Relaçòes entre o sistema imunológico e o reprodutor parecem depender de liberação de leucócitos pelo baço, através de comando do LH. Observar as figuras ilustrativas.

1. Opinion
Pituitary–ovary–spleen axis in
ovulation
Oliver R. Oakley1, Michele L. Frazer2 and CheMyong Ko1
1
Division of Clinical and Reproductive Sciences, Department of Clinical Sciences, College of Health Sciences,
University of Kentucky, Lexington, Kentucky 40536, USA
2
Hagyards Equine Medical Institute, 4250 Iron Works Pike, Lexington, Kentucky 40511, USA
Leukocytes are rapidly recruited to the preovulatory ovary examine trafficking of leukocytes into the ovary, the re-
and play a crucial role as facilitators of ovulation and luteal quirement for leukocytes in ovulation, and consider in
formation. In this article, recent findings on leukocyte depth the spleen as a source of leukocytes.
trafficking to the ovary, as well as the physiological role
of leukocytes in the ovary, will be summarized and dis- Trafficking of leukocytes into the ovary
cussed. We then explore the novel hypothesis that the The migration of leukocytes in response to chemokines has
hypothalamus–pituitary–ovary (HPO) axis might include been implicated in a plethora of normal and pathophysio-
the spleen as a reservoir of leukocytes by summarizing logical aspects of reproductive systems [8]. Multiple che-
recent reports on this topic, both in the fields of immu- moattractants including interleukin-8 (IL-8) and a variety
nology and reproductive biology. of their target populations of leukocytes have been shown
to play important roles in ovulation [9–11]. Here we sum-
Linking leukocytes with ovulation marize ovarian leukocyte populations, their function and
Ovulation, a key step in the propagation of life, has always factors that affect their infiltration into the ovary.
been a subject of human curiosity. This egg-releasing act of
the ovary is still a mysterious event and much about the Leukocyte populations and their localization within the
process has yet to be unveiled. Ovulation is a crucial step in ovary
reproduction and has become a key therapeutic target for Traditionally, immunohistochemical techniques have been
treating female infertility and various ovarian diseases. used to characterize ovarian leukocyte populations. Al-
Ovulatory failure is associated with the development of though these methods are effective for identifying the
ovarian disorders such as polycystic ovarian syndrome localization of leukocytes in ovarian tissues, determining
(PCOS), hemorrhagic cyst formation, and hormonal imbal- the precise leukocyte subsets that are present in the tissue
ance, all of which are major risk factors in women’s health has been challenging. Modern techniques such as flow
[1–3]. Furthermore, controlling ovulation has become a cytometry have made it possible to distinguish between
hallmark for contraception because blockage of ovulation CD4+ T cells, CD8+ T cells, B cells, natural killer (NK) cells,
ensures the absence of fertilizable eggs [4]. Understanding regulatory T cells or other cell types, each with different
the mechanisms that govern this ovulatory process, how- functions. Table 1 summarizes the leukocyte populations
ever, is challenging because there is interplay between the that have been identified in the ovary, their localization
reproductive system, the immune system, and possibly and possible functions. As shown in the table, most leuko-
other systems. Recently a comprehensive flow cytometry cyte subtypes are found in the ovary and are predominately
approach was applied to measure inflammation quantita- localized in the periphery of the follicle, interstitium, and
tively during ovulation by determining the spatiotemporal corpora lutea, but not inside follicles.
patterns of leukocyte infiltration in the ovaries of imma-
ture and adult rats. This effort led to the finding of massive Mechanism of leukocyte infiltration into the ovary
leukocyte infiltration into the ovary induced by the lutei- The initiator of an inflammatory event is often a discrete
nizing hormone (LH) surge or by human chorionic gonado- signal that is rapidly amplified by chemical signals pro-
tropin (hCG) injection during ovulation [5,6]. Surprisingly, duced by responding tissues and infiltrating cells. Unlike
ovarian leukocyte infiltration was accompanied by the during infection, where the inflammatory stimuli are ob-
release of millions of leukocytes into the bloodstream from vious, the initiating factors for an inflammatory response
the spleen, indicating that this immune organ might be a that occurs during a normal physiological event such as
source of leukocytes that infiltrate the preovulatory ovary. preovulatory inflammation are less clear. Infiltration and
Supporting this idea, recent studies showed that spleen distribution of leukocytes in the ovary are correlated with
leukocytes are recruited to injured heart tissues following hormonal changes associated with the estrous cycle
myocardial infarction [7]. Both of these studies demon- [12,13], indicating that reproductive hormones such as
strate the importance of the spleen as an immediate source ovarian steroids and gonadotropins can elicit inflammato-
of leukocytes for inflammatory events. In this article we ry responses in the ovary. The same adhesion molecules,
chemokines and cytokines observed in immune responses
Corresponding author: Ko, C. (cko2@uky.edu). to infectious agents are also found in the ovary during
1043-2760/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tem.2011.04.005 Trends in Endocrinology and Metabolism, September 2011, Vol. 22, No. 9 345

2. Opinion Trends in Endocrinology and Metabolism September 2011, Vol. 22, No. 9
Table 1. Ovarian leukocyte species, localization and functions
Cell type Location Possible function(s) References
Monocyte/macrophage Periphery of follicles, tunica Il-8 secretion, luteal regression [5,22,99–111]
albugenia, corpora lutea
Neutrophils Theca layer, corpora lutea Production of proteolytic enzymes, [5,22,110,112,113]
ECM degradation, follicle maturation,
ovulation, luteal formation
Lymphocytes Hilus, stroma, corpora lutea Selection of dominant follicle, [5,101,107,110]
luteal formation, luteal regression
NK-cells Follicle, corpora lutea Angiogenesis [114]
Mast cells Medulla, cortex, interstitium, ECM degradation, ovulation [109,115,116]
corpora lutea
Eosinophils Theca layer, corpora lutea ECM degradation, neovascularization [111,116,117]
estrous [5,14,15], suggesting a similar mechanism of leu- the ovary are low, but production increases in granulosa
kocyte infiltration in the preovulatory ovary. The cytokines cells and theca cells upon LH stimulation [11]. IL-8 is also
interleukins (IL)-1, IL-6 and IL-10 could contribute to known to increase vascular permeability [19,20], and this
increased cell adhesion molecule (CAM) expression during could facilitate the infiltration process. In addition, the
ovulation [16]. Furthermore, our recent data show in- infiltrating leukocytes can interact with ovarian endothe-
creased expression of ICAM-1 and E-selectin corresponds lial cells and other cell types via a multitude of chemokines
with increased infiltration of leukocytes into the ovary [5]. during ovulation [21–23]. Treatment with neutralizing
Accordingly, treatment with IL-1 receptor antagonist (IL- antibodies to either IL-8 [24] or neutrophils [1] significant-
1Ra) inhibits hCG-induced ovulation rates in rats by 40% ly reduces ovulation rates in animal models.
