Abstract
To determine whether glucocorticoids (GCs) play a role in regulating uterine function in cow, the present study examined the expression of mRNA encoding GC receptor (GC-R) α, 11β-hydroxysteroid dehydrogenase (11-HSD) type 1 and type 2, and the activity of 11-HSD1 in bovine endometrial tissue throughout the estrous cycle. We also studied the effects of cortisol on basal, oxytocin (OT)- and tumor necrosis factor-α (TNFα)-stimulated prostaglandin (PG) production. A quantitative real-time PCR analysis revealed that GC-Rα mRNA was expressed more strongly in the mid-luteal stage (days 8–12) than in the other stages. In contrast to GC-Rα mRNA expression, 11-HSD1 mRNA expression was greater in the follicular stage than in the other stages, whereas 11-HSD2 mRNA expression was lowest in the follicular stage. The activity of 11-HSD1 was greater in the follicular stage and estrus than in the other stages and was lowest in the mid-luteal stage. Cortisone was dose-dependently converted to cortisol in the cultured endometrial tissue. Although cortisol did not affect either the basal or OT-stimulated production of PGs in the cultured epithelial cells, the production of PGs stimulated by TNFα in the stromal cells was suppressed by cortisol (P < 0.05). Cortisol suppressed basal prostaglandin (PG)F2α without affecting basal PGE2 production in the stromal cells. The overall results suggest that the level of cortisol is locally regulated in bovine endometrium throughout the estrous cycle by 11-HSD1, and that cortisol could act as a luteoprotective factor by selectively suppressing luteolytic PGF2α production in bovine endometrium.
Introduction
Glucocorticoids (GCs) are involved in various physiological processes, including general metabolism (Wang 2005), immunological response (McKay & Cidlowski 1998, 1999), and female reproductive function (Brann & Mahesh 1991, Andersen 2002). At high concentrations, GCs suppress most immunological responses and are well known as anti-inflammatory agents limiting the production of cytokines and prostaglandins (PGs) in various target organs (Goppelt-Struebe et al. 1996, Goppelt-Struebe 1997, Hillier & Tetsuka 1998). Several uterine events such as menstruation, implantation, and parturition have been likened to an inflammatory process (Salamonsen & Lathbury 2000). Because GCs have been shown to exert specific effects on uterine physiology in rats (Rabin et al. 1990, Korgun et al. 2003), rabbits (Bigsby & Everett 1991), humans (Gellersen et al. 1994), and ewes (Monheit & Resnik 1981, Gupta et al. 2003), the uterus is considered to be a target organ for GCs in some species. However, the roles of GCs in the bovine endometrium remain unknown.
The endometrium is a complex tissue and mainly consists of epithelial and stromal cells (Fortier et al. 1988). Although both types of endometrial cells have the capacity to produce PGs, they have specific morphological and physiological properties (Asselin et al. 1997, Fortier et al. 1988, Miyamoto et al. 2000). We found that oxytocin (OT) stimulates PG production only in epithelial cells, while tumor necrosis factor-α (TNFα) stimulates PG production only in stromal cells (Skarzynski et al. 2000). This cell-specific response to OT and TNFα is a useful parameter for investigating the physiology and endocrine status of cultured bovine endometrial cells.
The biological action of GCs is mediated through the activation of intracellular GC receptors (GC-R). Two isoforms of GC-R, GC-Rα and GC-Rβ, have been identified (Giguere et al. 1986, Funder 1993). Access of GCs to GC-R in target tissues is regulated by two 11β-hydroxysteroid dehydrogenases (11-HSDs), a bidirectional 11-HSD type 1 (11-HSD1) that mainly converts cortisone to active cortisol (Stewart & Mason 1995) and 11-HSD type 2 (11-HSD2) that inactivates cortisol to cortisone (Albiston et al. 1994, Stewart et al. 1994). Therefore, cyclic changes of the expressions of GC-R and 11-HSDs mRNA could help to define the roles of GCs in uterine physiology.
