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a Department of Dairy and Animal Science, J.O. Almquist Research Center, Pennsylvania State University, University Park, Pennsylvania 16802
b Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Osaka 565, Japan
c PRESTO, Japan Science and Technology Corporation, Osaka 565, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The primary structure of GSH-independent PGD synthase indicates that it is a member of the superfamily of secretory transport proteins known as lipocalins, whose constituents are involved in the binding and transport of small lipophilic ligands such as retinoids, steroids, and pheromones [3]. It is the only enzyme among members of the lipocalin superfamily and has been termed lipocalin-type PGD synthase to distinguish it from GSH-dependent PGD synthase. It has been proposed that this enzyme is bifunctional, acting as a PGD2-producing enzyme within the central nervous system and as a potential lipophilic ligand transporter within CSF [4]. It has been recognized for several years that PGD2 plays a critical role in a number of physiological processes such as the control of the sleep-wake cycle, regulation of body temperature, pituitary hormone release, nociception, prevention of platelet aggregation, smooth muscle relaxation/contraction, and bronchoconstriction. In addition, PGD2 is the most abundant prostanoid produced by mast cells and is thought to function as an allergic and inflammatory mediator (for reviews see [5, 6]). PGD2 is also important because it readily undergoes dehydration in vitro and in vivo to produce prostaglandins of the J series such as PGJ2, 
12-PGJ2, and 15-deoxy-12,14-PGJ2 [7]. These have been reported to show various biological activities such as suppression of tumor growth, antiviral activity, and stimulation of osteogenesis [8]. PGD2 and some of its derivatives have been reported to be ligands for the peroxisome proliferator-activated receptors, which regulate genes involved in lipid metabolism [9, 10]. In addition, PGD2 may also be further converted to 9
,11ß-PGF2, a stereoisomer of PGF2, which exerts various pharmacological actions different from those induced by PGF2 (for review see [11]).
Whether lipocalin-type PGD synthase is functioning primarily as a conversion enzyme or as a lipophilic carrier within the male reproductive tract has not been determined. It is of interest to note that although only minute amounts of PGD2 have been found in seminal plasma [12], a positive correlation (P < 0.05) has recently been shown between concentrations of PGD synthase and PGD2 [13]. Lipocalin-type rat PGD synthase has been found to bind retinoids and other lipophilic substances with affinities similar to those of other lipocalins [4]. Likewise, the Xenopus counterpart of lipocalin-type PGD synthase also binds in vitro to retinal, retinoic acid, and thyroxine [14]. Retinoids (vitamin A derivatives) have been shown to be required for the normal development, maturation, and maintenance of a number of tissues, including most epithelia. They are essential for male fertility as sperm differentiation is arrested in animals maintained on vitamin A-deficient diets [15].
We previously detected lipocalin-type PGD synthase in the luminal fluids collected by cannulae from the bovine rete testis and cauda epididymidis, but not in fluid from the accessory sex glands [1]. The aim of the present study was to immunohistochemically localize lipocalin-type PGD synthase to the various cell types within the bovine testis and seven segments of the epididymis, and on ejaculated sperm, to gain further insight into the potential function of this enzyme in male reproduction. In addition, we made recombinant bovine lipocalin-type PGD synthase for use in this study and for other physiological studies in progress.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Holstein bulls used in this study had previously served as production sires for artificial insemination cooperatives in the northeastern United States. After they were retired from commercial service, the bulls were relocated to the J.O. Almquist Research Center at Pennsylvania State University (University Park, PA), where they remained until slaughter. All bulls had normal semen parameters and were in good health. Bull semen, collected via artificial vagina, was centrifuged at 1000 x g for 10 min at room temperature (RT). The seminal plasma recovered was then centrifuged at 10 000 x g for 1 h at 4°C.
Tissues were collected at a local abattoir from six bulls within 60 min of death. For Western blotting studies, the epididymis was divided into three regions: the caput, corpus, and cauda. For immunohistochemical studies, the epididymis was subdivided into seven regions [16] (Fig. 1) consisting of three regions of the caput epididymidis (A, B, and C), two of the corpus epididymidis (D and E), and two of the cauda epididymidis (F and G).
