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Characterization of epididymal sperm motion and its correlation with stages of target cells in rats given alpha-chlorohydrin, cyclophosphamide or nitrazepam.

Epididymal sperm motion in rats was characterized by computer-aided sperm motion analysis (CASA) with its correlation to testicular lesions in the 2-week treatment study, using three compounds which are known to affect different stages of germ cells. Mature male rats were treated daily for 2 weeks with alpha-chlorohydrin (alpha-CH, 5 mg/kg), cyclophosphamide (CP, 20 mg/kg) or nitrazepam (NZ, 20, 40, 60 mg/kg). Changes in sperm motion were detected only in the alpha-CH and 60-mg/kg NZ-treated groups. Of the sperm motion parameters, velocity and amplitude of lateral head displacement (ALH) were concomitantly reduced in these two groups with good correlation. With respect to the distribution of the values in parameters, however, alpha-CH shifted the values down within a small range with high percentages of motile sperm, while NZ distributed them over a wide range with low percentages of motile sperm. CP treatment showed no histopathological changes in advanced germ cells, though it showed a decrease in the number of early germ cells. NZ treatment affected round and elongating spermatids (approximately step 14) at doses of 20 and 40 mg/kg, and affected also more advanced spermatids (approximately step 19) at the dose of 60 mg/kg. alpha-CH treatment did not affect testicular histopathology. These findings indicate that 60-mg/kg NZ treatment reduced sperm motion as a result of lesions affected in elongated spermatids and alpha-CH reduced it by direct effects on epididymal spermatozoa. The present study indicates that in addition to percentage of motile sperm, the velocity and ALH can be useful to detect the changes in sperm motion caused by different actions of NZ and alpha-CH, though each compound showed a distinct distribution pattern of these parameters.

Animals↗

Functional significance of the sperm head morphometric size and shape for determining freezability in iberian red deer (Cervus elaphus hispanicus) epididymal sperm samples.

In the present study, computer-automated sperm head morphometry of epididymal samples was used to determine if sperm head area and shape are useful measurements for separating "good" and "bad" Iberian red deer freezers. A microscope slide was prepared from single diluted sperm fresh samples collected from 38 mature stags. Slides were air-dried and stained with Hemacolor. The sperm head area and shape (length/width) for a minimum of 145 sperm heads were determined for each male by means of the Sperm-Class Analyser. The remainder of each sample was frozen. After thawing, sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility and of plasma membrane and acrosome integrities. All sperm parameters evaluated at thawing were placed in a statistical database and a multivariate cluster analysis performed. Mean sperm parameters of the 2 clusters generated ("bad" and "good" freezers) were compared by ANOVA. Our results show that sperm quality at thawing for all sperm parameters evaluated was significantly higher (P < .01) for "good" freezers than for the "bad" ones (sperm motility index: 67.4 +/- 2.0 vs 57.1 +/- 2.8; NAR: 67.1 +/- 2.5 vs 54.5 +/- 3.5; viability: 68.8 +/- 2.0 vs 60.1 +/- 2.8; HOST: 71.3 +/- 2.2 vs 63.1 +/- 3.1). Additionally, differences (P < .01) in epididymal sperm head area and shape were found between "good" and "bad" freezers before freezing, with the smallest overall sperm head dimensions found in the "good" freezers group (area: 32.04 microm2 vs 34.42 microm2). Thus, the lower the sperm head area in the fresh samples, the greater the sperm cryoresistance. Our results show that the 2 groups of males also differ in sperm head shape in fresh samples (good: 1.96 vs poor: 1.72; P < .01). It is possible that sperm head area and shape influence total sperm volume, thus causing differences in heat exchange as well as in movements of water, ions, and cryoprotectants and, in turn, on sperm freezability.

Animals↗

Sperm characteristics and DNA integrity of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa frozen in the presence of enzymatic and nonenzymatic antioxidants.

