scholarly journals N-acetyl-L-cysteine Improves the Developmental Competence of Bovine Oocytes and Embryos Cultured In Vitro by Attenuating Oxidative Damage and Apoptosis

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 860
Author(s):  
Wu-Sheng Sun ◽  
Hoon Jang ◽  
Mi-Ryung Park ◽  
Keon Bong Oh ◽  
Haesun Lee ◽  
...  

Oxidative stress has been suggested to negatively affect oocyte and embryo quality and developmental competence, resulting in failure to reach full term. In this study, we investigated the effect of N-acetyl-L-cysteine (NAC), a cell-permeating antioxidant, on developmental competence and the quality of oocytes and embryos upon supplementation (0.1–10 mM) in maturation and culture medium in vitro using slaughterhouse-derived oocytes and embryos. The results show that treating oocytes with 1.0 mM NAC for 8 h during in vitro maturation attenuated the intracellular reactive oxygen species (ROS) (p < 0.05) and upregulated intracellular glutathione levels (p < 0.01) in oocytes. Interestingly, we found that NAC affects early embryonic development, not only in a dose-dependent, but also in a stage-specific, manner. Significantly (p < 0.05) decreased cleavage rates (90.25% vs. 81.46%) were observed during the early stage (days 0–2), while significantly (p < 0.05) increased developmental rates (38.20% vs. 44.46%) were observed during the later stage (from day 3) of embryonic development. In particular, NAC supplementation decreased the proportion of apoptotic blastomeres significantly (p < 0.05), resulting in enhanced hatching capability and developmental rates during the in vitro culture of embryos. Taken together, our results suggest that NAC supplementation has beneficial effects on bovine oocytes and embryos through the prevention of apoptosis and the elimination of oxygen free radicals during maturation and culture in vitro.

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Jolanta Opiela ◽  
Joanna Romanek ◽  
Daniel Lipiński ◽  
Zdzisław Smorąg

The objective of the present study was to evaluate the effect of hyaluronan (HA) during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC), and obtained blastocysts. COCs were maturedin vitroin control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P<0.001) was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P<0.01). Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higherBaxmRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.


2004 ◽  
Vol 16 (2) ◽  
pp. 282
Author(s):  
L.J. Royo ◽  
A. Rodriguez ◽  
A. Gutierrez-Adan ◽  
C. Diez ◽  
E. Moran ◽  
...  

Retinoic acid (RA) can induce cell differentiation and plays a role in controlling events within the cell cycle, but little is known of RA post-transcriptional modifications in the oocyte. Bovine oocyte and cumulus cells express most of RA receptors, and the presence of 9-cis-RA during in vitro prematuration and maturation (IVM) improves oocyte developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). This work analyzes the mRNA stability in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were cultured in defined medium with polyvinyl alcohol (DM). Those COCs undergoing prematuration were cultured for 24h in DM with 25μM roscovitine. For IVM, COCs were cultured in DM containing pFSH, LH and E2 for 24h, and some prematured COCs were then allowed to mature. Incubations were made at 39°C in 5% CO2 in air and high humidity. Within experiments, COCs were cultured with 5nM 9-cis-RA, in 1% ethanol (both as a vehicle and as an inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Groups of 10 COCs per treatment were cultured, and oocytes detached from cumulus cells were analyzed. Poly(A) mRNA quantification was based on the pyrophosphorylation property of the DNA polymerase (Klenow). ATP production was measured by luminometric assay as a function of numbers of poly(A) tails. Data (4 replicates) were analyzed by ANOVA and Duncan’s test (v,x,y,zP&lt;0.01; a,bP&lt;0.05), and poly(A) mRNA (pg oocyte−1) was expressed as LSM±SE. After prematuration, poly(A) mRNA contents differed between 9-cis-RA (125.7±4.8x) and untreated (95.5±4.8y) oocytes, as compared to 1% ethanol (72.2±4.8z) and immature (71.5±4.8z) oocytes. After IVM, untreated oocytes (23.0±2.2v) showed the lowest poly(A) mRNA amount, and poly(A) mRNA in 9-cis-RA (36.2±2.2y) basically equalled that in 1% ethanol (35.2±2.2y), while 3% (44.5±2.2yz) and 5% ethanol (52.0±2.2z) increased poly(A) mRNA levels. All groups of matured oocytes showed poly(A) mRNA contents lower than in immature (71.5±4.8x). After prematuration+maturation, poly(A) mRNA values were 34.2±2.2v (untreated+untreated), 36.5±2.2v (9-cis-RA+untreated), 49.5±2.2xa (untreated+9-cis-RA), 41.0±2.2vxb (9-cis-RA+9-cis-RA) and 59.0±2.2y (untreated+1% ethanol). Levels of poly(A) mRNA from prematured+matured oocytes were again lower than in immature (71.5±4.8x). Our study shows that beneficial effects of RA on the oocyte developmental competence can be represented in part as a gain in the quality of mRNAs stored. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).


