Biomedical subjects

A O Trounson

Publications and source records attributed to A O Trounson.

At least 19 recordsLinked to original sources

PubMed1992

Influence of a capacitation period on human sperm acrosome loss.

The present study investigates whether a 5 hour capacitation period modifies the ability of human spermatozoa to undergo induced acrosomal loss. Human sperm acrosomal loss was induced by treatment with either the calcium ionophore A23187, low concentrations of the phospholipid dilauroylphosphatidylcholine (PC12), or 2 hours incubation in conditioned medium prepared from human cumulus cells (CM/CC). The use of a dual staining method (FITC-ConA and Hoechst 33258) for simultaneous assessment of acrosomal status and viability demonstrated that induction of acrosomal loss with calcium ionophore was not dependent on a capacitation period. A short (5 hour) incubation period was not sufficient to induce acrosomal loss with CM/CC above spontaneous acrosome reaction rates in medium alone. A significant capacitation-dependent increase (P < 0.05) in acrosomal loss was observed when human spermatozoa were incubated with PC12. Induction of acrosomal loss of capacitated human spermatozoa with PC12 therefore provides a simple assay for the simultaneous assessment of human sperm capacitation and the acrosome reaction in vitro.

Acrosome↗

PubMed1992

Follicular steroidogenesis and oocyte maturation after superovulation of goats (Capra hircus) with gonadotrophins.

Follicles were sampled at three different times after treatment with 1200 iu pregnant mares' serum gonadotrophin (PMSG) or 12 mg ovine follicle-stimulating hormone (FSH), and from untreated control animals. The meiotic status and protein synthesis of the oocyte from each follicle was determined and correlated with the intrafollicular concentration of oestradiol and progesterone. Significantly higher amounts of oestradiol were present in PMSG-treated animals at sponge withdrawal than in FSH-treated and control goats. Twenty hours later, both oestradiol and progesterone concentrations in the PMSG group were higher than those in the FSH group, and were equivalent to control animals at the onset of oestrus. At 18 h after the administration of human chorionic gonadotrophin (hCG), oestradiol decreased markedly in all three treatment groups, whereas progesterone remained significantly higher in PMSG-treated follicles. Although these high concentrations of intrafollicular steroids were associated with a higher incidence of premature condensation of chromatin in oocytes, the two events were not causally related. Moreover, cytoplasmic maturation was not prematurely activated in these oocytes and a changed pattern of protein synthesis was observed in oocytes from all three treatment groups after the hCG injection. Whereas disturbances in follicular steroidogenesis of oestradiol and progesterone occur in vivo in goats superovulated with PMSG, they do not underlie the premature activation of the initial stages of nuclear maturation in oocytes but are associated with normal cytoplasmic maturation.

Administration, Intravaginal↗

PubMed1991

Staining of the inner acrosomal membrane of human spermatozoa with concanavalin A lectin as an indicator of potential egg penetration ability.

OBJECTIVE: To determine the sensitivity and functional significance of the fluorescein isothiocyanate concanavalin A (FITC-ConA) staining method of assessment of acrosomal status. DESIGN: Treatments were assessed for their ability to induce human sperm acrosomal loss. Penetration of zona-free human eggs by treated spermatozoa was subsequently determined. SETTING: Zona-free human eggs were obtained from the Infertility Medical Centre, Richmond, Victoria, Australia. PATIENTS, PARTICIPANTS: None. INTERVENTIONS: None. MAIN OUTCOME MEASURE: Acrosomal loss of human spermatozoa was determined at 4 hours and 10 hours of treatment incubation. Penetration of zona-free human eggs was assessed 16 hours after reinsemination. RESULTS: Human spermatozoa incubated in a strontium- or lanthanum-based medium, or T6 + 10% maternal human serum (HS) supplemented with 12 mM 8-bromo cyclic guanosine 3,5'-monophosphate and 10 mM imidazole for a 4-hour period before transfer to fresh T6 + 10% HS for a further 6 hours, demonstrated a significant increase (P less than 0.05) in acrosomal loss compared with T6 + 10% HS for a total 10-hour incubation. This increase in acrosomal loss with test treatments correlated with an increase in the development of pronuclei of zona-free human eggs (r = +0.98). CONCLUSIONS: The FITC-ConA staining procedure therefore reflects biological function as assessed by the penetration of zona-free human eggs and consequently provides a further research tool for the investigation of the human sperm acrosome reaction.

