Rift Valley Fever Virus Infection in Golden Syrian Hamsters

Rift Valley fever virus (RVFV) is a formidable pathogen that causes severe disease and abortion in a variety of livestock species and a range of disease in humans that includes hemorrhagic fever, fulminant hepatitis, encephalitis and blindness. The natural transmission cycle involves mosquito vectors, but exposure can also occur through contact with infected fluids and tissues. The lack of approved antiviral therapies and vaccines for human use underlies the importance of small animal models for proof-of-concept efficacy studies. Several mouse and rat models of RVFV infection have been well characterized and provide useful systems for the study of certain aspects of pathogenesis, as well as antiviral drug and vaccine development. However, certain host-directed therapeutics may not act on mouse or rat pathways. Here, we describe the natural history of disease in golden Syrian hamsters challenged subcutaneously with the pathogenic ZH501 strain of RVFV. Peracute disease resulted in rapid lethality within 2 to 3 days of RVFV challenge. High titer viremia and substantial viral loads were observed in most tissues examined; however, histopathology and immunostaining for RVFV antigen were largely restricted to the liver. Acute hepatocellular necrosis associated with a strong presence of viral antigen in the hepatocytes indicates that fulminant hepatitis is the likely cause of mortality. Further studies to assess the susceptibility and disease progression following respiratory route exposure are warranted. The use of the hamsters to model RVFV infection is suitable for early stage antiviral drug and vaccine development studies.     

Effects of Route of Inoculation and Viral Genetic Variation on Antibody Responses to Polyomavirus SV40 in Syrian Golden Hamsters

Genetic variants of polyomavirus SV40 are powerful agents with which to define viral effects on cells and carcinogenesis pathways. We hypothesized that differences in biologic variation among viral strains affect the process of viral infection and are reflected in antibody responses to the viral nonstructural large T-antigen (TAg) protein but not in neutralizing antibody responses against the inoculated viral particles. We analyzed the production of TAg antibody and neutralizing antibody in Syrian golden hamsters that were inoculated with SV40 viral strains by intracardiac, intravenous, or intraperitoneal routes and remained tumor free. Compared with the intraperitoneal route, intravascular (that is, intravenous, intracardiac) inoculation resulted in increased frequency of responsiveness to TAg but not in higher TAg antibody titers. The intravascular route was superior both for eliciting neutralizing antibody responses and for higher titers of those responses. Viruses with complex regulatory regions induced TAg antibody more often than did viruses with simple regulatory regions after intraperitoneal but not intravascular injections, with no differences in antibody titers. This viral genetic variation had no effect on neutralizing antibody production after intraperitoneal or intravascular inoculations or on neutralizing antibody titers achieved. These findings confirm that SV40 variants differ in their biologic properties. Route of inoculation combined with viral genetic variation significantly influence the development of serum antibodies to SV40 TAg in tumor-free hamsters. Route of inoculation—but not viral genetic variation—is an important factor in production of neutralizing antibody to SV40.Abbreviations: TAg, large T antigenPolyomavirus SV40 was discovered as an unrecognized contaminant of early poliovaccines³⁸ and was shown promptly to be an oncogenic virus.^9,15,17,18 Syrian golden hamsters are the small animal model that is susceptible to SV40 tumorigenicity.^{7,9-13,18,27,35} Since its discovery, SV40 has proven to be an outstanding tool for the discovery of mechanisms underlying carcinogenesis and for viral influences on cellular processes.^{1,3,5,19,23,30}Genetic variants of SV40 exist.^{16,20,26,28,33,34,36,37,41} This variation typically occurs in the viral regulatory region, in which some strains have duplications or rearrangements (or both) in the enhancer region, and in the C-terminal region of the large T-antigen (TAg) gene, in which nucleic acid variations may result in amino acid changes in the protein. TAg is an essential viral replication protein and the major viral oncoprotein. An important question is whether SV40 viral variants differ in their biologic properties, including in host responses to infections, as this could affect the spectrum of viral disease pathogenesis. We previously have shown that the viral regulatory region influences SV40 tumor induction in hamsters^35,40 and vertical transmission of the virus in hamsters²⁹ and that the route of inoculation influences SV40-mediated carcinogenesis.²⁷ Because TAg is not a component of the virus particle but instead is synthesized in virus-infected cells, we hypothesized that differences in antibody responses to TAg reflect biologic variation among viral strains with respect to the process of viral infection. In contrast, we expected that neutralizing antibody responses arise primarily against the injected viral particles, which represent a single serotype, and therefore are less informative about viral variation. We report here that an analysis revealed that the route of inoculation—in combination with viral genetic variation—significantly influences the development of serum antibodies to SV40 large TAg but not the titer of those antibodies in virus-exposed, tumor-free hamsters. The route of inoculation—but not viral genetic variation—influenced both the frequency of development and the titers of virus-neutralizing antibodies in the same animals.     

