Ultrastructural localization and distribution of Nardilysin in mammalian male germ cells.

BACKGROUND: NRD convertase, also termed Nardilysin, is a Zn(++) metalloendopeptidase that specifically cleaves the N-terminus of arginine and lysine residues into dibasic moieties. Although this enzyme was found located within the testis, its function in male reproduction is largely unknown. In addition, the precise distribution of this enzyme within germ cells remains to be determined. METHODS: To answer these questions, we developed an immuno-gold electron microscopy analysis to detect Nardilysin at ultrastructural level in mice. In addition, we performed a quantitative analysis of these gold particles to statistically estimate the distribution of Nardilysin in the different subcellular compartments of differentiating late spermatids/spermatozoa. RESULTS: Expression of Nardilysin in wild-type mice was restricted to germ cells and markedly increased during the last steps of spermiogenesis. In elongated spermatids, we found the enzyme mainly localized in the cytoplasm, more precisely associated with two microtubular structures, the manchette and the axoneme. No labelling was detected over the membranous organelles of the spermatids. To test whether this localization is dependent of the functional microtubules organization of the flagella, we analysed the localization into a specific mouse mutant ebo/ebo (ébouriffé) known to be sterile due to an impairment of the final organization of the flagellum. In the ebo/ebo, the enzyme was still localized over the microtubules of the axoneme and over the isolated cytoplasmic microtubules doublets. Quantification of gold particles in wild-type and mutant flagella revealed the specific association of the enzyme within the microtubular area of the axoneme. CONCLUSIONS: The strong and specific accumulation of Nardilysin in the manchette and axoneme suggests that the enzyme probably contributes either to the establishment of these specific microtubular structures and/or to their functional properties.

OBJECTIFS: La NRD convertase aussi appelée Nardilysine, une Zn++ metalloendopeptidase qui clive spécifiquement dans la région N terminale des résidus arginine et lysine des sites dibasiques, est impliquée dans la transformation/maturation des proprotéines. Le but de cette étude est de localiser cette enzyme durant la spermiogénèse afin de comprendre son rôle au cours de la maturation des spermatides. MÉTHODES: La Nardilysine est révélée par immunohistochimie au niveau ultrastructural chez des souris contrôles fertiles et chez un mutant stérile (ébouriffé : ebo/ebo). Des analyses quantitatives sont effectuées par comptage des grains d'or colloïdal qui permettent de détecter la localisation spécifique de l'enzyme au cours de la croissance des spermatides dans des régions particulières. RÉSULTATS: L'expression de la Nardilysine chez les souris sauvages et stériles ebo/ebo est limitée aux cellules germinales avec une augmentation significative dans les étapes ultimes de la spermiogénèse. L'enzyme est fortement exprimée dans le cytoplasme des spermatides allongées et dans les structures microtubulaires, la manchette et le flagelle. Aucun marquage n'est observé au niveau des organites cellulaires des spermatides. Chez le mutant ebo/ebo, dont le flagelle est anormal, l'enzyme est toujours présente sur les doublets de microtubules du flagelle. La quantification des particules d'or chez la souris sauvage et chez le mutant révèle une association spécifique de l'enzyme avec les microtubules du flagelle. CONCLUSIONS: L'accumulation spécifique de la Nardilysine au niveau de la manchette et du flagelle suggère que cette enzyme pourrait contribuer à l'établissement de ces structures microtubulaires particulières et/ou à leurs propriétés fonctionnelles.

The emerging role of connexin 43 in testis pathogenesis.

Direct intercellular communication is mediated by gap junctions and their constitutive proteins, the connexins, which are organized in a hexameric arrangement forming a channel between adjacent cells. Connexins are essential for cell homeostasis and are also involved in many physiological processes such as cell growth, differentiation and death. Spermatogenesis is a sophisticated model of germ cell proliferation, differentiation, survival and apoptosis, in which one connexin isoform, connexin 43, plays an essential role as evidenced by the targeted genetic deletion of Cx43 gene. A controlled balance of germ cell growth is a prerequisite to maintain either normal level of spermatozoa necessary for fertility and/or to limit an uncontrolled and anarchic germ cell proliferation, a major risk for germ cell tumor cell development. In the present review, we highlight the emerging role of connexins in testis pathogenesis, specifically in two intimately interconnected human testicular diseases: azoospermia with impaired spermatogenesis and testicular germ cell tumors, whose incidence increased during the last decades. This review proposes the gap junction protein connexin 43 as a new potential cancer diagnostic and prognostic marker, as well as a promising therapeutic target for testicular diseases.

