scholarly journals 303 EFFECT OF SERUM SUPPLEMENTATION AND ESTRUS CYCLE STAGE ON IN VITRO NUCLEAR MATURATION OF CANINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 302
Author(s):  
F.Y. Heru ◽  
H.J. Oh ◽  
M.K. Kim ◽  
J. Goo ◽  
M.S. Hossein ◽  
...  
Keyword(s):  
Luteal Phase ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Follicular Phase ◽  
Estrus Cycle ◽  
Immature Oocytes ◽  
Nuclear Maturation ◽  
Serum Supplementation ◽  
Maturation Rate

The present study investigated the effects of the estrus cycle stage and serum supplementation on nuclear maturation of canine oocytes. Ovaries were collected from a private clinic after ovariohysterectomy and classified into follicular, luteal, or anestrus stages through a combination of ovarian morphology and vaginal cytology. A total of 2214 oocytes from 196 ovaries (903 oocytes from 96 anestrus ovaries, 609 oocytes from 36 follicular ovaries, and 702 oocytes from 64 luteal ovaries) were used for experiments. The oocyte retrieval per ovary was 10, 19, and 12 for anestrus, follicular and luteal-phase ovaries, respectively. In Exp. 1, immature oocytes were cultured for 72 h in TCM-199 alone or TCM-199 supplemented with 10% canine anestrus (CAS), estrus (CES), or diestrus (CDS) serum or fetal bovine serum (FBS). In Exp. 2, immature oocytes were cultured for 72 h in TCM-199 supplemented with 0, 5, 10, or 20% CES. After staining with Hoechst 33342, chromatin state and position as well as spindle formation were evaluated to determine the stage of meiosis: germinal vesicle (GV) stage, germinal vesicle breakdown (GVBD), metaphase I (MI) stage, metaphase II (MII) stage. The experiments with anestrus and luteal-phase oocytes were repeated eight times and follicular-phase oocytes were repeated six times. Data were subjected to analysis of variance (ANOVA) and protected least significant difference (LSD) test to determine differences among experimental groups by using the Statistical Analysis System (SAS, SAS Institute, Inc., Cary, NC, USA) program. Statistical significance was determined where P value was less than 0.05. In Exp. 1, the in vitro maturation of oocytes up to MII stage was higher when oocytes were collected from ovaries in follicular phase. The maturation rate up to MII stage was 0.0 to 1.7%, 1.3 to 10.2%, and 1.0 to 3.2% for the oocytes collected from the anestrus, follicular, and luteal-phase ovaries, respectively, depending on the culture media used. In basic TCM media only, 0.0, 1.3, and 2.3% oocytes reached the MII stage for anestrus, follicular, and luteal-phase oocytes, respectively. A significantly higher rate of maturation was obtained when oocytes collected from follicular phase were cultured in TCM-199 supplemented with 10% CES (10.2%), compared to 10% CAS (4.0%), CDS (2.7%), FBS (1.3%), or the control (1.3%). In Exp. 2, supplementing with 10% CES induced the highest (P < 0.05) maturation rate to the MII stage in oocytes collected from follicular-stage ovaries (11.5%) compared to supplementing with 0% (1.0%), 5% (1.3%), or 20% CES (5.1%). Supplementing with CES (5, 10, or 20%) did not have a significant effect on nuclear maturation of canine oocytes collected from anestrus or luteal-stage ovaries. In conclusion, supplementing in vitro maturation medium with 10% CES increased nuclear maturation of canine oocytes, and canine oocytes collected from follicular-stage ovaries are the most suitable to complete nuclear maturation in vitro. This study was supported by grants from the Biogreen 21-1000520030100000.

scholarly journals Does the Apoptosis Value of Cumulus Cells Play a Role in Rescue Oocyte in Vitro Maturation?

2020 ◽  
pp. 1
Author(s):  
Tulay Irez ◽  
Sinem Ercan Dogan ◽  
Enver Ciraci ◽  
Saadet Busra Aksoyer ◽  
Muhammet Sait Toprak ◽  
...  
Keyword(s):  
Experimental Study ◽  
Luteinizing Hormone ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Immature Oocytes ◽  
Maturation Rate

<p><strong>OBJECTIVE:</strong> In this study, we aimed to investigate the role of the cumulus cell’s apoptosis parameter in the maturation of immature rescue oocytes. </p><p><strong>STUDY DESIGN:</strong> In this experimental study, donated immature germinal vesicle oocytes were cultured for, in vitro maturation, embryo development in matured germinal vesicle oocytes were compared with apoptotic properties of cumulus cells. </p><p><strong>RESULTS:</strong> In all of the immature oocytes after oocyte in vitro maturation, the maturation rate has been observed as 56.1% and 2PN rate as 63.0%. Afterin vitro maturation of germinal vesicle oocytes, there was no difference in apoptosis rates of the cumulus cells between mature and immature oocytes (p&gt; 0.05). The ratio of 2PN in matured germinal vesicle oocytes showing embryo development was 35.4%. A positive correlation was found between luteinizing hormone values on day 3 and E2 values during HCG days during oocyte maturation and embryo development (p=0.021, p=0.020). In addition, it has been observed that the germinal vesicle oocytes, which have completed their maturation and developed into embryos, have high E2 values during HCG days (p=0.020).</p><p><strong>CONCLUSION:</strong> In our study, it has been demonstrated that in vitro maturation in rescue oocytes from stimulated cycles, embryo development potential could not be explained by the apoptosis parameter.</p>

P–709 Dual stimulation in-vitro-maturation (Duostim IVM) for overcoming oocyte maturation arrest, resulting in embryo transfer and livebirth

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
S Hatirnaz ◽  
E Hatirnaz ◽  
M Dahan ◽  
B Ata ◽  
A Basbug ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Oocyte Maturation ◽  
Luteal Phase ◽  
In Vitro Maturation ◽  
Follicular Phase ◽  
Embryo Cryopreservation ◽  
Ovulatory Cycle ◽  
Maturation Arrest ◽  
Immature Oocytes

Abstract Study question Does luteal phase followed by follicular phase letrozole priming and dual oocyte retrieval for in-vitro maturation (IVM) overcome oocyte maturation arrest (OMA)? Summary answer Oocyte maturation, fertilization,embryo cryopreservation and livebirth can be achieved with letrozole priming IVM in rare cases of OMA. What is known already OMA is an intractable problem resulting in only immature oocytes being collected and to date no succesful treatment exists. Attempts to mature oocytes collected in stimulated IVF cycles with OMA have so far failed. Cases with OMA can be due to intrinsic oocyte defects, intrafollicular factors or resistance to stimulation. Study design, size, duration Six women with OMA in ≥ 2 prior stimulated IVF cycles were treated between March 2019 and December 2020. Participants/materials, setting, methods Participants had total of 18 (range 2 - 6) prior IVF cycles yielding only 166 immature oocytes. Letrozole 5mg was given days 15–18 of ovulatory cycle; SC decapeptyl 0.1mg trigger given at follicles 12 mm, 38 hours&lt;OPU. After menstruation, letrozole 5mg days 3–7; SChCG 250ug when follicles=12 mm 38 hours&lt;OPU. After in-vitro-maturation oocytes reaching MII were fertilized. Embryos from luteal collection were frozen and fresh embryo transfer was attempted after follicular phase collection. Main results and the role of chance Six women underwent DuoStim IVM, median (quartiles) 3.5 (0 - 9) GV and 0.5 (0 - 2) MI oocytes were collected from luteal phase OC and 0 (0 - 0) GV and 2(0 – 4.5) MI oocytes were collected from follicular phase OC. They had a total of 166 immature oocytes collected in prior IVF cycles. There were no MII oocytes at the time of collection in any cycles.0 (0 – 3.5) oocytes matured from luteal phase OC and 1 (0 – 4) from follicular phase OC. 0 (0 – 1.5) embryos were available from luteal phase and 0 (0 - 2) from follicular phase OC.Two subjects (29 and 33 years old) underwent fresh DET and the 29 year old with 2 previous failed IVF cycles achieved a livebirth (50% per ET and 16.7% per started cycle). None of the women who did not have an embryo for fresh transfer from the follicular phase collection had an embryo from the luteal phase collection. The same 29 year old has 2 luteal phase and 2 more follicular phase embryos vitrified. Limitations, reasons for caution OMA is a rare condition with a variety of etiologies. Different etiologies can require different managements. Wider implications of the findings: It may be possible to overcome OMA with letrozole IVM in rare cases. This case is the first recorded live birth. The value of dual stimulation overcoming OMA remains uncertain. Trial registration number This study is approved by the local ethical commitee of Medicana Samsun International Hospital by a Grant number of 02/05.02.2020: registration is not required due to retrospective status

336 MEIOTIC DEVELOPMENT AND CORTICAL GRANULES DISTRIBUTION IN CANINE OOCYTES DURING IN VITRO MATURATION

2010 ◽  
Vol 22 (1) ◽  
pp. 324 ◽  
Author(s):  
M. De los Reyes ◽  
D. Luna ◽  
J. Palomino
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Fluorescein Isothiocyanate ◽  
Cumulus Cells ◽  
Homogeneous Distribution ◽  
Cortical Granules ◽  
Dapi Staining ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Low development of IVM canine oocytes could be in part attributed to an impaired cytoplasmic maturation. In mammalian oocytes, migration and the redistribution of cortical granules (CGs) around the periphery of the oocyte contribute to the inhibition of polyspermy and it is an important criterion to evaluate cytoplasmic maturation. The state of nuclear maturation and the distribution of CGs were evaluated in canine oocytes cultured for different periods in order to compare the synchrony of nuclear and cytoplasmic maturation during in vitro maturation. Bitch ovaries at different stages of the estrous cycle were obtained following ovariectomy. COCs with compact cumulus cells showing a homogeneous cytoplasm were selected for experiments. Thirty-six COCs were processed at immature stage, placed in PBS medium until evaluation. A total of 275 COCs were matured in vitro for 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 penicillin, and 20 mg mL-1 streptomycin, at 38.5°C and 5% CO2. At each culture period, the oocytes were stained with Lens culinaris agglutinin (LCA), labeled with fluorescein isothiocyanate, and the CGs distributions were examined under a fluorescent microscope. The nuclear status of the denuded oocytes was determined by DAPI staining under a fluorescence microscope. For each treatment, at least four replicates were performed and the data was analyzed by ANOVA using Tukey’s test to determine the differences P < 0.05. Three types of CGs distribution were distinguished during canine oocyte maturation: (1) homogeneous distribution throughout the cytoplasm including the cortex; (2) heterogeneous (clusters) within the cytoplasm and (3) densely distributed beneath the oolemma. Nuclear stages were classified as immature or germinal vesicle (GV) stage; resumption of meiosis or germinal vesicle break down (GVBD); metaphase I to telophase I (MI toTel I); and mature or second metaphase (MII). The distribution patterns of GCs were different (P < 0.05) among oocytes cultured for different periods and the nuclear maturation status also differed between oocytes cultured for different intervals (P < 0.05). Most (>84%) of the immature oocytes at GV showed a uniform distribution of CGs throughout the cytoplasm. At 48 h of culture, CGs distribution was mainly Type 2 (25%) and 3 (61%) and the oocytes were at GVBD (33%) and MI-Tel I (33%) stages. Most nuclei of the type 3 oocytes were in the MI (40%) and MII (11%) stages, corresponding to those oocytes matured for 72 (88%) or 96 h (71%). These results indicate that canine oocytes migrate to the cortex during IVM and this process is not finished before 72 h of culture. In addition, although the re-distribution of the CGs occurred in parallel with nuclear maturation, the oocytes cannot always proceed to the MII stage; however, in such oocytes the CGs are distributed beneath the oolemma. Supported by Grant FONDECYT 1080618.

174 Effects of Melatonin on In Vitro Maturation of Bovine Oocytes and Gene Expression in Cumulus Cells: Preliminary Results

2018 ◽  
Vol 30 (1) ◽  
pp. 226
Author(s):  
F. C. Castro ◽  
L. Schefer ◽  
K. L. Schwarz ◽  
H. Fernandes ◽  
R. C. Botigelli ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Reverse Transcription ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Bovine Oocytes

Melatonin mediates several processes in animal reproduction and has drawn attention for its potent antioxidant, anti-apoptotic, anti-inflammatory action and, more recently, for its benefits on oocyte maturation and embryo development in vitro. The aim of this study was to assess the effect of melatonin during the in vitro maturation (IVM) on nuclear maturation of bovine oocytes and gene expression in their corresponding cumulus cells (CC). Bovine cumulus–oocyte complexes (COC) were obtained by aspiration of follicles (2-6 mm) from slaughterhouse ovaries, selected (grades I and II) and transferred to 4 well plates (25-30 COC/well) containing IVM medium [TCM-199 supplemented with sodium bicarbonate (26 mM), sodium pyruvate (0.25 mM), FSH (0.5 µg mL−1), LH (5.0 µg mL−1), 0.3% BSA, and gentamicin (50 µg mL−1)] with 0, 10−5, 10−7, 10−9 or 10−11 M melatonin and cultured for 24 h at 38.5°C and 5% CO2. At the end of IVM, oocytes were stained with Hoechst 33342 (10 μg mL−1) and evaluated for nuclear maturation rate. The CC were evaluated for the expression of antioxidant (SOD1, SOD2, GPX4), pro-apoptotic (P53, BAX) and expansion-related genes (PTX3, HAS1, HAS2). For transcript detection in CC, RNA isolation was performed with TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Relative quantification of transcripts was performed by RT-qPCR using 3 endogenous controls (β-actin, GAPDH, PPIA). Nuclear maturation rate and gene expression were tested by ANOVA and means were compared by Tukey’s test (6 replicates). In CC, the different concentrations of melatonin did not significantly alter expression of the investigated genes (P > 0.05), although all concentrations provided a numerical increase in the expression of the antioxidant SOD1 and of the expansion-related genes PTX3 and HAS2. Regarding the pro-apoptotic genes, concentrations of 10−11 and 10−9 M were able to reduce only numerically the expression of BAX and P53, respectively. In oocytes, the rate of nuclear maturation was not different among the tested treatments (P > 0.05), but it was numerically higher in the 10−7 M melatonin treated group compared with the control (69.71 ± 13.76% v. 88.1 ± 12.54%). In conclusion, under the studied conditions, melatonin was unable to improve maturation rate or to affect the expression of antioxidant, pro-apoptotic, and expansion-related genes in CC. Melatonin during IVM has shown variable results in different studies and appears to show different effects depending on culture conditions and parameters studied. In order to take advantage of the possible positive antioxidant effects of melatonin, other culture conditions and parameters should be investigated. In a next step, melatonin will be included during in vitro culture of embryos to evaluate its possible cytoprotective role, because such embryos are more exposed to oxidative stress during in vitro culture, and to investigate its benefits on developmental competence in vitro. This reaesrch was funded by FAPESP (2015/20379-0; 2014/17181-0).

43 WARMING TEMPERATURE AFFECTS THE VIABILITY AND MEIOTIC COMPETENCE OF IMMATURE PORCINE OOCYTES VITRIFIED IN A CHEMICALLY DEFINED SOLUTION

2014 ◽  
Vol 26 (1) ◽  
pp. 135
Author(s):  
D. Takahashi ◽  
H. Funahashi
Keyword(s):  
In Vitro Maturation ◽  
Fluorescent Microscopy ◽  
Dibutyryl Cyclic Amp ◽  
Nuclear Maturation ◽  
Porcine Oocyte ◽  
Different Temperatures ◽  
Maturation Rate ◽  
Post Hoc ◽  
Meiotic Competence

The aim of this study was to examine the viability and meiotic competence of porcine oocytes when immature porcine cumulus-oocyte complexes (COC) were pretreated for vitrification at different temperatures (25 and 39°C), vitrified in a chemically defined solution, and warmed at different temperatures (39 and 60°C). Cumulus-oocyte complexes were aspirated from middle-size follicles (3–6 mm in diameter) of abattoir-derived porcine ovaries. After collection, the COC were pretreated with cryoprotectants at different temperatures (25 and 39°C) and vitrified in a serum-free chemically defined solution containing 0.6 mg mL–1 of hydroxypropyl cellulose, basically according to a commercial protocol (Cryotop, Kitazato BioPharma Co. Ltd., Fuji, Japan). The vitrified COC were warmed in 1 M trehalose solution at 39 for 60 s or at 60°C for 30 s. The COC were cultured for in vitro maturation (IVM) in modified porcine oocyte medium (POM) supplemented with 50 μM β-mercaptoethanol, 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, and 1 mM dibutyryl cyclic AMP (dbcAMP) for 20 h and then in the fresh medium without hormonal supplements and dbcAMP for another 24 h. Viability of COC was evaluated under fluorescent microscopy after stain with fluorescein diacetate and propidium iodide. Nuclear maturation of the oocytes was evaluated after 44 h of IVM. Statistical analyses of results from 5 replicated trials were performed by ANOVA with a Bonferroni/Dunn post-hoc test (significance, P < 0.05). Although viabilities of vitrified oocytes after 44 h of IVM [6.0% (9/149) to 37.8% (59/155)] were significantly lower than fresh controls [98.8% (158/160)], the viabilities of vitrified oocytes warmed at 60°C [32.0% (49/160) to 37.8% (59/155)] were significantly higher than those warmed at 39°C [6.0% (9/149) to 10.0% (16/160)]. Maturation rates in vitrified oocytes [2.7% (4/149) to 19.8% (31/155)] were also significantly lower than fresh controls [74.8% (120/160)]. Regardless of temperature during pretreatment for vitrification (25 and 39°C), maturation rate of the oocytes warmed at 60°C after vitrification [16.4% (25/154) to 19.8% (31/155)] was significantly higher than that warmed at 39°C [3.1% (5/160) to 2.7% (4/149)]. In conclusion, these results demonstrate that warming at 60°C for 30 s maintains the viability and meiotic competence of immature porcine COC.

P–269 Effect of well-of-the-well culture as in vitro maturation system for human oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
B Dura. Lopez ◽  
I Moya ◽  
P Torres ◽  
M J Gomez-Torres ◽  
A Monzo ◽  
...  
Keyword(s):  
Culture System ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Study Groups ◽  
Culture Conditions ◽  
Human Oocytes ◽  
Nuclear Maturation ◽  
Secreted Factors ◽  
Experimental Group

Abstract Study question Can the Well-of-the-Well system (WOW), applied on denuded oocytes, improve germinal vesicle breakdown (GVBD) and maturation rate? Summary answer In vitro maturation (IVM) of denuded germinal vesicle (GV) oocyte using WOW culture system increases nuclear maturation competence when compared with droplet conventional culture What is known already Further research remains necessary to address the mechanism of oocyte maturation in order to refine culture conditions and improve the implantation rate of in vitro matured oocytes. Several studies on bovine oocytes have shown that oocyte-secreted factors (an uncharacterized mix of growth factors secreted by the oocyte) enhance oocyte developmental competence during in vitro maturation. These oocyte-secreted factors may accumulate at the bottom of the micro-well, as suggested for the WOW culture system. Previous reports suggested that diffusible factors secreted by individual oocytes probably accumulated in a micro-well WOW dish, may provide a suitable microenvironment for their in vitro maturation. Study design, size, duration A total of 879 GV collected between 2017 and 2019 were included in this study. They were randomly allocated into two experimental groups: (1) single-cultured oocytes (SC) that were cultured individually in micro-droplets, and (2) group-cultured oocytes (WOW) that were cultured in a microwell culture system using the WOW dish (culture dish for time lapse incubator). The nuclear maturation was assessed after 24 hours and 48 hours of IVM Participants/materials, setting, methods GV oocytes were obtained from 609 patients undergoing controlled ovarian stimulation cycles. Oocytes from the experimental group (1) were placed individually in conventional 25μl micro-droplets in a 35 mm dish. Oocytes from the experimental group (2) were placed in 80 μl droplet individually in each of 9 microwells of WOW dish. All GV oocytes were matured in a single step embryo culture medium, supplemented with human menopausal gonadotropin and synthetic serum substitute. Main results and the role of chance Mature oocyte (MII) was considered when we observed rupture of the GV and the presence of a first polar body in the perivitelline space during the first 24 or 48 hours of culture under inverted optical microscope. GVBD noted significant differences (p-valor = 0.000) between the study groups after culturing of 24 hours [GVBD: SC group; 70% (318/455) vs. WOW group; 83% (352/424)] and 48 hours [GVBD: SC group; 77% (319/416) vs. WOW group; 94% (398/424)]. The maturation rates (MR) showed significant differences (p-valor = 0.000) between the study groups after culturing of 24 hours [MR: SC group; 51% (233/455) vs. WOW group; 80% (338/424)] and 48 hours [MR: SC group; 71% (295/416) vs. WOW group; 91% (387/424)]. Limitations, reasons for caution There is no data on cleavage and blastocyst rates. There are no previous reports comparing the maturation rates in denuded human oocytes single-cultured in individually droplet or group-cultured in WOW dish. Wider implications of the findings: Our results must be taken into account in order to improve the culture conditions for the optimization of the in vitro maturation technique in human oocytes from stimulated cycles. We now provide evidence that group-cultured oocytes in WOW dish increase GVBD and maturation rates. Trial registration number Not applicable

scholarly journals Development of a reliable in vitro maturation system for zebrafish oocytes

Reproduction ◽  
2008 ◽  
Vol 135 (3) ◽  
pp. 285-292 ◽  
Author(s):  
Shinsuke Seki ◽  
Toshimitsu Kouya ◽  
Ryoma Tsuchiya ◽  
Delgado M Valdez ◽  
Bo Jin ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Saline Solution ◽  
Stage Iii ◽  
Hatching Rate ◽  
Cleavage Rate ◽  
Immature Oocytes ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

In zebrafish oocytes, it has been reported that a 60 or 75% Leibovitz L-15 medium or simple balanced saline solution containing 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) is effective for nuclear maturation. However, most of the oocytes that matured under these conditions were not fertilized and did not hatch. Thus, thesein vitromaturation methods could not support the cytoplasmic maturation of zebrafish oocytes. Therefore, we tried to develop a reliablein vitromaturation method for zebrafish oocytes, which supports their ability to be fertilized and to develop till hatching. When zebrafish oocytes at stage III were cultured in 50–100% Leibovitz L-15 medium supplemented with DHP, the highest rates of cleavage (24%) and hatching (12%) were obtained from oocytes matured in 90% Leibovitz L-15 medium. When we examined the suitable pH (7.5–9.5) of the 90% medium, higher rates of cleavage (45%) and hatching (33%) were obtained in oocytes matured at pH 9.0 than at pH 7.5, 8.5, or 9.5 (cleavage rate, 16–29%; hatching rate, 8–21%). In oocytes matured in 90% Leibovitz L-15 medium at pH 9.0, high rates of cleavage (70%) and hatching (63%) were obtained when oocytes were cultured for 270 min with 0.5 mg/ml BSA. Thus, 90% Leibovitz L-15 medium at pH 9.0 containing 0.5 mg/ml BSA was effective for normal maturation of zebrafish oocytes. This method will become a powerful tool for understanding the mechanism ofin vitromaturation in zebrafish oocytes and for the practical use of immature oocytes.

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