Biomedical subjects
Publications and source records attributed to L Nebel.
A second family with the autosomal recessive disorder now referred to as the limb/pelvis-hypoplasia/aplasia syndrome is reported. It is speculated that the gene for this rare skeletal dysplasia may be confined to the Middle East gene pool. The disorder has been shown to be diagnosable prenatally in a pregnancy at risk by using ultrasonography.
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Pregnant albino mice (ICR random-bred strain) received daily injections of 50 or 100 mg/kg body weight of D-amphetamine sulphate between days 9 and 11 of gestation. Parallel control animals were injected with saline solution. Treated mice were sacrificed on day 15 or 19 of gestation. The embryos were examined for gross malformations and direct embryonic ECG recordings were made; they were weighed, and their hearts were carefully dissected. Microscopic sections of the heart were stained with hematoxylin and eosin. It was found that high doses of D-amphetamine raised the incidence of mortality in the treated pregnant mice up to 40%. The resorption rate in the survivors was high (up to 58%) following the high dose of the drug. Up to 15% of the embryos from the treated groups showed gross malformations, including skeletal and eye malformations and exencephaly. The electrocardiogram (ECG) recordings in most 19-day-old embryos from both treated groups showed a pattern with prolonged Q-T interval (PQT), similar to that of control embryos in the intermediate developmental stages (days 14-16 of gestation). The ECG of control embryos (from day 17 on) resembled that of prenatal fetuses. Microscopically, the hearts of treated embryos showed a large number of undifferentiated cardiac myoblasts. It can be inferred that high doses of D-amphetamine affect embryonic development generally and delay the histodifferentiation of the myocardium, resulting in incomplete maturation of the cardiac muscles, thus leading to the immature ECG pattern, with PQT intervals.
Trophoblast was demonstrated to be unable to cause the production of interleukin-2 (IL-2) by allogeneic splenocytes in vitro in two ways: 1) The addition of lymphocyte-trophoblast culture-supernatant (LTC-SN) did not stimulate the proliferation of IL-2 dependent cytotoxic T lymphocyte (CTL-L cells; 2) When responder cells were cultured with heat-treated splenocytes (usually no CTL generation) an increase of CTL formation could be seen in the presence of mixed lymphocyte culture-supernatant (MLC-SN) but not of SN from the cultures in which trophoblast cells served as stimulators. In parallel, the trophoblast cells were found to be very poor stimulators of alloreactive CTL. The addition of interleukin-2-enriched media resulted in a significant amplification of trophoblast-induced CTL generation. The resulting killing lymphocytes were capable of destroying the only specific targets, did not lyse syngeneic target cells, and could not be generated in the absence of allogeneic trophoblast. The incubation of these lymphocytes with anti-Thy 1.2 monoclonal antibody in the presence of complement eliminated their killing effect. Lack of class II antigenic determinants on the surface of trophoblast and/or local immunosuppression of IL-2 production by trophoblast cells seem to be responsible for nondelivery of helper factor, which is essential for CTL production.
In order to investigate the functional organization of the progesterone receptor in chromatin we characterized the physical-chemical properties of the receptor bound chromatin fragments released by micrococcal nuclease and DNase I digestion. The crude nuclear fraction was isolated from T 47 D cells, previously exposed to 0.1 microM [3H]ORG 2058. The parameters determined in low and high salt concentrated buffers were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding abilities to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. Micrococcal nuclease digestion solubilized a receptor form sedimenting as a single peak at 4.4 S with a Rs = 7.78 nm and an estimated Mr = 144,000. About 53% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. 0.4 M KCl dissociated this receptor form into a smaller receptor sedimenting at 3.3 S with Rs = 5.53 nm and a calculated Mr = 76,000. A similar receptor form was extracted by 0.6 M KCl from the undigested crude nuclear fraction. DNase I digestion solubilized a receptor form sedimenting at 3.3 S with a Rs = 6.87 nm and a calculated Mr = 94,000. About 26% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. Dissociation of this receptor form by 0.4 M KCl resulted in a receptor sedimenting at 2.8 S with a Rs = 6.53 nm and an estimated Mr = 76,000. These results suggest: The progesterone receptor in chromatin is associated with several molecules probably proteins which complexed it to DNA. Some of these molecules still associated with the progesterone receptor could be released by nucleases digestion. Micrococcal nuclease releases a larger portion of these molecules than those release by DNase I.