Efficient Gene Targeting in Golden Syrian Hamsters by the CRISPR/Cas9 System

The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)—three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C—and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN) and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO) hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease.     

Oral Probiotic Microcapsule Formulation Ameliorates Non-Alcoholic Fatty Liver Disease in Bio F1B Golden Syrian Hamsters

The beneficial effect of a microencapsulated feruloyl esterase producing Lactobacillus fermentum ATCC 11976 formulation for use in non-alcoholic fatty liver disease (NAFLD) was investigated. For which Bio F₁B Golden Syrian hamsters were fed a methionine deficient/choline devoid diet to induce non-alcoholic fatty liver disease. Results, for the first time, show significant clinical benefits in experimental animals. Examination of lipids show that concentrations of hepatic free cholesterol, esterified cholesterol, triglycerides and phospholipids were significantly lowered in treated animals. In addition, serum total cholesterol, triglycerides, uric acid and insulin resistance were found to decrease in treated animals. Liver histology evaluations showed reduced fat deposits. Western blot analysis shows significant differences in expression levels of key liver enzymes in treated animals. In conclusion, these findings suggest the excellent potential of using an oral probiotic formulation to ameliorate NAFLD.     

Foodborne Transmission of Nipah Virus in Syrian Hamsters

Since 2001, outbreaks of Nipah virus have occurred almost every year in Bangladesh with high case-fatality rates. Epidemiological data suggest that in Bangladesh, Nipah virus is transmitted from the natural reservoir, fruit bats, to humans via consumption of date palm sap contaminated by bats, with subsequent human-to-human transmission. To experimentally investigate this epidemiological association between drinking of date palm sap and human cases of Nipah virus infection, we determined the viability of Nipah virus (strain Bangladesh/200401066) in artificial palm sap. At 22°C virus titers remained stable for at least 7 days, thus potentially allowing food-borne transmission. Next, we modeled food-borne Nipah virus infection by supplying Syrian hamsters with artificial palm sap containing Nipah virus. Drinking of 5×10⁸ TCID₅₀ of Nipah virus resulted in neurological disease in 5 out of 8 hamsters, indicating that food-borne transmission of Nipah virus can indeed occur. In comparison, intranasal (i.n.) inoculation with the same dose of Nipah virus resulted in lethal respiratory disease in all animals. In animals infected with Nipah virus via drinking, virus was detected in respiratory tissues rather than in the intestinal tract. Using fluorescently labeled Nipah virus particles, we showed that during drinking, a substantial amount of virus is deposited in the lungs, explaining the replication of Nipah virus in the respiratory tract of these hamsters. Besides the ability of Nipah virus to infect hamsters via the drinking route, Syrian hamsters infected via that route transmitted the virus through direct contact with naïve hamsters in 2 out of 24 transmission pairs. Although these findings do not directly prove that date palm sap contaminated with Nipah virus by bats is the origin of Nipah virus outbreaks in Bangladesh, they provide the first experimental support for this hypothesis. Understanding the Nipah virus transmission cycle is essential for preventing and mitigating future outbreaks.     

The Use of Space in Caged Golden Hamsters*

Das Verhalten des Goldhamsters in seinem Heimkäfig wurde untersucht. Obwohl die Tiere auf engem Raum lebten, war ihr Verhalten räumlich und zeitlich geordnet. Die Hamster fraßen und putzten sich bevorzugt am Schlafplatz, meistens während der Ruhezeit des Tages und besonders häufig kurz vor dem Einschlafen. Einige hatten einen besonderen, regelmäßig benutzten Freßplatz; auch putzten sie sich manchmal an anderen, anscheinend zufällig gewählten Orten. Kot entleerten sie überwiegend am Freßplatz, aber auch im ganzen übrigen Käfig verstreut. Zum Harnen suchten sie dagegen stets dieselbe, vom Schlafplatz abgelegene Stelle auf.     

