Gonadotropin-Induced 14-Reductase and 7-Reductase Gene Expression in Cumulus Cells during Meiotic Resumption of Porcine Oocytes
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内分泌学杂志 2005年第1期
Department of Applied Animal Science (Y.Y., M.N., T.T., M.S.), Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8528, Japan; and Department of Technology and Resources (N.I.), Graduate School of International Development and Cooperation, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8529, Japan
Address all correspondence and requests for reprints to: Masayuki Shimada, Ph.D., Department of Applied Animal Science, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8528, Japan. E-mail: mashimad@hiroshima-u.ac.jp.
Abstract
Progesterone is produced from cholesterol in cumulus cells during meiotic resumption of porcine oocytes. In follicular cells, it has been shown that exogenous lipoprotein-bound cholesterol ester can be used for steroid hormone production. However, in serum-free medium, progesterone is also secreted by FSH- and LH-stimulated cumulus-oocyte complexes, suggesting that progesterone could be produced from de novo synthesized cholesterol in cumulus cells. In the present study, we investigated the expression of 14-reductase and 7-reductase, which are the members of the superfamily that converts acetyl-CoA to cholesterol in cumulus cells. The expression of both genes was analyzed by RT-PCR. Both 14-reductase mRNA and 7-reductase mRNA in cumulus cells, cultured until 4 h, were under the level of detection limit. In response to gonadotropins, both mRNA levels were dramatically up-regulated, reaching a maximum at 20 h. To clarify the role of induced enzymes in cumulus cells, cumulus-oocyte complexes were cultured with either 14-reductase inhibitor, AY9944-A-7, or 7-reductase inhibitor, BM15.766. The results indicated that these inhibitors significantly suppressed the progesterone production in cumulus cells and meiotic progression of oocytes. The inhibitory effects reached a maximum at 1 μM AY9944-A-7 or 20 μM BM15.766. The addition of 20 ng/ml progesterone overcame the inhibitory effects of both drugs on meiotic resumption of oocytes. These results imply that gonadotropin-induced expression and function of 14-reductase and 7-reductase in cumulus cells contribute to oocyte meiotic resumption via a progesterone-dependent pathway.
Introduction
PROGESTERONE IS SYNTHESIZED from free cholesterol by cytochrome P450 cholesterol side-chain cleavage (P450scc) (1, 2) and 3?-hydroxysteroid dehydrogenase/isomerase (3?-HSD) (3). It has been shown that lipoprotein-bound cholesterol ester and stored cholesterol ester supply the major share of the cholesterol ester used for steroid hormone production (4, 5, 6). In our previous study, a 14 -demethylase inhibitor, ketoconazole, which suppressed the conversion of lanosterol to follicular fluid meiosis-activating sterol (FF-MAS) in a cholesterol biosynthetic pathway, was found to reduce both progesterone production in cumulus cells and germinal vesicle breakdown (GVBD) rate in cumulus-enclosed oocytes (7). The reduction of GVBD rate was overcome by the addition of progesterone to the gonadotropin and ketoconazole-containing medium. Moreover, Baranao and Hammond (8) have reported that FSH augments cholesterol synthesis in granulosa cells. As such, we hypothesized that progesterone is produced by de novo synthesized cholesterol in cumulus cells, which is stimulated by FSH and/or LH, with the newly synthesized progesterone playing an important role in meiotic resumption of porcine oocytes.
Numerous enzymes mediate the conversion of acetate to cholesterol in the sterol biosynthetic pathway (Fig. 1). Thaddeus and Strauss (9) have shown that exposure of cultured granulosa cells to 8-bromoadenosine cyclic 3',5'-phosphate results in a rapid increase in the content of the message for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. It has been reported that the levels of 14 -demethylase increased 6 times in rat ovary within 48 h after equine chorionic gonadotropin priming (10). Furthermore, a cAMP-responsive element (CRE) exists in the promoter region of the 14 -demethylase gene (11), and gene expression is dependent on the cAMP/CRE modulator-pathway (12). Thus, cAMP signaling stimulates both HMG-CoA reductase and 14 -demethylase gene expression. In fact, a high level of FF-MAS has been detected in gonadotropin-primed rabbit ovaries (13). Moreover, Baltsen (14) has reported an increased FF-MAS level after hCG stimulation, along with an increased level of progesterone in mouse ovary. The increasing level of progesterone in LH-stimulated rabbit ovary was significantly suppressed by 14-reductase inhibitor, AY9944-A-7 (15). The inhibitory effects of AY9944-A-7 on progesterone production were also observed in bovine luteinized cells (16) and porcine granulosa cells (17). These findings support our hypothesis that enzymes that play a role in the conversion from FF-MAS to cholesterol are also expressed in FSH- and LH-stimulated follicular cells, as well as HMG-CoA reductase and 14-demethylase. However, this hypothesis has not been confirmed.
FIG. 1. Simplified outline of the progesterone biosynthesis pathway. Three sources of cholesterol can be used as substrate: 1) exogenous cholesterol (HDL or LDL); 2) hydrolysis of stored cholesterol ester by cholesterol esterase; and 3) de novo synthesized cholesterol from acetate. Names of enzymes are in italics. Single arrow, Single enzymatic conversion; double arrow, multienzymatic step. AY9944-A-7 blocks the activity of both the 14-reductase and 7-reductase, and BM15.766 only blocks the activity of 7-reductase. T-MAS, Testicular-MAS.
In the present study, to investigate the role of the cholesterol biosynthetic pathway, especially from FF-MAS to cholesterol in progesterone production in cumulus cells, we analyzed the expression of 14-reductase gene and 7-reductase gene in cumulus cells when cumulus-oocyte complexes (COCs) were cultured with FSH and LH. We also examined the effects of AY9944-A-7 (14-reductase inhibitor) and BM15.766 (7-reductase inhibitor) on progesterone production and meiotic resumption of oocytes.
Materials and Methods
General protocol
Isolation of porcine COCs was described previously (18). The COCs were cultured in the maturation medium supplemented with or without 20 ng/ml highly purified porcine FSH [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Torrance, CA] and 0.5 μg/ml highly purified porcine LH (NIDDK). The maturation medium was modified NCSU37 (19), supplemented with 0.3% (wt/vol) polyvinylpyrrolidone (Sigma Chemical Co., St. Louis, MO), 2% (vol/vol) MEM amino acids (Life Technologies, Inc., Grand Island, NY), 1% (vol/vol) MEM nonamino acids (Life Technologies, Inc.), 7 mM Taurine (Sigma Chemical Co.), and 2 mM hypoxanthine (Sigma Chemical Co.). When COCs were cultured under the presence of serum, 10% (vol/vol) fetal calf serum (FCS) (Life Technologies, Inc.) was added to the maturation medium without PVP and amino acids (18). At selected time intervals, COCs were collected for RNA. Other COCs were cultured with 14-reductase inhibitor, AY9944-A-7 (kindly gifted by Dr. M. Abou-Gharbia, Wyeth Research, Madison, NJ), or 7-reductase inhibitor, BM15.766 (kindly gifted by S. Hauptmann, Roche Diagnostics GmbH, Mannheim, Germany). After cultivation, COCs were visualized using a phase-contrast microscope (Olympus IMT2, Olympus, Tokyo, Japan) and x10 objective. The cultured medium was used for the analysis of progesterone level according to our previous study (20, 21). The oocytes were fixed with acetic acids/ethanol (1:3) for 48 h and stained with aceto-lacmoid before examination under a phase-contrast microscope (x400) for evaluation of their chromatin configuration. Visualization of apoptotic cells (containing fragmented DNA) in chamber slides was performed by the terminal deoxynucleotidy transferase-mediated biotinylated deoxyuridine triphosphates nick end-labeling (TUNEL) method (In Situ cell Death Detection Kit, Roche Diagnostics GmbH) (22).
