Oocyte Nuclear Maturation Research Articles

Effect of Monosodium Glutamate on In vitro Oocyte Maturation and Embryonic Development after Parthenogenesis in Pigs

This study was designed to determine the effect of monosodium glutamate (MSG) on in vitro maturation (IVM) of oocytes and early development of parthenogenesis (PA) embryos in pigs. Each IVM and IVC medium was supplemented with various concentrations (0, 0.1, 0.5 and 5 mM) of MSG and non-essential amino acids (NEAA) depending on the experimental design. Immature pig oocytes were matured for 44 h and then oocytes reached metaphase II (MII) stage were electrically activated to induce parthenogenesis (PA). When immature oocytes were treated with MSG in the absence of NEAA during IVM, nuclear maturation (83.1-87.1%), intra-oocyte glutathione content, cumulus expansion, and cleavage (91.4-93.4%) of PA embryos were not influenced by MSG treatment at all concentrations. However, blastocyst formation of PA embryos was significantly increased by 5.0 mM MSG (45.3 ± 6.2%) compared to control (25.6 ± 3.4%). MSG treatment during IVM in the presence of NEAA did not show significant effect on nuclear maturation of oocytes and blastocyst formation after PA while 0.5 mM MSG (89.3 ± 1.9%) decreased (P < 0.05) cleavage of PA embryos compared to 0.1 mM MSG (94.6 ± 1.1%). When PA embryos were treated for 7 days with MSG during IVC, 5.0 mM MSG significantly decreased blastocyst formation (27.8 ± 4.9%) compared to no treatment (41.4 ± 1.9%) while no decrease in blastocyst formation was observed in 0.1 and 0.5 mM (37.4 ± 3.4% and 34.4 ± 2.6%, respectively). Our results demonstrated that 5 mM MSG in a NEAA-free chemically defined maturation medium showed positive effect on PA embryonic development while 5 mM MSG treatment during IVC was deleterious to PA embryonic development in pigs.

Effects of insulin-like growth factor-1 on the in vitro maturation of canine oocytes.

The maturation rate of canine oocytes during in vitro maturation (IVM) needs to be improved. The present study was designed to evaluate the effects of insulin-like growth factor-1 (IGF-1) on the IVM of canine oocytes. Ovaries were obtained by ovariohysterectomy and were sliced to release cumulus-oocyte complexes (COCs). In Experiment 1, the effects of different concentrations of IGF-1 on the nuclear maturation of oocytes was investigated. The COCs were cultured in a modified medium (mTCM199) with IGF-1 (0, 0.5, 5, 10, and 50 µg/ml). At the end of the 48 h culture, oocytes were fixed and stained to evaluate their nuclear stage. Supplementation with 50 µg/ml IGF-1 induced a significantly higher metaphase II (MII) rate (P < 0.05) compared to the 0 and 0.5 μg/ml IGF-1 groups. In Experiment 2, the expression levels of insulin receptor (INSR), IGF-1 receptor (IGF-1R), and IGF-2 receptor (IGF-2R) genes, localized to canine oocytes and cumulus cells, were investigated before and after IVM. The expression level of IGF-1R in cumulus cells after IVM was higher than that before IVM (P < 0.05). In Experiment 3, it was investigated whether an inhibitor of PTEN (phosphatase and tensin homolog), bpV, affects the nuclear maturation of oocytes. Regardless of bpV supplementation at a concentration of 0.2 to 200 µmol/l, there was no significant difference in the proportion of oocytes that reached the MII stage. These results indicated that IGF-1 has a favorable effect on the IVM of canine oocytes, possibly through the stimulation of the Ras/MAPK pathway via IGF-1R expressed in cumulus cells.

