmic components, including microfilaments and possibly endoplasmic reticulum, also become distributed around the pronuclei of the fertilizing ovum in a pattern similar to that of the mitochondrial redistribution. The mitochondrial clustering event is a normal process that is important for embryo development. This is clear because the hamster ova or embryos were flushed from the
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reproductive tracts, and stained within 15 min after collection, so the mitochondrial movements are not due to prolonged handling in vitro. In addition, mitochondrial relocation is concomitant with enhanced ability of ova to develop in vitro. Before mitochondrial relocation, approximately 3 h PEA, under present culture conditions only about 10% of hamster ova undergoing fertilization reach the morulalblastocyst stages. In contrast, when the pronuclei are surrounded by mitochondria, about 9 h PEA, over 65% of the ova can develop to morulae and blastocysts. Coincidentally, when the mitochondria are undergoing their relocation around the pronuclei, the Na+/H+ antiporter is being activated (8), resulting in an increased ability to maintain normal cytoplasmic pH, and this may reduce disturbances in mitochondrial clustering, as follows. We recently found that alterations in pHi also disrupts mitochondrial clustering, which could help explain inhibition of development when pHi is disturbed. Either trimethylamine (TMA) or dimethyloxazolidinedione (DMO), which respectively alkalinize or acidify the cytoplasm, were used to adjust pHi of hamster 2-cell embryos cultured in medium HECM-10 b. In both of these treatments, no embryos reached the morula or blastocyst stages, and this inhibition was strongly correlated with a disturbed pattern of mitochondrial clustering. There was a pronounced pattern of severely dispersed mitochondria in many embryos together with a striking decrease in the proportions of embryos showing normal, peri-nuclear clustering of mitochondria. We infer that, if the pHi of cultured embryos is perturbed by some anomaly of the culture environment, the resulting disturbance of the normal mitochondrial clustering pattern, which is perhaps associated with mitochondrial functions, could account for blocked embryo development. Even slight pHi changes, that may occur commonly in cultured embryos, might cause milder disturbance of the mitochondrial clustering that nevertheless compromises the ability of embryos to develop and/or their viability. Mitochondrial Redistribution in Ova of Primates and Cattle A similar technical approach to that described with hamster embryos was used to examine mitochondria in rhesus monkeys and cattle. Mitochondria in rhesus monkey ova were also found to relocate to some extent during fertilization, but there were differences compared to our c observations with hamster ova . Unlike in vivo fertilized hamster ova, IVF rhesus ova are heterogeneous in morphology and developmental competence. In some ova, the mitochondria concentrated in a narrow band between the pronuclei instead of surrounding them as in hamsters. In other ova, mitochondria were more or less homogeneously distributed in the cytoplasm. Our preliminary data indicate that the narrow band pattern of mitochondrial aggregation is consistent with competence of the ova to develop after IVF. The mitochondria in cattle germinal vesicle (GV) stage oocytes are mostly arranged in a cortical distribution (6) but relocate during in vitro maturation. In mature (metaphase II) ova, we observed two distinct patterns of mitochondria distribution. After maturation of oocytes in b J. Squirrell and M. Lane, unpublished data. C J. Squirrell and D. Schramm, unpublished data.
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medium with glucose and amino acids, which supports a high proportion of blastocyst development subsequent to IVF, mitochondria became located primarily in the center of the ovum, but in GV oocytes matured with glucose and lactate, which have poor embryo development after IVF, mitochondria stayed mostly in the cortex of the ovum. Thus, in cattle also the distribution pattern and location of the mitochondria appears to correlate with developmental competence, but the change is seen during oocyte maturation instead of during fertilization.
In summary, our data show that in three very different species, hamster, rhesus monkey and cattle, there are profound changes in distribution of mitochondria during oocyte maturation or during fertilization. The functional significance of these changes is unknown, but at least in hamsters, the relocation of mitochondria is normal and important for development. Because this redistribution is easily perturbed in culture, we infer that the oviduct somehow helps the embryo to maintain this important configuration during early embryo development. In the rhesus monkey, mitochondrial relocation also occurs during fertilization, but only in some ova, which again may be the ones capable of embryo development in vitro. The major relocation of mitochondria in cattle takes place during oocyte maturation in vitro, and it appears to correlate with oocyte developmental competence. Thus, studies on the pattern of mitochondrial distribution may provide a useful indication of the developmental competence of the ovum and embryo, as well as yielding insights into mechanisms of normal embryogenesis.
ACTIVATION OF MECHANISMS REGULATING EMBRYONIC pHj It seems self-evident that in order to maintain and control cellular functions, the plIj of cells must be maintained at an appropriate level. However, the question of how embryos regulate their pH j and what happens if this regulation is perturbed does not seem to have been addressed adequately until recently, when the elegant work by Baltz and his colleagues described in detail how mouse embryos re~ulate pHj (1,12). One of these studies (1) showed that mouse 2-cell embryos lack the Na+IH antiporter found in almost all cells, so by inference this effective H+exporting system must be activated later during embryo development. However, further investigations revealed that the lack of Na+IH+ antiporter in 2-cell embryos is mouse straindependent because this mechanism is active in 2-cell embryos from some other strains (5). Furthermore, this antiporter is active in hamster 2-cell embryos, and even in the ovum undergoing fertilization (9). Remarkably, the antiporter is not active in hamster unfertilized ova (8), which therefore regulate their pHj in some other, less conventional way until the Na+/Ft antiporter is activated.
