First proteomic analysis of diestrus and anestrus canine oocytes at the germinal vesicle reveals candidate proteins involved in oocyte meiotic competence

Several studies report that a two-step culture where mammalian oocytes are first kept under meiosis-arresting conditions (prematuration) followed by IVM is beneficial to embryo development. The most promising results were obtained by stratifying the oocyte population using morphological criteria and allocating them to different culture conditions to best meet their metabolic needs. In this study, horse oocytes were characterised to identify subpopulations that may benefit from prematuration. We investigated gap-junction (GJ) coupling, large-scale chromatin configuration and meiotic competence in compact and expanded cumulus–oocyte complexes (COCs) according to follicle size (<1, 1–2, >2cm) and season. Then we tested the effect of cilostamide-based prematuration in compact COCs collected from follicles <1 and 1–2cm in diameter on embryo development. Meiotic competence was not affected by prematuration, whereas COCs from follicles 1–2cm in diameter yielded embryos with a higher number of cells per blastocyst than oocytes that underwent direct IVM (P<0.01, unpaired Mann–Whitney test), suggesting improved developmental competence. Oocytes collected from follicles <1cm in diameter were not affected by prematuration. This study represents an extensive characterisation of the functional properties of immature horse oocytes and is the first report of the effects of cilostamide-based prematuration in horse oocyte IVM on embryo development.

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

Reproduction Fertility and Development ◽

10.1071/rd03006 ◽

2003 ◽

Vol 15 (4) ◽

pp. 223 ◽

Cited By ~ 15

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.

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The position of the germinal vesicle and the chromatin organization together provide a marker of the developmental competence of mouse antral oocytes

Reproduction ◽

10.1530/rep-09-0230 ◽

2009 ◽

Vol 138 (4) ◽

pp. 639-643 ◽

Cited By ~ 26

Author(s):

Michele Bellone ◽

Maurizio Zuccotti ◽

Carlo Alberto Redi ◽

Silvia Garagna

Keyword(s):

Germinal Vesicle ◽

Chromatin Organization ◽

Developmental Competence ◽

Cell Stage ◽

Morphological Marker ◽

Good Marker ◽

Chromatin Configuration ◽

Meiotic Competence

Based on their chromatin organization, antral oocytes can be classified into two classes, namely surrounded nucleolus (SN, chromatin forms a ring around the nucleolus), and not surrounded nucleolus (NSN, chromatin has a diffuse pattern). Oocytes of both classes are capable of meiotic resumption, but while SN oocytes, following fertilization, develop to term, NSN oocytes never develop beyond the two-cell stage. A recent study has shown that the position of the germinal vesicle (GV) can be used as a morphological marker predictive of oocyte meiotic competence, i.e. oocytes with a central GV have a higher meiotic competence than oocytes with an eccentric GV. In the present study, we have associated both markers with the aim of identifying, with more accuracy, the oocytes' developmental competence. Following their isolation, antral oocytes were classified on the basis of both SN and NSN chromatin configuration and their GV position, matured to metaphase II and fertilized in vitro. We demonstrated that the position of the GV is a good marker to predict the oocytes' developmental competence, but only when associated with the observation of the chromatin organization.

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Association between p34cdc2 levels and meiotic arrest in pig oocytes during early growth

Zygote ◽

10.1017/s0967199400002756 ◽

1995 ◽

Vol 3 (4) ◽

pp. 325-332 ◽

Cited By ~ 56

Author(s):

Yuji Hirao ◽

Youki Tsuji ◽

Takashi Miyano ◽

Akira Okano ◽

Masashi Miyake ◽

...

Keyword(s):

Germinal Vesicle ◽

Early Growth ◽

Catalytic Subunit ◽

The Other ◽

Antral Follicles ◽

Stages Of Growth ◽

Mean Diameter ◽

M Phase ◽

Chromatin Configuration ◽

Meiotic Competence

SummaryThe molecules involved in determining meiotic competence were determined in porcine oocytes isolated from preantral and antral follicles of different sizes. Oocytes isolated from preantral follicles had a mean diameter of 78 μm, contained diffuse filamentous chromatin in the germinal vesicle and were incapable of progressing from the G2 to the M phase of the cycle even after 72 h in culture. Oocytes from early antral follicles had a mean diameter of 105 μm, showed a filamentous chromatin configuration and about half resumed meiosis but arrested at metaphase I (MI) when cultured. Oocytes from mid-antral (3–4 mm) and large antral follicles (5–6 mm) had mean oocyte diameters of 115 and 119 μm respectively, contained condensed chromatin around the nucleolus and progressed to metaphase II (MII) in 48% and 93% of instances respectively. Analysis of p34cdc2, the catalytic subunit of maturation promoting factor (MPF), by immunoblotting indicates that the inability of small (78 μm) oocytes to resume meiosis is due, at least in part, to inadequate levels of the catalytic subunit of MPF. On the other hand, the inability of intermediate-sized (105 μm) oocytes from antral follicles to complete the first meiotic division by progressing beyond MI appears not to be limited by levels of p34cdc2, which are maximal by this stage. We postulate that an inadequacy of molecules other than p34cdc2 limits progression of MI to MII; the acquisition of these molecules during the final stages of growth may be correlated with the formation of the perinucleolar chromatin rim in the germinal vesicle.

