Protogynous Wrasse Research Articles

Expression patterns of sex differentiation-related genes during gonadal sex change in the protogynous wrasse, Halichoeres trimaculatus

The three-spot wrasse, Halichoeres trimaculatus, can change sex from female to male (i.e. protogyny) due to sharp decrease in endogenous estrogen. During the sex change, ovarian tissue degenerates and testicular tissue arises newly. Finally, ovarian tissue disappears completely and replaces into mature testis. In order to predict the molecular mechanisms controlling the processes of sex change, we investigated the expression patterns of four genes (rspo1, figla, sox9b and amh), which have been thought to be associated with ovarian/testicular differentiation in vertebrates. Expression levels of rspo1 and figla, which play important roles for ovarian differentiation in vertebrates, were stable until the middle stage of the sex change, and subsequently down-regulated. Therefore, it was indicated that decrease in rspo1 and figla could result from ovarian degeneration. On the other hand, basis on the expression pattern, it was indicated that sox9b and amh, which are involved in testicular differentiation in vertebrates, were implicated in testicular formation and spermatogenesis during the sex change as well. The present results could be fundamental information for investigating the relationship between these factors and E2 depletion, which is crucial trigger for sex change.

Expression profile of doublesex/male abnormal-3-related transcription factor-1 during gonadal sex change in the protogynous wrasse, Halichoeres trimaculatus.

Sex change in fish involves a dramatic transformation of gonadal tissue and a switch in gametogenesis. Doublesex/male abnormal-3-related transcription factor-1 (DMRT1), encoded by the DMRT1 gene, is involved in testicular differentiation in a wide range of vertebrates as well as in sexual differentiation and gonadal sex change. In the present study, we investigated changes in the expression of dmrt1 during artificial gonadal sex change in the three-spot wrasse, Halichoeres trimaculatus, by real-time quantitative PCR and immunolocalization, using an anti-wrasse-Dmrt1 antibody that we prepared. We found that dmrt1 expression was predominantly observed in the testes, and that Dmrt1 was expressed in Sertoli cells of testes and a few granulosa cells surrounding vitellogenic oocytes of the ovary. Additionally, the upregulation of dmrt1 expression was consistent with an increase in spermatogenic cyst quantity rather than proliferation of presumptive spermatogonia, suggesting that dmrt1 is involved in the progression of spermatogenesis during sex change. Changes in the localization of Dmrt1 during gonadal sex change further implied that Sertoli cells originate from somatic cells adjacent to gonial germ cells during testicular formation in the three-spot wrasse.

Characterization of gonadal soma‐derived factor expression during sex change in the protogynous wrasse, Halichoeres trimaculatus

Sex change in fishes provides a good experimental model for understanding the mechanisms and plasticity of sex determination and differentiation. The three-spot wrasse, Halichoeres trimaculatus is a protogynous hermaphrodite. During sex change from female to male, the ovary is replaced by the testis through the degeneration of oocytes and subsequent spermatogenesis. In the present study, we cloned a cDNA-encoding gonadal soma-derived factor (GSDF) from protogynous wrasse and examined its expression pattern in the sexually mature gonads and the sex-changing gonad induced experimentally by aromatase inhibition. Expression of gsdf was predominantly observed in the testis, and it was mainly localized to the supporting cells surrounding the spermatogonia. In the ovary, only slight expression of gsdf was observed in morphologically undifferentiated supporting cells in contact with oogonia. During sex change, strong expression of gsdf appeared first in the supporting cells surrounding the gonial germ cells before the onset of spermatogenesis. Thereafter, the expression of gsdf continually increased in the supporting cells surrounding the proliferating spermatogonia throughout the sex change. These results suggest that gsdf is involved in the proliferation of spermatogonia and subsequent spermatogenesis in both the testis and the gonad in the early stages of sex change.

