Protein D is differentially expressed and regulated in the rat epididymis.

We report the use of a sensitive and specific enyzme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) to study the expression of protein D, a major androgen-regulated sperm-binding glycoprotein at the protein and mRNA level in different anatomical regions of the rat epididymis. The concentration of protein D in the caput, corpus and cauda region of the epididymis was 10.2 +/- 0.67, 7.3 +/- 0.61 and 22.8 +/- 1.34 ng/micrograms total protein, respectively. The total RNA extracted from the caput, corpus and cauda regions of the rat epididymis was amplified by PCR with oligonucleotide primers specific for the 5' and 3' portion of protein D cDNA. Compared to the caput and cauda region, a significant reduction (greater than 82 +/- 3%) in the expression of protein D mRNA levels was observed for corpus epididymal RNA. This data demonstrates regional differences in the concentration of protein D and suggests that protein D expression may be regulated at the level of mRNA within the corpus epididymidis.     

Immunolocalization of androgen receptor in the epididymis of rats with dihydrotestosterone deficiency

Dihydrotestosterone (DHT), 5alpha-reduced metabolite of testosterone, is the most potent androgen in the epididymis. The conversion of T into DHT is carried out by 5alpha-reductase. The activity of 5alpha-reductase type 2, preferentially expressed in the epididymis can be inhibited by a finasteride (a steroid-based specific inhibitor of 5alpha-reductase type 2) which results in DHT deficiency. The aim of the study was to examine the morphology of epididymis and the immunolocalization of an androgen receptor (AR) in the initial segment, caput and cauda epididymis of rats treated with finasteride for 56 days. There were no morphological changes in the morphology of epididymal epithelium in the experimental rats. Immunostainable AR was localized in nuclei of epithelial cells, smooth muscle cells and mainly in the cytoplasm of interstitial cells in the epididymis of control rats. In the epididymis of experimental rats, AR immunostaining was noticed mainly in the cytoplasm of epithelial cells and interstitial cells. The single cells of the initial segment epithelium, basal cells and smooth muscle cells of cauda epididymis showed nuclear AR staining. In conclusion, finasteride affected the expression of the AR in the rat epididymis without changing the morphology of epididymal epithelium. Altered AR expression reflected the hormonal status within the epididymis.     

Expression and regulation of lipocalin-type prostaglandin d synthase in rat testis and epididymis

Lipocalin-type prostaglandin D synthase (L-PGDS), a bifunctional protein, is expressed in the male reproductive organs of many species. However, the expression and regulation of L-PGDS in rat are still uncertain. The present study investigated the regionalization and regulation of L-PGDS expression in rat testis and epididymis by in situ hybridization and immunohistochemistry under the conditions of sexual maturation, castration, and ethylene dimethane sulfonate (EDS) treatments. In sexually mature rats, L-PGDS mRNA was weakly expressed only in the testicular peritubular cells, whereas L-PGDS immunostaining was highly detected in the Leydig cells by Day 70 postpartum. During sexual maturation, L-PGDS mRNA expression was highly detected in the caput, corpus, and cauda of the epididymis 70 days after birth. Compared with normal L-PGDS expression in adult epididymis, both L-PGDS mRNA expression and protein immunostaining were significantly reduced in the caput, corpus, and cauda epididymis after castration. Testosterone propionate treatment induced a significant increase of L-PGDS expression in the epididymis of castrated rats. Compared with adult rat epididymis, L-PGDS mRNA and protein expression was down-regulated after EDS treatment. Testosterone propionate treatment could induce an increase of L-PGDS mRNA and protein expression in the epididymis of EDS-treated rats. In conclusion, both castration and EDS treatments caused a significant decrease of L-PGDS expression in the epididymis, whereas testosterone propionate treatment could induce an increase of L-PGDS expression in the epididymis of both castrated and EDS-treated rats, indicating that L-PGDS expression in the rat epididymis can be up-regulated by testosterone.     

