Ryo Nozu

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 microg 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.

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

Background: 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. Results: 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. Conclusions: 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. Developmental Dynamics 242:388–399, 2013.

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.

Cortisol Administration Induces Sex Change from Ovary to Testis in the Protogynous Wrasse, Halichoeres trimaculatus

Steroid hormones have been shown to play important roles in triggering sex change. However, the upstream mechanism that regulates the secretion of sex steroid hormones controlling sex change is not yet known. Cortisol, the primary glucocorticoid in teleost fish, is known to exhibit anti-stress action and is involved in many physiological functions, including regulation of steroidogenesis. Therefore, cortisol could be one of the candidate factors involved in the onset of sex change. In this study, we investigated the role of cortisol in sex change in the three-spot wrasse, Halichoeres trimaculatus, by prolonged administration of cortisol. Our results showed that gonads of all individuals treated with cortisol (1,000 µg/g diet) for 6 weeks contained spermatogenic germ cells. One of them exhibited matured testes with an ovarian cavity, indicating sex change. Additionally, the plasma estradiol-17β level in the cortisol treatment group was significantly lower than in the control group suggesting that cortisol plays a direct and/or indirect role in the regulation of estrogen production. These data imply that cortisol might be involved in the regulation of steroidogenesis by causing a decrease in the estrogen level, leading to the onset 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.

Role of estrogen in spermatogenesis in initial phase males of the three‐spot wrasse (Halichoeres trimaculatus): Effect of aromatase inhibitor on the testis

The three‐spot wrasse, Halichoeres trimaculatus, is a protogynous hermaphrodite. Under appropriate social conditions, female fish can become male. Previous studies indicated that estrogens are important regulators of sex change in this fish. However, the role of estrogen in the male is not known. To clarify the involvement of estrogen in spermatogenesis in hermaphrodite fish, we treated initial phase (IP) males for 10 weeks with exemestane, an aromatase inhibitor (AI), to block estrogen synthesis. Fish treated with AI exhibited decreases in gonadal weight, plasma estrogen levels, and spermatogonial proliferation in the testis, together with increases in androgen levels. Additionally, we confirmed that exogenous estrogen treatments stimulated the renewal and proliferation of spermatogonia in the testis of IP males. These results indicate that estrogens play an important role in regulating spermatogenesis in this fish. Developmental Dynamics, 2011.

How great white sharks nourish their embryos to a large size: evidence of lipid histotrophy in lamnoid shark reproduction

ABSTRACT The great white shark (Carcharodon carcharias) exhibits viviparous and oophagous reproduction. A 4950 mm total length (TL) gravid female accidentally caught by fishermen in the Okinawa Prefecture, Southern Japan carried six embryos (543-624 mm TL, three in each uterus). Both uteri contained copious amounts of yellowish viscous uterine fluid (over 79.2 litres in the left uterus), nutrient eggs and broken egg cases. The embryos had yolk stomachs that had ruptured, the mean volume of which was approximately 197.9 ml. Embryos had about 20 rows of potentially functional teeth in the upper and lower jaws. Periodic acid Schiff (PAS)-positive substances were observed on the surface and in the cytoplasm of the epithelial cells, and large, secretory, OsO4-oxidized lipid droplets of various sizes were distributed on the surface of the villous string epithelium on the uterine wall. Histological examination of the uterine wall showed it to consist of villi, similar to the trophonemata of Dasyatidae rays, suggesting that the large amount of fluid found in the uterus of the white shark was likely required for embryo nutrition. We conclude that: (1) the lipid-rich fluid is secreted from the uterine epithelium only in early gestation before the onset of oophagy, (2) the embryos probably use the abundant uterine fluid and encased nutrient eggs for nutrition at this stage of their development, and (3) the uterine fluid is the major source of embryonic nutrition before oophagy onset. This is the first record of the lipid histotrophy of reproduction among all shark species. Summary: Great white sharks (Carcharodon carcharias) nourish their embryos with a lipid-rich ‘milk’ secreted by uterine epithelium during the early gestation period, before the onset of oophagy.

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. Mol. Reprod. Dev. 82: 859–866, 2015.

Histological observation of doublesex-mab 3-related transcription factor 1 (DMRT1) localization in the adult testis of three-spot wrasse

Doublesex-mab 3-related transcription factor 1 (DMRT1) has been identified as the first conserved gene involved in the testicular differentiation of vertebrates. However, the precise role of DMRT1 in spermatogenesis has not been made clear. In this study, immunohistochemical method was used to observe DMRT1 protein localization in order to resolve cellular profile of DMRT1 in the adult testis of three-spot wrasse. DMRT1 protein was clearly and specifically localized in the Sertoli cells of all spermatogenic cells and epithelial cells comprising the efferent duct, but not in the germ cells. In addition, adult males were treated with aromatase inhibitor (AI) for investigating the role of estrogen on the transcription of DMRT1. AI treatment caused an increase in the levels of DMRT1 transcripts in the efferent duct region, concomitant with a decrease in spermatogonia and spermatocytes.

Dental ontogeny of a white shark embryo

Unlike most viviparous vertebrates, lamniform sharks develop functional teeth during early gestation. This feature is considered to be related to their unique reproductive mode where the embryo grows to a large size via feeding on nutritive eggs in utero. However, the developmental process of embryonic teeth is largely uninvestigated. We conducted X‐ray microcomputed tomography to observe the dentitions of early‐, mid‐, and full‐term embryos of the white shark Carcharodon carcharias (Lamniformes, Lamnidae). These data reveal the ontogenetic change of embryonic dentition of the species for the first time. Dentition of the early‐term embryos (∼45 cm precaudal length, PCL) is distinguished from adult dentition by 1) the presence of microscopic teeth in the distalmost region of the paratoquadrate, 2) a fang‐like crown morphology, and 3) a lack of basal concavity of the tooth root. The “intermediate tooth” of early‐term embryos is almost the same size as the adjacent teeth, suggesting that lamnoid‐type heterodonty (lamnoid tooth pattern) has not yet been established. We also discovered that mid‐term embryos (∼80 cm PCL) lack functional dentition. Previous studies have shown that the maternal supply of nutritive eggs in lamnoid sharks ceases during mid‐ to late‐gestation. Thus, discontinuation of functional tooth development is likely associated with the completion of the oophagous (egg‐eating) phase. Replacement teeth in mid‐term embryos include both embryonic and adult‐type teeth, suggesting that the embryo to adult transition in dental morphology occurs during this period. J. Morphol. 278:215–227, 2017.