Application of a zona pellucida binding assay (ZBA) in the domestic cat benefits from the use of in vitro matured oocytes

Background

Zona pellucida binding assays (ZBAs) have proven useful in determining the fertilising ability of spermatozoa in several species. Most ZBAs use fresh or salt-stored oocytes collected from fresh ovaries but because ovaries are not easy to obtain on a regular basis, chilled and frozen-thawed ovaries have been tested, with varying results. The present study tested the hypothesis that cat spermatozoa, either fresh or frozen-thawed, can bind to homologous zona pellucida of oocytes retrieved from frozen-thawed queen ovaries to a similar extent as they can bind to the zona pellucida of fresh, in vitro matured oocytes.

Methods

Ovaries were collected from queens after routine ovario-hysterectomy and either stored in NaCl at -20°C until use (treatment animals), or used fresh (controls). Cumulus-oocyte complexes (COCs) were retrieved by ovarian slicing from either source and used directly (immature oocytes from frozen-thawed ovaries; treatment animals) or after in vitro maturation (IVM) (fresh ovaries; controls) for 24 hours in TCM 199, supplemented with 1 IU hCG/mL and 0.5 IU eCG/mL and 0.5% bovine serum albumin (BSA). The oocytes were incubated for 4 hours in 5% CO₂ in air at 38°C and 100% humidity in the presence of 5 × 10⁶ fresh or frozen-thawed spermatozoa/mL. Representative samples of oocytes were processed for scanning electron microscopy (SEM).

Results

Both fresh and frozen-thawed spermatozoa bound to the in vitro matured zona pellucida but significantly fewer, or no, spermatozoa bound to frozen-thawed, immature zona pellucida (P < 0.001). Also, more fresh spermatozoa than frozen-thawed spermatozoa bound to the zona pellucida (P < 0.001). The zona pellucida surface differed in morphology (SEM), with in vitro matured oocytes showing a dense surface with few fenestrations in contrast to their frozen-thawed, immature counterparts, where fenestrations were conspicuously larger.

Conclusion

In conclusion, under the conditions of the present study, immature oocytes recovered from ovaries frozen immersed in NaCl at -20°C are less suitable for use in feline ZBA.     

Ultrastructural observations on gamete interactions using micromanipulated mouse oocytes

Cumulus-free mouse oocytes were subjected to zona opening by cracking with microhooks (ZC) or acid drilling (ZD) and fixed 30–90 min after insemination (10⁵ pre-capacitated motile sperms/ml). Ultrastructural observations were made on serially thin-sectioned oocytes: 15 ZC and 12 ZD. The zona lesion in ZC oocytes was a clean cut, whereas in ZD oocytes it formed a patchy area of partial zona loss, with reduced microvillar height on the underlying oocyte surface. Spermatozoa were observed within the perivitelline space and partially fusing with the oocyte after 30 min in both situations. Only acrosome-reacted sperm heads were observed to fuse: acrosome intact forms were generally in contact with the zona pellucida, either with the inner or outer surface. Acrosome-intact spermatozoa were also observed deeply embedded in the zona matrix, possibly indicating surface enzyme activity preceding the membrane fusion events of the acrosome reaction proper. The observations are consistent with the need for spermatozoa to make contact preferentially with the zona pellucida during the course of the acrosome reaction.     

The site of the acrosome reaction during in vivo penetration of the sheep oocyte

In vivo fertilization of sheep eggs has been studied by electron microscopy. Remnants of the acrosome reaction were present at the zona surface of every penetrated egg, indicating that the acrosome reaction in sheep occurs at the surface of the zona pellucida. To determine whether follicular oocytes could specifically bind spermatozoa, oocytes isolated from different size classes of antral follicles were transferred into the oviducts of mated ewes, recovered 4 hr 30 min later, and analyzed by electron microscopy. Oocytes from follicles up to 1 mm in diameter failed to bind spermatozoa and were not penetrated. In contrast, the zona of oocytes from follicles ? 2 mm in diameter induced the acrosome reaction. These oocytes were penetrated but failed to achieve cortical granule exocytosis and so to mount a block to polyspermy. Moreover, sperm nuclei incorporated into the ooplasm did not decondense although the sperm nuclear envelope was dispersed.     

In vitro fertilizing characteristics of bovine spermatozoa with multiple nuclear vacuoles: A case study

A study was designed to determine the in vitro fertilizing characteristics of bovine semen with a high percentage of spermatozoa with multiple nuclear vacuoles. In Experiment 1, a total of 620 oocytes was divided into 2 groups and inseminated with spermatozoa from 1 of 2 different bulls at a concentration of 2 × 10⁵/ml. After Percoll washes, 73.5 ± 3.0% of spermatozoa from Bull A contained multiple nuclear vacuoles, while no sperm cells from Bull B contained vacuoles. After 19.5 ± 0.5 h of co-incubation of oocytes with spermatozoa, loosely attached sperm cells were removed by washing, and the oocytes were fixed between 2 poly-l-lysine coated glass slides. Mean (±SD) percentage of fertilization was significantly lower (P < 0.05) in Bull A (19.7 ± 7.0%) than in Bull B (67.6 ± 4.5%). In one-third of the oocytes fertilized by spermatozoa from Bull A, sperm head decondensation was incomplete and normal male pronucleus formation did not occur. All oocytes fertilized by Bull B had normally decondensed sperm heads. Although fewer (P < 0.05) spermatozoa from Bull A were bound to the zona pellucida than from Bull B, the percentage of vacuolated sperm cells bound to the zona pellucida (73.3 ± 7.8%) did not differ from that in the inseminate. The mean number of sperm cells binding to fertilized oocytes was higher than to unfertilized oocytes for both bulls (P < 0.05). In Experiment 2, 748 salt-stored oocytes (zonae) were inseminated with semen from the same 2 bulls to determine the ability of spermatozoa to penetrate the zona pellucida. The percentage of zonae penetrated by spermatozoa from Bull A (69.9 ± 3.5%; a mean of 2.4 ± 2.3 spermatozoa) was lower (P < 0.05) than from Bull B (96.5 ± 14.7%; a mean of 11.3 ± 9.9). Although the proportion of vacuolated sperm cells from Bull A that bound to the zona pellucida did not differ from that in the inseminate, the proportion of those penetrating the zona pellucida (52.7%) was lower (P < 0.05). In summary, vacuolated sperm cells apparently gained access to the oocyte and bound to the zona pellucida, but they penetrated the zona pellucida at a lower rate and apparently did not form normal male pronuclei.     

Evidences for the presence of chymotrypsin-like activity in human spermatozoa with a role in the acrosome reaction

The effect of chymotrypsin inhibitors and substrates on the human sperm acrosome reaction stimulated by the human zonae pellucidae or follicular fluid were evaluated. Motile spermatozoa, selected by a Percoll gradient, were incubated at 1 × 10⁷ cells/ml, 37°C, and 5% CO₂, After 4.5 hr, the chymotrypsin inhibitor TPCK (N-Tosyl-L-Phenylalanine-Chloromethyl Ketone) or the substrate ATEE (N-Acetyl-L-Tyrosine Ethyl Ester) were added for 30 min. Then, four oocytes were added and the percentage of acrosome-reacted spermatozoa on the zona was determined. TPCK and ATEE inhibited the zona pellucida-induced acrosome reaction. The chymotrypsin inhibitors TPCK and chymostatin and the chymotrypsin substrates ATEE, BTEE (N-Benzoyl-L-Tyrosine Ethyl Ester), Succinyl-Ala-Ala-Phe-7-Amido-4-Methyl-Coumarin (Suc-Ala-Ala-Phe-AMC), and Succinyl-Leu-Leu-Val-Tyr-7-Amido-4-Methyl-Coumarin (Suc-Leu-Leu-Val-Tyr-AMC) inhibited the human follicular fluid-induced acrosome reaction. Sperm extracts exhibited hydrolytic activity toward Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. This enzyme activity was abolished by TPCK and chymostatin, was independent of Ca²⁺, and was not modified by 1,10 phenanthroline. In addition, the activity was present in the supernatant after the acrosome reaction was induced with calcium ionophore and in epididymal spermatozoa recovered from the cauda region. Electron microscopic observations indicated that the inhibitors prevented the membrane events of the acrosome reaction. These data suggest an association between human spermatozoa and chymotrypsin-like activity with a possible role in the acrosome reaction. © 1994 Wiley-Liss, Inc.     

