Intratesticular injection as a method to assess the potential toxicity of various agents and to study mechanisms of normal spermatogenesis

To better understand, to optimize, and to validate the technique of intratesticular (i.t.) injection, several parameters related to i.t. injection were examined. Volumes exceeding 50 microliters could be injected i.t.; however, testes frequently became excessively turgid and backflow of injected fluids occurred. Thus, a volume of 50 microliters or less was deemed optimal for injection. To determine the rate of distribution of substances throughout the testis, trypan blue was injected i.t. near the caudal pole of the testis, and the movement of dye was monitored. Within 2 min, the dye had spread approximately 1 cm from the site of injection, and in 5 min it had spread twice that distance. In 2 h, the dye had become distributed throughout the testis except at its extreme cranial pole. Seminiferous tubules did not take up dye, indicating that the spread of dye was via peritubular lymphatics. Seminiferous tubule histology appeared virtually unaffected by i.t. injection, even at regions adjacent to the site of injection, when a sterile 26-gauge or smaller bore needle was utilized. To determine disappearance from the testis, radiolabeled inulin was injected i.t. Half time for absorption was achieved at 1.75 h. Potential vehicles were explored in which compounds with a variety of physical properties could be injected. Gum tragacanth, normal saline, ethylene glycol, dimethyl sulfoxide (DMSO) mixed 1:1 with normal saline, sesame oil, and propylene glycol were found to be suitable injection vehicles, whereas ethanol, dissolved in normal saline in concentrations as low as 0.5% was found unsuitable. To assess vehicle efficiency, various vehicles were utilized with a known testicular toxin (taxol) and injected into one testis, and the histology was compared with the contralateral testis injected with vehicle alone. All vehicles, found suitable above, allowed dispersion of taxol to influence areas distant from the site of injection. Intratesticular injection assesses the potential of agents to directly affect the testis, and systemic metabolism is avoided. Their rapid spread throughout the lymphatics of the testes allows seminiferous tubules to be exposed to agents in innocuous vehicles more rapidly and in higher concentration than is often possible when using systemic injections.