Cyclic AMP and the preparation of the mouse uterus for implantation.

Experiments are described that demonstrate the ability of dibutyryl cyclic AMP (dcAMP) to induced oestrogen-like changes in the uteri of ovariectomized mice treated with progesterone. A single intraluminal injection of 6-14 micrograms dcAMP in 0-02 ml phosphate-buffered saline promoted cell division in the uterine stroma and sensitized the endometrium so that a decidual reaction could be induced by a subsequent injection of arachis oil into the uterine lumen. Since dcAMP has already been shown to induce implantation of diapausing mouse blastocysts in the absence of oestrogen, it is suggested that cyclic AMP is involved in egg implantation in rodents.     

Different lipolytic effects of theophylline and dibutyryl cyclic AMP in vivo and in vitro.

Both dcAMP and theophylline are known to promote lipolysis in vitro by increasing intracellular cAMP. Although theophylline stimulates FFA mobilization in vivo as well, a report of low circulating FFA levels in the rat given dcAMP suggested that dcAMP may inhibit lipolysis in the intact animal. To explore this possibility, a comparison of the in vitro and in vivo lipolytic effects of theophylline and dcAMP was made in the young dog. Circulating glycerol and FFA levels rose following the administration of theophylline. While glycerol and FFA fell slightly in puppies given dcAMP, only the FFA change was significant. Epinephrine infusions given alone produced sustained elevations of glycerol and FFA. When theophylline was given in conjunction with ongoing epinephrine infusions, plasma glycerol and FFA levels remained high. On the other hand, epinephrine-stimulated lipolysis was markedly inhibited by dcAMP, as shown by pronounced falls of glycerol and FFA from the elevated levels found with epinephrine alone. In vitro studies involving fragments of puppy adipose tissue reveal that epinephrine, theophylline, and dcAMP promoted glycerol release. In contrast to the in vivo observations, lipolysis was also stimulated by combinations of both epinephrine and theophylline as well as by epinephrine and dcAMP. Thus, theophylline stimulates lipolysis in vitro and in vivo in the puppy. In contrast, dcAMP stimulates lipolysis in vitro but inhibits this action in the intact animal. This important difference in the two pharmacologic agents suggests the need for caution when using them in in vivo studies involving the action of cAMP.     

Responsiveness of rat ovaries to dcAMP in the perinatal period: evidence for an inhibitory influence in vivo.

The progesterone production by rat ovaries from 18-day-old fetuses to 6-day-old neonates was measured in vitro in the presence of dibutyryl cAMP (dcAMP, 1 mM). A pronounced decline was observed at the end of fetal life. The 5-reductase activity did not seem sufficient to explain this decrease. Preculture of the ovaries for 48 h in the basal medium enhanced responsiveness to the nucleotide. Addition of spironolactone, an inhibitor of 17-hydroxylase to dcAMP did not modify this evolution.

3β-hydroxysteroid dehydrogenase activity, detectable in fetal ovaries in the absence of dcAMP was also increased after preculture. In the presence of spironolactone and trilostane, the pregnenolone production showed the same evolution as progesterone and was also enhanced after culture. These results suggest the existence of inhibitory factor(s) present in vivo at the end of fetal life.     

Activity of 3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal rat ovary

3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) was examined in rat fetal ovaries. The enzymatic activity was determined by measuring the conversion of radiolabeled pregnenolone to progesterone. 3 beta-HSD, present in 14-day old fetal ovaries showed a regular increase in the course of development. Pretreatment with dcAMP for 48 h enhanced the apparent maximal velocity of the enzyme by about 5-fold without increase in the apparent Km. The increase in 3 beta-HSD activity was not due to the synthesis of pregnenolone observed after dcAMP pretreatment, but it was dependent on protein synthesis. The present results indicate that (1) 3 beta-HSD activity is present in fetal female gonads and the absence of steroid biosynthesis cannot be related to a defect in this enzyme (2) 3 beta-HSD activity is enhanced in the presence of dcAMP. The absence of gonadotropic receptors in the rat ovary before birth could explain the low level of the enzymatic activity measured in fetal ovaries.     

Regulation by FSH and dibutyryl cyclic AMP of the formation of androgen-binding protein in Sertoli cell-enriched cultures.

Sertoli cell-enriched preparations from testes of 20-day-old rats were cultured in a defined medium in the presence and absence of FSH or dibutyryl cyclic AMP (dcAMP). Androgen-binding activity was assayed in the culture medium, and related to testicular androgen-binding protein (ABP). The production and secretion of ABP by the Sertoli cell-enriched preparation was increased after FSH or dcAMP treatment of the primary culture. It is concluded that ABP is produced by Sertoli cells. The possibility of involvement of other cell types in the testis in ABP production is discussed.     

