Introductory Overview to the Proceedings of the XXth North American Testis Workshop

Abstract

The central theme of the XXth North American Testis Workshop, “Testicular Function: Levels of Regulation,” reflected the many recent discoveries of new and complex levels of regulation of testicular functions. These included regulation of testicular development and the initial formation of the testis and male germ cells, as well as the subsequent differentiation of key components of the testis, including Sertoli cells, Leydig cells, and spermatogenic cells. Many different regulatory mechanisms responsible for these differentiative functions were explored. The roles of genes encoding key regulatory proteins, as well as signal transduction mechanisms, RNA-processing mechanisms, regulation by small noncoding RNAs, and mechanisms governing self-renewal and/or differentiation of spermatogonial stem cells were all discussed in this light. The workshop, which was held at the Hyatt Regency Philadelphia at Penn's Landing in Philadelphia, Pennsylvania, on April 1–4, 2009, featured 15 invited talks and 6 short talks selected from abstracts submitted for the 2 poster sessions. Manuscripts from 12 of the invited talks are presented in this volume. They are organized into 4 parts: “Regulation of Testis Development,” “Regulation of Testis Function,” “Regulation of Germ Cell Development,” and “Regulation of Gamete Development and Function.” “Part 1: Regulation of Testis Development” features 4 articles. The first, by Nel-Themaat et al, describes an elegant and cutting-edge approach to the study of differentiation of the testis involving expression of fluorescent markers in specific cell types within the developing testis as a means to visually follow the dynamics of testicular development and differentiation. This summary is preceded by a brief review of past studies aimed at elucidating the cellular dynamics associated with testicular development. The use of transgenes that differentially mark individual testicular cell types in conjunction with culture of developing testes and time-lapse imaging facilitates unprecedented insight into the developmental dynamics of testicular differentiation. This approach allows the investigators to understand aspects of testicular development that cannot be ascertained in any other way. In addition, this provides a very visual and, hence, very instructive tool that is attractive to both experts and nonexperts alike and should stimulate all readers to want to learn more about the genesis of the testis. The second article in this part is by Barsoum and Yao, and is focused on the origins of Leydig cells in the fetal testis. The authors note that these cells arise after the initial appearance of Sertoli cells induced by expression of the testis-determining Sry gene, and suggest that the origin of Leydig cells is likely based on or regulated by factors derived from Sertoli cells. They cite evidence suggesting that this process depends on a balance between differentiation-promoting and -suppressing mechanisms, such as paracrine signaling via Sertoli cell—derived Hedgehog ligands, cell-cell interactions involving Notch signaling, and intracellular transcription factors such as POD1. In the third article in part 1, Schlessinger et al present a fascinating review that provides a novel perspective on the genetic pathways that are responsible for initiating and/or maintaining the sexually dimorphic process of gonadal differentiation in each sex. A particularly novel feature of the thesis presented in this review is that it dispels the commonly accepted notion that the genetic pathways responsible for testis or ovary differentiation, respectively, are completely distinct. Rather, the authors point out that members of the gonadal differentiation pathway in one sex may also play a role in the gonadal differentiation of the other sex, and/or that testis-differentiation pathways may interact with ovary-differentiation pathways, or vice versa. With respect to the latter notion, this review also points out that the decision to follow the path toward testis or ovary differentiation is not as unilateral and stable as is often thought. Thus, the authors discuss the ramifications of defects in one gonadal differentiation pathway that can open the door to activity of an alternate pathway that normally functions only in the opposite sex. Finally, the fourth article in part 1, by Papaioannou and Nef, is an excellent, well-written, up-to-date and thorough review of the biology of microRNAs (miRNAs) in the testis. Studies during the last 5–10 years have revealed the abundant expression of small, noncoding RNAs in the testis, especially in spermatogenic cells. Among these are the miRNAs. Although the precise functions of these miRNAs (or of other small noncoding RNAs) during spermatogenesis are yet to be fully elucidated, studies to date, including several knockout studies, have clearly demonstrated that these RNAs are critically required for normal spermatogenesis and male fertility. This review presents a concise summary of the current state of knowledge about the expression and function of miRNAs in the testis. It provides a brief synopsis of the biogenesis of these RNAs, followed by a description of what is known about their functions or potential functions. Part 2 of this volume is entitled “Regulation of Testis Function,” and includes 2 articles. The first of these is by Yatsenko et al and reflects the research summarized by Dr Martin Matzuk in the keynote address at the XXth North American Testis Workshop. Over the past several years, Dr Matzuk's laboratory has conducted