A long noncoding RNA, LncMyoD, modulates chromatin accessibility to regulate muscle stem cell myogenic lineage progression.

Epigenetics regulation plays a critical role in determining cell identity by controlling the accessibility of lineage-specific regulatory regions. In muscle stem cells, epigenetic mechanisms of how chromatin accessibility is modulated during cell fate determination are not fully understood. Here, we identified a long noncoding RNA, LncMyoD, that functions as a chromatin modulator for myogenic lineage determination and progression. The depletion of LncMyoD in muscle stem cells led to the down-regulation of myogenic genes and defects in myogenic differentiation. LncMyoD exclusively binds with MyoD and not with other myogenic regulatory factors and promotes transactivation of target genes. The mechanistic study revealed that loss of LncMyoD prevents the establishment of a permissive chromatin environment at myogenic E-box-containing regions, therefore restricting the binding of MyoD. Furthermore, the depletion of LncMyoD strongly impairs the reprogramming of fibroblasts into the myogenic lineage. Taken together, our study shows that LncMyoD associates with MyoD and promotes myogenic gene expression through modulating MyoD accessibility to chromatin, thereby regulating myogenic lineage determination and progression.

Interaction of Influenza A Nucleoprotein with Host hnRNP-C Is Implicated in Viral Replication.

The host interactome of influenza viral proteins is ever-expanding. In this work, we report the identification of host heterogeneous nuclear ribonucleoprotein C (hnRNP-C) as an interacting partner of influenza A virus nucleoprotein (NP). We confirmed that this interaction exists across different influenza A subtypes and strains. Using biochemical methods, we determined that hnRNP-C interacts with NP via its C-terminal auxiliary domain. Further, we determined that the hnRNP-C is a negative regulator of influenza viral growth. Its interaction with NP is implicated in the promotion of host cell apoptosis during viral infection. It is the first time that the interaction between influenza nucleoprotein and host heterogeneous nuclear ribonucleoprotein C is characterized in detail. Overall, these findings not only characterize the interaction between NP and its host interacting partner hnRNP-C but also clarify the functional significance of this interaction. This work may lead to a new therapeutic target for the development of anti-influenza drugs.

Exchange protein directly activated by cAMP (Epac) 1 plays an essential role in stress-induced exercise capacity by regulating PGC-1α and fatty acid metabolism in skeletal muscle.

Exchange protein directly activated by cAMP (Epac) mediates cAMP-mediated cell signal independent of protein kinase A (PKA). Mice lacking Epac1 displayed metabolic defect suggesting possible functional involvement of skeletal muscle and exercise capacity. Epac1 was highly expressed, but not Epac 2, in the extensor digitorum longus (EDL) and soleus muscles. The exercise significantly increased protein expression of Epac 1 in EDL and soleus muscle of wild-type (WT) mice. A global proteomics and pathway analyses revealed that Epac 1 deficiency mainly affected "the energy production and utilization" process in the skeletal muscle. We have tested their forced treadmill exercise tolerance. Epac1-/- mice exhibited significantly reduced exercise capacity in the forced treadmill exercise and lower number of type 1 fibers than WT mice. The basal protein level of proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) was reduced in the Epac1-/- mice. Furthermore, increasing expression of PGC-1α by exercise was also significantly attenuated in the skeletal muscle of Epac1-/- mice. The expressions of downstream target genes of PGC-1α, which involved in uptake and oxidation of fatty acids, ERRα and PPARδ, and fatty acid content were lower in muscles of Epac1-/-, suggesting a role of Epac1 in forced treadmill exercise capacity by regulating PGC-1α pathway and lipid metabolism in skeletal muscle. Taken together, Epac1 plays an important role in exercise capacity by regulating PGC-1α and fatty acid metabolism in the skeletal muscle.

Sprouty4 mediates amphiregulin-induced down-regulation of E-cadherin and cell invasion in human ovarian cancer cells.

