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Biology of Reproduction 66, 906-916 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Onset of Steroidogenic Enzyme Gene Expression During Ovarian Follicular Development in Sheep1

Kathleen A. Logan2,a, Jennifer L. Juengela, and Kenneth P. McNattya

a Reproduction Group, AgResearch, Wallaceville Animal Research Centre, Ward Street, Upper Hutt 6007, New Zealand


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Steroidogenesis is a major function of the developing follicle. However, little is known about the stage of onset of steroid regulatory proteins during follicular development in sheep. In this study, several steroidogenic enzymes were studied by immunohistochemistry and/or in situ hybridization; cytochrome P450 side chain cleavage (P450scc), cytochrome P450 17{alpha}-hydroxylase (17{alpha}OH), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450 aromatase (P450arom), steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR), and LH receptor (LH-R). To define the stages of follicular growth, ovarian maps were drawn from serial sections of ovine ovaries, and follicles were located and classified at specific stages of growth based on morphological criteria. In this way, the precise onset of gene expression with respect to stages of follicular growth for all these proteins could be observed. The key findings were that ovine oocytes express StAR mRNA at all stages of follicular development and that granulosa cells in follicle types 1–3 express 3ß-HSD and SF-1. Furthermore, the onset of expression in theca cells of StAR, P450scc, 17{alpha}OH, 3ß-HSD, and LH-R occurred in large type 4 follicles just before antrum formation. This finding suggests that although the theca interna forms from the type 2 stage, it does not become steroidogenically active until later in development. These studies also confirm that granulosa cells of large type 5 follicles express SF-1, StAR, P450scc, LH-R, and P450arom genes. These findings raise new questions regarding the roles of steroidogenic regulatory factors in early follicular development.

follicular development, granulosa cells, ovary, steroid hormones, theca cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian follicular development is a complex process involving paracrine and autocrine signaling within the ovary and an exchange of endocrine signals between the ovary and pituitary gland [1]. Follicular development is a process involving both gonadotropin-independent and -dependent phases of growth, with an intermediate gonadotropin-responsive phase [2–4]. The gonadotropin-dependent phase of growth, particularly preovulatory follicular development, has been well studied in sheep [1, 5–9]. However, the relationships among the acquisition of gonadotropin receptors, gonadotropin responsiveness, and steroidogenic function in the earliest stages of follicular growth have not been well defined.

Recent studies have shown that small ovarian follicles in sheep express or are receptive to growth factors such as growth differentiation factor 9, bone morphogenetic protein (BMP) 15, type II and type IB BMP receptors, stem cell factor, and c-kit [10–13]. Thus, small preantral follicles, including primordial follicles, are not quiescent but rather are functionally active entities. Given that small follicles either synthesize or are receptive to growth factors, it is likely that during the early developmental phases locally produced growth factors regulate proliferation of granulosa and theca cells. However, at some stage of development many follicular cells become committed to a differentiation pathway and synthesize steroids [4]. To better understand this process, more precise information is needed with respect to the differentiation functions of follicular cells during follicular growth.

The synthesis of steroid hormones by the developing follicle is dependent upon the presence and activities of several key proteins, such as steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc), cytochrome P450 17{alpha}-hydroxylase (17{alpha}OH), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and cytochrome P450 aromatase (P450arom) [14–16]. In sheep, granulosa cells in preantral and antral follicles (i.e., types 2–5) express FSH receptor (FSH-R) mRNA and those in large antral follicles express P450scc, P450arom, and LH receptor (LH-R) [4, 7, 8, 17]. Moreover, theca cells of antral follicles express LH-R and the steroidogenic enzyme mRNAs for androgen synthesis [7–9]. However, the precise onset of expression of many of these factors together with those of StAR and SF-1 is not known. The purpose of this study was to determine the onset of expression for SF-1, StAR, P450scc, 17{alpha}OH, 3ß-HSD, P450arom, and LH-R in sheep follicles using a classification system defined by specific morphological criteria [18].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All procedures involving animals were approved by the Wallaceville Animal Ethics Committee.

Ovary Collection

To collect sufficient numbers of nonatretic follicles at all stages of follicular development from primordial (type 1/1a) to preovulatory, ovaries were collected from neonatal sheep (n = 5) and adult sheep during the luteal or follicular phases of the estrous cycle (n = 8). Ovaries from adult sheep were collected on Day 10 of the ovarian cycle either 24 or 36 h after a prostaglandin injection (n = 5) or without prostaglandin (n = 3) as representative of follicular and luteal phases tissues, respectively. Ovaries from all animals were removed and immersed overnight at 4°C in 4% paraformaldehyde following administration of an overdose of pentobarbitone (200 mg/kg body weight) to the animal via the jugular vein. Ovaries were then processed for histology using a standard protocol, embedded in paraffin, and stored at 4°C. Serial 5-µm-thick sections were cut, and every 10th section was stained with hematoxylin and eosin (HE).

