Hartmut Greven

The blood-testis barrier in vertebrates having different testicular organization

SummaryThe occurrence of tight junctions between Sertoli cells, providing the structural basis of the blood-testis barrier, has been studied using hypertonic fixative and lanthanum tracer in the testes of seven species of vertebrates having different testicular organization. In all cases inter-Sertoli tight junctions, establishing an effective barrier, appear either when meiosis is complete (teleosts and amphibians, both with cystic testes) or immediately after the onset of meiosis (reptiles and birds, both having testes consisting of seminiferous tubules). In the cystic testes, tight junctions are regularly associated with desmosomes, whereas in testes with seminiferous tubules, cisternae of the endoplasmic reticulum are present beneath the junctions (subsurface cisternae). The avian testes examined have, in addition, septate-like junctions between the Sertoli cells but before the tight junctions.

Comparative ultrastructural investigations of the uterine epithelium in the viviparous Salamandra atra Laur. and the ovoviviparous Salamandra salamandra (L.) (Amphibia, Urodela)

SummaryThe uterine epithelium of the viviparous Salamandra atra and the ovoviviparous Salamandra salamandra was studied in non pregnant and ovulating females and in females during different stages of pregnancy. The epithelium of both species is organized in a monolayer. The epithelial cells are characterized by a moderate secretory activity, a variable amount of apical granules which include PAS-positive material and by some apical and basal exo- or endocytotic vesicles. Adjacent cells are joined by junctional complexes. The lateral surfaces form a tortuous boundary with adjoining cells which suggest that the epithelium is involved in transport. Sporadic light cells possess highly variable cytoplasmic inclusions and are not joined with neighbouring cells. Possibly they represent migratory cells. The entire epithelium, except for a small cranial portion of the uterus in S. atra, undergoes no remarkable morphological changes during the different physiological stages examined except that flattened cells seem to be more numerous in pregnant females. The results are discussed with regard to the possible supply of the developing young by the mother.ZusammenfassungDas Uterusepithel des viviparen Alpensalamanders (Salamandra atra) und des ovoviviparen Feuersalamanders (Salamandra salamandra) wurde bei nichtträchtigen, ovulierenden und Weibchen in verschiedenen Trächtigkeitsstadien untersucht. Das Epithel beider Arten ist einschichtig. Die Epithelzellen sind durch eine mäßige sekretorische Aktivität, eine wechselnde Menge von apikalen PAS-positiven Grana und durch apikal und basal gelegene Exo- oder Endocytosevesikel gekennzeichnet. Benachbarte Zellen werden durch “junctional complexes” zusammengehalten. Ihre lateralen Zellmembranen sind stark gewunden und ihre Oberfläche durch Ausläufer vergrößert, eine Organisation, die an ein Transportepithel denken läßt. Vereinzelt vorkommende “helle” Zellen zeichnen sich durch sehr variable cytoplasmatische Einschlüsse aus und sind niemals mit benachbarten Zellen verbunden; möglicherweise sind sie amöboid beweglich. Im gesamten Epithel, mit Ausnahme eines kleinen cranial gelegenen Abschnittes im Uterus von S. atra, sind, abgesehen von einer wahrscheinlich größeren Anzahl abgeflachter Zellen bei trächtigen Weibchen, keine signifikanten morphologischen Veränderungen feststellbar, die in Zusammenhang mit der Trächtigkeit gebracht werden können. Die Ergebnisse werden im Hinblick auf eine mögliche Versorgung der heranwachsenden Jungen von seiten der Mutter diskutiert.

Observations on the blood-testis barrier in a frog and a salamander.

SummaryA blood-testis barrier has been demonstrated in a frog, Rana esculenta, and in a salamander, Salamandra salamandra, using lanthanum as an electron-dense marker during fixation. The tracer penetrates the interstices between somatic follicle or Sertoli cells and germ cells in regions of the testis containing spermatogonia and spermatocytes, up to the level of punctate tight junctions. The latter can be localized between the somatic cells that line seminiferous units containing spermatids and mature spermatozoa. The barrier thus appears to be established after meiosis in both species investigated, although spermatids of different developmental stages can be found in open compartments of the testis in S. salamandra.

Ultrahistochemical and autoradiographic evidence for epithelial transport in the uterus of the ovoviviparous salamander, Salamandra salamandra (L.) (Amphibia, Urodela)

SummaryThe uterine epithelium of pregnant females of the terrestrial ovoviviparous Salamandra salamandra is characterized by a considerable enlargement of its basolateral surface. Chloride and cations (among others sodium), preferentially within the intercellular spaces, can be demonstrated ultrahistochemically. There is indirect evidence of Na+-K+-ATPase activity along the basolateral plasma membranes of the epithelial cells using the Sr-technique for demonstration of a K+-NPPase and 3H-ouabain autoradiography. Preliminary measurements reveal a potential difference across the uterine wall of 15–25mV, the lumenal (mucosal) surface being negative with respect to the coelomic (serosal) surface, and a short circuit current of 200–300 μA. The possibly electrogenic ion transport is ouabain-sensitive. The results are in agreement with the model of a “forward” transporting, i.e. absorptive epithelium. An active transport of solute out of the uterine lumen across the epithelium to the subjacent connective tissue and the blood vessels may be involved in the regulation of an intrauterine milieu appropriate for the development of the offspring.ZusammenfassungDer Uterus des ovoviviparen Feuersalamanders Salamandra salamandra ist von Zellen ausgekleidet, die sich durch eine beträchtliche Vergrößerung ihrer basolateralen Oberfläche auszeichnen. In den Interzellular-räumen des Epithels sind mit Hilfe von simultaner Gewebefixierung und Ionenfällung Kationen (u.a. Natrium) und Anionen (Chlor) nachzuweisen. Basolateral der Epithelzellen wird mit zwei voneinander unabhängigen Methoden (Strontiummethode, 3H-Ouabain Autoradiographie) der indirekte Nachweis für das Vorhandensein einer Na+-K+-ATPase geführt. Erste Messungen der Potentialdifferenz zeigen, daß die Spannung über der Uteruswand bis zu 25 mV (lumenseits negativ), der Kurzschlußstrom etwa 200 bis 250 μA beträgt. Der wahrscheinlich elektrogene Ionentransport ist ouabainsensitiv. Die Ergebnisse sind mit den Vorstellungen über die Wirkungsweise ionenresorbierender Epithelien zu vereinbaren. Der Transport von Substanzen aus dem Uteruslumen in Richtung Bindegewebe und Blutgefäße könnte für die Regulierung eines für die Embryonen und Larven geeigneten intrauterinen Milieus bedeutsam sein.

Some ultrastructural observations on the midgut epithelium of Isohypsibius augusti (Murray, 1907) (Eutardigrada).

SummaryThe midgut epithelial cells of the eutardigrade Isohypsibius augusti are organized into a convoluted monolayer. Only a single cell type could be distinguished although this cell type displayed considerable morphological variation. The midgut begins with crescent-shaped cells. More distally the cells are of variable height depending on the stored amount of nutritional material. No regenerative cells are present. Adjoining cells are held together by apical zonulae continuae. All the cells are characterized by a striated border, some basal infoldings, cytosis vesicles, numerous mitochondria, and abundant rough endoplasmic reticulum. Dictyosomes occur in small numbers. Ovoid or spherical inclusions (spherites), often concentrically laminated, are common. The cells, especially those along the middle part of the gut, are filled with large amounts of nutritional storage which includes polysaccharide material. The possible functions of the midgut in Tardigrada are discussed.ZusammenfassungDas einschichtige, stark gewundene Mitteldarmepithel des Eutardigraden Isohypsibius augusti besteht aus nur einem Zelltyp. Der Mitteldarm beginnt mit halbmondförmigen Zellen; die Höhe der sich anschlie-ßenden Zellen schwankt je nach der Menge der eingelagerten Nahrungsreserven. Regenerative Zellen fehlen. Benachbarte Zellen sind apikal durch Zonulae continuae verbunden. Alle Mitteldarmzellen zeichnen sich durch einen apikalen Mikrovillisaum, Cytosevesikel, zahlreiche Mitochondrien und viel rauhes endoplasmatisches Retikulum aus. Dictyosomen sind nicht allzu häufig. Verbreitet sind ovoide oder runde, oft konzentrisch geschichtete Einschlüsse (Spherite). Besonders die Zellen des mittleren Darmabschnittes können mit großen Mengen von Nahrungsreserven gefüllt sein, die zum Teil Polysaccharide enthalten. Mögliche Funktionen des Mitteldarms der Tardigraden werden diskutiert.

Further freeze-fracture studies on the uterine epithelium of Salamandra salamandra (L.) (Amphibia, Urodela) using the antibiotic filipin.

