In vitro penetration of zona pellucida of salt-stored bovine oocytes before and after maturation by frozen-thawed spermatozoa

Bovine oocytes, before and after maturation in culture, were stored in PBS with 2 M-(NH(4))(2)SO(4) + 0.1% dextran or 2 M-(NH(4))(2)SO(4) + 40 mM-Hepes + 0.5% dextran and were inseminated with frozen-thawed spermatozoa in BO medium with caffeine (5 mM) and heparin (10 mug/ml). The penetration rates of mature oocytes were very low (19 to 24%) and not significantly different between the two salt solutions in which the oocytes were stored for 2 to 89 days. Significantly lower (P < 0.01) penetration rates were observed in immature (7 to 8%) than in mature (20 to 21%) oocytes stored in the two solutions. The synergistic effect of caffeine and heparin was observed in the penetration rate of fresh mature oocytes but not in the stored oocytes, indicating the difficulty of assessing sperm capacitation and/or acrosome reaction of salt-stored mature bovine oocytes under the present condition. Using 0.1% protease the solubility of the zonae decreased in salt-stored but not in fresh oocytes, but there was no significant difference between the immature and mature oocytes regardless of storage in the salt solutions. It appears from these results that some alteration was induced in the nature of zona glycoprotein by ammonium sulfate solution.     

Penetration of bovine follicular oocytes by frozen-thawed spermatozoa in the presence of caffeine and heparin

Bovine follicular oocytes matured in culture were inseminated with frozen-thawed spermatozoa which were either preincubated for 5-5.5 h or not preincubated in a medium with caffeine (5 mM) and heparin (10 micrograms/ml). When the oocytes with cumulus and corona cells were inseminated, spermatozoa started to penetrate oocytes 3 h later regardless of whether spermatozoa were preincubated or not. However, a significantly higher proportion of oocytes was penetrated by preincubated than non-preincubated spermatozoa. When the oocytes were freed from cumulus and corona cells, penetration was observed to start 1 h after insemination and there were no differences in penetration rates 1-5 h after insemination between preincubated and non-preincubated spermatozoa. This study demonstrates that capacitation and the acrosome reaction of bovine spermatozoa can be induced within 1 h in a medium containing both caffeine and heparin when denuded oocytes are inseminated.     

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 effect of heparin, caffeine and calcium ionophore A23187 on in vitro induction of the acrosome reaction in frozen-thawed bovine and caprine spermatozoa

The effect of heparin (5 IU), caffeine (5 mM) and calcium-ionophore A23187 (0.1 mM) on motility and in vitro induction of the acrosome reaction in glass wool filtered frozen-thawed bull and goat semen was studied. The motile spermatozoa fraction was obtained after glass wool filtration of frozen-thawed semen. The seminal plasma was removed from filtered semen by centrifugation, and the sperm pellet was resuspended in Sperm-TALP medium. Samples of treated and untreated control semen of both species were incubated at 37 degrees C. At 1, 15 and 30 min of incubation the proportions of progressively motile and acrosome-reacted spermatozoa were assessed. Trypan blue and Giemsa stain was used to differentiate live and dead spermatozoa having undergone acrosome reaction. Glass wool filtration enhanced the proportion of motile spermatozoa from 43% to 62% in the bovine and from 41% to 60% in the caprine. Whereas the effect of incubation with caffeine, heparin and calcium-ionophore on spermatozoan motility was negligible, the treatment of semen with calcium-ionophore resulted in a significantly improved percentage of live spermatozoa with true acrosome reaction at all stages of incubation, both in the bovine and the caprine.     

