Major proteins secreted by the epididymis of Lacerta vivipara. Isolation and characterization by electrophoresis of the central core.

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.

Major proteins secreted by the epididymis of Lacerta vivipara. Identification by electrophoresis of soluble proteins.

During their period of activity, epithelial cells of the lizard epididymis produce secretory granules containing highly insoluble central cores of protein nature (protein H). After centrifugation of the epididymal fluid at 15 000 X g, major soluble proteins were separated in the supernatant by polyacrylamide gel electrophoresis. These proteins were labelled by repeated injections of [3H]leucine into animals. In cylindrical gel electrophoreses, labelled proteins migrated as a single band towards the anode in the presence of SDS, and as two separate bands without SDS. The fastest component obtained in non-denaturing conditions was designated protein L. In two-dimensional gel electrophoreses, the two bands separated in the first dimension both migrated to the same position in the second dimension with SDS. Consequently it may be assumed that protein L is a monomer (molecular weight about 16 000-20 000) able to aggregate into polymers which can be dissociated with SDS. It was proved by hemicastration experiments that these soluble proteins did not originate from the testis. In addition, they were detected after short incubation of epididymal tissues in the presence of [3H]leucine. It is concluded that these proteins are elaborated by epithelial cells of the epididymis and discharged into the lumen. A possible role in the physiology of spermatozoa is briefly discussed.

Identification of an epididymal immunorelated protein family: sequential appearance under testosterone stimulation.

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.

Structural organization and regulation of the gene for the androgen-dependent glutathione peroxidase-like protein specific to the mouse epididymis.

Genomic clones containing the gene for the glutathione peroxidase-like androgen-regulated murine epididymal protein of 24 kilodaltons (arMEP24) were isolated. A 9-kilobase DNA fragment was sequenced and found to contain the entire coding region of the gene, which is divided into five exons. The exact sizes and boundaries of the exon blocks were deduced by comparison with the cDNA sequence. One major and four weak transcription initiation sites in the epididymis were localized by primer extension. The promoter of the gene does not contain a conventional TATA box immediately up-stream of the start site; rather, the sequence TATCA occurs at residue -35. Two CAAT boxes in opposite orientation and two putative binding sites for the transcription factor Sp1 were identified up-stream of the TATA-like box. To localize the cis-acting sequences responsible for androgen regulation of expression, fragments of the arMEP24 gene promoter region were cloned in front of the luciferase (LUC) reporter gene and cotransfected with an androgen receptor expression vector into CV-1 cells in a transient assay. LUC activities of CV-1 cells grown in the presence of various concentrations of 5 alpha-dihydrotestosterone were compared to LUC activities of untreated controls. The DNA fragment containing up to 200 nucleotides up-stream from the major transcription start site was sufficient for the full promoter activity, but not for the responsiveness to androgen induction. Depending on the 5 alpha-dihydrotestosterone concentration, a 2- to 4-fold induction of LUC activity was found if a -1797 to -167 arMEP24 gene fragment was used linked to the reporter gene driven by either the homologous promoter or the heterologous thymidine kinase promoter. Two or three copies of the imperfect palindromic sequence TGTTGAgagAGAACA, found at position -896 to -882 in the gene and resembling the consensus steroid hormone-responsive element, are able to confer androgen regulation to the thymidine kinase promoter independently of their orientation. These findings support evidence that transcriptional regulation of the arMEP24 gene occurs via the sequence TGTTGAgagAGAACA. Homologies found in the sequence up-stream of the promoter with several putative binding sites for erythroid-specific trans-acting regulatory proteins are discussed. Finally, the arMEP24 gene is located by in situ hybridization in the [A2-A4] region of mouse chromosome 13.

Cloning of the mouse gene encoding plasma glutathione peroxidase: organization, sequence and chromosomal localization.

