Role of androgens in survival of spermatozoa in epididymis of tammar wallaby (Macropus eugenii).

Studies of undiluted micropuncture samples of luminal fluid from the cauda epididymidis of the tammar indicated that spermatozoa are immotile in situ and spontaneously activate during collection or subsequent incubation in vitro. The suppression of sperm motility was related to the androgen status of the tammars and when this was increased by the use of Silastic implants of testosterone propionate, the spontaneous activation of samples was delayed for up to 2 h during incubation in vitro. Spermatozoa survived for up to 9 weeks when isolated in the cauda epididymidis between ligatures around the ductus. However, even after isolation for 3 weeks their viability was reduced compared with samples from the contralateral, unligated duct. Isolation of a length of ductus between ligatures also reduced the concentration of spermatozoa in the lumen of the duct and reduced the concentration of some proteins in the epididymal plasma. However, it did not affect the electrophoretic pattern of detergent extracts of spermatozoa. A study of the effects of orchidectomy and testosterone therapy indicated that sperm survival in the epididymis is androgen dependent. Orchidectomy reduced the concentration of spermatozoa in the luminal fluid and the volume of luminal fluid, and resulted in an increase in the concentration and a change in the electrophoretic pattern of protein in the fluid. The effects of orchidectomy were reduced or prevented by testosterone therapy. It is concluded that the cauda epididymidis of the tammar is at least as well adapted for sperm storage as it is in the eutherian mammals that have been studied.     

An autoradiographic study of macromolecular syntheses in the epithelium of the ductus epididymidis in the mouse

Summary Glycoprotein dynamism in the mouse epididymis was studied by means of histoautoradiography after injection of l-fucose-1-³H. The label was detected, at thirty minutes p.i., in the area occupied by Golgi apparatus in the epithelial cells. At 4 h p.i. the label was already present in the lumen of ductus epididymidis. At this time interval, the luminal labelling was highest in the initial segment of the epididymis and decreased against the more distal segments considerably.At ten days p.i. very high labelling was detected in the luminal contents in the terminal segment of the ductus epididymidis and in ductus deferens, the labelling in the proximal segments of the epididymis being much lower. These observations suggested a wave of labelled glycoprotein in epididymal plasma passing through the epididymis after a fucose pulse.Higher labelling was detected in so-called clear cells than in the neighboring principal cells.Association of the labelled material was seen in epididymal and uterine spermatozoa, mostly in sperm tail region.     

Evidence for the presence of oxytocin in the ovine epididymis

The testes of several species contain oxytocin and/or neurophysin, but the content or localization of oxytocin in epididymal tissue has not been studied. The present study was undertaken to localize oxytocin and neurophysin in epididymal tissue of the ram, and to quantify oxytocin in the ductus epididymidis and fluids entering and leaving the ductus epididymidis. Neurophysin was not detected in the epididymis; thus, synthesis of oxytocin by the epididymis is unlikely. Immunohistochemical localization of oxytocin was confined to the epithelium and capillaries. Oxytocin immunostaining was most intense for epithelium of the caput and declined in corpus and cauda regions. However, based on radioimmunoassay, no difference in oxytocin concentration was detected among regions of the epididymis. Since rete testis fluid entering and cauda epididymal fluid leaving the epididymis contained at least fourfold more oxytocin than testicular venous plasma, it was concluded that regional differences in epithelial concentration of oxytocin may have been masked by oxytocin contained in the luminal fluid. It was concluded further that the epididymis of the ram does not synthesize oxytocin, but about 22 ng/day enters the epididymis in rete testis fluid. Most of this luminal oxytocin apparently is absorbed by the epithelium of the caput epididymidis, with additional adsorption in the corpus and cauda. Although a role for oxytocin in ductal contractility cannot be excluded, it is more likely that the luminal oxytocin influences epithelial or sperm function.     

