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a Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada J2S 7C6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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FSH stimulated estradiol secretion and P450arom mRNA abundance; the effect was quadratic for both estradiol and P450arom mRNA. Estradiol secretion and P450arom mRNA levels were correlated. FSH stimulated progesterone secretion and P450scc mRNA abundance, although the minimum effective dose of FSH was lower for estradiol (0.1 ng/ml) than for progesterone (10 ng/ml) production. Insulin alone stimulated estradiol secretion and P450arom mRNA levels but not progesterone or P450scc mRNA abundance. We conclude that this cell culture system maintained both estradiol secretion and P450arom mRNA abundance responsiveness to FSH and insulin, whereas P450scc mRNA abundance and progesterone secretion were responsive to FSH but not insulin.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Estradiol is synthesized in granulosa cells from thecal androgens, by two successive enzyme-catalyzed reactions [1,2]. In cattle, androstenedione is the preferred substrate, which is converted to estrone by the cytochrome P450 aromatase complex (P450arom). Estrone is then converted to estradiol by the enzyme 17ß-hydroxysteroid dehydrogenase. Measuring estradiol in follicular fluid allows a good estimation of the estrogenic capability of a follicle. Estradiol concentrations in small antral follicles are modest relative to those in large growing follicles [3]. Peak follicular estradiol concentrations are generally obtained during the growth phase of the dominant follicle, and estradiol concentrations decrease considerably as the follicle reaches its maximum diameter [4,5]. The loss of estradiol synthetic capability is one of the early signs of follicle atresia [6,7] or regression [4,5,8].
Studies in vivo have shown that P450arom mRNA abundance increases in small follicles during the emergence of a nonovulatory follicle wave, then declines in all but the dominant follicle [9,10]. These data are consistent with the above-mentioned follicular fluid estradiol measurements. However, there are circumstances in which P450arom mRNA levels and estradiol concentrations are not so closely associated. For example, significant decreases in follicular estradiol concentrations were observed before P450arom mRNA levels decreased [9]; and during final preovulatory follicle maturation, there is increased follicular estradiol content in the absence of increased P450arom mRNA levels [11]. Dissociations between P450arom mRNA, protein levels, and aromatase activity have also been observed in rat gonadal tissues in vivo [12,13], and it has also been reported that P450arom mRNA levels were not significantly correlated with aromatase activity in human breast tumors [14]. Thus it should not be assumed that mRNA levels reflect enzyme activity for aromatase.
A problem with many such in vivo studies is the very complexity of follicular steroidogenesis. Numerous enzymes are involved, and a change in the activity of any one may affect precursor supply to another. Additionally, steroid synthesis is affected by several trophic hormones and probably paracrine factors as well. The study of granulosa cell function under defined conditions in vitro would permit a detailed examination of the actions of specific hormones on steroidogenesis, without the above-mentioned complications. However, the study of estradiol secretion and P450arom expression in ruminant granulosa cells has not been feasible until recently, as estradiol production [15–18] and P450arom mRNA levels are either not detectable [19] or decline rapidly [20] in these cells in vitro. A culture system has now been developed by Webb and colleagues, that permits the induction and maintenance of estradiol secretion in ovine [21] and bovine [22] granulosa cells in vitro.
The aim of the present study was to study P450arom mRNA abundance and estradiol secretion in bovine granulosa cells in vitro. We tested the hypotheses that FSH stimulates P450arom mRNA levels in nonluteinizing bovine granulosa cells, and that estradiol secretion and mRNA abundance are correlated in this culture system. Because granulosa cells secrete progesterone as well as estradiol [1,21,22], we also determined the effect of FSH on cytochrome P450 cholesterol side-chain cleavage (P450scc).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To estimate the time course of steroidogenic enzyme gene expression, granulosa cells were cultured for up to 8 days with 10 ng/ml bovine FSH (bFSH) (USDA-bFSH 17) and 100 ng/ml insulin. Cells were recovered on each of Days 2, 4, 6, and 8 of culture. Steroids were measured in the medium collected at the same time as the cells, and this measurement thus represents steroid released during the 48-h period before Day 2, Day 4, Day 6, and Day 8, respectively. This experiment was performed 3 times.
