Min Kyun Park - Academia.edu (original) (raw)

Papers by Min Kyun Park

The Journal of cell biology, 1990

The major nuclear pore protein p62 is modified by O-linked N-acetylglucosamine and functions in n... more The major nuclear pore protein p62 is modified by O-linked N-acetylglucosamine and functions in nuclear transport. We have cloned, sequenced, and expressed the full-length rat p62 cDNA. The rat p62 mRNA is 2,941 nucleotides long and encodes a protein of 525 amino acids containing 30% serine and threonine residues. The amino acid sequence near the amino-terminus contains unique tetrapeptide repeats while the carboxy-terminus consists of a series of predicted alpha-helical regions with hydrophobic heptad repeats. Heterologous expression of rat p62 in African Green Monkey Kidney COS-1 cells and CV-1 cells was detected using a species-specific antipeptide serum. When transiently expressed in COS-1 cells, rat p62 binds wheat germ agglutinin and concentrates at the spindle poles during mitosis. In CV-1 cells cotransfected with rat p62 cDNA and SV40 viral DNA, rat p62 associates with the nuclear membrane without interfering with the nuclear transport of SV40 large T antigen. The ability to...

General and Comparative Endocrinology, 1991

Endocrinology, 2006

GnRH was first identified as the hypothalamic decapeptide that promotes gonadotropin release from... more GnRH was first identified as the hypothalamic decapeptide that promotes gonadotropin release from pituitary gonadotropes. Thereafter, direct stimulatory and inhibitory effects of GnRH on cell proliferation were demonstrated in a number of types of primary cultured cells and established cell lines. Recently, the effects of GnRH on cell attachment, cytoskeleton remodeling, and cell migration have also been reported. Thus, the effects of GnRH on various cell activities are of great interest among researchers who study the actions of GnRH. In this study, we demonstrated that GnRH induces actin cytoskeleton remodeling and affects cell migration using two human prostatic carcinoma cell lines, TSU-Pr1 and DU145. In

Journal of Experimental Zoology, 1995

7% clarify the target cells of GnRH in the ovary, in vivo expression of ovarian GnRH receptor mRN... more 7% clarify the target cells of GnRH in the ovary, in vivo expression of ovarian GnRH receptor mRNA was examined histologically by in situ hybridization in immature rats treated with PMSG only or in combination with hCG. Strong hybridization signals were observed in the granulosa cells of atretic follicles. However, no significant signals were found in the granulosa cells of healthy small, preantral, or early antral follicles. Healthy Graafian and preovulatory follicles also showed intense signals in their mural granulosa cells, but no signals were detected in the cumulus oophorus cells. Corpora lutea showed only weak signals, but luteinizing follicles probably after atresia exhibited signals of moderate intensity in their luteinized and remaining granulosa cells. No signals were detected in the theca cells and oocytes in all the follicles. Interstitial cells sometimes exhibited hybridization signals of moderate intensity, when the cells were eosinophilic. Pretreatment with different combinations of gonadotropins yielded different ovarian histology, but this had no influence on the localization of hybridization signals. These results, showing that the authentic GnRH receptor mFtNA was expressed in a certain cell population in the rat ovary, suggest that the receptor is involved in the control of various ovarian functions including follicular development, atresia, ovulation, and luteinization after ovulation and follicular atresia.

Zoological Science, 2004

Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive ... more Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Studies on GnRH have demonstrated that GnRH has both stimulatory and inhibitory effects on cell proliferation depending on the cell type; however, the mechanisms of these effects remain to be elucidated. Against this background we used four human cell lines, TSU-Pr1, Jurkat, HHUA and DU145, and newly found that GnRH increased or decreased the colony-formation depending on the cell line. Moreover, we demonstrated that the stimulatory and inhibitory effects of GnRH exhibit distinct ligand selectivities. In order to investigate the molecular basis of these phenomena, analyses of the expression of human GnRH receptors were performed and, moreover, the effects of GnRH were analyzed under conditions in which human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH receptor, which had been suspected of being nonfunctional because of alterations in its sequence, is involved in the effects of GnRH on cell proliferation. In this article, the influence of the autocrine activities of the cells is also reviewed, focusing on the characteristics of substances secreted from the four cell lines. Based on recent studies of GnRH and its receptors and our up-to-date findings, the evolutionary implications of GnRH action are discussed.

Zoological Science, 2006

Thyroid hormones (THs) play crucial roles in various developmental and physiological processes in... more Thyroid hormones (THs) play crucial roles in various developmental and physiological processes in vertebrates, including squamate reptiles. The effect of THs on shedding frequency is interesting in Squamata, since the effects on lizards are quite the reverse of those in snakes: injection of thyroxine increases shedding frequency in lizards, but decreases it in snakes. However, the mechanism underlying this differential effect remains unclear. To facilitate the investigation of the molecular mechanism of the physiological functions of THs in Squamata, their two specific receptor (TRα α α α and β β β β ) cDNAs, which are members of the nuclear hormone receptor superfamily, were cloned from a lizard, the leopard gecko, Eublepharis macularius . This is the first molecular cloning of thyroid hormone receptors (TRs) from reptiles. The deduced amino acid sequences showed high identity with those of other species, especially in the C and E/F domains, which are characteristic domains in nuclear hormone receptors. Expression analysis revealed that TRs were widely expressed in many tissues and organs, as in other animals. To analyze their role in the skin, temporal expression analysis was performed by RT-PCR, revealing that the two TRs had opposing expression patterns: TRα α α α was expressed more strongly after than before skin shedding, whereas TRβ β β β was expressed more strongly before than after skin shedding. This provides good evidence that THs play important roles in the skin, and that the roles of their two receptor isoforms are distinct from each other.

