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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
,
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* Physiological Sciences, and
and
Departments of Veterinary Science and Microbiology,
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Physiology, and
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Animal Sciences, University of Arizona, Tucson 85724; and
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¶ Dairy Veterinary Services, Chandler, AZ 85225
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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-hydroxylase mRNA in theca interna were sevenfold higher (P < 0.01) on d 4 than on d 8. After 3 h in culture, secretion of androstenedione by theca interna collected on d 4 (236 ± 44 pg/µg of protein) tended to be lower (P = 0.055) compared with d 6 (517 ± 162 pg/µg protein) and was lower (P < 0.05) compared with d 8 (387 ± 51 pg/µg of protein). In granulosa cells, amounts of aromatase mRNA decreased (P < 0.05) on d 8 compared with d 6 but not d 4. In vitro secretion of estradiol was higher in granulosa cells collected on d 4 (3.5 ± 0.8 ng/[105 cells x 3 h]) compared with d 6 (1.8 ± 0.6 ng/[105 cells x 3 h]; P < 0.05) and tended to be higher on d 4 than on d 8 (2.2 ± 0.2 ng/[105 cells x 3 h]; P = 0.058). We conclude that the decrease in estradiol production observed during atresia of the dominant follicle is not due to lack of androgen substrate for aromatization or downregulated expression of the aromatase gene, but may be the direct result of decreased activity of the aromatase enzyme within granulosa cells.
Key Words: Bovine • Granulosa Cells • Ovary • Steroidogenesis • Theca interna
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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). The capacity of first-wave dominant follicles to secrete large amounts of estradiol is lost during the plateau phase of growth before signs of morphological regression are evident (Xu et al., 1995; Austin et al., 2001). Expression of genes involved in steroidogenesis is altered and concentrations of estradiol in follicular fluid of the dominant follicle decrease as early as d 6 of the first follicular wave following ovulation (Xu et al., 1995; Bao et al., 1997). However, concentrations of estradiol in follicular fluid represent the pooled accumulation of estradiol secreted by the follicle and may not accurately reflect the steroidogenic capacities of the theca interna and granulosa cells at specific time points during the lifespan of the dominant follicle. Estradiol biosynthesis depends on the activity of 17-hydroxylase in theca interna for production of androstenedione and aromatase in the granulosa cell, which converts androgen into estradiol (Rodgers, 1990). It is possible that concentrations of estradiol in follicular fluid decrease because theca interna cells stop producing adequate amounts of androgen for aromatization. Another possibility is that androgen supplies are adequate, but aromatase activity in granulosa cells decreases. To determine which of these mechanisms mediates the decrease in estradiol biosynthesis in the first-wave dominant follicle, theca interna and granulosa cells were characterized for their ability to produce androstenedione and estradiol, respectively, at three times during the first follicular wave following ovulation. The dynamic capacity of follicular cells to produce estradiol is critical to gaining a better understanding of how selected follicles achieve, maintain, and eventually lose dominance over contemporary subordinate follicles. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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All animal procedures were approved by the University of Arizona Care and Use Committee and were within guidelines established by the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999). Sexually mature Holstein heifers were housed in dry lots, fed (DM basis) high-quality alfalfa hay (55%) and steam-flaked corn (15%) with mineral supplement (3%) plus concentrate mix (
10% whole cottonseed, distiller’s grains, wheat bran, tallow, molasses, citrus pulp), and provided with constant access to water and shade. Experiments were conducted from October through August.
