Oocyte Formation Research Articles

Bait-subsidized diets and their effects on ovigerous North American lobsters (Homarus americanus)

Ovigerous American lobsters (Homarus americanus) display a protracted period of ovary maturation and maternal care when incubating their eggs, potentially influencing offspring fitness. Lobsters consume a wide range of food items; however, trap bait may comprise a larger proportion of their diet in some fished areas compared to non-fished areas, and the long-term consequences of a bait-based diet remain largely unexplored in lobsters. We tested the hypothesis that disproportionate amounts of bait in the diets of pre-ovigerous females affect the quality of their ovaries and eggs. We held pre-ovigerous lobsters (n = 29) over a period of ~ 300 days (range = 270–378) and fed them diets of herring bait, natural prey items (crab, mussel, urchin, macroalgae), or a combination of both diet types. Nutritional status, measured as biweekly blood indices and total glucose levels, suggest differences between lobsters fed a natural or combination diet and lobsters fed a bait-based diet (ANOVA; P < 0.05). We found that bait diets contained more protein (58.5%) and lipids (31.6%) compared to natural diets (34.5 and 13.2%, respectively) and lipid levels in ovaries and eggs significantly correlated with each other for all treatments (r = 0.76, n = 15, P = 0.028). Histopathological analysis indicates that ovaries contained more variable maturation in starved lobsters or those fed with bait, with some animals showing delayed gonad maturation. Results suggest that a varied diet promotes the overall fitness of ovigerous lobsters and the associated reserves that are used for ovarian development and subsequent oocyte formation.

Gametogenesis and reproductive pattern of the reef-building coral Acropora millepora in northwestern Philippines

ABSTRACTCoral populations continue to decline in many regions because of natural and anthropogenic disturbances; and the persistence of coral populations mainly relies on natural recruitment through coral reproduction. However, studies on the reproduction of many important reef-building coral species in one of the most biodiverse region, the Coral Triangle, are very limited. Here, we investigated the reproductive biology of the coral Acropora millepora in Bolinao-Anda Reef Complex, northwestern Philippines from February 2014 to March 2015, through in situ assessment of gamete maturity, histological examination of gamete development and ex situ spawning observations. Results showed that A. millepora exhibited an annual gametogenic cycle. Formation of oocytes took place within 8 months, whereas spermatocytes developed in 3 months before the gamete release. Abundance and size of eggs in A. millepora were greatest during the months nearest to the spawning period, which coincides with the rapid increase in sea surface temperature. Ex situ broadcast spawning occurred around the months of February–March, which suggests an extended spawning pattern. Results provide additional information on coral reproduction in the region and reveal the potential of A. millepora in providing coral propagules for the replenishment and recovery of degraded coral populations.

Effects of monoterpenes on mortality, growth, fecundity, and ovarian development of Bactrocera zonata (Saunders) (Diptera: Tephritidae).

The peach fruit fly (PFF), Bactrocera zonata, is a serious insect pest infesting fruits and vegetables. The insecticidal activity of three monoterpenes, namely, (R)-camphor, (R)-carvone, and (1R,2S,5R)-menthol, was evaluated on the second-instar larvae of B. zonata. In addition, the latent effects of monoterpenes on pupation, adult emergence, deformation, oviposition, adult longevity, and ovarian development were also examined. The three tested monoterpenes showed pronounced insecticidal activity against B. zonata larvae with (R)-carvone being the most potent toxicant. When the second-instar larvae of B. zonata were treated with monoterpenes at concentrations of 20, 50, and 70mg/kg for 72h, significant reduction in pupation and adult emergence was observed. The three monoterpenes caused complete suppression of adult emergence at 100mg/kg. Moreover, monoterpenes induced complete inhibition of egg deposition at all tested concentrations. Some adult deformations were also noticed at 20, 50, and 70mg/kg. However, (R)-carvone was more effective than (1R,2S,5R)-menthol and (R)-camphor on the examined biological parameters. On the other hand, histological examination of the ovaries of emerged females from larvae that fed on diet treated with (R)-carvone, (1R,2S,5R)-menthol, and (R)-camphor at 20 and 50mg/L indicated that both concentrations caused retardation in the development of ovarioles. It is clear that all the egg chambers are empty; the germarium region is constricted at base due to the failure of oocyte formation. Many vacant spaces were present between ovarioles.

