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BOR - Papers in Press, published online ahead of print January 23, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.065656
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BIOLOGY OF REPRODUCTION 78, 888–895 (2008)
DOI: 10.1095/biolreprod.107.065656
© 2008 by the Society for the Study of Reproduction, Inc.

Ovary; 

Cortisol Is a Suppressor of Apoptosis in Bovine Corpus Luteum1

Junichi Komiyama 3, Ryo Nishimura 3, Hwa-Yong Lee 3, Ryosuke Sakumoto 4, Masafumi Tetsuka 5, Tomas J. Acosta 3, Dariusz J. Skarzynski 6, and Kiyoshi Okuda 2 3

Laboratory of Reproductive Endocrinology,3 Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan Reproductive Biology Research Unit,4 National Institute of Agrobiological Sciences, Ibaraki 305-0901, Japan Department of Agricultural and Life Science,5 Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan Department of Reproductive Immunology,6 Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn 10-747, Poland

ABSTRACT

Glucocorticoid (GC) acts as a modulator of physiological functions in several organs. In the present study, we examined whether GC suppresses luteolysis in bovine corpus luteum (CL). Cortisol (an active GC) reduced the mRNA expression of caspase 8 (CASP8) and caspase 3 (CASP3) and reduced the enzymatic activity of CASP3 and cell death induced by tumor necrosis factor (TNF) and interferon gamma (IFNG) in cultured bovine luteal cells. mRNAs and proteins of GC receptor (NR3C1), 11beta-hydroxysteroid dehydrogenase type 1 (HSD11B1), and HSD11B2 were expressed in CL throughout the estrous cycle. Moreover, the protein expression and the enzymatic activity of HSD11B1 were high at the early and the midluteal stages compared to the regressed luteal stage. These results suggest that cortisol suppresses TNF-IFNG-induced apoptosis in vitro by reducing apoptosis signals via CASP8 and CASP3 in bovine CL and that the local increase in cortisol production resulting from increased HSD11B1 at the early and midluteal stages helps to maintain CL function by suppressing apoptosis of luteal cells.

apoptosis, cattle, corpus luteum, glucocorticoid, glucocorticoid receptor

INTRODUCTION

Glucocorticoid (GC), which is produced in the adrenal cortex, is involved in the regulation of a variety of physiological processes, including metabolism, immunological response, and female reproductive function [1–5]. Since ovulation is an inflammatory event characterized by increased synthesis of interleukins and prostaglandins (PGs) [6–8], increased generation of anti-inflammatory GC at ovulation helps to limit the ovarian inflammatory process [5, 9]. In sheep, stress has been shown to induce GC secretion and suppression of reproductive neuroendocrine function and ovarian cyclicity [10, 11]. Moreover, repeated administrations of a synthetic GC (betamethasone) during the luteal phase prolong luteal life span in cattle [12, 13]. Although it is possible that GC directly regulates corpus luteum (CL) function, the roles of GC in the CL remain unknown.

In the cow, luteolysis is a result of the pulsatile release of endometrial prostaglandin F2 alpha (PGF2 alpha), which initiates a complex cascade of events that finally interrupt steroidogenesis and induces structural regression of the CL [14]. During structural luteolysis, cells in the CL undergo apoptosis [15, 16]. Apoptosis occurs through two main pathways throughout FAS-induced cell death. In one pathway, high levels of CASP8 at the death-inducing signaling complex directly initiate cleavage of other downstream effector caspases, such as CASP3, thereby initiating the execution phase of apoptosis. In the other pathway, active CASP8 cleaves BID, a proapoptotic member of the BCL2 family. Cleaved BID stimulates the binding of proapoptotic members of the BCL2 family (e.g., BAX, BAK) to mitochondria and inhibits association of antiapoptotic members of the BCL2 family (e.g., BCL2). This causes leakage of cytochrome c from the mitochondria into the cytosol, which in turn promotes formation of the apoptosome and triggers activation of CASP3 [17]. GC inhibits FAS expression in bovine blood neutrophils via GC receptor (NR3C1) activation, possibly contributing to the cells' increased viability in culture [18]. Moreover, GC stimulates an increase in BCL2 protein levels and protects against serum deprivation- and forskolin-induced apoptosis in immortalized human granulosa cells [19].

