Steroids in equine testes: the identification of endogenous 19-hydroxy and 19-nor neutral steroids by gas chromatography–mass spectrometry.

The research examines the identification of specific types of steroids in horse testes, using various techniques like gas chromatography-mass spectrometry. It investigates the influence of age on steroidogenesis and identifies associated changes in different steroid fractions.

Methodology

• The research began with the homogenization of testicular tissue from stallions of varying ages – 1, 2, and 5 years old.
• The unconjugated and conjugated steroids were isolated using a combined solvent-solid extraction procedure.
• The conjugates went through further separation into glucuronides and sulphates using chromatography facilitated by Sephadex LH-20.
• Enzyme hydrolysis and solvolysis were applied to obtain three distinct extracts: unconjugated steroids, aglycones, and solvolysed sulphates.
• Kieselgel 60H columns were then used to purify these extracts, with five fractions obtained from each extract.
• By forming the methoxime-trimethylsilyl ethers, the steroids could be identified via gas chromatography-mass spectrometry.

Results

• The study found that within stallion testes, steroidogenesis functions through both the 4-ene and the 5-ene pathways.
• Age-related changes were discerned in both unconjugated and sulphoconjugated steroid fractions.
• Specific types of steroids, namely 19-hydroxy androgens and the 19-nor (C18) neutral steroids (19-norandrostenedione and 19-nortestosterone), were only found in the unconjugated fraction. Alternatively, isomers of oestradiol and 5(10)-oestrene-3,17-diol were uniquely identified in the sulphoconjugate fraction.
• The study suggests that the unconjugated 19-oxygenated androgens in stallion testes are converted into 19-nor neutral steroids through a reverse aldol reaction, with a proposed mechanism demonstrating the potential intermediates in their formation.

Interpretation and Conclusions

• The research provides essential insights into the presence and transformation of specific steroids in stallion testes.
• This knowledge enhances the understanding of endogenous steroid mechanics in horses, including reactions and conversions between different types of steroids.
• The study’s findings can contribute to broader research on equine health and reproduction, facilitating more effective treatments and interventions.