Treating boar sperm with cholesterol-loaded cyclodextrins widens the sperm osmotic tolerance limits and enhances the in vitro sperm fertilising ability

Treating sperm with cholesterol-loaded cyclodextrins (CLC) improves the cryosurvival of the sperm of different cold-shock sensitive species. However, the response of boar sperm to this treatment is not fully understood. The aim of this study was to determine how CLC and methyl-β-cyclodextrin (MβCD, not loaded with cholesterol) affect the parameters for boar sperm functionality, including sperm osmotic resistance, and the ability of the sperm to capacitate and to penetrate the sow's immature oocytes in vitro. Samples treated with CLC or MβCD prior to freezing exhibited similar percentages of motile sperm, live sperm and sperm with intact acrosomes as the control samples (P>0.05). In addition, these treatments did not alter the response of the boar sperm to capacitating conditions. However, when compared to the controls and the MβCD-treated samples, the CLC-treated sperm maintained greater percentages of motile sperm and live sperm in a wide range of osmotic solutions including hypo- (50, 75 and 150 mOsm/kg) and hyper-osmotic (600, 800 mOsm/kg) conditions (P<0.05). In addition, the CLC-treated sperm exhibited greater oocyte penetration ability than the control and the MβCD-treated sperm (P<0.0001). In conclusion, the pre-freezing treatment of boar sperm with CLC does not alter the ability of the sperm to respond to capacitating conditions. Despite not increasing the cryosurvival of the sperm, this treatment widens the sperm osmotic tolerance limits and enhances the in vitro sperm fertilising ability.     

Effect of sulfated glycoconjugates on capacitation and the acrosome reaction of bovine and hamster spermatozoa

The effects of sulfated glycoconjugates on the preparation of mammalian sperm for fertilization were investigated. The three sulfated glycoconjugates tested were heparin, dextran sulfate, and the fucose sulfate glycoconjugate (FSG) from the sea urchin egg jelly coat. In vivo, FSG induces the acrosome reaction in sea urchin sperm. Bovine sperm were found to be capacitated by heparin and FSG as judged both by ability of lysophosphatidylcholine (LC) to induce an acrosome reaction and by ability to fertilize bovine oocytes in vitro. The mechanism by which heparin or FSG capacitated bovine sperm appeared similar, since glucose inhibited capacitation by both glycoconjugates. In contrast to effects on bovine sperm, heparin and FSG induced the acrosome reaction in capacitated hamster sperm. When hamster sperm were incubated under noncapacitating conditions, heparin had no effect on capacitation or the acrosome reaction. Three molecular weights (MW) of dextran sulfate (5,000, 8,000, 500,000) were found to capacitate bovine sperm as judged by the ability of LC to induce an acrosome reaction. Whereas bovine sperm incubated with 5,000 or 8,000 M W dextran sulfate fertilized more bovine oocytes than control sperm (P <0.05), sperm treated with 500,000 M W dextran sulfate failed to penetrate oocytes. The high-MW dextran sulfate appeared to interact with the zona pellucida and/or sperm to prevent sperm binding. Results suggest that sulfated glycoconjugates may prepare sperm for fertilization across a wide range of species.     

Influence of a capacitation period on human sperm acrosome loss.

The present study investigates whether a 5 hour capacitation period modifies the ability of human spermatozoa to undergo induced acrosomal loss. Human sperm acrosomal loss was induced by treatment with either the calcium ionophore A23187, low concentrations of the phospholipid dilauroylphosphatidylcholine (PC12), or 2 hours incubation in conditioned medium prepared from human cumulus cells (CM/CC). The use of a dual staining method (FITC-ConA and Hoechst 33258) for simultaneous assessment of acrosomal status and viability demonstrated that induction of acrosomal loss with calcium ionophore was not dependent on a capacitation period. A short (5 hour) incubation period was not sufficient to induce acrosomal loss with CM/CC above spontaneous acrosome reaction rates in medium alone. A significant capacitation-dependent increase (P < 0.05) in acrosomal loss was observed when human spermatozoa were incubated with PC12. Induction of acrosomal loss of capacitated human spermatozoa with PC12 therefore provides a simple assay for the simultaneous assessment of human sperm capacitation and the acrosome reaction in vitro.     

Induction of epididymal boar sperm capacitation by pB1 and BSP-A1/-A2 proteins, members of the BSP protein family

A family of proteins designated BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, collectively called BSP (bovine seminal plasma) proteins, constitute the major protein fraction of bull seminal plasma. BSP proteins can stimulate sperm capacitation by inducing cholesterol and phospholipid efflux from sperm. Boar seminal plasma contains one homologous protein of the BSP family, named pB1; however, its physiological role is still unknown. In the current study, we report a novel method to purify pB1 from boar seminal plasma by chondroitin sulfate B-affinity chromatography and reverse-phase-high performance liquid chromatography. We also studied the effect of pB1, BSP-A1/-A2, and whole boar seminal plasma on boar sperm capacitation. Boar epididymal sperm were washed, preincubated in noncapacitating medium containing pB1 (0, 2.5, 5, 10 or 20 microg/ml), BSP-A1/-A2 (0 or 20 microg/ml) proteins, or whole seminal plasma (0, 250, 500, or 1000 microg/ml), then washed and incubated in capacitating medium. Acrosomal integrity was assessed by chlortetracycline staining. The status of sperm capacitation was evaluated by the capacity of sperm to undergo the acrosome reaction initiated by the addition of the calcium ionophore, A23187. The pB1 and BSP-A1/-A2 proteins increased epididymal sperm capacitation as compared with control (sperm preincubated without proteins). This effect reached a maximum level at 10 microg/ml pB1 and at 20 microg/ml BSP-A1/-A2 (2.3- and 2.2-fold higher than control, respectively). Whole boar seminal plasma did not induce sperm capacitation. In addition, pB1 bound to boar epididymal sperm and was lost during capacitation. These results indicate that BSP proteins and their homologs in other species induce sperm capacitation in a similar way.     

At least half of capacitated, motile mouse sperm can fertilize zona-free mouse oocytes.

The percentage of individual sperm capable of fertilizing zona pellucida-free mouse oocytes was investigated by placing motile sperm near zona-free oocytes with a micromanipulator. Incubation with one or two capacitated sperm per oocyte resulted in 50% and 70% fertilization, respectively, compared to 88% for cumulus intact (10(5) sperm/ml) and 87% for zona-free (2 x 10(3) sperm/ml) control oocytes. When sperm were treated with .1 microM calcium ionophore A23187 to facilitate the acrosome reaction, fertilization rates for single motile sperm were markedly lower than for capacitated, nontreated single sperm (4% and 35%, respectively). Similar fertilization rates resulted when one sperm was incubated per two ova (4% and 48% per sperm for A23187-treated and controls, respectively). When a lower dose of A23187 (.001 microM) was used to treat sperm, 7% of oocytes incubated with single sperm were fertilized. These experiments demonstrate that at least half of motile, capacitated mouse sperm are capable of fertilizing zona-free mouse oocytes in vitro, and that motile, A23187-treated mouse sperm resulted in poor fertilization rates.     

Sperm chromatin remodeling after intracytoplasmic sperm injection differs from that of in vitro fertilization

Intracytoplasmic sperm injection (ICSI) is a popular method used in assisted conception, and live offspring have been born from a variety of species, including humans. In ICSI, sperm chromatin is introduced into the oocyte together with the acrosome, a structure that does not enter the oocyte during normal fertilization. We compared sperm chromatin remodeling, the potential of embryos to develop in vitro, and DNA synthesis in mouse embryos obtained from in vitro fertilization (IVF) and ICSI. We also tested whether sperm pretreatment prior to ICSI (i.e., capacitation, acrosome reaction, membrane removal, and reduction of disulfide bonds in protamines) facilitates chromatin remodeling and affects embryo development. Sperm chromatin was examined on air-dried, Giemsa-stained preparations at 30-min intervals for up to 4.5 h postfertilization. In all experimental groups, the oocytes underwent activation and formed pronuclei with similar rates. However, the dynamics of sperm chromatin remodeling in ICSI and IVF embryos varied. In ICSI, chromatin remodeling was more asynchronous than in IVF. Sperm capacitation prior to injection enhanced remodeling asynchrony and resulted in delayed pronuclei formation and DNA synthesis. The removal of the acrosome prior to injection with calcium ionophore A23187 but not with detergent Triton X-100 allowed more synchronous chromatin remodeling, timely DNA synthesis, and good embryo development. Our data have significance for the refinement of the molecular and biologic mechanisms associated with ICSI for current and future applications.     

