Changes in equine endometrial oestrogen receptor alpha and progesterone receptor mRNAs during the oestrous cycle, early pregnancy and after treatment with exogenous steroids

Two experiments were performed to determine changes in the abundance of oestrogen and progesterone receptor (ER alpha and PR) mRNAs in equine endometrium during the oestrous cycle and early pregnancy, and under the influence of exogenous steroids. In Expt 1, endometrial biopsies were obtained from non-mated mares during oestrus and at days 5, 10 and 15 after ovulation, and from pregnant mares at days 10, 15 and 20 after ovulation. There were overall effects of day on the abundance of ER alpha (P = 0.0001) and PR (P = 0.0014) mRNAs. The amount of ER alpha mRNA decreased at day 10 of pregnancy, and PR mRNA was reduced at day 5 in non-mated mares and at day 15 of pregnancy, compared with oestrous values. Experiment 2 was conducted to determine the effects of exogenous steroids on endometrial ER alpha and PR mRNAs. Endometrial biopsies were obtained from 19 anoestrous mares that had been treated with vehicle, oestradiol, progesterone, or oestradiol followed by progesterone for either a short or a long duration. The steroid treatment affected the abundance of ER alpha mRNA (P = 0.0420), which was higher (P < 0.05) in the oestradiol group than in the group treated with oestradiol followed by long duration progesterone. The steroid treatment did not affect the abundance of PR mRNA. These results demonstrate that the amount of steroid receptor mRNA changes with the fluctuating steroid environment in the uterine endometrium of cyclic and early pregnant mares, and that the duration of progesterone dominance may affect ER alpha gene expression. In addition, factors other than steroids may regulate ER alpha and PR gene expression in equine uterine endometrium.     

Effects of progesterone and oestradiol on RNA and protein metabolism in the genital tract and on survival of embryos in the ovariectomized ewe.

The hormonal regulation of metabolism in the genital tract and the development of embryos during early pregnancy in the ewe have been examined. Ovariectomized ewes received injections of maintenance progesterone, oestrous oestradiol and priming progesterone according to schedules designed to simulate endogenous ovarian secretion during early pregnancy, around the time of oestrus and during the luteal phase of the oestrous cycle immediately preceding oestrus. The survival and development of embryos was dependent upon the dose of maintenaince progesterone and the duration of treatment at the time of transfer, but changes in progesterone dose did not change endometrial protein or RNA metabolism on particular days. Both priming progesterone and oestrous oestradiol were required for normal embryo development. Priming progesterone and oestrous oestradiol each increased endometrial RNA/DNA ratios during early pregnancy. There were no interactions between priming progesterone and oestrous oestradiol, their effects being simply additive. Neither maintenance nor priming progesterone had any effect on protein and RNA metabolism in the oviduct. It is suggested that in the intact ewe oestrogen secreted at oestrus and progesterone secreted prior to oestrus play important roles in the establishment of a uterine environment suitable for the subsequent normal development of embryos.     

Intracellular pH electrode : Experiments on the giant squid axon

A new, easy method to produce and calibrate a 1-μm tip intracellular pH electrode is described. This antimony electrode and a micro-calomel electrode were inserted into the giant axon of Loligo pealii. The potential obtained when the axon was bathed in seawater corresponded to a pH of 7.0 ± 0.2. It was found that acidification of the external perfusate induced a drop in axoplasmatic pH leading to changes in the membrane electrical properties. Changes of resting or action potentials did not influence intracellular pH.     

Changes in the density of peripheral benzodiazepine binding sites in genital organs of the female rat during the oestrous cycle

The affinity and the density of peripheral-type benzodiazepine binding sites (PBzS) in tissues from the genital organs of female rats were studied during the oestrous cycle. When comparing PBzS density on the day of oestrus to PBzS density on the day of pro-oestrus, a significant increase was observed in the ovary (1.9-fold), oviduct (2.4-fold) and uterus (1.7-fold), but not in the kidney. Serum oestradiol also increased to a maximum on the day of pro-oestrus. The ovarian and uterine PBzS density and serum concentrations of oestradiol and progesterone were measured every 8 h between the days of dioestrus and pro-oestrus. Ovarian and uterine PBzS density increased to a maximal value at 01:00 and 09:00 h, respectively, on the day of pro-oestrus. However, a significant increase in PBzS density occurred in the ovary (P less than 0.02) and uterus (P less than 0.001) at 09:00 h on the day of pro-oestrus as compared to 09:00 h on the day of dioestrus. These changes were associated with an increase in serum oestradiol and progesterone concentrations. The affinity of PBzS in all tissues examined remained unaltered during the oestrous cycle. This study demonstrates that changes associated with the oestrous cycle occur in the density of PBzS in various genital organs.     

