Characterization of Novel Long Non-coding RNAs and Their Roles as Indicators of Oocyte Quality in Cattle

Oocyte quality remains an undefined obscure topic in reproductive research. The goal of this research was to take a new approach to tackle this decades-old problem. As technology has increased more and more aspects of everyday life have started to utilize technology and take a turn more towards science. The goal was to incorporate molecular physiology with whole animal applications by investigating long noncoding RNAs (lncRNAs) and their potential roles in bovine reproduction specifically, oocyte quality. In the first year and a half of funding, I successfully verified the presence and differential expression of six highly abundant lncRNAs in single bovine oocytes aspirated from small and large follicles at varying developmental stages. More specifically, I documented the significance of the maturity stage in all six lncRNAs and graphically showed their accumulation and degradation during maturation. When examining each for the interaction of follicle size and maturity stage, there was a significant interaction in the expression of OOSNCR5 (P < 0.05) and a tendency for significant interaction in the expression of OOSNCR2. The literature indicates that follicle size can be an indicator of oocyte quality and that RNAs accumulated or degraded during maturation are crucial to achieving developmental competence. Therefore, lncRNAs OOSNCR2 and OOSNCR5 show promise to be linked with oocyte quality. 

My second year and a half of funding were met with challenges. As the pandemic continued to plague our world, researchers like everyone else have learned to roll with the punches and push forward.  Due to travel restrictions and various shutdowns, our lab had to shut down the in vitro embryo production portion of our lab. With the shutdown, we were unable to collect ovaries from the abattoir completely halting all projects. After a year and six months, we were finally approved to travel again. Sample collections began in September 2021.

In the last three months, we traveled bi-weekly to the commercial abattoir in hopes of producing data again. I successfully produced and quantified a tissue expression profile, verifying the oocyte-specificity of three lncRNAs and possibly an additional two. Various tissue samples (n = 26) were collected from freshly slaughtered beef cattle. Real-time qPCR analysis was performed and lncRNA expression was examined using a one-way ANOVA followed by Tukey's HSD post hoc test. In addition, follicular cells; cumulus, granulosa, and theca cells were collected from individual follicles. Follicular cells were subjected to qPCR analyzed using a one-sided t-test. When investigating lncRNA expression in various tissues, three lncRNAs were expressed solely in oocytes [fetal ovaries] with one lncRNA being detected in the adult ovary and one showing variable expression. Minimal expression was detected in the granulosa and theca cells. LncRNA expression in the cumulus cells (not included in the presented data) exhibits patterns similar to those seen in the maturity stage of single oocytes. Based on these data, we conclude lncRNAs OOSNCR1, OOSNCR3, and OOSNCR5 are oocyte-specific. Future investigations will focus on characterizing the mechanisms for the three oocyte-specific lncRNAs and their roles in oocyte maturation and early embryonic development.

Due to time constraints, objective 1 was unable to be completed. In situ hybridization is scheduled to start in the very near future. With regards to objective 3, it was unable to be completed, again due to time constraints and lack of sample collections for over a year. All supplies have been purchased and proper training has gotten underway to perform microinjection to elucidate the functional mechanisms utilized by specific lncRNAs. In the last month, preliminary data has been collected for three additional studies.

The first study utilized Brilliant Cresyl Blue (BCB) staining for the selection of immature oocytes before in vitro embryo production. Previously, oocytes have been selected as "good or bad" based on morphological assessment including the compactness and number of cumulus cells surrounding the oocyte, follicle size, cytoplasmic morphology, and oocyte size. All of which have proven to be highly subjective and difficult to replicate across laboratories. The need for a standardized measure of oocyte quality is more than obvious. However, a standardized measurement remains elusive. It is widely believed that glucose-6-phosphate dehydrogenase (G6PDH) is actively produced in growing oocytes, but its activity is decreased in oocytes that have finished their growth and achieved developmental competence. The enzyme G6PDH can degrade BCB thus fully grown [competent] oocytes show a blue cytoplasm (BCB+) after BCB staining while growing [immature] oocytes have high levels of G6PDH and exhibit a colorless cytoplasm (BCB-) after staining. BCB has previously been published in a wide variety of species as a marker of oocyte maturity. It has been published that BCB+ oocytes display a higher blastocyst developmental rate than BCB- and control oocytes in pig (Roca et al., 1998), goat (Rodriguez-Gonzalez et al., 2002)), sheep (Catala et al., 2011), mouse (Wu et al., 2007), dog (Rodrigues et al. 2009), and bovine (Bhojwani et al., 2007). Preliminary data suggests lncRNA expression displays an opposite pattern than expected with lower lncRNA expression in the BCB+ oocytes. Possibly suggests the selected lncRNA are necessary for oocyte growth. The data associated with BCB staining will be used as a puzzle piece that hopefully will help elucidate the functions of these unknown genes.

The second preliminary study conducted was to determine if the specific lncRNAs are maternal transcripts. During early embryogenesis, maternal mRNAs that accumulated in the oocyte during oogenesis, play important roles in embryonic genome activation (Hamatani et al., 2004). Some maternal transcripts are oocyte-specific and known as maternal effect genes which are required for the early cleavage events post-fertilization. Alpha amanitin is a selective inhibitor of RNA polymerase II. By culturing embryos in the presence of alpha amanitin maternal transcripts can be differentiated from embryonic transcripts. Preliminary data suggests a complicated relationship and a possible negative regulation by a maternal transcript. 

The third and final study on the horizon is to further investigate the bidirectional communication between the oocyte and its surrounding nurse [cumulus] cells. With the recent focus on transzonal projections (TZPs), there is compelling data to suggest that these cellular projections may be involved in the transporting and delivery of large molecules to the oocyte. It is well accepted that endogenous transcription of the oocyte is silent during oocyte maturation. Accumulation of transcripts in the oocyte during this time would support the potential transfer of material from the cumulus cells to the oocyte. Preliminary data collected under specific aim 2 supports the accumulation of predicted lncRNAs occurred during oocyte maturation. Those that were accumulated provide valid reasoning for selection to investigate their roles in the bidirectional communication between the cumulus cells and the oocyte. Data [unpublished] collected from immature and expanded cumulus cells display identical lncRNA expression accumulation and degradation patterns to those seen in single oocytes at corresponding maturity stages. Although preliminary, these data suggest a possible relationship between the two cell types that require further investigation.

This experiment may seem like a stretch for sustainable agriculture, especially discussing gene expression data. However, studies of this nature are the first steps necessary in determining potential bio-markers that can one day be utilized to predetermine which oocytes will produce viable embryos. By screening oocytes and embryos prior to fertilization and transfer farmers will be able to select the highest quality gametes. Once the basic research has been conducted, optimization techniques can be performed to translate what we've learned in the lab into a practical farm application. Although we will never fully understand the mechanism of how life begins, well-characterized biomarkers give both farmers and veterinarians the best possible scenario to invest their money in.