RNA isolation
After cumulus cells were separated from the oocytes, they were washed three times in PBS. Total RNA was extracted from cumulus cells using the SV Total RNA Isolation System (Promega, Madison, WI), according to the instruction manual, and dissolved in 20 μl nuclease-free water.
RT-PCR
RT-PCR was performed according to a coupled one-step procedure using Access RT-PCR System (Promega) with some modifications (23). Briefly, 10 ng total RNA was reverse transcribed at 48 C for 45 min, denatured at 94 C for 2 min, and amplified for 32 (?-actin) or 35 (14-reductase and 7-reductase) cycles of denaturation at 94 C for 30 sec, primer annealing at 56 C for 1 min, and extension at 68 C for 1 min, with a final extension step of 7 min at 68 C. The amplified products were analyzed by electrophoresis on 2% agarose gels.
Oligonucleotide primer used for amplification of the 14-reductase was from known cDNA sequences of human 14-reductase (GenBank accession no. AF096304). The upstream primer is identical with nucleotides 681–703 of the human cDNA, and the downstream primer represents the reverse complement of nucleotides 1135–1158 (Table 1). This primer pair predicts 430-bp DNA fragments. The primer used for amplification of the 7-reductase was from known cDNA sequences of human 7-reductase (GenBank accession no. AF034544). The upstream primer is identical with nucleotides 1–24 of human cDNA, and the downstream primer represents the reverse complement of nucleotides 537–560 (Table 1). This primer pair predicts 511-bp DNA fragments. ?-actin was used as a control for reaction efficiency and variations in concentrations of mRNA in the original RT reaction. The ?-actin primers were based on the mouse sequence (GenBank accession no. NM009609). The amplified cDNAs were directly sequenced according to our previous study (24).
TABLE 1. Primers used for determinations of the porcine 14-reductase, 7-reductase, and ?-actin mRNA by RT-PCR
Statistical analysis
Statistical analyses of all data from three or four separate culture experiments were carried out by one-way ANOVA followed by Duncan’s multiple-ranges test using STATVIEW (Abacus Concepts, Inc., Berkeley, CA). All percentage data were subjected to arc-sine transformation before statistical analysis.
Results
Time-dependent changes of 14-reductase gene and 7-reductase gene expression in cumulus cells of COCs
Partial cDNAs were amplified by RT-PCR from porcine cumulus cells using each primer set corresponding to the sequence for either human 14-reductase or 7-reductase, respectively (Table 1 and Fig. 2A). The similarity of the partial porcine 14-reductase sequence to human 14-reductase was 93.04%; and to bovine 14-reductase, it was 88.63% (Fig. 2B). The porcine 7-reductase sequence showed a high degree of similarity to human (91.06%), rat (88.55%), and mouse (86.23%) sequences (Fig. 2C). Sequence comparisons indicate that the partial porcine cDNAs amplified with either 14-reductase primer or 7-reductase primer conformed to the porcine 14-reductase region or the 7-reductase region, respectively.
FIG. 2. The expressions of 14-reductase and 7-reductase in cumulus cells of porcine COCs. A, RT-PCR analysis of 14-reductase mRNA and 7-reductase mRNA in cumulus cells of COCs cultured for 20 h with FSH and LH. B, Comparison of partial cDNA sequences of 14-reductase primers-RT-PCR with human 14-reductase (accession no. AF096304) or bovine 14-reductase (accession no. AF039681). C, Comparison of partial cDNA sequences of 7-reductase primers-RT-PCR with rat 7-reductase (accession no. AF071500), human 7-reductase (accession no. AF034544), or mouse 7-reductase (accession no. AF057368).
To investigate the time-dependent changes of both 14-reductase gene expression and 7-reductase gene expression in cumulus cells, COCs were cultured with or without both FSH and LH. The data showed low levels of both 14-reductase gene expression and 7-reductase gene expression in cumulus cells until 8-h cultivation (Fig. 3). However, in response to both FSH and LH, the dramatically increased levels of 14-reductase mRNA in cumulus cells were observed at later times of culture (12, 16, or 20 h) (Fig. 3A). The differences between the FSH+LH-treated group and control group were significant at 16 and 20 h. Significantly higher levels of 7-reductase mRNA were also observed at 20-h cultivation with FSH and LH as compared with those in cumulus cells cultured without gonadotropins (control) (Fig. 3B). When COCs were cultured with FSH and LH for 20 h under the presence of serum, the high levels of both genes expression were also detected in cumulus cells as compared with those in cumulus cells cultured without FSH and LH (Fig. 3C).
FIG. 3. Time-dependent changes of 14-reductase or 7-reductase mRNA level in cumulus cells cultured with FSH and LH. A, RT-PCR analysis of 14-reductase in cumulus cells of COCs cultured without FCS. The intensity of the amplified bands was quantified by densitometric scanning. The respective value of either 14-reductase was normalized according to those of ?-actin to evaluate arbitrary units of the relative abundance of the targets. *, The addition of FSH and LH to the medium significantly increased the level of 14-reductase mRNA in cumulus cells at 16 and 20 h (P < 0.05); control, COCs were cultured without gonadotropins. B, RT-PCR analysis of 7-reductase in cumulus cells of COCs cultured without FCS. The intensity of the amplified bands was quantified by densitometric scanning. **, The addition of FSH and LH to the medium significantly increased the level of 7-reductase mRNA in cumulus cells at 20 h (P < 0.01); control, COCs were cultured without gonadotropins. C, The effects of FCS on the expression of 14-reductase and 7-reductase. COCs were cultured with FCS, FSH, and LH. The intensity of the amplified bands was quantified by densitometric scanning. The addition of FSH and LH to the medium significantly increased the level of 14-reductase mRNA (*) or 7-reductase mRNA (**) in cumulus cells at 20 h (P < 0.05); control, COCs were cultured without gonadotropins.
The effects of AY9944-A-7 or BM15.766 on progesterone production by COCs and meiotic resumption of cumulus-enclosed oocytes
Both 14-reductase and 7-reductase were expressed in cumulus cells of COCs that were cultured with gonadotropins. In the next experiment, we examined the effects of 14-reductase inhibitor (AY9944-A-7) and 7-reductase inhibitor (BM15.766) on progesterone production and the meiotic progression of oocytes.
Low levels of progesterone were detected when COCs were cultured for 20 h without FSH and LH (control), whereas progesterone levels were significantly increased by the cultivation with FSH and LH (Fig. 4A). The higher progesterone levels in FSH+LH-medium were significantly decreased with increases in the AY9944-A-7 levels, and the inhibitory effect reached a plateau at 1 μM AY9944-A-7 (Fig. 4B). The treatment with BM15.766 also significantly reduced the level of progesterone levels in a dose-dependent manner (Fig. 4C).
FIG. 4. The effects of 14-reductase inhibitor (AY9944-A-7) or 7-reductase inhibitor (BM15.766) on progesterone production by COCs. A, The effects of adding FSH and LH (FSH+LH) on progesterone production by COCs cultured without FCS for 20 h. *, The effect of adding FSH+LH on progesterone production is significant (P < 0.05); control, COCs were cultured without gonadotropins. B, Effects of AY9944-A-7 on the progesterone production by COCs cultured without FCS for 20 h. a–c, Ratios with different letters are significant (P < 0.05). C, Effects of BM15.766 on the progesterone production by COCs cultured without FCS for 20 h. d–f, Ratios with different letters are significant (P < 0.05). D, The effects of adding 10% (vol/vol) FCS on progesterone production by COCs cultured with FSH and LH for 20 h. E, Effects of 1 μM AY9944-A-7 or 20 μM BM15.766 on progesterone production by COCs cultured with FCS, FSH, and LH for 20 h. P < 0.011, The effect of 1 μM AY9944-A-7 on the level of progesterone is significant (P < 0.01); P < 0.012, the effect of 20 μM BM15.766 on the level of progesterone is significant (P < 0.01).