Response of Gut Microbiota to Fasting and Hibernation in Syrian Hamsters

Although hibernating mammals wake occasionally to eat during torpor, this period represents a state of fasting. Fasting is known to alter the gut microbiota in nonhibernating mammals; therefore, hibernation may also affect the gut microbiota. However, there are few reports of gut microbiota in hibernating mammals. The present study aimed to compare the gut microbiota in hibernating torpid Syrian hamsters with that in active counterparts by using culture-independent analyses. Hamsters were allocated to either torpid, fed active, or fasted active groups. Hibernation was successfully induced by maintaining darkness at 4°C. Flow cytometry analysis of cecal bacteria showed that 96-h fasting reduced the total gut bacteria. This period of fasting also reduced the concentrations of short chain fatty acids in the cecal contents. In contrast, total bacterial numbers and concentrations of short chain fatty acids were unaffected by hibernation. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments indicated that fasting and hibernation modulated the cecal microbiota. Analysis of 16S rRNA clone library and species-specific real-time quantitative PCR showed that the class Clostridia predominated in both active and torpid hamsters and that populations of Akkermansia muciniphila, a mucin degrader, were increased by fasting but not by hibernation. From these results, we conclude that the gut microbiota responds differently to fasting and hibernation in Syrian hamsters.Some mammalian species have evolved with the physiological phenomenon of hibernation to survive unfavorable winter environments (9). Hibernation is realized by entering torpor in order to eliminate the need to maintain a constant, high body temperature. During torpor, typical hibernating mammals, such as hamsters and ground squirrels, lower their body temperature to only a few degrees above ambient temperatures to reduce energy expenditure. Torpor is interrupted by periods of intense metabolic activity. During these interbout arousals, physiological parameters are restored rapidly to near-normal levels. Thus, hibernators alternate between hypothermic and euthermic states during hibernation.Some hibernating mammals awake to forage during torpor, while food-storing hibernators such as hamsters eat cached food during interbout arousals. However, hibernation essentially involves periods of fasting. Fasting is known to affect the gut microbiota in nonhibernating mammals such as mice (12); therefore, it is possible that hibernation also influences the gut microbiota. Given that the gut microbiota plays important roles in mammalian tissue development and homeostasis (28), it was of interest to investigate the changes in the gut microbiota that may take place during hibernation. To date, this issue has received little attention; to our knowledge, there are only two reports on the gut microbiota in hibernating mammals. Schmidt et al. showed that although the total counts of coliforms, streptococci, and psychrophilic organisms in the feces of arctic ground squirrels held in a cold room at 3°C remained constant the composition changed, with a decrease in coliform count and a 1,000-fold increase in the number of aerobic psychrophilic gram-negative bacteria (31). Barnes and Burton reported that although there was some reduction in total numbers of viable bacteria in the cecum during hibernation, composition of the microbiota remained stable (6). In terms of amphibians, Banas et al. and Gossling et al. reported a reduction and compositional changes of the gut microbiota in hibernating leopard frogs (4, 5, 18, 19).Only 20 to 40% of bacterial species from the mammalian intestinal tract can be cultured and identified using classical culture methods (22, 34, 36). In contrast, culture-independent methods based on the amplification of bacterial 16S rRNA genes by PCR have revealed a great diversity of microbiota in environmental samples (3, 37). The present study compared the gut microbiota in hibernating torpid Syrian hamsters with that in active counterparts by using culture-independent analyses.     

Conspicuous sex difference in zona reticularis of the adrenal cortex of Syrian Hamsters

The adrenal weight of adult male hamsters was significantly heavier (p = less than 0.001) than those of age-matched females. The increased weight was due to an increased thickness of the adrenal cortex, especially of the zona reticularis. The width of the zona reticularis was approximately three times greater in males than females.     

