Circular RNA profiling in the oocyte and cumulus cells reveals thatcircARMC4is essential for porcine oocyte maturation

ABSTRACTThousands of circular RNAs (circRNAs) have been recently discovered in cumulus cells and oocytes from several species. However, the expression and function of circRNA during porcine oocyte meiotic maturation have been never examined. Here, we separately identified 7,067 and 637 circRNAs in both the cumulus cell and the oocyte via deep sequencing and bioinformatic analysis. Further analysis revealed that a faction of circRNAs is differentially expressed (DE) in a developmental stage-specific manner. The host genes of DE circRNAs are markedly enriched to multiple signaling pathways associated with cumulus cell function and oocyte maturation. Additionally, most DE circRNAs harbor several miRNA targets, suggesting that these DE circRNAs potentially act as miRNA sponge. Importantly, we found that maternalcircARMC4knockdown by siRNA microinjection caused a severely impaired chromosome alignment, and significantly inhibited first polar body extrusion and early embryo development. Taken together, these results demonstrate for the first time that circRNAs are abundantly and dynamically expressed in a developmental stage-specific manner in cumulus cells and oocytes, and maternally expressedcircARMC4is essential for porcine oocyte meiotic maturation and early embryo development.

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172 THE APOPTOTIC EFFECTS OF BISPHENOL A-INDUCED MITOCHONDRIAL-DERIVED REACTIVE OXYGEN SPECIES ON MATURATION OF PORCINE OOCYTES IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab172 ◽

2017 ◽

Vol 29 (1) ◽

pp. 194

Author(s):

S.-Y. Park ◽

H.-J. Park ◽

J.-W. Kim ◽

J.-Y. Park ◽

S.-G. Yang ◽

...

Keyword(s):

Oocyte Maturation ◽

Critical Role ◽

Cumulus Cell ◽

Cumulus Cells ◽

Meiotic Maturation ◽

Control Group ◽

Ros Production ◽

Maturation In Vitro ◽

Porcine Oocyte

Bisphenol A (BPA) is well known as oestrogen-like chemical and it is widely used in plastic products. Many studies have reported that BPA exposure has a well-known toxicity effect on reproduction function, such as reducing the number of ovulated oocytes, oocyte quality, and maturation rate. Recently, BPA induced mitochondrial-derived reactive oxygen species (mito-ROS) and disrupted mitochondrial homeostasis by increasing of superoxide anions production. In this study, we investigated how the regulation of mito-ROS production may play a critical role in meiotic maturation and expansion of cumulus cells during the in vitro maturation progression of porcine oocytes. Furthermore, we investigated the toxicity effect of BPA exposure on mitochondrial functions and mito-ROS production during porcine oocyte maturation in vitro. All results were analysed using a 1-way ANOVA followed by Bonferroni’s and Tukey’s Multiple Comparison Test and t-tests. First, porcine oocytes were matured in NCSU-23 medium supplemented with BPA (50, 75, and 100 µM) for 44 h. Our results indicated that the rates of matured oocytes were significantly decreased by BPA exposure in a dose-dependent manner (69.4 ± 5.1, 50.9 ± 6.3, and 29.9 ± 5.8% for BPA treatments of 50, 75, and 100 μM) compared with control group (70.2 ± 7.8%; P < 0.05). Next, we confirmed the secretion functions of oocyte and cumulus cell of cumulus-oocyte complex (COC) and ROS production. Cumulus cell secretion factors (has2, tnfaip6, and cx37) mRNA expression in COC were decreased in the BPA-treated (75 µM) group. In addition, mRNA expressions of mitochondrial-specific antioxidant enzymes (sod2, P < 0.001; prdx3, P < 0.01; prdx5, P < 0.001) and mitochondrial apoptosis genes (bax and caspase-3, P < 0.01) were significantly increased in COC of the BPA-treated (75 µM) group. We measured mitochondrial membrane potential and mito-ROS production using JC-1 analysis and Mito-SOX staining, respectively. The BPA treatment caused a rapid decrease of mitochondrial membrane potential maintenance and increase of mito-ROS production in porcine COC. Moreover, mitochondrial-specific ROS scavenger, Mito-Tempo (0.1 µM) treatment was significantly increased the meiotic maturation of porcine oocytes compared with control group (78.5 ± 3.5 v. 65.8 ± 5.0%; P < 0.05). Based on these results, we first confirmed that BPA exposure reduces the meiotic maturation and cumulus cells expansion of COC by increasing mito-ROS production during porcine oocyte maturation in vitro. Therefore, controlling of mito-ROS for mitochondrial function maintenance and apoptosis plays a critical role in improving porcine oocyte maturation in vitro. This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ01117604) and the Bio-industry Technology Development Program (316037–04–1-HD020) through the Rural Development Administration, the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.

