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D. Sakkas and A. O. Trounson

Summary. 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 < 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.

Keywords: co-culture; cleavage rate; embryo; oviduct; uterus; mouse

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A. O. TROUNSON and N. W. MOORE

Summary.

The effect of `Protease' on the zona pellucida of fertilized and unfertilized sheep eggs was examined, and the effects of the treatment and of mechanical removal of part of the zona on the subsequent development of fertilized eggs was assessed in vitro and in vivo.

The zonae of follicular eggs were digested by `Protease' within 25 min of exposure. In ovulated eggs, the resistance of the zona to digestion decreased with age. Zonae of eggs collected 2 days after oestrus (Day 2) were extremely resistant whereas nearly all the Day-5 eggs lost their zonae within 10 min. Fertilization had little effect upon the susceptibility of zonae to digestion by `Protease'.

Neither age of egg nor method of treating the zona had any effect on subsequent development of fertilized Day-2 and Day-6 eggs in culture, but there were effects of both factors on the proportion of eggs which developed to normal Day-25 embryos. `Protease' treatment of Day-2 and Day-3 eggs failed to digest the zona completely and a high proportion of such eggs developed to normal embryos whereas only a few eggs of similar age, in which part of the zona was removed mechanically, developed normally. The zonae of Day-4 to Day-6 eggs were invariably removed by `Protease' and the method of treatment had no effect on the proportion of eggs which developed to normal embryos. Irrespective of method of treatment, more Day-6 than Day-4 eggs developed to normal embryos. Culture for 2 days following treatment did not increase the survival of Day-6 eggs when they were transferred to recipients.

Ovulation and ageing of eggs appear to be associated with changes in the zona pellucida which influence its susceptibility to digestion by proteolytic enzymes. In early cleavage stage eggs, the major rôle of the zona may be protection of the inner cell mass from the uterine environment.

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Linda R. Mohr and A. O. Trounson

Summary. A hatched human blastocyst obtained after in-vitro fertilization and culture was examined by transmission electron microscopy and the ultrastructural features compared with hatched mouse and bovine blastocysts. The human blastocyst contained a continuous layer of trophoblast cells with apical junctional complexes, an inner cell mass and the beginning of a primitive endoderm layer. Certain ultrastructural features were common to the blastocysts of all 3 species; these included characteristic junction regions between adjacent trophoblast cells, an abundance of microvilli on the external surfaces of the blastocysts and the presence of well developed mitochondria and numerous ribosomes in the trophoblast cells. The features that were dissimilar included the extent of development of the endoderm layer, the appearance of the inner cell mass and the nature and extent of vesicular inclusions in the trophoblast cells.

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Linda R. Mohr and A. O. Trounson

Summary. Preimplantation mouse embryos that were exposed to fluorescein diacetate (FDA) accumulated intracellular fluorescein and fluoresced brightly under ultraviolet (u.v.) light. The rate at which intracellular fluorescein was lost from the cells was measured at 37, 28 and 4°C and the rate decreased as the storage temperature decreased. The rate at which intracellular fluorescein accumulated increased as FDA concentration increased until a maximum rate was attained. The ability to accumulate intracellular fluorescein could be removed by heating embryos at 56°C for 30 min or by damaging the cell membrane. Cells grown under inadequate culture conditions lost the ability to accumulate intracellular fluorescein. Exposure of 2-cell mouse embryos to FDA and u.v. light did not alter the rate of blastocyst formation in vitro, and exposure of blastocysts to FDA and u.v. light did not alter the rate of implantation or post-implantation development in vivo.

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R. M. Moor and A. O. Trounson

Summary. Oocytes removed from, or retained within, non-atretic and atretic follicles of different sizes were cultured for 24 h in the presence of a variety of hormones in an attempt to identify the factors affecting oocyte maturation in vitro. Resumption of meiosis was assessed morphologically; the developmental capacity of oocytes after culture was determined by transfer to the oviducts of inseminated ewes.

About 70% of oocytes cultured after removal from follicles of different sizes resumed meiosis in vitro, but they did not undergo normal development after transplantation.

Oocytes cultured within the follicle in hormone-free medium remained at the germinal vesicle stage. In the presence of FSH and LH some oocytes reached the second meiotic metaphase: 19% in small (2–3 mm diam.) and 73% in larger (3–5 mm diam.) non-atretic follicles, and 54% in small and 45% in larger atretic follicles.

Less than 5% of oocytes cultured in follicles developed into normal blastocysts after transplantation when either no hormone or only FSH and LH were added to the culture medium. The addition of oestradiol-17β to medium containing FSH (2 μg/ml) and LH (1 μg/ml) resulted in the development to blastocysts of 26% of oocytes from small non-atretic follicles, 46% from large non-atretic follicles and 50% from atretic follicles. Blastocyst formation was greatly depressed and fragmentation rate significantly increased with concentrations of 10 μg FSH/ml and 2 μg LH/ml.

