Search PubMed⌕ Search

Biomedical subjects

R D Lambert

Publications and source records attributed to R D Lambert.

At least 37 records · Page 2Linked to original sources
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

Androgen metabolites in bovine follicular fluid.

The values of C21-steroids, Delta4-androgens, estrogens as well as 5alpha-reduced steroids have been determined in follicular fluid obtained from superovulated and untreated cows. In the three cows treated with a hormone regimen to induce superovulation, the levels of progesterone and estradiol determined in 3 to 6 follicles per cow ranged from 65 to 448 ng/ml and 1.9 to 8.6 ng/ml, respectively while the concentrations of androstenedione and testosterone varied between 1.5 to 2.5 ng/ml. Low levels of dihydrotestosterone and androstane-3alpha, 17beta-diol (approximately 30 to 50% of Delta4-androgens) were found in the bovine follicular fluid. In untreated cows, the follicular steroid concentrations were divided into two groups on the basis of the ratio between estrogen and Delta4-androgen concentrations. In estrogen-rich follicles, the ratio of estrogens Delta4- androgens was higher than 1 and in estrogen-poor follicle, the ratio of estrogens Delta4- androgens was lower than 1. Pregnenolone, dehydroepiandrosterone, androst-5-ene-3beta, 17beta-diol, progesterone, androstenedione and testosterone levels were not significantly different in the two groups while the levels of estradiol and estrone were approximately 100-fold higher in the estrogen-rich group. The concentrations of 5alpha-reduced steroids particularly, dihydrotestosterone, androstane-3alpha, 17beta-diol and androsterone as well as their glucuronides which were found at values extremely low (under 1 ng/ml) were not significantly different in both groups. The results indicate that low levels of 5alpha-reduced steroids and their glucuronides are present in bovine follicular fluid and their concentrations remained fairly stable either in estrogen-rich or estrogen-poor groups.

Journal Article↗
PubMed1989

Relationship between steroid levels in peripheral serum and uterine tissue during pseudopregnancy in rabbit.

Determination of the main C21, C19 and C18 steroids in peripheral serum and uterine tissue was made to study the relationships of the steroids during pseudopregnancy in the rabbit. Tissue and serum progesterone levels rose significantly (P < 0.01) from estrus to Day 9 and then decreased by 81% (tissue) and 57% (serum) at Day 15, while pregnenolone levels in uterine tissue and serum remained unaffected. Estradiol serum levels remained fairly stable, whereas its concentration in uterine tissue decreased after estrus by 85%, followed by a significant (P < 0.05) increase from Day 9. Most of the C19 steroid values in the uterine tissue and in the serum were generally at levels below or near the limit of detection and did not vary significantly. Since progesterone and estradiol levels were high and/or varied significantly in tissue throughout pseudopregnancy, and since androgens were produced in small amounts, it is suggested that androgens might not play a significant role in uterine tissue during pseudopregnancy.

Journal Article↗
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↗
PubMed1988

Cytogenetic study of parthenogenetically activated bovine oocytes matured in vivo and in vitro.

