Sertoli-secreted FGF-2 induces PFKFB4 isozyme expression in mouse spermatogenic cells by activation of the MEK/ERK/CREB pathway

2012 ◽  
Vol 303 (6) ◽  
pp. E695-E707 ◽  
Author(s):  
Marta Gómez ◽  
Anna Manzano ◽  
Agnes Figueras ◽  
Francesc Viñals ◽  
Francesc Ventura ◽  
...  
Keyword(s):  
Gene Expression ◽  
Conditioned Medium ◽  
Cell Lines ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Hormonal Regulation ◽  
Neutralizing Antibodies ◽  
Spermatogenic Cell ◽  
Hormonal Stimulation ◽  
Paracrine Regulation

Sertoli cells play a central role in the control and maintenance of spermatogenesis by secreting growth factors, in response to hormonal stimulation, that participate in the paracrine regulation of this process. In this study, we investigated how the hormonal regulation of spermatogenesis modulates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) isozyme expression in two mouse spermatogenic cell lines, GC-1 spg and GC-2 spd (ts). For this purpose, TM4 Sertoli cells were used to obtain conditioned medium that was treated or not with dihydrotestosterone for 2 days [dihydrotestosterone conditioned medium (TCM) and basal conditioned medium (BCM), respectively]. We observed an increase in the expression of PFKFB4 along with a decrease in PFKFB3 in spermatogenic cell lines treated with TCM. These effects were inhibited by the antiandrogen drug flutamide and by heat-inactivated TCM, indicating the protein nature of the TCM mediator and its dependence on Sertoli cell stimulation by dihydrotestosterone. In addition, adult rat testes treated with the GnRH antagonist Degarelix exhibited a reduction in the expression of PFKFB4 in germ cells. Addition of exogenous FGF-2 mimicked the changes in the Pfkfb gene expression, whereas neutralizing antibodies against FGF-2 abolished them. Interestingly, similar effects on Pfkfb gene expression were observed using different MAPK inhibitors (U-0126, PD-98059, and H-89). Luciferase analysis of Pfkfb4 promoter constructs demonstrated that a putative CRE-binding sequence located at −1,463 relative to the transcription start site is required to control Pfkfb4 gene expression after TCM treatment. Pulldown assays showed the binding of the CREB transcription factor to this site. Altogether, these results show how the paracrine regulation orchestrated by Sertoli cells in response to testosterone controls glycolysis in germ cells.

scholarly journals Establishment and applications of male germ cell and Sertoli cell lines

Reproduction ◽  
2016 ◽  
Vol 152 (2) ◽  
pp. R31-R40 ◽  
Author(s):  
Hong Wang ◽  
Liping Wen ◽  
Qingqing Yuan ◽  
Min Sun ◽  
Minghui Niu ◽  
...  
Keyword(s):  
Cell Line ◽  
Sertoli Cell ◽  
Cell Lines ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Molecular Mechanisms ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Seminiferous Tubules ◽  
Male Germ Cells

Within the seminiferous tubules there are two major cell types, namely male germ cells and Sertoli cells. Recent studies have demonstrated that male germ cells and Sertoli cells can have significant applications in treating male infertility and other diseases. However, primary male germ cells are hard to proliferatein vitroand the number of spermatogonial stem cells is scarce. Therefore, methods that promote the expansion of these cell populations are essential for their use from the bench to the bed side. Notably, a number of cell lines for rodent spermatogonia, spermatocytes and Sertoli cells have been developed, and significantly we have successfully established a human spermatogonial stem cell line with an unlimited proliferation potential and no tumor formation. This newly developed cell line could provide an abundant source of cells for uncovering molecular mechanisms underlying human spermatogenesis and for their utilization in the field of reproductive and regenerative medicine. In this review, we discuss the methods for establishing spermatogonial, spermatocyte and Sertoli cell lines using various kinds of approaches, including spontaneity, transgenic animals with oncogenes, simian virus 40 (SV40) large T antigen, the gene coding for a temperature-sensitive mutant ofp53, telomerase reverse gene (Tert), and the specific promoter-based selection strategy. We further highlight the essential applications of these cell lines in basic research and translation medicine.

