Molecular characterization and expression of the feminization-1c(fem-1c)in the freshwater mussel(Hyriopsis cumingii)

Yyu Wng,Congdi Wu,Pengfei Guo,Guiling Wng,*,Jile Li,b

aKey Laboratory of Genetic Resources for Freshwater Aquaculture and Fisheries,Shanghai Ocean University,Shanghai,201306,China

bShanghai Engineering Research Center of Aquaculture,Shanghai,201306,China

1.Introduction

Hypriopsis cumingii,a unique freshwater pearl mussel of China,occupies a large proportion of the pearl breeding industry.The pearls produced by H.cumingii are characterized for their smoothness,bright color and fine texture.H.cumingii pearl production accounts for 80%of the total output value of freshwater pearls(Wang,Bai,Liu,&Li,2014).The pearl yield of the H.cumingii is affected by gender.The pearls produced by male mussels are superior to those produced by female mussels in terms of total weight,particle weight and diameter;thus,male mussels show a significant advantage in breeding pearls(Zhao et al.,2013).To date,there have been few studies on sex determination and sex differentiation in H.cumingii.Research on gender differentiation will help to improve the quality of breeding pearls and their economic value.

The fem gene is an important factor in gender regulation that was first identified in the nematode Caenorhabditis elegans,and its role is also very thorough(Zhang et al.,2013).The main characterized genes involved in nematode sex determination hierarchy are:her-1(hermaphroditization-1)＞tra-2(transformer-2)＞fem-1(feminization-1),fem-2(feminization-2),fem-3(feminization-3),Cul-2(Cullin-2)＞tra-1(transformer-1)(Zanetti&Puoti,2013).The specific regulatory mechanism is as follows:fem-1/2/3 genes act upon the downstream TRA-1 protein directly.TRA-1 protein is the terminal factor of the regulatory pathway that promotes the development of individuals into females(Kuwabara,2007;Zarkower&Hodgkin,1992).The X/A(number of X chromosomes per set of autosomes)ratios determined two types of chromosomes in the nematode:XX and XO(J¨ager,Schwartz,Horvitz,&Conradt,2004;Trent et al.,1991).A low X/A ratio activates the transcription of the her-1 gene,which was an inhibitory ligand of the TRA-2 protein.Freed from the influence of TRA-2 protein,the complexes formed by FEM-1/2/3 and CUL-2 proteins target the downstream transcription factor,TRA-1,for degradation and therefore,the individuals would develop into males.In contrast,a high X/A ratio represses the transcription of the her-1 gene,leaving the TRA-2 protein in the active form,which could bind to FEM-1/2/3 and CUL-2 to regulate the expression of TRA-1 protein.In this case,the organism would develop into a hermaphrodite(Arur,Ohmachi,Berkseth,Nayak,Hansen,&Zarkower,2011;Doniach&Hodgkin,1984;Hodgkin&Brenner,1977).Mutation of fem-1 in the nematode led to a sex change from male to hermaphrodite(Krakow,Sebald,King,&Cohn,2001;Schvarzstein&Spence,2006;Starostina et al.,2007).Thus,the fem-1 gene has a significant effect on the sex determination pathway of C.elegans.In addition,studies in silkworms,bees,and other invertebrates have confirmed that fem genes play a major role in gender regulation(Nagaraju,Gopinath,Sharma,&Shukla,2014;Sakai,Aoki,&Suzuki,2014).

Multiple fem-1 gene isoforms have been described.They are characterized by the existence of ankyrin repeat domains which comprises typically 30 amino acids and is one of the most common protein-protein interaction motifs in nature(Bork,1993;Du&Hu,2002).In vertebrates,three highly conserved fem-1 genes fem-1a,fem-1b,and fem-1c have been described(Itoh,Kampf,&Arnold,2009;Venturaholman,Seldin,Li,&Maher,1998).In humans,fem-1c is highly expressed in the testis and is associated with the formation of sperm(Venturaholman,Lu,Si,Izevbigie,&Maher,2003).In Locusta migratoria manilensis,expression of the fem-1c gene gradually increases with the development of the testis and thus may be related to spermatogenesis(Shi et al.,2013).However,in aquatic animals there have been fewstudies on fem-1c(Ma,Liu,Lin,Li,&Qiu,2016;Shi,Li,Gui,&Zhou,2015).In this study,we have isolated the fem-1c gene of H.cumingii,to understand its role in development and to provide a theoretical basis to study sex differentiation of H.cumingii.

