Differential expression of oar-mir-133, oar-mir-433-3p oar-mir-150, and oar-mir-376d in ram sperm associated with fertility

Year 2025, Volume: 2 Issue: 2, 67 - 71, 30.06.2025

Mustafa Hitit , Mustafa Bodu

https://doi.org/10.62425/crihs.1708690

Abstract

Ram fertility is a crucial determinant of reproductive efficiency in sheep production systems, yet the molecular mechanism of male fertility remains incompletely understood. Small non-coding RNAs (sncRNAs), particularly microRNAs (miRNAs), have been recognized as essential epigenetic regulators of spermatogenesis, sperm function, and early embryonic development. We aimed to investigate the differential expression and biological significance of four specific miRNAs; oar-miR-133 (log2FC = +3.30, p < 0.001), oar-miR-433-3p (log2FC = +5.72, p < 0.001), oar-miR-150 HF (log2FC = -3.49, p < 0.001), and oar-miR-376d (log2FC = -5.74, p < 0.001) in relation to ram sperm fertility phenotypes. We selected based on their statistically significant differential expression in a previously published small RNA sequencing dataset comparing spermatozoa from high-fertility (HF; n = 4; avg. 99.2% pregnancy) and low-fertility (LF; n = 4; avg. 73.6% pregnancy) rams. We showed that oar-miR-133 and oar-miR-433-3p were significantly upregulated in HF rams, while oar-miR-150 and oar-miR-376d were downregulated. Functional enrichment analysis of their predicted target genes revealed involvement in biological processes such as heterochromatin formation, cellular response to stress and cytokines, macromolecule metabolic regulation, and developmental processes. Protein–protein interaction (PPI) network analysis, using Maximal Clique Centrality (MCC), identified several central hub genes including DICER1, DROSHA, DDX10, TUFM, and EEF2. We showed that hub genes may be associated with RNA metabolism, mitochondrial function, and translational control processes critical for sperm integrity and fertilization capacity. These results highlight the potential of specific miRNAs and their gene networks as biomarkers for ram fertility.

Keywords

ram , sperm , fertility , miRNA , biomarker

Koc spermasında fertilite ile ilişkili olan oar-mir-133, oar-mir-433-3p, oar-mir-150 ve oar-mir-376d'nin farklılasmis gen ifadesi

Abstract

Koç fertilitesi, koyun üretim sistemlerinde üreme verimliliğinin kritik bir belirleyicisidir, ancak erkek fertilitesinin moleküler mekanizması henüz tam olarak anlaşılamamıştır. Küçük kodlama yapmayan RNA'lar (sncRNA'lar), özellikle mikro RNA'lar (miRNA'lar), spermatogenez, sperm fonksiyonu ve erken embriyonik gelişimin temel epigenetik düzenleyicileri olarak tanınmıştır. Dört spesifik miRNA'nın, yani oar-miR-133 (log2FC = +3.30, p < 0.001), oar-miR-433-3p (log2FC = +5.72, p < 0.001), oar-miR-150 HF (log2FC = -3.49, p < 0.001) ve oar-miR-376d (log2FC = -5.74, p < 0.001) ram sperm fertilite fenotipleriyle ilişkili olarak farklı ifade seviyeleri ve biyolojik önemlerini araştırmayı amaçladık. Yüksek fertiliteden (HF; n = 4; ort. %99.2 gebelik) ve düşük fertiliteden (LF; n = 4; ort. %73.6 gebelik) koçların spermatozoa'ları arasındaki karşılaştırmayı yapan daha önce yayımlanmış küçük RNA dizileme veri setinde istatistiksel olarak anlamlı farklı ifade seviyelerine dayalı olarak seçim yaptık. Biz, oar-miR-133 ve oar-miR-433-3p'nin HF koçlarında önemli ölçüde yukarıregüle olduğunu, oar-miR-150 ve oar-miR-376d'nin ise aşağıregüle olduğunu gösterdik. Tahmin edilen hedef genlerinin fonksiyonel zenginlik analizi, heterokromatin oluşumu, hücresel stres ve sitokinlere yanıt, makromolekül metabolizmasının düzenlenmesi ve gelişim süreçleri gibi biyolojik süreçlere katılımı ortaya koydu. Maksimal Clique Merkeziliği (MCC) kullanarak yapılan protein-protein etkileşim (PPI) ağ analizi, DICER1, DROSHA, DDX10, TUFM ve EEF2 gibi birkaç merkezi merkez genini tanımladı. Merkezi genlerin RNA metabolizması, mitokondriyal fonksiyon ve sperm bütünlüğü ve döllenme kapasitesi için kritik çeviri kontrol süreçleri ile ilişkili olabileceğini gösterdik. Bu sonuçlar, belirli miRNA'ların ve gen ağlarının koç fertilitesi için biyomarker olarak potansiyelini vurgulamaktadır.

