Pituitary-ovarian-splenic axis in ovulation

Abstract

Leukocytes are rapidly recruited to the preovulatory ovary and play a crucial role as facilitators of ovulation and luteal formation. In this article, recent findings on leukocyte trafficking to the ovary, as well as the physiological role of leukocytes in the ovary, will be summarized and discussed. We then explore the novel hypothesis that the hypothalamus-pituitary-ovarian (HPO) axis might include the spleen as a reservoir of leukocytes by summarizing recent reports on this topic, both in the fields of immunology and reproductive biology.

Linking leukocytes with ovulation

Ovulation, a key step in the propagation of life, has always been a subject of human curiosity. This egg-releasing act of the ovary is still a mysterious event and much about the process has yet to be unveiled. Ovulation is a critical step in reproduction and has become a key therapeutic target for treating female infertility and various ovarian diseases. Ovulatory failure is associated with the development of ovarian disorders such as polycystic ovarian syndrome (PCOS), hemorrhagic cyst formation, and hormonal imbalance, all of which are major risk factors in women’s health [1–3]. Furthermore, controlling ovulation has become a hallmark for contraception, as blockage of ovulation ensures the absence of fertilizable eggs [4]. Understanding the mechanisms that govern this ovulatory process, however, is challenging because there is interplay between the reproductive system, the immune system, and possibly other systems. Recently, a comprehensive flow cytometry approach was applied to quantitatively measure inflammation during ovulation by determining the spatiotemporal patterns of leukocyte infiltration in the ovaries of immature and adult rats. This effort led to the finding of massive leukocyte infiltration into the ovary induced by the luteinizing hormone (LH) surge or human chorionic gonadotropins (hCG) injection during ovulation [5, 6]. Surprisingly, ovarian leukocyte infiltration was accompanied by the release of millions of leukocytes to the bloodstream from the spleen, indicating that this immune organ might be a source of leukocytes that infiltrate the preovulatory ovary. Supporting this idea, recent studies showed that splenic leukocytes are recruited to injured heart tissues following myocardial infarction [7]. Both of these studies demonstrate the importance of the spleen as an immediate source of leukocytes for inflammatory events. In this article we examine trafficking of leukocytes into the ovary, the requirement of leukocytes for ovulation, and consider in depth the spleen as a source of leukocytes.

Trafficking of leukocytes into the ovary

The migration of leukocytes in response to chemokines has been implicated in a plethora of normal and pathophysiological aspects of reproductive systems [8]. Multiple chemoattractants such as interleukin-8 (IL-8) and a variety of their target populations of leukocytes have been shown to play important roles in ovulation [9–11]. Here, we summarize ovarian leukocyte populations, their function and factors that affect their infiltration into the ovary.

(1) Leukocyte populations and their localization within the ovary

Traditionally, immunohistochemical techniques have been used to characterize ovarian leukocyte populations. Whilst these methods are effective for identifying the localization of leukocytes in ovarian tissues, determining the precise leukocyte subsets that are present in the tissue has been challenging. Modern techniques such as flow cytometry have made it possible to distinguish between CD4⁺ T-cells, CD8⁺ T-cells, B-cells, natural killer (NK) cells, regulatory T-cells or other cell types, each with different functions. Table 1 summarizes the leukocyte populations that have been identified in the ovary, their localization and possible functions. As shown in Table 1, most leukocyte subtypes are found in the ovary and are predominately localized in the periphery of the follicle, interstitium, and corpora lutea, but not inside follicles.

Table 1.

Ovarian leukocyte species, localization and functions

Cell type	Location	Possible Function(s)	References
Monocyte/macrophage	periphery of follicles, tunica albugenia, corpora lutea,	IL-8 secretion, luteal regression	[5, 22, 99–111]
Neutrophils	theca layer, corpora lutea	production of proteolytic enzymes, ECM degradation, follicle maturation, ovulation, luteal formation	[5, 22, 110, 112, 113]
Lymphocytes	hilus, stroma, corpora lutea	selection of dominant follicle, luteal formation, luteal regression	[5, 101, 107, 110]
NK-cells	follicle, corpora lutea	angiogenesis	[114]
Mast cells	medulla, cortex, interstitium, corpora lutea	ECM degradation, ovulation	[109, 115, 116]
Eosinophils	theca layer, corpora lutea	ECM degradation, neovascularization	[111, 116, 117]

