Mechanism of Action of l-CDB-4022, a Potential Nonhormonal Male Contraceptive, in the Seminiferous Epithelium of the Rat Testis

The present study was conducted to elucidate the possible molecular mechanisms involved in the antispermatogenic activity of l-CDB-4022, an indenopyridine. In this study 45-d-old male Sprague-Dawley rats were treated with a single oral dose of l-CDB-4022 (2.5 mg/kg) or vehicle, and blood and testes were collected at various time points. The rate of body weight gain was not affected, but a significant loss of testes weight was induced by l-CDB-4022. Serum hormones were assayed using specific RIAs or ELISAs, and testicular protein and RNA were analyzed by Western blotting and RT-PCR, respectively. There was a significant decrease in inhibin B and concomitant increase in FSH in serum from l-CDB-4022-treated rats, but serum levels of activin A, testosterone, and LH were unchanged. Western analysis of testicular lysates from l-CDB-4022-treated rats exhibited phosphorylation of ERK1/2 at 4 h and later time points. Loss of nectin/afadin complex occurred at 48 h, but there was an increase in levels of integrin-β1, N-cadherin, α-catenin, and β-catenin protein at 24 h and later time points. Increase in expression of Fas ligand and Fas receptor was detected 8 and 24 h after l-CDB-4022 treatment. The ratio of the membrane to soluble form of stem cell factor mRNA was decreased. Immunohistochemical analysis of testicular sections indicated a dramatic disruption of the Sertoli cell microtubule network in l-CDB-4022-treated rats. Collectively, these results suggest that l-CDB-4022 activates the MAPK pathway, reduces expression of prosurvival factors such as the membrane form of stem cell factor, alters expression of Sertoli-germ cell adherens junction proteins, disrupts Sertoli cell microtubule structure, and induces the proapoptotic factor, Fas, culminating in germ cell loss from the seminiferous epithelium.

Download Full-text

Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor

International Journal of Molecular Sciences ◽

10.3390/ijms22126361 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6361

Author(s):

Eunyoung Lee ◽

Michelle Novais de Paula ◽

Sangki Baek ◽

Huynh Kim Khanh Ta ◽

Minh Tan Nguyen ◽

...

Keyword(s):

Stem Cell ◽

Ion Exchange ◽

Stem Cell Factor ◽

Transmembrane Domain ◽

Coli Strain ◽

Exchange Chromatography ◽

Soluble Form ◽

Human Stem Cell ◽

E Coli ◽

Human Stem Cell Factor

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b’a’ domain (PDIb’a’) tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.

Download Full-text

Role of stem cell factor in somatic–germ cell interactions during prenatal oogenesis

Zygote ◽

10.1017/s0967199400003373 ◽

1996 ◽

Vol 4 (04) ◽

pp. 349-351 ◽

Cited By ~ 6

Author(s):

Massimo De Felici ◽

Anna Di Carlo ◽

Maurizio Pesce

Keyword(s):

Stem Cell ◽

Germ Cells ◽

Stem Cell Factor ◽

Molecular Mechanisms ◽

Primordial Germ Cells ◽

Significant Progress ◽

Proliferation And Differentiation ◽

Complex Events ◽

Mechanisms Of Migration

During embryogenesis germ cells originate from primordial germ cells (PGCs). The development of mammalian PGCs involves a number of complex events (formation and segregation of PGC precursors, PGC migration and proliferation) which lead to the differentiation of oocytes or prospermatogonia (for a review see De Feliciet al., 1992). During recent years developments in methods for isolation, purification and culture of mouse PGCs have led to significant progress in the understanding of molecular mechanisms of migration, proliferation and differentiation of these cells (for reviews see De Felici, 1994; and De Felici & Pesce, 1994a). In this paper we describe the key role played by stem cell factor (SCF) in PGC development and early folliculogenesis.

Download Full-text

Molecular mechanisms involved in Sertoli cell adaptation to glucose deprivation

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00235.2009 ◽

2009 ◽

Vol 297 (4) ◽

pp. E907-E914 ◽

Cited By ~ 50

Author(s):

María F. Riera ◽

María N. Galardo ◽

Eliana H. Pellizzari ◽

Silvina B. Meroni ◽

Selva B. Cigorraga

Keyword(s):

