Showing papers on "Acrosome reaction published in 1995"

Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation.

Abstract: The molecular basis of mammalian sperm capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of M(r) 40,000-120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for capacitation, prevent the sperm from undergoing capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway.

Abstract: In the accompanying report (Visconti, P.E., Bailey, J.L., Moore, G.D., Pan, D., Olds-Clarke, P. and Kopf, G.S. (1995) Development, 121, 1129–1137) we demonstrated that the tyrosine phosphorylation of a subset of mouse sperm proteins of M(r) 40,000-120,000 was correlated with the capacitation state of the sperm. The mechanism by which protein tyrosine phosphorylation is regulated in sperm during this process is the subject of this report. Cauda epididymal sperm, when incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin do not display the capacitation-associated increases in protein tyrosine phosphorylation of this subset of proteins. This NaHCO3, CaCl2 or bovine serum albumin requirement for protein tyrosine phosphorylation can be completely overcome by the addition of biologically active, but not inactive, cAMP analogues. Addition of the active cAMP analogues to sperm incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin overcomes the inability of these media to support capacitation, as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. The effects of the cAMP analogues to enhance protein tyrosine phosphorylation and to promote capacitation appears to be at the level of the cAMP-dependent protein kinase (PKA), since two specific inhibitors of this enzyme (H-89 and Rp-cAMPS) block the capacitation-dependent increases in protein tyrosine phosphorylation in sperm incubated in media supporting capacitation. Capacitation, as assessed by the aforementioned endpoints, also appears to be inhibited by H-89 in a concentration-dependent manner. These results provide further evidence for the interrelationship between protein tyrosine phosphorylation and the appearance of the capacitated state in mouse sperm. They also demonstrate that both protein tyrosine phosphorylation and capacitation appear to be regulated by cAMP/PKA. Up-regulation of protein tyrosine phosphorylation by cAMP/PKA in sperm is, to our knowledge, the first demonstration of such an interrelationship between tyrosine kinase/phosphatase and PKA signaling pathways.

Reactive oxygen species, lipid peroxidation and enzymatic defence systems in human spermatozoa

Abstract: The reactive oxygen species, hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2 o- ), were generated with a xanthine-xanthine oxidase system and their effect on human sperm function was studied. The action of reactive oxygen species on selected human spermatozoa resulted in a decreased capacity for ionophore-induced acrosome reaction, a decrease in sperm motility, an increase in the concentration of lipid hydroperoxides and a loss of membrane polyunsaturated fatty acids. H 2 O 2 was the key intermediate of the deleterious effects exerted by the xanthine and xanthine oxidase. Among these parameters, the acrosome reaction appeared most susceptible to the reactive oxygen species generated by the xanthine-xanthine oxidase system, and was decreased without sperm motility being affected. Treatment with H 2 O 2 was shown to inactivate several enzymatic activities involved in the antioxidant defence of spermatozoa: glutathione peroxidase, superoxide dismutase and glucose-6-phosphate dehydrogenase. H 2 O 2 and O 2 o- were shown to be involved in the lipid alterations triggered by the xanthine-xanthine oxidase system. Singlet oxygen is proposed to intervene in the lipoperoxidation process. The inefficacy of mannitol in protecting spermatozoa suggests that hydroxyl radicals were not produced in the extracellular medium

Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm.

Abstract: Calcium flux is required for the mammalian sperm acrosome reaction, an exocytotic event triggered by egg binding, which results in a dramatic rise in sperm intracellular calcium. Calcium-dependent membrane fusion results in the release of enzymes that facilitate sperm penetration through the zona pellucida during fertilization. We have characterized inositol 1,4,5-trisphosphate (IP3)-gated calcium channels and upstream components of the phosphoinositide signaling system in mammalian sperm. Peptide antibodies colocalized G alpha q/11 and the beta 1 isoform of phospholipase C (PLC beta 1) to the anterior acrosomal region of mouse sperm. Western blotting using a polyclonal antibody directed against purified brain IP3 receptor (IP3R) identified a specific 260 kD band in 1% Triton X-100 extracts of rat, hamster, mouse and dog sperm. In each species, IP3R immunostaining localized to the acrosome cap. Scatchard analysis of [3H]IP3 binding to rat sperm sonicates revealed a curvilinear plot with high affinity (Kd = 26 nM, Bmax = 30 pmol/mg) and low affinity (Kd = 1.6 microM, Bmax = 550 pmol/mg) binding sites, reflecting among the highest receptor densities in mammalian tissue. Immunoelectron microscopy confirmed the acrosomal localization in rat sperm. The IP3R fractionated with acrosomes by discontinuous sucrose gradient centrifugation and was enriched in the medium of acrosome-reacted sperm. ATP-dependent 45Ca2+ loading of digitonin permeabilized rat sperm was decreased by 45% in the presence of 10 microM IP3. The IP3-mediated release of calcium was blocked by heparin. Thapsigargin, a sequiterpene lactone inhibitor of the microsomal Ca(2+)-ATPase, stimulated the acrosome reaction of mouse sperm to the same extent as the Ca2+ ionophore, A23187. The failure of caffeine and ryanodine to affect calcium accumulation suggested that thapsigargin acted through an IP3-sensitive store. The presence of G alpha q/11, PLC beta 1 and a functional IP3R in the anterior acrosomal region of mammalian sperm, as well as thapsigargin's induction of the acrosome reaction, implicate IP3-gated calcium release in the mammalian acrosome reaction.

