The bovine sex-determining region Y (Sry) gene and its mRNA transcript are present in Y sperm but not X sperm of bulls

in Animal Biology
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Abstract

The aims of this study were to establish whether the sex-determining region Y gene and its mRNA transcript are present in the Y sperm and X sperm of bulls and, if present, determine their cellular localization. Semen was collected from three bulls and sorted by flow cytometry into X- and Y-chromosome populations. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine Sry mRNA expression in X sperm and Y sperm. The presence and localization of Sry DNA and RNA were investigated by fluorescence in situ hybridization (FISH). RT-PCR detected a single Sry transcript of 142 bp in Y sperm but not in X sperm. In Y sperm, the FISH-positive rates for Sry DNA and Sry RNA did not differ significantly from the re-analyzed Y sperm purity. In further experiments, there were no significant differences between the FISH-positive rate for Sry RNA and the re-analyzed Y sperm purity for X-sorted, Y-sorted, or unsorted sperm. In conclusion, FISH analysis revealed that Sry transcripts are present at the edges of the sperm heads of Y sperm but are absent from X sperm.

The bovine sex-determining region Y (Sry) gene and its mRNA transcript are present in Y sperm but not X sperm of bulls

in Animal Biology

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References

Capra E.Turri F.Lazzari B.Cremonesi P.Gliozzi T.M.Fojadelli I.Stella A. & Pizzi F. (2017) Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between high- and low-motile sperm populations. BMC Genomics1814.

Chen X.Zhu H.Wu C.Han W.Hao H.Zhao X.Du W.Qin T.Liu Y. & Wang D. (2012) Identification of differentially expressed proteins between bull X and Y spermatozoa. J. Proteomics7759-67.

Clepet C.Schafer A.J.Sinclair A.H.Palmer M.S.Lovell-Badge R. & Goodfellow P.N. (1993) The human SRY transcript. Hum. Mol. Genet.22007-2012.

Das P.J.McCarthy F.Vishnoi M.Paria N.Gresham C.Li G.Kachroo P.Sudderth A.K.Teague S.Love C.C.Varner D.D.Chowdhary B.P. & Raudsepp T. (2013) Stallion sperm transcriptome comprises functionally coherent coding and regulatory RNAs as revealed by microarray analysis and RNA-seq. PLoS One8e56535. DOI:10.1371/journal.pone.0056535.

Dewing P.Chiang C.W.Sinchak K.Sim H.Fernagut P.O.Kelly S.Chesselet M.F.Micevych P.E.Albrecht K.H.Harley V.R. & Vilain E. (2006) Direct regulation of adult brain function by the male-specific factor SRY. Curr. Biol.16415-420.

Garner D.L. (2006) Flow cytometric sexing of mammalian sperm. Theriogenology65943-957.

Hacker A.Capel B.Goodfellow P. & Lovell-Badge R. (1995) Expression of Sry, the mouse sex determining gene. Development1211603-1614.

Hanley N.A.Hagan D.M.Clement-Jones M.Ball S.G.Strachan T.Salas-Cortes L.McElreavey K.Lindsay S.Robson S.Bullen P.Ostrer H. & Wilson D.I. (2000) SRY, SOX9, and DAX1 expression patterns during human sex determination and gonadal development. Mech. Dev.91403-407.

Harry J.L.Koopman P.Brennan F.E.Graves J.A. & Renfree M.B. (1995) Widespread expression of the testis-determining gene SRY in a marsupial. Nat. Genet.11347-349.

Hosken D.J. & Hodgson D.J. (2014) Why do sperm carry RNA? Relatedness, conflict, and control. Trends Ecol. Evol.29451-455.

Jodar M.Selvaraju S.Sendler E.Diamond M.P. & Krawetz S.A. (2013) The presence, role and clinical use of spermatozoal RNAs. Hum. Reprod. Update19604-624.

Johnson G.D.Sendler E.Lalancette C.Hauser R.Diamond M.P. & Krawetz S.A. (2011) Cleavage of rRNA ensures translational cessation in sperm at fertilization. Mol. Hum. Reprod.17721-726.