The addition of 10% (vol/vol) FCS to the maturation medium did not significantly affect the progesterone production by COCs as compared with that by COCs cultured without FCS (Fig. 4D). Under the FCS-supplemented condition, the treatment with either 1 μM AY9944-A-7 or 20 μM BM15.766 significantly suppressed the progesterone production by COCs (Fig. 4F).
The proportion of oocytes exhibiting GVBD was 20.72 ± 8.14% in oocytes cultured without FSH and LH for 20 h. The addition of FSH and LH to the medium significantly increased the proportion of oocytes exhibiting GVBD (62.33 ± 8.19%, Fig. 5A). This higher rate was significantly decreased by 0.5 or 1 μM AY9944-A-7 as well as progesterone production. However, the GVBD rate increased again with higher concentrations of AY9944-A-7 (2.5–10 μM) (Fig. 5A). When COCs were cultured in FSH+LH-medium supplemented with BM15.766 (0, 2, or 20 μM) for 20 h, the proportion of oocytes exhibiting GVBD was significantly decreased in a dose-dependent manner (Fig. 5B).
FIG. 5. The effects of 14-reductase inhibitor (AY9944-A-7) or 7-reductase inhibitor (BM15.766) on meiotic resumption of cumulus-enclosed oocytes. A, The effects of adding FSH and LH (FSH+LH) on the exhibition of GVBD in cumulus-enclosed oocytes for 20 h. *, The addition of FSH+LH to the medium significantly increased the proportion of oocytes exhibiting GVBD (P < 0.05); control, COCs were cultured without gonadotropins. B, The effects of AY9944-A-7 on the exhibition of GVBD in oocytes cultured for 20 h. a–c, Ratios with different letters are significant (P < 0.05). C, The effects of BM15.766 on the exhibition of GVBD in oocytes cultured for 20 h. d–f, Ratios with different letters are significant (P < 0.05).
The effects of adding progesterone to FSH+LH-medium containing 1 μM AY9944-A-7 or 20 μM BM15.766 on meiotic resumption and cumulus cell expansion
Treatment with 1 μM AY9944-A-7 or 20 μM BM15.766 suppressed both the progesterone production by COCs and the exhibition of GVBD. In this experiment, we examined whether the additional progesterone overcame the inhibitory effects of AY9944-A-7 or BM15.766 on meiotic resumption of oocytes.
The production of progesterone by COCs and the formation of progesterone receptor type A in cumulus cells were dramatically increased after 10-h cultivation (24, 25). After COCs were cultured with FSH+LH-medium containing 1 μM AY9944-A-7 or 20 μM BM15.766 for 10 h, the COCs were further cultured for 10 h with an additional 20 ng/ml progesterone. When COCs were cultured in FSH+LH-medium containing 1 μM AY9944-A-7 or 20 μM BM15.766, the rates of GVBD were 21.3 ± 7.2% or 13.2 ± 8.9%, respectively. The addition of 20 ng/ml progesterone to the medium overcame the inhibitory effects of both drugs in inducing meiotic resumption of oocytes (Fig. 6A). The rates of GVBD in oocytes were 55.0 ± 7.1% (FSH+LH+1 μM AY9944 + 20 ng/ml progesterone) and 51.7 ± 2.9% (FSH+LH+20 μM BM15.766 + 20 ng/ml progesterone).
FIG. 6. The effects of adding progesterone to FSH+LH-medium containing AY9944-A-7 or BM15.766 on meiotic resumption and cumulus cell expansion. A, The proportion of oocytes exhibiting GVBD. After COCs were cultured with FSH+LH-medium containing 1 μM AY9944-A-7 or 20 μM BM15.766 for 10 h, the COCs were further cultured for 10 h with an additional 20 ng/ml progesterone. C (control), COCs were cultured in FSH+LH-medium for 20 h. P4, COCs were cultured with 1 μM AY9944-A-7 or 20 μM BM15.766 in FSH+LH-medium for 10 h and further cultured with an additional 20 ng/ml progesterone for 10 h. P < 0.011, The effect of adding progesterone on GVBD in oocytes cultured with 1 μM AY9944-A-7 is significant (P < 0.01); P < 0.012, the effect of adding progesterone on GVBD in oocytes cultured with 20 μM BM15.766 is significant (P < 0.01). B, After COCs were cultured with FSH and LH for 40 h, COCs were visualized using phase-contrast microscopy and a x10 objective. a, COCs were cultured for 40 h with FSH and LH; a’, COCs were cultured for 40 h with FSH, LH, and progesterone; b, COCs were cultured for 40 h with 1 μM AY-9977-A-7, FSH, and LH; b’, COCs were cultured for 40 h with 1 μM AY-9977-A-7, progesterone, FSH, and LH; c, COCs were cultured for 40 h with 10 μM AY-9977-A-7, FSH, and LH; c’, COCs were cultured for 40 h with 10 μM AY-9977-A-7, progesterone, FSH, and LH; d, COCs were cultured for 40 h with 20 μM BM15.766, FSH, and LH; d’, COCs were cultured for 40 h with 20 μM BM15.766, progesterone, FSH, and LH.
We have reported that cumulus expansion appears to be regulated by a progesterone- and progesterone receptor (PR)-dependent pathway in porcine COCs (26). To examine the effects of 1 or 10 μM AY9944-A-7 or 20 μM BM15.766 on cumulus expansion of COC, COCs were cultured for 40 h. The culture with FSH and LH for 40 h induced fully expansion of COC as indicated by an increase of about 2-fold in diameter of the complex and the blurred image of the oocyte (Fig. 6Ba). The expansion was not affected by an additional 20 ng/ml progesterone (Fig. 6Ba’). However, the expansion was significantly suppressed when COCs were cultured with FSH, LH, and either AY9944-A-7 or BM15.766 for 40 h (Fig. 6Bb-d). In these nonexpanded COCs, the cumulus cells remained packed around the oocytes, with little visible expansion (Fig. 6B, b–d). The addition of 20 ng/ml progesterone to the medium overcame the inhibitory effects of 1 μM AY9944-A-7 or 20 μM BM15.766 in inducing cumulus expansion of COC (Fig. 6B, b’ and c’). However, the negative effects of 10 μM AY9944-A-7 on cumulus expansion were not improved by addition of progesterone (Fig. 6Bd’).
The effects of 1 or 10 μM AY9944-A-7 on apoptosis of cumulus cells during meiotic resumption of oocytes.
Although 1 μM AY9944-A-7 significantly decreased the GVBD rate of oocytes, further increasing the concentrations (2.5, 5 or 10 μM) induced meiotic resumption of oocytes. We tested whether the higher concentrations of AY9944-A-7 increased toxicity to cumulus cells and then caused apoptosis of cumulus cells. After COCs were cultured in FSH+LH-medium supplemented with 1 or 10 μM AY9944-A-7 for 28 h, apoptosis of cumulus cells were observed by the TUNEL-method.
When COCs were cultured with FSH+LH-medium for 28 h, a low proportion (9.1 ± 1.6%) of cumulus cells showed signs of apoptosis (TUNEL-positive). This proportion was significantly lower than that of cumulus cells cultured without gonadotropins (34.5 ± 5.1%) (Fig. 7). This gonadotropin-induced lower rate was not affected by the treatment with 1 or 10 μM AY9944-A-7. Thus, the survival of cumulus cells of COCs was not threatened by AY9944-A-7. However, the majority of oocytes failed to progress to the MII stage and actually degenerated after the COCs were cultured with 10 μM AY9944-A-7 for 44 h (Fig. 8).
