Developmental Competence Of Porcine Oocytes Research Articles

Pre-culture with transferrin-Fe3+ before in vitro maturation improves the developmental competence of porcine oocytes matured in vitro.

Since the developmental competence of oocytes cultured after in vitro maturation (IVM) is low, it is necessary to improve the IVM method for efficient offspring production. In this study, we revealed that transferrin (TF)-Fe3+ was accumulated in follicular fluid with increasing the follicular diameter, and that TF receptor (TFR1) was localized in granulosa cells of pig. Thus, we hypothesized that TF-Fe3+ would be a factor in the induction of developmental competence of porcine oocytes. To mimic the follicular development environment, cumulus-oocyte complexes (COCs) were cultured in pre-IVM medium (low dose of FSH) without or with Holo-TF (monoferric or diferric TF) or Apo-TF (non-iron bond TF). After pre-IVM without or with Holo-TF, COCs were cultured in IVM medium (high dose of FSH and EGF) without or with Holo-TF. Cultivation with Holo-TF increased the expression of follicular development maker (Cyp19a1 and Ccnd2), E2 production, and proliferative activity of cumulus cells, whereas cultivation with Apo-TF did not show these positive effects. The treatment with Holo-TF during pre-IVM, but not during IVM, dramatically induced oocyte maturation with increasing the blastocyst rate. We succeeded in showing for the first time that the cultivation with Holo-TF in pre-IVM can produce embryos in pig with high efficiency.

Copper deficiency affects the developmental competence of porcine oocytes matured in vitro.

The trace element Cu is required for the activity of various enzymes essential for physiological processes. In this study, we elucidated the copper transport system in porcine follicular cells and investigated the effect of Cu chelation during in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after parthenogenetic activation (PA). Cu chelation was induced by adding tetraethylenepentamine (TEPA) to the maturation media (TCM199-PVA). First, we identified the localization and relative levels of the copper transporter CTR1 in follicular cells. The level of CTR1 protein was the highest in mature cumulus cells; moreover, CTR1 was mainly localized in the cytoplasmic vesicular compartment in oocytes, whereas it was evenly distributed in the cytoplasm in cumulus cells. A total of 42 h after IVM, the TEPA-treated group showed reduced maturation rates compared to those of the control (p < 0.05). This negative effect of TEPA disappeared when it was added to the media with Cu (Cu + TEPA group). The TEPA treatment during IVM significantly increased the mRNA levels of the Has2 gene, which is related to cumulus expansion (p < 0.05). Both Cu supplementation and chelation significantly increased the reactive oxygen species (ROS) levels in porcine oocytes (p < 0.05). When we analyzed the transcript levels of folliculogenesis-related genes in Cu chelation conditions, only the expression of MAPK3 in cumulus cells significantly increased compared to that of the control. We also evaluated the subsequent embryonic development of PA embryos. TEPA-treated oocytes showed significantly decreased blastocyst formation rates compared to those of the control. The TEPA-induced toxic effect was alleviated when Cu was added with TEPA. Our findings suggest that the Cu transport system plays an important role in the porcine follicular development process and that the Cu deficiency negatively affects porcine oocyte maturation, as well as their subsequent developmental competence.

Idebenone relieves the damage of heat stress on the maturation and developmental competence of porcine oocytes.

