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Effect of high altitude exposure on spermatogenesis and epididymal sperm count in male rats.

The present study was designed to determine the effect of exposure to high altitude on spermatogenesis using transillumination technique and sperm count in male rats. In addition, the effect of oral intubation for intragastric administration of vehicle on testicular parameters in adult male rats in a schedule of 42 days was assessed. Male rats were exposed to Cerro de Pasco (Peru) at 4340 m for 3, 7, 14, 21, 28, 35 and 42 days resulting in a modification of the pattern of the seminiferous tubule stages. At day 3, stages I, IV-V, VI, VII and IX-XI were relatively shorter at high altitude than at sea level. At day 7, stages VIII, IX-XI, XII and XIII-XIV were reduced. At day 14, stages VII, VIII and IX-XI were reduced. At day 21 and 28, stages VIII, XII and XIII-XIV were significantly increased at high altitude. At day 35 an increase in stage XIII-XIV was observed. At day 42, stages II-III, IX-XI and XII were significantly increased at high altitude. Epididymal sperm count was significantly reduced at day 7 of exposure to high altitude and maintained low levels with respect to sea level up to 42 days. In conclusion, high altitude exposure affects spermatogenesis, particularly onset of mitosis and spermiation. This in turn affects epididymal sperm count.

Altitude↗

Characterization of mouse epididymal acrosin: comparative studies with acrosin from boar and human ejaculated spermatozoa.

Acrosin is an acrosomal protease believed to play a major role in fertilization. It is synthesized as an inactive precursor, proacrosin, which is processed via (auto)proteolysis into active form(s). In this paper, comparative studies on the characteristics of acrosin from mouse, boar and human are reported. The mouse proacrosin/acrosin was especially investigated to clarify whether or not the enzyme undergoes modifications during epididymal maturation. Acrosomal extracts from mature and immature mouse spermatozoa, as well as from ejaculated boar and human spermatozoa, were analysed by means of SDS-electrophoresis, Western blot and activity measurements. The studies showed that epididymal maturation produced a shift in the molecular weight of proacrosin. It was also observed that the activation kinetics differ strongly between the three species studied. Human proacrosin showed a constant substrate turnover, acrosin from boar showed sigmoidal activation kinetics and mouse acrosin, either from the caput or the cauda epididymides, showed a rapid decay in activity, suggesting the presence of an endogenous specific inhibitor.

Acrosin↗

alpha-Mannosidase from rat epididymal fluid is a ligand for phosphomannosyl receptors on the sperm surface.

This study demonstrates that alpha-mannosidase from rat epididymal fluid is a ligand for phosphomannosyl receptors on the sperm surface. This enzyme was bound to intact epididymal spermatozoa with high affinity and in saturable form, and the binding was inhibited by mannose-6-phosphate but not by phosphorylated derivatives of fructose. Treatment of the enzyme with sodium periodate inhibited the binding of alpha-mannosidase, confirming that a carbohydrate residue is involved in the interaction with spermatozoa. Evidence is also presented that the cation-independent phosphomannosyl receptors are responsible for the interaction with alpha-mannosidase. These findings suggest a new role for extracellular transport mediated by the mannose-6-phosphate receptor.

Animals↗

IL-10 is highly expressed in the cryptorchid cryptepididymal epithelium: a probable mechanism preventing immune responses against autoantigenic spermatozoa in the epididymal tubule.

The expression of several immunoregulatory adhesion proteins and cytokines was studied in the normal epididymis, cryptorchid cryptepididymis, the epididymis of oestrogen-treated mice and the epididymis of non-obese diabetic (NOD) mice at the protein level to see which of these immunoregulatory proteins may be involved in lymphocyte regulation in the normal or pathological epididymis and if cytokine balance in this organ is on the cellular or humoral side. The aim of the study was to characterize the immunological microenvironment of the epididymis to explain the survival of the autoantigenic spermatozoa in this site. In the 6-week-old BALB/c or NOD mouse epididymis there were some CD18 and CD44 expressing cells in the interstitial tissue. There were no differences between these strains in the expression of the studied antigens, except that some CD4 positive cells were present in the interstitial tissue of BALB/c mice. In the cryptorchid cryptepididymis CD4, CD8, CD18, CD44, CD54 and CD106 expressing cells were occasionally present in the connective tissue surrounding the epididymal tubule. In the epididymis of the oestrogen-treated mice these antigens were not expressed. In the cryptorchid cryptepididymis the epithelial cells expressed IL-10 highly and the myoid peritubular cells IL-6. The present results suggest that the epididymal epithelial IL-10 suppressing TH0, TH1 and TH2 immune responses may be involved in the protection of autoantigenic spermatozoa from immune destruction.

