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Regular Article |
4,5 Isomerase Compared to Normal Dominant Follicles
a Departments of Animal Sciences,
b Veterinary Medicine and Surgery,
c Biochemistry and Child Health, University of Missouri-Columbia, Columbia, Missouri 65211-5300
ABSTRACT
The objective was to compare ovarian steroids and expression of mRNAs encoding cytochrome P450 side-chain cleavage, cytochrome P450 17
-hydroxylase, cytochrome P450 aromatase, 3ß-hydroxysteroid dehydrogenase 
4,5 isomerase, LH, and FSH receptors and estrogen receptor-ß in ovaries of cows with dominant and nondominant ovarian follicular cysts and in normal dominant follicles. Estradiol-17ß, progesterone, and androstenedione concentrations were determined in follicular fluid using specific RIAs. Dominant cysts were larger than young cysts or dominant follicles, whereas nondominant cysts were intermediate. Estradiol-17ß (ng/ml) and total steroids (ng/follicle) were higher in dominant cysts than in dominant follicles. Expression of LH receptor and 3ß-hydroxysteroid dehydrogenase mRNAs was higher in granulosa cells of dominant cysts than in dominant follicles. Nondominant cysts had higher follicular concentrations of progesterone, lower estradiol-17ß concentrations, and lower expression of steroidogenic enzyme, gonadotropin receptor, and estrogen receptor-ß mRNAs than other groups. In summary, increased expression of LH receptor and 3ß-hydroxysteroid dehydrogenase mRNAs in granulosa and increased follicular estradiol-17ß concentrations were associated with dominant cysts compared to dominant follicles. Study of cysts at known developmental stages is useful in identifying alterations in follicular steroidogenesis.
estradiol receptor, follicle, follicular cyst, gene regulation, gonadotropin receptors, granulosa cells, steroidogenic enzymes, theca cells
INTRODUCTION
Follicular growth occurs in two or three waves during the normal bovine estrous cycle [1, 2]. Each wave is characterized by recruitment of (n = 2–6) follicles of 4–5 mm, followed by selection of one follicle to continue growth and become a dominant follicle (DF). In some cases, DFs fail to ovulate and continue to grow into ovarian follicular cysts (cysts).
In cows with normal estrous cycles, FSH receptor (FSHr) mRNA expression is localized to granulosa cells of follicles of all sizes starting at the preantral stage [3]. Expression of LH receptor (LHr) mRNA occurs in theca cells of healthy bovine follicles of all sizes but is expressed in granulosa cells of only DFs larger than 9 mm in size [3, 4]. Cytochrome P450 side-chain cleavage (P450scc) mRNA is expressed in theca cells of all healthy follicles but is only expressed in granulosa cells following recruitment of 5 mm follicles [5]. Cytochrome P450 17-hydroxylase (P450c17) mRNA is localized exclusively to bovine theca interna cells [5]. 3ß-Hydroxysteroid dehydrogenase (HSD) mRNA is localized to theca cells of healthy bovine follicles of all sizes but is only expressed in granulosa cells of DFs larger than 8 mm [6]. Cytochrome P450 aromatase (P450arom) mRNA is expressed in bovine granulosa cells after follicular recruitment [5]. Estradiol-17ß (E2) may increase responsiveness to gonadotropins at the level of the DF [7, 8] and increase gonadotropin receptor concentrations [9–11]. Estrogen receptor-ß (ER-ß) mRNA and protein have recently been detected in the granulosa layer of bovine follicles [12].
Follicular cysts have been defined as thin-walled structures of at least 25-mm diameter that persist for 10 days in the absence of a corpus luteum (CL) and fail to ovulate [13]. Ten to fifteen percent of dairy cows are affected by cysts each lactation [14, 15]. Recently, it was confirmed using transrectal ultrasonography that waves of follicular growth occur in cows with cysts [16]. In cows with normal estrous cycles [17] and in cows with cysts [16], each follicular wave is preceded by a transient increase in serum FSH concentrations. However, initiation of follicular waves may occur at longer or more irregular intervals in cows with cysts compared to cows with normal estrous cycles [16, 18]. Affected cows are infertile as long as cysts remain functional. However, cysts undergo one of three fates: cysts may remain dominant for a prolonged period without other follicular growth, cysts may lose dominance and be replaced by a cyst from a new follicular wave (cyst turnover), or cysts may lose dominance and a new DF may develop and ovulate [16, 19]. Low serum progesterone (P4) and high basal LH concentrations are characteristic of cows with cysts [16, 20, 21]. Serum E2 concentrations may reflect the stage of cyst development [16, 22].