Diurnal Sensitivity of the Neuroendocrine-Reproductive Axis to the Antigonadotrophic Influence of Melatonin in Male Syrian Hamsters with Experimentally Altered Cortisol Rhythms

Two different experimental models were used to test if a temporal relationship exists between the rhythm of adrenal steroid secretion and the vulnerability of the hamster reproductive system to short photoperiod exposure or to the daily afternoon injection of melatonin. In the first experiment adrenalectomized hamsters were implanted with a Cortisol pellet to provide a sustained, rather than rhythmic, level of the hormone. The animals were either placed in short photoperiod or given a daily afternoon melatonin injection. In both cases the gonads underwent atrophy. In the second experiment adrenalectomized hamsters were given a Cortisol injection either in the morning (approx. 8 hr before the subsequent afternoon injection of melatonin) or in the afternoon (approx. 1 hr before the subsequent melatonin injection). Measurements of testicular and accessory organ weights 7 weeks later indicated regression of the reproductive system in both the groups when compared with their appropriate controls. Depressed levels of plasma LH. PRL, testosterone and thyroxine (T4) in these animals confirmed the melatonin induced gonadal collapse. The results suggest that apparently there is no temporal correlation between the rhythm of secretion of the adrenal steroids and the responsiveness of the reproductive system to late afternoon injection of melatonin. Interestingly, all the adrenalectomized Cortisol injected control animals (not receiving melatonin) had depressed plasma LH and PRL while the testicular weights and plasma testosterone titers remain unaffected.     

Involvement of PSMD10, CDK4, and Tumor Suppressors in Development of Intrahepatic Cholangiocarcinoma of Syrian Golden Hamsters Induced by Clonorchis sinensis and N-Nitrosodimethylamine

Background

Clonorchis sinensis is a group-I bio-carcinogen for cholangiocarcinoma (CCA). Although the epidemiological evidence links clonorchiasis and CCA, the underlying molecular mechanism involved in this process is poorly understood. In the present study, we investigated expression of oncogenes and tumor suppressors, including PSMD10, CDK4, p53 and RB in C. sinensis induced hamster CCA model.

Methods

Different histochemical/immunohistochemical techniques were performed to detect CCA in 4 groups of hamsters: uninfected control (Ctrl.), infected with C. sinensis (Cs), ingested N-nitrosodimethylamine (NDMA), and both Cs infected and NDMA introduced (Cs+NDMA). The liver tissues from all groups were analyzed for gene/protein expressions by quantitative PCR (qPCR) and western blotting.

Principal Findings

CCA was observed in all hamsters of Cs+NDMA group with well, moderate, and poorly differentiated types measured in 21.8% ± 1.5%, 13.3% ± 1.3%, and 10.8% ± 1.3% of total tissue section areas respectively. All CCA differentiations progressed in a time dependent manner, starting from the 8^th week of infection. CCA stroma was characterized with increased collagen type I, mucin, and proliferative cell nuclear antigen (PCNA). The qPCR analysis showed PSMD10, CDK4 and p16INK4 were over-expressed, whereas p53 was under-expressed in the Cs+NDMA group. We observed no change in RB1 at mRNA level but found significant down-regulation of RB protein. The apoptosis related genes, BAX and caspase 9 were found downregulated in the CCA tissue. Gene/protein expressions were matched well with the pathological changes of different groups except the NDMA group. Though the hamsters in the NDMA group showed no marked pathological lesions, we observed over-expression of Akt/PKB and p53 genes proposing molecular interplay in this group which might be related to the CCA initiation in this animal model.

Conclusions/Significance

The present findings suggest that oncogenes, PSMD10 and CDK4, and tumor suppressors, p53 and RB, are involved in the carcinogenesis process of C. sinensis induced CCA in hamsters.     

Infectivity of Leishmania donovani Amastigotes and Promastigotes for Golden Hamsters*

SYNOPSIS. Intracardial injection of hamsters with from 5 to 114 million amastigotes or promastigotes of Leishmania donovani and screening of the 8th-day liver impression smears, provides a rapid and reproducible method for assaying infectivity. Amastigotes are at least 10X more infective than promastigotes, and log-phase promastigotes act as a single infective population for hamsters.     

