scholarly journals The Presence of Colony-Stimulating Factor-1 and Its Receptor in Different Cells of the Testis; It Involved in the Development of Spermatogenesis In Vitro

2021 ◽  
Vol 22 (5) ◽  
pp. 2325
Author(s):  
Alaa Sawaied ◽  
Eden Arazi ◽  
Ahmad AbuElhija ◽  
Eitan Lunenfeld ◽  
Mahmoud Huleihel
Keyword(s):  
Testicular Tissue ◽  
Culture Conditions ◽  
Human Testis ◽  
Seminiferous Tubules ◽  
Colony Stimulating Factor ◽  
Protein Levels ◽  
Stimulating Factor ◽  
Peritubular Cells ◽  
Testicular Macrophages

Spermatogenesis is a complex process, in which spermatogonial cells proliferate and differentiate in the seminiferous tubules of the testis to generate sperm. This process is under the regulation of endocrine and testicular paracrine/autocrine factors. In the present study, we demonstrated that colony stimulating factor-1 (CSF-1) is produced by mouse testicular macrophages, Leydig, Sertoli, peritubular cells and spermatogonial cells (such as CDH1-positively stained cells; a marker of spermatogonial cells). In addition, we demonstrated the presence of CSF-1 and its receptor (CSF-1R) in testicular macrophages, Leydig, Sertoli, peritubular cells and spermatogonial cells of human testis. We also show that the protein levels of CSF-1 were the highest in testis of 1-week-old mice and significantly decreased with age (2–12-week-old). However, the transcriptome levels of CSF-1 significantly increased in 2–3-week-old compared to 1-week-old, and thereafter significantly decreased with age. On the other hand, the transcriptome levels of CSF-1R was significantly higher in mouse testicular tissue of all examined ages (2–12-week-old) compared to 1-week-old. Our results demonstrate the involvement of CSF-1 in the induction the proliferation and differentiation of spermatogonial cells to meiotic and postmeiotic stages (BOULE- and ACROSIN-positive cells) under in vitro culture conditions, using methylcellulose culture system (MCS). Thus, it is possible to suggest that CSF-1 system, as a testicular paracrine/autocrine system, is involved in the development of different stages of spermatogenesis and may be used in the development of future therapeutic strategies for treatment of male infertility.

scholarly journals Interleukin-34, a Novel Paracrine/Autocrine Factor in Mouse Testis, and Its Possible Role in the Development of Spermatogonial Cells In Vitro

2020 ◽  
Vol 21 (21) ◽  
pp. 8143
Author(s):  
Alaa Sawaied ◽  
Eitan Lunenfeld ◽  
Mahmoud Huleihel
Keyword(s):  
Seminiferous Tubules ◽  
Isolated Cells ◽  
Proliferation And Differentiation ◽  
Endocrine Factors ◽  
First Time ◽  
Old Mice ◽  
Stimulating Factor ◽  
Autocrine Factor ◽  
Peritubular Cells

Spermatogenesis is the process of spermatogonial stem cell (SSC) proliferation and differentiation to generate sperm. This process is regulated by cell–cell interactions between Sertoli cells and developing SSCs by autocrine/paracrine and endocrine factors. It is also affected by cells in the interstitial compartment, such as Leydig cells and peritubular cells. Here, we demonstrate, for the first time, the presence of interleukin-34 (IL-34) in Leydig, Sertoli, and peritubular cells and in the premeiotic, meiotic, and postmeiotic cells. Its receptor, colony-stimulating factor-1 (CSF-1), has already been demonstrated in Leydig, Sertoli, premeiotic, and meiotic cells. IL-34 was detected in testicular homogenates and Sertoli cell-conditioned media, and was affected by mouse age. We showed that the addition of IL-34 in vitro to isolated cells from the seminiferous tubules of 7-day-old mice, using the methylcellulose culture system (MCS), increased the percentages and expression of the premeiotic cells (VASA), the meiotic cells (BOULE), and the meiotic/postmeiotic cells (ACROSIN) after four weeks of culture, when examined by immunofluorescence staining (IF) and qPCR analysis. It is possible to suggest that IL-34 is a novel paracrine/autocrine factor involved in the development of spermatogenesis. This factor may be used in future therapeutic strategies for the treatment of male infertility.