Zoological Science, 1998

Previously we reported that the experimental unilateral cryptorchidism altered the expression pat... more Previously we reported that the experimental unilateral cryptorchidism altered the expression pattern of LH receptor mRNA in abdominal testis. To clarify whether the change was due to exposure of Leydig cells to high temperature in the abdomen, we examined the effect of unilateral efferent duct ligation on the expression. Two weeks after the operation, a relatively decreased expression of 1.8 kb transcript, increased expression of other transcripts, and degeneration of germ cells were observed in the efferent ductligated testes. As these changes were similar to those seen in abdominal testes of cryptorchid rats, exposure of Leydig cells to high temperature was not responsible for the changes in expression pattern of LH receptor mRNA. To examine the correlation between the changes in the receptor mRNA expression and the degeneration of seminiferous epithelium, expression of the mRNA was analyzed in unilaterally cryptorchid rats for 28 days after the operation. In scrotal testes which showed no histological changes, a major, 1.8 kb and several minor, 6.5, 2.6, 1.4, 1.1 and 0.9 kb, transcripts were detected throughout the experimental period. In abdominal testes, the expression of 1.8 kb transcript declined rapidly within three days, while that of 6.5 kb and 2.6 kb was increased, reaching a maximum 14 days after the operation. Histological observations of cryptorchid and efferent duct-ligated testes revealed that these changes in the receptor mRNA expression paralleled the successive disappearance of spermatids and spermatocytes. These results suggest that the changes in expression pattern of LH receptor mRNA are closely related with degeneration of germ cells and that intratesticular paracrine factor(s) from seminiferous tubules might be concerned to the phenomenon.

Life Sciences, 2004

There have been numerous reports of the inhibitory effects of gonadotropin-releasing hormone (GnR... more There have been numerous reports of the inhibitory effects of gonadotropin-releasing hormone (GnRH) and its agonistic and antagonistic analogues on carcinomas derived from various organs, and in particular the direct inhibitory effects have been extensively studied. On the other hand, several studies have indicated that GnRH stimulates the proliferation of lymphoid tissues and cells, suggesting that GnRH is an immunomodulator. However, there have been few reports showing a stimulatory effect of GnRH on cell lines not derived from lymphoid tissues, and the mechanism of the stimulatory effect has not been investigated in detail. In this study, the stimulatory effect of GnRH (100 pM) on TSU-Pr1, a human prostatic carcinoma cell line, was demonstrated, and the dose-depedency of this effect of GnRH (3.125 fM∼20 nM) was observed by measuring colony-formation. RT-PCR analysis showed that both human GnRH receptor 1 and 2 are expressed in TSU-Pr1 cells, suggesting that this stimulatory effect of GnRH occurs through GnRH receptor(s). To our knowledge, this is the first report showing the stimulatory effect of GnRH on a prostatic carcinoma cell line. Moreover, we also examined the effect of conditioned medium of TSU-Pr1 cells and found that it inhibited the GnRH activity only on TSU-Pr1 cells. This characteristic of the conditioned medium of TSU-Pr1 cells is different from that of HHUA or Jurkat cells described in our previous study. TSU-Pr1 cells the proliferation of which is stimulated by GnRH can yield important clues about the mechanisms of the effects of GnRH on cell proliferation.

Zoological Science, 2007

Sexual differentiation in the amniote brain is believed to be regulated by gonadal sex steroid ho... more Sexual differentiation in the amniote brain is believed to be regulated by gonadal sex steroid hormones. Recently, however, the possibility of brain-autonomous sexual differentiation in avian and reptilian species has been reported. We conducted here an expressional analysis of genes related to sex steroid hormones in the chick-embryo brain before gonadal sexual differentiation. Female-specific P450 aromatase expression in the gonad was observed at day 6.5 of incubation, as previously reported, whereas the mRNAs of cholesterol side-chain cleavage enzyme, androgen receptor, and estrogen receptors α and β were clearly expressed in all brain samples of both male and female embryos from day 4.5 of incubation. P450 aromatase was expressed in some brain samples before day 5.5 of incubation and in all brain samples after day 6 of incubation. The mRNA of Ad4BP/SF-1, a transcription factor that regulates steroidogenic enzymes, showed higher expression levels in the male brain than in the female brain at day 5.5 of incubation. This gene was expressed in the ventromedial hypothalamic nucleus, a region important for reproductive behavior. Embryonic Ad4BP/SF-1 expression is reported to play an important role in the formation of this region. These results therefore suggest the involvement of a sex steroid hormone signaling system in brain-autonomous sexual differentiation.

Zoological Science, 1997

... During ovarian development, the follicular cells are differentiated from the cortical cord. W... more ... During ovarian development, the follicular cells are differentiated from the cortical cord. Woods et al. (1989) showed that the LH-binding cells are distributed both in the cortical cord and the cortical interstitial cells in 13.5-to 19.5-day embryos. ...

Life Sciences, 2002

A number of studies have demonstrated that GnRH has anti-proliferative effects on various carcino... more A number of studies have demonstrated that GnRH has anti-proliferative effects on various carcinomas of breast, ovary, endometrium, prostate, pancreas, and liver origin , , , , , , , , , , , , , , , and . In contrast, GnRH increases the proliferative activity of lymphoid tissues and cells, which suggests that GnRH is also an important immunomodulator , , , and . In a previous study, we demonstrated that the colony-forming efficiencies of HHUA (derived from human endometrial carcinoma) and Jurkat (derived from human mature leukemia) cells are affected by the GnRH agonist Buserelin, and that the conditioned media of HHUA and Jurkat cells severely affect the Buserelin activity [23]. The latter finding suggests that substances in the culture medium have some relation to the GnRH activity. Therefore, in the present study, to evaluate the effect of serum supplements on the colony-forming efficiency assay, the assay was performed using 3 lots of fetal bovine serum (FBS) and 2 lots of Nu-Serum I, a semi-synthetic serum supplement. The results showed that the colony-forming efficiencies of HHUA and Jurkat cells fluctuated greatly depending on the lot of FBS. In contrast, Buserelin significantly affected the colony-forming efficiency to similar extents in the media containing both the lots of Nu-Serum I. These results strongly suggest that the constituents of the serum supplements also influence the effect of GnRH on cell proliferation. For further studies about the relationship between substances in the culture medium and the GnRH effects on cell proliferation, it will be necessary to use a completely defined medium, and that a semi-synthetic serum supplement such as Nu-Serum I will also be useful.