Regulation of Intestinal Immune Response by Selective Removal of the Anterior,Posterior, or Entire Pituitary Gland in Trichinella spiralis Infected Golden Hamsters

The influence of anterior pituitary hormones on the gastrointestinal tract of humans and animals has been previously reported. Hypophysectomy (HYPOX) in the rat causes atrophy of the intestinal mucosa, and reduction of gastric secretion and intestinal absorption, as well as increased susceptibility to bacterial and viral infections. However, to our knowledge, no findings have been published concerning the immune response following HYPOX during worm infection, particularly that which is caused by the nematode Trichinella spiralis. The aim of this work was to analyze the effects of total or partial HYPOX on colonization of T. spiralis in the intestinal lumen, together with duodenal and splenic cytokine expression. Our results indicate that 5 days post infection, only neurointermediate pituitary lobectomy (NIL) reduces the number of intestinally recovered T. spiralis larvae. Using semiquantitative inmunofluorescent laser confocal microscopy, we observed that the mean intensity of all tested Th1 cytokines was markedly diminished, even in the duodenum of infected controls. In contrast, a high level of expression of these cytokines was noted in the NIL infected hamsters. Likewise, a significant decrease in the fluorescence intensity of Th2 cytokines (with the exception of IL-4) was apparent in the duodenum of control and sham infected hamsters, compared to animals with NIL surgeries, which showed an increase in the expression of IL-5 and IL-13. Histology of duodenal mucosa from NIL hamsters showed an exacerbated inflammatory infiltrate located along the lamina propria, which was related to the presence of the parasite. We conclude that hormones from each pituitary lobe affect the gastrointestinal immune responses to T. spiralis through various mechanisms.     

Effect of conjugated linoleic acid isomers on lipoproteins and atherosclerosis in the Syrian Golden hamster

Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of linoleic acid (LA, C18:2 cis-9, cis-12) that are reported to have important biological activities, including protection against atherosclerosis. In this study, the potential role of the individual cis-9, trans-11 and trans-10, cis-12 isomers of CLA in atherogenesis were compared with LA in the Syrian Golden hamster. Supplementation of a high-fat, high-cholesterol diet (HFHC) with 1% (w/w) cis-9, trans-11 CLA or trans-10, cis-12 CLA did not significantly affect plasma cholesterol levels compared to supplementation with 1% (w/w) LA. Very low density lipoprotein cholesterol (VLDL-C) was lower and plasma triglycerides (TG) were higher in diets where C18:2 fatty acid was added to the HFHC diet, but neither the cis-9, trans-11 CLA group nor trans-10, cis-12 CLA group was significantly different from the LA control group. CLA supplementation did not significantly affect low density lipoprotein cholesterol (LDL-C). Trans-10, cis-12 CLA increased high density lipoprotein cholesterol (HDL-C) levels compared to LA or cis-9, trans-11 CLA (P<0.02), and although the ratio of non-HDL-C:HDL-C in the cis-9, trans-11 CLA group (1.11+/-0.54) and the trans-10, cis-12 CLA group (1.11+/-0.21) was lower than the LA group (1.29+/-0.45), the reduction did not reach statistical significance. Atherosclerosis was assessed in the ascending aorta by measuring the number of aortic cross-sections containing Oil Red O-stained intimal lesions. Compared to the LA group (60+/-11%), both the cis-9, trans-11 CLA group (38+/-8%) and the trans-10, cis-12 CLA group (28+/-7%) had fewer sections displaying a fatty streak lesion, although the differences did not reach statistical significance. These results suggest that individual CLA isomers may reduce atherosclerotic lesion development in the hamster, but when compared to LA, the apparent atheroprotective effects do not correlate with beneficial changes in lipoprotein profile.     

Genetic studies of the Syrian hamster

A new mutant gene,anophthalmic white, is described for the Syrian hamster. The gene is inherited as a dominant to normal and, when homozygous, produces a characteristic syndrome of achromia and anophthalmia or microphthalmia. The heterozygote possesses white belly fur (instead of cream), a fine sprinkling of white hairs in the adult coat and a diminution of eye pigmentation. An occasional heterozygote may possess a small patch of unpigmented fur on the head or body. The new mutant does not appear to be linked with the gene forcream coat colour nor that forpiebald spotting. The significance of homologous mutants, with the above syndrome, in the mouse and hamster is briefly discussed.     

