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.     

Effect of cryopreservation of zona-binding capacity of canine spermatozoa in vitro

The hemizona assay (HZA) was used as a functional test for zona pellucida binding capacity of fresh and frozen-thawed canine spermatozoa. We investigated 30 ejaculates from 3 dogs with sperm motility > 70% and sperm concentration > 5.10(8) cells per ejaculate with up to 20% abnormal and dead spermatozoa. Fifteen ejaculates were each divided into 2 portions: one portion was used for analysis of fresh semen, the other for cryopreserved semen. On the day of the experiments, in vitro-matured canine oocytes were bisected into 2 equal hemizonae. One half of the hemizonae were coincubated with fresh capacitated (control) spermatozoa, the other half of the hemizonae were coincubated with frozen-thawed (tested) spermatozoa at final concentration of 1 to 2 x 10(6) cells/mL in 200 microL droplets of BSA-supplemented Toyoda, Yokojama and Hoshi (TYH) medium at 37 degrees C, 5%, CO2 for 1 h. Sperm suspensions were examined kinesigraphically for post capacitation type of movement. The Student's t-test was used to compare differences between semen parameters. The data on HZA binding activity of fresh and frozen-thawed canine semen were analyzed by ANOVA and then by the Newman-Keuls multiple range method. The results showed no differences in the initial semen quality parameters among the 3 dogs. After thawing, the semen from Dog 1 and Dog 2 demonstrated relatively uniform sperm parameters, while in Dog 3 sperm motility, and viability and the percentage of morphologically normal spermatozoa were significantly decreased. The binding activity of frozen-thawed spermatozoa from the 3 dogs was significantly reduced (29.40 +/- 9.02, 18.60 +/- 3.30, 8.20 +/- 4.49) compared with that (107.20 +/- 19.22, 109.80 +/- 20.75, 78.20 +/- 12.47; P < 0.01) of fresh spermatozoa. The results showed that semen samples with similar sperm parameters prior to cryopreservation displayed different sperm zona-binding capacity after freezing. The HZI (value of sperm binding capacity of frozen-thawed vs fresh semen samples) was higher in Dog 1 (27.43) than in Dog 2 (16.90) or Dog 3 (10.40), and thus confirmed the variation of zona binding activity after thawing between dogs. The freezability of individual dog semen is discussed. In conclusion HZA may be a valuable tool for evaluating the post-thaw fertilizing ability of canine spermatozoa.     

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   

Preliminary results of hemizona assay (HZA) as a fertility test for canine spermatozoa

The Hemizona assay (HZA) is considered to be an effective test for predicting the fertilizing potential of spermatozoa. It is a functional test that distinguishes the zona-binding capacity of spermatozoa from fertile and infertile males. The objective of this study was to validate the HZA for canine spermatozoa, as a test for diagnosing canine male fertility status. Various parameters that affect binding capacity were examined: the presence of an adequate number of capacitated and motile spermatozoa for an HZA, the influence of fertility status, sperm-binding variability within fertile dogs over 60 d, variability in sperm-binding capacity of different oocytes, the lower limit number of spermatozoa binding to a zona from the fertile control, and evaluation of HZI to determine the fertilizing capacity of spermatozoa. Hemizonae were obtained from frozen oocytes of spayed bitches. The oocytes were manually cut into nearly equal halves. Spermatozoa were capacitated by swim-up and 1 h incubation at 37 degrees C in modified Ham's F10 medium. Spermatozoa and hemizonae were co-incubated in 100-microL drops at 37 degrees C for 1 h. Spermatozoa from 7 fertile and 3 infertile dogs were used for this study. The optimal sperm concentration for hemizona insemination was 1 x 10(6)/mL capacitated and motile spermatozoa. A significant difference (P < 0.001) was found the number of tightly zonabound spermatozoa between fertile and infertile dogs. Although there was a small difference in zona binding capacity between ejaculates of the same fertile dog (44 +/- 18.24), the main cause for the difference mentioned above was that of poor zona pellucida-binding capacity of spermatozoa from infertile dogs. We found a maximum of 14.28% bad oocytes when we compared sperm samples from 3 fertile and 3 infertile dogs in 56 HZA replicates. To avoid the effect of bad zona on sperm binding we calculated 37 (95% CI) bound spermatozoa from infertile dogs in 56 replicates. Thus, an HZA experiment in which a control dog had < 37 zona bound spermatozoa was repeated. Based on a minimum of 37 bound spermatozoa for fertile males (controls), a differential zona-binding capacity and hemizona index (HZI) between fertile and infertile dogs and between 2 fertile dogs was determined. The binding differential between fertile and infertile dogs was 64.92 +/- 24.29, while between 2 fertile dogs it was 22.38 +/- 10.02 (P < 0.001). According to the HZI values, a value equal to or less than 41.11 indicated an infertile test dog, while an HZI value equal to or greater than 57.95 indicated a fertile test dog. Any value between these two could indicate either fertility or infertility. The evaluation of fertilizing potential of spermatozoa can be improved using the HZA protocols described above.     