Effects of 2-hydroxypropyl-beta-cyclodextrin and cholesterol on porcine sperm viability and capacitation status following cold shock or incubation

Porcine sperm are extremely sensitive to the damaging effects of cold shock. It has been shown that cholesterol-binding molecules, such as 2-hydroxypropyl-beta-cyclodextrin (HBCD), improve post-cooling porcine sperm viability when added to an egg yolk-based extender, but also enhance sperm capacitation in other species. The objective of this study was to determine the effects of HBCD and cholesterol 3-sulfate (ChS) on porcine sperm viability and capacitation following cold shock or incubation under conditions that support capacitation using a defined medium. We report here that porcine sperm incubated in medium containing both HBCD and ChS have significantly improved viability following cold shock (10 min at 10 degrees C) when compared to sperm incubated without HBCD or ChS, or with either component alone. Treatment with HBCD plus ChS also completely inhibited the increase in protein tyrosine phosphorylation induced by the cold shock treatment or by incubation for 3 hr under conditions that support capacitation. Two assays of sperm capacitation, the rate of calcium ionophore-induced acrosome reactions and chlortetracycline (CTC) staining, were not significantly altered by HBCD and ChS following cold shock. However, 3-hr incubation with HBCD plus ChS or with 1 mM ChS alone decreased the percentage of sperm undergoing the induced acrosome reaction without significantly affecting viability when compared to the control. These results indicate that the manipulation of sperm plasma membrane cholesterol content affects porcine sperm viability and capacitation status and could therefore be useful to protect sperm from cold shock during cryopreservation by improving viability without promoting premature capacitation.     

Macaque sperm fusion with zona-free hamster oocytes

Abstract: Semen from six cynomolgus macaques was washed twice by centrifugation in BWW media and resuspended in 1:2 (vol:vol) of Tes-Tris-buffered eggyolk extract (EXT) diluent and BWW (EXT-BWW). Caffeine and dbcAMP were added to induce capacitation. Sperm were treated with calcium ionophore, A23187, followed by the addition of EXT to recover motility, then washed into BWW. The percent motile sperm was similar in ionophore-treated and control sperm suspensions, but motility of treated sperm decreased significantly by 20 min after treatment. The percentage of viable, acrosome-reacted sperm was 56.3% following ionophore treatment versus 4.0% in control suspensions. When ionophore-treated sperm were coincubated with zona-free hamster oocytes, 51 % of oocytes had evidence of sperm fusion and no oocytes were penetrated after incubation with control sperm. These results are consistent with the hypothesis that macaque sperm are capable of fusion with zona-free hamster oocytes if sperm are viable and acrosome-reacted.     

Ubiquitination and its influence in boar sperm physiology and cryopreservation

Recent reports document the potential use of the ubiquitin protein as an indicator of mammalian sperm quality or fertility, based on poor morphology, sperm count, and other cellular qualities. However, its influence on cellular physiologic mechanisms and boar sperm cryopreservation are unknown. The objective of this research was to determine the influence of boar sperm ubiquitination (n=12 boars) on motility (using CASA), and flow cytometry and fluorescent probes (in parentheses) to evaluate mitochondrial activity (JC-1), plasma and acrosomal membrane integrity (PI and FITC-PNA), membrane fluidity (M540), and chromatin stability (TUNEL) for fresh and frozen-thawed samples. The effects of ubiquitination (determined flow cytometrically) on the ability of frozen-thawed boar sperm to capacitate (FLUO-3AM) and acrosome react (FITC-PNA) were also investigated using flow cytometry. Cryopreservation induced a decrease in the percentage of sperm that were ubiquitinated from 29 to 20% (P<0.0001), but no significant effects of ubiquitin on sperm quality (motility, membrane integrities and organization) were detected. The ability of sperm to capacitate and acrosome react was influenced by ubiquitination. Samples with more ubiquitinated boar sperm were able to maintain plasma membrane integrity (PMI) better and have fewer live acrosome-reacted cells over 120min of induced capacitation (P<0.05). In conclusion, frozen-thawed ubiquitinated boar sperm were better able to survive the physical stresses of induced capacitation, yet were still capable of capacitating and acrosome reacting, which may enable use of this assay for in the vitro evaluation of the quality of boar sperm.     

Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm.

Capacitation is a prerequisite for successful fertilization by mammalian spermatozoa. This process is generally observed in vitro in defined NaHCO3-buffered media and has been shown to be associated with changes in cAMP metabolism and protein tyrosine phosphorylation. In this study, we observed that when NaHCO3 was replaced by 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid (HEPES), hamster sperm capacitation, measured as the ability of the sperm to undergo a spontaneous acrosome reaction, did not take place. Addition of 25 mM NaHCO3 to NaHCO3-free medium in which spermatozoa had been preincubated for 3.5 h, increased the percentage of spontaneous acrosome reactions from 0% to 80% in the following 4 h. Addition of anion transport blockers such as 4,4'-diiso thiocyano-2, 2'-stilbenedisulfonate (DIDS) or 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) to the NaHCO3-containing medium inhibited the acrosome reaction, with maximal inhibition at 600 microM, and with an EC50 of 100 microM. Increasing either extracellular or intracellular pH did not induce the acrosome reaction in NaHCO3-free medium. In contrast, addition of 500 microM dibutyryl cAMP (dbcAMP), alone or together with 100 microM 1-methyl-3-isobutylxanthine (IBMX), induced the acrosome reaction in spermatozoa incubated in NaHCO3-free medium. These compounds also partially reversed the inhibition of the acrosome reaction caused by the DIDS or SITS in complete medium. In contrast to these results, IBMX or dbcAMP did not induce acrosome reactions in cells incubated in Ca2+-free medium. When hamster sperm were incubated in the absence of NaHCO3 or in the presence of NaHCO3 and DIDS, cAMP concentrations were significantly lower than the values obtained from sperm incubated in complete medium. Protein tyrosine phosphorylation has also been shown to be highly correlated with the onset of capacitation in many species. During the first hour of capacitation, an increase in protein tyrosine phosphorylation was observed in complete medium. In the absence of NaHCO3, the increase in protein tyrosine phosphorylation was delayed for 45 min, and this delay was overcome by the addition of dbcAMP and IBMX. The induction of the acrosome reaction by calcium ionophore A23187 in NaHCO3-free medium was delayed 2 h, as compared with control medium. This delay was not observed in the presence of dbcAMP and IBMX. Taken together, these results suggest that a cAMP pathway may mediate the role of NaHCO3 in the capacitation of hamster spermatozoa and that protein tyrosine phosphorylation is necessary but not sufficient for complete capacitation.     

Effect of cholesterol-loaded cyclodextrins on bull and goat sperm processed with fast or slow cryopreservation protocols

Cholesterol-loaded cyclodextrins (CLC) added to the sperm before cryopreservation enhance sperm quality after freeze-thawing in several cold shock-sensitive species, including cattle and goats. However, all studies conducted to date have used conventional protocols, in which sperm are cooled slowly to 5°C before freezing. As cholesterol plays a significant role in sperm cold shock resistance, it is possible that CLC-treated sperm can withstand cooling damage when the sperm are not cooled slowly to 5°C before freezing. In this study, we determined whether CLC-treated goat (1 mg CLC/120×10⁶ sperm) and bull (2 mg CLC/120×10⁶ sperm) sperm quality, after thawing, was different for sperm frozen using conventional protocols (including a slow cooling phase to 5ºC) and protocols in which the sperm were frozen from room temperature, without cooling the sperm slowly to 5°C before freezing. CLC-treated sperm exhibited higher percentages of plasma membrane-intact sperm than control sperm when cryopreserved using conventional protocols. In addition, CLC treatment enhanced both sperm motility and plasma membrane integrity when sperm were frozen directly from room temperature. However, this treatment did not fully prevent the damage of the sperm after cooling rapidly and subsequent freezing, as the sperm quality was lower than that presented by the samples frozen using the conventional protocol. The results are promising, but studies to optimize the protocols for freezing sperm directly from room temperature need to be conducted, as well as studies to determine how cryopreserving sperm in this manner affects other sperm functions.     

