The long-term objective of our lab is to determine how two types of programmed cell death, apoptosis (programmed cell death type I) and autophagy (programmed cell death type II), are utilized within reproductive tissues during mammalian development.  During macroautophagy (referred to as autophagy), macromolecules and organelles are encapsulated within autophagosomes (APs) that can fuse with lysosomes to form autolysosomes (ALs) which degrade their cargo.  My research program has developed unique mouse models that have illuminated roles for apoptosis and autophagy in the development and maintenance of the ovary, testis, and mammary gland for reproductive performance.  One of our models is based on the conditional gene deletion of Beclin1 (Becn1), a key regulator of autophagy.  Although our current models are based on tissue-specific gene deletion strategies within these reproduction organs, these models will be essential tools for the greater research community in the years to come.   

Autophagy in Female Reproduction.  In the mammalian ovary, the female is endowed with the highest numbers of germ cells before birth during gestation.  Many of these germ cells become surrounded by granulosa cells to form follicles, 99% of these follicles are lost during the female reproductive lifespan.  Although apoptosis has been considered the primary regulator of follicle loss, we have determined that autophagy plays a crucial role in this maintenance of female fertility.  We find that compromised autophagy, through the loss of Becn1 in the ovary, results in: 1) premature loss of female germ cells, 2) increased numbers of atretic follicles from loss of granulosa cells, and 3) reduced fertility.  Thus is appears that Becn1 maintains a requisite autophagic cell survival program in the murine female reproductive tract.

Autophagy in Male Reproduction.  Infertility affects 15% to 20% of couples in the United States, with male factors contributing 25% to 50% of the infertility problems predominantly due to low sperm number, reduced sperm motility, or altered sperm morphology.  A barrier in this field arises when the role of novel biological pathways in the process of fertility is completely unknown, such as autophagy.  In the testis, autophagy occurs within Sertoli cells, a cell population that provides nourishment and a protective environment for the germ cells during spermatogenesis.  One of the additional major characteristics of Sertoli cells is their ability to phagocytize damaged germ cells so that healthy germ cells can replenish the testis.  In order to characterize the impact of this major biological pathway on spermatogenesis, we generated a novel mouse model through a conditional knockout (cKO) approach that only renders the Sertoli cells autophagy-defective.  This induced mutation results in a complete male sterility phenotype by 10 weeks of age from the complete loss of germ cells within the testis. This has led us to hypothesize that Sertoli cells utilize autophagy during phagocytosis to eliminate damaged germ cells from the testis.  An additional model that causes ablation of autophagy in the Leydig cell population, a cell type that is responsible for the production of testosterone, results in acephalic (decapitated) sperm by 16 weeks of age and concomitant loss of fertility.  Therefore, we have two distinct cell populations within the male reproductive system that are reliant upon autophagy for the production of viable sperm and the maintenance of male fertility.

Autophagy and Pre-Term Birth.  Pre-term birth affects an estimated 13 million babies worldwide leading to approximately 1 million neonatal deaths per annum.  In addition to the emotional distress associated with pre-term birth, the economic burden is enormous, costing at least $26 billion annually in the U.S. alone. Currently, the vast majority of pre-term animal models studied in the reproductive field rely on the use of bacterial infections to induce an inflammatory response leading to an early parturition. We have developed a novel, genetic-based model for human pre-term birth through a conditional gene knockout (cKO) of the autophagy gene Beclin1 (Becn1) in ovarian granulosa cells.  Although the progesterone-secreting corpus luteum (CL) can initially establish pregnancy, a mid-gestation drop in progesterone leads to premature parturition, a phenotype reversible by progesterone administration.  In addition to determining the mechanism and biological effects of progesterone withdrawal on pregnancy, our lab seeks to uncover early diagnostic markers and more effective therapeutic strategies to reduce the rate of pre-term births.

Autophagy and Apoptosis in the Mammary Gland.  The mouse mammary gland serves as an ideal model to evaluate genes devoted to cell cycle regulation, proliferation, energy homeostasis, programmed cell death, and cancer. It is a highly dynamic organ that is dependent upon the secretory epithelia to provide sustenance to offspring.  These cells undergo massive levels of proliferation during pregnancy, secrete lipids and proteins into the milk during lactation, and succumb to programmed cell death during involution in order to remodel the gland to a non-pregnant state.  We have shown that the lactating mammary gland is poised to quickly undergo an involution state that is driven by apoptosis.  Mammary glands from a transgenic mouse designed to express a pro-apoptotic protein (BAX) cannot lactate and instead progress from pregnancy directly to an involution phase.  We also find that compromised autophagy in one of our conditional Becn1 mouse models results in a lactation failure phenotype.  Although the basic lobuloalveolar structure has developed during pregnancy, these mammary glands retain large, cytoplasmic lipid droplets and are unable to produce milk.  Thus, autophagy is essential in the transition of the pregnant gland into a functional lactating gland. 

The research efforts described above have helped define roles for autophagy in mammalian development, and have had direct impacts in the field of reproductive biology.  To sustain this impact and broaden my research program’s contributions, we will delve deeper into the mechanisms through which autophagy operates within our established models.  For example, in our Sertoli cell deficient model, we will need to determine whether the primary defect is from attenuated autophagy, reduced phagocytosis, or both.  If BECN1 is regulating the end stages of phagocytosis, there may be a mechanism similar to the BECN1-Rab5 pathway which Dr. Doug Green (St. Jude Children’s Research Hospital) has found within macrophages.  Since BECN1 has both autophagy-dependent as well as autophagy-independent roles, we can elucidate whether our established phenotypes are solely autophagy-based.  We have initiated this validation with another model in the autophagy field which is based on the conditional deletion of Atg7, a gene restricted to autophagy regulation.  In performing these experiments, we are finding that several of our phenotypes are due to autophagy-independent roles for BECN1.  Within the mammary gland and corpus luteum, there may be similarities in lipid transport that are defective in both organs when BECN1 is absent.  We will be examining the lipid profiles of these organs in collaboration with Dr. Andrew Morris (UK).  Since up to 40% of human primary breast tumors have a reduction in BECN1, we have initiated a project to determine the role of BECN1 and autophagy in a mouse model for breast cancer based on the overexpression of the HER2/NEU oncoprotein.  It is though that initially the loss of autophagy, which can act as a negatively regulator of cell growth, can provide a growth advantage to the tumor cells.  Once the tumor has achieved a critical mass, the interior portion of the tumor would need to activate autophagy as a stress survival mechanism.  This model should help clarify how autophagy is utilized by the developing tumor during its progression.  Finally, we have developed an additional novel reagent for the autophagy field to monitor autophagic flux.  In vivo autophagy is traditionally detected with a GFP-based transgenic mouse to assess autophagosomes (not autolysosomes) and only provides a ‘static’ measurement of a ‘dynamic’ process.  However, the biological role of autophagy in orchestrating the physiology and pathophysiology of development and disease can only be ascertained through tools that monitor autophagic flux.  Since there are currently no mouse models, we have developed a dual reporter system through a knock-in strategy in embryonic stem cells.  The generation of mice through these targeted cells will provide an important, additional resource for the scientific community.