Capacitation Of Hamster Spermatozoa Research Articles

Capacitation of hamster spermatozoa with the divalent cation chelators D-penicillamine, L-histidine, and L-cysteine in a protein-free culture medium.

By using a chemically defined (protein-free) culture medium that supports sperm viability but not capacitation or the acrosome reaction, we have determined that hamster spermatozoa can be chemically capacitated in vitro by the divalent cation chelators D-penicillamine, L-histidine, and L-cysteine in the absence of bovine serum albumin (BSA). Washed cauda epididymal spermatozoa were preincubated (1-2 x 10(6) sperm/ml) for 3, 4, or 6 hr at 37 degrees C in 5% CO2 in air. The basic culture medium used for sperm preincubation and for sperm:egg coincubation was a modified Tyrode's solution (protein-free) containing 10 mM sodium lactate, 100 microM sodium pyruvate, and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm viability was maintained in all preincubation and coincubation media with PHE (20 microM D-penicillamine, 100 microM hypotaurine, and 1.0 microM epinephrine). The low control sperm preincubation medium consisted of TLP-PVA. In some cases the high control preincubation medium also contained 3 mg/ml BSA (TALP-PVA). The experimental preincubation medium was TLP-PVA with additional D-penicillamine (125 or 500 microM), or L-histidine (10, 100, or 1,000 microM) or L-cysteine (25, 75, or 125 microM). After preincubation, sperm were coincubated (2 x 10(4) sperm/ml) with cumulus-free hamster eggs in TALP-PVA +/- additional D-penicillamine, L-histidine, or L-cysteine for 1.5 hr, fixed, and evaluated for percent egg penetration as an index of sperm capacitation. The results demonstrate that hamster spermatozoa can be chemically capacitated in vitro with D-penicillamine (500 microM: range of mean penetration values, 53.6%-84.3%), L-histidine (100 microM: range of mean values, 24.8%-56.3%) or L-cysteine (75 microM: 51.3%) in the absence of exogenous protein.

Structure of the cumulus matrix and zona pellucida in the golden hamster: a new view of sperm interaction with oocyte-associated extracellular matrices.

Hamster oocyte-cumulus complexes (OCC), with and without sperm, were structurally analyzed by light- and electron microscopy using freeze substitution. This method has yielded a clear picture of the extracellular oocyte investments, the cumulus cell matrix and the zona pellucida. The cumulus matrix has an overall homogeneous fibrillar structure which appears to attach to cumulus cells at their filopodial extensions. The matrix also extends into the outer regions of the zona pellucida. The zona pellucida has a distinct porous configuration throughout its entire structure. During gamete interaction experiments, capacitated hamster sperm with ultrastructurally intact acrosomes were found throughout the matrix. Sperm had dramatic effects on the matrix, resulting in compression and stretching. Sperm found on the zona pellucida had initiated or completed the acrosome reaction. During the initial stages of the acrosome reaction, the matrix was in contact with the sperm. At later stages of the acrosome reaction, there was a complete loss of matrix material in regions near the sperm.

A quantitative assay for capacitation: Evaluation of multiple sperm penetration through the zona pellucida of salt‐stored hamster eggs

