Polyol pathway along the bovine epididymis

During the epididymal transit, male gametes acquire new surface proteins necessary for their fertilizing ability. We have previously shown that membranous vesicles, called epididymosomes, interact with sperm surface within the epididymal fluid allowing transfer of some proteins to different subcellular compartments of spermatozoa. We previously showed that one of the major proteins associated with epididymosomes was an aldose reductase (gene: AKR1B5) and confirmed that aldose reductase is located in the epithelial cells bordering the intraluminal compartment of the epididymis. The present study shows that cytosolic aldose reductase activity was maximal in the proximal and middle segments of the epididymis and decreased in the distal epididymis. Western and Northern blot analysis confirmed the distribution pattern of aldose reductase and of the encoding mRNA. The optimal pH of epididymal aldose reductase was 6.0-6.5 when glucose was used as a substrate; this corresponds to the pH of the intraluminal epididymal fluid. In order to evaluate the possible involvement of sorbitol in sperm physiology, Western blot of tissue homogenates were probed with an anti-sorbitol dehydrogenase antibody. The amount of enzyme immunodetected was higher in the proximal and distal segments of the epididymis when compared to the amount detectable in the middle segment of the epididymis. Sorbitol dehydrogenase activity as well as the level of the encoding mRNA showed the same pattern of distribution. Furthermore, immunohistological studies using the anti-sorbitol dehydrogenase revealed that this enzyme was synthesized by the epididymal epithelial cells bordering the intraluminal compartment. Knowing the importance of sorbitol and fructose in sperm metabolism, we hypothesized that the polyol pathway is involved in the modulation of sperm motility within the epididymis.     

Seasonal changes in epididymis and the effects of FSH and testosterone in the spiny-tailed lizard,Uromastix hardwicki

Seasonal changes in epididymal weight and histology were studied in relation to testicular function in the adult spiny-tailed lizard, Uromastix hardwicki, over a period of 1 year. The eipdidymal weights, tubular diameter, and epithelial height increased in March, reaching a peak in April. This peak coincided with sperm maturation, elevated plasma testosterone levels, and release of sperm into the epididymis. The epididymal weights decreased in May following a sudden regression of the testis early in the month. The epididymal weights decreased further during June and remained low until February. The diameter of the duct and the height of the epithelial cells also decreased in May and the epididymal epithelium maintained a low histological profile from June to February. The fall testicular recrudescence was not accompanied by a change either in the weight or the histological structure of the epididymis. Administration of oFSH (0.1 mg) daily for 7 days during the sexually quiescent period induced a significant increase in the weight of the epididymis and epithelial height of the duct. Administration of testosterone alone, (2.0 mg) daily for the same period and under identical conditions, did not induce a change in the weight of epididymis or its histology. A possible permissive role of gonadotrophin in the hormonal regulation of the lizard epididymis has been suggested.     

Rabbit epididymal secretory proteins. I. Characterization and hormonal regulation

Analyses of samples of luminal fluid from the rete testis, distal efferent ducts, and epididymal regions 2-5 and 8 revealed that 91% of the fluid leaving the testis is reabsorbed by the efferent ducts, 79% of the remainder is reabsorbed proximal to epididymal regions 4 and 5, and there is a net secretion of fluid into the duct caudally. There is a net reabsorption by the efferent ducts of 73% of the protein leaving the testis and then a net secretion along the epididymis. SDS-PAGE of the luminal fluids indicated that four new protein bands that were not present in blood appeared in the efferent ducts, 5 in epididymal regions 1-5, 6 in regions 6 and 7, and one in region 8. Two bands in samples from the efferent ducts were absent caudally, and one band present in region 7 was absent in region 8. The rates of incorporation of (35)S-methionine into minced duct in vitro varied among regions when expressed per milligram of wet weight of tissue (region 2-5 > region 7 > region 6 > region 1 > region 8 > ductuli efferentes), and orchidectomy had little effect on the rates. Incorporation into four proteins that were secreted in vitro (M(r) 38 000, 20 000, 15 000, and 13 000) was reduced or abolished by orchidectomy and restored by testosterone therapy. The secretion of three proteins (M(r) 52 000, 23 000, and 22 000) was reduced or abolished by orchidectomy and not restored by testosterone therapy. SDS-PAGE of detergent extracts of sperm indicated that five proteins were lost and nine were gained during epididymal transit. Seven of the proteins gained were about the same molecular weight as proteins secreted by the epididymis (M(r) 94 000, 52 000, 38 000, 36 000, 22 000, 20 000, and 13 000) and were analyzed using N-terminal amino acid microsequencing.     

Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies

The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. Immunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bands of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.     

