Identification of the main gestagen metabolite in marmoset (Callithrix jacchus) urine by NMR and MS spectroscopy

Hydroxypregnanolone is the most abundant progesterone metabolite in the urine of marmoset monkeys (Callithrix jacchus). The substance is excreted as conjugate. The concentration of this steroid may be monitored by high performance, thin layer chromatography and postchromatographic derivatization. Hydroxypregnanolone was purified and subsequently identified by NMR spectroscopy and gas chromatography/mass spectroscopy. The exact chemical structure is 5 alpha-pregnane-3 alpha, 7 alpha-diol-20-one.     

Comparative anatomy and histology of the cervix uteri in non-human primates

The uterine cervix of rhesus macaque, crab-eating macaque, stump-tailed macaque, pig-tailed macaque, marmoset, baboon, patas, and squirrel monkeys was studied macroscopically and microscopically. Distribution of spermatozoa and leukocytes in the lumen and within the crypts and clefts was studied in rhesus and marmoset uterine cervix. The cervical canal of baboon, patas, marmoset, and stump-tailed monkeys is straight or slightly bent. The presence of variably developed ventral and dorsal colliculi in rhesus, crab-eating, and pig-tailed macaques causes the dorsoventral sinuosity of the cervical canal. The cervix of all investigated specimens is fundamentally fibrous tissue and the amount of muscle fibers increases toward the uterine corpus. The cervical mucosa of baboon, marmoset, and patas monkeys contains a large amount of clefts and tubular tunnels of variable structure, length, width, direction, and degree of branching. The cervical mucosa of macaques contains a large number of crypts of complex structure, length, width and degree of branching. The cervical crypts in macaques are usually longer in the ectocervix; whereas, in the midcervix, mucosa contains small crypts, clefts and long tunnels. Squamo-columnar junction is located near the external os in baboon, patas, marmoset, rhesus, crab-eating, and pig-tailed monkeys. In squirrel monkey, squamous epithelium is continuous through the external os, covers the vestibule and external surface of the cervical colliculi. In stump-tailed macaque, squamo columnar junction is located in the vagina, 1–3 cm from the external os of the cervix. The vaginal wall between the squamo-columnar junction and the external os of the cervix is covered with heavily branched mucosa lined with columnar epithelium. Ciliated cells of cervical epithelium occur in 9 to 19%. A large number of spermatozoa and a relatively low number of leucocytes have been found within crypts and clefts of cervical mucosa. The results are discussed in relation to the function of the cervix in different stages of the cycle, during pregnancy and parturition.This investigation was supported in part by Ford Foundation Grant No. 710-0287.     

“What’s wrong with my monkey?” Ethical perspectives on germline transgenesis in marmosets

The birth of the first transgenic primate to have inherited a transgene from its parents opens the possibility to set up transgenic marmoset colonies, as these monkeys are small and relatively easy to keep and breed in research facilities. The prospect of transgenic marmoset models of human disease, readily available in the way that transgenic laboratory mice are currently, prompts excitement in the scientific community; but the idea of monkeys being bred to carry diseases is also contentious. We structure an ethical analysis of the transgenic marmoset case around three questions: whether it is acceptable to use animals as models of human disease; whether it is acceptable to genetically modify animals; and whether these animals’ being monkeys makes a difference. The analysis considers the prospect of transgenic marmoset studies coming to replace transgenic mouse studies and lesion studies in marmosets in some areas of research. The mainstream, broadly utilitarian view of animal research suggests that such a transition will not give rise to greater ethical problems than those presently faced. It can be argued that using marmosets rather than mice will not result in more animal suffering, and that the benefits of research will improve with a move to a species more similar in phylogenetic terms to humans. The biological and social proximity of monkeys and humans may also benefit the animals by making it easier for scientists and caretakers to recognize signs of suffering and increasing the human motivation to limit it. The animal welfare and research impacts of the transition to marmoset use will depend very much on the extent to which researchers take these issues seriously and seek to minimize animal harm and optimize human benefit.     