[17]. Two chemokines, monocyte chemotactic protein-1
The classical four step process of tethering and rolling, (MCP-1, also known as chemokine C–C motif ligand
activation, firm adhesion and transmigration into the tis- CCL2) and thymus-expressed cytokine (TECK/CCL25),
sues is paramount to any event requiring infiltration of are well characterized in ovarian leukocyte infiltration.
leukocytes [18]; we anticipate ovulatory inflammation to be MCP-1 is a potent chemoattractant for monocytes and is
the same. Once the leukocytes are tethered on the endo- also effective in recruiting macrophages and T cells [25,26].
thelial cell wall, specific populations of leukocytes, charac- The major producers of MCP-1 are monocytes and macro-
terized by their chemokine receptor (CCR) expression, phages, although several other cell types including epithe-
migrate towards the source of the corresponding chemo- lial, endothelial, and smooth muscle cells have been shown
kine produced by theca cells (TC), granulosa cells (GC) or to produce this protein [27]. In addition to the chemotactic
resident ovarian leukocytes (Table 2). For instance, neu- properties, MCP-1 interacts with G-protein-coupled recep-
trophils infiltrate the ovary in response to an increased tors to induce multiple intracellular responses including
concentration gradient of IL-8 [9]. Amounts of basal IL-8 in activation and degranulation (a comprehensive review of
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/MF, cDC, BMDC, CCR-2a, b, T, Mo, baso, DC, NK [23,31,118–120]
GC, GLC, stromal fibroblast CCR-8, CCR-11
CCL-3 (MIP-1a) Mo/MF, cDC, BMDC, CCR-1, CCR-5 Mo/MF, T, baso, eos, neut, [118–120]
TE, TM, NK, B, GC, GLC DC, NK, GC, TC
CCL-4 (MIP-1b) Mo/MF, cDC, BMDC, CCR-5 Mo/MF, cDC, BMDC, B, T, [118–120]
TE, TM, NK, B NK, baso, eos, B, GC, TC
CCL-5 (RANTES) Mo/MF, cDC, BMDC, CCR-1, CCR-3, TE, TM, NK, Mo/MF, [118–120]
TE, GC, TM, NK CCR-5 DC, eos, baso, GC, TC
CCL-20(MIP-3a/LARC) Epithelial, GC CCR-6 PBMC, TE, TM, NKT, DC, B [118,121]
CCL25 (TECK) Mo/MF, cDC, BMDC, TC CCR-9 MF, thymocyte, DC, B [37,118]
CXCL-1 (Groa) Mo/MF, 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, CXCR1, CXCR2 Neut, baso, T, endo [20]
stromal fibroblast
CXCL-10 (IP-10) Osteoblasts, BM endo, GC CXCR3A CXCR3B T, NK, B, endo, Mo/MF, DC, GC [119,120]
CXCL-12 (SDF-1) BM reticular, GC, endo, CXCR4, CXCR7 CD34+ BM, thymocytes, [122,123]
stromal, fibroblast Mo/MF, TE, B, plasma,
neut, cDC, BMDC
CX3CL1 (Fractalkine) Mast cells, GC, TC and CX3CR1 NK, Mo, neut, mast, astrocytes, [118]
stromal fibroblasts, neurons, activated T cells
endothelium
Baso, basophil; BMDC, bone marrow-derived dendritic cell; cDC, conventional dendritic cell; DC, dendritic cell; endo, endothelial cell; GC, granulosa cell; GLC, granulosa-
lutein cell; NK, natural killer cell; neut, neutrophil; mast, mast cell; Mo, monocyte; MF, macrophage; PBMC, polymorphonuclear cell; TC, theca cell; TE, effector T cell; TM,
memory T cell.
346

3. Opinion Trends in Endocrinology and Metabolism September 2011, Vol. 22, No. 9
alternative functions for MCP-1 is given in [28]). Interest- Many immune mediators, such as cytokines, chemokines,
ingly, studies have demonstrated in humans and rats that cell surface receptors and adhesion molecules, are also
MCP-1 is involved in all aspects of ovarian function includ- substrates for MMP action [46]. In this regard, leukocyte-
ing follicular development [29,30], ovulation and luteolysis secreted MMPs might act both to break down the ECM and
[23,31]. Several studies have demonstrated that inhibiting to regulate the breakdown of chemotactic proteins, thereby
the production of monocytes/macrophages [32,33] or the limiting leukocyte infiltration
direct neutralization of ovarian macrophages [34] results Angiogenesis and neovascularization take place recur-
in reduced or inhibited ovulation rates in mice, further rently within the ovary, and vascular endothelial growth
supporting the role of monocytes/macrophages in ovula- factor (VEGF) expression closely correlates to the dynamic
tion. TECK was first described in the development of T changes that take place in the ovary. In particular, a dra-
cells in the thymus, but now has a well-accepted role as a matic increase in angiogenesis occurs before ovulation and a
chemokine that recruits cells to sites of inflammation fine network of capillaries develops and infiltrates the theca
[35,36]. Neutralization of TECK with specific antibodies layer. An increase also occurs immediately after ovulation to
inhibited leukocyte infiltration into the ovary by 85%, generate an extensive capillary network in the developing
resulting in a lack of ovulation. Interestingly, ovulatory corpus luteum (CL) [47]. The source of VEGF that drives this
failure has been attributed to the lack of infiltration of a active angiogenesis has not yet been clearly determined.
rare CD8a+ T cell population [37,38]. Consistent with this Although a body of literature suggests VEGF is expressed by
finding are reports that ovarian TECK expression is tightly TC, GC and the CL [48,49], monocytes, macrophages and
regulated by gonadotropins [39]. neutrophils also produce VEGF in many scenarios [50,51].
