Publications

There is a critical need to generate age- and sex-specific survival curves to characterize chronological aging consistently across nonhuman primates (NHP) used in biomedical research. Sex-specific Kaplan-Meier survival curves were computed in 12 translational aging models: baboon, bonnet macaque, chimpanzee, common marmoset, coppery titi monkey, cotton-top tamarin, cynomolgus macaque, Japanese macaque, pigtail macaque, rhesus macaque, squirrel monkey, and vervet/African green. After employing strict inclusion criteria, primary results are based on 12,269 NHPs that survived to adulthood and died of natural/health-related causes. A secondary analysis was completed for 32,616 NHPs that died of any cause. Results show a pattern of reduced male survival among catarrhines (African and Asian primates), especially macaques, but not platyrrhines (Central and South American primates). For many species, median lifespans were lower than previously reported. An important consideration is that these analyses may offer a better reflection of healthspan than lifespan since research NHPs are typically euthanized for humane welfare reasons before their natural end of life. This resource represents the most comprehensive characterization of sex-specific lifespan and age-at-death distributions for 12 biomedically relevant species, to date. These results clarify relationships among NHP ages and provide a valuable resource for the aging research community, improving human-NHP age equivalencies, informing investigators of expected survival rates, providing a metric for comparisons in future studies, and contributing to understanding of factors driving lifespan differences within and among species.

BACKGROUND: Common marmosets (Callithrix jacchus) are increasingly recognized as valuable nonhuman primates (NHPs) for biomedical research due to their small size and short reproductive cycle and lifespan relative to other NHP species. Maximizing the utility of captive research marmosets, including genetically manipulated animals, will require the use of assisted reproductive techniques (ART) including manipulation, storage, and sharing of marmoset sperm. Here, we identify characteristics of high-quality semen samples and validate a simple method for selecting high-quality sperm.

METHODS: Computer-assisted sperm analysis (CASA) was used to evaluate sperm quality in semen samples collected from 44 marmosets and assessed the use of the swim-up method for the selection of high-quality sperm was also tested in half the samples as a potential means to optimize in vitro fertilization or intrauterine insemination.

RESULTS: For each reference parameter, samples at or below the 5th percentile were categorized as abnormal sperm, while those above the 5th percentile were considered to be normal. Among normal samples, those at or above the 50th percentile were categorized as high-quality. High-quality semen samples exhibited the following characteristics: semen volume ≥ 30 µL; sperm count ≥ 107/ejaculate; total motility ≥ 35%; and normal morphology ≥ 5%. Sperm isolated by swim-up exhibited superior sperm progressive motility (19.7% ± 4.5 vs. 5.6% ± 2.1; P = 0.01) and normal morphology (13.1 ± 1.59 vs. 7.65 ± 1.1; P < 0.001) compared with unselected sperm.

CONCLUSION: This study defines robust, statistically supported reference values for evaluating marmoset semen samples to assist with the identification of optimal sperm donors and the selection of high-quality sperm samples for assisted reproduction. Ultimately, these reference values combined with a validated selection method will contribute to consistent standards for the international sharing of genetically diverse and/or gene-edited marmoset sperm for research and reproduction.

The marmoset is a fundamental nonhuman primate model for the study of aging, neurobiology, and many other topics. Genetic management of captive marmoset colonies is complicated by frequent chimerism in the blood and other tissues, a lack of tools to enable cost-effective, genome-wide interrogation of variation, and historic mergers and migrations of animals between colonies. We implemented genotype-by-sequencing (GBS) of hair follicle derived DNA (a minimally chimeric DNA source) of 82 marmosets housed at the Southwest National Primate Research Center (SNPRC). Our primary goals were the genetic characterization of our marmoset population for pedigree verification and colony management and to inform the scientific community of the functional genetic makeup of this valuable resource. We used the GBS data to reconstruct the genetic legacy of recent mergers between colonies, to identify genetically related animals whose relationships were previously unknown due to incomplete pedigree information, and to show that animals in the SNPRC colony appear to exhibit low levels of inbreeding. Of the >99,000 single-nucleotide variants (SNVs) that we characterized, >9800 are located within gene regions known to harbor pathogenic variants of clinical significance in humans. Overall, we show the combination of low-resolution (sparse) genotyping using hair follicle DNA is a powerful strategy for the genetic management of captive marmoset colonies and for identifying potential SNVs for the development of biomedical research models.

