Gender-typical olfactory regulation of sexual behavior in goldfish.

It is known that olfaction is essential for the occurrence of sexual behavior in male goldfish. Sex pheromones from ovulatory females elicit male sexual behavior, chasing, and sperm releasing act. In female goldfish, ovarian prostaglandin F2α (PGF) elicits female sexual behavior, egg releasing act. It has been considered that olfaction does not affect sexual behavior in female goldfish. In the present study, we re-examined the involvement of olfaction in sexual behavior of female goldfish. Olfaction was blocked in male and female goldfish by two methods: nasal occlusion (NO) which blocks the reception of olfactants, and olfactory tract section (OTX) which blocks transmission of olfactory information from the olfactory bulb to the telencephalon. Sexual behavior of goldfish was induced by administration of PGF to females, an established method for inducing goldfish sexual behavior in both sexes. Sexual behavior in males was suppressed by NO and OTX as previously reported because of lack of pheromone stimulation. In females, NO suppressed sexual behavior but OTX did not affect the occurrence of sexual behavior. Females treated with both NO and OTX performed sexual behavior normally. These results indicate that olfaction is essential in female goldfish to perform sexual behavior as in males but in a different manner. The lack of olfaction in males causes lack of pheromonal stimulation, resulting in no behavior elicited. Whereas the results of female experiments suggest that lack of olfaction in females causes strong inhibition of sexual behavior mediated by the olfactory pathway. Olfactory tract section is considered to block the pathway and remove this inhibition, resulting in the resumption of the behavior. By subtract sectioning of the olfactory tract, it was found that this inhibition was mediated by the medial olfactory tracts, not the lateral olfactory tracts. Thus, it is concluded that goldfish has gender-typical olfactory regulation for sexual behavior.

Sexual bipotentiality of behavior in male and female goldfish.

It is known that in goldfish Carassius auratus, a non-sex changing fish, prostaglandin (PG) treatment can induce female-typical sex behavior in males, and androgen treatment can induce male-typical sex behavior in females. These facts suggest that goldfish have a sexually bipotential brain even after attaining sexual maturity unlike mammals which have sexually differentiated brain. In the present study, in order to further characterize the brain function of goldfish, whether hormonal treatments which induce heterotypical sexual behavior suppress the occurrence of sex-typical behavior and whether sex-typical and heterotypical behavior can be induced in a relatively short time were examined. In the first series of experiments, male goldfish were shown to retain their ability to perform male-typical sex behavior within a week after being induced to perform female-typical behavior. Likewise, female goldfish were also shown to retain their female-typical sex behavior a week after being induced to perform male-typical behavior. In the second series of experiments, when PG-injected experimental males were placed with both PG-injected females and sexually mature males, the experimental males performed male- and female-typical behavior alternately with the females and the males, respectively during 90 min test period. When methyltestosterone-treated experimental females were injected with PG and placed with both PG-injected females and mature males, the experimental females performed male- and female-typical behavior alternately during 90 min test period. The results of the present study are consistent with the current knowledge that goldfish possess a sexually bipotential brain that can regulate both male and female-typical sex behaviors.

Histological observations of early gonadal development to form asymmetrically in the dwarf gourami Colisa lalia.

The asymmetrical and latitudinal formation of the gonads during early development in the dwarf gourami Colisa lalia was histologically observed using fish reared in laboratory. Hatching larvae obtained 33 hours after spawning possessed primordial germ cells (PGCs), which aggregated to the dorsal surface of the gut. In prolarvae on day 7, the gonadal anlagen were still situated on the dorsal surface of the gut, however, in those on day 10, the left gonadal anläge began to shift leftward, although the coelom did not develop sufficiently. In prolarvae on day 20, the right gonadal anlage also began to move leftward. During this period, the gut developed considerably in the right side of the coelom. On day 25, the developing gonads were located in the interspace of the developing spiral valve intestine. Sex differentiation of gonads appeared to begin by day 25. From those observations, we concluded that asymmetricity of gonads in C. lalia is caused by a limited space of the coelom opened on the left side of the body during the early phase of the formation of gonadal anlagen, which may be accurately determined by the position in which the gut develops.

Influences of establishment and maintenance of territory on reproductive activity in the male dwarf gourami Colisa lalia.

We examined the importance of establishment and maintenance of territory on reproductive activity in the male dwarf gourami, Colisa lalia. After three males had been forced to fight for territory (five sets) for three weeks, social status was divided into three classes: the territorially dominant male, which guarded the territory under the floating nest; the second male which remained near the nest and occasionally attacked the dominant male; and the third male which was non-aggressive and remained at a distance from the other two males. Comparing testicular size by gonadosomatic indices (GSI) after three weeks of aggression, GSI of the dominant male (1.19 ± 0.07) was significantly larger than that of the second (0.81 ± 0.15) and the third (0.62 ± 0.08) males, as well as the initial control (not involved in any experiments: 0.85 ± 0.10, n = 5), indicating that the testes of the dominant males enlarge during territory defense. Histological observations of testes revealed that sperm production in the dominant males was more active compared to males of other classes, although spermatogenesis was confirmed in all males examined, suggesting that dominance accelerates sperm production. Social-status dependent development of testes suggests an absence of sperm competition due to the lack of sneaking by subordinate males. Since non-territorial males do not engage in alternative tactics (e.g., sneaking) leading to emission of semen, male C. lalia must obtain and defend territory if they are to increase their reproductive success.

Characterization of an alternative chromatin remodeling to parasperm in a cottid fish, Hemilepidotus gilberti.

