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PubMed2000

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.

Animals↗
PubMed2001

Impact of epididymal maturation on the tyrosine phosphorylation patterns exhibited by rat spermatozoa.

As mammalian spermatozoa migrate through the epididymis, they acquire functionality characterized by the potential to express coordinated movement and the competence to undergo capacitation. The mechanisms by which spermatozoa gain the ability to capacitate during epididymal transit are poorly understood. The purpose of this study was to investigate the impact of epididymal maturation on the signal transduction pathways regulating tyrosine phosphorylation, because this process is thought to be central to the attainment of a capacitated state and expression of hyperactivated motility. Western blot and immunocytochemical analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues from the sperm head. As cells pass from the caput to the cauda epididymis, tyrosine phosphorylation becomes confined to a narrow band at the posterior margin of the acrosomal vesicle. Epididymal maturation of rat spermatozoa was also associated with an acquired competence to respond to high levels of intracellular cAMP by phosphorylating tyrosine residues on the sperm tail. Immature caput spermatozoa were incapable of exhibiting this response, despite the apparent availability of cAMP and protein kinase A. These findings help to clarify the biochemical changes associated with the functional maturation of spermatozoa during epididymal transit.

Acrosome↗
PubMed2001

Mouse epididymal Spam1 (PH-20) is released in vivo and in vitro, and Spam1 is differentially regulated in testis and epididymis.

The sperm adhesion molecule 1 (SPAM1 or PH-20) is an important sperm surface protein with a hyaluronidase activity and bifunctional roles in mammalian fertilization. Recently we reported that in the mouse, Spam1 is synthesized independently in the testis and the epididymis, where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Here we used mouse epididymal luminal fluid and cultured epididymal epithelial cells to demonstrate that epididymal Spam1 may be a secretory protein. Using a dual environment culture chamber system in which corpus or cauda epithelial cells are cocultured with their corresponding epididymal fibroblasts in medium supplemented with androgens and epidermal growth factor, we show that in 2- to 6-day cultures Spam1 can be detected immunocytochemically in the epithelial cells. The protein was also detected by Western blot analysis in extracts of the cultured cells and in their serum-free conditioned medium, as well as in luminal fluid from fresh caput, corpus, and caudal epididymis. Importantly, it was shown to have hyaluronidase activity, using hyaluronic acid substrate gel electrophoresis, and to be expressed in greater quantities in the corpus compared with the cauda and caput. The results not only confirm our previous finding that Spam1 is synthesized in the epididymis, but extend them by showing that it is released in the luminal fluid where it may effect posttesticular maturation and function of sperm. Results from transcript analysis indicate that epididymal and testicular Spam1 are under different transcriptional regulation.

Androgens↗
PubMed1998

Pathological epididymal obstruction unrelated to vasectomy: results with microsurgical reconstruction.

PURPOSE: We determine the success of end-to-side epididymovasostomy in patients presenting with obstructive azoospermia from primary epididymal obstruction and identify prognostic factors that could predict a successful outcome. MATERIALS AND METHODS: Of 256 consecutive patients undergoing end-to-side epididymovasostomy 49 had primary epididymal obstruction unrelated to prior vasectomy. Patients were excluded from study if they had a history of vasectomy, microsurgical reconstruction or ejaculatory duct obstruction. RESULTS: Followup was available in 43 of the 49 patients (88%). Patency rates were 87% in the bilateral epididymovasostomy group, 69% in the unilateral group and 81% overall. Pregnancy rates were 43% in the bilateral and 23% in the unilateral group. While higher patency rates were observed for proximal anastomoses, higher pregnancy rates were observed for distal anastomoses. Intraoperative epididymal fluid quality correlated with patency, that is motile sperm in the epididymal aspirate correlated with postoperative patency (p <0.05). There was no correlation between fluid quality and pregnancy rates. Postoperative semen analyses demonstrated higher sperm density and higher motility in the pregnant versus nonpregnant groups. CONCLUSIONS: Epididymovasostomy may be performed in patients with obstructive azoospermia unrelated to vasectomy with high patency and good pregnancy rates. Intraoperative epididymal fluid quality can be predictive of patency. There was a trend towards higher pregnancy rates for distal anastomosis. There were no pregnancies when anastomosis was at the caput epididymis on both sides.

