Intracytoplasmic Sperm Injection (ICSI) in B6D2F1 and CB6F1 Strains Mice Using Cauda Epididymal Spermatozoa

Abstract

Objective: Reproductive biotechnology studies focus on the long-term storage of embryos (cryopreservation), embryo cultures, ge-nome editing of embryos and embryo transfer. Micromanipulation techniques in reproduction biotechnologies have an important role, especially in studies investigating assisted reproductive tech-nology in laboratory animals. The aim of the present study was to investigate the effect of epididymal spermatozoa injected to oo-cyte by intracytoplasmic sperm injection (ICSI) in different mice strains. In this study, we evaluated the in vitro development of post-ICSI derived embryos using cauda epididymal sperm.

Material and Method: Female mice (8-10 weeks) were superovulated using pregnant mare serum gonadotropin/human chorionic gonadotropin (PMSG/hCG) and ~14h post hCG, the mice were sacrificed, and the oocytes were collected. Spermatozoa from the cau-da epididymal of a 12-week-old were used on the same strain for ICSI and the in vitro developmental potential was evaluated. Finally, the embryos were cultured for 120 hours at 5% CO2 with 37°C.

Results: The results showed that the two-cell embryo of the B6D2F1 strain (79.31%) was significantly higher than the CB6F1 (56.26%) (p<0.05). While the blastocyst rate was comparable between both the B6D2F1 strain (68.75%) and CB6F1 strain (69.57%) (p>0.05).

Conclusion: ICSI using cauda epididymal sperm is a suitable ap-plication for in vitro embryo development in B6D2F1 and CB6F1 strains. Finally, ICSI success of the B6D2F1 mice strains was found to be higher than CB6F1 mice strains.

Keywords

• Mouse
• oocyte
• ICSI
• development

B6D2F1 ve CB6F1 Irk Farelerde Kauda Epididimal Spermatozoa Kullanılarak İntrastoplazmik Sperm Enjeksiyonu (ICSI)

Abstract

Amaç: Üreme biyoteknolojisi alanındaki çalışmalar; embriyoların uzun süre saklanması (kriyoprezervasyon), embriyo kültürü, embriyoların genom düzenlenmesi araştırmaları ve embriyo transferi gibi konular üzerinde yoğunlaşmaktadır. Üreme biyoteknolojilerinde mikromanipülasyon teknikleri, özellikle laboratuvar hayvanlarında yardımcı üreme teknolojisinin araştırıldığı çalışmalarda önemli bir yere sahiptir. Bu çalışmanın amacı, farklı fare ırklarında intrastoplazmik sperm enjeksiyonu (ICSI) ile epididimal spermatozoanın oosite enjeksiyonunu araştırmaktır. Bu çalışmada, kauda epididimal fare spermi kullanılarak yapılan ICSI uygulaması sonrasında elde edilen embriyoların in vitro gelişimi değerlendirilmiştir.

Gereç ve Yöntem: Dişi fareler (8-10 hafta), gebe kısrak serum gonadotropini/insan koryonik gonadotropini (PMSG/hCG) kullanılarak süperovüle edilmiş, hCG'den ~14 saat sonra fareler sakrifiye edilerek oositler toplanmıştır. 12 haftalık erkek farenin kauda epididiminden alınan spermatozoa, aynı ırk oositle ICSI için kullanılmış ve in vitro gelişim potansiyeli değerlendirilmiştir. Son olarak tüm embriyolar 120 saat süre ile %5 CO2 ve 37°C’de kültüre edilmiştir.

Bulgular: Sonuçlar, B6D2F1 (%79,31) ırkının 2 hücreli embriyo gelişiminin CB6F1 (%56,26) ırklı farelerdeki 2 hücreli embriyo gelişiminden önemli ölçüde yüksek olduğunu göstermiştir (p<0,05). Blastosist oranı B6D2F1 (%68,75) ve CB6F1 (%69,57) ırkları arasında karşılaştırılmıştır (p>0,05).

Sonuç: B6D2F1 ve CB6F1 fare ırklarında, kauda epididimal sper-ma kullanılarak yapılan ICSI in vitro embriyo gelişimi için uygun bir yöntemdir. Sonuç olarak, B6D2F1 farelerde ICSI’nın başarısı, CB6F1 ırk farelere göre daha yüksektir.