Two fascinating aspects of ovarian leukocyte infiltration Macrophages isolated from human follicular aspirates upre-
are the speed and extent to which it takes place. In rats, gulate VEGF production more than fivefold upon stimula-
this increased expression of CAMs and corresponding tion with hCG or LH [52]. Although the function of MMPs
infiltration commences in as little as 1–3 hours after and VEGF appear mutually exclusive, several investigators
ovulatory gonadotropin stimulation. This is an extremely have shown a mutual regulation between MMPs and VEGF
fast event, particularly in contrast to inflammation caused [53]. It is expected that more detailed information on the
by infection in which infiltration of leukocytes occurs over leukocyte function in the ovary will emerge as new assay
several days [40]. By contrast, preovulatory leukocyte methods and animal models are developed.
infiltration is not an occasional event but a frequent one
because leukocyte infiltration takes place each time ovula- HPO axis in regulating ovulation
tion occurs, which is every 4–5 days in rodents and ap- The hypothalamus, pituitary and ovary have long been
proximately once per month in women. considered to constitute the axis of a regulatory loop that
controls ovulation. These three key organs, the HPO axis,
Roles of ovarian leukocytes in ovulation communicate between one another via hormonal signals.
Leukocytes are involved in three main aspects of ovarian The result is cyclic hormonal changes that result in the
function: (i) loosening of the follicular wall to facilitate periodic expulsion of eggs in the process known as ovula-
follicle growth and ovulation, (ii) tissue repair following tion. Gonadotropin releasing hormone (GnRH) secreted
follicle rupture, and (iii) luteal formation and regression. from the hypothalamus stimulates pituitary gonadotrophs
Ovulation, as well as the events that follow, requires major to synthesize and release the gonadotropins, follicle-stim-
modifications in the extracellular matrix (ECM), and these ulating hormone (FSH) and LH. These two hormones then
rearrangements of the ECM involve tightly controlled exert their effects on the ovary, leading to the growth and
production of tissue proteases. Matrix metalloproteinases maturation of follicles and the expulsion of the oocyte [54–
(MMPs) are a family of soluble and membrane type (MT- 57]. The ovary is a complex organ, comprised of follicles
MMPs) zinc-dependent endopeptidases [41]. Both follicu- that are at different stages of development including the
lar cells (GC and TC) and leukocytes are known to produce quiescent primordial, primary, small pre-antral, antral,
MMPs [42–44]. However, further studies are needed for and large antral (or preovulatory) follicles. During follicu-
better characterization of the cells types responsible for lar growth, GCs surround the oocyte, a basement mem-
production of each MMP subtype. brane forms, and a TC layer develops and surrounds the
Leukocytes were recently described as major producers of follicle. The two cell layers act cooperatively in the produc-
MMP-9, the most abundant MMP found in the preovulatory tion of steroid hormones. TCs produce androgens that
ovary [45]. MMP-producing cells were identified as mono- traverse the basement membrane to the GCs, where aro-
cytes/macrophages and granulosa cells are the major pro- matase converts these androgens to estrogens that regu-
ducers of inhibitors of MMPs (TIMPs). In inflamed tissues, late FSH and LH release from the pituitary [54].
as monocytes move through tissue they secrete MMPs that
digest matrix proteins, facilitating migration through tis- Inflammation and ovulation
sues to the sites of inflammation. It is feasible that ovarian Many hallmarks of inflammation are also observed in the
monocytes produce one class of MMPs for migration pur- ovary at the time of ovulation. Similar vascular changes
poses and, upon tissue specific differentiation, produce more include increased blood flow and vascular permeability,
potent MMPs that could further facilitate matrix break- and cellular events such as increased leukocyte extravasa-
down. Interestingly, although the major role of MMPs in the tion and activation occur at the site of inflammation and in
ovary is related to their function in ECM breakdown, the the ovulating ovary. In addition, chemical mediators in-
substrates for MMPs are not restricted to matrix proteins. cluding prostaglandins, vasoactive amines (histamine and
347

4. Opinion Trends in Endocrinology and Metabolism September 2011, Vol. 22, No. 9
serotonin), cytokines and chemokines are produced both in In particular, the ovary utilizes the same molecular signals
inflammatory responses and ovulation. The characteristic that attract leukocytes via the mechanism that governs
tissue damage, repair and remodeling that result from their infiltration at the site of infections or injuries
inflammation in non-ovarian tissues also occur in the [11,15,74,75]. Thus, does the spleen serve as a source of
ovary during ovulation [58–64]. Therefore, ovulation is infiltrating leukocytes during this period of ovulatory in-
now considered an outcome of acute inflammatory reac- flammation? To answer this question, a study recently
tions in the ovary. measured sequential changes of leukocyte content in the
Leukocytes as the main mediators of ovarian inflamma- ovary and spleen after inducing superovulation, by inject-
tory responses are a major target of investigation. Leuko- ing gonadotropins [a bolus injection with pregnant mare
cytes circulate in the blood, become attracted by serum gonadotropin (PMSG) to stimulate follicular growth
chemokines and adhesion molecules in inflamed tissues, followed 48 h later by hCG injection to induce ovulation]
and traverse the blood vessel wall infiltrating interstitial [5]. Flow cytometry was employed to count the actual
tissues to reach their sites of action. Cytokines that are numbers of leukocytes in these two distal organs using
initially released by the inflamed tissue play a crucial role CD45-specific fluorescent antibodies. This approach
in increasing adhesion molecule expression on endothelial revealed that as intraovarian leukocyte numbers in-
cells and in upregulating the corresponding receptor ex- creased, leukocyte numbers in the spleen decreased sharp-
pression on leukocytes, both of which greatly enhance ly. The same inverse relationship was observed in adult
leukocyte migration to the target tissues [65,66]. At sites rats during the period of proestrus to estrus, when ovula-
of inflammation, leukocytes and vascular endothelial cells tory inflammation occurs. Lower numbers of leukocytes
release chemokines and cytokines that accelerate leuko- infiltrated the ovary upon superovulation induction in
cyte recruitment and modulate leukocyte function. Even- splenectomized rats [5]. Together, these findings strongly
tually, however, the main function of leukocytes in the indicate that the spleen supplies leukocytes to the preovu-
ovary is exerted through release of proteases [67,68]. A latory ovary.
number of molecules, including diverse cytokines, chemo- These findings raise the interesting question, should the
kines, and proteases that are commonly associated with spleen be considered a key component of the reproductive
immunological responses, are present in the preovulatory axis in regulating ovulation? Are spleen leukocytes under
human ovary as well as in ovaries in animal models [60– the regulation of LH, progesterone and/or prostaglandins
63]. Therefore, the past 30 years of research have clearly whose ovarian functions are crucial for successful ovula-
demonstrated the significance of infiltrating leukocytes in tion? How does the HPO axis communicate with the spleen
ovarian function. Much of this work implies that the to trigger the preovulatory leukocyte release? Unfortu-
infiltration of leukocytes in periovulatory period is a key nately, none of these questions can be clearly answered
event in ovulation [69]. However, the origin of these infil- at present because very little research on the spleen has
trating leukocytes, their phenotype, and the mechanisms been done in relation to its reproductive function. However,
that govern their trafficking to the ovary are elusive. a glimpse of the role the spleen might play in ovulation can
be gathered from past and current literature.
