Oocyte Cytoplasmic Maturation Research Articles

175 Nobiletin enhances the quality of in vitro-matured bovine oocytes and blastocysts by altering the transcription of key developmental genes

One of the problems associated with in vitro production of embryos in bovine is the increase in reactive oxygen species (ROS), which leads to cell alterations and death. Nobiletin is a polymethoxyflavone isolated from citrus fruits with various beneficial effects on cell cycle regulation and inhibition of ROS production. In a preliminary study, we demonstrated that supplementation of 25 or 50 µM nobiletin to the in vitro maturation (IVM) medium reduces oxidative stress and improves oocyte nuclear and cytoplasmic maturation and embryo development. Thus, in this study, we aimed to evaluate the antioxidant activity of nobiletin during IVM on bovine matured oocytes, their cumulus cells (CC), and blastocysts by quantitative changes of gene expression. Immature cumulus oocytes complexes (COC) were aspirated from ovaries of slaughtered heifers. Selected COC underwent IVM in TCM-199+10% FCS and 10ng mL−1 epidermal growth factor (EGF; Control) supplemented with 25 µM (Nob25) or 50 µM (Nob50) nobiletin (MedChemExpress, Monmouth Junction, NJ, USA) or 0.001% dimethyl sulfoxide (DMSO control), a vehicle for nobiletin dilution, in 5% CO2 in air at 38.5°C. After 24h, 50 matured oocytes/group and their CC were snap-frozen in LN2 for gene expression analysis. The remaining oocytes were fertilized (Day 0) and cultured in vitro. Blastocysts (Day 7; n=50/group) were snap-frozen in LN2 for gene expression analysis (5 replicates). The mRNA abundance of candidate genes related with oxidative stress (SOD2, CYP51); apoptosis (BAX); quality (BMP15, BMP7, CLIC1, MAPK1, ABCB1); and cell junction (GJA1) was measured by quantitative PCR; H2AFZ and 18S rRNA were used as housekeeping genes. Statistical significance was assessed by one-way ANOVA. Supplementation of IVM medium with Nob25 or Nob50 produced changes in the expression levels of genes related to oxidative stress and apoptosis during IVM compared with controls. SOD2 and CYP51 were down-regulated in oocytes and CC (P<0.05) but not in blastocysts, whereas BAX was down-regulated only in CC (P<0.05). Nobiletin supplementation in IVM increased the expression of MAPK1 in oocytes and blastocysts (P<0.05); however, no differences were observed in CC. BMP15 for oocytes and their CC and GJA1 for CC were up-regulated in Nob25 and Nob50 groups compared with controls (P<0.05). The relative abundance of CLIC1 decreased in blastocysts from both nobiletin groups compared with controls (P<0.05). No significant differences in the expression in ABCB1 and BMP7 were detected. In conclusion, our results suggest that supplementation of 25 or 50 µM nobiletin to the IVM medium reduces oxidative stress in oocytes and CC, decreases CC apoptosis, and provokes positive changes in the expression of genes related to oocyte and embryo quality. This research was supported by Spanish MINECO (AGL2015-70140-R and AGL2015-66145-R). Y. N. Cajas was supported by a grant from SENESCYT-Ecuador.

Effect of carbohydrates on lipid metabolism during porcine oocyte IVM.

Porcine oocytes contain a large amount of endogenous lipid, which is thought to function as an intracellular source of energy. The aim of this study was to determine the effects of stimulating or inhibiting lipid metabolism using l-carnitine or etomoxir respectively on the IVM of porcine oocytes cultured in media of varying carbohydrate composition. In the presence of pyruvate and lactate, exclusion of glucose inhibited oocyte nuclear and cytoplasmic maturation compared with oocytes matured in media containing low (1.5mM) and high (4.0mM) concentrations of glucose. In the absence of pyruvate and lactate in low-glucose medium only, a greater proportion of l-carnitine-treated oocytes progressed to the MII stage compared with untreated oocytes. The inclusion of pyruvate and lactate significantly altered the distribution of cytoplasmic lipid droplets and elevated the ATP content of oocytes, whereas the l-carnitine treatment did not. Further, the inhibitory effect of etomoxir on nuclear maturation was decreased in high- compared with low-glucose medium. The results indicate that carbohydrate substrates are absolutely necessary for effective porcine oocyte maturation, and that l-carnitine supplementation can only partially compensate for deficiencies in carbohydrate provision.

