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Congenitally caused fused labia in the common marmoset (Callithrix jacchus).

In this paper, the occurrence of an external genital abnormality in female marmoset monkeys (fused labia) is discussed. This malformation was detected, for the first time, in a group of animals at the German Primate Center (GPC), Goettingen. The malformed vulva was completely sealed except for an opening of 1.5-2.5 mm around the urethra sufficient for urination. Because of this defect the animals were not able to copulate. As a consequence, the affected females were functionally infertile although they had a normal genital tract and a regular cycle. This vulvar abnormality was found in 12 females, offspring of 10 pairs in which either one or both came to the German Primate Center from two genetically related colonies in Munich, Germany, and one colony in Basel, Switzerland. The abnormality appeared to be recessive and inheritable from either parent. In pairs in which both animals were from one of the mentioned colonies, 45% of the female offspring were affected. In pairs where only one partner came from these colonies, 26% of female offspring had the malformation. These results indicate that avoidance of inbreeding, which is frequently performed in primate colonies, may reduce, but not eliminate the expression of abnormalities of genetic origin. Therefore selective breeding is required, and, in colonies where these recessive mutations are widespread, the development of genetic screening tests would be advantageous.

Animals↗

Ovarian stimulation of marmoset monkeys (Callithrix jacchus) using recombinant human follicle stimulating hormone.

To reduce the number of animals required for controlled studies of marmoset oocytes and early embryos, a superovulation protocol was developed for the common marmoset. Females were given up to 50 i.u./day recombinant human follicle stimulating hormone (FSH)--(r-hFSH) for 6 days. Ovaries were visualized by a modified laparoscopic technique and follicular aspiration was performed using a needle and suction apparatus inserted directly through an otoscope speculum. The number of follicles + ovulation points (+/- S.E.) was 2.9 (+/- 0.2) in controls and 14.1 (+/- 1.6; P < or = 0.001) in the 50 i.u. r-hFSH per day animals. Oocytes, typically at the germinal vesicle stage at collection, extruded a first polar body within 26 hours. In vitro fertilization was performed and embryos developed to the hatched blastocyst stage (34%). With many high quality oocytes and the ability to synchronize cycles, the marmoset is a valuable primate model for examining nuclear reprograming and early embryonic events.

Animals↗

Intraretinal axon diameters of a New World primate, the marmoset (Callithrix jacchus).

PURPOSE: Previously, measurements of retinal ganglion cell axon diameter have been used to make inferences about the physiology and clinical pathology of the visual pathway. However, few of these studies were able to unequivocally relate axon diameter to retinal ganglion cell type and other associated measurements. In this and our previous study we have examined intraretinal axon diameters to determine if differences in axon diameter may help to explain conduction velocity measurements found previously. METHODS: Individual retinal ganglion cells of a New World primate, the common marmoset (Collithrix jacchus) were injected iontophoretically with 2% Lucifer yellow and 4% neurobiotin. Labelled cells were visualized by horseradish peroxidase immunohistochemistry and diaminobenzidine and then retinae were mounted vitreal side up on a glass slide. Cell measurements were made with the aid of a camera lucida attachment and computer-aided morphometry Axons were photographed under x 100 oil immersion and measured at a final magnification of x 4600. RESULTS: A sample of 62 parasol cells, 22 midget cells, 16 hedge cells and 11 small bistratified cells were analysed. Dendritic field diameter of the different cell classes showed only moderate (non-significant) increases with eccentricity. Only the parasol cells demonstrated a significant increase in mean axon diameter with eccentricity. When the parasol class was examined more closely, it was found that only parasol cells of the superior, inferior and temporal retina (SIT group) showed significant positive correlations between different cell parameters (mean axon diameter, soma diameter, dendritic field diameter, eccentricity). Soma and dendritic field diameters of the SIT group were significantly larger than those of the nasal parasol cells. However, mean axon diameters of the SIT cells were not significantly different from nasal parasol cells. Axon diameters of nasal parasol cells were very variable and overlapped those of the midget and hedge cell classes to a large extent. CONCLUSIONS: The present data show that for marmoset parasol cells there may not be a clearly defined distinction between nasal and superior, inferior and temporal parasol cells on the basis of axon size. Of particular interest in the present analysis is the clear separation of superior, inferior and temporal parasol cells and nasal parasol cells when comparing soma and dendritic field diameters which is not reflected in the distribution of axon diameters. We suggest that changes in diameter along the length of an axon, differences between retinal quadrants and the variability between cells may be related to minimization of spatiotemporal dispersion necessary for accurate perception of motion within the visual world.

