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Biomedical subjects

R D Lambert

Publications and source records attributed to R D Lambert.

18 recordsLinked to original sources
PubMed1992

Ontogeny of Ha-ras and c-myc mRNA levels in rabbit embryo and extraembryonic tissues by quantitative in situ hybridization.

A large variety of proto-oncogenes are known to be of key importance in cellular growth and differentiation during embryonic development. Using quantitative in situ hybridization, we studied in detail the levels of the proto-oncogenes Ha-ras and c-myc mRNA in embryos and extraembryonic tissues (maternal and embryonic placentas, trophoblast, and endometrial epithelium) during prenatal life of rabbit. cDNA probes encoding for Ha-ras (fragment Kpn 1-BstE II of 883 bp) and c-myc (fragment Pst 1-Pst 1 of 490 bp) were used to detect specific transcripts in fixed cryostat sections. High levels of Ha-ras and c-myc mRNA were detected in the rabbit embryo as well as in the decidua and in the trophoblast as early as day 9 of gestation. At 12 and 15 days of gestation, Ha-ras and c-myc mRNA levels decreased in both embryonic and maternal placenta while in the embryo a significant increase of Ha-ras and c-myc expression was detected with particular evidence in the central nervous system. Finally, at 25 days of gestation the expression of the two proto-oncogenes, Ha-ras and c-myc, was greatly decreased in both the embryo and extraembryonic tissues, and was undetectable by 30 days of gestation. These results show that in rabbit the expression of the two proto-oncogenes Ha-ras and c-myc is localized in the same tissues with similar intensity and follows an unparallel temporal modulation in the embryo and in the extraembryonic tissues during prenatal development.

Animals↗
PubMed1992

Rabbit embryo-fetal fluid decreases the cell cycle activities of DU-145 cells.

Successful reproduction requires tight control of cell proliferation and differentiation. Rabbit blastocoelic fluid contains such regulatory factors. For instance, it inhibits tumour or transformed cell proliferation. In this study, DU-145 cells have been used to characterize further this inhibitory activity. Maximal inhibition of cell proliferation is observed at day 12 of embryo-fetal development and this is accompanied by a strong reduction of [3H]-thymidine incorporation. DNA specific staining and analysis by flow cytometry show that cells are not stopped at any specific stage of the cell cycle. Using bromodeoxyuridine incorporation in combination with propidium iodide labelling, it has been possible to estimate the percentage of labelled cells, the duration of the S phase of the cell cycle derived from their relative movement and also the proportion of cells participating to the cell cycle. In the presence of embryonic and fetal fluids collected on day 12 (EFF D-12) the duration of the S phase and the doubling time are considerably increased and the percentage of cells participating in the cell cycle is decreased. The results also show that treatment with EFF D-12 induces the release of the cells from the monolayer. Taken altogether, these results suggest that EFF D-12 increases the duration of the cell cycle. This reduction of the mitotic activities lead up to cell death with subsequent release of cells into the culture medium.

Animals↗
PubMed1992

Decrease in calmodulin concentrations during heparin-induced capacitation in bovine spermatozoa.

Concentrations of the intracellular Ca(2+)-mediator calmodulin (CaM), were measured by radioimmunoassay during heparin-induced capacitation of bull spermatozoa. Heparin reduced sperm CaM concentrations in a dose-dependent manner corresponding with an increase in in-vitro fertilization rates. Such reductions were observed after heparin treatment for 4-6 h, which is in agreement with the length of the capacitation period in bulls and was concomitant with an increase in CaM concentration in the incubation medium, suggesting translocation of CaM from the spermatozoa to the surrounding milieu. This CaM translocation was inhibited partly by the protease inhibitor benzamidine, suggesting a role for the sperm protease in this process.

Acrosome↗
PubMed1992

Possible implication of lysophosphatidylcholine in cell fusion accompanying implantation in rabbits.

