Synergistic Effect of Follicle-Stimulating Hormone and Luteinizing Hormone on Testicular δ-3β-Hydroxysteroid Dehydrogenase-isomerase: Application of a New Method for the Separation of Testicular Compartments*

Abstract

A relatively quick method is described for the separation of large quantities of interstitial tissue and seminiferous tubules of rat testes. This method involves a short treatment of decapsulated testes with collagenase (0.5 mg/g tissue) for 5 min at 34 C, followed by the wet dissection method. This procedure results in the complete separation of interstitial tissue and seminiferous tubules of one testis by one person in 30 min or less. The efficacy of separation of the new method is compared to two other widely used methods: collagenase treatment only and wet dissection only. [¹²⁵I]Iodo-hCG binding and δ⁵-3β-hydroxysteroid dehydrogenase-isomerase activity were used as markers for Leydig cells and sorbitol dehydrogenase activity was used as a marker enzyme for germ cells. Both the newly described method and the wet dissection method resulted in interstitial tissue free of contaminating germ cells and in seminiferous tubules essentially free of adhering Leydig cells. The advantage of the new method over the wet dissection method is speed and yield of tissue. Collagenase treatment resulted in a relatively small yield of Leydig cells, which were highly contaminated by germ cells, and seminiferous tubules, which were highly contaminated by adhering Leydig cells. The new method was used to investigate the role of gonadotropins in the maintenance of testicular δ⁵-3β-hydroxysteroid dehydrogenase-isomerase activity, a key enzyme in steroid hormone synthesis. Hypophysectomized adult rats were treated twice daily for 14 days with 20 μg NIH-LH-S19,50 μg NIH-FSH-Sll, or saline. Animals were killed on the morning of the 15th day. Microsomal fractions were prepared from either whole testes or isolated interstitial tissue and used for the determination of δ⁵-3β-hydroxysteroid dehydrogenase-isomerase activity. The following results were obtained with microsomal fractions of interstitial tissue. Hypophysectomy resulted in a 72% loss in enzyme activity 15 days after hypophysectomy. Administration of LH maintained enzyme activity at intact control values. FSH treatment had no effect on enzyme activity compared to hypophysectomized saline-injected control rats. However, when rats were treated with LH plus FSH, a synergistic effect of FSH and LH on δ⁵-3β-hydroxysteroid dehydrogenase-isomerase activity was demonstrated. Very different results were obtained when δ⁵-3β-hydroxysteroid dehydrogenase-isomerase was determined in microsomal fractions of whole testes from identically treated rats. The reason for this discrepancy is discussed.