Survival of ovarian somatic cells during sex change in the protogynous wrasse, Halichoeres trimaculatus

The three-spot wrasse (Halichoeres trimaculatus), which inhabits the coral reefs of Okinawa, changes sex from female to male. Sex change in this species is controlled by a social system. Oocytes disappear completely from the ovary, and male germ cells and somatic cells comprising testicular tissue arise a new during the sex change process. However, little is known of the fate and origin of the gonadal tissue-forming cells during sex change. In particular, the fate of ovarian somatic cells has not been determined, although the ovarian tissue regresses histologically. To approach this question, we analyzed apoptosis and cell proliferation in the sex-changing gonads. Unexpectedly, we found that few apoptotic somatic cells were present during sex change, suggesting that ovarian somatic cells might survive during the regression of the ovarian tissue. On the other hand, cell proliferation was detected in many granulosa cells surrounding the degenerating oocytes, a few epithelial cells covering ovigerous lamella and a few somatic cells associated with gonial germ cells at an early stage of sex change. Then, we found that proliferative ovarian somatic cells remained in the gonads late in the sex change process. Based on these results, we concluded that some functional somatic cells of the ovary are reused as testicular somatic cells during the gonadal sex change in the three-spot wrasse.

The Fate of Ovarian Somatic Cells during Sex Change in the Protogynous Wrasse, Halichoeres trimaculatus

The Fate of Ovarian Somatic Cells during Sex Change in the Protogynous Wrasse, Halichoeres trimaculatus

Expression and localization of forkhead transcriptional factor 2 (Foxl2) in the gonads of protogynous wrasse, Halichoeres trimaculatus.

BackgroundThree-spot wrasse, Halichoeres trimaculatus, is a marine protogynous hermaphrodite fish. Individuals mature either as initial phase (IP) males or females. Appropriate social cues induce the sex change from IP female to terminal phase (TP) male. However, the molecular mechanisms behind such a sex change remain largely unknown. Recently, the forkhead transcription factor 2 (Foxl2) was identified as an essential regulator of vertebrate ovarian development/function/phenotype. Inspired by this information, we characterized the expression patterns of Foxl2 in the protogynous wrasse assuming Foxl2 as the female-specific marker in this species.MethodsFirst, we clonedFoxl2 cDNA from ovary by reverse transcription polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). Next, we analysed expression pattern of Foxl2 messenger RNA (mRNA) and protein in gonads of different sexual phases by real time quantitative PCR assay and flour fluorescence immunohistochemical method, respectively. Additionally, we studied the changes in Foxl2 expression pattern during aromatase inhibitor (AI)-induced sex change.ResultsThe amino acid sequence (306 AA) of wrasse Foxl2, especially the forkhead domain, shows high identity with that of other reported teleost Foxl2s. Quite unexpectedly, no sexual dimorphism was observable between the testes and ovary in the expression pattern of Foxl2. In female phase fish, signals for Foxl2 protein were detectable in the granulosa cells, but not the theca cells. Transcript levels of Foxl2 in the testes of IP and TP males were identical to that in the ovaries of females and, further, Foxl2 protein was found to be localized in the interstitial cells including tubules and Leydig cells. Treatment with AI induced sex change in male gonads and an up-regulation was seen in the expression of Foxl2 in these gonads.ConclusionsUnlike in other vertebrates, including teleosts, Foxl2 may have a different role in the naturally sex changing fishes.