Acrosome↗

PubMed1991

Formulation of a complex serum-free medium (CSM) for use in the co-culture of mouse embryos with cells of the female reproductive tract.

Co-culture of pre-implantation embryos with cells of the reproductive tract requires a medium that is beneficial to both embryos and cells. However, many studies in this area utilize media originally formulated for specific cell lines. In the present study, a complex serum-free medium (CSM) was formulated on the basis of the ionic compositions of existing embryo culture media and mouse oviductal fluid as well as the concentrations of growth factors that appear to benefit mouse embryo development. The study began by investigating the effect of altering the concentrations of K+ ions (0-40 mM) and sulfate ions (0-10 mM) in embryo culture media on the development of 2-cell mouse embryos. Mouse embryos showed improved cell numbers at the blastocyst stage when cultured in 10 mM K+ compared with Whittingham's T6 medium. Embryos were also cultured in T6 supplemented with bovine serum albumin (BSA) containing various concentrations of insulin, insulin-like growth factors I and II, fibroblast growth factor, and epidermal growth factor. Insulin concentrations of 100 ng mL-1 significantly (P less than 0.05) improved the cell numbers of 2-cell embryos cultured to the morulae and blastocyst stages compared with those cultured in T6 + BSA alone. CSM was formulated on the basis of the results of these experiments and was found to support both improved development of 2-cell mouse embryos and the culture of mouse fibroblast and mouse oviduct cells.

Animals↗

PubMed1991

A simple rapid 4.5 M dimethyl-sulfoxide freezing technique for the cryopreservation of one-cell to blastocyst stage preimplantation mouse embryos.

This study applies a 4.5 M dimethyl-sulfoxide freezing procedure, developed for 2-cell mouse embryos, to pronuclear to hatched blastocyst stage mouse embryos. The embryos were plunged into liquid nitrogen after 3 min equilibration at room temperature, or 3-60 min equilibration at 0 degrees C. Equilibration at 0 degrees C gave survival rates as high as or higher than rates after equilibration at room temperature. Optimal blastocyst formation, or re-expansion, rates for embryos frozen after equilibration at 0 degrees C were 76% for pronuclear stage embryos and 96-100% for 2-cell to mid-blastocyst stage embryos. The optimal rates of fetus formation, per embryo frozen, ranged from 62 to 88% for pronuclear to mid-blastocyst stage embryos. These results compared favourably with non-frozen control embryos (80-100% blastocyst formation, and 67-78% fetus formation).

Analysis of Variance↗

PubMed1991

An association between chromosomal abnormalities in rapidly frozen 2-cell mouse embryos and the ice-forming properties of the cryoprotective solution.

This paper investigates the effect of straw handling on the viability of 2-cell mouse embryos rapidly frozen in dimethyl sulphoxide (DMSO) solutions. During the brief (3 min) equilibration step, straws were either rotated periodically to keep the embryos in suspension, or kept still to allow the embryos to settle onto the the inner surface of the straw. The effects of these straw movements were tested with cryoprotectant solutions containing 1.5, 3.0 or 4.5 M-DMSO. Rapidly cooled straws containing 4.5 M-DMSO vitrify throughout on cooling, but ice forms on warming. The survival and normality of embryos frozen in 4.5 M-DMSO was not influenced by straw handling as 91-92% formed blastocysts in vitro, 77-78% formed normal fetuses, and no chromosomal rearrangements were observed. In solutions containing less than 4.5 M-DMSO ice formation occurred throughout (1.5 M-DMSO), or in parts (3.0 M-DMSO) of the cryoprotectant during cooling. The viability of embryos frozen in 3.0 or 1.5 M-DMSO solutions was reduced both in vitro and in vivo and structural chromosome aberrations, predominantly tri- and quadri-radial rearrangements, were observed. The reduction in embryo viability, and the chromosomal damage was particularly pronounced in embryos frozen in 3.0 M-DMSO in straws which were rotated during the equilibration step (47% blastocysts, 15% fetuses, 77% chromosome rearrangements). The results indicate that rapid freezing of 2-cell mouse embryos in 4.5 M-DMSO is safe and efficient, whereas freezing at lower DMSO concentrations is associated with severe chromosome damage, and reduced viability in vitro and in vivo.