Sprouty (SPRY) proteins are well-characterized factors that inhibit receptor tyrosine kinase (RTK)-mediated activation of cellular signaling pathways. The down-regulation of SPRY4 expression has been reported in human ovarian cancer. However, the specific roles and mechanisms by which SPRY4 affects ovarian cancer progression are completely unknown. Amphiregulin (AREG) binds exclusively to the epidermal growth factor receptor (EGFR) and has been considered to be a dominant autocrine/paracrine EGFR ligand in ovarian cancer. In the present study, we first examined the effects of AREG on SPRY4 expression and the possible underlying molecular mechanisms involved in this process in two human ovarian cancer cell lines. Our results demonstrated that treatment with AREG up-regulated SPRY4 expression by activating the ERK1/2 signaling pathway. In addition, we showed that small interfering RNA (siRNA)-mediated knockdown of SPRY4 attenuated the AREG-induced down-regulation of E-cadherin by inhibiting the expression of SNAIL but not SLUG. In contrast, overexpression of SPRY4 enhanced AREG-induced down-regulation of E-cadherin by increasing the expression of SNAIL. Moreover, SPRY4 knockdown attenuated AREG-induced cell migration and invasion. Overexpression of SPRY4 enhanced AREG-induced cell invasion. This study reveals that SPRY4 is involved in EGFR-mediated human ovarian cancer progression.

Transforming growth factor-α induces human ovarian cancer cell invasion by down-regulating E-cadherin in a Snail-independent manner.

Transforming growth factor-α (TGF-α), like epidermal growth factor (EGF) and amphiregulin (AREG) binds exclusively to EGF receptor (EGFR). We have previously demonstrated that EGF, AREG and TGF-α down-regulate E-cadherin and induce ovarian cancer cell invasion, though whether these ligands use the same molecular mediators remains unknown. We now show that, like EGF, TGF-α- and AREG-induced E-cadherin down-regulation involves both EGFR and HER2. However, in contrast to EGF and AREG, the transcription factor Snail is not required for TGF-α-induced E-cadherin down-regulation. This study shows that TGF-α uses common and divergent molecular mediators to regulate E-cadherin expression and cell invasion.

Global chromatin accessibility profiling analysis reveals a chronic activation state in aged muscle stem cells.

Regulation of chromatin accessibility is critical for cell fate decisions. Chromatin structure responds to extrinsic environments rapidly. The traditional adult stem cell isolation approach requires tissue dissociation, which triggers stem cell activation and leads to alterations in chromatin structure. To preserve the in vivo chromatin states, we utilized the PFA-perfusion-based isolation approach and characterized the DNA regulatory landscapes during muscle stem cell quiescence exit and aging. We showed that aged SCs display a chronically activated chromatin signature. Detailed analysis of the chromatin accessibility profiles identified key enhancer elements for SC quiescence. Constant activation of the enhancer elements promotes stemness and prevents SCs from differentiation, whereas genetic deletion causes cell-cycle arrest and leads to defects in activation. Our comprehensive characterization of the chromatin accessibility and transcriptomic landscapes in SC quiescence and aging broadens our understanding of these processes and identifies key distal regulatory elements for SC function.

CPEB1 directs muscle stem cell activation by reprogramming the translational landscape.

Skeletal muscle stem cells, also called Satellite Cells (SCs), are actively maintained in quiescence but can activate quickly upon extrinsic stimuli. However, the mechanisms of how quiescent SCs (QSCs) activate swiftly remain elusive. Here, using a whole mouse perfusion fixation approach to obtain bona fide QSCs, we identify massive proteomic changes during the quiescence-to-activation transition in pathways such as chromatin maintenance, metabolism, transcription, and translation. Discordant correlation of transcriptomic and proteomic changes reveals potential translational regulation upon SC activation. Importantly, we show Cytoplasmic Polyadenylation Element Binding protein 1 (CPEB1), post-transcriptionally affects protein translation during SC activation by binding to the 3' UTRs of different transcripts. We demonstrate phosphorylation-dependent CPEB1 promoted Myod1 protein synthesis by binding to the cytoplasmic polyadenylation elements (CPEs) within its 3' UTRs to regulate SC activation and muscle regeneration. Our study characterizes CPEB1 as a key regulator to reprogram the translational landscape directing SC activation and subsequent proliferation.

Rapid effect of GNRH1 on follicle-stimulating hormone beta gene expression in LbetaT2 mouse pituitary cells requires the progesterone receptor.