Adult↗
PubMed2006

Fine needle aspiration of epididymal nodules in Chandigarh, north India: an audit of 228 cases.

BACKGROUND: Epididymal nodules are not infrequently encountered in surgical practice. These are generally small and slippery and fine needle aspiration cytology (FNAC) is not easy. But as it is rapid and less traumatic than a biopsy, this is a favoured technique in the assessment of epididymal nodules. PATIENTS AND METHODS: In the present study, all the cases of epididymal nodules aspirated from January 1998 to August 2004 were retrieved from the cytology files of the Department of Cytology, Post Graduate Institute of Medical Education and Research, Chandigarh, India. RESULTS: A total of 228 cases were retrieved and divided as follows: tuberculous epididymitis 70 (30.7%), non-specific inflammation 10 (4.4%), microfilaria 2 (0.9%), hydrocele 26 (11.4%), spermatocele 42 (18.4%), spermatic granulomas 12 (5.3%), adenomatoid tumour 3 (1.3%), leiomyosarcoma 1 (0.4%) and lipoma 1 (0.4%). Thirty-six (15.8%) cases were labelled as benign aspirate not otherwise specified. FNAC material was inadequate for opinion in 22 (9.65%) cases and three (1.3%) cases revealed evidence of a haematoma. CONCLUSIONS: FNAC was useful in the diagnosis of 90.3% of cases, thereby avoiding surgical biopsy and other investigations. Therefore, FNAC has an important role in the differential diagnosis of epididymal nodules as it can detect malignancy and benign conditions such as tuberculosis and acute and chronic epididymo-orchitis.

Biopsy, Fine-Needle↗
PubMed1980

Testicular and epididymal aldehyde dehydrogenase in rodents: modulation by ethanol and disulfiram.

The distribution of (newly found) NAD-dependent aldehyde dehydrogenase (ALDH) was determined in the rat testis and epididymis as a function of age. There were changes in specific activity of testicular ALDH as a function of age. Epididymal ALDH, which is found evenly distributed between the caput and cauda epididymis, increased initially from the 20th day after birth to th 50th day and remained little altered thereafter in both the caudet and the caput portion of the epididymis. Testicular and epididymal ALDH were found species-dependent with the hamster testis and the rat epididymis showing greater specific activities from the rest of the species studied, respectively. Determinations of testicular ALDH in various mouse strains indicate that it is strain-dependent. Intake of 25% (v/v) ethanol solution as the sole drinking fluid for 10 consecutive days inhibited testicular ALDH in the three mouse strains studied. Administration of disulfiram, 15 mg/kg/day for 60 consecutive days, exerted inhibitory action on testicular and epididymal ALDH in the rat. Intraperitoneal injection of pyrazol, 100 mg/kg once daily for 7 days, resulted in inhibition of epididymal but not of testicular ALDH. It is suggested that testicular and epididymal ALDH may play a role in the toxic action of ethanol-derived acetaldehyde on the gonad.

Alcohol Oxidoreductases↗
PubMed1983

Restricted entry of an anti-rat epididymal protein IgG into the rat epididymis.

A rat epididymal protein (MW 32000) was isolated and purified from the rat caudal epididymal fluid. Mono-specific antiserum against this protein was raised in rabbits, purified and labelled with 125I. The labelled IgG was infused intravenously into anaesthetized male rats, and the transfer of the labelled IgG across the luminally perfused cauda epididymidis was studied. It was found that during the 2 h infusion period, radioactivity in blood rose, but no radioactivity could be detected in the perfusates, irrespective of whether the epididymis was perfused with Krebs bicarbonate solution or a solution which resembled the rat caudal fluid in ionic composition. In some experiments, rats were given a single intravenous injection of labelled IgG and radioactivity in the epididymal content was measured 10 days later. It was found that despite a high IgG level in blood and liver, no radioactivity could be detected in epididymal fluid and sperm. It is concluded that the blood-epididymis barrier restricts the passage from blood to lumen, an immunoglobulin directed against an epididymal protein.

Animals↗
PubMed1994

Investigation of epididymal sperm maturation in the golden hamster.

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 +/- 12.2, 40.6 +/- 20.8, 144 [corrected] +/- 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 +/- 6.4 to 33.8 +/- 4.8 to 70 +/- 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 +/- 7.8% in the proximal caput epididymis to 57.2 +/- 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.