Effect of the hematopoietic growth factors erythropoietin and kit ligand on bovine oocyte in vitro maturation and developmental competence

The overall aim of this work was to study the influence of the hematopoietic growth factors erythropoietin (EPO) and kit ligand (KITL) during bovine oocyte in vitro maturation (IVM). The effect of adding different concentrations of EPO or KITL to maturation medium was evaluated analyzing oocyte nuclear maturation, cumulus cells apoptosis, embryo cleavage, reactive oxygen species (ROS) production in matured oocytes and cleaved embryos and the developmental competence to the blastocyst stage. No significant differences were observed in the percentage of oocytes that completed nuclear maturation among treatments, but the percentages of cleaved embryos and blastocysts obtained increased. With the addition of both hematopoietic growth factors the percentage of cumulus cells undergoing apoptosis decreased, the number of blastomeres per cleaved embryo was larger and ROS production per cleaved embryo increased. In conclusion, although the addition of EPO and KITL hematopoietic growth factors during bovine oocyte IVM had no impact on nuclear maturation, it had a positive effect on oocyte cytoplasmic maturation and developmental competence.

Heat waves impair cytoplasmic maturation of oocytes and preimplantation development in Korean native cattle (Hanwoo)

Heat waves impair cytoplasmic maturation of oocytes and preimplantation development in Korean native cattle (Hanwoo)

Is there a relation between the time to ICSI and the reproductive outcomes?

Does time to ICSI affect reproductive outcomes? Biochemical and clinical pregnancy diminish progressively as time between oocyte pick up (OPU) and ICSI increases after fresh embryo transfer. Appropriate oocyte cytoplasmic and nuclear maturation are of paramount importance to ensure an optimal embryonic developmental competence. While nuclear maturation is usually attained by the time an oocyte reaches OPU, cytoplasmic maturation cannot be readily assessed and might be incomplete. On the other hand, excessive in vitro culture of mature human oocytes can affect their ultrastructural characteristics and, in mice, induces alterations in gene expression and changes of chromatin and histone modification patterns. Retrospective consecutive cohort study including 1468 ICSI cycles carried out in a single center between December 2012 and September 2015. All cycles were with patient's own oocytes and fresh embryo transfer (ET). A radiofrequency-based system was used to record exact culture times, namely, OPU-denudation (DN); DN-ICSI and OPU-ICSI. We analyzed the effect of total and partial time intervals between procedures, from OPU to ICSI, on fertilization rate and biochemical, clinical, ongoing pregnancy and live birth rates. Differences in laboratory times between positive and negative biochemical, clinical, ongoing pregnancies and birth results were tested by Mann-Whitney U test. The likelihood of positive clinical outcomes was further modeled by locally weighted scatterplot smoothing (LOWESS) regression and logistic regression, adjusting for woman's age and BMI, number of transferred embryos; mean embryo morphological score, sperm origin and status, and number of mature oocytes obtained at OPU. Effect of time on fertilization rate was modeled by Generalized Linear Modeling (GLM) and LOWESS regression. The mean woman's age was 38.4 years (SD 4.6). Biochemical, clinical, ongoing pregnancy and live birth rates after the fresh ET were: 39.6, 33.1, 25.7 and 20.8%, respectively. Cumulative values for biochemical pregnancy and live birth were 46.4 and 26.3%, respectively. Mean times in hours for OPU-DN, DN-ICSI and OPU-ICSI were: 1.00 (SD 0.20); 3.86 (SD 1.93) and 4.87 (SD 1.96), respectively, and were not different for pregnant and non-pregnant patients. However, multivariate analyses showed that on average (anti-log transformed), each 1-h increase in the OPU-ICSI time reduced the likelihood of biochemical pregnancy by 7.3% (95% CI: 0.7-13.5%) and of clinical pregnancy by 7.7% (95% CI 0.8-14.1%), after the fresh ET. No effect of time was observed for ongoing pregnancy or live birth rates. Increasing OPU-ICSI time increased the fertilization rate (B = 0.052, 95% CI: 0.022, 0.082). The lack of relationship between incubation time of oocytes and live birth rates might be due to uncontrolled variables. Given the population analyzed, these results should not be extended to other ART protocols such as in vitro maturation of oocytes or classical IVF fertilization. This study indicates that in vitro ageing of mature oocytes significantly affects the chances to become pregnant. Effect on live birth rates, although not evident in this study, cannot be excluded. Limiting incubation time of mature oocytes in the embryology laboratory should improve reproductive results for patients using their own oocytes and with a transfer of fresh embryos. None. NA.

