scholarly journals Effects of C-type natriuretic peptide on meiotic arrest and developmental competence of bovine oocyte derived from small and medium follicles

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Zhenwei Jia ◽  
Xueli Wang
Keyword(s):  
Natriuretic Peptide ◽  
Basal Medium ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
The Novel ◽  
Bovine Oocyte ◽  
Meiotic Arrest ◽  
Follicle Size

Abstract The present study aimed to evaluate the effects of C-type natriuretic peptide (CNP) on meiotic arrest and developmental competence of bovine oocyte derived from follicles of different sizes. Collected immature cumulus-oocyte complexes from small follicles (< 3 mm) and medium follicles (3–8 mm) were cultured for 6 h in basal medium supplementated without or with 200 nM CNP. We observed that CNP effectively sustained meiotic arrest at germinal vesicle stage in in vitro cultured bovine oocytes from follicles of different sizes. Moreover, CNP treatment significantly improved the levels of cGMP in both cumulus cells and oocytes, as well as the levels of cAMP in oocytes regardless of follicle size. Based on the above results, we tested the effect of a novel in vitro maturation (IVM) system based on CNP-pretreatment, including a pre-IVM phase for 6 h using 200 nM CNP, followed by a extended IVM phase for 28 h, on developmental competence of bovine oocyte derived from small follicles (< 3 mm) and medium follicles (3–8 mm) compared to standard IVM system. The results showed that athough the novel IVM system based on CNP-pretreatment enhanced the developmental potencial of oocytes obtained from large follicles, but had no effect on the developmental comptence of oocytes obtained from small follicles.

Approaches to oocyte meiotic arrest in vitro and impact on oocyte developmental competence

2021 ◽  
Author(s):  
Dulama Richani ◽  
Robert B Gilchrist
Keyword(s):  
Protein Synthesis ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Antral Follicle ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Protein Synthesis Inhibitors ◽  
Meiotic Arrest ◽  
Oocyte Developmental Competence

Abstract Oocytes are maintained in a state of meiotic arrest following the first meiotic division until ovulation is triggered. Within the antral follicle, meiotic arrest is actively suppressed in a process facilitated by the cyclic nucleotides cGMP and cAMP. If removed from this inhibitory follicular environment and cultured in vitro, mammalian oocytes undergo spontaneous meiotic resumption in the absence of the usual stimulatory follicular stimuli, leading to asynchronicity with oocyte cytoplasmic maturation and lower developmental competence. For more than 50 years, pharmacological agents have been used to attenuate oocyte germinal vesicle (GV) breakdown in vitro. Agents which increase intra-oocyte cAMP or prevent its degradation have been predominantly used, however agents such as kinase and protein synthesis inhibitors have also been trialled. Twenty years of research demonstrates that maintaining GV arrest for a period before in vitro maturation (IVM) improves oocyte developmental competence, and is likely attributed to maintenance of bidirectional communication with cumulus cells leading to improved oocyte metabolic function. However, outcomes are influenced by various factors including the mode of action of the modulators, dose, treatment duration, species, and the degree of hormonal priming of the oocyte donor. Cyclic GMP and/or cAMP modulation in a prematuration step (called pre-IVM) prior to IVM has shown the greatest consistency in improving oocyte developmental competence, whereas kinase and protein synthesis inhibitors have proven less effective at improving IVM outcomes. Such pre-IVM approaches have shown potential to alter current use of artificial reproductive technologies in medical and veterinary practice.

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

Effect of follicle size on mRNA expression in cumulus cells and oocytes of Bos indicus: an approach to identify marker genes for developmental competence

2009 ◽  
Vol 21 (5) ◽  
pp. 655 ◽  
Author(s):  
Ester Siqueira Caixeta ◽  
Paula Ripamonte ◽  
Maurício Machaim Franco ◽  
José Buratini Junior ◽  
Margot Alves Nunes Dode
Keyword(s):  
Gene Expression ◽  
Bos Indicus ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Marker Genes ◽  
Oocyte Competence ◽  
Follicular Size ◽  
Follicle Size