Animals↗

Experimental hepatitis A virus (HAV) infection in Callithrix jacchus: early detection of HAV antigen and viral fate.

Common marmosets (Callithrixjacchus) were orally inoculated with a Brazilian strain (HAF-203) of hepatitis A virus (HAy). Three monkeys were euthanized at postinoculation hours 6, 12 and 24 to investigate the early events of HAV infection. Following others three inoculated and one control marmosets remained throughout the 46 day to evaluation of viral excretion. Different samples were collected to detect sequential presence of HAV RNA by nested reverse transcription-polymerase chain reaction (RT-PCR) in liver, saliva, bile and stools at 6 hours to 461h days postinoculation. Liver tissues were examined by immunofluorescence assay in a confocal laser-scanning microscope for the presence of HAV antigen. HAV RNA was detected in saliva during the course of the study, in bile from 24 hours to 46 days. in stools from 7 to 46 days and liver at 12 hours postinfection. In immunofluorescence of liver stained preparations, viral antigen was present at six hours after inoculation throughout the remainder of the 46-day study. The animals developed histological and biochemical acute hepatitis after second week postinoculation. Spleen, duodenum, and mesenteric lymph nodes specimens were negative for HAV antigens. This study supports the possibility that in Callithrixjacchus orally inoculated with hepatitis A virus the saliva route may be additional way of viral elimination. The viral replication in the liver was responsible for biliary HAV presence and latter HAV detection in fecal samples.

Animals↗

Functional response properties of neurons in the dorsomedial visual area of New World monkeys (Callithrix jacchus).

The dorsomedial visual area (DM), a subdivision of extrastriate cortex located near the dorsal midline, is characterized by heavy myelination and a relative emphasis on peripheral vision. To date, DM remains the least understood of the three primary targets of projections from the striate cortex (V1) in New World monkeys. Here, we characterize the responses of DM neurons in anaesthetized marmosets to drifting sine wave gratings. Most (82.4%) cells showed bidirectional sensitivity, with only 6.9% being strongly direction selective. The distribution of orientation sensitivity was bimodal, with a distinct population (corresponding to over half of the sample) formed by neurons with very narrow selectivity. When compared with a sample of V1 units representing a comparable range of eccentricities, DM cells revealed a preference for much lower spatial frequencies, and higher speeds. End inhibition was extremely rare, and the responses of many cells summated over distances as large as 30 degrees. Our results suggest clear differences between DM and the two other main targets of V1 projections, the second (V2) and middle temporal (MT) areas, with cells in DM emphasizing aspects of visual information that are likely to be relevant for motor control.

Action Potentials↗

Female marmoset monkeys (Callithrix jacchus) can be identified from the chemical composition of their scent marks.

The present study analyzed 42 organic solvent extracts of scent mark pools from five dominant female common marmosets by gas chromatography (GC) and combined GC and mass spectrometry. We determined whether there were qualitative or quantitative differences between the chemical composition of scent marks from individual females. Gas chromatography and mass spectral analysis detected the same 162 chemicals in 86% (36/42) of scent mark pools from five dominant females. This near identical chemical composition of scent marks suggested there were few, if any, qualitative differences between the chemical composition of scent marks from individual females. Instead, quantitative differences in scent may provide the key factor distinguishing individual females. Using the relative concentration of highly volatile chemicals detected by GC in scent marks, linear discriminant analysis classified scent mark pools to their correct donor approximately 91% of the time. Such highly reliable statistical matching of scent to donor suggested that each individual female common marmoset has a unique ratio of highly volatile chemicals in their scent marks which may permit individual identification of females from odors in their scent alone.

Animals↗

Meiotic abnormalities in in vitro-matured marmoset monkey (Callithrix jacchus) oocytes: development of a non-human primate model to investigate causal factors.

