Mechanisms regulating the specification, maintenance and loss of SSC fate
Spermatogonial stem cells (SSCs) sustain the seminiferous epithelium in the testis and maintain steady-state spermatogenesis by a balance between self-renewal and production of progenitor spermatogonia that enter the spermatogenic differentiation pathway. In collaboration with Drs. Brian Hermann at UTSA, Christopher Geyer at East Carolina University, and John Oatley at Washington State University, we are studying differential gene expression and related epigenetic programming associated with each of these spermatogonial subtypes. We have characterized subtype-specific gene expression by both bulk and single-cell RNA-seq, and associated epigenetic programming by multiparametric integrative epigenomic profiling including analyses of genome-wide patterns of DNA methylation, chromatin accessibility and six different histone modification patterns (H3K4me1/2/3, H3K9me3, H3K27me3, and H3K27ac). Our results suggest that SSCs and progenitors are distinct spermatogonial subtypes differentially programmed to either self-renew and maintain regenerative capacity as SSCs, or lose regenerative capacity as progenitors and initiate lineage commitment associated with spermatogenic differentiation.
Potential for Assisted Reproductive Technologies (ART) to disrupt epigenetic programming or genetic integrity
Previous reports from our lab and others have indicated that methods of ART may predispose epimutations in the ensuing offspring – particularly in imprinted genes. In collaboration with Drs. Alex Meissner from the Max Planck Institute in Berlin and Yukiko Yamazaki from the University of Hawaii in Honolulu, we have conducted a comprehensive analysis of gene expression and epigenetic programming in mice generated by either intracytoplasmic sperm injection (ICSI) or natural reproduction. Initial studies were focused on epigenetic programming at specific imprinted loci in mice produced by ICSI or natural mating. More recent studies have included genome-wide assessments of gene expression and DNA methylation patterns in mice produced by ICSI or natural mating. Reassuringly, our results indicate that genome-wide ART methods induce very few epimutations or aberrant gene expression patterns.
Effects of male lifestyle choices on the sperm epigenome and intergenerational epigenetic inheritance of paternal epimutations
We recently initiated a project that is part of a new Center for Male Reproductive Epigenomics which is one of the NICHD-sponsored National Centers for Translational Research in Reproduction and Infertility (NCTRI) and is headed by Dr. Wei Yan at UCLA and includes additional UCLA collaborators – Drs. Ron Swerdloff, Christina Wang and Harry Rossiter. The McCarrey lab project involves characterization of epigenetic profiling of the sperm epigenome in mice and men who have consumed either a healthy or a high fat diet, with or without an associated exercise regime. The male mice will be allowed to generate F1 offspring to determine if paternal diet and exercise can impact the male's offspring. We will also assess the extent to which any epimutations in the sperm epigenome associated with consumption of a high fat diet with no exercise can be reversed by transition to consumption of a healthy diet with regular exercise. This center also organizes a community engagement and education core designed to increase community awareness regarding the extent to which a man's lifestyle (diet and exercise) prior to conception may impact the health of his children.
• Healthy Lifestyle • Healthy Sperm • Healthy Children
Epigenetic reprogramming in a dish - an in vitro model of transgenerational epigenetic inheritance
Disrupting chemicals or other environmental effects can induce epimutations that can predispose development of disease states, and can be transmitted via inter- or transgenerational epigenetic inheritance to subsequent generations. These findings are the result of large-scale studies conducted in animals (e.g. mice or rats) that, while informative, afford low resolution of the underlying mechanism(s) and are time-, cost-, labor-, and animal-intense. We are developing a cell culture system in which the normal cycle of embryonic and germline epigenetic reprogramming can be recapitulated in vitro to facilitate high-resolution, time-, cost-, labor-, and animal-saving studies at the cellular and molecular levels to discern the manner in which exposure to environmental disruptors leads to the initial induction of epimutations, and how these epimutations subsequently escape, and/or circumvent the potentially corrective effects of epigenetic reprogramming such that they are transmitted to subsequent generations.
Differential epigenetic reprogramming potential among pluripotent, germ and somatic cell types
When different cell types are reprogrammed into induced pluripotent cells (iPSCs), they can retain residual epigenetic memory of their previous differentiated state. This residual epigenetic memory can influence the potential to fully reprogram and ultimately re-differentiate specific cell types for use in research or therapeutic applications. We are pursuing research to determine the extent to which residual epigenetic programming from source cell types limits normal epigenetic reprogramming in association with each of the following transitions in cell fate in vitro: 1) somatic to pluripotent, 2) pluripotent to early germ line, 3) early germ line to later germ line, and 4) germ line back to pluripotent. Our initial goal is to establish an in vitro system in which transitions among pluripotent, germ or somatic cell states can be induced and monitored, so that we can then study in vitro the epigenetic programming or reprogramming that normally accompanies similar transitions during development in vivo.
