A cytochemical study of oocyte growth in four species of millipedes

Summary The cytochemistry of oocyte growth was investigated in four species of millipedes; Narceus americanus, Oxidus gracilis, Cheiropus plancus, and a Pleuroloma species, probably P. cala. The oocyte of all four species produced a yolk nucleus which arose in contact with the nuclear membrane, subsequently detached, migrated into the central ooplasm and disrupted coincident with the appearance of protein yolk granules in the oocyte cytoplasm. Since the follicular epithelium did not display any morphological or cytochemical manifestations of secretory activity, it is suggested that direct incorporation of exogeneous proteins into yolk may play a lesser role in vitellogenesis in these forms than in insects and many other animal groups. Ooplasmic RNA levels achieved a maximum before vitellogenesis was initiated and then decreased. Similarly the nucleoli increased in size and RNA content up to the point at which cytoplasmic RNA levels began to decline. Basic proteins associated with RNA were present in the oocyte cytoplasm and the nucleoli. These achieved a peak concentration in the same size oocytes as RNA, but the intensity of staining decreased more rapidly than that of RNA during subsequent growth. The concentration of nucleoplasmic protein in oocytes of all four millipede species increased in the germinal vesicles during the course of oocyte growth. Coincident with the initiation of vitellogenesis, a class of cytoplasmic inclusions was developed which have been called concentric ring bodies. These inclusions consist of concentric layers of organic matrix material with crystallized calcium salts sandwiched between. These probably represent calcium reserves which are utilized in the formation of the exoskeleton by the embryo.Presented in partial fulfillment of the requirements for the Master of Science degree to the Graduate School of Arts and Sciences of the University of Florida.This work was supported by the following U.S. Public Health Service grants: Pathology Training Grant 5-T1-GM-1142-03, and to R. R. C. HD-1499-04 and Career Development Award K-3-HD-6176-04.     

Mallomonas transsylvanica,spec. nova (Chrysophyceae), light and electron microscopical studies

The new speciesMallomonas transsylvanica is described in detail. Its silica armour has been examined by light and electron microscopy. Differences and possible relationships with other species are discussed.     

Mouse epidermal growth factor: Light and electron microscopical localisation by immunocytochemical staining

Summary Epidermal Growth Factor (EGF) has been localised by immunostaining to granules of the convoluted duct cells of the submaxillary glands of mice. Improved techniques of freeze drying and formaldehyde vapour fixation have resulted in a light microscopical localisation sharper than was achieved by previous methods. EGF has also been identified by electron immunocytochemistry using the unlabelled antibody enzyme method. EGF is present in greater quantities in male mice than in female mice but in pregnant females the level of EGF in the submaxillary gland is equal to that of the male. It declines gradually during the three weeks of lactation. In view of the chemical similarity between mouse EGF and human Urogastrone these improved methods of identification may be useful in the localisation of the human substance.This work was presented at the first Symposium on Gastrointestinal Hormones held at Asilomar, California, USA in October 1976     

Melanocortins and opioids modulate early postnatal growth in rats

This study was undertaken to investigate the effects of melanocortins and opioids on rat early postnatal body and organ growth. Among melanocortins tested desacetyl-alpha-melanocyte-stimulating hormone (alpha-MSH) at dosages of 0.3 and 3 micrograms/g/day was effective in stimulating neonatal growth with a weight gain of 7 and 5.6%, respectively, after 2 weeks of treatment. Likewise, a weight rise of 4.2 and 3% was obtained with 3 micrograms/g/day of both alpha-MSH and Nle4-D-Phe7 alpha-MSH. As far as opioids were concerned, while N-acetyl-beta-endorphin (beta-End) was ineffective, the activity of beta-End was dependent on dosage. Indeed, newborns treated with 0.03 microgram/g/day showed a slight, but significant, increase in weight, whereas a marked decrease in growth followed treatment with 0.3 and, mainly, 3 micrograms/g/day, with a final weight loss of 3.4 and 5.5%, respectively. All melanocortins exerted a positive action on muscular and brain trophism and, in addition, desacetyl-alpha-MSH also induced a rise of fat deposits. On the contrary, while the 0.03 microgram/g/day beta-End dose caused an increase in muscular and brain weight, the higher dosages of the opioid were detrimental, not only for muscle and brain, but also for both liver and spleen weight. A slight, although significant (P < 0.05), enhancement of serum dehydroepiandrosterone sulfate (DHEAS) level was found after the injection of 0.3 microgram/g desacetyl-alpha-MSH, whereas both the 0.3 and 3 micrograms/g doses of desacetyl-alpha-MSH and the 3 micrograms/g dose of alpha-MSH determined the rise of plasma androstenedione (P < 0.05). All tested melanocortins and opioids failed to modify the concentrations of corticosterone. Our results suggest that melanocortins and opioids can modulate early postnatal growth in rats either by direct or indirect mechanisms.     

Formation and maturation of subneural apparatuses at neuromuscular junctions in postnatal rats: a scanning and transmission electron microscopical study

We examined the morphodifferentiation of subneural apparatuses at neuromuscular junctions with scanning and transmission electron microscopy (SEM and TEM) in the sternothyroid muscle of postnatal rats. As evidenced with SEM, primitive synaptic troughs found at birth were smooth cup-like depressions 5-6 micron in diameter. At the 5th postnatal day, low sarcoplasmic ridges appeared in the depression which successively grew and upheaved to remodel the depression into anastomosed gutters during the next 10 days. Subneural apparatuses attained almost the adult form by the 30th day, though synaptic troughs were smaller in size and exhibited a less complex pattern. At birth, the depression contained a few mostly pit-like or elongated oval invaginations:incipient junctional folds. By the 15th day, junctional folds rapidly developed, resulting in about an 18-fold increase in number per endplate with the parallel differentiation of slit-like junctional folds of adult form. At the 30th day, junctional folds were mostly slit-like, though pits still coexisted in a small proportion. As a shape factor, we measured the ratio of the length of the folds to their maximum width (L/W); the folds with L/W less than 2 were defined as pits, those with 2 less than or equal to L/W less than 5 as short slits, and those with L/W greater than or equal to 5 as long slits. At birth, pits occupied about 67% of the total number of the folds per endplate, which decreased to about 14% at the 30th day. Concomitantly, long slits remarkably increased from about 3 to 38%. Short slits increased from about 30 to 50% during the first 10 days but remained almost unchanged thereafter. The maximum L/W ratio was 12 at the 15th day and exceeded 20 after the 30th day. These quantitative data and the finding that pits were often closely associated with each other and also with a slit in a serial fashion indicate that the adjacent pits may fuse to each other and to the preformed slits. With TEM, a few incipient junctional folds were found at the 5th day, which extended into the subneural sarcoplasm with a depth less than 0.4 micron. At the 15th day, junctional folds increased both in number and in the maximum depth of about 0.8 micron. There also occurred a number of basal lamina-containing vacuoles identical in many respects to the transversely sectioned profiles of incipient junctional folds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mouse epidermal growth factor: light and electron microscopical localisation by immunocytochemical staining.

Epidermal Growth Factor (EGF) has been localised by immuno-staining to granules of the convoluted duct cells of the submaxillary glands of mice. Improved techniques of freeze drying and formaldehyde vapour fixation have resulted in a light microscopical localisation sharper than was achieved by previous methods. EGF has also been identified by electron immunocytochemistry using the unlabelled antibody enzyme method. EGF is present in greater quantities in male mice than in female mice but in pregnant females the level of EGF in the submaxillary gland is equal to that of the male. It declines gradually during the three weeks of lactation. In view of the chemical similarity between mouse EGF and human Urogastrone these improved methods of identification may be useful in the localisation of the human substance.     

The presence of gap junctions during early Patella embryogenesis: An electron microscopical study

The presence of gap junctions has been examined up to the sixth cleavage in the early Patella embryo. Gap junctions are located all over the blastomere borders. In 2-, 4-, and 8-cell embryos they were also observed at peripheral contacts. The frequency and size of the gap junctions increase at the 32-cell stage. The structure of gap junctions is similar in all stages investigated with hexagonally arranged equal-sized particles (11 nm) having a constant center-to-center spacing (13.0 nm). At the 32-cell stage formation plaques were observed, indicating an increase of gap junctions.     

Thyroliberin injected into pregnant heifers increase birthweight and stimulate postnatal growth of calves

Birthweight (51 +/- 2 kg) and mean daily weight gain (1,306 +/- 77 g; measured during the first 120 days of postnatal life) in 8 male calves born from eight primiparous heifers (mated with the same bull) given subcutaneous TRH injections (25 micrograms/kg body wt) on days 230, 240, 250 and 265 of gestation were significantly higher (+10%) than those measured in 7 control male calves born from the same bull (47 +/- 1 kg and 1147 +/- 89 g, respectively). TRH treatment also significantly increased plasma IGF1 concentrations both in term cows and in newborn calves.     

Amianthoid (asbestoid) transformation: electron microscopical studies on aging human costal cartilage

The present study reports on the fine structure of human costal cartilage at different ages in order to obtain information on the morphogenesis of amianthoid fibers. Our results reveal an overall increase of collagen fibril diameter with increasing age, even in areas with no signs of amianthoid transformation. Ultrastructural evidence is presented that this increase in diameter is due to a gathering of the preexisting collagen fibrils. The age-related change in collagen fibril diameter is paralleled by changes in the composition and ultrastructural appearance of cartilage proteoglycans (as revealed by acridine orange staining). Acridine-orange-positive filaments indicative for proteoglycans are markedly reduced in size with advancing age in centrally located regions of costal cartilage. Treatment with testicular hyaluronidase previous to acridine-orange staining leaves these small proteoglycan filaments unaffected. By contrast, the filaments visible after acridine-orange staining in the extracellular matrix near to the perichondrium are susceptible to hyaluronidase treatment. Infrequently, a sharp increase in collagen fibril diameter can be observed in territorial matrix areas of degenerating chondrocytes. This observation is conspicuous at ages of 10 and 20 years. Amianthoid transformation is characterized by the appearance of collagen fibrils strictly arranged in parallel. These amianthoid fibers are embedded in a matrix rich in small acridine-orange-positive filaments similar to the proteoglycan filaments observed in centrally located matrix regions. It can be concluded that extensive remodelling not only of the collagen fibrils but also of the cartilage proteoglycans is involved in the development of amianthoid transformation.     

CFU-gm assay, cytochemical and electron microscopic studies in agar in patients with preleukemic syndrome and aplastic anemia

Thirty-seven patients with chronic cytopenia were studied using a CFU-gm assay in agar. Cell proliferation was evaluated on days 2, 3, 5, 7, and 10 of incubation. Growth patterns were different in cultures of hematologically healthy persons versus patients with preleukemic syndrome (PL) and aplastic anemia (AA). Three types of PL syndrome and two types of AA (C1 and C2) were distinguished. Bone marrow dysfunction was evaluated further using cytochemistry and electron microscopy to morphologically study cell proliferation in vitro. Cytochemical staining performed in agar demonstrated well-defined maturation defects in myelopoietic precursor cells from the bone marrow of PL patients. Electron microscopic findings of Auer-body-like inclusions in "statu nascendi" in the vacuoles of preleukemic cells supported our results. PL patient groups at high risk for development of overt leukemia and patients with grave prognosis in AA were distinguished. Our results are relevant for the clinical diagnosis and prognosis of patients with cytopenia.     

A cytochemical, light and electron microscopical study of the leucocytes of the adult river lamprey, Lampetra fluviatilis (L. Gray)

The morphological features of the leucocytes from the blood of the river lamprey, Lampetra fluvimilis , were studied using light and electron microscopy. Four cell types were identified, namely granulocytes, lymphocytes, monocytes and thrombocytes. Enzyme cytochemical tests were also performed for the detection of acid phosphatase, β-glucuronidase and peroxidase. All the leucocyte types were positive for acid phosphatase and β-glucuronidase, to a variable extent, with the greatest activity seen in the granulocytes. None of the leucocyte types however, contained any peroxidase activity.
Only one type of granulocyte was found and this appears to be analogous to the mammalian neutrophil/heterophil. Characteristically, it has a cytoplasm containing a large number of morphologically heterogeneous granules (0.07–0.40 um in diameter). It is suggested that these granules, rather than belonging to several subpopulations, are in fact part of a single maturation series.
The results of this study show that precise identification of lamprey leucocytes can only be achieved using a combination of ultrastructural and cytochemical techniques.     

A fixation method for improved antibody penetration in electron microscopical immunoperoxidase studies.

A fixation method for electron microscopical immunoperoxidase staining has been developed, which (a) allows penetration of antibodies through cell membranes to intracellular antigen sites, (b) provides a reasonable cell preservation and (c) does not alter the antigenic structure in too great an extent. Penetration of the antibodies has been achieved by using saponin as a cell membrane attacking agent. The best results could be obtained after pretreatment of cell monolayers with a mixture of 0.05% saponin, 0.0125%-0.05% glutaraldehyde and 1% paraformaldehyde for 5 min at 4 degrees C, and postfixing them with the corresponding fixative without saponin for 45 min at 4 degrees C.     

Peripheral chemoreceptors: postnatal development and cytochemical findings in Sudden Infant Death Syndrome

The aim of the present study is to give a review of the postnatal development of peripheral chemoreceptors - carotid body, paraganglia, and pulmonary neuroendocrine cells (PNEC) - with implications in Sudden Infant Death Syndrome (SIDS). In the postnatal period, the hypoxic chemosensitivity of the carotid body gradually develops. Changes include proliferation of type I and II cells, increased numbers of dense core vesicles and K+ channels, and modifications of neurotransmitter/neuromodulator and receptor expression. Chromaffin paraganglia show increased expression of nitric oxide synthase and neuropeptides, and increased innervation. Innervation of PNEC develops fully only in the first postnatal period, after which their density falls. The neuropeptides produced by PNEC also changes, with increased expression of calcitonin gene-related peptide and neuropeptide YY and reduced expression of calcitonin and gastrin-releasing peptide. Most of the findings in the carotid body of SIDS victims, i.e., decrease in type I cells and dense cytoplasmic granules, and increase in progenitor cells, indicates immaturity of the carotid body, which may play a role in SIDS in the form of underlying biologic vulnerability. Aorticopulmonary paraganglia hyperplasia and increase of PNEC are also found in SIDS, and may be epiphenomena of alterations of the respiratory function with a pathogenetical role in SIDS. A comprehensive view of the pathogenesis of SIDS should also arise from the integration of peripheral chemoreceptors findings with neuro- and cardiopathologic ones.     

Follicle population, cumulus mucification, and oocyte chromatin configuration during the periovulatory period in the female dog

This study was designed to describe the follicular population present on the canine ovary (Canis familiaris) during the preovulatory period and essentially the changes in oocyte size, mucification, and chromatin configuration occurring from before the luteinizing hormone (LH) surge up to postovulation. In a first experiment, ovaries of beagle bitches were collected before (n = 21) or after LH surge but before ovulation (post-LH surge/preovulation stage, n = 24) as determined using hormone (LH, estradiol, progesterone) assays and ultrasonography. All large (>2 mm) follicles were measured and punctured. The numbers of oocytes collected per follicle and the degree of cumulus mucification were recorded. In a second experiment, ovaries were similarly collected before (n = 13) and after the LH surge but before ovulation (n = 11) as well as after ovulation as determined by ultrasonography (n = 9). Chromatin configuration of the oocytes was observed by DNA staining and confocal microscopy. In Experiment 1, before the LH peak, an average of 13.5 ± 0.7 follicles per bitch (total 284 follicles) were detected, and the maximal follicle diameter reached 6.5 mm. Large follicles were observed already in this period of the cycle and as early as when progesterone was still below 0.5 ng/mL. After the LH peak but before ovulation, 11.0 ± 0.7 follicles were present (total 264 follicles). Fully mucified cumulus cells were observed only in follicles larger than 4 mm. Multi-oocytic follicles represented 7% (before LH peak) and 4% (after LH peak) of the follicular population. In Experiment 2, all the oocytes were at the germinal vesicle (GV) stage, but three chromatin configurations could be distinguished: diffuse, partly grouped, and fully grouped chromatin. The proportion of oocytes with fully grouped chromatin increased with the follicular diameter and the time in estrus, the maximum being observed after the LH peak. These results suggest that (1) before LH peak, follicles are already of large diameter, similar to the ones at ovulation; (2) the ability for cumulus mucification is acquired during the late steps of follicular growth; (3) three GV patterns may be observed during the periovulatory period.