To identify the genes related to oocyte competence, we quantified transcripts for candidate genes in oocytes (H1Foo, H2A, H3A, GHR, GDF9, BMP15, OOSP1) and cumulus cells (FSHR, EGFR, GHR, PTX3, IGFII) using the follicle size model to select oocytes of better developmental quality. Follicles were dissected and distributed into four groups according to diameter as follows: 1.0–3.0, 3.1–6.0, 6.1–8.0 and ≥8.1 mm. Cumulus–oocyte complexes (COCs) were released, classified morphologically, matured, fertilised and cultured in vitro or denuded for measurement of diameter and determination of gene expression. Denuded germinal vesicle oocytes and their cumulus cells were used for gene expression analysis by reverse transcription–polymerase chain reaction. The blastocyst rate was highest for oocytes recovered from follicles >6 mm in diameter. In the oocyte, expression of the H2A transcript only increased gradually according to follicle size, being greater (P < 0.05) in oocytes from follicles ≥8.1 mm in diameter than in oocytes from follicles <6.0 mm in diameter. In cumulus cells, expression of FSHR, EGFR and GHR mRNA increased with follicular size. In conclusion, we confirmed the importance of H2A for developmental competence and identified important genes in cumulus cells that may be associated with oocyte competence.

249 FOLLICLE-STIMULATING HORMONE AND DIBUTYRYL cAMP TREATMENTS DURING IN VITRO MATURATION IMPROVE THE DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 204
Author(s):  
R. Oishi ◽  
Y. Isaji ◽  
H. Imai ◽  
M. Yamada
Keyword(s):  
In Vitro Maturation ◽  
Basal Medium ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Medium ◽  
Dibutyryl Camp ◽  
Mouse Oocytes ◽  
Junctional Communication

The high level of cyclic adenosine monophosphate (cAMP), which is provided to the oocytes from cumulus cells via gap junctional complexes in cumulus-enclosed oocytes (CEOs), is known to contribute to meiotic arrest at the germinal vesicle (GV) stage of CEOs. However, whether intraoocyte cAMP during the period of in vitro maturation (IVM) affects postfertilization developmental competence of mouse oocytes still remains unclear. The aim of this study was to examine the effects of FSH or dibutyryl cAMP (dbcAMP) treatment during IVM on in vitro development of mouse oocytes after in vitro fertilization (IVF). Whether a junctional association between cumulus cells and the oocyte would be essential for a cytoplasmic maturation-promoting effect was also examined. CEOs were isolated from and eCG-primed 3-week-old ICR mouse by rupturing preovulatory follicles with needles in M16 medium with 5% FCS and essential and nonessential amino acids (basal medium). IVM media used were basal medium without (control) or with 100 µm dbcAMP or 1 IU mL–1 FSH. Carbenoxolone (100 µm, CBX), an inhibitor of gap junction, was used to inhibit a junctional association between cumulus cells and the oocyte. Denuded oocytes (DOs) were prepared by repeatedly pipetting in basal medium with 0.2% hyaluronidase. CEOs and DOs were cultured in IVM media at 37�C under 5% CO2 in air for 16.5 h, and then transferred to TYH medium (a modified Krebs-Ringer bicarbonate medium) containing 0.4% BSA, followed by insemination with capacitated sperm. After 6 h of IVF, inseminated oocytes were cultured in KSOM medium with 0.3% BSA. Development to the 2-cell and blastocyst stages was estimated at 24 h and 120 h after IVF, respectively. All experiments were done in 3 replicates, and the statistical analysis was carried out by ANOVA and Fisher's protected least-squares difference (PLSD) test. When CEOs were matured in IVM media, the rates of postfertilization development to the 2-cell and blastocyst stages of oocytes matured in the control medium were very low(29% and 13%, respectively), whereas those of oocytes matured with FSH or dbcAMP significantly increased (FSH: 61% and 52%, dbcAMP: 63 and 57%, respectively, v. control; P < 0.05). Next, when CEOs were matured in basal medium with 1 IU mL–1 FSH and 100 µm CBX, the developmental rate to the 2-cell stage (56%) was similar to that in medium with FSH alone (61%) but the rate to the blastocyst stage (40%) was little lower compared with that in medium with FSH alone (52%), although not significantly different (P > 0.05). Furthermore, when DOs were matured in IVM media, the developmental rates to the blastocyst stage after IVF of the oocytes matured with FSH or dbcAMP significantly increased (FSH: 25%, dbcAMP: 15%; P < 0.05) compared with those in control medium (7%). Taken together, it is suggested that increasing the concentration of intraoocyte cAMP during the IVM period is important to improve the developmental competence after IVF of mouse oocytes, and that the competence is acquired in part in a cumulus-oocyte junctional communication-independent manner.

