Akira Kambegawa

Plasma 17α,20β-dihydroxy-4-pregnen-3-one levels during sexual maturation of amago salmon (Oncorhynchus rhodurus): Correlation with plasma gonadotropin and in vitro production by ovarian follicles

Plasma gonadotropin (GtH) and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-diOH-prog) levels were assessed by radioimmunoassay during the sexual maturation of female amago salmon (Oncorhynchus rhodurus). Both GtH and 17 alpha, 20 beta-diOHprog levels were low in vitellogenic females (June to September) and in those with full-grown immature oocytes and were elevated in mature and ovulated females to 40 ng/ml for GtH and 50-70 ng/ml for 17 alpha, 20 beta-diOHprog. In vitro production of 17 alpha, 20 beta-diOHprog by ovarian follicles and its stimulation by partially purified chinook salmon gonadotropin (SG-G100) was examined monthly using 18 hr incubations. In June and July, levels were too low to detect (less than 30 pg/ml) in media from all treatment groups. In August and September, SG-G100 at a concentration of 1 microgram/ml stimulated low levels of production (0.2-0.3 ng/ml). Full-grown, immature follicles in October showed a dose-response production of 17 alpha, 20 beta-diOHprog, averaging over 10 ng/ml when incubated with 1 microgram/ml SG-G100. Postovulatory follicles (1-2 days after ovulation) produced large amounts of 17 alpha, 20 beta-diOHprog, averaging over 100 ng/ml with 1 microgram/ml SG-G100. These results are discussed in relation to previous work on amago salmon and other species and together indicate that 17 alpha, 20 beta-diOHprog is the major steroid responsible for oocyte maturation in amago salmon, produced as the follicular mediator of gonadotropin.

17α,20β-Dihydroxy-4-pregnen-3-one: Plasma levels during sexual maturation and in vitro production by the testes of amago salmon (Oncorhynchus rhodurus) and rainbow trout (Salmo gairdneri)

The present study investigated seasonal changes in plasma 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOHprog) levels in precociously mature and fully grown mature male amago salmon (Oncorhynchus rhodurus) and in vitro 17α,20β-diOHprog production by the testes of amago salmon and rainbow trout (Salmo gairdneri) in response to chum salmon gonadotropin. Plasma 17α,20β-diOHprog levels were very low from June to September, and rapidly increased in October at the beginning of the spawning season in both precociously mature and fully grown mature male amago salmon. High levels of 17α,20β-diOHprog were maintained during the period of active spermiation in precociously mature males, and quickly declined in mid-November. Incubation of testicular fragments from spermiating rainbow trout with chum salmon gonadotropin or 17α-hydroxyprogesterone resulted in a highly significant increase in 17α,20β-diOHprog levels in the incubation medium. The chum salmon gonadotropin was also effective in stimulating 17α,20β-diOHprog production by testicular fragments obtained from amago salmon after the breeding season. These findings are discussed in relation to the possible involvement of 17α,20β-diOHprog in spermiation of salmonids.

Both mercury and cadmium directly influence calcium homeostasis resulting from the suppression of scale bone cells: the scale is a good model for the evaluation of heavy metals in bone metabolism.

To examine the effects of heavy metals such as cadmium and mercury on calcium homeostasis, plasma calcium and calcitonin were measured in goldfish. Cadmium induced hypocalcemia both at 4 and at 8 days. In methylmercury-treated goldfish, the plasma calcium level increased at 2 days and then decreased at 8 days. The plasma calcitonin level increased in correspondence with the increased plasma calcium by methylmercury treatment, although cadmium did not cause a significant change. To elucidate the mechanism in detail, fish scales, which have both osteoclasts and osteoblasts and are similar to mammalian membrane bone, were used in the present study. We measured tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) activity as respective indicators of activity in both types of cells. TRAP activity in the scales decreased by treatment of cadmium and methylmercury at 6u2009h incubation. Particularly, cadmium (even at 10−13u2009M) significantly suppressed TRAP activity, suggesting that this system is utilized as an acute biosensor for cadmium. ALP activity decreased after exposures of 64 and 96u2009h, although the activity did not change after 6, 18, and 36u2009h. In addition, mRNA expression of the estrogen receptor and insulin-like growth factor 1, which participate in osteoblastic growth and differentiation, was less than the control values by treatment with both metals. This study demonstrates that mercury directly acts on the bone cells and influences calcium homeostasis and indicates that, in a short-term exposure, mercury has a different action from that of cadmium and induces hypercalcemia.