Animals↗
PubMedDate not supplied

The effect of a synthetic progestine on the fine structure of the epididymal head (stereological analysis).

The alterations induced in principal cells of the epididymal head tissue of the rat by administration of a synthetic progestine (R2323, ethinyl-norgestrienone) were studied by electron microscopy. A total of 3 male, sex mature rats was applicated ethinyl-norgestrienone during a 3 months period in low dosage (60 mug) per day and was studied with 5 control animals. Stereological methods devised by Weibel (1969) and Rohr et al. (1976) were applied to light and electron micrographs. The calculated values are related to 1 cm3 of epididymal tissue, 1 cm3 of epididymal cell and 1 cm3 of epididymal cell cytoplasm. The Golgi apparatus was further subdivided; the values for the Golgi compartments are therefore refered to 1 cm3 of Golgi apparatus was further subdivided; the values for the Golgi compartments are therefore refered to 1 cm3 of Golgi apparatus. In the treated animals there was, related to the unit of epididymal tissue, a significant decrease in the volume density of the interductular tissue, whereas that for the lumina was significantly higher. The volume density of the glandular epithelium remains unchanged. Refered to the unit volume of principal cell cytoplasm the rough endoplasmic reticulum shows a signficant decrease by 6.5%, whereas the smooth endoplasmic retiuclum is increased by 6.3%. The volume density of the lysosomes was calculated to be 4.3% (Control 2.5%). The stereological data of the Golgi apparatus indicate a vacuolar transformation: Increased volume of vacuoles, decreased volume fraction of saccules and vesicles. However, the volume density of the whole Golgi apparatus remains unchanged. An impaired secretory activity of the principal cells could be assumed.

Animals↗
PubMedDate not supplied

Electrophoretic characterization of mouse epididymal esterases in inbred lines and in a natural population.

Esterase isozymes were studied in mouse epididymis of two inbred strains (C57BL, DBA/2) and in a natural population (Swiss OF1), by using vertical polyacrylamide gel electrophoresis and staining with alpha or beta-naphthyl acetate as a substrate. Eighteen (C57BL), 17 (DBA/2) or 16 (Swiss OF1) epididymal isozymes were separated; four were common to the testis, and five to both the testis and the serum. The use of different inhibitors showed that carboxylesterase activities account for the greater part of the total epididymis non-specific esterase activity. This comparative study revealed minor interspecies variations since only two isozymes were not expressed in the same manner in the three populations examined. Among the nine isozymes which appeared solely in the epididymis, the profiles varied between tissues and fluids as well as between the proximal part in which sperm maturation occurs and the distal part where sperm storage takes place. The variations proceeded from the relative activity of isozymes and the presence or absence of some of them; two characterized the proximal part and one the distal part in the three species. By comparing testis and epididymal tissues and fluids, it is suggested that the isozymes found in epididymal fluids originated from the testis, the epididymal epithelium or both. In addition to this epididymal secretory function, the lack in the fluid of the distal part of one isozyme identified in the testis, and two in the proximal part may also provide evidence for its reabsorptive function.

Animals↗
PubMed2006

Effects of egg yolk and cooling rate on the survival of refrigerated red deer (Cervus elaphus hispanicus) epididymal spermatozoa.

Egg yolk is a common component to sperm refrigeration for most of the deer species, the role of which is to protect sperm membranes against cold shock. In addition, there have been many studies of conservation of ejaculated semen from stags, but few have been reported for epididymal spermatozoa. This work was designed to investigate the combined effects of cooling rates (slow: 0.23 degrees C/min vs rapid: 4.2 degrees C/min) from room temperature to 5 degrees C, and egg-yolk concentration (0, 5 or 20%) in the extender on the survival of Iberian red deer epididymal spermatozoa refrigerated at 5 degrees C. Heterospermic sperm samples were diluted to a final sperm concentration approximately 400x10(6) sperm/ml with a Tris-citrate-fructose (TCF)-egg-yolk diluent. Sperm quality was in vitro judged by microscopic assessments of individual sperm motility [sperm motility index (SMI)], and of plasma membrane (hypo-osmotic swelling test) and acrosome (NAR) integrities. Our results first showed that the presence of egg yolk in the extender significantly improves (p=0.01) the viability and sperm motility after sperm dilution. In addition, acrosome and plasma membrane integrities post-refrigeration did not differ significantly between cooling procedures; however, the SMI differed significantly between cooling procedures (slow: 46.6% vs rapid: 50.0%; p=0.01). Our results also showed that sperm quality was significantly (p<0.01) affected by the combined effects of egg-yolk concentration and cooling procedure, being rapid cooling with 20% of egg yolk the most suitable combination for epididymal sperm refrigeration. In conclusion, egg-yolk improved red deer epididymal spermatozoa characteristics after dilution. Rapid cooling protocol using TCF with 20% egg-yolk significantly improved sperm motility of red deer epididymal spermatozoa after cooling.

