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-penicilla-mine, L-histidine, and L-cysteine in the absence of bovine serum albumin (BSA). Washed cauda epididymal spermatozoa were preincubated (1–2 × 10⁶ sperm/ml) for 3, 4, or 6 hr at 37°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 μM sodium pyruvate, and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm viability was maintained in all preincubation and coincubation media with PHE (20 μM D-penicillamine, 100 μM hypotaurine, and 1.0 μM 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 μM), or L-histidine (10, 100, or 1,000 μM) or L-cysteine (25, 75, or 125 μM). After preincubation, sperm were coincubated (2 × 10⁴ 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 μM: range of mean penetration values, 53.6%–84.3%), L-histidine (100 μM: range of mean values, 24.8%–56.3%) or L-cysteine (75 μM: 51.3%) in the absence of exogenous protein.     

Hamster zonae pellucidae cannot induce physiological acrosome reactions in chemically capacitated hamster spermatozoa in the absence of albumin

Cauda epididymal hamster spermatozoa were capacitated with D-penicillamine in a chemically defined (protein-free) medium (= "chemical" capacitation). Hamster zonae pellucidae were incapable of inducing functional acrosome reactions in chemically capacitated hamster sperm in a protein-free medium during sperm-egg coincubation. The culture medium used throughout incubation was a modified Tyrode's solution containing 10 mM sodium lactate, 100 microM sodium pyruvate and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm motility was maintained in all media with PHE (20 microM penicillamine, 100 microM hypotaurine, and 1.0 microM epinephrine). Additional D-penicillamine (125 or 500 microM) or 3 mg/ml bovine serum albumin (high control: TALP-PVA) was used to capacitate sperm during preincubation at 1-2 X 10(6) sperm/ml for 4.0 h at 37 degrees C in 5% CO2 in air. Sperm were then coincubated (2 X 10(4) sperm/ml) with cumulus-free hamster eggs in TALP-PVA or TLP-PVA +/- additional D-penicillamine (total: 500 or 125 microM) for 1.5 or 6.0 h. Percent egg penetration was used as the definitive index of sperm capacitation and functional acrosome reactions. Chemically capacitated sperm did not penetrate eggs (0.0 +/- 0.0%) in the absence of albumin during 1.5 h of sperm-egg coincubation. When sperm were chemically capacitated with 125 microM or 500 microM D-penicillamine, then coincubated with eggs for 6.0 h in the absence of albumin, only 18.8 +/- 28.6% and 23.7 +/- 29.7%, respectively, of eggs were penetrated. Significantly (p less than 0.05) more eggs (67.7 +/- 22.4%) were penetrated when coincubated with chemically capacitated sperm for 1.5 h in medium containing albumin. These results demonstrate that zonae pellucidae of hamster eggs require the presence of albumin to efficiently induce functional acrosome reactions in sperm that are chemically capacitated with D-penicillamine.     

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

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

The effects of taurine and hypotaurine on in vitro fertilization in the golden hamster

Taurine and hypotaurine were examined for their efficacy in replacing sperm motility factor (SMF), prepared from bovine adrenal cortex, for in vitro fertilization in the golden hamster. Combinations of these amino acids at concentrations of 0.001, 0.01, 0.1, and 1 mM together with 16 μM isoproterenol (a catecholamine β-agonist) were added to the sperm incubations. After three hours of sperm preincubation, oviductal eggs were added to the sperm suspensions and examined for penetration and stage of fertilization after three or five hours of culture. At 0.001 mM, neither taurine or hypotaurine was capable of maintaining motility of hamster sperm for four to 4½ hours or of inducing fertilization. With all other concentrations, both amino acids were found to maintain motility of sperm as well as SMF. Hypotaurine stimulated motility to a greater extent than taurine and both required isoproterenol for the greatest motility. A low proportion of cumulus-free ova were fertilized when sperm were preincubated with either amino acid alone over the range of 0.01 to 1 mM; however, over 80% fertilization was consistently obtained when isoproterenol was also present during sperm incubation. Proportions of ova fertilized with taurine or hypotaurine present during sperm preincubation were comparable to those achieved with SMF. The possibility that taurine or hypotaurine is the sperm motility factor is discussed. After three hours of sperm/egg incubation, a lag in the early events of fertilization was observed in experimental groups treated with one of the amino acids (0.01 mM) alone compared with groups treated with isoproterenol present. However, if sperm/egg incubation was extended from three to five hours, no increase in number of eggs penetrated was found. Therefore, the delay observed at three hours was considered a function of fewer numbers of capacitated sperm present in the absence of isoproterenol rather than of the need for an extended capacitation time.     

