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Developmental changes in luteinizing hormone/human chorionic gonadotropin steroidogenic responsiveness in marmoset granulosa cells: effects of follicle-stimulating hormone and androgens.

Factors regulating LH/hCG responsiveness in primate granulosa cells were examined in the marmoset monkey (Callithrix jacchus). Granulosa cells were isolated and pooled from small antral (0.5-1.0 mm) and large preovulatory (greater than or equal to 2 mm) follicles from mid- to late follicular phase ovaries of cyclic marmosets. The cells from small and large follicles were cultured in serum-free medium for 48 h in the absence or presence of increasing concentrations of hCG (0.1-100 ng/ml) with or without 0.1 microM androgen [testosterone or 5 alpha-dihydrotestosterone (DHT]). Granulosa cells from small follicles were also cultured in the absence or presence of a constant concentration of human FSH (30 ng/ml) with or without androgen for 48 h before exposure to hCG for an additional 48 h. Steroidogenic responsiveness was assessed by measuring progesterone accumulation in culture medium and aromatase activity in washed monolayers. Granulosa cells from large follicles showed dose-dependent increases in both progesterone accumulation and aromatase activity in response to treatment with hCG. In contrast, granulosa cells from small follicles were unresponsive to hCG. However, pretreatment of granulosa cells from small follicles for 48 h with FSH stimulated hCG responsiveness. The effects of both testosterone and DHT on hCG-stimulated aromatase activity and progesterone accumulation by granulosa cells from large preovulatory follicles were inhibitory. Testosterone and DHT also suppressed basal (no hCG) progesterone accumulation in these cells, but had no effect on basal aromatase activity. The effects of androgens on FSH-induced hCG responsiveness in immature granulosa cells were variable. The results show a development-related increase in marmoset granulosa cell responsiveness to LH/hCG and provide evidence that FSH and androgens interact to regulate the onset and expression of this critical event during preovulatory follicular development in the primate ovary.

Androgens↗

Prevention of thecal angiogenesis, antral follicular growth, and ovulation in the primate by treatment with vascular endothelial growth factor Trap R1R2.

This study was designed to investigate the effects of inhibition of thecal angiogenesis on follicular development in the marmoset monkey (Callithrix jacchus). To inhibit vascular endothelial growth factor (VEGF), a soluble combined truncated form of the fms-like tyrosine kinase (Flt) and kinase insert domain-containing receptor (KDR) receptor fused to IgG (VEGF Trap R1R2) was administered for 10 d during the follicular phase of the cycle. Changes in angiogenesis and follicular cell proliferation were quantified using immunocytochemistry for bromodeoxyuridine to obtain a proliferation index, CD31 to visualize endothelial cell area, and dual staining to distinguish thecal endothelial cell proliferation. The effects of the treatment on follicular development were assessed by morphometric analyses by measuring follicle diameter, thecal thickness, and a proliferation index for granulosa cells. Follicular atresia was detected and quantified using the terminal deoxynucleotidyltransferase-UTP nick end labeling method. Effects on gene expression of VEGF and its receptors, Flt and KDR, were studied by in situ hybridization. VEGF Trap R1R2 treatment resulted in a significant decrease in thecal proliferation and endothelial cell area, demonstrating the suppression of thecal angiogenesis. The absence of a normal thecal vasculature was associated with a significantly reduced thecal thickness. Antral follicular development was severely compromised, as indicated by decreased granulosa cell proliferation, decreased follicular diameter, and lack of development of ovulatory follicles. Furthermore, the rate of atresia was significantly increased. VEGF expression in granulosa and thecal cells increased after treatment, whereas Flt and KDR expressions in thecal endothelial cells were markedly decreased. These results show that VEGF Trap treatment is associated with the suppression of follicular angiogenesis, which results in the inhibition of antral follicular development and ovulation.

Angiogenesis Inhibitors↗

Relative effects of activin and inhibin on steroid hormone synthesis in primate granulosa cells.