To determine the effects of gonadotropin on steroidogenic enzyme gene expression and estradiol secretion, granulosa cells were cultured for 6 days in the absence or presence of 0.1, 1, 10, or 100 ng/ml bFSH. An additional treatment was culture with 100 ng/ml insulin in the absence of FSH. This experiment was performed 4 times.
To obtain sufficient RNA to measure P450arom mRNA, all cells from an entire plate were used for each treatment (time point or hormone dose). Cells of each plate were pooled for RNA and DNA extraction, and the medium from all wells of each plate was pooled. Conditioned medium was stored at -20°C until assayed for estradiol and progesterone.
Cell Culture
The cell culture system was based on that described by Campbell et al. [21]. All materials were obtained from Gibco BRL Canada (Burlington, ON, Canada) unless otherwise stated.
Bovine ovaries were collected from adult cows, irrespective of stage of the estrous cycle, at a local abattoir and were transported to the laboratory in M199 containing 25 mM Hepes, penicillin (100 IU/ml), streptomycin (100 µg/ml), and fungizone (1 µg/ml). Follicles were dissected free of surrounding tissue, and small follicles (2–4-mm diameter) were bisected into Dulbecco's PBS (without calcium and magnesium) at 37°C. Granulosa cells were recovered by passing the follicle walls repeatedly through a 1-ml disposable pipette. The follicle walls were allowed to sediment out under gravity, and the granulosa cell suspension was transferred to sterile centrifuge tubes. Cells were isolated by centrifugation at 800 x g for 5 min and washed 3 times in M199 containing 25 mM Hepes, penicillin (100 IU/ml), and streptomycin (100 µg/ml). Washed cells were resuspended in culture medium, and cell viability was estimated at 40% by Trypan blue exclusion.
Cells were seeded into 24-well tissue culture plates (Corning Glass Works, Corning, NY) at a density of 106 viable cells in 1 ml of Minimum Essential Medium (
-modification) with L-glutamine containing sodium bicarbonate (10 mM), Hepes (20 mM), protease-free BSA (0.1%), selenium (4 ng/ml), transferrin (2.5 µg/ml), androstenedione (10-7 M), insulin (10 ng/ml), human recombinant insulin-like growth factor I (10 ng/ml), nonessential amino acid mix (1.1 mM), penicillin (100 IU/ml), and streptomycin (100 µg/ml). Cultures were maintained at 37°C in 5% CO2 in air for up to 8 days, with 700 µl medium being replaced every 2 days.
Total RNA and DNA were extracted using Trizol (Gibco BRL) according to the manufacturer's instructions. Total DNA was quantified in duplicate by measuring fluorescence in the presence of Hoechst 33258 [23] and compared with a calf thymus DNA standard (Boehringer-Mannheim, Laval, PQ, Canada).
Hybridization
The relative abundance of mRNA for each protein was determined by Northern hybridization [24]. Electrophoresis of 15 µg RNA, performed through a 1% denaturing formaldehyde-agarose gel, was followed by overnight capillary transfer onto a nylon membrane (Hybond-N; Amersham, Oakville, ON, Canada). Membranes were UV cross-linked in a commercial UV chamber (Bio-Rad, Mississauga, ON, Canada) and incubated for 2 h in prehybridization solution containing 10% dextran sulfate, 5-strength saline-sodium phosphate-EDTE buffer (SSPE [single-strength SSPE is 0.75 M NaCl, 50 mM NaH2PO4·H2O, 5 mM EDTA, pH 7.4], 5-strength Denhardt's solution [single-strength Denhardt's is 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% BSA], 0.5% SDS, and herring sperm DNA [200 mg/ml]).
The bovine aromatase cDNA probe was prepared in our laboratory [25] and encompasses the entire heme-binding and I-helix regions of the open reading frame. The probe is specific for estrogen-secreting tissues, and it hybridizes to 3 bands of follicular total RNA at 6.5, 3.4, and 1.8 kilobases (kb) [25]. The bovine P450scc cDNA was a gift from Dr. M.R. Waterman (Vanderbilt University School of Medicine, Nashville, TN); it is a 1.7-kb cDNA containing the complete coding sequence [26], which hybridizes to a single band of 2 kb [27].
