Partenogenetik Aktivasyonun Vitrifiye Köpek Oositleri Üzerine Etkisi

Year 2023, Volume: 42 Issue: 2, 70 - 75, 31.12.2023

Rabia Gözde Özalp , Burcu Üstüner , Özge Bari , Ahmet Aktar , Ahmet Yavuz , Hakan Sağırkaya

https://doi.org/10.30782/jrvm.1326864

Abstract

Pet hayvanlarında biyoteknolojik çalışmalar son yıllarda hız kazanmaya başlamıştır. Köpeklerde başarısız yardımcı üreme teknikleriyle ilgili oluşan
sorular, muhtemelen köpek türlerinin reproduktif fizyolojisine ait yetersiz bilgiden kaynaklanmaktadır. Fakat diğer taraftan pet biyolojisindeki uygulamalar,
insan hastalıkları için model oluşturmaktadır. Bunun ötesinde gamet kriyopreservasyonunun gelişmesi, nesli tükenmekte olan türlerin
korunması ve genetik banka oluşturulması için önemlidir. Bu çalışmada, köpek oositlerindeki düşük maturasyon oranlarına rağmen, partenogenetik
aktivasyonun etkileri vitrifiye oositlerde test edildi.
Köpek oositleri, Yıldırım Belediyesi Sokak Hayvanları Bakım ve Rehabilitasyon merkezinden alınan, 20 adet sağlıklı köpekten toplandı. Ovaryumların
tekrarlı parçalanmasından sonra, seçilen COCs (kumulus oosit kompleksleri), 5% CO2 inkübatörde, mineral yağla kaplanmış 500 μl TCM-199
içeren dört-gözlü petrilerde, 39°C’de, 72 saat boyunca maturasyona bırakıldı. Maturasyondan sonra oositler, 0%, 10%, 20% etilen glikol içeren 50
ml PBl içinde sırasıyla, 10, 10 dakika ve 30 saniye muamele edildi. Oositler, 30 μl VS3 içeren kriyoviallere yerleştirilerek sıvı nitrojende donduruldu.
Bu grubun oositleri (n=257) ‘vitrifiye oosit-VO’ olarak gruplandı. Çözdürme sonrasında, oositler ionomisinle 5 dakika ve sikloheksimid ile 3 saat
muamele ederek partenogenetik aktivasyona bırakıldı. Sonrasında oositler 72 saat kültüre edilerek nükleer maturasyon değerlendirildi. Kontrol
grubu olarak kullanılan oositler (n=257), ‘non vitrifiye oosit-FO’ olarak gruplandırıldı. Maturasyondan sonra, oositler direkt olarak ionomisin ve sikloheksimid
ile muamele edilerek aktivasyona bırakıldı ve 72 saat kültüre edildi. Tüm oositler Hoechst33342 ile 30 dakika boyandıktan sonra nükleer
maturasyon oranları mikroskopta değerlendirildi.
Maturasyon oranları (MI+MII) gruplar arasında istatistiksel olarak anlamlı bulunmadı. (p>0,05). Gruplar arasında GV, GVBD, MI, ve MII oranlarında
da istatistiksel fark bulunmadı (p>0,05).
Maturasyon sonrasında, vitrifiye köpek oositlerinde partenogenetik aktivasyona bağlı nükleer değerlendirmeye çalışması bulunmamaktadır. Fakat
bu uygulamada elde edilen düşük maturasyon oranlarının, ileri moleküler çalışmalarla açıklanması gerektiği kanısındayız.

Keywords

Köpek oositleri, vitrifikasyon, in vitro maturasyon, partenogenetik aktivasyon

Effect of Parthenogenetic Activation on Vitrified Canine Oocytes

Abstract

Biotechnological research in pet animals has been run up in recent years. Raised questions about unsuccessful assisted reproductive technologies
in canids are probably related to poor information in the reproductive physiology of canids. But on the other hand, applications in pet biology are
accepted as a model for human diseases. Apart from this, the development of gamete cryopreservation is an important tool for genetic banking and
conservation of endangered species. In spite of the low maturation rates of canine oocytes, the results of parthenogenetic activation were tried to
maturated and vitrified-warmed canine oocytes in this study.
Oocytes were collected from 20 healthy bitches at Yıldırım Municipality Stray Animals Sterilization and Rehabilitation Center. After slicing of ovaries,
selected (COCs) cumulus-oocyte complexes were maturated for 72 h at 39°C in four-well petri dishes containing 500 μl TCM-199 under mineral
oil in a 5%CO2 incubator. After maturation, oocytes were exposed to 50 ml PBl containing 0%, 10%, 20% ethylene glycol for 10, 10 minutes, and
30 seconds, respectively. They were vitrified in cryovials containing 30 μl VS3 in liquid nitrogen. The oocytes in this group (n=257) were grouped as
‘vitrified oocyte-VO’. After warming, the oocytes were parthenogenetically activated with ionomycin for 5 minutes and followed by cycloheximide
for 3 h. Oocytes were then cultured for 72 h and assessed for nuclear maturation. The oocytes (n=257), grouped as ‘non-vitrified oocytes-FO’ were
used as the control group. After maturation, oocytes were directly incubated with ionomycin and cycloheximide for parthenogenetic activation and
cultured for 72 h. All oocytes were stained with Hoechst33342 for 30 min and nuclear maturation rates were assessed using a microscope.
Maturation rates (MI+MII) between groups had not been found statistically different (p>0,05). GV, GVBD, MI, and MII rates were also not statistically
different between the two groups (p>0,05).
To our knowledge, there is no information available about the influence of parthenogenetic activation on nuclear maturation after the vitrification of
maturated canine oocytes. However, low maturation rates should be clarified by further molecular studies.

