Identification and preliminary characterization of two distinct bovine seminal Pz-peptidases

Two peptidases hydrolyzing the Pz-peptide substrate were identified in bovine semen. Each Pz-peptidase was strongly inhibited by chelating agents, suggesting both were metallopeptidases. However, these peptidases could be distinguished by other properties and were designated Pz-peptidases A and B. Pz-peptidase A hydrolyzed the Pz-peptide at the Leu-Gly bond, was inhibited by tosylphenylethylchloromethylketone (TPCK) but not by phosphoramidon and had a pH optimum near 6, whereas Pz-peptidase B cleaved the Pro-Leu bond, was inhibited by phosphoramidon but not by TPCK and had a pH optimum near 7. Seminal plasma, light particulates and cytoplasmic droplets contained almost exclusively Pz-peptidase A, and Pz-peptidase A predominated in sperm extracts. Pz-peptidase B was found primarily in sperm extracts, but Pz-peptidase B activity was also present in ultralight particulates. Pz-peptidase A of spermatozoa required Triton X-100 for complete extraction, but Pz-peptidase B was solubilized from spermatozoa by nitrogen decompression without detergents. Pz-peptidase B was inhibited by several detergents. In particular, addition of 0.1% Hyamine 2389 to sperm extracts inhibited 99% of the Pz-peptidase B activity. Thus, Pz-peptidase B may have been overlooked in previous studies employing extraction of spermatozoa with Hyamine 2389. The properties of both seminal PZ-peptidases were different from those of purified bovine testicular PZ-peptidase, suggesting that PZ-peptidases from these sources were not identical.     

An acrosin inhibitor in ram spermatozoa that does not originate from the seminal plasma.

Ram seminal plasma, and ejaculated ram spermatozoa that have been washed with 0.25M sucrose, both contain acrosin inhibitor. The aim of this work was to determine whether the intracellular inhibitor originates from the seminal plasma. The amounts of inhibitor in ejaculated and epididymal spermatozoa were measured and compared with the amounts present in the seminal plasma of normal and vasectomized rams. One ejaculated ram spermatozoon contained 2.1 amol (2.1 X 10(-18) mol) of inhibitor and one epididymal spermatozoon contained 3.3 amol of inhibitor. (All molarities are mean values based on pooled ram semen or on single ejaculates from three vasectomized rams.) Calculations from results in earlier publications indicated that one ejaculated ram spermatozoon contains about 3 amol of acrosin; thus the inhibitor: acrosin ratio in washed ram spermatozoa is approximately 1. One ml of ram semen contains, on average, 3 X 10(9) spermatozoa and not more than 0.8 ml of seminal plasma. This number of ejaculated spermatozoa would contain 6.3 nmol of inhibitor, while the same number of epididymal spermatozoa would contain 9.9 nmol of inhibitor. These values exceed the quantities of inhibitor present in 0.8 ml of normal seminal plasma (approximately 1.6 nmol) or in 0.8 ml of seminal plasma from vasectomized rams (approximately 2.3 nmol). We conclude that seminal plasma is not a major source of the acrosin inhibitor that can be recovered from washed ejaculated ram spermatozoa.     

Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa.     

A major secretory protein from rat seminal vesicle binds ejaculated spermatozoa

The rat seminal vesicle produces large amounts of a protein-rich fluid that greatly contributes to semen volume. RSV IV, a protein abundantly secreted from this gland, binds in vitro to rat epididymal spermatozoa. However, there is no evidence that this protein may have an in vivo role as a sperm-coating antigen. We report in this paper that high-molecular-weight RSV IV immunologically related proteins can be detected on ejaculated spermatozoa, but not on epididymal spermatozoa. After incubation of purified RSV IV with ejaculated spermatozoa in freshly recovered semen or with epididymal spermatozoa in a medium containing the coagulating gland secretion, sperm-bound proteins with analogous properties were detected. These results support the hypothesis that RSV IV is modified at ejaculation to an high-molecular-weight, sperm-coating antigen.     

Seminal plasma proteins and the reaction of spermatozoa from intact boars and from boars without seminal vesicles to cooling.