Molecular basis of mammalian egg activation.

Abstract: Publisher Summary The chapter discusses signal transduction mechanisms in the activation of mammalian eggs, primarily in the mouse, and explains the sequence of events that accompanies fertilization in the mouse. This information provides a framework within which to examine known and proposed signaling mechanisms that regulate egg activation, helps understand the role of several possible second messengers in egg activation, and helps describe the sperm–egg interaction that may result in the production of second messengers. In the mammal, the first interaction between the sperm and egg is at the level of the zona pellucida (ZP), an extracellular coat that surrounds all mammalian eggs. In the mouse, the ZP is composed of three glycoproteins: ZP1, ZP2, and ZP3. Although acrosome-intact sperm establishes primary binding with ZP3 through an O-linked carbohydrate moiety, the identity of this moiety is controversial. Following sperm binding, ZP3 induces the acrosome reaction of the bound sperm.

Sperm capacitation in humans is transient and correlates with chemotactic responsiveness to follicular factors.

Abstract: In humans, only a small fraction (2-12%) of a sperm population can respond by chemoattraction to follicular factors. This recent finding led to the hypothesis that chemotaxis provides a mechanism for selective recruitment of functionally mature spermatozoa (i.e., of capacitated spermatozoa, which possess the potential to undergo the acrosome reaction and fertilize the egg). This study aimed to examine this possibility. Capacitated spermatozoa were identified by their ability to undergo the acrosome reaction upon stimulation with phorbol 12-myristate 13-acetate. Under capacitating conditions, only a small portion (2-14%) of the spermatozoa were found to be capacitated. The spermatozoa were then separated according to their chemotactic activity, which resulted in a subpopulation enriched with chemotactically responsive spermatozoa and a subpopulation depleted of such spermatozoa. The level of capacitated spermatozoa in the former was approximately 13-fold higher than that in the latter. The capacitated state was temporary (50 min < life span < 240 min), and it was synchronous with the chemotactic activity. A continuous process of replacement of capacitated/chemotactic spermatozoa within a sperm population was observed. Spermatozoa that had stopped being capacitated did not become capacitated again, which indicates that the capacitated state is acquired only once in a sperm's lifetime. A total sperm population depleted of capacitated spermatozoa stopped being chemotactic. When capacitated spermatozoa reappeared, chemotactic activity was restored. These observations suggest that spermatozoa acquire their chemotactic responsiveness as part of the capacitation process and lose this responsiveness when the capacitated state is terminated. We suggest that the role of sperm chemotaxis in sperm-egg interaction in vivo may indeed be selective recruitment of capacitated spermatozoa for fertilizing the egg.

Sperm motility hyperactivation facilitates penetration of the hamster zona pellucida.

Abstract: These experiments were conducted to determine whether or not sperm motility hyperactivation facilities penetration of the zona pellucida of the oocyte. Two approaches were used. For the first, hamster sperm were incubated for 4.0-4.25 h in a capacitating medium that contained either 2.9 or 25.0 mM sodium bicarbonate. In these media, sperm became equally capacitated as evidenced by their ability to undergo the acrosome reaction when exposed to lysophosphatidyl choline or intact zonae pellucidae; however, sperm became hyperactivated only in the medium containing 25.0 mM bicarbonate. When these sperm were added to cumulus-free oocytes in vitro, only 2 of 88 oocytes were penetrated by sperm preincubated in 2.9 mM bicarbonate, while 31 of 86 oocytes were penetrated by sperm in 25.0 mM bicarbonate. It was found that equal numbers of sperm were bound to the oocytes and that equal numbers were acrosome-reacted on the surface of the zonae in the two media. For the second approach, sperm were incubated in a capacitating medium containing 25 mM bicarbonate. When > 70% were hyperactivated, aliquots were added to three sets of oocytes. After 10 min had been allowed for sperm to attach and acrosome-react, inhibitors of hyperactivation were added and the sperm and oocytes were incubated for an additional 20 min before fixation and examination for zona penetration. In the dishes treated with the inhibitors verapamil or Cd2+, 1 of 42 and 0 of 42 oocytes were penetrated, respectively, compared with 25 of 40 in controls. Therefore, it appears that hyperactivation facilitates penetration of the hamster zona pellucida.