Although there have been previous histological and endocrine studies of bovine cysts, the studies have relied on slaughterhouse material where reproductive history and persistence of cysts was not known. There is a paucity of information on expression of mRNAs for the gonadotropin receptors, steroidogenic enzymes, and steroid receptors in cows with cysts. The current experiment was designed to compare expression of steroidogenic enzyme, gonadotropin receptor, and ER-ß mRNAs in cysts at known stages of development to normal DFs and to examine expression of these mRNAs after loss of cyst dominance.
MATERIALS AND METHODS
Induction and Detection of Cysts
All procedures were approved by the Institutional Animal Care and Use Committee of the University of Missouri-Columbia. Cysts occurred either spontaneously or were induced by injecting cows with E2 (30 mg; Sigma Chemical Company, St. Louis, MO) and P4 (75 mg; Sigma) daily for 7 days s.c. in absolute ethanol. Treatment started on Days 5–10 of the estrous cycle and 25 mg prostaglandin F2 (PGF2) i.m. (Lutalyse; Upjohn Company, Kalamazoo, MI). PGF2 was injected on the fourth day of steroid treatment. This protocol is similar to a protocol in which 50% of steroid-treated cows developed cysts approximately 40 days after treatment [19]. Following exogenous steroid treatment, ovaries were examined twice weekly by using transrectal ultrasonography until identification of cyst(s) (Aloka 210, 7.5-MHz probe; Corometrics Medical Systems Inc., Wallingford, CT). During this period, blood samples were taken twice weekly and serum was collected and stored at -20°C until analysis of P4 concentrations. The diagnosis of cysts was made when a single follicular structure greater than 20 mm or multiple structures >15 mm were present, and serum P4 concentrations were below 1.0 ng/ml for at least 7 days in the absence of a CL. Three cows developed cysts spontaneously without treatment with exogenous steroids. Following assignment of cows to the experiment, ovaries were examined daily by using transrectal ultrasonography to determine the time of emergence of follicular waves. Emergence of follicular waves in all animals was denoted by the appearance of a new cohort of follicles that grew beyond 4–5 mm in diameter.
Timing of Removal of Ovaries
Estrous cycles were synchronized in cows with normal estrous cycles with 25 mg PGF2. Ovaries were removed surgically by flank laparotomy on the fourth day after emergence of the first or second follicular wave (DF, n = 8) to obtain normal DFs.
Following initial diagnosis of cysts, ovaries were examined daily using transrectal ultrasonography. Three types of structures were collected. New, growing follicular structures were recovered 3–5 days after emergence of a follicular wave, in the presence of a regressing cyst and when P4 concentrations remained below 1 ng/ml. These were called young cysts (YCs, n = 4), as growth of new follicles in cows with cysts often results in the development of a new cyst [16]. Young cysts did not necessarily reach the size criteria defining the original cyst(s), as they were still growing. Initially, YCs grow at the same rate as DFs [16] but continue to grow past ovulatory size, the YC group was included in this experiment as it could possibly define a stage at which follicular alterations lead to development of cysts. Dominant cysts (n = 5) were recovered 6 or more days after emergence of a follicular wave, when P4 concentrations remained below 1 ng/ml and before emergence of a successive follicular wave. Nondominant cysts (NDCs, n = 8) were recovered after a loss of dominance as indicated by emergence of new follicular (cyst) wave(s). As cysts regress slowly, multiple NDCs that emerged initially from successive follicular waves could exist on the ovary [16].
Ovaries were brought to the laboratory in ice-cold saline. Follicles and cysts were dissected from ovaries within 30 min after ovariectomy and diameters were measured with a ruler. Tissues were frozen over liquid nitrogen and stored at -80°C. A small aliquot of follicular fluid was collected during freezing of the tissue for determination of steroid hormone concentrations.