numerous studies involving knockout or knockdown approaches to selectively inhibit the function of individual genes expressed in the testis and thereby delineate the normal function of each targeted gene, and this article summarizes the results from many of these studies. Genes expressed in the male germ line predominate those targeted by this approach. Such genes are involved in all phases of spermatogenesis, including those functioning in spermatogonia, spermatocytes, and spermatids. The authors' studies of genes expressed in spermatogonia have revealed functions contributing to spermatogonial stem cell function, whereas many of the genes expressed in spermatocytes were found to contribute to meiosis-specific or -related functions. Finally, most of the genes targeted in spermatids were found to contribute to differentiative processes that lead to unique structures in the mature spermatozoon. Yatsenko et al weave the results from their many individual studies into an informative overall functional insight into the biology of spermatogenesis from the perspective of “the immortal male germ line.” They conclude their article with a summary of how their findings based on studies of the basic biology of gene function in the male germ line can make significant contributions to clinical practice in the potential diagnosis and/or treatment of male infertility cases. Tsai-Morris et al authored the second article in part 2. It is focused on the gonadotropin-regulated testicular RNA helicase, GRTH/Ddx25, a member of the DEAD-box protein family. This protein functions as a testis-specific RNA helicase found in meiotic and postmeiotic germ cells as well as in Leydig cells. In germ cells, this protein serves as a posttranscriptional regulator and is required for normal fertility in the male. Interestingly, it appears to play a specific role in the formation of ribonucleoprotein particles and shuttling mRNAs from the nucleus to the cytoplasm for storage in the chromatoid body. This represents critical molecular insight into the function of the chromatoid body—a structure that has remained rather enigmatic until now. Part 3 is entitled “Regulation of Germ Cell Development.” The first of 3 articles in this part is by Updike and Strome, and describes P granule assembly and function in germ cells of the model organism C elegans. The P granules in germ cells of C elegans are similar to the germ plasm or germ granules found in germ cells of many metazoan species, including “polar granules” in Drosophila germ cells or perinuclear nuage observed in germ cells of mice and humans. This germ plasm consists of RNAs and proteins. The authors note that mutations in genes encoding components of germ granules in mice lead to male sterility. However, the manner in which the germ plasm functions in mammalian species is not well understood, despite research on this topic that began over 30 years ago. Thus, the use of a model organism such as C elegans affords a very useful approach to gain insight into the biology and function of germ plasm. In this article, the authors review the development of primordial germ cells (PGCs) in C elegans, and then discuss the structure, localization, and composition of the P granules within these cells. They then describe studies to deduce the functional roles of these granules, or specific components of these granules, during gametogenesis. They conclude that P granules play a critical role in the identity and maintenance of the germ line in C elegans, and thus are critical to fertility in this species. They also note that there are many similarities in the structure and composition of germ granules in C elegans and mammals, and suggest future parallel studies of these granules in model species from both groups. Dr Matsui provided the second article in part 2, which is an up-to-date review of aspects of the molecular mechanisms that regulate the specification and development of PGCs in mammals, with a particular emphasis on studies of these phenomena in the mouse. The author describes a nice collection of published observations from his own laboratory and from laboratories of other prominent investigators in the field, and accompanies these with well-labeled bits of speculation regarding the implications of the published findings. Overall, this review follows key changes in gene expression patterns in cells that function as the precursors of PGCs, as well as in the PGCs themselves and in the later gametogenic cells that derive from the PGCs. Key among these molecular events is the function of transcription factors that regulate PGC-specific genes such as the Mil-1 gene. Dr Matsui also describes the potential for PGCs to be converted into pluripotent stem cells upon activation of certain signaling pathways. Finally, part 3 concludes with an article by Sette et al focused on the function of the RNA-binding protein Sam68 and its role in facilitating the progression of spermatogenesis and, hence, male fertility. Sam68 belongs to the signal transduction and activation of RNA (STAR) family of RNA-binding proteins that appear to play critical roles during gametogenesis in both males and females. In the testis, Sam68 becomes phosphorylated during the meiotic divisions of spermatogenic cells, which allows it to interact with polysomes in late spermatocytes and round spermatids, and this, in turn, facilitates translation of target mRNAs. This protein is essential for male fertility, as shown by the oligospermic phenotype that accrues when this gene is deficient, and defects in the gene encoding this protein may be responsible for some cases of male infertility in human