Expression of transforming growth factor-β isoforms in the rat male accessory sex organs and epididymis

We studied the expression and distribution of transforming growth factor-β (TGF-β) isoforms in the rat male accessory sex glands and the epididymis. Our data demonstrate the expression of both TGF-β1 and -β3 isoforms in ventral prostate (VP), seminal vesicle (SV), coagulating gland (CG), and epididymis (E) by Northern blot analysis. In addition, there was differential expression of TGF-β3 in the three regions of epididymis, the corpus region being the highest. Immunostaining data showed intense staining for latent TGF-β1 in all the male accessory glands. In contrast, no staining using antibodies specific for active TGF-β1 was observed. No expression of TGF-β2 was evident either by immunohistochemistry or Northern blot analysis. The presence of mature TGF-β3 protein was observed in the secretory epithelium of VP, CG, and corpus E. There was no detectable staining of TGF-β3 in the seminal vesicle and caput and cauda regions of epididymis. These data suggest possible differential regulation of TGF-β isoform expression in the male reproductive system and predict unique roles for individual TGF-β isoforms in sperm maturation and maintenance.     

Expression of urocortin and its receptors in the rat epididymis

Urocortin (UCN; 40 aa) is a corticotrophin-releasing hormone (CRH)-related peptide. The biological actions of CRH family peptides are mediated by two types of G-protein-coupled receptors, CRH type 1 receptor (CRHR1) and CRH type 2 receptor (CRHR2). The biological effects of the peptides are mediated and modulated not only by CRH receptors but also by a highly conserved CRH-binding protein (CRHBP). The aim of the present study was to investigate the expression of UCN, CRHR1, CRHR2 and CRHBP by immunohistochemistry, Western blot, RT-PCR and real-time RT-PCR in the rat epididymis. Urocortin, CRHR1 and CRHR2, but not CRHBP, were expressed in all segments of the rat epididymis. Specifically, UCN- and CRHR2-immunoreactivities (IRs) were distributed in epididymal epithelial cells of the caput, corpus and cauda. CRHR1-IR was found in the fibromuscular cells surrounding the epididymal duct and in the smooth musculature of the blood vessels throughout the organ. UCN and CRHR2 mRNA expression levels were higher in the caput and corpus than in the cauda, while CRHR1 mRNA level was higher in the cauda than those in the caput and corpus. In summary, UCN, CRHR1 and CRHR2 are expressed in the rat epididymis. It is suggested that CRH-related peptides might play multiple roles in the maturation and storage of spermatozoa.     

Changes in distribution of phosphomannosyl receptors during maturation of rat spermatozoa

The aim of the present work was to study the distribution of the cation-independent (CI) and cation-dependent (CD) mannose-6-phosphate receptors (MPRs) in spermatozoa obtained from either rete testis or three regions of rat epididymis. We observed that both receptors underwent changes in distribution as spermatozoa passed from rete testis to cauda epididymis. CI-MPR was concentrated in the dorsal region of the head in rete testis sperm and that this labeling extended to the equatorial segment of epididymal spermatozoa. CD-MPR, however, changed from a dorsal distribution in rete testis, caput, and corpus to a double labeling on the dorsal and ventral regions in cauda spermatozoa. The percentages of spermatozoa that showed staining for either CI-MPR or CD-MPR increased from rete testis to epididymis. The observed changes were probably the result of a redistribution during transit rather than an unmasking of receptors. The fluorescence corresponding to CD-MPR and CI-MPR on the dorsal region disappeared when caudal spermatozoa underwent the acrosomal reaction. Receptors were localized on the plasmalemma of spermatozoa, as observed by immunoelectron microscopy. Changes in distribution may be related to a maturation process, which suggests new roles for the phosphomannosyl receptors.     

Identification and characterization of a new member of serpin family- Hon-grES1 in rat epididymis

A full length cDNA named HongrESl was isolated and cloned by screening rat epididymis cDNA library using a mouse EST as a probe and 5'RACE followed. It contained 1590bp nucleotides and its predicted protein had 415 amino acid residues including a serpin (serine protease inhibitor) conserved domain. Tissue distribution pattern showed it was specifically expressed in adult rat epididymis; moreover, in situ hybridyza-tion indicated this gene was expressed in a limited region of the cauda epididymis near vas deference. Such kind of expression pattern sugested that HongrESl had potential function in male reproduction.     

