NATIONAL INSTITUTE FOR BASIC BIOLOGY
LABORATORY OF REPRODUCTIVE BIOLOGY
- Professor:
- Yoshitaka Nagahama
- Research Associates:
- Michiyasu Yoshikuni
Masakane Yamashita
Minoru Tanaka
Tohru Kobayashi
- Monbusho Foreign Scientist:
- Alexander P. Scott
- JSPS Post-doctoral Fellows:
- Naoki Shibata
Takeshi Miura
Chiemi Miura
- Graduate Students:
- Akihiko Yamaguchi
Mika Takahashi
Toshinobu Tokumoto
Yoshinao Katsu
Shinji Onoe
Daisuke Kobayashi
Xiao-Tian Chang
- Visiting Scientists:
- Howard A. Bern 1)
Glen Van der Kraak 2)
Noriyoshi Sakat 3)
Michiya Matsuyama 4)
Masamichi Nakashima 5)
Jian-Quiao Jiang 6)
1) from University of California
2) from University of Guelph
3) from Fukui Prefectural University
4) from Mie University
5) from Tohoku University
6) from Wuhan University
The division of reproductive biology conducts research on the endocrine
regulation of differentiation, growth and maturation of germ cells in
multicellular animals, using fish as a primary study model.
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I. Endocrine regulation of oocyte differentiation, growth and
maturation
- Our research effort in previous years concentrated on the identification
and characterization of the molecules (gonadotropin hormones and
gonadal steroid hormones) that stimulate and control germ cell growth
and maturation. It was in 1985 that we identified, for the first time
in any vertebrate, 17a, 20B-dihydroxy-4-pregnen-3-0ne
(17a, 20B-DP) as the maturation-inducing hormone of amago
salmon (Oncorhynchus rhodurus). Along with estradiol-17B, which
was identified as the major mediator of oocyte growth, we now have two
known biologically important mediators of oocyte growth and maturation
in female salmonid fishes. It is established that the granulosa cells
are the site of production of these two mediators, but their production
by the ovarian follicle depends on the provision of precursor steroids
by the thecal cell (two-cell type model). A dramatic switch in the
steroidogenic pathway from estradiol-17B to 17a,
20B-DP occurs in ovarian follicle cells immediately prior to
oocyte maturation. This switch is a prerequisite step for the growing
oocyte to enter the maturation phase, and requires a complex and
integrated network of gene regulation involving cell-specificity,
hormonal regulation, and developmental patterning.
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- We have isolated and characterized the cDNA encoding several ovarian
steroidogenic enzymes of rainbow trout (Oncorhynchus mykiss) and medaka
(Oryzias latipes) which are responsible for estradiol-17B and
17a, 20B-DP biosynthesis: cholesterol side-chain cleavage
cytochrome P450 (P450scc), 3B-hydroxysteroid dehydrogenase
(3B-HSD), 17a-hydroxylase/C17, 20-lyase cytochrome P450
(P450cl7), P450 aromatase (P450arom) and 20B-hydroxysteroid
dehydrogenase (20B-HSD). These cDNA clones have been used for
Northern and whole mount in situ hybridization to investigate the
molecular basis of differential production of estradiol-17B and
17a, 20B-DP during oocyte growth and maturation in
rainbow trout and medaka. In both species, P450scc and P450c17 (also
3B-HSD in rainbow trout) mRNA transcripts were increased in
follicles towards the end of oocyte growth phase and during oocyte
maturation. Furthermore, incubations of isolated thecal layers with
gonadotropin resulted in the elevation of P450scc mRNA. The effect of
gonadotropin becomes more dramatic when the expression of P450scc mRNA
is examined in granulosa cells. P450scc mRNA is not detected in the
absence of gonadotropin, but markedly expressed in the presence of
gonadotropin. The increase in the amount of P450scc, 3B-HSD and
P450cl7 transcripts provide an explanation for the dramatic increase in
17a, 20B-DP production in follicles during naturally- and
gonadotropin-induced oocyte maturation. In contrast, Ievels of mRNA for
P450arom were high during oocyte growth, but rapidly decreased during
oocyie maturation. This decrease in P450arom mRNA Ievels appears to be
correlated with the decreased ability of maturing follicles to produce
estradiol-17B.
