Showing papers on "Sperm motility published in 1994"

Mesoscale sperm handling devices

Abstract: Devices and methods are provided for the clinical analysis of a sperm sample. The devices include a solid substrate, typically on the order of a few millimeters thick and approximately 0.2 to 2.0 centimeters square, microfabricated to define a sample inlet port and a mesoscale flow channel extending from the inlet port. In one embodiment, a sperm sample is applied to the inlet port, and the competitive migration of the sperm sample through the mesoscale flow channel is detected to serve as an indicator of sperm motility. In another embodiment, the substrate of the device is microfabricated with a sperm inlet port, an egg nesting chamber, and an elongate mesoscale flow channel communicating between the egg nesting chamber and the inlet port. In this embodiment, a sperm sample is applied to the inlet port, and the sperm in the sample are permitted to competitively migrate from the inlet port through the channel to the egg nesting chamber, where in vitro fertilization occurs. The devices of the invention may be used in a wide range of applications in the analysis of a sperm sample, including the analysis of sperm morphology or motility, to assess sperm binding properties, and for in vitro fertilization.

Anandamide (arachidonylethanolamide), a brain cannabinoid receptor agonist, reduces sperm fertilizing capacity in sea urchins by inhibiting the acrosome reaction

Abstract: Anandamide (arachidonylethanolamide) is an endogenous cannabinoid receptor agonist in mammalian brain. Sea urchin sperm contain a high-affinity cannabinoid receptor similar to the cannabinoid receptor in mammalian brain. (-)-delta 9-Tetrahydrocannabinol (THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. We now report that anandamide produces effects similar to those previously obtained with THC in Strongylocentrotus purpuratus in reducing sperm fertilizing capacity and inhibiting the egg jelly-stimulated acrosome reaction. Arachidonic acid does not inhibit the acrosome reaction under similar conditions. The adverse effects of anandamide on sperm fertilizing capacity and the acrosome reaction are reversible. The receptivity of unfertilized eggs to sperm and sperm motility are not impaired by anandamide. Under conditions where anandamide completely blocks the egg jelly-stimulated acrosome reaction, it does not inhibit the acrosome reaction artificially initiated by ionomycin, which promotes Ca2+ influx, and nigericin, which activates K+ channels in sperm. These findings provide additional evidence that the cannabinoid receptor in sperm plays a role in blocking the acrosome reaction, indicate that anandamide or a related molecule may be the natural ligand for the cannabinoid receptor in sea urchin sperm, and suggest that binding of anandamide to the cannabinoid receptor modulates stimulus-secretion-coupling in sperm by affecting an event prior to ion channel opening.

Gonadal hormones and semen quality in male runners : a volume threshold effect of endurance training

Abstract: Eleven high mileage runners (HR) (108.0 +/- 4.5 km.wk-1), 9 moderate mileage runners (MR) (54.2 +/- 3.7 km.wk-1) and 10 sedentary controls (SC) of similar age (28.3 +/- 1.5 yr) were studied to evaluate the effects of volume of endurance training on reproductive function in male runners. Levels of reproductive, adrenal and thyroid hormones were measured during a 1-hr period of serial blood sampling (q20 min) and urinary excretion of 24-hr luteinizing hormone (uLH) was determined on two separate days. Semen exams and sperm penetration of standard cervical mucus (Penetrak) were performed 2-5 times. Levels of total testosterone (TT) and free testosterone (FT) were significantly lower in HR (15.3 +/- 1.3 nmol.l-1 and 60.2 +/- 5.1 pmol.l-1) compared to MR (21.4 +/- 1.6 nmol.l-1 and 86.0 +/- 6.1 pmol.l-1) and SC (19.5 +/- 0.9 nmol.l-1 and 75.9 +/- 3.6 pmol.l-1). No differences (p > 0.05) were found in uLH, serum LH, follicle-stimulating hormone (FSH), and prolactin (PRL) among the three groups. No other hormonal differences (p > 0.05) were observed among the groups. Total motile sperm count and density were lower (p < 0.05) in HR than SC. Decreased (p < 0.0006) sperm motility and an increased (p < 0.004) population of immature sperm and round cells were observed in HR compared to MR and SC. Sperm penetration of bovine cervical mucus was also decreased (p < 0.024) in HR compared to SC. Volume of training, defined by km.wk-1 run, was significantly correlated to sperm motility, density and number of round cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Sperm recovery techniques to maximize fertilizing capacity

