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Articles |
a Reproductive Medicine Unit,
b Queen Elizabeth Hospital and Department of Obstetrics and Gynaecology, and
c Department of Animal Sciences, University of Adelaide, Adelaide, South Australia, Australia
d Department of Cell Biology and Immunology, Free University, Amsterdam, Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The distribution of macrophages within the ovary varies across the reproductive cycle, apparently influenced by gonadotropic and ovarian steroid hormones. In several species, macrophages have been shown to be most abundant within the theca of the follicle during follicular development [1–3], in atretic follicles [4,5], and in the corpus luteum [6–8]. In mice, immunohistochemical studies using macrophage-reactive antibodies such as Mac-1, F4/80, and CD18 have shown the presence of macrophages in the theca, stroma, and corpus luteum as well as in association with atretic follicles [9]. These variations in location and numbers during the different stages of the cycle suggest that macrophages may participate in the tissue restructuring and inflammatory-like processes that occur in the normal adult ovary. Many studies have provided substantial evidence that factors produced by macrophages, such as interleukin-1ß (IL-1ß) and tumor necrosis factor-
(TNF), can have stimulatory effects on ovulation [10,11] and act to regulate steroid synthesis in in vitro-cultured follicles [12,13] and in isolated granulosa and theca cells [14–16].
Together, these studies implicate macrophages in the modulation of follicle development, ovulation, and the formation and regression of the corpus luteum. The reproductive characteristics of the csfmop/csfmop mouse, which has a null mutation in the gene encoding colony-stimulating factor-1 (CSF-1), a cytokine that regulates both the development and differentiation of the macrophage lineage, provide convincing evidence to support this. These animals have few macrophages within the ovary, a significantly impaired ovulation rate, and an extended estrous cycle [4], suggesting that the ovarian macrophage population is important in the normal functions of the adult cycling ovary. However, since macrophages may not be the exclusive target of CSF-1 in the ovary, it cannot be concluded that local macrophage deficiency is the primary cause of ovarian dysfunction in csfmop/csfmop mice.
Liposomes are synthetic phospholipid spheres that have been used extensively to target a variety of aqueous compounds to the macrophages present in different organs of the body. After in vivo administration, liposomes are phagocytosed by macrophages, whereupon the liposomal membranes are degraded by lysosomal phospholipase membranes and the enclosed compound is released into the cytoplasm of the cell. The accumulation of clodronate liposomes in the cytoplasm of target macrophages leads to cell death through the apoptosis pathway [17]. Macrophages in the liver and spleen can be depleted within 24 h after a single i.v. injection of clodronate liposomes, and the population is not restored for 2 wk thereafter [18]. Macrophages in the testis and peritoneal cavity have also been depleted after direct injection of clodronate liposomes into the testis [19,20] and peritoneum [21], respectively. In this study, we set out to deplete the macrophage population from normal mouse ovaries using clodronate liposomes in an effort to determine whether these cells are required for the processes of follicular development and ovulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animals were handled in accordance with The Australian Code of Practise for the Care and Use of Animals for Scientific Purposes, and experiments were approved by the ethics committees of both The University of Adelaide and The Queen Elizabeth Hospital. Adult 8- to 11-wk-old C57Bl6 black female mice (from The University of Adelaide Central Animal House Facility) were maintained under controlled conditions (14L:10D cycle) with free access to food and water. The estrous cycles of the mice were synchronized with an i.p. injection of 20 µg LHRH agonist (Des-Gly10[D-Ala6]LHRH ethylamide; Sigma Aldrich, St. Louis, MO) at 0900 h on Day -4. At 1200 h on Day -2, animals were primed with an s.c. injection of 5 IU of eCG (Folligon; Intervet, Boxmeer, Holland), and ovulation was stimulated 48 h later (1200 h on Day 0) with an i.p. injection of 5 IU of hCG (Pregnyl; Organon, Oss, Holland; Fig. 1). Ovulation occurred 12–15 h after the hCG injection.
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Intrabursal Injection Technique
Animals were anesthetized using a mixture of fluothane, nitrous oxide, and oxygen gases. Anesthesia was then maintained throughout surgery with a continuous stream of the same gas mixture regulated by a Midget anesthetic machine (Commonwealth Industrial Gases [CIG], Adelaide, Australia). A single midline dorsal incision was made, then two small incisions into the peritoneum directly above the fat pad of the left and right ovaries. Each ovary was externalized through the respective incision, and the intrabursal injection was performed under microscopic magnification by inserting a 30-gauge needle through the ovarian fat pad into the ovarian bursa. In each animal, approximately 10 µl of either clodronate liposomes (containing dichloromethylene diphosphonate, CL2MDP, a gift from Boehringer Mannheim GmbH, Mannheim, Germany) saline liposomes (containing saline solution), or saline solution alone was delivered to both ovaries. All liposomes were prepared and supplied by N. van Rooijen as previously described [22]. Ovaries were returned to the peritoneal cavity, and the wound was sealed with a single Autoclip wound clip (Becton Dickinson, Franklin Lakes, NJ). Animals were initially placed in separate cages under a radiant heat source until normal behavioral activity was resumed and then housed separately under standard conditions until oocyte retrieval and tissue collection.
Treatment Groups and Oocyte Retrieval
Intrabursal injections were carried out as described above on either Day -3 or Day -1 (Fig. 1). In the first experiment, two groups of mice (n = 8 in each) received either 1) clodronate liposomes in one ovary and saline solution in the other (CL/S group) or 2) saline liposomes in one ovary and saline solution in the other (SL/S group). In a second experiment, mice received clodronate liposomes in both ovaries (CL/CL group, n = 7) or saline solution in both ovaries (S/S, n = 8 for Day -1 and n = 6 for Day -3). On the morning of Day 1 following ovulation, blood samples were taken via heart puncture, and animals were killed by cervical dislocation. Ovaries and oviducts were recovered, and the ovulated oocytes in the ampulla region were counted. In an additional experiment, animals treated as described above received either 1) saline solution in both ovaries (S/S, n = 6 for Day -1 and n = 10 for Day -3); 2) saline liposomes in both ovaries (SL/SL, n = 6 for Day -1 and n = 5 for Day -3); 3) clodronate liposomes in one ovary and saline liposomes in the other (CL/SL, n = 8 for Day -1 and n = 7 for Day -3; or 4) clodronate liposomes in both ovaries (CL/CL, n = 6 for Day -3). These animals were allowed to proceed to the subsequent "natural" ovulation following the induced ovulation. The day of estrus in these animals was monitored by daily vaginal smear tests [23]; and on the morning when 100% cornified epithelial cells were detected, indicating metestrus-1 (ME-1), blood samples were taken, animals were killed, and the ovulated oocytes in the ampulla region of the oviduct were counted. Progesterone levels were measured in serum samples using an automated chemiluminescence system (Ciba-Corning, Medfield, MA) as previously described [24].
Collection of Ovarian Tissue
Recovered ovaries were dissected free of fat and connective tissue, and snap-frozen in OCT (Tissue-Tek tissue freezing medium; Miles Inc., Elkhart, IN) at either the preovulatory stage, 1 h before expected ovulation, or on Day 1 postovulation, at the time of oocyte collection (Fig. 1). To establish that liposomes are able to penetrate the ovary, liposomes with the DiI fluorochrome (the lipophilic long-chain carbocyanine Di-I (DiIC18[3]; Molecular Probes, Eugene, OR) incorporated into the lipid bilayer of the liposome were injected into the bursa (n = 2 per time point), and ovaries were recovered from animals killed 2, 4, 6, 12, 24, or 48 h after surgery. Ten-micrometer sections from these ovaries were then examined using an Olympus (Woodbury, NY) Vanox microscope equipped for epifluorescence. Under conditions of green excitation (546 nm), the Di-I in the liposomes fluoresces red.
Ovarian Morphology and Immunohistochemistry
Serial sections (6 µm) were cut from both ovaries of each mouse, resulting in at least 140 consecutive sections from each ovary. Every ninth and tenth section was stained with hematoxylin and eosin (H&E) for morphological examination by light microscopy. Follicle size was measured in the H&E sections using video image analysis (VIA) software (Leading Edge Pty Ltd., Marion, South Australia), and adjacent sections containing preovulatory follicles (> 400 µm) were stained with primary antibodies reactive with the anti-major histocompatability complex (MHC) II antigen (Ia) TIB120 (reactive with activated macrophages and dendritic cells, from American Type Culture Collection, Rockville, MD), and with F4/80 (reactive with a surface glycoprotein specific to macrophages [25]) and FA/11 (reactive with macrosialin [26]), both kindly supplied by S. Gordon, University of Oxford. Sections were fixed in 96% alcohol (4°C for 10 min) and then incubated with primary antibody from culture supernatant diluted in PBS (GIBCO/BRL Life Technologies, Grand Island, NY) containing 10% normal mouse serum and 1% BSA (Boehringer Mannheim, Indianapolis, IN; PBS-NMS) at 4°C for 3 h. Ia was diluted 1:200, F4/80 1:10, and FA/11 1:600. After incubation, sections were washed in PBS and then incubated with biotinylated-rabbit-anti-rat secondary antibody (Dako, Carpenteria, CA) diluted in PBS-NMS (1:300) at 4°C for 2 h. After another PBS wash, sections were incubated with avidin-horseradish-peroxidase (Dako) diluted in PBS-NMS (1:400), and enzyme was then visualized using Sigma Fast DAB (diaminobenzidine) tablets (Sigma Aldrich). Uterus and spleen were used as positive control tissues, and negative controls included sections incubated without primary antibody or with irrelevant monoclonal antibodies. The area of positive stain in each section was evaluated by VIA and expressed as percentage of positivity (area of brown positive stain/area of total stain x 100). Three thecal regions in each preovulatory follicle and six stromal regions in each ovary were counted, and the mean percentage positivity value for each follicle and ovarian stroma was calculated.
Statistics
A paired Student's t-test was used to evaluate differences in the numbers of ovulations between the ovaries of animals within the CL/S and SL/S treatment groups as well as differences in the numbers of preovulatory follicles present in the ovaries of these animals at the time of ovulation and postovulation. A one-way ANOVA with Tukey-Kramer multiple comparisons was used to examine the differences in ovulation rates between treatment groups, and the length of the different stages of the estrous cycle in animals from the CL/CL and S/S groups. An unpaired Student's t-test was used to compare cycle length and serum progesterone levels in animals from the CL/CL and S/S groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the effect of administration of clodronate liposomes on ovulation rate, mice were killed 9 h after ovulation following treatment on either Day -1 or Day -3 before ovulation (Fig. 1). No significant difference in ovulation rate between clodronate liposome-treated and the contralateral saline solution-treated ovaries was observed in animals from the CL/S group when treatment was administered on Day -1 (number of oocytes ovulated, mean ± SE = 7 ± 1.7 and 6.4 ± 0.9 in clodronate liposome-treated and saline solution-treated ovaries, respectively). This ovulation rate did not differ significantly from that of animals in the S/S group (6.5 ± 1.3 and 5.8 ± 0.9 in left and right saline solution-treated ovaries, respectively). However, a significant reduction in the ovulation rate in clodronate liposome-treated ovaries was observed in animals from the CL/S group when treatment was administered on Day -3 (P < 0.05, Fig. 2a). No significant difference was seen in the ovulation rate in saline liposome-treated or saline solution-treated ovaries in the SL/S group when treatment was administered on Day -3. A large reduction in the ovulation rate of both clodronate liposome-treated ovaries was seen in the CL/CL group, while the ovulation rate in the S/S group was not diminished (Fig. 2b). When these results were pooled to give a single mean value for all saline solution-treated or clodronate liposome-treated ovaries, a significant reduction in ovulation rate was found in clodronate liposome-treated ovaries (number of oocytes ovulated = 8.5 ± 1.2 and 3.7 ± 0.8 in clodronate liposome-treated and saline solution-treated ovaries, respectively, P < 0.05). Progesterone levels measured in serum samples from animals in which treatment was administered on Day -3 showed no significant differences between any treatment groups, including those in which ovulation was affected (data not shown).
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Effect of Clodronate Liposome Treatment on the Ovarian Macrophage Population
To establish that liposomes were able to penetrate the ovarian epithelium from beneath the bursa and enter the ovarian tissue, liposomes containing the fluorescent marker DiI were injected into the bursal cavity, and animals were killed at several time points after injection. In sections from ovaries collected 2 h after injection, bright fluorescence could be seen in the stroma and thecal regions of the follicles in the ovary but not the granulosa or antrum (data not shown). Four hours after fluorescent liposome injection, the fluorescence was less bright, and 6 h later the fluorescence appeared significantly diminished. No significant difference in fluorescence intensity could be seen between untreated and fluorescent liposome-treated tissue at later time points.
To determine whether clodronate liposome treatment was effective in depleting macrophages from the ovary and to determine whether the observed reduction in ovulation rate was correlated with any decrease in thecal macrophage numbers, ovarian sections were incubated with antibodies specific for the macrophage antigens Ia, F4/80, and macrosialin. Macrophages reactive with all of these antibodies were present in all ovarian sections (Fig. 3). A comparison of the abundance of macrophages between the two ovaries from mice in the CL/S group treated on Day -1 indicates that clodronate liposome treatment caused a large but statistically insignificant reduction in mean F4/80 positivity in the stroma (Fig. 4a), with no apparent effect on the mean stromal positivity of the antigens Ia and macrosialin. In the theca of preovulatory follicles, a reduction in macrophage numbers was seen for all antigens, and this was statistically significant for the macrosialin-positive macrophages (P < 0.05, Fig. 4b). Comparison of the abundance of macrophages in the ovaries of the CL/S group in which treatment was administered on Day -3 indicates that clodronate liposome treatment had no apparent affect on the mean stromal positivity for any of the macrophage antigens (Fig. 4c). In the theca of preovulatory follicles, reductions were seen in the mean positivity for all antigens, with both the Ia and macrosialin antigens being significantly decreased by clodronate liposome treatment (P < 0.03, Fig. 4d).
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Effect of Clodronate Liposome Treatment on Ovarian Tissue Morphology
To determine whether the reduction in ovulation rate seen after clodronate liposome treatment on Day -3 was due to the prevention of follicular rupture or a reduction in the number of preovulatory follicles that developed, the total number of follicles of preovulatory size (diameter > 400 µm) and corpora lutea were counted in H&E sections of both the ovaries from 4 animals in the CL/S group. When ovaries were collected after ovulation, there were fewer corpora lutea and more preovulatory-sized, unruptured follicles in the clodronate liposome-treated ovaries than in the saline solution-treated ovaries, although these differences were not significant (Table 1). In ovaries collected immediately before ovulation, there were fewer preovulatory-sized follicles and more corpora lutea in the clodronate liposome-treated ovaries than in the saline solution-treated ovaries, although no significant differences were detected.
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Effect of Clodronate Liposome Treatment on Subsequent Natural Ovulation
Since clodronate liposome treatment may affect the numbers of follicles developing to the preovulatory size, we sought to determine the consequences of this for the next ovulatory event. In all animals allowed to progress to the next natural ovulation after treatment, ovulation rates were reduced in comparison to those of the stimulated cycle. No significant differences were detected regardless of the treatment group (SL/CL, SL/SL, CL/CL) or the time point of treatment (Day -1 or Day -3) (Table 2). However, it was noted that some animals receiving clodronate liposome treatment appeared to take longer to reach the subsequent natural ovulation. In animals treated on Day -1, no significant differences in cycle length were found between any of the treatment groups regardless of treatment, although the cycle length was extended beyond the expected 4–5 days in all of these animals (Table 2). In animals treated on Day -3 in the CL/S and CL/CL groups, cycle length was significantly extended over that in the SL/SL group (Table 2). Further breakdown of the cycle in this affected group revealed a significant delay in the metestrous-2/diestrous (ME-2/DE) stage (Fig. 5). The progesterone levels in serum obtained after ovulation were not significantly different between the treatment groups displaying differences in cycle length, and the estradiol levels were not detected consistently (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Examination of follicle numbers in the ovaries of this treatment group immediately before and after ovulation suggests that the growth of follicles to the preovulatory size as well as follicular rupture may be compromised by clodronate liposome treatment. This finding concurs with studies in the CSF-1-deficient csfmop/csfmop mice, in which severe reduction in macrophage numbers in the ovary is associated with significantly fewer antral and mature follicles present at the proestrous stage [27]. The difference in the extent of the effect in the two experimental systems might be related to the difference in the relative severity of the macrophage depletion achieved. Mice treated with clodronate liposomes 3 days before ovulation were also found to have delayed progression through the ME2/DE stage of the subsequent cycle. This stage is characterized by high numbers of macrophages within the ovary when compared to other stages of the cycle [5]. Interestingly, this stage of the ovarian cycle appears to be particularly susceptible to perturbations in macrophage population as both granulocyte-macrophage-CSF null mice (which have a reduced number of stromal Ia-positive cells in the ovarian stroma) and csfmop/csfmop mice exhibit delays in the diestrous stage of the estrous cycle [4].
Clodronate liposome treatment at the stage of late follicular development (Day -1) did not affect the numbers of oocytes ovulated. This suggests that macrophages may be less critical for the events immediately preceding ovulation or that the extent of depletion achieved was not sufficient to have a detectable effect on the process.
Although numerous authors have shown significant effects of macrophages on progesterone production in vitro [15,28–30], the levels of peripheral blood progesterone measured in this study were not altered by thecal macrophage depletion. The reduced number of macrophage cells in csfmop/csfmop mice also does not correlate with any change in the levels of progesterone produced during the luteal phase [4].
Although our results show that clodronate liposome treatment clearly influences ovarian function and alters macrophage numbers, complete depletion of the ovarian macrophage population, as has been seen elsewhere with systemic clodronate liposome treatment, was not achieved. This may be due to either insufficient or uneven distribution of clodronate liposomes through the ovarian tissue and might have been improved if it was possible to administer a larger volume of clodronate liposomes under the ovarian bursa. Alternatively, it is plausible that a high turnover of macrophages in the ovary may result in rapid replacement of those cells that are killed by clodronate treatment. Clearly, the surgery and anesthesia employed in the study had some nonspecific effect on ovarian function since all animals treated on Day -1 had extended cycles regardless of treatment type. We have been unable to determine the duration of the effect of macrophage depletion on the numbers of oocytes ovulated since the ovulation rate in the subsequent natural ovulation was much reduced in comparison to the ovulation rate of the first cycle regardless of the treatment type. This was probably due to either the exhaustion of the pool of growing follicles by the hyperstimulation regime employed to induce the first ovulatory event, or the physiological stress related to the surgery carried out.
It is interesting that macrophages in the vicinity of the thecal layer surrounding developing follicles appear to be preferentially targeted by clodronate liposome treatment. The reasons for this are not clear. The physical proximity of the developing follicle to the ovarian surface (and hence the bursal cavity) might be a contributing factor, and it may also be speculated that the composition of the extracellular matrix in this region is more conducive to diffusion of liposomes than the more dense collagenous structures of the ovarian stromal matrix. The results also indicate that, even within the thecal compartment, clodronate liposome treatment depletes macrosialin-positive and to a lesser extent Ia-positive macrophages in preference to F4/80-positive macrophages. This suggests that there is heterogeneity among the ovarian macrophage population and implies that some subsets might be more phagocytically active or otherwise more susceptible to the effect of clodronate liposome treatment. In view of the association of macrosialin with the phagocytic process, it might be speculated that strong macrosialin expression indicates high phagocytic activity and perhaps a greater capacity to take up liposomes from the extracellular milieu.
In conclusion, we have shown that local clodronate liposome treatment can be used to achieve a reduction in ovarian macrophage numbers. The technique has been successfully employed in the current study to generate data that suggest that thecal macrophages have a role in stimulating the growth of follicles to the preovulatory stage of development and in achieving follicular rupture. Further studies to define the phenotype and cytokine profile of the various macrophage populations residing in the ovary will help to elucidate the precise mechanisms through which they participate in regulating the different ovarian processes.
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FOOTNOTES |
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1 This research was funded by the National Health and Medical Research Council of Australia. 
2 Correspondence: R.J. Norman, Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville Rd., Woodville, SA, Australia 5011. FAX: 61 8 8222 7521; rnorman{at}medicine.adelaide.edu.au
Accepted: November 29, 1999.
Received: August 24, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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