HDAC8 drives spindle organization during meiotic maturation of porcine oocytes

ObjectivesHistone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood.Materials and methodsHDAC8‐targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI‐34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ‐tubulin dynamics and acetylation level of α‐tubulin.ResultsWe observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ‐tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI‐34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α‐tubulin.ConclusionsCollectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation.     

The C.elegans MAPK phosphatase LIP-1 is required for the G(2)/M meiotic arrest of developing oocytes

In the Caenorhabditis elegans hermaphrodite germline, spatially restricted mitogen-activated protein kinase (MAPK) signalling controls the meiotic cell cycle. First, the MAPK signal is necessary for the germ cells to progress through pachytene of meiotic prophase I. As the germ cells exit pachytene and enter diplotene/diakinesis, MAPK is inactivated and the developing oocytes arrest in diakinesis (G(2)/M arrest). During oocyte maturation, a signal from the sperm reactivates MAPK to promote M phase entry. Here, we show that the MAPK phosphatase LIP-1 dephosphorylates MAPK as germ cells exit pachytene in order to maintain MAPK in an inactive state during oocyte development. Germ cells lacking LIP-1 fail to arrest the cell cycle at the G(2)/M boundary, and they enter a mitotic cell cycle without fertilization. LIP-1 thus coordinates oocyte cell cycle progression and maturation with ovulation and fertilization.     

Apoptosis Maintains Oocyte Quality in Aging Caenorhabditis elegans Females

In women, oocytes arrest development at the end of prophase of meiosis I and remain quiescent for years. Over time, the quality and quantity of these oocytes decreases, resulting in fewer pregnancies and an increased occurrence of birth defects. We used the nematode Caenorhabditis elegans to study how oocyte quality is regulated during aging. To assay quality, we determine the fraction of oocytes that produce viable eggs after fertilization. Our results show that oocyte quality declines in aging nematodes, as in humans. This decline affects oocytes arrested in late prophase, waiting for a signal to mature, and also oocytes that develop later in life. Furthermore, mutations that block all cell deaths result in a severe, early decline in oocyte quality, and this effect increases with age. However, mutations that block only somatic cell deaths or DNA-damage–induced deaths do not lower oocyte quality. Two lines of evidence imply that most developmentally programmed germ cell deaths promote the proper allocation of resources among oocytes, rather than eliminate oocytes with damaged chromosomes. First, oocyte quality is lowered by mutations that do not prevent germ cell deaths but do block the engulfment and recycling of cell corpses. Second, the decrease in quality caused by apoptosis mutants is mirrored by a decrease in the size of many mature oocytes. We conclude that competition for resources is a serious problem in aging germ lines, and that apoptosis helps alleviate this problem.     

Hydrogen Sulfide Donor Protects Porcine Oocytes against Aging and Improves the Developmental Potential of Aged Porcine Oocytes

Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H₂S) in the process of porcine oocyte aging. H₂S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). We demonstrated that H₂S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H₂S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H₂S from a donor (Na₂S.9H₂O) significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H₂S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H₂S on MPF and MAPK activities were detected and the intracellular mechanism underlying H₂S activity remains unclear, our study clearly demonstrates the role of H₂S in the regulation of porcine oocyte aging.     

Cdc2-cyclin B triggers H3 kinase activation of Aurora-A in Xenopus oocytes

Xenopus oocytes are arrested in meiotic prophase I and resume meiotic divisions in response to progesterone. Progesterone triggers activation of M-phase promoting factor (MPF) or Cdc2-cyclin B complex and neosynthesis of Mos kinase, responsible for MAPK activation. Both Cdc2 and MAPK activities are required for the success of meiotic maturation. However, the signaling pathway induced by progesterone and leading to MPF activation is poorly understood, and most of the targets of both Cdc2 and MAPK in the oocyte remain to be determined. Aurora-A is a Ser/Thr kinase involved in separation of centrosomes and in spindle assembly during mitosis. It has been proposed that in Xenopus oocytes Aurora-A could be an early component of the progesterone-transduction pathway, acting through the regulation of Mos synthesis upstream Cdc2 activation. We addressed here the question of Aurora-A regulation during meiotic maturation by using new in vitro and in vivo experimental approaches. We demonstrate that Cdc2 kinase activity is necessary and sufficient to trigger both Aurora-A phosphorylation and kinase activation in Xenopus oocyte. In contrast, these events are independent of the Mos/MAPK pathway. Aurora-A is phosphorylated in vivo at least on three residues that regulate differentially its kinase activity. Therefore, Aurora-A is under the control of Cdc2 in the Xenopus oocyte and could be involved in meiotic spindle establishment.     

