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A simplified method for the isolation of sperm heads from the caput epididymidis of rodents and from ejaculated rabbit seminal plasma.

The present report describes a simple and rapid procedure to isolate highly purified sperm heads from mouse, rat and rabbit. The isolation procedure involves mincing of the caput epididymidis (CPE) of mice and rats to obtain a crude CPE suspension. This suspension is filtered to remove tubular fragments. The filtrate containing sperms and cellular contaminants is trypsinized, followed by sonication. To obtain rabbit sperm head suspension, ejaculated spermatozoa are briefly exposed to the cationic detergent CTAB-D, followed by trypsinization. Trypsin treatment of the suspension yields sperm heads and tails. The suspension containing sperm heads and tails is separated by discontinuous sucrose density gradient centrifugation. Highly purified and enriched sperm heads are recovered from the bottom of 2.5 M sucrose of the gradient. Enriched sperm heads contain less than 1% sperm tails.

Animals↗

The treatment of premature ejaculation: psychological and biological strategies.

This review provides criteria for defining premature ejaculation (PE) and identifying men with this dysfunction. Included are discussions of possible PE subtypes and causes of PE. Furthermore, two empirically supported cognitive-behavioral approaches for treating PE are described and salient issues surrounding these types of treatment are considered. Biologically based treatments available to PE men are presented, with attention to the relative effectiveness and limitations of the various alternatives. Finally, a model illustrating factors relevant to determining the best therapeutic strategy is described, concluding with a brief discussion of the value of combining biological and psychological modes of treatment.

Journal Article↗

The effect of ouabain on membrane potential and flagellar wave in ejaculated bull spermatozoa.

Ejaculated bull spermatozoa were exposed to various concentrations of ouabain to ascertain the effect on membrane potential, intracellular concentrations of sodium and potassium and motility. Membrane potenital, measured by electrophysiological methods, decreased. Intracellular potassium decreased and intracellular sodium increased. Progressive motility decreased. In addition, the motility change produced by ouabain was identified as a decrease both in beat frequency and in wave amplitude.

Animals↗

Fertilization in vitro of cattle follicular oocytes with ejaculated spermatozoa capacitated in a chemically defined medium.

Cattle follicular oocytes were collected from the ovarian follicles and cultured for 28 h in m-KRB solution in a CO2 incubator (5% CO2 in air at 37 degrees C, 95% humidity). After further culture for 15-18 h with spermatozoa, 78.6% of the oocytes had matured to the second metaphase. The highest fertilization rates (36-58%) were obtained when spermatozoa were collected 1 day before insemination, kept for 14-18 h at 20 degrees C in a test tube (10 X 10(8)/ml), preincubated in m-KRB solution (1-1.2 X 10(8)/ml) in a CO2 incubator for 0-12 h, and then inseminated at a concentration of 1.5-2.0 X 10(6)/ml. The optimum period of preincubation in the CO2 incubator was 8 h: 6/15 denuded oocytes (40%) were fertilized with spermatozoa preincubated for 8 h. No polyspermic fertilization was observed in any of the 139 penetrated oocytes. When spermatozoa were preincubated in the uteri of oestrous cows and gilts for 4-4.5 h instead of the m-KRB solution, the fertilization rates were 80 and 86%, respectively. However, in this system, 13 and 24% of the penetrated oocytes were polyspermic. These results indicate that ejaculated bull spermatozoa can be capacitated in a chemically defined isotonic medium, and about half of the oocytes matured in culture are normally fertilized in vitro.

Animals↗

In-vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa.

Pig oocytes matured in culture were inseminated with frozen-thawed ejaculated spermatozoa without preincubation in modified tissue culture medium (TCM) 199. High penetration rates (85-89%) and increased incidence of polyspermy were obtained at 25-100 x 10(6) spermatozoa/ml. Wide variation in penetration rates (16-89%) was observed in oocytes inseminated in medium containing 5mM caffeine and at 25-50 x 10(6) spermatozoa/ml obtained from 6 boars, regardless of sperm motility. At 25-50 x 10(6) spermatozoa/ml, penetration rates of oocytes were dependent upon the concentration of caffeine in the medium: there was no penetration without caffeine, but penetration was highest (89%) with 5mM caffeine. None of the oocytes was penetrated in the medium supplemented with heparin at 5-40 micrograms/ml. When heparin was included in the medium with 5mM caffeine, it inhibited the efficacy of caffeine to promote sperm penetration of oocytes.

