0021-972X/91/7304-0717$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 73, No. 4 Printed in U.S.A.
Expression of Aromatase Cytochrome P-450 in Premenopausal and Postmenopausal Human Ovaries: an Immunocytochemical Study* SANDRA E. INKSTER AND ANGELA M. H. BRODIE Dept. Pharmacology and Experimental Therapeutics University of Maryland, School of Medicine, Baltimore, Maryland 21201
stages of antrum formation (>700 ^m), and staining intensified as follicle diameter and antral cavity increased, being maximal in preovulatory follicles. GC aromatase was always found in the presence of TI immunostain. These two cell populations were separated by an unstained layer of TI cells giving the follicle walls a banded appearance. Immunostain was most intense in mural GC, was weaker in antral GC cells and was absent from the cumulus GC. Immunoreactive aromatase was also detected in functional corpora lutea (CL) but was absent from involuting CL's and corpora albicans. Our findings indicate that the immunostained cells of the CL are comprised of the former GC and possibly a subpopulation of former TI cells. In perimenopausal ovaries there was no evidence of any follicular or stromal aromatase immunostain. In postmenopausal ovaries no follicles were observed, but individual cells and clusters of cells in the stromal compartment of 3/7 specimens were found to have an aromatase immunostain reaction. In all cases, the aromatase immunostain reaction was cytoplasmic. The results provide the first direct evidence of the existence of TC aromatase, and of stromal cell aromatase in postmenopausal women. {J Clin Endocrinol Metab 73: 717-726,1991)
ABSTRACT. The cellular distribution of the aromatase cytochrome P-450 enzyme in human ovaries has been investigated immunocytochemically, using an aromatase-specific monoclonal antibody. Ovaries of females ranging in age from prepubertal infant girl through to postmenopausal adulthood were obtained from immediate autopsy or after surgery. The results have revealed temporal and spatial changes in expression of aromatase at different stages of development. No immunoreactive aromatase was detected in the ovary of the 2.5 month infant. In premenopausal ovaries, aromatase was absent from the stromal compartment, but in follicles, a consistent pattern in expression of aromatase was observed, related to their size and developmental stage. Aromatase was not expressed in primordial, primary, or small secondary follicles less than 250 /urn diameter. In slightly larger follicles (250-700 nm diameter) aromatase was first detected in a few thecal cells (TC). In more developed secondary through to large preovulatory follicles (>1 cm) TC aromatase immunostain increased in intensity and number of positive cells, and the reaction was localized to a band of theca interna (TI) cells at the Tl/theca externa interface. In granulosa cells (GC), aromatase was first detected in follicles in the initial
T
HE human menstrual cycle is a reflection of dynamic changes occurring in the structure and function of ovarian follicles. Little is known about the control mechanisms underlying follicular recruitment. However, once initiated during fetal life it becomes a continuous daily process that occurs at all stages of the cycle and is apparently independent of any pituitary gonadotropin influence(s). Follicular recruitment does not cease until the time of reproductive senescence, which is associated with exhaustion of the follicular pool, characterized by the menopause in women. As follicles develop antral cavities there is a large increase in the number and
activity of granulosa cells (GC). The thecal envelope becomes specialized into an outer fibrous layer, the theca externa (TE), and an inner, highly vascularized, glandular layer, the theca interna (TI) (1). Concomitant with antrum formation is a rise in serum estradiol (E2) which peaks before ovulation. In humans there is a secondary rise in serum E2 during the mid luteal phase. This is known to be produced by the corpus luteum (CL) but the precise cellular source has not been clearly established. Biochemical studies in numerous species have identified the GC as the major, if not exclusive, site of follicular estrogen synthesis, with GC of large preovulatory follicles having the highest levels of aromatase (estrogen synthetase) activity (2). Since most studies have used cultured GC obtained from either individual or pooled follicles, any intrafollicular variation in aromatase expression that may exist in vivo, as has been described for LH and FSH receptors (3), has not been examined. The currently accepted model of ovarian estrogen syn-
Received January 22,1991. Address correspondence and requests for reprints to: Angela Brodie, Ph.D., Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201. * Supported by NIH Grants CA-27440, HD-13909, and a University of Maryland Designated Research Initiative Award. Presented in part at the 71st Annual Meeting of the Endocrine Society, Seattle, WA, 1989.
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INKSTER AND BRODIE
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thesis is known as the two-cell two-gonadotropin hypothesis, the concept of which is that androgens, synthesized by thecal cells (TC) under the tonic influence of LH, are transported to the GC where their conversion to estrogens is mediated by FSH-regulated aromatase (4). The existence of human thecal aromatase is a controversial issue. Although the GC are recognized to be the major source of estrogen, there are a number of reports that unlike rodents, TC of humans and some other higher mammals do possess aromatase activity (5-8). However, the possibility of GC contamination of TC cultures cannot be unequivocally ruled out. Moreover, a recent study failed to detect any aromatase activity or messenger RNA (mRNA) in extracts of cultured human TC (9) and an immunocytochemical study in fixed human ovaries also did not find TC aromatase (10). In an attempt to resolve this discrepancy concerning thecal aromatase, we have applied our recently developed immunocytochemical method for detecting human aromatase in unfixed frozen sections (11), to premenopausal ovaries. The specific aims of our detailed study were to investigate: 1) the stage in follicular development at which aromatase is first expressed in GC, 2) whether GC are homogeneous or heterogeneous with respect to aromatase expression, 3) whether expression of aromatase is confined to the GC population, and 4) which cell(s) of the CL express aromatase. We have also examined aromatase expression in a single infant ovary (2.5 months), and have investigated what effects structural changes in the ovary associated with menopause (perimenopausal women, 45-54 yr) and the loss of reproductive function (postmenopausal women, >55 yr), have on ovarian aromatase expression.