Biochemical characterization of two ram cauda epididymal maturation-dependent sperm glycoproteins

Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.     

Sperm maturation in the domestic cat

The epididymis is essential for sperm development and maturation, and, subsequently, the ability of spermatozoa to penetrate and fertilize the female gamete. Functional differences in segments of the long tubule are reflected by histological differences among epididymal regions. The feline epididymis can be divided into six different regions according to their histological differences. A marked increase in sperm concentration occurs between regions 2 and 3, indicating resorption of fluid in region 2, a concept supported by the histological characteristics of the epithelium. At the transition between regions 4 and 5, located between the caput and corpus epididymides, histological characteristics change from being that of a maturation function to being typical of a storage function. Migration of the cytoplasmic droplet and induction of motility occur in this same region. Proteins are secreted from epithelial cells in the feline epididymis by merocrine and apocrine secretion, although the functions of different feline epididymal proteins have not been determined. Hypotaurine, taurine and, probably, alkaline phosphatase are produced by the feline epididymis. During epididymal transit the percentage of immature, unviable and morphologically abnormal spermatozoa decreases, indicating the existence of a mechanism that removes abnormal spermatozoa. In contrast, the percentage of spermatozoa with abnormal tails increases slightly during epididymal transit. Most of the distal droplets present on spermatozoa in the cauda epididymis are lost at or after ejaculation. Additional knowledge of the feline epididymis should be beneficial for developing sperm preservation protocols and advance the prospects for effective male contraceptive methods.     

Localization of a maturation-dependent epididymal sperm surface antigen recognized by a monoclonal antibody raised against a 135-kilodalton protein in porcine epididymal fluid.

A specific 135-kDa protein was purified from porcine cauda epididymal fluid. Analysis of its N-terminal amino acid sequence revealed it to be a new protein. Stable clones of hybridomas that produced monoclonal antibodies against the purified 135-kDa protein were established. A clone, B-11, reacting both with epididymal fluid and with sperm plasma membranes was selected and used in this study. Immunoblotting analysis showed that B-11 reacted only with a 135-kDa protein among epididymal fluid proteins. In contrast, B-11 did not recognize a similar 135-kDa sperm protein but did strongly react with a 27-kDa protein among sperm membrane proteins, extracted by NP-40 in the presence of protease inhibitors. B-11 also reacted only with a 27-kDa protein fragment among trypsin digests of the 135-kDa epididymal protein. The 135-kDa protein was first detected, by ELISA or immunoblotting analysis, at the beginning of the corpus epididymis. Maximal levels were reached in the distal corpus and levels were slightly decreased in the cauda epididymis. On the other hand, the surface of caput sperm were found to contain small amounts of antigen(s), the concentration of which gradually increased during epididymal transit. In immunocytochemical studies, the antigen was detectable in the epithelial cells from the initial segment to the corpus of the epididymis but not in the caudal cells. In the lumen, the presence of the 135 kDa protein was apparent in the corpus (at a maximum in the middle and distal corpus) and to a lesser degree in the caudal lumen. The 27-kDa protein was distributed all over the equatorial region of the acrosome of less than 10% of caput epididymal sperm. As sperm passed through the corpus epididymis, the percentage of immunoreactive cells increased and the protein was restricted to specific domains of the sperm head. Thus, on the mature sperm, antigen was localized in a crescent-shaped area of the equatorial segment just behind the anterior part of the acrosome and on the apical rim of the sperm head. This is the first observation of a sperm surface antigen derived from an epididymal protein as a proteolytic fragment that interacts with specific regions of the sperm membrane during the process of spermatozoa maturation.     

Germinal angiotensin I-converting enzyme is totally shed from the rodent sperm membrane during epididymal maturation

Acquisition of sperm fertilizing ability is due, in part, to the reorganization of plasma membrane proteins that occurs during epididymal sperm transit. Using polyclonal antibodies against angiotensin I-converting enzyme (ACE), we showed that this enzyme is immunolocalized mainly on the middle piece of rat and mouse testicular sperm and with less intensity along the initial part of the principal piece of the flagellum. In both species, only some sperm from the caput epididymis were still reactive, whereas no labeling was observed on cauda epididymal sperm. The 105- to 110-kDa germinal ACE was absent from the rat testicular fluid but appeared in the fluid of the anterior epididymis. Thereafter, its molecular weight shifted to 94 kDa in the corpus epididymal fluid and remained at this weight in the caudal region. The 105- to 110-kDa immunoreactive protein was present in testicular rat sperm extract but was completely absent from epididymal sperm extracts. Western blot analysis of testicular and epididymal tissue extracts from the rat and mouse also confirmed that the germinal enzyme was absent from the epididymal sperm cell. Our results demonstrated that the rodent germinal ACE is released from the testicular sperm membrane when sperm enter the epididymis, a process similar to that observed in domestic mammals. This result is discussed in view of the suggested role for this enzyme in sperm fertility.     

