IL-33 is required for disposal of unnecessary cells during ovarian atresia through regulation of autophagy and macrophage migration.

Physiological processes such as ovarian follicle atresia generate large amounts of unnecessary cells or tissue detritus, which needs to be disposed of rapidly. IL-33 is a member of the IL-1 cytokine gene family. Constitutive expression of IL-33 in a wide range of tissues has hinted at its role beyond immune defense. We have previously reported a close correlation between IL-33 expression patterns and ovarian atresia. In this study, we demonstrated that IL-33 is required for disposal of degenerative tissue during ovarian atresia using Il33(-/-) mice. Deletion of the Il33 gene impaired normal disposal of atretic follicles, resulting in massive accumulations of tissue wastes abundant with aging-related catabolic wastes such as lipofuscin. Accumulation of tissue wastes in Il33(-/-) mice, in turn, accelerated ovarian aging and functional decline. Thus, their reproductive life span was shortened to two thirds of that for Il33(+/-) littermates. IL-33 orchestrated disposal mechanism through regulation of autophagy in degenerating tissues and macrophage migration into the tissues. Our study provides direct evidence supporting an expanded role of IL-33 in tissue integrity and aging through regulating disposal of unnecessary tissues or cells.

Unique temporal and spatial expression patterns of IL-33 in ovaries during ovulation and estrous cycle are associated with ovarian tissue homeostasis.

Ovaries are among the most active organs. Frequently occurring events such as ovulation and ovarian atresia are accompanied with tissue destruction and repairing. Critical roles of immune cells or molecules in those events have been well recognized. IL-33 is a new member of the IL-1 cytokine gene family. Recent studies suggest its roles beyond immune responses. We systemically examined its expression in ovaries for its potential roles in ovarian functions. During ovulation, a high level of IL-33 was transiently expressed, making it the most significantly upregulated immune gene. During estrous cycle, IL-33 expression levels fluctuated along with numbers of ovarian macrophages and atresia wave. Cells with nuclear form of IL-33 (nIL-33(+) cells) were mostly endothelial cells of veins, either in the inner layer of theca of ovulating follicles during ovulation, or surrounding follicles during estrous cycle. Changes in number of nIL-33(+) cells showed a tendency similar to that in IL-33 mRNA level during estrous cycle. However, the cell number sharply declined before a rapid increase of macrophages and a surge of atresia. The decline in nIL-33(+) cell number was coincident with detection of higher level of the cytokine form of IL-33 by Western blot, suggesting a release of cytokine form of IL-33 before the surge of macrophage migration and atresia. However, IL-33 Ab, either by passive transfer or immunization, showed a limited effect on ovulation or atresia. It raises a possibility of IL-33's role in tissue homeostasis after ovarian events, instead of a direct involvement in ovarian functions.

Prolactin and lipopolysaccharide treatment increased apoptosis and atresia in rat ovarian follicles.

Follicular atresia is associated with the presence of increased macrophages within the follicle. What is not known is whether, in the adult rat, macrophages are instrumental in inducing apoptosis and/or atresia or whether they are simply secondary to a hormonally mediated event. As prolactin is an immunoreactive hormone and stimulates the expression of monocyte chemoattractant, the present experiments compared the effects of prolactin treatment with that of an immune challenge with lipopolysaccharide (LPS) on the invasion of macrophages into the follicular and luteal compartments of the ovary and the occurrence of apoptosis/atresia in relation to macrophage invasion. Rats were treated for 3 days with either prolactin or LPS and ovaries obtained at pro-oestrus or oestrus. Prolactin and LPS increased the number of atretic vs. healthy follicles (P < 0.008, chi2) in pro-oestrus ovaries and increased the mean number of apoptotic cells and macrophages (P < 0.05 for some groups). Macrophages were typically observed in the thecal layer, apoptotic cells in the granulosa cell layer, although 84% follicles which had macrophages within the granulosa cell layer also contained relatively high numbers of apoptotic nuclei. Prolactin and LPS treatment in vivo reduced the progesterone response to follicle stimulating hormone (FSH) (P < 0.001) in cultures of ovarian dispersates but did not inhibit the response to forskolin. In contrast, prolactin or LPS added in vitro to the cultures inhibited the progesterone response to forskolin. Results show that both prolactin and LPS increase follicular apoptosis and atresia and reduce the progesterone response to FSH.

Leukocytes in normal-cycling human ovaries: immunohistochemical distribution and characterization.

