scholarly journals Evaluation of equine oocyte developmental competence using polarized light microscopy

Reproduction ◽  
2017 ◽  
Vol 153 (6) ◽  
pp. 775-784 ◽  
Author(s):  
A Bertero ◽  
F Ritrovato ◽  
F Evangelista ◽  
V Stabile ◽  
R Fortina ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Zona Pellucida ◽  
Light Microscope ◽  
Polarized Light ◽  
Developmental Competence ◽  
Immature Oocytes ◽  
Oocyte Developmental Competence ◽  
Morphological Evaluation ◽  
Meiotic Competence

The purpose of this study was to observe in vitro-matured equine oocytes with an objective computerized technique that involves the use of a polarized light microscope (PLM) in addition to the subjective morphological evaluation obtained using a classic light microscope (LM). Equine cumulus-oocyte complexes (COCs, n = 922) were subjected to different in vitro maturation times (24, 36 or 45 h), however, only 36-h matured oocytes were analyzed using CLM. The 36-h matured oocytes that reached maturity were parthenogenetically activated to evaluate the quality and meiotic competence. Average maturation percentages per session in groups 1, 2 and 3 (24-, 36- and 45-h matured oocytes respectively) were 29.31 ± 13.85, 47.01 ± 9.90 and 36.62 ± 5.28%, whereas the average percentages of immature oocytes per session were 28.78 ± 20.17, 7.83 ± 5.51 and 22.36 ± 8.39% respectively. The zona pellucida (ZP) birefringent properties were estimated and correlated with activation outcome. ZP thickness and retardance of the inner layer of the zona pellucida (IL-ZP) were significantly increased in immature oocytes compared with mature oocytes (P < 0.001 and P < 0.01 respectively). The comparison between parthenogenetically activated and non-activated oocytes showed a significant increase in the area and thickness of the IL-ZP in parthenogenetically activated oocytes (P < 0.01). These results show that the 36-h in vitro maturation (IVM) protocol allowed equine oocytes to reach maturity, and PLM observation of ZP can be used to distinguish mature and immature oocytes as well as activated and non-activated oocytes.

351 POLARIZED LIGHT MICROSCOPY: DETECTION OF MICROTUBULES AND ITS EFFECTS ON THE VIABILITY OF IN VITRO-MATURED PORCINE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 332 ◽  
Author(s):  
I. Molina ◽  
M. Muñoz ◽  
C. Díez ◽  
E. Gómez ◽  
E. A. Martínez ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Polarized Light ◽  
Polarized Light Microscopy ◽  
Developmental Competence ◽  
Meiotic Spindle ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Oocyte Developmental Competence ◽  
Blastocyst Rate

The meiotic spindle in the oocyte is composed of microtubules and plays an important role during chromosome alignment and separation at meiosis. Polarized light microscopy (PLM) is used as a tool in human and, recently, in farm animals assisted reproductive technologies. PLM could be useful for a non-invasive evaluation of the meiotic spindle. The objectives of the present study were to assess the efficiency of PLM to detect microtubule-polymerized protein within in vitro-matured porcine oocytes and to examine the effects of PLM on the oocyte developmental competence. Cumulus-oocyte complexes from slaughterhouse ovaries were matured in vitro for 42 h as described by Gil et al. (2004 Theriogenology 62, 544-552). In the first experiment, a total of 97 oocytes from 6 replicates were placed individually in 10-μL drops of TCM-199-Hepes-FCS in a glass Petri dish. PLM was used to detect the presence of polymerized protein which could be forming a meiotic spindle. The presence of polymerized protein and a meiotic spindle was confirmed in individual oocytes by inmunostaining and chromatin detection as described by Morató et al. (2008 Mol. Reprod. Dev. 75, 191-201). In the second experiment, a total of 160 oocytes from 4 replicates were exposed or not (controls) to PLM for 10 minutes. Thereafter, the oocytes were parthenogenetically activated and cultured in vitro. Cleavage rate, total blastocyst rate, expanded blastocyst rate on Day 7 and total cell numbers in expanded blastocysts were assessed. Data were analyzed by GLM procedure of SAS. There was a positive correlation (r = 1; P < 0.0001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by inmunostaining. A positive PLM signal was detected in 98.9% of the oocytes. A barrel-shape spindle was observed in 94.8% of the individual samples by inmunostaining and all of these oocytes were positive to PLM. Moreover, oocytes exposed to PLM did not differ significantly from controls on cleavage rate (83.7 ± 1.5 v. 84.4 ± 1.5), total blastocyst rate (36.9 ± 3.6 v. 41.2 ± 3.6) and expanded blastocyst rate on Day 7 (21.9 ± 1.7 v. 26.2 ± 1.7), respectively. There were also no differences in total cell numbers counted in expanded blastocysts (32.8 ± 2.6 v. 35.6 ± 2.5). These results indicate that polarized light microscopy did not exert detrimental effects on porcine oocyte developmental competence and it seems an efficient system to detect polymerized protein in in vitro-matured porcine oocytes. Grant support: INIA: RZ2007-00013-00-00. I. Molina, M. Muñoz, B. Trigal and D. Martín are sponsored by INIA, RYC08-03454, Cajastur and PTA2007-0268-I, respectively.