Spermatozoa from intact boars and from boars without seminal vesicles were resuspected in diluent and cooled at different rates to 0 degrees C. Glutamic oxaloacetic transaminase and lactate dehydrogenase activities were greater in the diluents which had contained spermatozoa from intact boars than in those which contained spermatozoa from animals without seminal vesicles. The incubation of seminal plasma from an intact boar with spermatozoa from a vesiculectomized animal before cooling also increased the enzyme activity in the diluent. The factors responsible for this effect were associated with the basic protein fractions of boar seminal plasma, in particular the proteins with haemagglutinating activity which may have been adsorbed onto the spermatozoa. Spermatozoa were exposed to colloidal Fe(OH)2+ to determine by electron microscopy the charge on the surface of the plasma membrane of washed epididymal spermatozoa and ejaculated spermatozoa from intact and vesiculectomized boars. Epididymal spermatozoa bound the positively charged particles more readily than the ejaculated spermatozoa from the intact boars, due to the absence of membrane-bound protein.     

Metabolism of phospholipids by spermatozoa and seminal plasma

1. The hydrolysis of added (32)P-labelled phospholipids by whole ram and bull semen and the separated spermatozoal and plasma components was examined. 2. The ethanolamine phosphoglycerides were rapidly attacked by washed spermatozoa, forming predominantly glycerylphosphorylethanolamine, but with whole semen and seminal plasma a lysophosphatidylethanolamine was also detected. 3. The hydrolysis of lecithin by spermatozoa and plasma was very slow, and glycerylphosphorylcholine was the sole product detected. 4. Ram testicular spermatozoa were comparatively inactive in metabolizing both phospholipids, but ampulla contents showed the same activity as ejaculated semen. 5. Phosphatidylinositol was metabolized by spermatozoa obtained from any portion of the ram reproductive tract and also by seminal plasma. With testicular components, ampulla contents and washed ejaculated spermatozoa, inositol monophosphate, an unidentified phosphate ester and inorganic phosphate were the main products. In contrast, with whole semen and seminal plasma, glycerylphosphorylinositol was the predominant water-soluble phosphate ester. 6. Accessory-gland secretion obtained from vasectomized rams showed a pronounced phospholipase A activity towards ethanolamine phosphoglyceride. 7. On aerobic incubation of whole ram semen there was a decrease in the concentration of all phospholipid classes, although cardiolipin showed the greatest percentage decrease. In the choline phosphoglyceride fraction, this loss was confined to the plasmalogen component. This breakdown of phospholipids was decreased considerably when the spermatozoa were washed, and was not observed when whole bull semen was incubated under similar conditions.     

Activity of angiotensin-converting enzyme (ACE) in reproductive tissues of the stallion and effects of angiotensin II on sperm motility

A testis-specific isoform of angiotensin-converting enzyme (ACE) has been identified in a number of mammalian species. The purpose of this study was to characterize the activity of ACE in equine spermatozoa, seminal plasma, and testis. Activity of ACE was determined in seminal plasma, ejaculated and epididymal spermatozoa from mature stallions as well as from pre- and postpubertal testis. The effect of addition of angiotensin II on equine sperm motility was also evaluated.The activity of ACE in detergent extracted sperm plasma membrane was approximately 13-fold higher than that detected in seminal plasma (93.7 mU/mg versus 7.0 mU/mg protein, respectively). Activity of ACE in equine testis was significantly higher in postpubertal than in prepubertal males (3.0 mU/mg versus 0.4 mU/mg protein, respectively), and ACE activity was reduced (P<0.001) in a dose-dependent fashion by the addition of captopril.The effect of angiotensin II on sperm motility was evaluated by computer-assisted semen analysis in sperm incubated with angiotensin II (0, 1, 10, 100 nM) at 38.5 degrees C. There was no significant effect of angiotensin II on the percent motile sperm; however, there was a significant main effect of angiotensin II (P<0.01) on the kinematic parameters beat cross frequency (BCF), average path velocity (VAP), and curvilinear velocity (VCL), respectively. In addition, there were significant stallionxconcentration interactions for amplitude lateral movement (ALH), BCF, linearity (LIN), straightness (STR), and VCL.This study demonstrates that ACE activity is present in sperm membrane from ejaculated and epididymal spermatozoa and in postpubertal testis. Further studies are required to determine the role of this testis-specific enzyme.     

Effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa of the ram and bull during the cryopreservation process

Experiments were conducted to investigate the effect of seminal plasma on sperm motility during the cryopreservation process. Ejaculated and epididymal spermatozoa from the ram and the bull were washed by centrifugation and resuspended in either seminal plasma or a modified Tyrode's medium (TALP) prior to dilution in medium suitable for cryopreservation. Resuspension of washed ejaculated ram spermatozoa in seminal plasma resulted in higher percentages of motile spermatozoa than resuspension in TALP after the spermatozoa were cooled to 5 degrees C (52 vs 35%), and after thawing (14 vs 9%), respectively. Resuspension of epididymal ram spermatozoa in seminal plasma had no beneficial effect in maintaining sperm motility after cooling (78 vs 73%); however, seminal plasma was beneficial to epididymal ram spermatozoa after thawing (34 vs 3%), respectively. Resuspension of washed ejaculated bull spermatozoa in either seminal plasma or TALP had no effect on the percentage of motile spermatozoa after cooling to 5 degrees C (73 vs 75%) or after thawing (60 vs 60%), respectively. In addition, seminal plasma had no beneficial effect on the percentage of motile epididymal bull spermatozoa when compared with that of TALP-treated spermatozoa after cooling (75 vs 72%) or after thawing (66 vs 63%), respectively. Seminal plasma from different sires (ram and bull) affected epididymal sperm motility. The ability of sperm cells to withstand damage during cryopreservation, however, appears to reside in the sperm cells themselves, probably due to sperm cell composition.     

A bovine seminal plasma inhibitor of actin-stimulated myosin adenosine triphosphatase

During attempts to isolate bovine sperm actin, persistent low molecular weight proteinaceous (LMWP) contaminants were found. A LMWP fraction was prepared by gel filtration chromatography on Sephadex G150. The LMWP was found in extracts of washed bovine ejaculated spermatozoa and in clarified bovine seminal plasma. It was substantially reduced in amount in bovine epididymal spermatozoa, indicating that it originated from secondary sex gland secretions. The LMWP inhibited rabbit muscle actin-stimulated myosin adenosine triphosphatase (actin-myosin ATPase) activity. The LMWP:actin ratio for 50% inhibition of actin-myosin ATPase was 2.6 +/- 0.12 mg LMWP per mg actin. The LMWP interfered with actin inhibition of deoxyribonuclease, indicating that LMWP interacted with actin. The LMWP from seminal plasma had an estimated molecular weight of 8300 and consisted of several acidic components. It had negligible protease activity and its inhibition of actin-myosin ATPase was independent of divalent cations. The LMWP appears to readily aggregate with itself and other proteins, which may be related to its physiological role in semen.     

Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing

The suitability of ejaculated and epididymal stallion spermatozoa for cooled storage (5 degrees C) and cryopreservation was examined in 5 ejaculates from each of 6 stallions and in spermatozoa recovered from the cauda epididymidis after castration of these stallions. The percentage of progressively motile spermatozoa, examined by subjective estimation (cooled samples) or by computerized analysis (frozen-thawed samples), was used as parameter. In ejaculated semen samples containing 5 and 25% seminal plasma in a skim milk glucose extender, the lower amount of seminal plasma supported spermatozoal motility significantly better throughout storage at 5 degrees C. Addition of 5 or 25% seminal plasma to perfused epididymal spermatozoa (0% seminal plasma) resulted in a significant stimulation of spermatozoal motility by 25% seminal plasma at 0 h (P<0.05) and to a lesser extent at 24 and 48 h. Post-thaw motility of ejaculated as well as epididymal spermatozoa was not influenced by slow cooling to 15 degrees or 5 degrees C with or without glycerol prior to rapid freezing in liquid nitrogen vapor. During cooled storage, seminal plasma had a stimulatory effect on epididymal spermatozoa and depressed motility in ejaculated spermatozoa. Results on cryopreservation indicate that freezability of equine spermatozoa is already determined when spermatozoa leave the tail of the epididymis.     

