Koç Sperma Akrozom Bütünlüğünün Seminal Plazma Homosistein ve Nesfatin-1 Düzeyleri ile İlişkili Olarak İncelenmesi

Öz

Bu çalışmada homosistein, nesfatin-1 düzeyleri ile akrozom bütünlüğü arasındaki ilişkinin araştırılması amaçlanmıştır. Ejakülatlar, suni vajen yöntemiyle altı yetişkin koçtan alındı ve toplanan ejakülatlar beş gruba bölünerek 37 °C'de sulandırıldı. Sperma sulandırılmasında kolesterol yüklü siklodekstrin (CLC) 1.5 ve 2.5 mg, kolesterol yüklü 7-dehidrokolesterol (7-DCLC) 1.5 ve 2.5 mg içeren veya katkı maddesi içermeyen (kontrol) sulandırıcılar kullanıldı. Tüm gruplardaki sperma örnekleri 5 °C' de soğutulduktan sonra sıvı nitrojen buharında (-110~-120° C) donduruldu. Sıvı nitrojen içinde saklanan sperma örnekleri incelenmek için 38 °C ve 30 saniyede çözdürüldü. Akrozom bütünlüğü, FITC-PNA boyama ile incelendi. Homosistein (HCY) ve nesfatin-1 testleri ELISA yöntemi ile yapıldı. CLC 1.5 ve 2.5 mg gruplarında homosistein seviyeleri kontrol grubuna (1.36±0.9) göre daha düşük bulundu (0.67±0.11, 0.61±0.26) (p <0.05). Nesfatin-1 düzeylerinde gruplar arasında istatistiksel olarak fark gözlenmedi (p> 0.05). 2.5 mg CLC'de akrozom bütünlüğü bozulmamış sperma hücrelerinin oranları (65.88±2.84) kontrol grubuna (52.38±2.97) göre daha yüksek bulundu (p<0.05). Sperma akrozom bütünlüğü, çözüm sonu koç spermasında HCY seviyesi (r =-,630) ile negatif, nesfatin-1 seviyeleri (r =, 460) ile pozitif korelasyon gösterdi. Sonuç olarak, CLC'nin akrozom bütünlüğü üzerinde kriyoprotektif bir etki gösterdiği ve HCY seviyesi üzerinde faydalı olduğu belirlenmiştir.

Anahtar Kelimeler

• Akrozom bütünlüğü
• Siklodekstrin
• 7-dehidrokolesterol
• Homosistein
• Nesfatin-1

Investigation of Ram Sperm Acrosome Integrity in Relation with Seminal Plasma Homocysteine and Nesfatin-1 Levels

Öz

This study aimed to investigate the relationship between homocysteine, nesfatin-1 levels with acrosome integrity. Ejaculates were collected from six adult rams via artificial vagina, divided into five groups and diluted at 37 °C. The extenders, which contained cholesterol loaded cyclodextrin (CLC) 1.5 and 2.5 mg, cholesterol loaded 7-dehydrocholesterol (7-DCLC) 1.5 and 2.5 mg or no additive (control), were used for sperm dilution. Semen samples of all groups were cooled at 5°C and then frozen in liquid nitrogen vapor (-110~-120°C). Semen samples stored in liquid nitrogen then were thawed at 38°C for 30 seconds prior to examination. Acrosome integrity was examined with FITC-PNA staining. Homocysteine (HCY) and nesfatin-1 assays were performed with ELISA method. In the CLC 1.5 and 2.5 mg groups homocysteine levels were lower (0.67±0.11;0.61±0.26) compared to control group (1.36±0.9) (p<0.05). No statistical differences were observed between groups in nesfatin-1 levels (p>0.05). In CLC 2.5 mg, the values of spermatozoa with intact acrosome membrane (65.88±2.84) were higher (p<0.05) than in control group (52.38±2.97). Sperm acrosome integrity was negatively correlated with HCY level (r =-,630) and positive correlated with nesfatin-1 levels (r =,460) in thawed ram sperm. In conclusion, CLC has a cryoprotective effect on acrosome integrity and found to have beneficial effects on HCY level.

