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1.  Essential role of ubiquitin C-terminal hydrolases UCHL1 and UCHL3 in mammalian oocyte maturation 
Journal of cellular physiology  2012;227(5):2022-2029.
Ubiquitin C-terminal hydrolases (UCHs) comprise a family of deubiquitinating enzymes that play a role in the removal of multi-ubiquitin chains from proteins that are posttranslationally modified by ubiquitination to be targeted for proteolysis by the 26S proteasome. The UCH-enzymes also generate free monomeric ubiquitin from precursor multi-ubiquitin chains and, in some instances, may rescue ubiquitinated proteins from degradation. This study examined the roles of two oocyte-expressed UCHs, UCHL1 and UCHL3 in murine and rhesus monkey oocyte maturation. The Uchl1 and Uchl3 mRNAs were highly expressed in GV and MII oocytes, and were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Microinjection of the UCH-family enzyme inhibitor, ubiquitin-aldehyde (UBAL) to GV oocytes prevented oocyte meiotic progression beyond metaphase I in a majority of treated oocytes and caused spindle and first polar body anomalies. Injection of antibodies against UCHL3 disrupted oocyte maturation and caused meiotic anomalies, including abnormally long meiotic spindles. A selective, cell permeant inhibitor of UCHL3, 4, 5, 6, 7-Tetrachloroidan-1, 3-dione also caused meiotic defects and chromosome misalignment. Cortical granule localization in the oocyte cortex was disrupted by UBAL injected after oocyte maturation. We conclude that the activity of oocyte UCHs contributes to oocyte maturation by regulating the oocyte cortex and meiotic spindle.
doi:10.1002/jcp.22931
PMCID: PMC4316209  PMID: 21751213
oocyte; ubiquitin; proteasome; UCH
2.  Binge drinking prior to pregnancy detection in a nonhuman primate; behavioral evaluation of offspring 
Background
Minimal scientific information is available to inform public health policy on binge drinking prior to pregnancy detection. The nonhuman primate provides a valuable animal model for examining consequences to reproduction and offspring function that may result from this common pattern of alcohol abuse.
Methods
Adult female rhesus monkeys were dosed with 1.5 g/kg-d ethanol by gavage two days/week beginning seven months prior to mating and continuing to pregnancy detection at 19–20 days gestation. Postnatal evaluation of control (n=6) and ethanol treated (n=4) infants included a neonatal neurobehavioral assessment, a visual paired comparison (cognitive) test at 35 days of age and mother-infant interaction at 100–112 days of age.
Results
Alcohol-exposed neonates did not differ from controls in posture and reflex measures. Longer durations of visual fixation, suggesting slower visual processing, and greater novelty preference were seen in the alcohol group. At early weaning age, as infants spent more time away from their dams, more of the reunions between mother and infant were initiated by the mothers in the alcohol-exposed group, suggesting a more immature mother-infant interaction.
Conclusion
Intermittent high dose alcohol exposure (binge drinking) discontinued at early pregnancy detection in rhesus monkey can result in altered behavioral function in the infant. Mediating effects on ovum, reproductive tract and early embryo can be explored in this model. Studies of longer-term consequences in human populations and animal models are needed.
doi:10.1111/acer.12267
PMCID: PMC4201374  PMID: 24164332
3.  Rhesus monkey sperm cryopreservation with TEST-yolk extender in the absence of permeable cryoprotectant☆ 
Cryobiology  2008;58(1):20-27.
Recently, there has been increased interest in ultra-rapid freezing with mammalian spermatozoa, especially for vitrification in the absence of cryoprotectants. Sperm cryopreservation in non-human primates has been successful, but the use of frozen–thawed sperm in standard artificial insemination (AI) remains difficult, and removal of permeable cryoprotectant may offer opportunities for increased AI success. The present study intended to explore the possibility of freezing rhesus monkey sperm in the absence of permeable cryoprotectants. Specifically, we evaluated various factors such as presence or absence of egg yolk, the percentage of egg yolk in the extenders, and the effect of cooling and thawing rate on the success of freezing without permeable cryoprotectants. Findings revealed that freezing with TEST in the absence of egg yolk offers little protection (<15% post-thaw motility). Egg yolk of 40% or more in TEST resulted in decreased motility, while egg yolk in the range of 20–30% yielded the most motile sperm. Cooling at a slow rate (29 °C/min) reduced post-thaw motility significantly for samples frozen with TEST-yolk alone, but had no effect for controls in the presence of glycerol. Similarly, slow thawing in room temperature air is detrimental for freezing without permeable cryoprotectant (<2% motility). In addition to motility, the ability of sperm to capacitate based on an increase in intracellular calcium levels upon activation with cAMP and caffeine suggested no difference between fresh and frozen–thawed motile sperm, regardless of treatment. In summary, the present study demonstrates that ejaculated and epididymal sperm from rhesus monkeys can be cryopreserved with TEST-yolk (20%) in the absence of permeable cryoprotectant when samples were loaded in a standard 0.25-mL straw, cooled rapidly in liquid nitrogen vapor at 220 °C/min, and thawed rapidly in a 37 °C water bath. This study also represents the first success of freezing without permeable cryoprotectant in non-human primates.
doi:10.1016/j.cryobiol.2008.09.014
PMCID: PMC4259290  PMID: 18992734
Freezing; Semen; Egg yolk; Epididymal sperm; Macaca mulatta; Non-human primate
4.  Nuclear maturation and structural components of nonhuman primate cumulus-oocyte complexes during in vivo and in vitro maturation 
Fertility and sterility  2008;91(5 0):2043-2050.
