Preferential attachment of cock spermatozoa to the perivitelline layer directly over the germinal disc of the hen's ovum.

In vitro incubation of cock spermatozoa with perivitelline layer (PL) from recently ovulated ova of the hen resulted in binding of spermatozoa to the PL and activation of the acrosome reaction. A simple quantitative technique was developed for assessing these events. Following incubation of the PL (0.5 cm2 sections) with spermatozoa, the PL section was rinsed and stained with Schiff's reagent. Microscopic examination revealed holes in the PL that were assumed to be sites of spermatozoa penetration. Utilizing this technique, a correlation was demonstrated between sperm concentration and the number of spermatozoa attaching to the PL and undergoing an acrosome reaction. Pre-treatment of spermatozoa with solubilized PL inhibited spermatozoa binding to pieces of intact PL. The PL overlying the germinal disc and a similarly sized section of PL from another area of the ovum were removed and incubated separately with spermatozoa (1 x 10(5) sperm/100 microliters). Spermatozoa showed preferential attachment and digestion of the PL from the germinal disc area (809 sperm/mm2) as compared to PL from other areas of the ovum (608 sperm/mm2). Spermatozoa attached to the PL in a circular, doughnut-shaped fashion in the area directly over the germinal disc.     

Binding of chemotactic peptide to the outer surface and to whole human spermatozoa with different affinity states

Binding of N-formyl-methionyl-L-leucyl-[³H]phenylalanine (fML[³H]Ph) to human ejaculated spermatozoa and to its isolated plasma membrane was studied. Our data confirm the presence of specific receptors for f-MLPh in the human spermatozoa and suggest that whole spermatozoa receptors exist in two affinity states, one high-affinity, low-capacity specific receptor (K_d = 12.3 ± 0.5 nM, n = 22,285 ± 65,008 binding sites per sperm cell) and a second one (K_d = 700 ± 47 nM) that is not saturable, indicating a low-affinity, high-capacity nonspecific site. In contrast, sperm membrane showed only one class of binding site (K_d = 6.4 ± 0.12 nM), which was statistically different from that of the high-affinity binding site of intact spermatozoa. To explain this difference we discuss the possibility that first, the two binding affinities represent two interconvertible states of a single receptor population, which, depending on the metabolic activity of spermatozoa, may change its physicochemical properties; or second, they reflect two different processes, binding and/or transport into the spermatozoa.     

Oviduct binding ability of porcine spermatozoa develops in the epididymis and can be advanced by incubation with caudal fluid

The sperm reservoir is formed when spermatozoa bind to the epithelium of the uterotubal junction and caudal isthmus of the oviduct. It is an important mechanism that helps synchronize the meeting of gametes by regulating untimely capacitation and polyspermic fertilization. This study investigated the influence of epididymal maturation and caudal fluid on the ability of spermatozoa to bind to oviduct epithelium using a model porcine oviduct explant assay. Spermatozoa from the rete testis, middle caput (E2-E3), middle corpus (E6), and cauda (E8) of Large White or Large White × Landrace boars aged 10 to 14 months were diluted in modified Androhep solution and incubated with porcine oviduct explants. Results reported in this study support our hypothesis that testicular spermatozoa need to pass through the regions of the epididymis to acquire the ability to bind to the oviduct. There was a sequential increase in the number of spermatozoa that bound to oviduct explants from the rete testis to caudal epididymis. Binding of caudal spermatozoa to isthmic explants was the highest (15.0 ± 1.2 spermatozoa per 1.25 mm², mean ± standard error of the mean; P ≤ 0.05) and lowest by spermatozoa from the rete testis (2.0 ± 0.3 per 1.25 mm²), and higher to isthmus from sows compared to gilts (35.8 ± 6.7 per 1.25 mm² vs. 14.8 ± 3.0 per 1.25 mm²; P ≤ 0.05). Binding of ejaculated spermatozoa to porcine isthmus was higher than that for caudal spermatozoa (26.3 ± 1.4 per 1.25 mm² vs. 15.0 ± 0.8 per 1.25 mm²; P ≤ 0.05) and higher to porcine than to bovine isthmus (26.3 ± 2.3 per 1.25 mm² vs. 18.8 ± 1.9 per 1.25 mm²; P ≤ 0.05). Incubation of spermatozoa from the caput and corpus in caudal fluid increased the ability of spermatozoa to bind to the oviduct epithelium (P ≤ 0.05). In conclusion, the capacity of testicular spermatozoa to bind to the oviduct epithelium increases during their maturation in the epididymis and can be advanced by components of the caudal fluid.     

