Meeting Summary: 2008 Annual Meeting of the American Society of Andrology

Abstract

The 33rd American Society of Andrology Annual Meeting was held in Albuquerque, NM on April 12–15, 2008. Under the theme “Emerging Concepts and Technologies in Andrology,” the program was specifically designed and developed to appeal to the wide range of interests of ASA members, spanning both basic science and clinical medicine, and bridging bench work and patient care.

ASA Keynote Lecture. Stem Cells, Small RNAs and Self-Renewal in the Germline

Haifan Lin, Ph.D., Yale University School of Medicine

Dr Haifan Lin presented the ASA Keynote Lecture on Stem Cells, Small RNAs and Self-Renewal in the Germline. A recent focus of his research has been on small RNA-mediated epigenetic programming and translational regulation that are required for stem cell self-renewal in the germline. Piwi/argonaute genes represent the only known family of genes required for stem cell renewal in both animal and plant kingdoms. The Piwi (P-element induced wimpy testis) family binds a class of short RNAs called piRNAs (for Piwi associated RNAs) that is expressed primarily in the germline. The Drosophila protein Piwi is involved in stem cell maintenance; in piwi mutants stem cells differentiate without self-renewal. Dr Lin also reviewed the germ cell phenotypes of mutations in Piwi family members in a number of species including mouse and human. Piwi has been implicated in heterochromatin formation and epigenetic silencing of genes. piRNAs exist in large numbers, with over 60,000 species to date, and are transcribed from a limited number of regions in the genome. The precise role of piRNAs in the testis is unknown. Dr Lin's group has recently shown in Drosophila that Piwi proteins can have a role in transcriptional activation in addition to their role in transcriptional repression. His group showed that Piwi can bind to a subtelomeric heterochromatic region on chromosome 3 (known as 3R-TAS) and a piRNA uniquely mapped to 3R-TAS, leading to epigenetic activation of the 3R-TAS locus. His findings reveal an increased level of complexity of small RNA-mediated epigenetic regulation, i.e. that Piwi can exert opposite effects (activation versus repression) on different genomic regions. The physiological role(s) of the Piwi-piRNA system in stem cells is currently an active area of study.

AUA Lecture. Amniotic Cells as Stem Cell Source for Tissue Engineering

Anthony Atala, MD, Wake Forest University

Dr Atala gave an enlightened and informative plenary AUA lecture on regenerative medicine in urology and other fields. The talk began with a brief history of organ engineering beginning with kidney transplants almost 50 years ago. Dr Atala reviewed the 3 issues that have limited the field of organ and tissue regenerative medicine in the past, and explained how he and other are tackling these barriers. The first limitation is that it has historically been difficult to grow many cell types outside the body. The discovery and systematic isolation of committed targeted progenitor cells (the adult stem cells) in many organ systems as the key regenerative cell type has helped progress immensely. Second, there has been a great need for delivery vehicles on which to grow cells for organ regeneration. Dr Atala has studied and popularized the concept of a collagen scaffold on which endothelial and mesenchymal tissues can be grafted and grown before the organ is transplanted into the body for “terminal incubation” and final organ development. The third major barrier in regenerative medicine has been how to develop vascularity in the graft to allow the delivery of necessary nutrients and growth factors. With the assistance of the angiogenesis pioneer, Judah Folkman MD at Harvard, Dr Atala has developed several novel approaches to developing blood vessels and has found that endothelial cells and VEGF are required for success in whole organ regeneration. In grafts less than 1 cm in size, survival and proliferation is possible without creating a formal vascular substitute in most model and human organ systems.

Dr Atala then reviewed the experimental procedure that has evolved in the field of tissue engineering for regenerating animal and human organ systems. He and his colleagues have currently regenerated 18 organs, including urethra, bladder, vagina, uterus, penis, liver and cartilage. The general procedure involves in vivo biopsy of the affected organ, culture of the cells on a collagen scaffold in vitro for 4–6 weeks, followed by transplantation of the newly created, unfinished, organ into the body for “terminal incubation,” as the implant is not fully formed after in vitro culture but requires the in vivo setting to complete development.

