Dync1h1 was silenced in adult rat (~280-300 g body weight) testes in vivo by transfecting testes with Dync1h1 siRNA duplexes vs. non-targeting control using in vivo-jetPEI. In brief, siRNA duplexes (500 nM) and siGLO red transfection indicator (20 nM) were constituted in 100 µl of transfection solution containing 1.7 µl in vivo-jetPEI (an adult rat testis was at ~1.6 g in weight, with a volume of ~1.6 ml) with N/P=8. The transfestion solution in 100 µl was administered to each testis using a 28-gauge, 13-mm needle, attached to a 0.5 ml insulin syringe. Transfection efficiency was estimated to be ~50-60%.

In the mammalian testis, spermatogenesis is dependent on the microtubule (MT)-specific motor proteins such as dynein 1 that serve as the engine to support germ cell transport. Yet the underlying molecular mechanism(s) remain unknown. Herein, we used RNAi to knockdown dynein 1 heavy chain (Dync1h1) and an inhibitor ciliobrevin D to inactivate dynein in Sertoli cells in vitro and the testis in vivo, thereby probing the role of dynein 1 in spermatogenesis. Both treatments were shown to induce extensively disruption of MT organization across Sertoli cells in vitro and the testis in vivo. These changes also perturbed the transport of spermatids and other organelles (such as phagosomes) across the epithelium. These changes thus led to disruption of spermatogenesis. Interestingly, the knockdown of dynein 1 or its inactivation by ciliobrevin D also perturbed gross disruption of F-actin across the Sertoli cells in vitro and the seminiferous epithelium in vivo, illustrating there are cross-talks between the two cytoskeletons. In summary, these findings confirm the role of dynein 1 to support the transport of spermatids and organelles across the seminiferous epithelium during spermatogenesis.