The Rhesus Macaque (Macaca mulatta) Sperm Proteome

Mass spectrometry based proteomics has facilitated sperm composition studies in several mammalian species but no studies have been undertaken in non-human primate species. Here we report the analysis of the 1247 proteins that comprise the Rhesus macaque (Macaca mulatta) sperm proteome (termed the MacSP). Comparative analysis with previously characterized mouse and human sperm proteomes reveals substantial levels of orthology (47% and 40% respectively) and widespread overlap of functional categories based on Gene Ontology analyses. Approximately 10% of macaque sperm genes (113/1247) are significantly under-expressed in the testis as compared with other tissues, which may reflect proteins specifically acquired during epididymal maturation. Phylogenetic and genomic analyses of three MacSP ADAMs (A-Disintegrin and Metalloprotease proteins), ADAM18-, 20- and 21-like, provides empirical support for sperm genes functioning in non-human primate taxa which have been subsequently lost in the lineages leading to humans. The MacSP contains proteasome proteins of the 20S core subunit, the 19S proteasome activator complex and an alternate proteasome activator PA200, raising the possibility that proteasome activity is present in mature sperm. Robust empirical characterization of the Rhesus sperm proteome should greatly expand the possibility for targeted molecular studies of spermatogenesis and fertilization in a commonly used model species for human infertility.The application of mass spectrometry (MS) based proteomics, coupled with whole genome annotation of an increasing number of species, has greatly extended our knowledge of sperm composition. Traditional methods used to assess sperm composition, including the use of sperm-specific antibodies and 2D gel electrophoresis, have identified a limited number of sperm proteins. These traditional studies have been augmented in recent years by the use of high throughput and highly sensitive MS (shotgun proteomics) that have substantially increased the accuracy of peptide identification, resulting in a significant increase in proteome coverage. Indeed, advances in MS instrumentation, data acquisition, and the availability of genome annotations have, for example, increased sperm proteome coverage in Drosophila from 381 (1) to 1108 proteins (2) over a five year period.Two main MS based methodologies have been applied to study sperm composition, including (i) 2D PAGE followed by spot excision and MS and (ii) digestion of proteins, followed by MS/MS analysis of the resulting peptides (3). Although each method has its own advantages and disadvantages, a far greater level of proteome coverage is obtained using MS/MS (4). A previous comparative study found that each method identified proteins not found in the other and vice versa, and therefore it has been suggested that these methods should be used to complement each other (5). Thus, although no single methodology yet exists capable of producing a complete whole cell proteome, MS/MS methods provide deeper and broader coverage and are therefore the current method of choice. Shotgun proteomics has characterized sperm proteomes in a variety of taxa including plants, invertebrates and mammals such as human, mouse, rat, and bull (3, 6–11). These studies achieve varying levels of proteome coverage as a result of several factors including the choice of MS equipment, sample acquisition, purification, solublization, and fractionation schemes. Although these different approaches make direct comparisons difficult they nevertheless have provided invaluable information regarding the composition of sperm and have helped to identify novel proteins that play important roles in sperm function and reproduction.In this study we use MS based proteomics to elucidate the sperm proteome of a species of old world monkey, the Rhesus macaque (Macaca mulatta). Due primarily to their genetic and physiological similarities to humans, Rhesus macaques are the most widely used nonhuman primate model system for basic and applied biomedical research (12). Rhesus macaques are also used extensively as a model of human reproduction where numerous similarities at the molecular level have been observed between gametes of the two species, and why Rhesus macaques have become a useful model system for fertility and assisted reproductive technology research (13). A more complete knowledge of the sperm proteome will facilitate reproductive studies using the Rhesus macaque as a model organism. However, despite its widespread use in reproductive biology, the macaque sperm proteome (MacSP)¹ has yet to be characterized.Although insight into the MacSP will facilitate reproductive studies using the Rhesus macaque as a model organism, this knowledge can also be used to better understand the composition of human sperm. Sperm mature and gain fertilization competency as they traverse the epididymis, a specialized duct that connects the testis to the vas deferens (14). During the maturation process, sperm lose or modify a number of their surface proteins and gain additional transient or permanent surface proteins in a well-organized manner, and it is only after emerging from the cauda epididymis that sperm are motile and considered fertilization competent (14, 15).Proteomic studies of human sperm have been undertaken (3, 6, 10), identifying between 98–1760 sperm proteins, however these studies used sperm from ejaculates which complicates sperm proteome analysis. A previous study identified 923 proteins present in human seminal plasma (16), which is likely to be only a fraction of the seminal plasma proteome. Human sperm proteome data sets derived from human ejaculates makes it difficult to differentiate which of the identified proteins are sperm or seminal plasma constituents. For example, a major seminal protein family, the semenogelins are not expressed in the testis but are found in sperm proteomes determined from ejaculates (6, 10). Such highly abundant seminal proteins may mask lower abundance integral sperm proteins and inhibit their identification by MS. In order to avoid these problems, we collected mature sperm directly from the cauda epididymis of the Rhesus macaque, thus avoiding contamination from seminal plasma proteins.In the present study, sperm proteins were separated using 1D SDS-PAGE, digested and the resulting peptides analyzed by LC MS/MS. Using high stringency parameters for peptide identification, we conservatively identified 1247 proteins from purified samples of Rhesus macaque sperm. Given their close evolutionary relationship, the Rhesus macaque and human share 93% nucleotide homology (12). Data from this study can be used to complement what is currently known about the composition of human sperm and provides a more useful proxy of human sperm proteome composition than the proteomes of other non-primate mammals for which data is available. Studies of sperm composition, especially those in human, can be applied to develop novel molecular based clinical diagnostic tests of sperm quality, which is currently limited to evaluating parameters such as sperm count, morphology and motility. In addition, knowledge of sperm components can lead to the discovery of novel contraceptives and infertility treatments.     