Evidence suggesting a role for cyclic nucleotides in acrosome reactions of hamster sperm in vitro

There have been conflicting reports concerning the involvement of cyclic nucleotides in sperm capacitation. We have examined the effects of micromolar concentrations of dibutyryl cyclic AMP (Bt2cAMP) and of the phosphodiesterase inhibitors SQ20009 and ICI63,197 on hamster sperm incubated under in vitro capacitating conditions. Washed hamster sperm were incubated in a capacitation media containing bovine serum albumin, and a protein-free "motility-factor" from bovine adrenal cortex. Incubation for 3.5 hours was followed by addition of one of the compounds (0.1-10 microM) or control buffer. At the time of addition and after 30-120 minutes further incubation, sperm were examined by phase contrast microscopy. The final motility was similar to the initial motility (50-70%) and the same in incubation of controls or experimental compounds. Bt2cAMP, SQ20009, and ICI63,197 at these concentrations stimulated acrosome reactions to a statistically significant extent (P less than 0.005) compared to controls. Activation was stimulated to a varying degree by all three experimental compounds. These results suggest a role for cyclic nucleotides in capacitation and the acrosome reaction of hamster sperm.     

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

Studies 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.     

Use of salt-stored zonae pellucidae for assessing rabbit sperm capacitation for in vitro fertilization

Zonae pellucidae of tubal and follicular oocytes were collected and prepared for salt storage. Cumulus and corona radiata cells were removed from oocytes with hyaluronidase and a small bore pipette. The oocytes (referred to as zonae since vitelli were rendered nonfunctional) were stored in a salt solution at 4°C. Using in utero capacitated sperm, the penetrability of zonae from tubal and follicular oocytes stored immediately after collection was compared to controls, i.e., in vitro development of tubal ova to the 4-cell stage within 24 hr. The penetration rates were 100% (8 penetrated/ 8 inseminated), 77.8% (7 penetrated/ 9 inseminated), and 100% (10 fertilized/10 inseminated), respectively, and these were not statistically different. The mean (x ) numbers of sperm able to penetrate the zonae, into the perivitelline space (PVS) for tubal (34.0) and follicular (1.1) oocytes were significantly different (P < 0.01). However, following maturational incubation before salt storage, zonae of tubal and follicular origin showed no significant differences in penetrability of in utero capacitated sperm when assessed by percent penetration, or mean numbers of sperm cells reaching the PVS: tubal zonae, 100% (15/15), and follicular zonae, 100% (18/18), and mean number of sperm in the PVS (x [tubal zonae] = 12.4, and x [follicular zonae] = 11.8). The penetrability of tubal zonae with and without maturational incubation was compared, and no significant differences in penetrability by in utero capacitated sperm were present when assessed by percent penetration nonmatured 92.6% (25/27) and matured 93.3% (28/30) and mean number of sperm in the PVS (x [nonmatured] = 3.33 and x [matured] = 2.41). In vitro capacitation of ejaculated rabbit sperm by serum treatment was assessed by the penetration of salt-stored zonae, zonae-free hamster oocytes (ZFHO), and in vitro fertilization of freshly collected tubal oocytes. None of 60 salt-stored zonae and none of 31 tubal oocytes were penetrated, and these values were significantly (P < 0.005) smaller than the 9 of 78 (12%) zona-free hamster ova that were penetrated by sperm cells from the same sample. In vitro capacitation of ejaculated rabbit sperm by washing and preincubation was assessed by the penetration of salt-stored zonae, zona-free hamster oocytes (ZFHO), and fertilization of freshly collected tubal oocytes. Seventy-six of 80 salt-stored zonae were penetrated, and this was significantly (P < 0.005) greater than the 67 of 87 tubal oocytes fertilized and 30 of 35 ZFHO penetrated, which were not significantly different. The salt-stored zonae were more readily penetrated by capacitated sperm when compared to tubal oocytes. However, the ZFHO are more penetrable than salt-stored zonae and tubal oocytes when incompletely capacitated sperm is used. A useful role for this approach in studies dealing with sperm fertilizing ability is anticipated.     

