scholarly journals Effects of Stem Cell Factor/c-Kit Signaling on In Vitro Maturation of Porcine Oocytes and Subsequent Developmental Competence After Fertilization

2021 ◽  
Vol 8 ◽  
Author(s):  
Eunhye Kim ◽  
Lian Cai ◽  
Sang-Hwan Hyun
Keyword(s):  
Stem Cell ◽  
Mrna Expression ◽  
Stem Cell Factor ◽  
Polar Body ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Control Group ◽  
Secreted Factors ◽  
Polar Body Extrusion

Stem cell factor (SCF), also known as c-Kit ligand, plays an important role in the proliferation of primordial germ cells and the survival of oocytes during follicular development. The aim of this study was to investigate the effect of SCF/c-Kit signaling on in vitro maturation (IVM) of porcine oocytes by analyzing nuclear and cytoplasmic maturation, oocyte size, cumulus cell expansion, and developmental competence to the blastocyst stage. Moreover, mRNA expression patterns of porcine cumulus cells and oocytes were evaluated using qRT-PCR. Following 42 h of IVM, 10 and 50 ng/mL SCF-treated groups exhibited significantly (P < 0.05) increased polar body extrusion rates and intracellular glutathione levels compared with the control group. The cumulus expansion index significantly (P < 0.05) increased in all SCF-treated groups compared with the control samples. mRNA levels of the proapoptotic gene Bax and apoptosis-related cysteine peptidase Caspase3 were lower in SCF-treated cumulus cells than in the control group. Notably, the diameter of oocytes after IVM, the mRNA expression of well-known oocyte-secreted factors (GDF9 and BMP15), and an oocyte-specific protein essential for ovulation and oocyte health (YBX2) were significantly (P < 0.05) higher in SCF-treated than in non-treated oocytes. Inhibition of c-Kit during porcine IVM using ACK2, an antagonistic blocker of c-Kit, significantly (P < 0.05) decreased the polar body extrusion rate compared with the control, as well as blastocyst formation rate compared with the 10 ng/mL SCF-treated group. In conclusion, the effect of SCF/c-Kit-mediated signaling during porcine IVM could be ascribed to the reduced expression of apoptosis-related genes and higher expression of oocyte-specific/secreted factors.

133 VITRIFICATION CAUSES DAMAGE OF THE ANTIOXIDANT DEFENSE SYSTEM IN IVM PORCINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 184 ◽  
Author(s):  
T. Somfai ◽  
M. Ozawa ◽  
J. Noguchi ◽  
H. Kaneko ◽  
K. Ohnuma ◽  
...  
Keyword(s):  
Polar Body ◽  
Antioxidant Defense System ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Cleavage Rate ◽  
Sperm Penetration ◽  
Second Polar Body ◽  
Polar Body Extrusion

The present study investigated the ability of in vitro-matured (IVM) porcine oocytes to be fertilized in vitro after vitrification. Oocytes matured in vitro for 46 h according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) were cryopreserved by solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) or subjected to the steps of SSV without cooling (toxicity control, TC). Oocyte viability was assessed 2 h after treatment by morphology and fluorescein diacetate staining. Live oocytes were in vitro-fertilized (IVF) and cultured (IVC) for 6 days according to Kikuchi et al. (2002). Fertilization and pronuclear development of oocytes were assessed 10 h after IVF by aceto-orcein staining. Cleavage and blastocyst rates were recorded during IVC. Glutathione (GSH) and hydrogen peroxide levels in oocytes were analyzed by DTNB-glutathione disulfide reductase recycling assay and 20,70-dichlorofluorescein fluorescence assay, respectively. Data were analyzed by ANOVA and paired t-test. The rate of live oocytes after SSV was lower compared to the control and the TC groups (54.4%, 100%, and 100%, respectively; P < 0.05). Sperm penetration rates of SSV oocytes were lower than those of the control group (51.9% and 67.8%, respectively; P < 0.05). Significantly fewer penetrated oocytes in the SSV group formed male pronuclei than those in the control and the TC groups (66.7%, 96.5%, and 98.5%, respectively; P < 0.05). There were no differences in second polar body extrusion and monospermy rates between the treatment groups. The cleavage rate of SSV oocytes was significantly lower than that of the control and the TC groups (13.3%, 46.6%, and 47.7%, respectively; P < 0.05). Blastocyst rates of control and TC oocytes were similar (20.7% and 23.6%, respectively), whereas only a single embryo developed to the blastocyst stage in the SSV group. GSH content of SSV oocytes was significantly lower than that of the control oocytes (7.3 pM and 10.5 pM, respectively), whereas the peroxide level was higher in SSV oocytes than in the control oocytes (59.0 and 50.5 FIU, respectively; P < 0.05). Our results reveal a cryopreservation-related drop of intracellular GSH level in oocytes, which may cause their decreased ability to form a male pronucleus and their increased sensitivity to oxidative stress. These factors might contribute to the low developmental competence of vitrified oocytes. This work was supported by a grant-in-aid for the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers (P05648) and the Bilateral Scientific and Technological Collaboration Grant between Hungary and Japan (TET, no. JAP-11/02).

