Genetic Organization of the agouti Region of the Mouse

The agouti locus on mouse chromosome 2 acts via the hair follicle to control the melanic type and distribution of hair pigments. The diverse phenotypes associated with various agouti mutations have led to speculation about the organization of the agouti locus. Earlier studies indicated that two presumed agouti alleles, lethal yellow (Ay) and lethal light-bellied nonagouti (ax), are pseudoallelic. We present genetic data showing probable recombination between Ay and three agouti mutations (at, a, and ax), which suggest that Ay is a pseudoallele of the agouti locus. The close linkage of an endogenous ecotropic murine leukemia provirus, Emv-15, to Ay provides a molecular access to genes at or near the agouti locus. However, previous studies suggested that the Emv-15 locus can recombine with some agouti alleles and therefore we analyzed mice from recombinant inbred strains and backcrosses to measure the genetic distance between various agouti alleles and the Emv-15 locus. Our data indicate that the Emv-15 locus is less than 0.3 cM from the agouti locus. These experiments provide a conceptual framework for initiating chromosome walking experiments designed to retrieve sequences from the agouti locus and give new insight into the genetic organization of the agouti region.     

In vitro analysis of pre- and early postimplantation development of lethal yellow (Ay/Ay) mouse embryos

Preimplantation embryos from matings between yellow heterozygous (Ay/a) mice were recovered at 56 hours post coitum, cultured for five days, and compared with the development of embryos from three control matings (Ay/a female X a/a male, a/a female X Ay/a male, a/a female X a/a male). Most embryos were at the 8-cell stage at recovery; however fewer embryos from the experimental cross had developed to the 8-cell stage than embryos of control matings, indicating a developmental lag of experimental embryos (P less than 0.01). The yellow (Ay/a) uterus did not contribute (P = 0.05) to delayed development. Experimental and control embryos were equally capable of successful development in culture to the morula stage with no distinct morphological characteristics identifying the class of Ay/Ay mutants. However, significant differences were observed in the development from morulae to blastocysts; 9.4% (10/106) of the morulae in experimental crosses failed to undergo blastocyst formation as compared with 2.5% (10/398) of morulae in pooled control crosses (P = 0.010-0.025). In the experimental cross 25.0% (24/96) of embryos that developed successfully to the blastocyst stage failed to hatch from the zona pellucida; these are presumed to include the class of lethal yellow homozygotes. Abnormalities seen in cultured embryos consisted primarily of blastomere disintegration, blastomere arrest and exclusion, and embryo fragmentation.     

Quantitative trait loci that regulate plasma lipid concentration in hereditary obese KK and KK-Ay mice

To identify quantitative trait loci (QTLs) responsible for regulating plasma lipid concentration associated with obesity, linkage analysis was carried out on the 190 F2 progeny of a cross between C57BL/6J female and KK-Ay (Ay allele at the agouti locus congenic) male. In F2 a/a (agouti locus genotype) mice, two QTLs were identified on chromosome 1 and a QTL on chromosome 3 for total-cholesterol. A QTL for HDL-cholesterol was identified on chromosome 1 and a QTL for NEFA on chromosome 9. In F2 Ay/a mice, two QTLs for HDL-cholesterol were found on chromosome 1. Loci for other lipids with suggestive linkage were also identified. In both F2 mice, one QTL on chromosome 1 for total- and HDL-cholesterol was mapped near D1Mit150, in the vicinity of the apolipoprotein A-II (Apoa2) locus. Seven nucleotide substitutions out of 309 nucleotide apolipoprotein A-II cDNA sequences were identified between KK and C57BL/6J. The Ay allele may be an indication of the plasma lipid levels, but its influence was less apparent than in the case of weight control. The loci for lipids were not on identical chromosomes with those previously identified for obesity, suggesting that hyperlipidemia in KK does not coincidentally occur with obesity.     

Effects of dehydroepiandrosterone on obesity and glucose-6-phosphate dehydrogenase activity in the lethal yellow mouse (strain 129/Sv-Ay/Aw)