肝素处理山羊精子体外获能的研究

系统研究了作用浓度、时间和温度以及输卵管上皮细胞和卵丘细胞对肝素处理山羊精子体外获能后的精子活力、质膜完整性、顶体完整率、获能比例及受精和卵裂的影响,为改善山羊精子体外获能效果和研究获能机理提供了必要的数据。主要实验结果如下：1、在获能液中添加5、10、25、50和100μg/mL肝素处理45min时,添加50和100μg/mL肝素精子获能比率最高（分别为55%和56%）,但添加100μg/mL肝素处理后顶体完整率明显（P<0.05）低于对照组。说明山羊精子获能的最佳肝素浓度为50μg/mL。2、肝素作用时间（0, 10, 20, 30, 45, 60 和120 min）的延长,获能精子比例逐渐提高。其中,肝素处理45～120 min各组的获能精子比例差异不显著（P>0.05）,处理120 min组的精子活力和质膜完整率显著低于其它各组。说明50μg/mL肝素处理精子获能的最佳时间是45～60 min。3、在42℃和38.5℃下处理时,获能精子比例显著高于15℃和37℃,但42℃处理后精子活力和顶体完整率显著低于其它温度。因此,385℃为山羊精子获能的最佳温度。4、与输卵管上皮细胞共培养获能精子比例显著高于对照组和卵丘细胞组,但精子活力、质膜完整率和顶体完整率差异不显著。输卵管上皮组的受精率（91.3％）和卵裂率（72.2％）显著高于对照组（81.2％,65.0％）。说明与输卵管上皮细胞共培养能显著提高肝素处理山羊精子体外获能的效果。     

Na+/K+ATPase regulates sperm capacitation through a mechanism involving kinases and redistribution of its testis‐specific isoform

Incubation of bovine sperm with ouabain, an endogenous cardiac glycoside that inhibits both the ubiquitous (ATP1A1) and testis‐specific α4 (ATP1A4) isoforms of Na⁺/K⁺ATPase, induces tyrosine phosphorylation and capacitation. The objectives of this study were to investigate: (1) fertilizing ability of bovine sperm capacitated by incubating with ouabain; (2) involvement of ATP1A4 in this process; and (3) signaling mechanisms involved in the regulation of sperm capacitation induced by inhibition of Na⁺/K⁺ATPase activity. Fresh sperm capacitated by incubating with ouabain (inhibits both ATP1A1 and ATP1A4) or with anti‐ATP1A4 immunoserum fertilized bovine oocytes in vitro. Capacitation was associated with relocalization of ATP1A4 from the entire sperm head to the post‐acrosomal region. To investigate signaling mechanisms involved in oubain‐induced regulation of sperm capacitation, sperm preparations were pre‐incubated with inhibitors of specific signaling molecules, followed by incubation with ouabain. The phosphotyrosine content of sperm preparations was determined by immunoblotting, and capacitation status of these sperm preparations were evaluated through an acrosome reaction assay. We inferred that Na⁺/K⁺ATPase was involved in the regulation of tyrosine phosphorylation in sperm proteins through receptor tyrosine kinase, nonreceptor type protein kinase, and protein kinases A and C. In conclusion, inhibition of Na⁺/K⁺ATPase induced tyrosine phosphorylation and capacitation through multiple signal transduction pathways, imparting fertilizing ability in bovine sperm. To our knowledge, this is the first report documenting both the involvement of ATP1A4 in the regulation of bovine sperm capacitation and that fresh bovine sperm capacitated by the inhibition of Na⁺/K⁺ATPase can fertilize oocytes in vitro. Mol. Reprod. Dev. 77: 136–148, 2010. © 2009 Wiley‐Liss, Inc.     