2.Materials and methods

2.1.Animal rearing and tissue sampling

The larvae and two years old H.cumingii used in this study were collected from a breeding farm in Jinhua City,Zhejiang Province,China,and transported to the laboratory.All the mussels were kept at 26±2°C in cage for two days and regularly fed with chlorella in the cage.Various tissues,including the adductor muscle,foot,liver,gill,kidney,mantle,gonad were sampled from adult mussels.Embryos were collected from the gills of mussels.Fertilized mussels were placed in a cool dry place for 4-5 h,and placed back into water.Fertilized eggs were discharged from the gills,and approximately 200 were collected into a centrifuge tube using a pipette.The larvae of H.cumingii cannot be defined accurately;therefore,the approximate stages were deduced from the time of embryonic development.The embryonic period was divided into the following periods:0 day(fertilized eggs),3,6,9 and 10 days.For RNA isolation,all samples were collected and immediately frozen in liquid nitrogen.

2.2.RNA isolation and cDNA synthesis

Total RNA was extracted from the frozen preserved tissues using TRNzol(Tiangen,Shanghai,China),according to the manufacturer's instructions.A PrimeScript™ first-strand cDNA synthesis kit(TaKaRa,Dalian,China)was used to obtain cDNA,according to the manufacturer's protocol.

2.3.Cloning of Hcfem-1c

The full-length Hcfem-1c cDNA sequence was cloned by rapid amplification of cDNA ends(RACE).Primers for RACE were designed based on an expressed sequence tag(EST)sequence identified in GenBank under the accession number KY465663(Table 1).The open reading frame(ORF)was cloned by RT-PCR using the primers Fem-1c-F and Fem1c-R.A SMARTer RACE 5′/3′Kit(Clontech)was used to obtain the full-length cDNA sequence of Hcfem-1c,according to the manufacturer's instructions.Primers were designed according to the ORF of fem-1c;primers Fem-1c-3′and Fem-1c-5′were used to amplify the 5′and 3′untranslated regions(UTRs).For all PCRs,the program was:initial denaturation at 94°C for 3 min;35 cycles of denaturation at 94°C for 30 s,60°C for 30 s,and 72°C for 1 min;and a final extension at 72°C for 10 min.The PCR product was purified and ligated into vector PGEM-T vector(Promega,Madison,USA)at 16°C for 12 h,and then transformed into Escherichia coli DH5 α competent cells.The plasmid inserts of positive clones were sequenced by the sequencing service unit of Sangon Biotech(Shanghai,China).

2.4.Sequence and phylogenetic analysis

The sequence of the cloned cDNA was confirmed using the BLAST algorithm at the NCBI(http://www.ncbi.nlm.nih.gov/BLAST/).The ORF of Hcfem-1c gene was determined using ORF Finder(http://www.ncbi.nlm.nih.gov/projects/gorf/).The nucleotide and amino acid sequence identity and the prediction of conserved domains,were confirmed using the BLAST program against the nonredundant protein sequences database(GenBank,NCBI).The physical parameters of the HcFEM-1C protein were analyzed usingProtparam (http://www.expasy.org/tools/protparam.html).TMHMMprogramwas used to predict the transmembrane domains ofHcFEM-1C (http://www.cbs.dtu.dk/services/TMHMM/).The amino acid sequences encoded by the fem-1c genes from other species were retrieved from the NCBI GenBank database(http://www.ncbi.nlm.nih.gov/nucleotide/).Multiple comparisons of the amino acid and coding nucleotide sequences were performed using Clustalw1.8 and BioEdit(Xu,Shen,Yang,&Li,2011).Phylogenetic trees were constructed using the Neighbor-Joining(NJ)and in the MEGA 6 software,based on the amino acid sequences(Tamura et al.,2011).The number on the branch represents the bootstrap after repeating 1000 times.

