Transcriptomic signatures associated with underlying rapid changes in the early phase brain of bi-directional sex change in Trimma okinawae.

Teleost fish exhibit remarkable sexual plasticity and divergent developmental systems, including sequential hermaphroditism. One of the more fascinating models of sexual plasticity is socially controlled sex change, which is often observed in coral reef fish. The Okinawa rubble goby, Trimma okinawae, is a bi-directional sex-changing fish. It can rapidly change sex in either direction based on social circumstances. Although behavioural and neuroendocrine sex change occurs immediately and is believed to trigger gonadal changes, the underlying mechanisms remain poorly understood. In this study, we conducted a de novo transcriptome analysis of the T. okinawae brain and identified genes that are differentially expressed between the sexes and genes that were immediately controlled by social stimulation implicating sex change. Several genes showed concordant expression shifts regardless of the sex change direction and were associated with histone modification in nerve cells. These genes are known to function in the neuroendocrine control of reproduction in nerve cells. Overall, we identified genes associated with the initiation of sex change, which provides insight into the regulation of sex change and sexual plasticity.

Gonadal and cellular dynamics during protogynous sex change in the harlequin sandsmelt Parapercis pulchella.

Some teleost fishes change their sex, and some of these fishes have specific gonads known as "ovotestes," that is, gonads containing both ovarian and testicular tissues. In this study, we revealed the gonadal transformation process and cell dynamics during the female-to-male sex change in the harlequin sandsmelt, Parapercis pulchella (Pinguipetidae), in which females possess ovotestes. Histological observations revealed that although female ovotestes were composed of oocytes, a few cysts of male germ cells were observed among them. At the initial phase of sex change, male germ cells increased, and spermatogenesis proceeded. After that, oocytes decreased and finally disappeared, and the gonads became functional testes. Immunohistochemistry using antibodies against Pcna (proliferating cell nuclear antigen) as a cell proliferation marker revealed that spermatogonia were Pcna positive, whereas spermatocytes were negative, in female ovotestes. This suggests that spermatogenesis is arrested at the spermatocyte stage. In addition, some somatic cells surrounding oocytes, which were thought to be the female follicle cells, were Pcna positive during sex change, indicating that these cells proliferate during sex change and are reused in male testes after sex change. Also, immunostaining using antibodies against active cleaved-Caspase3a as an apoptosis marker demonstrated that oocytes degenerated through apoptotic cell death at the late transition stage. Together with previous findings in other fishes, these findings suggested that the histological processes in gonads during sex change, such as the order of developmental events, developmental fates of ovarian cavities, and ovotestis structures, are diversified among fish species. In contrast, cellular dynamics of female germ and somatic cells during sex change are common among protogynous species.

Bidirectional sex change and plasticity of gonadal phases in the goby Lubricogobius exiguus.

Of the ca. 500 known hermaphroditic fish species, bidirectional sex change and simultaneous hermaphroditism are currently known in 69 and 57 species, respectively. Both bidirectional sex change and simultaneous hermaphroditism are predicted to evolve when mating opportunities are limited, such as in cases of low-density distribution and low mobility of individuals. However, the plasticity of sex is adaptive in obtaining mating opportunities, especially when there is mate loss. Pair-rearing experiments and histological observations of gonads of the goby Lubricogobius exiguus, which has low-density distributions and low mobility, showed bidirectional sex change. The male-role individuals in pairs had gonads in which only the testis was functional (male-phase), whereas the female-role individuals had two types of gonads: only the ovary was functional (female-phase) or both testis and ovary were functional (simultaneously hermaphroditic phase, SH-phase). In addition, single-rearing experiments showed SH-phase gonads in all individuals, and some cyclic spawning but no self-fertilization occurred. These results revealed that L. exiguus has an unusual sexual pattern among hermaphroditic fishes because they undergo bidirectional sex change and some maintain SH-phase gonads. These findings indicate that the low-density distribution and low mobility of L. exiguus in their natural habitat may have influenced the evolution of this unique sexual pattern.

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria.

