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Neuroexcitatory Targets in the Female Reproductive System of the Nonhuman Primate (Macaca fascicularis)Health Canada, Toxicology Research Division, Ottawa, Ontario, Canada Correspondence: Address correspondence to: Santokh Gill, Health Canada, Toxicology Research Division, 251 Frederick Banting Dr, Tunney’s Pasture, Ottawa, Ontario, Canada K1A 0L2; e-mail:santokh_gill{at}hc-sc.gc.ca.
Glutamate receptors (GluRs) have been implicated in brain function and pathology. Their presence in peripheral tissues suggests a vital role in the pathophysiology of various organ systems. In earlier studies, the authors reported the differential distribution of ionotropic and metabotropic GluRs in neural and nonneural peripheral tissues of the rat. In this study, they investigated the presence and the localization of the GluRs in the reproductive organs of Macaca fascicularis. The data illustrate the presence of the GluR 2/3, metabotropic glutamate receptor 2/3, kainate 2, and N-methyl-D-aspartate receptor 1 (NMDAR 1). These are localized in the different structures of the ovaries, uterine cervix, myometrium, endometrium, and inflammatory cells. Smooth muscle of the myometrium and arterioles showed strong immunolabeling with anti-GluR 2/3 and, to a lesser intensity, with the other ionotropic glutamate receptor antibodies. NMDAR 1 showed the most widespread staining in all the structures. Mast cells showed strong immunolabeling with the anti-NMDA antibody. The demonstration and the differential expression of GluRs in the female reproductive system of nonhuman primate experimental models provide first evidence suggesting excitatory signaling in these tissues.
Key Words: glutamate receptors • female reproductive system • immunohistochemistry • nonhuman primate Macaca fascicularis Abbreviations: AMPA,
Glutamate and aspartate are the predominant excitatory neurotransmitters in the mammalian central nervous system (CNS). These 2 excitatory amino acids (EAAs) are found to be 1,000- to 10,000-fold higher concentrations than those of many other important neurotransmitters, including dopamine, serotonin, and acetylcholine. Although these EAAs are essential for central neural processing for cognition, memory, sensation, and movement, they are paradoxically also potent neurotoxins. A plethora of findings in the past 2 decades has provided direct and circumstantial evidence for abnormal neurotransmission due to glutamate (and its analogues) in the etiology and pathophysiology of syndromes and diseases such as epilepsy, stroke, schizophrenia, addiction, depression, anxiety, Alzheimer’s, Huntington’s, Parkinson’s, amyotrophic lateral sclerosis, and brain injury (Dawson et al., 1995; Kalariti et al., 2005). The EAAs are known to exert their physiologic action via 2 groups of glutamate receptors identified as (1) ionotropic glutamate receptors (iGluRs) and (2) metabotropic glutamate receptors (mGluRs). Ionotropic receptors contain integral cation-specific ion channels, and these are subdivided into (a) N-methyl-D-aspartate (NMDA), (b)  -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), and (c) kainate (Ka) receptors according to their selective agonists. Activation of these receptors leads to the opening of a group of ion channels that are typified by their different permeabilities to Na+, K+, and Ca+2 (Ferraguti and Shigemoto, 2006; Pinheiro and Mulle, 2006). Stimulation of these receptors underlies rapid glutamate-mediated excitatory synaptic transmission. The mGluRs are coupled to G-proteins and modulate the production of second messengers such as inositol phosphates and/or adenylate cyclase. The mGluRs function is predominantly the long-term aspects of cellular control operating via G proteins and several second-messenger systems. mGluRs are also subdivided into 3 groups based on agonist interactions and second messenger activation: (1) mGluR 1 and 5; (2) mGluR 2 and 3; and (3) mGluR 4, 6, 7, and 8 (Ferraguti and Shigemoto, 2006). There is growing evidence that the neurotoxic effects of EAAs are not limited to the brain. In peripheral tissues, there is a rich bed of nerve circuits, and many cells and tissues are capable of conducting excitatory impulses. We have recently reviewed the anatomical distribution of the glutamate receptors in peripheral tissues, their potential role, and the effects they can mediate (Gill et al., 2007; Gill and Pulido, 2001, 2005; Gill et al., 2000; Mueller et al., 2003). There is accumulating evidence indicating that GluRs also mediate excitatory neurotransmission in peripheral neural and nonneural tissues and that they are involved in various organ/tissue functions and pathologies (Gill and Pulido, 2001, 2005; Gill et al., 2000; Hayashi et al., 2003; Honoi et al., 2003). EAAs are known to exert a