A backpack system for long-term osmotic minipump infusions into unrestrained marmoset monkeys

A backpack system is described whereby osmotic minipumps are used to infuse gonadotrophin-releasing hormone (GnRH) subcutaneously in a pulsatile manner into infertile socially subordinate female marmoset monkeys (Callithrix jacchus jacchus). This procedure enables long-term infusion of GnRH without the necessity of repeated subcutaneous implantation of pumps and GnRH reservoirs. The backpack and cannulae system is inexpensive and can be constructed from commonly available materials. The GnRH treatment successfully overcame the suppression of pituitary luteinizing hormone secretion imposed by the low social status of female marmoset monkeys.     

Monitoring ovarian function in marmosets and tamarins by the measurement of urinary estrogen metabolites

Practical aspects of urinary estrogen analysis were considered with regard to establishing simple and reliable methods for monitoring ovarian function in marmosets and tamarins. Changes in the hormone:creatinine ratio in small volumes of urine from the common marmoset were significantly correlated with changes in 24-h excretion. Comparison of the metabolism and excretion of estrogens during the ovarian cycle in the common marmoset and cottontop tamarin revealed interesting species differences. High concentrations of conjugated estrone were measured in marmoset plasma, but estradiol 17β was the predominant estrogen in urine. In contrast, estrone was the most abundant estrogen measured in tamarin urine. Both species excreted very little estriol. Sulfates and glucuronides were present in urine in similar proportions before ovulation in the marmoset, although after ovulation sulfates were the more abundant. Conversely, most of the estrogens in tamarin urine appeared to be conjugated as glucuronides. Direct assay for estrone sulfate was applied to the measurement of urinary estrogen excretion during the ovarian cycle in a marmoset. The results compared well with those for total estradiol 17β after hydrolysis and ether extraction. The use of direct assays for conjugated estrogens in small volumes of urine is suggested as a practical method for monitoring ovarian function in marmosets and tamarins.     

LPS-induced lung inflammation in marmoset monkeys - an acute model for anti-inflammatory drug testing

Increasing incidence and substantial morbidity and mortality of respiratory diseases requires the development of new human-specific anti-inflammatory and disease-modifying therapeutics. Therefore, new predictive animal models that closely reflect human lung pathology are needed. In the current study, a tiered acute lipopolysaccharide (LPS)-induced inflammation model was established in marmoset monkeys (Callithrix jacchus) to reflect crucial features of inflammatory lung diseases. Firstly, in an ex vivo approach marmoset and, for the purposes of comparison, human precision-cut lung slices (PCLS) were stimulated with LPS in the presence or absence of the phosphodiesterase-4 (PDE4) inhibitor roflumilast. Pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-1 beta (MIP-1β) were measured. The corticosteroid dexamethasone was used as treatment control. Secondly, in an in vivo approach marmosets were pre-treated with roflumilast or dexamethasone and unilaterally challenged with LPS. Ipsilateral bronchoalveolar lavage (BAL) was conducted 18 hours after LPS challenge. BAL fluid was processed and analyzed for neutrophils, TNF-α, and MIP-1β. TNF-α release in marmoset PCLS correlated significantly with human PCLS. Roflumilast treatment significantly reduced TNF-α secretion ex vivo in both species, with comparable half maximal inhibitory concentration (IC(50)). LPS instillation into marmoset lungs caused a profound inflammation as shown by neutrophilic influx and increased TNF-α and MIP-1β levels in BAL fluid. This inflammatory response was significantly suppressed by roflumilast and dexamethasone. The close similarity of marmoset and human lungs regarding LPS-induced inflammation and the significant anti-inflammatory effect of approved pharmaceuticals assess the suitability of marmoset monkeys to serve as a promising model for studying anti-inflammatory drugs.     

Hemolytic complement in nonhuman primates

Hemolytic serum complement activity was quantitatively compared in baboons, squirrel monkeys, cebus monkeys, and cotton-top marmosets. Squirrel monkeys showed the highest activity, and marmosets had the lowest activity. The complement level in squirrel monkeys and tenfold greater than marmosets and almost four times higher than that of man. Cebus monkeys had levels most similar to that of man while the baboon exhibited activity almost as low as that of the marmoset.     