Study Objective
To compare cumulus cell structure and timing of oocyte maturation of in vitro and in vivo matured nonhuman primate oocytes.
Design
In vivo (VVM) and in vitro (IVM) maturation of oocytes.
Setting
Animal cell culture laboratory.
Animal(s)
48 female rhesus macaques.
Interventions
15 females were administered FSH and aspirated oocytes were cultured in vitro for 0, 3, 6, 12 or 24 hours (IVM). 33 females were administered FSH and hCG and oocytes were collected 3, 6, 12, or 28–30 hours following hCG (VVM).
Main Outcome Measures
Nuclear maturation and microtubule scores of oocytes and actin and tubulin transzonal processes of cumulus cells. Embryo development was observed for VVM oocytes.
Results
The rate of nuclear maturation was faster for IVM oocytes compared to VVM oocytes. Actin transzonal processes decreased 0 to 12 hours post hCG for VVM oocytes. Tubulin transzonal processes of IVM and VVM oocytes decreased from 0 to 24 hours and 0 to 3 hours respectively. Embryo development improved as VVM time increased.
Conclusions
Nuclear maturation and remodeling of cumulus oocyte complex structural components associated with IVM do not parallel that of oocyte maturation in vivo, indicating that in vitro culture conditions continue to be sub-optimal.
doi:10.1016/j.fertnstert.2008.05.013
PMCID: PMC4201365  PMID: 19108829
Rhesus monkey; tubulin; actin; zona pellucida; ovary; spindle
5.  Antioxidants, Oxyrase, and mitochondrial uncoupler 2, 4-dinitrophenol improved post-thaw survival of rhesus monkey sperm from ejaculates with low cryosurvival 
Fertility and sterility  2010;94(6):2359-2361.
Various antioxidant strategies such as supplementation of antioxidants, limiting oxygen concentration with Oxyrase, and reducing reactive oxygen species (ROS) through mild mitochondrial uncoupling had significant beneficial effects on sperm cryopreservation from rhesus monkeys with low cryoresistant ejaculates. Individuals or species that have higher sensitivity to cryodamage may derive the most benefit from these treatments.
doi:10.1016/j.fertnstert.2010.04.017
PMCID: PMC4201370  PMID: 20553783
Macaca mulatta; semen; reactive oxygen species; electron transport
6.  Bisphenol A and Reproductive Health: Update of Experimental and Human Evidence, 2007–2013 
Environmental Health Perspectives  2014;122(8):775-786.
Background: In 2007, an expert panel reviewed associations between bisphenol A (BPA) exposure and reproductive health outcomes. Since then, new studies have been conducted on the impact of BPA on reproduction.
Objective: In this review, we summarize data obtained since 2007, focusing on a) findings from human and animal studies, b) the effects of BPA on a variety of reproductive end points, and c) mechanisms of BPA action.
Methods: We reviewed the literature published from 2007 to 2013 using a PubMed search based on keywords related to BPA and male and female reproduction.
Discussion: Because BPA has been reported to affect the onset of meiosis in both animal and in vitro models, interfere with germ cell nest breakdown in animal models, accelerate follicle transition in several animal species, alter steroidogenesis in multiple animal models and women, and reduce oocyte quality in animal models and women undergoing in vitro fertilization (IVF), we consider it an ovarian toxicant. In addition, strong evidence suggests that BPA is a uterine toxicant because it impaired uterine endometrial proliferation, decreased uterine receptivity, and increased implantation failure in animal models. BPA exposure may be associated with adverse birth outcomes, hyperandrogenism, sexual dysfunction, and impaired implantation in humans, but additional studies are required to confirm these associations. Studies also suggest that BPA may be a testicular toxicant in animal models, but the data in humans are equivocal. Finally, insufficient evidence exists regarding effects of BPA on the oviduct, the placenta, and pubertal development.
Conclusion: Based on reports that BPA impacts female reproduction and has the potential to affect male reproductive systems in humans and animals, we conclude that BPA is a reproductive toxicant.
Citation: Peretz J, Vrooman L, Ricke WA, Hunt PA, Ehrlich S, Hauser R, Padmanabhan V, Taylor HS, Swan SH, VandeVoort CA, Flaws JA. 2014. Bisphenol A and reproductive health: update of experimental and human evidence, 2007–2013. Environ Health Perspect 122:775–786; http://dx.doi.org/10.1289/ehp.1307728
doi:10.1289/ehp.1307728
PMCID: PMC4123031  PMID: 24896072
7.  Prenatal exposure to bisphenol A impacts midbrain dopamine neurons and hippocampal spine synapses in non-human primates 
Neurotoxicology  2013;35:113-120.