Developmental capacity after culture was further demonstrated by the birth of lambs from 63% of blastocysts derived from oocytes matured in vitro; 52% of control blastocysts developed to lambs after transfer.

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A. W. N. Cameron, K. M. Battye and A. O. Trounson

Summary. The timing of ovulation in feral goats treated with 1200 i.u. PMSG ± 50 μg GnRH was studied by repeated laparoscopy. Experiment 1 established that superovulation began as early as 30 h after withdrawal of progestagen-impregnated sponges and was not completed at 54 h if goats received PMSG alone. GnRH synchronized ovulation, leading to 91% of ovulations appearing between 36 and 48 h after sponges were withdrawn. Experiment 2 established that superovulation continued until up to 77 h in goats treated only with PMSG. The stress of repeated laparoscopy appeared to delay or abolish ovulation in some females. The mean (±s.e.) ovulation rate was greater in goats treated with GnRH (12·7 ± 1·3) than in those that received PMSG only (9·7 ± 1·1; P < 0·05). Out of 47 of the females in Exp. 1, 43 had one or more corpora lutea at laparoscopy 24 h after withdrawal of progestagen. These early corpora lutea were associated with an increased concentration of plasma progesterone during the periovulatory period. Experiment 3 provided evidence that these corpora lutea arose before the withdrawal of progestagen-impregnated sponges.

Keywords: goats; PMSG; ovulation; laparoscopy; progesterone

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A. O. Trounson, S. M. Willadsen and L. E. A. Rowson

Summary.

Cow morulae cultured in a phosphate-buffered medium containing serum developed normally and retained viability when transferred to recipients. Unlike earlier cleavage stages, cow blastocysts tolerated cooling to 0°C and retained viability after storage for 48 hr at 0°C when transferred to recipients.

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A. O. Trounson, S. M. Willadsen and L. E. A. Rowson

Summary. Follicular oocytes were cultured for 24 h in vitro or obtained 6–24 h after HCG injection from cows pretreated with PMSG, and transferred to the oviducts of oestrous rabbits or heifers, inseminated with bull spermatozoa, to determine their capability for normal development.

More than 65% of oocytes cultured in fetal calf serum, equilibrated with 5% CO2 + 5% O2 + 90% N2, matured to metaphase II within 24 h. Fertilization was not obtained in the rabbit oviduct but about 8% of oocytes matured in vitro underwent parthenogenetic cleavage. Sperm penetration was observed in 49% of oocytes in the cow oviduct, but although 47% had undergone development 96 h after transfer few developed to morulae and blastocysts, confirming the impaired ability of oocytes matured in vitro, as assessed by nuclear change, to develop normally.

Oocytes obtained from heifers slaughtered 24 h after HCG developed normally when transferred to the oviduct of inseminated heifers. Of the oocytes with an activated cumulus, 39% developed to blastocysts and of 16 blastocysts transferred to suitable recipients, 13 developed to normal fetuses at 13–17 weeks gestation.

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A. O. Trounson, S. M. Willadsen and R. M. Moor

Summary. When 23 10–16-week-old Welsh Mountain lambs were treated with PMSG 19 ovulated, the number of eggs ovulated being directly correlated with the duration of progesterone pretreatment (0·5 ± 0·29 (S.E.M.) after 3 days; 7·8 ± 3·47 after 18 days). Injection of HCG at the time of the induced oestrus had no effect on ovulation. The eggs shed from immature ovaries became fertilized and developed normally when tested in the ligated rabbit oviduct for development to the morula stage and by transfer to adult ewes (1 live lamb). Luteal function in lambs with a single CL was similar to that in nonpregnant ewes; progesterone levels in entire lambs with multiple CL and in hysterectomized lambs remained elevated for at least 60 days.

The capacity of ovarian follicles from PMSG-primed lambs to secrete oestrogen, testosterone and progesterone in vitro was similar to that of follicles from adult ewes. However, oestrogen production by lamb follicles immediately after explantation was higher than that of adult follicles and the administration of progesterone to lambs before PMSG treatment decreased subsequent follicular testosterone production.

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P. L. Nayudu, L. E. Freemann and A. O. Trounson

Summary. A dilution series of a standard antiserum, raised in rabbit, against intact porcine oocytes was tested by an indirect immunofluorescent assay under a variety of conditions. Maximal antibody binding was obtained in phosphate-buffered saline with sucrose and sodium azide as a serum diluent and wash buffer, with relatively long incubation and wash periods, and in some cases heat-inactivation of sera.

Factors found to influence the antibody binding were: alteration of the zonae by fixation; the presence of sucrose, galactose or glycerol; addition of protein to the buffer; heat inactivation of serum; substitution of phosphate by borate buffer; and replacement of the labelled second antibody by labelled Protein A. Antisera produced against acid- and heat-solubilized zonae differed from the standard antiserum in their binding capacity. Control sera (anti-porcine spleen, adjuvant injected, and normal) were all completely negative.