Keywords

sperm , miRNA , biomarker , koc , fertilite

References

• Abu-Halima, M.; Hammadeh, M.; Schmitt, J.; Leidinger, P.; Keller, A.; Meese, E.; & Backes, C. (2013). Altered microRNA expression profiles of human spermatozoa in patients with different spermatogenic impairments. Fertility and Sterility, 99(5), 1249–1255.e1216. http://dx.doi.org/10.1016/j.fertnstert.2012.11.054
• Bodu, M.; Hitit, M.; Donmez, H.; Kaya, A.; Ugur, M.R.; & Memili, E. (2025a). Exploration of small non-coding rnas as molecular markers of ram sperm fertility. International Journal of Molecular Sciences, 26(6), 2690. https://doi.org/10.3390/ijms26062690
• Bodu, M.; Hitit, M.; & Memili, E. (2025b). Harnessing the value of fertility biomarkers in bull sperm for buck sperm. Animal Reproduction Science, 272, 107643. https://doi.org/10.1016/j.anireprosci.2024.107643
• Bodu, M.; Hitit, M.; Sari, A.; Kirbas, M.; Bulbul, B.; Ataman, M.B.; Bucak, M.N.; Parrish, J.; Kaya, A.; & Memili, E. (2025c). Sperm cellular and nuclear dynamics associated with ram fertility. Frontiers in Veterinary Science, 12. https://doi.org/10.3389/fvets.2025.1577004
• Capra, E.; Turri, F.; Lazzari, B.; Cremonesi, P.; Gliozzi, T.M.; Fojadelli, I.; Stella, A.; & Pizzi, F. (2017). Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between High- and Low-motile sperm populations. BMC Genomics, 18(1), 14. https://doi.org/10.1186/s12864-016-3394-7
• Carè, A.; Catalucci, D.; Felicetti, F.; Bonci, D.; Addario, A.; Gallo, P.; Bang, M.-L.; Segnalini, P.; Gu, Y.; Dalton, N.D.; Elia, L.; Latronico, M.V.G.; Høydal, M.; Autore, C.; Russo, M.A.; Dorn, G.W.; Ellingsen, Ø.; Ruiz-Lozano, P.; Peterson, K.L.; Croce, C.M.; Peschle, C.; & Condorelli, G. (2007). MicroRNA-133 controls cardiac hypertrophy. Nature Medicine, 13(5), 613–618. https://doi.org/10.1038/nm1582
• Hamatani, T. (2012). Human spermatozoal RNAs. Fertility and Sterility, 97(2), 275–281. https://doi.org/10.1016/j.fertnstert.2011.12.035
• Herbst-Damm, K. L., & Kulik, J. A. (2005). Volunteer support, marital status, and the survival times of terminally ill patients. Health Psychology, 24(2), ss. 225–229. https://doi.org/10.1037/0278-6133.24.2.225
• Hitit, M.; Kaya, A.; & Memili, E. (2024). Sperm long non-coding RNAs as markers for ram fertility. Frontiers in Veterinary Science, 11, 1337939. https://doi.org/10.3389/fvets.2024.1337939
• Jodar, M.; & Anton, E. (2018). Chapter 7 - Small RNAs present in semen and their role in reproduction. In J.A. Horcajadas & J. Gosálvez (Eds.), Reproductomics (pp. 109–123). Academic Press.
