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The epididymal epithelial cells of the lizard (Lacerta vivipara) produce large amounts of specific proteins under androgenic control. Amongst them, a major protein family that binds to the head of spermatozoa, the lizard epididymal secretory protein (LESP) family, has been identified as a member of the lipocalin superfamily. LESPs are composed of 9 elements that present an identical molecular mass of 18 000 Da but have a large range of pHi (3.5 to 9). The structural analysis of this protein family was performed by the determination and comparison of both the aminoterminal sequence of each element and the complete sequence of three specific LESP cDNA clones. When not identical, LESP elements present randomly dispatched nucleotide and amino acid substitutions, indicating the existence of at least five LESP mRNA populations encoded by a multigenic family. We determined that these LESP genes are differentially expressed during the annual epididymal cycle.

We have previously characterized and cloned a secreted sperm-bound selenium-independent glutathione peroxidase protein (GPX5), the expression of which was found to be restricted to the mouse caput epididymidis. Because of the lack of selenium (Se) in the active site of this enzyme, unlike the other animal GPXs characterized to date, it was suspected that GPX5 does not function in the epididymis as a true glutathione peroxidase in vivo. In the present report, following dietary selenium deprivation which is known to reduce antioxidant defenses and favor oxidative stress in relation with depressed Se-dependent GPX activities, we show that the epididymis is still efficiently protected against increasing peroxidative conditions. In this model, the caput epididymides of selenium-deficient animals showed a limited production of lipid peroxides, a total GPX activity which was not dramatically affected by the shortage in selenium availability and an increase in GPX5 mRNA and protein levels. Altogether, these data strongly suggest that the selenium-independent GPX5 could function as a back-up system for Se-dependent GPXs., (Copyright 1999 Wiley-Liss, Inc.)

We report here-using northern experiments, western blotting, and immunohistochemistry-on the findings that the plasma type glutathione peroxidase, GPx3, a major enzyme in reducing lipid hydroperoxides and hydrogen peroxide in plasma, is also expressed at significant levels in tissues of the male genital tract including epididymis and vas deferens. Within the epididymis and the kidney, the accumulation of the GPx3 mRNA and protein were investigated during postnatal development and found to be temporally regulated in a tissue-specific manner. Furthermore, we show here that androgen withdrawal by castration down regulates the expression of the GPx3 gene both in the epididymis and vas deferens while GPx3 expression in the kidney was found to be androgen-independent. Finally, immunohistochemistry data reveals that within the epididymis GPx3 distribution is quite peculiar suggesting the existence in this organ of complex traductional and/or transcriptional regulatory processes.

This report presents data that suggest that the tissue-restricted polyoma enhancer activator protein (PEA3) of the Ets oncogene family of DNA-binding proteins is a putative modulator of the epididymis-specific glutathione peroxidase 5 gene gpx5. Northern and polymerase chain reactions on reverse-transcribed epididymal RNAs were used to show that the PEA3 factor is spatially and temporally expressed within the mouse epididymis in a manner consistent with gpx5 characteristics of expression. Then, using contransfection experiments carried out in heterologous tissue-culture cells with various deletions of the gpx5 promoter driving a CAT reporter gene, we have shown that the transcriptional activity of the gpx5 promoter is modulated by the presence of the PEA3 protein. Subsequently, we have shown using gel-shift assays that DNA sequences located within the 5' flanking region of the gpx5 gene have the ability to bind specifically to the PEA3 protein. Finally, using Northern assays we present data that suggest that PEA3 mRNA accumulation in the mouse caput epididymidis is controlled by androgens and testicular factors. Altogether, these results strongly suggest that the PEA3 factor might participate in the transcriptional control of the murine epididymis caput-specific gpx5 gene.

Two glutathione peroxidase genes (gpx5 and gpx3) were found to be expressed in the mouse epididymis. Gpx5 was shown to be epididymis specific and restricted to the caput epididymidis, while gpx3 was found to be expressed in a wide array of tissues including the caput, corpus and cauda epididymides. Both single copy genes are regulated by androgens as well as being developmentally regulated during postnatal ontogenesis of the epididymis. In this report data collected to date concerning the mechanisms by which these genes are regulated in the mouse epididymis are summarized. The putative roles of these antioxidant enzymes in the sperm maturation process are discussed.