scholarly journals Ca2+ Signaling and IL-8 Secretion in Human Testicular Peritubular Cells Involve the Cation Channel TRPV2

2018 ◽  
Vol 19 (9) ◽  
pp. 2829 ◽  
Author(s):  
Katja Eubler ◽  
Carola Herrmann ◽  
Astrid Tiefenbacher ◽  
Frank-Michael Köhn ◽  
J. Schwarzer ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Transient Receptor Potential Channel ◽  
Ruthenium Red ◽  
Cation Channel ◽  
Angiogenic Factor ◽  
Human Testis ◽  
Seminiferous Tubules ◽  
Protein Levels ◽  
Peritubular Cells

Peritubular cells are part of the wall of seminiferous tubules in the human testis and their contractile abilities are important for sperm transport. In addition, they have immunological roles. A proteomic analysis of isolated human testicular peritubular cells (HTPCs) revealed expression of the transient receptor potential channel subfamily V member 2 (TRPV2). This cation channel is linked to mechano-sensation and to immunological processes and inflammation in other organs. We verified expression of TRPV2 in peritubular cells in human sections by immunohistochemistry. It was also found in other testicular cells, including Sertoli cells and interstitial cells. In cultured HTPCs, application of cannabidiol (CBD), a known TRPV2 agonist, acutely induced a transient increase in intracellular Ca2+ levels. These Ca2+ transients could be blocked both by ruthenium red, an unspecific Ca2+ channel blocker, and tranilast (TRA), an antagonist of TRPV2, and were also abolished when extracellular Ca2+ was removed. Taken together this indicates functional TRPV2 channels in peritubular cells. When applied for 24 to 48 h, CBD induced expression of proinflammatory factors. In particular, mRNA and secreted protein levels of the proinflammatory chemokine interleukin-8 (IL-8/CXCL8) were elevated. Via its known roles as a major mediator of the inflammatory response and as an angiogenic factor, this chemokine may play a role in testicular physiology and pathology.

P–125 Evaluation of N-acetylcysteine (NAC) effect on in vitro culture of immature mouse testis following vitrification

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
P Nikoosokhan ◽  
B Ebrahimi ◽  
A Alizadeh ◽  
S Hajiaghalou
Keyword(s):  
Stem Cells ◽  
Cell Survival ◽  
Culture Medium ◽  
Caspase 3 ◽  
Testicular Tissue ◽  
Culture Conditions ◽  
Seminiferous Tubules ◽  
Immature Mouse ◽  
Prepubertal Boys

