help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2005-1611
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
147/9/4273    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nishimura, R.
Right arrow Articles by Okuda, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nishimura, R.
Right arrow Articles by Okuda, K.
Endocrinology Vol. 147, No. 9 4273-4280
Copyright © 2006 by The Endocrine Society

Oxygen Concentration Is an Important Factor for Modulating Progesterone Synthesis in Bovine Corpus Luteum

Ryo Nishimura, Ryosuke Sakumoto, Yuko Tatsukawa, Tomas J. Acosta and Kiyoshi Okuda

Laboratory of Reproductive Endocrinology (R.N., Y.T., T.J.A., K.O.), Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan; and Department of Developmental Biology (R.S.), National Institute of Agrobiological Sciences, Ibaraki 305-0901, Japan

Address all correspondence and requests for reprints to: Dr. Kiyoshi Okuda, Laboratory of Reproductive Endocrinology, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan. E-mail: kokuda{at}cc.okayama-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Oxygen deficiency caused by a decrease in the blood supply is known to induce various responses of cells. Because luteal blood flow has been shown to decrease during luteolysis, a low-oxygen condition seems to be an integral part of the environment during luteolysis. To determine whether a low-oxygen condition is associated with functional luteolysis, we examined the influence of reduced oxygen tension on the luteal progesterone (P4) generating system in cultured bovine midluteal cells. Luteal cells obtained from midcycle corpus luteum (d 8–12) were incubated under different O2 concentrations (20, 10, 5, 3% O2) with or without LH for 24 h. P4 production decreased with decreasing O2 concentration but was significantly stimulated by LH regardless of O2 concentration. After 8 h of culture, both basal and LH-stimulated P4 production was significantly lower under 3% O2 than under 20% O2. Low-oxygen condition also inhibited pregnenolone production. Cytochrome P450 side-chain cleavage enzyme (P450scc) mRNA expression, measured by quantitative PCR, decreased under low-oxygen condition in both non-LH-treated and LH-treated cells. Low-oxygen condition did not affect the expressions of steroidogenic acute regulatory protein mRNA or protein, whereas steroidogenic acute regulatory protein mRNA expression was stimulated by LH during 4 h of culture. Low-oxygen condition also did not affect 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4 isomerase mRNA expression or the activity of the enzyme in the cells, regardless of the incubation period. The overall results indicate that a low-oxygen condition decreases P4 synthesis by attenuating P450scc production and P450scc activity in bovine luteal cells and suggest that oxygen deficiency is an essential condition for the progression of luteolysis in cattle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MAMMALS, LUTEOLYSIS consists of two phases, functional luteolysis and structural luteolysis (1). A rapid functional regression of corpus luteum (CL) is characterized by a decrease of progesterone (P4) production, followed by a phase of structural regression (1). In the cow, luteolysis is initiated by prostaglandin (PG)F2{alpha} released from the uterus at the late luteal stage or by an exogenous injection of PGF2{alpha} given at the midluteal stage. A decrease in luteal blood flow begins in parallel with systemic P4 concentrations during both spontaneous and PGF2{alpha}-induced luteolysis (2, 3, 4, 5, 6). Furthermore, the oxygen content in the blood collected from the ovarian vein began to decrease at the late luteal stage (6). Recently, blood flow within the CL during PGF2{alpha}-induced luteolysis has been shown to decrease significantly 8 h after PGF2{alpha} injection (7). The above findings suggest that a low oxygen condition is an integral part of the environment during luteolysis and that oxygen concentration is closely associated with P4 production.

The CL is the major site of P4 production during the luteal phase of the estrous cycle in cattle. Cholesterol mobilization is considered to be the rate-limiting step in the acute response of steroidogenic cells to tropic hormone stimulation (8). Steroidogenic acute regulatory protein (StAR) is known to transport cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane (9, 10, 11). Cytochrome P450 side-chain cleavage enzyme (P450scc) converts cholesterol into pregnenolone, and 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4 isomerase (3ß-HSD) converts pregnenolone into P4. Thus, P450scc and 3ß-HSD are recognized as the key enzymes in P4 biosynthesis (12, 13). Previous studies showed that StAR expression at both the mRNA and protein levels in the CL were lowest in the regressed stage (14) and that enzymatic activity and mRNA expression for both P450scc (14, 15, 16) and 3ß-HSD (17) were lower in the regressed stage than the developing stage in bovine CL. Based on these findings, we hypothesized that oxygen deficiency prevents P4 production during luteolysis by modifying StAR, P450scc, and 3ß-HSD expression and/or their activities.

In the present study, to determine the influence of oxygen deficiency on luteal function, we investigated the effects of reduced oxygen tension on P4 production and mRNA expressions of key steroidogenic factors (StAR, P450scc, and 3ß-HSD). In addition, we examined the effects of reduced oxygen tension on StAR protein expression and the enzymatic activity of P450scc and 3ß-HSD in cultured bovine luteal cells. Furthermore, to test whether a low oxygen condition used in the present study is hypoxic, we determined the expression of hypoxia-inducible factor-1{alpha} (Hif-1{alpha}) protein because Hif-1{alpha} is known to accumulate in cells and function specifically under hypoxic conditions (18, 19).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Collection of CL
Ovaries with a midstage CL from Holstein cows were collected at a local abattoir within 10–20 min after exsanguination. Midstage CLs were identified by macroscopic observation of the ovary and uterus as described previously (20). For cell culture experiments, the ovaries with CL were submerged in ice-cold physiological saline and transported to the laboratory.