Journal of Neuroscience Research, 2009

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in regulating the ... more Hypothalamic gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in regulating the reproductive function of vertebrates. These neurons are known to originate in the olfactory placode and migrate along olfactory-related axons to reach the forebrain during embryonic development. Although GnRH is suggested to be secreted during such migration, its physiological significance is unknown. This point is difficult to explore in vivo because recent studies suggest that GnRH is an important factor for normal brain development and that modification of the embryonic GnRH system by exogenous GnRH analogue or genetic methods would result in dysgenesis of the brain. Therefore, to study the role of GnRH in the migratory process of GnRH neurons, we established an in vitro chick embryonic olfactory nerve bundle explant model. Embryonic day 7.5–8 olfactory nerve bundles were cultured in a mixture of Matrigel and collagen gel. At day 3 of culture, GnRH neurons extended their unbranched neurites and migrated out from both edges of the explant. The nature of neurite extension and migratory behavior of GnRH neurons was well maintained in the gel containing 25% Matrigel and 50% collagen. With this culture system, we examined the effect of GnRH on the migrating GnRH neurons. Cetrorelix, a GnRH antagonist, was found to inhibit significantly neurite growth and neuronal migration of GnRH neurons, the effects of which were repressed by the addition of chicken GnRH-I. These results suggest that GnRH functions as one of the regulating factors of GnRH neuronal development by promoting neurite extension and neuronal migration. © 2009 Wiley-Liss, Inc.

Gene, 2005

In spite of their physiological significance, there is no available information about the nucleot... more In spite of their physiological significance, there is no available information about the nucleotide sequences of prolactin (PRL) and its receptor in reptilian species. In order to fill this gap, PRL and its receptor cDNAs were identified in a reptilian species, the leopard gecko Eublepharis macularius. The deduced leopard gecko PRL polypeptide showed high identities with the corresponding polypeptides of other reptiles. The leopard gecko PRL receptor (PRLR) was estimated to have tandem repeated regions in its extracellular domain, which had been originally found in avian PRLR. Molecular phylogenetic analysis suggests that these tandem repeated regions were generated by the duplication of the extracellular region in the latest common ancestor among reptiles and birds. In addition, tissue distributions of PRL and PRLR in the leopard gecko were examined by the reverse transcription-polymerase chain reaction (RT-PCR). PRLR mRNA was detected in all tissues examined and highly expressed in the whole brain, pituitary, intestine, kidney, ovary, oviduct and testis. Whereas, PRL mRNA was expressed in the whole brain, pituitary, ovary and testis. The co-expressions of PRL and its receptor in some extrapituitary organs suggest that PRL acts as an autocrine/paracrine factor in such organs of the leopard gecko.

Comparative Biochemistry and Physiology B-biochemistry & Molecular Biology, 2003

Sex steroid hormones play a central role in the reproduction of all vertebrates. These hormones f... more Sex steroid hormones play a central role in the reproduction of all vertebrates. These hormones function through their specific receptors, so the expression levels of the receptors may reflect the responsibility of target organs. However, there was no effective method to quantify the expression levels of these receptors in reptilian species. In this study, we established the competitive-PCR assay systems for the quantification of the mRNA expression levels of three sex steroid hormone receptors in the leopard gecko. These assay systems were successfully able to detect the mRNA expression level of each receptor in various organs of male adult leopard geckoes. The expression levels of mRNA of these receptors were highly various depending on the organs assayed. This is the first report regarding the tissue distributions of sex steroid hormone receptor expressions in reptile. The effects of environmental conditions on these hormone receptor expressions were also examined. After the low temperature and short photoperiod treatment for 6 weeks, only the androgen receptor expression was significantly increased in the testes. The competitive-PCR assay systems established in this report should be applicable for various studies of the molecular mechanism underlying the reproductive activity of the leopard gecko.

Journal of Reproduction and Development, 1994

Recent studies have shown that gonadotropin-releasing hormone (GnRH) can exert various effects on... more Recent studies have shown that gonadotropin-releasing hormone (GnRH) can exert various effects on the rat ovary by acting through its specific receptors. To determine the cell types responsive to the action of GnRH under physiological conditions in the ovary, distribution of the GnRH receptor mRNA was studied histologically by in situ hybridization in neonatal and adult rats. Expression of the luteinizing hormone receptor mRNA was also examined to judge the growing state of follicles and the corpora lutea. In neonatal rat ovaries, no significant GnRH receptor mRNA signal was detected until 5 days after birth. The expression was first observed at 10 days in the interstitial cells. At 15 days of age, the receptor mRNA was expressed in the granulosa cells of most preantral and early antral follicles, while no hybridization signal was detected in oocyte and theca cells. In adult cycling rats, GnRH receptor mRNA was detected mainly in the granulosa cells of most follicles and luteal cells. The granulosa cells of atretic follicles showed a very high level of the mRNA expression throughout their degenerating process. A strong hybridization signal was also detected in the mural granulosa cells of mature follicles. Newly formed (developing) corpora lutea exhibited signals with moderate intensity in the luteal cells, and the older ones showed weaker signals. The finding that the initial expression of GnRH receptor mRNA was seen in the interstitial cells of neonatal ovaries implies an unknown function of the ovarian GnRH receptor in ovarian development. The high level expression of GnRH receptor mRNA in atretic and mature follicles supports the putative roles of GnRH in the induction of follicular atresia and ovulation in rat ovaries.