Computer-assisted visualization of the rat epididymis: a methodological study based on paraffin sections autometallographically stained for zinc ions

A concept for the computer-assisted visualization of tubular organs is presented. Unmarked histological zinc-stained serial sections from the epididymis of the Wistar rat were aligned to demonstrate the concept. Virtual images were made through the aligned sections and served as controls for the alignment process. Animation of the serial sections and the virtual images revealed new information about the structure of the organ under investigation. The analysis was used to upgrade the anatomical knowledge of rat epididymis by describing how the epididymal duct runs through the structure. The proximal parts of the epididymis contain large communicating septa of connective tissue dividing the caput and the upper part of the corpus epididymidis into segments. The tortuousness was high in the caput with many turns within a small area of the epididymis, whereas longer loops were found in the lower part of the corpus and cauda epididymidis. The tube of the vas deferens was found to become an integrated part of the ductal system in the cauda epididymidis, although it was histologically easy to distinguish from the epididymal duct. The total number of cross-sections of the ductus epididymidis in the 2254, 15-mu m-thick, tissue sections analysed was 104 700, giving a minimum length of the ductal system of 1.5 m. © 1998 Chapman & Hall     

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 (6). 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.     

Expression and localization of PMCA4 in rat testis and epididymis

It has recently been shown in mice that the plasma membrane Ca²⁺-ATPase isoform 4 (PMCA4) is essential for sperm fertilization capacity. We analyzed whether sperm PMCA4 is formed in the rat during spermatogenesis or is synthesized in the epididymis and transferred onto sperm during sperm maturation. We could show that PMCA4 is conserved in sperm from testis to epididymis. In testis, PMCA4 mRNA was restricted to spermatogonia and early spermatocytes, while the PMCA4 protein was detected in spermatogonia, late spermatocytes, spermatids and in epididymal sperm. In epididymis PMCA4 mRNA was localized in basolateral plasma membranes of epithelial cells of the caput, corpus and cauda epididymidis. In contrast, the protein was only detectable in the epithelial cells of the caput, indicating that PMCA4 mRNA is only translated into protein in caput epithelium. In the epididymal corpus and cauda, PMCA4 mRNA and protein, respectively, was localized and in peritubular cells. Furthermore, we detected an identical distribution of PMCA4a and b splice variants in rat testis, epididymal corpus and cauda. In the caput epididymidis, where PMCA4 is located in the epithelium splice variant 4b was more prominent. Further experiments have to clarify the functional importance of the differences in the PMCA4 distribution.     

Regional expression and androgen regulation of carbonic anhydrase IV and II in the adult rat epididymis

Carbonic anhydrase (CA) is implicated in the acidification of epididymal fluid and thereby in the regulation of sperm maturation and motility. Among the CA isoenzymes, CA IV and II have been shown to be present in the rat epididymal duct epithelium. In the present study, we examined the expression and androgen regulation of CA IV and II mRNAs along the epididymal duct. Northern blot analysis revealed the presence of CA II mRNA in all regions of the epididymis with the strongest signal in the corpus region, while CA IV mRNA was expressed predominantly in the corpus epididymidis. Three days after bilateral castration, CA IV and II mRNAs were decreased by 80-90% in the corpus epididymidis. Testosterone (T) replacement maintained the expression of CA mRNAs at 50-60% of the control levels, indicating that circulating androgens alone are not sufficient to recover the CA expression in the corpus region. However, unilateral castration did not affect the mRNA levels of CA IV and II, suggesting that factors in testicular fluid do not play a major role in the regulation of CA expression in the corpus epididymidis. Immunoblot analysis showed that CA IV protein levels decreased 3 days after castration, while T administration maintained the protein expression virtually at the precastration levels. These data demonstrate that mRNAs for CA IV and II are predominantly expressed in the corpus region of the rat epididymis and can be regulated by androgens in that region. The present data suggest that the regulation of CA expression in the corpus epididymidis by androgens contributes to the known androgen effects on epididymal acidification.     