Abstract: Because seminal plasma contains factors that inhibit the fertilizing ability of spermatozoa, it is essential that spermatozoa be separated from it quickly and efficiently. Although the success of a sperm preparation method is often assessed by the yield of motile spermatozoa, the choice of a method also depends on its technical complexity, the materials and apparatus required and time costs. Any exposure of spermatozoa during preparation to factors that may cause iatrogenic sperm dysfunction must obviously be avoided. Consequently, methods involving centrifugal washing prior to the selection of motile spermatozoa should be avoided. Direct swim-up from semen is the simplest way to obtain highly motile sperm populations and can be a very rapid procedure with normal semen samples. Two-layer discontinuous Percoll gradients give excellent yields when the lower layer contains 81% (v/v) Percoll. However, for severely asthenozoospermic cases the results can be disappointing and a Nycodenz gradient may be better, although the 'mini-Percoll' technique might be useful if special care is taken to protect the spermatozoa from damage induced by free radicals. In such cases the migration-sedimentation approach can also be successful. Abnormal samples, especially those with increased viscosity, may benefit from prior dilution with culture medium, or even chymotrypsin-induced liquefaction, before density gradient centrifugation. Finally, pharmacological stimulation of sperm motility may increase yields but, for in vitro fertilization (IVF), such spermatozoa must be used to inseminate oocytes as soon as possible after exposure to the stimulant, although after its removal.

Enhanced recruitment of motile spermatozoa by prostasome inclusion in swim-up medium

Abstract: Prostasomes, which are prostate-derived organelles, were purified from human seminal plasma for inclusion in Earle's balanced salt solution (EBSS) medium with or without human serum albumin. These media were used for swim-up experiments and the subsequent analyses of sperm motility parameters at different incubation times. The yield of motile spermatozoa after swim-up in EBSS medium enriched with boiled prostasomes was increased by 32% compared with EBSS containing albumin. Native prostasomes were less active. Combinations of albumin and either prostasomes or boiled prostasomes significantly increased the recovery of motile spermatozoa and also increased the percentage of spermatozoa displaying progressive motility after 1 h of incubation. Media lacking albumin showed lower values regarding progressive motility after 22 h of incubation. A beneficial effect of prostasomes was noted on lateral head displacement and percentage of hyperactive spermatozoa during the first 6 h of incubation. These results suggest that inclusion of prostasomes, especially boiled prostasomes, in swim-up media may improve the recovery of hyperactive motile spermatozoa for up to 6 h in cases of established male factor infertility, and consequently increase the opportunities for fertilization.

Progesterone and RU486: opposing effects on human sperm.

Abstract: Progesterone induced a rapid influx of calcium in capacitated human sperm, followed by a long-lasting, dose-dependent increase of intracellular free calcium. Thereafter, progesterone increased the fraction of hyperactivated sperm and the acrosome reaction. On the contrary, the progesterone antagonist RU486 (mifepristone) induced an immediate and transient, dose-dependent decrease of intracellular free calcium and a drop in the values of sperm movement parameters related to hyperactivation. Moreover, RU486 counteracted the effects of progesterone on calcium influx, lateral sperm head displacement, and the acrosome reaction. Therefore, RU486 effects were opposite to those of progesterone. The nature of the membrane receptor(s) involved is unknown. Several steroids bearing 11 beta-phenyl substitutions, with different pharmacological profiles, were also investigated. It was concluded that the steroid structure and chemical groups added to the 11 beta-phenyl influence effects on calcium influx.

Evidence for presence of hyaluronan binding protein on spermatozoa and its possible involvement in sperm function.

Abstract: Hyaluronic acid, a major component of the extracellular matrix, plays an important role in the regulation of different cellular processes, e.g., locomotion, cell-cell interaction during morphogenesis, and differentiation. Distribution of hyaluronic acid with respect to the role of sperm hyaluronidase in sperm penetration and gamete interaction is well established. In order to elucidate this mechanism, in our current study we have identified and demonstrated, for the first time, the presence of a 68-kDa cell surface hyaluronic acid binding glycoprotein (HABP) in spermatozoa of different species (rat, mice, bull, and human) by immunoblot analysis and indirect immunofluorescence using the polyclonal antibodies raised against purified HABP. Furthermore, we were able to demonstrate a differential distribution of 68-kDa HA binding protein on the sperm head, midpiece, and tail of different species. To identify its role in sperm function, we observed its declining pattern during epididymal maturation and also the inhibition of sperm-oolemmal adherence by pretreatment of the sperms with anti-HABP antibodies. We have further observed its in vivo phosphorylation in motile spermatozoa. All our data clearly indicate that sperm hyaluronan binding protein may have a specific role in sperm maturation, motility, and fertilization processes.