Expression and regulation of H+K+ATPase in lysosomes of epithelial cells of the adult rat epididymis

Endocytosis is an important event in the epididymis as it contributes to a luminal environment conducive for sperm maturation. Principal and clear cells contain numerous lysosomes which degrade many substances internalized by endocytosis from the epididymal lumen. The interior of the lysosomes depends on low pH to activate the release of their enzymes and to activate their acid hydrolases. In the present study, H+K+ATPase was localized by light microscopy in the adult rat epididymis of intact and of orchidectomized animals supplemented with testosterone or not. In normal animals, numerous lysosomes of nonciliated cells of the efferent ducts were intensely reactive for anti-H+K+ATPase antibody. In the initial segment, only a few lysosomes of principal cells were reactive. In the intermediate zone of the epididymis, numerous lysosomes of principal cells were intensely reactive, while the number of intensely reactive lysosomes decreased progressively from the proximal caput to the distal caput with none being seen in the proximal corpus region. In the distal corpus and cauda regions, only a few lysosomes of some principal cells were reactive. In contrast, clear cells of all regions showed intense reactivity. Orchidectomy resulted in the abolishion of H+K+ATPase in lysosomes of principal cells of all regions except the initial segment. However, while clear cells of the caput and corpus regions also became unreactive, those of the cauda region remained as reactive as in controls. Orchidectomized animals supplemented with testosterone maintained a staining pattern similar to controls for both cell types. These observations demonstrate the presence in principal and clear cells of H+K+ ATPase which may have an important role in acidifying the interior of their lysosomes. However, there is a region-specific expression of H+K+ATPase in lysosomes of principal cells, unlike that for clear cells. In addition, H+K+ATPase expression in lysosomes of principal cells depends on testosterone in all regions except the initial segment. However, in the case of clear cells, only those of the caput and the corpus regions are dependent on testosterone, while those of the cauda region appear to be regulated by some other factor.     

The Rhox5 homeobox gene regulates the region-specific expression of its paralogs in the rodent epididymis

The mechanisms by which the region-specific expression patterns of clustered genes evolve are poorly understood. The epididymis is an ideal organ to examine this, as it is a highly segmented tissue that differs significantly in structure between closely related species. Here we examined this issue through analysis of the rapidly evolving X-linked reproductive homeobox (Rhox) gene cluster, the largest known homeobox gene cluster in metazoans. In the mouse, we found that most Rhox genes are expressed primarily in the caput region of the epididymis, a site where sperm mature and begin acquiring forward motility. This region-specific expression pattern depends, in part, on the founding member of the Rhox cluster--Rhox5--as targeted mutation of Rhox5 greatly diminishes the expression of several other family members in the caput region. In the rat, Rhox5 expression switches from the caput to the site of sperm storage: the cauda. All Rhox genes under the control of Rhox5 in the mouse epididymis display a concomitant change in their regional expression in the rat epididymis. Our results lead us to propose that widespread changes in the region-specific expression pattern of genes over evolutionary time can be the result of alterations of one or only a few master regulatory genes.     

Short-term effect of androgen deprivation on intraluminal pressure and contractility of the rat epididymis

Short-term effects of bilateral castration, cyproterone acetate and unilateral efferent duct ligation on intraluminal pressures and spontaneous contractions in different regions of the epididymis were studied in the rat. Ligation of the efferent ducts for 5 days did not alter pressures or spontaneous contractions in any region of the epididymis. However, bilateral castration produced time-dependent changes in pressures and contractions in different segments. In the caput, the amplitude, but not the basal pressure or the frequency, of spontaneous contractions increased by Day 1 after operation. In the corpus, increments in the basal pressure and the amplitude of contractions occurred by Day 5 whilst the frequency of contractions was not changed. Similar effects were observed in the cauda by 3 days after castration. Changes in all regions of the epididymis were also mimicked by cyproterone acetate treatment (10 mg/rat per day, s.c. for 21 days). In addition, this drug increased the amplitude of contractions in the cauda. The effect of castration was abolished by testosterone propionate (0.2 mg/kg per day, i.m. for 5 days). The results support the suggestion that an enhancement of sperm transport through the rat epididymis occurs shortly after castration. The results also suggest that, in normal rats, androgens suppress the contractility of the epididymal tubule to ensure an optimal rate of sperm transport.     

