262 MEIOTIC SPINDLE CONFORMATION ASSESSMENT BY POLARIZED LIGHT MICROSCOPY IN SHEEP AND GOAT OOCYTES

Polarized light microscopy (PLM) allows detection of microtubule-polymerized protein in in vitro-matured sheep and goat oocytes (Caamaño et al. 2011 Reprod. Fertil. Dev. 23, 226–227). Spindle birefringence, measured as the mean retardance value, has been proposed as a marker of meiotic spindle conformation in humans and mice. Because the conformation of the meiotic spindle cannot be readily assessed by PLM, in this study we aimed to measure the mean retardance value of normal and abnormal meiotic spindles from in vitro-matured prepubertal sheep and goat oocytes. Oocytes were matured in vitro for 27 h and were then individually assessed by PLM (Oosight System, Research Instruments Ltd., Falmouth, Cornwall, UK), and the mean retardance value was analysed in meiotic spindles using specific software (Oosight). Meiotic spindle conformation was determined in individual oocytes by immunostaining and chromatin detection, as described by Morató et al. (2008 Mol. Reprod. Dev. 75, 191–201). A barrel-shaped spindle was considered a normal spindle configuration. Only oocytes with meiotic spindles identified by both PLM and immunostaining were used in this study. The experiment was replicated four times. Data were analysed by the general linear models procedure of SAS (SAS Institute Inc., Cary, NC, USA). A normal barrel-shaped spindle was observed in 80.8% of the sheep oocytes (n = 47) and in 83.9% of the goat oocytes (n = 62). In sheep, the mean retardance value in oocytes with a normal meiotic spindle conformation did not differ from that in oocytes with abnormal spindles (4.42 ± 0.26 nm v. 3.92 ± 0.54 nm). Similar results were obtained with goat oocytes with normal (2.94 ± 0.20 nm) v. abnormal spindle conformation (2.77 ± 0.08 nm). These results indicate that the mean retardance value does not distinguish between oocytes with normal and abnormal meiotic spindle conformation, as assessed by subsequent immunostaining. Grant support from INIA-RZ2007-00013-00-00. M. Muñoz was sponsored by RYC08-03454.

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

Journal of Dental Research ◽

10.1177/00220345880670031101 ◽

1988 ◽

Vol 67 (3) ◽

pp. 582-584 ◽

Cited By ~ 8

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab351 ◽

2010 ◽

Vol 22 (1) ◽

pp. 332 ◽

Cited By ~ 2

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.

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257 DETECTION OF MICROTUBULES BY POLARIZED LIGHT MICROSCOPY IN SHEEP AND GOAT OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab257 ◽

2011 ◽

Vol 23 (1) ◽

pp. 226 ◽

Cited By ~ 1

Author(s):

J. N. Caamaño ◽

M. Catalá ◽

R. Romaguera ◽

C. Diez ◽

M. Muñoz ◽

...

Keyword(s):

