👤 G Dirami

Sertoli cells isolated from 6-day postpartum mouse testes were conditionally immortalized with the simian virus 40 large tumor antigen gene (SV40-LTAg) under the control of a promoter inducible with ponasterone A, an analog of ecdysone. This strategy produced 2 cell lines, which exhibited mixed phenotypes. We first tested the conditional expression of the LTAg gene in the presence or absence of ponasterone A. The results showed that both cell lines expressed LTAg when the inducer was present in the culture media. When ponasterone A was removed, the majority of the cells died. After 60 generations, however, the continued expression of LTAg in the absence of the hormone indicated that unknown changes may have occurred in the genome of the cells. One of the cell lines was further subcloned, resulting in 7 new lines exhibiting a morphology resembling that of Sertoli cells in tissue culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on RNA collected from each cell line in order to determine which cells were phenotypically similar to Sertoli cells in vivo. All cell lines expressed the products of the Sertoli cell-specific genes stem cell factor (SCF) and sulfated glycoprotein-2 (SGP-2), in addition to alpha-inhibin, GATA-1, and steroidogenic factor-1. Further, the lines express growth and differentiation factors known to act upon germ cells in vivo and in vitro such as leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-beta), and basic fibroblast growth factor (bFGF). Moreover, when used as feeder layers in cocultures, at least 2 of these lines are able to maintain the viability of type A spermatogonia for at least 7 days and to support the first steps of spermatogonial differentiation. Show less

Members of the Notch gene family have been shown to play an important role in the control of cell fate in many developmental systems. We hypothesized that the fate of the male germ line stem cells may also be mediated through the Notch signaling pathway. We therefore sought to determine whether the components of the Notch pathway are expressed in the mouse testis. Western blot analysis revealed the expression of three Notch receptors (Notch 1, Notch 2, and Notch 3), Notch ligands (Jagged 1, Jagged 2, and Delta 1), and presenilin 1 (PS1) in neonatal mouse testis. We then examined their cellular localization by immunohistochemical analysis of cocultures of spermatogonia and Sertoli cells. The 3 Notch receptors were found to be expressed in spermatogonia. Sertoli cells expressed only Notch 2 receptor. Among the Notch ligands, Delta 1 and Jagged 1 were localized exclusively in spermatogonia and Sertoli cells, respectively. PS1 was apparent in both spermatogonia and Sertoli cells. The presence of Notch receptors and Notch ligands in spermatogonia and Sertoli cells indicates that these cells are capable of responding to and eliciting Notch signaling during the process of spermatogenesis. Key words: Cell fate, delta, jagged, presenilin, spermatogenesis. Show less

In the mammalian testis, type A spermatogonia proliferate and differentiate into sperm under the tight control of both endocrine and paracrine factors. In order to study the complex process of spermatogenesis at the molecular level, an in vitro system must be devised in which type A spermatogonia can be cultured for a prolonged period of time. Therefore, cocultures including type A spermatogonia and Sertoli cells, which act as nurse cells to the developing germ cells, are desirable. We have developed a method for the specific isolation of type A spermatogonia using magnetic beads and antibodies that recognize the c-kit receptor or the homophilic adhesion molecule, Ep-CAM. Purified spermatogonia could survive for a period of 25 days when cocultivated on Sertoli cell monolayers. Moreover, we recently established Sertoli cell lines that produce growth factors that are essential for the maintenance of spermatogonia in a proliferative state. Some of these Sertoli cell lines are able to reorganize into tubular structures when cultivated on a layer of Matrigel as extracellular matrix. We show here that type A spermatogonia associate specifically with the Sertoli cell tubules, and are able to replicate their DNA in this environment. Thus, these in vitro culture systems could be used for the long-term culture of primary, nonimmortalized type A spermatogonia. Show less

Spermatogenesis is initiated with the divisions of the type A spermatogonial stem cells; however, the regulation of this stem cell population remains unknown. In order to obtain a better understanding of the biology of these cells, type A spermatogonia were isolated from 80-day-old pig testes by sedimentation velocity at unit gravity. The cells were cultured for up to 120 h in Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F12) or a potassium-rich medium derived by the simplex optimization method (KSOM). At the end of the 120-h culture period, 30-50% of the spermatogonia were viable in KSOM, whereas in DMEM/F12 very few cells survived. Using KSOM as the culture medium, the effects of stem cell factor (SCF) and granulocyte macrophage-colony stimulating factor (GM-CSF) were studied. SCF significantly enhanced the percentage of cell survival at 100 ng/ml but not at lower concentrations. In comparison, GM-CSF promoted survival at relatively low concentrations (0.01, 0.1, and 1 ng/ml). At a higher dose (10 ng/ml), a significant reduction in percentage of cell survival was observed. The combination of SCF with GM-CSF had no significant effect on the percentage survival of type A spermatogonial cells. These data indicate that SCF and GM-CSF play a role in the regulation of survival and/or proliferation of type A spermatogonia. Show less