Animals↗
PubMed1996

Different subtypes of alpha 1A-adrenoceptor mediating contraction of rat epididymal vas deferens, rat hepatic portal vein and human prostate distinguished by the antagonist RS 17053.

1. The alpha 1-adrenoceptor subtype mediating contraction of the rat hepatic portal vein to phenylephrine was characterized by use of competitive antagonists previously shown to have selectivity between the expressed alpha 1-subtype clones. Prazosin competitively antagonized the phenylephrine contractions with a pA2 value of 9.2, as did WB 4101 (pA2 9.4), 5-methyl urapidil (pA2 8.6), indoramin (pA2 8.4) and BMY 7378 (pA2 6.5). 2. The pA2 values on the rat portal vein correlated highly with their previously published pA2 values for the alpha 1A-adrenoceptors mediating contraction of the rat epididymal vas deferens and human prostate and poorly with those for the alpha 1B- and alpha 1D-adrenoceptors mediating contraction of the rat spleen and aorta, respectively. The antagonist pA2 values on the rat portal vein correlated highly with their previously published pK1 values for the expressed alpha 1a-clone and poorly with those for the expressed alpha 1b- and alpha 1d-clones. Therefore the results show that contraction of the rat portal vein to phenylephrine is mediated by alpha 1A-adrenoceptors. 3. The novel alpha 1-adrenoceptor antagonist RS 17053 had a relatively high affinity for the alpha 1A-adrenoceptors mediating contraction of the rat epididymal vas deferens (pA2 9.5) compared with the alpha 1B-adrenoceptors in the rat spleen (pA2 7.2) or the alpha 1D-adrenoceptors in the rat aorta (pKB 7.1), in agreement with its selectivity for the expressed alpha 1a-clone. However, RS 17053 had over 100 fold lower affinity for the alpha 1A-adrenoceptors mediating contraction of the rat portal vein (pKB 7.1) and human prostate (pKB 7.1) compared with its affinity for the alpha 1A-adrenoceptors in the rat epididymal vas deferens or the expressed alpha 1a-clone. 4. The difference in affinity of RS 17053 between the rat epididymal vas deferens and rat portal vein cannot be explained by a species difference in the receptor. Therefore RS 17053 may distinguish between subtypes of the alpha 1A-adrenoceptor in the rat portal vein and human prostate compared with those in the rat epididymal vas deferens or the expressed alpha 1a-clone.

Adrenergic alpha-1 Receptor Agonists↗
PubMed1995

Detection of antibodies toward epididymal sperm antigens--an obligatory step in evaluation of human immunologic infertility?

PROBLEM: To test the relative impact of epididymal versus ejaculated sperm in immunologic infertility. METHOD: Human antibody binding to epididymal and ejaculated spermatozoa was compared by flow cytometry (FCM) since it allows quantitative analysis of viable sperm while ignoring nonsperm cells. To select sera for FCM, GAT, TAT, and ELISA were applied on 145 sera from fertile men, idiopathically infertile and varicocele patients. RESULTS: All GAT/TAT-positive infertile patients, a representative group of varicocele patients and the fertile control, were assessed by FCM. Higher reactivity toward epididymal sperm revealed 18/22 sera while only four out of them bound to ejaculated sperm stronger than the control. All varicocele sera were positive against epididymal while negative against ejaculated spermatozoa. CONCLUSIONS: Epididymal sperm antigens may play a predominant role in some cases of immunologic infertility. Such patients might not be adequately diagnosed and respectively treated due to the limitations of diagnostic procedures applying only ejaculated spermatozoa.

