Resultados del Modelo por Estrato

These results indicate that Becn1 regulates the clearance of phagocytized differentiating germ cells. The Sertoli cell is a non-professional phagocyte within the seminiferous tubule and is responsible for the phagocytosis of apoptotic germ cells, in addition to, residual bodies. In H&E stained tissue, I observe the presence of sperm heads within Sertoli cells in close proximity to the basal lamina. Additionally, by ultrastructure analysis I observe the presence of elongated sperm heads and what appear to be spermatocytes within vesicles of the Becn1 cKO Sertoli cell. Moreover, these inclusions have a noticeable structure, with the ability to be identified, and are not present in WT Sertoli cells, indicating that they are not being degraded. These observations support my hypothesis that autophagy proteins are necessary for the clearance of phagocytic

vesicles.

I propose a four-step model for Sertoli cell failure in Becn1 cKO mice. First, Sertoli cells begin phagocytizing apoptotic, differentiating germ cells and residual bodies, but cannot fuse these to lysosomes. The BECN1 complex that binds to UVRAG has been shown to be necessary for endosomes containing EGFR and Cathepsin D to fuse to lysosomes

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(Zeng et al. 2006). One would expect an excess of lysosomes in Becn1 cKO Sertoli cells if this was the case, but the data showed the opposite. The lack of lysosomes could be due to decreased lysosome biogenesis that partly occurs from endosome-endosome fusion events (Nielsen et al. 2007), which I have implicated BECN1 in

previously. Either way, phagocytized vesicles fail to fuse to lysosomes. Second, Sertoli cells undergo a stress response due to having an accumulation of phagocytic vesicles and drop in available ATP. While the true implication of an accumulation of phagocytic vesicles is largely unknown, the accumulation of autophagosomes, endosomes or lysosomes is attributed to cell death. Thus, I suspect that the accumulation of

undigested phagocytic vesicles would be detrimental. Furthermore, Sertoli cells have a very high metabolism compared to other cells in the testis and obtain ATP from lipid metabolism or through the degradation of phagocytized material (Xiong et al. 2009).

Third, since Sertoli cells cannot further support phagocytosis, management over the incredibly high amount of apoptosis that occurs in the seminiferous tubule is lost. The observation of particles that resemble residual bodies in the epididymides of both Becn1 cKO and Atg7 cKO mice suggest that there is a failure in Sertoli cells to support further phagocytosis. Additionally, a role for autophagy in regulating the expression of

scavenger receptors on the surface of macrophages has been demonstrated (Bonilla et al. 2013). The specific scavenger receptor, scavenger-receptor, class B I (SR-BI) is partly responsible for the binding to and phagocytosis of membranes with exposed PS (e.g. apoptotic cells and residual bodies) (Nakanishi and Shiratsuchi 2004). Thus, loss of scavenger receptor trafficking by autophagy proteins in my conditional knockouts may be responsible for the apparent reduced phagocytosis. While SR-BI knockout males are fertile no study on the testis have been performed to determine what compensatory mechanisms exist (Rigotti et al. 1997). Fourth, with differentiating germ cells undergoing apoptosis there is an increase in inflammatory cytokines, such as TNFα, resulting in the

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degradation of the tubule and infiltration of leukocytes. Indeed, exposure of the seminiferous epithelium to TNFα disrupts the blood-testis-barrier leading to leukocyte infiltration (Li et al. 2006a). Additionally, recent insights have identified that TNFα

downregulates the expression of Coxsackievirus and adenovirus receptor in Sertoli cells, which facilitates germ cell-to-Sertoli cell junctions and results in sloughing of material into the lumens (Gao and Lui 2014). I detected an inflammatory response in the Becn1 cKO as increased Tnfa and ApoJ expression, and leukocyte infiltration into the

epididymides.

To determine if this model is correct, there are several experiments that need to be done. First, a molecular identification of the cytoplasmic material in the epipdymides to determine if these are residual bodies should be completed. Second, the

characterization of an accumulation of phagocytized material needs to be performed.

This could be done though feeding bacteria that block phagocytic vesicle fusion. Third, apoptotic indices of differentiating germ cells in mutant testes should be characterized to determine if increased apoptosis occurs with gene ablation. Fourth, to determine the apparent permeability of the blood-testis-barrier small molecule tracers could be injected into mutant testes. Characterization of the ability of mutant Sertoli cells to bind

molecules like PS can determine the propensity to initiate phagocytosis. Lastly, the presence of GNCA positive cells in Becn1 cKO tubules, suggests that Sertoli cells are minimally functioning. If the Sertoli cells are simply stressed, then this should be reversible through reintroduction of the gene that was knocked out. This could be done with the use of an inducible gene system, such as a TET-on system, where a

tetracycline-induced transcription factor induces the expression of a replacement transgene that is expressed only after Cre recombination, to limit the rescue to the original cells.

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4.4.3 Summary

Amhr2-Cre has also been shown to be expressed in interstitial cells of the testis, and any confounding effects from that expression might be of concern (Teixeira et al. 1996;

Jeyasuria et al. 2004; Tanwar et al. 2010). However, I did not observed recombination by GFP expression in Leydig cell and there was no phenotypic defect observed in Leydig cell steroidogenesis when comparing WT and Becn1 cKO mice. The

dysregulation of Becn1 in rat Leydig cells reduces testosterone production, and would have been an expected result if recombination occurred in Leydig cells. However, I did not detect a decrease in testosterone, as suggested by similar seminal vesicle weights.

Moreover, comparison of the seminal vesicles and prostate from 26-week-old Atg7 cKO mice and WT, suggest that there are increasing amounts of testosterone produced. In Becn1 cKO testis, it appears that Leydig cell hyperplasia does occur. However, at 12-weeks-old Becn1 cKO testes are smaller than WT testes, and their weight is half of WT testes, suggesting the apparent increase in Leydig cells numbers in any given section is caused by the decrease in total volume of the adult mutant testis. Similar observations have been made in other mouse models with defective Sertoli and/or Sertoli-germ functions (Meng et al. 2000; Papaioannou et al. 2009; Tanwar et al. 2010).

The conclusions from this study support a role for Becn1 in regulating Sertoli cell phagocytosis and function. Furthermore, the similar phenotype in Atg7 cKO mice demonstrate a role for autophagy in Sertoli cell function, although the ablation of Atg7 is less severe when compared to the deletion of Becn1. The conclusions from any study where a crucial developmental pathway is perturbed should be carefully analyzed, and this is true for this study. Further experiments are needed for broad and mechanistic conclusions, but I provide novel evidence that of autophagy is a necessary process for Sertoli cells.

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