Immunohistochemical Localization of the Interleukin-1 System in the ...

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vestigated immunohistochemically in the mouse ovary during follicular growth, ovulation, and ... of the mouse ovarian IL-i system in ovulation and luteinization.
BIOLOGY OF REPRODUCTION 50, 449-457 (1994)

Immunohistochemical Localization of the Interleukin-1 System in the Mouse Ovary during Follicular Growth, Ovulation, and Luteinization1 CARLOS SIMON, 2 ANA FRANCES, GARY PIQUETTE, and MARY LAKE POLAN Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, California94305 ABSTRACT The distribution of immunoreactive interleukin-l receptor type I (IL-IR tI), IL-la, and IL-IP, and of macrophages, was investigated immunohistochemically in the mouse ovary during follicular growth, ovulation, and luteinization. For this purpose, an indirect immunofluorescence technique, using specific monoclonal antibodies against mouse IL-1R tI, mouse IL-la, IL-l0, and macrophage antigens (CDllb/CD18) was used with sections of paraffin-embedded ovaries from eCG and eCG/hCG-treated 12wk-old B6C3F-1 female mice. During follicular development, IL-la, IL-1p, and IL-IR tI staining were confined to the thecainterstitial layer of growing follicles with one remarkable exception. Intense IL-1R tI staining was present in the cytoplasm and plasma membrane of the murine oocyte. During ovulation, IL-la and IL-IPi were still confined to the theca layer, but faint IL-1R tI staining was initiated in cumulus cells and in granulosa cells just before follicle rupture. Immediately after follicle rupture, granulosa cells stained positive for IL-IR tl, IL-la, and IL-1l. During luteinization, granulosa-luteal cells of the corpus luteum demonstrated strong IL-1R tI, IL-la, and IL-ll staining. Macrophages were detected in the theca layer and stroma, but never within the follicle before ovulation. Immediately after ovulation, there was a rapid entry of macrophages into the follicle, and macrophages were also present inside the corpus luteum. Our morphological results support a possible autocrine-paracrine role of the mouse ovarian IL-i system in ovulation and luteinization.

INTRODUCTION Paracrine cytokines produced by ovarian and lymphohematopoietic cells are increasingly implicated in the cyclic events of ovarian physiology. Interleukin-1 (IL-1), a family of polypeptides comprising IL-la, IL-13, and an inhibitor, interleukin-1 receptor antagonist (IL-lra) [1], is a major candidate for this cooperative action. Two IL-1 receptors have been identified and characterized: IL-1R type I (IL-1R tI [2]) and IL-1R type II (IL-1R tII [3]). The type I receptor is found on most cells and appears to be important for transducing the action of IL-1. The type II receptor is found primarily on B lymphocytes, neutrophils, and monocytes; its function is unclear. IL-1R tI recognizes both ligands, IL-la and IL-1{3, triggering similar responses in target cells [4]. IL-lra binds to IL-1R tI and appears to prevent signal transduction by blocking the binding of IL-1 [5]. In humans, preovulatory follicular fluid contains immunoreactive IL-1 [6-8], and the presence of a complete intraovarian IL-1 system has been demonstrated [9]. In the in vitro perfused rat ovary [10] and rabbit ovary [11], an ovulatory effect of IL-1 has been documented. IL-1 has also been implicated in the luteinization process, although this is controversial. Some researchers have reported a positive regulation of IL-1 on progesterone production and release from human granulosa-luteal cells [12, 13] and rat granuAccepted September 28, 1993. Received August 11, 1993. 'C.S. is a Postdoctoral Fellow supported by Subprograma General Extranjero grant from the Spanish Government, MEC, Madrid, Spain; on leave from the Department of Pediatrics, Obstetrics and Gynecology, Valencia University School of Medicine, Valencia, Spain. 2Correspondence: Carlos Sim6n, Instituto Valenciano de Infertilidad, Guardia Civil 23, 46020 Valencia, Spain.