Cell isolation
Luteal tissue was enzymatically dissociated and luteal cells were cultured as described previously (21). The luteal cells were suspended in a culture medium, DMEM, and Ham’s F-12 medium [1:1 (vol/vol); Sigma-Aldrich, Inc., St. Louis, MO; no. D8900] containing 5% calf serum (Life Technologies, Inc., Grand Island, NY; no. 16170-078) and 20 µg/ml gentamicin (Life Technologies; no. 15750-060). 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.

Cell culture
The dispersed luteal cells were seeded at 2.0 x 105 viable cells in 1 ml in 24-well cluster dishes (Costar, Cambridge, MA; no. 3524) for experiments 1, 3, and 5, or in a 80 cm2 culture flask (Greiner Bio-One, Frickenhausen, Germany; no. 658175) for experiments 2 and 4 and cultured in a humidified atmosphere of 5% CO2 in air at 37.5 C in a N2-O2-CO2-regulated incubator (ESPEC Corp., Osaka, Japan; no. BNP-110). After 12 h of culture, the medium was replaced with fresh medium (5% calf serum), and the following experiments were carried out. To culture cells under conditions with different levels of O2 (3, 5, 10, or 20%), an individual small culture chamber was used for each oxygen concentration and each time point. After setting the culture plates in the chamber, the atmospheric air was evacuated by a vacuum pump, and the chamber was refilled with a nonstandard gas mixture containing each level of O2 (3, 5, or 10%) and 5% CO2 in a N2 base. After repeating this evacuation and inflow five times, the incubations were started.

Experiment 1: effects of reduced oxygen tensions on P4 production
Luteal cells were incubated under various O2 concentrations (20, 10, 5, 3% O2) with or without LH (10 ng/ml, USDA-bLH-B6) for 24 h. Conditioned media were collected and stored at –30 C until assayed for P4.

Experiment 2: effects of reduced oxygen tension on Hif-1{alpha} protein expression
Hif-1{alpha} is known to accumulate in cells and function specifically under hypoxic conditions (18, 19). To test whether a condition with 3% O2 is hypoxic, we determined the expression of Hif-1{alpha} protein. Luteal cells were incubated under commonly used (normal) culture atmosphere (20% O2, 5% CO2, 75% N2) or low-oxygen concentration (3% O2, 5% CO2, 92% N2) for 2, 4, and 24 h. The cultured cells were scraped and placed in ice-cold homogenization buffer [25 mM Tris-HCl, 300 mM sucrose, 2 mM EDTA, Complete (protease inhibitor cocktail; Roche, Mannheim, Germany; no. 1697498) (pH 7.4)] and then frozen in liquid nitrogen and stored at –80 C until Hif-1{alpha} protein analysis by Western blotting.

Experiment 3: time-dependent effects of reduced oxygen tension
Luteal cells were incubated under a normal culture atmosphere (20% O2) or low-oxygen concentration (3% O2) with or without LH (10 ng/ml) for 1, 2, 4, 8, 12, and 24 h. Conditioned media from each culture period were collected and stored at –30 C until assayed for P4 and pregnenolone. After each incubation, total RNA was extracted for determination of StAR, P450scc, and 3ß-HSD mRNA expressions.

Experiment 4: effect of reduced oxygen tension on StAR protein expression
Luteal cells were incubated under a normal culture atmosphere (20% O2) or low-oxygen concentration (3% O2) with or without LH (10 ng/ml) for 8 and 24 h. The cultured cells were scraped and placed in ice-cold homogenization buffer and then frozen in liquid nitrogen and stored at –80 C until StAR protein analysis by Western blotting.

Experiment 5: time-dependent effect of reduced oxygen tension on 3ß-HSD activity
Luteal cells were incubated under a normal culture atmosphere (20% O2) or low-oxygen concentration (3% O2) with or without LH (10 ng/ml) in the presence or absence of pregnenolone (a substrate of 3ß-HSD, 1 µg/ml, no. P9129; Sigma) for 4, 8, 12, and 24 h. The conditioned media from each culture period were collected and stored at –30 C until assayed for P4.

P4 determination
Concentrations of P4 were determined directly from the cell culture media with an enzyme immunoassay (EIA) as described previously (22). The standard curve ranged from 0.391 to 100 ng/ml, and the effective dose of the assay for 50% inhibition (ED50) was 4.5 ng/ml. The intra- and interassay coefficients of variation were 7.0 and 10.8%, respectively.

RNA isolation and cDNA synthesis
Total RNA was prepared from cultured luteal cells using TRIZOL reagent according to the manufacturer’s directions (Invitrogen, Carlsbad, CA; no. 15596–026). Total RNA (1 µg) was reverse transcribed using a ThermoScript RT-PCR system (Invitrogen; no. 11146-016).