The actions of GC are mediated through an intracellular receptor, NR3C1, which is a member of the nuclear receptor family of ligand-dependent transcription factors [20]. The effects of GC on target tissues are modulated by 11beta-hydroxysteroid dehydrogenase (HSD11B) [21]. Two isoforms of the enzyme have been identified. The type 1 enzyme (HSD11B1) mainly converts cortisone to cortisol (the active form), while the type 2 isoform (HSD11B2) inactivates cortisol by metabolizing it to cortisone [22]. Although HSD11B1 and HSD11B2 mRNAs are expressed in bovine CL throughout the estrous cycle [23], enzymatic activity of the proteins was observed only at the early luteal stage [24]. A better understanding of the changes of 11beta-hydroxysteroid dehydrogenases activity should help to clarify the effect of cortisol on bovine CL.

In the present study, to determine whether cortisol plays a role in regulating bovine CL function, we examined 1) the effects of cortisol on apoptosis in the cultured luteal cells and 2) the patterns of expression of NR3C1, HSD11B1, and HSD11B2 at the mRNA and protein levels and HSD11B1 activity in bovine CL throughout the estrous cycle.

MATERIALS AND METHODS

Collection of CL

Ovaries from Holstein cows were collected at a local abattoir within 10–20 min after exsanguination. The stages of the estrous cycle were identified by macroscopic observation of the ovary and uterus as described previously [25]. For mRNA determination, CL tissues were collected from cows at five different stages of the estrous cycle (early: Days 2–3; developing: Days 5–6; mid: Days 8–12; late: Days 15–17; regressed luteal stage: Days 19–21). The CL tissues were immediately separated from the ovaries, frozen rapidly in liquid nitrogen, and stored at –80°C until processed for RNA and protein isolation. For tissue culture and cell culture experiments, the ovaries with CL were submerged in ice-cold physiological saline and transported to the laboratory.

Cell Isolation

Only those CLs classified in the midluteal stage were collected for the cell culture. Luteal tissue was enzymatically dissociated, and luteal cells were cultured as described previously [26]. Dissociated luteal cells from three CLs collected from three different cows were pooled. The luteal cells were suspended in a culture medium, Dulbecco modified Eagle medium (DMEM), and Ham F-12 medium (D/F; 1:1 [vol/vol]; Sigma-Aldrich, St. Louis, MO; No. D8900) containing 5% calf serum (Life Technologies, Grand Island, NY; No. 16170–078) and 20 µg/ml gentamicin (Sigma; No. G1397). Cell viability was greater than 85% as assessed by trypan blue exclusion. The cells in the cell suspension consisted of about 70% small luteal cells, 20% large luteal cells, 10% endothelial cells or fibrocytes, and no erythrocytes. Experiments with isolated cells were performed three to four times each with separate cell preparations.

Experiment 1: Effect of Cortisol on Fas Ligand-Mediated Cell Death in the Bovine Luteal Cells

