A. F. Holstein

Male germ cells and photoreceptors, both dependent on close cell–cell interactions, degenerate upon ClC‐2 Cl− channel disruption

The functions of some CLC Cl− channels are evident from human diseases that result from their mutations, but the role of the broadly expressed ClC‐2 Cl− channel is less clear. Several important functions have been attributed to ClC‐2, but contrary to these expectations ClC‐2‐deficient mice lacked overt abnormalities except for a severe degeneration of the retina and the testes, which led to selective male infertility. Seminiferous tubules did not develop lumina and germ cells failed to complete meiosis. Beginning around puberty there was a massive death of primary spermatocytes and later also of spermatogonia. Tubules were filled with abnormal Sertoli cells, which normally express ClC‐2 in patches adjacent to germ cells. In the retina, photoreceptors lacked normal outer segments and degenerated between days P10 and P30. The current across the retinal pigment epithelium was severely reduced at P36. Thus, ClC‐2 disruption entails the death of two cell types which depend on supporting cells that form the blood–testes and blood–retina barriers. We propose that ClC‐2 is crucial for controlling the ionic environment of these cells.

Auerbach's plexus of mammals and man: Electron microscopic identification of three different types of neuronal processes in myenteric ganglia of the large intestine from rhesus monkeys, guinea-pigs and man

SummaryGanglia from Auerbachs 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.

Organization and ultrastructural identification of the catecholamine nerve terminals in the neural lobe and pars intermedia of the rat pituitary.

SummaryThe central catecholamine innervation of the pituitary neural lobe and pars intermedia of the rat have been identified ultrastructurally and their organization has been investigated in a combined fluorescence histochemical and electron microscopical study. The dopamine analogues, 5-hydroxydopamine and 6-hydroxydopamine, were used to “label” the catecholamine terminals, and to enable the direct correlation between the fluorescence microscopical and the electron microscopical pictures.The fibre type that was identified as catecholamine-containing was ultrastructurally chiefly characterized by dense-cored vesicles, 500–1200 Å in diameter, intermingled with varying numbers of small empty vesicles. 5-hydroxydopamine was selectively accumulated in these fibres and caused an increased electron density of the granular vesicles as well as of some small normally agranular vesicles, and systemically administered 6-hydroxydopamine caused a selective degeneration of these fibres, most prominently within the neural lobe. The dopaminergic terminals of the neural lobe showed frequent close contacts (80–120 Å), without real membrane thickenings, to neurosecretory axons and to pituicyte processes. It is suggested that these close contacts might signify a direct dopaminergic influence on the neurosecretory axons and/or on the pituicyte processes. The identified central catecholamine fibres were also found to make common synapse-like contacts on the pars intermedia cells, whereas the innervation by neurosecretory fibres was very rare. This suggests that the direct central nervous control of the rat pars intermedia is exerted by the catecholamine neurons. A very special feature of the catecholamine fibres in the pituitary is the occurrence of peculiar, large dopamine-filled droplet-like swellings. Electron microscopically, such large axonal swellings (more than 2 μ in diameter) were found to contain, in addition to the characteristic vesicles and organelles, strongly osmiophilic lamellated membrane complexes resembling myelin bodies and multivesicular bodies encircling disintegrated vesicles, suggesting that these “droplet fibres” represent dilated stumps of spontaneously degenerating dopaminergic axons. It is suggested that the dopaminergic neural lobe fibres are undergoing continuous reorganization through degeneration—regeneration cycles, a phenomenon previously suggested for the neurosecretory axons of the neural lobe.

Understanding spermatogenesis is a prerequisite for treatment

Throughout spermatogenesis multiplication, maturation and differentiation of germ cells results in the formation of the male gamete. The understanding of spermatogenesis needs detailed informations about the organization of the germinal epithelium, the structure and function of different types of germ cells, endocrine and paracrine cells and mechanisms, intratesticular and extratesticular regulation of spermatogenesis. Normal germ cells must be discriminated from malformed, apoptotic and degenerating germ cells and tumor cells.Identification of the border line between normal and disturbed spermatogenesis substantiate the diagnosis of impaired male fertility. The profound knowledge of the complicate process of spermatogenesis and all cells or cell systems involved with is the prerequisite to develop concepts for therapy of male infertility or to handle germ cells in the management of assisted reproduction.

Arrangement, ultrastructure, and adrenergic innervation of smooth musculature of the ductuli efferentes, ductus epididymidis and ductus deferens of man