Ovarian Maps and Classification of Follicles

Outlines of ovaries and follicles were traced by placing HE-stained slides onto an Olympus BH2 microscope with a xenon lamp and a periscope with mirrors to project the outline onto a piece of paper. These maps were used to label follicles for subsequent identification and classification. Follicles were classified according to the criteria described by Lundy et al. [18]. These criteria are based on measurements of follicular diameter, the number of layers of granulosa cells, and the presence or absence of a theca interna in the largest cross section and/or the section through the oocyte nucleolus (SON). From the SON, type 1 follicles were classified as those with a single layer of flattened granulosa cells around the oocyte (primordial follicles); type 1a contained a mixture of flattened and cuboidal granulosa cells in a single layer around the oocyte; type 2 (primary) had one to less than two complete layers of cuboidal granulosa cells, with a theca interna evident in a proportion (35%) of the follicles; type 3 (small preantral) had two to less than four layers of granulosa cells, with a theca interna evident in all follicles; type 4 (large preantral) had four to approximately eight layers of granulosa cells, with a prominent theca interna but no antral spaces; ‘small type 5’ had small fluid-filled spaces between granulosa cells and a prominent theca interna; and type 5 (antral) contained a fully formed antrum. Type 5 follicles were further classified according to their maximal diameter across basement membranes. Several follicles were not classified because the serial sections available included neither the SON nor the maximal diameter; however, those with an antrum and a diameter of >3 mm were included as ‘large type 5’ follicles. All other unclassified follicles were excluded from this study. All follicles that had signs of degeneration (i.e., pyknotic granulosa cells or degenerate oocytes) also were excluded from this study. At each stage of follicular growth (except ‘large type 5’ follicles), there were at least six follicles for each product studied, obtained from at least three adults and three neonates.

In Situ Hybridization

In situ hybridization (ISH) was performed as previously described [13, 19]. Complementary DNA for the gene products was either developed in our laboratory (SF-1 and 17{alpha}OH) [19] or kindly donated by colleagues: ovine StAR, ovine 3ß-HSD, and ovine LH-R were provided by Dr. G. Niswender (Colorado State University, Fort Collins, CO) [20–22], bovine P450scc was provided by Dr. M. Waterman (Vanderbilt University, Nashville, TN) [23] as modified by Juengel et al. [24], and bovine P450arom was provided by Dr. E. Simpson (Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia) [25].

Hybridization buffer containing 45 000 cpm/µl of cRNA was added to each section, and sections were covered with a 22 x 22-mm coverslip for overnight hybridization at 55°C for homologous probes or 50°C for nonhomologous probes. Slides were then washed stringently and dried, dipped in emulsion (LM-1; Amersham Pharmacia Biotech, Auckland, New Zealand), stored for 2–4 wk at 4°C, developed in Kodak developer D-19 (Radiographic Supplies, Christchurch, New Zealand), and counterstained with hematoxylin.

Immunohistochemistry

Immunohistochemistry (IHC) was performed as previously described [13, 19] using a pressure cooker antigen-retrieval method. Following horseradish-peroxidase labeling of the secondary antibody (DAKO swine anti-rabbit IgG) with a DAKO ABC kit (both from Med-Bio Ltd., Christchurch, New Zealand), staining sensitivity was increased using NEN tyramide signal amplification (Invitrogen, Auckland, New Zealand). The chromagen was 3,3'-diaminobenzidine tetrahydrochloride (DAB; Invitrogen) with hematoxylin counterstaining. SF-1 (UpState Biotechnology, New York, NY) and 17{alpha}OH and 3ß-HSD (Prof. I. Mason, University of Edinburgh, Edinburgh, U.K.) were all localized using antibodies at a concentration of 10 µg/ml. Specificity of binding was assessed by incubating tissue sections with the nonimmune rabbit IgG at 10 µg/ml. Several antibodies raised against several species of StAR, LH-R, P450scc, and P450arom were tested, but none showed specific cross-reactivity with ovine tissues using the methods described above. Thus, we were unable to undertake IHC studies for these products.

