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)

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Nagahama, Y. (1993) Regulation of oocyte maturation in aquatic animals: the comparative and general aspects. Biology International Special Issue 28, 27-32.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.