Greg FitzHarris

Sperm-triggered [Ca2+] oscillations and Ca2+ homeostasis in the mouse egg have an absolute requirement for mitochondrial ATP production.

At fertilisation, repetitive increases in the intracellular Ca2+ concentration, [Ca2+]i, drive the completion of meiosis and initiate the development of the quiescent egg into an embryo. Although the requirement for an ATP supply is evident, the relative roles of potential ATP sources remains unclear in the mammalian egg, and the specific role of mitochondria in [Ca2+]i regulation as well as in the sperm-triggered [Ca2+] oscillations is unknown. We have used fluorescence and luminescence imaging to investigate mitochondrial activity in single mouse eggs. Simultaneous imaging of mitochondrial redox state (NADH and flavoprotein autofluorescence) and [Ca2+]i revealed that sperm-triggered [Ca2+] oscillations are transmitted to the mitochondria where they directly stimulate mitochondrial activity. Inhibition of mitochondrial oxidative phosphorylation caused release of Ca2+ from the endoplasmic reticulum because of local ATP depletion. Mitochondrial ATP production is an absolute requirement for maintaining a low resting [Ca2+]i and for sustaining sperm-triggered [Ca2+] oscillations. Luminescence measurements of intracellular [ATP] from single eggs confirmed that mitochondrial oxidative phosphorylation is the major source of ATP synthesis in the dormant unfertilised egg. These observations show that a high local ATP consumption is balanced by mitochondrial ATP production, and that balance is critically poised. Mitochondrial ATP supply and demand are thus closely coupled in mouse eggs. As mitochondrial ATP generation is essential to sustain the [Ca2+] signals that are crucial to initiate development, mitochondrial integrity is clearly fundamental in sustaining fertility in mammalian eggs.

Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei

In mammals, the sperm triggers a series of cytosolic Ca2+ oscillations that continue for ∼4 hours, stopping close to the time of pronucleus formation. Ca2+ transients are also seen in fertilized embryos during the first mitotic division. The mechanism that controls this pattern of sperm-induced Ca2+ signalling is not known. Previous studies suggest two possible mechanisms: first, regulation of Ca2+ oscillations by M-phase kinases; and second, regulation by the presence or absence of an intact nucleus. We describe experiments in mouse oocytes that differentiate between these mechanisms. We find that Ca2+ oscillations continue after Cdk1-cyclin B1 activity falls at the time of polar body extrusion and after MAP kinase has been inhibited with UO126. This suggests that M-phase kinases are not necessary for continued Ca2+ oscillations. A role for pronucleus formation in regulating Ca2+ signalling is demonstrated in experiments where pronucleus formation is inhibited by microinjection of a lectin, WGA, without affecting the normal inactivation of the M-phase kinases. In oocytes with no pronuclei but with low M-phase kinase activity, sperm-induced Ca2+ oscillations persist for nearly 10 hours. Furthermore, a dominant negative importin β that inhibits nuclear transport, also prevents pronucleus formation and causes Ca2+ oscillations that continue for nearly 12 hours. During mitosis, fluorescent tracers that mark nuclear envelope breakdown and the subsequent reformation of nuclei in the newly formed two-cell embryo establish that Ca2+ oscillations are generated only in the absence of a patent nuclear membrane. We conclude by suggesting a model where nuclear sequestration and release of a Ca2+-releasing activity contributes to the temporal organization of Ca2+ transients in meiosis and mitosis in mice.

Granulosa cells regulate intracellular ph of the murine growing oocyte via gap junctions : development of independent homeostasis during oocyte growth

Oocytes grow within ovarian follicles in which the oocyte is coupled to the surrounding granulosa cells by gap junctions. It was previously found that small growing oocytes isolated from juvenile mice and freed of their surrounding granulosa cells (denuded) lacked the ability to regulate their intracellular pH (pHi), did not exhibit the pHi-regulatory HCO3-/Cl- and Na+/H+ exchange activities found in fully-grown oocytes, and had low pHi. However, both exchangers became active as oocytes grew near to full size, and, simultaneously, oocyte pHi increased by approximately 0.25 pH units. Here, we show that, in the more physiological setting of the intact follicle, oocyte pHi is instead maintained at∼ 7.2 throughout oocyte development, and the growing oocyte exhibits HCO3-/Cl- exchange, which it lacks when denuded. This activity in the oocyte requires functional gap junctions, as gap junction inhibitors eliminated HCO3-/Cl- exchange activity from follicle-enclosed growing oocytes and substantially impeded the recovery of the oocyte from an induced alkalosis, implying that oocyte pHi may be regulated by pH-regulatory exchangers in granulosa cells via gap junctions. This would require robust HCO3-/Cl- exchange activity in the granulosa cells, which was confirmed using oocytectomized (OOX) cumulus-oocyte complexes. Moreover, in cumulus-oocyte complexes with granulosa cells coupled to fully-grown oocytes, HCO3-/Cl- exchange activity was identical in both compartments and faster than in denuded oocytes. Taken together, these results indicate that growing oocyte pHi is controlled by pH-regulatory mechanisms residing in the granulosa cells until the oocyte reaches a developmental stage where it becomes capable of carrying out its own homeostasis.

