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: In mammalian reproductive tract, the oviduct secretes
huge number of growth factors and cytokines that create an optimal
micro-environment for the initial stages of preimplantation embryos.
Secretion of these growth factors is stage-specific. Among them,
VEGF is a potent mitogen for vascular endothelium and stimulates
vascular permeability. Apart from angiogenesis, VEGF in the oviduct
may be involved in regulating the oocyte maturation and subsequent
developmental process during embryo production in vitro. In
experiment 1, to evaluate the effect of VEGF during IVM of porcine
COC and subsequent developmental ability after PA and SCNT. The
results from these experiments indicated that maturation rates among
the different VEGF concentrations were not significant different. In
experiment 2, total intracellular GSH concentrations of oocytes
matured with VEGF (5-50 ng/ml) were increased significantly
compared to a control and VEGF group (500 ng/ml). In experiment 3,
the blastocyst formation rates and total cell number per blastocyst
after parthenogenesis of oocytes matured with VEGF (5-50 ng/ml)
were increased significantly compared to a control and VEGF group
(500 ng/ml). Similarly, in experiment 4, the blastocyst formation rate
and total cell number per blastocyst after SCNT and IVF of oocytes
matured with VEGF (5 ng/ml) were significantly higher than that of
oocytes matured without VEGF group. In experiment 5, at 10 hour
after the onset of IVF, pronuclear formation rate was evaluated.
Monospermy was significantly higher in VEGF-matured oocytes than
in the control, and polyspermy and sperm penetration per oocyte
were significantly higher in the control group than in the VEGFmatured
oocytes. Supplementation with VEGF during IVM
significantly improved male pronuclear formation as compared with
the control. In experiment 6, type III cortical granule distribution in
oocytes was more common in VEGF-matured oocytes than in the
control. In conclusion, the present study suggested that
supplementation of VEGF during IVM may enhance the
developmental potential of porcine in vitro embryos through increase
of the intracellular GSH level, higher MPN formation and increased
fertilization rate as a consequence of an improved cytoplasmic
maturation.
Abstract: In the present study, the response of Nili Ravi buffalo
oocytes to recombinant human follicle stimulating hormone (rhFSH)
(Organon) on meiotic maturation in vitro was examined. Oocytes
were matured in vitro in medium containing either 0 or 0.05 IU/ ml
rhFSH and the stage of nuclear maturation recorded after 24 hours.
The percentage of oocytes in the control group undergoing germinal
vesicle breakdown (GVBD) observed after 24 hours of culture was
29 % whereas as in rhFSH group the percentage was 10 % were at
this stage (P< 0.001).Thus in the presence of rhFSH, a significantly
greater number of oocytes had progressed to the more advanced
stages of nuclear maturation. Indeed, the maturation of GV
(Germinal Vesicle) stage oocytes to the metaphase II (M II) stage
after 24 hours was significantly (P< 0.0001) increased by the
addition of rhFSH (82 % VS 47 %). The percentage of degenerated
oocytes after 24 hours of culture was 24 % in control group, whereas
in rhFSH group the percentage was 8 % after 24 hours. Degeneration
of the oocytes after 24 hours was not significantly (P = 0. 9361)
decreased.
Abstract: Our results showed that treatment with both
cyclooxygenase (COX1 or COX2) inhibitors impair reproduction
parameters of the medaka. Resveratrol (COX1 inhibitor) caused an
decrease in the number of spawning females at the first week of
feeding fish with experimental diets. In the group treated with NS-
398 (COX2 inhibitor) we found the lowest sperm velocity parameters
and decreased linearity of movement. The ovaries of the medaka fed
feed supplemented with Resveratrol or NS-398 were confirmed to
have a lower share of matured oocytes however during the
experiment (four weeks) the number of eggs spawned by females was
similar. Both inhibitors in fish diet (20 mg/kg body weight/day)
caused a decrease in the embryo survival. Our results revealed that
for the medaka female reproduction, activity of both COX enzymes
might be necessary whereas males reproduction competence, as
expressed by sperm motility parameters, might be related to COX2
activity.