Effects of pre-incubation with C-type natriuretic peptide on nuclear maturation, mitochondrial behavior, and developmental competence of sheep oocytes

In vitro produced mammalian embryos suffer from developmental failure, with a large proportion showing embryonic retardation, degradation, or apoptosis. This failure is due, in part, to incomplete oocyte cytoplasmic maturation. C-type natriuretic peptide (CNP) has been reported to act as a meiotic inhibitor. Here we explored the potential effects of CNP pre-treatment sheep oocytes on nuclear maturation, changes in mitochondrial behavior and developmental competence of in vitro fertilized embryos. Sheep cumulus–oocyte complexes (COCs) were aspirated from abattoir-derived ovaries. Nuclear progression was assessed using DAPI chromatin staining, the expression of natriuretic peptide receptor 2 (NPR2) was evaluated by RT-qPCR, active mitochondria localization was assessed with a confocal laser scanning microscopy using MitoTracker Red, and the developmental competence of sheep oocytes subjected to one-step IVM or two-step IVM with or without CNP pretreatment was also investigated. Our results showed that 200 nM CNP could effectively maintain meiotic arrest of sheep COCs in vitro within 4 h. Furthermore, NPR2 mRNA was mainly expressed in cumulus cells. COCs pre-treated with 200 nM CNP for 4 h followed by 24 h IVM showed significantly higher (P < 0.05) cleavage rate and blastocyst rate after in vitro fertilization (IVF), and significantly lower (P < 0.05) proportion of DNA-fragmented nuclei in blastocysts when compared to the conventional 24 h IVM (standard IVM). Non-matured oocytes mainly displayed brilliant circumferential and fine diffuse distribution of mitochondria throughout the cytoplasm. By comparison, 200 nM CNP pre-treated COCs for 4 h led to cytoplasmic mitochondrial granule localization to the peripheral and perinuclear regions. Moreover, oocytes pre-treated with 200 nM CNP for 4 h followed by 24 h IVM, showed mitochondrial organization were similar to those of conventional 24 h matured oocytes in which mitochondria were aggregated more toward the cortical regions of the oocytes, but with larger clumps of stained mitochondria. These results indicate that CNP pre-treatment improves the quality and developmental competence of sheep oocytes and has great potential for facilitating in vitro embryo production.

Endoplasmic Stress Inhibition during Oocyte Maturation Improves Preimplantation Development of Cloned Pig Embryos

Mitochondrial dysfunction is found in oocytes and transmitted to offspring due to maternal obesity. Treatment of obese mothers with endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL) can reverse the mitochondrial dysfunction and result in normal embryonic development. Pig oocytes have also shown ER stress mostly in metaphase II stage. ER stress in oocytes may hinder the in vitro production of pig embryos. This study investigated the effect of ER stress inhibition by SAL treatment during in vitro maturation (IVM) of porcine oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we tested various concentrations of SAL. SAL at 10 nM showed higher (P < 0.05) developmental competence to the blastocyst stage (55.6%) after parthenogenesis (PA) than control (44.2%) while not different from other concentrations (49.2, 51.6, and 50.8% for 1, 50, and 100 nM, respectively). Secondly, we performed time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h of IVM significantly improved PA embryonic development to the blastocyst stage compared to control (40.5, 46.3, 51.7 and 60.2% for control, 0 to 22 h, 22 to 44 h and 0 to 44 h of IVM, respectively, P < 0.05). Glutathione (GSH) content is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on developmental competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decreased ROS level (P < 0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (39.6% vs. 24.7%, P < 0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development PA and cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

Oocyte Cytoplasmic Gas6 and Heparan Sulfate (HS) are Required to Establish the Open Chromatin State in Nuclei During Remodeling and Reprogramming