Osteochondromatosis in a Rhesus macaque (Macaca mulatta)

A 5-y-old, male, rhesus macaque (Macaca mulatta) presented with a prominent mass slightly anteriomedial to the right stifle. On exam, multiple radiopaque masses were identified protruding from the mid- and distal femur. Lateral and anteroposterior radiographs of the right stifle region revealed multiple exophytic masses arising from the femur, with mild bony reaction of the proximal tibia. Histologic examination of biopsy tissue revealed woven and lamellar bone with granulation tissue and skeletal muscle. Because the macaque was exhibiting no lameness or signs of pain, we decided to monitor the progression of the masses. Minimal change was noted during the time prior to study termination at 6.5 y of age. Necropsy revealed that the bony masses were cartilage-capped lesions arising near the growth plate of the distal femur and midshaft of the femur and tibia. Histologic examination revealed chondro-osseous exophytic growths that blended imperceptibly with the cortex and spongiosa of the femur, consistent with a final diagnosis of multiple osteochondromas.     

A Hyper-Parasite of Iodamoeba buetschlii from the Rhesus Monkey, Macaca mulatta, and the Domestic Pig

SYNOPSIS. So called "inclusion" bodies in the cytoplasm of Iodamorba biretschlii cysts from rhesus monkeys ( Mocaca mulatta ) and domestic pips appeared to be hyper-parasites of unknown taxonomic position (posubly a protozoon or a fungus), They had a dot-like Feulgen-positive nucleus, cytoplasmic alkaline phosphatase, periodic acid Schiff -positive material, and proteins reacting to the bromophenol blue test. Their nuclei appeared to divide by budding and ultimately to produce six to eight minute, round or oval spore-like bodies. The Iodamoeba cysts mntaining these bodies had highly vacuolated cytoplasm, suggesting that the hyper-parasites had a degenerating effect on their host.     