Real-time PCR
Gene expression was measured by real-time PCR using a LightCycler (Roche Diagnostics, Mannheim, Germany) and the QuantiTect SYBR Green PCR system (QIAGEN GmbH, Hilden, Germany) starting with 1 ng reverse-transcribed total RNA. Standard curves of sample cDNA were generated using serial dilutions (1:2 to 1:1000). 18S ribosomal RNA expression was used as an internal control. Twenty-base pair primers with 50–60% GC contents were synthesized (Table 1Go). PCR conditions were: 95 C for 15 min, followed by 55 cycles of 94 C for 15 sec, 55 C for 30 sec, and 72 C for 30 sec. Use of the QuantiTect SYBR Green PCR system at elevated temperatures resulted in reliable and sensitive quantification of the PCR products with high linearity (Pearson correlation coefficient r > 0.99).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Primers used in real-time PCR

 
P450scc activity
P450scc activity was assessed by total pregnenolone production (nanograms per hour/2 x 105 cells) at various time points (0–4, 4–8, 8–12, and 12–24 h). Because pregnenolone is metabolized to P4, total pregnenolone production in an interval i (Pi) (nanograms per hour/2 x 105 cells) was calculated as follows:

Formula
where Apreg,i and AP4,i are the accumulated amounts of pregnenolone and P4 (nanograms per milliliter) in the interval, Mpreg and MP4, are their respective molecular weights (316.48 and 314.47, respectively), and ti is the length of the interval (4 or 12 h).

Pregnenolone concentration in conditioned media was measured by an EIA that was identical with the EIA for P4. Standards or samples (20 µl) were incubated with 100 µl polyclonal antibody (raised in a rabbit against pregnenolone-3-succ-BSA; Cosmo Bio Co., Tokyo, Japan; no. FKA316E) solution (1:10,000) and 100 µl pregnenolone-3-succ-horseradish peroxidase (HRP; 1:100,000; Cosmo Bio; no. FKA315) for 3 h at room temperature. The standard curve ranged from 0.391 to 100 ng/ml, and ED50 was 4.5 ng/ml. The intra- and interassay coefficients of variation were 6.7 and 9.6%, respectively. The cross-reactivities of the antibody were 100% for pregnenolone, 6% for P4, 1% for 17{alpha}-hydroxy-pregnenolone, 0.3% for 17{alpha}-hydroxy-progesterone, 0.01% for cortisol, 0.016% for cortisone, 0.02% for 4-androstenedione, and 0.03% for testosterone.

3ß-HSD activity
3ß-HSD activity was determined by measuring the conversion of pregnenolone to P4 in a bovine luteal cell culture. 3ß-HSD activity was assessed by subtracting the mean P4 concentration in the pregnenolone-untreated wells from the mean P4 concentration in the pregnenolone-treated wells. The activity was expressed as a percentage of the value in non-LH-treated cells incubated under 20% O2 for 4 h.

Hif-1{alpha} and StAR protein analysis
Hif-1{alpha} and StAR (9) protein levels in cultured bovine luteal cells were assessed by Western blotting analysis. The cultured cells (experiments 2 and 4) were ultrasonicated on ice in the homogenization buffer. For Hif-1{alpha} protein analysis, nuclei were isolated from the cell homogenates by centrifugation at 600 x g for 30 min. For StAR protein analysis, the resultant supernatant was recentrifuged at 9000 x g for 30 min to isolate mitochondria. Protein concentration was determined by the method of Osnes et al. (23), using BSA as a standard. The proteins were then solubilized in NuPAGE LDS sample buffer (Invitrogen; no. NP0007) and heated at 95 C for 10 min. Samples (Hif-1{alpha}: 50 µg protein; StAR: 25 µg protein) were subjected to electrophoresis on a NuPAGE 4–12% Bis-Tris gel (Invitrogen; no. NP0322BOX) for 45 min at 200 V.

The separated proteins were electrophoretically transblotted to a 0.2-µm nitrocellulose membrane (Invitrogen; no. LC2000) at 100 V for 2 h in transfer buffer [25 mM Tris-HCl, 192 mM glycine, 20% methanol (pH 8.3)]. The membrane was then washed in TBS-T [0.1% Tween 20 in Tris-buffered saline (TBS) (25 mM Tris-HCl [pH 7.5], 137 mM NaCl)] and cut into two pieces: one piece was used for a target protein [Hif-1{alpha} (120 kDa) or StAR (30 kDa)], and the other piece was used for ß-actin (42 kDa). The piece for Hif-1{alpha} was incubated in blocking buffer (4% nonfat dry milk in TBS-T) for 1 h at room temperature, whereas the pieces for StAR and ß-actin were incubated in blocking buffer overnight at 4 C. After the blocking incubation, the pieces of membrane were separately incubated with a primary antibody specific to each protein: Hif-1{alpha}antibody (Novus Biologicals, Inc., Littleton, CO; no. NB 100–105; 1:500 in blocking buffer, overnight at 4 C), StAR antibody (kindly donated by Dr. Stocco, Texas Tech Health Sciences Center; 1:1000 in blocking buffer, 1 h at room temperature), ß-actin antibody (Sigma; no. A2228; 1:1000 in TBS-T, 1 h at room temperature). After incubation, the membrane pieces were washed three times for 10 min in TBS-T at room temperature and then incubated with secondary antibody [StAR (1:20,000 in TBS-T): antirabbit Ig, HRP-linked whole antibody produced in donkey; Amersham Biosciences Corp., Piscataway, NJ; no. NA934; Hif-1{alpha} (1:2,000 in blocking buffer) and ß-actin (1:20,000 in TBS-T): antimouse Ig, HRP-linked whole antibody produced in sheep, Amersham Biosciences; no. NA931] for 1.5 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 (Hif-1{alpha}, StAR, ß-actin) in the cells was estimated by measuring the OD in the defined area by computerized densitometry using NIH Image (National Institutes of Health, Bethesda, MD).

