CDNA Cloning And Expression Patterns Of Medaka Gfr?1a/b And Their Roles In Spermatogonial Stem Cells

Spermatogonial stem cells(SSC)are the only adult stem cells for transmission genetics information to offsprings.The great biological significance has made them the focus of scientific research.A large number of studies have confirmed that glial cell line-derived neurotrophic factor(Gdnf)and its receptor complex Gfr?1(Gdnf family receptor anpha 1)/Ret signaling pathway are crucial for the self-renewal and maintenance of SSC in mammals.However,little is known about their roles in fish SSC.Our lab previous study found that medaka Gdnf recombinant protein play an important role in the self-renewal and maintenance in SSC.However,their regulatory mechanisms remain unknown.Our bioinformatic analysis found that Gfr?1 is widely distributed in teleost fish,moreover,there are two duplicated genes,namely Olgfr?1a and b.However,how about their expression patterns and their roles in SSC from teleost fish remains to be further investigated.Medaka features small size and fast breeding.Notably,it has the spermatogonial stem cell line(SG3),which is the only fish SSC line in the world up to date.This cell line has been cultured for 140 generations in vitro,and is still able to stably proliferate and maintain differentiation potential,thus providing an excellent research tool and platform for the research of fish SSC.In this study,the cDNA sequences and expression patterns of Gfr?1a and b from medaka and their roles in SSC were studied.The results are as follows:1.cDNA sequence and sequence analysisThe Olgfr?1a and Olgfr?1b contain a 1575-and 1401-bp open reading frame encoding 524-and 466-amino acid residues(aa),respectively.Both of the predicted Olgfr?1a and Olgfr?1b contain three cysteine-rich domains with a 18-or 17-aa putative signal peptide,3 potential N-glycosylation sites and 31 cysteine residues.Both have the characteristics of the Gdnf receptor family member,including three cysteine-rich domains(D1-3)and the mammalian GDNF-binding characteristic motifs MLF/RRR.Olgfr?1a and Olgfr?1b have identity 52% to each other,and over 52% and 55% to other indicated vertebrates,respectively.On the phylogenetic tree,fish Gfr?1 clustering with tetradpod Gfr?1 proteins form a separate clade,which is distinct from the clades formed by other members of Gfr? family,including Gfr?2,Gfr?3,Gfr?4,Gr?l(Gfr? like)and Gas.In the Gfr?1 clade,fish-specific Gfr?1a and Gfr?1b diverges and forms two fish-specific sub-clades.A cross-species comparison of chromosome neighboring genes reveals that both of Olgfr?1a-and Olgfr?1b-containing regions are syntenic to human Gfr?1-containing chromosome 10.These data support that Olgfr?1a and Olgfr?1b are co-homologs of mammalian GFR?1.2.The m RNA expression profileRT-PCR analyses reveal that the Olgfr?1a and Olgfr?1b transcripts are present in developing embryos through morula to fry.Meantime,both of them were readily detectable in SG3 cells and various adult tissues,high in the SG3 cells,eye,heart,intestine and testis,moderate in the liver,spleen,ovary and brain.In situ hybridization was used to detect the expression level and type of Olgfr?1a and b in the gonad.Our results revealed that Olgfr?1a and b was highly expressed in spermatogonia in the adult testis and was mainly found in the cytoplasm,decreased expression in primary spermatocytes and secondary spermatocytes,barely detectable in spermtids and sperm.At higher magnification,the Olgfr?1a RNAs were mainly distributed in the cytoplasm,whilst Olgfr?1b in both of the cytoplasm and nucleus.In the ovary,Olgfr?1a and b were observed in oocytes at different phases.These data suggest that Olgfr?1a and Olgfr?1b exhibit similar expression profile in the adult testis and are potential markers of medaka male germ cells at premeiotic stages.3.The role of Gfr?1 in SSC(1)The specificity of Gfr?1 Vivo-MorpholinoTo test the specificity of vivo-morpholino,1189 bp and 