The reproductive function of animals is often affected by climatic conditions. High-temperature conditions can cause damage to oocyte maturation and embryonic development in a variety of ways. The purpose of this study was to prove that supplementation idebenone (IDB) to the maturation medium can improve the maturation and development of porcine oocytes after heat stress (HS). Porcine cumulus-oocyte complexes (COCs) were cultured in the maturation medium with different concentrations of IDB (0, 0.1, 1 and 10μM) for 44hr at either 38.5°C or under the HS conditions. The cumulus oophorus expansion, nuclear maturation and blastocyst rate after parthenogenetic activation (PA) were measured. We found that HS (in vitro maturation 20-24hr, 42°C) exposure significantly reduced cumulus expansion index and maturation rate of oocytes and the blastocyst rate of PA embryos, while IDB supplementation significantly improved oocyte maturation and development to the blastocysts stage after PA. Moreover, the addition of IDB decreased the intracellular level of ROS and increased GSH content, hence enhancing the antioxidant capacity of oocytes under HS. Meanwhile, IDB treatment also obviously improved the mitochondrial membrane potential and ATP synthesis of oocytes under HS conditions. Furthermore, IDB treatment increased the expression of GDF9 and BMP15 in IVM oocytes which attribute to improve the quality and outcome of IVM oocytes and the development competence of PA embryos in pigs. In summary, we demonstrated that IDB supplementation into the maturation medium exerted protective effects and improved the ability of maturation and developmental competence of porcine oocytes exposed to HS.

The Role of MAPK3/1 and AKT in the Acquisition of High Meiotic and Developmental Competence of Porcine Oocytes Cultured In Vitro in FLI Medium.

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

Tributyltin Oxide Exposure During in vitro Maturation Disrupts Oocyte Maturation and Subsequent Embryonic Developmental Competence in Pigs.

Tributyltin oxide (TBTO), an organotin compound, has been demonstrated to have toxic effects on several cell types. Previous research has shown that TBTO impairs mouse denuded oocyte maturation. However, limited information is available on the effects of TBTO exposure on livestock reproductive systems, especially on porcine oocytes in the presence of dense cumulus cells. In the present research, we evaluated the effects of TBTO exposure on porcine oocyte maturation and the possible underlying mechanisms. Porcine cumulus-oocyte complexes were cultured in maturation medium with or without TBTO for 42 h. We found that TBTO exposure during oocyte maturation prevented polar body extrusion, inhibited cumulus expansion and impaired subsequent blastocyst formation after parthenogenetic activation. Further analysis revealed that TBTO exposure not only induced intracellular reactive oxygen species (ROS) accumulation but also caused a loss of mitochondrial membrane potential and reduced intracellular ATP generation. In addition, TBTO exposure impaired porcine oocyte quality by disrupting cellular iron homeostasis. Taken together, these results demonstrate that TBTO exposure impairs the porcine oocyte maturation process by inducing intracellular ROS accumulation, causing mitochondrial dysfunction, and disrupting cellular iron homeostasis, thus decreasing the quality and impairing the subsequent embryonic developmental competence of porcine oocytes.

The antioxidant dieckol reduces damage of oxidative stress-exposed porcine oocytes and enhances subsequent parthenotes embryo development.

This study investigated the effect of the antioxidant dieckol, a component of Ecklonia cava, on maturation and developmental competence of porcine oocytes exposed to oxidative stress in vitro. Oocytes were matured in in vitro maturation (IVM) medium containing various concentrations of dieckol. The blastocyst formation rate was highest in the 0.5 μM dieckol-treated (0.5 DEK) group. The reactive oxygen species level was decreased, and the level of glutathione and expression of antioxidant genes (NFE2L, SOD1, and SOD2) at metaphase II were increased in the 0.5 DEK group. Abnormal spindle organization and chromosome misalignment were prevented in the 0.5 DEK group. Expression of maternal markers (CCNB1 and MOS) and activity of p44/42 mitogen-activated protein kinase were increased in the 0.5 DEK group. After parthenogenetic activation, the total number of cells per blastocyst was increased and the percentage of apoptotic cells was decreased in the 0.5 DEK group. Expression of development-related genes (CX45, CDX2, POU5F1, and NANOG), antiapoptotic genes (BCL2L1 and BIRC5), and a proapoptotic gene (CASP3) were altered in the 0.5 DEK group. These results indicate that the antioxidant dieckol improves IVM and subsequent development of porcine oocytes and can be used to improve the quality of oocytes under peroxidation experimental conditions.

Melatonin Restores the Developmental Competence of Heat Stressed Porcine Oocytes, and Alters the Expression of Genes Related to Oocyte Maturation.