Keywords

ine oocytes, vitrification, in vitro maturation, parthenogenetic activation

References

• Kaynaklar 1. Mapletoft RJ, Hasler JF. Assisted reproductive technologies in cattle: a review. Rev Sci Tech. 2005; 24:393-403.
• 2. Amiridis GS, Cseh S. Assisted reproductive technologies in the reproductive management of small ruminants. Anim Reprod Sci. 2012; 130:152-161.
• 3. Dyck MK, Zhou C, Tsoi S, Grant J, Dixon WT, Foxcroft GR. Reproductive technologies and the porcine embryonic transcriptome. Anim Reprod Sci. 2014; 149:11-18.
• 4. Hewitson L. Primate models for assisted reproductive technologies. Reproduction. 2004; 128: 293–299.
• 5. Song HJ, Kang EJ, Kim MJ, Ock SA, Jeon BG, Lee SL, Rho GJ. Influence of parthenogenetic activation on nuclear maturation of canine oocytes. J Vet Med Sci. 2010; 72:887-892, 2010.
• 6. No J, Zhao M, Lee S, Ock SA, Nam Y, Hur TY. Enhanced in vitro maturation of canine oocytes by oviduct epithelial cell co-culture. Theriogenology. 2018; 105:66-74.
• 7. Renton JP, Boyd JS, Eckersall PD, Ferguson JM, Harvey MJ, Mullaney J, Perry B. Ovulation, fertilization and early embryonic development in the bitch (Canis familiaris). J Reprod Fertil. 1991; 93:221–231.
• 8. Songsasen N, Wildt DE. Oocyte biology and challenges in developing in vitro maturation systems in the domestic dog. Anim Reprod Sci. 2007; 98:2–22.
• 9. De los Reyes M, Palomino J, Parraguez VH, Hidalgo M, Saffie P. Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation. Theriogenology. 2011; 75:346–353.
• 10. Concannon PW. Reproductive cycles of the domestic bitch. Anim Reprod Sci. 2011; 200–210.
• 11. Turathum B, Saikhun K, Sangsuwan P, Kitiyanant Y. Effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes. Reproductive Biology and Endocrinology. 2010; 8:70.
• 12. Luvoni GC. Cryobanking of Oocytes and Ovarian Tissue in Cats and Dogs World Small Animal Veterinary Association World Congress Proceedings, New Zealand, 2013.
• 13. Fujihara M, Kaneko T, Inoue-Murayama M. Vitrification of canine ovarian tissues with polyvinylpyrrolidone preserves the survival and developmental capacity of primordial follicles. Scientific Reports. 2019; 9:3970
• 14. Kishimoto, T. Cell-cycle control during meiotic maturation. Curr Opin Cell Biol. 2003; 15: 654–663.
• 15. Kishimoto, T., Kuriyama, R., Kondo, H. and Kanatani, H. Generality of the action of various maturation-promoting factors. Exp Cell Res 1982; 137: 121–126.
• 16. Saint-Dizier M, Reynaud K, Chastant-Maillard S. Chromatin, microtubules, and kinases activities during meiotic resumption in bitch oocytes. Mol Reprod Dev 68(2):205-212, 2004.
• 17. Brunet S, Maro B. Cytoskeleton and cell cycle control during meiotic maturation of the mouse oocyte: integrating time and space. Reproduction. 2005; 130: 801–811.
• 18. Lee SR, Kim JW, Kim MO, Kim SH, Yoo DH, Shin MJ, Lee S, Park YS, Park YB, Ha JH, Ryoo ZY. The parthenogenetic activation of canine oocytes with Ca-EDTA by various culture periods and concentrations. Theriogenology. 2007; 67: 698–703.
• 19. Meo SC, Leal CL, Garcia JM. Activation and early parthenogenesis of bovine oocytes treated with ethanol and strontium. Anim Reprod Sci. 2004; 81:35–46.
• 20. Lee SR, Kim JW, Kim BS, Yoo DH, Park YS, Lee TH, Ha JH, Hyun BH, Ryoo ZY. Parthenogenetic induction of canine oocytes by electrical stimulation and CaEDTA. Reprod Domest Anim. 2009; 44: 740-744.
• 21. Luvoni GC, Chigioni S, Allievi E, Macis D. Factors involved in vivo and in vitro maturation of canine oocytes. Theriogenology. 2005; 63: 41–59.