Keywords

• Fare
• oosit
• ICSI
• gelişim

References

1. 1. Andersen ML, Winter LM. Animal models in biological and bio-medical research-experimental and ethical concerns. An Acad Bras Cienc 2019; 91. [CrossRef] google scholar
2. 2. Yanagimachi R. Intracytoplasmic injection of spermatozoa and spermatogenic cells: its biology and applications in humans and animals. Reprod Biomed Online 2005; 10(2): 247-88. [CrossRef] google scholar
3. 3. Li MW, Willis BJ, Griffey SM, Spearow JL, Lloyd KC K. Assessment of three generations of mice derived by ICSI using freeze-dried sperm. Zygote 2009; 17(3): 239-51. [CrossRef] google scholar
4. 4. Songsasen N, Leibo SP. Cryopreservation of mouse spermatozoa II. Relationship between survival after cryopreservation and os-motic tolerance of spermatozoa from three strains of mice. Cryo-biology 1997; 35: 255-69. [CrossRef] google scholar
5. 5. Sztein JM, Farley JS, Mobraaten LE. In vitro fertilization with cryo-preserved inbred mouse sperm. Biol Reprod 2000; 63: 1774-80. [CrossRef] google scholar
6. 6. Nishizono H, Shioda M, Takeo T, Irie T, Nakagata N. Decrease of fer-tilizing ability of mouse spermatozoa after freezing and thawing is related to cellular injury. Biol of Reprod 2004; 71: 973-8. [CrossRef] google scholar
7. 7. Dandekar PV, Glass RH. Development of mouse embryos in vitro is affected by strain and culture medium. Gamete Res 1978; 17: 279-85. [CrossRef] google scholar
8. 8. Ogonuki N, Mori M, Shinmen A, Inoue K, Mochida K, Ohta A, et al. The effect on intracytoplasmic sperm injection outcome of geno-type, male germ cell stage and freeze-thawing in mice. PLoS One 2010; 5(6): e11062. [CrossRef] google scholar

9. 9. Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 1998; 394, 369-74. [CrossRef] google scholar
10. 10. Taskin AC, Kocabay A, Gül S, Caglar-Erkal K. Comparison of parthe-nogenetic oocyte activation in different mouse strains on in vitro development rate and quality. Vet Hekim Der Derg 2021; 92(1): 42-8. google scholar
11. 11. Wang Y, Yamauchi Y, Wang Z, Zheng H, Yanagimachi R, Ward MA, et al. Both Cauda and Caput Epididymal Sperm Are Capable of Supporting Full-Term Development in FVB and CD-1 Mice. Dev Cell 2020; 55(6): 675-6. [CrossRef] google scholar
12. 12. Mallol A, Santalo J, Ibanez E. Psammaplin a improves develop-ment and quality of somatic cell nuclear transfer mouse embryos. Cell Reprogram 2014;16(5): 392-406. [CrossRef] google scholar
13. 13. Fernandez-Gonzalez R, Laguna R, Ramos-Ibeas P, Pericuesta E, Alcalde-Lopez V, Perez-Cerezales S, et al. Successful ICSI in Mice Using Caput Epididymal Spermatozoa. Front Cell Dev Biol 2019; 7: 346. [CrossRef] google scholar
14. 14. Stein P and Schultz RM. ICSI in the mouse. Methods Enzymol 2010; 476: 251-62. [CrossRef] google scholar
15. 15. Ohta H, Sakaide Y and Wakayama T. Age and substrain-dependent sperm abnormalities in BALB/c mice and functional assessment of abnormal sperm by ICSI. Hum Reprod 2009; 24(4): 775-81. [Cross-Ref] google scholar
16. 16. Ogonuki N, Mori M, Shinmen A, Inoue K, Mochida K, Ohta A, et al. The effect on intracytoplasmic sperm injection outcome of geno-type, male germ cell stage and freeze-thawing in mice. PLoS One, 2010; 5(6): e11062. [CrossRef] google scholar
17. 17. Kawase Y, Iwata T, Toyoda Y, Wakayama T, Yanagimachi R and Su-zuki H. Comparison of intracytoplasmic sperm injection for inbred and hybrid mice. Mol Reprod Dev 2001; 60(1): 74-8. [CrossRef] google scholar
18. 18. Fernandez-Gonzalez R, Laguna R, Ramos-Ibeas P, Pericuesta E, Alcalde-Lopez V, Perez-Cerezales S, et al. Successful ICSI in Mice Using Caput Epididymal Spermatozoa. Front Cell Dev Biol 2019; 7: 346. [CrossRef] google scholar
19. 19. Moreira PN, Giraldo P, Cozar P, Pozueta J, Jimenez A, Montoliu L, et al. Efficient generation of transgenic mice with intact yeast artificial chromosomes by intracytoplasmic sperm injection. Biol Reprod 2004; 71(6): 1943-7. [CrossRef] google scholar