Histone H1 kinase activity during in vitro fertilization of pig follicular oocytes matured in vitro

The present study was conducted to clarify the relationship between histone H1 kinase (H1K) activity and events associated with in vitro fertilization of pig follicular oocytes matured in vitro. Histone H1 kinase has been shown to be homologous with a maturation promoting factor (MPF). Cumulus-oocyte complexes obtained from prepubertal gilts were cultured for 46 h in a modified Waymouth's MB752/1 medium and were then inseminated in vitro with frozen-thawed and preincubated epididymal boar spermatozoa. At 4, 6, 8 and 10 h post insemination, the oocytes were stained with 10 microg/ml Hoechst-33342 and examined under a fluorescent microscope for the stage of fertilization, according to morphological changes of oocyte nuclear chromatin and the extent of sperm penetration. Sperm penetration was observed to occur within 4 h post insemination (20.5%), and the percentage of fertilized oocytes increased (P < 0.01) to 72.9% at 8 h post insemination. Pronuclear formation was observed from 6 h post insemination (3.3%) and the percentage increased (P < 0.01) to 46.8% at 10 h post insemination. In each examination period, H1K activities in unfertilized oocytes at metaphase-II remained unchanged (112.0 fmol/h/oocyte) and were higher (P < 0.01) than those in fertilized oocytes (30.1 fmol/h/oocyte). The H1K activity in fertilized oocytes such as oocytes emitting a second polar body, oocytes with an enlarging sperm head(s) and oocytes with multiple pronuclei did not differ significantly. These results suggest that MPF in pig oocytes is inactivated shortly after sperm penetration and is maintained at the basal level throughout pronuclear formation.     

Morphological events during in vitro fertilization of prepubertal goat oocytes matured in vitro

Experiments were carried out to study morphological changes temporally associated with in vitro fertilization (IVF) of prepubertal goat oocytes and to elucidate some of the abnormalities occurring during this process. The effects of different intervals of insemination on subsequent embryonic development were also studied. Prepubertal goat oocytes collected at slaughter were matured in TCM199 supplemented with estrous goat serum (20%), FSH (10 microg/ml), LH (10 microg/ml) and estradiol-17 beta (1 microg/ml) for 27 h at 38.5 degrees C. Matured oocytes were inseminated with freshly ejaculated spermatozoa following capacitation as described by Younis et al. (37) but with 100 microg/ml heparin. Representative oocytes were fixed every 2 to 4 h from 2 to 28 h after insemination for a study of sperm penetration, sperm head decondensation, meiotic activation, female chromosome decondensation, and male and female pronuclear formation. At the same intervals after insemination, some of the ova were co-cultured on granulosa cell monolayers for up to 9 d. Sperm penetration into the ooplasm was first observed at 4 h post insemination; decondensation of male and female chromatin and formation of male and female pronuclei occurred at 6 to 8 and 10 to 16 h after insemination, respectively. Highest proportions of oocytes were penetrated after exposure to spermatozoa for 8 h. There were no significant differences in ovum penetration after longer insemination intervals. Cleavage was first observed 24 h after insemination. Three types of abnormalities were observed. These were polyspermy, polygyny and asynchrony in the development of the female and male pronuclei, apparently due to a delay in the decondensation of the male pronucleus. Significantly higher proportions of oocytes cleaved (31.2 to 45.5%) after 20, 24 or 28 h insemination intervals than following shorter intervals of exposure to spermatozoa. However, the sperm exposure interval did not significantly affect subsequent embryonic development to the blastocyst stage. Embryos resulting from oocytes exposed to sperm cells for at least 12 h developed further than the 8-cell stage.     

Cryopreservation of unfertilized mouse oocytes from inbred strains by ultrarapid freezing

Unfertilized mouse oocytes from inbred strains (BALB/c, C3H/He and C57BL/6) were frozen ultrarapidly by direct plunging into liquid nitrogen, immediately after exposure to a highly-concentrated solution (DAP 213: 2 M dimethyl sulphoxide, 1 M acetamide, and 3 M propylene glycol in PB 1), and were later thawed in a 37 degrees C waterbath. After thawing, 76.8-90.9% of recovered oocytes were morphologically normal. Following fertilization in vitro of cryopreserved oocytes, the proportion of 2-cell embryos 24 h after insemination ranged from 70.7% to 83.4%. Nearly all 2-cell embryos obtained from cryopreserved oocytes were transferred to the oviducts of pseudopregnant recipients and 31.0-43.0% of 2-cell embryos developed into normal young.     