Germ Cell ◽

Glucose Uptake ◽

Sertoli Cell ◽

Sertoli Cells ◽

Molecular Mechanisms ◽

Lactate Concentration ◽

Glucose Deprivation ◽

Cell Development ◽

Germ Cell Development ◽

Glucose Levels

Sertoli cells provide the physical support and the necessary environment for germ cell development. Among the products secreted by Sertoli cells, lactate, the preferred energy substrate for spermatocytes and spermatids, is present. Considering the essential role of lactate on germ cell metabolism, it is supposed that Sertoli cells must ensure its production even in adverse conditions, such as those that would result from a decrease in glucose levels in the extracellular milieu. The aim of the present study was to investigate 1) a possible effect of glucose deprivation on glucose uptake and on the expression of glucose transporters in rat Sertoli cells and 2) the participation of different signal transduction pathways in the above-mentioned regulation. Results obtained show that decreasing glucose levels in Sertoli cell culture medium provokes 1) an increase in glucose uptake accompanied by only a slight decrease in lactate production, 2) an increase in GLUT1 and a decrease in GLUT3 expression, and 3) an activation of AMP-activated protein kinase (AMPK)-, phosphatidylinositol 3-kinase (PI3K)/PKB-, and p38 MAPK-dependent pathways. Additionally, by using specific inhibitors of these pathways, a possible participation of AMPK- and p38MAPK-dependent pathways in the regulation of glucose uptake and GLUT1 expression is shown. These results suggest that Sertoli cells adapt to conditions of glucose deprivation to ensure an adequate lactate concentration in the microenvironment where germ cell development occurs.

Download Full-text

Expression of Inhibin α, Glial Cell Line-Derived Neurotrophic Factor and Stem Cell Factor in Sertoli Cell-Only Syndrome: Relation to Successful Sperm Retrieval by Microdissection Testicular Sperm Extraction

Yearbook of Urology ◽

10.1016/s0084-4071(08)70387-7 ◽

2006 ◽

Vol 2006 ◽

pp. 227-228

Author(s):

C.K. Naughton

Keyword(s):

Stem Cell ◽

Cell Line ◽

Glial Cell ◽

Sertoli Cell ◽

Neurotrophic Factor ◽

Stem Cell Factor ◽

Testicular Sperm Extraction ◽

Testicular Sperm ◽

Sperm Retrieval

Download Full-text

Soluble stem cell factor in human serum

Blood ◽

10.1182/blood.v81.3.656.bloodjournal813656 ◽

1993 ◽

Vol 81 (3) ◽

pp. 656-660 ◽

Cited By ~ 3

Author(s):

KE Langley ◽

LG Bennett ◽

J Wypych ◽

SA Yancik ◽

XD Liu ◽

...

Keyword(s):

Stem Cell ◽

Human Serum ◽

Stem Cell Factor ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Immunoaffinity Chromatography ◽

Soluble Form ◽

Enzyme Linked Immunosorbent Assay ◽

Functional Importance ◽

Membrane Bound

Stem cell factor (SCF) is a recently described factor active in the early stages of hematopoiesis. It can exist in membrane-bound form and in proteolytically released soluble form. The levels and nature of SCF in human serum are described. As determined by an enzyme-linked immunosorbent assay performed for 257 samples, SCF level in serum averaged 3.3 +/- 1.1 ng/mL. The serum SCF was partially purified by immunoaffinity chromatography and analyzed by glycosidase treatments in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The results show that the SCF has N- linked and O-linked carbohydrate and corresponds to the soluble form, at or about 165 amino acids in length. The findings suggest functional importance for soluble SCF in humans.

Download Full-text

Soluble stem cell factor in human serum

Blood ◽

10.1182/blood.v81.3.656.656 ◽

1993 ◽

Vol 81 (3) ◽

pp. 656-660 ◽

Cited By ~ 89

Author(s):

KE Langley ◽

LG Bennett ◽

J Wypych ◽

SA Yancik ◽

XD Liu ◽

...

Keyword(s):

Stem Cell ◽

Human Serum ◽

Stem Cell Factor ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Immunoaffinity Chromatography ◽

Soluble Form ◽

Enzyme Linked Immunosorbent Assay ◽

Functional Importance ◽

Membrane Bound

Abstract Stem cell factor (SCF) is a recently described factor active in the early stages of hematopoiesis. It can exist in membrane-bound form and in proteolytically released soluble form. The levels and nature of SCF in human serum are described. As determined by an enzyme-linked immunosorbent assay performed for 257 samples, SCF level in serum averaged 3.3 +/- 1.1 ng/mL. The serum SCF was partially purified by immunoaffinity chromatography and analyzed by glycosidase treatments in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The results show that the SCF has N- linked and O-linked carbohydrate and corresponds to the soluble form, at or about 165 amino acids in length. The findings suggest functional importance for soluble SCF in humans.

Download Full-text

Signaling Through the Interaction of Membrane-Restricted Stem Cell Factor and c-kit Receptor Tyrosine Kinase: Genetic Evidence for a Differential Role in Erythropoiesis

Blood ◽

10.1182/blood.v91.3.879 ◽

1998 ◽

Vol 91 (3) ◽

pp. 879-889 ◽

Cited By ~ 62

Author(s):

Reuben Kapur ◽

Manus Majumdar ◽

Xiangli Xiao ◽

Monica McAndrews-Hill ◽

Karen Schindler ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Stromal Cells ◽

