Author ORCID Identifier

https://orcid.org/0000-0003-0419-284X

Semester

Spring

Date of Graduation

2023

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Animal and Nutritional Sciences

Committee Chair

Jianbo Yao

Committee Member

Robert Dailey

Committee Member

Ida Holásková

Committee Member

Melanie Clemmer

Committee Member

I-Chung Chen

Abstract

Early embryonic loss is a significant factor in livestock species' infertility, resulting in an economic deficit. In cattle, the in vivo fertilization rate is ~90%, with an average calving rate of about 55%, indicating an embryonic-fetal mortality rate of roughly 35%. Further, 70-80% of total embryonic loss in cattle occurs during the first three weeks after insemination, particularly between days 7-16. Growing evidence indicates that the oocyte plays an active role in regulating critical aspects of the reproductive process required for successful fertilization, embryo development, and pregnancy. However, defining oocyte quality remains enigmatic. Recently, many have abandoned the notion that one transcript or gene network modulates oocyte competence. Instead, it is speculated that a vast network of transcripts regulates gene expression.

With the advent of deep sequencing technology, it was discovered that roughly 1.2% of the human genome represents protein-coding exons, whereas the remaining classifies as non-coding RNA. What once was thought of as “genetic noise” from leaky transcriptional machinery has more recently come to the foreground of modern research in molecular biology due to its broad versatility in regulating gene expression. Specifically, long non-coding RNAs (lncRNAs) have been reported to play critical roles in various biological processes. Despite their gaining popularity, most lncRNA studies focus on identifying differentially expressed lncRNAs throughout bodily systems and are left to predict their functional roles using bioinformatic and comparative analyses. Recently, lncRNAs have been identified as critical regulators of embryonic genome activation in humans, mice, pigs, goats, and rabbits. Further investigations of lncRNAs in mouse oocytes and early embryos have revealed essential roles in regulating oocyte maturation and early embryonic development. However, the functional role of lncRNAs in bovine oocytes remains to be elucidated.

Previously, using RNA sequencing, our laboratory identified 1,535 lncRNAs present in bovine oocytes. The top three candidate genes, OOSNCR1, OOSNCR2, and OOSNCR3, were characterized in bovine somatic tissues, the cells within the ovarian follicle, and throughout early embryonic development. Our data revealed that OOSNCR1 and OOSNCR2 are oocyte-specific, with OOSNCR3 being highly abundant in the fetal ovary and detected at low levels in the spleen. Follicular cell expression revealed that all three lncRNAs were detected throughout the follicle. Further, all three lncRNAs were expressed highest in the oocyte, decreasing expression as the distance from the oocyte increased. Moreover, expression throughout oocyte maturation and early embryonic development revealed that OOSNCR1, OOSNCR2, and OOSNCR3 were highest during oocyte maturation, decreased at fertilization, and ceased altogether by the 16-cell stage. Collectively, the expression data suggested all three transcripts were maternal effect genes. Maternal origin was confirmed using an RNA polymerase II inhibitor, α-amanitin.

The functional role of OOSNCR1, OOSNCR2, and OOSNCR3 during oocyte maturation and early embryonic development was evaluated using siRNA-mediated knockdown. Injection of the cumulus-enclosed germinal vesicle (GV) oocyte did not affect cumulus expansion; however, oocyte survival at 12 hours post-insemination was significantly reduced following the microinjection procedure. Additionally, lncRNA knockdown decreased the relative abundance of maternal effect genes NPM2, GDF9, BMP15, and JY-1 and resulted in blastocyst rates close to zero. Using siRNA-mediated knockdown in the presumptive zygote, the percentage of embryos reaching the blastocysts stage was decreased by roughly half for all three lncRNAs.

The potential relationship between lncRNA expression and oocyte quality was investigated. In addition to OOSNCR1, OOSNCR2, and OOSNCR3, OOSNCR4 and OOSNCR5 were selected from the RNA sequencing dataset as highly abundant lncRNAs in bovine oocytes. All lncRNAs were quantified in oocytes of various qualities. Specifically, lncRNA expression was examined in oocytes (1) collected from small and large follicles before and after maturation, (2) differentially stained using brilliant cresyl blue (BCB), and (3) exposed to heat stress (410C) during oocyte maturation. Data revealed that OOSNCR1 and OOSNCR3 were accumulated during maturation, whereas OOSNCR2 and OOSNCR4 were degraded. Further, OOSNCR1, OOSNCR2, and OOSNCR4 were more abundant in oocytes collected from small follicles. Specifically, OOSNCR2 and OOSNCR4 were expressed highest in immature oocytes. Conversely, OOSNCR3 was more abundant in mature oocytes collected from large follicles. Following BCB staining, OOSNCR3 was expressed lower in BCB+ oocytes. Finally, maturation in a heat-stressed environment decreased cumulus cell expansion. Heat stress during maturation also caused OOSNCR1 to decrease expression, whereas OOSNCR3, OOSNCR4, and OOSNCR5 expression increased.

Overall, the data herein revealed dynamic expression profiles of novel lncRNAs and suggests a functional requirement of OOSNCR1, OOSNCR2, and OOSNCR3 during bovine oocyte maturation and early embryogenesis.

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