Differences between epididymal and ejaculated sperm characteristics in donkey

Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymal canal. In the epididymal caput and corpus spermatozoa undergo several biochemical and metabolic changes while the cauda of the epididymis should be considered as the primarily site for storage of the spermatozoa. In the horse spermatozoa from cauda epididymis were collected and frozen, and the fertility of semen assessed. However, no studies have detailed semen characteristics of spermatozoa collected from the cauda epididymis in the jackass. In this study sperm characteristics of spermatozoa in the cauda epididymis of the donkey was reported and a comparison with ejaculated spermatozoal characteristics was performed. Samples from 10 Martina Franca jackasses were collected and analyzed for viability (Propidium iodide/Sybr-14? fluorescent stain), mitochondrial activity (Mitotraker? fluorescent stain), objective motility characteristics (by Computer Assisted Sperm Analyzer - CASA) and morphology. A higher viability and mitochondrial activity in the cauda epididymis samples were reported in this paper. Samples reported in this paper were identified and the percentage of total and progressive spermatozoa was comparable, but trajectories were more rapid (higher VCL) with less progressiveness (higher ALH and lower STR and LIN) in the cauda epididymis. Sperm morphology showed a pronounced variability between jackasses, with comparable values for all morphological subclasses. In this study the loss of the distal cytoplasmic droplets happen close to or after ejaculation because the percentage fell to nearly 0% after ejaculation. As suggested for bulls, the presence of a similar percentage in sperm with proximal cytoplasmic droplet in epididymal and ejaculated semen is likely to indicate a failure in the maturation process.     

Influence of season on characteristics of epididymal and ejaculated semen in Meishan boars

Most of the epididymal spermatozoa collected in all the seasons examined maintained an ability to move progressively, had a cytoplasmic droplet in the distal site of the middle piece, and were morphologically normal. Reduced desire to mount a dummy was not observed during the experimental period. Characteristics of ejaculated semen were not significantly altered throughout the year. However, progressive motility and acrosomal integrity of spermatozoa ejaculated between July and September were more susceptible to storage at 4 degrees C than spermatozoa ejaculated during the other months and acrosomal integrity of spermatozoa ejaculated during the 3 months was to freezing-thawing. These results indicate that the reproductive activity of Meishan boars in Japan is only slightly influenced by season, but semen ejaculated during the summer is less suitable for storage than that ejaculated during the other seasons of the year.     

Seminal plasma non-heparin binding proteins (NHBP) reduce the cryoinjury to buffalo cauda epididymal spermatozoa induced by heparin binding proteins (HBP)

Previous cryopreservation studies with buffalo cauda epididymal spermatozoa have reported a deleterious effect of seminal plasma heparin binding protein (HBP). The amount of HBP used in these studies was meager compared to the normal level of HBP in the buffalo ejaculate, still the damage induced upon the spermatozoa was substantial when compared to that incurred to the spermatozoa during routine freezing of ejaculated semen. Thus there might be some factor(s) in the seminal plasma, which reduce the deleterious effect of HBP on spermatozoa during cryopreservation of ejaculated semen. This study was conducted to investigate for the presence of any such factor in buffalo seminal plasma. Seminal plasma proteins were separated on their heparin binding properties as heparin binding (HBP) and non-heparin binding (NHBP). The separated proteins were added to the extender of buffalo cauda epididymal semen for cryopreservation either alone or in combination. The spermatozoa were assessed for progressive motility, viability, acrosomal integrity and response to hypo-osmotic solution test (HOST) at prefreeze and post-thaw stages of cryopreservation. NHBP was found to provide some degree of protection to buffalo spermatozoa against cryopreservation stress as well as the deleterious effect of HBP during cryopreservation.     