Phosphatidylcholine-binding proteins of bovine seminal plasma modulate capacitation of spermatozoa by heparin.

Abstract: Bovine seminal plasma (BSP) contains four similar acidic proteins, previously designated as BSP-AI, BSP-A2, BSP-A3, and BSP30-kDa. These proteins are secreted by the seminal vesicles and coat the surface of the spermatozoa after ejaculation. The binding site of BSP proteins on the sperm surface has been identified as choline phospholipids on the plasma membrane. This study was undertaken to determine whether BSP proteins modulate capacitation of bovine spermatozoa induced by heparin. Bovine epididymal spermatozoa were washed and incubated in buffer containing BSP proteins and then washed and incubated with heparin. The percentage of capacitated spermatozoa was determined under the microscope after the acrosome reaction has been initiated with the addition of lysophosphatidylcholine. The results demonstrated that epididymal sperm undergo the acrosome reaction only in the presence of BSP proteins. This effect was concentration-dependent and reached a maximum level of a 3-5-fold increase at 20-40 g/ml BSP protein concentrations. In contrast, ribonuclease (purified from bovine seminal fluid) or seminal fluid proteins depleted of BSP proteins (by sequential absorption of BSP proteins on gelatin-Agarose and DEAE-Sephadex columns) showed no significant potentiating activity. The purified BSP proteins were more active than crude alcohol precipitates of bovine seminal plasma. These results indicate that BSP proteins are regulatory factors of capacitation.

Oligosaccharide Constructs with Defined Structures That Inhibit Binding of Mouse Sperm to Unfertilized Eggs in Vitro

Abstract: During fertilization in mice, free-swimming sperm bind to mZP3, an 83-kDa glycoprotein present in the egg extracellular coat, the zona pellucida [Wassarman, P. M. (1990) Development 108, 1-17]. Mouse sperm recognize and bind to a specific class of serine/threonine-linked (O-linked) oligosaccharides present on mZP3. After binding to mZP3, sperm undergo a form of cellular exocytosis, the acrosome reaction, thereby enabling them to penetrate the zona pellucida and fertilize the egg. Thus, gamete interactions in mice are carbohydrate-mediated. In this context, we tested 15 O-linked-related oligosaccharide constructs with defined structures for their ability to inhibit binding of mouse sperm to ovulated eggs and to induce sperm to undergo the acrosome reaction in vitro. Thirteen of the oligosaccharides were constructed and characterized in our laboratory [Seppo, A., Pentilla, L., Niemela, R., Maaheimo, H., Renkonen, O., & Keane, A. (1995) Biochemistry 34, 4655-4661]; two were obtained commercially. We found that, while none of the oligosaccharides induced sperm to undergo the acrosome reaction, a few of them inhibited binding of sperm to eggs at relatively low concentrations (ID50 < 5 microM). In certain cases, sperm formed head-to-head aggregates in the presence of the oligosaccharides. The results suggest that the ability of oligosaccharides to inhibit binding of sperm to eggs is dependent on several parameters, including the size and branching pattern of the oligosaccharide, as well as on the nature of the sugar residue at the nonreducing end of the oligosaccharide.

Sperm morphology assessment: historical review in relation to fertility

Abstract: Careful analysis of sperm morphology has always been an important part of a routine semen examination. However, the usefulness of sperm morphology assessment as a predictor of a man's fertilizing potential has often been challenged due to different classification systems, various slide preparation techniques and inconsistency of analyses within and between laboratories. Automated sperm morphology analysis instruments may overcome the subjective nature of visual assessments of sperm morphology, but the technical problems are numerous and the validity of these instruments has still to be proven. Having reviewed the literature, it seems clear that there is general agreement concerning the clinical relevance and predictive value of this single semen parameter in vivo and in vitro. Nevertheless, even in cases of severe teratozoospermia, fertilization may be possible. Studies on the acrosome reaction are very promising for patients with severe sperm morphology abnormalities that do not have major effects on the fertilizing potential. Most promising is the development of intracytoplasmic sperm injection (ICSI) as the treatment of first choice in cases of severe teratozoospermia with failed fertilization in vitro. Normal fertilization and pregnancy rates can be obtained with ICSI in the presence of extreme teratozoospermia, suggesting that sperm morphology may be important in spermatozoa-zona binding, penetration and spermatozoon-oocyte fusion but fails to be of any predictive value once the spermatozoon reaches the cytoplasm of the oocyte. To conclude, accurate and strict sperm morphology assessment is very useful in evaluating a patient's fertilizing potential, with the main advantage that the methods used to examine this parameter are easy to learn on equipment found in most laboratories.