Probes were labeled with [-32P]dCTP by random primer extension, using a kit from Boehringer-Mannheim to a specific activity of 1.5–3.0 x 109 dpm/µg, and purified by centrifugation through a minicolumn using the Wizard PCR Preps DNA purification system (Promega, Montréal, PQ, Canada). Hybridization to the membranes was performed overnight at 65°C. After hybridization, membranes were washed in double-strength SSPE-0.1% SDS, twice at room temperature (15 min each) and twice at 65°C (15 min each). Membranes were then stripped and rehybridized to a labeled human 28S ribosomal cDNA probe [28] for the standardization of RNA loading. The labeled membranes were exposed to Kodak X-Omat film at -70°C in the presence of an intensifying screen. Autoradiograms were scanned with a densitometer after 1–14 days exposure.
Steroid Assays
Estradiol was measured in conditioned medium in duplicate without extraction with the assay described by Bélanger et al. [29], with modifications [5]. Inter- and intraassay coefficients of variation were less than 12%. Progesterone was measured in duplicate as described [30] with inter- and intraassay coefficients of variation of less than 10%. The sensitivity of these assays was equivalent to 0.25 and 1 ng/ml medium for estradiol and progesterone, respectively.
Statistics
The density of hybridization signals was corrected for loading efficiency using hybridization to 28S ribosomal RNA. Steroid concentrations were corrected for cell number by expressing steroid mass per unit mass of DNA. Data were transformed to logarithms if they were not normally distributed (Shapiro-Wilk test). ANOVA was used to test main effects of FSH and of insulin treatments. Culture replicate was included in the model as an error term. Differences between groups were identified with the Tukey-Kramer HSD test. Correlations between steroid secretion and mRNA abundance were determined with the Pearson correlation coefficient. Analyses were performed with JMP software (SAS Institute, Cary, NC). The data are presented as means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Estradiol secretion from granulosa cells and the relative abundance of P450arom mRNA increased from Day 2 to Day 4 of culture (P < 0.01; Fig. 1A) but did not change significantly thereafter. Estradiol secretion and P450arom mRNA abundance were correlated between Days 2 and 6 of culture (r = 0.68, P < 0.05, n = 9) but were not correlated if the analysis included data from Day 8 (P > 0.05). As expected, the P450arom cDNA probe recognized three transcripts in freshly harvested granulosa cells, although one major band was detected in cultured cells. However, when signal strength was stronger, bands of lower molecular weight could be detected (Fig. 2).
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Progesterone secretion increased with time of culture (P < 0.01; Fig. 1B) and reached a maximum on Day 8. Relative abundance of P450scc mRNA did not change with time in culture (P > 0.05; Fig. 1B), and progesterone secretion and P450scc mRNA were not correlated (r = 0.42, P > 0.05, n = 12).
The mean DNA content of the cultures did not change with time in culture (4.9 ± 1.1, 4.1 ± 0.8, 5.1 ± 0.5, and 3.4 ± 1.2 µg/well, for Days 2, 4, 6, and 8, respectively; P > 0.05).
Effect of FSH on Steroid Secretion and Steroidogenic Enzyme mRNA Abundance
There were main effects of treatment on both estradiol secretion and relative P450arom mRNA abundance (P < 0.01), and no effect of culture replicate (P > 0.05). For cells cultured with 10 ng insulin but without FSH, estradiol secretion was low and was stimulated by as little as 0.1 ng/ml bFSH (Fig. 2). The addition of 1 ng/ml bFSH increased estradiol secretion above that observed with 0.1 ng/ml, although greater doses of FSH did not further affect estradiol secretion. There was a quadratic relationship between dose of FSH and estradiol secretion (P < 0.05). Insulin alone at 100 ng/ml stimulated estradiol secretion compared to insulin alone at 10 ng/ml (P < 0.01; Fig. 2).