Sperm protein (sp50) binds to acromosome and plasma membranes in a Ca2+-dependent manner: Possible role in acrosome reaction

Annexins are a family of Ca²⁺-binding proteins involved in the exocytotic process. The presence and the role of annexins in mammalian spermatozoa have not been well established. Two annexin-like proteins were obtained from guinea pig testis, a doublet of Mr 31–33 kD (p31/33) and a protein of Mr 50 kD (p50). Both proteins were able to bind to erythrocyte ghosts in a Ca²⁺-dependent fashion. Polyclonal antibodies against p31/33 reacted with two major proteins, Mrs 50 kD (sp50) and 42 kD (sp42), from mature and immature guinea pig spermatozoa. p50 and sp50 are likely the native proteins from testis and spermatozoa, respectively, and they are seemingly related. By immunofluorescence, sp50 was only found in the apical acrosome region of immature and capacitated and noncapacitated spermatozoa, and its location was intracellular. In spermatozoa undergoing acrosome reaction, sp50 was detected in the whole acrosome, while in spermatozoa that had undergone acrosome reaction sp50 was not detected. However, in the protein pattern of acrosome reaction vesicles, anti-p31/33 antibody revealed diffuse bands of Mr 35–38 kD. sp50 was able to bind to plasma membrane fragments and acrosome outer membrane from demembranated sperm in a Ca²⁺-dependent fashion. The presence of sp50 in the acrosome region, its distribution throughout the acrosome membrane just before the acrosome reaction, and its ability to bind both plasma and outer acrosome membranes in a Ca²⁺-dependent manner suggest that sp50 may participate in the acrosome reaction mechanism in guinea pig spermatozoa. © 1996 Wiley-Liss, Inc.     

SOB3, a human sperm protein involved in zona pellucida binding: Physiological and biochemical analysis,purification

LB5 antibody was selected from a monoclonal antibody (mAb) library directed against human sperm proteins. LB5 mAb detected the corresponding protein SOB3 in the neck region and the flagellum of most live ejaculated sperm while it labelled, in addition, the acrosome of about 10–20% of spermatozoa. The percentage of LB5 acrosome-stained sperm was significantly correlated with the percentages of either spontaneous or A23187-induced acrosome-reacted sperm. While SOB3 could not be detected in the testis, it appeared in spermatozoa from the corpus epididymis segment. LB5 mAb impaired neither sperm motion parameters, acrosomal reaction triggering, nor sperm binding to zona-free hamster oocytes. By contrast, LB5 Fab fragments (200 μg/ml) inhibited sperm binding to human zonae pellicidae by 35.7%. If sperm were induced to acrosome react with A23187 prior to LB5 treatment, the inhibitory effect shifted to 59.9%, while no significant effect was observed following A23187 incubation alone. Western blotting of human sperm and cauda epididymis extracts revealed two bands of 18 and 19 kDa. While no cross-reaction was observed with other tested organs, a similar 18-kDa band was revealed in erythocytes and one of 19 kDa in B-lymphocytes. No cross-reactivity could be evidenced in any animal sperm analyzed. SOB3 was first separated in a 17- to 20-kDa preparative electrophoresis fraction and finally purified by isoelectrofocusing according to its pI of 9.8. These results suggest that SOB3 is localized under the outer acrosomal membrane, that it participates in secondary sperm binding to the zona pellucida, and that it shares homologies with the immune system. Mol. Reprod. Dev. 49:286–297, 1998. © 1998 Wiley-Liss, Inc.     

Fertilization characteristics and in vitro embryo production with bovine sperm containing multiple nuclear vacuoles

