Ontogeny of Immunoreactive Lh and Fsh Cells in Relation to Early Ovarian Differentiation and Development in Protogynous Hermaphroditic Ricefield Eel Monopterus albus1

Abstract

Luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) control many aspects of gonadal development and function in teleosts. In the present paper, the specific antisera against ricefield eel Lhb (Lh beta subunit), Fshb (Fsh beta subunit), and Cga (the common pituitary glycoprotein hormone alpha subunit) were generated, and the cellular localization, initial appearance, and subsequent development of gonadotrophs in relation to early ovarian differentiation and development in the ricefield eel, a protogynous sex-changing teleost, were examined with immunochemistry. Lhb- and Fshb-immunoreactive signals were identified in distinct pituitary cells that occupied primarily the peripheral regions of the adenohypophysis. During ontogeny, Lhb-immunoreactive signals were first detected in the pituitary around 40 days after hatching (dah) when the oogonia transitioned into early primary growth oocytes, and the intensity of immunoreactivity increased concomitantly with the growth of primary oocytes from 60 to 140 dah. During overwintering from 170 to 230 dah, Lhb-immunoreactive signals were significantly decreased when a large proportion of perinucleolus oocytes contained intense Balbiani bodies. In contrast, Fshb-immunoreactive signals were not detectable in the pituitary until around 230 dah (in the spring after hatching) and slightly increased from 285 dah when the late perinucleolus oocytes began to enter the secondary growth phase. Both Lhb- and Fshb-immunoreactive cells were increased when the early cortical alveoli oocytes emerged at 300 dah. The mRNA expression of lhb and fshb coincided with their immunoreactive signals. Taken together, these results suggest that only Lh is involved in primary oocyte growth in ricefield eels, but both Fsh and Lh are important for the secondary ooctye growth.

Foxo3b but not Foxo3a activates cyp19a1a in Epinephelus coioides

“…The cDNA fragments were subcloned into the expression vector pET32a via NcoI and XhoI sites, and expressed in the host E. coli BL21 (DE3) as TRX fusion proteins by the induction of isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant EcFoxo3a and EcFoxo3b antigens were gel purified and used to immunize Balb/c mouse as previously reported (Wu et al 2012).…”

Scp3 expression in relation to the ovarian differentiation in the protogynous hermaphroditic ricefield eel Monopterus albus

“…To confirm the specificity of the anti-Scp3 antiserum, Western blot analysis was performed using the preadsorbed antiserum with excess recombinant ricefield eel Scp3 antigen. Immunohistochemistry Immunohistochemical analysis of Scp3 expression and localization in ricefield eels was performed essentially as previously described Wu et al 2012), with some modifications. Briefly, the sections were incubated at room temperature for 2 h with a 1:1000 dilution of the primary anti-Scp3 antiserum.…”

Cytoplasmic Localization of Lrh-1 Down-Regulates Ovarian Follicular cyp19a1a Expression in a Teleost, the Orange-Spotted Grouper Epinephelus coioides1

“…The recombinant Cyp19a1a antigen was gel purified from the inclusion bodies, while the Lrh-1 antigen was purified first by His Bind Purification Kit (Novagen, Merck KGaA) and then further by SDS-PAGE. The purified antigens were used to immunize rabbits and BALB/c mouse as previously reported [21].…”

“…The method was modified from a protocol used in our previous study [21]. The ovarian sections (6 lm) were deparaffinized, hydrated, and incubated with 3% hydrogen peroxide solution to quench the endogenous peroxidase activity, followed by antigen retrieval in 10 mM citrate buffer (pH 6.0) at 958C for 15 min and blocking in 0.01 M PBS containing 10% normal goat serum for 30 min at room temperature.…”

“…Double IHC was performed as described previously [21]. Briefly, deparaffinized sections were boiled for 15 min in 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval and blocked in 0.01 M PBS containing 10% normal goat serum for 30 min at room temperature.…”