76 EX VIVO MODEL TO STUDY THE EFFECT OF ACUTE EXPOSURE TO DI-(2-ETHYLHEXYL) PHTHALATE ON BOVINE OOCYTE MATURATION AND DEVELOPMENTAL COMPETENCE

2015 ◽  
Vol 27 (1) ◽  
pp. 131
Author(s):  
D. Kalo ◽  
R. Hadas ◽  
Z. Roth

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolites are environmental toxicants that potentially affect mammalian health. However, their effects on ovarian function, and in particular oocyte developmental competence, are less known. We established a model of acute exposure to DEHP to examine its immediate and long-term effects on follicles and oocyte competence. Lactating Holstein cows were synchronized and gavaged with DEHP (OXPLAST®O, ZAK, Kędzierzyn-Koźle, Poland; 100 mg kg–1 per day, n = 4) or water (n = 5) for 3 days. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed high DEHP metabolite levels in both the urine and plasma during DEHP exposure (acute phase), followed by relatively low levels through the subsequent 34 days (chronic phase). In the chronic phase, cows were synchronized with prostaglandin (PG)2α-gonadotropin-releasing hormone (GnRH) (day 0) and ovaries were monitored by ultrasonography (Aloka SSD-900, 7.5 MHz; Aloka, Tokyo, Japan) through an entire oestrous cycle. On Day 18 of the cycle, cows were administered with PG2α and within 30 h, follicular fluid (FF) was aspirated from the preovulatory follicle by ultrasonic scanner connected to a vaginal sector transducer (7.5-MHz, PieMedical, Maastricht, the Netherlands). For each group, the FF were pooled and analysed for DEHP metabolites (MEHP, MEHOH, MEHHP) by LC-MS/MS. These FF further served as maturation medium for in vitro embryo production. For all cows, FF aspirated before DEHP administration did not contain any of the examined metabolites. However, during the chronic phase, the level of MEHP (but not MEHOH, MEHHP) was higher (22.31 v. 0.00 nM) in the FF aspirated from treated cows (FF-DEHP, n = 4) relative to controls (FF-control, n = 5). To examine the effect of MEHP on oocyte maturation and developmental competence, cumulus-oocyte complexes aspirated from abattoir ovaries (n = 250 × 5 replicates) were matured (22 h, 38.5°C, 5% CO2) in FF-control or FF-DEHP, then in vitro fertilized (18 h, 38.5°C, 5% CO2) and cultured for 8 days in KSOM (38.5°C, 5% CO2, 5% O2). The proportion of oocytes with expanded cumulus cells (84.5 v. 81.2%) and distribution of oocyte within cortical granule types (I–III) did not differ between groups at the end of maturation. The proportion of oocytes with metaphase II plate and first polar body (i.e. nuclear-matured) was lower in the DEHP-treated group relative to the control (34.7 v. 64.3%, n = 100/group; P < 0.0001). Moreover, a decreased proportion of 2- to 4-cell-stage embryos (53.12 v. 67.61%; P < 0.04) and 7-day blastocysts (4.2 v. 12%; P < 0.08) was noted for the FF-DEHP group. In summary, the findings reveal long-lasting effects of DEHP exposure, expressed by MEHP incorporation into the FF, suggesting potential deleterious effects on developmental competence of the follicle-enclosed oocyte.

2017 ◽  
Vol 29 (1) ◽  
pp. 192
Author(s):  
P. Ferré ◽  
K. X. Nguyen ◽  
T. Wakai ◽  
H. Funahashi

