Specificity and partial purification of a factor in spent media from Sertoli cell-enriched cultures that stimulates steroidogenesis in Leydig cells

There is increasing evidence that factors derived from the seminiferous tubules influence Leydig cell function in a paracrine way. In previous experiments we demonstrated that conditioned media from Sertoli cell-enriched cultures contain a protein with stimulatory activity on prepubertal rat Leydig cells. In this paper we further studied the specificity of this factor. In addition we describe a simple but efficient partial purification procedure. It is demonstrated that Sertoli cell conditioned media contain a factor that stimulates the testosterone output from prepubertal and adult Leydig cells. The effects are evident within the first hour of incubation and can be observed in the presence as well as in the absence of LH. Peritubular cells do not produce a similar factor but enhance the production of the Leydig cell stimulating factor when cocultured with Sertoli cells. The Sertoli cell factor acts on rat as well as on mouse Leydig cells. It barely influences the adrenostenedione output of ovarian stromal cells or the corticosterone output of adrenal cells. The production of this factor is enhanced by dbcAMP, FSH, L-isoproterenol and glucagon but is not affected by androgens. The characteristics of the Sertoli cell factor have been compared with those of a Leydig cell stimulating factor in the medium from an established rabbit kidney cell line: RK13. It is shown that the active principle in RK13 conditioned medium is also a thermolabile trypsin-sensitive protein with a mol. wt of more than 10,000. Nonetheless, the RK13 and Sertoli cell derived factors act by different mechanisms since at maximally effective concentrations their effects are additive. Finally it is demonstrated that molecular weight fractionation of Sertoli cell conditioned medium using an Amicon ultrafiltration system results in a 50- to 130-fold increase in Sertoli cell factor activity in a fraction corresponding to a mol. wt of 10,000 up to 30,000.     

Kinetic analysis of short-term effects of alpha-agonists on gluconeogenesis in isolated rat hepatocytes

Isolated hepatocytes from fasted rats were perifused with glycerol as gluconeogenic substrate. Stimulation of gluconeogenesis with phenylephrine (10(-5) M) as alpha-adrenergic agonist consisted of two distinct phases. The first phase was a transient stimulation of gluconeogenesis and was accompanied by transient changes in cytosolic and mitochondrial redox state; this phase was abolished by the transaminase inhibitor aminooxyacetate. The second phase was a stable stimulation of less magnitude, without change in redox state and insensitive to addition of aminooxyacetate. It is concluded that the first phase is due to a transient enhancement of flux through the malate/aspartate shuttle and that the stable phase is probably due to a stimulation of mitochondrial glycerol-3-phosphate dehydrogenase and glycerol kinase.     

Nutrient dynamics in small mesotrophic fens surrounded by cultivated land

In a typical Dutch polder landscape the effects of nutrient transport from cultivated grassland to mesotrophic fen communities were studied. In a comparative approach, biomass production and nutrient (N, P and K) uptake were determined monthly in four fens and a hayfield differeing in productivity and species composition. The interstitial ground water was sampled every two weeks for determinations of inorganic nutrient concentrations.The differences in productivity between the fens were clearly reflected in the amount of N, P and K taken up in the above-ground vegetation. N and P proved to be limiting plant growth in the fens, whereas K was the main limiting factor in the hayfield. The ground water welling up from the sandy bottom into the fens proved to be rich in ammonia (3–5 ppm). There are strong indications that this continual seepage leads to a considerable input of N into the fens but not to a higher productivity, as the ammonia is absorbed by the lowermost peat layers covering the sand.At this moment, the differences in productivity between the fens must be caused by differences in the rates of mineralization of the superficial peat layer. The degree of fixation of the floating vegetation mat, determining whether or not low water levels lead to an aerated soil top layer, is important in this respect. Within a period of decades, however, the continuous inflow of ammonia may eventually cause an increase in the productivity and a change in the species composition of the fens.     

Cytological characterization of isolated sperm cells of perennial ryegrass (Lolium perenne L.)

