Amniotic fluid and decidual prolactin during pregnancy in rhesus macaques

In rhesus macaques, the concentration of immunoreactive prolactin in the amniotic fluid remains low during most of the first trimester of pregnancy and then increases abruptly at 60-80 days of gestation. During the second half of pregnancy, large amounts of prolactin accumulate in the amniotic fluid. Much of this amniotic fluid prolactin may originate from the superficial endometrium (decidua). This hypothesis is supported by the increasing amounts of decidual prolactin (dPRL) measured in endometrium obtained at early (50 days), mid-(80 days), and late (greater than or equal to 150 days) gestation. In culture, late pregnancy endometrium released more dPRL than did early pregnancy endometrium. When tissues were cultured in medium without progesterone, the amounts of dPRL measured in the medium declined steadily over 6 days, regardless of the gestational age of the endometrium. dPRL was consistently measured in medium harvested from cultures that received either progesterone or medroxyprogesterone; however, progesterone did not induce an increase in the amounts of dPRL released by cultures prepared from early pregnancy endometrium. This suggests that factors in addition to progesterone may stimulate the increase in dPRL that occurs at midgestation in rhesus macaques.     

Detection of a progesterone-dependent secretory protein synthesized by cat endometrium

Uterine flushings and culture media from endometrial explants incubated in the presence of radiolabeled amino acids were analyzed using one-(1-D) and two-dimensional (2-D) gel electrophoresis to identify proteins synthesized by the endometrium and subsequently released into the uterine lumen. 1-D and 2-D analyses of uterine flushings and culture media of endometrial explants obtained from 7- to 11-day pregnant cats (pre-implantation) showed a Mr 30,000 protein that appeared on 2-D gels as a family of macromolecules with isoelectric points between 6.5 and 7.0. This family of macromolecules was also present in the culture media of implantation-site tissue obtained from 12- to 16-day pregnant cats and of nonimplantation-site endometrium obtained form 12- to 28-day pregnant cats. The Mr 30,000 protein was absent in uterine flushings and culture media from estrous and 3- to 5-day-pregnant cats. In ovariectomized, steroid-treated animals, the Mr 30,000 protein was only detected in flushings and media from those animals treated with progesterone, regardless of the presence or absence of estradiol-priming and/or simultaneous estradiol treatment. In daily flushings obtained from ovariectomized, steroid-treated cats equipped with an indwelling uterine catheter, the Mr 30,000 protein was absent during the 14 days of estradiol treatment and was first detected 3-4 days after the onset of estradiol plus progesterone treatment. This protein was not detected in serum from estrous, 9-day pregnant, ovariectomized, and ovariectomized, steroid-treated animals. This study shows that 1) a progesterone-dependent protein, with an approximate molecular weight of 30,000 and an isoelectric point of 6.5-7.0, first appears within the uterine lumen soon after the arrival of the blastocyst and continues to be present during implantation; 2) the synthesis and release of the Mr 30,000 protein is dependent on progesterone regardless of the presence or absence of estradiol; and 3) the onset of secretion of the Mr 30,000 protein requires 3-4 days of continuous progesterone treatment in the estradiol-primed cat.     

Protein synthesis and secretion by the human endometrium during the menstrual cycle and the effect of progesterone in vitro

To identify markers of endometrial differentiation specimens of endometrium from the menstrual cycle were incubated in vitro with [35S]methionine, in the absence or presence of progesterone, and protein synthesis and secretion were studied by fluorographic analysis of one dimensional SDS/gradient polyacrylamide gels. Changes were demonstrated in the rate of synthesis and secretion of a number of endometrial proteins (EP) during the cycle and in response to progesterone. Endometrial proteins were classified into three groups: Group I-synthesized and secreted throughout the menstrual cycle and unaffected by progesterone exposure; Group II-synthesis and secretion associated with histological type of endometrium and unaffected by progesterone exposure, e.g. EP 13 (Mr 33,000) with proliferative, EP 15 (Mr 28,000) with secretory and EP 14 (Mr 32,000) with late secretory endometrium; Group III-synthesis and secretion regulated by progesterone exposure irrespective of source of endometrium, e.g. EP 9 (Mr 54,000) and 11 (Mr 45,000). The Group II proteins EP 14 and 15 were also the major secretory protein products of endometrium from first and second trimester pregnancy respectively, the native forms referred to as pregnancy-associated endometrial alpha 1- and alpha 2-globulins (alpha 1- and alpha 2-PEG). We conclude that EP 15 (alpha 2-PEG) represents a human analogue of uteroglobin.     

