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Regulation of intracellular pH during oocyte growth and maturation in mammals

Institute for Women's Health, University College London, 86-96 Chenies Mews, London WC1E 6HX, UK¹ Ottawa Health Research Institute and Departments of Obstetrics and Gynecology (Division of Reproductive Medicine) and Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada

Correspondence should be addressed to G FitzHarris; Email: g.fitzharris{at}ucl.ac.uk J M Baltz; Email: jmbaltz{at}ohri.ca

	Abstract

Top Abstract Introduction: growth and... Cellular homeostasis: the pH... Role of pH in... pH regulation in the... pH regulation mechanisms during... Conclusion Declaration of interest References

 
Regulation of intracellular pH (pH_i) is a fundamental homeostatic process essential for the survival and proliferation of virtually all cell types. The mammalian preimplantation embryo, for example, possesses Na⁺/H⁺ and HCO₃^–/Cl^– exchangers that robustly regulate against acidosis and alkalosis respectively. Inhibition of these transporters prevents pH corrections and, perhaps unsurprisingly, leads to impaired embryogenesis. However, recent studies have revealed that the role and regulation of pH_i is somewhat more complex in the case of the developing and maturing oocyte. Small meiotically incompetent growing oocytes are apparently incapable of regulating their own pH_i, and instead rely upon the surrounding granulosa cells to correct ooplasmic pH, until such a time that the oocyte has developed the capacity to regulate its own pH_i. Later, during meiotic maturation, pH_i-regulating activities that were developed during growth are inactivated, apparently under the control of MAPK signalling, until the oocyte is successfully fertilized. Here, we will discuss pH homeostasis in early mammalian development, focussing on recent developments highlighting the unusual and unexpected scenario of pH regulation during oocyte growth and maturation.

	Introduction: growth and maturation of the mammalian oocyte

Top Abstract Introduction: growth and... Cellular homeostasis: the pH... Role of pH in... pH regulation in the... pH regulation mechanisms during... Conclusion Declaration of interest References

 
Mammalian oocytes grow within ovarian follicles, each comprising a single oocyte enclosed within a surrounding shell of granulosa cells. For every reproductive cycle, a group of primordial follicles, consisting of a small non-growing primordial oocyte and a single layer of granulosa cells, is selected to begin growing. The oocyte increases in size from 20 µm in diameter to a species-dependent final size of 70–120 µm, with fully grown oocytes of the most common experimental model, the mouse, reaching about 75 µm by the end of the growth phase. Oocyte growth is an indispensable component of oogenesis, since in the mouse oocytes only become capable of resuming meiosis when they reach about 80% of their final size (Sorensen & Wassarman 1976, Wassarman et al. 1979). Oocyte growth is also absolutely dependent upon the presence of, and communication with, the surrounding granulosa cells (Eppig 1985, 1991, Buccione et al. 1990, Su et al. 2009). Granulosa cells are coupled to the enclosed oocyte via gap junctions that, at least in mouse, are composed principally of connexin 37 (Cx37) subunits (Simon et al. 1997, Kidder & Mhawi 2002). During growth, the granulosa cells proliferate rapidly, such that the follicle expands, and eventually forms a fluid-filled antrum from which the fully grown oocyte, surrounded by cumulus granulosa cells, is ovulated.

Throughout the growth phase, the oocyte is arrested in first meiotic prophase with a prominent prophase nucleus, termed the germinal vesicle (GV). The LH surge serves as the trigger for ovulation, and is also the signal for the oocyte to resume meiosis. The GV breaks down and the oocyte completes the first meiotic division (MI), ending in an unequal cell division forming a tiny nonviable first polar body (Pb1) and a large oocyte. The oocyte immediately enters the second meiotic division (MII), but arrests in metaphase until successfully fertilized (termed the MII arrest). It is around this time that the oocyte is expelled from the follicle and begins its journey along the oviduct (fallopian tube), and granulosa cells secrete hyaluronic acid matrix causing them to be physically uncoupled from the oocyte, including loss of gap junctional communication (Eppig 1982). Successful fertilization in the ampulla of the oviduct initiates a cascade of events culminating in the resumption of meiosis, extrusion of the Pb2, and initiation of embryonic division. The cleaving embryo differentiates into a blastocyst some 5 days after ovulation (in mouse), around which time the successful embryo attaches to the uterus marking the end of preimplantation (PI) development. Thus, having remained arrested from birth until it is recruited to grow at some point after puberty, the oocyte is transformed from a tiny primordial oocyte within the ovary to a partially differentiated embryo in a matter of a few weeks (see Fig. 1).