Statistical analysis
All experimental data are shown as the mean ± SEM. The statistical significance of differences in the concentration of P4, the amounts of StAR, P450scc, and 3ß-HSD mRNA, the activity of P450scc and 3ß-HSD, and the StAR protein levels was assessed by ANOVA followed by a Fisher protected least significant difference procedure (PLSD) as a multiple comparison test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1: effects of reduced oxygen tensions on P4 production
LH stimulated P4 production by luteal cells cultured for 24 h, regardless of the O2 concentration (Fig. 1Go; P < 0.05). P4 production was inhibited by decreasing O2 in a concentration-dependent manner in both non-LH-treated and LH-treated cells (Fig. 1Go; P < 0.001). Based on this result, further experiments were performed under 20% O2 and 3% O2 to more clearly determine the effects of low O2 on P4 synthesis in bovine luteal cells.


Figure 1
View larger version (51K):
[in this window]
[in a new window]
  		FIG. 1. Effects of reduced oxygen tensions on P4 production by cultured bovine midluteal cells. The cells were cultured under various oxygen tensions (20, 10, 5, 3% O2) with or without LH (10 ng/ml) for 24 h (n = 3). All values represent mean ± SEM of three separate experiments. Asterisks indicate that LH showed a significant effect on P4 production within each oxygen tension (P < 0.01) and different letters indicate significant differences (P < 0.01) among oxygen tensions within non-LH-treated cells (a–d) or within LH-treated cells (w–z), as determined by ANOVA followed by a Fisher PLSD as a multiple comparison test.

 
Experiment 2: time-dependent effects of reduced oxygen tension on Hif-1{alpha} protein expression
Hif-1{alpha} protein expression significantly increased under a condition with 3% O2 at 2, 4, and 24 h of culture (Fig. 2Go; P < 0.05).


Figure 2
View larger version (42K):
[in this window]
[in a new window]
  		FIG. 2. Time-dependent effects of reduced oxygen tension on Hif-1{alpha} protein expression in cultured bovine midluteal cells. The cells were cultured under 20% O2 or 3% O2 for 2, 4, and 24 h. A, Representative samples of Western blot for Hif-1{alpha} and ß-actin are shown in upper panels [lane 1, 20% O2 (2 h); lane 2, 3% O2 (2 h); lane 3, 20% O2 (4 h); lane 4, 3% O2 (4 h); lane 5, 20% O2 (24 h); lane 6, 3% O2 (24 h)]. The blot was incubated with primary antibodies against Hif-1{alpha} or ß-actin and then incubated with second antibody conjugated to HRP. The resultant signal was detected by chemiluminescence and quantitated by computer-assisted densitometry. The Hif-1{alpha} protein levels are expressed relative to the amounts of ß-actin protein (B; n = 3).

 
Experiment 3–1: time-dependent effect of reduced oxygen tension on P4 production
LH stimulated P4 production by cultured luteal cells regardless of the incubation period or O2 concentration (Fig. 3AGo; P < 0.05). As shown in Fig. 3AGo, a low-oxygen condition decreased basal and LH-stimulated P4 production by bovine luteal cells after 8, 12, and 24 h of culture (P < 0.05). Analysis of P4 production for each time interval revealed that the rate of P4 production was reduced by low-oxygen tension during 2–24 h in LH-treated cells and during 4–24 h in non-LH-treated cells (Fig. 3BGo; P < 0.05).


Figure 3
View larger version (42K):
[in this window]
[in a new window]
  		FIG. 3. Time-dependent effects of reduced oxygen tension on P4 production [accumulated value (A); production per hour (B)] by cultured bovine midluteal cells. The cells were cultured under 20% O2 or 3% O2 with or without LH (10 ng/ml) for different time periods (1, 2, 4, 8, 12, and 24 h; n = 5). All values represent mean ± SEM of five separate experiments. Different letters indicate significant difference (P < 0.05) within each culture period, as determined by ANOVA followed by a Fisher PLSD as a multiple comparison test.

 
Experiment 3–2: time-dependent effects of reduced oxygen tension on StAR, P450scc, and 3ß-HSD mRNA expressions
A real-time PCR analysis showed that LH increased the expression of StAR mRNA 2.7-fold under 20% O2 and 2.0-fold under 3% O2 at 4 h of culture (Fig. 4AGo; P < 0.01); no differences were observed between different oxygen tensions in any incubation period. After 24 h of culture of non-LH-treated cells, the expression of P450scc mRNA under 3% O2 (Fig. 4BGo, hatched bar on right) was only 31% of the expression under 20% O2 (white bar on right). Similarly, after 12 and 24 h of culture of LH-treated cells, the expressions of P450scc mRNA under 3% O2 (striped bars on right) were 47 and 27% of the expression under 20% O2 (black bars on right), respectively (P < 0.05). The expression of 3ß-HSD mRNA was not affected by O2 concentration or LH in any incubation period (Fig. 4CGo).


