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PubMed2006

Epididymal genomics and the search for a male contraceptive.

This report represents the joint efforts of three laboratories, one with a primary interest in understanding regulatory processes in the epididymal epithelium (TTT) and two with a primary interest in identifying and characterizing new contraceptive targets (DSJ and SAJ). We have developed a highly refined mouse epididymal transcriptome and have used it as a starting point for determining genes in the human epididymis, which may serve as targets for male contraceptives. Our database represents gene expression information for approximately 39,000 transcripts, of which over 17,000 are significantly expressed in at least one segment of the mouse epididymis. Over 2000 of these transcripts are up- or down-regulated by at least four-fold between at least two segments. In addition, human databases have been queried to determine expression of orthologs in the human epididymis and the specificity of their expression in the epididymis. Genes highly regulated in the human epididymis and showing high tissue specificity are potential targets for male contraceptives.

Animals↗
PubMed2005

Spermatozoa nucleus takes up lead during the epididymal maturation altering chromatin condensation.

Lead (Pb) alters sperm chromatin condensation (CC) and the mechanisms are investigated. During spermatogenesis, protamines replace histones and disulfide bonds formation during epididymal maturation condense the chromatin. We evaluated sperm Pb uptake in testis and epididymis and the effects on CC in mice (0.06% Pb(2+)/16 weeks/drinking water). Spermatozoa from caput epididymis (CP) and cauda epididymis-vas deferens (CE-VD) were obtained and CC was measured by SCSA. Lead levels in spermatozoa from CP were lower than those from CE-VD, and correlated with a decreased CC, while Pb in CE-VD spermatozoa correlated with an increased CC. Lead accumulation into the nucleus was observed and Pb binding to nuclear sulfhydryl groups decreased chromatin decondensation in vitro. Our results suggest that spermatozoa take up Pb during testicular development and epididymal transport and alter CC, depending of the timing of Pb incorporation into the sperm nucleus, which finally may interfere with the chromatin decondensation process after fertilization.

Animals↗
PubMed2005

Evaluation of boar sperm maturation after co-incubation with caput, corpus and cauda epididymal cultures [corrected].

Boar sperm from the proximal caput epididymis were co-incubated with 1, 4, 7, 10 and 14-day old caput, corpus and cauda epididymal cultures for 24, 48 and 72 h. Boar kidney epithelial cells (LLC-PK1) and ECM alone were used as negative controls. Sperm motility, morphology and membrane integrity were studied to evaluate boar sperm maturation in vitro. Our results showed that epithelial cell monolayers (10, 14-day old) create a suitable microenvironment for the survival of proximal caput sperm and the maintenance of sperm motility over a 72 h period. Moreover, corpus epididymal tubule fragments in culture (1, 4-day old) are capable of promoting the migration of the cytoplasmic droplet along the sperm tail after 24h of co-incubation.

Animals↗
PubMed2006

Freezing of epididymal spermatozoa from dogs after cool storage for 2 or 4 days.

An experiment was conducted to investigate the freezing ability of canine epididymal spermatozoa after cool storage at 5 degrees C for 2 or 4 days. Spermatozoa were collected from the caudae epididymidis from 16 dogs. Total motility, plasma membrane integrity and acrosome integrity were evaluated immediately on harvesting, and after 2 and 4 days of storage at 5 degrees C, and at 0 and 2 h post-thaw at 37 degrees C. Sperm motility decreased significantly during cold storage, compared to freshly harvested spermatozoa (P < 0.001). Although there was no significant effect of pre-freeze storage time on post-thaw motility, there was a tendency towards decreased motility in spermatozoa that had been stored for 4 days, compared to spermatozoa that were frozen immediately after collection (P = 0.09). The number of post-thaw spermatozoa with an intact plasma membrane was decreased in spermatozoa cold-stored for 4 days (P < 0.001). There was no significant effect of pre-freeze storage time on the acrosomal status of post-thaw spermatozoa. In conclusion, canine epididymal spermatozoa were stored at 5 degrees C for up to 4 days without a clear detrimental effect on post-thaw motility and acrosome integrity, but storage may have decreased post-thaw motility. Results were, however, generally low.

Acrosome↗
PubMed2006

Comparison of equilibration times when freezing epididymal sperm from African buffalo (Syncerus caffer) using Triladyl or AndroMed.

