8th European Workshop on Molecular and Cellular Endocrinology of the Testis, 1994

The 8th European Testis Workshop was held on March 27-31, 1994 in De Panne, Belgium. The main organizer was Prof. Guido Verhoeven of the Catholic University of Leuven, Belgium. The Workshop meets biannually in the Spring alternating with the American Testis Workshop. The Workshop has a special format, in which maximum available time is allocated for discussion of free communications. These are presented in the Workshop Proceedings in the form of miniposters (a full page extended abstract with a few figures and/or tables). The participants receive the miniposter book in advance and only the discussion of the presentations is conducted during the Workshop. In addition, a limited number of formal invited talks are presented during the Workshop.

The Workshop has become very popular during its existence of 16 years and the scientists working on molecular and cellular endocrinology of the testis attend it regularly. This time the number of participants exceeded 200, and about 160 miniposters were discussed with varying intensity and length during the 4.5 days of the Workshop. This was considered by many participants excessive and in this way the formerly cozy workshop has started to suffer from its own success. The Organizing Committee discussed the possible alterations needed in the format of future workshops and came to the conclusion that the number of miniposters to be formally discussed should be limited. It was also decided that an extra clinical day will be organized in connection with the Workshop, in response to the needs of clinical andrologists. However, it was the opinion of the organizing committee that the basic science nature of the Workshop should also be consciously maintained in the future. The 9th European Testis Workshop will be organized by Dr. Vidar Hansson in Lillehammer, Norway in Spring, 1996.

The invited lectures presented in De Panne will be published in the Schering Foundation Workshop Series (Springer Verlag), and are therefore not reviewed in this workshop summary. The following is a summary prepared by the chairmen of the discussion sessions on the main findings presented in the miniposters.

I. Testicular development

Two groups reported on cloning of the putative anti-müllerian hormone (AMH) receptor (di Clemente et al, France, and Baarends et al, The Netherlands). The receptor is structurally related to the family of transmembrane serine/threonine kinase receptors. It is expressed during male sexual differentiation in the mesenchymal cells surrounding the müllerian ducts, but not in the ducts themselves. After birth, major expression of the receptor gene is seen in Sertoli cells, and could be involved in autocrine actions of AMH.
Several miniposters dealt with the expression of inhibin, activin and their receptors during ontogeny. Krummen et al (USA) demonstrated binding of iodinated inhibin to interstitial cells throughout development. Several types of activin binding were demonstrated within the seminiferous tubules, including a stage-specific one. Rombauts et al (Belgium) presented evidence that in the chick embryo, the adrenal gland is a main source of circulating inhibin. Peritubular myoid cells secrete activin A in vitro, and this substance mimics the inhibin stimulating activity of P-Mod-S (de Winter et al, The Netherlands).

Two miniposters reported findings on the origin of precursor Leydig cells. After EDS treatment, a mesenchymal cell population remains within the interstitial space, and slowly attains its LH responsiveness and androgen producing capacity (Chemes et al, Argentina). Haider et al (Germany) used for these cells the term peritubular fibroblast-like cells, but it was agreed by the two authors that it is the same Leydig precursor cell type they refer to. Murphy and O'Shaughnessy (U.K.) presented further evidence that Leydig cell development may be an androgen dependent phenomenon, and inhibitory effects of TGF-ÿ on fetal rat Leydig cells were reported (Gautier et al, France).

Several reports presented findings on histological and functional markers of pathological testis development. Placental-like alkaline phosphatase-positive germ cells persist in the testes of intersex children (Kula et al, Poland). The c-kit/stem cell factor complex is involved in development and progression of germ cell tumors, and the c-kit gene products are a new marker of testicular carcinoma in situ (CIS) (Rajpert-de Meyts and Skakkebaek, Denmark). Joergensen et al (Denmark) demonstrated the presence of CIS markers in fetal germ cells and concluded that the latter is due to malignant transformation of fetal germ cells.

Finally, four miniposters reported on methodologies and findings on apoptosis in the testis. Apoptosis in testicular cells was found after EDS treatment (Flinterman et al, The Netherlands, Frasoldati et al, Modena), after GnRH antagonist treatment (Brinkworth et al, Germany), and during normal testicular function in early elongate spermatids and zygotene spermatocytes (Morris et al, U.K.).