167 Effect of Storage Time and Temperature of a Commercial Embryo Holding Medium on the Maturation Kinetics of Immature Bovine Oocytes

The effect of holding time and temperature during storage of immature bovine oocytes in a commercial embryo holding medium (EHM; Syngro® Ltd., Livingston, United Kingdom) was evaluated. Ovaries were collected at the local slaughterhouse and processed within 2 h. Cumulus-oocyte complexes (COC) were collected and allocated to groups of 60. The COC were held in 1-mL sterile glass osmometer tubes, filled to the top with the EHM to limit the amount of air. Vials were capped and covered with parafilm to ensure a tight seal and prevent leakage. Tubes were stored for 6 h at 4°C, room temperature (RT), or 38.5°C; for 10 h at 4°C and RT; and for 14 h at RT. Next, oocytes were fixed after storage in EHM (immature holding) or fixed after being held in EHM and subsequent 22-h maturation at 38.5°C in 5% CO2 in humidified air (mature holding). Maturation medium consisted of modified bicarbonate-buffered TCM-199 supplemented with gentamycin and epidermal growth factor. During all experiments, a control group was included each time. The control consisted of groups of 60 COC immediately fixed after collection or transferred to maturation medium for 22 h and subsequently fixed. Nuclear maturation of oocytes was assessed after Hoechst 33342 staining, using a 400× magnification fluorescence microscope. A total of 3043 COC were evaluated in 3 replicates. Oocytes maturation stages were classified as (1) oocytes in germinal vesicle stage, (2) oocytes in meiotic progression (diakinesis, metaphase I, or anaphase), (3) matured (telophase I or metaphase II), and (4) degenerated (degraded chromatin). Oocytes remained at the germinal vesicle stage when held in EHM (without subsequent maturation) regardless of holding time and temperature (P &gt; 0.05). When oocytes were held for 6 h and subsequently matured (Table 1), the number of matured oocytes was significantly lower for oocytes held at 38.5°C compared with the other groups (control, RT, and 4°C). When held for 10 h, the oocyte maturation rate was similar between the control and RT groups (P &gt; 0.05), but it was significantly lower in oocytes held at 4°C. Last, when compared with oocytes held at RT for 14 h, the maturation rate was higher in the control group (P &lt; 0.05). To conclude, immature bovine oocytes can be successfully held in EHM at RT for up to 10 h. Storing immature oocytes in EHM can delay oocyte maturation and concomitantly synchronize maturation. Table 1.Kinetics of cumulus-oocyte complex nuclear status after storage in embryo holding medium for different times and temperatures and subsequent 22-h maturation