Gonadotropin exposure,salt storage and storage duration affect penetration of domestic cat oocytes by homologous spermatozoa

Salt-stored domestic cat oocytes are routinely used to study sperm function in domestic and nondomestic felids. Our objectives were to assess the effects of in vitro maturation (IVM), salt storage and storage duration on penetration of domestic cat oocytes by homologous spermatozoa. In Experiment 1, domestic cat spermatozoa were coincubated with fresh immature oocytes, salt-stored (2-3 weeks) immature oocytes, or salt-stored (2-3 weeks) IVM oocytes matured in Minimum Essential Medium containing 0.1IU FSH and 0.1IU LH/ml (IVM1) or 0.5IU FSH and 2.2IULH/ml (IVM2). In Experiment 2, all oocytes were matured (IVM2) and inseminated fresh or after salt storage for 2-3 weeks, 2-3 months or 9 months. In Experiment 1, penetration of the outer zona pellucida (OZP) was greater (P<0.05) in salt-stored IVM2 oocytes than in salt-stored immature oocytes, whereas penetration of salt-stored IVM1 oocytes was intermediate (P>0.05). In Experiment 2, penetration of the OZP and inner zona pellucida (IZP) was higher (P<0.05) in fresh IVM2 oocytes than in salt-stored oocytes, and a higher (P<0.05) proportion of oocytes had IZP sperm after 2-3 weeks of storage than after 2-3 months. Penetration of the perivitelline space was higher (P<0.05) in fresh IVM2 oocytes than in oocytes stored for 2-3 weeks or 2-3 months. These results suggest that oocyte penetration is improved by IVM, but is impaired by exposure to salt-storage solution and prolonged storage duration.     

Synergistic effect of caffeine and heparin on in-vitro fertilization of cattle oocytes matured in culture

Frozen-thawed spermatozoa obtained from six different bulls were suspended in Brackett and Oliphant's (BO) medium (14), with or without 10 mM caffeine, after washing. A 50-mul aliquot of the sperm suspension was added to the 50-mul BO medium supplemented with bovine serum albumin (BSA, 20 mg/ml) and heparin (20 mug/ml) in which the bovine follicular oocytes matured in culture had been introduced previously. The proportion (35%) of oocytes penetrated in the presence of heparin alone 20 to 24 h after insemination was not significantly different from those (32%) penetrated in the presence of caffeine alone as reported previously (1). When heparin was added to the caffeine in the fertilization medium, the penetration rate of oocytes increased significantly to 68% (P < 0.001), indicating that both chemicals act sinergistically to induce capacitation and/or acrosome reaction of spermatozoa and stimulate in vitro fertilization of cattle oocytes. However, great variation in penetration rates (35 to 96%) was observed among the different bulls. The optimal concentration of heparin in the suspension medium in which the highest rate of oocyte penetration took place was 10 mug/ml.     

Birth of piglets by in vitro fertilization of zona-free porcine oocytes

The present experiments were conducted to optimize in vitro fertilization conditions for zona pellucida-free (ZP-free) oocytes and their subsequent development. The results demonstrated that: (1) maximal fertilization efficiency was achieved at 200 spermatozoa per ZP-free oocyte. At this sperm dose, there were no significant differences in penetration rates and polyspermy rates from controls (zona-intact oocytes with 1000 spermatozoa/oocyte), indicating that ZPs of in vitro matured pig oocytes failed to block polyspermy during in vitro fertilization. (2) In vitro development of zygotes from ZP-free oocytes showed that there was no difference in cleavage rates. The blastocyst rate was slightly lower in the ZP-free group than the control. However, there was no difference in cell number per blastocyst between the control and the ZP-free group. (3) Examination of acrosome status by a specific fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining procedure revealed that frozen-thawed pig spermatozoa could undergo acrosome reaction and penetrate oocytes without induction by ZP. These data suggested that there are alternative mechanistic pathways for acrosome reaction induction during the fertilization process than the widely accepted sperm-zona receptor models. Finally, the viability of ZP-free derived embryos was demonstrated by full-term development and the delivery of healthy piglets following embryo transfer. In conclusion, the present experiments showed for the first time in farm animals, that normal embryos could be produced by in vitro fertilization of ZP-free oocytes in optimized conditions and that they could develop normally to full-term.     

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.     

Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine

Effects of caffeine and adenosine on the function and in vitro penetration of frozen-thawed boar spermatozoa were examined. First, the effect on sperm function was determined by the chlortetracycline fluorescence assessment. Both caffeine and adenosine stimulated capacitation of spermatozoa. However, adenosine, but not caffeine, inhibited spontaneous acrosome loss. Second, sperm penetration into in vitro matured oocytes was compared among spermatozoa cultured in the absence or presence of caffeine or adenosine. Both caffeine and adenosine increased the penetration rate (99.1 +/- 0.9% in caffeine, 72.4 +/- 2.0% in adenosine vs. 54.8 +/- 5.1% in controls) but only caffeine decreased drastically the monospermic penetration rate (8.0 +/- 2.3% in caffeine vs. 75.4 +/- 4.8% in adenosine and 78.6 +/- 4.8% in controls). When oocytes were cocultured in various sperm concentrations, the proportion of monospermy changed in inverse proportion to sperm concentration in the presence of caffeine, but did not change in the presence of adenosine. A relatively high number of spermatozoa at the early stage of spontaneous acrosome reaction in the presence of caffeine may be one of the main causes of polyspermic penetration in porcine IVF system. These results indicate that replacement of caffeine with adenosine in fertilization medium improves monospermic penetration by frozen-thawed boar spermatozoa.     

A short sperm-oocyte incubation time ZBA in the dog

The fertilising capacity of a semen sample can be predicted by evaluation of spermatozoa with in vitro tests. The zona pellucida binding assay (ZBA) accounts for several parameters and interprets the interaction between the spermatozoa and the oocyte. The present study was made in two parts. The aim of the first experiment was to evaluate whether the sperm binding capacity of oocytes varies between different oocyte pools. Each zona binding was made with oocytes from different bitches, using pooled frozen-thawed semen from the same two dogs. The sperm-oocyte complexes were incubated for 1h. There was a significant difference between the six replicates in the number of sperm bound to the zona pellucida (ZP), which indicates that the sperm binding capacity of the ZP differs between oocyte pools. The aims of the second experiment were to evaluate the effects of five different treatments of the spermatozoa on the ZBA, and to evaluate two different incubation times of the sperm-oocyte complexes. ZBAs were made with: fresh semen; semen kept chilled for 1 or 2 days prior to the ZBA; and with semen that had been frozen with or without Equex. The oocytes and spermatozoa were incubated for 1 or 4h. For fresh semen and for semen frozen without Equex, incubation for 1h resulted in a higher number of bound spermatozoa per oocyte than incubation for 4h (P<0.0001). When the effect of the different sperm treatments on the number of spermatozoa bound to the ZP was evaluated, it was found that this number was higher for fresh spermatozoa than for chilled or frozen-thawed spermatozoa both after 1 and 4h of co-incubation (P<0.0001). After 1-h incubation of the sperm-oocyte complexes, spermatozoa chilled for 1 day showed better zona binding capacity than spermatozoa chilled for 2 days, and spermatozoa frozen without Equex had a better zona binding capacity than spermatozoa frozen with Equex. Sperm motility and sperm plasma membrane integrity were higher in fresh than in chilled and frozen-thawed semen. The acrosome integrity was high in all groups of treated semen. In conclusion, 1-h incubation of the sperm-oocyte complexes seems to be sufficient for fresh and chilled semen. Further studies are required to establish the optimal incubation time for sperm-oocyte complexes when frozen-thawed semen is evaluated, as a comparison between semen frozen with Equex and semen frozen without Equex gave different results depending on whether the incubation time was 1 or 4h (in the present study), or 6h [Str?m Holst B, Larsson B, Linde-Forsberg C, Rodriguez-Martinez H. Evaluating chilled and frozen-thawed dog spermatozoa using a zona pellucida binding assay.     

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.     

