scholarly journals The Expression Pattern of Prostaglandin-Endoperoxide Synthase-2 in Immature Oocytes and Surrounding Cumulus Cells May Explain A Disrupted Oocyte Maturation Process

2020 ◽  
pp. 1
Author(s):  
Goktan Kuspinar ◽  
Berrin Avcı
Keyword(s):  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Cumulus Cells ◽  
Control Group ◽  
Gene Expressions ◽  
Immature Oocytes ◽  
Prostaglandin Endoperoxide ◽  
Follicle Diameter ◽  
Prostaglandin Endoperoxide Synthase ◽  
Follicular Fluids

<p><strong>Objective:</strong> To evaluate the correlation between the gene expression of prostaglandin-endoperoxide synthase-2 in the cumulus-oocyte complex with the level of prostaglandin-endoperoxide synthase-2 in follicular fluid and follicle diameter, oocyte maturation, and fertilization capacity.</p><p><strong>Study Design:</strong> As the study group, 26 cumulus-oocyte complexes and 26 follicular fluids obtained from immature (n=10) or unfertilized mature oocytes (n=16) and as the control group, 26 cumulus complexes and 26 follicular fluids surrounding mature and fertilized oocytes were retrieved one by one totally from 32 patients in 32 intracytoplasmic sperm injection cycles. </p><p><strong>Results:</strong> There was no significant efficacy of prostaglandin-endoperoxide synthase-2 gene expressions in cumulus complexes and the level of prostaglandin-endoperoxide synthase-2 in follicular fluids in terms of oocyte maturation stage. The level of prostaglandin-endoperoxide synthase-2 in follicular fluids and follicle diameters showed a significantly positive correlation in the mature and fertilized oocyte group (r=0.414; p=0.035). </p><p><strong>Conclusions:</strong> Although the prostaglandin-endoperoxide synthase-2 gene expressions in immature oocytes and their cumulus cells were similar to those in oocytes that have completed their nuclear and cytoplasmic maturation, the level of prostaglandin-endoperoxide synthases-2 in the follicular fluid and follicle diameter correlation may lead to new clinical approaches in cases of premature follicular rupture before oocyte maturation is completed.</p>

338 EFFECTS OF CUMULUS CELLS AND FOLLICULAR FLUID ON PLASMINOGEN ACTIVATOR ACTIVITY AND OOCYTE MATURATION IN VITRO IN THE PIG

2006 ◽  
Vol 18 (2) ◽  
pp. 276
Author(s):  
C.-K. Park ◽  
J.-Y. An ◽  
S.-J. Sa ◽  
H.-T. Cheong ◽  
B.-K. Yang ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Sodium Dodecyl ◽  
Cumulus Cells ◽  
The Other ◽  
Tissue Type ◽  
Bromophenol Blue ◽  
Maturation In Vitro

Plasminogen activators (PAs) are serine proteases, known to be secreted by a large number of cell type. PAs are reported to play a role in variety of physiologic processes, including fibrinolysis, ovulation, mammary involution, implantation, and fertilization. The present study investigated the effects of cumulus cells and porcine follicular fluid (pFF) on PA activity and oocyte maturation in vitro in the pig. Porcine oocytes were harvested from slaughterhouse ovaries, selected, and matured in modified North Carolina State University-23 (NCSU-23) media. After culture, cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were separately put into microtubes containing 20 �L of sample buffer [5.0% (w:v) sodium dodecyl sulfate, 20% (v:v) glycerol, and 0.0025% (w:v) bromophenol blue in 0.125 M Tris-HCl buffer] and frozen at -80�C until used for zymographic analysis. Differences in data were evaluated by Duncan's multiple-range test using the General Linear Models procedure in the Statistical Analysis System (SAS Institue, Inc., Cary, NC, USA). To determine the effect of porcine follicular fluid (pFF) on PA activity in porcine oocytes during maturation, the COCs and DOs were incubated in NCSU-23 medium with or without 10% (v/v) pFF for 0, 24, or 48 h. In the presence of cumulus cells, the proportions of oocytes matured to metaphase-II stage were significantly (P < 0.05) higher in medium with pFF than without pFF (69.8% vs. 37.7%, respectively). When COCs and DOs were cultured in the presence of pFF, tissue-type PA (tPA), urokinase-type PA (uPA), and tPA-PA inhibitor (tPA-PAI) were observed in COCs, and PA activities were higher at 48 h than 24 h. However, no PA activity was detected in DOs. Under the same conditions, when COCs and DOs were cultured in the absence of pFF, tPA and tPA-PAI were observed in COCs, and PA activities were increased as duration of culture increased. However, no PA activity was detected in DOs. When porcine oocytes were cultured in the presence of pFF, the activities of tPA-PAI, tPA, and uPA were observed in conditioned medium with COCs and DOs cultured for 24 h and 48 h. In the absence of pFF, PA activities were observed only in conditioned medium with COCs, and no PA activities were detected in conditioned medium with DOs. On the other hand, three plasminogen-dependent lytic bands (tPA-PAI, tPA, and uPA) were observed in pFF cultures. Particularly uPA activity was higher than the other kinds of PA activity. When oocytes and cumulus cells were separated from porcine COCs at 0 h of cultrue, tPA-PAI, tPA, and uPA were detected in cumulus cells at 48-h culture, but no PA activities were in DOs. The presence of pFF and cumulus cells in maturation medium stimulated not only nuclear and cytoplasmic maturation in porcine COCs, but also PA production by cumulus cells and COCs. It is possible that PAs produced by cumulus cells migrated through the gap junction between oocyte and cumulus cells. These results suggest that porcine oocytes have no ability to produce PA themselves.

60 EFFECTS OF AY9944 A-7 ON MEIOTIC RESUMPTION OF PORCINE OOCYTES AND CUMULUS CELL EXPANSION

2016 ◽  
Vol 28 (2) ◽  
pp. 160
Author(s):  
S. Lee ◽  
C. Khoirinaya ◽  
J.-X. Jin ◽  
G. A. Kim ◽  
B.-C. Lee
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Cell Expansion ◽  
Cholesterol Biosynthesis ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Mitogen Activated Protein Kinase ◽  
Control Group ◽  
Cumulus Expansion

In vitro studies on mammalian oocytes have shown that follicular fluid-meiosis activating sterol (FF-MAS) can overcome the inhibitory effect of hypoxanthine (Hx) on the resumption of meiosis. FF-MAS, an intermediate in the cholesterol biosynthesis pathway, is converted to testis meiosis–activating sterol by a sterol Δ14-reductase. AY9944 A-7, an inhibitor of Δ14-reductase and Δ7-reductase, induces accumulation of FF-MAS by inhibiting its metabolism. The aim of this study was to evaluate the effects of AY9944 A-7 on meiotic resumption of porcine oocytes, cumulus cell expansion, and gene expression related to M-phase-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and oocyte maturation in oocytes and related to cumulus expansion in cumulus cells. In experiment 1, 1136 cumulus-oocyte complexes (COCs) were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in addition to a meiotic inhibitor (Hx, 4 mM) for 44 h. Oocytes treated with 10 and 20 μM AY9944 A-7 in the presence of Hx had significantly higher GVBD and M2 rates than the control group. However, 40 μM AY9944 A-7 significantly decreased GVBD and M2 rates and increased degeneration of oocytes compared with other groups. In experiment 2, 600 COCs were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in the absence of Hx for 44 h. Cumulus expansion of 40 μM AY9944 A-7 treated group was significantly decreased compared with other groups. In experiment 3, we evaluate the effects of AY9944 A-7 on gene expression, and the experiment was replicated four times. Data on gene expression were analysed using Student’s t-test. Oocytes treated with 10 μM AY9944 A-7 increased expression of genes involved in MPF (Cyclin B and Cdc2), MAPK (C-mos), and oocyte maturation (GDF9 and BMP15). Cumulus cells treated with 10 μM AY9944 A-7 decreased cumulus expansion-related genes (Has2, Tnfaip6, Ptgs2, and Ptx-3). In conclusion, our results suggest that although 10 μM AY9944 A-7 decreased cumulus expansion-related genes, there was no difference in cumulus expansion and it induced meiotic resumption of porcine oocytes with increased MPF, MAPK, and oocyte maturation-related genes. Further studies are needed to evaluate the effect of AY9944 A-7 on porcine embryo development. This study was supported by Ministry Of Trade, Industry & Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

scholarly journals Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro

Reproduction ◽  
2008 ◽  
Vol 136 (1) ◽  
pp. 9-21 ◽  
Author(s):  
Ikkou Kawashima ◽  
Tetsuji Okazaki ◽  
Noritaka Noma ◽  
Masahide Nishibori ◽  
Yasuhisa Yamashita ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Chorionic Gonadotropin ◽  
Follicular Fluid ◽  
Culture System ◽  
Expression Patterns ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Antral Follicles

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression ofLHCGRandPGRin cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-inducedLHCGRexpression in cumulus cells in culture. The expression ofLHCGRmRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation ofPGRandLHCGRin cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observedin vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.

scholarly journals TNFR1 is Upregulated and Mediates Apoptosis in Cumulus Cells of Women With Polycystic Ovarian Syndrome

2021 ◽  
Author(s):  
Yaping Jiang ◽  
Rui Jiang ◽  
Peng Zhang ◽  
qiong Yu ◽  
Hongping Ba ◽  
...  
Keyword(s):  
Polycystic Ovary Syndrome ◽  
Signaling Pathway ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Polycystic Ovary ◽  
Cumulus Cells ◽  
Control Group ◽  
Ovary Syndrome ◽  
Tnf Α ◽  
Pcos Group

Abstract Purpose To investigate the changes of human granulosa cell, TNFR1, TNFR2 and their downstream molecules in patients with polycystic ovary syndrome (PCOS) and the control group. Methods We recruited infertile women with polycystic ovary syndrome (n = 30) and compared them with infertility due to fallopian tube obstruction(n = 30, control group). The ovaries were stimulated with GnRH agonists and gonadotropins. Follicular fluid from large follicles ([14 mm]) was pooled and granulosa cells (GCs) were separated by a cellular filter. The TNF-α level of follicular fluid was measured by ELISA. TUNEL assay were used to detect the apoptosis of purified GCs. Real-time PCR and Western blotting were used to detect the expression of TNF-related signaling molecules in GCs. Results The rate of high quality embryos in the PCOS group was lower than that in the control group. There were higher percentages of apoptosis in GCs of PCOS patients than in the control group. TNF-α is upregulated in follicular fluid of PCOS patients. TNFR1 and caspase-3 mRNA level were signifificantly higher in PCOS group than in the control group. TNF-α-mediated apoptosis of PCOS granulosa cells was mainly dependent on TNFR1.The TNF-α/TNFR1 signaling pathway mediates apoptosis rather than survival in cumulus cells of PCOS patients. Conclusions TNF-α expression was upregulated in follicular fluid of PCOS patients, and TNFR1 overexpression in female granulosa cells of PCOS was associated with higher levels of apoptosis in these cells, suggesting that the TNF-α/TNFR1 signaling pathway may be a candidate for higher apoptosis in female granulosa cells of PCOS.

scholarly journals Intrafollicular and Systemic Dopamine, Noradrenaline and Adrenaline Concentrations in Cycling Mares

Animals ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1896
Author(s):  
Katiuska Satué ◽  
Esterina Fazio ◽  
Maria Dolores Rubio ◽  
Cristina Cravana ◽  
Pietro Medica
Keyword(s):  
Breeding Season ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Meat Production ◽  
Hormone Concentration ◽  
Blood Samples ◽  
Clinically Normal ◽  
Follicle Diameter ◽  
Noradrenaline And Adrenaline

In some species, catecholamines in follicular fluid (FF) are related to local physiological events responsible for the regulation of ovarian functions and oocyte maturation. The aim of the present study was to determine and compare intrafollicular and systemic concentrations of dopamine (DA), noradrenaline (NA) and adrenaline (AD) in cycling mares. Sixty ovaries were collected during breeding season from 30 mares raised for slaughterhouse meat production, with clinically normal reproductive tracts, were evaluated. Blood samples were collected prior to slaughter. Follicles were classified into three categories in relation to size: small (20–30 mm; n = 20), medium (≥31–40 mm; n = 20) and large (≥41 mm; n = 20). Follicular fluid (FF) samples were extracted from each follicle. Intrafollicular DA, NA and AD concentrations were significantly higher than the systemic concentrations (p < 0.05). Intrafollicular DA concentrations were higher in medium than small and large follicles (p < 0.05). Intrafollicular NA concentrations were higher in small than medium and large follicles (p < 0.05). Intrafollicular AD concentrations were higher in large than small and medium follicles (p < 0.05). Follicle diameter was significantly and negatively correlated with NA and AD (p < 0.05). A significant correlation of the same hormone concentration in FF and in systemic fluid was observed (p < 0.05). In summary, the FF can serve as an intraovarian catecholamine-storing compartment, with the ability to release neurotransmitters in a regulated way. These results provide novel insights into the neuronal nature of the follicle, suggesting the involvement of catecholamines in normal ovarian functions in mares.

