DESCRIPCIÓN DE LA ENTIDAD LOCAL

The data presented in this dissertation support that autophagy is required for embryonic oocyte survival and adult Sertoli cell function. Additionally, the data support that Becn1 is necessary for luteal cell steroidogenesis. The goal of this dissertation was to

determine the significance of autophagy in the ovary and testis though conditional deletion of two key autophagy genes, Atg7 and Becn1 in the mouse. The underlying hypothesis tested was that autophagy is necessary for cellular homeostasis and data presented in Chapters 2 and 4 data support this. Deficiency in either Atg7 or Becn1 resulted in a reduced oocyte endowment in females and the degeneration of the seminiferous tubule followed by infertility in males. However, in Chapter 3, the data support two contradictory conclusions, dependent on the gene studied. Becn1 is required for progesterone synthesis during pregnancy and gene deficiency resulted in reduced circulating progesterone followed by abortion or preterm labor. In contrast, Atg7 gene deficiency resulted in a 2-fold increased production of progesterone, and did not affect pregnancy outcome. Presented in this final chapter, is a discussion of how these findings relate to each other and possible future directions to take.

Dysregulation of an essential process such as autophagy is likely to have a detrimental phenotype. Indeed, knockout mice for several key regulators of autophagy cannot survive after birth or are embryonic lethal (reviewed by Hale et al. 2013). Additionally, many of the secondary and tertiary regulators of autophagy have been implicated in several human diseases (reviewed by Jiang and Mizushima 2014). For example, mutation of an Atg8-like protein that becomes conjugated to autophagosome membranes in neurons results in higher iron-ion accumulation and causes neuron damage leading to behavioral abnormalities (Haack et al. 2012). By obstructing

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autophagy through the ablation of Atg7 or Becn1, I expected a negative effect on cellular homeostasis that would inhibit the normal function of that cell, as well as the revelation of any or non-autophagy related functions. In the case of embryonic oocytes and Sertoli cells this notion was supported. I observed fewer oocytes at birth and seminiferous tubules degenerated in autophagy deficient mice. However, we must also consider that key regulators of essential pathways are under strong selection pressure. Furthermore, this selection pressure supports the development of functionally redundant pathways and proteins. It should be appreciated that autophagy is not only an animal process, but that it is deeply rooted in the eukaryote lineage, with a well-supported hypothesis that autophagy was developed in the last-common ancestor to all eukaryotes that last existed approximately 2 billion years ago (Hughes and Rusten 2007). This makes sense as eukaryotes have organelles, and autophagy is the only process characterized to remove and degrade organelles at any appreciable rate. Thus, it is reasonable that throughout time gene duplication events would have allowed for redundant genes and pathways to develop. Many of the single genes identified in yeast that participate in many aspects of autophagy have expanded to gene families in mammals. For example, Atg8 is

represented in mammals by three gene families for a total of 10 different proteins (reviewed by Shpilka et al. 2011).

Now let us consider all of the processes in a cell. Many of these processes are

unrelated, but occasionally we will find proteins that perform functions in several different pathways, also called pleiotropy. Additionally, proteins can develop new functional domains that can create dependence for that protein in an additional process.

Furthermore, some of these proteins are key regulators of essential processes, forming interaction networks that are subject to high selection pressure. However, more often we will find that there are cellular processes that rely upon essential processes to

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function. Think about ATP synthesis, RNA transcription or fatty acid hydrolysis that all generate products that are necessary for downstream cellular processes. There are models that try to explain how selection drives these interactions networks elsewhere, but I will not elaborate (Nowak et al. 1997). It is here that I will leave you with the implied hypothesis of this dissertation. By ablating the key regulators of autophagy Atg7 and Becn1 we dysregulate autophagy allowing for the potential discovery of processes that depend on either the gene, autophagy or both.

This dissertation provides evidence that Becn1 is necessary for endosome maturation, in addition, to its role in autophagy. The transmission electron micrographs presented in both Chapters 3 and 4 are great examples of this, demonstrating an accumulation of endosomes in Becn1 cKO luteal cells and Sertoli cells. Endosomes are a diverse group of vesicles within a cell. They can originate from the Golgi apparatus to secrete soluble proteins, the endoplasmic reticulum to deliver transmembrane signaling molecules and transporters or the plasma membrane to internalize extracellular components (Huotari and Helenius 2011). Moreover, there is evidence that not all endosomes are created equal. Different functional families of endosomes have a distinct protein-profile associated with their membranes and contents.