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Preparation of Crude Tissue Extracts
Samples of testis tissue and individual epididymal regions were homogenized in three volumes of ice-cold 20 mM potassium phosphate buffer, pH 6.0, containing 0.5 mM dithiothreitol with a Polytron homogenizer (Brinkman Instruments, Westbury, NY) in three 10-sec bursts at maximum speed and then centrifuged at 20 000 x g for 30 min at 4°C. The tissue homogenates were further centrifuged at 100 000 x g for 1 h at 4°C, and the resulting high-speed supernatant fractions were stored at -70°C. Protein concentrations were determined by the method of Lowry et al. [17], using BSA as a standard.
Preparation of Sperm Membranes in Solubilization Buffer
Sperm from each ejaculate were washed 3 times in warm PBS (1000 x g, 10 min) and then incubated (5 x 108 sperm per ml) for 1 h at 4°C in a membrane solubilization buffer containing 0.4% sodium deoxycholate in 0.264 M sucrose, 10 mM Tris-HCl (pH 8.5) [18]. After solubilization, each sample was centrifuged at 10 000 x g for 5 min. The supernatant containing the sperm membrane proteins was dialyzed extensively against 10 mM Tris-HCl (pH 7.4). Membranes were lyophilized and resuspended in double-distilled H2O, and protein concentrations were determined [17]. Previously, we demonstrated that this procedure removes only the plasma membrane [19].
Preparation of Recombinant Bovine Lipocalin-Type PGD Synthase
The cDNA for bovine lipocalin-type PGD synthase was isolated as reported previously [1]. The recombinant bovine lipocalin-type PGD synthase was expressed as a GSH-S-transferase fusion protein by using a pGEX-2T vector as reported [20]. Briefly, the coding region from the downstream residue, Ala29, of the signal peptide to the stop codon of bovine lipocalin-type PGD synthase was inserted into a BamHI/EcoRI site of pGEX-2T in which two cysteine residues of bovine lipocalin-type PGD synthase at the positions of 104 and 186 were replaced with alanine to avoid formation of a mismatched disulfide bridge [21]. Escherichia coli DH5 were transfected with the reconstituted plasmid and cultured in Luria broth with 0.6 mM isopropyl-1-thio-ß-D-galactopyranoside to produce a fusion protein of GSH S-transferase and bovine lipocalin-type PGD synthase. The fusion protein was purified by affinity chromatography with GSH-sepharose 4B. The recombinant bovine lipocalin-type PGD synthase was recovered from the resin by incubation with thrombin at RT overnight according to the manufacturer's instructions (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). It was further purified by Mono-S column chromatography in 10 mM sodium citrate (pH 4.5). The final purity of the recombinant bovine lipocalin-type PGD synthase was analyzed by SDS-PAGE and Western blotting with a resultant molecular mass of 20 kDa.
Preparation of Immune Reagents
Polyclonal antibody against recombinant bovine lipocalin-type PGD synthase was generated in New Zealand white rabbits. Recombinant lipocalin-type PGD synthase (1 mg in PBS) was mixed 1:1 with Freund's complete adjuvant and injected s.c. among 10 sites in the scapular region. Rabbits were boosted 4 wk later with 500 µg of recombinant lipocalin-type PGD synthase mixed 1:1 with Freund's incomplete adjuvant. Four weeks after the second injection, rabbits were bled from the marginal ear vein. Serum was obtained by centrifugation (1000 x g for 20 min) after clotting overnight at 4°C, and the IgG fraction was isolated by Protein A column chromatography (Sigma, St. Louis, MO) according to the manufacturer's instructions. Normal anti-rabbit IgG was purified in an identical manner from a pool of sera obtained from nonimmunized rabbits.
SDS-PAGE and Western Blot Analysis
One-dimensional (1D) SDS-PAGE separation of proteins was performed under denaturing conditions as previously described [22]. Gels (15%) were electrophoresed in a Bio-Rad mini-Protean II apparatus (Rockville Centre, NY) for 1.5 h at 100 V. Gels were then either silver-stained [23] or prepared for Western blotting.