Immunolocalization of α1A-adrenoceptors in rat and human epididymis

Immunohistochemistry was conducted to analyze the cellular localization of alpha(1A)-adrenoceptors along rat and human epididymis. ADR-A, a polyclonal antibody that recognizes the specific C-terminal region of alpha(1A)-adrenoceptors, immunostained this adrenoceptor subtype in smooth muscle cells surrounding the epididymal tubules and interstitial blood vessels and in subpopulations of epithelial cells from adult rat and human caput and cauda epididymidis. The same cell types from rat epididymidis were immunostained by ADR-1, a polyclonal antibody that recognizes a common region of the three alpha(1)-adrenoceptor subtypes, alpha(1A), alpha(1B), and alpha(1D). Immunostaining with both antibodies was also conducted in adult rat and human vas deferens and seminal vesicle used as positive controls because of the abundance of alpha(1A)-adrenoceptors in these tissues. ADR-A- and ADR-1-positive immunostaining was differentially distributed depending on the antibody, method of tissue fixation (Bouin-fixed and fresh frozen tissues), species (rat and human), tissue (caput and cauda epididymidis), and age (immature and adult rats) analyzed. This is the first report immunolocalizing alpha(1A)-adrenoceptor along rat and human epididymis. The presence of this adrenoceptor subtype in epididymal smooth muscle and epithelial cells indicates their contribution to smooth muscle contractile responses and a possible role in the absorptive and/or secretory activities of the epithelium lining the epididymal duct. Taken together, our results should contribute to a better understanding of the physiological role of alpha(1)-adrenoceptors in the epididymidis and the importance of the sympathetic nervous system for male (in)fertility.     

Structural features of the epididymis in a dasyurid marsupial (Antechinus stuartii)

Summary The ductus epididymidis of the marsupial mouse Antechinus stuartii was divided into caput, corpus, and caudal regions using several constant morphological landmarks. Tubule diameter and epithelial height increased gradually from caput to cauda. In contrast, the surface area of the lumen of the ductus epididymidis increased to a maximum in the distal caput region, but decreased markedly in the distal cauda in association with characteristic changes in lumen shape (from circular to slit-shaped) and epithelial height. Epithelial cells of the ductus epididymidis were generally similar in structure to those described in other mammalian species. Principal and basal cells were common throughout the epithelium. Clear and mitochondria-rich cells were also identified, but occurred less frequently. Regional variations in cell ultrastructure were observed only in principal cells. Numerous vesicular inclusions occurred in the apical cytoplasm of cells in caput segments, membrane-bounded, electron-dense bodies were common in distal corpus regions, and a brush border of microvilli characterized the luminal surface of principal cells in caudal segments. Sperm index increased in the proximal caput, declined to basal levels in the distal caput and proximal corpus, and then increased to a maximum in segment 9 of the distal corpus and remained at about this level throughout the cauda epididymidis. Nuclear rotation, loss of cytoplasmic droplets, and other sperm maturational changes were observed along the epididymis. Discarded cytoplasmic droplets collected in large masses interspersed between aggregates of spermatozoa throughout the distal regions of the duct. There was no evidence of phagocytosis by principal cells of cytoplasmic droplets. The epididymis of A. stuartii differs from that of other mammals. The unusual caudal region, which has little storage capacity for sperm, is an unusual adaptation in a species in which the male is known to be polygamous.     

Differential expression of the mouse mitochondrial genes and the mitochondrial RNA-processing endoribonuclease RNA by androgens.

Using subtractive hybridization to identify genes that are androgen regulated in the mouse epididymis, a number of cDNAs were identified that represented mitochondrial genes including cytochrome oxidase c subunits I, II, and III, cytochrome b, NADH dehydrogenase subunit 5, a region of the displacement loop, and the 16S rRNA. Northern blot analysis of RNA from intact, castrate, or testosterone-replaced epididymides confirmed that these mitochondrial mRNAs as well as the rRNA were androgen regulated with a 2- to 5-fold reduction in expression observed after 4 weeks castration with partial to full recovery to precastrate levels upon 4 weeks of testosterone replacement. In contrast to the mitochondrial genes, the expression of the RNA component of the mitochondrial RNA-processing endoribonuclease (RNAase MRP), a nuclear factor which is thought to be involved in the regulation of mitochondrial DNA synthesis, increased in the epididymis upon castration and then returned to precastrate levels after testosterone replacement. An examination of other androgen-responsive tissues showed that mitochondrial gene expression was also regulated by androgens in the kidney. The RNAase MRP RNA levels, however, showed an increase after castration only in the reproductive tissues (epididymis, vas deferens, and seminal vesicle) and not in the kidney. No correlative increase in mitochondrial DNA levels was observed for any of the tissues. Finally, an analysis of various mouse tissues as well as the different regions of the epididymis revealed large differences in mitochondrial mRNA levels. While for most tissues the mRNA levels correlated with the mitochondrial DNA content, the levels of the RNAase MRP RNA did not. Taken together, these findings not only show the large variations in mitochondrial gene expression between tissues but also demonstrate that the expression of mitochondrial genes and ultimately mitochondrial function are androgen regulated in the epididymis and kidney.     

