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Biology of Reproduction 62, 1335-1343 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Intrafollicular Concentrations of Steroids and Steroidogenic Enzymes in Relation to Follicular Development in the Mare1

François Belina, Ghylène Goudeta, Guy Duchampa, and Nadine Gérard2,a

a INRA-Haras Nationaux, Reproduction Equine, P.R.M.D., 37380 Nouzilly, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of the present study was to determine the changes in follicular fluid steroid concentrations and in granulosa cell steroidogenic enzyme expression during the follicular phase, in relation to follicular size and physiological status in the mare.

Follicular fluid and follicular cells were recovered by ultrasound-guided follicular punctures either around the time of emergence of the dominant follicle, at the end of the dominant follicle growth, or at the preovulatory stage, after injection of gonadotropin to induce ovulation. Cellular relative amounts of steroidogenic acute regulatory protein (StAR), P450-side chain cleavage (P450scc), 3ß-hydroxysteroid dehydrogenase (3ßHSD), 17{alpha}-hydroxylase, and aromatase were assessed by semiquantitative Western blot and densitometry. Follicular fluid was assayed for cholesterol concentrations by colorimetric assay and for progesterone, testosterone, and estradiol-17ß concentrations by RIA.

Intrafollicular concentrations of progesterone and estradiol-17ß significantly increased in the dominant follicle during growth. After injection of gonadotropin, follicular maturation was characterized by a decrease in estradiol-17ß concentrations and a further increase in progesterone concentrations. Granulosa cells from dominant follicles had increased levels of StAR, P450scc, 3ßHSD, and aromatase during growth, but decreased levels during maturation. Levels of StAR, P450scc, 3ßHSD, and aromatase, as well as progesterone and estradiol-17ß, were lower in granulosa cells from subordinate than from dominant follicles. We did not observe a relationship between the steroidogenic activity of follicles and the capacity of their enclosed oocytes to complete meiosis in vitro.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian folliculogenesis in mammals is mainly under the endocrine control of the pituitary gonadotropins FSH and LH, acting on specific receptors on ovarian cells (for review, see [1–3]). In response, follicular cells synthesize and secrete steroids that act as endocrine and paracrine factors, to regulate gonadotropin levels, ovarian activity, and folliculogenesis (for review, see [4, 5]). Steroid synthesis by gonads depends on the availability of cholesterol and the successive conversions to pregnenolone, progesterone, androgens, and estrogens (for review, see [6]). Cholesterol side-chain cleavage P450 (P450scc) is implicated in the conversion of cholesterol to pregnenolone. The availability of cholesterol to P450scc depends on steroidogenic acute-regulatory protein (StAR), which transfers cholesterol from the outer to the inner mitochondrial membrane, where the enzymatic reaction occurs. Pregnenolone can then be metabolized by two different routes, the {Delta}4 and {Delta}5 pathways. In equine follicles, the {Delta}4-pathway is preferred [7], in which pregnenolone is converted into progesterone by 3ß-hydroxysteroid dehydrogenase (3ßHSD). P450 17{alpha}-hydroxylase (P45017{alpha}) then catalyses, in the endoplasmic reticulum, the conversion of progesterone into androstenedione, which is converted afterwards into testosterone by 17ß-hydroxysteroid dehydrogenase (17ßHSD). Estrogen is synthesized from androgen by the activity of aromatase.

Steroid concentrations have been studied in plasma and follicular fluid from domestic mammals such as cows [8], pigs [9], and sheep [10], as well as from humans [11, 12]. In these species, it has been well documented that the dominant follicle is characterized by its ability to produce large amounts of estradiol-17ß, whereas follicles that undergo atresia produce decreasing amounts of estradiol-17ß. Ovarian expression of steroidogenic enzymes is related to follicular fluid steroid levels in cattle [13], pigs [14, 15], and humans [12]. Several studies have focused on the intrafollicular steroid levels in the mare [16–19], but no information is available on the in vivo expression of steroidogenic enzymes that bring about the ovarian steroid synthesis in the equine species. However, the ovarian physiology of the mare is an interesting model. Among domestic mammals, the estrous cycle of the mare displays some unique features: a long period of estrus (4–6 days, [20]), a large preovulatory follicle (>40 mm, [21]), and a progressive increase in LH concentration lasting many days and peaking 1–2 days after ovulation [22–24]. A better knowledge of steroidogenesis in equine follicles during the follicular phase is essential to understanding preovulatory follicular development and maturation.