Resetting the Annual Cycle with Timed Daily Injections of 5-Hydroxytryptophan and L-Dihydroxyphenylalanine in Syrian Hamsters

Daily injections of dihydroxyphenylalanine (DOPA), a precursor for dopamine synthesis, given 12 hr after daily injections of S-hydroxytryptophan (5-HTP), a serotonin precursor, induced uterine growth and increased serum thyroxine and luteinizing hormone (LH) concentrations in scotosensitive female hamsters maintained on short day lengths. On the other hand, daily injections of DOPA given at the same time as daily injections of S-HTP reduced uterine weights and serum concentrations of thyroxine and LH in scotorefractory female hamsters. These results indicate that the endogenous mechanism controlling seasonality (scotosensitivity and scotorefractoriness) in the Syrian hamster may be reset by drugs that influence serotonergic and catecholaminergic activity. This seasonal mechanism might involve two circadian systems that undergo seasonal changes in their phase relations.     

Testosterone Differentially Influences Sex-Specific Pheromone-Stimulated Fos Expression in Limbic Regions of Syrian Hamsters

The results of the present study indicate that (1) pheromones differentially stimulate neurons in males and females within a pathway that regulates copulatory behavior; and (2) testosterone (T) differentially regulates these sex differences. Exposure to the pheromones in FHVS (female hamster vaginal secretions) induces Fos immunoreactivity (Fos-IR) in the posterior subdivision of the medial nucleus of the amygdala (MeP) and the posteromedial subdivision of the bed nucleus of the stria terminalis (BNSTpm) of both sexes and stimulates the magnocellular subdivision of the medial preoptic nucleus (MPNmag) in males but not in females. Males also show more Fos in the MeP and BNSTpm than females. In the absence of T, gonadectomized males show greater FHVS-stimulated Fos-IR in the BNSTpm and MeP than gonadectomized females. T in females eliminates the sex difference in these regions. Only T-treated males show FHVS-stimulated Fos-IR within the MPNmag, and T has no effect on FHVS-stimulated Fos-IR within MPNmag in females. Thus, T influences FHVS-stimulated Fos-IR in the BNSTpm and MeP of females and the MPNmag of males. T also increases investigation (sniffing and licking) of FHVS in both males and females, but increases copulatory responses only in males. Our results indicate that T in the adult hamster differentially influences neural and behavioral responses to pheromone exposure in males and females. T only partially accounts for observed sex differences, and it is likely that neural organization during development also plays a role in influencing responses to pheromones.     

Effect of maternal hypercholesterolemia on fetal sterol metabolism in the Golden Syrian hamster.

The fetus obtains a significant amount of cholesterol from de novo synthesis. Studies have suggested that maternal cholesterol may also contribute to the cholesterol accrued in the fetus. Thus, the present studies were completed to determine whether diet-induced maternal hypercholesterolemia would affect fetal sterol metabolism. To accomplish this, maternal plasma cholesterol concentrations were increased sequentially by feeding hamsters 0.0%, 0.12%, 0.5%, and 2.0% cholesterol. At 11 days into a gestational period of 15.5 days, cholesterol concentrations and sterol synthesis rates were measured in the three fetal tissues: the placenta, yolk sac, and fetus. In the placenta and yolk sac, the cholesterol concentration increased significantly when dams were fed as little as 0.12% cholesterol (P < 0.0167), and sterol synthesis rates decreased in dams fed at least 0.5% or 2% cholesterol, respectively (P < 0.0167). In the fetus, changes in fetal cholesterol concentration and sterol synthesis rates occurred only when dams were fed at least 0.5% cholesterol, which corresponded to a greater than 2-fold increase in maternal plasma cholesterol concentrations. When the cholesterol concentration in the fetal tissues in each animal was plotted as a function of maternal plasma cholesterol concentration, a linear relationship was found (P < 0.001).These studies demonstrate that sterol homeostasis in fetal tissues, including the fetus, is affected by maternal plasma cholesterol concentration in a gradient fashion and that sterol metabolism in the fetus is dependent on sterol homeostasis in the yolk sac and/or placenta.