ALBERT

Notes: Abstract  Using RT-PCR, western blot and enzyme and fluorescence immunocytochemical techniques, the three isoforms of neurofilament proteins (NFPs), namely NF-L (NFP-68 kDa), NF-M (NFP-160 kDa) and NF-H (NFP-200 kDa) were found in Sertoli and Leydig cells of human testes. RT-PCR showed specific for the three NFP fragments in testicular tissue, in isolated seminiferous tubules and in isolated Leydig cells. In protein preparations from the same testicular components, western blot analysis detected bands with molecular weights characteristic for NF-H, NF-M and NF-L. Application of immunofluorescence and immunoenzyme methods on cryostat and paraffin sections resulted in differences in the staining pattern in Sertoli cells and Leydig cells. In these cells, the NFPs showed predominantly a perinuclear location from which bundles emerge that were directed towards the basal, apical and lateral extensions of the Sertoli cells as well as the periphery of Leydig cells. NF-H coexists with vimentin-type filaments as seen by dual staining and staining of conseccutive serial sections of material embedded in paraffin. In Sertoli cells, vimentin and NF-H showed distinct dynamic changes depending on the stage of spermatogenesis and some structural variations of seminiferous tubules. Although in some tubules both vimentin and NF-H immunoreactivity was present at high levels, in the Sertoli cells from most individuals an inverse relationship in the staining intensity of vimentin and NF-H was observed. The strongest NF-H immunoreactivity was detected in Sertoli cells associated with stage 3 spermatids, whereas vimentin immunoreactivity was most abundant in association with stage 5 spermatids. The leydig cells did not show functional changes of the NFP immunoreactivity. The results obtained provide new evidence for the heterogeneous phenotype of human Sertoli cells and raise the question of their exact nature and origin.

Notes: Summary Ganglia from Auerbach's plexus of the large intestine (caecum, appendix vermiformis, colon transversum and rectum) in man, rhesus monkey and guinea-pig are composed of nerve cells and their processes, typical Schwann cells and a vast neuropil. The neuropil consists of dendrites and axons of intrinsic nerve cell perikarya and axons of extrinsic neurons. Axonal profiles in large nerve fibre bundles are of uniform size and appearance, embedded in infoldings of Schwann cell cytoplasm and contain occasional large granular vesicles, mitochondria and neurotubules. Preterminal axons widen into vesicle filled varicosities, some of which establish synaptic contact with intrinsic nerve cell bodies. At least three different types of neuronal processes can be distinguished in the myenteric neuropil according to the size, appearance and commutual proportion of vesicles present in axonal varicosities, and their ability to accumulate exogenous 5- and 6-hydroxydopamine and 5-hydroxydopa: 1. Axonal enlargements containing a major population of small electron lucent “synaptic” vesicles (350–600 Å in diameter) together with a small number of membrane-bound, opaque granules (800–1,100 Å). These profiles have been identified as “cholinergic” axons. The boutons establish synaptic contacts with dendritic processes of intrinsic nerve cell bodies; membrane specializations are found at the preand postsynaptic sites. 2. Axonal beads of sometimes very large diameter, containing an approximately equal amount of large granular vesicles (850–1,600 Å) and small, electron lucent or faintly opaque vesicles (400–600 Å). The granular core of the large vesicles is of medium electron density and may either fill the entire vesicle or is separated from the limiting membrane by a more or less clear interspace. The fibres probably belong to intrinsic neurons, and because of the similarity of the large, membrane-bound vesicles with neurosecretory elementary granules, they have been designated “p-type fibres” (polypeptide fibres). The granular core of the vesicles in these fibres becomes more electron dense after treatment with 5-OH-dopa. The accumulation of an amine precursor analogue in combination with a possible storage of a polypeptide substance (or an ATP-like substance) resembles the situation in several diffusely distributed endocrine cell systems. 3. Varicosities of axons equipped with small (400–600 Å) empty or sometimes granular vesicles, medium sized (500–900 Å) vesicles with highly electron dense cores and occasional large (900–1,300 Å) granular vesicles. Pretreatment with 5-OH-dopamine increases the electron density in almost all medium-sized granular vesicles and some of the large granular vesicles; an osmiophilic core develops in some small vesicles. 6-hydroxydopamine results in degenerative changes in the varicosities of this type of neurons. Concomitantly, both catecholamine analogues markedly reduce neuronal noradrenaline in the large intestine, as demonstrated by fluorescence histochemistry and in fluorimetric determinations. The ultrastructural features of these varicosities and their reaction to 5- and 6-OH-dopamine indicate that they belong to adrenergic, sympathetic nerves. No membrane specializations could be detected at sites of close contact of the adrenergic boutons with dendrites and cell bodies of intrinsic nerve cells.

