Progesterone initiates tendril formation in the oviducal gland during egg encapsulation in cloudy catshark (Scyliorhinus torazame).

The diverse reproductive strategies of elasmobranchs (sharks, rays, and skates) have attracted research attention, but the endocrine control of reproduction is still incompletely known in elasmobranchs. By long-term monitoring of the egg-laying cycle in cloudy catsharks (Scyliorhinus torazame), we recently demonstrated a transient increase in plasma progesterone (P4) levels just prior to the appearance of the capsulated eggs in the oviducts. In the present study, we examined the in vivo effects of P4 administration in mature female cloudy catsharks. Although no capsulated eggs were observed following the implantation of P4-containing silicone tubing, we did find dark swollen oviducts in the abdominal cavity, in which clumps of long and coiled tendrils were observed. The tendril is an extension of the egg capsule, and the formation of the egg capsule begins with the tendril before main capsule formation. During the period of P4 implantation, the tendrils elongated, and their diameters were significantly increased on day 2 of treatment. Tendril formation was also confirmed on the day of endogenous P4 surge. Tendrils were not formed in catsharks implanted with estradiol-17ß or testosterone. Histological analysis of the oviducal gland revealed that P4 implantation induced the secretion of tendril materials from the secretory tubules in the baffle zone, while the tendril materials were stored in the cytoplasm of the secretory cells under low P4 condition. Morphometrically, the area of secreted luminal materials in the secretory tubules was highly correlated to the diameter of tendrils. Our results suggest that the P4 surge during the egg-laying cycle serves as a trigger for egg capsule formation in the oviducal gland of cloudy catshark, but the hormonal signals were incomplete as the main capsule was not formed. Further studies are required to identify the hormones required for ovulation and formation of the main egg capsule.

Ingestion of rubber tips of artificial turf fields by goldfish.

Marine microplastics are one of the global environmental issues. The present study examined whether rubber tips of artificial sports fields could be marine microplastics. We observed the migration of rubber tips from the artificial turf field to the surrounding ditch connected to sewer pipes and then examined the ingestion of rubber tips using the goldfish Carassius auratus. The rubber tips found in sediments in the ditch suggest that the rubber tips could be sent to the river and released into the ocean. The goldfish ingested rubber tips with or without fish feed, and rubber tips were found in the intestine. However, the fish discharged the rubber tips within 48 h after ingestion. These results indicate that ingestion of the rubber tips was not accidental but an active behavior. Therefore, artificial turf sports fields could be a source of marine microplastics and may cause hazardous effects on wild fishes through ingestion.

Evolutionally Conserved Function of Kisspeptin Neuronal System Is Nonreproductive Regulation as Revealed by Nonmammalian Study.

The kisspeptin neuronal system, which consists of a neuropeptide kisspeptin and its receptor Gpr54, is considered in mammals a key factor of reproductive regulation, the so-called hypothalamic-pituitary-gonadal (HPG) axis. However, in nonmammalian vertebrates, especially in teleosts, existence of kisspeptin regulation on the HPG axis is still controversial. In this study, we applied multidisciplinary techniques to a teleost fish, medaka, and examined possible kisspeptin regulation on the HPG axis. First, we generated knockout medaka for kisspeptin-related genes and found that they show normal fertility, gonadal maturation, and expression of gonadotropins. Moreover, the firing activity of GnRH1 neurons recorded by the patch clamp technique was not altered by kisspeptin application. Furthermore, in goldfish, in vivo kisspeptin administration did not show any positive effect on HPG axis regulation. However, as kisspeptin genes are completely conserved among vertebrates except birds, we surmised that kisspeptin should have some important nonreproductive functions in vertebrates. Therefore, to discover novel functions of kisspeptin, we generated a gpr54-1:enhanced green fluorescent protein (EGFP) transgenic medaka, whose gpr54-1-expressing cells are specifically labeled by EGFP. Analysis of neuronal projection of gpr54-1:EGFP-expressing neurons showed that these neurons in the ventrolateral preoptic area project to the pituitary and are probably involved in endocrine regulation other than gonadotropin release. Furthermore, combination of deep sequencing, histological, and electrophysiological analyses revealed various novel neural systems that are under control of kisspeptin neurons-that is, those expressing neuropeptide Yb, cholecystokinin, isotocin, vasotocin, and neuropeptide B. Thus, our new strategy to genetically label receptor-expressing neurons gives insights into various kisspeptin-dependent neuronal systems that may be conserved in vertebrates.