We investigated the anti-obesity effects of the adrenal androgen, dehydroepiandrosterone (DHEA), on genetically predisposed obese lethal yellow mice (Ay/Aw). Secondly, we tested the hypothesis that DHEA promotes its anti-obesity effects by decreasing the activity of glucose-6-phosphate dehydrogenase (G6PDH). We subjected four genotype-sex combinations of yellow and agouti (control) mice to four dietary treatments and determined weight changes, food consumption, and G6PDH activity. Although G6PDH activities of yellow mice were considerably decreased in the 0.4% DHEA treatment group, they were elevated in the 0.0 and 0.1% DHEA treatment groups. In contrast, G6PDH activities of DHEA-treated control agouti mice remained relatively constant. These studies confirm that DHEA prevents the Ay gene from promoting excess fat deposition via some mechanism(s) other than reduced dietary intake. However, the overall absence of agreement between weight change (gain or loss) and G6PDH activity suggests that the anti-obesity activity of DHEA is not mediated via G6PDH. Since yellow obese (Ay/Aw) mice were found to be more susceptible to DHEA's effects than their agouti (Aw/Aw) littermates, Ay appears to induce an altered metabolism in Ay/Aw mice which is more susceptible to the effects of DHEA than the normal metabolism of Aw/Aw mice.     

Postnatal development of corticosteroid function of the adrenals in C57BL/6J-Ay mice

We studied postnatal development of corticosteroid function of the adrenals in mice during the period of elevated activity of the hypothalamic-pituitary-adrenal system and the influence of mutant gene Ay on this process. Normally, a corticosterone peak in blood and increased basal and stimulated steroidogenesis in vitro are observed in 3-week old mice. In 3-week old Ay/a mice (hyperexpression of protein agouti) a corticosterone peak in blood is lowered and genotypic differences in steroidogenesis in vitro are absent, as compared to a/A mice (absence of agouti), while at the ages of 10 and 15 weeks, there were no genotypic differences in the blood level of corticosterone and steroidogenesis in vitro was elevated. Thus, a high level of corticosterone during the period of elevated activity of the hypothalamic-pituitary-adrenal system in 3-week old mice is determined by enhanced steroidogenic function of the adrenals. Mutant gene Ay in male mice affected the postnatal development of the adrenal function: the peak of corticosterone in blood was lowered during the period of elevated activity of the system.     

Differentiation of hairbulb pigment cell melanosomes in compound agouti and albino locus mouse mutants (Ay, a, c2J; C57BL/6J)

Our objective was to determine using electron microscopy how nonagouti (a), lethal yellow (Ay), and albino (c2J) genes affect the program of mouse hairbulb melanosome differentiation; 1,921 hairbulb melanosomes from four genotypes (a/a C/C = B,Ay/a C/C = Y, a/a c2J/c2J = BA, and Ay/a c2J/c2J = YA) were scored for developmental stage, length, and width. Qualitative and quantitative electron microscopy revealed the following. An albino locus-induced diminution of melanosome size suggests that the albino locus is involved in structural features of melanosomes not directly related to the synthesis and deployment of tyrosinase. Ratio data on melanosome length-to-width confirm that the agouti locus determines melanosome shape, either spherical or elliptical; melanization is not required for melanosomes to achieve their agouti-locus-determined shapes. YA (Ay/a c2J/c2J) melanosomes, characterized by poorly organized matrices, absence of active tyrosinase, unusually large membrane invaginations, and significantly smaller dimensions than those of BA (a/a c2J/c2J), showed additive effects of both Ay and c2J alleles. These data suggest that the albino locus plays a structural as well as functional (tyrosinase) role in the differentiation of mouse hairbulb melanosomes. The agouti locus, even in the absence of melanization, directs melanosome shape either via synthesis and deployment of agouti-locus-encoded matrix proteins or by other structural factors. The additive effects of Ay and c2J alleles in compound YA mutants document the importance of specific interactions both functional and structural between agouti and albino loci.     

Tyrosinase activity (TH, DO, PAGE-defined isozymes) and melanin production in regenerating hairbulb melanocytes of lethal yellow (Ay/a), black (a/a), agouti (AwJ/AwJ/) and albino (a/a/c2J/c2J) mice (C57BL/6J)

We compared tyrosinase activity (TH, DO, and native PAGE-defined isozymes) and melanin production in particulate and soluble fractions of hairbulb melanocytes of lethal yellow (Ay/a C/C), nonagouti black (a/a C/C), and albino (a/a c2J/c2J) of 3-, 6-, 9-, and 12-day regenerating hairbulbs. With respect to tyrosine hydroxylase (TH) and dopa oxidase (DO) activities, Ay/a melanocytes possessed only 25-35% of the activity of a/a; there were no genotype differences in either the subcellular distribution of activity in soluble and particulate fractions or in the relative increases of activity over the 12-day developmental period. TH data on wild-type agouti (AwJ/AwJ) mice over the 3-11 day regeneration interval showed an activity intermediate between that of a/a and Ay/a; the rate of TH increase reflected black and yellow phases of the agouti hair cycle. Analyses of the number and densities of dopa-sensitive bands following native PAGE of 3-, 6-, 9-, and 12-day hairbulb fractions of a/a and Ay/a mice suggested stage-dependent patterns. A comparison of rates and amounts of melanin production in 3-, 6-, 9-, and 12-day fractions showed consistent melanin production in Ay/a to be 10-20% that of a/a; however, fold increases in melanin production over the four stages were similar between genotypes. Overall, tyrosinase activity data support the notion that agouti locus modification of tyrosinase activity is a graded or quantitative rather than a qualitative phenomenon.     