Western blot analysis was performed after protein separation on 1D SDS-PAGE gels followed by electrophoretic transfer to 0.2 µm nitrocellulose membrane (Schleicher & Schuell, Keene, NH) using a Bio-Rad semidry transfer system. All gels were equilibrated in transfer buffer (0.125 M Tris, 0.096 M glycine) for 1 h at 4°C and then transferred at 25 V for 35 min. Immunodetection of lipocalin-type PGD synthase on Western blots was accomplished by incubating membranes with blocking buffer (50 mM Tris [pH 7.4], 500 mM NaCl [TBS] containing 5% normal goat serum [NGS] and 0.1% Tween-20) overnight at 4°C on a rocking platform. Membranes were then incubated with polyclonal antibodies in blocking buffer for 2 h at RT on a rocking platform. Control polyclonal antibodies included preimmune rabbit IgG and IgG to an unrelated seminal plasma protein used at the same concentration. After being washed 3 times for 20 min each with TBS containing 0.1% Tween-20 (TBST), membranes were incubated with goat anti-rabbit IgG coupled to horseradish peroxidase (Southern Biotech, Birmingham, AL) in TBST + 5% NGS for 30 min. Membranes were washed twice for 30 min each with TBST and once with TBS. Antibody binding to proteins was visualized using the enhanced chemiluminescence (ECL) detection system (Amersham) according to the manufacturer's instructions.
Immunohistochemical Staining for PGD Synthase
Bull testes and epididymides were perfused through the testicular artery with 1 L of PBS, pH 7.2, followed by 500 ml of 4% paraformaldehyde, and then 500 ml of Bouin's fixative, pH 2.5. Small pieces of tissue (1 cm2 by 0.5 cm) were excised and postfixed in Bouin's fixative for 40 h. Tissue was then transferred to 70% ethanol, which was changed several times, and then standard embedding procedures were followed by dehydration in alcohols, clearing in xylenes, and final paraffin embedding.
For immunolocalization of lipocalin-type PGD synthase, paraffin-embedded tissues were sectioned at 4–8 µm, floated on a 48°C water bath, applied to slides precoated with 3-aminopropyltrienthoxy-silane, dried in a 40°C oven overnight, and then stored in a cool, dark place until use. Sections were deparaffinized in xylene and rehydrated in ethanol with increasing concentrations of water. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 30 min, and tissues were pepsin-treated for 5 min. Nonspecific binding sites were blocked with 10% NGS for 1 h. Primary and secondary antibodies were diluted in a solution of PBS + 10% NGS. Tissue sections were exposed to primary antibodies (2 µg/ml) for 17 h at RT. After a 1-h incubation in secondary antibody (2 µg/ml), tissues were rinsed 3 times in PBS. The Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) with diaminobenzidine substrate was used according to the manufacturer's recommendations. Specificity of staining was checked by incubating sections with the polyclonal antibody preabsorbed with excess amounts of purified recombinant PGD synthase and with the IgG fraction (4 µg/ml) obtained from nonimmunized rabbits.
Testis tissue and efferent duct sections were counterstained by periodic acid-Schiff-hematoxylin (PAS-H; [24]). The eight stages of the bull spermatogenic cycle were determined according to previously published descriptions [25, 26]. Rete testis and epididymal tissue sections were counterstained only with hematoxylin. Sections were dehydrated, rinsed in xylene, and mounted in Entellen (Electron Microscope Sciences, Fort Washington, PA).
Indirect Immunofluorescence on Ejaculated Bull Sperm
Sperm were separated from seminal plasma by centrifugation at 700 x g for 10 min and washed once in warm PBS. Sperm were resuspended in PBS and treated with an equal volume of cold 4% paraformaldehyde (prepared fresh from formaldehyde) for 15 min on ice. All subsequent steps were conducted at RT. Fixed sperm were washed twice with PBS (10 000 rpm, 5 min) in an IEC Micromax (Needham, MA), and the pellet was resuspended in PBS containing 5% BSA. Samples of 1 ml, each containing 5 x 106 spermatozoa, were incubated overnight at 4°C with 30 µg of anti-recombinant bovine lipocalin-type PGD synthase IgG. The next day, after being washed 3 times in PBS-BSA, sperm were resuspended to 500 ml in Fc blocking medium (#NB-309, Accurate Chemical & Scientific Corp., Westbury, NY). Five microliters (0.5 mg/ml) of fluorescein isothiocyanate-conjugated goat anti-rabbit IgG Fab fragment (Southern BioTech) was added to 500 µl of sperm suspension for 1 h. After being washed 3 times in PBS-BSA as before, sperm were viewed with a Olympus microscope (BH2-RFC; Melville, NY) interfaced with a Sony Digital DKC-5000 camera (Carson, CA). Phase contrast and fluorescence images (x1250) were photographed for the same cell.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Native bovine PGD synthase in seminal plasma has a molecular mass of 26 kDa, which includes two N-linked glycosylation sites of 3 kDa in size [1]. Western blots of bull seminal plasma samples probed with anti-recombinant bovine PGD synthase were immunoreactive at 26 kDa (Fig. 2, lane 7). Interestingly, a larger molecular-size band of approximately 29 kDa, in addition to the band at 26 kDa, was evident in all extracts from testis and caput, corpus, cauda epididymidal tissue (Fig. 2, lanes 8–11), as well as a 23-kDa band in only the caput tissue extract (Fig. 2, lane 9). Differences in the band intensity for the three epididymal regions were evident, even though the same concentration of total protein was loaded in each lane. In each bull examined, the caput tissue extract stained with the greatest intensity, followed by the cauda and the corpus in order of decreasing intensity. Lanes of sperm membrane proteins from washed, ejaculated sperm were also immunoreactive at 26 kDa (Fig. 2, lane 12).
