In vitro penetration of zona pellucida of salt-stored bovine oocytes before and after maturation by frozen-thawed spermatozoa

1991 ◽  
Vol 36 (2) ◽  
pp. 209-219 ◽  
Author(s):  
R.C. Chian ◽  
K. Niwa ◽  
K. Okuda
Keyword(s):  
Zona Pellucida ◽  
Before And After ◽  
Bovine Oocytes

87 EFFECT OF CRYOPROTECTANT CONCENTRATION IN THE VITRIFICATION SOLUTION ON THE ZONA PELLUCIDA HARDENING AND SPERMATOZOA PENETRATION OF BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 149 ◽  
Author(s):  
A. Quiñones Martorello ◽  
G. Rios ◽  
A. Cano ◽  
R. H. Alberio
Keyword(s):  
Zona Pellucida ◽  
Calcium Influx ◽  
Oocyte Activation ◽  
High Concentration ◽  
Hoechst 33342 ◽  
Polar Bodies ◽  
Dmso Concentration ◽  
Sperm Penetration ◽  
Bovine Oocytes

In the murine model, it has been shown that the high concentration of cryoprotectants required for vitrification can activate the oocytes through a process mediated by calcium influx. This activation induces the zona pellucida (ZP) hardening and affects the sperm penetration. This study aimed to evaluate the effect of exposure of bovine oocytes to the vitrification solutions (VS1 and VS2) in calcium-free medium with 3 concentrations of etilenglycol (EG) and dimetylsulfoxide (DMSO) on the oocyte activation. Cumulus oocyte complexes (COC) were matured in vitro (22 h), partially denuded through pipetting in medium with hyaluronidase, and subject to four treatments: T1, untreated (control); T2, exposed to 20% EG+0% DMSO (VS1) and then 40% EG+0% DMSO (VS2); T3, 10% EG+10% DMSO (VS1) and then 20% EG+20% DMSO (VS2); and T4, 0% EG+20% DMSO (VS1) and then 0% EG+40% DMSO (VS2). The contact with each VS was 3 min and 30 s, respectively. After this, the COC were matured up to 24 h. In Expt. 1, COC were denuded and placed in a solution of pronase E in PBS (1 mg mL–1) to determine the number of oocytes with ZP digested after 9 min of exposure to the enzyme. In Expt. 2, COC were fertilized in TALP medium with 50 mg mL–1 heparin and 1 million mL–1 sperm. After 12 h, COC were denuded and stained with bisbenzimide (Hoechst 33342) and examined under epi-fluorescence. The number of oocytes indicating spermatic penetration was determined by presence of intact sperm heads, spermatic pro-nucleus, or 2 polar bodies. Data were analysed by the PROC GENMOD (SAS; see Table 1). In Expt. 1, there were no differences in the percentage of oocytes without ZP after pronase treatment in groups T1, T2, and T3. The T4 group had the lowest percentage of digestion, and T3 was not different from T4. In Expt. 2 there were no differences in the percentage of sperm penetration between T2, T3, and T4. All treatments had lower values than T1. In conclusion, bovine oocytes undergo hardening of the ZP when put in contact with the cryoprotectants, and this effect was significantly increased with the use of DMSO. Moreover, there was a decrease in sperm penetration in all treated groups, indicating that the natural blocking of polyspermy depends not only on the hardening of the ZP, but another process that could act at the plasma membrane. It is possible that cryoprotectants, regardless of their concentration, may trigger this early block through a mechanism that would be independent of calcium. Table 1.Effect of EG and DMSO concentration in the VS on the ZP hardening and sperm penetration of bovine oocytes exposed to these solutions Acknowledgment: the National Research Agency through the grant PICT 2007/1205.

360 EVALUATION OF QUALITY OF IN VITRO-MATURED BOVINE OOCYTES BY SEDIMENTATION WITH PERCOLL

2007 ◽  
Vol 19 (1) ◽  
pp. 295
Author(s):  
K. Yotsushima ◽  
M. Shimizu ◽  
H. Kon ◽  
Y. Izaike
Keyword(s):  
Specific Gravity ◽  
Simple Method ◽  
Serum Free ◽  
Class A ◽  
Serum Supplementation ◽  
Before And After ◽  
Class C ◽  
Bovine Oocytes