172 Effect of Melatonin on Meiosis Progression and Lipid Content in Bovine Oocytes Matured In Vitro

Melatonin (MLT) may have positive effects on oocyte nuclear maturation and may influence their lipid metabolism. The aim of this study was to assess the effect of MLT on nuclear maturation and lipid content inbovine oocytes during in vitro maturation (IVM). Cumulus-oocyte complexes (30-35/group) were submitted to IVM in TCM-199 supplemented with 3 mg mL−1 BSA (negative control; NC) or with added hormones: 1 µg mL−1 FSH (positive control; PC) or MLT (10−11, 10−9 and 10−7 M), to evaluate the individual action of each treatment on meiosis resumption (metaphase I, MI) at 9 h and progression to metaphase II (MII) at 24 h IVM. Lipid content in oocytes was also assessed. Oocytes were denuded and stained with Hoechst 33342 and Nile Red, and evaluated by epifluorescence microscopy to determine the nuclear maturation stage and lipid content, respectively. The fluorescence intensity (FI) was measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA). Data were analysed by ANOVA followed by Tukey’s test to compare effects of treatments on meiosis resumption and lipid contents at 9 h and on meiosis progression and lipid contents at 24 h; and t-test to compare the same treatment at 9 and 24 h IVM regarding lipid contents (4 replicates/treatment). Significance level was 5% (GraphPrism software; GraphPad Inc., San Diego, CA, USA). At 9 h IVM, meiosis resumption rate (MI) increased (P &lt; 0.05) for MLT 10−9 M (32.6 ± 14.8%, 45/138) and FSH groups (33.9 ± 18.1%, 38/112) compared with NC (4.2 ± 2.7%, 5/120). The other treatments did not differ in relation to NC (20.5 ± 12.5%, 27/132 and 14.9 ± 9.0%, 20/134 to MLT 10−11 and 10−7 M, respectively). Regarding lipid contents at 9 h, MLT 10−11 M showed higher lipid content (8.43 ± 7.61FI; P &lt; 0.0001) compared with other treatments [MLT 10−9 M (5.59 ± 4.13), MLT 10−7 M (4.87 ± 3.21), FSH (5.34 ± 3.66), and NC (4.27 ± 3.28)]. Maturation rate (MII) at 24 h IVM was highest in FSH group (70.1 ± 10.2%, 94/134). No differences were observed (P &gt; 0.05) between groups matured in different concentrations of MLT (65.9 ± 4.2%, 83/126; 62.2 ± 0.6%, 56/90 and 53.5 ± 10.0%, 61/114 for MLT 10−11, 10−9 and 10−7 M, respectively) compared with NC (86.3 ± 1.2%, 88/102; P &lt; 0.05). Similarly, lipid content at 24 h IVM was higher (P &lt; 0.05) in FSH group (10.72 ± 4.71 FI) compared with NC (7.98 ± 3.45 FI) or MLT treatments with 10−11 M (8.90 ± 3.91), 10−9 M (7.85 ± 3.56), and 10−7 M (8.29 ± 3.87). However, MLT 10−7 M, FSH, and NC groups had higher (P &lt; 0.0001) lipid content at 24 than at 9 h. In conclusion, MLT 10−9 M alone was able to stimulate meiosis resumption (9 h) at a rate similar to FSH, but was insufficient to stimulate progression to MII at 24 h, indicating that it may affect nuclear maturation only during the initial steps of meiosis. Lipid contents were increased during IVM but for lower MLT concentrations, this increase was not observed. Whether these observations have any impact on oocyte quality remains to be determined. Further studies are necessary to improve our knowledge of the role of MLT on lipid metabolism and by which mechanism it may affect meiosis resumption in bovine oocytes. This research was funded by FAPESP (HF- 2016/24884-3, scholarship; CLVL, financial support 2015/20379-0).

162 Supplementation of Fibroblast Growth Factor 2, Leukemia Inhibitory Factor, and Insulin-Like Growth Factor 1 (FLI) Enables Efficient Production of Competent Porcine Oocytes Without Gonadotropins in the Maturation Medium

In vitro maturation (IVM) of oocytes is a critical step in assisted reproductive technologies carried out in species such as cattle and swine, for generating oocytes capable of being fertilized in vitro and providing healthy offspring useful for biomedical and agricultural purposes. Cumulus-oocyte complexes (COC) collected from abattoir ovaries for IVM often respond poorly to gonadotropins, resulting in compromised oocyte competence. Our previous work demonstrated that the combination of fibroblast growth factor 2, leukemia inhibitory factor, and insulin-like growth factor 1 (FGF2, LIF, and IGF1; FLI) significantly improved nuclear maturation of porcine oocytes and their developmental competence. However, it is unclear whether the benefits of FLI are mediated through increased gonadotropin sensitivity of COC or improved downstream signalling. Here we investigated the effect of FLI supplementation of IVM medium with and without gonadotropins. The COC, collected from 2- to 6-mm follicles from prepubertal ovaries, were matured in 5% CO2/air at 38.5°C for 42 h in chemically defined TCM-199 medium (supplemented with 0.1% polyvinyl alcohol, 3.05 mM d-glucose, 0.91 mM pyruvate, 0.57 mM cysteine, 10 ng mL−1 epidermal growth factor), with or without FLI (40 ng mL−1 FGF2, 20 ng mL−1 LIF, and 20 ng mL−1 IGF1), and with or without gonadotropins (0.5 μg mL−1 LH, 0.5 μg mL−1 FSH), in a 2 × 2 factorial design experiment. After IVM, oocytes were fertilized in vitro and cultured under standard conditions until Day 6 when blastocyst formation was assessed. The experiment was replicated 4 times with a total of 792 oocytes. Percentage data were arcsin transformed and analysed by ANOVA to detect differences (significance, P &lt; 0.05). When FLI was absent from the maturation medium, oocytes matured in presence of gonadotropins demonstrated improved nuclear maturation (67.0 ± 2.2% v. 52.7 ± 5.3%) and blastocyst formation (23.7 ± 3.3% v. 10.2 ± 2.3%) relative to oocytes matured without gonadotropins, respectively. However, when FLI was present in the medium, the extent of oocyte maturation and subsequent blastocyst development was unaffected by gonadotrophin addition (maturation: 84.2 ± 1.8% with and 79.2 ± 3.4% without; blastocyst formation: 29.5 ± 4.3% with and 24.9 ± 4.3% without). In summary, these results suggest that FLI, rather than enhancing COC sensitivity to gonadotrophins, may activate certain downstream signalling pathways that are normally controlled by gonadotropins during IVM, thereby enhancing oocyte quality. Therefore, FLI appears able to substitute for gonadotropins in the maturation medium and supports porcine oocyte competence in the absence of FSH and LH. These observations will help us better understand the mechanisms whereby FLI enhances oocyte nuclear maturation and improves developmental competency. Supported by NIH R01HD69979, U42OD011140, and University of Missouri Food for the 21st Century Program.

