Takuya Wakai

High-resolution DNA methylome analysis of primordial germ cells identifies gender-specific reprogramming in mice

Dynamic epigenetic reprogramming occurs during mammalian germ cell development, although the targets of this process, including DNA demethylation and de novo methylation, remain poorly understood. We performed genome-wide DNA methylation analysis in male and female mouse primordial germ cells at embryonic days 10.5, 13.5, and 16.5 by whole-genome shotgun bisulfite sequencing. Our high-resolution DNA methylome maps demonstrated gender-specific differences in CpG methylation at genome-wide and gene-specific levels during fetal germline progression. There was extensive intra- and intergenic hypomethylation with erasure of methylation marks at imprinted, X-linked, or germline-specific genes during gonadal sex determination and partial methylation at particular retrotransposons. Following global demethylation and sex determination, CpG sites switched to de novo methylation in males, but the X-linked genes appeared resistant to the wave of de novo methylation. Significant differential methylation at a subset of imprinted loci was identified in both genders, and non-CpG methylation occurred only in male gonocytes. Our data establish the basis for future studies on the role of epigenetic modifications in germline development and other biological processes.

Ca2+ Signaling During Mammalian Fertilization: Requirements, Players, and Adaptations

Changes in the intracellular concentration of calcium ([Ca(2+)](i)) represent a vital signaling mechanism enabling communication among cells and between cells and the environment. The initiation of embryo development depends on a [Ca(2+)](i) increase(s) in the egg, which is generally induced during fertilization. The [Ca(2+)](i) increase signals egg activation, which is the first stage in embryo development, and that consist of biochemical and structural changes that transform eggs into zygotes. The spatiotemporal patterns of [Ca(2+)](i) at fertilization show variability, most likely reflecting adaptations to fertilizing conditions and to the duration of embryonic cell cycles. In mammals, the focus of this review, the fertilization [Ca(2+)](i) signal displays unique properties in that it is initiated after gamete fusion by release of a sperm-derived factor and by periodic and extended [Ca(2+)](i) responses. Here, we will discuss the events of egg activation regulated by increases in [Ca(2+)](i), the possible downstream targets that effect these egg activation events, and the property and identity of molecules both in sperm and eggs that underpin the initiation and persistence of the [Ca(2+)](i) responses in these species.

Regulation of inositol 1,4,5-trisphosphate receptor type 1 function during oocyte maturation by MPM-2 phosphorylation.

Egg activation and further embryo development require a sperm-induced intracellular Ca(2+) signal at the time of fertilization. Prior to fertilization, the eggs Ca(2+) machinery is therefore optimized. To this end, during oocyte maturation, the sensitivity, i.e. the Ca(2+) releasing ability, of the inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1), which is responsible for most of this Ca(2+) release, markedly increases. In this study, the recently discovered specific Polo-like kinase (Plk) inhibitor BI2536 was used to investigate the role of Plk1 in this process. BI2536 inactivates Plk1 in oocytes at the early stages of maturation and significantly decreases IP(3)R1 phosphorylation at an MPM-2 epitope at this stage. Moreover, this decrease in Plk1-dependent MPM-2 phosphorylation significantly lowers IP(3)R1 sensitivity. Finally, using in vitro phosphorylation techniques we identified T(2656) as a major Plk1 site on IP(3)R1. We therefore propose that the initial increase in IP(3)R1 sensitivity during oocyte maturation is underpinned by IP(3)R1 phosphorylation at an MPM-2 epitope(s).

Ca2+ homeostasis and regulation of ER Ca2+ in mammalian oocytes/eggs

The activation of the developmental program in mammalian eggs relies on the initiation at the time of fertilization of repeated rises in the intracellular concentration of free calcium ([Ca(2+)](i)), also known as [Ca(2+)](i) oscillations. The ability to mount the full complement of oscillations is only achieved at the end of oocyte maturation, at the metaphase stage of meiosis II (MII). Over the last decades research has focused on addressing the mechanisms by which the sperm initiates the oscillations and identification of the channels that mediate intracellular Ca(2+) release. This review will describe the up-to-date knowledge of other aspects of Ca(2+) homeostasis in mouse oocytes, such as the mechanisms that transport Ca(2+) out of the cytosol into the endoplasmic reticulum (ER), the Ca(2+) store of the oocyte/egg, into other organelles and also those that extrude Ca(2+). Evidence pointing to channels in the plasma membrane that mediate Ca(2+) entry from the extracellular milieu, which is required for the persistence of the oscillations, is also discussed, along with the modifications that these mechanisms undergo during maturation. Lastly, we highlight areas where additional research is needed to obtain a better understating of the molecules and mechanisms that regulate Ca(2+) homeostasis in this unique Ca(2+) signaling system.