Using immunohistochemistry and Western blotting analyses, we present a detailed study of the distribution of the glutathione peroxidase protein (GPX5) within the mouse epididymis. We have shown that the expression of the epididymis-specific protein is restricted to the caput and essentially localized to the apical cell border of the caput epithelium. Secretion of the protein was detected as early as the proximal segment of the caput and GPX5 was subsequently found in the lumen of corpus and cauda epididymis duct. Within the caput, Western blot analyses have shown that equivalent quantities of GPX5 protein were found in segments I, II, and III. During ontogenesis, GPX5 appeared at 20 days postnatal, before the completion of the morphological differentiation of the caput and concomitantly with the appearance of spermatozoa within the epididymis, in agreement with what was reported earlier regarding the transcription of its corresponding gene during epididymal ontogenesis (Faure et al., 1991). Hormonal privation by castration abolished the accumulation of the GPX5 protein confirming previous data obtained on GPX5 mRNA levels. Treatments such as testosterone replacement or hemicastration led to the restriction of the protein to the caput epithelium, suggesting that protein secretion partly depends both on the presence of testicular factors and on spermatozoa. Using electron microscopy, we have shown that the secreted protein binds to spermatozoa and is found predominantly on the sperm acrosomic region. Finally, we report here that the GPX5 protein can be detected in fluids recovered from the uterine horns of freshly mated female mice. These results suggest that GPX5 might play an important role in sperm maturation from the early events up to the onset of fertilization and therefore could potentially be used as a tool to monitor sperm quality.

We have reported earlier the cloning and the chromosomal localization of 2 GPX-encoding sequences expressed differentially within the mouse epididymis, gpx5 and gpx3. Here, we have mapped on the mouse chromosomes the third known murine GPX-encoding gene, the cytosolic GPX or gpx1. We have compared the degree of identity of the 3 GPX proteins, the respective organization of the 3 corresponding single copy genes and, using degenerated oligonucleotides designed in highly conserved domains of the proteins, we have analyzed the expression of GPX-encoding genes in the mouse epididymis as well as in control tissues known to express GPX proteins (the liver for GPX1 and the kidney for GPX3). The 3 genes characterized to date were found expressed in each of the tissues tested but in a highly tissue-restricted manner. Nucleotidic sequences comparisons were carried out on GPX-encoding sequences from various species and were used to draw a dendrogram. Phylogenetic evaluation of the sequence information, as well as the chromosomal localizations, suggest that the GPX genes have evolved by duplication events followed by random insertions from a single ancestral gene.

The complete sequence of the mouse epididymal protein (MEP24) was cloned. It contains a 663 bp open-reading frame that, after conceptual translation, shows extensive identity with proteins belonging to the glutathione peroxidase (GPX) family. However, a major difference between GPX5 (MEP24) and other known GPXs concerns a protein domain known to be critical for GPX function. To find out what could be the physiological function of such a protein in the mouse epididymis, we have used a mammalian expression system to overexpress the GPX5 protein. Cells constitutively expressing the GPX5 protein were generated and assayed for their ability to metabolize regular substrates of GPX enzymes. Data presented here show that the GPX5-expressing cells can metabolize hydrogen peroxide in a manner that is consistent with a peroxidase activity. However, the substrate preference of the GPX5-expressing cells and their apparent insensitivity to a regular inhibitor of GPX enzymes suggest that the GPX5 protein belongs to a particular class of GPX proteins. Involvement of this protein in the physiology of the mouse epididymis is discussed.

The sperm coating lizard epididymal secretory protein (LESP) family forms a complex of nine elements that are specifically synthesized under androgenic control and secreted by the epididymal epithelial cells of the lizard Lacerta vivipara. We report here the cloning and sequencing of an 806-base pair full-length cDNA (C731) encoding one of the elements of the LESP family. Southern blot hybridization analysis of lizard total genomic DNA revealed a complex band pattern, suggesting that LESPs are encoded by a multigenic family. The cDNA open reading frame of 516 nucleotides, starting at an ATG codon, encodes a protein precursor of 172 amino acids with a calculated M(r) = 19,500. The corresponding mature form of M(r) = 17,200 and pI = 5.2 has been identified as the element LESP IV, and presents significant similarities to the different members of the large lipocalin protein superfamily, and especially to mouse epididymal protein ESP I. Lipocalins are extracellular proteins that share a common basic framework for the transport of small hydrophobic molecules like retinoids, thus suggesting that LESPs could be such transporters into the epididymal fluid during the sperm maturation.