The CXCL12-CXCR4 signaling promotes oocyte maturation by regulating cumulus expansion in sheep

Gonadotropins and growth factors synergistically regulate folliculogenesis and oocyte development. C-X-C motif chemokine 12 (CXCL12) and its receptor CXCR4 are expressed in ovaries of sheep, cattle and other species, however, roles of this multifunctional signal axis in oocyte maturation are not defined. Using sheep as a model, we examined the expression patterns and functions of the CXCL12-CXCR4 axis during oocyte maturation. CXCL12 and CXCR4 mRNA and protein were present in oocytes and granulosa cells. Relative abundance of CXCR4 transcript was controlled by epidermal growth factor (EGF). Transient inhibition of CXCR4 suppressed oocyte nuclear maturation while supplementing recombination CXCL12 significantly increased percent of oocyte undergone metaphase I phase. Inhibition of CXCR4 function decreased cumulus expansion growth rate. Furthermore, granulosa cell migration was impaired and expression of hyaluronan synthase 2 (HAS2) and hyaluronan binding protein tumor necrosis factor-alpha-induced protein 6 (TNFAIP6) were downregulated by CXCR4 inhibition. These findings revealed a novel role of the CXCL12-CXCR4 signaling in oocyte development in sheep.

Effect of season on follicular population, quality and nuclear maturation of bovine oocytes under tropical conditions

The aim was to determine the effect of season of the year and the presence of a corpus luteum (CL) on follicular population (FP) and the quality of the oocytes, and of season on nuclear maturation of the bovine oocytes under tropical conditions. Three seasons were evaluated: hot-dry (March–June), hot-humid (July–October) and fresh-humid (November–February). In a first study, 1112 bovine ovaries were obtained from a local slaughterhouse. Follicles were classified as small (≤4mm), middle (4.1–8mm) and large (≥8.1mm); and the maximum diameter of the follicle (MDF) and CL (MDCL) were also recorded. The oocytes were collected by aspiration and classified as viable (grade I and II) and damaged (grade III and IV). In the second study, 2261 viable oocytes were matured in vitro, and then fixed and stained with Lacmoid to classify the stage of development as mature (metaphase II), immature or degenerate. Data were analyzed using analysis of variance and chi-square procedures. The largest FP of large follicles (0.67), MDF (1.18mm), MDCL (1.87mm), and the highest proportion of viable oocytes (34.19%) were obtained during the hot-humid season (P<0.05). The ovaries without CL had the greatest FP (10.34) with more viable oocytes (24.44%). The highest proportion of mature oocytes (76.92%) was also obtained in the hot-humid season. In conclusion, season influenced FP, MDF, MDCL, and the quality and nuclear maturation of oocytes. The presence of a CL in the ovary resulted in a decrease of FP and viability of oocytes.

The presence of acylated ghrelin during in vitro maturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development.

The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus-oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

Proportional oocyte nuclear maturation in relation to ICSI outcome

Proportional oocyte nuclear maturation in relation to ICSI outcome

An improved IVM method for cumulus-oocyte complexes from small follicles in polycystic ovary syndrome patients enhances oocyte competence and embryo yield.

