9th European Testis Workshop - Brief Report, 1996
The 9th European Workshop on the Molecular and Cellular Endocrinology of the Testis, organized by Vidar Hansson, Winnie Eskild, Trine B. Haugen, Tore Jahnsen, Finn Olav Levy, Rigmor Solberg, and Kjetil Taskén, (Institute of Medical Biochemistry, University of Oslo, Oslo, Norway), and by E. Martin Ritzén (Pediatric Endocrinology Unit, Karolinska Sjukhuset, Stockholm, Sweden), was held April 14-19, 1996, at Geilo, Norway.
A key feature of these meetings has been the presentation of scientific contributions in the form of miniposters, all printed in a book (1) and discussed in plenary session during the miniposter sessions of the meeting. The invited speakers, whose lectures are summarized below, also provided reviews of their recent work, published in a book that all participants received at the meeting (2). The printing of this book, as well as the organization of the meeting, was made possible by a generous grant from the Ernst Schering Research Foundation. For those specially interested, a limited number of the miniposter book (1) can be obtained from the organizers, and the book containing the reviews by the invited speakers (2) can be obtained by contacting one of the editors or the Ernst Schering Research Foundation. Below follows a brief summary of the meeting. A more extensive report will be published in Molecular and Cellular Endocrinology.
Robin Lovell-Badge (UK) gave the opening lecture on Sry and sex determination in mammals. He reviewed the work leading to the discovery, cloning and characterization of the Y-linked testis determining gene, Sry (previously called Tdy and TDF), a high mobility group (HMG)-box DNA-binding protein which turns out to be one of the most rapidly evolving genes known, and which has developed with the development of mammals. He further discussed the expression pattern of Sry during development, and its correlation to the formation of the genital ridge. He also addressed potential targets for Sry. Based on time of expression, the anti-Müllerian hormone (AMH) gene does not seem to be a direct target for Sry. The most likely target for Sry seems to be DAX-1, a gene linked to dosage-sensitive sex reversal, and also to congenital adrenal hypoplasia (AHC), and therefore also called AHC-1. DAX-1 is probably an ovary determining gene, the target of which may be an Sry-related HMG-box gene known as Sox-9. Sry may either interact directly with the expression of DAX-1 or it may inhibit the DAX-1 dependent inhibition of expression of Sox-9.
Keith Parker (USA) reviewed the role of steroidogenic factor 1 (SF-1) in reproductive function. He described the isolation and characterization of SF-1, an orphan receptor of the nuclear/steroid hormone receptor superfamily, which seems to be a global regulator of the steroid hydroxylases and plays key roles in both developmental and cell specific regulation of these enzymes, as well as in the differentiation of both adrenals and gonads. SF-1 "knock-out" revealed complete adrenal and gonadal agenesis, a male to female switch of XY offspring, and death in the absence of corticosteroid replacement. He also demonstrated interaction between SF-1 and the putative ovary determining gene DAX-1 and discussed the consequences of such interaction.
Michael Waterman (USA) reviewed the regulation of steroidogenesis, mainly by discussing the mechanism of peptide hormone action on steroidogenic cells. He discussed both chronic, i.e. regulation of steroid hydroxylase biosynthesis, and acute regulation of steroid hydroxylase activity, and mechanisms of cAMP action on these processes. Although similarly regulated by peptide hormones through cAMP and protein kinase A (PKA), the steroid hydroxylases each have developed their own cAMP responsive sequences, very different from the well known cAMP response element, CRE. There are also significant species specific differences in these systems. An important protein involved in acute regulation of steroidogenesis is the mitochondrial protein StAR (steroidogenic acute regulatory protein), and mutations in this protein lead to absence of steroid production, due to the crucial role of StAR in movement of cholesterol to the inner mitochondrial membrane. He also discussed the role of cytochrome b5 in regulation of the 17a-hydroxylase:C17,20 lyase.
Anthony Means (USA), through a review of the recent work on the calspermin gene, discussed the role of CRE-like motives in transcriptional regulation of postmeiotic male germ cell-specific genes. Calspermin is generated from the last two exons of the Ca2+-calmodulin-dependent protein kinase IV (CaMK IV) gene in postmeiotic germ cells, by the use of a tissue specific promoter containing two CRE elements and an alternatively spliced leader exon. Its expression is dependent on the transcription factor CREMt, and its role may be to keep calmodulin present in the cell, and protect it from degradation. He also presented new results on the role of CaMK IV in regulation of the function of the transcription factors CREMt and CREB.