The hamster experiments were done as follows. The rate of recovery from a challenge acidosis was measured in I-cell, 2-cell and 8-cell embryos and was similar across these stages, as were the resting pHj values (7.19, 7.21 and 7.22, respectively). However, after inhibiting Na+/It antiporter activity, either by using Na+.free culture medium or with specific inhibitors of the Na+IH+ antiporter, intracellular pH remained acidic (9). What is the functional importance of this antiporter? An acute acidification challenge is unlikely to happen in the oviduct, but the
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antiporter is important for baseline pHj maintenance, because when the it was inhibited by removing extracellular Na+, the cytoplasm immediately became acidic. In addition, when the Na+/H+ antiporter was blocked by an amiloride derivative, the ability of 2-cell embryos to develop in culture after an acidosis challenge was significantly reduced. Thus the Na+m+ antiporter is an important com;onent of the ion homeostasis mechanisms of the hamster 2-cell embryo. In contrut to the Na m+ antiporter, neither Na+-dependent HC03-/cr exchanger nor H+ATPase was found in these embryos. Additional studies showed that the Na+m+ antiporter is inactive in unfertilized hamster ova, and becomes activated during fertilization. These oocytes appear to lack any mechanism for regulating pHj in the acid range. Activity of the Na+m+ antiporterwas first detected at 5.5 h PEA with maximal activity evident by 7 h PEA (8). Parthenogenetically activated I-cell and 2-cell embryos also showed Na+m+ antiporter activity, indicating that it is maternally derived and activation of the egg triggers its appearance. Further experiments showed that activation of the "dormant" antiporter is regulated by protein kinase C. Using a similar approach, experiments with mouse ova showed that another major pHj regulatory mechanism, the HC03"/Cr exchanger, is quiescent until fertilization when it becomes from ova undergoing fertilization caused a marked fully active (12). Removing external alkalinization indicative of active HC0 3-/Cr exchanger, whereas the response observed in or using an inhibitor of anion transport unfertilized ova was very small. Absence of external (stilbene, DIDS), allowed a slow, incomplete pHi recovery that was unaffected by absence of external or by DIDS. Rather like the Na m+ antiporter in hamster ova undergoing fertilization, HC0 3"/Cr exchanger activity was first detectable about 4 h after sperm-egg incubation, and was maximal after about 8 h. Also, the activation of HC0 3-/cr exchanger during fertilization apparently occurred by activation of existing, inactive exchangers as it was unaffected by inhibition of protein synthesis, or by disruption of the Golgi apparatus and cytoskeleton. Very similar results were obtained with hamster ova showing that an active ability to regulate pHi in the alkaline range (i.e., above pH 7.18) was due to HC03-/Cr exchanger activity, and that this activity was not demonstrable in unfertilized ova or ova in the early stages of fertilization (7). Failure of embryos to restore normal pHi during an alkaline load inhibited their ability to reach the morula and blastocyst stages in vitro.
cr
cr
cr
In summary, in hamsters and some strains of mice, both the Na+m+ antiporter and the HC03Icr exchanger are involved in regulation ofpHj • These activities develop during fertilization as a result of ovum activation, and inhibition of these mechanisms while the embryos are being challenged by acid or alkaline loads, respectively, reduces or eliminates the formation of blastocysts. Data for other species are unfortunately very sparse, and research is urgently needed into the mechanisms of pHi regulation and consequences for embryo development of perturbations into these systems.
Theriogenology
624 CALCIUM HOMEOSTASIS IN DEVELOPING EMBRYOS
Hamster 2-cell embryos seem remarkably impervious to substantial changes in the divalent cation concentrations of the culture medium (11). However, this is not true of ova undergoing fertilization. One-cell ova collected from the oviducts of mated females showed striking sensitivity to extracellular calcium and magnesium (10). After culture for 24 h in standard medium (2.0 mM calcium, 0.5 mM magnesium), embryos showed almost a 3-fold increase in intracellular calcium concentration compared to in vivo controls, which suggests a reason for the poor development of these embryos. Intracellular magnesium was not affected. Altering the extracellular calcium and magnesium concentrations and ratios had a pronounced effect on embryo development, with the optimum being 1.0 and 2.0 roM, respectively, or an 8-fold decrease in the "nonnal" ratio present in most culture media. ROLE OF THE OVIDUCT IN SUPPORTING EARLY DEVELOPMENT Rather than providing a simple culture milieu for the autonomous embryo to develop and a passive conduit for its transport to the uterus, the oviduct likely has a dynamic relationship with the early embryo. This relationship may be especially critical for the earliest stages of development, i.e., fertilization and the first cleavage division. Indirect evidence for this comes from numerous observations that embryos produced by IVF and embryo culture are, in most cases, less developmentally competent and viable than their counterparts produced in vivo. For example, hamster IVF embryos or those undergoing fertilization in vivo, within 6 h of activation by spenn, are difficult to support in culture, only about 20% reaching the blastocyst stage. In contrast, ova collected from the oviduct about 9-10 h post egg activation have much higher success with around 60% able to develop into blastocysts. This difference obviously points to the inadequacy of present culture media for these very early stages of development, i.e., ova undergoing fertilization, with adverse consequences for subsequent embryo development. The change in the ability of the embryos to develop in culture corresponds to the time period when key events are taking place within the ovum, as discussed above, i.e., migration of the mitochondria and activation of pHi-regulatory mechanisms, to name but two events. From this, we may make some inferences: (i) the ability of ova to develop in vitro (in a suboptimal environment) is greatly reduced until these important changes have occurred, which then confer on the ova/embryos an increased tolerance to a suboptimal culture milieu; (ii) during these critical few hours at the beginning of fertilization, the oviduct is actively assisting development of the ovum; (iii) embryos produced in vitro by IVM/IVF of follicular ova are compromised because of the lack of exposure to the oviductal milieu; this would apply to domesticated animals (including cattle, pig, cat), primates (human and monkey) and endangered species. What could these putative influences of the oviduct be? One possibility is the abundance in oviductal epithelium of the enzyme carbonic anhydrase, which catalyzes conversion of carbonic acid to CO2 and water. Especially in the high HC0 3 - environment of the oviductal fluid, H+ extruded by the ovum/embryo and HC0 3- would immediately combine to fonn carbonic acid (H 2 C0 3), which is then split into CO 2 and H20 by the carbonic anhydrase. In this way, acid
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generated by metabolism of the embryo could be rapidly converted to products that are easily removed by the oviduct and the blood circulation. Lowering the extracellular [H+] would assist passive efflux of H+ from the ovum/embryo, at a time (early fertilization) when the embryo's ability to regulate its pHj is limited. However, placing such ova/embryos into culture media lacking carbonic anhydrase and with no system for rapidly removing excess H+ could impair pH j regulation and hence compromise development. Another possibility is that cumulus/ corona cells, which in several species remain associated with the ovum during early stages of fertilization, play an active role in removing H+ from the ovum, until they are no longer needed. A simple way that embryo developmental competence and viability may be supported by the oviduct is by providing optimal ion balance, which appears to be particularly important for hamster ova undergoing fertilization. While the concentrations and ratios of calcium and magnesium that we found were optimal for development of these embryos in vitro do not correspond to those found in the oviduct fluid, the pH of this fluid must be considered. Oviduct fluid is believed to have an unusually high pH, perhaps 7.8, which will favor binding of calcium to protein, thus lowering the effective (free) calcium concentration. This is the mechanism used in blood plasma to precisely regulate free calcium, and one reason why maintenance of plasma pH is so critical. In addition, the K+ concentration of the oviduct fluid, which is believed to be as high as 27 mM in rodents, will affect the resting membrane potential of the ovum/embryo, thus altering its permeability properties and other key characteristics. Plunging early embryos into culture media with relatively low K+ (about 3 mM) would have a striking effect on these properties and perhaps as a result diminish the capacity for normal development. How the oviduct might affect the migration of mitochondria to the pronuclei, seen in a dramatic manner in hamster ova and to a lesser extent in monkeys, is a mystery. But hamster ova collected from the oviduct about 3 h PEA, in the earliest stages of fertilization, are not able to organize their mitochondria in culture as effectively as seen in vivo. This difference indicates that the oviductal milieu is important in some way for proper organelle distribution in the developing embryo. SUMMARY During fertilization, from the beginning of ovum activation, major changes are occurring in subcellular structure/organization and homeostatic mechanisms. Experiments with cultured embryos have shown that these changes are critically important for normal development. It is suggested that the ovum is assisted by the oviduct to progress through the first hours of fertilization, before its cytoplasm is fully organized and the full complement of homeostatic mechanisms is activated. Absence of assistance from the oviduct may compromise the developmental ability of embryos derived from IVM and/or IVF follicular oocytes (e.g., cattle, pig, human). Establishing exactly how the oviduct and early embryo interact on a mechanistic level could help overcome some of the developmental difficulties frequently exhibited by cultured embryos, thus improving the "health" of embryos produced under in vitro conditions. In addition, deeper understanding of the changes undergone by early embryos that are necessary