Background/Aims: Previously, we found that silencing of growth arrest-specific gene 6 (Gas6) in oocytes impaired cytoplasmic maturation, resulting in failure of sperm chromatin decondensation (SCD) and pronuclear (PN) formation after fertilization. Thus, we conducted this study to determine the effect of Gas6 RNAi on downstream genes and to elucidate the working mechanism of Gas6 on oocyte cytoplasmic maturation and SCD. Methods: Using RT-PCR, Western blot and immunofluorescence, the expression levels of various target genes and the localization of heparan sulfate (HS) were analyzed after Gas6 RNAi. The roles of Gas6 in HS biosynthesis, production of ATP and GSH, ROS generation and ΔΨm were also investigated. SCD and micrococcal nuclease (MNase) analyses were used to examine the effects of HS on the open chromatin state in sperm and somatic cell nuclei, respectively. Results: Disruption of Gas6 expression led to the inhibition of HS biosynthesis through the reduction of several HS biosynthetic enzymes. The rescue experiment, HS treatment in vitro, significantly recovered SCD and PN formation, confirming that HS had the ability to induce sperm head remodeling during fertilization. Interestingly, excessive mitochondrial activation in Gas6-depleted MII oocytes caused ROS generation and glutathione (GSH) degradation via mitochondrial activation, such as elevated ΔΨm and ATP production. Indeed, HS-treated NIH3T3 cell nuclei showed an open chromatin state, as determined by diffuse DAPI staining and increased sensitivity to MNase. Conclusion: We propose that the addition of HS to sperm and/or oocyte maturation would improve the efficiency of in vitro fertilization and somatic cell nuclear transfer (SCNT) reprogramming.

158 The Effect of Follicular Wave Phase at Time of Ovum Pick-Up on Bovine Oocyte Cytoplasmic Maturation and Developmental Competence

During oocyte collection, follicular wave phase is unknown, although differences in follicle environment may have dramatic effects on oocyte quality. This project was performed to determine whether oocyte collection during different phases of the follicular wave affects oocyte competence. Oocytes were collected via transvaginal ultrasound guided oocyte aspiration from 18 cows, at 4, 8, and 12 days following dominant follicle removal, representing follicle wave emergence, peak, and atresia, respectively (160, 314, and 273 oocytes, respectively). Once recovered, oocytes were graded and assigned to either being held as immature or matured in vitro for 24 h. Oocytes were then stained in Mitotracker deep red, fixed and stained with an anti-IP3R1 primary antibody and an Alexa Fluor 488-conjugated secondary antibody, before being stained with DAPI, to identify mitochondria, inositol triphosphate receptor 1 (IP3R1), and chromatin respectively. Mitochondria were analysed based on cytoplasmic distribution and classified as peripheral (immature), diffuse, central (mature), or sparse. Expression of IP3R1 was measured as corrected total cell fluorescence in Image J (National Institutes of Health, Bethesda, MD, USA). Staining patterns were analysed using ANOVA. A subset of the matured oocytes was stained with Fluo-3 to measure cytoplasmic calcium levels. These oocytes were then parthenogenetically activated before being imaged again to view changes in calcium levels, and presumptive embryos were cultured for 4 days. Fluo staining was measured as intensity levels (none, slight, moderate, high) and differences in development and stain levels were analysed using the Kruskal-Wallis test. Although mitochondria location was unaffected by collection day, it was significantly affected by maturation status (P = 0.0036). However, oocytes showed incomplete mitochondrial maturation, with mitochondria residing in the diffuse orientation in the majority of oocytes. Expression of IP3R1 appeared to be more sensitive to treatment. Expression significantly increased as meiosis proceeded (P = 0.0081) and there was a significant difference in expression between oocyte collection days (P = 0.0026). The interaction between collection day and maturation status also had a significant effect (P = 0.048), with mature oocytes showing an increase in IP3R1 expression, most notable in those collected on Day 4. Oocyte quality had a notable effect on the ability of oocytes to progress through meiosis (P = 0.054) and on mitochondrial location (P = 0.053), with AB oocytes showing better maturation parameters in both respects. Although the day of collection did not affect embryo development, Fluo stain intensity was an indicator of embryo developmental potential (P = 0.053), with oocytes having decreased potential to develop if the initial calcium levels were moderate to high. Results suggest that oocyte collection during wave emergence yields a slight advantage in oocyte quality. Although IP3R1, necessary for Ca2+ spikes during fertilization, indicates competence, high levels of cytoplasmic Ca2+ at the time of activation appear to be detrimental to embryo development.