The main goal of this study was to investigate the potential protective effects of enzymatic and nonenzymatic antioxidants on cryopreservation injuries to red deer epididymal spermatozoa. In Experiment 1, the effects on sperm freezability of the enzymatic antioxidants catalase, superoxide dismutase, and a combination thereof were studied. In Experiment 2, sperm cryoresistance was evaluated when different nonenzymatic antioxidants, such as vitamin E, vitamin C, and butylated hydroxytoluene (BHT), were added to the freezing extender. Sperm quality was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and acrosome (ie, spermatozoa with normal apical ridges; % NAR) and membrane (by means of the HOS test) integrity. To address fully these topics, we incorporated a new set of functional sperm tests for mitochondrial function, membrane phospholipid disorder, and sperm chromatin stability. Samples were evaluated after freezing and thawing, and after a 2-hour period of incubation at 37 degrees C. The present study demonstrates that the addition of enzymatic antioxidants to freezing extenders improves sperm viability after cooling, and improves sperm motility, acrosome integrity, and mitochondrial status (P<.05) after thawing. After a 2-hour incubation period at 37 degrees C in the presence of enzymatic antioxidants, an improvement in membrane integrity (P<.05) was observed. However, when nonenzymatic antioxidants were present in the freezing diluents, no positive effects on thawed sperm parameters were noted. The chromatin stability test did not show significant differences between the treatments. We conclude that enzymatic antioxidants should be present in the early steps of cryopreservation of epididymal spermatozoa from red deer, so as to improve motility and acrosome integrity.

Animals↗

Potassium channel blockers inhibit anion secretion in cultured rat epididymal epithelium.

The effect of putative K channel blockers on anion secretion has been studied in primary monolayer cultures of rat epididymal cells using the short circuit current technique. Under basal conditions, monolayers had a transepithelial potential difference of about 2-3 mV, apical side negative and a short circuit current (SCC) of about 2 microA.cm-2. The transepithelial resistance was about 500 omega.cm2. Addition of adrenaline (0.23 microM, basolaterally) caused the SCC to rise to a peak value of about 10.5 microA.cm-2 and then stabilized at about 4 microA.cm-2 after 15 min. This rise in the short circuit current has previously been shown to be due to an increase in net anion secretion from the basolateral to the apical medium. In tissues stimulated with adrenaline, addition of barium (Ba) to the apical side did not affect the adrenaline-induced SCC, but addition to the basolateral side caused a dose-dependent inhibition of the current with an IC50 value (concentration required to inhibit 50% of the current) of 0.92 mM. At Ba concentration of 5 mM, the adrenaline-induced SCC was completely abolished. There was no effect on transepithelial resistance. Addition of tetraethylamonium (TEA) (16 mM) to the apical or basolateral side had no significant effect on the adrenaline-stimulated SCC. Lidocaine and quinidine inhibited the adrenaline-stimulated SCC when added either to the apical or basolateral bathing solution. The IC50 values for lidocaine were 0.42 mM and 0.35 mM for basolateral and apical application, respectively. The IC50 values for quinidine were 0.062 mM and 0.050 mM for basolateral and apical application, respectively. In all cases there was no change in tissue resistance. It is proposed that in the basolateral membrane of the epididymal cells, there is a component which is sensitive to putative K channel blockers. It is likely that it is a K channel. As in other secretory cells, this channel plays an important role in secretion.

Animals↗

Testicular development, epididymal sperm reserves and seminal quality in two-year-old Hereford and Angus bulls: effects of two levels of dietary energy.

The effect of high (HED) and medium energy diets (MED), fed to Hereford (H) and Angus (A) bulls from 6 through 24 mo of age, on scrotal circumference (SC), paired testes weight (PTW), epididymal sperm reserves (ESR) and seminal traits were examined. Over 3 yr, 120 bulls were involved. Angus exceeded H for both SC and PTW. Hereford bulls in yr 2 had smaller SC than in yr 1 or 3 but the response for A was consistent. Year affected PTW. In yr 2 Hereford bulls fed HED had 75% fewer ESR than MED-H bulls (9.3 vs 37.2 X 10(9]. Comparably treated A bulls had similar ESR numbers (29.2 vs 33.4 X 10(9]. In yr 3, epididymal sperm reserves of HED-H were depressed by 35% compared with MED-H (23.1 vs 35.7 X 10(9], whereas HED-A had 14% fewer ESR than did MED-A bulls (28.6 vs 33.1 X 10(9]. It was not obvious why H bulls were more susceptible to the effects of HED. Seminal quality of HED bulls was inferior to that of MED bulls, particularly with respect to progressive motility and the incidence of sperm in which a crater defect of the head was present at 2 yr of age. In yr 2 all seminal traits were severely depressed in 2-yr-old HED-H. Feeding HED to young H and A bulls reduced their reproductive potential.