The multiple-locus symmetric fertility model

Selection due to variation in the fecundity among matings of genotypes with respect to many loci each with two alleles is studied. The fitness of a mating depends only on the genotypic distinction between homozygote and heterozygote at each locus in the two individuals, and differences among loci are allowed. This symmetric fertility model is therefore a generalization of the multiple-locus symmetric viability model. The phenomena seen in the two-locus symmetric fertility model generalize—e.g., the possibility of joint stability of equilibria with linkage equilibrium and with linkage disequilibrium, and the existence of different types of totally polymorphic equilibria with the gametic proportions in linkage equilibrium. The central equilibrium with genotypic frequencies in Hardy-Weinberg proportions and gametic frequencies in Robbins proportions exists for all symmetric fertility models. For some symmetric fertility regimes additional equilibria exist with gametic frequencies in linkage equilibrium and with genotypic frequencies in Hardy-Weinberg proportions at all except one locus. These equilibria may exist in the dioecious symmetric viability model, and then they will be locally stable. For free recombination the stable equilibria show linkage equilibrium, but several of these with different numbers of polymorphic loci may be stable simultaneously.     

Molecular characterization of the mouse agouti locus.

The agouti (a) locus acts within the microenvironment of the hair follicle to regulate coat color pigmentation in the mouse. We have characterized a gene encoding a novel 131 amino acid protein that we propose is the one gene associated with the agouti locus. This gene is normally expressed in a manner consistent with a locus function, and, more importantly, its structure and expression are affected by a number of representative alleles in the agouti dominance hierarchy. In addition, we found that the pleiotropic effects associated with the lethal yellow (Ay) mutation, which include pronounced obesity, diabetes, and the development of neoplasms, are accompanied by deregulated overexpression of the agouti gene in numerous tissues of the adult animal.     

Molecular Markers for the agouti Coat Color Locus of the Mouse

The agouti (a) coat color locus of the mouse acts within the microenvironment of the hair follicle to control the relative amount and distribution of yellow and black pigment in the coat hairs. Over 18 different mutations with complex dominance relationships have been described at this locus. The lethal yellow (Ay) mutation is the top dominant of this series and is uniquely associated with an endogenous provirus, Emv-15, in three highly inbred strains. However, we report here that it is unlikely that the provirus itself causes the Ay-associated alteration in coat color, since one strain of mice (YBR-Ay/a) lacks the provirus but still retains a yellow coat color. Using single-copy mouse DNA sequences from the regions flanking Emv-15 we have detected three patterns of restriction fragment length polymorphisms (RFLPs) within this region that can be used as molecular markers for different agouti locus alleles: a wild-type agouti (A) pattern, a pattern which generally cosegregates with the nonagouti (a) mutation, and a pattern which is specific to Emv-15. We have used these RFLPs and a panel of 28 recombinant inbred mouse strains to determine the genetic linkage of these sequences with the agouti locus and have found complete concordance between the two (95% confidence limit of 0.00 to 3.79 centimorgans). We have also physically mapped these sequences by in situ hybridization to band H1 of chromosome 2, thus directly confirming previous assignments of the location of the agouti locus.     

Effects of the lethal yellow (Ay) and recessive yellow (e) genes on the population of epidermal melanocytes in newborn mice

Very few melanocytes can be detected by the DOPA reaction in the dorsal epidermis of newborn lethal yellow mice (Ay/a). Nevertheless, the epidermis contains a considerable number of melanoblasts (cells positive for the combined DOPA-premelanin reaction). On the other hand, numerous melanocytes as well as melanoblasts are found in the dorsal epidermis of black mice (a/a). The number of epidermal melanoblasts is smaller in (Ay/a than in a/a mice even though the same number of melanocytes is found in the dermis of these animals. It seems probable that the product of the A y gene suppresses either the differentiation or the proliferation of epidermal melanoblasts. The number of melanoblasts plus melanocytes in day-17 embryos from a cross between Ay/a and a/a mice shows a bimodal distribution. It seems possible that half of the embryos were Ay/a and possessed a reduced number of melanoblasts and melanocytes. This result seems to suggest that the Ay gene is active at this embryonic stage. In contrast to the case for the epidermis from Ay/a mice, numerous DOPA-positive melanocytes were detected in the epidermis from e/e mice. However, the total number of melanoblasts plus melanocytes in e/e epidermis did not differ from that in Ay/a epidermis, suggesting that the mode of action of the e gene in the epidermis is different from that of the Ay gene.     

