Effect of oestrous cycle and early pregnancy on uterine production and expression of immune regulatory factors in gilts.

This study was undertaken to characterize uterine immune factors involved in the establishment of pregnancy in gilts. Thirty crossbred Yorkshire-Landrace gilts of similar age and weight were observed twice a day for oestrous behaviour with intact boars. On the day of first standing oestrus (Day 0) and 12h later, 15 gilts were inseminated with pooled semen from Duroc boars of proven fertility. Pregnant gilts were slaughtered either on Days 10, 15 or 25 of gestation (n=5 per day). The other 15 gilts were not inseminated and were slaughtered on either Days 0, 10 or 15 of the oestrous cycle (n=5 per day). Immediately after slaughter, endometrial tissue samples from the mesometrial side were removed for gene expression using RNase protection assay and in situ hybridization methodologies. The other uterine horn was flushed with 20 ml of PBS to collect the uterine fluid. In pregnant gilts, endometrial interleukin (IL)-6 mRNA expression was higher on Day 15 than on Days 10 and 25 (P<0.01 and P<0.1, respectively). On Day 15, IL-6 expression was also significantly higher (P<0.01) in pregnant gilts than in cyclic gilts. In both pregnant and cyclic gilts, transforming growth factor (TGF)-beta2 in uterine fluid was significantly higher (P<0.0001) on Day 15 than on Day 10. At the gene expression level, TGF-beta2 also increased between Days 10 and 15 in both cyclic and pregnant gilts but differences were not significant. On Day 15, concentrations of interferon-gamma and prostaglandin E(2) (PGE(2)) in uterine fluid were markedly higher (P<0.001) in pregnant gilts than in cyclic gilts, whereas the total amount of TGF-beta2 in uterine fluid and its endometrial expression were approximately 70% higher although this increase was not significant. Finally, tumour-necrosis factor-alpha and granulocyte-macrophage/colony-stimulating factor mRNA expressions were undetectable in all endometrial samples. In conclusion, production and/or expression of uterine TGF-beta2, IL-6 and PGE(2) increased during the embryonic attachment period and are coincidental with embryonic interferon-gamma production.

Interferon-tau stimulates granulocyte-macrophage colony-stimulating factor gene expression in bovine lymphocytes and endometrial stromal cells.

Interferon-tau (IFN-tau), the antiluteolytic signal produced by the trophoblast prior to implantation in ruminants, exhibits immunomodulatory properties. It stimulates the production of prostaglandin (PG) E(2) in bovine endometrial cells via the induction of cyclooxygenase-2 (COX-2). We previously demonstrated that preconditioning lymphocytes with PGE(2) increases the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that promotes conceptus growth and survival. Our goal in the present study was to evaluate the impact of IFN-tau on the expression of GM-CSF in bovine peripheral blood lymphocytes (PBL) and endometrial epithelial and stromal cells. Changes in PGE(2) production and mRNA levels of COX-2 were also studied in PBL in response to IFN-tau. Gene expression was estimated by semiquantitative reverse transcription-polymerase chain reaction and Northern analysis. The expression of GM-CSF in PBL was stimulated by treatment with IFN-tau. Furthermore, GM-CSF mRNA levels were increased after preconditioning PBL for 3 days with IFN-tau, followed by a 12-h restimulation without IFN-tau. Inhibition rather than stimulation of PGE(2) production and COX-2 expression in PBL during treatment with IFN-tau suggests a direct effect on GM-CSF expression. Moreover, GM-CSF expression was stimulated in uterine stromal cells in response to IFN-tau. This study provides the first evidence for stimulation of GM-CSF expression by IFN-tau in both leukocytes and endometrial stromal cells. In view of the role of GM-CSF on fetal growth and survival, these results support the hypothesis that the conceptus mediates accommodation mechanisms in the uterus during early pregnancy by modulating the expression of beneficial cytokines at the fetomaternal interface.

CD8 membrane expression is down-regulated by transforming growth factor (TGF)-beta 1, TGF-beta 2, and prostaglandin E2.