Steroid synthesis by equine theca and granulosa cells from dominant follicles has been studied using follicular wall and isolated cells cultured for several days [18, 25]. However, such experiments may not accurately reflect in vivo conditions, considering the complexity of follicular fluid and its regulatory role in enzyme activity [26]. Recently we observed that intrafollicular concentrations of estradiol-17ß and progesterone increase during follicular growth [19, 27]. Follicular maturation after LH injection is associated with a decrease in estradiol-17ß and a further increase in progesterone levels [19, 27]. Subordinate follicles are characterized by low levels of these two steroids [27]. These observations suggest that variations in steroid levels could be associated with variations in steroidogenic enzyme levels.

The present study was undertaken to characterize the in vivo expression of P450scc, StAR, 3ßHSD, P45017{alpha}, 17ßHSD, and aromatase in follicular cells, as well as steroid concentrations in follicular fluid, in relation to follicular development and maturation at specific times of the follicular phase, in cyclic mares. Moreover, considering the link between the maturational events within the follicle and the oocyte, we have attempted to correlate the steroidogenic activity of follicles to the oocyte competence for in vitro maturation.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Design

Adult cyclic pony mares (n = 44) in good body condition, aged 3–19 yr, kept indoors, and fed with concentrates were used from March to May. The estrous cycle was synchronized by placement of an intravaginal sponge containing 0.5 g altrenogest (Regumate; Roussel UCLAF, Romainville, France) plus 50 mg estradiol benzoate (ß-estradiol 3-benzoate; Sigma, Chemical Corp., St. Louis, MO) for 1 wk [28]. On the day of sponge removal, mares received a luteolytic dose of prostaglandin F2{alpha} (cloprostenol, 250 µg/mare i.m.; Estrumate; Pitman-Moore, Meaux, France), and all follicles larger than 5 mm were punctured to eliminate atretic follicles from the ovaries. Subsequent follicular growth was then assessed and follicular diameter was recorded by routine rectal ultrasound scanning until the day of experimental follicular puncture [29].

Experimental Follicular Punctures

For each mare, follicular punctures were performed at a specific time during the follicular phase using an in vivo transvaginal, ultrasound-guided follicular aspiration technique with a 7.5-MHz sectorial probe (Kretz; Soframed, Truchtersheim, France) [30]. In group 1 (n = 12 mares), all follicles larger than 5 mm were punctured the day the largest follicle(s) reached a diameter >=20 mm (around the emergence of the dominant follicle); in group 2 (n = 12 mares), all follicles larger than 5 mm were punctured 24 h after the largest follicle reached a diameter >=30 mm (end of the dominant follicle growth); in group 3 (n = 20 mares), all follicles larger than 5 mm were punctured at the preovulatory stage, 34 h after an injection of crude equine gonadotropin (CEG; 25 mg i.v.), generally used to induce ovulation, when the largest follicle reached a diameter >= 33 mm [31]. CEG contains 3% FSH and 6% LH [32], and ovulation occurs between 34 and 40 h after injection [33].

At the start of each puncture session, mares were sedated with detomidine (0.6 mg/100 kg BW i.v.; Domosedan; Smith, Kline & French, Courbevoie, France), and relaxation of the rectum was induced with atropine sulfate (4 mg/100 kg BW i.v.; Chaix et du Marais, Paris, France). After puncture, mares were administered antibiotics (1 600 000 IU penicillin/100 kg BW, and 1.3 g dihydrostreptomycine/100 kg BW, i.m.; Intramicine; Rhône Mérieux, Lyon, France).

