scholarly journals Transient meiotic arrest maintained by DON (6-diazo-5-oxo-l-norleucine) enhances nuclear/cytoplasmic maturation of porcine oocytes

Reproduction ◽  
2019 ◽  
Vol 158 (6) ◽  
pp. 543-554
Author(s):  
Hae-Jun Yang ◽  
Sanghoon Lee ◽  
Bo-Woong Sim ◽  
Pil-Soo Jeong ◽  
Seon-A Choi ◽  
...  
Keyword(s):  
Formation Rate ◽  
Developmental Competence ◽  
Cortical Granules ◽  
Meiotic Arrest ◽  
Cellular Survival ◽  
Cytoplasmic Maturation ◽  
First Time ◽  
A Current

The developmental competence of in vitro-matured oocytes is still lower than that of the in vivo-matured oocytes due to precocious meiotic resumption and inappropriate cytoplasmic maturation. Although numerous efforts have been attempted to accomplish better in vitro maturation (IVM) condition, only limited progress has been achieved. Thus, a current study was conducted to examine the effects of 6-diazo-5-oxo-l-norleucine (DON, an inhibitor of hyaluronan synthesis) during the first half period of IVM on nuclear/cytoplasmic maturation of porcine oocytes and subsequent embryonic development. Based on the observation of the nucleus pattern, metaphase II (MII) oocyte production rate in 1 µM DON group was significantly higher than other groups at 44 h of IVM. The 1 µM of DON was suggested to be optimal for porcine IVM and was therefore used for further investigation. Meiotic arrest effect of DON was maximal at 6 h of IVM, which was supported by the maintenance of significantly higher intra-oocyte cAMP level. In addition, increased pERK1/2 levels and clear rearrangement of cortical granules in membrane of MII oocytes matured with DON provided the evidence for balanced meiosis progression between nuclear and cytoplasmic maturation. Subsequently, DON significantly improved blastocyst formation rate, total cell numbers, and cellular survival in blastocysts after parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Altogether, our results showed for the first time that 1 µM DON can be used to increase the yield of developmentally competent MII oocytes by synchronizing nuclear/cytoplasmic maturation, and it subsequently improves embryo developmental competence.

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

scholarly journals Antioxidant Nobiletin Enhances Oocyte Maturation and Subsequent Embryo Development and Quality

2020 ◽  
Vol 21 (15) ◽  
pp. 5340
Author(s):  
Yulia N. Cajas ◽  
Karina Cañón-Beltrán ◽  
Magdalena Ladrón de Guevara ◽  
María G. Millán de la Blanca ◽  
Priscila Ramos-Ibeas ◽  
...  
Keyword(s):  
Embryo Development ◽  
Biological Effects ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Cortical Granules ◽  
Cleavage Rate ◽  
Cytoplasmic Maturation

Nobiletin is a polymethoxylated flavonoid isolated from citrus fruits with wide biological effects, including inhibition of reactive oxygen species (ROS) production and cell cycle regulation, important factors for oocyte in vitro maturation (IVM). Therefore, the objective of the present study was to evaluate the antioxidant activity of nobiletin during IVM on matured bovine oocyte quality (nuclear and cytoplasmic maturation; oocyte mitochondrial activity; intracellular ROS and glutathione (GSH) levels) and their developmental competence, steroidogenesis of granulosa cells after maturation, as well as quantitative changes of gene expression in matured oocytes, their cumulus cells, and resulting blastocysts. Bovine cumulus-oocyte complexes were in vitro matured in TCM-199 +10% fetal calf serum (FCS) and 10 ng/mL epidermal growth factor (EGF) (Control) supplemented with 10, 25, 50, or 100 μM of nobiletin (Nob10, Nob25, Nob50, and Nob100, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for nobiletin dilution). A significantly higher percentage of matured oocytes in metaphase II was observed in Nob25 and Nob50 compared to other groups. Similarly, cleavage rate and cumulative blastocyst yield on Days 7 and 8 were significantly higher for Nob25 and Nob50 groups. Oocytes matured with 25 and 50 μM nobiletin showed a higher rate of migration of cortical granules and mitochondrial activity and a reduction in the ROS and GSH content in comparison with all other groups. This was linked to a modulation in the expression of genes related to metabolism (CYP51A1), communication (GJA1), apoptosis (BCL2), maturation (BMP15 and MAPK1), and oxidative stress (SOD2 and CLIC1). In conclusion, nobiletin offers a novel alternative for counteracting the effects of the increase in the production of ROS during IVM, improves oocyte nuclear and cytoplasmic maturation, and subsequent embryo development and quality in cattle.