In order to establish the rat testis as a model system for studying the human pregnancy-specific beta1-glycoprotein (PSG), expression and cellular distribution of PSG in rat testis were examined. Three partial PSG cDNAs, namely, rnCGM6, rnGCM7, and rnCGM8 were obtained when rat testis cDNA libraries were screened with a human placental PSG cDNA probe. Unlike the human PSGs, the rat PSGs show less nucleotide and amino acid sequence homology among family members. The rat PSGs also have multiple truncated leader sequences followed by immunoglobulin variable-like N domains while human PSGs have a single N domain. Examination of the testis, intestine, kidney, liver, lung, and muscle of male rats by reverse transcription-polymerase chain reaction (RT-PCR) with nested gene-specific primers showed that rnCGM6 was present only in the testis, while rnCGM8 was present in the testis, intestine and lung. On the other hand rnCMG7 was found in all tissues examined. Furthermore, rnCGM7 transcript was present in all somatic cells examined whereas rnCGM6 was predominantly in myoid cells and rnCMG8 in Leydig cells. These results suggest that there is cell-specificity in the expression of PSGs in the rat testis and that the rat testis is a good model for studying the biological activities of the PSGs. Show less

Although the role of basement membrane in the morphological and functional differentiation of Sertoli cells has been well characterized, very little is known about its involvement in Sertoli cell survival and maintenance throughout life. When cultured on laminin or Matrigel, 80-90% of Sertoli cells retained their viability. Sertoli cells prevented from attachment and basement membrane deposition by plating on plastic surfaces coated with polyhydroxyethylmethacrylate (poly-HEMA) exhibited a loss of viability by approximately 50% within 24 h. Addition of soluble laminin did not prevent the loss of viability of Sertoli cells, whereas soluble Matrigel enhanced the survival significantly when added at a concentration of 100 micrograms/ml or more. The addition of FSH, epidermal growth factor, testosterone, retinoic acid, or a mixture of insulin, transferrin, and selenium had no significant effect on the viability of Sertoli cells cultured on polyHEMA for up to 72 h. When all of these hormones and factors were added together, a significantly higher percentage of cell survival was observed at 24, 48, and 72 h, but the percent survival was significantly lower than that seen on either laminin or Matrigel. The nature of cell death occurring in the Sertoli cells plated on polyHEMA was determined by agarose gel analysis that revealed a ladder of approximately 200-base pair DNA multiple fragments. Flow cytometric analysis of propidium iodide-stained cells indicated that most of the cells were apoptotic. Freshly isolated Sertoli cells and adherent cells on basement membrane did not show internucleosomal DNA breakdown or an apoptotic peak in the flow cytometric analysis. These results suggest that basement membrane plays a crucial role in Sertoli cell survival in vitro when it is used as a solid substratum for culture, and in the absence of basement membrane, FSH and other regulators of Sertoli cell function cannot prevent Sertoli cell apoptosis. Show less

The objective of this study was to examine the expression and activation of the c-kit receptor, a specific receptor for kit ligand (stem cell factor, steel factor), in rat type A spermatogonia. Testes were obtained from 9-day-old rats, decapsulated, and then subjected to sequential enzymatic digestion. The mixture of testicular cell types was then separated by sedimentation velocity at unit gravity. The isolated type A spermatogonia were characterized by light and electron microscopy. They exhibited spherical nuclei containing several nucleoli and associated chromatin clumps and organelles generally in a perinuclear location similar to that found in the in vivo 9-day-old testis. The synthesis of the c-kit receptor by the spermatogonia was established by hybridization of total RNA with a specific cDNA for mouse c-kit receptor. Two mRNA transcripts migrating at 4.8 kb and 12 kb were observed. Localization of the c-kit receptor in the isolated cells was determined by immunocytochemistry using an antibody to c-kit protein. Specific staining for c-kit receptor was observed in the cytoplasm of the isolated type A spermatogonia. Furthermore, the presence of the c-kit receptor protein in the spermatogonia was confirmed by Western blot analysis using the same antibody. The antibody recognized the c-kit receptor at approximately 160 kDa. In an attempt to determine whether this receptor has a functional significance, we examined the effect of kit ligand on the phosphorylation of the c-kit receptor. The c-kit receptor appeared to be constitutively autophosphorylated on tyrosine at low basal levels, and upon stimulation with kit ligand, the amount of phosphorylated protein increased significantly. These observations indicate that kit ligand induces autophosphorylation of the c-kit receptor, which may lead to the activation of other cellular target proteins responsible for spermatogonial proliferation and/or differentiation. Show less