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losa cells [14]. However, others have shown that IL-la inhibits LH-induced luteinization and progesterone production of porcine granulosa cells [15, 16] and rat granulosa cells [17]. Resident ovarian macrophages have been immunohistochemically localized in the mouse ovary and constitute a substantial cellular component of the interstitial compartment [18]. IL-1 is an established immune mediator produced by resident macrophages, and these cells of the white blood cell series have been suggested as the main source of this cytokine in the ovary [19]. However, because macrophage-depleted human granulosa-luteal cells contain messenger RNAs for IL-la and IL-l1 [9], the intraovarian source of these cytokines remains unclear. Interestingly, the large body of literature implicating the IL-1 system in ovarian function contrasts with the absence of morphological data concerning the localization of the IL-1 system in the cellular components of the ovary. The present study was undertaken to examine the cellular localization of IL-1R tI, IL-la, IL-13, and macrophages in the mouse ovary during follicular growth, ovulation, and luteinization. MATERIAL AND METHODS Animals and Animal Treatment Twelve-week-old B6C3F-1 female mice were obtained from Charles River Breeding Laboratories, Inc (Wilmington, MA) and maintained at 22-240C on a 12L:12D cycle. Animals were monitored by daily vaginal smears. At proestrus, mice received i.p. injections of 5 IU eCG. Approximately 48 h after eCG, mice received i.p. injections of 5 IU hCG. Six animals from each stage were killed by cervical dislocation at 24, 12, and 2 h before hCG and at 2, 4, 6, 12, 14, 16, and

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TABLE 1. Summary of the immunohistochemical experiments identifying IL-1R tl, IL-1, IL-1a, and macrophages in the mouse ovary during follicular development. Ovarian follicle

IL-1R tl IL-1,8 IL-la CD-llb/CD18

Oocyte

Granulosa cells

Theca layer

Stroma

+++/+++a 0/0 0/0 0/0

0/0 0/0 0/0 0/0

+/++ +/+ +/+ 0/+

+/++ ++/++ +/++ +/++

aDesignations of + (weakly positive) to +++ (intensely positive) indicate the relative intensities of the signals averaged for the specimens during follicular development; variability between readers is indicated with a slash mark.

24 h after hCG administration. Ovaries and oviducts were immediately removed for fixation in 10% formalin in PBS. Tissue Processing Fixed tissue was embedded in paraffin, sectioned, and mounted on coated glass slides. Ten serial sections (6 ,m) from each sample were then prepared for immunohistochemistry, and the first and last section were stained with hematoxylin-eosin (H&E) and viewed with an Olympus 35mm camera and an Olympus BH2 microscope. Immunohistochemical Staining The immunostaining procedure was performed on mouse ovary paraffin-embedded sections by an indirect immunofluorescence technique. Sections were deparaffinized in xylene and rapidly rehydrated through graded alcohols. Excess liquid was removed, and sections were washed in PBS (pH 7.4) with 0.05% Tween-20 (PBS-T; Sigma Chemical Co., St. Louis, MO). To reduce the nonspecific binding, normal goat serum (1% in PBS) was applied to slides for 30 min at 37 0C. Sections were washed with PBS-T twice, and then incubated with the primary antibodies: monoclonal rat antimouse IL-1R tI antibody, monoclonal hamster anti-mouse IL-l1 antibody, monoclonal hamster anti-mouse IL-la antibody (all from Genzyme Corp., Cambridge, MA) at 50 jIg/ ml, 66.7 jig/ml, and 20 g/ml respectively, or rat monoclonal antibody to the mouse and human Mac-i antigen CDllb/CD18 (Anti-Mac-i, Boehringer-Mannheim, Indianapolis, IN) at 50 ILg/ml for 90 min at 37 0C on consecutive sections. Monoclonal hamster anti-mouse IL-lot does not cross-react with human IL-lao or IL-13, or with mouse IL11i, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, colony-stimulating factor, tumor necrosis factor-a (TNF-oa), or interferon-'y (IFN-y). Hamster anti-mouse IL-1P does not recognize human IL-laot or IL-1p3, and no detectable cross-reactivity was observed with mouse IL-loa, TNF-a, or IFN-y. Control incubations included deletion of the primary antibody. After being rinsed with PBS-T, sections were incubated with specific secondary antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-rat IgG mouse adsorbed for 60 min (16 g/ml

