Current Research Areas

Jonathan Tilly, PhD

The long-term goal of work in my laboratory is to improve women’s reproductive healthcare and overcome infertility by applying what we discover through basic and translational (preclinical) research. For over 15 years, a primary focus of my research group has been to understand the roles and regulation of the physiological cell death program termed apoptosis in the mammalian ovary (Nature Rev Mol Cell Biol 2001 2: 838-848). The majority of our work uses mouse models, which possess the advantage of relatively straightforward genetic manipulation. Since we started this work in the early 1990s, we have documented that premature ovarian failure and infertility resulting from conventional anti-cancer treatments involves the activation of apoptosis in oocytes (Nature Med 1997 3: 1228-1232; Genes Dev 1998 12: 1304-1314; Mol Endocrinol 1999 13: 841-850). We then validated an anti-apoptotic small molecule that protects the ovaries from side-effect damage caused by conventional caner treatments as a means to preserve normal reproductive function (Nature Med 1997 3:1228-1232; Nature Med 2000 6: 1109-1114; Nature Med 2002 8: 901-902), and we have recently taken this small molecule and its mimetics into preclinical testing using rhesus monkeys as a model.

In related work, we have demonstrated that polycyclic aromatic hydrocarbons (PAHs), which are toxic chemicals present at high abundance in the environment and in cigarette smoke, activate an orphan receptor-transcription factor (termed the aryl hydrocarbon receptor or AhR) in oocytes, leading to de novo expression of pro-apoptotic genes necessary for oocyte death (e.g., Bax) and early ovarian failure. Specifically, we reported that the AhR is abundantly expressed in oocytes, and that Ahr gene knockout in female mice leads to increased oocyte survival (Endocrinology 2000 141: 450-453). Moreover, in subsequent studies (Nature Genet 2001 28: 355-360; Endocrinology 2002 143: 615-620), we showed that the chemically-activated AhR induces Bax gene transcription in oocytes, and that expression of both the Ahr and Bax genes are functionally required for these chemicals to cause oocyte depletion and ovarian failure. Using a human ovarian xenograft model, we have further shown that an induction of Bax gene expression and apoptosis occurs in human primordial and primary oocytes following PAH exposure in vivo (Nature Genet 2001 28: 355-360). These data have been expanded on in a comprehensive study that details the global changes in expression of a large cassette of pro-apoptotic genes (in addition to Bax) in the ovaries following PAH exposure, and the requirement for p53 in working with the AhR to initiate oocyte apoptosis and follicle loss.
In addition to this work on pathological models of ovarian failure, we have shown that targeted inactivation of the pro-apoptotic Bax gene in mice prolongs ovarian lifespan into very advanced age, thereby eliminating the “mouse equivalent” of menopause (Nature Genet 1999 21: 200-203). This animal model has allowed us to explore, for the first time, the impact of sustained ovarian function on the aging female body, and to decipher the contribution of age-related ovarian failure versus the aging process itself to the manifestation of various health complications often observed in women after the menopause (Proc Natl Acad Sci USA 2007 104: 5229-5234).

Recently, the primary focus of our research changed from cell death to cell renewal, based on our studies that challenge one of the most basic doctrines in our field by demonstrating the existence of putative germline stem cells that support oocyte and follicle production in the ovaries of adult female mammals. For example, we recently provided evidence that juvenile and adult female mice retain proliferative germline cells that, based on rates of oocyte degeneration and clearance, are needed to continuously replenish the oocyte-containing follicle pool (Nature 2004 428: 145-150). This project is now aimed at fully characterizing female germline stem cell function, identifying the existence of such cells in humans, and developing new strategies based on stem cell transplantation technologies for enhancing fertility and perhaps delaying age-related ovarian failure.

Progress towards completion of these objectives is exemplified by a subsequent publication from our group documenting the presence of early germ cells in bone marrow and peripheral blood of adult female mice that are capable of generating immature oocytes in the ovaries of chemotherapy-sterilized or genetically-infertile adult female recipients following transplantation (Cell 2005 122: 303-315). Further, we have shown in a very recent paper that bone marrow transplantation rescues long-term fertility in adult female mice exposed to sterilizing doses of chemotherapy (J Clin Oncol 2007 25: 3198-3204). Moreover, infusions of young adult female bone marrow into aging female mice postpones the age-related onset of infertility and markedly improves the postnatal survival rates of offspring conceived by infused aged females (AGING 2009 1: 49-57). These studies, coupled with our efforts to identify specific genes and pathways that regulate the activity of germline stem cells in adult mammalian females (Cell Cycle 2007 6: 2678-2684), are providing the foundation for our ongoing development of a high throughput screening assay for identifying novel “oogenic” factors as potential therapeutics. Although this work remains controversial in some scientific circles, independent corroboration of these concepts from other laboratories is now available (reviewed in Biol Reprod 2009 80: 2-12).

