Expression of Markers of Semen Quality and Fertility in Stallions of the Crioulo Breed
DOI:
https://doi.org/10.22456/1679-9216.128772Abstract
Background: Mammalian spermatozoa contain a complex RNA population able to regulate spermatogenesis and play a role in the fertilization process. However, little is known about genetic factors and their role in fertility. Discovering novel molecular markers is necessary because semen quality parameters and routine exams still fail at detecting cases of subfertility. The objective of this study was to assess the relationship between the expression of the genes SPA17, TNF and TIMP2 by spermatozoa and semen quality, fertility, and motility parameters of sperm cells after thawing in stallions of the Crioulo breed.
Materials, Methods & Results: Analysis were performed on ejaculates from 40 stallions whose fertility was evaluated by checking their reproductive history, considering 30 inseminations for each animal. One mL of each ejaculate was reserved for fresh semen analysis, and the remaining volume was split into 2 samples; 1 of these samples was stored for gene expression analysis, and the other was cryopreserved. Sperm cell motility was analyzed using the computer-assisted semen analysis system. Sperm pathology analyses, hypoosmotic tests, and fluorescence tests were also performed. For gene expression analysis, mRNA was extracted for quantitation of expression of genes of interest by quantitative real-time polymerase chain reaction (qPCR). The results from qPCR assays were determined using an absolute standard curve [formula=10^(target ct - standard CT)/slope]. Statistical analysis was performed using Pearson correlation. Expression of SPA17 was positively correlated with functional integrity of the plasma membrane (r = 0.602; P = 0.004), structural integrity of the plasma membrane (r = 0.590; P = 0.004), conception rate (r = 0.454; P = 0,007), and total motility (r = 0.522; P = 0.001); it was negatively correlated with immobile sperm cells (r = -0.558; P = 0.006), and sperm cells with major defects (r = 0.4907; P = 0.012). Expression of TNF in sperm cells thawed after cryopreservation was positively correlated with curvilinear velocity (VCL) [r = 0.5147; P = 0.02], straight-line velocity (VSL) [r = 0.4714; P = 0.03], and average path velocity (VAP) [r = 0.4907; P = 0.02]. A positive correlation between TIMP2 expression and beat-cross frequency (BCF) was found [r = 0.408; P = 0.02].
Discussion: The positive correlations between SPA17 expression and the parameters total motility and conception rate may be related to the previously reported interaction of SPA17 with the zona pellucida, which facilitates penetration of the sperm cell into the oocyte. The positive correlations between expression of SPA17 and the parameters structural integrity of the plasma membrane and functionality of the plasma membrane are connected to characteristics important for viability of the sperm cell at the moment of conception, such as avoiding thermal shock and maintaining fluidity of the plasma membrane. Expression of TNF was positively correlated with sperm cell velocities after cryopreservation. TNF exerts a series of biological activities in different cell types. TNF regulates energy metabolism, especially in lipid homeostasis; it can be involved with plasma membrane phospholipid metabolism and reduce damage to the sperm cell during the cryopreservation process. We conclude that expression of SPA17 in equine sperm cells can be used as a biomarker for semen quality and fertility of stallions, while expression of TIMP2 can be used as a biomarker for beat-cross frequency. Expression of TNF was associated with better sperm cell survival rates after the cryopreservation process.
Keywords: stallion, fertility, concept rate, seminal quality, expression gene.
Título: Expressão de marcadores de qualidade seminal e fertilidade de garanhões da raça Crioula
Descritores: garanhão, fertilidade, taxa de prenhez, qualidade seminal, expressão gênica.
Downloads
References
Baumgart E., Lenk S.V., Loening S.A. & Jung K. 2002. Quantitative differences in matrix metalloproteinase (MMP)-2, but not in MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 or TIMP-2, in seminal plasma of normozoospermic and azoospermic patients. Human Reproduction. 17(11): 2919-2923. DOI: https://doi.org/10.1093/humrep/17.11.2919
Boerke A., Dieleman S.J. & Gadella B.M. 2007. A possible role for sperm RNA in early embryo development. Theriogenology. 68: S147-S155. DOI: https://doi.org/10.1016/j.theriogenology.2007.05.058
Brito L.F.C. 2007. Evaluation of Stallion Sperm Morphology. Clinical Techniques in Equine Practice. 6(4): 249-264. DOI: https://doi.org/10.1053/j.ctep.2007.09.004
Bueno V.L.C., Paul L.G., Araujo Bastos H.B., Larentis G.R., Mattos R.C. & Rechsteiner S.F. 2020. Características seminais pós-descongelamento em garanhões da raça crioula. Revista Brasileira de Reprodução Animal. 44(3): 100-107. DOI: https://doi.org/10.21451/1809-3000.RBRA2020.004
Carr D.W., Fujita A., Stentz C.L., Liberty G.A., Olson G.E. & Narumiya S. 2001. Identification of Sperm-specific Proteins That Interact with A-kinase Anchoring Proteins in a Manner Similar to the Type II Regulatory Subunit of PKA. Journal of Biological Chemistry. 276(20): 17332-17338. DOI: https://doi.org/10.1074/jbc.M011252200
CBRA. Colégio Brasileiro de Reprodução. 2013. Manual para exame andrológico e avaliação de sêmen animal. 3.ed. Belo Horizonte: Colégio Brasileiro de Reprodução Animal: 87p.