1055 bp containing Gfr?1a Mo and Gfr?1b Mo target sequence,respectively,were inserted in-frame to Ds Red open reading frame in p Ds Red backbone,resulting in construction of the reporter vectors p CV1a-Ds Red and p CV1b-Ds Red.The SG3 cells were transfected with the control p CV-Ds Red and the reporter vectors p CV1a-Ds Red and p CV1b-Ds Red by Trans IT-2020,respectively.At 2 h post transfection,vivo-morpholino oligos were added into the cells,respectively.Notably,Gfr?1a Mo and Gfr?1b Mo dramatically reduced the Ds Red signal of their corresponding reporter vector by over 90% rate.These data suggest that Gfr?1a Mo and Gfr?1b Mo show high specificity to their target sequence with high efficiency,respectively.(2)The proliferation activity of SG3The proliferative activity of SG3 cells was measured by Ed U incorporation staining.The proliferation activity of cells showed no significant reduction with the knockdown of Gfr?1a or Gfr?1b as compared with that of the control in 5N medium.In 5N containing medaka Gdnfa culture media,the proliferation activity of cells showed a significant reduction with the knockdown of Gfr?1a and/or Gfr?1b as compared with that of the control,while the lowest with the knockdown of Gfr?1a and Gfr?1b.In 5N containing medaka Gdnfb culture media,the proliferation activity of cells showed a significant reduction with the knockdown of Gfr?1a,and no significant reduction with the knockdown of Gfr?1b as compared with that of the control.In addition,it also suggests that Gfr?1a can bind Gdnfa or b,while Gfr?1b only binds Gdnfa but not Gdnfb.(3)The cell survival of SG3The SG3 cells were treated with 5 ?M Gfr?1a Mo and/or Gfr?1b Mo in 5N plus Gdnfa(10 ng/ml)and Gdnfb(10 ng/ml)medium for 48 h,and then were measured by Annexin V-FITC/PI staining assay.Notably,The cells showed a significant increase of apoptosis only with the knockdown both of Gfr?1a and Gfr?1b as compared with that of the control,whilst no significant increase of apoptosis with the knockdown either of Gfr?1a or Gfr?1b.Our results show that Gfr?1a and Gfr?1b have important effects on the survival of SG3,and the functions of the two can compensate each other.(4)The m RNA expression level of pluripotency and differentiation related genesThe m RNA expression levels of SG3 with the knockdown of Gfr?1a and/or Gfr?1b in ESM2.SG3 were administrated with Gfr?1a Mo and/or Gfr?1b Mo in ESM2 medium and then total RNAs were harvested after 7 days treatment for quantitative RT-PCR analyses.The m RNA expression levels of pluripotency-related genes including klf4,lin28 b,bcl6b,etv5 showed a significant decrease as compared with that of the control with the knockdown of Gfr?1a and/or Gfr?1b.Meanwhile,the m RNA expression levels of differentiation-related gene such as c-kit appeared significant increase only with the knockdown both of Gfr?1a and Gfr?1b.Our results suggest that Gfr?1a and Gfr?1b may mediate the self-renewal and maintenance of SG3 by up-regulating pluripotency-related genes and inhibiting the expression of differentiation genes.(5)The proliferation activity of spermatogonia in testisThe testicular tissues were treated with Gfr?1a Mo(2.5 ?M)and Gfr?1b Mo(2.5 ?M).After 3 days treatment,the proliferation activity was measured with Ed U incorpation assay.Con Mo(5 ?M)was added as the control.The proliferation activity of spermatogonial showed a significant reduction with the knockdown of Gfr?1a and Gfr?1b as compared with that of the control.Thus,Our results show that knockdown of Gfr?1a Mo and Gfr?1b Mo can inhibit the proliferation of spermatogonia in the testis.Our study firstly demonstrated that both Gfr?1a and b play an important role in the self-renewal and maintenance of SSC in teleost fish.This study will not only provides important reference for the study of proliferation and differentiation of SSC,but also promotes the application of SSC in transgenic animal production,genetic information preservation of rare and good fishes.