In the present study, to determine whether GCs play a role in regulating bovine uterine function, we examined 1) the temporal patterns of GC-Rα, 11-HSD1, and 11-HSD2 mRNA expressions, and 11-HSD1 activity in bovine endometrium throughout the estrous cycle and 2) the effects of cortisol on basal and OT- or TNFα-stimulated PG production in the cultured endometrial epithelial and stromal cells.
Materials and methods
Collection of endometrial tissues
Uteri of Holstein cows were obtained from a local abattoir in accordance with protocols approved by the local Institutional Animal Care and Use Committee. Apparently, healthy uteri without a visible conceptus were obtained within 10–20 min after exsanguination and immediately transported to the laboratory on ice. The stages of the estrous cycle were determined by macroscopic observation of the ovary and uterus as described previously (Okuda et al. 1988, Miyamoto et al. 2000). For mRNA determination, endometrial tissues (n = 8/stage) were collected from cows at six different stages of the estrous cycle (estrus, day 0; early luteal, days 2–3; developing luteal, days 5–6; mid-luteal, days 8–12; late luteal, days 15–17, and follicular stage, days 19–21). Intercaruncular endometrial tissues from the uterine horn, ipsilateral to the corpus luteum, were used for all experiments. The endometrial tissues were immediately frozen rapidly in liquid nitrogen and stored at −80 °C until processed for RNA isolation. For experiments involving tissue and cell cultures, the uterus was submerged in ice-cold physiological saline and transported to the laboratory.
Experiment 1: determination of GC-Rα, 11-HSDs mRNA expressions and 11-HSD1 activity throughout the estrous cycle
Reverse transcription and real-time PCR
Total RNA was extracted from endometrial tissue using TRIZOL reagent (Invitrogen) according to the manufacturer’s directions. One microgram of each total RNA was reverse transcribed using a ThermoScript RT-PCR System (Invitrogen) and 10% of the reaction mixture was used in each PCR using specific primers for GC-Rα and 11-HSDs from the bovine sequence. The primers were chosen using an online software package (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi).
Gene expression was measured by real-time PCR using the Mx3000P QPCR System (Stratagene, La Jolla, CA, USA) and the QuantiTect SYBR Green PCR system (Qiagen) starting with 2 ng reverse-transcribed total RNA as described previously (Sakumoto et al. 2005; Table 1). Briefly, GAPDH expression was used as an internal control. For quantification of the mRNA expression levels, the primer length (20 bp) and GC contents of each primer (50–60%) were selected, and PCR was performed under the following conditions: 95 °C for 15 min, followed by 55 cycles of 94 °C for 15 s, 55 °C for 20 s, and 72 °C for 15 s. Use of the QuantiTect SYBR Green PCR system at elevated temperatures resulted in reliable and sensitive quantification of the RT-PCR products with high linearity (Pearson’s correlation coefficient (r > 0.99)).
11-HSD1 activity
The level of 11-HSD reductase activity in endometrial tissue was determined by measuring the net conversion rate from cortisone to cortisol. Briefly, endometrial strips (30 mg) were placed in glass culture tubes (12 mm × 75 mm) containing 2 ml culture medium (Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12; 1:1 (v/v); Sigma) supplemented with 0.1% (w/v) BSA, 100 IU/ml penicillin, and 100 μg/ml streptomycin with 5% CO2 in air. The endometrial tissues were exposed to cortisone (0, 3, 30, 300, and 1000 nM) in a shaking water bath at 38 °C for 4 h. Media containing cortisone (0, 3, 30, 300, and 1000 nM) without tissues were incubated for 4 h for the blank value and to determine non-specific interconversion. At the end of incubation, 1 ml conditioned medium was collected and frozen at −30 °C until the cortisol assay. The tissues were blotted on filter paper and weighed. The specific conversion rate from cortisone to cortisol was calculated, and the blank values (defined as the amount of conversion in the absence of tissue) were subtracted and expressed as picogram of cortisol converted per milligram of tissue (pg/mg tissue). To examine the cyclic changes in 11-HSD1 activity throughout the estrous cycle, the endometrial tissues from the estrus, early luteal, developing luteal, mid-luteal, late luteal, and follicular stages (n = 4/stage) were exposed to cortisone (30 nM).