With selection for fecundity the mean fitness does not necessarily increase

A population with two alleles at one locus is considered. It is assumed that there is random mating of adults and that matings in which a particular pair of genotypes is involved may have a different mean number of offspring, or fecundity, than other types of matings. There is assumed to be no other selection. It is shown that the genotypic frequencies that maximize the mean fecundity of the population are not necessarily the same as the stable equilibrium frequencies. Thus, examples can be found for which the mean fecundity decreases from one generation to the next, and one such example is presented. An example in which there is no stable equilibrium, and the mean fecundity oscillates, is also given.     

The mouse lethal nonagouti (a(x)) mutation deletes the S-adenosylhomocysteine hydrolase (Ahcy) gene.

The lethal nonagouti (a(x)) mutation is a hypomorphic allele of the agouti coat color locus which, when homozygous, also leads to embryonic death around the time of implantation. To understand the molecular basis of these phenotypes, we identified and cloned a deletion breakpoint junction present in the ax chromosome. Long range restriction mapping demonstrated a simple deletion of approximately 100 kb, which does not affect agouti coding sequences, but begins only 4 kb 3' of the last exon, and thus may affect coat color by removing an agouti 3' enhancer. The Ahcy gene, which codes for the enzyme S-adenosylhomocysteine hydrolase (SAHase), is contained within a 20 kb region within the a(x) deletion. SAHase RNA and protein were detectable in early blastocysts and in embryonic stem cells, respectively, and analysis of embryos derived from an a(x)/a x a(x)/a embryo intercross indicated that a(x)/a embryos die between the late blastocyst and early implantation stages. Treatment of cultured embryos with an SAHase inhibitor, 3-deazaaristeromycin, or with metabolites that can result in elevated levels of cellular SAH, resulted in an inhibition of inner cell mass development, suggesting that loss of SAHase activity in a(x)/a(x) embryos is sufficient to explain their death around the time of implantation.     

The production of chimeric rats and their use in the analysis of the hooded pigmentation pattern

New, improved media and procedures for making rat chimeric embryos and culturing them in vitro have been developed. We have produced 27 rat chimeras: 20 males and 7 females. This ratio of males to females is consistent with that seen in mouse chimeras, suggesting that rat sex chimeras develop as phenotypic males. By aggregating embryos containing appropriate genetic markers for pigment cell differentiation, it is possible to produce chimeras that elucidate the site of action of the hooded gene. The coat color patterns of black ? black hooded chimeras display a white belly spot. In black ? albino hooded chimeras, small patches of white hair appear on the head and a large white spot occurs on the belly. Black ? agouti hooded chimeras display both agouti and nonagouti pigmentation over the entire surface of the chimera. These animals are fully pigmented with no white spots. In black ? albino non-hooded chimeras, rather small irregular patches of black and white hairs are distributed throughout the pelage. Histological examination of sections of hair follicles obtained from the white areas in the head of black ? albino hooded chimeras revealed amelanotic melanocytes. On the other hand, hair bulbs from the white belly spots do not contain any such melanocytes. Thus the white hairs of the head are due to the presence of albino melanocytes, but the white hairs of the belly are due to the total absence of melanocytes. All these observations are consistent with the conclusion that the hooded gene acts within melanoblasts, probably to retard their migration from the neural crest and/or to prevent their entrance into the hair follicles of the white areas of hooded rats.     

Sulfhydryl Compounds in Melanocytes of Yellow (Ay/a ), Nonagouti (a/a), and Agouti (A/A) Mice

CLEFFMANN (1953, 1963a,b) has reported that yellow but not black melanocytes of agouti (A/A) rabbits contained reducing sulfhydryl compounds. We have attempted to repeat CLEFFMANN's observations in mouse melanocytes of the lethal yellow (Ay/a), nonagouti (a/a) and agouti (A/A) genotypes. Our results contradict those of CLEFFMANN and reveal that yellow and black melanocytes, regardless of genotype, possess equivalent amounts of histochemically detectable sulfhydryl compounds. These results do not support the hypothesis that agouti-locus genes act by controlling the sulfhydryl metabolism of pigment cells.     