Gonadotropin-releasing hormone (GNRH) activates the progesterone receptor (PGR) in pituitary cells and accentuates gonadotropin expression. We show that GNRH1 increases Fshb mRNA levels in LbetaT2 mouse pituitary cells within 8 h and is three times more effective than GNRH2. By contrast, GNRH1 and GNRH2 do not affect Lhb gene expression in these cells. Within the same time frame, small interfering RNA (siRNA) knockdown of the PGR in LbetaT2 cells reduced GNRH1 activation of a PGR response element (PRE)-driven luciferase reporter gene and Fshb mRNA levels by >50%. Chromatin immunoprecipitation (ChIP) assays also demonstrated that PGR loading on the PRE within the Fshb gene promoter in LbetaT2 cells occurred within 8 h after GNRH1 treatment and was lost by 24 h. While the GNRH1-induced upregulation of the PRE reporter gene and Fshb mRNA levels was attenuated by cotreatment with protein kinase A (H-89) and protein kinase C (GF109203X) inhibitors, only GF109203X inhibited PGR phosphorylation at Ser249 in LbetaT2 cells. Immunoprecipitation assays also showed a progressive increase in the interaction between the PGR and its coactivator NCOA3 that peaked at 8 h coincident with the increase in Fshb mRNA after GNRH1 treatment. The siRNA-mediated knockdown of NCOA3 in LbetaT2 cells also reduced Fshb mRNA levels after GNRH1 treatment and loading of NCOA3 on the Fshb promoter PRE in a ChIP assay. We conclude that the rapid effect of GNRH1 on Fshb expression in LbetaT2 cells is mediated by PGR phosphorylation and loading at the PRE within the Fshb promoter together with NCOA3.

Characterization of inhibin alpha subunit (inha) in the zebrafish: evidence for a potential feedback loop between the pituitary and ovary.

Inhibin and activin are closely related disulphide-linked dimers that belong to the transforming growth factor beta superfamily. Although inhibin has been extensively studied in mammals, the information about its existence and function in lower vertebrates is very scarce. Using zebrafish as a model, the present study demonstrated that the inhibin-specific alpha subunit (inha) was predominantly expressed in the gonads and no transcript could be detected in other tissues including the pituitary and brain. In the ovary, the expression of inha was restricted to the somatic follicle cells surrounding the oocyte, together with the beta subunits (inhbaa and inhbb). This was further supported by the absence of its expression in the ovulated unfertilized eggs. During folliculogenesis, inha expression in the follicles slightly but steadily increased from primary growth to the mid-vitellogenic stage; however, its expression surged dramatically at the full-grown stage. Interestingly, the expression level of inha decreased significantly in the follicles whose oocytes were undergoing spontaneous maturation or germinal vesicle breakdown. When tested on cultured ovarian fragments, both goldfish pituitary extract and forskolin significantly stimulated inha expression. Further experiments showed that recombinant zebrafish FSH but not LH significantly increased inha expression in the same assay system. When tested in vitro, human inhibin A exhibited a slight but significant inhibitory effect on 17alpha, 20beta-dihydroxyprogesterone-induced oocyte maturation after 4 h incubation. The stimulation of inha expression by FSH and the potential inhibition of FSH by inhibin suggest a possible existence of a negative feedback loop between the pituitary and ovary in the zebrafish.

Temporal recruitment of transcription factors at the 3',5'-cyclic adenosine 5'-monophosphate-response element of the human GnRH-II promoter.

GnRH-II is a potent GnRH subtype involved in modulating OVCAR-3 cell proliferation and the invasive properties of JEG-3 cells, and an atypical cAMP-response element (CRE) in the human GnRH-II promoter influences its activation. We demonstrated that the GnRH-II promoter is activated by 8-bromoadenosine-cAMP in several cell lines including alphaT3, TE671, JEG-3, and OVCAR-3 cells and that cAMP enhances GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Moreover, 8-bromoadenosine-cAMP increases cAMP response element-binding protein (CREB) phosphorylation in JEG-3 and OVCAR-3 cells and augments CBP and CCAAT/enhancer-binding protein (C/EBP)-beta coimmunoprecipitation with phosphorylated CREB (p-CREB) in a temporally defined manner from nuclear extracts. When CREB, CBP, and C/EBPbeta levels were knocked down by small interfering RNA, reductions in any of these transcription factors reduced cAMP-enhanced GnRH-II promoter activity and GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Importantly, chromatin immunoprecipitation assay showed that p-CREB bound the CRE within the endogenous GnRH-II promoter within 1 h and that p-CREB association with C/EBPbeta occurs within 2 h of cAMP stimulation, coincident with the first appearance of C/EBPbeta at the CRE. By contrast, maximum interactions between p-CREB and CBP do not occur until at least 4 h after cAMP stimulation, and this is reflected in the progressive loading of CBP at the CRE at 2-4 h, as demonstrated by chromatin immunoprecipitation. Taken together, these data suggest that p-CREB, C/EBPbeta, and CBP are recruited to the CRE of the GnRH-II promoter in a temporarily defined manner to enhance its transcription in JEG-3 and OVCAR-3 cells in response to cAMP.