Animals↗

Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors.

Cells isolated from the ejaculates of a high proportion of patients exhibiting oligozoospermia are characterized by generation rates of reactive oxygen species that considerably exceed those obtained for the normal fertile population. The purpose of this study was to resolve the cellular source of this enhanced activity. Semen samples from a cohort of oligozoospermic patients and a group of fertile controls were fractionated on discontinuous Percoll gradients to generate three cell populations (0, 50 and 100%) of differing density. For each fraction, both the steady-state and the phorbol-ester-induced chemiluminescent signals were significantly (P less than 0.001) greater for the oligozoospermic samples than for the fertile controls. In the fertile donors, leucocytes comprised the major source of reactive oxygen species, particularly in the low-density Percoll fractions; in oligozoospermic patients, however, spermatozoa were identified as a second major source of reactive oxygen species. Particularly striking was an intense phorbol-ester-induced chemiluminescent signal generated by oligozoospermic spermatozoa, purified by passage through isotonic Percoll and free of leucocyte contamination, which was 167 times greater than the median signal generated by the corresponding fraction from the fertile controls (P less than 0.001). These results emphasize the importance of spermatozoa as a major source of reactive oxygen species in oligozoospermia and have implications for the diagnosis and treatment of this condition, as well as for the design of appropriate diagnostic strategies.

Cell Separation↗

Circannual inter-relationships among reproductive hormones, gross morphometry, behaviour, ejaculate characteristics and testicular histology in Eld's deer stags (Cervus eldi thamin).

Blood samples, morphometric measurements and behavioural data were collected weekly for 52 weeks from six adult Eld's deer stags exposed to natural fluctuations in photoperiod (38 degrees N latitude). Mean (+/- SEM) serum LH concentrations reached peak values in the autumn (October), three months before FSH and testosterone concentrations reached peak values in early winter (January). Prolactin concentrations were inversely related (r = -0.733, P < 0.001) to LH and directly related to daylength, and maximal concentrations were observed during mid-summer (July) and minimal concentrations during early winter (January). The temporal pattern of circulating inhibin was positively correlated with FSH (r = 0.88, P < 0.001), but lagged behind the seasonal FSH increase by 1-3 weeks. Antler length, body weight and chest girth were maximal during pre-rut (December-January). Maximal scrotal circumference and combined testes volume were observed in mid-winter (February), whereas peak neck girth and behavioural aggression occurred 1-3 months later (March-May). On the basis of quarterly electroejaculation results, motile spermatozoa were produced in all seasons. However, the greatest number of motile spermatozoa per ejaculate was observed during the winter and spring, whereas the highest incidence of sperm pleiomorphisms (> 80%) was detected in the autumn. Histological assessments of the regressed testis (July) revealed fewer germ cells undergoing spermatogenesis and an increased incidence of degenerating and abnormal cell types. In summary, Eld's deer exhibit a circannual hypothalamic-pituitary-gonadal cycle with onset of pituitary activation occurring during the autumn and winter, whereas gonadal activity peaks during the winter and spring as daylengths are increasing. Marked circannual variations in circulating prolactin suggest that Eld's deer may use photoperiodic cues to modulate seasonal fertility; however, the existence of an endogenous seasonal rhythm operating independently of photoperiod cannot be excluded.

Animals↗

Functional analysis using chlortetracycline fluorescence and in vitro fertilization of frozen-thawed ejaculated boar spermatozoa incubated in a protein-free chemically defined medium.