The anti-mitotic drug griseofulvin induces apoptosis of human germ cell tumor cells through a connexin 43-dependent molecular mechanism.

Griseofulvin, a widely used antifungal antimitotic drug has been proposed as an anti-tumoral treatment by way of in vitro experiments. Recently, in vivo demonstration of griseofulvin efficacy against multiple myeloma in mice argues for its potential as therapeutics for cancer. Nevertheless, the molecular mechanisms by which griseofulvin disrupts cancerous cell progression are far from being understood. In the present study, we found that griseofulvin inhibits human germ cell tumor cell growth through activation of mitochondrial caspase pathway (caspase 9 and 3) leading to the activation of apoptosis rather than an alteration of cell proliferation. Strikingly, we demonstrated that griseofulvin triggered the expression level of connexin 43 (mRNA and protein), a well described tumor-suppressor gene, known to participate in apoptosis regulation. Consistently, together with microtubule instability, a mechanism classically associated with cell death in response to griseofulvin, we observed a disruption of connexin 43/tubulin association concomitant of an enhanced translocation of connexin 43, or an immunoreactive fragment of the protein, from the cytoplasm to the nucleus. Finally, by using siRNA approaches we demonstrated the requirement of connexin 43 in the apoptotic induction of griseofulvin on our tumor cell model. Altogether, these results described a new molecular mechanism connexin 43-dependent targeted by griseofulvin leading to apoptosis of human germ cell tumor cells.

Chemical connexin impairment in the developing gonad associated with offspring infertility.

A dramatical decline in human male reproductive function has been reported for the past 20 years. Many recent epidemiological, clinical and experimental findings suggest that the reproductive dysfunction could result from prenatal and neonatal chemical compound exposure. Even if numerous studies argue for a relationship between male infertility and environmental and/or occupational exposure, the molecular mechanisms by which these anti-reproductive compounds act are still unclear. Recent findings showed that a family of transmembranous proteins, connexins, regulates numerous physiological functions involved in the development such as cell proliferation, differentiation, migration and apoptosis. In the testis and the ovary, connexins are known to be essential for the establishment and the maintenance of spermatogenesis in males and oogenesis and folliculogenesis in females. Moreover, mutation of connexin genes leads to several developmental human diseases (myelin-related diseases, hearing loss, congenital cataract, skin disorders or more complex syndromes such as the oculodendrodigital dysplasia....) and altered connexin expression, trafficking and degradation are often associated with the tumoral process. We propose, in the present work, to give an overview of connexin expression and intercellular gap junction coupling during development: in preimplantation, implantation and postimplantation embryos. Moreover, we underline the impact of maternal chemical exposure on connexin expression during fetal gonad development and we link this effect to future offspring fertility.

Reg I protein in healthy and seminoma human testis.

Regenerating gene (Reg), encodes a secretory protein with growth and differentiation stimulating effects mostly in digestive tissues. Overexpression of Reg proteins and specifically of Reg I, one member of the Reg family, is associated with several human diseases and cancers. In the present study we analyzed the expression of Reg I in normal rodent and human testes where germ cells normally proliferate and differentiate into spermatozoa, and in seminoma testis, the most common cancer of young men. Western blot analyses demonstrated the presence of a specific band at 19 kDa in human and rodent testis extracts. Immunofluorescence and deconvolution microscopy demonstrated that Reg I was present within the seminiferous tubules in both Sertoli and germ cells. By using a Sertoli cell line we demonstrated that Reg I was localized at the plasma membrane even in the absence of contact between neighboring cells and appeared before the tight junction associated protein ZO-1 was revealed at this location. Reg I was strongly expressed in human seminoma testis tissue and in a human tumor germ cell line where the immunoreactive signal was mainly detected at the plasma membrane level. These data showing for the first time the weak presence of Reg I in the normal testis and its strong expression in the testis cancer suggest a potential role of Reg I in normal and neoplastic germ cell proliferation.

Connexins as precocious markers and molecular targets for chemical and pharmacological agents in carcinogenesis.