BACKGROUND: Meiotic abnormalities are thought to be a major causal factor of low embryo development rates, for embryos developed from in vitro-matured oocytes. A new non-human primate model, in the common marmoset, is being developed to facilitate investigation of the mechanisms involved. METHODS: Oocytes were dissected from antral follicles from three size classes. They were allowed to mature in vitro for only 24 h, in order to focus the investigation on the rapidly maturing oocytes. Chromosome spreads were visualized with Giemsa staining, and spindles /chromosomes with fluorescently labelled anti-alpha-tubulin antibody combined with a DNA fluorochrome. RESULTS: 40% of the oocytes had reached metaphase II (MII) after 24 h. Of the MII oocytes selected for karyotyping, readable chromosomal spreads were obtained from 64%. Overall, 63% of these presented a normal haploid chromosome number of 23,X, with all abnormal karyotypes occurring in the oocytes from small follicles. For another group of MII oocytes, where meiotic spindles were visualized, only half of the MII oocytes displayed well-formed spindles and apparently correct chromosomal alignment. CONCLUSIONS: This work provides the first information on the normal and aneuploid MII meiotic chromosome sets for the marmoset oocyte, and demonstrates a high rate of chromosomal and spindle abnormality among rapidly maturing oocytes from small antral follicles.

Aneuploidy↗

Leukaemia inhibitory factor in the endometrium of the common marmoset Callithrix jacchus: localization, expression and hormonal regulation.

In the present study, changes in the immunohistochemical localization of leukaemia inhibitory factor (LIF) in the endometrium during various phases of ovarian cyclicity of the common marmoset have been reported. LIF was absent during the early and late follicular phases. LIF was observed mainly in the cytoplasm of the endometrial glands during the early luteal phase, reached maximum intensity during the mid-luteal phase and declined again during late luteal phase. In-situ hybridization also showed a similar cyclic pattern in the expression of LIF. Stromal cells only showed signals for LIF during the mid-luteal phase. In ovariectomized marmosets, graded dosages of oestradiol alone failed to induce the appearance of LIF protein. Progesterone treatment following oestradiol priming, however, induced distinct glandular localization of LIF, indicating that LIF is a progesterone-dependent protein. Thus endometrial LIF is under maternal control and is secreted in response to the increased progesterone concentrations in circulation. It is possible that high concentrations of LIF during mid-luteal phase may prepare the endometrium for blastocyst implantation in marmosets.

Animals↗

Clonal organization of proliferating spermatogonial stem cells in adult males of two species of non-human primates, Macaca mulatta and Callithrix jacchus.

The present study examines the existence of clonogenic patterns in the proliferation and differentiation of spermatogonial stem cells in two species of non-human primates, the marmoset and the rhesus monkey. We developed a novel approach to detect proliferating spermatogonial clones in whole mounts of seminiferous tubules. Dual fluorescence labeling of bromodeoxyuridine and acrosin in conjunction with confocal microscopy allows the description of the clonogenic and spatial arrangement of proliferating spermatogonia at specific stages of the seminiferous epithelial cycle. Cross-sections of paraffin-embedded tissue were labeled by the same approach. For both monkey species we demonstrate the presence of proliferating spermatogonial clones of variable size at specific stages of the cycle of the seminiferous epithelium. Detailed analysis of the rhesus monkey reveals proliferating Apale spermatogonia at stages VII and IX of the cycle of the seminiferous epithelium, and of proliferating B spermatogonia at stages II, IV, VI, and XII. Proliferating Apale spermatogonia at stages VII and IX of the cycle are organized in pairs or quadruplets. B1 spermatogonia appear as quadruplets or eight-cell clones, and B2 spermatogonia as 8- or 16-cell clones. We conclude that spermatogenesis in the rhesus monkey is initiated by two divisions of duplets or quadruplets of Apale spermatogonia: a first division at stage VII, after which the clones of Apale spermatogonia separate, and a second division at stage IX, which leads to clones of B1 spermatogonia as well as pairs and quadruplets of Apale spermatogonia replenishing the seminiferous epithelium to maintain the original size of the A spermatogonial population.

Animals↗

Luteinizing hormone/chorionic gonadotropin bioactivity in the common marmoset (Callithrix jacchus) is due to a chorionic gonadotropin molecule with a structure intermediate between human chorionic gonadotropin and human luteinizing hormone.