Endometrial Decidualization and Deciduosis in Aged Rhesus Macaques (Macaca mulatta)

Superficial decidualization of the endometrial stroma is an essential feature of the implantation stage of pregnancy in rhesus macaques and other primates. Decidualization involves proliferation of the endometrial stromal cells, with differentiation into morphologically distinct decidual cells. Previous reports involving nonpregnant rhesus monkeys have described localized and widespread endometrial decidualization in response to administration of progesterone and synthetic progestogens. Ectopic decidua or ‘deciduosis’ describes the condition in which groups of decidual cells are located outside of the endometrium, most often in the ovaries, uterus and cervix but also in various other organs. In humans, most cases of deciduosis are associated with normal pregnancy, and ectopic decidua can be found in the ovary in nearly all term pregnancies. Here we describe pronounced endometrial decidualization in 2 rhesus macaques. Both macaques had been treated long-term with medroxyprogesterone acetate for presumed endometriosis, which was confirmed in one of the macaques at postmortem examination. In one animal, florid extrauterine and peritoneal serosal decidualization was admixed multifocally with carcinomatosis from a primary colonic adenocarcinoma. Cells constituting endometrial and serosal decidualization reactions were immunopositive for the stromal markers CD10, collagen IV, smooth muscle actin, and vimentin and immunonegative for cytokeratin. In contrast, carcinomatous foci were cytokeratin-positive. To our knowledge, this report describes the first cases of serosal peritoneal decidualization in rhesus macaques. The concurrent presentation of serosal peritoneal decidualization with carcinomatosis is unique.Abbreviations: GnRH, gonadotropin-releasing hormone; PAS, periodic acid–Schiff; SMA, smooth-muscle actinSuperficial decidualization of the endometrial stroma is an essential feature of the implantation stage of pregnancy in rhesus macaques and other primates.^13,27,29,37 This process typically begins, and is most prominent, adjacent to the spiral arteries, eventually expanding to affect the endometrium uniformly.³⁵ The endometrial stroma surrounds and supports the endometrial glands and is composed mainly of endometrial stromal cells and blood vessels.³⁵ Decidualization involves proliferation of the endometrial stromal cells, with differentiation into morphologically distinct decidual cells.^7,27,38 Endometrial stromal cells transform into large, polyhedral, cytoplasm-rich cells with large amounts of stored glycogen and are often binucleated or polyploid in character.^{6,13,27,30,35} Ultrastructurally, decidualized cells have numerous ribosomes, prominent rough endoplasmic reticulum and Golgi complexes, and cytoplasmic accumulation of glycogen and lipid droplets.^13,35 Consistent with their stromal origin, decidualized cells express mesenchymal immunohistochemical markers, such as vimentin, desmin, and muscle-specific actin.^{6,7,14,16,20,22}Initiation of decidualization by attachment of the blastocyst to the uterine epithelium depends on previous sensitization by progesterone secretion, after a brief priming by estrogen.^12,13,27 Estrogen and progesterone regulate a series of complex interactions at the interface between the developing embryo and the cells in the stromal compartment, leading to the formation of a differentiated maternal tissue (decidua) that supports embryo growth and maintains early pregnancy.²⁷ Postovulatory levels of circulating progesterone increase and help maintain the differentiation of decidual cells.^{7,13,33,37,38}Ectopic decidua or ‘deciduosis’ describes the condition in which groups of decidual cells reside outside of the endometrium, most often in the ovaries, uterus, and cervix; the fallopian tubes, peritoneum, omentum, diaphragm, liver, skin, spleen, appendix, abdominal–pelvic lymph nodes, renal pelvis, and lungs of women have also been reported as affected.^{6,14,18,20,22,28,29,38} In humans, most cases of deciduosis are associated with normal pregnancy, and ectopic decidua have been reported in the ovary in 90.5% to 100% of term pregnancies.^{6-8,14,20,22,28-30,38} Occasional cases in nonpregnant or postmenopausal women have been attributed to progesterone-secreting active corpora lutea, progesterone secretion by the adrenal cortex, trophoblastic disease, exogenous progestational agents, and pelvic irradiation.^{6-8,14,18,20,22,28,38} Deciduosis is usually an incidental finding that regresses postpartum within 4 to 6 wk; rarely, florid reactions have been reported to cause peritonitis, adhesions, hydronephrosis and hematuria, acute bowel obstruction or perforation (or both), abdominal pain mimicking appendicitis, massive and occasionally fatal hemoperitoneum, vaginal bleeding, and pneumothorax.^{6,7,14,18,20,22,28,29,31}Previous reports involving nonpregnant rhesus macaques have described localized and widespread endometrial decidualization in response to the administration of progesterone, synthetic progestogens, or progesterone-releasing bioactive intrauterine devices and intravaginal rings and have referred to these changes as ‘pseudodecidualization’ to indicate the absence of pregnancy in these animals.^12,33,35,37 In macaques given low (but superphysiologic) levels of progestogens, decidual changes have been noted in localized regions (around spiral arteries and underneath superficial epithelium), whereas high doses of progesterone or synthetic progestagens can cause a more pronounced and extensive reaction.³⁵In cynomolgus macaques, extrauterine decidual cell plaques are rare histologic findings in the subcoelomic mesenchyme of the ovarian cortex.^8,30 Despite the frequency of the condition in women, deciduosis is postulated to be a rarely documented lesion in primates because it is most often observed in conjunction with pregnancy, and pregnant cynomolgus macaques are seldom used in toxicity studies.⁸ Here we describe the pronounced endometrial decidualization of 2 rhesus macaques, one of which also had florid extrauterine and peritoneal decidualization that was admixed multifocally with carcinomatosis. Both macaques had been treated long-term with medroxyprogesterone acetate for presumed endometriosis, which was confirmed in one of the macaques at postmortem examination. To our knowledge, this report describes the first cases of peritoneal decidualization in rhesus macaques as well as the concurrent occurrence of carcinomatosis, endometriosis and peritoneal decidualization in a macaque. The extensive intermixing of the cell populations presented a diagnostic challenge at pathologic examination, and accurate diagnosis was achieved only through the use of multiple immunohistochemical markers.     