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Localization of Lipocalin-Type PGD Synthase in the Bull Testis
A distinct cyclic variation in the intensity of staining was observed for lipocalin-type PGD synthase during the eight stages of the bull spermatogenic cycle. This variation is illustrated for a single section in Figure 3a. At higher magnifications, Figure 3b–g illustrates the variation in intensity and distribution of specific staining for lipocalin-type PGD synthase that was observed in tubules at different stages of the spermatogenic cycle. Lipocalin-type PGD synthase content (i.e., intensity of immune staining) was minimal in stages I–II (Fig. 3, b and c). The highest level of staining occurred during stages III–VII (Fig. 3, d–f) while moderate staining was observed in stage VIII (spermiation, Fig. 3g). Sa, Sb1, and Sb2 round spermatids had little or no staining for lipocalin-type PGD synthase. Also, spermatogonia and spermatocytes did not stain. Positive staining was evident in both spermatids and Sertoli cells. The cytoplasm of Sc and Sd1 spermatids undergoing progressive elongation stained most intensely for lipocalin-type PGD synthase. No immunoreactivity was observed within the interstitium found between adjacent seminiferous tubules.
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By initial preincubation of anti-PGD synthase antibody with recombinant lipocalin-type PGD synthase antigen in excess, positive immunoreactivity on tissue sections was eliminated. Additional control sections treated with rabbit preimmune serum substituted for the anti-recombinant lipocalin-type PGD synthase antibody also showed no reaction product in any tissue section examined (Fig. 3, h–k).
Localization of Lipocalin-Type PGD Synthase in the Rete, Efferent Ducts, and Epididymis
In the rete testis, intense reaction product was evident over the apical region of the low cuboidal epithelial cells and also within the lumen (Fig. 4a). This reaction appeared over the entire length of the rete testis. In addition, the apical portion of the epithelial cells lining the efferent ducts also stained for lipocalin-type PGD synthase (Fig. 4b).
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In the seven segments of the bull epididymis, specific staining for lipocalin-type PGD synthase was strikingly localized to the principal cells of the caput A and B epididymal regions in each of the six bulls examined and was much weaker or absent from the other regions of the epididymis depending on the individual bull. Bulls with minimal concentrations of lipocalin-type PGD synthase in their seminal plasma did not show immunostaining in the corpus or cauda epididymidis. Expression of lipocalin-type PGD synthase was limited to the epithelial principal cells in each of the segments, and there was no apparent immunoreactivity in any other cell types. Epididymal segments from one bull with a high amount of lipocalin-type PGD synthase in his seminal plasma are shown in Figure 4.
In the caput A and B regions, specific immune staining was evident throughout the cytoplasm of the principal cells but was most intense in the supranuclear regions. Stereocilia of the principal cells were also stained. Spermatozoa in the lumen did not appear to be stained, but reaction product droplets were observed within the lumen (Fig. 4, c and d). In the caput C region, overall staining intensity decreased, and there were obvious variations in staining between the principal cells (Fig. 4e). In corpus D and E regions, the principal cells displayed a checkerboard-like pattern of immunoreactivity, with some cells stained at varying degrees of intensity and other principal cells unreactive (Fig. 4, f and g). Reaction product was more evident in the epididymal lumen and appeared to increase in intensity from corpus to cauda. In the epithelium of the distal corpus and cauda, staining was localized on the stereocilia border. In the cauda, the principal cells were moderately stained, as were the stereocilia (Fig. 4, g and h). In cauda region G, the tubule lumen was also stained with reaction product, but it was not possible to determine whether there was specific staining of the epididymal spermatozoa because of high concentrations of spermatozoa (Fig. 4i). All positive immunostaining for lipocalin-type PGD synthase was eliminated by preabsorption of the anti-recombinant PGD synthase antibody with purified recombinant PGD synthase and was not detected with preimmune IgG (data not shown).
