Biomedical subjects

D Segretain

Publications and source records attributed to D Segretain.

At least 19 recordsLinked to original sources

PubMed2004

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.

Animals↗

PubMed2004

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.

Adult↗

PubMed2001

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.

Animals↗

PubMed2000

The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells.

The aim of the present study was to assess whether the whole meiotic process of spermatogenic cells is able to take place in vitro. Fragments of seminiferous tubules from 20- to 22- or 28-day-old rats were seeded in medium containing 0.2% fetal calf serum in bicameral chambers and then cultured for 4 weeks in a chemically defined medium. The differentiation of meiotic germinal cells was followed by four criteria: (i) ultramicroscopic examination of the different types of germ cells present in the cell layer throughout the culture period; (ii) determination of the changes in DNA content per nucleus of the cell population seeded with time in culture; (iii) assessment of the ability of germinal cells to transcribe genes expressed after completion of meiosis; and (iv) monitoring the fate of BrdU-labeled leptotene spermatocytes. The ultrastructural study showed that the overall organization of the cells in the culture well recalls that of the seminiferous epithelium throughout the culture period. Moreover the identification of young round spermatids 21 days after seeding suggested that these spermatids had been formed very recently in culture. Determination of DNA content per nucleus showed that a 1C cell population could be observed after several days of cultures reaching 6 to 10% of total cells. An exponential-like increase in the amounts of the mRNAs encoding for TP1 or TP2 occurred from the time when 1C cells appeared in the culture until the end of the experiment. Finally, BrdU-labeled leptotene spermatocytes differentiated into pachytene spermatocytes and then into secondary spermatocytes, and BdrU-labeled round spermatids were observed from Day 21 of culture onward. Taken together these results indicate that the whole meiotic process from leptotene spermatocyte to round spermatid can indeed occur in vitro under the present culture conditions.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

Targeted disruption of the murine junD gene results in multiple defects in male reproductive function.

JunD is one of three mammalian Jun proteins that contribute to the AP-1 transcription factor complex. Distinct regulation and functions have been proposed for each Jun member, but less is known about the biological functions of each of these proteins in vivo. To investigate the role of JunD, we have inactivated the murine gene by replacement with a bacterial lacZ reporter gene. Embryonic JunD expression was initially detected in the developing heart and cardiovascular system. Subsequent broadening phases of JunD expression were observed during embryonic development and expression in the adult was widespread in many tissues and cell lineages. Mutant animals lack JunD mRNA and protein and showed no evidence of upregulation of c-Jun and JunB mRNA levels. In contrast to the other two Jun members, homozygous JunD-/- mutant animals were viable and appeared healthy. However, homozygous JunD-/- animals showed a reduced postnatal growth. Furthermore, JunD-/- males exhibited multiple age-dependent defects in reproduction, hormone imbalance and impaired spermatogenesis with abnormalities in head and flagellum sperm structures. No defects in fertility were observed in JunD-/- female animals. These results provide evidence for redundant functions for members of the Jun family during development and specific functions for JunD in male reproductive function.

Animals↗

PubMed1999

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.

Animals↗

PubMed1998

The mouse Tsx gene is expressed in Sertoli cells of the adult testis and transiently in premeiotic germ cells during puberty.

Tsx is a gene of unknown function that was previously shown to be expressed specifically in the testis. In order to gain insight into the function of Tsx its pattern of expression was characterized with regard to both timing and cell type in the testis. Northern blot analysis of early postnatal testes showed not only that Tsx message was detectable shortly after birth, but that it increased substantially between 7 and 12 days postpartum (dpp), roughly coincident with the onset of meiosis in the mouse. Alternative Tsx transcripts, detected by RT-PCR, included a spliced form that first appeared at around 12 dpp. In situ hybridization revealed Tsx signal in the somatic Sertoli cells of the adult testis. Consistent with the data from Northern blots, in situ hybridization signal was first detectable in normal pubertal testes at 12 dpp. An anti-Tsx polyclonal antiserum specifically stained premeiotic germ cells in addition to Sertoli cells of pubertal testes at 16, 19, and 27 dpp. Tsx immunostaining in germ cells was nuclear, while Sertoli cells displayed signal throughout the cytoplasm and nucleus. In the adult, Tsx was detected exclusively in Sertoli cells. In contrast, in the adult testis of the oligotriche (olt) mutant, where spermatogenesis is blocked after meiosis, Tsx protein was still present in the spermatogonial nuclei of a subset of tubules. Taken together, these results demonstrate that Tsx expression is induced in both premeiotic germ cells and Sertoli cells during the first wave of spermatogenesis, but that expression is maintained at a detectable level only in Sertoli cells of the normal adult. The persistence of Tsx expression seen in spermatogonia of the adult olt mutant supports the hypothesis that during the first wave of normal spermatogenesis, the advent of a late-stage cell type, either elongating spermatid or spermatozoan, is responsible for extinguishing expression in spermatogonia in normal adult testis. To our knowledge, Tsx is the first gene to show a pattern of germ cell expression that is apparently specific to the pubertal testis.