Significance of regional difference in ion concentrations in lizard, Hemidactylus flaviviridis (Rüppell): assessment of ionic influence on sperm motility in vitro

A gradual increase in the concentration of Ca2+ from anterior to the posterior region was observed when mono- and divalent cations were estimated in different segments of the epididymis in wall lizard. Na+ and K+ levels increased from anterior to middle segment but declined significantly in the posterior segment. However, no significant difference in the levels of Mg2+ was observed in various segments. To study the influence of mono- and divalent cations on sperm motility in vitro, the spermatozoa from posterior region of the epididymis were incubated in medium with varying concentrations of Na+, K+, Ca2+ and Mg2+. Spermatozoa were non-motile when suspended in Na+-free medium. Addition of NaCl induced the acquisition of sperm motility in a concentration-dependent manner. Further, amiloride, a Na+-influx blocker, markedly reduced the Na+-induced forward progressive motility. Unlike Na+, the presence of K+ or Ca2+ in the incubation medium reduced the motility of spermatozoa even at very low concentrations. The inhibitory effect of Ca2+ decreased when nifedipine, a Ca2+-influx blocker, was added to the medium. Mg2+ at high concentrations only was able to reduce the forward progressive motility.     

An epididymal form of cauxin, a carboxylesterase-like enzyme, is present and active in mammalian male reproductive fluids

Mass spectrometric analysis of a prion protein (PrP)-containing complex isolated from ram cauda epididymal fluid revealed a protein that showed homology to a carboxylesterase-like protein previously identified in cat urine (cauxin). Using anti-cauxin antibodies, immunoreactive bands were detected in corpus and cauda epididymal fluid from all mammals tested (ram, boar, mouse, and cat). In the ram, the protein was also present in seminal fluid but not found to be associated with sperm. The bands reacting with the anti-cauxin antibody coincided with those having esterase activity in a zymographic assay and its levels paralleled the esterase activity of native epididymal fluids. A partial nucleotide sequence of 1143 bp, corresponding to 380 amino acids, was obtained by RT-PCR amplification from total RNA from the corpus epididymis (zone 6). The deduced protein sequence shows a high degree of homology (up to 90%) with the different cauxin proteins found in databases but only up to 60% with other known carboxylesterases. By PCR, strong mRNA expression was found in the corpus and cauda epididymis, while the testis, kidney, and caput epididymis had low expression. No mRNA was detected in the lung, heart, or liver. These data demonstrate that an epididymal form of the cauxin enzyme is secreted into mammalian epididymal fluid. In the ram, it is associated with a high molecular-weight PrP-associated complex and may be responsible for the majority of the esterase activity in the cauda epididymal fluid of this species.     

Two-dimensional electrophoresis of proteins in principal cells, spermatozoa, and fluid associated with the rat epididymis

Spermatozoa, fluids, and principal cells from different regions of the epididymis were characterized by two-dimensional electrophoresis. Rete testis fluid was collected after 36-h efferent duct ligation, and cauda epididymal fluid was collected by retrograde perfusion through the vas deferens. Spermatozoa were collected after their exudation from minced caput and corpus epididymal tissue. Principal cells were recovered after enzymatic disaggregation and centrifugal elutriation of epididymides. Two-dimensional polyacrylamide gel electrophoresis was used to prepare protein profiles of all samples. Comparison of the proteins found in rete testis fluid versus those found in cauda epididymal fluid revealed a dramatic change in composition, including the loss, addition, or retention of specific proteins as well as changes in the relative concentrations of certain proteins. Prominent cauda epididymal fluid proteins, possibly contributed by the epididymal epithelium, were detected at 16, 23, and 34 kDa. After epididymal transit, a considerable decrease was observed in the number of aqueous-soluble sperm proteins. Differences in the protein composition of epididymal epithelial principal cells from the caput versus corpus epididymidis were also noted, suggesting that functional differences exist for these epididymal regions. Of particular interest was the occurrence of a prominent protein of approximately 20-23 kDa found in all sperm samples, in fluids, and in caput and corpus principal cells. However, this protein was absent in cauda epididymal sperm after 36-h efferent duct ligation. The rapid loss of this protein from sperm after efferent duct ligation suggests that this surgical intervention may affect spermatozoa residing within the epididymis.     

