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.

Germ cells develop in a microenvironment created by the somatic cells of the gonad [1-3]. Although in males, the germ and somatic support cells lie in direct contact, in females, a thick extracellular coat surrounds the oocyte, physically separating it from the somatic follicle cells [4]. To bypass this barrier to communication, narrow cytoplasmic extensions of the follicle cells traverse the extracellular coat to reach the oocyte plasma membrane [5-9]. These delicate structures provide the sole platform for the contact-mediated communication between the oocyte and its follicular environment that is indispensable for production of a fertilizable egg [8, 10-15]. Identifying the mechanisms underlying their formation should uncover conserved regulators of fertility. We show here in mice that these structures, termed transzonal projections (TZPs), are specialized filopodia whose number amplifies enormously as oocytes grow, enabling increased germ-soma communication. By creating chimeric complexes of genetically tagged oocytes and follicle cells, we demonstrate that follicle cells elaborate new TZPs that push through the extracellular coat to reach the oocyte surface. We further show that growth-differentiation factor 9, produced by the oocyte, drives the formation of new TZPs, uncovering a key yet unanticipated role for the germ cell in building these essential bridges of communication. Moreover, TZP number and germline-soma communication are strikingly reduced in reproductively aged females. Thus, the growing oocyte locally remodels follicular architecture to ensure that its developmental needs are met, and an inability of somatic follicle cells to respond appropriately to oocyte-derived cues may contribute to human infertility.

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.

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.

Chromosome segregation errors in mammalian oocytes compromise development and are particularly prevalent in older females, but the aging-related cellular changes that promote segregation errors remain unclear [1, 2]. Aging causes a loss of meiotic chromosome cohesion, which can explain premature disjunction of sister chromatids [3-7], but why intact sister pairs should missegregate in meiosis-I (termed non-disjunction) remains unknown. Here, we show that oocytes from naturally aged mice exhibit substantially altered spindle microtubule dynamics, resulting in transiently multipolar spindles that predispose the oocytes to kinetochore-microtubule attachment defects and missegregation of intact sister chromatid pairs. Using classical micromanipulation approaches, including reciprocally transferring nuclei between young and aged oocytes, we show that altered microtubule dynamics are not attributable to age-related chromatin changes. We therefore report that altered microtubule dynamics is a novel primary lesion contributing to age-related oocyte segregation errors. We propose that, whereas cohesion loss can explain premature sister separation, classical non-disjunction is instead explained by altered microtubule dynamics, leading to aberrant spindle assembly.