Search

During the past 2 years our American collective has seen a return to political demonstrations and increased activities towards a deepening consciousness related to raciality. This article asks that we look at racism within the contexts of our collective American psychology and our personhood. This perspective considers Jungian psychology and its influences on the development of this form of psychoanalysis in America. Is this the work of psychology-to increase understanding and compassion among individuals of different ethnicities? The article explores the grievous and grief that is a necessary aspect of racial suffering for individuals of color. The individuation that Jungian psychology oftentimes references can be applied to the individual personhood of those within an Africanist cultural group. Discussion in the article acknowledges this inclusion as well as the idea of furthering a consciousness of Africanist people as valued members of American society.

Opioid use disorder (OUD) among adolescents and young adults (youth) is associated with drug use and sexual HIV-related risk behaviors and opioid overdose. This mixed methods analysis assesses risk behaviors among a sample of 15-21-year-olds (N = 288) who were being treated for OUD in a residential drug treatment program in Baltimore, Maryland. Participants were enrolled in a parent study in which they received either extended-release naltrexone (XR-NTX) or Treatment as Usual (TAU), consisting of outpatient counseling with or without buprenorphine, prior to discharge. At baseline, participants were administered the HIV-Risk Assessment Battery (RAB), and clinical intake records were reviewed to determine participants' history of sexual, physical, or other abuse, as well as parental and partner substance use. A sub-sample of study participants completed semi-structured qualitative interviews (N = 35) at baseline, three-, and six-month follow-up periods. This analysis identified gender (e.g., female IRR = 1.63, CI 1.10-2.42, p = .014), the experience of dependence (e.g., previous detoxification IRR = 1.08, CI 1.01-1.15, p = .033) and withdrawal (e.g., severe withdrawal symptoms IRR = 1.41, CI 1.08-1.84, p = .012), and the role of relationships (e.g., using with partner IRR = 2.45, CI 1.15-5.22, p = .021) as influencing high-risk substance use behaviors. Similarly, high-risk sex was influenced by gender (e.g., female IRR = 1.43, CI 1.28-1.59, p < .001), and the role of relationships (e.g., using with partner IRR = 0.78, CI 0.62-0.98, p = .036). These are key targets for future prevention, treatment, and intervention., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: A wide range of dilemmas encountered in the health domain can be addressed more efficiently by a transdisciplinary approach. The complex context of extreme prematurity, which is raising important challenges for caregivers and parents, warrants such an approach., Methods: In the present work, experts from various disciplinary fields, namely biomedical, epidemiology, psychology, ethics, and law, were enrolled to participate in a reflection. Gathering a group of experts could be very demanding, both in terms of time and resources, so we created a web-based discussion forum to facilitate the exchanges. The participants were mandated to solve two questions: "Which parameters should be considered before delivering survival care to a premature baby born at the threshold of viability?" and "Would it be acceptable to give different information to parents according to the sex of the baby considering that outcome differences exist between sexes?", Results: The discussion forum was performed over a period of nine months and went through three phases: unidisciplinary, interdisciplinary and transdisciplinary, which required extensive discussions and the preparation of several written reports. Those steps were successfully achieved and the participants finally developed a consensual point of view regarding the initial questions. This discussion board also led to a concrete knowledge product, the publication of the popularized results as an electronic book., Conclusions: We propose, with our transdisciplinary analysis, a relevant and innovative complement to existing guidelines regarding the decision-making process for premature infants born at the threshold of viability, with an emphasis on the respective responsabilities of the caregivers and the parents.

Research with animals and humans has suggested that acute and subacute use of 3,4-methylenedioxymethamphetamine (MDMA "ecstasy") may lead to memory impairment. However, research is limited by (1) low power due to small sample sizes, (2) the possible confound of polydrug use, and (3) the failure to consider intelligence as a covariate. The present study compared the memory performance on the Wechsler Memory Scale-III of 26 abstinent (2-wk. minimum) recreational MDMA users with 26 abstinent (2-wk. minimum) recreational polydrug users. Despite significantly greater polydrug use amongst these MDMA users, no significant group differences in memory were observed. Regression of total lifetime amount of MDMA use also did not predict memory performance after accounting for intelligence. In addition, the length of time since abstinence (at least 2 wk.) was not associated with an increase in memory performance. Greater total lifetime cocaine use, rather than total lifetime MDMA use, was significantly associated with greater decrements in General Memory and Delayed Verbal Memory performance.

Objective: The problem of iatrogenic multiple pregnancies has been addressed through an online multidisciplinary discussion forum with the primary aim of developing a transdisciplinary research and training tool., Study Design: The pedagogical approach was divided in three phases, unidisciplinary, multidisciplinary and multi/transdisciplinary. Evaluation of the level of transdisciplinarity reached by each of the students was based on their capacity to translate disciplinary knowledge, to establish a frank dialogue, to cross disciplinary barriers, to detach from their initial opinion, and to develop curiosity for others' perspectives and concerns., Results: Translation of disciplinary knowledge and frank dialogue were easily performed by all. Most students showed curiosity towards the others' point of view and started to be preoccupied with the concerns of other students., Conclusion: Online discussion forum is a particularly well-adapted tool for transdisciplinary research and training.

