Endoplasmic reticulum stress attenuation promotes bovine oocyte maturation in vitro

We have previously reported that regulation of endoplasmic reticulum (ER) stress during in vitro culture acutely increases bovine embryo developmental rate and cryotolerance; these data indicate that ER stress is a critical factor reducing the quality of in vitro-produced embryos. In the current follow-up study, we examined whether ER stress attenuation during in vitro maturation influences meiotic maturation, oocyte quality, and subsequent embryonic development. Bovine cumulus oocyte complexes (COCs) derived from slaughterhouse ovaries were matured with or without tauroursodeoxycholic acid (TUDCA), a selective inhibitor of ER stress (0, 50, 100, and 200 µM) for 22 h followed by in vitro fertilization, and zygotes were cultured for 8 days. Of the different doses of TUDCA, 100 μM TUDCA significantly increased the maturation rate, and decreased reactive oxygen species in denuded oocytes, and appeared lower number of apoptotic cells in matured COCs. Subsequently, treatment of TUDCA (100 µM) decreased the localization and amount of GRP78/BIP protein level as well as ER stress (GRP78/BIP, PERK, IER1, ATF4, and XBP1) and apoptosis (CHOP and BAX)-related gene expression, while it increased the anti-apoptotic gene BCL2 level in matured COCs. Moreover, addition of TUDCA (100 µM) during IVM significantly improved the blastocyst formation rate (43.6 ± 1.8% vs 49.7 ± 1.3%) and decreased the number of apoptotic cells (7.7 ± 1.1% vs 5.03 ± 0.6%) in blastocysts. These findings suggest that the presence of ER stress during maturation impairs the developmental competence of bovine COCs and that this process can be reversed by TUDCA.

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214 EFFECT OF OOCYTE SOURCE ON THE DEVELOPMENTAL CAPACITY OF SOUTH AFRICAN IN VITRO-PRODUCED INDIGENOUS CATTLE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab214 ◽

2009 ◽

Vol 21 (1) ◽

pp. 205

Author(s):

T. L. Nedambale ◽

M. B. Raito ◽

M. L. Mphaphathi

Keyword(s):

Treatment Group ◽

South African ◽

Formation Rate ◽

Developmental Rate ◽

Bovine Embryo ◽

Department Of Agriculture ◽

Indigenous Cattle ◽

Developmental Capacity ◽

Development In Vitro

The present study was undertaken to determine whether the source of oocytes (slaughterhouse ovaries from feedlot cows or naturally grazing indigenous cows) would affect in vitro bovine embryo production. Bovine oocytes (n = 1047), aspirated from slaughterhouse ovaries from feedlot cows and naturally grazing indigenous cows were randomly allocated to Sanyo, Forma, and Thermo 5% CO2 incubators. Oocytes were then in vitro matured in TCM-199 plus 10% fetal bovine serum, 1 μg mL–1 for both FSH and LH at 39°C for 24 h, and fertilized in Brackett and Oliphant (BO) medium per treatment group at 39°C. Presumptive zygotes were cultured in vitro per treatment group. Total cleavage and blastocyst rates were recorded postfertilization. Data were analyzed by ANOVA. Preliminary results demonstrated that there was no effect of incubator or source of oocytes on cleavage and 8-cell embryos. However, the cleavage and embryo developmental rate tended to be lower for the feedlot group, regardless of the incubator used (Table 1). In conclusion, this study suggests that slaughterhouse ovaries obtained from South African indigenous cows from a feedlot resulted in a lower blastocyst formation rate. Further studies are currently underway to count the cell numbers and to conduct embryo transfer. Table 1.Comparison of three different incubators and source of oocytes on embryo development in vitro This work was funded by the South African National Department of Agriculture, DST-PDP, and the National Research Foundation (NRF, Grant. Nos. RT21 and 24000).