Localization of Lipocalin-Type PGD Synthase on Ejaculated Spermatozoa
Immunoreactive lipocalin-type PGD synthase was concentrated at the apical segment of the acrosome (Fig. 5, a and b). There was no detectable fluorescence in any of the controls (Fig. 5, c and d). Not all the sperm in an ejaculate were immunoreactive. We observed that bulls with a greater amount of lipocalin-type PGD synthase in their seminal plasma had a higher percentage of immunofluorescent sperm.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, we demonstrated that lipocalin-type PGD synthase, a 26-kDa fertility-associated bull seminal plasma protein, was also present in cauda epididymal fluid. In rete testis fluid, this enzyme existed as multiple molecular-mass isoforms from 29 to 31 kDa [1]. In this study, we examined crude tissue extracts of the testis and the caput, corpus, and cauda epididymidis by Western blotting and again detected molecular variants of this protein ranging from 23 to 29 kDa, depending on which tissue was examined. Recent analyses of ram and stallion testicular and epididymal fluids have also shown the existence of multiple isoforms of this enzyme in the male genital tract [27]. Since only one lipocalin-type PGD synthase gene has been described to date [28], these isoforms are most likely due to post-translational modifications of the protein within the different tissues. In relation to the total proteins found in the three epididymal segments, the proportion of the total protein represented by lipocalin-type PGD synthase varied among the different segments. Total lipocalin-type PGD synthase concentrations were highest in the tissue extracts from the caput epididymidis in all bulls examined, followed by the cauda and corpus. These observations were consistent with the variations observed by Northern blot analysis of mRNA levels within the three epididymal segments of the bull [29].
It is well recognized that Sertoli cells secrete a wide variety of proteins at both their apical and basal poles in addition to proteases and inhibitors, hormones, and growth and paracrine factors of various types. Several of these have been shown to bind to the germ cells (e.g., SGP-2, transferrin, insulin-like growth factor), and in turn, the developing germ cells are known to synthesize a variety of intracellular and membrane-associated proteins that stimulate Sertoli cell secretion (reviewed by [30, 31]). The most intense staining for lipocalin-type PGD synthase was detected within the cytoplasm of the Sertoli cells of the testis and the cytoplasm of elongating spermatids (Sc, Sd1, and Sd2) at stages III–VIII of spermatid development. Beginning with stage III of the bull spermatogenic cycle, the nuclei of Sb2 spermatids begin to elongate and the acrosome starts to develop. In addition, an abundance of smooth endoplasmic reticulum from the Sertoli cell cytoplasm associates with the developing acrosome at this stage [25, 26]. Numerous reports have described the many finger-like processes existing along the Sertoli cell surface that penetrate the cytoplasm of the developing sperm and serve as a route for exchange of materials between the two cells (reviewed by [32, 33]).
There was no detectable staining for lipocalin-type PGD synthase in any of the cell types comprising the bull testicular interstitial tissue (i.e., Leydig cells, fibroblasts, macrophages, etc.). In situ hybridization studies using tissues from the same bulls were unable to detect lipocalin-type PGD synthase transcript within the testicular interstitium [29]. In contrast, lipocalin-type PGD synthase reportedly has been localized to the Leydig cells in both mice [34] and humans [13]. These discrepancies in localization of this protein may be due to variability between different animal species. Our findings (this paper and [29]) are consistent with the work of Blodorn et al. [35] and Sorrentino et al. [36], who localized transcripts for lipocalin-type PGD synthase mRNA to the Sertoli and germ cells of the human and rat testis, respectively, but did not detect transcripts within the interstitium.
The specific role of lipocalin-type PGD synthase within the Sertoli and germ cells of the testis remains to be established. Nearly all the reported expression sites for lipocalin-type PGD synthase are epithelial and located at or near blood-tissue barriers such as the blood-brain barrier [34]. The function of these barriers is to maintain a unique and carefully controlled homeostatic environment by separating specialized tissue fluids from blood and lymph. If functioning as a lipocalin within the testis, PGD synthase may act as a transmembrane carrier protein to establish or maintain the blood-testis barrier. Other testicular proteins with a similar function include androgen-binding protein, retinol-binding protein, or ß-lactoglobulin. The latter two proteins are also members of the lipocalin superfamily [3].