Mechanisms by which in vitro meiotic arrest and sexual maturity improve developmental potential of mouse oocytes

To study the relationship between chromatin condensation, gene transcription and developmental competence during oocyte maturation and to explore the mechanisms by which meiotic arrest maintenance (MAM) and sexual maturity improve oocyte competence, we examined effects of MAM with roscovitine or db-cAMP on chromatin condensation, gene transcription and developmental potential of NSN or SN oocytes from prepubertal or adult mice. MAM with roscovitine improved the developmental competence and global gene transcription of prepubertal NSN (prep-NSN) and adult-SN oocytes while having no effect on those of prep-SN oocytes. MAM with db-cAMP facilitated neither development nor transcription in any type of oocytes. MAM with either roscovitine or db-cAMP promoted chromatin condensation of prep-NSN oocytes. MAM with roscovitine promoted gene transcription and chromatin condensation simultaneously through inhibiting cyclin-dependent kinase (CDK) 5 and 2, respectively. The results suggested that MAM with roscovitine improved oocyte competence by promoting gene transcription via inhibiting CDK5. Oocyte cytoplasmic maturation is correlated with gene transcription but not with chromatin condensation. The difference in developmental competence between prepubertal NSN and SN oocytes and between prepubertal and adult SN oocytes was because while the former had not, the latter had completed or acquired the ability for transcription of important genes.

Meiotic arrest with roscovitine and follicular fluid improves cytoplasmic maturation of porcine oocytes by promoting chromatin de-condensation and gene transcription

The developmental capacity of in vitro matured oocytes is inferior to that of the in vivo matured ones due to insufficient cytoplasmic maturation. Although great efforts were made to accomplish better cytoplasmic maturation by meiotic arrest maintenance (MAM) before in vitro maturation (IVM), limited progress has been achieved in various species. This study showed that MAM of porcine oocytes was better achieved with roscovitine than with dibutyryl cyclic adenosine monophosphate (db-cAMP) or 3-isobutyl-1-methylxanthine. Oocyte developmental competence after IVM was significantly improved following MAM in 199 + FF medium (TCM-199 containing 10% porcine follicular fluid and 25 µM roscovitine) to a level even higher than that in control oocytes matured without pre-MAM. Observations on other markers further confirmed the positive effects of MAM in 199 + FF on oocyte cytoplasmic maturation. During MAM culture in 199 + FF, re-decondensation (RDC) of condensed chromatin occurred, and transcription of genes beneficial to cytoplasmic maturation was evident in some of the oocytes with surrounded nucleoli (SN). However, MAM with db-cAMP neither induced RDC nor improved oocyte developmental potential. Together, the results suggest that MAM in the presence of FF and roscovitine improved the developmental competence of porcine oocytes by promoting a pre-GVBD chromatin de-condensation and expression of beneficial genes.

ID3018 Direct reprogramming of mouse somatic cells into pluripotent stem sells by pig germinal vesicle oocytes extract

Genomic reprogramming factors in the cytoplasm of mature oocytes could be reprogrammed somatic cell cells to totipotency cells and full-term development (cloned animals). Since then, this technique has been considered an important toot not only for animal reproduction but also for regenerative medicine, concervation of endangered species, and for study of genes function and cell biology. Moreover, in order to produce nuclear transfer embryonic stem (ntES) cells using somatic cell nuclear transfer (SCNT), the SCNT technique requires donated fresh oocytes, which raises ethical problems for production in human cloned embryo. For this reason, the use of induced pluripotent stem (iPS) cells for genomic reprogramming and for regenerative medicine is currently a hot topic in this field. However, the use of iPS cells for human therapy by the technique of retroviruses used to insert the pluripotent genes into somatic cells could cause tumors in tissues grown from the host iPS cells. Recently, we found that genomic reprogramming factors in the cytoplasm of pig germinal vesicle (GV)-stage oocytes has been shown to improve the efficiency of producing cloned mouse offspring and could reprogram pig fibroblasts to stem-like cells. In this talk, we will discuss whether pig GV cytoplasmic extract could induce pluripotent stem cells from mouse fibroblast cells (interspecies reprogramming). We first established stem-like cells from mouse fibroblast cells treated with GV oocytes extracted (gviPS cells). We demonstrated that reactivation of Oct4 promoter in mouse Oct4-GFP fibroblast cells at day 10 after treated with pig GV oocyte cytoplasmic extract and the formation of colonies is observed at 3 weeks after treatment. In addition, mouse gviPS cells reprogrammed with pig GV cytoplasmic extract can in vitro re-differentiate into neuron-like cells. Interestingly, mouse gviPS cells injected into embryos of different mouse strain could be produced chimeric mice.

