The Testis Workshop sponsored by Serono Symposia USA, Inc. was convened in Louisville, Kentucky, April 7-10, 1999. There were more than 240 registrants, including 16 young investigators who received funding from a Conference Grant funded by the NICHD, NIA and NRR Institutes of NIH.
The Program, organized by Erwin Goldberg of Northwestern University, began Wednesday evening with a keynote address by Norman Hecht, University of Pennsylvania. In a forward-looking presentation Norm opened a window into future areas of research, both basic and applied, on the testis in the next century. This was followed by 2 1/2 days of talks and 2 evenings during which there were 93 poster presentations.
Session l: Testis Development and Differentiation. Lonnie Russell, Southern Illinois University, was Chair and submitted the following Summary:
The Testis Workshop started with a presentation by Blanche Capel from Duke University on SRY and formation of the testis. Dr. Capel presented an exciting experimental approach to understanding how the Y-linked SRY initiates organogenesis between 10.5 days and 12.5 days in the mouse. The SRY gene is expressed in Sertoli cells. A new finding is that proliferation in the coelomic epithelium, leads to the formation of Sertoli cells. The SRY gene is important in the migration of cells from the mesonephros into the developing gonad, cells that will eventually lead to the cord formation that characterizes male development.
Mary Lee from Harvard described the role of Mullerian inhibiting substance (MIS) in the developing testis. The well-known role of MIS is to cause regression of the Mullerian ducts, but since its presence is recorded postnatally, other functions are proposed. Both overexpression studies and knockouts suggest a role for MIS in maintaining Leydig cells. Studies described MIS type II receptors in progenitor Leydig cells to further suggest a role in Leydig cell maintenance.
Dirk de Rooij from Utrecht University spoke on regulation of the differentiation of undifferentiated spermatogonia. He showed that many agents interfere with the conversion of Aaligned cells to the differentiated A1 spermatogonia suggesting that this was a very vulnerable site in spermatogenesis. Transcription factor c-kit was found by de Rooij to be a marker for differentiated spermatogonia. A healthy debate ensured about localization of c-kit in spermatogonia and renewal of spermatogonia from stem cells in rodents and primates.
The session ended with a presentation from Peter Donovan. Dr. Donovan traced the primordial germ cell lineage and examined the various factors that could influence primordial germ cells.
Session II: Paracrine and Endocrine Interactions. Mathew Hardy, Population Council, was Chair.
Mike Griswold, Washington State University, updated his research on Clusterin (SGP-2), which is constitutively expressed in Sertoli cells. Mike described the putative molecular structure and its detergent like properties suggesting a functional role, binding Clusterin to hydrophobic molecules and complexes to promote their clearance and/or uptake into cells by association with a variety of cell surface receptors (excerpted from the author’s’s abstract).
Patricia Morris, Population Council, described the complex interrelationships between Sertoli cells and each of the germ cells. Pat’s data on signal transduction pathways utilizing the JAK family of protein kinases and the STAT proteins, reveals activation in response to a variety of polypeptide ligands including many cytokines, some growth factors and the interferons. These results suggest that the testicular JAK-STAT pathway affects spermatogenesis at multiple levels subserved by different intratesticular signaling molecules (excerpted from the author’s abstract).
The next two papers in this session considered hormone replacement therapy (HRT) for the aging male by Lisa Tenover, Emory University and T replacement in young males by Frederick C.W. Wu, University of Manchester. Lisa reviewed what is currently known about male HRT and discussed pros and cons of such therapy. Androgen target organs that show evidence of response in men over age 60 include : (l) increase in bone mineral density; (2) decline in fat mass; (3) increase in lean body mass (muscle); (4) increase in libido; (5) mood improvement; and (6) some aspects of cognitive function. There are potential adverse effects such as fluid retention, breast tenderness or enlargement, exacerbation of sleep apnea, development of polycythemia, exacerbation of benign or malignant prostate disease, or worsening of cardiovascular (atherosclerotic) disease. Many of these effects, at least in the short term, are predictable and manageable (excerpted from the author’s abstract).
Fred Wu pointed out that 6 decades of clinical experience with physiological androgen replacement in hypogonadic young males, has provided ample testimony to its undoubted efficacy and lack of serious side effects. Only recently have objective data on the multifaceted clinical effects of T action using modern methods of quantitative assessment been forthcoming; Wu discussed the monitoring and supervision of patients on T in terms of the benefits, side effects, disease risks and costs (excerpted from the author’s abstract).
