Marker Of Epididymal Function Research Articles

Modulatory effects of ghrelin on sperm quality alterations induced by a fructose-enriched diet

The objectives of this study were: 1) to evaluate the effects of a fructose enriched diet (FED) on rat sperm quality, epididymal function (i.e. oxidative stress and alpha-glucosidase expression) and testosterone concentrations; 2) to determine if the administration of ghrelin (Ghrl), reverses the effects induced by FED.After validating the protocol as an inductor of metabolic syndrome like-symptoms, adult male rats were assigned to one of the following treatments for 8 weeks: FED = 10% fructose enriched in water (v/v); FED + Ghrl = fructose enriched diet plus Ghrl (6 nmol/animal/day, s.c.) from week 6–8; or C = water without fructose (n = 5–10 animals/group).FED significantly decreased sperm concentration and motile sperm count/ml vs C (FED: 19.0 ± 1.6 × 106sperm/ml and 834.6 ± 137.0, respectively vs C: 25.8 ± 2.8 × 106 and 1300.4 ± 202.4, respectively; p < 0.05); ghrelin injection reversed this negative effect (23.5 ± 1.6 × 106sperm/ml and 1381.7 ± 71.3 respectively). FED resulted in hypogonadism, but Ghrl could not normalize testosterone concentrations (C: 1.4 ± 0.1 ng/ml vs FED: 0.8 ± 0.2 ng/ml and FED + Ghrl: 0.6 ± 0.2 ng/ml; p < 0.05). Ghrelin did not reverse metabolic abnormalities secondary to FED. FED did not alter epididymal expression of antioxidants enzymes (superoxido-dismutase, catalase and glutathione peroxidases –Gpx-). Nevertheless, FED + Ghrl significantly increased the expression of Gpx3 (FED + Ghrl: 3.47 ± 0.48 vs FED: 0.69 ± 0.28 and C: 1.00 ± 0.14; p < 0.05). The expression of neutral alpha-glucosidase, which is a marker of epididymal function, did not differ between treatments.In conclusion, the administration of Ghrl modulated the negative effects of FED on sperm quality, possibly by an epididymal increase in Gpx3 expression. However, Ghrl could not neither normalize the metabolism of FED animals, nor reverse hypogonadism.

Varicocele decreases epididymal neutral α‐glucosidase and is associated with alteration of nuclear DNA and plasma membrane in spermatozoa

To assess the relationship between a marker of epididymal function and both the fragmentation of the sperm nucleus and the integrity and maturity of the sperm membrane in patients with or without varicocele. Semen samples were obtained from men with varicocele grades II and III (n = 60) and from a control group with zoospermia defined as normal (n = 30). Samples were evaluated by a spermiogram, a hypoosmotic swelling test (HOST), neutral α-glucosidase (NAG) enzyme activity, sperm hyaluronan-binding assay (HBA) and DNA fragmentation using a sperm chromatin dispersion (SCD) test. Seminal plasma NAG levels, percentage of sperm bound to hyaluronic acid, HOST-positive cells and sperm quality were significantly lower in the varicocele compared with the control group. Higher levels of sperm DNA fragmentation, as measured by SCD, were also observed in the varicocele group compared with the control group. Seminal NAG activity levels showed a strong negative correlation with DNA fragmentation and a significant positive correlation with the HBA test and the HOST. Varicocele causes a reduction in NAG activity by the epididymis that is associated with damage to both the membrane and sperm nucleus and a reduction in the seminal parameters. NAG levels were correlated with the quality of the sperm membrane and nucleus. Data suggest that a reduction of fertilization capacity in men during varicocele can result from damage to both the testis and the epididymis.

Dendritic cells in semen of infertile men: association with sperm quality and inflammatory status of the epididymis

To determine whether dendritic cells (DCs) are present in semen and whether their abundance and activation correlate with sperm quality and inflammatory status of the male genital tract. Invitro prospective study. University hospital laboratory. Eighty infertile men and twelve control fertile men were enrolled in this study. None. The percentages of DCs, intracellular staining of cytokines, and spermatozoa DNA fragmentation index were assessed by flow cytometry. Seminal cytokines, neutral α-glucosidase, zinc, and fructose were measured with commercial kits. A significant number of CD11c(+) HLA-DR(+) DCs were detected in ejaculates from patients with chronic inflammation of genital tract, which was negatively correlated with spermatozoa motility, vitality, and DNA integrity. Intracellular staining of cytokines of seminal DCs showed enhanced ability to secrete inflammatory cytokines (interleukin [IL] 23p19, tumor necrosis factor [TNF]-related apoptosis-inducing ligand [TRAIL], and TNF-α). Furthermore, a significant correlation was found between DCs and the seminal concentrations of IL-6, IL-17, IL-23, TRAIL, and neutral α-glucosidase, the marker of epididymal function, in the inflammatory group but not in the noninflammatory and fertile groups. The abundance and activation of seminal DCs of infertile men may be closely associated with poor epididymal function and sperm quality.