Ultrasonography and Collection of Follicles
Estrous cycles of heifers were synchronized by injection of 25 mg of PGF2 (Lutalyse, Pharmacia & Upjohn, Kalamazoo, MI). Beginning on the day of estrus, blood samples were collected by coccygeal venipuncture, and ultrasonographic examination of ovaries was performed daily as previously described (Turzillo et al., 1990) using a real-time B-mode linear array ultrasound scanner equipped with a 7.5-MHz intrarectal probe (Aloka SSD-550V, Zug, Switzerland). Examinations were recorded on digital videotape (Handycam, Hi8 recording tape, Sony Electronics Inc., Park Ridge, NJ). Ovulation was identified by the disappearance of a large follicle preceded by estrus. Following ovulation, diameters and positions of all follicles 
4 mm in diameter were analyzed. Day 1 of the follicular wave was defined as the day two or more follicles 4 mm in diameter were first observed. These follicles continued to grow until one follicle deviated from the cohort to become dominant, while other follicles in the wave stopped growing and underwent regression.
The ovary bearing the dominant follicle was surgically removed via flank incision on d 4, 6, or 8 of the first follicular wave (n = 5/d). These days were chosen based on previous work (Xu et al., 1995; Bao et al., 1997) and were expected to encompass the dominant follicle’s transition from a healthy, estrogenic stage to decreased steroidogenic capacity without a significant change in diameter. Ovaries were placed in dissection medium (1x minimum Eagle’s essential medium with Earle’s salts and 25 mM HEPES, without L-glutamine; Life Technologies, Rockville, MD) and transported on ice 6.4 km to the laboratory. The dominant follicle was dissected from the ovarian stroma. Follicular diameter was measured using calipers and follicular fluid was aspirated and stored at –20°C. The collapsed follicle was cut into four pieces. Theca interna, along with the basement membrane and granulosa cells, was separated from the theca externa and remaining stroma using fine forceps. Using an angled, finely pulled Pasteur pipette, granulosa cells were scraped from the basement membrane and theca interna. Granulosa cells were collected in dissection medium, centrifuged for 15 min at 200 x g, resuspended in 1 mL of fresh dissection medium, and counted with a hemocytometer. Theca interna, attached to the basement membrane, and granulosa cells, following repelleting, were stored at –80°C until preparation of RNA or cultured as described below.
Template Generation and cRNA Probe Synthesis
Complementary DNA encoding bovine aromatase and bovine 17-hydroxylase was kindly provided by H. A. Garverick, University of Missouri (Xu et al., 1995). The 579-bp cDNA encoding aromatase (corresponding to bp 166 to 746 of GenBank Accession No. U18447) and a 407-bp BamHI fragment of the 17-hydroxylase cDNA (corresponding to bp 573 to 980 of GenBank Accession No. M12547) were ligated into the pBluescript SK plasmid (Stratagene, La Jolla, CA). A linear anti-sense template encoding 18S ribosomal RNA (pTRI RNA 18S) was purchased from Ambion, Inc. (Austin, TX). The identity and orientation of all cDNA were verified by dideoxy sequencing (Sanger et al., 1977).
To provide a shorter transcription product that could be used simultaneously with other cRNA probes for ribonuclease protection assay, the template for aromatase was linearized at sites within the cDNA insert using TaqI. The cDNA encoding 17-hydroxylase was linearized at an XbaI site within the vector. Anti-sense [32P] UTP-labeled cRNA probes were transcribed from linearized cDNA templates using T7 polymerase and the MAXiscript in vitro transcription kit (Ambion, Inc.) according to the manufacturer’s recommendations. Due to the abundance of 18S ribosomal RNA, the 18S antisense riboprobe was generated at one-tenth the specific activity of the other riboprobes. Antisense cRNA probes were purified on a denaturing acrylamide gel and used for hybridization within 1 d.