Animal Feed↗

Effects of prostaglandins and indomethacin on rat epididymal responses to norepinephrine and acetylcholine.

Prostaglandins E2 and F2alpha tromethamine salt caused an increase in amplitude and frequency of rat epididymal contractions. Moreover, they caused a significant potentiation of contractile responses of epididymal smooth muscle to stimulation by norepinephrine or acetylcholine. Indomethacin treatment caused no changes in the spontaneous motility, and this inhibitor of prostaglandins synthesis decreased the responses of epididymis to norepinephrine or acetylcholine. Spontaneous motility of epididymis is possible in the presence of smaller amounts of endogenous prostaglandins, and sudden contraction occurring during ejaculation, induced by sympathetic and parasympathetic nerves stimulation, could be influenced or modulated by prostaglandins.

Acetylcholine↗

Uptake of 3H-L-carnitine by isolated rat epididymal tubules.

The uptake of radiolabeled L-carnitine has been studied in isolated epididymal tubules from the rat. The uptake of 3H-L-carnitine increases with a temperature coefficient KT of 0.22 nmol carnitine.mg protein-1 in the intermal 22--31 degrees and with a low uptake at 4 degrees C. The uptake of radiolabeled carnitine (as percent) is reduced at high concentrations of L-carnitine, by deoxycarnitine but not by D-carnitine. This uptake mechanism is especially active in the distal caput and corpus segments of the epididymis. Thus, an uptake mechanism for carnitine is present in the epididymal cells which besides the carnitine uptake in spermatozoa is responsible for the dramatic increase in carnitine concentration in cauda epididymis.

Animals↗

Mode of entry of glycosaminoglycan sulfate during epididymal maturation of bull spermatozoa. I. Charge neutralization mechanism.

Heparin uptake by bull spermatozoa from each one of main epididymis regions was investigated to determine whether the heparin binding was modified during epididymal maturation, the heparin internalization differed during the maturation process, and the charge neutralization by histone or polylysine enhances heparin internalization. From our results we conclude that membrane binding of heparin is greater in the spermatozoa from the caput epididymis than in those of the cauda epididymis region (9.41 +/- 2.27 vs. 2.89 +/- 0.72 pmole of [3H]heparin) and that membrane binding of heparin occurs also at 4 degrees C (4.97 +/- 1.36 vs. 1.05 +/- 0.54 pmole of [3H]heparin). It was also clearly shown that heparin uptake requires active metabolism of the cell and that internalization decreases according to the progress of the epididymal maturation process [6.34 +/- 1.25 (caput) vs. 1.74 +/- 0.72 (cauda)]. Finally, charge neutralization of heparin by histone or polylysine did not enhance heparin internalization.

Animals↗

alpha-Glucosidase, sperm ATP concentrations, and epididymal function.

It has been suggested that alpha-glucosidase may be a marker of epididymal patency and function. Spermatozoal ATP concentrations decrease during passage through the epididymis, indicating efficient maturation. We correlated sperm motility with seminal plasma alpha-glucosidase activity and spermatozoal ATP. The sperm motility correlation with alpha-glucosidase activity was significantly positive, and the sperm motility correlation with spermatozoal ATP was significantly negative. It appears that high-alpha-glucosidase activity and low-spermatozoal ATP were present in semen with good sperm motility and could possibly indicate efficient epididymal function.

Adenosine Triphosphate↗

Identification of a rabbit epididymal protein gene.