The endpoint for sperm capacitation occurs when spermatozoa become able to penetrate intact zonae pellucidae of unfertilized homologous eggs. Activation of eggs stimulated by sperm fusion or by gamete aging initiates changes in zonae pellucidae that bar further sperm entry. This zona block mechanism reduces the usefulness of eggs as indicators for sperm capacitation. Egg storage in concentrated salt solutions destroys the zona block mechanism while retaining the biological sperm receptor/activator functions of the zona pellucida [Yanagimachi et al., Fertil Steril 31:562-574, 1979]. We have developed techniques for the quantitative assay of capacitation using multiple sperm penetration into zonae and have evaluated the sperm-response characteristics of hamster eggs stored in a modified salt solution (STOR). Sperm, preincubated in capacitating or noncapacitating treatments (CAP or NOCAP, respectively), were coincubated with unfertilized and fertilized STOR-treated zonae for 4 hr. Then zonae were stripped of externally bound sperm and the sperm heads in the perivitelline space (PVS) were fluorescently labeled with Hoechst dye 33342. Entry of CAP sperm into the PVS of STOR-treated unfertilized eggs was highly correlated with sperm concentration (Rw = .859) and was log linear from 1 X 10(3)-2 X 10(5) CAP sperm/ml. At 2 X 10(5) CAP sperm/ml, the mean number of PVS sperm was 63.5 and the maximum observed was 158. NOCAP sperm very rarely penetrated unfertilized zonae (2 sperm into 75 eggs). Few CAP sperm entered the PVS of STOR-treated fertilized eggs (two sperm into 54 eggs at 2 X 10(5) sperm/ml) unless the sperm concentration was raised to high levels. Differences between replicates were due to male but not female (ie, egg) differences. We conclude that 1) STOR-treated unfertilized zonae can be used to accurately quantitate differences between sperm capacitation and/or fertility states and 2) the availability of large numbers of homogeneous "shelf-stable" zonae will make it feasible to perform hamster sperm capacitation bioassays on a large scale.

Regulation of cAMP Levels during in Vitro Capacitation of Hamster Spermatozoa

Regulation of cAMP Levels during <i>in Vitro</i> Capacitation of Hamster Spermatozoa

Effect of in vivo oocyte aging on sperm chromatin decondensation in the golden hamster

AbstractAged spontaneously activated hamster oocytes recovered from adult females 18 and 24 hours after ovulation were at the pronuclear stage. These oocytes and fresh controls were inseminated in vitro with capacitated hamster spermatozoa and observed with the phase‐contrast microscope. The percentage of fertilization in fresh control oocytes was 98%, as compared to 36% and 18% when the oocytes were recovered 18 and 24 hours after ovulation, respectively. The mean number of sperm decondensations per egg in control oocytes was 10, and in the aged ones it was 0.69 and 0.12 when the oocytes were recovered 18 and 24 hours after ovulation, respectively. When similarly treated oocytes were studied with scanning and transmission electron microscopy, it was found that the degree of gamete membrane fusion was greater than that observed with the phase‐contrast microscope, but that most of the spermatozoa failed to decondense the chromatin. We suggest that parthenogenetic oocytes at the pronuclear stage are in a similar stage of the cell cycle as in fertilized eggs, in which the cytoplasm does not have the ability to decondense the sperm chromatin.

Glycosaminoglycans stimulate the acrosome reaction of previously capacitated hamster sperm.

The glycosaminoglycans (GAGs) hyaluronic acid and heparin were added (10 micrograms and 100 micrograms/ml to golden hamster sperm suspensions previously incubated for 4.5 h under capacitating conditions. After additions, sperm were incubated for 5-15 min and acrosome reactions (AR) assayed in motile sperm by phase contrast microscopy. Hyaluronic acid and heparin significantly stimulated AR over control levels. Hyaluronic acid did not stimulate AR 15 min after addition to sperm previously incubated for only 2.5 h. Pre-incubation of hyaluronic acid with streptomyces hyaluronidase destroyed the ability of that GAG to stimulate the AR. These results indicate that GAGs (at least one of which, hyaluronic acid, is present in the oocyte cumulus oophorous) can rapidly stimulate the acrosome reaction in motile previously capacitated hamster sperm.

Epinephrine decreases the potassium requirements of hamster sperm capacitation: Furosemide blocks the effect of epinephrine

We have investigated the possibility of an interaction between epinephrine and external potassium in the stimulation of the hamster sperm acrosome reaction and in vitro fertilizing ability. We have found that: 1) K+ is required late in capacitation and/or the acrosome reaction, but is not required for penetration of the zona pellucidae, 2) the presence of epinephrine (50 microM) in the capacitation medium lowers the K+ requirements of these processes and 3) furosemide, a cotransport inhibitor, blocks the acrosome reaction, in vitro fertilization, and inhibits the stimulation by epinephrine. These results support the idea that there is an interaction between epinephrine as a stimulus to the secretion of the acrosomal content, and K+ as a mediator of the stimulus signal. They further suggest that K+ influx through the cotransport mechanism occurs during capacitation, that this mechanism is activated late during the process, and that it probably mediates the stimulatory effects of epinephrine. We would like to propose that the activation of passive cation transport mechanisms is a general phenomenon in biological signal transduction.