Zika virus (ZIKV) infection during pregnancy poses significant threats to maternal and fetal health, leading to intrauterine fetal demise and severe developmental malformations that constitute congenital Zika syndrome (CZS). As such, the development of a safe and effective ZIKV vaccine is a critical public health priority. However, the safety and efficacy of such a vaccine during pregnancy remain uncertain. Historically, the conduct of clinical trials in pregnant women has been challenging. Therefore, clinically relevant animal pregnancy models are in high demand for testing vaccine efficacy. We previously reported that a marmoset pregnancy model of ZIKV infection consistently demonstrated vertical transmission from mother to fetus during pregnancy. Using this marmoset model, we also showed that vertical transmission could be prevented by pre-pregnancy vaccination with Zika purified inactivated virus (ZPIV) vaccine. Here, we further examined the efficacy of ZPIV vaccination during pregnancy. Vaccination during pregnancy elicited virus neutralizing antibody responses that were comparable to those elicited by pre-pregnancy vaccination. Vaccination also reduced placental pathology, viral burden and vertical transmission of ZIKV during pregnancy, without causing adverse effects. These results provide key insights into the safety and efficacy of ZPIV vaccination during pregnancy and demonstrate positive effects of vaccination on the reduction of ZIKV infection, an important advance in preparedness for future ZIKV outbreaks.

The common marmoset (Callithrix jacchus), a small South American monkey, is an important nonhuman primate model in the study of aging and age-related neurodegenerative disease, including Alzheimer's disease, Parkinson's disease, and related dementias. Thorough characterization of the wild type marmoset brain agingmodel, including biomarkers of aging and neural degeneration, will further the marmoset's utility in translational research. We measured serum concentration of four key biomarkers of neural degeneration [total tau (T-tau), glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), and ubiquitin C-terminal hydrolase-L1 (UCH-L1)] via single molecule array from 24 marmosets (female n = 13, male n = 11) ranging in age from 1.3 to 18.7 years. Aged marmosets (>7 years) had significantly higher GFAP, NfL, UCH-L1, and T-tau than adult marmosets. Sex differences were not detected for any of these biomarker concentrations. These data provide an important initial range of reference values for GFAP, NfL, T-tau, and UCH-L1 to evaluate aging and neural health in marmosets, as well as evaluation of therapeutics in clinical models of disease.

The common marmoset (Callithrix jacchus) is one of the most widely used nonhuman primate models of human disease. Owing to limitations in sequencing technology, early genome assemblies of this species using short-read sequencing suffered from gaps. In addition, the genetic diversity of the species has not yet been adequately explored. Using long-read genome sequencing and expert annotation, we generated a high-quality genome resource creating a 2.898 Gb marmoset genome in which most of the euchromatin portion is assembled contiguously (contig N50 = 25.23 Mbp, scaffold N50 = 98.2 Mbp). We then performed whole genome sequencing on 84 marmosets sampling the genetic diversity from several marmoset research centers. We identified a total of 19.1 million single nucleotide variants (SNVs), of which 11.9 million can be reliably mapped to orthologous locations in the human genome. We also observed 2.8 million small insertion/deletion variants. This dataset includes an average of 5.4 million SNVs per marmoset individual and a total of 74,088 missense variants in protein-coding genes. Of the 4956 variants orthologous to human ClinVar SNVs (present in the same annotated gene and with the same functional consequence in marmoset and human), 27 have a clinical significance of pathogenic and/or likely pathogenic. This important marmoset genomic resource will help guide genetic analyses of natural variation, the discovery of spontaneous functional variation relevant to human disease models, and the development of genetically engineered marmoset disease models.

The luteal-placental shift is an important milestone of mammalian pregnancy signifying when endocrine control of pregnancy shifts from the corpus luteum of the ovary to the placenta. The corpus luteum is maintained by chorionic gonadotropin (CG). Upon sufficient placental maturation, CG production wanes, the corpus luteum involutes, and control is shifted to the placenta, one consequence of which is a midgestational rise in glucocorticoid production, especially cortisol and cortisone, by both mother and fetus. Glucocorticoids are involved in initiating parturition, prenatal programming of offspring phenotype, and maturing fetal organs. Limited evidence from human pregnancy suggests that the timing of this shift is delayed in twin pregnancies, but little is known about the timing of the luteal-placental shift in litter-bearing monkeys from the primate family Callitrichidae. Here we provide evidence from cotton-top tamarins (Saguinus oedipus) and common marmosets (Callithrix jacchus) of longer duration of elevated CG associated with multiple infant births compared to single births. Urinary profiles from cotton-top tamarins demonstrate that the decline of the extended elevation of CG precedes the onset of the midpregnancy sustained rise in glucocorticoids; this shift occurs later with an increase from one to two fetuses carried to term. In the common marmoset, the onset of the sustained rise of glucocorticoids in maternal urine is also delayed with an increase in infant number. Total urinary glucocorticoid levels during the last half of gestation increase monthly but do not differ by infant number. The significant delay in the luteal-placental shift suggests a longer period of placental maturation is needed to support a greater number of fetuses.

