Oocyte Nuclear Maturation Research Articles

Vascular endothelial growth factor A improves quality of matured porcine oocytes and developing parthenotes

Vascular endothelial growth factor is a multipotent angiogenic factor implicated in cell survival and proliferation. The objective was to determine effects of exogenous recombinant human VEGFA (or VEGFA165) in culture media on porcine oocyte maturation and parthenote development. Adding 5 ng/mL VEGFA to the culture medium improved the maturation rate of denuded oocytes (P < 0.05), although 5, 50, or 500 ng/mL did not significantly affect nuclear maturation of oocytes. Parthenotes from oocytes cultured either in in vitro maturation or in vitro culture medium supplemented with 5 or 50 ng/mL VEGFA had an improved blastocyst rate and increased total numbers of cells (P < 0.05). Moreover, those treated with 5 ng/mL of VEGFA had a higher hatched blastocyst rate (average of 121 cells per blastocyst). All VEGFA-treated oocytes had reduced apoptotic indices (P < 0.05), except for those with a higher dose (500 ng/mL) of VEGFA which had more apoptotic cells (P < 0.05). Adding 5 ng/mL VEGFA to oocytes during the last 22 h of in vitro maturation improved (P < 0.05) blastocyst rates and total numbers of cells, with reduced apoptosis indices similar to that of long-term (44 h) culture. Furthermore, Axitinib (VEGFR inhibitor) reversed the effects of VEGFA on parthenote development (P < 0.05). Follicular fluids from medium (2–6 mm) to large (>6 mm) follicles contained 5.3 and 7.0 ng/mL vascular endothelial growth factor protein, respectively, higher (P < 0.05) than concentrations in small (<2 mm) follicles (0.4 ng/mL). Also, VEGFA and its receptor (VEGFR-2) were detected (immunohistochemistry) in growing follicles and developing blastocysts. In addition, VEGFA inhibited caspase-3 activation in matured oocytes (P < 0.05). In conclusion, this is apparently the first report that VEGFA has proliferative and cytoprotective roles in maturing porcine oocytes and parthenotes. Furthermore, an optimal VEGFA concentration promoted porcine oocyte maturation and subsequent development.

Effect of α-linolenic acid on oocyte maturation and embryo development of prepubertal sheep oocytes

The purpose of this study was to evaluate the effect of omega-3 α-linolenic acid (ALA) added to the IVM medium on embryo development of prepubertal sheep oocytes. Experiment 1 investigated the effect of ALA at different concentrations (0 [control], 50, 100, and 200 μM) and DMSO (100 μM) in IVM media on cumulus cell expansion and oocyte nuclear maturation and on synthesis of prostaglandins (PGE2 and PGF2α). Experiment 2 investigated the effects of ALA at different concentrations in the IVM medium on oocyte fertilization, cleavage, and developmental potential to blastocyst stage and changes in estradiol and progesterone concentrations in the spent IVM media. IVM oocytes were fertilized with frozen-thawed spermatozoa capacitated in a serum-free sperm medium. Presumptive zygotes were cultured 8 days in synthetic oviductal fluid (SOF) medium without serum. Blastocyst quality was assessed by counting total cell number and the number of apoptotic cells using Hoechst and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Nuclear maturation of oocytes and the number of fully expanded cumulus cells were reduced after treatment with 200 μM of ALA compared with other groups (P ≤ 0.05). Supplementation with ALA increased both PGE2 and PGF2α concentrations in the spent media (P ≤ 0.05). No differences were observed in blastocyst development among control (12.2%) and 50, 100, and 200 μM ALA groups (6.9%, 11.5% and 14.0%, respectively). However, the total cell number (46.50 ± 5.85, 67.94 ± 6.71, 45.20 ± 6.37, and 59.80 ± 5.51, respectively; P ≤ 0.05) and apoptotic cell number (6.45 ± 0.89, 2.48 ± 0.81, 4.02 ± 1.15, and 3.67 ± 1.15, respectively; P ≤ 0.05) were significantly improved. After IVM, estradiol concentration was lower and progesterone concentration was higher in ALA groups compared with the control group (P ≤ 0.05). In conclusion, these results revealed that ALA affects prepubertal sheep embryo quality associated with alteration of releasing reproductive hormones.

Insulin influences developmental competence of bovine oocytes cultured in α-MEM plus follicle-simulating hormone.