Animals↗

PubMed1990

Assessment of the human sperm acrosome reaction using concanavalin A lectin.

A method for assessment of the human sperm acrosome reaction is reported using fluorescein isothiocyanate (FITC)-conjugated Concanavalin A (ConA). The technique involved labelling prefixed spermatozoa, where only those spermatozoa that showed a complete loss of the acrosome bound FITC-ConA to the acrosomal region. Competitive sugar binding studies demonstrated that binding of ConA lectin to the acrosomal area of human spermatozoa was inhibited in the presence of 0.2 M D-mannose. Staining with the supravital stain Hoechst 33258 (H258) concomitantly with FITC-ConA allowed determination of only those spermatozoa that had undergone a true and not degenerative acrosomal loss. Incubation of human spermatozoa with 0, 1, 5, and 25 microM calcium ionophore, A23187, for 60 min demonstrated that changes in acrosomal status due to the different treatment protocols may be determined by the dual-staining method. Electron microscopy studies revealed that gold-conjugated ConA bound specifically to the surface of the inner acrosomal membrane of acrosome-reacted spermatozoa. A significant correlation (r = +.97) between transmission electron microscopy (TEM) and FITC-ConA labelling methods of acrosomal status assessment was achieved. The simple ConA labelling procedure reported here therefore provides a reliable method for quantitation of the physiological acrosome reaction of a population of human spermatozoa.

Acrosome↗

PubMed1990

Allocation of cells to the inner cell mass and trophectoderm of 3/4 mouse embryos.

The allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) was investigated at 6-h intervals from 78 h to 102 h after hCG injection in 3/4 mouse embryos to determine the effect of removal of a single blastomere at the 4-cell stage on early differentiation. The procedures used to produce 3/4 embryos had little effect on embryo development. Embryos that had a single blastomere removed and then re-aggregated (RA embryos) had the same total number of cells as untreated (UT) embryos except at 78 h (P less than 0.05) and 102 h (P less than 0.01) post hCG where there were slightly less cells in RA embryos. Three-quarter embryos always had significantly fewer cells than RA embryos (P less than 0.001), with an average of 74% of the total cell number of RA embryos. As expected, 3/4 embryos always had significantly fewer cells in the ICM and TE compared with RA embryos (P less than 0.001). However, the ICM:TE ratio was also significantly lower in 3/4 embryos compared with RA embryos at 84, 96, and 102 h post hCG, indicating that the allocation of cells to the ICM and TE was disturbed. The ICM:TE ratio of 3/4 embryos could not be manipulated if either an early- or late-dividing blastomere was selectively biopsied at the 4-cell stage; this suggests that the known preferential contribution of an early-dividing blastomere to the ICM is not cell autonomous.

Animals↗

PubMed1990

Maximum survival of frozen goat embryos is attained at the expanded, hatching and hatched blastocyst stages of development.

Embryos were collected 5, 6 and 7 days after ovulation to determine their optimum stage of development for cryopreservation. The embryos were frozen using either glycerol or dimethyl sulfoxide (DMSO) as cryoprotectant, and then thawed and cultured to determine their viability. The rate of survival increased as the stage of development of the embryos increased. All hatched blastocysts survived freezing regardless of which cryoprotectant was used, whereas none of the 22 morulae frozen with DMSO survived, and only 12 of the 63 morulae frozen with glycerol survived. At the early blastocyst stage there was a significantly different result between freezing procedures (P less than 0.05) with greater survival rates obtained using DMSO rather than glycerol. The viability of goat embryos obtained at the early to hatched blastocyst stages, when collected 7 days after ovulation, was assessed by transferring half the embryos singly to recipients and freezing the remainder before transfer. When pregnancy was tested at the 7th week of gestation, the survival rate of fresh embryos was 67/111 (60%) compared with 64/109 (59%) for frozen-thawed embryos.

Animals↗

PubMed1990

Premature condensation of chromatin induced in goat (Capra hircus) oocytes after gonadotrophin treatment.

In comparison with ovine follicle stimulating hormone (FSH), superovulation of goats with pregnant mare serum gonadotrophin (PMSG) produced premature ovulations within 48 h of drug administration. To test the hypothesis that this may be associated with a differential effect of the two drugs on oocyte maturation, we have compared the meiotic status of oocytes obtained at three different time intervals from animals treated with 1200 i.u. PMSG or 12 mg ovine FSH and from untreated control animals. Significantly more oocytes from PMSG-treated, compared with control and FSH-treated, animals showed premature condensation of chromatin at both the time of sponge withdrawal and 20 h later. The chromatin condensation was, however, not associated with germinal vesicle breakdown. In contrast, when oocytes were examined 6 h before the expected time of ovulation following human chorionic gonadotrophin (hCG) injection, no significant difference was found in the proportion of oocytes undergoing germinal vesicle breakdown between the three treatment groups, with most oocytes being at the metaphase I or II stage of meiosis. We conclude that superovulation of goats with PMSG at a dose resulting in a high incidence of premature ovulations is associated with premature activation of the initial stages of nuclear maturation in oocytes. In contrast, although treatment with 12 mg ovine FSH did not cause premature ovulations, it was not totally devoid of premature chromatin-condensing activity in oocytes.

Animals↗

PubMed1990

Cryopreservation.

Embryo cryopreservation is now firmly established as a routine component of in vitro fertilization (IVF) and other assisted conception techniques for the resolution of human infertility. Excess fertilized oocytes and embryos can be preserved for infertile couples avoiding the necessity to replace large numbers of embryos which reduces the incidence of multiple births and their sequelae. The additional pregnancies obtained from replacement of cryopreserved embryos has effectively increased the success rate of a cycle of IVF treatment for a couple by 1 to 10%. There is an increasing preference to cryopreserve fertilized pronuclear oocytes by slow cooling in 1,2-propanediol (PROH) although many clinics also cryopreserve early cleavage stage embryos by slow cooling in PROH or dimethyl sulphoxide (DMSO) with similar success rates to those for pronuclear oocytes. Numerous problems have appeared with the attempted cryopreservation of mature unfertilized oocytes, including nonreversible disassembly of the meiotic spindle during cooling, reduced fertilization rates due to the effects of cryoprotectants and cooling on the zona pellucida, parthenogenetic activation of oocytes by PROH, substantially increased polyploidy and aneuploidy when frozen-thawed oocytes are fertilized and increased cryoinjury to unfertilized oocytes when compared to fertilized oocytes. It is also necessary to address problems which have arisen through the disagreement of separated couples about the disposition of embryos in cryostorage.

Cryopreservation↗

PubMed1990

Co-culture of mouse embryos with oviduct and uterine cells prepared from mice at different days of pseudopregnancy.

Oviduct and uterine cell cultures were prepared from mice at different days of pseudopregnancy and their effects on the development of 1- and 8-cell mouse embryos in co-culture were examined. One-cell mouse embryos in co-culture with oviduct cells from 20 h to 120 h after hCG had a mean (+/- s.e.) cell number of 70.1 +/- 3.6, significantly (P less than 0.001) higher compared with those cultured in Whittingham's T6 medium supplemented with 5% fetal calf serum (T6 + 5% FCS) (30.4 +/- 1.6). Transfer of embryos, at 96 h after hCG, to synchronous pseudopregnant recipients showed that more embryos in oviduct co-culture formed fetuses than those cultured in T6 + 5% FCS. Co-culture of 1-cell embryos with uterine cells did not confer an advantage in cell numbers over T6 + 5% FCS. However, more 8-cell embryos formed blastocyst outgrowths after 100 h in co-culture with uterine cells prepared from mice at Day 3 of pseudopregnancy than with uterine cultures prepared from mice at Day 1 of pseudopregnancy or oviduct cells. In addition, there was further improvement when the Day 3 uterine co-cultures were supplemented with 1 or 10 ng progesterone/ml. These results highlight the importance of the oviduct and uterine cells during the different stages of preimplantation embryo development.