Gap junctions, intercellular channels structured by the connexin protein family, have been implicated in the control of cell homeostasis, proliferation, differentiation and death. A loss of the gap junction intercellular communication and/or connexin dysfunction are typical features of cancer per se and have been associated with the effect of many carcinogens. Indeed, many early human neoplasia of various organs and human tumor cell lines exhibit deficient connexin-mediated communication expression mainly related, in a large number of observations, with an aberrant cytoplasmic localization of this membranous protein. Restoration of normal phenotype in transformed cells by restoration of exogenous connexin gave rise to the concept that connexins may act as tumor suppressors. However, the mechanisms by which connexins mediate such a tumor suppressor effect are multiple. They may result from: formation of functional channels; hemichannels or are directly associated with connexin expression. In addition, the literature shows that they may be dependent upon the cell type and the connexin type. In the present review, we analyze all these aspects of connexin/gap junction involvement in the carcinogenesis process, in human cancers and discuss the possibility of using connexins as potential anti-oncogenic targets for cancer chemoprevention and/or chemotherapy.

Estrogen-induced growth inhibition of human seminoma cells expressing estrogen receptor beta and aromatase.

It is now well established that estrogens participate in the control of normal spermatogenesis and endogenous or environmental estrogens are involved in pathological germ cell proliferation including testicular germ cell tumors. Studying a human testicular seminoma cell line, JKT-1, we show here that 17beta-estradiol (10(-12) to 10(-6) M) induced in vitro a significant dose-dependent decrease of cell growth. This antiproliferative effect was maximum after 4 days of exposure at a physiologically intratesticular concentration of 10(-9) M, close to the K(d) of ER, and reversed by ICI 182780, an ER antagonist, suggesting an ER-mediated pathway. By RT-PCR and Western blot we were able to confirm that JKT-1, like tumoral seminoma cells and normal human testicular basal germ cells, expresses estrogen receptor beta (ERbeta), including ERbeta1 and ERbeta2, a dominant negative variant, but not ERalpha. Using immunofluorescence and confocal microscopy, ERbeta was observed as perinuclear intracytoplasmic spots in JKT-1 and tumoral seminoma cells without significant translocation of ERbeta into the nucleus, under 17beta-estradiol exposure. Double staining observed by confocal microscopy revealed that ERbeta colocalized in JKT-1 cells with cytochrome C, a mitochondrial marker. We report for the first time the expression of a functional aromatase complex in seminoma cells as assessed by RT-PCR, Western blot and enzymatic assay. Seminoma cells are able to respond to estrogens through a possible autocrine or paracrine loop. These preliminary results support estrogen-dependency of human testicular seminoma, the most frequent tumor of young men, and suggest potential pharmacological use. Whether this estrogen control, however, involves an ERbeta-mediated stimulation of cell apoptosis and/or an ERbeta-mediated inhibition of cell proliferation, remains to be further determined.

A proposed role for ZO-1 in targeting connexin 43 gap junctions to the endocytic pathway.

Gap junctions are intercellular channels organized in plaque that directly link adjacent cells. Connexins (Cx), the constitutive proteins of gap junctions are associated with several partner proteins (cytoskeletal, anchoring) which could participate in plaque formation and degradation. Coimmunoprecipitation and indirect immunofluorescence analyses showed that ZO-1, a tight junction-associated protein, was linked to Cx43 in the testis. By using gamma-hexachlorocyclohexane (HCH), known to induce gap junction endocytosis, we demonstrated that endocytosis increased Cx43/ZO-1 association within the cytoplasm of treated Sertoli cells. In control cells, the two proteins were present, as expected, at the plasma membrane level, but poorly colocalized. The increased intracytoplasmic Cx43/ZO-1 complex was associated with a shift towards increased levels of Cx43 P1 and P2 isoforms. The HCH induced Cx43 hyperphosphorylation was abolished by the ERK inhibitor PD98059 suggesting that this effect could be mediated through activation of the ERK pathway. These data strongly support a novel role for ZO-1 in the turnover of Cx43 during gap junction plaque endocytosis.

Functional characterization of Cx43 based gap junctions during spermatogenesis.