Using a reverse transcription coupled to PCR amplification strategy, with degenerated primers localized in highly conserved domains of known glutathione peroxidase (GPX) proteins, we have generated, from mouse epididymal RNA, a cDNA fragment which was subsequently used to isolate a genomic clone encoding mouse plasma GPX (GPX3). GPX3 is a major enzyme in reducing lipid hydroperoxides and hydrogen peroxide in plasma. We confirm here that the mouse epididymis is a new site of expression of GPX3 and report, together with the sequence, the structural analysis and the chromosomal localization of the mouse GPX3 single-copy gene to chromosome 11.

The lipocalin sperm coating lizard epididymal secretory protein family: mRNA structural analysis and sequential expression during the annual cycle of the lizard, Lacerta vivipara.

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.

Molecular cloning of a cDNA for androgen-regulated proteins secreted by the mouse epididymis.

A cDNA clone encoding androgen-dependent proteins secreted by the mouse epididymis was isolated by screening a cDNA library using differential hybridization, according to the selective expression of the mRNA in the normal but not in the castrated mouse. Translation of mRNA hybrid-selected by this 1.4 kb clone (M53) yielded proteins of Mr 26,000 which were processed in vitro in the presence of microsomal membranes into proteins of Mr 24,000. Northern blot analysis of epididymal total RNA revealed at least two populations of mRNA (1.4 and 1.8 kb) homologous to the M53 clone, which were restricted to the caput epididymidis. Studies in vivo demonstrated that testosterone regulates the concentration of these mRNA populations. Analysis of epididymal total RNA from ten individual animals provided no evidence that the M53 mRNA populations are the products of allelic variants of a gene. Southern analysis of mouse genomic DNA revealed single bands with most of the tested restriction enzymes. Furthermore, cross-hybridization to the M53 cDNA revealed homologous mRNA species in rat, human, rabbit, ram and boar epididymal RNA.

Histoautoradiographic study of rRNA and mRNA transcription activity related to secretory function in lizard epididymis.

The lizard epididymis is an androgen-dependent organ whose epithelial cells undergo marked changes in structure and secretory activity during the annual cycle. These changes are connected to fluctuations of testosterone levels. During the breeding season, the epididymis produces a major protein secretion, the L-proteins. In the present work we studied the fluctuations of RNA synthesis and accumulation during the annual cycle by means of histoautoradiographic methods. Total RNA synthesis was determined by uridine incorporation; accumulation of rRNAs and L-protein mRNAs were determined by in situ hybridization. Total RNA synthesis began during reorganization (Phase I), then increased gradually during differentiation and growth (Phase II). The synthesis peaked during maturation (Phase III), but stopped abruptly during hypersecretory activity (Phase IV). The rRNAs were very abundant from Phase II to Phase IV, which is related to the presence of many ribosomes as revealed by electron microscopy. The mRNAs of L-proteins were detected only during Phases III and IV in all epithelial cells. For every phase of the sexual cycle there exists a strong correspondence between the changes in transcriptional activity (rRNA and specific mRNA) of the epithelial cells and changes in the testosterone levels.

Characterization and hormonal regulation of 24 kDa protein synthesis by the adult murine epididymis.

The pattern of labelled proteins synthesized and secreted in vitro by the adult mouse epididymis was studied by one- and two-dimensional polyacrylamide gel electrophoresis. The presence of a major 24 kDa protein synthesized and secreted in a tissue-specific manner by the caput epididymidis was detected. For this molecular weight, two-dimensional analysis indicated several proteins including two polypeptides (pI 8.4 and 8.8) whose expression is under androgenic control. Partial amino acid sequence analysis showed a complete N-terminal identity between these two peptides. A polyclonal monospecific immune serum was raised against the two proteins. Only purified immunoglobulins precipitated them, showing that immunological affinity is restricted to these two proteins in the epididymis. Indirect immunofluorescence assay revealed specific binding of antibodies on the acrosomal region of spermatozoa isolated from the caput, corpus or cauda epididymides. Testicular spermatozoa were not labelled under the same conditions. To investigate the physiological role of androgens in the synthesis and secretion of the 24 kDa proteins, tissue slices of epididymides from adult mice which had been castrated, or castrated then injected with testosterone were incubated with [35S]methionine. Castration and testosterone replacement kinetics revealed that alterations in 24 kDa protein synthesis follow immediately upon androgenic privation and replacement.