Simple SummaryMelatonin improves the quality and in vitro maturation (IVM) of oocytes under heat stress. Melatonin treatment counteracts the adverse effects induced by heat stress (HS), such as the poor survival rate and maturation rate, distribution of α-tubulin and F-actin, expression of NRF2 and GDF9 mRNA. However, HS and melatonin have similar effects on increasing expression of HSP70 and NRF2 mRNA. Furthermore, HS inhibits expression of GDF9 mRNA.Melatonin enhances the quality and in vitro maturation (IVM) of oocytes under heat stress (HS), but the mechanism of melatonin in reducing HS injury on oocytes is not fully understood. In this study, porcine cumulus-oocyte complexes (COCs) were randomly divided into three groups. The COCs of the control group were cultured at 38.5 °C for 42 h, and the COCs of the HS group were cultured at 41.5 °C for 4 h, and then transferred into 38.5 °C for 38 h. The COCs of the HS + melatonin group were cultured with 10−9 M melatonin under the same conditions as the HS group. The survival rate, maturation rate, distribution of α-tubulin and F-actin of the oocytes were assessed. In addition, the expression profiles for genes related to the oocyte maturation, including heat shock protein 70 (HSP70), nuclear factor erythroid 2-related factor 2 (NRF2), cyclin-dependent kinase 1 (CDK1), growth differentiation factor 9 (GDF9) were analyzed by real-time quantitative PCR. The results showed that HS decreased the survival rate and maturation rate, distribution of α-tubulin and F-actin, but melatonin treatment could partly counteract these adverse effects. In addition, HS increased expression of HSP70 and NRF2 mRNA, and melatonin treatment had a similar effect on HSP70 expression, but had a contrary effect on NRF2 expression. Furthermore, HS inhibited expression of CDK1 and GDF9 mRNA, but melatonin treatment could weaken the effect on GDF9 expression induced by HS. In summary, melatonin treatment could attenuate the unfavorable effects induced by HS to enhance developmental competence of porcine oocytes during IVM.

In vitro maturation on ovarian granulosa cells encapsulated in agarose matrix improves developmental competence of porcine oocytes

In vivo, mammalian oocytes are surrounded by granulosa cells (GCs) that exist in a three-dimensional (3D) microenvironment with soft stiffness. The GCs play an important role for the in vivo growth and development of oocytes, through bidirectional communication between oocytes and GCs. To mimic the cellular microenvironment of a 3D organized follicle, this study designed a co-culture system using porcine ovarian GCs (pGCs) encapsulated in agarose matrix for in vitro maturation (IVM) of pig oocytes. We report the effects of our newly designed co-culture system on IVM and development of pig oocytes. Immature cumulus-oocyte-complexes (COCs) were matured on a 1% (w/v) agarose matrix encapsulated without or with pGCs. The number of pGCs within the agarose matrix was optimized by analyzing the in vitro development of parthenogenetic embryos. Moreover, the role of the ovarian stromal pGCs as feeder cells was assessed by analyzing the PA embryonic development. Subsequently, the effect of pGCs encapsulated in a 3D agarose matrix was evaluated for the developmental competence of pig oocytes by analyzing blastocyst formation after parthenogenetic activation (PA), intra-oocyte GSH and ROS contents, expression levels of BMP15 and BAX, TUNEL (terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling) assay, protein expression levels of BMP15, and intra-oocyte ATP levels. The optimized number of pGCs (5 × 104 cells/well) in a 3D agarose matrix led to a significantly higher blastocyst formation, increased BMP15 gene and protein expression, and intra-oocyte ATP levels; moreover, it induced significantly lower intra-oocyte ROS contents, pro-apoptotic BAX gene expression, and apoptotic index, compared to control. Our results demonstrate that application of pGCs as feeder cells encapsulated in the agarose matrix for IVM effectively increases the developmental competence of porcine oocytes.

