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

Abstract

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.

C-type natriuretic peptide regulates blood–testis barrier dynamics in adult rat testes

“…The integrity of the BTB was assessed in a functional assay by monitoring the ability of the BTB to restrict the diffusion of a small fluorescent probe across the barrier. In brief, adult rats (Ϸ270-300 g of body weight at Ϸ90-120 days of age) were treated with a low (0.35 g of peptide per testis; n ϭ 3 rats) or a high (3.5 g of peptide per testis; n ϭ 3 rats) dose of CNP-22 (CNP was prepared in saline, and each testis was at 1.6 Ϯ 0.13 g) via an intratesticular injection using a 27-gauge needle in a final volume of Ϸ150 l with Ϸ75 l per site (with a total of two sites per testis) (32,48) at time 0, and rats were used by day 1 and day 3 thereafter for FITC infusion (see below). At these doses, if the peptide had dispersed throughout the testes uniformly, and assuming a testicular volume of 1.6 ml, a concentration of 10 Ϫ7 M or 10 Ϫ6 M would have been achieved; however, this might not be the case given the M r of the peptide (see SI Discussion).…”

Complex gangliosides are essential in spermatogenesis of mice: Possible roles in the transport of testosterone

“…The intratesticular injection of [ 14 C]testosterone was performed as described (19). The injected testosterone is reported to enter the lymph space, overspread in the testis, and penetrate into the seminiferous tubules.…”

p-FAK-Tyr397 regulates spermatid adhesion in the rat testis via its effects on F-actin organization at the ectoplasmic specialization

“…Plasmid DNA was then treated with the Mira-CLEAN Endotoxin Removal Kit (Mirus, Madison, WI) to remove any bacterial endotoxin contaminant. Rats were transfected with corresponding plasmid DNA via intratesticular injection using a 29-gauge needle as described (28,30). On day 0, one testis of an adult rat (ϳ275-300 g body wt with a weight of ϳ1.6 g, assuming a volume of ϳ1.6 ml) received 15 g pCI-neo empty vector DNA plasmid while the other testis received the same amount of FAK phosphomimetic mutant Y397E plasmid DNA or FAK plasmid DNA for transfection.…”

“…Plasmid DNA (15 g, for the mutant, FAK, or empty vector) suspended in 20 l TE buffer (10 mM Tris, pH 8.0 at 22°C containing 1 mM EDTA) was combined with 140 l TransIT-EE delivery solution (Mirus Bio) to a total volume of 160 l and administered to each testis. Rats (n ϭ 7 rats/time point in both treatment and control groups) received intratesticular injection of plasmid DNA daily for 3 days at days 0, 1, and 2 using the procedure earlier described (28) and modified in our laboratory for gene silencing using short hairpin RNA or smallinterfering RNA for transfection in vivo (15,25). In brief, plasmid DNA in TransIT-EE delivery solution in 160 l was loaded on a 1-ml Insulin Syringe U-100 with a 29-gauge, 0.5-in.…”