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.     

High survival rate of unfertilized mouse oocytes after vitrification

Unfertilized mouse oocytes were cooled rapidly by directly plunging them into liquid nitrogen, immediately after exposure to a highly concentrated solution (modified VS1: 2.53 M-dimethyl sulphoxide, 2.36 M-acetamide, 1.19 M-propylene glycol, 5.4% (w/v) polyethylene glycol (Mr 8000) in PB1), and later warmed in a 37 degree C waterbath. After warming, 305 out of 348 oocytes (87.6%) were morphologically normal. After fertilization in vitro of cryopreserved oocytes, the proportions of pronuclear oocytes and 2-cell embryos 5 and 24 h after insemination were 81.6% (124/152) and 78.4% (120/153), respectively. All 2-cell embryos obtained from cryopreserved oocytes were transferred to the oviducts of pseudopregnant recipients and 45.8% (55/120) developed to normal young.     

Cryopreservation of mouse spermatozoa]

The spermatozoa of cauda epididymis of mature mice were suspended in 3% skim milk in distilled water supplemented with 12, 15, 18 or 21% (W/V) raffinose. The suspension of spermatozoa were frozen in liquid nitrogen gas for 10 min, then stored in liquid nitrogen (-196 degrees C). The frozen suspensions of spermatozoa were thawed by rapid warming in water bath at room temperature. For removing the cryopreservative solution, a pair of syringes connected with a three stop cock and a filter unit (pore size 0.45 mu) were used. Highest sperm motility was obtained after 1 hr of thawing from the cryopreservative solution containing 18% raffinose and 3% skim milk. These cryopreserved spermatozoa were used for fertilization in vitro. The proportion of pronuclear oocytes was 35.9% (74/206) 6 hr after insemination, and the proportion of 2-cell embryos was 33.6% (42/125) 28 hr after insemination. All 2-cell embryos were transferred to the oviducts of pseudopregnant recipients and 45.2% (19/42) developed to normal young.     

Effect of sperm concentration during preincubation in a defined medium on fertilization in vitro of pig follicular oocytes

Pig follicular oocytes cultured in a defined medium for 28-29 h were inseminated in vitro by epididymal or ejaculated boar spermatozoa that were preincubated in a modified KRB solution at various sperm concentrations for 4 h at 37 degrees C. Sperm concentration at insemination was 2 X 10(6) cells/ml. When epididymal spermatozoa were preincubated at concentrations of 4-16 X 10(8) cells/ml, 71-75% of oocytes were penetrated. In contrast, preincubation at a low concentration (0.8 X 10(8) cells/ml) resulted in a low penetration rate (11%). Epididymal spermatozoa preincubated at a concentration of 4 X 10(8) cells/ml could also penetrate denuded oocytes. None of the oocytes were penetrated by epididymal spermatozoa that were exposed to seminal plasma before preincubation or by ejaculated spermatozoa. After preincubation, whiplash motility was observed in the epididymal spermatozoa, but not in the ejaculated spermatozoa.     

Laparoscopical intrauterine insemination with different doses of fresh,conserved, and frozen semen for the production of ovine zygotes