Because of risks of disease transmission, it is not possible to move African buffalo (Syncerus caffer) within South Africa. Therefore, new ways must be found to enable exchange of genetic material and to increase genetic diversity. In this study epididymal sperm from 11 African buffaloes was exposed to 8 different pre-freezing equilibration times, using 2 different semen extenders. To test the influence of equilibration time and to find a practical way of freezing sperm in the field equilibration times between 2 and 9 h were compared. The extenders used were Triladyl and the totally defined extender AndroMed (both Minitüb, Tiefenbach, Germany). Post-thaw motility, longevity and acrosomal integrity were compared. Different equilibration times did not result in different post-thaw qualities. The use of Triladyl resulted almost always in higher post-thaw motilities and in better acrosomal integrity. Individual bulls had a significant influence on measured parameters. Results indicate that sperm flushed in the field can be stored in freezing medium for up to 9 h before being further processed and that Triladyl is superior to AndroMed when freezing epididymal African buffalo sperm. This knowledge is important to plan fieldwork, since working conditions are usually far from the ideal of a laboratory.

Animals↗
PubMed2006

Effect of egg yolk concentration on cryopreserving Spanish ibex (Capra pyrenaica) epididymal spermatozoa.

Tris-egg yolk based diluents provide adequate cryoprotection for the sperm of most wild species in which they have been tested. The objective of the current study was to evaluate various Tris-based diluents containing different concentrations of egg yolk, for the fertilizing ability of epididymal spermatozoa of the Spanish ibex (Capra pyrenaica) after freezing and thawing. For this purpose, we used heterologous in vivo fertilization by intrauterine insemination of domestic goats (Capra hircus). In Experiment 1, a Tris-citric acid-glucose (TCG) diluent containing 6% (v/v) egg yolk and a TCG extender containing 20% egg yolk were compared. In Experiment 2, a TCG-6% egg yolk extender was compared with Triladyl-20% egg yolk. Diluted samples were cooled slowly to 5 degrees C over 1 h and equilibrated at that temperature for 2 h. At that point, aliquots of samples were loaded into 0.25 ml straws, and frozen in nitrogen vapor for 10 min. The fertility of spermatozoa frozen in TCG-6% egg yolk was higher (P<0.05) than for those extended with TCG-20% egg yolk, and tended to be higher than for those frozen with Triladyl-20% egg yolk. From the results of this study, the use of Tris-based extenders containing low concentrations of egg yolk (6%) is recommended for cryopreserving Spanish ibex epididymal spermatozoa.

Animals↗
PubMed2006

Assessment of chromatin status (SCSA) in epididymal and ejaculated sperm in Iberian red deer, ram and domestic dog.

Abnormal chromatin condensation is not detected using classical techniques for sperm analysis. SCSA has demonstrated its usefulness in sperm chromatin analysis in several species (human, bull, stallion and boar). In this work, we studied sperm samples from red deer, ram and dog to analyze the differentiation of chromatin structure applying SCSA in epididymal and ejaculated spermatozoa. Epididymal samples were obtained from the caput, corpus and cauda by means of cuts, and ejaculated ones were obtained by electroejaculation (deer), artificial vagina (ram) and digital manipulation (dog). SCSA results suggested different critical points in sperm maturation (spermatozoa with loose chromatin to more condensed chromatin) among species: from corpus to cauda in ram and from caput to corpus in deer and dog. Moreover, we also detected differences in ruminants and dog, reflected in the appearance of SCSA plots. Indeed, ram and deer samples rendered two peaks within the sperm main population (sperm with condensed chromatin), whereas only one was detected in dog. Although some differences were observed between cauda and ejaculated samples, SCSA parameters indicated good chromatin condensation, making these samples suitable for germplasm banking. Some species-dependent modifications in the analysis of the results may be necessary to take full advantage of its analytical power.

Animals↗
PubMed2006

Extender osmolality and sugar supplementation exert a complex effect on the cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa.

