Regulation of Blood-Testis Barrier (BTB) Proteins in Sertoli-Germ Cell Nanotube Formation in EF-Treated Spermatogenesis

Nazli Ece Gungor¹*, Tugba Elgun², Mete Emir Ozgurses³, Bariscan Uzunkaya⁴ and Medine Tasdemir⁵

¹Biruni University, Medicine Faculty, Department of Histology and Embryology, Istanbul/Turkey
²Biruni University, Medicine Faculty, Department of Medical Biology, Istanbul/Turkey
³University of Illinois Chicago College of Medicine, Department of Physiology and Biophysics, Chicago, IL, US
⁴Acibadem Mehmet Ali Aydinlar University, Graduate School of Applied Sciences, Department of Biomaterials, Istanbul/Turkey
⁵The University of Chicago Medical Center, Department of Obstetrics and Gynecology, Chicago, IL, US

*Corresponding Author: Nazli Ece Gungor, Associate Professor, Biruni University, Medicine Faculty, Department of Histology and Embryology, Istanbul/Turkey.

Received: August 05, 2025; Published: October 14, 2025

Abstract

Objective: The aim of this study was to analyze visual data analysis of nanotubes formation and actin expression between Sertoli germ cells, gene silencing with the use of FAK siRNA and EF application, confirmation with mRNA levels, cell viability test and immunofluorescent staining associated ezrin, Fascin 1, FAK and N-cadherin. Expressions of blood-testicular barrier (BTB) proteins were evaluated.

Materials and Methods: As the experimental group of the research; control group (CG), in which intercellular nanotubes and cargo proteins were followed under normal culture conditions; Sertoli and germ cells co-culture; co-culture of testosterone (T) group, Sertoli and germ cells in which intercellular nanotubes and cargo proteins are tracked; The group in which actin organization and intercellular nanotubes and cargo proteins are monitored, and the group in which the focal adhesion kinase is suppressed with siRNA (FAK RNAi) and the co-culture of Sertoli and germ cells, the electromagnetic field applied group (EF), in which intercellular nanotubes and cargo proteins are followed under normal culture conditions, were used.

Results: In the control groups, nanotubes formations started at the 6th hour during the culture and increased at the 40th hour, while the number of nanotubes formation and disappearance was 52 in the Control group; 58 in the EF group; 44, 12 in the FAK RNAi group and 5 in the EF+ FAK RNAi group. It was shown that actin associated nanotubes formations were significantly decreased in FAK RNAi and EF+ FAK RNAi groups compared to control. Stable nanotubes formation rate but low disappearance rate was detected in the EF applied group. It was observed that there was a decrease in ezrin and Fascin 1 expressions in nanotubes formation regions, except for control and testosterone groups, and there was no significant difference in N-cadherin expression levels. It was determined that FAK, Ezrin and Fascin 1 cargo passage were significantly retained in the cytoplasm in the FAK RNAi groups.

Conclusions: With the results we obtained; It has been shown that the FAK molecule has an important role in the germ cell development process in vitro. It has been shown that in Sertoli-germ cell co-culture in which FAK gene is silenced and FAK RNAi and EF applied together, vesicle contents cannot be released by endocytosis and these molecules affect nanotubes formation due to decreasing the ratios of FAK, ezrin and Fascin 1 proteins. Based on our results, a research pattern and culture model were proposed for the detection of intercellular signaling due to the passage of regulatory proteins and nanotubes formation.

 Keywords: Focal Adhesion Kinase (FAK); Nanotubes; Spermatogenesis; Sertoli-Sertoli and Sertoli-germ