The M53 cDNA for Mr 24,000 androgen-regulated secretory proteins of the mouse caput epididymidis previously reported has been sequenced. This clone presents a 5'-incomplete open reading frame of 525 base pairs. The 3'-untranslated region of 946 base pairs contains a repetitive DNA element of the rodent B1 family just between two canonical polyadenylation signals AATAAA, upstream of the poly(A) track. The deduced amino acid sequence for the Mr 24,000 proteins reveals significant homologies of 66.6, 66, 65.2, 63.1 and 64.5% with mouse, rat, man, bull and rabbit cloned selenium-dependent glutathione peroxidases, respectively. The present results emphasize previous studies performed in numerous laboratories suggesting that the major protective system against oxidative damage in mouse spermatozoa could be an enzyme similar to glutathione peroxidase.

During the annual cycle of the lizard Lacerta vivipara dramatic changes in the secretory activity of the epididymis were observed. These changes and changes in morphology correlate with the plasma and epididymal testosterone concentrations. The secretory proteins contain a major group of immunorelated components referred to as LI to L1X. They consist of a group of nine proteins Mr 19,000 which can be separated according to pI 3.5 to 8.7. Post-translational modifications may be responsible for their pI diversity. All the L proteins are glycosylated (fucose, N-acetylgalactosamine and or N-acetylglucosamine) but only LVI glycosylation was inhibited with tunicamycin. Phosphorylation is unique to LV protein and none of the L proteins are sulfated. All L proteins appeared sequentially during the annual cycle and in organotypic culture when incubated in the presence of testosterone (150, 500, 1000 nM) in a time dependent manner.

Principal cells of the mouse caput epididymidis synthesize and secrete a 24 kDa protein able to bind to the head of the spermatozoa. Sequencing of several clones selected from cDNA and genomic libraries, combined with the microsequencing of the NH2 terminus of the protein allowed to reconstitute the entire primary structure of the mature 24 kDa protein. It revealed 81% homology with a human plasma glutathione peroxidase and 61% homology with a mouse erythrocyte glutathione peroxidase. This enzyme, once secreted in the epididymal fluid, might protect sperm membrane lipids, particularly those of the acrosomal part, against peroxidation.

During its reproductive period, the epididymis of the lizard Lacerta vivipara produces large amount of proteins among which "L" proteins are very prominent components. L proteins have been characterized as an androgen dependent protein family composed of 9 elements of identical MW and different pHi. An epididymal cDNA library was performed and a cDNA clone, C73 was isolated using a specific anti L immunoserum. We tested the tissue specificity and the androgen dependency of this clone in different physiological and experimental conditions by dot-blot analysis. The aminoacid deduced sequence of the C73 clone revealed that it strictly corresponds to the NH2 terminal sequence of the LIV element of the family. It consists of a 151 amino acids mature protein with a 17.2 kDa MW that present homologies with a rat epididymal protein supposed to be a retinoic acid binding protein.

Specific mRNAs for 24-kDa proteins specific to the caput epididymidis were quantified by filter hybridization, and cellular distribution was assessed by in situ hybridization of tissue sections. Messenger RNAs were detectable in 10-day-old animals, rapidly increased in quantity between 15 and 20 days, and reached a maximum at 40 days of age. The marked increase in concentration of mRNAs could be associated with the increase in epididymal testosterone content. Near 26 days of age, specific perinuclear and basal localization of mRNAs occurred in the principal cells of segment I, and a wide cytoplasmic distribution was observed in segment II. In the adult, mRNA levels decreased by 50% 3 days after castration and became undetectable within 30 days. Administration of testosterone to castrated mice caused an increase in mRNA levels, which reach almost normal levels after 3 days of treatment. Nevertheless, the particular organization of segment 1 was not restored. A similar observation was made after hemicastration or ligation of the efferent duct on the operated side. If expression of the mRNAs appears to be mostly under androgenic control, other testicular factors may be involved in the regulation of mRNA distribution in segment I.