Immunofluorescent localization, by use of anti-idiotypic antibody, of monoclonal anti-progesterone antibody in the mouse uterus before implantation

Mouse monoclonal anti-progesterone IgG1 antibody designated DB3 has an anti-fertility effect when injected into female mice shortly after mating. In BALB/c mice, pregnancy is blocked, probably as a result of progesterone withdrawal with inhibition of implantation. Rabbit polyclonal anti-idiotype raised against DB3 has been used in an indirect immunofluorescence method on frozen tissue sections to demonstrate the presence of DB3 on the surface of uterine luminal and glandular epithelia before implantation. DB3 was only detectable 30-60 h after a single parenteral injection (9 nmol antibody per mouse i.p. or i.v. at 32 h post coitum). Immunolocalization was both pregnancy-dependent and anti-progesterone antibody-specific, as it was not seen in pseudopregnant mice or mice treated with P3 (mouse myeloma IgG1 protein, using polyclonal anti-P3 anti-idiotype as a probe) or saline. The immunofluorescent reaction was completely blocked by addition of DB3 idiotype in vitro. The results indicate that anti-progesterone antibody binds to an antigen associated with luminal and glandular epithelia which may locally inhibit the uterine uptake of progesterone and disrupt the process of implantation.     

Effects of different schedules of progesterone treatment on mammary tumorigenesis and uterine adenomyosis in SHN virgin mice

As a possible step to estimate the factors controlling the effects of progesterone on mammary tumorigenesis, 3 groups of SHN virgin female mice were treated as follows beginning 2.5–4 months of age: Group A received the subcutaneous implantation of silastic tube containing progesterone (low dose) during the initial 4 months followed by progesterone pellet implantation (high dose) every 2 months. Group B was implanted with progesterone pellet throughout the experiment. Group C was given the vehicle only. Whereas there was little difference among groups in mammary tumorigenesis during the initial 4 months of treatments, tumorigenesis was significantly stimulated in group B thereafter. On the contrary, group A was different little from group C even after progesterone pellet implantation. The results indicate that the effects of progesterone on mammary tumorigenesis are affected by the ‘preceding’ progesterone conditions and that there is a critical period for manifestation of the effects of progesterone on mammary tumorigenesis, which is before 8 months of age at most. While all mice developing mammary tumors developed uterine adenomyosis in each group, the progression was enhanced by both low and high doses of progesterone.     

Relevance of c-fos proto-oncogene induction for the steroidogenic response to ACTH,dcAMP and phorbol ester in adrenocortical cells

We previously reported that ACTH, but not dibutyryl cAMP, rapidly induces the c-fos proto-oncogene in Y-1 adrenocortical cells.Here we show that PMA induces c-fos with similar kinetics when compared with ACTH (0.5–1 h peak) but reaches only 60% of the maximal ACTH induction and dcAMP is a weak c-fos inducer (15% of ACTH). However, combination of PMA and dcAMP has a synergistic effect leading to maximal c-fos induction. c-fos expression may play a role in the RNA synthesis-dependent corticosteroidogenesis response and/or growth regulation by ACTH.We also show that, in contrast to dcAMP, PMA is a poor steroidogenesis stimulator (15 to 17% of maximum ACTH-stimulated level), its activity being completely dependent on RNA synthesis. Combination of dcAMP and PMA yields an additive steroidogenesis stimulation, an effect that is also dependent on RNA synthesis. Although no strict correlation was found between c-fos induction and early steroidogenesis stimulation, particularly with respect to cAMP derivatives, the results suggest that a PKC pathway is likely to cooperate with the classical cAMP-PKA pathway in adrenal cells' RNA-dependent steroidogenesis.Abbreviations ACTH Adrenocorticotropic Hormone - PMA Phorbol-12-Myrystate-13-Acetate - dcAMP dibutyryl cyclic AMP - DME Dulbecco's Modified Eagle's minimal medium - FCS Fetal Calf Serum     

Differential hormonal regulation of leukemia inhibitory factor (LIF) in rabbit and mouse uterus