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.

On the basis of results obtained in vitro, we previously proposed a model in which signals from the conceptus, namely interferon-tau (IFN-tau) and prostaglandin E2, increase the expression of cyclooxygenase (COX)-2 or granulocyte-macrophage colony-stimulating factor (GM-CSF) in immune and nonimmune cells of the bovine endometrium. Two experiments were conducted to verify the validity of this hypothesis in vivo. In experiment 1, the in vivo expression of COX-2 and GM-CSF during early pregnancy was monitored. Uteri from heifers were collected at different days (d) of the estrous cycle and pregnancy (P). In experiment 2, the effects of intrauterine infusions of IFN-tau on the expression of COX-2 and GM-CSF were analyzed. Immunohistochemistry was performed on uterine sections, and image analysis was used to evaluate the staining intensity in the conceptus, the luminal epithelium (LE), and the subepithelial stroma. In experiment 1, staining for COX-2 was maximal between d18P and d24P, both in the LE and in the conceptus, whereas staining for GM-CSF reached a plateau between d18P and d30P in the LE. In experiment 2, in response to IFN-tau, COX-2 was up-regulated in the LE of the ipsilateral horn, whereas GM-CSF was enhanced in both uterine horns. The current report supports the view that the conceptus, through its secretion of IFN-tau, stimulates maternal epithelial expression of COX-2 and GM-CSF during the peri-attachment period in the cow.

The frequency of health problems in singleton assisted reproductive technologies (ART) babies is higher than in singletons from spontaneous gestations. Any of the following factors may be involved: in-vitro technology, ovarian stimulatory drugs and infertility itself. A literature review on premature birth, low birth weight, perinatal mortality and major birth defects in children conceived from infertility treatments was conducted. Only publications comparing the outcome of pregnancy in an infertile group of patients to a matched control group were selected. The analysis of the outcome of singleton pregnancies resulting from IVF versus artificial insemination, obtained with or without the use of ovarian stimulatory agents and obtained with or without the use of a semen donor, suggests that female infertility is an important risk factor. Criteria for screening at-risk infertile women have not yet been identified. Prospective studies designed to identify precisely the aetiology of health problems in singletons ART babies will have to be conducted. The absence of criteria correlating at-risk infertile women to health problems in their children does not allow a gynaecologist the opportunity to offer infertility treatments to the least susceptible patients.

Neurological sequelae and multiple birth defects have been observed in children conceived by IVF and ICSI. Multiple pregnancy is the most important risk factor. These health problems challenge the responsible practice of medicine. The core values of medicine and the deontology of the profession have been reviewed to define the responsible conduct of research and clinical practice. Professional associations have proposed guidelines to reduce health problems in assisted reproductive technology. Although these health problems could have been prevented, this response by the medical community is nonetheless an important step towards improving responsible medical practices that have become questionable over the years. Professional associations must find out means, not only to implement their guidelines, but also to prevent the recurrence of such episodes in the history of medicine.

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.

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 (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.

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.

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.

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.

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.

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.

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)

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.

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.

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.

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.

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.

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.

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)

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.

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).

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.

A commonly used and specific plasma LH radioimmunoassay was modified to provide reliable results for cow LH concentrations in blood within 4-5 h. Blood samples were collected at 2- or 4-h intervals from at least 24 h before the expected oestrus until after its onset. Thereafter, samples were collected at intervals of 2 or 4 days until the next oestrus. For the assay, the antigen-antibody reaction took place during a 2-h incubation at 37 degrees C. The immune complex was precipitated by addition of rabbit anti-gamma-globulin and polyethylene glycol followed by an incubation at 22 degrees C for 30 min. The coefficient of variation between this assay and the standard assay was less than 15%. From a study of 25 cycles from 12 animals, we obtained intervals of congruent to 27.3 h between the beginning of the LH peak and ovulation and congruent to 17.5 h between the end of the peak and ovulation. This modified technique can be used to predict rapidly and precisely the time of ovulation in cattle.

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.

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.

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.

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.

Different concentrations of rabbit or bovine follicular fluid were added to a defined medium or to rabbit oestrous oviduct fluid containing 13 hours in utero incubated sperm. With increasing concentration of follicular fluid, the percentage of fertilization was progressively and significantly lower than in oestrous tubal secretions or Brackett's medium, alone or containing up to 5% of follicular fluid. Utilization of ova, with or without cumulus clot, to the fertilization media, had no significant effect on fertilization of rabbit eggs. Both kinds of follicular fluid used did inhibit the motility of capacitated sperm.

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.

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.

Rabbit embryos at the stage of one-cell and morula were incubated in uterine secretions from rat. The uterine fluid was aspirated from uterus of freshly sacrificed estrus animals. We obtained a very good development of the morulas (mean of the maximum diameter : 0.79 +/- 0.02 mm); however only 35% of the one cell rabbit embryos reached the stage of 16 cells. This consequently suggest a uterine tropism in the rats similar to that previously found for the rabbit.

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.