Subunit profiling and functional characteristics of acetylcholine receptors in GT1-7 cells.

GnRH neurons form a final common pathway for the central regulation of reproduction. Although the involvement of acetylcholine in GnRH secretion has been reported, direct effects of acetylcholine and expression profiles of acetylcholine receptors (AChRs) still remain to be studied. Using immortalized GnRH neurons (GT1-7 cells), we analyzed molecular expression and functionality of AChRs. Expression of the mRNAs were identified in the order α7 > ß2 = ß1 â‰§ α4 â‰§ α5 = ß4 = Î´ > α3 for nicotinic acetylcholine receptor (nAChR) subunits and m4 > m2 for muscarinic acetylcholine receptor (mAChR) subtypes. Furthermore, this study revealed that α7 nAChRs contributed to Ca2+ influx and GnRH release and that m2 and m4 mAChRs inhibited forskolin-induced cAMP production and isobutylmethylxanthine-induced GnRH secretion. These findings demonstrate the molecular profiles of AChRs, which directly contribute to GnRH secretion in GT1-7 cells, and provide one possible regulatory action of acetylcholine in GnRH neurons.

Effect of sex steroid hormones on the number of serotonergic neurons in rat dorsal raphe nucleus.

Disorders caused by the malfunction of the serotonergic system in the central nervous system show sex-specific prevalence. Many studies have reported a relationship between sex steroid hormones and the brain serotonergic system; however, the interaction between sex steroid hormones and the number of brain neurons expressing serotonin has not yet been elucidated. In the present study, we determined whether sex steroid hormones altered the number of serotonergic neurons in the dorsal raphe nucleus (DR) of adult rat brains. Animals were divided into five groups: ovariectomized (OVX), OVX+low estradiol (E2), OVX+high E2, castrated males, and intact males. Antibodies against 5-hydroxytryptamine (5-HT, serotonin) and tryptophan hydroxylase (Tph), an enzyme for 5-HT synthesis, were used as markers of 5-HT neurons, and the number of 5-HT-immunoreactive (ir) or Tph-ir cells was counted. We detected no significant differences in the number of 5-HT-ir or Tph-ir cells in the DR among the five groups. By contrast, the intensity of 5-HT-ir showed significant sex differences in specific subregions of the DR independent of sex steroid levels, suggesting that the manipulation of sex steroid hormones after maturation does not affect the number and intensive immunostaining of serotonergic neurons in rat brain. Our results suggest that, the sexual dimorphism observed in the serotonergic system is due to factors such as 5-HT synthesis, transportation, and degradation but not to the number of serotonergic neurons.

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.

Production and characterization of recombinant Manchurian trout thyrotropin.

Thyrotropin (thyroid-stimulating hormone, TSH), a heterodimeric glycoprotein hormone produced in the pituitary, stimulates the thyroid gland and release of thyroid hormones. In contrast to a well-known efficacy of recombinant mammalian TSHs, there is no report about the production of teleost recombinant TSH and its biological activity. In this study, we report the production of a single-chain recombinant TSH (mtTSH) of Manchurian trout (Brachymystax lenok), by baculovirus in silkworm (Bombyx mori) larvae. The mtTSH was produced in silkworm larvae and characterized as a form of N-linked glycosylation. The cAMP signaling system in transiently transfected COS-7 cells revealed that the mtTSH was recognized by their cognate receptors, salmon TSHα and TSHß receptors, but not LH receptor. The thyrotropic potency of the mtTSH was examined by rainbow trout basibranchial tissues containing thyroid follicles. The height of follicle epithelial cells was significantly increased by treatments of mtTSH in vivo and in vitro. In conclusion, the present study suggests that the mtTSH produced by baculovirus-silkworm larvae is a biologically active recombinant TSH.