Dispersed luteal cells (2.0 x 105/ml) were cultured in 100 µl of D/F medium containing 5% calf serum in 96-well culture dishes (Iwaki, Chiba, Japan; No. 3860–096). After 18 h of culture, the medium was replaced with D/F without phenol red medium-BSA (DMEM and Ham F-12 without phenol red [D/F without phenol red; 1:1 (vol/vol); Sigma; No. D2906]) containing 0.1% BSA, 5 ng/ml sodium selenite (Sigma; No. S5261), and 5 µg/ml holo-transferrin (Sigma; No. T3400). The cells were then exposed to cortisol (hydrocortisone [10, 100, 1000 nM]; Sigma; No. H0888) in the presence or absence of TNF (2.9 nM; kindly donated by Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) and IFNG (2.5 nM; kindly donated by Dr. S. Inumaru, NIAH, Ibaraki, Japan) for 24 h. After 24 h of culture, the medium was replaced with the D/F without phenol red medium-BSA. The cells were then exposed to cortisol with or without TNF and IFNG in the presence or absence of 2.9 nM soluble recombinant human Fas ligand (FASLG; Upstate Biotechnology, Lake Placid, NY; No. 01–193) for 24 h. After the final 24 h of culture, the viability of the cells was determined by Dojindo Cell Counting Kit including WST-1 (Dojindo, Kumamoto, Japan; No. 345–06463) as described previously [27]. Briefly, WST-1, a kind of MTT [3-(4,5-dimethyl-2 thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide], is a yellow tetrazolium salt that is reduced to formazan by live cells containing active mitochondria. The culture medium was replaced with 100 µl D/F without phenol red medium-BSA, and a 10-µl aliquot (0.3% WST-1, 0.2 mM 1-methoxy PMS in PBS, pH 7.4) was added to each well. The cells were then incubated for 4 h at 38°C. The absorbance (A) was read at 450 nm using a microplate reader (Bio-Rad, Hercules, CA; Model 450). Percentage of cytotoxicity was determined by subtracting the mean A of cortisol-, TNF-, IFNG-, and/or FASLG-treated wells (Atest) from the mean A of untreated wells (Acontrol) and then dividing by the mean A of untreated wells (Acontrol). The mean A of wells in the absence of the cells was subtracted from the mean A of all experimental wells. The percent cytotoxicity was calculated as 100 x (Acontrol – Atest)/(Acontrol).

Experiment 2: Effect of Cortisol on FAS, CASP8, BCL2 Family, and CASP3 mRNA Expressions

Dispersed luteal cells were seeded at 2.0 x 105 viable cells in 1 ml of medium in 24-well culture dishes (Costar, Cambridge, MA; No. 3524). After 18 h of culture in D/F medium containing 5% CS, the medium was replaced with D/F without phenol red medium-BSA with or without cortisol (1000 nM), TNF, and IFNG. After 12 h of culture, total RNA was extracted from the cells using TRIZOL reagent (Invitrogen, Carlsbad, CA; No. 15596–026) according to the manufacturer's directions. One microgram of each total RNA was reverse transcribed using a ThermoScript RT-PCR System (Invitrogen; No. 11146–016). Gene expression was measured by real-time PCR using the MyiQ (Bio-Rad, Tokyo, Japan) and the iQ SYBR Green supermix (Bio-Rad; No. 170-8880) starting with 2 ng of reverse-transcribed total RNA as described previously [28]. Briefly, 18S rRNA 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 synthesized (Table 1) and were chosen using an online software package [29]. PCR was performed under the following conditions: 95°C for 3 min, followed by 45 cycles of 94°C for 15 sec, 55°C for 20 sec, and 72°C for 15 sec. Use of the iQ SYBR Green supermix at elevated temperatures resulted in reliable and sensitive quantification of the RT-PCR products with high linearity (Pearson correlation coefficient r > 0.99). The expression of each gene was evaluated on the basis of the 18S rRNA expression in the individual samples.


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  		TABLE 1. Primers used in real-time PCR.

Experiment 3: Effect of Cortisol on CASP3 Activity

Dispersed luteal cells were seeded at 2.0 x 105 viable cells in 100 µl of medium in 96-well culture dishes. After 18 h of culture in D/F medium containing 5% CS, the medium was replaced with D/F without phenol red medium-BSA with or without cortisol (1000 nM), TNF, and IFNG. After 48 h of culture, CASP3 activity was measured using a commercially available EnzoLyte homogeneous AMC caspase-3/7 assay kit (AnaSpec, San Jose, CA; No. 71118) according to the manufacturer's instructions.

Experiment 4: NR3C1, HSD11B1, and HSD11B2 mRNA Expressions

Total RNA was extracted from CL tissue using TRIZOL reagent according to the manufacturer's directions. Gene expression was measured by real-time PCR as described in experiment 2.