Kawano M.Kawaji H.Grandjean V.Kiani J. & Rassoulzadegan M. (2012) Novel small noncoding RNAs in mouse spermatozoa, zygotes and early embryos. PLoS One7e44542. DOI:10.1371/journal.pone.0044542.

Kempisty B.Antosik P.Bukowska D.Jackowska M.Lianeri M.Jaskowski J.M. & Jagodzinski P.P. (2008) Analysis of selected transcript levels in porcine spermatozoa, oocytes, zygotes and two-cell stage embryos. Reprod. Fertil. Dev.20513-518.

Koopman P.Munsterberg A.Capel B.Vivian N. & Lovell-Badge R. (1990) Expression of a candidate sex-determining gene during mouse testis differentiation. Nature348450-452.

Koopman P.Gubbay J.Vivian N.Goodfellow P. & Lovell-Badge R. (1991) Male development of chromosomally female mice transgenic for Sry. Nature351117-121.

Krawetz S.A.Kruger A.Lalancette C.Tagett R.Anton E.Draghici S. & Diamond M.P. (2011) A survey of small RNAs in human sperm. Hum. Reprod.263401-3412.

Lambard S.Galeraud-Denis I.Martin G.Levy R.Chocat A. & Carreau S. (2004) Analysis and significance of mRNA in human ejaculated sperm from normozoospermic donors: relationship to sperm motility and capacitation. Mol. Hum. Reprod.10535-541.

Larney C.Bailey T.L. & Koopman P. (2014) Switching on sex: transcriptional regulation of the testis-determining gene Sry. Development1412195-2205.

Martins R.P. & Krawetz S.A. (2005) RNA in human sperm. Asian J. Androl.7115-120.

Mayer A.Mosler G.Just W.Pilgrim C. & Reisert I. (2000) Developmental profile of Sry transcripts in mouse brain. Neurogenetics325-30.

Miller D. (2007) Spermatozoal RNA as reservoir, marker and carrier of epigenetic information: implications for cloning. Reprod. Domest. Anim.42(Suppl. 2) 2-9.

Miller D.Ostermeier G.C. & Krawetz S.A. (2005) The controversy, potential and roles of spermatozoal RNA. Trends Mol. Med.11156-163.

Modi D.Shah C.Sachdeva G.Gadkar S.Bhartiya D. & Puri C. (2005) Ontogeny and cellular localization of SRY transcripts in the human testes and its detection in spermatozoa. Reproduction130603-613.

Ninomiya Y. & Ichinose S. (2007) Subcellular distribution of mitochondrial ribosomal RNA in the mouse oocyte and zygote. PLoS One2e1241. DOI:10.1371/journal.pone.0001241.

Ostermeier G.C.Dix D.J.Miller D.Khatri P. & Krawetz S.A. (2002) Spermatozoal RNA profiles of normal fertile men. Lancet360772-777.

Ostermeier G.C.Miller D.Huntriss J.D.Diamond M.P. & Krawetz S.A. (2004) Reproductive biology: delivering spermatozoan RNA to the oocyte. Nature429154.

Ostermeier G.C.Goodrich R.J.Moldenhauer J.S.Diamond M.P. & Krawetz S.A. (2005) A suite of novel human spermatozoal RNAs. J. Androl.2670-74.

Pannetier M.Tilly G.Kocer A.Hudrisier M.Renault L.Chesnais N.Costa J.Le Provost F.Vaiman D.Vilotte J.L. & Pailhoux E. (2006) Goat SRY induces testis development in XX transgenic mice. FEBS Lett.5803715-3720.

Poole D.H.Ndiaye K. & Pate J.L. (2013) Expression and regulation of secreted phosphoprotein 1 in the bovine corpus luteum and effects on T lymphocyte chemotaxis. Reproduction146527-537.

Sachdeva G.Shah C.A.Kholkute S.D. & Puri C.P. (2000) Detection of progesterone receptor transcript in human spermatozoa. Biol. Reprod.621610-1614.

