Mohammad Ashraful Alam

Molecular cloning and quantitative expression of sexually dimorphic markers Dmrt1 and Foxl2 during female-to-male sex change in Epinephelus merra.

The honeycomb grouper (Epinephelus merra) is one of the smallest members of the Serranidae family and is often used to study protogynous sex change. To determine the role of the male-determining gene Dmrt1 and the ovarian-specific gene Foxl2 in sex change, we cloned these two markers from E. merra gonads by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Two isoforms, Dmrt1a and Dmrt1b, resulted from alternative splicing in the coding region, causing the insertion of one glutamine residue in Dmrt1b. RT-PCR revealed that Dmrt1 was expressed only in the gonads, with higher levels in the testis than in the ovary. cDNA encoding Foxl2 was isolated from the ovary; Foxl2 was expressed extensively in the brain, pituitary, gonads, and gill, with its highest level in the ovary, indicating a potential role for Foxl2 in the brain-pituitary-gonad axis. Real-time quantitative RT-PCR analyses showed that Foxl2 mRNA expression was significantly downregulated from the late transitional phase to the completion of sex change. Conversely, Dmrt1 expression increased with the progression of spermatogenesis and continued until the formation of the testis. The expression profiles of these two sex-specific marker genes corresponded closely with the histological process of sex change. The down-regulation of Foxl2 most likely facilitates oocyte degeneration, whereas the up-regulation of Dmrt1 causes the proliferation of gonial germ cells into spermatogina and initiates sex change.

Induction of Female-to–male Sex Change in the Honeycomb Grouper (Epinephelus merra) by 11-ketotestosterone Treatments

Abstract The honeycomb grouper, Epinephelus merra, is a protogynous hermaphrodite fish. Sex steroid hormones play key roles in sex change of this species. A significant drop in endogenous estradiol- 17β (E2) levels alone triggers female-to–male sex change, and the subsequent elevation of 11-ketotestosterone (11KT) levels correlates with the progression of spermatogenesis. To elucidate the role of an androgen in sex change, we attempted to induce female-to–male sex change by exogenous 11KT treatments. The 75-day 11KT treatment caused 100% masculinization of pre-spawning females. Ovaries of the control (vehicle-treated) fish had oocytes at various stages of oogenesis, while the gonads of the 11KT-treated fish had transformed into testes; these contained spermatogenic germ cells at various stages, including an accumulation of spermatozoa in the sperm duct. In the sex-changed fish, plasma levels of E2 were significantly low, while both testosterone (T) and 11KT were significantly increased. Our results suggest that 11KT plays an important role in sex change in the honeycomb grouper. Whether the mechanism of 11KT-induced female-to–male sex change acts through direct stimulation of spermatogenesis in the ovary or via the inhibition of estrogen synthesis remains to be clarified.

Sexually Dimorphic Expression of Gonadotropin Subunits in the Pituitary of Protogynous Honeycomb Grouper (Epinephelus merra): Evidence That Follicle-Stimulating Hormone (FSH) Induces Gonadal Sex Change

Recent studies have suggested that the hypothalamic-pituitary-gonadal axis is involved in gonadal sex change in sex-changing teleosts. However, its underlying mechanism remains largely unknown. In this study, we focused on the distinct roles of two gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), in the protogynous hermaphrodite teleost, honeycomb grouper (Epinephelus merra). First, we investigated the expression pattern of mRNAs for GTH subunits (cga, fshb, and lhb) in the pituitaries from fish at the different sexual phases. Real-time RT-PCR analyses showed that fhsb mRNA levels in the female pituitary were low. However, fshb transcripts increased dramatically in association with testis development. In contrast, levels of cga and lhb mRNAs did not significantly vary during sex change. In addition, immunohistochemical observations of Fshb- and Lhb-producing cells in the pituitary, through the use of specific antibodies for detections of teleost GTH subunits, were consistent with sexually dimorphic expression of Fshb. In order to identify the role of GTH in gonad of honeycomb grouper, we treated females with bovine FSH (50 or 500 ng/fish) or LH (500 ng/fish) in vivo. After 3 wk, FSH treatments induced female-to-male sex change and up-regulated endogenous androgen levels and fshb transcripts, whereas LH treatment had no effect on sex change. These results suggest that FSH may trigger the female-to-male sex change in honeycomb grouper.

Immunohistochemical evidence identifying the site of androgen production in the ovary of the protogynous grouper Epinephelus merra.

Androgen plays an important role in the developing ovaries of female fish. However, little is known regarding either the sites of production of androgen or its functional roles. In the present study, we investigated immunohistochemically the localization of cholesterol-side-chain-cleavage (P450scc) and cytochrome P45011β-hydroxylase (P45011β) with antibodies P450scc and P45011β in the ovary of the female honeycomb grouper Epinephelus merra during its reproductive cycle. Clusters of strongly immunopositive cells, with 100–1000 cells in each cluster, against both P450scc and P45011β, were observed throughout the annual reproductive cycle in tissue near blood vessels in the tunica ovary surrounding the outer periphery of the ovary. The ultrastructural characteristics of these cells showed that they were steroid-producing cells. In contrast, immunopositive cells against P450scc but not against P45011β were localized in the theca layer surrounding the outer periphery of oocytes. These results suggest that two distinct steroid biosynthesis sites exist in the ovary and that cells at the two sites differ functionally. The only cells that biosynthesize 11-ketotestosterone are found in clusters in the vicinity of blood vessels; they possibly play a physiological role in oocyte growth and gonadal restructuring during the sex change of individuals of this species.

Isolation, characterization and expression analyses of FSH receptor in protogynous grouper.

Follicle-stimulating hormone (FSH) and its receptor (FSHR) play important roles in spermatogenesis. We cloned and characterized the honeycomb grouper Epinephelus merra FSHR (EmFSHR) to elucidate its role in the protogynous sex change in groupers. Reverse transcription-polymerase chain reaction (RT-PCR) analysis suggested that EmFSHR was expressed exclusively in the gonads. In situ hybridization showed the distribution of EmFSHR in the granulosa cells of previtellogenic oocytes and Leydig cells in the testis. Quantitative reverse transcription PCR (RT-qPCR) analysis of gonadal EmFSHR transcripts during the process of sex change indicated that the lowest levels were found in the female phase before sex change. EmFSHR transcripts increased during the early transitional phase, when oocytes began to degenerate in parallel with the initiation of gonial germ cell differentiation into spermatogonia. A dramatic increase in EmFSHR transcription occurred during the late transitional phase, when the gonad contained numerous proliferating male germ cells and many degenerated oocytes. EmFSHR expression remained high until the transformation from ovary to testis was complete. The data reveal that female to male sex change is associated with the upregulation of EmFSHR transcripts, and that this upregulation may be responsible for the development of testicular tissue and the progression of spermatogenesis. Furthermore, how the upregulation of EmFSHR is controlled in the initiation of sex change remains to be elucidated.

Differentiation of steroid-producing cells and folliculogenesis in the developing ovary of the Nile tilapia Oreochromis niloticus.

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), 3&bgr;-hydroxysteroid dehydrogenase (3&bgr;HSD), 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 3&bgr;-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.