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.

Spermatogenesis of the lizard Lacerta vivipara: histological studies and amino acid sequence of a protamine lacertine 1.

The lizard Lacerta vivipara is a seasonal breeder with a well characterized reproductive cycle. An histological study of the lizard testis has been performed at different stages of spermatogenesis and the nuclear basic proteins content was assessed by electrophoretical analysis. Two protamines, lacertines 1 and 2, are present in spermatozoa in April and May. We have isolated lacertine1 and characterized a protamine with a mass of 4,963.7 Da. Amino acid sequence of this protamine (41 residues) was established from data provided by automated Edman degradation. It is characterized by a basic amino acid stretch in the N- and C-terminal regions and by a central part which only consists of 3 different intermingled amino acids. This protamine presents 62% homology with scylliorhinine Z3 from dog-fish Scylliorhinus caniculus and 58% homology with quail protamine. The reported lizard protamine sequence is the first reptilian protamine sequence available so far.

Identification of the most represented repeated motifs in Arabidopsis thaliana microsatellite loci.

The major simple sequence repeats present in the Arabidopsis genome were identified by Southern hybridizations with 49 oligonucleotide probes matching all the possible combinations of motifs up to 4 nucleotides long. The method used allowed us to perform all the hybridizations under the same temperature conditions. A good correlation was observed with the data obtained from database analysis, indicating that the method can be useful for identifying the major classes of microsatellite loci in species for which few or no sequence data are available. AG/CT, AAG/CTT, ATG/CAT and GTG/CAC are the major motifs present in the Arabidopsis genome that can be used as convenient probes to isolate microsatellite loci by screening libraries. AAG/CTT is the more frequent of these motifs, and its relative frequency in Arabidopsis is much higher than averagely found in the plant kingdom. About 8% of the cDNA clones from an immature silique library contains AG/CT, AAG/CTT or ATG/CAT microsatellite loci. Several microsatellite loci were isolated by screening genomic and cDNA libraries. Twenty-six tri-nucleotide loci were PCR amplified from four different ecotypes, and polymorphism was observed for 12 of them; 10 loci showing two alleles and 2 loci showing three alleles.

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.

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.

Molecular cloning and characterization of a cDNA encoding for the mature form of a specific androgen dependent epididymal protein.

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.

Structure and ultrastructure of the epididymis of the viviparous lizard during the annual hormonal cycle: Changes of the epithelium related to secretory activity.

This study deals with the structure and ultrastructure of the epithelial cells of the lizard (Lacerta vivipara Jacquin) epididymis as related to secretory activity. The epithelium contains only two types of cells, secretory cells and basal cells. The secretory cells undergo an annual cycle which has been divided into 10 stages. In its most active secretory state, epithelium forms 65.3% of the organ volume. The secretory cell is a tall columnar cell (from 55 ± 3.4 µm to 74.3 ± 2.4 µm height) with a basal nucleus and a supranuclear cytoplasm almost entirely occupied by numerous large secretory granules (5 to 7 µm in diameter). At the ultrastructural level, secretory cells contain rough endoplasmic reticulum (RER), Golgi complex, and secretory granules at various stages of synthesis before being discharged into the lumen. Each granule is membrane-limited and contains a spherical electron dense central core and a peripheral vacuole which varies in density. The secretory cell originates from small cubic cells (13.8 ± 0.7 µm) with few organelles (stage 1). The height of the cell increases gradually and free ribosomes appear first (stage 2), followed by scarce elements of RER (stage 3). The step preceding the secretion period (stage 4) is characterized by a conspicuous increase in volume of RER and Golgi complex. From stage 7 to stage 10, the cell undergoes a dramatic involution. After a transient hypertrophy of the RER, numerous autophagic vacuoles invade the cytoplasm. This degeneration can lead to a complete lysis of the cell and to its rebuilding after elimination of the greatest part of the cytoplasm. The volume of the epithelium falls to 15.6% of the total volume. With antibodies raised against the protein family which constitutes the main part of the secretion (L proteins of 19 kDa), it is shown by immunohistochemistry that these proteins are concentrated into secretory granules which are discharged into the lumen to finally bind to the heads of the spermatozoa.