This experiment was undertaken to assess the meiotic and developmental competences of oocytes derived from different sized follicles and denuded of cumulus cells 0, 20, and 44 h after the start of culture for in vitro maturation (IVM). Groups of 60 oocyte-cumulus complexes from small- (SF; <3 mm) and medium-sized follicles (MF; 3–6 mm) were cultured for IVM in porcine oocyte medium with 50 μM β-mercaptoethanol supplemented with 1 mM dibutyryl-cyclic adenosine monophosphate, 10 IU mL−1 of eCG, and 10 IU mL−1 of hCG for 20 h at 39°C and 5% CO2 in air. Then, after washing, they continued culture in fresh β-mercaptoethanol without dibutyryl-cyclic adenosine monophosphate and gonadotropins under the same conditions for another 24 h. At 0, 20, and 44 h of IVM, cumulus cells were removed with 0.1% (wt/vol) hyaluronidase and the denuded oocytes continued IVM culture following the protocol. Mature oocytes with the first polar body were selected, parthenogenetically activated with a single electrical pulse (DC: 1.2 kV/cm, 30 µs), incubated with 4% (wt/vol) BSA and 5 μM cytochalasin B for 4 h, and cultured in porcine zygote medium for 5 days. Cleavage and blastocyst formation rates were observed on Day 2 and 5, respectively. Blastocysts were stained with 4’,6-diamidino-2-phenylindole for cell count assessment. The experiment was replicated 5 times and analysed with a 1- or 2-way ANOVA. If P < 0.05 in ANOVA, a Tukey multiple comparisons test was performed. Regardless of the time of cumulus cell removal, oocytes from MF had significantly higher in rates of maturation, cleavage, and blastocyst rates, as compared with those from SF, whereas there were no significant differences in the cell number of blastocysts between SF and MF (32 v. 34 cells, respectively). When oocytes were denuded before IVM culture, rates of oocyte maturation (37.6% in SF and 50.8% in MF), and blastocyst formation (2.7% in SF and 27.3% in MF) were significantly lower than controls (51.2% in SF and 76% in MF; 25.8% in SF and 48.5% in MF, respectively). When oocytes were denuded 20 h after the start of IVM, oocyte maturation rates were significantly increased (64.1% in SF and 82.5% in MF) as compared with controls, whereas no significant differences were observed in cleavage and blastocyst formation rates in comparison with controls. These results conclude that removing cumulus cells from oocyte-cumulus complexes 20 h after the start of IVM improves the meiotic competence of oocytes derived from both SF and MF, without any reduction of developmental competence of the oocytes following parthenogenetical activation.


2011 ◽  
Vol 23 (1) ◽  
pp. 229
Author(s):  
M. J. Izquierdo-Rico ◽  
R. Romar ◽  
C. Kohata ◽  
H. Funahashi

Oocyte-specific transcripts play important roles in oocyte maturation, fertilization, and early embryonic development. Currently, oocytes from medium-size follicles have been used for different assisted reproductive techniques after in vitro maturation (IVM). The aim of this study was to compare the mRNA expression level in porcine oocytes collected from medium (3–6 mm) and small (<2 mm) size follicles. Genes were selected based on their described maternal effect (NALP9, HSF1), their identification as markers of oocyte maturation (AURK-A, AURK-B, MOS, and C-mos), their involvement in fertilization (ZP3, ZP4), and anti-apoptotic effect (Bcl-2). All transcripts were studied in oocytes just after collection [germinal vesicle (GV) stage] and after in vitro maturation (IVM; metaphase II stage). To ensure nuclear stage of immature oocytes, oocytes were mechanically denuded just after collection, centrifuged (10 000 rpm, 5 min, RT), and observed under the microscope (60×). Those oocytes with clear nucleolus and evident nuclear membrane were selected and stored (n = 10) until study. For metaphase II oocytes, only those exhibiting the extrusion of first polar body after IVM (n = 10) were selected. Total RNA was extracted from the pool of 10 immature and mature oocytes. One picogram of luciferase mRNA per oocyte was added as an exogenous standard. Total RNA was extracted from oocytes and cDNA was obtained and used as a template for quantitative PCR to analyse the level of different transcripts. The whole process was replicated 4 times. Data were normalized to the luciferase RNA and analysed by one-way ANOVA with maturational stage (GV or metaphase II) and follicle size (small or medium) as fixed factors. Results show that all transcripts were significantly decreased during IVM (P < 0.05). Therefore, after IVM, NALP9, AURK-A, MOS, C-mos, ZP3, ZP4, and Bcl-2 transcripts were significantly reduced in matured oocytes compared with immature ones irrespective of follicle diameter. Transcripts of AURKAB and HSF1 decreased after IVM in oocytes from medium follicles or small follicles, respectively. A significant effect of follicular size was only detected in MOS transcripts in GV-stage oocytes because those collected from middle follicles had a higher amount than the ones from small follicles (Table 1). These results suggest that the variations in the maternal store of RNA during IVM are not related with follicle diameter for the studied genes. Further investigations are necessary to determinate the developmental competence of oocytes that came from different types of follicles (small and medium follicles). Table 1.Variation of transcripts during in vitro maturation in porcine oocytes collected from small and medium follicles This study was supported by Okayama University. R. Romar was given a grant by JSPS (Ref. S-09210).