Anahtar Kelimeler

• Acrosome integrity
• Cyclodextrin
• 7-dehydrocholesterol
• Homocysteine
• Nesfatin-1

Kaynakça

1. Aksoy M, Akman O, Lehimcioğlu NC, Erdem H. Cholesterol-loaded cyclodextrin enhances osmotic tolerance and inhibits the acrosome reaction in rabbit spermatozoa. Anim Reprod Sci. 2010; 120(1-4): 166-172.
2. Amorim EAM, Graham JK, Spizziri B, Meyers M, Torres CA. Effect of cholesterol or cholesteryl conjugates on the cryo survival of bull sperm. Cryobiology. 2009; 58: 210-214.
3. Blommaert D, Franck T, Donnay I, Lejeune JP, Detilleux J, Serteyn D. Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm. Cryobiology. 2016; 72: 27-32.
4. Forges T, Monnier-Barbarino P, Alberto, JM, Gueant-Rodriguez RM, Daval JL, Gueant JL. Impact of folate and homocysteine metabolism on human reproductive health. Hum Reprod Update. 2007; 13(3): 225-238.
5. Gao X, Zhang K, Song M, Li X, Luo L, Tian Y, Zhang Y, Li Y, Zhang X, Ling Y, Fang F, Liu Y. Role of nesfatin-1 in the reproductive axis of male rat. Sci Rep. 2016; 6: e32877.
6. García-Galiano D, Pineda R, Ilhan T, Castellano JM, Ruiz-Pino F, Sánchez-Garrido M A Vazquez MJ, Sangiao-Alvarellos S, Romero-Ruiz A, Pinilla L, Diéguez C, Gaytán F, Tena-Sempere M. Cellular distribution, regulated expression, and functional role of the anorexigenic peptide, NUCB2/nesfatin-1, in the testis. Endocrinology. 2012;153(4): 1959-1971.
7. Glazar AI, Mullen SF, Liu J, Benson JD, Critser JK, Squires EL, Graham JK. Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol. Cryobiology. 2009; 59: 201–206.
8. Inanc ME, Uysal O, Ata A. Cryopreservation and evaluation of Akkaraman ram semen with 7-dehydrocholesterol. Vet J Ankara Univ. 2018; 65: 187-192.