Individual differences in capacitation of bull spermatozoa by heparin in vitro: relationship to fertility

Several parameters of motility and integrity of frozenthawed spermatozoa are compared with the ability of selected motile, intact spermatozoa to acrosome reaction induced by 0.005% hyamin. Between semen donors there exist distinct individual differences; however only the induced acrosome reaction after heparin treatment showed a significant correlation with fertility. For 12 bulls the nonreturn rates from 334 to 559 services correlated significantly with induced acrosome reaction (r = 0.607). The in vitro fertilization of 53 to 93 tubal bovine oocytes with frozen-thawed spermatozoa from five bulls yielded a correlation of r = 0.621 with the rate of induced acrosome reaction. The different capacity levels of heparin-treated spermatozoa to undergo acrosome reaction appears to correspond to the varying intensity and kinetics of heparinmediated head-to-head aggregation of motile cells. The applied functional parameters could be used for the selection of bulls with low fertility in artificial insemination programs, and for spermatozoa donors for in vitro fertilization.     

Progesterone treatment of boar spermatozoa improves male pronuclear formation after intracytoplasmic sperm injection into porcine oocytes

Boar spermatozoa were prepared for intracytoplasmic sperm injection (ICSI) by two different treatments to facilitate sperm chromatin decondensation and improve fertilisation rates after ICSI in pigs: spermatozoa were either frozen and thawed without cryoprotectants, or treated with progesterone. Morphological changes of the sperm heads after the treatments were examined and then the activation of oocytes and the transformation of the sperm nucleus following ICSI were assessed. After freezing and thawing, the plasma membrane and acrosomal contents over the apical region of sperm head were lost in all the spermatozoa. Following treatment with 1 mg/ml progesterone, the acrosome reaction was induced in 61% of spermatozoa. After injection of three types of spermatozoa, non-treated spermatozoa and progesterone-treated (i.e. acrosome-reacted) spermatozoa induced oocyte activation, but frozen-thawed spermatozoa induced oocyte activation at a significantly lower rate. Sixty-two per cent of sperm heads remained orcein-negative for 6 h, however, resulting in delayed sperm chromatin decondensation and low male pronuclear formation in the oocytes injected with a non-treated spermatazoon. Since the treatments of freezing and thawing and progesterone for spermatozoa accelerated the initial change in sperm chromatin and the latter treatment induced oocyte activation earlier, it is considered that the delay in oocyte activation and decondensation of sperm chromatin after injection of non-treated spermatozoa is caused by the existence of the sperm plasma membrane. These results show that progesterone treatment efficiently induces the acrosome reaction in boar spermatozoa without destroying their potency for oocyte activation, and the induction of the acrosome reaction results in the promotion of male pronuclear formation after ICSI.     

In-vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa.

Pig oocytes matured in culture were inseminated with frozen-thawed ejaculated spermatozoa without preincubation in modified tissue culture medium (TCM) 199. High penetration rates (85-89%) and increased incidence of polyspermy were obtained at 25-100 x 10(6) spermatozoa/ml. Wide variation in penetration rates (16-89%) was observed in oocytes inseminated in medium containing 5mM caffeine and at 25-50 x 10(6) spermatozoa/ml obtained from 6 boars, regardless of sperm motility. At 25-50 x 10(6) spermatozoa/ml, penetration rates of oocytes were dependent upon the concentration of caffeine in the medium: there was no penetration without caffeine, but penetration was highest (89%) with 5mM caffeine. None of the oocytes was penetrated in the medium supplemented with heparin at 5-40 micrograms/ml. When heparin was included in the medium with 5mM caffeine, it inhibited the efficacy of caffeine to promote sperm penetration of oocytes.     

Cryopreservation of equine oocytes by 2-step freezing

Immature equine oocytes were frozen-thawed with ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GL) in PBS and cultured to assess the rate of in vitro maturation (Experiment 1). Compact-cumulus oocyte complexes were collected from slaughterhouse ovaries and equilibrated for 10 min in the freezing medium containing 10% (V/V) cryoprotectant and 0.1 M sucrose. The 0.25-ml straws, loaded with 10 to 30 oocytes, were seeded at -6 degrees C and cooled to -35 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. The straws were thawed rapidly in a 37 degrees C waterbath for 20 sec. The proportions of frozen-thawed oocytes reaching Metaphase II (MII) stage after in vitro maturation of 32 h were 15.8% (EG), 5.8% (PD) and 0% (GL), while 63.3% of the nonfrozen control oocytes matured in vitro. The fertilizing ability of immature and mature oocytes after freezing in EG was tested by the insemination of zona-free oocytes with stallion spermatozoa (Experiment 2). Spermatozoa were preincubated for 3 h with 5 mM caffeine, treated with 0.1 mu M ionophore A23187, and inseminated for 20 h at the concentration of 1 to 2 x 10(7)/ml with 6 to 10 oocytes in 50 mu l of Brackett and Oliphant (BO) medium. Immature oocytes (Group 1) were matured in vitro after thawing and then their zona pellucida removed using 0.5% protease. The zona of mature oocytes were removed immediately after thawing (Group 2) or maturation (nonfrozen controls). The oocytes, which had mechanically damaged plasma membrane or lost by artifact, were not examined for insemination. Significantly more control oocytes exhibited a polar body at the time of insemination (53.5%) than either frozen-thawed immature or mature oocytes (25.8 and 27.3%, respectively). Similar proportion of frozen-thawed and control oocytes were penetrated by spermatozoa (71.8 to 79.1%) and exhibited 2 or more pronuclei (73.6 to 80.8%). The mean numbers of spermatozoa per penetrated oocyte were 1.9, 3.0 and 2.5, respectively, for Groups 1 and 2 and for the control oocytes. These results indicate that immature equine oocytes mature to the MII stage in vitro following freezing and thawing in EG or PD but not in GL. Stallion spermatozoa can penetrate zona-free immature and mature oocytes following freezing/thawing in EG and form morphologically normal pronuclei.     

Role of intra-acrosomal antigenic molecules acrin 1 (MN7) and acrin 2 (MC41) in penetration of the zona pellucida in fertilization in mice

In this study the role of two intra-acrosomal molecules, acrin 1 (MN7) and acrin 2 (MC41), during in vitro fertilization (IVF) was examined. The pertinent monoclonal antibodies mMN7 and mMC41 specifically recognize a 90 kDa protein (acrin 1) localized to the entire acrosome and a 200 kDa protein (acrin 2) localized to the cortex region of the anterior acrosome, respectively. Experiments were designed to assess the effects of mMN7 and mMC41 on fertilization in mice using TYH medium containing mMN7 or mMC41 at 0.0, 0.025, 0.05 and 0.1 mg ml-1. Under these conditions, capacitated spermatozoa inseminated the cumulus-invested oocytes. Acrosome-reacted spermatozoa inseminated the zona pellucida-free oocytes. The antibodies had no effect on sperm motility and primary binding to the zona pellucida, but significantly inhibited the rate of fertilization of zona pellucida-intact oocytes in a dose-dependent manner. A significantly small number of spermatozoa remained attached to the zona pellucida at 5 h after insemination in the presence of mMC41. mMC41 and mMN7 antibodies did not affect the fertilization rate of zona pellucida-free oocytes. Confocal laser scanning microscopy with indirect immunofluorescence traced the effect of the monoclonal antibodies on the zona pellucida-induced acrosome reaction, and revealed that mMN7 prevented completion of acrosomal matrix dispersal, whereas mMC41 did not affect the acrosome reaction. mMC41 appeared to inhibit secondary binding or some biochemical steps on the zona pellucida after the acrosome reaction but before penetration of the zona pellucida. Thus, the intra-acrosomal antigenic molecules acrin 1 and acrin 2 are essential for distinct events before sperm penetration of the zona pellucida in mice.     