Alpha-glucosidase activity and sperm motility

We correlated the activity of alpha-glucosidase in seminal plasma with the motility and differential motility of sperm. Significant positive correlations were found between the alpha-glucosidase activity and both motility and the percentage sperm with good forward progression. This supports the use of alpha-glucosidase in semen as a marker of epididymal function and specifically of the development of motility.

Neutral α-glucosidase activity in mouse: a marker of epididymal function?

Neutral alpha-glucosidase (NAG) activity is considered a functional epididymal marker in several species. Unlike the rat, no NAG activity has been detected in mice. The aims of the present study were to evaluate NAG secretory activity (the supernatant of the incubated tissue) in mouse epididymis and to determine whether it could be used as a functional epididymal marker. Epididymides (whole or in parts) were incubated in the presence or absence of testosterone (10(-5) m) and secretory NAG activity was compared with known positive controls. Furthermore, we compared enzyme activity in epididymides from well-fed and undernourished mice (50% food restriction for 21 days), a model that alters the epididymal maturation processes. Spectrophotometric analysis revealed NAG activity in mouse epididymis (22.6 +/- 3.7 mU g(-1) tissue; n = 4), being higher in the caput. NAG activity was statistically higher in the caput than in the corpus and in the cauda. No significant differences existed between the caput NAG activity and complete epididymis NAG activity. In undernourished mice, we confirmed changes in epididymal maturation observed previously (i.e. increased number of immature spermatozoa and diminution of the sperm concentration). Concordantly, the epididymides of undernourished mice exhibited decreased enzyme secretory activity, which increased to values similar to those seen in controls following incubation in the presence of testosterone (22.5 +/- 2.6, 12.5 +/- 1.0 and 22.4 +/- 3.7 mU g(-1) tissue, n = 9 in control (n = 7), undernourished (n = 9) and undernourished + testosterone groups (n = 9), respectively). In conclusion, NAG activity was detected in mouse epididymis. Although the present study supports the possibility of using NAG as an epididymal marker, more studies are necessary to effectively prove that NAG activity can be used as an epididymal marker.

Alpha-Glucosidase activity in the rat epididymis under different physiological conditions.

The estimation of alpha-glucosidase activity in semen is widely used as a marker of epididymal function. In the present studies, glucosidase activity was evaluated in the different segments of the rat epididymis under various physiological conditions. In addition, the effect of two known male antifertility agents, gossypol and alpha-chlorohydrin, on enzyme activity was evaluated. Enzyme activity was absent from the epididymis of rats aged 10 and 20 days but became detectable at 30 days of age when the adult pattern of distribution (highest activity in the caput epididymis) was established. Enzyme activity was reduced significantly in all segments of the epididymis at 7 days after castration and a significant decrease in activity was also observed following the administration of either gossypol or alpha-chlorohydrin. These findings are consistent with a role for alpha-glucosidase in sperm maturation in the epididymis.

The value of semen characteristics and tests of sperm function in selecting couples for intra-uterine insemination

In order to improve the selection of couples for intrauterine insemination (IUI) because of longstanding primary infertility of alleged male origin, we have performed a prospective study measuring conventional and advanced analysis of sperm characteristics, the hypoosmotic swelling test, the Shorr stain, the acidified aniline blue stain and α-glucosidase activity in seminal plasma, of 89 couples with no demonstrable abnormality of the female partner. Twenty-four couples attained spontaneous conception, 23 were successful within six cycles of IUI, and 42 remained without conception in spite of IUI during six unstimulated cycles. The proportion and concentration of spermatozoa with progressive motility was significantly lower ( P < 0.01) in the successful IUI cases than in the couples attaining spontaneous conception, and the lower quartile value was lower in the former than in the latter. There were less pregnancies among IUI treated couples when sperm concentration and motility were within the range of normal fertile men, or when the concentration of white blood cells was elevated. More pregnancies occurred when markers of epididymal function, namely the result of the Shorr stain and α-glucosidase measurement, were normal. Total progressive motility and the result of the Shorr stain were the only independent variables selected by logistic regression to discriminate between successful and failed IUI cases. It is concluded that only a limited group of couples may benefit from IUI.

Gene expression in the aging brown Norway rat epididymis.

