The germ cell lineage threads continuously from generation to generation via the sperm and egg. These gametes combine at fertilization to give rise to the totipotent zygote (one-cell embryo) of each succeeding generation. Therefore the germ cell lineage is immortal and has extraordinary developmental potential. The Orwig lab conducts research to identify and characterize the molecular mechanisms that control the development of germ cells during fetal, perinatal, juvenile and adult stages of life. Understanding the normal development of spermatogenesis, oogenesis and fertility provides a framework for understanding conditions that lead to fertility deficits in men, women, boys and girls.
Molecular Mechanisms Controlling Spermatogonial Stem Cell Biology
Ongoing studies in our lab have identified genes and proteins that are expressed by male germ cells at different stages of the life cycle and we are actively investigating their biological functions. CRISPR/CAS9 gene editing and microRNA gene knockdown technologies are used to target candidate genes in spermatogonial stem cell cultures. The function of the genetically modified stem cells is evaluated in vitro and in vivo after transplantation into infertile males.
Functions of candidate genes are also evaluated using transgenic and knockout animal models. Dr. Orwig and Dr. Sheng direct the Genome Editing, Transgenic and Virus Core of Magee-Womens Research Institute and have access to state of the art transgenic and gene editing technologies (Sheng et al., 2010; Zhang et al., 2013).
Testis and Germline
The spermatogonial stem cell transplantation technique provides a functional endpoint for evaluating stem cell activity and niche quality during fetal and postnatal testis development. Our results demonstrate that stem cells from newborn, young and adult testes have similar capacities to produce and maintain spermatogenesis. In contrast, testes of young animals provide a superior environment for donor germ cell engraftment and re-initiation of spermatogenesis. These principles educate us about how spermatogonial stem cells might be optimally deployed to preserve and restore male fertility. Similar stem cell transplant studies are being used to examine effects of disease, medical treatments (e.g., radiation, chemotherapy) or age on stem cell activity and niche quality (Ryu et al., 2006; Hermann et al., 2007).
Stem Cell Therapies for
Applying our discoveries on stem cell activity and niche quality in mouse and rat testes, it is now possible to restore fertility in infertile males by transplanting spermatogonial stem cells. The spermatogenic process is well conserved among mammals and techniques developed in rodents should translate to other species. To facilitate the extension to higher species, we optimized a primate to mouse xenotransplant technique as a routine biological assay for stem cell activity in nonhuman primate and human testes (Hermann et al., 2007; Hermann et al.,2009; Dovey et al., 2013; Valli et al., 2014).
Men or boys who receive chemotherapy or radiation treatments for cancer have few options to safeguard their fertility. Spermatogonial stem cell transplantation provides a potential therapeutic avenue. We recently demonstrated that transplanted spermatogonial stem cells could regenerate spermatogenesis and produce functional sperm in infertile male primates rendered infertile by chemotherapy treatment (Hermann et al., 2012). Ongoing studies will systematically evaluate the feasibility and safety of the stem cell transplant technology to preserve and restore the fertility of men or boys who will be rendered infertile due to disease or medical treatment. In addition to the spermatogonial stem cell transplantation technology, we are actively collaborating with several groups to investigate the potential of induced pluripotent stem cells (iPSCs) to produce transplantable germ cells or gametes (eggs or sperm) (Easley et al., 2012; Ramathal et al., 2014; Durruthy Durruthy et al., 2014; Dominguez et al., 2014).
Translating Lab Bench Discoveries
to the Clinic
In collaboration with our colleagues in the Center for Fertility and Reproductive Endocrinology, Pediatric Oncology and Urology, we established the Fertility Preservation Program in Pittsburgh. This is a multidisciplinary effort to educate patients and physicians about the reproductive consequences of diseases and medical treatments; provide access to established fertility preservation options; and develop new reproductive technologies for patients who currently have no options to preserve their fertility. The program currently has experimental protocols to freeze testicular tissue for men and boys who are not able to preserve a semen sample before initiating surgery or medical treatments that may compromise their fertility.
In collaboration with the Oncofertility Consortium at Northwestern University, we are freezing ovarian tissue for women and girls who are not able freeze eggs or embryos. We are freezing eggs for women who are not able or do not desire to freeze embryos. This method is now well established and ready to transition from “experimental” to standard of care.
Finally, we are evaluating the effects of chemotherapy on sperm so we can accurately counsel men about whether their treatment will increase their risk of having children with birth defects.
Preserving Ovarian Function
Unlike men who continually produce sperm throughout postpubertal life from a pool of spermatogonial stem cells, it is generally understood that women are born with all of the eggs that they will ever have. While some laboratories are working on stem cell technologies to treat female fertility, our lab is actively evaluating agents that can protect ovaries and the finite pool of follicles (containing eggs) from the damaging effects of chemotherapy. We demonstrated that granulocyte colony stimulating factor (G-CSF) protects the ovarian vasculature and ovarian follicles from busulfan and cyclophosphamide, which are two of the most toxic alkylating chemotherapies (Skaznik-Wikiel et al., 2013). We are actively investigating the mechanisms that lead to reduced fertility in men, women, boys and girls to gain insights about cell-based therapies or drugs that might be used to preserve or restore fertility in the future.
Are Rodents Similar to Humans?
While the basic elements of reproduction are similar among mammals, there are aspects of germ lineage development and hypothalamic, pituitary, gonadal function that are different between species (Hermann et al., 2010; Valli et al., 2014). For example, the reproductive life-span of humans is much longer than rodents, which places a unique demand on the pool of follicles in ovaries and stem cells in testes.
In contrast, humans have a long prepubertal period that does not exist in rodents. Understanding these species differences is important for responsibly translating lab bench discoveries to the clinic for diagnosis and treatment of infertility.