PROBLEM: CD8 T-cells are present at a lower frequency in human decidua than in peripheral blood. Because transforming growth factor (TGF)-beta 2 down-regulates CD4 membrane expression, its contribution, as well as the contribution of TGF-beta 1 and prostaglandin (PG) E2, to the modulation CD8 expression was studied using human peripheral blood lymphocytes (PBLs). METHOD OF STUDY: PBLs were cultured with TGF-beta 1, TGF-beta 2, PGE 2, PGI 2, or day-12 rabbit blastocoelic fluid (BF) that was or was not depleted of TGF-beta 2 and/or PGE 2. Quantum Simply Cellular Microbeads were then used to evaluate CD8 membrane expression levels. RESULTS: This study is the first demonstration that treatment of PBLs with TGF-beta 1, TGF-beta 2, and PGE 2 leads to a dose-dependent decrease in CD8 expression. A significant inhibition was observed at 2.5 mg/mL for TGF-beta 2, 5 ng/mL for TGF-beta 1, and 10 ng/mL for PGE 2. In contrast, PGI 2 had no effect. Treatment of PBLs with BF day-12 decreased CD8 expression. This effect, however, was not observed when BF was depleted of TGF-beta 2 and/or PGE 2. CONCLUSIONS: Our results suggest that TGF-beta s and PGE 2 are important modulators of CD8 membrane expression in human lymphocytes. Because TGF-beta 1, TGF-beta 2, and PGE 2 are produced by the conceptus and by uterine cells and because the effect is observed after only 3 days of treatment, the present data suggest that these substances can locally modulate the phenotype of lymphocytes at the fetomaternal interface. Such modulation may explain, at least partly, the changes observed in the population of decidual lymphocytes during pregnancy.

Prostaglandin E2 regulates both interleukin-2 and granulocyte-macrophage colony-stimulating factor gene expression in bovine lymphocytes.

Prostaglandin E2 (PGE2) is known to inhibit interleukin-2 (IL-2) production by human peripheral blood lymphocytes (PBL) and to increase granulocyte-macrophage colony-stimulating factor (GM-CSF). In many species with hemochorial placentation, down-regulation of IL-2 appears necessary to impede early embryonic demise, whereas up-regulation of GM-CSF increases embryonic growth and survival. It is not known whether the same mechanisms are involved in a species with a less invasive placenta. PGE2 is synthesized during early bovine gestation by the endometrium and by the embryo, and it may therefore be involved in regulating IL-2 and GM-CSF in this species. Our goal was to evaluate the impact of PGE2 on cellular proliferation and on IL-2 and GM-CSF gene expression in bovine PBL. Incorporation of [3H]thymidine was used to study DNA synthesis. Gene expression was estimated by semiquantitative polymerase chain reaction using bovine-specific primers and by Northern analysis using amplified bovine cDNAs as probes. The DNA synthesis and IL-2 mRNA levels of bovine PBL stimulated by concanavalin A (ConA) were greatly reduced by PGE2 in direct-treatment studies. Under the same conditions, GM-CSF gene expression was also inhibited. However, pretreatment of PBL for 72 h with ConA and PGE2, followed, after washing, by an incubation with ConA alone for 12 h resulted in reduced DNA synthesis, stable expression of IL-2, and a dramatic increase of GM-CSF mRNA levels. This is the first evidence in the bovine model that direct treatment with PGE2 down-regulates IL-2 and GM-CSF mRNA levels and that preconditioning with PGE2 stimulates GM-CSF gene expression. We propose that PGE2, either from embryonic or from endometrial compartments, induces bovine PBL to undergo functional changes, affecting cellular proliferation and cytokine production in order to accommodate the developing conceptus.

TGF-beta 2 and PGE2 in rabbit blastocoelic fluid can modulate GM-CSF production by human lymphocytes.

PROBLEM: During normal pregnancy, major changes occur in the production of Th2/Th1 cytokines at the feto-maternal interface. Th2 cytokines such as interleukin-4 (IL-4) or interleukin-10 (IL-10) are predominantly produced locally in the uterine and placental tissues, whereas the production of Th1 cytokines such as tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) are decreased. Because these modulation might be induced by the embryo, the current study was carried out to test the effect of rabbit blastocoelic fluid on the production of Th2/Th1 cytokines by lymphocytes, and to investigate the possible implication of transforming growth factor beta 2 (TGF-beta 2) prostaglandin E2 (PGE2) as modulators of the production of these cytokines. METHOD OF STUDY: Human peripheral blood lymphocytes (PBL) were cultured along with ConcanavalinA(Con A), and rabbit blastocoelic fluid was collected on day 12 of gestation (BF d-12). Concentrations of cytokines in culture media were determined by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: Addition of BF d-12 in the culture medium induced a strong inhibition of IL-2, TNF-alpha, IL-10, and granulocyte-macrophage colony-stimulating factor (GM-CSF) production. However, an initial pretreatment of the lymphocytes with BF d-12, followed by a Con A stimulation, led to a marked increase in GM-CSF production, whereas IL-2, TNF-alpha, and IL-10 secretions were inhibited. It was also demonstrated, for the first time, that a pretreatment of the lymphocytes with TGF-beta 2 and PGE2 increased GM-CSF production to the same level reached after the addition of BF d-12. Furthermore, removal of TGF-beta 2 and PGE2 from BF d-12 by affinity chromatography reduced the effect of BF d-12 on GM-CSF production. CONCLUSIONS: Taken together, these findings suggest that the embryo, in modulating harmful and beneficial cytokine production locally, plays an active role in its protection against maternal immune cellular assault. These results also emphasize the importance of growth factors for successfully maintaining pregnancy.