Chitosan nanoparticles enhance developmental competence of in vitro-matured porcine oocytes.

Oxidative stress is inevitable as it is derived from the handling, culturing, inherent metabolic activities and medium supplementation of embryos. This study was performed to investigate the protective effect of chitosan nanoparticles (CNPs) on oxidative damage in porcine oocytes. For this purpose, cumulus-oocyte complexes (COCs) derived from porcine slaughterhouse ovaries were exposed to different concentrations of CNPs (0, 10, 25 and 50µg/ml) during in vitro maturation (IVM). Oocytes treated with 25µg/ml CNPs showed significantly higher levels of GSH, along with a significant reduction in ROS levels compared to control, CNPs10 and CNPs50 groups. In parthenogenetic embryo production, the maturation rate was significantly higher in the CNPs25 group than that in the control and all other treated groups. In addition, when compared to the CNPs50 and control groups, CNPs25-treated oocytes showed significantly higher cleavage and blastocyst development rates. The highest concentration of CNPs reduced the total cell number and ratio of ICM: TE cells in parthenogenetic embryos, suggesting that there is a threshold where benefits are lost if exceeded. In cloned embryos, the CNPs25 group, as compared to all other treated groups, showed significantly higher maturation and cleavage rates. Furthermore, the blastocyst development rate in the CNPs25-treated group was significantly higher than that in the CNPs50-treated group, as was the total cell number. Moreover, we found that cloned embryos derived from the CNPs25-treated group showed significantly higher expression levels of Pou5f1, Dppa2, and Ndp52il genes, compared with those of the control and other treated groups. Our results demonstrated that 25µg/ml CNPs treatment during IVM improves the developmental competence of porcine oocytes by reducing oxidative stress.

Inhibition of miR-152 during In Vitro Maturation Enhances the Developmental Potential of Porcine Embryos.

Simple SummaryMiR-152 is a highly conserved miRNA across different species and plays a role in the regulation of cell differentiation, proliferation, and survival. However, the exact role of miR-152 in oocyte and embryo development is not yet known. In this study, we specifically manipulated the expression level of miR-152 in porcine cumulus-oocyte complexes (COCs) and monitored their developmental competence until the blastocyst stage. We mainly found that a suppressed expression of miR-152 during oocyte maturation significantly improved the blastocyst rate. Our results indicate that this negative correlation between miR-152 during oocyte maturation and the blastocyst rate in pigs could be through targeting IGF system components during oocyte development. These results provide more insights into the role of miRNAs during oocyte and embryonic development that could improve the in vitro production system for mammalian embryos. Oocyte developmental competence is regulated by various mechanisms and molecules including microRNAs (miRNAs). However, the functions of many of these miRNAs in oocyte and embryo development are still unclear. In this study, we managed to manipulate the expression level of miR-152 during oocyte maturation to figure out its potential role in determining the developmental competence of porcine oocytes. The inhibition (Inh) of miR-152 during oocyte maturation does not affect the MII and cleavage rates, however it significantly enhances the blastocyst rate compared to the overexpression (OvExp) and control groups. Pathway analysis identified several signaling pathways (including PI3K/AKT, TGFβ, Hippo, FoxO, and Wnt signaling) that are enriched in the predicted target genes of miR-152. Gene expression analysis revealed that IGF1 was significantly up-regulated in the Inh group and downregulated in the OvExp group of oocytes. Moreover, IGF1R was significantly upregulated in the Inh oocyte group compared to the control one and IGFBP6 was downregulated in the Inh oocyte group compared to the other groups. Blastocysts developed from the OvExp oocytes exhibited an increase in miR-152 expression, dysregulation in some quality-related genes, and the lowest rate of blastocyst formation. In conclusion, our results demonstrate a negative correlation between miR-152 expression level and blastocyst rate in pigs. This correlation could be through targeting IGF system components during oocyte development.

PS48 promotes in vitro maturation and developmental competence of porcine oocytes through activating PI3K/Akt signalling pathway.