Identification and characterization of a new member of serpin family- HongrES1 in rat epididymis

A full length cDNA named HongrES1 was isolated and cloned by screening rat epididymis cDNA library using a mouse EST as a probe and 5'RACE followed. It contained 1590bp nucleotides and its predicted protein had 415 amino acid residues including a serpin (serine protease inhibitor) conserved domain. Tissue distribution pattern showed it was specifically expressed in adult rat epididymis; moreover, in situ hybridyzation indicated this gene was expressed in a limited region of the cauda epididymis near vas deference. Such kind of expression pattern sugested that HongrES1 had potential function in male reproduction.     

Tissue and cell specificity of immobilin biosynthesis

The mechanisms for the initiation of sperm motility have been poorly understood until recently. Immobilin is a novel mucin glycoprotein of high molecular weight found in the cauda epididymis of the rat that, at concentrations equivalent to those found in native cauda epididymal fluid, reversibly inhibits sperm motility. In this study, immobilin was purified from rat cauda epididymal fluid to apparent homogeneity and used to generate polyclonal antibody in rabbits. The antibody was characterized by immunoblotting, and immunofluorescence was used to localize immobilin in paraffin sections of components of the reproductive system of adult male rats. Immobilin was not detectable in the efferent duct and was first detectable in the apical portion of some epithelial cells of the initial segment of the caput epididymis. Immobilin was detectable intracellularly only in cells of the caput epididymis. In the corpus and cauda epididymis immobilin was detectable only in the lumen of the tubules. Immunoprecipitation of immobilin radiolabeled in vitro confirmed that immobilin biosynthesis in the adult rat is restricted to the caput epididymis. Principal cells in the caput epididymis synthesize immobilin and secrete it into the lumen of the tubules to travel with the sperm into the cauda.     

Lactoferrin expression and secretion in the stallion epididymis

Lactoferrin is one of the most abundant proteins secreted by the stallion epididymis, but its cellular localization and regulation remain unknown. This study was designed to address the following objectives: (1) identify the epididymal cell types producing lactoferrin in pre-pubertal, peri-pubertal and post-pubertal animals; (2) demonstrate that lactoferrin binds to stallion sperm; and (3) determine if testosterone and estradiol regulate lactoferrin secretion in vitro. Using an immunohistochemical method, lactoferrin was localized in the cytoplasm of principal cells in the corpus and cauda of peri- and post-pubertal animals. The epididymis of pre-pubertal animals did not express lactoferrin. Immunolabeling of lactoferrin was also observed on the mid-piece and tail of the sperm. The role of estradiol and testosterone in regulating secretion of lactoferrin in the post-pubertal epididymis was investigated using tissue culture methods. Lactoferrin concentration in the culture media was determined by validated enzyme-linked immunosorbent assays (ELISA). Testosterone did not increase the concentration of lactoferrin in the media in any epididymal region. In contrast, estradiol-17β significantly increased the concentration of lactoferrin in the media containing tissue from the cauda. In conclusion, the expression of lactoferrin was found in the cytoplasm of principal cells in the corpus and cauda of the epididymis in peri- and post-pubertal stallions but not pre-pubertal stallions. Furthermore, lactoferrin binds to sperm, suggesting a biological role for protection or regulation of sperm in the corpus and cauda. In addition, estrogen appears to regulate lactoferrin secretion in the cauda of the epididymis in post-pubertal stallions.     