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a series of basic techniques are described to enable observations of the reproductive behavior of a wild-caught gobiid fish, as a model, kept in an aquarium. The method focuses on three steps: collection, transport, and observations of reproductive ecology of a substrate spawner. Essential aspects of live fish collection and transport are (1) preventing injury to the fish, and (2) careful acclimation to the aquarium. Preventing harm through injuries such as scratches or a sudden change of water pressure is imperative when collecting live fish, as any physical damage is likely to negatively affect the survival and later behavior of the fish. Careful acclimation to aquaria decreases the incidence death and mitigates the shock of transport. Observations during captive rearing include (1) the identification of individual fish and (2) monitoring spawned eggs without negative effects to the fish or eggs, thereby enabling detailed investigation of the study species' reproductive ecology. The subcutaneous injection of a visible implant elastomer (VIE) tag is a precise method for the subsequent identification of individual fish, and it can be used with a wide size range of fish, with minimal influence on their survival and behavior. If the study species is a substrate spawner that deposits adhesive eggs, an artificial nest site constructed from polyvinyl chloride (PVC) pipe with the addition of a removable waterproof sheet will facilitate counting and monitoring the eggs, lessening the investigator's influence on the nest-holding and egg-guarding behavior of the fish. Although this basic method entails techniques that are seldom mentioned in detail in research articles, they are fundamental for undertaking experiments that require the captive rearing of a wild fish.

Evolution of bidirectional sex change and gonochorism in fishes of the gobiid genera Trimma, Priolepis, and Trimmatom.

Size-advantage and low-density models have been used to explain how mating systems favor hermaphroditism or gonochorism. However, these models do not indicate historical transitions in sexuality. Here, we investigate the evolution of bidirectional sex change and gonochorism by phylogenetic analysis using the mitochondrial gene of the gobiids Trimma (31 species), Priolepis (eight species), and Trimmatom (two species). Trimma and Priolepis formed a clade within the sister group Trimmatom. Gonadal histology and rearing experiments revealed that Trimma marinae, Trimma nasa, and Trimmatom spp. were gonochoric, whereas all other Trimma and Priolepis spp. were bidirectional sex changers or inferred ones. A maximum-likelihood reconstruction analysis demonstrated that the common ancestor of the three genera was gonochoristic. Bidirectional sex change probably evolved from gonochorism in a common ancestor of Trimma and Priolepis. As the gonads of bidirectional sex changers simultaneously contain mature ovarian and immature testicular components or vice versa, individuals are always potentially capable of functioning as females or males, respectively. Monogamy under low-density conditions may have been the ecological condition for the evolution of bidirectional sex change in a common ancestor. As T. marinae and T. nasa are a monophyletic group, gonochorism should have evolved from bidirectional sex change in a common ancestor.

Accumulation of organotins in wharf roach (Ligia exotica Roux) and its ability to serve as a biomonitoring species for coastal pollution.

In this study, we measured the accumulation of tributyltin (TBT) in wharf roach (Ligia exotica Roux) and examined the species' ability to be used for TBT biomonitoring in coastal environments. In an exposure test, wharf roach were exposed to TBT via diet for 2d. TBT was accumulated in wharf roach, and its metabolite dibutyltin was detected. The concentrations of these compounds gradually decreased during the depuration period, but they were still detected 12d after exposure ceased (TBT 290±140ng/g; dibutyltin 1280±430ng/g). The biological half-life of TBT in wharf roach was estimated to be about 4d. In a field study conducted in 2011-2012, wharf roach were collected from 15 coastal sites in Japan and 3 sites in Manado, Indonesia. TBT was detected in both Japanese and Indonesian samples. The highest concentration of TBT was found in wharf roach collected at Bitung ferry port, Manado (57.9±16.5ng/g), which is close to a shipyard, and the highest concentration at a Japanese site was 12.3±6.2ng/g. Thus, we were able to detect organotins in the coastal environments by testing wharf roach, suggesting that L. exotica might serve as a good bioindicator for monitoring organotin pollution.