Experiment 5: NR3C1, HSD11B1, and HSD11B2 Protein Expressions

NR3C1, HSD11B1, and HSD11B2 protein levels in CL tissues collected from 20 different cows (n = 4 CLs for each of five stages) were assessed by Western blotting analysis. CL tissues were homogenized on ice in the homogenization buffer by a tissue homogenizer (Physcotron; Niti-on Inc., Chiba, Japan; NS-50), followed by filtration with a metal wire mesh (150 µm). For NR3C1 protein analysis, nuclei were isolated from the tissue homogenates by centrifugation at 600 x g for 30 min. Mitochondria were isolated from the resultant supernatant by centrifugation at 8000 x g for 30 min for HSD11B1 and HSD11B2 protein analyses. Protein concentration was determined by the method of Osnes et al. [30], using BSA as a standard. The proteins were then solubilized in SDS gel-loading buffer (10% glycerol, 1% β-mercaptoethanol [Wako Pure Chemical Industries, Ltd., Osaka, Japan; No. 137–06862], pH 6.8) and heated at 95°C for 10 min. Samples (30 µg protein) were subjected to SDS-PAGE (12%) for 1.5 h at 200 V. The separated proteins were electrophoretically transblotted to a nitrocellulose membrane (Amersham Biosciences Corp., Piscataway, NJ; No. RPN78D) for 3 h at 250 mA in transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20% methanol, pH 8.3). The membrane was washed in TBS-T (0.1% Tween 20 in TBS [25 mM Tris-HCl, pH 7.5, 137 mM NaCl]), incubated in blocking buffer (4% nonfat dry milk in TBS-T) overnight at 4°C, incubated at room temperature with a primary antibody specific to each protein (NR3C1 antibody [95 kDa; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; No. sc-1002; 1:1000, 1 h], HSD11B1 antibody [32 kDa; Cayman Chemical Co., Ann Arbor, MI; No. 10004303; 1:50, 2 h], HSD11B2 antibody [44 kDa; Cayman Chemical; No. 10004549; 1:200, 2 h], and beta-actin [ACTB] antibody [42 kDa; Sigma; No. A2228; 1:4000, 1 h]), incubated in blocking buffer for 10 min at room temperature, washed two times for 10 min in TBS-T at room temperature, incubated with secondary antibody (NR3C1, HSD11B1, and HSD11B2 [1:20 000]: anti-rabbit Ig, HRP-linked whole antibody produced in donkey, Amersham Biosciences; No. NA934; ACTB [1:40 000]: anti-mouse Ig, HRP-linked whole antibody produced in sheep, Amersham Biosciences; No. NA931) for 1 h, and washed three times in TBS for 10 min at room temperature. The signal was detected by ECL Western Blotting Detection System (Amersham Biosciences; No. RPN2109).

The intensity of the immunological reaction (NR3C1, HSD11B1, HSD11B2, and ACTB) in the tissues was estimated by measuring the optical density in the defined area by computerized densitometry using NIH Image (National Institutes of Health).

Experiment 6: HSD11B1 Activity

The level of 11beta-hydroxysteroid dehydrogenase reductase activity in CL tissue was determined by measuring the conversion rate of cortisone to cortisol as described previously [31]. Briefly, CL tissues (15–30 mg) were placed in culture glass tubes (12 x 75 mm) containing 2 ml of culture medium (D/F without phenol red) supplemented with 0.1% BSA, 0.5 mM ascorbic acid (Wako; No. 013–12061), 5 ng/ml sodium selenite, 5 µg/ml holo-transferrin, 2 µg/ml insulin (Sigma; No. I4011), and 20 µg/ml gentamicin (Sigma; No. G1397) with 5% CO2 in air. The CL tissues from three cows at the midluteal stage (n = 3) were exposed to cortisone (3, 30, 300 nM; Sigma; No. C2755) in a shaking water bath at 38°C for 2 h. For the blank value, the CL tissues were incubated for 2 h without cortisone. At the end of incubation, conditioned media were collected and frozen at –30°C until assayed for cortisol. 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 cortisol in the tissue) were subtracted and expressed as pmol cortisol converted/mg tissue. To examine HSD11B1 activity throughout the estrous cycle, the CL tissues from early, developing, mid-, late, and regressed luteal stages (n = 5 cows/stage) were exposed to cortisone (30 nM).