scholarly journals Association between human oocyte developmental competence and expression levels of some cumulus genes

Reproduction ◽  
2007 ◽  
Vol 134 (5) ◽  
pp. 645-650 ◽  
Author(s):  
Fabiana Cillo ◽  
Tiziana A L Brevini ◽  
Stefania Antonini ◽  
Alessio Paffoni ◽  
Guido Ragni ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Poor Quality ◽  
Developmental Competence ◽  
Human Oocyte ◽  
Transcript Levels ◽  
Oocyte Developmental Competence ◽  
Morphological Evaluation ◽  
Hyaluronic Acid Synthase ◽  
Morphological Criteria

At present, oocyte selection is mainly based upon morphological criteria but it is generally acknowledged that its reliability requires further improvement. The aim of this study was to determine whether transcript levels in cumulus cells can provide a useful marker of oocyte developmental competence in vitro. A retrospective study was performed on cumulus cells isolated from 90 oocytes retrieved from 45 patients. Upon fertilization, 35 oocytes originated good-quality embryos and 36 developed into poor-quality embryos, whereas 19 failed to be fertilized. Semi-quantitative measurement of hyaluronic acid synthase 2 (HAS2), gremlin1 (GREM1), and pentraxin 3 (PTX3) mRNAs was performed and data for all genes were obtained from all the samples. Cumulus cells isolated from oocytes that originated high-quality embryos on day 3 of culture had HAS2 and GREM1 transcript levels higher than those detected in cells from oocytes that did not fertilize or developed into poor-quality embryos. No differences were observed in PTX3 levels. Results indicate that the measurement of HAS2 and GREM1 levels in cumulus cells would reliably complement the morphological evaluation providing a useful tool for selecting oocytes with greater chances to be fertilized and develop in vitro.

160 ZONA PELLUCIDA BIREFRINGENCE CORRELATES WITH CUMULUS MORPHOLOGY AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE ACTIVITY OF EQUINE OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 192 ◽  
Author(s):  
A. Mohammadi-Sangcheshmeh ◽  
E. Held ◽  
D. Tesfaye ◽  
K. Schellander ◽  
M. Hoelker
Keyword(s):  
Light Microscopy ◽  
Zona Pellucida ◽  
Polarized Light ◽  
Polarized Light Microscopy ◽  
G6pd Activity ◽  
Developmental Competence ◽  
G6pdh Activity ◽  
Gentamicin Sulfate ◽  
Developmental Capacity ◽  
Glucose 6 Phosphate Dehydrogenase