PGE2, but not TGF beta 2, in rabbit blastocoelic fluid regulates the cytotoxic activities of NK and LAK cells.

Spontaneous and induced fetal resorptions have been associated with the infiltration and activation of GM1-positive natural killer (NK)-like cells. Predominance of these cells in the decidua and their reduced lytic activity suggest that regulation of their killing activity could be important for the survival of the fetus. It has therefore been hypothesized that the embryo was regulating NK lytic activity. To test this hypothesis, human and rabbit lymphocytes were cultured with various concentrations of interleukin-2. Their ability to kill 51Cr-labelled NK and lymphokine-activated killer (LAK)-sensitive targets was assessed in the presence of rabbit blastocoelic fluid taken at day-12 of pregnancy (BF D-12). BF D-12 dramatically suppressed the killing activity of NK and LAK cells. This effect was observed on K562 (NK-sensitive targets), P815 cells (LAK-sensitive targets), and freshly isolated cells in rabbit trophoblastic cell preparation. Elimination of prostaglandin E2 (PGE2), but not transforming growth factor beta 2 (TGF beta 2) or 6 keto prostaglandin F1 alpha (6KPGF 1 alpha), by affinity chromatography, completely abolished BF biological activity. These findings clearly suggest that PGE2 in BF regulates the killing activity of NK and LAK cells, and that the semiallograft embryo plays an active role in its own protection. To our knowledge, it is the first demonstration that PGE2 from the embryo inhibits NK and LAK cell lytic activity.

TGF beta 2 in rabbit blastocoelic fluid regulates CD4 membrane expression: possible role in the success of gestation.

PROBLEM: During pregnancy, major changes occur in the decidual cell population. One of these changes involves some phenotypical transformations of lymphocyte sub-populations. Since these variations might be due to the presence of the embryo, the current study was designed to investigate the implication of blastocoelic fluid (BF) in these changes and to determine the mechanism by which this phenomenon occurs. METHOD: Lymphocytes isolated from human peripheral blood (PBL) were cultured for 72 h in RPMI-FCS 10% and with or without BF day 12 (BF d-12) or Concanavalin A (ConA). After 72 h, T cells were labelled with anti-CD4 antibodies and Quantum Simply Cellular microbeads were used as a standard to evaluate the antibody binding capacity (ABC). RESULTS: Treatment of human PBL with BF d-12 decreases the percentage of CD4 and TCR positive cells, as compared to non-stimulated cells, but has no significant effect on CD2, CD3, and CD8 positive cells. It was also demonstrated, for the first time, that transforming growth factor beta-2 (TGF beta 2) in BF day 12 diminishes the percentage of CD4 positive cells by downregulating CD4 membrane expression on leucocytes. CONCLUSION: These findings suggest that the embryo plays a role in its own protection. Furthermore, it is predicted that any tissue producing TGF beta 2, such as certain types of tumor, downregulates the immune response, thus allowing tumor growth.

Modulation of postthaw motility, survival, calcium uptake, and fertility of bovine sperm by female genital products.

Because the different portions of the female genital tract act in many ways on sperm metabolism, the current study was undertaken to modulate the survival and fertilizing ability of bovine semen by incorporation of products from the oviduct or the follicle in extenders before freezing. Motility rates at 6 h in vitro showed a net positive effect when biological factors from total retentate or from a fraction of bovine follicular fluid (total retentate = 43%; fraction 2 = 54%), oviductal cell culture (total retentate = 43%; fraction 2 = 58%), or granulosa cell culture (total retentate = 43%; fraction 3 = 53%) were added to the extenders compared with the addition of BSA (31%). Fraction 3 of granulosa cell culture retentate also had a significant stimulatory effect on the number of sperm that penetrated mucus of cows in estrous compared with BSA (n = 205 vs. n = 159). The intracellular sperm Ca2+ concentrations were very different across treatments after thawing. Sperm from straws with BSA had the highest concentration. At 4 h, intracellular Ca2+ concentration increased for all treatments, except that for sperm treated with BSA and Ca alone, internal Ca2+ declined. Heparin plus Ca stimulated a greater internalization of Ca2+ than did Ca alone for retentate from bovine follicular fluid, oviductal cell culture, and BSA treatments: glucose consistently and significantly reduced internalization. In vitro fertilization rates were similar, and no significant differences were observed across treatments.