Unique regional distribution of delta 4-3-ketosteroid-5 alpha-oxidoreductase and associated epididymal morphology in the marsupial, Didelphis virginiana

The epididymal epithelial ultrastructure has been described in the adult male North American opossum, Didelphis virginiana. Morphological results have suggested that absorptive activity is prominent in the proximal epididymal region by virtue of numerous microvilli, an endocytotic complex, dense granules, and multivesicular bodies in the apical cytoplasm. In contrast, the middle and distal epididymal regions exhibit ultrastructural features indicative of protein synthesis such as large invaginated euchromatic nuclei, large nucleoli, and increased amounts of granular endoplasmic reticulum. It is in the middle and distal epididymal regions where sperm head rotation and sperm pairing take place. Epididymal delta 4-3-ketosteroid-5 alpha-oxidoreductase (5 alpha-reductase) activity also has been measured. It has been found that the level of enzyme activity differs significantly (p less than 0.01) between the proximal, middle, and distal epididymal regions. Enzyme-specific activity has been found to be highest in the middle region (47.6 +/- 5.4 picomoles 5 alpha-reduced androgens formed/b/mg protein), lower in the distal region (18.3 +/- 0.7 picomoles 5 alpha-reduced androgens formed/b/mg protein), with little activity (2.4 +/- 1.2 picomoles 5 alpha-reduced androgens formed/h/mg protein) found in the proximal epididymal region. This regional distribution of enzyme activity differs markedly from that reported for eutherian mammals. Both the suggested epididymal protein synthetic and secretory activity and the level of epididymal 5 alpha-reductase activity appear to correlate regionally with the morphological changes that occur in the opossum spermatozoa as they transit the epididymis.     

Characterization of hormonally regulated secretory proteins from the caput epididymidis of the rabbit.

1. The incorporation of [35S]methionine into protein was investigated in tissue minces from different regions of the rabbit epididymis incubated in vitro. Rates of synthesis were in the order: epididymal regions 2-5 greater than region 7 greater than region 6 greater than region 1 greater than region 8 greater than ductus deferens greater than ductuli efferentes. 2. Separation of labelled proteins on polyacrylamide gels containing sodium dodecyl sulphate followed by fluorography revealed that one protein (mol.wt. approx. 90 000) was characteristic of region 1, four proteins (one of mol.wt. 54 000 and three of mol.wt. 20 000) were synthesized principally in regions 2-5, and one protein (mol.wt. 22 500) was produced mainly in regions 6, 7 and 8. 3. Castration for 14 days decreased incorporation of [35S]methionine into total protein to less than 10% of that in controls in all regions of the epididymis. However, testosterone treatment for a further period of 14 days restored protein synthesis to normal values in regions 6, 7 and 8, but not in region 1 or regions 2-5. In regions 2-5 the synthesis of three proteins of mol.wt. 20 000 declined after castration, but was not stimulated by exogenous testosterone. Since the 20 000-mol.wt. proteins were major tissue proteins, accounting for 16-25% of the total synthesized, they were used as markers for investigating hormone action in the epididymis. 4. Castration followed immediately by testosterone treatment or ligation of the ductuli efferentes resulted in a decrease in their synthesis, suggesting that they are partially dependent on factors in testicular fluid. Purification and characterization showed them to be acidic glycoproteins with a number of biochemical and immunological properties in common. 5. It is suggested that there is a synergistic action between blood androgens and factors in testicular fluid in regulating protein synthesis in the proximal regions of the rabbit epididymis.     

Postnatal development and regulation of proteins secreted in the boar epididymis

The number of proteins secreted by the boar epididymis increased progressively from 1 mo of age to the adult period. The first specific secretory activity was revealed at 2 mo in the distal caput (hexosaminidase, clusterin, and lactoferrin) and in the corpus (train O/HE1). Train A and glutathione peroxidase specific to the proximal caput, and trains E and M specific to the corpus, appeared at 4 mo. At 5 mo, secretion of procathepsin L occurred in the middle caput and that of mannosidase and E-RABP in the distal caput. Approximately 48% of all the proteins secreted in the adult boar epididymis were dependent on the presence of androgens, either stimulated (33.6%) or repressed (14.4%); 47% were modulated by other factors, and 5% were unregulated. In the proximal caput, 50% of the specific secreted proteins were controlled essentially by factors emanating from the testis. In more distal regions, two proteins secreted in the corpus were regulated by factors from the anterior regions. The regionalization of the secretory activity of the epididymal epithelium resulted in a specific regulation for each protein, which was modulated according to the region of expression and influenced by either testicular or epididymal factors that remain to be identified.     

Histochemical localization of carbonic anhydrase in the testis and epididymis of the boar.