Pioglitazone administration alters ovarian gene expression in aging obese lethal yellow mice

Background  

Women with polycystic ovary syndrome (PCOS) are often treated with insulin-sensitizing agents, e.g. thiazolidinediones (TZD), which have been shown to reduce androgen levels and improved ovulatory function. Acting via peroxisome proliferator-activated receptor (PPAR) gamma, TZD alter the expression of a large variety of genes. Lethal yellow (LY; C57BL/6J Ay/a) mice, possessing a mutation (Ay) in the agouti gene locus, exhibit progressive obesity, reproductive dysfunction, and altered metabolic regulation similar to women with PCOS. The current study was designed to test the hypothesis that prolonged treatment of aging LY mice with the TZD, pioglitazone, alters the ovarian expression of genes that may impact reproduction.     

Action site of the lethal Ay gene in the mouse embryo.

We investigated the lethal effect of Ay gene in embryos at the preimplantation stage in vitro. First, the development until the blastocyst stage and the division of individual cells from 8-cell stage embryos were examined. No difference in development was detected between embryos from the experimental cross (Ay/a x Ay/a) and those from the control cross (a/a x a/a). Therefore, it seems that the abnormality of the Ay/Ay embryo does not appear until blastocyst formation in vitro. We subsequently examined the hatching from zona pellucida of the blastocysts. The hatching ratio of the embryos from the experimental cross was significantly lower than that of the control crosses (Ay/a x a/a, a/a x a/a: p < 0.05). Our observation indicates that deficiency of the Ay/Ay embryo can be detected in vitro at hatching. In order to elucidate the mechanism of the gene action of the Ay, we attempted to rescue the lethal embryos from decreased hatching ratio in vitro. When dbcAMP at the concentration of 1 mM was added to the culture medium, the hatching ratio of blastocysts from the experimental cross increased until the level of those from the control crosses. Since this result indicates that the cAMP content in Ay homozygote seemed to be lower than those in a/a and Ay/a, the cAMP content in individual blastocyst was quantified. It is found that Ay homozygosity was associated with lower level of cAMP. When adenylate cyclase was activated by forskolin and cholera toxin, the hatching ratio was increased. These results seem to suggest that Ay homozygote embryos possess a defect in signal transduction system mediated by adenylate cyclase during hatching.     

撒坝猪血清酯酶多态性与繁殖性能关系的研究

采用垂直板聚丙烯酰胺凝胶电泳法(PAGE), 对115头撒坝猪的血清酯酶(ES)多态性进行了检测,计算了该位点的基因型频率、基因频率和位点多态杂合度(h),并用二因素有互作的最小二乘模型对血清酯酶多态性与繁殖性能的关系进行了分析。结果表明,撒坝猪血清酯酶3种基因型AA、AB和BB的频率分别为0.2696、0.5826和0.1478。两个等位基因A和B的基因频率分别为0.5609和0.4391。该位点的杂合度为0.4926。在3种基因型中,不同基因型母猪的繁殖性能在产仔数、仔猪初生窝重、20日龄窝重、断奶仔猪数和断奶窝重等性状上存在着显著差异(P<0.05);公、母猪不同基因型交配组合在产仔数、断奶仔猪数、仔猪断奶体重和断奶窝重等性状上亦有显著差异(P<0.05)。显示出猪的血清酯酶多态性可望作为繁殖性能选种的遗传标记。 Abstract：　Serum esterase polymorphisms of 115 Saba pigs were investigated by using the method of vertical polyacrylamide gel electrophoresis. The genotype frequency, gene frequency and heterozygosity of this locus were calculated. The relationship between the serum esterase polymorphism and reproductive performance was analyzed by the least square analysis of two factors (ES genotype of boar and sow) with interaction. The results demonstrated that the genotype frequency of AA, ABandBBwas 0.2696, 0.5826 and 0.1478 respectively, the gene frequency of the alleleAandBwas 0.5609 and 0.4391 respectively. The heterozygosity of this locus was 0.4926. There are significant differences (P<0.05) on litter size, litter weight at birth, litter weight at 20 days, litter size at weaning and litter weight at weaning of different genotypic sows. The significant differences (P<0.05) were showed on litter size, litter size at weaning, weaning weight and litter weight at weaning of different genotypic mating combinations. It indicated that the serum esterase polymorphism was expected to be the genetic marker of pig reproductive performance.