Cumulus Cell and Oocyte Gene Expression in Prepubertal Gilts and Sows Identifies Cumulus Cells as a Prime Informative Parameter of Oocyte Quality

Abstract

Cumulus cells (CCs) are pivotal during oocyte development. This study aimed to identify novel marker genes for porcine oocyte quality by examining the expression of selected genes in CCs and oocytes, employing the model of oocytes from prepubertal animals being of reduced quality compared to those from adult animals. Total RNA was extracted either directly after follicle aspiration or after in vitro maturation, followed by RT-qPCR. Immature gilt CCs accumulated BBOX1 transcripts, involved in L-carnitine biosynthesis, to a 14.8-fold higher level (p < 0.05) relative to sows, while for CPT2, participating in fatty acid oxidation, the level was 0.48 (p < 0.05). While showing no differences between gilt and sow CCs after maturation, CPT2 and BBOX1 levels in oocytes were higher in gilts at both time points. The apparent delayed lipid metabolism and reduced accumulation of ALDOA and G6PD transcripts in gilt CCs after maturation, implying downregulation of glycolysis and the pentose phosphate pathway, suggest gilt cumulus–oocyte complexes have inadequate ATP stores and oxidative stress balance compared to sows at the end of maturation. Reduced expression of BBOX1 and higher expression of CPT2 in CCs before maturation and higher expression of G6PD and ALDOA after maturation are new potential markers of oocyte quality.

Epigenetic-related transcriptional reprogramming elucidated by identification and validation of a novel reference gene combination for RT-qPCR studies in porcine oocytes of contrasting quality

“…Genes encoding enzymes involved in glycolysis are considered good reference gene candidates [ 44 ]. PFKP, a rate limiting enzyme for glycolysis [ 45 ], did not demonstrate any significant change in transcript abundance during porcine COC maturation in a similar study [ 30 ]. NormFinder identified PFKP to be the most stably expressed single gene (Table 2 ) and ACTB and PFKP as the best combination of two reference genes, generating an acceptable stability value of 0.149.…”

“…They concluded, as in agreement with the present study, oocytes collected from premature animals had not completed accumulation of mRNA prior to in vitro maturation [ 1 , 31 ] nor DNA methylation reprogramming at the end of maturation [ 31 ]. Oocytes collected from prepubertal gilts indicated delayed metabolism compared to oocytes from sows [ 30 ], which could, in addition to the above-mentioned factors, influence the establishment of epigenetic modifications that require the availability of different metabolic intermediates [ 49 , 50 ]. The delayed accumulation of transcripts involved in epigenetic reprogramming in oocytes collected from premature animals could lead to failed fertilization, aneuploidy and arrested or abnormal embryonic development [ 3 , 4 ].…”

“…As a model for oocytes of low and high developmental competence [ 8 , 26 – 29 ], immature COCs were collected from prepubertal gilts and sows. Ovaries were selected as described in Haug et al [ 30 ]. The phrasing prepubertal gilts refers to young pigs that have not yet had a litter.…”