Prevalent use of bisphenol-A (BPA) in the manufacture of resins, plastics and paper products has led to frequent exposure of most people to this endocrine disruptor. Some rodent studies have suggested that BPA can exert detrimental effects on brain development. However as rodent models cannot be relied on to predict consequences of human exposure to BPA during development, it is important to investigate the effects of BPA on non-human primate brain development. Previous research suggests that BPA preferentially targets dopamine neurons in ventral mesencephalon and glutamatergic neurons in hippocampus, so the present work examined the susceptibility of these systems to low dose BPA exposure at the fetal and juvenile stages of development in non-human primates. Exposure of pregnant rhesus monkeys to relatively low levels of BPA during the final 2 months of gestation, induced abnormalities in fetal ventral mesencephalon and hippocampus. Specifically, light microscopy revealed a decrease in tyrosine hydroxylase-expressing (dopamine) neurons in the midbrain of BPA-exposed fetuses and electron microscopy identified a reduction in spine synapses in the CA1 region of hippocampus. In contrast, administration of BPA to juvenile vervet monkeys (14–18 months of age) was without effect on these indices, or on dopamine and serotonin concentrations in striatum and prefrontal cortex, or on performance of a cognitive task that tests working memory capacity. These data indicate that BPA exerts an age-dependent detrimental impact on primate brain development, at blood levels within the range measured in humans having only environmental contact with BPA.
doi:10.1016/j.neuro.2013.01.001
PMCID: PMC3660149  PMID: 23337607
Bisphenol-A; Dopamine; Hippocampus; Prenatal; Spine synapse; Substantia nigra
8.  Maternal Bisphenol A Exposure Impacts the Fetal Heart Transcriptome 
PLoS ONE  2014;9(2):e89096.
Conditions during fetal development influence health and disease in adulthood, especially during critical windows of organogenesis. Fetal exposure to the endocrine disrupting chemical, bisphenol A (BPA) affects the development of multiple organ systems in rodents and monkeys. However, effects of BPA exposure on cardiac development have not been assessed. With evidence that maternal BPA is transplacentally delivered to the developing fetus, it becomes imperative to examine the physiological consequences of gestational exposure during primate development. Herein, we evaluate the effects of daily, oral BPA exposure of pregnant rhesus monkeys (Macaca mulatta) on the fetal heart transcriptome. Pregnant monkeys were given daily oral doses (400 µg/kg body weight) of BPA during early (50–100±2 days post conception, dpc) or late (100±2 dpc – term), gestation. At the end of treatment, fetal heart tissues were collected and chamber specific transcriptome expression was assessed using genome-wide microarray. Quantitative real-time PCR was conducted on select genes and ventricular tissue glycogen content was quantified. Our results show that BPA exposure alters transcription of genes that are recognized for their role in cardiac pathophysiologies. Importantly, myosin heavy chain, cardiac isoform alpha (Myh6) was down-regulated in the left ventricle, and ‘A Disintegrin and Metalloprotease 12’, long isoform (Adam12-l) was up-regulated in both ventricles, and the right atrium of the heart in BPA exposed fetuses. BPA induced alteration of these genes supports the hypothesis that exposure to BPA during fetal development may impact cardiovascular fitness. Our results intensify concerns about the role of BPA in the genesis of human metabolic and cardiovascular diseases.
doi:10.1371/journal.pone.0089096
PMCID: PMC3934879  PMID: 24586524
9.  Bisphenol A Exposure Alters Developmental Gene Expression in the Fetal Rhesus Macaque Uterus 
PLoS ONE  2014;9(1):e85894.
Bisphenol A (BPA) exposure results in numerous developmental and functional abnormalities in reproductive organs in rodent models, but limited data are available regarding BPA effects in the primate uterus. To determine if maternal oral BPA exposure affects fetal uterine development in a non-human primate model, pregnant rhesus macaques carrying female fetuses were exposed orally to 400 µg/kg BPA or vehicle control daily from gestation day (GD) 50–100 or GD100–165. Fetal uteri were collected at the completion of treatment (GD100 or GD165); tissue histology, cell proliferation, and expression of estrogen receptor alpha (ERα) and progesterone receptor (PR) were compared to that of controls. Gene expression analysis was conducted using rhesus macaque microarrays. There were no significant differences in histology or in the percentage of cells expressing the proliferation marker Ki-67, ERα, or PR in BPA-exposed uteri compared to controls at GD100 or GD165. Minimal differences in gene expression were observed between BPA-exposed and control GD100 uteri. However, at GD165, BPA-exposed uteri had significant differences in gene expression compared to controls. Several of the altered genes, including HOXA13, WNT4, and WNT5A, are critical for reproductive organ development and/or adult function. We conclude that second or third trimester BPA exposure does not significantly affect fetal uterus development based on morphological, proliferation, and steroid hormone receptor assessments. However, differences in expression of key developmental genes after third trimester exposure suggest that BPA could alter transcriptional signals influencing uterine function later in life.
doi:10.1371/journal.pone.0085894
PMCID: PMC3900442  PMID: 24465770
10.  Fetal Exposure of Rhesus Macaques to Bisphenol A Alters Cellular Development of the Conducting Airway by Changing Epithelial Secretory Product Expression 
Environmental Health Perspectives  2013;121(8):912-918.
Background: Bisphenol A (BPA) exposure early in life results in organizational changes in reproductive organs, but the effect of BPA on conducting airway cellular maturation has not been studied. Late gestation is characterized by active differentiation of secretory cells in the lung epithelium.
Objective: We evaluated the hypothesis that BPA exposure disrupts epithelial secretory cell development in the fetal conducting airway of the rhesus macaque.
Methods: We exposed animals to BPA during either the second (early term) or the third (late term) trimester. There were four treatment groups: a) sham control early term, b) sham control late term, c) BPA early term (BPA-early), and d) BPA late term (BPA-late). Because cellular maturation occurs nonuniformly in the lung, we defined mRNA and protein expression by airway level using microdissection.