FIG. 7. The effects of AY9944-A-7 or BM15.766 on the apoptosis of cumulus cells cultured for 28 h. Free, COCs were cultured without FSH and LH for 28 h; COCs were cultured in FSH+LH-medium for 28 h; y-axis shows the proportion of TUNEL-positive cumulus cells. Over 300 cells in each treatment group were observed. Each experiment was repeated three times. P < 0.01, The addition of FSH and LH significantly decreased the proportion of apoptosis cells (TUNEL-positive cells).
FIG. 8. The effects of 10 μM AY9944-A-7 on progression to MII stage. COCs were cultured in FSH+LH-medium. *, The proportion of oocytes reached at the MII stage at 44 h is significantly decreased by the addition of 10 μM AY9944-A-7; **, the culture with 10 μM AY9944-A-7 for 44 h significantly increased the proportion of degenerated oocytes.
Discussion
The cholesterol biosynthetic pathway is regulated by the levels of cytochrome P450 superfamily enzymes, which are required for reduced nicotinamide adenine dinucleotide phosphate (NADPH) to function as a coactivator (27). It has been reported that 14-reductase and 7-reductase belong to the superfamily that catalyzes NADPH-dependent reduction in the conversion of acetyl-CoA to cholesterol (28, 29). The regulation of gene expression and the role played in steroidogenic cells, however, remain unclear. In the present study, we have, for the first time, shown that 14-reductase mRNA and 7-reductase mRNA are detected in cumulus cells and that the expression is markedly up-regulated by FSH and LH. During in vitro meiotic maturation of COCs, it has been shown in human (30), rat (31), cattle (32), and pig (20, 33, 34) that progesterone is produced by cumulus cells and that the level of progesterone is increased by stimulation with LH and FSH. Cytochrome P450scc, which cleaves the side chain of cholesterol, was also activated in gonadotropin-stimulated cumulus cells surrounding oocytes. Moreover, the messages for P450scc and 3 ?-HSD were induced in porcine cumulus cells of COCs that were cultured with FSH and LH (Y. Yamashita, M. Nishibori, and M. Shimada, unpublished data). Judging from these results, it is hypothesized that the gonadotropin stimulation induces the expression of 14-reductase, 7-reductase, P450scc, and 3 ?-HSD in cumulus cells, which results in an increase of progesterone production during meiotic resumption of oocytes.
We tested the role of 14-reductase and 7-reductase in cumulus cells during meiotic resumption of oocytes using inhibitors. AY9944-A-7 strongly inhibited 14-reductase and 7-reductase (36, 37, 38); the effects reached a plateau at 1 μM in rat liver cells (28). It has been reported that the inhibitory effects of BM15.766 on 7-reductase are linear to 20 μM in rat liver cells (39, 40). However, the concentrations in the medium cannot exceed 20 μM, because of the poor solubility of BM15.766, without producing solvent effects. The inhibitory effects of these drugs in progesterone production by porcine COCs have been found to reach a plateau at 1 μM AY9944-A-7 or 20 μM BM15.766. The levels of progesterone (AY9944-A-7, 5.53 ± 1.11 ng/ml; or BM15.766, 5.74 ± 1.06 ng/ml) are similar to those in medium where COCs have been cultured without gonadotropins. It has already been shown that exogenous lipoprotein-bound cholesterol ester can be used for progesterone production in ovarian follicular cells (4, 5). In corpus luteum, luteal cells contain numerous lipid droplets, and the stored cholesterol ester, which are hydrolyzed by cholesterol esterase, can be used as substrate for progesterone production (6). On the other hand, Armstrong (15) and Armstrong et al. (16) showed that the increase of progesterone production in rabbit ovary and bovine corpus luteum was exerted at a point subsequent to the biosynthesis of cholesterol. Thus, FSH- and LH-induced up-regulation of the cholesterol biosynthetic pathway in cumulus cells is also important to progesterone production during in vitro meiotic resumption of porcine oocytes.
The high levels of progesterone that are synthesized from cholesterol in cumulus cells accelerate meiotic resumption of porcine oocytes (7, 20). In the present study, the addition of 1 μM AY9944-A-7 or 20 μM BM15.766 to FSH+LH-medium significantly decreased the GVBD rate as well as progesterone production. Furthermore, the negative effects of 1 μM AY9944-A-7 or 20 μM BM15.766 on GVBD were overcome by the additional progesterone. These results suggest that up-regulation of the cholesterol biosynthetic pathway in cumulus cells is involved in meiotic resumption of porcine oocytes. Progesterone has been reported to mediate numerous physiological events of COCs during in vivo and in vitro maturation. We have recently shown that progesterone secreted by FSH- and LH-stimulated cumulus cells decreases the proliferative activity of cumulus cells and then induces differentiation, such as close of gap junctional communication and cumulus expansion (24, 25, 26). In the present study, the cumulus expansion of COCs was significantly suppressed when COCs were cultured with FSH, LH, and either 1 μM AY9944-A-7 or 20 μM BM15.766. The negative effects of both inhibitors on cumulus expansion were overcome by addition of 20 ng/ml progesterone. As such, we estimate that the expression and functions of 14-reductase and 7-reductase in cumulus cells are required for gonadotropin-induced cumulus cells differentiation and oocyte maturation via a progesterone-dependent pathway.
In contrast, in the present study, when COCs were cultured with FSH, LH, and high concentrations of AY9944-A-7 (10 μM), the GVBD rate was similar to that in oocytes cultured without the inhibitor. However, in the COCs treated with 10 μM AY9944-A-7, high levels of progesterone production and expansion of COC were not observed. We tested whether the higher concentrations of AY9944-A-7 increased toxicity to cumulus cells and then caused apoptosis of cumulus cells. The results showed that AY9944-A-7 (0, 1, or 10 μM) did not increase apoptosis of cumulus cells. Leonardsen et al. (41) reported that a high dose (5, 10, or 25 μM) of AY9944-A-7 accumulated Meiosis-activating sterols (FF-MAS), which induced meiotic resumption of oocytes. FF-MAS, which was first reported to be purified from human follicular fluid (4,4-dimethyl-5-cholesta-8,14,24-trien-3?-ol), was synthesized by COCs in response to FSH stimulation (42, 43). This type of sterol was found to be an intermediate in the cholesterol biosynthetic pathway produced by 14 -demethylase (35) and to be metabolized by 14-reductase (28). In the present study, meiotic resumption of oocytes was induced by treatment with a high dose of AY9944-A-7, whereas the further culture with AY9944-A-7 suppressed meiotic progression to the MII stage, and the majority of oocytes were degenerated. These results suggest that the functions of both 14-reductase and 7-reductase in cumulus cells support meiotic resumption and progression to the MII stage in porcine oocytes. However, further study is required for better understanding the effects of high doses of AY9944-A-7 and the role of FF-MAS in meiotic resumption of porcine cumulus-enclosed oocytes.
In conclusion, the expression of 14-reductase gene and 7-reductase gene is increased in FSH- and LH-stimulated cumulus cells. The addition of AY9944-A-7 or BM15.766 in FSH+LH-medium suppresses progesterone production in cumulus cells, cumulus cell expansion, and meiotic resumption of oocytes. The suppression of cumulus cell expansion and GVBD by AY9944-A-7 or BM15.766, however, can be overcome by additional progesterone. These results imply that progesterone, which is produced in FSH- and LH-stimulated cumulus cells though an intracellular cholesterol biosynthetic pathway, is involved in meiotic resumption of porcine oocytes.
Acknowledgments
Porcine FSH and LH were kindly provided by Dr. A. F. Parlow, the National Hormone and Pituitary Program, the National Institute of Diabetes and Digestive and Kidney Disease. AY9944-A-7 was kindly gifted by Dr. M. Abou-Gharbia, Wyeth Research; and BM15.766 was offered from Dr. S. Hauptmann, Roche Diagnostics GmbH, Germany. The authors are grateful to Dr. M. Fujita for technical advice on the use of HPLC-UV analysis. We thank Dr. J. Ito, Mr. T. Okazaki, and Mr. K. Kawahata for technical assistance, and the staff of the Meat Inspection Office in Hiroshima City for supplying the porcine ovaries.
