ILAR Journal Online, Volume 36(3/4) 1994: Advances in Gene Therapy

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ILAR Journal V36(3/4) 1994 [FORMERLY ILAR NEWS]
Advances in Gene Therapy

Animal Models

The First Documentation of Reproduction in a Genetically Obese Female fa/cp Rat
E. Bowie Kahle, Kenneth G. Butz, Otho E. Michaelis, Rudolph L. Leibel, and Carl T. Hansen

E. Bowie Kalale, Ph.D., is a professor in the Departments of Biology and Physiology at Marshall University School of Medicine, Huntington, West Virginia. Kenneth G. Butz, M.S., is a graduate assistant in the Department of Biology at Marshall University, Huntington, West Virginia. Otho E. Michaelis IV, is a research biologist at the Beltsville Human Nutrition Center, Agricultural Research Service, U.S. Department of Agriculture in Beltsville, Maryland, and adjunct professor in the Division of Renal Diseases, Department of Medicine, George Washington University, Washington, D.C. Rudolph L. Leibel, M.D., is professor of pediatrics and research biologist at the Laboratory of Human Behavior and Metabolism, The Rockefeller University, New York, New York. Carl T. Hansen, Ph.D., is a geneticist, NIH Animal Genetic Resource. Veterinary Resources Branch, The National Institutes of Health, Bethesda, Maryland.

INTRODUCTION

Prior to our study, no evidence of fertility has been published for genetically obese female fa/cp rats. Although Zucker and Zucker (1961) reported that obese female Zucker fa/fa rats were always sterile, Chelch and Edmonds (1981) found a small proportion of young obese Zucker fa/fa females (five of seventeen studied) to be fertile. Of the five obese females that became pregnant, only one actually delivered and successfully raised its litter.

Reproductive abnormalities causing sterility in obese female rats may include imbalances in estrogen secretion (Whitaker et al., 1983), reduced weight of the uteri and prolonged estrous cycles (Saiduddin et al., 1973).

Young genetically obese male rats are more likely to be fertile (Hemmes et al., 1978). Kava et al. (1990) reported that approximately 70% of obese males tested in the Zucker fatty rat colony at Vassar were fertile and willing breeders.

The fatty (fa) (Zucker and Zucker, 1961) and corpulent (cp) (Hansen, 1983) genes are two mutations in the rat genome that are known to cause obesity. The latter mutation was first observed in the Koletsky rat and designated f (Koletsky, 1973). Rats carrying the obesity trait from either mutation (fa or cp) are the product of an autosomal recessive mutation of a single gene (Zucker and Zucker, 1961; Hansen, 1983; Koletsky, 1973).

Interbreeding of heterozygous lean Zucker rats carrying the fa mutation (Zucker and Zucker, 1961) and Koletsky rats carrying the second mutation produced obese progeny (Yen et al., 1977). These progeny exhibited autosomal recessive obesity in a 3:1 lean:obese ratio. With these interstrain obese rats Yen et al. (1977) confirmed the single gene, recessive inheritance hypothesis and indicated that the two mutations appear to occur at the same genetic locus. These observations have not been repeated or confirmed with molecular genetic analysis.

The fa mutation, which arose spontaneously in the 13M Zucker rat (Zucker and Zucker, 1961), shows strong similarities to human obesity syndromes. The Zucker fatty is hyperlipemic, hypercholesterolemic (Zucker and Zucker, 1962, 1963), hyperinsulinemic (Zucker and Antoniades, 1972), and develops adipocyte hypertrophy and hyperplasia (Johnson et al., 1971). This outbred stock is now called ZUC-fa.

The cp mutation also arose spontaneously (Koletsky, 1973) and was introduced into the normotensive LA/N rat developed at the NIH, producing the congenic strain LA/N-cp (Hansen 1983). Obese LA/N-cp rats are normotensive and exhibit metabolic characteristics associated with type IV hyperlipoproteinemia (carbohydrate-sensitivity) in humans (Reiser et al., 1981) including normoglycemia or mild hyperglycemia, hypertriglyceridemia and basal hyperinsulinemia. Unlike the obese Zucker fatty rat, the lean tissue mass of obese LA/N-cp rats is similar to that of its lean littermates (Tulp, 1990).