Epigallocatechin-3-gallate added after thawing to frozen dog semen: Effect on sperm parameters and ability to bind to oocytes’ zona pellucida

Dog sperm cryopreservation is gaining importance both in breeding dogs for commercial purposes and for pet animals. Anyway, cryopreservation of mammalian spermatozoa, including dog ones, induces some negative effect on sperm fertility, leading to a lower use of this technique and limiting its widespread use. Therefore, studies to improve the quality of canine semen after cryopreservation could have a relevant impact on both the scientific advancement and the clinical practice. The aim of the present work was to investigate the putative ameliorative effect of Epigallochatechin-3-gallate (EGCG) addition to post thawing medium on dog sperm motility, mitochondrial activity, acrosome integrity and on zona-binding ability (zona binding assay). Spermatozoa were thawed in Tris-fructose-citrate medium supplemented with EGCG (0, 25 and 50 μM) and sperm motility, mitochondrial activity and acrosome integrity were assayed at 0.5, 1.5, 3 and 6 h after post thawing incubation at 37 °C. An aliquot of semen from each treatment group after 1.5 h post thawing incubation was washed and used to perform heterologous (using porcine oocytes) or homologous zona binding assay. The results obtained showed that no significant effect is exerted by EGCG on sperm parameters analysed neither at 0.5, 1.5, 3 or 6 h after thawing excepting for the reduction of the percentage of live cells with active mitochondria at the higher dose at 6 h; furthermore, both homologous or heterologous zona binding ability, was not influenced by EGCG. In conclusion, EGCG supplementation to thawing medium does not improve dog sperm quality or zona binding capacity.     

Protein tyrosine phosphorylation and zona binding ability of in vitro capacitated and cryopreserved buffalo spermatozoa

Many similarities between the changes associated with normal capacitation and cryocapacitation have been demonstrated. The present study was undertaken to determine whether similarities exist in the protein tyrosine phosphorylation pattern and zona binding ability between in vitro capacitated (heparin induced; 20 μg/ml) and frozen-thawed (cryocapacitated) buffalo spermatozoa. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris-egg yolk extender, equilibrated and frozen in liquid nitrogen. Localization of phosphotyrosine-containing protein was determined using an indirect immunoflourescence assay with anti-phosphotyrosine antibody. For zona binding assay, good quality oocytes collected by aspiration technique from fresh buffalo ovaries were used. The bound spermatozoa were stained with Hoechst 33342 dye and observed under fluorescent microscope. The results revealed sperm head associated protein tyrosine phosphorylation in both in vitro capacitated and frozen-thawed spermatozoa. In the zona binding assay, the mean number of bound spermatozoa was 90.6 ± 1.9 and 104.7 ± 2.2 in fresh semen after incubation in non capacitating media at 0 h and 3 h, respectively. But after incubation in capacitating media with heparin for 3 h, the mean number of spermatozoa attached to zona pellucida was 138.4 ± 2.6. The in vitro capacitated spermatozoa had significantly (P < 0.05) higher binding ability than that of fresh spermatozoa. After freezing and thawing, 2.5 fold reductions in the zona binding ability of cryopreserved spermatozoa was observed compared to in vitro capacitated spermatozoa. The binding ability of in vitro capacitated spermatozoa was significantly (P < 0.01) higher than that of frozen-thawed (cryocapacitated) spermatozoa. The study concluded that both in vitro capacitated and frozen-thawed (cryocapacitated) spermatozoa had similar immune-localization of tyrosine phosphorylated protein pattern, however, differed in the zona binding ability.     