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Cumulus cell-derived and maternal SIRT6 differentially regulates porcine oocyte meiotic maturation

Theriogenology ◽

10.1016/j.theriogenology.2019.09.048 ◽

2020 ◽

Vol 142 ◽

pp. 158-168

Author(s):

Zubing Cao ◽

Dandan Zhang ◽

Xu Tong ◽

Yiqing Wang ◽

Xin Qi ◽

...

Keyword(s):

Cumulus Cell ◽

Meiotic Maturation ◽

Porcine Oocyte ◽

Oocyte Meiotic Maturation

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Effects of nitric oxide synthase inhibitors on porcine oocyte meiotic maturation

Zygote ◽

10.1017/s0967199404002953 ◽

2005 ◽

Vol 13 (1) ◽

pp. 1-9 ◽

Cited By ~ 28

Author(s):

Yong Tao ◽

Huirong Xie ◽

Haiyan Hong ◽

Xiufen Chen ◽

Jie Jang ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Dna Fragmentation ◽

Cumulus Cell ◽

Cumulus Cells ◽

Meiotic Maturation ◽

Cumulus Expansion ◽

Wide Range ◽

Oxide Synthase ◽

Oocyte Meiotic Maturation

As an important biological messenger, nitric oxide (NO) exhibits a wide range of effects during physiological and pathophysiological processes, including mammalian oocyte meiotic maturation. The present study investigated whether NO derived from two nitric oxide synthase (NOS) isoforms, inducible NOS (iNOS) or endothelial NOS (eNOS), is involved in the meiotic maturation of porcine oocytes. Meanwhile, the cumulus cells' function in meiotic maturation and their interaction with oocyte development and degeneration were also investigated using cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs). Different inhibitors for NOS were supplemented to the medium. Cumulus expansion, cumulus cell DNA fragmentation and oocyte meiotic resumption were evaluated 48 h after incubation. Aminoguanidine (AG), a selective inhibitor for iNOS, suppressed cumulus expansion and inhibited CEOs to resume meiosis (p<0.05), but did not inhibit cumulus cell DNA fragmentation. Both Nω-nitro-L-arginine (L-NNA) and Nω-nitro-L-arginine methyl ester (L-NAME), inhibitors for both iNOS and eNOS, delayed cumulus expansion, inhibited cumulus cell DNA fragmentation and inhibited CEOs to resume meiosis. Such effects were not seen in DOs. These results indicate that iNOS-derived NO is necessary for cumulus expansion and meiotic maturation by mediating the function of the surrounding cumulus cells, and eNOS-derived NO is also involved in porcine meiotic maturation.

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228 EFFECT OF INSULIN ON BOVINE OOCYTE MATURATION, CUMULUS CELL APOPTOSIS, AND EARLY EMBRYO DEVELOPMENT IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab228 ◽

2015 ◽

Vol 27 (1) ◽

pp. 203

Author(s):

I. Lindgren ◽

P. Humblot ◽

D. Laskowski ◽

Y. Sjunnesson

Keyword(s):

