scholarly journals Marked extension of proliferation of rat Sertoli cells in culture using recombinant human FSH

Reproduction ◽  
2002 ◽  
pp. 633-641 ◽  
Author(s):  
JJ Buzzard ◽  
NG Wreford ◽  
Keyword(s):  
Sertoli Cells ◽  
Culture Media ◽  
Tritiated Thymidine ◽  
Enzymatic Digestion ◽  
Culture Vessel ◽  
Cells In Culture ◽  
Selective Depletion ◽  
Rodent Cell ◽  
Recombinant Human Fsh ◽  
Peritubular Cells

Previous studies indicate that proliferation of rat Sertoli cells in culture can only be maintained until the equivalent of days 10-12 after birth, irrespective of the age of the donor animal. This report describes methods for the isolation and culture of Sertoli cells from day 6 rat testes, which can proliferate in culture for 20-24 days (that is, until the equivalent of days 26-30 after birth). Cells were isolated by enzymatic digestion of seminiferous cords followed by selective depletion of contaminating peritubular cells by adhesion to a polystyrene surface. The purity of the Sertoli cells was assessed using a combination of markers to be > 99.5%. Proliferation was assayed using tritiated thymidine incorporation and further verified by bromodeoxyuridine histochemistry and flow cytometry. Sertoli cells proliferated at basal levels in Dulbecco's modified Eagle's medium (DMEM)-F12 media alone, and proliferation was stimulated further by addition of recombinant human FSH to the culture media. After 20-24 days in culture, proliferation rapidly ceased, and cells assumed abnormal morphology and detached from the culture vessel; these events are consistent with the cells undergoing classic rodent cell senescence. The method described provides a useful tool for investigating the control of Sertoli cell division. Furthermore, these findings indicate that the timely differentiation of Sertoli cells is not dependent solely on an intrinsic timing mechanism, as has been suggested previously.

scholarly journals Secretion of latent type IV procollagenase and active type IV collagenase by testicular cells in culture

1991 ◽  
Vol 279 (1) ◽  
pp. 75-80 ◽  
Author(s):  
M Ailenberg ◽  
W G Stetler-Stevenson ◽  
I B Fritz
Keyword(s):  
Molecular Mass ◽  
Sertoli Cells ◽  
Active Form ◽  
Cytochalasin D ◽  
Type Iv Collagenase ◽  
Active Type ◽  
Cells In Culture ◽  
Type Iv ◽  
Peritubular Cells ◽  
Latent Type

Testicular peritubular myoid cells, which have properties similar to those of vascular smooth-muscle cells, secrete a variety of metalloproteinases when maintained in culture in a chemically defined medium. The predominant metalloproteinases secreted were identified as latent type IV procollagenases having molecular masses of 72 kDa and 75 kDa, as detected in Western immunoblots with specific antibodies against type IV procollagenase. When peritubular cells were stimulated by dibutyryl cyclic AMP, forskolin or cholera toxin, they secreted increased amounts of type IV procollagenase. However, little if any of the active type IV collagenase, having a lower molecular mass of 66 kDa, could be detected under these conditions. Addition of low concentrations of cytochalasin D to peritubular cells in monoculture resulted in conversion of the latent type IV collagenase into its active form, assessed with antibody-specificity studies and by the appearance of the 66 kDa protein. In contrast, Sertoli cells in culture did not manifest an increased conversion of type IV procollagenase into type IV collagenase in the presence of cytochalasin D, even though cytochalasin D addition invariably resulted in a disruption of the microfilament assembly in each of these gonadal somatic cell populations. When peritubular cells were co-cultured with Sertoli cells, addition of cytochalasin D no longer resulted in formation of increased amounts of the active form of type IV collagenase. Sertoli cells and peritubular cells each secreted a tissue inhibitor of metalloproteinase type 2, detected with a specific antibody in a Western immunoblot to have a molecular mass of 21 kDa. We conclude that cytochalasin D acts on mesenchymal-type peritubular cells, but not on epithelial-type Sertoli cells, to enhance the conversion of latent type IV procollagenase into active type IV collagenase. This conversion of type IV procollagenase into type IV collagenase by peritubular cells was inhibited by factor(s) secreted by Sertoli cells. Interactions between Sertoli cells and peritubular cells are postulated to modulate net proteinase activities in discrete regions of the testis.

Effects of prostaglandins E1, E2, and F2alpha on the growth of leukaemia cells in culture

1976 ◽  
Vol 20 (1) ◽  
pp. 199-206
Author(s):  
T.J. Yang ◽  
J.B. Dale ◽  
R. Machanoff
Keyword(s):  
Tritiated Thymidine ◽  
Inhibitory Effect ◽  
Soft Agar ◽  
Significant Inhibition ◽  
Leucine Incorporation ◽  
Inhibitory Effects ◽  
Uridine Incorporation ◽  
Cells In Culture ◽  
Mouse Leukaemia

Prostaglandins E1, E2, and F2alpha (PGE1, PGE2, and PGF2alpha) were shown to inhibit the growth of mouse leukaemia lymphoblasts L5178Y in culture. The effects of PGE1 and PGE2 were greater than that of PGF2alpha. PGE1 and PGE2, at the concentration of 100 mug per ml showed significant inhibitory effects on the rates of incorporation of tritiated thymidine, uridine and leucine. At concentrations of 50 and 25 mug per ml, there was significant inhibition of thymidine and uridine incorporation, but not of leucine, PGF2alpha showed significant inhibition of thymidine and uridine incorporation but not leucine incorporation, in all 3 concentrations studied (100, 50, and 25 mug/ml). The ability of the cells to form colonies in soft agar was significantly inhibited by PGE1 and PGE2 at concentrations as low as 1–8 mug/ml. For F2alpha, however, a concentration as high as 56mug/ml was required to show inhibitory effect, but at 1–8 mug/ml it was found to be stimulatory.