scholarly journals 235RETINOID-DEPENDENT MRNA EXPRESSION IN BOVINE OOCYTES PREMATURED AND/OR MATURED IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 238
Author(s):  
E. Gomez ◽  
C. Diez ◽  
E. Moran ◽  
A. Rodriguez ◽  
L.J. Royo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mrna Expression ◽  
Cumulus Cells ◽  
Cyclin B1 ◽  
Defined Medium ◽  
Developmental Competence ◽  
Control Group ◽  
Free Oxygen Radicals ◽  
Bovine Oocytes

As a transcription factor, retinoic acid (RA) can activate or silence a wide number of genes, thus inducing differentiation in cell systems and playing a role in cell cycle regulation. However, little is known of RA-dependent gene expression in the oocyte. Bovine oocytes and cumulus cells express most RA receptors, and the presence of 9-cis-RA during in vitro maturation (IVM) is beneficial to oocyte development (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). The present work analyzes the relative abundance of various developmentally important gene transcripts in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were manipulated in defined medium with polyvinyl-alcohol (DM-PVA). Those COCs undergoing prematuration were cultured for 24h in DM-PVA with 25μM roscovitine. For IVM, some prematured COCs were cultured for 24h in DM-PVA containing pFSH, LH and E2. Incubations were made at 39°C in an atmosphere of 5% CO2 in air and high humidity. Within experiments, COCs were cultured with nM 9-cis-RA 5, in 1% ethanol (both as vehicle and inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Using Real Time PCR (10 oocytes per group) (Rizos et al., 2003 Biol. Reprod. 68, 236) we examined the relative mRNA expression of genes involved in protection against free oxygen radicals (Mn-superoxide dismutase, MnSOD), glucose metabolism (glucose-6-phosphate dehydrogenase, G6PDH) and cell cycle events (Cyclin B1 and H1). Data (of 4 replicates) were analyzed by ANOVA and Duncan test (P<0.05). Regarding immature oocytes, prematuration in 1% ethanol increased cyclin B1 expression and decreased cyclin H1, while 9-cis-RA increased G6PDH. Maturation without additives increased cyclin B1 and G6PDH, but decreased cyclin H1 and MnSOD expression;; opposite trends were observed under increasing ethanol dosages (3% and 5%). Maturation with 1% ethanol or 9-cis-RA enhanced cyclin B1 and G6PDH, while reducing cyclin H1 and MnSOD expressions. The presence of 9-cis-RA during both prematuration and maturation processes tended to show more prominent effects than the ones observed when it was present only during prematuration or maturation alone. In our study, in presence of 9-cis-RA during both prematuration and maturation processes, the expression of cyclin B1 and G6PDH tended to increase, while cyclin H1 and MnSOD tended to decrease. However, the differences with the control group without additives were not significant. Our study during both prematuration and maturation processes show that beneficial effects of RA on oocyte developmental competence may not be related to the alteration of mRNA expression of the four genes analyzed. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175; 2003-05783).