The mechanism of end-organ resistance to 1 alpha,25-dihydroxycholecalciferol in the common marmoset.

The common marmoset, a New World monkey, requires a large amount of cholecalciferol (110 i.u./day per 100g body wt.) to maintain its normal growth. In a previous report, we demonstrated that the circulating levels of 1 alpha, 25-dihydroxycholecalciferol [1 alpha,25(OH)2D3] in the marmosets are much higher than those in rhesus monkeys and humans, but the marmosets are not hypercalcaemic [Shinki, Shiina, Takahashi, Tanioka, Koizumi & Suda (1983) Biochem. Biophys. Res. Commun. 14, 452-457]. To compare the effect of the daily intake of cholecalciferol, two rhesus monkeys were given a large amount of cholecalciferol (900 i.u./day per 100g body wt). Their serum levels of calcium, 25-hydroxycholecalciferol and 24R,25-dihydroxycholecalciferol were markedly elevated, but the serum 1 alpha,25(OH)2D3 levels remained within a range similar to those in the rhesus monkeys fed the normal diet (intake of cholecalciferol 5 i.u./day per 100g body wt). Intestinal cytosols prepared from both monkeys contained similar 3.5 S macromolecules to which 1 alpha,25(OH)2D3 was bound specifically. However, the cytosols from the marmosets contained only one-sixth as many 1 alpha,25(OH)2D3 receptors as those from the rhesus monkeys. Furthermore, the activity of the 1 alpha,25(OH)2D3-receptor complex in binding to DNA-cellulose was very low in the marmosets. These results suggest that the marmoset possesses an end-organ resistance to 1 alpha,25(OH)2D3 and is a useful animal model for studying the mechanism of vitamin D-dependent rickets, type II.     

Development and annotation of shotgun sequence libraries from New World monkeys

The draft genome sequences of several primates are available, providing insights into evolutionary and anthropological research. However, genomic resources from New World monkeys are conspicuously lacking. To date, the genomes of only two platyrrhine species, the common marmoset and the Bolivian squirrel monkey, have been fully sequenced. This is especially limiting for comparative genomics research, considering that New World monkeys are the most speciose primate group, and platyrrhine genetic diversity is comparable to that of the catarrhines (i.e. apes and Old World monkeys). Here, we present the generation and annotation of numerous sequence reads from the genomes of Spider monkey (Ateles belzebuth), Owl monkey (Aotus lemurinus) and Uakari (Cacajao calvus), representing the three platyrrhine families, Atelidae, Cebidae and Pitheciidae, respectively. These sequencing reads were developed from gDNA shotgun libraries containing over 3000 individual sequences with an average length of 726 bps. Of these sequences, 1220 contain <20% repeats, and thus are potentially highly useful phylogenetic markers for other platyrrhine species. Among them, a large number of sequencing reads were found to match unique regions within the human (2462 sequences) and the marmoset (2829 sequences) genomes. In particular, the majority of these sequencing reads are from putatively neutrally evolving intergenic regions. Thus, they are likely to be highly informative for inferring neutral evolutionary patterns and genomic evolution for other New World monkeys.     

Observations on spontaneous hand use in the common marmoset (Callithrix jacchus)

Four male and four female marmoset monkeys were observed to make a total of 5,600 “right”, “left”, or “both” hand responses over seven categories of spontaneous behaviour. Significant and consistent hand preferences were shown for the majority of monkeys in two of the categories of behaviour, but not in the other five. The paper considers some of the methodological difficulties involved in recording “handedness” in a species such as the common marmoset.     

