Telomere Elongation During Pre-Implantation Embryo Development.

The primary mechanism of telomere elongation in mammals is reverse transcription by telomerase. An alternative (ALT) pathway elongates telomeres by homologous recombination in some cancer cells and during pre-implantation embryo development, when telomere length increases rapidly within a few cell cycles. The maternal and paternal telomeres in the zygote are genetically and epigenetically distinct, with differences in telomere length and in chromatin packaging. We discuss models for how these asymmetries may contribute to telomere regulation during the earliest embryonic cell cycles and suggest directions for future research.

Polystyrene nanoparticles incorporate into the endoplasmic reticulum and disturb translation during meiotic maturation in mouse oocytes.

Polystyrene (PS) is one of the most common polymers that cause plastic pollution after release into the environment. Although a growing body of evidence has shown the adverse effects of PS on living organisms including humans, their effects on mammalian oocytes have not been extensively studied. In this study, we investigated the effect of exposure to PS-nanoparticle (PS-NPs) on meiotic maturation in mouse oocytes. We found that exogenous PS-NPs internalized into oocytes after penetrating the zona pellucida and accumulated in the cytoplasm of oocytes during meiotic maturation. Exposure to PS-NPs did not affect meiotic resumption but inhibited meiotic maturation by impairing spindle assembly and chromosome alignment. Moreover, exposure to PS-NPs increased oxidative stress and mitochondrial aggregation during meiotic maturation. Notably, internalized PS-NPs localized around the endoplasmic reticulum (ER) and disturbed translation in oocytes. Therefore, it is suggested that PS-NPs impair meiotic maturation not only by increasing oxidative stress and mitochondrial dysfunction, but also by decreasing translation efficiency after incorporating into the ER during meiotic maturation in mouse oocytes.

TCTP overexpression reverses age-associated telomere attrition by upregulating telomerase activity in mouse oocytes.

A prolonged time span between ovulation and fertilization can cause postovulatory aging of oocytes, which impairs oocyte quality and subsequent embryo development. Telomere attrition has long been considered as the primary hallmark of aging or the cause of age-associated diseases. However, the status of telomere and its regulation during postovulatory oocyte aging are poorly understood. Here we found that oocytes experience telomere shortening during postovulatory aging, although they have the capacity to maintain telomere length. However, translationally controlled tumor protein (TCTP) overexpression could reverse age-associated telomere shortening by upregulating telomerase activity in mouse oocytes. Telomere length in mature oocytes gradually decreased with postovulatory aging, which was associated with a marked reduction in TRF1 expression, decreased telomerase activity, and decreased homologous combination (HR)-based alternative lengthening of telomeres (ALT) with a concomitant increase in oxidative stress. Surprisingly, however, overexpression of TCTP led to a remarkable increase in telomere length during postovulatory aging. Notably, neither TRF1 nor BRCA1 level was altered by TCTP overexpression. Moreover, TCTP-mediated telomere lengthening was not blocked by HR inhibition. In striking contrast, telomerase activity, as well as TERT and TERC levels, increased after TCTP overexpression. Importantly, unlike the chromosome-wide distribution of endogenous TCTP, overexpressed TCTP was ectopically localized at telomeres, implying that TCTP overexpression is required to increase telomerase activity. Collectively, our results demonstrate that TCTP prevents telomere attrition during postovulatory aging by upregulating telomerase activity in mouse oocytes.

TRF1 Depletion Reveals Mutual Regulation Between Telomeres, Kinetochores, and Inner Centromeres in Mouse Oocytes.

In eukaryotic chromosomes, the centromere and telomere are two specialized structures that are essential for chromosome stability and segregation. Although centromeres and telomeres often are located in close proximity to form telocentric chromosomes in mice, it remained unclear whether these two structures influence each other. Here we show that TRF1 is required for inner centromere and kinetochore assembly in addition to its role in telomere protection in mouse oocytes. TRF1 depletion caused premature chromosome segregation by abrogating the spindle assembly checkpoint (SAC) and impairing kinetochore-microtubule (kMT) attachment, which increased the incidence of aneuploidy. Notably, TRF1 depletion disturbed the localization of Survivin and Ndc80/Hec1 at inner centromeres and kinetochores, respectively. Moreover, SMC3 and SMC4 levels significantly decreased after TRF1 depletion, suggesting that TRF1 is involved in chromosome cohesion and condensation. Importantly, inhibition of inner centromere or kinetochore function led to a significant decrease in TRF1 level and telomere shortening. Therefore, our results suggest that telomere integrity is required to preserve inner centromere and kinetochore architectures, and vice versa, suggesting mutual regulation between telomeres and centromeres.

RASSF1A Regulates Spindle Organization by Modulating Tubulin Acetylation via SIRT2 and HDAC6 in Mouse Oocytes.

