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.

Selenium-independent epididymis-restricted glutathione peroxidase 5 protein (GPX5) can back up failing Se-dependent GPXs in mice subjected to selenium deficiency.

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.

GPx3: the plasma-type glutathione peroxidase is expressed under androgenic control in the mouse epididymis and vas deferens.

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.

The PEA3 protein of the Ets oncogene family is a putative transcriptional modulator of the mouse epididymis-specific glutathione peroxidase gene gpx5.

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.

Characterization, regulation of the expression and putative roles of two glutathione peroxidase proteins found in the mouse epididymis.

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.

Tissue and developmental distribution, dependence upon testicular factors and attachment to spermatozoa of GPX5, a murine epididymis-specific glutathione peroxidase.

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.

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.

Structural organization, chromosomal localization, expression and phylogenetic evaluation of mouse glutathione peroxidase encoding genes.

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.

In vitro expression of a mouse tissue specific glutathione-peroxidase-like protein lacking the selenocysteine can protect stably transfected mammalian cells against oxidative damage.

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.

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.

LESP, an androgen-regulated lizard epididymal secretory protein family identified as a new member of the lipocalin superfamily.

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.

Developmental expression and androgen regulation of 24 kDa secretory proteins by the murine epididymis.

Two peptides with a molecular weight of 24 kDa and a P(i) of 8.4-8.8 were found to be synthesized and secreted specifically by the caput epididymis of adult male mice under androgen control. The peptides can interact with spermatozoa. In the present study, the developmental pattern of [35S]-methionine-labelled proteins synthesized by the murine caput epididymis at 10, 20, 30 and 40 days of age were studied using two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and autoradiography. Active synthesis of the 24 kDa proteins was detected in the epididymis from 20 days of age, but secretion of the two peptides was only observed from 30 days of age onwards. To determine whether androgens influenced the active expression of 24 kDa proteins in the developing epididymis, their effect on [35S]-methionine incorporation into proteins was assessed using 2D PAGE. Mice were either castrated, castrated then testosterone injected or simply testosterone injected at 10, 20, 30 or 40 days of age. Androgen control of 24 kDa protein expression was also studies in vitro in epididymal organ culture over a 10-day period, with or without testosterone. Androgens were not involved in the initiation of synthesis of the 24 kDa proteins from days 10 to 20, as shown by in-vivo and in-vivo experiments. However, androgens appeared to be essential for maintaining synthesis and secretion of the proteins from 20 days of age onwards. Administration of excessive testosterone was only able to increase secretion of the 24 kDa proteins in intact male mice aged 40 days.

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.

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 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.

Sequence homology of androgen-regulated epididymal proteins with glutathione peroxidase in mice.

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.

Synthesis and post-translational modifications of an epididymal androgen dependent protein family.

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.

[Androgen-dependent protein secreted by mouse caput epididymis shows high homologies with different glutathione peroxidases]. / Une protéine androgéno-dépendante sécrétée par la tête de l'épididyme de souris présente des homologies élevées avec différentes glutathion peroxydases.

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.

Specific distribution of messenger ribonucleic acids for 24-kilodalton proteins in the mouse epididymis as revealed by in situ hybridization: developmental expression and regulation in the adult.

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.