Degree Type

Dissertation

Date of Award

2017

Degree Name

Doctor of Philosophy

Department

Animal Science

Major

Genetics and Genomics

First Advisor

Jason W. Ross

Abstract

This dissertation describes two processes that the oocyte and ovary potentially utilize to maintain reproductive competence: autophagy in response to heat stress and microRNA-21 function during meiotic maturation. Heat stress (HS) occurs when heat accumulation (from internal and external sources) exceeds heat dissipation. HS is associated with seasonal infertility and therefore is a production issue and profitability constraint in the swine industry. Autophagy is the process by which somatic cells recycle cellular components and it is activated by a variety of stressors. Therefore, characterizing autophagy in the ovary and oocyte is valuable because of the potential of autophagy to mitigate the detrimental effects of HS. Additionally, we also characterized the function of a specific microRNA (miRNA), mircroRNA-21 (MIR21), during oocyte maturation and the breakdown of the germinal vesicle (GVBD). The developmental competence of the oocyte is determined by molecular events that occur up to and during GVBD, though there is almost entirely no transcription occurring during GVBD. Thus, the maturing oocyte is reliant on post-transcriptional gene regulation (PTGR) and/or interactions with the surrounding cumulus cells to regulate the mRNA repertoire and proteome prior to fertilization. We hypothesized that miRNA is active within the oocyte and its biogenesis is dependent on GVBD.

To characterize the effect of HS on autophagy induction in the ovary and oocyte, we utilized both an in vivo model as well as in vitro model. Twelve gilts were synchronized and subjected to cyclical HS (n = 6) or thermal neutral (TN; n = 6) conditions for five days during the follicular phase. The abundance of autophagy-related proteins in total ovarian protein was compared between gilts that had either experienced TN conditions for 5 days or HS conditions for 5 days. Ovarian tissue was fixed and sectioned to compare the localization of autophagy-related proteins in the ovary between gilts that had either experienced TN or HS conditions for 5 days. Based on the effects of HS on cell morphology within the follicle and changes in autophagy-related proteins, autophagy induction occurs in response to HS during the follicular phase. To further characterize the autophagy response directly in the pig oocyte, cumulus-oocyte-complexes were aspirated from 2-4 mm follicles and subjected to different temperature treatments during in vitro maturation (IVM). The oocytes experienced either TN conditions throughout the entire IVM, or HS during the first or second half of IVM. The abundance of autophagy-related proteins was compared between metaphase II (MII) arrested oocytes, and the cleavage of LC3 was compared at different time points during IVM. This data suggests autophagy as a potential mechanism activated that the oocyte could use during environmental stress to recycle damaged cellular components to maintain developmental competence.

IVM was also utilized to characterize the function of a specific miRNA, mircroRNA-21 (MIR21), during oocyte maturation and GVBD. MiRNA, a class of functional small RNA, interact with the 3′ untranslated region (UTR) of target mRNA to affect their abundance and translational efficiency. Of particular importance is miRNA-21 due to its role in regulating programmed cell death 4 (PDCD4), and ultimately inhibiting apoptosis. To characterize the function of MIR21 in relation to GVBD, pig oocytes collected from aspirated 2-4 mm follicles experienced IVM at normal conditions or in the presence of a chemical inhibitor of GVBD, 3-isobutyl-1-methylxanthine (IBMX). Oocytes were collected at different time points of IVM with or without IBMX. The state of GVBD was compared to the abundance of mature MIR21, abundance of PDCD4, and activation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB). The cumulative results of this study suggest that MIR21 is activated and important during meiotic maturation of the oocyte, and that NF-κB is potentially driving MIR21 increase in the oocyte.

Copyright Owner

Benjamin J. Hale

Language

en

File Format

application/pdf

File Size

177 pages

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