Hormone Concentrations in Serum and Follicular Fluid
During cyst induction, a single daily blood sample was collected and analyzed to determine P4 concentrations by using a previously validated assay (Coat-A-Count; Diagnostic Products Corp., Los Angeles, CA) [23]. Sensitivity of the assay is 0.1 ng/ml. The intra-assay coefficient of variation (CV) was 8.0% and interassay CV was 12.2%. Concentrations of E2, P4, and androstenedione (A4) in follicular fluid were determined by using solid-phase assays (Coat-A-Count; Diagnostic Products Corp.) after dilution (1:100 to 1:1000) in PBS with 1% gelatin [5]. The kits provide standards prepared in human serum. Dilution curves for samples of bovine follicular fluid were parallel to the standard curves of E2 and P4 assays. In the single P4 assay, the intra-assay CV was 6.6% and the sensitivity was 0.02 ng/tube. In the E2 assays, intra-assay CV was 7.7% and interassay CV was 13.2%. Assay sensitivity was 0.008 ng/tube. Dilution curves for samples of bovine follicular fluid were not parallel to the standard curve of the A4 assay. Therefore, A4 standards were prepared in charcoal-treated follicular fluid. Intra-assay and interassay CVs were 8.9% and 9.8%, and the detection limit was 0.04 ng/tube. The volumes of follicular fluid were estimated by employing the formula for volume 4/3
r3, using the values derived from the diameters of dissected follicles/cysts. Volume (ml) was multiplied by the steroid values/ml to determine total steroids/follicle or cyst.
In Situ Hybridization
Messenger RNA transcripts encoding P450scc, P450c17, P450arom, 3ß-HSD, LHr, and FSHr were detected by in situ hybridization and quantified by computer image analysis (Bioquant image analysis system; R&M Biometrics, Nashville, TN) as previously validated in this laboratory [3–6]. Estrogen receptor-ß mRNA expression was determined as described previously [12]. Briefly, 14-µm sections were prepared in a cryostat, and two slides were incubated with the antisense cRNA probe, and one slide was incubated with the sense cRNA probe overnight at 55°C. Slides were washed, dipped in emulsion (Kodak, NB-2), and then allowed to develop for 3 days to 3 wk at 4°C. Specific hybridization intensity was defined as the mean hybridization of the antisense cRNA probe (percentage of white pixels in four defined areas/slide) minus the mean hybridization of the sense probe (four areas/slide). All sections were lightly counterstained with hematoxylin and eosin to enhance identification of granulosa and theca cell layers.
Thickness of the granulosa and theca cell layers was determined by measuring the image on the video screen after calibrating the video monitor to a slide micrometer. It was not possible to measure the thickness of the theca in NDCs because of difficulty in distinguishing theca externa from theca interna.
Statistical Analysis
Data were analyzed by using the general linear models procedure of Statistical Analysis Systems [24]; the pdiff procedure was used for mean separation. Data were pooled when multiple follicles/cysts with similar characteristics were present within one cow. LSMeans and LSMeans standard errors are reported in tables and text. Values from all classes of follicles/cysts, DFs, YCs, DCs, and NDCs, are included in analyses of size, granulosa and theca thickness, and intrafollicular hormone concentrations. Superscripts indicate differences among these groups in the tables. Data were subjected to log transformation when necessary to reduce variation among groups. For analyses of mRNA expression, NDC values were often much lower (often undetectable) and variance was heterogeneous between NDCs and other groups. Therefore, NDC values of mRNA expression were not included in the statistical analyses and are reported in the text only when above background values.
RESULTS
Follicle/Cyst Size at Ovariectomy
In the current study, one to three NDCs were found on the ovaries in four of five DCs and all YC cows. Log values were used for statistical analysis. Sizes of follicles/cysts (mm) were different (Table 1) among treatments. The DCs were larger than YCs and DFs (both P < 0.01). The NDCs were not different from the other groups. The thickness of the granulosa layer was similar between DCs, YCs, and DFs. The width of the granulosa layer was smaller (P < 0.05) in NDCs; where present, the granulosa was discontinuous and partly luteinized. The thickness of the theca layer was similar in DCs, YCs, and DFs (P > 0.39). Data are not reported from NDCs due to its indistinct nature.