II. Leydig cell function

Our understanding of regulation of steroidogenesis, in particular the hormone dependent transport of cholesterol, has been improved. D.M. Stocco (USA) showed that a rapidly turning over protein of 37 kDa molecular weight may assist in the hormone-dependent transfer of cholesterol from the outer mitochondrial membrane to the inner membrane, since steroid production in cells transfected with cDNA of this protein was enhanced. The peripheral type benzodiazepine receptor in conjunction with other yet unknown proteins can also play a role in the delivery of cholesterol to the cholesterol side chain cleavage enzyme inside the mitochondria (V. Papadopoulos, USA). At present it is not clear whether these regulatory systems have common path ways.

Several posters indicated that in addition to the protein kinase A pathway other signal transduction cascades play a role in regulation of steroidogenesis. Cross talk between these pathways may already occur immediately after LH receptor activation since the LH receptor can be coupled to cAMP and phospholipase C signaling pathways.

III. Paracrinology and blood flow

Local regulation of testicular cell functions has been intensively investigated in the last decade. The 22 miniposters discussed in this session dealt with several aspects of the paracrine/autocrine regulation of testicular somatic cell function. Sertoli cells or Sertoli cell conditioned medium (SCCM) increase human and rat Leydig cell testosterone production. This increased testosterone production by human Leydig cells (and) was associated with increased mRNA levels of the main steroidogenic enzymes (Lejeune et al, France). A steroidogenic factor has been purified and characterized from rat SCCM (Carreau, France). Several miniposters dealt with the local production and regulation of several factors (IGF-1, and its binding protein, TGF-beta, vasopressin, oxytocin, endothelin), the presence of growth factor receptor mRNA (EGF, FGF, TGF-beta) and the effects of some of these factors (IFG-1, EGF, TGF-beta, vasopressin, erythropoietin) in Leydig cell differentiated functions. The stimulatory effects of a peritubular myoid cell derived factor (P-Mod-S) on Sertoli cell function has been well established. Similar P-Mod-S-like activity is produced by several cells and cell lines both with and without smooth muscle characteristics (Hoeben et al, Belgium). Similarly, P-Mod-S-like activity was secreted by immortalized cell lines derived from testis and prostate stromal cells.

Three miniposters dealt with the production of interleukin (IL)-1 and IL-6 by somatic testicular cells (Syed, Gomez, Stéphan et al, France). Rat Sertoli cells secreted both cytokines, and their secretion was found to be regulated by low concentrations of testosterone, NGF, and TNF. Both human Leydig and Sertoli cells also secreted both ILs and their secretion was stimulated by lipopolysaccharide. In addition, hCG stimulated the secretion of both cytokines by Leydig cells whereas FSH stimulated only the secretion of IL-6. Finally, the presence of IL-1 receptor mRNA was demonstrated in rat peritubular and Sertoli cells and in human Leydig and Sertoli cells. Thus, somatic testicular cells are the site of production and reception of both cytokines.
The last seven miniposters dealt with the angioarchitecture of the human testis and the regulation in the rate of testicular blood flow and fluid reabsorption in the ductuli efferentes. Concerning the human testis, an interesting model was presented indicating that Leydig cell clusters are present in both the arterial and venous sites of the microcirculation of the interstitial tissue (Ergn and Holstein, Germany). In the rat, testicular blood flow can be modified by smoking, change in temperature, and several peptides including calcitonin gene related peptide, vasoactive intestinal peptide, endothelin and serotonin. To investigate fluid resorption in the efferent ducts, two new procedures were developed (Clulow, Hansen, and Jones, Australia). Using these methods it was demonstrated that sex steroids can modulate the fluid transport in the efferent ducts.

IV. Sertoli cell function

The identification of production and actions of the many factors acting on, and secreted by, Sertoli cells continues to be a major area of research. It is complicated further by the fact that because of the difficulties in obtaining pure viable adult Sertoli cells, most of the studies are restricted to immature cells. Interleukins and various growth factors are among the factors receiving attention. Guillaumont et al (France) demonstrated that TGF-beta is expressed in Sertoli cells and is under the control of prolactin. Work from Jégou's laboratory demonstrated that IL-1 and IL-6 are secreted from Sertoli cells and that there is a vectorial production of these compounds (Stéphan, Cudicini et al, France). Slegtenhorst-Eegdeman et al (The Netherlands) demonstrated that FSH increases a specific mRNA (C28) of unknown function, but which could be a useful marker for investigating the control of gene expression by FSH. Ungefroren and Ivell (Germany) found that alpha-inhibin and oxytocin genes in the ruminant testes are transcribed at defined stages during differentiation.