An in vitro fertilization and culture system was used to determine the effect of multiple nuclear vacuoles in bovine spermatozoa on fertilization and early embryonic development. After swim-up, semen parameters were similar between 2 bulls except that 60% of spermatozoa from bull A contained multiple nuclear vacuoles, whereas no spermatozoa from bull B (control) contained vacuoles. In Experiment 1, in vitro–matured (IVM) oocytes were inseminated with frozen-thawed semen from the 2 bulls to determine the ability of vacuolated sperm to bind with the zona pellucida. The mean number of spermatozoa bound to the zona pellucida was less (P< 0.05) for bull A (85.7 ± 5.7; n = 112) than for bull B (108.9 ± 5.4; n = 130). In Experiment 2, the percentages of zonae penetrated by spermatozoa from bull A (151 of 201; 75%) and bull B (116 of 150; 77%) were not different. However, the percentages of vacuolated spermatozoa from bull A bound to (43%) and penetrating the zona pellucida (34%) were lower than those in the inseminate (60%). In Experiment 3, fertilization rates, as evidenced by the presence of two pronuclei, were not different for bull A (101 of 136; 74%) and bull B (89 of 115; 77%). In Experiment 4, there was no significant difference in percentage cleavage (72.1% versus 76%) and morulae (29.2% versus 34.8%) or blastocyst production (7.2% versus 8.4 %) for bulls A and B, respectively. Data suggest that spermatozoa with multiple nuclear vacuoles are defective in zona binding. However, vacuolated spermatozoa gaining access to the ooplasm apparantly participate in fertilization and early embryonic development. Mol. Reprod. Dev. 50:328–333, 1998. © 1998 Wiley-Liss, Inc.     

Effect of oocyte-sperm co-incubation on acrosome reaction in the goat

The induction of acrosome reaction of goat spermatozoa was investigated. The acrosomal status of spermatozoa was determined by a triple-staining technique. The effect of the presence of goat oocytes on the proportion of acrosome-reacted spermatozoa was also determined. Ovulated oocytes were obtained from superstimulated adult goats. Other sources of oocytes were adult and prepubertal goats; oocytes from both sources were maturated in vitro. There was an increase in the percentage of acrosome-reacted spermatozoa from 4% +/- 0.98 to 9% +/- 1.41 when oocytes from adult females were used. Similar induction rates were measured with prepubertal and adult oocytes maturated in vitro (10.4% +/- 2.06 and 8.75% +/- 1.06, respectively). The influence of several qualities of cumulus oophorus as well as the presence of zona pellucida was also investigated. No significant differences were obtained with any cumulus oophorus or zona pellucida oocyte complexes. Although oocyte quality is important for high fertilization rates, it does not seem to be crucial for the induction of acrosome reaction.     

Ultrastructural localization of labeled acrosomal glycoproteins during in vivo fertilization in the rabbit

Rabbit spermatozoa were labeled predominantely in their acrosomal glycoproteins by 1-³H-glucosamine during spermiogenesis. Ova fertilized in vivo by spermatozoa labeled 22 days earlier were analyzed by fine-structure autoradiography for the localization of the label. The latter was found associated with 1) the fused membranes of the acrosomal cap remaining on the zona pellucida surface, 2) the material released on the zona surface after the acrosome reaction and possibly detectable after tannic acid fixation, 3) the equatorial segment of the sperm head and the preequatorial swellings, and 4) other sperm components, eg, the sperm tail. No labeling, on the other hand, was detected on the denuded leading edge of spermatozoa found either in the penetration slit or in the perivitelline space. Our observations suggest the involvement of acrosomal glycoproteins in different mechanisms of sperm/zona pellucida interaction but are not in favor of a major role of (enzymatic) glycoproteins bound to the inner acrosomal membrane during the penetration of the zona pellucida.     

Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro

The question of whether the acrosome reaction, which leads to fertilization, occurs in intact sperm bound to the zona pellucida of the egg or in intact sperm before contact with the egg, was addressed by assessing the effect of 3-quinuclidinyl benzilate (QNB) on the two types of acrosome reaction. QNB is a specific inhibitor of the fertilization of zona-intact mouse eggs by mouse sperm. Mouse spermatozoa in suspension underwent acrosome reactions at a low rate, which could be accelerated by addition of 5 μM divalent cation ionophore A23187; the occurrence of such acrosome reactions was not inhibited by QNB. The rate at which acrosome reactions occurred in sperm bound to the zona pellucida of cumulus-free eggs, bound to isolated zonae, or exposed to acid-solubilized zona components, was greatly accelerated relative to that observed in the absence of zonae. These acrosome reactions were strongly inhibited by QNB at concentrations which inhibit the fertilization of zona-intact mouse eggs in vitro. These data suggest that the zona pellucida can induce acrosome reactions in mouse spermatozoa and that these acrosome reactions are the ones which lead to the fertilization of zona-intact eggs. In contrast, the acrosome rection in sperm which are not in contact with the zona is not associated with fertilization of zona-intact eggs.     