Bo Rueda, PhD

Identification and functional characterization of gynecologic cancer stem cells.
Recent experiments conducted by our group in collaboration with Drs. Rosemary Foster and Dr. David Scadden (Harvard Stem Cell Institute) provide evidence to support the concept that both human endometrial and ovarian cancers contain a rare subpopulation of tumor-initiating cells, which have stem/progenitor like properties. We are actively optimizing strategies to better isolate these rare cells, define their stem like properties, determine how they are regulated, how their local environment may influence them, whether they are resistant to current radiation or chemotherapy and ultimately how we may target them.

Molecular interrogation of gynecologic tumors.
In collaboration with Drs. Darrell Borger and A. John Iafrate of the Translational Research Laboratory (MGH Cancer Center), we have been actively developing strategies for real-time identification of novel cell signaling pathways that contribute to malignant transformation, the pathology of the disease and/or chemoresistance and recurrence in gynecologic cancers. Once identified, we are actively testing new biologics and determining their efficacy in primary ovarian and/or endometrial tumor explant models and in short-term patient cell cultures derived from primary ovarian and/or endometrial tumors.

Delineating the contribution of novel genomic mutations to the pathology of gynecologic cancers.
We are currently conducting array CGH analysis of a large cohort of endometrial and ovarian gynecologic cancer samples with annotated clinical information in an attempt to detect novel mediators of malignant transformation and tumor growth, and expression with clinical response.

Defining the functional significance of mediators of cell proliferation and/or differentiation in ovarian and uterine biology.
We continue to focus effort into gaining a better overall understanding of steroid and growth factor induced cell proliferation. More recently, we have been identifying and characterizing Cables 1 interacting proteins to determine their functional significance in the regulation of the cell cycle, cell proliferation, differentiation and/or apoptosis. Moreover, we are assessing whether the biochemical interactions involving Cables 1 and the corresponding functional components are altered in the presence or absence of steroids and/or growth factors.

Identification of novel modulators in the pathogenesis of endometriosis.
Endometriosis is a polygenic disease with complex multifactorial etiologies affecting reproductive-aged women. Although this disorder is commonly treated in clinical practice, the mechanisms by which ectopic endometrium is disseminated and proliferates, is not completely understood. There are a number of different factors, which have been implicated in either the genesis or the propagation of endometriosis. They include but are not limited to prostaglandins, cytokines, growth factors, chemokines, cell adhesion molecules and steroid hormones. It is, however, difficult to investigate the functional role of these factors without adequate in vivo model systems. Dr. Styer and Dr. Rueda are continuing to develop mouse models which have been manipulated to ‘mimic’ the human disease in order that they may study the effect of specific factors on the growth of ectopic endometrium. The mouse model was chosen to incorporate the power of mouse genetics. Using mutant mouse strains that are devoid or over express one or more of the factors described above they should be able to delineate their cause an effect relationships with the pathogenesis of the disease. Using this strategy in tandem with gene and protein analysis Dr. Styer and Dr. Rueda hope to gain a better understanding of the underlying mechanisms of endometriosis. This information will also serve to develop more effective alternative treatment modalities.

Jose Teixeira, PhD

We are studying uterine development and function, particularly its differentiation from the primordial Müllerian ducts. We hypothesize that b-catenin plays a major role in postnatal uterine development and that its dysregulated function may be an underlying cause of leiomyomata, the most common gynecologic tumor. We are investigating the function of b-catenin in uterine development and have found that targeted deletion of b-catenin in mouse uterus leads to progressive replacement of smooth muscle with fat in the myometrium with estrous cycling. These results suggested that there is a regenerative cell in the uterus that is dependent on b-catenin function and is hormonally regulated. We propose to continue these studies by identifying the uterine smooth muscle cells that behave like the regenerative satellite cells in skeletal muscle. We will also attempt to determine the source of the cells that contribute to uterine smooth muscle. The studies will provide a better understanding of postnatal uterine development and should also provide clues to the etiology of uterine muscle pathologies such a leiomyomata, which are more commonly known as uterine fibroids and affect 25-40% of women.

Another focus of my laboratory is the in vitro maturation of human oocytes and their subsequent use for embryonic stem cell research. Human ES cells are currently derived from surplus embryos generated during in vitro fertilization (IVF) treatment. More recently, however, the technique of somatic cell nuclear transfer into unfertilized eggs offers the possibility of creating human ES cells whose genetic makeup matches that of the recipient. In addition to providing a powerful research tool for understanding human disease, this approach may eventually allow patients to be treated with an unlimited supply of new cells that will be recognized as 'self,' thereby avoiding the serious problem of rejection by the body's own immune defenses. In order for progress to be made on this front, a steady supply of human oocytes is needed. However, acquiring sufficient oocytes from donors undergoing requisite fertility treatments with no reproductive or medical benefit to themselves is problematic for both ethical and logistical reasons. Our contribution to this effort will be to isolate oocytes from the discarded ovaries of patients undergoing routine surgical procedures requiring removal of their ovaries. These isolated oocytes will be matured in vitro and stimulated to undergo parthenogenesis, with the ultimate goal of their development as recipients for somatic cell nuclear transfer.