Sperm exocytosis reconstructed in a cell-free system: evidence for the involvement of phospholipase C and actin filaments in membrane fusion

Abstract: We used a cell-free system to study membrane fusion during sperm exocytosis (acrosome reaction). Extracted bovine sperm plasma and outer acrosomal membranes were labeled with chlorophyll a or DCY, respectively. The occurrence of membrane fusion is indicated by the ability of the probes to diffuse from one membrane species to another which is revealed by resonance energy transfer between the two probes. We have previously shown using this system that the requirement of capacitation for sperm exocytosis is retained in cell-free membrane fusion, and that the pH and calcium dependence of the cell-free fusion mimics those of exocytosis in intact cells. In the present report we further characterize the fusion of sperm membranes which we observe in our assay. Phosphoproteins and phospholipases were found to be involved in the membrane fusion step of sperm exocytosis. Protein kinases, phosphatases, and Gi-like proteins, while involved in exocytosis in intact cells, are not involved specifically in the membrane fusion step of exocytosis. The role of membrane bound F-actin in regulating membrane fusion was also studied using fluorescently labeled phalloidin. The results show that cortical F-actin has two roles in regulating sperm exocytosis. One is to form a scaffolding to hold phospholipase C at the membrane. It also functions as a physical barrier to membrane fusion which is removed by the increases in intracellular calcium and pH which precede fusion.

Glycolipid migration from the apical to the equatorial subdomains of the sperm head plasma membrane precedes the acrosome reaction. Evidence for a primary capacitation event in boar spermatozoa

Abstract: In order to extend the static information of immunola belling sulphogalactolipids in fixed boar spermatozoa, a fluorescent sulphogalactolipid analogue, galactose(3sulphate)-β1-1′[( N-lissamine rhodaminyl)-12-aminododecanoyl]-sphingosine, was incorporated into plasma membranes of living spermatozoa and its lateral distribu tion over the sperm head was studied. The fluorescent lipid was enriched in the apical ridge subdomain of freshly ejaculated sperm cells. After sperm binding to the zona pellucida the lipid redistributed to the equatorial segment of the sperm surface. A similar shift occurred during capacitation in vitro with 2 mM CaCl2 or with 4% (w/v) bovine serum albumin. The desulphated derivative galactose-β1-1′[(N-lissamine rhodaminyl)-12-aminododecanoyl]-sphingosine was also incorporated into the plasma membrane of freshly ejaculated sperm cells and clearly stained the apical ridge subdomain and the (pre)-equatorial subdomains of the sperm heads. The desulphogalactolipid analogue showed a slightly faster migration to the equatori al segment of the sperm plasma membrane than did its sulphated counterpart. The measured fluorescence intensity distributions correlated linearly with the spatial probe dis tribution, which was checked by fluorescence lifetime imaging microscopy. The observed migration of the incorporated glycolipids precedes the acrosome reaction and is one of the underlying molecular events likely to be important in the process of sperm capacitation. The results of this study suggest that lipid phase segregation is an important driving force for the organization of the sperm head plasma membrane into subdomains.

The human sperm acrosome reaction: physiology and regulatory mechanisms. An update

Abstract: The acrosome reaction is a crucial step during gamete interaction in all species, including man. It allows spermatozoa to penetrate the zona pellucida and fuse with the oocyte membrane. Spermatozoa unable to undergo the acrosome reaction will not fertilize intact oocytes. This article concentrates on the characteristics and regulatory mechanisms of the acrosome reaction in human spermatozoa. During recent years, various entities found in the vicinity of the ovulated oocyte have been identified as stimulators of the acrosome reaction, of which zona protein is considered the prime physiological inducer in vivo. The steroid hormone progesterone has been shown to evoke critical responses in sperm cells leading to the acrosome reaction. Calcium has also been shown to play a central role during the acrosome reaction. Calcium flux is induced specifically by progesterone in capacitated and uncapacitated sperm cells, whereas only capacitated spermatozoa are able to subsequently complete the acrosome reaction. Progesterone as well as zona protein has been shown to evoke crucial responses within human spermatozoa, shedding light on the cascade of intracellular signalling events leading to the completion of the acrosome reaction. Furthermore, chemical agents which bring about the reaction in vitro, such as the ionophores ionomycin or A23187, have been used to shed light on its regulatory mechanisms. A number of molecules have been postulated to regulate the acrosome reaction in mammals, for example a galactosyl-transferase and a sperm protein tyrosine kinase. In addition, a novel protein, termed SAA-1, that was first detected on human spermatozoa is discussed with respect to its potential role as a regulatory protein closely involved in the initiation of the acrosome reaction.