A polyclonal monospecific immune serum was raised against androgen-regulated proteins with Mr 24000 secreted by the mouse caput epididymidis. Sections of frozen tissues from the different regions of the epididymis have been studied by indirect immuno- fluorescence. Results indicate that the antigens are secretory proteins produced by the epithelial cells of the caput epididymidis, essentially in the medial and distal segments. Accumulation of the antigens was observed in the lumen of the caput and the corpus epididymal duct. Subsequently, their association with the sperm surface occurred and persisted down to the cauda epididymidis.

Estrogen binding activity was investigated in the epididymis of the turtle, Chrysemys picta using DNA-cellulose affinity chromatography. A component binding estradiol-17 beta specifically with high affinity (Kd:8.0 X 10(-10) M) and limited capacity (20 fmol/mg protein) was demonstrated in the epididymal cytosol. In addition, binding of estradiol-17 beta was sensitive to excess (100-fold) diethylstilbestrol or natural estrogens (estradiol-17 beta, estrone, and estriol) but not to progesterone or androgens (testosterone and 5 alpha-dihydrotestosterone). The specific estrogen binding macromolecules eluted from DNA-cellulose columns sedimented at 4-5 S in linear 5-20% sucrose gradients. These characteristics suggest the presence of an estrogen receptor in this androgen target organ.

Testosterone binding to plasma proteins has been analyzed in the viviparous lizard by electrophoresis at steady state conditions and by equilibrium dialysis. Two binding systems are involved. The first system (S1) binds estradiol and testosterone, it is Sex Binding Protein like. The second one binds testosterone and dihydrotestosterone; the mains competitors are C21 steroids: progesterone and cortisone; estradiol doesn't perturb the equilibrium; this system is Corticosteroid Binding Globulin like. Androstenedione doesn't seem to be bound by these two systems. The high affinity (KA 4 degrees C = 1.28 X 10(8) M-1) and the high capacity (N = 1,18 X 10(-5) mole/litre) suggest that it is the second system that supports the main transport, buffer, reservoir role in the blood of viviparous lizard.

Study of the profile of androstenedione, testosterone and dihydrotestosterone elaboration was performed till 23 days of culture. Stimulation by hCG went through several phases which were expressed in terms of hormonal elaboration by successive steps of 1) progressive initiation 2) strong activity 3) fall 4) renewal of the secretion. The inital step corresponds to the restoration of cytoplasmic organels dealing with steroidogenesis. The fall could correspond to apparition of a transient refractoriness. This late event resembles desensitization which has been described in another models.

During the breeding season (April, May) the epididymis of the lizard Lacerta vivipara produces voluminous secretory granules which are abundantly discharged into the lumen of the duct where they mingle with spermatozoa. The mode of secretion appears quite unusual with respect to the method by which the cells discharge their products, the granules coming out of the cells like bullets out of a gun barrel. Spermatozoa come into close relationships with discharged granules, dipping into their outer layers. This is probably the way in which the heads of spermatozoa become covered with the epididymal soluble protein (protein L). This mode of secretion in Lacerta is discussed with regard to possible artifacts and compared with that encountered in the epididymis of some other species including mammals.

The lizard has a seasonal sexual cycle, during which the epididymis produces large amounts of proteins that mix with spermatozoa during the reproductive period. Through one-dimensional electrophoresis we identified among these proteins a band of major soluble Mr 19,000 proteins. In two-dimensional electrophoresis the Mr 19,000 protein molecules separated into four pHi groups ranging from 3.7 to 8.7. An immunoserum prepared against the most acidic fraction recognized all four protein groups. Immuno-electrophoresis confirmed that these proteins have similar immunological characteristics. The androgen dependence of each group was demonstrated in vitro with testosterone stimulation of tissue from castrated animals. The groups appear sequentially as a function of culture growth time.