Figure 4
View larger version (56K):
[in this window]
[in a new window]
  		FIG. 4. Time-dependent effects of reduced oxygen tension on the amounts of StAR (A), P450scc (B), and 3ß-HSD (C) mRNA in cultured bovine midluteal cells. The cells were cultured under 20% O2 or 3% O2 with or without LH (10 ng/ml) for different time periods (1, 2, 4, 8, 12, and 24 h). The amounts of StAR, P450scc, and 3ß-HSD mRNA are expressed relative to the amounts of 18S rRNA (n = 3). In each time period, letters (a) indicate significant differences between non-LH-treated and LH-treated cells within each oxygen tension (P < 0.05). Asterisks indicate significant differences between oxygen tensions within non-LH-treated cells or within LH-treated cells (P < 0.05). Significant differences were determined by ANOVA followed by a Fisher PLSD as a multiple comparison test.

 
Experiment 3–3: time-dependent effects of reduced oxygen tension on P450scc activity
P450scc activity was significantly stimulated by LH during 0–4 h of culture (Fig. 5Go; P < 0.05). On the other hand, reduced oxygen tension decreased P450scc activity during 4–24 h of culture in both non-LH-treated and LH-treated cells (Fig. 5Go; P < 0.05).


Figure 5
View larger version (20K):
[in this window]
[in a new window]
  		FIG. 5. Time-dependent effects of reduced oxygen tension on P450scc activity in cultured bovine midluteal cells. P450scc activity was assessed by total pregnenolone production (defined in Materials and Methods). The cells were cultured under 20% O2 or 3% O2 with or without LH (10 ng/ml) for different time periods (4, 8, 12, and 24 h). All values represent mean ± SEM of four separate experiments. Different letters indicate significant difference (P < 0.05) within each culture period, as determined by ANOVA followed by a Fisher PLSD as a multiple comparison test.

 
Experiment 4: effect of reduced oxygen tension on StAR protein expression
Under all four conditions (20% O2 and 3% O2, with and without LH treatment), two StAR-specific bands were expressed after both 8 and 24 h incubation (Fig. 6AGo), in agreement with previous results (14). Intensities of the bands, after normalization to ß-actin-specific bands, showed no effect of reduced oxygen tension or LH on StAR protein expression after either incubation period (Fig. 6BGo).


Figure 6
View larger version (57K):
[in this window]
[in a new window]
  		FIG. 6. Effects of reduced oxygen tension on StAR protein levels in cultured bovine midluteal cells. The cells were cultured under 20% O2 or 3% O2 with or without LH (10 ng/ml) for 8 and 24 h. A, Representative samples of Western blot for StAR and ß-actin are shown in upper panels [lane 1, non-LH-treated (20% O2); lane 2, non-LH-treated (3% O2); lane 3, LH-treated (20% O2); lane 4, LH-treated (3% O2)]. The blot was incubated with primary antibodies against StAR or ß-actin and then incubated with second antibody conjugated to HRP. The resultant signal was detected by chemiluminescence and quantitated by computer-assisted densitometry. The StAR protein levels are expressed relative to the amounts of ß-actin protein (B; n = 3).

 
Experiment 5: time-dependent effect of reduced oxygen tension on 3ß-HSD activity
Neither reduced oxygen tension nor LH had any significant effect on 3ß-HSD activity during any incubation period (Fig. 7Go).


Figure 7
View larger version (28K):
[in this window]
[in a new window]
  		FIG. 7. Time-dependent effects of reduced oxygen tension on 3ß-HSD activity in cultured bovine midluteal cells. The cells were cultured under 20% O2 or 3% O2 with or without LH (10 ng/ml) in the presence or absence of pregnenolone (1 µg/ml) for different time periods (4, 8, 12, and 24 h; n = 5). All values are expressed as a percentage of the activity of non-LH-treated cells incubated under 20% O2 for 4 h (defined in Materials and Methods).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated for the first time that reduced oxygen tension inhibits basal and LH-stimulated P4 production by bovine luteal cells. This inhibitory effect is closely associated with suppression of enzymatic activity and mRNA expression of P450scc. More than 20 yr ago, several researchers reported a decreased ovarian blood flow during spontaneous luteolysis in cows using electromagnetic flow probes implanted around the ovarian artery (5, 6). They also demonstrated a close correlation between the decrease of blood supply to the ovary and the concentration of P4 in the circulation (5, 6). Close temporal correlations between ovarian blood flow and plasma P4 concentration during luteolysis were also observed in sheep (2, 3) and guinea pigs (4). A recent study using color Doppler ultrasonography showed that the blood flow within the mid-CL significantly decreased 8 h after injection of a luteolytic dose of PGF2{alpha} and that circulating P4 concentration continuously decreased until 16 h after a significant reduction of blood flow during PGF2{alpha}-induced luteolysis in cattle (7). The present results provide strong evidence that a low-oxygen condition is involved in the inhibition of P4 synthesis by decreasing P450scc activity, which consequently reduced pregnenolone production in bovine luteal cells. The above findings suggest that a low-oxygen condition, caused by a decreased blood supply, is essential for the progression of the luteolytic cascade in cattle.