Thirty-one patients who became pregnant after in vitro fertilization were compared with a matched control group of 31 patients who underwent embryo transfer but did not conceive. During the follicular phase there was no difference in the mean daily estradiol (E2) levels between the pregnant and control groups. In 95% of the women who conceived, E2 continued to rise or plateau on the day after human chorionic gonadotropin (hCG) administration; this pattern was seen in only 62.5% of the control group. There were significantly more "regular" embryos (i.e., those with symmetric blastomeres and no visible cytoplasmic fragments) replaced in patients who became pregnant, compared with the control subjects (65.6% versus 47.0%, P less than 0.05). Embryo regularity was associated with preovulatory E2 pattern; falling E2 levels on the day after hCG administration were associated with transfer of only 18.8% of regular embryos in the control group, whereas in the conceptional cycles with rising E2 levels, 61.5% of the replaced embryos were morphologically regular (P less than 0.01). These results would indicate that changes in the follicular environment as reflected by peripheral E2 levels might affect the developmental potential of the human embryo.
The first step in fertilization is the interaction of the capacitated sperm with the zona pellucida. It has been proposed that the initial interaction, as in other types of cell adherence, is due to complementary interacting sites on the opposing surfaces of the gametes. This work intended to investigate the role of carbohydrates in sperm-egg binding in rats. Ejaculated sperm was collected from uterine horns of mated females. The sperm was suspended in Rat Fertilization Medium at final concentrations of 3-7 times 10(5) sperm/ml. After 5 1/2 h of sperm incubation, eggs were added to the sperm suspensions concomitantly with various carbohydrates to achieve a final concentration of up to 50 mM. The eggs were separated after 30 min, and the number of sperm bound to the zona pellucida was counted. Among a variety of monosaccharides tested at 50 mM concentration, it was found that alpha-methyl-mannoside was the most potent inhibitor (producing 80% inhibition); less potent was D-mannose and even less, L-fucose. A combination of alpha-methyl-mannoside and L-fucose showed a synergistic effect. Mannan was not more effective as an inhibitor than the monosugar mannose, while fucoidin was extremely potent, causing over 90% inhibition of binding at 0.1%. We assume the presence of macromolecules containing sugars on the zona pellucida because inhibition of sperm binding to this layer was observed: a) after preincubation of mannan or fucoidin with sperm, but not with the eggs; and b) after pretreatment of the egg with specific enzymes. The results obtained in this study in the rat are consistent with the hypothesis that carbohydrates are critical for the sperm-egg interaction.
The ability of sperm samples from male partners of infertile couples, with isolated teratospermia (ITS), to penetrate zona-free hamster eggs was examined. The ITS patients had a significantly lower proportion of normal spermatozoa than did a control group of men of proven fertility (23.6% and 47.8%, respectively; P less than 0.001), while the mean total sperm count and the % motility did not differ between the two groups. The mean hamster egg penetration rate of sperm from ITS patients was 2.64% compared to 31.1% in the control group. A significant negative correlation was found in the ITS group between the proportion of a) abnormal forms and the ability to penetrate zona-free hamster eggs, and b) pyriform-shaped sperm and the penetration rate. Of the sperm parameters which were examined, only the morphology could be correlated with the rate of penetration.