Three experiments were performed to evaluate the potential for parthenogenetic activation of bovine oocytes in in vitro fertilization systems and to determine the chromosome complement of the resulting parthenogenotes. In the first experiment, immature oocytes from slaughtered cattles were matured in vitro in Defined Medium (DM) for 24 h to simulate in vitro fertilization conditions. Subsequently, a portion was fixed, and the remainder were transferred to rabbit oviducts. Oocytes were then cultured for 6-8 h or for 24 h with Colcemid present during the last 6 to 8 h and fixed on slides and examined. In the second experiment, mature oocytes were collected from the preovulatory follicles, and the oocytes were subjected to the same culture as in experiment I. In the third experiment, oocytes were treated as in experiment II, except that instead of transfer to rabbit oviducts, they were cultured an additional 48 h in vitro. In experiment I, 131 oocytes were fixed after culture in DM. Of the 79 oocytes analyzed in the pre-rabbit group, 71 (90%) were at the second meiotic metaphase (MII), and 8 (10%) were at pre-MII stage; none were activated. After transfer to rabbits, 291 were fixed. Of these, 80 were analyzed; 37 (46.3%) were MII, 7 (8.6%) were pre-MII, and 36 (45%) were activated. Of the 36 activated oocytes, 26 (72.2%) were haploid, 4 (11.1%) were diploid, 1 (12.8%) was tetraploid, and 5 (13.8%) were in the process of endoreduplication. In experiment II, 51 oocytes were fixed after culture in DM of which 36 (70.6%) could be analyzed; 30 (83.3%) were MII, and 6 (16.7%) were pre-MII. After culture in the rabbit, 68 were fixed of which 27 (39.7%) could be analyzed. Of these 27, 20 (74.1%) were MII, and 7 (25.9%) were activated; 6 were haploid, and 1 was endoreduplicating. In experiment III, 30 oocytes were fixed at the end of the culture period; only 10 could be analyzed of which 8 (80%) were MII and 2 (20%) were pre-MII. In all, 46% of in vitro and 26% of in vivo matured oocytes were activated, based on chromosomal analysis. Of those activated, the majority (74.4%) were haploid, suggesting that activation occurs at or after completion of MII. Endoreduplication appears to be one of the mechanisms leading to the formation of diploid and polyploid parthenogenotes.

Animals↗
PubMed1988

The embryo influences adenylate cyclase activity and hormonal response in rabbit myometrium during early pregnancy.

The effect of pseudopregnancy (PSPG; days: 0 (estrus), 1, 6.5, 9 and 15) and pregnancy (PG; days: 6.5, 9 and 15) on adenylate cyclase (AC) activity was verified in rabbit myometrium. During PSPG, there was a time related decline in basal activity from 71 +/- 16.2 (D 0) to 13.1 +/- 1.6 (PSPG-D9) pmoles cAMP formed/mg prot-min. Stimulation of the enzyme by GTP, Isoproterenol (ISO), Prostaglandin E2 (PGE2) or Sodium Fluoride (NaF) followed a similar pattern. AC activity was compared in myometrial tissues of pregnant animals (PG) separated into embryonic (ES) and interembryonic (IES) sites. On days 6.5 and 9, AC activity measured in tissues from PG rabbits (ES and IES) was always higher than that found in tissues from PSPG animals on corresponding days. On day 6.5, AC activity was slightly higher (p less than 0.01) in ES than in IES. This was confirmed on day 9 where basal as well as GTP, ISO and PGE2 stimulated activities were higher in ES than in IES (p less than 0.001). Dose response to ISO, expressed as % of GTP, were similar on D0, 1, 6.5 and 15 of PSPG. However, on day 9, there was a striking diminution in response reflected by a lower stimulation at suboptimal dose (0.1 microM; p less than 0.05) from 115 +/- 2 on day 0 to 104 +/- 4 on day 9. These results suggest that protein content which is increased during pseudopregnancy could be responsible for the decline of AC activity. However, results obtained on day 9 and 15 suggest that other factors are involved. Dose responses to ISO during PG showed an alteration in response on days 6.5 and 9 at ES. It was reflected by a higher stimulation at suboptimal (0.1 microM) and optimal doses (100 microM). These results suggest that myometrial AC activity could be regulated by the presence of the embryo.

Adenylyl Cyclases↗
PubMed1988

Immunosuppressive effects of rabbit blastocoelic fluid and embryo culture medium.