Regulation of Sperm Motility: Emerging Evidence for a Major Role for Protein Phosphatases

Abstract: Lesions in the normal expression of sperm motility are associated with certain types of male infertility. Therefore, an understanding of the signal transduction pathways regulating sperm motility will be an important part of our understanding of the mechanisms underlying these types of male infertility. In this connection, the regulation of sperm fiagellar motility by second messengers is a rapidly expanding area of research in cell and reproductive biology. While abundant information can be found concerning protein kinases and phosphoproteins in relation to sperm motility, relatively little attention has been paid to the potential role for protein phosphatases in this process. This review will focus attention on protein phosphatases, in the context of current literature and more recent studies, as a growing area of interest in relation to the role that protein phosphorylation plays in the regulation of motility of sperm and other axoneme-containing cells.

Effects of antioxidants on human sperm preparation techniques.

Abstract: The effect of two different sperm preparation techniques, Percoll gradient centrifugation and swim-up from a washed pellet were tested on the functional competence of the selected spermatozoa. Percoll gradient centrifugation brought about an improvement in sperm motility parameters such as curvilinear velocity and straight-line velocity, an increase in the rates of hyperactivation and the acrosome reaction and an increase in the percentage of motile spermatozoa after 24 h of incubation compared to the centrifugation, swim-up procedure. The effects of antioxidants such as dithiothreitol (DTT) or reduced glutathione (GSH), and reactive oxygen species-scavenging enzymes such as catalase or superoxide dismutase (SOD) during the stage of centrifugation before the swim-up procedure were also studied. Though all of these agents prevented the fall in sperm motility after 24 h incubation, only DTT and SOD improved the rates of both hyperactivation and the acrosome reaction. GSH also improved the acrosome reaction, whereas catalase was without significant effect on the rates of hyperactivation or the acrosome reaction. These results indicate that Percoll gradient centrifugation selects spermatozoa with better functional competence than does centrifugation swim-up. The damage caused by the centrifugation can be prevented by the addition of antioxidants, suggesting that the differences noted with the Percoll gradient method was due to better protection against peroxidative damage due to the centrifugation of unselected spermatozoa. However, the use of DTT is limited by virtue of the fact that this sulphydryl reducing agent leads to destabilization of the sperm chromatin. In contrast, GSH and SOD could have therapeutic potential.

Effect of sperm washing on levels of reactive oxygen species in semen

Abstract: The possibility was evaluated that the production of reactive oxygen species (ROS) by human sperm is stimulated by the repeated cycles of centrifugation and resuspension involved in conventional sperm preparation. ROS generation by human sperm was monitored before and after the washing of sperm from 55 men (43 men with suspected subfertility and 12 normal volunteers). The ROS activity of all 55 specimens before washing was inversely correlated with original sperm motility (r = .278, p < .05). The mean level of ROS activity was significantly higher after washing than before processing (p < .05) for the 26 specimens with normal sperm motility, the 20 specimens with normal sperm morphology, and the 12 specimens with both normal motility and normal morphology. In contrast, the mean ROS level was not significantly changed after washing in the 27 specimens with poor sperm motility, the 16 specimens with abnormal sperm morphology, or the 13 specimens with both abnormal motility and abnormal morphology. It would appear that repeated centrifugation, resuspension, and vortexing cause excessive generation of ROS in the motile sperm population of the washed specimen. Washing procedures involving excessive manipulation of sperm may, in fact, cause the most harm to motile sperm, i.e., those that the method is trying to select. Procedures that minimize multiple centrifugation, resuspension, and vortexing steps should therefore be used for the preparation of semen specimens for assisted-reproduction techniques.