Dynamic quantification of intracellular calcium and protein tyrosine phosphorylation in cryopreserved boar spermatozoa during short-time incubation with oviductal fluid

Freshly ejaculated boar spermatozoa require several hours of exposure to capacitating conditions to undergo capacitation. We hypothesized that cryopreserved boar spermatozoa might elicit a capacitation response after a relatively shorter time of exposure to capacitating conditions. Washed, frozen-thawed boar spermatozoa were incubated separately with pre-ovulatory isthmic oviductal fluid (EODF), post-ovulatory ODF (MODF), capacitation medium (CM), and noncapacitating medium (NCM) for 60 minutes. Aliquots of spermatozoa were taken at 0, 5, 15, 30, and 60 minutes during incubation and sperm kinematics, intracellular calcium [Ca2⁺]i content, and protein tyrosine phosphorylation (PTP) were studied. The proportion of motile spermatozoa increased significantly after 5 minutes of incubation with EODF. A similar increase was not observed in the other groups. During the initial 5 minutes of incubation, the proportion of spermatozoa with high [Ca²⁺]i decreased significantly in all four groups. The proportion of tyrosine phosphorylated spermatozoa increased from 6.49 ± 1.93% to 15.42 ± 3.58% and 18.41 ± 1.57% in EODF and MODF groups, respectively, at 5 minutes of incubation. Neither CM nor NCM elicited any immediate effect on PTP in spermatozoa. There was a positive and significant correlation between [Ca²⁺]i and sperm motility (P = 0.009). It may be concluded that frozen-thawed boar spermatozoa undergo capacitation-associated changes after a relatively short exposure to EODF, and there are some subpopulations of spermatozoa that undergo PTP despite possessing low [Ca²⁺]i.     

Detection of a soluble acrosome reaction-inducing factor, different from serum albumin, associated with the ovulated egg-cumulus complex.

Soluble extracts of the ovulated hamster egg-cumulus complex (ECC) were tested on capacitated sperm for activity in inducing the physiological acrosome reaction (AR). Evidence for occurrence of the physiological AR included enhanced sperm penetration of intact homologous zonae pellucidae as well as induction of AR in nonattached and in zona-bound sperm following a brief coincubation with test compound. Since hamster serum albumin, a major protein of hamster body fluids, also induces spontaneous ARs under certain conditions, it was used as one of the comparators for the acrosome reaction inducing factor (ARIF; Westrick et al., Biol Reprod 32 [Suppl 1]. 213, 1985) activity in the ECC. Sperm exposure to concentrations of the soluble ECC extract ranging from 0.04 to 0.2 mg protein/ml significantly increased penetration of salt-stored zonae by 36%, mean numbers of penetrating sperm by 90%, ARs in nonattached sperm by 65%, and ARs in zona-bound sperm by 102%. Hamster serum albumin added after completion of capacitation had no significant effect on these parameters. We conclude that 1) the ovulated ECC contains a soluble ARIF that augments zona-induced ARs and sperm penetration and 2) the ARIF is not serum albumin.     

Tyrosine phosphoproteome of hamster spermatozoa: Role of glycerol‐3‐phosphate dehydrogenase 2 in sperm capacitation

Capacitation confers on the spermatozoa the competence to fertilize the oocyte. At the molecular level, a cyclic adenosine monophosphate (cAMP) dependent protein tyrosine phosphorylation pathway operates in capacitated spermatozoa, thus resulting in tyrosine phosphorylation of specific proteins. Identification of these tyrosine‐phosphorylated proteins and their function with respect to hyperactivation and acrosome reaction, would unravel the molecular basis of capacitation. With this in view, 21 phosphotyrosine proteins have been identified in capacitated hamster spermatozoa out of which 11 did not identify with any known sperm protein. So, in the present study attempts have been made to ascertain the role of one of these eleven proteins namely glycerol‐3‐phosphate dehydrogenase 2 (GPD2) in hamster sperm capacitation. GPD2 is phosphorylated only in capacitated hamster spermatozoa and is noncanonically localized in the acrosome and principal piece in human, mouse, rat, and hamster spermatozoa, though in somatic cells it is localized in the mitochondria. This noncanonical localization may imply a role of GPD2 in acrosome reaction and hyperactivation. Further, enzymatic activity of GPD2 during capacitation correlates positively with hyperactivation and acrosome reaction thus demonstrating that GPD2 may be required for sperm capacitation.     