Superoxide anion production by human spermatozoa as a part of the ionophore‐induced acrosome reaction process

Abstract: The involvement of superoxide anion (O 2 °− ) in human sperm capacitation and/or acrosome reaction was investigated. Addition of superoxide dismutase (SOD) to the medium at the beginning of the capacitation process or 15 min before induction of the acrosome reaction, decreased the level of ionophore-induced acrosome reaction. Hyperactivation was unaffected by the presence of SOD during the capacitation process. Addition of calcium ionophore to the sperm suspension increased production of O °− 2 by the spermatozoa by four to five-fold and induced the acrosome reaction. In the presence of SOD, superoxide anion could not be detected in the medium and the rate of inducedacrosome reaction was decreased greatly. The presence of an inhibitor of protein kinase C inhibited the production of O °− 2 in the medium and reduced the induced-acrosome reaction. The production of O °− 2 and the acrosome reaction were also increased by exposure of spermatozoa to 12-myristate 13-acetate phorbol ester, a specific activator of protein kinase C. While the level of spontaneous acrosome reaction was not increased by the direct addition of O °− 2 to the medium, its presence induced the release of unesterified fatty acids from membrane phospholipids. These findings suggest that the production of O °− 2 by spermatozoa could be involved in the ionophore-induced acrosome reaction, possibly through the de-esterification of membrane phospholipids. However, this production of superoxide anion is not sufficient on its own to induce the acrosome reaction

Capacitation In Vitro of Stallion Spermatozoa: Comparison of Progesterone‐Induced Acrosome Reactions in Fertile and Subfertile Males

Abstract: Mammalian sperm that have completed capacitation are capable of undergoing the acrosome reaction in response to a number of biological and chemical stimuli. In the present report, we have investigated the ability of progesterone to stimulate acrosome reactions of stallion sperm capacitated in vitro. Motile sperm were selected by a two-layer Percoll gradient centrifugation and were incubated in TALP medium modified by the 1:1 (v/v) addition of TEST-yolk medium for 5 hours at 39 degrees C, under 5% CO2 in humidified air. Sperm incubated in vitro in TALP-TEST medium had a higher percentage of acrosome reactions following the addition of progesterone (3.18 mumol/L) compared to controls (P < 0.05). Furthermore, sperm from stallions classified as fertile on the basis of breeding history had higher percentages of progesterone-induced acrosome reactions in comparison with stallions classified as subfertile (P < 0.05). Acrosome reactions were assessed routinely by fluoresceinated lectin binding, but the physiological appearance of induced acrosome reactions was confirmed at the ultrastructural level by transmission electron microscopy. We conclude that 1) TALP-TEST medium supports stallion sperm capacitation in vitro, 2) progesterone-induced acrosome reactions are physiological, and 3) sperm from fertile stallions may be more responsive to progesterone-induced acrosome reactions than those of subfertile stallions. This is the first report in a nonhuman species that differences exist between the sperm of fertile and subfertile males in the ability to capacitate and acrosome react in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure, function and biosynthesis of sperm-activating peptides and fucose sulfate glycoconjugate in the extracellular coat of sea urchin eggs