Collection of Cumulus-Oocyte Complexes (COCs), Follicular Cells, and Biological Fluids

After follicular fluid aspiration, the follicle wall was scraped with the puncture needle and flushed with PBS (Dulbecco "A"; Unipath, Dardilly, France) containing heparin (50 IU/ml; LEO S.A., St-Quentin Yvelines, France) at 37°C. Follicular fluid and flushing liquids were examined separately under a stereomicroscope for oocyte recovery and then centrifuged for 10 min at 1500 x g to pellet granulosa cells. Under our experimental conditions, pure follicular fluid could be obtained only from follicles larger than 15 mm in diameter, whereas follicular fluid from smaller follicles was diluted with PBS. A few follicles larger than 15 mm were collected after accidental dilution. Pure and diluted follicular fluid was individually stored at -20°C.

Blood samples were collected before each puncture session from the jugular vein. Serum and plasma were stored at -20°C before assays.

Oocyte Culture and Examination

At recovery, oocytes were individually cultured in a humidified atmosphere (95% air: 5% CO2) at 38.5°C for 30 h in 500 µl of maturation medium, as previously described [34]. After culture, the COCs were stripped of their cumulus cells with a small glass pipette in 500 µl of PBS supplemented with 87.5 IU/ml hyaluronidase (type III, 875 IU/mg; Sigma) at 37°C. Totally denuded oocytes were rinsed in PBS with 1% inactivated fetal calf serum (Gibco, Eragny, France) at 37°C, stained with 1 µg/ml bis-benzamide (Hoechst 33342; Sigma) in PBS for 5 min at 37°C for DNA detection, and observed in a drop on a slide under a fluorescence microscope. Oocytes were classified according to chromatin configuration as germinal vesicle, dense chromatin, metaphase I, metaphase II, and degenerated, as previously described [34].

Steroid Assays

Pure and diluted follicular fluids were assayed for progesterone [35] and estradiol-17ß [36] by RIAs as previously described. Testosterone was assayed in pure follicular fluid by RIA according to Hochereau de Reviers et al. [37]. Intraassay variability and limit of sensitivity were, respectively, 10.6% and 0.10 ng/ml for progesterone, 8.3% and 0.015 ng/ml for estradiol-17ß, and 6.6% and 0.1 ng/ml for testosterone. For the diluted follicular fluids, results were expressed only as the ratio of progesterone:estradiol-17ß, the dilution factor being unknown. Total cholesterol was measured in pure follicular fluids and in serum using a colorimetric detection system (Sigma). Intraassay variability was 6.4%, and interassay variability ranged between 4.3% and 10.4%. Plasma progesterone levels were determined as previously described [38]. For each steroid, all samples were assayed in the same assay to avoid interassay variability.

Granulosa Cell Extract Preparation

The pellets of granulosa cells were resuspended in 2 ml of PBS, layered onto 3 ml of Ficoll-paque (research grade; Pharmacia Biotech, Uppsala, Sweden), and centrifuged (10 min, 1500 x g, 4°C) in order to get rid of blood cells. Granulosa cells were recovered by aspiration at the interface layer. Cells were rinsed twice with PBS and suspended in 10 µl hypotonic storage buffer (KCl 10 mM, Tris 10 mM, EDTA 0.5 mM) containing 348 µg/ml PMSF (Sigma), 33.2 µg/ml N{alpha}-p-tosyl-L-lysine chloromethyl ketone (Sigma), and 34.1 µg/ml N-tosyl-L-phenylalanine chloromethyl ketone (Sigma). Samples were stored at -20°C until analyzed.

Gel Electrophoresis and Immunoblotting

Granulosa cell lysates were sonicated for 30 min and submitted to one-dimensional SDS-PAGE (12%) under reducing conditions [39]. Acrylamide-bisacrylamide solution was purchased from Serva GmbH & Co. (Heidelberg, Germany), and other reagents were purchased from Sigma. Prestained standards (range 6.9–202 kDa; Bio-Rad, Hercules, CA) were run simultaneously. Polypeptides were electrotransferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) overnight at 4°C.