168 Melatonin slightly alleviates the effect of heat shock on nuclear maturation of the bovine oocyte

2019 ◽  
Vol 31 (1) ◽  
pp. 209 ◽  
Author(s):  
C. Shimoni ◽  
D. Kalo ◽  
P. J. Hansen ◽  
Z. Roth
Keyword(s):  
Heat Shock ◽  
Distribution Pattern ◽  
Fluorescein Isothiocyanate ◽  
Fluorescence Microscope ◽  
Developmental Competence ◽  
Cortical Granules ◽  
Nuclear Maturation ◽  
Chi Squared ◽  
Cytoplasmic Maturation

Exposing bovine cumulus-oocyte complexes (COC) to heat shock during in vitro maturation (IVM) reduces oocyte developmental competence. Melatonin has been reported to improve cleavage of heat-shocked oocytes after fertilization. The current study examined whether protective activity of melatonin is associated with nuclear or cytoplasmic maturation events, or both. The COC were aspirated from Holstein ovaries collected at an abattoir and randomly divided into 3 experimental groups. The control COC were matured (22h) in standard oocyte maturation medium (OMM) at 38.5°C and 5% CO2; the treatment COC were matured (22h) in OMM at 41.5°C and 6% CO2 (HS) without or with 10−7 M melatonin (HS+M). Data are presented as least squares means±s.e.m. To examine nuclear maturation, COC were denuded of cumulus cells at the end of maturation, fixed in 2% (vol/vol) paraformaldehyde, and labelled with 10μg mL−1 4′,6-diamidino-2-phenylindole (DAPI) (200 COC, 3 replicates). The meiotic status of denuded oocytes was evaluated under inverted fluorescence microscope using Nis Elements software (Nikon, Tokyo, Japan). The proportion of oocytes that resumed meiosis and reached the metaphase II (MII) stage was lower in the HS group than in the control (13.7v. 55%, respectively; chi-squared, P<0.05); melatonin slightly alleviated this effect, as indicated by a higher proportion of MII-stage oocytes in HS+M than in the HS group (32v. 13.7%, respectively; chi-squared, P<0.05). Cytoplasmic maturation was evaluated according to the distribution pattern of cortical granules (CG). Matured COC were denuded and the oocytes were stained with 100μg mL−1 fluorescein isothiocyanate-conjugated Arachis hypogaea agglutinin (FITC-PNA) and counterstained with 10μg mL−1 DAPI. Oocytes were examined under an inverted fluorescence microscope and classified as 1 of 3 types according to their cortical distribution pattern (3 replicates; 200 COC). The proportion of oocytes classified as type 3, which is associated with cytoplasmic maturation, did not differ between the experimental groups, either when analysed for total oocytes or for nuclear matured oocytes (i.e. MII stage). To evaluate the level of reactive oxygen species (ROS), matured-denuded oocytes were labelled using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and the intracellular fluorescence of DCF was immediately measured by fluorescence microscopy (3 replicates, 425 COC). Heat shock increased ROS level relative to the control (68.8±5.32v. 33.9±4.08 pixels intensity, respectively; P<0.05) and melatonin tended to alleviate the effect of heat stress to some extent (56.0±4.78; P<0.06). In summary, heat shock impairs the process of nuclear maturation, expressed by reduced proportion of MII-stage oocytes at the end of maturation. Melatonin partly overcomes this effect, perhaps through its antioxidant activity.