Endocytosis or the process of internalizing extracellular components into endosomes, parallels autophagy in many aspects. First, cells can acquire energy and nutrients through endocytosis of macromolecules and proteins. Second, the end is the same, as a mature endosome fuses to a lysosome. An important function of endocytosis is the internalization of cell-surface receptors and their ligands, which is vital for a cell’s growth and communication, and can provide an example for endosome maturation.

Endocytosed material is internalized as endocytic vesicles that fuse together to form

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early endosomes. The function of the early endosome is to sort its contents for downstream processes. Cell-surface receptors inside can be recycled back to the plasma membrane, transferred to the Golgi network or remain in the endosome. The contents of an endosome can be directly recycled without digestion or can be

transported to multivesicular bodies, also called late endosomes where the continued fusing with intraluminal vesicles and themselves cause them to grow in size and gather contents. Late endosomes then fuse with lysosomes and the contents are digested and provided for use by the cell.

There are a number of proteins involved in transporting and identifying the different classes of endosomes. Of these the Rab-GTPases, Rab5 and Rab7 are best studied.

Once activated the Rab-proteins facilitate the binding to motor proteins to transport the endosome along microtubules. Additionally, the Rab-proteins recruit other proteins that facilitate endosome maturation including hydrogen ion pumps, which act to lower the pH inside a late endosome. Rab5 is found predominately on early endosomes and is necessary for early endosome fusion events, suggesting its role in sorting. On the other hand, Rab7 is predominately found on late endosomes and is necessary for

multivesicular body and lysosome fusion. Thus an exchange of Rab5 for Rab7 occurs during this transition that can be modeled (Binder and Holzhütter 2012).

In the Becn1 cKO luteal cells, I found an increased number of endosomes with a

concurrent decrease in progesterone production and neutral lipid storage. Cholesterol is the substrate for steroidogenesis and lipid droplet formation, and as discussed in

Chapter 3, it is possible that a decrease in the ability to transport cholesterol is

responsible for these phenotypes. I observed large endosomes that in many cases were empty, throughout a majority of the cytoplasm. There is no evidence that these

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endosomes fuse with multivesicular bodies and / or lysosomes because they are too big and are not electron dense structures. It is possible that these endosomes are early endosomes and their contents include the compliment of LDL and HDL cell-surface receptors. If we believe this, then receptors cannot be recycled back to the plasma membrane because early endosomes are not maturing. Therefore, the luteal cell cannot transport enough HDL and LDL to support progesterone production. If this is true, then there should be an accumulation of Rab5 on the endosomes in the luteal cells of Becn1 cKOs. I would also expect a phenotype in progesterone production when the receptor in question is inhibited. The LDLR knockout females have no apparent fertility defects (Ishibashi et al. 1993). As LDLR is the prominent lipoprotein receptor endocytosed and mice can compensate for its loss, this is not a likely candidate. In contrast, SR-BI females are infertile (Trigatti et al. 1999). This appears to be due to an increase of circulating HDL that in turn has a detrimental effect on oocyte competence (Miettinen et al. 2001). However, the SR-BI knockout females have been shown to produce 50% less progesterone during an induced pseudopregnancy (Jiménez et al. 2010) and knockdown of SR-BI in human differentiated granulosa cells results in reduced progesterone

synthesis (Kolmakova et al. 2010). This suggests that SR-BI function could be affected in the Becn1 cKO. I did not observe a reduction in Scarb1 mRNA in my study at P8.5 but it is possible the cellular distribution of SR-BI is not in the plasma membrane where it would be expected. While the relationship between SR-BI progesterone is suggestive of the interaction with BECN1, the endocytosis of SR-BI is not the primary function

described and might be rare. It would be important to characterize SR-BI endocytosis in mouse luteal cells during pregnancy. Additionally, I do not know what the contents of the observed endosomes are. The endosomes may be full of LDLR and LDL or SR-BI contributing to reduced cholesterol transport. Alternatively, the increased number of endosomes may cause cell stress leading to decreased steroidogenesis. A necessary

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experiment would be to detect Rab5 and Rab7 in isolated endosome fractions to determine if these are early or late endosomes. In the same study, the presence of the cell-surface receptor candidates can be tested.