Table 1 Primers used in this study.

Fig.1.cDNA and deduced amino acid sequences of fem-1c in H.cumingii.The start codon(ATG)and stop codon(TGA)are in bold.Putative polyadenylation signals(AATAAA)are shadowed.Ankyrin repeat regions are boxed.untranslated regions(UTRs)are shown by small letters;the coding region is represented by capital letters.

Fig.2.Three-dimensional structure of the HcFEM-1C protein.

2.5.Real-time PCR analysis

Primer5.0 was used to design the upstream primer Fem-1c-RT-F and the downstream primer Fem-1c-RT-R for qRT-PCR.Eflα expression in H.cumingii was analyzed as the internal reference.Each sample was analyzed in triplicate using a FastQuant RT Kit and SYBR®Green Super Real PreMix Plus(Tiangen,China).The 25-μL PCR reaction system contained Super Real PreMix Plus(12.5μL),primers(0.75 μL,10 mol/L),RNase-free water(10 μL),and cDNA(1.0μL).Amplification was performed using a BIO-RAD CFX96TMinstrument under the following conditions:95°C(pre-denaturation)for 15 min,95°C(degeneration)for 10 s,and 60°C(annealing)for 30 s(40 cycles),followed by dissociation curve analysis at 95°C for 15 s,60°C for 1 min and 95°C for 15 s to verify the amplification of a single product.The relative expression of the target gene and reference gene was calculated using the 2-△△CTmethod.Statistical analysis was performed using One-Way ANOVA in SPSS 16.0 software.The bar charts were drawn using Origin 9.0.

Fig.3.Neighbor-Joining phylogenetic tree analysis of the deduced HcFEM-1C protein with other metazoans.Accession numbers of sequences retrieved from GenBank are shown.Analysis was performed using the MEGA 6 software(The number on the branch represents the bootstrap after repeating 1000 times).

Fig.4.Tissue expression of Hcfem-1c in a two-year-old male and female H.cumingii.Values plotted represent the mean±SE(n=3).*significant difference(P＜0.05),**highly significant difference(P＜0.01).

2.6.In situ hybridization(ISH)

A three-year-old mature male and female H.cumingii were selected and their gonads removed.The tissue was fixed in 4%paraformaldehyde at 4°C for 2 h,and then in fresh 4%paraformaldehyde at 4°C overnight.The tissue was washed with 1×phosphate-buffered saline(PBS)two to three times and subsequently in methanol:PBS(1:3),methanol:PBS(1:1),with methanol:PBS(3:1)and with methanol:PBS(1:0)for 30 min in each step.The tissue was then frozen and sections were prepared using a microtome.

A 115 bp cDNA fragment of Hcfem-1c was amplified and subcloned into vector pGEM-T Easy vector(Promega,USA),and recombinant plasmid was linearized using NcoI(TaKaRa,Japan).The probe was then prepared using a DIG RNA Labeling Kit(SP6/T7)(Roche,Germany).The ISH experiment was carried out according to the manufacturer's instructions.

3.Results

3.1.Sequence analyses of fem-1c

The full-length cDNA sequence of Hcfem-1c was obtained after RACE amplification(Fig.1)and was submitted to GenBank under the accession number KY465663.The sequence was 2196 bp,comprising a 5′UTR of 10 bp,a 3′UTR of 320 bp,and an ORF of 1866 bp,encoding a putative protein of 621 amino-acid(aa)residues.The polyadenylation signal,AATAAA,was located in front 13 bp of the poly A tail.

The deduced protein had a molecular weight of 69812.76 Da,and an isoelectric point of 7.55.The protein molecular formula was C3091H4936N860O901S39.PROSITE analysis showed that HcFEM-1C's functional domains included seven ankyrin repeat regions,from aa40to aa62,aa82to aa114,aa115to aa147,aa148to aa180,aa181to aa213,aa486to aa531,and aa532to aa566.

No signal peptidr for HcFEM-1C was predicted according to the SignalP program analysis however ProtScale online analysis showed the amino acid residues of HcFEM-1C were hydrophilic.No potential transmembrane regions were also predicted using TMHMM programme.The predicted three-dimensional structure of HcFEM-1C was disordered 4%,alpha helix 56%,and no beta strand of predictable structure by Phyre2(Fig.2).

3.2.Phylogenetic analysis

An NJ phylogenetic tree was constructed based on the FEM-1C amino acid sequences of various species(Fig.3).The H.cumingii protein was clustered with H.schlegelii,and the closest relatives to the H.cumingii sequence were invertebrates,which are distant to the vertebrates.The C.elegans were furthest related to other species and became a separate group.

3.3.Expression of Hcfem-1c at different developmental stages

In two-year-old H.cumingii,the transcript abundance of the Hcfem-1c in the adductor muscle,foot,liver,gill,kidney,mantle and gonad tissues were analyzed by qRT-PCR(Fig.4).In males,the relative expression level of Hcfem-1c varied little among the tissues,with the highest expression in the mantle and the lowest in the foot.In females,Hcfem-1c was found highly expressed in the gonad and of low abundance in the kidney.When the expression in the male and female tissues was compared,transcript the relative abundance of Hcfem-1c in female gonads was strikingly higher than in the male gonads(P＜0.01).mRNA expression in the female adductor muscle and kidney were significantly lower(P＜0.05)than in those of the male.To investigate the effect of the Hcfem-1c gene on the sex determination and differentiation of H.cumingii,we selected embryos up to 8 months old and assessed expression in the gonad tissues by qRT-PCR(Fig.5).The result showed that expression of Hcfem-1c mRNA peaked at 5 months;thereafter,mRNA expression remained constant in subsequent developmental stages,being slightly lower at 8 months.

Fig.5.Expression level of Hcfem-1c mRNA during different developmental stages.Significant differences were determined by One-Way ANOVA,followed by multiple comparisons(P＜0.05);significant differences are indicated by different letters.D:days(0-10 days represents the embryonic period)M:months.

Fig.6.In situ hybridization expression of Hcfem-1c mRNA in two-year-old male and female gonads.Gonadal tissue sections were hybridized with the Hcfem-1 antisense RNA probe(A-D)and sense RNA probe(E,F).White arrows represent the Hcfem-1 mRNA signals.No signal was observed in the control sections hybridized with the sense probe(E,F).Fw,follicle wall;Oo,oocyte;Bar indicates 100μm.(A)female gonads;(B)magnified view of(A);(C)male gonads;(D)magnified view of(C).

3.4.In situ localization of Hcfem-1c in two-year-old male and female gonad

Hcfem-1c was expressed in the follicles in of male and female glands of H.cumingii(Fig.6).In the female gonads,ISH showed strong expression of Hcfem-1c concentrated in the cell membrane of the oocytes;however,in the follicular wall showed no obvious expression signals were detected(Fig.6 A,B).In the male gonads,strong expression signal was observed mainly in the follicular wall(Fig.6 C,D).No signal was detected when using the sense probe in the male and female gonads(Fig.6 E,F).