Microscopy

Slides were examined using bright and dark fields on an Olympus BX50 microscope. Photographs were taken using an Olympus PM-C35DX camera and PM-30 exposure control unit (Olympus New Zealand Limited, Lower Hutt, New Zealand). The onset of expression was deemed to occur when >5% of follicles of a particular classification showed silver grains (mRNA hybridization) or brown staining (protein immunolocalization) in any cell.

Hybridization of cRNA probes was determined using darkfield illumination, where silver grains in positive tissues were compared visually to negative tissues and sense-hybridized slides. When this signal was obviously above background levels, quantification of silver grain intensity was not recorded. However, in some cases (e.g., expression in oocytes of SF-1, StAR, and 3ß-HSD), where the signal appeared to be only slightly above background levels, image analyses were performed to confirm expression. The microscope image was projected via a video-camera attachment (JVC model TK1070E) to a Sony video monitor (Olympus New Zealand Limited), with a grid overlay calibrated for area measurements using a stage micrometer. The grid was used to estimate the area of oocytes, and silver grains were counted manually under 100x oil immersion magnification. Background silver grain density measurements were made in nonpositive tissues, except in the case of SF-1 where antral spaces were used since most ovarian cell types contained SF-1 (determined by IHC). Silver grain densities (net number of grains/µm2) were calculated by subtracting the mean background level on each slide from the densities measured in oocytes on that slide. To test for SF-1, 3ß-HSD, and StAR gene expression, silver grain densities were measured in oocytes of systematically sampled type 1 and type 1a follicles and in oocytes of all other follicles, without knowing which were treated with sense or antisense probes. The net silver grain densities in 3ß-HSD, SF-1, and StAR sense hybridized oocytes for each follicular type were compared with their respective antisense hybridized oocytes using ANOVA and, where appropriate, the Tukey-Kramer test (P < 0.05). Differences between neonatal and adult ovaries in the proportions of follicles first showing expression for SF-1, StAR, P450scc, 17{alpha}OH, 3ß-HSD, P450arom, and LH-R mRNA were tested by chi-square analysis.

To determine the proportion of follicles containing protein localization (e.g., 3ß-HSD protein in granulosa cells), a randomized sampling strategy was employed for counting some type 1 and type 1a follicles and all of the type 2 and larger follicles on each section. Those follicles with brown DAB staining (above the level of nonimmune IgG negative controls) in one or more cells were deemed to contain protein. The nonspecific binding of nonimmune rabbit IgG to oocytes in some negative control sections made it difficult to determine whether oocytes showed protein localization. Because of the variability of nonspecific binding, image analysis was not a viable option; therefore, the localization of protein in oocytes was not confirmed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
For all genes studied no significant differences between follicles from neonatal and those from adult ovaries were observed for the stage of onset of expression of SF-1, StAR, P450scc, 17{alpha}OH, 3ß-HSD, P450arom, and LH-R; therefore, the results were pooled.

Steroidogenic Factor 1

Both SF-1 mRNA and SF-1 protein were observed in several cell types in the ovary, including granulosa cells, theca cells, rete, surface epithelium, and stromal (interstitial) cells, especially those in the cortical regions (Figs. 1 and 2, A and B). During follicular development, SF-1 mRNA was first observed in granulosa cells of follicle types 1, 1a, and 2 (Fig. 1, A and C). SF-1 gene expression in follicle types 1 and 1a is denoted by silver grains in the vicinity of granulosa cells on the bright field microscope image (Fig. 1A), whereas sense (negative control) hybridization was minimal (Fig. 1B). SF-1 gene expression continued in granulosa cells of nonatretic follicles throughout all stages of growth (Fig. 1, D–H). SF-1 mRNA was not detectable in oocytes at any stage of follicular growth: no differences in silver grain densities were noted when comparing oocytes hybridized to sense and antisense cRNA (P > 0.2; e.g., Fig. 1, A and B). SF-1 mRNA was evident in the region of the newly forming theca interna of follicle types 2 and 3. However, because adjacent interstitial cells also expressed SF-1 mRNA, it was not possible to clearly distinguish between expression in theca cells and interstitial cells at these stages of follicular growth. However, in 78% (n = 32) of type 4 follicles (Fig. 1, E and F) and in 100% (n = 169) of type 5 follicles, SF-1 mRNA was clearly evident in the theca interna and this expression was intense in large nonatretic follicles (Fig. 1, G and H).