Animals↗

PubMed1989

Parthenogenetic activation of unfertilized mouse oocytes by exposure to 1,2-propanediol is influenced by temperature, oocyte age, and cumulus removal.

Cumulus-intact and -denuded unfertilized oocytes from two mouse strains were exposed to 1.5 M ethanol (EtOH) or two cryoprotectant solutions, 1.5 M propanediol (PROH) or 1.5 M dimethylsulfoxide (DMSO), for 4.5 min at 27 degrees C, and the proportion of activating or degenerating oocytes studied. Exposure to DMSO did not significantly increase activation above that of oocytes not exposed to DMSO. Treatment of oocytes in PROH resulted in the activation of up to 87% of viable oocytes. This was significantly higher (P less than .01) than in control oocytes and comparable to the rate of activation after treatment with EtOH (59-96% activation). In solutions at 1 degree C, 47% of control oocytes were activated, which was not significantly different from the rate of activation in EtOH (36%) or PROH (50%) at 1 degree C. Following treatment with PROH, up to 87% of oocytes degenerated within a period of 6 h in vitro. The age of the oocytes (h post hCG) and the time of cumulus removal with the enzyme hyaluronidase, relative to the time of exposure to the chemicals, influenced the level of degeneration in most groups. Significantly fewer oocytes degenerated when cumulus cells were removed before treatment (0-31%) than when the cumulus was left intact throughout the treatment and 6 h culture period (10-87%). Exposure to PROH at 1 degree C reduced oocyte degeneration to 5%. We conclude that PROH causes significantly greater losses of oocytes as a result of parthenogenetic activation and degeneration than of exposure to DMSO.

Animals↗

PubMed1989

Effect of dimethyl sulfoxide and protein concentration on the viability of two-cell mouse embryos frozen with a rapid freezing technique.

Two-cell mouse embryos were frozen by direct plunging into liquid nitrogen after a 3-min exposure to solutions containing 0.25 M sucrose with 1.5, 3, or 4.5 M dimethyl sulfoxide (Me2SO), and 0, 4, 8, 16, or 32 mg/ml bovine serum albumin (BSA). In the absence of BSA, significantly more embryos were lost or damaged during freezing and thawing. Increasing the BSA concentration from 4 to 32 mg/ml had no significant effect on subsequent embryo viability in vivo or in vitro. Blastocyst formation in vitro was greater than 90% in embryos exposed to the cryoprotective solutions only. Although development to blastocysts was not significantly different to nonfrozen controls in most groups frozen in 3 and 4.5 M Me2SO (up to 92% blastocysts), it was significantly reduced when embryos were frozen in 1.5 M Me2SO (up to 65% blastocysts). The development to fetuses of embryos frozen in 3 M Me2SO (64 to 74% fetuses) was not significantly different from nonfrozen controls (68 to 79% fetuses) or embryos frozen by a conventional slow cooling method (70%). Frozen thawed two-cell embryos developed into normal adults which were able to reproduce normally. We conclude that this freezing method can efficiently cryopreserve early cleavage stage mouse embryos.

Animals↗

PubMed1989

Successful single-cell biopsy and cryopreservation of preimplantation mouse embryos.

We have previously observed that preimplantation embryo biopsy in the mouse causes a reduction in implantation rate in utero. After minor modifications to the technique, we now find that sampling a single blastomere from the 4-cell mouse embryo does not compromise continued development in vitro or in vivo. When transferred to pseudopregnant foster mice, 60.3% and 64.3% of biopsied and control embryos, respectively, implanted into the uterine wall, and 52.6% and 52.4% of biopsied and control embryos, respectively, developed into fetuses. In a separate series of experiments, we have demonstrated that biopsied mouse embryos can be successfully cryopreserved by ultrarapid freezing even though they have a punctured zona pellucida. Biopsied (frozen-thawed), control (frozen-thawed), and nonfrozen embryos had an implantation rate of 81.1%, 74.3%, and 74.1%, respectively, and a fetal formation rate of 62.2%, 62.9%, and 66.7%, respectively.