We investigated, using an image analysis system, the immunohistochemical localization of leukocyte subpopulations and human leukocyte antigen (HLA)-DR in 30 normal-cycling human ovaries in order to better understand local immunological events in human ovaries. All subtypes of T lymphocytes examined (CD3+, CD4+ and CD8+ cells) were sporadically observed in the stroma and theca layers of follicles throughout the menstrual cycle (ranging from 4.32 to 6.25 cells/10(-7) m2, 1.67 to 3.33 cells/10(-7) m2 and 2.33 to 3.44 cells/10(-7) m2, respectively for the three subtypes), and subsequently, increased in number in atretic follicles (78.70 +/- 6.90, 31.13 +/- 2.54 and 43.31 +/- 3.35). After ovulation, the number of T lymphocytes was markedly low in the early and mid corpus luteum (13.88 +/- 1.62, 4.18 +/- 0.50 and 6.53 +/- 0.45). The number increased in the late corpus luteum, and was highest in the late degenerating corpus luteum (255.67 +/- 27.10, 102.12 +/- 7.80 and 137.34 +/- 12.50). HLA-DR was sporadically positive in fibroblasts in the stroma and theca layers of follicles (means ranged from 1.25 to 1.82 cells/10(-7) m2), and increased in atretic follicles (24.68 +/- 2.25). HLA-DR+ cells were markedly low in the early and mid corpus luteum (2.16 +/- 0.88), increased in the late corpus luteum, and reached a plateau in the late degenerating corpus luteum (121.84 +/- 17.73). The great majority of these increased HLA-DR+ cells were macrophages. Results of our study suggest that T lymphocytes and/or macrophages play important roles in luteal regression and follicular atresia in normal-cycling human ovaries.

Expression of Fas ligand in murine ovary.

Corresponding to the expression of Fas in the ovarian oocytes as previously reported (Guo et al., Biochem Biophys Res Commun 1994; 203:1438-1446; Mori et al., JSIR 1995; 9:49-50), the expression of Fas ligand (FasL) in the ovarian follicle was found to be restricted in the area of granulosa cells by the indirect immunofluorescence (IIF) test. Reverse transcriptase/polymerase chain reaction (RT/PCR) technique coupled with Southern blot hybridization analysis showed that the highest level of FasL mRNA was demonstrated in murine ovaries and granulosa cells 1 day after the administration of pregnant mare's serum gonadotropin (PMSG), while the level of FasL mRNA became very weak on the day 5, respectively. The observed gradual decrease in FasL mRNA could not be attributed to a generalized degradation of cellular RNA during atresia, as evidenced by the presence of constitutive expression of elongation factor 1 alpha (EF-1 alpha) mRNA in murine ovaries and granulosa cells treated with PMSG. Furthermore, in situ hybridization analysis with a FasL-specific probe confirmed that FasL was specifically localized in the granulosa cells of most follicles and its expression was regulated by PMSG administration. FasL localized in granulosa cells might possibly play an important role in the formation of the ovarian atretic follicles, most likely depending on PMSG administration.

Expression of Fas-Fas ligand system associated with atresia through apoptosis in murine ovary.

We examined the contribution of Fas and its ligand (FasL) in the process of follicular atresia using murine intraovarian follicles and pregnant mare's serum gonadotropin (PMSG)-hyperovulated eggs. Reverse transcriptase/polymerase chain reaction-Southern blot hybridization demonstrated positive expression of Fas in both intraovarian oocytes and hyperovulated eggs. In contrast, expression of FasL was only detected in granulosa cells. These findings were histologically confirmed by in situ hybridization using Fas- and FasL-specific probes. A time-course study showed that Fas mRNA was positive in atretic follicles through day 0 and day 2 of PMSG stimulation and negative thereafter. Levels of FasL mRNA were the highest on day 1 and tapered off toward day 5 of PMSG stimulation. Levels of elongation factor 1 alpha mRNA, a constitutive element, were constantly maintained throughout the experimental period. Coculture of ovulated eggs, intact and zona-free, and granulosa cells demonstrated positive TUNEL staining only in zona-free eggs. These findings indicate that follicular atresia is caused by apoptosis, and the apoptosis associated with internucleosomal DNA fragmentation is directly regulated by the Fas/FasL system.

New concepts in ovarian regulation: an immune insight.

The purpose of the ovary is to produce eggs. In the human, oocyte production occurs cyclically at monthly intervals. The control of egg production is provided by both the endocrine and immune systems. Successful oocyte production is the result of sophisticated communication between the two systems. These interactions allow adaptive processes necessary for continuation of the species.

Immunology of ovarian failure.

Ovarian failure is the result of depletion of ovarian follicles. Naturally occurring ovarian failure usually takes place around 50 years of age in the human. Premature ovarian failure occurs in 1% of women and is the result of acceleration of rate of ovarian follicular depletion in the majority of cases. Cytokines are involved in the mechanisms of ovarian follicular atresia, whether it occurs at a normal or accelerated rate. It is the balance between the actions of TGF alpha and TGF beta upon the granulosa cell that determines the fate of a nonluteinized follicle and between LH and INF gamma that determines destiny of a luteinized follicle. When granulosa cells express MHC antigens in response to IFN gamma or genetic stimulus, an autoimmune reaction ensures resulting in follicular atresia. If the immune processes proceed continuously rather than cyclically, premature ovarian failure occurs. Thus, not only do the immunologic and endocrinologic systems need to communicate to allow normal ovarian function, evidence exists to support the concept that they interact in the pathophysiology of ovarian failure.