Ovarian granulosa cells produce inhibin and activin, structurally related proteins with potentials to directly modulate follicular steroidogenesis. The aim of the present study was to compare development-related effects of inhibin-A and activin-A on steroidogenesis in marmoset monkey (Callithrix jacchus) granulosa cells. Granulosa cells from "immature" (< 1.0 mm diameter) and "mature" (> 2 mm diameter) follicles were incubated in serum-free culture medium for 96 h with and without peptide (1-100 ng/mL), in the presence and absence of gonadotropins [human (h) FSH or hLH] (10 ng/mL). Spent medium was collected and stored frozen for progesterone assay. Aromatase activity was determined by incubating cells for a further 6 h in the presence of 1 mumol testosterone and assaying accumulation of oestradiol. Granulosa cells from immature follicles showed characteristically low basal rates of steroid synthesis that were unaffected by treatment alone with either inhibin or activin. Treatment with hFSH stimulated both progesterone production and aromatase activity. Cotreatment with activin and hFSH further enhanced aromatase activity by up to 4-fold. The progesterone response to activin plus hFSH was related to the effect of hFSH in the absence of activin: high-level responsiveness to hFSH was suppressed by activin while low-level responsiveness was enhanced. Inhibin had no significant effect on FSH-responsive progesterone production, but at high concentrations (> 10 ng/mL) it caused slight (up to 30%) reduction in FSH-induced aromatase activity. Granulosa cells from mature follicles showed relatively high basal rates of steroidogenesis, and treatment with inhibin did not influence either basal or gonadotropin responsive steroidogenesis. Treatment with activin had divergent effects on aromatase activity and progesterone synthesis in that it increased both basal and hLH-responsive aromatase activity (up to 11-fold), had no effect on basal progesterone production, and markedly suppressed (by more than 50%) the progesterone response to hLH. These data reveal development-dependent effects of inhibin and activin on granulosa cell steroidogenesis that are likely to have physiological relevance to ovarian function in vivo.

Activins↗

Acoustic communication in noise: regulation of call characteristics in a New World monkey.

This study on common marmosets Callithrix jacchus is the first to examine noise-dependent mechanisms of vocal plasticity in a New World monkey. Since acoustic communication can be considerably impaired by environmental noise, some animals have evolved adaptations to counteract its masking effects. The studied marmosets increased the sound level of their spontaneous calls in response to increased levels of white noise broadcast to them. Possibly, such noise-dependent adjustment of vocal amplitude serves to maintain a specific signal-to-noise ratio that is favourable for signal production. Concurrently, the adjustment of vocal amplitude can maintain a given active space for communication. In contrast to some bird species, no noise-induced increase in the number of syllables per call series could be found, showing that an increased serial redundancy of vocal signals was not used to communicate under noisy conditions. Finally, we examined a possible noise-dependent prolongation of vocal signals. This approach was guided by the findings of perceptional studies, which suggest an increased detection probability of prolonged signals in noise by temporal summation. Marmosets indeed increased the duration of their call syllables along with increasing background noise levels. This is the first evidence of such mechanism of vocal plasticity in an animal communication system.

Adaptation, Physiological↗

Novel restraint system for neuroendocrine studies of socially living common marmoset monkeys.

We describe a novel soft jacket and sling-harness restraint that permits species-typical postures for small-bodied primates, such as the common marmoset (Callithrix jacchus), during long-term (>6 h), continuous restraint. The restraint system is straightforward to use and manipulate, it is easily repaired, and the materials used are readily available. The soft jacket allows for increased versatility and longevity, and the sling-harness provides for greater movement and much longer duration of continuous restraint (up to 3 days) compared to a previously described, more conventional chair restraint for small-bodied primates. The new restraint system prevents the normal diurnal decrease in plasma cortisol levels across the daylight hours; however, it does not disrupt ovulatory cycles. Unlike the previously available techniques, therefore, this new restraint system is applicable to many neurobiological and neuroendocrine studies involving small-bodied, non-human primates and is especially suited to investigations requiring the maintenance of relationships within social groups.

Animals↗

Effects of fibre on digestibility and passage time in callithricidae.