In order to understand the mechanism of the feto-maternal immune relationship, we assayed the immunosuppression activities of fresh blastocoelic fluid and decomplemented peripheral serum collected from day-9 pregnant white New Zealand rabbits and of rabbit embryo culture medium (ECM). Because the viability of the human lymphocytes was not affected by either of these biological fluids and since they were easy to obtain in sufficient quantities, they were used uniformly in all the experiments. Immunosuppressive effect was calculated by the relative inhibition of proliferation of Con A-stimulated lymphocytes. The immunosuppressive effect of blastocoelic fluid of the 9-day pregnant rabbits was significantly higher than that of autologous decomplemented serum (P less than 0.001). The inhibition by the serum was non-specific because sera from non-pregnant animals as well as sera from different stages of pregnancy and pseudo-pregnancy showed the same level of inhibition. The ECM of 6.5-7-day-old embryo showed a pronounced immunosuppressive effect. When embryos of 1,3 and 5 days were cultured and their culture media were assayed only with 5-day-old embryo the effect had begun to appear, but it was far less than that of 7-day-old embryo (P less than 0.02). The suppressive activity of both the blastocoelic fluid and ECM was not due to cytotoxic effect, since this fluid supported the in vitro growth of single-cell rabbit embryos up to the stage of blastocyst. These results suggest that the immunologic tolerance of the embryo might be due to the immunosuppressors secreted by the embryo and that there might be a localized effect at the implantation site rather than a maternal systemic immunosuppressive effect.

Animals↗
PubMed1987

Cell-specific localization of prostaglandin E2-sensitive adenylate cyclase in rabbit endometrium.

Epithelial and stromal cells were isolated from endometrium of Day 1 pseudopregnant rabbits by enzymatic digestion with trypsin or trypsin:collagenase:deoxyribonuclease. Dispersed cells were grown in RPMI 1640 supplemented with 10% whole or steroid-depleted fetal bovine serum (FBS). Epithelial and stromal cells reached confluency after 6 to 7 days in culture and showed specific characteristics. Cells could be differentiated according to morphology, growth patterns, electrophoretic patterns, and response to estrogen or progesterone. Hormonal stimulation of adenylate cyclase activity was measured in broken cell preparations by catalytic transformation of alpha-32P-adenosine triphosphate into 32P-adenosine 3'-5' cyclic monophosphate (cAMP). Adenylate cyclase activity was present in fresh endometrial tissue and in dispersed cells after 7 days in culture. The enzyme activity was significantly higher in stromal than in epithelial cells at all stimulation levels: basal (9.2 +/- 1.0 vs. 2.3 +/- 0.6, p less than 0.001) and guanosine triphosphate (GTP, 300 microM) (25.4 +/- 2.9 vs. 7.0 +/- 1.6, p less than 0.001). Net response to prostaglandin E2 (PGE2, 10 microM) was three times higher (p less than 0.001) in stromal (17 +/- 2) than in epithelial (5.0 +/- 1) cells. These results suggest that PGE2 can stimulate adenylate cyclase in rabbit endometrium and that the enzyme is preferentially localized in the stroma. Our results are in agreement with the hypothesis that cAMP formed in endometrium in response to PGE2 might be involved in the decidual reaction.

Adenylyl Cyclases↗
PubMed1986

Birth of calves after in vitro fertilisation using laparoscopy and rabbit oviduct incubation of zygotes.

The infertility of many cows could be treated by in vitro fertilisation. In the present study laparoscopy was utilised to recover the in vivo matured oocytes from the ovary of a standing donor. After the capacitation of fresh semen with high ionic strength medium and in vitro fertilisation, a rabbit oviduct was employed as an incubator for four to five days, in order to obtain sufficiently aged embryos to be transferred to the uterus of a recipient. Using surgical or non-surgical transfer six calves were obtained.

Animals↗
PubMed1985

In vitro fertilization of bovine follicular oocytes obtained by laparoscopy.

Bovine follicular oocytes (n = 454), obtained after laparoscopy, were used to study in vitro capacitation, fertilization, and embryo development. Capacitation was accomplished by treating bovine spermatozoa with high ionic strength medium. Maturation, fertilization, and development studies were carried out in Brackett's defined medium or in Ham's F-10. In vitro fertilization rates, ranging from 14% to 55%, were found to be influenced by individual variations among males. Brackett's defined medium was found to be superior to Ham's F-10 for oocyte maturation, fertilization, and growth, these media giving cleavage rates of 60% and 32%, respectively. Oocytes with expanded cumuli at the time of recovery cleaved at a rate of 43%, which is significantly different from oocytes recovered without granulosa cells (22%) or oocytes with compact cumuli and corona cells (5%). The in vitro development pattern of the in vitro-fertilized embryos was found to be similar to that observed in vivo. Embryos were observed at the 2-cell stage 44.5 +/- 6.3 h after in vitro insemination, 4-cell after 59.0 +/- 9.4 h, 8-cell after 74.8 +/- 12.7 h, and 16-cell after 96.2 +/- 13.9 h (observations at 12-h intervals). The procedures described here resulted in cleavage rates of up to 60% using follicular oocytes embedded in expanded cumuli cells and semen samples from selected males.