Oocyte maturation plays a vitally important role in porcine reproduction. Regrettably, the quality of oocytes matured in vitro is weakerthan that of in vivo matured oocytes. We collected and cultivated porcine cumulus oocyte complexes (COCs) in vitro with phosphoinositide-dependent kinase 1 (PDK1) activator 5-(4-chloro-phenyl)-3-phenyl-pent-2-enoic acid (PS48), whose concentrations were 0, 2, 5, 10 and 20µM to investigate whether the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signalling pathway would impact the oocyte quality. The results showed that 10µM PS48 increased the oocyte proportion of metaphase II (MII) stage and improved the expansion of cumulus cells (CCs). What's more, the activation of PI3K/Akt signalling pathway could regulate the expression of maturation-related genes and proteins. The results of quantitative real-time PCR showed that 10µM PS48 increased the mRNA and protein levels of Akt and regulated maturation-related genes, including cyclin B1, MOS, BMP15, GDF9, CDC2, mTOR, BAX, BCL2 and caspase-3. The results of Western blot indicated that 10µM PS48 increased the protein abundance of Akt, phosphorylation of Akt Thr308 (p-AktThr308 ) and cyclin B1, but decreased the protein abundance of pro-apoptotic BAX. These results suggested that adding 10µM PS48 to mature culture medium could promote the maturation of porcine oocytes, potentially through activating the PI3K/Akt signalling pathway.

Curcumin supplementation in the maturation medium improves the maturation, fertilisation and developmental competence of porcine oocytes.

This study was conducted to determine the effects of supplementing the maturation medium with the antioxidant curcumin on the in vitro maturation (IVM), fertilisation and development of porcine oocytes. Curcumin supplementation was performed at concentrations of 0, 5, 10, 20, and 40 µM. At concentrations of 5-20 µM, curcumin had significant positive effects (P < 0.05) on maturation and fertilisation rates compared to the non-treated group. Of the groups cultured with 5-20 µM curcumin, the number of oocytes with DNA-fragmented nuclei after IVM was significantly lower than in groups matured without curcumin. Moreover, curcumin supplementation at 10 µM also gave a significantly higher rate of blastocyst formation compared with oocytes matured without curcumin. Increasing the curcumin concentration to 40 µM yielded negative effects on fertilisation and embryonic development compared with the groups treated with lower concentrations of curcumin. Supplementation with 10 µM curcumin had beneficial effects on the oocyte maturation rate and DNA fragmentation index compared to the non-treated group both in the presence and absence of hydrogen peroxide. These results indicate that curcumin supplementation at a suitable concentration (10 µM) is potentially useful for porcine oocyte culture systems, in terms of protecting oocytes from various forms of oxidative stress.

Doubling of the cytoplasm volume improves the developmental competence of porcine oocytes injected with freeze-dried somatic cells

In the present study using pig cells, we examined the effect of the cryoprotectant trehalose on the DNA integrity of freeze-dried cells. We then investigated whether donor cell types and storage duration had impact on DNA integrity in freeze-dried cells or developmental competence of oocytes injected with freeze-dried somatic cells. We also examined whether double cytoplasm nuclear transfer (DCNT) would improve developmental competence of such oocytes. Furthermore, using a PCR-based method for sex identification, we determined whether the blastocysts obtained had actually been generated from the freeze-dried cells. It was found that, for a short storage duration at low temperature, trehalose had no beneficial effect on protection from DNA damage, and that donor cell type had no effect on the DNA integrity of freeze-dried somatic cells or the developmental competence of oocytes injected with them. We also confirmed that all of the blastocysts obtained following nuclear transfer were of freeze-dried somatic cell origin. Storage of freeze-dried somatic cells for up to 1 year at low temperature did not degrade DNA integrity in comparison with storage for 1 month, 1 week or 1 day. Following injection of freeze-dried cells, the proportion of oocytes that developed to blastocysts after storage for up to 1 year was similar to that after storage for 1 month, 1 week or 1 day. Moreover, DCNT significantly improved the developmental competence of oocytes treated in this way. In summary, using DCNT, we have demonstrated that freeze-dried porcine somatic cells subjected to long-term storage at 4 °C have nearly the same potential to develop to blastocysts as non-freeze-dried cells.

