6th Testis Workshop: Regulatory mechanisms of testicular cell differentiation, 2001

Feb 22-25, 2001 Newport Beach, California, USA

Session 1: Stem cell Differentiation and Commitment

The first session of the meeting was focused on stem cell biology; this is a topic that has generated a great deal of interest in the past few years. The standard definition of stem cells states that these are "cells that divide by mitosis to give rise to other stem cells and as well as to more differentiated cells". In view of the recent exciting new data in a number of fields, this definition may have to be reconsidered. A more up-to-date definition could be that a stem cell is " a master cell that retains the capacity to give rise to different lineages in the body".

The title of the session was Stem Cell Differentiation and Commitment. An alternate title that fits well most of the presentations is "What Keeps a Stem a Stem". In the first talk, entitled Regulation of Germ Cell Development in the Early Fetal Gonad, Dr. James Resnick reviewed his work on the role of growth factors in primordial germ cell survival. His work focused mostly on understanding two key events in the period following gonadal colonization, namely cessation of proliferation and differentiation. The second presentation entitled “Kit Receptor Signaling in Spermatogenesis" was given by Peter Besmer. Dr. Besmer pioneered this field in the early nineties when he discovered the kit gene and this quickly led to the development of a new ligand/receptor system that has proved to be active in many stem cell systems in the body including the gonads. Dr. Besmer reviewed his recent work where he showed that the PI 3-kinase signaling pathway is critical for spermatogenesis and oogenesis. Dr. Erika Matunis gave the third lecture entitled Regulation of Spermatogonial Stem cells in Drosophila. She uses Drosophila spermatogenesis as a model system since it parallels mammalian spermatogenesis. Her exciting new work demonstrated that STAT3 is actively involved in stem cell renewal in Drosophila. In the last presentation, Dr. Hans Schöler reviewed his work on Oct4 Gene Function and Regulation in Pluripotentt Stem Cells. He concluded that Oct4 is necessary for the maintenance of embryonic cell potency and it may play a role as a germ cell determinant by preventing these cells from differentiating. He is currently investigating the role of STAT3 in relation to Oct4.

Session 2: Chromosome and Chromatin Dynamics

Among the most remarkable transformations of spermatogenesis are those involving chromosomes. The speakers in this session presented new and exciting information about chromatin dynamics during both meiosis, when the chromosomes engage in pairing and recombination, and spermiogenesis, when the chromatin undergoes dramatic compaction and remodeling. Anne Villeneuve introduced the experimental advantages of C. elegans: genetic malleability and superior cytology of meiotic chromosomes, which can be viewed in the context of the intact nuclear three-dimensional structure. She has identified genes that control the establishment of meiotic chromosome pairing; one of these is a homolog of checkpoint kinases in other systems. Other genes control the stabilization and maintenance of homologous chromosome pairing, and include genes encoding proteins of the synaptonemal complex. The second speaker, Christer Hoog (Sweden), addressed an important component of the synaptonemal complex in mice, the SYCP3 protein. Male mice bearing a targeted null mutation in the Sycp3 gene are infertile and their spermatocytes undergo considerable asynapsis of chromosomes and ensuing apoptosis. Interestingly, this analysis has suggested the essential importance of elements of the cohesin complex in establishment of synapsis and recruiting proteins involved recombination. Subsequent to meiosis, during spermiogenesis, chromatin undergoes a complex rearrangement and compaction, and the second two talks addressed this process. Marvin Meistrich presented a detailed analysis of the roles of transition proteins 1 and 2 in condensation of chromatin in spermatids of the mouse. Knockout mutations revealed that neither protein alone is essential for testicular sperm production but spermiogenesis is abnormal when both are absent. This analysis suggests a compensatory mechanism. Surprisingly, the sperm missing these nuclear protein genes exhibited major defects in the cytoplasm and tail, revealing unsuspected function for the proteins or secondary effects of their absence. These phenomena may be related to some cases of human male infertility. Rod Balhorn noted that some cases of human male infertility are correlated with zinc deficiency. He presented a remarkably "user-friendly" description of complex analytical techniques for evaluation of interactions between protamine 2 and zinc in sperm chromatin. These analyses have enabled structural insight into the interactions between protamine 2 and zinc in relation to sperm maturation.