Oviduct binding ability of porcine spermatozoa develops in the epididymis and can be advanced by incubation with caudal fluid

The sperm reservoir is formed when spermatozoa bind to the epithelium of the uterotubal junction and caudal isthmus of the oviduct. It is an important mechanism that helps synchronize the meeting of gametes by regulating untimely capacitation and polyspermic fertilization. This study investigated the influence of epididymal maturation and caudal fluid on the ability of spermatozoa to bind to oviduct epithelium using a model porcine oviduct explant assay. Spermatozoa from the rete testis, middle caput (E2-E3), middle corpus (E6), and cauda (E8) of Large White or Large White × Landrace boars aged 10 to 14 months were diluted in modified Androhep solution and incubated with porcine oviduct explants. Results reported in this study support our hypothesis that testicular spermatozoa need to pass through the regions of the epididymis to acquire the ability to bind to the oviduct. There was a sequential increase in the number of spermatozoa that bound to oviduct explants from the rete testis to caudal epididymis. Binding of caudal spermatozoa to isthmic explants was the highest (15.0 ± 1.2 spermatozoa per 1.25 mm², mean ± standard error of the mean; P ≤ 0.05) and lowest by spermatozoa from the rete testis (2.0 ± 0.3 per 1.25 mm²), and higher to isthmus from sows compared to gilts (35.8 ± 6.7 per 1.25 mm² vs. 14.8 ± 3.0 per 1.25 mm²; P ≤ 0.05). Binding of ejaculated spermatozoa to porcine isthmus was higher than that for caudal spermatozoa (26.3 ± 1.4 per 1.25 mm² vs. 15.0 ± 0.8 per 1.25 mm²; P ≤ 0.05) and higher to porcine than to bovine isthmus (26.3 ± 2.3 per 1.25 mm² vs. 18.8 ± 1.9 per 1.25 mm²; P ≤ 0.05). Incubation of spermatozoa from the caput and corpus in caudal fluid increased the ability of spermatozoa to bind to the oviduct epithelium (P ≤ 0.05). In conclusion, the capacity of testicular spermatozoa to bind to the oviduct epithelium increases during their maturation in the epididymis and can be advanced by components of the caudal fluid.     

Atrial natriuretic peptide induces an acrosome reaction in giant panda spermatozoa and enhances their penetration of salt-stored porcine oocytes

Atrial natriuretic peptide (ANP) is a vasodilator peptide primarily produced in the heart. Locally synthesized ANP has been found in reproductive tissues of various mammals and humans, and plays an important role in rat oocyte maturation and human sperm function. The objective of the present study was to determine the effects of ANP on the function (acrosome reaction and zona penetration) of giant panda spermatozoa. In fresh and frozen-thawed spermatozoa that had been preincubated for 2.5h, treatment with ANP (for 60 min) significantly increased the proportion of acrosome-reacted spermatozoa; maximal response (an acrosome reaction in 18.3 and 21.8% of fresh and frozen-thawed spermatozoa, respectively) was detected at 1 nM ANP. Treatment with C-ANP-(4-23), an analogue of ANP and specific binder to natriuretic peptide receptors-C (NPRC), had no significant effect on the acrosome reaction. However, the cyclic guanosine 5'-monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor KT5823 completely abolished the effect of ANP on acrosome reaction. The effects of ANP, caffeine and heparin on frozen-thawed sperm function were studied by insemination of porcine salt-stored oocytes in a modified Tris-buffered medium (mTBM). The presence of ANP, caffeine or heparin in the insemination medium resulted in a higher proportion (P < 0.05) of oocytes with spermatozoa in the zona and perivitelline space (PVS), and a higher average number of spermatozoa/oocyte (P < 0.05) in the zona and PVS. However, in the absence of ANP, caffeine and heparin, there were no oocytes with a spermatozoon in the PVS. There were no differences among ANP, caffeine or heparin treatments for the proportion of oocytes penetrated or average number of spermatozoa/oocyte in the zona and PVS. In conclusion, we inferred that ANP induced the acrosome reaction of preincubated giant panda spermatozoa by a PKG pathway. Furthermore, ANP enhanced the penetrability of porcine salt-stored oocytes by frozen-thawed giant panda spermatozoa.     

