246 INDUCTION OF MEIOTIC ARREST IN IMMATURE FELINE OOCYTES WITH ROSCOVITINE AND DIBUTYRYL CYCLIC-AMP

2009 ◽  
Vol 21 (1) ◽  
pp. 221
Author(s):  
C. B. Hanna ◽  
T. M. Glover ◽  
C. R. Long
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Control Culture ◽  
Basic Medium ◽  
Meiotic Arrest ◽  
Chi Square ◽  
Exact Test ◽  
Chromatin Configuration

Successful application of some assisted reproduction technologies in feline species requires the use of competent, meiotically mature ova, which can be difficult to obtain in sufficient quantities. Meiotic arrest of immature feline oocytes would allow the accumulation of oocytes over time to maximize laboratory resources. Two meiosis inhibitors commonly used in other species, roscovitine (ROS) and dbcAMP, were evaluated for the ability to maintain a germinal vesicle (GV) in immature feline oocytes after 24 h of in vitro culture. Feline ovaries were obtained from routine ovariohysterectomies and oocytes with homogenous cytoplasm and at least 2 layers of cumulus cells were selected. All oocytes were cultured in a basic medium modified from Gomez et al. (2000 Reprod. Fertil. Dev.) consisting of TCM-199 with Earle’s salts, 0.3% fraction V bovine serum albumin, 2.0 mm L-glutamine, 1.12 mm L-cysteine, 2.2 mm calcium lactate, 0.36 mm sodium pyruvate, 100 μm cysteamine, 10 ng μL–1 epidermal growth factor, 1 μg mL–1 estradiol, and 50 μg mL–1 gentamicin, with or without meiotic inhibitor. After 24 h of culture, cumulus cells were removed; oocytes were fixed, permeabilized, and stained with 5 μg mL–1 of Hoechst 33342; and chromatin configuration was assessed under ultraviolet fluorescence. In 4 replicates of Experiment 1, oocytes were cultured with either 25 nm ROS, 1 mm dbcAMP, both ROS and dbcAMP (Both), or without inhibitor (Control). A greater proportion of oocytes retained the GV when cultured with Both compared with ROS, dbcAMP, or Control (90.6, 67.8, 25.0, and 14.8%, respectively; chi-square P < 0.05), and ROS alone was superior to dbcAMP or Control. Culture of oocytes in the base medium plus 0.5 IU mL–1 eCG and 1.0 IU mL–1 hCG after arrest showed that pretreatment with Both did not decrease their ability to resume meiosis (87.9 v. 87.0%, respectively). Given the low percentage of oocytes with a retained GV in the presence of 1.0 mm dbcAMP, Experiment 2 evaluated the concentration of dbcAMP required to inhibit meiosis. Oocytes were cultured over 4 replicates in the base media containing 0, 0.1, 0.5, 1.0, 2.0, and 10.0 mm dbcAMP. After 24 h of culture, a greater number of oocytes were arrested at the GV stage when cultured in 10 mM dbcAMP (39.8%) than in 0, 0.1, 1.0, and 2.0 mM dbcAMP (22.0, 20.4, 25.8, and 26.3%, respectively; Fisher’s exact test P < 0.05), but there was no difference from those cultured in 0.5 mm dbcAMP (28.1%). For many species, 1.0 mm dbcAMP is commonly used to inhibit meiosis successfully for 24 h. However, dbcAMP alone did not effectively arrest meiosis, but when combined with ROS, it tended to improve meiotic arrest. Experiment 2 indicates that at least 10.0 mm dbcAMP is required to show a significant effect of meiotic inhibition in feline oocytes. Under these culture conditions, dbcAMP at levels up to 10 mm were not effective at inducing meiotic arrest. Interestingly, ROS and dbcAMP may act synergistically to successfully induce a reversible inhibition of meiosis in a high percentage of feline oocytes.

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.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

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.

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.

