{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T15:19:01Z","timestamp":1790435941751,"version":"4.1.0"},"reference-count":43,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2024,2,10]],"date-time":"2024-02-10T00:00:00Z","timestamp":1707523200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"REPRODUCTION Hub (#111), Faculty of Health Sciences, Ben-Gurion University of the Negev"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Spermatogenesis is the process of proliferation and differentiation of spermatogonial cells to meiotic and post-meiotic stages and sperm generation. Normal spermatogenesis occurs in vivo at 34 \u00b0C to 35 \u00b0C, and high temperatures are known to cause male infertility. The aim of the present study was to examine the effect of temperature (35 \u00b0C compared to 37 \u00b0C) on the viability\/apoptosis of developed cells, on the development of different stages of spermatogenesis in 3D in vitro culture conditions, and the functionality of Sertoli cells under these conditions. We used isolated cells from seminiferous tubules of sexually immature mice. The cells were cultured in methylcellulose (as a three-dimensional (3D) in vitro culture system) and incubated in a CO2 incubator at 35 \u00b0C or 37 \u00b0C. After two to six weeks, the developed cells and organoids were collected and examined for cell viability and apoptosis markers. The development of different stages of spermatogenesis was evaluated by immunofluorescence staining or qPCR analysis using specific antibodies or primers, respectively, for cells at each stage. Factors that indicate the functionality of Sertoli cells were assessed by qPCR analysis. The developed organoids were examined by a confocal microscope. Our results show that the percentages and\/or the expression levels of the developed pre-meiotic, meiotic, and post-meiotic cells were significantly higher at 35 \u00b0C compared to those at 37 \u00b0C, including the expression levels of the androgen receptor, the FSH receptor, transferrin, the androgen-binding protein (ABP), and the glial-derived nerve growth factor (GDNF) which were similarly significantly higher at 35 \u00b0C than at 37 \u00b0C. The percentages of apoptotic cells (according to acridine orange staining) and the expression levels of BAX, FAS, and CASPAS 3 were significantly higher in cultures incubated at 37 \u00b0C compared to those incubated at 35 \u00b0C. These findings support the in vivo results regarding the negative effect of high temperatures on the process of spermatogenesis and suggest a possible effect of high temperatures on the viability\/apoptosis of spermatogenic cells. In addition, increasing the temperature in vitro also impaired the functionality of Sertoli cells. These findings may deepen our understanding of the mechanisms behind optimal conditions for normal spermatogenesis in vivo and in vitro.<\/jats:p>","DOI":"10.3390\/ijms25042160","type":"journal-article","created":{"date-parts":[[2024,2,12]],"date-time":"2024-02-12T03:50:27Z","timestamp":1707709827000},"page":"2160","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":19,"title":["Effect of Temperature on the Development of Stages of Spermatogenesis and the Functionality of Sertoli Cells In Vitro"],"prefix":"10.3390","volume":"25","author":[{"given":"Areej","family":"Jorban","sequence":"first","affiliation":[{"name":"The Shraga Segal Department of Microbiology, Immunology, and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 8410501, Israel"},{"name":"The Center of Advanced Research and Education in Reproduction (CARER), Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 8410501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eitan","family":"Lunenfeld","sequence":"additional","affiliation":[{"name":"Adelson School of Medicine, Ariel University, Ariel 4076414, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4246-9299","authenticated-orcid":false,"given":"Mahmoud","family":"Huleihel","sequence":"additional","affiliation":[{"name":"The Shraga Segal Department of Microbiology, Immunology, and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 8410501, Israel"},{"name":"The Center of Advanced Research and Education in Reproduction (CARER), Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 8410501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,2,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1459","DOI":"10.1098\/rstb.2010.0024","article-title":"The Biology of Spermatogenesis: The Past, Present and Future","volume":"365","author":"Cheng","year":"2010","journal-title":"Philos. Trans. R. Soc. B Biol. Sci."},{"key":"ref_2","first-page":"1","article-title":"Spermatogenesis and Cycle of the Seminiferous Epithelium","volume":"636","author":"Hess","year":"2008","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1152\/physrev.00025.2011","article-title":"The Germline Stem Cell Niche Unit in Mammalian Testes","volume":"92","author":"Oatley","year":"2012","journal-title":"Physiol. Rev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1007\/978-1-4899-0952-7_3","article-title":"Endocrine, Paracrine and Autocrine Regulation of Testicular Steroidogenesis","volume":"377","author":"Huhtaniemi","year":"1995","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3962","DOI":"10.1210\/jc.2006-1145","article-title":"The Relative Roles of Follicle-Stimulating Hormone and Luteinizing Hormone in Maintaining Spermatogonial Maturation and Spermiation in Normal