In the testis, spermatogenesis is a highly regulated process that includes germ cell multiplication and differentiation supported by Sertoli cells. Gap junction intercellular communication (GJIC), that is known to play an important role in the control of cell proliferation and differentiation, allows communication between adjacent cells. Gap junctions are present within the seminiferous epithelium but the precise nature of coupled cells is not yet identified. By applying a dye-transfer assay to testis, we demonstrated that coupling was basally located in the tubular compartment between adjacent Sertoli cells, between Sertoli cells and spermatogonia and early and late spermatocytes, but not between Sertoli cells and spermatids. Furthermore, no dye transfer occurred from germ cells to Sertoli cells. Specificity of the gap junction coupling was verified with known gap junction inhibitors such as oleamide, heptanol, and glycyrrhetinic acid. We developed a sophisticated assay that allows correlating the in vivo dye transfer with cell morphological identification and Cx43 expression. This approach demonstrated the Cx43 participation in the coupling. Interestingly Cx43 expression and dye-coupling varied with the stages of spermatogenesis. Our results suggest that Cx43 based gap junctions form a transversal and longitudinal intercellular communication network within seminiferous tubules, and that specific communication territories are formed within the seminiferous tubules to ensure the synchronization of germ cell proliferation and differentiation.

Disrupted traffic of connexin 43 in human testicular seminoma cells: overexpression of Cx43 induces membrane location and cell proliferation decrease.

Connexins, the constitutive proteins of gap junctions, are considered to be tumour suppressive agents and are often impaired in the tumourigenic processes. In the present study, the expression of connexin 43 (Cx43), which is involved in the control of spermatogenesis through Sertoli/germ cell coupling, has been investigated in human testicular seminoma cells (tumours and the JKT-1 cell line). Cx43 was immunolocalized in the Golgi apparatus without membrane expression and was detected by immunoblotting in JKT-1 as exclusive 70 kD bands. No mutation could be found by sequencing the transcript obtained by RT-PCR. Transfection with a Cx43-V5 vector reproduced the same gel shift, identifying these 70 kD bands as Cx43. The Cx43-70 kD bands were also expressed in normal testicular tissue, associated with the classical 43 kD isoforms. Stable transfection of JKT-1 with a Cx43-GFP vector allowed restoration of Cx43 membrane expression, functional cell coupling, and inhibition of the cell proliferation rate. Storage of Cx43 in the Golgi apparatus may correspond during spermatogenesis to an intermittent physiological process that becomes permanent in malignant seminoma cells as a result of the tumourigenic process. By preventing Cx43 membrane expression, this disrupted traffic may itself participate in tumour promotion.

Update on cryptorchidism: endocrine, environmental and therapeutic aspects.

Cryptorchidism is the most frequent developmental abnormality in boys, present in more than 1% of infants above three months of age. It is associated with an increased risk of infertility and testicular cancer. The etiological quest is often disappointing, except in bilateral cases or associated malformations. Recent focus is on genetic and environmental aspects. Animal models have revealed the role of genes encoding for proteins implicated in testicular migration (InsI3, Hoxa 10), but in humans results are less convincing. While some degree of endogenous hormonal abnormality is suspeeted in some patients, the endocrine disruptor hypothesis is also tested. It is unclear whether the incidence of cryptorchidism has really increased, or whether there is only a better screening for this condition. However, other male reproductive problems, such as subfertility, hypospadias and testicular cancer seem on the rise. This secular trend suggests the possible in utero impact of hormonally active environmental factors, such as pesticides with estrogenic or antiandrogenic effect, and is consistent with the increased risk of cryptorchidism observed in the sons of mothers exposed to diethylstilbestrol during pregnancy. From a therapeutic point of view, there is an agreement that the correction of cryptorchidism is needed, but there is controversy on the best medical and/or surgical approach and on the optimal timing. There is a recent trend in proposing early therapeutic intervention, before 1 yr of age, in the hope of improving fertility; however, there is no proof that such a strategy can reduce the risk of testicular cancer.

Connexin expression and gap junctional intercellular communication in human first trimester trophoblast.