4.Discussion

In this study,the full-length cDNA representing the Hcfem-1c gene was isolated from the H.cumingii.The 2196 bp ORF encoded a predicted protein of 622 amino acids,containing seven ANK domains.ANK repeat domains have multiple functions,including participation in protein interactions,signal transduction,or transcriptional regulation(Mehra,Gaudet,Heck,Kuwabara,&Spence,1999).In addition,ANK structures had been found in proteins such as cyclin-dependent kinase inhibitors,transcription regulators,Cytoskeleton organizers,Swi4/6,Resl/2,Cdc10(Swi6/Cdcl0 family members).These proteins are involved in cell signal transduction and transcriptional regulation.The ANK domain was confirmed to exist in the FEM-1C proteins of other species,such as the nine,nine,nine,and seven ankyrin repeats domains in human,zebra fish,Hyriopsis schlegelii,and C.elegans,respectively(Shi et al.,2015;Krakow et al.,2001;Xiong et al.,2014).In addition,it has been shown that the fem gene may regulate sex determination in the above species(Song,Cui,Min,Liu,&Li,2015).It is speculated that the fem gene would have a similar function in H.cumingii.Phylogenetic analysis revealed that H.cumingii FEM-1C grouped with that of H.schlegelii and clusteredin the same clade as the other invertebrate homologues.The C.elegans were furthest related to other species and became a separate group.

The qRT-PCR results revealed that the Hcfem-1c mRNA was expressedin all tissues of the twoyears old adult mussels.However,transcript abundance was strikingly different in the male and female gonads.The mRNA expression levels of Hcfem-1c in males were much lower than in females,indicating that Hcfem-1c has a more important role in female gonads suggesting that it may be involved in female gonad development.To explore the role of the Hcfem-1c gene in gonad development and sex determination,we investigated the expression of Hcfem-1c mRNA in the embryonic stages up to 8 months old.The transcripts of Hcfem-1c increased obviously in 5-month-old embryos and were significantly higher when compared to other stages.The early gonad tissue section showed that primordial germ cells appeared at 5 months,which indicated that this period was a stage of morphological sex differentiation(Xue,2016).Based on the experimental results,we hypothesized that Hcfem-1c is involved in the differentiation of gonad cells of 5-month-old H.cumingii.In two-year-old adults,expression of Hcfem-1c in the female gonad was higher than in the male gonad.Taking into account the experimental results for the two-year-old mussels,we conjectured that Hcfem-1c plays a dominant role the differentiation of female gonads as been shown for other species.In the early development of zebra fish embryos,fem-1c was detected at the seventh day after fertilization,which is the period of sex differentiation of zebra fish(Shi et al.,2015).In oriental river prawns,the fem-1c gene was only expressed in ovaries and embryos.The expression level was high in the early embryonic period,and decreased as the development of the embryos progressed.However,the expression level was significantly increased when the embryo developed into 2 cm larvae,which is the sex differentiation period of prawns.The fem-1c gene of shrimp is a maternal gene and is related to sex differentiation(Ma et al.,2016).These results were consistent with our study in H.cumingii,indicating that Hcfem-1c is related to female gonad differentiation.ISH experiments showed that both male and female gonads expressed Hcfem-1c(Fig.6 A-D).However,expression the pattern was distinct.Strong and specific signals for Hcfem-1c were concentrated in the female oocytes cell membrane and also in male follicle wall.The antisense hybridization signal was not detected in the sperm,but was localized at the edge of the cytoplasm of the oocyte,suggesting that the Hcfem-1c is notonly associated with female gonad differentiation,but also with oogenesis.Further experiments are required to test this hypothesis.

5.Conclusions

We have cloned the Hcfem-1c cDNA from H.cumingii.The encoded predicted protein contains seven ANK repeats in its structure which is similar to FEM-1C proteins from other species.QRT-PCR results revealed that Hcfem-1c expression might be associated with female gonad differentiation.ISH hybridization signal detected mainly in the female egg cell membrane and male follicle wall.We hypothesized that Hcfem-1c gene may be involved not only in female gonad differentiation,but also in egg development.

Acknowledgements

This work was supported by the National Natural Science Foundation of China[grant number 31772835],the Innovation Program of Shanghai Municipal Education Commission[grant number 13ZZ128],and the Shanghai Collaborative Innovation Center for Aquatic Animal Genetics and Breeding[grant number ZF1206].

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