Progestin regulation of protein synthesis in endometrial cancer

Protein synthesis in cancerous and normal human endometrium was investigated by the incorporation of [35S]methionine and analysis of products by two-dimensional polyacrylamide gel electrophoresis. Comparison of the cellular products from organ cultures of endometrial carcinoma, obtained from each subject before and after in vivo administration of medroxyprogesterone acetate (MPA) for 10-14 days revealed: (i) a decrease in the synthesis of tubulin and of a protein of molecular weight (mol. wt) 68 kDa, isoelectric point (pI) 6.0, and (ii) an increase in the synthesis of creatine kinase (CK) and of protein of mol. wt 36 kDa, pI 4, following MPA therapy. The 68 kDa protein was expressed at relatively reduced levels in organ cultures of normal human endometrium throughout the menstrual cycle. In primary cultures of normal human endometrium throughout the menstrual cycle. In primary cultures from cancerous endometrium endometrium established for several weeks the 68 kDa protein was not expressed but could be induced by heatshock. Primary cultures were also used to investigate the early events following progesterone stimulation which revealed a decrease in synthesis of a protein mol. wt 36 kDa, pI 8 at 8 h following administration.     

The effect of progesterone and human interferon alpha-2 on the release of PGF2 alpha and PGE from epithelial cells of human proliferative endometrium.

Progesterone and interferon-like trophoblastic proteins modulate prostaglandin (PG) synthesis from endometrium in early ovine and bovine pregnancy. Enriched epithelial cells were prepared from human endometrium removed in the proliferative phase of menstrual cycle (n = 8). Progesterone at a concentration of 1 microM suppressed PGE release from the cells during the first 24 hours in culture. After 48 hours in culture progesterone at a dose of 100 nM and 1 microM suppressed both the release of PGF2 alpha and PGE from the cells and this suppression was maintained for a further two days. Addition of exogenous 30 microM arachidonic acid (AA) abolished this effect of progesterone on both PGF2 alpha and PGE release. Interferon alpha-2 did not suppress the basal release of PGF2 alpha nor PGE. In the presence of progesterone, interferon alpha-2 attenuated the progesterone mediated suppression of PGF2 alpha but not PGE release from endometrial cells. These findings suggest that progesterone suppresses the basal release of PGs from human endometrium, but unlike the sheep, interferon alpha-2 does not exert this action on human endometrium.     

Role of progesterone and oestradiol in the regulation of uterine oxytocin receptors in ewes.

The interaction between oestrogen and progesterone in the regulation of the uterine oxytocin receptor in sheep was evaluated by measuring the binding of oxytocin to membrane preparations of caruncular and intercaruncular endometrium and myometrium. Ovariectomized ewes were assigned in groups of five to each cell of a 4 x 2 factorial design. The four treatments were (a) vehicle (maize oil) for 12 days, (b) progesterone (10 mg day-1) for 9 days, (c) progesterone for 9 days followed by maize oil until day 12 and (d) progesterone for 12 days. The two oestradiol treatments consisted of the administration of implants in the presence or absence of oestradiol. The ewes were killed on day 10 (group b) or day 13 (groups a, c and d) for collection of uterine tissues. The response of the caruncular and intercaruncular endometrium to the treatments was similar. In the absence of oestradiol, treatment with progesterone continuously for either 9 or 12 days reduced the concentration of the oxytocin receptor in comparison with both the control and the progesterone withdrawal group (in which values were similar). The presence of oestradiol reduced the receptor concentrations in control and both 9- and 12-day continuous progesterone treatment groups, but enhanced the concentration in the progesterone withdrawal group. The myometrial oxytocin receptors responded in a similar way to those in the endometrium to progesterone treatment alone, but the addition of oestradiol produced no further effect. In conclusion, progesterone and oestradiol caused downregulation of the endometrial oxytocin receptor. On the other hand, progesterone withdrawal, similar to that which occurs during luteolysis, increased receptor density in the presence of oestradiol. Progesterone may influence the response of the myometrium to oxytocin by causing a reduction in receptor density.     

