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A.R.C. Unit of Reproductive Physiology and Biochemistry,Cambridge
(Received 7th June 1974)
Previous publications (Boursnell & Roberts, 1974; Roberts, Boursnell & Brown, 1974; Roberts, Boursnell, Winsor & Mustill, 1974) have shown that the basic boar seminal haemagglutinin (Boursnell & Briggs, 1969) almost certainly combines with zinc in the seminal plasma (Boursnell, Baronos, Briggs & Butler, 1972) to produce reversible opalescence on cooling. The relative magnitude of the opalescence at any one temperature resembles the degree of damage observed when the spermatozoa are subjected to cold shock at that temperature. Boar spermatozoa also showed enhanced absorption of zinc protein at 4°C. Attempts were made to define the nature of the structures binding haemagglutinin. Nelson & Boursnell (1966) showed that substances of high molecular weight inhibited the haemagglutinin reaction, perhaps non-specifically, and no markedly inhibitory low molecular weight material could be found. Certain red cell species-specificities were, however, noted in some haemagglutinin fractions
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Summary.
Lactoferrin isolated from sow milk (about 0·6 mg/ml) was shown to be chromatographically homogeneous, an observation supported by electrophoresis and by reaction against monospecific anti-lactoferrin antiserum. Isoelectric focusing showed multiple forms of the protein (i.e.p., 9·3 to 10·0) converted by neuraminidase to one form (i.e.p., 9·65). Boar seminal plasma contains immunologically identical lactoferrin (0·1 to 0·5 mg/ml) which binds strongly to boar spermatozoa.
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Summary.
Zinc alone of the metals present in boar seminal plasma causes an opalescence in normal samples at room temperature. This opalescence is increased by cooling to 4° C, leading in some cases to a definite precipitate which redisperses on warming.
The normal opalescence can be minimized by addition of fractional millimolar amounts of EDTA equivalent to the zinc present. This treatment also abolishes the precipitate which occurs on cooling.
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A light microscope study of the choriovitelline (yolk sac) placenta of the dasyurid marsupial, Sminthopsis crassicaudata, and some comparative observations on that of the didelphid, Monodelphis domestica, were performed. In the former species, the placenta was composed of an invasive bilaminar, avascular, yolk sac and a non-invasive trilaminar, vascular yolk sac. The bilaminar yolk sac placenta had trophoblast giant cells that eroded the maternal epithelium, but there was no evidence of invasion of maternal capillaries; thus, an endotheliochorial placenta was present. In the trilaminar yolk sac placenta, the convoluted chorion followed the contours of the highly folded endometrial epithelium but did not erode it and, therefore, an epitheliochorial placenta was formed. In late pregnancy, the chorio-vitelline placenta of Monodelphis domestica also had two regions, but the fetal trophoblast did not invade the uterine epithelium in either region. Rather, there were discontinuous areas of adhesion between trophoblast giant cells and uterine epithelium in the trilaminar yolk sac placenta and some extensive areas of adhesion in the attenuated bilaminar yolk sac placenta. The yolk sac placenta in M. domestica, unlike that of S. crassicaudata, therefore appears to be epitheliochorial in the vascular and non-vascular regions.
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Summary.
Opalescence and precipitation of boar seminal plasma proteins, specifically promoted by zinc, have been shown to be due to a zinc-precipitable protein (ZPP). The properties of this purified protein cannot be distinguished from those of the seminal haemagglutinin and their identity is postulated. The response of seminal plasma opalescence to low temperature is especially marked between 14°C and 6°C. This is notably similar to the already recorded response of sperm survival to cooling. When semen is cooled to 4°C, ZPP, accompanied by zinc, is absorbed by spermatozoa.
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Summary.
The temperature-dependent opalescence caused by the presence of a zinc-precipitable protein (ZPP) in boar seminal plasma has been shown to be pH-dependent.