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Address all correspondence and requests for reprints to: Masayuki Shimada, Ph.D., Department of Applied Animal Science, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8528, Japan. E-mail: mashimad@hiroshima-u.ac.jp.
Abstract
Progesterone is produced from cholesterol in cumulus cells during meiotic resumption of porcine oocytes. In follicular cells, it has been shown that exogenous lipoprotein-bound cholesterol ester can be used for steroid hormone production. However, in serum-free medium, progesterone is also secreted by FSH- and LH-stimulated cumulus-oocyte complexes, suggesting that progesterone could be produced from de novo synthesized cholesterol in cumulus cells. In the present study, we investigated the expression of 14-reductase and 7-reductase, which are the members of the superfamily that converts acetyl-CoA to cholesterol in cumulus cells. The expression of both genes was analyzed by RT-PCR. Both 14-reductase mRNA and 7-reductase mRNA in cumulus cells, cultured until 4 h, were under the level of detection limit. In response to gonadotropins, both mRNA levels were dramatically up-regulated, reaching a maximum at 20 h. To clarify the role of induced enzymes in cumulus cells, cumulus-oocyte complexes were cultured with either 14-reductase inhibitor, AY9944-A-7, or 7-reductase inhibitor, BM15.766. The results indicated that these inhibitors significantly suppressed the progesterone production in cumulus cells and meiotic progression of oocytes. The inhibitory effects reached a maximum at 1 μM AY9944-A-7 or 20 μM BM15.766. The addition of 20 ng/ml progesterone overcame the inhibitory effects of both drugs on meiotic resumption of oocytes. These results imply that gonadotropin-induced expression and function of 14-reductase and 7-reductase in cumulus cells contribute to oocyte meiotic resumption via a progesterone-dependent pathway.
Introduction
PROGESTERONE IS SYNTHESIZED from free cholesterol by cytochrome P450 cholesterol side-chain cleavage (P450scc) (1, 2) and 3?-hydroxysteroid dehydrogenase/isomerase (3?-HSD) (3). It has been shown that lipoprotein-bound cholesterol ester and stored cholesterol ester supply the major share of the cholesterol ester used for steroid hormone production (4, 5, 6). In our previous study, a 14 -demethylase inhibitor, ketoconazole, which suppressed the conversion of lanosterol to follicular fluid meiosis-activating sterol (FF-MAS) in a cholesterol biosynthetic pathway, was found to reduce both progesterone production in cumulus cells and germinal vesicle breakdown (GVBD) rate in cumulus-enclosed oocytes (7). The reduction of GVBD rate was overcome by the addition of progesterone to the gonadotropin and ketoconazole-containing medium. Moreover, Baranao and Hammond (8) have reported that FSH augments cholesterol synthesis in granulosa cells. As such, we hypothesized that progesterone is produced by de novo synthesized cholesterol in cumulus cells, which is stimulated by FSH and/or LH, with the newly synthesized progesterone playing an important role in meiotic resumption of porcine oocytes.
Numerous enzymes mediate the conversion of acetate to cholesterol in the sterol biosynthetic pathway (Fig. 1). Thaddeus and Strauss (9) have shown that exposure of cultured granulosa cells to 8-bromoadenosine cyclic 3',5'-phosphate results in a rapid increase in the content of the message for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. It has been reported that the levels of 14 -demethylase increased 6 times in rat ovary within 48 h after equine chorionic gonadotropin priming (10). Furthermore, a cAMP-responsive element (CRE) exists in the promoter region of the 14 -demethylase gene (11), and gene expression is dependent on the cAMP/CRE modulator-pathway (12). Thus, cAMP signaling stimulates both HMG-CoA reductase and 14 -demethylase gene expression. In fact, a high level of FF-MAS has been detected in gonadotropin-primed rabbit ovaries (13). Moreover, Baltsen (14) has reported an increased FF-MAS level after hCG stimulation, along with an increased level of progesterone in mouse ovary. The increasing level of progesterone in LH-stimulated rabbit ovary was significantly suppressed by 14-reductase inhibitor, AY9944-A-7 (15). The inhibitory effects of AY9944-A-7 on progesterone production were also observed in bovine luteinized cells (16) and porcine granulosa cells (17). These findings support our hypothesis that enzymes that play a role in the conversion from FF-MAS to cholesterol are also expressed in FSH- and LH-stimulated follicular cells, as well as HMG-CoA reductase and 14-demethylase. However, this hypothesis has not been confirmed.
FIG. 1. Simplified outline of the progesterone biosynthesis pathway. Three sources of cholesterol can be used as substrate: 1) exogenous cholesterol (HDL or LDL); 2) hydrolysis of stored cholesterol ester by cholesterol esterase; and 3) de novo synthesized cholesterol from acetate. Names of enzymes are in italics. Single arrow, Single enzymatic conversion; double arrow, multienzymatic step. AY9944-A-7 blocks the activity of both the 14-reductase and 7-reductase, and BM15.766 only blocks the activity of 7-reductase. T-MAS, Testicular-MAS.
In the present study, to investigate the role of the cholesterol biosynthetic pathway, especially from FF-MAS to cholesterol in progesterone production in cumulus cells, we analyzed the expression of 14-reductase gene and 7-reductase gene in cumulus cells when cumulus-oocyte complexes (COCs) were cultured with FSH and LH. We also examined the effects of AY9944-A-7 (14-reductase inhibitor) and BM15.766 (7-reductase inhibitor) on progesterone production and meiotic resumption of oocytes.
Materials and Methods
General protocol
Isolation of porcine COCs was described previously (18). The COCs were cultured in the maturation medium supplemented with or without 20 ng/ml highly purified porcine FSH [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Torrance, CA] and 0.5 μg/ml highly purified porcine LH (NIDDK). The maturation medium was modified NCSU37 (19), supplemented with 0.3% (wt/vol) polyvinylpyrrolidone (Sigma Chemical Co., St. Louis, MO), 2% (vol/vol) MEM amino acids (Life Technologies, Inc., Grand Island, NY), 1% (vol/vol) MEM nonamino acids (Life Technologies, Inc.), 7 mM Taurine (Sigma Chemical Co.), and 2 mM hypoxanthine (Sigma Chemical Co.). When COCs were cultured under the presence of serum, 10% (vol/vol) fetal calf serum (FCS) (Life Technologies, Inc.) was added to the maturation medium without PVP and amino acids (18). At selected time intervals, COCs were collected for RNA. Other COCs were cultured with 14-reductase inhibitor, AY9944-A-7 (kindly gifted by Dr. M. Abou-Gharbia, Wyeth Research, Madison, NJ), or 7-reductase inhibitor, BM15.766 (kindly gifted by S. Hauptmann, Roche Diagnostics GmbH, Mannheim, Germany). After cultivation, COCs were visualized using a phase-contrast microscope (Olympus IMT2, Olympus, Tokyo, Japan) and x10 objective. The cultured medium was used for the analysis of progesterone level according to our previous study (20, 21). The oocytes were fixed with acetic acids/ethanol (1:3) for 48 h and stained with aceto-lacmoid before examination under a phase-contrast microscope (x400) for evaluation of their chromatin configuration. Visualization of apoptotic cells (containing fragmented DNA) in chamber slides was performed by the terminal deoxynucleotidy transferase-mediated biotinylated deoxyuridine triphosphates nick end-labeling (TUNEL) method (In Situ cell Death Detection Kit, Roche Diagnostics GmbH) (22).
RNA isolation
After cumulus cells were separated from the oocytes, they were washed three times in PBS. Total RNA was extracted from cumulus cells using the SV Total RNA Isolation System (Promega, Madison, WI), according to the instruction manual, and dissolved in 20 μl nuclease-free water.