A protein designated as BE-20 was purified from cauda epididymal fluid of the rabbit by preparative polyacrylamide gel electrophoresis and HPLC on a mono Q HR5/5 anion exchange column. The purified protein migrated with an estimated Mt of 20,000 when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequence of the N-terminus of the BE-20 protein was determined. The initial eight amino acid residues were His-Gly-Ala-Asp-Lys-Pro-Gly-Val. The corresponding 23 mer oligonucleotide (5'-CATGGCGCTGACAAGCCTGGGGT-3') was synthesized and used as sense primer with rabbit epididymal mRNA as template in the RT-PCR system. The purified BE-20 cDNA consisted of 499 bp with an open reading frame of 285 bp encoding a deduced polypeptide composed of 95 amino acids. The BE-20 cDNA had 78.5% identity in 479 bp overlap with human epididymis-specific HE4 cDNA. The amino acid sequences of the initial 30 amino acid residues of the N-terminus of the purified protein and the deduced polypeptides were as follows: N-His-Gly-Ala-Asp-Lys-Pro-Gly-Val-Cys-Pro-Gln-Leu-Ser-Ala-Asp-Leu-Asn-Cy s- Thr-Gln-Asp-Cys-Arg-Ala-Asp-Gln-Asp-Cys-Ala-Glu. The deduced polypeptide contained 16 cysteine residues and had partial sequence homology with proteins belonging to the four-disulfide core family of extracellular proteinase inhibitors. The BE-20 protein may play a role in sperm maturation and/or capacitation.

Amino Acid Sequence↗

Expression of the BE-20 epididymal protein gene: in situ hybridization.

A protein designated as BE-20 was purified from cauda epididymal fluid of male rabbits and the amino acid sequence of the N-terminus was determined. A 23-mer oligonucleotide coding the N-terminal eight amino acids of the BE-20 protein was synthesized. The oligonucleotide was used as sense primer with rabbit epididymal mRNA as template in the RT-PCR system. The BE-20 cDNA consisted of 499 bp with an open reading frame of 285 bp encoding a deduced polypeptide composed of 95 amino acids. Digoxigenin-labeled BE-20 cDNA was prepared and used as a hybridization probe to detect the specific mRNA. The probe interacted with a 1.2-kb mRNA prepared from rabbit epididymis; mRNAs prepared from rabbit testis gave negative reaction. Using tissue sections, the BE-20 mRNA was located in the epithelial cells of the cauda epididymis and proximal segment of the ductus deferens by in situ hybridization method. Sections of the corpus and caput epididymis, testis, and liver gave negative reaction. Polyclonal anti-BE-20 antibodies were raised and found to inhibit in vitro the capacity of human sperm to penetrate zona-free hamster ova. The results suggest that BE-20 protein may influence maturation of spermatozoa during its movement through the epididymis and/or the capacity of sperm to fertilize ova.

Animals↗

Evidence that reverse transcriptase is a component of murine epididymal fluid.

The sperm-free fluid fraction of epididymal semen obtained from males of four mouse strains contained a DNA polymerase activity with properties of murine retrovirus reverse transcriptase (RT). Epididymal fluids from two of the strains of mice, NIH Swiss and New Zealand Black (NZB), had an order of magnitude more enzyme activity per microgram of protein than did the fluids of C57Bl/6 and Balb/c males. Most of the enzyme activity in the Swiss and NZB males, but not in the C57Bl/6 and Balb/c males, banded in isopycnic sucrose gradients at the buoyant density of retrovirus particles. The males were fertile and free of tumor or other detectable disease up to 17 months of age. The evidence suggests RT activity is ubiquitous in the male reproductive tract of mice and may or may not be associated with virus particles.

Animals↗

[Trial of in vitro fertilization via epididymal duct aspirated sperm].