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Electrical maturation of the murine oocyte: an increase in calcium current coincides withacquisition of meiotic competence

Zygote ◽

10.1017/s0967199400001295 ◽

1993 ◽

Vol 1 (1) ◽

pp. 49-60 ◽

Cited By ~ 23

Author(s):

Joan M. Murnane ◽

Louis J. DeFelice

Keyword(s):

Polar Body ◽

Reversal Potential ◽

Germinal Vesicle ◽

Outward Current ◽

Ca Channels ◽

Oocyte Growth ◽

Inward Current ◽

First Polar Body ◽

Meiotic Competence

SummaryWe have used the whole-cell recording technique to compare three stages of primary and secondary oocytes from F1 hybrid mice (C57BL/6J x SJL/J):neonatal germinal vesicle (NGV) stage primary oocytes from 10- to 20-day-old, prepubescent mice; mature germinal vesicle (MGV) stage primary oocytes from 12-week-old, post-pubescent, superovulated mice; first polar body (FPB) stage secondary oocytes from 12-week-old, post-pubescent mice during the normal oestrus cycle or following superovulation. NGV, MGV and FPB oocytes all exhibit two voltage-dependent currents: an inward, rapidly activating/inactivating current, and an outward, slowly activating/non-inactivating current. In 1.5 mmol/1 external Ca the average peak inward current is − 2.9, − 12.4 and − 13.8 μA/cm2 in NGV, MGV and FPB oocytes, respectively. In 20 mmol/1 Ca these currents increase and the reversal potential shifts to the right. The outward current decreases slightly with growth and development: at 40 mV test potentials, NGV oocytes have average outward currents of 8.9 μA/cm2, and MGV and FPB oocytes have currents of 5.0 and 5.5 μA/cm2, respectively. Thus, MGV oocytes express FPB current patterns. The reversal potentials, kinetics and pharmacology of the currents indicate that Ca channels carry the inward current and K channels carry the outward current. During growth in vivo a gradual depolarisation accompanies maturation. Resting potentialsranged from − 45 to − 30 mV in NGV oocytes to − 35 to − 17 mV in MGV oocytes to − 20 mV to − 3 mV in FPB oocytes. These data suggest that a selective increase occurs in the number of Ca channels during oocyte growth. This increase precedes nuclear maturation and coincides with the acquisition of meiotic competence.

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Oocyte Meiotic Competence in the Domestic Cat Model: Novel Roles for Nuclear Proteins BRD2 and NPM1

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.670021 ◽

2021 ◽

Vol 9 ◽

Author(s):

Daniela R. Chavez ◽

Pei-Chih Lee ◽

Pierre Comizzoli

Keyword(s):

Embryo Development ◽

Germinal Vesicle ◽

Domestic Cat ◽

Nuclear Proteins ◽

Protein Levels ◽

Nuclear Factors ◽

Chromatin Configuration ◽

Mammalian Female ◽

Adult Cat ◽

Meiotic Competence

To participate in fertilization and embryo development, oocytes stored within the mammalian female ovary must resume meiosis as they are arrested in meiotic prophase I. This ability to resume meiosis, known as meiotic competence, requires the tight regulation of cellular metabolism and chromatin configuration. Previously, we identified nuclear proteins associated with the transition from the pre-antral to the antral follicular stage, the time at which oocytes gain meiotic competence. In this study, the objective was to specifically investigate three candidate nuclear factors: bromodomain containing protein 2 (BRD2), nucleophosmin 1 (NPM1), and asparaginase-like 1 (ASRGL1). Although these three factors have been implicated with folliculogenesis or reproductive pathologies, their requirement during oocyte maturation is unproven in any system. Experiments were conducted using different stages of oocytes isolated from adult cat ovaries. The presence of candidate factors in developing oocytes was confirmed by immunostaining. While BRD2 and ASRGL1 protein increased between pre-antral and the antral stages, changes in NPM1 protein levels between stages were not observed. Using protein inhibition experiments, we found that most BRD2 or NPM1-inhibited oocytes were incapable of participating in fertilization or embryo development. Further exploration revealed that inhibition of BRD2 and NPM-1 in cumulus-oocyte-complexes prevented oocytes from maturing to the metaphase II stage. Rather, they remained at the germinal vesicle stage or arrested shortly after meiotic resumption. We therefore have identified novel factors playing critical roles in domestic cat oocyte meiotic competence. The identification of these factors will contribute to improvement of domestic cat assisted reproduction and could serve as biomarkers of meiotically competent oocytes in other species.