(2) Mechanism of leukocyte infiltration into the ovary

The initiator of an inflammatory event is often a discrete signal that is rapidly amplified by chemical signals produced by responding tissues and infiltrating cells. Unlike during infection, where the inflammatory stimuli are obvious, the initiating factors for an inflammatory response that occurs during a normal physiological event, such as preovulatory inflammation are less clear. Infiltration and distribution of leukocytes in the ovary are correlated with hormonal changes associated with the estrous cycle [12, 13], indicating that reproductive hormones such as ovarian steroids and gonadotropins may elicit inflammatory responses in the ovary. The same adhesion molecules, chemokines and cytokines observed in immune responses to infectious agents are also found in the ovary during estrous [5, 14, 15], suggesting a similar mechanism of leukocyte infiltration in the preovulatory ovary. The cytokines (IL-1, IL-6 and IL-10) may contribute to increased cellular adhesion molecule (CAM) expression during ovulation [16]. Furthermore, our recent data show increased expression of ICAM-1 and E-selectin corresponds with increased infiltration of leukocytes into the ovary [5]. Accordingly, treatment with IL-1 receptor antagonist (IL-1Ra) inhibits hCG induced ovulation rates in rats by 40% [17].

The classical four step process of tethering and rolling, activation, firm adhesion and transmigration into the tissues is paramount to any event requiring infiltration of leukocytes [18]; we anticipate ovulatory inflammation to be the same. Once the leukocytes are tethered on the endothelial cell wall, specific populations of leukocytes, characterized by their chemokine receptor (CCR) expression, migrate towards the source of the corresponding chemokine, produced by theca cells (TC), granulosa cells (GC) or resident ovarian leukocytes (Table 2). For instance, neutrophils infiltrate the ovary in response to an increased concentration gradient of IL-8 [9]. Amounts of basal IL-8 in the ovary are low, but production increases in granulosa cells and theca cells upon LH stimulation [11]. IL-8 is also known to increase vascular permeability [19, 20], which may facilitate the infiltration process. In addition, the infiltrating leukocytes may interact with ovarian endothelial cells and other cell types via a multitude of chemokines during ovulation [21–23]. Treatment with neutralizing antibodies to either IL-8 [24] or neutrophils [1] significantly reduces ovulation rates in animal models.

Table 2.

List of chemokines, chemokine receptors and responsible cells present in the ovary

Chemokine	Producing cells	Chemokine receptor	Responding cells	References
CCL-2 (MCP-1)	Mo/MΦ, cDC, BMDC, GC, GLC, stromal fibroblast	CCR-2a, b, CCR-8, CCR-11	T, Mo, baso, DC, NK	[23, 31, 118–120]
CCL-3 (MIP-1α)	Mo/MΦ, cDC, BMDC, TE, TM, NK, B, GC, GLC	CCR-1, CCR-5	Mo/MΦ, T, baso, eos, neut, DC, NK, GC, TC	[118–120]
CCL-4 (MIP-1β)	Mo/MΦ, cDC, BMDC, TE, TM, NK, B	CCR-5	Mo/MΦ, cDC, BMDC, B, T, NK, baso, eos, B, GC, TC	[118–120]
CCL-5 (RANTES)	Mo/MΦ, cDC, BMDC, TE, GC, TM, NK	CCR-1, CCR-3, CCR-5	TE, TM, NK, Mo/MΦ, DC, eos, baso, GC, TC	[118–120]
CCL-20(MIP-3α/LARC)	Epithelial, GC	CCR-6	PBMC, TE, TM, NKT, DC, B	[118, 121]
CCL25 (TECK)	Mo/MΦ, cDC, BMDC, TC	CCR-9	MΦ, thymocyte, DC, B	[37, 118]
CXCL-1 (Groα)	Mo/MΦ, DC, BMDC, GC	CXCR1, CXCR2	neut, fibroblast	[20, 118, 120]
CXCL-5 (ENA-78)	BMDC	CXCR2	neut, endo, BMDC	[120]
CXCL-8 (IL-8)	Mast, GC, GLC, TC, stromal fibroblast	CXCR1, CXCR2	neut, baso, T, endo	[20]
CXCL-10 (IP-10)	Osteoblasts, BM endo, GC	CXCR3A CXCR3B	T, NK, B, endo, Mo/MΦ, DC, GC	[119, 120]
CXCL-12 (SDF-1)	BM reticular, GC, endo, stromal, fibroblast	CXCR4, CXCR7	CD34+ BM, thymocytes, Mo/MΦ, TE, B, plasma, neut, cDC, BMDC	[122, 123]
CX3CL1 (Fractalkine)	Mast cells, GC, TC and stromal fibroblasts, endothelium	CX3CR1	NK, Mo, neut, Mast, astrocytes, neurons, activated T cells	[118]