Endometrial protein secretion during early pregnancy in entire and ovariectomized ewes

The secretion and synthesis of protein in vitro by explants of endometrium were examined in entire ewes during the first 10 days of the oestrous cycle and during an equivalent interval in ovariectomized ewes which received injections of oestradiol and progesterone. The schedule of steroid injections given was designed to simulate endogenous ovarian secretion of progesterone during the luteal phase before oestrus, of oestradiol around oestrus and of progesterone during the luteal phase after oestrus. The rate of protein synthesis and tissue RNA:DNA and protein:DNA ratios in intercaruncular and caruncular endometrium were generally higher in entire than in ovariectomized ewes. In ovariectomized ewes oestradiol increased these activities at 2-4 days after oestrus, whereas progesterone preceding oestradiol caused increases at oestrus, but not thereafter. In entire ewes and in ovariectomized ewes receiving the full steroid treatment regimen, protein secretion was high at oestrus and declined markedly during the next 4-6 days. In ovariectomized ewes not receiving progesterone before oestradiol, secretion increased between 4 and 6 days after oestrus, or during the equivalent stage of treatment in ewes which did not show oestrus. The omission of this progesterone did not modify secretion by caruncular endometrium. Oestradiol increased protein secretion by both tissues. The data suggest that progesterone given before oestradiol (or its equivalent in entire ewes) inhibits the secretion, at about 4-7 days after oestrus, of uterine proteins which may impair embryo development in ovariectomized ewes which do not receive this progesterone.     

Expression of calbindin-D28k and its regulation by estrogen in the human endometrium during the menstrual cycle

Human endometrium resists embryo implantation except during the 'window of receptivity'. A change in endometrial gene expression is required for the development of receptivity. Uterine calbindin-D28k (CaBP-28k) is involved in the regulation of endometrial receptivity by intracellular Ca2+. Currently, this protein is known to be mainly expressed in brain, kidneys, and pancreas, but potential role(s) of CaBP-28k in the human uterus during the menstrual cycle remain to be clarified. Thus, in this study we demonstrated the expression of CaBP-28k in the human endometrium in distinct menstrual phases. During the human menstrual cycle, uterine expression levels of CaBP-28k mRNA and protein increased in the proliferative phase and fluctuated in these tissues, compared with that observed in other phases. We assessed the effects of two sex-steroid hormones, 17beta-estradiol (E2) and progesterone (P4), on the expression of CaBP-28k in Ishikawa cells. A significant increase in the expression of CaBP-28k mRNA was observed at the concentrations of E2 (10(-9 to -7) M). In addition, spatial expression of CaBP-28k protein was detected by immunohistochemistry. CaBP-28k was abundantly localized in the cytoplasm of the luminal and glandular epithelial cells during the proliferative phases (early-, mid-, late-) and early-secretory phase of menstrual cycle. Taken together, these results indicate that CaBP-28k, a uterine calcium binding protein, is abundantly expressed in the human endometrium, suggesting that uterine expression of CaBP-28k may be involved in reproductive function during the human menstrual cycle.     

The effect of clomiphene on the synthesis of prostaglandins (PGF2 alpha, PGE2, PGI2) in human endometrial cells in vitro with and without addition of estradiol-17 beta or progesterone

The production of prostaglandin F2 alpha in monolayer stromal cell cultures of proliferative human endometrium is enhanced by 10(-7) mol/l estradiol-17 beta or 10(-4) mol/l progesterone. Progesterone in high concentration (10(-4) mol/l) also enhanced the synthesis of prostaglandin E2. Clomiphene citrate reduced this increased prostaglandin production dose dependently. The synthesis of prostaglandin I2 was not influenced either by sex steroids or by clomiphene citrate.     