Chorionic gonadotropin (CG), a pregnancy-specific heterodimeric hormone found in primates, is responsible for CL rescue with pregnancy maintenance. Of the primates, the human and baboon gene sequences are the only structures so far determined. In order to study the structure and function of CG in other primates, we have isolated and sequenced the coding regions for the two subunits of marmoset CG (mCG) by the reverse transcription/polymerase chain reaction method. Study of multiple clones confirmed a high degree of homology with the human sequences (88% and 80% for the alpha and beta nucleotide sequences, respectively). Marmoset CG alpha has an extra four amino acids compared to hCG alpha, whereas the mCG beta sequence has a 3-bp deletion that maintains the reading frame and C-terminal amino acid sequence. Most of the differences between hCG beta and mCG beta peptides occur in the C-terminal region, which includes the loss of two of the O-linked glycosylation consensus sequences and the presence of an N-linked glycosylation consensus sequence. When mCG alpha and beta were co-expressed in CHO cells, assembly of biologically active hormone was confirmed by induced steroid secretion by MA10 cells. Partially purified mCG beta was used to raise anti-mCG antibodies. To date, an antibody has been obtained that is capable of detecting recombinant mCG beta, recombinant mCG dimer, and mCG dimer secreted by cultured marmoset trophoblast. Marmoset CG alpha and beta were also detectable at the transcriptional level in cultured trophoblast by in situ hybridization. This suggests that the LH/CG bioactivity reported from marmoset placentae and embryos is due to a molecule with structural features common to hLH (glycosylation pattern) and hCG (CG beta C-terminal structure).

Amino Acid Sequence↗

Patterns of relaxin and steroids in the reproductive cycle of the common marmoset (Callithrix jacchus): effects of prostaglandin F2 alpha on relaxin and progesterone secretion during pregnancy.

We measured the concentrations of relaxin (Rlx), progesterone, and estradiol-17 beta in serum samples obtained twice or three times weekly from marmosets during the estrous cycle and pregnancy. The cyclic patterns and concentrations of progesterone and estradiol-17 beta were similar to those reported by previous investigators. Rlx was not detected in individual serum samples ( < 0.62-1.25 ng/ml) obtained from nonpregnant marmosets. However, pooling of luteal serum from all animals permitted assay of much larger volumes of serum (0.4 ml vs. 0.1 ml), and a concentration of about 1 ng/ml was detected. Rlx was first detected in serum in the second or third week of the 21-wk marmoset pregnancy, rose to a peak during Weeks 10-14, and then declined slowly as the time of parturition approached. The pattern of Rlx was unlike that observed during pregnancy in Old World monkeys, chimpanzees, or women, and resembled, instead, that seen in rodents, carnivores, and equids. Progesterone and estradiol-17 beta likewise increased throughout pregnancy, and their patterns were similar to those previously described for marmosets by other investigators. The concentrations of the steroids and Rlx in serum of pregnant marmosets was 10-fold or more higher than those found in Old World monkeys, baboons, chimpanzees, or women. Spontaneous abortions in two of the marmosets were accompanied by precipitous falls in serum levels of progesterone, estradiol-17 beta, and Rlx. Following s.c. injection of the luteolytic agent prostaglandin F2 alpha (PGF2 alpha) into two marmosets at midpregnancy, serum progesterone and Rlx fell to low levels. These animals received a progestin, 17 alpha-ethyl-19-nortesterone, to preclude abortion. Serum progesterone rose again, but serum Rlx remained low for the duration of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Abortion, Veterinary↗

Metabolism of reproductive steroids during the ovarian cycle in two species of callitrichids, Saguinus oedipus and Callithrix jacchus, and estimation of the ovulatory period from fecal steroids.

Gonadal steroids were measured in daily fecal samples providing comparative data on steroid metabolism in two genera of New World primates. Circulating bioactive LH and progesterone concentrations and fecal progesterone, pregnanediol, estradiol, and estrone concentrations were measured by collecting blood and daily fecal samples from four captive common marmoset females and four cotton-top tamarin females for 30 days. High recoveries (> 80%) of labeled steroids that were added directly to the feces before extraction were recovered from feces of both species. Because of the presence of complex steroid conjugates, only one fifth the amount of estradiol was measured without solvolysis as compared to the amount measured with solvolysis. In tamarins, steroids were metabolized rapidly, with all postovulatory increases occurring within two days after the circulating LH peak (an increase of 2 SD higher than mean follicular levels). In marmosets, steroid excretion was slower; increased steroid levels occurred 2-4 days after the LH peak except in the case of estrone, which did not consistently increase after the LH peak. Circulating estrone and estradiol both contributed to the high excretion of estradiol in the feces from both species. The timing in the delay in excretion of fecal steroids was used to accurately determine the ovulatory period to within a 2-day window. This degree of accuracy is possible when the duration of the delay to the LH peak is known for a given species. Additionally, steroid concentrations were highly correlated between frozen and lyophilized fecal samples (0.81 +/- 0.07 SEM), indicating that fluid removal from the feces did not effectively alter steroid profiles.

Animals↗

A local oxytocin system is part of the luteinization process in the preovulatory follicle of the marmoset monkey (Callithrix jacchus).