血管内皮生长因子蛋白在大鼠睾丸和附睾的表达和定位研究

为探讨血管内皮生长因子(VEGF)在雄性生殖系精子发生发育和成熟过程中的调控作用,应用免疫组化、Periodic acid-Schiff(PAS)染色及蛋白质免疫印迹技术,检测VEGF蛋白在成年大鼠睾丸和附睾的表达和定位情况。Western-blots显示,在大鼠睾丸和附睾内均有VEGF蛋白(约45kD)的表达;免疫组化显示,睾丸内VEGF见于圆形和长形精子细胞、Sertoli细胞和Leydig细胞,免疫阳性产物位于细胞质内。精子细胞的VEGF表达伴随精子细胞项体发育的全过程,精子残余体呈强阳性。附睾内VEGF表达于附睾管上皮,且有区域和细胞特异性。附睾起始段的所有上皮主细胞内都有VEGF阳性颗粒;头、体、尾各段的VEGF阳性细胞多数与含PAS阳性颗粒的细胞重合,证明为亮细胞;近端附睾的管腔内可见精子头部呈VEGF阳性染色。睾丸、附睾间质血管内皮为VEGF阴性。上述结果表明,VEGF蛋白可由生殖细胞和附睾管上皮细胞直接产生,它可能以自分泌和／或旁分泌的形式共同作用于睾丸和附睾的生殖细胞和血管内皮,直接或间接影响精子的发生、发育和成熟过程,特别是精子顶体的形成过程,并可能与精子在附睾内的成熟有关。     

Expression and regulation of LRP‐2/megalin in epithelial cells lining the efferent ducts and epididymis during postnatal development

Low density lipoprotein receptor‐related protein‐2/megalin (LRP‐2) is a receptor belonging to the low density lipoprotein receptor family that mediates endocytosis and lysosomal degradation of a variety of ligands including apolipoprotein J (Apo J)/clusterin/SGP‐2. LRP‐2 has been shown to be expressed regionally in the adult rat epididymis. In this study, we describe the pattern of expression of LRP‐2 in the efferent ducts and epididymis during postnatal development of the rat and examine the role of testicular luminally derived substances on its expression. The expression of LRP‐2 was analyzed immunocytochemically in tissues of normal animals ranging in age from postnatal day 7–90 and in 15‐day‐old efferent‐duct‐ligated animals sacrificed at later ages. In the efferent ducts, LRP‐2 expression, appearing as a dense band on the apical surface of the nonciliated epithelial cells, was noted as early as day 7, well before the entry of sperm, Sertoli‐cell‐derived secretory products, and high levels of androgens. Efferent duct ligation studies further revealed that expression under this condition was comparable to controls at all later ages examined, suggesting that the factor regulating its expression was not a luminally derived testicular substance. In normal untreated animals, LRP‐2 expression was not apparent at any of the ages examined in the proximal initial segment of the epididymis. By comparison, the distal initial segment, although having no LRP‐2 expression from 7–15 days, showed expression in principal cells by day 21 which intensified at days 29 and 39. However, by day 49 and at later ages (56 and 90), LRP‐2 immunoreactivity over principal cells became spotty or with weak or moderate reactivity in some cells and none in others. LRP‐2 expression in the intermediate zone, proximal caput, corpus, and cauda regions also appeared in principal cells by day 21, intensified at days 29 and 39 and persisted as such at all later ages examined, correlating with high levels of androgens shown to occur by day 39. Although LRP‐2 expression in the distal caput region was evident in principal cells at days 21 and 29, it became spotty with weak, moderate, or absent reactivity over principal cells at all later ages. These data suggest that LRP‐2 expression is under the influence of both stimulatory and region‐specific inhibitory factors. Analysis of 15‐day‐old efferent‐duct‐ligated animals at all later ages examined revealed that there was no change in LRP‐2 expression along the entire epididymis, suggesting that both the stimulatory and inhibitory factors are not luminally derived testicular substances. The observed pattern of LRP‐2 expression in all regions of the epididymis, except the distal caput region, was similar to that described for Apo J internalization by principal cells during postnatal development, showing a correlation between LRP‐2 expression and its ligand, Apo J. In summary, LRP‐2 expression in the epididymis undergoes region‐specific changes during postnatal development and appears to be influenced by both stimulatory and inhibitory factors. Mol. Reprod. Dev. 53:282–293, 1999. © 1999 Wiley‐Liss, Inc.