Effect of C-type natriuretic peptide on maturation and developmental competence of immature mouse oocytes in vitro

2017 ◽  
Vol 29 (2) ◽  
pp. 319 ◽  
Author(s):  
Qiang Wei ◽  
Chuan Zhou ◽  
Min Yuan ◽  
Yuyang Miao ◽  
Xiaoe Zhao ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Oocyte Maturation ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Immature Oocytes ◽  
Mouse Oocytes ◽  
Immature Mouse ◽  
Cytoplasmic Maturation

In vitro maturation (IVM) of oocytes is an important assisted reproductive technology for infertility treatment and livestock breeding programs. Because of asynchronous nuclear and cytoplasmic maturation, the developmental competence of oocytes matured in vitro is compromised. C-Type natriuretic peptide (CNP), which has been proved to be an inhibitor of oocyte maturation, provides a new alternative to optimise synchronisation of nuclear and cytoplasmic maturation and improve developmental capacity of immature oocytes in vitro. To investigate the effect of temporary meiotic arrest mediated by CNP on maturation and subsequent development of immature oocytes, immature mouse oocytes from small antral follicles were temporarily arrested in meiosis by CNP (0, 5, 10 and 50 nM) for 24 h and then matured for 16 h. CNP treatment significantly increased the oocyte maturation rate from less than half to above 80%. After IVF, temporary meiotic arrest mediated by 10 and 50 nM CNP significantly improved fertilisation and blastocyst rate of oocytes matured in vitro up to approximately 55% and 30% respectively. Moreover, this positive effect of CNP was attributed, in part, to an increase in the number of mature oocytes with aligned chromosomes and a normal spindle. The present findings indicate the potential to use CNP to improve the efficiency of oocyte IVM.

scholarly journals Birth of pups after transfer of mouse embryos derived from vitrified preantral follicles

Reproduction ◽  
2002 ◽  
pp. 593-600 ◽  
Author(s):  
EC dela Pena ◽  
Y Takahashi ◽  
S Katagiri ◽  
EC Atabay ◽  
M Nagano
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Preantral Follicles ◽  
Cell Complexes ◽  
Vitrification Solution ◽  
First Time

Preantral follicles mechanically isolated from the ovaries of 12-day-old mice were exposed to 2 mol ethylene glycol l(-1) for 2 or 5 min and then to a vitrification solution containing 6 mol ethylene glycol l(-1) and 0.3 mol raffinose l(-1) for 0.5, 1.0 or 2.0 min before vitrification. The vitrified and fresh preantral follicles were treated with collagenase, and the oocyte-granulosa cell complexes (OGCs) obtained were cultured in vitro for 10 days in membrane inserts. Preantral follicles exposed to 2 mol ethylene glycol l(-1) for 5 min and then to the vitrification solution for 0.5 or 1.0 min showed the highest survival rates after warming. The follicular loss after warming was approximately 20%. After in vitro culture, the proportion of viable OGCs from the vitrified follicles was 10% lower than that of the fresh preantral follicles. There were no differences in the rates of maturation, fertilization and subsequent development to blastocysts between the oocytes derived from vitrified follicles and those derived from fresh preantral follicles; however, the developmental competence of the oocytes derived from both vitrified and fresh preantral follicles grown in vitro was lower than that of oocytes grown in vivo. One of the five recipient mice that received 20 blastocysts derived from vitrified preantral follicles gave birth to six live pups. The results of the present study demonstrate for the first time that mouse preantral follicles can be vitrified and that some of the embryos derived from vitrified preantral follicles can develop to live pups.

scholarly journals Hexapod Assassins’ Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 819
Author(s):  
Nicolai Rügen ◽  
Timothy P. Jenkins ◽  
Natalie Wielsch ◽  
Heiko Vogel ◽  
Benjamin-Florian Hempel ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Galleria Mellonella ◽  
Systemic Reactions ◽  
Venom Composition ◽  
Assassin Bug ◽  
Molecular Components ◽  
First Time ◽  
Tissue Digestion