Calcium influx into mouse spermatozoa activated by solubilized mouse zona pellucida, monitored with the calcium fluorescent indicator, fluo-3. inhibition of the influx by three inhibitors of the zona pellucida induced acrosome reaction: Tyrphostin A48, pertussis toxin, and 3-quinuclidinyl benzilate

Abstract: The fluorescent calcium indicator, fluo-3, was loaded as the membrane permeant tetraacetoxymethyl (AM) ester into cauda epididymal mouse sperm at 25 degrees C for 20 min in the absence of bovine serum albumin (BSA) and presence of the dispersant, Pluronic F-127. Excess indicator was removed by two centrifugation washes at 100g for 10 min, a procedure that did not impair sperm motility. Upon resuspension in medium containing 20 mg/ml BSA to promote capacitation, the sperm cells exhibited readily detectable fluorescence uniformly distributed in the cytoplasm. Cell fluorescence was stable over the time of the experiments and was responsive to changes in intracellular calcium concentration, [Ca2+]i. Initial [Ca2+]i was 231 +/- 58 nM (+/- SE, n = 43). Addition of heat-solubilized mouse zonae pellucidae to capacitated sperm increased [Ca2+]i by 106 +/- 19 nM (+/- SE, n = 18), the higher steady-state concentration being reached after 30 min. Subsequent addition of the non-fluorescent calcium ionophore Br-A23187 resulted in a further increase of 114 +/- 18 nM (+/- SE, n = 18), the higher steady-state concentration being reached after 6 min. The increase in [Ca2+]i induced by solubilized zonae pellucidae was largely blocked by 3-quinuclidinyl benzilate (QNB), an antagonist of muscarinic receptors that was earlier shown to block the zona pellucida induced acrosome reaction in mouse sperm (Florman and Storey, 1982: Dev Biol 91:121-130). This [Ca2+]i increase was completely blocked by the tyrosine kinase inhibitor, tyrphostin A48, and by the inactivator of G1 proteins, pertussis toxin. At the concentrations at which they blocked the zona pellucida-induced increase in [Ca2+]i, all three inhibitors also blocked the zona pellucida-induced acrosome reaction. These results indicate that [Ca2+]i increase in is an early, if not the initial, reaction in the sequence leading to zona pellucida induced acrosomal exocytosis in mouse sperm. The observation that the three inhibitors, each having a different mode of action, all block the zona pellucida induced [Ca2+]i suggests that the sperm plasma membrane receptors mediating the zona pellucida induced acrosome reaction may function as a complex, whose formation is activated by zona pellucida ligand binding.

Effect of cadmium and cigarette smoking on human semen quality

Abstract: OBJECTIVE: In recent years, there have been many nonconclusive studies on cigarette smoking and sperm quality. Few studies, if any, have attempted to implicate any decrease of sperm quality. The aim of this study was to examine the relationship between cigarette smoking, blood and seminal plasma concentrations of cadmium and lead, and sperm quality. METHODS: A total of 184 males who were undergoing initial screening for infertility were included in the study. Tests conducted included semen characteristics (volume, total sperm count, sperm viability, motility and morphology of spermatozoa), and blood and seminal plasma concentrations of lead and cadmium. RESULTS: More than 50% and 70% of the subjects had normal sperm density and motility, respectively. The mean concentrations of lead in blood (PbB) and seminal plasma (PbS) were 7.09 micrograms/dL and 12.98 micrograms/L, respectively, while the mean concentrations of cadmium in blood (CdB) and seminal plasma (CdS) were 0.95 micrograms/L, and 0.58 micrograms/L, respectively. Significant correlations were observed between CdB and cigarette-years and sperm density (negative). CdS was significantly correlated with cigarette-years and sperm volume (negative). Significant trends were observed for different categories of cigarette-years with CdB, CdS and sperm density. CONCLUSION: Cigarette smoking appears to affect sperm density, especially in heavy smokers. Cadmium (present) in cigarettes could be a possible causative agent for the low sperm density among smokers.

Antifertility effects of aqueous extract of Carica papaya seeds in male rats.