Regulation of leukocyte interleukin 2 and interleukin 2 receptor gene expression by rabbit blastocoelic fluid.

The mechanisms underlying the inhibition of lymphocyte proliferative response by rabbit blastocoelic fluid collected on day 12 of embryonic development were investigated. Treatment with blastocoelic fluid, even in the presence of concanavalin A, maintains lymphocytes in a quiescent state by preventing cell entry into the S phase of the cell cycle. Gene expression of interleukin 2 receptor is completely blocked by treatment with blastocoelic fluid as are the secretion and gene expression of interleukin 2. Addition of interleukin 2 to prestimulated interleukin 2 receptor positive lymphocytes failed to downregulate the expression of high-affinity interleukin 2 receptor and completely abolished the embryonic fluid-mediated inhibitory effect on [3H]thymidine incorporation. Taken together, these results suggest that embryonic fluid has differential inhibitory effects, depending on the activation state of the lymphocytes. Nevertheless, inhibition of interleukin 2 and interleukin 2 receptor expression by embryonic fluid restrains immune cell activity and therefore can be implicated in the survival of the fetal semi-allograft.

In vitro-cultured bovine granulosa and oviductal cells secrete sperm motility-maintaining factor(s).

Since bovine cumulus oophorous and oviductal cell cultures are known to support and maintain frozen-thawed bovine sperm viability and motility for extended time periods, we investigated whether granulosa cell (GC)- and oviductal cell (OC)-conditioned media have similar effects. GC and OC were cultured for 3 days in TCM-199 medium supplemented with 10% fetal calf serum. At that time, the supernatant was discarded from GC and the monolayers were covered with Sp-TALP medium containing 6 mg/ml bovine serum albumin, while the OC were recovered by centrifugation and transferred to culture bottles containing Sp-TALP. Two days later, GC-conditioned and OC-conditioned Sp-TALP were recovered and dialyzed, and their retentates were lyophilized. Bovine follicular fluid (BFF) was also dialyzed, and its retentate was lyophilized. When sperm were incubated in GC- or OC-conditioned media, motility remained above 62% and 42% at 6 hr and 30 hr, respectively, and motility was higher than that of the control both at 6 hr (39%; P < 0.001) and at 30 hr (9%; P < 0.0001). Similarly, when sperm were incubated in the lyophilized retentates of GC- and OC-conditioned media and in BFF at a dose of 0.1, 0.5, or 1.0 mg/ml, the motility rates were higher both at 6 hr (P < 0.05) and at 30 hr (P < 0.01) compared to the control. The increase in motility was dose dependent; a 1.0 mg/ml dose improved (P < 0.05) motility compared to a 0.1 mg mg/ml dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and morphological characterizations of DU-145 cell mortality in rabbit embryo-fetal fluid.

Rabbit embryo-fetal fluid (EFF) contains regulatory factors of cell proliferation which increase the duration of the cell cycle, induce a quiescent status in some cells and lead up to cell death in others. The objective of this study was to demonstrate which of the two processes, namely necrosis or apoptosis, was responsible for the cell death. Inhibitors of protein synthesis, and nuclease and phospholipase A2 activities did not restore the viability of the cells treated with EFF. Using a combination of DNA labelling and extraction, it was possible to show that a large proportion of DNA was fragmented in the cells released in the supernatant while only a very small portion of DNA was fragmented in the monolayer cells. EFF did not induce fragmentation of DNA into nucleosome-sized subunits as analysed using polyacrylamide gel electrophoresis. Nevertheless, using cytofluorometric analysis, it was possible to demonstrate that 50% of the cells released in the supernatant contained a lower quantity of DNA per cell than in the control cells. This was also observed with EFF-treated monolayer cells but not in the control monolayer cells. The reduction of the DNA content per monolayer cell became significant at 48 h of treatment with EFF. Electron microscopic analysis did not reveal blebbing of the cells. However, depletion of glycogen, condensation of mitochondria and increasing number of lysosomes and residual bodies were observed upon treatment with EFF. From these experiments we conclude that the DU-145 cells treated with EFF do not die by apoptosis, but rather seem to die by necrosis.

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.

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.

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.

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.

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.

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)

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).

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.