The considerable pH increase to which boar seminal plasma is subject on exposure to air is attributed to loss of CO2 from a system with a notable paucity of titratable groups in the physiological range pH 7 to 9. This markedly affects the temperature-dependent opalescence.
The ZPP has been purified and the temperature-dependent opalescence of this material can be demonstrated only within a narrow range of zinc concentration in the absence of other possible ligands present in whole seminal plasma.
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Summary. In Exp. 1 twice daily i.m. injections of 2 mg recombinant bovine IFN-αI1 (rboIFN-αI1) (N = 24) or placebo (N = 25) were administered to ewes from Day 12 to Day 16 during a normal oestrous cycle. Treatment did not increase (P > 0·10) oestrous cycle length (20·7 ± 1·2 versus 18·5 ± 1·4 days). In Exp. 2, ewes were injected twice daily with 2 mg IFN (N = 34) or placebo (N = 36) from Days 11 to 18 after natural mating. The rboIFN-αI1 significantly (P = 0·05) improved pregnancy rate (79% versus 58%) as determined by a failure of ewes to return to oestrus within 50 days. The number of ewes that lambed was greatest in the rboIFN-αI1-treatment group (71% versus 50%; P = 0·07), and no teratogenic effects were observed in the young born to IFN-treated ewes. The study was repeated a second year with a more fecund group of ewes (Exp. 3). More (P = 0·08) ewes injected with rboIFN-αI1 (58/65) than placebotreated ewes (48/61) were judged pregnant by ultrasound. Again more ewes lambed (55 versus 45) and more lambs were born (98 versus 80) from the rboIFN-αI1-treated group. Combining the data from both studies revealed a significant (P = 0·01) effect of treatment. The amount of antiviral activity in jugular vein blood of ewes injected with rboIFN-αI1 (2 mg) was determined over time in Exp. 4. Activity rose to a maximum (∼450 IRU/ml) within 1–2 h and declined by over 75% in 24 h. Single injections of 1, 2 and 5 mg in buffer or 2 mg emulsified in sesame oil all gave similar profiles of antiviral activity in jugular blood over a 48-h period. In Exp. 5, antiviral activity was measured in uterine vein, ovarian artery and jugular vein serum of untreated pregnant (N = 7) and non-pregnant (N = 11) ewes at Day 15 after mating. Activity was detected in the uterine vein (58 ± 19 IRU/ml) of all pregnant ewes. The observations in Exps 1–5 are consistent with a role for conceptus-derived IFN-α in maternal recognition of pregnancy and suggest that supplemental IFN-α might be useful in improving pregnancy success in sheep.
Keywords: α-interferon; oestrous cycle; pharmacokinetics; pregnancy; sheep
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Summary. Conceptuses were obtained from pony mares on each day of pregnancy between Days 12 and 28, and on Days 39, 45, 65 and 100. Endometrium was obtained from mares at Days 12, 14, 16, 18, 39, 45, 65 and 100 of pregnancy, and from non-pregnant mares during anoestrus, during transition into the breeding season, at oestrus, or during dioestrus. Tissues were incubated in vitro for 24 h with l-[3H]leucine. Proteins synthesized and released into the culture medium were analysed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and fluorography.
Conceptuses obtained before Day 14 after ovulation released a characteristic pattern of labelled proteins. These included two groups of apparent isoelectric variants of relative molecular weights (M r) 30 000–40 000 (pI values 4·5–5·5 and 6–7), one group of M r ∼22 000 (pI 6·5–7), and large protein(s) that did not enter the 10% polyacrylamide gel. After Day 14 the array of labelled proteins had changed and resembled that produced by isolated yolk sac at the later stages of pregnancy studied. Included amongst these were several acidic polypeptides with M r 20 000 (pI 5–6).
The endometrial samples released an array of non-dialysable polypeptides into the culture medium. Fluorograms could be assigned to one of three general groups, with endometrium from mares within each group producing similar patterns of labelled proteins. The first group consisted of anoestrous, transitional and ovariectomized mares, and mares at oestrus or Day 1 or Day 18 after ovulation. The second group was comprised of mares at Days 12–16 of dioestrus or Days 12–18 of pregnancy. Mares from Day 39 through 100 of pregnancy made up the third group.