profound stimulatory effect on the reproductive axis of several mammals (Mahesh and Brann, 2005). Furthermore, EAA’s neurotransmission is an essential component of the neuroendocrine transmission line that regulates anterior pituitary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. The preovulatory surge of gonadotropin-releasing hormone (GnRH) is essential for mammalian reproduction. Glutamate and its glutamate receptors are found in all key hypothalamic nuclei known to be important for GnRH release (Mahesh and Brann, 2005). Glutamate has also been implicated in the critical processes of puberty, hormone pulsatility, and sexual behavior (Olney, 1994). The administration of glutamate, NMDA, or kainate has been shown to increase the LH release mediated through the stimulation of hypothalamic GnRH release. Although GluRs agonists stimulate GnRH secretion both in vivo and in vitro, it is unclear whether GnRH neurons respond directly to glutaminergic excitation. In an earlier study, we demonstrated that in the rat female reproductive system, GluRs have a unique distribution within each organ/cell types. Each antibody had a differential affinity to specific structures in the ovaries, the fallopian tubes, the cervix, the myometrium, and the endometrium (Gill and Pulido, 2001, 2005). In the ovary, the distribution of GluRs within the follicles varied with the stages of follicular maturation. To determine if similar preferential distributions exist in higher mammals, we investigated the cellular distribution of both mGluRs and iGluRs in the ovary, uterus, and fallopian tubes of the nonhuman primate (Macaca fascicularis).
Tissue Preparation and Immunohistochemistry Formalin-fixed archived tissues from 5 female Macaca fascicularis of various age groups were used from a previous study (Arnold et al., 2001). These tissues were embedded in paraffin, and sections (4–5 µm) were mounted on salinized slides. The sections were then deparaffinized and passed through a series of 100% ethanol. Slides were placed in 10 mM sodium citrate buffer (pH 6.0) and microwaved for antigen retrieval for two 3-minute periods at 450 W with gentle agitation (Kenmore 900 W or H2200, Energy Beam Science Inc, East Granby, CT). Microwave-treated sections were washed in phosphate-buffered saline (PBS) and blocked for endogenous avidin and biotin in a 0.5% hydrogen peroxide/100% ethanol solution. The slides were rinsed in 95% ethanol and then in running distilled water (Pulido et al., 2004). The slides were then processed for immunohistochemistry using the avidin-biotin method. To obtain an optimal antibody dilution, a series of antibody dilutions in the range from 0.0005 to 0.02 µg/µl were made in 15% normal swine serum (Pulido et al., 2004) for NMDAR 1, mGluR 2/3, GluR 2/3 (Chemicon International Inc, Temecula, CA) and Ka 2 (Upstate Biotechnology Inc, Lake Placid, NY). After washing in PBS, slides were then incubated at room temperature in biotinylated F(ab’) swine-anti-rabbit secondary antibody (Dako Canada, Inc, Mississauga, ON, Canada) diluted in 15% normal serum. These slides were again washed in PBS and followed by an incubation of 30 minutes at room temperature in streptavidin (1:200) complex (Dako Canada, Inc). Slides were washed in PBS and 50 mM Tris buffer and then treated with 3,’3-diaminobenzidinetetrachloride (DAB, 80 mg/72 ul of 30% hydrogen peroxide) in 400 ml 50 mM Tris buffer (pH 7.2). The slides were then rinsed in running tap water and counterstained with hematoxylin. Slides were rehydrated and coverslipped with Micromount medium (Surgipath Canada, Winnipeg, MB, Canada). For anatomical references, some sections were stained with Mayer’s hematoxylin and eosin Y (H&E, Armed Forces Institute of Pathology, Washington, DC). Photographs were taken with an Axiophot Zeiss microscope (Carl Zeiss Canada, Toronto, Canada) equipped with a digital camera (Gill et al., 2007; Pulido et al., 2004).
Controls
Sections stained with H&E were used for anatomical reference as depicted in the photographs (Figures 2C, 3 A,C,F). For immunohistochemistry, the antibodies used were selected as markers of specific glutamate receptor subunits including NMDAR 1, GluR 2/3, Ka 2, and mGluR 2/3. Results show specific anatomical localization of selected subtypes of iGluRs and mGluRs throughout the reproductive system (Table 1; Figures 1, 2, 3). The distribution of the stain was specific to each antibody, varied with each organ or tissue, and showed marked contrast between positive and negative structures, supporting the specificity of the stain. Furthermore, after peptide absorption (10:1 peptide-to-antibody ratio), there was no signal in the rat brain or the monkey reproductive tissues. When the primary antibody was substituted with the diluent containing 15% normal swine serum, no staining was observed.