• 22. Funahashi H, Cantley TC, Stumpf TT, Terlouw SL, Day BN. In vitro development of in vitro matured porcine oocytes following chemical activation or in vitro fertilization. Biol Reprod. 1994; 50: 1072–1077.
• 23. Fulka J Jr, Motlík J, Fulka J, Jílek F. Effect of cycloheximide on nuclear maturation of pig and mouse oocyte. J Reprod Fertil. 1986; 77: 281–285.
• 24. Fulka JJ, Leibfried-Rutledge ML, First NL. Effect of 6-dimethyl-aminopurine on germinal vesicle breakdown of bovine oocytes. Mol Reprod Dev. 1991: 29: 379–384.
• 25. Yang X, Presicce GA, Moraghan L, Jiang SE, Foote RH. Synergistic effect of ethanol and cycloheximide on activation of freshly matured bovine oocytes. Theriogenology. 1994; 41: 395–403.
• 26. Watanabe N, Vande Woude GF, Ikawa Y, Sagata N. Specific proteolysis of the c-mos proto-oncogene product by calpain upon fertilization of Xenopus eggs. Nature. 1989; 342: 505–511.
• 27. Saeki K, Nagao Y, Kishi M, Nagai M, Iritani A. Timing of completion of the first meiotic division in bovine oocytes after maintenance of meiotic arrest with cycloheximide and their subsequent development. J Vet Med Sci. 1998; 60: 523– 526.
• 28. Luciano AM, Modina S, Vassena R, Milanesi E, Lauria A, Gandolfi F. Role of intracellular cyclic adenosine 3’-5’-monophosphate concentration and oocyte-cumulus cells cummunications on the acquisition of the developmental competence during in vitro maturation of bovine oocyte. Biol Reprod. 2004; 70: 465-472.
• 29. Sirard MA, First NL. In vitro inhibition of oocyte nuclear maturation in the bovine. Biol Reprod. 1998; 39: 229-234.
• 30. Smitz JE, Thompson JG, Gilchrist RB. The promise of in vitro maturation in assisted reproduction and fertility preservation. Semin Reprod Med. 2011; 29: 24-37.
• 31. Sutovský P, Fléchon JE, Fléchon B, Motlik J, Peynot N, Chesné P, Heyman Y. Dynamic changes of gap junctions and cytoskeleton during in vitro culture of cattle oocyte cumulus complexes. Biol Reprod. 1993; 49: 1277–1287.
• 32. Leibo SP, Brandley L. Comparative cryobiology of mammalian spermatozoa. In: C Gagnon, ed, The Male Gamet. Cache River Press, St Louis; 1999: 502-515.
• 33. Bucak MN, Tekin N. Kryoprotektanlar ve gamet hücrelerinin dondurulmasında kryoprotektif etki. Ankara Üniv Vet Fak Derg. 2007; 54: 67-72.
• 34. Abe Y, Suwa Y, Yanagimoto-Ueta Y, Suzuki H. Preimplantation development in Labrador retrievers. J Reprod Dev. 2008a; 54: 135–137.
• 35. Abe Y, Lee DS, Kim SK, Suzuki H. Vitrification of canine oocytes. J Mamm Ova Res. 2008b; 25: 32-36.
• 36. Abe Y, Asano T, Ali M, Suzuki H. Vitrification of canine cumulus–oocyte complexes in DAP213 with a cryotop holder. Reprod Med Biol. 2010; 9: 115–120.
• 37. Kasai M. Advaces in the cryopreservation of mammalian oocytes and embryos: development of ultrarapid vitrification. Reprod Med Biol. 2002; 1: 1-9.
• 38. Shaw JM, Oranratnachai A, Trounson AO. Fundamental cryobiology of mammalian ooctes and ovarian tissue. Theriogenology. 2000; 53: 59-72.
• 39. Merlo B, Iacono E, Regazzini M, Zambelli D. Cat blastocysts produced in vitro from oocytes vitrified using the cryoloop technique and cryopreserved electroejaculated semen. Theriogenology. 2008; 70: 126–130.
• 40. Murakami M, Otoi T, Karja NW, Wongsrikeao P, Agung B, Suzuki T. Blastocysts derived from in vitro-fertilized cat oocytes after vitrification and dilution with sucrose. Cryobiology. 2004; 48: 341–348.