Implantation in rabbits involves the cellular fusion of trophoblastic and uterine epithelial cells resulting in embryo penetration of the uterine endometrium. Since lysophospholipids, known to have fusigenic properties, could be responsible for this cell fusion, the metabolism of lysophospholipids was studied throughout gestation in blastocyst/yolk sac and extracoelic amnioallantoic fluids. Analysis of phospholipid composition revealed that lysophospholipids are present in blastocyst/yolk sac fluid. Their concentrations and haemolytic activity change during pregnancy. They increase and reach their highest values during days 7 to 9, the implantation days in rabbits. A clear correlation was observed between lysophosphatidylcholine concentrations in blastocyst/yolk sac fluid and haemolysis induced by this fluid. Phosphatidylcholine concentrations, phospholipase A2 activity, which generates lysophospholipids, and lysophospholipase A activity which hydrolyses lysophosphatidylcholine into fatty acid, were at their highest value at day 12. These data suggest that a transient accumulation of lysophospholipids could ensure local cell fusion. Moreover, we propose that the lysophospholipid concentrations in blastocyst/yolk sac fluid are dependent upon activities of phospholipase A2 and lysophospholipase.

Animals↗
PubMed1990

Local alteration in adenylate cyclase activity and stimulation response at implantation site in rabbit endometrium during early pregnancy.

Adenylate cyclase activity and its hormonal stimulation were measured in endometrial tissue, and sex steroid levels were quantified in uterine tissue collected from pregnant and estrous rabbits. The tissues from pregnant animals were separated into implantation (ES) and interimplantation (IES) sites. Adenylate cyclase activity was measured in broken cell preparations by enzymatic conversion of alpha-32P-adenosine triphosphate (ATP) into 32P-cyclic adenosine 3', 5'-monophosphate using Mg2(+)-ATP as a substrate. The activity was measured with no addition (basal) and after stimulation with guanosine triphosphate (GTP), NaF, or increasing doses (1 nM to 100 microM) of isoproterenol (ISO) and prostaglandin E2 (PGE2). The presence of GTP was necessary to observe a stimulation by ISO and PGE2. During pregnancy, adenylate cyclase activity was reduced compared to activity at estrus on Day 6.5 (IES and ES) and on Day 9 (IES); however, it reached its highest level at ES (Day 9). The regulation of isoproterenol response followed a similar pattern. Dose responses to PGE2 were markedly affected by physiological status. The response was higher during pregnancy than at estrus, and response (percent of GTP), as well as sensitivity, was higher in IES than in ES on Day 6.5 and even greater on Day 9. The levels of estradiol (E2) were reduced during pregnancy, but comparable in ES and IES; however, progesterone (P) levels were reduced in ES, and the E2/P ratio was significantly higher (p less than 0.01) in ES (15 +/- 1, 17 +/- 2) than in IES (8 +/- 1, 6 +/- 0.8) on Days 6.5 and 9, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1990

Decreased binding of calmodulin to bull sperm proteins during heparin-induced capacitation.

The 125I-calmodulin gel overlay procedure was used to evaluate the effect of a heparin treatment on the calmodulin-binding proteins of bull spermatozoa. At concentrations that increase the in vitro fertilization rate of in vitro-matured oocytes, heparin induced a decrease in the binding to calmodulin (CaM) in 3 sperm proteins of 28, 30, and 49 kDa. The binding of these proteins to CaM was higher when Ca2+ was absent from the overlay procedure, and this binding was negatively correlated to the fertilization rate. These results suggest that sperm capacitation is associated with a decrease in the binding of CaM to the 28, 30, and 49 kDa sperm CaM-binding proteins. Implications of such a decrease are discussed.

Animals↗
PubMed1989

Rabbit blastocoelic fluid regulation of tumor-cell proliferation in vitro.

To determine whether rabbit blastocoelic fluid could inhibit tumor-cell proliferation, day-9 and day-12 embryonic fluids, together with autologous and homologous sera, were collected from pregnant or pseudopregnant rabbits and tested against 13 different cell lines and on human carcinoma cells in primary culture. An inhibitory effect on cell proliferation was observed in the presence of blastocoelic fluids, but not with homologous or heterologous sera. This suppression was higher with samples collected at day 12 than at day 9 of pregnancy. No such inhibition could be detected on one-cell rabbit embryos or on freshly prepared uterine stromal or myometrial cells. In addition, the inhibitory activity on tumor cells was completely reversible upon removal of the fluids. Incorporation of 3H-thymidine, 3H-uridine and 35S-methionine revealed that, in the presence of blastocoelic fluids, both DNA and RNA syntheses were rapidly inhibited. Inhibition of protein synthesis did not occur before 24 hr of treatment. We conclude that rabbit blastocoelic fluid suppresses the proliferation of tumor cells via inhibition of RNA and DNA synthesis by a process which may involve the expression of growth-suppression gene(s).