NK, natural killer cell; neut, neutrophil; Mast, mast cell; Mo, monocyte; MΦ, macrophage; T_E, effector T-cell; T_M, memory T-cell; BMDC, bone marrow derived dendritic cell; cDC, conventional dendritic cell; GC, granulosa cell; TC, theca cell; GCL, granulosa-lutein cell; endo, endothelial cell; baso, basophil; PBMC, polymorphonuclear cell; DC, dendritic cell.

Two chemokines, monocyte chemotactic protein-1 (MCP-1/CCL2) and thymus-expressed cytokine (TECK/CCL25), are well characterized in ovarian leukocyte infiltration. MCP-1 is a potent chemoattractant for monocytes and is also effective in recruiting macrophages and T cells [25, 26]. The major producers of MCP-1 are monocytes and macrophages, although several other cell types including epithelial, endothelial, and smooth muscle cells have been shown to produce this protein [27]. In addition to the chemotactic properties, MCP-1 interacts with G-protein-coupled receptors to induce multiple intracellular responses including activation and degranulation (a comprehensive review of alternative functions for MCP-1 is given in [28]). Interestingly, studies have demonstrated in humans and rats that MCP-1 is involved in all aspects of ovarian function including follicular development [29, 30], ovulation and luteolysis [23, 31]. Several studies have demonstrated that inhibiting the production of monocytes/macrophages [32, 33] or the direct neutralization of ovarian macrophages [34] results in reduced or inhibited ovulation rates in mice, further supporting the role of monocytes/macrophages in ovulation. TECK was first described in the development of T-cells in the thymus, but now has a well-accepted role as a chemokine that recruits cells to sites of inflammation [35, 36]. Neutralization of TECK with specific antibodies inhibited leukocyte infiltration into the ovary by 85%, resulting in a lack of ovulation. Interestingly, ovulatory failure has been attributed to the lack of infiltration of a rare CD8α+ T cell population [37, 38]. Consistent with this finding are reports that ovarian TECK expression is tightly regulated by gonadotropins [39].

Two fascinating aspects of ovarian leukocyte infiltration are the speed and extent to which it occurs. In rats, this increased expression of CAMs and corresponding infiltration commences in as little as 1 to 3 hours after ovulatory gonadotropin stimulation. This is an extremely fast event, particularly in contrast to inflammation caused by infection in which infiltration of leukocytes occurs over several days [40]. In contrast, preovulatory leukocyte infiltration is not an occasional event but a frequent one as leukocyte infiltration takes place each time ovulation occurs, which is every four to five days in rodents and approximately once every month in women.

(3) Roles of ovarian leukocytes in ovulation

Leukocytes are involved in three main aspects of ovarian function: 1) loosening of the follicular wall to facilitate follicular growth and ovulation; 2) tissue repair following follicle rupture; and 3) luteal formation and regression. Ovulation, as well as the events that follow, requires major modifications in the extracellular matrix (ECM), and these rearrangements of the ECM involve tightly controlled production of tissue proteases. Matrix metalloproteinases (MMPs) are a family of soluble and membrane type (MT-MMPs) zinc dependent endopeptidases [41]. Both follicular cells (GC and TC) and leukocytes are known to produce MMPs [42–44]. However, further studies are needed for better characterization of the cells types responsible for production of each MMP subtype.

