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.
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.