Effects of tyrosine kinase inhibitor on the motility and ATP concentrations of fowl spermatozoa

The possible role of tyrosine kinase in the regulation of fowl sperm motility was investigated by using a stable analogue of erbstatin, methyl 2,5-dihydroxycinnamate (2,5-MeC), a specific inhibitor of tyrosine kinase. This inhibited the motility of intact spermatozoa at 30°C in a dose-dependent manner. In contrast, the motility of demembranated spermatozoa was not inhibited by the same concentrations of 2,5-MeC. At 40°C, both intact and demembranated spermatozoa were almost immotile with or without 2,5-MeC. Additionally, intact spermatozoa, stimulated by the addition of Ca²⁺ or calyculin A, a specific inhibitor of protein phosphatases, lost their motility with the subsequent addition of 2,5-MeC at 40°C. However, unlike the motility, the ATP concentrations of spermatozoa were maintained in about 30–35 nmol ATP/10⁹ cells during these incubation periods. The activity of tyrosine kinase of spermatozoa at 30°C, estimated by measuring the phosphorylation of a synthetic peptide substrate, RR-SRC, was 0.17 pmol/min per milligram of protein. This activity was lower than that of fowl testes or chick brain but higher than that of chick liver. These results suggest that tyrosine kinase activity, which is not retained in the axoneme and/or accessory cytoskeletal components, may be involved in the maintenance of flagellar movement of fowl spermatozoa at 30°C. Mol. Reprod. Dev. 49:196–202, 1998. © 1998 Wiley-Liss, Inc.     

Expanded cumuli induce acrosome reaction in boar sperm

The authors investigated acrosomal changes occurring in boar sperm that interact with the expanded cumulus matrix surrounding ovulated pig oocytes. Samples of washed boar sperm obtained from six donors were incubated for 4 hr under capacitating conditions and exposed either to solubilized zonae pellucidae (ZP) or solubilized expanded pig cumuli (SEC) obtained from IVM oocytes. Alternatively, hyaluronic acid, laminin, or fibronectin, components of the extracellular matrix (ECM) were added to capacitated sperm. Acrosomal integrity was evaluated 1hr later by using FITC-PSA staining. Solubilized cumuli induced acrosome reaction (AR) in a dose-dependent manner with a saturating effect exerted at 2.5 SEC/50 μl. Both 500 nM fibronectin and 500 nM laminin stimulated acrosomal exocytosis, the latter being more effective and inducing saturating levels of AR. By contrast, hyaluronic acid did not affect acrosomal status. Preincubation with anti-laminin antibodies completely prevented the inducing activity of SEC without affecting the activity of solubilized ZP. Consistent with these data, the integrin VLA-6, a receptor with high affinity for laminin, was detected by immunoblotting on the plasma membrane of capacitated boar spermatozoa. In addition, its immunoneutralization, obtained with the preincubation of capacitated sperm with the antibody raised against the α chain of VLA-6 integrin, prevented AR upon exposure to laminin or SEC (10.7 ± 3.2 and 10.2 ± 1.0% respectively), while the samples retained their responsiveness to ZP (29.6 ± 1.2%). The results demonstrate that the interaction between laminin, entrapped in the expanded cumuli, and specific integrins present on the sperm membrane can initiate AR, thus taking part in the process of sperm-egg recognition. Mol. Reprod. Dev. 51:445–453, 1998. © 1998 Wiley-Liss, Inc.     