scholarly journals Condition Medium of Glioblastoma Cell Lines Decreases the Viability of Glioblastoma Cells by Modulating Gene Expression Profile

2021 ◽  
Author(s):  
Mervenur Yavuz ◽  
Siddika Akgul ◽  
Egemen Kaya ◽  
Turan Demircan
Keyword(s):  
Gene Expression ◽  
Conditioned Medium ◽  
Cell Lines ◽  
Animal Experiments ◽  
Gene Expression Pattern ◽  
Incurable Disease ◽  
Initial Work ◽  
Migration Capacity ◽  
U87 Cells

Grade IV neoplasm of the central nervous system, GBM, is associated with poor prognosis and relatively short overall survival. Due to the current limitations in treatment methods, GBM is characterized as an incurable disease, and research to advance therapeutic options is required. Conditioned medium is commonly used in in-vitro studies complementary to animal experiments to simulate tumor microenvironment and has the potential to challenge and expand our current understanding of secretome effect on tumor characteristics. This study aimed to investigate the effects of conditioned mediums of GBM cell lines on each other. Conditioned mediums' cellular and molecular effects were evaluated using commonly employed techniques such as MTT assay, colony formation assay, wound healing assay, EdU labeling-based flow cytometry, and qRT-PCR. Our study demonstrated that conditioned medium harvested from U87 or LN229 cells at 48th h exhibited an anti-growth activity on each other by changing the gene expression pattern. Furthermore, the conditioned medium of LN229 decreased the migration capacity of U87 cells, and the conditioned medium of U87 cells significantly suppressed the LN229 proliferation. We believe that this initial work provides new insights for a better understanding of GBM cell lines' secretome roles and highlights the necessity of further studies to unveil the secretome content.

Testicular Estrogens and Male Reproduction

Physiology ◽  
2000 ◽  
Vol 15 (4) ◽  
pp. 195-198 ◽  
Author(s):  
Serge Carreau ◽  
Jérôme Levallet
Keyword(s):  
Gene Expression ◽  
Germ Cells ◽  
Hormonal Regulation ◽  
Reproductive Function ◽  
Male Reproduction ◽  
Active Protein ◽  
Widespread Distribution ◽  
Male Reproductive Function ◽  
Testicular Cells ◽  
Estrogen Production

Besides somatic cells, aromatase gene expression and its transduction in an active protein in germ cells provides evidence of an additional site for estrogen production within testes of some mammals. Together with the widespread distribution of estrogen receptors in testicular cells, these data illuminate the hormonal regulation of male reproductive function

020. HORMONAL REGULATION OF SPERMATOGENESIS

2010 ◽  
Vol 22 (9) ◽  
pp. 8
Author(s):  
D. Handelsman
Keyword(s):  
Germ Cells ◽  
Sertoli Cells ◽  
Hormonal Regulation ◽  
Intercellular Junctions ◽  
Cell Cytoplasm ◽  
Testicular Cells ◽  
Receptor Knockout Mouse ◽  
Protein Receptors ◽  
Pituitary Secretion ◽  
The Individual

Spermatogenesis is a spatially and temporally co-ordinated proliferation of the germinal epithelium in the semeniferous tubules. The germ cells are embedded in a scafolding formed by adjacent Sertoli cells linked tightly by intercellular junctions and with each germ cell enshrouded by elongations of Sertoli cell cytoplasm. Spermatogenesis comprises serial stages from the mitotic replication of the stem and early germ cells, followed by meiosis, the reductive division producing haploid, amorphous gametes which subsequently undergo spermiogenesis, the metamorphosis into terminally differentiated and functional spermatozoa. Although long known that all but the earliest stages are hormonally regulated by pituitary secretion of LH and FSH, it has remained difficult to separate gonadotrophin effects by classical endocrine ablate-replace methods as these two heterodimeric hormones have identical α and homologous β subunits, are secreted from the same pituitary gonadotrophs to target cognate receptors expressed on adjacent testicular cells as equally homologous, heptahelical G-coupled protein receptors. Over two decades our laboratory has developed a variety of complementary genetic and pharmacological approaches to dissect the individual and co-operative effects of LH, its main effector testosterone and FSH on spermatogenesis. Using the gonadotrophin and testosterone deficient hpg mouse, double transgenic human FSH secreting mouse and the androgen receptor knockout mouse lines together with steroidal depot homone delivery, we have explored systematically annd defined the individual primary actions of FSH and testosterone and their interactions in the regulation of testis growth, spermatogenesis and ultimately male fertility.