Increased positive selection pressure within the complementarity determining regions of the T‐cell receptor β gene in New World monkeys

Because of the long‐term co‐evolution of TCR and MHC molecules, numerous nucleotide substitutions have accumulated within the domains of TCRβ genes. We previously found that nonsynonymous nucleotide substitutions occurred more frequently in complementarity determining region (CDR)β than in CDRα, even though only a limited number of common marmoset (Callithrix jacchus) and human T‐cell receptor β variable (TRBV) sequences were compared. This interesting finding raised the question of whether the increased selective pressure within CDRβ was species‐specific. In this study, we identified 21 TRBV region sequences from the common marmoset and performed comparative sequence analyses of the T‐cell receptor α variable (TRAV) and TRBV regions from human, chimpanzee, rhesus monkey, cotton‐top tamarin, Ma's night monkey, and common marmoset. The ratios of the number of nonsynonymous nucleotide substitutions per site (d_N) to the d_S values (d_N/d_S) were less than 1 within the framework regions (FRs) of TRAV and TRBV region sequences, suggesting that purifying selection is largely dominant within the FRs. In contrast, the d_N values were statistically significantly greater for CDRβ than for CDRα only in New World monkeys. Also, increased d_N/d_S ratios (d_N/d_S>1) were observed within CDRβ between humans and New World monkeys and, interestingly, between New World monkeys, which share a relatively recent common ancestor. Moreover, phylogenetic analysis by maximum likelihood analysis provided firm evidence to support that positive selection occurred within CDRβ along New World monkey lineages. These results suggest that increased positive selection pressure within CDRβ is common in New World monkeys rather than being species‐specific. This study provides an intriguing insight into the co‐evolution of TCR and MHC molecules within primates. Am. J. Primatol. 73:1082–1092, 2011. © 2011 Wiley‐Liss, Inc.     

Molecular cloning and functional characterization of endogenous recombinant common marmoset monkey (Callithrix jacchus) follicle-stimulating hormone

Background Common marmoset monkeys (Callithrix jacchus) are readily used in biomedical research. However, superovulation for embryonic stem cell production and developmental research still remain difficult. Inexplicably, follicle‐stimulating hormone (FSH) as key player in superovulation has to be administered in extremely high dosages in this non‐human primate compared to human. Methods To evaluate whether marmoset FSH (cjFSH) is functionally more competent than its human homologue on the marmoset FSH receptor (cjFSHR), we established in vitro a homologous system characterizing homologous and recombinantly produced cjFSH. Results Upon stimulation of two cell lines stably expressing either the marmoset or the human FSH receptor (cj/hFSHR), respectively, the second messenger signaling of the activated receptor displayed no significant difference in ED₅₀ values. Thermostability of cjFSH was significantly prolonged by roughly 20% on both FSHRs. Conclusion High FSH dosage in marmoset superovulation cannot be explained by enhanced biopotency of the natural animal’s gonadotropin.     

Cross-genus adoption of a marmoset (Callithrix jacchus) by wild capuchin monkeys (Cebus libidinosus): case report

We report a case of interspecies adoption of an infant marmoset (Callithrix jacchus) by wild capuchin monkeys (Cebus libidinosus). The marmoset was an infant when it was first observed in the capuchin group on 3 March 2004. Since it first appeared it has been observed informally and frequently. In January 2005 systematic observations were made of the marmoset and a capuchin of similar age. Throughout its period of adoption the marmoset appeared to be socially integrated into the group, benefiting from nurturant behaviors exhibited by two successive adoptive "mothers" and pronounced tolerance from all members of the group. This case highlights the flexibility of both Callithrix and Cebus in accommodating variable social behaviors and other characteristics (including size) of social partners.     