Enriched endoplasmic reticulum-mitochondria interactions result in mitochondrial dysfunction and apoptosis in oocytes from obese mice

BackgroundMaternal obesity alters oocytes and subsequent fetal metabolism. An increasing number of studies have shown that the endoplasmic reticulums (ER) or mitochondria have important effects on oocyte quality, but there has been no study of the effect of mitochondria-associated ER membranes (MAMs) on oocyte quality. The present study was designed to assess whether the level of MAM and MAM-related proteins were different in oocytes from obese and control mice.ResultsFirst, oocytes from mice with high-fat-diet (HFD)-induced obesity had higher levels (either greater numbers or a higher proportion for the same numbers) of MAM than oocytes from control mice. The abundance of MAM-related proteins in oocytes from obese mice was significantly greater at both the messenger RNA and protein levels, including inositol 1,4,5-trisphosphate receptor, type 1 (IP3R1), inositol 1,4,5-trisphosphate receptor, type 2 (IP3R2) and phosphofurin acidic cluster sorting protein 2 (PACS-2). Further, there was an increase in mitochondrial Ca2+ ([Ca2+]m) which was associated with increased apoptosis and compromised cytoplasmic maturation in oocytes from obese mice. Down-regulation of MAM-related protein IP3R1 in oocytes from obese mice decreased [Ca2+]m and apoptosis and improved cytoplasmic maturation but did not reduce the overall MAM level. However, down-regulating MAM-related protein PACS-2 in oocytes from obese mice did reduce the level of MAM and [Ca2+]m, which decreased the rate of apoptosis and improved cytoplasmic maturation of oocytes from obese mice.ConclusionsIt is possible that enriched MAM could increase [Ca2+]m, and this increase has been found to be associated with increased apoptosis and compromised cytoplasmic maturation in oocytes from obese mice. This finding suggests a novel therapeutic target for obesity-induced oocyte defects.

Effect of urokinase type plasminogen activator on in vitro bovine oocyte maturation

This study examines the impacts of the urokinase-type plasminogen activator (uPA) on the in vitro maturation (IVM) of bovine oocytes. Cumulus-oocyte complexes in IVM medium were treated with uPA, amiloride (an uPA inhibitor), dimethyl sulfoxide (DMSO) or left untreated (control group). After 24 h of IVM, oocytes were recovered for testing or were in vitro fertilized and cultured to the blastocyst stage. The factors examined in all groups were: (i) oocyte nuclear maturation (Hoëscht staining); (ii) oocyte cytoplasmic maturation (cortical granules, CGs, distribution assessed by LCA-FITC); (iii) oocyte and cumulus cell (CC) gene expression (RT-qPCR); and (iv) embryo development (cleavage rate and blastocyst yield). Oocytes subjected to uPA treatment showed rates of nuclear maturation and CG distribution patterns similar to controls (P > 0.05), whereas lower rates of oocyte maturation were recorded in the amiloride group (P < 0.05). Both in oocytes and CC, treatment with uPA did not affect the transcription of genes related to apoptosis, cell junctions, cell cycle or serpin protease inhibitors. In contrast, amiloride altered the expression of genes associated with cell junctions, cell cycle, oxidative stress and CC serpins. No differences were observed between the control and uPA group in cleavage rate or in blastocyst yield recorded on Days 7, 8 or 9 post-insemination. However, amiloride led to drastically reduced cleavage rate (28.5% vs 83.2%) and Day 9 embryo production (6.0% vs 21.0%) over the rates recorded for DMSO. These results indicate that the proteolytic activity of uPA is needed for successful oocyte maturation in bovine.

"Bone Development" Is an Ontology Group Upregulated in Porcine Oocytes Before In Vitro Maturation: A Microarray Approach.