Summary1.There is a gradual proximo-distal increase in the thickness of the muscle coat of the human ductuli efferentes, duetus epididymidis and ductus deferens. Circularly arranged smooth muscle bundles predominate in the ductuli efferentes and ductus epididymidis of the caput section. Scanty strands of longitudinally and obliquely oriented smooth muscle bundles form an additional, incomplete outer muscle layer around the ductus epididymidis of the corpus. Small smooth muscle-like cells constitute the muscle elements of the upper sections of the excretory ducts (from the ductuli efferentes to the midcauda). At the transition of the corpus and cauda epididymidis ordinary large smooth muscle cells join the small contractile cells to form—in more distal sections of the cauda—a composed, thick subepithelial muscle coat. In most distal portions of the cauda, the two-layered muscle coat of the ductus epididymidis is transformed into a three-layered coat, a pattern of construction which is retained in the vas deferens.2.Electron microscopically, three types of contractile cells are distinguished in the human ductuli efferentes and ductus epididymidis: a) contractile cells of medium transparency containing exclusively thin myofilaments (60 Å in diameter), b) dark contractile cells containing bundles of thin myofilaments (60 Å in diameter) and single coarse filaments (140 Å in diameter), c) light contractile cells with loosely dispersed, interweaving thin and thick myofilaments. Commutual diameter changes at regular intervals are seen in individual myofilaments, giving the impression of structural periodicity not unlike that of filaments of striated muscle. Ordinary smooth muscle cells of the cauda epididymidis and vas deferens are characterized by uniformly sized, closely packed but evenly distributed thin myofilaments with numerous dense patches.3.Fluorescence microscopy performed on formaldehyde treated freeze dried tissues reveals that the contractile cells of the ductuli efferentes in man and monkey receive a low number of single adrenergic terminal fibres penetrating the depth of the muscle coat. The adrenergic innervation of the ductus epididymidis is restricted to small peritubular nerve fascicles running contiguous to the most superficially located bundles of smooth muscle-like cells. The adrenergic ground plexus is rather wide-meshed in the proximal cauda, becomes increasingly dense in more distal cauda sections and in initial, funicular portions of the vas deferens, and reaches maximum density in abdominal parts of the ductus. Perivascular and adventitial adrenergic plexuses are well developed at arteries of the caput and corpus epididymidis in man, monkey, rabbit, guinea-pig and rat.4.Electron microscopically, noradrenergic nerves have been identified by the presence of small granular vesicles in preterminal varicose axon dilatations. Nerve fibre swellings filled with small empty spherical vesicles have been considered to belong to “cholinergic” neurons whereas occasional varicosities equipped with some large membrane bound granules and abundant mitochondria may represent local expansions of sensory axons.5.Neuromuscular relationships in the upper sections of excretory ducts comprise adrenergic synapses by distance (more than 1000 Å), and a few intimate, ensheated close contacts, whereas the main type of contact of nerves to ordinary smooth muscle cells in the lower duct section is by means of close but not intimate approach (500–2000 Å).6.Adrenergic synapses in the ductus epididymidis and ductus deferens of the monkey resemble—what concerns their morphology, relationship to effectors and distribution pattern—those of man.7.In accordance with the total number of vascular and non-vascular adrenergic nerves, visualized by fluorescence microscopy, the amount of noradrenaline varied considerably in different sections of the human male internal genital organs: The lowest amounts were estimated in the testis (0.12±0.03 μg/g). Medium to high concentrations were detected in various sections of the caput and corpus epididymidis (ductuli efferentes 0.60±0.09 μg/g; ductuli efferentes and caput 0.72±0.13 μg/g; corpus epididymidis 1.04±0.25 μg/g; proximal cauda 0.95±0.17 μg/g; distal cauda 0.97±0.19 μg/g). The highest noradrenaline content was found in the human vas deferens (prox. vas deferens 1.11±0.21 μg/g; interm. vas deferens 1.20±0.42 μg/g; distal portion 1.43±0.39 μg/g).8.For comparison, the noradrenaline content of the testis and epididymis of the rhesus monkey, the epididymis of the rabbit and the vas deferens of the rabbit, mouse, guinea-pig and rat has been determined.9.Adrenaline of exogenous origin was detected in the vas deferens, cauda epididymidis and plexus pampiniformis of two cases who received this catecholamine as part of the local anaesthetic drug mixture. Due to methodological reasons, the presence of small amounts of adrenaline of endogenous source in adrenergic nerves of the human and monkey internal male genital organs cannot be excluded.10.The differences in motility behaviour of the ductus epididymidis (spontaneous, rhythmic contractions) and ductus deferens (absence of any spontaneous movements under conditions at rest) in vivo and in vitro have been correlated with the occurrence of specialized contractile cells in the upper segment (ductuli efferentes, ductus epididymidis of the caput, corpus and initial cauda) and ordinary large smooth muscle cells in the lower segment (ductus epididymidis of the distal cauda and the vas deferens) and furthermore correlated with differences in the pattern of the adrenergic innervation; the concept is advanced that progressive cytological differentiation of smooth muscle cells and the development of a dense direct adrenergic innervation suppresses autocontractility and, that the reverse condition may favour spontaneous motility of smooth muscle elements.

Adrenergic Innervation of the Human Testis, Epididymis, Ductus Deferens and Prostate : A Fluorescence Microscopic and Fluorimetric Study*

Summary1.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.