at 37°C; Sigma), and FITC-conjugated anti-hamster IgG mouse adsorbed for 60 min (100 jig/ml at 37°C; BoehringerMannheim). Slides were rinsed twice with PBS-T, mounted with Vectashield mounting media (Vector Labs., Burlingame, CA), and photographed by use of an Olympus 35 mm camera and an Olympus BH2 microscope fitted with a BH-RFL-W reflected light fluorescence attachment. Relative intensity of the immunostaining was evaluated by two of the authors in a double-blinded manner as absent (0), weakly positive (+), moderate (++), or intense (+++) for at least three different specimens from each time point. RESULTS IL-1 System in the Mouse Ovary during FollicularGrowth IL-1R tI was localized in the oocyte and theca cells during follicular development before hCG administration (Fig. 1, Table 1). In the oocyte, cytoplasm and plasma membrane stained strongly, but the germinal vesicle in the immature oocytes remained unstained (Fig. 1, A and B). IL-1R tI was not detected in either granulosa cells or cumulus cells in healthy follicles (Fig. 1, A and B). Only in those follicles that appeared morphologically abnormal could an increase of IL-1R tI be observed in granulosa and theca cells, accompanied by a decreased staining in the oocyte (Fig. 1C). During follicular development, localization of IL-la and IL-1P was not obvious within the follicle; only some cells of the theca layer and stroma stained positively for both cytokines (Fig. 1, D, E, and F). Macrophages were not present within the ovarian follicle before ovulation, isolated macrophages could be seen only in the theca layer, and these cells were a major cellular component of the interstitial tissue between developing follicles (Fig. 1G). IL-1 System in the Mouse Ovary during Ovulation Immediately before follicle rupture (10-12 h after hCG; Fig..2, Table 2), faint IL-1R tI staining could be observed in

FIG. 1. Immunohistochemical localization of IL-1R tl, IL-1, IL-la, and macrophages in the mouse ovary during follicular growth. A) Preantral follicle with two oocytes. IL-1R tl is strongly localized in the cytoplasm and plasma membrane of the oocyte, but the germinal vesicle remains unstained. IL-1R tl staining can also be seen in the theca layer and corpora lutea surrounding this follicle. B) Antral follicle. IL-1R tl staining is present in the same locations as in preantral follicles: cytoplasm and plasma membrane of the oocyte and theca layer. C) Morphologically. abnormal follicle (atretic follicle). Notice that the pattern for IL-1 Rtl is different. In the oocyte, there is obviously less staining in the cytoplasm, and staining is absent in the plasma membrane. In the granulosa cells, IL-R tl is present in the plasma membrane; this is never true of healthy follicles during follicular growth. In the theca layer, staining for IL-1R tl is increased. D) IL-I1 localization in a preantral follicle. E) IL-[ localization in preantral and antral follicles. F) IL-la staining in an antral follicle. In all cases (D-F), IL-1, and IL-la were not present within the follicle; only some cells of the theca layer and stroma stained positive for both cytokines. G) Immunohistochemical detection of mouse macrophages (CDllb/CD18) during follicular development. Isolated macrophages can be seen in the theca layer, and these cells are a major cellular component of the interstitial tissue between developing follicles. H) Negative control by deletion of the primary antibody. Bar = 10 im in all plates.

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INTERLEUKIN-1 SYSTEM IN THE MOUSE OVARY TABLE 2. Summary of the immunohistochemical experiments identifying IL-1R tl, IL-13, IL-la, and macrophages in the mouse ovary during ovulation immediately before follicle rupture. Ovarian follicle Oocyte IL-1R t IL-1p

Cumulus Granulosa Theca layer

Stroma

++/+++a 0/0

+/++ 0/0

+/0 0/0

++/++ +/++

++/+++ ++/+++

IL-la

0/0

0/0

0/0

+/++

+/++

CD-llb/CD18

0/0

0/0

0/0

+/++

+/++

'Designations of + (weakly positive) to +++ (intensely positive) indicate the relative intensities of the signals averaged for the specimens obtained 10-12 h after hCG administration; variability between readers is indicated with a slash mark.

the plasma membrane of cumulus cells surrounding the preovulatory oocyte and in granulosa cells (Fig. 2, A and B). Immunoreactive IL-13 (Fig. 2C) and IL-la (Fig. 2D) were not localized in granulosa cells or cumulus cells. Only some cells of the theca layer stained positively for both cytokines, and staining did not increase prior to ovulation. Macrophages were not present within the ovarian follicle before ovulation, but were a major cellular component of the interstitial tissue (Fig. 2E). Immediately after follicle rupture (14-16 h after hCG administration; Fig. 3, Table 3), IL-1R tI staining in granulosa cells was increased (Fig. 3A), and these cells were suddenly able to produce IL-la and IL-l1 (Fig. 3B) An entry of CDllb/CD18 antigen-positive blood cells representing macrophages/monocytes was also observed within the ovulated follicle (Fig. 3C). These cells of monocyte/macrophage lineage also exhibited intense staining for IL-1R tI (Fig. 3A), IL-la, and IL-1P (Fig. 3B). In the oviduct 24 h after hCG administration (Fig. 4), isolated cumulus cells surrounding the unfertilized oocyte exhibited increased staining for IL-1R tI in the plasma membrane (Fig. 4A); the unfertilized oocyte and its polar body within the oviduct also retained intense IL-1R tI staining in the cytoplasm (Fig. 4A). IL-la and IL-13 staining was initiated in both the oocyte and a few isolated cumulus cells (Fig. 4B). Macrophages were not detected in the oviduct at this time.

FIG. 3. Immunohistochemical localization of IL-1R tl, IL-13, and macrophages in the mouse ovary immediately after follicle rupture, 14 h after hCG administration. A) IL-R tl staining. Notice that IL-1 Rtl staining in granulosa cells is increased compared with the IL-1R tl staining present in cumulus cells. Entry of cells exhibiting intense staining for IL-R tl is also observed. B) IL-p staining (adjacent section to A). Granulosa cells abruptly acquire the capability to produce IL-18 (compare with IL-p staining present in the theca layer). The cells that recently entered the follicle exhibit intense staining for IL-1. C) CD11b/CD18 antigen staining in the adjacent section of B. Notice that a large group of new cells that invaded the follicle immediately after follicular rupture are immunohistochemically characterized as mouse macrophages. Bar = 10 m in all plates.

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TABLE 3. Summary of the immunohistochemical experiments identifying IL-1R tl, IL-lp, IL-la, and macrophages in the mouse ovary during ovulation immediately after follicle rupture. Ovarian follicle Oocvte

Cumulus

Granulosa

++/++

++/+++ +++/+++ +++/+++

Theca laver

Stroma

-

IL-IR t l IL-1p IL-la CD-I lb/CD18

+++/+++a

o/+ o/+

o/+ o/+

+++/+++

++/+++ +++/+++ +++/+++ +/++

++/+++

++/+++

++/+++

++/+++

"Designations of + (weakly positive) to +++ (intensely positive) indicate the relative intensities of the signals averaged for the specimens obtained 12-14 h after hCG administration; variability between readers is indicated with a slash mark

IL-1 System in the Mouse Ovary during Luteinization After hCG administration, as luteinization progressed (Fig. 5, Table 4), increased staining for IL-lR tI was seen in the cytoplasm of granulosa-luteal cells (Fig. 5A), and the mature corpus luteum exhibited even stronger IL-1R tI staining (Fig. 5B). Cytoplasmic IL-la and IL-IP staining was present in granulosa-luteal cells, with increased staining in the mature corpus luteum (Fig. 5, C and D). Some macrophages could also be detected within the corpus luteum (Fig. 5F). Although these cells also produced IL-la and IL-1P, macrophages were a relatively minor source of both cytokines and IL-1R tI in the corpus luteum compared with luteal cells (Fig. 5, E and F). DISCUSSION

The immunohistochemical observations in the present study demonstrate for the first time a simultaneous localization of IL-lR tI, IL-la, and IL-1P in different cell types of the mouse ovary, with differential expression during follicular growth, ovulation, and luteinization. During follicular development, IL-la, IL-1P and IL-1R tI staining were confined to the theca-interstitial layer of

growing follicles (Figs. 1 and 2). These observations are in agreement with the described presence of IL-IP mRNA in the theca-interstitial layer in the rat ovary [20] and the demonstrated effect of IL-la on theca cells in preovulatory hamster follicles [21]. In addition, similar morphological results regarding the absence of IL-la and IL-1P staining in granulosa cells from human preovulatory follicles have been reported by our laboratory [22].

FIG. 5. lmmunohistochemical localization of IL-1R tl. IL-la, IL-1P, and rnacrophages in the mouse ovary during luteinization. A) IL-R t l staining in a developing corpus luteum (20 h after hCG administration). Granulosaluteal cells exhibit progressively increased IL-1R tl staining; but during early luteinization, IL-1R tl staining is stronger in macrophages (arrow). B) IL-R tl staining in a mature corpus luteum (2 days after hCG administration). There is an increase IL-R t l staining in the cytoplasm of granulosa-luteal cells (compared with A, common reference oocyte staining). C) IL-la staining in a developing corpus luteum (adjacent section to A). Granulosa-Meal cells show progressively increasing IL-la staining. D) IL-p staining in a mature corpus luteum (2 days after hCG administration). Cytoplasmic IL-p staining is present in granulosa-luteal cells, showing a pattern similar to that of IL-IR tl. E) IL-R t l staining in granulosa-luteal cells 2 days after hCG administration, detail of B (arrow as a reference). F) Detail of macrophages staining in a mature corpus luteum 2 days after hCG. Bar = 10 Fm in all plates.

FIG. 4. lmmunohistochemical localization of IL-1R tl and IL-1p in the unfertilized oocyte within the oviduct 24 h after hCG administration. A) IL-lR tl staining in the mouse oviduct. The unfertilized oocyte retains IL-1R t l staining in the cytoplasm; notice that the polar body also stains positively for IL-IR tl. An obvious plasma membrane staining for IL-1R tl in cumulus cells is also observed. B) IL-lp staining in the mouse oviduct section adjacent to A. IL1p staining has started initiated in the oocyte and a few isolated cumulus cells. Bar = 10 pm in all plates.

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TABLE 4. Summary of the immunohistochemical experiments identifying IL-1R tl, IL-1, IL-la, and macrophages in the mouse ovary during luteinization. Corpus luteum Developing IL-1R tl+++a IL-1 IL-la CD-llb/CD18

Mature

+++/+++ +++/+++ ++/+++ +++/+++ ++/+++ (grouped) +++/+++ (scattered)+++/+++

Stroma ++/+++ ++/+++ ++/+++ ++/+++

aDesignations of + (weakly positive) to +++ (intensely positive) indicate the relative intensities of the signals averaged for the specimens obtained 20-48 h after hCG administration. Distinction of developing and mature corpora lutea are based on histologic criteria. Variability between readers is indicated with a slash mark.

Of particular interest is the fact that IL-1R tI at the protein level was strongly expressed in the murine oocyte independently of developmental follicular stage. Furthermore, this expression continued in oocytes found in the oviduct. Immunoreactive IL-iR tI detected in the oocyte could be due to sequestration instead of production; however, this is very unlikely since our finding is corroborated by the previously described presence of IL-1R tI mRNA within the murine oocyte by in situ hybridization [23]. The identification at the protein level of receptors in mammalian oocytes is certainly rare. As yet, only epidermal growth factor receptor protein has been localized in human oocytes [24]. Insulin-like growth factor-I receptor mRNA has been identified in the human [25] and murine oocyte [26], and the expression of the estradiol receptor gene has been identified in the mouse oocyte by use of the reverse transcriptase-polymerase chain reaction technique [27]. But none of these receptors has been identified as an immunoreactive protein in mammalian oocytes. Therefore, the remarkably high levels of IL-1R tI protein in the oocyte may have a previously unsuspected important physiological role(s) in oocyte maturation. This hypothesis is reinforced by the fact that only morphologically abnormal oocytes had decreased IL-1R tI staining (Fig. 1C). This observation may be most intriguing, considering that human preovulatory follicular fluid contains immunoreactive IL-1 [6-8], but according to our data, murine granulosa cells do not contain or produce IL-1 prior to ovulation. Other sources of IL-1 known at this time are thecal macrophages and peripheral monocytes. Our group has demonstrated that estradiol and progesterone modulate human monocyte IL-1 activity [28] and IL-l1 mRNA levels [29]. This loop provides an IL-1 feedback control by steroids according to the differential functional status of granulosa cells that may be relevant in oocyte maturation. The acquisition of IL-1R tI staining in granulosa cells in preovulatory follicles before the follicle rupture is interesting (Fig. 2B), and may be the mechanism whereby IL-1 participates in ovulation. However, immunoreactive IL-la (Fig. 2C) and IL-1 (Fig. 2D) were not localized in granulosa cells or cumulus cells at this time.

After ovulation, granulosa-luteal cells of the developing corpus luteum exhibited progressively increased IL-1R tI staining, and abruptly acquired IL-loa and IL-13 staining (Figs. 3 and 5). IL-1 bioactivity in human serum reaches maximal levels after ovulation [30], which is consistent with our observation that IL-1 production and action resided in the corpus luteum. However, whether IL-1 modulates progesterone production [12-17] or progesterone modulates IL-1 production by granulosa-luteal cells seems controversial. It is likely that both situations occur and that the IL-1 system is implicated in the corpus luteum formation and function. The present work provides morphological support for this hypothesis. Furthermore, we demonstrated that hematopoietic cells are not the predominant source of IL-1 in the corpus luteum. After ovulation, granulosa-luteal cells progressively acquire the capacity to produce and bind IL-1 in response to as yet unknown factors. Therefore, this cytokine may act as a paracrine-autocrine mediator in the formation and function of the corpus luteum. Interestingly, before ovulation the presence of monocyte-macrophages was detected in the theca layer and stroma, but never within the follicle itself (Figs. 1 and 2). Immediately after ovulation, macrophages that possessed IL-1R tI and both ligands IL-la and IL-113 migrated into the ovulated follicle (Fig. 3). Later on, macrophages were also detected within the corpus luteum (Fig. 5F). These results are in agreement with previous studies demonstrating the presence of macrophages in the mouse ovary [18], rat ovary [31], and human ovary [32]. Our morphological observations of an intimate relationship of macrophages to luteal cells support the demonstrated capability of macrophages to stimulate progesterone production in the murine corpus luteum [33,34] and human granulosa-luteal cells [35]. Together, this evidence invokes a cooperative role of monocyte-macrophages in luteal tissue function. In summary, our morphological study shows that the mouse ovary is a site for IL-la and IL-13 production and action through its receptor, IL-1R tI. During follicular development, both ligands and receptor were confined to the theca-interstitial layer of growing follicles. In addition, a dramatic increase of IL-1R tI, IL-la, and IL-i13 staining was observed in granulosa cells immediately before and after ovulation, and in the corpus luteum, supporting a possible autocrine-paracrine role for the mouse ovarian IL-1 system in both ovulation and luteinization. REFERENCES 1. Dinarello CA Biology of interleukin 1. FASEB J 1988; 2:108-115. 2. Sims JE, March CJ, Cosman D, Widmer MB, MacDonald HR, McMahan CJ, Grubin CE, WignallJM, Jackson JL, Call SM, Friend D, Alpert AR, Gillis S, Urdal DL, Dower SK. cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily. Science 1988; 241:585-589. 3. Horuk R,McCubreyJA. The IL-1 receptor in Raji human B-lymphorna cells. Molecular characterization and evidence for receptor-mediated activation of gene expression. Biochem J 1989; 260:657-663.

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19. Adashi EY. The potential relevance of cytokines to ovarian physiology: the emerging role of resident ovarian cells of the white blood cell series. Endo Reviews 1990; 11:454-464. 20. Hurwitz A, Ricciarelli E, Botero L, Rohan RM, Hernandez ER, Adashi EY Endocrine- and autocrine-mediared regulation of rat ovarian (theca-interstitial) interleukin-l gene expression: gonadotropin-dependent preovulatory acquisition. Endocrinology 1991; 129:3427-3429. 21. Nakamura Y, Kato H, Terranova PF. Interleukin-la increases thecal progesterone production of preovulatory follicles in cyclic hamsters. Biol Reprod 1990; 43:169173. 22. Polan ML, Loukides JA, Honig J. Interleukin-1 in human ovarian cells and in peripheral blood monocytes increases during the luteal phase: Evidence for a midcycle IL-1 surge in the human. Am J Obstet Gynecol 1994; (in press). 23. Deyerle KL, Sims JE, Dower SK, Bothwell MA. Pattern of IL-1 Receptor gene expression suggests role in noninflammatory processes. J Immunol 1992; 149:16571665. 24. Maruo T, Ladines-LLave CA, Samoto T, Matsuo H, Manalo AS, Ito H, Mochizuki M. Expression of Epidermal Growth Factor and its receptor in the human ovary during follicular growth and regression. Endocrinology 1993; 132:924-931. 25. Zhou J, Bondy C. Anatomy of the human ovarian insulin-like growth factor system. Biol Reprod 1993; 48:467-482. 26. Zhou J, Bondy C. Cellular pattern of IGF-1 and IGF-I receptor gene expression in the developing and mature follicle. Endocrinology 1991; 129:3281-3288. 27. Wu TC, Wang L, Wan YJ. Expression of estrogen receptor gene in mouse oocyte and during embryogenesis. Mol Reprod Dev 1992; 33:407-412. 28. Polan MJ, Daniele A, Kuo A. Gonadal steroids modulate human monocyte interleukin-1 (IL-1) activity. Fertil Steril 1988; 49:964-968. 29. Polan ML, Kuo A, Loukides J, Bottom L. Cultured human luteal peripheral monocytes secrete increased levels of interleukin-1. J Endocrinol Metab 1984; 70:480484. 30. Cannon JG, Dinnarello CA Increased interleukin-1 activity in women after ovulation. Science. 1985; 227:1247-1249. 31. Bulmer O. Histochemistry of ovarian macrophages in the rat. J Anat Lond 1964; 98:313-319. 32. Guillim SW, Christensen KA, McLennan CE. Fine structure of the human menstrual corpus luteum at its age of maximum secretory activity. Am J Anat 1969; 126:409-415. 33. Kirsch TM, Friedman AC, Vogel RL, Flickinger GL. Macrophages in the corpora lutea of mice: characterization and effects of steroid secretion. Biol Reprod 1981; 25:629-638. 34. Kirsch TM, Vogel RL, Flickinger GL. Macrophages: a source of luteotropic cybernins. Endocrinology 1983; 113:1910-1912. 35. Halme J, Hammond MG, Syrop CH, Talbert LM. Peritoneal macrophages modulate human granulosa-luteal cell progesterone production. J Clin Endocrinol Metab 1985; 61:912-916.