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- We have shown that 17a, 20B-DP acts via a receptor on the
plasma membrane of oocytes. 17a, 20B-DP receptor
concentrations increase during oocyte maturation. Our recent studies
suggest that inhibitory G-proteins are involved in the signal
transduction pathway of the maturational action of 17a,
20B-DP in fish oocyies. The early steps following 17a,
20B-DP action involve the formation of the major mediator of
this steroid, maturation-promoting factor or metaphase-promoting factor
(MPF). MPF activity cycles during 17a, 20B-DP-induced
oocyie maturation with the highest activity occurring at the first and
second meiotic metaphase. Studies from our laboratory and others have
shown that MPF activity is not species-specific and can be detected in
both meiotic and mitotic cells of various organisms, from yeast to
mammals.
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- Fish MPF, Iike that of amphibians, consists of two components,
catalyiic cdc2 kinase (34-kDa) and regulatory cyclin B (46- to 48-kDa).
Goldfish immature oocyies contain 35-kDa inactive cdc2 kinase. Although
immature oocytes contain mRNA for cyclin B, they do not contain cyclin
B protein. 17a, 20B-DP induces oocyies to synthesize
cyclin B. The preexixting 35-kDa inactive cdc2 kinase binds to de novo
synthesized cyclin B at first, then is rapidly converted into the
34-kDa active form. Introduction of a bacterially produced goldfish
cyclin B into immature goldfish oocyte extracts induces cdc2 kinase
activation, concurrent with the shift in apparent molecular weight of
cdc2 kinase from 35- to 34-kDa, as found in oocytes matured with
17a, 20B-DP. Phosphoamino acid analysis shows that
threonine (Thr) phosphorylation of the 34-kDa cdc2 kinase and serine
phosphorylation of cyclin B are associated with the activation. The same
phosphorylation is found in oocyies matured by 17a,
20B-DP. Cyclin B-induced cdc2 kinase activation is not observed
when threonine phosphorylation of cdc2 kinase and serine
phosphorylation of cyclin B are inhibited by protein kinase inhibitors,
although the binding of the 35-kDa cdc2 kinase to cyclin B occurs even
in the presence of the inhibitors. In contrast, cdc2 kinase is activated
by mutant cyclins that undergo no serine phosphorylation during the
activation. The site of threonine phosphorylation on cdc2 kinase was
mapped to residue Thr161. These findings indicate that the Thr161
phosphorylation of cdc2 kinase, but not serine phosphorylation of
cyclin B, is required for cdc2 kinase (MPF) activation in goldfish
oocyie
(Fig.1).
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- MPF activity decreases immediately after fertilization, coinciding with
the degradation of cyclin B protein. Purified goldfish 26S proteasome,
an ATP-dependent protease, can digest a bacterially produced goldfish
wild bype cyclin B, producing an intermediate cyclin B (42-kDa). In
contrast, various cyclin B mutants lacking the first 42, 68, and 96
N-terminal amino acids are not digested by the proteasome. Amino acid
sequence analysis of the 42-kDa intermediate cyclin B reveals that 26S
proteasome cleaves the C-terminal piptide bond of lysine 57. This is
the first evidence for the crucial role of 26S proteasome in cyclin B
degradation.
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II. Endocrine regulation of male germ cell development and
maturation
- We have identified two steroidal mediators of male germ cell development
in salmonid fishes (11-ketotestosterone for spermatogenesis and
17a, 20B-DP for sperm maturation). A steroidogenic switch,
from 11-ketotestosterone to 17a, 20B-DP, occurs in
salmonid testes around the onset of final maturation. In vitro
incubation studies using different testicular preparations have
revealed that the site of 17a, 20B-DP production is in
the sperm, but its production depends on the provision of precursor
steroids by somatic cells. The site of 11-ketotestosterone production
is in the testicular somatic cells. In the cultivated male Japanese eel
(Auguilla japonica), spermatogonia are the only germ cells
present in the testis. A serum-free, chemically defined organ culture
system developed for eel testes was used to investigate the effect of
various steroid hormones on the induction of spermatogenesis in vitro.
We obtained evidence that 11-ketotestosterone can induce the entire
process of spermatogenesis in vitro from premitotic spermatogonia to
spermatozoa within 21 days.