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  		FIG. 1. SF-1 mRNA expression. A) Silver grains denoting hybridization of antisense SF-1 cRNA to type 1 and type 1a follicles, stromal cells, and surface epithelium. B) Hybridization of the sense (negative control) SF-1 cRNA. C and D) Type 2 (C) and type 3 (D) follicles with silver grains showing SF-1 mRNA. Bars = 50 µm (A–D). E–H) Left panels are brightfield and right panels are darkfield views of a type 4 follicle with gene expression in both granulosa and theca cells (E and F) and a large type 5 follicle (4 mm in diameter) with mRNA expression in granulosa and theca cells (G and H). Bars = 200 µm (E–H). g, Granulosa cells; t, theca cells; se, surface epithelium

The nuclear localization of SF-1 protein in granulosa cells confirmed SF-1 expression in this cell type (Fig. 2A). Some oocytes appeared to show immunostaining with SF-1 antibody; however, nonimmune IgG also showed variable staining in some sections with oocytes, so it was not possible to demonstrate unequivocally the presence of SF-1 protein. Localization of SF-1 protein occurred in granulosa cells of all nonatretic follicles of types 1 (n = 145), 1a (n = 125), 2 (n = 22), 3 (n = 6), and larger (n = 106) (Fig. 2, A and B; some data not shown). In this study, we could not distinguish between theca and interstitial cells in the region of the developing theca interna of type 2 follicles. However, the localization of SF-1 protein in theca cells occurred in five of six type 3 follicles, 11 of 12 type 4 follicles, and 97% of all type 5 follicles (n = 94; Fig. 2B).



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  		FIG. 2. SF-1, 3ß-HSD, and 17{alpha}OH protein localization. A) SF-1 immunolocalization in small follicles (in the same region as Fig. 1, A and B) and localization to the surface epithelium and stromal cells. B) Widespread SF-1 localization in the cortical stroma, granulosa cells of all follicles, and theca interna of a type 5 follicle. C) Negative control in the same area as B incubated with nonimmune rabbit IgG. D) Protein localization of 17{alpha}OH in theca cells of a type 5 follicle but not a type 4 follicle or smaller follicles. The peach color in granulosa cells and oocytes is nonspecific background staining. E) 3ß-HSD in granulosa cells of small follicles. F) 3ß-HSD in theca but not granulosa cells of a large type 5 follicle (4 mm in diameter). Bars = 50 µm. g, Granulosa cells; t, theca cells; a, antrum; se, surface epithelium. Numbers (1, 1a, etc.) refer to the growth classification of each labeled follicle.

Steroidogenic Acute Regulatory Protein

StAR was the only product in this study that consistently showed mRNA expression in oocytes at all stages of development (Fig. 3, A–E). For each follicular type the number of silver grains per unit area was significantly greater in oocytes hybridized to the antisense StAR cRNA than in oocytes hybridized to the sense cRNA (P < 0.001). There was a significant increase in mean StAR density as follicles developed up to type 4 stage (P < 0.0001). The mean (±SEM;) density (net grains/µm2) was 0.016 ± 0.002 for type 1 follicles, 0.021 ± 0.002; for type 1a follicles, 0.017 ± 0.005 for type 2 follicles, 0.044 ± 0.006 for type 3 follicles, 0.066 ± 0.011 for type 4 follicles, 0.064 ± 0.011 for small type 5 follicles, and 0.060 ± 0.014 for other type 5 follicles.



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  		FIG. 3. StAR mRNA expression in the oocytes of follicle types 1 and 2 (A), type 3 (B), type 4 (C), and type 5 (D and E). Silver grains denoting StAR expression are also in the theca cells of the type 5 follicle (D, arrows). Bars (A–E) = 25 µm. F) Brightfield (upper panel) and darkfield (lower panel) views showing strong hybridization in theca cells of a large follicle (5 mm in diameter) with some expression in granulosa cells. Bar = 200 µm. a, Antrum; g, granulosa cells; t, theca cells; o, oocyte

Expression of StAR mRNA in the theca interna was first observed in large type 4 follicles (1/11) showing a few theca cells with faint hybridization (data not shown). By the small type 5 stage, 45% of the follicles (n = 29) contained theca cells expressing StAR mRNA. Of the type 5 follicles <=3 mm in diameter, 65% (n = 74) showed theca cell hybridization (arrows, Fig. 3D), increasing to 100% hybridization in follicles >3 mm in diameter (n = 7). Although not quantitated, the gene expression of StAR in theca cells of type 5 follicles generally increased with increasing size. Of seven large type 5 follicles (>3-mm diameter) expression was only observed in granulosa cells of the largest four follicles (those >=4.0-mm diameter; Fig. 3F).