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.

Experimental and computational study of inter- and intra- species specificity of gonadotropins for various gonadotropin receptors.

The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and their receptors play critical roles in vertebrate reproduction. In order to study intra- and interspecies ligand promiscuity of gonadotropins, COS-7 cells were transiently transfected with one of the gonadotropin receptor genes, FSHR or LHR, and tested for activation by gonadotropins from representative fish orders: Aquilliformes (eel; e), Salmoniformes (trout; tr), and Perciformes (tilapia; ta), and of mammalian origin: porcine (p), bovine (b) and human (h). The study reveals complex relations between the gonadotropin hormones and their receptors. Each gonadotropin activated its own cognate receptor. However, taLHR was also activated by hCG and eLHR was activated by hFSH, hCG, and trFSH. For FSHR, the only cross-reactivity detected was for hFSHR, which was activated by pFSH and bFSH. These findings are of great interest and applicability in the context of activation of various GTHRs by their ligands and by ligands from other vertebrates. Analysis of the three-dimensional models of the structures highlights the importance of residues outside of the currently established hormone-receptor interface region. In addition, the interface residues in taFSHR and the effect of exon duplication, which causes an insert in the LRR domain, are suggested to affect the interaction and binding of taFSH.

Steroidogenic response of carp ovaries to piscine FSH and LH depends on the reproductive phase.

The gonadotropins (GTHs) follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are the key regulators of reproduction. We determined the competence of heterologous recombinant GTHs at eliciting steroid secretion from carp ovaries at different reproductive stages. We collected carp ovaries at: early, mid and end vitellogenesis, when most of the oocytes still contained a germinal vesicle (GV) at a central stage, and mature ovaries with a migrating GV. Plasma estradiol (E2) levels at early vitellogenesis were high and decreased thereafter. Basal secretion levels of E2 increased with oocyte diameter and GSI value, whereas 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) was detected only in females with mature follicles. Carp ovary fragments were exposed to recombinant fish GTHs belonging to different teleost orders: Japanese eel (Anguilla japonica, Anguilliformes), Manchurian trout (Brachymystax lenok, Salmoniformes), and Nile tilapia (Oreochromis niloticus); to mammalian GTHs (pFSH and hCG), or to carp and tilapia pituitary extract (CPE and TPE, respectively). All of the recombinant GTHs tested stimulated steroid secretion. However, the steroid secretion differed according to the type of GTH and the developmental state of the ovary. CPE increased the secretion of both E2 and DHP at almost all stages of ovarian maturity. In mature ovarian fragments, DHP secretion was higher in response to recombinant LHs (eel and tilapia) than to recombinant FSH. Early- and mid-vitellogenic ovaries showed no secretion of DHP and high secretion of E2 in response to all recombinant GTHs tested. This is in line with the hypothesis that LH regulates the final stages of maturation, when the involvement of FSH is marginal. These results may contribute to understanding the mechanisms that determine differential activation of steroid secretion and specificity in fish.

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.

Immunohistochemical Localization of the Water Channels AQP4 and AQP5 in the Rat Pituitary Gland.

