Studies Of Functions And Molecular Mechanisms During Spermatogenesis Regulated By Rln3a In Tilapia

Relaxin has been a longstanding concern in the field of reproductive biology.It is reported that Relaxin3(RLN3),a primary neuropeptide,is expressed most dominantly in both the brain and the testis in mammals,and is involved in the regulation of stress response,memory,and appetite and so on.Previous studies proved that the RLN3 knockout mice possessed phenotypes including stress responses and anxiety-related behavior.RLN3 is used for clinical treatments for neurological disorders and depression.In teleosts,two rln3 paralogues emerged during fish specific genome duplication,which were designated as rln3 a and rln3 b.RNA-seq and our unpublished study revealed that rln3 a was dominantly expressed in both brain and testis.However,no data is available regarding the functions of rln3 a in reproductive regulation in vertebrates.In order to elucidate the roles of rln3 a in the fish reproduction,we identified the rln3 a gene,and analyzed its functions in this study by using tilapia as a model animal.Tilapia,an economically important specie for aquaculture,were an ideal model to study fish reproduction and development with a XX/XY sex determination system and the availability of mono sex fish and its open genome sequence,and gonadal transcriptomes at different developmental stages(5,30,90,180 dah).In addition,the effects of steroid hormones(11-KT,DHP),growth factors(IGF3,AMH)and transcription factors(Dmrt1,Sf1)were elucidated in tilapia spermatogenesis in previous research in our group.In this study,male F0 individuals carrying rln3 a gene mutations were crossed with wild type females to obtain F1 offsprings.Subsequently,the phenotypes of testicular changes and sperm were examined by immunohistochemistry,H.E.staining,flow cytometry and electron microscope.Transcriptome analysis of brain and testis were also performed to explore the possible molecular mechanisms of rln3 a mutation in fish spermatogenesis.The expression profiles were confirmed by Real-time PCR and IHC.Herein,the functions and molecular mechanisms of rln3 a in tilapia spermatogenesis was elucidated.The main results are as follows:1)Male F0 individuals carrying rln3 a gene mutations were crossed with wild type females to obtain F1 offsprings.The mutant F1 fish was screened by restriction digestion and sequencing for subsequent analysis.By 90 dah H.E.observation revealed that deficiency of rln3 a resulted in delayed spermatogenesis,disappeared primary spermatocyte and secondary spermatocyte in testis compared with control fish.It was shown that Cyp11b2(male specific marker gene in Leydig cells)and PCNA(marker gene of cell proliferation)was undetectable in the testis of male fish with rln3 a gene mutation by IHC.These expression patterns show that mutation of rln3 a gene led to the delay of initiation of spermatogenesis,and proliferation of spermatogonia and spermatocytes.2)H.E.staining was carried out to analyze the changes of testicular morphology in adults with rln3 a gene mutation.Results showed that deletion of rln3 a resulted in the disordered organization of seminiferous lobules,and decreased spermatid production compared with control fish.Consistently,compared with the control fish,abnormal cavity and smaller testis was observed in rln3 a gene mutated male by scanning electron microscope.Transmission electron microscope analysis revealed that mutation of rln3 a resulted in the severe reduction of spermatids and sperm.However,the morphology of spermatogonias and spermatocytes in rln3 a mutation was similar to control.3)Offsprings obtained by crossing rln3 a mutant XY fish with normal XX in F1 fish was lethal by 2 dah(blastocysts)nearly all suggesting the sterility of sperm from rln3 a gene mutant fish..To further investigate the effects of rln3 a mutation on sperm fertility in F1 fish,the semen were squeezed out and used for flow cytometry analysis.Our data indicated that mutation of rln3 a led to the diminishment of characteristics of sperm(FSC and SSC)comparing with the control.Through the sperm motility analysis,we found that sperm motility were significantly reduced in rln3 a mutation fish.Furthermore,the sperm count in rln3 a mutant testis was significantly decreased than control testis.At the same time,the level of serum 11-KT,FSH and LH were remarkably depressed in rln3 a mutant F1 fish than control group.4)Furthermore,transcriptomes of both brain and testis from rln3 a mutant fish and control were sequenced using Illumina HiseqTM technology.Comparative analysis of brain and gonadal transcriptome between the rln3 a mutation and control was carried out to screen differentially expressed genes(DEGs).To further assign putative functions to DEGs,we performed GO and KEGG pathway analyses.The expression pattern were studied by Real-time PCR and IHC.In summary,gene mutation and transcriptome analysis were performed to study the functions of Rln3 a on spermatogenesis initiation,differentiation of spermatids,and fertility in male tilapia.Taken together,these data demonstrated that rln3 a might be an essential factor to promote spermatogenesis and ensure fertility in male.