205 CUMULUS CELL MORPHOLOGY BEFORE AND AFTER IN VITRO MATURATION AS AN INDICATOR OF PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 260
Author(s):  
M. Bertoldo ◽  
P. K. Holyoake ◽  
G. Evans ◽  
C. G. Grupen
Keyword(s):  
Cell Morphology ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Oocyte Developmental Competence ◽  
Before And After ◽  
Follicle Size

Effective in vitro maturation (IVM) is essential for successful in vitro embryo production. The morphology of the cumulus investment before and after IVM may be a useful noninvasive indicator of oocyte quality. In pigs, oocyte developmental competence is reduced during the summer months. The aim of this study was to determine whether the morphology of cumulus-oocyte complexes (COC) before and after IVM are associated with oocyte quality, using COC collected from small and large follicles in summer and winter as models of poor and good oocyte quality. Ovaries were collected from sows slaughtered 4 days after weaning. The COC recovered from small (3-4 mm) and large (5-8 mm) antral follicles were morphologically graded and parthenogenetically activated following IVM during winter (n = 1419; 10 replicates) and summer (n = 2803; 10 replicates). Grade 1 and 2 COC had >2 layers of compact cumulus cells and a homogenous cytoplasm. Grade 3 COC were either partially or fully denuded, had a heterogeneous cytoplasm, or were vacuolated or dark in color. Grade 4 COC had expanded cumulus cells. Cumulus expansion was also assessed subsequent to IVM. The COC recorded as having a cumulus expansion index (CEI) of 1 had the poorest expansion with no detectable response to IVM, whereas those with a CEI of 4 had the greatest amount of expansion, including that of the corona radiata. Data were analyzed using a generalized linear mixed model in GenStat® (release 10, VSN International, Hemel Hempstead, UK). There was an effect of follicle size for Grade 1 COC, with COC from large follicles in both seasons yielding better quality COC (P < 0.05). The proportion of COC in Grade 2 was higher in small follicles during winter compared with large follicles, but there were no differences between follicle sizes during summer (P < 0.05). The proportion of COC with CEI 1 was highest in COC from small follicles during summer (P < 0.05). The proportion of COC from large follicles with CEI 2 was higher during summer compared with winter (P < 0.05). There were no seasonal or follicle size effects on COC with CEI 3 or 4 (P > 0.05). The proportion of oocytes that developed to blastocysts was greater in winter than in summer (39.06% ± 5.67 v. 22.27% ± 4.01; P < 0.05). Oocytes derived from large follicles had a greater ability to form blastocysts compared with those from small follicles (37.13% ± 5.65 v. 23.32% ± 4.56; P < 0.06). Morphological assessment of cumulus cells before and after IVM may be a useful tool to evaluate the effects of follicle size on oocyte developmental competence. However, the results of the present study indicate that cumulus cell morphology is not a good indicator of the effect of season on oocyte developmental competence.

168 INHIBITION OF BOVINE OOCYTE CUMULUS CELL PROGESTERONE SYNTHESIS DURING IN VITRO MATURATION AFFECTS CHROMOSOME ALIGNMENT AND SPINDLE INTEGRITY IN FERTILIZED EGGS AND EARLY EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 198
Author(s):  
E. Daly ◽  
A. G. Fahey ◽  
M. M. Herlihy ◽  
T. Fair
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Spindle Formation ◽  
Vitro Fertilization ◽  
Time Treatment