The testis and epididymis of sexually mature, fertile boars were studied for localization of carbonic anhydrase (CA) using a cobalt precipitation technique. In the testis, cytoplasmic CA was found in the Sertoli cells and in the capillaries surrounding the seminiferous tubules. The epididymal duct was divided into initial, middle and terminal segments, and regional differences in CA activity were observed. The cell membranes of the basal cells were stained in the initial and middle segments. Strong cytoplasmic CA staining was present only in the apical cells in the initial and middle segments. The basolateral cell membranes were stained in the principal cells of the terminal segment and the ductus deferens. As a rule the capillaries surrounding the epididymal duct were unstained. The enzyme, specifically localized in regions of the male genitalia acting as sperm reservoirs, might be related to the quiescence of the stored spermatozoa by influencing the acid-base status of the epididymal fluid.     

Recent advances in sperm maturation in the human epididymis

As spermatozoa move through the human epididymis they encounter a varied environment with respect to the proteins with which they come into contact. In the proximal epididymis sperm are subjected to the action of enzymes and exposure to proteins involved in membrane modification. In the middle region another set of proteins and enzymes predominates; those associated with sterol transport could modify the sperm membrane to permit the uptake of GPI-anchored zona binding proteins P34H and CD52. More distally sperm encounter increasing activities of lytic enzymes, proteins involved in both zona binding and oocyte fusion, the major maturation antigen CD52, antimicrobial activity and decapacitation factors, that help them to survive before ejaculation. Adherence of the proteins to different domains (e.g. anterior acrosome or equatorial acrosomal segment) may depend on the nature of the protein, the lipid composition of the particular membrane and the ionic environment in the epididymal lumen. The eventual location on a capacitated sperm (acrosomal membrane) or acrosome-reacted sperm (equatorial region) may dictate their role in, for example, zona-binding (P34H) or oocyte-binding (gp20). Both proteins and membranes may be modified during epididymal transit by the enzymes which may add to or remove carbo-hydrates and peptides from the sperm surface.     

Interactions of labeled epididymal secretory proteins with spermatozoa after injection of 35S-methionine in the mouse

The sequential interactions of epididymal secretory proteins with spermatozoa during epididymal transit were examined. Mice received injections of 35S-methionine, and the radiolabeled luminal fluid and sperm-associated proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis at various times after injection. The majority of the luminal fluid and sperm-associated proteins were found in the caput epididymidis at 8 h; by 7 days, many of these proteins had been transported to the cauda epididymidis. Two classes of epididymal protein-sperm interactions were distinguished on the basis of regional synthesis and secretion. The major class consisted of proteins that were synthesized, secreted, and bound to spermatozoa in the caput epididymidis. In this class, however, the binding of proteins to the spermatozoa was variable. For example, a protein of 25 kDa remained associated with spermatozoa in substantial amounts during epididymal transit, while proteins of 40 and 35 kDa decreased in amount. Other proteins such as a protein of 18 kDa did not remain associated with spermatozoa. Another class of proteins (54, 44, 29 kDa) were synthesized and secreted from all epididymal regions but bound only to caput spermatozoa. Most of the epididymal proteins appeared to be tightly bound to the spermatozoa since spermatozoa already saturated with the unlabeled protein in the distal epididymis remained so even though the spermatozoa were surrounded by labeled proteins in the luminal fluid. These studies demonstrate that a variety of specific interactions occur between epididymal secretory proteins and spermatozoa as they migrate and mature in the epididymis.     

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.     

Characterization and androgenic regulation of major mRNAs coding for epididymal proteins in a lizard (Lacerta vivipara)

During the reproductive period (spring) under the control of testosterone the epididymis of the viviparous lizard secretes a group of major proteins with an approximate Mr of 19,000 named L protein(s). These proteins are recognized by a specific immunoserum and bind to the heads of spermatozoa. During spring, translation in reticulocyte lysate of RNA from secreting epididymis (stage 6) produced 5 immunoprecipitable bands with Mr values from 21,500 to 25,000. Such synthesis is undetectable during sexual rest in summer (stage 1). The 5 bands disappear when translation is performed in the presence of dog pancreas microsomes although a new band of Mr 19 000 becomes prominent. This suggests that synthesis of L protein involves two steps, i.e. synthesis of precursors (L preproteins) followed by a maturation process. At least 11 translation products (including L-preproteins) are involved in annual variations that follow the differentiation of the epididymal epithelial cells and their androgen dependency was studied by castration and in-vitro stimulation by testosterone. In these conditions, testosterone is able to control accumulation of RNA corresponding to L preproteins and to a translation product of Mr 29 000.