Stem Cell Factor ◽

Progenitor Cell ◽

Erythropoietin Receptor ◽

Soluble Form

Abstract Mutations of the receptor tyrosine kinase c-kit or its ligand stem cell factor (SCF), which is encoded as a soluble and membrane-associated protein by the Steel gene in mice, lead to deficiencies of germ cells, melanocytes, and hematopoiesis, including the erythroid lineage. In the present study, we have used genetic methods to study the role of membrane or soluble presentation of SCF in hematopoiesis. Bone marrow–derived stromal cells expressing only a membrane-restricted (MR) isoform of SCF induced an elevated and sustained tyrosine phosphorylation of both c-kit and erythropoietin receptor (EPO-R) and significantly greater proliferation of an erythrocytic progenitor cell line compared with stromal cells expressing soluble SCF. Transgene expression of MR-SCF inSteel-dickie (Sld) mutants resulted in a significant improvement in the production of red blood cells, bone marrow hypoplasia, and runting. In contrast, overexpression of the full-length soluble form of SCF transgene had no effect on either red blood cell production or runting but corrected the myeloid progenitor cell deficiency seen in these mutants. These data provide the first evidence of differential functions of SCF isoforms in vivo and suggest an abnormal signaling mechanism as the cause of the severe anemia seen in mutants of the Sl gene.

Download Full-text

Growth Differentiation Factor 9 Is a Germ Cell Regulator of Sertoli Cell Function

Endocrinology ◽

10.1210/en.2008-1048 ◽

2008 ◽

Vol 150 (5) ◽

pp. 2481-2490 ◽

Cited By ~ 58

Author(s):

Peter K. Nicholls ◽

Craig A. Harrison ◽

Robert B. Gilchrist ◽

Paul G. Farnworth ◽

Peter G. Stanton

Keyword(s):

Tight Junction ◽

Germ Cell ◽

Sertoli Cell ◽

Cell Cultures ◽

Seminiferous Epithelium ◽

Inhibin B ◽

Receptor Type ◽

Type Ii ◽

Bmp Receptor ◽

Differentiation Factor

Oocyte-secreted growth differentiation factor (GDF) 9 and bone morphogenetic protein (BMP) 15 are critical regulatory factors in female reproduction. Together, they promote granulosa cell proliferation and stimulate the maturation of preovulatory follicles. Despite their importance in female fertility, GDF9 and BMP15 expression patterns and function during spermatogenesis have not been investigated. In this study we show that the expression and stage-specific localization of both factors are limited to the germ cells of the rat seminiferous epithelium, with GDF9 being principally localized in round spermatids and BMP15 in gonocytes and pachytene spermatocytes. To identify potential cellular targets for GDF9 actions, cells of the seminiferous tubule were isolated and screened for the expression of signaling receptors [activin-like kinase (ALK) 5, ALK6, and BMP receptor, type II)]. Individual receptor types were expressed throughout the seminiferous epithelium, but coexpression of ALK5 and BMP receptor, type II was limited to Sertoli cells and round spermatids. Based on the reproductive actions of related TGFβ ligands in the ovary and testis, GDF9 was assessed for its ability to regulate tight junction function and inhibin B production in rat Sertoli cell cultures. When recombinant mouse GDF9 was added to immature Sertoli cell cultures, it inhibited membrane localization of the junctional proteins claudin-11, occludin, and zonula occludens-1, thereby disrupting tight junction integrity. Concomitantly, GDF9 up-regulated inhibin subunit expression and significantly stimulated dimeric inhibin B protein production. Together, these results demonstrate that GDF9 and BMP15 are germ cell-specific factors in the rat testis, and that GDF9 can modulate key Sertoli cell functions.

Download Full-text

Stem cell factor affects fate determination of human gonocytes in vitro

Reproduction ◽

10.1530/rep-07-0161 ◽

2007 ◽

Vol 134 (6) ◽

pp. 757-765 ◽

Cited By ~ 18

Author(s):

Jiongjiong Tu ◽

Liqing Fan ◽

Ke Tao ◽

Wenbing Zhu ◽

Jianjun Li ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Factor ◽

Molecular Mechanisms ◽

Kinase Inhibitor ◽

Mitogen Activated Protein Kinase ◽

Tyrosine Kinase Receptor ◽

Dependent Manner ◽

Fate Determination

The stem cell factor (SCF), binding its tyrosine kinase receptor c-Kit, has been shown to play essential roles in the proliferation, differentiation, and survival of germline cells. However, few reports are available about the effect of SCF on the development of human gonocytes within the fetal testis. The objective of this study was to investigate whether SCF affects the biological behaviors of human gonocytes before or after they enter the mitotic arrest stage. Employing an organ culture system, we observed that addition of exogenous SCF could influence the morphology of human gonocytesin vitro. Moreover, SCF was able to trigger the colony formation of round gonocytes, which were characterized positive for alkaline phosphatase activity, Oct-4, SSEA-4, and c-Kit as well. We found that SCF exerted actions in a dose- and age-dependent manner, although the stimulatory effect lasted no more than 14 days. We also showed that SCF played a role in suppressing the apoptosis of human gonocytes. Blocking of SCF signaling with either phosphatidylinositol 3-kinase or mitogen-activated protein kinase inhibitor resulted in similar apoptotic features as well as the SCF-withdrawal cultures. Taken together, we report that SCF acts as a potent regulator in the fate determination of human gonocytes. Our studies should form the basis forin vitrostudies and facilitate investigation of the molecular mechanisms underlying this unique stage.

Download Full-text