Embryo Development ◽

Oocyte Maturation ◽

In Vitro Maturation ◽

Insulin Treatment ◽

Cumulus Cells ◽

Early Embryo ◽

Blastocyst Formation ◽

Early Embryo Development ◽

Maturation In Vitro

Dairy cow fertility has decreased during the last decades, and much evidence indicates that metabolic disorders are an important part of this decline. Insulin is a key factor in the metabolic challenge during the transition period that coincides with the oocyte maturation and may therefore have an impact on the early embryo development. The aim of this study was to test the effect of insulin during oocyte maturation on early embryo development by adding insulin during the oocyte maturation in vitro. In this study, abattoir-derived bovine ovaries were used and cumulus-oocyte complexes (n = 991) were in vitro matured for 22 h according to standard protocols. Insulin was added during maturation in vitro as follows: H (10 µg mL–1 of insulin), L (0.1 µg mL–1 of insulin), or Z (0 µg mL–1 of insulin). After maturation, oocytes were removed and fixed in paraformaldehyde before staining. Click-it TUNEL assay (Invitrogen, Stockholm, Sweden) was used for apoptotic staining and DRAQ5 (BioNordika, Stockholm, Sweden) for nuclear staining (n = 132). Cumulus-oocyte complexes were evaluated using laser scanning confocal microscope (Zeiss LSM 510, Zeiss, Oberkochen, Germany). Five levels of scans were used to assess oocyte maturation (MII stage) and apoptosis. Because of incomplete penetration of the TUNEL stain (3–5 layers of cumulus cells), only the outer 2 layers of the cumulus complex were investigated regarding apoptosis. Apoptotic index was calculated as apoptotic cells/total cells visualised. Remaining oocytes were fertilized and cultured in vitro until Day 8. Day 7 and Day 8 blastocyst formation was assessed as well as blastocyst stage and grade. Effect of insulin treatment on variables was analysed by ANOVA following arc sin √p transformation. Post-ANOVA comparisons between H+L group v. Z were performed by using the contrast option under GLM (Scheffé test). Results are presented as least squares means ± s.e. P-values ≤ 0.05 were considered as statistically significant. Insulin treatment during oocyte maturation in vitro had no significant effect on oocyte nuclear maturation or apoptotic index of the cumulus cells (Z: 0.052 ± 0.025, L: 0.039 ± 0.016, H: 0.077 ± 0.044, P > 0.05). No effect was seen on cleavage rates (Z: 0.85 ± 0.02, L: 0.85 ± 0.02, H: 0.89 ± 0.03, P > 0.05), but insulin treatment significantly decreased Day 7 rates from fertilized oocytes (Z: 0.19 ± 0.02, L: 0.14 ± 0.02, H: 0.12 ± 0.02, P < 0.05). This study also showed a significantly retarded developmental stage and decreased grade of blastocysts in insulin-treated groups taken together when compared with the control group (P < 0.05). In this study, no effect of insulin supplementation during in vitro maturation was seen on bovine oocyte maturation and apoptosis of cumulus cells, but blastocyst formation and development were negatively affected. Further studies are needed for understanding the relationship between the addition of insulin during maturation in vitro and impaired blastocyst formation. Insulin is a common supplement in the first phase of the first in vitro maturation medium for pig oocytes and is believed to have a beneficial effect on this species.Funding was received from Stiftelsen Nils Lagerlöfs Fond H12–0051-NLA.

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Small GTPase RhoA regulates cytoskeleton dynamics during porcine oocyte maturation and early embryo development

Cell Cycle ◽

10.4161/15384101.2014.952967 ◽

2014 ◽

Vol 13 (21) ◽

pp. 3390-3403 ◽

Cited By ~ 37

Author(s):

Yu Zhang ◽

Xing Duan ◽

Rui Cao ◽

Hong-Lin Liu ◽

Xiang-Shun Cui ◽

...

Keyword(s):

Embryo Development ◽

Oocyte Maturation ◽

Early Embryo ◽

Small Gtpase ◽

Early Embryo Development ◽

Porcine Oocyte ◽

Cytoskeleton Dynamics

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Inhibition of Rac1 GTPase activity affects porcine oocyte maturation and early embryo development

Scientific Reports ◽

10.1038/srep34415 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 4

Author(s):

Si-Jing Song ◽

Qiao-Chu Wang ◽

Ru-Xia Jia ◽

Xiang-Shun Cui ◽

Nam-Hyung Kim ◽

...

