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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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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    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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