Phospholipid composition of isolated guinea pig sperm outer acrosomal membrane and plasma membrane during capacitation in vitro

After capacitation of guinea pig spermatozoa in vitro, the plasma membrane was mechanically separated from the spermatozoa in the presence or absence of HgCl₂ and subsequently isolated by density gradient centrifugation. Examination of the spermatozoa by electron microscopy after homogenization in the presence of HgCl₂ revealed that plasma membrane was removed only from the acrosomal region and remained predominately intact posterior to the equatorial segment of the sperm head, as well as the midpiece and tail. In comparison, spermatozoa homogenized under similar buffer conditions but in the absence of HgCl₂ lose the large apical segment of the acrosome and the plasma membrane is removed essentially from the entire cell. If spermatozoa were homogenized in the absence of Hg²⁺, analysis of plasma membrane phospholipid composition revealed a complete loss of lysophosphatidylcholine (LPC) from the plasma membrane after incubation of spermatozoa in minimal capacitating medium (MCM-PL) for 2 hours. Under these culture conditions the addition of Ca²⁺ (5 mM) to the capacitated spermatozoa induced approximately 78 ± 5% (n = 3) of the motile spermatozoa to undergo acrosome reactions while still maintaining sperm motility (80 ± 5%) (n = 3). If the spermatozoa were homogenized in the presence of Hg²⁺, a time course study revealed that plasma membrane LPC loss occurred between 60 and 90 minutes of incubation. This complete loss of LPC was evident when approximately half of the capacitated spermatozoa had undergone acrosome reactions. Incubation of the spermatozoa with the metabolic and acrosome reaction inhibitor, 2-deoxyglucose (10 mM) for 2 hours, maintained the plasma membrane phospholipid composition similar to that in the noncapacitated state. These data provide evidence that changes in the plasma membrane phospholipid composition may be associated with guinea pig sperm capacitation.     

Effects of electrical stimulation before or after in vitro fertilization on sperm penetration and pronuclear formation of pig oocytes

The effects of exposure of pig oocytes to an electrical pulse on sperm penetration and pronuclear formation were determined before or after in vitro fertilization (IVF). After in vitro maturation (IVM) or after collection from oviducts of unmated gilts, pig oocytes either were not exposed or were exposed to an electrical pulse (a 10 sec pulse at 4.0 V mm^?1 AC followed by a 30 μsec pulse at 120 V mm^?1 DC), followed 30 min later by IVF. The incidence of male pronuclear formation of both IVM and in vivo-matured oocytes at 12 hr after insemination was decreased from 59% and 100%, respectively, to 2% and 36%, respectively, by the electrical pulse, but the penetration rates (88–100%) and polyspermic rates (79–100%) were not affected by exposure to an electrical pulse. Similarly, when pig IVM oocytes were exposed to an electrical pulse at 6 hr after insemination, electrical activation did not decrease penetration rates (93% vs. 90%), polyspermic rates (83% vs. 91%), or number of spermatozoa in penetrated oocytes (4.0 ± 0.5 vs. 4.6 ± 0.5) but did decrease the rate of male pronuclear formation from 58% to 18%. When oocytes were examined at 6 hr after insemination, 75% of them had been penetrated and resumed meiotic progression, but all sperm heads in penetrated oocytes were fully condensed or only partially decondensed. The percentage of penetrated eggs with multiple female pronuclei was increased when oocytes were exposed to an electrical pulse in all experimental series. In summary, electrical activation of pig oocytes before or just after IVF does not prevent sperm penetration but does inhibit male pronuclear formation and increases the formation of multiple female pronuclei. © 1993 Wiley-Liss, Inc.     

Evidence for nonmuscle myosin in bovine ejaculated spermatozoa

Mammalian spermatozoa contain nonmuscle actin and many of the components of regulatory systems thought to be involved in nonmuscle actin-myosin function. An actin-stimulated adenosine triphosphate hydrolase (ATPase) activity has been fractionated from bovine ejaculated spermatozoa by immobilized-actin affinity chromatography. The actinstimulated ATPase activity has a specific activity (0.04 ± 0.02 mM phosphate released/min/mg protein) similar to nonmuscle myosins from other mammalian cells and tissues, but it does not have appreciable K⁺-EDTA ATPase activity. The sperm actin-myosin may function in sperm morphogenesis in the seminiferous tubule, in capacitated spermatozoa undergoing an acrosome reaction, or in decondensation of the sperm nucleus after fertilization.     

Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.     

Cyclic-AMP receptors in the human spermatozoa membrane.

The binding properties of cyclic-AMP to human spermatozoa were studied. Incubation of whole human spermatozoa and of head and tail fractions with ¹⁴C-cyclic AMP induced the binding of 7.8^± 0.86 pmoles of the nucleotide per 5 × 10⁷ sperm cells showing an intrinsic association constant of 15 × 10⁻⁸ M. No significant inhibition of cyclic-AMP binding was caused by the addition of one hundred fold excess of AMP. However, when the excess AMP reached a thousand fold a slight but significant reduction (10%) was observed. These values were not modified by using sperm homogenates instead of intact sperm cells, which suggested that cyclic-AMP binding is to surface membranes and not to protein released from broken or damaged sperm. Binding was found to be unrelated with pH, between 6 to 8, but depended on the temperature of the incubation medium, showing a maximum at 20°C. The blocking of membrane sulfhydryl groups significantly inhibited (48%) cyclic-AMP binding to sperm cells.     

Urinary bladder, ionic composition of seminal fluid and urine with characterization of sperm motility in tench (Tinca tinca L.)

A small urinary bladder attached to the seminal duct in caudal part of the abdominal cavity was registered for the first time in dissected males of tench. The urinary bladder wall was of whitish color and the bladder contained 0.5–2 ml of urine. When collected in the experiment, the tench sperm was white‐colored. Spermatozoa density is highly variable due to contamination by urine, and the latter additionally activates spontaneous motility of the spermatozoa. Seminal fluid contains ions such as Na⁺ (18.4 ± 1.3 mm ), K⁺ (1.9 ± 0.6 mm ), Ca²⁺ (0.6 ± 0.2 mm ) and Mg²⁺ (0.5 ± 0.1 mm ), leading to osmolality of 230 ± 82 mOsmol kg^?1 depending on the dilution by urine. Urea was detected in urine samples uncontaminated by sperm with an osmolality of 85 ± 58 mOsmol kg^?1. Urine also contained high concentrations of ions such as Na⁺ (30.9 ± 8.9 mm ), K⁺ (4.3 ± 2.9 mm ), Ca²⁺ (0.9 ± 0.5 mm ) and Mg²⁺ (0.6 ± 0.2 mm ). The spontaneous sperm activation by urine was up to 100%, but could be prevented by collection in an immobilizing solution. Motility was observed for 90–100% spermatozoa just after their transfer to distilled water or in a swimming medium (SM, 30–45 mm KCl) with a velocity of 120–140 μm s^?1. A flagellar beat frequency of 60–70 Hz and forward motility lasted up to 80 s in distilled water, and up to 180 s in SM at room temperature.     

Magnetic-activated Cell Sorting before Cryopreservation Preserves Mitochondrial Integrity in Human Spermatozoa

Superparamagnetic annexin-V conjugated microbeads are able to eliminate spermatozoa with externalized phosphatidylserine, a membrane feature of apoptotic cells as well as spermatozoa with deteriorated plasma membrane. Our objective was to evaluate the effects of annexin-V Magnetic-Activated Cell Sorting (MACS) in cryopreservation–thawing protocols and on integrity of sperm mitochondrial transmembrane potential and mitochondrial integrity survival rate (MSR). Mature spermatozoa of 10 healthy donors were prepared by density gradient centrifugation and divided into 2 aliquots afterwards. The first one was subjected to annexin-V MACS followed by cryopreservation and thawing, while the second was cryopreserved–thawed without MACS to serve as control. Annexin-negative sperm separated by MACS showed significantly higher levels of intact mitochondria following cryopreservation–thawing (45.4±8.6%) compared to sperm that were not separated (15.8±4.6%, p<0.01). Separating a distinctive population of non-apoptotic spermatozoa with intact membranes may optimize cryopreservation–thawing outcome. MACS using annexin-V microbeads enhances the percentage of spermatozoa with intact transmembrane mitochondrial potential and mitochondrial integrity survival rates following cryopreservation.     