A simple method to evaluate the quality of in vitro-matured bovine oocytes is available for development of an in vitro embryo production system. Oocyte quality relates closely to oocyte fatty acid composition and mitochondrial distribution. The purpose of this study was to examine the influence of the quality of cumulus–oocyte complexes (COCs) and serum supplementation in IVM medium on the distribution of bovine oocyte specific gravities by sedimentation with Percoll before and after IVM. COCs were aspirated from abattoir-derived ovaries and were classified as classes A to D by the morphology of their cumulus cell layers as follows: class A, compact and more than 3 layers thick; class B, compact but <3 layers; class C, partially naked and <3 layers; and class D, naked or expanded. The classified COCs were cultured in TCM-199 supplemented with 0.1% BSA, 5 µg mL−1 insulin, 10 µg mL−1 transferrin, and 10 ng mL−1 transforming growth factor-α (M199-BITT) for 22–24 h. To evaluate the influence of serum supplementation, oocytes from classes A and B were also incubated in M199-BITT as serum-free culture or TCM-199 supplemented with 10% fetal calf serum as serum-supplemented culture. Percoll solutions were prepared by diluting Percoll with PBS supplemented with 0.3% BSA, 1 mg mL−1 glucose, and 0.2 mM sodium pyrvate to 20, 17.5, 15, 12.5, 10, 7.5, and 5% solutions. After removal of cumulus cells, denuded oocytes were put on the surface of Percoll solution for 3 min, and the precipitated oocytes were transferred to stepwise high density solution. The percent of Percoll solution just before buoyancy was considered as the oocyte specific gravity value. Statistical analysis was performed by one-way ANOVA. Oocytes from class A had the highest specific gravities before and after IVM in all classes (Table 1). After IVM, oocyte specific gravities from classes A and C were higher than those of oocytes before IVM (class A: P < 0.05, class C: P < 0.001). The specific gravities of in vitro-matured oocytes cultured in serum-free medium were higher than those cultured in serum-supplemented medium (15.3 ± 0.3%, n = 71, and 14.0 ± 0.3%, n = 58; P < 0.01). These results show that the specific gravity was affected by the morphological quality of COC, and the culture conditions for IVM may profile the metabolic activity of oocytes during IVM. Table 1.Specific gravities of the bovine oocytes classified by morphology of COC before and after IVM

Ultrastructural Imaging Analysis of the Zona Pellucida Surface in Bovine Oocytes

2019 ◽  
Vol 25 (4) ◽  
pp. 1032-1036 ◽  
Author(s):  
Francisco Báez ◽  
Álvaro A. Camargo ◽  
Gustavo D.A. Gastal
Keyword(s):  
Zona Pellucida ◽  
Ultrastructural Change ◽  
Early Embryo ◽  
Imaging Analysis ◽  
Mature Group ◽  
Immature Group ◽  
Imagej Software ◽  
Bovine Oocytes ◽  
Objective Methodology

AbstractThe aims of the present study were to: (i) evaluate the ultrastructural differences in the zona pellucida (ZP) surface between immature and mature bovine oocytes, and (ii) describe a new objective technique to measure the pores in the outer ZP. Intact cumulus–oocyte complexes (COCs) obtained from a local abattoir were immediately fixed (immature group) or submitted to in vitro maturation (IVM) at 38.5 °C for 24 h in a humidified atmosphere of 5% CO2 in air (mature group). Oocytes from both groups were morphologically evaluated via Scanning Electron Microscopy (SEM) and the images were processed in the Fiji/ImageJ software using a new objective methodology through the Trainable Weka Segmentation plugin. The average number of pores in ZP was greater (p < 0.05) in the mature group than the immature group. However, the size and circularity of pores in ZP did not differ (p > 0.05) between groups. In conclusion, it has been shown that the number of pores highlighted the main ultrastructural change in the morphology of the ZP surface of bovine oocytes during the IVM process. We have described an objective method that can be used to evaluate ultrastructural modifications of the ZP surface during oocyte maturation and early embryo development.

Coculturing cumulus oocyte complexes with denuded oocytes alters zona pellucida ultrastructure in in vitro matured bovine oocytes

2013 ◽  
Vol 80 (9) ◽  
pp. 1117-1123 ◽  
Author(s):  
Byung-Hyun Choi ◽  
Jae-Il Bang ◽  
Jong-In Jin ◽  
Seong-Su Kim ◽  
Hyun-Tae Jo ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Bovine Oocytes

Identification of glycoconjugates in the zona pellucida of in vitro matured and tubal unfertilized bovine oocytes by lectin histochemistry

