The presence of sex steroids and their receptors has been demonstrated in all vertebrate groups from Agnatha to Mammalia but not in invertebrates. In genomic analyses of urochordates, cytochrome P450 (CYP) genes important for biosynthesis of sex steroids are absent. In the present study, we confirmed the presence of estrogen, androgen, and progesterone by using radioimmunoassay in gonads of amphioxus, Branchiostoma belcheri, which is considered to be evolutionarily closer to vertebrates than other invertebrates. Furthermore, CYP genes encoding CYP11A, CYP17, and CYP19 and transcripts for 17beta-hydroxysteroid dehydrogenase were cloned from amphioxus ovaries. Among invertebrates, the presence of hydroxysteroid dehydrogenase enzymes and metabolized steroids was shown in paracytic Taenia and corals. However, CYPs metabolizing sex steroids have not been demonstrated in invertebrates, nor has an attempt been made to consider the entire pathway from cholesterol to estrogen. This study is the first evidence to suggest the presence of CYP enzymes in sex steroid production in invertebrates.

Sex steroids are essential hormones for reproduction in vertebrates. The existence of a sex steroidogenic pathway in invertebrates is controversial because cytochrome P450 (CYP) genes have not been detected in the genomes of an echinoderm and a urochordate. However, cloning and gene expressions of sex steroid-metabolizing enzymes have been reported in a cephalochordate. In this study, in vitro conversion of sex steroids from pregnenolone (P5) to progesterone (P4), from 17alpha-hydroxyprogesterone (17alpha-P4) to 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha, 20beta-P) and 17alpha, 20alpha-P, and from androstenedione (AD) to estrone (E1), estradiol-17beta (E2), and testosterone (T) were confirmed by an incubation experiment performed using (14)C-labeled precursors and mature ovarian homogenates of the amphioxus Branchiostoma belcheri. In amphioxus, the ability of immature ovaries to synthesize sex steroids was much lower than that of mature ovaries. Post-spawning, the mRNA of CYP11A significantly decreased in females. Transcripts of the CYP19 gene also declined in one-fourth of the females after spawning, although this trend was not supported statistically. The mRNAs of CYP17 and 17beta-HSD did not change before and after spawning. Our results indicate the presence of Delta(4) and another derivative pathway in the amphioxus ovary and suggest that the synthesis of sex steroids, particularly estrogen synthesis, may be low in females after spawning behavior.

We report two cases of envenomation by a Madagascan opisthoglyphous snake, Ithycyphus miniatus. In both cases, the snake bit the finger of a human who was preparing an experiment by tying a string around the snake body. Symptoms of the first case included temporal severe local pain and extensive bleeding. In the second case, severe pain accompanying obvious local swelling was caused and lasted for several hours. The present observations indicate that bite by I. miniatus potentially causes serious physiological effects in humans although the snake is basically calm and reluctant to bite

Adult individuals of amphioxus (Branchiostoma belcheri) were collected by dredging from a research vessel at selected stations in two areas off the coast of Japan in July 2000: Deyama and Takamatsu so named by local fishermen in the Enshu Nada Sea. The number of males collected exceeded that of females at all the stations in Takamatsu and at four of five stations in Deyama. The over all sex ratio (males : females) of the collected animals was 1.2 : 1. The animals showed various maturational stages of the gonad, and approximately 70% had mature gonads. However, post-spawning animals were identified only at two stations in Takamatsu. Mature animals were placed in laboratory tanks. These animals remained in good conditions for about two months, and many animals spontaneously spawned in the tanks. This is the first report of spontaneous spawning of B. belcheri in Japan.

No abstract

The formation of an acrosomal process at acrosomal exocytosis in spermatozoa of the amphioxus was described in the present report for the first time. A non-reacted acrosome was located in front of the nucleus, where a cup-shaped acrosomal vesicle covered a conical accumulation of subacrosomal material. When naturally spawned spermatozoa were treated with a calcium ionophore, ionomycin, the acrosomal vesicle opened at the apex and an acrosomal process was projected. The process exhibited a filamentous structure. The reaction followed the mode typically seen in marine invertebrates. These observations suggest that the features and function of the acrosome of amphioxus, whose position is on the border between invertebrates and vertebrates, reflect their ecological adaptation and phylogenic position.