The dimorphic sperm of Hemilepidotus gilberti, i.e., haploid eusperm and diploid parasperm, have different morphologies corresponding to their own roles in fertilization. To estimate how these specific sperm morphologies were established, we focused on the nuclear morphologies and examined their changing processes in dimorphic spermiogenesis. Electron microscopic observation revealed that, in euspermatids, chromatin condensation first appeared as a mosaic pattern of moderate electrodense material in the peripheral region of the round nucleus. Those materials spread across the whole area to form a uniformly condensed nucleus. Chromatin condensation began similarly in paraspermatids to that in euspermatids. These became localized to one side of a nucleus and further condensed to form strong electrodense chromatin clusters, which are a specific feature of parasperm. From the remodeled nuclei of eusperm and parasperm, we found five and three kinds of sperm-specific basic proteins (SBPs), respectively, substituted to histones. The N-terminus amino acid sequences of the SBPs suggest that, in parasperm, one major SBP and two minor ones were distinct from each other. In eusperm nuclei, two kinds of specific SBPs were detected in addition to the homologs of parasperm SBPs. The specific SBPs had homologous amino acid sequences with huge arginine clusters, and one of them was most dominant among the five kinds of SBPs. The different combinations of SBPs in the eusperm and parasperm may cause a specific pattern of chromatin condensation in the dimorphic sperm nuclei of H. gilberti.

Production of recombinant Japanese eel gonadotropins by baculovirus in silkworm larvae.

Recombinant follicle-stimulating hormone (reFSH) and luteinizing hormone (reLH) of the Japanese eel Anguilla japonica were produced by baculovirus in silkworm Bombyx mori larvae. cDNAs encoding Japanese eel gonadotropin subunits (i.e., FSH beta, LH beta, and common alpha) were introduced into the baculovirus, which was infected into silkworm larvae after propagation of the recombinant virus in B. mori culture cells. A 100ml solution of pooled hemolymph from silkworm larvae containing reFSH or reLH were obtained from approximately 250 infected larvae. Ten milliliters of hemolymph were applied to Ni-affinity choromatography, and 5.6 and 3.5mg of partially purified reFSH and reLH were obtained, respectively. Using Western blot analysis concentrations of reFSH and reLH in the original hemolymph was estimated to be 2.2 and 1.1mg/ml, respectively. Biological activities of reFSH and reLH were assessed in vitro and in vivo. Purified reFSH and reLH induced eel oocyte maturation in vitro, and administration of hemolymph containing reFSH or reLH induced spermatogenesis in vivo in sexually immature Japanese eel. The present study indicates that a baculovirus-silkworm system could produce large amounts of biologically active recombinant fish gonadotropins for use in investigations in reproductive endocrinology and/or aquaculture of fish.

Seasonal changes in sex ratio, maturation, and size composition of fresh water snail, Sinotaia quadrata histrica, in Lake Kasumigaura.

Sinotaia quadrata histrica is a fresh water viviparous snail distributing from the Kanto region to Kyushu Island, Japan. About 7000 snails were collected in Lake Kasumigaura (L. Nishiura and L. Kitaura) in 2001 and 2002, and the sex ratio, maturity in terms of the gonad-somatic index (GSI) and operculum diameter were determined. The total female proportion was 55.2% in 2001, 53.0% in 2002 in L. Nishiura, and that of L. Kitaura was 60.4% in 2002. Comparing the season, the female proportion was the highest during early summer in both 2001 (59.6%, July in L. Nishiura) and 2002 (61.6%, June in L. Nishiura, 65.8%, July in L. Kitaura). The GSI of females in L. Nishiura significantly increased from April to May and significantly decreased from June to August. The GSI of males was higher in spring, but significantly lower from June to August. The mean female operculum diameter was consistently larger than that of males for each month and year, and a particularly significant difference was found between females and males from April to August 2001, and from April to September 2002. The number of resting zones on the operculum correlates with the operculum diameter and the female proportion was larger in the snails, which have a high number of resting zones, suggesting a sex-dependent difference in age composition. This study estimated that the sex ratio and seasonal maturation of S. quadrata histrica and the sex-dependent difference in age composition might contribute to the population structure in L. Kasumigaura.

Ultrastructural observations of eu- and paraspermiogenesis in the cottid fish Hemilepidotus gilberti (Teleostei: Scorpaeniformes: Cottidae).

The developmental process of eu- and paraspermatozoa in the cottid fish, Hemilepidotus gilberti, was observed by electron microscopy. Euspermatozoa of H. gilberti consist of a thin disk-like sperm head (about 3 microm in length), a short middle piece, and a long flagellum, but lack an acrosome. On the other hand, during spermiogenesis, aberrant spermatids, rich in cytoplasm and possessing binuclei, develop into cysts containing spermatids. The developing aberrant spermatids connect with normal spermatids and euspermatozoa by intercellular bridges. The early phase of chromatin condensation in aberrant spermatids is almost identical to that in normal spermatids, but the nuclei in the later phase develop into a mass of highly electron-dense globules. Since the aberrant spermatids complete karyokinesis but not cytokinesis at telophase of the second meiotic division, they are considered to develop into hyperpyrenic cells due to incomplete cytokinesis of the second meiotic division. These spermatids are oval in shape (5-7 microm in diameter) and lack a flagellum. The aberrant spermatids of H. gilberti are shed along with euspermatozoa and amount to about 50% of semen in volume. Judging from their form and developmental process, aberrant spermatids produced in H. gilberti are considered hyperpyrenic paraspermatozoa.