Therlogenology for acquilition of full dovelopmental competence could load to now laboratory toatl for evaluatinj competence, avoiding the COlt and time needod for embryo tranlfer., and perhapi IUiiolt novel non-invasive tCltl for embryo viability that would allow lelection of the mOlt competent embryol for transfer. REFERENCES I. Baltz JM. BiUm JD and Lechene C. Apparent ahlence ofNa+/H+ antiport activity in tho 2-
eel! moule embryo. Dov Bioi 1990;38:421-429. 2. Barnett OK and Bavi.ter BD. What il tho rolationlhip botweon the metabolilm of preimplantation embryol and their development in vitro? Malec Reprod Dev 1996;43:105133. 3. Barnett OK. Clayton MK, Kimura J and Bavilter BD. Glucole and pholphate toxicity in ham.ter p",implantation embryol involvel dilruption of cellular orianization. includina diltribution of active mitochondria. Molec Reprod Dev 1997;48:227-237. 4. Barnett DK. Kimura J and Bavilter BD. Tranllocation of active mitochondria durina hamster preimplantation embryo development Itudied by confocal laser Icannina microlCopy. Dov Dynamici 1996;205:64-72. 5. Gibb CA, Poronnik P, Day ML and Cook DI. Control of cytoaolic pH in two-eeJl moulC embryo.: rolel ofW-lactate cotranlport and Na+/H+ exchange. Am J Phy.iol: Cell Phyliol 1997;273:C404-C419. 6. Kri.her RL and Bavilter BD. Correlation of mitochondrial oraanization with developmental competence in bovine oocyte8 matured in vitro. BioI Reprod 1997;56(Suppll):602. 7. Lane M, Baltz JM and Bavilter BD. Bicarbonate/chloride exchanae regulatoa intracellular pH of embryo. but not oocytel of tho hamlter. BioI Reprod 1999;61 :452-457. 8. Lano M. Baltz JM and Bavilter BD. Na+m+ antiporter activity in ham.tor embryo. i. activated durinl fertilization. Dev Bioi 1999;208:244-252. 9. Lano M, Baltz JM and Baviltor BD. Reaulation of intracellular pH in hamlter preimplantation embryos by the sodium hydroaon (Na+m+) antiportor. BioI Reprod 1998;~9: 1483-1490. 10. Lano M, Boatman DE. Albrecht RM and Bavilter BD. Intracellular divalent cation homeoltalil and developmental competence in the ham.ter preimplantation ombryo. Malec Reprod Dov 1998;50:443-450. 11. McKiernan, SH and Bavi8ter BD. Environmental variables influencing in vitro development of hamItor 2-cell embryos to the blastocylt Italo. Bioi Reprod 1990;43:404-413. 12. Philllpi KP and Baltz JM. Intracellular pH reaulation by HCOl"/el" exchango il activated during early moule zygote development. Dev Bioi 1999;208:392-40~. 13. SquirrcH JM, Wokosin DL, Bavilter BD and White JG. Long-torm multiphoton fluoreacenco imaiinl of mammalian embryos does not compromi.e viability. Nature Biotech 1999; 17:763-767.
INTERACTIONS BETWEEN EMBRYOS AND THE CULTURE MILIEU B.D. Bavister Department of Animal Health & Biomedical Sciences, University of Wisconsin-Madison, Madison, WI USA ABSTRACT Although in vitro production of embryos up to the blastocyst stage is now possible in numerous species, the quality and quantity of embryos are still not satisfactory. Clearly, culture conditions do not yet replace all of the benefits of development within the female reproductive tract. Analysis of the interactions between embryos and the components of culture media provides insights into regulatory mechanisms and how they are perturbed in vitro, and also offers some clues about the nature of the support provided to early embryos by the female tract. Further elucidation of these events and their underlying regulation will be helpful for improving culture media formulations to support normal embryo development in vitro. C 1m by S-Ier Sc:lence Inc.
Key words: preimplantation embryos, mitochondria, intracellular pH, calcium, oviduct INTRODUCTION During the early stages of embryo development, fundamental changes take place that are crucial for subsequent normal development. These changes include combination of the parental genomes, activation of the embryonic genome, alterations of energy-generating pathways, and at least in some species, activation of intracellular homeostatic mechanisms and reorganization of the cytoplasm and organelles. The significance of the cytoplasmic reorganizations, which have been described predominantly in the hamster (3,4,13), is unknown but these events occur in vivo as well as in vitro in this species, and correlate strongly with developmental ability of these embryos. To date, migration of active mitochondria to a central (peri-/pro-nuclear) position is the best documented structural change. Preliminary data indicate that similar though not identical changes occur in other species. Alteration of the normal pattern of mitochondrial distribution correlates strongly with developmental competence, although whether this disturbance is a cause or a symptom of perturbed development is not certain. When regulation of ion homeostasis is disturbed in cultured embryos, development is severely compromised or blocked completely. Most recently, it was found that alteration of intracellular pH (PHi) using weak acids or bases disrupted mitochondrial distribution and also severely inhibited embryo development (9t These Acknowledgments Work from my laboratory described here was supported by the NIH (grant no. HD22023) as part of the National Program on Non-Human In Vitro Fertilization and Embryo Development, and by the USDA (grant no. CSREES 9602156). a unpublished data. Thertogenology 63:619-626. 2000
o 1999 by ElHYIer SCIence Inc.
