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 function and potential of male germ cells during fetal, perinatal, juvenile and adult stages of life.  Studies are designed to provide insight into the fundamental characteristics of male germline stem cells as well as their regenerative potential to treat some cases of male infertility.  

Molecular Mechanisms Controlling Stem Cell Biology

A variety of ongoing studies are designed to identify the genes and proteins expressed by male germ cells at different stages of the life cycle and investigate their biological functions.  Genes encoding RNA-binding proteins are over represented in spermatogonial stem cells of the adult testis (Orwig et al., 2008). Studies are designed to confirm and examine the functions of candidate genes. In addition, comparative evaluation with stem cells in other tissues and species will reveal common themes in stem cell biology. Gene expression analyses are particularly powerful when pure target cell populations are available. We have identified pure germ cell populations with distinct developmental potentials (e.g., pluripotent and spermatogenic) from fetal newborn rat testes. Analysis of these pure cell populations (genetic or otherwise) will provide insight into the molecular determinants of developmental destiny.

Testis and Germline Development

The SSC 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 neonate, pup, and adult testes have similar capacities to produce and maintain spermatogenesis. In contrast, pup testes provide a superior environment for donor germ cell engraftment, supporting nearly 40-fold more donor spermatogenesis than adult testes from the same number of transplanted stem cells. Similar studies will examine the affects of environmental perturbations (e.g., radiation, chemotherapy, and age) on stem cell activity and niche quality.

Infertility and Regenerative Medicine

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 recently optimized a primate to nude mouse xenotransplant technique as a routine biological assay for primate SSCs (Hermann et al., 2007). 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. Systematic examination of donor and recipient testis models in an increasing repertoire of species will reveal important paradigms that will be instructive for translating the SSC transplant technique to for treatment of male infertility.

Transgenesis

Spermatogonial stem cells are unique among adult tissue stem cells because they pass genes to subsequent generations and thus, constitute a unique vehicle for modifying the germlines of animals. We and others have demonstrated that transgenic mice and rats can be generated by genetic modification and transplantation of male germline stem cells. Rats are widely used models of basic biology, human physiology and disease, but the study of genes and their functions in rats lags far behind progress in mice. The recent establishment of rat genomic resources (the Rat EST program and the Rat Genome program), combined with decades of phenotypic and physiological data provides unprecedented opportunities for establishing rat models of mammalian biology. Progress optimizing male germline-mediated transgenesis and other state of the art approaches in rats has implications for other species where current transgenic methods are inefficient and knockout technologies are not available.

MWRI Transgenic and Molecular Core Facility

The Transgenic and Molecular Core Facility of Magee-Womens Research Institute provides state of the art services for generating transgenic mice/rats and knockout mice. The facility also produces replication defective lentiviral vectors for delivering recombinant transgenes and performs teratoma and chimera analyses to evaluate stem cell developmental potential.