Potassium bromate disrupts mitochondrial distribution within murine oocytes during in vitro maturation.

PurposeAs disturbed mitochondrial distribution is thought to be a cause of the aging of oocytes, it was investigated whether oxidizing agents exert harmful effects on nuclear maturation and mitochondrial cluster formation in murine oocytes and whether antioxidants could rescue such harmful effects in vitro.MethodsOocytes were obtained from female Institute of Cancer Research mice 48 h after an intraperitoneal injection of 7.5 IU pregnant mare serum gonadotropin. The oocytes were cultured with potassium bromate, an oxidizing agent, in the presence or absence of the antioxidant, resveratrol. After 12 h, the nuclear phases and mitochondrial distribution were observed.ResultsSignificantly decreased rates of metaphase II (MII) oocytes were observed with 750 μM and 1000 μM of potassium bromate, while a significant increase in abnormal mitochondrial clusters was induced at 500 μM, 750 μM, and 1,000 μM. The addition of 10 μM or 20 μM resveratrol improved both MII maturity and the cluster formation rates in the presence of potassium bromate.ConclusionsThe addition of potassium bromate reduced MII maturity rates and induced abnormal mitochondrial cluster formation. This effect was alleviated by the antioxidant, resveratrol. The in vitro model used herein is useful for investigating the functions of antioxidants in the aging of oocytes.

Intracellular ice formation in human oocytes vs. cooling rate and temperature

Intracellular ice formation in human oocytes vs. cooling rate and temperature

Meiotic spindle formation in mammalian oocytes: implications for human infertility.

In the final stage of oogenesis, mammalian oocytes generate a meiotic spindle and undergo chromosome segregation to yield an egg that is ready for fertilization. Herein, we describe the recent advances in understanding the mechanisms controlling formation of the meiotic spindle in metaphase I (MI) and metaphase II (MII) in mammalian oocytes, and focus on the differences between mouse and human oocytes. Unlike mitotic cells, mammalian oocytes lack typical centrosomes that consist of two centrioles and the surrounding pericentriolar matrix proteins, which serve as microtubule-organizing centers (MTOCs) in most somatic cells. Instead, oocytes rely on different mechanisms for the formation of microtubules in MI spindles. Two different mechanisms have been described for MI spindle formation in mammalian oocytes. Chromosome-mediated microtubule formation, including RAN-mediated spindle formation and chromosomal passenger complex-mediated spindle elongation, controls the growth of microtubules from chromatin, while acentriolar MTOC-mediated microtubule formation contributes to spindle formation. Mouse oocytes utilize both chromatin- and MTOC-mediated pathways for microtubule formation. The existence of both pathways may provide a fail-safe mechanism to ensure high fidelity of chromosome segregation during meiosis. Unlike mouse oocytes, human oocytes considered unsuitable for clinical in vitro fertilization procedures, lack MTOCs; this may explain why meiosis in human oocytes is often error-prone. Understanding the mechanisms of MI/MII spindle formation, spindle assembly checkpoint, and chromosome segregation, in mammalian oocytes, will provide valuable insights into the molecular mechanisms of human infertility.

