N OCIETY' - the Society for Reproductive Biology

Cloning and Characterisation ofa Novel Testis Transcript with Homology toPhosphatidylethanolamine Binding Proteins - pebp2ISOLATION AND PARTIAL CHARACTERISATION OF THE OUTER DENSE FmRES FROM BRUSH·TAILED POSSUM SPERMATOZOAM. RiccI and W.O. BreedDepartment of Anatomical Sciences, The University of Adelaide, S.A., 5005Deborah M. Hickox l , John R. Morrison l , Kimberley L. Sebire l , Hooi-Hong Keah 2 , Milton T.W. Hearn 2 ,David M. de Kretser 1 , Moira K. O'BryanIllnstitute of Reproduction and Development and The Centre for Bioprocess Technology,2Department of Biochemistry, Monash University, Clayton, Victoria, 3168, Australia.IntroductionMany infertile men show an increased percentage of abnormally shaped and immotile sperm arising fromunknown factors. It is postulated that some of these abnormalities arise from mutations in genes criticallyrelated to the formation of the spermatozoa. To gain a better understanding of the mechanisms underlyingsperm development and function we set out to clone rat cDNAs involved in spermiogenesis. A previouslyunreported partial cDNA transcript was isolated and designated pebp2 based on the possession of a putativephosphatidylethanolamine binding protein (PEBP). The aims of the present study were to clone the fulllength rat pebp2 transcript, its mouse orthologue and to initiate a functional characterization.Materials and MethodsThe remainder of the rat pebp2 cDNA was obtained using a 5' rapid amplification of cDNA ends (RACE)method. The orthologous mouse sequence was obtained by screening a mouse testis lambda expressionlibrary. Isolated clone sequences and their predicted encoded proteins were compared against databaseentries for homology and _sequence motifs, using the BLAST and Prosite programs respectively. Peptidesspecific for pebp2 were produced using Fmoc-based chemistry and were linked to keyhole limpethaemocyanin (KLH). Peptide-KLH conjugates were immunized into rabbits to raise pebp2-specific antiserafor use in immunohistochemical techniques. Northern blotting and in situ hybridization were carried out todetermine the timing of pebp2 mRNA expression during spermatogenesis. RNA extracted from male andfemale Balb/c mice tissues were used for RT-PCR analysis oftissue distribution.Results5' RACE was successfully used to obtain an additional -1 kb of rat pebp2 cDNA sequence. Northernanalysis revealed 2 encoded transcripts of -1.6kb and -3.0kb which were up-regulated at day 25 and day 35post-partum, respectively. Screening of a mouse testis expression library identified a novel cDNA sequencewhich shared 93% homology in the coding region with the rat pebp2 sequence. In agreement with rat pebp2expression, mouse pebp2 mRNA was expressed in late pachytene spermatocytes and round spermatids. Thisresult was supported by immunohistochemical staining data which showed pebp2 protein in round andelongating spermatids and on sperm heads at spermiation. One of the six mouse pebp2 clones sequenced wasthought to represent an alternative splice variant which would result in an in-frame deletion of 14 aminoacids. This was supported by RT-PCR analyses. The long mouse isoform was predicted to have a Mr of-21.2kDa and an isoelectric point (pI) of -8.43. The short isoform was predicted to have a Mr of -19.9 kDaand pI of -8.77. Both the predicted mouse and rat proteins contained a PEBP family signature, severalpotential phosphorylation sites and a nuclear localization signal. Tissue expression studies revealed thepresence of both the short and long isoforms of mouse pebp2 in many tissues including brain, heart, kidney,liver, lung, skeletal muscle. In addition a putative human orthologue has been identified by searching theEST database. The predicted protein sequences from all three species were novel and more closely related toone another than other previously identified members of the PEBP family.ConclusionWe have identified novel rat and mouse testis transcripts and proteins which we have designated pebp2 basedon the possession of a PEBP motif. Mouse pebp2 is predicted to encode two potentially