Men","volume":"91","author":"Matthiesson","year":"2006","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"116","DOI":"10.4161\/spmg.1.2.16956","article-title":"Testosterone Signaling and the Regulation of Spermatogenesis","volume":"1","author":"Walker","year":"2011","journal-title":"Spermatogenesis"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e996025","DOI":"10.1080\/21565562.2014.996025","article-title":"Endocrine Control of Spermatogenesis: Role of FSH and LH\/Testosterone","volume":"4","author":"Ramaswamy","year":"2014","journal-title":"Spermatogenesis"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1016\/j.mce.2016.08.033","article-title":"Expression Profiling Identifies Sertoli and Leydig Cell Genes as Fsh Targets in Adult Zebrafish Testis","volume":"437","author":"Crespo","year":"2016","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1007\/BF00182039","article-title":"Paracrine Factors and the Regulation of Spermatogenesis","volume":"11","author":"Niederberger","year":"1993","journal-title":"World J. Urol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"16489","DOI":"10.1073\/pnas.0407063101","article-title":"Growth Factors Essential for Self-Renewal and Expansion of Mouse Spermatogonial Stem Cells","volume":"101","author":"Kubota","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"127","DOI":"10.3109\/01485018708986808","article-title":"Sonoda, Temperature Sensitivity of Human Spermatogonia and Spermatocytes in Vitro","volume":"19","author":"Nakamura","year":"1987","journal-title":"Arch. Androl."},{"key":"ref_12","first-page":"730","article-title":"Temperature Control of Spermatogenesis and Prospect of Male Contraception","volume":"2","author":"Liu","year":"2010","journal-title":"Front. Biosci. (Schol. Ed.)"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0015-0282(16)59640-X","article-title":"Role of Temperature in Regulation of Spermatogenesis and the Use of Heating as a Method for Contraception","volume":"49","author":"Kandeel","year":"1988","journal-title":"Fertil. Steril."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1111\/j.1365-2605.1995.tb00408.x","article-title":"Testicular Heating and Its Possible Contributions to Male Infertility: A Review","volume":"18","author":"Mieusset","year":"1995","journal-title":"Int. J. Androl."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"651","DOI":"10.21037\/tau.2019.11.18","article-title":"Mild Experimental Increase in Testis and Epididymis Temperature in Men: Effects on Sperm Morphology According to Spermatogenesis Stages","volume":"8","author":"Hadi","year":"2019","journal-title":"Transl. Androl. Urol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.theriogenology.2020.08.034","article-title":"Amelioration of Heat Stress-Induced Damage to Testes and Sperm Quality","volume":"158","author":"Shahat","year":"2020","journal-title":"Theriogenology"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2122","DOI":"10.1093\/humrep\/13.8.2122","article-title":"Occupational Heat Exposure and Male Fertility: A Review","volume":"13","author":"Thonneau","year":"1998","journal-title":"Hum. Reprod."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Jhun, H., Lee, W.Y., Park, J.K., Hwang, S.G., and Park, H.J. (2022). Transcriptomic Analysis of Testicular Gene Expression in a Dog Model of Experimentally Induced Cryptorchidism. Cells, 11.","DOI":"10.3390\/cells11162476"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"892342","DOI":"10.3389\/fendo.2022.892342","article-title":"Organotypic Rat Testicular Organoids for the Study of Testicular Maturation and Toxicology","volume":"13","author":"Sakib","year":"2022","journal-title":"Front. Endocrinol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1938","DOI":"10.1007\/s43032-022-01133-4","article-title":"Transcriptome Analysis in High Temperature Inhibiting Spermatogonial Stem Cell Differentiation In Vitro","volume":"30","author":"Gao","year":"2023","journal-title":"Reprod. Sci."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Gan, M., Jing, Y., Xie, Z., Ma, J., Chen, L., Zhang, S., Zhao, Y., Niu, L., Wang, Y., and Li, X. (2023). Potential Function of Testicular MicroRNAs in Heat-Stress-Induced Spermatogenesis Disorders. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24108809"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"111672","DOI":"10.18632\/oncotarget.22900","article-title":"Next-Generation Sequencing-Based MicroRNA Profiling of Mice Testis Subjected to Transient Heat Stress","volume":"8","author":"Rao","year":"2017","journal-title":"Oncotarget"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1207","DOI":"10.1016\/0093-691X(93)90291-C","article-title":"Effects of Elevated Testicular Temperature on Morphology Characteristics of Ejaculated Spermatozoa in the Bovine","volume":"40","author":"Vogler","year":"1993","journal-title":"Theriogenology"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Hirano, K., Nonami, Y., Nakamura, Y., Sato, T., Sato, T., Ishiguro, K.-i., Ogawa, T., and Yoshida, S. (2022). Temperature Sensitivity of DNA Double-Strand Break Repair Underpins Heat-Induced Meiotic Failure in Mouse Spermatogenesis. Commun. Biol., 5.","DOI":"10.1038\/s42003-022-03449-y"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"111216","DOI":"10.1016\/j.mce.2021.111216","article-title":"Heat Stress Response of Somatic Cells in the Testis","volume":"527","author":"Aldahhan","year":"2021","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Huleihel, M., and Lunenfeld, E. (2020). Approaches and Technologies in Male Fertility Preservation. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21155471"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1038\/aja.2011.112","article-title":"Differentiation of Murine Male Germ Cells to Spermatozoa in a Soft Agar Culture System","volume":"14","author":"Lunenfeld","year":"2012","journal-title":"Asian J. Androl."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1007","DOI":"10.1089\/scd.2017.0301","article-title":"Development of Postmeiotic Cells In Vitro from Spermatogonial Cells of Prepubertal Cancer Patients","volume":"27","author":"AbuMadighem","year":"2018","journal-title":"Stem Cells Dev."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"AbuMadighem, A., Solomon, R., Stepanovsky, A., Kapelushnik, J., Shi, Q., Meese, E., Lunenfeld, E., and Huleihel, M. (2018). Development of Spermatogenesis In Vitro in Three-Dimensional Culture from Spermatogonial Cells of Busulfan-Treated Immature Mice. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19123804"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"972","DOI":"10.4103\/1008-682X.154994","article-title":"Application of Three-Dimensional Culture Systems to Study Mammalian Spermatogenesis, with an Emphasis on the Rhesus Monkey (Macaca mulatta)","volume":"17","author":"Huleihel","year":"2015","journal-title":"Asian J. Androl."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1045","DOI":"10.1016\/j.fertnstert.2018.07.018","article-title":"Influence of Temperature, Serum, and Gonadotropin Supplementation in Short- and Long-Term Organotypic Culture of Human Immature Testicular Tissue","volume":"110","author":"Medrano","year":"2018","journal-title":"Fertil. Steril."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"17023","DOI":"10.1074\/jbc.272.27.17023","article-title":"Seminiferous Tubule Basement Membrane Composition and Organization of Type IV Collagen Chains, and the Linkage of A3(IV) and A5(IV) Chains","volume":"272","author":"Kahsai","year":"1997","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Abumadighem, A., Shuchat, S., Lunenfeld, E., Yossifon, G., and Huleihel, M. (2022). Testis on a Chip\u2014A Microfluidic Three-Dimensional Culture System for the Development of Spermatogenesis in vitro. Biofabrication, 14.","DOI":"10.1088\/1758-5090\/ac6126"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"896","DOI":"10.1002\/mrd.22358","article-title":"The Acrosomal Protein SP-10 (Acrv1) Is an Ideal Marker for Staging of the Cycle of Seminiferous Epithelium in the Mouse","volume":"81","author":"Osuru","year":"2014","journal-title":"Mol. Reprod. Dev."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"6289","DOI":"10.1074\/jbc.RA119.012375","article-title":"The Testis-Specific LINC Component SUN3 Is Essential for Sperm Head Shaping during Mouse Spermiogenesis","volume":"295","author":"Gao","year":"2020","journal-title":"J. Biol. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"G\u00f6b, E., Schmitt, J., Benavente, R., and Alsheimer, M. (2010). Mammalian Sperm Head Formation Involves Different Polarization of Two Novel LINC Complexes. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0012072"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1111\/j.1439-0272.2010.01095.x","article-title":"Developmental Expression of ACRV1 in Humans and Mice","volume":"44","author":"Tang","year":"2012","journal-title":"Andrologia"},{"key":"ref_38","first-page":"705","article-title":"Characterization of Cholesterol Metabolism in Sertoli Cells and Spermatogenesis (Review)","volume":"17","author":"Shi","year":"2018","journal-title":"Mol. Med. Rep."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"747","DOI":"10.1210\/er.2003-0022","article-title":"Sertoli-Sertoli and Sertoli-Germ Cell Interactions and Their Significance in Germ Cell Movement in the Seminiferous Epithelium during Spermatogenesis","volume":"25","author":"Mruk","year":"2004","journal-title":"Endocr. Rev."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1111\/j.1365-2605.1978.tb00501.x","article-title":"Temperature Dependence of Sertoli Cell Function","volume":"1","author":"Svensson","year":"1978","journal-title":"Int. J. Androl."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1095\/biolreprod.108.072827","article-title":"Effects of Different Temperatures on Testis Structure and Function, with Emphasis on Somatic Cells, in Sexually Mature Nile Tilapias (Oreochromis niloticus)","volume":"80","year":"2009","journal-title":"Biol. Reprod."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1344","DOI":"10.1111\/rda.14211","article-title":"Identification of Internal Reference Genes for Porcine Immature Sertoli Cells under Heat Stress","volume":"57","author":"Wang","year":"2022","journal-title":"Reprod. Domest. Anim."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1095\/biolreprod65.1.229","article-title":"Effects of Hyperthermia on Spermatogenesis, Apoptosis, Gene Expression, and Fertility in Adult Male Mice","volume":"65","author":"Rockett","year":"2001","journal-title":"Biol. Reprod."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/4\/2160\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T13:58:24Z","timestamp":1760104704000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/4\/2160"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,2,10]]},"references-count":43,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2024,2]]}},"alternative-id":["ijms25042160"],"URL":"https:\/\/doi.org\/10.3390\/ijms25042160","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,2,10]]}}}