Separation of ram spermatozoa bearing X and Y chromosome by centrifugal countercurrent distribution in an aqueous two-phase system

The availability of reliable quantification techniques of X and Y chromosome-bearing spermatozoa in a given insemination dose would allow further approaches to their separation, which is a topic of unquestionable interest in animal production. The aim of the current work was the development of a combined approach of polymerase chain reaction and countercurrent distribution to address both objectives. First, using Sac/polymorphisms for ZFX/ZFYloci in sheep deoxyribonucleic acid, a linear correlation has been established between the densitometric quantification of the restricted fragment length polymorphisms corresponding to the amplified loci ZFX/ZFY by polymerase chain reaction and the theoretical proportions of X and Y chromosomes in standard solutions. The method, subsequently applied to semen samples, estimated an equal proportion of spermatozoa bearing each chromosome. Second, by using centrifugal countercurrent distribution in a sensitive-charge aqueous two-phase system, we achieved the separation of a sperm population enriched in Y chromosome-bearing ram spermatozoa (75%) with a high viability rate (57%).     

In vitro effects of nonylphenol on motility,mitochondrial, acrosomal and chromatin integrity of ram and boar spermatozoa

The objective of this study was to determine the effects of nonylphenol (NP) on viability of ram and boar sperm in vitro. Ram or boar spermatozoa were exposed to 1, 10, 100, 250 and 500 μg NP ml^?1 for 1, 2, 3 or 4 h. Computer‐assisted sperm motility analysis (CASA) system was used to evaluate sperm motility characteristics. Flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity, while epifluorescent microscopy was used to determine sperm acrosomal status. Exposure of both species spermatozoa to 250 and 500 μg NP ml^?1 was detrimental to progressive motility (P < 0.05), and its adverse effect was significant at lower (100 μg NP ml^?1) concentration (P < 0.05). The percentages of ram and boar spermatozoa with high MMP declined drastically after exposures to ≥250 μg ml^?1 NP (P < 0.05). Unlike chromatin integrity, which did not appear to be altered by NP exposure, there were dose‐dependent NP effects (P < 0.05) on acrosomal integrity of both species at as low as 1 μg ml^?1 NP for boar spermatozoa and 10 μg ml^?1 NP for ram spermatozoa. These data show adverse effects of NP on ram and boar spermatozoa and thus its potential harmful effects on male reproduction as NP is found in fruits, vegetables, human milk, fish and livestock products.     

Immunocytochemical localization of acrosin on both acrosomal membranes and in the acrosomal matrix of porcine spermatozoa

Immunocytochemical techniques were employed to determine, at the ultrastructural level, the location of acrosin in porcine spermatozoa. Antisera to highly purified porcine acrosin was produced in rabbits. The (Fab')2 fragments of the immunoglobulins were purified and conjugated with horseradish peroxidase (HRP). Washed, formaldehyde-fixed spermatozoa were reacted with the labeled antiacrosin immunoglobulins, utilizing a direct staining technique. Electron microscopy revealed that the peroxidase reaction product of HRP-antiporcine acrosin was distributed evenly over the outer acrosomal membrane of spermatozoa with intact acrosomes. The labeled antibody was also distributed evenly over the inner acrosomal membrane of cells when the overlying acrosomal structures were absent. In some spermatozoa, labeling was noted throughout the acrosomal matrix. No significant labeling was observed in control specimens when spermatozoa were exposed to HRP-antiporcine acrosin immunoglobulins that had been adsorbed previously with excess purified acrosin or exposed to HRP-conjugated rabbit antiporcine immunoglobulins. This pattern of labeling is consistent with the hypothesis that acrosin may function as a zona lysin. The observation that the outer acrosomal membrane and acrosomal matrix are labeled suggests that acrosin is not exclusively located on the inner acrosomal membrane and, thus, could participate in physiologic events other than zona penetration.     

The fix vital stain method. Simultaneous determination of viability and acrosomal status of bovine spermatozoa.

A rapid, accurate and precise method for simultaneous determination of acrosomal status and viability of bull spermatozoa is introduced and evaluated. The method involves fixation of semen with glutaraldehyde and subsequent addition of the fluorescent dye Hoechst bisbenzimide 33258 (H33258). Wet mounts were examined using a combination of phase-contrast and fluorescence microscopy (x500) for simultaneous visualization of the acrosomal apical ridge, which is indicative of the presence of an intact acrosome, and H33258-labeled nuclei, which is indicative of membrane-damaged cells. This fix-vital stain method allows differentiation between true acrosome reactions and degenerative postmortem loss of acrosomal membranes. Incubation of frozen-thawed spermatozoa for 60 minutes at 37 degrees C in the presence of calcium ionophore A23187 resulted in an increase in the percentage of true acrosome-reacted spermatozoa. The fix-vital stain method does not contain any processing steps that result in loss, selection, or damage of spermatozoa and therefore allows evaluation of representative semen samples.     