Are meiotic and developmental competence of human oocytes from small (2-8 mm) antral follicles improved by applying an optimized IVM method involving a prematuration step in presence of C-Type Natriuretic Peptide (CNP) followed by a maturation step in presence of FSH and Amphiregulin (AREG)? A strategy involving prematuration culture (PMC) in the presence of CNP followed by IVM using FSH + AREG increases oocyte maturation potential leading to a higher availability of Day 3 embryos and good-quality blastocysts for single embryo transfer. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for the patients, but the approach is not widely used because of an efficiency gap compared to conventional ART. In vitro systems that enhance synchronization of nuclear and cytoplasmic maturation before the meiotic trigger are crucial to optimize human IVM systems. However, previous PMC attempts have failed in sustaining cumulus-oocyte connections throughout the culture period, which prohibited a normal cumulus-oocyte communication and precluded an adequate response by the cumulus-oocyte complex (COC) to the meiotic trigger. A first prospective study involved sibling oocytes from a group of 15 patients with polycystic ovary syndrome (PCOS) to evaluate effects of a new IVM culture method on oocyte nuclear maturation and their downstream developmental competence. A second prospective study in an additional series of 15 women with polycystic ovaries characterized and fine-tuned the culture conditions. Fifteen women with PCOS (according to Rotterdam criteria) underwent IVM treatment after 3-5 days of highly purified human menopausal gonadotropin (HP-hMG) stimulation and no human chorionic gonadotropin (hCG) trigger before oocyte retrieval. A first study was designed with sibling oocytes to prospectively evaluate the impact of an IVM culture method: 24 h PMC with CNP + 30 h IVM with FSH and AREG, on embryo yield, in comparison to the standard (30 h) IVM clinical protocol (Group I, n = 15). A second prospective study was performed in 15 women with polycystic ovaries, to characterize and optimize the PMC conditions (Group II, n = 15). The latter study involved the evaluation of oocyte meiotic arrest, the preservation of cumulus-oocyte transzonal projections (TZPs), the patterns of oocyte chromatin configuration and cumulus cells apoptosis following the 24 and 46 h PMC. Furthermore, oocyte developmental potential following PMC (24 and 46 h) + IVM was also evaluated. The first 20 good-quality blastocysts from PMC followed by IVM were analysed by next generation sequencing to evaluate their aneuploidy rate. PMC in presence of CNP followed by IVM using FSH and AREG increased the meiotic maturation rate per COC to 70%, which is significantly higher than routine standard IVM (49%; P ≤ 0.001). Hence, with the new system the proportion of COCs yielding transferable Day 3 embryos and good-quality blastocysts increased compared to routine standard IVM (from 23 to 43%; P ≤ 0.001 and from 8 to 18%; P ≤ 0.01, respectively). CNP was able to prevent meiosis resumption for up to 46 h. After PMC, COCs had preserved cumulus-oocyte TZPs. The blastocysts obtained after PMC + IVM did not show increased aneuploidy rates as compared to blastocysts from conventional ART. The novel IVM approach in PCOS patients was tested in oocytes derived from small antral follicles which have an intrinsically low developmental potential. Validation of the system would be required for COCs from different (larger) follicular sizes, which may involve further adjustment of PMC conditions. Furthermore, considering that this is a novel strategy in human IVM treatment, its global efficiency needs to be confirmed in large prospective randomized controlled trials. The further application in infertile patients without PCOS, e.g. cancer patients, remains to be evaluated. The findings of this pilot study suggest that the efficiency gap between IVM and conventional IVF can be reduced by fine-tuning of the culture methods. This novel strategy opens new perspectives for safe and patient-friendly ART in patients with PCOS. IVM research at the Vrije Universiteit Brussel has been supported by grants from: the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680); the Fund for Research Flanders (Fonds Wetenschappelijk Onderzoek-Vlaanderen-FWO, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69). The authors have no conflicts of interest.

Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in goat ovary and its effect on oocyte nuclear maturation in vitro

Adiponectin is an adipokine secreted primarily by adipocytes and is involved in the control of male and female reproductive functions. Circulating levels of adiponectin are inversely correlated with body fat mass, and its biological effects are predominantly mediated through two receptors, AdipoR1 and AdipoR2. The aim of the present study was to verify the expression of the adiponectin system (adiponectin and its receptors, AdipoR1 and AdipoR2) in goat ovary using qPCR and immunohistochemistry analyses and further investigate the in vitro effects of recombinant adiponectin (5 μg/mL and 10 μg/mL) on goat oocyte nuclear maturation. We demonstrated that the mRNA and proteins of the adiponectin system are present in goat ovary. Gene and protein expression of AdipoR1 and AdipoR2 was detected in follicular cells (oocyte, cumulus, granulosa and theca) of small and large antral follicles, while adiponectin mRNA was not detected in oocytes from small and large follicles or in large follicle cumulus cells. Finally, addition of various concentrations of adiponectin in maturation medium affected the number of oocytes that reached metaphase II. In conclusion, in the present study, we detected expression of adiponectin and its receptors AdipoR1 and AdipoR2 in goat ovarian follicles. Furthermore, we demonstrated that recombinant adiponectin increases nuclear maturation of goat oocytes in vitro.

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.

Treatment of Epidermal Growth Factor (EGF) enhances Nuclear Maturation of Porcine Oocytes and Stimulates Expression of ER/Golgi Transport Proteins.

ABSTRACTThis study was conducted to investigate stimulatory effect of epidermal growth factor (EGF) on nuclear maturation and the expression level of EGF-receptor (EGFR), GM-130 (a marker of Golgi apparatus), transport protein Sec61 subunit beta (Sec61β), and coatomer protein complex subunit gamma 2 (COPG2) in porcine oocytes. The cumulus-oocyte complexes were collected from follicle with 3-6 mm in diameter. They were incubated in medium with/without EGF for 22 h (IVMⅠ) and subsequently incubated hormone-free medium with/without EGF for 22 h (IVMⅡ). Nuclear maturation state was checked by aceto-orcein stain. Protein expression of EGFR, GM-130, Sec61β, and COPG2 were measured by immunofluorescence. In results, nuclear maturation of oocytes in EGF non-treated oocytes were significantly lower than EGF-treated groups at IVMⅠ or IVMⅡ stage (P<0.05), whereas maturational rate in EGF treatment groups at both of IVM stage was higher in among the all treatment groups (P<0.05). EGFR, GM-130, Sec61β and COPG2 were expressed in the cytoplasm of oocytes. Especially, GM-130 and EGFR were strongly expressed, but Sec61β and COPG2 were weakly expressed in cortical area of cytoplasm. The protein level of GM-130, Sec61β, and COPG2 were significantly higher in the EGF-treated groups (P<0.05). However EGFR was no difference between non EGF-treated groups and control. In conclusion, EGF plays an important role in the systems for oocyte maturation with endoplasmic reticulum and Golgi apparatus. In addition, the protein levels of Sec61β and COPG2 could be changed by EGF in the porcine oocytes during maturation.

Effect of Alpha-Linolenic Acid on Oocyte Maturation and Embryo Development in Pigs.

ABSTRACTThe aim of this study was to determine the effect of additional alpha-linolenic acid (ALA) supplementation during in vitro maturation (IVM) and culture (IVC) on nucleic maturation and embryo development of pigs. Cumulus-oocyte complexes (COCs) were incubated in IVM medium containing different concentration of ALA (25, 50 and 100 μM) for 44 h. After in vitro maturation, nuclear maturation of oocytes were evaluated by aceto-orcein stain. Mature oocytes with 50 μM ALA were fertilized and cultured in IVC medium with ALA (25, 50 and 100 μM) during early-embryogenesis (48 hours after fertilization). Then, embryos were cultured with 25 μM ALA during early embryogenesis and/or late embryogenesis (120 hours after early-embryogenesis). In results, oocyte maturation were significantly increased by 50 μM ALA treatment groups compared with control groups (p<0.05). Treatment of 25 μM ALA during early-embryogenesis enhanced cleavage rate of embryo compared with other groups (p<0.05), whereas formation and total cell number of blastocyst had no significant difference. Similarly, cleavage rate of embryos were increased by 25 μM ALA supplement during early- or late-embryogenesis than ALA treatment both stage of embryogenesis (p<0.05), but did not influence to blastocyst formation. Interestingly, total cell number of blastocyst were enhanced in ALA treatment group during early-embryogenesis. These findings indicated that ALA supplement enhance the nuclear maturation of oocyte and embryo development, however, excessive ALA could negatively influence. Therefore, we suggest that ALA is used for improvement of in vitro production of mammalian embryo and further study regarding with functional mechanism of ALA is needed.