Capacitation potential of the fallopian tube: A study involving surgical insemination and the subsequent incidence of polyspermy

In an attempt to demonstrate limitations in the capacitating potential of the Fallopian tube, ejaculated boar spermatozoa were introduced directly into the isthmus at varying intervals before ovulation. The incidence and degree of polyspermy subsequently observed were taken as indicators of the population of capacitated spermatozoa confronting the newly ovulated eggs: the more extensive the condition of polyspermy, the greater the number of capacitated spermatozoa presumed to have been available at the site of fertilization. Results are based on 673 eggs from 53 animals. When suspensions containing 2.21–3.87 × 10⁸ sperm per ml were introduced 36–40 hours and 26–30 hours before ovulation, 85% and 61% respectively of the eggs were polyspermic, such eggs exhibiting mainly dispermy and trispermy. By contrast, when comparable sperm suspensions from the same boar were instilled 17–18 hours before ovulation, 70% of the eggs were polyspermic but the degree of polyspermy had increased dramatically: most eggs contained 40 or more sperm heads in the vitellus, invariably forming swollen chromatin aggregates rather than male pronuclei. Surgical insemination at times closer to ovulation significantly reduced the incidence and degree of polyspermy, reaching a low of 2% with insemination 1–2 hours before ovulation. These results therefore support the concept of a limited capacitation potential of the Fallopian tube. In a separate series of observations, mating animals shortly before surgical insemination with sperm suspensions from the same boar markedly reduced the incidence of polyspermy. This latter observation may be of clinical significance in procedures of laparoscopic or transcervical insemination into the tubes to alleviate human infertility. The manner whereby myosalpingeal physiology could be modified in response to coital stimulation is discussed.     

In vitro penetration of hamster oocyte-cumulus complexes using physiological numbers of sperm

We have compared the ability of uncapacitated, capacitated acrosome intact, and acrosome-reacted hamster sperm to penetrate the cumulus and corona radiata of fresh hamster oocyte-cumulus complexes (OCC) in vitro. This was done using physiological numbers (1-20) of sperm so that cumulus and corona radiata cells did not disperse during challenge. Uncapacitated sperm did not penetrate to the zona pellucida surface; most (74%) uncapacitated sperm bound to cumulus cells at the periphery of the OCC. Capacitated acrosome-intact sperm penetrated to the zona pellucida surface; a significant percentage of these sperm arrived at the zona pellucida without showing evidence of initiating an acrosome reaction. Most capacitated acrosome-reacted sperm did not enter the extracellular matrix between cumulus and corona radiata cells; those which did penetrated to the zona surface with difficulty, if at all. These results suggest that the changes which occur in the sperm surface during capacitation are more important than the acrosome reaction in enabling hamster sperm to penetrate the cumulus and corona radiata. The effects of gold sodium thiomalate (GST) and polyphloretin phosphate (PPP) (inhibitors of hyaluronidase) on penetration of the OCC by capacitated sperm were also examined. Both synthetic inhibitors blocked sperm penetration to the zona pellucida, but the effective concentrations of inhibitors were far in excess of what was needed to block hyaluronidase activity. Reasons for concluding that the action of these inhibitors is nonspecific are discussed. These data show that hamster sperm with intact acrosomes can penetrate the cumulus and corona radiata cell layers of fresh OCC in vitro and support the hypothesis that the acrosome reaction occurs on the zona pellucida surface.     