Isolation and characterization of glycosaminoglycans from bovine follicular fluid and their effect on sperm capacitation

The majority of published studies have reported the use of commercial heparin to capacitate bovine sperm. However, heparin is not present in the female genital tract fluids. In this study, we purified large amounts of glycosaminoglycans (GAGs) from bovine follicular fluid (FF), characterized them and determined their potential to capacitate sperm. FF-GAGs were isolated by protease digestion, lipid extraction, and by different precipitation conditions and then purified by ion exchange chromatography. Two GAGs, heparan sulfate and chondroitin sulfate B, were present in FF. To determine the capacitation potential of FF-GAGs, bovine ejaculated sperm were incubated 5 hr with or without 12 or 24 microg/ml of each of the FF-GAG fractions or with heparin (12 microg/ml). The purified FF-GAGs and heparin did not stimulate sperm acrosome reaction (AR), but stimulated sperm capacitation. Fractions 1 and 2 (heparan sulfate) were more active to promote capacitation (stimulated up to 3.2-fold) than fractions 3 and 4 (mostly chondroitin sulfate B). Fractions 3 and 4 stimulated capacitation two times more than the control (without FF-GAGs or heparin). When the heparan sulfate impurity was removed from fractions 3 and 4 by acid hydrolysis, the capacitation-promoting activity associated with these fractions did not change significantly. When 24 microg/ml of fraction 1 or 2 were used, the percentage of sperm capacitation observed was similar to the capacitation with 12 microg/ml of heparin. Our results also indicated that the FF-GAGs interact strongly with the BSP proteins. Therefore, it is concluded that heparan sulfate is the GAG that is the most potent capacitating factor present in bovine FF.     

Capacitation of bovine sperm by heparin

Capacitation of bovine sperm was evaluated by determining the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction upon exposure to lysophosphatidylcholine (LC). Incubation of sperm with heparin (10 micrograms/ml) increased the percentage of oocytes fertilized, but this required exposing sperm to heparin for at least 4 h before adding them to oocytes. There was no effect on the percentage of motile or acrosome-reacted sperm after exposure of noncapacitated sperm to 100 micrograms/ml LC for 15 min. When sperm were incubated for 4 h with heparin, exposure to 100 micrograms/ml LC for 15 min had no effect on the percentage of sperm that were motile, but the percentage of acrosome-reacted sperm increased from less than 10% to over 70%. The acrosome reactions (ARs) induced by LC were synchronous, reached maximal levels within 15 min, and differed (p less than 0.001) between sperm incubated under capacitating (with heparin) and noncapacitating conditions (without heparin). The time course required for heparin to capacitate sperm as judged by in vitro fertilization and to render sperm sensitive to LC induction of the AR were found to be similar. The percentage of ARs induced by LC and percentage of oocytes fertilized by sperm were found to be heparin-dose-dependent, with the maximum responses occurring at 5-10 micrograms/ml heparin. The correlation between the mean fertilization and LC-induced AR percentages was 0.997 (p less than 0.01). These studies demonstrate capacitation of bovine sperm by heparin requires at least a 4-h exposure of sperm to heparin and suggest that plasma membrane changes prior to an AR can be detected by exposure of bovine sperm to LC.     

Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival

Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due to membrane alterations induced by the membrane changing from the fluid to the gel-state as the temperature is reduced lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane fluidity at low temperatures by adding cholesterol to the membrane. Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher percentages of motile sperm were maintained after thawing when 1.5 mg CLC was added to sperm from stallions whose sperm do not survive freezing well, compared to control sperm from those same stallions (67% vs. 50%; P<0.05). Addition of CLCs increased the percentages of membrane intact sperm surviving cryopreservation compared to untreated sperm for all stallions (P<0.05). The amount of cholesterol that incorporated into the membranes of the sperm cells increased in a polynomial fashion (R2=0.9978) and incorporated into all sperm membranes. In addition, there was a significant loss of cholesterol from sperm membranes after cryopreservation; however, addition of CLCs to sperm prior to cryopreservation maintained higher cholesterol levels in the sperm after freezing and thawing than untreated sperm (P<0.05). Addition of CLCs also resulted in more sperm binding to the zona pellucida of bovine oocytes after cryopreservation than control sperm (48 vs. 15; P<0.05). In conclusion, CLCs improved the percentage of post-thaw viability in equine sperm as well as increased the number of sperm that bind to zona pellucida. Addition of CLCs to stallion sperm prior to cryopreservation is a simple procedure that increases the cryosurvival of cells.     

Simple histochemical stain for acrosomes on sperm from several species

The acrosome reaction is an exocytotic process that enables a sperm to penetrate the zona pellucida and fertilize an egg. The process involves the fenestration and vesiculation of the sperm plasma membrane and outer acrosomal membrane releasing the acro somal contents. Many different methods have been devel oped to detect the acrosomal status of sperm. These techniques are sometimes complicated, costly, and can be used on only a few species. The aim of this study was to develop an efficient and inexpensive method to assess the acrosomal status of sperm from a variety of species. We prepared and fixed sperm from humans, cattle, swine, rabbits, guinea pigs, and mice and stained them with Coomassie G250. The acrosomes were stained intensely blue in color. Following capacitation, some sperm were incubated for 1 hr with 10 microM calcium ionophore A23187 to induce the acrosome reaction. They were also stained with Coomassie G-250. Ionophore-treated sperm lacked Coomassie staining over the acrosomal region. Differential interference contrast (DIC), bright field microscopy or Pisum sativum agglutinin staining confirmed that the acrosomes of sperm from these species were reacted in response to calcium ionophore treatment and the acrosome reaction frequencies matched results with Coomassie staining. These results demonstrate that the acrosomal status of mammalian sperm from several species can be determined easily and reliably using this simple Coomassie Blue G-250 staining method.     

Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa

Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.     

Penetration of zona-free hamster, bovine and equine oocytes by stallion and bull spermatozoa pretreated with equine follicular fluid, dilauroylphosphatidylcholine or calcium ionophore A23187

Experiments evaluated the ability of follicular fluid (FF), dilauroylphosphatidylcholine (PC12) and the calcium ionophore A23187 (A23187) to induce capacitation in stallion and bull spermatozoa, determined by the ability of the spermatozoa to penetrate zona-free hamster, bovine and equine oocytes. Spermatozoa suspensions were incubated at 37 degrees C in one of the following treatments: 1) a modified Tyrode's medium (BGM3) alone; 2) BGM3 + FF; 3) BGM3 + PC12; 4) BGM3 + FF + PC12; 5) BGM3 + A23187; and 6) BGM3 + FF + A23187. Treated spermatozoa were incubated with zona-free hamster, bovine and equine oocytes for 3 h, after which oocytes were stained to assess spermatozoa penetration. The number of hamster oocytes penetrated by spermatozoa incubated in BGM3 alone (1/30) or in presence of FF (2/31) was significantly lower (P < 0.05) than by spermatozoa treated with PC12 or A23187 (16/30 and 17/30, respectively). Processing stallion spermatozoa either by a swim-up procedure or by centrifugation through a Percoll gradient increased the percentages of motile spermatozoa in the final sample, and spermatozoa collected by both processes penetrated similar numbers of zona-free hamster oocytes (P > 0.05). Although treating spermatozoa with PC12 or A23187 enabled both stallion and bull spermatozoa to penetrate oocytes, higher numbers of bovine oocytes were penetrated by bull spermatozoa (25/30) than by stallion spermatozoa (4/30) regardless of spermatozoal treatment. However, the number of zona-free hamster and equine oocytes penetrated by bull spermatozoa (25/30 and 12/18 respectively) and stallion spermatozoa (17/30 and 15/21 respectively) were similar (P > 0.05). We conclude that both PC12 and A23187 capacitate stallion and bull spermatozoa sufficiently to permit the acrosome reaction to occur, enabling spermatozoa to penetrate homologous and heterologous zona-free oocytes.     