Antigens, Surface↗
PubMed2005

Sustained rise in triacylglycerol synthesis and increased epididymal fat mass when rats cease voluntary wheel running.

Four-week-old, Fischer-Brown Norway F1-generation male rats were given access to voluntary running wheels for 21 days, and then the wheels were locked for 5 (WL5), 10 (WL10), 29 (WL29), or 53 (WL53) hours. Two other groups (SED5 and SED10) had no access to voluntary running wheels and were killed at the same time as WL5 and WL10, respectively. Absolute and relative epididymal fat mass, mean cell volume, and amount of lipid per cell increased in WL53 relative to all other groups, with no change in cell number. C/EBPalpha protein levels in epididymal fat were 30% greater in SED5 than in WL5. The rate of triacylglycerol synthesis in epididymal fat was 4.2-fold greater in SED5 than in WL5, increased 14-fold between WLS and WL10, and was 79% lower in SED10 than in WL10. Triacylglycerol synthesis remained at this elevated level (at least 3.5-fold greater than SED5) through WL53. Thus, the rapid increase in epididymal fat mass with the cessation of voluntary wheel running is associated with a prolonged overshoot in epididymal fat triacylglycerol synthesis. Moreover, rats without running wheels had a 9.4% lower body mass after 21 days than those with running wheels. The individual mass of seven different muscles from the hindlimb, upper forelimb, and back were each lower in animals without running wheels, suggesting that physical activity in rapidly growing rats may be requisite for optimal muscle development.

Adipose Tissue↗
PubMed2003

Sperm retrieval procedures and intracytoplasmatic spermatozoa injection with epididymal and testicular sperms.

INTRODUCTION: Male infertility caused by azoospermia due to non-reconstructable obstruction or non-obstructive azoospermia can be treated by microsurgical epididymal aspiration (MESA) or testicular sperm extraction (TESE) followed by an intracytoplasmatic spermatozoa injection (ICSI). MATERIAL AND METHODS: From 9/93 to 6/01, we carried out 1,025 ICSI procedures with aspirated epididymal or testicular sperms in 684 cases. 163 ICSI cycles were performed with epididymal sperms and 862 ICSI cycles with testicular sperms or spermatids. The TESE was carried out by open biopsy, frequently in a multilocular technique. The aspirated spermatozoas were used after cryopreservation (frozen) or immediately after aspiration (fresh). RESULTS: 538 patients had obstructive azoospermia or ejaculation failure. In 487 cases the underlying cause of azoospermia was an impaired spermatogenesis, following maldescensus testis, chemotherapy, radiotherapy, or caused by Sertoli-cell-only syndrome, a genetic disorder or an unknown etiology. The transfer rates, pregnancy rates and birth rates per ICSI cycle showed no statistically significant differences between testicular and epididymal sperms in the cases of seminal obstruction (28% average birth rates in both cases). However, highly significant was the difference in birth rates with regard to the underlying cause of infertility. In contrast, in treating non-obstructive azoospermia we observed a birth rate of 19% per cycle. In all patient groups the birth rate with fresh spermatozoas did not differ from those with cryopreserved spermatozoa. 40% of patients after multilocular TESE showed clinical signs of testicular lesion. CONCLUSION: The underlying cause of azoospermia is the most important factor for the outcome of ICSI using epididymal and testicular sperms. In cases of non-obstructive azoospermia, the pregnancy rate is low compared with the results in cases of obstructive azoospermia. There is no difference between fresh and cryopreserved sperms. TESE with ICSI is the most efficient treatment of azoospermia caused by hypergonadotropic hypogonadism. The morbidity of the TESE procedure is highly relevant and must be considered if this technique is indicated.

Adult↗
PubMed2006

Regulation of spontaneous contractile activity in the bovine epididymal duct by cyclic guanosine 5'-monophosphate-dependent pathways.