Comparison of the ability of progesterone and heat solubilized porcine zona pellucida to initiate the porcine sperm acrosome reaction in vitro

It has been previously shown that progesterone can initiate the acrosome reaction (AR) of capacitated human and hamster sperm in vivo. We report here that progesterone can initiate a morphologically normal AR in porcine sperm that have undergone capacitation in a Hepes-buffered medium in vitro. In addition, we have compared the abilities of progesterone and heat-solubilized porcine zona pellucida (zona) to initiate the porcine sperm AR. Capacitated porcine sperm were treated with 1 m?g/ml progesterone, 150 m?g/ml porcine zona, or solvent control for 10 min. After treatment, sperm were incubated with the supravital dye Hoechst 33258, fixed and the acrosomal status determined in the previously viable sperm by fluorescence microscopy with fluorescein isothiocyanate-labeled Pisum sativum agglutini (FITC-PSA). There was no significant difference between the percentage of AR initiated by zona compared to that initiated by progesterone. In order to determine whether there was a synergistic interaction between the two AR initiators, both were added simultaneously to capacitated porcine sperm at optimal (1 m?g/ml progesterone, 150 m?g/ml zona) and suboptimal (75 ng/ml progesterone and 75 m?g/ml zona) concentrations. Simultaneous addition of the two AR-initiators at the two concentrations stimulated an additive AR-initating response, rather than a synergistic one. Several possible explanations for the additive results are discussed. © 1994 Wiley-Liss, Inc.     

Fusion of hamster and pig zona-free eggs stimulated by boar and guinea pig sperm at fertilization in vitro

In the course of in vitro fertilization of zona-free hamster and pig eggs by boar and guinea-pig spermatozoa it was observed that homologous and heterologous eggs fused together, forming cell hybrids between two or more cells. The fusogenic activity was attributed to spermatozoa and this was the hypothesis tested. The fusogenic activity (coinciding with sperm penetration activity) was dependent on the duration of sperm preincubation, which may be regarded as capacitation in vitro. Fusion occurred only after 3 hr of sperm preincubation and a narrow optimum was detected at 4–4.5 hr. Fusion of eggs was also dependent on sperm concentration. A relatively high proportion of fusions was observed at a sperm concentration of 4.0 × 10⁴ per ml and an optimum was attained at a concentration of 5.0 × 10⁵ per ml. The first fusions were observed at 90 min after semination. After 3 hr more than a half of the eggs reacted, and by 20 hr of incubation 80% of ova were fused. The fusability of eggs was tested and found to occur at 14 hr after ovulation. The fusion process was also studied using transmission electron microscopy. It is supposed that the process of egg fusion may be caused either by a similar mechanism to sperm-egg fusion, or by products released during the sperm acrosome reaction.     

Direct contact is required between serum albumin and hamster spermatozoa for capacitation in vitro

A dialysis unit was used to test whether direct physical contact between serum albumin and hamster spermatozoa is required for capacitation and/or the acrosome reaction. Sperm and bovine serum albumin (BSA) were incubated cither together (direct incubation) or separated by a dialysis membrane (indirect incubation). Sperm viability was supported with “sperm motility factors” (hypotaurine and epinephrine) and polyvinylalcohol (PVA). Spermatozoa became capacitated and underwent acrosome reactions when directly incubated in medium containing BSA (TALP-PVA), but did not undergo acrosome reactions when indirectly incubated with BSA (medium TLP-PVA). When sperm were first incubated for 4 hr indirectly with BSA, followed by 4 hr direct incubation with BSA, capacitation did not occur during indirect incubation. These findings indicate that an “intimate association” is necessary between serum albumin and spermatozoa to support capacitation under in vitro incubation conditions. The data are consistent with the concept of direct transfer of compounds from sperm to albumin and/or vice versa during sperm capacitation.     