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Steroid Hormone Concentrations in Follicular Fluid
Log values were used for statistical analysis of E2, P4, and A4 (ng/ml and ng/follicle). Follicular fluid concentrations of E2 calculated as ng/ml or ng/follicle were lower in NDCs than other groups (P < 0.001, Table 2). Concentrations of E2 in follicular fluid (ng/ml) were higher in DCs than in DFs (P < 0.04) but not different from YCs (P > 0.40). Total E2 content (ng/follicle) was also higher in DCs compared to DFs (P < 0.01) but not different than YCs (P > 0.10). Concentrations of P4 (ng/ml) in follicular fluid were higher (P < 0.001) in NDCs compared to all other groups, while concentrations of P4 in follicular fluid (ng/ml) were similar in DFs, YCs, and DCs. Total follicular content of P4 (ng/follicle) was not different between NDCs and DCs (P > 0.10), and both were higher than in DFs (P < 0.01), although DCs were not different from YCs (P < 0.05). Concentrations of A4 (ng/ml) did not differ among treatments. However, total follicular A4 content tended to be higher in DCs than in YCs, DFs, and NDCs (P < 0.07).
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Expression of LHr mRNA
The LHr mRNA was expressed in theca and granulosa cells (Fig. 1). Untransformed values were used for statistical analysis of expression in theca while log values were used for granulosa. Overall, expression of LHr mRNA was not different in theca of DCs, YCs, and DFs (P > 0.12, Table 3); however, DCs tended to have higher expression than DFs and YCs. Most DFs and YCs, but all DCs expressed LHr mRNA in the granulosa layer. There was an overall effect of treatment on expression of LHr mRNA in granulosa (P < 0.05). Hybridization intensity was higher in DCs compared to DFs (P < 0.02) and tended to be higher than YCs (P < 0.06). Only 3 of 17 NDCs expressed a detectable amount of LHr mRNA in theca cells and 1 of 17 NDCs expressed LHr mRNA in granulosa cells.
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Expression of FSHr mRNA
Expression of FSHr mRNA was confined to the granulosa cell layer of DFs, YCs, and DCs. Untransformed values were used for statistical analysis. Expression of FSHr mRNA was not different (P > 0.73) in granulosa cells of DF (28.4 ± 8.0%), YC (21.0 ± 11.3%), and DC groups (18.8 ± 10.1%). There was no specific hybridization of the antisense FSHr cRNA probe in NDCs.
Expression of P450scc mRNA
The P450scc mRNA was expressed in both theca and granulosa and granulosa cell layers of DFs, YCs, and DCs. Untransformed values were used for statistical analysis of expression in both theca and granulosa. There were no difference (P > 0.39) in expression of P450scc mRNA in theca of DFs (11.0 ± 2.4%), YCs (16.6 ± 3.4%), and DCs (14.4 ± 3.0%). Similarly, there were no differences (P > 0.72) in expression of P450scc mRNA in granulosa of DFs (10.5 ± 2.8%), YCs (12.5 ± 4.0%), and DCs (8.2 ± 3.6%). In NDCs, expression of P450scc in theca (0.6 ± 0.2%) and granulosa (1.8 ± 0.6%) was near background, except in a few cases of cysts with discontinuous luteinized layers.
Expression of 3ß-HSD mRNA
The mRNA for 3ß-HSD was expressed in both theca and granulosa cell layers of DFs, YCs, and DCs (Fig. 2). Untransformed values were used for statistical analysis of expression in theca, and log values were used for statistical analysis of expression in granulosa. Expression of 3ß-HSD mRNA was not different in the theca layer of DFs, YCs, and DCs (P > 0.99, Table 3). Expression of 3ß-HSD mRNA was higher in the granulosa layer of DCs compared to DFs (P < 0.01) and YCs (P < 0.04). Expression of 3ß-HSD mRNA was undetectable in granulosa cells of NDCs (1.2 ± 0.3%) except in patchy luteinized granulosa layers.