Changes of progesterone content of rat uterine flushings in relation to serum concentrations of progesterone during the oestrous cycle.

Uterine fluid was collected from four-day cyclic rats at each stage of the oestrous cycle and assayed for progesterone and protein content. Progesterone was determined by radioimmunoassay either after ethanol (or 2.5% NaOH) denaturation of proteins from uterine flushings ('total' progesterone) or without protein denaturation ('ether-extractable' progesterone). The amount of 'ether-extractable' progesterone in the lumen was constant from metoestrus to pro-oestrus (340 pg per uterus) but lower in oestrus (200 pg per uterus). However, 'total' progesterone content of uterine fluid was subject to cyclic variations and was highest in dioestrus (890 pg per uterus) and lowest in oestrus (350 pg per uterus), in contrast to serum progesterone which is lowest in dioestrus and highest in oestrus. Protein content of uterine flushings peaked to 780 micrograms per uterus in pro-oestrus then fell to about 140 micrograms per uterus until the end of the oestrous cycle. Changes in protein content of the lumen were followed by qualitative variations since the mean amount of 'bound' progesterone ('total' progesterone minus 'ether-extractable' progesterone) released per milligram of denatured lumen protein rose from 1.8 pmol in pro-oestrus to 18.2 pmol in dioestrus. The changes of luminal 'bound' progesterone during the oestrous cycle suggest that progesterone binding to luminal proteins could be an important modulator of progesterone action in rat uterus. Moreover, the variations in progesterone content of the lumen, irrespective of serum progesterone concentrations, are consistent with the hypothesis that progesterone synthesis occurs in the uterus.     

Concentrations of progesterone in the backfat of pigs during the oestrous cycle and after ovariectomy

Small samples of backfat were taken daily during one oestrous cycle and more frequently after ovariectomy from 12 gilts by means of a simple biopsy technique and the levels of progesterone were determined. Compared to the levels of progesterone in peripheral plasma changes in backfat levels during the oestrous cycle were delayed by 1-2 days. Maximal levels with 89.7 +/- 9.2 (mean +/- s.e.m) ng progesterone/100 mg backfat were recorded on Day 15 of the oestrous cycle. It was estimated that, on this day, a total amount of about 36 mg progesterone is stored in the adipose tissue, which is approximately 200 times that present in total blood and corresponds to the daily production of the corpora lutea of the sow on Day 11. Initial half-life of progesterone in backfat after ovariectomy was estimated to be about 34 h compared to an initial half-life of plasma progesterone of about 120 min. The exact calculation of half-lives was, however, confounded by an obvious effect of anaesthesia or surgery on progesterone levels. Changes in backfat or plasma progesterone concentrations were not affected by the fat-to-lean ratio of the gilts. Fat progesterone levels determined in 44 additional pregnant and non-pregnant sows 17 or 20 days after mating indicated that reliable diagnosis of non-pregnant sows was possible on Day 20. It is concluded that the endocrinology of the oestrous cycle in pigs is related to the enormous storage of progesterone in the fat.     

Variability in the response of the rabbit uterus to progesterone as influenced by prolactin

Ovariectomized rabbits from different breeders were treated at different times of the year with prolactin alone or with progesterone and the production of uteroglobin by the uterus was studied. There were seasonal, strain and dose variables in the uterine response to prolactin and progesterone. Treatment with prolactin (at 1 mg/day) plus progesterone generally induced higher levels of uteroglobin production than did treatment with progesterone alone. The differences were greatest in the winter for Tennessee animals and in the spring for animals from the New Mexico and North Carolina colonies. Ovariectomy produced a decrease (P less than 0.01) in the concentration of cytosolic oestrogen and progesterone receptors, and prolactin treatment restored the concentration to oestrous control values. However, there were no seasonally dependent changes in the concentration of the receptors for any of the treatment groups. Increased doses of prolactin (2 mg/day) induced high levels of uteroglobin production and new proteins to appear in uterine secretions of long-term ovariectomized rabbits but much lower levels (10-11%) when given to pregnant does. Additional ovulations were also noted plus adverse effects on the embryos.     