Regulation of gene expression and cellular localization of prostaglandin synthase by oestrogen and progesterone in the ovine uterus

Expression of the gene for prostaglandin synthase (PGS) was examined in whole endometrial tissue derived from ewes during the oestrous cycle (Days 4-14), on Day 15 of pregnancy and following ovariectomy and treatment with ovarian steroid hormones. Whilst no significant differences were seen in PGS mRNA concentrations analysed by Northern blot analysis in endometrial tissue during the oestrous cycle or in early pregnancy, treatment of ovariectomized (OVX) ewes with oestradiol-17 beta markedly reduced endometrial PGS mRNA concentration. There was no difference in PGS mRNA concentration in ewes treated with progesterone, either alone or in conjunction with oestrogen, from that in OVX controls. In contrast, differences in immunolocalization of PGS observed in uterine tissue from OVX-steroid-treated ewes were much more marked and reflected similar changes seen previously in the immunocytochemical distribution of endometrial PGS during the oestrous cycle. In OVX ewes and those treated with oestrogen, immunocytochemical staining for PGS was seen in stromal cells, but little immunoreactive PGS was located in the endometrial epithelial cells. However, in ewes treated with progesterone alone or with oestrogen plus progesterone, PGS was found in luminal and glandular epithelial cells and in stromal cells. Intensity of immunostaining for PGS in endothelial cells and myometrium did not differ between the treatments. Thus, whilst oestrogen lowers PGS mRNA in the endometrium, presumably in stroma, it may also increase the stability of the enzyme itself in the stromal cells. Although oestradiol-17 beta has no effect on PGS in endometrial epithelium, progesterone stimulates the production of PGS in endometrial epithelial cells without altering the overall abundance of PGS mRNA in the endometrium as a whole. Conceptus-induced changes in PGF-2 alpha release by ovine endometrium would not appear to be mediated via effects on PGS gene expression or protein synthesis.     

Platelet-activating factor in human luteal phase endometrium

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is one of the most potent mediators of vascular permeability. PAF levels change in the rabbit endometrium just prior to implantation, which suggests that PAF may be a key substance transducing preimplantation embryonic signals. To study whether PAF was present in the human endometrium, and if so, to determine the cellular origin and hormonal regulation of endometrial PAF, specimens were obtained from 14 women (aged 23-42 yr) undergoing elective hysterectomy during the luteal phase of the cycle (plasma progesterone levels greater than 2 ng/ml). No specimens were taken from women with malignant uterine pathology. Stromal cells and epithelial glandular cells were separated by collagenase and DNAse digestion, and then cultured to confluence in vitro in medium 199. Radioimmunoassays of prostaglandin F (PGF) and prolactin in the culture media were used to confirm cell type and viability. PGF release into the culture medium from stromal cells was low (control 1.52 +/- 0.20 ng/ml), and unchanged by hormone treatment. In contrast, release of PGF from unstimulated glandular cells was 6.05 +/- 0.52 ng/ml, and was significantly increased (p less than 0.05) by estradiol or progesterone plus estradiol, to 12.17 +/- 1.67, and 8.60 +/- 0.81, respectively. Progesterone alone was without effect. Prolactin was secreted by stromal cell cultures, increasing steadily from 24 to 120 h. The levels in the medium were increased by progesterone. PAF activity was assessed by rabbit platelet aggregation and serotonin-release bioassays after lipid extraction and separation by thin-layer chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin F and progesterone secretion by porcine endometrium and corpus luteum in vitro

Slices of porcine endometrium and corpus luteum tissue obtained from mature sows throughout the luteal phase of the oestrous cycle were incubated in culture medium which was analysed at regular intervals over a period of 8 hours for prostaglandin F and progesterone. Prostaglandin F secretion was greatest by endometrium obtained during the mid III to late I luteal stage of the cycle and the increased levels secreted by this tissue were paralleled by high levels of secretion from corpus luteum tissue. The addition of indomethacin (10 μg/ml) to the culture medium completely abolished prostaglandin F secretion by both endometrium and luteal tissue indicating that the high levels of the prostaglandin were due to synthesis. Progesterone secretion by the corpus luteum was maximal from early luteal tissue and had declined to considerably lower levels by late stage tissue when prostaglandin secretion was greatest. The possible physiological significance of luteal prostaglandin F secretion is discussed.     