The aim of this study was to evaluate the dose-response effect of insulin, plus follicle-simulating hormone (FSH) at a fixed concentration, in a serum-free defined culture medium (DCM) on the in vitro maturation of bovine cumulus-oocyte complexes (COCs). For oocyte nuclear maturation, the expression levels of GDF9, GLUT1, PRDX1 and HSP70.1 transcripts related to oocyte and embryo developmental competence were analysed. For in vitro maturation (IVM), cumulus-oocyte complexes from slaughterhouse ovaries were distributed into four groups based on insulin concentration added to serum-free DCM, which was composed of alpha minimum essential medium (α-MEM), as basal medium: (1) DCM control: 0 ng/ml; (2) DCM1: 1 ng/ml; (3) DCM10: 10 ng/ml; and (4) DCM100: 100 ng/ml. After IVM, the nuclear status of a sample of oocytes was analysed and the other oocytes were submitted for in vitro fertilization (IVF) and in vitro culture (IVC). Different concentrations of insulin did not affect significantly the nuclear maturation and cleavage rate (72 h post-insemination) across all groups. Blastocyst rate (192 h post-insemination) did not differ in DCM control (24.3%), DCM1 (27.0%) and DCM10 (26.3%) groups, but the DCM100 (36.1%) group showed a greater blastocyst rate (P 0.05) was observed at the different insulin concentrations. The results indicated that insulin added to DCM influenced levels of transcripts related to cellular stress (HSP70-1 and PRDX1) and oocyte competence (GDF9) in bovine oocytes and at higher concentrations enhanced blastocyst production.

Ghrelin accelerates in vitro maturation of bovine oocytes.

Ghrelin, apart from its metabolic role, is nowadays considered as a basic regulator of reproductive functions of mammals, acting at central and gonadal levels. Here, we investigated for possible direct actions of ghrelin on in vitro maturation of bovine oocytes and for its effects on blastocyst yield and quality. In experiment 1, cumulus oocyte complexes (COCs) were matured in the presence of four different concentrations of ghrelin (0, 200, 800 and 2000 pg/ml). In vitro fertilization and embryo culture were carried out in the absence of ghrelin, and blastocyst formation rates were examined on days 7, 8 and 9. In experiment 2, only the 800 pg/ml dose of ghrelin was used. Four groups of COCs were matured for 18 or 24 h (C18, Ghr18, C24 and Ghr24), and subsequently, they were examined for oocyte nuclear maturation and cumulus layer expansion; blastocysts were produced as in experiment 1. The relative mRNA abundance of various genes related to metabolism, oxidation, developmental competence and apoptosis was examined in snap-frozen cumulus cells, oocytes and day-7 blastocysts. In experiment 1, ghrelin significantly suppressed blastocyst formation rates. In experiment 2, more ghrelin-treated oocytes matured for 18 h reached MII compared with controls, while no difference was observed when maturation lasted for 24 h. At 18 and 24 h, the cumulus layer was more expanded in ghrelin-treated COCs than in the controls. The blastocyst formation rate was higher in Ghr18 (27.7 ± 2.4%) compared with Ghr24 (17.5 ± 2.4%). Differences were detected in various genes' expression, indicating that in the presence of ghrelin, incubation of COCs for 24 h caused over-maturation (induced ageing) of oocytes, but formed blastocysts had a higher hatching rate compared with the controls. We infer that ghrelin exerts a specific and direct role on the oocyte, accelerating its maturational process.

All‐trans retinoic acid improves goat oocyte nuclear maturation and reduces apoptotic cumulus cells during in vitro maturation

All-trans retinoic acid (t-RA) is a natural component and representative physiologically active metabolite of vitamin A, having multiple physiologic functions. The objective of this study was to evaluate the effect of t-RA on goat oocyte maturation and cumulus cell apoptosis during in vitro maturation (IVM). Immature goat cumulus-oocyte complexes (COCs) were matured in vitro in the absence or presence of t-RA at concentrations of 10 nmol/L, 100 nmol/L and 1000 nmol/L. Oocyte maturation and embryo development were assessed by polar body formation and parthenogenetic activation, respectively. Oocyte survival was checked by Trypan blue staining. Apoptosis of cumulus cells was analyzed by terminal deoxynucleotidyl transferase nick end labeling staining and quantitative real-time PCR. In comparison with the control group, 100 nmol/L and 10 nmol/L t-RA significantly improved goat nuclear oocyte maturation and survival (P < 0.05). Addition of 1000 nmol/L t-RA improved nuclear maturation (P < 0.05), but had no effect on survival of goat oocytes. t-RA had no positive effect on goat parthenogenetic embryonic cleavage, blastocyst formation or total cell numbers. However, t-RA inhibits the apoptosis of cumulus cells (P < 0.01). t-RA treatment up-regulated the expression of B-cell lymphoma 2 (BCL-2), catalase (CAT) (P < 0.05) and down-regulated the expression of Caspase-8 (P < 0.05). In conclusion, t-RA has positive effects on goat oocyte nuclear maturation and reduces apoptotic cumulus cells during IVM.