scholarly journals Culture patterns and sorting of rat Sertoli cell secretory proteins

1988 ◽  
Vol 89 (2) ◽  
pp. 175-188
Author(s):  
H. Ueda ◽  
L.L. Tres ◽  
A.L. Kierszenbaum
Keyword(s):  
Epithelial Cells ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Secretory Proteins ◽  
Spermatogenic Cells ◽  
Squamous Cells ◽  
Nylon Mesh ◽  
Continuous Sheet ◽  
Peritubular Cells ◽  
Cells Cultured

A cocultivation chamber and two types of permeable substrates have been used to study: (1) the culture patterns of rat Sertoli and peritubular cells, and Sertoli cells cocultured with spermatogenic cells or peritubular cells; and (2) the polarized secretion of Sertoli cell-specific proteins transferrin, S70 and S45-S35 heterodimeric protein. Substrates included a nylon mesh (with openings of 100 micron) coated with extracellular matrix (ECM) material and an uncoated microporous filter (with pores of 0.45 micron). Sertoli cells cultured on ECM-coated nylon mesh organized a continuous sheet of multilayered epithelial cells essentially devoid of spermatogenic cells while peritubular cells formed a layer of squamous cells. Sertoli cells cultured on uncoated microporous substrate formed a continuous sheet of cuboidal epithelial cells with numerous basal cytoplasmic processes projecting into the substrate and abundant apically located spermatogenic cells, while peritubular cells organized one or two layers of loose squamous cells. [35S]methionine-labelled secretory proteins resolved by two-dimensional polyacrylamide gel electrophoresis and autoradiography displayed cell-specific patterns that were slightly influenced by the type of substrate. Sertoli cells cocultured with peritubular cells on uncoated microporous substrate under conditions that enabled separation of apical and basal surfaces, secreted proteins in a polarized fashion. While transferrin was released bidirectionally, S45-S35 heterodimeric protein was released apically. S70 was detected in both apical and basal compartments. We conclude from these studies that: (1) the number of spermatogenic cells decreases when Sertoli-spermatogenic cell cocultures are prepared on ECM-coated nylon substrate; and (2) Sertoli cells in coculture with spermatogenic or peritubular cells on uncoated microporous substrate, organize continuous sheets displaying polarized protein secretion.

scholarly journals Secretion of glutathione S-transferase isoforms in the seminiferous tubular fluid, tissue distribution and sex steroid binding by rat GSTM1

1999 ◽  
Vol 340 (1) ◽  
pp. 309-320 ◽  
Author(s):  
Sikha Bettina MUKHERJEE ◽  
S. ARAVINDA ◽  
B. GOPALAKRISHNAN ◽  
Sushma NAGPAL ◽  
Dinakar M. SALUNKE ◽  
...  
Keyword(s):  
Cell Culture ◽  
Germ Cell ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Culture Media ◽  
Glutathione S Transferase ◽  
Steroid Binding ◽  
Spent Media

The seminiferous tubular fluid (STF) provides the microenvironment necessary for spermatogenesis in the adluminal compartment of the seminiferous tubule (ST), primarily through secretions of the Sertoli cell. Earlier studies from this laboratory demonstrated the presence of glutathione S-transferase (GST) in STF collected from adult rat testis and in the spent media of ST cultures. This study describes the cellular source, isoform composition and possible function of GSTs in the STF. The major GST isoforms present in STF in vivo share extensive N-terminal similarity with rat GSTM1 (rGSTM1), rGSTM2, rGSTM3 and rGST-Alpha. Molecular masses of rGSTM2, rGSTM3 and rGST-Alpha from liver and testis sources were similar, unlike STF-GSTM1, which was larger by 325 Da than its liver counterpart. Peptide digest analysis profiles on reverse-phase HPLC between liver and STF isoforms were identical, and N-terminal sequences of selected peptides obtained by digestion of the various isoforms were closely similar. The above results confirmed close structural similarity between liver and STF-GST isoforms. Active synthesis and secretion of GSTs by the STs were evident from recovery of radiolabelled GST from the spent media of ST cultures. Analysis of secreted GST isoforms showed that GST-Alpha was not secreted by the STs in vitro, whereas there was an induction of GST-Pi secretion. Detection of immunostainable GST-Mu in Sertoli cells in vitro and during different stages of the seminiferous epithelium in vivo, coupled with the recovery of radiolabelled GST from Sertoli cell-culture media, provided evidence for Sertoli cells as secretors of GST. In addition, STF of ‘Sertoli cell only’ animals showed no change in the profile of GST isoform secretion, thereby confirming Sertoli cells as prime GST secretors. Non-recovery of [35S]methionine-labelled GSTs from germ cell culture supernatants, but their presence in germ cell lysates, confirm the ability of the germ cells to synthesize, but not to release, GSTs. Functionally, STF-GSTM1 appeared to serve as a steroid-binding protein by its ability to bind to testosterone and oestradiol, two important hormones in the ST that are essential for spermatogenesis, with binding constants of < 9.8×10-7 M for testosterone and 9×10-6 M for oestradiol respectively.

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