The pituitary gland is composed of the adenohypophysis and neurohypophysis. The adenohypophysis contains endocrine cells, folliculo-stellate (FS) cells, and marginal layer cells, whereas the neurohypophysis mainly comprises axons and pituicytes. To understand the molecular nature of water transfer in the pituitary gland, we examined the immunohistochemical localization of the membrane water channels aquaporin-4 (AQP4) and AQP5 in rat tissue. Double immunofluorescence analysis of AQP4 and S100 protein, a known marker for FS cells, marginal layer cells, and pituicytes, clearly revealed that FS cells and marginal layer cells in the adenohypophysis and the pituicytes in pars nervosa are positive for AQP4. AQP5 was found to be localized at the apical membrane in some marginal layer cells surrounding the Rathke's residual pouch, in which AQP4 was observed to be localized on the basolateral membranes. These results suggest the following possibilities: 1) FS cells especially require water for their functions and 2) transepithelial water transfer could occur between the lumen of Rathke's residual pouch and the interstitial fluid in the adenohypophysis through the AQP4 and AQP5 channels in the marginal layer cells.

Expression of recombinant zebrafish follicle-stimulating hormone (FSH) in methylotropic yeast Pichia pastoris.

Pituitary gonadotropin follicle-stimulating hormone (FSH) was identified in fish two decades ago, but its functional importance in fish reproduction remains poorly defined, especially in non-salmonid species. This gap in our knowledge is partially due to the lack of the hormone in pure form in most of the species studied. We describe here the production of two different forms of biologically active recombinant zebrafish FSH (zfFSH and zfFSH(HIS)) using methylotrophic yeast, Pichia pastoris, as the bioreactor. One form (zfFSH) was produced as the molecule closer to the native form, with the two subunits (Cga and Fshb) expressed separately under different promoters. The other form (zfFSH(HIS)) was produced as a single polypeptide, with the cDNAs for the two subunits joined to form a fusion gene that contained a 6X His tag as part of the linker between the two subunits. The culture conditions were optimized for pH and incubation time for maximal production of the proteins. Using a zebrafish FSH receptor (Fshr)-based reporter gene assay, we tested and compared the biological activities of the two forms of recombinant zebrafish FSH. Our results provide useful information for the future production of recombinant gonadotropins in other fish species.

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.

Endocrine control of sexual behavior in teleost fish.

Sexual behavior is one of the most profound events during the life cycle of animals that reproduce sexually. After completion of gonadal development that is mediated by various hormones, oviparous teleosts perform a suite of behaviors, often termed as spawning behavior. This is particularly important for teleosts that have their gametes fertilized externally as the behavior patterns ensures the close proximity of both sexes for gamete release, fusion and ultimately the production of offspring. As in other vertebrates, sexual behavior of fish is also under the control of hormones. Testicular androgen is a requirement for male sexual behavior to occur in most fish species that have been studied. Unlike tetrapods, however, ovarian estrogen does not appear to be essential for the occurrence of female sexual behavior for fish that have their gametes fertilized externally. Prostaglandins produced in the ovary after ovulation act as a trigger in some teleosts to induce female sexual behavior. Potentiating effects of gonadotropin-releasing hormone in the brain on sexual behavior are reported in some species. Under endocrine regulation, male and female fish exhibit gender-typical behavior during spawning, but in some fish species there is also some plasticity in their sexual behavior. Sex changing fish can perform both male-typical and female-typical sexual behaviors during their lifetime and this sexual plasticity can also be observed in non-sex changing fish when undergoing hormonal treatment. Although the neuroanatomical basis is not clear in fish, results of field and laboratory observations suggest that some teleosts possess a sexually bipotential brain which can regulate two types of behaviors unlike most other vertebrates which have a discrete sex differentiation of their brain and can only perform gender-typical sexual behavior.

Effects of androgens on the development of nuptial coloration and chromatophores in the bitterling Rhodeus ocellatus ocellatus.