Cortisol Determination

The EIA for cortisol was done as described previously [32]. 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 interassay coefficients of variation were on average 3.7% and 6.9%, respectively. The cross-reactivities of the antibody (raised in a rabbit against corisol-3-CMO; Cosmo Bio Co., Tokyo, Japan; No. 13–2211) were 100% for cortisol, 5.7% for 11-deoxycortisol, 4.1% for 11-deoxycorticosterone, 1.2% for corticosterone, 0.7% for 17{alpha}-hydroxy progesterone, 0.6% for cortisone, and 0.02% for 20-dihydroxy progesterone.

Statistical Analysis

All experimental data are shown as the mean ± SEM. The statistical significance of differences in the cellular cytotoxicity and the amounts of FAS, CASP8, BCL2, BAX, CASP3, NR3C1, HSD11B1, and HSD11B2 mRNA, NR3C1, HSD11B1, and HSD11B2 protein and the enzymatic activity of CASP3 and HSD11B1 was assessed by analysis of variance (ANOVA) followed by a Fisher protected least-significant difference procedure (PLSD) as a multiple comparison test.

RESULTS

Experiment 1: Effect of Cortisol on Fas Ligand-Mediated Cell Death in the Bovine Luteal Cells

Treatment of the cells with TNF+IFNG and TNF+IFNG+FASLG significantly increased luteal cell death (killed 41% and 55%, respectively). Treatment of cells only with cortisol (10–1000 nM) did not affect luteal cell death. Cortisol (1000 nM) decreased luteal cell death 22% in the presence of TNF and IFNG and 11% in the presence of TNF, IFNG, and FASLG (Fig. 1; P < 0.05).


Figure 01
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  		FIG. 1. Effect of cortisol on bovine luteal cell death. The cells were treated with cortisol (10–1000 nM) in the presence or absence of cytokines (TNF: 2.9 nM, IFNG: 2.5 nM) for 24 h, followed by treatment with cortisol (10–1000 nM) with or without TNF and IFNG in the presence or absence of FASLG (2.9 nM) for 24 h. After the final 24 h of culture, the cell number was determined by optical density at 450 nm in a WST-1 assay. Experiments with isolated cells were performed four times each with separate cell preparations. Data are the mean ± SEM of four separate experiments. All values are expressed as a percentage of cytotoxicity in untreated control (defined in Materials and Methods). Different letters (a, b: TNF+IFNG-treated cells; X, Y: TNF+IFNG+FASLG-treated cells) indicate significant differences (P < 0.05) as determined by ANOVA followed by a Fisher PLSD as a multiple comparison test.

Experiment 2: Effect of Cortisol on FAS, CASP8, BCL2 Family, and CASP3 mRNA Expressions

Treatment of cells only with cortisol (1000 nM) did not affect the expression of FAS, CASP8, BCL2, BAX, BCL2/BAX, and CASP3 mRNA (Fig. 2). In cytokine (TNF+IFNG)-treated cells, cortisol (1000 nM) did not affect the expression of FAS (Fig. 2A), BCL2 (Fig. 2C), or BAX mRNA (Fig. 2D). Moreover, neither cytokines nor cortisol affected the ratio of BCL2 to BAX (BCL2/BAX) mRNA (Fig. 2E). However, cortisol (1000 nM) decreased the expression of CASP8 and CASP3 mRNA induced by cytokines (Fig. 2, B and F; P < 0.05).