Dr Atala discussed his work with embryonic stem (ES) cells, adult bone marrow (BM) stem cells and induced pluripotency stem (iPS) cells for tissue regeneration. He conveyed his belief about the relative clinical value of each of these for human regenerative medicine, and touched upon the FDA concerns regarding teratoma formation in ES cells that may are likely less of an issue in BM-derived or iPS cells. He has studied animal therapeutic cloning to better understand the risk or organ rejection due to maternal mitochondrial inheritance in the regenerated tissue and has found no immunological issues. Finally, Dr Atala reviewed his scientific work performed over the last 8 years using stem cells derived from amniotic fluid or placental tissue for tissue regeneration. Amniotic stem cells are neither ES nor adult stem cells, but lie somewhere in between. He has been able to differentiate these cells to fat, bone, and cartilage and has achieved phenotypes for cardiac tissue, bone marrow, endothelium, liver and pancreatic islet. He believes that if an amniotic stem cell bank can be created with 100,000 cell lines, that this will provide the potential for engineered tissue with perfect genetic matches for 99% of the US population. Overall, given our current understanding of this technology, he believes that heart, liver, pancreas and nerve are best suited for derivation from ES cells, whereas other organs are likely to be generated from patient-specific adult stem cells.

What regulates the spermatogonial stem cell niche

Dr Marie-Claude Hofmann, University of Illinois at Urbana-Champaign

The first presentation covered the regulation of spermatogonial stem cells (SSC) by the niche or surrounding microenvironment in mammalian testis. The testis has a complex architecture and function with numerous regulatory steps for renewal and differentiation of SCC. There is substantial evidence to implicate that Sertoli cells along with the basement membrane and extracellular matrix constitute the niche that directs SCC activity. The rest of this presentation covered various factors that regulate SSC niche activity. Sertoli cells produce a number of growth factors. Glial cell line-derived neurotrophic factor (GDNF) is a Sertoli cell protein product that acts through a receptor (GF Ralpha-RET) complex on the SSCs. Evidence shows that GDNF is essential for SSC self-renewal both in vitro and in vivo. GDNF promotes SSC proliferation through activation of members of the Src kinase family, and these kinases exert their action through a P I3K/Akt-dependent pathway to up-regulate N-myc expression. N-Myc serves as a marker for stem cell activity for research measures. A transcription factor variant gene-5 (Etv5) is expressed predominantly by Sertoli cells and is essential for SSC maintenance and self-renewal similar to GDNF. Of interest, FSH regulates the expression of GDNF mRNA, but not Etv5 mRNA. Fibroblast growth factor (FGF2) stimulates a time- and dose-dependent increase in Etv5 mRNA expression, with a maximal 8.3-fold increase at 6 h following 25 ng/ml FGF2 treatment. Similarly epidermal growth factor (EGF) stimulates Etv5 mRNA but not GDNF mRNA. TNFalpha and IL-1beta stimulate GDNF mRNA, but had no effect on Etv5 mRNA. Surprisingly, other hormonal regulators of Sertoli cells such as testosterone, triiodothyronine and activin-A did not influence Etv5 or GDNF mRNA expression. Dr Hoffman hypothesized on the potential of various chemokines to regulate the retention of SSCs in the niche, as documented using cell migration assays, and suggested that Etv5 may be the crucial factor for this regulation both directly and indirectly.

From stem cells to male germ cells

Dr Wolfgang Engel, University of Gottingen, Germany

This stimulating presentation was devoted to the potential for initiating male germ cells using embryonic stem (ES) cells, pluripotent stem (iPS) cells, and SSCs in vitro. Earlier studies showed that SSCs can convert to ES-like cells and then differentiate into cells of ecto-, endo-, and meso-derm origin. These researchers used stable transfected cell lines using Stra8 selection of SSCs, Stra8 being expressed in early stage spermatogonia. They also evaluated the methylation status of sperm that were produced in vitro and determined if there was any differences in their methylation pattern. One example given was Snrpn, which is paternally expressed and maternally imprinted and is unmethylated in sperm. They were able to demonstrate similar results in sperm differentiated from ES cells; currently these scientists are attempting to produce spermatozoa from iPS cells. In collaboration with other researchers, they have been able to create functional male germ stem cells, which spontaneously transdifferentiate into the three germ layer cells. These cells have been successfully transplanted into normal hearts of mice demonstrating that male SSCs are able to proliferate and differentiate. A grave concern of stem cell studies is that of malignancy; fortunately, no tumor formation has been identified for up to 1 month after cell transplantation in these studies so far.