Electron microscopic study of the in vivo zona pellucida binding ability of epididymal ram spermatozoa

In the ram, spermatozoa develop the ability to initiate pregnancy only after reaching the body of the epididymis. To determine the zona pellucida binding ability of ram spermatozoa collected from different levels of the epididymis, sufficient numbers of motile sperm cells of different epididymal origin were inseminated surgically below the uterotubal junction of ewes at the time of ovulation. Intratubal ova were recovered 24 hr later, and those having spermatozoa attached to the zona were examined by transmission electron microscopy to assess the characteristics of the bound spermatozoa. Data indicate that the ability of the capacitated spermatozoa to adhere to the zona pellucida depends on sperm egg binding sites that develop on the acrosomal membranes from the apex to equatorial segment during epididymal transit.     

Effect of the knobbed acrosome defect in bovine sperm on IVF and embryo production

An IVF and culture system was used to determine the effect of the knobbed acrosome defect in bovine spermatozoa on fertilization and early embryonic development. Three bulls affected with knobbed acrosomes were identified as K+ (flattened acrosome), K2+ (indented acrosome) or K3+ (deep indentation of the acrosome) based on the predominant type of acrosomal aberration present in sperm of the respective bulls. After swim-up, all semen traits, except for acrosome morphology, were similar between bulls with varying degrees of the knobbed acrosome defect and a control bull, C. The mean number of spermatozoa bound to the zona pellucida was lower (P< 0.05) for the bulls with the knobbed acrosome defects (40.3 +/- 2.3, 29.5 +/- 1.6, 14.6 +/- 1.3, respectively, for Bulls K+, K2+ and K3+) than for Bull C (52.3 +/- 2.3). The percentages of zonae pellucidae penetrated by spermatozoa from Bulls K+ (51.2%), K2+ (49.5%) and K3+ (37.1%) were lower than that of Bull C (84.5%). No sperm with knobbed acrosome defects were found to have penetrated the zona pellucida. Fertilization rates for bulls with the knobbed acrosome defect, K+ (63.0%), K2+ (62.7%) and K3+ (22.6%), were significantly lower than that of the control bull (82.8%). Percentages of cleaved embryos, morulae and blastocysts produced were also lower for the bulls with knobbed acrosomes than that of the control bull. Results indicate that sperm with the knobbed acrosome defect had a reduced ability to bind to the zona pellucida, depending upon the severity of the defect, and that these aberrant spermatozoa did not penetrate the zona pellucida. The apparently normal spermatozoa coexisting in the inseminate of bulls with a high percentage of knobbed spermatozoa were also functionally deficient; oocytes penetrated by these spermatozoa had a reduced potential for fertilization, and resulting zygotes had a reduced ability for cleavage and embryonic development to the blastocyst stage. The results of the present study do not support the hypotheses that the knobbed acrosome defect is compensable.     

Signal transduction pathways that regulate sperm capacitation and the acrosome reaction

Ejaculated spermatozoa must undergo physiological priming as they traverse the female reproductive tract before they can bind to the egg’s extracellular coat, the zona pellucida (ZP), undergo the acrosome reaction, and fertilize the egg. The preparatory changes are the net result of a series of biochemical and functional modifications collectively referred to as capacitation. Accumulated evidence suggests that the event that initiates capacitation is the efflux of cholesterol from the sperm plasma membrane (PM). The efflux increases permeability and fluidity of the sperm PM and causes influx of Ca²⁺ ions that starts a signaling cascade and result in sperm capacitation. The binding of capacitated spermatozoa to ZP further elevates intrasperm Ca²⁺ and starts a new signaling cascade which open up Ca²⁺ channels in the sperm PM and outer acrosomal membrane (OAM) and cause the sperm to undergo acrosomal exocytosis. The hydrolytic action of the acrosomal enzymes released at the site of sperm-egg (zona) binding, along with the hyperactivated beat pattern of the bound spermatozoon, are important factors in directing the sperm to penetrate the ZP and fertilize the egg. The role of Ca²⁺-signaling in sperm capacitation and induction of the acrosome reaction (acrosomal exocytosis) has been of wide interest. However, the precise mechanism(s) of its action remains elusive. In this article, we intend to highlight data from this and other laboratories on Ca²⁺ signaling cascades that regulate sperm functions.     

