T. Chuen-Im

Publications

2 Morphological Interaction of Porcine Oocyte and Cumulus Cells Study on in vitro Oocyte Maturation Using Electron Microscopy

Authors: M. Areekijseree, M. Pumipaiboon, T. Chuen-Im, W. Pongsawat, C. Thepsithar, S. Sengsai

Abstract:

Morphological interaction of porcine cumulus-oocyte complexes (pCOCs) was investigated on in vitro condition using electron microscope (SEM and TEM). The totals of 1,923 oocytes were round in shape, surrounded by Zona pellucida with layer of cumulus cells ranging between 59.29-202.14 μm in size. They were classified into intact-, multi-, partial cumulus cell layer oocyte, and completely denuded oocyte, at the percentage composition of 22.80% 32.70%, 18.60%, and 25.90 % respectively. The pCOCs classified as intact- and multi cumulus cell layer oocytes were further culturing at 37°C with 5% CO2, 95% air atmosphere and high humidity for 44 h in M199 with Earle’s salts supplemented with 10% HTFCS, 2.2 mg/mL NaHCO3, 1 M Hepes, 0.25 mM pyruvate, 15 μg/mL porcine follicle-stimulating hormone, 1 μg/mL LH, 1μg/mL estradiol with ethanol, and 50 μg/mL gentamycin sulfate. On electron microscope study, cumulus cells were found to stick their processes to secrete substance from the sac-shape end into Zona pellucida of the oocyte and also communicated with the neighboring cells through their microvilli on the beginning of incubation period. It is believed that the cumulus cells communicate with the oocyte by inserting the microvilli through this gap and embedded in the oocyte cytoplasm before secreting substance, through the sac-shape end of the microvilli, to inhibit primary oocyte development at the prophase I. Morphological changes of the complexes were observed after culturing for 24-44 h. One hundred percentages of the cumulus layers were expanded and cumulus cells were peeling off from the oocyte surface. In addition, the round-shape cumulus cells transformed themselves into either an elongate shape or a columnar shape, and no communication between cumulus neighboring cells. After 44 h of incubation time, diameter of oocytes surrounded by cumulus cells was larger than 0 h incubation. The effect of hormones in culture medium is exerted by their receptors present in porcine oocyte. It is likely that all morphological changes of the complexes after hormone treatment were to allow maturation of the oocyte. This study demonstrated that the association of hormones in M199 could promote porcine follicle activation in 44 h in vitro condition. This culture system should be useful for studying the regulation of early follicular growth and development, especially because these follicles represent a large source of oocytes that could be used in vitro for cell technology.

Keywords: In vitro, cumulus cells, porcine oocyte, electron microscopy (SEM and TEM)

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1 Leatherback Turtle (Dermochelys coriacea) after Incubation Eggshell in Andaman Sea, Thailand Study: Microanalysis on Ultrastructure and Elemental Composition

Authors: M. Areekijseree, M. Pumipaiboon, S. Nuamsukon, K. Kittiwattanawong, C. Thongchai, S. Sikiwat, T. Chuen-Im

Abstract:

There are few studies on eggshell of leatherback turtle which is endangered species in Thailand. This study was focusing on the ultrastructure and elemental composition of leatherback turtle eggshells collected from Andaman Sea Shore, Thailand during the nesting season using scanning electron microscope (SEM). Three eggshell layers of leatherback turtle; the outer cuticle layer or calcareous layer, the middle layer or middle multistrata layer and the inner fibrous layer were recognized. The outer calcareous layer was thick and porosity which consisted of loose nodular units of various crystal shapes and sizes. The loose attachment between these units resulted in numerous spaces and openings. The middle layer was compact thick with several multistrata and contained numerous openings connecting to both outer cuticle layer and inner fibrous layer. The inner fibrous layer was compact and thin, and composed of numerous reticular fibers. Energy dispersive X-ray microanalysis detector revealed energy spectrum of X-rays character emitted from all elements on each layer. The percentages of all elements were found in the following order: carbon (C) > oxygen (O) > calcium (Ca) > sulfur (S) > potassium (K) > aluminum (Al) > iodine (I) > silicon (Si) > chlorine (Cl) > sodium (Na) > fluorine (F) > phosphorus (P) > magnesium (Mg). Each layer consisted of high percentage of CaCO3 (approximately 98%) implying that it was essential for turtle embryonic development. A significant difference was found in the percentages of Ca and Mo in the 3layers. Moreover, transition metal, metal and toxic non-metal contaminations were found in leatherback turtle eggshell samples. These were palladium (Pd), molybdenum (Mo), copper (Cu), aluminum (Al), lead (Pb), and bromine (Br). The contamination elements were seen in the outer layers except for Mo. All elements were readily observed and mapped using Smiling program. X-ray images which mapped the location of all elements were showed. Calcium containing in the eggshell appeared in high contents and was widely distributing in clusters of the outer cuticle layer to form CaCO3 structure. Moreover, the accumulation of Na and Cl was observed to form NaCl which was widely distributing in 3 eggshell layers. The results from this study would be valuable on assessing the emergent success in this endangered species.

Keywords: Leatherback turtle (Dermochelys coriacea), SEM (SEI/EDX), turtle eggshell

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Abstracts

1 Morphological Interaction of Porcine Oocyte and Cumulus Cells Study on in vitro Oocyte Maturation Using Electron Microscopy

Authors: M. Areekijseree, M. Pumipaiboon, T. Chuen-Im, W. Pongsawat, C. Thepsithar, S. Sengsai

Abstract:

Morphological interaction of porcine cumulus-oocyte complexes (pCOCs) was investigated on in vitro condition using electron microscope (SEM and TEM). The totals of 1,923 oocytes were round in shape, surrounded by zona pellucida with layer of cumulus cells ranging between 59.29-202.14 µm in size. They were classified into intact-, multi-, partial cumulus cell layer oocyte, and completely denuded oocyte, at the percentage composition of 22.80% 32.70%, 18.60%, and 25.90 % respectively. The pCOCs classified as intact- and multi cumulus cell layer oocytes were further culturing at 37°C with 5% CO2, 95% air atmosphere and high humidity for 44 h in M199 with Earle’s salts supplemented with 10% HTFCS, 2.2 mg/mL NaHCO3, 1 M Hepes, 0.25 mM pyruvate, 15 µg/mL porcine follicle-stimulating hormone, 1 µg/mL LH, 1µg/mL estradiol with ethanol, and 50 µg/mL gentamycin sulfate. On electron microscope study, cumulus cells were found to stick their processes to secrete substance from the sac-shape end into zona pellucida of the oocyte and also communicated with the neighboring cells through their microvilli on the beginning of incubation period. It is believed that the cumulus cells communicate with the oocyte by inserting the microvilli through this gap and embedded in the oocyte cytoplasm before secreting substance, through the sac-shape end of the microvilli, to inhibit primary oocyte development at the prophase I. Morphological changes of the complexes were observed after culturing for 24-44 h. One hundred percentages of the cumulus layers were expanded and cumulus cells were peeling off from the oocyte surface. In addition, the round-shape cumulus cells transformed themselves into either an elongate shape or a columnar shape, and no communication between cumulus neighboring cells. After 44 h of incubation time, diameter of oocytes surrounded by cumulus cells was larger than 0 h incubation. The effect of hormones in culture medium is exerted by their receptors present in porcine oocyte. It is likely that all morphological changes of the complexes after hormone treatment were to allow maturation of the oocyte. This study demonstrated that the association of hormones in M199 could promote porcine follicle activation in 44 h in vitro condition. This culture system should be useful for studying the regulation of early follicular growth and development, especially because these follicles represent a large source of oocytes that could be used in vitro for cell technology.

Keywords: Electron Microscopy, In vitro, cumulus cells, porcine oocyte

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