Vitrification of carnation in vitro: Changes in ethylene production,ACC level and capacity to convert ACC to ethylene

Carnation tissue was allowed to vitrify in liquid culture and ethylene production, ACC content and capacity to convert ACC to ethylene were measured in comparison to tissue developing normally on solid medium. Flask atmospheres of liquid cultures accumulated ethylene at a higher rate during the first four days. Daily ethylene production by vitrifying material decreased later. Ethylene emission by vitrifying tissues always remained above controls when subcultured daily to fresh medium. Explants and microsomal preparations from vitrifying carnations converted ACC to ethylene at a higher degree from the first day in liquid medium. ACC level markedly increased in vitrifying tissues during the first two days of liquid culture. Raising the level of ethylene in the atmosphere of solid cultures did not induce vitrification symptoms nor did use of inhibitors of ethylene biosynthesis in liquid cultures prevent the process. The role of ethylene in vitrification is reappraised.     

Collagen synthesis in human fibroblasts: Effects of ascorbic acid and regulation by hydrocortisone

The effects of hydrocortisone and ascorbic acid on collagen and noncollagen protein synthesis, and on growth were examined in fibroblasts derived from normal human dermis. When the medium was supplemented with 0.28 mM ascorbic acid, the apparent rate of collagen production increased 2--3 fold over the culture cycle. Ascorbic acid also caused a small increase in the apparent rate of synthesis of noncollagen protein and an elevation in growth rate and maximum cell density. Growth was not required for the increase in collagen production since addition of ascorbate to confluent cultures induced a similar increase. Hydrocortisone (1.5 μM) blocked the ascorbate-related increase in collagen production during growth and in confluent cultures. The hormone simultaneously increased the apparent rate of noncollagen protein production and maximum cell density, suggesting that the effect on collagen synthesis was specific. Inhibition of collagen production by hydrocortisone was observed only in the presence of ascorbate, while the increase in growth and noncollagen protein production occurred in the presence and absence of the vitamin.     

Induction and inhibition of amphibian (Rana pipiens) oocyte maturation by protease inhibitor (TPCK)

We report for the first time that oocyte nuclear and cytoplasmic maturation are triggered in vitro in non-hormone-treated amphibian (Rana pipiens) ovarian follicles following transient exposure to synthetic chymotrypsin inhibitor Nα-tosyl-L-phenylalanine-chloromethyl ketone (TPCK). The mechanism of action of TPCK in regulating oocyte maturation was investigated and compared to that induced by progesterone or pituitary hormone. Follicular oocytes failed to mature following continuous exposure to the same doses of TPCK in the presence or absence of progesterone. Continuous treatment of follicles with lower levels of TPCK occasionally induced GVBD in the absence of progesterone and augmented maturational effects of low levels of progesterone. TPCK induced maturation of intrafollicular oocytes without stimulating progesterone production and also induced maturation of naked oocytes. Stimulation of follicular progesterone synthesis following gonadotropin stimulation or addition of pregnenolone was inhibited by TPCK, indicating that TPCK affects metabolic processes in both the somatic and germinal components of the ovarian follicle. Oocyte maturation induced by either TPCK or progesterone was inhibited by cycloheximide, calcium-deficient medium, and forskolin. Results suggest that TPCK induces oocyte maturation independent of steroidogenesis via mechanisms similar to those triggered by progesterone involving protein synthesis, formation of cytoplasmic maturation-promoting factor (MPF), and changes in cAMP levels. Our data indicate that a chymotrypsin-like protease plays a role(s) in regulating the oocyte meiotic maturation process.     

Establishment of callus and cell suspension cultures of raspberry (Rubus idaeus cv. Royalty)

Callus cultures were initiated from leaf sections of raspberry (Rubus idaeus L.) cv. Royalty. Explants from younger leaves produced significantly more calli than those from older leaves. Anderson's salt mixture was more efficient for callus induction than the Murashige-Skoog medium. The best propagation and growth of calli was observed on Anderson's medium supplemented with 9 M 2,4-dichlorophenoxyacetic acid, 4.9 M indolebutyric acid and 4.9 M 6-(dimethylallylamino)-purine. During a 28-day period, the fresh weight of calli increased approximately five times. The same medium without agar was used for establishing cell suspension cultures. Fresh weight of cells increased four times and dry weight approximately doubled during 10 days of culture.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - 2iP 6-(dimethylallylamino)-purine - IBA indolebutyric acid - MS Murashige & Skoog basal salt medium     