Epigenetic changes associated with oocyte aging

It is well established that the decline in female reproductive outcomes is related to postovulatory aging of oocytes and advanced maternal age. Poor oocyte quality is correlated with compromised genetic integrity and epigenetic changes during the oocyte aging process. Here, we review the epigenetic alterations, mainly focused on DNA methylation, histone acetylation and methylation associated with postovulatory oocyte aging as well as advanced maternal age. Furthermore, we address the underlying epigenetic mechanisms that contribute to the decline in oocyte quality during oocyte aging.     

Mitogen-activated protein kinase activity during goat oocyte maturation and the acquisition of meiotic competence

Changes in MPF and MAPK activities during meiotic maturation of goat oocytes were investigated. Detection of MPF activity occurred concomitantly with GVBD, increased at MI, decreased during anaphase-telophase I transition, and increased thereafter in MII oocytes. The appearance of MAPK activity was delayed compared to MPF activity. MAPK activity increased after GVBD and persisted during the MI-MII transition. Whether MAPK was implicated in goat oocyte meiotic competence was also investigated by using oocytes from different follicle size categories that arrest at specific stages of the maturation process (GV, GVBD, MI, and MII). Results indicate that the ability of goat oocytes to resume meiosis is not directly related to the presence of Erk2. The ability to phosphorylate MAPK is acquired by the oocyte during follicular growth after the ability to resume meiosis. GVBD-arrested oocytes exhibited a high level of MPF activity after 27 hr of culture. However, 28% of oocytes from this group contained inactive MAPK, and 72% exhibited high MAPK activity. In addition, 29% of GVBD-arrested oocytes contained a residual interphasic network without recruitment of microtubules around the condensed chromosomes; 71% of GVBD-arrested oocytes displayed recruitment of microtubules near the condensed chromosomes and contained asters of microtubules distributed throughout the cytoplasm. These results indicate that oocytes arrested at GVBD were not exactly at the same point in the meiotic cell cycle progression, and suggest that MAPK could be implicated in the regulation of microtubule organization. The data presented here suggest that in goat oocytes, MAPK is not implicated in the early events of meiosis resumption, but rather in post-GVBD events such as spindle formation and MII arrest. © 1996 Wiley-Liss Inc.     

HCO3 ?/Cl? Exchange Inactivation and Reactivation during Mouse Oocyte Meiosis Correlates with MEK/MAPK-Regulated Ae2 Plasma Membrane Localization

Background

Germinal Vesicle (GV) stage mouse oocytes in first meiotic prophase exhibit highly active HCO₃ ⁻/Cl⁻ exchange—a class of transport nearly ubiquitously involved in regulation of intracellular pH and cell volume. During meiosis, however, oocyte HCO₃ ⁻/Cl⁻ exchange becomes inactivated during first metaphase (MI), remains inactive in second metaphase (MII), and is reactivated only after egg activation. Previous work using pharmacological manipulations had indicated that activity of the MEK/MAPK signaling pathway was negatively correlated with HCO₃ ⁻/Cl⁻ exchange activity during meiosis. However, the mechanism by which the exchanger is inactivated during meiotic progression had not been determined, nor had the role of MEK/MAPK been directly established.