Cytochrome P450 Side Chain Cleavage

Follicle types 1–3 did not express P450scc mRNA in oocytes, theca cells, or granulosa cells. Expression of the P450scc gene was first detectable in theca cells of type 4 follicles (Fig. 4, A and B). Expression was observed in 35% of the type 4 follicles (n = 31), but the signal was faint and often limited to just a few theca cells. Subsequently, 57% of small type 5 (n = 21) and 93% of the other type 5 follicles (n = 104) expressed P450scc mRNA in theca cells. Although not quantitated, the expression levels in the theca interna appeared to increase with follicular size (Fig. 4, A–C). P450scc mRNA was not evident in granulosa cells of type 4, small type 5, or type 5 follicles <=2.20 mm in diameter (n = 17, upper right follicle in Fig. 4C). Expression was first observed in granulosa cells in five of the six type 5 follicles >2.20 mm but <2.50 mm in diameter (left follicle in Fig. 4C). All type 5 follicles >3 mm in diameter (n = 8) expressed the P450scc gene in granulosa cells (data not shown). Thus, P450scc mRNA was expressed in granulosa cells at a later stage than in theca cells of type 5 follicles and was not evident in oocytes at any stage of development.



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  		FIG. 4. Brightfield (A) and darkfield (B) views showing P450scc in the theca interna of a type 5 follicle 1.8 mm in diameter and fainter expression in theca cells of smaller antral follicles. C) P450scc in the theca interna of two type 5 follicles and in granulosa cells of the bigger follicle (2.4 mm in diameter, lower left) but not the smaller follicle (1.1 mm in diameter, upper right). Bar = 200 µm (A–C). g, Granulosa cells; t, theca cells. Numbers 4 and 5 refer to type 4 and type 5 follicles, respectively

Cytochrome P450 17{alpha}-Hydroxylase

Preantral follicles of types 1–3 did not express 17{alpha}OH mRNA. Furthermore, 17{alpha}OH mRNA was never observed in granulosa cells or oocytes. Messenger RNA for 17{alpha}OH was first observed in some theca cells in 38% of the large type 4 follicles (n = 39). Thereafter, 71% of the small type 5 follicles (n = 21) showed evidence of 17{alpha}OH gene expression, and theca cells of 96% of the other type 5 follicles (n = 94) showed evidence of expression (Fig. 5, A and B). Expression of this gene was most intense in large follicles (Fig. 5C).



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  		FIG. 5. Brightfield (A) and darkfield (B) views showing expression of the gene for 17{alpha}OH in type 5 follicles but not in a type 4 follicle. C) Strong 17{alpha}OH expression in the theca cells of a 5-mm-diameter (large type 5) follicle. Bar = 200 µm (A–C). g, Granulosa cells; t, theca cells. Numbers 4 and 5 refer to type 4 and type 5 follicles, respectively

Localization of 17{alpha}OH protein in theca cells first occurred in 54% of the type 4 follicles (n = 37), 96% of small type 5 follicles (n = 23), and 97% of the other type 5 follicles (n = 110), similar to the onset of expression of mRNA. The protein localization pattern was heterogeneous among cells within the theca interna layer (Fig. 2D).

3ß-Hydroxysteroid Dehydrogenase

Both 3ß-HSD protein and mRNA were localized in granulosa cells of follicle types 1–4 (Figs. 2E and 6, A–C, respectively). Table 1 shows the proportion of follicles of each type with 3ß-HSD mRNA or protein in any granulosa cells. Follicle types 1 and 2 generally showed gene expression or protein localization in all granulosa cells, whereas for types 3 and 4, expression, when present, was usually limited to a few cells near the oocyte (Fig. 6, A–C). No 3ß-HSD mRNA was observed in granulosa cells of type 5 follicles (Fig. 6D).


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  		TABLE 1. Percentage of follicles with 3ß-HSD expression in any granulosa cells, from a randomized sample of type 1 and type 1a follicles and from all type 2–5 follicles present



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  		FIG. 6. Silver grains denoting 3ß-HSD are located in the granulosa cells of type 1a and type 2 follicles (A) and a type 3 follicle (B). C) A type 4 follicle without signal in either the granulosa or theca cells. Bars = 50 µm (A–C). Numbers refer to the follicular growth stage. D) 3ß-HSD expression is limited to the theca cells of a 4-mm-diameter (large type 5) follicle. Bar = 200 µm. g, Granulosa cells; t, theca cells

Image analysis of silver grain density showed no significant differences between 170 antisense-hybridized oocytes and 132 sense-hybridized oocytes, from follicle types 1–3 (P > 0.3), suggesting no expression of the 3ß-HSD gene in oocytes.