Dynamic changes in microtubules during cell cycle progression are essential for spindle organization to ensure proper segregation of chromosomes. There is growing evidence that post translational modifications of tubulins are the key factors that contribute to microtubule dynamics. However, how dynamic properties of microtubules are regulated in mouse oocytes is unclear. Here, we show that tumor suppressor RASSF1A is required for tubulin acetylation by regulating SIRT2 and HDAC6 during meiotic maturation in mouse oocytes. We found that RASSF1A was localized at the spindle microtubules in mouse oocytes. Knockdown of RASSF1A perturbed meiotic progression by impairing spindle organization and chromosome alignment. Moreover, RASSF1A knockdown disrupted kinetochore-microtubule (kMT) attachment, which activated spindle assembly checkpoint and increased the incidence of aneuploidy. In addition, RASSF1A knockdown decreased tubulin acetylation by increasing SIRT2 and HDAC6 levels. Notably, defects in spindle organization and chromosome alignment after RASSF1A knockdown were rescued not only by inhibiting SIRT2 or HDAC6 activity, but also by overexpressing acetylation mimicking K40Q tubulin. Therefore, our results demonstrated that RASSF1A regulates SIRT2- and HDAC6-mediated tubulin acetylation for proper spindle organization during oocyte meiotic maturation.

Prevention of quality decline and delivery of siRNA using exogenous TCTP translocation across the zona pellucida in mouse oocytes.

The delivery of exogenous molecules into mammalian oocytes or embryos has been a challenge because of the existence of the protective zona pellucida (ZP) surrounding the oocyte membrane. Here we show that exogenous translationally controlled tumor protein (TCTP) is able to translocate into oocytes across the ZP and prevents quality deterioration during in vitro culture. Recombinant TCTP-mCherry added to culture media were incorporated into oocytes after passing through the ZP. After internalization, recombinant TCTP-mCherry were enriched at the cortex with wide distribution within the cytoplasm. This translocation capacity of TCTP is dependent on its N-terminal protein transduction domain (PTD). Moreover, translocated recombinant TCTP-mCherry reduced quality deterioration of oocytes during prolonged in vitro culture, which in turn improved fertilization and early embryo development. Furthermore, conjugates between PTD of TCTP and cyclin B1 siRNAs internalized into the cytoplasm of oocytes and downregulated cyclin B1 level. Therefore, our results are the first to show that TCTP has the ability to translocate into oocyte cytoplasm penetrating through the ZP, providing the possibility for preserving oocyte quality during extended in vitro culture and for delivering siRNAs into mouse oocytes.

Protective effects of ethanol extracts of Artemisia asiatica Nakai ex Pamp. on ageing-induced deterioration in mouse oocyte quality.

Following ovulation, oocytes undergo a time-dependent deterioration in quality referred to as post-ovulatory ageing. Although various factors influence the post-ovulatory ageing of oocytes, oxidative stress is a key factor involved in deterioration of oocyte quality. Artemisia asiatica Nakai ex Pamp. has been widely used in East Asia as a food ingredient and traditional medicine for the treatment of inflammation, cancer, and microbial infections. Recent studies have shown that A. asiatica exhibits antioxidative effects. In this study, we investigated whether A. asiatica has the potential to attenuate deterioration in oocyte quality during post-ovulatory ageing. Freshly ovulated mouse oocytes were cultured with 0, 50, 100 or 200 µg/ml ethanol extracts of A. asiatica Nakai ex Pamp. After culture for up to 24 h, various ageing-induced oocyte abnormalities, including morphological changes, reactive oxygen species (ROS) accumulation, apoptosis, chromosome and spindle defects, and mitochondrial aggregation were determined. Treatment of oocytes with A. asiatica extracts reduced ageing-induced morphological changes. Moreover, A. asiatica extracts decreased ROS generation and the onset of apoptosis by preventing elevation of the Bax/Bcl-2 expression ratio during post-ovulatory ageing. Furthermore, A. asiatica extracts attenuated the ageing-induced abnormalities including spindle defects, chromosome misalignment and mitochondrial aggregation. Our results demonstrate that A. asiatica can relieve deterioration in oocyte quality and delay the onset of apoptosis during post-ovulatory ageing.

TCTP regulates spindle assembly during postovulatory aging and prevents deterioration in mouse oocyte quality.

If no fertilization occurs for a prolonged time following ovulation, oocytes experience a time-dependent deterioration in quality both in vivo and in vitro due to processes called postovulatory aging. Because the postovulatory aging of oocytes has marked detrimental effects on embryo development and offspring, many efforts have been made to unveil the underlying mechanisms. Here we showed that translationally controlled tumor protein (TCTP) regulates spindle assembly during postovulatory aging and prevents deterioration in mouse oocyte quality. Spindle dynamics decreased with reduced TCTP level during aging of mouse oocytes. Knockdown of TCTP accelerated the reduction of spindle dynamics, accompanying with aging-related deterioration of oocyte quality. Conversely, overexpression of TCTP prevented aging-associated decline of spindle dynamics. Moreover, the aging-related abnormalities in oocytes were rescued after TCTP overexpression, thereby improving fertilization competency and subsequent embryo development. Therefore, our results demonstrate that TCTP-mediated spindle dynamics play a key role in maintaining oocyte quality during postovulatory aging and overexpression of TCTP is sufficient to prevent aging-associated abnormalities in mouse oocytes.