The effects of fibre in a pelleted diet on food intake, digestibility of crude fibre, dry matter and energy, on passage time and consistency of faeces were studied in 2 species of Callithricidae, Callithrix jacchus and Saguinus fuscicollis. Coarse cellulose, microcellulose, wheat bran and shrimp meal (chitin = crude fibre) were tested in diets containing 2,4 and 6% total crude fibre, respectively. Digestibility and passage time were determined by inclusion of 0.5% Cr2O3 in the diet. Both celluloses had little influence on the digestibility of energy and dry matter. Digestibility of crude fibre was very low. Wheat bran led to evident depression of energy and dry matter digestibility. High digestibility of crude fibre occurred at the higher levels of inclusion in the diet. Shrimp meal was highly digested with little influence on digestibility of energy and dry matter, indicating considerable degradation of chitin. Wheat bran showed a marked effect, while microcellulose had no effect on passage time, consistency and volume of faeces.

Animal Feed↗

Palatability and digestibility of soya- and milk proteins in Callithricidae.

4 protein sources were tested for palatability, digestibility, influence on bodyweight and consistency of faeces in Callithrix jacchus and Saguinus fuscicollis. The protein sources were incorporated at crude protein levels of 12, 17 and 22% in a pelleted diet. A banana supplement was also fed which reduced the total protein content to 10.7, 14.7 and 18.3%. The test diets, fed for 3 weeks, were well accepted with the milk proteins being slightly preferred. Consumption of the 12% protein pellets was greater than their 22% equivalents. True protein digestibility was calculated to be 97% for lactalbumin, 96% for casein, 90% for soy protein concentrate and 83% for soyabean meal. Changes in bodyweight were negligible. Dry matter of faeces varied between 25% (lactalbumin diets) and 28% (casein diets), but did not change with increasing protein intake. Increasing levels of cellulose, from 2.4 to 7.2% total crude fibre lowered the apparent protein digestibility from 84.3 to 79.2% and the digestibility of dry matter from 82.2 to 76.5%.

Animals↗

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.

Animals↗

Monitoring and controlling reproduction in captive common marmosets on the basis of urinary oestrogen metabolites.

Non-invasive methods for routine monitoring of reproductive states and reproduction control in large colonies of captive Callithrix jacchus have been developed. Immunoactive urinary oestrone-3-conjugates (E1C) were measured during non-conception cycles (n = 5) and pregnancy (n = 7). Using plasma progesterone levels to time ovulation, ovulation was quantitatively estimated by a) calculating the first E1C rise and b) by establishing an E1C threshold. Ovulation was thus defined as taking place 4 days preceding a) the first rise of E1C above follicular-phase levels, or b) a concentration > or = 4.5 micrograms E1C/mg creatinine. Early pregnancy could be determined after day 20 by continued luteal-phase levels of E1C. Secondly, the luteolytic effect of cloprostenol, injected over a wide range of doses and between days 1 and 64 after ovulation/conception, was analysed. Luteolysis was achieved when cloprostenol was administered after day 5 post-ovulation; the luteolytic effect was found not to be dose-dependent. The success of cloprostenol treatment was 87% as confirmed by endocrine monitoring. The methods described are effective and minimize intervention, and are therefore suitable for long-term applications, particularly in combination with behavioural studies.

Animals↗

Systemic AA amyloidosis in the common marmoset.

The common marmoset (Callithrix jacchus) is a small New World primate native to Brazil that has been used extensively in biomedical research. A retrospective analysis of archived hematoxylin and eosin-stained tissue sections and clinical records was conducted at the New England Primate Research Center on 86 marmosets more than 1 year of age that were euthanized during the past decade because of morbidity and failure to thrive. Approximately 17% (15 of 86) were found to have amyloid deposits in one or more organs, including the liver, adrenal glands, kidneys, and intestine. This material was shown by amino acid sequence analysis to be composed of serum amyloid A (SAA)-related protein. This type of amyloidosis, designated AA or "secondary," is associated typically with an inflammatory process that induces elevated levels of the SAA amyloidogenic precursor molecule. Notably, there were no significant pathologic differences or other distinguishing features in animals with amyloid versus those without; furthermore, on the basis of the limited number of serum specimens available for analysis, the SAA concentrations in the two groups were comparable, thus suggesting the possible inheritable nature of the disorder. In this respect, the common marmoset provides a unique experimental model for study of the pathogenesis and treatment of AA and other forms of systemic amyloidosis.