The oxygen concentration in bovine luteal tissue has not been reported yet. However, because mean oxygen concentration in the ovarian venous blood ipsilateral to the CL during the midluteal stage has been shown to be 15.1% (6), the oxygen concentration in the CL is presumably higher than 15.1%. In the present study, the amounts of P4 produced after 24 h of culture under the four oxygen concentrations were significantly different, indicating that luteal P4 synthesis is sensitive to oxygen concentration and suggesting that an oxygen deficiency caused by the decreased blood supply induces functional luteolysis. However, in the time-course experiments, low-oxygen tension (3% O2) did not affect P4 synthesis during 0–2 h of culture, although cellular responses to hypoxic condition have been suggested to be acute (24). We do not have a clear explanation for the lack of effects of low O2 during the first 2 h of culture. It might be due to the time lag for dissolved oxygen in the culture medium to be brought to the same level as the culture atmosphere. Under hypoxic conditions, Hif-1{alpha} protein is known to accumulate in the cells and enhance transcription of hypoxia-inducible genes (18, 19). Because the protein expression of Hif-1{alpha} was increased by low O2 (3% O2) starting at 2 h in the present study, the culture condition with 3% O2 is a hypoxic condition.

The effects of reduced oxygen tension on steroidogenesis were previously studied in human placental villi (25), rat (26), and porcine (27) granulosa cells and bovine (28, 29, 30), rabbit (31), and rat (32) adrenocortical cells. A low-oxygen condition (1% O2) did not significantly reduce cortisol production by cultured bovine adrenocortical cells after either short (2 h) or long (24 h) culture (30). Studies on rat (26) and porcine (27) granulosa cells revealed that a low-oxygen condition (1–2% O2) significantly reduced P4 production. In the present study, a low-oxygen condition (3% O2) significantly inhibited basal and LH-stimulated P4 production in cultured bovine luteal cells. Based on these findings, the biosynthesis of P4 seems to be more sensitive to low-oxygen concentrations than the biosynthesis of cortisol. Furthermore, the present results are coincident with those of Gafvels et al. (33), who used an isolated rat whole CL culture, demonstrating that LH-stimulated P4 production decreased under a low-oxygen condition. However, under a low-oxygen condition, P4 production of LH-treated cells was still significantly higher than that of non-LH-treated cells in the present study. Therefore, we assume that functional luteolysis is a result of both reduced oxygen and reduced LH delivery.

Our findings that a low-oxygen condition decreased P450scc mRNA expression in both non-LH-treated and LH-treated cells, but not mRNA expressions of StAR and 3ß-HSD, suggest that a low-oxygen condition inhibits P4 production, mainly by suppressing P450scc expression. In addition, the inhibition of P4 and pregnenolone production by a low-oxygen condition that we observed could be at least partly due to reduced P450scc activity. This is because P450scc is a key enzyme in the synthesis of pregnenolone so that pregnenolone production can act as an indicator of P450scc activity (32). Thus, the inhibition of P4 production by a low-oxygen condition observed in the present study could be due to the reduced P450scc activity at least. A low-oxygen condition has been suggested to inhibit the process of side-chain cleavage of cholesterol because molecular oxygen is required for this process (34), and the importance of oxygen in the process has been shown in different cell types (32, 35). Moreover, mRNA expression and enzymatic activity of P450scc in CL were less in the regressed stage than the developing stage in cattle (14, 15, 16). These findings suggest that a low-oxygen condition causes P4 suppression in functional luteolysis by inhibiting P450scc activity. In addition to P450scc activity, mRNA expression of this enzyme was decreased under a low-oxygen condition. Further studies are needed to clarify whether the amount of P450scc protein is inhibited by reduced oxygen tension.

In the present study, the activity of P450scc was decreased by a low-oxygen condition starting at 4–8 h of culture, whereas a low-oxygen condition did not start to decrease mRNA expression of P450scc until 12 h of culture. The reason for the decrease in enzyme activity occurred earlier than the decrease in mRNA expression is unclear. Protein synthesis was demonstrated to be strongly inhibited by a low-oxygen condition in rat hepatocytes in vitro (36, 37). Thus, it is possible that a low-oxygen condition also inhibits the synthesis of proteins, including P450scc, in bovine luteal cells, resulting in a decrease in P4 production. On the other hand, a low-oxygen condition (3% O2) did not affect the expression of StAR protein or 3ß-HSD activity in the present study, suggesting that StAR and 3ß-HSD do not have significant roles in P4 inhibition by low O2. Based on these findings, a low-oxygen condition seems to decrease P4 production mostly by regulating P450scc activity in bovine luteal cells.

Luteal regression is characterized by a phase of inhibition of P4 production (functional luteolysis), followed by a phase of decrease in luteal size (structural luteolysis), when cells of the CL undergo apoptosis (1). We previously demonstrated that CL volume significantly decreased 8 h after PGF2{alpha} injection when the blood flow within the CL was significantly decreased (7). In addition, we recently showed that a specific P4 receptor antagonist induced apoptosis of bovine luteal cells and suggested that P4 suppresses luteal cell apoptosis in bovine CL (38). Because a low-oxygen condition decreased P4 production in bovine luteal cells in the present study, oxygen deficiency may promote luteal cell apoptosis by inhibiting P4 production. Further studies are in progress to clarify whether a low-oxygen condition induces apoptosis in bovine luteal cells.