Abstract: A decapeptide (GFDLNGGGVG) isolated from the solubilized jelly layer of the sea urchin Hemicentrotus pulcherrimus stimulates the respiration and motility of H. pulcherrimus spermatozoa and, in addition, produces a number of biological effects on H. pulcherrimus spermatozoa including increases in cAMP and cGMP levels, activation of a Na+/H+ exchange system, and increases in intracellular pH (pHi) and [Ca2+] ([Ca2+]i). The peptide activates the metabolism of endogenous phosphatidylcholine and promotes the acrosome reaction as a specific co-factor of a major acrosome reaction-inducing substance, fucose sulfate glycoconjugate. The peptide also induces an electrophoretic mobility change in the guanylate cyclase of the sperm plasma membrane with concomitant dephosphorylation and inactivation of the enzyme. Seventy-four peptides producing similar biological effects, named sperm-activating peptide (SAP), have since been purified from the solubilized jelly layer of seventeen species of sea urchins distributed over five taxonomic orders. These peptides show essentially the same biological effects on sea urchin spermatozoa although their activity and structures are specific at the ordinal level. Equilibrium binding experiments using a radioiodinated SAP-I analogue [GGGY(125I)GFDLNGGGVG] to H. pulcherrimus spermatozoa suggests the presence of two classes of receptors (high affinity and low affinity) specific for SAP-I binding. Based on the Kd values and EC50's for SAP-I's biological activity, we presume that the high affinity receptor is associated with respiration-stimulating activity and elevations in pHi, while the low affinity receptor is coupled to elevations in cGMP and [Ca2+]i. The radioiodinated SAP-I analogue crosslinks to a 71 kDa protein which contains a single membrane-spanning domain at almost near C-terminus. A SAP-I precursor which is synthesized in the accessory cells contains five SAP-I and seven SAP-I-like decapeptides, each separated by a single lysine residue.

Bicarbonate/CO2 is not required for zona pellucida- or progesterone-induced acrosomal exocytosis of mouse spermatozoa but is essential for capacitation.

Abstract: We investigated whether bicarbonate/CO2 is required for capacitation or for acrosomal exocytosis triggered with zona pellucida or progesterone. Mouse spermatozoa, incubated for 90 min in a modified Tyrode's medium with bicarbonate and equilibrated with 5% CO2 in air, were washed in medium without bicarbonate but with Hepes, resuspended in media with or without bicarbonate, and then stimulated with 1 zona pellucida/microliter or 15 microM progesterone. Spermatozoa were able to undergo exocytosis, regardless of the medium in which they were resuspended, as ascertained via the chlortetracycline assay. If, however, spermatozoa were first incubated in a medium in which bicarbonate was replaced by Hepes and were then washed and resuspended in various media, they were unable to undergo exocytosis in response to zona pellucida or progesterone even when resuspended in bicarbonate-containing medium. This indicated that spermatozoa were not capacitated. Furthermore, the proportion of cells exhibiting the "B" pattern (characteristic of capacitated cells) after incubation in medium without bicarbonate was lower than that in cells incubated with the anion and the appropriate gas phase. Extended incubation in medium without bicarbonate (up to 3 h) did not increase the proportion of cells that exhibited the "B" pattern. These results demonstrate that bicarbonate is not required for acrosomal exocytosis but that it is essential for capacitation, exerting roles beyond its action as pH buffer.

New concepts in human sperm-zona pellucida interaction

Abstract: Binding of spermatozoa to the zona pellucida is an initial, crucial recognition event leading to fertilization. In the mouse, the best species characterized so far, the zona pellucida protein 3 (ZP3) has a central role as the specific, primary sperm receptor on the zona and as the inducer of the acrosome reaction. This sequence of events is not clearly understood as it relates to human gametes. The ideal test for the evaluation of sperm-zona pellucida interaction is one that can examine these events in a sequential fashion (i.e. binding followed by the acrosome reaction) in a standardized and specific bioassay. Here we have used the hemizona assay as an internally controlled test to examine human sperm-zona pellucida interaction. Results presented show that : (i) the hemizona assay has an excellent predictive power for IVF outcome and for the identification of male infertility ; (ii) using a specific anti-ZP3 antiserum in immunocyto-chemical studies, the hemizona assay allows for the identification of structural/functional anomalies of the protein backbone of human ZP3 (identification of oocyte anomalies) ; and (iii) glycobiological studies using the hemizona assay model indicate that the initial sperm-zona pellucida binding requires a selectin-like interaction between the human gametes. These efforts may help us to characterize the cellular and molecular mechanisms involved in human gametes and their dysfunctions in infertile patients.