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116 TAUROURSODEOXYCHOLIC ACID PREVENTS ENDOPLASMIC RETICULUM STRESS-INDUCED APOPTOSIS IN PREIMPLANTATION PIG EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab116 ◽

2009 ◽

Vol 21 (1) ◽

pp. 158

Author(s):

J.-S. Kim ◽

K.-S. Lee ◽

B.-S. Song ◽

J.-Y. Zhang ◽

Y.-K. Choo ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Blastocyst Stage ◽

Developmental Competence ◽

Preimplantation Embryos ◽

Control Group ◽

Tauroursodeoxycholic Acid ◽

Preimplantation Stage ◽

Induced Apoptosis

Apoptosis is an important determinant for the normal development of preimplantation embryos in vitro. Recently, endoplasmic reticulum (ER) stress-mediated apoptosis has been extensively investigated in a wide variety of diseases. The efficient functioning of the ER is essential for most cellular activities and survival. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has been reported to attenuate ER stress-mediated cell death by interrupting classic pathways of apoptosis. Therefore, we explored the anti-apoptotic effect of TUDCA on ER stress-induced apoptosis in preimplantation porcine embryos. Also, TM (tunicamycin; an ER stress inducing chemical reagent) was used to investigate the effect of ER-stress on pig embryo development. After in vitro maturation and fertilization, presumptive porcine embryos were cultured in NCSU23 medium supplemented with 200 μ g mL–1 TUDCA or 1 μg mL–1 (TM) for 6 days at 39°C, 5% CO2 in air. All data were analyzed by using Duncan test of ANOVA by Statistical Analysis System (SAS). When treated with TM during culture, only 8.2% (8/97) of the embryos developed to the blastocyst stage compared with 27.4% (28/102) of the embryos in the control group (P < 0.05). We also confirmed that TM stimulates up-regulation of ER stress response genes, such as XBP-1 mRNA, and induces a high rate of apoptosis. Whereas the frequency of blastocyst formation in the TUDCA-treated group was increased compared with that in the control group (32.8%, 49/149 v. 22.2%, 32/144), P < 0.05). Furthermore, the blastocyst cell number was enhanced (30.6 v. 39.5) and apoptosis reduced (TUNEL positive nuclei number, 6.0 v. 3.2) by TUDCA treatment in pig embryos. As the result of real-time quantitative RT-PCR analysis, the expression of anti-apoptotic Bcl-xl gene was increased in the blastocyst stage by TUDCA treatment, whereas expression of pro-apoptotic Bax was decreased. In addition, we also confirmed that TUDCA decreases the rate of TM-induced apoptosis in preimplantation stage pig embryos. Our results indicate that TUDCA improves the developmental competence of porcine embryos by modulating the ER stress-induced apoptosis during preimplantation stage.

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47 Inhibition of endoplasmic reticulum stress during invitro maturation improves the developmental competence of bovine cumulus-oocyte complexes

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab47 ◽

2020 ◽

Vol 32 (2) ◽

pp. 149

Author(s):

H. Khatun ◽

Y. Ihara ◽

K. Takakura ◽

Y. Wada ◽

K.-I. Yamanaka

Keyword(s):