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280 EFFECTS OF HOT SEASON ON BOVINE OOCYTE QUALITY: HOW TO BYPASS THE POOR OOCYTE QUALITY DURING THIS SEASON?

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab280 ◽

2015 ◽

Vol 27 (1) ◽

pp. 229

Author(s):

L. Boccia ◽

E. Iacono ◽

B. Rossi ◽

B. Merlo

Keyword(s):

Degradation Products ◽

Germinal Vesicle ◽

Oocyte Quality ◽

Female Reproductive Tract ◽

Bovine Oocyte ◽

Germinal Vesicle Breakdown ◽

Serum Replacement ◽

Hot Season ◽

Meiotic Competence

Many authors attribute the decline of reproductive activity in summer to the heat stress, a multifactorial problem in which hyperthermia affects cellular function in various tissues of the female reproductive tract (Hansen et al. 2001; De Rensis et al. 2003). In particular, the combination of high temperatures and high humidity for a long period causes a reduced blood flow to uterus, oviducts, and ovaries, leading to a rise in the concentration of the degradation products of cellular activity. Therefore, the aim of this work was to elucidate the negative effect of the hot season on bovine oocyte quality and evaluate the influence of different factors on the acquisition of meiotic competence. In particular, meiotic competence of bovine oocytes recovered from animals housed at 44°28′00″ N, 11°26′00″ E during spring (March, 4–13°C) and summer (June, 16–27°C) was evaluated. Likewise, in summer the effect of an antioxidant, myo-inositol, the use of serum replacement (SR), and the use of oocytes recovered from cycling heifers (16–18 months) as compared to cows (>24 months) were tested. A total of 1346 abattoir-derived oocytes, equally divided for different experimental groups (over 6 replicates), were in vitro matured in TCM 199 supplemented with EGF (25 ng mL–1), IGF1 (100 ng mL–1), ITS supplement, pFSH-LH (0.1 IU each), and 10% FBS. Myoinositol was added at a concentration of 0, 15, 30, and 50 mM, while 10% SR was used alternatively to FBS. At the end of maturation period (20–22 h), oocytes were denuded and stained with 10 μg mL–1 of Hoechst 33342 at room temperature in the dark. After 15 min they were mounted on glass slides for evaluation of nuclear status using a Nikon Eclipse E400 microscope equipped with fluorescence filters. Nuclear configurations were classified as (a) germinal vesicle (GV), (b) germinal vesicle breakdown (GVBD), (c) metaphase I (M-I), (d) metaphase II (M-II), and (e) degenerated (DEG). Data are expressed as mean ± s.e.m. and were analysed by ANOVA (IBM SPSS Statistics) considering significance at P < 0.05. Oocyte quality of summer oocytes was significantly lower than spring counterparts as result of a higher rate of DEG (8.2 ± 0.6 v. 0.7 ± 0.6) and GV (5.4 ± 0.3 v. 0.4 ± 0.4, respectively; P < 0.05). Myo-inositol supplementation in IVM medium did not significantly affect either oocyte quality or meiotic competence in the hot season, such as the use of SR. When the oocytes were collected from cycling heifers ovaries during summer, the recovery rate of COC/ovary was significantly higher as compared to cows (4.5 v. 2.0), and a lower rate of DEG (1.8 ± 0.2; 8.2 ± 0.6) and GVBD (0.9 ± 0.6; 6.1 ± 0.3) was found (P < 0.05), even if the rate of GV (22.4 ± 0.1 v. 5.4 ± 0.3) was higher (P < 0.05) compared with cow. In conclusion, the hot season negatively affects oocyte quality, myo-inositol does not affect nuclear maturation, and SR can be used alternatively to FBS. The lower age of oocyte donor positively influenced the number of recoverable oocyte and degeneration rate.

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Germinal vesicle chromatin configuration and meiotic competence is related to the oocyte source in canine

Animal Reproduction Science ◽

10.1016/j.anireprosci.2006.12.016 ◽

2008 ◽

Vol 103 (3-4) ◽

pp. 336-347 ◽

Cited By ~ 13

Author(s):

H.S. Lee ◽

X.J. Yin ◽

Y.X. Jin ◽

N.H. Kim ◽

S.G. Cho ◽

...