Purification and characterization of metallothionein from liver of cadmium exposed Rhesus monkeys (Macaca mulatta)

Metallothionein (MT) a low molecular weight, Cd-binding, cysteine rich, cytosolic protein has been isolated, purified and characterized from cadmium exposed Rhesus monkeys maintained on protein calorie malnourished (PCM) diet. Metallothionein was resolved into three isoforms i.e. MT_a, MT_b and MTV_c. The ratio of Cd, Zn and Cu varied in these isometallothioneins. MT_c was the major isometallothionein. UV Spectra of MT_c revealed the presence of mercaptide bonds and absence of aromatic amino acids. These observations were further confirmed by amino acid analysis of MT_c which demonstrated high cysteine content (22.6) followed by serine, glycine and lysine. The molecular weight of MT_c as determined by gel filtration and amino acid analysis was 13000 and 6398 daltons respectively. This demonstrates that MT_c is a non-globular ellipsoid polypeptide. MT_c showed a unique property of binding selenium. Monkey liver metallothionein was immunologically identical with human metallothionein. All the characteristics of MT_c obtained in the present study reveal a similarity between monkey and human metallothionein probably due to closer phylogenetic relationship between the two species.     

Intracranial Meningioma with Ophthalmoplegia in a Rhesus Macaque (Macaca mulatta)

A 21-y-old female rhesus macaque presented with signs of internal and external ophthamoplegia, including anisocoria and ptosis. Ophthalmoplegia is the paralysis or weakness of one or more intraocular or extraocular muscles that control the movement of eye; this condition can be caused by neurologic or muscle disorders. The macaque was euthanized due to progression of clinical symptoms, and postmortem gross examination revealed a mass at the base of the brain attached to the meninges. Histopathologic examination led to the diagnosis of intracranial meningioma. Here we describe a case of intracranial meningioma with internal and external ophthalmoplegia in a rhesus macaque (Macaca mulatta).Meningioma is a tumor that develops in the meninges, including the dura mater, arachnoid mater, and pia mater, all of which surround the brain and spinal cord. In humans, meningioma is the most frequently encountered primary intracranial neoplasia and accounts for approximately 35% of all reported primary brain tumors in the United States.^5,7 Clinical manifestations are dependent on the size and location of the tumor.^3,23 To our knowledge, only 3 cases of naturally occurring meningioma in nonhuman primates have been reported; these cases affected 2 baboons and 1 collared brown lemur.^17,19,21 The meningiomas of 2 of these previous cases were described only briefly as part of a disease survey of neoplasia in 100 free-ranging baboons and a captive prosimian population.^17,21 The third report focused on the pathologic features of the meningioma described with no mention of the clinical signs in the baboon, although its location was similar to that of the case we describe here.¹⁹In the current case report, we describe the clinical manifestations, postmortem examination, and diagnosis of an intracranial meningioma in a rhesus macaque (Macaca mulatta).     