The present study was conducted to determine the predictive value of zona pellucida birefringence (ZPB) measurement for developmental capacity of equine oocytes. In vitro developmental capacity of equine oocytes is related to cumulus morphology as well as glucose-6-phosphate dehydrogenase (G6PD) activity. Therefore, we classified equine oocytes according to cumulus morphology and G6PDH activity into groups of different developmental capacities and analysed the correlation between ZPB measured by polarized light microscopy and developmental competence. Ovaries were obtained from an abattoir and were transported to the laboratory within 2 to 4 h at 25 to 30°C in 0.9% saline. Cumulus–oocyte complexes (COC) were recovered by scraping the granulosa layer from opened follicles using a bone curette and were classified as having an expanded (Ex, n = 86) or compact (Cp, n = 93) cumulus. Other COC were subjected to 26 μM brilliant cresyl blue (BCB) in modified PBS for 90 min at 38.5°C and were categorised into BCB+ (blue cytoplasm, low G6PD activity, n = 89) and BCB– (colourless cytoplasm, high G6PD activity, n = 41) according to their ability to convert the BCB stain from blue to colourless. Following maturation for 28 to 30 h at 38.5°C in 5% CO2 in DMEM-F12 supplemented with 10% FCS, 5 mU mL–1 of FSH and 25 μg mL–1 of gentamicin sulfate, oocytes were imaged individually to assess zona thickness and ZPB by polarized light microscopy using OCTAX polarAIDE software (OCTAX, Bruckberg, Germany). Subsequently, oocytes were subjected to intracytoplasmic sperm injection using frozen-thawed stallion spermatozoa followed by culture in DMEM/F-12 medium (10% FCS and 25 μg mL–1 of gentamicin sulfate) under mineral oil (38.2°C, 5% CO2, 5% O2) for 8 days. Development rates were compared by chi-square test; variations in thickness and birefringence were analysed by Tukey's test and ANOVA. Higher maturation (58.4 vs 40.2%) and blastocyst rates (11.9 vs 3.8%) of Ex oocytes compared with Cp oocytes as well as higher proportions of BCB+ oocytes that matured (57.7% vs 28.1%), cleaved (45.9 vs 29.0%) and developed to the blastocyst stage (9.2 vs 1.4%) compared with BCB– oocytes (all P < 0.05) confirmed cumulus morphology and G6PDH activity as useful predictors for viability. Moreover, Ex oocytes had a thicker zona (18.2 ± 2.2 μm vs 17.3 ± 2.1 μm) and a higher birefringence (64.6 ± 5.2 vs 62.1 ± 4.2) compared with Cp oocytes (both P < 0.05). Concurrently, oocytes classified as BCB+ had a thicker zona (18.8 ± 2.4 μm vs 16.1 ± 2.0 μm) and higher values for birefringence (63.1 ± 4.5 vs 61.3 ± 3.3) compared with BCB– oocytes (both P < 0.05). Taken together, our results revealed that equine oocytes with higher developmental capacity have a thicker zona and greater birefringence scores compared with oocytes of lower developmental competence. Therefore, ZPB measurement provides a new, noninvasive method to assess the developmental competence of equine oocytes.

scholarly journals Evaluation of bovine zona pellucida characteristics in polarized light as a prognostic marker for embryonic developmental potential

Reproduction ◽  
2011 ◽  
Vol 141 (6) ◽  
pp. 779-787 ◽  
Author(s):  
M Koester ◽  
A Mohammadi-Sangcheshmeh ◽  
M Montag ◽  
F Rings ◽  
T Schimming ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Polarized Light ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Blue Staining ◽  
Developmental Potential ◽  
Preimplantation Embryo Development ◽  
Intensity Parameters ◽  
Bovine Oocytes

It has previously been demonstrated that zona pellucida imaging of human oocytes using polarized light microscopy is a clinically applicable method for the noninvasive assessment of oocyte quality. This study was designed to investigate whether zona pellucida characteristics of bovine oocytes and zygotes in polarized light may similarly serve as a useful marker for developmental competence in bovine reproductive biotechnologies. Zona birefringence intensity parameters of 2862 oocytes/zygotes were objectively evaluated with an automatic analysis system and correlated with oocyte/zygote quality. In detail, immature oocytes of good quality assessed with brilliant cresyl blue staining showed significantly lower zona birefringence than poor-quality counterparts (P<0.001). Afterin vitromaturation and classification according to maturational status, the birefringence intensity parameters were significantly different in those oocytes that reached metaphase II compared with arrested stages (P<0.001). Following either parthenogenetic activation or IVF with subsequentin vitroculture in a well-of-the-well system until day 9, superior development as determined by cleavage, blastocyst formation, and hatching ability was associated with lower zona birefringence intensity parameters. When early zygote-stage embryos were selected and assorted in groups based on zona birefringence (high/medium/low), the group of embryos derived from high-birefringence zygotes displayed a significantly compromised developmental potential compared with low-birefringence zygotes. These results clearly show that developmentally competent bovine oocytes/zygotes exhibit lower zona birefringence intensity parameters. Therefore, birefringence imaging of zona pellucida is a suitable technique to predict bovine preimplantation embryo development.

scholarly journals Cortical Granule Distribution and Expression Pattern of Genes Regulating Cellular Component Size, Morphogenesis, and Potential to Differentiation are Related to Oocyte Developmental Competence and Maturational Capacity In Vivo and In Vitro

Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 815
Author(s):  
Magdalena Kulus ◽  
Wiesława Kranc ◽  
Michal Jeseta ◽  
Patrycja Sujka-Kordowska ◽  
Aneta Konwerska ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Developmental Competence ◽  
Egg Cell ◽  
Cortical Granule ◽  
Distribution Analysis ◽  
Cortical Granules ◽  
Immature Oocytes ◽  
Component Size ◽  
Meiotic Competence