Results: BPA exposure of the dam during late term significantly accelerated secretory cell maturation in the proximal airways of the fetus; both Clara cell secretory protein (CCSP) and MUC5AC/5B mRNA and protein expression increased.
Conclusions: BPA exposure during late gestation accelerates secretory cell maturation in the proximal conducting airways. We identified a critical window of fetal susceptibility for BPA effects on lung epithelial cell maturation in the third trimester. This is of environmental health importance because increases in airway mucins are hallmarks of a number of childhood lung diseases that may be affected by BPA exposure.
doi:10.1289/ehp.1206064
PMCID: PMC3734491  PMID: 23757601
CC10; CC16; lung development; Macaca mulatta; mucin; prenatal; SCGB1A1
11.  A specific family of interspersed repeats (SINEs) facilitates meiotic synapsis in mammals 
Background
Errors during meiosis that affect synapsis and recombination between homologous chromosomes contribute to aneuploidy and infertility in humans. Despite the clinical relevance of these defects, we know very little about the mechanisms by which homologous chromosomes interact with one another during mammalian meiotic prophase. Further, we remain ignorant of the way in which chromosomal DNA complexes with the meiosis-specific structure that tethers homologs, the synaptonemal complex (SC), and whether specific DNA elements are necessary for this interaction.
Results
In the present study we utilized chromatin immunoprecipitation (ChIP) and DNA sequencing to demonstrate that the axial elements of the mammalian SC are markedly enriched for a specific family of interspersed repeats, short interspersed elements (SINEs). Further, we refine the role of the repeats to specific sub-families of SINEs, B1 in mouse and AluY in old world monkey (Macaca mulatta).
Conclusions
Because B1 and AluY elements are the most actively retrotransposing SINEs in mice and rhesus monkeys, respectively, our observations imply that they may serve a dual function in axial element binding; i.e., as the anchoring point for the SC but possibly also as a suppressor/regulator of retrotransposition.
doi:10.1186/1755-8166-6-1
PMCID: PMC3545902  PMID: 23276256
Meiosis; Synaptonemal complex; Chromatin Immunoprecipitation (ChIP); SINE; Synapsis; SYCP3; Mouse; Macaque
12.  The efficacy of ultrasound treatment as a reversible male contraceptive in the rhesus monkey 
Background
The use of therapeutic ultrasound as a contraceptive approach has involved nonhuman primates as well as rats and dogs. The current study was undertaken to determine whether this treatment could be a method for reversible contraception, using a model with testes size similar to adult humans.
Methods
Two methods of ultrasound exposure were used, either the transducer probe at the bottom of a cup filled with saline (Cup) or direct application to the surface of the scrotum (Direct). Four adult rhesus (Macaca mulatta) males with normal semen parameters were treated with therapeutic ultrasound at 2.5 W/cm(2) for 30 min. Treatment was given 3 times, one every other day on a Monday-Wednesday-Friday schedule. For each male, semen quality was evaluated a minimum of three times over several months prior to ultrasound exposure and weekly for two months following ultrasound treatment.
Results
Semen samples from all males, regardless of exposure method, exhibited a decrease in the percentage of motile sperm following ultrasound treatment. There was an average reduction in motility of 40% the week following treatment. Similarly, curvilinear velocity and the percentage of sperm with a normally shaped flagellum were also reduced in all males following ultrasound treatment. A significant reduction in the total number of sperm in an ejaculate (total sperm count) was only observed in males that received ultrasound via the cup method. Following treatment via the cup method, males exhibited up to a 91.7% decrease in average total sperm count (n = 2). Sperm count did not approach pre-treatment levels until 8 weeks following ultrasound exposure.
Conclusions
The sustained reduction in sperm count, percent motility, normal morphology, and sperm vigor with the cup exposure method provides proof of principle that testicular treatment with ultrasound can be an effective contraceptive approach in humans.
doi:10.1186/1477-7827-10-81
PMCID: PMC3447693  PMID: 22971106
Sperm morphology; Motility; Contraception; Testes; Male reproduction
13.  Ethanol, Acetaldehyde and Estradiol Affect Growth and Differentiation of Rhesus Monkey Embryonic Stem Cells 
Background
The timing of the origins of fetal alcohol syndrome have been difficult to determine, in part because of the challenge associated with in vivo studies of the peri-implantation stage of embryonic development. Because embryonic stem cells (ESCs) are derived from blastocyst stage embryos, they are used as a model for early embryo development.
Methods
Rhesus monkey ESC lines (ORMES-6 and -7) were treated with 0, 0.01%, 0.1%, or 1.0% ethanol, 1.0% ethanol with estradiol or 0.00025% acetaldehyde with or without estradiol for 4 weeks.
Results
Although control ESCs remained unchanged, abnormal morphology of ESCs in the ethanol and acetaldehyde treatment groups was observed before 2 weeks of treatment. Immunofluorescence staining of key pluripotency markers ( TRA-1- 81 and alkaline phosphatase) indicated a loss of ESC pluripotency in the 1.0% ethanol group. ORMES-7 was more sensitive to effects of ethanol than ORMES-6.