Session III: Genetics and Cell Biology of Spermatogenesis. Mary Ann Handel, University of Tennessee, was Chair and provided the following Summary:
This session provided a lively overview of new and emerging areas in genetic, cellular and molecular analysis of the processes of spermatogenesis. Two presentations on results using model organisms beautifully illustrated the power of genetic analysis. Steve L’Hernault (Emory University) introduced the search for spermatogenesis or "spe" mutations in the nematode C. elegans. Nine such genes expressed exclusively in the germ line have been cloned; some of these with human homologs. Analysis of several of these mutations shows fine genetic control over development of germ cell asymmetry and cytoplasmic partitioning during spermatogenesis. Interesting results will be forthcoming as interacting partners are identified and fine function elucidated and related to function of homologous proteins in normal and disease processes in our own species. Margaret Fuller (Stanford University) exploited a number of mutations of Drosophila melanagaster to dissect the process of transcriptional regulation directing the common and divergent pathways of meiotic division and post-meiotic spermiogenic differentiation. Interestingly, mutations in a number genes cause arrest at the G2/M boundary of the first meiotic division; in some mutants the spermiogenic differentiation can unfold in spite of lack of meiotic division. Genetic analysis is revealing a hierarchy of gene action and has identified genes as encoding transcriptional factors controlling a suite of spermatid differentiation genes. It appears that testis-specific forms of general transcription machinery proteins may regulate the coordinate expression of genes controlling differentiation. These results may ultimately be informing about human male maturation arrest phenotypes. James Bartles (Northwestern University) presented an elegant cellular and molecular analysis of espin, an actin-bundling protein involved in the organization and function of the ectoplasmic specialization of Sertoli cells, thought to anchor and position spermatids with relation to the Sertoli cells. Espin binds actin with high affinity and elegant mutational analysis has allowed identification of the actin-binding site of the protein. Such actin-bundling proteins are found in many sites, including stereocilia of ear hair cells, and continued analysis will be informative about a variety of basic cellular processes as well as about organization of spermatids in the seminiferous epithelium. Michael O’Rand (University of North Carolina) discussed the characterization of an interesting nuclear autoantigenic sperm nuclear protein (NASP). Cloning and characterization has revealed that NASP is a highly acidic protein with histone binding domains. Native mouse NASP is found in complexes with histones, and the nuclear localization sequence has been analyzed functionally. NASP is a member of a family of similar proteins, with other variants expressed somatically, including during embryogenesis. Predicted functions include a role in chromatin reorganization during spermiogenesis, and thus this protein may be critical to the formation of a fertilization-competent sperm cell. Finally, E. M. Eddy (NIEHS) discussed the roles of spermatid variants of HSP70 chaperone proteins in spermatogenesis. This provided a forum for an informative discussion of the difficulties, frustrations and rewards of testing hypotheses about gene function by analysis of targeted gene knock-outs. Transcripts and HSP70-2 protein are found in spermatocytes and male mice lacking HSP70-2 are sterile, lacking post-meiotic germ cells. This information led to the identification of a role for HSP70-2 as a chaperone required for association of the cell cycle regulators CDC2 and cyclin B1. In contrast, HSC70T, another member of the HSP70 heat-shock protein family, is expressed in post-meiotic cells. No obvious phenotypic effect of mutational disruption of the gene was detected, suggesting that other proteins can compensate for lack of HSC70T, or that the protein does not have an essential role in spermiogenesis. This presentation showed the power of induced mutations and compound heterozygotes for analysis of protein function in spermatogenesis.
Session IV: Epididymal Maturation and Fertilization. Terry Turner was Chair and provided the following Summary:
Dr. Barry Hinton of the University of Virginia reported on his work on the regulation of d-glutamyl transpeptidase (GGT) in the initial segment of the rat epididymis by lumicrine factors from the testis. Current data indicate that bFGF secreted by Sertoli cells in the testis acts in the epididymal epithelium through second messenger pathways such as the Ras/Raf/MAPK signal transduction pathway. MAPK presumably phosphorylates and activates PEA3, a transcription factor of the Ets family, which has been shown to activate the GGT mRNA-IV promotor in the initial segment. Dr. Hinton’s work suggests that the primary action of lumicrine factors in the initial segment is both to maintain gene transcription and to maintain mRNA stability. Thus, lumicrine factors are important to gene regulation and biological function of the initial segment epithelium.
Dr. Bernard Robaire of McGill University presented evidence from the Brown Norway rat that aging has striking effects on the epididymal epithelium. Morphological changes with advancing age included increases in intracellular lysosomes and vacuoles in principal cells and an increase in the number of halo cells in the epithelium over a two year period. Immunocytochemical studies of markers for the blood-epididymal barrier (e.g. occludin and E-cadherin) indicate a weakening of the barrier in aged animals in the corpus epididymis, specifically. Genes involved in androgen action, such as 5a-reductase, and in oxygen radical detoxification, such as glutathione S-transferase, have also been found to be altered in a region specific manner in aged animals. These results strongly suggest there is an intrinsic aging process associated with specific cell types in the epididymis, and oxidative stress may be a major factor mediating the aging process.
Dr. David Garbers of the University of Texas Southwestern Medical Center finished the session with a talk on the molecular basis of sperm-egg interaction. Dr. Garbers indicated that downstream signaling pathways in gametes appear similar or identical across species, and similar to somatic cells, as well. Thus, the cell and species specificity observed appears to arise from the recognition/receptor molecules on the cell surface and a few proteins immediately downstream of the receptors such as ion channels and transporters. There are still disagreements over the cellular events around sperm-egg interaction. It is conceivable that the acrosome reaction, for example, might be induced either by the cumulus cells, the zona pelucida, or be induced spontaneously through a variety of pathways all of which result in calcium ion mobilization. Some cells might respond to one stimulus, other cells to another; thus, in such a conceptual model, a single signaling pathway for successful fertilization would not exist. The pathway ultimately utilized by the fertilizing sperm is likely to be dependent on the status of the sperm cell at any given moment as well as the repertoire of signaling molecules in a particular sperm. New technological advances in protein identification may soon offer means by which studies of the individual sperm interacting with an individual egg may be undertaken. Such an eventuality may allow the dissection of the molecular processes within the individual fertilizing sperm as it interacts with the egg or the egg investments.
The Program concluded with Session V: Genetic Defects and Remedies. Michael McClure, NIEHS, was Chair. A summary will appear in the next issue.
Erwin Goldberg, USA