Circadian Changes in Serum Concentrations of Steroids in Japanese Char Salvelinus leucomaenis at the Stage of Final Maturation

Abstract Circadian changes in serum concentrations of testosterone (T), 11-ketotestosterone (11KT), estradiol-17β (E2), 17α,20β-dihydroxy-4-pregnen-3-one (DHP), 17α-hydroxyprogesterone (OHP), cortisol (F) and progesterone (P) were investigated in the spermiated/ovulated Japanese char Salvelinus leucomaenis for over three days using newly developed time-resolved fluoroimmunoassays. Testosterone and DHP in both sex and 11KT in male showed significantly (P<0.05) higher serum levels just before/after onset of darkness (15:00 or 18:00), and the levels during night and daytime were significantly (P<0.05) lower than those of the peak levels. Serum F levels in both sex during dark phase were significantly (P<0.05) higher than those levels during daytime. A surge of serum OHP concentrations in both sexes was observed at the time of twilight (03:00). The peak time of serum T, 11KT and DHP levels were approximately 6 hours prior to those of serum F and OHP levels. Serum E2 in female and P in both sex fluctuated intensely during sampling period, and did not show remarkable changes. These results strongly suggest the existence of circadian-like diel changes in serum T, DHP, F and OHP levels in both sex and 11KT in male, and no variations in serum E2 in female and P in both sex in spermiated/ovulated Japanese char under the stage of final maturation. Furthermore, relationship between circadian rhythms of steroid hormones and spawning behaviors are discussed in the present study.

Bisphenol A Influences the Plasma Calcium Level and Inhibits Calcitonin Secretion in Goldfish

Abstract In teleosts, it is well known that plasma calcium levels increase as a result of treatment with estrogen for at least during 2 weeks and that calcitonin secretion is induced by estrogen. The present study examined the influence of bisphenol A on calcium homeostasis in goldfish and compared the above known estrogenic action. In goldfish kept in water containing bisphenol A (10−6 M), the plasma calcium concentration increased significantly (P<0.001) at 4 days but decreased significantly (P<0.05) at 8 days. By the treatment of bisphenol A, calcitonin secretion was not induced until 4 days. At 8 days, however, plasma calcitonin, as well as calcium, decreased significantly (P<0.05), although vitellogenin was detected in the plasma. Therefore, bisphenol A influences plasma calcium levels, but its action is different from that of estrogen, which indicates that bisphenol A affects the calcium homeostasis and might bring about abnormal conditions in teleosts.

Sardine procalcitonin amino-terminal cleavage peptide has a different action from calcitonin and promotes osteoblastic activity in the scales of goldfish