Hamster sperm capacitation: role of pyruvate dehydrogenase A and dihydrolipoamide dehydrogenase

Recently, we demonstrated that pyruvate dehydrogenase A2 (PDHA2) is tyrosine phosphorylated in capacitated hamster spermatozoa. In this report, using bromopyruvate (BP), an inhibitor of PDHA, we demonstrated that hamster sperm hyperactivation was blocked regardless of whether PDHA was inhibited prior to or after the onset of hyperactivation, but the acrosome reaction was blocked only if PDHA was inhibited prior to the onset of the acrosome reaction. Further, inhibition of PDHA activity did not inhibit capacitation-associated protein tyrosine phosphorylation observed in hamster spermatozoa. It is demonstrated that the essentiality of PDHA for sperm capacitation is probably dependent on its ability to generate effectors of capacitation such as reactive oxygen species (ROS) and cAMP, which are significantly decreased in the presence of BP. MICA (5-methoxyindole-2-carboxylic acid, a specific inhibitor of dihydrolipoamide dehydrogenase [DLD]), another component of the pyruvate dehydrogenase complex (PDHc), also significantly inhibited ROS generation and cAMP levels thus implying that these enzymes of the PDHc are required for ROS and cAMP generation. Furthermore, dibutryl cyclic adenosine monophosphate could significantly reverse the inhibition of hyperactivation observed in the presence of BP and inhibition of acrosome reaction observed in the presence of BP or MICA. The calcium ionophore, A23187, could also significantly reverse the inhibitory effect of BP and MICA on sperm acrosome reaction. These results establish that PDHA is required for hamster sperm hyperactivation and acrosome reaction, and DLD is required for hamster acrosome reaction. This study also provides evidence that ROS, cAMP, and calcium are involved downstream to PDHA.     

Effect of heparin on in vitro capacitation of boar sperm

Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 microM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 microg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.     

Early pre-pubertal exposure to low-dose oral di(2-ethylhexyl) phthalate does not affect sperm plasma membrane stability, acrosomal integrity or chromatin structure in the post-pubertal boar

This study aimed to determine whether pre-pubertal exposure in boars to di(2-ethylhexyl) phthalate (DEHP), a plasticizer reported to have toxic effects on rodent reproduction, would affect the sperm ability to undergo capacitation and acrosome reaction (AR) in vitro or give rise to a higher degree of chromatin instability associated with acid-induced denaturation. Spermatozoa were collected from 16 boars (n=8/group) 8-9 months of age, exposed to 300mg/kg body weight of DEHP or placebo per os three times a week, from 3 to 7 weeks of age. The spermatozoa were cryopreserved and examined post-thaw by flow cytometry for their ability to capacitate in vitro when exposed to the effector bicarbonate and to acrosome-react when exposed to calcium ionophores, using the lipid stain Merocyanine-540 (m-540), and peanut agglutinin-fluorescein isothiocyanate, respectively, as probes. The ability of the DNA to sustain denaturation in vitro was tested using a sperm chromatin structure assay (SCSA). No significant differences between the DEHP-exposed group and controls were found for any of the sperm attributes examined. Frozen-thawed spermatozoa showed similar rates of non-capacitated cells between groups, and were capacitated at similar rates. Rates of induced ARs were also similar. Values of DNA denaturation were low and showed no differences between groups. In conclusion, pre-pubertal exposure to DEHP does not seem, under the conditions of the present experiment, to affect the ability of frozen-thawed spermatozoa collected post-puberty to capacitate or acrosome-react (the main requisites for fertilization) or to present damage in their nuclear genome.