SummaryThe apical portion of the uterine lining of the ovoviviparous fire salamander, Salamandra salamandra, was studied by the freeze-fracture technique in conjunction with the polyene antibiotic filipin. Filipin-sterol complexes were found in the luminal plasmalemma and in the membranes limiting the mucous secretory granules typical of this epithelium. In all females, but particularly in non-pregnant females, more or less discrete clusters of filipinsterol complexes were occasionally found overlying heavily affected secretory granules. The findings are discussed with regard to comparable results (Orci et al. 1980) based on the examination of collapsed and stretched urinary bladders of toads.ZusammenfassungDas apikale Plasmalemm und die Membranen der Sekretgrana im Uterusepithel des Feuersalamanders, Salamandra salamandra, sind nach Behandlung mit Filipin im Gefrierbruch unterschiedlich stark markiert. Bei trächtigen und nichtträchtigen Weibchen —hier wahrscheinlich vermehrt —finden sich gelegentlich Felder von Filipin-Sterol Komplexen im apikalen Plasmalemm unmittelbar unter ebenfalls meist stark markierten Sekretgrana. Die Befunde werden im Hinblick auf im wesentlichen vergleichbare Beobachtungen an kollabierten und gedehnten Harnblasen von Kröten (Orci et al. 1980) diskutiert.

Tight junctions in the ovotestis of the pond snail Lymnaea stagnalis (L.) (Gastropoda, Basommatophora)

Summary There is evidence to show that the blood-testis barrier in the hermaphroditic gonad of the pulmonate snail Lymnaea stagnalis might have its basis in tight rather than septate junctions as recently suggested by De Jong Brink et al. (De Jong Brink, M., De With, N.D., Hurkmans, P.J.M. and Bergamin Sassen, M.J.M., Cell Tissue Res., 235 (1984) 593–600). By means of conventional ultrathin sections and lanthanum impregnation very small punctate tight junctions could be demonstrated between sperm-associated Sertoli cells, but not between follicle cells surrounding female germ cells.

Nitrogen excretion in intra- and extrauterine larvae of the ovoviviparous salamander, Salamandra salamandra (L.) (amphibia, urodela)

Abstract 1. 1. Uterine fluid of pregnant females of the ovoviviparous Salamandra salamandra contains rather high amounts of urea (576mmol/l) as compared to non pregnant females (150 mmol/l). 2. 2. The same applies to the urea content of the plasma (pregnant female 19.4 mmol/l; non pregnant female 7.8 mmol/l). 3. 3. These data indicate ureotelism in intrauterine larvae. 4. 4. During extrauterine larval life ammonia excretion was found to be slightly higher than urea excretion. 5. 5. During metamorphosis ureotelism again becomes predominant. 6. 6. The capacity for urea synthesis in intrauterine larvae can be regarded as an adaptation to the prolonged stay in utero, where availability of fluid is greatly limited.

Morphological and biochemical studies on the innervation of the testis of Salamandra salamandra (L.) (Amphibia, Urodela)

The innervation of the male gonad of Salamandra salamandra was studied by fluorescence microscopy using the glyoxylic acid method, acetylcholinesterase histochemistry, electron microscopy using glutaraldehyde/osmium tetroxide and chromate/dichromate fixation, and high-performance liquid chromatography (HPLC) with electrochemical detection of biogenic amines in homogenates of the testicular tissue. Noradrenaline has been found to be the prevailing neurotransmitter in the testicular nerves; dopamine could be identified only in minor concentration. The relatively scarce noradrenergic innervation is mainly restricted to the connective tissue septa between the immature and the mature part of the testis and between the mature part and the glandular tissue. Most of the fluorescent fibers have a close connection to blood vessels. After chromate/dichromate fixation the nerve profiles contain in most cases small vesicles with electron-dense reaction products, indicating the presence of catecholamines. Varicosities are to be found near the capillaries, in the vicinity of vascular (in the wall of arterioles) and non-vascular (near the testicular surface) smooth muscle cells; no relationships were found between nerve fibers and glandular (steroid hormone-secreting) or germinal cells. Cholinergic fibers could not be identified, non-adrenergic/non-cholinergic fibers were present only in very small numbers.

Hepatic arginase activity in intra- and extrauterine larvae of the ovoviviparous salamander. Salamandra salamandra (L.) (Amphibia, Urodela).

The hepatic arginase activity of Salamandra salamandra was determined at three different stages of intra- and extrauterine larval development and at fully metamorphosed juveniles. The highest enzymatic activity was found in intrauterine larvae in November, the lowest in intrauterine larvae in June of the following year. These data can be correlated with the ureotelism of intrauterine larvae previously described and are discussed with respect to the metabolism of larval and juvenile fire salamanders.