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Title: Autophagy maintains the homeostatic environment in the male reproductive organs playing a key role in fertility
Author: Jaulim, Adil
ISNI:       0000 0004 8501 0442
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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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.
Supervisor: Masashi, Narita Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Autophagy ; fertilisation ; ATG5i model