We have previously demonstrated the importance of progesterone (P4) synthesis by cumulus cells during oocyte maturation in vitro (IVM) for bovine oocyte acquisition of developmental competence and subsequent embryo development (Aparicio et al. 2011 Biol. Reprod. 84). The aim of this study was to identify key processes that may be deregulated by the inhibition of P4 signalling in the cumulus–oocyte complex (COC) during IVM. To this end, good quality immature COC were placed in IVM medium [TCM-199 supplemented with 10% (vol/vol) FCS and 10 ng mL–1 epidermal growth factor] and cultured at 39°C for 22 h in a humidified atmosphere containing 5% CO2, in the presence or absence of 10 μM trilostane (which blocks P4 synthesis by inhibiting 3 β-hydroxysteroid dehydrogenase; Stegram Pharmaceuticals Ltd., Surrey, UK). Matured COC were washed and placed in 250 μL of fertilization medium (25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL–1 fatty acid-free BSA, and 10 mg mL–1 heparin). In vitro fertilization (IVF) was performed with 250 μL of frozen–thawed semen at a final concentration of 1 × 106 spermatozoa mL–1 at 39°C under 5% CO2 during 20 h. Presumptive zygotes were denuded, washed, and transferred to 25-μL culture droplets (SOF + 5% FCS) at 39°C under 5% CO2, 90% of N2, and 5% O2 atmosphere with maximum humidity. Subsets of presumptive fertilized eggs and developing embryos were recovered at 6, 72, 120, and 192 h postinsemination (hpi) and processed for confocal whole-mount immunocytochemistry. The meiotic and mitotic spindles and chromosomes were visualised by immunofluorescent labelling of α-tubulin and 4′,6-diamindino-2-phenylindole (DAPI), respectively, and classified as normal if the chromosomes were correctly aligned or appropriately segregated, or abnormal if lagging chromosomes or abnormal chromosome segregation were observed. Samples were collected from 5 replicates (n = 50 zygotes/embryos per treatment, per timepoint) and a total of 157 spindles were observed. Logistic regression analysis was conducted to determine the probability of abnormal spindle formation. The incidence of spindle abnormality was regressed on time, treatment, and treatment by time. For all time points, there was significant reduction in the odds of abnormal spindle formation in control samples versus trilostane-treated samples (P < 0.001). In conclusion, our data imply a role for P4 signalling in maintaining spindle integrity during oocyte meiotic maturation and progression through the initial mitotic divisions of early embryo development in cattle.

Nuclear morphology, diameter and meiotic competence of buffalo oocytes relative to follicle size

2003 ◽  
Vol 15 (4) ◽  
pp. 223 ◽  
Author(s):  
Muhammad Rizwan Yousaf ◽  
Kazim Raza Chohan
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Nuclear Morphology ◽  
Vitro Fertilization ◽  
Size Dependent ◽  
Size Groups ◽  
Follicle Size ◽  
Fetal Bovine ◽  
Meiotic Competence

The nuclear morphology, diameter and in vitro meiotic competence of buffalo oocytes was compared relative to follicle size. Cumulus–oocyte complexes (COCs) were collected from 1–<2, 2–<3, 3–<4, 4–<6 and 6–<8 mm follicles from abattoir ovaries. Cumulus cells were removed using 3 mg mL−1 hyaluronidase in saline and repeated pipetting. Denuded oocytes were measured, fixed in 3% glutaraldehyde, stained with 4,6-diamidoino-2-phenylindole and evaluated for nuclear morphology, namely the stage of germinal vesicle (GV) development before in vitro maturation (IVM). The COCs from >2-mm follicles were matured in vitro in their respective size groups for 24 h in Medium 199 supplemented with 10 μg mL−1 follicle-stimulating hormone, 10 μg mL−1 luteinizing hormone, 1.5 μg mL−1 oestradiol, 75 μg mL−1 streptomycin, 100 IU mL−1 penicillin, 10 mM HEPES and 10% fetal bovine serum. Matured oocytes were fixed, stained and evaluated for GV status and meiotic development. The number of oocytes collected from follicles 1–<8 mm in diameter averaged 1.82 per ovary. Oocytes from follicles 1–<2 mm (107.7 ± 1.6 μm), 2–<3 mm (108 ± 1.1 μm) and 3–<4 mm (114.6 ± 1.3 μm) in diameter were smaller in diameter (P < 0.05) than oocytes from follicles 4–<6 mm (124.4 ± 1.3 μm) and 6–<8 mm (131.9 ± 1.4 μm) in diameter. A majority of oocytes (P < 0.05) from <4-mm follicles was at the initial stages of GV development (GV-I, II and III), whereas oocytes from 4–<6- and 6–<8-mm follicles were at the final stages of GV-IV (35.0 and 21.6% respectively) and GV-V (49.1 and 67.5% respectively). Poor IVM rates of 32.0% and 32.7% to metaphase (M)-II were observed for oocytes isolated from 2–<3- and 3–<4-mm follicles, respectively, whereas significantly (P < 0.05) more oocytes from 4–<6- and 6–<8-mm follicles reached M-II (67.1% and 79.1% respectively). In conclusion, buffalo oocytes displayed a size-dependent ability to undergo meiotic maturation and we suggest that oocytes from >4-mm follicles should be considered in buffalo in vitro fertilization systems for better results.