1996 ◽  
Vol 43 (2-3) ◽  
pp. 99-111 ◽  
Author(s):  
A.Verini Supplizi ◽  
M. Monaci ◽  
G. Stradaioli ◽  
T. Greve ◽  
F. Parillo
Keyword(s):  
Zona Pellucida ◽  
Lectin Histochemistry ◽  
Bovine Oocytes

Zona pellucida birefringence correlates with developmental capacity of bovine oocytes classified by maturational environment, COC morphology and G6PDH activity

2012 ◽  
Vol 24 (4) ◽  
pp. 568 ◽  
Author(s):  
Eva Held ◽  
Eva-Maria Mertens ◽  
Abdollah Mohammadi-Sangcheshmeh ◽  
Dessie Salilew-Wondim ◽  
Urban Besenfelder ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Zona Pellucida ◽  
In Vitro Maturation ◽  
Immature Oocytes ◽  
Developmental Capacity ◽  
Bovine Oocytes ◽  
Scanning Electron

In the present study we aimed to analyse structural changes during in vitro maturation of the bovine zona pellucida (ZP) by scanning electron microscopy (SEM) ands zona pellucida birefringence (ZPB). Here we show that alterations during in vitro maturation invasively analysed by SEM are reflected in ZPB. In vivo-matured oocytes displayed significantly lower birefringence parameters and significantly higher blastocyst rates compared with in vitro-derived oocytes (39.1% vs 21.6%). The same was observed for in vitro-matured oocytes with cumulus–oocyte complex (COC) Quality 1 (Q1) compared with Q3-COCs with respect to zona birefringence and developmental capacity. Immature oocytes with Q1-COCs displayed higher ZPB values and a higher developmental capacity to the blastocyst stage (27.7% vs 16.9%) compared with immature Q3-COCs. Considering in vitro-matured oocytes, only those with Q1-COC showed a trend for ZPB similar to in vivo-matured oocytes. Therefore, a decreasing trend for ZPB during in vitro maturation seems to be typical for high-quality oocytes and successful cytoplasmic maturation. In accordance, fully-grown immature oocytes reached significantly higher blastocyst rates (32.0% vs 11.5%) and lower ZPB values compared with still-growing ones. In conclusion, we successfully evaluated the applicability of zona imaging to bovine oocytes: alterations during in vitro maturation invasively analysed by scanning electron microscopy were reflected in the birefringence of the zona pellucida of bovine oocytes affecting developmental capacity at the same value. Therefore ZPB measurement by live zona imaging has potential to become a new tool to assess correctness of in vitro maturation and to predict developmental competence.

Expression and cellular distribution of zona pellucida glycoproteins in canine oocytes before and after in vitro maturation

Zygote ◽  
2014 ◽  
Vol 23 (6) ◽  
pp. 863-873 ◽  
Author(s):  
Bartosz Kempisty ◽  
Hanna Piotrowska ◽  
Dorota Bukowska ◽  
Magdalena Woźna ◽  
Sylwia Ciesiółka ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Quantitative Polymerase Chain Reaction ◽  
Microscopic Analysis ◽  
Maturation Stage ◽  
Protein Distribution ◽  
Protein Levels ◽  
Peripheral Area ◽  
Before And After ◽  
Polymerase Chain

SummaryThis study was aimed at investigating zona pellucida glycoproteins (ZP) ZP2, ZP3 mRNA expression as well as ZP3, ZP4 (ZPB) protein distribution before and after in vitro maturation (IVM) in canine oocytes. The cumulus–oocyte complexes (COCs) were recovered from 27 anoestrous mongrel bitches and matured for 72 h in TCM199 medium. The canine COCs were analysed before and after IVM. Using real-time quantitative polymerase chain reaction (RQ-PCR), both groups of oocytes were analysed for detection of ZP2 and ZP3 mRNA profiles as well as using confocal microscopic analysis for observation of ZP3 and ZP4 protein distribution. In post-IVM canine oocytes an increase in transcript content of ZP2 and ZP3 genes as well as a decrease in ZP3 and ZP4 protein levels were observed when compared with pre-IVM oocytes. Moreover, the ZP4 protein before IVM was significantly distributed in the peripheral area of cytoplasm, whereas after IVM it was localized rather than in the entire cytoplasm. In contrast, the ZP3 protein was found both before and after IVM was distributed in the peripheral area of the cytoplasm. In conclusion, we suggest that the expression of ZP2 and ZP3 genes is associated with the maturation stage of canine oocytes, as higher mRNAs levels were found after IVM. However, a decreased expression of ZP3 and ZP4 proteins after IVM suggests maturation-dependent down-regulation of these protein translations, which may result in disturbed fertilization.

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