Relative P450arom mRNA abundance was very low in the absence of FSH and increased to a maximum in the presence of 1 ng/ml FSH (P < 0.05; Fig. 2). The relationship between FSH and relative P450arom mRNA abundance was quadratic (P < 0.01), and estradiol secretion and P450arom mRNA abundance were correlated (r = 0.57, P < 0.05). Insulin alone at 100 ng/ml stimulated P450arom mRNA abundance relative to insulin alone at 10 ng/ml (P < 0.05).
Progesterone secretion was not significantly stimulated by the addition of low doses of FSH (0.1 or 1 ng/ml) to cells cultured with 10 ng insulin (Fig. 3) but was stimulated with higher doses of FSH (10 and 100 ng/ml). Insulin alone at 100 ng/ml did not stimulate progesterone compared to insulin alone at 10 ng/ml.
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Relative abundance of P450scc mRNA increased with dose of FSH in a linear fashion (r = 0.88, P < 0.001; Fig. 3); however, insulin alone at 100 ng/ml did not stimulate P450scc mRNA abundance (P > 0.05). Progesterone secretion and P450scc mRNA abundance were correlated (r = 0.63, P < 0.001).
The DNA content of the cultures was not affected by dose of FSH (7.2 ± 0.6, 5.5 ± 0.3, 6.7 ± 0.5, 6.8 ± 0.8, and 5.9 ± 0.7 µg/well, for 0, 0.1, 1, 10, and 100 ng/ml FSH, respectively; P > 0.05) or insulin (4.6 ± 0.7 µg/well for 100 ng/ml insulin).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The initial expression of P450arom mRNA in small (< 4 mm) follicles in vitro in the present study was barely detectable, which is in agreement with in situ hybridization studies in vivo [9,10]. In vivo, P450arom mRNA abundance increased between Day 2 and Day 4 after the emergence of a follicle wave, but it did not increase further between Day 4 and Day 6 [9]. This temporal pattern of P450arom mRNA appearance is very similar to that observed in vitro (present study) and suggests that this is a good model for the study of follicle recruitment. It has been suggested [10] that the increase in P450arom mRNA abundance in medium-sized follicles during emergence of the follicle wave is stimulated by the transient rise in plasma FSH concentrations that precedes wave emergence [32]. The present data are the first to demonstrate a direct effect of FSH on P450arom mRNA abundance in cattle and thus support the role of FSH in this respect. It is interesting to note that physiological concentrations of FSH resulted in greatest stimulation of P450arom mRNA levels, whereas supraphysiological concentrations resulted in a somewhat muted response. Corresponding data are not available in vivo in cattle, although it is known that superovulation with FSH results in small follicles (3–5 mm) with P450arom mRNA content similar to that of nonstimulated preovulatory follicles [25]. In rats, a biphasic response of ovarian P450arom mRNA levels to FSH was observed in vivo [33], but not in vitro, when increasing concentrations of FSH (generally above 100 ng/ml) further stimulated estradiol secretion and P450arom mRNA levels [33,34].
The mechanism of the biphasic response of P450arom mRNA to FSH is not known, although it is clearly specific to P450arom; no such effect was observed for P450scc. Whereas estradiol secretion and P450arom mRNA levels are stimulated by FSH in rats, they are markedly inhibited by high levels of LH [33]. It has been proposed that FSH normally induces relatively small increases in intracellular cAMP, which stimulate steady-state P450arom mRNA levels, whereas LH induces much higher levels of cAMP accumulation, which in turn lead to luteinization and a reduction in steady-state P450arom mRNA levels [33]. Thus, in the present experiment, 10 and 100 ng/ml FSH may have resulted in concentrations of intracellular cAMP that were sufficient to down-regulate P450arom mRNA levels. It has been suggested that high LH levels may also activate the protein kinase-C second messenger pathway, leading to decreased P450arom mRNA levels [35]. As an alternative explanation, Rouillier et al. [36] proposed that the biphasic response of estradiol secretion to FSH may be mediated through a down-regulation of granulosa cell FSH receptors. This is less likely to be valid in the present study, as the higher doses of FSH did not down-regulate P450scc mRNA abundance.