Ingo Weyand (Germany) summarized the cloning and localization, the functional characteristics and the physiological role of cyclic nucleotide gated (CNG) channels in sperm. The channels, consisting of a and b subunits, are non-selective cation channels, which are permeable to calcium ions, and which are directly gated by cGMP and cAMP. Similar channels are present in retinal photoreceptors and olfactory sensory neurons, and at least the a subunit in sperm is identical to that expressed in cone photoreceptors. Their physiological function in sperm is unclear, but it is believed that the CNG channels may play a crucial role in regulating motility and chemoattraction of sperm.
Paolo Sassone-Corsi (France), in his talk "CREM: A transcriptional master switch governing the cAMP response in the testis", reviewed the function of the transcriptional activator CREMt, as well as the inducible cAMP early repressor ICER, which originates from the same gene by the use of an alternative promoter, and their roles during spermatogenesis. He went on to present the results of targeted disruption ("knock out") of the CREMt gene, which leads to an early block of spermiogenesis, apoptosis of germ cells, and block of postmeiotic gene expression.
Dr. G. Stanley McKnight (USA) presented recent results obtained through targeted disruption ("knock out") of two catalytic (Ca and Cb1) and four regulatory (RIa, RIb, RIIa and RIIb) subunits of protein kinase A in mice. Homozygous offspring was obtained in each case, except with RIa, where the homozygous null mice turned out to die in utero. All the other homozygous offspring were viable and fertile, except for the Ca knock out mice, which although relatively normal at birth, show severe growth retardation and usually die within the first six weeks of life. Both Cb1 null mutant mice and RIb null mutant mice showed defects in synaptic plasticity in the hippocampus (CA1 LTD deficiency and CA3 LTP deficiency), but apparently normal spatial learning. The RIIa null mutant mice, although clearly deficient in RIIa, appear as yet completely normal. The RIIb null mutant mice appear genetically lean, displaying a decrease in total body fat, as well as increased body temperature and oxygen consumption.
Norman Hecht (USA) addressed posttranscriptional regulation of postmeiotic gene expression. He emphasized translational control and introduced TB-RBP (testis-brain RNA-binding protein), a regulator of translation and cellular localization of mRNAs. TB-RBP specifically binds to conserved sequences in the 3'-untranslated region (3'-UTR) of protamine mRNAs, and the findings suggest that specific protein binding to highly conserved sequences present in the 3'-UTR of a group of translationally regulated testicular ("paternal") mRNAs is involved in their posttranscriptional regulation. TB-RBP is predominantly detected in round spermatids, requires phosphorylation for RNA binding, and is detected solely in the ribonucleoprotein fractions of a testicular postmitochondrial extract. Furthermore, TB-RBP from brain or testis (probably identical) bind specific mRNAs to microtubules in vitro. This GTP-dependent binding may, especially in the brain, serve an important role in storage and transport of mRNAs to specific intracellular sites, where they are translated.
Martin Dym (USA) reviewed the currently used technique for isolation and culture of immature rat type A spermatogonial stem cells. The main obstacle for this type of work has for many years been the lack of a specific marker for spermatogonial stem cells. This problem has now been solved by the discovery of and development of antibodies against the c-Kit or stem cell factor receptor, a receptor tyrosine kinase specific for stem cells, which reveals 95-100% pure type A spermatogonia. He also emphasized the ethical considerations connected with this type of research, and mentioned its potential uses and abuses, now that techniques for gene targeting in embryonic stem cells have become available.
Olli Jänne (Finland) discussed recent results obtained with deletion of functional domains of the androgen receptor, as well as interaction between androgen receptors and other steroid receptors during co-transfection experiments. By removing the ligand binding domain, for example, a constitutively active androgen receptor was obtained. He also gave examples of proteins negatively regulated by the androgen receptor. The mechanism of such down regulation is not well known. Besides actions of the androgen receptor at the level of transcription initiation, other mechanisms of action of the androgen receptor will be addressed in future research.