Each membrane was used for immunological detections of P450scc, actin, 3ßHSD, P45017{alpha}, aromatase, 17ßHSD, and StAR. After each immunological detection, the membranes were stripped of bound antibodies by incubation in stripping buffer (100 mM ß-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) at 50°C for 30 min, and rinsed twice in Tris-buffered saline (TBS)-Tween 20 (10 mM Tris, 150 mM NaCl, pH 7.4 containing 0.1% Tween 20), as recommended by the manufacturer (Amersham Corp., Arlington Heights, IL).

For immunological detection, membranes were washed in TBS-Tween 20 and incubated for 1 h in blocking solution (TBS containing 5% dry milk, 0.2% Igepal [Sigma], pH 7.4). Membranes were incubated for 3 h with the primary antiserum, rinsed twice in TBS-Tween 20, and incubated for 30 min in blocking solution before incubation for 1 h with horseradish peroxidase-conjugated antiserum as secondary antiserum. The enhanced chemiluminescence detection system (ECL; Amersham) was used to detect immunoreactive polypeptides. Membranes were exposed to Hyperfilm MP (Amersham) that were digitalized with an Eikonix 1412 scanner camera (Eastman Kodak, Rochester, NY). Patterns were quantified using Kepler software (Large Scale Biology Corporation, Rockville, MD).

The primary antibodies used were 1) rabbit antiserum raised against bovine P450scc (kindly provided by Dr. D.B. Hales, University of Illinois, Chicago); 2) mouse monoclonal antibody raised against chicken actin (Amersham); 3) rabbit antiserum raised against human placental 3ßHSD (kindly provided by Dr. V. Luu-The, Centre de Recherche en Endocrinologie Moléculaire, Québec, PQ, Canada); 4) rabbit antiserum raised against the purified porcine P45017{alpha} (kindly provided by Dr. A. Payne, University of Michigan, Ann Arbor, MI); 5) rabbit antiserum raised against equine aromatase (kindly provided by Dr. G.E. Séralini, IBBA Caen, France); 6) rabbit antiserum raised against purified human placental 17ßHSD1 (kindly provided by Dr. V. Luu-The); and 7) rabbit antiserum raised against the glutathione-S-transferase (GST)-fusion protein of mouse StAR protein (kindly provided by Dr. D.B. Hales). The peroxidase-conjugated second antiserum used was either rabbit anti-mouse IgG (Vector Laboratories, Burlingame, CA) for the actin immunological detection or goat anti-rabbit IgG (Institut Pasteur, Paris, France) for the other immunological detections.

The relative amount of P450scc, 3ßHSD, P45017{alpha}, aromatase, 17ßHSD and StAR in granulosa cells was expressed as a ratio to the amount of actin present within each lane.

Statistical Analysis

Data were expressed as mean ± SEM. Concentrations of steroids in follicular fluid and the results obtained by immunoblotting and densitometry were analyzed 1) by group, using ANOVA followed by a Student's t-test, and 2) by follicular diameter, using logistic regression analysis (Statistical Analysis Systems software, Cary, NC). Correlations between variables were assessed by correlation analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In the present study, a definition of dominance based on size was adopted. Follicles were considered dominant when larger than 20 mm in group 1 and larger than 30 mm in groups 2 and 3. Other follicles were considered subordinate.

Follicular Puncture Attempts and Plasma Progesterone Levels

During the 44 puncture sessions (12 in groups 1 and 2 and 20 in group 3), 262 follicles were punctured. In all but one mare, plasma progesterone concentration was low (<0.3 ng/ml) at the time of follicular puncture.