Adding the spleen to the HPO axis
Leukocytes are hematopoietic in origin and are produced in The impact of splenectomy on female fertility
the bone marrow. Upon release into the bloodstream they The removal of the entire spleen, splenectomy, has been a
circulate and infiltrate inflamed tissues or are stored in successful surgical procedure for many hematological, im-
lymphoid organs, such as the spleen, for future activation munological, and traumatic conditions. However, although
and release. The spleen releases leukocytes following in- the removal is advantageous in treating the specific dis-
duction of acute inflammation in the heart by ischemic orders, it leaves the patient with a significant defect in both
myocardial injury [7,70,71]. Using a sophisticated ap- innate and adaptive immune responses. As a consequence,
proach that involved the transplantation of spleens from splenectomized patients have a 60–100-fold increased risk
GFP mice into wild-type mice, it was demonstrated that of sepsis [76]. The ovulatory consequences of splenectomy
spleen leukocytes infiltrate heart tissues during acute in humans, however, are difficult to assess because other
inflammation. This finding indicates that at least one role treatment regimens such as chemotherapy or radiotherapy
of the spleen is to act as an immediate supplier of leuko- that often accompany the splenectomy procedure also
cytes for tissues that experience acute inflammation. This result in severe damage to ovulatory function. However,
concept is supported by the fact that, upon stimulation by there are several animal studies that indicate a role of the
an inflammatory signal, the bone marrow takes days to spleen in ovarian function. In particular, studies in rodents
produce leukocytes whereas spleen leukocytes reach sites and rabbits show that splenectomy results in either a delay
of inflammation within minutes to hours [7,72,73]. in ovulation [77], aberrant corpus luteal function [78], or an
Upon ovulatory gonadotropin stimulation the ovary absence of leuteolysis [79,80].
experiences an acute inflammatory response. This inflam-
mation is different from responses to infectious insults Proposed method of communication between the HPO
because ovulatory inflammation is in response to a normal axis and the spleen
physiological event, LH stimulation. However, the nature How would the HPO axis communicate with spleen to
and sequence of the inflammatory events occurring in the induce spleen leukocyte release? Because leukocytes are
preovulatory ovary are essentially identical to those reac- released as early as one hour after ovulatory LH surge, LH
tions taking place at the sites of infectious inflammation. could directly stimulate spleen (Figure 1). However, this is
348

5. [()TD$FIG] Opinion Trends in Endocrinology and Metabolism September 2011, Vol. 22, No. 9
Hypothalamus Hypothalamus
Anterior pituitary
GnRH
?
Pituitary
Spleen
E2 LH
?
Leukocytes
Ovary
TRENDS in Endocrinology & Metabolism
Figure 1. Proposed interplay between the HPO axis and spleen. Preovulatory rise of E2 (estradiol) stimulates release of LH into the bloodstream. LH then triggers leukocyte
release from the spleen either by acting directly on leukocytes or through indirect effects on spleen tissues. Spleen leukocytes released into the bloodstream migrate to the
ovary in response to cytokines and chemokines that act as leukocyte attractants; the cells then enter the tissue through interactions between leukocyte receptors and
adhesion molecules on the endothelial cells.
[()TD$FIG]
White pulp
Spleen
MZ
Red pulp
Theca cells
Ovary
Granulosa cells
Key: Chemokine receptors Chemokines Leukocyte expressed CAM Endothelial cell expressed CAM
TRENDS in Endocrinology & Metabolism
Figure 2. Spleen leukocyte trafficking to the ovary. Spleen 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 spleen reservoir leukocytes into the bloodstream. Upon arriving at the ovary, LH-mediated upregulation 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 follicle-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. MZ, marginal zone.
349

6. Opinion Trends in Endocrinology and Metabolism September 2011, Vol. 22, No. 9
unlikely because the presence of LH receptors in the spleen Concluding remarks
is reported only in poultry [81], and the spleen does not The immune system is not only important for battling
express LH receptors in mammals [82]. Instead, an endo- foreign invaders but is also essential for female reproduc-
crine molecule(s) produced by the ovary in response to LH tion. We propose here that the spleen can bridge the
stimulation could travel to the spleen via the circulation immune and reproductive systems by serving as a reser-
and stimulate leukocyte release (Figure 1). In the case of voir for the leukocytes required for the inflammatory
the myocardial injury model, angiotensin (Ang) II was events that regulate ovulation. Rigorous collaborative
shown to have such activity [7]. It will be interesting to studies between the fields of immunology and reproductive
determine if Ang II has this role in the periovulatory biology will be required to validate this hypothesis and
release of spleen leukocytes. In support of the potential elucidate the interaction between the reproductive organs
role of angiotensin II, studies indicate that ovarian Ang II and the spleen.
synthesis and secretion increases immediately after ovu-
latory LH/hCG stimulation and ovulation is inhibited if Acknowledgments
We thank Dr. Thomas E. Curry for critical comments and Mr. Tom Dolan
PD123319 or Saralasin, Ang II receptor antagonists, are
for the artwork. This work was supported by National Institutes of
injected into superovulation-induced rabbits and rats, re- Health grant RO1HD052694 (to C.K.).
spectively [83–85]. Other candidate molecules that could
originate from the ovary and induce spleen leukocyte References
release include chemokines and cytokines that are pro- 1 Urman, B. and Yakin, K. (2006) Ovulatory disorders and infertility. J.