Connexin (Cx) expression and gap junctional intercellular communication (GJIC) are involved in development and differentiation processes, and recently mutation of connexin genes has been implicated in pathologies. In the human placenta, two distinct differentiation pathways of cytotrophoblastic cells coexist and lead to a fusion phenotype (villous trophoblast) and a proliferative/invasive phenotype (extravillous trophoblast). Here we characterized in situ and in vitro the expression of Cx transcripts and proteins in the villous and extravillous trophoblast of first trimester placenta. In addition, the GJIC functionality was investigated using the gap-fluorescence recovery after photobleaching (gap-FRAP) method. We demonstrated in the villous trophoblast the presence of Cx43 mRNA and of Cx43 protein localized between cytotrophoblastic cells and between cytotrophoblastic cells and syncytiotrophoblast. In vitro, a transient functional gap junctional intertrophoblastic communication was demonstrated during the trophoblast fusion leading to the multinucleated syncytiotrophoblast. During the proliferative process of the extravillous trophoblast, Cx40 is expressed in the proximal part of the cell columns. When cytotrophoblastic cells were cultured on Matrigel for 2 days, alpha5beta1 integrin expression was observed concomitant with the presence of Cx40 mRNA and of Cx40 protein between the cells. No evidence for a GJIC was detected in this induced extravillous phenotype. In addition, Cx32 was detected between some aggregated cells after 72 h of culture. Our data show that the presence of Cx43 allows an inter-trophoblastic GJIC and is associated with the fusion process leading to the villous syncytiotrophoblast and that the presence of Cx40 does not allow GJIC and is associated with the extravillous phenotype.

Disruption of gap junctional intercellular communication by lindane is associated with aberrant localization of connexin43 and zonula occludens-1 in 42GPA9 Sertoli cells.

Lindane (gamma-hexachlorocyclohexane) is a lipid-soluble pesticide that exerts carcinogenic and reprotoxic properties. The mechanisms by which lindane alters testicular function are unclear. Sertoli cells control germ cell proliferation and differentiation through cell-cell communication, including gap junction intercellular communication. Using the 42GPA9 Sertoli cell line, we show that lindane, at a non-cytotoxic dose (50 microM), abolished gap junction intercellular communication (GJIC) between adjacent cells. This change was associated with a time-related diminution and redistribution of Cx43 from the membrane to the cytoplasmic perinuclear region. A similar alteration was observed for ZO-1, a tight junction component associated with Cx43, but not for occludin, an integral tight junction protein. After a 24 h lindane exposure, Cx43 and ZO-1 colocalized within the cytoplasm and no modification of non-phosphorylated and phosphorylated isoforms of Cx43 was observed. By double immunofluorescent labelling we demonstrate that the cytoplasmic Cx43 signal was not present in either the endoplasmic reticulum/Golgi apparatus or lysosomes. These results suggest that lindane inhibits GJIC between Sertoli cells and that aberrant Cx43/ZO-1 localization may be responsible for this effect. The alterations in gap junctions induced by lindane in 42GPA9 Sertoli cells are similar to those observed in tumour cells and may be involved in the pathogenesis of neoplastic seminomal proliferation.

Mapping of seminal plasma proteins by two-dimensional gel electrophoresis in men with normal and impaired spermatogenesis.

The present study analyses differential polypeptide expression of seminal plasma from fertile and infertile men by two-dimensional gel electrophoresis. Optimization of solubilization of seminal plasma was obtained by using [3-(3-(cholamidopropyl) dimethyl-ammonio)-1-propane sulphonate] and chaotropic agent mixture in lysis buffer before separation in immobilized pH gradient for isoelectric focusing. A two-dimensional map of seminal plasma from a fertile man allowed the detection of about 750 spots. Semi-preparative electrofocusing was performed. Analysis of tryptic fragments of two major spots by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectroscopy resulted in identification of prostatic acid phosphatase and prostate specific antigen. Three groups of spots and seven individual spots of isoelectric point from 4.6 to 6.2 and mol. wt from 41 to 18 kDa disappeared in the two-dimensional maps of seminal plasma of vasectomized men (n = 4) and of a patient with bilateral anorchidy as compared to that of fertile men (n = 5). Some of these polypeptides were also absent in seminal plasma of patients with alterations of seminiferous tubules showing Sertoli cell-only syndrome characteristics (n = 4) and could be potential diagnostic markers of spermatogenesis impairment.

Involvement of gap junctional communication and connexin expression in trophoblast differentiation of the human placenta.