Biochemical and Biophysical Research Communications, 2001

In this study, cell proliferation was examined at low and high cell densities, using HHUA and Jur... more In this study, cell proliferation was examined at low and high cell densities, using HHUA and Jurkat cell lines as experimental models for the antiproliferative and proliferation-enhancing effects of GnRH agonist, Buserelin, respectively. For efficient evaluation of Buserelin activity at low cell density, the colony-forming efficiency assay was adopted. Buserelin markedly affected colony-forming efficiency in a dose-dependent manner at low cell density; however, Buserelin had no effect at high cell density. The conditioned medium of HHUA cells inhibited the Buserelin action, whereas that of Jurkat cells mimicked it. These results suggest that each cell line secretes some substances which regulate cell proliferation, and that these substances can also change the effects of Buserelin. The measurement of colony-forming efficiency is a very effective way of eliminating autocrine and/or paracrine effects, and is a highly sensitive method for measuring GnRH activity.

General and Comparative Endocrinology, 2008

Sex steroid hormones are known to play a central role in vertebrate sex determination and differe... more Sex steroid hormones are known to play a central role in vertebrate sex determination and differentiation. However, the tissues in which they are produced or received during development, especially around the period of sex determination of the gonads, have rarely been investigated. In this study, we identified the cDNA sequence, including the full-length of the coding region of cholesterol side-chain cleavage enzyme (P450scc), from the leopard gecko; a lizard with temperature-dependent sex determination. Embryonic expression analysis of two steroidogenic enzymes, P450scc and P450 aromatase (P450arom), and four sex steroid hormone receptors, androgen receptor, estrogen receptor α and β, and progesterone receptor, was subsequently conducted. mRNA expression of both steroidogenic enzymes was observed in the brain and gonads prior to the temperature-sensitive period of sex determination. The mRNAs of the four sex steroid hormone receptors were also detected in the brain and gonads at all stages examined. These results suggest the existence of a gonad-independent sex steroid hormone signaling system in the developing leopard gecko brain.

General and Comparative Endocrinology, 2004

The gene for proopiomelanocortin (POMC), a common precursor of malanotropins, corticotropin, and ... more The gene for proopiomelanocortin (POMC), a common precursor of malanotropins, corticotropin, and β-endorphin, was isolated and analyzed in the squamata species, the leopard gecko, Eublepharis macularius. Leopard gecko POMC (lgPOMC) cDNA is composed of 1299 bp, excluding the poly(A) tail, and encodes 270 amino acids. The deduced amino acid sequence showed the same structural organization as that of other species and displayed identity with those of other vertebrates: 68% with mud turtles, 57/57% with African clawed frog A/B, 53% with chickens, and 45% with mice. In a phylogenic tree, the lgPOMC clustered with the sequences of the mud turtle POMC and python POMC. The lgPOMC gene comprises three exons and two introns and this structure is consistent with humans, rats, mice, African clawed frog and zebrafish. RT-PCR analysis revealed that the lgPOMC mRNA was expressed only in the whole brain, pituitary, and gonads. To analyze in more detail, a competitive assay system to quantify the expression levels of POMC mRNA was established. We measured the POMC mRNA expression levels in the leopard gecko testes following transfer from a condition of 29 °C, 16L/8D to 18 °C, 10L/14D over 6 weeks. This 6-week acclimation increased the POMC mRNA expression levels significantly. This suggests that the leopard gecko POMC-derived peptides play a role in the mediation of the effect of environmental factors on reproduction.

Zoological Science, 2004

Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive ... more Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Studies on GnRH have demonstrated that GnRH has both stimulatory and inhibitory effects on cell proliferation depending on the cell type; however, the mechanisms of these effects remain to be elucidated. Against this background we used four human cell lines, TSU-Pr1, Jurkat, HHUA and DU145, and newly found that GnRH increased or decreased the colony-formation depending on the cell line. Moreover, we demonstrated that the stimulatory and inhibitory effects of GnRH exhibit distinct ligand selectivities. In order to investigate the molecular basis of these phenomena, analyses of the expression of human GnRH receptors were performed and, moreover, the effects of GnRH were analyzed under conditions in which human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH receptor, which had been suspected of being nonfunctional because of alterations in its sequence, is involved in the effects of GnRH on cell proliferation. In this article, the influence of the autocrine activities of the cells is also reviewed, focusing on the characteristics of substances secreted from the four cell lines. Based on recent studies of GnRH and its receptors and our up-to-date findings, the evolutionary implications of GnRH action are discussed.

Zoological Science, 2004

GnRH (gonadotropin-releasing hormone) is well-known as the central regulator of the reproductive ... more GnRH (gonadotropin-releasing hormone) is well-known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Progress in studies on GnRH demonstrated that GnRH has both inhibitory and stimulatory effects on cell proliferation depending on the cell type, and the mechanisms of these effects have been intensively studied. However, even human GnRH receptors which mediate GnRH stimulation have not been completely identified. In the present study, we showed that the inhibitory and stimulatory effects of GnRH on colony-formation using four cell lines and have demonstrated that the inhibitory and stimulatory effects of GnRH exhibit distinctly different patterns of ligand sensitivity. This result strongly suggests that the two opposite effects of GnRH occur via different types of GnRH receptors, however expressional analyses of human GnRH receptors did not exhibit the significantly different pattern between negatively and positively responding cell lines. Then, in order to identify the GnRH receptors involved in the two opposite effects, effects of GnRH were analysed under the conditions that human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH receptor mediates GnRH stimulation and its splice variant determines the direction of the response to GnRH. These results are the first clear evidence for the functionality of human type II GnRH receptor. Therefore our novel findings are quite noticeable and will greatly contribute to the studies on the mechanisms of the effects of GnRH on cell proliferation in the future.