duced by the ovary upon LH stimulation. It will be inter- Reprod. Med. 51, 267–282
esting to determine whether receptors for these ovary- 2 Gibson, M. (1995) Reproductive health and polycystic ovary
syndrome. Am. J. Med. 98, 67S–75S
borne molecules are present in the spleen and if the 3 Mitwally, M.F. and Casper, R.F. (2006) Potential of aromatase
receptors are localized in leukocytes or spleen cells. It is inhibitors for ovulation and superovulation induction in infertile
however considered that the spleen leukocyte release women. Drugs 66, 2149–2160
could be a homeostatic response to the decrease in circu- 4 Cremer, M. et al. (2010) Recent innovations in oral contraception.
lating leukocytes because many of them infiltrate the Semin. Reprod. Med. 28, 140–146
5 Oakley, O.R. et al. (2010) Periovulatory leukocyte infiltration in the
ovary. Taken together, we propose the following path of rat ovary. Endocrinology 151, 4551–4559
spleen leukocyte trafficking to the ovary (Figure 2). The 6 Hedin, L. (2010) Invaders from the spleen: an unexpected origin of
leukocytes reside in steady-state equilibrium within the the leukocytes participating in ovulation. Endocrinology 151,
red pulp of the spleen where they are held via chemokine 4096–4099
7 Swirski, F.K. et al. (2009) Identification of splenic reservoir monocytes
receptors (CCRs). The LH surge could alter CCR expres-
and their deployment to inflammatory sites. Science 325, 612–616
sion on the leukocytes and/or induce chemokine expres- 8 Kitaya, K. and Yamada, H. (2010) Pathophysiological roles of
sion in the spleen endothelial cells, thereby stimulating chemokines in human reproduction: an overview. Am. J. Reprod.
mobilization of the leukocytes through the endothelial cell Immunol. 65, 449–459
layer of the blood vessel and into the circulation. Leuko- 9 Runesson, E. et al. (1996) The human preovulatory follicle is a source
of the chemotactic cytokine interleukin-8. Mol. Hum. Reprod. 2,
cytes circulate through the periphery until they reach the
245–250
ovary where an interaction with appropriate adhesion 10 Arici, A. et al. (1996) Interleukin-8 expression and modulation in
molecules on the endothelial cell wall occurs. Simulta- human preovulatory follicles and ovarian cells. Endocrinology 137,
neously, in the ovary the LH surge decreases blood flow by 3762–3769
dilating vessels and increasing the expression of leukocyte 11 Bukulmez, O. and Arici, A. (2000) Leukocytes in ovarian function.
Hum. Reprod. Update 6, 1–15
receptors such as adhesion molecules to promote the 12 Karaca, T. et al. (2008) Distribution and heterogeneity of mast cells in
interaction of leukocytes with receptors on ovarian endo- female reproductive tract and ovary on different days of the oestrus
thelial cells. cycle in Angora goats. Reprod. Domest. Anim. 43, 451–456
13 Smith, M.P. et al. (2005) Leukocyte origin and profile in follicular
Leukocytes in other reproductive organs aspirates at oocyte retrieval. Hum. Reprod. 20, 3526–3531
14 Bonello, N. et al. (2004) Periovulatory expression of intercellular
Leukocytes also play crucial roles in reproductive tissues adhesion molecule-1 in the rat ovary. Biol. Reprod. 71, 1384–1390
other than the ovary, including the uterus [86–88], pitui- 15 Rohm, F. et al. (2002) Correlation between expression of selectins and
tary [89,90], oviduct [91–93], testis [94], and vagina [95– migration of eosinophils into the bovine ovary during the
97]. Thus, implantation, pregnancy maintenance, embryo periovulatory period. Cell Tissue Res. 309, 313–322
16 Robker, R.L. et al. (2010) The inflammatory response at ovulation is
development, menstrual tissue shedding and many other
altered in ovaries of progesterone receptor null (PRKO) mice., In In
reproductive functions are regulated by leukocytes. With 43rd Annual Meeting of the Society for the Study of Reproduction,
the recent finding that the spleen is a reservoir for leuko- abstract #95
cytes that rapidly respond to perturbation, it is likely that 17 Simon, C. et al. (1994) Interleukin-1 receptor antagonist suppresses
the spleen could also serve as the leukocyte reservoir for human chorionic gonadotropin-induced ovulation in the rat. Biol.
Reprod. 51, 662–667
these reproductive tissues. In fact, we found a discrepancy
18 Langer, H.F. and Chavakis, T. (2009) Leukocyte–endothelial
between the numbers of leukocytes that leave the spleen interactions in inflammation. J. Cell. Mol. Med. 13, 1211–1220
upon LH stimulation and the combined increase of leuko- 19 Murayama, C. et al. (2010) Effect of VEGF (vascular endothelial
cyte numbers in the bloodstream and ovary [5], indicating growth factor) on expression of IL-8 (interleukin-8) IL-1beta and
that large numbers of spleen leukocytes migrate to other their receptors in bovine theca cells. Cell Biol. Int. 34, 531–536
20 Kawano, Y. et al. (2007) The effects of platelet-activating factor on the
organs – that could include uterus and oviduct where secretion of interleukin-8 and growth-regulated oncogene alpha in
increased leukocyte infiltration has been documented human immortalized granulosa cell line (GC1a). Am. J. Reprod.