The phagocytosis block in Sertoli cells presented in Chapter 4 parallels the endosome story. I identified elongated spermatids and degenerating spermatocytes within the Sertoli cells of Becn1 cKO but not in controls. This supports that phagocytized material is not entering the endosome pathway for further maturation and degradation.

Phagocytized vesicles are decorated with Rab-proteins and proceed to mature similar to early and late endosomes. A difference between endocytosis and phagocytosis being that cell-surface receptors are not recycled back to the plasma membrane but to the Golgi complex through the retromer complex. As introduced in Chapter 4, there is growing evidence that the Atg8-like protein, LC3, is recruited to phagocytic vesicles that contain Listeria monocytogenes (Lam et al. 2013), and this association is necessary for degradation. It is unknown if something similar occurs in Sertoli cells; however, cultured Sertoli cells have been shown to activate autophagy as recognized by LC3 conversion during phagocytosis (Yefimova et al. 2013). I attempted to look at this phenomenon by observing LC3 in the testes of GFP-LC3 mice; however, there was no detectable GFP puncta in WT or mutants (data not shown). This is surprising because autophagosomes have been observed by electron microcopy in Sertoli cells (Galdieri et al. 1981);

however, GFP puncta were also not observed in WT luteal cells, but were seen in oocytes and theca cells. As both the Sertoli cell and luteal cells are highly metabolic, it is possible that any autophagosome that is formed is small and short lived, making it difficult to observe puncta. Alternatively, it could be possible that fixation quenched the GFP signal in the testes. However, I did observed GFP puncta in the epididymal clear cells (data not shown). Additionally, different amounts of fixation did not bring about

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signal, suggesting that no puncta exist in the testis. I quantified LC3 by Western blot from whole testis but detected no difference between either Atg7 cKO or Becn1 cKO and WT (data not shown). This result could be because there is no difference, or that the population of Sertoli cells is too small to detect a difference. A better experiment would be to detect LC3 in a purified population of Sertoli cells. Now if we believe the GFP-LC3 results in the testis, that there are no puncta, this must mean either Sertoli cells do not use LC3 for incorporation into membranes or the LC3 is quickly degraded. Treatment of cultured GFP-LC3 Sertoli cells with bafilomycin A1, which blocks lysosome maturation, and thus causes an accumulation of autophagosomes, would be one way to test this.

Regardless of these suggested experiments, the data demonstrate the phenotype.

The ablation of either Becn1 or Atg7 in the Sertoli cells appear to have the same phenotype. I performed no mechanistic analysis to prove this, but the phenotypes are similar. It is possible that dysregulation of autophagy is the cause of the phenotype in both mutants, but the delayed onset in Atg7 cKO mice casts doubt. It is possible that Atg7-independent autophagy occurs in Sertoli cells to compensate for the loss of Atg7 and it merely takes longer for the phenotype to manifest itself. Alternatively, Becn1 is involved with the endosome maturation and this is essential for Sertoli cell function while in the Atg7 mutants the Sertoli cell succumbs to the loss of homeostasis. The latter is clearly the case in the luteal cells, where the loss of Atg7 showed the opposite

phenotype compared to the loss of Becn1. Not explored in this dissertation was the idea that compensation for the loss of Becn1 could be happening through Beclin-2. It would be easy enough to look at mRNA expression of Beclin-2 and protein expression of BECN2 in the Becn1 knockouts.

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So what is the evidence that BECN1 participates in endosome maturation? The receptor recycling of EGFR and the maturation of Cathepsin D are the best examples I currently know of. EGFR is a cell-surface receptor that binds to EGF and EGF-like ligands. Upon ligand binding, the receptor homodimerizes and results in the activation of multiple signaling pathways (e.g. MAPK, JAK/STAT, PI3K/AKT/mTOR, and

PLC/PAG/PKC) (Sui et al. 2014). Many of which were discussed in relation to the activation of the cumulus granulosa during the LH surge in Chapter 1. Once bound to ligand, the EGFR-ligand complex remains active and in order to downregulate the transduction pathways, the EGFR-ligand complex undergoes receptor-meditated endocytosis to either degrade the receptor-ligand complex or to degrade the ligand and recycle the receptors back to the plasma membrane. The presence of PtdIns3P on early endosomes that contain EGFR-ligand complexes is responsible for maturation and sorting to late endosomes by recruiting Rab7 (Petiot et al. 2003). It was later shown the presence of PtdIns3P is not a function of the plasma membrane class I PtdIns3k, but of the class III PtdIns3k, which also regulates autophagosome formation (Thoresen et al.