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

2021 ◽  
Author(s):  
Lijuan Liu ◽  
Shengting Zhang ◽  
Xiaodan Zheng ◽  
Hongmei Li ◽  
Qi Chen ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Fusobacterium Nucleatum ◽  
Living Cells ◽  
First Time ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

scholarly journals Fe3O4@Pt nanoparticles to enable combinational electrodynamic/chemodynamic therapy

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Tong Chen ◽  
Qiang Chu ◽  
Mengyang Li ◽  
Gaorong Han ◽  
Xiang Li
Keyword(s):  
Fenton Reaction ◽  
Pt Nanoparticles ◽  
Therapeutic Modality ◽  
Ros Generation ◽  
Tumor Treatment ◽  
Superior Efficacy ◽  
Platinum Nanocrystals ◽  
First Time

AbstractElectrodynamic therapy (EDT) has recently emerged as a potential external field responsive approach for tumor treatment. While it presents a number of clear superiorities, EDT inherits the intrinsic challenges of current reactive oxygen species (ROS) based therapeutic treatments owing to the complex tumor microenvironment, including glutathione (GSH) overexpression, acidity and others. Herein for the first time, iron oxide nanoparticles are decorated using platinum nanocrystals (Fe3O4@Pt NPs) to integrate the current EDT with chemodynamic phenomenon and GSH depletion. Fe3O4@Pt NPs can effectively induce ROS generation based on the catalytic reaction on the surface of Pt nanoparticles triggered by electric field (E), and meanwhile it may catalyze intracellular H2O2 into ROS via Fenton reaction. In addition, Fe3+ ions released from Fe3O4@Pt NPs under the acidic condition in tumor cells consume GSH in a rapid fashion, inhibiting ROS clearance to enhance its antitumor efficacy. As a result, considerable in vitro and in vivo tumor inhibition phenomena are observed. This study has demonstrated an alternative concept of combinational therapeutic modality with superior efficacy.

scholarly journals Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Miao-Miao Zhao ◽  
Wei-Li Yang ◽  
Fang-Yuan Yang ◽  
Li Zhang ◽  
Wei-Jin Huang ◽  
...  
Keyword(s):  
Drug Development ◽  
Cathepsin L ◽  
Human Cells ◽  
New Drugs ◽  
Disease Course ◽  
Promising Target ◽  
First Time

AbstractTo discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

scholarly journals Schistosoma mansoni eggs induce Wnt/β-catenin signaling and activate the protooncogene c-Jun in human and hamster colon

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jakob Weglage ◽  
Friederike Wolters ◽  
Laura Hehr ◽  
Jakob Lichtenberger ◽  
Celina Wulz ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Colorectal Carcinogenesis ◽  
Sub Saharan Africa ◽  
Interleukin 4 ◽  
Health Burden ◽  
Sub Saharan ◽  
Considerable Morbidity ◽  
First Time

AbstractSchistosomiasis (bilharzia) is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma, with considerable morbidity in parts of the Middle East, South America, Southeast Asia, in sub-Saharan Africa, and particularly also in Europe. The WHO describes an increasing global health burden with more than 290 million people threatened by the disease and a potential to spread into regions with temperate climates like Corsica, France. The aim of our study was to investigate the influence of S. mansoni infection on colorectal carcinogenic signaling pathways in vivo and in vitro. S. mansoni infection, soluble egg antigens (SEA) and the Interleukin-4-inducing principle from S. mansoni eggs induce Wnt/β-catenin signaling and the protooncogene c-Jun as well as downstream factor Cyclin D1 and markers for DNA-damage, such as Parp1 and γH2a.x in enterocytes. The presence of these characteristic hallmarks of colorectal carcinogenesis was confirmed in colon biopsies from S. mansoni-infected patients demonstrating the clinical relevance of our findings. For the first time it was shown that S. mansoni SEA may be involved in the induction of colorectal carcinoma-associated signaling pathways.

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