The objective of the present study was to increase the efficiency in the production of ovine zygotes suitable for microinjection via laparoscopical intrauterine insemination. In the first part of the study, 71 ewes of three different breeds were inseminated with one of two different insemination doses (50 x 10(6) or 300 x 10(6) sperm per inseminate) and semen was either freshly diluted, liquid conserved, or frozen/thawed, or females were mated by a fertile ram (controls). In the second part, a total of 46 ewes was inseminated with 300 x 10(6) freshly diluted sperm to verify the findings from part 1 and to unravel effects of breed and age of donor ewe. The oviducts were flushed 24-26 h after insemination and the success of insemination was assessed by microscopical examination. Recovery rates were 78.0+/-26.4 and 72.1+/-24.6% in parts 1 and 2 of the study, respectively. Of these oocytes 62.3 and 62.8% (parts 1 and 2, respectively) were fertilized. In part 1, the highest proportion (64.7%) of pronuclear stages was observed in the group inseminated with 300 x 10(6) freshly diluted semen and was significantly higher compared to the groups inseminated with 50 x 10(6) freshly diluted semen (25.5%, P<0.001), 300 x 10(6) liquid conserved semen (49.0%, P<0.001), or 50 x 10(6) frozen/thawed semen (39.6%, P<0.05). In the control group, the proportion of pronuclear stages amounted to 60.2%. Irrespective of the type of sperm conservation, the overall fertilization rate (zygotes plus 2-cell stages) was higher (P<0.05) following insemination with 300 x 10(6) sperm (68.2%) compared to 50 x 10(6) sperm (56.8%). In part 2, the proportion of pronuclear stages reached 54.2% with an overall fertilization rate of 62.9%. These results were affected by breed and age of the donor as crossbred and younger (<3 years) animals yielded the highest proportion of pronuclear stages. The present study shows that freshly diluted semen at a dosage of 300 x 10(6) sperm yields the highest fertilization rates, the greatest proportion of pronuclear stages and the lowest proportion of mature unfertilized oocytes. Further increases in yields of pronuclear stages can possibly be achieved by selection of sheep from the best suited breed and younger than 3 years of age.     

Offspring from intracytoplasmic sperm injection of aged mouse oocytes treated with caffeine or MG132

Postovulatory mammalian oocytes age significantly in culture. B6D2F1 or ICR strain mouse oocytes were collected 16 h after hCG injection and then cultured for up to 40 h post hCG at 37 °C under 5% CO(2) in air. After intracytoplasmic sperm injection (ICSI), B6D2F1 and ICR oocytes lost full-term developmental potential by 30 h and 26 h after hCG administration, respectively. However, using supplementation with 10 mM caffeine or 1-5 μM of MG132, we could obtain live offspring from oocytes at 34 h (BDF1, 5%-21%) or 28 h (ICR, 5%-18%), whereas none were obtained from untreated aged oocytes. Caffeine maintained normal meiotic spindle morphology, whereas MG132 maintained maturation-promoting factor activity. These treatments did not affect the potential of fresh oocytes for fertilization and subsequent development. Thus, it should be safe to use these chemicals in routine in vitro fertilization and offspring could be generated by ICSI of aged fertilization failed oocytes.     

Effects of the 7 21 Robertsonian translocation on fertilization rates and preimplantation development of bovine oocytes in vitro

A study was conducted to investigate the effect of the 7/21 Robertsonian translocation on fertilization and subsequent development of bovine oocytes matured in vitro. Semen from Japanese Black bulls, 2 with a normal karyotype (Bulls A and B) and 2 that were heterozygous for the 7/21 translocation (Bulls C and D), was used in this study. In vitro matured bovine oocytes were inseminated with frozen-thawed sperm capacitated with heparin. After insemination, oocytes were cultured at 38.5 degrees C on a monolayer of cumulus cells in TCM-199 supplemented with 5% superovulated cow serum and 0.5 mM sodium pyruvate in an atmosphere of 2% CO2 in air. Cleavage rate was evaluated at 54 h after insemination, and development of embryos to the blastocyst stage was observed 7 to 10 d post insemination. There was no difference in the fertilization rate among the 4 bulls. Although the cleavage rate of oocytes inseminated with semen from Bull C (heterozygote) was lower (P < 0.05) than that obtained with semen from Bull B (normal), the blastocyst formation rate did not differ among the 4 bulls. These results indicate that the 7/21 Robertsonian translocation had no effect on the fertilization and blastocyst formation rates of bovine in vitro-matured oocytes.     

Effects of electrical stimulation before or after in vitro fertilization on sperm penetration and pronuclear formation of pig oocytes