In theca cells, 3ß-HSD mRNA was first observed from the type 4 stage of growth: 26% of type 4 follicles (n = 19) contained a few cells with a low level of expression, whereas 52% of small type 5 (n = 23) and 87% of the other type 5 (n = 75) follicles showed clear evidence of mRNA expression (Fig. 6D). Protein was localized in theca cells in 1/18 type 4 and 2/23 small type 5 follicles, and the presence of protein increased to 64% of the other type 5 follicles <=3 mm in diameter (n = 69). All large type 5 follicles >3 mm in diameter (n = 8) showed 3ß-HSD immunostaining in theca cells, but there was no evidence for this protein in the granulosa cells of these follicles (Fig. 2F).

Cytochrome P450 Aromatase

Neither oocytes nor theca cells of any of the follicles studied expressed P450arom mRNA (data not shown). Moreover, P450arom was not observed in granulosa cells of any follicle <=3 mm in diameter (n = 226). However, gene expression in granulosa cells was observed in all eight large follicles (>3 mm in diameter) that were studied (n = 7 ewes; Fig. 7). Furthermore, there was very strong hybridization (denoted by relative intensity of silver grains) in six of these follicles.



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  		FIG. 7. Brightfield (left) and darkfield (right) views showing gene expression of P450arom limited to granulosa cells in this 4-mm-diameter type 5 follicle. Bar = 200 µm. g, Granulosa cells; t, theca cells

LH Receptor

There was no evidence for LH-R mRNA expression in oocytes of any of the follicles studied. Gene expression for LH-R was first observed in the theca interna of 20% of the type 4 follicles (n = 15), but it was limited to only a few cells (Fig. 8, A and B). In antral follicles, 63% of small type 5 follicles (n = 32) and 84% of the other type 5 follicles (n = 93) showed evidence of LH-R mRNA in the theca interna (Fig. 8, A and B). LH-R gene expression appeared to increase as follicles developed, and this increase appeared to be due to a greater number of theca cells with mRNA and an increase in the hybridization intensity. In follicles >3 mm in diameter, LH-R mRNA was also observed in granulosa cells in 3/5 follicles studied (Fig. 8C). There was no evidence for LH-R gene expression in granulosa cells of follicles <=3 mm in diameter.



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  		FIG. 8. LH-R gene expression. A) Brightfield view showing two type 4 and two type 5 follicles (labeled). B) Darkfield view with LH-R expression limited to theca cells of one type 4 follicle and the type 5 follicles. C) LH-R in both theca and granulosa cells of a 5-mm-diameter follicle. Bar = 200 µm (A–C). g, Granulosa cells; t, theca cells

Summary of Onset of Steroidogenic Regulatory Factors

The onset and pattern of gene expression for SF-1, StAR, P450scc, P45017{alpha}OH, 3ß-HSD, P450arom, and LH-R and protein localization for SF-1, 17{alpha}OH, and 3ß-HSD in nonatretic follicles are summarized in Table 2.


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  		TABLE 2. Onset of synthesis of steroidogenic regulatory factors and steroidogenic enzymes (i.e., protein and/or mRNA)a with respect to stage of follicular development (follicular type) and specific follicular cell type: granulosa cell (G), theca cell (T), or oocyte (O)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we discovered that ovine oocytes at all stages of development express StAR mRNA and that granulosa cells of primordial and small preantral follicles express 3ß-HSD concomitantly with SF-1. Moreover, the onset of steroidogenic capability occurs in ovine theca cells just before antrum formation, because the steroidogenic enzymes for androgen synthesis are first expressed in these cells in large type 4 follicles. Although the theca interna is first evident in some type 2 follicles and is present in all type 3 follicles [18], this tissue does not attain the ability to produce steroids until the type 4 stage of growth, suggesting that steroidogenesis occurs later in development than morphological differentiation.

Another key finding was that the stage of onset of expression for SF-1, StAR, P450scc, 17{alpha}OH, 3ß-HSD, P450arom, and LH-R was not significantly different between follicles recovered from neonatal and those recovered from adult ovaries. Where comparisons were possible (e.g., for SF-1, 3ß-HSD, and 17{alpha}OH), the mRNA and protein localizations and stage of follicular growth when first detected mirrored one another. The genes for StAR, the steroidogenic enzymes, and LH-R are expressed in the theca interna in an increasing proportion of follicles from the type 4 to the large type 5 stage. In studies of expression of these mRNAs in the development of antral follicles in the cow, expression of StAR, steroidogenic enzymes, and LH-R was observed in preantral follicles with a well-developed theca [16]. Although the precise onset of expression was not determined in the cow, this expression pattern appears very similar to that observed in sheep.