We have carried out two experiments to study the cryobiology of red deer epididymal spermatozoa and to improve freezing extenders: (1) effect of extender (Tris-citrate-fructose) osmolality (300-600 mOsm/kg), and (2) effect of sugar (0.4M) supplementation to the extender (no sugar, glucose, fructose, mannose, sucrose, maltose, threalose and raffinose). Sperm quality was assessed pre-freezing, post-thawing, and after 2h at 37 degrees C post-thawing: sperm motility index (SMI), acrosome integrity and membrane integrity (HOS test) were assessed subjectively; mitochondrial activity (JC-1) and membrane stability (merocyanine 540) were assessed by flow cytometry. In experiment 2, DNA status was assessed using acridine orange and flow cytometry. To find an optimal osmolality, we fitted the data to a quadratic curve. Four hundred Osmolal per kilogram rendered better results pre-freezing and post-thawing. However, post-thawing viability and most parameters after 2h incubation fitted a linear model. Osmolalities above 425 mOsm/kg were deleterious (P<0.05). In experiment 2, fructose had a positive effect respect to control after 2h of incubation at 37 degrees C post-thawing. Di and trisaccharides were deleterious. Trehalose showed impaired DNA status after 2h incubation. In conclusion, the osmolality of the extenders should be around 400 mOsm/kg, although the change from quadratic to lineal may indicate a complex effect that must be further studied. Monosaccharides may enhance red deer epididymal sperm cryopreservation, especially fructose, whereas di and trisaccharides may not be adequate.

Animals↗
PubMed2006

Postnatal exposure of the male mouse to 2,2',3,3',4,4',5,5',6,6'-decabrominated diphenyl ether: decreased epididymal sperm functions without alterations in DNA content and histology in testis.

2,2',3,3',4,4',5,5',6,6'-Decabrominated diphenyl ether (PBDE 209) is the second most used brominated flame retardant (BFRs) in constructed materials because it is considered less toxic than others, though other fire retardants, some congeners of PBDE 209, are reported to be toxic. This combined the fact that PBDE 209 has been found in high levels in human milk, blood, indoor environments as well as in foodstuffs has led us in this study attempt to find out whether PBDE 209, also known as decaBDE and decabrominated diphenyl oxide (DBDPO), has an adverse effect on this histology of testes and sperm in CD-1 male mice. The mice we studied were divided into groups and gavaged with 10, 100, 500 and 1500 mg/kg PBDE 209 in corn oil per day between postnatal Days 21 and 70. On Day 71, the mice were anesthetized and sperm function, testis DNA content, and histopathology were studied. We found in the 500- and 1500-mg/kg/day groups that neonatal exposure to PBDE 209 reduced sperm epididymal sperm mitochondrial membrane potential (MMP), reduced amplitude of the lateral head displacement (ALH) and induced the generation of hydrogen peroxide (H2O2) in the sperm of sexually mature male mice, without affecting the sperm count, motility, morphology, curvilinear velocity (VCL), angular progressive velocity (VAP), straight-line velocity (VSL), beat-cross frequency (BCF), sperm chromatin structure assay (SCSA), superoxide anion (O2-*) generation, DNA content in testis cells, or testicular histopathology. ALH was positively associated with an increase in MMP and negatively associated with generation of sperm H2O2. The reduction of MMP was negatively associated with an increase in generation of sperm H2O2. The presence of the relationships between sperm ALH, MMP, and generation of H2O2 indicate toxic action possibly resulting from PBDE 209-induced oxidative stress. In conclusion, this is the first study to report the lowest-observed-adverse-effect level (LOAEL) for sperm function to be 500 mg/kg of PBDE 209 in male mice. Decreased epididymal sperm MMP and ALH as well as induced generation of sperm H2O2 were some of the most serious effects of postnatal PBDE 209 exposure. Future investigations should be performed to study the effects of prenatal exposure of PBDE 209 and the mechanism behind PBDE 209-related oxidative stress in the fetal and pubertal stages of development.

Animals↗
PubMed2003

Differential localization of ADAM1a and ADAM1b in the endoplasmic reticulum of testicular germ cells and on the surface of epididymal sperm.

Although fertilin is a heterodimeric complex between ADAM1 (A Disintegrin And Metalloprotease 1, fertilin alpha) and ADAM2 (fertilin beta) located on the sperm surface, two different ADAM1 isoforms, ADAM1a and ADAM1b, are present in the mouse testis. In this study, we have examined the localization of ADAM1a and ADAM1b in testicular germ cells and epididymal sperm. ADAM1a was restrictedly present within the endoplasmic reticulum of germ cells, whereas epididymal sperm contained only ADAM1b on the cell surface. The precursors of ADAM1a and ADAM1b formed a heterodimeric complex with that of ADAM2 in the endoplasmic reticulum of germ cells. The heterodimeric complex between the mature forms of ADAM1b and ADAM2 was also found on the sperm surface. These data imply the potential roles of ADAM1a and ADAM1b in spermatogenesis and fertilization, respectively.

ADAM Proteins↗
PubMed1998

Arginine activates glycolysis of goat epididymal spermatozoa: an NMR study.