Cumulus-enclosed pig oocytes were matured in vitro, freed from cumulus cells, and inseminated with frozen-thawed ejaculated spermatozoa in a chemically defined protein-free medium containing 37.0 mmol NaHCO3 l-1 and 5 mmol caffeine l-1. When the medium was supplemented with 1 mg polyvinylalcohol (PVA) ml-1, more penetrated oocytes were observed 14 h after insemination with 7-12 x 10(6) cells ml-1 than with 4-5 x 10(6) cells ml-1 and the incidence of polyspermy reflected the sperm concentration used. Varying the NaHCO3 concentration but maintaining the sperm concentration at 8 x 10(6) cells ml-1 resulted in significantly more oocytes being penetrated in media containing 45.83-50.25 than 37.0-41.42 mmol NaHCO3 l-1; there were no significant differences in the incidence of either male pronuclear formation or polyspermy. In medium containing 45.83 mmol NaHCO3 l-1, the inclusion of PVA at 0-5 mg ml-1 had no effect on proportions of penetrated oocytes, male pronuclear formation or polyspermy. However, when spermatozoa from three different boars were evaluated, the penetration and male pronuclear formation rates were highly variable, unlike the incidence of polyspermy. Penetration of cumulus-free oocytes was first detected at 6 h. When spermatozoa were incubated for 6 h in the absence of oocytes, motility, but not vitality, decreased whether or not PVA was included in the medium. Chlortetracycline (CTC) fluorescence analysis of the capacitation state indicated a rapid decline in the proportion of live uncapacitated, acrosome-intact cells and a rapid rise in the proportion of live capacitated, acrosome-reacted cells during the first hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The protein encoded by cancer/testis gene D40/AF15q14 is localized in spermatocytes, acrosomes of spermatids and ejaculated spermatozoa.

We have previously identified and cloned a human gene, D40, that is preferentially expressed in testis among normal organs, while it is widely expressed in various human tumor cell lines and primary tumors derived from different organs. In this report, we have examined the expression and localization of this protein in human testis with an antibody specific to D40 protein. In Western analyses, the anti-D40 antibody recognized a major band with a molecular mass of 300 kDa and a minor band of 250 kDa. These bands were not observed in the testis lysates from patients with Sertoli-cell-only syndrome and with Kleinfelter syndrome, who lack germ cells of the testis, indicating that D40 protein is expressed in the germ cells of normal testis. Immunohistochemical studies have revealed that D40 protein is highly expressed in spermatocytes and in the pre-acrosome of round spermatids. In the acrosome, D40 protein expression is observed not inside but outside the acrosome membrane. This is consistent with the finding that the amino-acid sequence at the amino terminal of the D40 protein lacks a hydrophobic signal peptide that is required for proteins to translocate to the membrane. Expression of D40 protein is observed in the acrosome of ejaculated spermatozoa as well, although the level is low compared with that in the pre-acrosome of spermatids. These results suggest that D40 protein plays important roles in spermatogenesis, especially in the formation and maintenance of the acrosome.

Acrosome↗

Characterization of Mg2+- and Ca2+-ATPase activity in membrane vesicles from ejaculated ram seminal plasma.

The activity of divalent cation-stimulated adenosine triphosphatase (ATPase) has been studied in vesicular membranes isolated from ejaculated ram seminal plasma. This nonspecific acidic ATPase can be activated by millimolar concentration of any one of the following cations: Ca2+, Mg2+, Zn2+, or Mn2+ to give high specific activity (approximately 300 mumol/mg/hr), in absence of the other cations. Free Zn2+ inhibits activity of this ATPase. The Km for adenosonine triphosphate (ATP) ranged between 0.17 and 0.24 mM, and for the divalent cation ranged between 0.4 and 0.8 mM. When the ATPase is activated by Ca2+, two Kms for Ca2+ concentration were found: 0.8 and 0.08 mM. It is suggested that the seminal plasma membranes also contain alkaline ATPase, which is more specific for Ca2+.

Adenosine Triphosphatases↗

Enhancing detection of gonococcus in ejaculates of adult males using sperm dilution.

Specific cultures were used to detect growth of Neisseria Gonorrhoeae (NG) in 90 ejaculates of partners of childless marriages. Although no gonococcal growth was observed in undiluted semen, 9 out of 68 subjects with silent infection presented growth of NG in seminal plasma after dilution 1:2 with saline. It is concluded that semen dilution increases the chances of detection of NG in semen samples of asymptomatic gonococci carriers.

