Autoradiographic evidence for insulin and insulin-like growth factor binding to early mouse embryos.

Insulin binding to mouse oocytes and preimplantation embryos was assessed by light-microscopic autoradiography. Significant insulin binding was present on the cells of morulae and increased twofold at the blastocyst stage of development. Insulin binding was markedly decreased by native insulin and to a lesser extent by insulin-like growth factors (IGFs). No specific insulin binding was detected on oocytes or embryos throughout the eight-cell stage. Specific binding of IGF-I and IGF-II was also observed on the cells of blastocyst outgrowths. The findings demonstrate that specific binding of insulin and IGF is temporally expressed on the cells of pre- and peri-implantation mouse embryos. These results confirm and extend our previous immunofluorescence study.

Oogenesis: chromatin and microtubule dynamics during meiotic prophase.

Changes in the organization of germinal vesicle chromatin in mouse oocytes have been analyzed by fluorescence microscopy with respect to progressive stages of follicular development and the disposition of oocyte cytoplasmic microtubules. Four discrete patterns of chromatin organization exist in germinal vesicle (GV)-stage oocytes isolated from the ovaries of 21-25-day-old gonadotropin-primed mice. Analysis of ovarian cryosections stained with the DNA-binding fluorochrome Hoechst 33258 indicates that sequential changes in GV chromatin occur during folliculogenesis that result in the formation of a continuous perinucleolar chromatin sheath at the time of antrum formation. Specific alterations in the cytoplasmic microtubule complex of GV-stage oocytes were observed that correlate with chromatin patterns. The extensive cytoplasmic microtubule complex seen in oocytes of preantral follicles initially localizes to perinuclear areas of the ooplasm. This is followed by a progressive reduction in cytoplasmic microtubules and the appearance of prominent microtubule-organizing centers at the nuclear periphery. Coordinated nuclear and microtubular alterations also occur under in vitro conditions prior to progression of meiosis to prometaphase-1. The results are discussed with respect to the ongoing differentiation of the oocyte nucleus and the microtubule cytoskeleton during folliculogenesis in preparation for the resumption of meiosis.

Transitions in trophectoderm cellular shape and cytoskeletal organization in the elongating pig blastocyst.

Changes in cellular shape and filamentous actin (f-actin) organization within the trophectoderm of pig embryos have been studied by fluorescence microscopy during the transitions from spherical to filamentous blastocysts. Cells comprising the trophectoderm of spherical, ovoid, tubular, and filamentous blastocysts are distinctive in their shape, size, and organization of membrane-associated f-actin. Trophectodermal cells of spherical and ovoid embryos are both generally circular in shape. However, as the spherical embryo acquires an ovoid shape, uniformally distributed apical cell surface microvilli relocate to the apical intercellular margins of adjoining trophectodermal cells. Transitional modifications in cellular shape and f-actin organization are observed in tubular blastocysts when apical cell surface microvilli reappear. In elongating filamentous blastocysts, trophectodermal cells assume a spindle-shaped morphology. The f-actin associated with the apical surface is diminished whereas the associated with the basolateral membrane predominates, especially in constricted regions of the blastocyst. These observations, in conjunction with morphometric parameters of trophectodermal cells and whole blastocysts, are discussed in relation to the role of the actin cytoskeleton in processes that modify trophectodermal cell shape and function in the elongating pig blastocyst.

Stage-specific insulin binding in mouse preimplantation embryos.

Stage-specific insulin binding in the developing mouse embryo was demonstrated by an indirect immunofluorescence technique using an antibody against insulin. Concentration-dependent fluorescence labeling was observed in the morula and blastocyst stages of development, whereas no reactivity was seen in unfertilized oocytes or in 2-, 4-, and 8-cell embryos. The possible significance of these observations is discussed. This represents the first report of stage-specific insulin binding during mammalian preimplantation development.

Quality of oocytes from superovulated rhesus monkeys.

The parameters, serum oestradiol (E2) response, follicle size and cumulus morphology, which are commonly used to determine in-vivo oocyte maturation in human in-vitro fertilization and embryo transfer (IVF-ET) programmes, were shown to be unreliable predictors of maturation of rhesus oocytes. In two groups of rhesus, one stimulated with pregnant mares' serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) and the other with human menopausal gonadotrophin (HMG) and HCG, the three parameters varied widely within and between protocols. Triple fluorochrome staining (TFS) for chromatin, microtubules and filamentous actin (f-actin) in oocytes at the time of collection and following 24 h in culture showed major differences in their maturation both in vivo and in vitro following priming with PMSG and HMG. In evaluating IVF protocols, TFS provides a valuable assay for the meiotic status of fixed oocytes of non-human primates.

Toxicokinetic analysis of losartan during gestation and lactation in the rat.

Previous developmental and reproductive toxicity studies in rats with losartan, a potent AT1-selective angiotensin II (AII) receptor antagonist, correlated maternal treatment during gestation day (GD) 15-20 with irreversible renal abnormalities in the F1 generation (Spence et al., '95a,b). Continued treatment through lactation was also associated with increases in pup mortality and decreases in pup body weights that persisted through weaning. The studies presented here were undertaken to quantify fetal and neonatal exposure to losartan when administered to the dam by oral gavage during early gestation, late gestation, and lactation. Following daily oral dosing of 135 mg/kg/day on GD6-15, fetal drug levels were negligible. However, losartan and its active metabolite, EXP3174 (L-158,641) were readily detectable in fetal plasma on GD 20 (estimated AUC values, 50.70 and 167.70 micrograms/hr/ml, respectively) and maternal milk during lactation (1.61 and 1.67 micrograms/ml, respectively). These studies suggest that the relative increased sensitivity of the fetus as compared to the neonate for losartan-induced renal lesions is related to the degree of exposure which is dependent on the time of administration (early gestation vs. late gestation/lactation) and the route of exposure (transplacental or through the milk). Furthermore, the maximum exposure to losartan and EXP3174 correlates with the ontogeny of the renin angiotensin system on approximately GD 17 and the critical period for losartan-induced renal lesions (GD15-20). The data support the hypothesis that the observed adverse fetal and neonatal effects are pharmacologically mediated, presumably through the lack of AT1 receptor stimulation.