Leukemia inhibitory factor (LIF) has been shown to be essential for the implantation of mouse blastocysts. The present study was designed to determine how LIF protein was hormonally regulated in rabbit and mouse uterus using immunohistochemistry. In unmated rabbits, LIF protein was at a low level in the uterine epithelium and glands, and up-regulated by progesterone alone or estradiol-17β and progesterone combined. Estradiol-17β alone had no apparent effect. In ovariectomized mice, the level of LIF protein was very low in the uterine epithelium and glands, and was up-regulated by estradiol-17β alone or estradiol-17β and progesterone combined. Progesterone alone had no apparent effect. These results suggest that LIF protein is differentially regulated in rabbit and mouse uterus by progesterone and estrogen, respectively. This would explain the high level of LIF protein observed in uterine epithelium and glands prior to blastocyst implantation in the two species with different hormonal requirements for implantation. © 1996 Wiley-Liss, Inc.     

Modulation of stimulatory action of follicle stimulating hormone (FSH) and inhibitory action of epidermal growth factor (EGF) on aromatase activity in Sertoli cells by calcium

Aromatization of testosterone by cultured Sertoli cells isolated from immature rats was stimulated more than 7-fold by follicle stimulating hormone (FSH) or dcAMP. The effects of FSH and dcAMP could be partly inhibited by epidermal growth factor (EGF) in a dose-dependent manner (ID500.5 nM). The phorbol ester 4 beta-phorbol-12-myristate-13-acetate (PMA) could also inhibit aromatase activity in a fashion similar to EGF. When 3 mM EGTA was present in the culture medium, the inhibitory effect of EGF was abolished but the stimulatory effect of FSH or dcAMP was magnified. These results suggest that EGF exerts a negative control on aromatase via calcium and protein kinase C. The abolishment of the inhibitory effect of EGF and the enhancement of the stimulatory effect of FSH or dcAMP by a calcium deficiency may be an indication that growth factors produced by Sertoli cells negatively controls FSH-induced responses in an autocrine fashion.     

Role for cyclic adenosine monophosphate in modulating insulin-like growth factor binding protein secretion by muscle cells

The modulation of insulin-like growth factor-binding protein (IGFBP) secretion is an important variable affecting muscle cell metabolism, proliferation, and differentiation. We have previously shown that secretion of IGFBP-4 and IGFBP-5 by L6 and BC₃H-1 muscle cells was stimulated by treatment with either insulin, IGF-I, or IGF-II. Herein, these cells were used to further identify mechanisms involved in controlling IGFBP secretion. Agents that elevate intracellular cAMP concentrations (dcAMP, forskolin, isoproterenol, and prostaglandin [PGE₁]) increase secretion of IGFBP-4 and IGFBP-5 from L6 cells. Similar increases in IGFBP secretion were found by treatment with either insulin, IGF-I, or dcAMP. The effects of dcAMP and either insulin or IGF-I were additive, but the effects of insulin and IGF-I were not additive. These results suggest that insulin/IGF-I and dcAMP are acting via distinct mechanisms to stimulate IGFBP secretion. Indomethacin, which blocks endogenous prostaglandin synthesis, and progesterone, which decreases intracellular cAMP levels, decreased IGFBP-4 and IGFBP-5 secretion. IGFBP-5 secretion by BC₃H-1 cells was increased by either insulin or IGF-I. Agents which elevate intracellular cAMP concentrations did not increase IGFBP-5 secretion. Additionally, these agents were not synergistic with either insulin or IGF-I. However, indomethacin and progesterone depressed IGFBP-5 secretion by BC₃H-1 cells. In summary, there appear to be at least two intracellular signaling mechanisms controlling IGFBP-4 and IGFBP-5 secretion by L6 and BC₃H-1 muscle cells. IGFBP secretion by L6 cells is stimulated by both insulin/IGF-I and cAMP-dependent pathways, whereas IGFBP-5 secretion by BC₃H-1 cells is stimulated only by the insulin/IGF pathway. IGFBP secretion by both cell lines can be decreased by agents which depress cAMP levels. Our results suggest that two divergent but synergistic pathways modulate IGFBP production and these mechanisms can potentially modulate IGF activity during muscle cell proliferation and differentiation. J. Cell. Physiol. 174:293–300, 1998. © 1998 Wiley-Liss, Inc.     