[86,87,93,98]. Immunol. 58, 434–439
350

7. Opinion Trends in Endocrinology and Metabolism September 2011, Vol. 22, No. 9
21 Liptak, A.R. et al. (2005) Cooperative expression of monocyte 46 Van Lint, P. and Libert, C. (2007) Chemokine and cytokine processing
chemoattractant protein 1 within the bovine corpus luteum: by matrix metalloproteinases and its effect on leukocyte migration
evidence of immune cell–endothelial cell interactions in a coculture and inflammation. J. Leukoc. Biol. 82, 1375–1381
system. Biol. Reprod. 72, 1169–1176 47 Reisinger, K. et al. (2007) The gonadotropins: tissue-specific
22 Polec, A. et al. (2009) Cellular interaction regulates interleukin-8 angiogenic factors? Mol. Cell. Endocrinol. 269, 65–80
secretion by granulosa-lutein cells and monocytes/macrophages. 48 Chowdhury, M.W. et al. (2010) The expression of angiogenic growth
Am. J. Reprod. Immunol. 61, 85–94 factors and their receptors in ovarian follicles throughout the estrous
23 Dahm-Kahler, P. et al. (2009) Monocyte chemotactic protein-1 (MCP- cycle in the ewe. Theriogenology 73, 856–872
1), its receptor, and macrophages in the perifollicular stroma during 49 Stouffer, R.L. et al. (2007) Molecular control of ovulation and
the human ovulatory process. Fertil. Steril. 91, 231–239 luteinization in the primate follicle. Front. Biosci. 12, 297–307
24 Ujioka, T. et al. (1998) Interleukin-8 as an essential factor in the 50 Gargett, C.E. and Rogers, P.A. (2001) Human endometrial
human chorionic gonadotropin-induced rabbit ovulatory process: angiogenesis. Reproduction 121, 181–186
interleukin-8 induces neutrophil accumulation and activation in 51 Heissig, B. et al. (2010) Role of neutrophil-derived matrix
ovulation. Biol. Reprod. 58, 526–530 metalloproteinase-9 in tissue regeneration. Histol. Histopathol. 25,
25 Krensky, A.M. and Clayberger, C. (2009) Biology and clinical 765–770
relevance of granulysin. Tissue Antigens 73, 193–198 52 Guimera, M. et al. (2009) LH/HCG stimulation of VEGF and
26 Marcal, J.R. et al. (2010) T-helper cell type 17/regulatory T-cell adrenomedullin production by follicular fluid macrophages and
immunoregulatory balance in human radicular cysts and periapical luteinized granulosa cells. Reprod. Biomed. Online 18, 743–749
granulomas. J. Endod. 36, 995–999 53 Belotti, D. et al. (2008) Vascular endothelial growth factor stimulates
27 Riese, J. et al. (2004) Expression of interleukin-6 and monocyte organ-specific host matrix metalloproteinase-9 expression and
chemoattractant protein-1 by peritoneal sub-mesothelial cells ovarian cancer invasion. Mol. Cancer Res. 6, 525–534
during abdominal operations. J. Pathol. 202, 34–40 54 Channing, C.P. et al. (1980) Ovarian follicular and luteal physiology.
28 Yadav, A. et al. (2010) MCP-1: chemoattractant with a role beyond Int. Rev. Physiol. 22, 117–201
immunity: a review. Clin. Chim. Acta 411, 1570–1579 55 Bliss, S.P. et al. (2010) GnRH signaling, the gonadotrope and
29 Bornstein, S.R. et al. (2004) Cytokines and steroidogenesis. Mol. Cell. endocrine control of fertility. Front. Neuroendocrinol. 31, 322–340
Endocrinol. 215, 135–141 56 de la Iglesia, H.O. and Schwartz, W.J. (2006) Minireview: timely
30 Wong, K.H. et al. (2002) Expression, hormonal regulation, and cyclic ovulation: circadian regulation of the female hypothalamo–
variation of chemokines in the rat ovary: key determinants of the pituitary–gonadal axis. Endocrinology 147, 1148–1153
intraovarian residence of representatives of the white blood cell 57 Doufas, A.G. and Mastorakos, G. (2000) The hypothalamic–pituitary–
series. Endocrinology 143, 784–791 thyroid axis and the female reproductive system. Ann. N. Y. Acad. Sci.
31 Dahm-Kahler, P. et al. (2006) Monocyte chemotactic protein-1 in the 900, 65–76
follicle of the menstrual and IVF cycle. Mol. Hum. Reprod. 12, 1–6 58 Zeineh, K. et al. (2003) Possible modulators of IL-8 and GRO-alpha
32 Cohen, P.E. et al. (1997) Absence of colony stimulating factor-1 in production by granulosa cells. Am. J. Reprod. Immunol. 50, 98–103
osteopetrotic (csfmop/csfmop) mice disrupts estrous cycles and 59 Jokubkiene, L. et al. (2006) Assessment of changes in volume and
ovulation. Biol. Reprod. 56, 110–118 vascularity of the ovaries during the normal menstrual cycle using
33 Cohen, P.E. et al. (1999) Macrophages: important accessory cells for three-dimensional power Doppler ultrasound. Hum. Reprod. 21,
reproductive function. J. Leukoc. Biol. 66, 765–772 2661–2668
34 Van der Hoek, K.H. et al. (2000) Intrabursal injection of clodronate 60 Shimada, M. et al. (2007) Synaptosomal-associated protein 25 gene
liposomes causes macrophage depletion and inhibits ovulation in the expression is hormonally regulated during ovulation and is involved
mouse ovary. Biol. Reprod. 62, 1059–1066 in cytokine/chemokine exocytosis from granulosa cells. Mol.
35 Kunkel, E.J. et al. (2003) Chemokines in lymphocyte trafficking and Endocrinol. 21, 2487–2502
intestinal immunity. Microcirculation 10, 313–323 61 Son, D.S. and Roby, K.F. (2006) Interleukin-1alpha-induced
36 Youn, B.S. et al. (2002) Role of the CC chemokine receptor 9/TECK chemokines in mouse granulosa cells: impact on keratinocyte
interaction in apoptosis. Apoptosis 7, 271–276 chemoattractant chemokine, a CXC subfamily. Mol. Endocrinol. 20,
37 Zhou, C. et al. (2005) Transient expression of CC chemokine TECK in 2999–3013
the ovary during ovulation: its potential role in ovulation. Am. J. 62 Szlosarek, P.W. et al. (2006) Expression and regulation of tumor
Reprod. Immunol. 53, 238–248 necrosis factor alpha in normal and malignant ovarian epithelium.
38 Zhou, C. et al. (2009) Potential roles of a special CD8aa+ cell Mol. Cancer Ther. 5, 382–390
population and CC chemokine thymus-expressed chemokine in 63 Karstrom-Encrantz, L. et al. (1998) Selective presence of the
ovulation related inflammation. J. Immunol. 182, 596–603 chemokine growth-regulated oncogene alpha (GROalpha) in the
39 Foster, R. et al. (2010) A differential cytokine expression profile is human follicle and secretion from cultured granulosa-lutein cells at
induced by highly purified human menopausal gonadotropin and ovulation. Mol. Hum. Reprod. 4, 1077–1083
recombinant follicle-stimulating hormone in a pre- and postovulatory 64 Jasper, M.J. et al. (2000) Characterization of ovarian function in
mouse follicle culture model. Fertil. Steril. 93, 1464–1476 granulocyte-macrophage colony-stimulating factor-deficient mice.