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- To isolate the genes that are expressed or suppressed in eel testes
during HCG-induced spermatogenesis, we extracted mRNA from control
testes and testes that had been given a single injection of HCG one day
previously. Subtractive cDNA Iibraries were constructed to clone
specific cDNAs expressed at each stage. So far, three cDNA clones have
been isolated and characterized. From its deduced amino acid sequence,
one of the up-regulated cDNAs was identified as coding for the activin
B subunit. We used Northern blot analysis and in situ hybridization
techniques to examine sequential changes in transcripts of testicular
activin B during HCG-induced spermatogenesis. No transcripts for
activin B were found in testes prior to HCG injection. In contrast, 3.3
kb mRNA transcripts were prominent in testes one day after the
injection. The transcript concentration began to decrease three days
after the injection and there was a further sharp decrease by nine days.
The HCG-dependent activin B mRNA expression in the testes was confirmed
by in situ hybridization using a digoxigeninlabelled RNA probe: the
signal was restricted to Sertoli cells in testes treated with HCG for
one to three days. Taken together, these results suggest that activin
acts as a regulator of spermatogonial proliferation.
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- In salmonid fishes, spermatozoa taken from the testes are immotile, but
acquire motility during their passage through the sperm duct. Using
male masu salmon (Oncorhynchus masou), we found that
gonadotropin-induced testicular production of 17a, 20B-DP
is responsible for the acquisition of sperm motility; 17a,
20B-DP acts to increase sperm duct pH, which in turn increases
the cAMP content of sperm, allowing the acquisition of motility.
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- Our recently-initiated studies include work on the molecular mechanisms
of (1) sex determination and gonadal differentiation, (2) meiosis
initiation, and (3) vitellogenin uptake into the oocyie from the
vascular system.
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Publication List:
- Shibata, N., Yoshikuni, M. and Nagahama, Y. (1993) Vitellogenin
incorporation into oocytes of rainbow trout, Oncorhynchus mykiss, in
vitro: effect of hormones on dunuded oocyies. Develop. Growth Differ
35, 115-121.
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- Kamijo, M., Yasuda, H., Yau, P.M., Yamashita, M., Nagahama, Y. and
Ohba, Y. (1993) Preference of human cdc2 kinase for peptide substrate.
Peptide Res. 5, 281-285.
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- Yasuda, H., Nakata, T., Honda, R., Nakamura, M., Yamashita, M.,
Nagahama, Y. and Ohba, Y. (1993) Involvement of a cdc2-related cell
division kinase in S phase regulation in murine cdc2 kinase Ts mutant.
Somatic Cell Mole. Genet 18, 403-408.
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- Sakai, N., Tanaka, M., Takahashi, M. and Nagahama, Y. (1993) Isolation
and expression of rainbow trout (Oncorhynchus mykiss) ovarian cDNA
encoding 3B-hydroxysteroid dehydrogenase/ DELTA^5-4-isomerase.
Fish Physiol. Biochem. 11, 273-279.
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- Yoshikuni, M., Shibata, N. and Nagahama, Y. (1993) Specific bindings of
[3H] 17a, 20B-dihydroxy-4-pregnen-3-one to oocyte cortices
of rainbow trout (Oncorhynchus mykiss). Fish Physiol.Biochem. 11, 15-24.
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- Nagahama, Y., Yoshikuni, M., Yamashita, M., Sakai, N., Tanaka, M. and
Shibata, N. (1993) Molecular endocrinology of oocyte growth and
maturation in fish. Fish. Physiol. Biochem. 11, 3-14.
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- Natsuyama, S., Noda, Y., Yamashita, M., Nagahama, Y. and Mori, T. (1993)
Superoxide dismutase and thioiredoxin restore defective p34cdc2 kinase
activation in mouse two-cell block. Biochem. Biophys. Acta 1176, 90-94.
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- Nakamura, M., Specker, J.L. and Nagahama, Y. (1993) Ultrastructural
analysis of formation of a functional follicle during early vitellogenes
is in the tilapia, Oreochromis niloticus. Cell Tissue Res. 272, 33-39.
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- Nagahama, Y. (1993) Regulation of oocyte maturation in aquatic animals:
the comparative and general aspects. Biology International Special
Issue 28, 27-32.
