National Insitute for Basic Biology

LABORATORY OF REPRODUCTIVE BIOLOGY

Professor:
Yoshitaka Nagahama
Research Associates:
Michiyasu Yoshikuni
Minoru Tanaka
Tohru Kobayashi
JSPS Post-doctoral Fellows:
Takeshi Miura
Graduate Students:
Chiemi Miura
Yoshinao Katsu
Shinji Onoe
Daisuke Kobayashi
Xiao-Tian Chang
Yuichi Ooba
Institute Research Fellows:
Akihiko Yamaguchi
Toshinobu Tokumoto
Visiting Scientists:
Mishiya Matsuyama1)
Jian-quiao Jiang2)
Wei Ge3)
Technical Staffs:
Hiroko Kobayashi
Sachiko Fukada
1)from Kyushu University
2)from Wuhan University
3)from University of Alberta

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-dihy-droxy-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 oocyie 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-hy-droxysteroid dehydrogenase (3B-HSD), 17a-hydroxylase/C17,20-lyase cytochrome P450 (P450cl7), P450 aromatase (P450arom) and 20B-hydroxysteroid dehydrogenase (20B-HSD). We also isolated the structural gene encoding P450arom, for the first time from a non-mammalian vertebrate, the medaka. The medaka P450arom gene consisted of nine exons, but spans only 2.6 kb, being much smaller than the human P450arom gene (at least 70 kb), as the result of extremely small introns. Genomic Southern blots revealed the presence of a single medaka gene. Promoter analyses indicated two major transcription initiation sites 60 and 61 bp upstream from a putative initiation codon. The promoter region of medaka P450arom gene contain Ad4BP sites and estrogen responsive element half-sites.

The cDNA clones obtained 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 increases in the amount of P450scc, 3B-HSD and P450c17 transcripts provide an explanation for the dramatic increase in 17a,20B-DP production in follicles during naturally- and gonadotropin-induced oocyte maturation. In contrast, levels of mRNA for P450arom were high during oocyte growth, but rapidly decreased during oocyte maturation. This decrease in P450arom mRNA levels appears to be correlated with the decreased ability of maturing follicles to produce estradiol-17B (Fig. 1).

Fig.1

Fig.1
Whole mount in situhybridization showing P450 aromatase mRNA in medaka ovarian follicles.

Fig.2

Fig.2
Molecular mechanisms of 17 a ,20 B -dihydroxy-4-pregnen-3one induced MPF activation in goldfish oocytes.

17a,20B-DP acts via a receptor on the plasma membrane of oocytes. We have identified and characterized a specific 17a,20B-DP receptor from defolliculated oocytes of several fish species. Scatchard analysis reveled two different receptors: a high affinity with a Kd of 18 nM and a Bmax of 0.2 pmoles/mg protein, and a low affinity receptor with a Kd of 0.5 µM and a Bmax of 1 pmole/mg protein. 17a,20B-DP receptor concentrations increase during oocyte maturation. The interaction between 17 a ,20 B -DP receptors and G-proteins was examined. Pertussis toxin (PT) catalyzed the ADP ribosylation of a 40 kDa protein in crude membranes from rainbow trout oocyies. The 40 kDa protein was recognized by an antibody against a subunit of inhibitory G-protein. Treating the membrane fraction with 17 a ,20 B -DP decreased the PT-catalyzed ADP ribosylation of the 40 kDa protein. The specific binding of 17 a ,20 B -DP was decreased by PT. We conclude that the PT-sensitive Gi is involved in the signal transduction pathway of 17 a ,20 B -DP in fish oocytes.