Passage of spermatozoa through the epididymis is obligatory for sperm maturation processes and is based on spontaneous phasic contractions (SC) of the epididymal duct. Here, the functional role of cyclic GMP (cGMP) signaling in modulating SC in the bovine epididymal caput and corpus region was examined by muscle tension recording and immunological and autoradiographic techniques. The cGMP-analog 8-bromo (Br)-cGMP, as well as the nitric oxide (NO) donor sodium nitroprusside and the natriuretic peptides (NPs) atrial NP and C-type NP, displayed distally increasing SC-relaxant effects. In agreement, a distally increasing epididymal expression of the cGMP-dependent protein kinase I (PKG I), endothelial NO synthase (eNOS), and the atrial NP receptor was found. Immunoreactivity for PKG, soluble guanylate cyclase, and eNOS could be localized to the epididymal muscle cells as well as to the epithelial basal cells only at the corpus level. The SC-relevant action of NO and the NPs was cGMP dependent, and the action of 8-Br-cGMP, in turn, was modified by epithelial and luminal factors. The NOS inhibitor L-NAME (N(omega)-nitro-L-arginine methyl ester) caused an increase in SC frequency, indicating basal activity of NO generating enzymes. The SC-inhibitory effect of 8-Br-cGMP was clearly reduced by the PKG inhibitor Rp-8-Br-cGMPS as well as by iberiotoxin, thapsigargin, and indomethacin, pointing to PKG as main SC-relevant target of cGMP, and to large-conductance calcium-activated K(+) channels, the sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase and cyclooxygenase-1 as possible targets of PKG. These data support an essential role of cGMP signaling in the control of epididymal peristalsis, thereby enabling fine tuning of sperm transport and maturation.

Animals↗
PubMed1993

Evidence for a novel adenylyl cyclase in human epididymal sperm.

Since cAMP is considered to play a major role in the acquisition of maturation and fertilizing capacity of mammalian sperm, we investigated the expression of cAMP-synthesizing adenylyl cyclase (AC) in sperm retrieved directly from the human epididymis. Particulate fractions were prepared from purified epididymal sperm samples and AC was monitored by the direct conversion of ATP into cAMP. We report that in great contrast to human ejaculated sperm and other mammalian sperm cells, the human epididymal sperm do not express a Mn(2+)-sensitive AC. However, a functional AC was readily detectable in these sperm cells in the presence of saturating concentrations of Ca2+ (50mM) and bicarbonate (HCO3-, 50mM), a combination that causes maximal activation in human ejaculated sperm. Using these conditions, human epididymal sperm AC showed similar capacity to generate cAMP compared to human ejaculated sperm AC. When assays were performed in the presence of Mg2+ and a saturating concentration of GMP-P(NH)P (50 microM), the hydrolysis-resistant GTP analog, and forskolin (100 microM), no activity was detected indicating that the epididymal sperm AC differs from that in somatic cells. These data demonstrate that human epididymal sperm contain an AC that is unique and different from the enzyme system described in somatic cells and other mammalian sperm cells, including human ejaculated sperm.

Adenylyl Cyclases↗
PubMed1998

Molecular cloning and hormonal regulation of a murine epididymal retinoic acid-binding protein messenger ribonucleic acid.

A complementary DNA encoding the mouse epididymal secretory protein MEP 10 (mouse epididymal protein 10) was cloned and is now renamed murine epididymal retinoic acid binding protein (mE-RABP). The analysis of the predicted primary amino acid sequence showed that mE-RABP has a 75% identity with rat ESP I (epididymal secretory protein I), another epididymal retinoic acid-binding protein. The homology strongly suggests that mE-RABP is the mouse orthologue of rat ESP I. A computer analysis of the predicted three-dimensional structure confirmed that mE-RABP can accommodate retinoic acid as ligand. In the rat, ESP I messenger RNA (mRNA) is expressed in the efferent ducts and in the entire caput epididymidis. However, in the mouse, the expression of a 950-bp mE-RABP mRNA was detected only in principal cells of the mid/distal caput epididymidis, suggesting that the regulation of region-specific expression is different in rat and mouse. Northern blot analyses showed that mE-RABP gene expression is no longer detected 10 days after castration but progressively rebounds between days 15 and 60. However, mE-RABP protein could not be detected by Western blot 30 days after castration. Androgen replacement, begun 5 days after castration and continued for 4 days restored significant expression of mE-RABP mRNA. Efferent duct ligation for 10 days did not affect gene expression. Taken together, these results indicate that mE-RABP mRNA expression is regulated by androgens but not by testicular factors. The overall similarity in the primary amino acid sequence of mE-RABP with ESP I and other members of the lipocalin superfamily suggests that they are evolutionarily related.

Amino Acid Sequence↗