Thomas G. Kennedy

Dissociation of ovulatory and steroidogenic actions of luteinizing hormone in rabbits with indomethacin, an inhibitor of prostaglandin biosynthesis

Abstract The possible involvement of prostaglandins as mediators of ovarian responses to luteinizing hormone (LH) has been investigated in in vivo studies with the aid of indomethacin (1-[p-chlorobenzoyl]-5-methoxy-2-methylindole-3-acetic acid), an inhibitor of prostaglandin biosynthesis. LH (50 μg NIH-LH-B7) was injected i.v. 1 2 hour after i.v. injection of either indomethacin (20 – 40 mg/Kg) or phosphate buffer vehicle. Mean levels of progesterone and 20α-hydroxypregn-4-en-3-one (20α-OH-P) observed in peripheral plasma obtained by cardiac puncture 1 hour after LH were increased by 5.35 ± 1.48 and 26.0 ± 11.6 ng/ml respectively in phosphate buffer-treated controls, and by 5.60 ± 4.47 and 100.6 ± 37.1 ng/ml respectively in indomethacin-treated rabbits. The apparently greater increase in 20α-OH-P in indomethacin-treated rabbits could be attributed almost entirely to the larger amounts of interstitial tissue in the latter animals, as indicated by essential disappearance of this difference when covariance analyses were performed to correct for this difference in ovarian interstitial tissue weight. None of 12 indomethacin-treated, and 10 of 11 vehicle-treated control rabbits ovulated in response to this dosage of LH, as determined by flushing of oviducts to recover ova, and by gross examination of ovaries 1 or 3 days after LH treatment. Indomethacin did not prevent luteinization of follicles in LH-treated rabbits, as determined by gross and histologic examination of ovaries 8 days after treatment. These findings argue against a role of prostaglandins as mediators of the acute steroidogenic and luteinizing actions of LH in the rabbit ovary, but suggest an involvement in the process of ovulation.

PROSTAGLANDINS AND THE INITIATION OF BLASTOCYST IMPLANTATION AND DECIDUALIZATION

The process of blastocyst implantation in mammals is remarkably variable, especially in the extent of trophoblast invasion of the endometrium. In most species studied, the earliest macroscopically identifiable sign of blastocyst implantation is an increase in endometrial vascular permeability in areas adjacent to the blastocysts. This is followed in species with invasive implantation by decidualization, again localized to areas adjacent to the blastocysts. In some species, the application of a stimulus to the endometrium can result in increased endometrial vascular permeability and decidualization. Based initially on studies utilizing inhibitors of prostaglandin (PG) synthesis and more recently on studies using the techniques of transgenics, considerable evidence has accumulated indicating that PGs have an important role in the early events of implantation and artificially induced decidualization. However, which PGs are involved remains controversial. There may be differences between species, and different PGs may be involved at different times.

Suppression of lymphocyte alloreactivity by early gestational human decidua: I. Characterization of suppressor cells and suppressor molecules

We examined the immunosuppressor role of the first trimester human decidua on lymphocyte alloreactivity in vitro in order to identify (1) the major cell classes in the decidua mediating the suppressor effect; (2) the stages in the lymphocyte alloreactive responses susceptible to the suppressor effects of the decidua; and (3) the precise nature of the suppressor molecules. Irradiated (2800 R), Ficoll-Paque-separated nucleated cells of the collagenase-dispersed early gestational (6.5-9.5 weeks menstrual age) decidua containing 70-94% typical decidual cells (identified on the basis of distinctive morphology and numerous cytoplasmic or surface markers) or their plastic-nonadherent fractions further enriched for decidual cells (approximately 96% pure) caused a strong dose-dependent suppression of the one way mixed lymphocyte reaction (MLR, i.e., proliferative response measured on Days 3, 4, or 5), when added at the onset of the mixed lymphocyte cultures (MLC). As few as 10(3) decidual cells caused a detectable inhibition of the MLR exhibited by 10(5)-1.5 X 10(5) responder lymphocytes. A smaller degree of suppression was noted with the plastic-adherent fractions of the early decidua (which retained all macrophages and granulocytes, but still included many decidual cells) or unfractionated cells of later gestational (10-13 weeks) decidua containing a higher incidence of leukocytes, granulocytes, and macrophages in particular, or the plastic-adherent fraction thereof, enriched for macrophages. Thus, decidual cells seem to represent an important suppressor cell class in the early gestational human decidua; however, suppression by decidual leukocytes, macrophages in particular, was also evident. The suppressor effect was unrelated to the major histocompatibility phenotype of the responder or the stimulator cells. It was not caused by cell crowding, since an equivalent number of irradiated K562 erythroleukemia cells had little effect on the MLR. The effect was exerted during both the initiation and the progression of the MLR. A delay in the addition of regulator cells progressively minimized the effect on the Day 4 MLR, but did not abolish it completely even when added as late as on Day 3. The major class of mediator molecules was identified as prostaglandins, primarily PGE2, on the basis of the following results: (1) the presence of indomethacin (10(-5) M) or varying dilutions of an anti-PGE2 antibody abrogated this suppression substantially or completely. (2) Addition of pure PGE2 (3 X 10(-7) to 1.1 X 10(-5) M), but not PGF2 alpha, reproduced a dose-dependent suppressor effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Mimicking the Events of Menstruation in the Murine Uterus