Materials and Methods Tissues Thirty six fresh ovaries removed from women ranging in age from 17-68 yr, were obtained through the Human Tissue Resource service of the Pathology Department, University of Maryland School of Medicine, within 12 h of surgery or autopsy. Ovaries were classified as being 1) premenopausal, 17-45 yr, 2) perimenopausal, 45-54 yr, or 3) postmenopausal, greater than 55 yr. A single specimen was obtained from a 2.5 month infant. Since most premenopausal specimens were obtained from victims of accidental death, information on menstrual cycle status or relevant medication history (e.g. oral contraceptive usage) was not readily available. However, only specimens which appeared normal from gross pathological inspection were used. Most ovaries of peri- and postmenopausal women were obtained after surgery for nonovarian gynecological complaints (mainly for uterine fibroids). Ovaries were immediately rinsed in cold (4 C) 0.1 mol/L PBS, then dissected into smaller blocks with care being taken to maintain the integrity of any visible follicles. The specimens were coated with Tissue-Tek O.C.T. cry-
JCE & M • 1991 Vol 73 • No 4
oprotectant embedding medium (Miles Labs, Naperville, IL), immersed in liquid nitrogen, and then stored at —70 C. Fresh placental specimens obtained from routine full term deliveries were processed similarly and used as positive controls for aromatase. All tissues were collected under protocols approved by the Human Volunteers Research Committee of this institution. Immunocytochemistry. Frozen sections (6 /*m) of unfixed ovaries and placenta were thaw-mounted onto chrome-alum gel coated slides, then indirectly immunostained for aromatase. In initial studies our published protocol (11) based on the peroxidase-antiperoxidase (PAP) method (12) was used. However, in later experiments we applied a streptavidin modification of the avidin-biotinylated complex methodology (13). Both methods gave similar results and are briefly described below. Sections were rinsed in assay buffer (50 mmol/L Tris, 9 g/L NaCl, pH 7.6 at 25 C) and incubated at room temperature for 30 min with 30 g/L normal goat serum (NGS) (Vector Labs, Burlingame CA) to block nonspecific staining. Sections were then incubated at 4 C overnight with 1 mg/L anti-aromatase monoclonal antibody, generously provided by Drs. C. Mendelson and E. Simpson (Dallas TX). Details of the preparation and extensive characterization of these antibodies has been published (14). Negative control sections were incubated with either 10 g/L NGS, or with an equivalent dilution of monoclonal antibody specific to rat brain endothelium (courtesy of Dr. L. Sternberger, Baltimore, MD), both diluted in buffer. Second antibodies were applied for 30 min., 1:40 dilutions of goat antimouse (Cappel Labs., Durham, NC) or biotinylated goat anti-mouse immunoglobulin G (Amersham, Arlington Heights, IL) being used for PAP and streptavidin modification of avidinbiotinylated complex methods, respectively. After rinsing, sections were incubated for 1 h at room temperature with either 1:100 dilution of mouse PAP (gift of Dr. Sternberger) or with 1:50 dilution of streptavidin biotinylated peroxidase complex (Amersham, IL). The sections were rinsed before the location of aromatase-linked peroxidase complexes were visualized by 9-min reaction with 0.5 g/L 3,3-diaminobenzidine hydrochloride (DAB), 0.16 g/L hydrogen peroxide in buffer. The DAB reaction was terminated by rinsing in buffer and permanent slides were made following routine dehydration and mounting. Since our protocol uses unfixed sections, red blood cells (RBC), the main source of endogenous peroxidase activity, were washed away during the staining procedure, thus, we elected not to quench the endogenous peroxidase activity of immunostained sections. Instead we screened for presence of non-RBC endogenous peroxidase activity by incubating a single cryosection from each specimen with the DAB reagent for 9 min. A hematoxylin and eosin stained reference section was prepared for all specimens. Immunostained sections were routinely prepared without using counterstains since we have previously noted that they can lessen the intensity or even mask the DAB reaction product. However, to aid in the identification of ovarian structures, duplicate immunostained sections of some specimens were counterstained with 5 g/L toluidine blue. The sections were examined with a Nikon Microphot microscope
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LOCATION OF HUMAN OVARIAN AROMATASE equipped with Nomarski differential interference contrast (DIC) optic capabilities, and were photographically recorded.
Results The ovaries used in this study were obtained from women ranging in age from 2.5 months to 68 yr. Various stages of folliculogenesis, from the arrested primordial state to large preovulatory antral follicles, as well as CL and corpora albicans were examined. Follicular staging was based on the descriptions of Clement (1). As expected, primordial and preantral follicles were observed in all premenopausal ovaries; most contained a few small to large antral follicles, and only four preovulatory (>1 cm) follicles were encountered. The intact ovary of the 2.5 month infant was smooth surfaced, elongated, and flattened in appearance with approximate dimensions of 1.5 X 0.6 X 0.3 cm. Histological examination revealed it to be composed mainly of primordial follicles (40-60 fim diameter) situated in the cortex, and also a few scattered primary follicles (60-80 /im diameter) with expanded oocyte and one to two layers of cuboidal GC in the deeper cortex/medulla regions. There was no evidence of any aromatase immunostain throughout the entire structure (results not shown). Twenty-two premenopausal ovaries were examined. They were generally ovoid in shape with approximate dimensions of 4 X 3 x 2 cm. The external surfaces of the ovaries were visibly smoother in the younger women; the numbers of visible scars from previous ovulations increased with age. In most cases, fluid-filled spherical follicles of various sizes were evident beneath the ovary surface. In three ovaries a large CL with well vascularized outer surface was observed, one of which had a bloody stigma indicating the site of recent ovulation. Dissection usually revealed smaller antral follicles, involuting CL and corpora albicans situated in the deeper regions of the cortex. Histological examination of the ovaries supported the gross observations and also revealed primordial, primary and preantral follicles in the cortex of the ovaries; these immature follicles were more numerous in the younger premenopausal women and their incidence decreased with age. Specific staining of aromatase was detected in representative blocks of 17/22 premenopausal ovaries. From these, a pattern to the expression of the aromatase enzyme, related to the size and developmental stage of the follicle was evident (see Table 1). Aromatase could not be detected in primordial or primary follicles with one to four layers of GC (40-120 /um diameter) or in small secondary follicles (200 11 10 preantral 9 0 early antral 14 2 mid antral late antral 5 0 1 preovulatory 0
0 0 1 7
early CL midCL late CL Atretic follicles/corpora albicans
6 1
0
Theca and granulosa 0 0 0 2 6 4 1
1 2 5
0 0 5
0 0 0
0
15
15
0
0
1 2
could not be clearly distinguished; the GC of these follicles did not stain for aromatase (Fig. 1, a,b). In early antral follicles, thecal aromatase expression increased so that an immunostained circle of TI cells was evident, situated at the TI/TE interface; the TI cells immediately beneath the GC and the GC themselves were devoid of any immunostained products (Fig. 1, c,d,e). With further increase in follicular size, aromatase was still detected in cells at the TI/TE interface but now also started to be expressed in the GC. Granulosa cell staining was first detected in a 750 ixm diameter follicle (Fig. 2, a,b) and although there was some degree of overlap in the sizes of follicles in which the GC were positive or negative for aromatase (see Table 1), staining of GC was never found in the absence of TI cell aromatase. In fact, the two layers of immunostained cells (GC and TI) were separated from each other by an unstained layer of TI cells, giving the follicle wall a banded appearance. Regional variation of aromatase expression in the GC was evident with the mural GC having the strongest immunoreaction while the specialized GC of the oocyte cumulus oophorus complex did not express any aromatase protein (Fig. 2, b,d). In large preovulatory follicles, the level of stain in the GC increased and intensified so that it was deeper than the surrounding TC and was the major source of aromatase in the follicular-phase ovary (Fig. 2, e,f,g). Three CL were assessed as being recently formed and thus functional from the following criteria: 1) vascularization of their outer surfaces (in one case an obvious stigma was present), 2) sizes (~1.6, 1.5 and 1.3 cm diameter), 3) position at the ovary surface, and 4) intense yellow coloration of their folded inner layer, apparent at dissection. The 1.3 cm CL was estimated to be from very early in the luteal phase due to the presence of the stigma plug which normally persists for only a brief time. The structural organization of this CL was consistent with
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FIG. 1. Aromatase immunocytochemistry in 6 ^m unfixed frozen sections of human premenopausal ovaries (methods as described in text). Immunostained sections were photographed using Nomarski DIC optics to reveal structures of unstained cellular areas. Original magnifications in parentheses; bar = 100 /an. A, H + E stained section from the ovary of a 20-yr-old woman showing primordial (PO) and primary (P) follicles and a 320 /im diameter preantral follicle with distinct granulosa (G) and thecal (T) cell compartments (X200). B, Serial section of (A) showing aromatase immunostain localized to the cytoplasm of a few thecal cells (small arrowheads). Granulosa cells, PO and P follicles are unstained (X200). C, H + E stained section from the ovary of a 21-yr-old woman, showing an approximate 600 ^m follicle with developing antral cavity (A) and differentiated theca interna (TI) and theca externa (TE) layers (X160). D, Serial section of (C) immunostained with the control monoclonal antibody against rat brain endothelium (X200). E, Serial section of (C) showing aromatase immunostain localized to the cytoplasm of a ring of cells at the TI/TE interface (x200).
previously described transitional changes from follicle to mature CL (15). Aromatase immunostaining revealed an intense, homogeneous reaction of the inner layer of cells, presumably the luteinized GC. A more heterogeneous immunostain reaction occurred in the outer, thinner layer of cells (presumptive luteinized TI) which resembled the staining pattern of the follicles. Both cell laj^ers had characteristics of steroidogenic activity, that is they contained numerous vacuoles (lipid). The cells were of similar polygonal appearance, their dimensions ranging from 15-25 jum in the inner layer and 8-20 yum in the outer layer. In contrast to the pattern of staining in
antral follicles, no obvious basement membrane or discrete band of unstained TI cells was evident between the two layers which gradually merged into one another. The cells at the extreme periphery of the CL (presumptive TE) did not stain for aromatase (Fig. 3a). The larger CL (1.6, 1.5 cm) were estimated to be from the mid-luteal phase, and their cellular organization was very clearly structured. A thick, folded, inner layer of large, polygonal, steroidogenic cells (20-30 /xm) with spherical nuclei comprised the bulk of the CL. A thinner layer of morphologically distinct, smaller spindle-shaped cells with more elongated nuclei, surrounded the outer edges of the
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LOCATION OF HUMAN OVARIAN AROMATASE
FlG. 2. Aromatase immunocytochemistry in 6 /urn unfixed frozen sections of human premenopausal ovaries (methods as described in text). Immunostained sections were photographed using Nomarski DIG optics to reveal structures of unstained cellular areas except (A). Original magnifications in parentheses; bar = 100 /urn. A, Section from the ovary of a 17-yr-old woman immunostained for aromatase. Both granulosa and thecal cells of the secondary follicle (~850 Mm) contained immunoreactive aromatase. Note: Nomarski DIC optics not available at this magnification (X125). B, Detail of boxed area in (A) showing that TI cells immediately adjacent to the granulosa layer do not express immunoreactive aromatase (x200). C, H + E stained section from the ovary of 25-yr-old woman, showing a 1.65-mm antral follicle with oocyte (0) surrounded by specialized cumulus cells (C) of the granulosa layer (x50). D, Detail of the follicle wall in a serial section of (C) immunostained for aromatase, showing absence of stain in cumulus granulosa cells with patchy staining in some cells of the TI {arrowheads) (X200). E, H + E stained section through a greater than 1.3 cm follicle wall in the ovary of a 17-yr-old woman (x200). F, Serial section of (E) immunostained for aromatase showing cytoplasmic staining in both G and TI, separated by layer of unstained TI cells (X200). G, Serial section of (E) immunostained with the control monoclonal antibody (X200).