Santiso R.Tamayo M.Gosalvez J.Meseguer M.Garrido N. & Fernandez J.L. (2010) Swim-up procedure selects spermatozoa with longer telomere length. Mutat. Res.68888-90.

Schenk J.L.Suh T.K.Cran D.G. & Seidel G.E.Jr. (1999) Cryopreservation of flow-sorted bovine spermatozoa. Theriogenology521375-1391.

Sendler E.Johnson G.D.Mao S.Goodrich R.J.Diamond M.P.Hauser R. & Krawetz S.A. (2013) Stability, delivery and functions of human sperm RNAs at fertilization. Nucleic Acids Res.414104-4117.

She Z.Y. & Yang W.X. (2014) Molecular mechanisms involved in mammalian primary sex determination. J. Mol. Endocrinol.53R21-R37.

Tanaka S.S. & Nishinakamura R. (2014) Regulation of male sex determination: genital ridge formation and Sry activation in mice. Cell. Mol. Life Sci.714781-4802.

Ueno A.Takeda M.Hirasawa K.Itagaki S. & Doi K. (1997) Detection of viral RNA by electron microscopic in situ hybridization (ISH-EM) in the germinal epithelium of mice infected with encephalomyocarditis (EMC) virus. Exp. Anim.4679-81.

Wallis M.C.Waters P.D. & Graves J.A. (2008) Sex determination in mammals – before and after the evolution of SRY. Cell. Mol. Life Sci.653182-3195.

Welch G.R. & Johnson L.A. (1999) Sex preselection: laboratory validation of the sperm sex ratio of flow sorted X- and Y-sperm by sort reanalysis for DNA. Theriogenology521343-1352.

Wilhelm D.Martinson F.Bradford S.Wilson M.J.Combes A.N.Beverdam A.Bowles J.Mizusaki H. & Koopman P. (2005) Sertoli cell differentiation is induced both cell-autonomously and through prostaglandin signaling during mammalian sex determination. Dev. Biol.287111-124.

Yao C.Wang Z.Zhou Y.Xu W.Li Q.Ma D.Wang L. & Qiao Z. (2010a) A study of Y chromosome gene mRNA in human ejaculated spermatozoa. Mol. Reprod. Dev.77158-166.

Yao C.J.Xu W.J.Gong X.L.Zhou Y.Yan Z.Q.Zhu Z.J.Wang Z.X.Li Q.L.Guo X.B.Wang L.Y.Ma D. & Qiao Z.D. (2010b) The role of Dby mRNA in early development of male mouse zygotes. Asian J. Androl.12567-577.

Zhao L. & Koopman P. (2012) SRY protein function in sex determination: thinking outside the box. Chromosome Res.20153-162.

Figures

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    Sry and CD44 gene-specific primers.

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    RT-PCR and agarose gel electrophoresis for detection of Sry and CD44 in bovine sperm. Note: M: TaKaRa DL-2000 DNA marker; 1: negative control for Y sperm (no CD44); 2: Sry of Y sperm; 3: negative control for X sperm (no CD44); 4: Sry of X sperm; 5: negative control for unsorted-swim-up sperm (no CD44); 6: Sry of unsorted-swim-up sperm; 7: negative control for unsorted sperm (CD44); 8: Sry of unsorted sperm.

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    Re-analysis of Y-sorted sperm purity using Hoechst 33342 and detection of Sry DNA and RNA in Y-sorted sperm using fluorescence in situ hybridization.

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    Fluorescence in situ hybridization (FISH) of Sry DNA and mRNA in Y sperm (200×). (A) FISH of negative control DNA without probe. (B) FISH of Sry DNA. (C) FISH of negative control RNA with RNase A treatment. (D) FISH of Sry RNA. Green staining indicates the localization of Sry DNA or RNA detected by FISH.

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    Fluorescence in situ hybridization (FISH) of Sry mRNA in X-sorted (A), Y-sorted (B) and unsorted (C) sperm (200×). Green staining indicates the localization of Sry RNA detected by FISH.

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    Re-analysis of sperm purity using Hoechst 33342 and detection of Sry RNA using fluorescence in situ hybridization in X-sorted, Y-sorted and unsorted sperm.

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