Regulation of proliferation and differentiation of mouse spermatogonial stem cells

Dr Makoto Nagano, McGill University, Canada

This presentation focused on transplantation as a means to study spermatogenesis in a rat model. The authors attempted to study homing/migration and survival and proliferation mechanisms of SSCs after transplantation. Using this approach they quantified the number of SSCs that could home or migrate in pup and adult testis. Since transplantation techniques are laborious and time-consuming, these researchers developed an in vitro system in which single donor SSCs can generate three-dimensional structures of aggregated germ cells (clusters) within 6 days. This in vitro system can now be used reliably to study SSCs. The researchers observed delayed proliferation of SSCs in pups and adult mice with cryptorchidism. Studies showed that SSCs robustly expand in media containing GDNF and FGF2 and that GDNF and FGF2 are growth factors that promote differentiation of SSCs rather than self-renewal.

Lecture 1. Understanding the Basics of Male Fertility Using Proteomics in C. elegans

Diana Chu, PhD, University of San Francisco

Dr Chu spoke about using proteomics and Caenorhabditis elegans as a model organism to study molecular mechanisms of fertility. After comparing mammalian sperm with sperm from C. elegans, she discussed the need for sperm to package their DNA efficiently using DNA packaging proteins. One of the reasons that C. elegans is a good model to study fertility is that 70% of their body consists of gonads and the different stages of spermatogenesis can be easily visualized. Her research goal was to identify gamete-specific proteins by shotgun proteomics and characterize their functions using C. elegans with a focus on conserved proteins. Chromatin was purified from sperm and eggs using subcellular fractionation and subjected to MudPIT LC-MS to identify chromatin-associated proteins (CAPs). 1100 CAPs were identified from sperm and 814 CAPs were identified from eggs. The two lists were compared to eliminate shared factors and focus on 132 abundant sperm-specific CAPs. This technique also identified previously known chromatin proteins. Dr Chu used RNAi to knockdown candidate CAPs in C. elegans and characterized the worms' phenotypes. One example that she discussed was the sperm-specific variant of the histone H2A (HTAS-1). HTAS-1 was found associated with sperm chromatin from pachytene spermatocytes through to mature spermatozoa. Hermaphrodite and male worms with HTAS-1 knocked down exhibited decreased fertility. HTAS-1 did not seem to play a role in global DNA compaction, but rather, may function in gene regulation instead due to an increased number of post-translational modification sites in the HTAS-1 sequence.

Lecture 2. The Developmental Origin of Health and Disease

Kevin Sinclair, University of Nottingham

Dr Kevin Sinclair from the University of Nottingham provided a comprehensive review of the “Developmental Origins of Health and Disease” or DOHaD hypothesis and revealed new findings about unexpected critical windows of development that might be involved. Increased incidence of obesity, especially in English speaking countries has prompted research as to its basis, starting with the “Barker Hypothesis.” David Barker found an association between undernutrition during fetal development (low birth weight) and increased coronary artery disease 50–60 years later. Based on these observations he proposed that the fetus might be programmed to survive in lean times to have a “thrifty phenotype.” Subsequent research by Barker and others has been expanding this novel concept and exploring mechanisms associated with developmental plasticity. Dr Sinclair and colleagues have focused recently on the influence of prenatal diet on long term fertility and fecundity, including age at menopause, and have expanded the basic hypothesis to include the neonatal window of development. He explained the importance of folate, B vitamins and methionine balance for fetal development and reported recent studies in the ewe in which the pre-implantation phase of development may be impacted by dietary factors. Interestingly, the impact appears to be gender-specific. When ewes were put on a methionine-deficient diet prior to being superovulated and maintained on this diet for only 6 days after being inseminated, the male, but not the female, offspring exhibited, at two years of age, increased body size and body fat, poor performance on glucose tolerance test, and increased blood pressure. They are now studying DNA methylation and hence imprinting as an underlying molecular explanation for these novel observations.

Women in Andrology Lecture. Risks of ADHD and Autism in ART Offspring

Mary Croughan, PhD, University of California at San Francisco

Mary Croughan, PhD lectured on infertility and the likelihood that health problems related to infertility may be transmitted to offspring conceived by assisted reproductive technologies. Her work suggested that several conditions: autism, mental retardation, cerebral palsy, seizures and cancer were more likely to occur in children born to infertile couples treated with ART. She focused the talk on autism (4 times higher risk) and attention deficit hyperactivity disorder, which were markedly more common in children conceived by infertile couples. Nevertheless, the overall risk was still relatively low.