Luteinization is a complex differentiation process involving the interaction of extrinsic and intraovarian factors. The aim of this study was to examine the components of an intraovarian oxytocin (OT) system during the periovulatory period in the marmoset monkey, as well as the possible relationship of these components to other factors involved in the luteinization process, using immunohistochemistry and cell culture techniques. Ovaries were collected on Day 7 of the follicular phase (before the endogenous LH surge) and on Day 8 (22 h after an exogenous hCG application, but before ovulation). Before the endogenous LH increase, OT immunoreactivity was detectable at low levels in most antral follicles, where its presence was confined to antral granulosa cell (GC) layers. In contrast, immunoreactivity for the OT receptor (OTR) was localized primarily in the basal GC layer. After application of exogenous hCG, there was a marked enhancement in both the staining intensity and the number of cells positive for OT and the OTR in all GC layers of antral follicles, especially in the preovulatory follicle. Progesterone receptor and 3beta-hydroxysteroid dehydrogenase activity in GC were clearly present only when follicles were obtained after gonadotropin stimulation. Secretion of authentic OT was demonstrated from cultured GC obtained before the LH surge, with highest amounts in cells cultured from preovulatory as opposed to smaller antral follicles. OT production could be stimulated by the application of hCG to the GC cultured from preovulatory follicles, whereas the gonadotropin was without effect on GC from small follicles. FSH had no effect on OT production by GC from either follicle type. Application of OT to the cultures caused an increase in progesterone production by GC from large preovulatory follicles but was without effect on steroidogenesis by cells from small antral follicles. These results describing the presence and distribution of OT and OTR and their modulation by hCG, as well as the luteotrophic effect of OT in cultured GC from preovulatory follicles, implicate OT as a paracrine mediator in the luteinization process in the primate ovary.

3-Hydroxysteroid Dehydrogenases↗

Differential splicing and expression of the relaxin-like factor gene in reproductive tissues of the marmoset monkey (Callithrix jacchus).

The relaxin-like factor (RLF) is a novel member of the insulin/relaxin/insulin-like growth factor family of growth factors and hormones that is expressed predominantly in the reproductive system, with highest expression in the Leydig cells of the testis. Using a combination of molecular and immunological techniques, we have characterized the structure and expression of the RLF gene from a primate model, the marmoset monkey, with the intention of comparing this with recent results on the closely related hormone relaxin in this species. As in other species, including the human, RLF gene products can be detected maximally in Leydig cells and in the follicular theca interna cells and corpora lutea of the ovary. Reverse transcription-polymerase chain reaction analysis confirmed this expression and showed that in the corpus luteum, testis, and epididymis, a second, alternative RLF gene transcript was present that is expressed at low levels and that appears to be derived by differential splicing of a novel exon. Analysis of genomic DNA from the marmoset showed that in this species, the single-copy gene contains a longer intronic region separating the two exons described for the human. Alternative splicing introduces a novel exon 1A between exons 1 and 2, which leads to an altered open reading frame, with a new stop codon, such that if translated, the novel transcript will encode a truncated polypeptide comprising a C-terminally extended B-domain.

Alternative Splicing↗

Persistent Epstein-Barr virus infection in the common marmoset (Callithrix jacchus).

Epstein-Barr virus (EBV) infection of the common marmoset causes long-term infection, with production of antibodies to virus-induced antigens, without clinical illness. Attempts to show the presence of EBV DNA in saliva of infected animals by PCR were initially unsuccessful, although slot-blot hybridization analysis demonstrated that viral DNA was present. Further investigations showed that most samples of pilocarpine-induced saliva, and 33% of the samples of whole mouth fluids (WMF) tested, were inhibitory to PCR. Similar results were found using human WMF. A method of assessing samples of marmoset WMF for the presence of EBV, by PCR using an EBV BamHI W probe, and removing inhibition with Chelex 100, is described. A total of 202 samples from 21 EBV infected, and seven non-infected animals was tested. Five seropositive animals shed virus on every occasion, and 15 intermittently. Two marmosets, infected as neonates, showed progressively increasing humoral responses to viral antigens, and shed virus on every occasion tested over 3 years. When mated with uninfected animals, the latter seroconverted 4 and 6 weeks later, respectively, and later shed virus into their WMF. The naturally infected animals were paired with naive marmosets, and were able to pass on infection. These results establish that long-term, permissive EBV infection occurs in the common marmoset, and demonstrate again the similarities in the response to EBV between marmoset and man.

Animals↗