In Vitro Production of Sheep Embryos in CR1aa Medium Supplemented with L-Ascorbic Acid

In vitro embryo-production procedures developed for sheep have tremendously been improved from decades, but many factors influencing their efficiency still need to be investigated. The overall of this study showed the production of the sheep embryos till blastocyst stage from the ovaries of the slaughtered ewes for IVM, IVF of the oocytes and then IVC in the complex culture media like TCM-199, TCM-199-, TCM-199+ and CR1aa. The morula yield was significantly higher in the wheat peptone and BSA supplemented group. The nuclear maturation rates of ovine oocytes matured in FBS (69.15 ± 1.07) group was more compared to BSA (56.47 ± 0.73) and wheat peptone (35.26 ± 0.79). The nuclear maturation of oocytes was significantly higher in the FBS group when compared to BSA and wheat peptone supplemented groups. The lowest maturation rate was observed in wheat peptone supplemented group. The development rates in 2, 4, 8, 16 and morula stages of ovine embryos produced from BSA group were high (74.22 ± 2.56, 60.93 ± 2.55, 42.97 ± 2.20 and 24.21 ± 1.28) compared to FBS group (73.58 ± 2.92, 58.77 ± 2.01, 41.22 ± 1.68 and 20.17 ± 0.76) and wheat peptone (72.89 ± 1.09, 57.01 ± 0.76, 42.05 ± 0.36 and 24.29 ± 0.55). The cleavage, morula and blastocyst percent in CR1aa was found significantly high compared with TCM- 199, TCM-199- and TCM-199+. The cleavage, morula and blastocyst percent in L-Ascorbic acid at 100 μM with CR1aa is found more significant compared with other compositions used in the experimentation.

Effect of Epigallocatechin-3-Gallate on the In Vitro Developmental Potential of Porcine Oocytes and Embryos Obtained Parthenogenetically and By Somatic Cell Nuclear Transfer

The present study aimed to investigate the effects of epigallocatechin-3-gallate (EGCG) on the in vitro development of porcine oocytes, parthenogenetic activation embryos (PA), and somatic cell nuclear transfer (SCNT) embryos. In Experiment 1, 0 (control), 10, 30, and 50 μg/mL EGCG were added to in vitro maturation (IVM) medium to explore the effect of EGCG on IVM of pig oocytes. The matured oocytes were then used to produce PA and SCNT embryos. Either for nuclear maturation of oocytes or for the rates of cleavage and blastocyst of PA and SCNT embryos, no significant difference was found among all groups. However, the total cell number per cloned blastocyst was significantly lower in blastocysts derived from oocytes matured in 50 μg mL EGCG (P<0.05) as compared with the other groups. In Experiment 2, we cultured pig SCNT and PA embryos in medium containing various concentrations of EGCG to examine the effect of EGCG on preimplantation development. The cleavage and blastocyst rates and the total cell number per blastocyst did not significantly differ between PA and SCNT embryos among all groups. However, the reactive oxygen species level was significantly lower in the PA embryos cultured in 10 μg mL EGCG than the other groups (P<0.05). Our results suggest that high doses of EGCG in IVM are harmful to the oocytes as evidenced by the decreased quality of SCNT embryos, and EGCG has no beneficial effects on in vitro development of pig cloned embryos.