MamuSNP: a resource for Rhesus Macaque (Macaca mulatta) genomics

We developed a novel method for identifying SNPs widely distributed throughout the coding and non-coding regions of a genome. The method uses large-scale parallel pyrosequencing technology in combination with bioinformatics tools. We used this method to generate approximately 23,000 candidate SNPs throughout the Macaca mulatta genome. We estimate that over 60% of the SNPs will be of high frequency and useful for mapping QTLs, genetic management, and studies of individual relatedness, whereas other less frequent SNPs may be useful as population specific markers for ancestry identification. We have created a web resource called MamuSNP to view the SNPs and associated information online. This resource will also be useful for researchers using a wide variety of Macaca species in their research.     

Metastatic Hepatocellular Carcinoma in a Juvenile Rhesus Macaque (Macaca mulatta)

Neoplasia in juvenile (younger than 5 y) rhesus macaques has been estimated to represent only approximately 1.4% of all occurrences of spontaneous neoplasia. Here we report an unusual case of a 3.75-y-old primiparous female rhesus macaque that was euthanized due to poor prognosis associated with progressive anemia, marked hepatomegaly, and radiographic evidence of metastatic neoplasia. Postmortem examination revealed an invasive, hemorrhagic hepatic mass that effaced approximately 70% of the liver parenchyma and had evidence of metastatic spread to multiple abdominal organs, the lungs, and the pituitary gland. Neoplastic polygonal cells lined large necrohemorrhagic cavities and exhibited marked anisocytosis and anisokaryosis, with frequent multinucleate cells. There was no desmoplasia associated with the primary neoplasm or metastases. Immunohistochemical studies revealed the neoplastic cells to be diffusely reactive with pancytokeratin, cytokeratin 7, and cytokeratin 8/18 antibodies and rarely reactive with carcinoembryonic antigen antibodies. The cells did not react with vimentin, S100, CD31, or factor VIII antibodies. Tumor morphology and immunophenotype led to the diagnosis of anaplastic hepatocellular carcinoma. This report represents the first known case of metastatic liver neoplasia in a rhesus macaque. The young age of this animal and the aggressive nature of the neoplasm are highly unusual and reminiscent of adolescent onset hepatocellular carcinoma in humans.Abbreviations: CK, cytokeratin; HCC, Hepatocellular carcinomaNeoplasia in juvenile rhesus macaques (Macaca mulatta) is extremely uncommon.^17,18 In a recent review of spontaneous neoplasia in 2 colonies of rhesus macaques, animals younger than 5 y represented only 1.4% of the total number of cases, and primary hepatic tumors were uncommon.¹⁸ All 5 of the cases detected among 2660 macaques involved animals between 14 and 26.8 y of age, and none had evidence of metastasis. Primary hepatic tumors appear to be similarly infrequent occurrences in other nonhuman primates, with the notable exception of prosimians, in which tumors arising from the liver are common spontaneous neoplasms.^3,15,17,18 Malignant liver tumors account for only 1% of pediatric tumors in humans.^5,7,12 Approximately 80% of these are hepatoblastomas—neoplasms arising from liver progenitor cells—and hepatocellular carcinoma (HCC) represents the second most frequent diagnosis.^5,7,12The medicine department of the California National Primate Research Center evaluated a 3.75-y-old, sexually mature, female rhesus macaque for rapidly progressive disease associated with a hepatic mass. Anaplastic HCC with extensive metastasis was diagnosed after postmortem examination. The current report describes the clinical progression of disease, the gross and microscopic pathology of the affected macaque, and the immunohistochemical characterization of the neoplasm.     