Lizard epididymis produces large secretory granules (6 micrometer) which are discharged and mix with the spermatozoa. They consist of a dense central core and a peripheral vacuole. Central cores were prepared by two means: (1) homogenization of epididymal cells than isolation of a granular fraction by centrifugation on a discontinuous sucrose density gradient as a final step, and (2) collection of epididymal fluid containing both granules and spermatozoa, and separation of these elements by several steps of low speed centrifugations and washings. Purity of the different fractions was checked by microscopy. After complete dissolution in Triton X-100 (2.5%), the fractions containing central cores were submitted to SDS-polyacrylamide gel electrophoresis (15% acrylamide bisacrylamide). When apparently free from surrounding material, the dissolved central cores analyzed by electrophoresis showed only a main band representing a single protein (or a small group of proteins) of relative low mobility (molecular weight about 70 000). Other more mobile proteins have been identified in less purified fractions. They probably originate from the peripheral vacuole but this point is still under investigation. These two types of proteins do not originate from plasma or testis. Their androgen dependence is discussed.

Plasma testosterone binding activity was determined by an equilibrium dialysis method in the male of Lacerta vivipara during the annual cycle of activity (from March to September). Capacity showed significant variations during the sexual cycle although affinity remained constant. The variations of capacity were then compared with plasma testosterone levels measured by radioimmunoassay and plasma proteins content measured by the Lowry method. The three parameters showed parallel seasonal evolution except in May-June when binding activity increased although plasma testosterone and protein levels fell. The physiological meaning of this conspicuous phenomenon is discussed=an active mechanism of strengthening of the atrophy of accessory sexual organs is hypothesized. Furthermore, the increase of the binding activity which occurs in autumn and is parallel to the levels of testosterone must prevent these accessory organs from a full resumption of activity before the retreat since the lizard is a hibernating animal. These results emphasize the key role played by specific plasma binding proteins in the modulation of steroid hormone action.

The epididymis of lizards elaborates voluminous secretory granules made of a central core and a peripheral vacuole which in the species Lacerta vivipara contain respectively an insoluble protein (protein H) and a soluble protein (protein L). After their discharge these secretions mix with spermatozoa. In order to detect the presence of carbohydrates in these secretions, lectins isolated from Canavalia ensiformis (con A) and from eleven other plants (lentil, soja, pea, gorse and several mushrooms), conjugated to fluorescein isothiocyanate, have been utilized in light-microscopic histochemical investigations of frozen sections from Lacerta vivipara epididymis. Whereas lectins having affinity for L-fucose, lactose, D-galactose and N-acetyl-D-galactosamine bound to central cores, lectins having affinity to D-glucose, N-acetylglucosamine and chitobiose bound to the peripheral vacuole. D-mannose or D-glucose seem to be present both in central cores and in peripheral vacuoles.

From epididymal fluid samples taken at three different times during the reproductive period (early April, late April, mid-May), the soluble proteins were separated with one dimensional electrophoresis on polyacrylamide gel. Their evolution was studied: firstly quantitatively, after staining with Coomassie blue, or, for one protein (the "L" protein), by immunodetection; secondly, according to their glycosylation after transfer to nitrocellulose and treatment with a set of labelled lectins: from Wheat germ, Ricinus communis, Lens culinaris, Asparagus pea or Canavalia ensiformis, with or without use of their specific inhibitor sugars. At least 15 proteins underwent a quantitative and/or qualitative evolution, mainly during the month of April. Protein "L" (19 kDa), which is androgen dependent and which fixates on to spermatozoa during their epididymal transit, appears to be little or not glycosylated. By contrast its accumulation in the epididymal canal increases considerably during the month of April. Five other proteins proved to be especially interesting because of their evolution during this same period, notably the MW 94, 67, 35, 29 and 25.5 kDa proteins. With the exception of the 67 kDa all the others increased quantitatively. All were decisively enriched in mannose or in methyl-mannoside residues. The proteins of MW 29 and 25.5 kDa were also enriched in galactose or N-acetyl galactosamine residues. These findings are of physiological significance since they are set up concomitantly with the acquisition of maximum motility of spermatozoa in the distal segment of the epididymis, and they coincide with a very great increase in testosteronemia.