Effect of Hyaluronan on Developmental Competence and Quality of Oocytes and Obtained Blastocysts fromIn VitroMaturation of Bovine Oocytes

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

173 EFFECTS OF GLUCOSE METABOLISM DURING IN VITRO MATURATION ON CYTOPLASMIC MATURATION OF GOAT OOCYTES

It is well known that oocyte maturation consists of 2 processes: nuclear maturation and cytoplasmic maturation. Nuclear maturation refers to resumption of the first meiosis and extrusion of the first polar body (PB1), and cytoplasmic maturation is manifested as acquisition of the ability to complete pre-implantation development. Although it is recognised that energy supply is essential for oocyte maturation and there have been many reports on the effect of glucose metabolism on oocyte nuclear maturation, studies on the effect of glucose metabolism on ooplasmic maturation are limited. In the present study, goat oocytes recovered from slaughterhouse ovaries were cultured for 24 h in a simplified CR1 (sCR1) medium (NaCl, KCl, NaHCO3, CaCl2, BSA, and eCG) supplemented with glucose (10 mM) and/or lactate (3.5 mM) in the presence or absence of pentose phosphate pathway (PPP) inhibitor dehydroepiandrosterone (DHEA, 100 μM) or glycolysis inhibitor iodoacetate (1 μM). At the end of maturation culture, oocytes with PB1 were either activated by treatment with ionomycin plus 6-DMAP to observe embryo development, or assayed for total glutathione concentrations (GSX) and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios. Embryos were cultured for 9 days in CR1aa medium (NaCl, KCl, NaHCO3, calcium lactate, sodium pyruvate, glutamine, EAA, NEAA, and FCS) at 38.5°C under 5% CO2 in humidified air. In the absence of inhibitors, oocyte maturation rates of 82, 65, and 76%, and blastocyst rates of 7, 0, and 7%, were obtained, respectively, after oocytes were matured in sCR1 supplemented with glucose, lactate, or both. When oocytes were matured in sCR1 containing glucose and lactate in the presence of DHEA or iodoacetate, oocyte maturation rates were 69 and 67%, respectively, with no blastocyst produced in either case. However, whereas the presence of DHEA produced 12% morulae, no morulae were observed in the presence of iodoacetate. Furthermore, GSX concentrations (pmol/oocyte) were 8.5, 6.5, and 7.2, whereas GSH/GSSG ratios were 1.8, 0.3, and 0.5, respectively, after oocyte maturation without inhibitors or with 300 μM DHEA or 3 μM iodoacetate. The difference in GSX concentration was statistically significant (P &lt; 0.05; one-way ANOVA) between DHEA and iodoacetate. In conclusion, using a culture system (sCR1 containing 3.5 mM lactate) that sustained oocyte nuclear maturation but did not support blastocyst development, we have studied the effect of PPP and glycolysis of glucose metabolism on the cytoplasmic maturation of goat oocytes. The results suggest that both PPP and glycolysis are essential for ooplasmic maturation of goat oocytes, and that both promote oocyte cytoplasmic maturation by increasing glutathione synthesis and reduction. This study was supported by grants from the National Basic Research Program of China (Nos. 2012CB944403 and 2014CB138503) and the China National Natural Science Foundation (Nos. 31272444 and 30972096).

Beneficial effect of resveratrol on bovine oocyte maturation and subsequent embryonic development after in vitro fertilization

To analyze the potential beneficial effects and mechanisms of action of resveratrol on the maturation of bovine oocytes that were incubated in different concentrations of resveratrol (0.1, 1.0, or 10.0 μM) as germinal vesicle-stage oocytes. In vitro prospective study. University research laboratory. Animal models for human studies. In vitro culture in the presence of various concentrations of the antioxidant resveratrol. Parameters of hormone levels, oocyte nuclear maturation, cumulus expansion, levels of intracellular glutathione and reactive oxygen species, embryonic cleavage, blastocyst formation, gene expression associated with mature bovine oocytes and cumulus cells, and level of sirtuin 1 gene expression. Resveratrol statistically significantly increased progesterone secretion and decreased estradiol-17β secretion by cumulus cells. The elevated levels of progesterone activated the Mos/MEK/p42 mitogen-activated protein kinase (MAPK) cascade in the oocytes. At a concentration of 1.0 μM, resveratrol statistically significantly improved cumulus expansion, polar body formation, the (hatched) blastocyst rate, and the mean number of cells/blastocysts. Meanwhile, resveratrol statistically significantly reduced the level of reactive oxygen species (ROS) and increased the level of glutathione (GSH). For the first time, the expression of the sirtuin-1 gene was identified in granulosa cells, cumulus cells, oocytes, and blastocysts. Further studies revealed that resveratrol promoted sirtuin-1 gene expression. Resveratrol promoted bovine oocyte maturation and subsequent post-in vitro fertilization embryonic development by inducing progesterone secretion and an antioxidant effect, probably in a manner dependent on sirtuin-1.