Animals↗

PubMed1998

Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342.

Dictyostelium discoideum cells are highly resistant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size > 21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism.

Animals↗

PubMed1998

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.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1998

Characterization of a clonal Sertoli cell line using adult PyLT transgenic mice.

In the present study we report the isolation and characterization of a clonal Sertoli cell line (42GPA9) from sexually mature polyoma virus large T (PyLT) transgenic mice. The cells multiplied indefinitely and expressed large T antigen. The 42GPA9 cell line expressed biochemical features associated with normal Sertoli cells. Transferrin, sulfated glycoprotein-2, and the ligand of c-kit were detected by reverse transcription-polymerase chain reactions and Western blot analyses. Zymographic analysis indicated that the 42GPA9 cell line secreted tissue-type plasminogen activator. These cells also retained FSH receptors as suggested by their specific responsiveness to the gonadotropin (morphological and phagocytic changes, stimulation of cAMP production) and the detection of FSH receptor mRNAs. Another original aspect of the 42GPA9 cell line is its ability to form tight junctions at confluency as demonstrated by electron microscopic study and immunolocalization of the tight junction-associated protein zonula occludens 1. The 42GPA9 cell line, which has retained several important hallmarks of normal Sertoli cells, may prove useful for further studies on Sertoli cell behavior and on Sertoli-germ cell interactions in the mature testis.

Animals↗

PubMed1998

Stage-specific expression of the Kit receptor and its ligand (KL) during male gametogenesis in the mouse: a Kit-KL interaction critical for meiosis.

The Kit receptor and its ligand KL, which together constitute an essential effector at various stages of embryonic development, are both present during adult gametogenesis. In the testis, KL is expressed in Sertoli cells, and Kit in germ cells, starting at the premeiotic stages. A series of observations indicated previously a role in spermatogonia survival, without excluding a possible function at later stages. We identified a complex pattern of expression of the two components in the adult murine testis, suggestive of a role in the meiotic progression of spermatocytes. At stages VII-VIII of the cycle of the seminiferous epithelium, the time when spermatocytes enter meiosis, the membrane-associated form of KL extends on the Sertoli cell from the peripheral to the adluminal compartment of the tubule. We also found that the receptor is present on the surface of germ cells up to the pachytene stage. The availability of differentiated Sertoli cell lines, which express the KL protein and support part of the maturation of germ cells in coculture, allowed us to ask whether, in the in vitro reconstructed system, transit of spermatocytes through meiosis requires the Kit-KL interaction. Addition of a blocking monoclonal antibody against the Kit receptor (ACK2) inhibited extensively the appearance of haploid cells and the expression of a haploid-phase-specific gene (Prm1). Recognition of the supporting Sertoli cell by germ cells was not affected, indicating a requirement for the activity of the receptor for either entering or completing meiosis. Involvement of the membrane-associated form of the ligand was suggested by the observation that addition of the soluble form of KL was equally inhibitory.

Animals↗

PubMed1996

Characterization and localization of the rat, mouse and human testicular phosphatidylethanolamine binding protein.

A cytosolic 23kDa protein was initially purified from bovine brain and shown to bind phosphatidylethanolamine. Later, it was also characterized in rat and human brain, and it is now known to be widespread, having been found in numerous tissues in several species. Here, we report the high level of mRNA and phosphatidyl ethanolamine binding protein expression in rat testis and to a lesser extent in mouse testis. In human testis, although it was not detectable by Northern blot analysis, the mRNA was shown to be present when PCR amplification was performed. Immunohistochemical experiments revealed that the testicular phosphatidylethanolamine binding protein (tPBP) is principally expressed in the elongated spermatids of both rat and mouse testis. This finding, and the association of tPBP with cellular membranes, suggest its possible implication in membrane remodelling during spermatid maturation.