The Journal of cell biology, 1990

The major nuclear pore protein p62 is modified by O-linked N-acetylglucosamine and functions in n... more The major nuclear pore protein p62 is modified by O-linked N-acetylglucosamine and functions in nuclear transport. We have cloned, sequenced, and expressed the full-length rat p62 cDNA. The rat p62 mRNA is 2,941 nucleotides long and encodes a protein of 525 amino acids containing 30% serine and threonine residues. The amino acid sequence near the amino-terminus contains unique tetrapeptide repeats while the carboxy-terminus consists of a series of predicted alpha-helical regions with hydrophobic heptad repeats. Heterologous expression of rat p62 in African Green Monkey Kidney COS-1 cells and CV-1 cells was detected using a species-specific antipeptide serum. When transiently expressed in COS-1 cells, rat p62 binds wheat germ agglutinin and concentrates at the spindle poles during mitosis. In CV-1 cells cotransfected with rat p62 cDNA and SV40 viral DNA, rat p62 associates with the nuclear membrane without interfering with the nuclear transport of SV40 large T antigen. The ability to...

General and Comparative Endocrinology, 1991

Endocrinology, 2006

GnRH was first identified as the hypothalamic decapeptide that promotes gonadotropin release from... more GnRH was first identified as the hypothalamic decapeptide that promotes gonadotropin release from pituitary gonadotropes. Thereafter, direct stimulatory and inhibitory effects of GnRH on cell proliferation were demonstrated in a number of types of primary cultured cells and established cell lines. Recently, the effects of GnRH on cell attachment, cytoskeleton remodeling, and cell migration have also been reported. Thus, the effects of GnRH on various cell activities are of great interest among researchers who study the actions of GnRH. In this study, we demonstrated that GnRH induces actin cytoskeleton remodeling and affects cell migration using two human prostatic carcinoma cell lines, TSU-Pr1 and DU145. In

Journal of Experimental Zoology, 1995

7% clarify the target cells of GnRH in the ovary, in vivo expression of ovarian GnRH receptor mRN... more 7% clarify the target cells of GnRH in the ovary, in vivo expression of ovarian GnRH receptor mRNA was examined histologically by in situ hybridization in immature rats treated with PMSG only or in combination with hCG. Strong hybridization signals were observed in the granulosa cells of atretic follicles. However, no significant signals were found in the granulosa cells of healthy small, preantral, or early antral follicles. Healthy Graafian and preovulatory follicles also showed intense signals in their mural granulosa cells, but no signals were detected in the cumulus oophorus cells. Corpora lutea showed only weak signals, but luteinizing follicles probably after atresia exhibited signals of moderate intensity in their luteinized and remaining granulosa cells. No signals were detected in the theca cells and oocytes in all the follicles. Interstitial cells sometimes exhibited hybridization signals of moderate intensity, when the cells were eosinophilic. Pretreatment with different combinations of gonadotropins yielded different ovarian histology, but this had no influence on the localization of hybridization signals. These results, showing that the authentic GnRH receptor mFtNA was expressed in a certain cell population in the rat ovary, suggest that the receptor is involved in the control of various ovarian functions including follicular development, atresia, ovulation, and luteinization after ovulation and follicular atresia.

Zoological Science, 2004

Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive ... more Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Studies on GnRH have demonstrated that GnRH has both stimulatory and inhibitory effects on cell proliferation depending on the cell type; however, the mechanisms of these effects remain to be elucidated. Against this background we used four human cell lines, TSU-Pr1, Jurkat, HHUA and DU145, and newly found that GnRH increased or decreased the colony-formation depending on the cell line. Moreover, we demonstrated that the stimulatory and inhibitory effects of GnRH exhibit distinct ligand selectivities. In order to investigate the molecular basis of these phenomena, analyses of the expression of human GnRH receptors were performed and, moreover, the effects of GnRH were analyzed under conditions in which human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH receptor, which had been suspected of being nonfunctional because of alterations in its sequence, is involved in the effects of GnRH on cell proliferation. In this article, the influence of the autocrine activities of the cells is also reviewed, focusing on the characteristics of substances secreted from the four cell lines. Based on recent studies of GnRH and its receptors and our up-to-date findings, the evolutionary implications of GnRH action are discussed.

Zoological Science, 2006

Thyroid hormones (THs) play crucial roles in various developmental and physiological processes in... more Thyroid hormones (THs) play crucial roles in various developmental and physiological processes in vertebrates, including squamate reptiles. The effect of THs on shedding frequency is interesting in Squamata, since the effects on lizards are quite the reverse of those in snakes: injection of thyroxine increases shedding frequency in lizards, but decreases it in snakes. However, the mechanism underlying this differential effect remains unclear. To facilitate the investigation of the molecular mechanism of the physiological functions of THs in Squamata, their two specific receptor (TRα α α α and β β β β ) cDNAs, which are members of the nuclear hormone receptor superfamily, were cloned from a lizard, the leopard gecko, Eublepharis macularius . This is the first molecular cloning of thyroid hormone receptors (TRs) from reptiles. The deduced amino acid sequences showed high identity with those of other species, especially in the C and E/F domains, which are characteristic domains in nuclear hormone receptors. Expression analysis revealed that TRs were widely expressed in many tissues and organs, as in other animals. To analyze their role in the skin, temporal expression analysis was performed by RT-PCR, revealing that the two TRs had opposing expression patterns: TRα α α α was expressed more strongly after than before skin shedding, whereas TRβ β β β was expressed more strongly before than after skin shedding. This provides good evidence that THs play important roles in the skin, and that the roles of their two receptor isoforms are distinct from each other.