Recent analyses of the lipocalin-type PGD synthase gene in both rats [37] and humans [38] have identified a thyroid hormone (3,3',5-triiodothyronine, T3) response element in the 5'-flanking region. It is well known that T3 is important for the growth, maturation, and function of the testicular Sertoli cells. Together with FSH, thyroid hormone is considered a major endocrine regulator of seminiferous epithelium development in the prepubertal animal [39]. Both Sorrentino et al. [36] and Hoffman et al. [34] have reported a 2-fold increase in lipocalin-type PGD synthase expression at the time of puberty, which would correspond to the high concentrations of hormone activity at this stage of development.
Interestingly, the T3 response element in the lipocalin-type PGD synthase gene also confers regulation by retinoic acid [37], a vitamin A derivative required by the testis for spermatogenesis and by the epididymis during sperm maturation [40]. As mentioned previously, lipocalin-type PGD synthase has been shown to bind 9-cis and all-trans retinoic acid in vitro with an affinity comparable to that of other retinoic acid-binding proteins [4]. The mechanism by which retinoids affect the development of germ cells is not known. Their action is mediated, at least in part, through nuclear retinoid receptors that are known to regulate the transcription of retinoid-responsive genes. There are two known families of retinoid receptors: the retinoic acid receptors (RARs), which recognize all-trans-retinoic acid, and the retinoid X receptors (RXRs), which recognize 9-cis retinoic acid. Each family consists of three types of receptors: , ß, and 
(reviewed in [41]). Retinoid-associated binding proteins and nuclear receptors have been shown to be expressed in both the testis and germ cells and to vary according to the stage of the spermatogenic cycle. For example, Sertoli cells express cellular retinol binding protein (CRBP), RAR, RARß, and RAR, whereas germ cells express cellular retinoic acid binding protein (CRABP) and RAR [42, 43].
Lipocalin-type PGD synthase was also immunolocalized to the cuboidal epithelial cells of the rete testis and efferent ducts. These tissues have an active role in fluid-phase, adsorptive, and receptor-mediated endocytosis. The lumen of the rete testis stained intensely for lipocalin-type PGD synthase, which was not surprising since we had previously reported the existence of this protein in rete testis fluid [1]. The immunostaining observed within the cuboidal cells of the efferent ducts may arise either because of lipocalin-type PGD synthase synthesis or because of absorption of the protein by these cells.
Within the epididymis, lipocalin-type PGD synthase was localized in the caput epididymidis while moderate to weak staining was observed in the remaining epididymal segments. Histologically, the bull epididymis is composed of a number of cell types including principal, basal, apical, halo, clear, and narrow cells, each of which vary in number and size along the epididymal duct. In all epididymal regions, expression of lipocalin-type PGD synthase was specific to the epithelial principal cells; no immunoreactivity was apparent in the other cell types. Within the principal cells, there were also differences in staining pattern and intensity for lipocalin-type PGD synthase depending on the epididymal segment. Strong uniform staining was observed throughout the principal cells in the caput epididymidis and also on the stereocilia. In comparison, a weaker staining, concentrated at the apical portion of the principal cells, was observed for the remainder of the epididymis. Interestingly, there were essentially undetectable levels of staining in the corpus or cauda segments for two of the six bulls examined.