Sexually mature male bitterlings, Rhodeus ocellatus ocellatus, exhibit distinct nuptial color, whereas females maintain a body color similar to that of juveniles. In the present study, body color and chromatophores were compared between male and female bitterlings, and the effects of androgens on body color and chromatophore densities were examined in females to clarify the role of androgen in the development of nuptial coloration and chromatophores. Males showed green, blue, and red color in specific regions of their skin and red color on the dorsal and caudal fins; females showed a subdued silver body color. For chromatophores, small greenish-type iridophores were observed in the green color region in the skin of males, whereas females had large spindle-shaped silvery-type iridophores in corresponding regions. Many erythrophores were observed in males in blue and red color regions in the skin and red color regions in the fins, but females possessed xanthophores in corresponding regions. The melanophore density of the skin was not different between males and females, but the distribution of melanophores in the fins was different between them. Treatment with 11-ketotestosterone or methyltestosterone induced male-type nuptial coloration in the female skin and fins. The distribution of chromatophores in androgen-treated females was similar to that in sexually mature males: an increase in the number of greenish-type iridophores and erythrophores was also observed in the skin. These results indicate that androgen induces male-type nuptial coloration in the bitterling and that the responses of chromatophores to androgen differ with the type and distribution site of the chromatophores.

Biological activities of recombinant Manchurian trout FSH and LH: their receptor specificity, steroidogenic and vitellogenic potencies.

Gonadotropins (GTHs), FSH and LH, play central roles in vertebrate reproduction. Here, we report the production of biologically-active recombinant FSH (r-mtFSH) and LH (r-mtLH) of an endangered salmon species, Manchurian trout (Brachymystax lenok), by baculovirus in silkworm (Bombyx mori) larvae. The biological activities of the recombinant hormones were analyzed using COS-7 cell line transiently expressing either amago salmon FSH or LH receptor. The steroidogenic potency of the r-mtFSH and r-mtLH was examined by a culture system using rainbow trout follicles in vitro. In vivo, bioactivity was assessed by measuring ovarian weight, oocyte diameter, and plasma steroid hormone levels in female rainbow trout. Moreover, inducing potency of milt production were examined in vivo using goldfish. Our results demonstrated that the r-mtFSH and r-mtLH were successfully produced in the baculovirus-silkworm system and recognized by their cognate receptors specifically in vitro. The production of estradiol-17beta (E2) and testosterone (T) was stimulated by the r-mtFSH and r-mtLH respectively, from the full-grown follicles of rainbow trout, whereas both E2 and T were increased by relatively higher doses of the recombinant hormones from the follicles of the maturing stage. In in vivo assay, injection of the r-mtFSH but not r-mtLH increased ovarian weight, oocyte diameter, and plasma E2 levels in immature rainbow trout. Injection of both r-mtFSH and r-mtLH induced milt production in male goldfish. In conclusion, the present study strongly suggests that the r-mtFSH and r-mtLH have distinct biological properties, such as a specific responsiveness for the cognate receptor, steroidogenic, and vitellogenic activities for ovarian follicles in salmonids. These recombinant FSH and LH may be applied for future studies on the gonadal development and maturation in fishes as well as the endangered salmon species.

Production of low-estrogen goldfish diet for in vivo endocrine disrupter test.

A low-estrogenic diet for goldfish Carassius auatus was produced for an in vivo estrogen activity test, because commercial fish feed has estrogenic activity and may affect the results of estrogen assays. The newly produced diet (FD5) was formulated with defatted rice bran and casein, and did not contain any soybean meal or fish meal. Phytoestrogen contents (genistein, daidzein, equol, and coumestrol) of FD5 were measured by liquid chromatography-mass spectroscopy/mass spectroscopy (LC-MS/MS) and compared with those of the commercial trout diet (TD) and carp diet (CD). The genistein, daidzein, and coumestrol contents of TD and CD were much higher (5-2000 times) than those of FD5, but equol was detected only in FD5. Estrogenic activity of the fish diets was estimated in vitro by the yeast estrogen-screen assay (YES assay). The estrogenic activity was detected in TD and CD, but not in FD5. The in vivo estrogenic activity of the diets was examined by determining the production of vitellogenin in male goldfish. When male goldfish were fed TD or CD, plasma vitellogenin levels increased, but fish that were fed FD5 maintained low vitellogenin levels. These results indicate that FD5 produced in the present study has a low estrogenic activity, and FD5 would be suitable for the in vivo estrogen activity test using goldfish.