Amino Acid Sequence↗

PubMed1996

Developmental and stage-dependent expression of melanin-concentrating hormone in mammalian germ cells.

Melanin-concentrating hormone (MCH) is a cyclic peptide predominantly expressed in the hypothalamus of mammals. This peptide modulates the stress response and regulates many goal-oriented behaviors in the rat brain. MCH mRNA and peptides generated from the precursor, namely MCH and neuropeptide (N) glutamic acid (E) isoleucine (I) amide (NEI), were also found in rodent peripheral tissues including those in adult testis. In the present study, we first examined the cellular distribution and content of MCH gene products and peptide in the testes of adult rats. Using reverse-transcription polymerase chain reaction with MCH gene primers and in situ hybridization with specific 33P-labeled oligoprobes, we characterized the MCH RNA species. Pro-MCH products were revealed through use of immunoperoxidase detection and an RIA with specific MCH and NEI antisera. Both MCH gene transcripts and MCH peptide were found within germ cells at the periphery of some of the seminiferous tubules of adult rats. We further investigated temporal expression of MCH in 7-microns sections of testes from adult rats and mice. MCH was predominantly found in nuclei of spermatogonia at stages II-IV of spermatogenesis and in nuclei of leptotene and zygotene spermatocytes (at stages IX- XIII). MCH was markedly absent in preleptotene spermatocytes at the stage VII-VIII, and MCH immunoreactivity was no longer detectable as spermatocytes underwent the pachytene step. MCH immunoreactivity was also found in some peritubular cells, mainly at stages II-IV. Finally, we studied MCH expression during puberty in the rat, in sterile mutant mice, and in one adult man. We found predominant staining with MCH antiserum over nuclei of immature germ cells as early as 10 days postpartum in the rat and further confirmed the absence of staining except for that in spermatogonia and early spermatocytes during rat postnatal development. MCH distribution was found to be similar in normal and sterile mutant mice, suggesting that MCH expression is not dependent upon the early steps of spermiogenesis. MCH immunoreactivity was found to be confined to the nuclei of spermatogonia and early spermatocytes in the adult man. Our results indicate that MCH is present predominantly within nuclei of spermatogonia and primary spermatocytes in three mammalian species and that its expression is under strong stage-specific and developmental regulation. This peptide may play a role during stem cell renewal and/or differentiation of early spermatocytes.

Adult↗

PubMed1996

Expression of HLA class I genes in meiotic and post-meiotic human spermatogenic cells.

In human spermatogenic cells, in contrast to somatic cells, expression of major histocompatibility complex (MHC) class I molecules is undetectable. This lack of expression may contribute to the absence of female immune reaction against spermatozoa and may be necessary for gamete fusion. Among the molecular repressor mechanisms that may be used at the DNA level, we investigated 5' CpG methylation of the different class Ia and class Ib loci in meiotic pachytene spermatocytes and postmeiotic round spermatids, which had been purified from human testes by centrifugal elutriation. These results were compared with those obtained with mature spermatozoa and peripheral blood mononuclear cells. Using methylation-sensitive restriction enzymes and DNA locus-specific probes, we found that HLA-A, HLA-B/C, and HLA-E loci were similarly unmethylated in the germ and somatic cells tested, whereas HLA-F and HLA-G were even less methylated in the former cells. Together with the observation that spermatozoon DNA contains class I genes that are transfectable and able to direct transcription and protein synthesis in murine L cells, these data suggest that HLA class I genes are in an active conformation in male germ cells. We indeed found that both spermatocytes and spermatids contained low levels of class Ia and class Ib mRNA. Using reverse transcriptase-polymerase chain reaction, followed by DNA sequencing, we also detected three HLA-G transcriptional isoforms, resulting from alternative splicings, which suggested that this class Ib gene may have a potential function in these germ cells. Although intracellular expression of beta2-microglobulin (the light chain that associates with HLA class I heavy chains) was found in spermatocytes but not in round spermatids, no membrane-bound nor intracellular translated HLA class I heavy chain was detected in either germ cell type, when monomorphic anti-HLA class I monoclonal antibodies were used. Thus, lack of expression of HLA class I proteins in the male germ line is likely to involve post-transcriptional mechanisms of regulation.