patients. The final part of this volume is part 4, entitled “Regulation of Gamete Development and Function.” The first of 4 articles in this part is by Okada et al and reviews recent findings pertaining to the expression and function of the histone demethylase JHDM2A, primarily in spermatogenic cells, but also in certain somatic cell types. Recent studies from several laboratories have revealed that posttranslational modifications of histones directly influence chromatin structure and transcriptional activity. It is well established that there are dramatic changes in both of these parameters during spermatogenesis, and that the proper orchestration of these changes is critical to male fertility. Thus, investigations of factors that regulate changes in histone modifications during spermatogenesis are very well warranted. This manuscript focuses on one particular modulator of histone modifications—the JHDM2A histone deacetylase that is normally expressed during spermatogenesis. Much of the manuscript describes the effects of ablating the gene encoding this histone deacetylase. A key ultimate effect of knocking out this gene is male infertility. This review summarizes data from several studies investigating the specific molecular pathways or interactions that become disrupted in the knockout model in an attempt to decipher the molecular etiology of the male infertility that results from ablation of this gene. This review provides significant insight into how histone deacetylases may normally function in general, and into how the JHDM2A histone deacetylase functions primarily during spermatogenesis in particular. In the second article in part 4, Zheng et al summarize the contribution of X-linked genes to fertility in male mammals. An earlier study by the senior author of this article revealed that an abundance of spermatogenesis-specific genes are X-linked in the mouse. This article summarizes recent data on the specific function of several individual X-linked, spermatogenesis-specific genes. One interesting finding is that ablation of several of these genes leads to spermatogenic defects manifest during meiosis, even though transcription of these genes is suppressed in spermatocytes by meiotic sex-chromosome inactivation. Thus, it appears that whereas many of these genes are transcribed in premeiotic spermatogonia, the function of the proteins they encode is normally most significant in meiotic spermatocytes. The authors point out that X-linked, spermatogenesis-specific genes are potentially relevant candidates for studies of fertility or male contraception because they are hemizygous in males such that loss of function of a single copy will result in a complete ablation of function of the encoded gene product. The final article in this volume, by Goldberg et al, is a thorough and thoroughly enjoyable review of studies of the testis-specific LDHC isozyme spanning over 45 years of research that could only have been written by Dr Goldberg and his colleagues. This stands not only as an excellent review of information about this particular testis-specific isozyme, but also as a wonderful example of how research on a particular topic centered on the testis can progress over the years. Indeed, the journey that is artfully depicted in this manuscript reveals how the constant ongoing development of new techniques and approaches can be brought to bear to advance our knowledge of a particular biological function. Regarding that function, the role of tissue-specific isozymes encoded by tissue-specific members of gene families has become increasingly important to our understanding of the biology of the testis. In this regard, the studies of the testis-specific LDHC isozyme encoded by the testis-specific Ldh3 gene have paved the way. We now know that there are at least hundreds of testis-specific genes encoding testis-specific proteins, many of which belong to gene/protein families that feature members expressed in other tissues as well. Thus, the LDHC system has exemplified the evolution of these testis-specific genes and their products and the contribution that these make to the normal function of the testis. The XXth North American Testis Workshop was made possible by the generous contributions of time and effort by many people. We gratefully acknowledge the essential participation of the invited plenary speakers, the speakers selected from poster abstracts to give short talks, and everyone presenting posters. We also thank the other attendees for contributing to the vigorous discussions throughout the formal and informal gatherings at the workshop that made this such an informative and stimulating meeting. Other key contributors included the members of the Testis Workshop Executive Committee (Erwin Goldberg, Bernard Robaire, Barry Zirkin, Norman Hecht, Michael Griswold, and Mary Ann Handel) and the members of the Program Committee (John Aitken, Robert Braun, Marco Conti, Ina Dobrinski, Mary Ann Handel, Norman Hecht, Brigitte Le Magueresse-Battistoni, Deborah O'Brien, and Tony Plant). The meeting would not have been possible without the generous support and contributions by the American Society of Andrology, the National Institutes of Health (NIEHS and NIDDK), the American Society for Reproductive Medicine, CONRAD, and the Serono Research Institute. Our personal thanks go to Debbie Roller and Ann Marie Bray from Weiser Associates for management of the financial, organization, and logistical issues so vital for making the meeting run smoothly and to Mel Clifton and the other members of the Journal of Andrology Editorial Office for helping us to pull this publication together.