Animals↗

Actin polymerisation during morphogenesis of the acrosome as spermatozoa undergo epididymal maturation in the tammar wallaby (Macropus eugenii).

In the tammar wallaby (Macropus eugenii), post-testicular acrosomal shaping involves a complex infolding and fusion of the anterior and lateral projections of the scoop-shaped acrosome into a compact button-like structure occupying the depression on the anterior end of the sperm nucleus. The present study has generated cytochemical and histological evidence to demonstrate that the occurrence of actin filaments (F-actin, labelled by Phalloidin-FITC) in the acrosome of tammar wallaby spermatozoa is temporally and spatially associated with the process of acrosomal shaping in the epididymis, through a pool of monomeric actin (G-actin, labelled by Rh-DNase I) present in the acrosome throughout all stages of epididymal maturation. F-actin was not detected in the acrosome of testicular spermatozoa, but was found in the infolding and condensing acrosome of caput and corpus epididymal spermatozoa. When the spermatozoa completed acrosome shaping in the cauda epididymidis, F-actin disappeared from the acrosomal area. The strong correlation between the occurrence of F-actin and the events of acrosomal shaping suggested that the post-testicular shaping of the acrosome might depend on a precise succession of assembly and disassembly of F-actin within the acrosome as the spermatozoa transit the epididymis. Thus, actin filaments might play a significant role in the acrosomal transformation, as they are commonly involved in morphological changes in somatic cells.

Acrosome↗

Distribution of DNA, nuclear micro-heterogeneities and compaction of the chromatin in rabbit epididymal spermatozoa. Ultrastructural evaluation of the Feulgen-like technique using osmium ammine.

An adaptation of the Feulgen procedure to visualise DNA at the ultrastructural level, using osmium ammine instead of Schiff reagent, was applied to ultrathin sections of rabbit epididymal spermatozoa, known to display increasing chromatin compaction as they progress through the epididymis. In contrast with the somatic cell nuclei of the epididymal epithelium, which display classical staining, the chromatin of spermatozoa is partly or fully destroyed during the hydrolysis step of the technique. The sperm chromatin resistance towards destruction is a function of the initial sperm nuclear compaction and of the duration of hydrolysis prior to Feulgen-like staining. For a given nucleus, the maximum staining intensity is only obtained after an optimal duration of hydrolysis. However, because of this duration and local differences in chromatin compaction, the total DNA of a section is never completely visualized. The most compact parts of the nuclei are not yet stained, when the less compact parts have already been destroyed. This gives rise to a sperm-specific pattern which corresponds to the enhancement of microheterogeneities present in all sperm nuclei and to the local depolymerisation of the nuclear material during hydrolysis. The depolymerised parts of nuclei sit over the sections, and they also bind uranyl acetate, ethidium bromide and anti-protamine antibodies. Therefore, in sperm nuclei, the Feulgen-like staining at the ultrastructural level does not reveal the true DNA distribution. However, the amount of staining can be quantified to evaluate sperm chromatin compaction.

Animals↗

Epididymal and sex accessory gland secretions in transfusion-dependent beta-thalassemic patients: evidence of an impaired prostatic function.

Neutral alpha-glucosidase levels as epididymal marker, fructose levels as vesicular marker, zinc, citric acid and prostate specific antigen levels as prostatic markers were measured in the seminal plasma of eight transfusion-dependent beta-thalassemic patients in order to study epididymal and sex accessory gland secretions (eighteen subjects served as controls). FSH and LH as well as total and free testosterone were detected displaying unaltered serum values. Ejaculate of patients showed normal sperm count and low sperm motility, in the meantime seminal plasma exhibited unaltered both neutral alpha-glucosidase and fructose values but low levels of zinc, citric acid and prostate specific antigen were noticed as well. These data suggest an impaired prostatic secretion in the thalassemic patients studied. A local iron toxicity on the prostatic tissue could be supported by the decrease of its specific markers observed only in the subgroup of patients with high ferritin serum levels.

Adolescent↗

Epididymal constituents and related substances in the storage of spermatozoa: a review.