Progesterone treatment in vitro enhances prostaglandin E and forskolin-promoted cyclic AMP production in human endometrial stromal cells

Confluent human endometrial stromal cell cultures were exposed to steroids for up to 72 h and then stimulated with agonists of adenylate cyclase for 60 min. Neither steroid alone or in combination had significant effect on cyclic AMP production. However, when stromal cell adenylate cyclase was stimulated with a receptor-dependent agonist (prostaglandin E), or with forskolin (which acts at a post-receptor site), progesterone in oestradiol-primed cells markedly enhanced (P less than 0.05) the effect of both agonists. The presence of phenol red, a weak oestrogenic compound, in the standard culture medium was sufficient to allow the progesterone effect to be manifest. Moreover, while oestradiol alone had no significant effect on prostaglandin E or forskolin-stimulated cyclic AMP production, the simultaneous exposure of cells to oestradiol and progesterone was the most effective treatment. Short-term incubation (up to 120 min) with progesterone had no effect on agonist-induced cyclic AMP accumulation, indicating that progesterone elicits its effect by the classic nuclear mechanism of action. It is suggested that the potentiation by progesterone of prostaglandin E-promoted production of cyclic AMP represents an important aspect of the functional role progesterone plays in the preparation of the endometrium for implantation.     

Induction of the synthesis of the pregnancy-specific protein p70 in the endometrium by intramuscular injection of recombinant bovine interferon-alpha I1 in nonpregnant ewes.

We examined the effect of recombinant bovine interferon-alpha I1 (rboIFN-alpha I1) or recombinant bovine trophoblast protein-1 (rbTP-1) on protein synthesis by endometrial explants from Day-13 cyclic ewes and studied the ability of rboIFN-alpha I1 injected i.m. to influence subsequent protein secretion by endometrial tissue explants. In Expt 1, ewes were injected with either 2 mg rboIFN-alpha I1 or vehicle alone at 12 h intervals beginning on Day 11 of the oestrous cycle and ending on the morning of Day 13; 8 h after the last injection, ewes were hysterectomized and endometrial explant cultures were prepared. Explants were cultured for 24 h in leucine-deficient medium supplemented with 250 microCi L-[3H]leucine per culture. For Expt 2, additional explants were prepared from Expt 1 controls. Explants were cultured in the presence of 0, 20 or 200 ng/ml of either rboIFN-alpha I1 or rbTP-1 for 24 h in leucine-deficient medium supplemented with 250 microCi L-[3H]leucine per culture. Secreted proteins were analysed by two-dimensional electrophoresis and fluorography. There was a marked enhancement of a 70 kDa acidic protein, p70, in explants cultured in the presence of rboIFN-alpha I1 or rbTP-1. This polypeptide is a product of the gravid uterine horn from Day 14 to Day 20 of pregnancy and is a useful marker of the action of interferon-alpha (IFN-alpha) on endometrium. Enhanced production of p70 also occurred in ewes injected i.m. with rboIFN-alpha I1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of prostaglandin synthesis by progesterone in the bovine corpus luteum

The objective of the present study was to investigate the influence of progesterone on prostaglandin synthesis by the corpus luteum (CL). Corpora lutea were obtained from dairy cows on days 4, 6, 10, and 18 of the estrous cycle, dissociated, and placed in serum-free culture. The addition of luteinizing hormone (LH) resulted in a slight, but non-significant (p greater than 0.05), increase in levels of 6-keto-PGF1 alpha, and had no effect on PGF2 alpha. Progesterone treatment caused a significant, dose-dependent decrease in both PGF2 alpha and 6-keto-PGF1 alpha in 6-day and 10-day corpora lutea, but not in 4-day or 18-day corpora lutea. In the 6- and 10-day corpora lutea, progesterone treatment resulted in a greater inhibition of PGF2 alpha than 6-keto-PGF1 alpha production. Therefore, progesterone treatment brought about an increase in the 6-keto-PGF1 alpha to PGF2 alpha ratio in these cells (12.9 vs. 21.3). It is concluded from these studies that progesterone can modulate luteal prostacyclin and PGF2 alpha synthesis, suggesting an interaction of progesterone and prostaglandin production within the corpus luteum.