In conclusion, our results indicate that a low-oxygen condition inhibits P4 production by suppressing P450scc activity in bovine luteal cells and suggest that an oxygen deficiency caused by a decreased blood supply is an important condition for progression of the luteolytic cascade in cattle.


   Acknowledgments
 
We are grateful to Dr. Albert F. Parlow (National Hormone and Pituitary Program, University of Maryland School of Medicine, and the National Institute of Diabetes and Digestive and Kidney Disease) for bovine LH (USDA-bLH-B6) and Dr. Douglas M. Stocco (Texas Tech Health Sciences Center) for the StAR antiserum.


   Footnotes
 
This research was supported by Grants-in-Aid for Scientific Research (18380166 and 18658114) of the Japan Society for the Promotion of Science (JSPS). R.N. is supported by a JSPS Research Fellowship (no. 03589).

Disclosure summary: all authors have nothing to declare.

First Published Online June 1, 2006

Abbreviations: CL, Corpus luteum; EIA, enzyme immunoassay; Hif-1{alpha}, hypoxia-inducible factor-1{alpha}; HRP, horseradish peroxidase; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; P4, progesterone; PG, prostaglandin; PLSD, protected least significant difference procedure; P450scc, P450 side-chain cleavage enzyme; StAR, steroidogenic acute regulatory protein; TBS, Tris-buffered saline; TBS-T, Tween 20 in TBS.

Received December 17, 2005.