Gene silencing reveals multiple functions of Na+/K+-ATPase in the salmon louse (Lepeophtheirus salmonis)

Na+/K+-ATPase has a key function in a variety of physiological processes including membrane excitability, osmoregulation, regulation of cell volume, and transport of nutrients. While knowledge about Na+/K+-ATPase function in osmoregulation in crustaceans is extensive, the role of this enzyme in other physiological and developmental processes is scarce. Here, we report characterization, transcriptional distribution and likely functions of the newly identified L. salmonis Na+/K+-ATPase (LsalNa+/K+-ATPase) α subunit in various developmental stages. The complete mRNA sequence was identified, with 3003 bp open reading frame encoding a putative protein of 1001 amino acids. Putative protein sequence of LsalNa+/K+-ATPase revealed all typical features of Na+/K+-ATPase and demonstrated high sequence identity to other invertebrate and vertebrate species. Quantitative RT-PCR analysis revealed higher LsalNa+/K+-ATPase transcript level in free-living stages in comparison to parasitic stages. In situ hybridization analysis of copepodids and adult lice revealed LsalNa+/K+-ATPase transcript localization in a wide variety of tissues such as nervous system, intestine, reproductive system, and subcuticular and glandular tissue. RNAi mediated knock-down of LsalNa+/K+-ATPase caused locomotion impairment, and affected reproduction and feeding. Morphological analysis of dsRNA treated animals revealed muscle degeneration in larval stages, severe changes in the oocyte formation and maturation in females and abnormalities in tegmental glands. Thus, the study represents an important foundation for further functional investigation and identification of physiological pathways in which Na+/K+-ATPase is directly or indirectly involved.

Effect of pre-in vitro maturation with cAMP modulators on the acquisition of oocyte developmental competence in cattle.

The administration of follicle-stimulating hormone (FSH) prior to oocyte retrieval improves oocyte developmental competence. During bovine embryo production in vitro, however, oocytes are typically derived from FSH-unprimed animals. In the current study, we examined the effect of pre-in vitro maturation (IVM) with cAMP modulators, also known as the second messengers of FSH, on the developmental competence of oocytes derived from small antral follicles (2–4 mm) of FSH-unprimed animals. Pre-IVM with N6,2ʹ-O-dibutyryladenosine 3′,5′-cyclicmonophosphate (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX) for 2 h improved the blastocyst formation in oocytes stimulated by FSH or amphiregulin (AREG). Furthermore, pre-IVM enhanced the expression of the FSH- or AREG-stimulated extracellular matrix-related genes HAS2, TNFAIP6, and PTGS2, and epidermal growth factor (EGF)-like peptide-related genes AREG and EREG. Additionally, pre-IVM with dbcAMP and IBMX enhanced the expression of EGFR, and also increased and prolonged cumulus cell-oocyte gap junctional communication. The improved oocyte development observed using the pre-IVM protocol was ablated by an EGF receptor phosphorylation inhibitor. These results indicate that pre-IVM with cAMP modulators could contribute to the acquisition of developmental competence by bovine oocytes from small antral follicles through the modulation of EGF receptor signaling and oocyte-cumulus/cumulus-cumulus gap junctional communication.

Involvement of Kif4a in Spindle Formation and Chromosome Segregation in Mouse Oocytes.

Kif4a, a member of the kinesin superfamily, has been reported to participate in a series of cellular processes such as chromosome condensation and cytokinesis during mitosis. However, the roles of KIF4a in meiosis are still unknown. In present study we found that the Kif4a protein expression decreased in maternal aged mouse oocytes. We then explored the roles of Kif4a in mouse oocyte meiosis by knockdown analysis. Kif4a was enriched at the spindle during mouse oocyte maturation. By specific knock down of the Kif4a using morpholino microinjection, we found that the disruption of Kif4a caused the failure of polar body extrusion. Further analysis indicated that Kif4a might affect the spindle morphology and chromosome alignment in the mouse oocytes, and this might be due to the regulation of tubulin acetylation. Moreover, our results showed that an increased proportion of aneuploidy in the Kif4a knock down oocytes, and this might be due to the loss of kinetochore-microtubule attachment. Taken together, these results suggested that Kif4a possibly regulated mouse oocyte meiosis through its effects on the spindle organization and accurate chromosome segregation, and the loss of Kif4a might be related with aneuploidy of aging oocytes.

