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.     

Effects of diapause on lethal yellow (Ay/Ay) mouse embryos.

One of the problems in studying early acting recessive lethal genes is recognizing the homozygotes prior to their demise. Molecular probes can assist in this task, but their use generally requires removal of cells and consequent damage to the embryo, which may compromise its subsequent development. The present study was undertaken in an attempt to distinguish intact, living, lethal yellow (Ay/Ay) embryos from Ay/ae and ae/ae littermates before implantation by placing them in implantation delay or diapause. After 2 days in the reproductive tract of prepubertal females, the great majority of presumptive lethal Ay/Ay embryos has failed to hatch from the zona pellucida and they exhibited a marked deficiency of cells relative to controls, particularly in the inner cell mass. This argues against a stage-specific role for the gene in implantation.     

Effects of the lethal yellow (Ay) mutation in mouse aggregation chimeras

The Ay allele is a recessive lethal mutation at the mouse agouti locus, which results in embryonic death around the time of implantation. In the heterozygous state, Ay produces several dominant pleiotropic effects, including an increase in weight gain and body length, a susceptibility to hepatic, pulmonary and mammary tumors, and a suppression of the agouti phenotype, which results in a yellow coat color. To investigate the cellular action of Ay with regard to its effects upon embryonic viability and adult-onset obesity, we generated a series of aggregation chimeras using embryos that differ in their agouti locus genotype. Embryos derived from Ay/a x Ay/a matings were aggregated with those derived from A/A x A/A matings, and genotypic identification of the resultant chimeras was accomplished using a molecular probe at the Emv-15 locus that distinguishes among the three different alleles, Ay, A, and a. Among 50 chimeras, 25 analyzed as liveborns and 25 as 9.5 day embryos, 29 were a/a in equilibrium A/A and 21 were Ay/a in equilibrium A/A. The absence of Ay/Ay in equilibrium A/A chimeras demonstrates that Ay/Ay cells cannot be rescued in a chimeric environment, and the relative deficiency of Ay/a in equilibrium A/A chimeras suggests that, under certain conditions, Ay heterozygosity may partially affect cell viability or proliferation. In the 25 liveborn chimeras, Ay/a in equilibrium A/A animals became obese as adults and a/a in equilibrium A/A animals did not. There was no correlation between genotypic proportions and rate of weight gain, which shows that, with regard to its effects on weight gain, Ay heterozygosity is cell non-autonomous.     

Investigation of the tissue specificity of the lethal yellow (Ay) gene in mouse embryos

The tissue specificity of the lethal yellow mutant was investigated by separation of blastocyst tissues. Embryos from experimental (Ay/ae X Ay/ae) and control (ae/ae X Ay/ae) crosses of the AG/CamPa inbred strain were recovered at 3.5 days post coitum, cultured for 24 hours, and then mechanically dissected into the component tissues of the blastocyst, the inner cell mass (ICM), and trophectoderm. These fragments were then cultured separately, with or without a feeder layer of inactivated fibroblasts, for an additional 3-5 days. Comparisons between experimental and control crosses indicated that the lethal Ay/Ay embryos were among the blastocysts successfully dissected but that both the ICM and trophectoderm from lethal embryos failed to develop further in vitro, either with or without feeders. With retrospective identification of the lethal embryos, it was found that at 4.5 days, after 1 day of culture, they had formed morphologically normal blastocysts but were frequently more fragile upon dissection and had smaller ICMs. Although none had hatched from the zona pellucida, some had ruptured it and were halfway out. With culture, lethal ICMs showed no development, and lethal trophectoderm usually attached but showed very limited outgrowth. Thus, no rescue of lethal tissue was shown with dissection and in vitro culture, and results are consistent with the gene affecting both tissues of the late blastocyst.     

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.     

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.     

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.     

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.     

Dermal-epidermal interactions and the site of action of the yellow (Ay) and nonagouti (a) coat color genes in the mouse

The alleles at the agouti locus in mice determine whether eumelanin or pheomelanin is synthesized by the follicular melanocytes. Previous studies have indicated the dermis as the site of action of the agouti alleles, while implying that the epidermis plays only a passive role. Using methods of dermal-epidermal recombinations of embryonic yellow (A^y) and nonagouti (a) mouse skin, the study reported here indicates that the epidermis, as well as the dermis, plays a role in the action of the agouti alleles. When yellow dermis is recombined with nonagouti epidermis, the hairs produced contain only pheomelanin, thus substantiating the role of the dermis. However, the reciprocal combination of nonagouti dermis and yellow epidermis also produces hairs containing pheomelanin, indicating a more important role for the epidermis. The role of the dermal-epidermal interactions in the action of the alleles at the agouti locus is discussed.     