Effect of women's age on embryo morphology, cleavage rate and competence-A multicenter cohort study.

This multicenter cohort study on embryo assessment and outcome data from 11,744 IVF/ICSI cycles with 104,830 oocytes and 42,074 embryos, presents the effect of women’s age on oocyte, zygote, embryo morphology and cleavage parameters, as well as cycle outcome measures corrected for confounding factors as center, partner’s age and referral diagnosis. Cycle outcome data confirmed the well-known effect of women’s age. Oocyte nuclear maturation and proportion of 2 pro-nuclear (2PN) zygotes were not affected by age, while a significant increase in 3PN zygotes was observed in both IVF and ICSI (p<0.0001) with increasing age. Maternal age had no effect on cleavage parameters or on the morphology of the embryo day 2 post insemination. Interestingly, initial hCG value after single embryo transfer followed by ongoing pregnancy was increased with age in both IVF (p = 0.007) and ICSI (p = 0.001) cycles. For the first time, we show that a woman’s age does impose a significant footprint on early embryo morphological development (3PN). In addition, the developmentally competent embryos were associated with increased initial hCG values as the age of the women increased. Further studies are needed to elucidate, if this increase in initial hCG value with advancing maternal age is connected to the embryo or the uterus.

Insulin-Like Growth Factor-I Concentration in the Follicular Fluid of Bali Cattle and Its Role in the Oocyte Nuclear Maturation and Fertilization Rate

The objective of this study was to determine the concentration of IGF-I in the follicular fluid (FF) of Bali cattle and its role in the nuclear maturation and fertilization rate. The follicular fluid was collected by the aspiration technique, then it was centrifuged at 1500 g for 30 min at 24oC. The supernatant was collected and stored at -20oC until being used in the experiment for analysis of IGF-1. A total of 1105 oocytes were used in this study. The oocytes were matured in M199 without supplementation of bovine serum albumin, with supplementation of BSA, and with supplementations of 10% FF (v/v) from the follicle with diameter O<4 mm, 4≤O<6 mm, 6≤O<8 mm, and O≥8 mm at the luteal phase and then fertilized. The results showed that the concentrations of IGF-I in the FF obtained during the luteal phase was significantly higher (P<0.05) compared to those obtained during follicular phase. The IGF-I concentrations in the follicular fluid of follicle with diameter smaller than 6 mm were significantly higher (P<0.05) compared to those with diameters larger than 6 mm. The percentage of nuclear maturation rate of oocytes cultured with FF obtained from follicle with diameter <4 mm was significantly higher (P<0.05) compared to those obtained from the other groups of follicle diameters. The supplementation of maturation media with BSA and FF were able to improve fertilization rate significantly (P<0.05) compared to maturation media without BSA. In conclusion, the concentration of IGF-I in the follicular fluid obtained during the luteal phase was higher compared to those obtained during the follicular phase. The IGF-I concentrations in the follicular fluid of smaller follicles (diameter <6 mm) were higher compared to those in the large follicles (diameter ≥6 mm). The supplementation of FF can improve the nuclear maturation and fertilization rate

Effect of eicosapentaenoic acid on bovine cumulus-oocyte complex in vitro.