Author(s):  
Sicong Yu ◽  
Lepeng Gao ◽  
Yang Song ◽  
Xin Ma ◽  
Shuang Liang ◽  
...  

Abstract Mitochondria play an important role in controlling oocyte developmental competence. Our previous studies showed that glycine can regulate mitochondrial function and improve oocyte maturation in vitro. However, the mechanisms by which glycine affects mitochondrial function during oocyte maturation in vitro have not been fully investigated. In this study, we induced a mitochondrial damage model in oocytes with the Bcl-2-specific antagonist ABT-199. We investigated whether glycine could reverse the mitochondrial dysfunction induced by ABT-199 exposure and whether it is related to calcium regulation. Our results showed that ABT-199 inhibited cumulus expansion, decreased the oocyte maturation rate and the intracellular glutathione (GSH) level, caused mitochondrial dysfunction, induced oxidative stress, which was confirmed by decreased mitochondrial membrane potential (Δ⍦m) and the expression of mitochondrial function-related genes (PGC-1α), and increased reactive oxygen species (ROS) levels and the expression of apoptosis-associated genes (Bax, caspase-3, CytC). More importantly, ABT-199-treated oocytes showed an increase in the intracellular free calcium concentration ([Ca 2+]i) and had impaired cortical type 1 inositol 1,4,5-trisphosphate receptors (IP3R1) distribution. Nevertheless, treatment with glycine significantly ameliorated mitochondrial dysfunction, oxidative stress and apoptosis, glycine also regulated [Ca 2+]i levels and IP3R1 cellular distribution, which further protects oocyte maturation in ABT-199-induced porcine oocytes. Taken together, our results indicate that glycine has a protective action against ABT-199-induced mitochondrial dysfunction in porcine oocytes.


Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 416-425 ◽  
Author(s):  
Yan Yun ◽  
Peng An ◽  
Jing Ning ◽  
Gui-Ming Zhao ◽  
Wen-Lin Yang ◽  
...  

SummaryOocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.


Author(s):  
Batara Sirait ◽  
Budi Wiweko ◽  
Ahmad Aulia Jusuf ◽  
Dein Iftitah ◽  
R. Muharam

Oocyte developmental competence is one of the determining factors that influence the outcomes of an IVF cycle regarding the ability of a female gamete to reach maturation, be fertilized, and uphold an embryonic development up until the blastocyst stage. The current approach of assessing the competency of an oocyte is confined to an ambiguous and subjective oocyte morphological evaluation. Over the years, a myriad of biomarkers in the cumulus-oocyte-complex has been identified that could potentially function as molecular predictors for IVF program prognosis. This review aims to describe the predictive significance of several cumulus-oocyte complex (COC) biomarkers in evaluating oocyte developmental competence. A total of eight acclaimed cumulus biomarkers are examined in the study. RT-PCR and microarray analysis were extensively used to assess the significance of these biomarkers in foreseeing oocyte developmental competence. Notably, these biomarkers regulate vital processes associated with oocyte maturation and were found to be differentially expressed in COC encapsulating oocytes of different maturity. The biomarkers were reviewed according to the respective oocyte maturation events namely: nuclear maturation, apoptosis, and extracellular matrix remodeling, and steroid metabolism. Although substantial in vitro evidence was presented to justify the potential use of cumulus biomarkers in predicting oocyte competency and IVF outcomes, the feasibility of assessing these biomarkers as an add-on prognostic procedure in IVF is still restricted due to study challenges.


Reproduction ◽  
2001 ◽  
pp. 51-75 ◽  
Author(s):  
A Trounson ◽  
C Anderiesz ◽  
G Jones

Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.


Reproduction ◽  
2006 ◽  
Vol 132 (6) ◽  
pp. 859-867 ◽  
Author(s):  
Xiao-Qian Meng ◽  
Ke-Gang Zheng ◽  
Yong Yang ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
...  