Comparative Analysis of the Effects of Neurotrophic Factors CDNF and GDNF in a Nonhuman Primate Model of Parkinson’s Disease

Cerebral dopamine neurotrophic factor (CDNF) belongs to a newly discovered family of evolutionarily conserved neurotrophic factors. We demonstrate for the first time a therapeutic effect of CDNF in a unilateral 6-hydroxydopamine (6-OHDA) lesion model of Parkinson’s disease in marmoset monkeys. Furthermore, we tested the impact of high chronic doses of human recombinant CDNF on unlesioned monkeys and analyzed the amino acid sequence of marmoset CDNF. The severity of 6-OHDA lesions and treatment effects were monitored in vivo using ¹²³I-FP-CIT (DaTSCAN) SPECT. Quantitative analysis of ¹²³I-FP-CIT SPECT showed a significant increase of dopamine transporter binding activity in lesioned animals treated with CDNF. Glial cell line-derived neurotrophic factor (GDNF), a well-characterized and potent neurotrophic factor for dopamine neurons, served as a control in a parallel comparison with CDNF. By contrast with CDNF, only single animals responded to the treatment with GDNF, but no statistical difference was observed in the GDNF group. However, increased numbers of tyrosine hydroxylase immunoreactive neurons, observed within the lesioned caudate nucleus of GDNF-treated animals, indicate a strong bioactive potential of GDNF.     

Body mass growth in common marmosets: Toward a model of pediatric obesity

While much is known about adult obesity in nonhuman primates, very little is known regarding development of childhood adiposity. As small monkeys that are easy to handle and have a relatively fast life history, common marmoset monkeys (Callithrix jacchus) offer interesting opportunities to examine the question of fat versus lean mass growth in a nonhuman primate. This article provides an overview of our understanding of early life growth in mass in marmoset monkeys, based primarily upon our past 20 years of research, culminating in our recent findings on early life obesity in this species. Common marmosets display variance in early life growth patterns that is related to both pre‐ and postnatal factors and the marmoset uterine environment is exquisitely designed to reflect resources available for the gestation of multiple offspring, making them an interesting model of developmental programming. We have demonstrated that obesity can be generated in very early life in captive marmosets, with excess adiposity evident by one month of age, making this species a potentially valuable model in which to study pediatric obesity and its sequelae. Birth weight is associated with adiposity in animals vulnerable to obesity. Early life exposure to higher fat diets enhances the chances of postweaning obesity development. However, overall higher food consumption is also associated with obesity development at later ages. One unexpected finding in our studies has been the relatively high body fat percentage of neonatal (12–18%) marmosets suggesting that hypotheses regarding the uniqueness of high human neonatal adiposity merit further examination. Am J Phys Anthropol, 2013. © 2012 Wiley Periodicals, Inc.     

Immunodiffusion systematics of the primates. Part V. The Platyrrhini.

Evolutionary relationships between New World monkeys and marmoset genera and the place of the Ceboidea within the primates are considered in terms of the immunological specificity of ceboid proteins. Antigenic distances between the New World primates are measured using antisera produced in rabbits to nine ceboid genera: Alouatta, Aotes, Ateles, Callicebus, Cebus, Chiropotes, Lagothrix, Saimiri and Saguinus. A cladogram constructed on the basis of increasing degrees of antigenic distance between branches depicts Ceboidea as a monophyletic assemblage within which Alouatta is grouped with the Atelinae genera, Lagothrix and Ateles, Chiropotes joins Cacajao and Cebus joins Saimiri. The joining of the cebid genera Aotes and Callicebus with callithricid genera Callimico and Saguinus into a single complex lineage suggests that Cebidae is a paraphyletic or polyphyletic taxon. A phylogenetic taxonomy for Platyrrhini is proposed.     

The chromosomal location of rDNA in selected lower primates.

Hybridization in stiu was used to identify the chromosomes that carry rDNA in representative lower primates, including the baboons, Papio cynocephalus and Papio hamadryas; the colobus monkey, Colobus polykomos; the tree shrew, Tupaia glis; the lemur, Lemur fulvis; the saki, Pithecia pithecia; the marmoset, Saguinus nigricollis, and the spider monkey, Ateles geoffroyi. The marker chromosome, common to the Cercopithecines studied to date, carries the rDNA in the baboons. Another marker chromosome carries rDNA in a South American species, the spider monkey. A multichromosomal distribution of rDNA was demonstrated in the tree shrew, lemur, saki, and marmoset. None of the rDNA-containing chromosomes in the prosimians and New World monkeys show homology to the chromosomes that carry rDNA in the Hominids, Pongids, or Old World monkeys.     