Conclusions
Estradiol appeared to increase sensitivity to ethanol in the ORMES-6 and -7 cell line. The morphological changes and labeling for pluripotency, proliferation and apoptosis demonstrated that ethanol affects how these early cells develop in culture, their differentiation state in particular. The effects of ethanol may be mediated in part through metabolic pathways regulating acetaldehyde formation, and while potentially accentuated by estradiol in some individuals, how remains to be determined.
doi:10.1111/j.1530-0277.2011.01490.x
PMCID: PMC3139714  PMID: 21438889
Primate; fetal alcohol syndrome; embryo development
14.  The primate preimplantation embryo is a target for relaxin during early pregnancy 
Fertility and sterility  2011;96(1):203-207.
OBJECTIVE
To determine if preimplantation embryos are targets for relaxin secreted from the corpus luteum of the menstrual cycle.
DESIGN
Rhesus monkey oocytes obtained from females undergoing controlled ovarian hyperstimulation were inseminated and the resulting embryos were cultured in medium with or without recombinant human relaxin (20 ng/ml) for 8 days.
SETTING
Research laboratory.
ANIMALS
Rhesus monkey.
INTERVENTIONS
Controlled ovarian stimulation to obtain oocytes for in vitro produced embryos that were cultured with or without human recombinant relaxin.
MAIN OUTCOME MEASURES
The rate of blastocyst development and the percentage of blastocysts and ICM/TE ratio were measured on Day 8 of culture. The presence of relaxin receptor (RXFP1) mRNA in 8 cell embryos was observed by array hybridization.
RESULTS
RXFP1 receptor expression was localized to the inner cell mass of blastocysts as shown by immunohistochemistry. The percentage of embryos that developed to blastocyst and the inner cell mass/ trophectoderm cell ratio was unchanged with relaxin supplementation, however the relaxin-treated embryos developed into blastocysts significantly sooner than untreated embryos.
CONCLUSIONS
These results are the first evidence that the preimplantation primate embryo is a target for relaxin and that the addition of relaxin to in vitro culture medium enhances rhesus monkey embryo development.
doi:10.1016/j.fertnstert.2011.05.016
PMCID: PMC3129389  PMID: 21645893
gene expression; granulosa cells; blastocyst; rhesus macaque
15.  Maturation and Fertilization of Non-Human Primate Oocytes are Compromised by Oral Administration of a COX2 Inhibitor 
Fertility and sterility  2011;95(4):1256-1260.
Objective
To determine if oral administration of a cyclooxygenase-2 (COX2) inhibitor affects oocyte nuclear maturation and fertilization in non-human primates.
Design
Laboratory research study.
Setting
Medical school.
Animals
Adult female cynomolgus monkeys (Macaca fascicularis).
Interventions
Monkeys received gonadotropins to stimulate multiple follicular development. An ovulatory dose of human chorionic gonadotropin (hCG) was administered either alone or concomitant with oral celecoxib, a COX2 inhibitor; oocytes were retrieved 36 hours later and exposed to sperm in vitro.
Main Outcome Measures
Oocytes were assessed for nuclear status at retrieval, resumption of meiosis in vitro, and success of in vitro fertilization.
Results
Treatment with hCG alone yielded oocytes which were primarily at the meiosis II (MII) stage of nuclear maturation (72.9%); few oocytes were obtained at the germinal vesicle (GV) and germinal vesicle break down (GVBD) stages. Treatment with hCG and celecoxib yielded fewer mature (MII) oocytes (35.6%) and more oocytes at less mature stages when compared to oocytes from monkeys treated with hCG alone. The majority (68.3±15.9%) of MII oocytes from monkeys treated with hCG alone fertilized in vitro, compared with only 11.0±5.9% of MII oocytes from monkeys treated with hCG and celecoxib.
Conclusions
Oral administration of a COX2 inhibitor reduced the rate of oocyte nuclear maturation and the success of in vitro fertilization. Drugs of this class may block multiple essential steps in female reproduction and be effective contraceptives for women.
doi:10.1016/j.fertnstert.2010.12.048
PMCID: PMC3053529  PMID: 21236424
Monkey; Prostaglandin; Contraception; Ovary
16.  Ontological aspects of pluripotency and stemness gene expression pattern in the rhesus monkey 
Gene expression patterns : GEP  2011;11(3-4):285-298.
Two essential aspects of mammalian development are the progressive specialization of cells toward different lineages, and the maintenance of progenitor cells that will give rise to the differentiated components of each tissue and also contribute new cells as older cells die or become injured. The transition from totipotentiality to pluripotentiality, to multipotentiality, to monopotentiality, and then to differentiation is a continuous process during development. The ontological relationship between these different stages is not well understood. We report for the first time an ontological survey of expression of 45 putative “stemness” and “pluripotency” genes in rhesus monkey oocytes and preimplantation stage embryos, and comparison to the expression in the inner cell mass, trophoblast stem cells, and a rhesus monkey (ORMES6) embryonic stem cell line. Our results reveal that some of these genes are not highly expressed in all totipotent or pluripotent cell types. Some are predominantly maternal mRNAs present in oocytes and embryos before transcriptional activation, and diminishing before the blastocyst stage. Others are well expressed in morulae or early blastocysts, but are poorly expressed in later blastocysts or ICMs. Also, some of the genes employed to induce pluripotent stem cells from somatic cells (iPS genes) appear unlikely to play major roles as stemness or pluripotency genes in normal embryos.
doi:10.1016/j.gep.2011.02.001
PMCID: PMC3109727  PMID: 21329766
stem cell; cell lineage; embryo; trophoblast
17.  Frozen-thawed rhesus sperm retain normal morphology and highly progressive motility but exhibit sharply reduced efficiency in penetrating cervical mucus and hyualuronic acid gel 
Cryobiology  2010;62(1):15-21.