Summary The cytoplasmic content and the distribution of intramembrane particles (IMPs) of the plasma membrane of isolated sperm cells of perennial ryegrass (Lolium perenne L.) have been characterized using flow cytometry, transmission electron microscopy, confocal scanning laser microscopy and freeze-fracture studies. The isolated haploid sperm cells contain a variety of cell organelles with the exception of microtubules. Proplastids and plastids with starch were observed, although only rarely. Vacuoles containing remnants of organelles and stacked lamellae of endoplasmic reticulum with cytoplasmic inclusions were observed frequently, indicating that autophagy takes place. The number of mitochondria varies from 11 to 26 with an average of 17. Generally, the nucleus has a lobed shape and displays various interphasic stages of chromatin condensation. The analysis of the number of mitochondria and the nuclear state did not show evidence of sperm cell dimorphism. The cytological variability observed, could be explained by differences in developmental stages already present in vivo at the moment of isolation. No correlation between the number of mitochondria and the nuclear cross-sectioned area and/or the condensation state of the chromatin could be found. The density of intramembrane particles of the plasma membrane on the exoplasmic fracture face is more than twice that on the protoplasmic fracture face. That is the opposite of what was found for sporophytic cells of perennial ryegrass. These results are discussed in relation to the potential use of these cells for biotechnology and developmental studies.     

Flow Cytometric Determination of Nuclear Replication Stage in Seed Tissues

Flow cytometric determination of DNA levels in embryos of fullymatured seeds of various plant species revealed large amountsof 2C DNA signals, indicating that most cells had arrested thecell cycle at the presynthetic G₁ phase of nuclear division.The accumulation of cells at G₁ was found both in orthodox andin recalcitrant (i.e. Castanea sativa) seed species. As recalcitrantseeds are characterized by the absence of maturation drying,the arrest of the cell cycle in the presynthetic phase may notbe linked to the seed water status. Apart from the 2C signal, 4C values were found in the embryoof some seed species (e.g. Raphanus sativus) indicating thatcells were arrested in G₂ Cells arrested in G₂ were primarilylocated in the root-tip region of the embryo. In addition, combinationsof higher C values (i.e. 8C, 12C, 16C and 64C) were observedin the endosperm of Solanum melongena and Lycopersicon esculentum,and in the root-tip cells of Phaseolus vulgaris and Spinaciaoleracea. These mixtures of polyploid nuclei (also called 'polysomaty')may arise from a developmentally controlled cellular endoreduplicationand indicates that in each cell type of the seed the amountof DNA is regulated both spatially and temporally.Copyright1993, 1999 Academic Press Endive, Cichorium endiva, lettuce, Lactuca sativa, egg-plant, Solanum melongena, pepper, Capsicum annuum, tomato, Lycopersicon esculentum, radish, Raphanus sativus, bean Phaseolus vulgaris, spinach, Spinacia oleracea, chestnut, Castanea sativa, beech, Fagus sylvatica, pine, Pinus nigra, DNA content, flow cytometry, seed, nuclear replication stage, C levels, storage     

Proviral tagging in E mu-myc transgenic mice lacking the Pim-1 proto-oncogene leads to compensatory activation of Pim-2.

The Pim-1 proto-oncogene is one of the most potent collaborators of the myc proto-oncogenes in inducing lymphomagenesis in mice. Contrary to the profound effects when overexpressed in vivo, Pim-1-deficient mice showed only subtle phenotypic alterations, which could indicate the presence of redundantly acting genes. In line with this, a PCR-based screen has led to the identification of a closely homologous gene, Pim-2. The X-linked Pim-2 gene is 53% identical to Pim-1 at the amino acid level and shares substrate preference and the usage of non-AUG initiation codons with Pim-1. We have used these data to test whether the strong synergistic interaction between Pim-1 and c-myc can be utilized to gain access to Pim-1 compensatory pathways. We reasoned that, upon proviral tagging in compound mutant mice (E mu-myc/Pim-1-/- mice), the selective advantage of cells carrying provirally activated genes, that act downstream from or parallel to Pim-1, would increase. We show here that this is the case. A dramatic increase (from 15 to 80%) was found in the frequency of proviral activation of the Pim-2 gene. These data show that the described strategy of 'complementation tagging' represents a powerful new tool to identify components of pathways involved in processes as complex as multistep tumorigenesis.     

Quantification of energy consumption in platelets during thrombin-induced aggregation and secretion. Tight coupling between platelet responses and the increment in energy consumption.

The involvement of metabolic energy in platelet responses was investigated by measuring the energy consumption during aggregation and secretion from dense, alpha- and acid-hydrolase-containing granules. Gel-filtered human platelets were stimulated with different amounts of thrombin (0.05-5.0 units X ml-1). At various stages during aggregation and secretion the energy consumption was measured from the changes in metabolic ATP and ADP following abrupt arrest of ATP resynthesis. Stimulation with 5 units of thrombin X ml-1 increased the energy consumption from 6.2 +/- 0.9 to 17.8 +/- 0.4 mumol of ATPeq. X min-1 X (10(11) platelets)-1 during the first 15 s. It decreased thereafter and returned to values found in resting cells after about 30 s. With 0.05 unit of thrombin X ml-1, the energy consumption accelerated more slowly and took at least 3 min before it normalized. A strong positive correlation was found between the velocities of the three secretion responses and the concurrent energy consumption (a) at different stages of the responses induced by a given dose of thrombin, and (b) at different secretion velocities initiated by different amounts of thrombin. When, at different stages of the responses, the extent of secretion was compared with the amount of energy that had been consumed, a strong linear correlation was found with the increment in energy consumption but not with the total energy consumption. This correlation was independent of the concentration of thrombin and indicated that complete secretion from dense, alpha- and acid-hydrolase-containing granules was paralleled by an increment of 4.0, 6.5 and 6.7 mumol of ATPeq. X (10(11) platelets)-1, respectively. An energy cost of 0.7 mumol of ATPeq. X (10(11) platelets)-1 was calculated for separate dense-granule secretion, whereas the combined alpha- and acid-hydrolase granule secretion required 5.3 mumol of ATPeq. X (10(11) platelets)-1. There was no correlation between energy consumption and optical aggregation. In contrast, the rate of single platelet disappearance, which is a measure for the early formation of small aggregates, correlated closely with the rate of energy consumption. Compared with secretion, however, the energy requirement of single platelet disappearance was minor, since 2mM-EDTA completely prevented this response but decreased the energy consumption only slightly. An increase of 0.5-1.0 mumol of ATPeq. X (10(11) platelets)-1 was seen before single platelet disappearance and the three secretion responses were initiated, indicating an increase in energy consuming processes that preceded these responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Simultaneous increase in the aromatization of testosterone into estrogens and secretion of inhibin by Sertoli cells in culture

17 beta oestradiol and inhibin production by Sertoli cells has been investigated in vitro. In basal conditions, 17 beta-oestradiol secretion is weak whereas inhibin production is undetectable on days 7 and 9 of culture. Addition of PMSG (FSH-like gonadotrophin) and testosterone to the culture medium induces a simultaneous increase in 17 beta-oestradiol and inhibin production. PMSG alone has no effect neither on inhibin nor on 17 beta-oestradiol secretion. Testosterone alone significantly increases 17 beta-oestradiol and inhibin production. Human chorionic gonadotrophin (LH-like gonadotrophin) does not modify inhibin secretion. Dihydrotestosterone stimulates inhibin production without affecting oestrogens secretion. Thus, stimulation of aromatization of testosterone into 17 beta-oestradiol is associated with an increase of inhibin production, but this effect seems to be due to a direct action of androgens.     

fMet-Leu-Phe-induced activation of phospholipase D in human neutrophils. Dependence on changes in cytosolic free Ca2+ concentration and relation with respiratory burst activation.

In this study, we have investigated the Ca2+ requirements for the activation of phospholipase D by the tripeptide fMet-Leu-Phe (fMLP) in human neutrophils. EGTA inhibited the activation of phospholipase D (PLD) by 55% (n = 4). When the initial transient rise in [Ca2+]i was prevented by loading the cells with limited amounts of the Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), PLD activation was inhibited by 92% (n = 4). In the presence of both chelators, PLD activation was only 4% of control. In electropermeabilized neutrophils, too, the activation of PLD after the addition of fMLP strongly depends on the Ca2+ concentration, being almost absent with 100 nM free Ca2+ present and reaching maximum activation with a free [Ca2+] of 500 nM. We subsequently investigated the relationship between PLD activation and the activation of the respiratory burst. In neutrophils loaded with BAPTA/AM (10 microM), in which PLD activation was almost absent, a respiratory burst could be induced by fMLP, albeit with a much longer lag time. A respiratory burst could also be elicited by fMLP in electropermeabilized neutrophils incubated with 100 nM free Ca2+. This response, however, was strongly enhanced in the presence of 1 microM Ca2+. Our results indicate that changes in [Ca2+]i are essential for the activation of PLD by fMLP, but probably do not constitute the sole activation signal. In addition, our data provide evidence that PLD activation is important, but not necessary, for activation of the neutrophil respiratory burst.