Autologous embryo–cumulus cells co-culture and blastocyst transfer in repeated implantation failures: a collaborative prospective randomized study

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 173-180 ◽  
Author(s):  
M. Benkhalifa ◽  
A. Demirol ◽  
T. Sari ◽  
E. Balashova ◽  
M. Tsouroupaki ◽  
...  
Keyword(s):  
Embryo Development ◽  
Randomized Study ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Prospective Randomized Study ◽  
Human Embryos ◽  
Feeder Cells ◽  
Positive Role ◽  
Significant Difference

SummaryIn repeated implantation failure, the co-culture of human embryos with somatic cells has been reported to promote the improvement of embryos quality, implantation and pregnancy rate. It was reported that feeder cells can be more beneficial to the oocyte and embryo by detoxifying the culture medium and supporting embryo development via different pathways. In this study, 432 patients, each with a minimum of three repeated implantation failures, were accepted for a prospective randomized study with or without autologous cumulus cell embryo co-culture and transfer at day 3 or day 5–6. We also investigated the expression of leukaemia inhibitor factor (LIF) and platelet activating factor receptor (PAF-R) on day 3 confluent cumulus cells. The statistic analysis of the data showed significant difference of implantation and clinical pregnancy rates between classical culture and day 3 compared with co-culture and day 5–6 transfer. The molecular analysis showed that cumulus cells express the LIF and the PAF-R genes and confirmed the possible positive role of growth factors and cytokines in early embryo development. Embryo co-culture systems with autologous cells can be beneficial in routine in vitro fertilization for embryo selection and implantation improvement. More molecular investigations need to be done to improve elucidation of the complex dialogue between the embryo and feeder cells prior to implantation and to understand the involved biological function and molecular process during embryo development.

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

168 INHIBITION OF BOVINE OOCYTE CUMULUS CELL PROGESTERONE SYNTHESIS DURING IN VITRO MATURATION AFFECTS CHROMOSOME ALIGNMENT AND SPINDLE INTEGRITY IN FERTILIZED EGGS AND EARLY EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 198
Author(s):  
E. Daly ◽  
A. G. Fahey ◽  
M. M. Herlihy ◽  
T. Fair
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Spindle Formation ◽  
Vitro Fertilization ◽  
Time Treatment

We have previously demonstrated the importance of progesterone (P4) synthesis by cumulus cells during oocyte maturation in vitro (IVM) for bovine oocyte acquisition of developmental competence and subsequent embryo development (Aparicio et al. 2011 Biol. Reprod. 84). The aim of this study was to identify key processes that may be deregulated by the inhibition of P4 signalling in the cumulus–oocyte complex (COC) during IVM. To this end, good quality immature COC were placed in IVM medium [TCM-199 supplemented with 10% (vol/vol) FCS and 10 ng mL–1 epidermal growth factor] and cultured at 39°C for 22 h in a humidified atmosphere containing 5% CO2, in the presence or absence of 10 μM trilostane (which blocks P4 synthesis by inhibiting 3 β-hydroxysteroid dehydrogenase; Stegram Pharmaceuticals Ltd., Surrey, UK). Matured COC were washed and placed in 250 μL of fertilization medium (25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL–1 fatty acid-free BSA, and 10 mg mL–1 heparin). In vitro fertilization (IVF) was performed with 250 μL of frozen–thawed semen at a final concentration of 1 × 106 spermatozoa mL–1 at 39°C under 5% CO2 during 20 h. Presumptive zygotes were denuded, washed, and transferred to 25-μL culture droplets (SOF + 5% FCS) at 39°C under 5% CO2, 90% of N2, and 5% O2 atmosphere with maximum humidity. Subsets of presumptive fertilized eggs and developing embryos were recovered at 6, 72, 120, and 192 h postinsemination (hpi) and processed for confocal whole-mount immunocytochemistry. The meiotic and mitotic spindles and chromosomes were visualised by immunofluorescent labelling of α-tubulin and 4′,6-diamindino-2-phenylindole (DAPI), respectively, and classified as normal if the chromosomes were correctly aligned or appropriately segregated, or abnormal if lagging chromosomes or abnormal chromosome segregation were observed. Samples were collected from 5 replicates (n = 50 zygotes/embryos per treatment, per timepoint) and a total of 157 spindles were observed. Logistic regression analysis was conducted to determine the probability of abnormal spindle formation. The incidence of spindle abnormality was regressed on time, treatment, and treatment by time. For all time points, there was significant reduction in the odds of abnormal spindle formation in control samples versus trilostane-treated samples (P < 0.001). In conclusion, our data imply a role for P4 signalling in maintaining spindle integrity during oocyte meiotic maturation and progression through the initial mitotic divisions of early embryo development in cattle.