Reactive Oxygen Species ◽

Endoplasmic Reticulum ◽

Embryonic Development ◽

Er Stress ◽

Mrna Expression ◽

Reactive Oxygen ◽

Developmental Competence ◽

Apoptotic Cells ◽

Oxygen Species ◽

Maturation Rate

Endoplasmic reticulum (ER) stress, a dysfunction in protein-folding capacity of ER, is involved in many physiological responses including embryonic development. Evidence shows that the ER stress-induced unfolded protein response signaling pathway is associated with impairment of oocyte maturation and pre-implantation embryonic development; supplementation of culture medium with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, improved the developmental process of oocytes and embryos by attenuating ER stress. However, no reports are available on the role of TUDCA in reducing ER stress during IVM of bovine oocytes. Therefore, the aim of this study was to examine the mechanism of TUDCA on reducing ER stress in maturation of bovine cumulus-oocyte complexes (COCs) and whether inhibition of ER stress during maturation can promote subsequent embryonic development. Bovine ovaries were collected from a local slaughterhouse, and after aspiration COCs were matured with/without TUDCA (50, 100, and 200 µM) for 22h at 38.5°C in a humidified atmosphere of 5% CO2. After IVM, we examined the maturation rate, reactive oxygen species, apoptosis, protein/mRNA expression levels, and subsequent embryonic development after IVF. The data were analysed using analysis of variance followed by the Tukey-Kramer multiple comparison test. As a result, the dose-dependent experiment shows that a 100μM concentration of TUDCA significantly increased the maturation rate and decreased the percentage of apoptotic cells in COCs and reactive oxygen species levels in denuded oocytes. Subsequently, the expression of ER stress inducible protein GRP78/BIP significantly decreased in COCs treated with 100 µM TUDCA compared with the control COCs. In addition, the mRNA expression of ER stress and pro-apoptotic markers (GRP78/BIP, PERK, IER1, ATF4, XBP1, CHOP, and BAX) in COCs were significantly decreased by TUDCA (100 µM) treatment, whereas it increased anti-apoptotic BCL2 expression. Moreover, we show that TUDCA (100 µM) supplementation enhances embryonic development by significantly increasing the blastocyst formation rate (43.6±1.8% vs. 49.7±1.3%) and decreasing the number of apoptotic cells (7.7±1.1% vs. 5.03±0.6%) in blastocysts. These findings suggest that existence of ER stress during maturation alters the developmental competence of bovine COCs. Therefore, for the first time, we demonstrate that application of TUDCA during IVM plays a crucial role in reducing ER stress and improves the meiotic maturation, oocyte quality, and subsequent embryonic development invitro.

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151. LIPOTOXICITY MEDIATED ENDOPLASMIC RETICULUM STRESS, MITOCHONDRIAL DYSFUNCTION AND APOPTOSIS CONTRIBUTE IMPAIRED OOCYTE QUALITY IN RESPONSE TO OBESITY

Reproduction Fertility and Development ◽

10.1071/srb09abs151 ◽

2009 ◽

Vol 21 (9) ◽

pp. 69

Author(s):

L. L. Y. Wu ◽

X. Yang ◽

K. R. Dunning ◽

R. J. Norman ◽

R. L. Robker

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Potential ◽

Er Stress ◽

Mitochondrial Dysfunction ◽

Granulosa Cells ◽

Oocyte Quality ◽

Developmental Competence ◽

Tunel Staining ◽

Marker Genes ◽

Er Homeostasis

In obesity, accumulation of lipid in non-adipose tissues, a process termed lipotoxicity, is associated with endoplasmic reticulum (ER) stress, mitochondrial dysfunction and ultimately apoptosis . We have previously shown that diet-induced obesity in mice causes impaired oocyte developmental competence, but whether this is due to activation of lipotoxicity pathways in the ovary is not known. The present study examined the hypothesis that diet-induced lipid accumulation in the cumulus oocyte complex (COC) disrupts ER homeostasis and mitochondrial membrane potential which leads to apoptosis. COCs and mural granulosa cells were collected from ovaries of adult mice fed a high fat (HFD) or control diet for 4 weeks. ER homeostasis was assessed by measuring expression of known ER stress marker genes, GRP78, ATF4 and CHOP. COCs from mice fed HFD showed significantly increased expression of GRP78 and ATF4. There was a similar trend towards increased expression in granulosa cells. Mitochondrial function was assessed by measuring membrane potential using the dual emission probe JC-1. In COCs from mice fed HFD there were reduced numbers of active mitochondria but instead large aggregated clusters of inactive mitochondria. Apoptosis in granulosa cells was determined by DNA laddering assay which showed significantly increased DNA fragmentation in cells from mice fed HFD. Apoptosis was also assessed by TUNEL staining of paraffin embedded ovaries from identical treatment groups. Ovaries from HFD mice appeared to have increased TUNEL positivity in both granulosa and cumulus cells. Our results demonstrate that the ER stress, mitochondrial dysfunction and apoptosis are markedly increased in granulosa cells and COCs from mice fed HFD, suggesting that lipotoxicity contributes to the impaired oocyte quality and reduced fertility observed in response to obesity.

