Summary. A quantitative indirect immunofluorescent assay developed for the measurement of antibodies produced in rabbits against porcine zonae pellucidae was modified and used to detect autoantibodies specific to human zonae pellucidae. An undetermined number of human serum constituents which appear to vary quantitatively between individuals had the capacity to bind non-specifically the porcine zonae used as antigen in this assay. Binding was reduced or eliminated by altering the chemical composition of the assay system until the only positive results were for sera from infertile, sub-fertile and menopausal women. These positive sera were adsorbed with washed porcine blood cells, spleen cells, and liver cells. Each tissue completely removed the positive binding to porcine zonae. The positive sera were also tested against human oocytes with and without porcine tissue adsorption, and no positive fluorescence was detected. It is therefore unlikely that the antibodies in human sera which bind to porcine zonae pellucidae could be a causal factor in infertility in women.
P. L. Nayudu, L. E. Freemann and A. O. Trounson
J. Kumar, J. C. Osborn, A. W. N. Cameron and A. O. Trounson
Summary. 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.
Keywords: follicles; oocytes; maturation; steroidogenesis; superovulation; gonadotrophins; goat
K. M. Battye, R. J. Fairclough, A. W. N. Cameron and A. O. Trounson
Summary. Feral does of various ages were treated with intravaginal progestagen sponges for 16 days to synchronize oestrus. On Day 2 before sponge removal the goats were given 1200 i.u. PMSG to induce superovulation: 6 of the goats were also injected every 12 h with flunixin meglumine, a prostaglandin (PG) synthetase inhibitor, from Day 3 to 7 of the synchronized oestrous cycle. Jugular blood samples were collected from all females into heparinized syringes at daily intervals over the 2 days before sponge removal, twice daily for the next 2 days, then at hourly intervals from 09:00 to 17:00 h for 2 days and then twice daily for a further 2 days, for measurement of plasma progesterone and the PGF metabolite 13,14-dihydro-15-keto-PGF (PGFM) by radioimmunoassay. Intermittent surges in plasma PGFM concentrations were observed in hourly samples collected from 4/4 untreated females but in only 2/6 of the inhibitor-treated females(P < 0·05), and the peak plasma PGFM concentrations were reduced in these 2 inhibitor-treated goats compared with the control goats. The corpora lutea (CL) of the inhibitor-treated females appeared to be functional as indicated by the plasma progesterone profile and endoscopic examination of CL. In the control females, however, there was evidence of premature regression of CL. These results suggest that the premature release of PGF-2α may be the cause of premature regression of CL in nanny goats induced to superovulate.
Keywords: corpus luteum; goat; prostaglandin; superovulation; progesterone
A. O. Trounson, S. M. Willadsen, L. E. A. Rowson and R. Newcomb
The survival and development of cow eggs in the rabbit oviduct after storage at room temperature and after cooling and storage at 0-7·5°C was examined. In PBS medium at room temperature 88% of Day-5 and 85% of Day-3 eggs showed normal development, but in TCM 199, 71% of Day-5 and only 49% of Day-3 eggs showed normal development. Duration of storage (1½-2 hr or 6½-7½ hr) and cleavage stage before storage had no appreciable effect on development. Some retardation of development occurred in Day-3 eggs after 96 hr in the rabbit oviduct when compared to Day-5 eggs after 48 hr. Cooling of Day-5 and Day-6 eggs to 0-7·5°C resulted in degeneration of a large proportion of eggs. Of the factors examined, storage medium (PBS or PBS+20% FCS), storage time (2 min, 30 min, 24 hr) and storage temperature (0, 2, 5 or 7·5°C) had little effect, but slower cooling rates tended to improve survival of eggs although the differences were not significant. More morulae (>32 cells) than 8- to 24-celled eggs developed normally.
J. M. Shaw, I. Kola, D. R. MacFarlane and A. O. Trounson
Summary. 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 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 <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.
Keywords: dimethyl sulphoxide; rapid freezing; ice formation; handling; chromosome rearrangements; mouse; embryos
W. R. Allen, Francesca Stewart, A. O. Trounson, M. Tischner and W. Bielański
Agricultural Research Council Unit of Reproductive Physiology and Biochemistry, Animal Research Station, Cambridge, U.K. and Academy of Agriculture, Institute of Applied Animal Physiology, 30-059 Krakow 2, al. Mickiewicza 24128, Poland
Sheep and cattle embryos continue to develop normally in the ligated oviducts of rabbits (Adams, Moor & Rowson, 1968) and retain viability when transferred to ewes or cows, provided they are removed from the rabbit within 3-4 days (Lawson, Adams & Rowson, 1972; Lawson, Rowson & Adams, 1972). The rabbit genital tract was first used for long-distance transport of embryos by Hunter, Bishop, Adams & Rowson (1962), who obtained normal lambs after transfer to recipient ewes. In this paper, we describe the development and viability of horse embryos after long-distance transport in rabbits.
