Abstracts of the presentations selected for 2004 JSAR Outstanding Presentation Award

Abstracts of the presentations selected for 2004
JSAR Outstanding Presentation Award

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 OKUDA¹

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 F_2α (PGF_2α; 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 PGF_2α, 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 PGF_2α, NA or GH. Although PGF_2α, 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 cells.

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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 OKUDA¹
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 F_2α (PGF_2α; 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 PGF_2α, 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 PGF_2α, NA or GH. Although PGF_2α, 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 cells.