Chiriva-Internati M., Gagliano N., Donetti E., Costa F., Grizzi F., Franceschini B., Albani E., Levi-Setti P.E., Gioia M., Jenkins M., Cobos E. & Kast W.M. 2009. Sperm protein 17 is expressed in the sperm fibrous sheath. Journal of Translational Medicine. 7(1): 1-5. DOI: https://doi.org/10.1186/1479-5876-7-61
Chen X., Xun K., Chen L. & Wang Y. 2009. TNF-α, a potent lipid metabolism regulator. Cell Biochemistry and Function. 27(7): 407-416. DOI: https://doi.org/10.1002/cbf.1596
Colenbrander B., Gadella B. & Stout T. 2003. The Predictive Value of Semen Analysis in the Evaluation of Stallion Fertility. Reproduction in Domestic Animals. 38(4): 305-311. DOI: https://doi.org/10.1046/j.1439-0531.2003.00451.x
De S.K., Chen H.L., Pace J.L., Hunt J.S., Terranova P.F. & Enders G.C. 1993. Expression of tumor necrosis factor-alpha in mouse spermatogenic cells. Endocrinology. 133(1): 389-396. DOI: https://doi.org/10.1210/endo.133.1.8319585
Desai N., Mahfouz R., Sharma R., Gupta S. & Agarwal A. 2010. Reactive oxygen species levels are independent of sperm concentration, motility, and abstinence in a normal, healthy, proven fertile man: a longitudinal study. Fertility and Sterility. 94(4): 1541-1543. DOI: https://doi.org/10.1016/j.fertnstert.2009.12.041
Díaz‐Pérez E. & Meizel S. 1992. Importance of mammalian sperm metalloendoprotease activity during the acrosome reaction to subsequent sperm‐egg fusion: Inhibitor studies with human sperm and zona‐free hamster eggs. Molecular Reproduction and Development. 31(2): 122-130. DOI: https://doi.org/10.1002/mrd.1080310206
Garner D.L., Pinkel D., Johnson L.A. & Pace M.M. 1986. Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses. Biology of Reproduction. 34(1): 127-138. DOI: https://doi.org/10.1095/biolreprod34.1.127
Gil M.C., García-Herreros M., Barón F.J., Aparicio I.M., Santos A.J. & García-Marín L.J. 2009. Morphometry of porcine spermatozoa and its functional significance in relation with the motility parameters in fresh semen. Theriogenology. 71(2): 254-263. DOI: https://doi.org/10.1016/j.theriogenology.2008.07.007
Grizzi F., Chiriva–Internati M., Franceschini B., Hermonat P.L., Soda G., Lim S. H. & Dioguardi N. 2003. Immunolocalization of Sperm Protein 17 in Human Testis and Ejaculated Spermatozoa. Journal of Histochemistry & Cytochemistry. 51(9): 1245-1248. DOI: https://doi.org/10.1177/002215540305100916
Holt W.V. 2000. Basic aspects of frozen storage of semen. Animal Reproduction Science. 62(1-3): 3-22. DOI: https://doi.org/10.1016/S0378-4320(00)00152-4
Intasqui P., Agarwal A., Sharma R., Samanta L. & Bertolla R.P. 2017. Towards the identification of reliable sperm biomarkers for male infertility: A sperm proteomic approach. Andrologia. 50(3): e12919. DOI: 10.1111/and.12919 DOI: https://doi.org/10.1111/and.12919
Kareskoski A.M., Palviainen M., Johannisson A. & Katila T. 2020. Upregulation of CRISP‐3 and kallikrein in stallion seminal plasma is associated with poor tolerance of cooled storage. Reproduction in Domestic Animals. 55(4): 496-502. DOI: https://doi.org/10.1111/rda.13643
Kasimanickam V., Kasimanickam R., Arangasamy A., Saberivand A., Stevenson J.S. & Kastelic J.P. 2012. Association between mRNA abundance of functional sperm function proteins and fertility of Holstein bulls. Theriogenology. 78(9): 2007-2019.e2. DOI: https://doi.org/10.1016/j.theriogenology.2012.07.016
Lagares M. A., Petzoldt R., Sieme H. & Klung E. 1998. Preservação do sêmen fresco equino: avaliação da integridade da membrana espermática sob condições hiposmóticas. Arquivos da Faculdade de Veterinária da UFRGS. 26(1): 29-42.
Lea I.A., Widgren E.E. & O’Rand M.G. 2004. Association of sperm protein 17 with A-kinase anchoring protein 3 in flagella. Reproductive Biology and Endocrinology. 2(1): 57. DOI: 10.1186/1477-7827-2-57 DOI: https://doi.org/10.1186/1477-7827-2-57
McCauley T.C., Zhang H.M., Bellin M.E. & Ax R.L. 2001. Identification of a heparin‐binding protein in bovine seminal fluid as tissue inhibitor of metalloproteinases‐2. Molecular Reproduction and Development. 58(3): 336-341. DOI: https://doi.org/10.1002/1098-2795(200103)58:3<336::AID-MRD12>3.0.CO;2-Z
Morris L.H.A. & Allen W.R. 2002. Reproductive efficiency of intensively managed Thoroughbred mares in Newmarket. Equine Veterinary Journal. 34(1): 51-60. DOI: https://doi.org/10.2746/042516402776181222
Moura A.A., Koc H., Chapman D.A. & Killian G.J. 2006. Identification of proteins in the accessory sex gland fluid associated with fertility indexes of dairy bulls: a proteomic approach. Journal of andrology. 27(2): 201-211. DOI: https://doi.org/10.2164/jandrol.05089
Ostermeier G.C., Dix D.J., Miller D., Khatri P. & Krawetz S. A. 2002. Spermatozoal RNA profiles of normal fertile men. The Lancet. 360(9335): 772-777. DOI: https://doi.org/10.1016/S0140-6736(02)09899-9
Panner Selvam M.K., Agarwal A., Pushparaj P.N., Baskaran S. & Bendou H. 2019. Sperm Proteome Analysis and Identification of Fertility-Associated Biomarkers in Unexplained Male Infertility. Genes. 10(7): 522. DOI: 10.3390/genes10070522 DOI: https://doi.org/10.3390/genes10070522
Pascarelli N.A., Fioravanti A., Moretti E., Guidelli G.M., Mazzi L. & Collodel G. 2017. The effects in vitro of TNF-α and its antagonist “etanercept” on ejaculated human sperm. Reproduction, Fertility and Development. 29(6): 1169-1177. DOI: https://doi.org/10.1071/RD16090
Papa F.O., Melo C.M., Fioratti E.G., Dell’Aqua Jr. J.A., Zahn F.S. & Alvarenga M.A. 2008. Freezing of stallion epididymal sperm. Animal Reproduction Science. 107(3-4): 293-301. DOI: https://doi.org/10.1016/j.anireprosci.2008.05.003
Richardson R.T., Yamasaki N. & O’Rand M.G. 1994. Sequence of a Rabbit Sperm Zona Pellucida Binding Protein and Localization during the Acrosome Reaction. Developmental Biology. 165(2): 688-701. DOI: https://doi.org/10.1006/dbio.1994.1285
Suliman Y., Becker F. & Wimmers K. 2018. Implication of transcriptome profiling of spermatozoa for stallion fertility. Reproduction, Fertility and Development. 30(8): 1087-1098. DOI: https://doi.org/10.1071/RD17188
Verstegen J., Iguer-Ouada M. & Onclin K. 2002. Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology. 57(1): 149-179. DOI: https://doi.org/10.1016/S0093-691X(01)00664-1
Viana A.G.A., Martins A.M.A., Pontes A.H., Fontes W., Castro M.S., Ricart C.A. O., Sousa MV., Kaya A., Topper E., Memili E. & Moura A.A. 2018. Proteomic landscape of seminal plasma associated with dairy bull fertility. Scientific Reports. 8(1): 1-13. DOI: https://doi.org/10.1038/s41598-018-34152-w
Additional Files
Published
How to Cite
Issue
Section
License
Copyright (c) 2023 Isabela Trevisan Roese, Verônica La Cruz Bueno, Henrique Boll de Araujo Bastos, Rodrigo Costa Mattos, Sandra Fiala Rechsteiner
This work is licensed under a Creative Commons Attribution 4.0 International License.
This journal provides open access to all of its content on the principle that making research freely available to the public supports a greater global exchange of knowledge. Such access is associated with increased readership and increased citation of an author's work. For more information on this approach, see the Public Knowledge Project and Directory of Open Access Journals.
We define open access journals as journals that use a funding model that does not charge readers or their institutions for access. From the BOAI definition of "open access" we take the right of users to "read, download, copy, distribute, print, search, or link to the full texts of these articles" as mandatory for a journal to be included in the directory.
La Red y Portal Iberoamericano de Revistas Científicas de Veterinaria de Libre Acceso reúne a las principales publicaciones científicas editadas en España, Portugal, Latino América y otros países del ámbito latino