Although it is known that calcium plays an important role in Leydig cell and Sertoli cell functions, the mechanisms controlling its level are still unclear. Gorczynska and Handelsman (Australia) have made the interesting observation that testosterone can increase the level of calcium in Sertoli cells from immature rats and furthermore proposed that this action was mediated via a non-genomic membrane effect.
The extracellular matrix plays an important role in maintaining cells in vivo and has dramatic effects on cells grown in vitro, especially Sertoli cells. Carreau et al (France) presented evidence that proteoglycans in the extracellular matrix may repress FSH dependent estradiol synthesis in Sertoli cells and that phosphodiesterases may be involved. Integrins may also be important because Salanova et al (Italy) found that they are expressed in specialized cell-cell contacts within the seminiferous epithelium. Matrigel is a reconstituted basement membrane extract and is extensively used for Sertoli cell cultures. Surprisingly, Dirami et al (USA) have found that transferrin, inhibin and ABP-like proteins are present in Matrigel and thus may account for some changes in Sertoli cell functions when these cells are grown on Matrigel, in addition to the effects of the matrix itself. These workers also demonstrated that ECM components have a chronic effect on steroidogenic enzymes when cultured with immature Leydig cells.

V. Gonadotropins and their receptors

One of the unusual features of the expression of the FSH and LH receptor is the formation by alternative splicing of numerous mRNA transcripts. Recent research has focused on the nature of these transcripts, their ontogeny, and the possible functions of their translated products.

Rannikko and Huhtaniemi (Finland) have demonstrated that the mRNA coding for the extracellular domain of the FSH receptor (R) is expressed in the immature rat before the mRNA coding for the full length receptor, i.e. changes in alternative splicing precede the onset of FSHR protein translation. A similar conclusion was reached by the same group (and by work from Dr. Teerds´ laboratory, presented in her lecture at this meeting) for the LHR. The mRNA coding for the extracellular LHR domain but not for the full length receptor, is expressed in Leydig cell precursors. This is based on evidence from ethylene dimethane sulphonate (EDS) treated rats in which all the mature Leydig cells had been destroyed.

There is increasing evidence for the expression of both gonadotropins at sites in addition to their classical locations. The mRNAs for both the LHb and a subunits have been found in the rat testis (Zhang and Huhtaniemi, Finland), and the mRNA coding for the truncated LHR in the rat pituitary. Kranewitter et al (Austria) presented evidence for the formation of prolactin in the human testis.

The phenomenon of homologous and heterologous desensitization of the responses to LH and FSH in their target cells is well established, although the mechanisms have not been elucidated. Chuzel et al (France) reported that LH-induced losses in LH/hCG binding sites in pig Leydig cells parallel the decreases in LHR mRNA. The latter were found to be related to post-transcriptional changes rather than modulation of the transcription of the LHR gene. They proposed that specific proteins were involved that specifically degrade LHR mRNAs. In contrast, heterologous desensitization may be controlled at the transcriptional level.
The elucidation of the functions and mechanisms involved in the control of full-length and truncated LHR will be helped by the reported development by Pallikaros et al (UK) of antipeptide antibodies to the N- and C-terminal sequences of the LHR. These antibodies are able to detect LHRs in Leydig and luteal cell membranes.

VI. Regulation of spermatogenesis

The major issue discussed was the identification of growth factors involved in the control of germ cell proliferation. It has been shown that bFGF may be involved in the regulation of rat gonocyte in vitro proliferation (van Dissel-Emiliani et al, The Netherlands), and that leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are important factors for gonocyte survival but not for their differentiation in vitro (de Miguel et al, The Netherlands). The same group presented data confirming that in the vitamin A-deficient rat, A spermatogonia are arrested before the S-phase of the A1 spermatogonia (van Pelt and de Rooij, The Netherlands). By studying the proliferating cell nuclear antigen (PCNA) as a marker of S-phase DNA duplication, it was observed that FSH is more effective than hCG in inducing S-phase in immature monkey A-pale spermatogonia and causing the loss of this cell type (Schlatt et al, Germany). It was demonstrated (Kula et al, Poland) that combined action of testosterone and estradiol increases the efficacy of the transition between A1 spermatogonia and B spermatogonia in the rat seminiferous epithelium. In rat tubular fragments cultured in vitro, IL-6 induces a significant inhibition of premeiotic DNA synthesis (Hakovirta et al, Finland).

Data were presented concerning possible pathways of interaction of germ cells with the extracellular environment. Further evidence demonstrating that PACAP-38 (pituitary adenylate cyclase activating peptide) and VIP can stimulate cyclic AMP and regulate protein secretion by rat isolated spermatocytes has been presented. This further suggests a role for PACAP in the testis (West and Sharpe, UK).

