Histologic, morphometric, and immunocytochemical analysis of myometrial development in rats and mice: I. Normal development

Myometrial development from the prenatal to adult period was examined in rats and mice 1) by histologic and immunocytochemical methods with anti-actin, -vimentin, and -laminin to assess cytodifferentiation of smooth muscle and fibroblastic cells; and 2) by morphometric procedures to assess quantitatively the expression of cellular orientation in the emerging inner circular myometrial layer. Uterine mesenchymal cells initially were uniformly vimentin-positive, undifferentiated, and randomly oriented during the late fetal period. By the early neonatal period, three mesenchymal layers became recognizable histologically, the middle one of which (prospective circular myometrium) developed distinct circular orientation and differentiated into a layer composed of actin-positive smooth muscle cells. The cells of the inner mesenchymal layer initially exhibited radial orientation. By 10 days postpartum, the outer longitudinal mesenchymal layer differentiated into bundles of smooth muscle cells representing the longitudinal myometrium. The inner mesenchymal layer remained vimentin-positive and differentiated into the randomly ordered endometrial stroma. The cells of the middle and outer mesenchymal layers that were destined to form myometrium initially expressed vimentin throughout and then coexpressed vimentin and actin, but with time vimentin staining disappeared in the maturing smooth muscle cells as they expressed actin.     

Effects of the plasticiser DEHP on lung of newborn rats: catalase immunocytochemistry and morphometric analysis

Experimental administration of di-(2-ethylexyl)-phthalate (DEHP), a plasticiser employed in the fabrication of polyvinyl chloride (PVC), causes increases in lipid metabolising enzymes along with marked peroxisomal proliferation. The effects are found in several mammalian tissues, of which the rodent liver is the most responsive target. Leakage of DEHP from PVC devices is favoured by high temperature and contact with lipid-containing biological fluids. Since preterm babies are currently ventilated through endotracheal PVC tubes, it seemed worthwhile to investigate DEHP effects on immature mammalian lung. In this research, female rats were fed with DEHP in the last week of pregnancy and after delivery, and lungs were excised from 2-day-old pups. At this age, in fact, rat lung histological features closely resemble those found in 24- to 36-week-old human fetuses. In treated animals, morphometric analysis of histological parameters revealed a dramatic decrease in the number of parenchymal airspaces, together with significant increases in their mean size. Moreover, cytochemical detection of the peroxisomal marker catalase revealed an increase in the number of type II pneumocytes. Our findings closely resemble abnormal histological features observed in autoptic lung specimens from children affected with chronic lung diseases.     

Effects of prenatal exposure to 50 Hz magnetic fields on development in mice: II. Postnatal development and behavior

To investigate the potential of magnetic fields to act as a behavioral teratogen, pregnant CD1 mice were exposed or sham-exposed for all of gestation to a 50 Hz/20 mT magnetic field. Maturation of offspring was assessed using a range of standard developmental indices (eye opening, pinna detachment, hair coat, tooth eruption, sexual maturity, and weight) and simple reflexive behaviors (air righting, surface righting, forepaw grasp, cliff avoidance, and negative geotaxis). Activity and coordination levels were explored in juvenile and adult mice using an open field arena, a head-dip board, an accelerating Rotarod, and a residential activity wheel. All assessments were carried out without knowledge of exposure condition. Results from 168 sham-exposed mice from 21 litters and from 184 exposed mice from 23 litters were compared using survival analysis techniques and multivariate regression methods. Three possible field-dependent effects were found: Exposed animals performed the air righting reflex earlier (P < 0.01); exposed males (but not females) were significantly lighter in weight (P = 0.008) at 30 days of age; and exposed animals remained on a Rota-rod for less time as juveniles (P = 0.03). Some of these results have not been reported in other studies and may reflect spurious statistical significance, although some effect of magnetic field exposure cannot be ruled out. Overall, these results suggest that prenatal exposure to a 50 Hz magnetic field does not engender any gross impairments in the postnatal development or behavior of mice. This does not preclude such exposure affecting more subtle aspects of behavior. Published 1994 by Wiley-Liss, Inc.     

Gap junction modulation in rat uterus. II. Effects of antiestrogens on myometrial and serosal cells

The ability of several triphenylethylene antiestrogens to affect the modulation of gap junctions in rat uterine myometrial and serosal cells was examined in animals 60 days following hypophysectomy. Five daily injections of enclomiphene, zuclomiphene, tamoxifen, nafoxidine, CI 628 or CI 680 (500 micrograms per injection) promote uterine luminal epithelial cell hypertrophy characteristic of exogenous estrogen administration. These same compounds, however, fail to induce myometrial cell or increase the number of serosal cell gap junctions, respectively, which is also characteristic of exogenous estrogen treatment. Pretreatment of animals with antiestrogens blocks the ability of estradiol benzoate (E2 B) to induce gap junction formation in myometrial cells when followed by combined injections of E2 B and antiestrogens (both 250 micrograms) administered daily for 5 days. Therefore, with respect to the parameter of myometrial cell gap junction stimulation, all of the antiestrogens examined act as pure estrogen antagonists. These same antiestrogen pretreatments only weakly antagonized the ability of E2 B to modulate serosal cell gap junction membrane. These studies indicate the presence of different mechanisms for the estrogenic modulation of gap junctional membrane in myometrial and serosal cells.     

Effects of postnatal DES treatment on uterine growth, development, and estrogen receptor levels

The neonatal rodent appears to be an appropriate animal model for estrogen toxicity in the developing reproductive tract. Newborn rats were treated with diethylstilbestrol (DES) at human therapeutic doses (approx 1 mg/kg) during two ontogenetic periods (postnatal days 1-5 and 1-25). Treatment on days 1-5 doubled uterine wt by day 5; however, these uteri failed to grow after discontinuation of DES treatment. In contrast, uterine wt was 4-fold higher and DNA content was 2-fold higher than controls on days 10-25 with continued DES treatment. Total uterine estrogen receptor levels, depressed 60% by day 5 of DES treatment, partially recovered after discontinuation of DES treatment but remained 25% below controls on day 25. Receptor levels following DES on days 1-25 decreased to about 15% of the controls by day 15. Short-term DES treatment approximately halved uterine gland content while continued treatment almost completely inhibited gland appearance. DES effects on glands appear related to continued hypertrophy of the luminal epithelium, from which uterine glands are derived. Subsequent failure of uterine growth caused by DES treatment on days 1-5 is similar to clinical findings of hypoplastic uteri in DES-treated patients. Disruption of the normal ontogenetic patterns of estrogen receptor by DES may be involved. These data demonstrate abnormal patterns of growth, estrogen receptor levels and morphogenesis in uteri of rats treated postnatally with DES.     

Proximal skeletal muscle alterations in streptozotocin-diabetic rats: a histochemical and morphometric analysis.

The response of rat quadriceps muscle fibers to chronic streptozotocin (STZ) diabetes was studied. Transverse sections of rectus femoris muscle from diabetic and weight-matched control rats were assayed for myofibrilar adenosine triphosphatase (ATPase) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR). A quantitative analysis was carried out by an automatic interactive analysis system focused on the fiber type size and distribution. STZ-induced diabetes caused important effects in this muscle, with changes in the distribution of oxidative enzyme reactions, type I fiber hypertrophy, and type II fiber atrophy, which was greater in type IIB than in type IIA. It is concluded that hypoinsulinism produces morphological alterations in proximal skeletal muscle fibers that are similar to those of neurogenic myopathy. Thus the pathological changes in these mammalian muscle fibers could explain the clinical syndrome seen in diabetic patients called "diabetic symmetrical proximal motor neuropathy," perhaps the least understood of the major neuropathic complications of diabetes.     

A morphometric analysis of craniofacial growth in cleft lip and noncleft mice.

Differences in face shape are considered a factor in cleft lip malformation. The purpose of this study was to analyze craniofacial growth in two strains: A/WySn with 28% cleft lip and C57BL/6J without cleft lip. Standardized photographs of 27 A/WySn and 25 C57BL/6J embryos with 34-46 somites (S) were taken in the superior, frontal, and lateral views. Landmarks were located and digitized for computerized analysis of growth change relative to somite number and at stages of face development before, during, and after primary palate closure. The results showed that both strains had similar overall growth patterns with increases in head width and face width, and decreases in nasal pit width. During early palatal closure in C57BL/6J mice, the nasal pit width was unchanged as brain width increased rapidly; and then later, the nasal pit width decreased as brain width increased slowly. However, during early closure in A/WySn mice, the nasal pit width decreased rapidly as brain width increased slowly; and then later, the nasal pit width was unchanged as brain width increased more rapidly. During early palatal closure, the narrower nasal pit width in A/WySn mice appeared to result from delayed growth of the supporting forebrain as the nasal pits become more medially positioned with normal face development. From the lateral view, the maxillary prominence depth was also smaller in the A/WySn strain during early palatal closure. This deficient forward growth of the maxillary prominences and the narrower positioning of the medial nasal prominences in A/WySn embryos appear to reduce the contact between the prominences and thus predispose this strain to cleft lip malformation.     

Ultrastructural, immunocytochemical and morphometric characterization of liver peroxisomes in gray mullet, Mugil cephalus

Peroxisomes of the hepatocytes of gray mullets, Mugil cephalus, were characterized cytochemically and immunocytochemically using antibodies against the peroxisomal proteins catalase and palmitoyl-coenzyme A (CoA) oxidase. In addition, morphometric parameters of peroxisomes were investigated depending on the hepatic zonation, the age of the animals and the sampling season. Mullet liver peroxisomes were reactive for diaminobenzidine, but presented a marked heterogeneity in staining intensity. Most of the peroxisomes were spherical or oval in shape, although irregular forms were also observed. Their size was heterogeneous, with profile diameters ranging from 0.2 to 3 microm. Peroxisomes tended to occur in clusters, usually near the mitochondria and lipid droplets. They also showed a very close topographical relationship to the smooth endoplasmic reticulum. Mullet liver peroxisomes did not contain cores or nucleoids as rodent liver peroxisomes, but internal substructures were observed in the matrix, consisting of small tubules about 60 nm in diameter and larger semicircles 120 nm in diameter. The volume density of peroxisomes was higher in periportal hepatocytes of mullets sampled in summer than in pericentral hepatocytes, indicating that mullet peroxisomes vary depending on physiological and environmental conditions. By immunoblotting, the mammalian antibodies cross-react with the corresponding proteins in whole homogenates of mullet liver. Paraffin sections immunostained with the antibodies against catalase and palmitoyl-CoA oxidase showed a positive reaction corresponding to peroxisomes localized in the hepatocyte cytoplasm. In agreement, the ultrastructural study revealed that catalase and palmitoyl-CoA oxidase are exclusively localized in the peroxisomal matrix in fish hepatocytes, showing a dense gold labeling. The presence of the peroxisomal beta-oxidation enzyme palmitoyl-CoA oxidase in peroxisomes indicated that these organelles play a key role in the lipid metabolism of fish liver.     

Further immunocytochemical study on the localization of aromatase in the ovary of rats and mice

The precise localization of estrogen biosynthesis in the ovary of rats and mice were immunocytochemically studied using new antisera against aromatase cytochrome P-450. The positive reaction for aromatase was detected mainly on the granulosa cells of large, apparently preovulatory follicles. In addition, the cells of some corpora lutea showed very weak positive reaction but most corpora lutea were negative to the staining. Those cells such as the granulosa cells of smaller follicles, the theca interna cells, the interstitial gland cells, oocytes, peritoneal epithelial cells were entirely negative. These results indicate that in the ovary of rats and mice, the granulosa cells of preovulatory follicles are the main site for synthesis of estrogen from androgen which is provided by the theca interna cell and the interstitial gland cell.     

Further immunocytochemical study on the localization of aromatase in the ovary of rats and mice

Summary The precise localization of estrogen biosynthesis in the ovary of rats and mice were immunocytochemically studied using new antisera against aromatase cytochrome P-450. The positive reaction for aromatase was detected mainly on the granulosa cells of large, apparently preovulatory follicles. In addition, the cells of some corpora lutea showed very weak positive reaction but most corpora lutea were negative to the staining. Those cells such as the granulosa cells of smaller follicles, the theca interna cells, the interstitial gland cells, oocytes, peritoneal epithelial cells were entirely negative. These results indicate that in the ovary of rats and mice, the granulosa cells of preovulatory follicles are the main site for synthesis of estrogen from androgen which is provided by the theca interna cell and the interstitial gland cell.This study was supported by Grants from the Ministry of Education, Science and Culture, Japan, and from USPHS Research Grants HD04945, USA.     

Influence of unilateral castration and increased plane of nutrition on sexual development of Holstein bulls. II. Histologic development of the testes

Sixty-five Holstein bull calves were assigned in an experiment to determine the effects of unilateral castration (UC) at 1 week of age and of two levels of nutrition after 6 months on reproductive development to 16 months. Five animals were killed at 1 wk and half the remainder UC at that age. Groups of 5 in all factorial groups were killed at 2, 4, 8 and 16 months. Tubular diameter increased with age (P<.01) and at 8 and 16 months with UC (P<.01). Epithelial area at 8 and 16 months increased with age and UC (P<.01). The percents of tubular and intertubular tissue varied with age (P<.01) with the tubular tissue having its highest value at 8 months. Indexes of both total tubular and intertubular tissue were increased with age and UC (P<.01). The number of type A spermatogonia per cross section of stage 1 tubules of 16-month bulls was increased by UC (P<.05).     

Effects of diosgenin on myometrial matrix metalloproteinase-2 and -9 activity and expression in ovariectomized rats

Diosgenin, a traditional Yam extraction, has been used in hormone replacement for menopausal women. We aimed to investigate the influences of diosgenin administration upon the MMP-2 and -9 activity and expression and reproductive hormones of ovariectomized (OVX) rats, a model of menopausal status. Seven-week old female Wistar rats with bilateral OVX or sham operation (controls) were divided and administered different dosages of diosgenin (0, 10, 50, or 100 mg/kg/day) for 8 weeks. Serum was then sampled for progesterone (P4) and estradiol (E2) assay and uterine horns harvested. Myometrial MMP-2 and -9 activity and expression were surveyed and myometrial collagen expression was also assayed. The results show higher body weight in OVX rats across the 8 weeks post surgery and no significant differences were noted among OVX or Sham rats with diosgenin supplements. There were lower P4 and E2 concentrations in OVX rats compared to Sham rats, and higher P4 concentration of Sham rats post diosgenin supplement. MMP-2 and -9 mRNA expression and activity was lower in OVX rats, although higher MMP-2 and lower MMP-9 activity/mRNA expression was observed in OVX rats post diosgenin supplementation. Collagen mRNA expression was higher in OVX rats compared to Sham controls, and diosgenin administration decreased collagen mRNA expression in OVX rats. In conclusion, diosgenin is associated with gelatinase expression and collagen metabolism in OVX rats. Diosgenin administration can partially reverse the effects of OVX upon MMP functions and hormone status. Adequate diosgenin supplement might modulate myometrial gelatinase expression and collagen metabolism in menopausal subjects.     

The chick optic tectum developmental stages. A dynamic table based on temporal‐ and spatial‐dependent histogenetic changes: A structural,morphometric and immunocytochemical analysis

Development is often described as temporal sequences of developmental stages (DSs). When tables of DS are defined exclusively in the time domain they cannot discriminate histogenetic differences between different positions along a spatial reference axis. We introduce a table of DSs for the developing chick optic tectum (OT) based on time‐ and space‐dependent changes in quantitative morphometric parameters, qualitative histogenetic features and immunocytochemical pattern of several developmentally active molecules (Notch1, Hes5, NeuroD1, β‐III‐Tubulin, synaptotagmin‐I and neurofilament‐M). Seven DSs and four transitional stages were defined from ED2 to ED12, when the basic OT cortical organization is established, along a spatial developmental gradient axis extending between a zone of maximal and a zone of minimal development. The table of DSs reveals that DSs do not only progress as a function of time but also display a spatially organized propagation along the developmental gradient axis. The complex and dynamic character of the OT development is documented by the fact that several DSs are simultaneously present at any ED or any embryonic stage. The table of DSs allows interpreting how developmental cell behaviors are temporally and spatially organized and explains how different DSs appear as a function of both time and space. The table of DSs provides a reference system to characterize the OT corticogenesis and to reliably compare observations made in different specimens. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.