Light Microscopy ◽

In Vitro Maturation ◽

Polarized Light ◽

Petri Dish ◽

Polarized Light Microscopy ◽

Farm Animals ◽

Meiotic Spindle ◽

Efficient System ◽

Positive Correlation

The meiotic spindle in the oocyte is composed of microtubules and plays a key role in normal chromosome alignment and segregation during meiosis. In oocytes from farm animals, the meiotic spindle cannot be detected by conventional light microscopy due to the characteristic of their cytoplasm. Conventional methods to image the meiotic spindle rely on fixation of the oocytes. Polarized light microscopy (PLM) allows noninvasive evaluation of the meiotic spindle of metaphase oocytes. The aim of this study was to assess the efficiency of polarized light microscopy to detect microtubule-polymerized protein within in vitro matured prepubertal sheep and goat oocytes. We carried out 2 studies. In the first one, cumulus–oocyte complexes from slaughterhouse sheep ovaries were matured in vitro for 27 h. After in vitro maturation, oocytes (n = 77) were denuded of cumulus cells and placed individually in 10-μL drops of TCM-199-HEPES-BSA in a glass Petri dish. Polarized light microscopy was used to detect the presence of polymerized protein, which could be associated with the forming of a meiotic spindle. To confirm the presence of the polymerized protein and the meiotic spindle, each individual oocyte was subjected to immunostaining and chromatin detection as described by (Morató et al. 2008 Mol. Reprod. Dev. 75, 191–201). The experiment was replicated 4 times. The correlation analysis was performed using the Proc Corr procedure of SAS. There was a positive correlation (r = 0.87; P < 0.001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by immunostaining. A positive PLM signal was detected in 87.0% of the oocytes, and 69.0% of the oocytes reached the metaphase II (MII) stage after in vitro maturation. A barrel-shaped spindle was observed in 77.3% of the MII oocytes. In the second study, we performed a similar experiment but used goat oocytes. A total of 78 oocytes were used, and PLM and immunostaining were performed in each individual oocyte as it was described with sheep oocytes. There was also a positive correlation (r = 1; P < 0.001) between the signal obtained by PLM and the presence of microtubule-polymerized protein. A positive PLM signal was detected in 98.7% of the oocytes, and 80.7% of the oocytes reached the MII stage after in vitro maturation. A barrel-shaped spindle was observed in 92.0% of the MII oocytes. These results indicate that PLM is an efficient system to detect polymerized protein in in vitro matured sheep and goat oocytes. This work was supported by the following grant: INIA: RZ2007-00013-00-00. M. Muñoz and D. Martín are sponsored by RYC08-03454 and PTA2007-0268-I, respectively.

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242 DETECTION OF MICROTUBULES BY POLARIZED LIGHT MICROSCOPY IN BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab242 ◽

2009 ◽

Vol 21 (1) ◽

pp. 219 ◽

Cited By ~ 1

Author(s):

J. N. Caamaño ◽

C. Díez ◽

M. Muñoz ◽

D. Martin ◽

R. Morató ◽

...

Keyword(s):

Light Microscopy ◽

Calf Serum ◽

Polarized Light ◽

Reproductive Technologies ◽

Polarized Light Microscopy ◽

Farm Animals ◽

Meiotic Spindle ◽

Efficient System ◽

Bovine Oocytes

Polarized light microscopy (PLM) is being used as a tool in assisted reproductive technologies in humans and, more recently, in farm animals. Polarized light microscopy may allow for a quick localization or removal of nuclear structures, or both in the oocyte without fluorochrome staining and ultraviolet exposure, and may reduce nuclear damage when performing intra-cytoplasmic sperm injection. The aim of this study was to assess the efficiency of PLM to detect microtubule-polymerized protein within in vitro-matured bovine oocytes. Cumulus–oocyte complexes from slaughterhouse ovaries were matured in vitro for 23 h in bicarbonate-buffered TCM-199, FSHp (1 μg mL–1), LH (5 μg mL–1), 17β-estradiol (1 μg mL–1), and 10% fetal calf serum at 38.7°C in 5% CO2 in air with high humidity. After in vitro maturation, oocytes (n = 98) were denuded with 0.5% hyaluronidase and were placed individually in 10-μL drops of TCM-199-HEPES-BSA in a glass Petri dish. Polarized light microscopy was used to detect the presence of polymerized protein that could be forming a meiotic spindle. To confirm the presence of the polymerized protein and the meiotic spindle, each individual oocyte was subjected to immunostaining and chromatin detection as described by Morató et al. 2008 Mol. Reprod. Dev. 75, 191–201. The experiment was replicated 6 times. There was an absolute positive correlation (r = 1; P < 0.0001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by immunostaining. A barrel-shaped spindle was observed in 40% of the individual samples, whereas disorganized microtubule structures were obtained in 55% of the samples. The biological implication of these findings needs to be explored. However, PLM seems to be an efficient system to detect polymerized protein in in vitro-matured bovine oocytes.