Recent Insights into Spindle Function in Mammalian Oocytes and Early Embryos

ABSTRACT Errors in chromosome segregation in oocytes and early embryos lead to embryo aneuploidy, which contributes to early pregnancy loss. At the heart of chromosome segregation is the spindle, a dynamic biomechanical machine fashioned from microtubules, which is tasked with gathering and sorting chromosomes and dispatching them to the daughter cells at the time of cell division. Understanding the causes of segregation error in the oocyte and early embryo will undoubtedly hinge on a thorough understanding of the mechanism of spindle assembly and function in these highly specialized cellular environments. The recent advent of live imaging approaches to observe chromosome segregation in real-time in oocytes and embryos, paired with gene-silencing techniques and specific inhibition for assessing the function of a protein of interest, has led to a substantial advance in our understanding of chromosome segregation in early mammalian development. These studies have uncovered numerous mechanistic differences between oocytes, embryos, and traditional model systems. In addition, a flurry of recent studies using naturally aged mice as the model for human aging have begun to shed light on the increased levels of aneuploidy seen in embryos from older mothers. Here we review these recent developments and consider what has been learned about the causes of chromosome missegregation in early development.

Kinetochore microtubule establishment is defective in oocytes from aged mice

Errors in chromosome segregation in mammalian oocytes increase in number with advancing maternal age, and are a major cause of pregnancy loss. Why chromosome segregation errors are more common in oocytes from older females remains poorly understood. In mitosis, accurate chromosome segregation is enabled by attachment of kinetochores to microtubules from appropriate spindle poles, and erroneous attachments increase the likelihood of mis-segregation. Whether attachment errors are responsible for age-related oocyte aneuploidy is unknown. Here we report that oocytes from naturally aged mice exhibit substantially increased chromosome misalignment, and fewer kinetochore pairs that make stable end-on attachments to the appropriate spindle poles compared with younger oocytes. The profile of mis-attachments exhibited is consistent with the types of chromosome segregation error observed in aged oocytes. Loss of chromosome cohesion, which is a feature of oocytes from older females, causes altered kinetochore geometry in meiosis-I. However, this has only a minor impact upon MT attachment, indicating that cohesion loss is not the primary cause of aneuploidy in meiosis-I. In meiosis-II, on the other hand, age-related cohesion loss plays a direct role in errors, since prematurely individualized sister chromatids misalign and misattach to spindle MTs. Thus, whereas cohesion loss leading to precocious sister chromatid separation is a direct cause of errors in meiosis-II, cohesion loss plays a more minor role in the etiology of aneuploidy in meiosis-I. Our data introduce altered MT-kinetochore interactions as a lesion that explains aneuploidy in meiosis-I in older females.

A shift from kinesin 5-dependent metaphase spindle function during preimplantation development in mouse

Microtubules within meiotic and mitotic spindles continually move towards spindle poles in a process termed poleward flux, which is essential for spindle integrity and faithful chromosome segregation. Kinesin 5 is a longstanding candidate for a molecular motor that might drive poleward flux, and has been shown to drive flux and to be necessary for spindle bipolarity in Xenopus egg extracts. However, kinesin 5 is not necessary for poleward flux or for maintaining metaphase spindle bipolarity in intact mammalian cells, and the reason for the different results in these systems is unknown. The experiments presented here test the hypothesis that these results might reflect developmental differences in spindle function by examining the role of kinesin 5 in mouse eggs and preimplantation embryos. In contrast to cultured somatic cells, poleward flux in mouse eggs is critically dependent upon kinesin 5. Inhibition of poleward flux leads to spindle shortening as a result of continued microtubule depolymerisation at the pole, and eventual loss of spindle bipolarity. Spindle bipolarity is also dependent upon kinesin 5 during the first three embryonic cleavages, but becomes kinesin 5-independent in the majority of spindles by the blastocyst stage. This switch occurs asynchronously in different blastomeres but is independent of clonal cell heritage and of whether the blastomere is within the inner cell mass or the trophoectoderm. These experiments reveal a novel developmental switch in the requirements for spindle function and chromosome segregation during preimplantation development.