Session 3: Cell Signaling in Reproductive Cells

This session included scientific talks presented by Ilpo Huhtaniemi (Finland), Marco Conti, Jochen Buck, Stuart Moss and Gregory Kopf. This exciting session explored several novel aspects of intracellular metabolic and ionic sensing mechanisms as well as intracellular signal transduction in the gonads and sperm. Their investigations focused on: (1) genetic and animal models of gonadotropin action, (2) hormone- and developmental-regulated changes in intracellular signaling and metabolism, (3) the role of soluble adenylyl cyclase (sAC), (4) the regulation of cAMP activity by phosphodiesterases (PDE), and (5) the functions of adaptor and scaffolding proteins in the testis and spermatozoa. Dr. Huhtaniemi began the session by placing the data learned from both activating and inactivating mutations of gonadotropins and their receptors and the studies with transgenic and knockout models into a physiological context. In addition, he presented data from LHR KO mice showing that both sexes have normal sexual differentiation until birth. In the male, such data are consistent with the postulate that fetal Leydig cell androgen production is independent of LH. However, postnatal sexual development was blocked in both sexes. In KO males, spermatogenesis proceeds until round spermatid stage, consistent with the hypothesis that progression through meiosis is independent of LH activation of testosterone. In animals with both gonadotropins missing, spermatogenesis fails before meiosis, indicating that in the mouse FSH is needed for the male germ cell to progress, develop and/or survive meiotic events. Using both in vitro and PDE4KO murine models, Dr. Conti provided scientific evidence for the regulation by FSH of Sertoli cell PDE4 at the transcriptional and post-transcriptional levels. Dr. Buck studies demonstrate that in the testis, male germ cells show the highest expression of soluble AC (sAC). sAC is directly regulated by bicarbonate ions. The working hypothesis is that sAC may function as a physiological carbon dioxide chemosensor critical to the survival of germ cells in the testis. Dr. Conti also presented evidence that two forms of sAC exist in spermatids and perhaps pachytene spermatocytes. High levels of bicarbonate exist in the tubule fluid and round spermatid cAMP levels are regulated by sodium bicarbonate. These data imply that spermiogenesis can be effected by sAC-mediated mechanisms in addition to those transcriptional regulation mediated by cAMP signaling. The regulatory (RII) subunit of protein kinase A (PKA) can be either found in an intracellular soluble form or tethered by A-Kinase Anchor Proteins (AKAPs) to subcellular organelles. Dr. Moss’s studies showed that only those haploid spermatids containing an X chromosome express the Akap4 gene. Studies examined Akap4 transcription and translation of two transcripts that arise by alternative splicing. Although expression of both transcripts occurred in early spermiogenesis, only one transcript was translated in spermatids. Interestingly, their findings suggest that the protein expressed must be shared among adjacent X and Y chromosome-bearing spermatids. Such a novel transfer of protein information between conjoined spermatids provides compelling data supporting their intimate communication in vivo. Dr. Kopf discussed sperm function with data that supported the relevance of compartmentalized metabolic and signaling pathways to integrate ionic changes and activation of ATP production. His studies suggest that the molecular mechanisms underlying sperm fertilization competence and activation are subserved by specific scaffolding, anchoring and adaptor proteins.

Section 4: Genetic Approaches to Reproduction

Significant advances in our understanding of the molecular and cellular regulation of reproduction have resulted from genetic analyses of defects in gametogenesis. Two presentations highlighted the advantages of model organisms for identifying key regulators of developmental processes. Steven Wasserman identified Boule, a Drosophila gene that encodes an RNA-binding protein required for meiotic entry during spermatogenesis. Boule is an ortholog of Dazl, a family of proteins that are expressed in germ cells and required for fertility in vertebrates. The Boule protein acts in a common pathway of cell cycle regulators governing the G2/M transition in spermatocytes, and appears to regulate the translation of Twine, a Cdc25-type phosphatase that is also required for meiotic entry. Barbara Wakamoto used the Drosophila model system to identify a class of paternal effect genes that exert their effects after fertilization. Flies with these mutations in these genes (including snky, K81, and pal) produce motile sperm that can enter the egg, but then disrupt early stages of development. Further analyses of these mutations have identified specific processes immediately following fertilization that depend upon sperm components. Using both forward and reverse genetic approaches in mice, Robert Braun described analyses of 3 genes involved in germ cell formation and differentiation. Mutations in Fancc, a Fanconi anemia locus, reduce the number of primordial germ cells during embryonic development. Luxoid, a spontaneous mouse mutation, causes defects in spermatogonia stem cell specification and renewal. Tenr encodes a germ cell-specific RNA-binding protein, although disruption of this gene in knockout mice did not affect male fertility. Patricia Hunt analyzed causes of aneuploidy in human and mouse oocytes and in mouse preimplantation embryos. Her studies indicate that increases in nondisjunction during meiosis may be caused by age-related changes in ovarian microenvironment, and that the first two cleavage divisions are particularly error-prone.

Session 5: New Millennial Techniques

Major advances in knowledge have often sprung from the development of new methods and technologies. Speakers in this session described three particularly exciting examples. Lance Liotta discussed how laser-capture microdissection is used for identifying and harvesting individual cells from tissue sections and how small pools of these cells can be analyzed for patterns of gene expression, using PCR and microarray approaches, or for protein content using quantitative proteomic methods, including solid phase protein-binding chips and antibody microarrays. Ryuzo Yanagimachi traced the progression of his pioneering work on the development and application of intracytoplasmic sperm injection. He described his success with this method to study the remarkable stability of the sperm nucleus, to determine the role of the sperm perinuclear materials in oocytes activation, and to test the ability of nuclei from spermatids and spermatocytes to produce offspring when injected into oocytes. Monica Justice reported how she and her colleagues are using ENU mutagenesis in combination with embryonic stem cells and high throughput screening to carry out large-scale studies of gene function in the mouse. These methods are being used, along with some very clever genetic tricks, to rapidly identify recessive mutations with effects on a wide variety of phenotypes, including fertility. (See www.mouse-genome.bcm.tmc.edu.)

M. Dym, E.M.Eddy, M.A.Handel, N.B. Hecht, P.Morris, and D.O'Brien , USA