The effects of exposure of pig oocytes to an electrical pulse on sperm penetration and pronuclear formation were determined before or after in vitro fertilization (IVF). After in vitro maturation (IVM) or after collection from oviducts of unmated gilts, pig oocytes either were not exposed or were exposed to an electrical pulse (a 10 sec pulse at 4.0 V mm^?1 AC followed by a 30 μsec pulse at 120 V mm^?1 DC), followed 30 min later by IVF. The incidence of male pronuclear formation of both IVM and in vivo-matured oocytes at 12 hr after insemination was decreased from 59% and 100%, respectively, to 2% and 36%, respectively, by the electrical pulse, but the penetration rates (88–100%) and polyspermic rates (79–100%) were not affected by exposure to an electrical pulse. Similarly, when pig IVM oocytes were exposed to an electrical pulse at 6 hr after insemination, electrical activation did not decrease penetration rates (93% vs. 90%), polyspermic rates (83% vs. 91%), or number of spermatozoa in penetrated oocytes (4.0 ± 0.5 vs. 4.6 ± 0.5) but did decrease the rate of male pronuclear formation from 58% to 18%. When oocytes were examined at 6 hr after insemination, 75% of them had been penetrated and resumed meiotic progression, but all sperm heads in penetrated oocytes were fully condensed or only partially decondensed. The percentage of penetrated eggs with multiple female pronuclei was increased when oocytes were exposed to an electrical pulse in all experimental series. In summary, electrical activation of pig oocytes before or just after IVF does not prevent sperm penetration but does inhibit male pronuclear formation and increases the formation of multiple female pronuclei. © 1993 Wiley-Liss, Inc.     

Pregnancies from vitrified equine oocytes collected from super-stimulated and non-stimulated mares

The objectives were to compare embryo development rates after transfer into inseminated recipients, vitrified thawed oocytes collected from super-stimulated versus non-stimulated mares. In vivo matured oocytes were collected by transvaginal, ultrasound guided follicular aspiration from super-stimulated and non-stimulated mares 24-26 h after administration of hCG. Oocytes were cultured for 2-4 h prior to vitrification. Cryoprotectants were loaded in three steps before oocytes were placed onto a 0.5-0.7 mm diameter nylon cryoloop and plunged directly into liquid nitrogen. Oocytes were thawed and the cryoprotectant was removed in three steps. After thawing, oocytes were cultured 10-12 h before transfer into inseminated recipients. Non-vitrified oocytes, cultured 14-16 h before transfer, were used as controls. More oocytes were collected from 23 non-stimulated mares (20 of 29 follicles), than 10 super-stimulated mares (18 of 88 follicles; P < 0.001). Of the 20 oocytes collected from non-stimulated mares, 12 were vitrified and 8 were transferred as controls. After thawing, 10 of the 12 oocytes were morphologically intact and transferred into recipients resulting in one embryonic vesicle on Day 16 (1 of 12 = 8%). Fourteen oocytes from super-stimulated mares were vitrified, and 4 were transferred as controls. After thawing, 9 of the 14 oocytes were morphologically intact and transferred into recipients resulting in two embryonic vesicles on Day 16 (2 of 14 = 14%). In control transfers, 7 of 8 oocytes from non-stimulated mares and 3 of 4 oocytes from super-stimulated mares resulted in embryonic vesicles on Day 16. The two pregnancies from vitrified oocytes resulted in healthy foals.     

The effects of spermatozoal irradiation with X-rays on chromosome abnormalities and on development of mouse zygotes after delayed fertilization

The chromosome complements of zygotes derived from oocytes aged post ovulation and fertilized in vivo with X-ray-irradiated sperm were studied. Ovulation was induced by an injection of luteinizing hormone-releasing hormone (LHRH) at pro-estrus and fertilization was achieved by artificial insemination at 13 h and 24 h after LHRH in order to obtain embryos from unaged and aged (12 h post-ovulation) oocytes respectively. Post-ovulatory aging prior to fertilization did not significantly affect the percentage of zygotes with irradiation-induced chromosome abnormalities. However, post-ovulatory aging had a negative effect on the morphology of male as well as female pronuclear chromosomes of the first cleavage metaphase. When fertilized with control spermatozoa this effect was apparent in both the male and the female pronucleus. When unaged oocytes were fertilized with X-irradiated spermatozoa chromosome morphology was also adversely affected in both pronuclei. In zygotes from aged oocytes, there was an extra negative effect of X-rays on the male pronuclear chromosomes only. After fertilization with X-irradiated sperm 27% of zygotes from aged oocytes were arrested at interphase compared to 7% from unaged oocytes. We suggest that post-ovulatory aging and X-rays affect the male and female pronuclear chromatin structure after fertilization. These chromatin alterations could interact with DNA lesions induced in the spermatozoa prior to fertilization, such that development to first cleavage can be blocked.     