Molecular cloning of two cDNAs for related secretory proteins in lizard epididymis: gene expression during androgen-induced cell growth and secretion.

Lizard epididymis is an androgen-dependent tissue which produces notably ten related secretory proteins (L-proteins, Mr 19,000) during the reproductive period. These proteins were synthesized in vitro as preproteins (Mr 25,000, 24,000, 23,500). A cDNA library in the plasmid pBr322 was constructed and two cDNA clones were isolated by differential hybridization according to the differential expression of the mRNAs in stages 1 and 6 of the annual reproductive cycle. Translations of mRNAs hybrid-selected by two clones (LV123, LV132) yielded proteins which were immunoprecipitated by the L-antiserum. These preproteins were processed in vitro into six peptides; four were encoded by mRNAs selected with the LV123 clone, the others by the LV132 clone. Only three bands were detected using Northern blot analysis suggesting that the L-family could be derived from various mRNAs and from post-translational maturations. Southern analysis of genomic DNA suggests that the L-mRNAs were encoded by at least two distinct genes which could exist in numerous copies. The L-gene expression was studied under various physiological conditions and was found to be androgen-dependent. Furthermore, the results suggest the presence of a translational regulation in the newly differentiated epithelial cells.

Regulation of the epididymal glutathione peroxidase-like protein in the mouse: dependence upon androgens and testicular factors.

The protein MEP24 was previously described as a glutathione peroxidase-like molecule specifically secreted by the mouse caput epididymidis. Recently, its binding to the head of spermatozoa was demonstrated. Here, the regulation of MEP24 expression was studied by analyzing transcriptional and translational activities in the epididymis (1) of adult mice castrated on day 60 and given various substitutive testosterone (T) treatments from day 90 and (2) of hemicastrated adult animals. In castrated mice, T treatment induced a significant rise in plasma T and 5 alpha-dihydrotestosterone (DHT) concentrations that greatly exceeded the control values. Owing to efficient regulation, however, the epididymal T and DHT levels were never higher than those of the controls. The restoration of MEP24 mRNA accumulation was complete when the epididymal DHT content returned to its normal value. However, when estimated in a cell-free system, the in vitro translatable MEP24 mRNA level never exceeded 70% of control values, even though the DHT and accumulated mRNAs were restored by 100% or more. In hemicastrates, the T content was normal on the castrated side, while the DHT content exhibited a significant decrease (47%). In this case, the MEP24 mRNA accumulation reached 88% of the normal value, but the translation rate, both in vitro and in vivo, was only about 50%. Ultrastructural studies showed that the normal rough endoplasmic reticulum organization in segment I cells is dependent upon the presence of testicular fluid in the epididymal duct lumen. Thus, this report shows that the MEP24 mRNA steady-state level is completely recovered in the presence of a normal epididymal DHT content, while restoration of the regulation of translation is just partial. This could be related to the cell organization but seems mainly dependent upon the presence of specific mRNA-associated factors which are probably under the control of androgens and/or molecules carried by the testicular fluid.

Characterization of an androgen response element within the promoter of the epididymis-specific murine glutathione peroxidase 5 gene.

We have shown in earlier studies, using a mouse model, that the expression of the glutathione peroxidase 5 protein (GPX5) is restricted to the epididymis and that the accumulation of its corresponding mRNA is hormonally, spatially and temporally regulated throughout postnatal development. We report here, using run-on assays, transient expression experiments as well as gel-shift and footprinting analyses on the findings that at least part of the androgenic control of the GPX5 expression is exerted at the transcriptional level via an androgen response element localized in the distal promoter region of the GPX5 gene. The gpx5 androgen response element (ARE) is found to be consistent with the consensus palindromic steroid-receptor target sequence 5'-AGWACWnnnTGTYCT-3' but exhibits a quite weak conservation in the left half site. The data presented here further expand the diversity of sequence able to confer androgen responsiveness.