The preservation of the genetic diversity of captive populations of rhesus monkeys is critical to the future of biomedical research. Cryopreservation of rhesus macaque sperm is relatively simple to perform, yields high post-thaw motility, and theoretically, provides via artificial insemination (AI) a way to easily transfer genetics among colonies of animals. In the interest of optimizing semen cryopreservation methods for use with vaginal AI, we evaluated the ability of frozen-thawed rhesus sperm to penetrate periovulatory cervical mucus (CM). Motile sperm concentration of pre–freeze (“fresh”) and post-thawed (“thawed”) samples from 5 different males were normalized for both computer assisted sperm motion analysis and CM penetration experiments. Sperm samples were deposited into slide chambers containing CM or gel composed of hyaluronic acid (HA) as a surrogate for CM and numbers of sperm were recorded as they entered a video field a preset distance from the sperm suspension-CM (or HA) interface. Fresh and thawed sperm were dried on glass slides, “Pap”-stained, and assessed for changes in head dimensions and head and flagellar shape. While retaining better than 80% of fresh sperm progressive motility, thawed sperm from the same ejaculate retained on average only 18.6% of the CM penetration ability. Experiments using HA gel yielded similar results only with reduced experimental error and thus improved detection of treatment differences. Neither the percentage of abnormal forms nor head dimensions differed between fresh and thawed sperm. While findings suggests that sperm-CM interaction is a prominent factor in previous failures of vaginal AI with cryopreserved macaque sperm, neither sperm motility nor morphology appears to account for changes in the ability of cryopreserved sperm to penetrate CM. Our data points to a previously unidentified manifestation of cryodamage which may have implications for assessment of sperm function beyond the cervix and across mammalian species.
doi:10.1016/j.cryobiol.2010.11.004
PMCID: PMC3044926  PMID: 21112322
spermatozoa; cervical mucus; hyaluronic acid; artificial insemination; monkey; cryopreservation
18.  Dynamics of intra-follicular glucose during luteinization of macaque ovarian follicles 
Molecular and cellular endocrinology  2010;332(1-2):189-195.
Glucose is important to the maturation of the oocyte and development of the embryo, while hyperglycemia results in profound reproductive and developmental consequences. However, the normal physiology of glucose in the ovary remains poorly understood. The goal of this study was to determine intra-follicular glucose dynamics during the periovulatory interval in non-human primates undergoing controlled ovarian stimulation protocols. Follicular fluid and mural granulosa cells were isolated before or up to 24 hr after an ovulatory hCG bolus, and the human granulosa-lutein cell line hGL5 was used. Intra-follicular glucose increased 3 hr after hCG, and remained at that level until 12 hr when levels decline back to pre-hCG concentrations. Pyruvate and lactate concentrations in the follicle were not strongly altered by hCG. Mural granulosa cell expression of hexokinase 1 and 2, and glucose-6-phosphate dehydrogenase mRNA decreased following hCG, while glycogen phosphorylase (liver form) increased following hCG. Glucose uptake by hGL5 cells was delayed until 24 hr following stimulation. In summary, intra-follicular glucose increases following an ovulatory stimulus and mural granulosa cells do not appear able to utilize it, sparing the glucose for the cumulus-oocyte complex.
doi:10.1016/j.mce.2010.10.011
PMCID: PMC3011036  PMID: 20969917
macaque; (granulosa cells); glucose; glycolysis; luteinization
19.  The role of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in comparison with whole egg yolk for sperm cryopreservation in rhesus monkeys 
Asian Journal of Andrology  2011;13(3):459-464.
Low-density lipoprotein (LDL) extracted from hen egg yolk has recently been considered to be superior to whole egg yolk in sperm cryopreservation of various animal species. Meanwhile, there was a notion that high-density lipoprotein (HDL) in egg yolk may have a negative effect on post-thaw survival. The role of LDL and HDL in sperm cryopreservation of rhesus monkeys has not been explored. The present study evaluates their effect in comparison with egg yolk with or without the addition of permeable cryoprotectant (glycerol) on sperm cryopreservation of rhesus macaques. In addition, various additives intended to change the lipid composition of LDL–sperm membrane complex have also been tested for their effectiveness in preserving post-thaw viability. Our findings indicated that LDL is the main component in egg yolk that is responsible for its protective role for sperm cryopreservation in rhesus monkeys. Regardless of the presence or absence of glycerol, the protective role of LDL is similar to that of egg yolk and we did not observe any superiority in post-thaw survival with LDL when compared to egg yolk. Modifying the lipid composition of LDL–sperm membrane complex with the addition of cholesterol, cholesterol loaded cyclodextrin and phosphatidylcholine also did not yield any improvements in post-thaw survival; while addition of methyl-β-cyclodextrin reduced post-thaw motility. HDL plays a neutral role in sperm cryopreservation of rhesus monkeys. The present study suggests that egg yolk may still hold advantages when compared with LDL as effective components in extenders for sperm cryopreservation in rhesus monkeys.