As for the cyclic changes observed in the seminiferous epithelium, evidence has been presented suggesting that duration of the epithelial cycle can be experimentally altered in the adult rat (Rosiepen et al, Germany). In addition, it has been demonstrated that in the rat, of the four different genes coding for the cAMP specific phosphodiesterases (PDEs), expression of rat PDE3 gene in Sertoli cells undergoes conspicuous changes during the seminiferous epithelial cycle, and that rat PDE1 and PDE2 are preferentially expressed in spermatocytes and spermatids at defined stages of their differentiation (Morena et al, Italy).

The expression in the testis of proteins previously described to be present in the brain has been reported by several groups. In particular, a phosphatidylethanolamine-binding protein, previously purified from bovine brain, has been described to be secreted by rat tubules cultured in vitro (Turner et al, UK), and to be present in late spermatids and adjacent Sertoli cells (Segretain et al, France). A 74 kDa microtubule associated protein 2-like protein is present in the immature and adult rat brain and is expressed in the adult rat testis (Hayes et al, Australia).

VII. Spermatids, sperm and sperm action

Before the era of molecular biology, gene transcription in male germ cells was thought to be confined to premeiotic cells, while the DNA of maturing spermatocytes and spermatids is gradually condensed and inactivated. Now, spermatids have been shown to produce a large number of substances. At the workshop, evidence was presented for higher levels of mRNA for two different X chromosomal genes, Ube1x and HHR6A, in spermatids (Hendriksen et al, The Netherlands). The method used for quantitation of mRNA was in this study (as well as in many others) RT-PCR, which raised questions as to how reliable were the observed differences.

The spermatid was the focus of interest of a number of investigators. Martti Parvinen (Finland) showed microcinematography of the movements of the chromatoid body in early spermatids. He proposed that these bodies, so far with unknown function, act as carriers for haploid gene products from various sites at the nuclear envelope to the site of translation. Three different groups studied the acrosome reaction of spermatozoa in vitro; it was found to be stimulated by ATP in vitro (Rossato et al, Italy) and modified by the progesterone antagonist RU486 (Serres et al, France). Baldi et al (Italy) reported that platelet activating factor (PAF), previously shown to be produced by spermatozoa, causes protein phosphorylation during the course of acrosome reaction. Another phenomenon (so far with unknown function) was demonstrated: GABA, which is also produced in the testis, shows specific binding to human spermatozoa (Aanesen et al, Sweden).

There are now numerous factors shown to be produced and/or bound to receptors in the testis. In most cases, the experiments have been performed in vitro, in more or less artificial media. In other instances, mRNA for a substance has been identified by PCR, possibly compared to other tissues by RT-PCR. It seems that the next step should involve demonstration of such phenomena to occur also in vivo, to alter the functions of the testes compared to a situation where the putative stimulating or inhibiting factor is absent. After such demonstrations, it is time to include the knowledge into textbooks!

Jones and coworkers (Australia) demonstrated that glucose uptake into marsupial epididymal spermatozoa was inhibited by a sugar, N-acetyl D-glucosamine, that is normally present in prostatic sections of this animal. They also showed that rat spermatozoa in undiluted cauda epididymal fluid are activated by calcium, and are able to use both endogenous and exogenous substrates for energy metabolism. The energy supply may be crucial for the activation of motility of the spermatozoa. An epididymal protein of the surface of sperm was tentatively identified as a cytokeratin (Bou et al, France). Progress was also made in isolation and identification of the proteins of the sperm fibrous sheath (Kim et al, Australia), and perinuclear proteins of mammalian sperm (Hess et al, Germany). The latter have been named 'cylicins', a family of proteins present in both bull and man. They do not show any homology with hitherto identified proteins.

VIII. Testicular immunology

The immunological milieu in the testis has become the subject of numerous studies. This was also reflected in the present Testis Workshop. It was shown that mouse Sertoli cells express low levels of the lymphocyte binding protein ICAM-1 on their surface (Riccioli et al, Italy). This expression is increased by treatment of the Sertoli cells with TNF-a and interferon-g. Although ICAM-1 is known to cause binding of lymphocytes, it is unclear if lymphocytes come in contact with Sertoli cells in vivo. Possibly, it has other functions in the testis.