In vertebrate animals, the duration of storage of viable spermatozoa in the epididymis varies from a few hours in birds to many months in reptiles and bats. The available information on the unusual composition of the epididymal luminal fluid is summarized, and the effect of the various constituents on sperm motility is described. Spermatozoa would probably be best stored in an immotile state and some constituents of epididymal luminal fluid may be able to inhibit the motility of mammalian spermatozoa during storage in vitro. If this effect can then be removed at the time of insemination, by changing the spermatozoa to a different medium, such a procedure may allow storage of spermatozoa at room or even body temperature for extended periods.

Animals↗

Testicular and epididymal sperm content in grazing Cashmere bucks: seasonal variation and prediction from measurements in vivo.

Each month, for 15 months, the testes and epididymides were recovered from five Australian cashmere bucks selected at random from a group of mature bucks (initial n = 116) at pasture (location 29 degrees S, 153 degrees E). The extent of seasonal change in testicular and epididymal sperm reserves was determined and indirect methods for predicting these reserves were developed. Paired testicular weight exhibited clear seasonal variation from 137 g in August to 269 g in February. The total elongated spermatid content of the testes, determined by homogenization, showed a very similar seasonal pattern, ranging from 13.8 x 10(9) in September to 36.2 x 10(9) in March. Seasonal changes of similar timing were observed for paired epididymal weight and sperm content. Although changes in total testicular spermatid content were largely the result of change in testicular weight (R2 = 0.72, P < 0.001), the elongated spermatid content of testicular parenchyma (mean, 127 x 10(6) g-1) exhibited significant seasonal variation with elevated values between February and June, suggestive of an increase in the efficiency of spermatogenesis. Daily sperm production, calculated by means of a spermatogenic time divisor for sheep, ranged from 2.76 x 10(9) in September to 7.23 x 10(9) in February. Scrotal circumference, scrotal volume and testicular length x diameter2 were identified as accurate predictors of testicular weight (R2 > or = 0.87) and sperm content (R2 > or = 0.70). The results demonstrate that: (a) cashmere bucks exhibit considerable seasonal variation in spermatogenesis associated primarily with changes in testicular mass but also with changes in the efficiency of spermatogenesis; and (b) indirect measures of testicular size are good predictors of testicular elongated sperm content. When the correct spermatogenic time divisor for goats is determined, such indirect measures may be used to predict daily sperm production.

Animals↗

Influence of hyperthyroidism induced at prepuberty on the epididymal lipids, number and motility of spermatozoa in rats.

Fifteen prepubertal rats were divided into 3 groups of 5 each: (1) hyperthyroid group (TH), the rats were treated with thyroxine (T4) (25 micrograms/100g body weight/day) for 60 days from the age of day 31 to day 90; (2) T4-withdrawal group (TH-T4), the rats were given T4 for 30 days from the age of day 31 to day 60 and then the T4 treatment was withdrawn for the following 30 days from the age of day 61 to day 90 and the animals were given vehicle during this period; and (3) control group, the rats were given vehicle for 60 days from the age of day 31 to day 90. All were killed at the age of day 91. Serum levels of testosterone, T4 and triiodothyronine increased in the TH group (P < 0.001). The levels decreased to the euthyroid status in the TH-T4 group. Hyperthyroidism caused various changes in the levels of epididymal lipids. The levels changed further or were restored differentially in the TH-T4 group. The number of spermatozoa decreased, in caput epididymis (CpE) as well as in cauda epididymis (CdE), in the TH group (P < 0.001). The number was not restored in the TH-T4 group. The forward motility of the spermatozoa, determined in CdE, decreased in the TH group (P < 0.001) and was not restored in the TH-T4 group. The study showed that chronic hyperthyroidism, induced at the prepubertal age, causes various changes in epididymal lipid composition and sperm parameters. The study may be helpful in revealing the reasons for male infertility in hyperthyroid patients.

Animals↗

High-affinity sites for beta-D-galactosidase on membrane-bound vesicles isolated from rat epididymal fluid.

Glycosidases in rat epididymal fluid are secreted under androgen stimulation and possess receptors on the sperm surface. One of these enzymes, beta-D-galactosidase (gal), was found in the epididymal fluid as a soluble enzyme and also in a heterogeneous population of membrane bound vesicles (mbv). beta-D-Galactosidase was specifically localized to a subpopulation of larger, electron-dense mbv. The aim of this study was to analyze the high-affinity sites for gal on the membrane of mbv using two different methods: classical fluorometric assay (used in previous papers) and colloidal gold (20 nm) conjugated to gal as a marker in ultrastructural studies. beta-D-Galactosidase bound to mbv with high-affinity (Kd in a nanomolar range) are in a saturable form. Furthermore, 25 mM fructose-1,6-diphosphate (f-1,6-dip), a sugar that competes for the binding site, showed 50% inhibition of the binding. The gold conjugates were mostly observed on the surface of the large, electron-dense mbv but not on the small, electron-lucent mbv. Gold particles were also observed on the larger vesicles, but less frequently in the presence of f-1.6-dip. Larger mbv possesses high-affinity sites for gal on their membrane.