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Inhibition of Demineralization at Restoration Margins of Z100 and Tetric EvoCeram Bulk Fill in Dentin and Enamel

Bioengineering ◽

10.3390/bioengineering6020036 ◽

2019 ◽

Vol 6 (2) ◽

pp. 36

Author(s):

Cristina Leon-Pineda ◽

Kevin Donly

Keyword(s):

Light Microscopy ◽

Polarized Light ◽

In Vitro Study ◽

The Other ◽

Solution Ph ◽

Class V ◽

The Mean ◽

Student’S T ◽

Student’S T Test

Recurrent caries is still considered the main reason restorations need to be replaced. There are different materials available now that promise to reduce the possibility of recurrent caries by releasing fluoride and inhibiting restoration marginal caries. The purpose of this in vitro study was to evaluate the demineralization inhibition potential of a non-fluoride-releasing resin (Z100TM 3M, St. Paul, MN, USA) and a glass containing resin-based composite (Tetric EvoCeram Bulk Fill, Ivoclar/Vivadent AG, Schaan, Liechtenstein), which contains fluoride. Class V preparations were placed on 22 premolars; the gingival margin was below the cementoenamel junction and the occlusal margin was placed above the cemento-enamel junction. Ten teeth were randomly selected to be restored with Z100 while the other 10 were restored with Tetric EvoCeram Bulk Fill. Both groups were restored following manufacturer’s instructions. All teeth had an acid resistant varnish placed within one millimeter of the preparation margins. Both groups were placed in artificial caries challenge solution (pH 4.4). At the end of the 4 days; 100 µm buccolingual sections were obtained for each tooth; these were photographed under polarized light microscopy and the demineralized areas adjacent to the restorations were measured and quantified. The mean (±S.D.) area (µm2) of demineralization from the occlusal margin (enamel) and dentin margin were: Z100 2781.889 ± 1045.213; 3960.455 ± 705.964 and for Tetric EvoCeram Bulk Fill 1541.545 ± 1167.027; 3027.600 ± 512.078. Student’s t-test indicated that there was significantly less enamel and dentin demineralization adjacent to Tetric EvoCeram Bulk Fill compared to Z100; there was significantly less demineralization in enamel compared to dentin in both Tetric EvoCeral Bulk Fill and Z100. Tetric EvoCeram Bulk Fill performed better inhibiting demineralization at restoration margins when compared to Z100 and provided better demineralization inhibition in enamel than cementum/dentin.

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Polarized Light Microscopy for Analyzing Tissue Mechanics During In Vitro Acupuncture

ASME 2008 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2008-192595 ◽

2008 ◽

Author(s):

Lowell Taylor Edgar ◽

Margaret Julias ◽

David I. Shreiber ◽

Helen M. Buettner

Keyword(s):

Connective Tissue ◽

Light Microscopy ◽

Type I Collagen ◽

Polarized Light ◽

Tissue Mechanics ◽

Polarized Light Microscopy ◽

Tissue Response ◽

Type I ◽

Collagen Gels

Acupuncture is a traditional therapy originating in China almost 2000 years ago. Acupuncture has slowly been growing in popularity in the West, and clinical evidence has shown the potential for acupuncture as a low-cost ‘alternative’ therapy for an assortment of ailments [1]. The practice of acupuncture involves inserting fine needles into the skin followed by needle manipulation, usually by rotation. Recent studies by Langevin et al demonstrate that this rotation causes the subcutaneous connective tissue to couple to and wind around the needle [2–4], which suggests that mechanotransduction in the connective tissue might play a role in the therapeutic mechanisms that underlay acupuncture [2, 3]. To begin to decompose and quantify this complex mechanism at the tissue level in a controlled setting, we have simulated acupuncture in type I collagen gels in vitro, and have developed algorithms to quantify the tissue response following imaging with polarized light microscopy (PLM).

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Active Phagocytosis and Diachronic Processing of Calcium Oxalate Monohydrate Crystals in an in vitro Macrophage Model

Kidney & Blood Pressure Research ◽

10.1159/000501965 ◽

2019 ◽

Vol 44 (5) ◽

pp. 1014-1025

Author(s):

Atsushi Okada ◽

Hiromasa Aoki ◽

Daichi Onozato ◽

Taiki Kato ◽

Tadahiro Hashita ◽

...