In contrast to the effects on P450arom mRNA levels, FSH did not have a biphasic effect on estradiol secretion at the dosages evaluated. Thus, we did not observe the inhibition of estradiol secretion reported for high doses of FSH [18,22,31,36]. This discrepancy may be the result of the use of different preparations of FSH of different biopotencies between laboratories. Another potential explanation includes the source of the cells. In previous studies, cells were obtained from large (> 8-mm-diameter) follicles [18,31,36], whereas the cells in this report were obtained from small (2–4-mm-diameter) follicles. Cells from small follicles were also studied by Gutiérrez et al. [22], and in the presence of insulin, FSH had no significant effect on estradiol secretion. As estradiol secretion did not decrease in concert with the decrease in P450arom mRNA abundance observed with high doses of FSH, it is possible that FSH modulates aromatase activity at two levels: transcription and translation/enzyme activity.
The linear effect of FSH on progesterone secretion and P450scc mRNA abundance is similar to that previously observed in rat, pig, and bovine granulosa cell cultures [18,31,37,38]. Interestingly, estradiol secretion was more sensitive to FSH compared to progesterone secretion; 1 ng/ml FSH induced near-maximum estradiol secretion, whereas this dose did not significantly affect progesterone secretion. This divergent effect of FSH on estradiol and progesterone secretion is similar to, though not as marked as, that observed for human granulosa cells [39].
In the present study, increasing insulin concentrations from 10 to 100 ng/ml significantly increased estradiol secretion and P450arom mRNA abundance but had no effect on P450scc mRNA abundance or progesterone secretion. This is not fully in agreement with the literature, as it is generally considered that the secretion of both estradiol and progesterone from bovine granulosa cells is stimulated by insulin in vitro (reviewed in [40]). However, in many of these studies, the cells are spontaneously luteinizing and/or estradiol secretion is not responsive to FSH [41,42]. In a study with FSH-responsive granulosa cells, insulin alone at 100 ng/ml stimulated progesterone but not estradiol secretion [31]. In the first report of the culture system used herein for bovine granulosa cells, insulin stimulated estradiol secretion [22], although the effects of insulin on progesterone secretion were not reported. Thus these data suggest that there is a developmental switch in the role of insulin, from stimulation of estradiol secretion in nonluteinizing cells to stimulation of progesterone in luteinizing cells.
We conclude from these data that the present cell culture technique permits the stimulation of P450arom mRNA accumulation as well as estradiol secretion from granulosa cells from small bovine follicles. Both estradiol secretion and P450arom mRNA abundance were responsive to FSH and insulin, whereas P450scc mRNA abundance and progesterone secretion were responsive to FSH but not insulin. This cell culture system may provide a good model for studying the mechanisms of follicle recruitment in vitro.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from NSERC Canada and CONACYT, Mexico. 
2 Correspondence: C.A. Price, CRRA, Faculté de médecine vétérinaire, Université de Montréal, 3200 Sicotte, St-Hyacinthe, PQ, Canada J2S 7C6. FAX: 450 778 8103; pricec{at}medvet.umontreal.ca
Accepted: August 24, 1999.
Received: May 18, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit and -ß dimer) in morphologically dominant follicles during their growing and regressing phases of development in cattle. Biol Reprod 1993; 48:268–276.[Abstract]-hydroxylase, and cytochrome P450 aromatase in bovine follicles during the first follicular wave. Endocrinology 1995; 136:981–989.[Abstract]-hydroxylase, but not P450 aromatase. Endocrinology 1995; 136:5102–5110.[Abstract]-hydroxylase and P450 aromatase in preovulatory bovine follicles decrease after the luteinizing hormone surge. Endocrinology 1993; 132:2339–2245.-hydroxylase and aromatase messenger ribonucleic acids in cattle undergoing superovulation. Biol Reprod 1996; 55:1419–1426.[Abstract] in heifers. Biol Reprod 1985; 33:1113–1119.[Abstract]This article has been cited by other articles:
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