Effect of mouse strain variations and cortisone treatment on the establishment and growth of primary Echinococcus multilocularis hydatid cysts.

The main purpose of this study was to determine the effects of cortisone on the number and size of primary Echinococcus multilocularis cysts developing in a moderately resistant strain of mice, i.e., C3H/HeJ. Computerized image analysis was used to measure the surface area occupied by hydatid cysts 10 wk after inoculation of the mice with E. multilocularis eggs. Our second objective was to compare the infectivity of primary E. multilocularis hydatid cysts in C57BL/6J-Ay/a (lethal yellow) mice with that in C57BL/6J-a/a (non-agouti black) mice. The data obtained show no difference between the C3H/HeJ and C57BL/6J-a/a strains of mice; yet, the image analysis method was able to detect a slight increase in the total cyst size within the Ay/a mutant of the C57BL/6J strain. Treatment of C3H/HeJ mice with cortisone drastically increased both the number of cysts and the average size of each cyst when the treatment occurred early in the infection.

Agouti-related maturation and tissue distribution of alpha-Melanocyte Stimulating Hormone in wild-type (AwJ/AwJ) and mutant (Ay/a,a/a) mice.

Because of ectopic overproduction of agouti protein, yellow alleles (A(y) and A(vy)) of the murine agouti gene may secondarily modulate the synthesis, maturation (i.e., acetylation), and/or tissue deployment of alpha-Melanocyte Stimulating Hormone (MSH). We used HPLC to test the hypothesis that A(y)/a mice exhibit altered concentrations of desacetyl-, monoacetyl-, and diacetyl-alpha-MSH in pituitaries, sera, and telogen hair bulbs when compared to black (a/a) mice. We also used RIA to measure total MSH in those same tissues of A(y)a,a/a, and white-bellied agouti (A(wJ)/A(wJ)) mice (Strain C57BL/6J). We found no evidence that A(y)/a mice possessed an imbalance of des-, mono-, and diacetylated alpha-MSH species. However, radioimmunoassay (RIA) analyses of total MSH suggest that wild-type agouti mice (A(wJ)/A(wJ)) exhibited significantly decreased (P < 0.05) tissue levels of total alpha-MSH in pituitaries, sera, and regenerating hair bulbs when compared to those of mutant A(y)/a and a/a mice.

Agouti alleles alter cysteine and glutathione concentrations in hair follicles and serum of mice (A y/a, A wJ/A wJ, and a/a).

Ectopic overexpression of the agouti protein in the lethal yellow (A y/a) mouse causes a yellow coat as well as the lethal yellow syndrome. Presence of thiols like glutathione (GSH) or cysteine (Cys) may regulate the conversion of dopaquinone to phaeomelanin in hair follicle melanocytes. GSH also plays important roles in cellular health and maintenance. Cys and GSH were measured using high-performance liquid chromatography in hair follicles and serum of A wJ/A wJ (agouti), A y/a (yellow), and a/a (black) mice over a 20-d hair growth regeneration period. Agouti alleles modulate thiol concentrations. A y/a hair follicles exhibited higher total thiol levels and an increased ratio of Cys to GSH. A wJ/A wJ mice showed intermediate levels, while a/a mice had lowest total thiol concentrations and a decreased ratio of Cys to GSH. Hair follicle cysteine concentrations showed yellow > agouti > black (p < 0.01). In all genotypes, unplucked skin and day 0 hair follicles showed GSH as the major thiol, but a shift to predominantly Cys on peak melanogenic days was seen. Presence of high concentrations of free cysteine support the hypothesis of phaeomelanin synthesis via cysteinyldopas. The A y/a mouse had the most dramatic follicular thiol changes as well as a depression in serum thiols. An altered thiol metabolism in these and other A y/a tissues might impair normal cell functioning to contribute to the lethal yellow syndrome.

Agouti alleles influence thiol concentrations in hair follicles and extrafollicular tissues of mice (Ay/a, AwJ/AwJ, a/a).

Agouti protein (AP) expression in the wild-type agouti mouse (AwJ/AwJ) coincides with a switch in hair follicle melanogenesis from black (eumelanin) to yellow (pheomelanin). Ectopic overexpression of AP in the lethal yellow (Ay/a) mouse cause a pure yellow coat and the lethal yellow syndrome. Thiol concentrations may control the conversion of dopaquinone to pheomelanin in hair follicle melanocytes. Glutathione (GSH) also plays important roles in cellular health and protection. Using HPLC, cysteine and GSH were measured in 1) hair follicles, liver and serum of Ay/a, AwJ/AwJ, and a/a (black) mice, and 2) adipose and spleen tissues of Ay/a and a/a mice on day 9 of regenerating hair growth (late pheomelanin phase). Agouti locus alleles influence thiol metabolism in hair follicles and in other systemic tissues. Ay/a hair follicles and serum showed highest cysteine and lowest GSH levels. AwJ/AwJ mice showed intermediate levels, while a/a hair follicles and serum had lowest cysteine and highest GSH concentrations. In the hair follicle, cysteine (likely derived from enzymatic degradation of GSH) appears to be the primary pheomelanogenic thiol. Agouti locus alleles may also directly or indirectly affect thiol concentrations in systemic tissues like liver and spleen. Cysteine in spleen extracts showed Ay/a > a/a (P < 0.01). An Ay-induced imbalance of thiol metabolism (altering GSH concentrations in multiple tissues) may contribute to the pleiotropic defects of the lethal yellow syndrome.