Amino Acid Sequence↗

The common marmoset monkey as a model for implantation and early pregnancy research.

This chapter describes methods used to investigate implantation in the common marmoset monkey, Callithrix jacchus. A reverse-transcriptase polymerase chain reaction-strategy with which to detect transcripts for steroid receptors and enzymes involved in estradiol biosynthesis is described, and an immunohistochemistry approach for detecting proteins within the implantation site is presented.

17-Hydroxysteroid Dehydrogenases↗

Random wiring in the midget pathway of primate retina.

The present study addresses the questions of how topographically organized neuronal populations are connected, and whether there is anatomical evidence for color-selective wiring in retinal pathways for red-green color vision. The connectivity of OFF midget bipolar and OFF midget ganglion cells was studied in the peripheral retina of dichromatic ("red-green color blind") and trichromatic ("color normal") marmosets (Callithrix jacchus). Midget bipolar cells were identified immunohistochemically. Midget ganglion cells were retrogradely labeled from the lateral geniculate nucleus and photofilled. Comparable results were obtained from all retinas studied. Between 3 and 16 bipolar terminals converge onto each ganglion cell. Nearly all bipolar terminals investigated show regions of colocalization (areas of presumed synaptic contacts) with ganglion cell dendrites. This contact area makes up approximately 14% of the axon surface area for a typical midget bipolar cell. The output from individual midget bipolar axons is often shared between midget ganglion cells so that, on average, <70% of the axon terminal area of a midget bipolar cell shows overlap with the dendritic field of a given midget ganglion cell. We conclude that there is no morphological evidence of red-green color selectivity in the connections between midget bipolar and midget ganglion cell mosaics. Furthermore, the results suggest that convergence is based on local interactions between axons and dendrites rather than cell-by-cell recognition between members of each mosaic.

Animals↗

Plasticity in primary auditory cortex of monkeys with altered vocal production.

Response properties of primary auditory cortical neurons in the adult common marmoset monkey (Callithrix jacchus) were modified by extensive exposure to altered vocalizations that were self-generated and rehearsed frequently. A laryngeal apparatus modification procedure permanently lowered the frequency content of the native twitter call, a complex communication vocalization consisting of a series of frequency modulation (FM) sweeps. Monkeys vocalized shortly after this procedure and maintained voicing efforts until physiological evaluation 5-15 months later. The altered twitter calls improved over time, with FM sweeps approaching but never reaching the normal spectral range. Neurons with characteristic frequencies <4.3 kHz that had been weakly activated by native twitter calls were recruited to encode self-uttered altered twitter vocalizations. These neurons showed a decrease in response magnitude and an increase in temporal dispersion of response timing to twitter call and parametric FM stimuli but a normal response profile to pure tone stimuli. Tonotopic maps in voice-modified monkeys were not distorted. These findings suggest a previously unrecognized form of cortical plasticity that is specific to higher-order processes involved in the discrimination of more complex sounds, such as species-specific vocalizations.

Acoustic Stimulation↗

GnRH-II receptor-like antigenicity in human placenta and in cancers of the human reproductive organs.

We have recently demonstrated that the antiproliferative activity of GnRH-II on human endometrial and ovarian cancer cell lines is not mediated through the GnRH-I receptor. A functional receptor for human GnRH-II has not yet been identified. In this study, we have generated a polyclonal antiserum to the putative human GnRH-II receptor using a peptide (YSPTMLTEVPPC) corresponding to the third extracellular domain coupled to keyhole limpet haemocyanin via the Cys residue. A database search showed no identical peptide sequences in any other human gene. To avoid cross-reactions against two similar amino acid sequences the antiserum was pre-absorbed using these peptides. Immune histological sections of human placenta and human endometrial, ovarian and prostate cancers using rabbit anti-human GnRH-II receptor antiserum showed GnRH-II receptor-like staining. Western blot analysis of cell membrane preparations of human endometrial and ovarian cancer cell lines yielded a band at approximately 43 kDa whereas Western blot analysis of cell membrane preparations of ovaries obtained from the marmoset monkey (Callithrix jacchus) yielded a band at approximately 54 kDa. To identify the GnRH-II receptor-like antigen we used the photo-affinity labelling technique. Photochemical reaction of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II (10(-9) M) with cell membrane preparations of human endometrial and ovarian cancer cells yielded a band at approximately 43 kDa. In competition experiments, the GnRH-I agonist Triptorelin (10(-7) M) showed a weak decrease of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II binding to its binding site. The GnRH-I antagonist Cetrorelix (10(-7) M) showed a clearly stronger decrease, whereas GnRH-II agonist [d-Lys(6)]-GnRH-II (10(-7) M) was the most potent competitor. Western blot analysis of the same gel using rabbit anti-human GnRH-II receptor antiserum identified this band as GnRH-II receptor-like antigen.