Jahnukainen et al (Finland) showed that leukemic cells injected into rats preferentially occupy the testes, similar to the situation in human acute lymphoblastic leukemia. Cryptorchidism increased the relative proportion occupied by leukemic cells, inactivation by estradiol caused the opposite. This suggests that the functional state of the testis is of importance for the invasion of leukemia into the testis.

It has been known that the rat testis constitutes an immunologically privileged site; allografts transplanted into rat testes show an increased survival compared to other organs. However, Setchell et al (Sweden) were unable to demonstrate a survival of thyroid allografts in the testes of monkeys. This implied that the primate testis does not show the same immune tolerance as the rat testis.

Saari et al (Finland) demonstrated that auto-antigens on the outside of the blood-testis barrier in the seminiferous tubules are identified by circulating antibodies. The same group (Jahnukainen et al) has also mapped the number of mononuclear leukocytes (MNC) in the human testis of varying ages, normal and abnormal. They found that in boys younger than one year of age, MNC were more numerous than later, indicating a less tight endothelial barrier.

In addition to the previously known immunologically active peptides in the testis, interferon (INF)-a and g were shown to be constituents of the rat testis; INF-a in Sertoli and germ cell culture media and INF-g in early spermatids (Jégou et al, France). The function of INFs in the testis is so far unknown.

IX. Genes, gene expression and transgenic animals

Two very interesting miniposters focusing on transgenic mice bearing the rat androgen-binding protein (ABP) gene were presented. The first poster by Larriba et al (France and Spain) showed that overexpression of rABP in mice caused a decrease in serum testosterone and an increase in [3H]-DHT binding of testicular homogenates. Moreover, the same mice presented an increase in Sertoli cell number, abnormal Sertoli cell morphology and reduced fertility. According to the miniposter of Esteban et al (Spain), this reduced fertility resulted from germ cell maturation defects, as clearly evidenced by morphometric and flow cytometric analyses and by TEM examination.

The three following miniposters presented the results of several elegant genetic manipulations. In the first poster (Markkula et al, Finland) a toxigene approach, which consists of the selective ablation of a transgene-expressing cell lineage by antiherpes drugs, was used to ablate FSH producing cells in the pituitary of transgenic mice. By treating pregnant females with the antiherpes drugs, it was possible to lower pituitary FSH levels in their transgenic pups. This constitutes a new model for studying the role of FSH in testicular development and maturation. The second poster (Kananen et al, Finland) established that by transfecting under the SV 40 promoter the rat FSH receptor gene into a Sertoli cell line that does not express this receptor (MSC-1) it is possible to create an FSH responsive cell line (elevation of cAMP production under FSH stimulation). In the third miniposter, Hofmann et al (USA) reported the establishment of two germ cell lines, one of which is able to undergo meiosis: production of early and late spermatids in vitro. This presents us with the precious opportunity to study Sertoli cell-spermatid interactions under culture conditions which are completely controlled.

With regard to Sertoli cell transferrin, Suire et al (France) have nicely demonstrated that the stimulation of transferrin mRNA levels by FSH is due, at least in part, to an increase in transcription. Furthermore, by studying the promoter of human transferrin gene, these authors have identified two regions (PR I and PR II) which are essential for FSH action. In a second miniposter, the same colleagues have clearly shown that, in vitro, rat Sertoli cell transferrin production greatly increased between 11 and 17 days of age due to specific stabilization of mRNA transcripts and to acute transduction rate.

Two other original miniposters dealt with proto-oncogenes in the mouse and in dogfish testes. In the mouse testis, Dolci et al (Italy) showed that the rat protooncogene encoding a transmembrane tyrosine-kinase receptor presents a similar pattern of expression to that of c-kit in the germ cell line and in embryonic gonadal tissue. According to Fasano et al (France and Italy), in contrast to the situation existing in mammals where c-mos (a protooncogene with a serine/threonine kinase activity) is expressed in germ cells, in the dogfish testis, this protooncogene is exclusively present in the interstitial tissue. This indicates that during evolution, the site of protooncogene expression has changed.

With CRISP-1 and CRISP-3 (acidic epididymal proteins), Haendler et al (Germany) discovered two new androgen-dependent mouse genes with different tissue distribution, whose function is not yet known. Finally, Guillaudeux et al (France) demonstrated the existence of differences in the methylation pattern of HLA class I genes in human spermatozoa, pachytene spermatocytes and early spermatids, when compared to human somatic tissue.

Ilpo Huhtaniemi, Finland; Focko Rommerts, The Netherlands; Jose Saez, France; Brian Cooke, UK; Mario Stefanini, Italy; Martin Ritzén, Sweden; and Bernard Jégou, France