Isolation of endometrial cells
For cell culture, endometrial tissues were obtained at the early luteal phase (days 2–5). The epithelial and stromal cells from the bovine endometrium were separated using a modification of procedures described previously (Skarzynski et al. 2000). A polyvinyl catheter was inserted into the side of the oviduct and the ends of the horn were tied in order to retain trypsin solution for detaching the epithelial cells as described below. The uterine lumen was washed thrice with 30–50 ml sterile Ca2+- and Mg2+-free Hanks’ balanced salt solution (HBSS) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.1% (w/v) BSA (Roche Diagnostics). Thirty to fifty milliliters of sterile HBSS containing 0.3% (w/v) trypsin (Sigma) was then infused into the uterine lumen through the catheter. Epithelial cells were isolated by incubation at 38 °C for 60 min with gentle shaking.
After collection of the epithelial cells, the uterine lumen was washed with sterile HBSS supplemented with antibiotics and 0.1% (w/v) BSA. The horn was then cut transversely with scissors into several segments, which were slit to expose the endometrial surface. Intercaruncular endometrial strips were dissected from the myometrial layer with a scalpel and washed once in 50 ml sterile HBSS containing antibiotics. The endometrial strips were then minced into small pieces (1 mm3). The minced tissues ( = 5 g) were digested by stirring for 60 min in 50 ml sterile HBSS containing 0.05% (w/v) collagenase (Sigma), 0.005% (w/v) DNase I (Sigma) and 0.1% (w/v) BSA. The dissociated cells were filtered through metal meshes (100 and 80 μm) to remove undissociated tissue fragments. The filtrate was washed thrice by centrifugation (10 min at 100 g) with DMEM (Sigma) supplemented with antibiotics and 0.1% (w/v) BSA. After the washes, the cells were counted with a hemocytometer. Cell viability was higher than 85% as assessed by 0.5% (w/v) trypan blue dye exclusion.
Culture of endometrial cells
The final pellets of the isolated stromal or epithelial cells were resuspended in culture medium (DMEM/Ham’s F-12; 1:1 (v/v); Sigma) supplemented with 10% (v/v) calf serum (Sigma), 20 μg/ml gentamicin (Invitrogen), and 2 μg/ml amphotericin B (Sigma; Skarzynski et al. 2000). The stromal cells were seeded at a density of 1 × 105 viable cells/ml in 48-well cluster dishes (Costar, Cambridge, MA, USA), and the epithelial cells were seeded at a density of 1 × 105 viable cells/ml in culture flasks (Nunc) and cultured at 38 °C in a humidified atmosphere of 5% CO2 in air. To purify the stromal preparation, the medium was changed 2 h after plating, by which time selective attachment of stromal cells had occurred (Fortier et al. 1988, Skarzynski et al. 2000). Alternatively, since the epithelial cells attached 24–48 h after plating, the medium in the epithelial cell culture was replaced 48 h after plating. The medium was changed every 2 days until the cells reached confluence. When the epithelial cells were confluent, 0.02% (w/v) trypsin solution was added to the cells to remove the other cells. After removal of the other cells, 0.25% (w/v) trypsin solution was then added to the epithelial cells to collect the pure epithelial cells. The cells were removed, adjusted to a density of 1 × 105 cells/ml, and placed in 48-well cluster dishes for DNA quantification in fresh DMEM/Ham’s F-12 supplemented with 10% (v/v) calf serum, 20 μg/ml gentamicin, and 2 mg/ml amphotericin B until the cells reached confluence. The homogeneity of stromal and epithelial cells was evaluated using immunofluorescent staining for specific markers of epithelial (cytokeratin) and stromal cells (vimentin; Murakami et al. 2003) as described previously. The epithelial cell contamination of stromal cells was about 1% and stromal cell contamination of epithelial cells was <1%. When cells of each type were confluent (6–7 days after the start of the culture), the medium was replaced with fresh DMEM/Ham’s F-12 supplemented with 0.1% (w/v) BSA, 5 ng/ml sodium selenite (Sigma), 0.5 mM ascorbic acid (Wako Pure Chemical Industries, Ltd, Osaka, Japan), 5 μg/ml transferrin (Sigma), 2 μg/ml insulin (Sigma), and 20 μg/ml gentamicin. The cells were then exposed to various stimulants for Experiment 2.