Intrafollicular and Circulating Concentrations of Cholesterol

Total cholesterol concentration was assayed in pure follicular fluids collected (n = 87; mean = 0.5 ± 0.1 ng/ml) and in corresponding sera (n = 44; mean = 1.15 ± 0.02 ng/ml). No clear relationship was detected between the cholesterol concentration in serum and follicular fluid for each mare. The intrafollicular concentration of cholesterol was not significantly different among groups, classes of follicular diameter, or physiological status of the follicle (dominant or subordinate).

Intrafollicular Level of Steroids

The intrafollicular concentration of testosterone was not significantly different when groups, follicular diameters, or physiological status of the follicle (dominant/subordinate) were compared.

In the population of follicles larger than 15 mm from which pure follicular fluid had been obtained, the intrafollicular concentration of progesterone was significantly higher in follicular fluid recovered at the preovulatory stage (group 3) than in fluids recovered at earlier stages (P < 0.01 each). In contrast, the intrafollicular concentration of estradiol-17ß was not significantly different among the three groups.

In dominant follicles only, we observed that the levels of progesterone and estradiol-17ß were lower in group 1 (20–24 mm) than in the other two groups (>30 mm; Fig. 1, A and B). Moreover, the intrafollicular concentration of progesterone in the dominant follicle was significantly higher after gonadotropin injection (group 3) than before (group 2; Fig. 1A), whereas the intrafollicular concentration of estradiol-17ß was significantly lower (Fig. 1B). Consequently the progesterone:estradiol-17ß (P:E) ratio was higher in group 3 than in groups 1 and 2 for dominant follicles (Fig. 1C).



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  		FIG. 1. Intrafollicular concentrations of A) progesterone and B) estradiol-17ß in relation to the group and the follicular diameter; C) ratio of the intrafollicular concentrations of progesterone:estradiol-17ß. Group 1, emergence of the dominant follicle; group 2, end of the follicular growth; group 3, preovulatory stage (34 h after injection of CEG). a,b,c, Within each class, values with different letters differ significantly (P < 0.05 at least). na, Not available (follicular fluid diluted with PBS)

In subordinate follicles, the P:E ratio was significantly higher in fluids collected at the preovulatory stage (group 3) than in those collected at earlier stages (P < 0.01 each). These significant differences were observed within each class of follicular diameter studied (Fig. 1C).

When dominant and subordinate follicles were compared, no significant difference in intrafollicular concentrations of progesterone and estradiol-17ß was observed in group 1 (Fig. 1, A and B). In groups 2 and 3, the intrafollicular concentrations of progesterone and estradiol-17ß were higher in dominant than in subordinates follicles (Fig. 1, A and B). Moreover, within groups 1 and 2, the P:E ratio significantly decreased with an increase in follicular diameter (P < 0.01 each), whereas no such variation was observed in group 3. In group 2, this decrease was due to an increase in intrafollicular concentration of estradiol-17ß with the increase in follicular diameter (P < 0.01, Fig. 1B).

Steroidogenic Enzymes and StAR in Granulosa Cells

Samples of granulosa cells from 190 follicles were analyzed for StAR, P450scc, 3ßHSD, P45017{alpha}, 17ßHSD-1, aromatase, and actin. The expected band for actin at 42 kDa was visualized in most samples (Fig. 2A); nevertheless, 40 samples did not contain a sufficient amount of proteins to visualize this signal, and data from these samples were not used for analysis. The expected bands were visualized at 52 kDa for P450scc (Fig. 2B), 45 kDa for 3ßHSD (Fig. 2C), 79 kDa for P45017{alpha} (Fig. 2D), 55 kDa for aromatase (Fig. 2E), and 30 kDa for StAR (Fig. 2F). No band was observed for the immunodetection of the enzyme 17ßHSD-1, even though different incubation times and different antibody concentrations were tested.