Regulation of endoplasmic reticulum Ca2+ oscillations in mammalian eggs

Summary Changes in the intracellular concentration of free calcium ([Ca2+]i) regulate diverse cellular processes including fertilization. In mammalian eggs, the [Ca2+]i changes induced by the sperm unfold in a pattern of periodical rises, also known as [Ca2+]i oscillations. The source of Ca2+ during oscillations is the endoplasmic reticulum ([Ca2+]ER), but it is presently unknown how [Ca2+]ER is regulated. Here, we show using mouse eggs that [Ca2+]i oscillations induced by a variety of agonists, including PLC&zgr;, SrCl2 and thimerosal, provoke simultaneous but opposite changes in [Ca2+]ER and cause differential effects on the refilling and overall load of [Ca2+]ER. We also found that Ca2+ influx is required to refill [Ca2+]ER, because the loss of [Ca2+]ER was accelerated in medium devoid of Ca2+. Pharmacological inactivation of the function of the mitochondria and of the Ca2+-ATPase pumps PMCA and SERCA altered the pattern of oscillations and abruptly reduced [Ca2+]ER, especially after inactivation of mitochondria and SERCA functions. We also examined the expression of SERCA2b protein and found that it was expressed throughout oocyte maturation and attained a conspicuous cortical cluster organization in mature eggs. We show that its overexpression reduces the duration of inositol-1,4,5-trisphosphate-induced [Ca2+]i rises, promotes initiation of oscillations and enhances refilling of [Ca2+]ER. Collectively, our results provide novel insights on the regulation of [Ca2+]ER oscillations, which underlie the unique Ca2+-signalling system that activates the developmental program in mammalian eggs.

Regulation of inositol 1,4,5-trisphosphate receptor function during mouse oocyte maturation

At the time of fertilization, an increase in the intracellular Ca2+ concentration ([Ca2+]i) underlies egg activation and initiation of development in all species studied to date. The inositol 1,4,5‐trisphosphate receptor (IP3R1), which is mostly located in the endoplasmic reticulum (ER) mediates the majority of this Ca2+ release. The sensitivity of IP3R1, that is, its Ca2+ releasing capability, is increased during oocyte maturation so that the optimum [Ca2+]i response concurs with fertilization, which in mammals occurs at metaphase of second meiosis. Multiple IP3R1 modifications affect its sensitivity, including phosphorylation, sub‐cellular localization, and ER Ca2+ concentration ([Ca2+]ER). Here, we evaluated using mouse oocytes how each of these factors affected IP3R1 sensitivity. The capacity for IP3‐induced Ca2+ release markedly increased at the germinal vesicle breakdown stage, although oocytes only acquire the ability to initiate fertilization‐like oscillations at later stages of maturation. The increase in IP3R1 sensitivity was underpinned by an increase in [Ca2+]ER and receptor phosphorylation(s) but not by changes in IP3R1 cellular distribution, as inhibition of the former factors reduced Ca2+ release, whereas inhibition of the latter had no impact. Therefore, the results suggest that the regulation of [Ca2+]ER and IP3R1 phosphorylation during maturation enhance IP3R1 sensitivity rendering oocytes competent to initiate oscillations at the expected time of fertilization. The temporal discrepancy between the initiation of changes in IP3R1 sensitivity and acquisition of mature oscillatory capacity suggest that other mechanisms that regulate Ca2+ homeostasis also shape the pattern of oscillations in mammalian eggs. J. Cell. Physiol. 227: 705–717, 2012.

Ca2+ influx and the store-operated Ca2+ entry pathway undergo regulation during mouse oocyte maturation

In preparation for fertilization, mammalian oocytes undergo optimization of the mechanisms that regulate calcium homeostasis. Among these changes is the increase in the content of the Ca(2+) stores ([Ca(2+)]ER), a process that requires Ca(2+) influx. Nevertheless, the mechanism(s) that mediates this influx remains obscure, although is known that [Ca(2+)]ER can regulate Ca(2+) influx via store-operated Ca(2+) entry (SOCE). We find that during maturation, as [Ca(2+)]ER increases, Ca(2+) influx decreases. We demonstrate that mouse oocytes/eggs express the two molecular components of SOCE--stromal interaction molecule 1 (Stim1) and Orai1--and expression of human (h) Stim1 increases Ca(2+) influx in a manner that recapitulates endogenous SOCE. We observe that the cellular distribution of hStim1 and hOrai1 during maturation undergoes sweeping changes that curtail their colocalization during the later stages of maturation. Coexpression of hStim1 and hOrai1 enhances influx throughout maturation but increases basal Ca(2+) levels only in GV oocytes. Further, expression of a constitutive active form of hStim1 plus Orai1, which increases basal Ca(2+) throughout maturation, disturbs resumption of meiosis. Taken together, our results demonstrate that Ca(2+) influx and SOCE are regulated during maturation and that alteration of Ca(2+) homeostasis undermines maturation in mouse oocytes.