Additionally, I have a long-standing collaboration with Dr. Patricia Donahoe to study the molecular mechanisms of MIS (also known as anti-Müllerian hormone or AMH) signaling and function. MIS is expressed shortly after differentiation of the fetal testis and is required for Müllerian duct regression during male fetal development. In females, the absence of MIS allows the Müllerian duct to develop into the female internal reproductive tract structures. By the end of puberty, the level of MIS expression has decreased substantially in males and is no longer sexually dimorphic because females have begun expressing MIS at levels that are indistinguishable from males. Why is MIS still expressed at such high levels in males well after Müllerian duct regression has occurred? Why does decreased expression of MIS herald puberty in males? Why do females begin expressing MIS? What role does MIS play in the gonad? Does MIS (or lack thereof in females) contribute to any predisposition to or protection from developmental errors in metabolism? MIS has been shown to be a strong inhibitor of ovarian, prostate, and breast cancer cell proliferation. Does MIS control proliferation of these tissues physiologically? These questions are largely unanswered and form the basis of our current and future efforts to understand the continued postnatal expression of MIS and its consequence to normal health and development, and as a possible cancer therapeutic.

Antony Wood, PhD

Cell signaling and embryonic development
The embryonic period, during which the fertilized egg, or zygote, develops into a fully-formed fetus, is among the most fascinating, yet enigmatic, phenomena in biology. It is also among the most sensitive stages of human development, as perturbations during this period underlie a majority of congenital birth defects. It is therefore a primary goal of reproductive biologists to more fully understanding the factors controlling embryonic development. One of the biggest barriers to understanding human embryogenesis is the simple fact that human embryos, like those of all mammals, must develop within the protective confines of the womb, and thus cannot be observed directly. To circumvent this problem, Dr. Wood employs a small tropical fish, called “zebrafish”, to study the genetic control of embryogenesis. Zebrafish are remarkably versatile creatures: they are highly similar to humans at the genetic level, yet develop externally of the mother, allowing researchers to study every stage of embryonic development in remarkable detail. During the last decade, the zebrafish has established itself at the forefront of embryology research, providing researchers with unparalleled insights into genes and pathways regulating embryonic development.

Dr. Wood has been using the zebrafish embryo to explore the embryonic functions of the insulin-like growth factor (IGF) signaling pathway, which has long been known to be important for human growth. However, the functions of this pathway during the embryonic phase of development have remained obscure, due to the inherent difficulties of studying mammalian embryos. Through genetic studies in the zebrafish embryo, Dr. Wood has now established an essential role for IGF signaling in development of the urogenital system (reproductive organs and kidney), and the population of cells that give rise to bones and muscles of spinal column. Experimentally suppressing the expression of IGF genes not only causes embryos to grow more slowly, but also leads to developmental defects resembling a spectrum of human birth defects known as holoprosencephaly. Dr. Wood is now pursuing the possibility of a genetic “dialogue” between IGF signaling and other genes linked to holoprosencephaly. He is optimistic that his findings will contribute to the search for clinical strategies to ensure the development and birth of healthy babies.

Genetic control of de novo oogenesis
Research at the Vincent Center for Reproductive Biology has led to the remarkable discovery that adult female mammals may have the capacity to generate new eggs during postnatal life, a controversial finding that has challenged one of the central dogmata of modern reproductive biology. It has been accepted for decades that, among invertebrates and lower vertebrates, females contain mitotically active ovarian germline stem cells in adult life, whereas evolution has inexplicably eliminated or inactivated this population of cells in higher vertebrates, including humans. While extensive research has characterized germline stem cells in model invertebrates, little work has been done on lower vertebrate model species, which may serve as a useful bridge to validating the existence and/or functionality of germline stem cells in adult mammals. Dr. Wood, with his postdoctoral fellow Yvonne Brown, is employing the zebrafish as a model for ovarian germline stem cell research: adult female zebrafish produce hundreds of eggs on a weekly basis throughout adult life, and are therefore viewed as good candidates in which to identify and characterize ovarian germline stem cells.

Aaron Styer, MD

The focus of our group’s work is the study of endometriosis and uterine fibroids, and the ultimate development of consistently effective medical therapies for this benign gynecologic disorder. Our studies have been based in the development and application of a novel immunocompetent, sygeneic mouse endometriosis model. Use of this model with mutant and wild type animals has been instrumental in characterizing the attachment and maintenance of endometriosis-like lesions in vivo. Notably, our recent work in this animal model has elucidated the functional role of the cytokine leptin, which has also been implicated in the pathogenesis of human disease. Specifically, we have demonstrated the mitogenic and neoangiogenic significance of leptin during the establishment of ectopic endometrial lesions and the early recruitment of vasculature (Endocrinology 2008; 149(2):506-14).

Ongoing studies have focused on the anti-inflammatory and anti-angiogenic role of omega three fatty acids during the incipient stages of endometriosis-like lesion development. Through continued investigation of our in vitro and in vivo models, we hope to increase our understanding of the potential application of this dietary supplement as an adjuvant therapy for human disease. In addition to animal studies, we will initiate gene express profile studies of banked human endometriosis and fibroids, with a long term goal to clearly understand the underlying mechanisms of disease development and recurrence.