Scanning electron microscope study of in vitro prepenetration gamete interactions

AbstractHamster and mouse capacitated spermatozoa were interacted in vitro with hamster and mouse eggs in homologous and heterologous combinations. Also, fertilized and trypsin treated unfertilized hamster eggs, and unfertilized rat eggs were made to interact with capacitated hamster spermatozoa. The surface of the zona pellucida was then examined with the scanning electron microscope. It was found that sperm attachment, followed by sperm binding and penetration through the zona pellucida, was observed only when homologous gamete combinations were used. Binding of the spermatozoa to the zona was evidenced by the lytic effect of the acrosomal enzymes on the zona substance. When fertilized eggs and trypsin‐treated unfertilized hamster eggs were mixed with capacitated hamster spermatozoa as well as in the heterologous gamete combinations, we found that the spermatozoa were able to establish attachment but not binding. Under these conditions the outer surface of the zona pellucida was never found to have penetration tracks made by the spermatozoa. Failure of heterologous spermatozoa to cross the foreign zona pellucida is believed to be associated with the inability of the foreign spermatozoa to establish binding and to the inability of their acrosomal enzymes to digest the zona. A similar mechanism is believed to work in zona‐reacted and in trypsin‐treated hamster eggs inseminated in vitro with homologous spermatozoa.

Changes at the hamster oocyte surface from the germinal vesicle stage to ovulation

AbstractImmature and ovulated hamster oocytes were studied with the scanning electron microscope. Immature oocytes at the germinal vesicle stage have their surface uniformly covered by microvilli. When meiosis has progressed to the first meiotic metaphase the overlying surface shows the differentiation of a circular area 19 μm in diameter with a low density of microvilli. Later, from this region the first polar body emerges, and the oocyte surface at the point from which it was extruded shows a cluster of cytoplasmic, conical projections. When the zona‐free oocytes are cultured at 37°C for 5 minutes these projections disappear and the oocyte surface at that point becomes smooth. However, when the oocytes remain in the oviduct for several hours after ovulation these projections remain unchanged. The in vitro interactions of capacitated hamster sperm with the immature oocyte was always seen at microvillus surfaces and never associated with the differentiated regions.

Effects of the postovulatory oviduct contents and exogeneous glucose on capacitation of hamster spermatozoa in vitro.

Effects of the postovulatory oviduct contents and exogeneous glucose on capacitation of hamster spermatozoa in vitro.

Sperm interaction with the zona-free hamster egg.

The interaction of sperm with the zona-free hamster egg was studied. Hamster sperm were capacitated in Tyrodes media, containing 50% heat-inactivated serum and used to inseminate zona-free eggs in BWW containing 10% serum. Capacitated sperm began fusing with eggs within the first hour of insemination and by 3 h penetration had ceased as indicated by the absence of further changes in the mean number of sperm incorporated per egg. Penetration by capacitated hamster sperm was linearly related to the log of the motile sperm concentration at concentrations above 10(4) cells/ml. The viability of sperm and eggs in culture was limited in these studies to approximately 3-5 h. The existence of a block to unlimited sperm penetration of the zona-free egg was sought in reinsemination experiments. A relatively low sperm concentration was used to initiate egg responses, followed, at timed intervals, by reinsemination with a high challenge concentration of sperm. Subsequent polyspermy levels reflected the presence or absence of the egg's block to polyspermy response. In order to circumvent the problems arising from the rapid aging of hamster sperm in culture, mouse sperm were employed, a convenience afforded by the lack of species specificity in this egg. Reinseminated eggs incorporated additional sperm during the challenge; therefore, the hamster egg is not capable of preventing unlimited sperm penetration. The implications of these findings to the use of the zona-free hamster egg test in fertility evaluation is discussed.