Gap junctional intercellular communication (GJIC) permits coordinated cellular activities during development and differentiation processes, and its dysfunction or mutation of connexin genes have been implicated in pathologies. In the human placenta, two distinct differentiation pathways of cytotrophoblastic cell coexist leading to a double model: fusion phenotype (villous trophoblast) and proliferative/invasive phenotype (extravillous trophoblast). This review focuses on current knowledge on the connexin expression and the implication of GJIC in trophoblastic differentiation. Experimental evidence obtained in human placenta demonstrates the involvement of connexin 43-gap junctions in the trophoblastic fusion process and of a connexin switch during the spatially and temporally controlled proliferation/invasion process.

Connexin43 gene expression and regulation in the rodent seminiferous epithelium.

Connexin43 (Cx43) is one of the most predominant gap junction proteins found in the testis. We used in situ hybridization and indirect immunofluorescence to study the distribution of Cx43 mRNA and protein in the rodent seminiferous epithelium. During mouse testis maturation, Cx43 mRNA and its corresponding protein were first detected in the adluminal compartment of the growing seminiferous tubules (early postnatal age: Day 12) to become progressively located in the basal compartment at later ages (Days 16, 19, 27). In seminiferous tubules of sexually mature animals, the intensity of the hybridization signal was stage-dependent, with a maximum at Stage VII compared with Stages V and IX of the spermatogenic cycle (p<0.05). The highest expression of Cx43 mRNA was observed in the supporting Sertoli cells and, to a lesser extent, in the most basally located and less mature germ cells (spermatogonia and spermatocytes). Consistent with these observations, in situ dye coupling was observed between Sertoli cells and basal germ cells. In a mutant mouse deficient for the retinoid X receptor beta, which exhibited abnormal spermatogenesis due to altered Sertoli cell function, Cx43 transcripts were markedly decreased in the seminiferous epithelium (p<0.01). The immunoreactive signal for Cx43 was significantly reduced in seminiferous tubules of the 3-month-old mutant mice (p<0.05) and undetectable in older animals. These data provide new information about the precise localization of Cx43 mRNA and protein in seminiferous tubules of immature and mature rodent testes. Moreover, they suggest that retinoids, through the RXRbeta receptors, could be involved in the control of Cx43 gene expression in Sertoli cells.

Modified expression of testicular gap-junction connexin 43 during normal spermatogenic cycle and in altered spermatogenesis.

In order to better understand the implications of gap junction proteins in spermatogenesis, connexin 43 (Cx43), the most abundant connexin in the testis, was evaluated in testes of wild-type mice and of two mutants with impaired spermatogenesis (ebo/ebo, and jun-d-/-mice). Reverse transcription-polymerase chain reaction (RT-PCR) amplification revealed a constitutive expression of mRNA for Cx43 in both wild-type mice and infertile mutants. In the seminiferous tubules of wild-type mice, indirect immunofluorescence revealed that Cx43 expression was stage-dependent and that the signal was mainly located in the region of Sertoli cell occluding junctions. Colocalization of Cx43 and of the tight-junction-associated protein zonula occludens 1 (ZO-1) was demonstrated in seminiferous tubules by using dual-label immunofluorescence in conjunction with confocal microscopy. The Cx43 staining analyzed by high-resolution confocal microscopy appeared as continuous, anastomozed ribbons and thin dots. The level of Cx43 immunoreactivity was reduced in seminiferous tubules of ebo/ebo and jun-d-/- mutants as compared to the respective wild-type mice. No staining for Cx43 was detected in Sertoli cell-only seminiferous tubules observed sometimes in jun-d-/- mice. The present study represents one of the first in vivo examples of alteration of seminiferous tubule Cx43 in testes with impaired spermatogenesis.

Anti-Müllerian hormone as a seminal marker for spermatogenesis in non-obstructive azoospermia.