doi:10.1038/aja.2010.145
PMCID: PMC3739331  PMID: 21423197
non-human primates; sperm cryopreservation; low-density lipoprotein; high-density lipoprotein; egg yolk
20.  Growth Hormone and Gene Expression of In Vitro-Matured Rhesus Macaque Oocytes 
Growth hormone (GH) in rhesus macaque in vitro oocyte maturation (IVM) has been shown to increase cumulus expansion and development of embryos to the 9–16 cell stage in response to 100 ng/ml recombinant human GH (r-hGH) supplementation during IVM. Although developmental endpoints for metaphase II (MII) oocytes and embryos are limited in the macaque, gene expression analysis can provide a mechanism to explore GH action on IVM. In addition, gene expression analysis may allow molecular events associated with improved cytoplasmic maturation to be detected. In this study, gene expression of specific mRNAs in MII oocytes and cumulus cells that have or have not been exposed to r-hGH during IVM was compared. In addition, mRNA expression was compared between in vitro and in vivo-matured MII oocytes and germinal vesicle (GV)- stage oocytes. Only two of 17 genes, insulin-like growth factor 2 (IGF2) and steroidogenic acute regulator (STAR), showed increased mRNA expression in MII oocytes from the 100 ng/ml r-hGH treatment group compared with other IVM treatment groups, implicating insulin-like growth factor (IGF) and steroidogenesis pathways in the oocyte response to GH. The importance of IGF2 is notable, as expression of IGF1 was not detected in macaque GV-stage or MII oocytes or cumulus cells.
doi:10.1002/mrd.21152
PMCID: PMC2830295  PMID: 20043319
non-human primate; growth hormone; oocyte; gene expression
21.  Similarity of Bisphenol A Pharmacokinetics in Rhesus Monkeys and Mice: Relevance for Human Exposure 
Environmental Health Perspectives  2010;119(4):422-430.
Objective
Daily adult human exposure to bisphenol A (BPA) has been estimated at < 1 μg/kg, with virtually complete first-pass conjugation in the liver in primates but not in mice. We measured unconjugated and conjugated BPA levels in serum from adult female rhesus monkeys and adult female mice after oral administration of BPA and compared findings in mice and monkeys with prior published data in women.
Methods
Eleven adult female rhesus macaques were fed 400 μg/kg deuterated BPA (dBPA) daily for 7 days. Levels of serum dBPA were analyzed by isotope-dilution liquid chromatography–mass spectrometry (0.2 ng/mL limit of quantitation) over 24 hr on day 1 and on day 7. The same dose of BPA was fed to adult female CD-1 mice; other female mice were administered 3H-BPA at doses ranging from 2 to 100,000 μg/kg.
Results
In monkeys, the maximum unconjugated serum dBPA concentration of 4 ng/mL was reached 1 hr after feeding and declined to low levels by 24 hr, with no significant bioaccumulation after seven daily doses. Mice and monkeys cleared unconjugated serum BPA at virtually identical rates. We observed a linear (proportional) relationship between administered dose and serum BPA in mice.
Conclusions
BPA pharmacokinetics in women, female monkeys, and mice is very similar. By comparison with approximately 2 ng/mL unconjugated serum BPA reported in multiple human studies, the average 24-hr unconjugated serum BPA concentration of 0.5 ng/mL in both monkeys and mice after a 400 μg/kg oral dose suggests that total daily human exposure is via multiple routes and is much higher than previously assumed.
doi:10.1289/ehp.1002514
PMCID: PMC3080921  PMID: 20855240
biomonitoring; bisphenol A; endocrine disruption; pharmacokinetics; xenobiotic metabolism
22.  Expression of the IGF and insulin systems in the luteinizing macaque ovarian follicle 
Fertility and sterility  2009;93(5):1421-1429.
Objective
To determine intrafollicular hormone levels and characterize mRNA expression of the IGF receptors, IGF binding proteins (IGFBP), and pregnancy-associated plasma protein-A (PAPP-A) in granulosa cells before and after an ovulatory hCG stimulus.
Design
Experimental animal study.
Setting
Academic medical center.
Animal(s)
Adult rhesus macaques.
Intervention(s)
Animals received exogenous FSH to promote the development of multiple preovulatory follicles. Follicles were aspirated before (0 hr), 3, 6, 12, or 24 hr after an ovulatory hCG bolus.
Main Outcome Measure(s)
IGF1, IGF2, and insulin levels in follicular fluid were determined by radioimmunoassay. Messenger RNA (mRNA) levels in granulosa cells were determined by real-time RT-PCR. IGFBPs and PAPP-A in follicular fluid were determined by Western blot analysis and ELISA.
Result(s)
IGF1, IGF2 and insulin in follicular fluid did not change during luteinization. IGF1R, IGFBP1 and IGFBP2 mRNAs were unchanged by hCG. IGF2R, IGFBP3, 5, 6 and PAPP-A mRNAs increased following hCG, while insulin receptor and IGFBP4 mRNAs decreased following hCG treatment. IGFBP 3 and 6 and PAPP-A protein increased following hCG.
Conclusion(s)
Dynamic changes in the expression of the IGFBPs and PAPP-A suggest tight regulation of IGF action during ovulation and corpus luteum formation.
doi:10.1016/j.fertnstert.2008.12.096
PMCID: PMC2859722  PMID: 19243760
luteinization; granulosa cell; IGF; IGFBP; primate
23.  Interactions among pre-cooling, cryoprotectant, cooling, and thawing for sperm cryopreservation in rhesus monkeys 
Cryobiology  2009;59(3):268-274.