Preantral follicle culture and oocyte quality

The formation of high-quality oocytes depends on complex stage-specific interactions between the germ cell and the somatic compartment involving endocrine, paracrine, and autocrine regulation. Cooperativity in bidirectional signalling and metabolism in response to factors in the microenvironment drive growth, proliferation, cell cycle regulation, spindle formation and the establishment of epigenetic marks in oocytes. This is essential to ensure faithful chromosome segregation and to achieve high oocyte quality, with far-reaching consequences for embryo survival, development and the health of offspring. Oocytes reach developmental capacity throughout early meiotic stages up to full growth and acquisition of nuclear and cytoplasmic maturational competence during folliculogenesis. Improved preantral follicle culture in which ideally intimate contacts between oocyte and somatic cells are retained provides unique opportunities to assess the effects of microenvironment, growth factors, hormones, cryopreservation and environmental exposure on folliculogenesis and oocyte quality. An optimised follicle culture can contribute to the generation of high-quality oocytes for use in fertility preservation in cancer patients, animal breeding and the preservation of endangered species. The past decade has brought about major advances in follicle culture from different species. Recent advances in preantral follicle culture are discussed to assess the effects of environment, adverse exposure, cryopreservation and age on oocyte quality.

Intersectin 2 controls actin cap formation and meiotic division in mouse oocytes through the Cdc42 pathway.

Intersectins (ITSNs), an evolutionarily conserved adaptor protein family, have been implicated in multiple biologic processes; however, their functions in mammalian oocytes have not been addressed. Here, we report delayed meiotic resumption and defective cytokinesis upon specific depletion of ITSN2 in mouse oocytes. In particular, abnormal spindle, misaligned chromosomes, and loss of cortical actin cap are readily observed in ITSN2-depleted oocytes. Similarly, a small molecule that targets the Cdc42-ITSN interaction also disrupts oocyte maturation and actin polymerization. Moreover, we find that ITSN2 depletion reduces the activity of Cdc42 in oocytes and, of note, that forced expression of the dominant-positive mutant of Cdc42, in part, prevents the effects of ITSN2 knockdown on actin cap formation. In addition, the localization of WASP and Arp2, the downstream effector proteins of Cdc42, is altered in ITSN2-depleted oocytes accordingly. In summary, our data support a model in which ITSN2 depletion induces the inactivation of Cdc42, which, in turn, influences the distribution and function of Arp2/3 and WASP, consequently disrupting oocyte polarity establishment and meiotic division.-Zhang, J., Ma, R., Li, L., Wang, L., Hou, X., Han, L., Ge, J., Li, M., Wang, Q. Intersectin 2 controls actin cap formation and meiotic division in mouse oocytes through the Cdc42 pathway.

The blood vessels development, morphogenesis and blood circulation are three ontologic groups highly up-regulated in porcine oocytes before in vitro maturation

AbstractThe mammalian oocytes undergo significant biochemical and structural modifications during maturation both in vitro and in vivo. These changes involve chromatin reorganization and modification within metabolic status of cytoplasmic organelles. After oocytes’ successful maturation the substantially increased storage of RNA was observed. Moreover, the early embryo interaction with maternal endometrial tissue after fertilization is up to now considered as the main marker of proper embryo implantation and early growth. In this study, we first investigated the expression profile of genes involved in blood vessel formation and blood circulation in porcine oocytes before and after in vitro maturation.The cumulus-oocyte complexes were collected from pubertal Landrace gilts and classified as before in vitro maturation (in Vivo) or after in vitro maturation (in Vitro). The RNA was isolated from these two experimental groups and analyzed using Affymetrix microarrays.We found an increased expression of genes involved in ontological groups such as “blood circulation” (TPM1, ECE1, ACTA2, EPHX2, EDNRA, NPR2, MYOF, TACR3, VEGFA, GUCY1B3), “blood vessel development” (ANGPTL4, CYR61, SEMA5A, ID1, RHOB, RTN4, IHH, ANGPT2, EDNRA, TGFBR3, MYO1E, MMP14), and “blood vessels morphogenesis” (ANGPT2, as well as other common transcripts) in in Vivo group as compared to decreased expression of these genes in in Vitro group of oocytes.It has been suggested that investigated genes undergo significant expression before in vitro maturation, when enhanced storage of large amount of RNA takes place. Creating templates for synthesis of proteins is required for formation of fully mature gametes and early embryo growth. Therefore we hypothesized that the processes of vascularization and/or angiogenesis reach a high activity in immature oocytes and are distinct from achievement of maturational stage by oocytes in pigs.