In vitro fertilization and embryo development in vitro and in vivo in the tiger (Panthera tigris)

A study was conducted to evaluate the adaptability to the tiger of an in vitro fertilization/embryo culture system previously developed in the domestic cat. In Trial I (July 1989), 10 female tigers were treated with either 2,500 (n = 5) or 5,000 (n = 5) IU eCG i.m. and with 2,000 IU hCG i.m. 84 h later. In Trial II (January 1990), 6 females (5 of which were treated in Trial I) were given 2,500 IU eCG i.m. and 2,000 IU hCG i.m. 84 h later. Twenty-four to twenty-six hours after hCG treatment, all tigers were subjected to laparoscopy, and oocytes were aspirated transabdominally. On the basis of follicular development (follicles greater than or equal to 2 mm in diameter), all females responded to exogenous gonadotropins (range, 6-52 follicles/female). Follicle number and oocyte recovery rate were unaffected (p greater than 0.05) by eCG dose or time of year. A total of 456 oocytes were collected from 468 follicles (97.4% recovery; mean, 28.5 +/- 3.4 oocytes/female). Of these, 378 (82.9%) qualified as mature, 48 (10.5%) as immature, and 30 (6.6%) as degenerate. During Trial I, 8 electroejaculates were collected from 7 male tigers, and in Trial II, 3 semen samples were collected from 3 males. Motile sperm were recovered on each occasion; the overall mean (+/- SEM) ejaculate volume was 7.5 +/- 0.7 ml, the number of motile sperm/ejaculate was 105.9 +/- 20.6 x 10(6), and the percentage of structurally normal sperm/ejaculate was 81.4 +/- 2.0%. After swim-up processing, 0.05 x 10(6) motile sperm were co-cultured with 10 or fewer tiger oocytes in a humidified atmosphere (38 degrees C) of 5% CO2 in air. Of the 358 mature oocytes inseminated, 227 (63.4%) were fertilized. Oocytes from 2 females became contaminated in culture and, therefore, were excluded from embryo cleavage calculations. Of the remaining 195 fertilized oocytes, 187 (95.9%) cleaved to the two-cell stage. No parthenogenetic cleavage was observed in noninseminated control oocytes (n = 20). Eighty-six good-to-excellent-quality two- to four-cell embryos were transferred surgically into the oviducts of 4 of the original oocyte donors in Trial I and 2 females in Trial II. A pregnancy occurred in 1 female in Trial II, and 3 live-born cubs were delivered by Caesarean section 107 days after embryo transfer. Of the 56 cleaved embryos cultured in vitro in Ham's F10 for 72 h, 14 (25.0%) were at the sixteen-cell stage, and 15 (26.8%) were morulae.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

A quantitative and qualitative study of craniofacial development in the rat embryo aged 302 hours to 374 hours (days 12.5 to 15.5)

One-hour mated Sprague-Dawley rats underwent a normal pregnancy, which was terminated at hours 302, 326, 350, and 374. After appropriate processing and scanning electron microscopy of the embryos, absolute measurements of the craniofacial area, especially of the snout, maxillary process, eye, lower face, and brain were taken in the 302- and 326-hr embryos. Additionally, growth ratios were established in all stages. The greatest growth and developmental changes occurred between 302 and 326 hr, when 1) the snout developed from the nasal anlagen and the maxillary process, 2) the eye lens replaced the eyepit, and 3) the hindlimb developed. Between 326 and 350 hr, the optic pit developed. The results of the present study can markedly enhance precision in interpretation of results of teratological studies in the rat. The detailed quantitative data established allow reporting of subtle departures from the normal hitherto impossible.     

Effect of adrenalectomy on weight gain and body composition of yellow obese mice (Ay/a).

It has been reported that the adrenal gland is essential to the development of obesity if Ay/a Yellow obese mice (Hausberger and Hausberger 1960). Since the actual body composition data to support this report has not been published, we attempted to duplicate this observation by adrenalectomy of the Yellow mice before the onset of obesity. Two groups of Yellow mice (Ay/a) and normal mice (a/a) were either sham operated or adrenalectomized at two months of age and at four months of age. Body weight was monitored until body gain had stopped. At that time the animals were sacrified and checked for completeness of adrenal removal. Body composition of dry matter, fat, protein, and ash was determined. Adrenalectomy caused a reduction of body fat of 33% and 30% in both the a/a Control and the Ay/a Yellow mice, respectively. The adrenalectomized Yellow mice were still fatter than adrenalectomized Controls (25.3% fat vs. 10.6% fat). Yellow and control adrenalectomized mice showed similar depression in growth rate. These data suggest that while the adrenal gland is essential for complete expression of the genetic potential for fat deposition, it may not be necessary for partial expression indicating a secondary role in the development of obesity in the Ay/a Yellow mouse.