Notes: Summary In the proximal part of the epididymis of adult rabbits the heads of the spermatozoa exhibit a brilliant green autofluorescence. It has been investigated by means of histochemical, pharmacological and microspectrographical methods. The results show that the green fluorescent substance is thermolabile. Its UV-stability is pH-dependant. Pretreatment of the animals with Reserpine does not influence the intensity of the fluorescence. The green fluorescence does not disappear after treatment of the tissue with sodiumborohydride. Therefore the fluorescence is not due to the presence of biogenic catecholamines. By means of enzymatic methods it is shown that the fluorescence is dependant on the presence of TPNH; after oxidation of TPNH the fluorescence changes from a bright green to a deep orange red.

Notes: Summary 1. The localization of catecholamines and 5-hydroxytryptamine has been investigated in the human testis, epididymis, ductus deferens and prostate obtained at operations on patients aged 26–79 years. 2. In fluorimetric determinations noradrenaline was found to be present in varying concentrations in the genital tract. The lowest amount (0.07 μg/g) was found in the testis. In proximal direction along the ejaculatory duct system there was a progressive increase in the noradrenaline level, reaching a maximum of 1.43 μg/g in ductus deferens. The prostatic tissue (obtained from hyperplastic glands) contained only 0.15 μg/g noradrenaline. No dopamine was present. Adrenaline occurred, by all probability as an artefact, only in tissues from one single case to which this amine had been administered as part of the local anaesthetic mixture given before operation. 3. Fluorescence microscopic analysis of formaldehyde-treated genital organs revealed the presence of adrenergic nerves, the number of which well agreed with the noradrenaline concentrations. Only vascular nerves were found in the testis. The bulk of the adrenergic nerve terminals in the epididymis, ductus deferens and prostate innervated the smooth muscle coats, whereas only a smaller portion of the adrenergic nerve population was of vasomotor nature. 4. Microspectrophotometric characterization of the formaldehyde-induced fluorescence in the tissue sections confirmed that the adrenergic nerves contained noradrenaline. 5. The finding of fluorescent ganglion cells in the prostate indicated that part — if not all — of the male genital tract also in humans is innervated by way of so-called short adrenergic neurons, originating in peripherally located sympathetic ganglia. 6. Flask-shaped cells, morphologically resembling the enterochromaffin cells and emitting a yellow indole fluorescence, were present in the epithelium of the prostatic mucosa. 7. Apart from the specific, formaldehyde-induced monoamine fluorescence, a considerable amount of autofluorescence of varying colour and related to different structures occurred both in the epithelial layers and in the connective tissue.

Notes: Summary It is shown that in the region of the musculus sphincter ani internus nerve cells are absent in the submucosa as well as in the connective tissue between the smooth muscles. Taking into consideration that nerve cells are also absent in the narrow segment of the gut in Hirschsprung's disease, the present findings are thought to represent a morphological basis for anal continence.

Notes: Summary The testis and epididymis of the swan were investigated by means of Falck's and Hillarp's method for fluorescence microscopical detection of catechol- and tryptamines. In the testis a dense plexus of green fluorescent nerve fibres was found to course between the tubuli seminiferi and to surround clusters of Leydig-cells. Electronmicroscopically numerous bare axons containing dense core vesicles were observed between individual Leydig-cells. These results favour the assumption that in contrast to what has been found in most mammals the testicular interstitial cell system of the swan is supplied by sympathetic nerve fibres. The adrenergic innervation of the excretory ducts was also found to behave different from that in mammals: The innervation is most dense in the proximal portions of the ductuli efferentes. From the results of microspectrofluorimetric measurements it is concluded that the transmitter is most likely noradrenaline. Fluorimetric determinations reveal the presence of high amounts of noradrenaline (5,16–5,50 μg/g).

Notes: Summary The spermatogenesis in the spiny dogfish (Squalus acanthias) was investigated by means of light- and electronmicroscopy. In a single histologic section the reproduction and differentiation of germ cells — located in so called ampullae — can be studied, proceeding from early spermatogonial cells up to mature spermatozoa. 9 zones of ampullae in different stages of development can be distinguished in a single transverse section from the testis. 13 generations of spermatogonial cells can be found (2 type A, 11 type B). 2 meiotic divisions are followed by the spermatohistogenesis. Each ampulla contains 424 bundles, and each bundle normally consists of 64 spermatozoa. The central lumen of the ampulla is bordered by prismatic cells during the multiplication phase of the germ cells. They originate from mesenchymal cells, correspond to the Sertoli cells of higher vertebrates and show a biphasic behaviour during the spermatogenesis. During the multiplication phase of the germ cells the mesenchymal cells become prismatic epithelial cells and produce a secretion product consisting of vesicles and osmiophilic droplets which is delivered into the lumen of the ampulla. During the differentiation phase these cells produce lipoid droplets; they are considered to behave like supporting cells in favour of the spermatozoa. The morphological and functional changes of these cells proceed during the initial stages of the prophase of the meiosis. This functional and morphological transformation is considered to be the result of a local control of the spermatogenesis. The possibility that the secretion product of the prismatic cells might be an androsteroid influencing the whole body of the animal — known to lack specialized Leydig cells — is discussed.