The role of L-type calcium channels in mouse oocyte maturation, activation and early embryonic development

Calcium ion fluctuation is closely related to the transformation of cell cycle. However, little is known about the function of L-type calcium channel in mammalian oocyte and embryo development. We thus studied the roles of L-type calcium channel in mouse oocyte meiotic maturation, parthenogenetic activation and early embryonic development. We used the antagonist Amlodipine to block L-type calcium channel. Oocytes or zygotes were cultured to different time points with 0 μM, 10 μM, 30 μM and 50 μM Amlodipine. Then we checked the rate of first polar body extrusion, spindle formation, asymmetric division parthenogenetic activation and early embryo cleavage. The results showed that Amlodipine treatment did not affect germinal vesicle breakdown, but caused disruption of cytoskeleton organization, symmetric division, formation of mature oocytes with a large polar body, or reduced the first polar body extrusion, depending on its concentrations. Amlodipine treatment also resulted in decreased parthenogenetic activation and arrested early embryonic development. Overall, these data suggest that proper function of L-type calcium channel is critical for oocyte maturation, activation, and early embryonic development.

Chlorogenic acid supplementation during in vitro maturation improves maturation, fertilization and developmental competence of porcine oocytes.

Chlorogenic acid (CGA) is a quinic acid conjugate of caffeic acid, and a phytochemical found in many fruits and beverages that acts as an antioxidant. The present study investigated the effects of CGA supplementation during in vitro maturation (IVM), on in vitro development of porcine oocytes, to improve the porcine in vitro production (IVP) system. Oocytes were matured either without (control) or with CGA (10, 50, 100 and 200μM). Subsequently, the matured oocytes were fertilized and cultured in vitro for 7day. The rates of maturation, fertilization and blastocyst formation of oocytes matured with 50μM CGA were significantly (p<.05) higher than those of the control oocytes. Hydrogen peroxide (H2 O2 ) is one of the reactive oxygen species and induces DNA damage in porcine oocytes. When oocytes were matured with 1mM H2 O2 to assess the protective effect of CGA, 50μM CGA supplementation improved the maturation rate and the proportion of DNA-fragmented nuclei in oocytes compared with control oocytes matured without CGA. Moreover, when oocytes were matured with either 50μM CGA (control) or caffeic acid (10, 50 and 100μM), the rates of maturation, fertilization and the blastocyst formation of oocytes matured with 50μM CGA were similar to those of oocytes matured with 10 and 50μM caffeic acid. Our results suggest that CGA has comparable effects to caffeic acid, and IVM with 50μM CGA is particularly beneficial to IVP of porcine embryos and protects oocytes from DNA damage induced by oxidative stress. Supplementation of CGA to the maturation medium has a potential to improve porcine IVP system.

Epigenetic regulation during the differentiation of stem cells to germ cells.