2010). Moreover, the knockdown of individual components of the PtdIns3k complex (e.g. VPS34, VPS15, BECN1, UVRAG and BIF-1) results in inhibited EGFR sorting and recycling (Thoresen et al. 2010).

The evidence with Cathepsin D is similar, but from a different source of endosome. I have only described endocytosis, thus far, but endosomes can mature from the Golgi-network, as well. Cathepsin D is an aspartic protease that is a member of the milieu of lysosome proteases. To inhibit random protease activity, many lysosome proteases are translated in an inactive form that only become active after entering a lysosome.

Cathepsin D is such a protein that must undergo maturation by cleavage after entering a lysosome. Thus, tracking of Cathepsin D through the endosome maturation is possible

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by detecting the different forms. Using experiments similar to what I just described, Cathepsin D was identified as another protein that did not translocate correctly in endosomes when trying to elucidate how EGFR is sorted in VPS34 deficient cells (Row et al. 2001). In the same experiments already discussed for EGFR, Cathepsin D

maturation was equally, negatively affected, suggesting a similar mechanism (Thoresen et al. 2010). The identification of the Rab7 interacting protein, Rubicon, that also

interacts with BECN1, demonstrated a mechanism for how the BECN/PtdIns3K complex could interact with endosomes (Liang et al. 2008). Later a “feed-forward loop” between UVRAG, Rubicon and C-VPS/HOPS (C vacuolar protein sorting complex / homotypic vacuole fusion and protein sorting) was found to exist in human cell culture that activates Rab7 and subsequent endosome maturation (Sun et al. 2010). Thus, the

BECN1/PtdIns3k complex is present on endosomes during endosome maturation.

However, this appears to only influence some endosomes, such as those containing Cathepsin D and EGFR, as other aspects of endosome maturation were reported to be normal in the referenced studies. So, is there a commonality between phagocytic vesicles, vesicles containing potential lipid substrate and the EGFR/Cathepsin D

endosomes? I am not sure, but will speculate that the scavenger receptors involved with phagocytosis in Sertoli cells and endocytosis in luteal cells are similar to

EGFR/Cathepsin D. It would be an attractive experiment to follow such receptors in the mutants described in this dissertation.

Within this dissertation, I have presented data supporting my hypotheses that autophagy is necessary for oocyte survival and Sertoli cell function. Additionally, the data provide evidence that Becn1 has an essential role for steroidogenesis in luteal cells. A theme that Becn1 has pleiotropic roles to autophagy is also prevalent in the data, further supporting that the BECN1/PtdIns3K complex plays a role in endosome maturation,

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regulating the degradation of phagocytic vesicles and perhaps lipoprotein-receptor mediated endocytosis. Overall, I have shown that autophagy is a process necessary for the function of the reproductive system and is an attractable model to study. The

preterm labor mouse may provide insights into progesterone production in humans, and demonstrates that there is conservation of autophagy regulation on steroidogenesis between Leydig and luteal cells. This provides us with a hypothesis that Becn1 deficiency in placenta will negatively impact steroidogenesis, and may lead to preterm birth. Additionally, this opens up the opportunity to test the autophagy-steroidogenesis interaction in other cell types, like the adrenal corticotrophs. The implications for the Sertoli cell work may be important for future study of phagocytosis, trafficking and degradation of the phagocytized material. Knowing that phagocytosis machinery such as BECN1 is conserved among several different phagocytes, allows for a comparative approach to studying disease models such as, macular degeneration. Regardless of these outcomes, the data within this dissertation provide a better understanding of the role of autophagy in oocytes, luteal cells and Sertoli cells and provides new evidence to ask new questions in the field of reproductive biology.

Copyright © Thomas Raymond Gawriluk 2014

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