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   

Penetration by bull spermatozoa into the zona pellucida of dead bovine oocytes recovered from frozen-thawed ovaries

The present study was carried out to determine if the zona pellucida of dead bovine oocytes obtained from ovaries stored at -196 degrees C could be used to assess penetrability of capacitated bull spermatozoa. Follicular oocytes were recovered from bovine ovaries which were frozen slowly in a box containing dry ice, plunged into liquid nitrogen, and thawed at 37 degrees C. The dead oocytes were inseminated with various concentrations of spermatozoa preincubated for 0 to 4 h. Sperm penetration rates of the dead oocytes were significantly altered by sperm concentration and preincubation time. Dead and living oocytes matured in vitro (control) gave similar patterns of penetrability based on sperm preincubation time. When sperm concentration was increased, the rate of multiple sperm penetration into the dead oocytes also increased significantly, but the rate of penetration into living oocytes did not alter significantly. All dead oocytes from ovaries stored at -196 degrees C for 1 d to 3 mo were penetrated at similar rates by spermatozoa preincubated for 1-h. Thus, we conclude that dead follicular oocytes recovered from frozen ovaries are useful for the assessment of sperm capacitation and/or the acrosome reaction in cattle.     

Maturation and sperm penetration of canine ovarian oocytes in vitro.

Canine ovarian oocytes were cultured in a medium consisting of TC medium 199, fetal calf serum and antibiotics. Ninety-nine percent of the apparently healthy oocytes were in the germinal vesicle (dictyate) stage when recovered from the ovaries; 25% of them reached metaphase I or II by 72 hours of culture. Washed ejaculated spermatozoa were added to BWW medium containing oocytes which had either been removed directly from the follicles or which had been cultured for 24--72 hours. The earliest acrosome reaction and zona penetration by spermatozoa were seen at seven hours after insemination. Seventy-four percent of the oocytes examined between 11 and 24 hours after insemination showed evidence of zona penetration by spermatozoa. Neither the condition of the oocyte vitellus nor the stage of nuclear maturation influenced the incidence of zona penetration. Decondensing sperm nuclei were found in the vitellus of 27% of the oocytes which had not been cultured and in the vitellus of 20% of those which had been cultured for 24--72 hours and were in various stages of maturation. These results indicate that (1) canine ovarian oocytes can be matured in vitro, (2) the spermatozoa require capacitation which takes approximately seven hours in vitro and (3) maturation of the oocytes is not required for sperm passage through the zona pellucida or entry into the vitellus nor for sperm nuclear decondensation.     

Calcium dependence of human sperm fertilizing ability

The Ca2+ dependency of human-sperm fertilizing ability was investigated using a modified Tyrode's medium either containing 2.4 mM CaCl2 (CA medium) or with the CaCl2 replaced by SrCl2 and 0.1 mM EGTA added to chelate any residual Ca2+ ions (SREG-medium). Ten washed sperm populations incubated in either medium for 0, 6, and 22 hours showed the same occurrence of acrosome reactions (by fluorescent lectin labelling and triple stain). A further 3-hour incubation after washing into fresh CA medium resulted in only a slight increase in acrosome reactions in both media. Eight sperm populations preincubated overnight in CA and SREG media were coincubated for 1 hour with previously salt-stored human zonae pellucidae also in the same media. Significantly more motile spermatozoa were bound to more of the zonae in CA medium (53.9% vs. 27.6% of zonae with 13.8 vs. 4.3 sperm/bound zona). In three hamster egg penetration test (HEPT) experiments, sperm populations preincubated overnight in either CA or SREG media were coincubated with hamster oocytes prepared in the same media. Only 2.1% of oocytes (1.0 polyspermy) were penetrated in SREG medium, cf., 30.9% of oocytes (1.3 polyspermy) in CA medium. These results demonstrate that while Sr2+ ions can substitute fully for Ca2+ in the capacitation and acrosome reaction of human spermatozoa, sperm-zona, and sperm-oolemma interaction seem to involve some more Ca2+-specific process(es). Furthermore, the increased HEPT fertilizing ability of human spermatozoa using overnight preincubation in SREG medium and CA medium for the test cannot be explained on the basis of differential kinetics of capacitation or the acrosome reaction.