Accepted for publication May 22, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. McCracken JA, Custer EE, Lamsa JC 1999 Luteolysis: a neuroendocrine-mediated event. Physiol Rev 79:263–323[Abstract/Free Full Text]
  2. Niswender GD, Reimers TJ, Diekman MA, Nett TM 1976 Blood flow: a mediator of ovarian function. Biol Reprod 14:64–81[Abstract]
  3. Nett TM, McClellan MC, Niswender GD 1976 Effects of prostaglandins on the ovine corpus luteum: blood flow, secretion of progesterone and morphology. Biol Reprod 15:66–78[Abstract]
  4. Azmi TI, O’Shea JD, Lee CS, Rodgers RJ 1982 Effects of a synthetic prostaglandin analogue, cloprostenol, on the corpus luteum of the guinea pig. Prostaglandins 24:519–526[CrossRef][Medline]
  5. Ford SP, Chenault JR 1981 Blood flow to the corpus luteum-bearing ovary and ipsilateral uterine horn of cows during the oestrous cycle and early pregnancy. J Reprod Fertil 62:555–562[Abstract]
  6. Wise TH, Caton D, Thatcher WW, Barron DH, Fields MJ 1982 Ovarian function during the estrous cycle of the cow: ovarian blood flow and progesterone release rate. J Anim Sci 55:627–637[Abstract/Free Full Text]
  7. Acosta TJ, Yoshizawa N, Ohtani M, Miyamoto A 2002 Local changes in blood flow within the early and midcycle corpus luteum after prostaglandin F2{alpha} injection in the cow. Biol Reprod 66:651–658[Abstract/Free Full Text]
  8. Pon LA, Orme-Johnson NR 1986 Acute stimulation of steroidogenesis in corpus luteum and adrenal cortex by peptide hormones. Rapid induction of a similar protein in both tissues. J Biol Chem 261:6594–6599[Abstract/Free Full Text]
  9. Clark BJ, Wells J, King SR, Stocco DM 1994 The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem 269:28314–28322[Abstract/Free Full Text]
  10. Clark BJ, Soo SC, Caron KM, Ikeda Y, Parker KL, Stocco DM 1995 Hormonal and developmental regulation of the steroidogenic acute regulatory protein. Mol Endocrinol 9:1346–1355[Abstract]
  11. Stocco DM 1997 A StAR search: implications in controlling steroidogenesis. Biol Reprod 56:328–336[Abstract]
  12. Juengel JL, Niswender GD 1999 Molecular regulation of luteal progesterone synthesis in domestic ruminants. J Reprod Fertil Suppl 54:193–205[Medline]
  13. Niswender GD 2002 Molecular control of luteal secretion of progesterone. Reproduction 123:333–339[Abstract]
  14. Pescador N, Soumano K, Stocco DM, Price CA, Murphy BD 1996 Steroidogenic acute regulatory protein in bovine corpora lutea. Biol Reprod 55:485–491[Abstract]
  15. Rodgers RJ, Waterman MR, Simpson ER 1986 Cytochromes P-450scc, P-450(17){alpha}, adrenodoxin, and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase in bovine follicles and corpora lutea. Changes in specific contents during the ovarian cycle. Endocrinology 118:1366–1374[Abstract]
  16. Rodgers RJ, Waterman MR, Simpson ER 1987 Levels of messenger ribonucleic acid encoding cholesterol side-chain cleavage cytochrome P-450, 17 {alpha}-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor in bovine follicles and corpora lutea throughout the ovarian cycle. Mol Endocrinol 1:274–279[Abstract]
  17. Couet J, Martel C, Dupont E, Luu-The V, Sirard MA, Zhao HF, Pelletier G, Labrie F 1990 Changes in 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4 isomerase messenger ribonucleic acid, activity and protein levels during the estrous cycle in the bovine ovary. Endocrinology 127:2141–2148[Abstract]
  18. Semenza GL 1999 Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol 15:551–578[CrossRef][Medline]
  19. Wenger RH 2002 Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J 16:1151–1162[Abstract/Free Full Text]
  20. Miyamoto Y, Skarzynski DJ, Okuda K 2000 Is tumor necrosis factor {alpha} a trigger for the initiation of endometrial prostaglandin F2{alpha} release at luteolysis in cattle? Biol Reprod 62:1109–1115[Abstract/Free Full Text]
  21. Okuda K, Miyamoto A, Sauerwein H, Schweigert FJ, Schams D 1992 Evidence for oxytocin receptors in cultured bovine luteal cells. Biol Reprod 46:1001–1006[Abstract]
  22. Okuda K, Uenoyama Y, Lee KW, Sakumoto R, Skarzynski DJ 1998 Progesterone stimulation by prostaglandin F2{alpha} involves the protein kinase C pathway in cultured bovine luteal cells. J Reprod Dev 44:79–84
  23. Osnes T, Sandstad O, Skar V, Osnes M, Kierulf P 1993 Total protein in common duct bile measured by acetonitrile precipitation and a micro bicinchoninic acid (BCA) method. Scand J Clin Lab Invest 53:757–763[Medline]
  24. Hochachka PW, Buck LT, Doll CJ, Land SC 1996 Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack. Proc Natl Acad Sci USA 93:9493–9498[Abstract/Free Full Text]
  25. Gabbe SG, Villee CA 1971 The effect of hypoxia on progesterone synthesis by placental villi in organ culture. Am J Obstet Gynecol 111:31–37[Medline]
  26. Koos RD, Feiertag MA 1989 The effect of reduced oxygen tension on progesterone accumulation in rat granulosa cell cultures. Steroids 54:553–562[CrossRef][Medline]
  27. Basini G, Bianco F, Grasselli F, Tirelli M, Bussolati S, Tamanini C 2004 The effects of reduced oxygen tension on swine granulosa cell. Regul Pept 120:69–75[CrossRef][Medline]
  28. Raff H, Kohandarvish S 1990 The effect of oxygen on aldosterone release from bovine adrenocortical cells in vitro: PO2 versus steroidogenesis. Endocrinology 127:682–687[Abstract]
  29. Brickner RC, Jankowski B, Raff H 1992 The conversion of corticosterone to aldosterone is the site of the oxygen sensitivity of the bovine adrenal zona glomerulosa. Endocrinology 130:88–92[Abstract]
  30. Chabre O, Defaye G, Chambaz EM 1993 Oxygen availability as a regulatory factor of androgen synthesis by adrenocortical cells. Endocrinology 132:255–260[Abstract]
  31. Papanek PE, Jankowski BM, Raff H 1996 Aldosterone release from adrenal cells is inhibited by reduced oxygen levels in vitro during maturation in rabbits. Reprod Fertil Dev 8:1131–1136[CrossRef][Medline]
  32. Bruder ED, Nagler AK, Raff H 2002 Oxygen-dependence of ACTH-stimulated aldosterone and corticosterone synthesis in the rat adrenal cortex: developmental aspects. J Endocrinol 172:595–604[Abstract]
  33. Gafvels M, Selstam G, Damber JE 1987 Influence of oxygen tension and substrates on basal and luteinizing hormone stimulated progesterone production and energy metabolism by isolated corpora lutea of adult pseudopregnant rats. Acta Physiol Scand 130:475–482[Medline]
  34. Lieberman S, Lin YY 2001 Reflections on sterol side-chain cleavage process catalyzed by cytochrome P450(scc). J Steroid Biochem Mol Biol 78:1–14[CrossRef][Medline]
  35. Raff H, Jankowski B 1995 O2 dependence of pregnenolone and aldosterone synthesis in mitochondria from bovine zona glomerulosa cells. J Appl Physiol 78:1625–1628[Abstract/Free Full Text]
  36. Lefebvre VH, Van Steenbrugge M, Beckers V, Roberfroid M, Buc-Calderon P 1993 Adenine nucleotides and inhibition of protein synthesis in isolated hepatocytes incubated under different pO2 levels. Arch Biochem Biophys 304:322–331[CrossRef][Medline]
  37. Tinton SA, Buc-Calderon PM 1999 Hypoxia increases the association of 4E-binding protein 1 with the initiation factor 4E in isolated rat hepatocytes. FEBS Lett 446:55–59[CrossRef][Medline]
  38. Okuda K, Korzekwa A, Shibaya M, Murakami S, Nishimura R, Tsubouchi M, Woclawek-Potocka I, Skarzynski DJ 2004 Progesterone is a suppressor of apoptosis in bovine luteal cells. Biol Reprod 71:2065–2071[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
R. Nishimura, J. Komiyama, Y. Tasaki, T. J. Acosta, and K. Okuda
Hypoxia Promotes Luteal Cell Death in Bovine Corpus Luteum
Biol Reprod, March 1, 2008; 78(3): 529 - 536.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
147/9/4273    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nishimura, R.
Right arrow Articles by Okuda, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nishimura, R.
Right arrow Articles by Okuda, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals