The Effect of Cryopreservation on Human Sperm Growth Factors Expression: An Experimental Study
DOI:
https://doi.org/10.22100/ijhs.v11i4.1244Keywords:
Cryopreservation, Normozoospermic, Nerve growth factors, Fibroblast growth factors, Growth factor receptorsAbstract
Background: Cryopreservation is a widely employed assisted reproductive technology (ART) for the treatment of infertility. However, numerous sperm functions are compromised during the cryopreservation process. Considering the pivotal roles of nerve growth factor (NGF) and fibroblast growth factor (FGF) in sperm physiology and function, this study aimed to investigate the impact of cryopreservation on the expression and quality of these growth factors and their respective receptors.
Methods: Semen samples were collected from 30 normozoospermic donors, with each sample divided into fresh and frozen-thawed groups. Sperm parameters were evaluated using computer-assisted sperm analysis (CASA). Real-time polymerase chain reaction (PCR) and Western blotting techniques were employed to quantify the gene and protein expression levels of growth factor receptors. Apoptosis was assessed using Annexin V staining, and enzyme-linked immunosorbent assay (ELISA) was conducted to measure levels of NGF and FGF2 in seminal plasma.
Results: The findings demonstrated that the frozen-thawed group exhibited significantly reduced levels of fibroblast growth factor receptor 2 (FGFR2) and a trend towards decreased expression of tropomyosin receptor kinase A (TrkA) compared to the fresh group, although the latter was not statistically significant. Notably, the percentage of cells undergoing late apoptosis was markedly higher in the cryopreservation group relative to the fresh group. Furthermore, cryopreservation resulted in substantial reductions in NGF and FGF2 concentrations within the seminal plasma.
Conclusions: The detrimental effects of freezing and thawing on the expression of growth factors and their receptors in sperm cells suggest that the integration of these factors during the cryopreservation process may enhance sperm quality post-thaw. Further research is warranted to explore the potential clinical applications of these findings.
References
L. K. Thomson, S. D. Fleming, R. J. Aitken, G. N. De Iuliis, J. A. Zieschang and A. M. Clark. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Human reproduction (Oxford, England) 2009;24:2061-70. doi: 10.1093/humrep/dep214
S. Saeednia, et al. Nerve growth factor in human semen: Effect of nerve growth factor on the normozoospermic men during cryopreservation process. Iran J Basic Med Sci 2015; 18:292-9.
O. A. Bogle, et al. Identification of protein changes in human spermatozoa throughout the cryopreservation process. Andrology 2017; 5:10-22. doi: 10.1111/andr.12279
S. Saeednia, M. Shabani Nashtaei, H. Bahadoran, A. Aleyasin and F. Amidi. Effect of nerve growth factor on sperm quality in asthenozoosprmic men during cryopreservation. Reproductive biology and endocrinology: RB&E 2016; 14:29. doi: 10.1186/s12958-016-0163-z
S. Chatterjee and C. Gagnon. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing. Molecular Reproduction and Development: Incorporating Gamete Research 2001; 59:451-8. doi: 10.1002/mrd.1052
C. Aurich. Factors affecting the plasma membrane function of cooled-stored stallion spermatozoa. Animal reproduction science 2005; 89:65-75. doi: 10.1016/j.anireprosci.2005.06.025
J. Schiller, J. Arnhold, H. J. Glander and K. Arnold. Lipid analysis of human spermatozoa and seminal plasma by MALDI-TOF mass spectrometry and NMR spectroscopy - effects of freezing and thawing. Chemistry and physics of lipids 2000; 106:145-56. doi: 10.1016/S0009-3084(00)00148-1
N. K. Duru, M. S. Morshedi, A. Schuffner and S. Oehninger. Cryopreservation-Thawing of fractionated human spermatozoa is associated with membrane phosphatidylserine externalization and not DNA fragmentation. Journal of andrology 2001; 22:646-51. doi: 10.1002/j.1939-4640.2001.tb02225.x
D. Dominguez-Fandos, M. I. Camejo, J. L. Ballesca and R. Oliva. Human sperm DNA fragmentation: correlation of TUNEL results as assessed by flow cytometry and optical microscopy. Cytometry Part A: the journal of the International Society for Analytical Cytology 2007; 71:1011-8. doi: 10.1002/cyto.a.20484
J. K. Critser, B. W. Arneson, D. V. Aaker, A. R. Huse-Benda and G. D. Ball. Cryopreservation of human spermatozoa. II. Postthaw chronology of motility and of zona-free hamster ova penetration. Fertility and sterility 1987; 47:980-4. doi: 10.1016/S0015-0282(16)59233-4
N. L. Cross and J. W. Overstreet. Glycoconjugates of the human sperm surface: distribution and alterations that accompany capacitation in vitro. Gamete research 1987; 16:23-35. doi: 10.1002/mrd.1120160104
W. Jin, A. Tanaka, G. Watanabe, H. Matsuda and K. Taya. Effect of NGF on the motility and acrosome reaction of golden hamster spermatozoa in vitro. Journal of Reproduction and Development 2010:1005270273-. doi: 10.1262/jrd.09-219N
R. T. Uren and A. M. Turnley. Regulation of neurotrophin receptor (Trk) signaling: suppressor of cytokine signaling 2 (SOCS2) is a new player. Frontiers in molecular neuroscience 2014; 7:39. doi: 10.3389/fnmol.2014.00039
C. Li, et al. Detection of nerve growth factor (NGF) and its specific receptor (TrkA) in ejaculated bovine sperm, and the effects of NGF on sperm function. Theriogenology 2010; 74:1615-22. doi: 10.1016/j.theriogenology.2010.06.033
C. Li, L. Zheng, C. Wang and X. Zhou. Absence of nerve growth factor and comparison of tyrosine kinase receptor A levels in mature spermatozoa from oligoasthenozoospermic, asthenozoospermic and fertile men. Clinica chimica acta; international journal of clinical chemistry 2010; 411:1482-6. doi: 10.1016/j.cca.2010.06.002
L. Zhang, et al. NGF induces adult stem Leydig cells to proliferate and differentiate during Leydig cell regeneration. Biochemical and Biophysical Research Communications 2013; 436:300-5. doi: 10.1016/j.bbrc.2013.05.098
L. Saucedo, et al. Deficiency of fibroblast growth factor 2 (FGF-2) leads to abnormal spermatogenesis and altered sperm physiology. J Cell Physiol 2018; 233:9640-51. doi: 10.1002/jcp.26876
L. Saucedo, et al. Fibroblast growth factor receptors (FGFRs) in human sperm: expression, functionality and involvement in motility regulation. PLoS One 2015;10: e0127297. doi: 10.1371/journal.pone.0127297
S. Asadian, et al. Circulating endothelial progenitor cells in pregnant women with premature rupture of membranes: potential association with placental disorders. Reproduction, Fertility and Development 2018;30:1689-98. doi: 10.1071/RD17523
M. Bagheri, M. Fazli, S. Saeednia, M. G. Kharanagh and N. Ahmadiankia. Sulforaphane modulates cell migration and expression of β-catenin and epithelial mesenchymal transition markers in breast cancer cells. Iranian Journal of Public Health 2020; 49:77. doi: 10.18502/ijph.v49i1.3054
M. Bagheri, M. Fazli, S. Saeednia, A. Kor and N. Ahmadiankia. Pomegranate peel extract inhibits expression of β-catenin, epithelial mesenchymal transition, and metastasis in triple negative breast cancer cells. Cellular and molecular biology (Noisy-le-Grand, France) 2018;64:86-91. doi: 10.14715/cmb/2018.64.7.15
J. Kopeika, A. Thornhill and Y. Khalaf. The effect of cryopreservation on the genome of gametes and embryos: principles of cryobiology and critical appraisal of the evidence. Human reproduction update 2015; 21:209-27. doi: 10.1093/humupd/dmu063
M. O'connell, N. Mcclure and S. Lewis. The effects of cryopreservation on sperm morphology, motility and mitochondrial function. Human reproduction 2002; 17:704-9. doi: 10.1093/humrep/17.3.704
M. Giraud, C. Motta, D. Boucher and G. Grizard. Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa. Human Reproduction 2000; 15:2160-4. doi: 10.1093/humrep/15.10.2160
H.-J. Glander and J. Schaller. Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage. MHR: Basic science of reproductive medicine 1999; 5:109-15. doi: 10.1093/molehr/5.2.109
B. Sion, L. Janny, D. Boucher and G. Grizard. Annexin V binding to plasma membrane predicts the quality of human cryopreserved spermatozoa. International journal of andrology 2004; 27:108-14. doi: 10.1046/j.1365-2605.2003.00457.x
D. Mutter, R. Middendorff and M. S. Davidoff. Neurotrophic factors in the testis. Biomedical Reviews 1999; 10:25-30. doi: 10.14748/bmr.v10.4
W. Jin, et al. Cellular localization of NGF and its receptors trkA and p75LNGFR in male reproductive organs of the Japanese monkey, Macaca fuscata fuscata. Endocrine 2006; 29:155-60. doi: 10.1385/ENDO:29:1:155
C. Castellini, et al. Role of NGF on sperm traits: A review. Theriogenology 2020; 150:210-4. doi: 10.1016/j.theriogenology.2020.01.039
C. Li, L. Zheng, C. Wang and X. Zhou. Absence of nerve growth factor and comparison of tyrosine kinase receptor A levels in mature spermatozoa from oligoasthenozoospermic, asthenozoospermic and fertile men. Clinica Chimica Acta 2010; 411:1482-6. doi: 10.1016/j.cca.2010.06.002
M. Regueira, et al. FSH and bFGF regulate the expression of genes involved in Sertoli cell energetic metabolism. General and Comparative Endocrinology 2015; 222:124-33. doi: 10.1016/j.ygcen.2015.08.011
M. Gómez, et al. Sertoli-secreted FGF-2 induces PFKFB4 isozyme expression in mouse spermatogenic cells by activation of the MEK/ERK/CREB pathway. American Journal of Physiology-Endocrinology and Metabolism 2012;303: E695-E707. doi: 10.1152/ajpendo.00381.2011
H. P. Pui and Y. Saga. Gonocytes-to-spermatogonia transition initiates prior to birth in murine testes and it requires FGF signaling. Mechanisms of development 2017; 144:125-39. doi: 10.1016/j.mod.2017.03.002
Z.-J. Lan, J. C. Labus and B. T. Hinton. Regulation of gamma-glutamyl transpeptidase catalytic activity and protein level in the initial segment of the rat epididymis by testicular factors: role of basic fibroblast growth factor. Biology of Reproduction 1998; 58:197-206. doi: 10.1095/biolreprod58.1.197
D. G. Azúa, et al. Fibroblast growth factor 2 (FGF2) is present in human spermatozoa and is related with sperm motility. The use of recombinant FGF2 to improve motile sperm recovery. Andrology 2017; 5:990-8. doi: 10.1111/andr.12398
Published
Issue
Section
License
The Copyright Form should be downloaded and signed by corresponding author in the fourth step "upload supplementary files" during submission process.
After acceptance, copyright form should be downloaded and signed by all authors one by one ( "summery --> supp. file" part and click on "add a supplementary file" link).
https://orcid.org/0000-0002-0424-9722