Keyword(s):

Embryo Development ◽

Oocyte Maturation ◽

Early Embryo ◽

Gtpase Activity ◽

Early Embryo Development ◽

Porcine Oocyte ◽

Rac1 Gtpase

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Sonic hedgehog promotes porcine oocyte maturation and early embryo development

Reproduction Fertility and Development ◽

10.1071/rd08277 ◽

2009 ◽

Vol 21 (6) ◽

pp. 805 ◽

Cited By ~ 27

Author(s):

Ngoc Tan Nguyen ◽

David Pei-Cheng Lin ◽

Shih-Ying Yen ◽

Jung-Kai Tseng ◽

Jui-Fen Chuang ◽

...

Keyword(s):

Embryo Development ◽

Sonic Hedgehog ◽

Oocyte Maturation ◽

Calcium Release ◽

Cyclin B1 ◽

Early Embryo ◽

Control Group ◽

Early Embryo Development ◽

Porcine Oocyte

In the present study, we investigated the effects of the Sonic hedgehog (Shh) protein on porcine oocyte maturation and early embryo development. Immunohistochemistry showed activation of Shh signalling in cumulus–oocyte complexes (COCs), as reflected by Patched (Ptc), Smoothened (Smo) and Gli1 expression in oocytes, cumulus cells and granulosa cells, particularly those of small follicles (<2 mm in diameter). Western blot analysis showed Smo expression in COCs and in denuded oocytes derived from small and medium (3–7 mm)-sized follicles. Small follicles contained the highest concentration of Shh in follicular fluid compared with medium-sized and large (>7 mm in diameter) follicles. Supplementation with Shh (0.5 or 1 μg mL–1) enhanced oocyte maturation compared with the control group (92.4% and 90.4% v. 81.9%, respectively; P < 0.05). This effect was reversed by the simultaneous addition of cyclopamine (1–2 μm), an Shh inhibitor. Similar to intact COCs, denuded COCs showed enhanced maturation following Shh supplementation. Furthermore, cyclin B1 content, extracellular signal-regulated kinase 1/2 phosphorylation, intracellular calcium release, blastocyst rate and total cell numbers were greater (P < 0.05) in oocytes matured in the presence of 0.5 and 1 μg mL–1 Shh compared with control oocytes. The findings of the present study provide the first evidence that the Shh signalling pathway is active, or at least partially activated, in the porcine ovary and is likely to promote oocyte cytoplasmic and nuclear maturation, as well as subsequent in vitro development, although the underlying mechanisms remain to be elucidated.

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HASPIN kinase mediates histone deacetylation to regulate oocyte meiotic maturation in pigs

Reproduction ◽

10.1530/rep-18-0447 ◽

2019 ◽

Vol 157 (6) ◽

pp. 501-510 ◽

Cited By ~ 2

Author(s):