Methodology/Principal Findings

Expression of a constitutively active form of MEK (MAP kinase kinase), which prevented the normal downregulation of MAPK after egg activation, also prevented reactivation of HCO₃ ⁻/Cl⁻ exchange. Conversely, suppression of endogenous MAPK activity with dominant negative MEK activated the normally quiescent HCO₃ ⁻/Cl⁻ exchange in mature MII eggs. A GFP-tagged form of the HCO₃ ⁻/Cl⁻ exchanger isoform Ae2 (Slc4a2) was strongly expressed at the GV oocyte plasma membrane, but membrane localization decreased markedly during meiotic progression. A similar pattern for endogenous Ae2 was confirmed by immunocytochemistry. The loss of membrane-localized Ae2 appeared selective, since membrane localization of a GFP-tagged human dopamine D1 receptor did not change during meiotic maturation.

Conclusions

Direct manipulation of MAPK activity indicated that GFP-tagged Ae2 localization depended upon MAPK activity. Inactivation of HCO₃ ⁻/Cl⁻ exchange during the meiotic cell cycle may therefore reflect the loss of Ae2 from the oocyte plasma membrane, downstream of MEK/MAPK signaling. This identifies a novel role for MEK/MAPK-mediated cytostatic factor (CSF) activity during meiosis in membrane protein trafficking in mouse oocytes, and shows for the first time that selective retrieval of membrane proteins is a feature of meiosis in mammalian oocytes.     

NAMPT reduction‐induced NAD+ insufficiency contributes to the compromised oocyte quality from obese mice

Maternal obesity is associated with multiple adverse reproductive outcomes, whereas the underlying molecular mechanisms are still not fully understood. Here, we found the reduced nicotinamide phosphoribosyl transferase (NAMPT) expression and lowered nicotinamide adenine dinucleotide (NAD⁺) content in oocytes from obese mice. Next, by performing morpholino knockdown assay and pharmacological inhibition, we revealed that NAMPT deficiency not only severely disrupts maturational progression and meiotic apparatus, but also induces the metabolic dysfunction in oocytes. Furthermore, overexpression analysis demonstrated that NAMPT insufficiency induced NAD⁺ loss contributes to the compromised developmental potential of oocytes and early embryos from obese mice. Importantly, in vitro supplement and in vivo administration of nicotinic acid (NA) was able to ameliorate the obesity‐associated meiotic defects and oxidative stress in oocytes. Our results indicate a role of NAMPT in modulating oocyte meiosis and metabolism, and uncover the beneficial effects of NA treatment on oocyte quality from obese mice.     

Controlled X‐chromosome dynamics defines meiotic potential of female mouse in vitro germ cells

The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X‐chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X‐inactivation and reactivation dynamics using a tailor‐made in vitro system of primordial germ cell‐like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X‐inactivation in PGCLCs in vitro and in germ cell‐competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X‐inactivation is followed by step‐wise X‐reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X‐inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine‐tuned X‐chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis.     

Coenzyme Q10 restores oocyte mitochondrial function and fertility during reproductive aging

Female reproductive capacity declines dramatically in the fourth decade of life as a result of an age‐related decrease in oocyte quality and quantity. The primary causes of reproductive aging and the molecular factors responsible for decreased oocyte quality remain elusive. Here, we show that aging of the female germ line is accompanied by mitochondrial dysfunction associated with decreased oxidative phosphorylation and reduced Adenosine tri‐phosphate (ATP) level. Diminished expression of the enzymes responsible for CoQ production, Pdss2 and Coq6, was observed in oocytes of older females in both mouse and human. The age‐related decline in oocyte quality and quantity could be reversed by the administration of CoQ10. Oocyte‐specific disruption of Pdss2 recapitulated many of the mitochondrial and reproductive phenotypes observed in the old females including reduced ATP production and increased meiotic spindle abnormalities, resulting in infertility. Ovarian reserve in the oocyte‐specific Pdss2‐deficient animals was diminished, leading to premature ovarian failure which could be prevented by maternal dietary administration of CoQ10. We conclude that impaired mitochondrial performance created by suboptimal CoQ10 availability can drive age‐associated oocyte deficits causing infertility.