• Kasimanickam, V.; Kumar, N.; & Kasimanickam, R. (2022). Investigation of sperm and seminal plasma candidate microRNAs of bulls with differing fertility and in silico prediction of miRNA-mRNA interaction network of reproductive function. Animals, 12(18), 2360. https://doi.org/10.3390/ani12182360
• Kastelic, J.; & Thundathil, J. (2008). Breeding soundness evaluation and semen analysis for predicting bull fertility. Reproduction in Domestic Animals, 43(S2), 368–373. https://doi.org/10.1111/j.1439-0531.2008.01186.x
• Luo, L.; Tang, Y.; Sun, L.; Li, S.; Liu, H.; Chen, Z.; & Li, G. (2023). SAP30 targeted by miR-133b was involved in the process of nuclear decondensation in Chinese mitten crab (Eriocheir sinensis) sperm. Aquaculture Reports, 29, 101540. https://doi.org/10.1016/j.aqrep.2023.101540
• Luo, L. F.; Hou, C. C.; & Yang, W.X. (2016). Small non-coding RNAs and their associated proteins in spermatogenesis. Gene, 578(1), 141–157. https://doi.org/10.1016/j.gene.2015.12.020
• Salas-Huetos, A.; James, E.R.; Aston, K.I.; Jenkins, T.G.; Carrell, D.T.; & Yeste, M. (2019). The expression of miRNAs in human ovaries, oocytes, extracellular vesicles, and early embryos: A systematic review. Cells, 8(12), 1564. https://doi.org/10.3390/cells8121564
• Sendler, E.; Johnson, G.D.; Mao, S.; Goodrich, R.J.; Diamond, M.P.; Hauser, R.; & Krawetz, S.A. (2013). Stability, delivery and functions of human sperm RNAs at fertilization. Nucleic Acids Research, 41(8), 4104–4117. https://doi.org/10.1093/nar/gkt132
• Sharma, U. (2019). Paternal contributions to offspring health: role of sperm small RNAs in intergenerational transmission of epigenetic information. Frontiers in Cell and Developmental Biology, 7, 215. https://doi.org/10.3389/fcell.2019.00215
• Shen, J.; Hung, M. C. (2015). Signaling-mediated regulation of microRNA processing. Cancer Research, 75(5), 783–791. https://doi.org/10.1158/0008-5472.Can-14-2568
• Song, P.; Yue, Q.; Chen, X.; Fu, Q.; Zhang, P.; & Zhou, R. (2023). Identification of ID1 and miR-150 interaction and effects on proliferation and apoptosis in ovine granulosa cells. Theriogenology, 212, 1–8. https://doi.org/10.1016/j.theriogenology.2023.08.029
• Xu, Z.; Xie, Y.; Zhou, C.; Hu, Q.; Gu, T.; Yang, J.; Zheng, E.; Huang, S.; Xu, Z.; Cai, G.; Liu, D.; Wu, Z.; & Hong, L. (2020). Expression pattern of seminal plasma extracellular vesicle small RNAs in boar semen. Frontiers in Veterinary Science, 7, 585276. https://doi.org/10.3389/fvets.2020.585276
• Zhang, S.; Yu, M.; Liu, C.; Wang, L.; Hu, Y.; Bai, Y.; Hua, J. (2012). MIR-34c regulates mouse embryonic stem cells differentiation into male germ-like cells through RARg. Cell Biochemistry and Function, 30(7), 623–632. https://doi.org/10.1002/cbf.2922