Analogous to the blood barriers found within the brain, retina, and testis, the epididymis also has a blood barrier that partitions the lumen containing sperm and fluid secretions from the rest of the body [44]. As sperm pass through the epididymal duct, they progressively acquire the capacity for motility in the caput [45] and the ability to bind to the zona pellucida in the corpus [46, 47] and to fertilize the oocyte in the cauda [48]. Epididymal fluid components include organic solutes, ions, and proteins, which change in composition as the fluid moves from the initial caput segment to the cauda epididymidis. Epididymal proteins include transferrin, albumin, SGP-2 (clusterin), immobilin, metalloproteins, and enzymes such as glycosyltransferases, glycosidases, and glutathione peroxidase (reviewed in [49]). Obvious differences in staining patterns and intensity among the epididymal segments have been noted for quite a number of unique proteins synthesized by the epididymal epithelium. Some proteins are localized to specific regions of the epididymis and affect sperm maturation either by becoming associated with the sperm membrane or by changing the glycosylation sites of integral sperm membrane proteins. These proteins may also be important in the regulation of the structural and functional integrity of the epididymis. Several of these have been shown to be associated with spermatozoa, suggesting a role in sperm maturation and/or sperm-egg interactions [50]. Significant amounts of retinoic acid and its binding proteins and nuclear receptors are abundant in the epididymis [51]. For example, epididymal retinoic acid-binding protein (E-RABP), another member of the lipocalin superfamily, has been localized to the principal cells of the initial segment, proximal caput, and cauda. Both 9-cis and all-trans retinoic acid are found in high concentrations in the epididymis and serve as ligands for E-RABP [52]. Also, CRBP and its mRNA was found in highest concentration in the cytoplasm of the principal cells of the initial segment and proximal caput, even exceeding the levels found in the liver or testis. Similar patterns were observed for levels of RAR; highest levels were observed in the proximal caput, with dramatically lower concentrations observed in the distal caput and corpus [53, 54]. The selective localization of lipocalin-type PGD synthase in the proximal caput region of the epididymis in all bulls examined suggests that this protein plays a particular functional role at this site. It is generally believed that most of the physiological changes of spermatozoa associated with maturation are initiated in the caput epididymidal region, including the development of sperm motility and morphologic changes in the acrosome.
Clearly, lipocalin-type PGD synthase is associated with the plasma membrane of the ejaculated sperm head. It is not seen on acrosome-reacted bull sperm. The acrosome is known to contain numerous hydrolytic enzymes and proteinases that may function during the acrosome reaction [55]. Two other members of the lipocalin superfamily, lizard epididymal secretory protein (LESP) and rat epididymal protein (ESPI), also bind to the plasma membrane of spermatozoa [56, 57]. If lipocalin-type PGD synthase functions to transport retinoic acid, it is of interest to note that retinoic acid has been shown to induce disorders in phospholipid bilayers and especially to increase the membrane permeability to different ions (K+ and I-) and to glucose [58]. This may play a functional role in the membrane permeability modifications that occur during sperm maturation and/or the membrane fusion events of the acrosome reaction.
Further work is needed to elucidate the role of lipocalin-type PGD synthase in the male reproductive organs. Its relationship to overall male fertility may be the result of its ability to function as a transmembrane lipophilic carrier protein to maintain the blood-testis and blood-epididymal barriers. Overall, the specific localization of lipocalin-type PGD synthase to the spermatids and Sertoli cells of the testis and to the principal cells of the epididymis, especially within the proximal caput segment, suggests that it plays an integral role in both the development and maturation of sperm.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This research was supported by a fellowship from the Japan Society for the Promotion of Science (R.L.G.) and USDA grants #93-37203-9069 and #97-35203-4806 (G.J.K.); in part by the Grants-in-Aid for Scientific Research Program of the Ministry of Education, Science and Culture of Japan (07558108 and 07457033 to Y.U.); and by grants from the Sankyo Foundation and the Cell and Science Foundation (Y.U.). 
2 Correspondence: Gary J. Killian, J.O. Almquist Research Center, Fox Hollow Road, The Pennsylvania State University, University Park, PA 16802. FAX: 814 863 0833; lwj{at}psu.edu
Accepted: October 15, 1999.
Received: July 21, 1999.
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 K. Fujimori, K. Kadoyama, and Y. Urade Protein Kinase C Activates Human Lipocalin-type Prostaglandin D Synthase Gene Expression through De-repression of Notch-HES Signaling and Enhancement of AP-2{beta} Function in Brain-derived TE671 Cells J. Biol. Chem., May 6, 2005; 280(18): 18452 - 18461. [Abstract] [Full Text] [PDF]
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 H. Zhu, H. Ma, H. Ni, X.-H. Ma, N. Mills, and Z.-M. Yang Expression and Regulation of Lipocalin-Type Prostaglandin D Synthase in Rat Testis and Epididymis Biol Reprod, April 1, 2004; 70(4): 1088 - 1095. [Abstract] [Full Text] [PDF]
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 R. Sakai, T. Kinouchi, S. Kawamoto, M. R. Dana, T. Hamamoto, T. Tsuru, K. Okubo, and S. Yamagami Construction of Human Corneal Endothelial cDNA Library and Identification of Novel Active Genes Invest. Ophthalmol. Vis. Sci., June 1, 2002; 43(6): 1749 - 1756. [Abstract] [Full Text] [PDF]
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