Determination of fatty acid profile in ram spermatozoa and seminal plasma

Fatty acids are important in male reproductive function because they are associated with membrane fluidity, acrosome reaction, sperm motility and viability, but limited information exists about the fatty acid profile of ram semen. Our aim was to determine the fatty acid composition in ram spermatozoa and seminal plasma. Sixty ejaculates were obtained from three ram (20 ejaculates/ram) using artificial vagina. Ram spermatozoa (RS) and seminal plasma (SP) were separated using centrifugation, and the fatty acids were analysed by gas chromatography. Total lipids obtained in ram spermatozoa were 1.8% and 1.6% in seminal plasma. Saturated fatty acid (SFA) was proportionally major in SP (66.6%) that RS (49.9%). The highest proportions of SFA corresponded to C4:0 (RS = 16.3% and SP = 28.8%) and C16:0 (RS = 16.3% and PS = 20%). The most important unsaturated fatty acid (UFA) was docosahexaenoic acid (DHA), 44.9% in RS and 31.5% in SP. The profile of fatty acid and their proportions showed differences between spermatozoa and seminal plasma.     

Preincubation in peritoneal fluid decreases the follicular fluid-induced acrosomal reactivity of human spermatozoa

Summary. The aim of this study was to determine the effects of preincubation in peritoneal fluid on the follicular fluid-induced acrosomal reactivity of human spermatozoa in vitro. Thirty women participating in our IVF-EL program were given a GnRH-analogue, highly purified FSH and hCG in order to induce superovulation. Peritoneal and follicular fluids were aspirated during pick-up laparoscopy, centrifuged, filtered and frozen until use. An aliquot of swim-up suspension from nor-mospermic semen specimens ( n =30) was incubated with peritoneal fluid or HAM-F10 for 30–180 min, and follicular fluid (in volumetric proportion approximately 50/50 with peritoneal fluid) was subsequently added. The percentage of acrosomally-reacted spermatozoa was assessed using the FITC-conjugated Pisum sativum lectin before and after incubation in peritoneal fluid or control medium, as well as after follicular fluid addition. Peritoneal fluid was not able to stimulate acrosomal reactivity; further, preincubation in peritoneal fluid decreased, but not abolished, the follicular fluid-induced acrosomal reactivity. A longer pre-incubation in peritoneal fluid was associated with a lower percentage of reacted spermatozoa in response to the addition of follicular fluid. In conclusion, our data suggest that peritoneal fluid acts maintaining spermatozoa in an unreacted status in the upper female genital tract. After mixing with follicular fluid, a phenomenon that is likely to occur at ovulation, peritoneal fluid reduces, but does not abolish, the stimulating effect of follicular fluid on acrosomal reactivity.     

Effect of the cryopreservation process on the activity and immunolocalization of antioxidant enzymes in ram spermatozoa

In this study, certain enzymes in ram semen involved in reactive oxygen species elimination and their changes during the cryopreservation process were characterized in order to investigate the hypothesis that the antioxidant defense system is involved in the maintenance of frozen sperm quality. Glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activities were quantified in ram sperm samples subjected to cooling and freezing/thawing processes. In addition, their distribution on the sperm surface and the changes due to cryoinjury were determined by indirect immunofluorescence. SOD showed the highest antioxidant activity, which was also twice as high in fresh and cooled samples as in frozen/thawed ones. Enzymatic activity of GPx and GR showed no significant change throughout the freezing process. Seminal plasma proteins (SPPs) added alone or with other compounds showed a protective effect and accounted for an increase in the sperm quality parameters and enzyme activity levels not only in the fresh sample but also after cooling and freezing/thawing. These antioxidant enzymes were distributed over several sperm regions, and we were able to define several subpopulations according to the obtained sperm immunofluorescence patterns. The sperm membrane distribution of SOD, GPx, and GR changed considerably during cryopreservation, and the type and percentage of the immunofluorescence patterns found in fresh samples were severely modified. This remodeling was strongly affected by the use of different cryoprotectants. The mixture of SPPs, oleic/linoleic acids, and vitamin E was able to partly maintain and recover the fresh enzyme distribution, particularly of SOD.     