Epigenetic Regulation of Testis-Specific Gene Expression

John McCarrey, PhD, University of Texas, San Antonio

Epigenetics refers to mitotically and/or meiotically heritable changes in gene function and allows the programming of differential, tissue-specific gene expression. Dr McCarrey has been studying factors that regulate the testis-specific expression of phosphoglycerate kinase 2 (Pgk2) to better understand how stage- and cell type-specific expression occur in developing male germ cells. The transcription of Pgk2 is activated in primary spermatocytes to provide a source of phosphoglycerate kinase that is critical for the motility and fertility of spermatozoa. He has recently shown that the testis-specific homeodomain factor PBX4 and its cofactor PREP1 bind the Pgk2 enhancer in cells in which the gene is expressed. Thus it is suggested that PBX4, PREP1 along with CREM and SP3 direct stage- and cell type-specific transcription of the Pgk2 gene during spermatogenesis. Within the same time window a number of histone modifications take place including acetylation of H3 and H4. Using transgene constructs he has shown that enhancer sequences in addition to the core promoter of Pgk2 are needed for the histone modifications to take place. One of the earliest epigenetic events however, beginning in the late fetal stages, is demethylation over 2 kb in the promoter region of the gene. Specific sequences in the promoter are required for this demethylation event to occur; modification of this sequence prevents demethylation, suggesting that demethylation is required for Pgk2 expression. Ongoing studies are designed to determine the endogenous factors involved in demethylation of the Pgk2 promoter in fetal germ cells.

Epigenetic Regulation of Self Renewal and Differentiation

Robert Blelloch, MD, University of California, San Francisco

Global regulation of gene expression involves a number of factors including DNA methylation, histone modifications, and small RNAs. The focus of Dr Blelloch's talk was the role of miRNAs in stem cell differentiation. In embryonic stem (ES) cells in which there is a global loss of miRNAs, the cells proliferate more slowly and accumulate in the G1 phase of the cell cycle. By adding back miRNAs one by one to the ES cells with a global loss in miRNAs, a subset of miRNAs, all expressing the same seed sequence, were found that could rescue the proliferation phenotype. P21 was found to be one of the targets of the miRNAs, acting via the 3′UTR. miRNAs appear to be needed for ES cell differentiation since in their absence ES cells are arrested between renewal and differentiation, i.e. they do not turn off genes required for self renewal at the same time as they are turning on some genes for differentiation. In essence certain miRNAs are needed to silence pluripotency.

Epigenetic Transgenerational Actions of Endocrine Disruptors on Reproduction and Disease: The Ghosts in your Genes

Michael Skinner, PhD, Washington State University

Dr Skinner introduced the possibility that epigenetic mechanisms can underlie environmental impact on disease. His laboratory has employed a rat model wherein exposure of male embryos to the fungicide vinclozolin between embryonic (E) days 8–14 leads to a number of postnatal pathologies, and transmission of these pathologies across a number of generations. In particular, amongst the exposed males, in the postnatal period there was an increased incidence of apoptosis in the testis, a decrease in spermatid numbers and altered spermatozoal motility. The spermatogenic defect was transmitted to the fourth generation despite no further exposure to vinclozolin. His lab found a number of alterations in DNA methylation at many sites across the genome that may provide the basis for a potential underlying epigenetic mechanism for the effects of vinclozolin in this model. Ongoing studies are pursuing the DNA methylation defects and examining alterations in gene expression in the testes of the exposed and unexposed rats.

Clinical Debate: Is the detection of sperm DNA damage clinically useful?

Pro: Armand Zini, MD, McGill University

Con: Mark Sigman, MD, Brown University

The advent of assisted reproductive technologies (ARTs), particularly ICSI (intracytoplasmic sperm injection), has revolutionized the treatment of male-factor infertility. However, there are many unanswered questions regarding the safety of these techniques. These safety concerns are relevant because (1) these technologies often bypass the barriers to natural selection, (2) experimentally, sperm DNA damage has been shown to adversely affect the developing embryo and (3) infertile men possess substantially more sperm DNA damage than do fertile men.

The presentation briefly reviewed our current understanding of the etiology of sperm DNA damage, described a number of different tests used to detect sperm DNA damage and summarized what is known about the potential impact of sperm DNA damage on pregnancy outcomes. A systematic review of the literature indicates that sperm DNA damage is associated with lower natural, IUI and IVF pregnancy rates, but not with ICSI pregnancy rates. The literature also suggests that sperm DNA damage is associated with an increased risk of pregnancy loss in those couples undergoing IVF or ICSI. To date, the true clinical utility of sperm DNA damage tests remains to be established as the available studies are small, few in number and the study characteristics are heterogeneous. Nonetheless, the data suggest that those couples with high levels of sperm DNA damage should proceed to IVF or ICSI, although these same couples may experience higher rates of pregnancy loss.