Exposure of bovine oocytes to EGF during maturation allows them to develop to blastocysts in a chemically-defined medium

When cumulus-enclosed bovine oocytes were cultured for 24 h in serum-free medium containing 0 to 50 ng/ml EGF, the proportions of oocytes reaching metaphase II were higher (P < 0.05) in the presence of 30 ng/ml EGF (88.1 +/- 1.3%) than under control conditions (65.5 +/- 3.5%) or in the presence of 10 ng/ml (73.9 +/- 4.5%) and 50 ng/ml (73.6 +/- 4.0%) EGF. When oocytes matured under these conditions were inseminated in vitro, the proportions of oocytes penetrated were higher (P < 0.05) in 10 to 50 ng/ml EGF (96.7 +/- 3.3 to 100%) than in its absence (77.9 +/- 8.9%). However, the proportions of penetrated oocytes with male and female pronuclei did not differ among the different groups (96.7 +/- 3.3 to 100%). When oocytes were matured under the same conditions, fertilized in vitro, and cultured until 192 h post insemination in a chemically-defined medium, the proportion of embryos at the >/=2-cell stage was higher (P < 0.05) in the groups treated with 30 ng/ml (96.1 +/- 2.5%) and 50 ng/ml (90.6 +/- 3.5%) EGF than in the controls (71.8 +/- 3.1%) at 48 h post insemination. Although there were no differences in the proportions (37.3 +/- 5.3 to 47.2 +/- 5.8%) of >/=morulae at 144 h post insemination among treatments, the proportion of embryos developing to the blastocyst stage was higher (P < 0.05) in the presence of 10 to 50 ng/ml EGF (16.5 +/- 2.0 to 20.8 +/- 4.9%) than in control medium (3.4 +/- 2.1%). The mean blastocyst cell number at 192 h post insemination did not differ between culture media in the presence (91 to 107 cells) and the absence (116 cells) of EGF (10 to 50 ng/ml) during maturation. Thus, higher proportions of oocytes matured in serum-free medium with EGF than without EGF could develop to the blastocyst stage in a chemically-defined medium after in vitro fertilization. These results indicate that EGF can induce not only nuclear maturation but also cytoplasmic maturation of cumulus-enclosed bovine oocytes in vitro.     

Effect of semen preparation on IVF of prepubertal goat oocytes

The aim of these experiments was to study the effects of different methods of washing and selection of spermatozoa on the IVF of IVM oocytes from prepubertal goats. Fresh ejaculates from 3 males of proven fertility were processed according to the following treatments: 1) centrifugation in TALP, 2) centrifugation in sucrose-based Ficoll medium, 3) centrifugation in Percoll gradients at 40 and 80%, 4) by swim-up and 5) by dilution of spermatozoa (1:40) in (1:1) TALP. In all 5 treatments spermatozoa were incubated for 45 min with 100 microg/mL of heparin and then added to Fert-TALP. Oocytes were matured for 27 h in TCM-199 supplemented with 20% estrous goat serum (EGS), FSH, LH and estradiol-17beta. Spermatozoa (4x10(6) cells/mL) were coincubated with oocytes in 100 microL of Fert-TALP with hypotaurine for 24 h, after which the oocytes were transferred to a granulosa cells monolayer in TCM-199 plus 10% of EGS for 24 h (48 h post insemination). At 17 h post insemination a sample of sperm-exposed oocytes was taken and stained in lacmoid to observe sperm penetration and the formation of pronuclei. At 48 h post insemination the cleavage rate of oocytes was evaluated. Motility, viability and acrosome status of the spermatozoa were evaluated immediately after the mixing of the ejaculates, after washing and selection treatments, and after incubation with heparin and at 17 h post insemination. The different ejaculate treatments did not affect the penetration and cleavage rates of oocytes. At 48 h post insemination the cleavage rate was 46.9, 36.6 and 29.0% for dilution, Ficoll and swim-up preparations, respectively. Only the swim-up protocol improved sperm motility and viability compared with that of the initial semen sample and with the other sample treatments. At 17 h post insemination the semen parameters were the same for all sperm sample treatments.     