Acquired amegakaryocytic thrombocytopenia purpura in a Rhesus macaque (Macaca mulatta)

A 10-y-old multiparous rhesus macaque presented for an annual routine physical examination. Clinically, the animal had pale mucous membranes, petechial and ecchymotic hemorrhages in multiple sites, and a laceration at the tail base. Severe pancytopenia was noted on hematologic evaluation. The monkey was seronegative for SIV, simian T-lymphotropic virus, simian retrovirus type D, and Macacine herpesvirus 1. Bone marrow evaluation revealed a paucity of megakaryocytic precursors in a hypercellular marrow with marked erythroid hyperplasia. In light of these findings, the diagnosis was acquired amegakaryocytic thrombocytopenia purpura. Due to the poor prognosis of the syndrome and clinical deterioration of the monkey, euthanasia was elected. A definitive cause of the thrombocytopenia was not identified; however, the syndrome may have developed secondary to a recent spontaneous abortion. To our knowledge, this case represents the first reported observation of acquired amegakaryocytic thrombocytopenia purpura in a rhesus monkey.     

Alternative Activation of Macrophages in Rhesus Macaques (Macaca mulatta) with Endometriosis

Endometriosis is one of the most frequently encountered gynecologic diseases and a common cause of chronic pelvic pain and infertility. The pathophysiology of this syndrome can best be described as the presence of ectopic endometrium and a pelvic inflammatory process with associated immune dysfunction and alteration in the peritoneal environment. Macrophages play an important role in the progression and propagation of endometriosis. Alternative macrophage activation occurs in rodents and women with endometriosis but had not been examined previously in nonhuman primates. This case–control study aimed to characterize macrophage polarization in the ectopic and eutopic endometrial tissue of nonhuman primates with and without endometriosis. In addition, circulating cytokines in endometriosis cases and normal controls were investigated in an effort to identify serum factors that contribute to or result from macrophage polarization. Endometriosis lesions demonstrated increased infiltration by macrophages polarized toward the M2 phenotype when compared with healthy control endometrium. No serum cytokine trends consistent with alternative macrophage activation were identified. However, serum transforming growth factor α was elevated in macaques with endometriosis compared with healthy controls. Findings indicated that the activation state of macrophages in endometriosis tissue in nonhuman primates is weighted toward the M2 phenotype. This important finding enables rhesus macaques to serve as an animal model to investigate the contribution of macrophage polarization to the pathophysiology of endometriosis.Abbreviations: HLA, human leukocyte antigen; Iba1, ionized calcium binding adaptor molecule 1; M1, classically activated macrophage; M2, alternatively activated macrophage; sCD40L, soluble cluster of differentiation 40 ligand; TGF, transforming growth factor; VEGF, vascular endothelial growth factorEndometriosis is a common cause of chronic pelvic pain and infertility and affects more than 5.5 million women in North America alone.⁴¹ Although endometriosis is one of the most frequently encountered gynecologic health problems among women of reproductive age, the pathophysiology of this disease remains elusive due to its complexity and multifactorial etiology. The presence of functional endometrial glands and stroma outside the uterine cavity defines endometriosis. Currently, the most widely accepted theory for the origin of ectopic endometrial tissue is a combined effect of retrograde menstruation and associated implantation of endometrial fragments at an ectopic site. Progression of endometriosis lesions is thought to then be supported by peritoneal factors that allow cell adhesion and growth.⁴⁴ Although endometriosis is not a neoplastic disease, it exhibits aggressive features such as cellular proliferation, invasion, and vascular proliferation.¹² Strong evidence indicates that endometriosis involves a pelvic inflammatory process, with immune dysfunction and alteration in the peritoneal environment.^13,27 Numerous studies have demonstrated marked increases in macrophage populations and activity in the peritoneum of endometriosis patients.^6,54,59 Although macrophages are integral to homeostasis of the peritoneal environment, during endometriosis they mediate inflammation and facilitate the establishment and maintenance of the disease.Macrophages can be classified into 2 main populations: classically activated macrophages (M1), whose activating stimuli include IFNγ and LPS, and alternatively activated macrophages (M2), whose activating stimuli includes IL4, IL13, IL10, and transforming growth factor (TGF) β.