Experimental manipulation of embryo implantation in the marmoset monkey and exotic equids

Embryos from marmoset monkeys were studied in vivo and in vitro to examine their requirements during the peri-implantation period. Marmoset embryos were recovered surgically, frozen and transferred to synchronized recipients, with a high rate of successful births. Embryos from Przewalski's horses and common zebras were transferred to domestic horses and donkeys to explore the potential of interspecies embryo transfer.The results obtained indicate that marmoset embryos can be frozen and transferred without a major loss in normal rates of embryo survival. The domestic horse is able to sustain Przewalski's horse and zebra fetuses to term.     

Development of prolactin levels in marmoset males: from adult son to first-time father

Previous studies have found a clear relationship between prolactin (prl) and paternal care in various vertebrate taxa. In New World monkeys, it has been demonstrated in several species that fathers have high prolactin levels even during periods without infant rearing. In this study, we followed the reproductive careers of common marmoset males as they transitioned from being an adult son within their native family to fathering their own offspring for the first time. Specifically, we examined the first experience of elevated prolactin levels in marmoset males. Additionally, we investigated the effects of the total number of experienced births as well as of age on prolactin levels. Our results show that common marmoset males did not experience an increase in prolactin secretion after pairing or shortly before birth of their first infants. However, prolactin levels rose more than twofold after the birth of their first infants and had lowered again 2.5 months after this event. We found no correlation between prolactin levels and the number of previous births experienced or age. Our study demonstrates that further work about a possible enhancing effect of prolactin on paternal care, by means of experimentally reducing hormonal levels, should be conducted in common marmosets using first-time fathers before males experience the first paternal increase in prolactin levels.     

Phylogenomics of species from four genera of New World monkeys by flow sorting and reciprocal chromosome painting

Background

The taxonomic and phylogenetic relationships of New World monkeys (Platyrrhini) are difficult to distinguish on the basis of morphology and because diagnostic fossils are rare. Recently, molecular data have led to a radical revision of the traditional taxonomy and phylogeny of these primates. Here we examine new hypotheses of platyrrhine evolutionary relationships by reciprocal chromosome painting after chromosome flow sorting of species belonging to four genera of platyrrhines included in the Cebidae family: Callithrix argentata (silvered-marmoset), Cebuella pygmaea (pygmy marmoset), Callimico goeldii (Goeldi's marmoset) and Saimiri sciureus (squirrel monkey). This is the first report of reciprocal painting in marmosets.

Results

The paints made from chromosome flow sorting of the four platyrrhine monkeys provided from 42 to 45 hybridization signals on human metaphases. The reciprocal painting of monkey probes on human chromosomes revealed that 21 breakpoints are common to all four studied species. There are only three additional breakpoints. A breakpoint on human chromosome 13 was found in Callithrix argentata, Cebuella pygmaea and Callimico goeldii, but not in Saimiri sciureus. There are two additional breakpoints on human chromosome 5: one is specific to squirrel monkeys, and the other to Goeldi's marmoset.

Conclusion

The reciprocal painting results support the molecular genomic assemblage of Cebidae. We demonstrated that the five chromosome associations previously hypothesized to phylogenetically link tamarins and marmosets are homologous and represent derived chromosome rearrangements. Four of these derived homologous associations tightly nest Callimico goeldii with marmosets. One derived association 2/15 may place squirrel monkeys within the Cebidae assemblage. An apparently common breakpoint on chromosome 5q33 found in both Saimiri and Aotus nancymae could be evidence of a phylogenetic link between these species. Comparison with previous reports shows that many syntenic associations found in platyrrhines have the same breakpoints and are homologous, derived rearrangements showing that the New World monkeys are a closely related group of species. Our data support the hypothesis that the ancestral karyotype of the Platyrrhini has a diploid number of 2n = 54 and is almost identical to that found today in capuchin monkeys; congruent with a basal position of the Cebidae among platyrrhine families.