Animals↗
PubMed1989

Modulation of the immunosuppressive activities by blastocoelic fluid during rabbit pregnancy.

Temporal variation in immunosuppressive activity was determined in biological samples such as embryo-foetal fluids (blastocoelic- or amino-allantoic fluid) and blood collected from pregnant and pseudopregnant rabbits. Each of the fluids to be analyzed was pre-incubated with mitogen stimulated human lymphocytes for 48 h and then inhibition of [3H]thymidine incorporation or IL-2 receptor expression was estimated. Both means of assessing immunosuppression indicated variations in the suppressive activity throughout pregnancy. This was observed in embryo-foetal fluids but not in autologous peripheral blood nor in homologous pseudopregnant blood. At days 9-13 of pregnancy, the immunosuppressive effects of blastocoelic fluids were higher than that of the autologous sera, reached a peak at days 12 and 13 and declined thereafter, to reach the lowest levels. In order to further characterize the biological activity of day-12 blastocoelic fluid and autologous serum, they were submitted to ultracentrifugation. No suppressive activity could be demonstrated in the lipoprotein fractions. But all the activity was found in the protein fraction. Precipitation with cold ethanol confirmed that the biologically active compound was a protein. Furthermore, results obtained after ultrafiltration suggest biologically active compounds of high mol. wt (greater than 300 kDa). From the above findings, we can suggest that in the rabbit, there is no pregnancy specific systemic immunosuppression. We can also infer that (1) the immuno-tolerance of the mother towards the embryo is more due to a localized effect; (2) this effect decreases with the progression of gestation and (3) a high mol. wt factor is responsible for the immunosuppression.

Allantois↗
PubMed1989

Local effect of the rabbit embryo-foetus on uterine progesterone and pregnenolone levels.

Rabbit peripheral serum and uterine tissue (embryonic (EZ) and interembryonic (IEZ) zones) were assayed for the main C21, C19 and C18 steroids throughout pregnancy and pseudopregnancy (PSPG). Pregnenolone concentrations in PSPG and IEZ were comparable and remained relatively stable, while its level in EZ increased, reaching a peak value of 18.2 +/- 0.8 ng/g by day 15, and decreasing thereafter to a level comparable to oestrus by day 25. Tissue concentrations of progesterone were comparable in PSPG and IEZ, reached their maximal level on days 6.5 and 9, and decreased significantly (P less than 0.01) on day 15. In EZ, progesterone level was significantly lower than in IEZ and decreased on day 9 compared to day 6.5. A further decrease was observed from days 9 to 15 but no difference between tissues was observed on the latter day. Thus, the blastocyst-foetus exerts a local effect by decreasing progesterone content and increasing pregnenolone level in the uterine tissue adjacent to its implantation (EZ). The conversion of progesterone in uterine tissue to less-active metabolites does not appear to occur towards the C19 and C18 steroids.

Animals↗
PubMed1989

Regulation of adenylate cyclase activity and stimulation response in relation to endometrial receptivity in the rabbit.