160 Exposure to Cadmium Affects Oocyte and Embryo Competence in Cattle

2018 ◽  
Vol 30 (1) ◽  
pp. 219
Author(s):  
C. De Canditiis ◽  
N. Pagano ◽  
V. Franco ◽  
I. Paradiso ◽  
É. C. Dos Santos ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Dapi Staining ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Vitro Model ◽  
Hoechst Staining

There is a growing worldwide concern regarding the increased release of the heavy metal cadmium (Cd) in the environment, due to several industrial processes, as it is known to affect health. Among other heavy metals, Cd is widely recognised to influence the reproductive system at different levels, interfering with both gametes and embryo functions in several species (Thompson and Bannigan, 2008 Reprod. Toxicol. 25, 304-315). The in vitro model can be used to mimic environmental conditions allowing us to evaluate their effect on oocyte maturation and early embryo development. Therefore, the aim of this study was to evaluate the influence of different Cd concentrations on nuclear maturation, apoptosis in cumulus cells, and cleavage and blastocyst yields in cattle. For this purpose, abattoir-derived bovine oocytes were in vitro matured, fertilized, and cultured according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). In particular, oocytes were matured with 0 (control; n = 126), 0.1 μM (n = 139), 1 μM (n = 134), and 10 μM of Cd (n = 135), at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2. For each replicate, after 22 h of maturation, a representative sample of oocytes (n = 10 per each group) was used to evaluate nuclear maturation by 4′,6-diamidino-2-phenylindole (DAPI) staining and another sample (n = 10 per each group) to assess cumulus-cells complex apoptosis by TUNEL/Hoechst staining (Pocar et al. 2005 Reproduction 130, 857-868). The remaining oocytes were in vitro fertilized and cultured with 0 (n = 106), 0.1 μM (n = 119), 1 μM (n = 114), and 10 μM (n = 115) Cd. The experiment was repeated 3 times. On Day 8 post-IVF, the blastocyst yields were recorded. Differences among groups were analysed by ANOVA, with the least significant difference method used as a post hoc test. Data are presented as means ± SE. Unexpectedly, the exposure of oocytes to Cd during IVM did not affect the percentage of oocytes undergoing nuclear maturation (on average 96.3 ± 2.3). In contrast, concentrations of 1 and 10 μM Cd increased the percentage of apoptotic cumulus-cells in cumulus–oocyte complexes (COC) compared with the control (3.4 ± 0.4, 10.6 ± 1.8, 15.0 ± 0.9, 16.7 ± 4.0, respectively, with 0, 0.1, 1, and 10 μM; P < 0.05). It is worth pointing out that with the highest concentration, cumulus expansion did not occur and cumulus cells appeared detached from the oocyte. Likewise, 1 and 10 μM Cd decreased cleavage rates compared with the control (68.7 ± 1.8, 54.3 ± 5.0, 58.5 ± 4.2 and 2.8 ± 2.6, respectively, with 0, 0.1, 1, and 10 μM Cd; P < 0.01). Finally, blastocyst yields decreased when oocytes were treated with 0.1 μM Cd and no development to blastocyst was observed at the 2 higher concentrations (35.1 ± 1.7, 26.2 ± 3.1, 0, 0, respectively, with 0, 0.1, 1, and 10 μM; P < 0.01). In conclusion, exposure to Cd during maturation negatively affects bovine COC, as indicated by the increased apoptotic index in cumulus cells, without influencing the nuclear maturation process. Furthermore, the presence of Cd during in vitro fertilization and culture severely impairs both the fertilization and post-fertilization embryo development.