2015 ◽  
Vol 27 (1) ◽  
pp. 204
Author(s):  
G. Singina ◽  
I. Lebedeva ◽  
T. Taradajnic ◽  
N. Zinovieva

The competence for embryonic development acquired during the oocyte maturation attenuates during the subsequent oocyte aging both in vivo and in vitro. Thus, the successful control of the female fertility requires information regarding factors responsible for the oocyte protection from early aging. The aim of the present research was to study the pattern and pathways of actions of two closely related pituitary hormones, prolactin (PRL), and growth hormone (GH), on the developmental potential of bovine oocytes during their aging in vitro. Therefore, we analysed (1) effects of PRL and GH during the prolonged culture of bovine oocytes on their subsequent development up to the blastocyst stage and (2) the role of cumulus cells (CC) and tyrosine kinases, the well-known mediators of PRL and GH signalling, in these effects. Bovine cumulus-enclosed oocytes (CEO) were cultured for 22 h in the following maturation medium: TCM 199 containing 10% fetal calf serum (FCS), 10 μg mL–1 of porcine FSH, and 10 μg mL–1 of ovine LH. After IVM, CEO or denuded oocytes (DO) were transferred to the aging medium consisting of TCM 199 supplemented with 10% FCS and cultured for 10 h in the absence (Control) or presence of 50 ng mL–1 bovine PRL or 10 ng mL–1 recombinant bovine GH and/or 10 μg mL–1 genistein (a non-selective inhibitor of tyrosine kinases). Genistein was not applied in the case of aging DO, since their developmental potential was not affected by both hormones. Following the prolonged culture, oocytes underwent IVF and IVC. Embryos were cultured in CR1aa medium until Day 5 post-insemination and then transferred to the same medium supplemented with 5% FCS and cultured up to Day 8. The embryo development was evaluated at Days 2 and 8 for cleavage and blastocyst formation. The data from 5 to 6 replicates using 135–184 oocytes per treatment were analysed by ANOVA. Aging of oocytes in the control medium had no effect on the cleavage rate, but caused the blastocyst yield to decline (P < 0.001) from 31.1 ± 2.3% (CEO fertilized immediately after maturation) to 10.5 ± 2.4% (aged CEO) and 7.9 ± 1.9% (aged DO). Cleavage rates of aging CEO and DO were unaffected by both PRL and GH. In the case of CEO, the addition of PRL (but not GH) to the aging medium raised the blastocyst yield from 8.2 ± 0.9% to 15.2 ± 2.1% (P < 0.05), whereas the removal of CC abolished this effect, reducing the yield up to 9.1 ± 2.7% (P < 0.05). At the same time, genistein did not influence the blastocyst yield in the PRL-treated group. The findings demonstrate that PRL can inhibit the attenuation of the developmental competence of bovine oocytes aging in vitro, with this effect being achieved via cumulus cells. Tyrosine kinases are unlikely to mediate the beneficial action of PRL on the CEO capacity for embryonic development. Meanwhile, closely related GH does not affect the developmental competence of aging bovine oocytes.This research was supported by RFBR (project No. 13-04-01888).