Molecular Regulation of Cellular Quiescence: A Perspective from Adult Stem Cells and Its Niches.

Cellular quiescence is a reversible growth arrest state. In response to extracellular environment, quiescent cells are capable of resuming proliferation for tissue homeostasis and tissue regeneration. Subpopulations of adult stem cells remain quiescent and reside in their specialized stem cell niches. Within the niche, they interact with a repertoire of niche components. Niche integrates signals to maintain quiescence or gear stem cells toward regeneration. Recent studies provide insights into the regulatory components of stem cell niche and their influence on residing stem cells. Aberrant niche activities perturb stem cell quiescence and activation, compromise stem cell functions, and contribute to tissue aging and disease pathogenesis. This review covers current knowledge regarding cellular quiescence with a focus on original and emerging concepts of how niches influence stem cell quiescence.

Sprouty2 inhibits amphiregulin-induced down-regulation of E-cadherin and cell invasion in human ovarian cancer cells.

Similar to Drosophila Sprouty (SPRY), mammalian SPRY proteins inhibit the receptor tyrosine kinase-mediated activation of cellular signaling pathways. SPRY2 expression levels have been shown to be down-regulated in human ovarian cancer, and patients with low SPRY2 expression have significantly poorer survival than those with high SPRY2 expression. In addition, epidermal growth factor receptor (EGFR) is overexpressed in human ovarian cancer and is associated with more aggressive clinical behavior and a poor prognosis. Amphiregulin (AREG), the most abundant EGFR ligand in ovarian cancer, binds exclusively to EGFR and stimulates ovarian cancer cell invasion by down-regulating E-cadherin expression. However, thus far, the roles of SPRY2 in AREG-regulated E-cadherin expression and cell invasion remain unclear. In the present study, we show that treatment with AREG up-regulated SPRY2 expression by activating the EGFR-mediated ERK1/2 signaling pathway in two human ovarian cancer cell lines, SKOV3 and OVCAR5. In addition, overexpression of SPRY2 attenuated the AREG-induced down-regulation of E-cadherin by inhibiting the induction of the E-cadherin transcriptional repressor, Snail. Moreover, SPRY2 overexpression attenuated AREG-stimulated cell invasion and proliferation. This study reveals that SPRY2 acts as a tumor suppressor in human ovarian cancer and illustrates the underlying mechanisms that can be used as possible targets for the development of novel therapeutics.

Loss of Sprouty2 in human high-grade serous ovarian carcinomas promotes EGF-induced E-cadherin down-regulation and cell invasion.

Sprouty (SPRY) proteins are well-characterized factors that inhibit receptor tyrosine kinase signaling. Our Human Exonic Evidence-Based Oligonucleotide (HEEBO) microarray results showed that the mRNA levels of SPRY2, but not of SPRY1 or SPRY4, are down-regulated in high-grade serous ovarian carcinoma (HGSC) tissues and epithelial ovarian cancer (EOC) cell lines. Molecular inversion probe (MIP) copy number analysis showed the deletion of the SPRY2 locus in HGSC. Overexpression of SPRY2 reduced EGF-induced cell invasion by attenuating EGF-induced E-cadherin down-regulation. Moreover, a positive correlation between SPRY2 and E-cadherin protein levels was observed in HGSC tissues. This study reveals the loss of SPRY2 in HGSC and indicates an important tumor-suppressive role for SPRY2 in mediating the stimulatory effect of EGF on human EOC progression.

Amphiregulin induces human ovarian cancer cell invasion by down-regulating E-cadherin expression.

Aberrant epidermal growth factor receptor (EGFR) activation is associated with ovarian cancer progression. In this study, we report that the EGFR ligand amphiregulin (AREG) stimulates cell invasion and down-regulates E-cadherin expression in two human ovarian cancer cell lines, SKOV3 and OVCAR5. In addition, AREG increases the expression of transcriptional repressors of E-cadherin including SNAIL, SLUG and ZEB1. siRNA targeting SNAIL or SLUG abolishes AREG-induced cell invasion. Moreover, ERK1/2 and AKT pathways are involved in AREG-induced E-cadherin down-regulation and cell invasion. Finally, we show that three EGFR ligands, AREG, epidermal growth factor (EGF) and transforming growth factor-α (TGF-α), exhibit comparable effects in down-regulating E-cadherin and promoting cell invasion. This study demonstrates that AREG induces ovarian cancer cell invasion by down-regulating E-cadherin expression.