Histological observation of the urogenital papillae in the bi-directional sex-changing gobiid fish, Trimma okinawae.

The gobiid fish Trimma okinawae changes its sex bi-directionally according to its social status. Morphological changes in the urinogenital papillae (UGP) of this fish have been reported during sex change. However, there have been no detailed observations of such changes. Here, we histologically examined the UGP structure of male- and female-phase fish. UGPs of fish in female and male phase contained both oviducts and sperm ducts. Both ducts were coalesced into one duct within the posterior region of the UGP. Female-phase fish had many longitudinal folds in the hypertrophied tunica mucosa of the oviduct, which was found to be responsible for the transport of eggs and the removal of follicular cells from the oocyte. In contrast, male-phase fish had an immature oviduct and a mature sperm duct in the UGP. In the male-phase fish, the co-existence of spermatozoa and fibrillar secretions was observed in the sperm duct during spermiation.

Sex change in the Gobiid fish is mediated through rapid switching of gonadotropin receptors from ovarian to testicular portion or vice versa.

Sex-changing fish Trimma okinawae can change its sex back and forth from male to female and then to male serially, depending on the social status in the harem. T. okinawae is well equipped to respond to its social status by possessing both ovarian and testicular tissues even though only one gonad remains active at one time. Here we investigated the involvement of gonadotropins in sex change by determining the changes in gonadotropin receptor (GtHR) gene expression during the onset of sex change from female to male and male to female. The expression of the GtHR was found to be confined to the active gonad of the corresponding sexual phase. During the sex-change from female to male, initially the ovary had high levels of FSHR and LHR, which eventually went up in the testicular tissue if the fish was bigger. Changing of the gonads started with switching of GtHR expression discernible within 8-12 h of the visual cue. Further in vitro culture of the transitional gonads with a supply of exogenous gonadotropin (human chorionic gonadotropin) revealed that the to-be-active gonad acquired the ability to produce the corresponding sex hormone within 1 d of the activation of GtHR. Conversely, the to-be-regressed gonad did not respond to the exogenous gonadotropin. Our findings show that the gonads of successive sex-changing fish possess the intrinsic mechanism to respond to the social cue differentially. Additionally, this location switching of GtHR expression also could substantiate the importance of the hypothalamo-pituitary-gonadotropic axis.

Molecular cloning and expression of Ad4BP/SF-1 in the serial sex changing gobiid fish, Trimma okinawae.

Involvement of Ad4BP/SF-1 was investigated using the serial sex changing goby Trimma okinawae. First, a cDNA encoding Ad4BP/SF-1 was cloned from ovarian follicles. The open reading frame of goby Ad4BP/SF-1 encodes a protein of 489 amino acids. Ad4BP/SF-1 was expressed in gonadal tissues, brain, and kidney. Second, transcript levels of Ad4BP/SF-1 were measured in the ovary and testis of the same individuals. Expressions were related to sexual phases. Moreover, ovarian expression of Ad4BP/SF-1 increased during vitellogenesis and declined sharply during the post-vitellogenic period. Finally, the expression profile of Ad4BP/SF-1 was measured during serial sex change in the ovary. Ad4BP/SF-1 increased in parallel with the onset of the female-phase and decreased as female became male. Therefore, Ad4BP/SF-1 probably acts as an important indirect regulator of oocyte growth and maturation at female-phase of serial sex changing gobiid fish T. okinawae.

Aromatase immunoreactivity and the role of enzymes in steroid pathways for inducing sex change in the hermaphrodite gobiid fish Trimma okinawae.