Leukocytes were recently described as major producers of MMP-9, the most abundant MMP found in the preovulatory ovary [45]. MMP-producing cells were identified as monocytes/macrophages and granulosa cells are the major producers of inhibitors of MMPs (TIMPs). In inflamed tissues, as monocytes move through tissue, they secrete MMPs that digest matrix proteins, facilitating easier migration through tissues to the sites of inflammation. It is feasible that ovarian monocytes produce one class of MMPs for migration purposes and, upon tissue specific differentiation, produce more potent MMPs that may further facilitate matrix breakdown. Interestingly, although the major role of MMPs in the ovary is related to their function in ECM breakdown, the substrates for MMPs are not restricted to matrix proteins. Many immune mediators, such as cytokines, chemokines, cell surface receptors and adhesions molecules also are substrates of MMP action [46]. In this regard, leukocyte-secreted MMPs might act both to breakdown the ECM, and to regulate the breakdown of chemotactic proteins thereby limiting leukocyte infiltration

Angiogenesis and neovascularization occur with great frequency within the ovary. As such, vascular endothelial growth factor (VEGF) expression closely correlates to the dynamic changes that take place in the ovary. In particular, a dramatic increase in angiogenesis occurs prior to ovulation and a fine network of capillaries develops and infiltrates the theca layer. An increase also occurs, immediately after ovulation forming a massive capillary network in the developing CL [47]. The source of VEGF that drives the active angiogenesis is not clearly determined. Whilst a body of literature suggests VEGF is expressed by TC, GC and the CL [48, 49], monocytes, macrophages and neutrophils also produce of VEGF in many scenarios [50, 51]. Macrophages isolated from human follicular aspirates upregulate VEGF production more than five-fold upon stimulation with hCG or LH [52]. Although the function of MMPs and VEGF appear mutually exclusive, several investigators have shown a mutual regulation between MMPs and VEGF [53]. It is expected that more detailed information on the leukocyte function in the ovary will emerge as new assay methods and animal models are developed.

HPO axis in regulating ovulation

The hypothalamus, pituitary and ovary have long been considered to constitute the axis of a regulatory loop that controls ovulation. These three key organs, the HPO axis, communicate between one another via hormonal signals. The result is that cyclic hormonal changes that result in the periodic expulsion of eggs in the process known as ovulation. Gonadotropin releasing hormone (GnRH) secreted from the hypothalamus stimulates pituitary gonadotrophs to synthesize and release the gonadotropins, follicle stimulating hormone (FSH) and LH. These two hormones then exert their effects on the ovary, leading to the growth and maturation of follicles and the expulsion of the oocyte [54–57]. The ovary is a complex organ, comprised of follicles that are at different stages of development including the quiescent primordial, primary, small pre-antral, antral, and large antral (or preovulatory) follicles. During follicular growth, granulosa cells (GCs) surround the oocyte, a basement membrane forms, and a theca cell (TC) layer develops and surrounds the follicle. The two cell layers act cooperatively in the production of steroid hormones. TC produce androgens that traverse the basement membrane to the GCs, where aromatase converts these androgens to estrogens that regulate FSH and LH release from the pituitary [54].

Inflammation and ovulation

Many hallmarks of inflammation are also observed in the ovary at the time of ovulation. Similar vascular changes include increased blood flow and vascular permeability, and cellular events such as increased leukocyte extravasation and activation occur at the site of inflammation and in the ovulating ovary. In addition, chemical mediators such as prostaglandins, vasoactive amines (histamine and serotonin), cytokines and chemokines are produced both in inflammatory responses and ovulation. The characteristic tissue damage, repair and remodeling that result from inflammation in non-ovarian tissues also occur in the ovary during ovulation [58–64]. Therefore, ovulation is now considered an outcome of acute inflammatory reactions in the ovary.

Leukocytes as the main mediators of ovarian inflammatory responses are a major target of investigation. Leukocytes circulate in the blood, become attracted by chemokines and adhesion molecules in inflamed tissues and traverse the blood vessel wall infiltrating interstitial tissues to reach their sites of action. Cytokines that are initially released by the inflamed tissue play a crucial role in increasing adhesion molecule expression on endothelial cells and in up-regulating the corresponding receptor expression on leukocytes, both of which greatly enhance leukocyte migration to the target tissues [65, 66]. At sites of inflammation, leukocytes and vascular endothelial cells release chemokines and cytokines that accelerate leukocyte recruitment and modulate leukocyte function. Eventually, however, the main function of leukocytes in the ovary is exerted through release of proteases [67, 68]. A number of molecules, including various cytokines, chemokines, and proteases that are commonly associated with immunological responses, are present in the preovulatory human ovary as well as ovaries in animal models [60–63]. Therefore, the last 30 years of research has clearly demonstrated the significance of infiltrating leukocytes in ovarian function. Much of this work implies that the infiltration of leukocytes in periovulatory period is a key event in ovulation [69]. However, the origin of these infiltrating leukocytes, their phenotype, and the mechanisms that govern their trafficking to the ovary are elusive.