0093-691X100l$-lee front matter PII 50093-691 X(99)OO262-9
Theriogenology
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results suggest that (i) maintenance of pHj is critically important for development, (ii) pHi is easily disturbed in culture, so mechanisms that regulate the H+ gradient can be ovetWhelmed or attenuated, (iii) pHj and mitochondrial distribution are linked, so perhaps loss of pHj regulation alters mitochondrial function, i.e., oxidative metabolism, (iv) in designing culture media for preimplantation embryos, we should endeavor to assist embryos through the critical early development period in ways that mimic the help normally provided by the oviduct. ORGANIZATIONAL CHANGES IN EARLY EMBRYOS Changes in Distribution of Active Mitochondria During Early Development. Metabolic studies on embryos of several species indicate that mitochondria are less important in the earliest stages of embryo development, since oxidative metabolism becomes much more pronounced at the morula and blastocyst stages (2). Nevertheless, the mitochondria in the oocyte, fertilized ovum and/or cleavage stage embryos may be already preparing for later role(s) in development. For example, in the golden hamster, soon after egg activation the mitochondria change from a homogeneous distribution in the cytoplasm and cluster around the pronuclei. Although the functional significance of this change is unknown, in hamsters it occurs during normal development in vivo, and it correlates strongly with the developmental competence of the egg or early embryo. Mitochondrial re-distribution has also been described in other species, including cattle and primates, although the timing and distribution patterns differ from that described for the hamster. Distribution of Active Mitochondria in Fertilized Hamster Ova Our laboratory has used confocal microscopy to examine ova and embryos stained with Rhodamine 123 or Mitotracker, fluorescent dyes that stain active mitochondria. The measured response is the pixel intensity calculated by the image processing program. Either ova undergoing fertilization or cleaved embryos were flushed from the reproductive tracts of mated female hamsters, and stained within 15 min in gas-equilibrated culture medium. The mitochondria are uniformly distributed in the cytoplasm in unfertilized ova, but by about 6 h post-egg activation (PEA) by sperm, they migrate towards the center of the ovum (4). By the time the pronuclei have become apposed just before syngamy, that is about 12 h PEA, the mitochondria have become arranged in a dense cluster encircling the pronuclei. This clustering of mitochondria persists around the nuclei of the cleaving embryo at least up to the 4-cell stage. In hamsters, this relocation of is remarkably consistent: all the embryos from all females examined exhibited this event during the same collection time period. However, this is not true with embryos from other species that are generated from in vitro matured and/or fertilized ova, as discussed later. Other cytoplasmic components, including microfilaments and possibly endoplasmic reticulum, also become distributed around the pronuclei of the fertilizing ovum in a pattern similar to that of the mitochondrial redistribution. The mitochondrial clustering event is a normal process that is important for embryo development. This is clear because the hamster ova or embryos were flushed from the
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reproductive tracts, and stained within 15 min after collection, so the mitochondrial movements are not due to prolonged handling in vitro. In addition, mitochondrial relocation is concomitant with enhanced ability of ova to develop in vitro. Before mitochondrial relocation, approximately 3 h PEA, under present culture conditions only about 10% of hamster ova undergoing fertilization reach the morulalblastocyst stages. In contrast, when the pronuclei are surrounded by mitochondria, about 9 h PEA, over 65% of the ova can develop to morulae and blastocysts. Coincidentally, when the mitochondria are undergoing their relocation around the pronuclei, the Na+/H+ antiporter is being activated (8), resulting in an increased ability to maintain normal cytoplasmic pH, and this may reduce disturbances in mitochondrial clustering, as follows. We recently found that alterations in pHi also disrupts mitochondrial clustering, which could help explain inhibition of development when pHi is disturbed. Either trimethylamine (TMA) or dimethyloxazolidinedione (DMO), which respectively alkalinize or acidify the cytoplasm, were used to adjust pHi of hamster 2-cell embryos cultured in medium HECM-10 b. In both of these treatments, no embryos reached the morula or blastocyst stages, and this inhibition was strongly correlated with a disturbed pattern of mitochondrial clustering. There was a pronounced pattern of severely dispersed mitochondria in many embryos together with a striking decrease in the proportions of embryos showing normal, peri-nuclear clustering of mitochondria. We infer that, if the pHi of cultured embryos is perturbed by some anomaly of the culture environment, the resulting disturbance of the normal mitochondrial clustering pattern, which is perhaps associated with mitochondrial functions, could account for blocked embryo development. Even slight pHi changes, that may occur commonly in cultured embryos, might cause milder disturbance of the mitochondrial clustering that nevertheless compromises the ability of embryos to develop and/or their viability. Mitochondrial Redistribution in Ova of Primates and Cattle A similar technical approach to that described with hamster embryos was used to examine mitochondria in rhesus monkeys and cattle. Mitochondria in rhesus monkey ova were also found to relocate to some extent during fertilization, but there were differences compared to our c observations with hamster ova . Unlike in vivo fertilized hamster ova, IVF rhesus ova are heterogeneous in morphology and developmental competence. In some ova, the mitochondria concentrated in a narrow band between the pronuclei instead of surrounding them as in hamsters. In other ova, mitochondria were more or less homogeneously distributed in the cytoplasm. Our preliminary data indicate that the narrow band pattern of mitochondrial aggregation is consistent with competence of the ova to develop after IVF. The mitochondria in cattle germinal vesicle (GV) stage oocytes are mostly arranged in a cortical distribution (6) but relocate during in vitro maturation. In mature (metaphase II) ova, we observed two distinct patterns of mitochondria distribution. After maturation of oocytes in b J. Squirrell and M. Lane, unpublished data. C J. Squirrell and D. Schramm, unpublished data.