The role of aromatase (Arom) in the process of bi-directional sex change in the gobiid fish Trimma okinawae was investigated by immunohistochemical methods. Irrespective of sexual phase, gonads comprised both ovarian and testicular tissues. In each sexual phase of females, the 2nd (2DF-M) and 4th (4DF-M) days after initiation of sex change to male, males, and the 2nd (2DM-F), 4th (4DM-F) and 6th (6DM-F) days after the initiation of reversion from male to female, ovarian and testicular histological observations were made. During the female, 2DF-M, 4DF-M and 6DM-F phases, the ovary contained vitellogenic and previtellogenic oocytes, compared with previtellogenic oocytes in the other phases. Although sperm was found in the testis in every phase, sperm ducts were apparent in the male phase, but not the female phase. Arom immunoreactivity was detected in the interstitial cells between the oocytes in all phases. On the other hand, it was localized in the thecal and granulosa cells of the follicular layer enclosing the oocytes in the female, 2DF-M, 4DF-M and 6DM-F phases. Activity of Arom in the thecal and granulosa cells is thought to be important for the development of oocytes and subsequent sex change.

Gonadal structure of the serial-sex changing gobiid fish Trimma okinawae.

In order to obtain basic information about the role played by endogenous sex hormones in bringing about sex changes in the serial-sex changing gobiid fish Trimma okinawae, the gonadal structure of male and female phases were observed histologically. Steroid-producing cells (SPC; Leydig cells in a testis) were observed ultrastructurally in the ovaries and testes of both female-phase and male-phase fish. In addition, gonadal expression of P450 cholesterol side-chain-cleavage (scc) was examined immunohistochemically. Gonads of fish in female and male phases were observed to have both ovaries and testes simultaneously. Female-phase fish had matured with many developed vitellogenic oocytes, while male-phase individuals had immature ovaries with many numbers of previtellogenic oocytes at the perinucleolus stage. Testes of fish in different sexual phases had active spermatogenic germ cells. Organellae of SPC in the ovaries of female-phase fish had active structures of steroid production. In contrast, SPC in the ovaries of male-phase fish did not show active structures of steroid production. Immunopositive reactions against the scc antibody in the ovaries of female-phase fish were very strong, but immunoreactions in the ovaries of male-phase fish were very weak. In the testis, moderate immunopositive signals were obtained from dual-phase male/females.

Promoter analysis of two aromatase genes in the serial-sex changing gobiid fish, Trimma okinawae.

Despite numerous endocrine studies on sex change in teleost, no general mechanism that mediates sex change has emerged. The gobiid fish, Trimma okinawae, can change sex in both directions repeatedly. This phenomenon of sex change in goby assigns it as an excellent animal model to elucidate the understanding mechanisms of sex change. In hermaphrodite fishes, estrogen plays a particularly important role in natural and experimentally induced sex change. To investigate the role of estrogen in the serial-sex changing fish T. okinawae, we cloned and analyzed the 5'-flanking regions of P450arom genes from goby genome DNA. Both regions have consensus sequences of TATA, CRE and ERE. Ad4 binding site was restricted in the region of P450aromA. These findings indicate that different regulators control the expression of the two P450arom genes.

Characterization of two types of cytochrome P450 aromatase in the serial-sex changing gobiid fish, Trimma okinawae.

To investigate the role of estrogen in the serial-sex changing fish Trimma okinawae, we isolated complementary DNAs encoding two distinct cytochrome P450 aromatase isoforms from adult ovary and brain (termed P450aromA and P450aromB, respectively). Sequence and phylogenic analyses showed that the goby P450arom forms belong to two separate CYP19 subfamilies. Transient expression of these cDNAs in HEK293 cells caused conversion of exogenous testosterone to estradiol-17beta. RT-PCR showed that P450aromA was expressed in the brain, spleen, testis and ovary. P450aromB was expressed in the brain, liver, testis and ovary. In situ hybridization studies showed that P450aromA mRNA, but not P450aromB mRNA, was present in both ovary and testis. Positive signals were restricted to granulosa cells of vitellogenic follicles and interstitial cells of mature testis. Ovarian expression of both P450arom genes during the spawning cycle was examined by quantitative real-time RT-PCR. P450aromA transcripts increased during vitellogenesis and decreased prior to spawning. In contrast, P450aromB transcripts were barely detectable and did not correlate with ovarian development. These findings suggest that P450aromA, but not P450aromB, is involved in regulating ovarian vitellogenesis in goby.