Zubing Cao ◽

Tengteng Xu ◽

Xu Tong ◽

Dandan Zhang ◽

Chengxue Liu ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Histone H3 ◽

Germinal Vesicle ◽

Early Embryo ◽

Histone Deacetylation ◽

Meiotic Maturation ◽

Metaphase Chromosomes ◽

Germinal Vesicle Breakdown ◽

Porcine Oocyte ◽

Oocyte Meiotic Maturation

HASPIN kinase-catalyzed phosphorylation of histone H3 on threonine 3 (H3T3p) directs the activity and localization of chromosomal passenger complex (CPC) and spindle assembly checkpoint (SAC) to regulate chromosome condensation and segregation in both mitosis and meiosis. However, the function of HASPIN kinase in the meiotic maturation of porcine oocytes is not yet known. Here, we found that HASPIN mRNA is constantly expressed in porcine oocyte maturation and subsequent early embryo development. H3T3p is highly enriched on chromosomes at germinal vesicle breakdown (GVBD) stage and thereafter maintains a low level in progression through metaphase I (MI) to metaphase II (MII). Correspondingly, H3T3p was completely abolished in oocytes treated with an inhibitor of HASPIN kinase. Functionally, inhibition of HASPIN activity led to a significant reduction in the rate of oocyte meiotic maturation and the limited cumulus expansion. Additionally, HASPIN inhibition caused both spindle disorganization and chromosome misalignment in oocytes at MI and MII stage. Importantly, HASPIN inhibition severely prevented deacetylation of several highly conserved lysine (K) residues of histone H3 and H4 including H3K9, H3K14, H4K5, H4K8, H4K12 and H4K16 on the metaphase chromosomes during oocyte meiotic maturation. Taken together, these results demonstrate that HASPIN kinase regulates porcine oocyte meiotic maturation via modulating histone deacetylation.

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265 HYALURONAN SYNTHESIS ABILITY OF PORCINE CUMULUS - OOCYTE COMPLEXES DERIVED FROM SMALL FOLLICLES

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab265 ◽

2013 ◽

Vol 25 (1) ◽

pp. 280

Author(s):

M. Nakakoji ◽

H. Funahashi

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Cumulus Cell ◽

Cumulus Cells ◽

Cumulus Expansion ◽

Elisa Kit ◽

Porcine Oocyte ◽

Post Hoc ◽

Metaphase Ii Oocytes

The degree of cumulus expansion, an important step in oocyte maturation, of porcine cumulus–oocyte complexes (COC) derived from small follicles (SF: 1 to 2 mm in diameter) is known to be lower than those derived from middle follicles (MF: 3 to 6 mm in diameter). The objective of this study was to compare the abilities of hyaluronan (HA) synthesis of COC from SF and MF. Furthermore, the effect of oestradiol during pre-incubation of COC on proliferation of the cumulus cells was examined. Cumulus–oocyte complexes from SF and MF of porcine ovaries were cultured for in vitro maturation [IVM, in modified porcine oocyte medium (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213) supplemented with 50 µM β-mercaptoethanol, 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, and 1 mM dbcAMP for 20 h and then in the fresh medium without those supplements for another 24 h]. Hyaluronan production was quantified at 20 h after the start of IVM with a commercial HA-ELISA kit (20 COC/tube × 4 times). The number of cumulus cells was assessed 0 and 20 h after the start of IVM (50 COC × 4 times). Furthermore, proliferation of cumulus cells was examined after pre-culture of COC (n = 40 COC × 5 times) in modified porcine oocyte medium with various concentrations of oestradiol (0, 0.1, 1, and 10 ng mL–1) for 6 h. Statistical analyses of results from 4 to 5 replicated trials were performed by ANOVA with a Bonferroni-Dunn post-hoc test (significance, P < 0.05). The degree of cumulus expansion of COC from MF (n = 152) was higher than that of COC from SF (n = 156). The incidence of metaphase-II oocytes was significantly lower in COC from SF (n = 133; 48.9%) than in COC from MF (n = 148; 74.7%). The HA content of COC was higher in those from MF (20.8 µg/COC) than in those from SF (10.8 µg/COC), whereas the content per cumulus cell was not different because the numbers of cumulus cells at 0 and 20 h were also higher in COC (n = 200 in each group) from MF (3.0 × 103 and 3.3 × 103 cells, respectively) than from SF (2.0 × 103 and 2.5 × 103 cells, respectively). Cumulus cells proliferated significantly in the presence of oestradiol, regardless of the concentration, during pre-incubation for 6 h (2.5 to 2.8 × 103 cells), as compared with the oestradiol-free controls (2.2 × 103 cells). These results demonstrate that the different abilities of cumulus expansion between COC (n = 200 in each group) from SF and MF may be due to the number of cumulus cells per COC. Pre-incubation in the presence of oestradiol stimulates the proliferation of cumulus cells and may improve the oocyte maturation of COC derived from SF.

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