Ribonuclease Protection Assays
Following homogenization for 30 s with a hand-held Tissue Tearor (Biospec Products, Inc., Bartlesville, OK), total RNA was extracted from theca interna and granulosa cells by the guanidinium isothiocyanate-phenol-chloroform extraction procedure (Chomczynski and Sacchi, 1987) using TRIzol reagent (Life Technologies). Optical densities at 260 and 280 nm were measured and used to determine the quantity and purity of RNA samples. Ribonuclease protection assays were carried out using the RPA III ribonuclease protection assay kit (Ambion, Inc.) according to the manufacturer’s recommendations. Assays for aromatase mRNA, 17-hydroxylase mRNA, and 18S ribosomal RNA were done simultaneously using 3 µg of RNA isolated from granulosa cells or theca interna. All hybridizations were carried out at 50°C for 15 h, followed by incubation with a 1:50 dilution of RNase A/TI cocktail (Ambion, Inc.) and resolution of protected fragments on a 5% acrylamide/8 M urea gel. Following electrophoresis at 260 V for 2 h, gels were transferred to filter paper, covered with plastic wrap, and placed in an InstantImager Electronic Autoradiography System (Packard Instrument Co., Meriden, CT) for 15 min to quantify size and relative abundance of all protected fragments. Gels were also exposed with one intensifying screen overnight to Hyperfilm MP autoradiography film (Amersham Pharmacia Biotech Inc., Piscataway, NJ) at –80°C. Relative amounts of mRNA are expressed as counts per minute (cpm) ± SEM.
Theca Interna Culture
Techniques used in our laboratory for culture of theca interna have been described previously (Sanders et al., 2002). All materials for culture of theca interna and granulosa cells were obtained from Life Technologies unless otherwise noted. Theca interna from each dominant follicle was cut into 63 to 85 pieces (based on the calculated surface area of the follicles, which ranged in diameter from 13.5 to 16 mm). Tissue was placed in 24-well plates (three pieces per well, three wells per follicle) and cultured in 0.5 mL of medium consisting of 1x minimum Eagle’s essential medium with Earle’s salts (without L-glutamine) supplemented with 50 µg/ mL of penicillin/streptomycin, 2 mM L-glutamine, 0.1 mM nonessential AA, 5 µg/mL of transferrin (Collaborative Biomedical Products, Bedford, MA), 1 µg/mL of insulin, and 40 ng/mL of cortisol (Steraloids, Inc., Newport, RI) at 37°C with 5% CO2. At 3, 6, 12, and 24 h of culture, 0.5 mL of medium was collected from each well and replaced with fresh medium. Total protein was isolated from theca interna following the final collection of media by homogenation in lysis buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.35% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethanesulfonyl fluoride) with a hand-held Tissue Tearor (Biospec Products, Inc.). The lysate was centrifuged twice at 6,610 x g for 10 min at 4°C. Protein concentration was determined using the bicinchoninic acid protein assay kit (Pierce, Inc., Rockford, IL) according to the manufacturer’s recommendations.
Granulosa Cell Culture
Techniques used in our laboratory for culture of granulosa cells have been described previously (Sanders et al., 2002). The medium for granulosa cell cultures was identical to that used for theca interna cultures, except that 1% fetal bovine serum was added in the presence or absence of 10–7 M testosterone (Steraloids) as substrate for aromatase and estradiol production. Granulosa cells were cultured in 6-well plates (1 x 106 cells per well, three wells per treatment for each follicle) in 3 mL of medium per well. Cells were incubated at 37°C with 5% CO2. At 3, 6, 12, and 24 h of culture, 3 mL of medium were removed from all culture wells and replaced with fresh media.
Hormone Assays
Concentrations of progesterone in serum were measured using the Coat-A-Count progesterone RIA (Diagnostic Products Corp., Los Angeles, CA) according to the manufacturer’s recommendations. This assay was previously validated in our laboratory (Sanders et al., 2002). Serum was not extracted before assay. Sensitivity of the assay, calculated as two standard deviations below the mean cpm at maximum binding, was 0.02 ng/mL. The intraassay CV was 15.2%.