Isolation and characterization of DMRT1 and its putative regulatory region in the protogynous wrasse, Halichoeres tenuispinis

A full-length cDNA of doublesex and mab-3-related transcription factor 1 gene ( DMRT1) from wrasse testis was isolated by cDNA library screening. Wrasse DMRT1 was 3116 bp in size and contained the DM domain, with a zinc finger DNA-binding motif, and the male-specific motif. Northern blot analysis identified a 3.2-kb transcript approximately equal in size to the DMRT1 nucleotide sequence detected in the testis, but not in the ovary, confirming that this sequence is male-specific in protogynous wrasse. Southern blot analysis suggested that the wrasse genome contains two copies of the DMRT1 gene. The ORF consisted of five exons and four introns with conserved donor–acceptor splice sites at all exon–intron junctions. The 5′-flanking region of the wrasse DMRT1 gene was isolated by DNA walking, and putative regulatory sites were identified by searching data bases. The 5′-flanking region was divided into 9 elements, then 17 DMRT–luciferase chimeric plasmids were constructed. By transient transfection into Cos-1 and TM4 cells, distal element I which contains GATA-binding sites and proximal element B containing the sex-determining region on Y chromosome gene ( SRY) binding site were revealed to have an important role in transcriptional regulation of the wrasse DMRT1 when an enhancer sequence was provided.

Short term treatment with aromatase inhibitor induces sex change in the protogynous wrasse, Halichoeres trimaculatus

The purpose of this study was to specify the time when individuals are committed to female to male sex change in the protogynous wrasse, Halichoeres trimaculatus, induced by treatment with the nonsteroidal aromatase inhibitor (AI) Fadrozole. In this study, treatment with AI was carried out by providing adult females with a diet containing 500 μg AI/g food for 3 (AI-3), 5 (AI-5), and 10 days (AI-10). We examined the gonadal structure of the fishes histologically at the end of the AI treatment and 30 days after the start of the experiment. At the end of the AI treatment, all individuals in the AI-3 treated group had gonads with degeneration of yolky oocytes, indicating the onset of sex change. Most individuals in the AI-5 treated group had gonads with atretic vitellogenic oocytes, like those in AI-3 treated group, whereas most individuals in the AI-10 treated group had gonads with testicular tissue. At 30 days after the onset of the experiment, approximately 70% of the individuals in the AI-3 treated group had mature ovaries, whereas all fishes in AI-5 and AI-10 treated groups had mature testes, indicating sex change. Therefore, treatment with AI for only 5 days resulted in complete sex change. Our results also indicate that crucial events for testicular differentiation occur within 5 days from the start of AI treatment. Thus, we conclude that females are committed to change into males after 5 days of AI treatment.

Group spawning results from the streaking of small males into a sneaking pair: male alternative reproductive tactics in the threespot wrasse Halichoeres trimaculatus

In many protogynous wrasses, large males with bright coloration (terminal phase males, TP males) establish mating territories and pair-spawn with females. In contrast, small primary males with drab coloration (initial phase males, IP males) are non-territorial and adopt three alternative reproductive tactics—group spawning, streaking, and sneaking. We investigated how IP males of the threespot wrasse Halichoeres trimaculatus use these tactics in different situations. The mating frequency of the IP males was positively correlated with their courtship frequency, but not with their body size. Larger IP males tended to attack the smaller ones at the mating sites. This indicates that the larger IP males attempted to exclude the smaller ones from mating with the intention of minimizing the number of IP males involved in group spawning and ultimately leading to pair spawning (sneaking). However, the larger IP males were unable to completely exclude the smaller males because the intensity of the attack by the larger IP males was weak. Consequently, the smaller IP males could easily streak into the sneaking of larger IP males, thereby resulting in group spawning.

Examination of the Ability of Gonadal Sex Change in Primary Males of the Diandric Wrasses Halichoeres poecilopterus and Halichoeres tenuispinis: Estrogen Implantation Experiments

Primary males that function as males throughout their lives are often found among protogynous fishes such as wrasses and parrotfishes. However, the issue of whether the sexuality of primary males involves gonochorism or hermaphroditism remains uncertain. To clarify this, we implanted estradiol-17beta (E2) into the body cavities of primary males of two protogynous wrasses, Halichoeres poecilopterus and Halichoeres tenuispinis. At 51-63 days after implantation, primary males with E2 treatment in both wrasses were observed to develop ovarian tissues. These results suggest that primary males of Halichoeres wrasses potentially have the ability to change sex and that estradiol-17beta is related to gonadal transitions in primary males.