Figure 02
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  		FIG. 2. Effect of cortisol on FAS (A), CASP8 (B), BCL2 (C), BAX (D), and CASP3 (F) mRNA expressions and the ratio of BCL2/BAX mRNA expression (E) in cultured bovine luteal cells. The cells were treated with TNF (2.9 nM) and IFNG (2.5 nM) with or without cortisol (1000 nM) for 12 h. Experiments with isolated cells were performed three times each with separate cell preparations. Data are the mean ± SEM of three separate experiments and are expressed as the relative levels to 18S rRNA levels. Different letters indicate significant differences (P < 0.05) among treatments as determined by ANOVA followed by a Fisher PLSD as a multiple comparison test.

Experiment 3: Effect of Cortisol on CASP3 Activity

TNF+IFNG increased CASP3 activity (172.6% of the control) in cultured luteal cells after 48 h of incubation (Fig. 3). Cortisol (1000 nM) decreased CASP3 activity induced by cytokines (P < 0.05).


Figure 03
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  		FIG. 3. Effect of cortisol on CASP3 activity in cultured bovine luteal cells. The cells were treated with TNF (2.9 nM) and IFNG (2.5 nM) with or without cortisol (1000 nM) for 48 h. Experiments with isolated cells were performed four times each with separate cell preparations. Data are the mean ± SEM of four separate experiments. Asterisk indicates significant differences (P < 0.05) among treatments as determined by ANOVA followed by a Fisher PLSD as a multiple comparison test.

Experiment 4: NR3C1, HSD11B1, and HSD11B2 mRNA Expressions

Specific transcripts for NR3C1, HSD11B1, and HSD11B2 were detected in bovine CL throughout the estrous cycle. A real-time PCR analysis of NR3C1, HSD11B1, and HSD11B2 mRNA in the CL tissue during the estrous cycle is shown in Figure 4. The expression of mRNA for NR3C1, HSD11B1, and HSD11B2 was greater at the developing luteal stage than at the other luteal stages (Fig. 4, A and B; P < 0.05).


Figure 04
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  		FIG. 4. Changes in relative amounts of (A) NR3C1 and (B) HSD11B1 and HSD11B2 mRNA in bovine CL throughout the estrous cycle (early, Days 2–3; developing [Dev], Days 5–6; mid, Days 8–12; late, Days 15–17; regressed luteal stages [Regress], Days 19–21). Data are the mean ± SEM for six samples/stage and are expressed as the relative ratio of NR3C1, HSD11B1, and HSD11B2 mRNA to 18S rRNA. A) Different letters indicate significant differences (P < 0.05), and B) different letters (a–d: HSD11B1; X–Z: HSD11B2) indicate significant differences (P < 0.05) as determined by ANOVA followed by a Fisher PLSD as a multiple comparison test.

Experiment 5: NR3C1, HSD11B1, and HSD11B2 Protein Expressions

NR3C1, HSD11B1, and HSD11B2 proteins were detected in bovine CL throughout the estrous cycle (Fig. 5). The expression of NR3C1 protein was greater at the developing luteal stage than at the other luteal stages (Fig. 5B; P < 0.05). Moreover, the expression of HSD11B1 protein was the greatest at the early luteal stage and decreased toward the regressed luteal stage (Fig. 5C). Significant differences were observed between the early and the regressed luteal stages or between the mid- and the regressed luteal stages (P < 0.05). The expression of HSD11B2 protein did not change throughout the estrous cycle (Fig. 5C).


Figure 05
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  		FIG. 5. Changes in relative amounts of NR3C1, HSD11B1, and HSD11B2 protein in bovine CL throughout the estrous cycle (early, Days 2–3; developing [Dev], Days 5–6; mid, Days 8–12; late, Days 15–17; regressed luteal stages [Regress], Days 19–21). Representative samples of Western blot for NR3C1, HSD11B1, HSD11B2, and ACTB are shown in upper panels (A). Data are the mean ± SEM for four samples/stage and are expressed as the relative ratio of (B) NR3C1 and (C) HSD11B1 and HSD11B2 protein to ACTB protein. Different letters indicate significant differences (P < 0.05) among luteal stages as determined by ANOVA followed by a Fisher PLSD as a multiple comparison test.