Phenotypic characteristics often vary among strains of obese animals carrying the cp mutation (Koletsky rats, LA/ N-cp, SHR-cp, and WKY-cp) (Michaelis et al., 1988). In contrast to the LA/N-cp rats, obese male SHR/N-cp rats are mildly hypertensive and exhibit some metabolic and histopathological characteristics (Michaelis et al., 1988) associated with non-insulin dependent diabetes mellitus in humans (National Diabetes Data Group, 1979).

This paper presents the first documentation of reproduction in a genetically obese female fa/cp rat. The protocol during which the accidental mating of an obese female and her brother occurred was designed to evaluate the relationship between the fa and cp gene mutations through a series of interstrain crosses involving ZUC-fa and LA/N-cp rats.

MATERIALS AND METHODS

Heterozygous Vc:ZUC-fa/+) females (provided by Vassar College, Poughkeepsie, New York) were bred with LA/N-cp/+) males (provided by C. T. Hansen, NIH Animal Genetic Resources, Veterinary Resources Branch, The National Institutes of Health, Bethesda, Maryland). Crosses were conducted within husbandry guidelines established by the Marshall University Animal Care Committee. Progeny of this interstock cross were weaned at 31 days. The obese phenotype was determined by visual inspection at 5 weeks of age. In a deviation from the protocol, the team failed to separate male and female pups at 6 weeks, and inbreeding occurred.

RESULTS AND DISCUSSION

The progeny of eight fa x cp crosses approximated the 3:1 ratio typical of an autosomal recessive trait yielding 68 lean and 18 obese progeny.

Two weeks after separation, an obese fa/cp female (Figure 1) was observed with pups. Her ten-pup litter included four lean (one female, three males) and six obese (two females, four males) progeny. Representative male and female progeny are shown with the dam in Figure 2. The accidental cross between this fa/cp female and her brother produced a 2:3 ratio of lean:obese progeny. While the genotype of the sire is unknown, the observed ratio of offspring does not differ significantly from a 1:1 ratio, and suggests he was heterozygous. Obese and lean progeny are compared in Figure 3 (male) and Figure 4 (female).

Data from both the first and second generation crosses appear to support the postulate of Yen et al. (1977) that both fa and cp are inherited through autosomal recessive single genes and that the two mutations occur at the same genetic locus. However, with advances in molecular genetic analysis, alternative hypotheses are possible. The fa and cp genes could be mutations in two genes that are tightly linked, or they may be two mutations within the same gene locus. Either of these postulates may account for physiological and anatomical differences noted between the LA/N-cp and ZUC-fa obese phenotypes.

The primary molecular nature of the fa and cp genes may soon be identified. Using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques, the fa gene has been mapped to rat chromosome 5 (Truett et al., 1991). This work suggests that the fa gene is homologous to the db (diabetes) mutation on mouse chromosome 4 based on the location of two flanking genes used as markers, the Ifa and the Glut 1 genes (Truett et al., 1991). Rat chromosome 5 and mouse chromosome 4 have been shown to possess synteny homology with a region of human chromosome 1 (1 p31-34) (Searle et al., 1989). Attempts to clone the gene(s) are underway.

The fertility of this obese fa/cp interstock female suggests that normal fertility can be unmasked by crossing fa and cp stocks. Our study confirms the earlier work of Chelch and Edmonds (1981) that obese females carrying two autosomal recessive obesity genes can be fertile, in this case producing predominantly obese progeny.

REFERENCES

Chelch, A. M., and E. S. Edmonds. 1981. Copulatory behavior and reproductive capacity of the genetically obese female Zucker rat. Physiol. Behav. 27:331-335.