Experiment 2: effect of cortisol on basal and OT- or TNFα-stimulated PGF2α and PGE2 production by epithelial and stromal cells
Epithelial cells were exposed to cortisol (0.1–100 nM), OT (100 nM; Teikoku Hormone MFG Co., Tokyo, Japan), or cortisol in combination with OT for 24 h. Stromal cells were exposed to cortisol (0.1–100 nM), TNFα (0.06 nM; Dainippon Pharmaceutical Co., Ltd, Osaka, Japan), or cortisol in combination with TNFα for 24 h. The concentrations of OT and TNFα were based on a previous study (Skarzynski et al. 2000). Media with supplements without stimulants incubated with cells were used as controls.
After culture, the conditioned media were collected in 1.5 ml tubes containing 5 μl of a stabilizer solution (0.3 M EDTA, 1% (w/v) acid acetyl salicylic, pH 7.3) and frozen at −30 °C until the PGs assay. The DNA content, estimated by the spectrophotometric method of Labarca & Paigen (1980), was used to standardize the results.
PG and cortisol determination
The concentrations of PGF2α and PGE2 in the culture medium were determined by enzyme immunoassay (EIA) as described previously (Woclawek-Potocka et al. 2004). The PGF2α standard curve ranged from 0.016 to 4 ng/ml and the ED50 of the assay was 0.25 ng/ml. The intra- and inter-assay coefficients of variation were on average 2.8 and 7.7% respectively. The PGE2 standard curve ranged from 0.39 to 100 ng/ml and the ED50 of the assay was 6.25 ng/ml. The intra- and inter-assay coefficients of variation were on average 3.1 and 8.6% respectively. The EIA for cortisol was done as described previously (Acosta et al. 2002). The standard curve ranged from 0.1 to 400 ng/ml and the ED50 of the assay was 1.6 ng/ml. The intra- and inter-assay coefficients of variation were on average 5.4 and 6.0% respectively. The cross-reactivities of the polyclonal antibody (raised in a rabbit against cortisol-3-carboxymethyloxime (CMO); Cosmo Bio Co., Tokyo, Japan) were 100% for cortisol, 0.6% for cortisone, 5.7% for 11-deoxycortisol, 0.5% for 21-deoxycortisol, 4.1% for 11-deoxycorticosterone, 1.2% for corticosterone, 0.7% for 17α-hydroxy progesterone, and 0.02% for 20-dihydroxy progesterone.
Statistical analysis
Experimental data are shown as the mean ± s.e.m. of values obtained in four to five separate experiments, each performed in triplicate. Endometrial cells and tissues collected from different cows were cultured separately. Data on the effects of cortisol, TNFα, and OT on absolute concentrations of PGF2α and PGE2 were statistically analyzed and are shown as a fold change of the control. The statistical significance of differences in concentrations of PG in culture media between the control and treated groups and the mRNA expressions was assessed by one-way ANOVA followed by Fisher’s protected least-significant difference procedure (PLSD) as a multiple comparison test by StatView (Version 4.58; Abacus Concepts, Inc. Berkeley, CA, USA).
Results
mRNAs for GC-Rα, 11-HSD1, and 11-HSD2 during the estrous cycle
Specific transcripts for GC-Rα, 11-HSD1, and 11-HSD2 were detected in bovine endometrium throughout the estrous cycle. A real-time PCR analysis of GC-Rα, 11-HSD1, and 11-HSD2 mRNA in the endometrial tissue during the estrous cycle is shown in Fig. 1. The level of mRNA for GC-Rα was greater in the mid-luteal stage (days 8–12) than in the other stages (Fig. 1A; P < 0.05). In contrast, the level of mRNA for 11-HSD1 was greater in the follicular stage than in the other stages (Fig. 1B; P < 0.05), whereas the level of mRNA for 11-HSD2 was lowest in the follicular stage (Fig. 1C; P < 0.05).