Porcine follicular fluid derived from > 8 mm sized follicles improves oocyte maturation and embryo development during in vitro maturation of pigs.

The aim of the present study was to investigate the effects of porcine follicular fluid (pFF) from large-sized (LFF; >8 mm in diameter) and medium-sized (MFF; 3-6 mm in diameter) follicles on the maturation and developmental competence of porcine oocytes. Cumulus-oocyte complexes (COCs) were collected from follicles 3-6 mm in diameter. The collected COCs were incubated for 22 h with LFF or MFF (in vitro maturation (IVM)-I stage) and were incubated subsequently for 22 h with LFF or MFF (IVM-II stage). Cumulus expansion was confirmed after the IVM-I stage and nuclear maturation was evaluated after the IVM-II stage. Intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured and embryonic development was evaluated. Relative cumulus expansion and GSH levels were higher in the LFF group compared with in the MFF group after the IVM-I stage (P < 0.05). After the IVM-II stage, the numbers of oocytes in metaphase-II were increased in the LFF group and GSH content was higher in all of the LFF treatment groups compared with in the MFF treatment groups during both IVM stages (P < 0.05). ROS levels were reduced by LFF treatment regardless of IVM stage (P < 0.05). Blastocyst formation and the total numbers of cells in blastocysts were increased in all LFF treatment groups compared with the control group (P < 0.05). These results suggested that pFF from large follicles at the IVM stage could improve nucleic and cytoplasmic maturation status and further embryonic development through reducing ROS levels and enhancing responsiveness to gonadotropins.

Single cell RNA-seq reveals molecular pathways altered by 7, 12-dimethylbenz[a]anthracene treatment on pig oocytes

Oocytes of better quality and developmental competence are highly demanded, which is affected by many intrinsic and external factors, including environmental pollutants. We have previously demonstrated that 7, 12-dimethylbenz [a]anthracene (DMBA) reduces the developmental competence of porcine oocytes, by desynchronizing nuclear and ooplasmic maturation. However, the underlying molecular mechanism remains obscure. Here we performed single cell RNA-seq to study the transcriptome changes in DMBA-treated porcine MII oocytes, and identified 19 protein-coding genes and 156 novel long non-coding RNAs (lncRNAs) with abundance to be significantly different (P < 0.05), which enriched in signaling pathways such as glycosphingolipid biosynthesis, nicotine addiction, basal transcription factors and nucleotide excision repair. RT-qPCR on oocyte pools confirmed ornithine aminotransferase (Oat) and serine/arginine-rich splicing factor 4 (Srsf4) to be significantly up- and down-regulated, respectively (P < 0.05). Treating porcine COCs with MAPK and PLC pathway inhibitors suppressed DMBA’s effects on increasing PB1 extrusion rate. In addition, DMBA co-incubation with 250 μM vitamin C derivative (l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, AA2P) and 100 μM co-enzyme Q10 (CoQ10) could significantly reduce the DMBA-induced high ROS level, and partially alleviate the DMBA-induced high PB1 rate, whereas the cleavage and blastocyst rates of parthenotes derived from treated mature oocytes remained to be low. Collectively, our findings indicate that single cell RNA-seq can help reveal the dynamics of molecular signaling pathways for porcine oocytes treated by DMBA, and supplement of anti-oxidative reagents could not sufficiently rescue DMBA-induced defects of porcine oocytes.