[Characteristics of testosterone binding to plasma proteins during the sexual cycle in a seasonally-reproductive animal, the male viviparous lizard]. / Particularité de la liaison de la testostérone aux protéines plasmatiques au cours du cycle sexuel chez un animal à reproduction saisonnière, le lézard vivipare mâle.

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.

[Incorporation of radioactive leucine into lizard epididymis proteins. Study during the sexual cycle and after injection of testosterone to untreated or castrated animals]. / Mise en évidence par incorporation de leucine radioactive d'une élaboration protéique dans l'épididyme de Lézard. Etude au cours due cycle sexuel et après injection de testostérone chez l'animal entier et castré

Lizard epididymis activity during the reproductive cycle and after testosterone administration results in a marked increase of radioactive leucine incorporation in vivo and in vitro. The electrophoretic distribution of soluble proteins shows different peaks of radioactivity. Comparison between histological response and pattern of electrophoresis suggests that one of these peaks may correspond to an activity of secretion.

[Ultrastructural study of the pseudopregnant rat luteal cell following the effect of luteinizing hormone and of 2 inhibitors of synthesis (amino-glutethimide and cycloheximide)]. / Etude ultrastructurale de la cellule lutéale de la ratte pseudogestante après action de l'hormone lutéinisante et de deux inhibiteurs de synthèse (aminoglutéthimide et cycloheximide

Incubation of ovaries from pseudopregnant rats have been performed with the gonadotropin LH and with two inhibitors, aminoglutéthimide and cycloheximide for periods comprised between 1 and 6 hours. Then the luteal cells have been studied with electron microscope. Measurements in have been used to express the variations in volume of two organels, mitochondria and lipid droplets. The principal modifications induced by these compounds bear on the mitochondria. Treatment with LH is followed by an increase in the volume of individual mitochondria resulting probably of fusion of organels. Treatments with the inhibitors are followed by reduction in the volume of individual mitochondria and in the total volume of mitochondrial apparatus. Mixed treatments using LH and an inhibitor allow to obtain organels of intermediate size. Volume of lipids is increased by incubation with LH and probably decreased by the inhibitors. Smooth endoplasmic reticulum shows rearrangements with the various treatments. It seems not convenient to extend incubations over 4 hours in order to avoid cytolysis.

[Histoautoradiographic study of two target organs (epididymis and femoral glands) after injection of 3H testosterone in a non-mammalian vertebrae, the viviparous lizard]. / Etude histoautoradiographique de deux organescibles (épididyme et glandes fémorales) après injection de testostérone 3H chez un vertébré non mammalien, le Lézard vivipare.

After 3H testosterone injection into castrated males of the Lizard Lacerta vivipara, the radioactive compound is detected by radioautography of epididymis, femoral glands, gut and liver. Between 1 hr. 30 min. to 12 hrs. of retention the 3H material concentrates progressively into nuclei of the glandular cells of epididymis and femoral organs although no particular concentration occurs in gut and liver cells. This submammalian model is consistent with those previously described for mammals.

[Histo-autoradiographic study of a testosterone target organ, the epididymis of the lizard (Lacerta vivipara), after administration of 3H-17 beta-estradiol]. / Etude histo-autoradiographique d'un organe-cible de la testostérone, l'épididyme de Lézard (Lacerta vivipara), après administration de 17 beta-oestradiol 3H.

[6,7 3H] oestradiol-17 beta was injected into castrated male viviparous lizards in order to compare retention of this steroid to that of testosterone during the period of sexual activity. Retention of the isotope in epididymis was greater than in blood, lung and stomach. Radioautographs of epididymis indicated that oestradiol or a metabolite was concentrated in cell nuclei and over discharged secretory granules as was observed with testosterone. Reason of such binding is not yet known.

[Binding of testosterone to plasma proteins in the viviparous male lizard]. / Liaison de la testostérone aux protéines plasmatiques chez le lézard vivipare mâle.

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.