scholarly journals The Number of LH Receptor could Predict the Success of Oocyte Maturity in the Process of In Vitro Maturation

2016 ◽  
Author(s):  
Adek Amansyah
Keyword(s):  
In Vitro Maturation ◽  
Clinical Laboratory ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Maturity ◽  
Lh Receptor ◽  
First Polar Body ◽  
Significant Difference

Objective: To evaluate the relationship between the number of LH receptor and the success of oocyte maturity in the process of in vitro maturation (IVM). Method: This experimental study was conducted in the Permata Hati Infertility Clinical Laboratory, Dr. Sardjito General Hospital, Yogyakarta, with the samples of 300 oocytes obtained through collecting immature cow’s oocytes from the abattoir and grouped the oocytes into 3 (three) groups based on the pattern of oocyte cumulus cells on the vesicle germinal stage 2 - 8 mm with three layers of cumulus cell. The sample of the cumulus cells from these three groups were taken and the LH receptor examination was done with immunohistochemistry. After that, the IVM process was performed to the three groups and its development for 24 hours was evaluated. Its maturation quality was evaluated with the emergence of the first polar body (1PB) and compared to the other groups and related to the number of LH receptor in the three groups. Result: The result of this study indicated that the oocyte cumulus cells showed a difference of function during IVM process. The maturity rate in this study showed that the number of LH receptor was related to the morphological pattern of oocyte cumulus cells with oocyte maturity. The maturity of the cumulus cells which 100% covered the oocyte was higher than that of the cumulus cells which > 50% and < 30% covered the oocytes, namely, 74% compared to 60% and 12%. The result of this study also showed that the average number of LH receptors in the three groups (A, B, and C) was 183.4, 78.8, and 24.0 respectively. A significant difference was found in the three groups (p < 0.0001). When related to IVM maturity, this difference showed that the bigger number of oocyte cumulus cells influenced the oocyte maturity. Conclusion: The number of LH receptor can be used as a prediction to determine the success of oocyte maturation in the process of in vitro maturation. [Indones J Obstet Gynecol 2013; 1-4:183-7] Keywords: IVM, LH receptor, oocyte cumulus cell

scholarly journals MiR-191-5p is upregulated in culture media of implanted human embryo on day fifth of development

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Ricardo Josué Acuña-González ◽  
Mercedes Olvera-Valencia ◽  
Jorge Skiold López-Canales ◽  
Jair Lozano-Cuenca ◽  
Mauricio Osorio-Caballero ◽  
...  
Keyword(s):  
Embryo Development ◽  
Human Embryo ◽  
Poor Prognosis ◽  
Culture Media ◽  
Uterine Cavity ◽  
Human Embryos ◽  
Common Criteria ◽  
Significant Difference ◽  
The Media