Reconstitution of germ cell fate from pluripotent stem cells provides an opportunity to understand the molecular underpinnings of germ cell development. Here, we established robust methods for induced pluripotent stem cell (iPSC) culture in the common marmoset (Callithrix jacchus [cj]), allowing stable propagation in an undifferentiated state. Notably, iPSCs cultured on a feeder layer in the presence of a WNT signaling inhibitor upregulated genes related to ubiquitin-dependent protein catabolic processes and enter a permissive state that enables differentiation into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs in vivo. Induction of cjPGCLCs is accompanied by transient upregulation of mesodermal genes, culminating in the establishment of a primate-specific germline transcriptional network. Moreover, cjPGCLCs can be expanded in monolayer while retaining the germline state. Upon co-culture with mouse testicular somatic cells, these cells acquire an early prospermatogonia-like phenotype. Our findings provide a framework for understanding and reconstituting marmoset germ cell development in vitro, thus providing a comparative tool and foundation for a preclinical modeling of human in vitro gametogenesis.

Researchers and veterinarians often use hematology and clinical chemistry to evaluate animal health. These biomarkers are relatively easy to obtain, and understanding how they change across healthy aging is critical to clinical care and diagnostics for these animals. We aimed to evaluate how clinical biomarkers from a chemistry profile and complete blood count (CBC) change with age in common marmosets (Callithrix jacchus). We assessed blood samples collected during routine physical exams at the Southwest National Primate Research Center and the University of Texas Health San Antonio marmoset colonies from November 2020-November 2021. We found that chemistry and CBC profiles varied based on facility, sex, and age. Significant changes in albumin, phosphorus/creatinine ratio, albumin/globulin ratio, amylase, creatinine, lymphocyte percent, hematocrit, granulocytes percent, lymphocytes, hemoglobin, red cell distribution width, and platelet distribution width were all reported with advancing age. Aged individuals also demonstrated evidence for changes in liver, kidney, and immune system function compared with younger individuals. Our results suggest there may be regular changes associated with healthy aging in marmosets that are outside of the range typically considered as normal values for healthy young individuals, indicating the potential need for redefined healthy ranges for clinical biomarkers in aged animals. Identifying animals that exhibit values outside of this defined healthy aging reference will allow more accurate diagnostics and treatments for aging colonies.

Zika virus (ZIKV) infection during pregnancy causes severe developmental defects in newborns, termed congenital Zika syndrome (CZS). Factors contributing to a surge in ZIKV-associated CZS are poorly understood. One possibility is that ZIKV may exploit the antibody-dependent enhancement of infection mechanism, mediated by cross-reactive antibodies from prior dengue virus (DENV) infection, which may exacerbate ZIKV infection during pregnancy. In this study, we investigated the impact of prior DENV infection or no DENV infection on ZIKV pathogenesis during pregnancy in a total of four female common marmosets with five or six fetuses per group. The results showed that negative-sense viral RNA copies increased in the placental and fetal tissues of DENV-immune dams but not in DENV-naïve dams. In addition, viral proteins were prevalent in endothelial cells, macrophages, and neonatal Fc receptor-expressing cells in the placental trabeculae and in neuronal cells in the brains of fetuses from DENV-immune dams. DENV-immune marmosets maintained high titers of cross-reactive ZIKV-binding antibodies that were poorly neutralizing, raising the possibility that these antibodies might be involved in the exacerbation of ZIKV infection. These findings need to be verified in a larger study, and the mechanism involved in the exacerbation of ZIKV infection in DENV-immune marmosets needs further investigation. However, the results suggest a potential negative impact of preexisting DENV immunity on subsequent ZIKV infection during pregnancy in vivo.