SF-1 was first identified as a transcription factor regulating P450 enzymes [26, 27]. Subsequently, more functions for SF-1 have been discovered [28, 29], including the regulation of StAR gene expression [30], inferring that SF-1 has a wider role in the regulation of steroidogenesis [28, 29]. Consistent with this role, SF-1 protein was localized specifically to theca cells of type 3 follicles, which preceded the onset of steroidogenic enzyme gene expression. The apparent relative increase in intensity of SF-1 mRNA and protein in both granulosa and theca cells of progressively larger type 5 follicles is consistent with the notion that the level of SF-1 is related to the level of steroidogenic activity [30]. The role of SF-1 protein in follicle types 1–3 is not clear. SF-1 mRNA and protein are present in the fetal gonad as it first differentiates from the mesonephros. Expression continues in the early differentiating ovary after morphological sexual differentiation and exists in the smallest differentiated ovarian follicle, the type 1 structure [19, 31]. Thus, its progression in type 1 follicles may be related in some way to the function(s) of SF-1 in the developing gonad.

StAR is an important factor in the acute regulation of steroidogenesis [15]. For the cow, expression of StAR is limited to theca cells in nonatretic antral follicles [16]. In contrast, in sheep, we observed expression in theca cells of antral follicles extending to granulosa cells of large follicles, 4.0–5.3 mm in diameter. Overall the pattern of expression for StAR was similar to that observed in many other mammals [32–35].

Although StAR is a regulator of steroid synthesis [15], the finding of StAR mRNA in oocytes from follicles of types 1–5 without the concomitant expression of steroidogenic enzymes suggests that its presence in oocytes is related to an alternative function. StAR mRNA has been reported in human renal distal tubule epithelia, Sertoli cells, and fetal human oocytes, none of which express P450scc mRNA [35], and the function of StAR in these cells is not known. Cholesterol is used as a precursor lipid for products other than steroid hormones [35, 36]. Therefore, it is possible that in the oocyte StAR is used for transport of cholesterol precursors to regulate cellular metabolic processes other than steroidogenesis.

Huet et al. [7] reported the localization of P450scc protein in theca cells of 1- to 2-mm-diameter ovine follicles and that immunostaining intensity increased significantly with increasing follicular diameter. Our study extends these findings to show that the onset of P450scc mRNA in theca cells occurs before antrum formation in large type 4 follicles. In granulosa cells, the onset of P450scc gene expression was at a diameter of 2.2 mm, and no obvious increase in expression during follicular growth was noted. Although Huet et al. [7] referred to a significant increase in granulosa cell expression during follicular growth, their comparison was between granulosa cells in 1- to 2-mm-diameter follicles with no expression and granulosa cells in 3.5- to 5-mm-diameter follicles with positive expression; these observations are consistent with those of the present study.

In sheep, 17{alpha}OH protein has been localized to the theca interna of small antral follicles (1–2 mm in diameter) [7, 9]. The present study extends these findings to show that the onset of 17{alpha}OH mRNA and protein occurs in large type 4 follicles. We also confirmed that 17{alpha}OH gene and protein expression are localized exclusively to theca cells. Collectively, these findings indicate that by the time the antrum forms, the theca interna has the ability to convert progestins to androgens.

Conley et al. [9] reported that localization of 3ß-HSD protein in ovine follicles was restricted to theca cells of antral follicles (type 5 follicles). Our study extends this finding by showing that the onset of 3ß-HSD mRNA and protein in theca cells occurs before antrum formation, i.e., in large type 4 follicles. No evidence was found that oocytes express 3ß-HSD mRNA or protein. An unexpected observation was the presence of 3ß-HSD mRNA and protein in most granulosa cells of follicle types 1, 1a, and 2 and in some granulosa cells close to the oocytes of follicle types 3 and 4. In the developing ovary of fetal sheep, there is evidence of 3ß-HSD expression without concomitant expression of other steroidogenic enzymes (e.g., P450scc), and it has been suggested that some of these cells may be destined to become the granulosa cells of type 1 follicles, which continue to express this enzyme [19, 31]. Because granulosa cells in follicle types 1, 1a, and 2 do not express the steroid regulatory factor StAR or other steroidogenic enzymes it is unlikely that these small follicles synthesize steroids. One possibility is that 3ß-HSD may be metabolizing steroids such as dehydroepiandrosterone or pregnenolone to remove them from the vicinity of the oocyte, which may be important in preventing oocyte maturation and/or regulating growth.