phosphorylationregulatedisoforms of -21 and 20kDa which are expressed during spermiognesis. Further, pebp2 isofonnsappear to be integral component of several tissues throughout the body. The involvement of pebp2 in spermepididymal maturation, capacitation and fertilization is the subject of further research.IntroductionIn all therian mammals, nine prominent cytoskeletalstructures, the outer dense fibres, surround theaxoneme of the midpiece and principle piece of thesperm flagellum (Figure 1). The possible function(s)of the outer dense fibres may be to either maintain thepassive elastic recoil of the sperm flagellum (1)and/or to protect the flagella against shearing forcesduring epididymal transit (2). The isolation andcharacterisation of the outer dense fibres from rat,bull and human sperm have been previously reported(3, 4, 5), but as yet no study has been published forany marsupial species. The aim of the present study,which is part of a broader investigation of theevolution of eutherian and marsupial gametes, was toisolate and determine the protein composition of theouter dense fibres for the first time in a marsupialspecies, the brush-tailed possum, Trichosurusvulpecula-.Materials & MethodsSpermatozoa obtained from the cauda epididymidesof 10 adult possums were decapitated by sonication,layered over a 20%,40% and 60% (w/v) sucrose stepgradient and centrifuged at 2500 x g for 120 minutesat 4°C. Sperm flagella fractions were collected fromthe 40-60% interface and purity was assessed byNomarski microscopy. Sperm flagella were pelletedand incubated in 1% sodium dodecyl sulfate (SDS),2mM dithiothrietol (DIT), 25mM Tris-HCI (pH 8.0),and shaken at room temperature for approximately 90minutes. The resultant suspension was layered over a20%, 40% and 60% (w/v) sucrose step gradient,centrifuged, and the outer dense fibres were collectedfrom the 40-60% interface. The isolated outer densefibres were either fixed for routine transmissionelectron microscopy and/or solubilised with 2% SDS­5% B-mercaptoethanol for 5 minutes at 100°C andrun on linear gradient (7.5-15%) SDS polyacrylamidegels which were stained with Coomassie.Results and DiscussionSeparation of flagella from the sperm head bysonication and subsequent sucrose density gradientcentrifugation resulted in a high yield of flagella fromthe 40-60% interface. Incubation of the sperm flagellain SDS-DIT for 90 minutes resulted in the completesolubilisation of the axoneme, mitochondria andfibrous sheaths but the outer dense fibres remainedintact (Figure 1). SDS-polyacrylamide gels of theouter dense fibre fractions revealed 5 majorpolypeptide bands with molecular masses of 73, 57,52, 42 and 16 kDa, the last of which was the mostprominent band (Figure 2). In comparison, rat, bulland human sperm outer dense fibres contain 7, 3 and2 major polypeptides respectively (3, 4, 5), and themost prominent bands in rat and bull outer densefibres are 27 kDa and 33 kDa in mass (3, 4).f'~8·"'.tt." I'.., '•. '.·.,.< ~:;1 ;Figure 1. Electron micrograph of isolated possum outer dense fibres; in~et:transverse section of principle piece of possum sperm flagellum showmgouter dense fibres (arrows).Figure 2. SDS-PAGE of isolated possum outer dense fibres (ODF) andmolecular weight standards (STD).ConclusionsThe isolation and molecular weights of the proteins ofthe outer dense fibres has been described for the fIrsttime in a marsupial species, the brush-tailed possum.Polyc1onal antibodies are currently being raisedagainst the major possum outer dense fibrepolypeptides and these will be used toimmunocytochemically investigate the developmentand localisation of these cytoskeletal structures. Inaddition, techniques are currently being developed tosimilarly isolate and characterise the proteins of theother major cytoskeletal component of the possumsperm flagellum, the fibrous sheath.References(1) Phillips DM (1972) J. Cell Biol.53: 561-573.(2) Baltz JM et al. (1990). Biol. Reprod. 43: 485­491.(3) Oko R (1988) BioI. Reprod. 39: 169-182.(4) Brito et ai. (1986) Gamete Res. 15: 327-336.(5) Henkel et al. (1992). BioI. Chern. Hoppe-Seyler373: 685-689.2108109