Adding the spleen to the HPO axis

Leukocytes are hematopoietic in origin and are produced in the bone marrow. Upon release into the bloodstream, they circulate and infiltrate inflamed tissues or they are stored in lymphoid organs, such as the spleen, for future activation and release. The spleen releases leukocytes following induction of acute inflammation in the heart by ischemic myocardial injury [7, 70, 71]. Using a sophisticated approach that involved the transplantation of spleens from GFP mice into wild type mice, it was demonstrated that splenic leukocytes infiltrate heart tissues during acute inflammation. This finding indicates that at least one role of the spleen is to act as an immediate supplier of leukocytes for tissues that experience acute inflammation. This concept is supported by the fact that upon stimulation by an inflammatory signal, the bone marrow takes days to produce leukocytes whereas splenic leukocytes reach sites of inflammation within minutes to hours [7, 72, 73].

Upon ovulatory gonadotropin stimulation, the ovary experiences an acute inflammatory response. This inflammation is different from responses to infectious insults, as ovulatory inflammation is in response to a normal physiological event, LH stimulation. However, the nature and sequence of the inflammatory events occurring in the preovulatory ovary are essentially identical to those reactions taking place at the sites of infectious inflammation. In particular, the ovary utilizes the same molecular signals that attract leukocytes via the mechanism that governs their infiltration at the site of infections or injuries [11, 15, 74, 75]. Thus, does the spleen serve as a source of infiltrating leukocytes during this period of ovulatory inflammation? To answer this question, a study recently measured sequential changes of leukocyte content in the ovary and spleen after inducing superovulation, by injecting gonadotropins (a bolus injection with PMSG to stimulate follicular growth followed 48 hours later by hCG injection to induce ovulation) [5]. Flow cytometry was employed to count the actual numbers of leukocytes in these two distal organs using CD45 specific fluorescent antibodies. This approach revealed that as intraovarian leukocyte numbers increased, the leukocyte numbers in the spleen sharply decreased. The same inverse relationship was observed in adult rats during the period of proestrus to estrus, when ovulatory inflammation occurs. Lower numbers of leukocytes infiltrated the ovary upon superovulation induction in splenectomized rats [5]. Together, these findings strongly indicate that the spleen supplies leukocytes to the preovulatory ovary.

These findings raise the interesting question, should the spleen be considered a key component of the reproductive axis in regulating ovulation? Are splenic leukocytes under the regulation of LH, progesterone and/or prostaglandins whose ovarian functions are critical for successful ovulation? How does the HPO axis communicate with the spleen to trigger the preovulatory leukocyte release? Unfortunately, none of these questions can be clearly answered at present since very little research on the spleen has been done in relation to its reproductive function. However, a glimpse of the role the spleen might play in ovulation can be gathered from past and current literature.

(1) The impact of splenectomy on female fertility

The removal of the entire spleen, splenectomy, has been a successful surgical procedure for many hematological, immunological, and traumatic conditions. However, although the removal is advantageous in treating the specific disorders, it leaves the patient with a significant defect in both innate and adaptive immune responses. As a consequence, splenectomized patients have a 60- to 100-fold increased risk of sepsis [76]. The ovulatory consequences of splenectomy in humans, however, is difficult to assess as other treatment regimens such as chemotherapy or radiotherapy that often accompany the splenectomy procedure also result in severe damage to ovulatory function. However, there are several animal studies that indicate a role of the spleen in ovarian function. In particular, studies in rodents and rabbits show that splenectomy results in either a delay in ovulation [77], aberrant corpus luteal function [78] or an absence in leuteolysis [79, 80].