Zona pellucida binding ability and responsiveness to ionophore challenge of cryopreserved dog spermatozoa after different periods of capacitation in vitro

The present study was aimed to evaluate the functional status of cryopreserved dog spermatozoa after different periods (2, 8 and 24 h) of capacitation in vitro. Sperm motility, viability and binding capacity to the zona pellucida of canine oocytes derived from frozen-thawed ovaries were evaluated at each time point. Sperm viability was assessed by flow cytometry using the Ca(2+)-sensitive indicator Fluo 3 AM and PI, to simultaneously detect the proportion of live spermatozoa and the existence of live sperm subpopulations with different intracellular Ca(2+) content. In addition, the acrosome reaction frequency in ionophore-treated aliquots of spermatozoa incubated in capacitating (CCM) versus non-capacitating (NCM) medium, were evaluated by using eosin-nigrosin staining at the same time intervals. The number of spermatozoa bound to the zona pellucida decreased in about 50% (from 18.61 +/- 14.40 to 7.7 +/- 6.97) when sperm incubation was prolonged from 2 to 8h, however, sperm motility, viability and the subpopulation of live spermatozoa with higher intracellular Ca(2+) concentration decreased in lower extent (10-15%). In CCM-incubated samples, the rate of acrosomal exocytosis in response to ionophore challenge was high (>80%), independently of the evaluation period. NCM-incubated sperm were not affected by ionophore treatment, however, their intracellular Ca(2+) concentration was no different than that observed in CCM-incubated spermatozoa. It was concluded that, after being capacitated, motile and viable spermatozoa seem to lose their ability to bind to the zona pellucida, but this loss is not accompanied by a reduced response to ionophore challenge and it may not be related with changes in the intracellular Ca(2+) concentration of spermatozoa.     

Hormonal regulation of zona-binding ability and fertilizing ability of rat epididymal spermatozoa

Rat spermatozoa from the proximal caput, the proximal corpus, the middle corpus, and the distal cauda epididymidis were examined for their ability to bind to the zona pellucida after a 1-, 2.5-, or 4.5-h incubation at 34°C with rat eggs in cumulus. Caput spermatozoa did not bind to the zona after 1, 2.5, or 4.5 h of incubation. Corpus spermatozoa did bind to the zona, but the percentage of eggs with bound spermatozoa and number of bound spermatozoa per egg increased with the length of incubation. Cauda spermatozoa bound readily to the zona pellucida, and their zona binding ability did not change with longer incubations. It thus appears that rat spermatozoa gradually acquire the ability to bind to the zona pellucida in the corpus epididymidis. The zona-binding capacity of cold immobilized cauda spermatozoa, defined as the percentage of eggs with bound spermatozoa, increased with the number of spermatozoa incubated and reached a plateau characteristic of the endocrine status of the animal. After castration, zona-binding ability is progressively lost from day 3 until day 10 where it is nil. Testosterone supplementation maintains zona-binding ability to control levels. Similarly, fertilizing ability declines from day 5 after castration until day 10. Testosterone prevents this loss of fertilizing ability. It thus appears that the development of zona-binding ability during epididymal transit is, like the development of fertilizing ability, under androgen regulation. The close correlation between the onset of fertilizing ability and zona-binding ability during maturation, the loss of fertilizing ability and zona-binding ability after castration, and the recovery of both fertilizing ability and zona-binding ability with testosterone treatment suggests that the androgen-dependent development of zona-binding ability is an important component of the acquisition of sperm fertilizing ability during epididymal transit.     