Isolation and Partial Characterization of a Glycoprotein Complex with Sperm-Receptor Activity from Ciona intestinalis Ovary1

Sperm-egg interaction in the ascidian Ciana intestinalis is mediated by a fucosyl-glycoprotein (FP) component of the egg vitelline coat. FP are responsible for sperm binding, sperm activation and the acrosome reaction. In this paper we report a detailed biochemical and functional characterization of FP purified from the ovaries by affinity chromatography. thic component with sperm receptor activity is a high molecular weight glycoprotein complex (>10⁷) with a protein-carbohydrate ratio of 2:1, which inhibits the binding of the spermatozoa to the vitelline coat and induces sperm activation and the acrosome reaction. Exhaustive proteolytic digestion of FP yields high molecular weight glycopeptides (> 4×10⁵), which contain N-acetylgalactosamine, fucose, galactose and rhamnose. These glycopeptides retain some receptor activity, thus raising the question of the involvement of the polypeptide backbone in the sperm-egg binding process. However, the glycopeptide fraction fails to induce the acrosome reaction: we suggest that the polypeptide fraction plays a role in the induction of sperm activation and the acrosome reaction.     

Cholesterol efflux from bovine sperm: II. Effect of reducing sperm cholesterol on penetration of zona-free hamster and in vitro matured bovine ova

Several reports have indicated that sperm capacitation includes loss of membrane cholesterol (Chol) with a concomitant decrease in the Chol-to-phospholipid (PL) ratio. Methods were developed for quantifiable removal of bovine sperm Chol, which predisposed sperm to induction of the acrosome reaction upon addition of lysophosphatidylcholine (LPC). The objective of this study was to evaluate the effect of Chol removal from bovine sperm on penetration of zona-free hamster and intact bovine ova in vitro. Washed ejaculated bovine sperm were incubated (2 h, 39°C) in a modified Tyrode's solution (TALP) containing (1) Chol-free liposomes (—Chol, 50 × 10⁶ sperm and 600 nmol phospholipid/ml); (2) liposomes containing 30 mol% Chol (+ Chol, 2 × 10⁸ sperm and 300 nmol total lipid/ml); or (3) no liposomes (Control). We have previously shown that net Chol efflux from sperm is 31% of the total sperm Chol with —Chol liposomes and less than 1% with control media. Sperm were then washed twice and challenged with LPC bound to bovine serum albumin (BSA) using celite as a carrier. Treated sperm (25 × 10⁶) were incubated immediately with either zona-free hamster ova (HO) or in vitro matured bovine ova (BO) in 50-μl droplets of TALP under medical fluid in an atmosphere of 5% CO₂ in air (3 h, 39°C). Ova were fixed in ethanol:acetic acid, stained with 1% orcein, and examined. Percent penetration (%P) of HO (X ± SEM) for 30 and 40 μg of LPC/mg BSA was 59.4 ± 5.3 and 82.9 ± 5.4; 38.5 ± 5.6 and 52.3 ± 4.7; and 16.0 ± 4.6; and 23.2 ± 5.6 for —Chol, Control, and + Chol treatments, respectively (n = 3). %P of BO (X ± SEM) for 30, 35, and 40 μg of LPC/mg BSA was 43.3 ± 5.4, 70.7 ± 7.5, and 81.5 ± 5.1 for —Chol and 16.4 ± 6.9, 36.2 ± 6.9, and 44.2 ± 8.6 for Control treatments, respectively (n = 3). In a second set of experiments %P of BO (X ± SEM) was 63.6 ± 6.8, 31.8 ± 4.9, and 10.5 ± 3.4 for —Chol, Control, and +Chol treatments, respectively, when 40 μg LPC/mg BSA was added (n = 2). %P and the number of sperm per fertilized ovum were consistently higher for the —Chol treatment for both HO and BO (P < .01). These results demonstrate that Chol removal from bovine sperm facilitates penetration of ova in vitro suggesting a potential role in bovine sperm capacitation.     