Effect of pinealectomy on heat-induced endocrine changes in female rats

Exposure of pinealectomized rats to high ambient temperature (35 degrees C; PXH) brought about a diminution in pituitary weight and LH content when compared to their sham-operated peers (35 degrees C) or to pinealectomized controls (22 degrees C). Serum corticosterone level of PXH rats was significantly depressed while heat or pinealectomy alone had no effect. Mean oestrous cycle length was prolonged and blood serum progesterone was increased in the heat-exposed rats. However, the extended oestrous cycles and elevated serum progesterone levels of heat-exposed rats were depressed or abolished by pineal ablation. Thus, the pineal appears to exert a moderating effect on heat-induced endocrine changes in female rats. No changes were noticed in uterine and ovarian weights corrected for body weights either on the day of vaginal opening, at occurrence of the oestrous phase expressed as percentage of total oestrous cycle, or in N-acetyltransferase and hydroxyindole-O-methyltransferase activities.     

Characterization of Low pH-induced Catecholamine Secretion in the Rat Adrenal Medulla

Abstract: Catecholamine (CA) secretion was evoked when the isolated rat adrenal gland was perfused with HEPES-buffered Krebs solution acidified by the addition of HCI or by gassing with 95% O₂/5% CO₂. The secretion was detectable at pH 7.0 and increased with decreasing pH until at ～6.4. The low pH-induced CA secretion consisted of two phases, an initial transient response followed by a sustained phase. An intracellular Ca²⁺ antagonist, 3,4,5-trimethoxybenzoic acid 8-(N,N-diethylamino)octyl ester, selectively inhibited the initial phase of secretion. Both of the responses were resistant to nifedipine, a blocker of voltage-gated Ca²⁺ channel, but were completely inhibited in Ca²⁺-free (1 mM EGTA containing) solution. Adrenaline was an exclusive component in CAs released by low pH. The time course and extent of intracellular acidification caused either by low pH in the external medium or by the offset of a transitory NH₄CI application had no correlation with those of the secretory responses in the corresponding period. These results suggest that extracellular acidification preferentially activates adrenaline secretive cells to evoke CA secretion and that this low pH-induced CA secretion may be mediated by dihydropyridine-insensitive Ca²⁺ influx. Furthermore, the initial transient phase of the low pH-induced CA secretion might be caused by a Ca²⁺ release from intracellular stores, which is also induced by the Ca²⁺ influx.     

Characterisation of proton fluxes across the cytoplasmic membrane of the yeast Saccharomyces cerevisiae.

We have tested the efficacy of fluorescent probes for the measurement of intracellular pH in Saccharomyces cerevisiae. Of the compounds tested (fluorescein, carboxyseminaphthorhodafluor-1 (C.SNARF-1) and 2',7'bis(carboxyethyl)-5(6')-carboxyfluorescein), C.SNARF-1 was found to be the most useful indicator of internal pH. Fluorescence microscopy showed that in Saccharomyces cerevisiae strain DAUL1, C.SNARF-1 and fluorescein had a heterogeneous distribution, with dye throughout the cytoplasm and concentration of the dye to an area close to the cell membrane. This region was also labeled by quinacrine, which is known to accumulate in acidic regions of the cell. Saccharomyces cerevisiae BJ4932, which carries a defect in vacuolar acidification, did not show the same degree of dye concentration, suggesting that the site of C.SNARF-1 and fluorescein localisation in DAUL1 is the acidic vacuole. Changes in intracellular pH could be monitored by measuring changes in the fluorescence intensity of C.SNARF-1. The addition of glucose caused an initial, rapid decrease in fluorescence intensity, indicating a rise in cellular pH. This was followed by slow acidification. Fluorescence intensity changes were similar in all strains studied, suggesting that the localisation of dye to acidic regions does not affect the measurement of intracellular pH in DAUL1. The changes in intracellular pH on the addition of glucose correlated well with glucose-induced changes in external pH. Preincubation of cells in the presence of the plasma membrane H(+)-ATPase inhibitor diethylstilbestrol reduced extracellular acidification and intracellular alkalinisation on the addition of glucose. Both amiloride and 5-(N-ethyl-N-isopropyl)amiloride also inhibited glucose-induced proton fluxes. Phorbol 12-myristate 13-acetate had no effect on the activity of the plasma membrane ATPase.     

