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Identification of a unique mu-class glutathione S-transferase in mouse spermatogenic cells
K D Fulcher1, J E Welch, D G Klapper
1Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Abstract:
The fibrous sheath is a major cytoskeletal structure in the principal piece of the mammalian sperm flagellum. Two peptide sequences obtained from a tryptic digest of mouse fibrous sheath proteins exhibited high homology with mu-class glutathione S-transferases (GSTs). Using a DNA probe amplified from degenerate polymerase chain reaction (PCR) primers predicted from these two peptide sequences, a approximately 1.1 kb cDNA clone for fibrous sheath component 2 (Fsc2) was isolated which had 84% nucleic acid and 89% amino acid sequence identity with a previously reported mu-class human GST gene (hGSTM3; Campbell et al., 1990: J Biol Chem 265:4188-9193). Sequences corresponding to those of the two fibrous sheath peptides were present in the protein encoded by the Fsc2 cDNA. Northern analysis with the full length Fsc2 cDNA detected a approximately 1.1 kb mRNA in 12 of 15 somatic tissues examined, as well as in testis and isolated spermatogenic cells. However, 5'(nt--96 to 12) or 3'(nt 637 to 808) Fsc2 probes, containing mostly noncoding sequences, detected a approximately 1.1 kb mRNA abundant in testis and isolated spermatogenic cells, but absent or present at low levels in somatic tissues. Northern analysis with RNA from testes of mice of different postnatal ages and purified spermatogenic cell populations indicated that this transcript is first present during the meiotic phase of germ cell development. These results suggest that a previously unreported mu-class GST gene (mGSTM5.) is expressed at a specific time during the development of spermatogenic cells in the mouse. Immunoblot analysis indicated that a mu-class GST protein is associated with the fibrous sheath, suggesting that it becomes an integral part of the mouse sperm cytoskeleton.
Insights
A novel mu-class glutathione S-transferase (GST) gene, mGSTM5, is identified in mouse sperm fibrous sheath. This GST protein is integral to the sperm cytoskeleton and specifically expressed during spermatogenic cell development.
Area of Science:
- Spermatogenesis research
- Molecular biology
- Cytoskeletal protein identification
Background:
- The fibrous sheath is crucial for mammalian sperm function.
- Previous studies identified peptide sequences in fibrous sheath proteins with homology to glutathione S-transferases (GSTs).
Purpose of the Study:
- To identify and characterize the specific GST gene involved in the mouse sperm fibrous sheath.
- To determine the expression pattern of this gene during spermatogenesis.
Main Methods:
- Degenerate polymerase chain reaction (PCR) and cDNA cloning were used to isolate the gene.
- Northern blot analysis was employed to examine mRNA expression in various tissues and cell types.
- Immunoblot analysis confirmed protein association with the fibrous sheath.
Main Results:
- A novel mu-class GST gene, designated mGSTM5, was identified, sharing high homology with human GSTs.
- A specific transcript for mGSTM5 was abundant in testis and spermatogenic cells, but not in most somatic tissues.
- The mGSTM5 transcript was first detected during the meiotic phase of germ cell development.
- A mu-class GST protein was found to be associated with the sperm fibrous sheath.
Conclusions:
- A previously unreported mu-class GST gene (mGSTM5) is expressed during specific stages of mouse spermatogenic cell development.
- This GST protein is an integral component of the mouse sperm cytoskeleton, specifically within the fibrous sheath.