40 Buchweitz, J.P. et al. (2007) Time-dependent airway epithelial and Biol. Reprod. 62, 704–713
inflammatory cell responses induced by influenza virus A/PR/8/34 in 65 Kluger, M.S. (2004) Vascular endothelial cell adhesion and signaling
C57BL/6 mice. Toxicol. Pathol. 35, 424–435 during leukocyte recruitment. Adv. Dermatol. 20, 163–201
41 Szklarczyk, A. and Conant, K. (2010) Matrix metalloproteinases, 66 Radi, Z.A. et al. (2001) Cell adhesion molecules, leukocyte trafficking,
synaptic injury, and multiple sclerosis. Front. Psychiatry 1, 12 and strategies to reduce leukocyte infiltration. J. Vet. Intern. Med. 15,
42 Jo, M. and Curry, T.E., Jr (2004) Regulation of matrix 516–529
metalloproteinase-19 messenger RNA expression in the rat ovary. 67 Korpos, E. et al. (2009) Multiple roles of the extracellular matrix in
Biol. Reprod. 71, 1796–1806 inflammation. Curr. Pharm. Des. 15, 1349–1357
43 Curry, T.E., Jr and Osteen, K.G. (2003) The matrix metalloproteinase 68 Weber, C. and Koenen, R.R. (2006) Fine-tuning leukocyte responses:
system: changes, regulation, and impact throughout the ovarian and towards a chemokine ‘interactome’. Trends Immunol. 27, 268–273
uterine reproductive cycle. Endocr. Rev. 24, 428–465 69 Pate, J.L. et al. (2010) The interface of the immune and reproductive
44 Duffy, D.M. and Stouffer, R.L. (2003) Luteinizing hormone acts systems in the ovary: lessons learned from the corpus luteum of
directly at granulosa cells to stimulate periovulatory processes: domestic animal models. Am. J. Reprod. Immunol. 64, 275–286
modulation of luteinizing hormone effects by prostaglandins. 70 Robbins, C.S. and Swirski, F.K. (2010) The multiple roles of monocyte
Endocrine 22, 249–256 subsets in steady state and inflammation. Cell. Mol. Life Sci. 67,
45 Fedorcsak, P. et al. (2010) Differential release of matrix 2685–2693
metalloproteinases and tissue inhibitors of metalloproteinases by 71 Leuschner, F. et al. (2010) Angiotensin-converting enzyme inhibition
human granulosa-lutein cells and ovarian leukocytes. prevents the release of monocytes from their splenic reservoir in mice
Endocrinology 151, 1290–1298 with myocardial infarction. Circ. Res. 107, 1364–1373
351

8. Opinion Trends in Endocrinology and Metabolism September 2011, Vol. 22, No. 9
72 Popovich, P.G. and Hickey, W.F. (2001) Bone marrow chimeric rats 100 Bowen, J.M. et al. (1999) Luteal regression in the normally cycling rat:
reveal the unique distribution of resident and recruited macrophages apoptosis, monocyte chemoattractant protein-1, and inflammatory
in the contused rat spinal cord. J. Neuropathol. Exp. Neurol. 60, cell involvement. Biol. Reprod. 60, 740–746
676–685 101 Hameed, A. et al. (1995) Perforin expression in human cell-mediated
73 Jovcic, G. et al. (1993) Acute sterile inflammation – correlation luteolysis. Int. J. Gynecol. Pathol. 14, 151–157
between cellular changes and extramedullary-produced regulators 102 Olson, K.K. and Townson, D.H. (2000) Prolactin-induced expression of
in vivo. Ann. Hematol. 66, 195–201 intercellular adhesion molecule-1 and the accumulation of monocytes/
74 Brannstrom, M. et al. (1993) Localization of leukocyte subsets in the macrophages during regression of the rat corpus luteum. Biol.
rat ovary during the periovulatory period. Biol. Reprod. 48, 277–286 Reprod. 62, 1571–1578
75 Godiska, R. et al. (1997) Human macrophage-derived chemokine 103 Olson, K.K. et al. (2001) Actions of prostaglandin F2alpha and
(MDC), a novel chemoattractant for monocytes, monocyte-derived prolactin on intercellular adhesion molecule-1 expression and
dendritic cells, and natural killer cells. J. Exp. Med. 185, 1595–1604 monocyte/macrophage accumulation in the rat corpus luteum. Biol.
76 Tracy, E.T. and Rice, H.E. (2008) Partial splenectomy for hereditary Reprod. 64, 890–897
spherocytosis. Pediatr. Clin. North Am. 55, 503–519 x 104 Nagaosa, K. et al. (2002) Determination of cell type specificity and
77 Matsuyama, S. et al. (1987) The critical period in which splenectomy estrous cycle dependency of monocyte chemoattractant protein-1
causes functional disorder of the ovary in adult rats. Endocrinol. Jpn. expression in corpora lutea of normally cycling rats in relation to
34, 849–855 apoptosis and monocyte/macrophage accumulation. Biol. Reprod. 67,
78 Saito, S. et al. (1988) Involvement of splenocytes in the control of 1502–1508
corpus luteum function in the rat. Endocrinol. Jpn. 35, 891–898 105 Bowen, J.M. et al. (1996) Prolactin-induced regression of the rat
79 Endo, T. and Kanayama, K. (1998) Effects of splenectomy on luteal corpus luteum: expression of monocyte chemoattractant protein-1
function in pseudopregnant rabbits. J. Int. Med. Res. 26, 93–97 and invasion of macrophages. Biol. Reprod. 54, 1120–1127
80 Nariai, K. et al. (1995) Effects of splenectomy on luteolysis in 106 Kuranaga, E. et al. (1999) Fas/Fas ligand system in prolactin-induced
pseudopregnant rabbits. J. Vet. Med. Sci. 57, 503–505 apoptosis in rat corpus luteum: possible role of luteal immune cells.