Fernand Labrie (Canada) reviewed the 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) gene family. The 3beta-HSD family is rather complicated, with different numbers of isoenzymes in different species. In humans, there are two genes encoding 3beta-HSD, type I and type II, as well as three pseudogenes. Rats have four subtypes of 3beta-HSD, and mice have six. Functionally, there is also significant diversity. For example, rat types I and IV 3beta-HSD also have 17beta-HSD activity, and rat type III and mouse types IV and V 3beta-HSD have 3-ketosteroid reductase activity. Dr. Labrie also described the molecular genetics of human 3beta-HSD deficiency.
Thomas Mustelin (USA) gave an overview of the Src family of protein tyrosine kinases (PTKs). Of the ten currently known families of non-receptor PTKs (Src, Brk, Abl, Fes, Syk, Tec, Jak, Fak, Ack and Csk), the Src family is the largest one with nine members (Src, Yes, Fyn, Yrk, Fgr, Lyn, Lck, Hck and Blk). As opposed to receptor tyrosine kinases, the focus of the following talk, the non-receptor tyrosine kinases lack a transmembrane domain. The Src family of PTKs contain four functional domains, called SH1, SH2, SH3 and SH4. SH1 is the catalytic domain, SH2 is important for protein-protein interaction and for regulation of the catalytic activity by phosphorylation, the SH3 domain is important for protein-protein interaction and the SH4 domain is involved in membrane attachment. Src and Yes are widely expressed, whereas the other family members show tissue-specific expression, mostly in subsets of leukocytes. All the Src family PTKs are activated by dephosphorylation, typically by the transmembrane protein tyrosine phosphatase CD45, and inactivated by phosphorylation by another non-receptor tyrosine kinase, Csk. As yet, very little is known about the role of different non-receptor tyrosine kinases in the testis.
Deborah Burks (USA) reviewed work in Patricia Saling's laboratory on the sperm-zona pellucida (ZP) interaction. A 95 kDa sperm protein with characteristics of a tyrosine kinase, zona receptor kinase (ZRK), which serves as a receptor for one of the three main glycoproteins, ZP3, of the zona pellucida has been identified. By molecular cloning, they have isolated a cDNA from human testis, hu9, encoding a receptor PTK probably representing human ZRK. Human ZP3 stimulates acrosomal exocytosis as well as the tyrosine kinase activity of ZRK, and both processes are blocked by a specific tyrosine kinase inhibitor. Examining the signaling pathways utilized by ZRK, they have demonstrated interaction of ZRK with phospholipase C gamma (PLCg), and with PI 3-K. Thus, the primary signaling molecules responsible for sperm activation and triggering of acrosomal exocytosis seem to be identified, and the further molecular mechanisms involved in these processes are under investigation.
Simon Fishel (UK) talked about the use of spermatids for human conception - a controversial topic. The development of intracytoplasmic sperm injection into oocytes (ICSI) has completely revolutionized the treatment of male infertility, and combined with different techniques to obtain sperm from the epididymis or even from the testis, requires only a few very weak and barely twitching sperm for normal fertilization and pregnancy. But even these may not be available in azoospermic patients due to incomplete spermatogenesis, e.g. maturation arrest and Sertoli cell only syndrome, and to allow these men to have their own genetic offspring, the possibility of using immature haploid germ cells, spermatids, for conception has been addressed. In a time span of only three to four years, the concept of using spermatids for conception has evolved through initial studies in mouse and hamster (1993), live birth in mouse and rabbit (1994), fertilization of oocytes by spermatid injection in humans, ongoing pregnancy and live births (1995).
Dr. Fishel reviewed the progress in this research. He went through some aspects which need careful consideration, such as the changes in DNA and nucleoproteins during spermatogenesis, the role of genomic imprinting, the role of oocyte activation and the role of the sperm centrosome. With the demonstration that the approach is feasible, and the development of techniques for separation, culture and cryopreservation of spermatids, he made the point that the fundamental processes required for successful conception are oocyte activation, the presence of a centrosome and appropriately formed haploid DNA, and that possibly the future will see the microinjection "cocktail" as a mixture of paternal haploid DNA, reconstituted purified oscillogen combined with a centrosome. After his talk, Dr. Fishel was challenged with several questions raising both technical and ethical concerns about this research, and especially about its rapid pace. In addition to the points brought up by Dr. Fishel himself, several other concerns were raised: Possible oxidative damage to the spermatid DNA; possible unknown genetic reasons for azoospermia or teratozoospermia, with unknown consequences for the offspring; the experience from several "knock out" mice, with no apparent phenotype at birth, but subtle phenotypes revealed by careful examination; and ethical considerations regarding high miscarriage rate in some of the trials. It was also pointed out that compared to the extreme care required from pharmaceutical companies when introducing new drugs, the introduction of ICSI and now possibly human conception by spermatid injection has been astonishingly rapid. On the other hand, it was pointed out that the use of spermatids may be as safe as the use of obviously morphologically aberrant sperm from patients with oligozoospermia.
