DNA Synthesis In Type Research Articles

Role of c-kit in mammalian spermatogenesis.

The tyrosine-kinase receptor c-kit and its ligand, stem cell factor (SCF), are essential for the maintenance of primordial germ cells (PGCs) in both sexes. However, c-kit and a post-meiotic-specific alternative c-kit gene product play important roles also during post-natal stages of spermatogenesis. In the adult testis, the c-kit receptor is re-expressed in differentiating spermatogonia, but not in spermatogonial stem cells, whereas SCF is expressed by Sertoli cells under FSH stimulation. SCF stimulates DNA synthesis in type A spermatogonia cultured in vitro, and injection of anti-c-kit antibodies blocks their proliferation in vivo. A point mutation in the c-kit gene, which impairs SCF-mediated activation of phosphatidylinositol 3-kinase, does not cause any significant reduction in PGCs number during embryonic development, nor in spermatogonial stem cell populations. However males are completely sterile due to a block in the initial stages of spermatogenesis, associated to abolishment of DNA-synthesis in differentiating A1-A4 spermatogonia. With the onset of meiosis c-kit expression ceases, but a truncated c-kit product, tr-kit, is specifically expressed in post-meiotic stages of spermatogenesis, and is accumulated in mature spermatozoa. Microinjection of tr-kit into mouse eggs causes their parthenogenetic activation, suggesting that it might play a role in the final function of the gametes, fertilization.

In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage-specific fashion.

The effects of FSH on stage-specific apoptosis and DNA synthesis in the adult rat seminiferous epithelium were studied in vitro. Seminiferous tubular segments from stages I, V, VIIa, and VIII-IX were cultured for 24, 48, and 72 h in different concentrations of FSH. Apoptotic cells were detected by in situ end labeling of DNA strands and quantified from squash preparations. After 48 h of culture, a FSH concentration of 2 ng/ml prevented apoptosis of early (steps 1-3) spermatids. In stage VIII-IX tubules cultured for 72 h, FSH decreased the apoptosis of pachytene spermatocytes. An apoptotic type of cell death of germ cells was confirmed by DNA laddering, electron microscopy, supravital acridine orange staining, and phase contrast microscopy of unstained living cells. The effects of FSH on stage-specific DNA synthesis were studied using the same culture system. FSH increased [3H]thymidine incorporation specifically at stages I and VIII-IX, and autoradiography confirmed stimulation of mitotic and meiotic DNA synthesis in type B spermatogonia and preleptotene spermatocytes, respectively. Increased thymidine incorporation also suggested that FSH stimulated DNA synthesis of type A and intermediate spermatogonia. Most effects exerted by FSH were seen in stages containing high levels of FSH receptors and FSH-stimulated cAMP production. In conclusion, the results suggest that FSH, probably acting via Sertoli cells, has a regulatory function in spermatogenic apoptosis and DNA synthesis in stages previously demonstrated to be preferentially dependent on FSH stimulation.

Hepatocyte growth factor stimulates DNA synthesis in alveolar epithelial type II cells in vitro.

Hepatocyte growth factor (HGF) and its receptor, the product of c-MET proto-oncogene, are highly expressed in both fetal and adult lung, though their physiologic functions in the lung are largely unknown. In the present study, we examined whether alveolar type II cells in the lung are the target of HGF and whether HGF has any effects on growth of these cells. The alveolar epithelial type II cells were isolated from the lungs of adult male Sprague-Dawley rats by elastase digestion, and the cells were used to determine whether they express HGF and c-MET mRNAs and whether recombinant HGF has any effect on their DNA synthesis in primary culture. The effects were further compared with those induced by epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), transforming growth factor-alpha (TGF-alpha), and transforming growth factor-beta 1 (TGF-beta 1). Northern blot analysis and in situ hybridization revealed that type II cells express c-MET mRNA but not HGF mRNA. HGF stimulated [3H]thymidine incorporation into type II cells in primary cultures. An increase was also seen in labeling index as determined by nuclear immunostaining of bromodeoxyuridine-incorporated DNA. While aFGF (200 ng/ml) exerted an effect comparable to HGF (25 ng/ml) on DNA synthesis in type II cells, EGF (20 ng/ml) and TGF-alpha (100 ng/ml) had lesser effects. TGF-beta 1, a potent inhibitor of epithelial cell proliferation, at 0.25 to 2 ng/ml, did not inhibit HGF-induced [3H]thymidine incorporation into type II cells. The results indicate that HGF exerts its effects on type II cells as a potent mitogen by a paracrine mode of action.

Follicle-Stimulating Hormone Induction of Steel Factor (SLF) mRNA in Mouse Sertoli Cells and Stimulation of DNA Synthesis in Spermatogonia by Soluble SLF

Follicle-stimulating hormone (FSH) and its intracellular mediator, cAMP, increase the mRNA levels for the Steel factor (SLF, the c-kit ligand) in cultured primary mouse Sertoli cells. The inductive effect of cAMP is more evident in cultures from 13-day-old animals than in cultures from 18-day-old animals. Analysis through the polymerase chain reaction (PCR) indicates that (Bu) 2cAMP or FSH treatment increases the levels of the mRNAs for both the potentially soluble form and the transmembrane form of SLF in cultured Sertoli cells. The ratio between mRNAs encoding the potentially soluble form and the transmembrane form of SLF increases during postnatal testis development, and it is higher in cultured Sertoli cells with respect to total testis, suggesting that, under the in vitro conditions, SLF could be produced by Sertoli cells mainly as a soluble factor. Soluble recombinant SLF stimulates, in a dose-dependent fashion, thymidine incorporation in cultures of isolated germ cell populations enriched in the mitotic stages (spermatogonia), independently of the presence of serum, whereas cAMP analogs have no effect. Autoradiographic analysis shows that SLF selectively stimulates DNA synthesis in type A spermatogonia.

Hypertrophic Alveolar Type II Cells from Silica-treated Rats Are Committed to DNA Synthesis In Vitro

Alveolar type II cell hyperplasia and hypertrophy are common reparative responses of the alveolar epithelium after silica-induced lung injury. We studied in vitro DNA synthesis in type II cells isolated after silica instillation in the rat to determine the proliferative potential of silica type II cells in primary culture and to correlate alveolar type II cell size with the level of in vitro DNA synthesis. To determine if the alveolar lining fluid is a source of growth factors that stimulate alveolar type II cell proliferation, we also examined the mitogenic effect of bronchoalveolar lavage fluid (BALF) from silica-treated rats on type II cells in primary culture. Alveolar type II cells were isolated from rats 1, 2, 3, and 4 wk after intratracheal silica instillation, cultured in DME supplemented with 10% fetal bovine serum, and labeled with [3H]thymidine from day 1 to day 3 in culture. DNA synthesis was determined by [3H]thymidine incorporation and autoradiographic labeling index. The level of thymidine incorporation increased progressively from 22.3 +/- 5.4 x 10(3) dpm/well 7 d after silica instillation to 34.4 +/- 5.0 x 10(3) dpm/well at 28 d. Type II cells isolated 14 d after silica instillation were separated into groups of increasing cell size by centrifugal elutriation. The plating efficiency and alveolar type II cell purity (greater than 88%) were the same in all groups of elutriated cells. The hypertrophic type II cells had a higher level of thymidine incorporation (22.0 +/- 2.8 x 10(3) dpm/well) than the normotrophic type II cells (11.1 +/- 0.7 x 10(3) dpm/well) [P less than 0.01]).(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophages stimulate DNA synthesis in rat alveolar type II cells.

Proliferation of alveolar type II cells after lung injury is crucial for repair of the epithelium. Because an influx of macrophages occurs as part of the inflammatory response associated with acute lung injury and macrophages produce mitogenic factors for a variety of cell types, experiments were conducted to determine if macrophages stimulated DNA synthesis in type II cells. Dialyzed medium conditioned by macrophages consistently stimulated type II cell DNA synthesis, whereas medium conditioned by a variety of other cell types did not. A SV40-transformed macrophage cell line, produced in our laboratory, also secreted substance(s) that enhanced 3H-thymidine incorporation into type II cells. In addition, coculturing rat alveolar macrophages with type II cells stimulated DNA synthesis in the epithelial cells. As determined by autoradiography, the addition of macrophages to type II cells cultured on plastic or on an endothelial cell extracellular matrix increased the labeling index of the epithelial cells from 1 to 15% and from 19 to 51%, respectively. The culture conditions that promoted the greatest increase in DNA synthesis, as well as an increase in cell number, occurred with type II cells plated on an extracellular matrix in medium containing macrophage-conditioned medium, cholera toxin, insulin, and epidermal growth factor. The results suggest that substances secreted by macrophages play a role in regulating alveolar type II cell proliferation in vivo.

Stimulation of DNA synthesis in cultured rat alveolar type II cells.

Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated 3H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of 3H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture.