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.

Efficiency and kinetics of the in vitro fertilization process in bovine oocytes with different meiotic competence

Zygote ◽  
2007 ◽  
Vol 15 (3) ◽  
pp. 251-256
Author(s):  
J. Horakova ◽  
K. Hanzalova ◽  
Z. Reckova ◽  
P. Hulinska ◽  
M. Machatkova
Keyword(s):  
Growth Phase ◽  
Cumulus Cells ◽  
Vitro Fertilization ◽  
Fertilization Process ◽  
Follicular Wave ◽  
Follicle Size ◽  
Kinetics Of ◽  
Meiotic Competence ◽  
Dominant Phase

SummaryThe aim of the study was to investigate the efficiency and kinetics of fertilization in oocytes with different meiotic competence, as defined by the phase of the follicular wave and follicle size. Oocytes were recovered from cows with synchronized estrus cycles, slaughtered in either the growth (day 3) or the dominant (day 7) phase, separately from large, medium and small follicles. The oocytes were matured and fertilized by a standard protocol. Twenty-four hours after fertilization, the oocytes were denuded from cumulus cells, fixed and stained with bisbensimid Hoechst–PBS. Fertilization was more efficient and the first cleavage was accelerated in growth phase-derived oocytes, as shown by significantly higher (p ≤ 0.01) proportions of both normally fertilized and cleaved oocytes (68.8 and 25.1%), in comparison with dominant phase-derived oocytes (44.2 and 10.3%). In the growth-phase derived oocytes, proportions of normally fertilized and cleaved oocytes were significantly higher (p ≤ 0.01) in oocytes from large (100.0 and 36.4%) and medium (83.3 and 36.5%) follicles than in those from small (54.8 and 14.6%) follicles. The dominant phase-derived oocytes showed higher proportions of normally fertilized and cleaved oocytes in the populations recovered from small (51.5 and 10.0%) and medium (43.1 and 12.0%) follicles than in those from large (25.0 and 0%) follicles; however, the differences were not significant. It can be concluded that: (i) efficiency and kinetics of fertilization differ in relation to oocyte's meiotic competence; (ii) improved development of embryos from oocytes with greater meiotic competence is associated with a more effective fertilization process.

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.

scholarly journals MicroRNA-378 regulates oocyte maturation via the suppression of aromatase in porcine cumulus cells

2015 ◽  
Vol 308 (6) ◽  
pp. E525-E534 ◽  
Author(s):  
Bo Pan ◽  
Derek Toms ◽  
Wei Shen ◽  
Julang Li
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cell ◽  
Specific Effect ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Cumulus Expansion ◽  
Vitro Fertilization ◽  
And Function

We sought to investigate whether miR-378 plays a role in cumulus cells and whether the manipulation of miRNA levels in cumulus cells influences oocyte maturation in vitro. Cumulus-oocyte complexes (COCs) from ovarian follicles had significantly lower levels of precursor and mature miR-378 in cumulus cells surrounding metaphase II (MII) oocytes than cumulus cells surrounding germinal vesicle (GV) oocytes, suggesting a possible role of miR-378 during COC maturation. Overexpression of miR-378 in cumulus cells impaired expansion and decreased expression of genes associated with expansion ( HAS2, PTGS2) and oocyte maturation ( CX43, ADAMTS1, PGR). Cumulus cell expression of miR-378 also suppressed oocyte progression from the GV to MII stage (from 54 ± 2.7 to 31 ± 5.1%), accompanied by a decrease of growth differentiation factor 9 ( GDF9), bone morphogenetic protein 15 ( BMP15), zona pellucida 3 ( ZP3), and CX37 in the oocytes. Subsequent in vitro fertilization resulted in fewer oocytes from COCs overexpressing miR-378 reaching the blastocyst stage (7.3 ± 0.7 vs. 16.6 ± 0.5%). miR-378 knockdown led to increased cumulus expansion and oocyte progression to MII, confirming a specific effect of miR-378 in suppressing COC maturation. Aromatase (CYP19A1) expression in cumulus cells was also inhibited by miR-378, leading to a significant decrease in estradiol production. The addition of estradiol to IVM culture medium reversed the effect of miR-378 on cumulus expansion and oocyte meiotic progression, suggesting that decreased estradiol production via suppression of aromatase may be one of the mechanisms by which miR-378 regulates the maturation of COCs. Our data suggest that miR-378 alters gene expression and function in cumulus cells and influences oocyte maturation, possibly via oocyte-cumulus interaction and paracrine regulation.

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.