Animals↗
PubMed1985

Pregnancies after in-vitro fertilization of cow follicular oocytes, their incubation in rabbit oviduct and their transfer to the cow uterus.

Cow embryos fertilized in vitro (1-8-cell) (n = 113) were transferred surgically to the ligated oviduct of pseudopregnant female rabbits (31 +/- 4 h after 75 i.u. hCG). Rabbits were killed 99 +/- 5 h later and 67 embryos were recovered: 45 (67%) had cleaved at least once, 15 had reached the morula stage and 2 blastocysts were obtained. Transfer of cleaved embryos (2-8-cell) led to a high recovery rate (84%) compared to 39% for one-cell embryos. Of the embryos incubated for more than 99 h in the rabbit oviduct, 41% were at the morula stage. Embryos incubated in vivo (n = 21) (8-cell to blastocyst) were transferred to the uterus of 14 synchronized recipient heifers by a surgical (N = 5) or a non-surgical (N = 9) procedure: 6 pregnancies resulted (4 from the non-surgical procedures). In addition, 27 (2-8-cell) cow embryos developed in vitro were transferred to the oviduct of synchronized heifers by a paralumbar surgical approach on a standing animal, but no pregnancies resulted. It is therefore concluded that (1) the rabbit oviduct can be used to obtain cow eggs at an embryonic stage sufficiently advanced to permit transfer to the uterus of a synchronized recipient; and (2) the pregnancy rate after in-vitro fertilization and incubation in the rabbit oviduct are similar to results with fertilization in vivo.

Animals↗
PubMed1984

Laparoscopic investigation of the bovine ovary in the periovulatory phase of the cycle.

Laparoscopy, in combination with a rapid radioimmunoassay for plasma-LH determination, has been used to predict and observe ovulation in heifers. Experiments on three animals with typical progesterone levels, LH levels and apex formation are described. Ovulation was observed from 25 h to 29 h after the beginning of LH rise and from 17 h to 19 h after LH peak. The LH peak lasted for 9 h to 11 h. Cow ovulation was observed and photographed. The preovulatory follicle, apex formation, ovulation and freshly ruptured follicles are illustrated. The results presented here demonstrate that laparoscopy could offer valuable diagnostic assistance in clinical veterinary medicine.

Journal Article↗
PubMed1984

[Ovulation prediction in cattle by rapid measurement of plasma LH and by laparoscopic observation].

The last hours of intrafollicular maturation are important to preserve the potential of oocytes for fertilization and development. To collect mature oocytes it is consequently essential to predict accurately the time of ovulation. Therefore we have modified a regular and specific plasma LH radioimmunoassay to a convenient assay providing reliable results within four to five hours. Using this technique we have been able to detect the LH peak with a delay small enough to predict the moment of ovulation; then we have been able to observe and photograph ovulation. The antigen-antibody reaction takes place during a two hour incubation at 37 degrees C. The immune complex is precipitated by addition of a rabbit anti-gamma-globulin and incubation at 22 degrees C for 30 minutes. The variation between fast and regular assays is lower than 15%. From a study of 25 cycles in 12 animals we suggest intervals of approximately 27 hours between the beginning of the LH peak and ovulation and of about 17 hours between the end of the peak and ovulation. Intervals between the beginning of heat and ovulation appear irregular and cannot be used with certainty as a reference point to predict ovulation. Five ovulations were observed closely and in the five cases, the formation of an apex was noted Our results clearly show that with the technique mentioned above we can predict rapidly and precisely bovine ovulation.

Animals↗