^
steroidogenic cells. This layer also ensheathed the vascular septa which extended into the core of the CL (Fig. 3, b,d). When immunostained for aromatase only the large cells were found to express the enzyme, resulting in a clear demarcation between the cell types (Fig. 3, c,e). Higher magnification revealed projections of smaller cells apparently penetrating the margins of the large cell areas, and isolated unstained cells, clearly of the smaller variety, were found among the aromatase-positive cells, presumably related to progressive vascularization of the structure. It should be noted that the area of aromatase immunostaining in both the early and mid stage CL was
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II lE
TO
more extensive than in preovulatory follicles. Although ages of older corpora lutea/albicans could not be determined very accurately, they were assessed as being in the regressive state from earlier menstrual cycles if, 1) they were not obvious on gross inspection of the ovary, 2) their size was less than 1 cm diameter, 3) they were situated more deeply in the cortex of the ovary, and 4) their inner yellow margins were relatively shrunken. Histologically, these structures were distinctly different from the functional CL. The dense inner layer of vacuolated cells had been replaced by an involuted layer of fibrous connective tissue with a thin, vascularized layer
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INKSTER AND BRODIE
JCE & M • 1991 Vol 73 • No 4
PCT
FIG. 3. Aromatase immunocytochemistry in 6 fim unfixed frozen sections of human premenopausal ovaries (methods as described in text). Immunostained sections were photographed using Nomarski DIC optics to reveal structures of unstained cellular areas. Original magnifications in parentheses; bar = 100 nm. A, Section through the wall of a transitional follicle/early corpus luteum (>1.5 cm diameter) in the ovary of a 21yr-old woman, immunostained for aromatase: note the intense homogeneous aromatase immunostain in the luteinized granulosa (LG) layer next to the central cavity (cav), with more heterogeneous reaction in the presumptive luteinized TI (pLTI) -derived component of the follicle/CL wall, and a lack of a distinct border (basement membrane) separating these layers (X200). B, H + E stained section through a mature functional CL in the ovary of a 37-yr-old woman. Two cell populations with distinct borders are apparent; darkly stained cells of the presumed luteinized granulosa (LG) and lightly stained cells of the presumed luteinized theca (LT) (X200). C, Serial section of (B) immunostained for aromatase plus nuclear toluidine blue counterstain. Immunoreactive aromatase is restricted to the cells of the LG population (X200). D, H + E stained detail of the CL shown in (B) showing that the LG is composed of large polygonal cells with rounded/ovoid nuclei and vacuolated cytoplasm, while spindle shaped LT cells have elongated nuclei and penetrate the edges of the LG cell areas (see arrowheads) (X400). E, Matched area of (D) immunostained for aromatase and counterstained with toluidine blue. The non-immunoreactive cells are clearly of LT morphology (see arrowheads) (X400). F, H + E stained section through the wall of a regressing CL/corpora albicans (6 days) of third passage human TC do not contain either aromatase activity or mRNA. It is possible that the absence of aromatase in these TC cultures may be a consequence of the lengthy period of culture in isolation from other regulatory ovarian cell components, and/or previous exposure to the high levels of gonadotropins administered during IVF procedures, which could have initiated irreversible changes in cell metabolism. Thus, the data is not directly comparable with our immunocytochemical study, which we believe is more representative of the in vivo situation. In GC, our immunocytochemical findings are in accord with biochemical data that aromatase levels are highest in preovulatory follicles (18). Furthermore, they reveal that mural GC have a higher level of expression than antral GC and indicate that aromatase is absent from specialized GC of the cumulus region. Whereas the physiological significance of the stratified expression of aromatase within GC populations and in TC has yet to be established, regional specialization within human follicles has been previously suggested for other systems (19-21). The distribution of immunoreactive aromatase described here is analogous to those observed in immunocytochemical studies on the cholesterol side chain cleavage cytochrome P-450 enzyme (P-450SCCl) in rat ovary (22), and 3/3-hydroxysteroid dehydrogenase in human ovaries (23). We do not believe that the similarity of these findings are due to antibody cross-specificity since the purified antigens used to generate the antibodies for each study were derived from mitochon-
JCE & M • 1991 Vol 73 • No 4
drial (P-450scc) and microsomal placental fractions (P450arom = Mr 55,000; 3/?-hydroxysteroid dehydrogenase = Mr 45,000). Obviously, the significance of our results is dependent on the specificity of aromatase monoclonal antibody. We are confident in the monospecificity of the aromatase monoclonal antibody since it has been shown to react with only a single 55 kilodalton band (authentic aromatase) in placental microsomes and was used in immunoaffinity columns to purify functional aromatase enzyme to homogeneity (14). Also, it was subsequently used to isolate cDNA sequences that hybridize with mRNA encoding human placental aromatase. This ultimately allowed for sequencing and structural analysis of the aromatase gene (the aromatase gene has
Vol. 73, No. 4 Printed in U.S.A.