Keyword(s):

Light Microscopy ◽

Calcium Oxalate ◽

Polarized Light ◽

Calcium Oxalate Monohydrate ◽

In Vitro Models ◽

Polarized Light Microscopy ◽

Transmission Electron ◽

Imaging Cytometry ◽

Acidic Organelles

Background: We previously discovered that renal macrophages (Mφs) phagocytose renal calcium oxalate monohydrate (COM) crystals. This study investigated the processing of engulfed crystals using in vitro models. Methods: J774.1 mouse Mφs were exposed to COM crystals and observed for 24 h using polarized light microscopy with/without cytochalasin B (CB), an inhibitor of phagocytosis, to confirm active crystal phagocytosis. LysoTracker and immunohistochemical staining using transmission electron microscopy for lysosomal-associated membrane protein 1 were used to confirm engulfed COM crystal uptake into lysosomes. Diachronic tracking of specific Mφs was performed to capture the entire course of engulfed COM crystal processing using polarized light microscopy. Follow-up studies of fluorescent COM (f-COM) crystals using imaging cytometry were performed in the presence and absence of nigericin to dissipate the pH gradient in acidic organelles. Results: Phagocytosis rates increased with COM density and were significantly lower in cells treated with CB (p < 0.01). We observed that engulfed crystals colocalized within lysosomes of the Mφs; moreover, diachronic observation indicated that the engulfed COM crystals were subdivided during Mφ division and eliminated by the 7th day of culture. Additionally, imaging cytometry showed that the fluorescence level of f-COM crystals in the nigericin (–) group after 48 h was significantly lower than that in the nigericin (+) group. Conclusions: This study confirmed active phagocytosis and lysosomal processing of engulfed COM crystals by Mφs. This discovery is expected to contribute to the development of future drugs that enhance the COM crystal phagocytic ability of Mφs.

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Overheating is detrimental to meiotic spindles within in vitro matured human oocytes

Zygote ◽

10.1017/s0967199404002631 ◽

2004 ◽

Vol 12 (1) ◽

pp. 65-70 ◽

Cited By ~ 30

Author(s):

Xiao-Fang Sun ◽

Wei-Hua Wang ◽

David L. Keefe

Keyword(s):

High Temperature ◽

Light Microscope ◽

Polarized Light ◽

Germinal Vesicle ◽

Meiotic Spindle ◽

Human Oocytes ◽

Living State ◽

Meiotic Spindles ◽

Metaphase I

The present study was designed to examine the effects of overheating on meiotic spindle morphology within in vitro matured human oocytes using a polarized light microscope (Polscope). Immature human oocytes at either germinal vesicle or metaphase I stage were cultured in vitro for 24–36 h until they reached metaphase II (M-II) stage. After maturation, oocytes at M-II stage were imaged in the living state with the Polscope at 37, 38, 39 and 40 °C for up to 20 min. After heating, oocytes were returned to 37 °C and then imaged for another 20 min at 37 °C. The microtubules in the spindles were quantified by their maximum retardance, which represents the amount of microtubules. Spindles were intact at 37 °C during 40 min of examination and their maximum retardance (1.72–1.79) did not change significantly during imaging. More microtubules were formed in the spindles heated to 38 °C and the maximum retardance was increased from 1.77 before heating to 1.95 at 20 min after heating. By contrast, spindles started to disassemble when the temperature was increased to 39 °C for 10 min (maximum retardance was reduced from 1.76 to 1.65) or 40 °C for 1 min (maximum retardance was reduced from 1.75 to 1.5). At the end of heating (20 min), fewer microtubules were present in the spindles and the maximum retardance was reduced to 0.8 and 0.78 in the oocytes heated to 39 °C and 40 °C, respectively. Heating to 40 °C also induced spindles to relocate in the cytoplasm in some oocytes. After the temperature was returned to 37 °C, microtubules were repolymerized to form spindles, but the spindles were not reconstituted completely compared with the spindles imaged before heating. These results indicate that spindles in human eggs are sensitive to high temperature. Moreover, maintenance of an in vitro manipulation temperature of 37 °C is crucial for normal spindle morphology.

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