Animals↗

Partial hepatectomy of marmoset: clinical and pathological effects and utility in microsomal enzyme analysis.

Liver biopsy based on a partial hepatectomy technique (shearing) was performed in 10 common marmosets (Callithrix jacchus). This is a preliminary study to evaluate the effects of drugs on hepatic microsomal enzymes: cytochrome P-450 and T4 uridine diphosphate glucuronyl transferase (T4-UDPGT), by comparing post-treatment values with pre-treatment values individually with a limited number of animals. The effects of the biopsy on clinical findings and liver pathology were evaluated during the first 5 post-surgical weeks. Although the plasma aspartate aminotransferase (AST) activities tended to decrease from 1 to 4 weeks post-surgery, no abnormality was noted in clinical sign, body weight, the hematocrit value or other blood chemical values. At necropsy, adhesion of the sheared site of the liver to the parietal peritoneum or the small intestine was evident in 2 of the 4 marmosets. Microscopic examination revealed focal fibrosis in the liver, but it was localized around the sheared site. Based on the above results, it was concluded that liver biopsy must be performed more than one month before administration of the drug to be tested. The biopsy samples and the whole liver samples obtained at autopsy were subjected to analysis of microsomal protein content, cytochrome P-450 content and T4-UDPGT activity. In comparison with the values from the whole liver samples, those from the biopsy samples showed no significant difference. Furthermore, there was a significant correlation rather than difference between matched values. This suggested that partial hepatectomy is a useful method for obtaining pretreatment values in liver biochemistry to evaluate the effects of drug-treatment in individual animals.

Alkaline Phosphatase↗

The haemoculture of Trypanosoma minasense Chagas, 1908.

Trypanosoma minasense was isolated for the first time in blood axenic culture from a naturally infected marmoset, Callithrix penicillata, from Brazil. The parasite grew profusely in an overlay of Roswell Park Memorial Institute medium plus 20% foetal bovine serum, on Novy, McNeal and Nicolle medium (NNN), at 27 degrees C, with a peak around 168 hr. The morphometry of cultural forms of T. minasense, estimates of cell population size and comparative growth in four different media overlays always with NNN, were studied. The infectivity of cultural forms to marmosets (C. penicillata and C. jacchus) and transformation of epimastigotes into metacyclic-like forms in axenic culture in the presence of chitin derivates (chitosan) were evaluated.

Animals↗

Polymorphism in trypomastigotes of Trypanosoma (Megatrypanum) minasense in the blood of experimentally infected squirrel monkey and marmosets.

Experimental infections by Trypanosoma (Megatrypanum) minasense were performed in primates - Saimiri sciureus and Callithrix penicillata - with the objective of searching for morphological variations of the blood trypomastigotes with respect to hosts and time of infection. We carried out morphological and morphometric analysis of blood trypomastigotes. Illustrations are given. Both the squirrel monkey and marmoset became infected after the injection of blood trypomastigotes of T. minasense, although the parasitaemia were briefer in the squirrel monkey. The parasites detected in the later host were narrower and shorter than those found in the inoculated marmoset. In the marmoset, the blood stream parasites derived from culture metacyclic trypomastigotes were considerably smaller than those derived from the inoculation of infected blood. Stronger evidence of polymorphism was found when, at the same time of infection, the blood trypomastigotes found in squirrel monkey had smaller length, body width and the distance from posterior end of the body to the kinetoplast almost four times smaller than the parasite found in the marmoset. Therefore, conflicting results on morphology and morphometry of T. minasense obtained by previous investigators could be due to polymorphism.

Animals↗