Concentrations of estradiol were measured in follicular fluid and media samples using the double antibody estradiol RIA (Diagnostic Products Corp.) according to the manufacturer’s recommendations, as previously validated for use in our laboratory (Sanders et al., 2002). Sensitivity of the assay, calculated as two standard deviations below the mean cpm at maximum binding, was 1.5 pg/mL. The intra- and interassay CV were 5.91 and 9.5%, respectively. Production of estradiol in culture media is expressed as the difference in estradiol concentrations between samples incubated with and without testosterone as nanograms of estradiol produced/(105 granulosa cells of dominant follicles x 3 h). Hormone production was expressed per 3 h to normalize data for length of time in culture and thereby facilitate comparison of production across the various time points (e.g., the 3- and 6-h time points were each collected after 3 h of culture, whereas the 12- and 24-h time points were collected after 6 and 12 h of culture, respectively).
Concentrations of androstenedione in media samples were measured using the Coat-A-Count androstenedione RIA (Diagnostic Products Corp.), according to the manufacturer’s recommendations and previously validated for use in our laboratory (Sanders et al., 2002). Sensitivity of the assay method was 0.05 ng/mL. The intra- and interassay CV were 3.51 and 5.55%, respectively. Androstenedione production in culture media is expressed as picograms of androstenedione/(microgram of protein x 3h).
Statistical Analyses
One- and two-way ANOVA were used to analyze differences among groups (SAS Inst., Inc., Cary, NC). The Shapiro-Wilk statistic and Fmax tests were used to assess normality and heterogeneity of variances, respectively. Means were separated by Duncan’s multiple range test; P < 0.05 was considered significant. All data are presented as mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Mean circulating concentrations of progesterone were undetectable on d 0 and increased to 2.4 ± 2.0, 5.2 ± 1.5, and 7.7 ± 2.0 ng/mL on d 4, 6, and 8 of the first follicular wave, respectively, indicating normal luteal function following ovulation. The first follicular wave was initiated 1.25 ± 0.11 d (range = 1 to 2 d) following ovulation, and in each heifer a single dominant follicle was selected from the recruited cohort. Emergence of the second wave of follicular development was not evident. Follicular diameters were similar among follicles collected on d 4, 6, or 8 (Table 1).
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). In theca interna, amounts of mRNA encoding 17-hydroxylase were lower (P < 0.05) in d 8 follicles than in those collected on d 4 (Table 1). Aromatase mRNA was not detectable in theca interna of any follicles (data not shown). In granulosa cells, amounts of mRNA encoding aromatase decreased (P < 0.05) in follicles collected on d 8 compared with those collected on d 6. Messenger RNA encoding 17-hydroxylase was not detected in granulosa cells of any follicles (data not shown). Androstenedione Production
After 3 h of culture, androstenedione production tended to be lower (P = 0.055) in theca interna collected on d 4 compared with d 6, and was lower on d 4 compared with d 8 (P < 0.05; Figure 1). At 6, 12, and 24 h of culture, androstenedione production was lower (P < 0.05) in theca interna collected on d 4 compared with d 6 and 8.
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). Amounts of androstenedione produced by theca interna collected on d 4 did not differ between the 12-and 24-h time points. In theca interna collected on d 6, androstenedione production decreased between 3 and 6 h, 6 and 12 h, and 12 and 24 h (P < 0.05). Androstenedione secretion by theca interna collected on d 8 did not differ between 3 and 6 h, but decreased (P < 0.05) between 6 and 12 h and 12 and 24 h of culture. Estradiol Production
After 3 h in culture, secretion of estradiol was higher in granulosa cells collected on d 4 (3.5 ± 0.8 ng/[105 cells x 3 h]) compared with d 6 (1.8 ± 0.6 ng/[105 cells x 3 h]; P < 0.05) and tended to be higher on d 4 than on d 8 (2.2 ± 0.2 ng/[105 cells x 3 h]; P = 0.058; Figure 2). At 6, 12, and 24 h of culture, secretion of estradiol did not differ among follicles collected on d 4, 6, and 8.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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). Estradiol exerts negative feedback on FSH secretion in cattle (Price and Webb, 1988). Therefore, the decrease in estradiol secretion by the first-wave dominant follicle may play an important physiological role by stimulating increased FSH concentrations temporally associated with emergence of the second wave of follicular development (Adams et al., 1992). The goal of this study was to gain insight into the cellular events leading to the decrease in estradiol production during the first follicular wave. To our knowledge, this is the first report describing steroidogenic capacities of individual follicular cell populations in the first-wave dominant follicle. As observed previously (Skinner and Osteen, 1988; Roberts and Skinner, 1990), steroidogenesis by both theca interna and granulosa cells decreased over the 24-h culture period, most likely due to dedifferentiation of the cells as they remain in culture. It is likely that the in vivo environment is most closely reflected by the earliest time point in vitro. Therefore, this discussion will focus primarily on differences in steroidogenesis after 3 h of culture.