11-HSD1 activity throughout the estrous cycle
Endometrial tissue has the capacity to convert cortisone to cortisol as indicated by a significant increase in cortisol content in the medium incubated with tissue compared with those incubated without tissue. The concentration of converted cortisol in the media increased with the dose of cortisone (Fig. 2A). The activity of 11-HSD1 was lowest in the mid-luteal stage and greater in the follicular stage and estrus than in the other stages (Fig. 2B). It was shown that maximal cortisol concentration was reached at a cortisone concentration of 300 nM.
Effect of cortisol on basal and OT-stimulated PGs production in epithelial cells
Oxytocin significantly increased both PGF2α and PGE2 production (P < 0.05) compared with the basal level. Cortisol (0.1–100 nM) did not affect basal or OT-stimulated production of PGF2α or PGE2 by epithelial cells (Fig. 3A and B).
Effect of cortisol on basal and TNFα-stimulated PGs production in stromal cells
Cortisol decreased basal production of PGF2α from stromal cells at concentrations of 10 and 100 nM, but did not affect PGE2 production (Fig. 4A and B). TNFα stimulated both PGF2α and PGE2 production (P < 0.05). The production of TNFα-stimulated PGF2α and PGE2 was inhibited by cortisol in a dose-dependent manner.
Discussion
The present study demonstrated for the first time the temporal pattern of GC-Rα, 11-HSD1, and 11-HSD2 mRNA expression in the bovine endometrium throughout the estrous cycle. In addition, cortisol inhibited basal and TNFα-stimulated PGF2α production without affecting basal PGE2 production in the cultured stromal cells, whereas it did not affect basal or OT-stimulated PG output in the epithelial cells. These findings suggest that GCs play a role in regulating PG production in bovine endometrial stromal cells.
Cortisol, mainly synthesized in the adrenal cortex, reaches the target organs in one of two forms. The majority is bound to plasma proteins and only a small fraction is free and unbound. The steroid-binding proteins reduce alterations in the levels of biologically active free cortisol, maintaining its level relatively constant (Munck et al. 1984, Escher et al. 1997). The biological activity of cortisol seems to be confined to the free unbound fraction, which is available for movement out of capillaries and into cells, where it may initiate a biological response (Hryb et al. 1990). The biological action of GC is mediated through intracellular GC-R. Two isoforms of GC-R (GC-Rα and GC-Rβ), which originate from the same gene by alternative splicing of the GC-R primary transcript, have been identified (Hollenberg et al. 1985, Encio & Detera-Wadleigh 1991, Oakley et al. 1996). Since the ligand-dependent GC-Rα stimulates gene transcription in GC target tissues, GC-Rα is thought to be the active receptor isoform (Hollenberg et al. 1985). The levels of GC-Rα mRNA data obtained in the present study were inversely correlated with the levels of PGF2α output by bovine endometrial tissue that we found in our previous studies (Miyamoto et al. 2000, Murakami et al. 2001). Plasma concentrations of cortisol are low during the luteal phase (days 7–16; McCann & Hansel 1986). Therefore, the differential expression of GC-Rα during the estrous cycle may be important for GC actions controlling endometrial PG production. Since cortisol inhibited basal and TNFα-stimulated PGF2α production in the stromal cells in the present study, an increase of GC-Rα may be responsible for the low endometrial PGF2α production during the mid-luteal phase. Cortisol may down-regulate its own receptor to prevent an exaggerated response to cortisol, when cortisol is abundant in the stromal cells. It is also possible that the low PG production in the mid-luteal phase is due to other mechanisms, such as the down-regulation of oxytocin receptor by progesterone, the availability of arachidonic acid, or a decrease in the expression or activity of PGHS. Since PGF2α is synthesized from PGE2 by 9K-PGR, or from PGD2 or PGH2 by PGFS (Asselin & Fortier 2000, Madore et al. 2003), GC may also decrease the expression or activity of the enzymes in bovine endometrium. Further studies on mRNA, protein expressions or activities of the above enzymes are necessary.