We made a trial of our method by which sperm are collected directly from epididymal duct, then subjected to in vitro fertilization. The subjects were a total of 22 cases comprising 14 of obstructed azoospermia and 8 of severe oligoasthenozoospermia, on which the surgery was performed 26 times. Using the microscope, an epididymal duct was directly punctured to make a suction of the fluid or was slightly incised to make a suction of the outflow. Sperms thus collected were preincubated for 4 to 6 hours, then subjected to in vitro fertilization. Condition of collected sperms; 32 x 10(4)-15300 x 10(4) sperms were obtained in real number. Sperm motility rate was 0-82%, averaging 25.0%. Sperm forward motility were fairly good in 18 of 26 test cases. Sperm condition was so poor as to make the insemination impossible in 4 test cases (16%). Fertility rate for cases of obstructed azoospermia was 53% (8/15 test cases) and 63% (55/87 eggs) in the test cases and the number of eggs, respectively, compared with 33% (2/6 test cases) and 5.4% (2/37 eggs), respectively for cases of oligo-asthenozoospermia. Pregnancy; Successful pregnancy was obtained in 2 of the cases of obstructed azoospermia, in each of which sperms were collected from the head of the epididymis, then passed into the maternal body by zygote intra fallopian tubal transfer (ZIFT); with the one case resulting in uneventful delivery in November 1991 and the other in a miscarriage. The above results suggested the present method would become an epoch-making approach in the treatment of severe male infertility.

Epididymis↗

[Study on the fertility of epididymal sperms].

One approach to the treatment of obstructive azoospermic cases in whom either vasovasostomy cannot be performed or the vas deferens is absent is to construct an artificial spermatocele in the epididymis. By using this method to recover sperms for use in AIH, we have succeeded in achieving pregnancy in only 2 of 35 cases, leading us to investigate the fertility of the recovered sperms. Sperm fertility was investigated by the hypoosmotic swelling (HOS) test, observation by confocal laser-scanning microscope (CLSM), flow cytometry and hamster egg sperm penetration test. The percentage of swollen sperm determined by the HOS test was 15.0-83.3%, with a mean of 47.6 +/- 16.1%, significantly lower than the values of the normal group and the infertile male group, excluding cases of obstructed azoospermia. Examination by confocal laser-scanning microscope of samples stained with PI and FITC-PSA or PI and FITC-Con A stain revealed viable and dead spermatozoa as well as viable and dead acrosome-reacted spermatozoa. In addition, fertility was evaluated from the distribution of spermatozoa in each area from the flow cytometry percentage. This method was shown previously to be useful for the evaluation of fertility as it demonstrated the presence of numerous spermatozoa in the fertile area of cases who did not succeeded in pregnancy. The hamster egg sperm penetration test yielded a fertility rate of 0-25% with a mean of 8.2 +/- 10.0%, which was significantly lower than the value of the normal group, but was not significantly different from the infertile group. The findings of this study indicated that the fertility of epididymal sperms is low, thereby pointing to the need for studies to improve the materials used in the artificial spermatocele as well as the method of sperm recovery. Furthermore, our findings suggest that flow cytometry may be used to select the epididymal sperms with the highest fertility for sperm recovery.

Animals↗

Abnormal echogenicity of the testicle caused by epididymal cysts.

Ultrasonography of the scrotum has proven to be highly accurate for the diagnosis of testicular and other scrotal abnormalities, particularly for defining focal lesions. False-positive examinations are unusual. Three cases are presented where large epididymal cysts compressed the testes and the remainder of the epididymis. This led to an abnormal echogenic area in testes of two patients and a hypoechoic defect of the testis suggesting malignancy in the thirds. The patients were sonographically and anatomically normal without testicular abnormality during and after operation, following removal of the epididymal fluid collections.

Adult↗

[Bilateral simultaneous epididymal leiomyoma: a case report].

Primary epididymal leiomyoma is relatively rare. We experienced a case of bilateral simultaneous primary epididymal leiomyoma. A 61-year-old man visited our clinic, with the chief complaint of bilateral painless nodules in the scrotum. Each nodule was palpated at the tail of each epididymis (the diameters of these nodules were 12 mm on left and 5 mm on right). We followed the patient for 18 months. Since the nodules were increasing in size, the nodules were surgically removed. Histological examination revealed primary leiomyomas of the epididymis.

Epididymis↗