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medium with glucose and amino acids, which supports a high proportion of blastocyst development subsequent to IVF, mitochondria became located primarily in the center of the ovum, but in GV oocytes matured with glucose and lactate, which have poor embryo development after IVF, mitochondria stayed mostly in the cortex of the ovum. Thus, in cattle also the distribution pattern and location of the mitochondria appears to correlate with developmental competence, but the change is seen during oocyte maturation instead of during fertilization.
In summary, our data show that in three very different species, hamster, rhesus monkey and cattle, there are profound changes in distribution of mitochondria during oocyte maturation or during fertilization. The functional significance of these changes is unknown, but at least in hamsters, the relocation of mitochondria is normal and important for development. Because this redistribution is easily perturbed in culture, we infer that the oviduct somehow helps the embryo to maintain this important configuration during early embryo development. In the rhesus monkey, mitochondrial relocation also occurs during fertilization, but only in some ova, which again may be the ones capable of embryo development in vitro. The major relocation of mitochondria in cattle takes place during oocyte maturation in vitro, and it appears to correlate with oocyte developmental competence. Thus, studies on the pattern of mitochondrial distribution may provide a useful indication of the developmental competence of the ovum and embryo, as well as yielding insights into mechanisms of normal embryogenesis.
ACTIVATION OF MECHANISMS REGULATING EMBRYONIC pHj It seems self-evident that in order to maintain and control cellular functions, the plIj of cells must be maintained at an appropriate level. However, the question of how embryos regulate their pH j and what happens if this regulation is perturbed does not seem to have been addressed adequately until recently, when the elegant work by Baltz and his colleagues described in detail how mouse embryos re~ulate pHj (1,12). One of these studies (1) showed that mouse 2-cell embryos lack the Na+IH antiporter found in almost all cells, so by inference this effective H+exporting system must be activated later during embryo development. However, further investigations revealed that the lack of Na+IH+ antiporter in 2-cell embryos is mouse straindependent because this mechanism is active in 2-cell embryos from some other strains (5). Furthermore, this antiporter is active in hamster 2-cell embryos, and even in the ovum undergoing fertilization (9). Remarkably, the antiporter is not active in hamster unfertilized ova (8), which therefore regulate their pHj in some other, less conventional way until the Na+/Ft antiporter is activated.
The hamster experiments were done as follows. The rate of recovery from a challenge acidosis was measured in I-cell, 2-cell and 8-cell embryos and was similar across these stages, as were the resting pHj values (7.19, 7.21 and 7.22, respectively). However, after inhibiting Na+/It antiporter activity, either by using Na+.free culture medium or with specific inhibitors of the Na+IH+ antiporter, intracellular pH remained acidic (9). What is the functional importance of this antiporter? An acute acidification challenge is unlikely to happen in the oviduct, but the
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Therlogenology
antiporter is important for baseline pHj maintenance, because when the it was inhibited by removing extracellular Na+, the cytoplasm immediately became acidic. In addition, when the Na+/H+ antiporter was blocked by an amiloride derivative, the ability of 2-cell embryos to develop in culture after an acidosis challenge was significantly reduced. Thus the Na+m+ antiporter is an important com;onent of the ion homeostasis mechanisms of the hamster 2-cell embryo. In contrut to the Na m+ antiporter, neither Na+-dependent HC03-/cr exchanger nor H+ATPase was found in these embryos. Additional studies showed that the Na+m+ antiporter is inactive in unfertilized hamster ova, and becomes activated during fertilization. These oocytes appear to lack any mechanism for regulating pHj in the acid range. Activity of the Na+m+ antiporterwas first detected at 5.5 h PEA with maximal activity evident by 7 h PEA (8). Parthenogenetically activated I-cell and 2-cell embryos also showed Na+m+ antiporter activity, indicating that it is maternally derived and activation of the egg triggers its appearance. Further experiments showed that activation of the "dormant" antiporter is regulated by protein kinase C. Using a similar approach, experiments with mouse ova showed that another major pHj regulatory mechanism, the HC03"/Cr exchanger, is quiescent until fertilization when it becomes from ova undergoing fertilization caused a marked fully active (12). Removing external alkalinization indicative of active HC0 3-/Cr exchanger, whereas the response observed in or using an inhibitor of anion transport unfertilized ova was very small. Absence of external (stilbene, DIDS), allowed a slow, incomplete pHi recovery that was unaffected by absence of external or by DIDS. Rather like the Na m+ antiporter in hamster ova undergoing fertilization, HC0 3"/Cr exchanger activity was first detectable about 4 h after sperm-egg incubation, and was maximal after about 8 h. Also, the activation of HC0 3-/cr exchanger during fertilization apparently occurred by activation of existing, inactive exchangers as it was unaffected by inhibition of protein synthesis, or by disruption of the Golgi apparatus and cytoskeleton. Very similar results were obtained with hamster ova showing that an active ability to regulate pHi in the alkaline range (i.e., above pH 7.18) was due to HC03-/Cr exchanger activity, and that this activity was not demonstrable in unfertilized ova or ova in the early stages of fertilization (7). Failure of embryos to restore normal pHi during an alkaline load inhibited their ability to reach the morula and blastocyst stages in vitro.