Isolation and characterization of renal erythropoietin-producing cells from genetically produced anemia mice

Understanding the nature of renal erythropoietin-producing cells (REPs) remains a central challenge for elucidating the mechanisms involved in hypoxia and/or anemia-induced erythropoietin (Epo) production in adult mammals. Previous studies have shown that REPs are renal peritubular cells, but further details are lacking. Here, we describe an approach to isolate and characterize REPs. We bred mice bearing an Epo gene allele to which green fluorescent protein (GFP) reporter cDNA was knocked-in (Epo(GFP)) with mice bearing an Epo gene allele lacking the 3' enhancer (Epo(Δ3'E)). Mice harboring the mutant Epo(GFP/Δ3'E) gene exhibited anemia (average Hematocrit 18% at 4 to 6 days after birth), and this perinatal anemia enabled us to identify and purify REPs based on GFP expression from the kidney. Light and confocal microscopy revealed that GFP immunostaining was confined to fibroblastic cells that reside in the peritubular interstitial space, confirming our previous observation in Epo-GFP transgenic reporter assays. Flow cytometry analyses revealed that the GFP fraction constitutes approximately 0.2% of the whole kidney cells and 63% of GFP-positive cells co-express CD73 (a marker for cortical fibroblasts and Epo-expressing cells in the kidney). Quantitative RT-PCR analyses confirmed that Epo expression was increased by approximately 100-fold in the purified population of REPs compared with that of the unsorted cells or CD73-positive fraction. Gene expression analyses showed enrichment of Hif2α and Hif3α mRNA in the purified population of REPs. The genetic approach described here provides a means to isolate a pure population of REPs, allowing the analysis of gene expression of a defined population of cells essential for Epo production in the kidney. This has provided evidence that positive regulation by HIF2α and negative regulation by HIF3α might be necessary for correct renal Epo induction.     

Region-specific expression and secretion of the fibrinogen-related protein, fgl2, by epithelial cells of the hamster epididymis and its role in disposal of defective spermatozoa

The cauda epididymidis functions in the storage and protection of mature, fertile spermatozoa. We previously identified a region-specific secretory glycoprotein (termed HEP64) of the hamster proximal cauda epididymidis that specifically bound and coated the nonviable, but not the viable, spermatozoa within the epididymal lumen. In this study we employed expression screening of a hamster epididymal cDNA library to obtain the full-length sequence of HEP64 and to identify it as the fibrinogen-like protein fgl2. Northern blot analysis demonstrated that fgl2 mRNA is highly expressed by the proximal cauda epididymidis in comparison to other hamster tissues examined, and, in situ hybridization analysis of the epididymis revealed that fgl2 mRNA exhibited a region- and principal cell-specific expression pattern. Immunohistochemistry confirmed the association of fgl2 with abnormal spermatozoa in the cauda epididymidis and revealed smaller fgl2-containing particles. Immunoelectron microscopy revealed that fgl2 was distributed throughout an amorphous, "death cocoon," complex assembled onto abnormal spermatozoa and that the smaller fgl2 aggregates consisted of the amorphous material with embedded sperm fragments, organelles, and membrane vesicles. A protocol was developed to isolate an enriched death cocoon fraction. SDS-PAGE and microsequence analyses revealed that the Mr 64,000 fgl2 monomer was assembled into two disulfide-linked oligomers of Mr 260,000 and 280,000. These data demonstrate that the epididymis possesses a specific mechanism to identify and envelop defective spermatozoa with a protein complex containing the fibrinogen-like protein fgl2. We propose that this represents an important protective mechanism not only to shield the viable sperm population from potentially deleterious enzymes released by dying spermatozoa but also to prevent the release of sperm proteins that could initiate an immune response if they escaped the epididymal environment.     