⁵⁵ These polar phenotypes are not expressed together, but the activation state of tissue macrophages can change over time. This phenotypic switch is possible because macrophages retain plasticity, resulting in macrophage polarization that is transient and reversible.⁴⁰ A key component in determining the phenotype of the differentially activated macrophage is their response to microenvironmental signals, and this response allows for expression of a spectrum ranging from the M1 to M2 extremes.⁵¹ M1- and M2-activated macrophages perform different functions by producing pro- or antiinflammatory factors. M1 macrophages have enhanced endocytic functions and an enhanced ability to kill intracellular pathogens; they also secrete large amounts of proinflammatory cytokines such as IL1α, IL6, IL12, and TNFα.⁷ In contrast, M2 macrophages are involved in resolution of inflammation and promotion of tissue repair, and they secrete antiinflammatory and immunosuppressive cytokines including IL10 and TGFβ.³² M2 cells also express proangiogenic factors, such as coagulation factor XIII and vascular endothelial growth factor (VEGF) and have been associated with a high degree of vascularization in vivo.¹ The pathogenesis of endometriosis is therefore a likely combination of inappropriate or sustained polarization, leading to tissue damage (increased M1 response) and immune dysfunction (increased M2 response) and allowing for persistence of ectopic endometrial tissue.The use of animal models in endometriosis research is crucial. Work done with rodents involves the study of induced disease.⁵³ Despite this caveat, rodent models have been the basis for important contributions. Global macrophage depletion in a rat model of endometriosis effectively inhibits the initiation and growth of endometriosis implants.¹⁵ Attenuation of endometriosis has recently also been demonstrated in a mouse model of endometriosis.⁴ In that study, systemic depletion of macrophages was associated with failure of endometrial lesion development and defective angiogenesis of established lesions. Further evaluation of specific roles of differentially activated macrophages in that study⁴ showed that adoptive transfer of alternatively activated macrophages (M2) was associated with enhanced endometriosis progression. Conversely, adoptive transfer of inflammatory macrophages (M1) was associated with abrogated progression. In addition to evaluating murine lesions, the authors of the cited study⁴ investigated markers for alternative macrophage activation in women with endometriosis and matched controls which revealed increased expression of CD163 and CD206 (2 markers of M2 polarized macrophages) in endometriosis lesions as compared with disease-free peritoneum. Although many studies have been published about the pivotal role of macrophages in the pathophysiology of endometriosis, only a few have dealt with activation of the M1 and M2 macrophage phenotypes.^4,57 Furthermore, few studies have examined tissue infiltration of macrophages in eutopic endometrium of human subjects with endometriosis.^6,23 An exhaustive literature search failed to identify studies that investigate the role of M1 and M2 macrophage populations in eutopic endometrium.The current study uses rhesus macaques, which have been studied extensively in reproductive medicine.⁵⁸ Because spontaneous development of the disease requires menstrual shedding, endometriosis occurs naturally only in some nonhuman primate species, making development of lesions more comparable to the establishment of disease in humans.¹⁴ Compared with rodents, the nonhuman primate model of endometriosis is advantageous due to a close recapitulation of human disease and physiology. Work characterizing M1 and M2 macrophage activation in a species with spontaneous disease development may reflect a closer immunologic characterization to humans. In the current study, macrophage populations were evaluated in archival tissue collected from rhesus macaques with a diagnosis of endometriosis as confirmed by histologic examination. To characterize the phenotype of endometrial tissue macrophages in ectopic endometriosis lesions and eutopic endometrium of both cases and controls, immunohistochemistry was used to quantify cells expressing M1- and M2-specific markers. We hypothesized that endometriosis lesions and eutopic endometrium of rhesus macaques would be associated with a polarized macrophage infiltration consisting of increased numbers of M2 macrophages. This increase in M2 response may cause reduced immune clearance of ectopic endometrial cells, facilitating their implantation and growth. Further we speculated that M2 polarization would be associated with increased serum cytokines including IL10 and VEGF and decreased production of IL6, IL12, and TNFα. The lack of findings that support our hypotheses may suggest that the micro- or peritoneal environment is more important for lesion development or that another component of the systemic milieu is the determining factor in the development of endometriosis.     