Immunochemical localization and association with spermatozoa of androgen-regulated proteins of MR 24000 secreted by the mouse epididymis.

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.

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.

Changes in epididymal protein synthesis during the sexual cycle of the lizard, Lacerta vivipara.

During its annual cycle, the lizard epididymis undergoes strong modifications of the secretory epithelium. These modifications previously were classified into 10 stages. The present study gives the biochemical basis of these modifications. Several parameters, such as the quantity of soluble proteins, rates of protein synthesis, and electrophoretic profiles of newly synthesized proteins and of in vitro RNA translation products were compared at 8 stages. Two-dimensional gel electrophoresis of newly synthesized tissue proteins showed that the synthesis of about 20 proteins fluctuated during the cycle. Furthermore, it revealed that the protein band L of molecular weight 19,000 identified in one-dimensional (1-D) electrophoresis was composed of at least 10 proteins. Their rate of synthesis paralleled the concentrations of their mRNA evaluated with in vitro translation. This could indicate that in this system protein synthesis is regulated by mRNA concentrations. The present analysis has confirmed that 4 different phases characterize the annual evolution of the lizard epididymis: regeneration, onset of secretory activity, hypersecretion and involution. Well-defined, newly synthesized proteins would characterize some of these phases, and could be used as markers for future detailed analysis of epididymis control.

Production and glycosylation of sperm constitutive proteins in the lizard Lacerta vivipara. Evolution during the reproductive period.

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.

Evolution and testosterone content of the epididymis during the annual cycle of the lizard Lacerta vivipara.

The lizard epididymis provides a model for studying the control, by testosterone, of a secretory activity related to the physiology of spermatozoa. To evaluate seasonal changes and to establish chronological correlations between the structure of the epididymis and its testosterone content, lizards (Lacerta vivipara) were killed between March (emergence) and October (retreat). The epididymal tissue was examined histologically and assayed for testosterone content. Ten stages of development were defined, mainly on the basis of the epithelial structure and the morphological features of secretory activity. Degeneration of the epithelium after the breeding period and its subsequent renewal also were considered. Increased epithelial height and secretory activity coincided with a progressive rise of the testosterone level, and a severe atrophy followed a sudden reduction of blood testosterone. Reorganization of the epithelium takes place when testosterone is at its lowest level, and the hormonal dependency of this stage is questionable. This study confirms in vivo, during a sexual cycle, experimental evidence previously obtained concerning testosterone's control of the secretory activity of the lizard epididymis.

Acquisition of sperm motility and its maintenance during storage in the lizard, Lacerta vivipara.

Lizard spermatozoa, which are non-motile in the testis, develop the ability to swim as they pass along the excurrent duct. The addition of caffeine, a phosphodiesterase inhibitor, induced forward motility in spermatozoa from the caput epididymidis and increased the velocity of spermatozoa from the distal part of the epididymis. Caffeine had no effect on the motility of testicular spermatozoa. This suggests that sperm motility in this species is cyclic AMP-dependent but this factor alone is not sufficient to induce testicular sperm motility. In samples from the distal region of the epididymis, sperm motility was maximal in April just after the breeding season and then decreased significantly during the following months. A parallel can be drawn between these data and the levels of testosterone in the plasma. In the lizard, as in mammals, the epididymis may play an important role in the maturation of spermatozoa.

Histochemical study of epididymal secretions in the lizard, Lacerta vivipara. Localization of lectin-binding sites.

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.

Scanning electron-microscopical study of epididymal secretions in the lizard Lacerta vivipara (Reptilia, Squamata) and of their relationships with spermatozoa.

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.

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.

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.