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- Specker, J.L., Kishida, M., Huang, L., King, D.S., Nagahama, Y., Ueda,
H. and Anderson, T.R. (1993) Immunocytochemical and immunogold
localization of two prolactin isoforms in the same pituitary cells and
in the same granules in the tilapia (Oreochromis mossambicus). Gen.
Comp. Endocrinol. 89, 28-38.
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- Takahashi, M., Tanaka, M., Sakai, N., Adachi, S., Miller, W.L. and
Nagahama, Y. (1993) Rainbow trout ovarian cholesterol side-chain
cleavage cyiochrome P450 (P450scc): cDNA cloning and mRNA expression
during oogenesis. FEBS Letters 319, 45-48.
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- Nakamura, M., Tsuchiya, F., Iwasaki, M. and Nagahama, Y. (1993)
Reproductive characteristics of precociously mature triploid male masu
salmon, Oncorhynchus masou. Zoof Sci. lO, 117-125.
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- Hasumi, M., Iwasawa, H. and Nagahama, Y. (1993) Seasonal changes in
plasma concentrations of sex steroids in the salamander Hynobius
nigrescens. Gen. Comp. EndocrinoL 90, 51-57.
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- Lin, D., Black, S.M., Nagahama, Y. and Miller, W.L. (1993) Steroid
17a-hydroxylase and 17, 20 Iyase activities of P450cl7:
contributions of serinel06 and P450 reductase. Endocrinology 132,
2498-2506.
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- Kobayashi, T., Sakai, N., Adachi, S., lwasawa, H., Asahina, K. and
Nagahama, Y. (1993) 17a, 20B-Dihydroxy-4-pregnen-3-0ne is
the naturally occurring spermiation-inducing hormone in the testis of a
frog, Rana nigromaculata. Endocrinology 133, 321-327.
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- Nakamura, M. and Nagahama, Y. (1993) Ultrastructural study on the
differentiation and development of steroid-producing cells during
ovarian differentiation in the amago salmon, Oncorhynchus rhodurus.
Aquaculture 112, 237-251.
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- Tokumoto, T., Yamashita, M., Yoshikuni, M. and Nagahama, Y. (1993)
Changes in the activity and protein levels of proteasomes during oocyte
maturation in goldfish (Carassius auratus). Biomed Res. 14, 305-308.
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- Tokumoto, T., Kajiura, H., Yoshikuni, M., Yamashita, M. and Nagahama,
Y. (1993) Purification of ubiquitin from goldfish (Carassius auratus)
oocyie cytosol. Biomed Res. 14, 309-312.
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- Iwao, Y., Sakamoto, N., Takahara, K., Yamashita, M. and Nagahama, Y.
(1993) The egg nucleus regulates the behavior of sperm nuclei as well
as cycling of MPF in physiologically polyspermic newt eggs. Develop.
Biol. 160, 15-27.
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- Katsu, Y., Yamashita, M., Kajiura, H. and Nagahama, Y. (1993) Behavior
of the components of maturation-promoting factor, cdc2 kinase and
cyclin B, during oocyie maturation of goldfish. Develop. Biol. 160,
99-107.
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- Kajiura, H., Yamashita, M., Katsu, Y. and Nagahama, Y. (1993) Isolation
and characterization of goldfish cdc2, a catalytic component of
maturationpromoting factor. Develop. Growth Differ. 35, 647-654.
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- Yamashita, M., Jiang Jianqiao, Onozato, H., Nakanishi T. and Nagahama,
Y. (1993) A tripolar spindle formed at meiosis I assures the retention
of the original ploidy in the gynogenetic triploid crusian carp,
ginbuna Carassius auratus langsdorfii. Develop. Growth Differ. 35,
631-636.
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- Onoe, S., Yamashita, M., Kajiura, H., Katsu, Y., Jiang, J. and Nagahama,
Y. (1993) A fish homolog of the cdc2-related protein p40^MO15: its cDNA
cloning and expression in oocytes. Biomed Res. 14, 441-444.
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- Iwamatsu, T., Toya, Y., Sakai, N., Terada, Y., Nagata, R. and Nagahama,
Y. (1993) Effect of 5-hydroxy-tryptamine on steroidogenesis and oocyte
maturation in preovulatory follicles of the medaka Oryzias latipes.
Develop. Growth Differ 35, 625-630.