cr
cr
cr
In summary, in hamsters and some strains of mice, both the Na+m+ antiporter and the HC03Icr exchanger are involved in regulation ofpHj • These activities develop during fertilization as a result of ovum activation, and inhibition of these mechanisms while the embryos are being challenged by acid or alkaline loads, respectively, reduces or eliminates the formation of blastocysts. Data for other species are unfortunately very sparse, and research is urgently needed into the mechanisms of pHi regulation and consequences for embryo development of perturbations into these systems.
Theriogenology
624 CALCIUM HOMEOSTASIS IN DEVELOPING EMBRYOS
Hamster 2-cell embryos seem remarkably impervious to substantial changes in the divalent cation concentrations of the culture medium (11). However, this is not true of ova undergoing fertilization. One-cell ova collected from the oviducts of mated females showed striking sensitivity to extracellular calcium and magnesium (10). After culture for 24 h in standard medium (2.0 mM calcium, 0.5 mM magnesium), embryos showed almost a 3-fold increase in intracellular calcium concentration compared to in vivo controls, which suggests a reason for the poor development of these embryos. Intracellular magnesium was not affected. Altering the extracellular calcium and magnesium concentrations and ratios had a pronounced effect on embryo development, with the optimum being 1.0 and 2.0 roM, respectively, or an 8-fold decrease in the "nonnal" ratio present in most culture media. ROLE OF THE OVIDUCT IN SUPPORTING EARLY DEVELOPMENT Rather than providing a simple culture milieu for the autonomous embryo to develop and a passive conduit for its transport to the uterus, the oviduct likely has a dynamic relationship with the early embryo. This relationship may be especially critical for the earliest stages of development, i.e., fertilization and the first cleavage division. Indirect evidence for this comes from numerous observations that embryos produced by IVF and embryo culture are, in most cases, less developmentally competent and viable than their counterparts produced in vivo. For example, hamster IVF embryos or those undergoing fertilization in vivo, within 6 h of activation by spenn, are difficult to support in culture, only about 20% reaching the blastocyst stage. In contrast, ova collected from the oviduct about 9-10 h post egg activation have much higher success with around 60% able to develop into blastocysts. This difference obviously points to the inadequacy of present culture media for these very early stages of development, i.e., ova undergoing fertilization, with adverse consequences for subsequent embryo development. The change in the ability of the embryos to develop in culture corresponds to the time period when key events are taking place within the ovum, as discussed above, i.e., migration of the mitochondria and activation of pHi-regulatory mechanisms, to name but two events. From this, we may make some inferences: (i) the ability of ova to develop in vitro (in a suboptimal environment) is greatly reduced until these important changes have occurred, which then confer on the ova/embryos an increased tolerance to a suboptimal culture milieu; (ii) during these critical few hours at the beginning of fertilization, the oviduct is actively assisting development of the ovum; (iii) embryos produced in vitro by IVM/IVF of follicular ova are compromised because of the lack of exposure to the oviductal milieu; this would apply to domesticated animals (including cattle, pig, cat), primates (human and monkey) and endangered species. What could these putative influences of the oviduct be? One possibility is the abundance in oviductal epithelium of the enzyme carbonic anhydrase, which catalyzes conversion of carbonic acid to CO2 and water. Especially in the high HC0 3 - environment of the oviductal fluid, H+ extruded by the ovum/embryo and HC0 3- would immediately combine to fonn carbonic acid (H 2 C0 3), which is then split into CO 2 and H20 by the carbonic anhydrase. In this way, acid
Theriogenology
625
generated by metabolism of the embryo could be rapidly converted to products that are easily removed by the oviduct and the blood circulation. Lowering the extracellular [H+] would assist passive efflux of H+ from the ovum/embryo, at a time (early fertilization) when the embryo's ability to regulate its pHj is limited. However, placing such ova/embryos into culture media lacking carbonic anhydrase and with no system for rapidly removing excess H+ could impair pH j regulation and hence compromise development. Another possibility is that cumulus/ corona cells, which in several species remain associated with the ovum during early stages of fertilization, play an active role in removing H+ from the ovum, until they are no longer needed. A simple way that embryo developmental competence and viability may be supported by the oviduct is by providing optimal ion balance, which appears to be particularly important for hamster ova undergoing fertilization. While the concentrations and ratios