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  		FIG. 2. Western immunoblots. Representative profiles of A) actin, B) P450scc, C) 3ßHSD, D) P45017{alpha}, E) aromatase, and F) StAR

Relative Amount of StAR

The protein StAR was observed in 2 out of 10 follicles larger than 20 mm from group 1, in 9 out of 15 follicles larger than 30 mm from group 2, and in all follicles larger than 25 mm from group 3 (Fig. 3). The expression of StAR was significantly lower in follicles from group 1 than in follicles from groups 2 and 3 (P < 0.01 each). Moreover, in dominant follicles, the relative amount of StAR was higher at the end of the follicular growth before injection of gonadotropin (group 2) than after (group 3, P < 0.05).



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  		FIG. 3. Semiquantitative analysis of the expression of StAR in equine granulosa cells. Groups are as described in the legend to Figure 1. a,b,c, Within each class, values with different letters differ significantly (P < 0.05 at least)

Relative Amount of P45017{alpha}

The enzyme P45017{alpha} was detected in only 23% of the follicular cell lysates analyzed. No significant difference was observed among groups, classes of follicular diameter for each group, or the physiological status of follicles.

Relative Amounts of P450scc, 3ßHSD, and Aromatase in Relation to Experimental Groups

Within the entire follicular population studied, we observed that the expression of P450scc was not significantly different among the three groups. The expression of 3ßHSD was significantly higher in granulosa cells recovered around the emergence of dominance (group 1) than in cells recovered at the end of the follicular growth (group 2, P < 0.05) as well as at the preovulatory stage (group 3, P < 0.05). The expression of aromatase was significantly higher in granulosa cells recovered around the emergence of dominance (group 1) than in cells recovered at the end of the follicular growth (group 2, P < 0.01), and was significantly lower in cells recovered at the preovulatory stage (group 3) than in the two previous groups (P < 0.05).

Relative Amounts of P450scc, 3ßHSD, and Aromatase in Relation to Physiological Status of Follicles

At the end of the follicular growth (group 2) and at the preovulatory stage (group 3), the relative amounts of P450scc, 3ßHSD, and aromatase were significantly higher in granulosa cells recovered from dominant follicles (> 30 mm) than in cells from subordinate ones (5–29 mm) (group 2, P < 0.05, P < 0.01, and P < 0.05, respectively; group 3, P < 0.01, P < 0.05, and P < 0.01, respectively).

Moreover, the relative amounts of P450scc and 3ßHSD significantly increased with an increase in follicular diameter regardless of the experimental group (P < 0.05 each; Fig. 4, A and B). The relative amount of aromatase similarly increased in follicles collected at the end of follicular growth (group 2) and at the preovulatory stage (group 3; P < 0.01 each; Fig. 4C).



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  		FIG. 4. Semiquantitative analysis of the expression in equine granulosa cells of A) P450scc, B) 3ßHSD, C) aromatase. Groups are as described in the legend to Figure 1. a,b,c, Within each class, values with different letters differ significantly (P < 0.05 at least)

Relative Amounts of P450scc, 3ßHSD, and Aromatase in Dominant Follicles

In dominant follicles, the relative amounts of P450scc, 3ßHSD, and aromatase in granulosa cells were significantly higher before injection of gonadotropin (group 2) than after (group 3, Fig. 4). Moreover, the expression of P450scc, 3ßHSD, and aromatase in cells from dominant follicles from group 1 was lower than those from groups 2 and 3 (Fig. 4).

Relative Amounts of P450scc, 3ßHSD, and Aromatase in Subordinate Follicles

In subordinate follicles, the expression of P450scc, 3ßHSD, and aromatase around the time of emergence of a dominant follicle (group 1) was higher than at the end of the follicular growth before the injection of gonadotropin (group 2) within each size class (Fig. 4). The expression of aromatase at the end of the follicular growth was higher before (group 2) than after gonadotropin injection (group 3) within each size class (Fig. 4). For subordinate follicles smaller than 20 mm, the expression of P450scc at the end of the follicular growth was lower before (group 2) than after (group 3) injection of gonadotropin to induce ovulation.