Body color change and serum steroid hormone levels throughout the process of sex change in the adult wrasse, Pseudolabrus sieboldi

Gonadal sex steroid hormones are the principal factors that directly control the gonadal and morphological alterations during sex change in hermaphrodite fish; however, the physiological mechanism of action by which these hormones govern body coloration is poorly understood. The protogynous wrasse Pseudolabrus sieboldi is a good model for understanding the physiological mechanisms of gonadal and body color change during sex change in hermaphrodite fish. To obtain information on the relationship between sex steroids and body color change during the process of gonadal sex change, we analyzed body color, gonadal histology, and serum levels of sex steroids. Body color was analyzed using a quantitative analytical method based on the hue value. Compared to other body parts of the fish, the anal fin changed color the most, becoming increasingly redder in association with gonadal changes that converted ovaries to testes. Levels of serum 11-ketotestosterone (11KT) increased as the gonadal sex change proceeded, whereas no significant change was observed in estradiol-17β (E2) levels. Moreover, we found a significant correlation between the hue value of the anal fin and serum 11KT levels, but not E2 levels. These results suggest that androgen, but not estrogen, plays a principle role in the changes in both gonadal morphology and body color in the transformation from female to male in this species. To our knowledge, this is the first quantitative demonstration of the relationship between body color and serum steroid levels during sex change in fish.

Molecular cloning of cytochrome P450 aromatases in the protogynous wrasse, Halichoeres tenuispinis

P450 aromatase (P450arom, CYP19), a CYP19 gene product, is a member of the cytochrome P450 superfamily that catalyzes the formation of aromatic C 18 estrogen from C 19 androgen. To begin to understand the molecular mechanisms of P450 aromatase action in the protogynous wrasse, we isolated two cDNAs: one encoding CYP19a from ovary and the other encoding CYP19b from brain. The full-length cDNA of wrasse CYP19a, isolated from ovary cDNA library, is 2020 bp long and encodes 519 amino acids. The amino acid sequence of CYP19a has 62–83% identity with ovary-type aromatases of other teleosts. The full-length cDNA of wrasse CYP19b obtained using 5′ and 3′ RACE consists of 2666 bp, and its open reading frame encodes 496 amino acids. The deduced amino acid sequence has 62–83% identity with brain-type aromatases of other teleosts. Northern blot analysis identified a single 2.2-kb transcript in the ovary (CYP19a), and a single 2.6-kb transcript in the brain (CYP19b), suggesting that there are single forms of CYP19a and CYP19b, respectively, in the wrasse. RT-PCR assay showed that two CYP19 genes were expressed ubiquitously in various tissues, although each CYP19 subtype was expressed at highest level in the ovary and brain of the wrasse. These results suggest that CYP19 genes act in diverse tissue types, in addition to their effects on the physiological and reproductive functions of estrogen.

Role of steriod hormones in sex change of protogynous wrasse

The endocrine mechanisms of sequential hermaphroditism were investigated in protogynous wrasse, Halichoeres trimaculatus. Administration of aromatase inhibitor and androgen both stimulated sex change, while estrogen suppressed the induced sex inversion. The results suggest that removal of estrogen is a key step to initiate sex change.

Bi-directional sex change and its steroidogenesis in the wrasse, Pseudolabrus sieboldi

The protogynous wrasse, Pseudolabrus sieboldi, is a good model for studying the physiological mechanism of sex change in teleosts. We established a way to induce sex change bidirectionally by controlling the social conditions in captivity. This is the first report of a reversed sex change in a promiscuous species. After identifying all the steroid hormones produced in the ovarian follicles and testis, and clarifying their synthetic pathways, we examined the function of the sex steroids in the sex change of P. sieboldi. We concluded that the activation of 17β-HSD-III is the first step in inducing morphological and functional change from an ovary to a testis.