Failure of platelet-activating factor (PAF-acether) to induce decidualization in mice and failure of antagonists of PAF to inhibit implantation

The possible role of platelet-activating factor (PAF) in the uterine responses associated with implantation was investigated. Attempts to trigger a decidual cell response in the uteri of hormonally sensitized, ovariectomized mice by instilling PAF-acether (1-1000 ng) intraluminally were unsuccessful. The effect of PAF antagonists on implantation was investigated in females ovariectomized on Day 3 of pregnancy and treated with progesterone. Implantation was induced in these females by injection of 10 ng oestradiol-17 beta on Day 8. Hourly intraperitoneal injections of three PAF antagonists (WEB 2086, CV 3988 and BN 52021 at doses of 1.2-1.4 mg/kg) given over a 24-h period starting 1 h before the injection of oestradiol-17 beta had no significant effect on the occurrence of implantation sites. Intraluminal injection of WEB 2086 (15 micrograms) or BN 52021 (5 micrograms) either 3 h before or 6 h after the nidatory oestradiol also had no significant inhibitory effect on implantation. SRI 63-441 given once daily over the first 4 days of pregnancy at a dose of 40 micrograms/30 g body weight had no inhibitory effect on the establishment of pregnancy. These results are not consistent with a critical role for PAF in implantation in mice.     

Estradiol-17 β and cAMP: in vitro studies on the cervicovaginal epithelium of neonatal mice

Summary In organ culture of the cervicovaginal epithelium from neonatal mice, the epithelium synthesizes a material with specific antigenic properties (CVA). CVA was studied with immunofluorescence and the amount estimated semiquantitatively. In line with earlier studies, adenosine 3,5-cyclic monophosphate (dibutyryl derivative) (dcAMP), increased the amount of CVA, but adenosine 2,3-cyclic monophosphate did not. Addition of histamine to the culture medium moderately increased the amount of CVA, whereas the antihistamine diphenhydramine (H₁-antagonist) slightly reduced the strong increase induced by dcAMP and estradiol in combination. No effect was seen under similar conditions using the H₂-antagonist metiamide. Taken together with earlier results it is considered possible that the action of histamine and diphenhydramine is related to effects on the cell membranes. Phentolamine had no effect. The dcAMP effect was inhibited by actinomycin D and cycloheximide.This investigation was supported by grants from the Norwegian Research Council for Science and the Humanities     

The effect of progesterone, oestradiol and HCG on cell-mediated immunity in pregnant mice.

During pregnancy in mice, cell-mediated immunity as measured by a contact allergic reaction to picryl chloride was diminished (P less than 0.001). Mice in which delay of implantation was maintained by progesterone, and mice with progesterone- and oestradiol-maintained pregnancies, also showed a reduction in the inflammatory response. The response of pseudopregnant mice did not differ from that of the non-pregnant controls. Young mice sensitized before complete immunological competence gave a 50% response. The response doubled in animals given a second sensitization. The extent of the response in females with delay of implantation varied inversely with the dose of progesterone. A range of oestrogen doses gave the same depression in the response when given to pseudopregnant animals. Administration of HCG to pseudopregnant mice also reduced the inflammatory response.     

Correlation of increased concentration of ovine endometrial cyclooxygenase 2 with the increase in PGE2 and PGD2 in the late luteal phase

The effect of intraoviductal embryos on endometrial receptivity was studied by intraendometrial and intrauterine embryo transfer. Five-week-old female ICR mice were mated after superovulation; a vaginal plug confirmed day 1 of pregnancy. On day 4 (90 h after hCG injection), blastocysts were collected and transferred to pseudopregnant female mice and to recipient mice in which the uterotubal junction had been ligated bilaterally on day 1 of pregnancy. Three embryos per uterine horn, a total of six embryos per recipient mouse at days 1-6, were transferred to the endometrium or uterine cavity and implantation and pregnancy rates were calculated. The implantation rate for intraendometrial embryo transfer to recipients of days 3, 5 and 6 was significantly higher for uterotubal junction-ligated mice (72.2, 20.8 and 9.7%, respectively) than for pseudopregnant mice (55.0, 8.3 and 0.0%, respectively). The implantation rate for intrauterine embryo transfer to recipients at days 2, 5 and 6 was significantly higher for uterotubal junction-ligated mice (11.1, 25.0 and 8.3%, respectively) than for pseudopregnant mice (0.0, 3.3 and 0.0%, respectively). Uterotubal junction-ligated mice achieved implantation and bore neonates by intrauterine embryo transfer on days 2 and 6, whereas no implantation was achieved in pseudopregnant mice. The difference in implantation rate could not be explained by a difference in progesterone concentration between the groups. The distribution of proliferating cells in the endometrium was also studied immunohistochemically by use of anti-proliferating cell nuclear antigen (PCNA) antibody in the recipient mice. PCNA-positive cells were more abundant in uterotubal junction-ligated mice and demonstrated a marked extension from the epithelium to the stroma over time, in contrast to those in pseudopregnant mice. These findings indicate that an intraoviductal embryo exerts a biological effect by sending a signal to the endometrial epithelium and stroma, thus facilitating endometrial receptivity to the embryo and improving the rate of implantation.     