Effect of seminal plasma fractions from entire and vasectomized rams on the motility characteristics, membrane status, and in vitro fertility of ram spermatozoa

Whole seminal plasma from ram semen collected before and after vasectomy was separated into 2 fractions, supernatant and pellet of vesicles, and their protein profiles characterized by one-dimensional (1D) gel electrophoresis. The effects of autologous whole seminal plasma and these fractions on motility characteristics (assessed subjectively and by computer-assisted sperm analysis), membrane status (assessed by chlortetracycline staining patterns), and in vitro fertility (assessed by fertilization success and timing of fertilization events) of washed frozen-thawed ram spermatozoa were studied. Regardless of vasectomy, whole seminal plasma and supernatant displayed similar protein patterns. These fractions, when included in the postthaw buffer, improved the motility characteristics (59.6% +/- 6.21% and 39.6% +/- 6.21% vs 31.7% +/- 6.46% and 15.5% +/- 6.46% total motility) and membrane integrity (36.6% +/- 8.52% and 31.2% +/- 8.19% vs 30.3% +/- 11.49% and 21.6% +/- 10.28% B staining pattern [characteristic of capacitated acrosome-intact cells] for whole seminal plasma and supernatant vs control at 3 and 6 hours of postthaw incubation, respectively) of frozen-thawed spermatozoa and improved their ability to fertilize in vitro-matured oocytes compared with control buffer without seminal plasma fractions (25.3%, 47.4%, and 37.4% vs 12.3%, 20.2%, and 20.5% oocytes fertilized for spermatozoa incubated with supernatant vs control at 2, 6, and 18 hours after insemination, respectively). Vesicles were absent from semen collected after vasectomy. Pellets of vesicles collected before vasectomy had no effect on spermatozoa at their normal protein concentration but marginally improved both motility characteristics and in vitro fertility, possibly due to contamination from supernatant proteins, when their concentration in the postthaw medium was increased by threefold. It was concluded that the vesicle-free supernatant fraction of seminal plasma, but not the seminal plasma membrane vesicles, improved the function and fertility of frozen-thawed ram spermatozoa when added to the postthaw medium.     

The fate of acrosomal staining during the acrosome reaction of human spermatozoa as revealed by a monoclonal antibody and PNA-lectin

A monoclonal anti-human sperm antibody, raised against an acrosomal antigen and indicated to recognize in boar sperm the serine protease, acrosin, stained in human spermatozoa a 50 Kd antigen and several others in the region 24-34 Kd by immunoblotting. The 50 Kd band and the region of 30-34 Kd showed proteolytic activity by zymographic enzyme detection. The fate of the antigen was studied in the acrosome reaction induced by the calcium ionophore A23187. In control incubations 69.5 +/- 14.2% (mean +/- SD) of the spermatozoa had intact acrosomal staining according to indirect immunofluorescence using this antibody whereas in acrosome-reacted samples only 21.0 +/- 2.0% of the sperm were stained. Another marker for the acrosome, peanut agglutinin-lectin (PNA), was used to detect the acrosome with similar results. Acrosome reactions were verified by electron microscopy. The present results indicate that the corresponding antigen, evidently acrosin, and PNA-positive material are liberated during the acrosome reaction which suggests that they are not bound to the inner acrosomal membrane but are components of the acrosomal matrix.     

The effect of melatonin implants on blood testosterone and acrosin activity in spermatozoa of the ram

In a series of consecutive blood sampling in 15 days intervals over 15 weeks after implantation of melatonin in rams an increased mean value, basal level and number of peaks of testosterone was observed in samples of the third fortnight (45th day). This increase was greater in the autumn (breeding season) than in spring (non-breeding season). Total acrosin activity in spermatozoa was increased between days 35-56 (autumn) and days 49-70 (spring) after implantation and the relative increase was higher in autumn than in spring. The increase of acrosin activity was independent of the changes of testosterone. An increase of acrosin activity by melatonin, in cases of low activity, might improve fertilization rates in sheep not only during the breeding season, but also during the non-breeding season (after oestrus induction).     

Calpain and calpastatin are located between the plasma membrane and outer acrosomal membrane of cynomolgus macaque spermatozoa

Mammalian sperm must undergo an acrosome reaction prior to penetration of the zona pellucida and subsequent fusion with an oocyte. Sperm gain the capability to acrosome react after a period of capacitation, which primarily involves biochemical changes in the sperm membranes. The morphological events of the acrosome reaction have been well-documented, but the underlying cellular mechanisms that regulate capacitation and the acrosome reaction remain unclear. Antibodies to the 2 ubiquitous calpains, mu and m, as well as the small subunit, which associates with both calpains, were localized at the ultrastructural level to the region between the plasma membrane and the outer acrosomal membrane of cynomolgus macaque sperm. After the acrosome reaction, all of the anti-calpain antibodies labeled the acrosomal shroud, suggesting that calpains are located throughout the cytoplasmic area between the 2 outer sperm membranes. Calpastatin is an endogenous modulator of calpain activity and is also localized within the same cytoplasmic region as calpains. The antibodies used for ultrastructural localization were also used to probe Western blots of sperm extracts. Antibodies to either the mu- or m-calpain recognized an 80-kd protein, which is similar to the molecular weights of other ubiquitous calpains described. The small subunit (30 kd) was also recognized with a specific monoclonal antibody. An antibody to calpastatin recognized a major band at 78 kd and a lighter band at 45 kd, while the antibody to the testis-specific isoform of calpastatin (TCAST) recognized a 110-kd protein. We hypothesize that this cysteine protease system may be functional in cynomolgus macaque sperm during capacitation, the acrosome reaction, or both.     

Effect of male reproductive tract fluids and proteins on the metabolism and motility of ram spermatozoa

Cauda epididymal fluid (CEF) greatly stimulated the oxygen uptake of washed ejaculated ram spermatozoa; the effect was evident within 1 h and persisted over the 8 h of the experiment. The stimulus was comparable to that produced by 10 mM glucose and the effects were not additive, that is, CEF and CEF plus glucose elicited about the same oxygen uptake. This suggested that CEF suppressed the oxidation of added glucose and this was confirmed by measuring the amount of glucose oxidized in the presence and absence of CEF. Cauda epididymal fluid improved the motility of washed ram spermatozoa but it was somewhat less than that produced by glucose and usually only became evident after about 6 h or incubation. Electrophoretic analysis of cauda epididymal spermatozoa incubated in radioiodinated CEF showed that these cells absorb fluid components in the zone 82 to 56 kD. However, a molecular weight fraction less than 5 kD obtained by passing CEF through a Sephadex G-25 column, was not effective in stimulating the oxygen uptake of ram spermatozoa. The effects of CEF on the metabolism of ram spermatozoa could be mimicked by 2.5-4.0 mg/ml bovine serum albumin (BSA). Stimulation of oxygen uptake was apparent within 1 h and persisted over the 8 h of the experiment. As with CEF, stimulation of oxygen uptake by BAS was less than with 10 mM glucose but the effects were not additive. Like CEF, BSA reduced the amount of glucose oxidized. Bovine serum albumin also improved the motility of ram spermatozoa over 8 h. After passage through Sephadex G-25 to remove any low molecular weight contaminants (less than 5 kD), BSA was still effective in stimulating the oxygen uptake of spermatozoa over 4 h. Ram blood plasma and especially ram seminal plasma were also effective after passage through the Sephadex. Human serum albumin (HSA) was as effective as BSA in stimulating the oxygen uptake of ram spermatozoa but defatting decreases its effectiveness. The motility score of the spermatozoa was also adversely affected by this treatment. It is concluded that the stimulating effects of CEF and of the other fluids and proteins are due to substrates, at least some of which are present as or associated with macromolecules.     

Effect of gossypol-acetic acid on calcium transport and ATPase activity in plasma membranes from ram and bull spermatozoa

The effects of gossypol acetic acid on the activity of Mg-ATPase and Ca-Mg-ATPase and on calcium uptake by plasma membranes from ram and bull spermatozoa were examined. The three parameters were almost completely inhibited by 10 microM gossypol for both ram and bull sperm. In order to assess the effects of higher gossypol concentrations isolated membrane vesicles were loaded with calcium by operating the ATP-dependent calcium pump after which gossypol was added and calcium uptake followed. At 10 microM gossypol, additional calcium uptake was 85% inhibited while at 40 microM a release of the accumulated calcium was observed. The inhibitory effect of 10 microM gossypol was almost completely reversible by simple dilution of gossypol-treated membranes, whilst at 40 microM the effect was only 50% reversible. The data show a high degree of similarity between bull and ram, suggesting minimal differences between the two species as far as the structure and function of the sperm plasma membrane is concerned.     

Characterization of the kisspeptin system in human spermatozoa

Kisspeptin, the product of the KISS1 gene, plays an essential role in the regulation of spermatogenesis acting primarily at the hypothalamic level of the gonadotropic axis. However, the presence of kisspeptin and its canonical receptor, KISS1R, in spermatozoa has not been explored nor the direct effects of kisspeptin on sperm function have been studied so far. In the present study, we analysed the expression of kisspeptin and its receptor in sperm cells by western blot and immunocytochemistry assays and evaluated the effects of exposure to kisspeptin on sperm intracellular Ca(2+) concentration, [Ca(2+)]i, sperm motility, sperm hyperactivation and the acrosome reaction. Changes in [Ca(2+)]i were monitored using Fura-2, sperm kinematic parameters were measured using computer-assisted sperm analysis (CASA), and the acrosome reaction was measured using fluorescein isothiocyanate-coupled Pisum sativum agglutinin lectin (FITC-PSA method). We found that kisspeptin and its receptor are present in sperm cells, where both are mainly localized in the sperm head, around the neck and in the flagellum midpiece. Exposure to kisspeptin caused a slow, progressive increase in [Ca(2+)]i, which reached a plateau about 3-6 min after kisspeptin exposure. In addition, kisspeptin modulated sperm progressive motility causing a biphasic (stimulatory and inhibitory) response and also induced transient sperm hyperactivation. The effects of kisspeptin on sperm motility and hyperactivation were inhibited by the antagonist of KISS1R, peptide 234. Kisspeptin did not induce the acrosome reaction in human spermatozoa. These data show for the first time that kisspeptin and its receptor are present in human spermatozoa and modulate key parameters of sperm function. This may represent an additional mechanism for their crucial function in the control of male fertility.     

Comparison of two techniques to evaluate the acrosomal status of zona pellucida bound sperm in humans

In this work, we have compared two procedures that evaluate the acrosomal status of human sperm bound to the human zona pellucida. Motile sperm, selected by a Percoll gradient, were capacitated by incubation at 37 degrees C, 5% CO2, for 4.5 h, at 20 x 10(6) cells ml(-1). Then, the sperm were incubated with nonviable human oocytes for 10 min at 37 degrees C, 5% CO2. The oocytes with bound sperm were transferred to 500 microl phosphate-buffered saline (PBS) and washed to remove loosely bound sperm. The oocytes were then processed according to the procedures of Cross et al. (1986) or Liu & Baker (1996). In the Cross's procedure, the sperm were labelled while they were bound to the zona. In the Liu's procedure, the sperm were first dislodged from the zona into a droplet of PBS and labelled in there. Both procedures gave equivalent percentages of acrosome-reacted sperm. However, the total number of zona-bound sperm available for assessment with the procedure of Liu & Baker was greater than that of Cross et al. We suggest to use the former procedure to evaluate the acrosomal status of zona-bound sperm in humans. Moreover, this procedure also provided information about sperm ability to bind to the zona pellucida.     

Lack of correlation between human y-positive spermatozoa and acrosomal abnormalities and variations in intensity of fluorescence of sperm stained with quinacrine compounds

Seminal fluid from 170 men was examined for acrosomal abnormalities of sperm (Papanicolaou procedure X 1000) and for y-positive cells (quinacrine-stained smears). No correlation was found between these parameters, suggesting that acrosomal abnormalities are similarly distributed among x- and y-bearing spermatozoa. The proportion of y-positive sperm was found to be low (23.6 + 10.7% (SD) for the oligozoospermic specimens and 23.7 +/- 9.8% for specimens with sperm counts above 40 million/ml). Three degrees of fluorescence intensity were observed--weak, moderate, and strong--the strong fluorescence being associated with the highest percentage of y-bodies (30.6 +/- 12.7%, 36.6 +/- 5.7%) and the weak fluorescence with the lowest (16.7 +/- 7.0%, 15.0 +/- 9.5%). It is suggested that structural abnormalities and/or metabolic alterations in either the DNA molecule or the chromosomal proteins may be responsible for variability in the ability to bind quinacrine compounds.     

Purification of an acrosomal antigen recognized by a monoclonal antibody and antifertility effects of isoimmune serum

A highly conserved acrosomal antigen reactive to a monoclonal antibody (HS-63), generated against human sperm, was purified to homogeneity with a combination of conventional procedures and immunoaffinity chromatography using a soluble extract of mouse and rabbit testes. The molecular weight of the purified antigen was 42-50 kD when analysed by sodium dodecylsulphate polyacrylamide gel electrophoresis. The high specificity of the purified antigen to monoclonal antibody HS-63 was shown by indirect immunofluorescent inhibition assay, enzyme-linked immunosorbent assay, Western blot analysis and radioimmunosorbent assay. The purified antigen was used for isoimmunization of mice and rabbits. Following successive immunizations, antisera of high titres were raised and reacted specifically with antigen on the sperm acrosome and in testes of several mammalian species, but not with somatic tissues. These isoimmune sera exhibited strong inhibition on mouse in-vitro fertilization and human sperm penetration of zona-free hamster eggs. The results of this study suggest that the sperm-specific acrosomal antigen reacting with HS-63 could be a good candidate for the development of immunocontraceptive vaccines in humans and in other animals.