Diurnal rhythm of steroid biosynthesis in the testis of terminal phase male of protogynous wrasse, Pseudolabrus sieboldi, a daily spawner

The wrasse, Pseudolabrus sieboldi, is a diandric protogynous labrid fish. Spawning is performed by a terminal phase (TP) male and an initial phase (IP) female between 6:00 and 9:00 h daily during two-month-long spawning season. In the present study, to investigate the roles of steroid hormones in the diurnal spermatogenesis of the P. sieboldi TP male, all steroid hormones produced in the testis were identified and the synthetic pathways of these steroids were determined. Furthermore, the circulating levels of the major steroids produced were analyzed throughout a day at 3-hour intervals during spawning season. In the testis, 11-ketotestosterone (11-KT), estradiol-17β (E2), 17,20β-dihydoxy-4-pregnane-3-one (17,20β-P) and 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) were synthesized as the major metabolites. In vitro steroid biosynthesis experiments showed similar results to the circulation profiles of the major steroids. This study is the first to clarify the complete steroidogenic pathways in the gonads of a diandric protogynous species throughout its life, when combined with the results of the steroidogenesis in the ovarian follicles. This is also the first report of a clear diurnal rhythm of the steroid production corresponding to the spermatogenic process in the testis of a male teleost.

Sequence and expression of androgen receptor and estrogen receptor gene in the sex types of protogynous wrasse, Halichoeres trimaculatus

Sex steroid hormones play important roles in sex change and behavior of wrasses. Their actions are considered to be mediated through the nuclear hormone receptors. In this study, to elucidate the roles of estrogen receptor (ER) and androgen receptor (AR) in the reproduction of the protogynous wrasse, Halichoeres trimaculatus, AR and ER genes were partially cloned using 5 ′- and 3 ′-RACE and their transcript levels in the gonads and the brain were measured by competitive RT-PCR. The amino acid sequence (563 a.a) deduced from 5 ′ truncated cDNA encoding wrasse AR shows about 81%, 69%, 66%, 64%, and 58% identity with those of red seabream ( Chrysophrys major) androgen receptor subtype, AR1, and rainbow trout ( Oncorhynchus mykiss) ARα, ARβ, Japanese eel ( Anguilla japonica) ARα, and ARβ, respectively. The amino acid sequence (458 a.a) deduced from 5 ′ truncated cDNA encoding wrasse ER shows about 81%, 79%, 73%, 66%, and 63% identity with those of red seabream estrogen receptor subtype, ERα, Atlantic croaker ( Micropogonias undulatus) ERα, tilapia ( Oreochromis niloticus) ERα, rainbow trout ERα, and catfish ( Ictalurus punctatus) ERα, respectively. Among the various tissues tested, AR and ER mRNAs were highly expressed in the gonads and brains. When the transcript levels of ER were measured in the gonads and the brains of females (F), initial phase male (IP), and terminal phase male (TP), no significant changes in the gene expression were observed. The transcript levels of AR in the gonads did not change among different sex types, while those in the brains of TP were higher than F and IP. These results suggest that higher expression of AR in the brains of TP is strongly correlated with behavioral change.

Reversed Sex‐Change in the Protogynous Reef Fish Labroides dimidiatus

Protogynous hermaphroditism, or female‐to‐male sex change, is known for many reef fishes including wrasses (family Labridae) in which large males monopolize mating. When the dominant male disappears from a polygynous group, the largest female may change sex within a few weeks. Such social control of sex change was first documented in harems of the cleaner wrasse Labroides dimidiatus almost 30 yr ago. To examine whether change of social status would induce males of L. dimidiatus to perform reversed sex‐change, we conducted experiments: (i) releasing single males near lone males whose mates have been removed in the field; and (ii) keeping two males in a tank. Smaller males changed back to females when they became subordinate: it took 53–77 d (n=3) for them to complete gonadal sex change and release eggs in the aquarium. The male–male pairs performed spawning behavior, with the smaller male in the female role already 5–58 d before completion of gonadal sex change. This is the first report of reversed sex‐change among protogynous wrasses. Moreover, we conducted another experiment, keeping a pair of a male and a larger female in a tank (n=1). We found sex change by both mates, which has not been reported from any fishes. Thus, the sex of L. dimidiatus is strictly determined by social status whenever it changes after mate loss.