The aim of the present study was to investigate the effects of eicosapentaenoic acid (EPA) supplementation during in vitro maturation (IVM) of bovine oocytes. The concentrations tested in all experiments were 1 nM, 1 μM, and 1 mM EPA. The effect of EPA was evaluated on cumulus-oocyte complexes (COC) by oocyte maturation (cumulus expansion area and oocyte nuclear maturation), genotoxicity [single cell gel electrophoresis (SCGE)], and cytotoxicity (apoptosis, viability, and MTT assays) end points. The maturation parameters were affected by exposure of COC to different EPA concentrations in the IVM medium. Cumulus expansion area increased in the presence of 1 nM EPA (P < 0.05) whereas addition of 1 nM EPA (P < 0.05) decreased cumulus expansion after 24 h of IVM. Moreover, the maturation rate significantly decreased when 1 mM of EPA was assayed (P < 0.001). EPA at 1 nM induced genotoxic and cytotoxic effects on bovine cumulus cells (CC) and primary DNA lesions (P < 0.001). A significant increase in the frequency of apoptotic (P < 0.01) and necrotic (P < 0.001) cells was observed after 24 h of treatment with 1 nM, 1 μM, and 1 mM EPA. Mitochondrial activity was altered with 1 mM EPA (P < 0.001). We inferred that optimal oocyte quality was partially dependent on the presence of adequate EPA concentrations; EPA could be beneficial to improve oocyte quality in the maturation process, because low concentration tested (1 nM EPA) improved cumulus expansion.

Research Article: Effects of endocrine-disrupting chemical exposure on zebrafish ovarian follicles

The process of oocyte maturation involves both nuclear and cytoplasmic events that are essential for the production of viable eggs and subsequent fertilization in sexually reproducing animals. In zebrafish, oocyte nuclear maturation (meiotic resumption) is marked by a transition from opaque to translucent ovarian follicles. This transition is called clearing and results from germinal vesicle breakdown (GVBD) and the transformation of vitellogenin into yolk proteins. Oocyte maturation can be perturbed by endocrine-disrupting chemicals (EDCs). These EDCs have been broadly shown to interfere with reproductive development. Many herbicides and pesticides used agriculturally act as EDCs. These chemicals can enter aquatic ecosystems via runoff and erosion. To evaluate the effects of EDCs on zebrafish oocyte maturation, zebrafish ovarian follicles were cultured and exposed to various concentrations of either atrazine, 2,4-D, or diazinon. Follicles were evaluated at 2, 3, and 4 hours for size and viability, but clearing was assessed only at 4 hours post-exposure. Atrazine and diazinon significantly reduced follicle clearing at the tested concentrations. No effect was observed with 2,4-D exposure. At low enough concentrations, none of the chemicals produced endocrine-disrupting effects. The results suggest that at or above environmentally relevant concentrations, some EDCs impair oocyte maturation in exposed zebrafish follicles. The results support the need to monitor EDC exposure to prevent harmful effects not only on aquatic organisms, but also on humans and wildlife that are also exposed to these contaminants.

Steroid hormones interact with natriuretic peptide C to delay nuclear maturation, to maintain oocyte-cumulus communication and to improve the quality of in vitro-produced embryos in cattle.

In vivo, oocyte maturation is triggered by the ovulatory LH surge, whereas in vitro it is precociously induced when the cumulus-oocyte complex is removed from the follicle. Natriuretic peptide C (NPPC) delays germinal vesicle breakdown (GVBD) while increasing oocyte-cumulus communication during in vitro maturation (IVM) in cattle. In the present study we first tested the hypothesis that steroids secreted by the follicle (17β-oestradiol, progesterone and androstenedione) interact with NPPC to delay GVBD and to maintain oocyte-cumulus communication as assessed by transfer of a dye (Lucifer Yellow) from the oocyte to cumulus cells. Then, we assessed the effects of steroid hormones and NPPC, alone and in combination in a pre-IVM culture, on embryo production. The combination of NPPC with steroids delayed GVDB, increased natriuretic peptide receptor 2 (NPR2) mRNA abundance in cumulus cells during culture, and maintained oocyte-cumulus communication at levels not different from non-cultured controls. The addition of steroids and/or NPPC to a pre-IVM culture did not alter blastocyst rates after IVF, but supplementation with steroids increased blastocyst total cell number. The present study provides evidence, for the first time in cattle, that steroids interact with NPPC to regulate oocyte nuclear maturation and oocyte-cumulus communication, and improve oocyte developmental competence.