Effects of exogenous MSH on the transformation from phaeo- to eumelanogenesis within C57BL/6J-Ay/a hairbulb melanocytes.

The extent to which alpha melanocyte-stimulating hormone (MSH) is a true in vivo regulator of melanogenesis in mice is unknown. The objective of this study was to determine if MSH-induced eumelanogenesis in hairbulb melanocytes of yellow (Ay/a) mice mimics the natural program of eumelanogenesis occurring in genetically black (a/a) hairbulb melanocytes. We conducted quantitative transmission electron microscopy on melanosome differentiation within MSH-treated regenerating 9-d hairbulbs of Ay/a and a/a mice. Results of exogenous alpha-MSH injections (5 d at 0.15 mM MSH) showed that the striking visual darkening of hair was accompanied by an incomplete transformation of phaeo- to eumelanogenesis. Ontogenetic data on developmental stages I-IV of 3678 melanosomes based on geometric considerations (length, width, shape, and area) showed that MSH did not induce a complete transformation from spherical phaeomelanosomes to elliptical eumelanosomes. Also, observations on the number of vesiculoglobular bodies and matrix organization reveled that MSH-treated Ay/a melanosomes retained distinct features of phaeomelanogenesis even after 5 d of MSH treatment. Thus, MSH induced a partial but incomplete pattern of eumelanogenesis in regenerating hairbulb melanocytes of Ay/a mice. The continued investigation of the dynamics of melanin synthesis in MSH-induced Ay/a mice melanocytes possessing "mosaic" melanosomes could be productive in understanding fundamental relationships between tyrosinase activity, matrix function, matrix structure, and regulation of melanin (phaeo- and/or eumelanin) synthesis.

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.

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.

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.

Effects of reciprocal ovary transplantation on reproductive performance of lethal yellow mice (Ay/a; C57BL/6J).

This study was conducted to determine whether reproductive failures in ageing, obese lethal yellow (Ay/a) females are due primarily to defects within Ay/a ovaries or to systemic defects which may operate outside the ovaries. Reciprocal ovary transplantation between control (a/a) and lethal yellow (Ay/a) females provided an experimental system to test the reproductive potential of not only Ay/a ovaries in control (a/a) females but also control (a/a) ovaries in mutant (Ay/a) females. Results on reproductive performance of all four combinations of grafts between Ay/a and a/a mice proved that Ay-induced reproductive failures are not due to intrinsic ovarian lesions but rather to defects operating extrinsically to the ovary. The hypothalamo-pituitary axis is a likely site for this reproductive lesion.

Progressive infertility in female lethal yellow mice (Ay/a; strain C57BL/6J).

Obese Ay/a females of 120 days or older, when compared to age-matched a/a controls (strain C57BL/6J), exhibited abnormal oestrous cyclicity characterized by reduced frequencies of true oestrous-stage smears, decreased mating success to proven a/a males, lowered uterine weights, and depressed ovulation rates. Exogenous gonadotrophins (PMSG/hCG) partly restored ovulation in obese Ay/a females to near control levels, demonstrating the sensitivity of Ay/a ovarian tissues to FSH and LH, at least at superovulatory levels. Concentrations of endogenous gonadotrophins and/or sensitivity of ovarian target cells to gonadotrophins may therefore be impaired in obese Ay/a females. Aberrant copulatory behaviour, reduced uterine weights, and depressed conception rates strongly suggest ovarian steroid deficiencies, perhaps secondary effects of reduced endogenous gonadotrophin activity. As in other obese rodent syndromes e.g. ob/ob, db/db, and fa/fa), a possible fundamental Ay-induced hypothalamic lesion is consistent with our data.

Development of velvet coat (Ve/Ve), another early lethal mutation in the house mouse.

A new semidominant mutation in the house mouse, velvet coat (Ve), is described. Ve homozygotes, recognizable on day 5 of gestation by their deficiency of ectodermal cells, never produce mesoderm and are resorbed by days 9-10. Primary Ve action may occur during the formation of the blastocyst or during determination and differentiation of the inner cell mass, or both. Accordingly, Ve/Ve embryos may provide a useful model for investigating primary gene action during blastocyst formation and subsequent differentiation.

Histological analyses of lethal yellow mouse embryos (Ay/Ay) at 90 and 132 h post coitum.

Strain C57BL/6J-Ay/Ay embryos are developmentally retarded in utero at 90 hpc and are resorbed by 132 hpc. The Ay/Ay developmental delay previously observed in vitro has now been confirmed in vivo. Apparently Ay is involved in some aspect of cleavage stage metabolism.

Latent effects on in vitro development following cytochalasin B treatment of 8-cell mouse embryos.