PI3K inhibitor reduces in vitro maturation and developmental competence of porcine oocytes

The phosphatidylinositol -3- kinase (PI3K) signaling pathway is critical for the cell proliferation, apoptosis, metabolism, DNA repair and protein synthesis. Significant effort has focused on elucidating the relationship between PI3K signaling pathway and other nuclear signal transducers; However, little is known about the connection between PI3K signaling pathway and porcine oocyte meiotic maturation. In this study, we investigated the function of PI3K signaling pathway in porcine oocytes. PI3K signaling pathway was important during oocyte maturation. Furthermore, the PI3K signaling pathway inhibitor LY-294002 blocked porcine oocyte maturation, reducing the percentage of oocytes that first polar body (PBI) extrusion. LY-294002 also decreased the expression of oocyte proliferation-related gene PCNA and reduced the mRNA and protein levels of PI3K. What’s more, LY-294002 also decreased other maturation-related genes that are predominantly expressed duringporcine oocyte maturation, including bone morphogenetic protein 15 (BPM15), growth differentiation factor 9 (GDF9), cell division cycle protein 2 (CDC2), phosphatase and tensin homolog (PTEN), CyclinB1, MOS and Akt. LY-294002 treatment decreased the developmental potential of blastocysts following parthenogenetic activation, increased the level of cell apoptosis and reduced the level of cell-cycle. This study revealed that inhibiting the PI3K signaling pathway could reduce in vitro maturation and developmental competence of porcine oocytes, probably by reducing cell cycle arrest and proliferation, promoting the oocyte apoptosis, and altering the expression of other maternal genes.

In vitro maturation on an agarose matrix improves the developmental competence of porcine oocytes

Oocytes in vivo generally mature in ovarian follicles that are soft, whereas oocytes that mature in vitro are on the hard surface of culture dishes. Embryonic ontogeny through organogenesis has greater ability in in vivo matured oocytes than it does in in vitro matured oocytes, indicating the importance of a soft culture matrix. In this study, we report the effect of using an agarose matrix as a culture substrate on the development of pig oocytes derived from medium antral follicles. The cumulus-oocyte complexes (COCs) retrieved from medium antral follicles were matured on noncoated (control) culture dishes or dishes coated with 1% and 2% (w/v) agarose matrices. Subsequently, the effect of the soft culture matrix on the developmental competence of porcine oocytes was assessed by analyzing cumulus expansion, blastocyst formation after parthenogenetic activation (PA), gene expression levels (ACTN4, BMP15, BAX, HIF1A, PFKP and VEGFA), TUNEL indices, BMP15 protein expression levels, cortical granule (CG) distribution, and intraoocyte ATP levels. In vitro maturation (IVM) of pig COCs using a 1% (w/v) agarose matrix resulted in significantly higher blastocyst formation, cumulus expansion, gene expression of BMP15, HIF1A and VEGFA, protein expression of BMP15, and intraoocyte ATP levels, and there was significantly reduced expression of a pro-apoptotic gene and ACTN4 gene and a reduction in TUNEL indices. These results demonstrate that the developmental competence of porcine oocytes can be effectively improved through IVM on a soft culture matrix made of agarose over what is observed using hard culture dishes.

Effects of cobalamin on meiotic resumption and developmental competence of growing porcine oocytes