RUNX2, GPX3 and PTX3 gene expression profiling in cumulus cells are reflective oocyte/embryo competence and potentially reliable predictors of embryo developmental competence in PCOS patients

BackgroundPolycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women. The developmental competence of oocytes and embryos in PCOS patients is reduced to a certain extent (comparing to non-PCOS patients, the high quality embryo rate was decreased by 16% from the data of our centre) during the in vitro fertilization (IVF) process. Cross-talk between the oocyte and cumulus cells is critical for oocyte maturation and embryo competence. In this study, we have evaluated the transcription of specific genes in cumulus cells harvested from pre-ovulatory follicles of PCOS patients before IVF, according to individual oocyte nuclear maturity and developmental competence. Seven genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, and ITGB5) were targeted from our previous cDNA microarray data which isolated genes related to oocyte nuclear maturation in PCOS patients. Two additional genes which had been found to be associated with oocyte maturation or embryo quality in non-PCOS patients (GPX3 and PTX3) were also studied.MethodsThe mRNA expression levels of cumulus cells were detected by qRT- PCR.ResultsConsistent with our previous cDNA microarray data, with the exception of GPX3 and PTX3, the selected 7 genes were related to oocyte nuclear maturation in PCOS patients. Noticeably, the expression level of RUNX2 was lower in cumulus cells derived from oocytes that could develop into blastocysts than the level of expression from oocytes that could not. The PTX3 expression level was significantly lower in cumulus cells from oocytes with two normal pronuclei than that from oocytes that formed >2 pronuclei (MPN) after fertilization. GPX3 mRNA levels were decreased in cumulus cells isolated from oocytes that developed into blastocysts with high potential development competence.ConclusionsSeveral cumulus cell genes were associated with oocyte maturation, fertilization and embryo quality in PCOS patients. RUNX2 and GPX3 are candidate genetic markers in the monitoring of embryo quality for PCOS patients, whereas PTX3 mainly played a role in fertilization process. Together with morphological evaluation, cumulus cells genes may serve as biomarkers of oocyte and embryo selection during the IVF process for PCOS patients and may advance our understanding of PCOS.

Influence of manganese on apoptosis and glutathione content of cumulus cells during in vitro maturation in bovine oocytes

We have investigated the effect of different Mn concentrations on (1) DNA integrity of cumulus cells by olive tail moment (OTM); (2) cumulus cells apoptosis by Annexin V staining assay; (3) intracellular total glutathione (GSH-GSSG) content; and (4) oocyte nuclear maturation and embryo cleavage after in vitro fertilisation (IVF). For this purpose, 0 (control), 2 (Mn1), 5 (Mn2) and 6 ng/mL (Mn3) Mn concentrations were added to IVM medium. Comet assay analysed by OTM was significantly higher in cumulus cells arising from COCs matured without Mn (control, P < 0.01) respect to cumulus cells obtained from COCs matured with Mn (control: 5.18 ± 2.3; Mn1: 2.93 ± 2.2; Mn2: 2.63 ± 2.4; Mn3: 2.92 ± 2.4). The frequency of apoptotic cells was higher in the control group (control: 6.63 ± 0.59; Mn1: 5.05 ± 0.5; Mn2: 4.61 ± 0.49; Mn3: 3.33 ± 0.42). Intracellular concentration of GSH-GSSG increased in oocytes and cumulus cells matured in the presence of Mn (P < 0.01). There were no differences in percentages of nuclear maturation when Mn was added to IVM medium at any concentration, but at 6 ng/mL Mn a higher cleavage rate was observed respect to the control group (P < 0.05). In conclusion, deficiency in Mn concentration during in vitro maturation increased the damage in the DNA molecule and the frequency of apoptotic cumulus cells. However, the addition of an adequate Mn concentration (6 ng/mL Mn) to IVM medium improved the health of cumulus-oocyte complexes and produced more cleaved embryos 48 h after IVF.