Abstract Background Morphological features are the most common criteria used to select human embryos for transfer to a receptive uterine cavity. However, such characteristics are not valid for embryos in cellular arrest. Even aneuploid embryos can have normal morphology, and some euploid embryos have aberrant morphology. The aim of this study was to quantify the expression profile of hsa-miR-21-3p, -24-1-5p, -191-5p, and -372-5p in culture media on day 5 of in vitro embryo development, and compare the profiles of two groups of media classified by outcome: successful (n = 25) or unsuccessful (n = 25) implantation pregnancy. Methods Fifty patients were accepted in the Department of Reproductive Biology of a Hospital in México City, based on the Institutional inclusion criteria for in vitro fertilization. Embryos were transferred to the women on day 5 of cultivation, and the culture media were collected. RNA was isolated from each culture medium with TRIzol reagent, and microRNA (miRNA) expression was detected through RT-PCR with specific primers. Expression bands were quantified by reading optical density. Results There was a 5.2-fold greater expression of hsa-miR-191-5p in the pregnancy-related culture media (p ≤ 0.001) and a 1.6-fold greater level of hsa-miR-24-1-5p (p = 0.043) in the media corresponding to non-pregnant women. No significant difference existed between the two groups hsa-miR-21-3p (p = 0.38) or hsa-miR-372-5p (p = 0.41). Conclusions Regarding adequate in vitro embryo development, hsa-miR-191-5p could possibly represent a positive biomarker, while has-miR-24-1-5p may indicate poor prognosis. This former miRNA modulates IGF2BP-1 and IGF2R, associated with the implantation window. On the other hand, hsa-miR-24-1-5p may be related to a poor prognosis of human embryo development.

Unravelling the needs of singly in vitro-produced bovine embryos: from cumulus cell co-culture to semi-defined, oil-free culture conditions

2012 ◽  
Vol 24 (8) ◽  
pp. 1084 ◽  
Author(s):  
I. G. F. Goovaerts ◽  
J. L. M. R. Leroy ◽  
A. Langbeen ◽  
E. P. A. Jorssen ◽  
E. Bosmans ◽  
...  
Keyword(s):  
Embryo Development ◽  
Apoptotic Cell ◽  
Specific Interaction ◽  
Cell Monolayer ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
In Vitro Production ◽  
Small Surface

Producing bovine in vitro embryos individually is a challenge as it generally leads to impaired embryo development. Earlier research optimised a single embryo in vitro production (IVP) protocol using serum, cumulus cells and oil during culture. As some of these factors are undesirable in certain circumstances, the present study investigated their necessity and possible interactions, and defined their role during single-embryo culture. Although the cumulus cell monolayer produced progesterone, it appeared not to be a key factor in supporting single-embryo development. Because in vitro culture in large medium volumes was shown to impair single-embryo development, two new oil-free culture protocols were tested. Using a 30-µL droplet of medium in 96-well plates with a small surface area resulted in comparable blastocyst rates to those obtained under oil. When serum was used, co-culture with cumulus cells seems necessary, leading to consistently high blastocyst rates. Finally, a serum-free, oil-free culture system using insulin, transferrin, selenium and BSA resulted in embryos with similar total cell numbers and apoptotic cell ratios, but blastocyst rates did not equal those obtained with serum and co-culture. This research additionally stresses the fact that specific interaction mechanisms between somatic cells and a developing in vitro embryo are far from unravelled.

88 EFFECTS OF REMOVAL OF CUMULUS CELLS AND pb1 PRESENCE OF IN VITRO-MATURED BUFFALO OOCYTES PRIOR TO IVF ON CLEAVAGE RATE AND SUBSEQUENT EMBRYO DEVELOPMENT

2014 ◽  
Vol 26 (1) ◽  
pp. 158
Author(s):  
J.-H. Shang ◽  
H.-Y. Zheng ◽  
C.-Y. Yang ◽  
F.-X. Huang ◽  
B.-Z. Yang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Natural Science ◽  
Polar Body ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Lower Efficiency ◽  
Significant Difference