Eight-cell mouse embryos when treated with 4.0 microgram/ml cytochalasin B (CB) in vitro undergo a reversible developmental arrest. Upon rinsing of embryos and subsequent culture in control medium, normal morphogenetic processes such as compaction of 8-cell embryos, cavitation, and post-blastocyst attachment and outgrowth are restored. However, the effects of CB on mouse embryos are not completely reversible; latent post-blastocyst defects become increasingly more prevalent as CB treatment duration increases. The present study was conducted to quantitatively determine latent effects of CB on post-blastocyst embryos by comparing their ability to attach and to sustain the growth and differentiation of ICM and trophoblast tissues. Groups of 8-cell embryos were cultured in Brinster's BMOC-3 medium containing 4.0 microgram/ml cytochalasin B for 6, 12, 18, and 24 h. Following treatment, embryos were rinsed and cultured until 190 h post coitum (h.p.c.) in Eagle's MEM/10% fetal calf serum modified to contain optimal levels of essential amino acids. Blastocysts generally attached to the surface of the plastic substratum by 120 h.p.c. At selected time periods after attachment (130, 160, and 190 h.p.c.), embryos were scored for outgrowth size, ICM size, extent of peripheral hyaloplasmic fan, and number of trophoblast nuclei per outgrowth. Analyses of variance (ANOVAs) were conducted for each of the four parameters listed above. Rates of attachment were analyzed by chi2 test. Results show that the treatments affect (P less than 0.01) embryo attachment, number of trophoblast nuclei per outgrowth, hyaloplasmic fan production, and ICM growth in a duration-dependent manner. Interestingly, since treatment effects on outgrowth areas are nonsignificant apparently CB does not significantly change total outgrowth area. But CB treatment does cause abnormal fan production and decreased trophoblast nuclei numbers. However, trophoblast cells are apparently more resistant than ICM to CB as is evident by the high incidence of trophoblast outgrowths devoid of ICM. CB (4.0 microgram/ml) treatments at 8-cell stages for relatively short durations (6 and 12 h) induce latent effects on post-blastocyst embryos. Finally, there exists a definite 4.0 microgram/ml CB duration response over the 68-190 h.p.c. observation interval.

Enhanced identification of lethal yellow (Ay/Ay) mouse embryos by means of delayed development of four-cell stages.

At present, experimental studies on the primary genetic lesion in Ay/Ay embryos are restricted by the absence of techniques designed to identify Ay homozygotes prior to the onset of secondary defects. This paper describes a methodology to enrich the yields of Ay homozygotes in experimental populations by taking advantage of an early homozygous Ay expression-lagging development in 4-cell embryos.

Ultrastructural analysis of preimplantation lethal yellow (Ay/Ay) mouse embryos.

Embryos recovered at 62 and 80 h post coitum from reproductive tracts of yellow (Ay/a) and black (a/a) female mice mated to Ay/a males were examined ultrastructurally to define the developmental defect of lethal Ay homozygotes. No abnormalities were observed in five embryos from control matings (female a/a x male Ay/a). Of 24 morulae and blastocysts from Ay/a x Ay/a matings, six were observed to possess some morphological aberration. Two of the six abnormal embryos were morulae and contained isolated blastomeres which had developmental features typical of younger embryos; remaining cells of these embryos were normal. The third, an early blastocyst, contained a degenerating trophoblast cell' other cells of this embryo were also abnormal but not in an advanced stage of degeneration. The fourth abnormal embryo (late cleavage stage) was in an advanced stage of degeneration affecting all blastomeres. Finally, the remaining two abnormal morulae had a unique nucleolar morphology and an unusual abundance of intra-cisternal A particles. Presumably, one or more of the six abnormal embryos from Ay/a x Ay/a matings were Ay homozygotes. However, no single ultrastructural alteration characteristic of Ay/Ay embryos was found.

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.

Differential concanavalin A-induced agglutination of eight-cell preimplantation mouse embryos before and after compaction.

Eight-cell mouse embryos undergo a morphogenetically significant process termed compaction during which blastomeres flatten against one another maximizing their areas of cell-cell contact. Data presented here show striking differences (P less than 0.01) in Con A-induced agglutination between groups of uncompacted and compacted embryos suggesting that the compaction process is accompanied by changes in blastomere surface properties.

Identification of eight-cell tw32 homozygous lethal mutants by aberrant compaction.

Early cleavage stage (4- and uncompacted 8-cell) embryos from experimental (+/tw32 X +/t32) and control (male +/tw32 X female +/T) matings were recovered at 56 hours post coitum, cultured, separated into uncompacted and compacted 8-cell embryo groups, and either analyzed histologically for cytoplasmic lipids or cultured through the morula-to-blastocyst transformation. Abnormal compaction was associated with excess cytoplasmic lipids at the 8-cell stage (P less than 0.01). Also, 89.2% (33/37) of the original uncompacted experimental embryos underwent developmental arrest characteristic of tw32 homozygous lethals. Thus, aberrant compaction, a newly reported symptom of the tw32/tw21 syndrome, affords a visual means to identify tw32 homozygotes at 8-cell stages.