scholarly journals Effect of Heat Stress on Developmental Competence of In Vitro Matured Oocytes of Camelus Dromedaries with Different Qualities

2020 ◽  
Vol 10 (4) ◽  
pp. 658-664
Author(s):  
G Ashour ◽  
Ashraf El-Sayed ◽  
M Khalifa ◽  
Nasser Ghanem
Keyword(s):  
Heat Stress ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Cleavage Rate ◽  
Developmental Capacity ◽  
Polar Body Extrusion

The deleterious effect of heat stress on cumulus-oocytes complexes (COCs) competence is well recognized in different livestock species. Therefore, the present study aimed to investigate the effect of physiologically relevant heat stress on the developmental competence of camel COCs during in vitro maturation (IVM). A total of 1548 COCs were divided into six groups in this study. The groups were named K1 and K2 representing good and low-quality COCs incubated at 38.5oC for 30 hours. While K3 and k4 represent good and low-quality COCs exposed to 41oC for the first 6 hours of IVM. Finally, K5 and k6 represent the groups of good and low-quality COCs exposed to 42oC for the first 6 hours of IVM. After exposure of COCs to heat stress at 41°C and 42°C during the first 6 hours of in vitro maturation, the COCs were incubated at 38.5°C for 24 hours of IVM. The in vitro matured COCs were activated to cleave using ethanol followed by 4 mM 6-DMAP and developed embryos were cultured in vitro for 7 days post parthenogenetic activation. The results of this study indicated that heat stress at 42oC significantly decreased the Pb (polar body) extrusion rate in K4 and K6, compared to other groups. Additionally, the embryo cleavage rate was significantly lower for good and low-quality oocytes exposed to heat stress (K2, K3, K4, K5, and K6), compared to good quality COCs of the control group (K1). The cleavage rate was lower for low quality (K2; 63 ± 1.28) than good quality COCs (K1; 53 ± 1.85). The percentages of oocytes that developed to the blastocyst stage were lower for K2, K3, K4, K5, and K6 than K1. Moreover, the blastocyst rate was lower for K2 (9 ± 0.22) than K1 (15 ± 0.22). The results of this study indicated that exposure of camel oocytes to heat stress for 6 hours during in vitro maturation severely reduced extrusion of polar body, cleavage, and blastocyst rates. The low-quality camel COCs were reduced developmental capacity than good quality oocytes.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

2018 ◽  
Vol 30 (1) ◽  
pp. 189
Author(s):  
L. Landeo ◽  
R. S. Molina ◽  
M. E. Zuñiga ◽  
T. R. Gastelu ◽  
C. Sotacuro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Streptomyces Griseus ◽  
Petri Dish ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Morula Stage

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

Impact of oocyte-secreted factors on its developmental competence in buffalo

Zygote ◽  
2017 ◽  
Vol 25 (3) ◽  
pp. 313-320 ◽  
Author(s):  
Swati Gupta ◽  
Sriti Pandey ◽  
Mehtab S. Parmar ◽  
Anjali Somal ◽  
Avishek Paul ◽  
...  
Keyword(s):  
Cumulus Cell ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Marker Genes ◽  
Enabling Factors ◽  
Secreted Factors ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Follicle Size