Anti-NMDAR 1 showed the strongest stain intensity and the widest distribution in all the tissues tested from each animal. Histopathological evaluation of the ovary showed features that were consistent with animals during an active reproductive age. The most characteristic of these features are the constellation of follicles at different stages of maturation, active luteinization of the theca, and fully formed corpora lutea (Figure 1A–H). This figure illustrates the cellular distribution of anti-NMDAR 1, anti-mGluR 2/3, anti-Ka 2, and anti-GluR 2/3 to the different structures of the ovary. Affinity for the corpus luteum and lutein cells is clearly evident. The intensity of the stain varied with the antibody, being particularly strong with the anti-NMDAR1, anti-GluR 2/3, and anti-Ka 2, respectively. All, except for the anti-Ka 2, have moderate to strong affinity for primordial and primary follicles. Anti-NMDAR 1 and anti-mGluR 2/3 have strong immunolabeling for the oocyte, the theca, and granulosa cells. None of the antibodies stained the atretic follicles, corpora albicans, or the stroma. Anti-GluR 2/3 and anti-Ka 2, but not anti-NMDAR 1 or anti-mGluR 2/3, show strong immunolabeling of the smooth muscle in the wall of the arterioles within the stroma and the hilus of the ovary. Figure 2A–F shows the histopathology of the cervix with moderate to severe inflammation in the birth canal. Anti-GluR 2/3 bound to the smooth muscle of the wall of the cervix, myometrium, and arterioles (Figure 2 A,B). Anti-GluR 2/3 also showed strong affinity for inflammatory cells and mild affinity for the squamous epithelium (Figure 2D), whereas anti-NMADR 1 immunolabeling was evident in the inflammatory infiltrate and to lesser extent in the most superficial layers of the squamous epithelium. Intense immunostaining with anti-NMDAR 1 was seen in the basal layer of the squamous epithelium, areas of squamous metaplasia in the endocervical glands, and within the inflammatory infiltrate (Figures 2E–F and 3B). Squamous metaplasia of the endocervical glands was visualized by its strong staining with anti-NMDAR 1 in contrast to the mucus-secreting columnar epithelium (Figure 3B). Except for the areas of transition and squamous metaplasia, the endocervical glands and columnar epithelium of the endocervix remained unstained with any of the antibodies tested (Figure 3 A,B). Figure 3 (D and F) shows strong affinity for anti-NMDAR 1 within the foci of adenomyosis, the surrounding inflammatory infiltrate and the endometrial glands. The endometrium (Figure 3 E,F) shows secretory endometrial glands characteristic of the luteal phase of the menstrual cycle and consistent with the presence of corpora lutea in the ovary. The stroma of the endometrium shows mast cells with strong anti-NMDAR 1 immunolabeling (Figure 3F).
The histology of the ovary correlates strongly with the stages of the endometrium within the estrous cycle, as is expected for the protracted age of the animals. Previously, we showed the differential distribution of GluRs in the reproductive organs of male and female rats (Gill and Pulido, 2001). In this study, we illustrate that these receptors are similarly distributed in the reproductive organs of the female Macaca fascicularis monkey. Each receptor subtype has a differential and preferential distribution within the ovaries, the cervix, the myometrium, and the endometrium. As in our previous studies, the GluR subtype NMDAR 1 had the widest distribution. The distribution of GluRs in the ovaries varies according to the different stages of follicle maturation but showed preferential distribution to primordial, primary, and mature follicles including the oocyte. The immunolabeling is increasingly less apparent in follicles in stages of atresia. Ovaries from older monkeys show similar immmunolabeling compared to younger animals. However, in older animals, the age-related abundance of corpora albicans and atretic follicles that do not stain with any of the antibodies gives the overall impression of a more limited distribution. Luteal cells in the corpora lutea show binding to the antibodies tested, supporting the assumption of an active functional role. The oocyte of both rat and monkey are labeled by anti-NMDAR 1 and anti-mGluR 2/3 (Gill and Pulido, 2001). The affinity of the antibodies for the theca and granulosa cells within growing follicles is higher in the M. fascicularis than in the rat. All of the antibodies show strong to moderate affinity for these structures except for the Ka 2. Anti-GluR 2/3 and anti-Ka 2 showed strong affinity for the smooth muscle of the myometrium and arterioles, which seems to be more specific in the M. fascicularis than in the rat. In the Macaca fascicularis, anti-Ka 2 selectively stains the corpus luteum, but not the oocyte or follicles, whereas in the rat, anti-Ka 2 has an affinity for both the corpus luteum and the oocyte. These species’ differences may be attributed to their specific reproductive/estrous cycle.