Quality and fertilizing ability of electroejaculated cat spermatozoa frozen with or without Equex STM Paste

An optimal protocol for cat semen cryopreservation has not yet been defined. Addition of Equex STM Paste has been tested for epididymal cat spermatozoa but not for ejaculated cat spermatozoa. Furthermore, the effect of Equex STM Paste on fertilizing ability of cryopreserved semen has never been evaluated in that species. Therefore, the aims of the current study were to investigate if addition of Equex STM Paste to a freezing extender for electroejaculated cat (Felis catus) semen would improve postthaw sperm quality and if sperm fertilizing ability after cryopreservation with or without Equex STM Paste was preserved. Semen was collected by electroejaculation and frozen in a Tris-glucose-citrate egg yolk extender supplemented with (0.5% vol/vol) or without Equex STM Paste. In Experiment 1, sperm motility, membrane integrity, and acrosomal status were determined immediately after collection and at 0, 3, and 6 h postthaw. In Experiment 2, frozen semen from the two groups was used for in vitro fertilization (IVF) of in vitro-matured cat oocytes. Cleavage rate was recorded 30 h after IVF, and embryo development was evaluated on Days 6 and 7 of culture. In Experiment 1, the rate of motile spermatozoa after freezing-thawing was higher when Equex STM Paste was added to the freezing extender, but progressive motility score was not influenced (P > 0.05). Sperm membrane integrity was positively affected (P < 0.05) by the addition of the detergent. Intact acrosomes after thawing were similar (P > 0.05) between groups. Even if the decreasing rates of motility and membrane integrity were more rapid in presence of Equex than those in controls, total motility and sperm viability were similar at 3 and 6 h after thawing (P > 0.05). In Experiment 2, there was no difference in fertilizing ability and embryo development between the two groups (P > 0.05). The results of this study demonstrate that the addition of Equex STM Paste in the freezing extender avoids the loss of motile spermatozoa and maintains fertilizing ability of frozen-thawed spermatozoa.     

The application of in vitro sperm competition test to evaluate the impact of ZP-derived peptides on fertilization capacity of cat sperm

The present study aimed to establish a sensitive in vitro assay to assess the binding capacity of cat spermatozoa. Cat oocytes and epididymal sperm cells were isolated from gonads and cultured for in vitro fertilization. Before fertilization, the sperm cells were incubated either in 10 microM green dye Fluo-3-AM or 10 microM orange dye CellTracker Orange CMTMR (Molecular Probes), respectively. After removing the dyes by washing, sperm cells stained with each dye were added to medium drops containing oocytes in various proportions and cultured for 16 h at 37 degrees C, 5% CO(2). The oocytes were examined using fluorescence microscopy. Sperm bound to oocytes, and stained with different colors, were counted. When fresh epididymal sperm were mixed in at a specific proportion, the number of sperm bound to the zona pellucida (ZP) of oocytes reflected the proportion of differently colored sperm in the medium. This indicated that neither dye influenced the binding capacity of cat sperm. Mixing fresh and cryopreserved sperm, however, resulted in a higher number of fresh sperm bound to the oocyte surface in comparison to frozen-thawed sperm. Also, the pre-incubation of cat sperm cells with ZP derived peptide reduced the sperm binding capacity by 40%. In conclusion, the presented sperm competition assay allows assessment of fertilizing capacity of cat spermatozoa in vitro when a mixture of two different populations is used. The applied supravital fluorescence dyes do not affect motility and binding capacity of sperm cells and were clearly distinguishable under fluorescence microscopy. We demonstrate that the assay can be used to study the impact of sperm treatment, such as cryopreservation or pre-incubation in bioactive peptides, on fertilizing capacity.     