The early steps following 17 a ,20 B -DP action involve the formation of the major mediator of this steroid, maturation-promoting factor or metaphase-promoting factor (MPF). MPF activity cycles during 17 a ,20 B -DP-induced oocyte 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, catalytic cdc2 kinase (34 kDa) and regulatory cyclin B (46- to 48 kDa). Immature goldfish oocytes contain only monomeric 35 kDa cdc2 and do not stockpile cyclin B, although immature oocytes contain mRNA for cyclin B. In maturing oocytes, activation of cdc2 is associated with its phophorylation of threonine 161 (Thr161) after binding to cyclin B, producing 34 kDa cdc2. Using mutant cdc2, we showed that Thr161 phosphorylation is required for both the down-ward shift of cdc2 (35- to 34 kDa) and the kinase activation. Since thyrosine 15 of cdc2 is not phosphorylated after binding to cyclin B, it does not require dephosphorylation. This situation is obviously different from that in immature Xenopus oocytes, in which the cdc2-cyclin B complex preexists with cdc2 phosphorylated on both Tyr15 and Thr161, thereby requiring Tyr15 dephosphorylation catalyzed by cdc25 phosphatase for MPF activation. Recently we have isolated a cDNA clone encoding a goldfish homolog of p40M015 the catalytic subunit of a protein kinase which activates cdc2 kinase through phosphorylation of Thr161, from a goldfish oocyte cDNA library. Northern and Western blot analyses revealed that both p40M015 mRNA and protein are already present in goldfish immature oocytes and do not exhibit any changes during hormonally induced maturation.

Immediately prior to the transition from metaphase to anaphase, M-phase-promoting factor (MPF) is inactivated by degradation of cyclin B. We investigated the role of proteasomes (a nonlysosomal large protease) in cyclin degradation, using Eschelicia coli-produced goldfish cyclin B and purified goldfish proteasomes (20S and 26S). The purified 26S proteasome, but not 20S proteasome, cleaved both monomeric and cdc2-bound cyclin B at lysine 57 (K57) restrictively in vitro, and produced a 42 kDa N-terminal truncated cyclin B, which was transiently detected at the initial phase of the normal egg activation. The 42 kDa cyclin B, as well as full-length one, was degraded in Xenopus egg extracts, but a mutation on K57 (K57R, the 26S proteasome-catalyzed digestion site K57 was substituted with arginine) inhibited both the digestion by 26S proteasome and the degradation in Xenopus egg extracts. These findings strongly suggest the involvement of 26S proteasome in cyclin degradation through the first cleave on its N-terminus.

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 17 a ,20 B -DP for sperm maturation). A steroidogenic switch, from 11-ketotestosterone to 17 a ,20 B -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 17 a ,20 B -DP production is in the sperm, but its production depends on the provision of precursor steroids by somatic cells. The site of 1 1-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.

We have used subtractive hybridization to identify genes that are expressed differentially in eel testes in the first 24 hr after HCG treatment in vivo, which ultimately induces spermatogenesis. One up-regulated cDNA was isolated from subtractive cDNA libraries derived from mRNA extracted from control testes and testes one day after a single injection of HCG. From its deduced amino acid seqence, this clone was identified as coding for the activin BB subunit. Using Northern blot analysis and in situ hybridization techniques, we examined sequential changes in transcripts of testicular activin BB during HCG-induced spermatogenesis. No transcripts for activin BB 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, followed by a further sharp decrease by nine days. The HCG-dependent activin BB mRNA expression in the testes was confirmed by in situ hybridization using a digoxigenin-labelled RNA probe: the signal was restricted to Sertoli cells in testes treated with HCG for one to three days. A marked stimulation of activin B production, but not either activin A or activin AB, was observed in testes after HCG and 11-ketotestosterone treatment. Addition of recombinant human activin B induced spermatogonial proliferation in vitro. Taken together, these findings suggest the following sequence of the hormonal induction of spermatogenesis in the eel. Gonadotropin stimulates the Leydig cells to produce 11-ketotestosterone, which, in turn, activates the Sertoli cells to produce activin B. Activin B then acts on spermatogonia to induce mitosis leading to the formation of spermatocytes.

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 17 a ,20 B -DP is responsible for the acquisition of sperm motility; 17 a ,20 B -DP acts to increase sperm duct pH, which in turn increases the cAMP content of sperm allowing the acquisition of motility.