Impact of oocyte-secreted factors on its developmental competence in buffalo

Zygote ◽  
2017 ◽  
Vol 25 (3) ◽  
pp. 313-320 ◽  
Author(s):  
Swati Gupta ◽  
Sriti Pandey ◽  
Mehtab S. Parmar ◽  
Anjali Somal ◽  
Avishek Paul ◽  
...  
Keyword(s):  
Cumulus Cell ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Marker Genes ◽  
Enabling Factors ◽  
Secreted Factors ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Follicle Size

SummaryOocyte-secreted factors (OSFs) play an important role in the acquisition of oocyte developmental competence through bidirectional cross-talk between oocyte and cumulus cells via gap junctions. Thus, the present study was designed to investigate the effect of two OSFs, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the developmental competence of buffalo oocytes derived from two different follicle sizes. Cumulus–oocyte complexes (COCs) from large follicles (LF, >6 mm) or small follicles (SF, <6 mm) were collected and matured in vitro either in the presence of GDF9 or BMP15, or both, or with the denuded oocytes (DOs) as a source of native OSFs. Cleavage and blastocyst rates were significantly (P < 0.05) higher in LF-derived than SF-derived oocytes. Cleavage and blastocyst rates were significantly higher (P < 0.05) in the DOs and the combination groups compared with the control, GDF9 alone and BMP15 alone groups, both in LF-derived and SF-derived oocytes, although the cleavage and blastocyst rates did not differ significantly (P > 0.05) between DOs and combination groups. Relative mRNA analysis revealed significantly higher (P > 0.05) expression of the cumulus cell marker genes EGFR, HAS2, and CD44 in LF-derived than SF-derived oocyte; the expression of these markers was significantly higher (P > 0.05) in DOs and combination groups, irrespective of the follicle size. These results suggested that LF-derived oocytes have a higher developmental competence than SF-derived oocytes and that supplementation of GDF9 and BMP15 modulates the developmental competence of buffalo oocytes by increasing the relative abundance of cumulus-enabling factors and thereby increasing cleavage and the quality of blastocyst production.

Genomic expression profiles in cumulus cells derived from germinal vesicle and MII mouse oocytes

2016 ◽  
Vol 28 (11) ◽  
pp. 1798 ◽  
Author(s):  
Li Shao ◽  
Ri-Cheng Chian ◽  
Yixin Xu ◽  
Zhengjie Yan ◽  
Yihui Zhang ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Expression Patterns ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Genomic Expression ◽  
Oocyte Competence ◽  
Before And After

Cumulus cells (CCs) are distinct from other granulosa cells and the mutual communication between CCs and oocytes is essential for the establishment of oocyte competence. In the present study we assessed genomic expression profiles in mouse CCs before and after oocyte maturation in vitro. Microarray analysis revealed significant changes in gene expression in CCs between the germinal vesicle (GV) and metaphase II (MII) stages, with 2615 upregulated and 2808 downregulated genes. Genes related to epidermal growth factor, extracellular matrix (Ptgs2, Ereg, Tnfaip6 and Efemp1), mitochondrial metabolism (Fdx1 and Aifm2), gap junctions and the cell cycle (Gja1, Gja4, Ccnd2, Ccna2 and Ccnb2) were highlighted as being differentially expressed between the two development stages. Real-time polymerase chain reaction confirmed the validity and reproducibility of the results for the selected differentially expressed genes. Similar expression patterns were identified by western blot analysis for some functional proteins, including EFEMP1, FDX1, GJA1 and CCND2, followed by immunofluorescence localisation. These genes may be potential biomarkers for oocyte developmental competence following fertilisation and will be investigated further in future studies.