Correlates of Male Consortship Rate in Free-Ranging Rhesus Macaques (Macaca mulatta)

Male–male competition for access to receptive females can take the form of nonrecurring fights and/or a sustained contest over mating opportunities. Male physical condition has been linked to dominance rank and reproductive success in species characterized by intrasexual fights for dominance and access to females. In group-living species characterized by endurance rivalry, however, factors contributing to male reproductive success are less well understood. In such species, particularly seasonally breeding ones with low sexual dimorphism and seniority-based rank, age and social factors other than rank may prove important. In the absence of genetic data, male mate guarding or consortship can serve as an indicator of male reproductive success. To evaluate the contribution of age and intragroup sociality to male consortship rate, I collected behavioral data during one nonmating and one mating season in two social groups of free-ranging rhesus macaques that experience no predation or food scarcity. Higher-ranking males, younger males, or males that exhibited lower rate of intrasexual aggression had higher consortship rates. Male–female dyads that groomed outside consortship did not form consortships as often as dyads that did not engage in nonconsort grooming. This is the first study to identify the significance of male–male aggression and male–female affiliation to male consortship rate in a species characterized by endurance rivalry, high male rank stability, and strong female mate choice. Social behaviors and male age may be particularly important in determining male reproductive success in populations experiencing high food availability and a lack of predation, which are typical of an increasing number of vertebrates in areas densely populated by humans.     

A Behavioral Taxonomy of Loneliness in Humans and Rhesus Monkeys (Macaca mulatta)

Social relationships endow health and fitness benefits, but considerable variation exists in the extent to which individuals form and maintain salutary social relationships. The mental and physical health effects of social bonds are more strongly related to perceived isolation (loneliness) than to objective social network characteristics. We sought to develop an animal model to facilitate the experimental analysis of the development of, and the behavioral and biological consequences of, loneliness. In Study 1, using a population-based sample of older adults, we examined how loneliness was influenced both by social network size and by the extent to which individuals believed that their daily social interactions reflected their own choice. Results revealed three distinct clusters of individuals: (i) individuals with large networks who believed they had high choice were lowest in loneliness, (ii) individuals with small social networks who believed they had low choice were highest in loneliness, and (iii) the remaining two groups were intermediate and equivalent in loneliness. In Study 2, a similar three-group structure was identified in two separate samples of adult male rhesus monkeys (Macaca mulatta) living in large social groups: (i) those high in sociability who had complex social interaction with a broad range of social partners (putatively low in loneliness), (ii) those low in sociability who showed tentative interactions with certain classes of social partners (putatively high in loneliness), and (iii) those low in sociability who interacted overall at low levels with a broad range of social partners (putatively low or intermediate in loneliness). This taxonomy in monkeys was validated in subsequent experimental social probe studies. These results suggest that, in highly social nonhuman primate species, some animals may show a mismatch between social interest and social attainment that could serve as a useful animal model for experimental and mechanistic studies of loneliness.     

Coat Color Variation and Pigmentation Gene Expression in Rhesus Macaques (Macaca mulatta)

Light-dark coat color variation is a common aspect of color diversity within and across mammalian taxa. This variation in pelage brightness is associated with aspects of evolutionary ecology, particularly for primates, but little is known about the genetic mechanisms underlying light-dark differences in pelage pigmentation. Previous work, focusing particularly on macaques (Genus Macaca), has found no clear relationship between color variation and coding sequences of key pigmentation genes. This suggests that other loci and/or gene regulatory differences underlie this variation and raises the question of how patterns of gene expression differ in light verses dark hair follicles. Here, we examine relative expression levels of pigmentation genes in hair follicles from free-ranging rhesus macaques (Macaca mulatta) showing stark light-dark coat color variation. We quantified the brightness (reflectance) of plucked hair tufts using a spectrophotometer. We extracted RNA from the follicles and used quantitative RT-PCR to measure the relative amounts of gene product (mRNA) for seven candidate pigmentation genes (MITF, MC1R, MGRN1, ATRN, SLC24A5, TYRP1, and DCT). Expression values were normalized with the house-keeping gene ACTB. All candidate genes were expressed at similar levels in dark, intermediate, and light hair, and thus, light-dark variation in macaque coat color is unlikely to be due to differences in the expression of these key pigmentation genes. This study represents the first examination of gene expression and natural color variation in a non-human primate population. Our results indicate that even in a system, like pigmentation, where a candidate-gene approach is promising, identifying important intra-specific gene regulatory differences remains challenging.