Experiment 6: HSD11B1 Activity

CL tissue had the capacity to convert cortisone to cortisol as indicated by a significant increase in cortisol content in the media, in which midluteal tissue was incubated with cortisone compared with those incubated without cortisone. The concentration of cortisol in the media increased with the dose of cortisone (Fig. 6A). The activity of HSD11B1 was greater at the early and the midluteal stages than at the developing and the regressed luteal stages (Fig. 6B; P < 0.05).


Figure 06
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  		FIG. 6. A) Cortisol accumulation by the mid-CL tissue in the presence of different levels of cortisone (mean ± SEM, n = 3). After CL tissues were treated with cortisone (0–300 nM) for 2 h, cortisol levels accumulated in the media were determined by an EIA (defined in Materials and Methods). B) Changes in HSD11B1 activity in CL tissue throughout the estrous cycle (mean ± SEM, n = 5/stage). HSD11B1 activity assessed by measuring cortisol levels in the media after CL tissues (15–30 mg) were cultured in the presence of cortisone (30 nM) for 2 h. Different letters indicate significant differences (P < 0.05) among treatments (A) or among luteal stages (B) as determined by ANOVA followed by a Fisher PLSD as a multiple comparison test. Cont, Control.

DISCUSSION

The present study showed that cortisol suppresses luteal cell apoptosis, which is known to occur during structural luteolysis. Cortisol synthesized in the adrenal cortex systemically affects many organs. The circulating levels of cortisol are relatively constant throughout the estrous cycle in cattle [33–35]. However, local cortisol concentration has been known to be modulated by HSD11B1 and HSD11B2 in different organs of several species [22]. In the present study, since mRNA expressions of HSD11B1 and HSD11B2 were higher at the developing stage compared to the other stages in bovine CL and since the enzymatic activity of HSD11B1 in bovine CL tissue was high at the early and the midluteal stages compared to the regressed stage, cortisol locally increased by HSD11B1 may play a role in maintaining CL function by suppressing apoptosis of luteal cells.

During structural luteolysis, cells of the CL undergo apoptosis [15, 16], which occurs through two main pathways throughout FAS-induced cell death [17]. In one pathway, high levels of CASP8 directly initiate cleavage of CASP3, whereas in the other pathway, active CASP8 cleaves BID, a proapoptotic member of the BCL2 family. In bovine CL, CASP3 mRNA expression increases during PGF2 alpha-induced luteolysis [36, 37]. Moreover, we have previously shown that FAS-mediated apoptosis of bovine luteal cells is associated with the action of cytokines, especially TNF and IFNG [27], which are secreted by immune cells whose numbers are greatly increased in CL at the time of luteolysis [38]. In the present study, cortisol significantly decreased luteal cell death and CASP3 mRNA expression and CASP3 activity induced by the cytokines. Thus, cortisol may exert an antiapoptotic action on bovine luteal cells by reducing both the expression and the activity of CASP3. In addition, cortisol significantly attenuated CASP8 mRNA expression but did not affect the expressions of FAS, BCL2, or BAX mRNA or the ratio of BCL2 to BAX (BCL2/BAX) mRNA. These findings suggest that cortisol inhibits apoptosis by suppressing apoptosis signals via CASP8 and CASP3 without affecting BCL2 and BAX.

In vitro experiments using a specific progesterone (P4) antagonist (onapristone) demonstrates that P4 suppresses apoptosis in bovine luteal cells by inhibition of CASP3 activity [39]. The ligand-binding domains of human P4 receptor (PGR) and NR3C1 have 55% homology at the amino acid level [40]. Since cortisol has been demonstrated not to bind to PGR in the calf uterine cytosol [41], we believe that the antiapoptotic effect of cortisol is not mediated via PGR. Moreover, cortisol slightly increased the secretion of P4 in cultured bovine granulosa cells [42], suggesting that cortisol enhances the luteoprotective (antiapoptotic) action of P4 in bovine CL. In fact, cortisol has a luteotropic role during maternal recognition of pregnancy in human CL [43], although the physiological role of cortisol in bovine CL is not known. Further studies are needed to clarify the physiological relevance of cortisol in bovine CL.

