The anemonefish, Amphiprion clarkii, is a protandrous hermaphrodite. Under appropriate social conditions, male fish can become female. Previous studies indicated that estrogens are important ...regulators of sex change in this fish. However, the mechanism of sexual plasticity in the gonad of this fish is still unknown. To elucidate the mechanisms underlying the sexual plasticity in the ovary of female anemonefish, an aromatase inhibitor (AI, 500 μg/g diet) was administered to the functional female fish for 80 days.
The levels of estradiol-17β (E2) in the fish treated with AI were significantly lower than those in the control group. Three out of five fish had ambisexual gonads with active spermatogenic germ cells in the ovarian tissue. However, female fish in the AI-treated group prior to treatment and those in the control group displayed no testicular characteristics in their developed ovaries. This result strongly suggests that germ cells with bipotentiality or spermatogonial cells remain in the functional ovaries of anemonefish following sex change from functional males to functional females. There is a possibility that estrogen depletion due to AI treatment might have caused the opposite-directional sex change from functional female to male in the anemonefish.
The anemonefish keeps their high sexual bipotential in the ovary after sex change.
To clarify the cause of sex change recovery after the withdrawal of androgen treatment, immature female Malabar grouper were fed a diet containing 17alpha-methyltestosterone (MT) at 50 μg/g for 7 mo ...and then a normal diet for 6 mo. The MT brought about precocious sex change from immature ovaries to mature testes with active spermatogenesis, including the development of spermatozoa, and sex change reversed soon after MT treatment withdrawal. This result indicates that precocious sex change in immature Malabar grouper with oral MT treatment is impermanent. The expression of three steroidogenic enzymes (Cyp11a, Cyp19a1a, and Cyp11b) in the gonads of the Malabar grouper were analyzed immunohistochemically at the end of the 7-mo treatment. No apparent differences were seen in the expression pattern of these enzymes between the mature testes of MT-treated fish and the immature ovaries of control fish. In addition, serum estradiol-17beta and 11-ketotestosterone levels in treated fish were the same as those in control fish. These results indicate that in the case of immature Malabar grouper MT might have little effect on endogenous steroidogenesis during precocious sex change even though it induced active spermatogenesis in the gonads of treated fish. From these results, we also concluded that MT might have little effect on the steroidogenic endocrine pathway, and this is one cause of sex change recovery after treatment withdrawal.
Three-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.
First, 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.
The 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.
Unlike in other vertebrates, including teleosts, Foxl2 may have a different role in the naturally sex changing fishes.
Differentiation and development of steroid-producing cells (SPCs) and folliculogenesis during ovarian differentiation in the Nile tilapia Oreochromis niloticus were immunohistochemically and ...ultrastructurally examined. Clusters of immunopositive cells (IPCs) against antibodies (ABs) of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD), and cytochrome P450aromatase (P450arom) only appeared in the area near blood vessels in the fish ovaries at 50-60 days after hatching (dah). Ultrastructural results showed that differentiation and development of SPCs from undifferentiated to maturation occurred in the area near blood vessels, indicating that it would be the original site of SPCs. At 70-80 dah, IPC clusters invaded the interstices among oocytes at the perinucleolar stage from the area near the blood vessels. IPCs increased in number in the interstices among the previtellogenic oocytes, and some clusters began to enclose the outer thecal layer of the previtellogenic oocytes at 90 dah. The process of folliculogenesis was ultrastructurally observed. SPCs enclosed by fibroblastic cells invaded the interstitial areas among oocytes and some reached the surfaces of oocytes. The upper portions of these elongations opened and began to enclose the outer surfaces of developed oocytes to become thecal layer. Later, newly migrated SPCs reach the thecal layer to become thecal cells. These results indicate that steroid-producing thecal cells originate from the SPCs in the area near blood vessels. After thecal layer formation, an immunopositive reaction against P450arom AB, but not against P450scc or 3β-HSD ABs, appeared first in the granulosa cells enclosing the vitellogenic oocytes at 100 dah. At this time, estrogen production in serum levels rapidly increased. Thus, folliculogenesis could be essential for active production of estrogen in the ovary.
To investigate how estrogen and androgen affect each other in inducing sex reversal in the medaka, O. Iatipes, 17β-estradiol (E2) and 17α-methyldihydrotestosterone (MDHT) were co-administered by a ...convenient method for hormonal treatment, in which freshly fertilized eggs were immersed for 24 h in saline containing either or both of the two sex steroids in different concentrations and/or ratios. The minimal concentrations of E2 and MDHT sufficient to induce the maximal rate of sex reversal from male to female and from female to male were 500 ng/ml and 2.5 ng/ml, respectively, both of which were referred to as the most efficacious dose (MED), and each equivalent for the inducing potency in sex reversal. E2 and MDHT, when simultaneously administered at MED, greatly suppressed each other to induce each corresponding sex reversal. Thus, the present experimental results indicate that E2 and DMHT are antagonists that induce corresponding sex reversal, and suggest that genotypic sex in the medaka might be modified through an unknown factor of common affinity to both sex steroids, by which the pathway of differentiation of either sex could be switched at the early stages of development far before gonadal sex differentiation.
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.
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.
To understand the mechanism of sex differentiation in the protogynous Malabar grouper Epinephelus malabaricus, we performed an immunohistochemical investigation of the expression of three ...steroidogenic enzymes, cholesterol-side-chain-cleavage enzyme (CYP11a), aromatase (CYP19a1a), and cytochrome P45011beta-hydroxylase (CYP11b), in the gonads during ovarian differentiation. Strong positive immunoreactivity against CYP11a, the key enzyme of steroidogenesis, and CYP19a1a which is essential for estrogen (17beta-estradiol) production, appeared first in the somatic cells surrounding gonial germ cells in undifferentiated gonads and throughout ovarian differentiation. However, positive immunoreactivity against CYP11b, which is important for androgen (11-ketotestosterone) production, first appeared in the cluster of somatic cells in the ovary tunica near the dorsal blood vessel after differentiation. CYP19a1a and CYP11b did not co-localize in any cells. These results indicate that there are two types of steroid-producing cells, estrogen-producing cells and androgen-producing cells, in the gonads of this fish, and they are distributed differently, suggesting that these cells are derived from different somatic cells. Estrogen-producing cells appeared prior to ovarian differentiation, while androgen-producing cells were first detected after ovarian differentiation. These results suggest that endogenous estrogen is involved in ovarian differentiation.
Eels are animals commonly used in zoological research, as these species have a unique catadromous life history and belong to a phylogenetically ancient group of Teleostei. However, eel reproduction ...is difficult to investigate, since mature samples are not easily obtainable in the wild. In this study, we tested daggertooth pike conger (Muraenesox cinereus), an Anguilliformes species, as a potential model for the investigation of the reproductive biology of eels. Seventy individuals were caught between June and October, which is supposed to be their spawning season, from inshore of the Seto Inland Sea.
The lengths and ages of samples ranged from 510 to 1239 mm and three to nine years, respectively, and the sex ratio was skewed towards females (96 % of the total sample). The gonado-somatic index of the females peaked in July. Histological observation revealed that these ovaries were similar to those of other eel species and contained matured oocytes (migratory-nucleus stage), suggesting that pike conger spawn inshore in July. The plasma concentrations of sex steroid hormones (estradiol-17β and 11-keto-testosterone) in females gradually increased during maturation and decreased after spawning, indicating the involvement of these hormones in oogenesis of pike conger.
The present study is the first to report on characteristics of natural oogenesis in pike conger. Because naturally maturing samples can easily be captured, daggertooth pike conger may represent an excellent model for the study of reproduction in Anguilliformes.
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