SummaryOocyte-secreted factors (OSFs) play an important role in the acquisition of oocyte developmental competence through bidirectional cross-talk between oocyte and cumulus cells via gap junctions. Thus, the present study was designed to investigate the effect of two OSFs, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the developmental competence of buffalo oocytes derived from two different follicle sizes. Cumulus–oocyte complexes (COCs) from large follicles (LF, >6 mm) or small follicles (SF, <6 mm) were collected and matured in vitro either in the presence of GDF9 or BMP15, or both, or with the denuded oocytes (DOs) as a source of native OSFs. Cleavage and blastocyst rates were significantly (P < 0.05) higher in LF-derived than SF-derived oocytes. Cleavage and blastocyst rates were significantly higher (P < 0.05) in the DOs and the combination groups compared with the control, GDF9 alone and BMP15 alone groups, both in LF-derived and SF-derived oocytes, although the cleavage and blastocyst rates did not differ significantly (P > 0.05) between DOs and combination groups. Relative mRNA analysis revealed significantly higher (P > 0.05) expression of the cumulus cell marker genes EGFR, HAS2, and CD44 in LF-derived than SF-derived oocyte; the expression of these markers was significantly higher (P > 0.05) in DOs and combination groups, irrespective of the follicle size. These results suggested that LF-derived oocytes have a higher developmental competence than SF-derived oocytes and that supplementation of GDF9 and BMP15 modulates the developmental competence of buffalo oocytes by increasing the relative abundance of cumulus-enabling factors and thereby increasing cleavage and the quality of blastocyst production.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

scholarly journals Possible mechanism for acceleration of meiotic progression of bovine follicular oocytes by growth factors in vitro

Reproduction ◽  
2002 ◽  
pp. 135-142 ◽  
Author(s):  
M Sakaguchi ◽  
T Dominko ◽  
N Yamauchi ◽  
ML Leibfried-Rutledge ◽  
T Nagai ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Kinase Activity ◽  
Polar Body ◽  
Control Group ◽  
Meiotic Cell ◽  
Treatment Groups ◽  
Igf I ◽  
Polar Body Extrusion

The mechanism for the accelerating effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the meiotic cell cycle of bovine oocytes cultured in vitro was investigated. Cumulus-oocyte complexes (COCs) were obtained from small (< or = 3 mm in diameter), medium (4-6 mm in diameter) or large (7-10 mm in diameter) ovarian follicles and cultured with or without a combination of EGF and IGF-I (growth factors). Growth factors significantly increased the frequency of first polar body extrusion of oocytes derived from small follicles at 16 h of culture (PB16 oocytes; with growth factors: 75%; without growth factors: 55%), but did not increase the frequency in oocytes from medium or large follicles. COCs from small follicles were cultured with individual growth factors and sampled for kinase activity. The frequencies of polar body extrusion in EGF only (67%) and EGF + IGF-I (75%) treatment groups were significantly higher than those in the control (no growth factor) group (49%), but not significantly higher than in the IGF-I only group (63%). The H1 kinase activity at 6-8 h of culture in each group increased significantly from the baseline value at 0 h of culture, and the H1 kinase activities in the EGF only, IGF-I only and EGF + IGF-I treatment groups were significantly higher than those in the control group at 8 h of culture. MAP kinase activity was significantly higher than the baseline value and significantly higher than that in the control group at 6 h of culture in the EGF treatment group only. In conclusion, EGF and IGF-I act on COCs from small follicles to accelerate the meiotic cell cycle of the oocytes. This accelerating effect may be related to increased H1 and MAP kinase activities during the early stages of maturation.

scholarly journals 301EFFECT OF THE MII-AGE AND ACTIVATION PROTOCOL ON THE PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 270
Author(s):  
I. Lagutina ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Cytochalasin B ◽  
Average Rate ◽  
Polar Body ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Average Cell ◽  
Electric Impulse ◽  
Polar Body Extrusion