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MicroRNA-210 Regulates Endoplasmic Reticulum Stress and Apoptosis in Porcine Embryos

Animals ◽

10.3390/ani11010221 ◽

2021 ◽

Vol 11 (1) ◽

pp. 221

Author(s):

Muhammad Rosyid Ridlo ◽

Eui Hyun Kim ◽

Geon A. Kim

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Embryo Development ◽

Caspase 3 ◽

Formation Rate ◽

Embryo Production ◽

Embryo Survival ◽

Expression Levels ◽

In Vitro Embryo Production

Endoplasmic reticulum (ER) stress can be triggered during in vitro embryo production and is a major obstacle to embryo survival. MicroRNA (miR)-210 is associated with cellular adaptation to cellular stress and inflammation. An experiment was conducted to understand the effects of miR-210 on in vitro embryo development, ER stress, and apoptosis; to achieve this, miR-210 was microinjected into parthenogenetically activated embryos. Our results revealed that miR-210 inhibition significantly enhanced the cleavage rate, blastocyst formation rate, and total cell number (TCN) of blastocysts, and reduced expression levels of XBP1 (p < 0.05). miR-210 inhibition greatly reduced the expression of ER stress-related genes (uXBP1, sXBP1, ATF4, and PTPN1) and Caspase 3 and increased the levels of NANOG and SOX2 (p < 0.05). A miR-210-mimic significantly decreased the cleavage, blastocyst rate, TCN, and expression levels of XBP1 compared with other groups (p < 0.05). The miR-210-mimic impaired the expression levels of uXBP1, sXBP1, ATF4, PTPN1, and Caspase 3 and decreased the expression of NANOG and SOX2 (p < 0.05). In conclusion, miR-210 plays an essential role in porcine in vitro embryo development. Therefore, we suggest that miR-210 inhibition could alleviate ER stress and reduce apoptosis to support the enhancement of in vitro embryo production.

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Adiponectin Improves In Vitro Development of Cloned Porcine Embryos by Reducing Endoplasmic Reticulum Stress and Apoptosis

Animals ◽

10.3390/ani11020473 ◽

2021 ◽

Vol 11 (2) ◽

pp. 473

Author(s):

Muhammad Rosyid Ridlo ◽

Eui Hyun Kim ◽

Anukul Taweechaipaisankul ◽

Byeong Chun Lee ◽

Geon A. Kim

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Early Stage ◽

Formation Rate ◽

Cell Number ◽

Control Group ◽

Cleavage Rate ◽

Cell Stage ◽

The Impact

The main factor of embryonic demise is endoplasmic reticulum (ER) stress. Successful attenuation of ER stress results in an improvement in embryo development. We studied the impact of adiponectin in the in vitro culture (IVC) of porcine embryos derived from parthenogenetic activation and somatic cell nuclear transfer (SCNT). The first experiment revealed that 15 and 30 μg/mL adiponectin treatments improved cleavage, blastocyst rates, and total cell number (TCN) of parthenogenetic embryos and reduced the expression of XBP1 compared to the 5 μg/mL adiponectin treatment and control groups (p < 0.05). The second experiment showed that cleavage rate, blastocyst formation rate, and TCN of blastocysts were improved in the 15 μg/mL adiponectin treatment group compared with the control group, with significantly reduced XBP1 expression in ≥4-cell stage SCNT embryos and blastocysts (p < 0.05). Treatment with 15 μg/mL adiponectin significantly improved the expression of XBP1 and reduced the expression of ER stress-related genes (uXBP1, sXBP1, PTPN1, and ATF4), increased the expression levels of pluripotency-related genes (Nanog and SOX2), and decreased apoptosis-related gene expression (Caspase-3). These results suggest that 15 μg/mL adiponectin enhanced the in vitro developmental capacity of early-stage SCNT porcine embryos by reducing ER stress and apoptosis.

