|Changes in Pulsatile Pattern of
Growth Hormone Secretion during Estrous Cycle in Shiba Goats.
|Tomohiro YONEZAWA, Keitaro YAMANOUCHI and Masugi NISHIHARA
|Department of Veterinary Physiology, Veterinary Medical
Science, The University of Tokyo, Tokyo, Japan.
||ABSTRACT. Growth hormone (GH) is secreted in a pulsatile manner,
the pattern of which regulates growth and metabolism. Sex steroids are
also involved in the metabolic regulation during the estrous cycle. In
the present study, the changes in GH pulsatility and metabolic transition
during the inherent estrous cycle were studied in Shiba goats. From ovariectomized
(OVX) and intact females, plasma samples were taken every 15 min for 24
h, and plasma GH was measured by radioimmunoassay. In the early luteal
phase, GH was secreted in a distinct pulsatile manner, the pattern of
which was similar to that in OVX goats. In the late luteal phase, GH pulse
frequency, amplitude and the area under the curve (AUC) were decreased.
In the follicular phase, these parameters were increased. By means of
the approximate entropy analysis, the regularity of GH secretion in the
late luteal phase was found to be lower than those in the other phases.
Both of plasma insulin-like growth factor-I (IGF-I) and free fatty acid
levels on the day of estrus were higher than those in the luteal phase.
Moreover, subcutaneous injection of estradiol to OVX goats increased GH
pulse amplitude and AUC, whereas the implantation of progesterone for
five days decreased those parameters. These results suggest that GH pulsatility
in goats alters during the estrous cycle with sex steroid levels, and
thereby affects IGF-I secretion and lipolysis to attain appropriate metabolic
milieu for each reproductive stage.
|Expression of Anti-Apoptotic Factor
(cFLIP) in Granulosa Cells of Porcine Ovarian Follicles.
|Fuko MATSUDA-MINEHATA1, Yasufumi GOTO2,
Naoko INOUE3 and Noboru MANABE4
|1Laboratory of Animal Breeding, Graduate
School of Agricultural and Life Sciences, The University of Tokyo, Tokyo,
2Unit of Anatomy and Cell Biology, Department of Animal Sciences,
Kyoto University, Kyoto, 3Laboratory of Animal Morphology and
Function, Graduate School of Bioagricultural Sciences, Nagoya University,
Nagoya, 4Research Unit for Animal Life Sciences, Animal Resource
Science Center, The University of Tokyo, Ibaraki-Iwama, Japan
||ABSTRACT. In mammalian ovaries, less than 1% of follicles reach
ovulation, while the remainder undergoes atresia. Granulosa cell apoptosis
plays a crucial role in the atretic process, the mechanisms of which are
still largely unclear. Cellular Flice (procaspase-8) inhibitory protein
(cFLIP) is an intracellular anti-apoptotic factor. Its structure is similar
to that of procaspase-8, but it has no proteolytic activity. cFLIP interacts
with the adapter protein, FADD, and precursor protease, procaspase-8,
and inhibits cell-death receptor-dependent apoptosis induction. We identified
porcine cFLIP genes, the cFLIP-long and -short forms, which show high
homology with those of humans and mice, and examined the contribution
of cFLIPs on granulosa cell apoptosis during follicular atresia. cFLIP
mRNA was detected in granulosa cells by real time RT-PCR, and cFLIP protein
expression was confirmed by Western blotting. mRNA and protein expression
levels were higher in the granulosa cells of healthy follicles than those
of atretic follicles. Immunohistochemistry for cFLIP also revealed that
cFLIP was localized in granulosa cells. These data indicate that cFLIP
may contribute to the survival of healthy follicles. Then, to evaluate
the anti-apoptotic activity of cFLIP, we performed an in vitro
experiment. Porcine cFLIP (pcFLIP) expression vector was constructed and
transfected into HeLa cells or KGN cells, and then apoptosis was induced
by adding anti-Fas antibody and cycloheximide to the transfected cells.
The apoptosis induction rate in pcFLIP-transfected cells was lower than
that in empty vector-transfected cells, indicating that pcFLIP acts as
a functional anti-apoptotic factor, the as human or murine cFLIP. In conclusion,
cFLIP acts as a survival factor and plays important roles in follicle
selection in porcine ovaries.
|Influence of the Cell Shape and
Cell-to-Cell Contact in Oxytocin Secretion by Cultured Bovine Luteal Cells.
|Masami SHIBAYA1, Katarzyna M. DEPTULA2,
Anna KORZEKWA2, Dariusz J. SKARZYNSKI2 and Kiyoshi
|1Laboratory of Reproductive Endocrinology,
Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan, 2Division
of Reproductive Endocrinology and Pathophysiology, Institute of Animal
Reproduction & Food Research, PAS, Tuwima-St 10, Olsztyn 10-747, Poland
||ABSTRACT. A number of substances have been implicated in the
regulation of oxytocin (OT) secretion from bovine corpus luteum (CL)
in vivo. However, isolated bovine luteal cells cultured in a monolayer
lose the ability to secrete OT in response to stimulatory substances.
The present study investigated how cell-to-cell contact and the cytoskeleton
affect OT secretion by isolated bovine luteal cells. In Exp. 1, bovine
mid-luteal cells (Days 8-12 of the estrous cycle) were stimulated with
prostaglandin F2α (PGF2α; 1µM),
noradrenaline (NA; 10µM) or growth hormone (GH; 5nM) in two culture
systems: In one system cell monolayers were incubated in 24-well culture
plates and in the other system aggregates of cells were incubated in glass
tubes in a shaking water bath. The cells cultured in a monolayer underwent
considerable spreading and showed a variety of shapes, whereas the cells
cultured in glass tubes remained fully rounded during the experimental
period and soon formed aggregates of cells. Although PGF2α,
NA and GH did not stimulate OT secretion by the monolayer cells, all tested
substances stimulated OT secretion by the aggregated cells (p<0.01).
In Exp. 2, the monolayer cells were pre-exposed for 1 h to an anti-microfilament
agent (cytochalasin B; 1µM) or two anti-microtubule agents (colchicine
or vinblastine; 1µM) before stimulation with PGF2α,
NA or GH. Although PGF2α, NA, and GH did not stimulate
OT secretion by the monolayer cells in the presence of colchicine or vinblastine,
they all stimulated OT secretion in the presence of cytochalasin B (p<0.001).
The overall results show that OT secretion by bovine luteal cells depends
on microfilament function and the cell shape. Moreover, the aggregate
culture system that allows three-dimensional cell-to-cell contact seems
to be a good model for studying OT secretion by isolated bovine luteal