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Expression of P450c17 mRNA
Expression of P450c17 mRNA was confined to the theca cell layer of DFs, YCs, and DCs. Untransformed values were used for statistical analysis of expression. Expression of P450c17 mRNA was not different (P > 0.21) in theca cells of DFs (36.6 ± 9.3%), YCs (59.8 ± 13.1%), and DCs (60.9 ± 11.7%). In NDCs, expression of mRNA for P450c17 was undetectable in most cases.
Expression of P450arom mRNA
Expression of P450arom mRNA was confined to the granulosa layer of DFs, YCs, and DCs. Untransformed values were used for statistical analysis. Expression of P450arom mRNA was not different (P > 0.63) in granulosa cells of DFs (55.1 ± 10.5%), YCs (63.0 ± 14.8%), and DCs (71.3 ± 13.3%). In NDCs, there was no specific binding of the antisense P450arom cRNA probe.
Expression of ER-ß mRNA
Expression of ER-ß mRNA was lower than expression of most steroidogenic or gonadotropin receptor mRNAs. Log values were used for statistical analysis. Expression of ER-ß mRNA was localized only to the granulosa cell layer and expression was not different (P > 0.08) in DFs (7.6 ± 1.4%), YCs (5.1 ± 1.9%), and DCs (2.3 ± 1.7%). In NDCs, there was no specific binding of the antisense ER-ß cRNA probe.
DISCUSSION
Most previous studies of histological and endocrine characteristics of bovine cysts have relied on slaughterhouse material, where reproductive history and persistence of cysts was not known. In the current study, cysts were recovered at known times after cyst emergence or turnover that permitted a more accurate analysis of functional state and steroidogenic potential of cysts. Characteristically, cows with cysts have higher mean LH concentration, LH pulse amplitude, and LH pulse frequency than cows with normal estrous cycles [16, 21, 25]. Frequent GnRH or LH pulses lead to prolonged growth of follicles [26, 27] and increased follicular E2 synthesis [26, 28]. Dominant cysts were larger in diameter than DFs and YCs in the current study. However, the thickness of theca and granulosa layers did not differ between DFs, YCs, and DCs. Concentrations of E2, P4, and A4 in follicular fluid were higher on a ng/follicle basis in DCs than in DFs. Synthesis of E2 in ovine follicles cultured in vitro is correlated with the number of granulosa cells [29]. Increased total steroid hormone content in DCs may be a function of follicular diameter, as granulosa cell number and theca cell mass increase with follicular size [30–32].
Most cows with cysts had multiple estrogenic (DCs or YCs) and nonestrogenic (NDC) cysts. Short [33] reported that intrafollicular concentrations of E2 and P4 may change in cysts over time, and that cows may have concurrent cysts with different hormone profiles. Cows in the current experiment had multiple NDC follicles, suggesting that most cysts are replaced over time and undergo atresia or limited luteinization as they age. The presence of multiple large estrogenic follicles in the current study is consistent with observations of increased twin births or twin ovulations in cows with cysts observed in previous studies [16, 34, 35]. Because cows normally develop a single DF, the growth of multiple estrogenic follicles suggests that there may be an alteration in follicular selection or dominance in cows with cysts.
Increased expression of LHr mRNA was observed in granulosa cells of DCs compared to DFs. These results are in agreement with observation of increased LHrs in persistent follicles that remain estrogenic for prolonged periods when exposed to low P4/high LH pulse frequency [31]. The number of LHrs in theca cells is correlated with greater testosterone and E2 synthesis of follicles cultured in vitro, and the number of granulosa LHrs is correlated with E2 production in vitro [36]. Although thecal LHr mRNA expression was numerically but not significantly increased in DCs compared to DFs, greater LH stimulation [16, 21] in cows with cysts and greater expression of granulosa LHr mRNA may contribute to increased follicular steroidogenesis.
Expression of 3ß-HSD mRNA was higher in granulosa cells of DCs compared to DFs and YCs. Al-Dahash and David [37] reported high 3ß-HSD activity in granulosa cells and weak 3ß-HSD activity in undifferentiated theca cells of cysts. Although expression of 3ß-HSD mRNA is not higher in persistent bovine follicles compared to DFs [32], mRNA expression does increase in bovine theca and granulosa cells of preovulatory follicles following luteolysis [38]. Conceivably, exposure to high LH stimulation could increase expression of 3ß-HSD mRNA in granulosa cells of DCs.