Keywords: embryo; horse; proteins; endometrium; electrophoresis
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The efficiency of cloning by somatic cell nuclear transfer (SCNT) is poor in livestock with ~5% of transferred cloned embryos developing to term. SCNT is associated with gross placental structural abnormalities. We aimed to identify defects in placental histology and gene expression in failing ovine cloned pregnancies to better understand why so many clones generated by SCNT die in utero. Placentomes from SCNT pregnancies (n = 9) and age matched, naturally mated controls (n = 20) were collected at two gestational age ranges (105–134 days and 135–154 days; term = 147 days). There was no effect of cloning on total placental weight. However, cloning reduced the number of placentomes at both gestational ages (105–134 days: control 55.0 ± 4.2, clone 44.7 ± 8.0 and 135–154 days: control 72.2 ± 5.1, clone 36.6 ± 5.1; P < 0.001) and increased the mean individual placentome weight (105–134 days: control 10.6 ± 1.3 g, clone 18.6 ± 2.8 g and 135–154 days: control 6.6 ± 0.6 g, clone 7.0 ± 2.0 g; P < 0.02). Placentomes from cloned pregnancies had a significant volume of shed trophoblast and fetal villous hemorrhage, absent in controls, at both gestational age ranges (P < 0.001) that was shown to be apoptotic by activated caspase-3 immunoreactivity. Consequently, the volume of intact trophoblast was reduced and the arithmetic mean barrier thickness of trophoblast through which exchange occurs was altered (P < 0.001) at both gestational age ranges in clones. In addition, cloning reduced placental expression of key genes in placental differentiation and function. Thus, cloning by SCNT results in both gross and microscopic placental abnormalities. We speculate that trophoblast apoptosis, shedding, and hemorrhage may be causal in fetal death in ovine clones.
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Priority Research Centre for Reproductive Science, School of Biomedical Sciences and Pharmacy, University of Newcastle, Newcastle, New South Wales, Australia
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Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland, Australia
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Australian Research Council Centre for Excellence in Nanoscale Biophotonics, Adelaide, South Australia, Australia
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The preimplantation embryo in vivo is exposed to numerous growth factors in the female reproductive tract, which are not recapitulated in embryo culture media in vitro. The IGF2 and plasminogen activator systems facilitate blastocyst development. We hypothesized that the addition of IGF2 in combination with urokinase plasminogen activator (uPA) and plasminogen could improve rates of blastocyst hatching and implantation in mice. B6BcF1 and CBAB6F2 mouse embryos were divided into one of four supplemented culture media treatment groups: (1) control (media only); (2) 12.5 nM IGF2; (3) 10 µg/mL uPA and 5 µg/mL plasminogen; or (4) a combination of IGF2, uPA and plasminogen treatments. Embryo development to blastocyst stage and hatching were assessed before transfer to pseudopregnant recipient females and implantation, pregnancy rates and postnatal growth were assessed. After 90.5 h of culture, IGF2 + U + P treatment increased the percentage of B6BcF1 embryos that were hatching/hatched and percentage developing to blastocyst stage compared with controls (P < 0.02). Following B6BcF1 embryo transfer, IGF2 + U + P treatment increased implantation sites at day 8 of pregnancy compared with controls (P < 0.05). Replication in the CBAB6F2 mouse strain showed significant improvements in pregnancy rates at days 8 and 18 but not in blastocyst development. No adverse effects were seen on gestational age, litter size or birthweight, or the reproductive capacity of offspring of IGF2 + U + P treated embryos. For embryos susceptible to detrimental effects of in vitro culture, IGF2, uPA and plasminogen supplementation of culture media can improve pregnancy success, but the effect of treatment is dependent on the mouse strain.