Maternal Liver Damage Delays Meiotic Resumption in Bovine Oocytes through Impairment of Signalling Cascades Originated from Low p38MAPK Activity in Cumulus Cells

The main objective of the present study is to investigate the molecular mechanism underlying the delay in progression of nuclear maturation in oocytes derived from cows with damaged livers (DL cows), which was previously reported. In present study, delayed progression of nuclear maturation of oocytes derived from DL cows relative to oocytes derived from cows with healthy livers (HL cows) was accompanied by low maturation promoting factor (MPF) activity (0.43 fold, p<0.05). When cumulus cells were removed from cumulus-oocyte complexes and the denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two liver conditions. In addition, gap junctional communication (GJC) between the oocyte and cumulus cells was higher in DL cows than in HL cows at 3 and 7h of in vitro maturation (IVM) (p<0.05). Supplementation of IVM medium with epidermal growth factor (EGF) increased the ratio of germinal vesicle breakdown (GVBD) of oocytes derived from DL cows to the level seen in oocytes derived from HL cows. Additionally, the level of p38MAPK phosphorylation at 0h of IVM was significantly lower in cumulus cells derived from DL cows than in cumulus cells derived from HL cows (HL cows, 53.5%; DL cows, 28.9%; p<0.05). Thus, a low level of p38MAPK phosphorylation in cumulus cells induced slow GJC closure between oocyte and cumulus cells, which resulted in slow meiotic maturation of oocytes derived from DL cows.

Granulocyte-macrophage colony stimulating factor (GM-CSF) enhances cumulus cell expansion in bovine oocytes

BackgroundThe objectives of the study were to characterize the expression of the α- and β-subunits of granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in bovine cumulus cells and oocytes and to determine the effect of exogenous GM-CSF on cumulus cells expansion, oocyte maturation, IGF-2 transcript expression and subsequent competence for embryonic development.MethodsCumulus-oocyte complexes (COC) were obtained by aspirating follicles 3- to 8-mm in diameter with an 18 G needle connected to a vacuum pump at −50 mmHg. Samples of cumulus cells and oocytes were used to detect GM- CSF receptor by immunofluorescence. A dose–response experiment was performed to estimate the effect of GM-CSF on cumulus cell expansion and nuclear/cytoplasmic maturation. Also, the effect of GM-CSF on IGF-2 expression was evaluated in oocytes and cumulus cells after in vitro maturation by Q-PCR. Finally, a batch of COC was randomly assigned to in vitro maturation media consisting of: 1) synthetic oviductal fluid (SOF, n = 212); 2) synthetic oviductal fluid supplemented with 100 ng/ml of GM-CSF (SOF + GM-CSF, n = 224) or 3) tissue culture medium (TCM 199, n = 216) and then subsequently in vitro fertilized and cultured for 9 days.ResultsImmunoreactivity for both α and β GM-CSF receptors was localized in the cytoplasm of both cumulus cells and oocytes. Oocytes in vitro matured either with 10 or 100 ng/ml of GM-CSF presented a higher (P < 0.05) cumulus cells expansion than that of the control group (0 ng/ml of GM-CSF). GM-CSF did not affect the proportion of oocytes in metaphase II, cortical granules dispersion and IGF-2 expression. COC exposed to 100 ng/ml of GM-CSF during maturation did not display significant differences in terms of embryo cleavage rate (50.4% vs. 57.5%), blastocyst development at day 7 (31.9% vs. 28.7%) and at day 9 (17.4% vs. 17.9%) compared to untreated control (SOF alone, P = 0.2).ConclusionsGM-CSF enhanced cumulus cell expansion of in vitro matured bovine COC. However, GM-CSF did not increase oocyte nuclear or cytoplasmic maturation rates, IGF-2 expression or subsequent embryonic development.

Cysteamine supplementation during in vitro maturation (IVM) of rabbit oocyte improves the developmental capacity after intracytoplasmic sperm injection.

Current approaches to in vitro maturation (IVM) may result in low efficiency and inadequate quality of the oocytes due to insufficient cytoplasmic maturation. Although positive effects of the cysteamine supplementation in IVM medium for oocyte nuclear maturation or male pronuclear formation have been confirmed, it is still controversial whether the cysteamine addition affects embryo development after IVM. We aimed here to confirm the effect of cysteamine addition into IVM medium for subsequent embryo development in vitro. We administered the cysteamine to the IVM culture of rabbit immature oocytes at various concentrations and observed the developmental rate, speed to reach blastocyst stage and cell numbers at the blastocyst stage. Cysteamine supplementation improved developmental rate to blastocyst stage of the IVM oocytes. On the other hand, addition of glutathione (GSH) inhibitor buthionine sulfoximine inhibited GSH accumulation in the oocytes and subsequent embryo development to the blastocyst stage. Controlling the GSH quantity of IVM oocytes may be an important factor for success of embryo development, and it is quite probable that a cysteamine supplementation can contribute to an increase of GSH content in oocyte.