Despite the increased LH pulse frequency to which cysts are exposed and the higher follicular content of E2, P450arom mRNA expression is not higher in DCs compared to DFs. This is, however, in agreement with the lack of increase of P450arom mRNA expression in preovulatory follicles following luteolysis [38], when serum LH and E2 concentrations increase. It has been suggested that expression of P450arom mRNA [38, 39] or aromatase activity [39–41] may not be the primary limitation in follicular E2 synthesis.
Although expression of both P450scc and P450c17 mRNAs increase following luteolysis [38], expression does not increase in persistent follicles compared to DFs [32]. In the current experiment, neither P450scc nor P450c17 mRNAs differed in expression between cysts and DFs.
Estrogen receptor-ß appears to be more highly expressed in granulosa cells than ER- [42], and expression of both ER-ß protein and mRNA has been detected previously in bovine granulosa cells [12]. However, there were no differences in expression of granulosa ER-ß mRNA in DFs and cysts in the current experiment. Expression of ER-ß mRNA appears to be downregulated in the ovary by treatment with exogenous gonadotropins [42, 43] and during preovulatory maturation in rat follicles [43]. Therefore, ERs may not be involved in late stages of bovine follicular growth.
Young cysts were similar in size to DFs and less mature than DCs, which may explain why follicular steroid concentrations, expression of gonadotropin receptors, and steroidogenic enzyme mRNAs were more similar between YCs and DFs than between YCs and DCs. It is apparent that in cows with cysts, changes in expression of LHr and 3ß-HSD mRNAs have not occurred in newly growing follicles by the time they reach the size of a normal DF. It has been reported that follicles in cows with cysts may ovulate and form normal CLs in response to treatment with GnRH or hCG [44]. In this case, it may be that newly developing follicles are not initially defective in cows with cysts, and that a prolonged period of growth in a high LH and low P4 environment may be necessary to alter expression of gonadotropin receptor and steroidogenic enzyme mRNAs. Therefore, a defective endocrine milieu may play an earlier role in cyst development than follicular alterations in mRNA expression.
Nondominant cysts fit the description of chronic cysts with degenerated granulosa cell layers, no basement membrane, and theca layers containing hyalinized collagen and luteinized zones [45]. Low intrafollicular E2 concentrations and lack of P450arom mRNA expression in NDCs were likely related to degeneration of the granulosa cell layer, as indicated in earlier studies [46–48]. Additionally, an absence of aromatase and 17-hydroxylase activity was identified in cysts without granulosa layers in earlier studies [47, 48]. Reduced LH and FSH binding was observed previously in chronic (estrogen-inactive) cysts [49]. Low numbers of LHrs in NDCs could explain the failure of some cows to respond to exogenous GnRH or hCG that are often used to treat cysts. The decreased expression of mRNAs for gonadotropin receptors, weak expression of 3ß-HSD, and P450scc mRNAs in luteinized granulosa cells and increased intrafollicular P4 concentrations is consistent with the morphological observations (current study) that NDCs are undergoing atresia [3, 6, 30, 50].
In summary, expression of LHr and 3ß-HSD mRNAs was higher in granulosa cells of DCs compared to normal bovine DFs. Under higher LH stimulation, increased expression of LHr and 3ß-HSD mRNAs in cyst granulosa cells may be responsible for the prolonged period of growth and increased steroidogenesis of cysts compared to DFs. Loss of dominance in cysts is associated with atresia of granulosa cells and decreased expression of steroidogenic enzyme and gonadotropin receptor mRNAs.
FOOTNOTES
First decision: 18 October 2000.
1 Correspondence: H. Allen Garverick, Department of Animal Sciences,
163 Animal Science Research Center, 920 E. Campus Dr., University of
Missouri-Columbia, Columbia, MO 65211-5300. FAX: 573 882 6827;garvericka{at}missouri.edu 
2 Current address: Departments of Obstetrics and Gynecology and Physiology,
University of Western Ontario, London, ON, Canada N6A 5C1.
3 Current address: Department of Anatomy and Physiology, College of Veterinary
Medicine, Kansas State University, Manhattan, KS 66506-5602.
Accepted: March 22, 2001.
Received: September 15, 2000.
REFERENCES
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