This study was performed to explore the effects of cobalamin treatment during in vitro maturation (IVM) of porcine oocytes. Specifically, the effects of cobalamin exposure on nuclear and cytoplasmic maturation of oocytes, diameter, glutathione (GSH) and reactive oxygen species (ROS) levels of matured oocytes, as well as development and gene expression of porcine parthenogenetic and cloned embryos were assessed. Cumulus–oocyte complexes were exposed to 200 pM cobalamin for 22 h or incubated for 22 h without cobalamin (controls). The mean diameter of cobalamin-treated oocytes was greater than that of control oocytes (160.0 vs. 154.5 μm; p < 0.05). GSH level increased but ROS level decreased in the cobalamin-treated oocyte group. Parthenogenetic embryos derived from cobalamin-treated oocytes showed improved oocyte maturation (91.3% vs. 83.8%), cleavage (88.9% vs. 82.1%), and blastocyst formation (38.7% vs. 31.9%) rates compared with control embryos (p < 0.05). Similarly, cloned embryos derived from cobalamin-treated oocytes showed higher oocyte maturation (89.2% vs. 82.2%), cleavage (87.5% vs. 80.3%), and blastocyst formation (30.0% vs. 23.8%) rates than control embryos (p < 0.05). Furthermore, in parthenogenetic and cloned embryos, total cell number, inner cell mass (ICM), trophectoderm (TE) expression, and ICM: TE ratio were higher in the cobalamin-treated group compared to that in the control group (p < 0.05). Cloned embryos in the cobalamin-treated group showed higher mRNA expression levels of POU5F1, DPPA2, and NDP52IL than control group embryos. Together, these results demonstrate that cobalamin treatment during IVM improves the developmental competence of porcine oocytes by neutralizing the free radicals produced in the IVM medium.

Improve the developmental competence of porcine oocytes from small antral follicles by pre-maturation culture method

The oocytes from small antral follicle have low developmental potential to reach blastocyst due to incomplete cytoplasmic maturation during in vitro maturation (IVM). Thus, we developed an in vitro culture system for porcine oocytes derived from small antral follicles with l-ascorbic acid supplement during pre-maturation (pre-IVM) to support their development to blastocyst stage. Besides that, how l-ascorbic acid effect on the developmental competence of porcine oocytes with a special focus on histone modifications will be elucidated.The in vitro culture process consisted of two steps. The first step is 22 h of pre-IVM and the second step is 42 h of IVM. We utilized dibutyryl-cyclicAMP (dbcAMP) with L-ascorbic supplement during pre-IVM. Based on the result of this procedure, we proposed that the best culture condition in which hormone human chorionic gonadotropin (hCG) be added during the last 7 h of pre-IVM and continued culture to complete IVM. We observed that, in this culture system, the meiotic competence of porcine oocytes derived from small follicles was as high as those derived from large follicles after undergoing IVM. In addition, our study suggested that l-ascorbic acid supplementation at 100 μg/mL sharply enhanced the developmental potential of porcine oocytes. Interestingly, oocytes from small antral follicles treated with l-ascorbic acid could obtain the blastocyst quantity and quality as high as that of large antral follicles. The treated groups showed a significantly higher number of blastomeres compared to those in non-treated groups in both small and large follicle groups. Besides that, = The increasing levels of acetylation of histone H3 at lysine 9 (H3K9) and methylation of histone H3 at lysine 4 (H3K4) in blastocyst derived from small and large antral follicle under the present of l-ascrobic acid lead to a significant positive effect on the developmental competence and improvement in quality of porcine embryos.

Characterization of porcine oocytes stained with Lissamine Green B and their developmental potential in vitro.

Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus-oocyte complex, polar body and meiotic spindle. This study is focused on the differences between the morphological assessment of oocyte quality, the assessment based on Lissamine Green B (LB) staining and the analysis of oocytes using a proteomic approach. We evaluated the effectiveness of electrochemical and chemical parthenogenetic activation under our laboratory conditions and evaluated the applicability of Lissamine Green B staining of cumulus-oocyte complexes (COCs) as a non-invasive method for predicting the maturational and developmental competence of porcine oocytes cultured in vitro. We determined that chemical parthenogenetic activation using ionomycin and 6-dimethylaminopurine was slightly more effective than electrochemical activation. After oocyte selection according to LB staining, we found significant differences (P<0.05) between the LB- group and LB+ group and the control group in their maturation, cleavage rate and rate of blastocysts. Proteomic analyses identified a selection of proteins that were differentially expressed in each group of analysed oocytes. Oocytes of the LB- group exhibited an increased variability of proteins involved in transcription regulation, proteosynthesis and the protein folding crucial for oocyte maturation and further embryonic development. These results found a better competence of LB- oocytes in maturation, cleavage and ability to reach the blastocyst stage.