Animals↗

Galvanotactic response of mouse epididymal sperm: in vitro effects of zinc and diethyldithiocarbamate.

This study was conducted to evaluate the galvanotactic response of mouse epididymal sperm as well as the in vitro effects of Zn-acetate and diethyldithiocarbamate (DEDTC) on the galvanotaxis of sperm. The galvanotaxis of sperm was observed in a direct current (DC) electric field between 0 and 10 V/cm. The sperm were treated with Zn-acetate or DEDTC at concentrations of 0.02, 0.20, and 2.00 mM before the observations. The sperm exhibited galvanotaxis toward both cathode and anode in the fields between 1 and 9 V/cm. The number of sperm that accumulated at both electrodes was found to reach the highest level between 5 and 7 V/cm. In comparison with the results from untreated sperm, the number of accumulated sperm at the electrodes increased with the addition of 0.20 and 2.00 mM Zn-acetate, but decreased with the addition of DEDTC at all 3 concentrations. The galvanotaxis of mouse epididymal sperm is related to the field strengths, and the guidance of sperm migration may be influenced by the sex chromosomes. The galvanotaxis of the sperm may be improved by addition of Zn-acetate but depressed by DEDTC in the presence of DC electric fields.

Animals↗

Expression and phosphorylation of mitogen-activated protein kinases during spermatogenesis and epididymal sperm maturation in mice.

The expression and phosphorylation/dephosphorylation of mitogen-activated protein (MAP) kinases during mouse spermatogenesis and epididymal sperm maturation have been investigated by immunoblotting and immunohistochemical staining with commercially available anti-ERK2 and anti-Active MAPK antibodies. Two forms of MAP kinases, p42ERK2 and p44ERK1, were expressed in a similar amount in spermatogenic cells at different stages. ERK1 and ERK2 were phosphorylated (activated) in early spermatogenic cells from primitive spermatogonia to zygotene primary spermatocytes, while only a small quantity of phosphorylated MAP kinases could be detected in pachytene primary spermatocytes and spermatids. MAP kinase activity in primative spermatogonia and preleptotene primary spermatocytes was the highest among spermatogenic cells. ERK1 and ERK2 were also present in epididymal spermatozoa, and their phosphorylation was increased while spermatozoa pass through epididymis and vas deferens for maturation. It would appear that MAP kinase activation may contribute to the mitotic proliferation of primative spermatogonia, an early phase of spermatogenic meiosis, and, later, sperm motility acquirement.

Animals↗

Epididymal and systemic phospholipidosis in rats and dogs treated with the dopamine D3 selective antagonist PNU-177864.

Repeat dose oral toxicity studies were conducted in rat and dog to assess the safety for human clinical testing of the potent dopamine D3 receptor antagonist, PNU-177864. Systemic phospholipidosis was the principal treatment-related change with epididymal epithelial cell phospholipidosis being the most prominent finding in rats and dogs. Epididymal epithelial cells had no histologic evidence of degeneration; sperm density and morphology were normal histologically in both species; and sperm concentration, morphology, and motility in the dog were comparable to dogs given vehicle. Other sites with phospholipidosis included lymphoid tissues (lymph nodes, Peyer's patches, and/or spleen), pulmonary alveolar macrophages, and peripheral blood lymphocytes in rats and dogs and adrenal cortex, liver, pituitary, hair follicles, bone marrow lymphocytes and plasma cells, and skeletal muscle in rats only. The phospholipidosis was resolved after a 6-week recovery period in all tissues but epididymis. There was no evidence of cell injury in tissues that had phospholipid accumulations except in rat skeletal muscle that had multifocal myofiber degeneration and necrosis. For clinical trials, serum AST and CK and peripheral blood lymphocyte vacuolation were considered potential safety biomarkers for skeletal muscle degeneration and phospholipidosis, respectively.

Animals↗

Follicular lymphoma with bilateral testicular and epididymal involvement: case report and review of the literature.

