Differential expression patterns of the two paralogous Rec8 from Nile tilapia and their responsiveness to retinoic acid signaling.

REC8 (meiotic recombination protein 8) is an essential component of meiotic cohesion complexes. Interestingly, two paralogous rec8 genes happen to exist in the stra8 (stimulated by retinoic acid gene 8)-absent fishes but not in stra8-existing fishes. Stra8 is usually considered as the prerequirement during RA (retinoic acid)-mediated meiosis initiation in mammals. However, how RA triggers meiosis in the stra8-absent fishes just like Nile tilapia (Oreochromis niloticus) remains elusive. Here we characterized the two paralogous rec8 genes in Nile tilapia (Onrec8a and Onrec8b), and investigated their expression patterns and responsiveness to RA signaling by treatment of ex vivo testicular culture and promoter luciferase reporter assay. OnRec8a and OnRec8b share 36% identity to each other and are true orthologs of REC8. Their expression was predominantly restricted to meiotic germline cells with differential spatiotemporal patterns. During spermatogenesis, OnRec8b predominantly exhibited nuclear expression in spermatocytes from 60 dah (days after hatching), while OnRec8a exhibited cytoplasmic expression from 90 dah. During oogenesis, OnRec8a was expressed from 30 dah, while OnRec8b from 90 dah. Further study shows that RA signaling could upregulate the expression of both Onrec8a and Onrec8b. Collectively, our data implies that OnRec8a and OnRec8b might have differential function during meiosis and be involved in RA-mediated meiosis program.

Metabolic Profile of Dendrobine in Rats Determined by Ultra-high-performance Liquid Chromatography/Quadrupole Time-of-flight Mass Spectrometry.

AIMS: Dendrobine is a major alkaloid present mainly in dendrobium nobile Lindl. It has been reported to have analgesic, antipyretic, lower heart rate and blood pressure and other pharmacologic activities. Despite its critical pharmacological function, its metabolite profiling is still unclear. METHODS: In this study, the in vivo metabolite profiling of dendrobine in rats was investigated using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS). The metabolites were predicted using MetabolitePilotTM software with a mass defect filter (MDF) technique. These predicted metabolites were further analyzed by MS2 spectra and compared with the detailed fragmentation pathway of the dendrobine standard and literature data. RESULTS: Total of 59 metabolites were identified for the first time in rat plasma and urine after oral administration of dendrobine. Demethylated, dehydrogenated, hydroxylated, ketonizated and glucuronide were the major metabolic pathways. CONCLUSION: This research provides scientific and reliable support for full understanding of the metabolic fate of dendrobine in vivo.

Establishment of a stem Leydig cell line capable of 11-ketotestosterone production.

The deficiency or insufficiency of androgen can trigger a range of reproductive diseases as well as other symptoms. Stem Leydig cells (SLCs) are critical for the formation and maintenance of a functional androgen-producing cell (Leydig cell, LC) population throughout adult male life. However, to date, our knowledge about SLCs is poor. Here we report the derivation and characterisation of a clonal stem LC line (designated as TSL) capable of 11- ketotestosterone (11-KT) production from a 3-month-old Nile tilapia (Oreochromis niloticus) testis. The cells retained stable proliferation after 77 generations with normal karyotype and growth factor dependency. They expressed platelet-derived growth factor receptor-α (pdgfrα), nestin and chicken ovalbumin upstream promoter transcription factor II (coup-tfIIa), which are characteristic of SLCs. Upon induction in defined medium, TSLs could undergo differentiation into steroidogenically active LCs and produce 11-KT. When implanted into recipient Nile tilapia testes from which endogenous LCs had been eliminated by ethane dimethanesulphonate (EDS) treatment, the PKH26-labelled TSLs could colonise the interstitium, subsequently express steroidogenic genes and restore 11-KT production. Taken together, our data suggest that TSLs possess the ability of continuous proliferation and potential of differentiation into functional LCs invitro and invivo. To the best of our knowledge TSL might represent the first stem LC line capable of 11-KT production to date. Our study may offer new opportunities for investigating the self-renewal of SLCs and steroidogenesis invitro, and provide an invaluable invitro model for investigating endocrine disruptors.

Both Gfrα1a and Gfrα1b Are Involved in the Self-renewal and Maintenance of Spermatogonial Stem Cells in Medaka.

Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRα1) plays a crucial role in the self-renewal and maintenance of spermatogonial stem cells (SSCs) from mammals. However, to date, our knowledge about its role in fish SSCs is limited. In the present study, the medaka (Oryzias latipes) gfrα1 duplicate genes, Olgfrα1a and Olgfrα1b, were cloned and characterized. Furthermore, their expression profile and biological activity were investigated. OlGfrα1a and OlGfrα1b predict 524 and 466 amino acid residues, respectively. Both are orthologous to mammalian Gfrα1 by sequence analyses and appear high in spermatogonia by in situ hybridization assay. The knockdown of OlGfrα1a and/or OlGfrα1b via Vivo-Morpholino oligos significantly inhibited the self-renewal and maintenance of SSCs, as evidenced by the decreased proliferation activity of SG3 cells (a spermatogonial stem cell line derived from adult medaka testis) as well as spermatogonia in the testicular organ culture and by the decreased survival rate and expression levels of pluripotency-related genes (klf4, lin28b, bcl6b, and etv5) in SG3 cells. Additionally, our study indicates that OlGfrα1a might function by binding either Gdnfa or Gdnfb (the two medaka Gdnf homologs), whereas OlGfrα1b function by binding Gdnfa not Gdnfb. Taken together, our study indicates that both OlGfrα1a and OlGfrα1b are involved in the self-renewal and maintenance of SSCs by binding Gdnfa and/or Gdnfb, respectively. These findings suggest that the GDNF/GFRα1 signaling pathway might be conserved from mammals to fish species.