Intracellular calcium increase and acrosome reaction in response to progesterone in human spermatozoa are correlated with in-vitro fertilization

Abstract: In this study we have investigated responsiveness to progesterone in spermatozoa from a group of unselected male partners of couples undergoing in-vitro fertilization (IVF). We evaluated progesterone-stimulated intracellular Ca2+ ([Ca2+]i) and percentage increase in acrosome reaction in the same sperm sample used for oocyte inseminations. [Ca2+]i was measured with a fluorimetric method, while the acrosome reaction was assessed using a fluorescent probe (fluorescein isothiocyanate-labelled peanut lectin). The average percentage [Ca2+]i as well as the rate of increase in the frequency of acrosome reaction following progesterone challenge were significantly lower (P < 0.005) in the group of patients with a fertilization rate < 50%. In addition, significant correlations between the fertilization rate and the progesterone-stimulated [Ca2+]i and acrosome reaction increases (r = 0.78 and r = 0.79 respectively) were observed. Furthermore, in cases of fertilization failure, no increase of [Ca2+]i or acrosome reaction was observed in response to progesterone with the exception of one case. Our results indicate that [Ca2+]i and acrosome reaction increases in response to progesterone can be of value in the prediction of sperm fertilizing ability. As the two parameters were significantly correlated to each other (r = 0.86), the two assays have similar IVF predictive value and might be used interchangeably as a diagnostic tool in the assignment of male patients to the different kinds of assisted fertilization techniques.

Mapping the mouse ZP3 combining site for sperm by exon swapping and site-directed mutagenesis.

Abstract: During fertilization in mice, sperm bind to mouse ZP3 (mZP3), a M(r) approximately 83,000 glycoprotein present in the ovulated egg extracellular coat, or zona pellucida. Sperm recognize and bind to specific serine/threonine-linked (O-linked) oligosaccharides present at the mZP3 combining site for sperm. Binding to mZP3 induces sperm to undergo a form of exocytosis, the acrosome reaction. To map the mZP3 combining site for sperm, we examined the effect of exon swapping and site-directed mutagenesis on the glycoprotein's two activities, sperm binding and induction of the acrosome reaction. Stably transfected embryonal carcinoma cell lines were established that synthesized recombinant glycoproteins and secreted them into the culture medium. The glycoproteins were partially purified from culture medium and assayed for sperm-binding and acrosome reaction-inducing activities. Results of these assays suggest that glycosylation of one or more of five serine residues, clustered together in a polypeptide region encoded by mZP3 gene exon 7, is required for activity. Interestingly, this polypeptide region exhibits considerable sequence divergence during evolution and may be related to the proposed role for oligosaccharides in species-specific gamete adhesion during mammalian fertilization.

Identification of a sperm penetration factor in the oviduct of the golden hamster.

Abstract: Previously, we found oviductal eggs to be significantly more penetrable and fertilizable in vitro than ovulated eggs collected from the ovarian bursa, while bursal eggs were comparable to mature (unovulated) follicular eggs. Incubation of follicular eggs with a soluble eluate of oviductal egg cumulus complexes (COF) increased sperm penetration: the activity was macromolecular, was destroyed at 56°C, and was produced in the oviduct. We now report purification of this oviductal factor that enhances penetration of follicular eggs and have identified it as oviductin (OVN). Oviducts, 1-1.5 h post-LH from eCG-primed females, were homogenized and the cytosolic fraction was chromatographed on a Helix pomatia lectin affinity column; specific proteins were eluted with 0.2 M N-acetyl-D-galactosamine. Fractions were monitored by dot-blot assay using as the primary antibody monoclonal antibody (mAb) 1C4 against OVN. Proteins were resolved by one-dimensional SDS-gel electrophoresis, followed by electrotransfer and immunostaining of Western blots. OVN fractions were indexed to COF by quantitative dot-blot assay, and activity was bioassayed by penetration of follicular eggs within 1 h of coincubation with precapacitated sperm ± factors: COF and BSA (high and low controls, respectively) and fractions from the lectin-isolated peak. The mean penetration rates for three isolations were 17 4.0 a , 51.7 ± 5 .0 b, and 49 + 2.7 b% for BSA, COF, and column fractions, respectively (p 0.05). Purified OVN bound to follicular zonae during culture. Acrosome-intact sperm heads bound OVN during 30 min of incubation both before (t = 0 h) and after capacitation (t = 5.5 h) (visualized by indirect immunofluorescence). We conclude that OVN enhances penetration and fertilization by altering both sperm and eggs.

Effect of bovine ampullary and isthmic oviductal fluid on motility, acrosome reaction and fertility of bull spermatozoa