In the present study, the profile of 11-HSD1 mRNA expression during the estrous cycle contrasted with that of 11-HSD2. 11-HSD1 mRNA remained low during the estrus, early, developing, mid-, and late luteal phases, and markedly increased in the follicular phase, whereas the expression of 11-HSD2 mRNA was at the lowest level in the follicular phase. The change in 11-HSD1 activity in bovine endometrial tissue throughout the estrous cycle has not been previously reported. The increase in 11-HSD1 mRNA was temporally coincident with the increase in the basal release of PGF2α during the estrous cycle (Miyamoto et al. 2000, Murakami et al. 2001). PGF2α has been demonstrated to stimulate 11-HSD1 activity in human chorionic trophoblasts to generate biologically active cortisol (Alfaidy et al. 2001). Therefore, it is possible that the increased PGF2α production by the endometrium in the late luteal and the follicular stages stimulates 11-HSD1 activity. The increased 11-HSD1 activity may then enhance the conversion of cortisone to cortisol in the bovine endometrium to reduce PGF2α production in the following stage of the estrous cycle. In fact, the PGF2α concentration in the ovarian–uterine venous plasma is high before the luteinizing hormone (LH) surge and drops as the LH surge approaches (Acosta et al. 2000). The decrease in PGF2α concentration in the follicular phase observed in our previous studies (Miyamoto et al. 2000, Murakami et al. 2001) was temporally associated with the highest 11-HSD1 mRNA expression and activity in the follicular stage. Thus, cortisol may play a physiologically relevant role in preventing excessive uterine PG production during the follicular phase. Furthermore, 11-HSD1 and 11-HSD2 may be directly involved in the cyclic changes in cortisol action to control endometrial PG production. However, since the cellular levels of enzyme cofactors such as NADP+ and NADPH have also been demonstrated to influence the activities of 11-HSDs (Michael et al. 2003), conversion of cortisone to cortisol in the present study may be influenced by the levels of enzyme cofactors such as NADP+ and NADPH in the bovine endometrium. Further studies are needed to clarify the role of NADP+ and NADPH during the estrous cycle.
In ruminants, PGF2α originating from the endometrium is responsible for luteolysis (McCracken et al. 1999), whereas PGE2 is thought to exert actions opposite to those of PGF2α, i.e. luteoprotective actions, for establishing pregnancy (Pratt et al. 1977, Magness et al. 1981). Furthermore, TNFα has been demonstrated to affect the length of the estrous cycle through controlling uterine PG production in the cow (Skarzynski et al. 2003). A 30-min infusion of 1 μg TNFα into the posterior aorta abdominalis on day 14 induced luteolysis and shortened the estrous cycle in cattle (Skarzynski et al. 2003), whereas 10 μg TNFα extended the estrous cycle. The changes in the length of the estrous cycle may have resulted from a preferential stimulation of PGF2α by a low dose of TNFα and a preferential stimulation of PGE2 by a high dose of TNFα. In the present study, cortisol inhibited TNFα-stimulated PGE2 and PGF2α production in a dose-dependent manner. In addition, cortisol inhibited basal PGF2α, whereas it did not affect basal PGE2 production in the stromal cells. These findings strongly suggest that cortisol mainly acts as an antiluteolytic factor suppressing basal and TNFα-stimulated PGF2α production in bovine endometrial stromal cells. Furthermore, the fact that cortisol did not affect basal and OT-stimulated PG production in epithelial cells provides direct evidence for a cell type-specific modulatory action of cortisol on PG production in stromal cells. Since the endometrium apparently consists of many more stromal cells than epithelial cells, the cortisol-inhibited PGF2α production by stromal cells could be of physiological relevance inhibiting the initiation of luteolysis.
In conclusion, the overall results suggest that the level of cortisol is locally regulated in non-pregnant bovine endometrium by 11-HSD1, and lead us to hypothesize that cortisol mainly acts as a luteoprotective factor by suppressing luteolytic PGF2α production in bovine endometrium.