Stimulation of hamster sperm capacitation and acrosome reaction in vitro by glucose and lactate and inhibition by the glycolytic inhibitor α‐chlorohydrin

AbstractStudies were made of the effects of D(+)‐glucose, L‐lactate and pyruvate on in vitro capacitation and acrosome reactions (AR) of hamster sperm using a more “defined” medium that that used in previous similar studies. In the absence of glucose or lactate, sperm underwent very few AR and activation (whiplash‐like motility characteristic of capacitated hamster sperm) was reduced compared to those events in sperm preincubated in the presence of glucose plus lactate plus pyruvate. Glucose and pyruvate supported more AR than glucose alone, but less than glucose, lactate, and pyruvate. The glycolytic inhibitor α‐chlorohydrin (10 μm) inhibited AR by 50% and reduced activation by less. When glucose was added to sperm incubated 2 hr with pyruvate and lactate, the number of AR observed after 4 hr was the same as that obtained when glucose was present throughout the incubation. When glucose was added after 3.5 hr, AR were delayed for 1 hr and lower numbers of sperm underwent AR. In the presence of lactate and pyruvate, 0.38 mM glucose was able to support activation and AR as well as 3.24 mM glucose. These results indicate that exogenous glucose and lactate are necessary for in vitro capacitation and AR of hamster sperm; only low levels of exogenous glucose are required; exogenous glucose is not required during the first 2 hr of capacitation; and glycolytic activity is necessary for capacitation and the AR.

Nature and fate of the factors released during early contact interactions between hamster sperm and egg prior to fertilization in vitro

Initial attempts have been made to characterize three factors (Hartmann and Hutchison, 1977, J. Cell Physiol., 93, 41) which are released in vitro at 2, 31, and 50 min after capacitated hamster sperm make contact with but prior to penetration of the zona pellucida. A fourth factor is known to be released at 20–25 min but in the experiments described no effort was made toward its characterization. The assay for the factors is based upon their ability to induce early binding between gametes. Because the release of each of the factors occurs at a different time they can be harvested by sampling the supernatant at the appropriate time. The short-lived activity of these factors, which normally disappears soon after their release, was stabilized by buffering the medium to pH 7.0–7.4 with Tris or TES and removing the cells. Under these conditions, the factors released at 31 and 50 min were stable when incubated at 37°C in the absence of cells for at least 60 min, but the activity of the factor released at 2 min was erratic under similar incubation conditions. The factors released at 31 and 50 min passed unimpeded through filters with molecular weight cutoffs of 2000, and both eluted off Bio-Gel P-2 columns as single peaks of activity in regions corresponding to molecular weights of at least 1800 (void volume) and approximately 1400, respectively. The 2-min factor passed unimpeded through a filter capable of excluding molecules of molecular weight larger than 5000, but 50–86% of the activity was recovered after passing through a filter with a molecular weight cutoff at 2000. The release of the 2- and 31-min factors was inhibited 48 and 74%, respectively, by macromolecular trypsin inhibitors at concentrations which also blocked penetration of the egg by the sperm; these inhibitors had little or no effect on the release of the 50-min factor. The activity of ultrafiltered and pH-stabilized 31- and 50-min factor was destroyed by the proteases subtilisin and leucine aminopeptidase but was unaffected by trypsin or glycosidases. The disappearance of the 31- and 50- min factors after their release was investigated by incubating each factor with each of the cell types present in the drop and it was found that activity only declined in the presence of the eggs. Taken together, these results provide evidence that (1) at least two populations of peptides are released in a time-dependent manner when capacitated sperm make contact with the zona pellucida and (2) these peptides may disappear from the supernatant through an egg-mediated mechanism.