View larger version (38K): [in this window] [in a new window]   Figure 1 Regulation of intracellular pH changes in mammalian oogenesis and embryogenesis. Cartoon outlining the major milestones of oocyte development and early embryogenesis in mammals, indicating the relative level of pH regulation capacity displayed by the oocyte. Note that times indicated are for mouse, the most closely studied mammal in terms of pHi regulation. Granulosa cells are indicated in green, and the germinal vesicle as a dotted line. Further details are in text and in Table 1.

	Cellular homeostasis: the pH regulation toolkit

Top Abstract Introduction: growth and... Cellular homeostasis: the pH... Role of pH in... pH regulation in the... pH regulation mechanisms during... Conclusion Declaration of interest References

 
Virtually all cells possess the ability to regulate against unwelcome perturbations in pH_i. Most cellular processes are acutely pH sensitive, and so it is unsurprising that impairment of pH_i regulation can critically compromise cell function and viability. Classic experiments in cultured somatic cells showed that growth and proliferation are impaired if pH_i regulation is compromised and pH_i is disturbed (Grinstein et al. 1989, Kapus et al. 1994), and pH_i dysregulation has been shown to jeopardize cell survival (Pouyssegur et al. 1984). The mammalian cell has therefore been equipped to maintain a normal pH_i in response to changing environments and metabolic acid generation. To correct alkalosis, most mammalian cells possess HCO₃^–/Cl^– exchangers of the anion exchange (AE) family (AE – three isoforms with various splice variants), which export HCO₃^– in exchange for Cl^–, thereby raising pH_i (Alper 1991, Romero et al. 2004). The principal means for correcting acidosis are Na⁺/H⁺ exchangers (NHEs) of the NHE gene family, which export protons in exchange for Na⁺, thereby raising pH_i (Orlowski & Grinstein 2004). Out of the nine known NHE isoforms, five (NHE1–5) are known to reside at least partly in the plasmalemma, and may therefore regulate pH_i (Orlowski & Grinstein 2004, Romero et al. 2004). Dependent upon the cell type, these two main players may be backed by a supporting cast including Na⁺, HCO₃^–/Cl^– exchangers (NBC) that relieve acidosis in some cell types by importing HCO₃^– (Romero et al. 2004), and V-type H⁺-ATPases (V-ATPases) that export protons across the plasmalemma in some cells (Merzendorfer et al. 1997, Nelson & Harvey 1999, Kawasaki-Nishi et al. 2003). The study of cellular pH_i regulation has been facilitated considerably by commercially available pH-sensitive fluorophores, such as BCECF and SNARF, which allow pH_i measurement during experimental manipulations in living cells. For example, real-time recording of the recovery of a cell from an induced acidosis or alkalosis can be performed, and identification of the transport systems responsible for the recovery can be achieved by inhibition of each system.

	Role of pH in early embryo development

Top Abstract Introduction: growth and... Cellular homeostasis: the pH... Role of pH in... pH regulation in the... pH regulation mechanisms during... Conclusion Declaration of interest References

 
It is well established that pH_i regulation is of importance in early mammalian development. The PI mouse embryo has very active HCO₃^–/Cl^– exchange for mitigating alkalosis (Baltz et al. 1991, Zhao et al. 1995, Zhao & Baltz 1996, Phillips & Baltz 1999), which is also seen in human (Dale et al. 1998, Phillips et al. 2000), hamster (Lane et al. 1999a), and, to a lesser extent, bovine embryos (Lane & Bavister 1999). The effectiveness of these exchangers is substantial; one-cell mouse embryos can efficiently progress to blastocyst in conditions of pH 8.2 (normal embryo culture media pH 7.3), provided HCO₃^–/Cl^– exchange is functional (Zhao et al. 1995).

