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Protein kinase activity is central to rat germ cell apoptosis induced by methoxyacetic acid
1Reproductive Toxicology Group, National Toxicology Program, NIEHS, Research Triangle Park, North Carolina, USA.
Abstract:
Methoxyacetic acid (MAA) is a major metabolite of ethylene glycol monomethyl ether (EGME). Previous investigations of the testicular lesion induced by EGME have found that dividing meiotic cells are the most sensitive, although several stages of spermatocytes are also vulnerable. Preliminary data from this lab suggested the involvement of protein kinase activity in the development of this lesion, a hypothesis explored in the present studies. We used cultured seminiferous tubules (STs) from juvenile rats (25-day-old), exposed in vitro to MAA and several inhibitors of protein kinases. Nineteen h following a 5-h exposure to 5 mM MAA (the plasma level in vivo after a toxic dose of EGME), apoptotic spermatocytes were seen in early- and late-stage STs. Cell death was prevented by cotreatment with broad-spectrum inhibitors of protein kinases such as H-7, H-8, K-252a, W-7, and genistein. In corroboration, immunocytochemistry with antibodies to various kinases (PKCmu, zeta, and gamma, AKAP220, CaMKII, MLCK, and Src) showed increased staining around dying spermatocytes following EGME treatment in vivo. 2D-PAGE, autoradiography, and nanospray mass spectrometry was used to separate and identify proteins whose phosphorylation status was most greatly changed following exposure to MAA. One protein was identified by sequence analysis as being glucose-regulated protein 94 (grp94). Westem blotting and immunocytochemistry confirmed this finding. The data we present implicate kinase activities in the pathogenesis of this lesion and suggest the involvement of Sertoli cells.
Insights
Methoxyacetic acid (MAA), a metabolite of ethylene glycol monomethyl ether (EGME), causes testicular damage. Protein kinase inhibitors prevented MAA-induced spermatocyte apoptosis, implicating kinases in this toxicity.
Area of Science:
- Reproductive Toxicology
- Molecular Cell Biology
- Biochemistry
Background:
- Ethylene glycol monomethyl ether (EGME) induces testicular lesions, primarily affecting meiotic cells and spermatocytes.
- Methoxyacetic acid (MAA) is a key toxic metabolite of EGME.
- Preliminary evidence suggests protein kinase activity involvement in EGME-induced testicular damage.
Purpose of the Study:
- To investigate the role of protein kinase activity in the pathogenesis of MAA-induced testicular lesions.
- To identify specific kinases and downstream targets involved in MAA toxicity to spermatocytes.
Main Methods:
- In vitro culture of rat seminiferous tubules (STs) exposed to MAA and protein kinase inhibitors.
- Assessment of spermatocyte apoptosis using morphological and immunocytochemical methods.
- In vivo immunocytochemistry to detect kinase localization in testicular tissue after EGME exposure.
- Proteomic analysis (2D-PAGE, mass spectrometry) to identify proteins with altered phosphorylation.
Main Results:
- In vitro exposure to MAA induced apoptosis in early- and late-stage spermatocytes.
- Broad-spectrum protein kinase inhibitors (H-7, H-8, K-252a, W-7, genistein) prevented MAA-induced cell death.
- In vivo EGME treatment increased the staining of several kinases (PKCmu, zeta, gamma, AKAP220, CaMKII, MLCK, Src) around dying spermatocytes.
- Proteomic analysis identified glucose-regulated protein 94 (grp94) as a protein with significantly altered phosphorylation status after MAA exposure.
Conclusions:
- Kinase activities are implicated in the mechanism of MAA-induced testicular toxicity.
- Sertoli cells may play a role in the pathogenesis of these lesions.
- Targeting specific kinase pathways could offer a therapeutic strategy against EGME/MAA testicular toxicity.