Sperm binding capacity and ultrastructure of the zona pellucida of stored canine oocytes

Sperm binding to the zona pellucida is a prerequisite for fertilization, and tests that evaluate this function have been described for several species. When carrying out such tests in the canine species, ovaries or oocytes have to be stored to obtain a sufficient number of oocytes at the time of testing. In the present study, the sperm binding capacities of salt-stored oocytes and oocytes from deep frozen ovaries were measured and compared with that of fresh oocytes. Two different procedures for washing the sperm-oocyte complexes (gentle and tough) were used before evaluating the number of bound spermatozoa. The total number of oocytes that bound spermatozoa was significantly lower for both salt-stored and deep frozen oocytes compared with fresh oocytes. Significantly fewer spermatozoa bound to stored oocytes than to fresh oocytes (P 相似文献   

Effect of in vitro storage of bovine spermatozoa on acrosomal integrity,proteolytic activity,binding, and initiation of penetration of oocytes

During a 10-day 5°C storage and subsequent 4–6-hr 37°C incubation, both percent live spermatozoa and percent spermatozoa with an intact acrosome decreased, and percent spermatozoa with a late-reacted or without an acrosome increased. When stored spermatozoa were mixed with oocytes, no decrease in percent of oocytes with spermatozoa bound or percent of oocytes with spermatozoa starting to penetrate occurred as storage time increased. A 58% decrease in acrosin gelatinolytic activity and a 56% decrease in acrosin esterolytic activity but no decrease in nonacrosin proteolytic activity were evident over the 10-day storage. These studies show that a change in acrosomal morphology as well as a loss of acrosin may be responsible for the decreased fertility following extended in vitro storage of mammalian spermatozoa.     

Normospermic versus teratospermic domestic cat sperm chromatin integrity evaluated by flow cytometry and intracytoplasmic sperm injection

Teratospermia (>60% of morphologically abnormal spermatozoa) is well documented in felids. Even morphologically normal spermatozoa from teratospermic ejaculates have reduced ability to undergo tyrosine phosphorylation, acrosome react, and bind and penetrate oocytes compared with normospermic (<40% abnormal spermatozoa) counterparts. However, it is unknown whether fertilization deficiencies originate at a nuclear level. This study examined whether fertilization failure also was attributable to abnormal sperm chromatin, using the sperm chromatin structure assay (SCSA), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). Aliquots of unprocessed and swim-up-processed (to isolate morphologically normal spermatozoa) spermatozoa from teratospermic and normospermic domestic cats were analyzed by the flow cytometric SCSA. Swim-up-processed sperm were incubated with in vivo-matured oocytes or used for ICSI. Teratospermic ejaculates expressed more (P < 0.05) chromatin heterogeneity (abnormal chromatin structure) than their normospermic counterparts, both in unprocessed and swim-up-processed samples. Fertilization success in vitro was higher (P < 0.05) from normo- compared with teratospermic inseminates. Similar (P > 0.05) proportions of oocytes fertilized after ICSI using spermatozoa from normo- and teratospermic cats. Results reveal that teratospermia in the cat is expressed at the nuclear level as increased sperm chromatin heterogeneity, but ICSI showed that this does not apparently affect fertilization rates if the zona pellucida and oolemma can be bypassed.     

Sperm-egg interaction in the mouse using live and glutaraldehyde-fixed eggs

A study of interaction between gametes in the mouse, using live and glutaraldehyde-fixed eggs, showed that, while in live eggs the binding is a two-step process (“early” and “late” binding), the process is one step when the spermatozoa interact with fixed eggs. The second point that emerged from this study is that uncapacitated and capacitated spermatozoa bind the zonae pellucidae of live and fixed eggs in the same way, but only the capacitated spermatozoa bound to live eggs undergo a complete acrosome reaction and penetrate the zona pellucida. Moreover, it has been shown that binding to fixed eggs, just as to live eggs, is Ca²⁺-dependent, and it can be reversed by EGTA. Fixed eggs thus are a good model to study only one step of the sperm-egg interaction removed from all the other events of the fertilization process.