PI mouse embryos also exhibit NHE activity for correcting acidosis (Gibb et al. 1997, Lane et al. 1998, Steeves et al. 2001, Harding et al. 2002). Oddly, NHE activity in mouse embryos varies widely among mouse strains, and in at least some strains it is not robust (Steeves et al. 2001). As is the case for HCO₃^–/Cl^– exchanger activity, NHE activity is present in the PI embryos of human (Phillips et al. 2000), hamster (Lane et al. 1998), and bovine (Lane & Bavister 1999), and inhibition impairs PI development (Lane et al. 1998). In addition, early embryos possess monocarboxylate transporters that cotransport H⁺ and can influence pH_i of the embryo (Harding et al. 1999), although this is not pH regulation per se, but a manifestation of passive transport of H⁺ as lactic acid across the plasma membrane. In short, the mammalian PI embryo is well equipped with pH_i regulation machinery that is essential for embryo development.

It is worth also noting that pH_i-regulatory exchangers perform a second role in embryos distinct from pH_i regulation, which is to promote the formation of the blastocoel (embryo cavitation). Formation and expansion of the blastocoel are the result of an osmotic gradient driven by Na⁺ accumulation within the cavity, which can be prevented by NHE inhibitors (Manejwala et al. 1989). Immunolocalization of NHE3 (SLC9A3) on the inner surface of the blastocoel (Barr et al. 1998) and inhibition of blastocoel formation using a NHE3-selective inhibitor (Kawagishi et al. 2004) are very consistent with NHE3 playing the dominant role in blastocoel formation in mouse. In contrast, AE2 (SLC4A2)-mediated HCO₃^–/Cl^– exchange is necessary for Cl^– efflux from the blastocoel (Zhao et al. 1997). The molecular identities (i.e. which isoforms) of the exchangers that regulate against pH changes during PI development are yet to be reported.

	pH regulation in the growing oocyte

Top Abstract Introduction: growth and... Cellular homeostasis: the pH... Role of pH in... pH regulation in the... pH regulation mechanisms during... Conclusion Declaration of interest References

 
In stark contrast to embryos, a recent series of studies has revealed that ovarian oocytes that are growing and have yet to achieve their fully grown size lack the ability to regulate pH_i. These studies took advantage of the fact that, in mice, a wave of follicular development occurs shortly after birth such that oocyte size and follicular development are closely related to age during post-natal days 5–20. Thus, the ability of oocytes of different sizes to regulate their pH_i could be examined. Small oocytes (<50 µm in diameter) that were isolated from the ovary and freed of granulosa cells (denuded) assumed a much lower ‘resting’ pH when placed in a standard culture medium than their fully grown (70–75 µm diameter) counterparts. Moreover, small oocytes apparently lacked both HCO₃^–/Cl^– and Na⁺/H⁺ exchange activities, but these became increasingly active and capable of regulating pH_i with increasing oocyte size. This activation of the transporters occurred mainly over the range 60–65 µm, mirroring the increase in resting pH within the oocyte, implying that the lower pH_i of smaller oocytes was a result of inadequate pH_i regulation (Erdogan et al. 2005).

Crucially, however, these initial experiments were unable to determine whether pH_i of growing oocytes behaves similarly within the physiological environment of the ovarian follicle. Key to addressing this question were experiments in which the pH_i of denuded and follicle-enclosed oocytes could be reliably and directly compared. This was achieved by microinjecting a pH-sensitive fluorescent dye into both types of oocytes, allowing the pH_i of the follicle-enclosed oocyte to be measured without any signal from the follicle cells (see Fig. 2A, for example). As in the previous study, small denuded oocytes adopted a much lower pH (pH6.8), than fully grown denuded oocytes (pH7.2). Strikingly, however, follicle-enclosed oocytes adopt essentially the same resting pH_i regardless of their size (pH7.2). Furthermore, fully grown denuded oocytes could efficiently recover from an experimentally induced acidosis or alkalosis, whereas in small oocytes normal pH_i was only re-established when the oocyte was still enclosed within the follicle. Thus, granulosa cells provide the small growing oocyte with the ability to regulate against pH_i changes, thereby allowing it to maintain a steady pH_i during growth. This ability of the granulosa cells to ‘assist’ the oocyte in regulating its pH_i is dependent upon gap junctions, since gap junction inhibition prevents small follicle-enclosed oocytes from dealing with induced pH changes, and causes the resting pH_i to drop (FitzHarris & Baltz 2006, FitzHarris et al. 2007).