In vitro fertilizing characteristics of bovine spermatozoa with multiple nuclear vacuoles: A case study

A study was designed to determine the in vitro fertilizing characteristics of bovine semen with a high percentage of spermatozoa with multiple nuclear vacuoles. In Experiment 1, a total of 620 oocytes was divided into 2 groups and inseminated with spermatozoa from 1 of 2 different bulls at a concentration of 2 × 10⁵/ml. After Percoll washes, 73.5 ± 3.0% of spermatozoa from Bull A contained multiple nuclear vacuoles, while no sperm cells from Bull B contained vacuoles. After 19.5 ± 0.5 h of co-incubation of oocytes with spermatozoa, loosely attached sperm cells were removed by washing, and the oocytes were fixed between 2 poly-l-lysine coated glass slides. Mean (±SD) percentage of fertilization was significantly lower (P < 0.05) in Bull A (19.7 ± 7.0%) than in Bull B (67.6 ± 4.5%). In one-third of the oocytes fertilized by spermatozoa from Bull A, sperm head decondensation was incomplete and normal male pronucleus formation did not occur. All oocytes fertilized by Bull B had normally decondensed sperm heads. Although fewer (P < 0.05) spermatozoa from Bull A were bound to the zona pellucida than from Bull B, the percentage of vacuolated sperm cells bound to the zona pellucida (73.3 ± 7.8%) did not differ from that in the inseminate. The mean number of sperm cells binding to fertilized oocytes was higher than to unfertilized oocytes for both bulls (P < 0.05). In Experiment 2, 748 salt-stored oocytes (zonae) were inseminated with semen from the same 2 bulls to determine the ability of spermatozoa to penetrate the zona pellucida. The percentage of zonae penetrated by spermatozoa from Bull A (69.9 ± 3.5%; a mean of 2.4 ± 2.3 spermatozoa) was lower (P < 0.05) than from Bull B (96.5 ± 14.7%; a mean of 11.3 ± 9.9). Although the proportion of vacuolated sperm cells from Bull A that bound to the zona pellucida did not differ from that in the inseminate, the proportion of those penetrating the zona pellucida (52.7%) was lower (P < 0.05). In summary, vacuolated sperm cells apparently gained access to the oocyte and bound to the zona pellucida, but they penetrated the zona pellucida at a lower rate and apparently did not form normal male pronuclei.     

Cholesterol efflux from bovine sperm. I. Induction of the acrosome reaction with lysophosphatidylcholine after reducing sperm cholesterol

Methods were developed to quantitatively reduce the cholesterol (Chol)/phospholipid (PL) ratio of bovine sperm and to determine the effectiveness of this treatment in capacitating sperm. Washed sperm (2 × 10⁸) were incubated in 1.0 ml of modified Tyrode's solution (TS) containing unilamellar liposomes of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and [¹⁴C]-Chol (35:35:30 molar ratio, 300 nmol total PL). [³H]-triolein was included as a nonexchangable marker. After 90 min at 39°C, a 13% net exchange of [¹⁴C]-Chol from liposomes to sperm was observed (n = 4), and sperm motility was 80%. Sperm were then washed and 50 × 10⁶ sperm were incubated as before with PC/PE liposomes containing no Chol. After 90 min, sperm were separated from liposomes by centrifugation. Measurement of [¹⁴C]-Chol in the liposomes (supernatant) and parallel gas chromatographic analysis of extracted, saponified liposomes (n = 4) indicated that 30% of sperm Chol was removed by this procedure. Chol efflux decreased percent motile sperm by less than 10% but reduced sperm velocity by more than 50%. Sperm incubated with no liposomes (control), with liposomes containing Chol ( + Chol), and with Chol-free liposomes (—Chol) were washed and resuspended in TS with 0.2% BSA and 30 μg lysophosphatidylcholine (LPC)/mg bovine serum albumin (BSA). Percent sperm undergoing the acrosome reaction (AR) upon incubation with LPC-BSA was used as a measure of sperm capacitation. After 60 min of exposure to LPC-BSA at 39°C, the mean (± SE) percent motile sperm for control, +Chol, and —Chol treatments was 57.0 ± 4.9, 60.0 ± 4.7, and 57.0 ± 6.8, respectively. Corresponding values for percent AR were 14.0 ± 3.4, 20.3 ± 4.4, and 39.7 ± 1.2. These results suggest that loss of Chol from bovine sperm may be an early step in sperm capacitation in this species.     