(2) Proposed method of communication between HPO axis and spleen

How would the HPO axis communicate with spleen to induce splenic leukocyte release? Since leukocytes are released as early as one hour after ovulatory LH surge, LH may directly stimulate spleen (Fig. 1). However, this is unlikely because the presence of LH receptors in the spleen is reported only in poultry [81], and the spleen does not express LH receptors in mammals [82]. Instead, an endocrine molecule(s) produced by the ovary in response to LH stimulation may travel to spleen via the circulation and stimulate leukocyte release (Fig. 1). In the case of the myocardial injury model, angiotensin II was shown to have such activity [7]. It will be interesting to determine if angiotensin II has this role in the periovulatory release of splenic leukocytes. In support of the potential role of angiotensin II, studies indicate that ovarian angiotensin II synthesis and secretion increases immediately after ovulatory LH/hCG stimulation and ovulation is inhibited if PD123319 or Saralasin, receptor antagonists, are injected into superovulation-induced rabbits and rats, respectively [83–85]. Other candidate molecules that may originate from the ovary and induce splenic leukocyte release include chemokines and cytokines that are produced by the ovary upon LH stimulation. It will be interesting to determine whether receptors for these ovary-borne molecules are present in the spleen and if the receptors are localized in leukocytes or splenic cells. It is however considered that the splenic leukocyte release could be a homeostatic response to the decrease in circulating leukocytes as many of them infiltrate the ovary. Taken together, we propose the following path of splenic leukocyte trafficking to the ovary (Fig. 2). The leukocytes reside in steady state equilibrium within the red pulp of the spleen where they are held via chemokine receptors (CCRs). The LH surge may alter CCR expression on the leukocytes and/or induce chemokine expression in the splenic endothelial cells stimulating mobilization of the leukocytes through the endothelial cell layer of the blood vessel and into the circulation. Leukocytes circulate through the periphery until reaching the ovary where an interaction with appropriate adhesion molecules on the endothelial cell wall occurs. Simultaneously, in the ovary, the LH surge decreases blood flow by dilating vessels and increases expressions of leukocyte receptors such as adhesion molecules so that leukocytes interact with receptors on ovarian endothelial cells.

Figure 1. Proposed interplay between HPO axis and spleen.

Preovulatory rise of E2 (estradiol) stimulates release of LH into the bloodstream. Then LH triggers leukocyte release from the spleen either by directly acting on leukocytes or through indirect effects on splenic tissues. Splenic leukocytes released into the bloodstream migrate to the ovary in response to cytokines and chemokines that act as leukocyte attractants, the cells enter the tissue through interactions between leukocyte receptors and adhesion molecules on the endothelial cells.

Figure 2. Splenic leukocyte trafficking to the ovary.

Splenic reservoir leukocytes are tethered in the open blood system of the red pulp through either specific chemokine-chemokine receptor or adhesion molecule interactions. Upon LH surge, changes in leukocyte CCR expression or the reduced chemokine production by reticular fibroblast within the red pulp results in the mobilization of splenic reservoir leukocytes into the bloodstream. Upon arriving at the ovary, LH mediated up-regulation of adhesion molecule expression on ovarian endothelial cells, together with LH mediated vasodilation, increases leukocyte adherence to the endothelial cells. Once tethered to the ovarian endothelial cells, leukocytes respond to follicular produced chemokines. The migration of leukocytes through the interstitial space towards the source (GC, TC or ovarian leukocytes) results in the production of MMPs to facilitate movement through the ECM. At the mature follicle, leukocytes become activated by locally produced cytokines and produce reactive oxygen species (ROS), MMPs and proteolytic granules that weaken the basement membrane of the follicle, enabling the release of the oocyte.

Leukocytes in other reproductive organs

Leukocytes also play critical roles in reproductive tissues other than the ovary, such as the uterus [86–88], pituitary [89, 90], oviduct [91–93], testis [94], and vagina [95–97]. Thus, implantation, pregnancy maintenance, embryonic development, menstrual tissue shedding and many other reproductive functions are regulated by leukocytes. With the recent finding that the spleen is a reservoir for leukocytes that rapidly respond to perturbation, it is likely that the spleen may also serve as leukocyte reservoir for these reproductive tissues. In fact, we found a discrepancy between the numbers of leukocytes that leave spleen upon LH stimulation and the combined increase of leukocyte numbers in the bloodstream and ovary [5], indicating that large numbers of splenic leukocytes migrate to other organs that may include uterus and oviduct where increased leukocyte infiltration have been documented [86, 87, 93, 98].

Concluding remarks

The immune system is not only important for battling foreign invaders but is also essential for female reproduction. Here, we propose that the spleen may bridge the immune and reproductive systems serving as a leukocyte reservoir required for the inflammatory events that regulate ovulation. For validation of this hypothesis and determination of the interaction between the reproductive organs and the spleen, rigorous collaborative studies between the fields of immunology and reproductive biology should follow.