The effect of Equex STM paste and sperm morphology on post-thaw survival of cat epididymal spermatozoa

Cryopreservation of epididymal spermatozoa is a potentially valuable tool for preserving genetic material from individuals of endangered species that die accidentally. Improvement of sperm-freezing protocols would increase the efficacy of gene banking from endangered felids, and the domestic cat can be used as a model for the wild felids. Addition of the detergent Equex STM paste to semen freezing extenders has been found to improve post-thaw survival and longevity of spermatozoa from various species but has never been tested for cat spermatozoa. Spermatozoa from cats with a high percentage of morphologically abnormal spermatozoa are more susceptible for cold injury and osmotic stress than spermatozoa from normozoospermic cats. Therefore, the aims of this study were to investigate: (a) if addition of Equex STM paste to a semen freezing extender would improve post-thaw sperm survival, and (b) if there is a relation between the percentage of morphologically normal spermatozoa and cryopreservation induced damage in cat epididymal spermatozoa. Spermatozoa harvested from epididymides of 10 male cats were frozen in a Tris egg yolk extender with or without the addition of Equex STM paste (0.5%, v/v). Sperm motility, membrane integrity and acrosomal status were evaluated immediately after harvesting, and at 0, 2, 4 and 6 h post-thaw. Sperm membrane integrity and acrosomal status were also evaluated after cooling to 4 degrees C, just before freezing. Cooling did not cause significant damage to the spermatozoa, whereas freezing damaged sperm membranes and acrosomes. Addition of Equex to the freezing extender had a significant positive effect on the percentage of intact acrosomes immediately after thawing (P > 0.05), but had a negative effect on the longevity of the spermatozoa; the percentages of membrane intact and motile spermatozoa being significantly lower in the presence of Equex than in the controls at 6h after thawing. The percentage of morphologically normal spermatozoa was not found to be correlated with either cryopreservation induced acrosome or plasma membrane damage, or with post-thaw motility (P > 0.05). The results clearly show that addition of Equex STM paste in the freezing extender protects the acrosomes of cat epididymal spermatozoa during the freezing--thawing process, but reduces the sperm longevity during in vitro incubation at 38 degrees C. Our results also indicate that the percentage of morphologically normal epididymal spermatozoa is not correlated with cryopreservation induced sperm damage using the described freezing protocol.     

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.     

Effects of equex STM paste on viability of frozen-thawed dog spermatozoa during in vitro incubation at 38 degrees C

In the canine, artificial insemination with cryopreserved semen generally yields lower pregnancy rates with vaginal deposition than with uterine deposition, one of the reasons being the shortened life span of frozen-thawed spermatozoa. The incubation of spermatozoa at body temperature partially mimics the situation in vivo, and evaluation of the kinetics of viability loss under these conditions can be used to measure the damage caused by freezing and thawing procedures. In this study, 2 aliquots were separated from split ejaculates collected from 7 dogs and were frozen by lowering the straws, in 3 steps, into an LN(2) tank after dilution with egg yolk Tris-citrate-glucose extender with or without the addition of 0.5% Equex STM paste. Motility and plasma membrane integrity (evaluated with the combined fluorescent probes 6-carboxyfluorescein diacetate and propidium iodide) were assessed immediately after thawing and over the next 3 h at 38 degrees C. The addition of Equex STM paste significantly increased the proportion of spermatozoa having an intact plasmalemma immediately after thawing compared with the control. It also increased the longevity of the thawed spermatozoa, prolonging the maintenance of both motility and plasma membrane integrity.     

Effects of Equex STM Paste on the quality of frozen-thawed epididymal dog spermatozoa

This study was carried out to investigate the cryoprotective efficacy of Equex STM Paste on the quality of canine post-thaw epididymal spermatozoa. Following castration, spermatozoa were flushed from the cauda epididymides. Epididymal spermatozoa from 13 of 16 dogs with a sperm motility of >70% were frozen in an egg yolk-Tris extender, supplemented with Equex STM Paste (0.5%, v/v); the extender free of Equex STM Paste served as a control cryoprotective diluent. The quality of spermatozoa, judged by its motility, plasma membrane integrity and acrosome integrity, was evaluated on four occasions, immediately after collection, after equilibration and at 0 and 2h post-thaw. Reducing the temperature to 4 degrees C for 2h prior to freezing decreased sperm motility (P=0.001), but had no effects on membrane integrity or acrosome integrity. Immediately after thawing, the percentage of acrosome-intact spermatozoa significantly decreased in samples frozen without Equex STM Paste compared to freshly collected or Equex-treated samples. After incubation at 37 degrees C for 2h post-thaw, a greater percentage of motile spermatozoa (P=0.018) and spermatozoa with intact acrosomes (P=0.001) were observed in Equex-treated samples compared with the control. The percentage of membrane-intact spermatozoa did not differ significantly between Equex-treated and control samples at any time. Supplementation with Equex STM Paste in the semen extender was effective for freezing canine epididymal spermatozoa because it protected acrosome integrity against damage induced by cryopreservation and it prolonged post-thaw sperm motility during in vitro incubation at 37 degrees C.     

Use of frozen-thawed oocytes for efficient production of normal offspring from cryopreserved mouse spermatozoa showing low fertility

Freezing of spermatozoa and unfertilized oocytes is a useful tool for the conservation of mouse genetic resources. However, the proportion of frozen-thawed oocytes fertilized with spermatozoa in vitro is low because spermatozoa, especially those frozen-thawed, can not penetrate into oocytes because of hardening of the zona pellucida following premature release of cortical granules. To produce offspring efficiently from cryopreserved transgenic mouse gametes, we fertilized frozen-thawed gametes by using intracytoplasmic sperm injection (ICSI) and assessed pre- and postimplantation development of embryos. Compared with fresh unfertilized oocytes, frozen-thawed unfertilized oocytes were highly tolerant to damage by injection, as the survival rates after injection of frozen spermatozoa were 51 and 78%, respectively. Frozen-thawed oocytes that survived after sperm injection developed normally to the blastocyst stage and gave rise to offspring. Moreover, offspring with transgenes also were obtained from frozen gametes fertilized by ICSI. These results demonstrate that ICSI is an efficient technique for producing offspring from transgenic spermatozoa showing low fertility and that use of frozen-thawed oocytes leads to conservation of genetic resources because suboptimally preserved gametes are not wasted.     

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.     

Zona pellucida resistance to sperm penetration before the completion of human oocyte maturation

Human oocytes exposed to capacitated spermatozoa in vitro when at metaphase of the 1st meiotic division (metaphase I) were not penetrated, even though some subsequently progressed to metaphase of the 2nd meiotic division (metaphase II). When the non-penetrated oocytes that had reached metaphase II during the incubation with spermatozoa were freed from the zona pellucida and reinseminated, two or more pronuclei developed in most of them. By contrast, no penetration was observed when the oocytes were reinseminated in the zona-intact state. When compared with metaphase II oocytes, metaphase I oocytes had a similar zona-binding capacity for spermatozoa, but fewer spermatozoa were found within the zona. These results indicate that the zona pellucida of human oocytes undergoes important maturational changes during the transition from metaphase I to metaphase II. Ultrastructural and previous histochemical findings suggest that these changes involve secretions from both the oocyte and cumulus cells and that the increased zona resistance at metaphase I may be due to relative insufficiency of cumulus cell-secreted 'softening' factors. If the integrity of the cumulus oophorus is disturbed at this stage, this condition becomes irreversible.     

Tracking diffusion of GM1 gangliosides and zona pellucida binding molecules in sperm plasma membranes following cholesterol efflux

The molecules on mammalian spermatozoa that mediate recognition and binding to the zona pellucida of the egg are still not understood. Current concepts favour their assembly into multimolecular complexes in the plasma membrane in response to cholesterol efflux, an important step during sperm capacitation. Here, we track in real time diffusion of cross-linked clusters containing zona-binding molecules and GM1 gangliosides in the plasma membrane of live boar spermatozoa before and after cholesterol reduction. Both GM1 gangliosides and zona-binding molecules partition into a low density Triton X100 resistant phase suggesting their association with lipid rafts. Initially, GM1 and zona-binding molecules localize to the apical ridge on the acrosome but following cholesterol efflux with methyl-β-cyclodextrin, clusters containing zona-binding molecules diffuse randomly over the acrosomal domain. Diffusing clusters of either type do not access the postacrosome. Spermatozoa agglutinated head-to-head show contact-induced coalescence of GM1 gangliosides (but not zona-binding molecules) suggestive of a specific mechanosensitive response. Thus, cholesterol efflux initiates diffusion (and possibly formation) of novel lipid raft-like structures containing zona-binding molecules over the sperm acrosome. We hypothesise that in combination with contact coalescence, these mechanisms concentrate important molecules to the appropriate site on the sperm surface to mediate zona binding.     

Effects of exposure of epididymal boar spermatozoa to seminal plasma on the binding of zona pellucida proteins during in vitro capacitation

The purpose of the investigation was to determine whether seminal plasma plays a role in the increase during in vitro capacitation of the number of boar spermatozoa with enhanced binding of zona pellucida proteins. Ejaculated spermatozoa and spermatozoa collected from the caudae epididymides of boars were incubated at 39 degrees C in a Tyrode's IVF medium. During incubation, the zona binding ability of individual spermatozoa was assessed with fluorescein-conjugated solubilized zona pellucida proteins (FITC-sZP), using a flow cytometer. Propidium iodide (PI) was included to simultaneously monitor cell viability. During incubation of ejaculated spermatozoa, a percentage of the spermatozoa expressed enhanced binding of FITC-sZP. The percentage of viable spermatozoa with enhanced binding reached a maximum of 37% (S.D.=8, averaged over five boars) after 2-3 h. In epididymal sperm, a similar maximum was observed after incubation in vitro, but a longer time of incubation was needed (6 h). Also, the rate of cell death of epididymal sperm was much lower than that of ejaculated sperm. When epididymal spermatozoa was exposed to seminal plasma in vitro, the time needed to reach a maximal percentage of viable spermatozoa with enhanced FITC-sZP binding was similar to that in ejaculated semen. However, the rate of cell death was still much lower than in ejaculated sperm. We concluded that the binding sites on the sperm surface that are involved in the increased binding of zona proteins during incubation under IVF conditions were not derived from the seminal plasma. The cellular processes leading to the increased binding capacity were accelerated by exposure of the sperm to seminal plasma.     

The chaperonin containing TCP1 complex (CCT/TRiC) is involved in mediating sperm-oocyte interaction

Sperm-oocyte interactions are among the most remarkable processes in cell biology. These cellular recognition events are initiated by an exquisitely specific adhesion of free-swimming spermatozoa to the zona pellucida, an acellular matrix that surrounds the ovulated oocyte. Decades of research focusing on this interaction have led to the establishment of a widely held paradigm that the zona pellucida receptor is a single molecular entity that is constitutively expressed on the sperm cell surface. In contrast, we have employed the techniques of blue native-polyacrylamide gel electrophoresis, far Western blotting, and proximity ligation to secure the first direct evidence in support of a novel hypothesis that zona binding is mediated by multimeric sperm receptor complex(es). Furthermore, we show that one such multimeric association, comprising the chaperonin-containing TCP1 complex (CCT/TRiC) and a zona-binding protein, zona pellucida-binding protein 2, is present on the surface of capacitated spermatozoa and could account for the zona binding activity of these cells. Collectively, these data provide an important biochemical insight into the molecular basis of sperm-zona pellucida interaction and a plausible explanation for how spermatozoa gain their ability to fertilize.