Defining oligosaccharide specificity for initial sperm-zona pellucida adhesion in the mouse

The identity of the sperm surface protein(s) responsible for sperm-zona pellucida binding in the mouse, as well as the characteristics of the oligosaccharide groups on zona pellucida glycoprotein 3 (ZP3) having ligand activity toward this receptor, remain controversial. Conflicting results from several groups have made interpretation of the current data difficult. By developing a quantitative binding assay to evaluate the molecular interactions between mammalian sperm and the zona pellucida during initial gamete interactions, we directly quantified sperm-ZP binding interactions at the molecular level for the first time. The ZP binding assay demonstrated that live, capacitated mouse sperm bind solubilized ¹²⁵I-labeled ZP glycoproteins in a concentration-dependent manner characterized by a rapid forward rate constant of 3.0 × 10⁷ M⁻¹ min⁻¹. Following the initial characterization, the binding assay was used to examine the roles of the sperm surface enzymes galactosyltransferase (GalTase) and fucosyltransferase (FucTase) in sperm-zone pellucida binding in the mouse. These data indicate that substrates for FucTase, but not for GalTase, inhibit sperm-ZP binding, in contrast to earlier reports in which GalTase substrates significantly inhibited sperm binding to intact ZPs. A model is presented which resolves conflicting results between assays using intact ZPs and the results obtained here using soluble ¹²⁵I-ZPs. Assuming a complex binding/recognition site, monosaccharides that could occupy part of the binding site would have a dramatic effect on sperm-ZP binding to the intact ZP, since they need only occupy the binding sites for a short time (∼ 100 msec) to disrupt binding. The current results suggest that the sperm ZP3 receptor binding site minimally recognizes the galβ1,3GlcNAc moiety also recognized by FucTases. The current data do not exclude the possibility that additional sugar residues form part of the ligand oligosaccharide group and are recognized by a yet-to-be-identified sperm surface protein which serves as the ZP3 receptor. © 1996 Wiley-Liss, Inc.     

Caffeine, dibutyryl cyclic-AMP and heparin affect the chemotactic and phagocytotic activities of neutrophils for boar sperm in vitro

The objective was to examine the effects of caffeine, dibutyryl cyclic AMP, and heparin on the chemotaxis and/or phagocytosis of PMNs for porcine sperm. The chemotactic activity of PMNs, determined in a blind well chamber, increased (P < 0.05) when fresh serum was added to the medium (control containing BSA, 1109.5 cells/mm² vs serum, 1226.3 cells/mm²), regardless of the presence of sperm (control, 1121.1 cells/mm² vs serum, 1245.8 cells/mm²), whereas heat-inactivated serum did not affect activity (without sperm, 1099.4 cells/mm² and with sperm, 1132.6 cells/mm²). Regardless of live and dead sperm and of the origin of PMNs (boars vs sows), the phagocytotic activity of PMNs, as determined by co-culture of PMNs with sperm for 60 min, increased (P < 0.05) in the presence of fresh serum containing active complement (46.7 and 43.0%, respectively), but stimulation was decreased (P < 0.05) when 1 mM or higher concentrations of caffeine was added to the medium (from 40.7 to 20.8-31.6%). The origin of PMNs (sows vs boars) did not significantly affect phagocytotic activity. The percentage of PMNs that phagocytized polystyrene latex beads decreased when 2 mM caffeine was added to the medium containing porcine serum (from 43.7 to 21.5%). Serum-stimulated chemotactic activity of PMNs (1089.9 cells/mm²) was also reduced (P < 0.05) with 2 mM caffeine (942.5 cells/mm²). Furthermore, dibutyryl cAMP at ≥ 0.1 mM or heparin at ≥ 100 μg/mL decreased phagocytotic activity, in a concentration-dependent manner (P < 0.05). Supplementation of PMNs with heparin at 100 or 500 μg/mL decreased (P < 0.05) chemotactic activity in the presence of serum (from 1137.1 cells/mm² to 1008.8-1026.3 cells/mm²). We inferred that opsonization in the presence of active complement stimulated phagocytotic and chemotactic activities of PMNs, whereas supplementation with caffeine and dibutyryl cAMP (which could be associated with the intracellular cAMP level of PMNs) or adding heparin decreased serum-stimulated phagocytotic and chemotactic activities.     