The efficiency of oocyte maturation and embryo production in vitro in buffalo is relatively poor when compared with that in cattle. The percentage of oocytes selected by pb1 (the 1st polar body) presence for somatic cell nuclear transfer (SCNT) ranged from 50 to 70% in our laboratory, which meant that 30 to 50% oocytes have been abandoned. The present study was designed to identify the effect of cumulus cells removal and pb1 presence or absence before the IVF of matured buffalo oocytes on cleavage rate and subsequent embryo development and to try to reuse those oocytes without pb1 for embryo in vitro production. In vitro-matured oocytes enclosed with cumulus cells were randomly selected and denuded mechanically, then the denuded oocytes (DO) were divided into 3 groups by non-selection (pb1 ± ), selection of pb1 presence (pb1+) and absence (pb1–). Intact cumulus–oocyte complexes (COC, control) and pb1 ± , pb1+, and pb1– DO (treatments) were inseminated with motile buffalo sperm in Tyrode's medium for 24 h. The presumed zygotes were washed 3 times and transferred into 50-μL droplets of IVC medium (TCM 199 + 10% fetal bovine serum) and co-cultured with buffalo cumulus cells monolayer for more than 10 days to evaluate the developmental ability of embryos. Cleavage rate (CR) and blastocyst rate (BR) were assessed at 48 h and 240 h after fertilization (0 h). The results indicated that CR and BR for COC (61.69 ± 9.22% and 34.07 ± 7.61%) and pb1+ (66.59 ± 15.50% and 35.96 ± 10.87%) were significantly higher (P < 0.01) than those for pb1 ±  (49.11 ± 6.83% and 21.88 ± 8.17%) and pb1– (35.09 ± 9.17% and 13.16 ± 5.38%). In addition, there was a significant difference (P < 0.05) in the CR and BR between pb1 ±  and pb1– but no difference (P > 0.05) between COCs and pb1+ DO. These data show that removal of cumulus cells before IVF significantly reduces the overall developmental competence to cleavage and blastocyst stage and this negative effects mainly caused by the immature oocytes (indicated by the absence of pb1), but there was no effect on mature oocytes (presence of pb1). However, the oocytes without pb1 can still be used for in vitro embryo production even with lower efficiency when compared with intact COC. This research was supported by grants from the National Natural Science Foundation of China (31160456), the Natural Science Foundation of Guangxi, China (2011GXSFB018045, 2013GXNSFAA019075).

150 PROTEOME OF BOVINE CUMULUS CELLS AS RELATED TO OOCYTE MORPHOLOGY AND IN VITRO EMBRYO PRODUCTION

2017 ◽  
Vol 29 (1) ◽  
pp. 183
Author(s):  
I. C. Velez ◽  
M. M. Ramirez ◽  
A. I. Chica ◽  
R. Urrego ◽  
A. A. Moura ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Silico ◽  
Triosephosphate Isomerase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Type I ◽  
Type Ii

The present study was conducted to study the effect of cumulus-oocyte complex (COC) morphology on subsequent in vitro embryo development and to assess the proteome of their corresponding cumulus cells (CC). Cow ovaries were obtained at an abattoir and COC aspirated from 3–8 mm follicles. The COC were defined as type I (TI): homogeneous ooplasma and ≥4 layers of compact CC; type II (TII): granular ooplasma and ≥4 layers of slight expanded CC. Fifty COC had ~500,000 CC. Cumulus cells were frozen in ammonium bicarbonate and immediately lyophilized for proteome analysis. Other selected COC were matured in vitro in TCM-199-supplemented media for 24 h. After maturation, CC were collected (T24) and processed as described above. The remaining COC were fertilized with sperm from a fertile bull and zygotes, cultured in vitro until Day 7. Ten blastocysts per group were stained (Hoechst 33342) and blastomeres, counted for assessment of embryo quality. The CC proteins were obtained from the following groups: immature type I (TIT0) and type II (TIIT0), and in vitro matured type I (TIT24) and type II (TIIT24). For protein extraction, we used sonication (30 min, 4°C), freezing and unfreezing in liquid nitrogen, and maceration. The CC proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by ESI-MS/MS. Differences in cleavage, embryo rates, and blastomere numbers were analysed by t-test. Protein expression difference was set at 2.5-fold (P < 0.05). In silico protein interactions were investigated using STRING v. 10.0. There were no differences in cleavage (88 ± 4 v. 89 ± 8%) and embryo rates (36 ± 7 v. 33 ± 8%) between COC of TI (n = 220) and TII (n = 161), respectively. Blastomeres were also similar in TI (101) and TII (104) groups. Major proteins expressed in all CC were α-enolase, β-actin, oestradiol 17-β-dehydrogenase 1, glutathione S-transferase, glyceraldehyde 3-phosphate dehydrogenase, heat shock protein β-1, histone H2B type 1-N, histone H4, mitochondrial malate dehydrogenase 2, protein disulfide isomarase A6, triosephosphate isomerase, tubulin α-1C chain, and vimentin. Glyceraldehyde 3-phosphate dehydrogenase appeared to be more expressed in TIT0, whereas tubulin α-1C chain and vimentin had greater expression in TIIT24. As evidenced by in silico analysis, most CC proteins interact among themselves, participating in complex networks involving intracellular signalling and other events. In conclusion, there are no difference in embryo development when using compact and early-expanded COC, indicating that both types can be selected for IVP. Protein profile of cumulus cell may serve as a marker for in vitro embryo competence.