View larger version (53K): [in this window] [in a new window]   Figure 2 Follicle-enclosed oocytes recover from an induced acidosis unless Na+/H+ exchanger isoforms 1 and 3 are simultaneously inhibited. (A) Example of follicle-enclosed oocytes isolated from the ovaries of 10-day-old mice and microinjected with the pH-sensitive fluorophore SNARF-dextran (10 kDa). Note that the 10 kDa dextran prevents the indicator from diffusing through gap junctions, which permit the passage of molecules <1 kDa, thereby allowing oocyte pH to be measured directly without any contaminating fluorescence from the granulosa cells. (B) Summary of known locations of active NHEs within murine follicles. (C) The ability of the oocyte to recover from acidosis is assessed using the NH4-pulse assay. The 10 min pulse of NH4Cl causes a net acidosis. Note that following re-addition of Na+, pHi recovers normally in the presence of 10 µM cariporide, a concentration that inhibits all Na+/H+ exchange attributable to NHE1 (SLC9A1). However, recovery from acidosis is completely inhibited by 10 µM S3226, which inhibits both isoforms 1 and 3. Recovery was not inhibited by 1 µM S3226, which would be expected to inhibit NHE3 (SLC9A3) but not NHE1 (not shown; see Schwark et al. 1998, Masereel et al. 2003 regarding selective NHE inhibitors). Thus, inhibition of NHE isoforms 1 and 3 is necessary to prevent recovery.

To further address the question, we have sought to exploit the different array of transporters in the two cell types. Granulosa cells express both NHE1 and NHE3, and simultaneous inhibition of both isoforms is necessary to suppress the ability of granulosa cells to recover from acidosis. Fully grown denuded oocytes, on the other hand, regulate against acidosis using Na⁺/H⁺ exchange provided exclusively by NHE1 (see Table 1; FitzHarris et al. 2007). We reasoned that if regulation against oocyte acidosis in small follicle-enclosed oocytes is performed by Na⁺/H⁺ exchange within the granulosa cells, then inhibition of both NHE1 and NHE3 should be necessary to prevent acidosis recovery in follicle-enclosed oocytes. Conversely, if granulosa cells activate Na⁺/H⁺ exchange native to the oocyte, then inhibition of NHE1 alone should be sufficient to prevent acid correction in small follicle-enclosed oocytes. The results of these experiments are shown in Fig. 2C. Oocytes within follicles exposed to high concentrations of cariporide, a NHE1-specific inhibitor, recover from an induced acidosis as normal, whereas simultaneous inhibition of NHE1 and NHE3 using relatively high concentrations of S3226 prevents oocyte recovery in the follicle, implying that pH_i regulation in the granulosa cells has to be inhibited to prevent pH_i regulation in the follicle-enclosed oocyte. Although the possibility that granulosa cells provide a signal to the growing oocyte that activates Na⁺/H⁺ exchange other than NHE1 cannot be formally ruled out, this seems unlikely given that the fully grown oocyte utilizes only NHE1. Thus, we strongly favour a model in which the small growing oocyte, which is incapable of regulating its own pH, has instead ‘outsourced’ this function to the granulosa cells.

That granulosa cells provide physiological support for the growing oocyte is not without precedent. It has long been known that metabolic cooperativity between oocyte and granulosa cells underpins oocyte energy production. Although oocytes utilize glucose poorly, granulosa cells readily metabolize glucose and supply the oocyte with substrates that it can use (Biggers et al. 1967). The granulosa cells also facilitate uptake of some amino acids and nucleotides, which are ineffectively taken up by denuded oocytes, an effect dependent upon gap junctions (Cross & Brinster 1974, Heller & Schultz 1980, Colonna & Mangia 1983, Haghighat & Van Winkle 1990, Su et al. 2009). Recent work has shown that in the case of some amino acids, granulosa cells can respond to the increased demands of the oocyte by upregulating transporters in order to provide the oocyte with more substrate (Eppig et al. 2005). pH_i regulation distinguishes itself from these mechanisms insofar as granulosa cells provide rapid homeostatic support to the oocyte in the form of an acute (seconds to minutes) response to pH_i change using pre-existing transporters. It remains to be seen whether granulosa cells also take up the slack on behalf of the oocyte in terms of other aspects of ionic homeostasis, although a similar phenomenon has been noted for Na⁺/Ca⁺ exchange in immature Xenopus laevis oocytes (Supplisson et al. 1991).

	pH regulation mechanisms during oocyte maturation and fertilization

Top Abstract Introduction: growth and... Cellular homeostasis: the pH... Role of pH in... pH regulation in the... pH regulation mechanisms during... Conclusion Declaration of interest References

 
As outlined above, by the time it is fully grown, the GV stage mouse oocyte has developed robust mechanisms for regulating its own pH_i (Erdogan et al. 2005, FitzHarris & Baltz 2006, FitzHarris et al. 2007). Similarly, the PI embryo from the pronuclear one-cell stage onwards exhibits robust ability to regulate its own pH_i (see above). Thus, given that developmental stages occurring both before and after are each capable of regulating pH_i, it was surprising that the ovulated MII oocyte exhibits little or no ability to regulate its pH_i.