The effects of diet, overfeeding and moderate dietary restriction on Sprague-Dawley rat survival, disease and toxicology.

Overfeeding by ad libitum (AL) food consumption is the most significant, uncontrolled variable affecting the outcome of the current rodent bioassay. The correlation of food consumption, the resultant adult body weight and the 2-y survival in Sprague-Dawley rats is highly significant. Feeding natural ingredient diets that varied in protein, fiber and metabolizable energy content did not improve low 2-y survival if Sprague-Dawley rats were allowed AL food consumption. Moderate dietary restriction (DR) of all diets tested significantly improved survival and delayed the onset of spontaneous degenerative disease (i.e., nephropathy and cardiomyopathy) and diet-related tumors. By 2 y, moderate DR resulted in an incidence of spontaneous tumors similar to that seen with AL consumption; however, the tumors were more likely to be incidental and did not result in early mortality. There was a decreased age-adjusted incidence in pituitary and mammary gland tumors, but tumor volume and growth time were similar in the AL and DR groups, indicating a similar tumor progression with a delay in tumor onset. Moderate DR did not significantly alter drug-metabolizing enzyme activities or the toxicologic response to five pharmaceuticals tested at maximum tolerated doses (MTD). However, moderate DR did require higher doses of compounds to be given before classical MTD were produced with four pharmaceutical drug candidates. Toxicokinetic studies of two of these compounds demonstrated steady-state systemic exposures that were equal or higher in moderate DR-fed rats. These and other data indicate that moderate DR is the most appropriate method of dietary control for rodent bioassays used to assess human safety of candidate pharmaceuticals.

The effects of diet, ad libitum overfeeding, and moderate dietary restriction on the rodent bioassay: the uncontrolled variable in safety assessment.

Ad libitum (AL) overfeeding is the most significant, uncontrolled variable affecting the outcome of the current rodent bioassay. There is a highly significant correlation between AL food consumption, the resultant obesity and body weight, and low 2-yr survival in rodents. AL feeding of diets with lowered protein, metabolizable energy (ME), and increased fiber does not improve survival. Only dietary restriction (DR) of all diets tested significantly improves survival and delays the onset of spontaneous degenerative disease (i.e., nephropathy and cardiomyopathy) and diet-related tumors. Moderate DR results in an incidence of spontaneous tumors similar to AL-fed rats, but the tumors are found incidentally and do not cause early mortality. There is a decreased age-adjusted incidence of pituitary and mammary gland tumors in moderate DR-fed rats, but tumor growth time is similar between AL and DR rats with only a delay in tumor onset time seen in DR-fed groups. Moderate DR does not significantly alter drug-metabolizing enzyme activities nor the toxicologic response to 5 pharmaceuticals tested at maximum tolerated doses (MTDs). However, moderate DR-fed rats did require much higher doses of 4 additional pharmaceutical compounds before classical MTDs were produced. Toxicokinetic studies of 2 of these compounds demonstrated equal or higher steady-state systemic exposures to parent drug and metabolites in moderate DR-fed rats. Markers of oxidative stress (lipid peroxidation, protein oxidation) are decreased and cytoprotective anti-oxidant markers are preserved in moderate DR-fed rats. But moderate DR does not delay reproductive senescence in female rats. Only marked DR delays reproductive senescence compared to AL and moderate DR-fed female rats. These and other data indicate that moderate DR is the most appropriate method of dietary control for the rodent bioassay when used to assess pharmaceuticals for human safety and compounds for risk assessment.

Diet, overfeeding, and moderate dietary restriction in control Sprague-Dawley rats: II. Effects on age-related proliferative and degenerative lesions.

This study compared the effects of ad libitum (AL) overfeeding and moderate dietary restriction (DR) of 2 different diets on Sprague-Dawley (SD) rat survival and spontaneous, age-related proliferative and degenerative lesions. SD rats were fed Purina Rodent Chow 5002 or a modified Rodent Chow 5002-9 containing lower protein, fat, metabolizable energy, and increased fiber by AL or by DR at 65% of the AL amount by measurement or time (6.5 hr). At 106 wk, rats fed the 5002-9 diet AL did not have significantly improved survival over rats fed the 5002 diet AL. The 5002 diet fed DR by time (6.5 hr) improved survival for males but not females. Only DR by measurement of both diets resulted in lower mortality for both sexes. By 106 wk rats fed either diet by AL had the same brain weights as DR fed rats, but AL fed rats had greater body weight, body fat content, and increased heart, lung, kidney, liver, adrenal, thyroid, and pituitary weights that correlated with an increased incidence and severity of degenerative and/or proliferative lesions in these organs. Moderate DR delayed the progression of chronic nephropathy by delaying the early development of glomerular hypertrophy that initiates the development of glomerular sclerosis and nephron loss in AL overfed rats. Moderate DR lowered the incidence, severity, and progression of cardiomyopathy and other degenerative, age-related lesions and appeared to delay the development of reproductive senescence in SD females. The conclusion from this study is that moderate DR delayed onset and progression of degenerative lesions, and death due to cardiovascular or renal disease, and thus potentially improves the bioassay to detect compound-specific chronic toxicity.