311 TEMPERATURE DURING OVERNIGHT HOLDING IN MEIOSIS INHIBITOR-FREE MEDIUM AFFECTS CHROMATIN CONFIGURATION AND MEIOTIC RESUMPTION IN EQUINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 244
Author(s):  
N. A. Martino ◽  
M. E. Dell'Aquila ◽  
M. F. Uranio ◽  
R. Lampignano ◽  
G. M. Lacalandra ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Chi Squared ◽  
Chromatin Configuration ◽  
Higher Temperature ◽  
Meiotic Inhibitors ◽  
Multiple Comparison Methods ◽  
And Control

Immature equine oocytes may be held overnight in an Earle's/Hanks' M199-based medium in the absence of meiotic inhibitors (EH medium) to schedule the onset of in vitro maturation. Holding in EH has been shown not to affect meiotic or developmental competence of equine oocytes (Choi et al. 2006 Theriogenology 66, 955–963). However, no studies have been performed to identify the mode by which this medium suppresses meiosis. We hypothesised that holding temperature may affect oocyte meiotic arrest. The effect of 3 holding temperatures (25, 30, 38°C) on chromatin status was investigated after Hoechst 33258 staining (Hinrichs et al. 2005 Biol. Reprod. 72, 1142–1150). Oocytes were recovered by scraping of follicles from slaughterhouse-derived ovaries. Data were analysed by Chi-squared test and one-way ANOVA followed by Dunn's or Holm-Sidak Multiple Comparison methods. A level of P < 0.05 was considered significant. There were no significant differences in chromatin configuration between oocytes held overnight at 25°C (25°C-held) and controls (immediately-fixed oocytes); the proportion of oocytes showing meiotic resumption was 1/27, 4% and 0/26, 0%, respectively (not significant, NS). In contrast, holding at higher temperature significantly increased meiosis resumption (14/38, 37% and 14/28, 50%, at 30 and 38°C, respectively; P < 0.01) and reduced the proportion of oocytes showing the most meiotically-competent germinal-vesicle (GV) configuration (condensed chromatin, CC; 24 to 29% v. 65 to 70% for control and 25°C-held, respectively; P < 0.05). Based on these results, a subsequent experiment was performed in which oocyte meiotic stage and mitochondrial (mt) potential of 25°C-held (n = 29) and control (n = 36) oocytes was evaluated. Nuclear chromatin, mt activity (MitoTracker orange), intracellular reactive oxygen species (ROS) levels (2′,7′-dichlorodihydrofluorescein diacetate, DCDHFDA), and mt/ROS colocalization (Pearson's coefficient) were analysed by epifluoscence and confocal microscopy (Martino et al. 2012 Fertil. Steril. 97, 720–728). Meiotic arrest after EH treatment at 25°C was confirmed (0/29, 0% v. 5/36, 14% for meiotic resumption in 25°C-held and controls, respectively; NS). At any GV stage, 25°C-held treatment had no effect on mt activity, ROS levels, or mt/ROS colocalization. For example, in CC oocytes, values for control and 25°C-held, respectively, were: MitoTracker, 547.8 ± 499.5 v. 722.9 ± 390.3; DCF fluorescence intensity, 278.5 ± 179.3 v. 378 ± 185, and mt/ROS colocalization, 0.5 ± 0.1 v. 0.5 ± 0.2; these were not significantly different (NS). In conclusion, EH holding at 25°C maintains meiotic arrest, viability, and mt potential of equine oocytes.

255 SPERM NUCLEAR DECONDENSATION ABILITY OF IN VITRO MATURED CANINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 225
Author(s):  
M. De los Reyes ◽  
J. Vergara ◽  
J. Palomino
Keyword(s):  
Cumulus Cells ◽  
Sperm Nucleus ◽  
Culture Conditions ◽  
Sperm Chromatin ◽  
Chromatin Decondensation ◽  
Chi Square ◽  
Cytoplasmic Maturation ◽  
Sperm Nuclei ◽  
Nuclear Decondensation