Protective effects of antioxidants on linoleic acid–treated bovine oocytes during maturation and subsequent embryo development

Linoleic acid (LA; n-6, 18:2) is the most abundant polyunsaturated fatty acid in the ovarian follicular fluid and is known to inhibit oocyte maturation and its subsequent development. In the present study, we investigated how its effects on cumulus cell expansion, oocyte nuclear maturation, and blastocyst development are altered by supplementation of the media with vitamin E (VE; 100 μM) and glutathione peroxidase (GPx; 1 μM) either alone or in combination, and whether it has any effect on the mRNA expression of GPx1, GPx4, or superoxide dismutase (SOD2) in the bovine cumulus oocyte complexes (COCs). LA supplementation of the culture media significantly (P ≤ 0.05) reduced the percentage of COCs exhibiting full cumulus cell expansion and the percentage of oocytes reaching metaphase II stage, and lowered the blastocyst rate compared with controls. And these inhibitory effects were associated with a reduction in the relative mRNA expression of GPx1 and SOD2 but not of GPx4 compared with controls. However, VE and GPx, both alone and in combination, completely abrogated the inhibitory effects of LA on nuclear maturation of oocytes and blastocyst rate but failed to do so for cumulus cell expansion. In conclusion, these data suggest that the detrimental effects of LA on oocyte developmental competence are mediated, at least in part, by a reduction in GPx1 and SOD2 mRNA expression. Moreover, VE and GPx may provide protection to most of the inhibitory effects of LA.

Comparative Importance of Fatty Acid Beta-Oxidation to Nuclear Maturation, Gene Expression, and Glucose Metabolism in Mouse, Bovine, and Porcine Cumulus Oocyte Complexes1

The objective of these experiments was to evaluate the importance of fatty acid beta-oxidation (FAO) in the cumulus oocyte complex (COC) during in vitro maturation (IVM) to oocyte nuclear maturation and gene expression in both the oocyte and cumulus cells in three species with differing amounts of oocyte intracellular lipids (mouse, low; bovine, moderate; porcine, high). We inhibited FAO using etomoxir at 0, 10, 25, 100, or 250 μM. Completion of oocyte nuclear maturation was inhibited after COC exposure to 250 μM etomoxir in mouse oocytes, 100 μM etomoxir in bovine oocytes, and as little as 10 μM etomoxir in porcine oocytes (P < 0.05). When FAO was inhibited in mouse and porcine COCs resulting in inhibition of meiosis, the abundance of FAO, glycolytic, and oxidative stress gene transcripts were decreased in oocytes and cumulus cells (P < 0.05), although to a much greater extent in the pig. In bovine oocytes and cumulus cells, FAO gene transcripts were increased and glycolytic gene expression altered following meiotic inhibition due to etomoxir. Etomoxir, at doses that did not inhibit nuclear maturation in bovine and murine COCs, increased glucose consumption (P < 0.05), suggesting glucose metabolism is increased to meet the metabolic demands of the COCs when fatty acid metabolism is compromised. Our data demonstrates that FAO is essential to oocyte nuclear maturation in all three species. Sensitivity of nuclear maturation to FAO inhibition reflects the amount of lipid present in the ooplasm and may suggest a relative reliance on this metabolic pathway.

Similar patterns of embryo development in canine oocytes cultured in vitro at oxygen tensions of 5 and 20%

The influence of two oxygen tensions (5 and 20%) on fertilization, cleavage, development, and morphological quality of canine embryos produced in vitro was investigated. To assess embryo production, canine oocytes (N = 608) were matured in vitro at 20% oxygen tension in TCM 199 supplemented with glucose (11 mM) and 0.1% polyvinyl alcohol. Oocytes and sperm were coincubated at 37 °C for 24 hours in serum-free medium. Subsequently, presumptive zygotes were cultured in vitro in synthetic oviductal fluid in either 5% CO2 in air (20% oxygen; N = 298) or 5% O2, 5% CO2 and 90% N2 (5% oxygen; N = 310) for 7 days. Regardless of the oxygen concentration (5 vs. 20%), rates of pronucleus formation, cleavage, and embryo development up to the eight-cell stage did not differ (46/310 [14.8%] vs. 35/298 [11.7%], respectively; P > 0.05). Moreover, similar proportions of embryos developed in 20 and 5% oxygen tensions (18/298 vs. 27/310). The oocyte nuclear maturation (metaphase II), in terms of decondensed sperm heads, pronucleus formation, cleavage, and embryo development, was similar (P = 0.299) between the atmospheric (20% O2; 12.4% [37/298]) and reduced oxygen tensions (5% O2; 15.8% [49/310]) at all steps of the in vitro culture (IVC) after in vitro fertilization (IVF). To our knowledge, this was the first study that demonstrated that canine embryos can be produced using a low-oxygen in vitro culture system. Both oxygen tensions (5 and 20%) resulted in similar embryonic development and therefore were feasible for IVC of canine oocytes.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Influence of cattle genotype (Bos indicus vs. Bos taurus) on oocyte and preimplantation embryo resistance to increased temperature1,2