Cellular architecture of the lamina propria of human seminiferous tubules

SummaryThe lamina propria of human seminiferous tubules is composed of 5 to 7 cellular layers separated by laminae of extracellular connective-tissue components. By means of immunocytochemical methods the different nature of the cellular layers could be defined for the first time. Based on the light-microscopic demonstration of both desmin-like and vimentin-like immunoreactivity in the inner 3 to 4 layers of the lamina propria, these cells can be identified as myofibroblasts. The outermost one or two cellular layers, on the contrary, only show a vimentin-like immunoreactivity indicating the pure fibroblastic nature of these cells. Therefore, the outermost cellular layers are suggested to be derivatives of the interstitium. In cases of disturbed spermatogenesis, the lamina propria is frequently considerably thickened by an increase in the extracellular matrix components between the cellular layers. Whereas the ultrastructural localization of laminin-, collagen type-IV- and fibronectin-like immunoreactivity remains unaffected in the thickened lamina propria, the desmin-like immunoreactive cells of the inner layers strongly decrease in number and staining intensity. Most probably, the myofibro-blasts lose their myoid characteristics to participate in the secretion of increased amounts of extracellular matrix components, which in turn presumably block the mediation of the lamina propria between the interstitium and the germinal epithelium. It is still unclear whether the thickened lamina propria provokes the disturbance of spermatogenesis or vice versa.

The Leydig cell of the human testis- a new member of the diffuse neuroendocrine system

A number of marker substances for neuronal and neuroendocrine cells have been demonstrated in the cytoplasm of the interstitial Leydig cells of human testes using basic immunocytochemical methods and some of their modifications. We were able to reveal immunoreactivity for enzymes involved in the synthesis of the catecholamines dopamine and noradrenaline (tryosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine-β-hydroxylase), for the indolamine 5-hydroxytryptamine (serotonin), as well as for a number of wellknown neuronal markers such as the neurofilament protein 200, synaptophysin, chromogranin A+B, the neural cell-adhesion molecule (N-CAM), the microtubule-associated protein (MAP-2), and the calcium-binding proteins: S-100, calbindin and parvalbumin. Immunoreactivity for these substances was found in the majority of the interstitial cells although differences in the staining intensity among the individual Leydig cells and among Leydig cells from different patients were observed. At the electron-microscopic level the Leydig cell cytoplasm was seen to contain microtubules, intermediate- and microfilaments as well as clear (40–60 nm) and dense-core (100–300 nm) vesicles, providing a morphological correlate for some of the immunocytochemical results. Although individual marker substances are not absolutely specific for nerve and neuroendocrine cells, the results obtained, together with the already established neuronspecific enolase-, substance P-, methionine-enkephalinand proopiomelanocortin (POMC)-derived peptide-like immunoreactivity, provide strong evidence for the neuroendocrine (paraneuronal, APUD-like) nature of the Leydig cells of the human testis.

On structural patterns of the lamina propria of human seminiferous tubules

SummaryThe ultrastructure of the lamina propria of human seminiferous tubules was analyzed in normal specimens and compared to biopsies showing great thickenning of this area in light microscopy.The contractile cells are stellate in shape, the intercellular gaps between their branchings being less than 150 Å. The cytoplasmic features of these cells are similar to those described by Ross and Long (1966) and do not differ significantly in the pathological cases examined.The intercellular components, namely collagen fibers, microfibrils and an incomplete basement membrane-like coating of the contractile cells, are strikingly increased in the thickenned lamina propria, although the number of layers making up this structure needs not be increased. Occasionally, the intercellular space is occupied by only one of these materials.The distribution of collagen permits identification of two main patterns in the thickenned lamina propria: a) one where the basement membrane of the seminiferous epithelium is separated from the first layer of contractile cells by a wide collagen zone, and b) another case where the layer displaying greater thickness because of increased collagen deposition is located further away from the germinal epithelium.The functional activity of the contractile cells, the physiological implication of structural alterations of the lamina propria and the necessity to correlate these observations to andrological findings, are discussed.

Chemical degeneration of indolamine axons in rat brain by 5,6-dihydroxytryptamine

SummaryEvidence has been obtained by electron microscopy of a direct cytotoxic effect of intraventricularly administered 5,6-dihydroxytryptamine (5,6-DHT) on unmyelinated axons in the rat brain. Ultrastructural signs of axonal damage were observed in areas rich in indolamine nerve terminals as early as 2 hrs after injection. By 6–24 hrs, characteristic and more dramatic signs of degeneration developed, involving coalescence of all axonal constituents—often in combination with a uniform osmiophilic impregnation of the axoplasm—accompanied by engulfment of the dystrophic structures by glial processes. During the next five days, the degenerating axons and axon terminals appeared to be removed by glial cell phagocytosis, whose equivalents were the inclusion of axonal residues into membrane-bound lysosome-like bodies. Concomitantly, there was a progressively increasing number of extremely large and dilated axons in all regions analysed. These axonal swellings, which have an ultramorphology similar to that of dilated stumps of mechanically severed monoamine axons, correspond most probably to proximal, dilated portions of drug-damaged axons.The present results, in combination with biochemical and fluorescence microscopical data, indicate that within a proper dose range the 5,6-DHT-induced degeneration is largely restricted to indolamine axons and axon terminals. However, unselective effects on other unmyelinated axons, on myelin, and on glial cells were observed in narrow subependymal zones close to the lateral ventricles, i.e. close to the injection cannula.