The mammalian epididymis is the site where spermatozoa are matured and then stored. Though many studies have described epididymal functions and their regulation, little is known about how aging affects this tissue. The Brown Norway rat, which does not show the many age-related pathologies common to other rat strains, was used as a model to study aging of the epididymis. The present study was designed to determine the effect of aging on the mRNA levels for selected markers of epididymal function. Brown Norway rats ranging in age from 6 to 30 months were examined at 6-month intervals; epididymides were sectioned into caput-corpus and cauda regions. Relative mRNA concentrations were assessed using Northern blot analysis and specific cDNAs for the rat 5 alpha-reductase isozymes, types 1 and 2; proenkephalin; the androgen receptor; epididymal proteins B/C and D/E; and sulfated glycoprotein-2 (SGP-2, clusterin). Northern blots were quantitated by densitometric scanning. In the caput-corpus epididymidis, 5 alpha-reductase type 1 and type 2 mRNA levels decreased significantly by 43% and 33%, respectively, between 6 and 12 months and by 64% and 40%, respectively, between 6 and 30 months. No significant change, however, was found in the expression of the 5 alpha-reductase mRNAs in the cauda epididymidis. Interestingly, proenkephalin mRNA was only detected in the caput-corpus epididymidis of 6-month-old rats. In marked contrast to the 5 alpha-reductase isozymes and proenkephalin, no significant age-related changes were observed in the mRNA levels for the androgen receptor, protein B/C, or protein D/E. No age-related changes in mRNA expression for SGP-2 occurred in the caput-corpus epididymidis. However, in the cauda epididymidis, SGP-2 mRNA levels rose by twofold between 6 and 18 months and then decreased sharply by 75% between 18 and 30 months. We conclude that as the epididymis ages, the expression of genes for certain specific markers of epididymal function is affected in a region-specific manner. Further, the decrease in the concentrations of the mRNAs for the 5 alpha-reductase isozymes and proenkephalin in the epididymis between 6 and 12 months is thus far the earliest marker for aging in the male reproductive tract of the Brown Norway rat.

Improvement in the assessment of human epididymal function by the use of inhibitors in the assay of α‐glucosidase in seminal plasma

Seminal alpha-glucosidase has been used clinically as a marker of epididymal function. In this study enzyme inhibitors were used to increase the specificity of the human seminal alpha-glucosidase assay and improve its diagnostic value as an indicator of distal epididymal occlusion in cases of azoospermia. Sodium dodecylsulphate was added to the sample to eliminate the interfering acid isoenzyme secreted by the prostate gland, and castanospermine was used with semen pools to provide a semen blank for the assay by eliminating non-glucosidase-regulated degradation of the substrate. With both inhibitors included in the assay, glucosidase activity in semen samples from 17 fathers was measured to provide reference values for the clinic (lower threshold 18 mU per ejaculate). With the improved assay glucosidase was non-detectable in 8 out of 11 cases of proven and 5 out of 8 cases of suspected ductal obstruction; other azoospermic patients with distal occlusion had values below 11 mU per ejaculate.

Further characterization of a model system for the study of human epididymal physiology and its relation to sperm maturation.

Some preliminary speculations about the possible participation of epididymal antigens in sperm function may be supported by the above data. On the one hand, the reduction in the amount of antigens and their abnormal localization on spermatozoa from infertile patients may be coincident with our view about participation of epididymal antigens in the development of zona pellucida binding ability and fertilizing capacity by spermatozoa during maturation. This hypothesis is derived from experiments showing that immature hamster spermatozoa gain their ability to recognize and bind to zona pellucida and to penetrate homologous oocytes when exposed to preparations enriched in androgen-dependent epididymal secretory proteins or preincubated in conditions that favor their interaction with these proteins. Supporting our viewpoint for such a role in humans is evidence showing the progressive development of the ability to interact with hamster denuded oocytes as human spermatozoa pass along the epididymis. On the other hand, the apparent correlation between the loss of epididymal antigens during capacitation and the increased fertilization of human oocytes in vitro may be reminiscent of the removal of a decapacitation or acrosome stabilizing factor known to occur in many species and that must be removed prior to fertilization. Pending further understanding of their physiological role, the androgen-dependent epididymal proteins may become a useful marker of epididymal function and/or of sperm capacitation in humans. Within this context, we wish to stress the potential value of the model system that we have developed for the study of human epididymal physiology.

Purification and properties of neutral α-1,4 glucosidase from human seminal plasma

Following a preliminary fractionation of neutral alpha-glucosidase (E.C. 3.2.1.20) from human seminal plasma, we have shown by ion exchange chromatography, Sephadex G-200 filtration, and adsorption chromatography that this alpha-glucosidase activity corresponded to two isoenzymes having the same ability to hydrolyse p-nitrophenyl-alpha-D-glucopyranoside. Both isoenzymes present a heat-stable fraction at 60 degrees C, require the presence of divalent cations in the incubation medium to demonstrate their glycolytic activity, and are inhibited by maltotriose and maltose. They have a molecular weight of approximately 200,000 daltons and different sedimentation profiles on sucrose density gradient. This basic knowledge appears to be the prerequisite for further studies dealing with the importance of such isoenzymes as markers of epididymal function in male fertility.