Correlation Between Follicular Fluid Levels of Steroid and Granulosa Cell Expression of Steroidogenic Enzymes

Correlation analyses were performed on samples of pure follicular fluid that had matching steroidogenic enzyme levels (n = 35). Intrafollicular estradiol-17ß concentration was positively correlated to cellular relative amount of aromatase (r = 0.67; P < 0.01), and intrafollicular progesterone concentration was positively correlated to cellular relative amount of 3ßHSD (r = 0.61; P < 0.01). In the population of dominant follicles, no obvious correlation was observed between 3ßHSD and progesterone.

Relation Between the Steroidogenic Activity of Follicle and the Ability of Oocyte to Mature In Vitro

From the 262 follicles punctured, 143 COCs were collected. The relative amounts of StAR, P450scc, 3ßHSD, and aromatase in granulosa cells and the intrafollicular concentrations of progesterone, testosterone, and estradiol-17ß were not significantly different between the nuclear stages of the corresponding oocytes after in vitro culture.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we determined the relationship between follicular growth and preovulatory maturation, and intrafollicular concentrations of cholesterol, progesterone, testosterone, and estradiol-17ß, as well as levels of steroidogenic enzymes. We report for the first time changes in cellular levels of StAR, P450scc, 3ßHSD, P45017{alpha}, and aromatase in granulosa cells of individual equine follicles at specific times of the follicular phase, in the presumptive ovulatory follicle as well as in subordinate follicles.

As previously observed in humans [40, 41] and horses [42], we observed a higher circulating than intrafollicular concentration of cholesterol. No variation in intrafollicular cholesterol level was observed during follicular growth and maturation of the preovulatory dominant follicle, or during growth of the subordinate follicles. The intrafollicular level of testosterone did not vary with follicular size or physiological status, as previously observed in the mare [43, 44]. In contrast, the growth of the dominant follicle was characterized by an increase in intrafollicular concentrations of estradiol-17ß and progesterone, whereas follicular maturation after LH administration was associated with a decrease in estradiol-17ß and a further increase in progesterone concentrations, which is consistent with our previous findings [19, 27]. Low levels of estradiol-17ß and progesterone were observed in subordinate cohort follicles. The latter were probably atretic [27].

It has previously been demonstrated that the theca interna layer plays only a minor role in estradiol-17ß and progesterone production in the mare, but it is the predominant site for androstenedione synthesis in equine preovulatory follicles [25]. It has been previously shown in cows [45], sows [6, 13], ewes [46], and mares [47] that P45017{alpha}, which synthesizes androstenedione from progesterone, is present only in theca cells. In the present study, only 23% of the follicular cell lysates analyzed exhibited P45017{alpha}, at very low levels, confirming that the cellular population recovered during in vivo follicular punctures consisted almost exclusively of granulosa cells [48]. In the present study, no signal was observed for 17ßHSD, the enzyme that converts androstenedione to testosterone. It is possible that 17ßHSD is not present in equine granulosa cells or that equine 17ßHSD does not cross-react with the human antiserum used. Since 17ßHSD has been localized in granulosa cells, and not theca cells, from rat [49] and human [50] ovary, further studies are needed to confirm our observations.

Steroidogenic activity in early dominant follicles was relatively low compared with that in later stages. Increased estradiol-17ß and progesterone production during growth of the dominant follicle may be the result of increased levels of steroidogenic enzymes, as we observed for P450scc, 3ßHSD, and aromatase in the present study. This is consistent with findings in pigs [14, 51, 52] and cattle [13, 53, 54]. Additionally, the decrease in intrafollicular estradiol-17ß concentration during the LH-induced maturation of the presumptive ovulatory follicle may be due, at least in part, to a decrease in aromatase level rather than to a decrease in androgen availability as observed in sheep [55], cattle [56], and humans [57]. The decrease in aromatase expression after LH administration was observed in the rat [58, 59], sheep [60], and pig [61] ovary. In the present study, the decrease in P450scc and 3ßHSD expression after gonadotropin injection, along with the increasing follicular fluid progesterone concentration, was unexpected. Nevertheless, similar observations have been made at the mRNA and/or protein levels in the porcine [14], bovine [54, 62], and human species [63]. Thus, two hypotheses could be proposed to explain such a discrepancy: an increase in enzymatic activities instead of enzyme levels after LH injection, or the production of progesterone, at the preovulatory stage, not only by granulosa cells but also by theca cells, which are the only contaminating cells of samples in the present study (see above). The second hypothesis is in accordance with studies performed on bovine and human ovaries [54, 64], but appears contradictory to previous reports demonstrating that 3ßHSD activity was exclusively localized in the granulosa cell layer in the mare [65].