Mammalian cumulus-oocyte complexes (COCs) reach full developmental capability during folliculogenesis and oogenesis. It is well recognized that only gametes achieving MII stage after in vivo or in vitro maturation (IVM) are successfully fertilized by a single spermatozoon. Although the process of oocyte nuclear and/or cytoplasmic maturation in pigs is well determined, there exist many differences that promote these processes in vivo and in vitro. Therefore, this study aimed to investigate the differences in RNA expression profiles between porcine oocytes before and after IVM using microarray and real-time quantitative polymerase chain reaction (RT-qPCR) assays. Experiments were performed on oocytes isolated from 55 pubertal crossbred Landrace gilts. The oocytes were analyzed both before and after IVM and only Brilliant Cresyl Blue (BCB)-positive gametes were used for subsequent microarray analysis (Affymetrix) and RT-qPCR analysis. The microarray assay, which measures expression of 12,258 transcripts, revealed 419 differentially expressed transcripts in porcine oocytes, from which 379 were downregulated and 40 were upregulated before IVM compared to those analyzed after IVM. After DAVID analysis, we found eight different transcripts, including IHH, BMP1, WWTR1, CHRDL1, KLF10, EIF2AK3, MMP14, and STC1. Their expression is related to the "bone development" ontology group and was further subjected to hierarchical clusterization. Using RT-qPCR analysis, we confirmed the results of the microarray assay, showing increased expression of the eight genes in oocytes before IVM compared to oocytes after maturation in vitro. It has been suggested that "bone development" belongs to one ontological group involving genes substantially upregulated in porcine oocytes before IVM. We suggest that the gamete mRNA expression profile before IVM may comprise stored transcripts, which are templates for protein biosynthesis following fertilization. We also hypothesize that these mRNAs may be a specific "fingerprint" of folliculogenesis and oogenesis in pigs.

Effect of lipopolysaccharide on developmental competence of oocytes

In postpartum dairy cows, lipopolysaccharide (LPS) derived from gram-negative bacteria such as Escherichia coli causes uterine inflammation resulting in low fertility. The aim of this study was to determine the effect of LPS on the developmental competence of bovine oocytes in vitro. LPS perturbed the nuclear maturation of bovine oocytes by inhibiting meiotic progression. Although LPS did not affect the copy number of mitochondrial DNA, it decreased mitochondrial membrane potential in matured oocytes. LPS inhibited mitochondrial redistribution throughout the cytoplasm. Oocytes matured under LPS treatment showed decreased development to the blastocyst stage. Moreover, the trophoblast cell number of blastocysts was significantly lower when the oocytes were matured in the presence of LPS. Our findings suggest that LPS might impair the nuclear and cytoplasmic maturation of oocytes and obstruct subsequent embryonic development in dairy cows.

Zona pellucida gene mRNA expression in human oocytes is related to oocyte maturity, zona inner layer retardance and fertilization competence.

Do the mRNA expression levels of zona pellucida (ZP) genes, ZP1, 2, 3 and 4 in oocyte and cumulus cells (CC) reveal relevant information on the oocyte? The ZP mRNA expression in human oocytes is related to oocyte maturity, zona inner layer (IL) retardance and fertilization capacity. ZP structure and birefringence provide useful information on oocyte cytoplasmic maturation, developmental competence for embryonic growth, blastocyst formation and pregnancy. In order to understand the molecular basis of morphological changes in the ZP, in the current study, the polarized light microscopy (PLM) approach was combined with analysis of the expression of the genes encoding ZP1, 2, 3 and 4, both in the oocytes and in the surrounding CC. This is a retrospective study comprising 98 supernumerary human cumulus oocyte complexes (COC) [80 Metaphase II (MII), 10 Metaphase I (MI) and 8 germinal vesicle (GV)] obtained from 39 patients (median age 33.4 years, range 22-42) after controlled ovarian stimulation. Single oocytes and their correspondingCC were analysed. Oocytes were examined usingPLM, and quantitative RT-PCR was performed for ZP1, 2, 3 and 4 in these individual oocytes and their CC. Ephrin-B2 (EFNB2) mRNA was measured in CC as a control. Presence of ZP3 protein in CC and oocytes was investigated using immunocytochemistry. Data were analysed using one-parametric and multivariate analysis and were corrected for the potential impact of patient and cycle characteristics. Oocytes contained ZP1/2/3 and 4 mRNA while in CC only ZP3 was quantifiable. Also ZP3 protein was detected in human CC. When comparing mature (MII) and immature oocytes (MI/GV) or their corresponding CC, ZP1/2 and 4 expression was lower in mature oocytes compared to the expression in immature oocytes (all P < 0.05) and ZP3 expression was lower in the CC of mature oocytes compared to the expression in CC of immature oocytes (P < 0.05). This coincided with a significantly smallerIL-ZP area and thickness in mature oocytes than in immature oocytes (all P < 0.05). In mature oocytes, IL-ZP retardance was significantly correlated with the expression of all four ZP mRNAs (all P < 0.05). The oocyte ZP3 expression was the main predictor of the fertilization capacity, next to IL-retardance and IL-thickness. Using stepwise regression analysis, IL-thickness combined with EFNB2 expression in CC and the patient's ovarian response resulted in a noninvasive oocyte fertilization prediction model. Not applicable. This is a retrospective study and the relation of oocyte mRNA levels to fertilization capacity is indirect as oocyte gene expression analysis required lysis of the oocyte. Overall relations between PLM observations, mRNA expression changes and intrinsic oocyte competence were successfully documented. As such PLM and CC gene expression are confirmed as valuable noninvasive techniques to evaluate oocyte competence. This study was funded by University of Torino, Italy, WFWG UZ-Brussel and Agentschap voor Innovatie door Wetenschap en Technologie IWT 110680, Belgium. All authors declare that their participation in the study did not involve actual or potential conflicts of interests.