Abstract Menstruation and endometrial regeneration occur during every normal reproductive cycle in women and some Old World primates. Many of the cellular and molecular events of menstruation have been identified by correlative or in vitro studies, but the lack of a convenient model for menstruation in a laboratory animal has restricted functional studies. In this study, a mouse model for menstruation first described by Finn in the 1980s has been modified for use in a commonly used inbred strain of mouse. A decidual stimulus was applied into the uterine lumen of appropriately primed mice and leukocyte numbers and apoptosis were examined over time following progesterone withdrawal. Endometrial tissue breakdown was initiated after 12–16 h, and by 24 h, the entire decidual zone had been shed. Re-epithelialization was nearly complete by 36 h and the endometrium was fully restored by 48 h. Leukocyte numbers increased significantly in the basal zone by 12 h after progesterone withdrawal, preceding stromal destruction. Stromal apoptosis was detected by TUNEL staining at 0 and 12 h but decreased by 16 h after progesterone withdrawal. This mouse model thus mimics many of the events of human menstruation and has the potential to assist in elucidation of the functional roles of a variety of factors thought to be important in both menstruation and endometrial repair.

Suppression of lymphocyte alloreactivity by early gestational human decidua: II. Characterization of the suppressor mechanisms☆

We have earlier shown that first trimester human decidual cells (typical decidual cells and decidual macrophages) suppress lymphocyte alloreactivity (MLR and CTL generation) in vitro in an MHC-unrestricted manner and that this suppression is mediated by PGE2. The present study explored the mechanisms underlying this suppression by noting the effects of decidual cells (+/- indomethacin or anti-PGE2 antibody) or chemically pure PGE2 on numerous T lymphocyte activation events following allogeneic stimulation in mixed lymphocyte culture (MLC) or polyclonal activation with Con A. The results revealed that this suppression was the net result of an action of PGE2 on at least two events during lymphocyte activation: (i) a down-regulation of IL-2 receptor development on lymphocytes, quantitated with a radioimmunoassay and radioautography; this was noted in MLC or Con A-stimulated lymphocyte cultures in the presence of decidual cells (reversible in the presence of indomethacin or anti-PGE2 antibody), or PGE2, but not PGF2 alpha; (ii) an inhibition of IL-2 production in the MLC, measured with a bioassay using an IL-2-dependent T cell line (CTLL-2) and a recombinant IL-2 standard. These effects blocked cell proliferation and eventual generation of killer cells in the MLC. Decidual cells or PGE2 did not interfere with IL-2-dependent proliferation of CTLL-2 cells, which require an interaction between IL-2 receptors on these cells and IL-2. Finally, neither agent interfered with the lytic function of CTL, once generated. These results indicate that the PGE2-mediated immunosuppressor function of early gestational human decidual cells is accomplished by an afferent blockade of the early events in T lymphocyte activation.

The concentration of 6-keto-prostaglandin F1α is markedly elevated at the site of blastocyst implantation in the rat

The initiation of blastocyst implantation in the rat is indicated by localized increases in endometrial vascular permeability at the sites where blastocysts are present. The concentrations of 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), a stable metabolite of prostaglandin I2 (PGI2), were measured by gas chromatography-mass spectrometry in the areas of increased endometrial vascular permeability (uterine dye sites), and compared with those in the remainder of the uterus (uterine non-dye sites). For rats killed either on the evening of Day 5 of pregnancy or on the morning of Day 6, measurable amounts of 6-keto-PGF1alpha were found in the dye sites of all animals, whereas 1 of 6 and 4 of 6 rats killed on Days 5 and 6, respectively, had undetectable amounts (less than 1 ng) in non-dye site tissue. It was estimated that, on average, the concentration of 6-keto-PGF1alpha in dye sites on the evening of Day 5 is at least 40-fold that in non-dye sites. The possible role of PGI2 in the initiation of blastocyst implantation is discussed.

Uterine Sensitization-Associated Gene-1: A Novel Gene Induced Within the Rat Endometrium at the Time of Uterine Receptivity/Sensitization for the Decidual Cell Reaction

Abstract For successful implantation, the embryo must develop to the blastocyst stage and the endometrium must attain a state that is receptive to the implanting blastocyst. In rodents, the timing, duration, and hormonal regulation of this receptive state has been well defined. However, the molecular cascade of events involved in the onset of the receptive phase remains unclear. In the present study, we sought to identify genes involved in the onset of the receptivity using the technique of suppressive subtraction hybridization. Herein we report the isolation, cloning, and characterization of a novel gene, uterine sensitization-associated gene-1 (UASG-1), that is preferentially expressed within the maximally sensitized/receptive rat endometrium. USAG-1 mRNA encodes a putative protein of 206 amino acids that contains a possible N-terminal secretion signal and a C-terminal cystine knotlike motif. Northern blot analysis revealed that induction of USAG-1 mRNA was restricted to the Day 5 pregnant or pseudopregnant uterus. In situ hybridization experiments demonstrated that this induction was restricted to the uterine glandular epithelial cells. Given the remarkably tight restriction of its expression, USAG-1 may be involved in the onset of endometrial receptivity for implantation/sensitization for the decidual cell reaction.