Characteristics of the oestrous cycle of Iberian red deer (Cervus elaphus hispanicus) assessed by progesterone profiles

This study examines the length of the oestrous cycle in 16 Iberian red deer females assessed by means of changes in progesterone concentrations, along with the changes in the profile of this hormone. Samples were collected three occasions per week from the week after calving (15 May to 15 June) up to May of the following year. The oestrous cycle lasted 19.57+/-0.29 days (range 10-27 d) calculated in 130 oestrous cycles examined. Progesterone titres did not rise above 0.5 ng/ml in the follicular phase, except in four samples. The maximum peak in progesterone concentration during the luteal phase remained above 1 ng/ml in most cases. Twenty-five percent of the individuals studied (4 out of 16) showed an oestrous cycle lasting shorter than the mean (15.2+/-0.30 days) before the start of the reproductive season, followed by a period of sexual inactivity. The standard progesterone profile in natural oestrous cycles rose from basal levels to those above 0.5 ng/ml four days after onset of oestrus, reached a peak of 1.71+/-0.07 ng/ml and then declined to less than 0.2 ng/ml after day 20. Following the rapid decline of progesterone after day 14, the concentration remained around the baseline level of 0.1 to 0.2 ng/ml during the immediate pre- and post-ovulatory phase of the cycle.     

Peripheral plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2α and progesterone around luteolysis and during early pregnancy in the goat

Peripheral plasma concentrations of 13,14-dihdyro-15-keto-prostaglandin F_2α (PGFM) and progesterone were determined during both luteolysis in the oestrous cycle and early pregnancy in four goats. Luteal regression, characterised by decreasing progesterone concentrations, began on day 12 or 13. PGFM concentrations showed a pulsatile pattern around this time, with peak concentrations increasingly markedly as progesterone levels fell and oestrus approached. During early pregnancy progesterone concentrations did not fall after day 12 and no marked elevation of PGFM above basal values of 50–150 pg/ml was detected.     

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.     

Composition of the bovine uterine proteome is associated with stage of cycle and concentration of systemic progesterone

Early embryonic loss accounts for over 70% of total embryonic and foetal loss in dairy cattle. Early embryonic development and survival is associated with the concentration of systemic progesterone. To determine if the uterine proteome is influenced by stage of cycle or systemic progesterone concentrations, uterine flushings were collected from the ipsi‐ and contralateral uterine horns of beef heifers on Days 7 (n = 10) and 15 (n = 10) of the oestrous cycle. Animals were separated into low or high progesterone groups based on plasma progesterone concentrations on Day 5 of the cycle. Samples were albumin depleted before iTRAQ® labeling and subsequent strong cation exchange‐LC‐MS/MS analyses. A total of 20 proteins were up to 5.9‐fold higher (p < 0.05) and 20 were up to 2.3‐fold lower on Day 15 compared to Day 7. In addition, the expression of a number of proteins on Day 7 and/or 15 of the cycle was correlated with progesterone concentrations during Days 3–7 or the rate of change in progesterone between Days 3 and 7. This study highlights the dynamic changes occurring in the microenvironment surrounding the embryo during this period. The findings here also support the hypothesis that progesterone supports embryonic development by altering the maternal uterine environment.     

A quantitative study of some lysosomal enzymes in the bovine endometrium during early pregnancy.

Endometrium was obtained from cattle slaughtered at various stages of early pregnancy and of the oestrous cycle. Analyses for protein, RNA, DNA, glucose and some lysosomal enzymes were carried out on this tissue. The results are considered with respect to the general influence of the hormonal status of pregnancy and the specific influence of the hormonal status of pregnancy and the specific influence of the blastocyst in one uterine horn.     