Human exhaled air can efficiently support in vitro maturation of porcine oocytes and subsequent early embryonic development.

Air phase is an indispensable environmental factor affecting oocyte maturation and early embryo development. Human exhaled air was previously proved to be a reliable and inexpensive atmosphere that sustains normal early development of mouse and bovine embryos. However, whether human exhaled air can support in vitro maturation (IVM) of porcine oocytes is not yet known. To evaluate the feasibility of maturing oocytes in human exhaled air, we examined oocyte morphology, BMP15 expression, nuclear and cytoplasmic maturation. We found that cumulus expansion status, expression levels of BMP15 important for cumulus expansion and the rate of first polar body emission were similar among human exhaled air, 5% O2 or 20% O2 in air after IVM of 44 h. Furthermore, the percentage of metaphase II (MII) oocytes showing normal cortical and sub-membranous localization of cortical granules and diffused mitochondrial distribution patterns is comparable among groups. The cleavage, blastocyst rate and total cell number were not apparently different for parthenogenetic activated and somatic cloned embryos derived from MII oocytes matured in three air phases, suggesting oocytes matured in human exhaled air obtain normal developmental competence. Taken together, human exhaled air can efficiently support in vitro maturation of porcine oocytes and subsequent early embryonic development.

The Role of Glucose Metabolism on Porcine Oocyte Cytoplasmic Maturation and Its Possible Mechanisms.

In the present study, we investigated the potential role of glucose and pyruvate in the cytoplasmic maturation of porcine oocytes by investigating the effect of glucose and/or pyruvate supplementation, in the presence or absence of 10% porcine follicular fluid (PFF), on meiotic maturation and subsequent embryo development. In the absence of 10% PFF, without exogenous addition of glucose and pyruvate, the medium seemed unable to support maturation. In the presence of 10% PFF, the addition of 5.6 mM glucose and/or 2 mM pyruvate during in vitro maturation of cumulus enclosed oocytes increased MII oocyte and blastocyst rates. In contrast, oocytes denuded of cumulus cells were not able to take full advantage of the glucose in the medium, as only pyruvate was able to increase the MII rate and the subsequent early embryo developmental ability. Treatment of cumulus enclosed oocytes undergoing maturation with 200 μM dehydroepiandrosterone (DHEA), a pentose phosphate pathway inhibitor, or 2 μM iodoacetate (IA), a glycolysis inhibitor, significantly reduced GHS, intra-oocyte ATP, maternal gene expression, and MPF activity levels. DHEA was also able to increase ROS and reduce the levels of NADPH. Moreover, blastocysts of the DHEA- or IA-treated groups presented higher apoptosis rates and markedly lower cell proliferation cell rates than those of the non-treated group. In conclusion, our results suggest that oocytes maturing in the presence of 10% PFF can make full use of energy sources through glucose metabolism only when they are accompanied by cumulus cells, and that pentose phosphate pathway (PPP) and glycolysis promote porcine oocyte cytoplasmic maturation by supplying energy, regulating maternal gene expression, and controlling MPF activity.

Progesterone influences cytoplasmic maturation in porcine oocytes developing in vitro.

Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and development in vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p < 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p < 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression.