Structural and functional changes in ovaries from adult mice treated with diethylstilboestrol in the neonatal period

Ovaries from 8-week-old female NMRI mice in different stages of the oestrous cycle, or from females neonatally treated with the synthetic oestrogen diethylstilboestrol (DES; 5-10(-6) micrograms daily for 5 days), were studied histologically and for the ability to synthesize steroids from [3H]pregnenolone in vitro. Daily doses of 10(-4) micrograms DES or higher resulted in absence of corpora lutea. In ovaries lacking corpora lutea, the interstitial tissue dominated and the cells in this compartment were large with a clear cytoplasm. The steroids synthesized in ovarian homogenates were separated with thin-layer chromatography. The homogeneity of the steroids was checked in recrystallization experiments. Daily doses of 5-10(-4) micrograms DES in the neonatal period resulted in pronounced deviations in the pattern of ovarian steroids synthesized as compared with control ovaries. In DES-exposed ovaries, the synthesis of androstenedione and, above all, progesterone was high while the synthesis of 17 alpha-hydroxyprogesterone and testosterone was reduced compared with controls. These results could argue for a difference in activities of 17 alpha-hydroxylase and 17 beta-ol-dehydrogenase in ovaries from DES-treated females compared with controls. After transplantation of DES-exposed ovaries to ovariectomized control females, the steroid pattern changed to that typical for control ovaries. Control ovaries transplanted to DES-treated females had a steroid pattern similar to that of DES-exposed ovaries.     

Immunocytological studies on an estradiol sensitive antigen in the cervicovaginal epithelium of neonatal mice

Summary Cells of the cervicovaginal epithelium of neonatal mice underwent morphological changes in response to estradiol injection. On the luminal border, estradiol treatment caused development of distinct microvilli and a prominent surface coat of delicate filamentous material. Very deep nuclear folds appeared, and the border between adjacent cells became strongly interdigitated. The cells developed a pronounced smooth and rough endoplasmic reticulum, and dark-stained membrane-bounded granules accumulated in the apical part of the cells.Estradiol promoted increased production of an antigenic material specific for the cervicovaginal epithelium (CVA). Immunofluorescence studies demonstrated CVA in the most apical part of the cells, in the extracellular material on the epithelial surface, and in the intercellular spaces between adjacent epithelial cells. This was confirmed by immunoferritin methods, which revealed that the antigen was localized to the surface coat and to material adhering closely to the exterior of the cell membrane, the part facing the lumen and also the part facing intercellular spaces. Within the cells, ferritin tagging was recognized around the membranes enclosing the dark-stained granules in the apical part of the cells and also on the inside of the luminal cell membrane. This is so interpreted that CVA acquires its antigenic properties when passing out from the dark-stained granules through the surrounding envelope. CVA apparently forms part of the glycocalyx of the cervicovaginal cells.This investigation was supported by grants from the Norwegian Cancer Society (Landsforeningen mot Kreft) and the Norwegian Research Council.     

Studies on the differentiation pattern and hormonal sensitivity of an antigenic material specific for the cervicovaginal epithelium in fetal and neonatal mice.

By use of an indirect mixed haemagglutination method for tissue sections, the Müllerian cervicovaginal epithelium of fetal and neonatal mice has been shown to contain a material (CVA) with antigenic properties specific for this epithelium. The method is highly sensitive and permits a semiquantitative estimation of CVA in the epithelium. The studies showed that the cervicovaginal epithelium undergoes a multiphasic differentiation pattern. An estradiol injection 1 day prior to killing the animals strongly increased the amount of demonstrable CVA. The quantitative response to estradiol varied with the age of the animals. Notably, estradiol given as early as the day of birth stimulated CVA accumulation in the vaginal epithelium. Differences between the cervical epithelium and the vaginal epithelium regarding the response to estradiol are described.     

Dynamics of alpha-fetoprotein concentration in mice of different genotypes during the neonatal period]

A comparative assay of the alpha-fetoprotein (alpha-FP) level in mice of various genotypes (CBA, C3H, C57BL/Se/Sn, BALB/c, CC57W, AKR and nude--nu/nu) was conducted in the course of 3 weeks of postnatal period. The concentration of alpha-FP reached the following levels: the first day 2(-10)-2(-9); the 5th day 2(-8); the 8th day 2(-7); the 15th day 2(-4); on the 22nd day the level was zero. Nude mice which showed the alpha-FP concentration of 2(-2) on the 15th day were an exception. A conclusion was drawn that the alpha-FP synthesis was based not on the athymia of nude mice per se, but upon other unknown factors.     

Effect of epidermal chalones on induction and growth of tumors of the cervicovaginal epithelium of mice

Tumors of the cervicovaginal epithelium were induced in BALB/c female mice with local application of dimethylbenzanthra cene. In the course of inducing tumors or after their appearance the mice were treated intravaginally with 55-81% ethanolic extract of the rat skin or liver (control) containing 1% chalones. The skin extract rather than liver one delays slightly the growth and incidence of cervical and vaginal tumours, and increases the survival of animals.     

Pulmonary vascular resistance in adult rats exposed to hypoxia in the neonatal period

Newborn rats were exposed to hypoxia (10% O2 + N2) from 24 h to day 6 of neonatal life and then returned to room air until 45 days of age (experimental). The rats were anaesthetized, heparinized, and exsanguinated. The chest was opened and the lungs were perfused with diluted autologous blood at a constant flow rate (Q). The pulmonary arterial pressure (Pa) and venous pressure (Pv) were monitored. The properties of the pulmonary vasculature were assessed by measuring baseline vascular resistance, PVR = (Pa-Pv)/Q, segmental pressure gradients (double occlusion technique), pressure-flow relationship, hypoxic pressor response (HPR, 3% O2), and the response to 0.5 microgram bolus of angiotensin II (AII). These were compared with similar measurements on age-matched control animals never exposed to hypoxia. The perfusate hematocrit and gases were not significantly different between the two groups. The PVR normalized to body weight was 30% higher in the experimental groups (p less than 0.005). The double occlusion results (obtained at a flow rate of 13 mL/min) revealed that this increase in resistance was primarily due to the increase in the postcapillary resistance. HPR was primarily in the upstream segment in both groups but was larger in the experimental group. In contrast, the response to AII occurred in both the upstream as well as in the downstream vascular segments and did not differ between the two groups. We conclude that adult rats exposed to hypoxia in the neonatal period have elevated pulmonary vascular resistance and increased vascular reactivity to hypoxia.     

Developmental changes of cardiac function and mass assessed with MRI in neonatal, juvenile, and adult mice

Cardiovascular transgenic mouse models with an early phenotype or even premature death require noninvasive imaging methods that allow for accurate visualization of cardiac morphology and function. Thus the purpose of our study was to assess the feasibility of magnetic resonance imaging (MRI) to characterize cardiac function and mass in newborn, juvenile, and adult mice. Forty-five C57bl/6 mice from seven age groups (3 days to 4 mo after birth) were studied by MRI under isoflurane anesthesia. Electrocardiogram-gated cine MRI was performed with an in-plane resolution of (78-117 microm)(2). Temporal resolution per cine frame was 8.6 ms. MRI revealed cardiac anatomy in mice from all age groups with high temporal and spatial resolution. There was close correlation between MRI- and autopsy-determined left ventricular (LV) mass (r = 0.95, SE of estimate = 9.5 mg). The increase of LV mass (range 9.6-101.3 mg), cardiac output (range 1.1-14.3 ml/min), and stroke volume (range 3. 2-40.2 microl) with age could be quantified by MRI measurements. Ejection fraction and cardiac index did not change with aging. However, LV mass index decreased with increasing age (P < 0.01). High-resolution MRI allows for accurate in vivo assessment of cardiac function in neonatal, juvenile, and adult mice. This method should be useful when applied in transgenic mouse models.     

Morphological changes related to the climateric period of the adult Cuban women

The aim of the present study is to analyze the morphologic changes occurring along the period of a woman's life thatareknown as climacterium. Our sample consists of 648 women from different provinces of Cuba, but who lived in Havana for at least 15 years. Morphological variables such as height, weight, biliocristal and biachromial diameters and six subcutaneous fat skinfolds were measured. With regard to the menopausic condition, the sample was divided into three subpopulations: premenopausic, naturally menopausic and surgically menopausic (when the two ovaries had been removed). Age at menopause was calculated by both the “retrospective” (49.45±0.49) and “status quo” (48.83±0.02) methods. The results obtained point towards morphological differences between pre- and postmenopausic women. We observed greater accumulations of fatty tissue in the suprailiac regions of postmenopausal females. Lean body mass represents a bigger fraction of the total body weight in women who are still menstruating. In the case of surgically menopausic women, they are at a mid-distance between the premenopausic and naturally menopausic subpopulations. A discriminant analysis was carried out which confirmed the results previously observed. From this analysis it appeared the body mass index and the subcutaneous skinfolds are the variables that discriminante, that is, separate, the three subsamples.     

Homeobox gene expression in the intestinal epithelium of adult mice.

Using a polymerase chain reaction-based strategy, we have detected the expression of nine different homeobox genes in adult mouse intestine. Included among these are the recently described intestine-specific Cdx-1 gene and a new, related gene, Cdx-2. Southern blot experiments show that Cdx-2 is present in a single copy in the mouse genome. Of several adult mouse tissues assayed, intestine was the only one that contained detectable levels of Cdx-2 mRNA. Expression of all nine homeobox genes in different regions of the intestine was quantitated by RNase protection analysis, which revealed a unique expression profile for each gene. These observations suggest that homeobox gene expression may play an important role in cellular differentiation in the adult intestine.     

Differential effects of sublethal irradiation on the humoral immune responses of adult and neonatal mice.

Adult and 13 day old neonatal mice were given 500 r total body irradiation and immediately immunized with T independent antigens. Whereas 500 r sublethal irradiation markedly depressed the adult plaque forming cell response, exposure to 500 r only marginally reduced the response of neonates. When antigen was delayed 2 days after 500 r, the response of the exposed neonates was equivalent to the unirradiated controls. In a study of the kinetics of the neonatal response, it was seen that irradiation resulted in an early depressed number of plaque forming cells; however, 6 days after challenge the irradiated neonates were fully recovered. These experiments characterize a sub-population of B lymphocytes by its ability to quickly recover from the effects of irradiation. This subpopulation is restricted to the neonatal period of life, and it may have important consequences in the regulation of the immune response.     

Decreased and disproportionate T-cell population in adult mice after neonatal exposure to diethylstilbestrol

Female Balb/c mice neonatally treated with diethylstilbestrol show persistent impairment of several immunological parameters. The distribution of different classes of lymphocytes in the spleen has been determined at 6, 12, and 18 weeks of age. DES resulted in a decreased percentage of T cells in spleen while the number of B cells was normal. Utilizing Lyt antisera the T-cell subpopulations were found to be imbalanced with an increase in Lyt 123 cells and a concomitant decrease in Lyt 1 cells. Ovariectomy did not influence the diethylstilbestrol-induced alterations in the T-lymphocyte population.     

Localization of calcitonin gene-related peptide in cardiomyocytes: comparison of neonatal and dedifferentiating cells to adult myocytes

The purpose of this study was to localize sites of calcitonin gene-related peptide binding in neonatal, freshly isolated and dedifferentiated adult cardiac myocytes in order to help us elucidate the mechanisms of action of this neuropeptides. Previous work has shown that treatment with calcitonin gene-related peptide results in dramatic changes in calcium transients, so we carried out multi-channel acquisitions of fluorescently labeled images to reveal where calcitonin gene-related protein and the L-type calcium channel were localized. Calcitonin gene-related protein was sparse and randomly distributed in rod-like adult cardiomyocytes, found in abundance in areas of the cell where striations were apparent and not where adhesion proteins predominated in dedifferentiating adult myocytes, and in a large perinuclear concentration, with some spreading into the cytoplasm in neonatal cells. Subsequent modeling demonstrated that calcitonin gene-related peptide and the L-type calcium channel protein were closely associated in each of the three myocyte types, suggesting that while the peptide has dramatic and different effects on intracellular calcium levels of the various cardiomyocytes, the action is probably via diverse mechanisms as a result of effects on different channels or pump proteins due to alterations in intracellular calcium concentrations.