Dual compartment (bicameral) culture: role of basement membrane in epithelial differentiation

A number of years ago we reported that tight junctions between adjacent Sertoli cells subdivide the seminiferous epithelium into two compartments, basal and adluminal, thus forming the morphological basis of the blood-testis barrier. It is now generally believed that the special milieu created by the Sertoli cells in the adluminal compartment is essential for germ cell differentiation. In order to duplicate the compartmentalization that occurs in vivo, Sertoli cells were cultured in bicameral chambers on Millipore filters impregnated with a reconstituted basement membrane. Confluent monolayers of these cells were tall columnar (40–60 µ in height) and highly polarized. These Sertoli cell monolayers established electrical resistance that peaked when the Sertoli-Sertoli tight junctions developed in culture. In addition, the monolayers formed a permeability barrier to ³H-inulin and lanthanum nitrate. The bicameral chambers were utilized in a number of studies on protein secretion, and it was revealed that numerous proteens are secreted in a polarized manner. In another study, hormone- stimulated aromatase activity was measured in Sertoli cells grown on plastic culture dishes, plastic dishes coated with laminin or Matrigel, and in the bicameral chambers. Cell culture on basement membrane substrate decreased the FSH-dependent estrogen production. No estrogen production was observed when the Sertoli cells were cultured in the bicameral chambers. These results are in accord with the hypothesis that differentiated Sertoli cells lose their ability to metabolize androgen to estrogen in an hormone-dependent manner, whereas undifferentiated cells in culture, or in vivo, have a very active FSH-dependent aromatase activity. This bicameral culture system could serve as an important model system to examine various functions of Sertoli cells including interactions of Sertoli cells with germ, Leydig, and myoid cells.     

Sterol carrier protein 2 (non-specific lipid transfer protein) is localized in membranous fractions of Leydig cells and Sertoli cells but not in germ cells.

The cellular and subcellular distribution of sterol carrier protein 2 (SCP2; nsL-TP) was reinvestigated in rat testicular cells by Western blotting and immunocytochemistry, using the affinity purified antibody against rat liver SCP2. Western blot analysis revealed high levels of the protein in the somatic cells of the testis, e.g., Leydig and Sertoli cells whereas it could not be detected in germ cells. This cellular localization of SCP2 was confirmed by Northern blotting. Immunocytochemical techniques revealed that in Leydig cells, immunoreactive proteins were concentrated in peroxisomes. Although SCP2 was also detected in Sertoli cells, a specific subcellular localization could not be shown. SCP2 was absent from germ cells. Analysis of subcellular fractions of Leydig cells showed that SCP2 is membrane bound without detectable amounts in the cytosolic fraction. These results are at variance with data published previously which suggested that in Leydig cells a substantial amount of SCP2 was present in the cytosol and that the distribution between membranes and cytosol was regulated by luteinizing hormone. The present data raise the question in what way SCP2 is involved in cholesterol transport between membranes in steroidogenic cells but also in non-steroidogenic cells.     

Ovarian substance induces steroid production in cultured granulosa cells

Summary The rat ovary produces an apparently low molecular weight substance that mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induces temporal cell rounding and, later on, intensive progestin production. However, unlike FSH, OS does not induce accumulation of cyclic AMP (cAMP) in the granulosa cells. The ovarian factor cannot be cAMP as its action is not abolished by phosphodiesterase (PDE) treatment. Neither is it a possible PDE inhibitor, as it does not augment cAMP accumulation in granulosa cells or Friend erythroleukemic cells induced by FSH or PGE₁, respectively. The factor is still active after heating for 20 min at 90° C but is rapidly inactivated by alkali treatment. In addition, treatment with various proteases did not abolish the steroidogenic activity. These findings suggest a possible novel intraovarian regulator of the granulosa cell function. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This work was supported by the United States-Israel Binational Science Foundation, Grant 2656/81. This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.     

Intraepithelial lymphocytes in the male reproductive tract of rats and rhesus monkeys.