341 IN VITRO MATURATION AND IN VITRO FERTILIZATION OF ALPACA (VICUGNA PACOS) OOCYTES: EFFECT OF TIME OF INCUBATION ON NUCLEAR MATURATION AND CLEAVAGE

2010 ◽  
Vol 22 (1) ◽  
pp. 327 ◽  
Author(s):  
W. Huanca ◽  
R. Condori ◽  
J. Cainzos ◽  
M. Chileno ◽  
L. Quintela ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Hypodermic Needle ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Maturation Time ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Maturation Medium

Experiments were carried out to evaluate the effect of incubation time on nuclear maturation (Experiment 1) and determine the cleavage rate of alpaca oocytes after of IVF time (Experiment 2) In Experiment 1, CCOs were collected from slaughterhouse ovaries and transported to the laboratory in a thermos flask containing a saline solution 0.9% with antibiotic antimycotic at 35°C. CCOs were aspirated from follicles >2 mm and pooled in a conical tube to sedimentation previous to evaluation under stereomicroscope and CCOs with a cytoplasm homogeneous and 2 or more layers of cumulus cells were transferred to plates with a 40-μL drop of maturation medium TCM-199 supplemented with 10% FCS (v : v) plus 0.5 μg mL-1 FSH, 10 μg mL-1 hCG, 0.2 mM sodium pyruvate, 50 μg mL-1 gentamicine, and 1 μg mL-1 Estradiol under mineral oil with 10-12 oocytes/drop. Oocytes were incubated under the following maturation times: 30, 34, and 38 h at 39°C in an atmosphere of 5% CO2 and high humidity. After each maturation time, CCOs were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1 mgmL-1 of hyaluronidase and fixed in ethanol: acetic acid (3 : 1). Oocytes were placed on the slide with minimum medium and stained with 1% orcein for 5 min The slides were examined under a phase contrast microscope at × 400 to evaluate status of nuclear maturation and classified as germinal vesicle (GV); metaphase I (M-I), anaphase-telophase; metaphase II (M-II) and degenerated. Experiment 2: The same maturation method as Experiment 1 was used. Testes were collected of mature males from slaughterhouse and transported to the laboratory. Caudal epididymide was isolated. A prick was made on the convoluted tubules with a sterile hypodermic needle and the fluid, rich in spermatozoa, was aspirated in syringes containing 2 mL of Tris-fructose egg yolk extender. Motile spermatozoa were obtained by centrifugation: 700 g on a Percoll discontinuous gradient (22.5 :45.0%) for 25 min. The supernatant was removed by aspiration and pellet (containing viable spermatozoa) was resuspended in TL stock. Spermatozoa and oocytes were co-incubated for 18-20 h at 39°C with 5% CO2 and then cultivated in TCM-199 supplemented with 10% FCS (v: v), 0.2 mM sodium pyruvate, and 50 μg mL-1 gentamicine and evaluated at 48 h. Data were subjected to ANOVA. For Experiment 1, the proportions of oocytes reaching M-II stage was 18.9 ± 15.7, 42.9 ± 16.2, and 65.8 ± 8.1% for the 30, 34, and 38 h of culture, respectively, with difference to maturation time (P < 0.05). For Experiment 2, the cleavage rate was 9.5, 7.7, and 15.4% to 30, 34, and 38 h after of fertilization time 48 h culture. These results indicate that 38 or more h is required for the maturation and fertilization of alpaca oocytes. Grant 064 FINCyT-PIBAP 2008.

Methodological approaches for vitrification of bovine oocytes

Zygote ◽  
2020 ◽  
pp. 1-11
Author(s):  
Linda Dujíčková ◽  
Alexander V. Makarevich ◽  
Lucia Olexiková ◽  
Elena Kubovičová ◽  
František Strejček
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Oocyte Vitrification ◽  
Factors Affecting ◽  
Oocyte Stage ◽  
Follicle Size ◽  
Oocyte Donors ◽  
Methodological Approaches ◽  
Bovine Oocytes

Summary Numerous factors affect vitrification success and post-thaw development of oocytes after in vitro fertilization. Therefore, elaboration of an optimal methodology ensuring higher cryotolerance of oocytes and subsequent blastocyst yield is still of great interest. This paper describes and evaluates critical factors affecting the success of oocyte vitrification. In particular, an appropriate oocyte stage such as maturation status (germinal vesicle stage, metaphase II stage), presence/absence of cumulus cells before vitrification, and the effect of follicle size, as well as different culture systems and media for in vitro production of embryos, the types and concentrations of cryoprotectants, and cooling and warming rates at vitrification are considered. Special attention is paid to various cryocarriers used for low-volume vitrification, which ensures safe storage of oocytes/embryos in liquid nitrogen and their successful post-thaw recovery. At the end, we focussed on how age of oocyte donors (heifers, cows) influences post-thaw development. This review summarizes results of recently published studies describing different methodologies of cryopreservation and post-thaw oocyte development with the main focus on vitrification of bovine oocytes.