Abstract Study question Can the Culture of cryopreserved immature mouse testicular tissue in the presence of NAC improves the developmental process and prevent apoptosis induction during the culture? Summary answer An appropriate dosage of NAC in the culture medium of immature mouse testicular tissue was associated with increased cell survival and spermatogonia stem cell regeneration. What is known already Spermatogonial stem cells (SSCs) are the most advanced type of stem cells in the testes of prepubertal boys which are the main targets of oncological treatments. Therefore, the only possible alternative to maintain fertility in prepubertal boys is to preserve SSCs before their depletion by cryopreserving the testicular tissue. Despite the possibility of obtaining viable spermatozoa using cryopreserved testicular tissue cultivated in vitro,cryopreservation methods and damages caused by the culture procedure would be obstacles for maintaining the testicular tissueand it seems that optimizing the culture condition is vital. Study design, size, duration Testis tissues were harvested from 6-days-old immature NMRI male mice (n = 100) after cervical dislocation and vitrified. After 3 days testicular biopsies were warmed and distributed into control, culture Ӏ (not supplemented with NAC) and culture ӀӀ (supplemented with NAC) groups. To determine the appropriate NAC concentration 8 different dosages of NAC were evaluated in terms of cell survival and the best dose, a culture medium containing 125mmol/L NAC was selected to continue the study. Participants/materials, setting, methods Vitrified-warmed fragments (2mm3) obtaining from immature NMRI mice were cultured in vitro for 7 days on agar gel. The effects of culture conditions were assessed by Morphological evaluation of seminiferous tubules (using Hematoxylin-eosin staining). Cell viability, protein expression (caspase–3), and gene expression (Bax, Bcl2, Caspase–3, plzf) were evaluated by flow cytometry, immunofluorescence staining, and real time polymerase chain reaction respectively. Additionally, Malondialdehyde (MDA) concentration in the culture medium was measured by MAD Assay Kit. Main results and the role of chance Significant (p < 0.01) increase in cell viability was observed in the culture ӀӀ group after 7 days of culture compared to the culture Ӏ. Bax/Bcl2 ratio was significantly (p < 0.01) lower in the culture ӀӀ group compared to the control and culture Ӏ group. The expression of caspase–3 showed a significant (p < 0.001) increase in the culture ӀӀ group while immunofluorescence analysis showed low expression of it in all groups. These results were consistent with the high level of Bcl2expression that inhibited Caspase–3 expression and consequently the inhibition of apoptosis, and on the other hand, the presence of NAC showed that plzf expressions significantly (p < 0.001) increased in culture ӀӀ group compared to the control and culture Ӏ group. Although the presence of NAC did not inhibit all the deleterious effects of culture medium on tissue morphology, NAC was able to maintain better integrity of tissue and seminiferous tubules within central regions compared to the group without NAC. The decrease in MDA level in the presence of NAC (culture ӀӀ) was also a good indicator to confirm the desired results obtained from the presence of NAC in the culture medium. Limitations, reasons for caution Although the findings of the study were satisfactory in mice tissue after 1 week of culture, it is essential to replicate the experiments using human tissue and evaluate the quality and reproductive potential of surviving spermatogonia after long-term storage to become clinically applicable. Wider implications of the findings: This study highlights the necessity for further experiments to improve the testicular tissue culture conditions for better spermatogonial survival and differentiation to sperm, as the prepubertal fertility restoration methods are promising to be implemented in the clinic in the near future. Trial registration number Not applicable

scholarly journals Irradiation Causes Acute and Long-Term Spermatogonial Depletion in Cultured and Xenotransplanted Testicular Tissue from Juvenile Nonhuman Primates

Endocrinology ◽  
2007 ◽  
Vol 148 (11) ◽  
pp. 5541-5548 ◽  
Author(s):  
Kirsi Jahnukainen ◽  
Jens Ehmcke ◽  
Mirja Nurmio ◽  
Stefan Schlatt
Keyword(s):  
Organ Culture ◽  
Type A ◽  
Childhood Cancer Survivors ◽  
Testicular Tissue ◽  
Human Testis ◽  
Seminiferous Tubules ◽  
Preclinical Model ◽  
Acute Response ◽  
Dose Dependent Decrease

Infertility is a serious late effect in childhood cancer survivors. Little is known about acute irradiation effects in immature primate testis. Radiation defects have previously only been studied in postpubertal primates. Here we use the juvenile rhesus monkey as a preclinical model. We expose fragments of testicular tissue to 0, 0.5, 1.0, and 4.0 Gy irradiation in vitro. We then maintain the fragments in organ culture for 24–48 h or xenograft the fragments into nude mice for 4 months. Histological endpoints were determined to explore the cellular responses to the irradiation. At the highest dose, irradiation provoked an acute depletion of A-spermatogonia and a rise of apoptotic germ and Sertoli cells in organ culture. A dose-dependent decrease in the number of seminiferous tubules containing type A dark and type A pale spermatogonia was observed in irradiated xenografts. The number of Sertoli-cell only tubules increased respectively. Outgrowth of grafts was affected by the 4-Gy dose. Our observations reveal that irradiation evoked an immediate and sustained depletion of A-spermatogonia. We conclude that spermatogonia in the juvenile primate testis are highly sensitive to irradiation and that spermatogonial depletion and cessation of proliferation is an acute response. In contrast to adult testes, where such damage is immediately visible, this damage in immature testes becomes apparent only when spermatogonial insufficiency leads to spermatogenic failure, and thus infertility, at the onset of puberty. Our methods are applicable to immature human testis and might serve as powerful tool to study irradiation toxicity in the juvenile human testis.

