In order to elucidate the ultrastructural dynamics of endometrium differentiation, uterine samples of fetuses aged 14 to 22 weeks of gestation (WG) were analyzed. Samples were processed for light (LM), transmission (TEM) and field-emission scanning electron microscopy (FE-SEM). Initial stratification of the uterine wall occurred at 14 WG: endometrial, myometrial, and perimetrial primordia were identified. At this age, the endometrial epithelium was simple columnar to pseudostratified and consisted of microvillous cells. Blood capillaries developed mainly in the stroma and between the myometrium and perimetrium primordia. At 18-20 WG the endometrial epithelium became clearly pseudostratified, with active ciliogenesis and a predominance of microvillous cells. Primordia of tubular glands were present at 20 WG. Microvillous cells still predominated in the endometrial epithelium at 21-22 WG and showed morphological features of apoptosis. The endometrial stroma at this stage was organizing into a thick lamina propria provided with subepithelial capillary plexuses. However, the stroma was formed by still undifferentiated mesenchymal cells during the whole period of study. Our data showed that the epithelial differentiation and distribution in the uterus occur in the human fetus in a similar way as in the adult. The above events are likely the expression of an early developmental patterning and related to future reproductive processes, such as the regulation of gamete passage and blastocyst implantation. Because the structure of the adult uterus is determined by the degree of paramesonephric duct fusion, septum absorption, and differentiation of the uterine primordial layers, our study may contribute toward clarifying to normal urogenital development.

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An experimental design was developed for morphometric analysis of the subcutaneous connective tissue after the subcutaneous injection of 0.1 ml of saline or a histamine solution (0.01, 0.1 or 1% histamine dihydrochloride in saline). The subcutaneous connective tissue of 4-week-old rats, originally 170.0 ± 13.6 μm in thickness, swelled 5.2-fold at 15 min, 3.0-fold at 2 h, and 1.2-fold at 6 h after the injection of saline. The total cross sectional area of both blood and lymphatic vessels increased when compared to that at pre-injection (0.0186 ± 0.0030 mm² in 6-mm-long specimen), 1.4-fold at 15 min, 2.2-fold at 2 h, and 1.1-fold at 6 h post-injection, while the total number of these vessels increased 1.1-fold at 2 h. The total cross sectional area of lymphatic vessels (0.0006 ± 0.0002 mm² in 6-mm-long specimen) alone surged 7.7-fold at 15 min, 4.8-fold at 2 h, and 7.3-fold at 6 h. Collagen fibers were respectively highly, moderately, and mildly disorganized in arrangement at 15 min, 2 h, and 6 h after the saline injection. Histamine elicited an earlier, longer, and more pronounced vasodilatation, particularly at high concentrations. The transvascular permeability of Evans blue increased depending on the concentration of histamine. These findings indicate that the subcutaneous connective tissue has the ability to expand and store a considerable amount of fluid and reversibly returns to normal steady-state conditions by increasing fluid excretion into the blood and lymphatic vessels. It was also strongly suggested that the blood vessels are deeply involved in the excretion and volume regulation of the tissue fluid.

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This paper introduces an aqueous solution of platinum blue (Pt-blue) as an alternative to uranyl acetate (UA) for staining in transmission electron microscopy (TEM). Pt-blue was prepared from a reaction of cis-dichlorodiamine-platinum (II) (cis-platin) with thymidine. When Pt-blue was dried on a microgrid and observed by TEM it showed a uniform appearance with tiny particles less than 1 nm in diameter. The effect of Pt-blue as an electron stain was then examined not only for positive staining of conventional ultrathin resin sections and counterstaining of post-embedding immuno-electron microscopy but also for negative staining. In ultrathin sections of the rat liver and renal glomerulus, Pt-blue provided good contrast images, especially in double staining combined with a lead stain (Pb). Almost all cell organelles were clearly observed with high contrast in these sections. Glycogen granules in the hepatic parenchymal cells were particularly electron dense in Pt-blue stained sections compared with those treated with UA. In longitudinal and transverse sections of budding influenza A viruses, a specific arrangement of rod-like structures, which correspond to the ribonucleoprotein complexes, was clearly shown in each virion stained with Pt-blue and Pb. When post-embedding immunoelectron microscopy was performed in ultrathin sections of HeLa cells embedded in Lowicryl K4M, the localization of Ki-67 protein was sufficiently detected even after Pt-blue and Pb staining. The present study also revealed that Pt-blue could be used for the negative staining of E. coli, allowing the visualization of a flagellum. These findings indicate that Pt-blue is a useful, safe, and easily obtainable electron stain that is an alternative to UA for TEM preparations.

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The reeler mouse is an autosomal recessive mutant mouse caused by mutation of the reelin gene and characterized by cerebellar ataxia. To determine whether the distribution pattern of precerebellar nuclei neurons in the brainstem of the reeler mouse changes, we injected a small volume of a replication-defective recombinant adenovirus carrying E. coli β-galactosidase (lacZ) into the cerebellar cortex of normal and reeler mice. Five days later, the mice were transcardially perfused by a fixative solution. X-gal staining of coronal or sagittal sections of the brainstem revealed that many origins for reticulocerebellar, cuneocerebellar, trigeminocerebellar, and pontocerebellar projections were retrogradely labeled, but only a few olivocerebellar neurons were labeled. Retrogradely labeled neurons in the lateral reticular nucleus tended to locate more laterally and be more condensed into a small compartment in the reeler compared with their normal counterparts. Retrogradely labeled neurons in the external cuneate nucleus were more dorsally shifted in the reeler mice compared with their normal counterparts. We could not find any differences between the normal and reeler mice in the distribution patterns of their trigeminocerebellar projection neurons. Retrogradely labeled pontocerebellar neurons in the basilar pons of the reeler mouse were reduced in number compared with their normal counterparts in addition to being more ventrally and laterally shifted. These findings strongly suggest that the migration of some precerebellar nuclei neurons from the rhombic lip to their final loci may be obstructed in the reeler mice.

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C-src deficiency is characterized by osteopetrosis due to impaired bone resorption by hypofunctional osteoclasts and the resultant failure of tooth eruption. In preliminary observations, we frequently encountered erupted molars in c-src deficient mice unlike in other osteopetrotic animals. Here we examine the effects of c-src deficiency on the development of molar teeth with an emphasis on the spatial relation of growing teeth with the surrounding bones.
In c-src deficient mice, the magnitude of tooth impaction differed considerably among the types of molars; all maxillary 1st molars were totally impacted deep in the alveolar sockets, whereas most mandibular 1st molars fully erupted into oral cavity. Distribution of osteoclasts in the alveolar bone was identical among all types of molars, and electron microscopy revealed signs of bone resorbing activity in these osteoclasts despite the absence of a ruffled border. From early development, the alveolar space was much narrower in the upper molar tooth germs than in the lower ones in both wild type and homozygous animals, and particularly so in the upper 1st molars.
Current observations thus indicate a significant contribution of “hypofunctional osteoclasts” in c-src deficient mice in molar tooth development except for the upper 1st molars, which appear to require highly functional osteoclasts to gain sufficient space for them to grow normally. Taken together, these findings on the seemingly tooth-type specific effects of c-src deficiency on the development and eruption of molar teeth in c-src deficient mice can be attributed to the given differential spatial relation of the respective tooth germs with the surrounding bones in the presence of hypofunctional osteoclasts.

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