Developing the baboon as a model system for cell-based therapies
Stem cell-based therapies have the potential to dramatically improve the treatment and prognosis of patients suffering from a variety of debilitating or degenerative diseases, or from injury or battlefield trauma by restoring cell or tissue function. Yet, the advancement of stem cell-based therapies requires optimization of efficacy and safety of these novel methodologies, and this requires preclinical validation in model organisms prior to transition into the clinic. Nonhuman primate (NHP) species provide the most accurate recapitulation of the human condition for use in preclinical testing. The baboon (Papio anubis) represents the most attractive NHP species for assessments of stem cell therapies because they are similar to humans in size and anatomy, as well as in development, physiology and neurological functions. In addition, baboons possess an immunogenetic system that is more like that of humans than that found in other NHPs. We have validated baboon ESCs and iPSCs using gene expression and epigenetic profiling analyses, and we are optimizing the use of a "scorecard" method to assess pluripotency and differentiation potential of baboon stem cell lines. Our long-term goal is to establish the baboon as a model NHP species for testing and optimizing the efficacy and safety of stem cell-based therapeutic approaches.
For several decades, research in the McCarrey laboratory is centered on mammalian germ cells and stem cells. We have made use of several experimental models, including the mouse, baboon, and opossum. Dr. McCarrey discovered the first example of a functional, germ-cell-specific retroposon in the human genome. He has published several papers on mechanisms that regulate germ-cell-specific gene expression in mammals.
Dr. McCarrey has also published several papers on mechanisms of epigenetic programming that function during embryogenesis, germ cell development and gametogenesis, as well as papers focused on mechanisms governing X-chromosome activity or inactivity in germ cells and early embryos, the effects of cloning and assisted reproductive technologies on genetic integrity, epigenetic programming in gametes and embryos, the relationship between pluripotency and maintenance of genetic integrity, and the development of nonhuman primate model systems for studies of stem cell research and regenerative medicine. Finally, he also maintains an active interest in the evolution of genetic and epigenetic mechanisms of gene regulation in mammals.
In 1987, Dr. McCarrey reported the first example of a functional retroposon in the human genome – the autosomal PGK2 gene. Evidence suggests that this intronless member of the PGK gene family arose as a reverse-transcribed copy of an mRNA from the intron-containing, X-linked PGK1 gene. This is an example of Susumu Ohno's theory of "Evolution by Gene Duplication." Following its origin, the PGK2 retrogene evolved from a ubiquitously expressed housekeeping gene to a tightly regulated tissue-specific gene expressed only during spermatogenesis in male eutherian mammals and encoding a protein that functions uniquely in sperm.
Studies of the spermatogenesis-specific Pgk2 gene in the mouse during the 1980s and 90s and early 2000s yielded novel information about the manner in which a spermatogenesis-specific gene promoter functions, including protein-DNA interactions, the identification of key transcription factors regulating spermatogenesis-specific expression, and changes in DNA methylation, histone modifications, nucleosome positioning, and chromatin structure associated with transcriptional activation of gene expression in spermatogenic cells.
Throughout the 1990s – 2000s, Dr. McCarrey was engaged in collaborative studies with Dr. Howard Cedar at the Hebrew University in Jerusalem describing epigenetic reprogramming during germline development, including methylation patterns of testis-specific genes, reprogramming of DNA methylation patterns genome-wide, germline-specific reprogramming of imprinted genes. Studies regarding normal and disrupted epigenetic programming in the germ line have continued in the McCarrey lab and are currently ongoing.
During meiosis in male mammals the X and Y sex chromosomes undergo a transient transcriptional inactivation in spermatocytes termed meiotic sex chromosome inactivation (MSCI). We previously showed that the Xist gene is expressed in spermatogenic cells but is not required to initiate MSCI. We then showed that MSCI occurs in both eutherian and metatherian mammals. Finally, in collaboration with Dr. Wei Yan we showed that while no X-linked mRNA-encoding genes have been shown to escape MSCI, many X-linked microRNA-encoding genes do escape MSCI.
In collaboration with Dr. Christi Walter at UT Health San Antonio, we have shown that as predicted by the "Disposable Soma Theory," both germ cells and pluripotent cells maintain genetic integrity at elevated levels relative to somatic or differentiated cells, respectively, and that this is based on enhanced expression of DNA repair and cell death genes regulated in concert with genes driving pluripotency or germ cell fate.
Disruptive environmental effects including exposure to certain chemicals, or aberrant dietary effects or stress or other conditions can induce epimutations that can then be transmitted from the exposed individual to his or her descendants over multiple generations. In collaboration with Dr. Michael Skinner at Washington State University, we have characterized this transgenerational epigenetic inheritance, especially with respect to how it is mediated by the germ line.