The present study explores the mechanism underlying the action of L-arginine on the metabolic activity of spermatozoa. Goat epididymal spermatozoa were incubated with different concentrations of L-arginine to determine its effect on the utilization of glucose, fructose, and pyruvate. NMR techniques have been applied to elucidate the effect of L-arginine, L-lysine, and L-ornithine on the glycolysis of epididymal goat spermatozoa. Whereas 31P NMR has been used to estimate the change of pH in the presence of different concentrations of L-arginine, 13C NMR has been used to estimate the substrate consumption and lactate production. At optimal concentration of L-arginine, the forward metabolic rates have been found to increase by two to three times over control experiments. Arginine is not consumed in these reactions, but acts as an activator. Longitudinal relaxation time (T1) measurements indicate that the guanidino group of L-arginine plays an active role in binding to cells. The amino acid L-lysine is less effective, and L-ornithine is ineffective.

Animals↗
PubMed1975

Vaso-epididymal anastomosis.

Our experiences with 343 vaso-epididymal anastomosis operations carried out for the relief of obstructive azoospermia in the past 20 years were reported. The etiologic factors in obstructive azoospermia were discussed. Smallpox was the single most common factor responsible for the obstructive lesion in our series. Obstructive azoospermia due to congenital anomalies was very infrequent. The pathologic physiology of obstructive azoospermia was reviewed. In 37 patients vaso-epididymostomy could not be carried out because of some genital pathology or congenital abnormality. Vaso-epididymal anastomosis was performed in the remaining 281 patients. The operation was succesful in 137 patients (48.75%), and pregnancies occurred in 40 instances (14.23%). However, even after successful operations, the postoperative sperm counts were above 20 million/ml in only 56 patients (19.93%). Persistent necrospermia resistant to treatment was noted in 48 patients (35.04%). In 71 patients (51.83%), spermatozoa appeared in the semen within the first 3 months after operation, but, in 17 patients (12.41%), the appearance of spermatozoa in the semen was delayed for 1 year or more. Twenty patients (7.11%) again became azoospermic after a variable length of time. In this series, bilateral operations were performed on 185 patients and unilateral operations were performed on 96 patients. Our results indicated beyond doubt that bilateral operation is the method of choice. In addition, an analysis of our data showed that operations performed with an internal nylon splint yield more satisfactory results.

Biopsy↗
PubMed1979

Effect of prolactin on the calcium binding and/or transport of ejaculated and epididymal human spermatozoa.

The prolactin concentration in human seminal plasma and in human epididymal cauda fluid was assessed by radioimmunoassay. The prolactin concentration in cauda plasma was found to be similar to that found in male blood serum (5.5 to 9.1 ng/ml) and significantly lower than the concentration of the hormone found in seminal plasma obtained either from euspermic (48 +/- 12 ng/ml) or from vasectomized volunteers (50 +/- 10.2 ng/ml) (P less than 0.001). Calcium binding and/or transport in ejaculated spermatozoa were found to be little (0.29 +/- 0.08 nmoles/10(8) cells) and dependent on a quickly saturable process. The addition of 200 ng of human prolactin/ml induced a 60% increase in this parameter, while 50 ng of prolactin/ml were ineffective. Epididymal human spermatozoa differ from ejaculated sperm cells in showing greater, time-dependent, calcium binding and/or transport under basal conditions (2.00 +/- 0.35 nmoles/10(8) cells/hour), and in being more susceptible to the stimulating action of prolactin (4.4 +/- 0.68 nmoles/10(8) cells/hour in the presence of 50 ng of prolactin/ml).

Binding Sites↗
PubMed1979

Studies on factors in hamster caudal epididymal plasma and other sources which inhibit sperm dilution damage.

We observed that hamster caudal epididymal (HCE) plasma could inhibit dilution damage in HCE sperm. Here it is shown that in the absence of HCE plasma survival factors (SF), dilution of HCE sperm led to apparently simultaneous lysis and loss of motility. However, in the presence of HCE plasma, lysis did not occur when HCE sperm motility was blocked by palytoxin. Using a newly developed microassay for SF, significant amounts of SF activity were detected in dog and bovine caudal epididymal plasma, hamster testes exudate, HCE sperm cytosol, human seminal plasma, hamster and bovine adrenal extracts, hen's egg, and human serum. The SF activity of HCE plasma could tolerate restricted periods of boiling or pH extremes but was destroyed by trypsin and protease. Unlike human serum, HCE plasma did not significantly alter HCE sperm respiration or ATP content.