Expression of Aromatase Cytochrome P-450 in Premenopausal and Postmenopausal Human Ovaries: an Immunocytochemical Study* SANDRA E. INKSTER AND ANGELA M. H. BRODIE Dept. Pharmacology and Experimental Therapeutics University of Maryland, School of Medicine, Baltimore, Maryland 21201
stages of antrum formation (>700 ^m), and staining intensified as follicle diameter and antral cavity increased, being maximal in preovulatory follicles. GC aromatase was always found in the presence of TI immunostain. These two cell populations were separated by an unstained layer of TI cells giving the follicle walls a banded appearance. Immunostain was most intense in mural GC, was weaker in antral GC cells and was absent from the cumulus GC. Immunoreactive aromatase was also detected in functional corpora lutea (CL) but was absent from involuting CL's and corpora albicans. Our findings indicate that the immunostained cells of the CL are comprised of the former GC and possibly a subpopulation of former TI cells. In perimenopausal ovaries there was no evidence of any follicular or stromal aromatase immunostain. In postmenopausal ovaries no follicles were observed, but individual cells and clusters of cells in the stromal compartment of 3/7 specimens were found to have an aromatase immunostain reaction. In all cases, the aromatase immunostain reaction was cytoplasmic. The results provide the first direct evidence of the existence of TC aromatase, and of stromal cell aromatase in postmenopausal women. {J Clin Endocrinol Metab 73: 717-726,1991)
ABSTRACT. The cellular distribution of the aromatase cytochrome P-450 enzyme in human ovaries has been investigated immunocytochemically, using an aromatase-specific monoclonal antibody. Ovaries of females ranging in age from prepubertal infant girl through to postmenopausal adulthood were obtained from immediate autopsy or after surgery. The results have revealed temporal and spatial changes in expression of aromatase at different stages of development. No immunoreactive aromatase was detected in the ovary of the 2.5 month infant. In premenopausal ovaries, aromatase was absent from the stromal compartment, but in follicles, a consistent pattern in expression of aromatase was observed, related to their size and developmental stage. Aromatase was not expressed in primordial, primary, or small secondary follicles less than 250 /urn diameter. In slightly larger follicles (250-700 nm diameter) aromatase was first detected in a few thecal cells (TC). In more developed secondary through to large preovulatory follicles (>1 cm) TC aromatase immunostain increased in intensity and number of positive cells, and the reaction was localized to a band of theca interna (TI) cells at the Tl/theca externa interface. In granulosa cells (GC), aromatase was first detected in follicles in the initial
T
HE human menstrual cycle is a reflection of dynamic changes occurring in the structure and function of ovarian follicles. Little is known about the control mechanisms underlying follicular recruitment. However, once initiated during fetal life it becomes a continuous daily process that occurs at all stages of the cycle and is apparently independent of any pituitary gonadotropin influence(s). Follicular recruitment does not cease until the time of reproductive senescence, which is associated with exhaustion of the follicular pool, characterized by the menopause in women. As follicles develop antral cavities there is a large increase in the number and
activity of granulosa cells (GC). The thecal envelope becomes specialized into an outer fibrous layer, the theca externa (TE), and an inner, highly vascularized, glandular layer, the theca interna (TI) (1). Concomitant with antrum formation is a rise in serum estradiol (E2) which peaks before ovulation. In humans there is a secondary rise in serum E2 during the mid luteal phase. This is known to be produced by the corpus luteum (CL) but the precise cellular source has not been clearly established. Biochemical studies in numerous species have identified the GC as the major, if not exclusive, site of follicular estrogen synthesis, with GC of large preovulatory follicles having the highest levels of aromatase (estrogen synthetase) activity (2). Since most studies have used cultured GC obtained from either individual or pooled follicles, any intrafollicular variation in aromatase expression that may exist in vivo, as has been described for LH and FSH receptors (3), has not been examined. The currently accepted model of ovarian estrogen syn-
Received January 22,1991. Address correspondence and requests for reprints to: Angela Brodie, Ph.D., Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201. * Supported by NIH Grants CA-27440, HD-13909, and a University of Maryland Designated Research Initiative Award. Presented in part at the 71st Annual Meeting of the Endocrine Society, Seattle, WA, 1989.
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INKSTER AND BRODIE
718
thesis is known as the two-cell two-gonadotropin hypothesis, the concept of which is that androgens, synthesized by thecal cells (TC) under the tonic influence of LH, are transported to the GC where their conversion to estrogens is mediated by FSH-regulated aromatase (4). The existence of human thecal aromatase is a controversial issue. Although the GC are recognized to be the major source of estrogen, there are a number of reports that unlike rodents, TC of humans and some other higher mammals do possess aromatase activity (5-8). However, the possibility of GC contamination of TC cultures cannot be unequivocally ruled out. Moreover, a recent study failed to detect any aromatase activity or messenger RNA (mRNA) in extracts of cultured human TC (9) and an immunocytochemical study in fixed human ovaries also did not find TC aromatase (10). In an attempt to resolve this discrepancy concerning thecal aromatase, we have applied our recently developed immunocytochemical method for detecting human aromatase in unfixed frozen sections (11), to premenopausal ovaries. The specific aims of our detailed study were to investigate: 1) the stage in follicular development at which aromatase is first expressed in GC, 2) whether GC are homogeneous or heterogeneous with respect to aromatase expression, 3) whether expression of aromatase is confined to the GC population, and 4) which cell(s) of the CL express aromatase. We have also examined aromatase expression in a single infant ovary (2.5 months), and have investigated what effects structural changes in the ovary associated with menopause (perimenopausal women, 45-54 yr) and the loss of reproductive function (postmenopausal women, >55 yr), have on ovarian aromatase expression.