Gametogenesis is an essential process to ensure the transfer of genetic information from one generation to the next. It also provides a mechanism by which genetic evolution can take place. Although the genome of primordial germ cells (PGCs) is exactly the same with somatic cells within an organism, there are significant differences between their developments. For example, PGCs eventually undergo meiosis to become functional haploid gametes, and prior to that they undergo epigenetic imprinting which greatly alter their genetic regulation. Epigenetic imprinting of PGCs involves the erasure of DNA methylation and the reestablishment of them during sperm and oocyte formation. These processes are necessary and important during gametogenesis. Also, histone modification and X-chromosome inactivation have important roles during germ cell development. Recently, several studies have reported that functional sperm or oocytes can be derived from stem cells in vivo or in vitro. To produce functional germ cells, induction of germ cells from stem cells must recapitulate these processes similar to endogenous germ cells, such as epigenetic modifications. This review focuses on the epigenetic regulation during the process of germ cell development and discusses their importance during the differentiation from stem cells to germ cells.

Putative germline and pluripotent stem cells in adult mouse ovary and their in vitro differentiation potential into oocyte-like and somatic cells.

According to classical knowledge of reproductive biology, in the ovary of female mammals there is a limited number of oocytes and there is no possibility of renewal if the oocytes are lost due to disease or injury. However, in recent years, the results of some studies on renewal and formation of oocytes and follicles in the adult mammalian ovary have led to the questioning of this opinion. The aim of our study is to demonstrate the presence of putative germline and pluripotent stem cells in the adult mouse ovary and their differentiation potential into germ and somatic cells. In ovary tissues and cells harvested from pre-differentiation step, the expression of pluripotent and germline stem cell markers was analysed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and western blotting. Embryoid bodies that formed in this step were analysed using immunofluorescence staining and transmission electron microscopy. Ovarian stem cells were induced to differentiate into oocyte, osteoblast, chondrocyte and neural cells. Besides morphological observation, differentiated cells were analysed by RT-PCR, histochemical and immunofluorescence staining. Expression of germline and pluripotent stem cell markers both in mRNA and at the protein level were detected in the pre-differentiated cells and ovary tissues. As a result of the differentiation process, the formation of oocyte-like cells, osteoblasts, chondrocytes and neural cells was observed and characteristics of differentiated cells were confirmed using the methods mentioned above. Our study results revealed that the adult mouse ovary contains germline and pluripotent stem cells with the capacity to differentiate into oocyte-like cells, osteoblasts, chondrocytes and neural cells.

Morphogenesis-related gene-expression profile in porcine oocytes before and after in vitro maturation.

Mammalian oocyte maturation is achieved when oocytes reach metaphase II (MII) stage, and accumulate mRNA and proteins in the cytoplasm following fertilization. It has been shown that oocytes investigated before and after in vitro maturation (IVM) differ significantly in transcriptomic and proteomic profiles. Additionally, folliculogenesis and oogenesis is accompanied by morphogenetic changes, which significantly influence further zygote formation and embryo growth. This study aimed to determine new transcriptomic markers of porcine oocyte morphogenesis that are associated with cell maturation competence. An Affymetrix microarray assay was performed on an RNA template isolated from porcine oocytes before (n = 150) and after (n = 150) IVM. The brilliant cresyl blue (BCB) staining test was used for identification of cells with the highest developmental capacity. DAVID (Database for Annotation, Visualization, and Integrated Discovery) software was used for the extraction of the genes belonging to a cell morphogenesis Gene Ontology group. The control group consisted of freshly isolated oocytes. In total, 12,000 different transcripts were analysed, from which 379 genes were downregulated and 40 were upregulated in oocytes following IVM. We found five genes, SOX9, MAP1B, DAB2, FN1, and CXCL12, that were significantly upregulated in oocytes after IVM (in vitro group) compared with oocytes analysed before IVM (in vivo group). In conclusion, we found new transcriptomic markers of oocyte morphogenesis, which may be also recognized as significant mediators of cellular maturation capacity in pigs. Genes SOX9, MAP1B, DAB2, FN1, and CXCL12 may be involved in the regulation of the MII stage oocyte formation and several other processes that are crucial for porcine reproductive competence.