In comparison with late dominant and preovulatory follicles, in subordinate follicles we observed a reduced steroidogenic capability. Actually, significantly lower expression of P450scc, 3ßHSD, and aromatase was observed in subordinate follicles, along with decreased levels of estradiol-17ß and progesterone. This is in agreement with previous observations in the mare [27, 66] and demonstrates that in the mare, as in other species, the fall in steroidogenic capability could be an early physiological consequence of atresia.

In the present study, the expression of aromatase in granulosa cells increases with an increase in follicular diameter, which confirms our recent data [48]. The same results have been obtained by Almadhidi et al. [67] by using immunohistochemistry. This is in agreement with findings in the rat [58], pig [52], and sheep [68]. Moreover, mRNA for aromatase increased with follicular diameter in porcine [52] and bovine [54] follicles.

The protein StAR has been recently characterized in the mouse [69], and until very recently, no information was available for the equine species [70]. We have shown that the protein StAR is expressed by equine follicular cells, which are, under our experimental conditions, mainly granulosa cells. The granulosa cell expression of StAR has been confirmed by an immunoblot study using cumulus cells (not shown). Kiriakidou et al. [71] have shown that in the human ovary the protein StAR was expressed only in theca cells in the preovulatory follicle and in the luteal cells. No expression of StAR was observed in bovine granulosa cells [72] at all. In contrast, granulosa cell expression of StAR was observed in porcine granulosa cells [73, 74]. Of note is the fact that we demonstrated that in equine species the expression of this protein is linked to the follicular growth. A low expression was observed around the time of emergence of the dominant follicle, a maximal expression in the dominant follicle before injection of exogenous CEG to induce ovulation, and a decrease at the preovulatory stage, after injection. Surprisingly, this observation is not consistent with previous data documented in equine granulosa cells by Northern blot [70]. Further studies are needed to clarify this discrepancy.

In conclusion, the present findings demonstrate that in the equine ovary, P450scc, 3ßHSD, and aromatase are expressed in granulosa cells from all follicles from 5 mm until the preovulatory stage. Their expression is influenced by the follicular diameter and the stage of follicular development, but it is not related to oocyte competence for in vitro maturation. Finally, the changes in intrafollicular patterns of steroids during the follicular phase are mainly determined by the expression levels of the various steroidogenic enzymes.


   ACKNOWLEDGMENTS
 
We are grateful to Isabelle Couty and the staff of the experimental farm for technical assistance, to D. André and the staff of the Laboratoire de Dosages Hormonaux (INRA, Nouzilly, France) for RIA of steroids, to Alain Beguey and Odile Moulin for photographic work, and to Peter Daels for correction of the manuscript. We wish to thank Monique Ottogalli for technical assistance and the preparation of CEG, Dr. Daniel Guillaume for help with statistical analysis, and Dr. Dale Buchanan Hales, Dr. Van Luu-The, Dr. Anita Payne, and Dr. Gilles Eric Séralini for generously providing the different antibodies. F. Belin wishes to thank Eric Palmer for accepting him in the Lab.


   FOOTNOTES
 
First decision: 29 September 1999.

1 This work was supported by grants from the Institut National de la Recherche Agronomique (INRA, France) and the Haras Nationaux (France). Back

2 Correspondence. FAX: 33 2 47 42 77 43; gerard{at}tours.inra.fr Back

Accepted: January 5, 2000.

Received: August 18, 1999.


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 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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