Animals↗

PubMed1989

In vivo cleavage rates and viability obtained for early cleavage mouse embryos in co-culture with oviduct cells.

The cleavage rate and development of two-cell mouse embryos to the morulae stage in co-culture with mouse oviduct cells was studied in vitro and compared with those achieved in vivo. Embryos were cultured in Whittingham's T6 (T6), T6 supplemented with fetal calf serum (FCS) and in co-culture with either Dulbecco's Modified Eagles Medium supplemented with sodium lactate (DMEM + 1a) or a modification of T6 medium containing vitamins and amino acids (T6 + v + aa). Co-culture of oviductal cells with DMEM + la medium supported two-cell mouse embryo development to eight cells at a rate significantly better (P less than 0.001) than T6, but the rate of embryo development was not equivalent to that in vivo. DMEM + la alone was inadequate as an embryo culture medium. Co-cultures using T6 + v + aa with mouse oviductal cells were prepared from mice at days 1, 2 or 3 of pseudopregnancy. Day 2 and 3 co-cultures allowed two-cell embryos to develop at a rate comparable to that in vivo up to the mid eight-cell stage (68 h after hCG), but by 76 h after hCG embryos were retarded. Transfer to pseudopregnant recipients of embryos co-cultured with day 2 oviductal cells until 68 h after hCG resulted in a rate of fetal development equivalent to that of embryos grown in vivo. Our results show that co-culture of early cleavage-stage embryos with mouse oviductal cells allows embryos to retain cleavage rates and viability comparable to in vivo development.

Animals↗

PubMed1989

Biopsy of preimplantation mouse embryos: development of micromanipulated embryos and proliferation of single blastomeres in vitro.

We have developed a technique to sample the preimplantation embryo, which may, in the future, be applied to prenatal diagnosis of genetic disease. Using micromanipulation, we aspirated a single blastomere from 4-cell mouse embryos. This procedure had no effect on in vitro development; 98% of control and 94% of biopsied embryos reached the blastocyst stage after 48 h in culture. Furthermore, after transfer to pseudopregnant recipient mice, the rate of fetal development of biopsied embryos was not significantly different from control embryos, although implantation rate was significantly reduced (mean +/- SD: biopsied 53.1 +/- 4.0, control 81.8 +/- 8.4, p less than 0.001). For the first time we have produced monolayer cell cultures derived from single preimplantation blastomeres. Individual biopsied blastomeres were cultured in vitro on different extracellular matrix components. Significantly greater cell proliferation was obtained in wells coated with fibronectin (FN), laminin (LN), and a complex of laminin and nidogen (LNC) than in a less specific matrix of swine skin gelatin (SSG). Mean (+/- SE) cell nuclei number per well after 6 days in culture was 6.4 +/- 2.1, 11.9 +/- 1.5, 19.8 +/- 2.9, and 20.9 +/- 2.6 in wells coated with SSG, LN, FN, and LNC respectively.

Animals↗

PubMed1988

The effects of ultrarapid freezing on meiotic and mitotic spindles of mouse oocytes and embryos.

Preovulatory mouse oocytes and 2-cell embryos were frozen with dimethyl sulfoxide and propanediol by an ultrarapid method. The survival of frozen oocytes was low (33-34%) compared to that of 2-cell embryos (78-79%) with either cryoprotectant. Development to blastocysts after postthaw culture was about 7-15% for oocytes and 79-80% for the embryos. Ultrarapid freezing preserves cell structure quite well as revealed by electron microscopy, but meiotic oocytes and late 2-cell embryos undergoing mitosis showed evidence of spindle disorganization involving loss or clumping of microtubules resulting in some scattering of chromosomes. Embryos developed from frozen eggs showed clear evidence of micronuclear formation and incomplete incorporation of chromosomal material into main nuclei. These experiments confirm our observations on freezing of human oocytes and show that spindle microtubules are sensitive to freeze-thawing and that cryopreservation could cause chromosomal aberrations during early development. A cautious approach to the introduction of oocyte freezing in human in vitro fertilization (IVF) programs is advocated.

Animals↗