Effect of intrauterine infusion of Escherichia coli on hormonal patterns in gilts during the oestrous cycle

The aim of this study was to determine the effect of intrauterine Escherichia coli infusion on the patterns of plasma LH, prolactin, progesterone, androstenedione, testosterone, oestrone, oestradiol-17beta, cortisol and 13,14-dihydro-15-keto-prostaglandin F2alpha (PGFM) in gilts during the oestrous cycle. On day 4 of the oestrous cycle (day 0), 25 mL of saline or 25 mL of Escherichia coli suspension, containing 10(7) colony forming units x mL(-1), was infused once into the each uterine horn in group I or II respectively. The control gilts developed a new oestrous cycle at the expected time but not bacteria-treated. Endometritis and vaginal discharge developed in all gilts after Escherichia coli infusion. The administration of Escherichia coli resulted in a reduction of plasma levels of LH, prolactin, oestrone and oestradiol-17beta (P < 0.05-0.001), mainly on days 15-18 after treatment (expected perioestrous period). During this time, the plasma androstenedione level was elevated (P < 0.05-0.001) after bacteria infusion. In the gilts receiving bacteria, progesterone concentration decreased from day 8 after treatment and was low until the end of the study (P < 0.05-0.001). On days 8-12 after bacteria administration, the level of PGFM was higher (P < 0.001) than that found in the control group. These results suggest that the developing inflammatory process of the endometrium in gilts following Escherichia coli infusion significantly affects the pituitary-ovarian axis function as well as prostaglandin production leading to anoestrus.     

Protons and calcium alter gating of the hyperpolarization-activated cation current (I(h)) in rod photoreceptors

We investigated the effects of protons and calcium ions on the voltage-dependent gating of the hyperpolarization-activated, nonselective cation channel current, I(h), in rod photoreceptors. I(h) is a cesium-sensitive current responsible for the peak-plateau sag during the rod response to bright light. The voltage dependence of I(h) activation shifted about 5 mV per pH unit, with external acidification producing positive shifts and alkalinization producing negative shifts. Increasing external [Ca(2+)] from 3 to 20 mM resulted in a large (approximately 17 mV) positive shift in I(h) activation. External [Ca(2+)] (20 mM) blocked pH-induced shifts in activation. Cytoplasmic acidification produced by 25 mM sodium acetate led to a negative shift in inactivation (-9 mV) and internal alkalinization produced with 20 mM ammonium chloride resulted in a positive shift (+6 mV). Surface charge binding and screening theory (Gouy-Chapman-Stern) accounted for the observed shifts in I(h) activation, with the best fit achieved when protons and calcium ions were assumed to bind to distinct sites on the membrane. Since light induces changes in the retinal ionic environment, these results permit us to gauge the degree to which rod light responses could be modified via alterations in I(h) activation.     

Progesterone Receptor Expression Declines in the Guinea Pig Uterus during Functional Progesterone Withdrawal and in Response to Prostaglandins

Progesterone withdrawal is essential for parturition, but the mechanism of this pivotal hormonal change is unclear in women and other mammals that give birth without a pre-labor drop in maternal progesterone levels. One possibility suggested by uterine tissue analyses and cell culture models is that progesterone receptor levels change at term decreasing the progesterone responsiveness of the myometrium, which causes progesterone withdrawal at the functional level and results in estrogen dominance enhancing uterine contractility. In this investigation we have explored whether receptor mediated functional progesterone withdrawal occurs during late pregnancy and labor in vivo. We have also determined whether prostaglandins that induce labor cause functional progesterone withdrawal by altering myometrial progesterone receptor expression. Pregnant guinea pigs were used, since this animal loses progesterone responsiveness at term and gives birth in the presence of high maternal progesterone level similarly to primates. We found that progesterone receptor mRNA and protein A and B expression decreased in the guinea pig uterus during the last third of gestation and in labor. Prostaglandin administration reduced while prostaglandin synthesis inhibitor treatment increased progesterone receptor A protein abundance. Estrogen receptor-1 protein levels remained unchanged during late gestation, in labor and after prostaglandin or prostaglandin synthesis inhibitor administration. Steroid receptor levels were higher in the non-pregnant than in the pregnant uterine horns. We conclude that the decreasing expression of both progesterone receptors A and B is a physiological mechanism of functional progesterone withdrawal in the guinea pig during late pregnancy and in labor. Further, prostaglandins administered exogenously or produced endogenously stimulate labor in part by suppressing uterine progesterone receptor A expression, which may cause functional progesterone withdrawal, promote estrogen dominance and foster myometrial contractions.