Adenosine Triphosphate↗
PubMed1980

Daily spermatozoal production and epididymal spermatozoal reserves of the human male.

Testicular and epididymal spermatozoal reserves were determined for 11 healthy men, a vasectomized man and 12 other men. For healthy men testis weight (19 testes) averaged 16.9 plus or minus 1.2 gm. and daily spermatozoal production was 4.25 times 10(6) per gm. (range equals 1.4 to 6.3 times 10(6) per gm.). Based on our total sample (23 patients) 67 per cent of the men 20 to 50 years old probably produce between 45 times 10(6) and 207 times 10(6) spermatozoa per day. Daily spermatozoal production was normal in 1 vasectomized man. Epididymal reserves of healthy men totaled 182 times 10(6) spermatozoa per epididymis, of which 26 per cent were in the caput, 23 per cent in the corpus and 52 per cent in the cauda. The interval between any previous ejaculation and death was unknown but we assumed that these values are typical for men copulating or masturbating every 1 to 7 days. Extragonadal spermatozoal reserves totaled approximately 440 times 10(6), of which less than 225 times 10(6) would be available for ejaculation in the ductuli deferentia and caudae epididymides. Transit time of spermatozoa through the caput, corpus and cauda epididymidis was estimated as 0.72, 0.71 and 1.76 days. In a vasectomized man extragonadal reserves totaled 7 times 10(9) spermatozoa or about 75 days of production by the testes. Thus, spermatozoal production in man is much less efficient than in other mammals, the extragonadal reserves of spermatozoa are small and maturation of spermatozoa in the caput plus corpus epididymidis occurs in less than 2 days. In terms of these quantitative characteristics the reproductive capacity of man is considerably less than that of a rhesus monkey.

Animals↗
PubMed1988

Clinical aspects in the surgical treatment of varicocele in subfertile men. II. The role of the epididymal factor.

Ninety-six couples, consulting for primary (n = 73) or secondary (n = 23) infertility, and thought to be infertile due to a varicocele in the husband associated with sperm abnormalities, were subjected to a follow-up study. Of these, 57 were surgically corrected and 39 were not, depending only on the patient's choice. All women were intensively treated where necessary. All patients who were corrected were subjected to scrotal exploration in a search for epididymal anomalies. From our data we conclude that (1) on a statistical basis, comparing observed with expected pregnancy rates, varicocelectomy is useful in primary infertility and unnecessary in secondary infertility and (2) on an individual basis, patients with primary infertility and 'pure' varicoceles have a better prognosis than patients with concomitant epididymal dysfunction.

Epididymis↗
PubMed2003

Testicular and epididymal percutaneous sperm aspiration in men with either obstructive or nonobstructive azoospermia.

OBJECTIVES: To investigate our experience with epididymal and testicular sperm obtained by percutaneous aspiration to determine the effectiveness of these procedures in patients with obstructive or nonobstructive azoospermia. We also report on the pooled outcome of intracytoplasmic sperm injection, performed at six in vitro fertilization centers, using sperm from these two patient populations. METHODS: A method of percutaneous aspiration of sperm was developed to facilitate diagnostic biopsy and therapeutic aspiration. A total of 265 aspirations were performed for diagnostic and therapeutic purposes from August 1995 through July 2001. RESULTS: Mature sperm were found in 34 (47%) of 73 men with nonobstructive azoospermia, who underwent testicular sperm aspiration for diagnostic purposes. In 56 procedures for men with nonobstructive azoospermia who underwent testicular sperm aspiration for intracytoplasmic sperm injection, an adequate number of viable sperm was obtained from 46 (82%), with an oocyte fertilization and clinical pregnancy rate of 55% and 44%, respectively. In men with obstructive azoospermia who underwent percutaneous epididymal sperm aspiration, viable sperm were found in 91 (97%) of 94 procedures, with an oocyte fertilization and clinical pregnancy rate of 58% and 39%, respectively. Sperm were obtained in 18 (100%) of 18 procedures in men with obstructive azoospermia who underwent testicular sperm aspiration, with a corresponding fertilization and clinical pregnancy rate of 51% and 38%, respectively. A small (less than 1 cm in diameter) subcapsular hematoma was the only complication. CONCLUSIONS: Percutaneous aspiration of sperm is an effective, safe, and reproducible means of obtaining adequate sperm for diagnosis or intracytoplasmic sperm injection in patients with obstructive or nonobstructive azoospermia. Our experience indicates that similar results can be obtained in both patient groups.

Adult↗