Adult↗

Effect of osmolality and glycosaminoglycans on motility, capacitation, acrosome reaction, and in vitro fertilizability of bovine ejaculated sperm.

Bovine ejaculated semen was placed in a modified Tyrode's medium with albumin, lactate, and pyruvate. The sperm were washed three times and subjected to nine treatment in a 3 X 3 factorial arrangement. Treatments consisted of osmolality (exposure to 380 mOsmol/kg medium for 5 min, exposure to 340 or 295 mOsmol/kg medium for the entire incubation period), and the presence or absence of glycosaminoglycans (100 micrograms/ml chondroitin sulfate A or 10 micrograms/ml heparin). Sperm were examined at 4.5 h, 8 to 9 h, and 24 to 25 h of incubation (37 degrees C, 5% CO2, and 95% air). Heparin caused head-to-head agglutination of sperm, raised the percent sperm without seminal antigens over the acrosome (capacitated) by 20% at 4.5 h, and doubled the percent of acrosome-reacted sperm. However, this stimulation did not improve in vitro fertilizability. Chondroitin sulfate A tended to maintain motility, but did not affect capacitation or the acrosome reaction, possibly due to glucose inhibition. Both high osmolality treatments tended to reduce motility, especially after 24 h of incubation when the 340 osmolality treatment reduced motility by 14% over the 295 treatment. No consistent effect on capacitation was observed. The 340 and 380 osmolality treatments induced 8.6 and 6.1% more acrosome reactions by 24 h than the 295 treatment. The 340 mOsmol/kg treatment yielded insignificantly higher in vitro fertilization rates, as evidenced by development of zygotes to the two-cell stage. Lack of statistical significance was due to high variation with in vitro fertilization rates.

Acrosome↗

A VR based therapy for the treatment of impotence and premature ejaculation.

The use of psycho-dynamic psychotherapy integrating virtual reality (VR) dealt with in this study on the treatment of erection dysfunctions and premature ejaculation started several years ago, after having seen the scarce results we obtained using exclusively a psycho-dynamic approach (accompanied by pre-recorded sound and music). Considering the particular way that full-immersion VR involves the subject who experiences it, we hypothesized that better results could be obtained during therapy for these sexual disorders and in particular regarding the nature of erection dysfunction, commonly referred to as impotence "a persistent or recurrent inability to attain, or to maintain until completion of the sexual activity, an adequate erection." The plan for therapy consisted of 12 hour-long sessions over a 25-week period, and the methods involved the use of a VR helmet, joystick and miniature television screens that projected specially-designed CD-ROM programs on psychological development.

Adult↗

Morphological and biochemical analysis of cell death in human ejaculated spermatozoa.

We have analyzed the type of cell death occurring in human normal ejaculated spermatozoa. Sperm cells were prepared either by centrifugation alone (group 1) or by density gradient centrifugation (group 2) and were cultured for 24 hrs. Cells were examined after 4 and 24 hrs. By comparison unprepared spermatozoa were used as a control group. Necrosis was investigated by intra-cellular vital stain penetration and electron microscopy. Apoptosis was researched by DAPI staining, annexin V-binding, electron microscopy, DNA fragmentation and PARP cleavage. In group 1, after 4 hrs., there was a mixture of spermatozoa dead either by necrosis or apoptosis while after 24 hrs., necrosis was prominent. Similar findings were observed in the control group. In contrast, in group 2 apoptosis was the major form of cell death of spermatozoa after 24 hrs. of culture. These findings suggest that apoptosis can be an important factor when spermatozoa are used for assisted reproductive technologies.

Adult↗

Bicarbonate stimulation of boar sperm motility via a protein kinase A-dependent pathway: between-cell and between-ejaculate differences are not due to deficiencies in protein kinase A activation.