Primers for real-time PCR
| Primer | Sequence | Accession no. | Product (bp) | |
|---|---|---|---|---|
| Gene | ||||
| GC-Rα | Forward | 5′-CCATTTCTGTTCACGGTGTG-3′ | AY238475 | 132 |
| Reverse | 5′-CTGAACCGACAGGAATTGGT-3′ | |||
| 11-HSD1 | Forward | 5′-ACTCTGCGCCAAGATGAAGT-3′ | AF548027 | 149 |
| Reverse | 5′-TAGCCCTCAGGAAGTGCCTA-3′ | |||
| 11-HSD2 | Forward | 5′-CCTAGACCGGATCCTTCTCC-3′ | AF074706 | 114 |
| Reverse | 5′-ACCTTGGGGGTCAGAATACC-3′ | |||
| GAPDH | Forward | 5′-CACCCTCAAGATTGTCAGCA-3′ | BC102589 | 103 |
| Reverse | 5′-GGTCATAAGTCCCTCCACGA-3′ | |||
Changes in relative amounts of (A) GC-Rα, (B) 11-HSD1, and (C) 11-HSD2 mRNA in bovine endometrium throughout the estrous cycle. Data are the mean ± s.e.m. for eight samples/stage and are expressed as the relative ratio of GC-Rα and 11-HSDs mRNA to GAPDH mRNA (estrus, day 0; early luteal, days 2–3; developing luteal, days 5–6; mid-luteal, days 8–12; late luteal, days 15–17; and follicular stage, days 19–21). Different superscript letters indicate significant difference (P < 0.05), as determined by ANOVA followed by a Fisher’s PLSD as a multiple comparison test.
Citation: Journal of Endocrinology 193, 1; 10.1677/joe.1.06975
(A) Dose-dependent effects of cortisone on 11-HSD1 activity by endometrial tissue from the follicular stage. (B) Endometrial tissues were exposed to cortisone (0–1000 nM) for 4 h. Changes in 11-HSD1 activity in endometrial tissue throughout the estrous cycle (mean ± s.e.m., n = 4/stage). 11-HSD1 activity was determined using cortisone as substrate; endometrial tissues (30 mg) were exposed to cortisone (30 nM) for 4 h. Different superscript letters indicate significant difference (P < 0.05), as determined by ANOVA followed by a Fisher’s PLSD as a multiple comparison test.
Citation: Journal of Endocrinology 193, 1; 10.1677/joe.1.06975
Effects of cortisol on basal or OT-stimulated (A) PGF2α and (B) PGE2 production by cultured bovine epithelial cells (mean ± s.e.m., n = 5 experiments). Cortisol (0.1–100 nM) with or without OT (100 nM) was added 24 h before the end of culture. The concentrations of PGF2α and PGE2 in untreated controls were used to calculate the baseline. All values are expressed as the mean fold change of a percentage of the baseline. The concentrations of PGF2α and PGE2 in the control were 27.54 ± 9.34 pg/μg DNA and 1.03 ± 0.54 ng/μg DNA in epithelial cells respectively. Different superscript letters indicate significant difference (P < 0.05), as determined by ANOVA followed by a Fisher’s PLSD as a multiple comparison test. Cont, control.
Citation: Journal of Endocrinology 193, 1; 10.1677/joe.1.06975
Effects of cortisol on basal or TNFα-stimulated (A) PGF2α and (B) PGE2 production by cultured bovine stromal cells (mean ± s.e.m., n = 5 experiments). Cortisol (0.01–100 nM) with or without TNFα (0.06 nM) was added 24 h before the end of culture. The concentrations of PGF2α and PGE2 in untreated controls were used to calculate the baseline. All values are expressed as a percentage of the baseline. The concentrations of PGF2α and PGE2 in the control were 77.71 ± 19.09 pg/μg DNA and 3.48 ± 1.36 ng/μg DNA in stromal cells respectively. Different superscript letters indicate significant difference (P < 0.05), as determined by ANOVA followed by a Fisher’s PLSD as a multiple comparison test. Cont, control.
Citation: Journal of Endocrinology 193, 1; 10.1677/joe.1.06975
This work was supported by a Grant-in-Aid for Scientific (B) no. 18380166 of the Japan Society for the Promotion of Science (JSPS). H-Y Lee is supported by a scholarship from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank Dr S Ito of Kansai Medical University, Osaka, Japan, for antisera of PGF2α and PGE2, Teikoku Hormone MFG Co. (Tokyo, Japan) for synthetic OT, and Dainippon Pharmaceutical Co. Ltd for recombinant human TNFα. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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