scholarly journals 184 PRELIMINARY FINDINGS ON CARBOHYDRATE METABOLISM OF INTACT EQUINE CUMULUS-OOCYTE COMPLEXES DURING IN VITRO MATURATION

2016 ◽  
Vol 28 (2) ◽  
pp. 223 ◽  
Author(s):  
N. Lewis ◽  
K. Hinrichs ◽  
D. Brison ◽  
R. Sturmey ◽  
D. Grove-White ◽  
...  
Keyword(s):  
Carbohydrate Metabolism ◽  
Dna Content ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Glucose Consumption ◽  
Cumulus Cells ◽  
Significant Difference ◽  
Spent Media

Production of equine embryos in vitro is gaining popularity, and many differences exist in composition of in vitro maturation (IVM) media. Metabolism of the cumulus-oocyte complex (COC) is essentially unknown in the horse. Here, we describe preliminary data on carbohydrate metabolism of the equine COC during IVM. COC, collected by scraping of the granulosa layer of all visible follicles of abattoir-derived ovaries, were held overnight (12–18 h) at room temperature (~20°C) and then placed in Maturation Medium (M199 with Earle’s salts, 10% FBS, with 25 μg mL–1 gentamicin and 5 mU mL–1 FSH). They were incubated singly in 10-μL droplets under mineral oil for 30 h at 38.3°C in 5% CO2 in air. Control droplets without COC were incubated in the same dish. After incubation, COC were removed and spent media kept at –80°C for later analysis. Oocytes were denuded of cumulus cells by pipetting in the presence of hyaluronidase and evaluated by light microscopy at 500×. Those with a visible polar body were classified as metaphase II (MII); oocytes with an intact oolemma and no polar body were classified as immature intact (INT) and those with an irregular or unapparent oolemma, or shrunken cytoplasm, were classed as degenerating (DEG). To adjust for variation in cumulus cell number, the stripped cumulus cells were frozen at –20°C and later analysed for DNA content using Picogreene. The spent media was analysed for depletion of glucose and appearance of lactate on a BMG Fluostar spectrophotometer using an enzyme-linked ultrafluorometric method. Data were expressed as pmol/ng DNA/hr and analysed by t-test, x2 and logistic regression. Thirty COC were cultured and analysed; 14 were classified as MII, 2 INT and 14 DEG. Seven COC (23%) depleted all the available glucose, indicating that the rate of glucose consumption in those 7 complexes was ≥1866 pmol/COC per hour. DNA content was positively correlated with glucose depletion (P = 0.02). In the COC that did not deplete available glucose, the ratio of glucose consumption:lactate production was 1.82, indicating that the major fate of exogenous glucose was production of lactate by glycolysis. In the 7 oocytes that depleted all the glucose, the ratio of glucose consumption:lactate production was 1.22. One explanation for this may be that when glucose was no longer available, it was conserved for other pathways. It was noteworthy that these COC had more cumulus cells (P < 0.01) and the maturation rate was 4/7 (57%). In the group of COC that did not deplete all of the glucose, there was no significant difference in glucose consumption (13.17 v. 12.25 pmol/ng DNA per hour; P > 0.4) or lactate production (21.48 v. 20.28 pmol/ng DNA per hour; P > 0.4) between COC in which the oocyte reached MII (10/23; 43%), and those which contained a degenerated oocyte at the end of culture, respectively. To the best of our knowledge, this is the first report documenting the metabolism of equine COC. These data underline the importance of further studies to determine optimal conditions for in vitro maturation of equine COC, especially in terms of glucose availability.