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In an in vitro fertilization and embryo transfer program composed mainly of patients with tubal infertility, the incidence of fertilized oocytes with multiple pronuclei was 4.3%. An attempt was made to fix such oocytes with supernumerary pronuclei in order to assess the chromosome constitution of the gametes. Nine multipronuclear oocytes containing a total of 29 pronuclei were successfully fixed before the first cleavage division, and another oocyte containing 3 pronuclei was fixed before the second cleavage division. On analysis, chromosome counts could be obtained for 28 of the 29 total pronuclei, and 24 gave informative results. Nineteen pronuclei had a normal haploid chromosome count, 2 pronuclei contained one extra chromosome, and 3 pronuclei had one chromosome missing. The presence of a Y chromosome in six pronuclei identified their paternal origin; two of the six sperm pronuclei had a 22,Y,-E chromosome constitution. Only three pronuclei could be conclusively ascertained to be maternal in origin, yet two of these were aneuploid; one pronucleus had a 24,X,+D karyotype and the second had only 22 chromosomes. Multipronuclear oocytes present ideal material for analyzing the chromosome constitution of those human gametes which can undergo fertilization in vitro.
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Embryo transfer probably produces a lower incidence of implantation than the physiological incidence despite all other factors seeming similar. The only factor known to be present physiologically and absent in embryo transfer is the presence of sperm in the uterine cavity. Implantation and decidualization are often considered a modified form of inflammatory reaction. Semen contains factors which excite an inflammatory response. This project attempted to determine whether insemination would affect the implantation rate of transferred blastocysts in the rat. The figures showed a significantly increased implantation rate after insemination at day 4 of pseudopregnancy as compared to controls.
In using both a 51Cr-release assay and a single cell technique we measured different parameters of the effector lymphocytes killing process in pregnant and control women on a cell population and single cell level. Total NK activity was lower in pregnancy, but the difference was not significant. Parity showed no cumulative effect. Pregnant women had normal percentages of potentially cytotoxic target binding cells, however, the relative number of lymphocytes that could kill bound targets (i.e. active NK cells) was significantly depressed in pregnancy when compared with normal controls (p less than 0.05). This diminished number of active NK lymphocytes was, however, normal in all phases of the lytic procedure. Interferon (IFN) treatment of effector cells in pregnant women in vitro did not alter target cell binding, but did increase the percentage of active NK cells. The level of stimulation by IFN was the same in pregnancy and control patients. The kinetics of response were the same for both cell populations and different doses of interferon caused a similar level of augmentation. In conclusion we suggest that the here demonstrated deficiency of active NK lymphocytes in pregnancy may represent a function of pregnancy associated immunoregulatory molecules which prevent a population of pre-NK cells to express their cytotoxic potential.
This study was performed utilizing a standard 51Cr-release cytotoxicity and a single cell assay against K-562 NK-sensitive target cells. Pregnancy sera and amniotic fluids, were found to decrease human natural killer (NK) activity. Pregnancy sera were able to inhibit overall NK activity of partly purified peripheral blood lymphocytes in a dose dependent fashion. However, the exposure of effector cells to concentrations higher than 20% did not further increase the serum's suppressive activity. Sera taken from preeclamptic pregnancies had a similar inhibitory effect as those from normal pregnancies. Amniotic fluid was found to be even more suppressive than pregnancy serum. Using a single cell cytotoxicity assay we determined that neither serum nor amniotic fluid affected potentially cytotoxic target binding cells ("pre"-NK). The relative number of conjugates with dead target was, however, significantly depressed under the influence of pregnancy serum and to an even greater extent by amniotic fluid. Both pregnancy substances were also able to decrease the number of conjugates with dead target in an interferon-augmented population of lymphocytes. Our investigation indicates that the suppressive effect of amniotic fluid and pregnancy serum on NK activity is based on a blocking effect exerted at the killing capability of the active NK cells.
Thirty-one patients with severe mechanical infertility were treated with clomiphene citrate and human chorionic gonadotropin (HCG) at the start of our in vitro-fertilization and embryo-transfer program. Laparoscopies were performed 34 to 36 h after HCG administration in 32 cases, oocytes were recovered in 29 (91%) and in 19 (59%) at least one oocyte was fertilized. Embryos were transferred to the uterus in 12 cases (38%) and a pregnancy was established in a woman in whom 3 embryos were transferred. This pregnancy terminated at the 39th week of gestation with the delivery of a healthy female baby.