RT-PCR
RT-PCR was performed according to a coupled one-step procedure using Access RT-PCR System (Promega) with some modifications (23). Briefly, 10 ng total RNA was reverse transcribed at 48 C for 45 min, denatured at 94 C for 2 min, and amplified for 32 (?-actin) or 35 (14-reductase and 7-reductase) cycles of denaturation at 94 C for 30 sec, primer annealing at 56 C for 1 min, and extension at 68 C for 1 min, with a final extension step of 7 min at 68 C. The amplified products were analyzed by electrophoresis on 2% agarose gels.
Oligonucleotide primer used for amplification of the 14-reductase was from known cDNA sequences of human 14-reductase (GenBank accession no. AF096304). The upstream primer is identical with nucleotides 681–703 of the human cDNA, and the downstream primer represents the reverse complement of nucleotides 1135–1158 (Table 1). This primer pair predicts 430-bp DNA fragments. The primer used for amplification of the 7-reductase was from known cDNA sequences of human 7-reductase (GenBank accession no. AF034544). The upstream primer is identical with nucleotides 1–24 of human cDNA, and the downstream primer represents the reverse complement of nucleotides 537–560 (Table 1). This primer pair predicts 511-bp DNA fragments. ?-actin was used as a control for reaction efficiency and variations in concentrations of mRNA in the original RT reaction. The ?-actin primers were based on the mouse sequence (GenBank accession no. NM009609). The amplified cDNAs were directly sequenced according to our previous study (24).
TABLE 1. Primers used for determinations of the porcine 14-reductase, 7-reductase, and ?-actin mRNA by RT-PCR
Statistical analysis
Statistical analyses of all data from three or four separate culture experiments were carried out by one-way ANOVA followed by Duncan’s multiple-ranges test using STATVIEW (Abacus Concepts, Inc., Berkeley, CA). All percentage data were subjected to arc-sine transformation before statistical analysis.
Results
Time-dependent changes of 14-reductase gene and 7-reductase gene expression in cumulus cells of COCs
Partial cDNAs were amplified by RT-PCR from porcine cumulus cells using each primer set corresponding to the sequence for either human 14-reductase or 7-reductase, respectively (Table 1 and Fig. 2A). The similarity of the partial porcine 14-reductase sequence to human 14-reductase was 93.04%; and to bovine 14-reductase, it was 88.63% (Fig. 2B). The porcine 7-reductase sequence showed a high degree of similarity to human (91.06%), rat (88.55%), and mouse (86.23%) sequences (Fig. 2C). Sequence comparisons indicate that the partial porcine cDNAs amplified with either 14-reductase primer or 7-reductase primer conformed to the porcine 14-reductase region or the 7-reductase region, respectively.
FIG. 2. The expressions of 14-reductase and 7-reductase in cumulus cells of porcine COCs. A, RT-PCR analysis of 14-reductase mRNA and 7-reductase mRNA in cumulus cells of COCs cultured for 20 h with FSH and LH. B, Comparison of partial cDNA sequences of 14-reductase primers-RT-PCR with human 14-reductase (accession no. AF096304) or bovine 14-reductase (accession no. AF039681). C, Comparison of partial cDNA sequences of 7-reductase primers-RT-PCR with rat 7-reductase (accession no. AF071500), human 7-reductase (accession no. AF034544), or mouse 7-reductase (accession no. AF057368).
To investigate the time-dependent changes of both 14-reductase gene expression and 7-reductase gene expression in cumulus cells, COCs were cultured with or without both FSH and LH. The data showed low levels of both 14-reductase gene expression and 7-reductase gene expression in cumulus cells until 8-h cultivation (Fig. 3). However, in response to both FSH and LH, the dramatically increased levels of 14-reductase mRNA in cumulus cells were observed at later times of culture (12, 16, or 20 h) (Fig. 3A). The differences between the FSH+LH-treated group and control group were significant at 16 and 20 h. Significantly higher levels of 7-reductase mRNA were also observed at 20-h cultivation with FSH and LH as compared with those in cumulus cells cultured without gonadotropins (control) (Fig. 3B). When COCs were cultured with FSH and LH for 20 h under the presence of serum, the high levels of both genes expression were also detected in cumulus cells as compared with those in cumulus cells cultured without FSH and LH (Fig. 3C).
FIG. 3. Time-dependent changes of 14-reductase or 7-reductase mRNA level in cumulus cells cultured with FSH and LH. A, RT-PCR analysis of 14-reductase in cumulus cells of COCs cultured without FCS. The intensity of the amplified bands was quantified by densitometric scanning. The respective value of either 14-reductase was normalized according to those of ?-actin to evaluate arbitrary units of the relative abundance of the targets. *, The addition of FSH and LH to the medium significantly increased the level of 14-reductase mRNA in cumulus cells at 16 and 20 h (P < 0.05); control, COCs were cultured without gonadotropins. B, RT-PCR analysis of 7-reductase in cumulus cells of COCs cultured without FCS. The intensity of the amplified bands was quantified by densitometric scanning. **, The addition of FSH and LH to the medium significantly increased the level of 7-reductase mRNA in cumulus cells at 20 h (P < 0.01); control, COCs were cultured without gonadotropins. C, The effects of FCS on the expression of 14-reductase and 7-reductase. COCs were cultured with FCS, FSH, and LH. The intensity of the amplified bands was quantified by densitometric scanning. The addition of FSH and LH to the medium significantly increased the level of 14-reductase mRNA (*) or 7-reductase mRNA (**) in cumulus cells at 20 h (P < 0.05); control, COCs were cultured without gonadotropins.
The effects of AY9944-A-7 or BM15.766 on progesterone production by COCs and meiotic resumption of cumulus-enclosed oocytes
Both 14-reductase and 7-reductase were expressed in cumulus cells of COCs that were cultured with gonadotropins. In the next experiment, we examined the effects of 14-reductase inhibitor (AY9944-A-7) and 7-reductase inhibitor (BM15.766) on progesterone production and the meiotic progression of oocytes.
Low levels of progesterone were detected when COCs were cultured for 20 h without FSH and LH (control), whereas progesterone levels were significantly increased by the cultivation with FSH and LH (Fig. 4A). The higher progesterone levels in FSH+LH-medium were significantly decreased with increases in the AY9944-A-7 levels, and the inhibitory effect reached a plateau at 1 μM AY9944-A-7 (Fig. 4B). The treatment with BM15.766 also significantly reduced the level of progesterone levels in a dose-dependent manner (Fig. 4C).
FIG. 4. The effects of 14-reductase inhibitor (AY9944-A-7) or 7-reductase inhibitor (BM15.766) on progesterone production by COCs. A, The effects of adding FSH and LH (FSH+LH) on progesterone production by COCs cultured without FCS for 20 h. *, The effect of adding FSH+LH on progesterone production is significant (P < 0.05); control, COCs were cultured without gonadotropins. B, Effects of AY9944-A-7 on the progesterone production by COCs cultured without FCS for 20 h. a–c, Ratios with different letters are significant (P < 0.05). C, Effects of BM15.766 on the progesterone production by COCs cultured without FCS for 20 h. d–f, Ratios with different letters are significant (P < 0.05). D, The effects of adding 10% (vol/vol) FCS on progesterone production by COCs cultured with FSH and LH for 20 h. E, Effects of 1 μM AY9944-A-7 or 20 μM BM15.766 on progesterone production by COCs cultured with FCS, FSH, and LH for 20 h. P < 0.011, The effect of 1 μM AY9944-A-7 on the level of progesterone is significant (P < 0.01); P < 0.012, the effect of 20 μM BM15.766 on the level of progesterone is significant (P < 0.01).