Effects of exposure of epididymal boar spermatozoa to seminal plasma on the binding of zona pellucida proteins during in vitro capacitation

The purpose of the investigation was to determine whether seminal plasma plays a role in the increase during in vitro capacitation of the number of boar spermatozoa with enhanced binding of zona pellucida proteins. Ejaculated spermatozoa and spermatozoa collected from the caudae epididymides of boars were incubated at 39 degrees C in a Tyrode's IVF medium. During incubation, the zona binding ability of individual spermatozoa was assessed with fluorescein-conjugated solubilized zona pellucida proteins (FITC-sZP), using a flow cytometer. Propidium iodide (PI) was included to simultaneously monitor cell viability. During incubation of ejaculated spermatozoa, a percentage of the spermatozoa expressed enhanced binding of FITC-sZP. The percentage of viable spermatozoa with enhanced binding reached a maximum of 37% (S.D.=8, averaged over five boars) after 2-3 h. In epididymal sperm, a similar maximum was observed after incubation in vitro, but a longer time of incubation was needed (6 h). Also, the rate of cell death of epididymal sperm was much lower than that of ejaculated sperm. When epididymal spermatozoa was exposed to seminal plasma in vitro, the time needed to reach a maximal percentage of viable spermatozoa with enhanced FITC-sZP binding was similar to that in ejaculated semen. However, the rate of cell death was still much lower than in ejaculated sperm. We concluded that the binding sites on the sperm surface that are involved in the increased binding of zona proteins during incubation under IVF conditions were not derived from the seminal plasma. The cellular processes leading to the increased binding capacity were accelerated by exposure of the sperm to seminal plasma.     

Carnitine, acetylcarnitine and the activity of carnitine acyltransferases in seminal plasma and spermatozoa of men, rams and rats.

The concentration of total carnitine (i.e. carnitine plus acetylcarnitine) was measured in seminal plasma and spermatozoa of men and rams. In ram semen, there was a close correlation between the concentration of spermatozoa and that of total carnitine in the seminal plasma, indicating that the epididymal secretion was the sole source of seminal carnitine. The percentage of total carnitine present as acetylcarnitine was 40% in seminal plasma and 70-80% in spermatozoa. The acetylation state of carnitine in seminal plasma was apparently not influenced by the metabolic activity of spermatozoa in ejaculated ram semen as no change was found in the plasma concentration of carnitine or acetylcarnitine up to 45 min after ejaculation. In spermatozoa, the activity of carnitine acetyltransferase (EC 2.3.1.7) was approximately equivalent to that of carnitine palmitoyltransferase (EC 2.3.1.21); and the activity of these enzymes was similar in ram and human spermatozoa but greater in rat spermatozoa. It is concluded that there is no correlation between the content of either total carnitine or the carnitine acyltransferases and the respiratory capacity of spermatozoa.     

Prostaglandin F2α concentration in genital tract secretions of dairy bulls

Prostaglandin F_2α (PGF_2α) concentrations in genital tract secretions of conscious dairy bulls were determined by radioimmunoassay procedures and compared with peripheral blood plasma levels. The mean (± SD) PGF_2α concentration of coccygeal venous blood plasma from four bulls was 0.14 ± 0.05 ng/ml. Values for rete testis fluid and seminal plasma were the same, namely 0.17 ± 0.01 ng/ml (n = 5) and 0.17 ± 0.02 ng/ml (n = 4), respectively. However, the PGF_2α level in cauda epididymal plasma was 1.61 ± 0.41 ng/ml, or over 8 to 10 times (P < 0.01) the concentration of any other fluid studied.Added PGF_2α had no effect on the endogenous oxygen consumption of washed cauda epididymal spermatozoa or on the oxidative and glycolytic activities of washed ejaculated spermatozoa in vitro. No evidence was obtained suggesting that the prostaglandin may interact with the stimulatory effect of added testosterone or phosphatidylinositol (PI) on the motility, respiration or glucose uptake of ejaculated spermatozoa.     