Effects of breeding season and mating on total number and distribution of spermatozoa in the epididymis of the brown marsupial mouse, Antechinus stuartii

Changes in the number and distribution of spermatozoa in the epididymis of the adult brown marsupial mouse were examined during July/August in mated and unmated males. The effects of mating on epididymal sperm populations were studied in 2 groups of males each mated 3 times and compared with the number and distribution of spermatozoa in the epididymides of 4 unmated control groups. One testis and epididymis were removed from each animal (hemicastration) either before or early in the mating season to provide information on initial sperm content and distribution. The contralateral side was removed later in the mating season to examine the effects of mating or sexual abstinence on epididymal sperm distribution. Epididymal sperm number peaked in both the distal caput and distal corpus/proximal cauda epididymidis in late July. The total number of spermatozoa, including those remaining in the testis, available to each male at the beginning of the mating season in early August was approximately 4.4 x 10(6)/side. Although recruitment of spermatozoa into the epididymis from the testis continued until mid-August, sperm content of the epididymis reached a peak of about 3.5 x 10(6)/epididymis in early August. At this time approximately 0.9 x 10(6) spermatozoa remained in the testis which had ceased spermatogenic activity. Throughout the mating season, epididymal spermatozoa were concentrated in the distal corpus/proximal cauda regions of the epididymis and were replenished by spermatozoa from upper regions of the duct. Relatively few spermatozoa were found in the distal cauda epididymidis, confirming a low sperm storage capacity in this region. A constant loss of spermatozoa from the epididymis, probably via spermatorrhoea, occurred throughout the mating season and very few spermatozoa remained in unmated males in late August before the annual male die-off. Mating studies showed that an average of 0.23 x 10(6) spermatozoa/epididymis were delivered per mating in this species, but the number of spermatozoa released at each ejaculation may be as few as 0.04 x 10(6)/epididymis when sperm loss via spermatorrhoea is taken into account. We suggest that the unusual structure of the cauda epididymidis, which has a very restricted sperm storage capacity, may function to limit the numbers of spermatozoa available at each ejaculation and thus conserve the dwindling epididymal sperm reserves in order to maximize the number of successful matings which are possible during the mating season.     

Fine structure distribution of non-specific acid phosphatase in the head region of mouse spermatozoa from various regions of the male reproductive tract.

The fine structure distribution of non-specific acid phosphatase was determined in the head region of mouse spermatozoa from the testes, the caput, corpus and cauda epididymidis and the ductus deferens. Enzymatic localization was achieved by the Gomori technique. The postacrosomal dense lamina, the nuclear side of the inner acrosomal membrane and the space between the plasmalemma and the outer acrosomal membrane showed reaction product in spermatozoa from the testis and caput epididymidis. Spermatozoa from the cauda epididymidis exhibited reaction product only between the plasmalemma and the outer acrosomal membrane. Spermatozoa from the corpus epididymidis and from the ductus deferens showed no reaction product in the head region. The changes observed in the distribution of acid phosphatase in the sperm head during epididymal transport may reflect maturational events.     