Fibroblast growth factor 2 induces E-cadherin down-regulation via PI3K/Akt/mTOR and MAPK/ERK signaling in ovarian cancer cells.

Fibroblast growth factor 2 (FGF2) is produced by ovarian cancer cells and it has been suggested to play an important role in tumor progression. In this study, we report that FGF2 treatment down-regulated E-cadherin by up-regulating its transcriptional repressors, Slug and ZEB1, in human ovarian cancer cells. The pharmacological inhibition of phosphatidylinositol-3-kinase (PI3K), mammalian target of rapamycin (mTOR), and MEK suggests that both PI3K/Akt/mTOR and MAPK/ERK signaling are required for FGF2-induced E-cadherin down-regulation. Moreover, FGF2 up-regulated Slug and ZEB1 expression via the PI3K/Akt/mTOR and MAPK/ERK signaling pathways, respectively. Finally, FGF2-induced cell invasion was abolished by the inhibition of the PI3K/Akt/mTOR and MAPK/ERK pathways, and the forced expression of E-cadherin diminished the intrinsic invasiveness of ovarian cancer cells as well as the FGF2-induced cell invasion. This study demonstrates a novel mechanism in which FGF2 down-regulates E-cadherin expression through the activation of PI3K/Akt/mTOR and MAPK/ERK signaling, and the up-regulation of Slug and ZEB1 in human ovarian cancer cells.

Integrin ß1 mediates epithelial growth factor-induced invasion in human ovarian cancer cells.

Integrins function as cell-extracellular matrix adhesion proteins and have been implicated in tumor progression. In ovarian tumors, elevated integrin ß1 expression correlates with high clinical stage and poor patient survival. In this study, we report that EGF treatment up-regulated integrin ß1 mRNA and protein levels in ovarian cancer cells. Moreover, pharmacological inhibition of MEK totally abolished EGF-induced integrin ß1 up-regulation and cell invasion suggesting that MAPK/ERK signaling is required for EGF-induced integrin ß1 up-regulation and cell invasion. Furthermore, we found that knockdown of integrin ß1 expression reduced the intrinsic invasiveness of ovarian cancer cells and the EGF-induced cell invasion. Finally, we found that overexpression of integrin ß1 was sufficient to promote ovarian cancer cell invasion. This study demonstrates that integrin ß1 mediates EGF-induced cell invasion in ovarian cancer.

Gonadotropin-releasing hormone-I-mediated activation of progesterone receptor contributes to gonadotropin alpha-subunit expression in mouse gonadotrophs.

In pituitary cells, cross talk between GnRH-I and the progesterone receptor accentuates gonadotropin production. We show that GnRH-I activates a progesterone response element (PRE)-driven luciferase reporter gene at 8 h and gonadotropin alpha-subunit (gsu alpha) gene expression at 24 h in two mouse gonadotrope cell lines, alpha T3-1 and L beta T2. In alpha T3-1 cells, progesterone had an additive effect on GnRH-I-induced PRE-luciferase reporter gene activity but not on GSU alpha mRNA levels. However, progesterone had no synergistic effect on the GnRH-I-induced expression of these genes in L beta T2 cells. Up-regulation of the PRE-luciferase reporter gene by GnRH-I was attenuated by pretreatment with protein kinase A (H89) and protein kinase C (GF109203X) inhibitors in both cell lines, whereas only GF109203X inhibited GnRH-I-induced GSU alpha mRNA levels. Most important, in both cell lines within the same time frame, knockdown of progesterone receptor levels by small interfering RNA reduced GnRH-I activation of GSU alpha mRNA levels by approximately 40%. We conclude that the effect of GnRH-I on gsu alpha expression in both alpha T3-1 and L beta T2 cells is mediated by ligand-independent activation of progesterone receptor and that this contributes to the self-priming effect of GnRH-I in the pituitary.

Gonadotropin-releasing hormone and ovarian cancer: a functional and mechanistic overview.