Zoological Science, 1998

Previously we reported that the experimental unilateral cryptorchidism altered the expression pat... more Previously we reported that the experimental unilateral cryptorchidism altered the expression pattern of LH receptor mRNA in abdominal testis. To clarify whether the change was due to exposure of Leydig cells to high temperature in the abdomen, we examined the effect of unilateral efferent duct ligation on the expression. Two weeks after the operation, a relatively decreased expression of 1.8 kb transcript, increased expression of other transcripts, and degeneration of germ cells were observed in the efferent ductligated testes. As these changes were similar to those seen in abdominal testes of cryptorchid rats, exposure of Leydig cells to high temperature was not responsible for the changes in expression pattern of LH receptor mRNA. To examine the correlation between the changes in the receptor mRNA expression and the degeneration of seminiferous epithelium, expression of the mRNA was analyzed in unilaterally cryptorchid rats for 28 days after the operation. In scrotal testes which showed no histological changes, a major, 1.8 kb and several minor, 6.5, 2.6, 1.4, 1.1 and 0.9 kb, transcripts were detected throughout the experimental period. In abdominal testes, the expression of 1.8 kb transcript declined rapidly within three days, while that of 6.5 kb and 2.6 kb was increased, reaching a maximum 14 days after the operation. Histological observations of cryptorchid and efferent duct-ligated testes revealed that these changes in the receptor mRNA expression paralleled the successive disappearance of spermatids and spermatocytes. These results suggest that the changes in expression pattern of LH receptor mRNA are closely related with degeneration of germ cells and that intratesticular paracrine factor(s) from seminiferous tubules might be concerned to the phenomenon.

Life Sciences, 2004

There have been numerous reports of the inhibitory effects of gonadotropin-releasing hormone (GnR... more There have been numerous reports of the inhibitory effects of gonadotropin-releasing hormone (GnRH) and its agonistic and antagonistic analogues on carcinomas derived from various organs, and in particular the direct inhibitory effects have been extensively studied. On the other hand, several studies have indicated that GnRH stimulates the proliferation of lymphoid tissues and cells, suggesting that GnRH is an immunomodulator. However, there have been few reports showing a stimulatory effect of GnRH on cell lines not derived from lymphoid tissues, and the mechanism of the stimulatory effect has not been investigated in detail. In this study, the stimulatory effect of GnRH (100 pM) on TSU-Pr1, a human prostatic carcinoma cell line, was demonstrated, and the dose-depedency of this effect of GnRH (3.125 fM∼20 nM) was observed by measuring colony-formation. RT-PCR analysis showed that both human GnRH receptor 1 and 2 are expressed in TSU-Pr1 cells, suggesting that this stimulatory effect of GnRH occurs through GnRH receptor(s). To our knowledge, this is the first report showing the stimulatory effect of GnRH on a prostatic carcinoma cell line. Moreover, we also examined the effect of conditioned medium of TSU-Pr1 cells and found that it inhibited the GnRH activity only on TSU-Pr1 cells. This characteristic of the conditioned medium of TSU-Pr1 cells is different from that of HHUA or Jurkat cells described in our previous study. TSU-Pr1 cells the proliferation of which is stimulated by GnRH can yield important clues about the mechanisms of the effects of GnRH on cell proliferation.

Zoological Science, 2007

Sexual differentiation in the amniote brain is believed to be regulated by gonadal sex steroid ho... more Sexual differentiation in the amniote brain is believed to be regulated by gonadal sex steroid hormones. Recently, however, the possibility of brain-autonomous sexual differentiation in avian and reptilian species has been reported. We conducted here an expressional analysis of genes related to sex steroid hormones in the chick-embryo brain before gonadal sexual differentiation. Female-specific P450 aromatase expression in the gonad was observed at day 6.5 of incubation, as previously reported, whereas the mRNAs of cholesterol side-chain cleavage enzyme, androgen receptor, and estrogen receptors α and β were clearly expressed in all brain samples of both male and female embryos from day 4.5 of incubation. P450 aromatase was expressed in some brain samples before day 5.5 of incubation and in all brain samples after day 6 of incubation. The mRNA of Ad4BP/SF-1, a transcription factor that regulates steroidogenic enzymes, showed higher expression levels in the male brain than in the female brain at day 5.5 of incubation. This gene was expressed in the ventromedial hypothalamic nucleus, a region important for reproductive behavior. Embryonic Ad4BP/SF-1 expression is reported to play an important role in the formation of this region. These results therefore suggest the involvement of a sex steroid hormone signaling system in brain-autonomous sexual differentiation.

Zoological Science, 1997

... During ovarian development, the follicular cells are differentiated from the cortical cord. W... more ... During ovarian development, the follicular cells are differentiated from the cortical cord. Woods et al. (1989) showed that the LH-binding cells are distributed both in the cortical cord and the cortical interstitial cells in 13.5-to 19.5-day embryos. ...

Life Sciences, 2002

A number of studies have demonstrated that GnRH has anti-proliferative effects on various carcino... more A number of studies have demonstrated that GnRH has anti-proliferative effects on various carcinomas of breast, ovary, endometrium, prostate, pancreas, and liver origin , , , , , , , , , , , , , , , and . In contrast, GnRH increases the proliferative activity of lymphoid tissues and cells, which suggests that GnRH is also an important immunomodulator , , , and . In a previous study, we demonstrated that the colony-forming efficiencies of HHUA (derived from human endometrial carcinoma) and Jurkat (derived from human mature leukemia) cells are affected by the GnRH agonist Buserelin, and that the conditioned media of HHUA and Jurkat cells severely affect the Buserelin activity [23]. The latter finding suggests that substances in the culture medium have some relation to the GnRH activity. Therefore, in the present study, to evaluate the effect of serum supplements on the colony-forming efficiency assay, the assay was performed using 3 lots of fetal bovine serum (FBS) and 2 lots of Nu-Serum I, a semi-synthetic serum supplement. The results showed that the colony-forming efficiencies of HHUA and Jurkat cells fluctuated greatly depending on the lot of FBS. In contrast, Buserelin significantly affected the colony-forming efficiency to similar extents in the media containing both the lots of Nu-Serum I. These results strongly suggest that the constituents of the serum supplements also influence the effect of GnRH on cell proliferation. For further studies about the relationship between substances in the culture medium and the GnRH effects on cell proliferation, it will be necessary to use a completely defined medium, and that a semi-synthetic serum supplement such as Nu-Serum I will also be useful.