Terminal phase males stimulate ovarian function and inhibit sex change in the protogynous wrasse Thalassoma duperrey.

Under experimental conditions, the probability of sex change in the protogynous wrasse Thalassoma duperrey is determined largely by an individual's relative size within a social group. Natural populations, however, contain two distinct male phenotypes that may also play a role in regulating sex change. To investigate potential effects of male phenotype, the ability to change sex, ovarian histology and serum estradiol-17 beta levels were examined in females maintained under controlled social settings. Large females housed with smaller or larger terminal phase males had significantly larger gonadosomatic indices than females housed singly, with other females or with smaller initial phase males. Similarly, ovaries of females housed with terminal phase males showed no histological evidence of sex change, whereas large females from other social groupings were in advanced stages of sex change. These results demonstrate terminal phase males inhibit sex change regardless of their size relative to the female. Furthermore, gonadosomatic indices, ovarian histology, and serum estradiol-17 beta levels of females housed with terminal phase males indicate normal ovarian function whereas ovaries of other treatment groups appear quiescent or are undergoing sex change. Consequently, terminal phase males may be required for normal ovarian development which may, in turn, inhibit sex change in T. duperrey.

Immunocytochemical and Ultrastructural Identification of Pituitary Cell Types in the Protogynous Thalassoma duperrey during Adult Sexual Ontogeny

Abstract Protogynous wrasses (Thalassoma duperrey): females (F), primary males (PM) along with a few terminal-phase males (TM) and sex-changed males (SM), were used to characterize the topographical organization of the pituitary. In general, immunocytochemical and ultrastructural features of the adenohypo-physeal cell types of the saddleback wrasse pituitary resemble those of other teleosts. In the rostral pars distalis (RPD), corticotropic cells were found bordering the neurohypophysis (NH) and surrounding the centroventrally located prolactin cells. Thyrotropic cells formed a small group in the anteriodorsal part of the rostral and proximal pars distalis (PPD). The somatotropic cells were distributed in large clusters, mostly organized in cell cords around the interdigitations of the NH of the dorsal PPD. Cells containing gonadotropin Ib subunit were localized in the dorsal parts of the PPD, in close association with somatotropic cells and gonadotropin IIb subunit containing cells were seen in the centr...

Diurnal Spermatogenesis and Spawning in the Secondary Male of a Protogynous Wrasse, Pseudolabrus japonicus (Teleostei, Labridae)

Abstract The bambooleaf wrasse, Pseudolabrus japonicus, exhibits diandric protogyny, and the secondary male performs pair spawning with a female who enters his territory. We examined diurnal spermatogenesis and spawning in the secondary male of a protogynous wrasse. In captivity, a single secondary male spawned daily over one month between 06:00 and 09:00 from October to November. Number of B-type spermatogonia and spermatocytes showed the lowest level at 00:00, increased gradually thereafter, peaked at 15:00, and decreased rapidly from 21:00 to 00:00. Spermatid number did not change significantly throughout the day. The number of spermatozoa increased gradually from 18:00, reached a maximum at 06:00, just prior to spawning, and thereafter decreased markedly at 09:00, after spawning. These results clearly showed that spermatogonial proliferation and meiosis occurred between 00:00 and 15:00, and spermiation occurred between 18:00 and 06:00. Thus, the secondary male of bambooleaf wrasse exhibits a diurnal r...