Segmental expression of the bradykinin type 2 receptor in rat efferent ducts and epididymis and its role in the regulation of aquaporin 9

Water and solute transport in the efferent ducts and epididymis are important for the establishment of the appropriate luminal environment for sperm maturation and storage. Aquaporin 9 (AQP9) is the main water channel in the epididymis, but its regulation is still poorly understood. Components of the kinin-kallikrein system (KKS), leading to the production of bradykinin (BK), are highly expressed in the lumen of the male reproductive tract. We report here that the epididymal luminal fluid contains a significant amount of BK (2 nM). RT-PCR performed on epididymal epithelial cells isolated by laser capture microdissection (LCM) showed abundant BK type 2 receptor (Bdkrb2) mRNA expression but no type 1 receptor (Bdkrb1). Double-immunofluorescence staining for BDKRB2 and the anion exchanger AE2 (a marker of efferent duct ciliated cells) or the V-ATPase E subunit, official symbol ATP6V1E1 (a marker of epididymal clear cells), showed that BDKRB2 is expressed in the apical pole of nonciliated cells (efferent ducts) and principal cells (epididymis). Triple labeling for BDKRB2, AQP9, and ATP6V1E1 showed that BDKRB2 and AQP9 colocalize in the apical stereocilia of principal cells in the cauda epididymidis. While uniform Bdkrb2 mRNA expression was detected in the efferent ducts and along the epididymal tubule, marked variations were detected at the protein level. BDKRB2 was highest in the efferent ducts and cauda epididymidis, intermediate in the distal initial segment, moderate in the corpus, and undetectable in the proximal initial segment and the caput. Functional assays on tubules isolated from the distal initial segments showed that BK significantly increased AQP9-dependent glycerol apical membrane permeability. This effect was inhibited by BAPTA-AM, demonstrating the participation of calcium in this process. This study, therefore, identifies BK as an important regulator of AQP9.     

Effect of vasectomy on gene expression in the epididymis of cynomolgus monkey

Vasectomy has been shown to affect the pattern of mRNA expression of P34H, a human sperm protein added to the acrosomal cap during epididymal transit. It has been reported that vasectomy alters the histology of the reproductive tract in various species as a result of the increased pressure in the epididymis. The aim of this study was to evaluate if other epididymis-specific mRNAs, which are expressed in different patterns along the duct, are altered by vasectomy as well. We analyzed the expression of P31m (a monkey homologue of human P34H) and three different HE-like (HE-l) mRNAs along the epididymis in the cynomolgus monkey (Macaca fascicularis). Sexually mature cynomolgus monkeys were vasectomized unilaterally; then the epididymides were surgically removed at different time points. The ipsilateral normal epididymis was used as a control. Histomorphometric measurements showed that the height of the epididymal epithelial cells started to be affected only at 14 wk postsurgery. However, Northern blot and in situ hybridization analysis showed that the expression pattern of P31m, HE1, and HE5-like mRNA along the epididymis was not affected by vasectomy. Only the HE2-like mRNA predominantly expressed in the normal corpus epididymidis was significantly lowered 14 wk after vasectomy. Thus, ductal obstruction differentially alters mRNA expression along the epididymis of the cynomolgus monkey.     

Immunocytochemical localization of the major glycoprotein of epididymal fluid from the cauda in the epithelium of the mouse epididymis

Summary The most abundant protein in fluid from the mouse cauda epididymidis, designated CP 27, is a glycoprotein that migrates at approximately 27000 daltons on SDS-polyacrylamide gels. Samples of CP 27 were isolated by preparative gel electrophoresis and were used to raise a guinea-pig polyclonal antiserum, which reacted with a single band on western blots of caudal epididymal fluid. This antiserum was used for immunocytochemical localization of CP 27 in histological sections of mouse epididymis using the peroxidase-antiperoxidase and protein A-gold methods. The most proximal staining with anti-CP 27 was in segment 6 of the distal caput epididymidis, where the lumen and a portion of the supranuclear cytoplasm of principal cells were stained. In contrast, in the distal corpus and cauda epididymidis (segments 8–11), there was pronounced staining of the luminal contents, stereocilia, and scattered cells identified as the light cells of the epididymal epithelium. Although CP 27 was found in the epididymal lumen of all segments distal to segment 6, the intensity of staining appeared to decline distally in the cauda epididymidis. Control sections exposed to pre-immune serum instead of anti-CP 27 showed no reaction. The results suggest that CP 27, the major glycoprotein of cauda epididymal fluid, is synthesized by principal cells of segment 6 of the distal caput epididymidis. CP 27 may be among the substances absorbed from the lumen by the light cells of the distal epididymis.     

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.