The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) is well known for its role in the control of pituitary gonadotropin secretion, but the hormone and receptor are also expressed in extrapituitary tissues and tumor cells, including epithelial ovarian cancers. It is hypothesized that they may function as a local autocrine regulatory system in nonpituitary contexts. Numerous studies have demonstrated a direct antiproliferative effect on ovarian cancer cell lines of GnRH and its synthetic analogs. This effect appears to be attributable to multiple steps in the GnRH signaling cascade, such as cell cycle arrest at G(0)/G(1). In contrast to GnRH signaling in pituitary gonadotropes, the involvement of G(alpha q), protein kinase C and mitogen-activated protein kinases is less apparent in neoplastic cells. Instead, in ovarian cancer cells, GnRH receptors appear to couple to the pertussis toxin-sensitive protein G(alpha i), leading to the activation of protein phosphatase, which in turn interferes with growth factor-induced mitogenic signals. Apoptotic involvement is still controversial, although GnRH analogs have been shown to protect cancer cells from doxorubicin-induced apoptosis. Recently, data supporting a regulatory role of GnRH analogs in ovarian cancer cell migration/invasion have started to emerge. In this minireview, we summarize the current understanding of the antiproliferative actions of GnRH analogs, as well as the recent observations of GnRH effects on ovarian cancer cell apoptosis and motogenesis. The molecular mechanisms that mediate GnRH actions and the clinical applications of GnRH analogs in ovarian cancer patients are also discussed.

Zebrafish gonadotropins and their receptors: II. Cloning and characterization of zebrafish follicle-stimulating hormone and luteinizing hormone subunits--their spatial-temporal expression patterns and receptor specificity.

Gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) play critical roles in vertebrate reproduction. In the present study, we cloned and characterized zebrafish FSHbeta (fshb), LHbeta (lhb), and GTHalpha (cga) subunits. Compared with the molecules of other teleosts, the cysteine residues and potential glycosylation sites are fully conserved in zebrafish Lhb and Cga but not in Fshb, whose cysteines exhibit unique distribution. Interestingly, in addition to the pituitary, fshbeta, lhbeta, and cga were also expressed in some extrapituitary tissues, particularly the gonads and brain. In situ hybridization showed that zebrafish fshbeta and lhbeta were expressed in two distinct populations of gonadotrophs in the pituitary. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that all the three subunits increased expression before ovulation (0100-0400) when the germinal vesicles in the full-grown follicles were migrating toward the periphery, but the levels dropped at 0700, when ovulation occurred. Recombinant zebrafish FSH (zfFSH) and LH (zfLH) were produced in the Chinese hamster ovary (CHO) cells and their effects on the cognate receptors (zebrafish Fshr and Lhr) tested. Interestingly, zfFSH specifically activated zebrafish Fshr expressed together with a cAMP-responsive reporter gene in the CHO cells, whereas zfLH could stimulate both Fshr and Lhr. In conclusion, the present study systematically investigated gonadotropins in the zebrafish in terms of their structure, spatial-temporal expression patterns, and receptor specificity. These results, together with the availability of recombinant zfFSH and zfLH, provide a solid foundation for further studies on the physiological relevance of FSH and LH in the zebrafish, one of the top biological models in vertebrates.

Zebrafish gonadotropins and their receptors: I. Cloning and characterization of zebrafish follicle-stimulating hormone and luteinizing hormone receptors--evidence for their distinct functions in follicle development.

In the present study, we cloned and characterized zebrafish FSH receptor (Fshr) and LH receptor (Lhr). Both fshr and lhr were abundantly expressed in the zebrafish gonads; however, they could also be detected in the kidney and liver, respectively. When overexpressed in mammalian cell lines together with a cAMP-responsive reporter gene, zebrafish Fshr responded to goldfish pituitary extract but not hCG, whereas Lhr could be activated by both. It was further demonstrated that Fshr was specific to bFSH, while Lhr could be stimulated by both bovine FSH and LH. Low level of fshr expression could be detected in the immature ovary, but the level steadily increased during vitellogenesis of the first cohort of developing follicles. In contrast, the expression of lhr could barely be detected in the immature ovary, but it became detectable at the beginning of vitellogenesis and steadily increased afterward with the peak level reached at the full-grown stage. At the follicle level, the expression of fshr was very weak in the follicles of primary growth stage but significantly increased with the follicles entering vitellogenesis. However, after reaching the maximal level in the midvitellogenic follicles, the level of fshr expression dropped slightly but significantly at the full-grown stage. In comparison, the expression of lhr obviously lagged behind that of fshr. Its expression became detectable only when the follicles started to accumulate yolk granules, but the level rose steadily afterward and reached the peak at the full-grown stage before oocyte maturation. These results suggest differential roles for Fshr and Lhr in zebrafish ovarian follicle development.