Microfilaments (actin filaments) regulate various dynamic events during meiotic maturation. Relatively, little is known about the regulation of microfilament organization in mammalian oocytes. Proline-rich tyrosine kinase2 (Pyk2), a protein tyrosine kinase related to focal adhesion kinase (FAK) is essential in actin filaments organization. The present study was to examine the expression and localization of Pyk2, and in particular, its function during rat oocyte maturation. For the first time, by using Western blot and confocal laser scanning microscopy, we detected the expression of Pyk2 in rat oocytes and found that Pyk2 and Try402 phospho-Pyk2 were localized uniformly at the cell cortex and surrounded the germinal vesicle (GV) or the condensed chromosomes at the GV stage or after GV breakdown. At the metaphase and the beginning of anaphase, Pyk2 distributed asymmetrically both in the ooplasm and the cortex with a marked staining associated with the chromosomes and the region overlying the meiotic spindle. At telophase, Pyk2 was observed in the cleavage furrows in addition to its cortex and cytoplasm localization. The dynamics of Pyk2 were similar to that of F-actin, and this kinase was found to co-localize with microfilaments in several developmental stages during rat oocyte maturation. Microinjection of Pyk2 antibody demolished the microfilaments assembly and also inhibited the first polar body (PB1) emission. These findings suggest an important role of Pyk2 for rat oocyte maturation by regulating the organization of actin filaments.


2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Jolanta Opiela ◽  
Joanna Romanek ◽  
Daniel Lipiński ◽  
Zdzisław Smorąg

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 maturedin vitroin 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 higherBaxmRNA 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.


2014 ◽  
Vol 26 (1) ◽  
pp. 136
Author(s):  
T. Somfai ◽  
K. Kikuchi ◽  
K. Yoshioka ◽  
F. Tanihara ◽  
H. Kaneko ◽  
...  

Development to term of vitrified porcine follicular oocytes is reported in the present study. Immature cumulus-oocyte complexes (COC) were collected from slaughtered prepubertal gilts and were vitrified according to our method published recently (Somfai et al. 2013 J. Reprod. Dev., in press). Briefly, after pretreatment with 7.5 μg mL–1 of cytochalasin B (CB) for 30 min in modified NCSU-37 (a basic medium, BM) at 38.5°C, groups of 88 to 121 COC were equilibrated in a mixture of 2% ethylene glycol (EG), 2% propylene glycol (PG), and 7.5 μg mL–1 CB for 13 to 15 min. Then, COC were washed in vitrification solution (17.5% EG, 17.5% PG, 5% polyvinyl pyrrolidone, and 0.3 M trehalose in BM) and then dropped with 2 μL of vitrification solution onto the surface of aluminum foil floating on liquid nitrogen (LN2). Microdroplets (each containing 10–25 COC) were transferred into cryotubes. After storage in LN2 for 2 to 4 weeks, the oocytes were warmed by dropping the microdroplets directly into 2.5 mL of warming solution (0.4 M trehalose in BM) kept in a 35-mm Petri dish on a 42°C hotplate for less than 1 min. Then, the warming dish was placed on a 38°C hotplate and COC were consecutively transferred for 1-min periods into BM containing 0.2, 0.1, or 0.05 M trehalose at 38°C. The COC were matured in vitro for 44 h using porcine oocyte medium (POM) supplemented with 10% follicular fluid (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Then, oocytes were denuded, and their live/dead status and nuclear maturation were determined by their morphology and the presence of the first polar body, respectively. To assess their developmental competence, vitrified and non-vitrified (control) oocytes were in vitro fertilized (IVF; Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) and then in vitro cultured in porcine zygote medium-5 (PZM-5; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Blastocyst rates were recorded on Days 5, 6, and 7 of culture (Day 0 = the day of IVF). The experiment was replicated 4 times. Data were analysed with 1-way ANOVA and the Tukey test. The results revealed that 86.4% (364/424) of oocytes survived after vitrification, which was significantly lower (P < 0.05) than that of controls [100% (326/326)]. Live oocytes in vitrified and control groups did not differ statistically in terms of nuclear maturation (63.9 v. 65.3%). Blastocyst rates of surviving vitrified oocytes were significantly lower compared with controls on Days 5 (2.4 v. 12.7%), 6 (4.8 v. 17.6%), and 7 (5.6 v. 18.4%). To test their ability to develop to term, 16 and 27 blastocysts on Day 5 developing from vitrified COC were transferred into 2 recipients. Both recipients became pregnant and farrowed a total of 10 live piglets (4 and 6 piglets, respectively). These data demonstrate that large groups of immature porcine oocytes could be cryopreserved by this method showing high survival and maturation rates. Furthermore, despite a low rate of blastocyst development, transfer of Day-5 blastocysts generated from vitrified oocytes resulted in piglet production for the first time in the world. Partially supported by JSPS and HAS under the Japan-Hungary Research Cooperative Program.


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