The sperm chromatin decondensation occurs when a spermatozoon enters into an oocyte during fertilization, and the effectiveness of this process is connected to the grade of oocyte cytoplasmic maturation. In this study chromatin sperm decondensation was evaluated after IVF of canine oocytes matured in vitro (IVM), comparing different durations of maturation. Cumulus–oocytes complexes (COC) for IVM were obtained from bitch ovaries after ovariohysterectomy, selecting those COC with compact cumulus cells and a homogeneous dark cytoplasm. The COC were matured in vitro for 0, 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM HEPES, 10% FCS, 0.25 mM pyruvate, 10 IU mL–1 of hCG, 300 IU mL–1 of penicillin, and 20 mg mL–1 of streptomycin at 38.5°C and 5% CO2. Fresh ejaculates from 3 adult dogs were centrifuged, and the sperm pellet was resuspended in fert-TALP medium. In each replicate, 100-μL fert-TALP drops containing 10 to 12 IVM oocytes after each culture time were co-culture with 2.5 × 106 spermatozoa mL–1 for 24 h under culture conditions. Soon after co-culture, all oocytes were denuded from cumulus cells and fixed in 3% paraformaldehyde. The nuclear stage of the oocytes and the appearance of the sperm nucleus were determined by 4′,6-diamidino-2-phenylindole staining under a fluorescence inverted microscope. For each treatment, at least 4 replicates were performed, and the data were compared statistically by chi-square test, using InfoStat Professional Program. A total of 800 oocytes were evaluated, and the percentages of oocytes with sperm penetration were 58% (138/238), 61% (108/177), 72% (118/165), and 70% (153/220) at 0, 48, 72, and 96 h of IVM, respectively. The percentage of sperm nuclear decondensation at each time point significantly increased up to 72 h of culture, showing 12, 34, 81, and 85% of sperm nuclei deconsated, respectively. The percentages of nuclear maturation also increased (P ≤ 0.05) with time, showing 0, 8, 20, and 27% of oocytes at second metaphase (MII) stage at 0, 48, 72, and 96 h of culture. The percentage of MII stage was much lower than that of chromatin decondensation in all maturing groups. These results suggest that canine oocytes matured in vitro are able to decondense the sperm chromatin during IVF, and this ability increases up to 72 h of culture. Nevertheless, cytoplasmic maturation, as evaluated by sperm chromatin decondensation, in canine oocytes matured in vitro may not be completely connected with nuclear development. This work was supported by Grant FONDECYT 1080618.

265 INHIBITION OF CLASS III PHOSPHATIDYLINOSITOL-3-KINASE, BY 3-METHYLADENINE, REVERSIBLY ARRESTS PORCINE OOCYTES AT GERMINAL VESICLE STAGE

2011 ◽  
Vol 23 (1) ◽  
pp. 230
Author(s):  
M. R. Park ◽  
M. K. Gupta ◽  
S. J. Uhm ◽  
S. T. Shin ◽  
Y. M. Han ◽  
...  
Keyword(s):  
Specific Inhibitor ◽  
Indirect Evidence ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Class Iii ◽  
Cleavage Rate ◽  
Chi Square ◽  
Meiotic Progression ◽  
Phosphatidylinositol 3

Phosphatidylinositol-3-kinases (PI3K) play pivotal roles in the meiotic progression of oocytes from metaphase I to metaphase II stage. This study evaluated the effect of 3-methyladenine (3MA), a specific inhibitor of Class III PI3K, on the meiotic progression of porcine oocytes. Immature porcine oocytes (n = 4744) retrieved from abattoir-derived oocytes were cultured in the absence or presence (10 mM) of 3MA for 22 h and evaluated for meiotic progression by florescent Hoechst 33342 staining. Data were analysed by chi-square test or ANOVA using SPSS software, and differences at P < 0.05 were considered significant. Results showed that 3MA treatment arrested the immature porcine oocytes at germinal vesicle (GV) stage in the presence (99.2 ± 0.8 v. 54.0 ± 10.1%) or absence (96.5 ± 1.8 v. 41.0 ± 17.6%) of cumulus cells. Furthermore, a significantly high proportion (60.9 ± 13.8%) of 3MA-treated oocytes acquired a nucleolus completely surrounded by a rim of highly condensed chromatin (GV-II stage). When immature oocytes, arrested at GV stage for 22 h by 3MA, were further cultured for 22 h in the absence of 3MA, 96.1 ± 1.5% of oocytes reached metaphase II stage at 42 h of in vitro maturation and did not differ (P > 0.05) from nontreated control oocytes with respect to their ability to fertilize, cleave (74.1 ± 1.2 v. 72.7 ± 2.8%), and form blastocyst (15.4 ± 1.5 v. 12.7 ± 0.6%) upon IVF or parthenogenetic activation (cleavage rate: 89.8 ± 1.7 v. 84.6 ± 5.1%; blastocyst rate: 44.3 ± 12.4 v. 45.1 ± 7.6%). These data suggest that 3MA reversibly blocks and synchronizes the meiotic progression of porcine oocytes at GV stage without affecting their ooplasmic maturation in terms of post-fertilization/activation in vitro embryonic development. Our data also provide indirect evidence for the likely participation of Class III PI3K in meiotic maturation of porcine oocytes beyond GV stage. This work was supported by grants (Code #200901FHT010305191 and #20070401034017) from BioGreen 21 program of RDA, Republic of Korea.

Phosphatidylinositol 3-kinase in cumulus cells is responsible for both suppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes

2003 ◽  
Vol 179 (1) ◽  
pp. 25-34 ◽  
Author(s):  
M Shimada ◽  
J Ito ◽  
Y Yamashita ◽  
T Okazaki ◽  
N Isobe
Keyword(s):  
High Activity ◽  
Cell Function ◽  
Kinase Inhibitor ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Basic Medium ◽  
Phosphatidylinositol 3 Kinase ◽  
Germinal Vesicle Breakdown ◽  
Phosphatidylinositol 3

In this study, we investigated the mechanisms of protein kinase B (PKB) activation and its role in cumulus cells during in vitro meiotic resumption of porcine oocytes. PKB activity in cumulus cells was significantly decreased by 12 h cultivation of cumulus-oocyte complexes (COCs) in basic medium. However, the addition of phosphodiesterase inhibitors, hypoxanthine or 3-isobutyl-1-methylxanthine, maintained the level of PKB activity in cumulus cells at comparable with that in cumulus cells just after collection from their follicles. When COCs were cultured with phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, PKB activity was significantly decreased, and both caspase 3 activity and the proportion of apoptotic cells were significantly increased as compared with those in cumulus cells just after collection from their follicles. Moreover, the inhibitory effect of hypoxanthine on spontaneous meiotic resumption was overcome by addition of LY294002. On the other hand, markedly high activity of PKB and high intensity of the phosphorylated PKB band were observed in cumulus cells of COCs which were cultured with FSH. The addition of 20 microM LY294002 to FSH-containing medium induced an apoptosis of cumulus cells, whereas little apoptotic-positive signal was detected in COCs cultured with 5 microM LY294002 and FSH. However, the inhibitory effects of LY294002 on progesterone production by cumulus cells and germinal vesicle breakdown in oocytes reached a maximum at 5 microM. Thus, high activity of the PI 3-kinase-PKB pathway in cumulus cells plays an important role in FSH regulation of cell function. Judging from these results, it is estimated that PI 3-kinase in cumulus cells is required for both the suppression of spontaneous meiotic resumption and the induction of gonadotropin-stimulated meiotic resumption.

scholarly journals 288 RELATIONSHIP BETWEEN CHROMATIN ORGANIZATION AND OOCYTE - CUMULUS CELL COMMUNICATION IN GERMINAL VESICLE STAGE BOVINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 294
Author(s):  
V. Lodde ◽  
C. Galbusera ◽  
S. Modina ◽  
M.S. Beretta ◽  
A. Lauria ◽  
...  
Keyword(s):  
Chromatin Condensation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Hoechst 33342 ◽  
Oocyte Growth ◽  
The Status ◽  
Chromatin Configuration ◽  
Bovine Oocytes

Chromatin configuration in the germinal vesicle (GV) undergoes dynamic changes during oocyte growth, and the progressive chromatin condensation has been related to the acquisition of embryonic developmental potential. However, little is known about the mechanisms that regulate chromatin remodeling. In immature mouse oocytes, chromatin condensation and redistribution around the nucleolus are associated with transcriptional repression in both in vivo-derived and in vitro-cultured oocytes in the presence of an intact cumulus oophorus (de la Fuente et al. 2001 Dev. Biol. 229, 224). It is widely accepted that oocyte communication with the somatic cell compartment is essential for both oocyte growth and acquisition of meiotic competence (Eppig et al. 1997 Hum. Reprod. 12, 127). In particular, cumulus cells play an active role in modulating the levels of transcription in the nucleoplasm and in perinuclear domains as well as in chromatin configuration of GV stage oocytes. In cattle, a heterogeneous population of cumulus-oocyte complexes (COCs) has been found after isolation from the follicle, and this is characterized by a different functional degree of gap junction-mediated communication (Luciano et al. 2004 Biol. Reprod. 70, 465). This study was aimed at investigating the possible correlation between the chromatin configuration of immature bovine oocytes and the status of communication between the oocyte and cumulus cells, and oocyte developmental competence. In the first experiment, 138 COCs, isolated from follicles 2–6 mm in diameter, were injected with a 3% solution of Lucifer Yellow to assess the communication status between oocytes and cumulus cells. Successively, COCs were freed of cells, and denuded oocytes (DOs) were stained with Hoechst 33342 to determine the chromatin configuration. In a second experiment, 330 COCs were denuded and stained with Hoechst 33342 in order to assess chromatin configuration and then matured in vitro according to their GV stage. After IVM, DOs were fertilized, and presumptive zygotes were cultured for 7 days at which time blastocyst rate was assessed. Data were analyzed by ANOVA and Fisher's PLSD test. Three stages of GV oocytes were identified: GVI, with filamentous chromatin distributed in the nucleoplasm; GVII, with chromatin condensed into thick clumps; and GVIII, with chromatin condensed into a single clump. The GVIII stage showed a lower proportion of functional open communication than the GVI and GVII groups (8.5 vs. 45.7 and 46.1, respectively, P < 0.05). However, when compared with each other, the GVI stage oocytes showed lower embryonic developmental competence (12.9 in GVI vs. 22.1 and 24.2 in GVII and GVIII, respectively, P < 0.05). Our findings indicate that the status of communication between oocytes and cumulus cells could be related to the chromatin organization in immature bovine oocytes. A direct correlation between the communications grade, the modulation of oocyte transcriptional activity, and the acquisition of oocyte developmental competence remain to be confirmed. This work was supported by a 2003 UniMi Grant.