Polyspermia is an adverse phenomenon during mammalian fertilization when more than one sperm fuses with a single oocyte. The egg cell is prepared to prevent polyspermia by, among other ways, producing cortical granules (CGs), which are specialized intracellular structures containing enzymes that aim to harden the zona pellucida and block the fusion of subsequent sperm. This work focused on exploring the expression profile of genes that may be associated with cortical reactions, and evaluated the distribution of CGs in immature oocytes and the peripheral density of CGs in mature oocytes. Oocytes were isolated and then processed for in vitro maturation (IVM). Transcriptomic analysis of genes belonging to five ontological groups has been conducted. Six genes showed increased expression after IVM (ARHGEF2, MAP1B, CXCL12, FN1, DAB2, and SOX9), while the majority of genes decreased expression after IVM. Using CG distribution analysis in immature oocytes, movement towards the cortical zone of the oocyte during meiotic competence acquisition was observed. CGs peripheral density decreased with the rise in meiotic competence during the IVM process. The current results reveal important new insights into the in vitro maturation of oocytes. Our results may serve as a basis for further studies to investigate the cortical reaction of oocytes.

Light microscopy of ceramics

1993 ◽  
Vol 51 ◽  
pp. 908-909
Author(s):  
Walter C. McCrone
Keyword(s):  
Refractive Index ◽  
Light Microscopy ◽  
Light Microscope ◽  
Polarized Light ◽  
Refractive Indices ◽  
Complete Coverage ◽  
The Subject ◽  
Lower Refractive Index

An excellent chapter on this subject by V.D. Fréchette appeared in a book edited by L.L. Hench and R.W. Gould in 1971 (1). That chapter with the references cited there provides a very complete coverage of the subject. I will add a more complete coverage of an important polarized light microscope (PLM) technique developed more recently (2). Dispersion staining is based on refractive index and its variation with wavelength (dispersion of index). A particle of, say almandite, a garnet, has refractive indices of nF = 1.789 nm, nD = 1.780 nm and nC = 1.775 nm. A Cargille refractive index liquid having nD = 1.780 nm will have nF = 1.810 and nC = 1.768 nm. Almandite grains will disappear in that liquid when observed with a beam of 589 nm light (D-line), but it will have a lower refractive index than that liquid with 486 nm light (F-line), and a higher index than that liquid with 656 nm light (C-line).

Defective zonae pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development

Development ◽  
2001 ◽  
Vol 128 (7) ◽  
pp. 1119-1126 ◽  
Author(s):  
T.L. Rankin ◽  
M. O'Brien ◽  
E. Lee ◽  
K. Wigglesworth ◽  
J. Eppig ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Embryonic Stem ◽  
Single Copy ◽  
Targeted Mutagenesis ◽  
Developmental Competence ◽  
Normal Male ◽  
Null Mouse ◽  
Developmental Potential ◽  
Early Embryos

All vertebrate eggs are surrounded by an extracellular matrix. This matrix is known as the zona pellucida in mammals and is critically important for the survival of growing oocytes, successful fertilization and the passage of early embryos through the oviduct. The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2 and ZP3), each encoded by a single copy gene. Using targeted mutagenesis in embryonic stem cells, Zp2-null mouse lines have been established. ZP1 and ZP3 proteins continue to be synthesized and form a thin zona matrix in early follicles that is not sustained in pre-ovulatory follicles. The abnormal zona matrix does not affect initial folliculogenesis, but there is a significant decrease in the number of antral stage follicles in ovaries isolated from mice lacking a zona pellucida. Few eggs are detected in the oviduct after stimulation with gonadotropins, and no two-cell embryos are recovered after mating Zp2-null females with normal male mice. The structural defect is more severe than that observed in Zp1-null mice, which have decreased fecundity, but not quite as severe as that observed in Zp3-null mice, which never form a visible zona pellucida and are sterile. Although zona-free oocytes matured and fertilized in vitro can progress to the blastocyst stage, the developmental potential of blastocysts derived from either Zp2- or Zp3-null eggs appears compromised and, after transfer to foster mothers, live births have not been observed. Thus, in addition to its role in fertilization and protection of early embryos, these data are consistent with the zona pellucida maintaining interactions between granulosa cells and oocytes during folliculogenesis that are critical to maximize developmental competence of oocytes.