Alternative Splicing↗

PubMed1996

Male sterility caused by sperm cell-specific structural abnormalities in ebouriffé, a new mutation of the house mouse.

We have investigated the male sterility associated with a new recessive mutation of the house mouse: ébouriffé (ebo). All spermatozoa present in the epididymis showed severe malformations, mostly of the tail. Light and electron microscopy showed a drastic decrease of the spermatid population, whereas spermatogonia and spermatocytes seemed moderately affected. This suggests that the mutation affects mostly spermiogenesis. Defects appeared during formation of the acrosome: the acrosomal granule was frequently vacuolated at stages II-III, giving rise first to abnormal acrosomes (stages VI-VII) with dilations and perforations, and then to an abnormal head and acrosome shape (stages IX-XI). However, the most common malformations affected the flagella in elongated spermatids. Sometimes the centriole doublet did not move into the implantation fossa, causing an unattached and isolated flagellum. The major defect occurred in the midpiece region of differentiating spermatozoa: flagellar components were present but highly disorganized, and mitochondria were aggregated in a compact mass. Even though we have no evidence that the ebo gene is a testis structural gene or a regulatory gene that disrupts the complex spermatogenesis process, this mutation may provide an interesting tool for studying the late stages of spermatogenesis. Using an interspecific backcross, we localized the ebo mutation on chromosome 2, with no recombination out of 44 meioses with locus D2Mit32.

Acrosome↗

PubMed1996

NRD convertase: a putative processing endoprotease associated with the axoneme and the manchette in late spermatids.

N-arginine dibasic convertase is a novel metalloendopeptidase which selectively cleaves at the N terminus of arginine residues in paired basic amino acids. Although present in brain and several other tissues, NRD convertase is particularly abundant in testis, where its expression appeared to be restricted to germ cells. Low levels of both mRNA and its corresponding protein were detected early in spermatogenesis. However, a marked accumulation of the protein was observed during late steps (14 to 19) of spermiogenesis. By electron microscopy, the NRD convertase immunoreactivity was localized in the cytoplasm of elongating and elongated spermatids, with a noticeable concentration at the level of two microtubular structures, i.e. the manchette and the axoneme. These observations strongly support the hypothesis that NRD convertase is involved in processing events potentially associated with the morphological transformations occurring during spermiogenesis.

Animals↗

PubMed1995

Lead affects steroidogenesis in rat Leydig cells in vivo and in vitro.

Lead is known to impede the male reproductive function, however, the mechanisms through which the adverse effects are mediated are not clearly elucidated. In order to get insight into those mechanisms, we have examined the effects of lead on the biosynthesis of steroid hormones by Leydig cells in the rat. To determine whether lead has a direct action on Leydig cells, we have compared the concentrations of testosterone secreted by Leydig cells in ex vivo experiments after animals had been injected with high doses of lead and in vitro experiments with Leydig cells from normal rats maintained in culture in presence or absence of lead. In ex vivo experiments male Spargue-Dawley rats were injected i.p. with lead acetate (8 mg lead/kg/day, 5 days a week for 5 weeks) or with sodium acetate. Testosterone production by Leydig cells isolated and maintained in culture for 48 h was then assessed under basal conditions or after stimulation by human chorionic gonadotrophin (hCG). Both basal and hCG-stimulated testosterone production dropped by 59% and 37%, respectively, with Leydig cells from lead-exposed rats. For in vitro experiments, cultures of Leydig cells from control rats were exposed to various concentrations of lead acetate for different periods. Dose and time-dependent reductions of testosterone level were observed in the culture medium. The effective doses of hCG for maximal and half-maximal testosterone production did not change, indicating that the sensitivity of Leydig cells to hCG was not impaired by exposure to lead in vitro. Progesterone production was also decreased after this exposure. The negative effect of lead on testosterone and progesterone production was correlated with the lower expression of the enzymes cytochromes P450scc (CYP11A1) and P450c17 (CYP17) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) involved in steroid hormone biosynthesis, as shown by immunohistochemistry. Ultrastructural alterations of the smooth endoplasmic reticulum observed after lead administration might be correlated with the lower expression of the microsomal enzymes P450c17 and 3 beta-HSD. Our results indicate that lead can adversely affect the Leydig cell function by impairing directly steroidogenesis.

3-Hydroxysteroid Dehydrogenases↗