During steroidogenesis in bovine follicles, cholesterol is converted to pregnenolone by cytochrome P450 side-chain cleavage in theca interna. Pregnenolone is then converted to dehydroepiandrosterone by P450 17-hydroxylase. Dehydroepiandrosterone is converted to androstenedione, which diffuses into granulosa cells and is used as substrate for estradiol synthesis by cytochrome P450 aromatase (Rodgers, 1990). We found that in theca interna, mRNA encoding 17-hydroxylase decreased by 50% between d 4 and 6 of the first follicular wave. However, this decrease was variable, and was not significant until d 8. Xu et al. (1995) also reported a decrease in 17-hydroxylase mRNA abundance during the first wave using in situ hybridization, but noted a significant decrease earlier in the wave between d 4 and 6. Based on decreases in concentrations of estradiol in follicular fluid, abundance of P450 side-chain cleavage mRNA, and amounts of P450 17-hydroxylase mRNA between d 4 and 6, Bao and Garverick (1998) suggested that the reduction in the ability of dominant follicles to produce estradiol on d 6 is due to insufficient androgen production by thecal cells. However, despite evidence that 17-hydroxylase mRNA decreases after d 4, we found that production of androstenedione after 3 h in vitro was actually higher by theca interna of follicles collected on d 6 compared to d 4. Although reasons for discordance between amounts of mRNA and hormone secretion are unclear, Carriere et al. (1996) reported that activity of 17-hydroxylase did not differ among growing dominant, nongrowing dominant, and nongrowing, nondominant bovine follicles. Taken together, these results support the claim that the decline in follicular fluid estradiol between d 4 and 6 of the wave is not caused by insufficient supply of androstenedione from theca interna.
Cytochrome P450 aromatase is the enzyme responsible for converting androstenedione to estradiol in granulosa cells of the bovine follicle. We found that amounts of mRNA encoding aromatase in granulosa cells of dominant follicles were similar on d 4 and 6 of the first follicular wave, but decreased between d 6 and 8. These results are consistent with Xu et al. (1995), who reported similar changes in aromatase mRNA during the first wave using in situ hybridization. The fall in aromatase mRNA occurred after the drop in concentrations of follicular fluid estradiol on d 6 in both studies; therefore, it seems that decreased expression of the aromatase gene is not the underlying cause of decreased estradiol production between d 4 and 6. After 3 h in culture, estradiol biosynthesis was lower in granulosa cells collected on d 6 compared with those collected on d 4. Therefore, despite high thecal production of androstenedione and maintenance of aromatase mRNA, it seems that decreased activity of the aromatase enzyme is the underlying cause of the fall in follicular fluid estradiol observed between d 4 and 6. Unfortunately, aromatase activity has not been measured in dominant follicles collected according to day of wave, and additional studies involving inhibition of P450 aromatase in granulosa cells are needed to confirm that activity of this enzyme is indeed the rate-limiting step in the production of estradiol, independent of aromatase mRNA expression. Carriere et al. (1996) reported that between d 5 and 18 of the estrous cycle, aromatase activity in bovine granulosa cells was lower in nongrowing, nondominant follicles compared with growing dominant and nongrowing dominant follicles. Rhodes et al. (2001) observed a greater than twofold decrease in concentrations of estradiol follicular fluid of dominant follicles between d 3 and 5 after ovulation, without a concomitant change in estrogen secretion in vitro. However, it is important to note that dominant follicles in their study were still growing (4.7 mm larger diameter on d 5 vs. d 3), whereas the dominant follicles in the present study had already reached maximal diameter. This