An in vitro Evaluation of Artificial Caries-like Lesions on Restored Overdenture Abutments

1988 ◽  
Vol 67 (3) ◽  
pp. 582-584 ◽  
Author(s):  
P.P. Kambhu ◽  
R.L. Ettinger ◽  
J.S. Wefel
Keyword(s):  
Light Microscopy ◽  
Polarized Light ◽  
Polarized Light Microscopy ◽  
The Other ◽  
Type Ii ◽  
Restorative Materials ◽  
Abutment Teeth ◽  
The Mean ◽  
Materials Used

An acidified dialyzed gelatin gel system was used to determine the caries resistance of a variety of restorative materials used to obturate the canal orifice of overdenture abutment teeth. The restorative materials used were Tytin, Tytin + Copalite, P30 + Scotchbond, Fuji Ionomer-Type II, and Miracle Mix. Polarized light microscopy and microradiography were used to examine the caries-like lesions adjacent to the restorations. The lesions formed in the Fuji Ionomer-Type II and Miracle Mix groups appeared arrested at the wall adjacent to the restoration, and did not penetrate apically down the wall as did those associated with the other restorative materials. The mean depths of lesions adjacent to Fuji Ionomer-Type II and Miracle Mix restorations were significantly less than those of Tytin, Tytin + Copalite, or P30 + Scotchbond.

scholarly journals Oocyte Competence Biomarkers Associated With Oocyte Maturation: A Review

2021 ◽  
Vol 9 ◽  
Author(s):  
Batara Sirait ◽  
Budi Wiweko ◽  
Ahmad Aulia Jusuf ◽  
Dein Iftitah ◽  
R. Muharam
Keyword(s):  
Oocyte Maturation ◽  
Blastocyst Stage ◽  
Current Approach ◽  
Developmental Competence ◽  
Matrix Remodeling ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Oocyte Developmental Competence ◽  
Cumulus Oocyte Complex

Oocyte developmental competence is one of the determining factors that influence the outcomes of an IVF cycle regarding the ability of a female gamete to reach maturation, be fertilized, and uphold an embryonic development up until the blastocyst stage. The current approach of assessing the competency of an oocyte is confined to an ambiguous and subjective oocyte morphological evaluation. Over the years, a myriad of biomarkers in the cumulus-oocyte-complex has been identified that could potentially function as molecular predictors for IVF program prognosis. This review aims to describe the predictive significance of several cumulus-oocyte complex (COC) biomarkers in evaluating oocyte developmental competence. A total of eight acclaimed cumulus biomarkers are examined in the study. RT-PCR and microarray analysis were extensively used to assess the significance of these biomarkers in foreseeing oocyte developmental competence. Notably, these biomarkers regulate vital processes associated with oocyte maturation and were found to be differentially expressed in COC encapsulating oocytes of different maturity. The biomarkers were reviewed according to the respective oocyte maturation events namely: nuclear maturation, apoptosis, and extracellular matrix remodeling, and steroid metabolism. Although substantial in vitro evidence was presented to justify the potential use of cumulus biomarkers in predicting oocyte competency and IVF outcomes, the feasibility of assessing these biomarkers as an add-on prognostic procedure in IVF is still restricted due to study challenges.

scholarly journals Impact of L-carnitine supplementation on the in vitro developmental competence and cryotolerance of buffalo embryos

2021 ◽  
pp. 3164-3169
Author(s):  
Mohamed M. M. El-Sokary ◽  
Al-Shimaa Al-H. H. El-Naby ◽  
Amal R. Abd El Hameed ◽  
Karima Gh. M. Mahmoud ◽  
T. H. Scholkamy
Keyword(s):  
Embryo Culture ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Effective Concentration ◽  
Developmental Competence ◽  
Carnitine Supplementation ◽  
Oocyte Developmental Competence ◽  
High Lipid ◽  
Significant Difference

Background and Aim: Despite many trials, buffalo embryos have poor cryosurvivability because of their high lipid content. L-carnitine was found to be a lipid-reducing agent when added to oocyte and embryo culture media. The study aimed to determine the most effective concentration of L-carnitine to improve the oocyte developmental competence and cryotolerance of buffalo embryos. Materials and Methods: In vitro maturation and embryo culture media were supplemented with four concentrations of L-carnitine: 0 (control), 0.25, 0.5, and 1 mM. Good-quality embryos on 7 days were vitrified using mixtures of dimethyl sulfoxide and ethylene glycol at two concentrations (3.5 and 7 M). Results: The result showed that the cleavage and morula rates were significantly (p<0.05) higher in the 0.5 mM group. Blastocyst rates were significantly (p<0.05) higher at both 0.5 and 1 mM. The rates of viable embryos directly after thawing were significantly (p<0.05) increased in the 0.5 mM group. No significant difference was found in embryos cultured for 24 h after warming among all the groups. Conclusion: The addition of L-carnitine at a concentration of 0.5 mM to the culture media improves the oocyte developmental competence and cryotolerance of buffalo embryos directly after warming but not after 24 h of culture. Nevertheless, further studies must identify how L-carnitine exerts its beneficial micromechanisms.

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