184 INTERCELLULAR COUPLING AND CHROMATIN CONFIGURATION STATE IN HORSE OOCYTE - CUMULUS CELL COMPLEXES OF DIFFERENT ORIGINS

2013 ◽  
Vol 25 (1) ◽  
pp. 241
Author(s):  
V. Lodde ◽  
S. Colleoni ◽  
F. Franciosi ◽  
C. Dieci ◽  
I. Tessaro ◽  
...  
Keyword(s):  
Lucifer Yellow ◽  
Chromatin Condensation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Cumulus Oophorus ◽  
Follicle Diameter ◽  
Chromatin Configuration ◽  
Different Origins

Fewer follicles and of variable size are found at any time on the mare ovary compared with other livestock species, also influenced by seasonal variation. This is reflected on the population of cumulus–oocyte complexes (COC) collected that is characterised by a high heterogeneity. Recovered immature oocytes presumably need different culture conditions. The main factors contributing to this high heterogeneity are the follicle diameter, the status of the cumulus oophorus, and the reproductive seasonality. Therefore, the aim of the present study was to assess the nuclear chromatin configuration and the oocytes–cumulus cell gap junction-mediated communication (GJC) functionality in COC of different origins because these parameters are indicative of the oocyte’s metabolic state and should be taken into account when designing IVM strategies. The COC with compact (Cp) or expanded (Ex) cumulus oophorus were collected from follicles of different diameters (<1, 1–2, and >2 cm) in October–November, January–February, and April–May. The GCJ functionality was assessed by Lucifer Yellow microinjection and chromatin configuration was evaluated by Hoechst and Lacmoid staining, after cumulus cells removal. Data were obtained from a total of 1003 oocytes and were analysed by chi-squared test. Overall, GJC functionality was impaired in the majority of Ex COC in each follicle category, even though a certain proportion of them had open GJC (% of Ex COC with open GJC was 39.7, 29.3, and 39.3 in <1, 1–2, and >2 cm follicles, respectively). Moreover, the proportion of Ex COC with open GJC did not differ significantly between periods (% of Ex COCs with open GJC in October–November, January–February, and April–May was, respectively, 43.3, 28.6, and 41.7 in <1 cm follicles; 45.5, 19.3, and 26.47 in 1–2 cm follicles; 66.7, 50, and 16.7 in >2 follicles). On the contrary, the majority of Cp COC from follicles <1 and 1–2 cm, showed open GCJ in October–November and April–May, whereas they decreased significantly in January–February. This tendency was not maintained in Cp COC from follicles >2 cm, where GJC functionality did not differ significantly between periods (% of Cp COC with open GJC in October–November, January–February, and April–May was, respectively, 74.4, 35.7, and 75 in <1 cm follicles; 73.8, 42.1, and 67.7 in 1–2 cm follicles; 58.3, 58.3, and 68.6 in >2-cm follicles). Chromatin configuration analysis revealed that the highest proportion (23.9%) of oocytes with fibrillar chromatin was found in Cp oocytes from <1 cm follicles, whereas the proportion of oocytes with fibrillar chromatin ranged from 5.4 to 12.5% in the other groups. Moreover, the increase in follicle diameter was generally associated with an increase of chromatin condensation in Cp COC. Interestingly, the chromatin configuration distribution did not differ significantly among seasons. Our data could be useful in setting up new in vitro cultural strategies aimed to improve horse-assisted reproductive technology efficiency as well as in the understanding of horse oocyte biology. Funding: Grant no. 26096200 ‘Ex Ovo Omnia’ from Regione Sardegna & Lombardia and ‘Dote Ricerca Applicata’ (VL and IT).

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