Huet et al. [7] described P450arom protein in sheep follicles between 3.5 and 5 mm in diameter. In the current study, P450arom mRNA was detected exclusively in granulosa cells of follicles >3 mm in diameter (i.e., large type 5 follicles), which is consistent with P450arom protein localization and previous reports measuring aromatase activity or estradiol in follicular fluid [6, 7, 37]. This observation is also similar to reports of P450arom in dominant follicles only [7, 16, 37, 38]. Thus, the onset of P450arom mRNA expression is consistent with the long-held view that estradiol is a product of a large nonatretic follicle in the final phases of preovulatory maturation [2, 3].

The onset of LH-R expression in theca cells was at the large type 4 stage of development. In the cow, the onset of LH-R in the theca interna has been reported to occur around the time of antrum formation [16]. In both species, the interaction of LH with its receptor is known to be important in regulating steroidogenesis by, for example, upregulating StAR and steroidogenic enzyme expression [39].

Expression of LH-R in granulosa cells was observed in only three follicles >=3.9 mm in diameter and not in two follicles >3 mm in diameter that expressed P450arom. Although the numbers studied were small, this result suggests that, as in cows [16], the onset of LH-R mRNA expression in ovine granulosa cells occurs at a later stage of growth than that of P450arom.

McNatty et al. [40] reported low but detectable levels of progesterone and androstenedione production by large type 4 to small type 5 follicles (i.e., antrum-forming follicles, 0.1–0.2 mm in diameter) in vitro. Their study also showed that steroid production increased markedly as follicles increased to 0.44 mm in diameter (i.e., small antral follicles). Moreover, as follicles increased beyond 0.5 mm in diameter, they synthesized estradiol-17ß after 48 h in culture. Collectively, the results from these studies, where follicles were exposed to both LH and FSH in vitro, are consistent with those of the present study for the theca interna, where the onset of LH-R, SF-1, StAR, P450scc, 17{alpha}OH, and 3ß-HSD protein and/or mRNA synthesis occurred in large type 4 follicles. However, the in vitro findings differed from those of the present study with respect to estradiol synthesis. The discrepancy between the onset of P450arom in vivo and the apparent early onset of estradiol synthesis in vitro may be due to differences in sensitivity between the RIA method used to detect estradiol and the in situ technique of detecting P450arom mRNA, which was optimized to minimize background silver grains. In sheep, the synthesis of FSH receptor mRNA is known to occur in granulosa cells from the type 2 to type 3 follicular stage of development [4, 17]. Moreover, ovine follicles 0.5–1.0 mm in diameter respond to FSH to synthesize cAMP in vitro [40]. Thus, exposure to FSH in vitro may have induced the onset of P450arom mRNA expression in the granulosa cells earlier than it would normally occur in vivo [41].

One of the most important aspects of this study was the extension of knowledge regarding small preantral follicles. In previous studies with follicle types 1 and 1a, these small "nongrowing" structures were functionally active in synthesizing growth factors or growth factor receptors [4, 10–13, 42]. In the present study, these follicles also synthesized SF-1, StAR, and 3ß-HSD. Thus, there is increasing evidence for the notion that type 1 and 1a (i.e., primordial) follicles and type 2 (primary) follicles have a much more complex biochemistry with respect to growth and steroidogenic regulatory factors than was previously thought. The presence of SF-1, 3ß-HSD, and StAR in granulosa cells or oocytes of follicle types 1, 1a, and 2 suggests possible links among cholesterol metabolism, intraovarian steroidogenesis, and the viability and/or mediation of action of growth factors in these structures.

With this study, we extended current knowledge regarding the expression of steroidogenic enzymes and regulatory factors and defined the precise onset of follicular steroidogenic capability during follicular development in sheep. In addition, we highlight the potential for SF-1, 3ß-HSD, and StAR to play important roles in the regulation of primordial and small growing follicles.


   ACKNOWLEDGMENTS
 
We thank the Reproduction Group, especially Norma Hudson, Anne O'Connell, and Peter Smith, for collection of ovarian tissue and Lynn O'Donovan and Lee-Ann Still for processing and sectioning ovaries.


   FOOTNOTES
 
First decision: 16 August 2001.

1 This work was supported by the New Zealand Foundation for Research, Science and Technology. Back

2 Correspondence: Kathleen Logan, AgResearch, Wallaceville Animal Research Centre, P.O. Box 40-063, Ward Street, Upper Hutt 6007, New Zealand. kathleen.logan{at}agresearch.co.nz Back

Accepted: October 31, 2001.

Received: July 9, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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