MII mouse oocytes lack both HCO₃^–/Cl^– exchanger activity and the ability to recover from experimentally induced alkalosis (Phillips & Baltz 1999). A similar lack of HCO₃^–/Cl^– exchange activity has been shown in MII hamster oocytes (Lane et al. 1999a). The lack of HCO₃^–/Cl^– exchange activity in mature unfertilized eggs may not be a property of all mammals, however, as human MII eggs are able to recover from alkalosis, and recovery is inhibited by DIDS (Dale et al. 1998, Phillips et al. 2000).

Similar to HCO₃^–/Cl^– exchange, there is no detectable NHE activity in the MII oocytes of hamster (Lane et al. 1999b) or mouse (G FitzHarris, C Zhou & JM Baltz, unpublished data). Unlike for HCO₃^–/Cl^– exchange, human MII oocytes also lack NHE activity as well, and only exhibit this activity in embryos after fertilization, similar to mouse and hamster (Phillips et al. 2000). Therefore, somewhat unexpectedly, the robust pH_i-regulatory capacity, which is developed during oocyte growth and evident in the fully-grown GV oocyte, is downregulated during meiotic maturation, and then reactivated following fertilization.

The most in-depth investigations of this phenomenon have been carried out on the HCO₃^–/Cl^– exchanger in the mouse. HCO₃^–/Cl^– exchange activity and the ability to regulate pH_i against alkalosis begin to decrease after release from first meiotic prophase arrest, and decrease slowly during MI until exchanger activity becomes almost undetectable by the MI/MII transition (Phillips et al. 2002) and is undetectable in the arrested MII egg. However, after egg activation either by fertilization or experimentally induced parthenogenetic activation, HCO₃^–/Cl^– exchanger activity begins to reappear about 2–3 h post-egg activation and reaches a maximal level several hours later, when the zygotic pronuclei appear (Phillips & Baltz 1999, Phillips et al. 2002, Cetinkaya & Erdogan 2008). A similar time course of activation of NHE activity after egg activation has been seen in the hamster egg (Lane et al. 1999b). Thus, pH_i regulation is ‘turned off’ during meiotic maturation and is quiescent in the ovulated MII egg. It is then reactivated following fertilization, remaining present throughout subsequent PI embryogenesis.

Since pH_i regulation varies so dramatically during meiotic maturation, subsequent studies have focussed on the question of whether the same mechanisms that control meiotic progression and release from meiotic arrests may also participate in silencing the pH_i regulation machinery. GV arrest is mediated by high cAMP maintained by an activated G-protein-coupled receptor (Mehlmann et al. 2004). Thus, GV oocytes have high cAMP levels, which then decrease to allow meiotic resumption. However, raising cAMP levels in oocytes after meiotic resumption did not reactivate HCO₃^–/Cl^– exchange in MII eggs, indicating that high cAMP was not sufficient to maintain pH_i-regulatory activity (Phillips et al. 2002). Similarly, the intracellular Ca²⁺ oscillations that follow fertilization to trigger the release of the MII arrest are not required for reactivation of HCO₃^–/Cl^– exchange (Phillips et al. 2002). Reactivation is also independent of protein synthesis (Phillips & Baltz 1999).

MII arrest is maintained by cytostatic factor (CSF), a cytoplasmic signal that is due, at least in part, to the MOS/MEK/MAPK pathway, (Colledge et al. 1994, Hashimoto et al. 1994). The temporal variation of MAPK activity during meiotic maturation inversely parallels the activity of pH_i-regulatory mechanisms described above. Manipulations that induce ectopically high MAPK activity in GV oocytes or maintain high levels in activated eggs (Moos et al. 1995) reveal that high MAPK activity is not compatible with the activity of HCO₃^–/Cl^– exchange in mouse oocytes (Phillips et al. 2002). Conversely, inhibiting MAPK activity using the specific MEK inhibitor U0126 in MI oocytes activated the normally quiescent HCO₃^–/Cl^– exchanger (Phillips et al. 2002). Unpublished experiments in which a constitutively activated form of MEK (Moos et al. 1996) was expressed in parthenogenetically activated eggs to more specifically maintain high MAPK activity after egg activation has shown that maintaining high MAPK activity prevented reappearance of HCO₃^–/Cl^– exchange, confirming a role for MEK and MAPK (C Zhou & JM Baltz, unpublished data). Thus, the MAPK cascade that is necessary for CSF activity appears to have a novel role in controlling pH_i-regulatory mechanisms during meiosis.