Granulosa cells regulate oocyte intracellular pH against acidosis in preantral follicles by multiple mechanisms

Mammalian oocytes grow within ovarian follicles in which the oocyte is coupled to surrounding granulosa cells by gap junctions. We report here that growing oocytes isolated from mouse preantral follicles are incapable of recovering from an experimentally induced acidosis, and that oocytes acquire the ability to manage acid loads by activating Na+/H+ exchange during growth. By contrast, granulosa cells from similar preantral follicles possess substantial Na+/H+ exchange capacity, which is attributable to the simultaneous action of two Na+/H+ exchanger isoforms: NHE1 and NHE3. Granulosa cells were also found to possess a V-type H+-ATPase that drives partial acidosis recovery when Na+/H+ exchange is inactivated. By monitoring intracellular pH (pHi) in small follicle-enclosed oocytes, we found that the oocyte has access to each of these acidosis-correcting activities, such that small follicle-enclosed oocytes readily recover from acidosis in a manner resembling granulosa cells. However, follicle-enclosed oocytes are unable to access these activities if gap-junction communication within the follicle is inhibited. Together, these experiments identify the NHE isoforms involved in regulating oocyte pHi, indicate that gap junctions allow granulosa cells to exogenously regulate oocyte pHi against acidosis until the oocyte has acquired endogenous pHi regulation, and reveal that granulosa cells possess multiple mechanisms for carrying out this function.

MCAK regulates chromosome alignment but is not necessary for preventing aneuploidy in mouse oocyte meiosis I

Errors in chromosome segregation in mammalian oocytes lead to aneuploid eggs that are developmentally compromised. In mitotic cells, mitotic centromere associated kinesin (MCAK; KIF2C) prevents chromosome segregation errors by detaching incorrect microtubule-kinetochore interactions. Here, we examine whether MCAK is involved in spindle function in mouse oocyte meiosis I, and whether MCAK is necessary to prevent chromosome segregation errors. We find that MCAK is recruited to centromeres, kinetochores and chromosome arms in mid-meiosis I, and that MCAK depletion, or inhibition using a dominant-negative construct, causes chromosome misalignment. However, the majority of oocytes complete meiosis I and the resulting eggs retain the correct number of chromosomes. Moreover, MCAK-depleted oocytes can recover from mono-orientation of homologous kinetochores in mid-meiosis I to segregate chromosomes correctly. Thus, MCAK contributes to chromosome alignment in meiosis I, but is not necessary for preventing chromosome segregation errors. Although other correction mechanisms may function in mammalian meiosis I, we speculate that late establishment of kinetochore microtubules in oocytes reduces the likelihood of incorrect microtubule-kinetochore interactions, bypassing the requirement for error correction.

Regulation of intracellular pH during oocyte growth and maturation in mammals

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 HCO(3)(-)/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.

Anaphase B Precedes Anaphase A in the Mouse Egg

Segregation of chromosomes at the time of cell division is achieved by the microtubules and associated molecules of the spindle. Chromosomes attach to kinetochore microtubules (kMTs), which extend from the spindle pole region to kinetochores assembled upon centromeric DNA. In most animal cells studied, chromosome segregation occurs as a result of kMT shortening, which causes chromosomes to move toward the spindle poles (anaphase A). Anaphase A is typically followed by a spindle elongation that further separates the chromosomes (anaphase B). The experiments presented here provide the first detailed analysis of anaphase in a live vertebrate oocyte and show that chromosome segregation is initially driven by a significant spindle elongation (anaphase B), which is followed by a shortening of kMTs to fully segregate the chromosomes (anaphase A). Loss of tension across kMTs at anaphase onset produces a force imbalance, allowing the bipolar motor kinesin-5 to drive early anaphase B spindle elongation and chromosome segregation. Early anaphase B spindle elongation determines the extent of chromosome segregation and the size of the resulting cells. The vertebrate egg therefore employs a novel mode of anaphase wherein spindle elongation caused by loss of k-fiber tension is harnessed to kick-start chromosome segregation prior to anaphase A.