References

1. Petersen C and Soder O. “The Sertoli cell--a hormonal target and 'super' nurse for germ cells that determines testicular size”. Hormone Research4 (2006): 153-61.
2. Sharpe RM., et al. “Inhibin B levels in plasma of the male rat from birth to adulthood: effect of experimental manipulation of Sertoli cell number”. Journal of Andrology1 (1999): 94-101.
3. Meachem SJ., et al. “Neonatal exposure of rats to recombinant follicle stimulating hormone increases adult Sertoli and spermatogenic cell numbers”. Biological Reproduction1 (1996): 36-44.
4. Griswold MD., et al. “Stimulation by follicle-stimulating hormone of DNA synthesis and of mitosis in cultured Sertoli cells prepared from testes of immature rats”. Molecular and Cellular Endocrinology 2 (1977): 151-165.
5. Sharpe RM., et al. “Abnormalities in functional development of the Sertoli cells in rats treated neonatally with diethylstilbestrol: a possible role for estrogens in Sertoli cell development”. Biological Reproduction 5 (1998): 1084-1094.
6. Buzzard JJ., et al. “Thyroid hormone, retinoic acid, and testosterone suppress proliferation and induce markers of differentiation in cultured rat Sertoli cells”. Endocrinology9 (2003): 3722-3731.
7. Meng X., et al. “Regulation of cell fate decision of undifferentiated spermatogonia by GDNF”. Science5457 (2000): 1489-1493.
8. Kubota H., et al. “Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells”. Proceedings of the National Academy of Sciences of the United States of America 47 (2004): 16489-16494.
9. Hu J., et al. “Glial cell line-derived neurotropic factor stimulates Sertoli cell proliferation in the early postnatal period of rat testis development”. Endocrinology8 (1999): 3416-3421.
10. Wu Z. “Effects of glial cell line-derived neurotrophic factor on isolated developing mouse Sertoli cells in vitro”. Journal of Anatomy2 (2005): 175-184.
11. Rudnicka D., et al. “Intrusive HIV-1-infected cells”. Nature Immunology9 (2009): 933-934.
12. Xu W., et al. “HIV-1 evades virus-specific IgG2 and IgA responses by targeting systemic and intestinal B cells via long-range intercellular conduits”. Nature Immunology9 (2009): 1008-1017.
13. Arkwright PD., et al. “Fas stimulation of T lymphocytes promotes rapid intercellular exchange of death signals via membrane nanotubes”. Cell Research1 (2010): 72-88.
14. Watkins SC and Salter RD. “Functional connectivity between immune cells mediated by tunneling nanotubules”. Immunity3 (2005): 309-318.
15. Salter RD and Watkins SC. “Dynamic properties of antigen uptake and communication between dendritic cells”. Immunology Research1-3 (2006): 211-220.
16. Veranic P., et al. “Different types of cell-to-cell connections mediated by nanotubular structures”. Biophysics Journal9 (2008): 4416-4425.
17. Davis DM and Sowinski S. “Membrane nanotubes: dynamic long-distance connections between animal cells”. Nature Reviews Molecular Cell Biology6 (2008): 431-436.
18. Plotnikov EY., et al. “Cytoplasm and organelle transfer between mesenchymal multipotent stromal cells and renal tubular cells in co-culture”. Experimental Cell Research15 (2010): 2447-2455.
19. Domhan S., et al. “Intercellular communication by exchange of cytoplasmic material via tunneling nano¬tube like structures in primary human renal epithelial cells”. PLoS One6 (2011): e21283.
20. Gungor-Ordueri NE., et al. “Fascin 1 is an actin filament-bundling protein that regulates ectoplasmic specialization dynamics in the rat testis”. American Journal of Physiology-Endocrinology and Metabolism9 (2014): E738-753.
21. Gungor-Ordueri NE., et al. “Ezrin is an actin binding protein that regulates Sertoli cell and spermatid adhesion during spermatogenesis”. Endocrinology 10 (2015): 3981-3995.
22. Gungor-Ordueri NE., et al. “Ezrin: a regulator of actin microfilaments in cell junctions of the rat testis. Asian Journal of Andrology4 (2015): 653-658.
23. Tanaka TM., et al. “The luteinizing hormone-testosterone pathway regulates mouse spermatogonial stem cell self-renewal by suppressing WNT5A expression in Sertoli cells”. Stem Cell Reports2 (2014): 279-291.
24. Tuszynski JA., et al. “Ionic wave propagation along actin filaments”. Biophysics Journal4 (2004): 1890-1903.
25. Minten J., et al. “Differences in high-energy phosphate catabolism between the rat and the dog in a heart preservation model”. The Journal of Heart and Lung Transplantation 1 (1991): 71-78.
26. Gautreau A., et al. “Function and regulation of the endosomal fusion and fission machineries”. Cold Spring Harbor Perspectives in Biology 6 (2014).
27. Collinet C and Lecuit T. “Stability and dynamics of cell-cell junctions”. Progress in Molecular Biology and Translational Science 116 (2013): 25–47.
28. Xiao X., et al. “N-WASP is required for structural integrity of the blood-testis barrier”. PLoS Genetics 10 (2014): e1004447.
29. Su L., et al. “A peptide derived from laminin-γ3 reversibly impairs spermatogenesis in rats”. Nature Communication 3 (2012): 1185.
30. Xiao X., et al. “Differential effects of c-Src and c-Yes on the endocytic vesicle-mediated trafficking events at the Sertoli cell blood-testis barrier: an in vitro study”. American Journal of Physiology-Endocrinology and Metabolism 307 (2014): E553–E562.
31. Xiao X., et al. “c-Yes regulates cell adhesion at the blood-testis barrier and the apical ectoplasmic specialization in the seminiferous epithelium of rat testes”. The International Journal of Biochemistry & Cell Biology 43 (2011): 651-665.
32. Xiao X., et al. “c-Yes regulates cell adhesion at the apical ectoplasmic specialization-blood-testis barrier axis via its effects on protein recruitment and distribution”. American Journal of Physiology-Endocrinology and Metabolism 304 (2013): E145–E159.

Citation

Citation: Nazli Ece Gungor., et al. “Regulation of Blood-Testis Barrier (BTB) Proteins in Sertoli-Germ Cell Nanotube Formation in EF-Treated Spermatogenesis”.Acta Scientific Medical Sciences 9.11 (2025): 21-29.

Copyright

Copyright: © 2025 Nazli Ece Gungor., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.