Cleavage development of bovine oocytes fertilized by sperm injection

Whole in vitro capacitated bovine spermatozoa were microinjected directly into the ooplasm of in vitro matured bovine oocytes in order to determine whether oocytes fertilized by sperm injection could undergo normal pronuclear formation and cleavage development. Immature oocytes recovered from follicles (2-5 mm) of unstimulated ovaries were cultured for 24-25 h in modified TCM 199 medium supplemented with heat-treated day 20 cow serum, luteinizing hormone (LH), and estradiol 17-B. In vitro capacitated, frozen-thawed spermatozoa were injected into the ooplasm, and the injected oocytes were cultured for an additional 24-28 h. Twenty-one percent (21/101) of the sperm-injected oocytes contained a sperm within the ooplasm; however, only 2% (2/101) cleaved. The remaining oocytes either did not contain a sperm or had degenerated. After oocyte activation induced by a 5 min incubation in 1 microM A23187, sperm nuclear decondensation occurred in the A23187-activated, injected oocytes but not in the unactivated, injected controls (37% vs. 0% after 3 h). Those injected, activated oocytes that contained a male pronucleus also exhibited a female pronucleus and second polar body. Furthermore, a significantly higher number (28%, 6/21) of the injected, activated oocytes cleaved to a two- to four-cell stage after 48 h than did the injected, unactivated oocytes (4%). These results indicate that, unlike hamster and rabbit oocytes, bovine oocytes are not sufficiently stimulated by the injection procedure to complete meiosis, but, upon activation by calcium ionophore, they will undergo normal-appearing cleavage development following fertilization by sperm injection.     

In vitro fertilization rate of horse oocytes with partially removed zonae

Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 mu M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1-2 x 10(7)/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stained with 1% orcein. The sperm penetration rate of zona-free oocytes was 83%, whereas the sperm penetration rate was very low (1 to 3%) in the cumulus-enclosed or zona-intact oocytes. In Experiment 2, denuded zona-intact oocytes were placed in PBS supplemented with 10% fetal bovine serum 1 h before the end of in vitro maturation. The zona pellucida was micromanipulated with a metal microblade under x 100 magnification within 20 min of treatment with 0.3 M sucrose. For partial zona dissection, a slit in the zona pellucida was made. For partial zona removal, oocytes were transferred to protein-free PBS to fix the oocytes on the bottom of the Petri-dish and to remove a piece of the zona pellucida. Micromanipulated oocytes were subjected to in vitro fertilization as described above. Zona-intact and zona-free oocytes treated with sucrose solution for 20 min were used as controls. The penetration rates were 4 (2 57 ), 12 (7 58 ), 52 (31 60 ), and 86% (44 51 ) for zona-intact, partially zona dissected, partially zona removed, and zona-free oocytes, respectively. Proportions of oocytes with monospermic penetration were 100 (2 2 ), 57 (4 7 ), 58 (18 31 ), and 34% (15 44 ), respectively. In Experiment 3, sperm penetration and male pronucleus formation in the partially zona removed oocytes were examined at 2.5 to 20.0 h of insemination. Sperm penetration started 2.5 h post-insemination (22%, 11 49 ), and increased to 38% (21 55 ) at 5 h, to 46% (23 50 ) at 10 h, and to 56% (27 48 ) at 20 h. The transformation of sperm heads into male pronuclei was first observed 10 h post insemination. These results indicate that assisted fertilization techniques may be a useful tool for achieving fertilization and embryo production in vitro in horses.     

Sequential transformations of human sperm nucleus in human egg

In-vitro insemination of human zona-free oocytes prepared from oocytes that failed to fertilize in an in-vitro fertilization programme was used as an experimental model to study the time course and morphological events during the development of sperm nuclei into male pronuclei. At 30 min after insemination, 22 eggs were cultured in a CO2 incubator for further 3.5 h and 17 eggs were placed individually between a slide and coverslip for randomly repeated microscopical observations in a controlled environment for at least 3.5 h. Simultaneous arrest of maternal meiosis and sperm nuclear development occurred in 36.4% (8/22) eggs cultured in the CO2 incubator and 47.1% (8/17) of those cultured between a slide and coverslip. Sequential transformation of the human sperm nucleus in human eggs was studied in 6 eggs that showed continuous development of sperm nuclei into male pronuclei during at least 3.5 h after insemination. The early sperm nuclear development in human egg ooplasm can be divided into three phases: the sperm nucleus first decondenses (phase 1) then partly recondenses (phase 2) before expanding again to form an early male pronucleus (phase 3). The prepronuclear stages (phases 1 and 2) took about 60 min each and the pronuclear formation (phase 3) began between 120 and 170 min after insemination. Early pronuclear formation was associated with the occurrence of dense outline material, probably a precursor of the future pronuclear membrane, around the recondensed nucleus in re-expansion (phase 3). Between 30 and 60 min after the beginning of phase 3, numerous (greater than 20) dense grains, considered as nucleolar precursors, were clearly visible inside the growing male pronucleus. Moreover, we have examined sperm nuclear changes in some eggs in which the progression of late meiosis was abnormal. Meiotic arrest of maternal chromatin was always associated with arrest of sperm head development. In 75% (6/8) of the eggs arrested in the metaphase II stages and in 87.5% (7/8) of the eggs arrested in late anaphase II, sperm nuclear development was stopped at the decondensed and recondensed stages, respectively. We have always observed male pronuclei when a maternal pronucleus was present in the egg. These observations suggested that maternal chromatin and sperm nuclear development are probably regulated by common factor(s).     