Caffeine alleviates the deterioration of Ca2+ release mechanisms and fragmentation of in vitro‐aged mouse eggs

The developmental competence of mammalian eggs is compromised by postovulatory aging. We and others have found that in these eggs, the intracellular calcium ([Ca2+]i) responses required for egg activation and initiation of development are altered. Nevertheless, the mechanism(s) underlying this defective Ca2+ release is not well known. Here, we investigated if the function of IP3R1, the major Ca2+ release channel at fertilization, was undermined in in vitro‐aged mouse eggs. We found that in aged eggs, IP3R1 displayed reduced function as many of the changes acquired during maturation that enhance IP3R1 Ca2+ conductivity, such as phosphorylation, receptor reorganization and increased Ca2+ store content ([Ca2+]ER), were lost with increasing postovulatory time. IP3R1 fragmentation, possibly associated with the activation of caspase‐3, was also observed in these eggs. Many of these changes were prevented when the postovulatory aging of eggs was carried out in the presence of caffeine, which minimized the decline in IP3R1 function and maintained [Ca2+]ER content. Caffeine also maintained mitochondrial membrane potential, as measured by JC‐1 fluorescence. We therefore conclude that [Ca2+]i responses in aged eggs are undermined by reduced IP3R1 sensitivity, decreased [Ca2+]ER, and compromised mitochondrial function, and that addition of caffeine ameliorates most of these aging‐associated changes. Understanding the molecular basis of the protective effects of caffeine will be useful in elucidating, and possibly reversing, the signaling pathway(s) compromised by in vitro culture of eggs. Mol. Reprod. Dev. 78:684–701, 2011.

Phosphorylation of inositol 1,4,5-triphosphate receptor 1 during in vitro maturation of porcine oocytes.

During fertilization in mammalian species, a sperm-induced intracellular Ca(2+) signal ([Ca(2+)](i)) mediates both exit of meiosis and oocyte activation. Recently, we demonstrated in mouse oocytes that the phosphorylation levels of inositol 1,4,5 trisphosphate receptor type1 (IP(3)R1), the channel responsible for Ca(2+) release and oscillations during fertilization, changed during maturation and fertilization. Therefore, we examined the expression and phosphorylation of IP(3)R1 during in vitro maturation of pig oocytes. Here, our present study shows that expression of IP(3)R1 protein did not change during maturation, although the phosphorylation status of the receptor, specifically at an MPM-2 epitope, did. We found that while at the beginning of maturation IP(3)R1 lacked MPM-2 immunoreactivity, it became MPM-2 reactive by 24 h and reached maximal reactivity by 36 h. Interestingly, the acquisition of MPM-2 reactivity coincided with the activation of p34(cdc2) kinase and mitogen-activated protein kinase (MAPK), which are involved in meiotic progression. Following completion of maturation, inactivation of MAPK by U0126 did not affect IP(3)R1 phosphorylation, although inactivation of p34(cdc2) kinase by roscovitine dramatically reduced IP(3)R1 phosphorylation. Neither inhibitor affected total expression of IP(3)R1. Altogether, our results show that IP(3)R1 undergoes dynamic phosphorylation during maturation and this might underlie the generation of oscillations at fertilization.

Molecular characteristics of horse phospholipase C zeta (PLCζ).

A sperm-specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca(2+) ]i ) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT-PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ complementary RNA (cRNA) into mouse oocytes induced long-lasting [Ca(2+) ]i oscillations, and most of the injected oocytes formed pronuclei (PN). The injection of cRNAs encoding horse, mouse, human and cow PLCζ into mouse oocytes showed that ePLCζ had the highest [Ca(2+) ]i oscillation-inducing activity among the species tested. Mutation of D202R, which renders the protein inactive, abrogated the activity of ePLCζ. The nuclear translocation ability of ePLCζ was defective when expressed in mouse oocytes. Taken together, our findings show for the first time that ePLCζ has highest activity of the mammalian species studied to date. Our findings will be useful for the improvement of reproductive technologies in the horse.