Peroxiredoxins are required for spindle assembly, chromosome organization, and polarization in mouse oocytes.

Peroxiredoxins (Prxs) are highly conserved antioxidant enzymes and are implicated in multiple biological processes; however, their function in oocyte meiosis has not been studied. Here we show that inhibition of Prx I and II results in spindle defects, chromosome disorganization, and impaired polarization in mouse oocytes. Prx I was specifically localized at the spindle, whereas Prx II was enriched at the oocyte cortex and chromosomes. Inhibition of Prx activity with conoidin A disturbed assembly of the microtubule organizing center (MTOC) through Aurora A regulation, leading to defects in spindle formation. Moreover, conoidin A impaired actin filament and cortical granule (CG) distribution, disrupting actin cap and CG formation, respectively. Conoidin A also increased DNA damage without significantly increasing reactive oxygen species (ROS) levels, suggesting that the effects of conoidin A on meiotic maturation are not likely associated with ROS scavenging pathways. Therefore, our data suggest that Prxs are required for spindle assembly, chromosome organization, and polarization during meiotic maturation.

Eccentric localization of catalase to protect chromosomes from oxidative damages during meiotic maturation in mouse oocytes.

The maintenance of genomic integrity and stability is essential for the survival of every organism. Unfortunately, DNA is vulnerable to attack by a variety of damaging agents. Oxidative stress is a major cause of DNA damage because reactive oxygen species (ROS) are produced as by-products of normal cellular metabolism. Cells have developed eloquent antioxidant defense systems to protect themselves from oxidative damage along with aerobic metabolism. Here, we show that catalase (CAT) is present in mouse oocytes to protect the genome from oxidative damage during meiotic maturation. CAT was expressed in the nucleus to form unique vesicular structures. However, after nuclear envelope breakdown, CAT was redistributed in the cytoplasm with particular focus at the chromosomes. Inhibition of CAT activity increased endogenous ROS levels, but did not perturb meiotic maturation. In addition, CAT inhibition produced chromosomal defects, including chromosome misalignment and DNA damage. Therefore, our data suggest that CAT is required not only to scavenge ROS, but also to protect DNA from oxidative damage during meiotic maturation in mouse oocytes.

Beclin-1 knockdown shows abscission failure but not autophagy defect during oocyte meiotic maturation.

Cytokinesis is the final step in cell division that results in the separation of a parent cell into daughter cells. Unlike somatic cells that undergo symmetric division, meiotic division is highly asymmetric, allowing the preservation of maternal resources for embryo development. Beclin-1/BECN1, the mammalian homolog of yeast Atg6, is a key molecule of autophagy. As part of a class III phosphatidylinositol 3-kinase (PI3K-III) complex, BECN1 initiates autophagosome formation by coordinating membrane trafficking. However, emerging evidence suggests that BECN1 regulates chromosome segregation and cytokinesis during mitosis. Thus, we investigated the function of BECN1 during oocyte meiotic maturation. BECN1 was widely distributed during meiotic maturation forming small vesicles. Interestingly, BECN1 is also detected at the midbody ring during cytokinesis. Depletion of BECN1 impaired the cytokinetic abscission, perturbing the recruitment of ZFYVE26 at the midbody. Similar phenotypes were observed when PI3K-III activity was inhibited. However, inhibition of autophagy by depleting Atg14L did not disturb meiotic maturation. Therefore, our results not only demonstrate that BECN1 as a PI3K-III component is essential for cytokinesis, but also suggest that BECN1 is not associated with autophagy pathway in mouse oocytes.

TCTP regulates spindle microtubule dynamics by stabilizing polar microtubules during mouse oocyte meiosis.

Dynamic changes in spindle structure and function are essential for maintaining genomic integrity during the cell cycle. Spindle dynamics are highly dependent on several microtubule-associated proteins that coordinate the dynamic behavior of microtubules, including microtubule assembly, stability and organization. Here, we show that translationally controlled tumor protein (TCTP) is a novel microtubule-associated protein that regulates spindle dynamics during meiotic maturation. TCTP was expressed and widely distributed in the cytoplasm with strong enrichment at the spindle microtubules during meiosis. TCTP was found to be phosphorylated during meiotic maturation, and was exclusively localized to the spindle poles. Knockdown of TCTP impaired spindle organization without affecting chromosome alignment. These spindle defects were mostly due to the destabilization of the polar microtubules. However, the stability of kinetochore microtubules attached to chromosomes was not affected by TCTP knockdown. Overexpression of a nonphosphorylable mutant of TCTP disturbed meiotic maturation, stabilizing the spindle microtubules. In addition, Plk1 was decreased by TCTP knockdown. Taken together, our results demonstrate that TCTP is a microtubule-associating protein required to regulate spindle microtubule dynamics during meiotic maturation in mouse oocytes.