297 EFFECT OF CUMULUS CELL REMOVAL DURING IN VITRO MATURATION OF PORCINE CUMULUS–OOCYTE COMPLEXES ON THE APOPTOTIC STATUS AND MEIOTIC PROGRESSION OF THE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 237
Author(s):  
P. Ferré ◽  
H. Funahashi
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Annexin V ◽  
Cumulus Cells ◽  
Dapi Staining ◽  
Meiotic Progression ◽  
Viability Assay ◽  
Significant Difference ◽  
Porcine Oocyte

This study was undertaken to examine the apoptotic status and meiotic progression of oocytes from small (SF) and medium follicles (MF) when the oocytes were denuded from cumulus cells (CC) before, during and after culture for in vitro maturation (IVM). Cumulus-oocyte complexes (COC) were aspirated from SF (0.5–2 mm in diameter) or MF (3–6 mm in diameter) of slaughtered prepubertal gilt ovaries. Only COC with a good morphology of the surrounding cumulus cells were cultured for IVM in modified porcine oocyte medium supplemented with 50 µM β-mercaptoethanol, 1 mM dibutyryl c-AMP, 10 IU mL–1 of eCG, and 10 IU mL–1 of hCG for 20 h at 39°C and 5% CO2 in air and then continued culture in the absence of dibutyryl c-AMP, eCG, and hCG in the same medium for another 24 h. Before and 20 h after the start of IVM culture, some of the oocytes were denuded of CC and the oocytes continued the IVM culture. After IVM culture, oocyte viability and meiotic progression were examined by the annexin V/PI viability assay and DAPI staining. Statistical analyses of 5 replicate data were performed with a 2-way ANOVA and a Tukey's multiple comparisons test. Before IVM culture, there was no significant difference between the viability of SF and MF oocytes, but the incidence of oocytes at the GV0 stage was higher in specimens from SF than MF (24.8 v. 3.3%), and that of oocytes at the GVI stage was the opposite (57.8 in MF v. 22.7% in SF). After IVM culture, apoptotic status of oocytes was only affected by the decumulation timing. The percentage of normal live oocytes was significantly higher when CC were removed after 20 and 44 h of IVM in both SF (39.7 and 39.3 v. 17.7%) and MF (45.4 and 37 v. 22.2%). The incidence of early and late apoptotic oocytes was significantly higher when the CC were removed before IVM culture in both SF (74.3 and 7.4%) and MF (69.4 and 6.7%). The incidence of mature live oocytes was significantly affected by both the origin of COC and the decumulation timing. Although the percentage of mature oocytes was higher in MF, maturation rates were significantly higher when oocytes were denuded at 20 h of IVM culture (SF 65.4%, MF 83.1%) as compared at 0 (SF 27.9%, MF 32.3%) and 44 h (SF 41%, MF 68.5%). However, the percentage of oocytes with normal spindle morphology was significantly higher when oocytes were denuded at 44 h of IVM culture (SF 70.6%, MF 91.5%) than 20 h (SF 66.8%, MF 73%). In summary, regardless of COC from SF and MF, removal of CC at 20 h of IVM culture seems to promote meiotic progression of the oocytes to the MII stage, but factor(s) from or communication with CC during the latter half of IVM culture may be needed to obtain a normal spindle morphology in mature oocytes.

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