Publication List:

Fukada, S., Sakai, N., Adachi, S. and Nagahama, Y. (1994) Steroidogenesis in the ovarian follicle of medaka (Oryzias latipes, a daily spawner) during oocyte maturation. Develop. Growth Differ. 36, 81-88.

Furukawa, K., Inagaki, H., Naruge, T., Tabata, S., Tomida, T., Yamaguchi, A., Yoshikuni, M., Nagahama, Y. and Hotta, Y. (1994) cDNA cloning and functional characterization of a meiosis-specific protein (NMS1) with apparent nuclear association. Chromosome Res. 2, 99-113.

lwamatsu, T., Nakashima, S., Onitake, K., Matsuhisa, A. and Nagahama, Y. (1994) Regional differences in granulosa cells of preovulatory medaka follicles. Zool. Sci. 11, 77-82.

Matsuyama, M., Fukada, T., Ikeura, S., Nagahama, Y. and Matsuura, S. (1994) Spawning characteristics and steroid hormone profiles in the wild female Japanese sardine Sardinops melanostictus. Fish. Sci. 60, 703-706.

Mita, M., Yoshikuni, M. and Nagahama, Y. (1994) G-proteins and adenylate cyclase in ovarian granulosa cells of amago salmon ( Oncorhynchus rhodurus). Mol. Cell. Endocrinol. 105, 83-88.

Miura, T., Kobayashi, T. and Nagahama, Y. (1994) Hormonal regulation of spermatogenesis in the Japanese eel (Anguilla japonica). Perspecitive in Comparative Endocrinology, pp. 631-635.

Nagahama, Y. (1994) Molecular biology of oocyte maturation in fish. Perspective in Comparative Endocrinology, 193-198.

Nagahama, Y. (1994) The onset of spermatogenesis in fish. In Germline Development, Ciba Foundation Symposium No. 182, 255-270.

Nagahama, Y. (1994) Endocrine regulation of gametogenesis in fish. Int. J Develop. Biol. 38, 217-229.

Nagahama, Y., Yamashita, M., Tokumoto, T. and Katsu, Y. (1994) Regulation of oocyie maturation in fish. Current Topics in Developmental Biology, 30.

Nagahama, Y., Yoshikuni, M., Yamashita, M. and Nagahama, Y. (1994) Regulation of oocyte maturation in fish. In Molecular Endocrinology of Fish, Fish Physiology XIII (N.M. Sherwood and C.L. Hew, eds.), Academic Press, New York, pp.393-439.

Nakamura, M., Mariko, T. and Nagahama, Y. (1994) Ultrastructure and in vitro steroidogenesis of the gonads in the protandrous anemonefish Amphiprion frenatus. Japan. J. Ichthyol. 41, 47-56.

Sakai, N., Tanaka, M.,Takahashi, M., Fukada, S., Mason, J.I. and Nagahama, Y. (1994) Ovarian 3B-hydroxysteroid dehydrogenase/Æ5-4-isomerase of rainbow trout: its cDNA cloning and properties of the enzyme expressed in a mammalian cell. FEBS Lett. 350, 309-313.

Yamashita, M., Kajiura, H., Tanaka, T., Onoe, S. and Nagahama, Y. (1994) Molecular mechanisms of the activation of maturation-promoting factor during goldfish oocyie maturation. Develop. Biol. 166 (in press).

Yoshikuni, M. and Nagahama, Y. (1994) Involvement of an inhibitory G-protein in the signal transduction pathway of maturation-inducing hormone (17 a ,20 B -dihydroxy-4-pregnen-3-0ne) action in rainbow trout (Oncorhynchus rhodurus) oocyies. Develop. Biol. 166, 615-622.

Yoshikuni, M., Matsushita, H., Shibata, N. and Nagahama, Y. (1994) Purification and characterization of 17 a ,20 B -dihydroxy-4-pregnen-3-0ne-binding protein from plasma of rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 96, 189-196.