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Dehydrodiisoeugenol inhibits colorectal cancer growth by endoplasmic reticulum stress-induced autophagic pathways

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01915-9 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Changhong Li ◽

Kui Zhang ◽

Guangzhao Pan ◽

Haoyan Ji ◽

Chongyang Li ◽

...

Keyword(s):

Colorectal Cancer ◽

Endoplasmic Reticulum ◽

Cell Growth ◽

Er Stress ◽

Cancer Cells ◽

Tumor Xenograft ◽

Anticancer Activities ◽

Colorectal Cancer Cells

Abstract Background Dehydrodiisoeugenol (DEH), a novel lignan component extracted from nutmeg, which is the seed of Myristica fragrans Houtt, displays noticeable anti-inflammatory and anti-allergic effects in digestive system diseases. However, the mechanism of its anticancer activity in gastrointestinal cancer remains to be investigated. Methods In this study, the anticancer effect of DEH on human colorectal cancer and its underlying mechanism were evaluated. Assays including MTT, EdU, Plate clone formation, Soft agar, Flow cytometry, Electron microscopy, Immunofluorescence and Western blotting were used in vitro. The CDX and PDX tumor xenograft models were used in vivo. Results Our findings indicated that treatment with DEH arrested the cell cycle of colorectal cancer cells at the G1/S phase, leading to significant inhibition in cell growth. Moreover, DEH induced strong cellular autophagy, which could be inhibited through autophagic inhibitors, with a rction in the DEH-induced inhibition of cell growth in colorectal cancer cells. Further analysis indicated that DEH also induced endoplasmic reticulum (ER) stress and subsequently stimulated autophagy through the activation of PERK/eIF2α and IRE1α/XBP-1 s/CHOP pathways. Knockdown of PERK or IRE1α significantly decreased DEH-induced autophagy and retrieved cell viability in cells treated with DEH. Furthermore, DEH also exhibited significant anticancer activities in the CDX- and PDX-models. Conclusions Collectively, our studies strongly suggest that DEH might be a potential anticancer agent against colorectal cancer by activating ER stress-induced inhibition of autophagy.

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Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

Reproduction Fertility and Development ◽

10.1071/rd17543 ◽

2018 ◽

Vol 30 (10) ◽

pp. 1342 ◽

Cited By ~ 4

Author(s):

Zhao-Bo Luo ◽

Long Jin ◽

Qing Guo ◽

Jun-Xia Wang ◽

Xiao-Xu Xing ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Formation Rate ◽

Epigenetic Reprogramming ◽

Developmental Competence ◽

Abnormal Development ◽

Control Group ◽

Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

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P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

Human Reproduction ◽

10.1093/humrep/deab130.218 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Á Martíne. Moro ◽

I Lamas-Toranzo ◽

L González-Brusi ◽

A Pérez-Gómez ◽

P Bermejo-Álvarez

Keyword(s):

Cumulus Cell ◽

Cumulus Cells ◽

Oocyte Quality ◽

Blastocyst Stage ◽

Early Embryo ◽

Developmental Competence ◽

Success Rates ◽

Developmental Potential ◽

Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p > 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

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Induction of autophagy protects against extreme hypoxia-induced damage in porcine embryo

Reproduction ◽

10.1530/rep-20-0311 ◽

2021 ◽

Vol 161 (4) ◽

pp. 353-363

Author(s):

Mun-Hyeong Lee ◽

Pil-Soo Jeong ◽

Bo-Woong Sim ◽

Hyo-Gu Kang ◽

Min Ju Kim ◽

...

Keyword(s):

Oxygen Tension ◽

Early Phase ◽

Inner Cell Mass ◽

Formation Rate ◽

Cell Mass ◽

Female Reproductive Tract ◽

Developmental Competence ◽

Early Embryos ◽

Developmental Parameters

In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0–2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2–4 days) and late phases (4–6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

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