Abstract: The influence of the crude aqueous extract of Carica papaya L. (Caricaceae) seeds has been studied on semen profile, fertility, body and organ weight response, and toxicology in male albino rats. The extract was administered at the dose regimens of 10 and 50 mg/animal/day orally for 30, 60, and 90 days and 0.1 and 1.0 mg/animal/day intramuscularly for 15 and 30 days. Cauda epididymal sperm motility and count was reduced significantly at low and high dose regimens both in the oral as well as the intramuscular groups. The reduced sperm motility was associated with morphological defects. Testicular sperm counts were also reduced in all the treatment groups except the low dose intramuscular group. Fertility tests showed dose- and duration-dependent reduction and zero fertility was observed at high dose regimens of the oral and intramuscular groups following 60 and 30 days of treatment, respectively. Testicular weight was reduced in all the treatment groups, whereas accessory sex organs showed a variable response. Body weight and toxicological observations did not show any untoward response. Fertility and all other associated changes returned to normal within 45 and 30 days of treatment cessation in the oral and intramuscular groups, respectively. The data revealed that reversible sterility could be induced in male rats by papaya seeds aqueous extract treatment without adverse effects on libido and toxicological profile.

Sequential acquisition of chemotactic responsiveness by human spermatozoa.

Abstract: Recent studies have indicated that human spermatozoa respond to follicular fluid by attraction to chemotactic factor(s) in the fluid, accompanied by enhancement of motility and ultimately hyperactivation. In this study, we quantified the sperm response. We exposed spermatozoa to a gradient of a chemotactically active fraction of follicular fluid (denoted as "the attractant") and separated the spermatozoa that accumulated in the attractant and those that did not. We thus obtained two subpopulations: one enriched with chemotactically responsive spermatozoa, and one deficient in such spermatozoa. The fraction of the responsive spermatozoa out of the total sperm population was 2-12% at any measured time point. With time, the responsive spermatozoa lost their ability to be attracted, while such activity was gradually acquired by the subpopulation originally deficient in responsive spermatozoa. These results indicate that the identity of responsive spermatozoa is continuously changing. If the in vitro results are representative of the physiological conditions in vivo, they imply that the role of sperm chemotaxis combined with enhanced motility may be to select capacitated spermatozoa and bring them to the egg. Such a mechanism may, over an extended period of time, increase the prospect that an egg will meet capacitated spermatozoa as soon as it ovulates.

Role of cAMP in the reactivation of demembranated ram spermatozoa

Abstract: Ejaculated ram sperm were demembranated with Triton X-100, separated from the detergent-soluble matrix, and reactivated [San Agustin and Witman (1993): Cell Motil. Cytoskeleton 24:264–273]. The percent motility of models prepared from freshly washed sperm was comparable to that of the washed sample before demembranation, regardless of whether cAMP was included in the reactivation medium. However, demembranated models derived from aging or metabolically inhibited sperm exhibited a lower percent reactivation and required cAMP to attain the level of motility of freshly washed sperm. Cyclic AMP was ∼100 times more effective than cGMP. The requirement for cAMP could be bypassed by addition of porcine heart cAMP-dependent protein kinase (PKA) catalytic subunit to the reactivation medium, demonstrating that cAMP was acting via PKA. The cAMP stimulation of reactivation was not affected by inclusion of the PKA inhibitor PKI(5–24) in the reactivation medium, but was decreased when the models were preincubated with PKI(5–24) prior to reactivation. The cytosol-free models retained >90% of the sperm PKA activity; therefore, the PKA appears to be anchored to internal sperm structures. This PKA could not be extracted by cAMP or Triton X-100 alone, but only by cAMP and Triton X-100 in combination. We conclude that cAMP-dependent protein phosphorylation is critical for sperm motility, but that the essential protein phosphate sites turn over slowly under our reactivation conditions, so that the cAMP requirement is apparent only in models prepared from sperm having a low internal ATP or cAMP content. Interestingly, reactivation was rapidly blocked by the peptide arg-lys-arg-ala-arg-lys-glu, which has been reported to be a selective inhibitor of cGMP-dependent protein kinase. © 1994 Wiley-Liss, Inc.

Effect of platelet-activating factor on motility and acrosome reaction of human spermatozoa.

Abstract: The effect of platelet-activating factor (PAF) on motility parameters and induction of the acrosome reaction in human spermatozoa was investigated in 36 unselected men with different degrees of initial sperm motility. The characteristics of sperm movement were assessed by computer-assisted sperm analysis (Hamilton-Thorn Motility Analyser) and the percentage of acrosome-reacted spermatozoa was evaluated after 1 h incubation with PAF (10 nM) and staining with fluorescent peanut lectin. We found that short-term (4 h max) incubation with PAF significantly enhanced total and progressive sperm motility as well as acrosome reaction. An increase of sperm motility in response to PAF was present in 16 out of the 25 subjects studied (defined as responders) and was inversely correlated with basal motility. In the 11 samples (six responders and five non-responders) where the incubation with PAF was prolonged overnight, an increase of sperm motility was present in all the subjects studied. Similarly, an increase in numbers of acrosome reactions in response to 10 nM PAF was present in 20 out of the 26 subjects examined, and was inhibited by the PAF receptor antagonist L659 989. Our results indicate a possible physiological role for PAF in fertilization and suggest a potential use of PAF in in-vitro fertilization techniques in cases of reduced sperm motility.