Properties of the first and second factors released after hamster sperm make contact with the zona pellucida prior to fertilization in vitro

AbstractAn investigation was made as to the nature of two of the factors, termed S1, released within the first 30 minutes after contact is made between capacitated hamster sperm and the zona pellucida in vitro. Previous studies showed that these S1 factors were detected two and 20 to 25 minutes after the gametes were combined and that, based on filtration studies, the former possessed a molecular weight of less than 5,000 daltons. The present results show that the quantity of the 20–25‐minute S1 factor released into the supernatant increased linearly as a function of the sperm concentration. This factor passed unimpeded through a filter with a 5,000 molecular weight cutoff but only 42% of the activity traversed a filter with a cutoff of 2,000 daltons. The two‐minute S1 factor, in the virtual total absence of cells, was stable for 10 to 15 minutes, but lost significant activity upon longer incubation. Under the same conditions, the 20–25‐minute factor lost approximately 25% of its activity within 15 minutes, but remained stable at this level for at least 45 minutes of incubation. Both S1 factors were not affected by a mixture of glycosidases, but were inactivated by subtilisin, trypsin, and leucine aminopeptidase which was contaminated with endopeptidases. The activity of the two‐minute S1 factor appeared more susceptible to the action of the proteases than that of the 20–25‐minute S1 factor. In contrast to previous results obtained with the two‐minute S1 factor, the release of the 20–25‐minute S1 factor was not inhibited by the inclusion of soybean trypsin inhibitor a t concentrations which are known to inhibit penetration of the zona by the sperm. The results suggest that the two‐ and 20–25‐minute S1 factors are peptides which are not identical.

Taurine maintains and stimulates motility of hamster sperm during capacitation in vitro

AbstractIt is known that hamster sperm require an unidentified low molecular weight “factor” found in several cells and tissue types in order to remain strongly motile during in vitro capacitation. The β‐amino acid taurine (2‐aminoethane sulfonic acid) is present in a partially purified “factor” from bovine adrenal glands and can substitute for that “factor” in maintaining and stimulating hamster sperm motility. Incubation of washed hamster sperm with bovine serum albumin and 2 × 10−3 to 2 × 10−5M taurine for periods of approximately 5 hr. resulted in a higher number of strongly motile sperm compared to controls in the absence of exogenous taurine. Incubation of sperm with (‐)epinephrine in the absence of taurine resulted in nearly all sperm becoming immotile. Activation (whiplash flagellar movement characteristic of capacitated hamster sperm) did not occur in the absence of taurine, but high percentages of activation occurred in the presence of 2 × 10−3, 2−10−4 and 2 × 10−5M taurine. Acrosome reactions occurred in the presence of taurine, even in the absence of (‐)‐epinephrine. However, (‐)‐epinephrine, previously shown to stimulate activation and acrosome reactions, was required for the maximum number of the latter. These results suggest that in addition to its importance for motility taurine might stimulate capacitation. The mechanism through which taurine supports and stimulates motility is not yet known, but its use results in the first relatively “defined” in vitro capacitation medium for hamster sperm.

Procedures for obtaining high percentages of viable in vitro capacitated hamster sperm

AbstractSuspensions of nearly 100% viable golden hamster sperm were prepared by passing washed cauda epididymal sperm through a column of 0.25–0.3 mm glass beads. Incubations of these viable sperm under in vitro capacitation conditions in volumes of 0.1 or 1 ml (2–2.5 × 106/ml) resulted in 85–92% viable sperm after four hours and 45 minutes of incubation. More than 70% of these sperm were judged to have been capacitated after four hours and 45 minutes of incubation on the basis of their having undergone acrosome reactions and the presence of high numbers of sperm exhibiting the activated motility characteristic of capacitated hamster sperm.Thus, for the first time, procedures are available that will yield large numbers of viable capacitated sperm for biochemical analysis and that will also allow other studies of hamster sperm capacitation with minimum interference from molecules released from dead sperm.

