Colorado State University
Analysis of equine zygote development after intracytoplasmic sperm injection.
Degree: PhD, Biomedical Sciences, 2016, Colorado State University
Intracytoplasmic sperm injection (ICSI) is an established and widely used method to achieve oocyte fertilization in equine reproductive assisted technologies. However, not all the oocytes fertilized by ICSI undergo cleavage and develop into viable embryos. Limited knowledge on equine zygote development after ICSI is available, and reasons why developmental failure occurs after ICSI have been only partially studied and need further investigation. Fertility decline and early embryo loss is associated with maternal aging in the mare, and it is concomitant with reduced oocyte quality. Relatively little is known about the effect of maternal aging and zygote developmental failure or success in the mare. Effects of in vitro maturation of the oocyte or zygote development in the mare still need to be clarified and further studied. The overall objective of this dissertation was to study equine zygote development after ICSI using confocal microscopy. Objectives were to: (1) compare cytoskeletal and nuclear changes during progression of equine zygote development after ICSI for in vivo versus in vitro matured oocytes; (2) compare changes in cytoskeletal and chromosomal configurations after ICSI between oocytes from young and old mares to define maternal-aging related alterations; (3) determine cytoskeletal and nuclear alterations associated with fertilization failure in ICSI-produced presumptive zygotes in young and old mares; (4) determine cell-aging and cell donor-aging effects on cytoskeleton and chromatin configurations. Specifically, in our studies we evaluated the tubulin and actin cytoskeleton, chromatin, and kinetochores/centromeres. Immunostaining and confocal imaging of the equine zygotes was performed using a spinning disk confocal microscope. After ICSI, five distinct events of development were observed with no major differences over time whether oocytes matured in vivo or in vitro. Oocytes matured in vivo appeared to reach the pronucleus stage earlier after ICSI compared to in vitro matured oocytes. Abnormal phenotypes associated with fertilization failure were more significant in oocytes matured in vitro than in vivo. When ICSI was performed in oocytes from young and old mares, similar stages of zygote development were observed, and the number of zygotes reaching the pronucleus stage was similar between the two age groups. Nucleolus like bodies, sites of ribosomal RNA involved in embryonic genome activation, were observed only in zygotes at the pronucleus stage from young mares; no nucleolus-like bodies were observed in pronuclei of zygotes from old mares. Pronuclei morphology, based on CREST staining, and DNA localization, also differed between pronuclei of young and old mares. Actin vesicles were observed significantly more often within zygotes from old mares compared to young mares during all stages of zygote developmental progression. When potential zygotes were analyzed after failure of cleavage after ICSI, actin vesicles were greater in area, perimeter and number in oocytes from old mares than those…
Advisors/Committee Members: Carnevale, Elaine (advisor), Clay, Colin (advisor), Albertini, David (committee member), DeLuca, Jennifer (committee member), Seidel, George (committee member).
Subjects/Keywords: cytoskeleton; intracytoplasmic sperm injection; zygote; equine; confocal microscopy; maternal aging
to Zotero / EndNote / Reference
APA (6th Edition):
Ruggeri, E. (2016). Analysis of equine zygote development after intracytoplasmic sperm injection. (Doctoral Dissertation). Colorado State University. Retrieved from http://hdl.handle.net/10217/173335
Chicago Manual of Style (16th Edition):
Ruggeri, Elena. “Analysis of equine zygote development after intracytoplasmic sperm injection.” 2016. Doctoral Dissertation, Colorado State University. Accessed April 18, 2021.
MLA Handbook (7th Edition):
Ruggeri, Elena. “Analysis of equine zygote development after intracytoplasmic sperm injection.” 2016. Web. 18 Apr 2021.
Ruggeri E. Analysis of equine zygote development after intracytoplasmic sperm injection. [Internet] [Doctoral dissertation]. Colorado State University; 2016. [cited 2021 Apr 18].
Available from: http://hdl.handle.net/10217/173335.
Council of Science Editors:
Ruggeri E. Analysis of equine zygote development after intracytoplasmic sperm injection. [Doctoral Dissertation]. Colorado State University; 2016. Available from: http://hdl.handle.net/10217/173335