9. Kelly TL, Neaga OR, Schwahn BC, Rozen R, Trasler JM. Infertility in 5,10- methylenetetrahydrofolate reductase (MTHFR)-deficient male mice is partially alleviated by lifetime dietary betaine supplementation. Biol Reprod. 2005; 72: 667–677.
10. Khan J, Tahir MZ, Khalid A, Sattar A and Ahmad N. Effect of cholesterol-loaded cyclodextrins on cryo survival of dog spermatozoa. Reprod Domest Anim. 2017; 52: 265–268.
11. Kim J, Yang H. Nesfatin-1 as a new potent regulator in reproductive system. Dev Reprod 2012; 16(4): 253-264.
12. Kralikova M, Crha I, Huser M, Melounova J, Zakova J, Matejovicova M, Ventruba P. The intracellular concentration of homocysteine and related thiols is negatively correlated to sperm quality after highly effective method of sperm lysis. Andrologia. 2017; 49(7): e12702.
13. López-Revuelta A, Sánchez-Gallego JI, García-Montero AC, Hernández-Hernández A, Sánchez-Yagüe J, Llanillo M. Membrane cholesterol in the regulation of aminophospholipid asymmetry and phagocytosis in oxidized erythrocytes. Free Radic Biol Med. 2007; 42(7): 1106-1118.
14. Maejima Y, Sedbazar U, Suyama S, Kohno D, Onaka T, Takano E, Yoshida N, Koike M, Uchiyama Y, Fujiwara K, Yashiro T, Horvath TL, Marcelo OD, Tanaka S, Dezaki K, Oh-I S, Hashimoto K, Shimizu H, Yada T. Nesfatin-1-regulated oxytocinergic signaling in the paraventricular nucleus causes anorexia through a leptin-independent melanocortin pathway. Cell Metab. 2009; 10(5): 355–365.
15. Maxwell WMC, Watson PF. Recent Progress in The Preservation of Ram Semen. Anim Reprod Sci. 1996; 42: 55-65.
16. Mocé E, Blanch E, Tomás C, Graham JK. Use of cholesterol in sperm cryopreservation: present moment and perspectives to future. Reprod Domest Anim. 2010; 45: 57-66.
17. Moore AI, Squires EL, Graham JK. Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival. Cryobiology. 2005; 51: 241-249.
18. Moraes EA, Matos WCG, Graham JK, Ferrari Jr WD. Cholestanol-loaded-cyclodextrin improves the quality of stallion spermatozoa after cryopreservation. Anim Reprod Sci. 2015; 158: 19-24.
19. Nagy S, Jansen J, Topper EK, Gadella BM. A triple-stain flow cytometric method to assess plasma and acrosome membrane integrity of cryopreserved bovine sperm immediately after thawing in presence of egg-yolk particles. Biol Reprod. 2003; 68: 1828-1835.
20. Naseer Z, Ahmad E, Aksoy M, Küçük N, Serin İ, Ceylan A, Boyacıoğlu M, Kum C. Protective effect of cholesterol-loaded cyclodextrin pretreatment against hydrogen peroxide-induced oxidative damage in ram sperm. Cryobiology. 2015; 71: 18–23.
21. Pinho RO, Lima DM, Shiomi HH, Siqueira JB, Silveira CO, Faria VR, Lopes PS, Guimarães SE, Guimarães JD. Effect of cyclodextrin-loaded cholesterol conjugates on plasma membrane viability of Piau swine breed frozen/thawed spermatozoa. Cryobiology. 2016; 73: 1-6.
22. Purdy P, Graham J. Effect of cholesterol-loaded cyclodextrin on the cryo survival of bull sperm. Cryobiology. 2004; 48: 36–45.
23. Ranjan A, Choubey M, Yada T, Krishna A. Direct effects of neuropeptide nesfatin-1 on testicular spermatogenesis and steroidogenesis of the adult mice. Gen Comp Endocrinol. 2019; 271: 49-60.
24. Renard P, Grizard GG, Sion JF, Boucher B, Lannou DDL. Improvement of motility and fertilization potential of post thawhuman sperm using glutamine. Cryobiology. 1996; 33: 311–319.
25. Salamon S, Maxwell WMC. Storage of ram semen. Anim Reprod Sci. 2000; 62(1-3): 77-111.
26. Sibrian-Vazquez M, Escobedo JO, Lim S, Samoei GK, Strongin RM. Homocystamides promote free-radical and oxidative damage to proteins. Proc Natl Acad Sci. (2010); 107(2): 551-554.
27. Tamer SA, Yildirim A, Köroğlu MK, Çevik Ö, Ercan F, Yeğen BÇ. Nesfatin-1 ameliorates testicular injury and supports gonadal function in rats induced with testis torsion. Peptides. 2018; 107: 1-9.
28. Tomás C, Blanch E, Hernandez M, Gil MA, Roca J, Vazquez JM, Martinez EA, Mocé E Treating boar sperm with cholesterol-loaded cyclodextrins widens the sperm osmotic tolerance limits and enhances the in vitro sperm fertilizing ability. Anim Reprod Sci. 2011; 129: 209-220.
29. Trimeche A, Yvon JM, Vidament M, Palmer E, Magistrini M. Effects of glutamine, proline, histidine and betaine on post-thaw motility of stallion spermatozoa. Theriogenology. 1999; 52: 181-191.
30. Voutilainen S, Morrow JD, Roberts LJ, Alfthan G, Alho H, Nyyssonen K, Salonen JT. Enhanced in vivo lipid peroxidation at elevated plasma total homocysteine levels. Arterioscler Thromb Vasc Biol. 1999; 19: 1263-1266.
31. Wallock LM, Tamura T, Mayr CA, Johnston KE, Ames BN, Jacob RA. Low seminal plasma folate concentrations are associated with low sperm density and count in male smokers and nonsmokers. Fertil Steril. 2001;75(2):252-259.
32. Watson PF. The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci. 2000; 60:481-492.
33. Yeagle PL. Cholesterol and the cell membrane. Biochim Biophys Acta Rev Biomembranes. 1985; 822: 267-287.
34. Yurdakök B, Tekin K, Daskin A, Filazi A. Effect of polychlorinated biphenyls 28,30 and 118 on bovine spermatozoa in vitro. Reprod Domest Anim. 2015; 50: 41-47.
35. Zidovetzki R, Levitan I. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. Bioch Bioph Acta. 2007; 1768(6): 1311-1324.