of calcium and magnesium that we found were optimal for development of these embryos in vitro do not correspond to those found in the oviduct fluid, the pH of this fluid must be considered. Oviduct fluid is believed to have an unusually high pH, perhaps 7.8, which will favor binding of calcium to protein, thus lowering the effective (free) calcium concentration. This is the mechanism used in blood plasma to precisely regulate free calcium, and one reason why maintenance of plasma pH is so critical. In addition, the K+ concentration of the oviduct fluid, which is believed to be as high as 27 mM in rodents, will affect the resting membrane potential of the ovum/embryo, thus altering its permeability properties and other key characteristics. Plunging early embryos into culture media with relatively low K+ (about 3 mM) would have a striking effect on these properties and perhaps as a result diminish the capacity for normal development. How the oviduct might affect the migration of mitochondria to the pronuclei, seen in a dramatic manner in hamster ova and to a lesser extent in monkeys, is a mystery. But hamster ova collected from the oviduct about 3 h PEA, in the earliest stages of fertilization, are not able to organize their mitochondria in culture as effectively as seen in vivo. This difference indicates that the oviductal milieu is important in some way for proper organelle distribution in the developing embryo. SUMMARY During fertilization, from the beginning of ovum activation, major changes are occurring in subcellular structure/organization and homeostatic mechanisms. Experiments with cultured embryos have shown that these changes are critically important for normal development. It is suggested that the ovum is assisted by the oviduct to progress through the first hours of fertilization, before its cytoplasm is fully organized and the full complement of homeostatic mechanisms is activated. Absence of assistance from the oviduct may compromise the developmental ability of embryos derived from IVM and/or IVF follicular oocytes (e.g., cattle, pig, human). Establishing exactly how the oviduct and early embryo interact on a mechanistic level could help overcome some of the developmental difficulties frequently exhibited by cultured embryos, thus improving the "health" of embryos produced under in vitro conditions. In addition, deeper understanding of the changes undergone by early embryos that are necessary
Therlogenology for acquilition of full dovelopmental competence could load to now laboratory toatl for evaluatinj competence, avoiding the COlt and time needod for embryo tranlfer., and perhapi IUiiolt novel non-invasive tCltl for embryo viability that would allow lelection of the mOlt competent embryol for transfer. REFERENCES I. Baltz JM. BiUm JD and Lechene C. Apparent ahlence ofNa+/H+ antiport activity in tho 2-
eel! moule embryo. Dov Bioi 1990;38:421-429. 2. Barnett OK and Bavi.ter BD. What il tho rolationlhip botweon the metabolilm of preimplantation embryol and their development in vitro? Malec Reprod Dev 1996;43:105133. 3. Barnett OK. Clayton MK, Kimura J and Bavilter BD. Glucole and pholphate toxicity in ham.ter p",implantation embryol involvel dilruption of cellular orianization. includina diltribution of active mitochondria. Molec Reprod Dev 1997;48:227-237. 4. Barnett DK. Kimura J and Bavilter BD. Tranllocation of active mitochondria durina hamster preimplantation embryo development Itudied by confocal laser Icannina microlCopy. Dov Dynamici 1996;205:64-72. 5. Gibb CA, Poronnik P, Day ML and Cook DI. Control of cytoaolic pH in two-eeJl moulC embryo.: rolel ofW-lactate cotranlport and Na+/H+ exchange. Am J Phy.iol: Cell Phyliol 1997;273:C404-C419. 6. Kri.her RL and Bavilter BD. Correlation of mitochondrial oraanization with developmental competence in bovine oocyte8 matured in vitro. BioI Reprod 1997;56(Suppll):602. 7. Lane M, Baltz JM and Bavilter BD. Bicarbonate/chloride exchanae regulatoa intracellular pH of embryo. but not oocytel of tho hamlter. BioI Reprod 1999;61 :452-457. 8. Lano M. Baltz JM and Bavilter BD. Na+m+ antiporter activity in ham.tor embryo. i. activated durinl fertilization. Dev Bioi 1999;208:244-252. 9. Lano M, Baltz JM and Baviltor BD. Reaulation of intracellular pH in hamlter preimplantation embryos by the sodium hydroaon (Na+m+) antiportor. BioI Reprod 1998;~9: 1483-1490. 10. Lano M, Boatman DE. Albrecht RM and Bavilter BD. Intracellular divalent cation homeoltalil and developmental competence in the ham.ter preimplantation ombryo. Malec Reprod Dov 1998;50:443-450. 11. McKiernan, SH and Bavi8ter BD. Environmental variables influencing in vitro development of hamItor 2-cell embryos to the blastocylt Italo. Bioi Reprod 1990;43:404-413. 12. Philllpi KP and Baltz JM. Intracellular pH reaulation by HCOl"/el" exchango il activated during early moule zygote development. Dev Bioi 1999;208:392-40~. 13. SquirrcH JM, Wokosin DL, Bavilter BD and White JG. Long-torm multiphoton fluoreacenco imaiinl of mammalian embryos does not compromi.e viability. Nature Biotech 1999; 17:763-767.