2009 ◽  
Vol 15 (1) ◽  
pp. 16-19
Author(s):  
Ioan GROZA ◽  
Simona CIUPE ◽  
Mihai CENARIU ◽  
Emoke PALL ◽  
Anamaria PETREAN

The objective of the present study was to asses the quality of various cultivation media used for the maturation of bovine oocytes that are prepared for IVF. Upon collection from slaughtered bovine ovaries and after morphological evaluation, a total number of 513 viable oocytes have been selected for cultivation, being divided into 3 batches, 171 oocytes / batch. The oocytes belonging to batch 1 were cultivated in TCM 199 NaHCO3 + 10% FCS + FSH 20 μl/ml. The oocytes belonging to batch 2 were cultivated in TCM 199 NaHCO3 + 10% FCS + HCG 2.3 x 103 UI/ml + FSH 8 μl/ml + pyruvate 0.25 mM + 17β estradiol 1 μl/ml. The oocytes belonging to batch 3 were cultivated in TCM 199 NaHCO3 + 10% FCS + 17β estradiol 1 μl/ml + FSH 20 μl/ml. The cultivation conditions, for all three batches, were: 24 hours at 39°C, 5% CO2. Spermatozoa have been prepared using the Percoll method and IVF of the matured oocytes has been performed. Embryonic development has been assessed 72 hours and then up to 10 days after IVF. The results showed the superior quality of the oocytes belonging to batch 2 and matured using TCM 199 NaHCO3 + 10% FCS + HCG 2.3x103 UI/ml + FSH 8 μl/ml + pyruvate 0.25 mM + 17β estradiol 1 μl/ml, as their use for IVF yielded the highest number of viable embryos.


2007 ◽  
Vol 59 (2) ◽  
pp. 280-287 ◽  
Author(s):  
F. Perecin ◽  
S.C. Méo ◽  
C.L.V. Leal ◽  
J.M. Garcia

The efficiency of bohemine and roscovitine in combination with ionomycin on parthenogenetic activation and initial embryonic development of bovine oocytes was studied. Two experiments were performed: in the first, different concentrations (0, 50, 75 or 100µM) and different exposure periods (2, 4 or 6 hours) to bohemine or roscovitine were tested for activation rates of in vitro matured (IVM) bovine oocytes, which were pre-exposed to ionomycin. The best treatments, 75µM bohemine and 50µM roscovitine, both for 6h, were used in the second experiment, in which IVM bovine oocytes were exposed to ionomycin, followed or not by bohemine or roscovitine treatment, and evaluated for nuclear status, activation rate and blastocyst development were assessed. The combined treatments (ionomycin + cyclin-dependent kinases inhibitors - CDKIs) showed better results for activation rates (77.3%) and initial embryonic development (35.2%) than the single ionomycin treatment (69.4% for activation and 21.9% for development); and also lead to a more uniform activation (nearly 90% single pronucleus development). The results showed that CDKIs improve the effects of ionomycin on parthenogenetic activation and blastocyst development in bovine oocytes and could help to achieve more efficient activation protocols, increasing the developmental competence of embryos obtained by reproductive biotechniques.


Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 2048
Author(s):  
Élisa Fonseca ◽  
Carla Cruz Marques ◽  
Jorge Pimenta ◽  
Joana Jorge ◽  
Maria Conceição Baptista ◽  
...  

Oxidative stress and mitochondrial dysfunction have been associated with the age-related decline of oocyte quality and strategies for their prevention are currently quested. Urolithin A (UA) is a natural metabolite with pro-apoptotic and antioxidant effects, capable of preventing the accumulation of dysfunctional mitochondria in different aged cells. UA has never been tested in bovine oocytes. Our aim was to study the effect of UA on the developmental potential of cumulus-oocyte-complexes (COCs) and granulosa cells’ (GCs) expression of important genes related to reproductive competence. Nuclear maturation progression, mitochondrial membrane potential (MMP) and developmental competence of physiologically mature (22 h) and in vitro aged oocytes (30 h of IVM) obtained from prepubertal and adult females, either supplemented with UA or not were assessed. Additionally, the amount of mRNA of several genes (NFE2L2, NQO1, and mt-DN5) and the number of mt-ND5 DNA copies were quantified in cultured GCs from prepubertal and adult females, either supplemented with UA or not. Our study confirmed the harmful effect of oocyte aging on the nuclear maturation progression, MMP, developmental competence and gene expression levels. UA treatment during in vitro maturation enhanced (p < 0.05) the maturation rate and subsequent developmental capacity of aged oocytes. A positive effect (p < 0.05) of UA on physiological maturation, MMP and embryonic development was also identified. UA also interfered on the expression profile of NFE2L2 and NQO1 genes in GCs cultures. Our findings demonstrate that UA supplementation is an effective way to prevent oocyte aging and improves the subsequent bovine embryonic development.