Testicular involvement with indolent lymphoma is extremely rare, particularly in the absence of transformation to an aggressive histology. We report a case of a 64-year-old man who presented with cervical lymphadenopathy. Staging CT scans revealed extensive lymphadenopathy as well as bilateral testicular and epididymal masses. Histologic examination of lymph node, bone marrow, and testicular/epididymal biopsies revealed involvement with grade I follicular lymphoma. The patient was started on chemotherapy with cyclophosphamide, vincristine, prednisone, and rituximab in addition to intrathecal methotrexate and testicular radiation. He is now 6 months into therapy and responding well. A review of the literature demonstrated this to be the first confirmed case of testicular and epididymal involvement with grade I follicular lymphoma.

Antineoplastic Combined Chemotherapy Protocols↗

Epididymal sperm transport in normal and recent spinal cord injured Sprague Dawley rats.

Causes of poor semen quality following spinal cord injury (SCI) are not known. One possible reason, based upon studies that reported improved semen quality in SCI men after several induced ejaculations, is delayed epididymal sperm transport. Our study was designed to establish baseline epididymal sperm transport values in the Sprague Dawley rat and evaluate effects of SCI on this process. Spermatozoa protamine was labeled with tritiated arginine, and the rats were sacrificed various times after injection. Each epididymis was divided into six equal sections from proximal to distal. Sperm tails were dissolved with 8 molar (M) urea in the presence of 2 mM dithiothreitol (DTT); sperm heads were collected by centrifugation (3,000 rpms, 10 min.). The radioactivity in sperm heads from each section was counted and expressed as counts per million sperm heads. To account for different rates of labeled arginine incorporation, the percentage of counts per million sperm heads in each section was calculated relative to the total number of counts in all six sections. Our results showed there was an orderly progression of sperm through the epididymis. It took 8 days for labeled sperm to enter the epididymis and 28 days to peak in the caudal (tail) section in non-SCI rats. Stasis was present 10 days after T-9 SCI in rats compared with transport in sham controls. This was evidenced by a significant increase in the percentage of labeled sperm in proximal sections of the epididymis (sections 1, 2, and 4) in T-9 transected animals (p < 0.01). If similar stasis occurs in SCI men, it could obviously contribute to poor semen quality. However, it remains to be determined how long this stasis persists after SCI in rats.

Animals↗

Sheep testicular and epididymal angiotensin converting enzyme: inhibitions by captopril, lisinopril and enalapril.

Inhibition of angiotensin converting enzyme(ACE) in presence of captopril(C), lisinopril(L) and enalapril(E) were investigated in testis and epididymis of sheep using Hip-His-Leu as substrate. Captopril, lisinopril and enalapril were competitive inhibitors of the enzyme from both tissues. Differences in the I50 and Ki values using these three inhibitors reflects the affinities of these inhibitors for the ACE. In addition, the relative potencies of captopril, lisinopril and enalapril were different for testicular ACE(C > L > E) and epididymal ACE(L > C > E). This observation suggests differences between the active sites of the testicular and epididymal ACE which may reflect on their functions in vivo.

Angiotensin-Converting Enzyme Inhibitors↗

Sperm retrieval and fertilization in repeated percutaneous epididymal sperm aspiration.

Percutaneous epididymal sperm aspiration (PESA) for retrieval of spermatozoa for intracytoplasmic sperm injection (ICSI) is a new simplified technique in the treatment of men with obstructive azoospermia. There has been a fear that the PESA procedure, being blind, could cause damage to the epididymal duct system and make it impossible to retrieve spermatozoa if a repeated procedure is required. We report here on repeated PESA procedures from the same unilateral epididymis. Twenty-seven men with obstructive azoospermia were investigated retrospectively regarding sufficiency of the number of motile spermatozoa for ICSI, fertilization rate (FR) and possibility of collecting spermatozoa for cryopreservation in repeated PESA procedures. Sufficient motile spermatozoa for ICSI were found in a similar proportion of men at the first two attempts: 91 and 89% respectively. Fertilization rate and the possibility of collecting spermatozoa for cryopreservation were also similar at the first two PESA procedures: 62 versus 67% and 33 versus 33% respectively. At the third procedure, motile spermatozoa for ICSI were retrieved in 86% (6/7), FR was 47% and spermatozoa were cryopreserved in one case. Two men underwent a fourth PESA. In both cases, a sufficient number of motile spermatozoa for ICSI was found and FR was 62%. This study shows that in men with obstructive azoospermia, PESA can be repeated on the same unilateral epididymis up to three times, with good opportunity of retrieving sufficient motile spermatozoa for ICSI.

Adult↗