Anti-Müllerian hormone (AMH) also known as Müllerian inhibiting substance or factor, is a Sertoli cell-secreted glycoprotein responsible in male embryos for Müllerian duct regression. However, its role in adults remains unknown. AMH seminal concentrations have been evaluated using an enzyme-linked immunoassay in three groups of young men: group 1, fertile donors (n = 18); group 2, obstructive azoospermia (n = 9) after vasectomy or associated with deferent duct agenesia; and group 3, non-obstructive azoospermia with spermatogenesis deficiency and normal karyotype (n = 23). AMH was present in seminal plasma of most fertile donors at concentrations ranging from undetectable (<3.5 pmol/l) up to 543 pmol/l (geometric mean: 153 pmol/l), higher than the serum level (range <3.5 up to 67 pmol/l, geometric mean: 10.7 pmol/l, n = 13). Seminal AMH concentrations were undetectable in all obstructive azoospermic patients, confirming its testicular origin. In non-obstructive azoospermia (group 3), seminal AMH concentration was lower (range <3. 5-68.5 pmol/l, geometric mean: 17 pmol/l) than in fertile donors (P < 0.003) without correlation with plasma follicle stimulating hormone values. In group 3, comparison of seminal AMH concentration and the results of histological analysis of testicular biopsies revealed that undetectable AMH found in 14 cases was associated in 11 of them with lack of spermatozoa, while detectable concentrations of AMH (10-68.5 pmol/l) found in nine cases were associated in seven of them with persistent spermatogenesis. In the adult, AMH is secreted preferentially towards the seminiferous lumen. Although its relationship with spermatogenesis requires further investigation, our results suggest that seminal AMH may represent a non-invasive marker of persistent hypospermatogenesis in cases of non-obstructive azoospermia which may indicate the likely success of testicular spermatozoa recovery before intracytoplasmic sperm injection.

Regulation of tissue-type plasminogen activator and its inhibitor (PAI-1) by lipopolysaccharide-induced phagocytosis in a Sertoli cell line.

The plasminogen activator (PA) system is thought to play a major role in the proteolytic events associated with spermatogenesis. The mechanisms controlling the expression of PA and of its major physiological inhibitor, plasminogen activator inhibitor type-1 (PAI-1), in the seminiferous epithelium are still unknown. In the present study we analyzed the expression of PA and PAI-1 in a murine Sertoli cell line (42GPA9) in response to stimulation by lipopolysaccharides (LPS) used to activate the phagocytic activity of these cells. Immortalized Sertoli cells cultured under basal conditions secreted predominantly tissue-type PA (tPA) as demonstrated by zymographic analysis and the presence of tPA transcripts. In zymographic experiments a larger molecular weight proteolytic band corresponding to the formation of PA-PAI-1 complex was also observed. The stimulation of immortalized Sertoli cells by LPS resulted in both alteration of the apparent tPA molecular weight to a higher form and transient increase in PAI-1 biosynthesis. The phorbol ester TPA stimulates similarly PAI-1 synthesis in the Sertoli cell line, while 8-bromo-cAMP has no effect. These results suggest for the first time the existence of a direct linkage between molecular events triggered by phagocytosis and regulation of tPA and PAI-1 in Sertoli cells.

Ultrastructural and biochemical evidence for gap junction and connexin 43 expression in a clonal Sertoli cell line: a potential model in the study of junctional complex formation.

To clarify the exact role of Sertoli cells in testicular intercellular communications, a murine Sertoli cell line (42GPA9) has recently been established. Electron-microscopy studies indicate that the morphology of these immortalized cells strongly resembles that of mouse Sertoli cells in vivo with an indentend nucleus, elongated mitochondria and numerous lysosome-like structures. Ultrastructure analysis has also revealed that 42GPA9 cells form gap junctions as demonstrated by the presence of small electron-dense bridges that connect the plasma membranes of adjacent cells. The gap junction protein connexin 43 (Cx43) has been identified in cultured 42GPA9 cells by immunofluorescence and Western blot analysis. No immunostaining is detected in the absence of apparent intercellular contact. The anti-Cx43 antibody labels the contacts between 42GPA9 cells at confluency. This specific staining appears as small dots forming isolated rows of dots or surrounding the entire cell, suggesting that Cx43 is assembled into membrane plaques. The gap junctional communication capacity of the 42GPA9 cell line has been demonstrated by the dye-transfer technique. Exposure of 42GPA9 cells for 24 h to cAMP and 12-O-tetradecanoylphorbol-13-acetate greatly reduces the Cx43 staining at cell-cell contacts and concomitantly increases the cytoplasmic staining, suggesting that these agents alter the trafficking of Cx43 to the plasma membrane. Thus, the 42GPA9 line may provide a useful in vitro model for studying gap junction communication between Sertoli cells.