Keyword(s):

Germinal Vesicle ◽

Chromatin Configuration ◽

Meiotic Competence

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Transcriptional activity associated with meiotic competence in fully grown mouse GV oocytes

Zygote ◽

10.1017/s0967199402004069 ◽

2002 ◽

Vol 10 (4) ◽

pp. 327-332 ◽

Cited By ~ 71

Author(s):

Honglin Liu ◽

Fugaku Aoki

Keyword(s):

Transcriptional Activity ◽

Polar Body ◽

Germinal Vesicle ◽

Cumulus Cells ◽

Meiotic Maturation ◽

Germinal Vesicle Breakdown ◽

First Polar Body ◽

Active Transcription ◽

Subsequent Activation ◽

Meiotic Competence

The involvement of cumulus cells and chromatin organisation in transcriptional activity was investigated. In addition, the relationship between transcriptional activity and meiotic competence in fully grown mouse oocytes was surveyed. Transcriptional activity was detected in fully grown oocytes in which chromatin did not surround the nucleolus in the germinal vesicle (NSN-type oocytes), but not in oocytes in which chromatin surrounded the nucleolus (SN-type oocytes). Cumulus cells seemed to downregulate transcriptional activity in NSN-type oocytes, since transcriptional activity was 3 times greater in the denuded NSN-type oocytes free of cumulus cells (DO oocytes) than in NSN-type oocytes enclosed in cumulus cells (COC oocytes). Higher transcriptional activity corresponded to lower germinal vesicle breakdown (GVB) competence of fully grown oocytes in culture. Although GVB occurred in nearly all (99%) the SN-type oocytes, it occurred in 88% of COC/NSN-type oocytes (cumulus-oocyte complex with SN-type configuration) and in 61% of DO/NSN-type oocytes (denuded oocytes with NSN-type configuration). There was a negative correlation between transcriptional activity and the capacity of a cell to complete the progression to the second metaphase (MII). In GVB oocytes, the percentage of first polar body (PBI) extrusion differed among COC/NSN-type (81%), DO/SN-type (66%), COC/NSN-type (47%) and DO/NSN-type (29%) oocytes. After activation with 10 mM Sr2+, the frequency of parthenogenetic activation was greater in SN-type oocytes (46.9%) than in transcriptionally active NSN-type oocytes (27.5%). These results suggest that transcriptional activity has a detrimental effect on the competence of meiotic maturation and subsequent activation in fully grown GV oocytes. Alternatively, active transcription in the fully grown oocytes suggests that they are still in the process of synthesising substances required for meiotic maturation and are not yet competent for these processes.

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Proteomic analysis of porcine oocytes during in vitro maturation reveals essential role for the ubiquitin C-terminal hydrolase-L1

Reproduction ◽

10.1530/rep-07-0079 ◽

2007 ◽

Vol 134 (4) ◽

pp. 559-568 ◽

Cited By ~ 26

Author(s):

Andrej Susor ◽

Zdenka Ellederova ◽

Lucie Jelinkova ◽

Petr Halada ◽

Daniel Kavan ◽

...

Keyword(s):

Proteomic Analysis ◽

Essential Role ◽

Specific Inhibitor ◽

Germinal Vesicle ◽

Cyclin B1 ◽

Cyclin Dependent Kinase ◽

Proteomic Approach ◽

Metaphase I

In this study, we performed proteomic analysis of porcine oocytes during in vitro maturation. Comparison of oocytes at the initial and final stages of meiotic division characterized candidate proteins that were differentially synthesized during in vitro maturation. While the biosynthesis of many of these proteins was significantly decreased, we found four proteins with increased biosynthetic rate, which are supposed to play an essential role in meiosis. Among them, the ubiquitin C-terminal hydrolase-L1 (UCH-L1) was identified by mass spectrometry. To study the regulatory role of UCH-L1 in the process of meiosis in pig model, we used a specific inhibitor of this enzyme, marked C30, belonging to the class of isatin O-acyl oximes. When germinal vesicle (GV) stage cumulus-enclosed oocytes were treated with C30, GV breakdown was inhibited after 28 h of culture, and most of the oocytes were arrested at the first meiosis after 44 h. The block of metaphase I–anaphase transition was not completely reversible. In addition, the inhibition of UCH-L1 resulted in elevated histone H1 kinase activity, corresponding to cyclin–dependent kinase(CDK1)–cyclin B1 complex, and a low level of monoubiquitin. These results supported the hypothesis that UCH-L1 might play a role in metaphase I–anaphase transition by regulating ubiquitin-dependent proteasome mechanisms. In summary, a proteomic approach coupled with protein verification study revealed an essential role of UCH-L1 in the completion of the first meiosis and its transition to anaphase.

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