Abstract: Motility, acrosome reaction and oocyte fertilizing ability were assessed for bull spermatozoa after incubation in regional (isthmic or ampullary), bovine oviductal fluid, pooled by stage of the oestrous cycle. Oviductal fluids collected daily from isthmic and ampullary cannulae implanted in the same oviduct were divided into pools, representing two oestrous cycle stages, based on daily serum progesterone concentrations. Ejaculated bull spermatozoa were incubated for 0\p=n-\6h in each type of oviductal fluid. Incubation in isthmic oviductal fluid collected during the nonluteal stage, including oestrus and ovulation, decreased overall sperm motility (from 71.7% motile spermatozoa to 34.0% and both path (78 \g=m\ms \m=-\1versus 86\p=n-\89\g=m\m s\m=-\1) and progressive (74 \g=m\ms\m=-\1versus 83\p=n-\85\g=m\m s\m=-\1) velocities of spermatozoa motion. Spermatozoa incubated in isthmic, non-luteal oviductal fluid had a higher rate and extent of sperm acrosome reaction (213% of control versus 136\p=n-\161%of control by 2 h incubation) compared with spermatozoa incubated in other oviductal fluid types. However, incubation in nonluteal ampullary fluid increased the number of spermatozoa, which were both acrosome reacted and live, and able to fertilize bovine ova (88.7% fertilized versus 75\p=n-\81%).Glycosaminoglycan concentrations were similar among types of oviductal fluid (0.77\p=n-\0.88mg ml \m=-\1). These findings indicate that oviductal fluid differentially affects sperm function, depending on the oviduct region and the stage of the oestrous cycle at which the fluid was obtained.

Sequence and localization of the mouse sperm autoantigenic protein, Sp17.

Abstract: The present study characterizes the sperm protein Sp17 in the mouse. Sp17 is a mammalian testis- and sperm-specific protein that has been isolated, sequenced, and characterized from rabbit testis and spermatozoa. In this study, a rabbit Sp17 cDNA probe representing the entire protein coding region was used to screen a mouse testis cDNA library to obtain the mouse Sp17 sequence. The mouse mRNA for Sp17 encodes a 149-amino acid protein with a predicted molecular weight of 17 296. The mouse Sp17 (MSp17) cDNA sequence is 82% identical to the rabbit Sp17 cDNA sequence while the MSp17 protein sequence is 74% identical to the rabbit protein sequence. The presence of native Sp17 in mouse spermatozoa and testis was demonstrated by Western blot analysis, immunoprecipitation, and immunolocalization. After SDS-PAGE, the native Sp17 has an apparent molecular mass of 24 kDa. The sequence of the native Sp17 was confirmed by Western blots of mouse testis and spermatozoa probed with two anti-peptide antibodies-one, anti-G22C, made against amino acids 6182 in the rabbit sequence (61-83 in the mouse), and a second, anti-K18C, made against amino acids 120-136 in the C-terminal region in the human sequence (118-134 in the mouse sequence). In the absence of proteolytic inhibitors, part of the C-terminal of native MSp17 is cleaved, giving rise to an 18-kDa band. Sp17 is present in spermatocytes and spermatids in the testis. In spermatozoa, Sp17 is not available to bind antibody on the surface of live, acrosome-intact spermatozoa, but it is present on the equatorial surface of live, acrosome-reacted spermatozoa. In fixed spermatozoa, staining is observed along the length of the principal piece, weakly along the midpiece, and over the acrosomal region of the head. When the acrosome reaction begins, acrosomal staining is seen throughout the equatorial region of the acrosome. Using mimotope analysis, this study has also demonstrated that native Sp1 7 is a sperm autoantigen and that recombinant mouse Sp17 is immunogenic in males with a highly restricted linear epitope.

Intracellular calcium reaches different levels of elevation in hyperactivated and acrosome‐reacted hamster sperm

Abstract: Calcium plays a role in sperm mo- tility hyperactivation and the acrosome reaction, but the relationship between cytoplasmic calcium (Ca",,) levels in the two states was heretofore unknown. The Ca2+ indica- tor indo-1 was used to detect Ca2+in in moving hamster sperm in two sets of experiments. In the first experiment, activated, hyperactivated, and zona pellucida-induced acrosome-reacted/hyperactivated sperm were analyzed at the time of peak of activity for each state. In the second experiment, sperm in all states were analyzed at one time point. In both sets, mean Ca2+i, in the acrosomal region, postacrosomal region, and flagellar midpiece was greater in hyperactivated sperm than in activated sperm, and in acrosome-reacted/hyperactivated sperm than in unreacted/hyperactivated sperm (P < 0.001). Ca2+in had increased to a greater extent in the midpiece than in the head in hyperactivated sperm, while the reverse was true for acrosome-reacted sperm. Oscillations at the frequency of the flagellar beat cycle were detected chiefly in the proximal flagellar midpiece of acrosome-reacted sperm, as they had been previously reported to occur in activated and hyperactivated sperm. Thus, Ca2+,, may be main- tained at two different elevated levels in sperm, and contin- ues to oscillate after the acrosome reaction. 0 1995 Wiley-Liss, Inc.