Adenylate cyclase activity was measured in broken cell preparations of whole endometrial tissue from rabbits on Days 0, 1, 6.5, 9 and 15 of pseudopregnancy and in endometrial epithelial and stromal cells on Days 1 and 6.5 to assess the specific response of individual cell types. In dispersed cells, adenylate cyclase activity was higher (P less than 0.01) in stromal than in epithelial cells and reduced on Day 6.5 compared to Day 1 in both cell types. The response of adenylate cyclase to isoproterenol appeared more important relative to the PGE-2 response in epithelial than in stromal cells and strongly reduced in the former on Day 6.5. In endometrium, the overall adenylate cyclase activity was increased significantly on Day 1 of pseudopregnancy compared to Day 0 (oestrus), only 18 h after injection of hCG. On the following days, the activity decreased progressively on Days 6.5 and 9 and exhibited a recovery on Day 15. Adenylate cyclase response to isoproterenol (% over GTP) was comparable on Days 0, 1 and 6.5, abolished on Day 9 and recovered on Day 15. Maximal response to PGE-2 (% over GTP) was observed on Day 6.5, at the time of implantation, maintained on Day 9 and reduced on Day 15 towards the low levels measured in oestrus and Day 1 of pseudopregnancy. Our results demonstrate a dramatic alteration of adenylate cyclase activity in rabbit endometrium during pseudopregnancy. It suggests a possible involvement of catecholamines and prostaglandin E-2 in the regulation of endometrial receptivity through a cAMP-mediated process.

Adenylyl Cyclases↗
PubMed1989

Effect of heparin on the expression of calmodulin-binding proteins in bull spermatozoa.

A 125I-labelled calmodulin gel overlay procedure in the presence and the absence of Ca2+ was used to evaluate bull spermatozoa calmodulin-binding proteins. Frozen spermatozoa were thawed, washed and incubated for 6 h before being processed for SDS polyacrylamide gel electrophoresis and the 125I-labelled calmodulin gel overlay procedure. In non-incubated spermatozoa, up to 14 binding proteins were detected. Some exhibited greater calmodulin binding in the presence of Ca2+ while others exhibited greater binding when Ca2+ was absent. When heparin (2 micrograms/ml) was present in the incubation medium, a decrease in the calmodulin binding to the proteins of Mr 28,000 and 30,000 was detected in the presence of Ca2+ and EGTA. This effect of heparin was time- and dose-dependent and was increased by the presence of the acrosin inhibitor benzamidine. Sperm capacitation could thus be related to a decrease in the binding of calmodulin to these proteins.

Animals↗
PubMed1979

[Confirmation of the presence of uteroglobin in tubal secretions].

The acrylamide gel electrophoresis of rabbit serum, of rabbit tubal secretions at the oestrus stage (STO) and at the luteal stage (STL), followed by immunodiffusion with anti-serum, was done to detect any tubal proteins which were not found in the rabbit serum. The anti-rabbit STO goat anti-serum was absorbed with rabbit plasma in order to purify antibodies to proteins found specifically in tubal secretions. This technique revealed the presence of a protein (STx) in STO and STL but not in the rabbit serum. The electrophoretic migration (Rf = 0.72) of the tubal protein (STx) is similar to uteroglobin (Rf = 0.74). The tubal protein was detected immunologically only in tissues where uteroglobin had been found by other workers: lungs, semen, Fallopian tubes and uterine secretions (5 days post-ovulation); further more a complete identity for the precipitating line existed between these extracts. These results corroborate the presence of uteroglobin or of a very similar protein in oestrus of luteal tubal secretions and in some other rabbit tissues.

Animals↗
PubMed1978

[Effect of coitus on sperm transport and fertilization in rabbits].

The number of spermatozoa into the Fallopian tubes and levels of in vivo fertilization were measured following different treatment. The results show that copulation by the artificially inseminated animal has a strong influence on the transport of the rabbit spermatozoa and "in vitro" fertilization. This constitutes some more evidence about the physiological role played by the utero-tubal junction.

Animals↗
PubMed1975

In vitro fertilization of rabbit eggs in oviduct secretions from different days before and after ovulation.

In vitro fertilization of rabbit eggs in oviduct secretions has been studied through different days of the psudopregnant cycle. The appearance of the pronuclei and cleavage into two cells, which were the criteria of fertilization, occurred significantly less frequently when in vitro fertilization was attempted in the oviduct secretions obtained during the estrous (preovulatory) period of the cycle than it was in the secretions obtained during pseudopregnancy or in Brackett's medium plus 20% heated rabbit blood serum. Whether fertilization was attempted in ampulla secretions or in whole oviduct secretions had no effect on the success rate. The effects of oviduct secretions from the estrous period of the cycle is probably due mainly to an effect on the egg, involving the very first development processes during and after fertilization.

Animals↗