The nucleotide sequence of a sardine preprocalcitonin precursor has been determined from their ultimobranchial glands in the present study. From our analysis of this sequence, we found that sardine procalcitonin was composed of procalcitonin amino-terminal cleavage peptide (N-proCT) (53 amino acids), CT (32 amino acids), and procalcitonin carboxyl-terminal cleavage peptide (C-proCT) (18 amino acids). As compared with C-proCT, N-proCT has been highly conserved among teleosts, reptiles, and birds, which suggests that N-proCT has some bioactivities. Therefore, both sardine N-proCT and sardine CT were synthesized, and their bioactivities for osteoblasts and osteoclasts were examined using our assay system with goldfish scales that consisted of osteoblasts and osteoclasts. As a result, sardine N-proCT (10-7M) activated osteoblastic marker enzyme activity, while sardine CT did not change. On the other hand, sardine CT (10-9 to 10-7M) suppressed osteoclastic marker enzyme activity, although sardine N-proCT did not influence enzyme activity. Furthermore, the mRNA expressions of osteoblastic markers such as type 1 collagen and osteocalcin were also promoted by sardine N-proCT (10-7M) treatment; however, sardine CT did not influence their expressions. The osteoblastic effects of N-proCT lack agreement. In the present study, we can evaluate exactly the action for osteoblasts because our scale assay system is very sensitive and it is a co-culture system for osteoblasts and osteoclasts with calcified bone matrix. Both CT and N-proCT seem to influence osteoblasts and osteoclasts and promote bone formation by different actions in teleosts.

Amino Acid Sequences of N-Terminal Procalcitonin of Some Vertebrates

In the present study, the complete amino acid sequence of N-terminal procalcitonins (N-proCTs) of stingray (an elasmobranch) and spotlined sardine (a teleost), as well as the partial amino acid sequence of N-proCTs of goldfish (a teleost; three types of N-proCTs) and bullfrog (an amphibian) have been determined. These N-proCTs have some common features to 9 N-proCTs sequenced to date. These features were summarized as follows: 1) The first amino acid residue is Ala or Val. 2) There is no Cys residue in the sequence of N-proCTs. 3) Seven residues (Pro 3 , Leu 19 , Leu 28 , Val 32 , Gln 37 , Gln 45 and Ser 56 ) are conserved in the sequence of N-terminal procalcitonin (N-proCT), at least judging from the present data. 4) Insertion/deletion points are present in the sequence of N-proCTs at the 2nd position, from the 10th to 14th and from 46th to 52nd. We examined whether the N-proCTs can be categorized into several groups by similarity of amino acid residues conserved in the molecules. As a result, the N-proCTs were roughly categorized into 2 groups: a mammalian group, including 2 subgroups and a non-mammalian group, including 3 subgroups. This classification of N-proCTs coincides with that of calcitonin. Sardine N-proCT has no accelerating effects on mitosis and cell differentiation of human osteosarcoma cells, as does the homologous human N-proCT. Further studies are needed to elucidate the biological activity of N-proCTs.

α-Melanocyte-stimulating hormone directly increases the plasma calcitonin level and involves calcium metabolism in goldfish

The effects of α-melanocyte-stimulating hormone (α-MSH) on calcium metabolism were examined with goldfish. The scales on the left side of goldfish bodies were removed to allow the regeneration of scales under anesthesia. Thereafter, the influences of α-MSH injection (low dose: 0.1xa0μg/g body weight; high dose: 1xa0μg/g body weight) on plasma calcitonin (calcium-regulating hormone) and the calcium content of the scales were investigated. Ten days after removing the scales, we measured the plasma calcitonin and calcium content of both regenerating scales on the left side and ontogenic scales on the right side. At both doses of α-MSH injection, plasma calcitonin concentrations in the α-MSH-treated group were significantly higher than those in the control group. The mRNA expressions of α-MSH-receptors were detected in the ultimobranchial glands (secretory organ of calcitonin), indicating that α-MSH directly functions in ultimobranchial glands and promotes calcitonin secretion. Furthermore, we found that the calcium content of regenerating scales in α-MSH-treated goldfish was higher than that in control goldfish, while the calcium content of ontogenic scales on the right side was significantly decreased by α-MSH injection. There was a significant co-relationship between plasma calcitonin and the calcium content of regenerating scales. The mRNA expression of calcitonin receptors in regenerating scales was remarkably higher than that in ontogenic scales. These results imply that calcitonin functions to promote scale regeneration resulting from the inhibition of bone resorption because calcitonin suppresses osteoclastic activity. Thus, we are the first to demonstrate the interaction between α-MSH and calcitonin in teleosts.