Pyriproxyfen treatment terminates Halyomorpha halys reproductive diapause, with an indirect mortality effect on its egg parasitoid Trissolcus japonicus

The success of Halyomorpha halys (Stal) as an invasive species in temperate climates is partly due to its ability to survive winter in a state of reproductive diapause. In H. halys, the termination of juvenile hormone (JH) production by the corpora allata, in response to a decline in day length, leads to the initiation of diapause and suspension of reproductive activity. Results of this study showed that topical application of the JH analog pyriproxyfen at doses of 0.05–100 μg per insect resulted in the resumption of oocyte formation and oviposition in a dose-dependent manner, with the ED50 estimated at 1.14 μg per insect. Eggs induced by pyriproxyfen application had significantly lower hatchability than control eggs. The indirect effects of host exposure to pyriproxyfen on the hymenopteran parasitoid Trissolcus japonicus (Ashmead), a candidate biological control agent of H. halys, were evaluated. When presented with eggs produced by pyriproxyfen-treated females, T. japonicus was observed ovipositing, but no adult parasitoids emerged from host eggs. Exposure to pyriproxyfen has the potential to lower the reproductive potential of H. halys populations by inducing the production of eggs with reduced viability during periods of low resource availability. The complete mortality of T. japonicus developing on pyriproxyfen-induced eggs suggests that insect growth regulators which interfere with host reproduction may exert a strong indirect effect on beneficial egg parasitoids, and may not be compatible with egg parasitoid biological control programs.

Caffeine delays oocyte aging and maintains the quality of aged oocytes safely in mouse.

Caffeine, as an oocyte aging inhibitor, was used in many different species to control or delay oocyte aging. However, the safety of caffeine and developmental competence of aged oocytes inhibited by caffeine has not been studied systematically. So we detected the spindle morphology, distribution of cortical granules, zona pellucida hardening and pronucleus formation to assess oocyte quality of caffeine treated oocytes. We found that aged oocytes treated by caffeine maintained weak susceptibility to activating stimuli and regained normal competent after aged further 6 hr. Caffeine maintained the spindle morphology, changed cortical granules distribution of aged oocytes and could not prevent zona pellucida hardening. Furthermore, caffeine increased pronucleus formation of aged oocytes and decreased fragmentation after fertilization. These results suggested that caffeine could maintain the quality of aged oocytes safely in mouse.

DNA Double-Strand Breaks Induce the Nuclear Actin Filaments Formation in Cumulus-Enclosed Oocytes but Not in Denuded Oocytes

As a gamete, oocyte needs to maintain its genomic integrity and passes this haploid genome to the next generation. However, fully-grown mouse oocyte cannot respond to DNA double-strand breaks (DSBs) effectively and it is also unable to repair them before the meiosis resumption. To compensate for this disadvantage and control the DNA repair events, oocyte needs the cooperation with its surrounding cumulus cells. Recently, evidences have shown that nuclear actin filament formation plays roles in cellular DNA DSB repair. To explore whether these nuclear actin filaments are formed in the DNA-damaged oocytes, here, we labeled the filament actins in denuded oocytes (DOs) and cumulus-enclosed oocytes (CEOs). We observed that the nuclear actin filaments were formed only in the DNA-damaged CEOs, but not in DOs. Formation of actin filaments in the nucleus was an event downstream to the DNA damage response. Our data also showed that the removal of cumulus cells led to a reduction in the nuclear actin filaments in oocytes. Knocking down of the Adcy1 gene in cumulus cells did not affect the formation of nuclear actin filaments in oocytes. Notably, we also observed that the nuclear actin filaments in CEOs could be induced by inhibition of gap junctions. From our results, it was confirmed that DNA DSBs induce the nuclear actin filament formation in oocyte and which is controlled by the cumulus cells.