The presence of GluRs in the follicles and the corpus luteum in the ovary of rats and monkeys suggests a possible female sex steroid-GluRs interaction. It is possible that the glutaminergic excitation system may play a role in the up- or down-regulation of estrogen and progesterone. Granulosa follicular cells in the mature follicule and lutein cells in the corpus luteum are known sources of progesterone, estrogen, and inhibin (Gartner and Hiatt, 2001). Estrogen and progesterone may either be up- or down-regulated by GluRs-gated chloride channels via allosteric sites. This interaction could influence the development of GluRs in the CNS, the reproductive organs, and other peripheral tissues responsive to steroidal hormones. For example, pregnane steroids (steroids that have sedative and neuroprotective effects), particularly 3 Our examination of the uterus reveals some interesting findings with functional and pathophysiological implications. These include the presence of the GluRs in the smooth muscle of the myometrium, wall of the cervix, and arterioles. The excitability and the hormonal response of these structures is an integral component of parturition. The presence of GluRs in the endometrium, particularly evident in the glandular epithelium during the luteal phase, suggests (a) a possible hormone receptor interplay and/or (b) a possible role in the implantation of the fertilized ovum. From the above, we hypothesized that GluRs play an important role in female reproductive functions, which include steroidal sex hormone regulation, ovulation, and excitability of the myometrium and the cervix. The presence of the glutamate receptors in the monkey suggests that they play an important role in the regulation of uterotubal and ovarian functions in the nonhuman primate. The findings described may have important therapeutic and toxicological implications. Pharmaceuticals and food chemicals that have affinity for these receptors can potentially modulate the reproductive functions and ovarian development. It is probable that the GluRs, like the GABA receptors, may play a major role during pregnancy. These receptors and possibly others could be involved in the modulation (suppression) of uterine contraction and dilations (Lan et al., 1990; Erdo, 1990). The differential distribution of GluRs in the reproductive organs and the placental hemorrhage, torsion, and uterine ruptures observed with the intra-abdominal delivery in pregnant sea lions that died from intoxication of domoic acid (Silvagni et al., 2005; Gulland, 1998; Scholin et al., 2000) suggest a cause-effect relationship. More recently, Tsibris et al. (2002) showed that GluR 2 was up-regulated in the leiomyomata relative to the myometrium. This up-regulation may be correlated with tumor progression (Maas et al., 2001). Studies by Sun et al. (1991) showed that MSG-treated rats showed sex-specific impairment in sexual behavior. Results illustrated that neonatal treatment resulted in severe and widespread neuron destruction in the basomedial hypothalamus of both sexes. In addition, the suprachiasmatic nucleus was affected only in male rats. In these animals, there was a decline in sexual behavior. These authors attributed the various sex-specific abnormalities to hypothalamic-adenohypophyseal dysfunction. However, the presence of glutamate receptors in the reproductive tissues could also explain these results. It has been shown that neurons are found in ovaries of rats, pigs, and nonhuman primates. They are also found in ovaries of humans ranging in age from 24 weeks of gestation to 10 months postpartum (Anesetti et al., 2001). The preferential distribution of glutamate receptors in the reproductive tissues opens new possibilities for therapeutic manipulation in reproductive endocrinology. Further investigation on the toxicity on EAAs and their impact on fertility and reproduction will provide further information for safety assessment of food and for the development of therapeutic products.
We would like to thank Dr. Doug Arnold for his generous donation of the monkey tissues; Peter Smyth, Ian Greer, and Joann Clausen for their technical expertise in the preparation of the slides; and Dr. M. Barker for collecting tissues during necropsies. In addition, we also thank Dr. G. Cooke for reading the preliminary draft of this article.
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This version was published on April
1, 2008 Toxicologic Pathology, Vol. 36, No. 3,
478-484 (2008)
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Abstract
Introduction