In vitro fertilization of porcine oocytes with fresh and frozen-thawed ejaculated or frozen-thawed epididymal semen obtained from identical boars

The objective of this study was to compare the in vitro fertilizing capacity of porcine spermatozoa from fresh and frozen-thawed semen and frozen-thawed epididymal spermatozoa obtained from identical boars. Prior to IVF, fresh spermatozoa were capacitated in TCM 199. Frozen semen samples were stored in 0.25-ml plastic straws using a lactose/glycerol/orvus-es-paste extender. Cumulus-oocyte-complexes (COC) obtained from superovulated prepuberal gilts were fertilized in vitro within 2 h after aspiration with one of the semen samples. After final dilution for IVF, frozen-thawed epididymal semen samples showed motility rates (72.2 +/- 5.6%) similar to those of spermatozoa in fresh semen (76.4 +/- 4.5%), while sperm motility decreased in frozen-thawed ejaculated semen (40.2 +/- 9.4%). Considerable individual differences in sperm motility between boars were observed for ejaculated semen but not for epididymal semen. Enhanced fertilizing capacity of frozen-thawed epididymal spermatozoa was confirmed by pronucleus formation and cleavage rates, with significantly more embryos developing to the 2- and 4-cell stages compared with the groups fertilized with fresh or with frozen-thawed ejaculated semen (59.7 vs 14.6 and 16%). In conclusion, consistent in vitro fertilization rates with minimal semen variability are obtained using frozen-thawed epididymal semen. Following a modified freezing protocol, epididymal spermatozoa can easily be frozen in small containers for IVF, with higher resultant motility and fertilization rates than with ejaculated semen.     

Sperm treatment affects capacitation parameters and penetration ability of ejaculated and epididymal boar spermatozoa

This work was designed to study how this ability is affected by different sperm treatments routinely used for in vitro fertilization (IVF) assay. In this study, boar sperm samples from epididymal or ejaculated origin were processed by three different methods: left unwashed (NW group), washed in Dulbecco's phosphate-buffered saline supplemented with 0.1% BSA (BSA group), and washed on a Percoll^® gradient (PERCOLL group). After preparation of semen samples, changes in motility patterns were studied by CASA, calcium uptake by spectrofluorimetry, and ROS generation, spontaneous acrosome reaction, and lipid disorder by means of flow cytometry. Finally IVF assays were also performed with the different semen samples and penetrability results evaluated at 2 and 4 h post insemination (hpi). Independently of the sperm treatment, epididymal spermatozoa showed higher values of progressive motility, percentage of live cells with low lipid disorder, and penetration ability at 4 hpi than the corresponding ejaculated spermatozoa. Ejaculated spermatozoa showed higher levels of calcium uptake, ROS generation and percentage of spontaneous acrosome reaction than epididymal sperm. Regarding sperm treatments, PERCOLL group showed the highest values for some motility parameters (linearity of the curvilinear trajectory, straightness, and average path velocity/curvilinear velocity), ROS generation and penetration ability at 2 and 4 hpi; however this same group showed the lowest values for sperm curvilinear velocity and lateral head displacement. From all experimental groups, ejaculated-PERCOLL-treated spermatozoa showed the highest fertilization ability after 2 hpi. Results suggest that capacitation pathways can be regulated by suitable treatments making the ejaculated sperm able to reach capacitation and fertilize oocytes in similar levels than epididymal spermatozoa, although most of the studied capacitation-associated changes do not correlate with this ability.     

Arylsulfatases are present in seminal plasma of several domestic mammals.

Mammalian spermatozoa and seminal plasma both contain high levels of arylsulfatases (AS), enzymes that remove sulfate from sulfated glycoconjugates. In ejaculated semen of boars, 85% of AS was found in seminal plasma whereas only 13% was found in spermatozoa. A comparable distribution of AS between spermatozoa and seminal plasma was observed in other domestic mammals. The presence of AS in seminal plasma was not due to leakage from spermatozoa because sperm cells had intact acrosomes and plasma membranes after their separation from seminal plasma, and because 84% of the acrosomal marker enzyme hyaluronidase was retained in washed spermatozoa. Spermatozoa in boar semen diluted with Beltsville Thawing Solution (BTS) deteriorated faster during storage at 17 degrees C than spermatozoa stored in BTS without seminal plasma. This suggests that seminal plasma has a deleterious effect on mammalian spermatozoa. We propose that (1) sulfated glycoconjugates stabilize sperm plasma membranes; (2) AS present in seminal plasma contribute to the deterioration of spermatozoa by desulfating these glycoconjugates; and (3) AS present in seminal plasma could well play a role in sperm capacitation.