scholarly journals The Differential Metabolomes in Cumulus and Mural Granulosa Cells from Human Preovulatory Follicles

2021 ◽  
Author(s):  
Er-Meng Gao ◽  
Bongkoch Turathum ◽  
Ling Wang ◽  
Di Zhang ◽  
Yu-Bing Liu ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Cumulus Cells ◽  
Cholesterol Transport ◽  
Vitro Fertilization ◽  
Expression Of Genes ◽  
Preovulatory Follicles ◽  
Morphological Differences

AbstractThis study evaluated the differences in metabolites between cumulus cells (CCs) and mural granulosa cells (MGCs) from human preovulatory follicles to understand the mechanism of oocyte maturation involving CCs and MGCs. CCs and MGCs were collected from women who were undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. The differences in morphology were determined by immunofluorescence. The metabolomics of CCs and MGCs was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by quantitative polymerase chain reaction (qPCR) and western blot analysis to further confirm the genes and proteins involved in oocyte maturation. CCs and MGCs were cultured for 48 h in vitro, and the medium was collected for detection of hormone levels. There were minor morphological differences between CCs and MGCs. LC-MS/MS analysis showed that there were differences in 101 metabolites between CCs and MGCs: 7 metabolites were upregulated in CCs, and 94 metabolites were upregulated in MGCs. The metabolites related to cholesterol transport and estradiol production were enriched in CCs, while metabolites related to antiapoptosis were enriched in MGCs. The expression of genes and proteins involved in cholesterol transport (ABCA1, LDLR, and SCARB1) and estradiol production (SULT2B1 and CYP19A1) was significantly higher in CCs, and the expression of genes and proteins involved in antiapoptosis (CRLS1, LPCAT3, and PLA2G4A) was significantly higher in MGCs. The level of estrogen in CCs was significantly higher than that in MGCs, while the progesterone level showed no significant differences. There are differences between the metabolomes of CCs and MGCs. These differences may be involved in the regulation of oocyte maturation.

scholarly journals A Brief Incubation of Cumulus-Enclosed Mouse Eggs in a Calcium-Free Medium Containing a High Concentration of Calcium-Chelator Markedly Improves Preimplantation Development

2020 ◽  
Vol 17 (10) ◽  
pp. 3505
Author(s):  
Valeria Merico ◽  
Silvia Garagna ◽  
Maurizio Zuccotti
Keyword(s):  
In Vitro Fertilization ◽  
Mammalian Species ◽  
Developmental Rate ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Preimplantation Development ◽  
High Concentration ◽  
Vitro Fertilization ◽  
Positive Effects

The presence of cumulus cells (CCs) surrounding ovulated eggs is beneficial to in vitro fertilization and preimplantation development outcomes in several mammalian species. In the mouse, this contribution has a negligible effect on the fertilization rate; however, it is not yet clear whether it has positive effects on preimplantation development. Here, we compared the rates of in vitro fertilization and preimplantation development of ovulated B6C3F1 CC-enclosed vs. CC-free eggs, the latter obtained either after a 5 min treatment in M2 medium containing hyaluronidase or after 5–25 min in M2 medium supplemented with 34.2 mM EDTA (M2-EDTA). We found that, although the maintenance of CCs around ovulated eggs does not increment their developmental rate to blastocyst, the quality of the latter is significantly enhanced. Most importantly, for the first time, we describe a further quantitative and qualitative improvement, on preimplantation development, when CC-enclosed eggs are isolated from the oviducts in M2-EDTA and left in this medium for a total of 5 min prior to sperm insemination. Altogether, our results establish an important advancement in mouse IVF procedures that would be now interesting to test on other mammalian species.

scholarly journals Localization and gene expression of steroid sulfatase by RT-PCR in cumulus cells and relationship to serum FSH levels observed during in vitro fertilization

2005 ◽  
Vol 2 (1) ◽  
Author(s):  
Yukiko Otsuka ◽  
Atsushi Yanaihara ◽  
Shinji Iwasaki ◽  
Junichi Hasegawa ◽  
Takumi Yanaihara ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Cumulus Cells ◽  
Steroid Sulfatase ◽  
Rt Pcr ◽  
Vitro Fertilization
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