Induction of implantation by aromatase inhibitors in ovariectomized mice

The ability of aromatase inhibitors to induce implantation in mice was tested in animals in which implantation was delayed by ovariectomy and progesterone treatment. Implantation was consistently induced by 7 mg 4-hydroxyandrostene-3,17-dione (4-OH-A), 7 X 5 mg 1,4,6-androstatriene-3,17-dione (ATD) or 15 mg 4-acetoxyandrostene-3,17-dione, an activity comparable to that of 1 mg testosterone. In intact mice treated with 2 or 10 mg 4-OH-A or ATD/day from Day 2 of pregnancy (Day 1 = vaginal plug), the number and size of implantation sites were not affected. These results may not be necessarily due to inhibitory effects of the compounds on aromatase.     

Uterine RGS2 expression is regulated by exogenous estrogen and progesterone in ovariectomized mice,and downregulation of RGS2 expression in artificial decidualized ESCs inhibits trophoblast spreading in vitro

Embryo implantation is a complicated event that relies on two critical factors: the competent blastocyst and the receptive uterus. Successful implantation results from tight coordination of these two factors. The maternal hormone environment of the uterus and molecular cross‐talk between the embryo and uterine tissue play pivotal roles in implantation. Here we showed that regulator of G‐protein signaling 2 (RGS2), a member of ubiquitous family of proteins that regulate G‐protein activation, plays an important role in embryo implantation by interfering in the cross‐talk between the embryo and uterine tissue. RGS2 expression increased during the implantation process, and was higher in the implant site than at the nonimplantation site. Meanwhile, ovariectomized (OVX) mice exhibited higher expression of RGS2 in the uterus. Exogenous 17β‐estradiol and progesterone in OVX mice downregulated the expression of RGS2. Treatment with exogenous 17β‐estradiol alone caused uterine RGS2 messenger RNA levels of OVX mice to return to those of normal female mice; when these mice were treated with progesterone or 17β‐estradiol plus progesterone, RGS2 levels rose. Downregulation of Rgs2 by small interfering RNA in an in vitro coculture system of decidualized endometrial stromal cells and blastocysts inhibited blastocyst outgrowth by restricting trophoblast spreading, suggesting a mechanism by which RGS2 regulates embryo implantation.     

Lysophosphatidic receptor, LPA3, is positively and negatively regulated by progesterone and estrogen in the mouse uterus

Reciprocal interactions between blastocysts and receptive uteri are essential for successful implantation. This process is regulated by the timely interplay of two ovarian hormones, progesterone and estrogen. However, the molecular targets of these hormones are largely unknown. We showed recently that a small bioactive lysophospholipid, lysophosphatidic acid, plays a pivotal role in the establishment of implantation via its cellular receptor, LPA(3). Here we demonstrate that LPA(3) expression is positively and negatively regulated by steroid hormones in mouse uteri. The LPA(3) mRNA level in the uteri increased during early pseudopregnancy, peaking around 3.5 days post coitus (3.5 d.p.c.), then, decreased to the basal level on 4.5 d.p.c. LPA(3) expression remained at a low level in ovariectomized mice, and administration of progesterone to ovariectomized mice up-regulated LPA(3) mRNA expression. In addition, simultaneous administration of estrogen counteracted the effect of progesterone. These results show that progesterone and estrogen cooperatively regulate LPA(3) expression, thereby contributing to the receptivity of uteri during early pregnancy.     

Plasma gonadotrophins and progesterone concentrations during various degrees of underfeeding in pregnant mice

Daily reduction of the normal (ad libitum) food consumption by as little as 35% significantly reduced (P less than 0.05) the percentage of mice with implantation sites at Days 7 and 9 of gestation. Underfeeding decreased body weight and reduced the weight of the ovaries and uterus. Plasma progesterone was decreased (P less than 0.05) as dietary intake was restricted and was associated with regression of the corpora lutea. No significant alterations in the plasma values of LH and FSH were observed in mice underfed between Days 1 and 9 of pregnancy. The decrease in plasma progesterone in the absence of reduced LH values may indicate that progesterone secretion between Days 5 and 9 of gestation is not controlled solely by LH.