Journal of Neuroscience Research, 2009

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in regulating the ... more Hypothalamic gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in regulating the reproductive function of vertebrates. These neurons are known to originate in the olfactory placode and migrate along olfactory-related axons to reach the forebrain during embryonic development. Although GnRH is suggested to be secreted during such migration, its physiological significance is unknown. This point is difficult to explore in vivo because recent studies suggest that GnRH is an important factor for normal brain development and that modification of the embryonic GnRH system by exogenous GnRH analogue or genetic methods would result in dysgenesis of the brain. Therefore, to study the role of GnRH in the migratory process of GnRH neurons, we established an in vitro chick embryonic olfactory nerve bundle explant model. Embryonic day 7.5–8 olfactory nerve bundles were cultured in a mixture of Matrigel and collagen gel. At day 3 of culture, GnRH neurons extended their unbranched neurites and migrated out from both edges of the explant. The nature of neurite extension and migratory behavior of GnRH neurons was well maintained in the gel containing 25% Matrigel and 50% collagen. With this culture system, we examined the effect of GnRH on the migrating GnRH neurons. Cetrorelix, a GnRH antagonist, was found to inhibit significantly neurite growth and neuronal migration of GnRH neurons, the effects of which were repressed by the addition of chicken GnRH-I. These results suggest that GnRH functions as one of the regulating factors of GnRH neuronal development by promoting neurite extension and neuronal migration. © 2009 Wiley-Liss, Inc.

Gene, 2005

In spite of their physiological significance, there is no available information about the nucleot... more In spite of their physiological significance, there is no available information about the nucleotide sequences of prolactin (PRL) and its receptor in reptilian species. In order to fill this gap, PRL and its receptor cDNAs were identified in a reptilian species, the leopard gecko Eublepharis macularius. The deduced leopard gecko PRL polypeptide showed high identities with the corresponding polypeptides of other reptiles. The leopard gecko PRL receptor (PRLR) was estimated to have tandem repeated regions in its extracellular domain, which had been originally found in avian PRLR. Molecular phylogenetic analysis suggests that these tandem repeated regions were generated by the duplication of the extracellular region in the latest common ancestor among reptiles and birds. In addition, tissue distributions of PRL and PRLR in the leopard gecko were examined by the reverse transcription-polymerase chain reaction (RT-PCR). PRLR mRNA was detected in all tissues examined and highly expressed in the whole brain, pituitary, intestine, kidney, ovary, oviduct and testis. Whereas, PRL mRNA was expressed in the whole brain, pituitary, ovary and testis. The co-expressions of PRL and its receptor in some extrapituitary organs suggest that PRL acts as an autocrine/paracrine factor in such organs of the leopard gecko.

Comparative Biochemistry and Physiology B-biochemistry & Molecular Biology, 2003

Sex steroid hormones play a central role in the reproduction of all vertebrates. These hormones f... more Sex steroid hormones play a central role in the reproduction of all vertebrates. These hormones function through their specific receptors, so the expression levels of the receptors may reflect the responsibility of target organs. However, there was no effective method to quantify the expression levels of these receptors in reptilian species. In this study, we established the competitive-PCR assay systems for the quantification of the mRNA expression levels of three sex steroid hormone receptors in the leopard gecko. These assay systems were successfully able to detect the mRNA expression level of each receptor in various organs of male adult leopard geckoes. The expression levels of mRNA of these receptors were highly various depending on the organs assayed. This is the first report regarding the tissue distributions of sex steroid hormone receptor expressions in reptile. The effects of environmental conditions on these hormone receptor expressions were also examined. After the low temperature and short photoperiod treatment for 6 weeks, only the androgen receptor expression was significantly increased in the testes. The competitive-PCR assay systems established in this report should be applicable for various studies of the molecular mechanism underlying the reproductive activity of the leopard gecko.

Journal of Reproduction and Development, 1994

Recent studies have shown that gonadotropin-releasing hormone (GnRH) can exert various effects on... more Recent studies have shown that gonadotropin-releasing hormone (GnRH) can exert various effects on the rat ovary by acting through its specific receptors. To determine the cell types responsive to the action of GnRH under physiological conditions in the ovary, distribution of the GnRH receptor mRNA was studied histologically by in situ hybridization in neonatal and adult rats. Expression of the luteinizing hormone receptor mRNA was also examined to judge the growing state of follicles and the corpora lutea. In neonatal rat ovaries, no significant GnRH receptor mRNA signal was detected until 5 days after birth. The expression was first observed at 10 days in the interstitial cells. At 15 days of age, the receptor mRNA was expressed in the granulosa cells of most preantral and early antral follicles, while no hybridization signal was detected in oocyte and theca cells. In adult cycling rats, GnRH receptor mRNA was detected mainly in the granulosa cells of most follicles and luteal cells. The granulosa cells of atretic follicles showed a very high level of the mRNA expression throughout their degenerating process. A strong hybridization signal was also detected in the mural granulosa cells of mature follicles. Newly formed (developing) corpora lutea exhibited signals with moderate intensity in the luteal cells, and the older ones showed weaker signals. The finding that the initial expression of GnRH receptor mRNA was seen in the interstitial cells of neonatal ovaries implies an unknown function of the ovarian GnRH receptor in ovarian development. The high level expression of GnRH receptor mRNA in atretic and mature follicles supports the putative roles of GnRH in the induction of follicular atresia and ovulation in rat ovaries.