Materials and Methods Tissues Thirty six fresh ovaries removed from women ranging in age from 17-68 yr, were obtained through the Human Tissue Resource service of the Pathology Department, University of Maryland School of Medicine, within 12 h of surgery or autopsy. Ovaries were classified as being 1) premenopausal, 17-45 yr, 2) perimenopausal, 45-54 yr, or 3) postmenopausal, greater than 55 yr. A single specimen was obtained from a 2.5 month infant. Since most premenopausal specimens were obtained from victims of accidental death, information on menstrual cycle status or relevant medication history (e.g. oral contraceptive usage) was not readily available. However, only specimens which appeared normal from gross pathological inspection were used. Most ovaries of peri- and postmenopausal women were obtained after surgery for nonovarian gynecological complaints (mainly for uterine fibroids). Ovaries were immediately rinsed in cold (4 C) 0.1 mol/L PBS, then dissected into smaller blocks with care being taken to maintain the integrity of any visible follicles. The specimens were coated with Tissue-Tek O.C.T. cry-
JCE & M • 1991 Vol 73 • No 4
oprotectant embedding medium (Miles Labs, Naperville, IL), immersed in liquid nitrogen, and then stored at —70 C. Fresh placental specimens obtained from routine full term deliveries were processed similarly and used as positive controls for aromatase. All tissues were collected under protocols approved by the Human Volunteers Research Committee of this institution. Immunocytochemistry. Frozen sections (6 /*m) of unfixed ovaries and placenta were thaw-mounted onto chrome-alum gel coated slides, then indirectly immunostained for aromatase. In initial studies our published protocol (11) based on the peroxidase-antiperoxidase (PAP) method (12) was used. However, in later experiments we applied a streptavidin modification of the avidin-biotinylated complex methodology (13). Both methods gave similar results and are briefly described below. Sections were rinsed in assay buffer (50 mmol/L Tris, 9 g/L NaCl, pH 7.6 at 25 C) and incubated at room temperature for 30 min with 30 g/L normal goat serum (NGS) (Vector Labs, Burlingame CA) to block nonspecific staining. Sections were then incubated at 4 C overnight with 1 mg/L anti-aromatase monoclonal antibody, generously provided by Drs. C. Mendelson and E. Simpson (Dallas TX). Details of the preparation and extensive characterization of these antibodies has been published (14). Negative control sections were incubated with either 10 g/L NGS, or with an equivalent dilution of monoclonal antibody specific to rat brain endothelium (courtesy of Dr. L. Sternberger, Baltimore, MD), both diluted in buffer. Second antibodies were applied for 30 min., 1:40 dilutions of goat antimouse (Cappel Labs., Durham, NC) or biotinylated goat anti-mouse immunoglobulin G (Amersham, Arlington Heights, IL) being used for PAP and streptavidin modification of avidinbiotinylated complex methods, respectively. After rinsing, sections were incubated for 1 h at room temperature with either 1:100 dilution of mouse PAP (gift of Dr. Sternberger) or with 1:50 dilution of streptavidin biotinylated peroxidase complex (Amersham, IL). The sections were rinsed before the location of aromatase-linked peroxidase complexes were visualized by 9-min reaction with 0.5 g/L 3,3-diaminobenzidine hydrochloride (DAB), 0.16 g/L hydrogen peroxide in buffer. The DAB reaction was terminated by rinsing in buffer and permanent slides were made following routine dehydration and mounting. Since our protocol uses unfixed sections, red blood cells (RBC), the main source of endogenous peroxidase activity, were washed away during the staining procedure, thus, we elected not to quench the endogenous peroxidase activity of immunostained sections. Instead we screened for presence of non-RBC endogenous peroxidase activity by incubating a single cryosection from each specimen with the DAB reagent for 9 min. A hematoxylin and eosin stained reference section was prepared for all specimens. Immunostained sections were routinely prepared without using counterstains since we have previously noted that they can lessen the intensity or even mask the DAB reaction product. However, to aid in the identification of ovarian structures, duplicate immunostained sections of some specimens were counterstained with 5 g/L toluidine blue. The sections were examined with a Nikon Microphot microscope
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LOCATION OF HUMAN OVARIAN AROMATASE equipped with Nomarski differential interference contrast (DIC) optic capabilities, and were photographically recorded.
Results The ovaries used in this study were obtained from women ranging in age from 2.5 months to 68 yr. Various stages of folliculogenesis, from the arrested primordial state to large preovulatory antral follicles, as well as CL and corpora albicans were examined. Follicular staging was based on the descriptions of Clement (1). As expected, primordial and preantral follicles were observed in all premenopausal ovaries; most contained a few small to large antral follicles, and only four preovulatory (>1 cm) follicles were encountered. The intact ovary of the 2.5 month infant was smooth surfaced, elongated, and flattened in appearance with approximate dimensions of 1.5 X 0.6 X 0.3 cm. Histological examination revealed it to be composed mainly of primordial follicles (40-60 fim diameter) situated in the cortex, and also a few scattered primary follicles (60-80 /im diameter) with expanded oocyte and one to two layers of cuboidal GC in the deeper cortex/medulla regions. There was no evidence of any aromatase immunostain throughout the entire structure (results not shown). Twenty-two premenopausal ovaries were examined. They were generally ovoid in shape with approximate dimensions of 4 X 3 x 2 cm. The external surfaces of the ovaries were visibly smoother in the younger women; the numbers of visible scars from previous ovulations increased with age. In most cases, fluid-filled spherical follicles of various sizes were evident beneath the ovary surface. In three ovaries a large CL with well vascularized outer surface was observed, one of which had a bloody stigma indicating the site of recent ovulation. Dissection usually revealed smaller antral follicles, involuting CL and corpora albicans situated in the deeper regions of the cortex. Histological examination of the ovaries supported the gross observations and also revealed primordial, primary and preantral follicles in the cortex of the ovaries; these immature follicles were more numerous in the younger premenopausal women and their incidence decreased with age. Specific staining of aromatase was detected in representative blocks of 17/22 premenopausal ovaries. From these, a pattern to the expression of the aromatase enzyme, related to the size and developmental stage of the follicle was evident (see Table 1). Aromatase could not be detected in primordial or primary follicles with one to four layers of GC (40-120 /um diameter) or in small secondary follicles (200 11 10 preantral 9 0 early antral 14 2 mid antral late antral 5 0 1 preovulatory 0
0 0 1 7
early CL midCL late CL Atretic follicles/corpora albicans
6 1
0
Theca and granulosa 0 0 0 2 6 4 1
1 2 5
0 0 5
0 0 0
0
15
15
0
0
1 2
could not be clearly distinguished; the GC of these follicles did not stain for aromatase (Fig. 1, a,b). In early antral follicles, thecal aromatase expression increased so that an immunostained circle of TI cells was evident, situated at the TI/TE interface; the TI cells immediately beneath the GC and the GC themselves were devoid of any immunostained products (Fig. 1, c,d,e). With further increase in follicular size, aromatase was still detected in cells at the TI/TE interface but now also started to be expressed in the GC. Granulosa cell staining was first detected in a 750 ixm diameter follicle (Fig. 2, a,b) and although there was some degree of overlap in the sizes of follicles in which the GC were positive or negative for aromatase (see Table 1), staining of GC was never found in the absence of TI cell aromatase. In fact, the two layers of immunostained cells (GC and TI) were separated from each other by an unstained layer of TI cells, giving the follicle wall a banded appearance. Regional variation of aromatase expression in the GC was evident with the mural GC having the strongest immunoreaction while the specialized GC of the oocyte cumulus oophorus complex did not express any aromatase protein (Fig. 2, b,d). In large preovulatory follicles, the level of stain in the GC increased and intensified so that it was deeper than the surrounding TC and was the major source of aromatase in the follicular-phase ovary (Fig. 2, e,f,g). Three CL were assessed as being recently formed and thus functional from the following criteria: 1) vascularization of their outer surfaces (in one case an obvious stigma was present), 2) sizes (~1.6, 1.5 and 1.3 cm diameter), 3) position at the ovary surface, and 4) intense yellow coloration of their folded inner layer, apparent at dissection. The 1.3 cm CL was estimated to be from very early in the luteal phase due to the presence of the stigma plug which normally persists for only a brief time. The structural organization of this CL was consistent with
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FIG. 1. Aromatase immunocytochemistry in 6 ^m unfixed frozen sections of human premenopausal ovaries (methods as described in text). Immunostained sections were photographed using Nomarski DIC optics to reveal structures of unstained cellular areas. Original magnifications in parentheses; bar = 100 /an. A, H + E stained section from the ovary of a 20-yr-old woman showing primordial (PO) and primary (P) follicles and a 320 /im diameter preantral follicle with distinct granulosa (G) and thecal (T) cell compartments (X200). B, Serial section of (A) showing aromatase immunostain localized to the cytoplasm of a few thecal cells (small arrowheads). Granulosa cells, PO and P follicles are unstained (X200). C, H + E stained section from the ovary of a 21-yr-old woman, showing an approximate 600 ^m follicle with developing antral cavity (A) and differentiated theca interna (TI) and theca externa (TE) layers (X160). D, Serial section of (C) immunostained with the control monoclonal antibody against rat brain endothelium (X200). E, Serial section of (C) showing aromatase immunostain localized to the cytoplasm of a ring of cells at the TI/TE interface (x200).
previously described transitional changes from follicle to mature CL (15). Aromatase immunostaining revealed an intense, homogeneous reaction of the inner layer of cells, presumably the luteinized GC. A more heterogeneous immunostain reaction occurred in the outer, thinner layer of cells (presumptive luteinized TI) which resembled the staining pattern of the follicles. Both cell laj^ers had characteristics of steroidogenic activity, that is they contained numerous vacuoles (lipid). The cells were of similar polygonal appearance, their dimensions ranging from 15-25 jum in the inner layer and 8-20 yum in the outer layer. In contrast to the pattern of staining in
antral follicles, no obvious basement membrane or discrete band of unstained TI cells was evident between the two layers which gradually merged into one another. The cells at the extreme periphery of the CL (presumptive TE) did not stain for aromatase (Fig. 3a). The larger CL (1.6, 1.5 cm) were estimated to be from the mid-luteal phase, and their cellular organization was very clearly structured. A thick, folded, inner layer of large, polygonal, steroidogenic cells (20-30 /xm) with spherical nuclei comprised the bulk of the CL. A thinner layer of morphologically distinct, smaller spindle-shaped cells with more elongated nuclei, surrounded the outer edges of the
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LOCATION OF HUMAN OVARIAN AROMATASE
FlG. 2. Aromatase immunocytochemistry in 6 /urn unfixed frozen sections of human premenopausal ovaries (methods as described in text). Immunostained sections were photographed using Nomarski DIG optics to reveal structures of unstained cellular areas except (A). Original magnifications in parentheses; bar = 100 /urn. A, Section from the ovary of a 17-yr-old woman immunostained for aromatase. Both granulosa and thecal cells of the secondary follicle (~850 Mm) contained immunoreactive aromatase. Note: Nomarski DIC optics not available at this magnification (X125). B, Detail of boxed area in (A) showing that TI cells immediately adjacent to the granulosa layer do not express immunoreactive aromatase (x200). C, H + E stained section from the ovary of 25-yr-old woman, showing a 1.65-mm antral follicle with oocyte (0) surrounded by specialized cumulus cells (C) of the granulosa layer (x50). D, Detail of the follicle wall in a serial section of (C) immunostained for aromatase, showing absence of stain in cumulus granulosa cells with patchy staining in some cells of the TI {arrowheads) (X200). E, H + E stained section through a greater than 1.3 cm follicle wall in the ovary of a 17-yr-old woman (x200). F, Serial section of (E) immunostained for aromatase showing cytoplasmic staining in both G and TI, separated by layer of unstained TI cells (X200). G, Serial section of (E) immunostained with the control monoclonal antibody (X200).
^
steroidogenic cells. This layer also ensheathed the vascular septa which extended into the core of the CL (Fig. 3, b,d). When immunostained for aromatase only the large cells were found to express the enzyme, resulting in a clear demarcation between the cell types (Fig. 3, c,e). Higher magnification revealed projections of smaller cells apparently penetrating the margins of the large cell areas, and isolated unstained cells, clearly of the smaller variety, were found among the aromatase-positive cells, presumably related to progressive vascularization of the structure. It should be noted that the area of aromatase immunostaining in both the early and mid stage CL was
721
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TO
more extensive than in preovulatory follicles. Although ages of older corpora lutea/albicans could not be determined very accurately, they were assessed as being in the regressive state from earlier menstrual cycles if, 1) they were not obvious on gross inspection of the ovary, 2) their size was less than 1 cm diameter, 3) they were situated more deeply in the cortex of the ovary, and 4) their inner yellow margins were relatively shrunken. Histologically, these structures were distinctly different from the functional CL. The dense inner layer of vacuolated cells had been replaced by an involuted layer of fibrous connective tissue with a thin, vascularized layer
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PCT
FIG. 3. Aromatase immunocytochemistry in 6 fim unfixed frozen sections of human premenopausal ovaries (methods as described in text). Immunostained sections were photographed using Nomarski DIC optics to reveal structures of unstained cellular areas. Original magnifications in parentheses; bar = 100 nm. A, Section through the wall of a transitional follicle/early corpus luteum (>1.5 cm diameter) in the ovary of a 21yr-old woman, immunostained for aromatase: note the intense homogeneous aromatase immunostain in the luteinized granulosa (LG) layer next to the central cavity (cav), with more heterogeneous reaction in the presumptive luteinized TI (pLTI) -derived component of the follicle/CL wall, and a lack of a distinct border (basement membrane) separating these layers (X200). B, H + E stained section through a mature functional CL in the ovary of a 37-yr-old woman. Two cell populations with distinct borders are apparent; darkly stained cells of the presumed luteinized granulosa (LG) and lightly stained cells of the presumed luteinized theca (LT) (X200). C, Serial section of (B) immunostained for aromatase plus nuclear toluidine blue counterstain. Immunoreactive aromatase is restricted to the cells of the LG population (X200). D, H + E stained detail of the CL shown in (B) showing that the LG is composed of large polygonal cells with rounded/ovoid nuclei and vacuolated cytoplasm, while spindle shaped LT cells have elongated nuclei and penetrate the edges of the LG cell areas (see arrowheads) (X400). E, Matched area of (D) immunostained for aromatase and counterstained with toluidine blue. The non-immunoreactive cells are clearly of LT morphology (see arrowheads) (X400). F, H + E stained section through the wall of a regressing CL/corpora albicans (6 days) of third passage human TC do not contain either aromatase activity or mRNA. It is possible that the absence of aromatase in these TC cultures may be a consequence of the lengthy period of culture in isolation from other regulatory ovarian cell components, and/or previous exposure to the high levels of gonadotropins administered during IVF procedures, which could have initiated irreversible changes in cell metabolism. Thus, the data is not directly comparable with our immunocytochemical study, which we believe is more representative of the in vivo situation. In GC, our immunocytochemical findings are in accord with biochemical data that aromatase levels are highest in preovulatory follicles (18). Furthermore, they reveal that mural GC have a higher level of expression than antral GC and indicate that aromatase is absent from specialized GC of the cumulus region. Whereas the physiological significance of the stratified expression of aromatase within GC populations and in TC has yet to be established, regional specialization within human follicles has been previously suggested for other systems (19-21). The distribution of immunoreactive aromatase described here is analogous to those observed in immunocytochemical studies on the cholesterol side chain cleavage cytochrome P-450 enzyme (P-450SCCl) in rat ovary (22), and 3/3-hydroxysteroid dehydrogenase in human ovaries (23). We do not believe that the similarity of these findings are due to antibody cross-specificity since the purified antigens used to generate the antibodies for each study were derived from mitochon-
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drial (P-450scc) and microsomal placental fractions (P450arom = Mr 55,000; 3/?-hydroxysteroid dehydrogenase = Mr 45,000). Obviously, the significance of our results is dependent on the specificity of aromatase monoclonal antibody. We are confident in the monospecificity of the aromatase monoclonal antibody since it has been shown to react with only a single 55 kilodalton band (authentic aromatase) in placental microsomes and was used in immunoaffinity columns to purify functional aromatase enzyme to homogeneity (14). Also, it was subsequently used to isolate cDNA sequences that hybridize with mRNA encoding human placental aromatase. This ultimately allowed for sequencing and structural analysis of the aromatase gene (the aromatase gene has