scholarly journals The Expression Levels and Cellular Localization of Pigment Epithelium Derived Factor (PEDF) in Mouse Testis: Its Possible Involvement in the Differentiation of Spermatogonial Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1147
Author(s):  
Noy Bagdadi ◽  
Alaa Sawaied ◽  
Ali AbuMadighem ◽  
Eitan Lunenfeld ◽  
Mahmoud Huleihel
Keyword(s):  
Cellular Localization ◽  
Pigment Epithelium ◽  
Testicular Tissue ◽  
Seminiferous Tubules ◽  
Target Cells ◽  
Isolated Cells ◽  
Expression Levels ◽  
Cellular Origin ◽  
Pigment Epithelium Derived Factor

Pigment epithelium derived factor (PEDF) is a multifunctional secretory soluble glycoprotein that belongs to the serine protease inhibitor (serpin) family. It was reported to have neurotrophic, anti-angiogenic and anti-tumorigenic activity. Recently, PEDF was found in testicular peritubular cells and it was assumed to be involved in the avascular nature of seminiferous tubules. The aim of this study was to determine the cellular origin, expression levels and target cells of PEDF in testicular tissue of immature and adult mice under physiological conditions, and to explore its possible role in the process of spermatogenesis in vitro. Using immunofluorescence staining, we showed that PEDF was localized in spermatogenic cells at different stages of development as well as in the somatic cells of the testis. Its protein levels in testicular homogenates and Sertoli cells supernatant showed a significant decrease with age. PEDF receptor (PEDF-R) was localized within the seminiferous tubule cells and in the interstitial cells compartment. Its RNA expression levels showed an increase with age until 8 weeks followed by a decrease. RNA levels of PEDF-R showed the opposite trend of the protein. Addition of PEDF to cultures of isolated cells from the seminiferous tubules did not changed their proliferation rate, however, a significant increase was observed in number of meiotic/post meiotic cells at 1000 ng/mL of PEDF; indicating an in vitro differentiation effect. This study may suggest a role for PEDF in the process of spermatogenesis.

scholarly journals A GM-colony-stimulating factor (CSF) activated ribonuclease system transregulates M-CSF receptor expression in the murine FDC-P1/MAC myeloid cell line.

1992 ◽  
Vol 3 (5) ◽  
pp. 535-544 ◽  
Author(s):  
B C Gliniak ◽  
L S Park ◽  
L R Rohrschneider
Keyword(s):  
Protein Synthesis ◽  
Cell Line ◽  
Transcriptional Activation ◽  
Mrna Degradation ◽  
Receptor Expression ◽  
Metabolic Inhibitors ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Stimulating Factor

The murine myeloid precursor cell line FDC-P1/MAC simultaneously expresses receptors for multi-colony-stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, and macrophage (M)-CSF. Growth of FDC-P1/MAC cells in either multi-CSF or GM-CSF results in the posttranscriptional suppression of M-CSF receptor (c-fms proto-oncogene) expression. We use the term transregulation to describe this control of receptor expression and have further characterized this regulatory process. The removal of FDC-P1/MAC cells from GM-CSF stimulation resulted in the re-expression of c-fms mRNA independent of M-CSF stimulation and new protein synthesis. Switching FDC-P1/MAC cells from growth in M-CSF to GM-CSF caused the selective degradation of c-fms mRNA within 6 h after factor switching. Blocking protein synthesis or gene transcription with metabolic inhibitors effectively prevented GM-CSF stimulated degradation of c-fms mRNA. These results suggest that the transregulation of c-fms transcripts by GM-CSF requires the transcriptional activation of a selective mRNA degradation factor. In vitro analysis, the use of cytoplasmic cell extracts, provided evidence that a ribonuclease is preferentially active in GM-CSF stimulated cells, although the specificity for mRNA degradation in vitro is broader than seen in vivo. Together, these data suggest that GM-CSF can dominantly transregulate the level of c-fms transcript through the transcriptional activation of a ribonuclease degradation system.

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