The completion of porcine oocyte nuclear maturation (MII) in vitro, characterized by the time of polar body extrusion, starts at about 32h of maturation and lasts more than 12h. This leads to the simultaneous presence in the population of matured oocytes with differing abilities to be activated. We investigated age-dependent changes in pig oocyte maturation, activation and development in SOFaa in response to electric impulse (EL) in the presence of cytochalasin B (CB) and EL in combination with cycloheximide and cytochalasin B (EL+CHX+CB). Oocytes were matured in TCM 199 with 10% FCS, cysteine, LH, FSH (Pergovet, Serono, Geneva, Switzerland) for 36h and then decumulated. Matured oocytes were activated at 40 and 44h by double pulse of 30μs DC 1, 5kVcm−1 and cultured in 5μgmL−1 CB for 4h or by EL followed by incubation in 10μgmL−1 CHX+5μgmL−1 CB for 4h. According to the MII-age before activation oocytes were divided into 2 age classes: 3–7 and 7–11h after polar body extrusion. Embryos were cultured in SOFaa in 5% CO2, 5% O2 at 38.5°C. The rates of cleavage, blastocyst formation and cell number of BL on Day 7 (BLD7) were recorded. Our results showed that the average rate of maturation at 44h was 72% (n=1377). About 50% and 87% of oocytes, that eventually matured, extruded the polar body at 37 and 40h, respectively. The average cell number of BLD7 developed in SOFaa was 80±36 (n=52) and was not affected by activation protocol. Seventy-nine and 27% of BL had more than 50 and 100 cells per BL, respectively. Porcine oocytes activated by EL acquired their developmental competence gradually, achieving the highest rates of cleavage and blastocyst formation 7h after polar body extrusion. By contrast, oocytes activated by EL+CHX+CB showed their maximal developmental competence earlier (3–7h group). In conclusion, we demonstrate that electric impulse in combination with CHX+CB treatment permits earlier efficient activation of porcine oocytes (3–7h after polar body extrusion).

167 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES DERIVED FROM SMALL- OR MEDIUM-SIZED FOLLICLES AND DENUDED OF CUMULUS CELLS BEFORE AND DURING IN VITRO MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 192
Author(s):  
P. Ferré ◽  
K. X. Nguyen ◽  
T. Wakai ◽  
H. Funahashi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
First Polar Body

This experiment was undertaken to assess the meiotic and developmental competences of oocytes derived from different sized follicles and denuded of cumulus cells 0, 20, and 44 h after the start of culture for in vitro maturation (IVM). Groups of 60 oocyte-cumulus complexes from small- (SF; <3 mm) and medium-sized follicles (MF; 3–6 mm) were cultured for IVM in porcine oocyte medium with 50 μM β-mercaptoethanol supplemented with 1 mM dibutyryl-cyclic adenosine monophosphate, 10 IU mL−1 of eCG, and 10 IU mL−1 of hCG for 20 h at 39°C and 5% CO2 in air. Then, after washing, they continued culture in fresh β-mercaptoethanol without dibutyryl-cyclic adenosine monophosphate and gonadotropins under the same conditions for another 24 h. At 0, 20, and 44 h of IVM, cumulus cells were removed with 0.1% (wt/vol) hyaluronidase and the denuded oocytes continued IVM culture following the protocol. Mature oocytes with the first polar body were selected, parthenogenetically activated with a single electrical pulse (DC: 1.2 kV/cm, 30 µs), incubated with 4% (wt/vol) BSA and 5 μM cytochalasin B for 4 h, and cultured in porcine zygote medium for 5 days. Cleavage and blastocyst formation rates were observed on Day 2 and 5, respectively. Blastocysts were stained with 4’,6-diamidino-2-phenylindole for cell count assessment. The experiment was replicated 5 times and analysed with a 1- or 2-way ANOVA. If P < 0.05 in ANOVA, a Tukey multiple comparisons test was performed. Regardless of the time of cumulus cell removal, oocytes from MF had significantly higher in rates of maturation, cleavage, and blastocyst rates, as compared with those from SF, whereas there were no significant differences in the cell number of blastocysts between SF and MF (32 v. 34 cells, respectively). When oocytes were denuded before IVM culture, rates of oocyte maturation (37.6% in SF and 50.8% in MF), and blastocyst formation (2.7% in SF and 27.3% in MF) were significantly lower than controls (51.2% in SF and 76% in MF; 25.8% in SF and 48.5% in MF, respectively). When oocytes were denuded 20 h after the start of IVM, oocyte maturation rates were significantly increased (64.1% in SF and 82.5% in MF) as compared with controls, whereas no significant differences were observed in cleavage and blastocyst formation rates in comparison with controls. These results conclude that removing cumulus cells from oocyte-cumulus complexes 20 h after the start of IVM improves the meiotic competence of oocytes derived from both SF and MF, without any reduction of developmental competence of the oocytes following parthenogenetical activation.

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