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Leptin is expressed by adipocytes and is thought to play a role in regulating food intake and in reproduction. It has been demonstrated that acute leptin administration to immature gonadotrophin-primed rats in vivo inhibits ovulation and causes a decline in food intake. However, feed restriction alone does not inhibit ovulation. Two experiments were designed to investigate the mechanism of leptin-induced inhibition of ovulation. In the first experiment, which was prompted by the importance of ovarian leucocytes in ovulation, the role of leucocytes in leptin-induced inhibition of ovulation was investigated. The second experiment investigated whether high leptin concentrations could inhibit other factors important to ovulation, such as meiotic competence of oocytes, granulosa cell proliferation, steroid or PGE(2) release, and interleukin 1beta production, in vitro. In the first experiment, the populations of neutrophils and monocytes-macrophages in the preovulatory follicles of gonadotrophin-primed, leptin-treated and -untreated rats were examined. A decrease in food intake, as a result of either leptin treatment or feed restriction, specifically reduced the numbers of neutrophils and monocytes-macrophages infiltrating the theca interna of preovulatory follicles without affecting the numbers found in the stroma. The findings show that reduced infiltration of thecal neutrophils and macrophages into preovulatory follicles is a response to reduced food intake. Furthermore, this reduction is not the direct cause of the leptin-induced inhibition of ovulation. In the second experiment, ovarian follicles were cultured for 4 or 12 h in the presence or absence of the following hormones: FSH (500 miu), insulin-like growth factor I (IGF-I) (50 ng ml(-1)), LH (100 ng ml(-1)) and leptin (300 ng ml(-1)). The results demonstrated that high concentrations of leptin in follicle culture do not affect meiotic maturation or steroid release, but tend to inhibit release of PGE 2 (although this result was not significant). DNA synthesis in granulosa cells was not inhibited by leptin in FSH- and IGF-I-supplemented culture media. These results are in agreement with previous studies that have shown that leptin inhibits the stimulatory effects of IGF-I on FSH-stimulated oestradiol production in rat granulosa cells without affecting progesterone production. In summary, leptin does not appear to have an adverse effect on the components of ovulation tested in this study, and therefore must impact on the ovulatory cascade in a way that remains to be defined.

To investigate the role of the ovarian macrophage population in ovulation, we examined the effect of depleting this population using liposome-encapsulated clodronate. Clodronate liposomes, saline liposomes, or saline alone was injected under the ovarian bursa in gonadotropin-primed adult mice, either 84 h (Day -3) or 36 h (Day -1) before ovulation. Ovulation rates were determined by counting the number of oocytes released. The numbers of graafian follicles and corpora lutea were also counted immediately before and after ovulation. Macrophage distribution within the theca and stroma of preovulatory ovaries was examined by immunohistochemistry with specific monoclonal antibodies to the macrophage antigens macrosialin, major histocompatability complex class II (Ia), and F4/80. Injection of clodronate liposomes on Day -1 did not affect ovulation rates, whereas administration on Day -3 caused a significant reduction in ovulation rate (mean oocytes ovulated = 5. 25 +/- 0.6 from clodronate liposome-treated ovaries and 9.13 +/- 0.9 from saline-treated ovaries, respectively, P < 0.05). The numbers of macrosialin-positive macrophages present in the theca at ovulation were reduced by treatment with clodronate liposomes on Day -1, and treatment on Day -3 reduced the numbers of Ia-positive and macrosialin-positive macrophages present in the theca. When the subsequent ovarian cycles were examined by vaginal smearing, the metestrous-2/diestrous stage was found to be extended in clodronate liposome-treated animals (7.5 +/- 1.3 days vs. 3.4 +/- 0.4 days for saline liposome-treated animals, P < 0.05). These results suggest that thecal macrophages may be involved in the regulation of follicular growth and rupture, as well as being important for the normal progression of the estrous cycle.

During the estrous cycle and early pregnancy, lymphohemopoietic cytokines and chemokines contribute to the regulation of ovarian function by orchestrating the recruitment and activation of leukocytes associated with the ovulatory follicle and corpus luteum. The purpose of this study was to investigate the physiological role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the ovary, utilizing mice genetically deficient in GM-CSF. Our results show that the mean duration of the estrous cycle in GM-CSF-deficient (GM-/-) mice was extended by 1.5 days (mean +/- SE, 4.9 +/- 0.3 vs. 6.5 +/- 0.5 days for GM+/+ and GM-/- mice, respectively). Similar ovulation rates were observed in immature superovulated mice (31.8 +/- 7.7 vs. 28.9 +/- 6.4 oocytes per mouse) and adult naturally cycling mice (10.4 +/- 0.8 vs. 10.3 +/- 0.8 oocytes per mouse). Furthermore, comparable numbers of oocytes were released from GM+/+ and GM-/- ovaries in an in vitro perfusion model. However, ovaries in pregnant GM-/- mice were found to comprise fewer cells and synthesize less progesterone (141.6 +/- 10.3 vs. 116.5 +/- 6 nM plasma), although the duration of pseudopregnancy was unaltered by GM-CSF deficiency (11.0 +/- 0.2 vs. 11.0 +/- 0.5 days). Immunohistochemical staining of leukocytes in the ovary during the periovulatory period indicated that the size and composition of ovarian leukocyte populations were unaltered in the absence of GM-CSF. However, an effect of GM-CSF deficiency on the activation phenotype of ovarian leukocytes was indicated by a 57% increase in mean secretion of nitric oxide in in vitro-perfused GM-/- ovaries, and diminished major histocompability complex (MHC) class II (Ia) expression in ovarian macrophages and/or dendritic cells (30.5 +/- 7. 2% vs. 9.1 +/- 1.8% positive stain in GM+/+ and GM-/- ovaries, respectively). Furthermore, ovarian macrophages and neutrophils were diminished in number after parturition, with significantly decreased CD11b+ (Mac-1) staining in the stromal region of postpartum GM-/- ovaries (6.7 +/- 0.6 vs. 3.6 +/- 0.7% positive stain). In summary, GM-CSF does not appear to be essential for ovarian function but may play a role in fine-tuning the activation status and adhesive properties of ovarian myeloid leukocytes. Aberrant activation of these cells appears to compromise the luteinization process and the steroidogenic capacity of the corpus luteum during early pregnancy in GM-CSF-deficient mice.

Evidence that cytokines have important roles in ovulation is accumulating, with various cytokines having been found to influence the ovulatory cascade. Interleukin (IL)-6 is a pluripotent cytokine involved in inflammatory reactions, and it has been demonstrated in high concentrations in human ovarian follicular fluid and in vitro in secretions from the ovary. We set out to determine the effect this cytokine has on ovulation rate and steroidogenesis in the in vitro-perfused rat ovary. Preovulatory ovaries were taken from eCG-primed animals, and ovulation was induced by LH (100 ng/ml) alone or in combination with cytokine. Ovaries in the IL-6/LH groups (IL-6 concentration of 0.19 nM or 1.9 nM) did not have ovulation rates different from ovaries in the LH-only group. Ovaries in the LH/IL-1beta group ovulated more oocytes than ovaries in the LH-only group (LH/IL-1beta =11+/-1.8 oocytes; LH alone=4.9+/-1.1; p=0.015) and the IL-6/LH/IL-1beta group (LH/IL-1beta/IL-6 [0.19 nM]=4+/-1.40; LH/IL-1beta=11+/-1.8; p=0.009). We have found that 1) exogenous IL-6 did not significantly alter the LH-induced ovulation rate but significantly reduced the LH/IL-1beta-induced ovulation rate; 2) exogenous IL-6 did not alter LH-induced progesterone levels measured at time points during the perfusion period, but the average increase in progesterone over basal level was stimulated by IL-6; 3) exogenous IL-6 did not affect LH-induced estradiol production; 4) exogenous IL-6 did not affect LH-induced androstenedione production but increased LH/IL-1beta-induced production; 5) exogenous IL-6 did not affect LH-induced prostaglandin E2 production. This study demonstrates that IL-6 does not play a role in regulating ovulation induced by LH in vitro but is capable of reducing LH/IL-1beta-enhanced ovulation rates. In addition, IL-6 may play a role in the regulation of ovarian steroid production.

The disciplines of reproduction and immunology, once quite discrete, are now closely associated, with compelling evidence to suggest that immune mechanisms play important roles in the cervix, uterus, fallopian tubes and ovary. Cells and mediators classically described as part of the immune system are found throughout the reproductive tract. Disorders of reproduction, including pre-eclampsia, unexplained infertility, endometriosis, recurrent miscarriage and disturbed fetal growth almost certainly have some of their origins in the dysfunction of immune regulation. There appears to be some evidence that immune disorders, such as rheumatoid arthritis and scleroderma, can manifest as infertility, before clinical disease becomes apparent.

Macrophages represent a major immune cell type in reproductive tissues and are thought to regulate multiple aspects of reproduction, including ovarian function. We have previously shown a distinctive phenotype of ovarian thecal macrophages present around the preovulatory follicle, such that secreted cytokines are uniquely regulated within these cells across the oestrus cycle. C-fms is a macrophage-specific gene that encodes the receptor for colony-stimulating factor-1 (CSF-1), and that regulates macrophage proliferation, differentiation and migration, as well as pro-inflammatory responses.1,2 We acquired transgenic mice (from DA Hume, Institute for Molecular Bioscience, University of Queensland) that express green fluorescent protein (GFP) exclusively in macrophages under direction of the c-fms gene promoter.3 In ovaries from these animals we have previously reported that macrophages constitutively positive for macrophage markers, F4/80 and MHCII, exhibited spatially regulated expression of GFP (c-fms); being GFP+ within the stroma surrounding small follicles, particularly atretic follicles, but GFP– in theca surrounding preovulatory follicles and healthy corpora lutea (CL), further reinforcing the concept that these macrophages are not classically activated but have a unique resident phenotype. Further examination of the GFP+ ovarian macrophage population has revealed that the highest levels of GFP expression were in macrophages associated with TUNEL+ regressing CL and, even though CSF-1 typically induces proliferation, the GFP+ macrophages within the regressing CL did not incorporate BrdU label nor express cyclin D1. This indicates that in the murine ovary c-fms expression may not regulate ovarian macrophage proliferation or migration but more likely represents a subset of classically activated ovarian macrophages that are actively differentiating or phagocytically active. (1)Fixe P and Praloran V (1998) Cytokine 10, 32–37.(2)Pixley FJ and Stanley ER (2004) Trends in Cell Biology 14, 628–38.(3)Sasmono RT et al. (2003) Blood 101, 1155–63.

Polycystic ovary syndrome (PCOS) is associated with anovulatory infertility and metabolic disturbances. PCOS has an unknown aetiology but is involved with aberration of substances that lead to follicular growth. The ovarian leukocyte/cytokine network is important in the ovary and has not been adequately examined in PCOS. The aim of the study was to look at the distribution of leukocytes in the ovaries of women with PCOS and to look at expression of cytokine and chemokine mRNA in follicular cells from these patients. Ovaries were obtained from PCOS (n�=�5) and non-PCOS (n�=�4) women undergoing gynaecological surgery for non-ovarian conditions prior to the menopause. They were immunostained for a wide variety of leukocyte markers and distribution counted using visual imaging software. Luteinising granulosa cells were obtained prior to ovulation in women undergoing in vitro fertilization with (n�=�11) and without (n�=�22) PCOS and mRNA studied using quantitative RT-PCR for various cytokines and chemokines. The CD45RO subset of leukocytes (principally activated/memory T-lymphocytes) were significantly decreased in ovaries from PCOS women compared to non-PCOS women. The other leukocytes were not different in distribution and numbers. Transcripts for CSF-1, IL-1β, IL-6, IL-8, IL-10, MCP-1 and TNFα were not different between PCOS and non-PCOS women whilst GM-CSF mRNA was not detectable in either group. There was an association between high testosterone levels and high IL1β and low TNFα transcripts. Women who became pregnant following IVF had higher levels of IL-10 mRNA. The role of T-lymphocytes in PCOS needs further examination, and if the leukocyte/cytokine network in PCOS is important, other cells and cytokines need examination. This is the first study to definitively describe the leukocyte/cytokine network within polycystic ovaries. While other cells and substances may be important in PCOS and intervention procedures such as ovarian drilling, it does not appear as though macrophages, neutrophils, B-lymphocytes and a variety of cytokines are involved in the aetiology of PCOS.

IL-1beta (IL-1β), TNF alpha (TNFα), nitric oxide (NO) and macrophages have been shown to be important in stimulating the ovulatory event [1-4]. Since macrophages produce these mediators elsewhere, it was our aim to determine if ovarian macrophages are a source of these known ovulatory mediators. Ovaries from gonadotrophin-primed immature mice were removed at various times and enzymatically digested. Macrophages were tagged with the specific antibodies F480 or anti-MHC II (anti- Ia) and isolated using antibody panning. mRNA was isolated from some macrophages and analysed by quantitative RT-PCR; other macrophages were cultured and secreted protein levels measured. mRNA levels, expressed as fold changes from the first isolation time point and normalised to the housekeeper gene HPRT, show that ovarian macrophages exhibit regulated cytokine mRNA profiles in response to hCG. Both IL-1β and TNFα mRNA increased significantly (*) post hCG injection (IL-1β:Ia. 7.3 ± 2*, F480 5.1 ± 0.8*, TNFα:Ia 2.39 ± 0.5, F480 2.47 ± 0.21*). Secreted TNFα protein increased 6 h post hCG and increased further 24 h post ovulation and then returned to pre-hCG levels 48 h after ovulation. Prior to hCG administration IL-1β was not detectable in conditioned media, but could be detected in media from Ia cells following hCG administration. No changes in total NO activity were detected across the stimulated cycle. Therefore, ovarian macrophages exhibit regulated cytokine expression, particularly of TNFα, in response to hCG. Since TNFα is known to modulate follicular rupture, its production by ovarian macrophages may contribute to the ovulatory process.