Cell Interactions in the Reproductive System

1978 ◽  
Vol 36 (3) ◽  
pp. 643-650
Author(s):  
D.W. Fawcett
Keyword(s):  
Germ Cells ◽  
Granulosa Cells ◽  
Sertoli Cells ◽  
Supporting Cells ◽  
Hormonal Stimulation ◽  
Germ Cell Differentiation ◽  
Occluding Junctions ◽  
Fetal Ovary ◽  
Gonadotropic Hormones ◽  
Shape Changes

Gametogenesis in both sexes involves a nurse-cell relationship between an epithelium and the developing gametes. In the female, a lifetime supply of oocytes is formed in the embryo and no new germ cells are formed postnatally. The granulosa cells of the ovarian follicles are believed to be responsible for maintaining the arrest of the oocytes in the fetal ovary in the dictyate stage of meiosis, in which stage they remain until shortly before ovulation many months or years later depending upon the species. Responding to gonadotropic hormones, the granulosa cells then terminate their inhibition, permit maturation of the oocyte, and participate in the reorganization of the follicle that is necessary for ovulation. In the male, the Sertoli cells which are also responsive to hormonal stimulation, maintain in the seminiferous epithelium a special microenvironment which is essential for germ cell differentiation. The cooperative motor activity and associated shape changes of these supporting cells is responsible for the adluminal translocation of the developing germ cells and for the separation of individual spermatozoa from long chains of interconnected cell bodies during sperm release. This paper will review the types and locations of the communicating and occluding junctions that are essential for the complex and highly integrated activities of the epithelia in the mammalian gonads.

scholarly journals Establishment of adult mouse Sertoli cell lines by using the starvation method

Reproduction ◽  
2013 ◽  
Vol 145 (5) ◽  
pp. 505-516 ◽  
Author(s):  
Yoko Sato ◽  
Kaoru Yoshida ◽  
Shiari Nozawa ◽  
Miki Yoshiike ◽  
Michiko Arai ◽  
...  
Keyword(s):  
Growth Factor ◽  
Sertoli Cell ◽  
Cell Lines ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Transforming Growth Factor ◽  
Cultured Cells ◽  
Transforming Growth Factor Β1 ◽  
Leukaemia Inhibitory Factor ◽  
Specific Gene

Sertoli cells were isolated from the testes of 6-week-old mice and stable Sertoli cell lines with higher proliferation rates were subcloned after starvation of primary cultured cells. After two rounds of this subcloning, 33 subcloned lines were selected on the basis of their proliferation rates. In addition, these subclones were screened according to their phagocytic activity and the characteristics of mature Sertoli cells, such as the expression of androgen receptors (ARs) and progesterone receptors, by using western blotting and immunocytochemical analysis, in addition to their morphology and proliferation rates. After the third round of subcloning, 12 subclones were selected for the final selection using RT-PCR for identification of genes specifically expressed by various testicular cells. Three clones were selected that expressed Sertoli-cell-specific genes, i.e. stem cell factor, clusterin, AR, α-inhibin, transferrin, Wilms' tumour-1, Müllerian inhibitory substance, sex-determining region Y-box 9, FSH receptor (Fshr) and occludin; however, these clones did not express globulin transcription factor 1, steroidogenic factor or androgen-binding protein. These clones also expressed growth and differentiation factors that act on germ cells, such as leukaemia inhibitory factor, transforming growth factor β1 and basic fibroblast growth factor 2, but did not express c-kit (specific for germ cells), LH receptor and 3β-hydroxyl-dehydrogenase (specific for Leydig cells). Immunocytochemical data confirmed the expression of clusterin in these clones. Furthermore, the Bromodeoxyuridine incorporation assay confirmed the proliferation activity of these clones throughFshrafter treatment with FSH. These clones are considered to be valuable tools for the study of Sertoli cell-specific gene expression and function.

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