The addition of 10% (vol/vol) FCS to the maturation medium did not significantly affect the progesterone production by COCs as compared with that by COCs cultured without FCS (Fig. 4D). Under the FCS-supplemented condition, the treatment with either 1 μM AY9944-A-7 or 20 μM BM15.766 significantly suppressed the progesterone production by COCs (Fig. 4F).
The proportion of oocytes exhibiting GVBD was 20.72 ± 8.14% in oocytes cultured without FSH and LH for 20 h. The addition of FSH and LH to the medium significantly increased the proportion of oocytes exhibiting GVBD (62.33 ± 8.19%, Fig. 5A). This higher rate was significantly decreased by 0.5 or 1 μM AY9944-A-7 as well as progesterone production. However, the GVBD rate increased again with higher concentrations of AY9944-A-7 (2.5–10 μM) (Fig. 5A). When COCs were cultured in FSH+LH-medium supplemented with BM15.766 (0, 2, or 20 μM) for 20 h, the proportion of oocytes exhibiting GVBD was significantly decreased in a dose-dependent manner (Fig. 5B).
FIG. 5. The effects of 14-reductase inhibitor (AY9944-A-7) or 7-reductase inhibitor (BM15.766) on meiotic resumption of cumulus-enclosed oocytes. A, The effects of adding FSH and LH (FSH+LH) on the exhibition of GVBD in cumulus-enclosed oocytes for 20 h. *, The addition of FSH+LH to the medium significantly increased the proportion of oocytes exhibiting GVBD (P < 0.05); control, COCs were cultured without gonadotropins. B, The effects of AY9944-A-7 on the exhibition of GVBD in oocytes cultured for 20 h. a–c, Ratios with different letters are significant (P < 0.05). C, The effects of BM15.766 on the exhibition of GVBD in oocytes cultured for 20 h. d–f, Ratios with different letters are significant (P < 0.05).
The effects of adding progesterone to FSH+LH-medium containing 1 μM AY9944-A-7 or 20 μM BM15.766 on meiotic resumption and cumulus cell expansion
Treatment with 1 μM AY9944-A-7 or 20 μM BM15.766 suppressed both the progesterone production by COCs and the exhibition of GVBD. In this experiment, we examined whether the additional progesterone overcame the inhibitory effects of AY9944-A-7 or BM15.766 on meiotic resumption of oocytes.
The production of progesterone by COCs and the formation of progesterone receptor type A in cumulus cells were dramatically increased after 10-h cultivation (24, 25). After COCs were cultured with FSH+LH-medium containing 1 μM AY9944-A-7 or 20 μM BM15.766 for 10 h, the COCs were further cultured for 10 h with an additional 20 ng/ml progesterone. When COCs were cultured in FSH+LH-medium containing 1 μM AY9944-A-7 or 20 μM BM15.766, the rates of GVBD were 21.3 ± 7.2% or 13.2 ± 8.9%, respectively. The addition of 20 ng/ml progesterone to the medium overcame the inhibitory effects of both drugs in inducing meiotic resumption of oocytes (Fig. 6A). The rates of GVBD in oocytes were 55.0 ± 7.1% (FSH+LH+1 μM AY9944 + 20 ng/ml progesterone) and 51.7 ± 2.9% (FSH+LH+20 μM BM15.766 + 20 ng/ml progesterone).
FIG. 6. The effects of adding progesterone to FSH+LH-medium containing AY9944-A-7 or BM15.766 on meiotic resumption and cumulus cell expansion. A, The proportion of oocytes exhibiting GVBD. After COCs were cultured with FSH+LH-medium containing 1 μM AY9944-A-7 or 20 μM BM15.766 for 10 h, the COCs were further cultured for 10 h with an additional 20 ng/ml progesterone. C (control), COCs were cultured in FSH+LH-medium for 20 h. P4, COCs were cultured with 1 μM AY9944-A-7 or 20 μM BM15.766 in FSH+LH-medium for 10 h and further cultured with an additional 20 ng/ml progesterone for 10 h. P < 0.011, The effect of adding progesterone on GVBD in oocytes cultured with 1 μM AY9944-A-7 is significant (P < 0.01); P < 0.012, the effect of adding progesterone on GVBD in oocytes cultured with 20 μM BM15.766 is significant (P < 0.01). B, After COCs were cultured with FSH and LH for 40 h, COCs were visualized using phase-contrast microscopy and a x10 objective. a, COCs were cultured for 40 h with FSH and LH; a’, COCs were cultured for 40 h with FSH, LH, and progesterone; b, COCs were cultured for 40 h with 1 μM AY-9977-A-7, FSH, and LH; b’, COCs were cultured for 40 h with 1 μM AY-9977-A-7, progesterone, FSH, and LH; c, COCs were cultured for 40 h with 10 μM AY-9977-A-7, FSH, and LH; c’, COCs were cultured for 40 h with 10 μM AY-9977-A-7, progesterone, FSH, and LH; d, COCs were cultured for 40 h with 20 μM BM15.766, FSH, and LH; d’, COCs were cultured for 40 h with 20 μM BM15.766, progesterone, FSH, and LH.
We have reported that cumulus expansion appears to be regulated by a progesterone- and progesterone receptor (PR)-dependent pathway in porcine COCs (26). To examine the effects of 1 or 10 μM AY9944-A-7 or 20 μM BM15.766 on cumulus expansion of COC, COCs were cultured for 40 h. The culture with FSH and LH for 40 h induced fully expansion of COC as indicated by an increase of about 2-fold in diameter of the complex and the blurred image of the oocyte (Fig. 6Ba). The expansion was not affected by an additional 20 ng/ml progesterone (Fig. 6Ba’). However, the expansion was significantly suppressed when COCs were cultured with FSH, LH, and either AY9944-A-7 or BM15.766 for 40 h (Fig. 6Bb-d). In these nonexpanded COCs, the cumulus cells remained packed around the oocytes, with little visible expansion (Fig. 6B, b–d). The addition of 20 ng/ml progesterone to the medium overcame the inhibitory effects of 1 μM AY9944-A-7 or 20 μM BM15.766 in inducing cumulus expansion of COC (Fig. 6B, b’ and c’). However, the negative effects of 10 μM AY9944-A-7 on cumulus expansion were not improved by addition of progesterone (Fig. 6Bd’).
The effects of 1 or 10 μM AY9944-A-7 on apoptosis of cumulus cells during meiotic resumption of oocytes.
Although 1 μM AY9944-A-7 significantly decreased the GVBD rate of oocytes, further increasing the concentrations (2.5, 5 or 10 μM) induced meiotic resumption of oocytes. We tested whether the higher concentrations of AY9944-A-7 increased toxicity to cumulus cells and then caused apoptosis of cumulus cells. After COCs were cultured in FSH+LH-medium supplemented with 1 or 10 μM AY9944-A-7 for 28 h, apoptosis of cumulus cells were observed by the TUNEL-method.
When COCs were cultured with FSH+LH-medium for 28 h, a low proportion (9.1 ± 1.6%) of cumulus cells showed signs of apoptosis (TUNEL-positive). This proportion was significantly lower than that of cumulus cells cultured without gonadotropins (34.5 ± 5.1%) (Fig. 7). This gonadotropin-induced lower rate was not affected by the treatment with 1 or 10 μM AY9944-A-7. Thus, the survival of cumulus cells of COCs was not threatened by AY9944-A-7. However, the majority of oocytes failed to progress to the MII stage and actually degenerated after the COCs were cultured with 10 μM AY9944-A-7 for 44 h (Fig. 8).
FIG. 7. The effects of AY9944-A-7 or BM15.766 on the apoptosis of cumulus cells cultured for 28 h. Free, COCs were cultured without FSH and LH for 28 h; COCs were cultured in FSH+LH-medium for 28 h; y-axis shows the proportion of TUNEL-positive cumulus cells. Over 300 cells in each treatment group were observed. Each experiment was repeated three times. P < 0.01, The addition of FSH and LH significantly decreased the proportion of apoptosis cells (TUNEL-positive cells).
