A novel Atg5-shRNA mouse model enables temporal control of Autophagy in vivo.

Macroautophagy/autophagy is an evolutionarily conserved catabolic pathway whose modulation has been linked to diverse disease states, including age-associated disorders. Conventional and conditional whole-body knockout mouse models of key autophagy genes display perinatal death and lethal neurotoxicity, respectively, limiting their applications for in vivo studies. Here, we have developed an inducible shRNA mouse model targeting Atg5, allowing us to dynamically inhibit autophagy in vivo, termed ATG5i mice. The lack of brain-associated shRNA expression in this model circumvents the lethal phenotypes associated with complete autophagy knockouts. We show that ATG5i mice recapitulate many of the previously described phenotypes of tissue-specific knockouts. While restoration of autophagy in the liver rescues hepatomegaly and other pathologies associated with autophagy deficiency, this coincides with the development of hepatic fibrosis. These results highlight the need to consider the potential side effects of systemic anti-autophagy therapies

Autophagy maintains the homeostatic environment in the male reproductive organs playing a key role in fertility

Autophagy is an evolutionarily conserved recycling process that plays a crucial role in maintaining cellular homeostasis. It has been reported that autophagy is closely involved in the physiological process of spermatogenesis and the regulation of sperm survival. However, the role of autophagy during the phase of sperm maturation in the epididymis and seminal fluid production from the accessory sex organs remains unknown. Using a doxycycline-inducible short hairpin (shRNA) mouse model (called ATG5i) targeting Atg5, we created a mouse model that demonstrates ubiquitous inhibition of autophagy. With this model, we investigated the effects of autophagy inhibition and restoration in the environment of male reproductive organs using a combination of histological and transcriptomics methods. Interestingly, ATG5i mice spermatogenesis remained unimpaired as doxycycline does not penetrate the blood-testis barrier. This gave us a unique window to assess the effects of autophagy inhibition on the quality of sperm post-spermatogenesis. ATG5i male mice displayed a progressive reduction in the size of their reproductive organs after 6 weeks on doxycycline, which we called a ‘castration-like’ phenotype. This was consistent with the reported phenotype of conventional Atg5 knockout (KO) mouse models, which show hypogonadism, leading to a reduced level of testosterone. However, our model showed no changes in serum testosterone levels. Despite this, we found gross cellular changes in the epididymis and evidence of fibrosis following autophagy inhibition. Semen analysis revealed low counts and poor motility with significantly reduced fertilisation rates during in vitro fertilisation (IVF), as well as evidence of subfertility following in vivo natural copulation. Finally, transcriptomic analysis of the seminal vesicles revealed a subset of androgen-responsive genes coding for seminal fluid protein that were strongly downregulated. The findings from this study suggest that the apparent ‘castration-like phenotype’ associated with systemic inhibition of autophagy is not merely due to hormonal influence. In this homeostasis model, knockdown of autophagy affects the epididymal environment leading to poor sperm quality. Moreover, the seminal vesicles are smaller as autophagy is crucial for seminal fluid production, highlighting a critical post-testicular role for autophagy in male fertility. Restoration of autophagy, through the removal of doxycycline food, led to a recovery in the seminal vesicle and epididymal phenotypes as well as the overall fertilisation rates. Finally, we developed a second ATG5i model (1654 model), using the second most potent shRNA against Atg5. Initial data showed phenotypic effects comparable to the ATG5i model, suggesting that our findings in the male reproductive organs were not off-target effects. Future work will look to identify the mechanistic role of autophagy in the epididymal cells following both autophagy depletion and restoration

Autophagy maintains the homeostatic environment in the male reproductive accessory organs playing a key role in fertility

Autophagy has been implicated in male fertility but its specific role in the post-testicular organs remains unclear. Here, we investigate this in mice expressing a doxycycline-inducible RNAi against Atg5 (Atg5i). Systemic autophagy inhibition in Atg5i mice resulted in the morphological and functional abrogation of the male accessory sex organs, leading to male subfertility. However, the testis was largely protected, likely due to the limited permeability of doxycycline through the blood-testis barrier. Interestingly, restoration of autophagy by doxycycline withdrawal in Atg5i mice led to substantial recovery of the phenotype in the accessory organs. This model offers a unique opportunity to dissect the pre- and post-testicular roles of autophagy, highlighting the non-autonomous impact of autophagy on male fertility

A novel <i>Atg5</i>-shRNA mouse model enables temporal control of Autophagy <i>in vivo</i>

<p>Macroautophagy/autophagy is an evolutionarily conserved catabolic pathway whose modulation has been linked to diverse disease states, including age-associated disorders. Conventional and conditional whole-body knockout mouse models of key autophagy genes display perinatal death and lethal neurotoxicity, respectively, limiting their applications for <i>in vivo</i> studies. Here, we have developed an inducible shRNA mouse model targeting <i>Atg5</i>, allowing us to dynamically inhibit autophagy <i>in vivo</i>, termed ATG5i mice. The lack of brain-associated shRNA expression in this model circumvents the lethal phenotypes associated with complete autophagy knockouts. We show that ATG5i mice recapitulate many of the previously described phenotypes of tissue-specific knockouts. While restoration of autophagy in the liver rescues hepatomegaly and other pathologies associated with autophagy deficiency, this coincides with the development of hepatic fibrosis. These results highlight the need to consider the potential side effects of systemic anti-autophagy therapies.</p