The enzymatic activity of HSD11B1 was low at the developing and the regressed luteal stages in the present study. Estrogen has been demonstrated to down-regulate the enzymatic activity of HSD11B1 in the rat kidney [44]. In cattle, intraovarian estrogen concentration is high before ovulation, declines after that, and then increases around Days 4–5 of the cycle in coincidence with the first wave of follicular growth [45–47]. Furthermore, estrogen receptors are expressed in bovine CL throughout the estrous cycle [48]. Thus, the declining enzymatic activity of HSD11B1 at the developing and the regressed luteal stages observed in the present study may be due to increased estrogen levels in the ovary. In addition to estrogen, HSD11B1 may also be activated by PGF2 alpha in human granulosa-lutein cells [49]. Since intraluteal PGF2 alpha concentration has been shown to be high for a few days after ovulation in cattle [47], this high level of PGF2 alpha may be responsible for the increasing enzymatic activity of HSD11B1 at the early CL observed in the present study. PGF2 alpha production is less in the midluteal cells than in the early luteal cells [50]. However, HSD11B1 activity was high at the midluteal stage in the present study. The increasing activity of HSD11B1 might be modulated by factor(s) other than PGF2 alpha at the midluteal stage.

In the present study, NR3C1 mRNA and protein expressions were greater at the developing luteal stage than at the other luteal stages. NR3C1 mRNA and protein expressions have been demonstrated to be up-regulated by hypoxia in human renal proximal tubular epithelial cells [51]. We have found that the protein expression of hypoxia-inducible factor 1 alpha (HIF1A), a well-known marker of hypoxia, in bovine CL is greater at the early and the developing luteal stages than at the other luteal stages (Nishimura and Okuda, unpublished results). Therefore, the high level of NR3C1 mRNA and protein expressions at the developing stage observed in the present study could be due to hypoxia. Furthermore, cytokines produced by immune cells, such as TNF, interleukin 1 (IL1), IL2, and IL4, have been shown to increase NR3C1 mRNA and protein expressions in human HeLaS3, CEMC7, and peripheral blood mononuclear cells [52, 53]. Since the number of immune cells in bovine CL increases between Days 1 and 5 of the estrous cycle [38], cytokines produced by immune cells may enhance NR3C1 expression during luteal development. However, the same study also demonstrated that a high number of macrophages are present between Days 19 and 21 [38], whereas NR3C1 expression was lower at the regressed luteal stage than at the developing luteal stage in the present study. The reason that the NR3C1 expression decreased in spite of the increase in the number of macrophages at the regressed luteal stage is unclear. Populations of immune cells other than macrophages change between the early and the regressed luteal stages [38]. Therefore, it is possible that local concentrations of cytokines in bovine CL are different between the early and the regressed luteal stages. Moreover, since the functions of cytokines depend on their concentrations [54], the reason that the NR3C1 expression decreased in spite of the increase in the number of macrophages at the regressed luteal stage may be due to different local concentrations of cytokines. Further studies are needed to clarify the regulation of NR3C1 expression by cytokines in bovine CL.

The overall findings suggest that cortisol locally increased by HSD11B1 in bovine CL suppresses apoptosis of luteal cells by reducing apoptosis signals via CASP8 and CASP3 in order to maintain CL function at the early and the midluteal stages.

FOOTNOTES

1Supported by a Grant-in-Aid for Scientific Research (No. 18380166) of the Japan Society for the Promotion of Science (JSPS). R.N. was supported by a JSPS Research Fellowship (No. 03589). H.-Y.L. is supported by a scholarship from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Back

Correspondence: 2FAX: 81 86 251 8333; e-mail: kokuda{at}cc.okayama-u.ac.jp

Received: 19 September 2007.

First decision: 20 October 2007.

Accepted: 11 January 2008.

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