Biochemical and Biophysical Research Communications, 2001

In this study, cell proliferation was examined at low and high cell densities, using HHUA and Jur... more In this study, cell proliferation was examined at low and high cell densities, using HHUA and Jurkat cell lines as experimental models for the antiproliferative and proliferation-enhancing effects of GnRH agonist, Buserelin, respectively. For efficient evaluation of Buserelin activity at low cell density, the colony-forming efficiency assay was adopted. Buserelin markedly affected colony-forming efficiency in a dose-dependent manner at low cell density; however, Buserelin had no effect at high cell density. The conditioned medium of HHUA cells inhibited the Buserelin action, whereas that of Jurkat cells mimicked it. These results suggest that each cell line secretes some substances which regulate cell proliferation, and that these substances can also change the effects of Buserelin. The measurement of colony-forming efficiency is a very effective way of eliminating autocrine and/or paracrine effects, and is a highly sensitive method for measuring GnRH activity.

General and Comparative Endocrinology, 2008

Sex steroid hormones are known to play a central role in vertebrate sex determination and differe... more Sex steroid hormones are known to play a central role in vertebrate sex determination and differentiation. However, the tissues in which they are produced or received during development, especially around the period of sex determination of the gonads, have rarely been investigated. In this study, we identified the cDNA sequence, including the full-length of the coding region of cholesterol side-chain cleavage enzyme (P450scc), from the leopard gecko; a lizard with temperature-dependent sex determination. Embryonic expression analysis of two steroidogenic enzymes, P450scc and P450 aromatase (P450arom), and four sex steroid hormone receptors, androgen receptor, estrogen receptor α and β, and progesterone receptor, was subsequently conducted. mRNA expression of both steroidogenic enzymes was observed in the brain and gonads prior to the temperature-sensitive period of sex determination. The mRNAs of the four sex steroid hormone receptors were also detected in the brain and gonads at all stages examined. These results suggest the existence of a gonad-independent sex steroid hormone signaling system in the developing leopard gecko brain.

General and Comparative Endocrinology, 2004

The gene for proopiomelanocortin (POMC), a common precursor of malanotropins, corticotropin, and ... more The gene for proopiomelanocortin (POMC), a common precursor of malanotropins, corticotropin, and β-endorphin, was isolated and analyzed in the squamata species, the leopard gecko, Eublepharis macularius. Leopard gecko POMC (lgPOMC) cDNA is composed of 1299 bp, excluding the poly(A) tail, and encodes 270 amino acids. The deduced amino acid sequence showed the same structural organization as that of other species and displayed identity with those of other vertebrates: 68% with mud turtles, 57/57% with African clawed frog A/B, 53% with chickens, and 45% with mice. In a phylogenic tree, the lgPOMC clustered with the sequences of the mud turtle POMC and python POMC. The lgPOMC gene comprises three exons and two introns and this structure is consistent with humans, rats, mice, African clawed frog and zebrafish. RT-PCR analysis revealed that the lgPOMC mRNA was expressed only in the whole brain, pituitary, and gonads. To analyze in more detail, a competitive assay system to quantify the expression levels of POMC mRNA was established. We measured the POMC mRNA expression levels in the leopard gecko testes following transfer from a condition of 29 °C, 16L/8D to 18 °C, 10L/14D over 6 weeks. This 6-week acclimation increased the POMC mRNA expression levels significantly. This suggests that the leopard gecko POMC-derived peptides play a role in the mediation of the effect of environmental factors on reproduction.

Zoological Science, 2004

Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive ... more Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Studies on GnRH have demonstrated that GnRH has both stimulatory and inhibitory effects on cell proliferation depending on the cell type; however, the mechanisms of these effects remain to be elucidated. Against this background we used four human cell lines, TSU-Pr1, Jurkat, HHUA and DU145, and newly found that GnRH increased or decreased the colony-formation depending on the cell line. Moreover, we demonstrated that the stimulatory and inhibitory effects of GnRH exhibit distinct ligand selectivities. In order to investigate the molecular basis of these phenomena, analyses of the expression of human GnRH receptors were performed and, moreover, the effects of GnRH were analyzed under conditions in which human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH receptor, which had been suspected of being nonfunctional because of alterations in its sequence, is involved in the effects of GnRH on cell proliferation. In this article, the influence of the autocrine activities of the cells is also reviewed, focusing on the characteristics of substances secreted from the four cell lines. Based on recent studies of GnRH and its receptors and our up-to-date findings, the evolutionary implications of GnRH action are discussed.

Zoological Science, 2004

GnRH (gonadotropin-releasing hormone) is well-known as the central regulator of the reproductive ... more GnRH (gonadotropin-releasing hormone) is well-known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Progress in studies on GnRH demonstrated that GnRH has both inhibitory and stimulatory effects on cell proliferation depending on the cell type, and the mechanisms of these effects have been intensively studied. However, even human GnRH receptors which mediate GnRH stimulation have not been completely identified. In the present study, we showed that the inhibitory and stimulatory effects of GnRH on colony-formation using four cell lines and have demonstrated that the inhibitory and stimulatory effects of GnRH exhibit distinctly different patterns of ligand sensitivity. This result strongly suggests that the two opposite effects of GnRH occur via different types of GnRH receptors, however expressional analyses of human GnRH receptors did not exhibit the significantly different pattern between negatively and positively responding cell lines. Then, in order to identify the GnRH receptors involved in the two opposite effects, effects of GnRH were analysed under the conditions that human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH receptor mediates GnRH stimulation and its splice variant determines the direction of the response to GnRH. These results are the first clear evidence for the functionality of human type II GnRH receptor. Therefore our novel findings are quite noticeable and will greatly contribute to the studies on the mechanisms of the effects of GnRH on cell proliferation in the future.