Changes in the distribution of intramembranous particles and filipin-reactive membrane sterols during in vitro capacitation of golden hamster spermatozoa

Membrane alterations accompanying in vitro capacitation of hamster spermatozoa were examined using the freeze-fracture technique with or without use of filipin, a sterol-binding probe. In the spermatozoa prior to or at 10 min after start of incubation in capacitating medium, large (about 11 nm) and small (8–9 nm) intramembranous particles (IMPs) were present in the periacrosomal region of the sperm plasma membrane (PAPM). Filipin sterol complexes (FSCs) were densely (about 500/μ²) distributed in the PAPM prior to incubation. The density of FSCs in the PAPM was reduced by 70–80% of the original density by 2 hr of incubation. At the same time, small patches of IMP-free areas appeared in the plasma membrane above the equatorial and middle segments of the acrosome. By the end of 3 hr of incubation, the majority of small IMPs had disappeared from the PAPM. Remaining large and small IMPs tended to aggregate in the PAPM. During incubation in capacitation medium, “cords,” or linear arrangements of closely packed IMPs, appeared near the posterior ring of the sperm head. These observations strongly suggest that the acrosome reaction of the hamster spermatozoa is preceded by the removal (deletion) of filipin-reactive sterols (FRSs) and the disappearance of small IMPs from the lipid bilayer of PAPM.     

Medium effects on capacitation and sperm penetration through the zona pellucida in inbred BALB/c spermatozoa

Inbred BALB/c mice are one of the most difficult inbred strains to fertilize in vitro. In this study we examined the abilities of various media used for mouse in vitro fertilization (IVF) to support capacitation and sperm penetration through the zona pellucida (ZP) of inbred BALB/c spermatozoa. Media examined were TYH, M16, CZB, mWhitten medium, T6, modified Tyrode's solution (mTyrode's), mKSOM, MEM and TCM199. Modified human tubal fluid (mHTF) was used as a control medium. When sperm were capacitated and inseminated in the same medium, mHTF showed the best fertilization (approximately 80%) scored by male pronuclear formation (<26%) at 5h post-insemination (PI). When sperm were capacitated in various media and inseminated in mHTF, sperm capacitated in TYH solution (93%) but no other media (<45%) showed a significantly higher level of sperm nuclear decondensation (SND) than mHTF at 2 h PI (approximately 65%). When sperm were capacitated in mHTF and inseminated in various media, only mTyrode's (52%) was not significantly lower than mHTF (66%) in terms of SND at 2h PI (<49%). Sperm capacitation also was examined by chlortetracycline (CTC) staining. Sperm capacitated in TYH solution showed a significantly higher percentage of capacitation (46%) than those treated in HTF (28%) and other media (<24%). These results indicate that the best approach for IVF in the BALB/c strain is capacitation in TYH and insemination in mHTF. Poor fertilization of BALB/c may result from suboptimal conditions of sperm capacitation and insemination, and overall IVF success may differ depending on strains used.     

In vitro capacitation of boar ejaculated spermatozoa: Effect of conditioned media prepared from preincubated sperm suspension

The fertilizing ability of boar ejaculated spermatozoa was examined in vitro after prcincubation at a concentration of 2.5 × 10⁸/ml for 4 hr in several conditioned media (CM). For preparation of CM, boar spermatozoa were incubated in a modified Krebs-Ringer bicarbonate solution (TYH) at concentrations of 20 to 40 × 10⁸/ml for several hours up to 4 hr; then their supernatant fluids were collected by centrifugation. When boar ejaculated spermatozoa were preincubated in TYH alone, 14.1% of oocytes were penetrated by them as we reported previously. On the other hand, preincubating them with CM, their fertilizing ability was elevated according as the incubation time of CM preparation was lengthened. The fertilization rate reached 75.0%, using 4 hr-incubated CM for the preincubation medium. The effect of CM was not deteriorated by heat treatments (56°C, 30 min, or 100°C, 5 min). The components of CM were separated at a molecular weight of 25,000 by ultrafiltration, and high fertilization rate (69.8%) was obtained when low molecular weight fraction was used for the preincubation medium. Sperm extracts prepared from directly frozen-thawed sperm suspension and 0.1–10 mM of taurine or hypotaurine had no effect on the fertilizing ability of boar spermatozoa. These results suggest that substances stimulating boar sperm capacitation were accumulated from viable spermatozoa into the medium during incubation and that the effective substances were heat-stable and of low molecular weight and were not taurine and hypotaurine.