Eberhard Nieschlag (Germany) raised concern about the lack of scientific foundation and controlled clinical trials for many of the treatments currently in use in andrology, in his talk called "Evidence-based andrology: The importance of controlled clinical trials". He suggested a classification of andrological therapies into rational, preventive, symptomatic and empirical, and gave examples of commonly used empirical therapies, for conditions such as varicocele, idiopathic infertility and immunological infertility, for which randomized, placebo-controlled clinical trials have failed to demonstrate significant beneficial effects of the treatment over placebo. He concluded that standards of clinical treatment in andrology should meet requirements similar to those in other medical disciplines, and that any therapeutic procedures whose effectiveness has not been verified by placebo-controlled studies should be applied only in the context of clinical studies until their effectiveness are indeed proven.
The molecular genetics and neurobiology of Kallmann's syndrome, a human genetic disorder characterized by the association of anosmia (lack of sense of smell) and hypogonadotropic hypogonadism was reviewed by Pierre Bouloux (UK) The characterization of the KAL locus, followed by the cloning of the candidate gene, KALIG-1 or ADMLX, later called KAL, the characterization of the KAL protein and the current hypothesis of KAL protein function were presented. He concluded that KallmannÕs syndrome is a developmental defect caused by defective targeting of olfactory axons to the olfactory bulbs and failed migration of GnRH neurons into the septo-optic nucleus.
Ilpo Huhtaniemi (Finland) gave a talk on the polymorphisms of gonadotropin action, its molecular basis and clinical implications. First, he pointed out that although there is microheterogeneity in the carbohydrate side chains of the gonadotropins, the majority of the previously observed differences in the ratios of the biological to immunological activities (B/I ratios) are due to nonlinearity of the dose-response curves of the conventional RIA measurements, and that the diagnostic value of LH in vitro bioassay is less important than previously assumed. Second, he described a common polymorphism in the LHbeta subunit gene, widely distributed in different ethnic groups (9-29%), consisting of a combined Trp8Arg and Ile15Thr mutation, probably leading to altered glycosylation. The resulting variant LH is slightly more potent, but has a shorter half-life in circulation, compared to the wild-type hormone. The variant LH may explain certain subtypes of PCOD in women and slow progression of puberty in boys. Third, he reviewed activating and inactivating mutations of the gonadotropin receptor genes, and described a recently discovered inactivating mutation in the FSH receptor gene, associated with hypergonadotropic ovarian dysgenesis (ODG).
As in previous testis workshops, the miniposters covered essentially all aspects of testis function, and a summary of these is beyond the scope of this report. Topics covered were (alphabetically, number of miniposters in parentheses): ABP (3), the acrosome (2), androgen-regulated genes (1), apoptosis (6), blood flow (3), clinical aspects (11), cytokines (9), the cytoskeleton (3), drug resistance (2), estrogen effects/environmental estrogens (5), germ cell-specific gene expression (17), germ cells (2), inhibin/activin (2), Leydig cell development (6), Leydig cell regulation (9), lipid peroxidation/oxidative stress (3), paracrinology (9), peritubular cells (2), proteases (7), retinoids (2), Sertoli cell regulation (3), signal transduction/cAMP (9), stages of the seminiferous epithelium (5), testicular pathophysiology (4), tumor/carcinogenesis (7), and viral defense (1).
1. Solberg, R., Hansson, V. (Eds.) Miniposter Book, 9th European Workshop on the Molecular and Cellular Endocrinology of the Testis. University of Oslo, 1996, ISBN 82-993237-1-1
2. Hansson, V., Levy, F.O., TaskŽn, K. (Eds.) Signal Transduction in Testicular Cells. Ernst Schering Research Foundation Workshop, Supplement 2, Springer-Verlag, Heidelberg, 1996.
Finn Olav Levy, Kjetil Taskén and Vidar Hansson, Norway