253 OOCYTE PREMATURATION IN THE PRESENCE OF MILRINONE IMPROVES NUCLEAR BUT NOT CYTOPLASMIC MATURATION OF MACAQUE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 224 ◽  
Author(s):  
E. C. Curnow ◽  
J. P. Ryan ◽  
D. M. Saunders ◽  
E. S. Hayes
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Molecular Probes ◽  
Developmental Competence ◽  
Oocyte Growth ◽  
North West ◽  
Significant Difference ◽  
Cytoplasmic Maturation

During oocyte growth chromatin configuration of the germinal vesicle (GV) oocyte undergoes modification in relation to changes in transcriptional activity crucial for conferring meiotic as well as developmental competence on the oocyte. In the macaque oocyte, there are 3 distinct GV states: GV1, noncondensed chromatin; GV2, an intermediate state; and GV3, condensed chromatin. The aim of this study was to test the effects of a prematuration culture (PMC) system, using the phosphodiesterase type 3 inhibitor milrinone (MIL), on the synchronization of GV chromatin to the GV3 stage and assess metaphase II (MII) oocyte reduced glutathione (GSH) content as a measure of cytoplasmic maturation. Reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. To assess the effect of PMC on GV chromatin status, immature oocytes retrieved from unstimulated ovaries were either fixed (2% paraformaldehyde+0.1% Triton-X100) immediately after follicular aspiration (t = 0) or after culture in a humidified atmosphere of 6% CO2 in air at 37°C for 24 h in modified Connaught Medical Research Laboratories medium (mCMRL) supplemented with 10% FCS (Hyclone, Logan, UT, USA) and 12.5 μM MIL in the absence (MILNil) or presence of 1.0 IU of FSH (MILFSH). For chromatin assessment, fixed GV oocytes were stained with 5 μg mL–1 of 4′,6-diamidino-2-phenylindole (Molecular Probes, Leiden, the Netherlands) and imaged using confocal microscopy. Following PMC, MILFSH oocytes were transferred to fresh mCMRL+FCS supplemented with 1.0 IU of recombinant human FSH and 1.0 IU of hLH and cultured for a further 30 h. Control and MILFSH oocytes were denuded of cumulus cells and assessed for maturation. The MII oocytes were prepared for GSH analysis, and total GSH content was determined using a commercial 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay kit (North-West Life Science). The MII rates were compared using chi-square. Differences in oocyte GSH content were compared using t-test. Significant differences were determined at P < 0.05. There was no significant difference in the proportion of oocytes remaining at the GV stage following 24 h of PMC in MILNil or MILFSH (42/44, 96% v. 32/35, 91%, respectively). However, there was a significant reduction in GV1 chromatin (15/49, 31% v. 28/54, 52% and 22/58, 38%) and a significant increase in GV3 chromatin (23/49, 47% v. 14/54, 26% and 16/58, 28%) observed in MILFSH oocytes compared with both MILNil and t = 0 oocytes, respectively. The MII rate of MILFSH oocytes following in vitro maturation was significantly higher compared with the MII rate of control in vitro matured oocytes (91/167, 55% v. 83/243, 34%). There was no significant difference in the GSH content of GV oocytes from the time of oocyte collection (t = 0) or GV oocytes following PMC in MILFSH (3.69 ± 0.16 and 4.14 ± 0.28 pmol/oocyte, n = 39–49 oocytes). The GSH content of control in vitro matured MII oocytes was significantly greater than that of MILFSH-treated MII oocytes (3.13 ± 0.16 v. 2.02 ± 0.04 pmol/oocyte, n =53–54 oocytes). The PMC supported high rates of nuclear maturation, but cytoplasmic maturation, assessed by GSH content, was negatively affected. Further assessment following fertilization and development is required to determine the practical utility of PMC in a primate in vitro maturation setting.

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