Because poorly motile sperm samples can often be stimulated by treatments that increase intracellular levels of cyclic adenosine monophosphate (cAMP), it has been supposed that such samples are unable to maintain an adequate supply of the cyclic nucleotide with which to activate protein kinase A (PKA). To investigate this hypothesis, we incubated boar sperm samples with bicarbonate (a stimulator of adenylyl cyclase) and compared its effect with that of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate (cBIMPS, a highly permeable and stable cAMP analog). Videomicroscopy assessment of motility was followed by computer analysis of the sperm tracks to produce motility descriptor values for many individual cells in each sample, whence "cluster" analysis of these data identified groups of spermatozoa that differed in motility characteristics. Bicarbonate stimulation of motility was characterized by an increase in the linearity (LIN) and progressive velocity of part of the sperm population only. The size of this "fast linear" subpopulation varied considerably between ejaculates. However, treatment with cBIMPS did not induce significantly more "fast linear" sperm than treatment with bicarbonate. In further experiments investigating the role of protein kinases in motility control, bicarbonate stimulation was greatly inhibited by H89 (a specific inhibitor of PKA), whereas GF109203X and lavendustin A (inhibitors of protein kinase C [PKC] and protein tyrosine kinase [PTK], respectively) had essentially no effect. While inclusion of the protein phosphatase inhibitor calyculin stimulated motility, it failed to increase the overall percentage of "fast linear sperm" induced by bicarbonate. We conclude that intersperm and interejaculate differences in boar sperm motility are not due to inadequacy in cAMP supply or to ineffective PKA activity.

Animals↗

Vibratory Stimulation of Ejaculation Yields Increased Motile Spermatozoa, Compared with Electroejaculation, in Squirrel Monkeys (Saimiri boliviensis).

Semen sample collection in squirrel monkeys has traditionally relied on electroejaculation (EE) via the rectum; how- ever, the yield is low when anesthesia is used. An alternative method has been advocated for men with spinal cord injury that relies on penile vibratory afferent stimulation. Vibratory stimulation (VS) physiologically coordinates emission and ejaculation without requiring anesthesia; this procedure was investigated in squirrel monkeys. During the breeding season, 10 donor males were evenly allotted to two groups; one received VS, and the other received EE. Three days later, treatments were reversed. For VS, a clinical unit was adapted to hold a 1.8-ml microcentrifuge tube as an artificial vagina. Unanesthetized, manually restrained animals were positioned in ventral recumbency. Vibratory stimulation consisted of a I-mm amplitude, 75-Hz stimulation for 1 min, rest for 1 min, then continued stimulation at 1.8-mm amplitude, 90 Hz for 1 min. For EE, animals were anesthetized by administration of Telazol(3.5 mg/kg of body weight, IM) and stimulated by EE via the rectum, using a 5- to 7.5-V, 60-Hz sine wave for 1 minute of 1 sec on/ I sec off and a probe with two longitudinal electrodes and a temperature monitor. Stimulation was repeated after 1 minute s rest. Semen samples were diluted, incubated, centrifuged, and evaluated for spermatozoal count and motility. Nine of 10 monkeys responded to VS with semen samples, all of which contained spermatozoa. Mean motility was 79.8% and total mean motile spermatozoa yield was 26.1 (range, 0.4 to 110) million. By use of EE, all monkeys produced semen samples, but only 6 samples contained spermatozoa, with mean motility of 70% and low yield of 0.534 (range, 0.038 to 2.2) million motile spermatozoa. In conclusion, penile VS was superior with regard to quantity of motile spermatozoa obtained. Additionally, no anesthesia was necessary for this relatively benign procedure.

Journal Article↗

[Ejaculate microflora sensitivity to lactoferrin in chronic prostatitis].

To determine an antimicrobial action of lactoferrin on bacterial flora of ejaculate in chronic prostatitis, we examined 124 patients with various diseases of male reproductive system at the age between 17 and 56. A total of 280 strains were used as test microorganisms (MO). Sensitivity of the latter to lactoferrin was studied with a human lactoferrin apoform. MO isolated from patients with marked dysbiosis were resistant to lactoferrin (94%). MO, not leading in cenosis, were more sensitive to lactoferrin than MO of the main colonies grown after seeding. Reproductive dysfunction was accompanied by higher occurrence of strains resistant to lactoferrin. The least sensitivity to lactoferrin was registered in asthenozoospermia. Lactoferrin resistance of MO may raise MO pathogenicity. When lactoferrin-resistant strains predominate in microbiocenosis, it is an unfavourable prognostic sign in relation to development of complications, primarily subfertility.

Adolescent↗