The present study evaluated the interactions among pre-cooling, cryoprotectant, cooling, and thawing for rhesus monkey sperm using a four-way factorial design. Specifically, pre-cooling and thawing were evaluated for two conditions: slow vs. fast. Cooling was evaluated at four rates of 5, 29, 200, and 400 °C/min. The types of cryoprotectant involved combinations of egg yolk and glycerol, egg yolk and ethylene glycol, and egg yolk alone without permeable cryoprotectants or buffer alone with glycerol but without egg yolk. Our findings showed strong interactions among cryoprotectants, cooling, and thawing rates, but not pre-cooling rate, on post-thaw motility and forward progression. The optimal combination of cooling and thawing for maximum post-thaw survival depended on the types of cryoprotectant. When glycerol was used as a permeable cryoprotectant in the presence of egg yolk, slow thawing yielded similar success as fast thawing in some males. However, when glycerol was replaced with ethylene glycol for the same treatment, post-thaw motility was significantly lower in samples that were thawed slowly than those that were thawed rapidly. In the absence of permeable cryoprotectant but the presence of egg yolk, fast cooling was always favorable. On the contrary, in the absence of egg yolk but the presence of permeable cryoprotectant (glycerol), post-thaw motility was significantly reduced especially when samples were thawed slowly. Generally, fast thawing was superior to slow thawing regardless of the types of cryoprotectant or cooling rates, and glycerol in the presence of egg yolk yielded the highest post-thaw motility in all treatment groups.
doi:10.1016/j.cryobiol.2009.08.002
PMCID: PMC2789560  PMID: 19686717
cryopreservation; sperm; rhesus monkey; Macaca mulatta
24.  Differential Effects of Follistatin on Nonhuman Primate Oocyte Maturation and Pre-Implantation Embryo Development In Vitro1 
Biology of Reproduction  2009;81(6):1139-1146.
There is a vital need to identify factors that enhance human and nonhuman primate in vitro embryo culture and outcome, and to identify the factors that facilitate that objective. Granulosa and cumulus cells were obtained from rhesus monkeys that had either been FSH-primed (in vitro maturation [IVM]) or FSH and hCG-primed (in vivo maturation [VVM]) and compared for the expression of mRNAs encoding follistatin (FST), inhibin, and activin receptors. The FST mRNA displayed marginally decreased expression (P = 0.05) in association with IVM in the granulosa cells. The ACVR1B mRNA was more highly expressed in cumulus cells with IVM compared with VVM. Cumulus-oocyte complexes from FSH-primed monkeys exposed to exogenous FST during the 24-h IVM period exhibited no differences in the percentage of oocytes maturing to the metaphase II stage of meiosis compared to controls. However, embryos from these oocytes had significantly decreased development to the blastocyst stage. The effect of FST on early embryo culture was determined by exposing fertilized VVM oocytes to exogenous FST from 12 to 60 h postinsemination. FST significantly improved time to first cleavage and embryo development to the blastocyst stage compared with controls. The differential effects of exogenous FST on embryo development, when administered before and after oocyte maturation, may depend on the endogenous concentration in cumulus cells and oocytes. These results reveal evolutionary conservation of a positive effect of FST on embryogenesis that may be broadly applicable to enhance in vitro embryogenesis, with potential application to human clinical outcome and livestock and conservation biology.
Follistatin supplementation of culture medium after fertilization improves primate embryo development, while addition during in vitro maturation decreases oocyte developmental potential.
doi:10.1095/biolreprod.109.077198
PMCID: PMC2802231  PMID: 19641179
embryo; gene expression; gene regulation; granulosa cells; oocyte development; ovary; rhesus macaque
25.  Mammalian oocytes are targets for prostaglandin E2 (PGE2) action 
Background
The ovulatory gonadotropin surge increases synthesis of prostaglandin E2 (PGE2) by the periovulatory follicle. PGE2 actions on granulosa cells are essential for successful ovulation. The aim of the present study is to determine if PGE2 also acts directly at the oocyte to regulate periovulatory events.
Methods
Oocytes were obtained from monkeys and mice after ovarian follicular stimulation and assessed for PGE2 receptor mRNA and proteins. Oocytes were cultured with vehicle or PGE2 and assessed for cAMP generation, resumption of meiosis, and in vitro fertilization.
Results
Germinal vesicle intact (GV) oocytes from both monkeys and mice expressed mRNA for the PGE2 receptors EP2, EP3, and EP4. EP2 and EP4 proteins were detected by confocal microscopy in oocytes of both species. Monkey and mouse oocytes responded to PGE2 as well as agonists selective for EP2 and EP4 receptors with elevated cAMP, consistent with previous identification of EP2 and EP4 as Gαs/adenylyl cyclase coupled receptors. Incubation of mouse GV stage oocytes with PGE2 delayed oocyte nuclear maturation in vitro, but PGE2 treatment did not alter the percentage of mouse oocytes that fertilized successfully. PGE2 treatment also decreased the percentage of monkey oocytes that resumed meiosis in vitro. In contrast with mouse oocytes, the percentage of monkey oocytes which fertilized in vitro was lower after treatment with PGE2. Monkey oocytes with intact cumulus showed delayed nuclear maturation, but fertilization rate was not affected by PGE2 treatment.
Conclusions
Monkey and mouse oocytes express functional PGE2 receptors. PGE2 acts directly at mammalian oocytes to delay nuclear maturation. Surrounding cumulus cells modulate the effect of PGE2 to alter subsequent fertilization.
doi:10.1186/1477-7827-8-131
PMCID: PMC2988801  PMID: 21040553

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