A new test for the assessment of sperm- zona pellucida penetration: relationship with results of other sperm tests and fertilization in vitro

Abstract: The spermatozoa of some patients attending for in-vitro fertilization (IVF) fail to penetrate the zona pellucida in vitro. A test has been devised to identify these cases. It is based on the number of spermatozoa penetrating into the zona pellucida, which were counted after removing spermatozoa bound to the zona surface by vigorous aspiration of each oocyte through a narrow gauge (120 microns) glass pipette. The oocytes were collected from 197 patients undergoing IVF treatment with their own gametes; 79 with no oocytes fertilized and 118 with some oocytes fertilized. Sperm motility, morphology and DNA normality (acridine orange stain) were also measured. The relationships between sperm test results and IVF rate were examined by logistic regression. The proportions of penetrated zonae, normal sperm morphology and normal DNA were the most significant factors related to IVF rate in the whole group. Also, in patients with > or = 30 spermatozoa bound per zona pellucida or with normal sperm morphology > or = 30%, the proportion of penetrated zonae and normal DNA were most significant. Oocytes from 42 patients who had zero fertilization and low sperm-zona binding (average, 2.2 spermatozoa/zona pellucida) were re-incubated with normal donor spermatozoa: large numbers of spermatozoa bound (average, 88 spermatozoa/zona pellucida) and each zona was penetrated by at least one spermatozoon. In conclusion, the percentage of zonae penetrated was the variable most significantly correlated with IVF rate. Penetration of the zona was also strongly related to fertilization rates in patients without defects of sperm morphology and sperm-zona binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Implication that potassium flux and increase in intracellular calcium are necessary for the initiation of sperm motility in salmonid fishes

Abstract: Flux of K+ and changes in intracellular Ca2+ in the sperm of salmonid fishes were measured with spectrophotometry, ion electrode, microscopic fluorometry, and radioisotope accumulation. Release of K+ occurred at the initiation of sperm motility which is induced by decrease in external K+ and the K+ efflux and sperm motility were inhibited by K+ channel blockers. Intracellular Ca2+ increased within a short period in K(+)-free condition, and the accumulation of 45Ca in sperm cells was higher in motile sperm than that in immotile sperm. The efflux of K+ and the increase in intracellular Ca2+ were suppressed when external K+ concentration increased, i.e., sperm remained immotile. These results suggest that efflux of K+ through K+ channel and subsequent increase in intracellular Ca2+ are prerequisite for the initiation of sperm motility.

Effect of macromolecules from oviductal conditioned medium on bovine sperm motion and capacitation.

Abstract: The effect of macromolecules from oviductal conditioned medium (CM) on sperm motility and capacitation was studied. Sperm pooled from three bulls was incubated in either luteal isthmic CM, luteal ampullary CM, estrual isthmic CM, estrual ampullary CM, or control medium (no CM) for 4 h. Sperm capacitation and motility were assessed at 10 min and 4 h. Estrual isthmic CM capacitated significantly more spermatozoa at 4 h than estrual ampullary CM or control medium. CM also affected lateral head movement (ALH) and beat cross-frequency (BCF) of sperm. In a second experiment, the glycosaminoglycan (GAG) content of the different types of oviductal CM was quantified. Estrual isthmic CM contained more GAG than estrual ampullary CM. Among luteal samples, no difference in GAG concentration between the isthmic and ampullary CM was found. Heat treatment (100 degrees C) of oviductal CM before coincubation with sperm significantly reduced, but did not eliminate, the capacitating ability. Because heat treatment denatures proteins and decreases the capacitating ability of certain GAG, we concluded that the capacitating effect of estrual isthmic CM may be associated with proteins, GAG, and proteoglycans in the CM. Isthmic secretions may play a major role during in vivo sperm capacitation, given that bovine spermatozoa may reside in the oviduct isthmus for up to 18 h before fertilization.