discrepancy is indicative of fundamental differences in follicular dynamics between the beef heifers used by Rhodes et al. (2001) and the dairy heifers in the present study. Badinga et al. (1992) reported that aromatase activity, defined in their studies as conversion of [1,2-3H] testosterone into 3H2O and estrogens in follicle wall of first-wave dominant follicles, did not differ between d 5 and 8 of the estrous cycle, corresponding roughly to d 4 and 7 of the wave respectively. However, the presence of theca interna in follicle wall complicates comparison of these results with those derived from isolated granulosa cells. Because thecal supply of androgen does not seem to be limited on d 6 of the wave, we cannot rule out the possibility that in vivo, theca interna contributes some factor other than androgen that stimulates aromatase activity.
During the normal bovine estrous cycle, the dominant follicle of the first follicular wave inevitably undergoes atresia. Cessation of estradiol production is one of the earliest events in the atretic process (Xu et al., 1995), and atresia in bovine follicles is characterized by apoptosis of granulosa cells (Jolly et al., 1994). The goal of the current study was not to characterize apoptosis but to understand the cellular basis for the dramatic decrease in estradiol biosynthesis by the dominant follicle of the first wave. However, in related studies we have found that the incidence of apoptosis, as measured by annexin-V staining, in bovine granulosa cells tends to increase between d 4 and 6 of the first follicular wave (K. E. Valdez and A. M. Turzill, unpublished results). In the same studies, the percentage of nonviable granulosa cells (positively stained with propidium iodide) increased between d 4 and 6 and remained elevated on d 8. The temporal association among increased apoptosis and/or death of granulosa cells, the threefold decrease in concentration of estradiol in follicular fluid, and the approximately 50% decrease in estradiol biosynthesis by granulosa cells in vitro leads us to agree with previous speculation (Bao and Garverick, 1998) that atresia of the bovine dominant follicle is initiated between d 4 and 6 of the first follicular wave, several days before morphological regression is evident.
In summary, our findings indicate that decreased ability of granulosa cells to produce estradiol precedes a loss in aromatase mRNA and is independent of thecal contribution of androstenedione. Thus it seems that disruption of the steroidogenic pathway in bovine dominant follicles of the first wave is initiated in the granulosa cells rather than the theca interna and may be the direct result of decreased activity of the aromatase enzyme. Future studies are needed in first-wave dominant follicles to verify the loss of aromatase activity in granulosa cells, identify its cause, and thereby provide further insight into mechanisms that mediate the dynamic pattern of estradiol biosynthesis during early luteal phase in cattle.
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Footnotes |
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-hydroxylase and the Univ. of Arizona Dairy Research Facility for providing daily care and management of the animals used in this study. 
2 This research was supported by National Research Initiative Competitive Grant 00-35203-9167 from the USDA Cooperative State Research, Education, and Extension Service.
3 Correspondence: U.S. FDA, Center for Veterinary Medicine, 7500 Standish Place, Rockville, MD 20855 (phone: 301-251-3315; fax: 301-594-2298; e-mail: adele.turzillo{at}fda.hhs.gov).
Received for publication August 15, 2004. Accepted for publication November 29, 2004.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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-hydroxylase and cytochrome P450 aromatase in bovine follicles during the first follicular wave. Endocrinology 136:981–989.[Abstract]
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