81 You, S. et al. (2000) Three different turkey luteinizing hormone Biochem. Biophys. Res. Commun. 260, 167–173
receptor (tLH-R) isoforms I: characterization of alternatively 107 Wu, R. et al. (2007) Ovarian leukocyte distribution and cytokine/
spliced tLH-R isoforms and their regulated expression in diverse chemokine mRNA expression in follicular fluid cells in women with
tissues. Biol. Reprod. 62, 108–116 polycystic ovary syndrome. Hum. Reprod. 22, 527–535
82 Leuschner, C. and Hansel, W. (2005) Targeting breast and prostate 108 Gaytan, F. et al. (1997) Role of prolactin in the regulation of
cancers through their hormone receptors. Biol. Reprod. 73, 860–865 macrophages and in the proliferative activity of vascular cells in
83 Yoshimura, Y. et al. (1994) Gonadotropin stimulates ovarian renin- newly formed and regressing rat corpora lutea. Biol. Reprod. 57,
angiotensin system in the rabbit. J. Clin. Invest. 93, 180–187 478–486
84 Pellicer, A. et al. (1988) Blockage of ovulation by an angiotensin 109 Brannstrom, M. and Enskog, A. (2002) Leukocyte networks and
antagonist. Science 240, 1660–1661 ovulation. J. Reprod. Immunol. 57, 47–60
85 Kuji, N. et al. (1996) Involvement of angiotensin II in the process of 110 Kucharski, J. and Jana, B. (2005) Immuno-endocrine mechanisms
gonadotropin-induced ovulation in rabbits. Biol. Reprod. 55, 984–991 connected with the creation of corpora lutea persistent in animal
86 Gomez-Lopez, N. et al. (2010) Invasion of the leukocytes into the fetal- ovaries. Pol. J. Vet. Sci. 8, 255–259
maternal interface during pregnancy. J. Leukoc. Biol. 88, 625–633 111 Gaytan, F. et al. (2003) Effects of indomethacin on ovarian leukocytes
87 Nagamatsu, T. and Schust, D.J. (2010) The contribution of during the periovulatory period in the rat. Reprod. Biol. Endocrinol.
macrophages to normal and pathological pregnancies. Am. J. 1, 26
Reprod. Immunol. 63, 460–471 112 Das, S. et al. (2008) Follicular fluid expression of alpha-defensins and
88 Christiaens, I. et al. (2008) Inflammatory processes in preterm and their role in ovulation. J. Assist. Reprod. Genet. 25, 83–87
term parturition. J. Reprod. Immunol. 79, 50–57 113 El-Nefiawy, N. et al. (2005) Role of prostaglandin E2 receptor
89 Barbieri, F. et al. (2007) Role of stromal cell-derived factor 1 (SDF1/ subtypes in ovarian follicle growth in the rat in vivo Correlation
CXCL12) in regulating anterior pituitary function. J. Mol. with interleukin-8 and neutrophils. Histol. Histopathol. 20, 825–831
Endocrinol. 38, 383–389 114 Fainaru, O. et al. (2010) CD56brightCD16– natural killer cells
90 Callewaere, C. et al. (2007) Chemokines and chemokine receptors in accumulate in the ovarian follicular fluid of patients undergoing in
the brain: implication in neuroendocrine regulation. J. Mol. vitro fertilization. Fertil. Steril. 94, 1918–1921
Endocrinol. 38, 355–363 115 Karaca, T. and Simsek, N. (2007) Effects of spirulina on the number of
91 Rodriguez-Martinez, H. et al. (2005) Boar spermatozoa in the oviduct. ovary mast cells in lead-induced toxicity in rats. Phytother. Res. 21,
Theriogenology 63, 514–535 44–46
92 Matsuda, H. et al. (1983) Tissue concentrations of eosinophilia in the 116 Gaytan, F. et al. (1991) Estrous cycle-related changes in mast cell
bovine oviduct and uterus of different stages of the oestrous cycle. Res. numbers in several ovarian compartments in the rat. Biol. Reprod. 45,
Vet. Sci. 34, 369–370 27–33
93 van Bogaert, L.J. et al. (1978) The percentage of granulocyte-like cells 117 Vogel, B. et al. (2005) Bovine eotaxin (CCL11) – an unusual member of
in human oviduct epithelium. Br. J. Obstet. Gynaecol. 85, 373–375 the eotaxin group – attracts eosinophils in vitro but is not responsible
94 Hedger, M.P. (1997) Testicular leukocytes: what are they doing? Rev. for eosinophilia in the ovary. Vet. Immunol. Immunopathol. 107,
Reprod. 2, 38–47 67–77
95 Chen, B. et al. (2005) Elastin metabolism in pelvic tissues: is it 118 Yuan, G.H. et al. (2001) Role of chemokines/chemokine receptor
modulated by reproductive hormones? Am. J. Obstet. Gynecol. 192, systems in cartilage degradation. Drug News Perspect. 14, 591–600
1605–1613 119 Skinner, M.K. et al. (2008) Regulation of granulosa and theca cell
96 Givan, A.L. et al. (1997) Flow cytometric analysis of leukocytes in the transcriptomes during ovarian antral follicle development. Mol.
human female reproductive tract: comparison of fallopian tube, Reprod. Dev. 75, 1457–1472
uterus, cervix, and vagina. Am. J. Reprod. Immunol. 38, 350–359 120 Eberlein, J. et al. (2010) Comprehensive assessment of chemokine
97 Hill, J.A. and Anderson, D.J. (1992) Human vaginal leukocytes and expression profiles by flow cytometry. J. Clin. Invest. 120, 907–923
the effects of vaginal fluid on lymphocyte and macrophage defense 121 Williams, I.R. (2006) CCR6 and CCL20: partners in intestinal
functions. Am. J. Obstet. Gynecol. 166, 720–726 immunity and lymphorganogenesis. Ann. N. Y. Acad. Sci. 1072,
98 Li, T.C. et al. (1990) Histological and clinical features of menstruation 52–61
induced by the antiprogestin mifepristone (RU486) compared to 122 Dewan, M.Z. et al. (2006) Stromal cell-derived factor-1 and CXCR4
menstruation occurring spontaneously. J. Obstet. Gynaecol. receptor interaction in tumor growth and metastasis of breast cancer.
(Lahore) 10, 411–414 Biomed. Pharmacother. 60, 273–276
99 Bowen, J.M. and Keyes, P.L. (1999) The proestrous prolactin surge is 123 Kalinkovich, A. et al. (2009) Blood-forming stem cells are nervous:
not the sole initiator of regressive changes in corpora lutea of normally direct and indirect regulation of immature human CD34+ cells by the
cycling rats. Biol. Reprod. 61, 1208–1215 nervous system. Brain Behav. Immun. 23, 1059–1065
352