Although lymphocytes are never present in 'normal ' seminiferous epithelium, they are found in the terminal portions of the seminiferous tubles near their junctions with the tubuli recti. Intraepithelia lymphocytes are also found in the tubuli recti testis, ductuli efferentes, epididymis and ductus deferens. The ultrastructural morphology of these cells closely resembles that of the intraepithelial lymphocytes in the intestinal mucosa and those obtained from the lymph nodes, spleen blood and thoracic duct. The mucleus is spherical and is characterized by clumps of chromatin near the nuclear membrane. A thin rim of cytoplasm is usually found, and is remarkably free of most cell organelles except for free ribosomes. Frequently, a blunt cytoplasmic process can be seen extending from one end of the cell. Membrane-bounded granules and other dense bodies are occasionally encountered in the cytoplasm. The possible functional significance of intraepithelial lymphocytes in the male reproductive tract is discussed.     

In vitro effects of calcium phosphate biomaterials on fibroblastic cell behavior

The effect of synthetic granular hydroxyapatite (HAP) on cultured fibroblastic cells (L929, human bone and gingiva cells) was studied. Phagocytosis of HAP particles and resulting morphological cell changes were demonstrated by microscopic examinations. Cell counts and [3H]thymidine uptake indicated significant increases in cell proliferation and DNA synthesis. These results could account for some of the alterations of the fibroblast behavior induced by changes in intracellular levels of calcium ions released from the material.     

Evolutionary history of the European free-tailed bat,a tropical affinity species spanning across the Mediterranean Basin

The Mediterranean Basin is a global biodiversity hotspot, hosting a number of native species belonging to families that are found almost exclusively in tropical climates. Yet, whether or not these taxa were able to survive in the Mediterranean region during the Quaternary climatic oscillations remains unknown. Focusing on the European free-tailed bat (Tadarida teniotis), we aimed to (a) identify potential ancient populations and glacial refugia; (b) determine the post-glacial colonization routes across the Mediterranean; and (c) evaluate current population structure and demography. Mitochondrial and nuclear markers were used to understand T. teniotis evolutionary and demographic history. We show that T. teniotis is likely restricted to the Western Palearctic, with mitochondrial phylogeny suggesting a split between an Anatolian/Middle East clade and a European clade. Nuclear data pointed to three genetic populations, one of which is an isolated and highly differentiated group in the Canary Islands, another distributed across Iberia, Morocco, and France, and a third stretching from Italy to the east, with admixture following a pattern of isolation by distance. Evolutionary and demographic reconstruction supports a pre-Last Glacial Maximum (LGM) colonization of Italy and the Anatolian/Middle East, while the remaining populations were colonized from Italy after the Younger Dryas. We also found support for demographic expansion following the Iberian colonization. The results show that during the LGM T. teniotis persisted in Mediterranean refugia and has subsequently expanded to its current circum-Mediterranean range. Our findings raise questions regarding the physiological and ecological traits that enabled species with tropical affinities to survive in colder climates.     

The phosphatidylinositol‐transfer protein Nir2 binds phosphatidic acid and positively regulates phosphoinositide signalling

Phosphatidic acid (PA) and phosphoinositides are metabolically interconverted lipid second messengers that have central roles in many growth factor (GF)‐stimulated signalling pathways. Yet, little is known about the mechanisms that coordinate their production and downstream signalling. Here we show that the phosphatidylinositol (PI)‐transfer protein Nir2 translocates from the Golgi complex to the plasma membrane in response to GF stimulation. This translocation is triggered by PA formation and is mediated by its C‐terminal region that binds PA in vitro. We further show that depletion of Nir2 substantially reduces the PI(4,5)P2 levels at the plasma membrane and concomitantly GF‐stimulated PI(3,4,5)P3 production. Finally, we show that Nir2 positively regulates the MAPK and PI3K/AKT pathways. We propose that Nir2 through its PA‐binding capability and PI‐transfer activity can couple PA to phosphoinositide signalling, and possibly coordinates their local lipid metabolism and downstream signalling.