High environmental temperatures during the hot months of the year reduce reproductive performance in cattle. Summer heat stress depression in fertility is a multifactorial problem; however, there is evidence that the bovine germinal vesicle and maturing oocyte, as well as the early embryo, are major targets of the deleterious effects of heat stress. Such adverse effects are less pronounced in heat-tolerant breeds (Bos indicus) than heat-sensitive breeds (Bos taurus). This genetic variation results from the greater thermoregulatory ability and cellular thermoresistance of heat-tolerant breeds. Heat-induced oocyte cellular damage occurs in both cytoplasmic and nuclear compartments. Heat shock has been shown to reduce oocyte nuclear maturation, induce apoptosis, compromise oocyte cytoskeleton, and impair oocyte mitochondrial function and developmental competence. However, the oocyte cytoplasm is more susceptible to heat shock than the nucleus. This effect is greater for Bos taurus than Bos indicus oocytes. The detrimental effects of heat shock are also critical during the first cleavage divisions when most of the embryonic genome is inactive; however, the bovine embryo becomes more resistant to increased temperature as it proceeds through development. Several studies demonstrated that Bos indicus embryos are more thermotolerant than Bos taurus embryos. Adaptive changes involved in acquisition of thermotolerance are likely derived from changes in gene expression and (or) activity of biochemical molecules that control cellular functions against stress. Recently, molecules such as IGF-I and caspase inhibitor z-DEVD-fmk have been shown to exert a thermoprotective role, rescuing heat-induced oocyte and embryo cellular damage and developmental competence. Therefore, cattle genotype and thermoprotective molecules can be considered as an alternative to modulate the effects of increased temperature in reproductive function.

Oocyte maturation and in vitro hormone production in small antral follicles (SAFs) isolated from rhesus monkeys

The small antral follicles (SAFs) from the ovarian medulla can be a potential source of oocytes for infertility patients, but little is known about their ability to yield mature oocytes. This study evaluated the response of these SAFs to a stimulatory bolus of human corionic gonadotropin (hCG) in vitro. Oocyte nuclear maturation and hormone production (estradiol [E2], progesterone [P4]), antimullerian hormone [AMH]) by individual intact SAFs (n = 91; >0.5mm; n = 5 monkeys) was evaluated after 34h of culture in the absence (control) or presence of hCG. Of the total cohort (n = 91), 49% of SAFs contained degenerating oocytes. The percentage of healthy oocytes able to reinitiate meiosis to the metaphase I (MI) and MII was greater (p < 0.05) after hCG compared to controls. E2, P4 and AMH levels were higher (p < 0.05) in SAF cultures containing germinal vesicle (GV) oocytes compared to those with MII oocytes regardless of hCG exposure. SAF with MI oocytes produced more E2, but less (p < 0.05) P4 and AMH compared to SAFs containing GV oocytes (p < 0.05). Follicles ≥1mm produced more (p < 0.05) E2, whereas follicle diameter did not correlate with P4 or AMH levels. Only P4 increased (p < 0.05) in response to hCG, regardless of follicle size or oocyte maturity. SAFs containing degenerating oocytes produced similar levels of E2, P4 and AMH compared to SAFs containing healthy oocytes. These data indicate, for the first time, that oocytes within primate SAFs can reinitiate meiosis in vitro in the absence of hCG, but nuclear maturation is enhanced in SAFs cultured with hCG. Oocyte nuclear maturation within SAFs in is associated with decreased E2, P4 and AMH levels. Furthermore, hormone content within the culture media does not necessarily reflect oocyte quality.