Prostaglandin-mediated inactivation of natural killer cells in the murine decidua☆

Previous studies from this laboratory have demonstrated a large influx of null lymphocytes into the murine decidua during pregnancy. We had also shown that trophoblast cells of the murine placenta bear target structures recognized by NK cells. Since NK lineage cells belong to the null category of lymphocytes, we examined whether cells of this lineage appear in the murine decidua, and if so, whether their activity is locally regulated by NK suppressor cells. We further investigated the identity of the suppressor cells as well as their suppressor products. NK lineage cells, irrespective of their activation status, were identified morphologically in radioautographic preparations as the non-T, non-B (null) lymphocytes capable of binding YAC-1 lymphoma targets. NK activity of nucleated cells was measured with a 4-hr 51Cr-release assay against labeled YAC-1 targets. Studies with outbred CD1 mice, and to a smaller extent, inbred CBA mice revealed that the incidence of NK lineage cells remained fairly constant within the decidua throughout pregnancy, but their activity decreased steadily to negligible levels by Day 12-14 of gestation. This was found to result from an inactivation caused by NK-suppressor cells in the decidua. A mixing of Ficoll-Paque-separated nucleated cells of the decidua with normal splenic effector cells (at 1:1 ratio) led to a suppression of their NK activity tested immediately or after a 20-hr coculture. This suppression was MHC unrestricted. Suppressor cells were identified both in plastic nonadherent fraction highly enriched for typical decidual cells as well as in the plastic adherent fraction containing decidual cells and macrophages. Addition of indomethacin (10(-5) M), an inhibitor of prostaglandin synthesis, or anti PGE2 antibody, revived the NK activity in the mixed population, as well as in the decidua, suggesting a PGE2-mediated suppression. High levels of PGE2 were detectable in decidual cell supernatants with a sensitive radioimmunoassay. Addition of pure PGE2 (10(-7)-10(-6) M) but not PGF2 alpha (10(-6) M) during the NK assay or to the effector cells for a 20-hr period prior to the assay led to an inhibition of NK activity. These results reveal that NK cells appearing in the murine decidua are progressively inactivated by PGE2 produced by decidual cells and decidual macrophages.

Effect of prostaglandin E2 on rate of decidualization in rats.

Based on morphology, it has been suggested that prostaglandin E2 (PGE2) accelerates the process of endometrial stromal cell differentiation to decidual cells in the rat. The present study investigated this possibility, using changes in uterine weight and in endometrial alkaline phosphatase (ALP) activity as indicators of decidualization. Rats were ovariectomized and treated with one of two steroid protocols; the first was identical to that previously used for the study of morphology, the second a modified protocol which results in greater uterine sensitization for decidualization, producing larger amounts of decidual tissue, thereby making it easier to detect differences between treatments. On the day of uterine sensitization, rats within each treatment protocol were given a unilateral intrauterine infusion of phosphate-buffered saline (PBS) or PGE2 plus indomethacin, and killed 24, 48 or 72 h later. The time-courses for the increases in uterine weight and ALP activity in uterine horns infused with PBS or PGE2 plus indomethacin differed between steroid protocols, but within a protocol were statistically indistinguishable. The results do not support the hypothesis that PGE2 accelerates the process of decidualization but do provide additional support for the notion that PGE2 is a physiological rather than pharmacological mediator of decidualization.

Induction of Glucose-Regulated Protein 78 in Rat Uterine Glandular Epithelium During Uterine Sensitization for the Decidual Cell Reaction

Abstract Endometrial receptivity for implantation and sensitization for decidualization in rodents is a transient state under the control of the ovarian steroids estrogen and progesterone. It is unclear, however, what molecular events mediate the onset of uterine receptivity. Messenger RNA differential display was performed on endometrial RNA from ovariectomized rats differentially sensitized for decidualization. Maximally sensitized uteri were at the equivalent of Day 5 of pseudopregnancy, and temporally nonsensitized uteri at Day 4 or 6; hormonally nonsensitized uteri were from animals on Day 5 treated with low or high doses of estradiol on Day 4. A cDNA with endometrial expression restricted to maximally sensitized uteri was isolated, cloned, and sequenced. The cDNA matched the sequence for glucose-regulated protein 78 (GRP78), a heat shock 70-related protein that resides in the lumen of the endoplasmic reticulum (ER) and has roles in several cellular processes including multimeric protein assembly, the degradation of proteins, and the storage and regulation of ER luminal calcium. Northern blot analysis indicated a dramatic increase in GRP78 mRNA levels restricted to the sensitized, Day 5 endometrium, suggesting a role in the onset of the sensitized phase. In situ hybridization and immunohistochemistry experiments localized the up-regulation of GRP78 within the receptive endometrium to the glandular epithelium.