FIG. 8. The effects of 10 μM AY9944-A-7 on progression to MII stage. COCs were cultured in FSH+LH-medium. *, The proportion of oocytes reached at the MII stage at 44 h is significantly decreased by the addition of 10 μM AY9944-A-7; **, the culture with 10 μM AY9944-A-7 for 44 h significantly increased the proportion of degenerated oocytes.
Discussion
The cholesterol biosynthetic pathway is regulated by the levels of cytochrome P450 superfamily enzymes, which are required for reduced nicotinamide adenine dinucleotide phosphate (NADPH) to function as a coactivator (27). It has been reported that 14-reductase and 7-reductase belong to the superfamily that catalyzes NADPH-dependent reduction in the conversion of acetyl-CoA to cholesterol (28, 29). The regulation of gene expression and the role played in steroidogenic cells, however, remain unclear. In the present study, we have, for the first time, shown that 14-reductase mRNA and 7-reductase mRNA are detected in cumulus cells and that the expression is markedly up-regulated by FSH and LH. During in vitro meiotic maturation of COCs, it has been shown in human (30), rat (31), cattle (32), and pig (20, 33, 34) that progesterone is produced by cumulus cells and that the level of progesterone is increased by stimulation with LH and FSH. Cytochrome P450scc, which cleaves the side chain of cholesterol, was also activated in gonadotropin-stimulated cumulus cells surrounding oocytes. Moreover, the messages for P450scc and 3 ?-HSD were induced in porcine cumulus cells of COCs that were cultured with FSH and LH (Y. Yamashita, M. Nishibori, and M. Shimada, unpublished data). Judging from these results, it is hypothesized that the gonadotropin stimulation induces the expression of 14-reductase, 7-reductase, P450scc, and 3 ?-HSD in cumulus cells, which results in an increase of progesterone production during meiotic resumption of oocytes.
We tested the role of 14-reductase and 7-reductase in cumulus cells during meiotic resumption of oocytes using inhibitors. AY9944-A-7 strongly inhibited 14-reductase and 7-reductase (36, 37, 38); the effects reached a plateau at 1 μM in rat liver cells (28). It has been reported that the inhibitory effects of BM15.766 on 7-reductase are linear to 20 μM in rat liver cells (39, 40). However, the concentrations in the medium cannot exceed 20 μM, because of the poor solubility of BM15.766, without producing solvent effects. The inhibitory effects of these drugs in progesterone production by porcine COCs have been found to reach a plateau at 1 μM AY9944-A-7 or 20 μM BM15.766. The levels of progesterone (AY9944-A-7, 5.53 ± 1.11 ng/ml; or BM15.766, 5.74 ± 1.06 ng/ml) are similar to those in medium where COCs have been cultured without gonadotropins. It has already been shown that exogenous lipoprotein-bound cholesterol ester can be used for progesterone production in ovarian follicular cells (4, 5). In corpus luteum, luteal cells contain numerous lipid droplets, and the stored cholesterol ester, which are hydrolyzed by cholesterol esterase, can be used as substrate for progesterone production (6). On the other hand, Armstrong (15) and Armstrong et al. (16) showed that the increase of progesterone production in rabbit ovary and bovine corpus luteum was exerted at a point subsequent to the biosynthesis of cholesterol. Thus, FSH- and LH-induced up-regulation of the cholesterol biosynthetic pathway in cumulus cells is also important to progesterone production during in vitro meiotic resumption of porcine oocytes.
The high levels of progesterone that are synthesized from cholesterol in cumulus cells accelerate meiotic resumption of porcine oocytes (7, 20). In the present study, the addition of 1 μM AY9944-A-7 or 20 μM BM15.766 to FSH+LH-medium significantly decreased the GVBD rate as well as progesterone production. Furthermore, the negative effects of 1 μM AY9944-A-7 or 20 μM BM15.766 on GVBD were overcome by the additional progesterone. These results suggest that up-regulation of the cholesterol biosynthetic pathway in cumulus cells is involved in meiotic resumption of porcine oocytes. Progesterone has been reported to mediate numerous physiological events of COCs during in vivo and in vitro maturation. We have recently shown that progesterone secreted by FSH- and LH-stimulated cumulus cells decreases the proliferative activity of cumulus cells and then induces differentiation, such as close of gap junctional communication and cumulus expansion (24, 25, 26). In the present study, the cumulus expansion of COCs was significantly suppressed when COCs were cultured with FSH, LH, and either 1 μM AY9944-A-7 or 20 μM BM15.766. The negative effects of both inhibitors on cumulus expansion were overcome by addition of 20 ng/ml progesterone. As such, we estimate that the expression and functions of 14-reductase and 7-reductase in cumulus cells are required for gonadotropin-induced cumulus cells differentiation and oocyte maturation via a progesterone-dependent pathway.
In contrast, in the present study, when COCs were cultured with FSH, LH, and high concentrations of AY9944-A-7 (10 μM), the GVBD rate was similar to that in oocytes cultured without the inhibitor. However, in the COCs treated with 10 μM AY9944-A-7, high levels of progesterone production and expansion of COC were not observed. We tested whether the higher concentrations of AY9944-A-7 increased toxicity to cumulus cells and then caused apoptosis of cumulus cells. The results showed that AY9944-A-7 (0, 1, or 10 μM) did not increase apoptosis of cumulus cells. Leonardsen et al. (41) reported that a high dose (5, 10, or 25 μM) of AY9944-A-7 accumulated Meiosis-activating sterols (FF-MAS), which induced meiotic resumption of oocytes. FF-MAS, which was first reported to be purified from human follicular fluid (4,4-dimethyl-5-cholesta-8,14,24-trien-3?-ol), was synthesized by COCs in response to FSH stimulation (42, 43). This type of sterol was found to be an intermediate in the cholesterol biosynthetic pathway produced by 14 -demethylase (35) and to be metabolized by 14-reductase (28). In the present study, meiotic resumption of oocytes was induced by treatment with a high dose of AY9944-A-7, whereas the further culture with AY9944-A-7 suppressed meiotic progression to the MII stage, and the majority of oocytes were degenerated. These results suggest that the functions of both 14-reductase and 7-reductase in cumulus cells support meiotic resumption and progression to the MII stage in porcine oocytes. However, further study is required for better understanding the effects of high doses of AY9944-A-7 and the role of FF-MAS in meiotic resumption of porcine cumulus-enclosed oocytes.
In conclusion, the expression of 14-reductase gene and 7-reductase gene is increased in FSH- and LH-stimulated cumulus cells. The addition of AY9944-A-7 or BM15.766 in FSH+LH-medium suppresses progesterone production in cumulus cells, cumulus cell expansion, and meiotic resumption of oocytes. The suppression of cumulus cell expansion and GVBD by AY9944-A-7 or BM15.766, however, can be overcome by additional progesterone. These results imply that progesterone, which is produced in FSH- and LH-stimulated cumulus cells though an intracellular cholesterol biosynthetic pathway, is involved in meiotic resumption of porcine oocytes.
Acknowledgments
Porcine FSH and LH were kindly provided by Dr. A. F. Parlow, the National Hormone and Pituitary Program, the National Institute of Diabetes and Digestive and Kidney Disease. AY9944-A-7 was kindly gifted by Dr. M. Abou-Gharbia, Wyeth Research; and BM15.766 was offered from Dr. S. Hauptmann, Roche Diagnostics GmbH, Germany. The authors are grateful to Dr. M. Fujita for technical advice on the use of HPLC-UV analysis. We thank Dr. J. Ito, Mr. T. Okazaki, and Mr. K. Kawahata for technical assistance, and the staff of the Meat Inspection Office in Hiroshima City for supplying the porcine ovaries.
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