Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion

Mouse oocytes exposed to 1 microgram Hoechst 33342 (H-33342)/ml and then fertilized in vitro developed normally into blastocysts and blastocyst outgrowths. After penetration of the zona, the fertilizing spermatozoon showed intense fluorescence upon fusion with the vitelline membrane. Due to fluorochrome leakage from the perivitelline space a faint fluorescence was detected in zona-bound spermatozoa. This fluorescence of zona-bound spermatozoa intensified with increased fluorochrome concentration (10 micrograms/ml), obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 micrograms H-33342/ml) fluoresced within 10 min of insemination, provided the zonae were removed mechanically. Removal by protease digestion induced leakage of fluorochrome, so that all spermatozoa in the vicinity of an oocyte pre-loaded with 10 micrograms H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 microgram H-33342/ml. The technique could not be applied to zona-free hamster oocytes under our conditions, since the fluorochrome leaked freely from the oocytes whether the zona was removed mechanically or enzymically.     

Porcine sperm viability, oocyte fertilization and embryo development after staining spermatozoa with SYBR-14

The objective of these experiments was to determine the efficacy of the new membrane permeant nucleic acid stain, SYBR-14, for assessing boar sperm viability and to determine it's effect on fertilization and early embryonic development using the pig as a model. We examined the staining patterns of SYBR-14 and another vital stain, Hoechst 33342, both in combination with the dead cell stain, propidium iodide (PI), to quantify the proportion of living and dead spermatozoa in ejaculated and epididymal semen. Flow cytometry analyses of semen from 4 boars revealed significant differences among boars for the proportion of SYBR-14-stained spermatozoa in both epididymal and ejaculated samples, but not for Hoechst 33342 or PI stained spermatozoa. Gilts were inseminated with unstained spermatozoa or spermatozoa stained with 2 levels of SYBR-14 or 2 levels of the reference stain, Hoechst 33342. Embryos recovered at 42 to 48 h postinsemination were morphologically evaluated, and only 4 to 8-cell embryos were continued in culture. Overall, fluorescent staining of boar spermatozoa with SYBR-14 or Hoechst 33342 neither affected their ability to fertilize oocytes, nor the developmental competence of the resultant embryos.     

Changes in the reciprocal position of the first polar body and oocyte chromosome set in golden hamsters

The golden hamster is an attractive model organism for studying reproductive physiology, oncology, genetics and virology. In an effort to establish experimental protocols necessary for cloning golden hamsters, we examined changes in the reciprocal position of the FPB (first polar body) and chromosome set of MII (the second meiotic metaphase) oocytes of golden hamsters. Oocytes were collected under three different conditions: (i) oocyte direct recovery from the oviduct of hormonally treated donor; (ii) oocyte recovery from the oviduct of hormonally treated donor followed by 5 h/10 h in vitro culture; and (iii) oocyte recovery from ovaries of hormonally treated donors and in vitro maturation. Then oocyte recovery was performed from the oviduct of hormonally treated donors, followed by 5 h in vitro culture with colchicine and/or CB (cytochalasin B). Denuded oocytes were stained with Hoechst 33342 and propidium iodide and evaluated under a microscope. Our results demonstrate that the change in FPB position in relation to the MII oocyte chromosome set increases with age of in vivo-matured oocytes. Cumulus cells can protect the FPB of in vitro-cultured oocytes from degeneration but do not significantly affect its repositioning, and in vitro-matured oocytes age slower. The colchicine has a stronger effect on cytoplasmic protrusions of golden hamster oocytes when compared with CB. These results define conditions for changes in FPB position relative to the MII oocyte chromosome set. Early ovulated oocytes, in vitro-matured oocytes and oocytes treated with colchicine should improve the effectiveness of the cloning procedure in golden hamsters as an animal model for human diseases.     

Effect of short periods of sperm-oocyte coincubation during in vitro fertilization on embryo development in pigs

The present study was conducted to determine if the efficiency of in vitro pig embryo production could be improved by a reduction in the period of time that oocytes are exposed to sperm during in vitro fertilization. A total of 1596 immature cumulus-oocyte complexes from five replicates were matured in vitro and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) for 10, 30, 60 min or 6h (control group). The oocytes from short coincubation times were washed three times in fertilization medium to remove spermatozoa not bound to the zona and transferred to another droplet of the same medium (containing no sperm) for 6h. After 6h, the oocytes from each group were cultured in embryo culture medium for another 6h to assess fertilization parameters and for 7 days to assess embryo development. After each period of coincubation, some oocytes were stained with Hoechst-33342 to count zona-bound sperm. Although the number of zona-bound sperm increased with the coincubation time (34.1 +/- 1.7, 46.8 +/- 2.8, 62.8 +/- 3.8 and 139.5 +/- 6.1 for 10, 30, 60 min and 6h, respectively, P < 0.02), the penetration rate was not significantly different among groups (61.3-68.2%). However, the efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) increased (P < 0.04) as the coincubation time was increased (26.6 +/- 2.9%, 29.0 +/- 4.4%, 39.5 +/- 6.2%, and 49.3 +/- 3.0% for 10, 30, 60 min and 6h, respectively). Nevertheless, there were no significant differences among groups in blastocyst formation rates (17.5-25.5%). These results demonstrate that although a sperm-oocyte coincubation time of as little as 10 min results in fertilization rates similar to a 6-h coincubation, the reduction in the period of time of sperm-oocyte coincubation does not improve the efficiency of in vitro pig embryo production.     

哺乳动物精子质量的评价方法

精子的各种功能状态反应了精子的受精能力。检测精子质膜完整性的荧光探针有SYBR－14,SYTO－17,CFDA、CDMFDA、CAM、PI、Hoechst33258、Hoechst33342,其中以SYBR－14结合PI使用效果最好,检测线粒体活性的荧光探针有JC－1、MITO、Rh123,JC-1比MITO和Rh123更适用于检测精子线粒体功能,检测顶体状态的荧光探针有PNA－FITC、PSA－FITC、LYSO－G及CTC等。检测获能状态的荧光探针有CTC。此外,还可以通过检测精子与透明带的结合能力、精子穿入去透明带卵子的能力以及使卵子受精的能力和其后胚胎的发育能力等方面来评价精子的功能状态。     

A technique for the evaluation of sperm penetrating ability and quality of bovine semen processed in an extender made with Brackett-Oliphant medium and egg yolk

Egg yolk-sodium citrate (EYC) semen extender was compared with an extender made of Brackett-Oliphant medium and egg yolk (BOEY). Ejaculates were divided into equal portions, processed and frozen. Semen was thawed and evaluated for quality. Additional semen was thawed, stained with Hoechst 33342 and the spermatozoa capacitated, after which they were co-incubated with zona-free hamster oocytes to determine their penetrating ability. Sperm penetration of non-compressed, unfixed oocytes was evaluated using an optical sectioning technique on a standard research microscope. Sperm penetration was considered successful if a fluorescing sperm head was observed within the living oocyte in a hanging drop of fertilization medium. There were small differences in percentage of secondary abnormalities and percentage of progressive motility immediately after thawing between spermatozoa extended in EYC or BOEY diluent. There were no differences due to by extender composition in percentage of spermatozoa with intact acrosomes or percent of progressively motile after a 3 h incubation at 37 degrees C, nor the percentage of spermatozoa with head abnormalities. While there were significant correlations between all seminal quality characteristics, no quality measurements were correlated to percentage of oocyte penetration. The new penetration evaluation method allowed for examination of the fertilized oocytes using fluorescent microscopy initially and again after re-incubation for further development.     

Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO(2) in air) for 1h in the following treatments either 500 microL of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1h either FM or FM with alpha L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) alpha L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) alpha L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 x 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha L-PGDS: (1) 26.4+/-3.0; (2) 25.6+/-3.0; (3) 59.7+/-3.0; (4) 56.4+/-3.0; and (5) 57.1+/-3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P<0.05) compared with the control: (1) 89.2+/-2.0%; (2) 87.5+/-2.0%; (3) 19.4+/-2.0%; (4) 27.2+/-3.1%; and (5) 14.1+/-3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization.     

Brief coincubation of gametes in porcine in vitro fertilization: role of sperm:oocyte ratio and post-coincubation medium

In this study, a short coincubation time of 10 min was used to determine the effect of different sperm:oocyte ratios during in vitro fertilization (IVF), and different periods of post-coincubation in a medium that is not appropriate for IVF, on fertilization parameters. In the first experiment, a total of 1624 in vitro matured oocytes, from 4 replicates, were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000, 1500, 1000 and 500 sperm:oocyte) and coincubated for 10 min or 6 h. The oocytes from 10 min of coincubation were washed in IVF medium to remove spermatozoa not bound to the zona pellucida and transferred to another droplet of the same medium (containing no spermatozoa) for 6h. The oocytes from the other group remained with the spermatozoa for 6h. Oocytes from both groups were then cultured in embryo culture medium (IVC) for 12h to assess fertilization parameters. In the second experiment, 1872 in vitro matured oocytes, in 3 replicates were inseminated with frozen-thawed spermatozoa using the same sperm:oocyte ratios as in the first experiment. The oocytes were coincubated for 10 min and transferred directly to IVC medium for 18 h (group A), to IVF medium (containing no sperm) only for 2h and then to IVC medium for 16 h (group B), or to IVF medium (containing no sperm) for 6h and then to IVC medium for 12 h (group C or control). There was an effect of sperm:oocyte ratio on all fertilization parameters in experiment 1. The efficiency of IVF (number of monospermic oocytes/total number inseminated) was higher (P<0.05) for oocytes coincubated with spermatozoa for 10 min and inseminated with 1500 and 1000 sperm:oocyte (35.8+/-3 and 37.6+/-2.7%, respectively) and for those coincubated for 6h with 500 spermatozoa per oocyte (37.2+/-3.1%). In experiment 2, the penetration and efficiency rates obtained in group A were poor (between 3 and 15%) irrespective of the sperm:oocyte ratio. However, in group B the fertilization parameters were similar to the controls and were also affected by the sperm:oocyte ratio. These results demonstrate that coincubation time may be reduced to 10 min to increase the efficiency of fertilization depending on the sperm:oocyte ratio, and that the spermatozoa bound to the zona pellucida require a maximum of 2h in an appropriate medium to penetrate the oocytes.     

Prognostic value of canine frozen-thawed semen parameters on in vitro sperm-oocyte interactions

Combining the data from conventional semen analysis with oocyte penetration assays should improve the assessment of the fertilizing ability of a semen sample. Thus, the objective of the present study was to evaluate the prognostic value of various semen parameters on the in vitro interactions between frozen-thawed canine sperm and homologous oocytes. Ten ejaculates from five stud dogs (two ejaculates/dog) were collected by digital manipulation. Semen samples were evaluated, extended in Tris-egg yolk-glycerol, frozen and stored in liquid nitrogen, and thawed several weeks later. Samples were evaluated for motility and sperm populations by computer-aided semen analysis (CASA), plasma membrane integrity (carboxy-fluorescein diacetate and propidium iodide), and sperm morphology (Bengal Rose). Thawed spermatozoa were also incubated with homologous oocytes for 18 h in an atmosphere of 5% CO(2) and 95% air at 38 degrees C and sperm-oocyte interactions were evaluated. Simple linear regression models were calculated, with sperm parameters as independent variables and sperm-oocyte interactions as the dependent variable. There were significant associations between: percentage of oocytes bound to spermatozoa and beat cross frequency (BCF; R(2)=63%); percentage of oocytes that interacted with spermatozoa and BCF (R(2)=73%); and number of penetrated spermatozoa and velocity average pathway (VAP; R(2)=64%) and velocity straight line (VSL; R(2)=64%). Although plasma membrane integrity and sperm morphology had little prognostic value for in vitro interactions between canine frozen-thawed sperm and homologous oocytes, some motility patterns (evaluated by CASA) were predictive of in vitro sperm-oocyte interactions.     

Effects of sperm preparation and bull fertility on in vitro penetration of zona-free bovine oocytes

The objectives of this study were to develop and validate a zona-free bovine oocyte penetration assay for detecting relative differences in bovine sperm fertility and to determine the effect of different sperm preparation methods on oocyte penetration. Oocytes were incubated with heparin-capacitated spermatozoa which either were or were not induced to acrosome-react with lysophosphatidylcholine. Heparin-capacitated spermatozoa treated with lysophosphatidyl-choline penetrated more oocytes and had more penetrations per oocyte than spermatozoa capacitated in heparin but not induced to acrosome-react with lysophosphatidylcholine. Spermatozoa stained with Hoechst 33342, fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate, alone or in combination, penetrated similar numbers and percentages of zona-free bovine oocytes as the similar to non-stained spermatozoa. When spermatozoa from the same ejaculate were stained with either fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate and competed in penetrating the same oocytes, the number of penetrations generated by the 2 differently stained spermatozoa was similar. Spermatozoa from bulls of differing in vivo fertilities were labeled with different fluorescent dyes, and their relative abilities to penetrate the same oocytes were assessed. Comparisons between spermatozoa from high and low fertility bulls demonstrated that high fertility spermatozoa had a significant oocyte penetrating advantage over low fertility spermatozoa in 13 of 16 paired competitions. We concluded that the results of the competitive penetration of zona-free bovine oocytes by fluorochrome-labeled spermatozoa from bulls of different fertilities were indicative of their relative in vivo fertility.     

A supravital staining technique for honey bee spermatozoa

ABSTRACT A new supravital staining technique is described for honey bee, Apis mellifera L., spermatozoa using the fluorochromes, propidium iodide and Hoechst 33342 (H342), a bis-benzimidazole derivative. Propidium iodide binds to the DNA of sperm which lack membrane integrity and H342 binds to the DNA of all sperm. This assay is a simple and rapid method for determining the percentage nonviabiiity of a male honey bee's sperm. The recommended staining procedure is to incubate sperm in a solution of 5 μ.g/ml H342 and 10 μ.g/ml propidium iodide in modified Kiev solution for 15–20 min. After incubation, wet mounts of the sperm-stain suspension are examined using fluorescence microscopy. Percentage nonviabiiity is determined by the ratio of propidium iodide stained sperm to H342 stained sperm.     

Exposure of in vitro-matured porcine oocytes to SYBR-14 and fluorescence impairs their developmental capacity

Staining with Hoechst 33342 followed by ultraviolet irradiation is frequently used to aid or confirm the enucleation of recipient oocytes in porcine somatic cell nuclear transfer programs. However, the procedure has a clearly deleterious effect on the developmental ability of oocytes. This study evaluated the effectiveness of a longer-wavelength fluorochrome (SYBR-14) for visualizing maternal chromosomes in in vitro-matured porcine oocytes and the effects of this dye in combination with fluorescence excitation on the subsequent in vitro fertilization and embryo development of the oocytes. In the first experiment, the oocytes were exposed to different concentrations (1, 3, 5 and 7 μg/mL) of SYBR-14 at different incubation times (5, 10 and 30 min) in a 4 × 3 factorial design. The optimal condition for proper metaphase-II plate and first polar body visualization was a 10-min incubation with 5 μg/mL of SYBR-14. In the second experiment, the degeneration rate of the oocytes 18 h after exposure to SYBR-14 (5 μg/mL for 10 min) and fluorescence excitation for 5 or 30s was significantly higher (p<0.002) than that obtained for non-exposed oocytes. The fertilization parameters were not influenced by the treatments. The cleavage and blastocyst rates during culture were lower (p<0.001) for the oocytes exposed to SYBR-14 and fluorescence than for those in the non-exposed group. These results indicate that the exposure of mature oocytes to SYBR-14 and fluorescence for periods as short as 5s increased the rate of oocyte degeneration and limited their subsequent developmental competence.     

The binding kinetics and interaction of DNA fluorochromes used in the analysis of nuclei and chromosomes by flow cytometry

The interactions and binding characteristics of DNA dyes used in the flow cytometric analysis of chromatin were studied using human chromosomes and mouse thymocyte nuclei. The kinetics of dye binding and the relationship between fluorescence intensity and dye concentration are presented. Under the conditions used, Hoechst 33258, propidium iodide and chromomycin A3 reach an equilibrium with thymocyte nuclei after approximately 5 min, 20 min and more than 1 h, respectively. The same binding kinetics are observed with Hoechst 33258 and chromomycin when nuclei are stained with a mixture of the two dyes. Sodium citrate, which improves the resolution of flow karyotypes, causes a rapid increase in Hoechst and propidium iodide fluorescence, but a decrease in the fluorescence of chromomycin. The relative peak positions of chromosomes in a flow karyotype are unaffected by sodium citrate addition. The spectral interaction between Hoechst and chromomycin is quantified. There is variation among the human chromosome types in the amount of energy transferred from Hoechst to chromomycin. By measuring the Hoechst and chromomycin fluorescence of each chromosome after Hoechst excitation, it is shown that the amount of energy transferred is correlated to the ratio of the amount of Hoechst to chromomycin bound. Although the energy transfer between the two dyes is considerable, this has little effect on the reproducibility of flow karyotype measurements. The relative peak positions of all human chromosomes in a 64 X 64 channel flow karyotype, except for the 13 and Y chromosomes, vary in the order of 0.5 channel over a 16-fold change in either Hoechst or chromomycin concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ability of hamster oolemma to fuse with spermatozoa: its acquisition during oogenesis and loss after fertilization.

To determine when the growing hamster oocyte gains the ability to fuse with the spermatozoon, oocytes at various stages of development were collected from ovaries, and zona-pellucida-free oocytes were inseminated in vitro with acrosome-reacted spermatozoa. Very small primary oocytes were unable to fuse with spermatozoa. Oocytes first became competent to fuse with spermatozoa when they had grown to about 20 microns in diameter. The acquisition of fusibility coincided with the first appearance of zona pellucida material and oolemma microvilli. The fusibility of the oolemma increased as the oocyte grew, reaching a maximum when the oocyte reached the metaphase of the second meiosis. The fusibility of the oolemma was reduced drastically after fertilization, and was lost completely by the 8-cell stage. The appearance and subsequent disappearance of a putative fusion-mediating molecule in the oolemma is proposed. Since this molecule is fairly resistant to proteinase digestion, at least in the hamster, it could be a cryptic protein or a glycolipid.     

Effect of Hoechst 33342 staining on developmental competence of prepubertal goat oocytes

This study was undertaken to evaluate the effects of Hoechst staining on nuclear maturation and fertilisation when used at different stages of in vitro maturation (IVM) in prepubertal goat oocytes. Oocytes were matured in TCM1999 supplemented with 10% fetal bovine serum, 10 microg LH/ml, 10 microg FSH/ml and 1 microM 17beta-estradiol for 27 h. Frozen-thawed sperm cells were prepared by centrifugation in a discontinuous Percoll gradient and resuspended in DMH medium with 20% steer serum. Oocytes were fertilised in DMH medium with 7.75 mM calcium lactate. During IVM oocytes were exposed to 0.5 microg/ml of Hoechst 33342 staining and to ultraviolet light for a mean time of 3 s at 0 h, 8 h, 15 h, 20 h and 27 h. The percentage of metaphase II oocytes decreased significantly when oocytes were stained with Hoechst dye at 0 h, 8 h and 15 h of IVM. There was a decrease in total fertilisation rate and normal fertilisation rate of Hoechest-stained oocytes, independently of the time of Hoechst staining. Hoechst staining produces a significant reduction in oocyte viability when it is used in the early stages of in vitro maturation.     

The binding kinetics and interaction of DNA fluorochromes used in the analysis of nuclei and chromosomes by flow cytometry

Summary The interactions and binding characteristics of DNA dyes used in the flow cytometric analysis of chromatin were studied using human chromosomes and mouse thymocyte nuclei. The kinetics of dye binding and the relationship between fluorescence intensity and dye concentration are presented. Under the conditions used, Hoechst 33258, propidium iodide and chromomycin A3 reach an equilibrium with thymocyte nuclei after approximately 5 min, 20 min and more than 1 h, respectively. The same binding kinetics are observed with Hoechst 33258 and chromomycin when nuclei are stained with a mixture of the two dyes. Sodium citrate, which improves the resolution of flow karyotypes, causes a rapid increase in Hoechst and propidium iodide fluorescence, but a decrease in the fluorescence of chromomycin. The relative peak positions of chromosomes in a flow karyotype are unaffected by sodium citrate addition. The spectral interaction between Hoechst and chromomycin is quantified. There is variation among the human chromosome types in the amount of energy transferred from Hoechst to chromomycin. By measuring the Hoechst and chromomycin fluorescence of each chromosome after Hoechst excitation, it is shown that the amount of energy transferred is correlated to the ratio of the amount of Hoechst to chromomycin bound. Although the energy transfer between the two dyes is considerable, this has little effect on the reproducibility of flow karyotype measurements. The relative peak positions of all human chromosomes in a 64×64 channel flow karyotype, except for the 13 and Y chromosomes, vary in the order of 0.5 channel over a 16-fold change in either Hoechst or chromomycin concentration. This implies that, with the present flow cytometers, variation in staining conditions will have minimal effects on the reproducibility of the relative peak positions in flow karyotypes.In honour of Prof. P. van Duijn     

Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model

Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine residues. The procedure delivers two sperm fractions: annexin V-negative (nonapoptotic) and annexin V-positive (apoptotic). Our aim was to determine whether the sperm fertilizing potential can be improved by selecting a nonapoptotic fraction using MACS. Semen samples (n = 35) were subjected to separation on a density gradient followed by MACS. Extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labeled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and DNA fragmentation using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay. The sperm fertilization potential was assessed using hamster oocyte penetration assay and hamster oocyte-intracytoplasmic sperm injection (ICSI). Annexin V-negative sperm displayed superior quality in terms of high motility, low caspase 3 activation, MMP integrity, and small extent of DNA fragmentation. Annexin V-negative sperm demonstrated higher oocyte penetration capacity but comparable sperm chromatin decondensation (SCD) following ICSI. Conversely, the annexin V-positive sperm presented with poor quality and fertilization potential. The oocyte penetration rate was negatively correlated with apoptotic marker expression, whereas SCD following ICSI was only associated with apoptosis on sperm-damaged membranes. We conclude that apoptosis appears to impact sperm-oocyte penetration rate; however, it does not seem to affect early stages of fertilization such as SCD in spermatozoa of healthy donors. The selection of nonapoptotic sperm by MACS may be used to enhance results of in vitro fertilization by increasing sperm-oocyte penetration.     

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.     

Fertilization and development of quail oocytes after intracytoplasmic sperm injection

The present study was conducted to establish the intracytoplasmic sperm injection (ICSI) method for in vitro fertilization and development in quail. The efficiency of fertilization of oocytes was compared 1) between spontaneous and premature ovulation and 2) among testicular round spermatids, elongated spermatids, and immature and mature spermatozoa. The oocytes were injected with a single spermatozoon or spermatid and cultured for 24 h. Cell division was histologically observed with hematoxylin-eosin (HE) and a nucleus-specific fluorescent dye (DAPI). Five of 30 (16.6%) and 4 of 30 (13.3%) oocytes injected with mature sperm were fertilized in the spontaneous and induced ovulation group, respectively. Those embryos showed development at stages II-VII. Half the number (three of six) of the oocytes injected with testicular spermatozoa were fertilized and developed to stages IV-VII, and two of five oocytes injected with elongated spermatids were fertilized and developed to stage VI. All ooocytes injected with round spermatids were unfertilized. The results demonstrate that intracytoplasmic injection of a single sperm into quail oocyte can activate the oocyte and lead to fertilization. Oocytes prematurely ovulated are capable of fertilizing with mature sperm as are those spontaneously ovulated. In addition, the results suggest that the testicular round spermatids may not possess sufficient oocyte-activating potency but that the elongated spermatids and immature spermatozoa are competent to participate in fertilization and early embryonic development in quail.     

A flow-cytometric method for the separation and quantitation of normal and apoptotic thymocytes.

Using flow cytometry, we describe a method for separating and quantifying normal and apoptotic thymocytes. Apoptosis was induced in isolated thymocytes from immature rats by treatment with the glucocorticoid dexamethasone or the antitumor agent etoposide. Subsequent incubation with the vital bisbenzimidazole dye Hoechst 33342 and the DNA intercalating agent propidium iodide enabled three distinct populations of cells to be identified and sorted by flow cytometry. Dead cells fluoresced red due to propidium iodide whereas normal and apoptotic cells fluoresced blue due to Hoechst 33342. Apoptotic cells were distinguished from normal thymocytes both by their higher intensity of blue fluorescence and by their smaller size as determined by a reduction in forward light scatter. The larger cells, with low blue fluorescence, showed normal thymocyte morphology by electron microscopy and the absence of any DNA fragmentation as measured by agarose gel electrophoresis. In contrast, the smaller cells showed both the morphological characteristics of apoptosis and extensive internucleosomal fragmentation of DNA to multiples of approximately 180 bp. Using this method, a time-dependent induction of apoptosis by dexamethasone, which was inhibited by cycloheximide, actinomycin D, and aurin tricarboxylate, was observed. The method should facilitate mechanistic studies on the induction of apoptosis in thymocytes.