Development of in vitro matured and fertilized bovine embryos, cultured from days 1-5 post insemination in either Menezo-B2 medium or in HECM-6 medium

Bovine embryos were produced by in vitro maturation and fertilization of abattoir oocytes. The embryos were randomly allocated either to coculture with bovine oviduct cells in Menezo-B2 medium (control group), or to culture in the defined HECM-6 medium. At Day 5 after insemination the HECM-6 embryos were transferred to Menezo-B2 medium with (HECM-B2/BOEC) or without (HECM-B2) oviduct cells for further culture. The proportion of cleaved embryos and blastocysts, the morphology and the speed of development were compared for the control and HECM groups. Significantly more HECM-6 embryos than control embryos cleaved (88 +/- 3% vs 76 +/- 5% (+/- SD)). Significantly fewer blastocysts developed in the HECM-B2 than in the control group (28 +/- 2% vs 35 +/- 3%), in addition the speed of development was delayed and the morphology was impaired. In the HECM-B2/BOEC group no differences in neither morphology, blastocyst rates (31 +/- 8%) nor speed of development could be demonstrated, when compared with the control group. A portion of the control and HECM-B2 embryos were vitrified at Days 7-8, but no differences were noted in survival or morphology at 48 and 72 h post thawing. It can be concluded, that the defined medium HECM-6 can support bovine embryonic development through the 8-16 cell in vitro block stage without the use of coculture in a reliable way. In our system it was however necessary to transfer the embryos at Day 5 to coculture in Menezo-B2 medium to ensure optimal continuation of development.     

Presence of granulosa cells during oocyte maturation improved in vitro development of IVM-IVF bovine oocytes that were collected by ultrasound-guided transvaginal aspiration

The effects of granulosa cells in maturation media on male pronuclear formation and in vitro development of in vitro-matured and fertilized (IVM-IVF) bovine oocytes were examined. In Experiment 1, cumulus-oocyte complexes (COCs) were aspirated from follicles of slaughterhouse ovaries and classified into 4 morphological categories according to the surrounding cumulus cells: Grade 1 (> 4 layers), Grade 2 (3 to 4 layers), Grade 3 (1 to 2 layers) and Grade 4 (denuded). Oocytes were co-cultured with or without granulosa cells (1 x 10(6) cells/ml) for 21 to 22 h. At 18 and 192 h after insemination, the abilities of oocytes to form a male pronucleus and develop up to the blastocyst stage in vitro were determined, respectively. The presence of granulosa cells during maturation did not affect (P < 0.05) the ability of oocytes in Grades 1 and 2 to form a male pronucleus and to develop to the blastocyst stage in Grades 1 and 4. However, the incidence of male pronuclear formation in Grades 3 and 4 and in vitro development to the blastocyst stage in Grades 2 and 3 was higher (P < 0.05) when COCs were cultured in the presence of granulosa cells than when cultured in the absence of granulosa cells. In Experiment 2, COCs collected by ultrasound-guided aspiration were co-cultured with or without granulosa cells, fertilized and cultured as described above. The incidence of blastocysts at 192 h after insemination was higher (P < 0.05) when COCs were cultured for maturation in the presence of granulosa cells (24%) than in the absence of granulosa cells (12%). These results demonstrate that supplementation of maturation medium with granulosa cells improves the quality of oocytes with relatively few cumulus cell layers, as determined by male pronuclear formation and in vitro development. We also conclude that this supplementation effectively improves the developmental ability of bovine IVM-IVF oocytes that were collected by ultrasound-guided transvaginal aspiration.