Hansen, C.T. 1983. Two new congenic rat strains for nutrition and obesity research. Fed. Proc. Fed. Am. Soc. Exp. Biol. 42:573.

Hemmes, R. B., S. Hubsch, H. M. Pack. 1978. High dosage of testosterone propionate increases litter production of the genetically obese male Zucker rat. Proc. Soc. Exp. Biol. Med. 159:424-427.

Johnson, P. R., L. M. Zucker. J. A. Cnace. and J. Hirsch. 1971. Cellularity of adipose depots in the genetically obese Zucker rat. J. Lipid Res. 12:706-714.

Kava, R., M. R. C. Greenwood, and P. R. Johnson. 1990. Zucker (fa/fa) rat. ILAR News 32(3):4-8.

Koletsky, S. 1973. Obese spontaneously hypertensive rats--a model for study of atherosclerosis. Exp. Mol. Pathol. 19:53-60.

Michaelis IV, O. E., N. Carswell, and K. C. Ellwood. 1988. Metabolic characteristics of the LA/N-corpulent and SHR/N-corpulent rat strains. Pp. 13-15 in New Models of Genetically Obese Rats for Studies in Diabetes, Heart Disease and Complications of Obesity. Bethesda, Md.: National Institutes of Health.

National Diabetes Data Group. 1979. Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes 28:1039-1057.

Reiser,.S., E. Bobri, J. Halif'tisch, O. E. Michaelis IV, M. Keeney, and E. S. Prather. 1981. Serum insulin and glucose in hyperinsulinemic subjects fed three different levels of sucrose. Am. J. Clin. Nutr. 34:2348-2358.

Saiduddin, S., G. A. Bray, D. York, and R. S. Swerdloff. 1973. Reproductive function in the genetically obese "fatty" rat. Endocrinology 93:1251-1256.

Searle, A. G., J. Peters, M. F. Lyon, J. G. Hall, E. P. Evans, J. H. Edwards, and V. J. Buckle. 1989. Chromosome maps of man and mouse. IV. Ann. Hum. Ganet. 54:89-140.

Truett, G. E., N. Bahary, J. M. Friedman, and R. L. Leibel. 1991. Rat obesity gene fatty (fa) maps to chromosome 5: Evidence for homology with the mouse gene diabetes (db). Proc. Nat. Acad. Sci. USA 88:7806-7809.

Tulp, O.L. 1990. Characteristics of thermogenesis, obesity, and longevity in the LA/N-cp rat. ILAR News 32(3):32-38.

Whitaker, E. M., M. A. Shaw, and G. R. Hervey. 1983. Plasma oestradiol-17 beta and testosterone concentrations as possible causes of the infertility of congenitally obese Zucker rats. J. Endocrinol. 99:485-490.

Yen, T. T., W. N. Shaw, P-L Yu. 1977. Genetics of obesity in Zucker rats and Koletsky rats. Heredity 38(3):373-377.

Zucker, L. M., and T. F. Zucker. 1961. Fatty, a new mutation in the rat. J. Hered. 52:275-278.

Zucker, L. M., and T. F. Zucker. 1962. Hereditary obesity in the rat associated with high senam fat and cholesterol. Proc. Soc. Exp. Biol. Med. 110:165-171.

Zucker, T. F., and L. M. Zucker. 1963. Fat accretion and growth in the rat. J. Nutr. 80:6-19.

Zucker, L. M., and H. N. Antoniades. 1972. Insulin and obesity in the Zucker genetically obese rat "fatty." Endocrinol. 90:1320-1330.

FIGURE 1 F1 obese fa/cp dam (19.2 months of age).

FIGURE 2 Three F2 obese fa/cp male progeny (from left), obese dam F1 (fourth from left), one F2 obese fa/cp female (far right). F2 progeny are 17 months of age.

FIGURE 3 F2 obese (fa/cp) and lean (genotype unknown) male progeny (17 months of age).

FIGURE 4 F2 obese (fa/cp) and lean (genotype unknown) female progeny (17 months of age).