Interaction of isolated components from mammalian sperm and egg

In order to identify those proteins from the plasma membrane of hamster spermatozoa that exhibit an affinity for components of the zona pellucida we have used the Western blot technique. Zonae pellucidae from postovulatory hamster oocytes were solubilized by exposure to an acidic pH and then radiolabeled using the Bolton-Hunter reagent. These ¹²⁵I-zona pellucida proteins retain their immunoreactivity and migrate in three heterogeneous bands when submitted to SDS-PAGE electrophoresis. Membrane proteins from epididymal spermatozoa of mature hamsters were extracted by treatment with Nonidet P-40 and then submitted to electrophoresis (SDS-PAGE). The proteins in the gel were electrophoretically transferred to a nitrocellulose membrane and then probed with the radiolabelled zone pellucida proteins. ¹²⁵I-zona pellucida proteins bind preferentially to a sperm protein with a molecular weight of 26,400 ± 1,400 daltons (n = 9). Using a similar procedure it was shown that this protein also binds ¹²⁵I-Concanavalin A. The interaction between the sperm protein and the ¹²⁵I-zona pellucida proteins shows species specificity as demonstrated by the fact that the hamster ¹²⁵I-zona pellucida proteins do not bind to proteins extracted from ram, bull, and stallion spermatozoa. Whether this sperm protein could be implicated be implicated in the process of sperm-egg interaction is under investigation.     

Follicular fluid proteins stimulate nitric oxide (NO) synthesis in human sperm: A possible role for NO in acrosomal reaction

Nitric oxide (NO) is a free radical involved in the regulation of several functions of the male genitourinary system. It is produced by neurons and the endothelium and epithelia of reproductive system; it mediates penile erection and regulates sperm motility, viability, and metabolism. Here we show that human spermatozoa exhibit a detectable NO synthase (NOS) activity, measured both as ability of the intact sperm and cell lysate to convert L-[³H]arginine into L-[³H]citrulline and as 24 h accumulation of extracellular nitrite in intact sperm suspensions. NOS activity (identified as an endothelial isoform) was inhibited by L-canavanine and N^G-monomethyl-L-arginine, and nitrite accumulation was inhibited by the NO scavenger hemoglobin; both enzyme activity and nitrite production were increased by a 24 h incubation of spermatozoa with protein-enriched extracts of human follicular fluid (PFF); a significant increase of citrulline synthesis was observed only after a 4 h incubation with 40% PFF, a time period during which acrosomal reactivity was significantly increased. PFF-induced acrosomal reaction was inhibited by L-canavanine and hemoglobin, and the NO donors sodium nitroprusside (SNP), S-nitroso-N-acetyl-penicillamine (SNAP), and DETA NONOate were able to increase the percentage of reacted spermatozoa. Our results suggest that NO synthesized by human sperm may play a role in follicular fluid–induced acrosomal reaction. J Cell Physiol 178:85–92, 1999. © 1999 Wiley-Liss, Inc.