Seven maiden 2- and 3-year-old Welsh Mountain Pony mares maintained in Cambridge were used as embryo donors and six mixed-breed mares aged 2 to 8 years
T. T. Peura, M. W. Lane, G. Vajta and A. O. Trounson
The use of cryopreserved in vitro produced bovine embryos as nuclear transfer donors was assessed. Day 4 or 5 morulae were vitrified and warmed using the open pulled straw method and used as donors for nuclear transfer. Although the proportion of morulae and blastocysts that developed from nuclear transfer embryos derived from day 5 vitrified embryos did not differ from that derived from fresh embryos (16.7 and 24.3%, respectively), development to blastocysts was reduced when vitrified donor cells were used (8.3 and 19.1%, respectively). Likewise, development to morulae and blastocysts was not different between nuclear transfer embryos derived from day 4 vitrified embryos allowed to recover for 24 h, and day 5 vitrified embryos allowed to recover for 1–2 h (27.7 and 15.6%, respectively), but the development to blastocysts was reduced when day 5 vitrified donor cells were used (23.2 and 10.0%, respectively). However, in nuclear transfer embryos derived from either day 4 vitrified or day 5 fresh donors, no differences were observed in development rates to morulae and blastocysts (34.3 and 36.3%, respectively) or to blastocysts alone (20.2 and 18.1%, respectively). Nor were there differences in the development rates of fresh or day 4 or day 5 vitrified in vitro produced (non-nuclear transfer) embryos (47.9, 51.0 and 35.5% developing to blastocysts at day 7, respectively). In vitro produced embryos and nuclear transfer embryos derived from day 4 vitrified or day 5 fresh donors were transferred to recipients at morula or blastocyst stage at day 6 or 7. The pregnancy rates were similar in both groups of nuclear transfer embryos, but higher in the control group consisting of in vitro produced embryos (47, 42 and 67%, respectively). In conclusion, if vitrified donor embryos are allowed to recover for 24 h after warming, their use in nuclear transfer results in similar efficiencies to those achieved with fresh embryos.
A. O. Trounson, Linda R. Mohr, C. Wood and J. F. Leeton
Summary. Oocytes were obtained from patients with tubal infertility at fixed times after the onset of the endogenous LH rise or hCG injection, and were inseminated immediately after recovery or after periods of 4–4½, 5–5½ and 6–6½ h in culture in vitro. Delayed insemination resulted in a marked increase in the proportion of oocytes that were fertilized and developed to normal embryos and maximum rates occurred after 5–5½ h in culture (0–½ h, 26%; 4–4½ h, 50%; 5–5½ h, 89%; 6–6½ h, 69%). The range and mean (± s.d.) intervals from insemination for the pronuclear and early cleavage stages were 27–43 (35·6 ± 4·4) h for 2-cell stages, 36–65 (45·7 ± 8·3) h for 4-cell stages, 45–73 (54·3 ± 12·6) h for 8-cell stages and 68–85 h for the 16-cell stage. In 7/50 patients receiving 1 or 2 embryos at the 2-, 4- and 8-cell stages, fetal development was normal and 2 women had twin pregnancies (36% success compared with 8% for single embryos). All pregnancies were from the groups in which insemination was delayed for 5–6½ h. It is concluded that a short period of culture in vitro may allow the completion of oocyte maturation, and improve the results of in-vitro fertilization.
C. G. Tsonis, Helen Quigg, V. W. K. Lee, Lorraine Leversha, A. O. Trounson and J. K. Findlay
Summary. Inhibin activity was measured by bioassay in follicular fluid of 99 individual ovine follicles ranging from 1·4 to 6·8 mm diameter (used to calculate volume) and in various stages of atresia. Treatment of samples before assay with charcoal concentrations of > 1 mg/ml resulted in significant loss of inhibin activity.
The inhibin content of follicular fluid from individual follicles varied with follicular fluid volume but not with the degree of atresia, as assessed by morphological criteria. Inhibin concentration was not related to atresia, but was correlated with follicular fluid volume. However, aromatase activity in granulosa cells and oestradiol-17β concentration of follicular fluid, considered to be good indices of atresia, were highly correlated with both inhibin content and concentration in follicles ≥ 3·5 mm diameter.
Inhibin in ovine follicular fluid shows marked variation between follicles and it is suggested that this reflects a combination of the number and activity of granulosa cells within the follicle and the exit rate of inhibin from the follicle.