Stimulation of in vitro activation and the acrosome reaction of hamster spermatozoa by catecholamines

Capacitation and the acrosome reaction of mammalian spermatozoa are essential for fertilization. In vitro results are presented that demonstrate that catecholamines stimulate activation (a whiplash flagellar movement characteristic of capacitated hamster spermatozoa) and the acrosome reaction. Protein-free ultrafiltrates of bovine adrenal cortex and medulla preparations stimulated motility, activation, and acrosome reactions of hamster spermatozoa in the presence of bovine serum albumin. The medulla preparation was more effective than the cortex preparation in the stimulation of activation and acrosome reactions. Epinephrine (0.5-50 muM) and norepinephrine (50.0 muM) in the presence of bovine serum albumin and a partially purified protein-free cortex preparation also stimulated activation and the acrosome reactions. Both activation and acrosome reactions in the presence of epinephrine were inhibited by the adrenergic antagonists phentolamine and propranolol, suggesting the involvement of alpha- and beta-adrenergic receptors in the stimulation of capacitation and the acrosome reaction. In addition, phenylephrine, an alpha-adrenergic agonist, was as potent as epinephrine in the stimulation of acrosome reactions, but activation was reduced. Isoproterenol, a beta-adrenergic agonist, was as potent as epinephrine in the stimulation of activation, but acrosome reactions were reduced. High percentages of both activation and acrosome reactions were observed only in the presence of epinephrine, norepinephrine, or phenylephrine and isoproterenol together.

Contact between hamster spermatozoa and the zona pellucida releases a factor which influences early binding stages.

Summary. The studies reported here demonstrate that isolated hamster zonae pellucidae affect the binding between capacitated hamster spermatozoa and eggs included in the same drop. Mouse zonae pellucidae fail to affect binding between hamster gametes. These results suggest that binding, which occurs only between homologous spermatozoa and isolated zonae pellucidae causes a factor (S1 factor) to be released. The SI factor is diffusible and influences the two successive binding steps (B1 and B2) previously shown to occur between capacitated spermatozoa and eggs. The B2 step is delayed by the action of the SI factor during the early part of the interval between B1 and B2 suggesting that the effect may be mediated through Bl. It is proposed that the SI factor is also released during the first binding interaction (B1) between the spermatozoa and the egg which appears to be an analogous but terminated form of the binding to the isolated zona pellucida. The data are consistent with the possibility that the B1 step is terminated by the product of the interaction between the S1 factor and a factor from the vitellus or perivitelline space.

The effect of fluid from the cauda epididymidis, serum components and caffeine upon the survival of diluted epididymal hamster spermatozoa.

Summary. When epididymal hamster spermatozoa were diluted in increasing amounts of Tyrode's solution, their survival diminished markedly. If, instead, they were diluted in 20% `cauda epididymidis' fluid (CEF), their survival increased. The active factor of CEF was found to be of low molecular weight and heat stable. If the spermatozoa were diluted in the presence of caffeine or a capacitating medium derived from human serum, their survival was not significantly increased but the rate of those moving was markedly stimulated. Human serum components, but not caffeine\p=m-\albuminor epididymal fluid\p=m-\albumin,capacitated hamster spermatozoa. The survival-promoting factor of epididymal fluid did not appear to act through sperm cyclic AMP metabolism.

Penetration of guinea-pig spermatozoa into hamster eggs in vitro.

With some important exceptions (Chang & Hancock, 1967), fertilization of mammalian eggs is species-specific in that the eggs of a species can be penetrated only by spermatozoa of the same species. The analysis of the species-specificity of fertilization is very difficult when in-vivo systems are used. In-vitro systems, on the other hand, eliminate many factors and enable us to examine directly the interactions between the eggs and spermatozoa. Barros (1968) inseminated rat eggs in vitro with capacitated golden hamster spermatozoa and found that only one out of 294 eggs had been penetrated. In this egg, two spermatozoa had penetrated the zona pellucida but failed to enter the vitellus. On the other hand, none of 280 mouse eggs inseminated in vitro with capacitated hamster spermatozoa were found to be penetrated.