Zygote ◽  
2014 ◽  
Vol 23 (6) ◽  
pp. 933-942 ◽  
Author(s):  
Marwa S. Faheem ◽  
E. Baron ◽  
I. Carvalhais ◽  
A. Chaveiro ◽  
K. Pavani ◽  
...  

SummaryImmature bovine oocytes were vitrified using the cryotop method and their post-warming survivability and capability to undergo in vitro maturation, fertilization and subsequent embryonic development were evaluated. In addition throughout the embryonic 2-cell, 4-cell, morula and blastocyst stages, the expression of four developmentally important genes (Cx43, CDH1, DNMT1 and HSPA14) was analysed using the real-time polymerase chain reaction (PCR). Immature oocytes (n = 550) were randomly assigned to non-vitrified (fresh) or cryotop vitrification groups using ethylene glycol (EG) with 1,2 propanediol (PROH) or dimethylsulphoxide (DMSO). After warming, oocytes survivability, embryo cleavage and embryonic developmental rates were not statistically different between the two cryoprotectants groups. However, the DMSO group had a lower (P < 0.05) oocyte maturation rate compared with the fresh and PROH groups. For morula and blastocyst rates, the DMSO group achieved a lower (P < 0.05) morula rate compared with the fresh group, while at the blastocyst stage, there were no differences between fresh and both cryoprotectants groups. For molecular analysis, at the 4-cell stage, most studied genes showed an inconsistent pattern of expression either from the PROH or DMSO groups. Noteworthily, these differences were limited at the morula and blastocyst stages. In conclusion, the cryotop method is sufficient for vitrification of immature bovine oocytes, both for embryonic developmental competence and at the molecular level. Moreover, PROH showed some advantage over DMSO as a cryoprotectant.


2010 ◽  
Vol 22 (1) ◽  
pp. 159
Author(s):  
D. Paul ◽  
W. Sonnet ◽  
R. Rezsohazy ◽  
I. Donnay

HOX genes encode transcription factors known to play a major role in patterning the main body axis of vertebrate embryos from the gastrulation stage onward. A few studies have provided evidence that some HOX genes might be expressed before implantation in mammalian embryos. Translation of maternally inherited transcripts is regulated by modifications of the poly(A) tail length until embryonic genome activation (EGA), occurring during the 4th cell cycle in the bovine. The objective of this work was to establish the expression pattern of various HOX genes and to study the polyadenylation of their transcripts during oocyte maturation and early embryonic development. Pools of 20 bovine oocytes before and after in vitro maturation and 20 in vitro-produced embryos at different stages of development up to the blastocyst stage were collected. Three to 12 pools were used for each stage. RNA was extracted and reverse transcribed (RT) using random hexamers. Quantitative real-time PCR (qPCR) was performed to establish expression profiles of 4 HOX genes: HOXD1, HOXA3, HOXB9, and HOXC9. Two distinct patterns of expression were observed. First, relative amounts of HOXD1, HOXA3, and HOXC9 were lower in morulae and blastocysts than in oocytes. On the other hand, relative expression of HOXB9 increased between the 5 to 8 cell stage and the morula stage (Mann-Whitney, P < 0.05). Those expression patterns were not modified when embryos were cultured in presence of α-amanitin, a RNA polymerase II inhibitor, indicating the maternal origin of the transcripts until EGA. Total amount of mRNAs, estimated by RT-qPCR with random hexamers, was stable for all studied genes during oocyte maturation. The relative amount of polyadenylated GAPDH mRNAs, estimated by RT-qPCR with poly(dT), decreased greatly in mature oocytes compared with immature oocytes indicating massive deadenylation of those transcripts. The relative amount of polyadenylated HOXC9 transcripts decreased slightly but significantly during oocyte maturation (Mann-Whitney, P < 0.05).The relative amount of polyadenylatedm RNAs corresponding to HOXD1, HOXA3, and HOXB9 was stable during oocyte maturation. This indicates that those transcripts escape the default deadenylation pathways followed by housekeeping genes. This experiment has been repeated 3 to 4 times. In conclusion, we confirmed the presence of HOXD1, HOXA3, HOXB9, and HOXC9 transcripts in bovine oocytes and early-stage embryos. Their role during oocyte maturation and the first stages of embryonic development will be investigated through loss of function studies. This work is funded by the Fonds National de la Recherche Scientifique (Belgium).


2016 ◽  
Vol 28 (2) ◽  
pp. 213
Author(s):  
T. Suttirojpattana ◽  
T. Somfai ◽  
S. Matoba ◽  
T. Nagai ◽  
R. Parnpai ◽  
...  