Changes of follicular fluid composition during estrous cycle: The effects on oocyte maturation and embryo development in vitro

2019 ◽  
Author(s):  
A.A. Mohammed ◽  
T. Al-Shaheen ◽  
S. Al-Suwaiegh
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Extracellular Fluid ◽  
Cumulus Cells ◽  
Fluid Composition ◽  
Lh Surge ◽  
Antral Follicles ◽  
Positive Effects

Oocytes are bathed in extracellular fluid of the antral follicles, which is termed follicular fluid (FF). Follicular fluid is synthesized from secretions of theca, granulosa, and cumulus cells and from a transudate of blood plasma. Oocytes persist in meiotic arrest in antral follicles until luteinizing hormone (LH) surge or removal the oocytes from the ovarian follicles. This suggests that FF before LH surge might contain meiosis inhibiting factor(s). The microvasculatory bed of the follicular wall and the composition of FF undergo changes during follicular growth and development, which is important for oocyte maturation and subsequent embryo development. Therefore, it is expected that FF composition and components might change according to timing of FF aspiration from follicles. Hence, negative or positive effects could be expected when FF supplemented during oocyte maturation in vitro. Nutrition effects on microvasculatory bed of follicles and their sizes. Thus, the nutritional status of animals is a factor affected on oocyte maturation and embryo development. The present article reviews and discusses these effects.

Effects of ghrelin on activation of Akt1 and ERK1/2 pathways during in vitro maturation of bovine oocytes

Zygote ◽  
2017 ◽  
Vol 25 (2) ◽  
pp. 183-189 ◽  
Author(s):  
Thomas-Markos Chouzouris ◽  
Eleni Dovolou ◽  
Fotini Krania ◽  
Ioannis S. Pappas ◽  
Konstantinos Dafopoulos ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Treated Group ◽  
Control Group ◽  
Dependent Manner ◽  
Nuclear Maturation ◽  
Bovine Oocytes ◽  
Phosphorylation Rate ◽  
Matured Oocyte

SummaryThe purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus–oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml–1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P < 0.05) matured oocyte were found at 24 h. Oocyte maturation for 24 h in the presence of ghrelin resulted in substantially reduced (P < 0.05) blastocyst yield(16.3%) in comparison with that obtained after 18 h (30.0%) or to both control groups (29.3% and 26.9%, for 18 and 24 h in maturation, respectively). Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

scholarly journals 295 COMPARISON OF TWO METHODS TO AVOID MOVEMENT OF BOVINE OOCYTES DURING IN VITRO MATURATION

2005 ◽  
Vol 17 (2) ◽  
pp. 298 ◽  
Author(s):  
M.M. Petersen ◽  
B. Avery ◽  
T. Greve ◽  
I.B. Bøgh
Keyword(s):  
Oocyte Maturation ◽  
Laser Scanning ◽  
Early Stage ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Laser Scanning Microscopy ◽  
Control Group ◽  
Exact Test ◽  
Standard Procedures

Development of two-photon laser scanning microscopy (TPLSM) has made it possible to conduct several recordings over time of early stage embryos without compromising viability. To use TPLSM to study structures within the oocyte it is necessary to remove at least part of the cumulus cells to prevent emitted light from being blocked. Aspiration of cumulus oocyte complexes (COC) through a denudation pipette creates a “window” through which the emitted light can escape and be recorded. To allow repeated recordings of the same location within an object it is important to avoid movement of the object. Gelatine (Gel) and poly-l-lysine (PLL) have previously been used to promote adhesion of cells in culture. The aim of our study was to develop a method to avoid movement during IVM of partially denuded COCs without compromising oocyte viability. Previous experiments in our lab showed that partial denudation of COC had no effect on embryo development (unpublished). Bovine COCs were obtained from abattoir ovaries. In the control group COCs were placed in non-treated dishes. In the experimental groups, they were placed in Gel- or PLL-coated dishes, either intact or partially denuded, where the length of cumulus cell “tails” was shortened to around 200 μm on each side of the oocyte. The coated dishes were prepared 24 h prior to IVM with 200 μL of 0.1% Gel (Sigma, Copenhagen, Denmark, G2500) or 200 μL 0.01% PLL (Sigma, P-4832). Partial denudation of COCs was performed with a 127–129 μm diameter denudation pipette. Standard procedures were used for IVM (23 h in DMEM with 5% serum and eCG/hCG), IVF (23 h in TALP), and IVC (SOF with 10% serum); IVM and IVF were incubated at 38.5°C in 5% CO2 in air, and IVC at 5% CO2 in 5% O2. The study was based on a total of 1151 oocytes and 3 replicates. Day 8 blastocyst (BL) rates, BL kinetics, and morphology were used as endpoints to assess oocyte maturation. Kinetics/morphology were graded by a scoring system: hatched/excellent 3, expanded/good 2, non-expanded/poor 1. COCs placed in Gel- or PLL-coated dishes did not move during handling of the dishes. The BL rates in the Gel group were 37%, 25%, and 17%, and in the PLL group 24%, 21%, and 12%, for the control, intact, and partially denuded COCs, respectively. In the Gel group the BL rates showed a decreasing trend (P < 0.0036), whereas in only the PLL group the BL rates from the partially denuded COC differed from the control and the intact COCs (P < 0.008). No significant differences were seen between blastocyst kinetics (Gel/PLL 1.9/1.9, 1.8/1.9, 1.6/1.7) or morphology (Gel/PLL 2.2/2.4, 2.0/2.5, 2.2/2.1) in the control, intact or partially denuded groups. Fisher's exact test used. We conclude that it is possible to avoid movement of COCs during IVM without compromising oocyte maturation in dishes coated with Gel or PLL, if the cumulus layer is intact. The BL rates are compromised if COCs are partially denuded and the “cumulus tails” shortened before IVM in Gel or PLL coated dishes, whereas kinetics and morphology are unaffected. This research was funded by the Danish Research Agency, no. 23-023-0133.

176 IN VITRO MATURATION OF DOG OOCYTES IN CANINE FOLLICULAR FLUID

2014 ◽  
Vol 26 (1) ◽  
pp. 202
Author(s):  
K. Reynaud ◽  
S. Canguilhem ◽  
S. Thoumire ◽  
S. Chastant-Maillard
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Control Group ◽  
Limiting Factor ◽  
Cytoplasmic Maturation

In the canine species, assisted reproductive technologies, especially in vitro maturation (IVM) and IVF, are still ineffective. The main limiting factor remains the immaturity of the oocytes collected from anestrus ovaries. The ability of an oocyte to reach the MII stage in vitro is linked to the diameter of its follicle and anestrus oocytes, collected from small (<1 mm) follicles, are profoundly immature (De Lesegno et al. 2008). The objective of this study was to improve cytoplasmic quality by mimicking in vivo conditions; that is, to test the effect of pure preovulatory follicular fluid (FF) on survival and IVM rates of anestrus dog oocytes, in order to improve the nuclear and cytoplasmic maturation of these immature oocytes. Follicular fluids samples were collected from 54 Beagle bitches at 2 stages: before the LH peak (n = 23 bitches) and after the LH peak (n = 31 bitches). Only follicular fluid samples from large (>4 mm) follicles were collected and pooled by stage. Control oocytes were matured in 20% FCS/M199 medium. Groups of 5 oocytes were in vitro matured in 30 μL of follicular fluid, in half-area 96-well plates (5% CO2, 38°C). After 72 h of IVM, oocytes were denuded, fixed, and stained for DNA and tubulin before observation by confocal microscopy, and nuclear stages were classified as GV-A to GV-E, MI, and MII (Reynaud et al. 2012). A total of 460 oocytes were collected from 13 anestrus bitches and allocated to either the control medium (n = 155), the Pre-LH FF (n = 145) or the Post-LH FF (n = 160) groups. After 72 h of IVM, the morphology of the cumulus–oocyte complexes (COC) in the post-LH group was different from that of the others: cumulus cells appeared more compact and darker. Analysis of the nuclear stages showed that the degeneration rate was significantly higher (P < 0.05) in the post-LH group (58.7%) than in the pre-LH (40.9%) or in the control group (34.4%). No significant differences (P > 0.05) were observed between the 3 groups in the rate of immature GVA-B oocytes (36.4, 28.5, and 25.3% in the control, Pre-LH, and Post-LH groups, respectively), in the rate of meiotic resumption (GV-C/D/E, MI, MII stages, 44.4, 51.9, and 38.7% in the control, Pre-LH, and Post-LH groups, respectively). Metaphase II rates were not significantly different (12.1, 8.6, and 4.8% in the control, Pre-LH, and Post-LH groups, respectively). In conclusion, canine COC may survive when exposed to IVM in pure follicular fluid, but the degeneration rate was higher in the post-LH group. The presence of follicular fluid did not inhibit meiosis resumption, but did not significantly improve IVM rates. To better mimic in vivo conditions, IVM in a sequence of media, such as IVM in follicular fluid followed by IVM in oviducal fluid remains to be tested.

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