Several questions remain to be answered. For example, what is the mechanism by which MAPK signalling inactivates the transporters during oocyte maturation? Although, in other cells, NHE1 is known to be regulated by MAPK via its effector P90rsk, MAPK activity normally activates Na⁺/H⁺ exchange, rather than inactivating it (Orlowski & Grinstein 2004), and little is known about the effect of MAPK signalling on HCO₃^–/Cl^– exchangers. Alternatively, perhaps MAPK triggers a process of transporter internalization and/or degradation during oocyte maturation, which is reversed following fertilization. Thus, the mechanism by which CSF inactivates transport in eggs requires further study. In addition, the physiological benefit of inactivating pH regulation in the unfertilized egg is obscure. One plausible possibility relates to the repetitive pulsatile increases in [Ca²⁺]_i, which occur after fertilization to trigger development. The re-activation of pH_i regulation (when MAPK activity decreases) corresponds well with the cessation of Ca²⁺ oscillations (Jones et al. 1995, Marangos et al. 2003). Some pH-regulatory transporters are sensitive to Ca²⁺ or its downstream pathways, and thus inactivation of pH_i-regulatory transporters may prevent Ca²⁺-triggered pH_i fluctuations following fertilization. Another possibility relates to cell volume regulation. In many cells, functionally coupled Na⁺/H⁺ and HCO₃^–/Cl^– exchangers act to protect cells against unwanted shrinkage by importing NaCl to increase intracellular osmotic pressure (Jiang et al. 1997, Hoffman et al. 2009). Early embryos possess a novel mechanism for regulating their cell volume that relies on accumulation of glycine rather than inorganic ions (Steeves et al. 2003). This mechanism becomes activated during meiotic maturation (Tartia et al. 2009), i.e. during the period when Na⁺/H⁺ and HCO₃^–/Cl^– exchanger is inactivated. Thus, it is possible that the inactivation of pH_i-regulatory mechanisms during meiosis occurs to allow the switch to glycine-based control in embryos. These possibilities remain to be investigated.

	Conclusion

Top Abstract Introduction: growth and... Cellular homeostasis: the pH... Role of pH in... pH regulation in the... pH regulation mechanisms during... Conclusion Declaration of interest References

 
In summary (Fig. 1 and Table 1), the small growing oocyte lacks the ability to regulate pH_i, and therefore has to rely upon the surrounding granulosa cells. Paradoxically, the fully grown oocyte apparently has the machinery necessary to regulate its own pH, but specifically inactivates this at the same time as it uncouples itself from the granulosa cells that were previously providing support (gap junction inactivation and cumulus expansion). Only after fertilization are these transporters re-activated. A summary of the transport mechanisms that have been identified so far as active in mammalian oocytes and embryos is presented in Table 1. The challenge now is to understand the precise mechanisms that control the inactivation and reactivation of pH_i-regulatory mechanisms, and to ascertain how the changing pH of the external environment (follicle, oviduct, and uterus) and the changes in the manner in which the oocyte/embryo handles acid and alkali together impact pH_i to affect the metabolism of the oocyte and embryo throughout PI development. Improved in vitro oocyte growth and maturation remain an important hurdle in the clinic, and understanding the interaction between the oocyte and its environment during these developmental milestones will likely be key in the development of improved culture systems.

	Declaration of interest

Top Abstract Introduction: growth and... Cellular homeostasis: the pH... Role of pH in... pH regulation in the... pH regulation mechanisms during... Conclusion Declaration of interest References

 
There is no conflict of interest that could be perceived as prejudicing the impartiality of the research/work reported.

	Funding

 
Work by the authors has been supported by the Canadian Institutes of Health Research in the form of a programme grant to J M Baltz and a fellowship to G FitzHarris.

Received March 25, 2009
First decision May 20, 2009
Accepted June 11, 2009

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