The objective of this study was to clarify the effect of the temperature during liquid storage in in vitro matured (IVM) bovine oocytes. IVM bovine oocytes were stored in Eppendorf tube containing 1 mL HEPES TCM-199 supplemented with 10% (v/v) new born calf serum at different temperatures (4°C, 15°C, 25°C, and 38.5°C) for 20 h. The developmental rates of stored and not stored (control) oocytes to the blastocyst stage, cell numbers in resultant blastocysts, and fertilization normality were evaluated after in vitro fertilization and in vitro culture. The ATP content, reduced glutathione (GSH) content, and apoptosis rates in oocytes were also determined in stored and control groups. At least 3 replicates were conducted for each experiment. The data were analysed by 1-way ANOVA followed by post hoc Fisher’s protected least significantly difference test. Percentage data were transformed to arc-sine before analysis. All of the storage groups (4, 15, 25, and 38.5°C groups, respectively) showed significantly lower blastocyst developmental rates (8.5, 14.9, 19.3, and 24.5%, respectively) compared with the control group (39.8%; P < 0.05). Within the storage groups, the 25°C and the 38.5°C groups exhibited the greatest rate of blastocyst formation. In contrast, the total cell number of the 38.5°C group was significantly lower than that of control group (P < 0.05), whereas that of the 25°C group was similar with the control group. The frequency of normal emission of the second polar body (2PB) was significantly greater in the control group compared with the storage groups (P < 0.05). The 2PB emission rate was significantly lower in the 38.5°C group compared with the 4°C group (P < 0.05) but not different from those of the 15°C and 25°C storage groups. The percentage of male pronuclear formation in the control group was significantly higher than those in the stored groups (P < 0.05) except for the 25°C group. During storage at 4°C, the ATP content was significantly decreased compared with the control group (1.3 v. 1.7 pmol; P < 0.05); however, in the 25°C and 38.5°C groups, the ATP content (2.0 and 1.9 pmol, respectively) was significantly higher than that in the control group (1.7 pmol; P < 0.05); whereas the 15°C group showed the same ATP level compared with the control group. Storage of oocytes for 20 h reduced the GSH content compared with the control group without storage (P < 0.05); however, there were no significant differences among storage groups. Annexin-V staining revealed increased incidences of early apoptotic oocytes in the 4°C and 15°C groups (P < 0.05) compared with other groups. In conclusion, based on the embryo developmental competence of stored oocytes and quality of resultant blastocysts, 25°C was determined as the most suitable temperature for temporal storage of matured bovine oocytes. The study was supported by the NARO Institute of Livestock and Grassland Science, Japan (N32G4126), and the Royal Golden Jubilee-PhD scholarship (2.B.TS/53/F.2).


2019 ◽  
Vol 31 (2) ◽  
pp. 272 ◽  
Author(s):  
M. A. Abdel-Ghani ◽  
Y. Yanagawa ◽  
A. Z. Balboula ◽  
K. Sakaguchi ◽  
C. Kanno ◽  
...  

In this study we investigated the effect of astaxanthin (Ax), which exhibits strong antioxidant activity, during invitro growth (IVG) on the developmental competence of oocytes and steroidogenesis of granulosa cells derived from early antral follicles. Bovine oocyte–cumulus–granulosa complexes collected from early antral follicles were cultured for 12 days in the presence or absence (control) of 500µM Ax. The viability of oocytes and antrum formation in the granulosa cell layer during IVG culture were greater in the presence than absence of Ax (P&lt;0.05). Regardless of Ax treatment, 17β-oestradiol production increased during IVG culture; however, progesterone production was significantly lower in the presence than absence of Ax (P&lt;0.05). Reactive oxygen species levels were lower in Ax-treated oocytes than in controls after IVG (P&lt;0.05). Although nuclear maturation and cleavage rates did not differ between the Ax-treated and control groups, Ax treatment led to weaker cathepsin B activity in oocytes and better blastocyst rates than in controls (P&lt;0.05). Accordingly, Ax treatment during IVG increased the total number of cells in blastocysts (P&lt;0.05). These results indicate that Ax supplementation of IVG medium improves the quality of bovine oocytes due to its antioxidative effects on growing oocytes and its suppression of the luteinisation of granulosa cells.


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